Small molecule trail gene induction by normal and tumor cells as an anticancer therapy
13 claims: 5 independent, 8 dependent
- 1癌を有するかまたは有するリスクがある被験体を処置するための組成物であって、薬学的に有効な量 の化 合物 NSC350625 、またはその薬学的に許容され得る塩;および薬学的に許容され得るキャリアを含む、組成物。
- 2前記被験体が、脳腫瘍を有するかまたは有するリスクがある被験体である、請求項1に記載の組成物。
- 3前記被験体が、結腸癌、乳癌、多形神経膠芽腫、および直腸結腸癌からなる群から選択される癌を有するかまたは有するリスクがある被験体である、請求項1に記載の組成物。
- 4前記組成物が第2の治療薬と組み合わせて前記被験体に投与されることを特徴とし、前記第2の治療薬は抗癌剤を含む、請求項1に記載の組成物。
- 5前記組成物が第2の治療薬と組み合わせて前記被験体に投与されることを特徴とし、前記第2の治療薬は抗血管新生剤を含む、請求項1に記載の組成物。
- 6前記抗癌剤は有糸分裂抑制剤である、請求項4に記載の組成物。
- 7前記抗癌剤は、パクリタキセル、ドセタキセルおよびそれらの組み合わせからなる群から選択される、請求項4に記載の組成物。
- 8前記抗血管新生剤は、ベバシズマブである、請求項5に記載の組成物。
- 9経口投与されることを特徴とする、請求項1 ~8のいずれか1項 に記載の組成物。
- 10直腸、鼻、肺、硬膜外、眼、耳、動脈内、心臓内、脳室内、皮内、静脈内、筋肉内、腹腔内、骨内、髄腔内、膀胱内、皮下、局所的、経皮的、経粘膜的、舌下、頬側、膣および吸入の投与経路からなる群から選択される投与経路により投与されることを特徴とする、請求項1 ~8のいずれか1項 に記載の組成物。
- 11前記処置の有効性が評価されることを特徴とする、請求項1 ~10のいずれか1項 に記載の組成物。
- 12前記処置の有効性の評価が、前記被験体から得られた生体サンプル中のTNF関連アポトーシス誘導リガンドをアッセイすることを含む、請求項1 1 に記載の組成物。
- 13前記生体サンプルが、血液、血清、血漿、および脳脊髄液からなる群から選択される、請求項12に記載の組成物。
Independent claims13
114 paragraphs, as filed
0001Cross-reference to related applications This application claims the priority of US Provisional Patent Application No. 61 / 480,743 filed on April 29, 2011, and the entire contents of this US Provisional Patent Application are incorporated herein by reference. ..
0002Reference to support This application was made with government support under grant number U54CA105008 awarded by the National Institutes of Health. The United States Government reserves certain rights to the present invention.
0003Field of invention The present invention generally relates to methods and compositions for treating a proliferative disease such as cancer in a subject requiring treatment of such disease.
0004Background of the invention TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that selectively induces apoptosis in cancer cells.
0005TRAIL is a wide range of human cancer cells mediated by cell surface apoptosis-promoting death receptor 4 (DR4; TRAIL-R1) and death receptor 5 (DR5; TRAIL-R2) due to the involvement of exogenous or endogenous apoptosis pathways. It is a potent inducer of apoptosis in strains. TRAIL plays a direct role in tumor suppression by immune surveillance, but this antitumor mechanism is lost during the course of the disease. The ability of TRAIL to selectively induce apoptosis in cancer cells is ongoing with the administration of recombinant TRAIL and longer-lived TRAIL agonist antibodies targeting either of its two pro-apoptotic death receptors. We are in the process of clinical trials.
0006Recombinant TRAIL, despite its potential, has properties that limit its effectiveness (eg, short serum half-life, stability, cost and delivery). Delivery of recombinant TRAIL or TRAIL agonist antibodies to the brain is limited by the inability of recombinant TRAIL and TRAIL agonist antibodies to cross the blood-brain barrier.
0007Anti-cancer compositions and methods continue to be needed.
<p num="0008"> Abstract of the invention According to aspects of the invention, also referred to herein as TIC10.</p><p num="0009"><chemistry num="1-1"><img id="000002" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, A pharmaceutical composition comprising a pharmaceutically acceptable derivative, salt, ester, amide, hydrate, solvate and / or prodrug; and a pharmaceutically acceptable carrier. The composition has utility in treating such diseases in subjects in need of treatment of the disease, including human subjects as well as subjects of other species. The composition has utility in treating cancer in subjects in need of treatment for cancer, including human subjects as well as subjects of other species.</p><p num="0010"> According to aspects of the invention, TIC10, a pharmaceutically acceptable derivative thereof, a salt, an ester, an amide, a hydrate, a solvate and / or a prodrug; a pharmaceutically acceptable carrier; and a second treatment. Pharmaceutical compositions comprising the drug are provided.</p><p num="0011"> According to aspects of the invention, TIC10, a pharmaceutically acceptable derivative thereof, salt, ester, amide, hydrate, solvate and / or prodrug; pharmaceutically acceptable carrier; and a second anticancer agent. Provided are pharmaceutical compositions comprising, where TIC10, a pharmaceutically acceptable derivative thereof, salts, esters, amides, hydrates, solvates and / or prodrugs are first anti-cancer agents. ..</p><p num="0012"> According to aspects of the invention, TIC10, a pharmaceutically acceptable derivative thereof, a salt, an ester, an amide, a hydrate, a solvate and / or a prodrug; a pharmaceutically acceptable carrier; and mitosis inhibition.<u style="single">Agent</u>Pharmaceutical compositions comprising.</p><p num="0013"> According to aspects of the invention, TIC10, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs; pharmaceutically acceptable carriers; and paclitaxel, docetaxel or Pharmaceutical compositions containing a combination thereof are provided.</p><p num="0014"> According to aspects of the invention, TIC10, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs; pharmaceutically acceptable carriers; and anti-angiogenic agents. A pharmaceutical composition comprising the above is provided.</p><p num="0015"> According to aspects of the invention, TIC10, a pharmaceutically acceptable derivative thereof, a salt, an ester, an amide, a hydrate, a solvate and / or a prodrug; a pharmaceutically acceptable carrier; and a pharmacy comprising bevacizumab. Compositions are provided.</p><p num="0016"> According to aspects of the invention, oral administration comprising TIC10, a pharmaceutically acceptable derivative thereof, salts, esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers. Pharmaceutical compositions formulated for use are provided.</p><p num="0017"> According to aspects of the invention, a method of treating a subject in need of treatment is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, an ester, an amide, and the like. Includes the step of administering hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers.</p><p num="0018"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers.</p><p num="0019"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of administering a pharmaceutically effective amount of TIC10; and a pharmaceutically acceptable carrier. Including the process.</p><p num="0020"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of a pharmaceutically acceptable derivative of TIC10; and pharmaceutically acceptable. Includes the step of administering an acceptable carrier to.</p><p num="0021"> According to aspects of the invention, a method of treating a subject having or at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable salt, ester thereof, Includes the step of administering amides, hydrates and / or solvates; and pharmaceutically acceptable carriers.</p><p num="0022"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10 or a pharmaceutically acceptable salt thereof, hydration thereof. Includes the step of administering a product or solvate; and a pharmaceutically acceptable carrier.</p><p num="0023"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers; in addition, TNF-related apoptosis-inducing ligands in samples obtained from the subject. Includes the step of assaying and assessing the effectiveness of the treatment.</p><p num="0024"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers; in addition, blood, serum, plasma or cerebrospinal obtained from the subject. Includes the step of assaying TNF-associated apoptosis-inducing ligands in serum samples and assessing the effectiveness of the treatment.</p><p num="0025"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers; and further includes the step of administering a therapeutically effective amount of a second anti-cancer agent. , Where TIC10, its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs are the first anti-cancer agents.</p><p num="0026"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers; and further includes the step of administering therapeutically effective amounts of anti-thread fission agents. To do.</p><p num="0027"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers; in addition, therapeutically effective amounts of paclitaxel, docetaxel, bevacizumab or any two of them. Includes the step of administering one or more.</p><p num="0028"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. esters, amides, hydrates, solvates and / or flop including as well as oral administration of carriers that may be pharmaceutically acceptable; prodrug.</p><p num="0029"> According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; as well as pharmaceutically acceptable carriers, where the administration is rectal, nasal, lung, epidural,. Eyeball, ear, intraarterial, intracardiac, intraventricular, intradermal, intravenous, intramuscular, intraperitoneal, intraosseous, intramedullary, intravesical, subcutaneous, topical, percutaneous, transmucosal, sublingual Administration by a route selected from the group consisting of buccal, vaginal and inhalation routes of administration.</p><p num="0030"> According to aspects of the invention, a method of treating a subject who has or is at risk of having a brain tumor is provided, the method of which is a pharmaceutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, a salt, and the like. Includes the step of administering esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers.<u style="single">In a preferred embodiment of the present invention, for example, the following items are provided.</u><u style="single">(Item 1)</u><chemistry num="14-1"><img id="000003" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><u style="single">, A pharmaceutical composition comprising a pharmaceutically acceptable derivative, salt, ester, amide, hydrate, solvate and / or prodrug; and a pharmaceutically acceptable carrier.</u><u style="single">(Item 2)</u><u style="single">The pharmaceutical composition according to item 1, further comprising a second therapeutic agent.</u><u style="single">(Item 3)</u><u style="single">The pharmaceutical composition according to item 2, wherein the second therapeutic agent is an anticancer agent.</u><u style="single">(Item 4)</u><u style="single">The pharmaceutical composition according to item 3, wherein the anticancer agent is a mitosis inhibitor.</u><u style="single">(Item 5)</u><u style="single">The pharmaceutical composition according to item 3, wherein the anticancer agent is selected from the group consisting of paclitaxel, docetaxel and combinations thereof.</u><u style="single">(Item 6)</u><u style="single">The pharmaceutical composition according to item 2, wherein the second therapeutic agent is an anti-angiogenic agent.</u><u style="single">(Item 7)</u><u style="single">The pharmaceutical composition according to item 6, wherein the anti-angiogenic agent is bevacizumab.</u><u style="single">(Item 8)</u><u style="single">The pharmaceutical composition according to item 1, which is formulated for oral administration.</u><u style="single">(Item 9)</u><u style="single">A method of treating a subject in need of treatment,</u><u style="single">Of a pharmaceutically effective amount</u><chemistry num="12"><img id="000004" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><u style="single">A method comprising administration of a pharmaceutically acceptable derivative, salt, ester, amide, hydrate, solvate and / or prodrug; and a pharmaceutically acceptable carrier.</u><u style="single">(Item 10)</u><u style="single">9. The method of item 9, further comprising the step of assaying a TNF-related apoptosis-inducing ligand in a sample obtained from said subject.</u><u style="single">(Item 11)</u><u style="single">9. The method of item 9, wherein the subject has or is at risk of having cancer.</u><u style="single">(Item 12)</u><u style="single">The method of item 11, further comprising administration of an additional anti-cancer agent.</u><u style="single">(Item 13)</u><u style="single">The method of item 12, wherein the additional anti-cancer agent is an anti-mitotic agent.</u><u style="single">(Item 14)</u><u style="single">12. The method of item 12, wherein the additional anti-cancer agent is paclitaxel, docetaxel, bevacizumab or any two or more of them.</u><u style="single">(Item 15)</u><u style="single">The method of item 10, wherein the increased TNF-related apoptosis-inducing ligand is assayed in a blood sample obtained from said subject.</u><u style="single">(Item 16)</u><u style="single">9. The method of item 9, wherein the administration is oral administration.</u><u style="single">(Item 17)</u><u style="single">The administration is rectal, nasal, lung, epidural, eyeball, ear, intraarterial, intracardiac, intraventricular, intracutaneous, intravenous, intramuscular, intraperitoneal, intraosseous, intrathecal, intravesical, subcutaneous. 6. The method of item 6, selected from the group consisting of topical, percutaneous, transmucosal, sublingual, buccal, vaginal and inhalation routes of administration.</u><u style="single">(Item 18)</u><u style="single">A method of treating a subject who has or is at risk of having a brain tumor.</u><u style="single">Of a pharmaceutically effective amount</u><chemistry num="13"><img id="000005" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><u style="single">A method comprising administration of a pharmaceutically acceptable derivative, salt, ester, amide, hydrate, solvate and / or prodrug; and a pharmaceutically acceptable carrier.</u><u style="single">(Item 19)</u><u style="single">A method of treating a subject in need of treatment,</u><u style="single">Of a pharmaceutically effective amount</u><chemistry num="14"><img id="000006" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><u style="single">, A method comprising administration of a pharmaceutically acceptable salt, ester, amide, hydrate or solvate; and a pharmaceutically acceptable carrier.</u><u style="single">(Item 20)</u><u style="single">To manufacture medicines to treat cancer,</u><chemistry num="15"><img id="000007" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><u style="single">Use of its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs.</u><u style="single">(Item 21)</u><u style="single">Compounds for treating cancer</u><chemistry num="16"><img id="000008" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry><u style="single">, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs.</u><u style="single">(Item 22)</u><u style="single">A pharmaceutical composition substantially as described herein.</u><u style="single">(Item 23)</u><u style="single">A method of treatment substantially as described herein.</u></p>
0031<figref num="1">Figure 1 shows the HCT116 Bax under transcriptional regulation of the first 504 base pairs of the human TRAIL gene promoter upstream of transcription initiation.<sup>-/-</sup>It is a graph which shows the activity of a luciferase reporter in a cell;</figref><figref num="2">Figure 2 shows HCT116 p53<sup>-/-</sup>Graph showing RT-qPCR analysis of TRAIL mRNA levels in cells;</figref><figref num="3">Figure 3 is a graph showing TIC10-induced surface TRAIL levels in a panel of cancer cells;</figref><figref num="4">Figure 4 shows the HCT116 p53 after TIC10 treatment at the indicated conditions and time points.<sup>-/-</sup>It is a graph showing the surface TRAIL level in cells;</figref><figref num="5">Figure 5 shows HCT116 p53 by flow cytometry 72 hours after the start of TIC10 treatment.<sup>-/-</sup>TRAIL is a graph showing surface levels;</figref><figref num="6">Figure 6 shows the TIC10 processed HCT116 p53.<sup>-/-</sup>And shows the cell cycle profile of human foreskin fibroblasts (HFF) cells;</figref><figref num="7">Figure 7 is a graph showing the quantification of colonization assays for cancer cells treated with TIC10;</figref><figref num="8">FIG. 8 is a graph showing a similar experiment as in FIG. 7 except that HFF cells were counted at the end;</figref><figref num="9">Figure 9 shows HCT116 WT, p53 after treatment with DMSO, TIC10 or rhTRAIL (25 ng / mL).<sup>-/-</sup>And Bax<sup>-/-</sup>It is a graph showing sub-G1 analysis of cells;</figref><figref num="10">Figure 10 is an image showing the results of Western blot analysis;</figref><figref num="11">FIG. 11 is a graph showing a sub-G1 analysis of TIC10 treated cancer cells pre-incubated with or without zVAD-fmk;</figref><figref num="12">Figure 12 is a graph showing sub-G1 analysis of MDA-MB-231 cells in which TRAIL was stably knocked down by short hairpin RNA;</figref><figref num="13">FIG. 13 is a graph demonstrating MDA-MB-231 shTRAIL knockdown by flow cytometric analysis of TIC10 treated cells;</figref><figref num="14">FIG. 14 is a graph showing a sub-G1 analysis of TIC10-induced cell death in H460 cells with overexpression of endogenous DR5, or a DR5 construct in which its death domain has been replaced by EGFP;</figref><figref num="15">FIG. 15 is a graph showing sub-G1 analysis of HCT116 cells treated with DMSO, TIC10 or rhTRAIL in the presence or absence of the TRAIL sequestering antibody RIK-2;</figref><figref num="16">FIG. 16 is a graph showing TIC10-induced surface TRAIL when using freshly resected human colon cancer cells;</figref><figref num="17">FIG. 17 is a graph showing the results of the cell survival assay for primary colon cancer cells of FIG. 16 treated with DMSO, TIC10 or 5-FU;</figref><figref num="18">FIG. 18 is a graph showing that TIC10 or rhTRAIL can reduce the cell viability of HCT116 cells after 1 hour of preincubation at the indicated temperature;</figref><figref num="19">Figure 19 shows the HCT116 p53 treated with TIC10, TRAIL or vehicle.<sup>-/-</sup>It is a graph showing xenografts;</figref><figref num="20">Figure 20 shows a luciferase-infected HCT116 p53 treated with TIC10 or vehicle.<sup>-/-</sup>It is a graph showing the results of bioluminescence imaging of xenografts;</figref><figref num="21">Figure 21 is a graph showing RKO xenografts treated with TIC10, TRAIL or vehicle;</figref><figref num="22">FIG. 22 is a boxplot of tumor volume 9 days after initiation of treatment with TIC10, TRAIL or vehicle in MDA-MB-231 vector or shTRAIL xenografts;</figref><figref num="23">FIG. 23 is a graph showing the relative tumor volume of DLD-1 xenografts treated with TRAIL, TIC10 or DMSO;</figref><figref num="24">FIG. 24 is a graph showing a comparison of TIC10 ip and oral administration in SW480 xenografts;</figref><figref num="25">FIG. 25 is a graph showing TIC10 or vehicle administered as a single oral dose in HCT116 xenografts;</figref><figref num="26">FIG. 26 is a graph showing the body weight of single-dose TIC10-treated athymic female nude mice;</figref><figref num="27">FIG. 27 is a graph showing the body weight of C57 / B6 female mice at the end of the 4th week of treatment with oral TIC10;</figref><figref num="28">FIG. 28 is a graph showing overall survival of Εμ-myc treated with oral TIC10 weekly between weeks 9-12;</figref><figref num="29">FIG. 29 is a graph showing the cell viability of TIC10-treated DLD-1 cells with paclitaxel;</figref><figref num="30">FIG. 30 is a graph showing the cell viability of SW620 cells treated with TIC10 along with paclitaxel;</figref><figref num="31">Figure 31 is a graph showing the cell viability of TIC10-treated DLD-1 cells with Taxotere;</figref><figref num="32">FIG. 32 is a graph showing the cell viability of SW620 cells treated with TIC10 with Taxotere;</figref><figref num="33">FIG. 33 is a graph showing the percentage of cohorts that retain tumor tissue in H460 xenografts with TIC10 or taxotere alone, in combination, or after treatment with a vehicle;</figref><figref num="34">FIG. 34 is a graph showing a plot of tumor volume relative to FIG. 33;</figref><figref num="35">Figure 35 is a graph showing the percentage of cohorts that retain tumor tissue in H460 xenografts with TIC10 or paclitaxel alone, in combination, or after treatment with a vehicle;</figref><figref num="36">FIG. 36 is a graph showing a plot of tumor volume relative to FIG. 35;</figref><figref num="37">Figure 37 shows HCT116 p53 in the cecum.<sup>-/-</sup>A graph showing the percentage of cohorts in which tumors were transplanted and treated with TIC10, bevacizumab or a combination of TIC10 and bevacizumab and had obvious tumors at the primary and distal sites at termination;</figref><figref num="38">Figure 38 shows intracecal HCT116 p53 treated with vehicle, TIC10, bevacizumab or a combination of TIC10 and bevacizumab.<sup>-/-</sup>It is a graph showing the body weight of mice transplanted with a tumor;</figref><figref num="39">FIG. 39 is a graph showing TRAIL serum levels in tumor-free mice after TIC10 or doxorubicin;</figref><figref num="40">FIG. 40 is a graph showing the absorbance profile of TIC10 with a peak absorbance at 239 nm;</figref><figref num="41">FIG. 41 is a graph showing a calibration curve for TIC10 added to mouse plasma and quantified using area under the curve (AUC) by HPLC analysis;</figref><figref num="42">FIG. 42 is a graph showing the plasma concentration of TIC10 after intravenous administration to C57 / B6 female mice;</figref><figref num="43">FIG. 43 is a graph showing analysis of surface TRAIL of HFF cells after TIC10 treatment (0, 2.5, 5 or 10 μM from left to right);</figref><figref num="44">Figure 44 shows the HCT116 p53.<sup>-/-</sup>It is a graph showing the sub-G1 analysis of the co-culture of cells and pretreated HFF;</figref><figref num="45">FIG. 45 is a graph showing surface TRAIL in GBM cell lines after incubation with TIC10;</figref><figref num="46">FIG. 46 is a graph showing the GI50 values extrapolated from the cell life-and-death assay of the indicated GBM cell lines 72 hours after treatment with TIC10 or DMSO;</figref><figref num="47">Figure 47 shows the results of a cell viability assay for freshly resected glioblastoma tissue treated with DMSO, TIC10 or temozolomide;</figref><figref num="48">FIG. 48 is a graph showing subcutaneous xenografts of T98G in mice that received a single dose of vehicle, TIC10 or bevacizumab;</figref><figref num="49">FIG. 49 is a graph showing overall survival of mice with SF767 intracranial tumors treated with a single oral vehicle, TIC10, bevacizumab or TIC10 and bevacizumab;</figref><figref num="50">Figure 50 shows HCT116 p53 48 hours after TIC10 treatment compared to DMSO.<sup>-/-</sup>Graph showing transcriptional changes associated with FOXO signaling from cellular gene expression profiling;</figref><figref num="51">FIG. 51 is an image of a Western blot analysis of DR5 in HCT116 cells treated with TIC10 or DMSO;</figref><figref num="52">FIG. 52 is a graph showing flow cytometric analysis of surface DR5 levels in TIC10 treated cancer and normal cells;</figref><figref num="53">FIG. 53 is an image of Western blot analysis of whole cell solubilized (W) and cytoplasmic (C) and nuclear (N) extracts of DMSO or TIC10 treated HCT116 cells;</figref><figref num="54">Figure 54 shows HCT116 p53<sup>-/-</sup>Image of chromatin immunoprecipitation assay results for TIC10-induced translocation of Foxo3a to the TRAIL promoter 48 hours after TIC10 treatment in cells (0, 2.5, 5 or 10 μM from left to right);</figref><figref num="55">Figure 55 shows HCT116 p53 with siRNA.<sup>-/-</sup>It is a graph showing the results of flow cytometric analysis of cell surface TRAIL levels induced by TIC10 with or without transient knockdown of Foxo1 and / or Foxo3a in cells;</figref><figref num="56">FIG. 56 is a graph showing sub-G1 analysis of TIC10-induced cell death with or without stable knockdown of Foxo3a in HCT116 cells;</figref><figref num="57">FIG. 57 is a graph showing flow cytometric analysis of surface TRAIL induced by TIC10 with or without stable knockdown of Foxo3a in HCT116 cells;</figref><figref num="58">FIG. 58 is a graph showing tumor volume of HCT116 xenografts with or without stable knockdown of Foxo3a after a single oral dose of vehicle or TIC10;</figref><figref num="59">Figure 59 shows the HCT116 p53 treated with TIC10 (2.5, 5, 10 μM) for 72 hours.<sup>-/-</sup>Image of Western blot analysis of cells;</figref><figref num="60">Figure 60 shows HCT116 p53 processed with TIC10.<sup>-/-</sup>Image of Western blot analysis of cells;</figref><figref num="61">FIG. 61 is a graph showing the time course of protein expression levels of TIC10-induced effects as measured by Western blot densitometry of repeated experiments as in FIG. 60;</figref><figref num="62">FIG. 62 is an image of a Western blot analysis of the effect induced by TIC10 on Foxo3a in DLD1 human colon cancer cells, MDA-MB-468 human breast cancer cells and T98G human polymorphic glioblastoma cell lines;</figref><figref num="63">FIG. 63 is an image of Western blot analysis showing overexpression of myr-Akt;</figref><figref num="64">FIG. 64 is a graph showing flow cytometric analysis of surface TRAIL in HCT116 cells overexpressing empty vector or myristilated Akt (myr-Akt) treated with TIC10;</figref><figref num="65">FIG. 65 is a graph showing the sub-G1 content of TIC10 treated, empty vector or myr-Akt overexpressing HCT116 cells;</figref><figref num="66">Figure 66 shows HCT116 p53 after incubation with A6730 (Akt inh), U0126 monoethanolate (MEK inh), or both.<sup>-/-</sup>It is a graph showing RT-qPCR analysis of TRAIL mRNA in cells;</figref><figref num="67">FIG. 67 is a graph showing the induction of surface TRAIL as in FIG. 66 with or without stable knockdown of Foxo3a;</figref><figref num="68">FIG. 68 is a graph showing sub-G1 analysis of MDA-MB-231 with or without TRAIL knockdown by shRNA after incubation with Akt inh, MEK inh or both;</figref><figref num="69">Figure 69 shows HCT116 p53 after incubation with A6730 (Akt inh), U0126 monoethanolate (MEK inh), or both.<sup>-/-</sup>It is a graph showing the surface TRAIL analysis of cells;</figref><figref num="70">Figure 70 shows HCT116 p53<sup>-/-</sup>Graph showing RT-qPCR analysis of TRAIL mRNA levels after transient knockdown of Akt and / or ERK in cells;</figref><figref num="71">Figure 71 is an image showing confirmation of Akt and ERK knockdowns by Western blot analysis;</figref><figref num="72">FIG. 72 is a graph showing surface TRAIL analysis after transient knockdown of Akt and / or ERK in HCT116 cells.</figref>
0032Detailed description of the invention The scientific and technical terms used herein are intended to have meanings commonly understood by those skilled in the art. Such terms have been found to be defined and used in the context of various standard references, such as J. Sambrook and DWRussell, exemplarily. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; FM Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; DL Nelson and MMCox, Lehninger Principles of Biochemistry, 4th Ed., WH Freeman & Company, 2004; Engelke, DR, RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A.Nagy, M.Gertsenstein, K .Vintersten, R. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; December 15, 2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002; 185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 9780470151808.
0033The singular terms "a", "an" and "the" are not intended to be limiting and indicate that something else is not explicitly stated or that the context is clearly different. Includes multiple referents unless otherwise noted.
0034Methods according to aspects of the invention because p53 is often inactivated in late stage cancers, which causes resistance to many standard-of-care therapies such as 5-FU and doxorubicin. And the composition relates to TRAIL-inducing compound 10 (TIC10) identified by us as a small molecule transcription inducer of the TRAIL gene by screening for TRAIL-inducing compounds that upregulate the TRAIL gene by a p53-independent mechanism.
0035TIC10 induces TRAIL expression in both normal and cancerous cells. Expression of the term "TRAIL" used herein to describe the effects of TIC10 or its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs. "Induce", "TRAIL induced by TIC10" and their grammatical equivalents are TIC10 or its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or pros. It refers to the fact that cells in contact with the drug result in a detectable increase in TRAIL. The detectable increase in TRAIL can be measured by assaying for TRAIL protein or TRAIL nucleic acid using well-known protein or nucleic acid assay methods.
0036TIC10-induced TRAIL is sustained in cancer cells and normal cells and sera, resulting in a TRAIL-mediated bystander effect on cancer cells and tumors. TIC10 inactivates Akt and ERK, leading to nuclear translocation of Foxo3a and induction of TRAIL transcription.
0037TIC10-induced TRAIL depends on Foxo3a, which upregulates the TRAIL death receptor DR5 among other targets, allowing sensitization of some TRAIL-resistant tumor cells. Induction of TRAIL caused by TIC10 is sustained in tumor cells, stromal cells and host cells.
0038According to aspects of the invention, pharmaceutical compositions containing TIC10 or pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs, and for use thereof. The method is provided.
0039According to aspects of the invention, a pharmaceutical composition comprising a compound of structure (I) is provided.
0040<chemistry num="2-1"><img id="000009" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry> Compounds of structure (I) are also referred to herein as TRAIL-induced compound 10 (TIC10) and NSC350625.
0041Compound (TIC10) of structure (I) can be obtained commercially or can be synthesized using standard chemical synthesis methods.
0042The pharmaceutical composition according to an aspect of the present invention can also be a pharmaceutically acceptable derivative, salt, ester, amide, hydrate, solvate and / or prodrug of the compound of structure (I).
0043Pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs of the compounds of structure (I) are commercially available or standard chemicals. It can be synthesized using a synthesis method.
0044The term "pharmaceutically acceptable derivative" used with respect to a compound of structure (I) is any substitution in which the compound of structure (I) substantially retains the indicated activity of inducing expression of TRAIL in the cell. It is a compound of structure (I) further substituted at possible positions. For example, compounds of structure (I) include: F, Cl, Br, lower alkyl groups, lower alkoxy groups or fluorinated lower alkyl groups (eg CF).<sub>3</sub>) Is further replaced as needed at any replaceable position.
0045"Pharmaceutically acceptable" salts, esters, amides, hydrates, prodrugs or solvates are suitable and intended for use in a subject without undue toxicity or irritation. It is effective for the usage to be done.
0046Pharmaceutically acceptable salts include pharmaceutically acceptable acid and base addition salts. Pharmaceutically acceptable salts are well known in the art and are, for example, the salts detailed in SMBerge et al., J. Pharm. Sci., 66: 1-19, 1977. An exemplary pharmaceutically acceptable salt is a salt that is suitable for use in a subject without undue toxicity or irritation to the subject and is effective for their intended use. These salts are inorganic acids (eg hydrochloric acid, hydrobromic acid, hydroiodic acid, nitrate, phosphoric acid, sulfuric acid and sulfamic acid); organic acids (eg acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid). , Benzensulfonic acid, benzoic acid, 2-acetoxybenzoic acid, butyric acid, gypsum acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, formic acid, fumaric acid, glutamate, glycolic acid, glycerophosphate Sulfate (hemisulfic) acid), heptanic acid, hexanoic acid, 2-hydroxyethanesulfonic acid (isethionic acid), lactic acid, maleic acid, hydroxymaleic acid, malic acid, malonic acid, mandelic acid, mesitylenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, Nicotinic acid, 2-naphthalene sulfonic acid, oxalic acid, pamoic acid, pectinic acid, phenylacetic acid, 3-phenylpropionic acid, picric acid, pivalic acid, propionic acid, pyruvate, pyruvate, salicylic acid, stearic acid, succinic acid, Sulfanilic acid, tartaric acid, p-toluenesulfonic acid, trichloroacetic acid, trifluoroacetic acid and undecanoic acid); inorganic bases (eg ammonia, hydroxides of ammonium, carbonates and hydrogen carbonates); organic bases (eg first Primary, secondary, tertiary and quaternary amine compounds, ammonium, arginine, betaine, choline, caffeine, diolamine, diethylamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, dicyclohexylamine , Dibenzylamine, N, N-dibenzylphenethylamine, 1-ephenamine, N, N'-dibenzylethylenediamine, ethanolamine, ethylamine, ethylenediamine, glucosamine, histidine, hydrabamine, isopropylamine, 1h-imidazole, lysine , Methylamine, N-ethylpiperidine, N-methylpiperidin, N-methylmorpholin, N, N-dimethylaniline, piperazin, trolamine, methylglucamine, purine, piperidine, pyridine, theobromine, tetramethylammonium compound, tetraethylammonium compound , Trimethylamine, triethylamine, tripropylamine and tributylamine) and metal cations (eg, aluminum, calcium, copper, iron, lithium, magnesium, manganese, potassium, sodium and zinc).
0047Examples of pharmaceutically acceptable solvates include hydrates, etanolates, and metanolates.
0048Exemplary pharmaceutically acceptable amides include amides obtained from ammonia, primary C1-C6 alkylamines and secondary C1-C6 dialkylamines (amides in the form of 5- or 6-membered nitrogen-containing heterocycles). Including).
0049The TIC10 prodrug is a form of TIC10 covalently attached to a moiety released from TIC10 that results in intact active TIC10. The forms of prodrugs are Sloan, KB, Prodrugs, M. Dekker, New York, 1992; and Testa, B. and Mayer, JM, Hydrolysis. It is well known in the art as illustrated in in drug and prodrug metabolism: chemistry, biochemistry, and enzymology, Wiley-VCH, Zurich, 2003.
0050As the first remedy
0051<chemistry num="3-1"><img id="000010" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; pharmaceutically acceptable carriers; and pharmaceuticals including anti-cancer agents and other second therapeutic agents. The composition is provided.
0052According to aspects of the invention, a method of treating a subject in need of treatment is provided, the method of which is in a pharmaceutically effective amount:
0053<chemistry num="4-1"><img id="000011" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; and administration of pharmaceutically acceptable carriers.
0054A method of treating a subject in need of treatment is provided, the method of which is in a pharmaceutically effective amount effective in inducing the expression of TRAIL in that subject.
0055<chemistry num="5"><img id="000012" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; and administration of pharmaceutically acceptable carriers.
0056By assaying the TRAIL protein in a test sample obtained from a subject, TIC10-induced expression of TRAIL can be detected.
0057Enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELIFA), flow cytometry, immunoblot, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescence immunoassay (LIA), fluorescence immunoassay (FIA) and radioimmunoassay Immunoassay methods, including, but not limited to, can be used to assay TRAIL in a sample. Assay methods can be used to obtain qualitative and / or quantitative results. Specific details of assay methods suitable for both qualitative and quantitative assay of samples can be found in standard references, such as E. Harlow, exemplary. and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling and S. Duebel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H. Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Basics: From Background to Bench, BIOS Scientific Publishers, 2000; BKCLo, Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; FM Ausubel et al., Eds. , Short Protocols in Molecular Biology, Current Protocols, Wiley, 2002; S. Klussman, Ed., The Aptamer Handbook: Functional Oligonucleotides and Their Applications, Wiley, 2006; Ormerod, MG, Flow Cytometry: a practical approach, Oxford University Press, 2000; Givan, AL, Flow Cytometry: first principles, Wiley, New York, 2001; Gorczyca, W., Flow Cytometry in Neoplastic Hematology: morphologic-immunophenotypic correlation, Taylor & Francis, 2006 Crowther, JR, The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000; Wild, D., The Immunoassay Handbook, 3rd Edition, Elsevier Science, 2005 and J. Sambrook and DWRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Ed., 2001.
0058Aptamers can be used to assay samples for TRAIL. The term "aptamer" refers to a peptide and / or nucleic acid that binds substantially specifically to a particular substance. In the case of nucleic acid aptamers, the aptamers are characterized by binding interactions with targets other than Watson / Crick base pairing or triple helix binding with second and / or third nucleic acids. Such bond interactions may include, for example, van der Waals interactions, hydrophobic interactions, hydrogen bonds and / or electrostatic interactions. Similarly, peptide-based aptamers are characterized by specific binding to a target, where the aptamer is not a naturally occurring ligand for that target. Techniques for the identification and production of peptide and nucleic acid aptamers and their use are described, for example, in FMAusubel et al., Eds., Short Protocols in Molecular Biology, Current Protocols, Wiley, 2002; S. Klussman, Ed., The Aptamer. Handbook: Functional Oligonucleotides and Their Applications, Wiley, 2006; and J. Sambrook and DWRussell, Molecular Cloning: A It is known in the art as described in Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Ed., 2001.
0059Spectroscopic analysis is used to assay the sample for TRAIL. For example, mass spectrometry can be used in assays according to aspects of the invention. Mass spectrometry is performed using, for example, time-of-flight (TOF) mass spectrometry or Fourier transform ion cyclotron resonance mass spectrometry. Mass spectrometric techniques are known in the art, and an exemplary detailed description of methods for protein and / or peptide assays can be found in Li J. et al., Clin Chem., 48 (8): 1296-304, 2002; Hortin, GL, Clinical Chemistry 52: 1223-1237, 2006; Hortin, GL, Clinical Chemistry 52: 1223-1237, 2006; ALBurlingame et al. (Eds.), Mass Spectrometry in Biology and Medicine, Humana Press, 2000; and DM Desiderio, Mass Spectrometry of Found in Peptides, CRC Press, 1990.
0060By assaying the localization of TRAIL on the surface of cells, the effects of the pharmaceutical compositions of the present invention can be detected. Detection of TRAIL localization can be performed by immunoassays such as flow cytometry as well as immunohistochemistry.
0061The test sample is any biological fluid, cell or tissue of the subject (eg, blood, plasma, serum, urine, saliva, ascites, cerebrospinal fluid, ventricular fluid, pleural fluid, lung and bronchi. Alveolar lavage fluid sample, mucus, sweat, tears, semen, bladder lavage sample, sheep's water, lymph, ascites (peritoneal fluid), synovial fluid, bone marrow puncture, tumor cells or tissues, organ cells or tissues (eg, biopsy material) Including). In a preferred embodiment, the test sample is blood, plasma or serum.
0062Subject-derived test samples are optionally purified for assaying TRAIL or other biomarkers. The term "purified" in the context of a test sample refers to the separation of TRAIL or another biomarker from at least one other component present in the test sample. Purification of test samples is exemplified by electrophoresis methods (eg, gel electrophoresis and 2-D gel electrophoresis); chromatography methods (eg, HPLC, ion exchange chromatography, affinity chromatography, size exclusion chromatography, etc.) Achieved by techniques involving thin layers and paper chromatography).
0063The TRAIL assay can be performed on cells and tissues. For example, immunohistochemical methods and in situ hybridization can be used to assay TRAIL proteins and / or nucleic acids in cell or tissue test samples.
0064One or more criteria can be used to allow quantitative measurement of TRAIL in a sample.
0065The TRAIL assay in the test sample can be compared to the TRAIL assay in the control sample. Control samples may be obtained, for example, from one or more normal subjects.
0066According to aspects of the invention, assays for TRAIL are used to monitor subjects. Thus, for example, test samples are obtained from a subject prior to treatment with the pharmaceutical compositions of the invention and at one or more time points during and / or after treatment to assess the effectiveness of the treatment. .. In a further example, test samples are obtained from the subject at various time points to assess the course or progression or cure of the disease.
0067In certain embodiments, one or more additional biomarkers are assayed in test samples obtained from the subject to aid in monitoring treatment with the pharmaceutical compositions of the invention. For example, a test sample obtained from a subject in which one or more of the localization and / or phosphorylation of phospho-ERK, phospho-Akt, Foxo3a aids in monitoring treatment with the pharmaceutical compositions of the present invention. Assayed at. Such additional biomarkers are assayed by immunoassay methods such as those described herein.
0068TRAIL nucleic acids can be assayed in test samples obtained from subjects to detect expression of TIC10-induced TRAIL. Assays for detecting TRAIL nucleic acids, especially mRNA or cDNA, include, but are limited to, polymerase chain reaction (PCR) (eg, RT-PCR), dot blots, in situ hybridization, Northern blots and RNase protection. Not done.
0069According to the present invention, methods and compositions for treating cancer are provided.
0070According to aspects of the invention, a method of treating a subject who has or is at risk of having cancer is provided, the method of which is in a pharmaceutically effective amount.
0071<chemistry num="6"><img id="000013" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; and administration of pharmaceutically acceptable carriers.
0072A method of treating a subject who has or is at risk of having cancer is provided, and the method is in a pharmaceutically effective amount effective in inducing the expression of TRAIL in that subject.
0073<chemistry num="7"><img id="000014" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; and administration of pharmaceutically acceptable carriers.
0074Cancers treated using the methods and compositions described herein are aberrant cell proliferation, including but not limited to pre-neoplastic hyperproliferation, carcinoma in situ, neoplasms and metastases. Characterized by. The methods and compositions of the present invention can be used for the prevention and recovery of signs and / or symptoms of cancer. The terms "treat" and "treatment" used to refer to the treatment of cancer in a subject include the prevention, inhibition, or recovery of cancer in that subject (eg, cancer progression). And / or reducing or ameliorating the signs or symptoms of cancer).
0075A pharmaceutically effective amount of the composition of the invention is an amount that has a beneficial effect on the subject being treated. Due to pre-neoplastic hyperproliferation, carcinoma in situ, neoplasms, metastases, tumors, benign growth, or abnormal cell proliferation including, but not limited to, other conditions responsive to the compositions of the invention. In a subject who has or is at risk of having cancer, such as a condition characterized, a pharmaceutically effective amount of the composition of the invention ameliorate one or more signs and / or symptoms of that condition. Or effective for prevention. For example, pharmaceutically effective amounts of compositions of the invention increase apoptosis to a detectable extent and / or abnormal cell proliferation (pre-neoplastic hyperproliferation, carcinoma in situ, neoplasm, metastasis, etc.) It is effective in reducing cell proliferation of cancerous conditions characterized by tumors, benign growth, or other conditions that are responsive to the compositions of the invention, but not limited to these.
0076TIC10 has the broader activity described herein in primary patient samples and cell lines that are resistant to conventional treatments, which means that the therapeutic effects of TIC10 usually change in cancer. It suggests that it is exclusively independent of the molecule (eg, EGFR, Her2, KRAS or PTEN). Elucidation of the therapeutic cellular mechanism of TIC10 identifies the resistance mechanism described herein (eg, overactivated Akt) and serves as a correlated biomarker of therapeutic activity of TIC10 in cancer. , Phosphor-ERK, phospho-Akt, Foxo3a localization and phosphorylation, as well as surface and serum TRAIL.
0077Therefore, according to aspects of the invention, one or more correlated biomarkers of TIC10 therapeutic activity in cancer are assayed to evaluate treatment with the pharmaceutical compositions of the invention.
0078Subjects treated according to the methods of the invention and with the compositions of the invention can be mammalian or non-mammalian. The mammalian subject can be any mammal, such as humans; non-human primates; rodents (eg, mice, rats or guinea pigs); domesticated pets (eg, cats or dogs); Examples include, but are not limited to, horses, cows, pigs, sheep, goats or rabbits. The non-mammal subject can be any non-mammal, including, but not limited to, birds (eg, ducks, geese, chickens or turkeys). The subject may be of any gender and of any age. In an aspect of a method comprising administering to a subject the pharmaceutical composition of the present invention, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.
0079Pharmaceutical compositions according to the invention typically contain approximately 0.1-99% TIC10, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs; and pharmaceuticals. Includes carriers that are acceptable. Combinations of TIC10 in pharmaceutical compositions with at least one pharmaceutically acceptable derivative, salt, ester, amide, hydrate, solvate and / or prodrug thereof are also within the scope of the invention. It is believed that there is. Furthermore, combinations of at least two of its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and prodrugs in pharmaceutical compositions are also within the scope of the invention. Conceivable.
0080According to aspects of the invention, a combination of therapeutic agents is administered. According to aspects of the invention, methods of treating cancer in a subject include TIC10, a pharmaceutically acceptable derivative thereof, salts, esters, amides, hydrates, solvates and / or prodrugs; and at least one. Includes administration of pharmaceutical compositions of two additional therapeutic agents. According to aspects of the invention, methods of treating cancer in a subject include TIC10, a pharmaceutically acceptable derivative thereof, salts, esters, amides, hydrates, solvates and / or prodrugs; and at least 2 Includes administration of pharmaceutical compositions of two additional therapeutic agents.
0081The term "additional therapeutic agent" refers to a chemical, a mixture of chemicals, a biological polymer (eg, nucleic acid, antibody, protein or part thereof, eg, peptide), or locally or systemically in a subject. Prepared from biological materials (eg, cells or tissues of bacteria, plants, fungi or animals (particularly mammals)) that are biologically, physiologically or pharmacologically active substances (s) that act. As used herein to refer to an extract.
0082Additional therapeutic agents included according to aspects of the methods and compositions of the invention include antibiotics, antiviral agents, anti-neoplastic agents, analgesics, antipyretics, antidepressants, anti-psychiatric agents, anti-cancer agents, anti-histamines, anti-hysterinics Examples include osteoporosis agents, anti-osteone crosis agents, anti-inflammatory agents, anti-anxiety agents, chemotherapeutic agents, diuretics, growth factors, hormones, non-steroidal anti-inflammatory drugs, steroids and vasoactive agents. , Not limited to these.
0083Combination therapy with TIC10, its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs and one or more additional therapeutic agents may be synergistic. , For example, the present invention comprising TIC10 as a monotherapy alone, its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs or one or more additional therapeutic agents. Can exhibit a therapeutic effect greater than the effect that can be observed when using the pharmaceutical compositions of.
0084According to aspects, the combination therapy is (1) formulated together as a single composition with one or more additional therapeutic agents, the TIC10 of the invention, a pharmaceutically acceptable derivative thereof, a salt, an ester, and the like. Pharmaceutical compositions comprising pharmaceutical compositions containing amides, hydrates, solvates and / or prodrugs; and (2) TIC10 of the invention, pharmaceutically acceptable derivatives thereof, salts, esters, amides thereof. Co-administration of a pharmaceutical composition comprising a hydrate, solvate and / or prodrug with one or more additional therapeutic agents (where, TIC10 of the invention, a pharmaceutically acceptable derivative thereof, A pharmaceutical composition comprising a salt, ester, amide, hydrate, solvate and / or prodrug and one or more additional therapeutic agents (not formulated in the same composition). When using separate formulations, one or more pharmaceutical compositions comprising TIC10 of the invention, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs. Administration of one or more additional therapeutic agents at the same time, at intermittent times, at staggered times, prior to administration of the one or more additional therapeutic agents. Can be administered after or in combination thereof.
0085Combination treatments include TIC10 of the invention, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs and one or more additions used in the methods of the invention. It may be possible to reduce the effective dose of the pharmaceutical composition comprising the therapeutic agent of and increase the therapeutic index.
0086According to aspects, the combination therapy is (1) formulated together as a single composition with one or more additional anti-cancer agents, the TIC10 of the invention, pharmaceutically acceptable derivatives thereof, salts, esters, amides thereof. , A pharmaceutical composition comprising a pharmaceutical composition comprising a hydrate, a solvate and / or a prodrug; and (2) the TIC10 of the invention, a pharmaceutically acceptable derivative thereof, salt, ester, amide, Co-administration of a pharmaceutical composition comprising a hydrate, solvate and / or prodrug with one or more additional anti-cancer agents (where TIC10 of the invention, a pharmaceutically acceptable derivative thereof, salt, A pharmaceutical composition comprising an ester, amide, hydrate, solvate and / or prodrug and one or more additional therapeutic agents (not formulated in the same composition). When using separate formulations, one or more pharmaceutical compositions comprising the TIC10 of the invention, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates, solvates and / or prodrugs. At the same time, at intermittent times, at staggered times, before the administration of the one or more additional anti-cancer agents, and after the administration of the one or more additional anti-cancer agents, with respect to the administration of the additional anti-cancer agents. Or in combination thereof, it can be administered.
0087Antineoplastic agents are described, for example, in Goodman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
0088Examples of anti-cancer agents include ashibicin, acralubicin, acodazole, acronine, adzelesin, aldesroykin, alitretinoin, alloprinol, alretamine, ambomycin, amethanthrone, and amyhostin. (amifostine), aminoglutetimide, amsacrine, anastrosol, anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa , Bebasizumab, bicartamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin, blecinal, bropyrimin, busulfan, cactinomycin, carsterone, capecitabine, cladribine, carsterone, capecitabine, carasemido, carvetimer, carboplatin, carmustine Carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylatemesylate), cyclophosphamide, citarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, dexormaplatin, dezaguanin, dezaguanine mesylate, diazicon, docetaxel, doxorubicin, droloxyphene, dromostanolone (duazomycin), edatrexate, eflomithine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, erbulozole, esorubicin (esorubicin) , Ethanidazole, etopocid, etoprine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabin, fluorouracil, flurocitabine, flurocitabine, hosquidone, hosteriecin , Idalbisin, iphosphamide, ilmofosine, interferon II (IL-2 containing recombinant interferon II or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta -Ia, interferon gamma-Ib, iproplatin, irinotecan, lanleotide, retrosol, leuprolide, liarozole, lometrexol, romustin, losoxantrone, masoprocol, masoprocolnehydrochlride), megestol, melengestrol acetate, melphalan, menogalil, mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitocromin (mitogillin), mitomalcin, mitomycin, mitosper, mittane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel, pe Gaspargase, peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promethane, plomestane, porphimer Porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, riboprine, rogletimide, safingol, semstrazene, simtrazene, sparphosate Sparsomycin, spirogermanium, spiromustine, spiroplatin, streptnigrin, streptozosine, sulofenur, talisomycin, tamoxyphene, tecogalan, tegafur, teloxantroneTeloxantrone, temoporfin, teniposide, teroxirone, test lactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamin, topotecan, tremiphen, trestolone Trimetrexate, triptorelin, tubulozole, urasyl mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglycinate (Vinleurosine), vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, dinoplatin, zoledronate and sorbicin.Examples include vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, dinostatin, zoledronic acid and sorbicin.Examples include vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, dinostatin, zoledronic acid and sorbicin.
0089Pharmaceutical composition containing TIC10 and one or more additional anti-cancer agents (eg, one or more mitotic inhibitors)<u style="single">Agent</u>And / or the synergistic effect of concomitant treatment with one or more anti-angiogenic agents) has unexpectedly been found as described herein.
0090According to aspects of the invention, methods of treating subjects with or at risk of having cancer are therapeutically effective amounts of TIC10, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates. , Solvates and / or prodrugs; and mitotic inhibition<u style="single">Agent</u>Includes administration of.
0091According to aspects of the invention, methods of treating subjects with or at risk of having cancer are therapeutically effective amounts of TIC10, pharmaceutically acceptable derivatives thereof, salts, esters, amides, hydrates. , Solvates and / or prodrugs; and taxane mitosis inhibition<u style="single">Agent</u>Includes administration of (eg, but not limited to paclitaxel and docetaxel).
0092According to aspects of the invention, the method of treating a subject with or at risk of having cancer is a therapeutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, salt, ester, amide, hydrate. , Solvates and / or prodrugs; and administration of anti-angiogenic agents.
0093According to aspects of the invention, the method of treating a subject with or at risk of having cancer is a therapeutically effective amount of TIC10, a pharmaceutically acceptable derivative thereof, salt, ester, amide, hydrate. , Solvates and / or prodrugs; as well as administration of anti-angiogenic agents (eg, but not limited to bevacizumab).
0094Compositions of the Invention In certain embodiments, the amount of co-antineoplastic agent administered is a therapeutically effective amount of the structure (I), a pharmaceutically acceptable derivative thereof, a salt, an ester, an amide, a hydrate, a solvate. And / or less than the amount of adjuvant anti-cancer agent required to achieve a therapeutic effect when administered without administration of a prodrug. Thus, in certain aspects of the compositions of the invention, the amount of adjuvant anti-cancer agent in a unit dose of the composition is a therapeutically effective amount of structure (I), a pharmaceutically acceptable derivative thereof, salt, ester, amide, and the like. At least 5%, at least 10%, at least 15%, at least 20%, at least more than the amount of adjuvant anti-cancer agent required to achieve a therapeutic effect when administered without hydrates, solvates and / or prodrugs 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% Few.
0095According to aspects of the invention, TRAIL, in addition to administration of TIC10, to one or more histone deacetylase (HDAC) inhibitors (eg, Nebbioso, A. et al., 2005, Nat Med 11,77-84). Vorinostat described; one or more TRAIL agonist antibodies (eg, lexatumumab and mapatumumab); and / or recombinant TRAIL (eg, Abdulghani, J. et al., 2010, Exp. It can be induced or provided by methods or compositions such as administration of adenovirus TRAIL) as described in Opin.Ther.Targets 14: 1091-1108.
0096If necessary, methods of treating a subject who has or is at risk of having cancer further include adjuvant anti-cancer treatment. The adjuvant anti-cancer treatment can be a radiation treatment of the subject or the affected area of the subject's body.
0097TRAIL expression induced by a subject by administration of the pharmaceutical composition of the invention is detectable in a sample obtained from that subject (eg, a blood sample obtained from that subject).
0098Aspects of the invention include upregulation of the TRAIL gene by normal and tumor tissues in which serum levels of secreted TRAIL are sustained for 3-4 days after a single dose of TIC10. Usually, the serum half-life of TRAIL protein is 20-30 minutes.
0099TIC10 has a mass calculation value of 387.21 and crosses the blood-brain barrier. Administration of TIC10 allows the induction of TRAIL in cells of the central nervous system, including, for example, glial cells and neurons of the brain and spinal cord. In addition, administration of pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates or prodrugs of TIC10 across the blood-brain barrier allows the induction of TRAIL in cells of the central nervous system. ..
0100According to aspects of the invention, there is provided a method of treating a subject who has or is at risk of having cancer of the central nervous system (CNS), the method of which is by route of administration other than direct administration to the CNS. A pharmaceutically effective amount:
0101<chemistry num="8"><img id="000015" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; and administration of pharmaceutically acceptable carriers.
0102According to aspects of the invention, CNS metastases (also referred to herein as brain tumors) of primary and non-CNS cancers are treated. Primary CNS cancers treated according to aspects of the invention include, but are not limited to, gliomas, meningiomas, pituitary adenomas and schwannomas. Polymorphic glioblastoma is a primary CNS cancer treated according to aspects of the invention. Oligodendroglioma is a primary CNS cancer treated according to aspects of the invention.
0103The method of the present invention is oral, rectal, nose, lung, epidural, eyeball, ear, intraarterial, intracardiac, intraventricular, intradermal, intravenous, intramuscular, intraperitoneal, intraosseous, intrathecal. Administration of the pharmaceutical compositions of the invention by routes of administration including, but not limited to, intravesical, subcutaneous, topical, transdermal and transmucosal (eg, sublingual, buccal, vaginal and inhalation). Including.
0104According to aspects of the invention, a method of treating a subject in need of treatment is provided, the method of which is in a pharmaceutically effective amount formulated for oral administration:
0105<chemistry num="9"><img id="000016" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>Includes oral administration of pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs thereof.
0106The pharmaceutical compositions of the present invention are in any dosage form suitable for administration to a subject (eg, solid, semi-solid and liquid dosage forms (eg, tablets, capsules, powders, granules, suppositories). , Suppositories, solutions, suspensions, ointments, lotions, creams, gels, pastes, sprays and aerosols). Liposomes and emulsions are well-known types of pharmaceutical formulations that can be used to deliver pharmaceuticals, especially hydrophobic pharmaceuticals. The pharmaceutical compositions of the present invention typically include pharmaceutically acceptable carriers (eg, excipients, diluents and / or vehicles). A delayed release formulation of the composition and a delayed release system (eg, a semipermeable matrix of solid hydrophobic polymers) can be used.
0107The pharmaceutical preparation of the composition of the present invention may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is suitable for use in a subject without undue toxicity or irritation to the subject and with other ingredients contained in the pharmaceutical composition. Refers to a carrier that is compatible.
0108Pharmaceutically acceptable carriers, methods for preparing pharmaceutical compositions and various dosage forms, and modes of administration are described, for example, in Pharmaceutical Dosage Forms: Tablets, eds. HALieberman et al., New York: Marcel Dekker, Inc. ., 1989; and LV Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, PA: Lippincott, Williams & Wilkins, 2004; ARGennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed., 2005, especially chapter 89; and JG Hardman et al., Goodman & Gilman's The Pharmacological Basis of It is well known in the art as detailed in Therapeutics, McGraw-Hill Professional, 10th ed., 2001.
0109The pharmaceutical composition according to an aspect of the present invention is formulated for oral administration.
0110Solid dosage forms for administration or solid dosage forms for suspension in liquid prior to administration include, exemplary, capsules, tablets, powders and granules. In such solid dosage forms, one or more active substances are at least one carrier (eg, a buffer (eg, sodium citrate or alkali metal phosphate (eg, sodium phosphate)). , Potassium phosphate and calcium phosphate)); Fillers (eg starch, lactose, sucrose, glucose, mannitol and silicic acid); Binding agents (eg carboxymethyl cellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and Acacia); Moisturizer (eg glycerol); Disintegrant (eg agar, calcium carbonate, vegetable starch (eg potato or tapioca starch), alginic acid, certain complex silicates and sodium carbonate); Dissolution retarder (eg) , Paraffin); Absorption enhancer (eg, quaternary ammonium compound); Wetting agent (eg, cetyl alcohol, glycerol monostearate and glycol); Adsorbent (eg, kaolin and bentonite); Lubricating agent (eg, talc, Calcium stearate, magnesium stearate, solid polyethylene glycol or sodium lauryl sulfate); preservatives (eg, antibacterial and antifungal agents (including, for example, sorbic acid, gentamycin and phenol)); and stabilizers (eg, sucrose, EDTA). , EGTA and antioxidants) are mixed).
0111The solid dosage form optionally includes a coating such as an enteric coating. The enteric coating is typically a polymeric material. Preferred enteric coating materials are characterized by biodegradable, gradual hydrolyzable and / or gradual water soluble polymers. The amount of coating material applied to the solid formulation generally affects the time interval between ingestion and drug release. A coating is applied that is thick enough that the entire coating is insoluble in gastric acid-related gastrointestinal fluids with a pH of less than 3, but is soluble above pH 3 in the environment of the small intestine. Any anionic polymer that exhibits a pH-dependent solubility profile is expected to be readily used as an enteric coating in the practice of the present invention to deliver the active agent to the lower gastrointestinal tract. The choice of specific enteric coating material has properties (eg, resistance to gastric disintegration; impermeability to gastric juice and diffusion of active substances during their presence in the stomach; ability to dissipate in the target intestinal region; during storage Depends on physical and chemical stability; non-toxicity; and ease of application).
0112Suitable enteric coating materials include, for example, cellulose polymers (eg, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl). Methyl cellulose phthalate, hydroxypropyl methyl cellulose succinate and sodium carboxymethyl cellulose); acrylic acid polymers and acrylic acid copolymers, preferably acrylic acid, methacrylic acid, methyl acrylate, ammonium methyl acrylate, ethyl acrylate, methyl methacrylate and / Or those formed from ethyl; vinyl polymers and vinyl copolymers (eg, polyvinylpyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinyl crotonic acid acetate copolymers and ethylene-vinyl acetate copolymers); Shelac; and The combination thereof can be mentioned. Specific enteric coating materials include, for example, acrylic acid polymers and acrylic acid copolymers described in US Pat. No. 6,136,345.
0113The enteric coating optionally contains a plasticizer that prevents the formation of pores and cracks that allow gastric juice to penetrate into the solid dosage form. Suitable plasticizers are, for example, triethyl citrate (Citroflex2), triacetin (glyceryl triacetate), acetyltriethyl citrate (Citroflec). A2), Carbowax400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglyceride, glycerol, fatty acid ester, propylene glycol and dibutyl phthalate. In particular, coatings composed of anionic carboxyacrylic polymers typically contain approximately 10% to 25% by weight of plasticizers, in particular dibutyl phthalate, polyethylene glycol, triethyl citrate and triacetin. The coating is used to solubilize or disperse the coating material, as well as to improve coating performance and coated products, such as other coating excipients (eg, detackifiers, defoaming). Agents, lubricants (eg magnesium stearate) and stabilizers (eg hydroxypropyl cellulose, acids or bases) may also be included.
0114Liquid dosage forms for oral administration include one or more active substances and pharmaceutically acceptable carriers, which are formulated as emulsions, solutions, suspensions, syrups or elixirs. Liquid dosage forms of the compositions of the present invention may include colorants, stabilizers, wetting agents, emulsifiers, suspending agents, sweeteners, flavors or fragrances.
0115For example, a composition for parenteral administration can be formulated as an injectable liquid. Examples of suitable aqueous and non-aqueous carriers are water, ethanol, polyols (eg, propylene glycol, polyethylene glycol, glycerol, etc.), suitable mixtures thereof; vegetable oils such as olive oil; and injectable such as ethyl oleate. Organic ester. Proper fluidity is maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion and / or by using a surfactant such as sodium lauryl sulfate. Can be done. Stabilizers (eg, sucrose, EDTA, EGTA and antioxidants) are included as needed.
0116For topical administration, the composition can be formulated for administration to the skin for topical effects and / or as a "patch" formulation for transdermal delivery. Pharmaceutical formulations suitable for topical administration include, for example, ointments, lotions, creams, gels, pastes, sprays and powders. Ointments, lotions, creams, gels and pastes, in addition to one or more active substances, are bases (eg, absorbent bases, water-removable bases, water-soluble bases or oil-based bases) and excipients. Agents (eg, thickeners, gelling agents, colorants, stabilizers, emulsifiers, suspending agents, sweeteners, flavors or fragrances) may be included.
0117The transdermal preparation may contain a transdermal absorption enhancer (eg, acetone, azone, dimethylacetamide, dimethylformamide, dimethyl sulfoxide, ethanol, oleic acid, polyethylene glycol, propylene glycol and sodium lauryl sulfate). Percutaneous delivery can be facilitated by using iontophoresis and / or sonophoresis.
0118Powders and sprays for topical administration of one or more active substances may contain excipients (eg, talc, lactose and one or more silicic acids). The spray may include a pharmaceutical spray (eg, a fluorinated hydrocarbon spray, carbon dioxide or a suitable gas). Alternatively, the spray can be delivered from a pump-type spray device that does not require a spray. The spraying device uses, for example, a valve for controlling the delivery amount to deliver the quantitative composition contained therein.
0119An ophthalmic (Opthalmic) preparation of one or more active substances may contain ingredients such as preservatives, buffers and thickeners.
0120Suitable surfactants useful as pharmaceutically acceptable carriers or excipients in the pharmaceutical compositions of the present invention are nonionic, cationic and / or nonionic, cationic and / or having good emulsifying, dispersing and / or wetting properties. Alternatively, an anionic surfactant may be mentioned. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surface active agents. Suitable soaps are natural available from alkali metal or alkaline earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), such as oleic acid or stearic acid, or coconut oil or tallow oil. It is a sodium salt or a potassium salt of a fatty acid mixture of. Synthetic surfactants include sodium or calcium salts of polyacrylic acid; fatty sulfonates and sulfates; Benzimidazole derivatives and alkylaryl sulfonates are included. Fat sulfonates and sulfates are usually alkali metal or alkaline earth metal salts, unsubstituted ammonium salts, or alkyl or acyl radical substituted ammonium salts having 8 to 22 carbon atoms, such as lignosulfonic acid or Sodium or calcium salts of dodecyl sulfonic acid, or mixtures of fatty alcohol sulfates obtained from natural fatty acids, alkali metal salts or alkaline earth metal salts of sulfate or sulfonic acid esters (eg, sodium lauryl sulfate) and fatty alcohols. / It is in the form of sulfonic acid as an ethylene oxide adduct. Suitable sulfonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylaryl sulfonates are dodecylbenzene sulfonic acid or dibutyl-naphthalene sulfonic acid or naphthalene-sulfonic acid / formaldehyde condensate sodium salt, calcium salt or alkanolamine salt. Corresponding phosphates, such as salts of phosphate esters, as well as adducts or phospholipids of p-nonylphenol with ethylene and / or propylene oxide are also suitable. Suitable phospholipids for this purpose are natural phospholipids (originating from animal or plant cells) or synthetic phospholipids of the kephalin or lecithin type (eg, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerin, lysolecithin, etc.) Cardiolipin, dioctanylphosphatidylcholine, dipalmitoylphoshatidyl-choliner and mixtures thereof.
0121Suitable nonionic surfactants useful as pharmaceutically acceptable carriers or excipients in the pharmaceutical compositions of the present invention include alkylphenols, aliphatic alcohols, fatty acids, and at least 12 carbon atoms in the molecule. Polyethoxylated and polypropoxylated derivatives of aliphatic amines or amides, alkylarene sulfonates and dialkylsulfosuccinates (eg, polyglycol ether derivatives of aliphatic and alicyclic alcohols, saturated and unsaturated fatty acids and alkylphenols). (Preferably, the derivative contains 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol). Be done. More suitable nonionic surface active substances are polyethylene oxide and poylypropylene glycol, a water-soluble adduct of ethylenediaminopolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain (these adducts are: Contains 20-250 ethylene glycol ether groups and / or 10-100 propylene glycol ether groups). Such compounds typically contain 1-5 ethylene glycol units per propylene glycol unit. Representative examples of nonionic surfactants are nonylphenol-polyethoxyethanol, castor oil polyglycol ethers, polypropylene / polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (eg, polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable nonionic surfactants.
0122Suitable cationic surfactants useful as pharmaceutically acceptable carriers or excipients in the pharmaceutical compositions of the present invention include quaternary ammonium salts, preferably halos, phenyls, substituted phenyls as needed. Or a halide having four hydrocarbon radicals substituted with hydroxy; for example, at least one C8-C22 alkyl radical as an N-substituent (eg, cetyl, lauryl, palmityl, myristyl, oleyl, etc.), and further substitutions. Examples include quaternary ammonium salts containing unsubstituted or halide lower alkyl, benzyl and / or hydroxy-lower alkyl radicals.
0123More detailed descriptions of surfactants suitable for this purpose are described, for example, in "McCutcheon's Detergents and Emulsifiers Annual" (MC Publishing Crop., Ridgewood, New Jersey, 1981), "Tensid-Taschenbuch", 2nd ed. (Hanser Verlag). , Vienna, 1981) and Encyclopaedia of Surfactants (Chemical Publishing Co., New It may be found in York, 1981).
0124Structure-forming agents, thickeners or gel-forming agents can be included in the pharmaceutical compositions and combination preparations of the present invention. Suitable such substances are, in particular, highly dispersed silicic acid (eg, commercially available product under the trade name Aerosil); bentonite; tetraalkylammonium salt of montmorillonite (eg, commercially available under the trade name Bentone). Available products) (each of those alkyl groups may contain 1-20 carbon atoms); in cetostearyl alcohols and processed castor oil products (eg, commercially available products under the trade name Antisettle). is there.
0125In certain embodiments, the pharmaceutically acceptable carriers are lipid particles containing granular carriers (eg, liposomes, micelles, monolayer or multilamellar vesicles; polymer particles (eg, hydrogel particles, polyglycolic acid particles or). Polylactic acid particles); inorganic particles (eg, calcium phosphate particles, such as those described in US Pat. No. 5,648,097); and granular inorganic / organic carriers (eg, as described in US Pat. No. 6,630,486). It is)).
0126Granular pharmaceutically acceptable carriers can be selected from lipid particles; polymer particles; inorganic particles; and inorganic / organic particles. Particle-type mixtures can also be included as granular pharmaceutically acceptable carriers.
0127Granular carriers are typically formulated so that the particles have an average particle size in the range of about 1 nm to 10 microns. In certain embodiments, the granular carriers are formulated such that the particles have an average particle size in the range of about 1 nm to 100 nm.
0128The dosage of the pharmaceutical composition of the present invention is a factor (eg, route of administration; age, health, gender and weight of the subject to whom the composition is administered; the nature and extent of the subject's symptoms, if any. And can vary based on (but not limited to) the desired effect. The dose can be adjusted depending on whether the treatment is short-term or continuous. One of ordinary skill in the art can determine a pharmaceutically effective amount in light of these and other considerations specific to medical practice.
0129Generally, the daily dose of the pharmaceutical composition of the present invention is contemplated to range from about 0.001 to 100 milligrams per kilogram of subject body weight. The daily dose may be administered in two or more divided doses to obtain the desired effect. The pharmaceutical compositions of the present invention can also be formulated for sustained release to obtain the desired effect.
0130For more information on the usual ingredients, equipment and processes for preparing dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. HALieberman et al., New York: Marcel Dekker, Inc., 1989; and LV Allen, Jr. et al., Ansel's. Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, PA: Lippincott, Williams & Wilkins, 2004; ARGennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed ,, 2005, especially chapter 89; and JG Hardman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Found in Professional, 10th ed., 2001.
0131Commercial packages according to aspects of the invention include the pharmaceutical compositions described herein. According to aspects of the invention, instructions for administering the pharmaceutical composition are included.
0132According to aspects of the invention, the commercial packaging
0133<chemistry num="10"><img id="000017" he="46" wi="114" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></chemistry>, Its pharmaceutically acceptable derivatives, salts, esters, amides, hydrates, solvates and / or prodrugs; and pharmaceutically acceptable carriers.
0134One or more auxiliary components (eg, buffers or diluents) are optionally included in the commercial packaging of the present invention.
0135Aspects of the compositions and methods of the invention are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limiting to the scope of the compositions and methods of the invention.
<p num="0136"> Reagents and cell-based assays HCT116 Bax from Bert Vogelstein (Johns Hopkins University, Baltimore, MA)<sup>-/-</sup>And HCT116 p53<sup>-/-</sup>Cells and Akiva Mintz (Wake Forrest) All cell lines except GBM cell lines from University, Winston-Salem, NC) were obtained from ATCC. Lentivirus infection was performed using MDA-MB-231 cells using TRAIL shRNA or vector and using HCT116 using Foxo3a shRNA or vector purchased from Sigma-Aldrich (St. Louis, MO). It was. H460 by inserting amino acids 1-298 of the human DR5 gene into the pEGFP-N1 vector expressing the DR5 (1-298) fusion protein using cDNA encoding a death domain-free DR5 fragment. DR5ΔDD-EGFP cells were constructed. The fusion construct was transfected into H460 cells with Lipofectamine 2000 (Invitrogen) and selected with G418. Positive clones were confirmed by fluorescence microscopy and Western blot analysis. Bioluminescent high-throughput screening using NCI diversity set II was performed stably to express the firefly luciferase construct under the transcriptional regulation of the first 504 base pairs of the TRAIL promoter upstream of the transcription initiation of the human TRAIL gene. Perfect HCT116 Bax<sup>-/-</sup>Performed in cells. Compounds were tested at working concentrations of 20 nM, 200 nM, 500 nM and 1 μM and bioluminescence assessment of transcriptional activity was performed 12, 24, 36 and 48 hours after treatment. Details of this screening method are described in Wang et al., 2006, PNAS 103: 11003-11008). TIC10 (NSC350625) was obtained from NCI DTP, reconstituted in DMSO at 20 mM, divided equally and stored at -20 ° C. A6730 and U0126 monoethanolates were obtained from Sigma. Purified recombinant TRAIL was prepared as described in Kim et al., 2004, J. of Biol. Chem. 279: 40044-40052. RIK-2 antibody (Santa-Cruz Biotechnology) was used at 1 μg / mL and zVAD-fmk (Promega) was used at 20 μM.</p><p num="0137"> Primary specimen from human patient All primary specimens from human patients are received immediately after excision, manually digested in complete DMEM and filtered through 100 μm nylon mesh for use in the examples described herein. , 2x10 in Complete DMEM<sup>5</sup>Plated with cells / mL.</p><p num="0138"> mouse For subcutaneous xenografts, 1 x 10 on each dorsal abdomen of 4-6 week old female athymic nu / nu mice (Charles River Laboratories)<sup>6</sup>Pieces (2.5 x 10 for T98G)<sup>6</sup>Cell lines labeled (1) were inoculated as a suspension of 200 μL of 1: 1 Matrigel (BD): PBS. All intraperitoneal and intravenous injections were performed in a total volume of 200 μL. The oral formulation of TIC10 was administered using oral gastrointestinal feeding as a 200 μL suspension containing 20% Cremophor EL® (Sigma), 10% DMSO and 70% PBS. Tumors were monitored using digital calipers at the time of display. All subcutaneous tumors are about 125 mm 1 to 4 weeks after injection and before the start of treatment<sup>3</sup>It was established until it reached the volume of. Reduction of tumor tissue volume was monitored for 3 weeks after tumor disappearance and confirmed by visual inspection after euthanasia.</p><p num="0139"> Intracecal transplantation was performed as described in Cespedes, MV et al., Am J Pathol, 2007, 170 (3): p.1077-1085.</p><p num="0140"> For intracranial xenograft, 2 × 10 as a 25 μL suspension of serum and antibiotic-free RPMI in anesthetized athymic nude mice<sup>5</sup>Three SF767 cells were transplanted. The injection site was a burr hole made 1 mm lateral to the midline of the skull and 1 mm anterior to the coronal suture. The injection was gradually administered over a 5-minute period using a Hamilton syringe, and the burr was used to close the burr hole. Tumor engraftment was assessed by bioluminescence imaging 2 weeks after transplantation. Bioluminescence imaging of tumors was performed in the IVIS imaging system as described in Wang et al., 2003, PNAS 100: 15095-15100.</p><p num="0141"> Near-infrared imaging of mice was performed in the Pearl Impulse Imaging System (LI-COR) after tail vein injection of AngioSense® 680 (VisEn Medical, Woburn, MA) according to the manufacturer's protocol. Six-week-old Εμ-myc mice were obtained from The Jackson Laboratory (B6.Cg-Tg (IghMyc) 22Bri / J).</p><p num="0142"> For CBC / differential and serum chemistry assays, 1 mL of blood was collected from anesthetized mice by terminal cardiac puncture of the left ventricle. For serum chemistry, 500 μL was placed in a microtube, coagulated at room temperature for 30 minutes and then centrifuged. Any additional blood clots were removed by removing the serum and centrifuging again and the serum was sent for analysis. For CBC / differential, 500 μL of blood was collected in EDTA tubes and analyzed.</p><p num="0143"> Statistical analysis. For pair comparisons, Excel (Microsoft) was used to analyze the data by Student's two-sided t-test. Log rank statistical analysis was performed using a web-based script that works with statistics package R.</p><p num="0144"> RT-qPCR Total RNA was extracted using RNeasy Minikit (Qiagen) according to the manufacturer's instructions. SuperScript II (Invitrogen) was used with 1 μg RNA and oligodT to generate cDNA. Primers were TRAIL forward (CAGAGGAAGAAGCAACACATT, SEQ ID NO: 1), TRAIL reverse (GGTTGATGATTCCCAGGAGTTTATTTG, SEQ ID NO: 2), GAPDH forward (CCACATCGCTCAGACACCAT, SEQ ID NO: 3), GAPDH reverse (GGCAACAATATCCACTTTACCAGAGT, SEQ ID NO: 4). PCR amplification was performed using the Applied Biosystems 7900HT Fast Real-time Detection System. Samples were standardized to 10 ng / μl, then 20 ng of cDNA per sample was added to SYBR Green Master Mix (Qiagen). Used as a template for real-time PCR using Corp, USA). Samples were normalized to GAPDH used under the same conditions. For quantification, GAPDH as an endogenous control for normalization and the 2ΔΔCt method that intersects the thresholds described in Livak et al., 2001, Methods.2001 Dec; 25 (4): 402-8 were used. The reaction was performed on a 384-well optical plate in a 7900HT instrument (Applied Biosystems) using a reaction volume of 10 μl. Data analysis used ABI PRISM 7900 Sequence Detection System 2.2 software. To rule out the possibility of genomic DNA contamination, control PCR reactions were also performed on each gene-specific primer set using control samples that did not contain the cDNA template and RT. A quadruple of each PCR reaction was performed and the data obtained were averaged.</p><p num="0145"> Immunofluorescence The indicated cell lines were grown for 72 hours in log phase growth in a 6-well plate in the presence of the indicated working concentration of TIC10 in the absence. Cells were fixed and permeabilized using Cytofix / Cytoperm solution (BD Biosciences, San Jose, CA). Cells in Perm / Wash solution (BD Biosciences) with anti-TRAIL (ab2435, Abcam, Cambridge, MA) at 1: 100 or anti-active caspase-3 (559565, BD Pharmingen, San Diego, MA) at 1: 250. Incubated with CA) for 1 hour in the absence of light. Anti-rabbit Alexa Fluor488 was incubated in Perm / Wash solution for 20 minutes at room temperature at 1: 200 and rinsed with PBS. Hoechst33342 (Invitrogen) was used as nuclear counterstain according to the manufacturer's protocol. Axiovert Inverted Microscope (Carl Zeiss) Using iVision Imaging System (Biovision) Fluorescence imaging was performed in Micro Imaging).</p><p num="0146"> Flow cytometry and cell death assay Coulter-Beckman Elite floating and adherent cells for all flow cytometric analyzes Analyzed with an Epics hemocytometer. For surface TRAIL experiments, adherent cells were harvested by short trypsin treatment, fixed in 4% paraformaldehyde in PBS for 20 minutes, incubated with anti-TRAIL antibody for 2 hours (Abcam), washed and anti-rabbit. Incubated for 30 minutes with Alexafluor 488 (Invitrogen) and analyzed. Debris and dead cells were excluded from the analysis by gating the cells forward and sidescatter. Surface TRAIL data are expressed as median fluorescence intensity relative to the control sample unless otherwise indicated. For Sub-G1 and cell cycle profile experiments, all cells were pelleted, fixed in ethanol and then stained with propidium iodide (Sigma) in the presence of RNAse. Cell Titer-Glo® (Promega) was used according to the manufacturer's protocol to perform a cell viability assay on a 96-well black-walled clear bottom plate. Imaging and quantification of these assays was performed on the IVIS Imaging System (Xenogen).</p><p num="0147"> Colonization assay The indicated cell lines were plated with 500 cells per well and treated with fresh complete medium the next day after adhesion. After 3 days of treatment, the medium was replaced with drug-free medium and fresh medium was given once every 3 days to allow cells to grow for 10 days. At the end of the 10 days, cells were washed with PBS, fixed with methanol, stained with Coomassie blue, rinsed and dried for quantification.</p><p num="0148"> Tissue analysis. Mice were humanely sacrificed at the time of labeling and resected normal tissue or tumors were fixed overnight in 4% paraformaldehyde / PBS at 4 ° C. If a plasma sample was desired, 500 μL of blood was collected in an EDTA-Vacutainer tube (BD) by terminal cardiac puncture under anesthesia. Serum samples were collected in the same manner except for the microcentrifuge tube, and then incubated at room temperature for 30 minutes to coagulate. Serum was then removed after 5 minutes of centrifugation. Paraffin-embedded blocks, serial section slides and hematoxylin and eosin staining were prepared according to standard procedures. TUNEL staining was performed using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Millipore). For IHC analysis, slides were removed in xylene and ethanol was gradually reduced and hydrated. Antigen recovery was performed by boiling in 10 mM citric acid (pH 6.0) for 6 minutes. Streptavidin and biotin blocking solution and goat serum (Vector) Laboratories) was used to block the sample. The primary antibody was incubated overnight at 4 ° C in a humidity chamber. Incubation with biotinylated secondary antibody and DAB deposition were performed according to the manufacturer's protocol (Vector Laboratories DAB Substrate Kit for Peroxidase). Samples were counterstained with hematoxylin (DAKO) for 6 minutes and dH<sub>2</sub>Rinse in O for 5 minutes, rinse with PBS, dehydrate and seal with cover glass. Images were recorded under an Axioskop microscope using QCapture software (QImaging).</p><p num="0149"> Co-culture HCT116 p53<sup>-/-</sup>And HFF cells were co-cultured in a 1: 1 mixture of complete DMEM and McCoy's 5A medium. For fluorescence images, the two types of cells were labeled separately using the Fluorescent Cell Linkers Kits for gene cell membrane labeling (Sigma) according to the manufacturer's protocol. Cells were counterstained with Hoechst 33342 as described in the immunofluorescence section. For flow cytometric analysis of cell death, two populations of cells were discriminated by differences in light scattering and analyzed as described for sub-G1 analysis in the Cell Death Assay section.</p><p num="0150"> ELISA ELISA for TRAIL was performed using the Quantikine® TRAIL / TNFSF10 kit according to the manufacturer's protocol (DTRL00, R & D systems, Minneapolis, MN). The absorbance at 540 nm was used to make an optical correction as suggested by the manufacturer. Absorbance was measured using a DTX880 plate reader (Beckman Coulter).</p><p num="0151"> Pharmacokinetic analysis of TIC10 The absorbance profile of TIC10 was measured on a Gene Spec III spectrometer (Hitachi Solutions American, South San Francisco, CA). HPLC analysis was performed by absorbance detection at 239 nm on an Agilent 1200 series system (Agilent, Santa Clara, CA) using an Eclipse XDB-C18 column (Agilent) and a 100 μL injection loop. Isocratic elution at 1 mL / min, dH<sub>2</sub>It was performed with .1% trifluoroacetic acid in O. Acetonitrile (ACN) gradient was applied for elution at 15-20% ACN at 0-5 minutes, 20-23% at 5-12 minutes, and 25% at 12-18 minutes. A calibration curve was generated by adding a concentration of TIC10 in plasma recovered from athymic nude mice in an unrelated experiment. Blood was obtained by terminal cardiac puncture of the left ventricle for all plasma samples and collected in EDTA tubes (BD). The sample was centrifuged at 500 g for 10 minutes. Plasma was deproteinized by adding 30 μL of perchloric acid to 100 μL of sample, vortexed for 15 seconds, centrifuged for 2 minutes, and the supernatant was immediately injected into the HPLC. AUC was normalized to internal serum peaks with a retention time of 8.1 minutes. AUC data for time, formula AUC = Ae<sup>-αt</sup>+ Be<sup>-βt</sup>(Where t = time, where A and B are extrapolated concentrations at the start of the two phases (distribution and excretion)) 2 compartment open with primary excretion from the central compartment using Fitted to the model. Half-life t<sub>1 / 2α</sub>= .693 / α and t<sub>1 / 2β</sub>Calculated as = .693 / β. Another formula used for the calculation is CL = dose / AUC<sub>0-∞</sub>And V<sub>d</sub>= Dose / (AUC<sub>0-∞</sub>× β) can be mentioned.</p><p num="0152"> Gene expression analysis HCT116 p53<sup>-/-</sup>Cells were grown in log phase and treated with DMSO or TIC10 (10 μM). After 48 hours, RNA was isolated using the RNeasy Mini Kit (Qiagen). Microarray analysis was performed using the Illumina HT-12 Beadchip (Illumina). RNA quality and concentration were evaluated using the Agilent 2100 Bioanalyzer with RNA Nano Lab Chip® (Agilent). TotalPrep according to the manufacturer's instructions<sup>TM</sup>CRNA was synthesized from 500 ng of RNA by Amplification (Ambion). Reverse transcription with T7 oligo (dT) as a primer was used to generate first strand cDNA. Next, the cDNA was subjected to second-strand synthesis and RNA degradation by DNA polymerase and RNase H, followed by filtration and purification. In vitro transcription (IVT) was used to generate multiple copies of biotinylated cRNA. The labeled cRNA was purified using filtration and quantified by NanoDrop to adjust the volume to a total of 750 ng / sample. The sample was fragmented, denatured and hybridized at 58 ° C for 18 hours. After hybridization, the bead chips were washed and fluorescently labeled. Bead chips were scanned using a BeadArray Reader (Illumina). The obtained scan data was imported into Genome Studio 1.0 (Illumina) to create a project. The result is Gene Spring Gxll (Agilent) Exported to Technologies). Measurements less than 0.01 were then set to 0.01, the array was normalized to the 50th percentile, and individual genes were normalized to the median control. Data sets were analyzed using Ingenuity Pathway Analysis software (Ingenuity Systems) for network analysis of transcriptional changes induced by TIC10.</p><p num="0153"> Western blot analysis Western blot analysis was performed using NuPAGE4-12% Bis-Tris as described in Wang, W. et al., PNAS 103,11003-11008,2006, and Supersignal West Femto (Thermo Scientific) and X-ray film. Visualized using. Cytoplasmic lysis buffer (10 mM) HEPES, 10 mM KCl and 2 mM MgCl<sub>2</sub>, 1 mM DTT) followed by karyolysis buffer (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl)<sub>2</sub>, 250 μM EDTA, 25% glycerol) was used to prepare nuclei and cytoplasmic extracts. For all lysis buffers, a fresh protease inhibitor (Roche) and 1 mM sodium orthovanadate were added just prior to use.</p><p num="0154"> Chromatin immunoprecipitation assay ChIP-grade antibody against Foxo3a (Abcam) or equal concentration of rabbit IgG as a non-specific control, as described for the TRAIL promoter in Nebbioso, A. et al., Nat Med, 11 (1), 77-84, 2005. Chromatin immunoprecipitation (ChiP) assay was performed using (Southern Biotech).</p><p num="0155"> TIC10 triggers p53-independent transcriptional induction of the TRAIL gene Cell-based bioluminescence reporter screening performed on TRAIL-resistant Bax null HCT116 human colon cancer cells using the TRAIL gene promoter yielded the small molecule TIC10 as a TRAIL-inducing compound.</p><p num="0156"> TIC10 induces TRAIL promoter-dependent transcriptional activity of the luciferase reporter construct under regulatory control of the first 504 base pairs of the TRAIL promoter, which excludes the p53 DNA-binding response element identified in Takimoto et al., 2000, Oncogene 19, 1735-1743. did. Figure 1 shows the HCT116 Bax under transcriptional regulation of the first 504 base pairs of the human TRAIL gene promoter upstream of transcription initiation.<sup>-/-</sup>It is a graph which shows the activity of a luciferase reporter in a cell (n = 3). The error bars indicate the sd of the iterative experiment. Between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0157"> TIC10 caused a dose-dependent increase in TRAIL messenger RNA. Figure 2 shows HCT116 p53<sup>-/-</sup>It is a graph which shows RT-qPCR analysis of TRAIL mRNA level in a cell (48 hours, n = 4). The error bars indicate the sd of the iterative experiment. TIC10 caused a dose-dependent increase in TRAIL protein localized on the cell surface of some cancer cell lines in a p53-independent manner. FIG. 3 is a graph showing TIC10-induced surface TRAIL levels in a panel of cancer cells (10 μM, 72 hours, n = 3). The error bars indicate the sd of the iterative experiment. Between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0158"> TIC10 exposure results in the significant and persistent presence of TRAIL on the cell surface of cancer cells. Over time analysis revealed that TRAIL was localized on the cell surface as a delayed event, but this induction could be sustained over time even after removal of TIC10 from the medium. Figure 4 shows the HCT116 p53 after TIC10 treatment at the indicated conditions and time points.<sup>-/-</sup>It is a graph which shows the surface TRAIL level in a cell (n = 3). The error bars indicate the sd of the iterative experiment. Between display conditions and controls<sup>*</sup>P <0.05. Figure 5 shows HCT116 p53 by flow cytometry 72 hours after the start of TIC10 treatment.<sup>-/-</sup>It is a graph showing the TRAIL surface level (5 μM, n = 3). Cells were treated for the indicated time of preincubation and then replaced with drug-free medium for the remaining time until analysis after 72 hours. The error bars indicate the sd of the iterative experiment. Between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0159"> TIC10 induces TRAIL-mediated apoptosis TIC10 does not alter the cell cycle profile of normal fibroblasts at equal doses, TRAIL-sensitive HCT116 p53<sup>-/-</sup>Induced the DNA content of sub-G1 suggesting cell death in cells. Figure 6 shows the TIC10 processed HCT116 p53.<sup>-/-</sup>And the cell cycle profile of HFF cells (5 μM, 72 hours, n = 3).</p><p num="0160"> TIC10 reduced clonogenic survival of cancer cell lines while retaining normal fibroblasts. FIG. 7 is a graph showing the quantification of a colonization assay for cancer cells treated with TIC10 (10 μM, 72 hours, n = 3). Error bars indicate the standard deviation (sd) of repeated experiments. FIG. 8 is a graph showing a similar experiment as in FIG. 7 except that HFF cells were counted at the end (n = 3). Error bars indicate the standard deviation (sd) of repeated experiments.</p><p num="0161"> TIC10 induced sub-G1 content in a p53-independent and Bax-dependent manner. Figure 9 shows HCT116 WT, p53 after treatment with DMSO, TIC10 (1, 5 or 10 μM) or rhTRAIL (25 ng / mL) for 72 hours.<sup>-/-</sup>And Bax<sup>-/-</sup>It is a graph showing the sub-G1 analysis of cells (n = 3). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0162"> HCT116 p53 treated with 5 μM TIC10 for 72 hours, consistent with apoptotic cell death<sup>-/-</sup>HCT116 p53 treated by immunofluorescence assay in cells and with 1 μM, 2.5 μM, 5 μM or 10 μM TIC10 for 72 hours<sup>-/-</sup>TIC10 increased the level of active caspase-3, as shown by Western blot analysis in cells. FIG. 10 is an image showing the results of Western blot analysis. The sub-G1 content induced by TIC10 was significantly inhibited by incubation with the pan caspase apoptosis inhibitor zVAD-fmk. FIG. 11 is a graph showing sub-G1 analysis of TIC10 treated cancer cells pre-incubated with or without zVAD-fmk (10 μM, 72 hours, n = 3). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0163"> Apoptosis induced by TIC10 appears to be specifically mediated by TRAIL, as indicated by inhibition of TIC10-induced cytotoxicity after stable knockdown of TRAIL by shRNA. FIG. 12 is a graph showing sub-G1 analysis of MDA-MB-231 cells in which TRAIL was stably knocked down by short hairpin RNA (72 hours, n = 3). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05. FIG. 13 is a graph showing the demonstration of MDA-MB-231 shTRAIL knockdown by flow cytometric analysis of TIC10 treated cells (5 μM, 72 hours, n = 3). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0164"> Additional evidence for the need for TRAIL in TIC10-induced tumor cell death was observed after disruption of the DR5 death domain, which regulates apoptosis-promoting TRAIL signaling. FIG. 14 is a graph showing sub-G1 analysis of TIC10-induced cell death in H460 cells with overexpression of endogenous DR5, or a DR5 construct in which its death domain was replaced by EGFP (10 μM, 72). Time, n = 3). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0165"> Experimental isolation of TRAIL by using blocking antibodies showed that TRAIL was required for TIC10-induced tumor cell death. FIG. 15 is a graph showing sub-G1 analysis of HCT116 cells treated with DMSO, TIC10 (10 μM) or rhTRAIL (25 ng / mL) in the presence or absence of the TRAIL sequestering antibody RIK-2. There is (72 hours, n = 3). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0166"> When the activity of TIC10 in freshly resected colon tumor cells from human patients was examined, it was found that TIC10 induces TRAIL and a potent cytotoxic effect different from 5-FU. FIG. 16 is a graph showing TIC10-induced surface TRAIL using freshly resected colon cancer cells (10 μM, 72 hours). The tissue was mucinous adenocarcinoma removed from an 85-year-old female patient. The data are expressed as median fluorescence intensity. FIG. 17 shows the results of the cell survival assay for primary colon cancer cells of FIG. 16 treated with DMSO, TIC10 (.6, 1.25, 2.5, 5, 10, 20 μM) or 5-FU (5 μM). The graph is (n = 3). Error bars indicate the sd of the iterative experiment.</p><p num="0167"> The cytotoxic activity of TIC10 is thermally stable, unlike TRAIL. FIG. 18 is a graph showing that TIC10 (5 μM) or rhTRAIL (25 ng / mL) can reduce cell viability in HCT116 cells after 1 hour preincubation at the indicated temperature (72 hours). , N = 3). Error bars indicate the standard deviation (sd) of repeated experiments.</p><p num="0168"> TIC10 is a potent TRAIL-mediated antitumor agent in vivo TIC10 is comparable to that observed with TRAIL when both are administered in multiple doses, HCT116 p53<sup>-/-</sup>Caused tumor regression in xenografts. Figure 19 shows HCT116 p53 treated with 3 TIC10 (ip), TRAIL (iv) or vehicle (ip) doses administered on days 0, 3 and 6 as indicated by the gray vertical bars.<sup>-/-</sup>It is a graph showing a xenograft (n = 10). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05 and<sup>**</sup>P <.005.</p><p num="0169"> Single-dose experiments in mice with HCT116 WT) and RKO human colon cancer xenografts support the potent antitumor activity of TIC10 and underscore the superiority of TRAIL in RKO xenografts under given conditions. did. Figure 20 shows a luciferase-infected HCT116 p53 that received a single ip injection of TIC10 or vehicle.<sup>-/-</sup>It is a graph which shows the result of bioluminescence imaging of a xenograft (n = 6). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05 and<sup>**</sup>P <.005.</p><p num="0170"> FIG. 21 is a graph showing RKO xenografts with a single dose of TIC10 (ip), TRAIL (iv) or vehicle (ip, n = 10). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05 and<sup>**</sup>P <.005.</p><p num="0171"> TIC10 induced regression of MDA-MB-231 human breast cancer xenografts, an effect significantly inhibited by stable knockdown of TRAIL, while TRAIL-treated tumors progressed. Figure 22 shows a box of tumor volumes 9 days after initiation of treatment with a single dose of TIC10 (ip), TRAIL (iv) or vehicle (DMSO, ip) in the MDA-MB-231 vector or shTRAIL xenograft. It is a beard diagram (n = 8). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05 and<sup>**</sup>P <.005. TUNEL staining of tumors from MDA-MB-231 vector and shTRAIL xenografts 2 days after treatment with 50 mg / kg or 100 mg / kg TIC10 was treated with vector but with shTRAIL. Shows increased TUNEL staining in non-existing cells.</p><p num="0172"> This indicates that the antitumor activity of TIC10 is superior to that of TRAIL when administered as a single dose under these conditions and is at least partially regulated by TRAIL produced by tumor cells. Prove directly. In DLD-1 xenografts, TIC10 induced tumor stagnation one week after treatment, whereas TRAIL-treated tumors progressed after a single dose. FIG. 23 shows the relative tumor volume of DLD-1 xenografts treated with TRAIL (iv), TIC10 (ip) or DMSO (ip) as a single dose at the indicated concentration on day 0. The graph is (n = 8).</p><p num="0173"> TIC10 as a single dose by intraperitoneal or oral delivery also induced sustained regression of SW480 xenografts, suggesting favorable bioavailability. FIG. 24 is a graph showing a comparison between a single dose of TIC10 at 30 mg / kg treated on day 0 and oral administration in SW480 xenografts (n = 6).</p><p num="0174"> Dose setting of a single dose of TIC10 administered orally in the HCT116 xenograft model revealed sustained antitumor efficacy at 25 mg / kg. FIG. 25 is a graph showing TIC10 or vehicle administered as a single oral dose in HCT116 xenografts (n = 6). Error bars indicate the standard deviation (sd) of repeated experiments. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05 and<sup>**</sup>P <.005.</p><p num="0175"> In addition to having no adverse effects on body weight or histological examination of the liver, the lack of obvious toxicity in multiple doses delivered at doses four times higher than this therapeutic dose in previous xenografts is It suggests that TIC10 has a wide therapeutic window. FIG. 26 is a graph showing the body weight of athymic female nude mice treated with a single dose of TIC10 (100 mg / kg, ip). FIG. 27 is a graph showing the body weight of C57 / B6 female mice at the end of week 4 of treatment with weekly oral TIC10 administration (25 mg / kg) over 4 weeks. Histological analysis of liver from athymic female nude mice recovered 3 days after treatment with TIC10 (100 mg / kg, ip) by H & E staining showed that TIC10 had no apparent toxicity.</p><p num="0176"> Prolonged exposure of immunoqualified mice to 25 mg / kg oral TIC10 weekly for 4 weeks did not cause any changes in the panel of serum chemical markers, as shown in Tables IA and IB.</p><p num="0177"> Tables IA and IB show serum chemistry of C57 / B6 mice treated with vehicle or TIC10 (25 mg / kg) weekly for 4 weeks.</p><p num="0178"><tables num="1"><img id="000018" he="91" wi="144" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> To test the efficacy of TIC10 in an immunoeligible preclinical cancer model, we used Εμ-Myc transgenic mice that spontaneously develop lymphoma. The same oral dosing schedule as above, which proved safe at 9-12 weeks of age, was used. TIC10 significantly prolonged the survival of these mice for 4 weeks. FIG. 28 is a graph showing overall survival of Εμ-myc treated with oral TIC10 (25 mg / kg) weekly between weeks 9-12. The P value was determined by the log rank test. For relative tumor volume plots, tumor size is expressed relative to day 0 tumor size, which is defined as the treatment start date. Histological analysis of axillary lymph nodes of Εμ-myc and WT C57 / B6 at 14 weeks of age by H & E staining showed that TIC10 had no apparent toxicity.</p><p num="0179"> Synergistic combination of TIC10 and chemotherapeutic agent Surprisingly (Syrprisingly), an in vitro synergy between TIC10 and the taxanes paclitaxel and docetaxel (trade name Taxotere) is observed. FIG. 29 is a graph showing the cell viability of DLD-1 cells treated with TIC10 together with paclitaxel under the indicated conditions (72 hours, n = 3). Error bars indicate the sd of the iterative experiment. FIG. 30 is a graph showing the cell viability of SW620 cells treated with TIC10 together with paclitaxel under the indicated conditions (72 hours, n = 3). Error bars indicate the sd of the iterative experiment. FIG. 31 is a graph showing the cell viability of DLD-1 cells treated with TIC10 together with taxotere under the indicated conditions (72 hours, n = 3). Error bars indicate the sd of the iterative experiment. FIG. 32 is a graph showing the cell viability of SW620 cells treated with TIC10 with taxotere under the indicated conditions (72 hours, n = 3). Error bars indicate the sd of the iterative experiment.</p><p num="0180"> The cooperation of TIC10 in combination with either paclitaxel or docetaxel for taxanes resulted in sustained cure in H460 non-small cell lung cancer xenografts. Figure 33 shows tumors in H460 xenografts as a single dose, TIC10 (30 mg / kg, ip) or taxotere (20 mg / kg, iv) alone, in combination, or after treatment with vehicle (DMSO, ip). It is a graph showing the percentage of the cohort holding the tissue (n = 8). FIG. 34 is a graph showing a plot of tumor volume relative to FIG. 33. Error bars indicate the sd of the iterative experiment.</p><p num="0181"> Figure 35 shows tumors in H460 xenografts as a single dose, treated with TIC10 (30 mg / kg, ip) or paclitaxel (20 mg / kg, iv) alone, in combination, or after treatment with vehicle (DMSO, ip). It is a graph showing the percentage of the cohort holding the tissue (n = 8). FIG. 36 is a graph showing a plot of tumor volume relative to FIG. 35. Error bars indicate the sd of the iterative experiment.</p><p num="0182"> In this example, when both TIC10 and bevacizumab were given once a week in a metastatic orthotopic mouse model of p53-deficient colorectal cancer, TIC10 worked with bevacizumab to provide primary cecal tumors and distant tumors. It was found to reduce the incidence of tumors at metastatic sites (including lungs, liver, lymph nodes and peritoneum). Figure 37 shows HCT116 p53 in the cecum.<sup>-/-</sup>It is a graph showing the percentage of cohorts in which tumors were transplanted and had obvious tumors at the primary and distal sites at the end (n = 5). As indicated by the time series, treatment is performed once a week starting 2 weeks after transplantation and in a cohort of vehicle, TIC10 (25 mg / kg, oral), bevacizumab (bev, 10 mg / kg, iv). ) Or a combination of TIC10 and bevacizumab.</p><p num="0183"> TIC10 alone and in combination with bevacizumab were well tolerated when using this multiple dose regimen and did not significantly change body weight at termination. FIG. 38 is a graph showing the weight of mice at the end. Error bars indicate the sd of the iterative experiment.</p><p num="0184"> TIC10 causes tumor-specific cell death by direct and bystander effects mediated by TRAIL HCT116 after a single dose of TIC10 (100 mg / kg, ip) on day 0 p53<sup>-/-</sup>Immunohistochemical (IHC) analysis of xenograft tumors revealed elevated protein levels of TRAIL and truncated caspase-8, an initiator caspase involved in TRAIL-mediated apoptosis.</p><p num="0185"> Fragmented nuclei observed by histological examination and increased TUNEL (TdT-mediated dUTP nick terminal labeling) staining further confirmed that TIC10 induced apoptosis in treated tumors. In addition, HCT116 p53 after treatment with TIC10 (100 mg / kg, ip) or vehicle 2 days after treatment.<sup>-/-</sup>As shown by H & E and IHC analysis for TRAIL at the border between tumor and stromal fibroblasts in heterologous transplant tumors, TIC10 not only TRAIL in tumors but also in stromal fibroblasts that border the tumor. Induced.</p><p num="0186"> Focusing on the expression of TIC10-induced TRAIL in fibroblasts, soluble TRAIL was assayed in TIC10-treated tumor-free mice to determine if normal cells secrete TRAIL in response to TIC10. TIC10 rapidly raises serum levels of TRAIL in a manner that lasts longer than 72 hours, which is longer than the serum half-life of recombinant TRAIL (about 30 minutes). FIG. 39 is a graph showing TRAIL serum levels in tumor-free mice after TIC10 (100 mg / kg, iv) or doxorubicin (30 mg / kg, ip) (n = 2). Error bars indicate the sd of the iterative experiment.</p><p num="0187"> Serum TRAIL induced by TIC10 was detected as early as 2 hours after dosing, which is more rapid than the kinetics observed in vitro in the examples described herein. Pharmacokinetic analysis revealed that TIC10 was immediately distributed and had a plasma half-life of approximately 6.5 hours. Table II shows the results of pharmacokinetic analysis of TIC10 in plasma of C57B6 mice. FIG. 40 is a graph showing the absorbance profile of TIC10 having a peak absorbance at 239 nm. FIG. 41 is a graph showing a calibration curve for TIC10 added to mouse plasma and quantified using area under the curve (AUC) by HPLC analysis. FIG. 42 is a graph showing the plasma concentration of TIC10 after intravenous administration at 25 mg / kg to C57 / B6 female mice (n = 3). The error bar shows the average value of the standard error of the iterative experiment.</p><p num="0188"><tables num="2"><img id="000019" he="30" wi="142" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> TIC10 has a longer half-life than recombinant TRAIL, and the effect of TIC10, i.e. induction of TRAIL, is sustained in vivo over several days as seen in vitro.</p><p num="0189"> It is clear from IHC analysis of normal tissues of tumorless athymic nude mice after administration of TIC10 (100 mg / kg, iv) on day 0, as determined by histological examination and TUNEL staining. It was revealed that TRAIL was upregulated at protein levels in the brain, kidney and spleen of mice without toxicity. Upregulation of TRAIL in response to TIC10 was not significant in other tissues, including the liver, at any time point.</p><p num="0190"> The effect of TIC10 on normal fibroblasts and its selectivity on normal cells was tested in this example. TIC10 selectively induced apoptosis in p53-deficient tumor cells, but treated with TIC10 (10 μM) or DMSO for 3 days HCT116 p53<sup>-/-</sup>And normal fibroblasts in co-culture experiments with HFF cells did not induce apoptosis.</p><p num="0191"> TIC10 induces a significant but moderate amount of TRAIL on the surface of normal fibroblasts. FIG. 43 is a graph showing analysis of surface TRAIL of HFF cells after TIC10 treatment (0, 2.5, 5 or 10 μM from left to right) (72 hours, n = 3). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0192"> To test whether normal cells contribute to the antitumor efficacy of TIC10 through the TRAIL-mediated bystander effect, normal fibroblasts pre-incubated with TIC10 were p53-deficient colon cancer cells. It was transplanted into a co-culture with. This resulted in a moderate but significant increase in TRAIL-specific cell death in a subpopulation of cancer cells. Figure 44 shows the HCT116 p53.<sup>-/-</sup>It is a graph showing the sub-G1 analysis of the co-culture of cells and pretreated HFF (24 hours, n = 3). Pretreatment of HFF consisted of a 72-hour incubation with TIC10 (10 μM) or DMSO. These experiments were performed in the presence or absence of the TRAIL isolation antibody (RIK-2). The scale bar is 100 μm. Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0193"> Thus, as demonstrated herein, TIC10 has a favorable therapeutic index and induces TRAIL in tumor cells, stromal cells and normal cells, which is a direct mechanism as well as a bystander mechanism. It may contribute to the antitumor efficacy of TIC10 through the introduction.</p><p num="0194"> TIC10 is an effective antitumor agent in polymorphic glioblastoma (GBM) TIC10 induces TRAIL in the brain and is useful as an antitumor agent for brain tumors. In this example, the activity of TIC10 in GBM cell lines was tested and found that TIC10 induces TRAIL and has a p53-independent GI50 in the low micromolarity range comparable to other cancer cell lines. Found. FIG. 45 is a graph showing surface TRAIL in GBM cell lines after incubation with TIC10 (5 μM, 72 hours, n = 3). Between display conditions and controls<sup>*</sup>P <0.05. FIG. 46 is a graph showing the GI50 value extrapolated from the cell life-and-death assay of the indicated GBM cell line 72 hours after treatment with TIC10 or DMSO (n = 3).</p><p num="0195"> TIC10 is temozolomide resistant and has a cytotoxic effect on freshly isolated GBM cells pre-irradiated in this example. FIG. 47 shows the results of a cell viability assay for freshly resected glioblastoma tissue treated with DMSO, TIC10 or temozolomide (TMZ, 10 μM) (72 hours, n = 3). The tissue was grade IV glioblastoma containing components of oligodendroglioma cells taken from a 38-year-old female patient who had previously undergone tumor reduction and radiation.</p><p num="0196"> TIC10 was tested in a preclinical model of GBM as a monoagent and in combination with bevacizumab. TIC10 exhibits p53-independent cytotoxicity against a panel of GBM cell lines (including glioblastoma-resistant GBM cell lines such as T98G) and is similar to bevacizumab when given as a single oral dose. Induced continuous regression of subcutaneous T98G xenografts to the extent of. FIG. 48 is a graph showing subcutaneous xenografts of T98G in mice that received a single dose of vehicle, TIC10 (30 mg / kg, PO) or bevacizumab (10 mg / kg, iv) on day 0 ( n = 8). Between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0197"> Single-dose TIC10 significantly doubles overall mouse survival and has such brain tumors by cooperating with bevacizumab as a single agent in invasive intracranial xenograft of human GBM using the SF767 cell line. The survival time of mice was tripled.</p><p num="0198"> Figure 49 shows a single oral vehicle (n = 8), TIC10 (25 mg / kg, n = 7), bevacizumab (10 mg / kg, iv, n = 6) or TIC10 and bevacizumab 2 weeks after transplantation. FIG. 5 is a graph showing overall survival of mice with SF767 intracranial tumor treated with (n = 7).</p><p num="0199"> Table III shows changes in overall survival in a mouse cohort with SF767 intracranial tumors.</p><p num="0200"><tables num="3"><img id="000020" he="48" wi="140" file="JP6132833B2_D0001.tif" img-format="tif" img-content="drawing" /></tables> TIC10-induced TRAIL upregulation is Foxo3a dependent HCT116 p53 treated with TIC10 to identify molecular events that support upregulation of TIC10-induced TRAIL<sup>-/-</sup>The gene expression profile in the cells was measured. Transcription factors of the FOXO family (Modur, V. et al., 2002, J. Biol. Chem. 277: 47928-47937) previously regulated the TRAIL gene promoter at the binding site contained within the selected region. Transcriptional changes in the target genes (including Foxo3a shown in) were observed. Figure 50 shows HCT116 p53 48 hours after TIC10 treatment (10 μM) compared to DMSO.<sup>-/-</sup>It is a graph showing the transcriptional changes associated with FOXO signaling from cellular gene expression profiling (n = 3). All of these changes were P <.05 between the DMSO and TIC10 treatment groups. Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0201"> The FOXO target gene DR5 was upregulated by TIC10 in some cancer cell lines and, if not so much, normal cells, which was also observed in TIC10 treated tumors. FIG. 51 is an image of a Western blot analysis of DR5 in HCT116 cells treated with TIC10 or DMSO at the indicated concentrations for 72 hours. Ran is shown as a filling control. FIG. 52 is a graph showing flow cytometric analysis of surface DR5 levels in TIC10 treated cancer and normal cells (72 hours, n = 3). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0202"> IHC analysis of DR5 in HCT116 xenograft tumors treated with vehicle (ip) or TIC10 (100 mg / kg, ip) is consistent with in vitro observations and DR5 in xenograft tumors treated with TIC10. It shows that the high expression of was obvious.</p><p num="0203"> Foxo3a, a member of the FOXO family, was measured by immunofluorescence and Western blot analysis of Foxo3a in HCT116 cells and immunofluorescence analysis of Foxo3a in H460 and SW480 cells treated with DMSO or TIC 10,10 μM for 48 hours. It caused nuclear translocation in response to TIC10 (but did not cause Foxo1a).</p><p num="0204"> FIG. 53 is an image of Western blot analysis of whole cell solubilized (W) and cytoplasmic (C) and nuclear (N) extracts of HCT116 cells treated with DMSO or TIC10 (48 hours, 10 μM). .. β-actin and lamin B1 have been shown as cytoplasmic and nuclear filling controls, respectively.</p><p num="0205"> A TIC10 dose-dependent increase in the amount of Foxo3a localized to the TRAIL promoter was found, as indicated by the chromatin immunoprecipitation assay. Figure 54 shows the HCT116 p53<sup>-/-</sup>Image of chromatin immunoprecipitation assay results for TIC10-induced translocation of Foxo3a to the TRAIL promoter 48 hours after TIC10 treatment (0, 2.5, 5 or 10 μM from left to right) in cells.</p><p num="0206"> Transient knockdowns of Foxo3a and Foxo1 revealed that Foxo3a specifically mediates upregulation of TIC10-induced TRAIL. Figure 55 shows HCT116 p53 with siRNA.<sup>-/-</sup>Graph showing the results of flow cytometric analysis of cell surface TRAIL levels induced by TIC10 (10 μM) with or without transient knockdown of Foxo1 and / or Foxo3a in cells (72 hours, n = 3). Knockdown is confirmed by Western blot analysis. Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0207"> Stable knockdown of Foxo3a significantly inhibited upregulation of TIC10-induced TRAIL production and subsequent tumor cell death. FIG. 56 is a graph showing sub-G1 analysis of TIC10-induced cell death with or without stable knockdown of Foxo3a in HCT116 cells (10 μM, 72 hours, n = 3). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05. FIG. 57 is a graph showing flow cytometric analysis of surface TRAIL induced by TIC10 with or without stable knockdown of Foxo3a in HCT116 cells (10 μM, 72 hours, n = 3). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05. The results of stable knockdown of Foxo3a were confirmed by Western blot analysis.</p><p num="0208"> Stable knockdown of Foxo3a in tumor cells also significantly inhibited the antitumor activity of TIC10 and the characteristics of TIC10-induced TRAIL-mediated apoptosis in tumors in vivo. FIG. 58 is a graph showing tumor volume of HCT116 xenografts with or without stable knockdown of Foxo3a after a single oral dose of vehicle or TIC10 (25 mg / kg) on day 0. There is (n = 10). Error bars indicate the sd of the iterative experiment. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0209"> Three days after a single dose of TIC10 (25 mg / kg, oral), IHC analysis and TUNEL staining of HCT116 tumors with or without stable knockdown of Foxo3a were performed, and they were stable for Foxo3a in tumor cells. Knockdown was shown to significantly inhibit the antitumor activity of TIC10 and the characteristics of TIC10-induced TRAIL-mediated apoptosis in tumors in vivo.</p><p num="0210"> Double inactivation of Akt and ERK by TIC10 cooperatively induces TRAIL TIC10-induced changes in Foxo3a regulators (eg, IKK, Akt and ERK) were measured. Figure 59 shows the HCT116 p53 treated with TIC10 (2.5, 5, 10 μM) for 72 hours.<sup>-/-</sup>It is an image of Western blot analysis of cells.</p><p num="0211"> It was found that both pAkt and pERK levels were eliminated in a dose-dependent manner by TIC10 treatment co-dephosphorylation of each phosphorylation site on Foxo3a. Over time analysis revealed that TIC10-induced Akt and ERK inactivation, a kinetics coordinated with Foxo3a dephosphorylation and TRAIL upregulation, occurred after 48 hours. Figure 60 shows the HCT116 p53 treated with TIC10 (10 μM) over the indicated time.<sup>-/-</sup>It is an image of Western blot analysis of cells. FIG. 61 is a graph showing the time course of protein expression levels of TIC10-induced effects as measured by Western blot densitometry of repeated experiments as in FIG. 60 (n = 3). The data is represented for the control sample for each time point and normalized to Ran. TRAIL was quantified by flow cytometry as a parallel experiment (n = 3).</p><p num="0212"> These TIC10-induced effects on Foxo3a include human cancer cell lines with diverse genetic backgrounds, including carcinogenic changes in several cancer cell lines of various tumor types (p53, KRAS, PTEN, etc.). ) Was obvious. FIG. 62 is an image of a Western blot analysis of the effect induced by TIC10 on Foxo3a in DLD1 human colon cancer cells, MDA-MB-468 human breast cancer cells and T98G human polymorphic glioblastoma cell lines (10 μM, 72). time).</p><p num="0213"> Akt has been found to be a determinant of cytotoxic susceptibility to TIC10 and its TRAIL upregulation, and has been treated with TIC10 (10 μM, 48 hours), empty vector or myristylated Akt (myr-Akt). Overactivation of Akt can suppress even basal levels of TRAIL, as shown by immunofluorescence analysis of Foxo3a in overexpressing HCT116 cells. Confirmation of overexpression of myr-Akt by Western blot analysis is shown in Figure 63. FIG. 64 is a graph showing flow cytometric analysis of surface TRAIL in HCT116 cells overexpressing empty vector or myristylated Akt (myr-Akt) that received TIC10 treatment (10 μM, 48 hours). .. FIG. 65 is a graph showing the sub-G1 content of TIC10 treated HCT116 cells overexpressing empty vector or myr-Akt (10 μM, 72 hours, n = 3).</p><p num="0214"> Double inhibition of the Akt and MAPK pathways cooperatively results in Foxo3a nuclear translocation followed by TRAIL upregulation. A6730 and U0126 monoethanolates are commercially available, Akt1 / 2 (Desplat, V. et al., 2008, J.Enz.Inhib.Med.Chem., 23: 648-658) and MEK (Favata,). MF et al., 1998, J. Biol. Chem., 273: 18623-18632), which are previously reported inhibitors, in which double inhibition of the Akt and MAPK pathways cooperatively translocates Foxo3a to the nucleus. Each was used in this example to determine if it would result in and subsequent TRAIL upregulation. The combination of MEK and Akt inhibitors was found to synergistically induce Foxo3a-dependent TRAIL upregulation and synergistically induce TRAIL-mediated cell death. Figure 66 shows 10 μM A6730 (Akt inhibitor (Akt inh)), U0126 monoethanolate (MEK inhibitor (MEK)). HCT116 p53 after incubation with inh))) or both<sup>-/-</sup>It is a graph which shows RT-qPCR analysis of TRAIL mRNA in a cell (48 hours, n = 3). For Akt + MEK inh, P <.05 compared to all other conditions. Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0215"> FIG. 67 is a graph showing the induction of surface TRAIL as in FIG. 66 with or without stable knockdown of Foxo3a (n = 3). Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05.</p><p num="0216"> FIG. 68 is a graph showing sub-G1 analysis of MDA-MB-231 with or without TRAIL knockdown by shRNA after incubation with 10 μM Akt inh, MEK inh or both for 48 hours (n). = 3). Unless otherwise indicated, between display conditions and controls<sup>*</sup>P <0.05. Figure 69 shows the HCT 116 p53 after incubation with 10 μM A6730 (Akt inh), U0126 monoethanolate (MEK inh) or both.<sup>-/-</sup>It is a graph showing the surface TRAIL analysis of cells (48 hours, n = 3).</p><p num="0217"> SiRNA experiments in this example show that TRAIL can be cooperatively upregulated by inhibiting ERK and Akt. Figure 70 shows HCT116 p53<sup>-/-</sup>It is a graph showing RT-qPCR analysis of TRAIL mRNA levels after transient knockdown of Akt and / or ERK in cells (48 hours after knockdown) (n = 3). For the combination of siERK and siAkt, P <.05 compared to all other conditions.</p><p num="0218"> FIG. 71 is an image showing confirmation of Akt and ERK knockdown by Western blot analysis. Error bars indicate the sd of the iterative experiment. FIG. 72 is a graph showing surface TRAIL analysis of Akt and / or ERK after transient knockdown (48 hours after knockdown) in HCT116 cells (n = 3).</p><p num="0219"> TIC10 cooperatively results in nuclear translocation of their mutual substrate Foxo3a, which transcribes the TRAIL gene as a unique target gene that enhances cell death and potent antitumor effects in vivo. ) Causes double inactivation.</p><p num="0220"> Any patent or publication referred to herein is incorporated herein by reference to the same extent as if each individual publication was explicitly and individually indicated to be incorporated by reference. ..</p><p num="0221"> The compositions and methods described herein are currently representative and exemplary of preferred embodiments and are not intended to be limiting to the scope of the invention. Changes and other uses herein will be apparent to those skilled in the art. Such modifications and other uses may be made without departing from the scope of the invention as set forth in the claims.</p>
91 sheets
Sheet 1 Sheet 2 Sheet 3 Sheet 4 Sheet 5 Sheet 6 Sheet 7 Sheet 8 Sheet 9 Sheet 10 Sheet 11 Sheet 12 Sheet 13 Sheet 14 Sheet 15 Sheet 16 Sheet 17 Sheet 18 Sheet 19 Sheet 20 Sheet 21 Sheet 22 Sheet 23 Sheet 24 Sheet 25 Sheet 26 Sheet 27 Sheet 28 Sheet 29 Sheet 30 Sheet 31 Sheet 32 Sheet 33 Sheet 34 Sheet 35 Sheet 36 Sheet 37 Sheet 38 Sheet 39 Sheet 40 Sheet 41 Sheet 42 Sheet 43 Sheet 44 Sheet 45 Sheet 46 Sheet 47 Sheet 48 Sheet 49 Sheet 50 Sheet 51 Sheet 52 Sheet 53 Sheet 54 Sheet 55 Sheet 56 Sheet 57 Sheet 58 Sheet 59 Sheet 60 Sheet 61 Sheet 62 Sheet 63 Sheet 64 Sheet 65 Sheet 66 Sheet 67 Sheet 68 Sheet 69 Sheet 70 Sheet 71 Sheet 72 Sheet 73 Sheet 74 Sheet 75 Sheet 76 Sheet 77 Sheet 78 Sheet 79 Sheet 80 Sheet 81 Sheet 82 Sheet 83 Sheet 84 Sheet 85 Sheet 86 Sheet 87 Sheet 88 Sheet 89 Sheet 90 Sheet 91
Every citation, both ways
| Document | Relation | Office | Cited during |
|---|---|---|---|
| US10239877B2 | Cited by | United States of America | Applicant |
| JP2005509414A | Cites | Japan | – |
| WO2010149357A1 | Cites | World Intellectual Property Organization (WIPO) | – |
| WO2010009985A1 | Cites | World Intellectual Property Organization (WIPO) | – |
| JP2009526009A | Cites | Japan | – |
| JE Allen, et al.,Abstract 4502: The small molecule TIC10 has potent anticancer efficacy mediated by induction of TRAIL production in normal and tumor cells ,Cancer Res,2011年 4月15日,Vol.71, No.8(Supplement),p4502 | Non-patent | – | – |
| Chen J,Cisplatin-enhanced sensitivity of glioblastoma multiforme U251 cells to adenovirus-delivered TRAIL in vitro,Tumour Biol.,2010年 7月11日,Vol.31, No.6,p613-622 | Non-patent | – | – |
| Hetschko H, et al.,Upregulation of DR5 by proteasome inhibitors potently sensitizes glioma cells to TRAIL-induced apoptosis,FEBS J.,2008年 4月,Vol.275, No.8,p1925-1936 | Non-patent | – | – |
| Nagane M, et al.,Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxicity with tumor necrosis factor-related apoptosis-inducing ligand in vitro and in vivo,Cancer Res.,2000年 2月15日,Vol.60, No.4,p847-53 | Non-patent | – | – |
| Siegelin MD, et al.,KAAD-cyclopamine augmented TRAIL-mediated apoptosis in malignant glioma cells by modulating the intrinsic and extrinsic apoptotic pathway,Neurobiol Dis.,2009年 2月 6日,Vol.34, No.2,p259-266 | Non-patent | – | – |
| Yoshida T, et al.,Kaempferol sensitizes colon cancer cells to TRAIL-induced apoptosis,Biochem Biophys Res Commun.,2008年 8月 3日,Vol.375, No.1,p129-133 | Non-patent | – | – |
| Sung B, et al.,Celastrol, a triterpene, enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and up-regulation of death receptors,J Biol Chem.,2010年 4月 9日,Vol.285, No.15,p11498-11507 | Non-patent | – | – |
| Yoshida T, et al.,Lipoxygenase inhibitors induce death receptor 5/TRAIL-R2 expression and sensitize malignant tumor cells to TRAIL-induced apoptosis,Cancer Sci.,2007年 7月17日,Vol.98, No.9,p1417-1423 | Non-patent | – | – |
| Bevis KS, et al.,Anti-tumor activity of an anti-DR5 monoclonal antibody, TRA-8, in combination with taxane/platinum-based chemotherapy in an ovarian cancer mode,Gynecol Oncol.,2011年 1月 5日,Vol.121, No.1,p193-199 | Non-patent | – | – |
62 members in 18 offices
Priority claims3
| Document | Office | Kind | Date |
|---|---|---|---|
| 61480743 | United States of America | – | |
| 201161480743 | United States of America | P | |
| 2012035831 | United States of America | W |
Members62
| Document | Office | Kind | |
|---|---|---|---|
| CA2832342A1 | Canada | A1 | |
| US2012276088A1 | United States of America | A1 | |
| WO2012149546A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO2012149546A3 | World Intellectual Property Organization (WIPO) | A3 | |
| EP2701708A2 | European Patent Office (EPO) | A2 | |
| US8673923B2 | United States of America | B2 | |
| JP2014514326A | Japan | A | |
| MX2013012346A | Mexico | A | |
| US2014248264A1 | United States of America | A1 | |
| EP2701708A4 | European Patent Office (EPO) | A4 | |
| US9061032B2 | United States of America | B2 | |
| US9072744B1 | United States of America | B1 | |
| US2015202206A1 | United States of America | A1 | |
| US2015265619A1 | United States of America | A1 | |
| US9452165B2 | United States of America | B2 | |
| JP2016199580A | Japan | A | |
| US2017000790A1 | United States of America | A1 | |
| USRE46290E | United States of America | E | |
| US9629842B2 | United States of America | B2 | |
| JP6132833B2This record | Japan | B2 | |
| US2017224690A1 | United States of America | A1 | |
| JP2019019145A | Japan | A | |
| MX365365B | Mexico | B | |
| MX2019006379A | Mexico | A | |
| CA2832342C | Canada | C | |
| EP2701708B1 | European Patent Office (EPO) | B1 | |
| DK2701708T3 | Denmark | T3 | |
| PT2701708T | Portugal | T | |
| LT2701708T | Lithuania | T | |
| RS60186B1 | Serbia | B1 | |
| SMT202000272T1 | San Marino | T1 | |
| HRP20200636T1 | Croatia | T1 | |
| EP3679934A1 | European Patent Office (EPO) | A1 | |
| SI2701708T1 | Slovenia | T1 | |
| HUE049207T2 | Hungary | T2 | |
| ES2786033T3 | Spain | T3 | |
| PL2701708T3 | Poland | T3 | |
| CY1123010T1 | Cyprus | T1 | |
| JP2021185210A | Japan | A | |
| US2022288079A1 | United States of America | A1 | |
| JP2023060196A | Japan | A | |
| EP4335511A2 | European Patent Office (EPO) | A2 | |
| JP2024051080A | Japan | A | |
| EP4335511A3 | European Patent Office (EPO) | A3 | |
| EP3679934B1 | European Patent Office (EPO) | B1 | |
| FI3679934T3 | Finland | T3 | |
| US12036223B2 | United States of America | B2 | |
| DK3679934T3 | Denmark | T3 | |
| RS65813B1 | Serbia | B1 | |
| PT3679934T | Portugal | T | |
| LT3679934T | Lithuania | T | |
| SMT202400421T1 | San Marino | T1 | |
| HRP20241214T1 | Croatia | T1 | |
| JP7600286B2 | Japan | B2 | |
| ES2992934T3 | Spain | T3 | |
| US2024415841A1 | United States of America | A1 | |
| PL3679934T3 | Poland | T3 | |
| HUE068623T2 | Hungary | T2 | |
| MX379539B | Mexico | B | |
| SI3679934T1 | Slovenia | T1 | |
| JP2025159099A | Japan | A | |
| EP4335511B1 | European Patent Office (EPO) | B1 |
18 legal events, as the office reported them to INPADOC
Over the term
Point at a mark for the eventEvents
| Event | Code | |
|---|---|---|
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Receipt of annual feesJAPANESE INTERMEDIATE CODE: R250R250 | R250 | |
| Certificate of patent or registration of utility modelJAPANESE INTERMEDIATE CODE: R150R150 | R150 | |
| First payment of annual fees (during grant procedure)JAPANESE INTERMEDIATE CODE: A61A61 | A61 | |
| Written decision to grant a patent or to grant a registration (utility model)JAPANESE INTERMEDIATE CODE: A01A01 | A01 | |
| Decision of grant or rejection writtenTRDD | TRDD | |
| Request for written amendment filedJAPANESE INTERMEDIATE CODE: A821A521 | A521 | |
| Notification of reasons for refusalJAPANESE INTERMEDIATE CODE: A131A131 | A131 | |
| Request for written amendment filedJAPANESE INTERMEDIATE CODE: A523A521 | A521 | |
| Written request for extension of timeJAPANESE INTERMEDIATE CODE: A601A601 | A601 | |
| Notification of reasons for refusalJAPANESE INTERMEDIATE CODE: A131A131 | A131 | |
| Written request for application examinationJAPANESE INTERMEDIATE CODE: A621A621 | A621 | |
| Request for written amendment filedJAPANESE INTERMEDIATE CODE: A523A521 | A521 |
Numbers
- Publication
- 6132833
- Application
- 2014508181
Titles2
- Japanese
- 抗癌療法としての正常細胞および腫瘍細胞の小分子TRAIL遺伝子誘導
- English
- Small molecule TRAIL gene induction in normal and tumor cells as an anti-cancer therapy
Classification
- CPC, 18
- A61K31/519
- A61K39/39558
- A61K31/337
- A61K31/4188
- A61K31/4545
- A61K31/513
- A61P35/00
- A61P43/00
- G01N2800/52
- G01N2333/52
- A61K39/3955
- A61K45/06
- A61K2039/505
- G01N33/6863
- A61K9/0019
- A61K9/0053
- C07K16/22
- C07K2317/24
- IPC, 7
- A61K31 519
- A61K45 00
- A61K31 337
- A61K39 395
- A61P35 00
- C12N15 09
- C12Q1 68
