PRENYLATED CHALCONE AND FLAVONOID COMPOSITIONS FOR USE IN THE TREATMENT OF CANCER - Patent application

Abstract

Compositions comprising xanthohumol and isoxanthohumol and their uses in the treatment of cancer, for example, tyrosine kinase inhibitor-resistant cancers, are provided.

Description

[Background technology]

[0001] Currently, cancer treatments mainly consist of surgery, chemotherapy, radiation therapy, targeted therapy, gene therapy, and immunotherapy, which traditionally target a single biological target. However, the curative efficacy of these treatments has so far been limited by certain tumor characteristics.

[0002] Drug resistance inevitably limits the efficacy of all targeted therapies. For tumor cells, drug resistance to tyrosine kinase inhibitors (TKIs) represents a major obstacle in both solid tumors and leukemia / lymphoma. Tumor cells can be TKI-sensitive or TKI-resistant, exhibiting intrinsic or acquired resistance and accumulating alterations within or outside the target that promote their survival.

[0003] Xanthohumol is a prenylated chalcone derived from hops, more specifically female hop plants. Xanthohumol exhibits a wide range of biological activities, including the regulation of key transcription factors, but is primarily known for its antioxidant activity. It is therefore believed to be useful in the treatment of diseases associated with oxidative stress, such as cancer, diabetes, and dyslipidemia. Summary of the Invention [Problem to be solved by the invention]

[0004] Current xanthohumol-containing products have at least two drawbacks. First, the bioavailability of xanthohumol in these products is very low due to its poor water solubility. Second, xanthohumol products often contain significant amounts of isoxanthohumol, which results from the degradation of xanthohumol during the manufacturing (e.g., heating process) or storage of the xanthohumol-containing product. Therefore, there is a need to develop new formulations that contain large amounts of bioavailable xanthohumol for the treatment of cancers, including those that are drug resistant. [Means for solving the problem]

[0005] Disclosure Summary The present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of xanthohumol and isoxanthohumol, and at least one pharma- ceutically acceptable carrier. In one embodiment, the composition further comprises 6-prenylnaringenin and 8-prenylnaringenin. In another embodiment, xanthohumol is present in an amount of at least 50-99% w / w, isoxanthohumol is present in an amount of 1-15% w / w, 6-prenylnaringenin is present in an amount of 0.0-5%, and 8-prenylnaringenin is present in an amount of 0.0-5%.

[0006] In one embodiment, at least one of xanthohumol, isoxanthohumol, 6-prenylnaringenin, or 8-prenylnaringenin is extracted from a plant. In another embodiment, the plant is hops, spent hops, or a product containing hops. In another embodiment, at least one of xanthohumol, isoxanthohumol, 6-prenylnaringenin, or 8-prenylnaringenin is chemically synthesized.

[0007] In one embodiment, the composition of the present disclosure includes a pharma- ceutically acceptable carrier that is at least one of a binder, a disintegrant, a surfactant, or a lubricant. In another embodiment, the binder is one or more of starch 1500, polyvinylpyrrolidone, and microcrystalline cellulose. In another embodiment, the binder is present in an amount of 10-30% w / w. In another embodiment, the disintegrant is croscarmellose sodium. In another embodiment, the disintegrant is present in an amount of 1-5% w / w. In another embodiment, the surfactant is sodium dodecyl sulfate. In another embodiment, the surfactant is present in an amount of 1-5% w / w. In another embodiment, the lubricant is magnesium stearate. In another embodiment, the lubricant is present in an amount of 0.5-3% w / w.

[0008] The present disclosure also provides a method for preparing a composition comprising xanthohumol and isoxanthohumol from hops, comprising the steps of: suspending the hops plant material in n-heptane to remove non-polar impurities; Evaporating the heptane and filtering the residue; Extracting the residue with ethyl acetate and collecting the xanthohumol-containing fractions on a rotary evaporator; Extraction of xanthohumol with organic acid solution containing 0.05M ZnCl2, 5% NaHCO3 and saline; Dry the organic layer over anhydrous Na2SO4; Precipitating xanthohumol from a mixture containing ethyl acetate; and drying the precipitated xanthohumol and isoxanthohumol. The present disclosure also relates to a composition comprising xanthohumol and isoxanthohumol obtained by this method.

[0009] The present disclosure also relates to a method of agonizing farnesoid X receptor activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition described herein.In one embodiment, agonizing farnesoid X receptor activity results in the treatment of cancer.In another embodiment, the cancer is resistant to tyrosine kinase inhibitors.

[0010] The present disclosure also relates to a method for inhibiting NFκB activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition described herein.In one embodiment, inhibiting NFκB activity results in the treatment of cancer.In another embodiment, the cancer is resistant to tyrosine kinase inhibitors.

[0011] The present disclosure also relates to a method of modulating expression and / or activation of nuclear factor erythroid 2-related factor 2 (NRF2) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition described herein. In one embodiment, modulating expression and / or activation of nuclear factor erythroid 2-related factor 2 (NRF2) results in the treatment of cancer. In another embodiment, the cancer is resistant to a tyrosine kinase inhibitor.

[0012] The present disclosure also relates to a method for inducing apoptosis in a cell, comprising contacting the cell with an effective amount of the composition described herein.In one embodiment, the cell is a leukemia cell.In another embodiment, the cell comprises bcr-abl gene mutation.In another embodiment, the cell is a patient cell with chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), or acute myelogenous leukemia (AML).

[0013] The present disclosure also relates to a method for treating chronic myeloid leukemia (CML) in a subject, comprising administering to the subject a therapeutically effective amount of the composition described herein.In one embodiment, the CML is resistant to treatment with a tyrosine kinase inhibitor (TKI).In another embodiment, the TKI is imatinib, dasatinib, or ponatinib.

[0014] The present disclosure also relates to a method of reducing the incidence of secondary tyrosine kinase inhibitor (TKI) resistance in a subject, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0015] The present disclosure also relates to a method for treating BCR-ABL-independent resistant cancer in a subject, comprising administering to the subject a therapeutically effective amount of the composition described herein. In one embodiment, the BCR-ABL-independent resistance is caused by inhibiting CRKL and STAT5 phosphorylation, or inhibiting the persistent phosphorylation of the translation regulator ribosomal protein S6 (RPS6), which indicates activation of mTOR complex 1 (mTORC1).

[0016] In one embodiment of the present disclosure, the method further comprises administering a TKI to the subject.In another embodiment, the TKI is administered after the composition.In another embodiment, the TKI is imatinib, dasatinib, ponatinib or nilotinib.

[0017] In one embodiment of the disclosure, the method further comprises administering a cannabinoid to the subject. In another embodiment, the cannabinoid is administered after the composition. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows a block diagram of a process for purifying drug substance, including xanthohumol, from remyelin. [Figure 2A-2C] 2A-2C show the particle size distributions of XN-54 (FIG. 2A), XN-63-10 (FIG. 2B), and XN-87 (FIG. 2C). [Figure 3A-3B] 3A-3B show differential scanning calorimetry (DSC) of batch XN-54 (FIG. 3A) and DS batch XN-87 (FIG. 3B). [Figure 4A-4B] 4A-4B show the thermogravimetric analysis of batch XN-54 (FIG. 4A) and batch XN-87 (FIG. 4B). [Figure 5A-5B] 5A-5B show the Fourier transform infrared analysis (FTIR) of batches XN-54 (FIG. 5A) and XN-87 (FIG. 5B). [Figure 6A-6B] 6A-6B show the powder X-ray diffraction grams of batch XN-54 (FIG. 6A) and batch XN-87 (FIG. 6B). [Figure 7A-7B] 7A-7B show pCrkl (FIG. 7A) and BCR-ABL (FIG. 7B) levels in K562-DR 1000 nM cells. [Figure 8A-8B] Figures 8A-8B show the effect of increasing concentrations of XN-54 or dasatinib on cell viability at 48 hours (Figure 8A) and 72 hours (Figure 8B) in K562-IS, -IR, and -DR cells. [Figure 9A-9B] 9A-9B show XN-54-induced apoptosis in K562-IS, -IR, and -DR cells, measured at 48 hours (FIG. 9A) and 72 hours (FIG. 9B) using early apoptotic markers. [Figure 10A-10B] 10A-10B show XN-54-induced apoptosis in K562-IS, -IR, and -DR cells, measured at 48 hours (FIG. 10A) and 72 hours (FIG. 10B) using late apoptotic markers. [Figure 11A-11B]Figures 11A-11B show the effect of the combination of XN-54 and ponatinib on cell viability in K562-IR cells based on either log10(M) XN-54 (Figure 11A) or ponatinib (Figure 11B). [Figure 12A-12B] Figures 12A-12B show the effect of the combination of XN-54 and nilotinib on cell viability in K562-IR cells based on either log10(M) nicotinib (Figure 12A) or XN-54 (Figure 12B). [Figures 13A-13C] Figures 13A-13C show the effect of a combination of XN-54 (Compound 1) and cannabidiol (CBD) (Compound 2) on cell viability in K562-DR (Figure 13A), K562-IS (Figure 13B), and K562-IR cells (Figure 13C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description The present disclosure relates to compositions comprising xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin, and their use in the treatment of cancer. In some embodiments, the compositions are useful in the treatment of leukemia and solid tumors. In other embodiments, the compositions are useful in the treatment of drug-resistant cancer. General definition So that this disclosure may be more readily understood, certain terms are first defined. As used herein, unless expressly defined otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the specification.

[0020] It should be noted that the terms "a" or "an" refer to one or more of that entity; for example, a "feed medium" is understood to represent one or more feed media. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0021] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or elements, regardless of the presence or absence of the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0022] Whenever an embodiment is described herein using the term "comprising," it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0023] 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 disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary of many of the terms used herein.

[0024] Units, prefixes, and symbols are shown in the accepted format of the International System of Units (SI). Numerical ranges include the numerical values ​​that define the range. The headings provided herein are not intended to limit the various aspects of the disclosure, which aspects may be understood by reference to the entire specification. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0025] The use of the alternatives (e.g., "or") should be understood to mean either, both, or a combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to indicate "one or more" of any recited or enumerated components.

[0026] The term "about" or "essentially includes" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measurement system. For example, "about" or "essentially includes" may mean within or more than one standard deviation per practice, as is customary in the art. Alternatively, "about" or "essentially includes" may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude greater or up to 5 times the value. When a particular value or composition is provided in the application and claims, unless otherwise specified, the meaning of "about" or "essentially includes" should be assumed to be within an acceptable error range for that particular value or composition.

[0027] As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise specified, should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers).

[0028] Pharmaceutical Compositions In the context of the present disclosure, the term "xanthohumol" should be understood to mean a prenylated chalconoid obtained from plants such as hops, spent hops, or hop products, and is represented by the following formula: [ka]

[0029] Xanthohumol as used in the present disclosure may be a commercially available pure form of the molecule or may be a (concentrated) extract obtained from a suitable source such as a plant.

[0030] In the context of the present disclosure, the term "isoxanthohumol" should be understood as the corresponding prenylated flavanone of xanthohumol, represented by the following formula: [ka]

[0031] 6-Prenylnaringenin is a trihydroxyflavanone with the structure of naringenin prenylated at C-6. It is a trihydroxyflavanone, a member of the 4'-hydroxyflavanone family, and a (2S)-flavan-4-one. It is derived from (S)-naringenin. It has the following formula: [ka]

[0032] 8-Prenylnaringenin or Sophoraflavanone B is a trihydroxyflavanone, which is (S)-naringenin with a prenyl group at the 8-position. It has a role as a platelet aggregation inhibitor and a plant metabolite. It is a trihydroxyflavanone, a member of the 4'-hydroxyflavanones, and a (2S)-flavan-4-one. It is derived from (S)-naringenin. It is the conjugate acid of Sophoraflavanone B(1-). It has the following formula: [ka]

[0033] As used herein, terms such as xanthohumol, isoxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin include derivatives such as hydroxylated and sulfated prenylated flavonoids, or may be polymorphs or may be in solid or liquid physical form. For example, the compounds may be in crystalline form, amorphous form, and may have any particle size. The particles may be micronized, agglomerated, particulate granules, powders, oils, oily suspensions, or any other form that is in solid or liquid physical form.

[0034] The methods described in the literature for extracting and purifying xanthohumol, isoxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin from plant materials have many drawbacks. For example, large amounts of organic solvents, especially highly toxic solvents (dichloromethane, hexane, chloroform), are required for efficient extraction. Current methods also use large amounts of expensive materials such as silica gel. This method causes irreversible decomposition of xanthohumol to produce by-products (mainly isomeric isoxanthohumol) and polymeric oxidative degradation products (extraction with aqueous solutions of strong bases, e.g., NaOH, KOH). Countercurrent chromatography techniques have low process efficiency and scalability. Most importantly, it is nearly impossible to obtain xanthohumol with high pharmaceutical purity (minimum 95% by weight). However, the present disclosure provides a method for preparing xanthohumol with an HPLC purity of more than 95%.

[0035] Known methods for obtaining xanthohumol, etc., by chemical synthesis are inefficient and not very scalable, and furthermore, the final stage of production does not allow for economically viable preparation of large quantities of xanthohumol of pharmaceutical purity.

[0036] In some embodiments, the present disclosure provides a method for preparing xanthohumol from a plant. In some embodiments, the plant is hops. Hops or Humulus lupulus is a climbing vine that belongs to the Cannabaceae family, Urticales order, and the Hops genus. Early taxonomists included the genus Humulus in the mulberry family (Moraceae).

[0037] Hops are dioecious perennial plants native to the Northern Hemisphere. They are found in shrubs and forest edges where there is sufficient water and can reach a maximum height of 7–8 metres (23–26 ft). Many female flowers form inflorescences called stalks, which consist of membranous stipules and bracts attached to a zigzag, hairy axis. Hops bracts and stipules contain polyphenols; their medicinal odour and taste are due mainly to highly complex secretions contained in the lupulin glands.

[0038] After harvest, the inflorescences are immediately dried to a moisture content of about 10% to stabilize them. Depending on environmental conditions, hops are kept refrigerated for part or all of the process from harvest to the final product. Bitter compounds are known to decompose rapidly during storage, and without refrigeration, their concentration decreases by 50-70% in just six months.

[0039] 25% of the hop berries are extracted with ethanol or supercritical carbon dioxide to extract as many alpha acids as possible. Ethanol and carbon dioxide occur naturally during the brewing process, so there are no problems with using these solvents.

[0040] Due to the enormous biomass production, the inflorescence (cone) is the only part of the hop plant that is used. The stems, leaves, rhizomes, and roots are not used by humans, except for the use of some of the shoots eaten in salads. The aerial parts are composted and used as fertilizer for fields. However, using current processes, xanthohumol and other molecules are purified from hops, hop waste (spent hops) generated from other processes such as beer production, or products that contain hops in biologically active and safe proportions.

[0041] In certain aspects, the compositions of the present disclosure are formulated for administration in a therapeutically effective amount. The terms "effective amount", "pharmaceutical effective amount" and "therapeutically effective amount" refer to an amount effective to induce a desired biological or medical response, including an amount of a compound that is sufficient to affect the treatment of a disease when administered to a subject for treating the disease. The effective amount varies depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. The effective amount can include a range of amounts. Furthermore, the effective amount includes the amount of a drug that is effective when combined with other drugs.

[0042] In some embodiments, an effective amount of the composition is combined with one or more pharma- ceutically acceptable vehicles. The term "pharmaceutical acceptable" refers to a material that is biologically or otherwise undesirable, e.g., a material that can be incorporated into a pharmaceutical composition administered to a patient without causing significant undesirable biological effects or adversely interacting with any of the other components of the composition in which it is contained. Pharmaceutically acceptable vehicles (e.g., carriers, adjuvants, and / or other excipients) preferably have met the required standards of toxicological and manufacturing testing and / or are included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0043] The term "carrier" or "pharmaceutical acceptable carrier" refers to diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, and other excipients and vehicles with which a compound is administered. Carriers are generally described herein and in "Remington's Pharmaceutical Sciences" by E. W. Martin. Examples of carriers include, but are not limited to, aluminum monostearate, aluminum stearate, carboxymethylcellulose, sodium carboxymethylcellulose, croscarmellose sodium, crospovidone, glyceryl isostearate, glyceryl monostearate, hydroxyethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxyoctacosanyl hydroxystearate, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, lactose monohydrate, magnesium stearate, mannitol, microcrystalline cellulose, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 188, poloxamer 237, poloxamer 407, povidone, silicon dioxide, colloidal silicon dioxide, silicone, silicone adhesive 4102, and silicone emulsion. However, it should be understood that the carrier selected for the pharmaceutical composition and the amount of such carrier in the composition may vary depending on the formulation method (e.g., dry granulation formulation, solid dispersion formulation).

[0044] The term "diluent" refers to a compound used to dilute the compound of interest before delivery. Diluents also serve to stabilize the compound. Non-limiting examples of diluents include starch, sugars, disaccharides, sucrose, lactose, lactose monohydrate, polysaccharides, cellulose, cellulose ethers, hydroxypropyl cellulose, microcrystalline cellulose, sugar alcohols, xylitol, sorbitol, maltitol, compressible sugars, calcium or sodium carbonate, dicalcium phosphate, dibasic calcium phosphate dihydrate, mannitol, and tribasic calcium phosphate.

[0045] The term "binder" as used herein refers to any pharma- ceutically acceptable film that can be used to bind the active and inactive components of the carrier together to maintain the cohesive and discrete portions. Non-limiting examples of binders include hydroxypropyl cellulose, hydroxypropyl methylcellulose, povidone, copovidone, and ethyl cellulose.

[0046] The term "disintegrant" refers to a substance that, when added to a solid formulation, promotes its breakup or disintegration after administration, allowing the release of the active ingredient as efficiently as possible and dissolving quickly. Non-limiting examples of disintegrants include corn starch, sodium starch glycolate, croscarmellose sodium, crospovidone, microcrystalline cellulose, modified corn starch, sodium carboxymethyl starch, povidone, pregelatinized starch, and alginic acid.

[0047] The term "lubricant" refers to a substance added to a powder blend to prevent the compressed powder mass from sticking to the equipment during the tabletting or encapsulation process. Lubricants aid in the ejection of the tablet from the die and improve powder flow. Non-limiting examples of lubricants include magnesium stearate, stearic acid, silica, fats, calcium stearate, polyethylene glycol, sodium stearyl fumarate, or talc; and lauric acid, oleic acid, C8 / C 10 Solubilizers such as fatty acids, for example fatty acids.

[0048] The term "film coating" refers to a thin, uniform film on the surface of a substrate (e.g., a tablet). Film coatings are particularly useful for protecting active ingredients from photodegradation. Non-limiting examples of film coatings include polyvinyl alcohol-based, hydroxyethyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate film coatings.

[0049] The term "glidant" refers to substances used in tablet and capsule formulations to improve flow characteristics during tablet compression and to create an anti-caking effect. Examples of glidants include colloidal silicon dioxide, talc, fumed silica, starch, starch derivatives, and bentonite.

[0050] How to use Prenylated chalcones and flavonoids have attracted increasing attention in disease prevention as well as nutrition due to their biological and molecular activities in humans. In one aspect of the present disclosure, the compositions described herein are useful for the treatment / prevention of various diseases and conditions, including cancer, particularly leukemia, such as TKI-resistant chronic myeloid leukemia (CML).

[0051] CML is a myeloproliferative neoplasm associated with a molecular alteration in the fusion gene BCR-ABL1, which encodes the tyrosine kinase oncoprotein BCR-ABL1. This led to the development of TKIs, with imatinib being the first TKI approved for treatment. Although the majority of CML patients respond to imatinib, resistance to this targeted therapy contributes to treatment failure and relapse.

[0052] Resistance to targeted therapy is a complex and multicomponent process that ultimately leads to the selection of cancer clones with the ability to evade treatment. In CML, TKI resistance mechanisms are usually subdivided into BCR-ABL1-dependent and independent mechanisms (Morozova EV et al. Biomark. Insights. 2015;10:43-47). However, treatment guidelines only consider BCR-ABL1-related mechanisms for dose adjustment and TKI switching (Baccarani M. et al. 2013. Blood. 2013;122:872-884). Persistence of leukemia stem cells (LSCs) and LSC-like phenotypes based on suppression of BCR-ABL1 protein have also been reported as major TKI resistance mechanisms (Baykal-Koese S. et al. PLoS ONE. 2020;15:e0229104).

[0053] Without being bound to the mechanism, in some embodiments, the compositions of the present disclosure are useful for stimulating the farnesoid X receptor.The farnesoid X receptor (FXR) belongs to a family of receptors known as nuclear hormone receptors.FXR is a bile acid-activated nuclear receptor and is widely involved in tumorigenesis.

[0054] Activation of FXR downregulates breast cancer target genes: local estrogen producer aromatase and transporters MDR3, MRP-1, solute carrier transporter 7A5 (SLC7A5), inhibiting cell proliferation (Bishop-Bailey, D., et al. Cancer Res (2006) 66 (20): 10120-10126). It also induces the expression of known FXR target genes SHP, IBABP, MRP2. It has been shown to regulate several cell signaling pathways such as EGFR / ERK, NF-κB, p38 / MAPK, PI3K / AKT, Wnt / β-catenin, and JAK / STAT, along with their targets such as caspases; MMPs, cyclins; tumor suppressor proteins such as p53, C / EBPβ, p-Rb; various cytokines; EMT markers; and others.

[0055] In some embodiments, the compositions of the present disclosure are useful for inhibiting NFκB activity in cells. Nuclear factor kappa B (NF-κB) is an ancient protein transcription factor (Salminen, A., Huuskonen, et al. (2008). Ageing Res. Rev. 7, 83-105) and is considered a regulator of innate immunity (Baltimore, D. (2009). Cold Spring Harb. Perspect. Biol. 1:a000026). NFκB is also an important signaling pathway involved in the development and treatment of cancer (Xia, L., et al. Onco Targets Ther. (2018) 11:2063-2073).

[0056] In some embodiments, the compositions of the present disclosure are useful for regulating the expression / activity of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). The Nrf2 / Keap1 pathway is a key signaling cascade responsible for resistance to oxidative damage caused by exogenous chemicals. It exerts anti-inflammatory and anti-cancer activities by maintaining redox homeostasis and regulating multiple downstream cytoprotective genes, thereby playing a key role in cell survival. Interestingly, in recent years, evidence has been accumulating suggesting that Nrf2 plays opposing roles in cancer. Aberrant activation of Nrf2 is associated with poor prognosis. Constitutive activation of Nrf2 in various cancers promotes cancer cell proliferation by inducing pro-survival genes and reprogramming metabolism, inhibiting apoptosis of cancer cells, and enhancing the self-renewal capacity of cancer stem cells. More importantly, Nrf2 has been demonstrated to contribute not only to inflammation-induced carcinogenesis but also to chemotherapy resistance and radioresistance of cancer cells.

[0057] In some embodiments, the compositions of the present disclosure are administered in combination with other therapeutic agents. In one embodiment, the compositions of the present disclosure are administered in combination with one or more tyrosine kinase inhibitors. Tyrosine kinase inhibitors (TKIs) are a group of pharmacologically active agents that disrupt protein kinase signaling pathways by several modes of inhibition. Mutations, dysregulation, and overexpression of protein kinases are involved in many disease processes. Approximately one in 40 human genes encodes a protein kinase, and nearly half of these genes have been mapped to either disease loci or cancer amplicons. Interest in protein kinase inhibitors began with the FDA approval of the tyrosine kinase inhibitor (TKI) imatinib in 2001. Imatinib is an oral chemotherapy drug designed to target the BCR-Abl hybrid protein, a tyrosine kinase signaling protein produced in patients with Philadelphia chromosome-positive chronic myeloid leukemia.

[0058] Overall, tyrosine kinases phosphorylate specific amino acids on substrate enzymes, which then alter signal transduction, leading to downstream changes in cell biology. Downstream signaling initiated by TKs can alter cell growth, migration, differentiation, apoptosis, and cell death. Constitutive activation or inhibition by mutation or other means can lead to dysregulation of signal cascades, resulting in malignancies and other pathologies. Thus, blocking these initial signals via TKIs can prevent the aberrant behavior of mutated or dysfunctional TKs.

[0059] Kinase inhibitors can be irreversible or reversible. Irreversible kinase inhibitors tend to covalently bind and block the ATP site, causing irreversible inhibition. Reversible kinase inhibitors can be further subdivided into four major subtypes based on the identification of the binding pocket and DFG motif.

[0060] In some aspects, the compositions of the present disclosure are administered in combination with imatinib, dasatinib, or ponatinib.

[0061] An unavoidable barrier limiting the efficacy of TKI therapy is the problem of resistance, a pervasive challenge for long-term disease management today. Cancer, at its core, is a microcosm of evolution. Its survival depends on the long-term accumulation of genetic diversity and mutations, which are subject to the selective pressure of TKI therapy. These elementary but complex principles are the basis of the intractability of TKI resistance, traditionally classified as primary (intrinsic) or secondary (acquired). In primary resistance, patients never experience any therapeutic response to targeted therapy. In secondary resistance, patients initially achieve some clinical benefit but then experience disease progression. With the discovery of the respective oncogenic drivers and targeted inhibitors, an increasingly numerous and diverse set of resistance mechanisms are being defined.

[0062] In one embodiment of the present disclosure, the compositions described herein are administered to subjects with primary or secondary TKI resistance, either alone or in combination with TKI.In one embodiment, the compositions of the present disclosure are administered as first-line drugs to avoid the development of secondary TKI resistance.In some embodiments, the compositions of the present disclosure are administered in combination with one or more TKIs, either simultaneously or in any order.

[0063] In some embodiments, the composition of the present disclosure is administered in combination with cannabinoid, simultaneously or in any order.In one embodiment, the cannabinoid is a phytocannabinoid, an endocannabinoid, or a synthetic cannabinoid.In one embodiment, the cannabinoid is cannabidiol (CBD), tetrahydrocannabinol (THC), or cannabigerol (CBG) or other cannabinoids.

[0064] A "synthetic cannabinoid" is a compound that has a cannabinoid or cannabinoid-like structure and is produced using chemical means rather than from the plant.

[0065] Phytocannabinoids can be obtained as neutral (decarboxylated) or carboxylated forms, depending on the method used to extract the cannabinoids. For example, it is known that heating the carboxylated form will decarboxylate most of the carboxylated form to the neutral form. EXAMPLES

[0066] Purification of biologically active drug substances Input raw materials Green-grey dust and granules (production waste, "leaps") with a xanthohumol content of 0.3-1.0 wt.%. The raw material contains large amounts of non-polar and polar impurities that are difficult to remove by conventional extraction processes (aqueous extraction / water-immiscible organic solvents).

[0067] Extraction procedure The plant material was suspended in n-heptane and subjected to a continuous extraction process at 20-30 °C to remove non-polar impurities. The extraction temperature was increased to 50-60 °C (at this temperature some components of the plant material melt) and the process was repeated. The heptane was evaporated and the yellow residue was filtered through a cotton filter in a press. The process was repeated as in n-heptane extraction, except that the dry residue was transferred to an extraction reactor, ethyl acetate was added and the semi-product was collected in a flask on a rotary evaporator. At the end of the process, the xanthohumol content in the yellowish precipitate was about 10-12% by weight.

[0068] The precipitate was dissolved in a mixture of an appropriate amount of organic solvent and liquid alkane to obtain xanthohumol at a concentration of about 0.05M. The resulting dark solution was subjected to an extraction process against 0.2M organic acid solution, 0.05M ZnCl2, 5% NaHCO3, and brine. After drying the organic layer with anhydrous Na2SO4 and evaporating the organic solvent, a solid mixture with a xanthohumol content of 20-25% was obtained. The mixture was then dissolved in a sufficient amount of ethyl acetate to obtain a concentration of xanthohumol of 0.05-0.1M, and then the solution was filtered and a base-acid extraction was carried out in stream using a liquid-liquid separator. Briefly, the ethyl acetate solution of xanthohumol was extracted with 0.05-0.1M NaOH solution, the organic layer was discarded, and the xanthohumol (as sodium salt) in the aqueous layer was added to a 0.05M-0.1M organic acid solution, resulting in the precipitation of Xn, which was extracted into ethyl acetate and dried with anhydrous Na2SO4. Ethyl acetate and brine were added to the residue. After filtering off the inorganic salts, most of the ethyl acetate was evaporated and the calculated amount of n-heptane was added to initiate the precipitation of xanthohumol from the solvent mixture. The distillation process was continued until all of the xanthohumol had precipitated. Alcohol was added (to remove traces of ethyl acetate) and the evaporation process was continued. The precipitate was filtered off, washed with n-heptane and dried under vacuum to give the final product as a pale yellow powder (HPLC purity >95.0%).

[0069] Drug Substance Characterization Three batches of drug substances (XN-54, XN-63-10, and XN-87) containing xanthohumol, isoxanthohumol, 6-prenylnaringenin, and 8-prenylnaringenin were prepared by the above process and analyzed.

[0070] Particle size distribution (PSD) PSD testing was performed by laser diffraction using a Mastersizer 2000 by Malvern Analytical Instruments. Table 1 shows the average values ​​(μm) for the three batches tested: XN-54 (samples were ground in a mortar before analysis), XN-63-10, and XN-87. Table 2 summarizes the results for batch XN-63 and its micronized derivatives. The particle size distributions for XN-54 (Figure 2A), XN-63-10 (Figure 2B), and XN-87 (Figure 2C) are shown.

[0071] Table 1. PSD results for drug substance, batches: XN-54, XN-63-10 and XN-87 [Table 1]

[0072] Table 2. PSD results for drug substance, batch XN-63-10 and its micronized derivatives [Table 2]

[0073] Differential Scanning Calorimetry (DSC) Differential scanning calorimetry analysis was performed on a TA Instruments Differential Scanning Calorimetry (DSC), Type DSC Q20 V24.10 Build 122. DSC analysis of the two DS batches showed two different melting point results: DS batch XN-54 had a melting point of 153.22 °C (Figure 3A), and DS batch XN-87 had a melting point of 171.86 °C (Figure 3B).

[0074] Thermogravimetric analysis (TGA) Thermogravimetric analysis was performed on a TA Instruments Thermogravimetric Analyzer (TGA), Type Q50 V20.13 Build 39. The TGA of drug substance batch XN-54 is shown in Figure 4A, and the TGA of batch XN-87 is shown in Figure 4B.

[0075] Fourier Transform Infrared Analysis (FTIR) Fourier transform infrared analysis was performed on the following FTIR spectrophotometers: Shimadzu, type IRTracer-100 (DS, batch XN-54) and JASCO FT / IR-6200 (DS, batch XN-63-10). Figures 5A and 5B show the results of the FTIR analysis of DS batches XN-54 and XN-87, respectively.

[0076] X-ray Powder Diffraction (XRPD) Powder X-ray diffraction analysis demonstrated the crystalline nature of the drug substance, revealing two different crystalline forms for two different batches. The diffractogram of XN-54 is shown in Figure 6A, and the diffractogram of XN-8 is shown in Figure 6B.

[0077] Solubility of drug substance Solubility tests of the drug substance were performed in five solvents: water, 0.1N hydrochloric acid, acetate buffer pH=4.5, phosphate buffer pH=6.8 and artificial saliva. The effect of the addition of various surfactants on the solubility of the materials was also investigated. Tests were performed at room temperature on two samples of each material in each medium using a magnetic stirrer (except for the preliminary study, an orbital shaker was used). Buffer solutions at pH=4.5 and pH=6.8 were prepared according to the European Pharmacopoeia 10.5, 5.17.1. Artificial saliva was prepared according to the following publication: Artificial saliva and its use in biological experiments J. Pytko-Polonczyk1, A. Jakubik, A. Przeklasa-Bierowiec, B. Muszynska, Journal of Physiology and Pharmacology 2017, 68, 6, 807-813. The content of the drug substance in the solution was analyzed by HPLC.

[0078] Table 3 shows the HPLC conditions for determining the content of drug substance in the samples from the solubility test. Table 3. HPLC method parameters for measuring dissolved drug substance [Table 3] * 3μl drug substance solution / 1μl granule solution

[0079] Pharmaceutical Formulation XN87 underwent formulation development using the three formats shown in Table 4. Table 4. Formulation of drug substance [Table 4] *Added as powder **Added as a 3% solution

[0080] How to use The effect of the drug substance on chronic myeloid leukemia (CML) cells was tested using the K562 cell line model. Three cell lines were used: K562-IR (imatinib-resistant), and K562-IS (imatinib-sensitive), purchased from the American Type Culture Collection (ATCC); and K562-DR 1000nM (imatinib- and dasatinib-resistant), developed by continuous treatment with TKIs and clonal selection. Basal protein expression was confirmed by Western blotting for α / β-tubulin and GAPDH (data not shown). pCrkl levels in K562-DR 1000nM were decreased, whereas BCR-ABL levels were similar between dasatinib-treated cells (Figures 7A and 7B, respectively), indicating that dual resistance is not dependent on BCR-ABL. Treatment of K562-IS cells with 1000 nM imatinib did not significantly alter pCrk1 levels compared with untreated controls (data not shown).

[0081] To measure the effect of XN-54 (compound 1) on the K562 cell line, viability experiments were performed using the CellTiter-Glo 2.0 system (Promega) according to the manufacturer's instructions. Briefly, opaque-walled multi-well plates containing K562 cells in medium were incubated with XN-54 at room temperature for the desired time. A volume of CellTiter-Glo reagent equal to the volume of cell culture medium was added to each well. Luminescence was measured after the luminescence signal stabilized.

[0082] Cell cultures were performed in 96-well plate format in 200 μl of medium specific for each cell line. Cells were cultured at the following densities: 5 x 10 5 / ml, 2.50 x 10 5 / ml, 1.25 x 10 5 Cells were seeded at 1000 x g / ml for 24, 48, or 72 h. Viability in response to increasing concentrations of XN-54 or dasatinib (control) at both 48 and 72 h is shown in Figure 8. As shown in Figure 8, XN-54 (compound 1) showed significant activity in both K562 imatinib-sensitive and K562 imatinib-resistant cells. To determine the mechanism by which XN-54 affects viability, cells were analyzed for apoptosis using both early (annexin V) and late (annexin V / propidium iodide) markers (BD Pharmingen cat. 556547). As shown in Figures 9 and 10, XN-54 induced apoptosis in all cell lines using both early (Figure 9) and late apoptotic (Figure 10) markers.

[0083] Combination therapy The effect of XN-54 in combination with TKIs was also analyzed in K562 cells. Viability was measured for dose-response combinations of XN-54 and ponatinib in K562-IR cells. As shown in Figure 11, the combination of XN-54 and ponatinib had a significant effect on cell viability based on the log 10(M) of either XN-54 (Figure 11A) or ponatinib (Figure 11B). Similar results were seen in K562-IS cells using XN-54 in combination with nilotinib (Figure 12).

[0084] The effects of XN-54 (compound 1) and cannabidiol (compound 2) were assayed using the cell viability method described above. As shown in Figure 13, the combination of XN-54 and CBD dramatically affected cell viability in K562-DR (Figure 13A), K562-IS (Figure 13B), and K562-IR cells (Figure 13C).

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of xanthohumol and isoxanthohumol and at least one pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, further comprising 6-prenylnaringenin and 8-prenylnaringenin.

3. The pharmaceutical composition according to claim 2, wherein xanthohumol is present in an amount of at least 50 to 99% w / w, isoxanthohumol is present in an amount of 1 to 15% w / w, 6-prenylnaringenin is present in an amount of 0.0 to 5%, and 8-prenylnaringenin is present in an amount of 0.0 to 5%.

4. The pharmaceutical composition according to claim 2, wherein at least one of xanthohumol, isoxanthohumol, 6-prenylnaringenin, or 8-prenylnaringenin is extracted from a plant.

5. The pharmaceutical composition according to claim 4, wherein the plant is hop, used hop, or a product containing hop.

6. The pharmaceutical composition according to claim 2, wherein at least one of xanthohumol, isoxanthohumol, 6-prenylnaringenin, or 8-prenylnaringenin is chemically synthesized.

7. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable carrier is at least one of a binder, a disintegrant, a surfactant, or a lubricant.

8. The pharmaceutical composition according to claim 7, wherein the binder is one or more of starch 1500, polyvinylpyrrolidone, and microcrystalline cellulose.

9. The pharmaceutical composition according to claim 8, wherein the binder is present in an amount of 10 to 30% w / w.

10. The pharmaceutical composition according to claim 7, wherein the disintegrant is croscarmellose sodium.

11. The pharmaceutical composition according to claim 10, wherein the disintegrant is present in an amount of 1 to 5% w / w.

12. The pharmaceutical composition according to claim 7, wherein the surfactant is sodium dodecyl sulfate.

13. The pharmaceutical composition according to claim 12, wherein the surfactant is present in an amount of 1 to 5% w / w.

14. The pharmaceutical composition according to claim 7, wherein the lubricant is magnesium stearate.

15. The pharmaceutical composition according to claim 14, wherein the lubricant is present in an amount of 0.5 to 3% w / w.

16. A method for preparing a pharmaceutical composition containing xanthohumol and isoxanthohumol from hop, Suspending hop plant material in n-heptane to remove non-polar impurities; Evaporating the heptane and filtering the residue; Extracting the residue with ethyl acetate and collecting the xanthohumol-containing fraction with a rotary evaporator; Extract with an organic acid solution containing xanthohumol, 0.05 M ZnCl 2 , 5% NaHCO 3 and saline; Dry the organic layer with anhydrous Na 2 SO 4 ; Precipitating xanthohumol from the mixture containing ethyl acetate; and Drying the precipitated xanthohumol and isoxanthohumol; A method comprising.

17. A pharmaceutical composition comprising xanthohumol and isoxanthohumol obtained by the method according to claim 16.

18. A pharmaceutical composition according to any one of claims 1 to 15 and 17, for treating a disease treated by agonizing farnesoid X receptor activity, in a therapeutically effective amount.

19. The pharmaceutical composition according to claim 18, wherein the disease is cancer.

20. The pharmaceutical composition according to claim 19, wherein the cancer is resistant to tyrosine kinase inhibitors.

21. A pharmaceutical composition according to any one of claims 1 to 15 and 17, for treating a disease treated by inhibiting NFκB activity, in a therapeutically effective amount.

22. The pharmaceutical composition according to claim 21, wherein the disease is cancer.

23. The pharmaceutical composition according to claim 22, wherein the cancer is resistant to tyrosine kinase inhibitors.

24. A pharmaceutical composition according to any one of claims 1 to 15 and 17, for treating a disease treated by increasing the expression and / or activation of nuclear factor erythroid 2-related factor 2 (NRF2), in a therapeutically effective amount.

25. The pharmaceutical composition according to claim 24, wherein the disease is cancer.

26. The pharmaceutical composition according to claim 25, wherein the cancer is resistant to tyrosine kinase inhibitors.

27. An effective amount of a pharmaceutical composition according to any one of claims 1 to 15 and 17, for treating a disease treated by inducing apoptosis in cells.

28. The pharmaceutical composition according to claim 27, wherein the cells are leukemia cells.

29. The pharmaceutical composition according to claim 27, wherein the cells contain a bcr-abl gene mutation.

30. The pharmaceutical composition according to claim 28, wherein the cells contain a bcr-abl gene mutation.

31. The pharmaceutical composition according to any one of claims 27 to 30, wherein the cell is a cell of a patient with chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or acute myeloid leukemia (AML).

32. The pharmaceutical composition according to any one of claims 1 to 15 and 17, in a therapeutically effective amount for treating chronic myeloid leukemia (CML).

33. The pharmaceutical composition according to claim 32, wherein CML is resistant to treatment with a tyrosine kinase inhibitor (TKI).

34. The pharmaceutical composition according to claim 33, wherein the TKI is imatinib, dasatinib, or ponatinib.

35. The pharmaceutical composition according to any one of claims 1 to 15 and 17, in a therapeutically effective amount for reducing the incidence of secondary tyrosine kinase inhibitor (TKI) resistance.

36. The pharmaceutical composition according to any one of claims 1 to 15 and 17, in a therapeutically effective amount for treating BCR-ABL-independent resistant cancer.

37. The pharmaceutical composition according to claim 36, wherein BCR-ABL-independent resistance is caused by inhibition of CRKL and STAT5 phosphorylation, or inhibition of sustained phosphorylation of the translational regulator ribosomal protein S6 (RPS6), which activates mTOR complex 1 (mTORC1).

38. The pharmaceutical composition according to claim 37, further comprising administering a TKI.

39. The pharmaceutical composition according to claim 38, wherein the TKI is administered after the pharmaceutical composition.

40. The pharmaceutical composition according to claim 38, wherein the TKI is imatinib, dasatinib, ponatinib, or nilotinib.

41. The pharmaceutical composition according to claim 38, further comprising administering a cannabinoid.

42. The pharmaceutical composition according to claim 41, wherein the cannabinoid is administered after the pharmaceutical composition.

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