US 20110268722A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0268722 A1 Siegelin et al. (43) Pub. Date: Nov. 3, 2011 (54) COMBINATION THERAPIES WITH Publication Classification MTOCHONORAL-TARGETED (51) Int. Cl ANT-TUMIORAGENTS A638/17 (2006.01) CI2N 5/071 (2010.01) (76) Inventors: Markus D. Siegelin, Weehawken, A6IP35/00 (2006.01) NJ (US); Dario C. Altieri, A 6LX 39/395 (2006.01) Worcester, MA (US) C07D 225/06 (2006.01) (52) U.S. Cl. ..................... 424/130.1; 514/18.9; 540/461; (21) Appl. No.: 13/092,475 435/375 (57) ABSTRACT (22) Filed: Apr. 22, 2011 Described are mitochondria-targeted anti-tumor agents, death receptor agonists, autophagy inhibitors, and NF-KB Related U.S. Application Data signaling pathway inhibitors, and methods of making and (60) Provisional application No. 61/326,872, filed on Apr. using the same for the treatment of disorders associated with 22, 2010. unwanted cell proliferation. Patent Application Publication Nov. 3, 2011 Sheet 1 of 13 US 2011/0268722 A1 120% 8: 8: 8 CE 60% : 5 P_GA s : RA 2:: : P-GA-RA : : 8 N3 FAS Figure 1A Patent Application Publication Nov. 3, 2011 Sheet 2 of 13 US 2011/0268722 A1 igure 1B ---, igure 2A Figure 2B Patent Application Publication Nov. 3, 2011 Sheet 3 of 13 US 2011/0268722 A1 8. s::::::: se: -83.383.3 ---sixxxxx:----- --~~~x-x-x-xxii. : s: 8 i88 s: sig ::::::: ...i Figure 2C Figure 2D Figure 3A Figure 3B Patent Application Publication Nov. 3, 2011 Sheet 4 of 13 US 2011/0268722 A1 ::: :*:: & : : isix; {S3.g3-8 : Exx8:8-3 : {kari 333333. ::::: :: 3 8: 8 : x : x: 88: : S. 8 st::::: 34 gas $3. Figure 3D Patent Application Publication Nov. 3, 2011 Sheet 5 of 13 US 2011/0268722 A1 . f w i i : f Days O S 8 7 8 a death Figure 4 eige exte: vehicle PBS es s: RA -3- 888; 3. Figure 5A Patent Application Publication Nov. 3, 2011 Sheet 6 of 13 US 2011/0268722 A1 388838: After treatert Air: - -3.3888-89: & 8 223-8-3-3F 3888: '3's vehicle (PBS R & R& Ras 888 8 s. 3.88-3 as . .38F Figure 5B 25x100 20x1007 5x100 0x007 5.0x 106 Figure 6A Patent Application Publication Nov. 3, 2011 Sheet 7 of 13 US 2011/0268722 A1 ge:38 x 18 8. Ox of 8.0x108 40x1087 2x 18 Figure 6B 50 an Vehicle sks RA 100 ...s. pp x RA-PP 50 ine Figure 7 Patent Application Publication Nov. 3, 2011 Sheet 8 of 13 US 2011/0268722 A1 Figure 8 Patent Application Publication Nov. 3, 2011 Sheet 9 of 13 US 2011/0268722 A1 Figure 9 Patent Application Publication Nov. 3, 2011 Sheet 10 of 13 US 2011/0268722 A1 inst Figure 10 A. B C G Target siRNA 2. - as 100 7 E 53 3 25 . Ctraig5 (Ctrl ag5 Figure 11 B Figure 12 Patent Application Publication Nov. 3, 2011 Sheet 11 of 13 US 2011/0268722 A1 A 3. 2 8. Figure 13 A B 6 15 as & 4 10 2 E 5 i. 17AAC 7AA Figure 14 Patent Application Publication Nov. 3, 2011 Sheet 12 of 13 US 2011/0268722 A1 A B Citri siRNA target siRNA N Ox& xS& occces c lcHOP -acti Fores is st s's 8 ss& NF -- Fig. Figure 15 Target cMA Figure 16 Patent Application Publication Nov. 3, 2011 Sheet 13 of 13 US 2011/0268722 A1 Figure 17 US 2011/0268722 A1 Nov. 3, 2011 COMBINATION THERAPES WITH 0006. Also provided are methods of treating of a prolif MTOCHONORAL-TARGETED erative disorder in a Subject that include administering to the ANT-TUMIORAGENTS Subject at least one mitochondrial-targeted chaperone inhibi tor and at least one agent selected from the group of a death CROSS-REFERENCE TO RELATED receptor agonist, an autophagy inhibitor, and a NF-KB Sig APPLICATIONS naling pathway inhibitor, where the at least one mitochon 0001. This application claims priority to U.S. provisional drial-targeted chaperone inhibitor and the at least one agent application 61/326,872, filed Apr. 22, 2010, the contents of are administered in an amount Sufficient to treat of a prolif each of which are incorporated herein by reference in their erative disorder in the subject. entirety. 0007 Also provided are methods for enhancing cancer or tumor cell death that include administering to the Subject at TECHNICAL FIELD least one mitochondrial-targeted chaperone inhibitor and at 0002. This invention relates to the use of at least one mito least one agent selected from the group of a death receptor chondria-targeted inhibitor of molecular chaperones in com agonist, an autophagy inhibitor, and a NF-KB signaling path bination with one or more death receptoragonists, autophagy way inhibitor, where the at least one mitochondrial-targeted inhibitors, and/or NF-kB signaling pathway inhibitors for the chaperone inhibitor and the at least one agent are adminis treatment of disorders associated with unwanted cell prolif tered in an amount Sufficient to enhance cancer or tumor cell eration. death in a Subject. 0008. Some embodiments of the methods described BACKGROUND hereinfurther include identifying a subject having cancer or a 0003 Tumor cells exhibit an enhanced ability to survive tumor and/or determining whether cells of the cancer or and proliferate in highly unfavorable environments. They tumor have increased mitochondrial concentrations of a have been shown to down-regulate many of the cellular path chaperone as compared to a control cell. In some embodi ways that prevent normal (i.e., non-cancerous) cells from ments of the methods described herein, the at least one mito dividing in a hostile environment, and they also inactivate chondrial-targeted chaperone inhibitor and the at least one apoptotic pathways that bring about cell death in many nor agent are administered simultaneously to the Subject. In some mal tissues under adverse conditions. Tumor cells are also embodiments of the methods described herein, theat least one believed to up-regulate pathways required to maintain active agent is administered to the subject prior to the at least one proliferation. For example, many tumor cells activate the mitochondrial-targeted chaperone inhibitor, or the at least cellular stress-response pathway that allows tumor cells to one mitochondrial-targeted chaperone inhibitor is adminis synthesize and maintain the protein machinery they need to tered to the Subject prior to the at least one agent. In some continue proliferating. The activated stress response in embodiments of all of the methods described herein, the tumors includes up-regulation of heat-shock proteins (Hsps), proliferative disorder is a cancer (e.g., a cancer selected from which are ATPase-directed molecular chaperones. In particu the group of Small-cell lung cancer, non-Small cell lung can lar, Hsp90 is upregulated in many cancerous tissues. Hsp90 cer, colon cancer, colorectal cancer, and pancreatic cancer). controls the balance between folding/maturation and protea Somal destruction of a restricted number of client proteins, 0009. Also provided are methods of treating atherapeutic Some of which are involved in signal transduction and cell resistant cancer in a subject that include administering to a proliferation. Subject having cancer cells resistant to a cancer therapeutic at least one mitochondrial-targeted chaperone inhibitor and the SUMMARY cancer therapeutic, where the at least one mitochondrial targeted chaperone inhibitor and the cancer therapeutic are 0004. The present invention is based, at least in part, on the administered in amount Sufficient to treat the therapeutic discovery that inhibition of molecular chaperones in tumor resistant cancer. Some embodiments of these methods, fur cell mitochondria using mitochondrial-targeted chaperone ther comprise identifying a subject as having a therapeutic inhibitors potentiated the effect of death receptor agonists to resistant cancer. In some embodiments, the Subject is induce cell death, e.g., in cancer cells, and that mitochondrial identified by detecting an increase in NF-KB signaling activ targeted chaperone inhibitors induce autophagy and decrease NF-KB signaling pathway activity in cancer cells. ity in a cancer cell in the Subject. 0005 Provided herein are methods of enhancing apoptosis 0010 Also provided is the use of at least one mitochon in one or more mammalian cells that include contacting the drial-targeted chaperone inhibitor for the preparation of a one or more cells with at least one mitochondrial-targeted medicament for treatment of a proliferative disorder. Also chaperone inhibitor and at least one agent selected from the provided is the use of at least one death receptor agonist, group of a death receptor agonist, an autophagy inhibitor, autophagy inhibitor, and NF-KB signaling pathway inhibitor and a NF-kB signaling pathway inhibitor, where the at least in the preparation of a medicament for treatment of a prolif one mitochondrial-targeted chaperone inhibitor and the at erative disorder, characterized in that the treatment includes least one agent are in an amount Sufficient to enhance apop administering at least one mitochondrial-targeted chaperone tosis in the one or more cells. In some embodiments, the one inhibitor. Also provided is the use of at least one mitochon or more cells are contacted sequentially with the at least one drial-targeted chaperone inhibitor in the preparation of a mitochondrial-targeted chaperone inhibitor and the at least medicament for treatment of a proliferative disorder, charac one agent. In some embodiments, the one or more cells are terized in that the treatment includes administering at least contacted simultaneously with the at least one mitochondrial one death receptor agonist, autophagy inhibitor, and NF-KB targeted chaperone inhibitor and the at least one agent. signaling pathway inhibitor. US 2011/0268722 A1 Nov.