Therapeutic compositions for treating liver cancer

Abstract

The present invention is directed to therapeutic compositions for treating liver cancer comprising a cyclohexenone compound and one or more antibodies of programmed cell death-1 (PD-1) or its ligand (PD-L1).

Description

THERAPEUTIC COMPOSITIONS FOR TREATING LIVER CANCER BACKGROUND OF THE INVENTION

[0001] Liver cancer, also known as hepatic cancer, is a cancer that originates in the liver. Primaryliver cancer is globally the sixth most frequent cancer, and the second leading cause of cancer death. The most frequent liver cancer, accounting for approximately 75% of all primary liver cancers, is hepatocellular carcinoma (HCC) (also named hepatoma, which is a misnomer because adenomas are usually benign). HCC is a cancer formed by liver cells, known as hepatocytes that become malignant. Hepatocellular carcinoma accounts for more than half million deaths annually worldwide.

[0002] Immunotherapy is a promising approach for the treatment of liver cancer, involving usingmedications that stimulate the body's own immune system to recognize and destroy cancer cells. SUMMARY OF THE INVENTION

[0003] In one aspect provided herein are compositions comprising a cyclohexenone compoundR3CH3O R4having the structure: or a pharmaceutically acceptable salt, metabolite, solvate orantibodies of programmed cell death-1 (PD-1) or its ligand (PD-L1), wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one ormore substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12.

[0004] In one aspect provided herein are pharmaceutical compositions for use in treating livercancer in a subject comprising a cyclohexenone compound having the structure: R3CH3a pharmaceutically acceptable salt, metabolite, solvate or prodrugof programmed cell death-1 (PD-1) or its ligand (PD-L1),wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one ormore substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12.

[0005] In another aspect provided herein are uses of a therapeutically effective amount of aR3CH3O R4cyclohexenone compound having the structure: or a pharmaceutically acceptable salt, metabolite, solvate or moreantibodies of programmed cell death-1 (PD-1) or its ligand (PD-L1), disclosed herein, in the manufacture of a medicament for treating liver cancer, wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one ormore substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12.

[0006] In another aspect provided herein are methods for treating liver cancer in a subjectcomprising administering the subject in need thereof of a cyclohexenone compound having the R3CH3R4structure: a pharmaceutically acceptable salt, metabolite, solvate orof programmed cell death-1 (PD-1) or its ligand (PD-L1), wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6;R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one ormore substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12. INCORPORATION BY REFERENCE

[0007] All publications, patents, and patent applications mentioned in this specification are hereinincorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features of the invention are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present invention will be obtained 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:

[0009] FIG. 1 provides a chart of the study results of In vivo antitumor efficacy of anti-PD-1 Ab,exemplary Compound 1 (antroquinonol (ANQ)), and combinations in immune-competent (Hepa1- 6 cells implanted subcutaneous into C57BL / 6 mice; N=10; Mean ± SD) liver cancer model over tumor volume.

[0010] FIG. 2 provides a chart of the study results showing the enhancing anti-cancer efficacy ofexemplary Compound 1 (antroquinonol (ANQ)) with anti-PD-1 antibody in the Hepa1-6 / C57BL / 6 subcutaneous HCC model over survival fraction.

[0011] FIG. 3 provides a chart showing the results of monitoring of body weight at seven-dayintervals (N=10; Mean ± SD) of exemplary Compound 1 (antroquinonol (ANQ)) with anti-PD-1 antibody in the Hepa1-6 / C57BL / 6 subcutaneous HCC model.

[0012] FIG. 4 provides a chart of the study results of In vivo antitumor efficacy of anti-PD-1 Ab,exemplary Compound 1 (antroquinonol (ANQ)), and combinations in immune-competent (Hep- 53.4 cells implanted subcutaneous into C57BL / 6 mice; N=5-10; Mean ± SD) liver cancer model over tumor volume.

[0013] FIG. 5 provides a chart showing the results of monitoring of body weight at seven-dayintervals (N=5-10; Mean ± SD) of exemplary Compound 1 (antroquinonol (ANQ)) with anti-PD- 1 antibody in the Hep-53.4 liver cancer cell line / C57BL / 6 subcutaneous HCC model.

[0014] FIG. 6 provides a chart of the study results of tumor volume at Day 23 after varioustreatments (*, p < 0.05; ***, p < 0.001, two-tailed unpaired Student’s t test) by exemplary Compound 1 (antroquinonol (ANQ)) with anti-PD-1 antibody in the Hep-53.4 / C57BL / 6 orthotopic HCC model. Bars represent the mean ± standard deviation (N = 5).

[0015] FIG. 7 provides a chart showing the results of monitoring of body weight at seven-dayintervals (N=5; Mean ± SD) of exemplary Compound 1 (antroquinonol (ANQ)) with anti-PD-1 antibody the Hep-53.4 / C57BL / 6 orthotopic HCC model. DETAILED DESCRIPTION OF THE INVENTION

[0016] Cancer is a leading cause of death worldwide, accounting for an estimated 10 million deathsin 2020, of which 830,000 deaths were due to liver cancer(http: / / www.who.int / mediacentre / factsheets / fs297 / en / ). The development of new systemictherapies for patients with hepatocellular carcinoma (HCC) is challenging because of the highintrinsic resistance of the tumors, underlying liver cirrhosis, and impaired functional reserves of most patients with advanced disease, as well as the lack of identified driver mutations (Hsu C, et al. 2017). Multiple co-stimulatory and inhibitory interactions of immune checkpoints are known to regulate T-cell responses in tumor microenvironments (Wei SC, et al.2018). The discovery of immune checkpoint interactions might lead to the identification of more inhibitory checkpoints that can be targeted for cancer immunotherapy or a better understanding of tumor immune evasion mechanisms, enabling the development of a better approach to determine the subgroup of patients that will respond to the particular therapy.

[0017] Anti-programmed cell death protein 1 (anti-PD1) or its ligand, anti-programmed cell deathligand-1 (anti-PD-L1) therapy have become the mainstay of oncology new drug development in the past few years and the momentum is rapidly accumulating for exploration of combination regimens (Tang J, et al, 2018). Anti-PD1 / anti-PD-L1 agents are tested in combination with a vast variety of novel immune modulators, molecular targeted agents, as well as conventional systemic or local anticancer therapies. Combination with anti-angiogenic therapy has shown promising antitumor activity, in terms of high response rate and long progression-free survival, in multiple cancer types, such as renal cell carcinoma and hepatocellular carcinoma (Atkins MB, et al. 2018; Choueiri TK, et al. 2018). This strategy is now extensively pursued by many pharmaceutical companies. Combination therapy using anti-PD1 / anti-PD-L1 ICI as the backbone is the most notable advance in systemic therapy for unresectable HCC (Yau T, et al. 2022; Kelley RK, et al. 2022; Ren Z, et al. 2021; Cheng AL, et al. 2022; Finn RS, et al. 2020; Qin S, et al. 2022; Abou- Alfa GK, et al. 2022; Finn RS, et al. 2020;). Two important findings from pivotal clinical trials are instrumental in the future development of novel systemic therapy. Firstly, the efficacy of anti- PD1- / anti-PD-L1-based combination therapy appears to have plateaued, with overall survival of about 20 months, progression-free survival of 7–8 months, and objective tumor response, according to RECIST 1.1, of about 25%. Further improvements in efficacy may depend on the identification of agents with novel mechanisms of action. Secondly, exploratory analyses ofsurvival outcomes in these trials suggest different levels of efficacy of ICI-based systemic therapy for HCC patients with different etiologies of the underlying liver diseases. These findings clearly illustrate the critical role of translational research in new drug development for HCC.

[0018] An immunosuppressive tumor microenvironment and chronic inflammation are the mainfeatures of HCC, which make the introduction of ICIs a reasonable strategy for new therapy development (El-Khoueiry AB, et al. 2017). For example, the two anti-PD-1 antibodies, nivolumab and pembrolizumab, received accelerated approval from the US FDA (Yau T, et al. 2019; El-Khoueiry AB, et al.2017). Overall response rates assessed in patients previously treated with sorafenib were approximately 14% and 17%, and the median overall survival (OS) was approximately 12 months. This makes PD-1 / PD-L1 blocking therapy a mainstream target for HCC drug development. However, neither anti-PD-1 trial (nivolumab versus sorafenib or pembrolizumab versus placebo) achieved a statistically significant improvement in OS. In addition, serious adverse events (grade 3-4, approximately 50%) were reported for tyrosine kinase inhibitor therapy and ICI therapy, leading to discontinuation (approximately 10%). Therefore, combination therapy is the next logical attempt to further improve the therapeutic effect against HCC. The combination of anti-PD1 (nivolumab) and anti-CTLA4 (ipilimumab) therapy received accelerated approval from the US FDA in 2020 and has a minimum OS of 28 months (from the last patient randomization to cutoff) (He AR, et al, 2020). In the same year, trials applied anti-PD- L1 (atezolizumab) plus anti-VEGF (bevacizumab); the median OS of patients who received the combination therapy was not reached during the trials, compared to 13.2 months for those who received sorafenib. Moreover, 36% of the patients experienced grade 3-4 adverse events (Galle PR, et al. 2021). In the updated report, the median OS of patients who received combination therapy was 19.2 months compared to 13.4 months for those who received sorafenib (Cheng AL, et al.2022). This is the first time a new treatment strategy challenging sorafenib obtained a positive result. Therefore, the development of a combination strategy may prove to be the right direction. On October 24, 2022, the FDA approved durvalumab (anti-PD-L1) + tremelimumab (anti- CTLA4), approximately 31% of patients treated with the combination were still alive after 3 years vs 20% of patients on sorafenib for the treatment of adult patients with unresectable hepatocellular carcinoma (Abou-Alfa GK, et al.2022).

[0019] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation279), is a protein on the surface of T and B cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.

[0020] PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms.First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).

[0021] PD-1 inhibitors, a new class of drugs that block PD-1, activate the immune system to attacktumors and are used to treat certain types of cancer. The PD-1 protein in humans is encoded by the PDCD1 gene. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2, which are members of the B7 family. Several lines of evidence suggest that PD-1 and its ligands negatively regulate immune responses.

[0022] A method for liver protection from chemically induced injury in a subject comprisingadministering the subject in need thereof a cyclohexanone compound such as Antroquinonol wasreported in U.S. patent number 7,456,225. In addition, Antroquinonol has shown to reduceproliferation of both hepatitis B virus-DNA positive (PLC / PRF / 5 and Hep3B) and negative (HepG2, Mahlavu, and SK-Hep1) hepatocellular carcinoma cell lines.

[0023] It is found unexpectedly a synergistic effect existed in a combination therapy comprising aR3CH3O R4cyclohexenone compound having the structure: or a pharmaceutically acceptable salt, metabolite, solvateone or more antibodies of programmed cell death-1 (PD-1), or its ligand (PD-L1), or a combination thereof, providing synergistic effects compared with the mono therapy; wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2and R3independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one ormore substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8 haloalkyl; n=1-12.In certain embodiments, the cyclohexenone compound is Antroquinonol.

[0024] In some embodiments, provided herein are compositions for use in treating liver cancer ina subject comprising a cyclohexenone compound having the structure:R3CH3O R4n a pharmaceutically acceptable salt, metabolite, solvate or prodrug of programmed cell death-1 (PD-1), or its ligand (PD-L1),. compounds, in some embodiments, are obtained from extracts of natural products and in some other embodiments, are prepared synthetically. See for example, U.S. patent No. 9365481. In certain embodiments, the cyclohexenone compound is Antroquinonol.

[0025] In some embodiments, there are provided methods for treating liver cancer in a subjectcomprising administering the subject in need thereof of a cyclohexenone compound having the R3CH3O R4structure: a pharmaceutically acceptable salt, metabolite, solvate orof programmed cell death-1 (PD-1) or its ligand (PD-L1), wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one or more substituentsselected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12. In certain embodiments, the cyclohexenone compound is Antroquinonol.

[0026] In some embodiments, there are provided uses of a therapeutically effective amount of aR3CH3R4compound having the a pharmaceutically acceptable salt, metabolite, solvate orof programmed cell death- 1 (PD-1), or its ligand (PD-L1) or a combination thereof, in the manufacture of a medicament for treating liver cancer in a subject wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6;R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with one ormore substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12. In certain embodiments, the cyclohexenone compound is Antroquinonol.

[0027] In some embodiments, the cyclohexenone compound having the structureR3CH3O R4n prepared synthetically or semi-synthetically from any suitable embodiments, the cyclohexenone compound is prepared byor example, Compounds 1, 3 and 4 are isolated from organic solvent extracts or prepared synthetically or semi-synthetically. The non-limited exemplary compounds are illustrated below. OCH3. In someembodiments, R1 is a hydrogen or methyl. In certain embodiments, R2 is a hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R3is a hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R4 is hydrogen. In some embodiments, R4 is C1- C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl, optionally substituted with one or moreselected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl. In certain embodiments, R4 is CH2CH=C(CH3)2. In certain embodiments, the compound is CH3CH3CH3CH3antroquinonol).Certain Pharmaceutical and Medical Terminology

[0029] Unless otherwise stated, the following terms used in this application, including thespecification and claims, have the definitions given below. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed. In this application, the use of “or” or “and” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0030] An “alkyl” group refers to an aliphatic hydrocarbon group. The alkyl group may be asaturated alkyl group (which means that it does not contain any carbon-carbon double bonds or carbon-carbon triple bonds) or the alkyl group may be an unsaturated alkyl group (which means that it contains at least one carbon-carbon double bonds or carbon-carbon triple bond). The alkyl moiety, whether saturated or unsaturated, may be branched, or straight chain.

[0031] The “alkyl” group may have 1 to 12 carbon atoms (whenever it appears herein, a numericalrange such as “1 to 12 refers to each integer in the given range; e.g., “1 to 12 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 12 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group of the compounds described herein may be designated as “C1-C8alkyl” or similar designations. By way of example only, “C1- C8 alkyl” indicates that there are one, two, three, four, five, six, seven or eight carbon atoms in the alkyl chain. In one aspect the alkyl is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, hexyl, allyl, but-2-enyl, but-3-enyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like. In one aspect, an alkyl is a C1-C8 alkyl.

[0032] The term “alkylene” refers to a divalent alkyl radical. Any of the above mentionedmonovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from the alkyl. In one aspect, an alkylene is a C1-C12alkylene. In another aspect, an alkylene is a C1- C8alkylene. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2- , -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like.

[0033] As used herein, the term “aryl” refers to an aromatic ring wherein each of the atoms formingthe ring is a carbon atom. Aryl rings are formed by five, six, seven, eight, nine, or more than nine carbon atoms. Aryl groups are optionally substituted. In one aspect, an aryl is a phenyl or anaphthalenyl. In one aspect, an aryl is a phenyl. In one aspect, an aryl is a C6-C10aryl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). In one aspect, an arylene is a C6-C10 arylene. Exemplary arylenes include, but are not limited to, phenyl- 1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene.

[0034] The term “aromatic” refers to a planar ring having a delocalized ^-electron systemcontaining 4n+2 ^ electrons, where n is an integer. Aromatic rings can be formed from five, six, seven, eight, nine, ten, or more than ten atoms. Aromatics are optionally substituted. The term “aromatic” includes both carbocyclic aryl (“aryl”, e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes monocyclic or fused- ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups.

[0035] The term “halo” or, alternatively, “halogen” or “halide” means fluoro, chloro, bromo oriodo.

[0036] The term “alkenyl” as used herein, means a straight, branched chain, or cyclic (in whichcase, it would also be known as a “cycloalkenyl”) hydrocarbon containing from 2-10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. In some embodiments, depending on the structure, an alkenyl group is a monoradical or a diradical (i.e., an alkenylene group). In some embodiments, alkenyl groups are optionally substituted. Illustrative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2- propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-cecenyl.

[0037] The term “alkynyl” as used herein, means a straight, branched chain, or cyclic (in whichcase, it would also be known as a “cycloalkynyl”) hydrocarbon containing from 2-10 carbons and containing at least one carbon-carbon triple bond formed by the removal of four hydrogens. In some embodiments, depending on the structure, an alkynyl group is a monoradical or a diradical (i.e., an alkynylene group). In some embodiments, alkynyl groups are optionally substituted. Illustrative examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and the like.

[0038] The term “cycloalkyl” as used herein, means a monocyclic or polycyclic radical thatcontains only carbon and hydrogen, and includes those that are saturated, partially unsaturated, or fully unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Representative examples of cyclic include but are not limited to, the following moieties:, , , , , ,on .include alkyl, alkenyl, alkynyl and alkoxy structures in which at least one hydrogen is replaced with a halogen atom. In certain embodiments in which two or more hydrogen atoms are replaced with halogen atoms, the halogen atoms are all the same as one another. In other embodiments in which two or more hydrogen atoms are replaced with halogen atoms, the halogen atoms are not all the same as one another. The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. In certain embodiments, haloalkyls are optionally substituted.

[0040] The term “acceptable” with respect to a formulation, composition or ingredient, as usedherein, means having no persistent detrimental effect on the general health of the subject being treated.

[0041] Antrodia is a genus of fungi in the family Meripilaceae. Antrodia species have fruitingbodies that typically lie flat or spread out on the growing surface, with the hymenium exposed to the outside; the edges may be turned so as to form narrow brackets. Most species are found in temperate and boreal forests, and cause brown rot.

[0042] The term “carrier,” as used herein, refers to relatively nontoxic chemical compounds oragents that facilitate the incorporation of a compound into cells or tissues.

[0043] The terms “co-administration” or the like, as used herein, are meant to encompassadministration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

[0044] The term “diluent” refers to chemical compounds that are used to dilute the compound ofinterest prior to delivery. Diluents can also be used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can providepH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.

[0045] The terms “effective amount” or “therapeutically effective amount,” as used herein, referto a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.

[0046] A “metabolite” of a compound disclosed herein is a derivative of that compound that isformed when the compound is metabolized. The term “active metabolite” refers to a biologically active derivative of a compound that is formed when the compound is metabolized. The term “metabolized,” as used herein, refers to the sum of the processes (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes) by which a particular substance is changed by an organism. Thus, enzymes may produce specific structural alterations to a compound. For example, cytochrome P450 catalyzes a variety of oxidative and reductive reactions while uridine diphosphate glucuronyltransferases catalyze the transfer of an activated glucuronic- acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines and free sulphydryl groups. Metabolites of the compounds disclosed herein are optionally identified either by administration of compounds to a host and analysis of tissue samples from the host, or by incubation of compounds with hepatic cells in vitro and analysis of the resulting compounds.

[0047] The term “pharmaceutical combination” as used herein, means a product that results fromthe mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g. a compound (i.e., a cyclohexenone compound described herein) and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g. a compound (i.e., a cyclohexenone compound described herein) and a co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

[0048] The term “pharmaceutical composition” refers to a mixture of a compound (i.e., acyclohexenone compound described herein) with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients.The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0049] The term “subject” or “patient” encompasses mammals. Examples of mammals include,but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one embodiment, the mammal is a human.

[0050] The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating orameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and / or therapeutically. Routes of Administration and Dosage

[0051] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal,aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0052] In certain embodiments, a compound as described herein is administered in a local ratherthan systemic manner, for example, via injection of the compound directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in other embodiments, the drug is delivered in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody. In such embodiments, the liposomes are targeted to and taken up selectively by the organ. In yet other embodiments, the compound as described herein is provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. In yet other embodiments, the compound described herein is administered topically.

[0053] In some embodiments, the cyclohexenone compound, or a pharmaceutically acceptablesalt, metabolite, solvate or prodrug thereof, is administered parenterally or intravenously. In other embodiments, the cyclohexenone compound, or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof, is administered by injection. In some embodiments, the cyclohexenonecompound, or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof, is administered orally.

[0054] In the case wherein the patient’s condition does not improve, upon the doctor’s discretionthe administration of the compounds may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient’s life in order to ameliorate or otherwise control or limit the symptoms of the patient’s disease or condition. In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the compounds may be given continuously or temporarily suspended for a certain length of time (i.e., a “drug holiday”).

[0055] The foregoing ranges are merely suggestive, as the number of variables in regard to anindividual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages may be altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0056] Toxicity and therapeutic efficacy of such therapeutic regimens can be determined bystandard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. In certain embodiments, the CH3CH3CH3CH3cyclohexenone compound is , CH3CH3CH3CH3CH3CH3CH3CH3O CH3. herein are formulated into pharmaceuticalcompositions are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are used as suitable to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[0058] Provided herein are pharmaceutical compositions comprising a compound (i.e., acyclohexenone compound described herein) and a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). In certain embodiments, the compounds described are administered as pharmaceutical compositions in which a compound (i.e., a cyclohexenone compound described herein) is mixed with other active ingredients, as in combination therapy. Encompassed herein are all combinations of actives set forth in the combination therapies section below and throughout this disclosure. In specific embodiments, the pharmaceutical compositions include one or more compounds (i.e., a cyclohexenone compound described herein).

[0059] A pharmaceutical composition, as used herein, refers to a mixture of a compound (i.e., acyclohexenone compound described herein) with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the compound to an organism. In some embodiments, practicing the methods of treatment or use provided herein, therapeutically effective amounts of compounds (i.e., a cyclohexenone compound described herein) are administered in a pharmaceutical composition to a mammal having a disease or condition to be treated. In specific embodiments, the mammal is a human. In certain embodiments, therapeutically effective amounts vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The compoundsdescribed herein are used singly or in combination with one or more therapeutic agents as components of mixtures.

[0060] In one embodiment, a compound (i.e., a cyclohexenone compound described herein) isformulated in an aqueous solution. In specific embodiments, the aqueous solution is selected from, by way of example only, a physiologically compatible buffer, such as Hank’s solution, Ringer’s solution, or physiological saline buffer. In other embodiments, a compound (i.e., a cyclohexenone compound described herein) is formulated for transmucosal administration. In specific embodiments, transmucosal formulations include penetrants that are appropriate to the barrier to be permeated. In still other embodiments wherein the compounds described herein are formulated for other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and / or excipients.

[0061] In another embodiment, compounds described herein are formulated for oraladministration. Compounds described herein, including a compound (i.e., a cyclohexenone compound described herein), are formulated by combining the active compounds with, e.g., pharmaceutically acceptable carriers or excipients. In various embodiments, the compounds described herein are formulated in oral dosage forms that include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like.

[0062] In certain embodiments, pharmaceutical preparations for oral use are obtained by mixingone or more solid excipients with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragees cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as: for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In specific embodiments, disintegrating agents are optionally added. Disintegrating agents include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0063] In one embodiment, dosage forms, such as dragee cores and tablets, are provided with oneor more suitable coating. In specific embodiments, concentrated sugar solutions are used for coating the dosage form. The sugar solutions, optionally contain additional components, such as by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs and / or pigments are also optionally added to the coatings for identification purposes.Additionally, the dyestuffs and / or pigments are optionally utilized to characterize different combinations of active compound doses.

[0064] In certain embodiments, therapeutically effective amounts of at least one of the compoundsdescribed herein are formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In specific embodiments, push-fit capsules contain the active ingredients in admixture with one or more filler. Fillers include, by way of example only, lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In other embodiments, soft capsules, contain one or more active compound that is dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

[0065] In other embodiments, therapeutically effective amounts of at least one of the compoundsdescribed herein are formulated for buccal or sublingual administration. Formulations suitable for buccal or sublingual administration include, by way of example only, tablets, lozenges, or gels. In still other embodiments, the compounds described herein are formulated for parental injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, formulations for injection are presented in unit dosage form (e.g., in ampoules) or in multi-dose containers. Preservatives are, optionally, added to the injection formulations. In still other embodiments, the pharmaceutical compositions of a compound (i.e., a cyclohexenone compound described herein) are formulated in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles. Parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In specific embodiments, pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In additional embodiments, suspensions of the active compounds are prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. In certain specific embodiments, aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension contains suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0066] In one aspect, compounds (i.e., cyclohexenone compounds described herein) are preparedas solutions for parenteral injection as described herein or known in the art and administered withan automatic injector. Automatic injectors, such as those disclosed in U.S. Patent Nos.4,031,893, 5,358,489; 5,540,664; 5,665,071, 5,695,472 and WO / 2005 / 087297 (each of which are incorporated herein by reference for such disclosure) are known. In general, all automatic injectors contain a volume of solution that includes a compound (i.e., a cyclohexenone compound described herein) to be injected. In general, automatic injectors include a reservoir for holding the solution, which is in fluid communication with a needle for delivering the drug, as well as a mechanism for automatically deploying the needle, inserting the needle into the patient and delivering the dose into the patient. Exemplary injectors provide about 0.3 mL, 0.6mL, 1.0mL or other suitable volume of solution at about a concentration of 0.5 mg to 50 mg of a compound (i.e., a cyclohexenone compound described herein) per 1 mL of solution. Each injector is capable of delivering only one dose of the compound.

[0067] In still other embodiments, the compounds (i.e., cyclohexenone compounds describedherein) are administered topically. The compounds described herein are formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0068] In still other embodiments, the compounds (i.e., cyclohexenone compounds describedherein) are formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted.

[0069] In certain embodiments, pharmaceutical compositions are formulated in any conventionalmanner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients is optionally used as suitable and as understood in the art. Pharmaceutical compositions comprising a compound (i.e., a cyclohexenone compound described herein) may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0070] Pharmaceutical compositions include at least one pharmaceutically acceptable carrier,diluent or excipient and at least one compound (i.e., cyclohexenone compounds described herein) described herein as an active ingredient. The active ingredient is in free-acid or free-base form, orin a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type of activity. All tautomers of the compounds described herein are included within the scope of the compounds presented herein. Additionally, the compounds described herein encompass unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein. In addition, the pharmaceutical compositions optionally include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, buffers, and / or other therapeutically valuable substances.

[0071] Methods for the preparation of compositions comprising the compounds described hereininclude formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions and creams. The form of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.

[0072] In some embodiments, pharmaceutical composition comprising at least compound (i.e.,cyclohexenone compounds described herein) illustratively takes the form of a liquid where the agents are present in solution, in suspension or both. Typically when the composition is administered as a solution or suspension a first portion of the agent is present in solution and a second portion of the agent is present in particulate form, in suspension in a liquid matrix. In some embodiments, a liquid composition includes a gel formulation. In other embodiments, the liquid composition is aqueous.

[0073] In certain embodiments, pharmaceutical aqueous suspensions include one or morepolymers as suspending agents. Polymers include water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein include a mucoadhesive polymer, selected from, for example, carboxymethylcellulose, carbomer (acrylicacid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0074] Pharmaceutical compositions also, optionally include solubilizing agents to aid in thesolubility of a compound (i.e., cyclohexenone compounds described herein). The term “solubilizing agent” generally includes agents that result in formation of a micellar solution or a true solution of the agent. Certain acceptable nonionic surfactants, for example polysorbate 80, are useful as solubilizing agents, as can ophthalmically acceptable glycols, polyglycols, e.g., polyethylene glycol 400, and glycol ethers.

[0075] Furthermore, pharmaceutical compositions optionally include one or more pH adjustingagents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0076] Additionally, pharmaceutical compositions optionally include one or more salts in anamount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0077] Other pharmaceutical compositions optionally include one or more preservatives to inhibitmicrobial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0078] Still other pharmaceutical compositions include one or more surfactants to enhancephysical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

[0079] Still other pharmaceutical compositions may include one or more antioxidants to enhancechemical stability where required. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0080] In certain embodiments, pharmaceutical aqueous suspension compositions are packaged insingle-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.

[0081] In alternative embodiments, other delivery systems for hydrophobic pharmaceuticalcompounds are employed. Liposomes and emulsions are examples of delivery vehicles or carriersherein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also employed. In additional embodiments, the compounds described herein are delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials are useful herein. In some embodiments, sustained-release capsules release the compounds for a few hours up to over 24 hours. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0082] In certain embodiments, the formulations described herein include one or moreantioxidants, metal chelating agents, thiol containing compounds and / or other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof. General Consideration for Combination Treatments

[0083] In general, the compositions described herein and, in embodiments where combinationaltherapy is employed and described herein, other agents do not have to be administered in the same pharmaceutical composition, and in some embodiments, because of different physical and chemical characteristics, are administered by different routes. In some embodiments, the initial administration is made according to established protocols, and then, based upon the observed effects, the dosage, modes of administration and times of administration is modified by the skilled clinician.

[0084] In some embodiments, therapeutically-effective dosages vary when the drugs are used intreatment combinations. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient. For combination therapies described herein, dosages of the co-administered compounds vary depending on the type of co-drug employed, on the specific drug employed, on the disease, disorder, or condition being treated and so forth.

[0085] It is understood that in some embodiments, the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, is modified in accordance with a variety of factors. These factors include the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, in other embodiments, the dosageregimen actually employed varies widely and therefore deviates from the dosage regimens set forth herein. Examples Example 1. Preparation of the exemplary cyclohexenone compounds

[0086] Via a particular herbal purification procedure: the following procedure is for illustrationpurposed only. The other purification methods may be used as well.

[0087] One hundred grams of mycelia, fruiting bodies or mixture of both from Antrodiacamphorata were placed into a flask. A proper amount of water and alcohol (70-100% alcohol solution) was added into the flask and were stirred at 20-25° C for at least 1 hour. The solution was filtered through a filter and 0.45 μm membrane and the filtrate was collected as the extract.

[0088] The filtrate of Antrodia camphorata was subjected to High Performance Liquidchromatography (HPLC) analysis. The separation was performed on a RP18 column, the mobile phase consisted of methanol (A) and 0.3% acetic acid (B), with the gradient conditions of 0-10 min in 95% - 20% B, 10-20 min in 20%-10% B, 20-35 min in 10%-10% B, 35-40 min in 10%-95% B, at the flow rate of 1 ml / min. The column effluent was monitored with a UV-visible detector.

[0089] The fractions collected at 25 to 30 min were collected and concentrated to yield 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone (compound 1), a product of pale yellow brown liquid. The analysis of compound 1 showed the molecular formula of C 24 H 38 O4, molecular weight of 390 with melting point of 48 to 52oC. NMR spectra showed that1H-NMR (CDCl3) δ (ppm)=1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.68, 4.05, 5.07, and 5.14;13C-NMR (CDCl3) δ (ppm)=12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 40.27, 43.34, 59.22, 60.59, 120.97, 123.84, 124.30, 131.32, 135.35, 135.92, 138.05, 160.45, and 197.12. CH3CH3CH3CH36-methyl-5-(3,7,11-trimethyldodeca-2,6,10- trienyl)cyclohex-2-enone (Antroquinonol) Other compounds where R4is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionallysubstituted with one or more substituents selected from C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl,C3-C8cycloalkyl, and C1-C8haloalkyl were obtained as well.

[0090] Alternatively, the exemplary compounds may be prepared synthetically. See for example,U.S. Patent No.9365481. R3CH3O R4n Similarly, other cyclohexenone compounds having the are isolated from Antrodia camphorata or prepared thesuitable starting materials. An ordinary skilled in the art conditions for such synthesis. Example 2. Exploring the immunomodulatory effects of exemplary Compound 1 (i.e., Antroquinonol) for the treatment of hepatocellular carcinoma to show antitumor synergy between Antroquinonol and immune checkpoint inhibitors

[0091] In order to explore the immunomodulatory effects of exemplary Compound 1 (i.e.,Antroquinonol, ANQ) for the treatment of hepatocellular carcinoma, an immune-competent mouse model of HCC is used to clarify the mechanisms of the antitumor efficacy between Compound 1 and anti-PD1 therapy. Two anti-PD1 drugs, nivolumab and pembrolizumab, were granted accelerated approval for HCC patients after sorafenib therapy, based on promising antitumor activity seen in single-arm studies. However, in a randomized trial comparing pembrolizumab with placebo as second-line therapy for advanced HCC (KEYNOTE-240), the therapeutic benefit of pembrolizumab did not reach the pre-defined statistical significance (hazard ratio 0.78, (95% CI 0.611-0.998, p=0.0238) for overall survival and 0.78 (95% CI 0.61-0.99, p=0.0209) for progression-free survival) (Merck news release, 19 February 2019). Therefore, exploration of more active regimens of combination therapy is critical for future development in HCC.

[0092] The purpose of this pre-clinical study is to explore the immunomodulatory effects ofAntroquinonol for the treatment of hepatocellular carcinoma and to evaluate the antitumor synergy between Antroquinonol and immune checkpoint inhibitor. Results from this project provides proof-of-concept evidence for rationale of combination therapy.

[0093] Objectives: To find potential combination of antitumor synergy between immunecheckpoint inhibitor and Antroquinonol.

[0094] Materials and Methods. Compound 1 is provided by Golden Biotechnology Corporation.Anti-PD-1 antibody (Clone: RMP1-14, catalog no. BE0146) and isotype control (Clone: 2A3, catalog no. BE0089) antibodies were purchased from Bio X Cell (West Lebanon, New Hampshire, USA).

[0095] Murine liver cancer models and cell lines.

[0096] The protocol for the animal experiments in this study (Approval no. IACUC- 20230373)was approved by the Institutional Animal Care and Use Committee (IACUC) of the College of Medicine, National Taiwan University, and conformed to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

[0097] The mouse HCC cell lines Hepa1-6 (ATCC) and Hep-53.4 (NCIt: C38756) were used inthis study. The following three immuno-competent in vivo models were used in this project: (1) Hep-53.4 liver cancer cell line / C57BL / 6 mice (orthotopic); (2) Hepa1-6 liver cancer cell line / C57BL / 6 mice (heterotopic); (3) Hep-53.4 liver cancer cell line / C57BL / 6 mice (heterotopic). For the subcutaneous model (Hepa1-6 liver cancer cell line / C57BL / 6 mice), 2×106cells were subcutaneously injected into the right flank, and drug treatment was started when tumor volume was about 100 mm3(calculated by the formula 0.5×length×width2) and the HCC-bearing mice were randomized into each treatment group. For the subcutaneous model (Hep-53.4 liver cancer cell line / C57BL / 6 mice), 2×106cells were subcutaneously injected into the right flank, and drug treatment was started when tumor volume was about 200 mm3(calculated by the formula 0.5×length×width2) and the HCC-bearing mice were randomized into each treatment group. For the orthotopic model (Hep-53.4 liver cancer cell line / C57BL / 6 mice), about 3×105cells were injected into the subcapsular area of the left liver lobe, and the mice were randomized to each treatment group 5 days after tumor cell injection. The antitumor efficacy and safety of drug treatment were measured by change in tumor volume, body weight, or animal survival. The definition of animal death once the tumor volume is larger than 2,000 mm3.

[0098] General Treatment Plan

[0099] The subcutaneous studies in general follow the treatment plan below. Compound 1 (i.e.,Antroquinonol, 30, 45, and / or 60 mg / kg), is administered by oral gavage twice daily, the 2nddose is given 6 hours after the 1stone, and 5 days / 1week) and anti-PD-1 Ab (200ug, i.p., day 1, 3, 5, 10, 14) is administrated starting tumor volume reaches ~100 or ~200 mm3. Antroquinonol alone (e.g., high dose) or in combination with anti-PD-1 Ab (low dose of Antroquinonol + anti-PD-1 Ab and high dose of Antroquinonol + anti-PD-1 Ab) will be evaluated the therapeutic effects. Also, the animal survival is analyzed. Vehicle control treatment will be set as the control group after confirmation that the vehicle solutions for test compound and the isotype control IgG. Assessment of tumor growth, survival, or safety is done.

[0100] The orthotopic study in general follow the treatment plan below. Compound 1 (i.e.,Antroquinonol, 45 mg / kg), is administered by oral gavage twice daily, and 5 days / 1week and anti- PD-1 Ab (200ug, i.p., day 1, 3, 5, 8, 14) is administrated 5 days after tumor cell injection. Antroquinonol alone or in combination with anti-PD-1 Ab will be evaluated the therapeutic effect.Vehicle control treatment will be set as the control group after confirmation of the vehicle solutions for test compound and the isotype control IgG. Assessment of tumor volume and body weight is conducted.

[0101] Detailed Plans

[0102] Model 1: ANQ+Anti-PD-1 Ab in Hepa 1-6 cell line / C57BL / 6 (subcutaneous HCCmodels): the antitumor synergy between ANQ and anti-PD-1 Ab was evaluated over tumor growth (tumor volume) and safety (animal body weight).

[0103] Groups: G1: Vehicle control (vehicle solution for ANQ+ Isotype Ab)G2: anti-PD-1 (200ug / IP injection x 5) G3: ANQ-45 BID (Antroquinonol 45 mg / kg BID (total 90 mg / kg / day)) G4: ANQ-30 BID (Antroquinonol 30 mg / kg BID (total 60 mg / kg / day)) + anti- PD-1 G5: ANQ-45 BID + anti-PD-1 Administration: ANQ or vehicle solution: Oral gavage / 5 days / 1 week, for 4 weeks Anti-PD-1 or isotype Ab: IP injection / on day 1, 3, 5, 10, 14 (It will be administrated starting tumor volume reaches ~100 mm3) Mice = 10 / group

[0104] Administration scheme.

[0105] For the subcutaneous model (Hepa1-6 liver cancer cell line / C57BL / 6 mice), 2×106 cellswere subcutaneously injected into the right flank, and drug treatment was started when tumor volume was about 100 mm3(calculated by the formula 0.5×length×width2) and the HCC-bearing mice were randomized into each treatment group. Antroquinonol (30 and 45 mg / kg, is administered by oral gavage twice daily, the 2nd dose is given 6 hours after the 1st one, and 5 days / 1week, for 4 weeks) and anti-PD-1 Ab (200ug, i.p., day 1, 3, 5, 10, 14) were administrated when tumor volume reached ~100 mm3. Antroquinonol alone (e.g., high dose) or in combination with anti-PD-1 Ab (low dose of Antroquinonol + anti-PD-1 Ab and high dose of Antroquinonol + anti-PD-1 Ab) were evaluated the therapeutic effects. Vehicle control treatment was set as the control group after confirmation that the vehicle solution (corn oil) for test compound and the isotype control IgG. Assessment of tumor growth, survival, or safety was done. At day 60 after various treatments, tumors were harvested.

[0106] Model 2: ANQ+Anti-PD-1 Ab in Hep-53.4 cell line / C57BL / 6 (subcutaneous HCCmodels): the antitumor synergy between ANQ and anti-PD-1 Ab was evaluated over tumor growth (tumor volume) and safety (animal body weight).

[0107] Groups: G1: Vehicle control (vehicle solution for ANQ+ Isotype Ab)G2: anti-PD-1 (200ug / IP injection x 5) G3: ANQ-45 BID (Antroquinonol 45 mg / kg BID (total 90 mg / kg / day)) G4: ANQ-45 BID + anti-PD-1 G5: ANQ-60 BID (Antroquinonol 60 mg / kg BID (total 120 mg / kg / day)) + anti-PD-1 Administration: ANQ or vehicle solution: Oral gavage / 5 days / 1 week, for approximately 4 weeks Anti-PD-1 or isotype Ab: IP injection / on day 1, 3, 5, 10, 14 (It will be administrated starting tumor volume reaches ~200 mm3) Mice = 5-10 / group

[0108] Administration scheme.

[0109] For the subcutaneous model (Hep-53.4 liver cancer cell line / C57BL / 6 mice), 2×106 cells weresubcutaneously injected into the right flank, and drug treatment was started when tumor volume was about 200 mm3(calculated by the formula 0.5×length×width2) and the HCC-bearing mice were randomized into each treatment group. Antroquinonol (45 and 60 mg / kg, is administered by oral gavage twice daily, the 2nd dose is given 6 hours after the 1st one, and 5 days / 1week, for approximately 4 weeks) and anti-PD- 1 Ab (200ug, i.p., day 1, 3, 5, 10, 14) were administrated when tumor volume reached ~200 mm3. Antroquinonol alone (e.g., 45 mg / kg BID) or in combination with anti-PD-1 Ab (Antroquinonol 45 mg / kg BID + anti-PD-1 Ab and Antroquinonol 60 mg / kg BID + anti-PD-1 Ab) were evaluated the therapeutic effects. Vehicle control treatment was set as the control group after confirmation that the vehicle solution (corn oil) for test compound and the isotype control IgG. Assessment of tumor growth and safety was done. At day 28 after various treatments, tumors were harvested.

[0110] Model 3: ANQ+Anti-PD-1 Ab in Hep-53.4 cell line / C57BL / 6 (orthotopic HCCmodels): the antitumor synergy between ANQ and anti-PD-1 Ab was evaluated over tumor growth (tumor volume) and safety (animal body weight).

[0111] Groups: G1: Vehicle control (vehicle solution for ANQ+ Isotype Ab)G2: anti-PD-1 (200ug / IP injection x 5) G3: ANQ-45 BID (Antroquinonol 45 mg / kg BID (90 mg / kg / day)) G4: ANQ-45 BID + anti-PD-1 Administration: ANQ or vehicle solution: Oral gavage / 5 days / 1 week, for approximately 3 weeks Anti-PD-1 or isotype Ab: IP injection / on day 1, 3, 5, 8.14; Mice = 5 / group

[0112] Administration scheme.

[0113] For the orthotopic model (Hep-53.4 liver cancer cell line / C57BL / 6 mice), about 3×105cells were injected into the subcapsular area of the left liver lobe, and the mice were randomized to each treatment group 5 days after tumor cell injection. Antroquinonol (45 mg / kg, is administered by oral gavage twice daily, and 5 days / 1week, for approximately 3 weeks) and anti-PD-1 Ab (200ug, i.p., day 1, 3, 5, 8, 14) were administrated. Antroquinonol alone or in combination with anti-PD-1 Ab were evaluated the therapeutic effect. Vehicle control treatment was set as the control group after confirmation that the vehicle solution (corn oil) for test compound and the isotype control IgG. Assessment of tumor volume and body weight was done. At day 23 after various treatments, tumor samples were harvested.

[0114] Statistical analysis. A two-tailed Student’s t-test with equal variance wasperformed to compare two experimental groups. A one-way analysis of variance (ANOVA) with Tukey’s post hoc test was performed to evaluate the difference between multiple groups. The data in this study are presented as the mean and error bars represent the standard deviation. *p < 0.05 was considered significant differences among the treatment groups.

[0115] Study Results

[0116] Model 1: ANQ + anti-PD-1 Ab in Hepa1-6 liver cancer cell line / C57BL / 6(subcutaneous HCC model)

[0117] Based on the study results, it is evident that the combination treatment of ANQ-45BID and anti-PD-1 Ab has effectively enhanced antitumor efficacy while also extending the survival time of mice. The evidence for example can be found form FIG. 1 which provides the study results showing the enhancing anti-cancer efficacy of exemplary Compound 1 with anti-PD- 1 antibody in the Hepa1-6 / C57BL / 6 subcutaneous HCC model over tumor volume. It is also found that the combination treatment extends the survival time of mice based on the results shown in FIG. 2 showing evaluation of treatment efficacy based on mice survival (N=10 in each treatmentgroup). There is no significant difference between ANQ-45 BID monotherapy group and anti-PD-1 Ab monotherapy group for antitumor effect. Additionally, safety evaluation indicated nosignificant changes in mouse body weight among groups (see FIG. 3)

[0118] Model 2: ANQ + anti-PD-1 Ab in Hep-53.4 liver cancer cell line / C57BL / 6(subcutaneous HCC model)

[0119] Based on the study results, it is clear that the combination treatment of ANQ-45BID or ANQ-60 BID with anti-PD-1 Ab markedly enhances antitumor efficacy. The combinations of ANQ and anti-PD-1 Ab (e.g., ANQ-45 BID + anti-PD-1 Ab and ANQ-60 BID + anti-PD-1 Ab) demonstrated statistically significant tumor-suppressing effects compared to ANQ monotherapy or anti-PD-1 Ab monotherapy (see FIG.4). There is no significant difference between ANQ-45 BID monotherapy group and anti-PD-1 Ab monotherapy group for antitumor effect. Additionally,safety evaluation indicated no significant changes in mouse body weight among groups (see FIG. 5).

[0120] Model 3: ANQ + anti-PD-1 Ab in Hep-53.4 cell line / C57BL / 6 (orthotopic HCCmodel)

[0121] Based on the study results, it is clear that the combination treatment of ANQ-45 BIDwith anti-PD-1 Ab markedly enhances antitumor efficacy. The combination of ANQ-45 BID and anti-PD-1 Ab demonstrated statistically significant tumor-suppressing effects compared to ANQ- 45 BID monotherapy or anti-PD-1 Ab monotherapy (see FIG.6). There is no significant difference between ANQ-45 BID monotherapy group and anti-PD-1 Ab monotherapy group for antitumoreffect. Additionally, safety evaluation indicated no significant changes in mouse body weightamong groups (see FIG.7).

[0122] Based on the study results, it can be shown that the combination treatment of ANQ-45 BID and anti-PD-1 Ab significantly augments in three HCC mouse models. Furthermore, the combination treatment of ANQ-45 BID plus anti-PD-1 Ab exhibited a significant antitumor effect compared to ANQ-45 BID monotherapy or anti-PD-1 Ab monotherapy. Additionally, ANQ-45 BID and anti-PD-1 Ab combination treatment extends the survival time of mice. Safety evaluation indicated no significant changes in mouse body weight among groups.

[0123] While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS 1. A method for treating liver cancer in a subject comprising administering to saidsubject a therapeutically effective amount of a cyclohexenone compound having the structure: R3CH3O R4n a pharmaceutically acceptable salt, metabolite, solvate or antibodies of programmed cell death-1 (PD-1) or its ligand, wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2and R3independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with oneor more substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12; or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof.

2. The method of claim 1, wherein said subject is human.

3. The method of claim 1, wherein R is a hydrogen, C(=O)C3H8, C(=O)C2H5, orC(=O)CH3.

4. The method of any one of claims 1-3, wherein each of R1, R2 and R3independently is a hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

5. The method of any one of claims 1-4, wherein R1 is a hydrogen or methyl.

6. The method of any one of claims 1-4, wherein R2 is a hydrogen or methyl.

7. The method of any one of claims 1-6, wherein R4 is C1-C8alkyl optionallysubstituted with one or more substituents selected from C1-C8 alkyl, C2-C8 alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl.

8. The method of claim 7, wherein R4 is CH2CH=C(CH3)2.

9. The method of any one of claims 1-8, wherein said cyclohexenone compound isCH3CH3CH3CH3.

10. The method of any one of claims 1-9, wherein said cyclohexenone compound, ora pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof, is administered orally, parenterally, intravenously or by injection.

11. A composition comprising a cyclohexenone compound having the structure:R3CH3O R4n a pharmaceutically acceptable salt, metabolite, solvate or antibodies of programmed cell death-1 (PD-1) or its ligand, wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2 and R3 independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with oneor more substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12.

12. The composition of claim 11, wherein R is a hydrogen, C(=O)C3H8, C(=O)C2H5,or C(=O)CH3.

13. The composition of claim 12, wherein each of R1, R2 and R3 independently is ahydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

14. The composition of claim 13, wherein R4 is H, or C1-C8alkyl optionallysubstituted with one or more substituents selected from C1-C8 alkyl, C2-C8 alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, or C1-C8haloalkyl.

15. The composition of claim 14, wherein R4 is CH2CH=C(CH3)2.

16. The composition of any one of claims 11-15, wherein said compound isCH3CH3CH3CH317. A pharmaceutical composition for use in treating liver cancer in a subjectR3CH3O R4n comprising a cyclohexenone compound having the a pharmaceutically acceptable salt, metabolite, solvate orantibodies of programmed cell death-1 (PD-1) or its ligand , wherein each of X and Y independently is oxygen; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R2and R3independently is a hydrogen, methyl or (CH2)m—CH3, m=0-6; R4 is H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, or aryl optionally substituted with oneor more substituents selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl; n=1-12.

18. The pharmaceutical composition for use in according to claim 17, wherein saidcyclohexenone compound is antroquinonol.

19. Use of the composition of claim 11 in the manufacture of a medicament fortreating liver cancer.

20. The use of claim 19, wherein said cyclohexenone compound is antroquinonol.

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