Methods and compositions for the treatment of cancer metastases

专利摘要:
P41300887SE00 !IHS¶¶{Äk(Ûf (IF ¶¶¶E1I)IS(ÉLÅ)SIJI{EI[00242] The present invention provides methods and cornpositions for treating cancer rnetastasis by cyclohexenone cornpounds. _6g_
公开号:SE1350211A1
申请号:SE1350211
申请日:2013-02-21
公开日:2013-08-24
发明作者:Sheng-Yung Liu；Wu-Che Wen
申请人:Golden Biotechnology Corp；
IPC主号:

专利说明:

[2] [0002] Cancer metastasis is the primary cause of post-operation or post-therapy recurrence incancer patients. For example, bone metastases, or metastatic bone disease, a class of cancermetastases that results from primary tumor invasion to bone among cancer metastasis, is one ofthe most common ones of metastasis of various types of human cancers (e.g., breast, lung,prostate and thyroid cancers). The occurrence of bone metastases causes serious morbidity dueto intractable pain, high susceptibility to fracture, nerve compression and hypercalcemia. Cancermetastasis remains substantially refractory to therapy despite intensive efforts to developtreatments .
[6] [0006] In another aspect provides herein methods of treating osteolytic lesions in a subject,coniprising adniinistering to the subject in need thereof a therapeutically effective amount of acyclohexenone conipound having the structure:Rs cHsO / R4nWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and Rg independently is a hydrogen, n1ethyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-nienibered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-n1en1bered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or n1ore substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-Cs alkenyl, CZ-Csalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and RÖ is independently a hydrogen or Cl-Csalkyl;R7 is a Cl-Csalkyl, OR5 or NR5R6;ni = l-l2; andn=l-12; or a pharrnaceutically acceptable salt, nietabolite, solvate or prodrug thereofP41300887SE00INCORPORATION BY REFERENCE[0007] A11 publications, patents, and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if each individual publication, patent, orpatent application was specifically and individually indicated to be incorporated by reference.
[8] [0008] The novel features of the invention are set forth with particularity in the appendedclaims. A better understanding of the features and advantages of the present invention will beobtained by reference to the following detailed description that sets forth illustrativeembodiments, in which the principles of the invention are utilized, and the accompanyingdrawings of which:
[9] [0009] Figures la and lb show relative change (%) in body weight during the study (meaniSD)(la) and at sacrifice (lb) of Groups 1-5. Figure la: Relative change (%) in body weight duringthe study (meaniSD). Body weight at sacrifice, maximum bodyweight and weight loss frommaximum body weight were statistically analyzed and the results are shown in figures lb, 2 and2b (not shown), respectively. The rise in mean body weight of control group at day 25 is due toeuthanasia of moribund animals at day 24. Figure lb: Body weight at sacrifice (relative changefrom day 0, mean+SD). Statistical analysis was performed using One way ANOVA withlo garithmic transformation followed by Dunnett°s-test for comparison against the control group.One asterisk (*) indicates a statistically significant difference with a p-value < 0.05. Animasl ingroup 5 had higher body weight at sacrifice.
[10] [0010] Figure 2 shows maximum body weight obtained during the study (relative change fromday 0, mean+SD). Statistical analysis was performed using One way ANOVA with lo garithmictransformation followed by Dunnett”s-test for comparison against the control group. Oneasterisk (*) indicates a statistically significant difference with a p-value < 0.05 and letter “a”indicates a trend with p-value < 0.1. Group 5 obtained more weight during the study.
[11] [0011] Figures 3a, 3b and 3c show illustrative results of total osteolytic lesion area (mmz) atsacrifice (mean+SD) of Groups 1-5; mean osteolytic lesion area (mmz) at sacrifice (mean+SD);and number of osteolytic lesions at sacrifice (mean+SD), respectively. Figure 3a: Totalosteolytic lesion area (mmz) at sacrifice (mean+SD). The results are shown as the sum of areasof bone lesions in right and left tibia and femur / animal. Statistical analysis was performedusing Kruskal Wallis test followed by Mann-Whitney test for comparison against the controlgroup. One asterisk (*) indicates a statistical significance with p-value <0.05, two asterisks (**)a statistical significance with p-value <0.0l and three asterisks (***) a statistical significancewith p-value <0.00l. Osteolysis was inhibited in groups 2, 4 and 5. Figure 3b: Mean osteolytic_4_P4l300887SE00lesion area (mmz) at sacrifice (mean+SD). The results are shown as the mean areas of individualbone lesions in right and left tibia and femur / animal. Statistical analysis was perforrned usingKruskal Wallis test followed by Mann-Whitney test for comparison against the control group.One asterisk (*) indicates a statistical significance with p-value <0.05, two asterisks (**) astatistical significance with p-value <0.0l and three asterisks (***) a statistical significance withp-value <0.001. NS= Non-significant. Osteolytic lesions were smaller in groups 2, 4 and 5.Figure 3c: Number of osteolytic lesions at sacrifice (mean+SD). The results are shown as thecount of individual bone lesions in right and left tibia and femur / animal. Statistical analysiswas performed using Kruskal Wallis test followed by Mann-Whitney test for comparison againstthe control group. One asterisk (*) indicates a statistical significance with p-value <0.05, twoasterisks (**) a statistical significance with p-value <0.0l and three asterisks (***) a statisticalsignificance with p-value <0.001. NS= Non-significant. There were less osteolytic lesions ingroups 2 and 5, but more in group 3.
[12] [0012] Figure 4 shows illustrative results of whole body tumor burden (mmz) at sacrifice(mean+SD). The results are shown as the average of fluorescent area in two pictures (prone andsupine positions) of each animal. Statistical analysis was performed using Kruskal Wallis test.Statistically significant differences were not observed (p=0.25 1).
[13] [0013] Figure 5 shows illustrative results of relative change (%) in serum TRACP 5b activityduring the study (meaniSD).
[14] [0014] Figures 6a and 6b show illustrative results of relative change from day -4 to day l7 inserum TRACP 5b activity (mean+SD) and relative change from day -4 to day 24 in serumTRACP 5b activity (mean+SD), respectively. Figure 6a: Relative change from day -4 to day 17in serum TRACP 5b activity (mean+SD). Statistical analysis was performed using One wayANOVA with lo garithmic transformation followed by Dunnett”s-test for comparison against thecontrol group. Three asterisks (***) indicate a statistical significance with p-value <0.001.TRACP 5b activity was decreased in group 2. Figure 6b: Relative change from day -4 to day 24in serum TRACP 5b activity (mean+SD). Statistical analysis was performed using One wayANOVA with lo garithmic transformation followed by Dunnett”s-test for comparison against thecontrol group. Three asterisks (***) indicate a statistical significance with p-value <0.001.TRACP 5b activity was decreased in group 2.
[15] [0015] Figure 7 shows illustrative results of relative change (%) in serum procollagen I N-terrninal propeptide (PINP) values during the study (meaniSD).
[16] [0016] Figures 8a and 8b show illustrate relative change from day -4 to day 17 in serum PINPvalues (mean+SD) and relative change from day -4 to day 24 in serum PINP values (mean+SD),respectively. Figure 8a: Relative change from day -4 to day 17 in serum PINP values_5_P4l300887SE00(mean+SD). Statistical analysis was performed using One way ANOVA with lo garithmictransformation followed by Dunnett”s-test for comparison against the control group. Oneasterisk (*) indicates a statistical significance with p-Value <0.05. PINP activity was decreasedin group 2. Figure 8b: Relative change from day -4 to day 24 in serum PINP values (mean+SD).Statistical analysis was performed using One way ANOVA with lo garithmic transformationfollowed by Dunnett”s t-test for comparison against the control group.
[17] [0017] Figure 9 shows illustratiVe results of cortical bone area (mmz, mean +SD). Statisticalanalysis was performed using Oneway ANOVA followed by Dunnett”s test for pairwisecomparison against the control group. Statistically significant differences were not observed.[0018] Figure 10 shows illustrative results of intraosseus area (bone marrow space withtrabecular bone included). Statistical analysis was performed using Oneway ANOVA followedby Dunnett°s test for pairwise comparison against the control group. One asterisk (*) indicates astatistically significant difference with a p-Value < 0.05 and two asterisks (**) with a p-Value <0.01. Intraosseous area was increased in groups 2 and 3.
[19] [0019] Figure 11 shows illustrative results of trabecular bone area (relative to the intraosseousarea). Statistical analysis was performed using Oneway ANOVA with lo garithmictransformation followed by Dunnett°s test for pairwise comparison against the control group.Three asterisks (***) indicate a statistically significant difference with a p-Value < 0.001.Trabecular bone area was increased in group 2.
[20] [0020] Figure 12 shows illustratiVe results of tumor area (relative to the intraosseous area).Statistical analysis was performed using Kruskall Wallis test followed by Mann-Whitney U-testfor pairwise comparison. Tumor area was increased in group 3.
[21] [0021] Figure 13 shows illustrative results of number of osteoclasts at tumor-bone interface(relatiVe to the tumor-bone interface length, #/mm). Statistical analysis was performed usingKruskal-Wallis test. Statistically significant differences were not observed.
[22] [0022] Figures 14a and 14b show illustratiVe results of relatiVe change (%) in body weight ofGroups l-5 during the study (meaniSD), and body weight at sacrifice, respectively. Figure 14a:Relative change (%) in body weight during the study (meaniSD). Body weight at sacrifice,maximum bodyweight and weight loss from maximum body weight were statistically analyzedand the results are shown in figures 14b, 15a and 15b, respectively. Figure 14b: Body weight atsacrifice (relative change from day 0, mean+SD). Statistical analysis was performed using Oneway ANOVA with lo garithmic transformation followed by Dunnett°s t-test for comparisonagainst the control group. Statistically significant differences were not observed.
[23] [0023] Figures 15a and 15b show illustrative results of maximum bodyweight of Groups l-5and weight loss from maximum body weight, respectively. Figure 15a: Maximum body weight_6_P41300887SE00obtained during the study (relative change from day 0, mean+SD). Statistical analysis wasperformed using One way ANOVA with logarithmic transformation followed by Dunnett°s t-testfor comparison against the control group. Two asterisks (**) indicate a statistically significantdifference with a p-value < 0.01. Group 4 obtained more weight during the study. Figure 15b:Weight loss from maximum body weight (relative change from maximum body weight,mean+SD). Statistical analysis was performed using Kruskal Wallis test followed by Mann-Whitney test for comparison against the control group. Two asterisks (**) indicate a statisticallysignificant difference with a p-value < 0.01. Groups 4 and 5 lost more weight.
[24] [0024] Figures 16a and 16b show illustrative results of total osteolytic lesion area (mmz) at day28 (mean+SD) and total osteolytic lesion area (mmz) at sacrifice (mean+SD), respectively.Figure 16a: Total osteolytic lesion area (mmz) at day 28 (mean+SD). The results are shown asthe sum of areas of bone lesions in right and left tibia and femur / animal. Statistical analysiswas performed using Kruskal Wallis test followed by Mann-Whitney test for comparison againstthe control group. One asterisk (*) indicates a statistical significance with p-value <0.05 andthree asterisks (***) statistical significance with p-value <0.00l. Total osteolytic lesion area wasdecreased in groups 2 and 4. Figure 16b: Total osteolytic lesion area (mmz) at sacrifice(mean+SD). The results are shown as the sum of areas of bone lesions in right and left tibia andfemur / animal. Statistical analysis was performed using Kruskal Wallis test followed by Mann-Whitney test for comparison against the control group. One asterisk (*) indicates a statisticalsignificance with p-value <0.05, two asterisks (**) a statistical significance with p-value <0.0land three asterisks (***) a statistical significance with p-value <0.00l. Total osteolytic lesionarea was decreased in groups 2 and 4.
[25] [0025] Figures 17a and 17b show illustrative results of mean osteolytic lesion area (mmz) at day28 (mean+SD) and mean osteolytic lesion area (mmz) at sacrifice (mean+SD), respectively.Figure 17a: Mean osteolytic lesion area (mmz) at day 28 (mean+SD). The results are shown asthe mean areas of individual bone lesions in right and left tibia and femur / animal. Statisticalanalysis was performed using Kruskal Wallis test followed by Mann-Whitney test forcomparison against the control group. One asterisk (*) indicates a statistical significance with p-value <0.05 and three asterisks (***) a statistical significance with p-value <0.00l. Osteolyticlesions were smaller in groups 2 and 5 at day 28. Figure 17b: Mean osteolytic lesion area (mmz)at sacrifice (mean+SD). The results are shown as the mean areas of individual bone lesions inright and left tibia and femur/ animal. Statistical analysis was performed using Kruskal Wallistest followed by Mann-Whitney test for comparison against the control group. One asterisk (*)indicates a statistical significance with p-value <0.05 and three asterisks (***) a statisticalsignificance with p-value <0.00l. Osteolytic lesions were smaller in groups 2 and 4 at sacrifice._7_P4l300887SE00
[26] [0026] Figures 18a and 18b show illustrative results of number of osteolytic lesions at day 28(mean+SD) and number of osteolytic lesions at sacrifice (mean+SD), respectively. Figure 18a:Number of osteolytic lesions at day 28 (mean+SD). The results are shown as the count ofindividual bone lesions in right and left tibia and femur/ animal. Statistical analysis wasperformed using Kruskal Wallis test followed by Mann-Whitney test for comparison against thecontrol group. One asterisk (*) indicates a statistical significance with p-value <0.05. There weremore osteolytic lesions in group 2 because individual lesions were prevented from fusing witheach other. Figure 18b: Number of osteolytic lesions at sacrifice (mean+SD). The results areshown as the count of individual bone lesions in right and left tibia and femur / animal.Statistical analysis was performed using Kruskal Wallis test followed by Mann-Whitney test forcomparison against the control group. Two asterisks (**) indicate a statistical significance withp-value <0.0l. There were more osteolytic lesions in group 2 because individual lesions wereprevented from fusing with each other.
[27] [0027] Figure 19 shows illustrative results of cortical bone area (mmz, mean +SD). Statisticalanalysis was performed using Oneway ANOVA with square root transformation followed byDunnett”s test for pairwise comparison against the control group. Cortical bone area wasincreased in group 2.
[28] [0028] Figure 20 shows illustrative results of intraosseus area (bone marrow space withtrabecular bone included). Statistical analysis was performed using Oneway ANOVA followedby Dunnett°s test for pairwise comparison against the control group. Statistically significantdifferences were not observed.
[29] [0029] Figure 21 shows illustrative results of trabecular bone area (relative to the intraosseousarea). Statistical analysis was performed using Kruskal Wallis test followed by Mann-Whitneytest for comparison against the control group. Three asterisks (***) indicate a statisticallysignificant difference with a p-value < 0.001 and one asterisk (*) with a p-value < 0.05.Trabecular bone area was increased in all treatment groups.
[30] [0030] Figures 22a and 22b show illustrative results of intraosseous tumor area (relative to theintraosseous area) and total tumor area, respectively. Figure 22a: Intraosseous tumor area(relative to the intraosseous area). Statistical analysis was performed using Kruskal-Wallis testfollowed by Mann- Whitney U-test for pairwise comparison against the control group. Twoasterisks (**) indicate a statistically significant difference with a p-value < 0.01 and one asterisk(*) with a p-value < 0.05. Intraosseous tumor area was decreased in groups 2 and 4. Figure 22b:Total tumor area. Statistical analysis was performed using Kruskal- Wallis test followed byMann-Whitney U-test for pairwise comparison against the control group. Two asterisks (**)P41300887SE00indicate a statistically significant difference With a p-value < 0.001. Total tumor area Wasdecreased in group 2.
[31] [0031] Figure 23 shows illustrative results of number of osteoclasts at tumor-bone interface(relative to the tumor-bone interface length, #/mm). Statistical analysis Was perforrned usingOneway ANOVA With lo garithmic transforrnation followed by Dunnett°s test for pairwisecomparison against the control group. Three asterisks (***) indicate a statistically significantdifference With a p-value < 0.001. The number of osteoclasts at tumor-bone interface Wasdecreased in group 2.
[32] [0032] Common treatments for cancer metastasis include palliative care, surgery, chemotherapy,and radiation therapy. However, cancer metastasis remains substantially refractory to therapydespite intensive efforts to develop treatments. Many synthetic anticancer agents used inchemotherapy cause discomfort or toxicity issues. The invention cyclohexenone compounds, insome embodiments, are obtained from extracts of natural products and provide reducedcomplications and/or side effects. In some embodiments, provided herein are methods for thetreatment of a disease or condition associated With bone metastasis by administering acyclohexenone compound provided herein to a subject (e.g. a human). In some embodiments,provided herein are cyclohexenone compounds provided herein for use in the treatment of adisease or condition associated With bone metastasis. The cyclohexenone compounds providetherapeutic benefit to a subject being treated a disease or condition associated With bonemetastasis (see Examples l-4).
[33] [0033] Bone metastases, or metastatic bone disease, is a class of cancer metastases that resultsfrom primary tumor invasion to bone. Bone-originating cancers like osteosarcoma,chondrosarcoma, and EWing's sarcoma are rare. And, unlike hematological malignancies thatoriginate in the blood and form non-solid tumors, bone metastases generally arise from epithelialtumors and form a solid mass inside the bone. Bone metastases cause severe pain, characterizedby a dull, constant ache With periodic spikes of incident pain. The occurrence of bone metastasescauses serious morbidity due to intractable pain, high susceptibility to fracture, nervecompression and hypercalcemia.
[34] [0034] Hypercalcaemia or hypercalcemia is an elevated calcium level in the blood. It can be anasymptomatic laboratory finding, but because an elevated calcium level is often indicative ofother diseases, a Workup should be undertaken if it persists. It can be due to excessive skeletalcalcium release, increased intestinal calcium absorption, or decreased renal calcium excretion.
[35] [0035] Bone is one of the most common locations for metastasis. While any type of cancer iscapable of forrning metastatic tumors Within bone, the microenvironment of the marroW tends tofavor particular types of cancer, including prostate, breast, and lung cancers. Particularly inprostate cancer, bone metastases tend to be the only site of metastasis.
[36] [0036] In some embodiments, there are provided methods treating a disease or conditionassociated With bone metastasis in a subject comprising administering to the subject in needthereof a therapeutically effective amount of a cyclohexenone compound having the structure:R3 CH3O / R4nRzWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and Rg independently is a hydrogen, methyl or (CHflm-CHg;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-CS alkenyl, CZ-Csalkynyl, C3-C8 cycloalkyl, and Cl-CS haloalkyl;each of R5 and R6 is independently a hydrogen or Cl-Csalkyl;Ry is a Cl-Csalkyl, OR5 or NR5R6;m = l-12; andn=l-12; or a pharrnaceutically acceptable salt, metabolite, solvate or prodrug thereof Insome embodiments, the subject has breast cancer or prostate cancer. In someembodiments, the subject is human. See Examples l-4.
[37] [0037] In some embodiments, the cyclohexenone compound having the structureR3 CH3O / R4nRz is prepared synthetically or semi-synthetically from any suitablestarting material. In other embodiments, the cyclohexenone compound is prepared byferrnentation, or the like. For example, Compound 1 (also known as AntroquinonolTM or_10-P41300887SE00“Antroq”) or Cornpound 3, in some instances, is prepared from 4-hydroxy-2,3-dirnethoxy-ó-rnethylcyclohexa-LS-dienone. The non-limited exernplary cornpounds are illustrated below.oHs oHs oHs oHsXO / / CHBH3CO CH3 CH3 CH3 CH3OCH3 1O / / / CH3H3Co oHsoHg, 2oHs oHs oHs oHs/ / /oHsoHOH 4CH3 CH3 CH3 CH3 CH3 CH3o / / oH o /cHs oozH HoH3Co oH 3 o oHoo o5oHs oHs oHß oHso / / ooH3oHsHsu0 O OH oHs oHs oHs oHs O / / /CH3 7 CH3Hßcfi oHoCH3 8oHs oHsO / OH oHs oHsHsce O /° °” Q ÛCHB 9 3 o oHO_11_P4l300887SE00cHs cHs cHs cHscH3 14o / / NHZ o / NcHs cHs cHs cHs19
[38] [0038] In other embodiments, the cyclohexenone compound having the structureRs cHsO / R4nX ORYRz is iso lated from the organic so lVent extracts of Antrodiacamphorata. In some embodiments, the organic solVent is selected from alcohols (e. g.,methanol, ethanol, propanol, or the like), esters (e. g., methyl acetate, ethyl acetate, or the like),alkanes (e. g., pentane, hexane, heptane, or the like), halogenated alkanes (e. g., chloromethane,chloroethane, chloroforrn, methylene chloride, and the like), and the like. For example,exemplary Compounds 1-7 are isolated from organic solVent extracts. In certain embodiments,the organic solVent is alcohol. In certain embodiments, the alcohol is ethanol. In someembodiments, the cyclohexenone compound is isolated from the aqueous extracts of Antrodíacamphorata._12_P41300887SE00
[39] [0039] In some embodiments, R is a hydrogen, C(=O)C3Hg, C(=O)C2H5, or C(=O)CH3. In someembodiments, R1 is a hydrogen or methyl. In certain embodiments, R; is a hydrogen, methyl,ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, Rg is a hydrogen, methyl, ethyl,propyl, butyl, pentyl or hexyl. In some embodiments, R4 is halogen, NHg, NHCHg, N(CH3)2,OCHg, OC2H5, C(=O)CH3, C(=O)C2H5, C(=O)OCH3, C(=O)OC2H5, C(=O)NHCH3,C(=O)NHC2H5, C(=O)NH2, OC(=O)CH3, OC(=O)C2H5, OC(=O)OCH3, OC(=O)OC2H5,OC(=O)NHCH3, OC(=O)NHC2H5, or OC(=O)NH2. In some embodiments, R4 isC2H5C(CH3)2OH, C2H5C(CH3)2OCH3, CHQCOOH, C2H5COOH, CHgOH, C2H5OH, CHgPh,C2H5Ph, CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=O)CH3), 5 or 6-membered lactone, Cl-CSalkyl, CZ-Cgalkenyl, CZ-Cgalkynyl, aryl, and glucosyl, Wherein the 5 or 6-membered lactone, C1-CS alkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl are optionally substituted With one ormore substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,Cl-Cs alkyl, CZ-Cs alkenyl, CZ-Cs alkynyl, C3-C8 cycloalkyl, and Cl-Cg haloalkyl. In certainembodiments, R4 is CH2CH=C(CH3)2. In certain embodiments, the compound isCH3 CH3 CH3 CH3O / / / CH3H3C“o oHoeCH3
[40] [0040] In some embodiments, there are provided methods for inhibiting hypercalcemia ofmalignancy in a subject comprising administering to the subject in need thereof a therapeuticallyeffective amount of a cyclohexenone compound having the structure:R3 CH3O / R4nWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and Rg independently is a hydrogen, methyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,P41300887SE00OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-Cs alkenyl, CZ-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and RÖ is independently a hydrogen or Cl-Csalkyl;R7 is a Cl-Csalkyl, OR5 or NR5R6;m = 1-12; andn=l-12; or a pharrnaceutically acceptable salt, metabolite, solvate or prodrug thereof Insome embodiments, the subject has breast cancer or prostate cancer. In someembodiments, the subject is human. See Examples 1-4.
[41] [0041] In some embodiments provide methods for inhibiting bone resorption in a subjectcomprising administering to the subject in need thereof a therapeutically effective amount of aRs cHsO / R4nR“x oRYcyclohexenone compound having the structure: RzWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and Rg independently is a hydrogen, methyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-Cs alkenyl, CZ-Csalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and RÖ is independently a hydrogen or Cl-Csalkyl;R7 is a Cl-Csalkyl, OR5 or NR5R6;m = 1-12; andn=l-12; or a pharrnaceutically acceptable salt, metabolite, solvate or prodrug thereof Insome embodiments, the subject has breast cancer or prostate cancer. In someembodiments, the subject is human. See Examples l-4._14_P4l300887SE00
[42] [0042] In some embodiments provide methods of treating osteolytic lesions in a subjectcomprising administering to the subject in need thereof a therapeutically effective amount of aRs cHsO / R4nR“x oRYcyclohexenone compound having the structure: Rzwherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and Rg independently is a hydrogen, methyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted with one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-CS alkenyl, CZ-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and R6 is independently a hydrogen or Cl-Csalkyl;Ry is a Cl-Csalkyl, OR5 or NR5R6;m = l-l2; andn=l-12; or a pharrnaceutically acceptable salt, metabolite, solvate or prodrug thereof Insome embodiments, the subject has breast cancer or prostate cancer. In someembodiments, the subject is human. See Examples l-4.[0043] Osteolytic lesions also called osteoclastic lesions or lytic lesions for short, arecharacteristic areas of damage caused by myeloma. When myeloma invades bone tissue, itcauses weak areas to form. In addition, the myeloma cells release chemicals that also lead tobone breakdown. The result is lesions with a specific “punched-out” appearance that may occurin any bone in the body, but are most often noted in the spine, skull, pelvis and ribs.Certain Pharmaceutical and Medical Terminology[0044] Unless otherwise stated, the following terms used in this application, including thespecif1cation and claims, have the def1nitions given below. It must be noted that, as used in thespecif1cation and the appended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unless otherwise indicated, conventionalmethods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinantDNA techniques and pharrnacology are employed. In this application, the use of “or” or “and”means “and/or” unless stated otherwise. Furthermore, use of the term ““including” as well as_15-P4l300887SE0077 CCother forms, such as “include , includes,” and “included,” is not limiting. The section headingsused herein are for organizational purposes only and are not to be construed as limiting thesubject matter described.
[45] [0045] An “alkyl” group refers to an aliphatic hydrocarbon group. The alkyl group may be asaturated alkyl group (Which means that it does not contain any carbon-carbon double bonds orcarbon-carbon triple bonds) or the alkyl group may be an unsaturated alkyl group (Which meansthat it contains at least one carbon-carbon double bonds or carbon-carbon triple bond). The alkylmoiety, Whether saturated or unsaturated, may be branched, or straight chain.
[46] [0046] The “alkyl” group may have l to 12 carbon atoms (whenever it appears herein, anumerical range such as “l to 12 refers to each integer in the given range; e. g., “l to 12 carbonatoms” means that the alkyl group may consist of l carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 12 carbon atoms, although the present definition also covers theoccurrence of the terrn “alkyl” Where no numerical range is designated). The alkyl group of thecompounds described herein may be designated as “Cl-Cg alkyl” or similar designations. ByWay of example only, “C1-Cg alkyl” indicates that there are one, two , three, four, five, six, sevenor eight carbon atoms in the alkyl chain. In one aspect the alkyl is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typicalalkyl groups include, but are in no Way limited to, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, hexyl, allyl, but-2-enyl, but-3-enyl,cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like. Inone aspect, an alkyl is a Cl-Cg alkyl.
[47] [0047] The terrn “alkylene” refers to a divalent alkyl radical. Any of the above mentionedmonovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from thealkyl. In one aspect, an alkylene is a Cl-Clgalkylene. In another aspect, an alkylene is a C1-Cgalkylene. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, andthe like.
[48] [0048] As used herein, the terrn “aryl” refers to an aromatic ring Wherein each of the atomsforming the ring is a carbon atom. Aryl rings are formed by five, six, seven, eight, nine, or morethan nine carbon atoms. Aryl groups are optionally substituted. In one aspect, an aryl is a phenylor a naphthalenyl. In one aspect, an aryl is a phenyl. In one aspect, an aryl is a C6-C10aryl.Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylenegroup). In one aspect, an arylene is a C6-C10 arylene. Exemplary arylenes include, but are notlimited to, phenyl-1,2-ene, phenyl-l,3-ene, and phenyl-l,4-ene._16-P4l300887SE00
[49] [0049] The terrn “aromatic” refers to a planar ring having a delocalized Jt-electron systemcontaining 4n+2 Jr electrons, where n is an integer. Aromatic rings can be formed from five, six,seven, eight, nine, ten, or more than ten atoms. Aromatics are optionally substituted. The terrn“aromatic” includes both carbocyclic aryl (“aryl”, e. g., phenyl) and heterocyclic aryl (or“heteroaryl” or “heteroaromatic”) groups (e. g., pyridine). The terrn includes monocyclic orfused-ring polycyclic (í.e., rings which share adjacent pairs of carbon atoms) groups.
[50] [0050] The terrn “halo” or, altematively, “halogen” or “halide” means fluoro, chloro, bromo oriodo.
[51] [0051] The terrn “lactone” refers to a cyclic ester which can be seen as the condensation productof an alcohol group -OH and a carboxylic acid group -COOH in the same molecule. It ischaracterized by a closed ring consisting of two or more carbon atoms and a single oxygen atom,with a ketone group =O in one of the carbons adjacent to the other oxygen.
[52] [0052] The terrn "heterocycle" or “heterocyclic” refers to heteroaromatic rings (also known asheteroaryls) and heterocycloalkyl rings (also known as heteroalicyclic groups) containing one tofour heteroatoms in the ring(s), where each heteroatom in the ring(s) is selected from O, S andN, wherein each heterocyclic group has from 4 to l0 atoms in its ring system, and with theproviso that the any ring does not contain two adj acent O or S atoms. Non-aromatic heterocyclicgroups (also known as heterocycloalkyls) include groups having only 3 atoms in their ringsystem, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. Theheterocyclic groups include benzo-fused ring systems. An example of a 3-memberedheterocyclic group is aziridinyl. An example of a 4-membered heterocyclic group is azetidinyl.An example of a 5-membered heterocyclic group is thiazolyl. An example of a 6-memberedheterocyclic group is pyridyl, and an example of a 10-membered heterocyclic group isquinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl,aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl,diazepinyl, thiazepinyl, l,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, l,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl,dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.l.0]hexanyl, 3-azabicyclo[4.l.0]heptanyl, 3H-indolyl and quinolizinyl. Examplesof aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, fiJryl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl,indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,_ 1 7-P4l300887SE00thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups may be C-attached or N-attached where such is possible. For instance, a group derived from pyrro le maybe pyrrol-l-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazo lemay be imidazol-l-yl or imidazol-S-yl (both N-attached) or imidazol-2-yl, imidazol-4-yl orimidazol-5-yl (all C-attached). The heterocyclic groups include benzo-fiased ring systems. Non-aromatic heterocycles may be substituted with one or two oxo (=O) moieties, such as pyrro lidin-2-one.
[53] [0053] The term “alkenyl” as used herein, means a straight, branched chain, or cyclic (in whichcase, it would also be known as a “cycloalkenyl”) hydrocarbon containing from 2-10 carbonsand containing at least one carbon-carbon double bond formed by the removal of two hydrogens.In some embodiments, depending on the structure, an alkenyl group is a monoradical or adiradical (i.e., an alkenylene group). In some embodiments, alkenyl groups are optionallysubstituted. 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-l-heptenyl, and 3-cecenyl.
[54] [0054] The term “alkynyl” as used herein, means a straight, branched chain, or cyclic (in whichcase, it would also be known as a “cycloalkenyl”) hydrocarbon containing from 2-10 carbonsand containing at least one carbon-carbon triple bond formed by the removal of four hydrogens.In some embodiments, depending on the structure, an alkynyl group is a monoradical or adiradical (i.e., an alkynylene group). In some embodiments, alkynyl groups are optionallysubstituted. Illustrative examples of alkynyl include, but are not limited to, ethynyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, and the like.
[55] [0055] The term “alkoxy” as used herein, means an alkyl group, as defined herein, appended tothe parent mo lecular moiety through an oxygen atom. Illustrative examples of alkoxy include,but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.
[56] [0056] The term “cycloalkyl” as used herein, means a monocyclic or polycyclic radical thatcontains only carbon and hydrogen, and includes those that are saturated, partially unsaturated,or fully unsaturated. Cycloalkyl groups include groups having from 3 to l0 ring atoms.
[58] [0058] The term “glucosyl” as used herein, include D- or L-form glucosyl groups, in Which theglucosyl group is attached via any hydroxyl group on the glucose ring.
[59] [0059] The term ““acceptable” With respect to a forrnulation, composition or ingredient, as usedherein, means having no persistent detrimental effect on the general health of the subject beingtreated.
[60] [0060] Antrodía is a genus of fiangi in the family Meripilaceae. Antrodía species have fruitingbodies that typically lie flator spread out on the growing surface, With the hymenium exposed tothe outside; the edges may be tumed so as to form narrow brackets. Most species are found intemperate and boreal forests, and cause brown rot. Some of the species in this genus are havemedicinal properties, and have been used in TaiWan as a Traditional medicine.
[61] [0061] The term “carrier,” as used herein, refers to relatively nontoxic chemical compounds oragents that facilitate the incorporation of a compound into cells or tissues.
[62] [0062] The terms “co-administration” or the like, as used herein, are meant to encompassadministration of the selected therapeutic agents to a single patient, and are intended to includetreatment regimens in Which the agents are administered by the same or different route ofadministration or at the same or different time._19-P4l300887SE00
[63] [0063] The terrn “diluent” refers to chemical compounds that are used to dilute the compound ofinterest prior to delivery. Diluents can also be used to stabilize compounds because they canprovide a more stable environment. Salts dissolved in buffered solutions (Which also can providepH control or maintenance) are utilized as diluents in the art, including, but not limited to aphosphate buffered saline solution.
[64] [0064] The terms “effective amount” or “therapeutically effective amount,” as used herein, referto a sufficient amount of an agent or a compound being administered Which Will relieve to someextent one or more of the symptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. For example, an “effective amount” for therapeutic uses is theamount of the composition comprising a compound as disclo sed herein required to provide aclinically significant decrease in disease symptoms. An appropriate “effective” amount in anyindividual case may be deterrnined using techniques, such as a dose escalation study.
[65] [0065] The terms “enhance” or “enhancing,” as used herein, means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancing the effect of therapeuticagents, the term “enhancing” refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An “enhancing-effective amount,”as used herein, refers to an amount adequate to enhance the effect of another therapeutic agent ina desired system.
[66] [0066] A ““metabolite” of a compound disclosed herein is a derivative of that compound that isformed When the compound is metabolized. The term “active metabolite” refers to a bio lo gicallyactive derivative of a compound that is formed When the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes (including, but not limited to,hydrolysis reactions and reactions catalyzed by enzymes) by Which a particular substance ischanged by an organism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidative and reductivereactions While uridine diphosphate glucuronyltransferases catalyze the transfer of an activatedglucuronic-acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines andfree sulphydryl groups. Metabolites of the compounds disclosed herein are optionally identifiedeither by administration of compounds to a host and analysis of tissue samples from the host, orby incubation of compounds With hepatic cells in vitro and analysis of the resulting compounds.[0067] The term “pharrnaceutical combination” as used herein, means a product that resultsfrom the mixing or combining of more than one active ingredient and includes both fixed andnon-fixed combinations of the active ingredients. The term “fixed combination” means that theactive ingredients, e.g. a compound (i.e., a cyclohexenone compound described herein) and a co-_20-P41300887SE00agent, are both administered to a patient simultaneously in the form of a single entity or dosage.The terrn “non-fixed combination” means that the active ingredients, e. g. a compound (i.e., acyclohexenone compound described herein) and a co-agent, are administered to a patient asseparate entities either simultaneously, concurrently or sequentially With no specific interveningtime limits, Wherein such administration provides effective levels of the two compounds in thebody of the patient. The latter also applies to cocktail therapy, e.g. the administration of three ormore active ingredients.
[68] [0068] The term “pharrnaceutical composition” refers to a mixture of a compound (i.e., acyclohexenone compound described herein) With other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.The pharrnaceutical composition facilitates administration of the compound to an organism.Multiple techniques of administering a compound exist in the art including, but not limited to:intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
[69] [0069] The term “subject” or “patient” encompasses mammals. Examples of mammals include,but are not limited to, any member of the Mammalian class: humans, non-human primates suchas chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep,goats, sWine; domestic animals such as rabbits, dogs, and cats; laboratory animals includingrodents, such as rats, mice and guinea pigs, and the like. In one embodiment, the mammal is ahuman.
[70] [0070] The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abatingor ameliorating at least one symptom of a disease or condition, preventing additional symptoms,inhibiting the disease or condition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease or condition, relieving acondition caused by the disease or condition, or stopping the symptoms of the disease orcondition either prophylactically and/or therapeutically.
[71] [0071] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal,aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transderrnal, vaginal, otic, nasal, andtopical administration. In addition, by Way of example only, parenteral delivery includesintramuscular, subcutaneous, intravenous, intramedullary injections, as Well as intrathecal, directintraventricular, intraperitoneal, intralymphatic, and intranasal injections.
[72] [0072] In certain embodiments, a compound as described herein is administered in a local ratherthan systemic manner, for example, via injection of the compound directly into an organ, oftenin a depot preparation or sustained release forrnulation. In specific embodiments, long actingformulations are administered by implantation (for example subcutaneously or intramuscularly)_21_P41300887SE00or by intramuscular injection. Furthermore, in other embodiments, the drug is delivered in atargeted drug delivery system, for example, in a liposome coated With organ-specific antibody.In such embodiments, the liposomes are targeted to and taken up selectively by the organ. In yetother embodiments, the compound as described herein is provided in the forrn of a rapid releaseformulation, in the forrn of an extended release forrnulation, or in the forrn of an interrnediaterelease forrnulation. In yet other embodiments, the compound described herein is administeredtopically.
[73] [0073] In some embodiments, the cyclohexenone compound, or a pharrnaceutically acceptablesalt, metabolite, solvate or prodrug thereof, is administered parenterally or intravenously. Inother embodiments, the cyclohexenone compound, or a pharrnaceutically acceptable salt,metabolite, solvate or prodrug thereof, is administered by injection. In some embodiments, thecyclohexenone compound, or a pharrnaceutically acceptable salt, metabolite, solvate or prodrugthereof, is administered orally.
[74] [0074] In some embodiments provide compounds having the structure:Rs cHsO / R4nRzWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and R3 independently is a hydrogen, methyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, Cz-CS alkenyl, Cz-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and R6 is independently a hydrogen or Cl-Csalkyl;R7 is a Cl-Csalkyl, OR5 or NR5R6;m = 1-12; and n=l-l2; or a pharrnaceutically acceptable salt, metabolite, solvate orprodrug thereof[0075] In some embodiments, R is a hydrogen, C(=O)C3Hg, C(=O)C2H5, or C(=O)CH3. In someembodiments, each of R1, R; and R3 independently is a hydrogen, methyl, ethyl, propyl, butyl,_22_P4l300887SE00pentyl hexyl, heptyl, or octyl. . In certain embodiments, R1 is a hydrogen or methyl. In certainembodiments, R; is a hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In certainembodiments, Rg is a hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In someembodiments, R4 is halogen, NHg, NHCHg, N(CH3)2, OCHg, OC2H5, C(=O)CH3, C(=O)C2H5,C(=O)OCH3, C(=O)OC2H5, C(=O)NHCH3, C(=O)NHC2H5, C(=O)NH2, OC(=O)CH3,OC(=O)C2H5, OC(=O)OCH3, OC(=O)OC2H5, OC(=O)NHCH3, OC(=O)NHC2H5, orOC(=O)NH2. In certain embodiments, R4 is C2H5C(CH3)2OH, C2H5C(CH3)2OCH3, CHQCOOH,C2H5COOH, CHgOH, C2H5OH, CHgPh, C2H5Ph, CH2CH=C(CH3)(CHO),CH2CH=C(CH3)(C(=O)CH3), 5 or 6-membered lactone, aryl, Cl-CS alkyl, or glucosyl, Wherein5 or 6-membered lactone, aryl, Cl-CS alkyl, and glucosyl are optionally substituted With one ormore substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,Cl-Cs alkyl, CZ-Cs alkenyl, CZ-Cs alkynyl, C3-C8 cycloalkyl, and Cl-Cg haloalkyl. In certainembodiments, R4 is CHzCOOH, C2H5COOH, CHQOH, C2H5OH, CHgPh, C2H5Ph,CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=O)CH3), 5 or 6-membered lactone, aryl, orglucosyl, Wherein the 5 or 6-membered lactone, aryl, and glucosyl are optionally substitutedWith one or more substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5,C(=O)NR5R6, Cl-Cs alkyl, CZ-Cs alkenyl, CZ-Cs alkynyl, C3-C8 cycloalkyl, and Cl-Cg haloalkyl.
[76] [0076] In certain embodiments, the compound is selected from group consisting ofCH3 CH3 CH3 CH3O / / /CH3H3CO OHSCH3 ,CH3 CH3 CH3 CH3O / X XCH3H3CO OHOH ,CH3 CH3 CH3 CH3O / / OHCH3H Cï3 o oHOïCH3_23-P41300887SE00CH3 CH3 CH3 CH3 CH3 CH3o / o / / ocHsCOZH CH3o / / NHZ o / NHscx _24_P41300887SE00CH3 , and
[77] [0077] In certain ernbodirnents, the cornpound is selected from group consisting ofcHs cHs cHs cHsO / / /_25-P41300887SE00o // // NH2 o // NH c H c3 o oH 3 s oH OcH3 , cH3 ,cH3 cH3 cH3 cH3O / / / FH3co oHOCH3 , and
[78] [0078] In some embodiments provide pharrnaceutical compositions comprising a therapeuticallyeffective amount of a cyclohexenone compound having the structure:R3 cH3O / R4nWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8a1ky1;each of R1, R; and Rg independently is a hydrogen, methyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,_26-P41300887SE00OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-CS alkenyl, CZ-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and RÖ is independently a hydrogen or Cl-Csalkyl;Ry is a Cl-Csalkyl, OR5 or NR5R6;m = 1-12; and n=1-12; or a pharrnaceutically acceptable salt, metabolite, solvate orprodrug thereof; and a pharrnaceutically acceptable excipient.
[79] [0079] In some embodiments, the compounds described herein are forrnulated intopharrnaceutical compositions. In specific embodiments, pharrnaceutical compositions areformulated in a conventional manner using one or more physio lo gically acceptable carrierscomprising excipients and auxiliaries Which facilitate processing of the active compounds intopreparations Which can be used pharrnaceutically. Proper forrnulation is dependent upon theroute of administration chosen. Any pharrnaceutically acceptable techniques, carriers, andexcipients are used as suitable to forrnulate the pharrnaceutical compositions described herein:Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, MackPublishing Co., Easton, Pennsylvania 1975; Liberrnan, H.A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and PharmaceuticalDosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).[0080] Provided herein are pharrnaceutical compositions comprising a compound (i.e., acyclohexenone compound described herein) and a pharrnaceutically acceptable diluent(s),excipient(s), or carrier(s). In certain embodiments, the compounds described are administered aspharrnaceutical compositions in Which a compound (i.e., a cyclohexenone compound describedherein) is mixed With other active ingredients, as in combination therapy. Encompassed hereinare all combinations of actives set forth in the combination therapies section beloW andthroughout this disclosure. In specific embodiments, the pharrnaceutical compositions includeone or more compounds (i.e., a cyclohexenone compound described herein).
[81] [0081] A pharrnaceutical composition, as used herein, refers to a mixture of a compound (i.e., acyclohexenone compound described herein) With other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.In certain embodiments, the pharrnaceutical composition facilitates administration of thecompound to an organism. In some embodiments, practicing the methods of treatment or useprovided herein, therapeutically effective amounts of compounds (i.e., a cyclohexenonecompound described herein) are administered in a pharrnaceutical composition to a mammalhaving a disease or condition to be treated. In specific embodiments, the mammal is a human. In_27-P4l300887SE00certain embodiments, therapeutically effective amounts vary depending on the severity of thedisease, the age and relative health of the subject, the potency of the compound used and otherfactors. The compounds described herein are used singly or in combination With one or moretherapeutic agents as components of mixtures.
[82] [0082] In one embodiment, a compound (i.e., a cyclohexenone compound described herein) isformulated in an aqueous solution. In specific embodiments, the aqueous solution is selectedfrom, by Way of example only, a physiologically compatible buffer, such as Hank°s solution,Ringer°s solution, or physiological saline buffer. In other embodiments, a compound (i.e., acyclohexenone compound described herein) is forrnulated for transmucosal administration. Inspecific embodiments, transmucosal forrnulations include penetrants that are appropriate to thebarrier to be perrneated. In still other embodiments Wherein the compounds described herein areformulated for other parenteral injections, appropriate forrnulations include aqueous ornonaqueous solutions. In specific embodiments, such solutions include physiologicallycompatible buffers and/or excipients.
[83] [0083] In another embodiment, compounds described herein are forrnulated for oraladministration. Compounds described herein, including a compound (i.e., a cyclohexenonecompound described herein), are forrnulated by combining the active compounds With, e.g.,pharrnaceutically acceptable carriers or excipients. In various embodiments, the compoundsdescribed herein are forrnulated in oral dosage forms that include, by Way of example only,tablets, poWders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions andthe like.
[84] [0084] In certain embodiments, pharrnaceutical preparations for oral use are obtained by mixingone or more solid excipients With one or more of the compounds described herein, optionallygrinding the resulting mixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparationssuch as: for example, maize starch, Wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calciumphosphate. In specific embodiments, disintegrating agents are optionally added. Disintegratingagents include, by Way of example only, cross-linked croscarrnellose sodium,polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[85] [0085] In one embodiment, dosage forms, such as dragee cores and tablets, are provided Withone or more suitable coating. In specific embodiments, concentrated sugar solutions are used forcoating the dosage forrn. The sugar solutions, optionally contain additional components, such as_28-P41300887SE00by Way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs and/or pigments are also optionally added to the coatings for identificationpurposes. Additionally, the dyestuffs and/or pigments are optionally utilized to characterizedifferent combinations of active compound doses.
[86] [0086] In certain embodiments, therapeutically effective amounts of at least one of thecompounds described herein are forrnulated into other oral dosage forrns. Oral dosage forrnsinclude push-fit capsules made of gelatin, as Well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. In specific embodiments, push-fit capsules contain theactive ingredients in admixture With one or more filler. Fillers include, by Way of example only,lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In other embodiments, soft capsules, contain one or more activecompound that is dissolved or suspended in a suitable liquid. Suitable liquids include, by Way ofexample only, one or more fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition,stabilizers are optionally added.
[87] [0087] In other embodiments, therapeutically effective amounts of at least one of thecompounds described herein are forrnulated for buccal or sublingual administration.Forrnulations suitable for buccal or sublingual administration include, by Way of example only,tablets, lozenges, or gels. In still other embodiments, the compounds described herein areformulated for parental injection, including forrnulations suitable for bolus injection orcontinuous infusion. In specific embodiments, forrnulations for injection are presented in unitdosage form (e. g., in ampoules) or in multi-dose containers. Preservatives are, optionally, addedto the injection formulations. In still other embodiments, the pharrnaceutical compositions of acompound (i.e., a cyclohexenone compound described herein) are forrnulated in a form suitablefor parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueousvehicles. Parenteral injection formulations optionally contain forrnulatory agents such assuspending, stabilizing and/or dispersing agents. In specific embodiments, pharrnaceuticalformulations for parenteral administration include aqueous solutions of the active compounds inWater-soluble forrn. In additional embodiments, suspensions of the active compounds areprepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles foruse in the pharrnaceutical compositions described herein include, by Way of example only, fattyoils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, orliposomes. In certain specific embodiments, aqueous injection suspensions contain substancesWhich increase the viscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension contains suitable stabilizers or agents Which_29-P4l300887SE00increase the solubility of the compounds to allow for the preparation of highly concentratedsolutions. Altematively, in other embodiments, the active ingredient is in powder forrn forconstitution With a suitable vehicle, e. g., sterile pyrogen-free Water, before use.
[88] [0088] In one aspect, compounds (i.e., cyclohexenone compounds described herein) areprepared as solutions for parenteral injection as described herein or knoWn in the art andadministered With an automatic inj ector. Automatic injectors, such as those disclosed in U.S.Patent Nos. 4,031,893, 5,358,489; 5,540,664; 5,665,071, 5,695,472 and WO/2005/087297 (eachof Which are incorporated herein by reference for such disclosure) are known. In general, allautomatic injectors contain a volume of solution that includes a compound (i.e., acyclohexenone compound described herein) to be injected. In general, automatic injectorsinclude a reservoir for holding the solution, Which is in fluid communication With a needle fordelivering the drug, as Well as a mechanism for automatically deploying the needle, inserting theneedle into the patient and delivering the dose into the patient. Exemplary injectors provideabout 0.3 mL, 0.6mL, 1.0mL or other suitable volume of solution at about a concentration of 0.5mg to 50 mg of a compound (i.e., a cyclohexenone compound described herein) per 1 mL ofsolution. Each injector is capable of delivering only one dose of the compound.
[89] [0089] In still other embodiments, the compounds (i.e., cyclohexenone compounds describedherein) are administered topically. The compounds described herein are forrnulated into avariety of topically administrable compositions, such as solutions, suspensions, lotions, gels,pastes, medicated sticks, balms, creams or ointments. Such pharrnaceutical compositionsoptionally contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.[0090] In yet other embodiments, the compounds (i.e., cyclohexenone compounds describedherein) are forrnulated for transderrnal administration. In specific embodiments, transderrnalformulations employ transderrnal delivery devices and transderrnal delivery patches and can belipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive. In various embodiments, such patches are constructed for continuous, pulsatile, oron demand delivery of pharrnaceutical agents. In additional embodiments, the transderrnaldelivery of a compound (i.e., a cyclohexenone compound described herein) is accomplished bymeans of iontophoretic patches and the like. In certain embodiments, transderrnal patchesprovide controlled delivery of a compound (i.e., a cyclohexenone compound described herein).In specific embodiments, the rate of absorption is sloWed by using rate-controlling membranesor by trapping the compound Within a polymer matrix or gel. In altemative embodiments,absorption enhancers are used to increase absorption. Absorption enhancers or carriers includeabsorbable pharrnaceutically acceptable solvents that assist passage through the skin. Forexample, in one embodiment, transderrnal devices are in the form of a bandage comprising a_3()_P41300887SE00backing member, a reservoir containing the compound optionally With carriers, optionally a ratecontrolling barrier to deliver the compound to the skin of the host at a controlled andpredeterrnined rate over a prolonged period of time, and means to secure the device to the skin.[0091] Transderrnal forrnulations described herein may be administered using a variety ofdevices Which have been described in the art. For example, such devices include, but are notlimited to, U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097,3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307,4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378, 5,837,280,5,869,090, 6,923,983, 6,929,801 and 6,946,144.
[92] [0092] The transderrnal dosage forms described herein may incorporate certainpharrnaceutically acceptable excipients Which are conventional in the art. In one embodiment,the transderrnal forrnulations described herein include at least three components: (1) aformulation of a compound (i.e., a cyclohexenone compound described herein); (2) a penetrationenhancer; and (3) an aqueous adjuvant. In addition, transderrnal forrnulations can includeadditional components such as, but not limited to, gelling agents, creams and ointment bases,and the like. In some embodiments, the transderrnal forrnulations further include a Woven ornon-Woven backing material to enhance absorption and prevent the removal of the transderrnalformulation from the skin. In other embodiments, the transderrnal forrnulations described hereinmaintain a saturated or supersaturated state to promote diffusion into the skin.
[93] [0093] In other embodiments, the compounds (i.e., cyclohexenone compounds described herein)are forrnulated for administration by inhalation. Various forms suitable for administration byinhalation include, but are not limited to, aerosols, mists or poWders. Pharmaceuticalcompositions of a compound (i.e., a cyclohexenone compound described herein) areconveniently delivered in the form of an aerosol spray presentation from pressurized packs or anebuliser, With the use of a suitable propellant (e. g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Inspecific embodiments, the dosage unit of a pressurized aerosol is deterrnined by providing avalve to deliver a metered amount. In certain embodiments, capsules and cartridges of, such as,by Way of example only, gelatins for use in an inhaler or insufflator are forrnulated containing apowder mix of the compound and a suitable powder base such as lactose or starch.
[94] [0094] Intranasal forrnulations are knoWn in the art and are described in, for example, U.S. Pat.Nos. 4,476,116, 5,116,817 and 6,391,452, each of Which is specifically incorporated herein byreference. Forrnulations, Which include a compound (i.e., a cyclohexenone compound describedherein), Which are prepared according to these and other techniques Well-knoWn in the art areprepared as solutions in saline, employing benzyl alcohol or other suitable preservatives,_31-P4l300887SE00fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example,Ansel, H. C. et al., Pharmaceutical Dosage Forrns and Drug Delivery Systems, Sixth Ed. (1995).Preferably these compositions and forrnulations are prepared with suitable nontoxicpharrnaceutically acceptable ingredients. These ingredients are found in sources such asREMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005, astandard reference in the field. The choice of suitable carriers is highly dependent upon the exactnature of the nasal dosage form desired, e. g., solutions, suspensions, ointments, or gels. Nasaldosage forms generally contain large amounts of water in addition to the active ingredient.Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents,preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizingagents may also be present. Preferably, the nasal dosage form should be isotonic with nasalsecretions.
[95] [0095] For administration by inhalation, the compounds described herein, may be in a form asan aerosol, a mist or a powder. Pharmaceutical compositions described herein are convenientlydelivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant, e. g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurizedaerosol, the dosage unit may be deterrnined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler orinsufflator may be forrnulated containing a powder mix of the compound described herein and asuitable powder base such as lactose or starch.
[96] [0096] In still other embodiments, the compounds (i.e., cyclohexenone compounds describedherein) are forrnulated in rectal compositions such as enemas, rectal gels, rectal foams, rectalaerosols, suppositories, j elly suppositories, or retention enemas, containing conventionalsuppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such aspolyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally incombination with cocoa butter is first melted.
[97] [0097] In certain embodiments, pharrnaceutical compositions are forrnulated in anyconventional manner using one or more physiologically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the active compounds into preparationswhich can be used pharrnaceutically. Proper forrnulation is dependent upon the route ofadministration chosen. Any pharrnaceutically acceptable techniques, carriers, and excipients isoptionally used as suitable and as understood in the art. Pharmaceutical compositionscomprising a compound (i.e., a cyclohexenone compound described herein) may be_32-P41300887SE00manufactured in a conventional manner, such as, by Way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or compression processes.
[98] [0098] Pharmaceutical compositions include at least one pharrnaceutically acceptable carrier,diluent or excipient and at least one compound (i.e., cyclohexenone compounds describedherein) described herein as an active ingredient. The active ingredient is in free-acid or free-baseform, or in a pharrnaceutically acceptable salt form. In addition, the methods and pharrnaceuticalcompositions described herein include the use crystalline forms (also known as polymorphs), asWell as active metabolites of these compounds having the same type of activity. All tautomers ofthe compounds described herein are included Within the scope of the compounds presentedherein. Additionally, the compounds described herein encompass unsolvated as Well as solvatedforms With pharrnaceutically acceptable solvents such as Water, ethanol, and the like. Thesolvated forms of the compounds presented herein are also considered to be disclosed herein. Inaddition, the pharrnaceutical compositions optionally include other medicinal or pharrnaceuticalagents, carriers, adjuvants, such as preserving, stabilizing, Wetting or emulsifying agents,solution promoters, salts for regulating the osmotic pressure, buffers, and/or othertherapeutically valuable substances.
[99] [0099] Methods for the preparation of compositions comprising the compounds described hereininclude forrnulating the compounds With one or more inert, pharrnaceutically acceptableexcipients or carriers to form a solid, semi-solid or liquid. Solid compositions include, but arenot limited to, poWders, tablets, dispersible granules, capsules, cachets, and suppositories.Liquid compositions include solutions in Which a compound is dissolved, emulsions comprisinga compound, or a solution containing liposomes, micelles, or nanoparticles comprising acompound as disclosed herein. Semi-solid compositions include, but are not limited to, gels,suspensions and creams. The form of the pharrnaceutical compositions described herein includeliquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid priorto use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic,auxiliary substances, such as Wetting or emulsifying agents, pH buffering agents, and so forth.[00100] In some embodiments, pharrnaceutical composition comprising at leastcompound (i.e., cyclohexenone compounds described herein) illustratively takes the form of aliquid Where the agents are present in solution, in suspension or both. Typically When thecomposition is administered as a solution or suspension a first portion of the agent is present insolution and a second portion of the agent is present in particulate form, in suspension in a liquidmatrix. In some embodiments, a liquid composition includes a gel forrnulation. In otherembodiments, the liquid composition is aqueous._33_P4l300887SE00
[101] [00101] In certain embodiments, pharrnaceutical aqueous suspensions include one ormore polymers as suspending agents. Polymers include Water-soluble polymers such ascellulosic polymers, e. g., hydroxypropyl methylcellulose, and Water-insoluble polymers such ascross-linked carboxyl-containing polymers. Certain pharrnaceutical compositions describedherein include a mucoadhesive polymer, selected from, for example, carboxymethylcellulose,carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil,acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.
[102] [00102] Pharmaceutical compositions also, optionally include solubilizing agents to aid inthe solubility of a compound (i.e., cyclohexenone compounds described herein). The term“solubilizing agent” generally includes agents that result in formation of a micellar solution or atrue solution of the agent. Certain acceptable nonionic surfactants, for example polysorbate 80,are useful as solubilizing agents, as can ophthalmically acceptable glycols, polyglycols, e. g.,polyethylene glycol 400, and glycol ethers.
[103] [00103] Furthermore, pharrnaceutical compositions optionally include one or more pHadjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic,phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodiumborate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane;and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids,bases and buffers are included in an amount required to maintain pH of the composition in anacceptable range.
[104] [00104] Additionally, pharrnaceutical compositions optionally include one or more saltsin an amount required to bring osmolality of the composition into an acceptable range. Suchsalts include those having sodium, potassium or ammonium cations and chloride, citrate,ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite andammonium sulfate.
[105] [00105] Other pharrnaceutical compositions optionally include one or more preservativesto inhibit microbial activity. Suitable preservatives include mercury-containing substances suchas merfen and thiomersal; stabilized chlorine dioxide; and quatemary ammonium compoundssuch as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.[00106] Still other pharrnaceutical compositions include one or more surfactants toenhance physical stability or for other purposes. Suitable nonionic surfactants includepolyoxyethylene fatty acid glycerides and vegetable oils, e. g., polyoxyethylene (60)hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, eg., octoxynoll0, octoxynol 40._34-P41300887SE00
[107] [00107] Still other pharrnaceutical compositions may include one or more antioxidants toenhance chemical stability Where required. Suitable antioxidants include, by Way of exampleonly, ascorbic acid and sodium metabisulfite.
[108] [00108] In certain embodiments, pharrnaceutical aqueous suspension compositions arepackaged in single-dose non-reclosable containers. Altematively, multiple-dose reclosablecontainers are used, in Which case it is typical to include a preservative in the composition.[00109] In altemative embodiments, other delivery systems for hydrophobicpharrnaceutical compounds are employed. Liposomes and emulsions are examples of deliveryvehicles or carriers herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also employed. In additional embodiments, the compounds describedherein are delivered using a sustained-release system, such as semiperrneable matrices of solidhydrophobic polymers containing the therapeutic agent. Various sustained-release materials areuseful herein. In some embodiments, sustained-release capsules release the compounds for a fewhours up to over 24 hours. Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for protein stabilization may be employed.
[110] [00110] In certain embodiments, the forrnulations described herein include one or moreantioxidants, metal chelating agents, thiol containing compounds and/or other general stabilizingagents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% toabout 2% W/v glycerol, (b) about 0.1% to about 1% W/v methionine, (c) about 0.1% to about 2%W/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2%W/v ascorbic acid, (f) 0.003% to about 0.02% W/v polysorbate 80, (g) 0.001% to about 0.05%W/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosanpolysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n)combinations thereofCombination Treatments
[111] [00111] In general, the compositions described herein and, in embodiments Wherecombinational therapy is employed, other agents do not have to be administered in the samepharrnaceutical composition, and in some embodiments, because of different physical andchemical characteristics, are administered by different routes. In some embodiments, the initialadministration is made according to established protocols, and then, based upon the observedeffects, the dosage, modes of administration and times of administration is modified by theskilled clinician.
[112] [00112] In some embodiments, therapeutically-effective dosages vary When the drugs areused in treatment combinations. Combination treatment further includes periodic treatments thatstart and stop at various times to assist With the clinical management of the patient. For_35_P41300887SE00combination therapies described herein, dosages of the co-administered compounds Varydepending on the type of co-drug employed, on the specific drug employed, on the disease,disorder, or condition being treated and so forth.
[113] [00113] It is understood that in some embodiments, the dosage regimen to treat, prevent,or ameliorate the condition(s) for which relief is sought, is modified in accordance with a Varietyof factors. These factors include the disorder from which the subject suffers, as well as the age,weight, sex, diet, and medical condition of the subject. Thus, in other embodiments, the dosageregimen actually employed Varies widely and therefore deviates from the dosage regimens setforth herein.
[114] [00114] Combinations of compounds (i.e., the cyclohexenone compound describedherein) with other anti-cancer agents are intended to be covered. In some embodiments,examples of anti-cancer agents include, but are not limited to, the following: cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VPl6), tamoxifen, raloxifene, estrogen receptorbinding agents, taxol, gemcitabine, naVelbine, famesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, Vincristin, Vinblastin and methotrexate, other topoisomeraseinhibitors (e.g., irinotecan, topotecan, camptothecin, etc.) or any deriVatiVe related agent of theforegoing.
[115] [00115] The combinations of the cyclohexenone compounds and other anti-cancer agentsdescribed herein encompass additional therapies and treatment regimens with other agents insome embodiments. Such additional therapies and treatment regimens can include another anti-cancer therapy in some embodiments. Altematively, in other embodiments, additional therapiesand treatment regimens include other agents used to treat adjunct conditions associated with thecancer or a side effect from such agent in the combination therapy. In further embodiments,adjuvants or enhancers are administered with a combination therapy described herein.
[116] [00116] Additional anti-cancer therapies include chemotherapy, radiotherapy,immunotherapy, gene therapy, surgery or other therapies that are capable of negatively affectingcancer in a patient, such as for example, by killing cancer cells, inducing apoptosis in cancercells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases,reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancercells, promoting an immune response against cancer cells or a tumor, preVenting or inhibitingthe progression of cancer, or increasing the lifespan of a subject with cancer._36_P41300887SE00
[117] [00117] In some embodiments provide compositions for “creating a disease or conditionassociated With bone metastasis comprising a therapeutically effective amount of acyclohexenone compound having the structure:Rs cHsO / R4nWherein each of X and Y independently is oxygen, NR5 or sulfur;R is a hydrogen or C(=O)C1-C8alkyl;each of R1, R; and Rg independently is a hydrogen, methyl or (CH2)m-CH3;R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein the 5or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-Cs alkenyl, CZ-Csalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl;each of R5 and R6 is independently a hydrogen or Cl-Csalkyl;Ry is a Cl-Csalkyl, OR5 or NR5R6;m = 1-12; and n=1-12; or a pharrnaceutically acceptable salt, metabolite, solvate orprodrug thereof; and one or more anti-cancer agents.
[118] [00118] One hundred grams of mycelia, fruiting bodies or mixture of both from Antrodiacamphorata Were placed into a flask. A proper amount of Water and alcohol (70-100% alcoholsolution) Was added into the flask and Were stirred at 20-25° C for at least 1 hour. The solutionWas f1ltered through a filter and 0.45 um membrane and the f1ltrate Was collected as the extract.[00119] The filtrate of Antrodia camphorata Was subj ected to High Performance Liquidchromatography (HPLC) analysis. The separation Was performed on a RP1 8 column, the mobilephase consisted of methanol (A) and 0.3% acetic acid (B), With the gradient conditions of 0-10min in 95% - 20% B, 10-20 min in 20%-10% B, 20-35 min in 10%-10% B, 35-40 min in 10%-95% B, at the floW rate of 1 ml/min. The column effluent Was monitored With a UV-visibledetector._37_P41300887SE00
[120] [00120] The fractions collected at 21.2 to 21.4 min were collected and concentrated toyield con1pound 5, a product of pale yellow liquid. Conipound 5 was analyzed to be 4-hydroxy-5 -(1 1-hydroxy-3 ,7, 1 1-trin1ethyldodeca-2,6-dienyl)-2,3 -din1ethoxy-6-n1ethylcyclo hex-2-enonewith n1olecular weight of 408 (Molecular forrnula: C24 H40O5). 1H-NMR(CDCl3) ö (ppn1)=1.21, 1.36, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.68, 4.05, 5.71 and5.56. ”c-NMiucoclgexppmy 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74,27.00, 30.10, 40.27, 43.34, 59.22, 60.59, 71.8, 120.97, 123.84, 124.30, 131.32,134.61, 135.92, 138.05, 160.45, and 197.11.
[121] [00121] The fractions collected at 23.7 to 24.0 niin were collected and concentrated toyield con1pound 7, a product of pale yellow liquid. Conipound 7 was analyzed to be 4-hydroxy-2,3 -din1ethoxy-5 -(1 1-n1ethoxy-3 ,7, 1 1-trin1ethyldodeca-2,6-dienyl)-6-n1ethylcyclo hex-2-enonewith n1olecular weight of 422 (C 25 H 42 05). lH-NMR (CDClg) ö (ppm) = 1 .21, 1.3 6, 1 .71,1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.24, 3.68, 4.05, 5.12, 5.50, and 5.61. ”C-NMR(CDCl3)ö(ppn1): 12.31,16.1, 16.12, 17.67, 24.44, 26.44, 26.74, 27.00,37.81, 39.81, 40.27, 43.34, 49.00, 59.22, 60.59, 120.97, 123.84, 124.30, 135.92,138.05, 160.45 and 197.12.cHs cHs cHs cHso / / ocHscHsH3co oH3 7Con1pound 7: 4-hydroxy-2,3-din1ethoxy-5-(11-n1ethoxy-3,7,11-trin1ethyldodeca-2,6-dienyl)-6-n1ethylcyclohex-2-enone
[122] [00122] The fractions collected at 25 to 30 niin were collected and concentrated to yield4-hydroxy-2,3 -din1ethoxy-6-n1ethyl-5 -(3 ,7, 1 1-trin1ethyldodeca-2,6, 10-trienyl)cyclohex-2-enone(con1pound 1), a product of pale yellow brown liquid. The analysis of con1pound 1 showed then1olecular forniula of C 24 H 38 O4, n1olecular weight of 390 with nielting point of 48 to 52 °C.NMR spectra showed that lH-NMR (CDClg) ö (ppn1)=1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07,2.22, 2.25, 3.68, 4.05, 5.07, and 5.14; 13C-NMR(CDCl3) ö (ppn1)=12.31, 16.1, 16.12, 17.67,_3g_P41300887SE0025.67, 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.cHs cHs cHs cHsO / / /cHsH3CO OHOCH3 1Compound 1: 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,1 1-trimethyldodeca-2,6, 10-trienyl)cyclohex-2-enone
[123] [00123] Compound 6, a metabolite of compound 1, Was obtained from urine samples ofrats fed With Compound 1 in the animal study. Compound 6 Was deterrnined to be 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3 -methyl-2-hexenoic acid)cyclohex-2-enone With molecular Weightof 3 12 (C16 H24 06). Compound 4 Which Was deterrnined as 3,4-dihydroxy-2-methoxy-6-methyl-5-(3,7,1 1-trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone (molecular Weight of 376,C23H36O4), Was obtained When compound 1 Was under the condition of above 40 °C for 6 hours.
[125] [00125] Patients With breast cancer typically develop metastases to bone. Almost allbreast cancer patients With end-stage disease have bone metastases. Bone metastases caused bybreast cancer are osteolytic, causing severe bone lo ss that leads to highly increased risk offractures. Although the primary tumor could be successfully removed, the disease may have_39_P41300887SE00already Spread to bone. In this case, the disease can develop further visceral and bonemetastases. Currently, there is no efficient cure for prevention or treatment of bone metastases.[00126] The objective of this study was to test the efficacy of three different doses of theexemplary compound 1 from Example 1 on breast cancer bone metastases in female athymicnude mice in a 25-day study in preventive setting. The following 5 experimental groups wereincluded in the study:1. Control group receiving vehicle (po twice a day)2. Reference group receiving zoledronic acid (sc at days 1 and 14) + vehicle (po twice a day)3. Test group receiving 15 mg/kg of test compound (po twice a day)4. Test group receiving 30 mg/kg of test compound (po twice a day)5. Test group receiving 45 mg/kg of test compound (po twice a day)
[127] [00127] The vehicle was com oil. All groups included 15 mice that are 5 weeks of age atthe beginning of the study. Based on the body weights the animals were randomized to fivegroups. On day 0, the animals were given intracardiac inoculation of human breast cancer cells.Body weights were determined twice a week. At sacrifice, the osteolytic lesions and whole bodytumor burden were quantified by x-ray and fluorescence imaging, respectively. The testcompound and vehicle were administered by oral gavage twice a day. Blood samples foranalysing TRACP 5b were collected at days 0, 17 and 24. The animals were sacrificed at day 25,or earlier if they became moribund. Tissue samples were collected for histology and embeddedin paraffin from left and right tibia and femur for possible future histomorphometric analyses.Gross necropsy was performed to all animals at the end of the study, and all macroscopic signswere recorded.
[129] [00129] Zoledronic acid was used as a reference compound. 100ul of Zometa® (NovartisPharma GmbH, Nümberg, Germany) 4 mg / 5ml infusion concentrate was diluted in 3.9 ml ofsterile saline (0.9% NaCl) to obtain the administration concentration of 0.02 mg/ml. Thissolution was prepared once during the study and stored at +4°C. The dosing volume was 5_40-P41300887SE00ml/kg, resulting in the dose of 0.1mg/kg. The reference compound was administered SC twice inthe study: at days 1 and 14.
[130] [00130] The vehicle was corn oil (Sigma-Aldrich).
[132] [00132] This experiment utilized an animal model that closely resembles thecharacteristics of breast cancer metastatic disease or condition in humans. This model can beeffectively used for testing the effects of cancer drug candidates on the metastasis of breastcancer cells to bone. In this model, human breast cancer cells are inoculated into the left cardiacventricle of 5 Weeks old female athymic nude mice. This mouse strain is immunodeficient,allowing rapid spreading of the human cancer cells. Within two weeks after the inoculation, theanimals develop osteolytic bone metastases that can be visualized by X-ray radiography. Inaddition, soft tissue metastases are observed in approximately 40% of the animals, especially inadrenal glands. At 3 weeks, the bone metastases are clearly visible in X-ray radiographs and theanimals have developed cachexia and lost weight substantially. The animals are sacrificedtypically at 3-4 weeks after inoculation, and their hindlegs are collected for furtherhistomorphometric analysis.
[133] [00133] The bone metastasis animal model provided herein can be conveniently used totest drug candidates that have been shown in preliminary studies to affect one or more of thefollowing: 1) growth of cancer cells; 2) invasion of cancer cells; 3) migration of cancer cells; 4)angiogenesis; 5) function or differentiation of osteoclasts. The model can also be used forfinding new indications to existing drugs, allowing the pharrnaceutical companies a way offinding new indications to patented drugs that have already proved efficient in some otherindications.
[134] [00134] Female Athymic nude mice (Hsd: Athymic nude-nu, obtained from Harlan, TheNetherlands) were used for this study. The age of the animals was 5 weeks, and their bodyweights were approximately 16-22g at the beginning of the study. The mice were specificpathogen free (SPF) and isolator-reared animals. Correct age and good clinical health were_41_P41300887SE00qualifications for the study. The quarantine and acclimatization period for the mice was oneweek. A110 cation to groups was performed by randomization procedure based on body weight.The animals were marked with ear marks. The mice were housed in Scantainer®, 5 mice percage. For intracardiac inoculations and imaging, the mice were anesthetized with ip inj ections ofXylazin (4-5 mg/ml) and Ketamine (75-92 mg/ml). Analgesia (buprenorphine 3 mg/kg po indrinking water) was used for the last 5 days of the study.
[135] [00135] MDA-MB-23 lSA-GFP6 human breast cancer cells (105 cells in 0.1 ml of PBS)were inoculated into the left cardiac ventricle of the mice at day 0, leading to development ofbone metastases typical to the disease.
[136] [00136] The animals were weighed twice a week and doses were adjusted accordingly.For the last 5 days of the study, the animals were weighed and observed daily to monitor theprogression of disease. Appearance of any clinical signs was recorded on follow-up forms.Analgesic was given to all animals for the last 5 days of the experiment.
[137] [00137] 10 animals were found dead or had to be euthanized due to breathing difficulties(see Table 1). At necropsy, punctured esophagus and oil in thoracic cavity was found. Oneanimal was euthanized due to paraplegia before day 22 (see Table 1) 15 animals had to beeuthanized between days 22 and 24 of the experiment (see Table 1). They were weighed andradiographed, and bone samples were prepared.
[138] [00138] Two animals were removed from the final analysis due to excessive growth ofcancer cells in lungs and thoracic cavity, indicating an unsuccessful inoculation of cancer cells(see Table 1).
[139] [00139] One animal in control group was removed because it had no observable bonelesions (cut-off limit 0.5 mmz). Proportionally, one animal from groups 2 and 4 with noobservable bone lesions was removed from analysis (see Table 1). There were no animals underthe cut-off limit in groups 3 and 5.
[140] [00140] Table 1: The results of animal monitoring - Final N with breast cancer cells.Group Animal Treatment Final N Dead Time of Notesnumbers animals death(id) (study day)1 1-15 Control 15 9 25 No observable bonelesions.2 1-15 Zoledronic 9 1 25 No observable boneacid 5 15 lesions.8 25 Esophageal pundture.9 25 Lung metastasis.1 1 15 Lung metastasis._42_P41300887SE0012 19 Esophageal pundture.Esophageal pundture.3 1-15 Compound 14 6 19 Esophageal pundture.1 30mg/kg/d4 1-15 Compound 12 3 25 No observable bone1 60 9 21 lesions.mg/kg/ d 11 9 Paraplegic, euthanizedbefore d22.Esophageal pundture.5 1-15 Compound 10 2 13 Esophageal pundture.1 90 3 14 Esophageal pundture.mg/kg/ d 5 13 Esophageal pundture.8 13 Esophageal pundture.13 15 Esophageal pundture.Blood sampling and bone marker analyses[00141] Blood samples for analyzing tartrate-resistant acid phosphatase isoforrn 5b(TRACP 5b) were collected from saphenous vein before the inoculation of cancer cells and atdays 14 and 24. The serum samples were collected to 100 ul serum gel tubes, prepared withinone hour from sampling, divided to two aliquots and stored at -70°C. Serum TRACP 5b wasanalyzed using MouseTRAPTM kit (IDS Ltd, Boldon, UK).
[142] [00142] Metastases were monitored by imaging the fluorescence emitted by the MDA-MB-231SA-GFP6 cells using the LT 9 GFP-imaging system LT-MACIMSYSPLUSC(Lightools Research, Encinitas, USA). The used excitation wavelength was 470/40 nm andemission wavelength 515 nm. The animals were imaged in prone and supine positions and theGFP fluorescent area (an average of the two pictures) was deterrnined from the images withMetaMorph image analysis software.
[143] [00143] At the end of the study (at days 22-25), the animals were weighed and sacrificedwith cervical dislocation under anesthesia. Necropsy was carried out in all animals. Macroscopicfindings were recorded on the fo llow-up forms. Ex vívo tissue samples from hind limbs (left andright tibiae and femur) were collected for possible histomorphometric analysis in future. Thebone samples were fixed in 10% neutral-buffered forrnalin for 2-3 days, then decalcified in 10%EDTA for two weeks, and finally processed with conventional paraff1n technique forhistological analyses.
[144] [00144] Statistical analysis was performed with SPSS (version 15.0). The mean andstandard deviation of each parameter were deterrnined. One animal in control group with no_43-P4l300887SE00observable bone lesions (cut-off limit 0.5 mmz) was removed from the analysis. Proportionally,one animal from groups 2 and 4 With no observable bone lesions was removed from analysis.There were no animals under the cut-off limit in groups 3 and 5. All statistical analyses wereperformed as two-sided tests. Normal distribution of residuals and homo geneity of variancewere checked before further analyses. In case of violating these assumptions, either logtransformation or other appropriate transformation (e.g. square root, reciprocal) was applied. Ifthe assumptions were fulf1lled as such or after transformation, one-way ANOVA was used tostudy if the values obtained between groups are statistically different (with p<0.05). Ifdifferences were found, Dunnett°s t-test was used for comparison against the control group. Ifthe assumptions were not fulf1lled even after the transforrnations described above, rank-transforrnation was applied and the non-parametric Kruskal-Wallis followed by Mann-WhitneyU-test was used. Fisher s Exact test was used for frequency data.
[145] [00145] The following deviations from the original protocol were performed during thestudy:[00146] Deviation l: At the intracardial inoculation the animals were anesthetized withXylazin (4-5 mg/ml) and Ketamine (75-92 mg/ml) ip instead of isoflurane due to malfunction ofthe isoflurane vaporizer. This change does not affect the results of this study.
[147] [00147] Deviation 2: Animals ll-l5 in group 2 received one dose of Compound l(l5mg/kg) at study day 8 due to human error. The results of these animals were compared to therest of group 2, and since they did not differ, these animals were not excluded from the group.RESULTSBody Weight
[148] [00148] Figure la shows relative change (%) in body weight during the study (meaniSD)of Groups 1-5. In addition, body weight at sacrifice, maximum bodyweight and weight lossfrom maximum body weight were statistically analyzed and the results are shown in figures lb,2 and 2b (not shown), respectively. The rise in mean body weight of control group at day 25 isdue to euthanasia of moribund animals at day 24. Group l received vehicle (com oil) 20ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.l mg/kg sc at days land 14; groups 3, 4 and 5 received Compound ldoses 30, 60 and 90 mg/kg/d po, respectively.In figure 2, Group l received vehicle (com oil) 20 ml/kg/d po; group 2 received vehicle 20ml/kg/d po and zoledronic acid 0.l mg/kg sc at days l and l4; groups 3, 4 and 5 receivedCompound ldoses 30, 60 and 90 mg/kg/d po, respectively._44_Paraplegia and cachexia
[149] [00149]P41300887SE00Animals With loss of function in any limb were considered paraplegic. Animalswere considered cachectic when 2 out of 3 conditions were met: curved spine, dehydrationand/or a 20% or more reduction from the maximum weight. Statistical analysis was performedusing Fischer Exact test. Statistically significant differences wer not observed.
[150] [00150]Groups: Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2 receivedvehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4 and 5received Compound ldoses 30, 60 and 90 mg/kg/d po, respectively.
[151] [00151]Control Zoledronic Compound 1 Compound 1 Compound 1acid 30 mg/kg/d 60 mg/kg/d 90 mg/kg/dCachectic % 21.4 11.1 21.4 16.7 20p-value 1.000 1.000 1.000 1.000Significance NS NS NS NSParaplegic 28.6 22.2 57.1 8.3 20.0%p-value 1.000 0.252 0.330 1.000Significance NS NS NS NSRadiographic analysis
[152] [00152]Figure 3a shows total osteolytic lesion area (mmz) at sacrifice (mean+SD) ofGroups 1-5. Osteolysis was inhibited in groups 2, 4 and 5. Figure 3b shows mean osteolyticlesion area (mmz) at sacrifice (mean+SD). Osteolytic lesions were also smaller in groups 2, 4and 5. Figure 3c shows number of osteolytic lesions at sacrifice (mean+SD). There were lessosteolytic lesions in groups 2 and 5, but more in group 3. Group 1 received vehicle (com oil) 20ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1and 14; groups 3, 4 and 5 received Compound ldoses 30, 60 and 90 mg/kg/d po, respectively.
[153] [00153]Figure 4 shows whole body tumor burden (mmz) at sacrifice (mean+SD) ofGroups 1-5. The results are shown as the average of fluorescent area in two pictures (prone andsupine positions) of each animal. Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4and 5 received Compound ldoses 30, 60 and 90 mg/kg/d po, respectively._45-P41300887SE00Serum TRACP 5b
[154] [00154] Tartrate-resistant acid phosphatase (TRACP) is an enzyme that is expressed inhigh amounts by bone resorbing osteoclasts, inflammatory macrophages and dendritic cells.Two forms of TRACP circulate in human blood, TRACP 5a derived from macrophages anddendritic cells, and TRACP 5b derived from osteoclasts. Recent data have demonstrated theutility of TRACP 5b as a marker of osteoclast number and bone resorption, and serum TRACP5a as a marker of inflammatory conditions.
[155] [00155] Figure 5 shows relative change (%) in serum Tartrate-resistant acid phosphatase5b (TRACP 5b) activity during the study (meaniSD) of Groups 1-5. Results of statisticalanalyzes are presented in figures 6a and 6b. Group 1 received vehicle (com oil) 20 ml/kg/d po;group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14;groups 3, 4 and 5 received Compound 1doses 30, 60 and 90 mg/kg/d po, respectively.
[156] [00156] Figure 6a shows relative change from day -4 to day 17 in serum TRACP 5bactivity (mean+SD). TRACP 5b activity was decreased in group 2. Figure 6b shows relativechange from day -4 to day 24 in serum TRACP 5b activity (mean+SD). TRACP 5b activity wasdecreased in group 2. Compared to Group 1 (Control), Groups 4 and 5 showed decreasedTRACP 5b activity at day 24, although not as significant as Group 2. Group 1 received vehicle(corn oil) 20 ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kgsc at days 1 and 14; groups 3, 4 and 5 received Compound 1doses 30, 60 and 90 mg/kg/d po,respectively.
[157] [00157] Serum procollagen I N-terrninal propeptide (PINP) is a sensitive bone formationmarker in humans. Serum PINP has emerged as a reliable marker of bone turnover in humansand is routinely used to monitor bone formation.
[158] [00158] Figure 7 shows relative change (%) in serum PINP values during the study(meaniSD) of Groups 1-5. Results of statistical analyzes are presented in figures 8a and 8b.Figure 8a illustrates relative change from day -4 to day 17 in serum PINP values (mean+SD).PINP activity was decreased in group 2 (recorded at day 17). Figure 8b illustrates relativechange from day -4 to day 24 in serum PINP values (mean+SD). Compared to Group 1(control), Groups 2 and 5 showed a lower PINP activity at day 24. Group 1 received vehicle(corn oil) 20 ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kgsc at days 1 and 14; groups 3, 4 and 5 received Compound 1doses 30, 60 and 90 mg/kg/d po,respectively._46-P41300887SE00Histomorphometry
[159] [00159] Figure 9 shows cortical bone area (mmz, mean +SD) of Groups 1-5. Figure 10shows illustrative results of intraosseus area (bone marrow space With trabecular bone included).Intraosseous area was increased in groups 2 and 3. In addition, figure 11 illustrates trabecularbone area (relative to the intraosseous area) and figure 12 illustrates tumor area (relative to theintraosseous area). Trabecular bone area was increased in group 2 and Tumor area was increasedin group 3. Figure 13 shows number of osteoclasts at tumor-bone interface (relative to thetumor-bone interface length, #/mm). Statistically significant differences were not observed.Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2 received vehicle 20 ml/kg/d po andzoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4 and 5 received Compound 1doses 30,60 and 90 mg/kg/d po, respectively.
[160] [00160] Table 3 provides summary of the results shown before. An upwards arrow (T)indicates increase and a downwards arrow (i) a decrease. One asterisk (*) indicates astatistically significant difference with a p-value < 0.05, two asterisks (**) with a p-value < 0.01,and three asterisks (***) with a p-value < 0.001. NS = Non-significant._47-P4l300887SE00Table 3.
[161] [00161] In conclusion, zoledronic acid prevented the forrnation of osteolytic lesions anddecreased TRACP 5b. PINP Was decreased at day 17. Trabecular bone area Was increased.Zoledronic acid exhibited no effect on body Weight, Whole body tumor burden or intraosseoustumor area, as expected. Compound 1 dose 90 mg/kg/d increased the maximum Weight obtainedduring the study and the body Weight at sacrifice. Compound 1 decreased the total osteolyticarea and mean osteolytic lesion size at doses 60 and 90 mg/kg/d. The number of osteolyticlesions Was decreased at Compound 1 dose 90 mg/kg/d. Statistically significant effects on theWho le body tumor burden Were not found. Compound 1 dose 30 mg/kg/d increased and higherdoses had no effect on intraosseous tumor area. Compound 1 did not have any effect on corticalor trabecular bone area as analysed by histomorphometry.
[162] [00162] Patients With prostate cancer typically develop metastases to bone. Almost allprostate cancer patients With end-stage disease have bone metastases. Bone metastases causedby prostate cancer are mainly osteoblastic, causing severe bone pain and also increasing the riskof fractures. Although the primary tumor could be successfully removed, the disease may havealready spread to bone. In this case, the disease can develop further visceral and bonemetastases. Currently, there is no efficient cure for prevention or treatment of bone metastases.[00163] The objective of this study Was to test the efficacy of three different doses of thetest compound 1 on prostate cancer bone metastases in male athymic nude mice in a S-Weekstudy in preventive setting. The following 5 experimental groups Were included in the study:_49-P41300887SE001. Control group receiving vehicle (po twice a day)2. Reference group receiving zoledronic acid (sc at days 1 and 14) + vehicle (po twice a day)3. Test group receiving 15 mg/kg of test compound (po twice a day)4. Test group receiving 30 mg/kg of test compound (po twice a day)5. Test group receiving 45 mg/kg of test compound (po twice a day)
[164] [00164] The vehicle was com oil. All groups included 15 mice that were 7 weeks of ageat the beginning of the study. Based on the body weights the animals were randomized to fivegroups. On day 0, the animals were given an intratibial ino culation of human prostate cancercells. Body weights were deterrnined twice a week. The development of osteoblastic andosteolytic lesions were followed by x-ray. The test compound and vehicle were administered byoral gavage twice a day. The animals were sacrificed at day 35. Tissue samples were collectedfor histology and embedded in paraff1n from left and right tibia and femur for possible futurehistomorphometric analyses. Gross necropsy was performed to all animals at the end of thestudy, and all macroscopic signs were recorded.
[165] [00165] Test compound 1 (99.85%, specific density: 1.005) was obtained in liquid form.Three 50x stock solutions were prepared in com oil (75 mg/ml, 150 mg/ml and 225 mg/ml) andstored them in dark at +4°C. The dosing solutions of 1.5, 3.0 and 4.5 mg/ml were prepared fromthe stock solutions by diluting the stock solutions in com oil 1:50. The dosing solutions wereprepared once a week and stored in dark at +4°C. The dosing volume was 10 ml/kg. The testcompound was administered by oral gavage twice a day with dosing intervals of 10+14 hours.The dosings were started at day 1 and performed at the same time every day.
[166] [00166] Zoledronic acid was used as a reference compound. 100ul of Zometa® (NovartisPharma GmbH, Nümberg, Germany) 4 mg / 5ml infusion concentrate was diluted in 3.9 ml ofsterile saline (0.9% NaCl) to obtain the administration concentration of 0.02 mg/ml. Thissolution was prepared once during the study and stored at +4°C. The dosing volume was 5ml/kg, resulting in the dose of 0.1mg/kg. The reference compound was administered SC twice inthe study: at days 1 and 14.
[167] [00167] The vehicle was com oil (Sigma-Aldrich).
[168] [00168] Male athymic nude mice (Hsd: Athymic nude-nu, obtained from Harlan, TheNetherlands) and PC-3 human prostate cancer cells (ATCC) were used for this study._50_P41300887SE00
[169] [00169] In this model, human prostate cancer cells are inoculated into the left proximaltibia of 5-7 week old male athymic nude mice. This mouse strain is immunodeficient, allowingrapid spreading of the human cancer cells. The bone lesions are mixed, being mainly osteolyticbut also containing an osteoblastic component. Within four weeks after the inoculation part ofthe animals have already developed some bone lesions, that can be visualized by X-rayradiography. At 7 weeks, the bone metastases are clearly visible. The animals are sacrificed at 7-8 Weeks after inoculation, and their bones are collected for further histomorphometric analysis.[00170] The bone metastasis model provided herein can be conveniently used to test drugcandidates that have been shown in preliminary studies to affect one or more of the following: 1)growth of cancer cells; 2) angiogenesis; 3) fianction or differentiation of osteoclasts. The modelcan also be used for finding new indications to existing drugs, allowing the pharrnaceuticalcompanies a way of finding new indications to patented drugs that have already proved efficientin some other indications.
[171] [00171] Male athymic nude mice (Hsd: Athymic nude-nu, obtained from Harlan, TheNetherlands) were used for this study. The age of the animals was 7 weeks, and their bodyweights were approximately 18-25 g at the beginning of the study. The mice were specificpathogen free (SPF) and isolator-reared animals. Correct age and good clinical health werequalifications for the study. The minimum quarantine and acclimatization period for the micewas 7 days. Allocation to groups was performed by randomization procedure based on bodyweight. The animals were marked with tail and ear marks. The mice were housed inScantainer®, 5 mice per cage. In case of fighting, some of the animals had to be isolated. Forintratibial inoculations the mice were anesthetized with i.p. inj ections of Xylazin (5-6 mg/kg)and Ketamine (92-109 mg/kg). For x-ray imaging the mice were anesthetized with inhalation ofisoflurane. Analgesia (buprenorphine: either 0.1 mg/kg, s.c. twice a day or 3 mg/kg po indrinking water) was used for 2 days after the intratibial inoculation (first dose s.c., then indrinking water) and for the last 5 days of the study (in drinking water).
[172] [00172] PC-S human prostate cancer cells (106 cells in 20 ul of PBS) were inoculated intothe left proximal tibia of the mice at day 0, leading to development of bone metastases. The rightplacement of the needle will be verified by X-ray before inoculating the cells. In case ofmisplacement, the cells were inoculated to the right proximal tibia instead.
[173] [00173] The animals were weighed twice a week and doses were adjusted accordingly.
[174] [00174] 6 animals Were found dead or had to be euthanized due to breathing difficulties(see Table 4). At necropsy, punctured esophagus and oil in thoracic cavity Was found. Oneanimal Was euthanized due to hemorrhage in the tumor at day 30 (see Table 4). It Was Weighedand bone samples Were prepared.
[175] [00175] Table 4: The results of animal monitoring - Final N With prostate cancer cells.Group Animal Treatment Final N Dead Time of Notesnumbers animals death(id) (study day)l l- l 5 Control l5 -2 l- l 5 Zoledronic l5 -acid3 l- l 5 Compound 1 l4 l5 23 Esophageal30 mg/kg/d puncture.4 l- l 5 Compound 1 l4 l0 30 Hemorrhage60 mg/kg/d l5 23 in the tumorEsophagealpuncture.5 l- l 5 Compound 1 l l 7 25 Esophageal90 mg/kg/ d 9 l2 puncture.l2 23 Found dead.l5 23 Esophagealpuncture.Esophagealpuncture.Esophagealpuncture.
[176] [00176] The development of osteoblastic and osteolytic lesions Was monitored by x-rayradiography at 4 Weeks and prior to the sacrif1ce of the animals. The animals Were anesthetizedand x-rayed in an anteroposterior position With the Faxitron Specimen Radiographic SystemMX-20 Dl2 (Faxitron Corp. Illinois, USA) using Faxitron Dicom 3.0 -software. At least oneradiograph of both hind limbs per animal Was taken on each x-ray occasion (3l kV, l0 seconds,magnification 2x). The lesion number and area in hind limbs Was deterrnined from the imagesWith MetaMorph image analysis software.
[177] [00177] At the end of the study (at day 35), the animals Were Weighed and sacrif1ced Withcervical dislocation under anesthesia. Terminal blood sample Was draWn for possible futureanalyses. Necropsy Was carried out in all animals. Macroscopic findings Were recorded on the_52-P41300887SE00fo llow-up forms. Ex vívo tissue samples from hind limbs (left and right tibiae and femur) werecollected for possible histomorphometric analysis in future. The bone samples were fixed in10% neutral-buffered forrnalin for 2-3 days, then decalcif1ed in 10% EDTA for two weeks, andfinally processed with conventional paraff1n technique for possible histological analyses.Statistical analysis
[178] [00178] Statistical analysis was perforrned with SPSS (version 19.0). The mean andstandard deviation of each parameter were deterrnined. All statistical analyses were perforrnedas two-sided tests. Norrnal distribution of residuals and homo geneity of variance were checkedbefore further analyses. In case of violating these assumptions, either log transforrnation or otherappropriate transforrnation (e. g. square root, reciprocal) was applied. If the assumptions werefulfilled as such or after transforrnation, one-way ANOVA was used to study if the valuesobtained between groups are statistically different (with p<0.05). If differences were found,Dunnett°s t-test was used for comparison against the control group. If the assumptions were notfulf1lled even after the transforrnations described above, rank-transformation was applied andthe non-parametric Kruskal-Wallis followed by Mann-Whitney U-test was used. Fischer Exacttest was used for frequency data.
[179] [00179] The following deviations from the original protocol were perforrned during thestudy:[00180] Deviation 1: The study was terrninated at day 35 instead of day 56 for ethicalreasons (large tumors and massive osteolysis with severe fractures). This change does not affectthe results of the study because the desired end point was reached.
[181] [00181] Figure 14a shows relative change (%) in body weight of Groups 1-5 during thestudy (meaniSD). Body weight at sacrif1ce, maximum bodyweight and weight loss frommaximum body weight were statistically analyzed and the results are shown in figures 14b, 15aand 15b, respectively. Group 4 obtained more weight during the study and Groups 4 and 5 lostmore weight. Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2 received vehicle 20ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4 and 5 receivedCompound 1 doses 30, 60 and 90 mg/kg/d po, respectively.
[182] [00182] Table Sa provides data of fractures and osteoblastic lesions at day 28. The x-rayimages were evaluated qualitatively (yes or no) for osteoblastic lesions (OB) and fractures (FR).
[183] [00183] Table Sa: Fractures and osteoblastic lesions at day 28.Day 28 Control Zoledronic Compound 1 Compound 1 Compound 1acid 30 mg/kg/d 60 mg/kg/d 90 mg/kg/dOsteoblastic 0.0 0.0 0.0 0.0 9.1lesionsp-value 1.000 1.000 1.000 0.423Significance NS NS NS NSFractures 33.3 0.0 0.0 0.0 36.4p-value 0.042 0.042 0.042 1.000Significance * * * NS
[184] [00184] Table Sb: Fractures and osteoblastic lesions at sacrif1ce.
[185] [00185] Figure 16a shows illustrative results of total osteolytic lesion area (mmz) at day28 (mean+SD). The results are shown as the sum of areas of bone lesions in right and left tibiaand femur/ animal. Total osteolytic lesion area at day 28 Was decreased in groups 2 and 4._54-P41300887SE00Group 5 also showed a similar trend. Figure 16b shows illustrative results of total osteolyticlesion area (mmz) at sacrifice (mean+SD). The results are shown as the sum of areas of bonelesions in right and left tibia and femur / animal. Total osteolytic lesion area at sacrif1ce wasdecreased in groups 2 and 4. Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4and 5 received Compound 1 doses 30, 60 and 90 mg/kg/d po, respectively.
[186] [00186] Figure 17a provides illustrative results of mean osteolytic lesion area (mmz) atday 28 (mean+SD). The results are shown as the mean areas of individual bone lesions in rightand left tibia and femur / animal. Osteolytic lesions were smaller in groups 2 and 5 at day 28.Group 4 also showed a similar trend. Figure 17b provides illustrative results of mean osteolyticlesion area (mmz) at sacrif1ce (mean+SD). The results are shown as the mean areas of individualbone lesions in right and left tibia and femur / animal. Osteolytic lesions were smaller in groups2 and 4 at sacrif1ce. Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2 received vehicle20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4 and 5 receivedCompound 1 doses 30, 60 and 90 mg/kg/d po, respectively.
[187] [00187] Figure 18a shows number of osteolytic lesions at day 28 (mean+SD). The resultsare shown as the count of individual bone lesions in right and left tibia and femur/ animal.There were more osteolytic lesions in group 2 because individual lesions were prevented fromfusing with each other. Figure 18b shows number of osteolytic lesions at sacrif1ce (mean+SD).The results are shown as the count of individual bone lesions in right and left tibia and femur/animal. There were more osteolytic lesions in group 2 because individual lesions wereprevented from fusing with each other. Group 1 received vehicle (com oil) 20 ml/kg/d po; group2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4and 5 received Compound 1 doses 30, 60 and 90 mg/kg/d po, respectively.
[188] [00188] Figure 19 shows illustrative results of cortical bone area (mmz, mean +SD) ofGroups 1-5. Cortical bone area was increased in group 2. Group 1 received vehicle (com oil) 20ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1and 14; groups 3, 4 and 5 received Compound 1 doses 30, 60 and 90 mg/kg/d po, respectively.[00189] Figure 20 shows data of intraosseus area (bone marrow space with trabecularbone included) of Groups 1-5. Statistically significant differences were not observed. Group 1received vehicle (com oil) 20 ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronicacid 0.1 mg/kg sc at days 1 and 14; groups 3, 4 and 5 received Compound 1 doses 30, 60 and 90mg/kg/d po, respectively._55-P41300887SE00
[190] [00190] Figure 21 shows illustrative results of trabecular bone area (relative to theintraosseous area) of Groups 1-5. Trabecular bone area was increased in all treatment groups.Group 1 received vehicle (com oil) 20 ml/kg/d po; group 2 received vehicle 20 ml/kg/d po andzoledronic acid 0.1 mg/kg sc at days 1 and 14; groups 3, 4 and 5 received Compound 1 doses 30,60 and 90 mg/kg/d po, respectively.
[191] [00191] Figure 22a shows intraosseous tumor area (relative to the intraosseous area) andFigure 22b provides data of total tumor area. Intraosseous tumor area was decreased in groups 2and 4 and total tumor area was decreased in group 2. Group 1 received vehicle (com oil) 20ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc at days 1and 14; groups 3, 4 and 5 received Compound 1 doses 30, 60 and 90 mg/kg/d po, respectively.[00192] Figure 23 shows illustrative data of Groups 1-5 re number of osteoclasts attumor-bone interface (relative to the tumor-bone interface length, #/mm). . The number ofosteoclasts at tumor-bone interface was decreased in group 2. Group 1 received vehicle (comoil) 20 ml/kg/d po; group 2 received vehicle 20 ml/kg/d po and zoledronic acid 0.1 mg/kg sc atdays 1 and 14; groups 3, 4 and 5 received Compound 1 doses 30, 60 and 90 mg/kg/d po,respectively.
[193] [00193] Table 6 shows summary of the results. An upwards arrow (T) indicates increaseand a downwards arrow (i) a decrease. One asterisk (*) indicates a statistically significantdifference with a p-value < 0.05, two asterisks (**) with a p-value < 0.01, and three asterisks(***) with a p-value < 0.001. NS = Non-significant.
[194] [00194] Table 6: Summary of the resultsMETHOD / Zoledronic Compound 1 Compound 1 Compound 1PARAMETER acid 30 mg/kg/d 60 mg/kg/d 90 mg/kg/dBODY WEIGHTRelative change of body NS NS NS NSweight at sacrificeRelative maximum weight NS NS T* NSStudy day of maximum NS NS NS NSweightRelative loss of body weight NS NS Tff* Tff*from maximum weightRADIOGRAPHICANALYSIS_56_P4l300887SE00Osteoblastic lesions, d28 NS NS NS NSOsteoblastic lesions, sacr. i* * * NS i* NSFractures, d28 i* i* i* NSFractures, sacr. i* * * i* i* NSTotal osteolytic lesion area, i* * * NS i* NSd28Total osteolytic lesion area, i* * * NS i* * NSsacr.
[195] [00195] Zoledronic acid inhibited the formation of osteolytic and osteoblastic lesions andfractures. The increase in osteolytic lesion count is a result of individual lesions prevented fromfusing together. Zoledronic acid increased both cortical and trabecular bone area as quantifiedby histomorphometry and decreased the number of osteoclasts at the tumor-bone interface.Zoledronic acid had no effect on body Weight. Zoledronic acid decreased both intraosseous andtotal tumor area. Compound 1 increased maximum body Weight obtained at the dose 60ngkgdTmflæsdbmwwmguwæaßommæædammmmhomewmmflmmpmgmqßreceiving 60 and 90 mg/kg/d of Compound 1. However, the Weight loss Was only around 6%MmhßmqmmmgwlCmmmmfldwflwmflgflæwææümommflßbmmmæ.Compound 1 doses 30 and 60 mg/kg/d inhibited fractures at day 28 and at sacrifice. The dose 60mg/kg/d inhibited also osteoblastic reactions at sacrifice. All Compound 1 doses increased theHwæmmmmmmmmfimdæflwmflgüwæmmmüdñww.Qmwmfilmmwfiflwmmæmfimfimmmmmmmmwfiw(mmwmlmæwmflgflæmmwthe intraosseous tumor area, but had no effect on the total tumor area. In conclusion, Compound1 had positive, bone protective effects in this aggressive model for prostate cancer bonemetastasis and it also decreased the intraosseous tumor area at the dose level on 60 mg/kg/d.Example 4: Safetv and Efficacv of Compound l in Patients With Breast Cancer With MetastaticBone Lesions
[196] [00196] The objective of this study is to assess the clinical benefit of two different dosingschedules of Compound 1 in patients With metastatic bone lesions from breast cancer.
[197] [00197] .AnnualOvenfllSkdeüdhdoflndüylme(Sh4R)[TnnePræne:l2rnonflß][Designated as safety issue: No]
[198] [00198] The SMR is computed by summing all Skeletal Related Event(s) (SREs)Whichoccurre during the observation period and dividing it by the ratio "days of observation period/365.25", for each participant. SRE is defined as: pathologic bone fracture, spinal cordcompression, surgery to bone both curative and prophylactic, radiation therapy to bone, orhypercalcemia of malignancy.
[199] [00199] Percentage of Participants Experiencing Skeletal Related Event(s) (SREs) [TimeFrame: 12 month] [Designated as safety issue: No]
[200] [00200] Skeletal Related Events (SREs) are defined as a:0 pathologic bone fracture such as non-vertebral and vertebral compressionfractures_5g_P4l300887SE000 spinal cord compression identified by positive diagnosis documented by X-rayevidence0 surgery to bone both curative and prophylactic0 radiation therapy to bone including palliative, therapeutic or prophylactic0 hypercalcemia of malignancy, defined as a corrected serum calcium > 12 mg/dl(3.00 mmol/l) or a lower level of hypercalcemia which is symptomatic andwhich requires active treatment other than rehydration.[00201] Annual Incidence of Any Skeletal Related Events (SREs) [Time Frame: 12months] [Designated as safety issue: No][00202] Skeletal Related Events (SREs) are defined as a:0 pathologic bone fracture such as non-vertebral and vertebral0 spinal cord compression identified by X-rays evidence0 surgery to bone both curative and prophylactic0 radiation therapy to bone including palliative, therapeutic or prophylactic0 hypercalcemia of malignancy, defined as a corrected serum calcium > 12 mg/dl(3.00 mmol/l) or a lower level of hypercalcemia which is symptomatic andwhich requires active treatment other than rehydration. Annual incidence foreach SRE was computed in the same way as annual overall SMR.[00203] Median Time to First Skeletal Related Event(s) (SRE) [Time Frame: 12 month][Designated as safety issue: No][00204] Median Time to first skeletal related event (SRE) is defined as the time fromrandomization to the date of first occurrence of any SRE which includes at least one of thefollowing: radiation therapy to bone, pathologic bone fracture, spinal cord compression, surgeryto bone, and hypercalcemia of malignancy (HCM). Due to the few numbers of SRE, Kaplan-Meier estimate never reaches a failure probability >=25%; so median time, 25th and 75thpercentiles are not deterrnined. For this reason only the estimated percentage of patient SRE freeare reported at each time point.[00205] Percentage of Participants Skeletal Related Event (SRE) Free [Time Frame: 12months] [Designated as safety issue: No][00206] Percentage of participants SRE free is defined as the Kaplan-Meier estimate ofparticipants free of any Skeletal Related Events(SRE) at each time point.[00207] Skeletal Related Events (SREs) are:0 pathologic bone fracture; non-vertebral and vertebral0 spinal cord compression identified by X-rays_59_P41300887SE000 surgery to bone both curative and prophylactic0 radiation therapy to bone (palliative, therapeutic or prophylactic)0 hypercalcemia of malignancy, defined as a corrected serum calcium > 12 mg/dl(3.00 mmo 1/l) or a loWer level Which is symptomatic and requires treatmentother than rehydration.
[208] [00208] Composite Bone Pain Score According to the Brief Pain Inventory (BPI)Questionnaire [Time Frame: At Baseline, Month 3, Month 6, Month 9 and Month 12][Designated as safety issue: No]
[209] [00209] Bone pain is assessed by means of a pain score obtained using the Brief PainInventory (BPI) questionnaire. The BPI can produce three pain scores: Worst pain, a compositepain score, and a pain interference score. The composite pain score, Which is the average ofquestions 3, 4, 5 and 6 of the questionnaire is used in this study. Pain is rated on a scale of 0 (nopain) to 10 (pain as bad as you can imagine). The outcome is given as the median score forparticipants at baseline, and 3, 6, 9 and 12 months of treatment.
[210] [00210] Evaluation of Pain According to Verbal Rating Scale (VRS) Based on MedianScore Value [Time Frame: At Baseline, Month 3, Month 6, Month 9 and Month 12] [Designatedas safety issue: No]
[211] [00211] Pain intensity at rest and on movement is rated by the patient by means of avalidated 6-point Verbal Rating Scale (VRS) and refers to the pain occurred during the last Weekbefore the assessment. Median score value is the median of all the observed scores (none=0,very mild=1, mild=2, moderate=3, severe=5 and very severe=6) at each time point.
[212] [00212] Use Of Analgesic Medications According to the Analgesic Score Scale [TimeFrame: At Baseline, Month 3, Month 6, Month 9 and Month 12] [Designated as safety issue:No]
[213] [00213] The analgesic score used for this study is modified from the Radiation TherapyOncology Group (RTOG) analgesic score scale. The scale represents type of medicationadministered from 0 to 4 Where:
[214] [00214] 0 = None; 1 = Minor analgesics (aspirin, NSAID, acetaminophen, propoxyphene,etc.); 2 = Tranquilisers, antidepressants, muscle relaxants, and steroids; 3 = Mild narcotics(oxycodone, meperidine, codeine, etc.); 4 = Strong narcotics (morphine, hydromorphone, etc.)The outcome is given a median score for the participants at Baseline and 3, 6, 9 and 12 monthsof treatment.
[215] [00215] Assessment of the Eastem Cooperative Oncology Group (ECOG) PerformanceScore [Time Frame: At Baseline, Month 3, Month 6, Month 9 and Month 12] [Designated assafety issue: No]_6()_P4l300887SE00
[216] [00216] ECOG Performance Score has 4 grades. 0 = Fully active, able to carry out all pre-disease activities; l = Restricted in strenuous activity but ambulatory and able to carry out Workof light or sedentary nature; 2 = Ambulatory and capable of all self-care but unable to carry outWork activities. Active about 50% of Waking hours; 3 = Capable of limited self-care, conf1ned tobed/chair more than 50% of Waking hours; 4 = Completely disabled; cannot carry on self-care.
[217] [00217]ArmsAssigned InterventionsExperimental: Every 3 monthsCompound 1 as a 15-minute (at least)intravenous (i.v.) infusion every threemonths. The dose of study drug Will be thesame administered before the study entry,that is 50 mg or a reduced dose.Randomized patients Will receive amaximum of 4 infusions in this group.
[218] [00218] Ages Eligible for Study:
[219] [00219] Genders Eligible for Study: Female[00220] Accepts Healthy Volunteers: NoCriteria
[221] [00221]given; Histologically confirmed Stage IV breast cancer With at least one bone metastasisradio logically confirrns.
[222] [00222]Inclusion criteria: Female patients 2 18 years of age; Written inforrned consentEastem Cooperative Oncology Group (ECOG) performance status S2 ._61-P41300887SE00
[223] [00223] Life expectancy 2 1 year.
[224] [00224] Exclusion criteria: Treatments with other cyclohexenone compounds thanCompound 1 at any time prior to study entry.
[225] [00225] Serum creatinine > 3 mg/dL (265 umol/L) or calculated (Cockcroft-Gaultformula) creatinine clearance (CLCr) < 30 mL/min CrCl = ({[140-age (years)] x weight(l
[226] [00226] Corrected (adjusted for serum albumin) serum calcium < 8 mg/dl (2 mmol/L) or> 12 mg/dL ( 3.0 mmol/L).
[227] [00227] Current active dental problem including infection of the teeth or j awbone(maxilla or mandibular); dental or f1xture trauma, or a recurrent or prior diagnosis ofosteonecrosis of the j aw (ONJ), of exposed bone in the mouth, or of slow healing after dentalprocedures.
[228] [00228] Recent (within 6 weeks) or planned dental or j aw surgery (e. g. extraction,implants).
[229] [00229] Pregnant patients (with a positive pregnancy test prior to study entry) or lactatingpatients. Women of childbearing potential not using effective methods of birth control (e. g.abstinence, oral contraceptives or implants, IUD, vaginal diaphragm or sponge, or condom withsperrnicide).
[230] [00230] History of non-compliance to medical regimens or potential unreliable behavior.[00231] Known sensitivity to study drug(s) or class of study drug(s).
[232] [00232] Patients with severe medical condition(s) that in the view of the investigatorprohibits participation in the study[00233] Use of any other investigational agent in the last 30 days.
[234] [00234] To prepare a parenteral pharrnaceutical composition suitable for administrationby injection, 90 mg of a compound or its salt described herein is dissolved in DMSO and thenmixed with 10 mL of 09% sterile saline. The mixture is incorporated into a dosage unit formsuitable for administration by injection.
[235] [00235] To prepare a pharrnaceutical composition for oral delivery, 90 mg of anexemplary Compound 1 was mixed with 90 mg of com oil. The mixture was incorporated intoan oral dosage unit in a capsule, which is suitable for oral administration.
[236] [00236] In some instances, 90 mg of a compound described herein is mixed with 750 mgof starch. The mixture is incorporated into an oral dosage unit for, such as a hard gelatin capsule,which is suitable for oral administration._62-P4l300887SE00Example 7: Sublingual (Hard Lozenge) Forrnulation
[237] [00237] To prepare a pharrnaceutical composition for buccal delivery, such as a hardlozenge, mix 90 mg of a compound described herein, with 420 mg of powdered sugar mixed,with 1.6 mL of light com syrup, 2.4 mL distilled water, and 0.42 mL mint extract. The mixtureis gently blended and poured into a mold to forrn a lozenge suitable for buccal administration.
[238] [00238] To prepare a pharrnaceutical composition for inhalation delivery, 20 mg of acompound described herein is mixed with 50 mg of anhydrous citric acid and l00 mL of 09%sodium chloride solution. The mixture is incorporated into an inhalation delivery unit, such as anebulizer, which is suitable for inhalation administration.
[239] [00239] To prepare a pharrnaceutical composition for rectal delivery, 90 mg of acompound described herein is mixed with 2.5 g of methylcelluose (1500 mPa), l00 mg ofmethylparapen, 5 g of glycerin and l00 mL of purified water. The resulting gel mixture is thenincorporated into rectal delivery units, such as syringes, which are suitable for rectaladministration.
[240] [00240] To prepare a pharrnaceutical topical gel composition, 90 mg of a compounddescribed herein is mixed with l.75 g of hydroxypropyl cellulo se, l0 mL of propylene glycol,l0 mL of isopropyl myristate and l00 mL of purified alcohol USP. The resulting gel mixture isthen incorporated into containers, such as tubes, which are suitable for topical administration.[00241] While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, and substitutions will nowoccur to those skilled in the art without departing from the invention. It should be understoodthat various altematives to the embodiments of the invention described herein may be employedin practicing the invention. It is intended that the following claims define the scope of theinvention and that methods and structures within the scope of these claims and their equivalentsbe covered thereby._63_

权利要求:
Claims (14)
[1] 1. A cyc1ohexenone compound having the structure: Rs cHs O / R4n Wherein each of X and Y independently is oxygen, NR5 or sulfur; R is a hydrogen or C(=O)C1-C8a1ky1; each of R1, R; and Rg independently is a hydrogen, methy1 or (CH2)m-CH3; R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered 1actone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, ary1, g1ucosy1, Wherein 5 or6-membered 1actone, Cl-Csalkyl, CZ-Csalkenyl, CZ-Csalkynyl, ary1, and g1ucosy1 areoptiona11y substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 a1ky1, CZ-CS a1keny1, CZ-CSa1kyny1, Cg-CS cyc1oa1ky1, and Cl-CS ha1oa1ky1; each of R5 and R6 is independently a hydrogen or Cl-Csalkyl; Ry is a Cl-Csalkyl, OR5 or NR5R6; m = 1-12; and n=1-12; or a pharrnaceutica11y acceptable sa1t, metabo1ite, so1vate or prodrug thereof, for use in the treatment of a disease or condition associated With bone metastasis.
[2] 2. A cyc1ohexenone compound having the structure: Rs cHs O / R4 HRß X OR YRz Wherein each of X and Y independently is oxygen, NR5 or sulfur; R is a hydrogen or C(=O)C1-C8a1ky1; each of R1, R; and Rg independently is a hydrogen, methy1 or (CH2)m-CH3; R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, ha1ogen, 5 or 6-membered 1actone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, ary1, g1ucosy1, Wherein 5 or 6-membered 1actone, Cl-Csalkyl, CZ-Csalkenyl, CZ-Csalkynyl, ary1, and g1ucosy1 are optiona11y substituted With one or more substituents se1ected from NR5R6, OR5, _64- P4l300887SE00 OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-CS alkenyl, CZ-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl; each of R5 and RÖ is independently a hydrogen or Cl-Csalkyl; R7 is a Cl-Csalkyl, OR5 or NR5R6; ni = l-l2; and n=l-12; or a pharrnaceutically acceptable salt, nietabolite, solvate or prodrug thereof, for use in the inhibition of hypercalceniia of n1alignancy.
[3] 3. A cyclohexenone conipound having the structure: Rs cHs O / R4 nRR X OR YRz Wherein each of X and Y independently is oxygen, NR5 or sulfur; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R; and Rg independently is a hydrogen, n1ethyl or (CH2)m-CH3; R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-n1en1bered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein 5 or6-n1en1bered lactone, Cl-Csalkyl, CZ-Csalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-CS alkenyl, CZ-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl; each of R5 and RÖ is independently a hydrogen or Cl-Csalkyl; R7 is a Cl-Csalkyl, OR5 or NR5R6; ni = l-l2; and n=l-12; or a pharrnaceutically acceptable salt, nietabolite, solvate or prodrug thereof, for use in the inhibition of bone resorption.
[4] 4. A cyclohexenone conipound having the structure: Rs cHs O / R4 HRR X OR YRz P4l300887SE00 Wherein each of X and Y independently is oxygen, NR5 or sulfur; R is a hydrogen or C(=O)C1-C8alkyl; each of R1, R; and R3 independently is a hydrogen, methyl or (CH2)m-CH3; R4 is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, halogen, 5 or 6-membered lactone, Cl-Csalkyl, CZ-Cgalkenyl, CZ-Csalkynyl, aryl, glucosyl, Wherein 5 or6-membered lactone, Cl-Csalkyl, CZ-Csalkenyl, CZ-Csalkynyl, aryl, and glucosyl areoptionally substituted With one or more substituents selected from NR5R6, OR5,OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,C1-C8 alkyl, CZ-CS alkenyl, CZ-CSalkynyl, Cg-CS cycloalkyl, and Cl-CS haloalkyl; each of R5 and R6 is independently a hydrogen or Cl-Csalkyl; R7 is a Cl-Csalkyl, OR5 or NR5R6; m = l-l2; and n=l-12; or a pharrnaceutically acceptable salt, metabolite, solvate or prodrug thereof, for use in the treatment of osteolytic lesions.
[5] 5. The compound of any of claims l-4, Wherein the subject has breast cancer or prostate cancer.
[6] 6. The compound of any one of claims 1-5, Wherein R is a hydrogen, C(=O)C3Hg,C(=O)C2H5, OI'
[7] 7. The compound of any one of claims l-6, Wherein each of R1, R; and R3 independently is a hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.
[8] 8. The compound of any one of claims 7, Wherein R1 is a hydrogen or methyl.
[9] 9. The compound of any one of claims l-8, Wherein R; is a hydrogen or methyl.
[10] l0. The compound of any one of claims l-9, Wherein R4 is halogen, NHg, NHCHg,N(CH3)2, OCHg, OC2H5, C(=O)CH3, C(=O)C2H5, C(=O)OCH3, C(=O)OC2H5, C(=O)NHCH3,C(=O)NHC2H5, C(=O)NH2, OC(=O)CH3, OC(=O)C2H5, OC(=O)OCH3, OC(=O)OC2H5,OC(=O)NHCH3, OC(=O)NHC2H5, or OC(=O)NH2.
[11] ll. The compound of any one of claims l-9, Wherein R4 is C2H5C(CH3)2OH, C2H5C(CH3)2OCH3, CHZCOOH, CgHgCOOH, CHgOH, C2H5OH, CHgPh, C2H5Ph,_66_ P41300887SE00 CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=O)CH3), 5 or 6-n1en1bered lactone, aryl, orglucosyl, Wherein 5 or 6-n1en1bered lactone, aryl, and glucosyl are optiona11y substituted Withone or n1ore substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5,C(=O)NR5R6, Cl-Cs alkyl, CZ-Cs a1keny1, CZ-Cs alkynyl, C3-C8 cyc1oa1ky1, and Cl-Cg ha1oa1ky1.
[12] 12. The conipound of c1ain1 11, Wherein R4 is Cl-Csalkyl optiona11y substituted Withone or n1ore substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, Cl-Cs alkyl, CZ-Cs a1keny1, CZ-Cs alkynyl, C3-C8 cyc1oa1ky1, and Cl-Cs ha1oa1ky1.
[13] 13. The conipound of c1ain1 12, Wherein R4 is CH2CH=C(CH3)2.
[14] 14. The conipound of any one of c1ain1s 1-5, Wherein said conipound isoHs oHs oHs oHsO / / / CH3H3C“o oHOoHs _67_

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法律状态:
2016-02-23| NAV| Patent application has lapsed|

优先权:

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申请号 | 申请日 | 专利标题

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US201261602489P| true| 2012-02-23|2012-02-23|

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