TWI846676B - Therapeutic compositions for treating pancreatic cancer - Google Patents

Abstract

The present invention is directed to therapeutic compositions for treating pancreatic cancer comprising a cyclohexnone compound and one or more anti-cancer agents.

Description

Therapeutic compositions for treating pancreatic cancer

The present invention relates to a therapeutic composition for treating pancreatic cancer, comprising a cyclohexanone compound and one or more anticancer agents.

Pancreatic cancer develops when cells in the pancreas begin to multiply uncontrollably and form clumps. These cancer cells have the ability to invade other parts of the body. Pancreatic cancer is the fourth leading cause of cancer death in Western countries and the tenth leading cause of cancer death in Taiwan. About 60% of pancreatic cancers start in the head of the pancreas, and about 21% invade the entire pancreas.

Pancreatic cancer can be divided into two main types. The vast majority of cases (about 99%) occur in the part of the pancreas that produces digestive enzymes and are called exocrine pancreatic cancer. There are several subtypes of exocrine pancreatic cancer, but their diagnosis and treatment have many similarities. A small number of pancreatic cancers occur in the hormone-producing (endocrine) tissue of the pancreas.

Only about one in five (20%) new cases are likely to undergo surgery with curative intent. Although CT scans are available in practice, it can be difficult to determine whether a tumor can be completely removed (its "resectability"), and it may only become clear during surgery that it cannot be successfully removed without damaging other vital tissue. Even when surgery appears to have been successful, cancer cells are often found around the edges ("margins") of the removed tissue. After surgery, if the patient is well enough after a recovery period of 1 to 2 months, adjuvant chemotherapy with gemcitabine or 5-FU may be given. In people who are not good candidates for curative surgery, chemotherapy may be used to prolong life or improve their quality of life.

其中X和Y中的每一個獨立地為氧或硫；R為氫或C(=O)C₁-C₈烷基；R₁、R₂和R₃中的每一個獨立地為氫、甲基或(CH₂)_m-CH₃；R₄為H、C₁-C₈烷基、C₂-C₈烯基、C₂-C₈炔基或任選地被一個或多個取代基取代的芳基，該取代基選自C₁-C₈烷基、C₂-C₈烯基、C₂-C₈炔基、C₃-C₈環烷基和C₁-C₈鹵代烷基；n=1-12。 In one aspect, provided herein is a composition for treating pancreatic cancer in a subject, the composition comprising a compound having the following structure or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof and one or more anticancer agents:

wherein each of X and Y is independently oxygen or sulfur; R is hydrogen or C(═O)C ₁ -C ₈ alkyl; each of R ₁ , R ₂ and R ₃ is independently hydrogen, methyl or (CH ₂ ) _m -CH ₃ ; R ₄ is H, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl or aryl optionally substituted with one or more substituents selected from C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₈ cycloalkyl and C ₁ -C ₈ halogenated alkyl; and n=1-12.

In another aspect, provided herein is a method for treating pancreatic cancer in a subject, comprising administering to a subject in need thereof a compound having the following structure or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof and one or more anticancer agents:

wherein each of X and Y is independently oxygen or sulfur; R is hydrogen or C(=O)C ₁ -C ₈ alkyl; each of R ₁ , R ₂ and R ₃ is independently hydrogen, methyl or (CH ₂ ) _m -CH ₃ ; R ₄ is H, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl or aryl optionally substituted with one or more substituents selected from C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₈ cycloalkyl and C ₁ -C ₈ halogenated alkyl; and n=1-12.

In another aspect, provided herein is a method for treating a pancreatic cancer patient that is resistant, refractory or unresponsive to gemcitabine, paclitaxel or a combination thereof, comprising administering to a patient in need thereof a compound having the following structure or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof:

wherein each of X and Y is independently oxygen or sulfur; R is hydrogen or C(═O)C ₁ -C ₈ alkyl; each of R ₁ , R ₂ and R ₃ is independently hydrogen, methyl or (CH ₂ ) _m -CH ₃ ; R ₄ is H, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl or aryl optionally substituted with one or more substituents selected from C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₈ cycloalkyl and C ₁ -C ₈ halogenated alkyl; and n=1-12.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are incorporated herein 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.

The novel features of the present invention are specifically described in the attached patent application scope. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative specific examples utilizing the principles of the present invention and the accompanying drawings, in which:

Figure 1A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated for 48 hours (1A) and 72 hours (1B) with exemplary compound 1. Cytotoxic activity was measured using the MTT assay.

Figure 2A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated with erlotinib for 48 hours (2A) and 72 hours (2B). Cytotoxic activity was measured using the MTT assay.

Figure 3A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated with 5-FU for 48 hours (3A) and 72 hours (3B). Cytotoxic activity was measured using the MTT assay.

Figure 4A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated with gemcitabine for 48 hours (4A) and 72 hours (4B). Cytotoxic activity was measured using the MTT assay.

Figure 5A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated with irinotecan for 48 hours (5A) and 72 hours (5B). Cytotoxic activity was measured using the MTT assay.

Figure 6A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated with oxaliplatin for 48 hours (6A) and 72 hours (6B). Cytotoxic activity was measured using the MTT assay.

Figure 7A/B provides the results of cytotoxic activity in AsPC-1 pancreatic cancer cells treated with paclitaxel for 48 hours (7A) and 72 hours (7B). Cytotoxic activity was measured using the MTT assay.

Figure 8 shows the effect of compound 1 combined with chemotherapy drugs _paclitaxel , gemcitabine, 5-FU, oxaliplatin, erlotinib or irinotecan on the cytotoxicity of AsPC-1 pancreatic cancer cell line for 48 hours. Cytotoxic activity was measured using MTT assay. Values are mean ± SEM of survival rate. Different letters (ae) indicate significant differences among various treatments (P<0.05).

Figure 9 shows the cytotoxic activity of compound 1 in combination with chemotherapy drugs paclitaxel, gemcitabine, 5-FU, oxaliplatin, erlotinib and irinotecan. AsPC-1 pancreatic cancer cells were treated with antroquinol or combination preparations for 72 hours. Cytotoxic activity was measured using the MTT assay. Values are mean survival rates ± SEM. Different letters (ag) indicate significant differences among the various treatments (P<0.05).

Figures 10A-C show the dose-effect of compound 1 (also known as androquinol, Aq) in combination with paclitaxel (Px), gemcitabine (Ge) in pancreatic cancer cell lines. The graphs show the Fa values for treatment with androquinol in combination with paclitaxel, androquinol in combination with gemcitabine, androquinol plus paclitaxel and gemcitabine in AsPC-1 (10A), CaPan-2 (10B) and Panc-1 (10C) pancreatic cancer cell lines. The x-axis represents the drug dose in μmol/L, and the y-axis represents Fa, i.e., the fraction of affected cells (growth inhibition).

Figures 11A-C show the results of the analysis of compound 1 combined with paclitaxel and gemcitabine in pancreatic cancer cell lines treated for 72 hours. CI is plotted for androquinol (Aq) combined with paclitaxel (Px), androquinol combined with gemcitabine (Ge), androquinol plus paclitaxel and gemcitabine in AsPC-1 (11A), CaPan-2 (11B) and Panc-1 (11C) pancreatic cancer cell lines. The x-axis represents Fa (fraction of affected cells) and the y-axis represents CI (combination index). CI = 1, <1 and >1 represent additive effects, synergistic effects and antagonistic effects, respectively.

FIG12 shows the results of changes in mouse body weight during the anti-tumor activity test of Compound 1 combined with chemotherapy on the AsPC-1 subcutaneous xenograft model.

Figure 13 shows the effect of compound 1 plus chemotherapy on the anti-tumor activity of the AsPC-1 subcutaneous xenograft model. Tumor size was measured weekly, and the average relative tumor volume was plotted as a function of time after the start of treatment. Each point represents the mean of the tumor volume.

Figure 14 shows the effect of compound 1 plus chemotherapy on the AsPC-1 subcutaneous xenograft model. Compared with the control group (T1, corn oil), the percentage of tumor volume in the treatment group containing the combination of compound 1 was significantly reduced.

Figure 15 shows the analysis results of the percentage of tumor growth inhibition (TGI) of AsPC- 1 tumor volume growth by compound 1 alone or in combination with chemotherapy drugs paclitaxel and gemcitabine. Each bar represents the mean value of tumor growth inhibition. The values are mean ± SEM. Different upper letters indicate significant differences between the treatment groups (P < 0.05).

FIG. 16 illustrates the comparison results of representative images of AsPC-1 pancreatic tumor xenografts before and after treatment with Compound 1 and/or Compound 1 plus chemotherapy, respectively.

FIG. 17 depicts the comparison results of representative ultrasound images of AsPC-1 pancreatic tumor xenografts before and after treatment with Compound 1 and/or Compound 1 plus chemotherapy, respectively.

Figure 18A/B shows images (18A) and diagrams (18B) of tumors excised when male nude mice bearing subcutaneous AsPC-1 pancreatic tumor xenografts were sacrificed 28 days after treatment. Statistical significance was analyzed using ANOVA. *P<0.05 was considered statistically significant when compared with the control group (T1, corn oil group).

Figure 19 shows the results of the inhibitory effect of compound 1 alone or compound 1 combined with chemotherapy drugs paclitaxel and gemcitabine on the growth of AsPC-1 solid tumors. Each bar represents the mean value of tumor inhibition rate (TIR). The values are TIR mean ± SEM. Different upper letters indicate significant differences between treatment groups (P < 0.05).

Only about one in five new cases (20%) may undergo surgery with curative intent. Although by the 1980s curative surgery no longer resulted in very high mortality rates, a large proportion of people (about 30-45%) still must receive treatment for postoperative illness that is not caused by the cancer itself. The most common complication of surgery is difficulty emptying the stomach. After surgery, if the patient is well enough after a 1 to 2 month recovery period, adjuvant chemotherapy with gemcitabine or 5-FU can be given. Gemcitabine was approved by the U.S. Food and Drug Administration (FDA) in 1997 after clinical trials reported improved quality of life and a 5-week increase in median survival in patients with advanced pancreatic cancer. Chemotherapy with gemcitabine alone has been the standard for about a decade because many trials testing it in combination with other drugs have failed to show significantly better results.

The four-drug FOLFIRINOX chemotherapy regimen was found to be more effective than gemcitabine but has significant side effects and is therefore only indicated for people with good performance status. The same is true for protein-bound paclitaxel (nab-paclitaxel), which was approved by the FDA in 2013 for use with gemcitabine in pancreatic cancer. However, advances in the past few years have resulted in only a few months’ extension of survival.

A method for treating pancreatic cancer in a subject is reported in U.S. Patent No. 8,236,860, the method comprising administering a cyclohexanone compound to a subject in need thereof. An exemplary compound for treating pancreatic cancer, anthroquinol, was studied using an orthotopic PANC-1 human pancreatic cancer xenograft model. Four groups of mice were treated with 30 mg/kg, 60 mg/kg, 90 mg/kg, and a vehicle control group, respectively. Treatment with 30, 60, and 90 mg/kg anthroquinol produced effective antitumor activity compared to the vehicle control group, with statistically significantly smaller mean tumor volumes and tumor weights at all three dose levels. Although Androquinol has been shown to be an excellent candidate for the treatment of pancreatic cancer, it is not being considered for use in any combination therapy.

However, it has been unexpectedly discovered that the synergistic effects present in combination therapy comprising a compound having the following structure or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof and one or more anticancer agents (such as gemcitabine, paclitaxel or a combination thereof) provide synergistic effects compared to single therapies;

wherein each of X and Y is independently oxygen or sulfur; R is hydrogen or C(=O)C ₁ -C ₈ alkyl; each of R ₁ , R ₂ and R ₃ is independently hydrogen, methyl or (CH ₂ ) _m -CH ₃ ; R ₄ is H, C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl or aryl optionally substituted with one or more substituents selected from C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₈ cycloalkyl and C ₁ -C ₈ halogenated alkyl; and n=1-12.

In some embodiments, such combination therapy also includes the administration of an immunotherapy agent.

的化合物和一種或多種抗癌劑的用於治療受試者中的胰腺癌的組合物(參見實施例1-3)。在一些具體例中，該環己烯酮化合物從天然產物的提取物獲得，而在其他一些具體例中，通過合成來製備。參見例如美國專利號9365481。 In some embodiments, the present invention provides a method comprising:

Compositions of a compound and one or more anticancer agents for treating pancreatic cancer in a subject (see Examples 1-3). In some embodiments, the cyclohexenone compound is obtained from an extract of a natural product, while in other embodiments, it is prepared by synthesis. See, for example, U.S. Patent No. 9,365,481.

In some embodiments, the one or more anticancer agents include gemcitabine, paclitaxel, idarubicin/cytarabine, etoposide phosphate, glievec (imatinib), temozolomide, bortezomib, letrozole, cetuximab, bevacizumab, nab-paclitaxel, docetaxel, erlotinib, pemetrexed, pemetrexed/carboplatin, paclitaxel (paxlitaxel)/carboplatin, letrozole/cyclophosphamide, temsirolimus, bevacizumab/temsirolimus, ipilimumab, RAD001, parvovir, tadalafil ... Zopanib, FOLFIRI, BKM120, GSK1120212, PF-05212384/irinotecan, AZD2171, PF-04691502, cyclophosphamide, cisplatin, cytarabine/daunorubicin, temsirolimus, erlotinib/temsirolimus, capecitabine, tamoxifen, bortezomib, trastuzumab, docetaxel/capecitabine, trastuzumab/tipifarnib, tipifarnib/gemcitabine, tootecan, or a combination thereof. In certain specific examples, the one or more anticancer agents are gemcitabine, paclitaxel, or a combination thereof. In certain specific examples, the one or more anticancer agents are a combination of gemcitabine and paclitaxel.

For example, paclitaxel is classified as a "plant bioalkaloid," "taxane," and "antimicrotubule agent." It is used to treat several types of cancer, such as ovarian cancer, breast cancer, lung cancer, Kaposi's sarcoma, cervical cancer, and pancreatic cancer. Albumin-bound paclitaxel (trade name Abraxane, also known as nab-paclitaxel) is an alternative formulation in which paclitaxel is bound to albumin nanoparticles. Paclitaxel is known to have some common side effects, including nausea and vomiting, loss of appetite, changes in taste, thinning or brittle hair, pain in the joints of the arms or legs that lasts two to three days, changes in nail color, and tingling in the hands or toes. Paclitaxel is one of several cytoskeletal drugs that target tubulin. Paclitaxel-treated cells had defects in mitotic spindle assembly, chromosome segregation, and cell division. Unlike other tubulin-targeting drugs such as colchicine, which inhibit microtubule assembly, paclitaxel stabilizes microtubule polymers and protects them from disassembly.

Another exemplary anticancer drug, gemcitabine, is a nucleoside analog used as chemotherapy to treat a variety of types of cancer, including breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, and bladder cancer. Common side effects include bone marrow suppression, liver and kidney problems, nausea, fever, rash, shortness of breath, and hair loss. Use during pregnancy may cause harm to the baby. Gemcitabine belongs to the nucleoside analog drug family. It works by preventing the production of new DNA, which leads to cell death.

In some embodiments, a method for treating pancreatic cancer in a subject is provided, comprising administering a combination therapy composition described herein to a subject in need thereof. In some embodiments, a therapeutically effective amount of a combination therapy composition described herein is provided for use in the preparation of a medicament for treating pancreatic cancer in a subject.

的化合物或其藥學上可接受的鹽、 代謝物、溶劑化物或前藥。在一些具體例中，所述方法進一步包括施用免疫治療劑。在一些具體例中提供了治療有效量的具有結構

的化合物或其藥學上可接受的鹽、代謝物、溶劑化物或前藥在製備用於治療對吉西他濱、紫杉醇或其組合具有抗性、難治性或無反應性的胰腺癌患者的藥物中的用途。 In some embodiments, a method for treating a pancreatic cancer patient that is resistant, refractory, or unresponsive to gemcitabine, paclitaxel, or a combination thereof is provided, comprising administering to a patient in need thereof a composition having a structure

A compound or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof. In some embodiments, the method further comprises administering an immunotherapeutic agent. In some embodiments, a therapeutically effective amount of a compound having a structure

Use of a compound or a pharmaceutically acceptable salt, metabolite, solvate or prodrug thereof in the preparation of a medicament for treating pancreatic cancer patients who are resistant, refractory or unresponsive to gemcitabine, paclitaxel or a combination thereof.

In certain embodiments, the cancer is resistant, refractory or unresponsive to a drug selected from the group consisting of gemcitabine, paclitaxel, idarubicin/cytarabine, etoposide phosphate, glievec, temozolomide, bortezomib, letrozole, cetuximab, bevacizumab, nab-paclitaxel, docetaxel, erlotinib, pemetrexed, pemetrexed/carboplatin, paclitaxel/carboplatin, letrozole/cyclophosphamide, temsirolimus, bevacizumab/temsirolimus, ipilimumab, RAD00 1. Pazopanib, FOLFIRI, BKM120, GSK1120212, PF-05212384/irinotecan, AZD2171, PF-04691502, cyclophosphamide, cisplatin, cytarabine/daunorubicin, temsirolimus, erlotinib/temsirolimus, capecitabine, tamoxifen, bortezomib, trastuzumab, docetaxel/capecitabine, trastuzumab/tipifarnib, tipifarnib/gemcitabine, topotecan or a combination thereof. In some specific examples, the cancer is resistant, refractory or unresponsive to gemcitabine or paclitaxel. In some specific examples, the cancer is resistant, refractory or unresponsive to a combination of gemcitabine and paclitaxel.

的環己烯酮化合物由任何合適的起始材料以合成或半合成方式製備。在其他具體例中，該環己烯酮化合物通過發酵等製備。例如，化合物1、3和4從有機溶劑提 取物中分離或以合成或半合成方式製備。以下示出了非限制性的示例化合物。 In some specific examples, the structure

The cyclohexenone compound is prepared synthetically or semi-synthetically from any suitable starting material. In other specific examples, the cyclohexenone compound is prepared by fermentation, etc. For example, compounds 1, 3 and 4 are isolated from organic solvent extracts or prepared synthetically or semi-synthetically. Non-limiting exemplary compounds are shown below.

In some embodiments, R is hydrogen, C(=O)C ₃ H ₇ , C(=O)C ₂ H ₅ or C(=O)CH ₃ . In some embodiments, R ₁ is hydrogen or methyl. In some embodiments, R ₂ is hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R ₃ is hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R ₄ is hydrogen. In some embodiments, R ₄ is C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, or aryl optionally substituted with one or more substituents selected from C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, C ₃ -C ₈ cycloalkyl, and C ₁ -C ₈ halogenated alkyl. In some embodiments, R ₄ is CH ₂ CH=C(CH ₃ ) ₂ . In some embodiments, the compound is

In some embodiments, the methods for treating pancreatic cancer described herein include administering an immunotherapeutic agent. The immunotherapeutic agent includes, but is not limited to, any living immune cells that can be administered to a patient, and/or antibodies specific to target cells (e.g., tumor cells, such as pancreatic cancer cells). Preferably, the immunotherapeutic agent is a NK cell or a T cell, or a modified or derivative thereof.

Immunotherapy

Immunotherapy is "the treatment of disease by inducing, enhancing, or suppressing an immune response." Immunotherapies designed to elicit or amplify an immune response are classified as activating immunotherapies, while those designed to reduce or suppress it are classified as suppressive immunotherapies.

In some specific examples, the immunotherapeutic agent is an anticancer antibody. Non-limiting examples include trastuzumab (Herceptin®), bevacizumab (Avastin®), cetuximab (Erbitux®), pantuzumab (Vectibix®), ipilimumab (Yervoy®), rituximab (Rituxan®), alemtuzumab (Campath®), ofatumumab (Arzerra®), gemtuzumab ozogamicin (Mylotarg®), brentuximab vedotin (Adcetris®), ⁹⁰ Y-ibritumomab tiuxetan (Zevalin®), ¹³¹ I-tositumomab (Bexxar®), anti-programmed death 1 (anti-PD-1) antibodies such as nivolumab, pembrolizumab, etc.

Certain pharmaceutical and medical terms

Unless otherwise indicated, the following terms used in this application (including the specification and the claims) have the definitions given below. It must be noted that, as used in this specification and the attached claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Conventional methods of mass spectrometry, NMR, HPLC, proteochemistry, biochemistry, recombinant DNA technology, and pharmacology are used unless otherwise indicated. In this application, the use of "or" or "and" means "and/or" unless otherwise indicated. In addition, the use of the term "includes" and other forms such as "includes", "contains", and "includes" is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

"Alkyl" refers to an aliphatic hydrocarbon group. An alkyl group can be a saturated alkyl group (meaning it does not contain any carbon-carbon double bonds or carbon-carbon triple bonds), or an alkyl group can be an unsaturated alkyl group (meaning it contains at least one carbon-carbon double bond or carbon-carbon triple bond). The alkyl portion, whether saturated or unsaturated, can be branched or straight chain.

"Alkyl" can have 1-12 carbon atoms (whenever a numerical range such as "1-12" appears herein, it refers to each integer in the given range; for example, "1-12 carbon atoms" means that the alkyl group can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 12 carbon atoms, but the present definition also encompasses the presence of the term "alkyl" without specifying a numerical range). The alkyl group of the compounds described herein can be designated as "C ₁ -C ₁₂ alkyl", "C ₁ -C ₈ alkyl" or similar designations. By way of example only, "C ₁ -C ₈ alkyl" means that there are 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in the alkyl chain. In one aspect, the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are by no means limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, allyl, but-2-enyl, but-3-enyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. In one aspect, the alkyl group is a C ₁ -C ₈ alkyl group.

The term "alkylene" refers to a divalent alkyl group. Any of the above monovalent alkyl groups can be converted to an alkylene group by abstracting the second hydrogen atom from the alkyl group. In one aspect, the alkylene group is a C ₁ -C ₁₂ alkylene group. In another aspect, the alkylene group is a C ₁ -C ₈ alkylene group. Typical alkylene groups include, but are not limited to, -CH ₂ -, -CH(CH ₃ )-, -C(CH ₃ ) ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH(CH ₃ )-, -CH ₂ C(CH ₃ ) ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH ₂ CH _{2 CH 2} -, and the like.

As used herein, the term "aryl" refers to an aromatic ring in which each atom forming the ring is a carbon atom. The aryl ring is composed of five, six, seven, eight, nine or more than nine carbon atoms. The aryl group is optionally substituted. In one aspect, the aryl group is phenyl or naphthyl. In one aspect, the aryl group is phenyl. In one aspect, the aryl group is a C ₆ -C ₁₀ aryl group. Depending on the structure, the aryl group can be a monovalent group or a divalent group (i.e., an arylene group). In one aspect, the arylene group is a C ₆ -C ₁₀ arylene group. Exemplary arylene groups include, but are not limited to, phenyl-1,2-subunit, phenyl-1,3-subunit, and phenyl-1,4-subunit.

The term "aromatic" refers to a planar ring having a delocalized π-electron system containing 4n+2 π electrons, where n is an integer. Aromatic rings can be composed of five, six, seven, eight, nine, ten, or more than ten atoms. Aromatic rings are optionally substituted. The term "aromatic" includes 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 that share adjacent pairs of carbon atoms) groups.

The term "halogenated" or alternatively "halogen" or "halogen" means fluorine, chlorine, bromine or iodine.

As used herein, the term "alkenyl" refers to a straight chain, branched chain, or cyclic (in this case, it is also referred to as a "cycloalkenyl") hydrocarbon containing 2-10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. The alkenyl groups of the compounds described herein may be represented as " _C2 - _C10 alkenyl,"" _C2 - _C8 alkenyl," or similar designations. By way of example only, " _C2 - _C8 alkenyl" means that there are two, three, four, five, six, seven, or eight carbon atoms in the alkenyl chain. In some embodiments, alkenyl groups are monovalent or divalent (i.e., alkenylene), depending on the structure. 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.

As used herein, the term "alkynyl" refers to a straight chain, branched chain, or cyclic (in this case, it is also referred to as a "cycloalkynyl") alkane containing 2-10 carbons and containing at least one carbon-carbon triple bond formed by the removal of four hydrogens. In some embodiments, the alkynyl group is monovalent or divalent (i.e., alkynylene), depending on the structure. The alkynyl group of the compounds described herein can be represented as " _C2 - _C10 alkynyl", " _C2 - _C8 alkynyl", or similar designations. By way of example only, " _C2 - _C8 alkynyl" means that there are two, three, four, five, six, seven, or eight carbon atoms in the alkynyl chain. In some embodiments, the alkynyl group is optionally substituted. Illustrative examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and the like.

，

，

。在一些具體例中，根據結構，環烷基基基團是單價或雙價的(即，亞環烷基)。 The term "cycloalkyl" as used herein refers to a monocyclic or polycyclic group containing only carbon and hydrogen, and includes saturated, partially unsaturated, or fully unsaturated groups. Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative examples of rings include, but are not limited to, the following moieties:

,

,

In some embodiments, depending on the structure, a cycloalkyl group is monovalent or divalent (i.e., cycloalkylene).

As used herein, the terms "haloalkyl", "haloalkenyl", "haloalkynyl" and "haloalkoxy" include alkyl, alkenyl, alkynyl and alkoxy structures in which at least one hydrogen is replaced by a halogen atom. In certain embodiments in which two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are all identical to one another. In other embodiments in which two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are not all identical to one another. The terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups, respectively, in which the halogen is fluorine. In certain embodiments, the haloalkyl is optionally substituted.

As used herein, the term "acceptable" with respect to a formulation, composition or ingredient means having no persistent adverse effect on the general health of the subject being treated.

Antrodia is a genus of fungi in the family Meripilaceae. The fruiting bodies of species of Antrodia generally lie flat or spread out on the growing surface, with the hymenium exposed; the edges may be rolled up, forming narrow brackets. Most species are found in temperate and boreal forests and cause brown rot.

As used herein, the term "carrier" refers to a relatively nontoxic chemical compound or agent that facilitates the introduction of a compound into cells or tissues.

As used herein, the term "co-administration" or similar terms is intended to include the administration of multiple selected therapeutic agents to a patient, and is intended to include treatment regimens in which multiple agents are administered by the same or different routes of administration or at the same or different times.

The term "diluent" refers to a chemical compound used to dilute a compound of interest prior to delivery. Diluents can also be used to stabilize compounds because they can provide a more stable environment. Salts dissolved in a buffered solution (which can also provide pH control or maintenance) are used as diluents in the art, including but not limited to phosphate buffered saline solutions.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent or compound administered that is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic applications is the amount of a composition comprising a compound disclosed herein that is required to cause a clinically significant reduction in disease symptoms. In any individual case, an appropriate "effective" amount can be determined using techniques such as dose escalation studies.

A "metabolite" of a compound disclosed herein is a derivative of the compound formed when the compound is metabolized. The term "active metabolite" refers to a biologically active derivative of the compound formed when the compound is metabolized. As used herein, the term "metabolism" refers to the sum of processes (including but not limited to hydrolysis reactions and enzyme-catalyzed reactions) by which a particular substance is changed by an organism. Thus, an enzyme can produce a specific structural change to a compound. For example, cytochrome P450 catalyzes a variety of oxidation and reduction reactions, while UDP-glucuronosyltransferase catalyzes the transfer of activated glucuronic acid molecules to aromatic alcohols, fatty alcohols, carboxylic acids, amines, and free hydroxyls. Metabolites of the compounds disclosed herein are optionally identified by administering the compound to a host and analyzing a tissue sample from the host, or by incubating the compound with hepatocytes in vitro and analyzing the resulting compound.

As used herein, the term "pharmaceutical combination" means a product obtained by mixing or combining more than one active ingredient, and includes fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredient, such as a compound (i.e., a cyclohexenone compound described herein) and a co-agent are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredient, such as a compound (i.e., a cyclohexenone compound described herein) and a co-agent are administered to a patient simultaneously, concurrently or sequentially as separate entities without specific time interval restrictions, wherein such administration provides effective levels of both compounds in the patient's body. The latter also applies to cocktail therapy, such as the administration of three or more active ingredients.

The term "pharmaceutical composition" refers to a mixture of a compound (i.e., the cyclohexenone compound described herein) and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients. The pharmaceutical composition facilitates the administration of the compound to an organism. There are a variety of techniques for administering compounds in the art, including but not limited to: intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, pigs; livestock such as rabbits, dogs and cats; experimental animals, including rodents such as rats, mice and guinea pigs, etc. In a specific example, the mammal is a human.

As used herein, the term "treat" or "treatment" includes prophylactically and/or therapeutically alleviating, eliminating or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, such as arresting the development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, relieving a condition caused by a disease or condition, or terminating symptoms of a disease or condition.

Route of administration and dosage

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

In certain embodiments, the compounds described herein are typically administered locally rather than systemically, for example, by injecting the compound directly into an organ, in a depot preparation or sustained release preparation. In certain embodiments, the long-acting preparation is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. In addition, in other embodiments, the drug is delivered in a targeted drug delivery system, for example, in liposomes coated with organ-specific antibodies. In such embodiments, the liposomes are targeted to the organ and selectively taken up by the organ. In other embodiments, the compounds described herein are provided in the form of a rapid release preparation, in the form of an extended release preparation, or in the form of an immediate release preparation. In other embodiments, the compounds described herein are administered locally.

In some embodiments, the cyclohexenone compound or its pharmaceutically acceptable salt, metabolite, solvate or prodrug is administered parenterally or intravenously. In other embodiments, the cyclohexenone compound or its pharmaceutically acceptable salt, metabolite, solvate or prodrug is administered by injection. In some embodiments, the cyclohexenone compound or its pharmaceutically acceptable salt, metabolite, solvate or prodrug is administered orally.

In the event that the patient's condition does not improve, at the physician's discretion, administration of the compound may be continued chronically, i.e., for an extended period of time, including throughout the patient's life, in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition. In the event that the patient's condition does improve, administration of the compound may be continued or temporarily discontinued for a period of time (i.e., a "drug holiday"), at the physician's discretion.

The foregoing ranges are only suggestive, as the number of variables regarding individual treatment regimens is large, and considerable deviations from these recommendations are not uncommon. Such dosages may vary depending on many variables, including, but not limited to, the activity of the compound used, the disease or condition being treated, the route of administration, the requirements of the subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

The toxicity and therapeutic efficacy of such treatment regimens can be determined by standard pharmaceutical procedures in cell culture 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 to 50% of the population). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, and it can be expressed as the ratio between _LD50 and _ED50 . Compounds showing high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the _ED50 and have minimal toxicity. The dosage may vary within this range, depending on the dosage form used and the route of administration used. In certain embodiments, the cyclohexenone compound is

In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. In certain embodiments, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants, which facilitate processing of the active compounds into pharmaceutically acceptable preparations. Suitable formulations depend on the chosen route of administration. Any pharmaceutically acceptable techniques, carriers, and excipients may be suitably used to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy , 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms , Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999).

Provided herein are pharmaceutical compositions comprising a compound (i.e., a cyclohexenone compound described herein) and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, as in combination therapy, the compound is administered in the form of a pharmaceutical composition in which the compound (i.e., a cyclohexenone compound described herein) is mixed with other active ingredients. Included herein are all combinations of active ingredients described in the following combination therapy section and throughout the disclosure. In certain embodiments, the pharmaceutical composition comprises one or more compounds (i.e., a cyclohexenone compound described herein).

As used herein, a pharmaceutical composition refers to a mixture of a compound (i.e., a cyclohexenone compound described herein) and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients. In certain embodiments, the pharmaceutical composition facilitates the administration of the compound to an organism. In certain embodiments, to implement the treatment or use methods provided herein, a therapeutically effective amount of the compound (i.e., a cyclohexenone compound described herein) is administered in the form of a pharmaceutical composition to a mammal suffering from a disease or condition to be treated. In certain embodiments, the mammal is a human. In certain embodiments, the therapeutically effective amount varies according to the severity of the disease, the age and relative health of the subject, the efficacy of the compound used, and other factors. In addition, the compounds described herein are used alone or in combination with one or more therapeutic agents as components of a mixture.

In one embodiment, the compound (i.e., the cyclohexenone compound described herein) is formulated into an aqueous solution. In a specific embodiment, the aqueous solution is selected from a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or a physiological saline buffer, by way of example only. In other embodiments, the compound (i.e., the cyclohexenone compound described herein) is formulated for transmucosal administration. In a specific embodiment, the transmucosal formulation includes a permeabilizer suitable for the barrier to be permeated. In other embodiments of formulating the compounds described herein for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In a specific embodiment, such solutions include physiologically compatible buffers and/or excipients.

In another embodiment, the compounds described herein are formulated for oral administration. The compounds described herein, including the compounds (i.e., the cyclohexenone compounds described herein), are formulated by mixing the active compound with, for example, a pharmaceutically acceptable carrier or excipient. In various embodiments, the compounds described herein are formulated into oral dosage forms, including, by way of example, tablets, powders, pills, sugar tablets, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

In certain embodiments, a pharmaceutical preparation for oral use is obtained by mixing one or more solid excipients with one or more compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable excipients, if desired, to obtain tablets or dragee cores. In particular, suitable excipients are: fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or other substances, such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In certain embodiments, a disintegrant is optionally added. By way of example only, disintegrants include sodium cross-linked carboxymethylcellulose, polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate.

In one embodiment, dosage forms such as sugar tablet cores and tablets have one or more suitable coatings. In a particular embodiment, a concentrated sugar solution is used to coat the dosage form. The sugar solution optionally contains additional ingredients, such as, for example, gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes and/or pigments are also optionally added to the coating for identification purposes. In addition, dyes and/or pigments are optionally used to indicate different combinations of active compound doses.

In certain embodiments, a therapeutically effective amount of at least one compound described herein is formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and plasticizers (such as glycerol or sorbitol). In certain embodiments, push-fit capsules contain active ingredients mixed with one or more fillers. By way of example only, fillers include lactose, binders such as starch, and/or lubricants such as talc or magnesium stearate, and optional stabilizers. In other embodiments, soft capsules contain one or more active compounds dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oils, liquid parathion or liquid polyethylene glycols. In addition, stabilizers are optionally added.

In other embodiments, a therapeutically effective amount of at least one compound described herein is formulated for buccal or sublingual administration. By way of example only, formulations suitable for buccal or sublingual administration include tablets, troches, or gels. In yet other embodiments, the compounds described herein are formulated for parenteral injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, the formulation for injection is provided in a unit dosage form (e.g., in an ampoule) or in a multi-dose container. A preservative is optionally added to the injection formulation. In other specific examples, the pharmaceutical composition of the compound (i.e., the cyclohexenone compound described herein) is formulated as a sterile suspension, solution or emulsion in an oily or aqueous vehicle in a form suitable for parenteral injection. Parenteral injection preparations optionally contain formulation agents, such as suspending agents, stabilizers and/or dispersants. In specific specific examples, pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds in water-soluble form. In other specific examples, the suspension of the active compound is prepared into a suitable oily injection suspension. By way of example only, suitable lipophilic solvents or vehicles used in the pharmaceutical compositions described herein include 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 that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension contains suitable stabilizers or reagents that increase the solubility of the compound to allow the preparation of highly concentrated solutions. Alternatively, in other embodiments, the active ingredient is in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

In one aspect, the compound (i.e., the cyclohexenone compound described herein) is prepared as a solution for parenteral injection as described herein or known in the art and administered using an autoinjector. Autoinjectors are known, for example, the autoinjectors 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 is incorporated herein by reference to these disclosures). Generally, all autoinjectors contain a certain volume of solution to be injected containing the compound (i.e., the cyclohexenone compound described herein). Typically, an autoinjector includes: a reservoir for containing a solution, which is fluidly connected to a needle for drug delivery; and a mechanism for automatically deploying the needle, inserting the needle into a patient, and delivering a dose into the patient's body. Exemplary syringes provide about 0.3 mL, 0.6 mL, 1.0 mL, or other appropriate volumes of solution, with a concentration of about 0.5 mg to 50 mg of the compound (i.e., the cyclohexenone compound described herein) per 1 mL of solution. Each syringe is capable of delivering only one dose of the compound.

In other specific examples, the compound (i.e., the cyclohexenone compound described herein) is applied topically. The compounds described herein are formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, sticks, balms, creams or ointments. Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancers, buffers and preservatives.

In yet other embodiments, the compound (i.e., the cyclohexenone compound described herein) is formulated as a rectal composition, such as an enema, rectal gel, rectal foam, rectal aerosol, suppository, jelly suppository, or retention enema containing a conventional suppository base such as cocoa butter or other glycerides and a synthetic polymer such as polyvinylpyrrolidone, PEG, etc. In the suppository form of the composition, a low melting point wax, such as but not limited to a mixture of fatty acid glycerides optionally combined with cocoa butter, is first melted.

In certain embodiments, the pharmaceutical composition is formulated in any conventional manner using one or more physiologically acceptable carriers containing excipients and adjuvants that facilitate processing of the active compound into a pharmaceutically acceptable preparation. The appropriate formulation depends on the selected route of administration. Any pharmaceutically acceptable techniques, carriers and excipients are optionally used as appropriate and as understood in the art. The pharmaceutical composition containing the compound (i.e., the cyclohexenone compound described herein) can be produced in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, tableting, milling, emulsifying, encapsulating, capturing or pressing processes.

The pharmaceutical composition comprises at least one pharmaceutically acceptable carrier, diluent or excipient and at least one compound described herein (i.e., the cyclohexenone compound described herein) as an active ingredient. The active ingredient is in the form of a free acid or a free base, or in the form of a pharmaceutically acceptable salt. In addition, the methods and pharmaceutical compositions described herein include the use of crystalline forms (also referred to as polymorphs) and active metabolites of these compounds with the same active type. All tautomers of the compounds described herein are also included in the scope of the compounds presented herein. In addition, the compounds described herein include non-solvated forms and solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. The solvated forms of the compounds presented herein are also considered to be disclosed herein. In addition, the pharmaceutical composition optionally contains other medicinal or pharmaceutical agents, carriers, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts for regulating osmotic pressure, buffers and/or other substances with therapeutic value.

Methods for preparing compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semisolid, or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles, or nanoparticles containing the compounds as disclosed herein. Semisolid compositions include, but are not limited to, gels, suspensions, and creams. The forms of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to use, or as emulsions. These compositions optionally also contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc.

In some embodiments, the pharmaceutical composition comprising at least one compound (i.e., the cyclohexenone compound described herein) is illustratively in liquid form, wherein the agent is present in a solution, a suspension, or both. Generally, when the composition is administered as a solution or a suspension, the first portion of the agent is present in a solution, and the second portion of the agent is suspended in a liquid matrix in the form of particles. In some embodiments, the liquid composition includes a gel formulation. In other embodiments, the liquid composition is aqueous.

In certain embodiments, the aqueous drug suspension comprises one or more polymers as a suspending agent. The polymers include water-soluble polymers such as cellulose polymers (e.g., hydroxypropylmethylcellulose) and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain drug compositions described herein comprise a mucoadhesive polymer selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate, and dextran.

The pharmaceutical composition also optionally contains a solubilizing agent to aid in the solubility of the compound (i.e., the cyclohexenone compound described herein). The term "solubilizing agent" generally includes an agent that causes the formation of a micellar solution or a true solution of the agent. Certain acceptable nonionic surfactants, such as polysorbate 80, can be used as solubilizing agents, and ophthalmically acceptable glycols, polyglycols (e.g., polyethylene glycol 400), and glycol ethers can also be used.

In addition, the pharmaceutical composition optionally contains one or more pH adjusters or buffers, including: acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trihydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

In addition, the pharmaceutical composition optionally contains one or more salts in an amount required to bring the molar osmotic pressure concentration of the composition into an acceptable range. Such salts include salts containing 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.

Other pharmaceutical compositions optionally contain one or more preservatives to inhibit the activity of microorganisms. Suitable preservatives include: mercury-containing substances, such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as hydroxychloroaniline, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

Other pharmaceutical compositions contain one or more surfactants to enhance physical stability or for other purposes. Suitable non-ionic surfactants include: polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10 and octoxynol 40.

Still other pharmaceutical compositions may contain one or more antioxidants to enhance chemical stability where necessary. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

In certain embodiments, the aqueous suspension of the pharmaceutical composition is packaged in a non-reclosable single-dose container. Alternatively, a reclosable multi-dose container is used, in which case a preservative is typically included in the composition.

In alternative embodiments, other delivery systems for hydrophobic drug compounds are used. Liposomes and emulsions are examples of delivery vehicles or carriers herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also used. In other embodiments, sustained release systems, such as semipermeable matrices of solid hydrophobic polymers containing therapeutic agents, are used to deliver the compounds described herein. Various sustained release materials are useful herein. In some embodiments, sustained release capsules release the compound for several hours, up to 24 hours. Depending on the chemical nature and biological stability of the therapeutic agent, additional protein stabilization strategies may be used.

In certain embodiments, the formulations described herein contain one or more antioxidants, metal chelators, hydroxyl-containing compounds, and/or other common stabilizers. Examples of such stabilizers 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) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

General considerations for combined therapy

Generally, the compositions described herein and (in embodiments where combination therapy is employed and described herein) other agents need not be administered in the same drug composition, and in some embodiments, due to differences in physical and chemical properties, they are administered by different routes. In some embodiments, initial administration is performed according to an established regimen, and then the dosage, mode of administration, and time of administration are modified by a skilled clinician based on the observed effects.

In some embodiments, when the drugs are used in a therapeutic combination, the therapeutically effective dose varies. Combination therapy further includes periodic treatments that start and end at different times to aid in the clinical management of the patient. For combination therapies described herein, the dose of the co-administered compounds varies depending on the type of combination drug used, the specific drug used, the disease, disorder or condition being treated, etc.

It should be understood that in some embodiments, the dosage regimen used to treat, prevent or improve the condition for which relief is sought varies according to a variety of factors. These factors include the condition from which the subject suffers, as well as the subject's age, weight, sex, diet and medical condition. Therefore, in other embodiments, the dosage regimen actually used varies widely and therefore deviates from the dosage regimen described herein.

Embodiment Example 1. Preparation of Exemplary Cyclohexenone Compounds

Purification procedures using specific herbs: The following procedures are for illustrative purposes only. Other purification methods may also be used.

100 g of mycelium, fruiting body or a mixture of the two from Antrodia camphorata was placed in a flask. Appropriate amounts of water and alcohol (70%-100% alcohol solution) were added to the flask and stirred at 20-25°C for at least 1 hour. The solution was filtered through a filter and a 0.45 μm filter membrane, and the filtrate was collected as an extract.

The filtrate of Antrodia cinnamomea was analyzed by high performance liquid chromatography (HPLC). Separation was performed on an RP18 column with a mobile phase consisting of methanol (A) and 0.3% acetic acid (B) with a gradient of 0-10 minutes in 95%-20% B, 10-20 minutes in 20%-10% B, 20-35 minutes in 10%-10% B, and 35-40 minutes in 10%-95% B at a flow rate of 1 ml/min. The column effluent was monitored with a UV-visible detector.

The fractions collected at 25 to 30 minutes were collected and concentrated to obtain 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone (Compound 1) - a light yellow-brown liquid product. Analysis of Compound 1 showed a molecular formula of C ₂₄ H ₃₈ O ₄ , a molecular weight of 390, and a melting point of 48 to 52° C. NMR spectrum showed: ¹ H-NMR (CDCl ₃ ) 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; ¹³ C-NMR (CDCl ₃ )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.

Compound 1: 4-Hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone

Other compounds are also obtained wherein _R4 is H, _C1 - _C8 alkyl, C2-C8 alkenyl, _C2 - _C8 alkynyl, or aryl optionally substituted with one or more substituents selected from _C1 - _C8 alkyl, _C2 - _{C8 alkenyl} , _{C2 -C8 alkynyl} , _C3 - _C8 cycloalkyl, and _C1 - _C8 halogenated alkyl.

Alternatively, exemplary compounds can be prepared by synthesis. See, e.g., U.S. Patent No. 9,365,481.

的環己烯酮化合物從牛樟芝中分離或由適合的起始材料以合成或半合成方式製備。本領域普通技術人員將會容易地利用合適的條件進行這樣的合成。 Similarly, other structures

The cyclohexenone compound is separated from Antrodia cinnamomea or prepared by synthesis or semi-synthesis from suitable starting materials. A person skilled in the art will easily perform such synthesis using suitable conditions.

Example 2: Study of Compound 1 in combination with clinical chemotherapy for human pancreatic cancer in the AsPC-1 cell culture system and severe combined immunodeficient mouse xenograft tumor model

To evaluate the efficacy of combination therapy comprising exemplary compound 1 and an anticancer agent (e.g., any clinically used drug) for treating pancreatic cancer progression, in vitro studies and in vivo xenograft studies were conducted using exemplary compound 1 and selected current clinical chemotherapy drugs.

For in vitro studies, Compound 1 and each of the selected six (6) clinical chemotherapeutic drugs were used on human pancreatic cancer AsPC-1 and/or CaPan-2, PANC-1 in a cell culture system.

In the in vivo xenograft mouse model, gemcitabine and/or paclitaxel were selected to be administered via the caudal vein twice a week, and for the human pancreatic cancer AsPC-1 xenograft model, compound 1 was orally administered once a day for a total of 28 doses.

In vitro testing:

Human pancreatic cancer cell lines AsPC-1, PANC-1, and CaPan-2 were seeded at 3 x 10 ⁴ cells per well and cultured in forty-eight (48) well cell culture dishes (0.95 cm ² growth area per well). Sixteen (16) hours later, cells were treated with Compound 1 alone or in combination with clinical chemotherapy drugs. Methylthiazolidine tetrazolium (MTT) assay (Supplementary Protocol-1) was performed 48 or 72 hours after treatment to measure the effect of treatment on cell viability during proliferation.

In vivo testing:

The human pancreatic cancer cell line AsPC-1 was cultured and the cells were injected subcutaneously into the flank of 60 mice. After three days, tumors were palpable in the mice. When the tumors reached approximately 100-200 mg (mm ³ ), a total of fifty-one (51) tumor-bearing mice were randomly selected and divided into one of six (6) groups. Vehicle (corn oil) treated mice were used as negative control groups. Compound 1 was administered orally once daily in combination with gemcitabine or paclitaxel, and/or intravenously twice a week for four (4) weeks. Tumor measurements were recorded weekly using a handheld caliper and a Visualsonic Vevo 2100 imaging system. Individual mouse weights were measured once a week during the dosing period. Mice were monitored daily for signs of toxicity and morbidity; any abnormal findings were recorded. The study was terminated four (4) weeks after the start of drug administration.

Mice were euthanized when individual mice had tumors of 2,000 mg (cm ³ ) in one (1) measurement. Mice were considered moribund when tumors prevented the ability of mice to feed, drink, or walk, or if mice lost more than 20% of their original body weight. Individual groups were euthanized when tumors reached an average target size of 1,500 mg per group in two (2) consecutive measurements. The Study Coordinator should be notified of any unplanned euthanasia. The study was terminated four (4) weeks after the start of drug administration.

Test system

A. Cell lines:

Human pancreatic cancer AsPC-1 (ATCC catalog number CRL-1682).

Human pancreatic cancer PANC-1 (ATCC catalog number CRL-1469).

Human pancreatic cancer CaPan-2 (ATCC catalog number HTB-80).

B. Cell preparation

1. Remove and discard the culture medium.

2. Rinse the cell layer briefly with 5 mL of HBSS. Swirl gently and remove the HBSS.

3. Add 5 mL of trypsin-EDTA to a 15 cm2 ^polystyrene culture dish and incubate at 37°C in a humidified incubator with a 95% air/5% _CO2 atmosphere for 5 minutes.

4. Add 5 mL of complete growth medium (DMEM medium containing 10% FBS) and remove the cells by gently pipetting.

5. Count the viable cells using a hemocytometer using the trypan blue exclusion test (Supplementary Protocol-2).

6. For in vitro studies, cells were seeded at 3 x ¹⁰⁴ cells per well and cultured in forty-eight (48) well cell culture dishes for sixteen (16) hours until treatment.

7. Incubate the culture at 37°C.

C. Animal species/strain:

a． Mouse, BALB/cAnN.Cg- Foxnl ^nu /CrlNarl

b. Female, athymic nude mice (nu/nu), 4-6 weeks old.

c. Weight: 15 to 20g on the day of tumor cell inoculation.

D. Human pancreatic cancer xenograft model:

1. To create a subcutaneous xenograft model, athymic female nude mice (4-6 weeks of age) were subcutaneously inoculated with 1 × ¹⁰⁷ AsPC-1 cells in 0.1 mL of serum-free DMEM containing 20% Matrigel (BD Biosciences, Bedford MA).

2. Tumor-bearing animals were divided into the following 6 groups, as shown in Table 1.

3. Monitor the activity and physical condition of mice daily, and determine body weight and measure tumor quality once a week. Take tumor images with a digital camera and Visualsonic Vevo 2100 imaging system. Determine tumor quality by measuring two perpendicular diameters of the implant with calipers and calculating using the formula (4/3)X (π X a X b ² ), where "a" represents the long diameter and "b" represents the short diameter.

E. Test material:

Clinical chemotherapy drugs were purchased from Sigma-Aldrich Co. LLC and prepared in the following manner:

- Erlotinib (stock concentration is 100mM in DMSO, Sigma catalog number E4997).

- 5-Fluorouracil (stock concentration is 100mM, Sigma catalog number F6627).

- Gemcitabine (stock concentration is 100 mM in DMSO, Sigma catalog number G6423).

- Irinotecan (stock concentration is 100 mM in DMSO, Sigma catalog number I1406).

- Oxaliplatin (stock concentration is 50mM in DMSO, Sigma catalog number O9512).

- Paclitaxel (stock concentration is 10mM in DMSO, Sigma catalog number T7402).

For in vivo testing, gemcitabine (G) was purchased from Eli Lilly (Indianapolis, IN, USA), and paclitaxel (P) was purchased from Sigma-Aldrich.

F. In vitro experimental design:

a. Cytotoxicity of test articles (TC ₅₀ ): AsPC-1 were inoculated at 3 x 10 ⁴ cells per well (counted by cell counter, LUNA ^TM automated cell counter or hemocytometer) and cultured in 48-well cell culture dishes. Cells were treated with 400 μM or 800 μM of the individual articles serially diluted to 0 μM. After 48 or 72 hours of treatment, MTT assay was performed to measure the effect of treatment on AsPC-1 cell viability during proliferation. The absorbance at 595 nm was measured using an ELISA reader (BioTEK, Synergy ^TM H1). Article sensitivity curves and TC ₅₀ values were calculated using GraphPad Prism 4.0 software.

b. Combination therapy of compound 1 and chemotherapeutic drugs: This study also includes the cytotoxicity of compound 1 plus the selected chemotherapeutic drugs to investigate the efficacy of this combination. Compound 1 was combined with each of paclitaxel, gemcitabine, 5-FU, oxaliplatin, erlotinib, and irinotecan. After 72 hours of treatment, MTT assay was performed to measure the effect of treatment on the viability of AsPC-1 cells during proliferation. The absorbance at 595 nm was measured using an ELISA instrument.

c. Evaluation of drug interactions: MTT assays were also performed to test the effects of drug combinations on the activity of AsPC-1, PANC-1, and CaPan-2 cell lines. The combination index (CI) isobologram method of Chou and Talalay (Chou and Talalay, 1984; Chang and Chou, 2000) based on the median effect principle was used to calculate the synergistic or antagonistic effects of the combined drug effects. The dose-effect curves of each drug alone or in combination at serial dilution concentrations were plotted using the median effect equation and plot (Chou, 1991) and the CI equation and plot (Chou et al., 1994). The CI values and isobolograms at different effect and dose levels were automatically generated using the computer software CompuSyn (Chou and Martin, 2005). Using this method, additive, synergistic, or antagonistic effects are indicated by CI values equal to 1, <1, and >1, respectively.

G. In vivo experimental design:

a. Cell injection: 1 x 10 ⁷ live human pancreatic cancer AsPC-1 cells were injected subcutaneously into the right thoracic cavity of mice. A total of 60 mice were injected, and approximately 36 tumor-bearing mice were used in this study.

b. Animal Selection and Grouping: This study included six (6) groups, and each group included eight (8) or nine (9) mice, respectively. When tumors reached a target window size of approximately 100-200 mg (mm ³ ), 54 tumor-bearing mice were randomly selected and assigned to one of the six groups. Group 1 was a solvent-treated negative control group and included mice that received Treatment-1. Groups 2, 3, 4, 5, and 6 included mice that received Treatment-2, Treatment-3, Treatment-4, Treatment-5, and Treatment-6, respectively. Table 1 lists the test article concentrations, solvent preparations, and dosing schedules.

c. Dosage preparation:

c-1: Compound 1 (A)

i. Weigh an appropriate amount of compound 1 and add it to a 50 mL tube.

ii. Add an appropriate amount of corn oil (Sigma-Aldrich) to a 50 mL tube. Vortex the preparation until completely homogenized. Take out the preparation into a disposable syringe and transfer it to a 4 mL brown tube, 3.2 mL/tube.

iii. Store the suspension under refrigerated conditions (-20°C) until use.

iv. Thaw the preparation completely in a 37°C water bath on the day of intended use.

c-2: Gemcitabine (G)

i. Store gemcitabine stock solution as a 50 mg/mL solution in sterile PBS at -20°C (Ito et al., 2006; Awasthi et al., 2013).

ii. Prior to animal studies, weigh the appropriate amount of gemcitabine into a 4 mL tube.

iii. Add an appropriate volume of Vehicle-I (50% ethanol and 50% Tween-80 (v/w)) into a 4 mL tube and shake until the material is completely dissolved.

iv. Add an appropriate volume of saline to the 4 mL tube and move the tube in parallel to homogenize the solution completely.

c-3: Paclitaxel (P)

i. Dissolve the stock solution of paclitaxel in 100% ethanol to a final concentration of 10 mg/mL (Shi et al., 2000; Chang et al., 2006; Awasthi et al., 2013).

ii. Prior to animal studies, weigh the appropriate amount of paclitaxel into a 4 mL tube.

iii. Add appropriate volume of Vehicle- ^Ib to the 4 mL tube and shake until the material is completely dissolved.

iv. Add an appropriate volume of physiological saline to the 4mL tube and move the tube in parallel to homogenize the solution completely.

Observation and examination of body systems:

A. Clinical Observations: Clinical observations were performed on each study animal and observed at least daily (including weekends and holidays) for signs of morbidity, mortality, and test substance toxicity. Morbidity includes signs of illness such as, but not limited to, emaciation, dehydration, lethargy, huddled posture, unkempt appearance, labored breathing, and discolored urine or feces. All abnormal findings were recorded.

B. Animal Weights: Individual animals were weighed once (1) week before/after dosing. Body weights were recorded prior to cell injection and continued throughout the study.

C. Tumor Measurements: Monitor the injection site of each animal once (1) week for indications of tumor growth. Throughout the study, measure the length (a) and width (b) of any developing tumor using a vernier caliper in millimeters (mm), where a is the longer of the two (2) dimensions. Calculate the applicable tumor size in millimeters (mm) or centimeters (cm) by using the formula related to the long ellipsoid: M = (4/3) X (π X a X b ² ); and record these data.

D. Evaluation of drug synergistic effects by tumor growth inhibition (TGI) and tumor inhibition rate (TIR): The tumor growth inhibition (TGI) of each group was calculated according to the following formula: Tumor growth inhibition (%) = (1-[TT ₀ ]/[CC ₀ ]) x 100 T and T ₀ are the average tumor volumes of the experimental group at the end of treatment and day 1, respectively. C and C ₀ are the average tumor volumes of the control group at the end of the study and at the beginning, respectively. For TIR, mice were sacrificed at week 4 after treatment, and tumors were carefully ablated and weighed after washing away any remaining blood with PBS. The tumor inhibition rate was calculated by the following formula: Tumor inhibition rate (%) = [(W _control - W _treatment ) / W _control ] x 100% W _treatment and W _control are the average tumor weights of treated mice and control mice, respectively.

D. Hematological parameters: Cardiac blood was collected in EDTA for analysis of hematological parameters such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration (HB), hematocrit, mean corpuscular hemoglobin and concentration, platelet distribution width, platelet count, neutrophils, lymphocytes, monocytes, eosinophils. This was determined using a hematological counter (Abbott Cell-Dyn 3700, IL).

Statistical analysis: All data are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to determine the significance between groups. For multiple comparisons of more than two groups, ANOVA analysis was used, followed by post hoc tests with Bonferroni correction. A value of P < 0.05 was considered significant.

Illustration of the daily schedule for xenotransplantation studies:

result :

In vitro cytotoxicity : To evaluate the combined therapeutic efficiency of exemplary compound 1 , the viability of the treated AsCp-1 cell line was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxic concentrations (TC ₅₀ ) of compound 1 (i.e., antroquinol) and chemotherapeutic drugs on the AsPC-1 cell line were studied. Figures 1 to 7 and Table 2 show the cytotoxicity of compound 1 and chemotherapeutic drugs on the AsPC-1 cell line. Based on these results, it was confirmed that the effects of compound 1 and the combined treatment of compound 1 with chemotherapeutic drugs on the viability of AsPC-1 cells followed the drug concentrations in Table 3. AsCP-1 cells were incubated with compound 1 alone or with each of the drug combinations for 48 hours and 72 hours. In Figures 8 and 9, the viability and MTT assay of AsPC-1 cells incubated with compound 1, each chemotherapeutic drug, and their combination for 48 hours and 72 hours, respectively, were measured. AsPC-1 cells treated with compound 1 or chemotherapeutic drugs alone showed 75% to 80% viability after incubation for 48 hours or 72 hours. Based on these results, the combination therapy of compound 1 with the selected chemotherapeutic drugs may have a synergistic effect in controlling the viability of control AsPC-1 cells. Furthermore, these results also demonstrated that the combination of the tested clinical drugs and compound 1 had more extreme effects on AsPC-1 human pancreatic cancer cells compared with the corresponding single-drug treatments.

^aTC₅₀：細胞活力降至50%時的化合物濃度。

^a _TC50 : Compound concentration at which cell viability drops to 50%.

The combination of compound 1 with paclitaxel and/or gemcitabine was evaluated for synergistic effects against pancreatic cancer. These combinations were tested using three pancreatic cancer cell lines, AsPC-1, CaPan-2, and Panc-1, by an intermediate efficacy assay.

Evaluation of the therapeutic effect of compound 1 in combination with the chemotherapy drugs gemcitabine and paclitaxel

The CI of each drug combination formulation was calculated using the median effect analysis of Chou and Talalay (Chou and Talalay, 1984). Therefore, the CI value was used to determine whether there was a synergistic effect between compound 1 and gemcitabine and/or paclitaxel in this study. In AsPC-1, CaPan-2, and Panc-1 cell lines, the combination of compound 1 with two or three compounds of the chemotherapeutic drugs paclitaxel and gemcitabine had a strong synergistic effect (CI<1, Figure 11) over the entire dose range (see, for example, Figures 10 and 11). These results indicate that the exemplary compound (i.e., compound 1 ) combined with a second or third currently used chemotherapeutic drug strongly inhibits the growth of pancreatic cancer, especially the combination of these three compounds.

In vivo antitumor activity in the AsPC-1 subcutaneous xenograft model

The anti-tumor activity of the combination therapy comprising exemplary compound 1 was evaluated on nude mice carrying AsPC-1 tumors. AsPC-1 cells were injected subcutaneously 7 days before the start of treatment in mice (Table 1). These animals then received administration of a variety of drug combinations daily for 28 days. On the 28th day, all test mice were sacrificed and the corresponding tumors were excised for further study. As shown in Figures 13 and 14, at the end of the experiment, mice treated with a combination of compound 1 and gemcitabine (T3) or a combination of compound 1, gemcitabine and paclitaxel (T6) showed very obvious tumor regression and slow tumor volume growth. Compared with the T3 and T6 groups, mice in the other four treatment groups showed severe tumor growth. In the combination of compound 1 and gemcitabine (T3) or the combination of compound 1 , gemcitabine and paclitaxel (T6) ( FIG. 15 ), the tumor growth inhibition rate was significantly increased compared to the T2, T4 and T5 groups ( FIG. 16 ).

Figure 17 depicts the treatment results represented by ultrasound images of AsPC-1 pancreatic tumor xenografts during treatment. Photos of tumors from sacrificed mice are shown in Figure 18A. It can be seen that the tumor volume of mice treated with compound 1 and gemcitabine is significantly smaller than that of mice treated with other combinations. Specifically, Figure 18B shows that the tumor weight of mice treated with compound 1 and gemcitabine is much smaller than that of tumors in other groups. All these results prove that the combination therapy described herein does have an outstanding tumor growth inhibitory effect. The tumor inhibition rate (TRI) in each group was also calculated based on the final tumor weight (Figure 19). In the groups of compound 1 plus gemcitabine ( FIG. 19 , T3) and compound 1 plus the other two drugs ( FIG. 19 , T6), tumor growth was significantly inhibited.

To confirm that the therapeutic efficacy of the combination therapy based on the exemplary compound 1 is much better than that of a single chemotherapy drug, the synergistic effect of the selected drug combination was evaluated by the treatment: control ratio (TCR) of tumor volume, tumor growth inhibition (TGI) and tumor inhibition rate (TIR). In AsPC-1 xenografts, the monotherapy of compound 1 was able to inhibit tumor growth with a treatment: control ratio (TCR) of 0.55. The combination of compound 1 and gemcitabine (Table 4, Ge+Aq) produced significantly enhanced antitumor activity (TCR 0.36; Table 4; Figure 13) compared to other single or two compound combination therapies. Meanwhile, the three-drug combination containing compound 1 (Table 4, Px+Ge+Aq) also induced strong antitumor activity compared with single or other combination therapy (TCR 0.46; Table 4; Figure 13). According to these results, compound 1 combined with gemcitabine or combined with additional paclitaxel has significantly enhanced antitumor activity.

In addition, no significant weight loss was observed in tumor-bearing mice treated with various combinations, indicating that compound 1 and the other two chemotherapeutic drugs had negligible side effects on tumor treatment at the doses used (Figure 12). Consistently, compound 1 and its combination did not affect blood cell counts except for blood glucose in AsPC-1 xenografts (Table 5). Based on these data, it is suggested that compound 1 and/or its combination has a low oral safety risk.

Table 6: Hematological values for each treatment group in the AsPC-1 xenotransplantation study.

As shown in the research results, the unexpected superior benefits of using a combination comprising exemplary compound 1 and current clinical chemotherapy drugs such as paclitaxel and gemcitabine for pancreatic cancer are clearly shown. Cell viability studies have shown that treatment with such a combination induces much higher inhibitory activity than using compound 1 or other chemotherapy drugs alone, suggesting synergistic and/or additive effects. Such an effect is confirmed by the combination of compound 1 with gemcitabine alone and with additional paclitaxel, which showed significant tumor growth inhibition in tumor-bearing mice.

Example 3: Phase I study to determine the maximum tolerated dose (MTD) and evaluate the safety/tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy of compound 1 combined with nab + paclitaxel + gemcitabine in first-line metastatic pancreatic cancer

Introduction: In 2011, it was noted that the multidrug combination of leucovorin, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) provided an increased median survival of 4.3 months compared with gemcitabine; however, given its side effect profile, it was indicated only for a select group of patients with advanced pancreatic cancer. Patients were randomly assigned to receive FOLFIRINOX or gemcitabine. The median overall survival (OS) was 11.1 months in the FOLFIRINOX group compared with 6.8 months in the gemcitabine group (hazard ratio [HR] for death = 0.57; 95% confidence interval [CI], 0.45 to 0.73; p < 0.001). The median progression-free survival (PFS) was 6.4 months in the FOLFIRINOX group and 3.3 months in the gemcitabine group (HR for disease progression = 0.47; 95% CI, 0.37-0.59; p < 0.001).

Recently, the combination of gemcitabine plus nab-paclitaxel (a 130-nm albumin-bound nanoparticle formulation of paclitaxel) was shown to prolong median survival by 1.8 months and increase 1- and 2-year OS; adverse events were reasonable and included cytopenias and peripheral neuropathy. The multicenter, international, phase III MPACT trial included 861 patients with previously untreated metastatic pancreatic adenocarcinoma. Patients were randomized to receive gemcitabine and nab-paclitaxel or gemcitabine alone. The median OS was 8.5 months in the nab-paclitaxel/gemcitabine group compared with 6.7 months in the gemcitabine group (HR for death = 0.72; 95% CI, 0.62-0.83; p < 0.001). The median PFS was 5.5 months in the nab-paclitaxel/gemcitabine group and 3.7 months in the gemcitabine group (HR for disease progression = 0.69; 95% CI, 0.58-0.82, p < 0.001).

Current National Comprehensive Cancer Network recommendations recommend first-line chemotherapy combinations for patients with a good performance status (ie, Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1), based on patient preference and available support systems. These combinations include FOLFIRINOX for patients with a better/favorable comorbidity profile, or gemcitabine plus nab-paclitaxel, nab-paclitaxel + gemcitabine, and gemcitabine plus erlotinib for patients with an adequate/acceptable comorbidity profile. For patients with an ECOG PS of 2 or a comorbidity profile that precludes other options, gemcitabine alone is recommended; the addition of capecitabine or erlotinib may be offered. Therefore, guidance for selecting appropriate treatment regimens for patients with metastatic pancreatic cancer remains unclear, and in the absence of randomized trials directly comparing combination regimens, the most appropriate first-line treatment for these patients remains unclear.

As shown in Example 2, exemplary compound 1 exhibited antitumor activity and a low toxicity profile in preclinical in vitro and in vivo animal experiments. In a separate nonclinical study conducted in 2012 using compound 1 and an orthotopic PANC-1 human pancreatic cancer xenograft model, four groups of mice were treated with 30 mg/kg, 60 mg/kg, 90 mg/kg of compound 1 and a vehicle control group, respectively, to test the in vivo therapeutic efficacy of compound 1. Tumor volume and tumor weight were measured 10 days after drug treatment (19 days after tumor implantation). Treatment with 30, 60, and 90 mg/kg of compound 1 produced potent antitumor activity, with statistically significantly smaller mean tumor volumes and tumor weights at all three dose levels compared to vehicle control. Tumor-bearing mice tolerated all three doses of androquinol well; no severe weight loss was observed during the study.

In an early reported clinical trial study, daily administration of androquinol (i.e., compound 1 ) at dose levels of 50, 100, 200, 300, 450, and 600 mg for 4 weeks was generally safe and well tolerated, as no specific safety issues or DLTs were found in the study. The data generated from the safety and pharmacokinetic (PK) profiles in this study support further studies of androquinol in patients with advanced malignant tumors to determine the maximum tolerated dose (MTD) and efficacy in a given patient population.

Research purposes

The primary objective of this study was to determine the MTD or maximum feasible dose (MFD) of compound 1 in combination with nab + paclitaxel + gemcitabine in subjects with metastatic pancreatic cancer. Although compound 1 has been evaluated for cancer treatment, many cancer drugs are known in the field to be less effective in treating metastatic cancer.

Queue expansion : evaluating the antitumor activity of antroquinolone in combination with nab+paclitaxel+gemcitabine in subjects with metastatic pancreatic cancer.

The secondary aims of this study are:

Dose escalation:

． To evaluate the safety and tolerability of the combination of antroquinolone and nab + paclitaxel + gemcitabine.

． Characterize the PK of antroquinolone and nab+paclitaxel+gemcitabine.

． Evaluating the activity of Androquinol by routine pancreatic cancer monitoring.

． To investigate the preliminary antitumor activity of androquinol in combination with nab+paclitaxel+gemcitabine in subjects with metastatic pancreatic cancer.

Cohort expansion : To evaluate the safety and tolerability of the MFD of the combination of androquinol and nab+paclitaxel+gemcitabine; to characterize the PK of androquinol and nab-paclitaxel-gemcitabine; to evaluate the activity of androquinol by conventional pancreatic cancer monitoring.

Study End Point

Main destination

Dose escalation:

‧DLT occurred during the first 28-day treatment cycle. DLT definition (graded according to NCI Common Terminology Criteria for Adverse Events [CTCAE] v.4.03):

‧Non-hematologic toxicity of grade 3 or above, excluding: grade 3 diarrhea, nausea, or vomiting that resolves to grade 1 within 3 days of onset or resolves to baseline level with appropriate treatment

48小時 ‧Grade 4 thrombocytopenia or persistent neutropenia

48 hours

3級發熱性嗜中性粒細胞減少 ‧

Grade 3 febrile neutropenia

‧Grade 3 thrombocytopenia with bleeding

‧Persistent toxicity of grade 2 or higher resulting in delayed dosing for more than 14 days.

AEs that the investigator considered to be related to underlying disease conditions, concomitant medications, or to new unrelated medical events or treatments were not defined as DLTs.

Trial expansion :

Primary endpoint: To evaluate the antitumor activity of antroquinolone in combination with nab + paclitaxel + gemcitabine in subjects with metastatic pancreatic cancer.

Secondary endpoint:

Safety: Safety will be assessed using AEs, physical examinations, laboratory test results (including clinical chemistry, hematology, and urinalysis), vital signs (including blood pressure and pulse), and electrocardiograms (ECGs).

Efficacy: Objective response rate (ORR), duration of response (DoR), disease control rate (DCR), and PFS and OS using investigator-assessed RECIST 1.1 (assessments were performed every 8 weeks).

Pharmacokinetics: Plasma concentrations and PK parameters of androquinolone and the combination of nab + paclitaxel + gemcitabine will be evaluated.

Biomarkers: Evaluation of the activity of androquinol by routine pancreatic cancer monitoring.

Research Project

Description of overall research design and plan

This is a single-arm, open-label, multicenter Phase I study of compound 1 in combination with nab-paclitaxel and gemcitabine as first-line treatment in treatment-naive subjects with stage IV metastatic pancreatic cancer.

The first part of this study will follow a 3+3 dose escalation design to determine the MTD or MFD (based on PK and capsule strength) of the standard dosing regimen of androquinol in combination with nab-paclitaxel and gemcitabine. Two dose levels of androquinol three times daily (TID) are planned based on dose escalation data and capsule strength. The final number of subjects will be based on the number of dose levels studied and the occurrence of DLTs. Up to 12 subjects may be treated in this part of the study. A Safety Monitoring Committee (SMC) will be established for this study to review DLTs and overall safety data and to judge the relevance of events to the dose escalation regimen.

After the MTD/MFD is determined, enrollment will continue at a fixed dose of Androquinol (MTD or MFD). A maximum of 40 evaluable subjects are planned in this cohort expansion portion of the trial.

Test products, dosages, and routes of administration

‧Increasing doses of Compound 1 (200 to 300 mg) were administered orally three times daily until unacceptable toxicity or disease progression, with no discontinuation criteria.

· 125 mg/m ² nab-paclitaxel and 1000 mg/m ² gemcitabine by intravenous (IV) infusion, both on days 1, 8, and 15 of each 28-day cycle (i.e., 1 cycle = once weekly for 3 weeks followed by 1 week off) until unacceptable toxicity or disease progression.

DLT Definition and Evaluation

Dose-limiting toxicity is defined as the occurrence of any toxic event outlined below during the DLT evaluation period that is determined to be related or possibly related to the investigational medicinal product (IMP) combination. The DLT evaluation period will be the first 28-day treatment cycle. Toxicity grading is determined using the NCI CTCAE version 4.03. Dose-limiting toxicity will be defined as:

‧Non-hematologic toxicity of grade 3 or above, excluding:

─ Grade 3 diarrhea, nausea, or vomiting that resolves to Grade 1 within 3 days of onset or resolves to baseline levels with appropriate treatment

‧Grade 4 thrombocytopenia or neutropenia persisting for 248 hours

‧Grade 3 febrile neutropenia

‧Grade 3 thrombocytopenia with bleeding

‧Persistent toxicity of grade 2 or higher resulting in delayed dosing for more than 14 days.

AEs that the investigator considered to be related to underlying disease conditions, concomitant medications, or to new unrelated medical events or treatments were not defined as DLTs.

Selection of study population

Inclusion Criteria: Subjects eligible to participate in this study must meet the following criteria:

18歲或以上的男性和女性受試者。 1.

Male and female subjects aged 18 years and above.

2. Histologically or cytologically confirmed metastatic pancreatic ductal adenocarcinoma that is measurable according to RECIST 1.1.

3. Metastatic disease must be diagnosed within 6 weeks before randomization.

4. Treatment-naïve subjects with metastatic pancreatic ductal adenocarcinoma who have not received prior systemic therapy (excluding adjuvant or neoadjuvant therapy if progression occurred >6 months from the last therapy or surgery, respectively, and who have not previously received nab-paclitaxel).

5. Adequate hematological, liver and kidney function, including:

9g/dL - Hemoglobin

9 g/dL

1500/mm³ - Absolute neutrophil count

1500/mm ³

100 000/mm³ - Platelet count

100 000/mm ³

1.5 x正常值上限(ULN)，但不包括被證明有吉伯特症候群(Gilbert’s syndrome)的受試者(>3 x ULN) - Total bilirubin

1.5 x upper limit of normal (ULN), excluding subjects with proven Gilbert's syndrome (>3 x ULN)

2.5 x ULN；對於肝轉移的受試者，ALT和AST

5 x ULN - Alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

2.5 x ULN; for subjects with liver metastases, ALT and AST

5 x ULN

3mg/dL， - Albumin

3 mg/dL,

1.5mg/dL，或者如通過Cockcroft-Gault方程確定的，計算的肌酐清除率

50mL/分鐘， - Serum creatinine

1.5 mg/dL or calculated creatinine clearance as determined by the Cockcroft-Gault equation

50mL/min,

- ECOG is 0 or 1.

6. For women of childbearing potential, serum pregnancy test results are negative at screening and on day 1.

7. Willing to use two medically recognized and effective contraceptive methods (male and female, as appropriate) selected from the following list during the study and continue for 3 months after the last dose of study drug:

- Established use of oral, injectable or implantable hormonal contraceptive methods

- Placement of an intrauterine device or intrauterine system

- Barrier contraceptive methods: condoms or occlusive caps (diaphragms or cervical/vault caps) with spermicidal foam/gel/film/cream/suppositories

- Male sterility (with appropriate evidence of absence of sperm in ejaculate after vasectomy)

- True abstinence: when this is consistent with the subject's preferred and daily lifestyle.

8. Sign the ICF.

12周。 9. Life expectancy

12 weeks.

Exclusion Criteria: Subjects meeting any of the following criteria will not be eligible to participate in this study:

1. Pancreatic islet cell tumor or locally advanced disease.

2. Toxicity from chemotherapy, hormone or immunotherapy or study drug, and/or previous anticancer therapy that is considered clinically relevant persists within 4 weeks or 5 half-lives (whichever is shorter) from the date of first administration of study drug.

3. Treatment with any drug known to be a strong inhibitor or inducer of cytochrome P450 (CYP) 2C19, CYP3A4, CYP2C8, and CYP2E1 within 14 days from the date of the first administration of study drug and during the study treatment period.

4. Other confirmed malignant tumors in the past 5 years (except for curatively treated cervical carcinoma in situ, non-melanoma skin cancer, superficial bladder tumor Ta [non-invasive tumor] and TIS [carcinoma in situ], or stage 1 to 2 non-metastatic prostate cancer that has been previously treated with surgery or radiotherapy and serum prostate-specific antigen is within the normal range [for trials within the past 12 months before the first dose of study drug]).

5. Subjects with any serious active infection (i.e., requiring intravenous antibiotics, antifungals, or antivirals).

6. Subjects with known human immunodeficiency virus, active hepatitis B, or active hepatitis C.

7. Subjects with any other life-threatening diseases or organ system dysfunctions that, in the opinion of the researchers, may compromise the safety of the subjects or interfere with the evaluation of the safety of the study drugs.

8. Known or suspected drug abuse or alcohol abuse.

9. Uncontrolled intermittent illness, including but not limited to: persistent or active infection, symptomatic congestive heart failure, uncontrolled hypertension, unstable angina, arrhythmia, interstitial lung disease, or psychiatric/social conditions that would limit compliance with study requirements, significantly increase the risk of AEs caused by study treatment, or impair the subject's ability to provide written informed consent.

10. Inability to swallow oral medications or recent acute gastrointestinal illness with diarrhea, such as Crohn's disease, malabsorption, or diarrhea of any etiology > CTCAE grade 2 at baseline.

11. Pregnant or breastfeeding female subjects, or male or female subjects of reproductive potential who are not using effective contraceptive methods.

Withdrawal of Subjects: Subjects may withdraw their consent to participate in the study and the investigator may withdraw a subject at any time without prejudice. A subject will be withdrawn from the study or study treatment if any of the following occurs:

1. Record disease progression according to response criteria.

2. Unacceptable toxicity.

3. The subject decides to withdraw his/her informed consent.

4. The researcher believes that the subject is no longer physically and/or psychologically able to remain in this study.

Replacement Procedure : During the dose escalation phase, subjects who have not experienced a DLT in the first cycle and who meet any of the following criteria should be replaced by new subjects:

． All planned dosing of the trial treatment was not completed within the first 28-day cycle.

. All planned dosing of trial treatments was completed with/without delay; however, 90% of the total planned dose of any one drug was not administered during the first cycle.

Guidance for Investigators on When to Terminate Study Treatment : Subjects may withdraw their consent to participate in the study, and investigators may withdraw subjects at any time. Subjects will be withdrawn from the study or study treatment if any of the following conditions are met:

1. Lost contact during the visit.

2. Any AE or medical condition that the investigator or sponsor determines may jeopardize the safety of the subject if he or she continues to participate in the study.

3. Pregnant or trying to become pregnant.

4. Noncompliance by a subject who, in the opinion of the investigator or sponsor, withdraws from the study drug (e.g., refusal to attend scheduled visits).

5. Start alternative anticancer treatment, including another experimental agent.

6. Confirmation of disease progression and the investigator determines that the subject no longer benefits from the study treatment.

7. The sponsor terminates the study. The reasons for terminating the study may include but are not limited to the following: the incidence or severity of AEs in this study or other studies indicates potential health hazards to the subjects; the selection of subjects is not satisfactory.

Subjects who stop following the study treatment prematurely or withdraw from the study:

All subjects will participate in a safety follow-up visit 28 days after the last administration of study treatment. Subjects who prematurely discontinue study drug will be asked to return to the clinic for an early termination visit and may receive a follow-up assessment. The primary reason for premature discontinuation of study drug should be recorded on the appropriate electronic case report form (eCRF).

Treatment of subjects

1/6的受試者具有DLT，則將劑量增至300mg TID。在該試驗的佇列擴展部分中研究一個劑量(MTD或MFD)。 Treatments Administered: Compound 1 will be administered at 200 to 300 mg (planned dose level) three times daily, given as 100 mg capsules (to achieve a daily dose of 600 mg to 900 mg). Additional dose levels may be studied as determined by the study SMC. Study drug will be filled into size 2 capsules (100 mg) and then packaged in light-tight polyethylene bottles and sealed with a polyethylene cap liner that fits in the cap for each dispensing at Visits 1 (Day 0), 3 (Day 28), 4 (Day 42), 5 (Day 56), and 6 (Day 84) and at subsequent visits for subjects entering the extension period. Study drug will be labeled according to specific national requirements. Several doses of androquinol are planned for the first dose escalation part of this trial. Dose escalation will follow a 3+3 design based on the occurrence of DLTs. The starting dose of androquinol is 200 mg TID, which is the dose being tested in the ongoing Phase II single-agent trial in NSCLC. If there are no DLTs or

If 1/6 subjects had a DLT, the dose was escalated to 300 mg TID. One dose (MTD or MFD) was studied in the queuing expansion portion of the trial.

Subjects will receive Androquinol as long as they continue to demonstrate clinical benefit, at the investigator's discretion, and there are no discontinuation criteria. Androquinol should be taken every 8 hours, approximately 15 minutes after a meal or snack, and not within ±1 hour of consuming an ethanol-containing beverage such as alcohol. Subjects who forget or are unable to take their medication at the scheduled time should take it as soon as directed. If they do not remember or are unable to take their medication before the next scheduled dose, they should take the scheduled dose without making up for the missed dose. The date and time of each study drug administration should be recorded in the subject's diary.

The recommended dose of nab-paclitaxel in combination with gemcitabine is 125 mg/m ² by 30-minute intravenous infusion on Days 1, 8, and 15 of each 28-day cycle. The recommended dose (RD) of concomitant gemcitabine is 1000 mg/m ² by 30-minute intravenous infusion on Days 1, 8, and 15 of each 28-day cycle immediately after completion of nab-paclitaxel administration. Treatment with nab-paclitaxel + gemcitabine will continue until unacceptable toxicity or disease progression occurs. Based on clinical practice and local regulations, treatment administration on Day 15 may be omitted in the event of subject toxicity. In this case, subsequent cycles will begin earlier, on Day 22 of Cycle 1 dosing (except during the DLT period of Cycle 1). The tumor assessment schedule will not change (ie, continue to be every 8 weeks).

Dose selection and scheduling for each subject: In the dose escalation portion of the study, subjects will be enrolled and treated sequentially according to a 3+3 dose escalation design. In the queue expansion portion, subjects will be enrolled and treated in parallel.

Efficacy and safety variables

Efficacy evaluation: The subjects' response to treatment was evaluated by determining ORR, DoR, DCR, PFS, 3-month PFS, and 6-month PFS using RECIST 1.1 criteria. Based on the revised response evaluation criteria in the Solid Tumor Guidelines Version 1.1 (adapted from the original publication with supplementary instructions), RECIST 1.1 guidelines and objective tumor response criteria (complete response [CR], partial response [PR], stable disease [SD], or progressive disease) for measurable, unmeasurable, target, and non-target lesions were proposed. OS, 6-month OS, and 12-month OS were also evaluated.

Baseline assessments should be performed no more than 28 days before the start of study treatment and ideally as close to the start of study treatment as possible. Efficacy will be assessed in all subjects by objective tumor assessments every 8 weeks for the first 12 months and every 12 weeks thereafter until objective disease progression is confirmed. If an unscheduled assessment is performed and the subject has not progressed, every effort should be made to obtain a follow-up assessment at his or her scheduled visit.

A confirmatory scan is required after initial confirmation of disease progression. The confirmatory scan should preferably be performed at the next scheduled visit and no earlier than 4 weeks after the initial assessment of disease progression in the absence of clinically significant worsening. Treatment will continue between the initial assessment of progression and confirmation of progression. If a subject discontinues treatment (and/or receives subsequent anticancer therapy) prior to progression, the subject should continue to be followed until objective disease progression is confirmed.

Objective tumor response (CR or PR) should preferably be confirmed at the next scheduled visit and no less than 4 weeks after the visit at which the response was first observed.

After confirming progress, the subjects' survival status should continue to be followed up every 2 months (8 weeks). In addition, all subjects will be contacted within the week after the data cutoff to confirm the survival status.

Safety assessment: Monitor the safety of all subjects throughout the study. Use the safety analysis set to analyze safety data.

Adverse events:

Definition: The term "adverse event" used by the sponsor is synonymous with the term "adverse experience" used by the FDA.

An AE is any adverse, undesirable, unexpected clinical event in the form of a sign, symptom, disease, or laboratory or physiological observation, regardless of causality, occurring in a human participant in a clinical study using the Sponsor's test article. This includes the following:

. Any clinically significant worsening of pre-existing condition

． Note: The appearance of new pathogens associated with clinical events during treatment at sites other than the initial infection site will be considered an AE

. Any recurrence of pre-existing conditions

. AEs that occur as a result of overdosing (whether accidental or intentional) of a sponsored study drug (i.e., a dose that is higher than that prescribed by a healthcare professional for clinical reasons)

． AEs caused by misuse of sponsored study drugs (i.e., use for non-clinical reasons)

. AEs associated with discontinuation of sponsored study drugs.

Note: A medical procedure is not an AE, but the cause of a medical procedure may be an AE.

Pre-existing conditions are clinical conditions (including conditions being treated) that were diagnosed before the subject signed the ICF and were recorded as part of the subject's medical history.

Questions regarding whether the condition was present before the start of the active period of the study and whether it increased in severity and/or frequency will be used to determine if an event is a TEAE. An AE is considered treatment-emergent if (1) the AE was not present at the start of the active period of the study and was not a chronic condition that was part of the subject's medical history, or (2) the AE was present at the start of the active period of the study or was part of the subject's medical history but increased in severity or frequency during the active period. The active period of a study begins with the first dose of study drug. The active period of a study ends at the follow-up visit.

Reporting of Adverse Events: At each visit, the investigator or representative will determine if any AEs have occurred. Subjects will be questioned in the usual manner and will not mention specific symptoms. If any AEs have occurred, they will be recorded in the AE section of the eCRF and in the subject's medical record. The diagnosis, if known, should be recorded in preference to listing individual signs and symptoms.

Reporting of adverse events begins at the time of informed consent and ends 30 days after the last dose of IMP.

Severity Assessment: The severity of AEs was assessed using the AE severity rating scale of the NCI CTCAE (version 4.03). For AEs not specifically listed in the NCI CTCAE, the following definitions were used:

. Grade 1: Mild; no symptoms or mild symptoms; clinical or diagnostic observation only; or no intervention is indicated.

. Grade 2: Moderate; minimal, local, or noninvasive intervention indicated; or limitation of age-appropriate instrumental activities of daily living.

. Grade 3: severe or medically important but not immediately life-threatening; hospitalization or prolonged hospitalization indicated; disability; or limiting self-care activities of daily living.

. Level 4: Life-threatening consequences or urgent intervention indicated.

. Level 5: Death related to AE.

Relationship to Study Treatment: Investigators will determine the relationship of AEs to study drug using a four-category system based on the following guidance:

. Unrelated : The clinical event has an incompatible temporal relationship to drug administration and is either explained by the underlying disease or other drugs or chemicals, or is undisputedly unrelated to the study drug.

Unlikely to be related : The temporal relationship between the clinical event and drug administration makes causation uncertain, but the event is plausibly explained by the underlying disease or by another drug or chemical.

Possibly Related : The clinical event has a reasonable temporal relationship to study drug administration but could also be explained by concurrent illness or other drugs or chemicals.

Definitely related : The clinical event has a reasonable temporal relationship to the administration of the study drug and cannot be explained by concurrent illness or other drugs or chemicals.

Tracking of Adverse Events: It is important for the investigator to consider information about underlying disease, concomitant medications, and the temporal relationship of the event to the timing of study drug administration and re-challenging consequences when making causality decisions. The investigator is responsible for proactively tracking the consequences of each AE until the condition has resolved or stabilized and starting alternative treatment for pancreatic cancer 6 months after the last dose of study drug is completed or follow-up is lost, whichever occurs first. If severe or study drug-related toxicity occurs, the subject should be followed until resolution or stabilization. Safety follow-up data may be collected by telephone contact every 3 months after the end of the study visit.

Serious Adverse Events: A serious adverse event (SAE) is any AE occurring at any dose that meets one or more of the following criteria:

．Cause death

． Life-threatening (see below)

． The subject requires hospitalization or an extension of existing hospitalization (see below)

. Causing persistent or marked disability or incapacity (see below)

．Causing congenital abnormalities or birth defects

. Leading to a major medical event (see below).

In addition, medically important events that may not result in death, be life-threatening, or require hospitalization may be considered serious adverse events when, based on appropriate medical judgment, they may harm the subject and may require medical or surgical intervention to prevent one of the outcomes listed above. Examples of such events include allergic bronchospasm requiring intensive treatment in the emergency room or at home, dyscrasia or seizures that do not require hospitalization, or the development of drug dependence or drug abuse.

A life-threatening adverse event is any adverse event that places the subject at an immediate risk of death from the event at the time of the event. Life-threatening events do not include events that could have resulted in death if the event had occurred in a more severe form, but which did not result in an immediate risk of death at the time of the event. For example, drug-induced hepatitis without supporting evidence of liver failure would not be considered life-threatening, even though a more severe form of drug-induced hepatitis could be fatal. Hospitalization is considered to be an overnight stay only.

Hospitalization or prolonged hospitalization is the criterion for considering an adverse event serious. In the absence of adverse events, participating investigators are not required to report hospitalization or prolonged hospitalization.

In addition, hospitalizations planned prior to study entry for pre-existing conditions that had not worsened did not constitute serious adverse events (e.g., elective hospitalization for total knee replacement because the pre-existing condition of osteoarthritis of the knee had not worsened during the study).

Disability is defined as a substantial impairment of an individual's ability to carry out normal life functions. If there is any doubt as to whether a case constitutes an adverse event or a severe adverse event based on the available information, the case should be considered a severe adverse event.

Alternatively, in cases where a major medical event may not be immediately life-threatening or result in death, hospitalization, disability, or incapacity, medical and scientific judgment should be used to determine whether the case is serious in these situations. These events include those that may harm the subject or may require medical intervention to prevent one or more of the outcomes listed in the definition of a serious event.

Pharmacokinetic analysis

Noncompartmental PK analysis will be performed on individual plasma concentration data. PK analysis will be performed using commercial software such as Phoenix ^TM WinNonlin® Version 6.4 or higher (Certara USA, Inc.). Maximum plasma concentration ( _Cmax ), trough (pre-dose) concentration ( _Ctrough ), and _Cmax time ( _Tmax ) will be obtained directly from the observed data. The area under the plasma concentration-time curve (AUC _τ ) will be calculated using the linear trapezoidal rule. PK parameters will be listed for each individual and summarized by treatment group using descriptive statistics (sample size [N], arithmetic mean, standard deviation, coefficient of variation [CV%], median, minimum, maximum, and geometric mean). Individual concentration data will be listed and summarized by treatment group with descriptive statistics (N, arithmetic mean, standard deviation, median, minimum, maximum, geometric mean, and CV%). Mean and individual plasma concentration-time curves will be presented graphically on both linear and semi-logarithmic scales.

Actual dose administration times and sample collection times will be used for the analysis recorded in the case report form. Plasma concentrations below the limit of quantitation will be set to zero for this analysis. Pharmacokinetic parameter estimates will be presented to three or four significant figures. No attempt will be made to estimate missing data. Additional parameters and data processing may be added as appropriate. Only subjects who received active androquinolone and had an evaluable concentration-time curve will be included in the analysis. All statistical analyses will use non-rounded parameter estimates.

Population pharmacokinetics (PopPK) model development and analysis can be performed using mixed-effects methods. Dosing, sampling, demographic, and laboratory data from each subject will be assembled into a database suitable for population analysis. Nonlinear mixed-effects models will be developed using NONMEM (Globomax, Ellicot City, MD). Various structural models will be evaluated as indicated by the data. Covariate effects (age, sex, race, body size, smoking history, etc.) will be evaluated and incorporated into the model using univariate or multivariate methods. Once the best model is developed, it will be validated using standard methods such as visual prediction checks and bootstrap analysis.

Potential exposure-exposure/concentration relationships can be assessed based on post hoc estimates of androquinol exposure from the PopPK model described above or from observed concentrations (i.e., model-predicted _Cmax , _Ctrough , and _AUCτ ).

Statistical methods

General considerations: For the purpose of this research report, the following aspects will be considered:

． Descriptive statistics and graphical presentation will be the main analytical tools.

. Subject disposition for each dose level will be reported using the full set of subjects from both study phases, and subject profiles will be presented only for the dose escalation phase of the study.

. For all analyses, results and graphical representations of individual subject data will be presented by dose level. The dose level assigned to the subject at the first treatment will be shown on all output values.

Descriptive statistics and graphical representations will be used to summarize the data for each dose level. Unless otherwise stated, the following summary statistics will be used to summarize the trial data for each dose level based on their nature:

. Continuous variables: number of nonmissing observations (N), mean, standard deviation, median, minimum and maximum values; 95% CI will be presented when appropriate.

． Categorical variables: frequency and percentage.

The details of the statistical analysis will be listed in the statistical analysis plan.

Efficacy analysis

Main outcome measures:

Dose-escalation Phase: The number and proportion of subjects experiencing a DTL at each dose level during Cycle 1 will be assessed in the DLT Analysis Set.

16周])以及95% CI的受試者的描述性總結。 Trial expansion phase: In the efficacy analysis set, the best overall response (CR, PR, SD and disease progression) in each category, objective response rate (CR+PR), disease control rate (CR+PR+SD [SD is

16 weeks]) and 95% CIs.

The solid tumor criteria (RECIST) version 1.1 will be performed for median OS time, and PFS time with 95% CI will be estimated using the Kaplan-Meier method on the full analysis set. OS is defined as the time from the first dose of study drug to the death of the subject. PFS is defined as the time from the first dose of study drug to the onset of disease progression or the death of the subject, whichever occurs first.

Analysis of Secondary Outcome Measures: Secondary endpoint data will be presented by dose level, subject, and visit (if applicable). Secondary endpoints will be analyzed descriptively by dose level in the relevant analysis set.

Safety and Tolerability Analyses: The number and proportion of subjects who experienced TEAR; drug exposure; clinically significant changes in laboratory parameters, vital signs, physical examination, weight, ECOG performance status, ECG, and/or pulse oximetry values judged to be related to the trial drug; and the number and cause of death will be summarized.

Safety evaluation will include the incidence of AEs (or TEAEs), laboratory test results, vital signs, ECG results, and physical examination results. All safety data summaries will be based on the safety analysis set. No formal statistical analysis of safety data will be performed.

A summary table of all adverse events (AEs) will be presented by dose level and Regulatory Activities Medical Dictionary SOC and preferred terminology when applicable. The incidence and type of the following AEs will be analyzed.

An AE was considered “treatment-emergent” if it occurred after the first dose of study treatment or if it occurred before the first dose of study treatment and worsened thereafter.

The TEAE summary will include subject counts. Therefore, if a subject experiences more than one episode of a particular AE, that subject is counted only once for that event. If a subject has more than one AE labeled by the same preferred term, that subject is counted only once for that preferred term. Similarly, if a subject has more than one AE within a SOC, that subject is counted only once in that SOC.

All deaths and deaths within 30 days after the last dose of study treatment and the cause of death will be tabulated by dose level. Drug exposure will be summarized by dose level.

Clinically important laboratory test variables will be summarized by dose level and visit (if applicable) using descriptive statistics (number of subjects, mean, standard deviation, minimum, maximum, and mean change from baseline, standard deviation of the mean and standard error of the mean change, minimum, median, maximum, and number and percentage of subjects in a given category). Change tables (i.e., cross-tabulations of values below the lower limit of the normal range, within the limit of the normal range, and above the upper limit of the normal range at baseline relative to the prespecified visit) will be presented by laboratory test (if applicable). Laboratory tests whose categorical results cannot be analyzed by change from baseline or change table analysis will not be included in these summaries but will be listed. Data obtained from laboratory tests not required by the protocol are not summarized but are presented.

Descriptive statistics of vital signs, weight, and ECG findings at each visit will be presented by treatment group. Physical examination findings will be listed for each subject. Overall response rate and DCR will be assessed, along with 95% CIs calculated by the method of Clopper and Pearson by dose level.

Pharmacokinetic analysis

Pharmacokinetic sampling: All subjects enrolled in Phase 1 underwent PK sampling on Day 0 and Day 28 at the following time points:

. Day 0: (approximately 5 mL per sample, 60 mL total) 30 minutes before the first dose, and 0.5, 1, 2, 3, 4, 6, and 8 hours thereafter.

. Day 28: (approximately 5 mL per sample, 60 mL total) Immediately before the first dose on Day 28, and 0.5, 1, 2, 3, 4, 6, and 8 hours thereafter.

In all subjects enrolled in Phase 2, sparse PK sampling will be performed on Days 28, 42, and 56. At least two samples will be collected at each time point, one of which will be the trough concentration (30 minutes before the first dose on Days 28, 42, and 56, and approximately 8 hours after the last dose on the previous day). At least one sample per subject will coincide in time with the peak concentration (3 hours after the first dose). The remaining samples may be collected at any time during the dosing interval.

Following administration of androquinol, each blood sample was analyzed for androquinol plasma concentration using a fully validated bioanalytical method (copies of the analytical method validation report and bioanalytical report will be included in the clinical study report) to determine PK parameters.

Pharmacokinetic Sampling Procedure

Blood Samples: At the pre-scheduled sampling time, venous blood samples were obtained in 5 mL sodium heparin ^Vacutainer® tubes. Immediately after collection, the tubes were gently inverted 8 to 10 times to mix the anticoagulant with the blood sample. All samples will be processed and placed in the refrigerator within one (1) hour of collection. The plasma portion was separated by centrifugation at 3000 rpm in a refrigerated centrifuge (4°C) for 10 minutes. The plasma portion was removed by pipette and divided into two polypropylene cryotubes (each tube receiving approximately equal aliquots). All sample collection and cryotubes will be clearly labeled with the subject number, study period, and collection time. The labels were attached to the cryotubes in a way that prevented them from falling off after freezing. All plasma samples were placed in a -70°C freezer within 1 hour of collection.

Analytical Methods: Concentrations of study drug are determined from plasma samples using validated analytical methods. Details of method validation and sample analysis will be included in the final clinical study report.

Preliminary results:

Preliminary results from patients using a daily dose of 600 mg showed that after the end of the third cycle (8 weeks of treatment), the patient's tumor size (mm) was reduced. The tumor size of one patient decreased from 91 mm to 84 mm, a decrease of 7.6%, and the tumor size of another patient decreased from 50 mm to 39 mm, a decrease of 22%. The preliminary reduction in tumor size further supports the superior and unexpected benefits of the composition of the present invention comprising exemplary compound 1 and current clinical chemotherapy drugs such as paclitaxel and gemcitabine for pancreatic cancer, as shown in animal studies.

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

Claims (6)

Use of a therapeutically effective amount of a composition for preparing a medicament for treating pancreatic cancer in a subject, the composition comprising a compound having the following structure, or a pharmaceutically acceptable salt thereof, and one or more anticancer agents:

The one or more anticancer agents are erlotinib, 5-FU, oxaliplatin, irinotecan, gemcitabine, paclitaxel or a combination thereof. The use as described in claim 1, wherein the one or more anticancer agents are gemcitabine, paclitaxel or a combination thereof. The use as described in claim 1 further comprises an immunotherapeutic agent. Use of a therapeutically effective amount of a compound having the following structure or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pancreatic cancer patients who are resistant, refractory or unresponsive to gemcitabine, paclitaxel or a combination thereof:

The use as described in claim 1 or claim 4, wherein the compound or a pharmaceutically acceptable salt thereof is administered orally, parenterally, intravenously or by injection. The use as described in claim 4, wherein the medicament further comprises an immunotherapeutic agent.