NZ616474B2 - Combination of anti-clusterin oligonucleotide with hsp90 inhibitor for the treatment of prostate cancer - Google Patents

Abstract

The disclosure relates to the use of an oligonucleotide which reduces clusterin expression and a Heat Shock Protein 90 (Hsp90) inhibitor of the formula shown in the abstract figure for the treatment of prostate cancer. The disclosure also relates to pharmaceutical compositions comprising an amount of an oligonucleotide which reduces clusterin expression, and an Hsp90 inhibitor. f an oligonucleotide which reduces clusterin expression, and an Hsp90 inhibitor.

Description

/000696 COMBINATION OF ANTI-CLUSTERIN OLIGONUCLEOTIDE WITH HSPQO INHIBITOR FOR THE TREATMENT OF TE CANCER This application claims priority' of U.S. Provisional Application No. 61/453,102, filed. March 15, 2011, the contents of which are hereby incorporated by reference.

Throughout this ation, various publications are nced, including referenced in hesis. Full citations for publications referenced in parenthesis may be found listed in alphabetical order at the end of the specification immediately preceding the claims. The disclosures of all referenced publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention ns.

Field of the Invention The t invention relates to combination therapy for treating prostate cancer.

Background of the Invention Prostate cancer (PCa) is the most common cancer and the third most common cause of cancer related. mortality in men in the United States (Jemal et al., 2006). Androgen on remains the standard effective therapy for patients with advanced PCa, inhibiting proliferation and inducing apoptosis in tumor cells (Kyprianou et al., 1990). Unfortunately, after short—term remissions, surviving tumor cells recur with castrate resistant prostate cancer (CRPC) and death usually within 3 years in most men (Gleave et al., 1999).

CRPC progression results from rechanisms attributed to re— activation of androgen receptor axis (Knudsen et al., 2009), alternative mitogenic growth factor pathways (Miyake et al., 2000; Culig et al., 2004), and stress-induced vival gene (Gleave et al., 1999; Miyake et al., 1999) and Cytoprotective Chaperone networks (Rocchi et al., 2004; Miyake et al., 2000). To significantly improve survival in men with PCa, new therapeutic gies to inhibit the appearance of this phenotype must be developed. It has been observed that numerous ns are expressed in increased amounts by prostate tumor cells following androgen withdrawal. At least some of these proteins are assumed to be associated. with the observed. apoptotic cell death which is observed upon androgen awal. (Raffo et al., 1995; Krajewska et al., 1996; McDonnell et al., 1992). The functions of many of the proteins, however, is not completely understood“ Clusterin (also known as sulfated glycoprotein—2 (SGP—Z) or TRPM~2) is within this latter category.

Clusterin Clusterin is a cytoprotective chaperone protein that promotes cell al and confers broad—spectrum ance to cancer treatments (Chi et al. 2005). In Sensibar et al., Cancer Research 55: 2431— 2437, 1995, the authors reported on LNCaP cells transfected with a gene encoding clusterin, and watched to see if expression of this n altered the effects of tumor necrosis factor a (TNFd), to which LNCaP cells are very sensitive. Treatment of the transfected LNCaP cells with TNFd was shown to result in a transient increase in clusterin levels for a period of a few hours, but these levels had dissipated by the time DNA fragmentation preceding cell death was observed.

As bed in U.S. Patent No. 7,534,773, the contents of which are incorporated. by reference, enhancement of castration—induced tumor cell death and delay of the progression of en—sensitive cancer cells to androgen—independence may be achieved by inhibiting the expression of clusterin by the cells.

Custirsen Custirsen is a second—generation antisense ucleotide that inhibits clusterin expression. Custirsen is designed specifically to bind to a portion of clusterin mRNA, resulting in the inhibition of W0 2012/123823 the production of clusterin protein. The structure of custirsen is available, for example, in U.S. Patent No. 6,900,187, the contents of which are incorporated herein by reference. A broad range of studies have shown that custirsen potently regulates the expression of U] clusterin, facilitates apoptosis, and izes cancerous human prostate, breast, ovarian, lung, renal, bladder, and melanoma cells to herapy (Miyake et al. 2005), see also, U.S. Patent Application Publication No. 2008/0119425 A1. In a clinical trial for androgen—dependent prostate cancer, the drugs flutamide and buserelin were used together in combination with custirsen, increasing te cancer cell sis (Chi et al. 2004; Chi et al., 2005).

Heat shock protein 90 (Hsp90) is an —dependent molecular chaperone required for protein folding, maturation and conformational stabilization of many “client" proteins ' et al., 2000; Kamal et al., 2003). Hsp90 interacts with several proteins involved in CRPC, including growth factor receptors, cell cycle regulators and signaling kinases like Akt, androgen or (AR) or Raf—l, (Whitesell. et al., 2005; Takayama. et al., 2003).

Tumor cells s higher Hsp90 levels compared with benign cells (Kamal et al., 2003; Chiosis et al., 2003), and Hsp90 inhibition has emerged as an exciting target in CRPC and other cancers. Many Hsp90 inhibitors were developed targeting its ATP—binding pocket, ing natural compounds such as geldanamycin and its analogs, or synthetic compounds. These agents have been shown to t Hsp90 function and induce apoptosis in preclinical studies of colon, breast, PCa and other cancers (Kamal et al., 2003; Solit et al., 2003; Solit et al., 2002).

Combination Therapy The administration of two drugs to treat a given condition, such as prostate cancer, raises a number of potential problems. In vivo ctions between two drugs are complex. The effects of any single drug are related to its absorption, distribution, and W0 2012/123823 elimination. When two drugs are introduced into the body, each drug can affect the absorption, bution, and. elimination of the other and hence, alter the effects of the other. For instance, one drug may inhibit, activate or induce the production of enzymes ed in. a metabolic route of elimination of the other drug (Guidance for Industry. In Vivo drug' metabolism/drug interaction studies — study , data is, and recommendations for dosing" and. labeling). Thus, when two drugs are administered. to treat. the same condition, it is unpredictable whether* each. will complement, have no effect on, or interfere with, the eutic ty of the other in a human subject.

Not only may the interaction between two drugs affect the intended eutic activity of each drug, but the interaction may increase the levels of toxic metabolites (Guidance for Industry. In vivo drug metabolism/drug interaction studies - study design, data analysis, and recommendations for dosing and labeling). The interaction may also heighten or lessen the side effects of each drug. Hence, upon administration of two drugs to treat a disease, it is unpredictable what change will occur in the profile of each drug.

Additionally, it is difficult to accurately predict when the effects of the interaction between the two drugs will become manifest. For example, metabolic ctions between drugs may become apparent upon the initial administration of the second drug, after the two have reached. a steady~state concentration or upon discontinuation of one of the drugs (Guidance for Industry. In vivo drug metabolism/drug interaction s — study design, data 3O analysis, and recommendations for dosing and labeling).

Thus, the success of one drug or each drug alone in an in Vitro model, an animal model, or in humans, may not correlate into efficacy when both drugs are administered to humans.

Summarx of the Invention In one aspect of the present invention there is provided a use of i) an nse or RNAi oligonucleotide that is complementary to the sequence of clusterin of a mammalian subject, and that s clusterin expression; and ii) a Heat Shock Protein 90 (Hsp90) inhibitor comprising 4—(6,6— Dimethyl—4—oxo—3—trifluoromethyl—4,5,6,7—tetrahydro—indazol—l~y1)— 2—(4~hydroxy—cyclohexylamino)—benzamide, or a pharmaceutically acceptable salt thereof, or a prodrug that is metabolized to release 4—(6,6—Dimethyl—4—oxo—3—trifluoromethyl—4,5,6,7—tetrahydro— l—l—yl)—2-(4—hydroxy—cyclohexylamino)~benzamide in the manufacture of a medicament for treating prostate cancer in the mammalian subject.

The present invention provides a method for treating a mammalian subject affected by prostate cancer sing administering to the ian subject i) an oligonucleotide which reduces clusterin expression and ii) a Heat Shock Protein 90 (Hsp90) inhibitor having the structure: 0 NH2 or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—C14 alkyl, C1-C10 haloalkyl, C3-C7 lkyl, heterocycloalkyl, C1—C5 acyl, aryl, or heteroaryl, [followed by page 5a] wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is optionally substituted with from 1-4 groups that are independently C1—C6 alkyl, C1—C6 alkoxy, halogen, y, amino, mono- or di-(Cy4k)alkylamino, nitro, halo(C1—C6)alkyl, halo(C1-C6)alkoxy, or carboxamide, wherein when R1 is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are ally replaced independently by R4, carbonyl, ethenyl, ethynyl or a moiety selected from N, O, S, 302, or SO, with the proviso that two 0 atoms, two S atoms, or an O and S atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, [followed by page 6] W0 2012/123823 (ii) aryl, (iii) saturated or unsaturated C3—C3 cycloalkyl, or (iv) saturated or unsaturated C2—Cm heterocycloalkyl, each aryl, aryl, saturated or unsaturated lkyl, or saturated or unsaturated heterocycloalkyl, independently, is optionally substituted with at least one group, which independently is hydroxy, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, —S—(C1-C6) alkyl, -SOz(C1—C6) alkyl, -SOg— aryl, 1—C6)alkyl, yl, —SOZNH2, ’SOzNH— (C1‘C6) alkyl, —SOZNH-aryl, (C1- C6)alkoxy, or mono- or di—(Cl— Cm)alkylamino; and R4 is optionally fused to a C6—Cm aryl group, C5-C8 ted cyclic group, or' a C4—C10 heterocycloalkyl group; and R1 is optionally substituted at any available position with C1—Cm alkyl, C1—Cm haloalkyl, C2~Cm alkenyl, C2— Cm alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro, —SH, ~S—(C1—C6)alkyl, —SO;—(C1~ yl, —SO7NH;, ~SO;NH(C1-C6)alkyl, -SO;NH—ary1, —SO;- aryl, ~SO—(C1C6)alkyl, ~SO;—aryl, C1—C6 alkoxy, C2-Cx alkenyloxy, C2-Cm alkynyloxy, mono— or di*(C1— Cm)alkylamino, —C;-Cm alkyl—Z, —OC1—Cm alkyl—Z, or Ra wherein Z is ORC or —N(Ra2, wherein each R6 is independently -H or C1—C6 alkyl, or N(Ra2 represents pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, 1,3— or 1,4— diazepanyl, or morpholinyl, each of which is optionally substituted with hydroxy, amino, aminoalkyl, C1—C6 alkyl, mono— or di(Cl—C6)alkylamino, C1—C6 , or halogen, and U1 DU0 is —H, ~C1—C10 alkyl, —C2—C10 alkenyl, —c2—C-_c alkynyl, aryl, heteroaryl, or —C1-C6 acyl; R5 is (l) heteroaryl , (2) aryl, (3) saturated. or unsaturated. C5-Cm cycloalkyl, or (4) saturated. or unsaturated C5—Cm heterocycloalkyl, and the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, —SH, -S-(C1-C5)alkyl, Cl—C6)alkyl, —SOz-aryl, ~SO—(C1—C5) alkyl, yl, 2, -SOZNH— (C1—C6) alkyl, -802NH—aryl, (C1—C6)alkoxy, or mono— or di-(Cl-Cm)alkylamino; R2 is H, Cl, halogen, 0E5, CHE), CH3, C1~Cm alkyl, or 1—C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CHM or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

Hsp90 inhibitors having structure above are described in U.S.

Patent No. 7,928,135, the entire contents of which are hereby incorporated herein by reference.

The present invention es a HEthOd for treating a mammalian t affected by prostate cancer comprising administering to the mammalian subject i) an oligonucleotide which reduces clusterin expression and ii) a Hsp90 inhibitor, which inhibitor is other than Hsp90i—l, each in an amount that when in combination with the other is ive to treat the mammalian subject.

The present invention provides a method for treating a mammalian subject ed by prostate cancer comprising administering to the mammalian. subject i) an oligonucleotide which reduces clusterin expression and. ii) a Hsp9O tor which binds to Hsp90d and Hsp9OB with a Ka of less than 50 nmol/L, or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

The present ion provides a pharmaceutical composition comprising an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor having the structure: 0 NW 0 or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CM alkyl, C1—Cm haloalkyl, C3—C7 cycloalkyl, heterocycloalkyl, C1—C6 acyl, aryl, or aryl, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is ally substituted with from 1—4 groups that are independently C1—C6 alkyl, C1—C6 alkoxy, halogen, hydroxy, amino, mono— or di—(Cl— C6)alkylamino, nitro, halo(C1—C6)alkyl, halo(C1—C6)alkoxy, or carboxamide, wherein when R; is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are optionally U1 replaced independently by R4, carbonyl, ethenyl, l or a moiety selected from N, O, S, 802, or 80, with the proviso that two 0 atoms, two 8 atoms, or an O and 8 atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii) saturated. or unsaturated C3—C3 cycloalkyl, or (iv) saturated or unsaturated. C2*Cx cycloalkyl, wherein each aryl, heteroaryl, saturated or unsaturated cycloalkyl, or saturated or unsaturated heterocycloalkyl, independently, is optionally tuted with at least one group, which independently is hydroxy, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, -S—(C1-C6) alkyl, —807(C1-C5) alkyl, -SO;- aryl, —SO—(C1—C6)alkyl, yl, ~SO;NH” —802NH—(Cl-C5)alkyl, —SOzNH—aryl, (C1— C6)alkoxy, or mono— or di—(Cl— Cm)alkylamino; and R4 is optionally fused to a C6—Cm aryl group, C5—Cg saturated cyclic group, or' a C4—C10 heterocycloalkyl group; and R1 is optionally substituted at any available position with C1—C3 alkyl, C1—Cm haloalkyl, C2—Cm alkenyl, C2— Cm alkynyl, hydroxy, y, carboxamido, oxo, halo, W0 2012/123823 amino, cyano, nitro, —SH, ~S—(C1—C6)alkyl, —SOz—(Cl— C5)alkyl, —SOZNH2, —SOZNH(C1-C6)alkyl, —SOZNH-aryl, -SOZ— aryl, —SO—(C1C6)alkyl, -SOz-aryl, C1—C6 alkoxy, C2—C3 loxy, C2—Cm alkynyloxy, mono— or di—(Cl— ylamino, —C;-Cm alkyl—Z, —OC1—Cm alkyl—Z, or Rm wherein Z is ORG or —N(Raz, wherein each R6 is independently —H or C1‘C6 alkyl, or N(RQ2 represents pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, l,3~ or 1,4— diazepanyl, or Hmrpholinyl, each. of which is optionally substituted with y, amino, aminoalkyl, C1-C6 alkyl, mono— or di(C1-C6)alkylamino, C1—C6 alkoxy, or halogen, and R0 is ~H, —C1-Cm alkyl, —C2-Cm alkenyl, —C2-Cm alkynyl, aryl, heteroaryl, or -%h—C6 acyl; R5 is 2O (l) heteroaryl, (2) aryl, (3) saturated. or rated C5~Cx cycloalkyl, or (4) saturated. or unsaturated. C5—C3 heterocycloalkyl, the R5 groups are optionally tuted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, —SH, ~S*(C1—C5)alkyl, —SOz—(C1—C6)alkyl, —802—aryl, -SO—(C1—C6) alkyl, —SO—aryl, —SOZNH2, —SOZNH— (C1-C5) alkyl, —SOzNH—aryl, (C1-C6)alkoxy, or mono- or di—(Cl-Cm)alkylamino; is H, Cl, halogen, CF3, CHF2, CH3, C1—Cm alkyl, or halo(C1—C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CH3 or a prodrug thereof, for use in treating a ian subject affected by prostate cancer.

Hsp90 inhibitors having structure above are described in U.S.

Patent No. 7,928,135, the entire contents of which are hereby incorporated herein by reference.

The present invention provides a pharmaceutical composition comprising an amount of an oligonucleotide which s clusterin sion, and a Hsp90 inhibitor, which inhibitor is other than Hsp90i—l, for use in treating a mammalian subject affected by prostate cancer.

The present invention provides a pharmaceutical composition sing an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor which binds to Hsp90d and Hsp9OB with a Ka of less than 50 nmol/L, or a prodrug thereof, for use in treating a mammalian subject affected by prostate cancer.

The present ion provides an oligonucleotide which reduces clusterin sion for use in combination with a Hsp90 inhibitor having the ure: or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CM alkyl, C1-Cw haloalkyl, C3-C7 cycloalkyl, cycloalkyl, C1—C6 acyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is optionally substituted with from U} 1-4 groups that are independently C1—C6 alkyl, C1—06 alkoxy, halogen, hydroxy, amino, mono— or di—(Cl— C6)alkylamino, nitro, halo(C1—C6)alkyl, 1—C5)alkoxy, or carboxamide, wherein when R1 is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are optionally replaced independently by RA, carbonyl, ethenyl, ethynyl or a moiety selected from N, O, S, 802, or SO, with the proviso that two 0 atoms, two S atoms, or an O and S atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii) saturated or unsaturated C3-C3 cycloalkyl, or (iv) saturated or unsaturated, C2-Cx cycloalkyl, wherein each aryl, heteroaryl, ted or unsaturated cycloalkyl, or saturated or unsaturated heterocycloalkyl, independently, is optionally substituted with at least one group, which independently is hydroxy, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, ~S-(C1-C6) alkyl, -SOZ(C1-C6) alkyl, —SO;— aryl, -SO—(C1-C6)alkyl, —SO-aryl, -SO2NHh -SOZNH-(C1-C5)alkyl, -aryl, (C1- C6)alkoxy, or mono— or — Cm)alkylamino; and R4 is optionally fused to a C6-C13 aryl group, C5-C8 saturated cyclic group, or a C4-C10 heterocycloalkyl group; and R1 is optionally substituted at any available position with C1—C3; alkyl, C1—C10 haloalkyl, C2-C10 alkenyl, C2— Clo alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro, —SH, —S—(C1—C6)alkyl, —SOz—(C1- C5)alkyl, -SOZNH2, wSOgNH(Cl-C6)alkyl, ~SOgNH—aryl, —SOZ— aryl, -SO—(C1C6)alkyl, -SOZ-aryl, C1~Cb , C2“C’_O loxy, C2—C10 alkynyloxy, mono— or di— (C1— Clo)alkylamino, —C-_—C10 alkyl—Z, ~OC1—Cw alkyl—Z, or R5, wherein Z is ORG. or —N(R6)2, wherein each R6 is independently —H or C1—C6 alkyl, or N(R6)2 represents pyrrolidinyl, piperidinyl, zinyl, azepanyl, 1,3- or 1,4- anyl, or morpholinyl, each of which is optionally substituted with hydroxy, amino, aminoalkyl, C1-C6 alkyl, mono- or di (C1-C6) alkylamino, C1-C6 , or halogen, and R0 is -H, -C1-Clo alkyl, -C2-C10 alkenyl, -s2~C-_o alkynyl, aryl, heteroaryl, or -C1-C6 acyl; (l) heteroaryl, (2) aryl, (3) saturated or unsaturated C5“C;o lkyl, or (4) saturated or unsaturated C5-C;o heterocycloalkyl, the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, -SH, -S-(Cl-C5)alkyl, cl—c6)a1ky1, —SOZ—aryl, 1—C5) alkyl, -SO-aryl, -SOZNH2, —SOZNH- (C1-C6) alkyl, -aryl, (C1-C6)alkoxy, or mono— or -Cm)alkylamino; U‘ R2 is H, Cl, halogen, CFa CHE}, CH3 C1—Cm alkyl, or halo(C1—C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CH“ or a prodrug thereof, in treating a mammalian t ed by prostate cancer.

Hsp90 inhibitors having structure above are described in U.S.

Patent No. 7,928,135, the entire contents of which are hereby incorporated herein by reference.

The present invention provides an oligonucleotide which reduces clusterin expression for use in combination with a Hsp90 inhibitor, which inhibitor is other than Hsp90i-l, in treating' a mammalian subject affected by prostate cancer.

The present invention provides an oligonucleotide which reduces clusterin expression for use in ation with a Hsp90 tor which binds to Hsp90d and Hsp905 with a Ka of less than 50 nmol/L, or a prodrug thereof, in treating a mammalian subject affected by prostate cancer.

The present invention provides a composition for treating a mammalian subject affected, by prostate cancer comprising' i) an oligonucleotide which reduces clusterin expression and ii) a Hsp9O inhibitor having the structure: 2012/000696 or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CN alkyl, C1-Cu; haloalkyl, C3‘Cy cycloalkyl, heterocycloalkyl, C1—C6 acyl, aryl, or heteroaryl, n each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is optionally substituted with from 1—4 groups that are independently C1—C6 alkyl, C1~C6 alkoxy, halogen, hydroxy, amino, mono- or — C6)alkylamino, nitro, halo(C1-C6)alkyl, halo(C1—C6)alkoxy, or carboxamide, wherein when R1 is a C1-Cm alkyl group, up to five of the carbon atoms in the alkyl group are optionally replaced independently by R4, carbonyl, ethenyl, ethynyl or a moiety selected from N, O, S, 802, or 80, with the o that two 0 atoms, two S atoms, or an O and 8 atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii)saturated or unsaturated C3~Cm cycloalkyl, or (iv) saturated, or unsaturated. C2-C3 heterocycloalkyl, each aryl, heteroaryl, saturated or unsaturated cycloalkyl, or saturated or unsaturated heterocycloalkyl, VV()2012/123823 independently, is optionally substituted with at least one group, which independently is hydroxy, halo, amino, cyano, y, carboxamido, nitro, oxo, —S—(C1—C6) alkyl, ~SOZ(C1—C6) alkyl, —SOZ— aryl, 1—C5)alkyl, —SO—aryl, —SOZNH2, — ) alkyl, —SOZNH—aryl, (Cl— C6)alkoxy, or mono— or di—(Cl~ C10)alkylamino; and R4 is optionally fused to a C6—Cm aryl group, C5—C8 saturated cyclic group, or a. C4—C10 heterocycloalkyl group; and R1 is optionally substituted at any available position with C1—C;o alkyl, C1—C10 haloalkyl, C2—C10 alkenyl, C2~ Cm alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro, -SH, —S—(C1—C6)alkyl, -SOz—(C1— C6)alkyl, —SOZNH2, —SOZNH(C1—C6)alkyl, —SOZNH-aryl, —SOZ- aryl, —SO-(01C6)alkyl, ~SOZ-aryl, C1-C6 alkoxy, CZ-Cx alkenyloxy, C2—Cm alkynyloxy, mono— or di-(Cl— Cm)alkylamino, ~C;—Cm alkyl—Z, ~OC1—Cw alkyl—Z, or Rm wherein Z is ORO or —N(Rd2, wherein each R6 is independently -H or C1—C6 alkyl, or N(Ra2 represents pyrrolidinyl, piperidinyl, zinyl, azepanyl, 1,3— or 1,4— diazepanyl, or erpholinyl, each of which is optionally tuted with hydroxy, amino, aminoalkyl, C1—C6 alkyl, mono— or C6)alkylamino, C1-C6 alkoxy, or halogen, and R0 is —H, —C1—Cm alkyl, —C2—Cm alkenyl, —C2—Cm alkynyl, aryl, heteroaryl, or -{h—C6 acyl; R5 is (l) heteroaryl, -16~ WO 23823 (2) aryl, (3) ted or unsaturated CS—Cx cycloalkyl, or (4) saturated or unsaturated C5—Cm heterocycloalkyl, the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, “SH, -C5)alkyl, —SOg—(C1~C6)alkyl, -SOZ~aryl, —SO—(C1—Cb) alkyl, —SO—aryl, —SOZNH2, 430an— (C1—C6) alkyl, —802NH—aryl, (C1—C6)alkoxy, or mono— or —Cm)alkylamino; R2 is H, Cl, halogen, CFy CHE” CH3, C1—Cm alkyl, or halo(C1—C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CHW or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

The present invention es a composition for treating a mammalian subject affected. by prostate cancer comprising i) an oligonucleotide which reduces clusterin expression and ii) a Hsp90 inhibitor, which inhibitor is other than Hsp90i—l, each in an amount that when in combination with the other is effective to treat the mammalian subject.

The present invention provides a composition for treating a mammalian subject ed, by prostate cancer comprising' i) an oligonucleotide which s clusterin expression and ii) a Hsp90 inhibitor which binds to HspBOd and Hsp9OB with a Ka of less than 50 nmol/L, or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

W0 2012/123823 Brief Description of the Drawings Figure 1. Hsp90i-l and Hsp90i-2 induce HSPs and clusterin (CLU) sion 511 prostate cancer(PCa) cells in vitro. PC—3 and LNCaP cells were treated with lnM Hsp90i-2 (A) or 1 HM Hsp90i—l (C) for the ted time points. In parallel, PC—3 and LNCaP cells were treated for 48h with Hsp90i-2 for the indicated doses (B). Protein ts were analyzed for CLU, Hsp70, Akt and vinculin. Tumor cells were treated for 24h with lpM Hsp90i—2 or luM Hsp90i—l (D). mRNA extracts were analyzed. by ime PCR for CLU, Hsp90 and Hsp70. ***, p<0.0Dl.

Figure 2. Hsp901-2 induces HSP and CLU expression in PCa afts. Mice were treated for 6 weeks with 50mg/kg' Hsp90iPRO (the prodrug' of Hsp90i-2) or vehicle (Control). A, tumors were collected and CLU and Hsp70 were evaluated by immunohistochemical analysis. B, total proteins were extracted from the xenograft tumors and CLU expression was analyzed by western blotting. The relative levels were normalized with GAPDH and ted in densitometric units. ***, p<0.00l.

Figure 3. CLU ion following Hsp90 inhibitor treatment is cytoprotective via an increase of HSF-l activity. A, LNCaP cells were treated with indicated concentrations of Hsp90i—l or Hsp90i—2 for 48h. B, LNCaP cells were transiently transfected with indicated concentrations of CLU~plasmid for 48h.

Total amount of plasmid DNA transfected was normalized to Zug per well by the addition of an -18— W0 2012/123823 empty vector. C (Top), LNCaP cells were transfected with ZOnM CLU siRNA or control siScr, followed of Hsp90i—l or Hsp90i—2 treatment (luM) for 48h. C (bottom), LNCaP cells were treated twice with 300nM custirsen. or control Sch ASO. D, LNCaP and. PC—3 cells were d. twice with 300nM custirsen or control SchB ASO, followed by lpM of —l or Hsp90i~2 for 48h. Cells were harvested, and. HSE- luciferase activity or western blotting analyses were performed. Means of at least three independent experiments done in triplicate. ***, p<0.00l; *, p<0.05; ns, not significant.

Figure 4. Increased potency of CLU knockdown and Hsp90 inhibitor ation treatment in PCa cells. A, LNCaP cells were treated twice with 300nM custirsen or control Sch ASO, followed by the indicated concentration of Hsp90i-l or Hsp90i—2 for 48h. Cell growth was determined by crystal violet and compared with control. B, dose dependent effects and combination index (CI) values calculated by CalcuSyn software were assessed in LNCap cells treated for 48h with custirsen alone, Hsp90i—2 alone or combined treatment at indicated concentration with constant ratio design between both drugs. The CI for EDW and EDm was 0.4 and 0.75, respectively, indicative of a combination effect of this ed ent. C and D, LNCaP cells were treated twice with 300nM custirsen or control Sch, followed by lpM Hsp90i-l or -2 for 48h. Cells were harvested, and n blotting analyses were med (C). The proportion of cells in subGl, GO-Gl, S, GZ-M was W0 2012/123823 determined by propidium iodide staining and e— 3 activity was determined on the cell s and the results are expressed in arbitrary units and corrected. for protein content (D).All experiments U1 were ed at least . $$$, p<0.001; ***, p<0.00l; **, p<0.0l *, p<0.05.

Figure 5. Increased potency of custirsen + Hsp90i-1 combination in PC-3 aft model. Mice were treated IP with 25mg/kg Hsp90i-l and lSmg/kg sen starting when tumors reached 300mm as described in Example 6. A, The mean tumor volume of mice custirsen + Hsp90i—l was compared with control Sch ASO + Hsp90i—l i SEM (n=7). **, p<0.01. B, in Kaplan-Meier curve, cancer~specific survival was compared between mice treated with custirsen + Hsp90i-l and control Sch ASO + Hsp90i-l over a 72—d period. *, p<0.05. C, tumors were collected after 72-d and CLU, Ki67 and TUNEL were evaluated by immunohistochemical analysis (original magnification: x200).

Figure 6. sed potency of custirsen + Hsp90iPRO combination in LNCaP xenograft model. Mice were treated with 25mg/kg Hsp90i-2—PRO and lSmg/kg custirsen starting when serum PSA values ed to pre—castration levels. The mean tumor volume (A) and the serum PSA level (B) were compared between the 4 groups i SEM (n=lO). ***, p<0.00l. C, PSA doubling time and velocity were calculated as described in Example 6. *, p<0.05. D, in —Meier curve, cancer-specific survival was compared between the 4 -20_ W0 2012/123823 groups over a 62—d period. ***, p<0.00l.

Progression—free survival was defined. as time for the first tumor volume doubling.

Figure 7. Increased potency of custirsen + Hsp90i—2—PRO combination treatment apoptosis levels in CRPC LNCaP tumors. A, tumors were collected after 57 days and CLU, Ki67, AR, AKT and TUNEL were evaluated. by immunohistochemical analysis (original magnification: x200). 3, total proteins were extracted from the xenograft tumors and CLU, AR, Akt and PSA were analyzed by western blotting. The relative levels were normalized with. vinculin and estimated in densitometric units rSEM.

Figure 8. rin protects tumor cells to Hsp90 tors via a regulation of HSF-l. A, PC—3 cells were transfected to overexpress CLU compared to wt—PC—3 and treated with indicated concentrations of Hsp90i~ 2 for 48h. Cell growth. was determined. by crystal Violet and compared. with. l. **, pS0.0l. 3, tumor cells were treated with ZOnM HSF—l siRNA vs control Scr siRNA and treated with uM Hsp90i—2 for 48h. Total proteins were extracted and, western blotting and caspase 3/7 activity were med. C, PC-3 cells were d with ZOnM CLU siRNA vs control Scr siRNA and treated with luM Hsp90i—1 for 24h. HSF—l localization was assessed by immunofluorescence ng.

Figure 9. LNCaP and PC—3 cells were treated twice with 300nM custirsen or control Sch ASO, followed by luM of 2012/000696 Hsp90i~l or Hsp90i—2 for 48h. Cells were harvested, and HSE-luciferase activity or western blotting analyses were performed.

Detailed Description of the Invention The t invention provides a method for treating a mammalian subject affected by prostate cancer comprising administering to the mammalian subject i) an oligonucleotide which reduces rin expression and ii) a Heat Shock Protein 90 (Hsp90) inhibitor having the structure: 0 NH2 or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CM alkyl, C1-Cm haloalkyl, C3-C7 cycloalkyl, heterocycloalkyl, C1—C6 acyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is optionally tuted with from 1-4 groups that are ndently C1~C6 alkyl, C1-C6 , halogen, hydroxy, amino, mono— or di-(Qr C6)alkylamino, nitro, 1*C6)alkyl, halo(C1-C5)alkoxy, or carboxamide, wherein when R1 is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are optionally replaced independently by R4, carbonyl, ethenyl, ethynyl or a moiety selected from N, O, S, 802, or SO, with the proviso that two 0 atoms, two 8 atoms, or an O and S atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii) saturated or unsaturated C3—Cx cycloalkyl, or (iv) saturated or rated Cg‘Cx U] heterocycloalkyl, wherein each aryl, heteroaryl, saturated or rated cycloalkyl, or ted or unsaturated heterocycloalkyl, independently, is optionally substituted with at least one group, which ndently is hydroxy, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, ~S-(C1—C6) alkyl, —SOZ(C1—C6) alkyl, —SOZ— aryl, ~SO—(C1—C6)alkyl, -SO—aryl, ~SOZNHE -SOzNH-(C1~C6)alkyl, -SOZNH—aryl, (Cl- C6)alkoxy, or mono- or di-(Cl- ylamino; and R4 is optionally fused to a C6-Cm aryl group, C5-C3 saturated cyclic group, or a Ch-Clo heterocycloalkyl group; and R1 is optionally substituted at any ble position with (h-C;o alkyl, C1—Clo kyl, C2—C10 alkenyl, C2- Cm alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro, —SH, —S—(C1~C5)alkyl, —SO;—(C1— Cg)alkyl, -807NH7, -SO;NH(C1-C6)alkyl, -507NH-aryl, —SO;- aryl, —SO~(C1C6)alkyl, —SOQ—aryl, C1—C5 alkoxy, C2—C1 alkenyloxy, C2—Cw alkynyloxy, mono- or di—(Cl— Cm)alkylamino, —C;—Cm alkyl~Z, —OC1—Cm alkyl—Z, or Ra wherein Z is ORo or —N(Raz, wherein each R6 is independently —H or C1—C6 alkyl, or N(Ra2 represents pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, 1,3— or 1,4— diazepanyl, or erpholinyl, each of which is ally substituted with hydroxy, amino, aminoalkyl, C1-C6 alkyl, mono— or di(C1—C6)alkylamino, C1—C6 alkoxy, or n, and R0 is —H, —C1—C10 alkyl, —C2—C10 alkenyl, —C2-C-_o l, aryl, heteroaryl, or ~C1-C6 acyl; (l) heteroaryl, (2) aryl, (3) saturated or unsaturated. C5-Cm cycloalkyl, or (4) saturated or unsaturated. C5—Cx heterocycloalkyl, and the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, -SH, -S-(C1-C5)alkyl, -SOz-(C1-C6)alkyl, —SOz-aryl, —SO—(C1—C5) alkyl, —SO—aryl, —SOZNH2, —SOZNH— (Cl—C6) alkyl, —SOZNH—aryl, (C1—C6)alkoxy, or mono— or di—(Cl-Cm)alkylamino; H, Cl, halogen, CFg, CHFz, CH3, C1—C2“; alkyl, or halo(C1-C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CHy or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

The present ion provides a method for treating a mammalian t affected by prostate cancer comprising administering to the mammalian subject i) an oligonucleotide which s clusterin expression and ii) a Hsp90 inhibitor, which inhibitor is other than Hsngi—l, each in an amount that when in combination with the other is effective to treat the mammalian subject.

The present invention provides a method for ng a ian subject affected by prostate cancer comprising administering to the ian subject i) an oligonucleotide which reduces clusterin sion and. ii) a Hsp90 inhibitor which binds to Hsp90d and HspQOB with a Ka of less than 50 nmol/L, or a prodrug f, each in an amount that when in combination with the other is effective to treat the mammalian subject.

In some embodiments, the Hsp90 inhibitor binds to Hsp90d and/or Hsp9OB with a Ka of less than about 70, 60, 50, 40, 35, 30, 25, 20, , 10, or 5 nmol/L.

In some embodiments, the cancer is androgen-independent. te cancer.

In some embodiments, the amount of the oligonucleotide and the amount of the Hsp90 inhibitor when taken together is more effective to treat the subject than when each agent is administered alone.

In some embodiments, the amount of the oligonucleotide in combination with the amount of the Hsp90 inhibitor is less than is clinically effective when administered alone.

In some embodiments, the amount of the Hsp90 inhibitor in combination with the amount of the oligonucleotide is less than is clinically effective when administered alone.

In some embodiments, the amount of the oligonucleotide and the amount of the Hsp90 tor when taken together is effective to reduce a clinical symptom of prostate cancer in the subject.

In some embodiments, the mammalian subject is human.

In some embodiments, the oligonucleotide is an antisense oligonucleotide.

In some embodiments, the antisense oligonucleotide spans either the translation initiation site or the termination site of clusterin- encoding mRNA.

In some embodiments, the antisense oligonucleotide comprises nucleotides in the sequence set forth in SEQ ID NO: to 11.

In some embodiments, the antisense oligonucleotide ses nucleotides in the sequence set forth in SEQ ID NO: 3.

In some embodiments, the antisense oligonucleotide is modified to enhance in vivo ity relative to an unmodified oligonucleotide of the same sequence. _n some ments, the oligonucleotide is custirsen.

In some embodiments, the amount of custirsen is less than 640mg.

In some embodiments, the amount of custirsen is less than 480mg.

In some embodiments, the amount of custirsen is administered intravenously once in a seven day .

In some embodiments, the amount of the Hsp90 inhibitor is less than 50mg/kg.

In some embodiments, the amount of the Hsp90 inhibitor is 25mg/kg or less.

In some embodiments, the Hsp90 inhibitor is Hsp90i—2.

PCT/IB2012l000696 In some embodiments, a prodrug of the Hsp90 inhibitor is administered. to the mammalian subject which. prodrug is Hsp90i—2— PRO.

In some embodiments, a prodrug of the Hsp90 inhibitor is administered to the ian subject which prodrug is Hsp90i—2— PROZ.

In some embodiments, the combination of the oligonucleotide and the Hsp90 inhibitor is effective to t the proliferation of prostate cancer cells.

The t invention es a pharmaceutical composition comprising an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor having the ure: 0 NW or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CM alkyl, C1-Cm haloalkyl, C3—C7 cycloalkyl, heterocycloalkyl, C1-C6 acyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is ally substituted with from 1—4 groups that are independently C1-C6 alkyl, C1-C6 alkoxy, halogen, hydroxy, amino, mono- or di—(Cl— C6)alkylamino, nitro, halo(C1—C6)alkyl, halo(C1-C5)alkoxy, or carboxamide, wherein when R1 is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are optionally replaced independently by R4, carbonyl, l, ethynyl or a moiety selected from N, O, S, 802, or 80, with the proviso that two 0 atoms, two S atoms, or an —28- O and S atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii) ted or unsaturated C3—C3 cycloalkyl, or (iv) saturated. or unsaturated. C2~Cx heterocycloalkyl, wherein each aryl, heteroaryl, saturated or unsaturated cycloalkyl, or ted or unsaturated heterocycloalkyl, independently, is optionally tuted with at least one group, which independently is hydroxy, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, —S-(C1—C6) alkyl, —SOZ(C1—C6) alkyl, —SOZ— aryl, —SO—(C1—C6)alkyl, -SO—aryl, —SOzNH2, ~~SOgNH—(C1-C6)al}<yl, -SOZNH-aryl, (C1— C6)alkoxy, or mono— or — Cm)alkylamino; and R4 is optionally fused to a C6—Cm aryl group, C5—C8 saturated cyclic group, or at C4*C10 heterocycloalkyl group; and R1 is optionally substituted at any available position with C1—Cm alkyl, C1—Cw haloalkyl, C2—Cm alkenyl, C2— Cm alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, amino, cyano, nitro, —SH, —S—(C1—C6)alkyl, —SOZ—(C1— C6)alkyl, —SO2NH2, (C1-C6)alkyl, —SOZNH-aryl, —SOZ— aryl, —SO—(C1C6)alkyl, —SO;—aryl, C1—C6 alkoxy, C2—C3 alkenyloxy, C2—Cw alkynyloxy, mono— or di—(Cl— Cm)alkylamino, -C;-Cw alkyl—Z, w alkyl-Z, or Rm wherein Z is ORG or —N(Ra2, wherein W0 2012/123823 each R6 is independently -H or C1—C6 alkyl, or N(Raz represents pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, l , 3- or 1 , 4— diazepanyl, or Inorpholinyl, each of which is optionally substituted with hydroxy, amino, aminoalkyl, C1—C6 alkyl, mono— or di (C1-C6) alkylamino, C1—C6 , or halogen, and RO is —H, 0 alkyl, —C2—C10 alkenyl, —CZ—C»_0 alkynyl, aryl, heteroaryl, or —C1—C6 acyl; (l) heteroaryl, (2) aryl, (3) saturated or unsaturated. C5-Cm cycloalkyl, or (4) saturated or unsaturated C5—C;o heterocycloalkyl, the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, —SH, -S~(C1—C5)alkyl, —SOZ~(Cl—C6)alkyl, ~SOg—aryl, ~SO—(C1-C6) alkyl, —SO-aryl, ~SOZNH2, —SOZNH- (C1—C6) alkyl, —SO;NH—aryl, (C1—C6)alkoxy, or mono— or (11— (C1—Cm)alkylamino; R2 is H, Cl, halogen, GEE, CHE}, CH3, C1—Cu; alkyl, or 1—C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CH3 or a prodrug' thereof, for use in ng’ a mammalian subject affected by te cancer. -30_ WO 23823 The present invention provides a pharmaceutical composition comprising an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor, which inhibitor is other than Hsp90i—l, for use in treating a mammalian subject affected by te cancer.

The present invention provides a pharmaceutical composition comprising an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor which binds to Hsp900t and Hsp9OB with a Ka of less than 50 nmol/L, or a prodrug thereof, for use in treating a ian subject affected by prostate cancer.

The present invention provides an oligonucleotide which reduces clusterin expression for use in combination with a Hsp90 tor having the structure: or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CM alkyl, C1—Cm haloalkyl, C3—C7 cycloalkyl, heterocycloalkyl, C1—C6 acyl, aryl, or heteroaryl, n each alkyl, lkyl, heterocycloalkyl, aryl, and heteroaryl group is optionally substituted with from 1—4 groups that are independently C1—C6 alkyl, C1—C6 alkoxy, halogen, hydroxy, amino, mono— or di—(Cl— Cdalkylamino, nitro, halo(C1—C6)alkyl, halo(Cl~C6)alkoxy, or carboxamide, wherein when R1 is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are optionally replaced independently by R4, carbonyl, ethenyl, ethynyl or a moiety selected from N, O, S, 802, or SO, with the proviso that two 0 atoms, two S atoms, or an O and 8 atom are not immediately nt each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii) saturated or unsaturated Cj—Cw cycloalkyl, or (iv) saturated. or unsaturated. C2—Cx heterocycloalkyl, wherein each aryl, heteroaryl, saturated or unsaturated cycloalkyl, or saturated or unsaturated heterocycloalkyl, independently, is optionally substituted with at least one group, which independently is hydroxy, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, -S-(C1—C6) alkyl, ~SOZ(C1—C6) alkyl, —SOZ— aryl, —SO-(C1—C6)alkyl, —SO—aryl, —SOZNH2, ~802NH-(C1—C6)alkyl, -SOZNH-aryl, (C1— C6)alkoxy, or mono- or di—(Cl— Cm)alkylamino; and R4 is optionally fused to a C6—Cm aryl group, C5-C8 saturated cyclic group, or a CM~C10 heterocycloalkyl group; and R1 is optionally substituted at any available position with C1—C;o alkyl, C1—C10 haloalkyl, Ch—Clo alkenyl, C2— Cm alkynyl, hydroxy, y, carboxamido, oxo, halo, amino, cyano, nitro, —SH, ~C6)alkyl, —SOz—(C1— C6)alkyl, —SOZNH2, —SO;NH(C1—C6)alkyl, —aryl, —802— aryl, 1C6)alkyl, —SOz—aryl, C1—C6 , C2—C3 alkenyloxy, C2—Cm alkynyloxy, mono- or di—(Cl— W0 2012/123823 Cw)alkylamino, ~C;-Cm alkyl-Z, —OC1—Cm alkyl—Z, or Ry wherein Z is ORG or —N(Ra2, wherein each R6 is independently —H or C1—C6 alkyl, or N(R@2 represents pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, 1,3— or 1,4— diazepanyl, or linyl, each. of which is optionally substituted with y, amino, lkyl, C1~Cb alkyl, mono— or di(Cl—C6)alkylamino, C1—C6 alkoxy, or halogen, and R0 is —H, —C1—Cw alkyl, ~C2~Cm alkenyl, —o2—Cm alkynyl, aryl, heteroaryl, or -%h-C6 acyl; R5 is (l) heteroaryl, (2) aryl, (3) saturated or rated C5-C3 cycloalkyl, or (4) saturatedv or unsaturated. C5—Cm heterocycloalkyl, the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, —SH, —S—(C1-Cg)alkyl, —SO;—(C1-C6)alkyl, ~Sop—aryl, -SO-(C1-C6) alkyl, ~SO—aryl, —SOZNH2, —SOZNH~ (C1~C6) alkyl, —aryl, (C1—C6)alkoxy, or mono— or di—(Cl-Cm)alkylamino; R2 is IL Cl, halogen, CF3, CHE” CH” Cy4hc alkyl, or halo(C1—C6)alkyl; and X is N or CR3, wherein R3 is H, halogen, or CHa W0 2012/123823 or a prodrug f, in treating a mammalian subject ed by prostate cancer.

The present invention es an oligonucleotide which reduces clusterin expression for use in combination with a Hsp90 inhibitor, which inhibitor is other than Hsp90i—l, in treating' a mammalian subject affected by te cancer.

The present invention provides an oligonucleotide which reduces clusterin expression for use in ation with a Hsp90 inhibitor which binds to Hsp90d and Hsp9OB with a Ka of less than 50 , or a prodrug thereof, in treating a mammalian subject affected by prostate cancer.

The present invention provides a composition for treating a mammalian subject affected. by te cancer comprising i) an oligonucleotide which reduces clusterin expression and ii) a Hsp90 inhibitor having the structure: or a pharmaceutically acceptable salt thereof, wherein R1 is H, C1—CM alkyl, C1—Cm haloalkyl, C3—C7 cycloalkyl, heterocycloalkyl, C1—C6 acyl, aryl, or heteroaryl, wherein each alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl group is optionally substituted with from 1—4 groups that are ndently C1-C6 alkyl, C1~C5 alkoxy, halogen, hydroxy, amino, mono— or di—(Cy' C6)alkylamino, nitro, halo(Cl—C6)alkyl, halo(Cl—C5)alkoxy, or carboxamide, wherein when R1 is a C1—CM alkyl group, up to five of the carbon atoms in the alkyl group are optionally replaced independently by R4, carbonyl, ethenyl, ethynyl or a moiety selected from N, O, S, 802, or 80, with the proviso that two 0 atoms, two S atoms, or an O and 8 atom are not immediately adjacent each other, wherein R4 is (i) heteroaryl, (ii) aryl, (iii) saturated or unsaturated C3—C3 cycloalkyl, or (iv) ted. or unsaturated. C2—C3 heterocycloalkyl, each aryl, heteroaryl, saturated or unsaturated cycloalkyl, or saturated or unsaturated heterocycloalkyl, independently, is optionally substituted with at least one group, which independently is y, halo, amino, cyano, carboxy, carboxamido, nitro, oxo, -S-(C1-C6) alkyl, -SO;(C3*C6) alkyl, -SO;- aryl, —SO—(C1—C6)alkyl, ~SO—aryl, —SO;NH7, —(C1—C6)alkyl, —SO2NH—aryl, (C1— C6)alkoxy, or mon0* or — Cm)alkylamino; and R4 is optionally fused to a C6~Cm aryl group, C5—C3 saturated cyclic group, or a Ch-Clo heterocycloalkyl group; and R1 is optionally substituted at any available on with C1—C;o alkyl, C1—C10 haloalkyl, C2—C10 alkenyl, C2— Cm alkynyl, hydroxy, carboxy, carboxamido, oxo, halo, W0 2012/123823 amino, cyano, nitro, —SH, —S—(C1—C6)alkyl, -SOZ—(C1- C6)alkyl, -SOZNH2, (C1—C6)alkyl, —SOZNH—aryl, -SOZ- aryl, —SO—(C1C6)alkyl, -SOZ-aryl, C1—C6 alkoxy, C2—C3) alkenyloxy, Cz‘Cio loxy, mono— or di- (C1- Clo)alkylamino, "C;"C10 alkyl—Z, 10 alkyl—Z, or R5, wherein Z is ORG or —N(R6)2, wherein each R6. is independently —H or C1—C6 alkyl, or N(Rb)2 represents pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, 1,3— or 1, 4— diazepanyl, or morpholinyl, each of which is optionally substituted with hydroxy, amino, aminoalkyl, C1-C6 alkyl, mono— or di (C1—C6) alkylamino, C1—C6 alkoxy, or halogen, and R0 is ~H, o alkyl, —C2—C10 alkenyl, —v2-C-_o alkynyl, aryl, heteroaryl, or —C1—C6 acyl; R5 is (l) heteroaryl, (2) aryl, (3) saturated or unsaturated C5-C;o cycloalkyl, or (4) saturated or unsaturated C5~C—_o heterocycloalkyl, the R5 groups are optionally substituted at least one group which is independently hydroxy, oxo, halo, amino, cyano, nitro, —SH, —S—(C1—C5)alkyl, -SOz—(C1—C6)alkyl, ~802—aryl, —SO—(C1—C6) alkyl, —SO—aryl, ~SOZNH2, —802NH— (C1~C6) alkyl, —802NH—aryl, (C1—C6)alkoxy, or mono- or di-(Cl-Cm)alkylamino; is H, Cl, halogen, CFy CHE” CHM C1—ClC alkyl, or halo (C1-C6) alkyl; and -36— X is N or CR3, wherein R3 is H, halogen, or CHy or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

The present invention provides a composition for ng a mammalian subject affected, by prostate cancer comprising i) an oligonucleotide which reduces clusterin expression and ii) a Hsp9O inhibitor, which inhibitor is other than Hsp90i—l, each in an amount that when. in ation with the other is ive to treat the mammalian subject.

The present invention provides a ition for treating a mammalian subject affected. by prostate cancer comprising* i) an oligonucleotide which reduces clusterin expression and ii) a Hsp90 inhibitor which binds to Hsp90a and Hsp9OB with a Ka of less than 50 nmol/L, or a prodrug thereof, each in an amount that when in combination with the other is effective to treat the mammalian subject.

In some embodiments, the combination of the oligonucleotide and the Hsp90 inhibitor is effective to inhibit the proliferation of prostate cancer cells.

In some embodiments, Hsp90 inhibitor—mediated. induction of rin expression is attenuated by custirsen, wherein the combination of the Hsp90 inhibitor and custirsen delays the progression of CRPC. In some embodiments, the combination of the Hsp90 inhibitor and custirsen inhibits tumor growth in the ian subject. In some embodiments, the combination. of the Hsp90 inhibitor and custirsen prolongs the al of the mammalian subject.

W0 2012/123823 PCT/IB20122'000696 An aspect of the invention provides pharmaceutical composition comprising an amount of an oligonucleotide which reduces clusterin expression, and a Hsp90 inhibitor for use in treating a mammalian subject affected by prostate .

An aspect of the invention provides oligonucleotide which reduces clusterin expression for use in ation with a Hsp90 inhibitor in treating a mammalian subject affected by prostate cancer.

An aspect of the invention provides a composition for treating a mammalian subject affected. by te cancer comprising' i) an oligonucleotide which reduces rin expression and ii) a Hsp90 inhibitor each in an amount that when in combination with the other is effective to treat the mammalian subject.

Aspects of the invention e the sed potency of a combination treatment comprising an oligonucleotide that targets clusterin expression and an Hsp90 inhibitor compared to ucleotide or Hsp90 inhibitor monotherapy. In some embodiments of the invention, the combination of an oligonucleotide that targets clusterin expression and an HSP9O inhibitor increases prostate cancer cell apoptosis and/or decreases prostate cancer cell proliferation compared to oligonucleotide or Hsp90 inhibitor monotherapy. In some embodiments, the combination of an oligonucleotide that targets clusterin expression and an Hsp90 inhibitor decreases the protein expression and/or a on of HSF—l ed to oligonucleotide or Hsp90 inhibitor monotherapy.

Aspects of the invention provide ed strategies employing an 3O oligonucleotide which reduces clusterin expression in combination with Hsp90 inhibitors to improve patient outcome in castration— resistant prostate cancer.

The present invention relates to a method for ng a mammalian t affected by prostate cancer comprising i) an 2012/000696 oligonucleotide which s clusterin expression and ii) a Heat Shock Protein 90 (Hsp90) inhibitor, each in an amount that when in combination with the other is effective to treat the mammalian subject.

In some embodiments, the Hsp90 inhibitor is Hsp90i—l.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed ments. Thus, all combinations of the various elements described herein are within the scope of the invention.

It is understood. that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2—5 mg/kg/day" includes 0.2 day, 0.3 mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day etc. up to .0 mg/kg/day.

Terms As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

As used. herein, ” in the context of a numerical value or range means i10% of the numerical value or range recited or claimed.

As used. in the specification and. claims of this application, the term "clusterin" refers to a glycoprotein present in s, including humans, and nated as such in the humans. The sequences of numerous clusterin species are known. For example, the sequence of human clusterin is described by Wong et al., Eur. J.

Biochem. 221 (3),9l7—925 (1994), and in NCBI sequence accession number NM_001831 (SEQ ID NO: 43). In this human sequence, the coding sequence spans bases 48 to 1397.

As used herein, “oligonucleotide which reduces clusterin expression” is an ucleotide with a sequence which is effective to reduce clusterin expression in a cell. The oligonucleotide which reduces clusterin expression may be, for e, an antisense oligonucleotide or an RNA interference inducing le.

As used herein, “antisense oligonucleotide” refers to a Ai oligonucleotide that reduces clusterin expression and that has a sequence complementary to clusterin mRNA. Antisense ucleotides may be antisense oligodeoxynucleotides (ODN). Exemplary sequences which can be employed as antisense molecules in the invention are disclosed in PCT Patent ation WO 00/49937, U.S. Patent Publication US—2002—0128220-Al, and U.S. Pat. NO. 6,383,808, all Of which are incorporated herein by reference. Specific antisense sequences are set forth in the t application as SEQ ID NOS: 1 to 11, and may be found in Table 1.

Table 1. Sequence Identification Numbers for Antisense Oligonucleotides SEQ ID NO: Sequence l—‘I—‘KOCDQONUlItP-UJNH HO iGCACAGCAGG AGAATCTTCA H ‘TGGAGTCTTT GCACGCCTCG G CAGCAGCAGA GTCTTCATCA H ATTGTCTGAG ACCGTCTGGT O CCTTCAGCTT TGTCTCTGAT H AGCAGGGAGT CGATGCGGTC W lATCAAGCTGC GGACGATGCG D ‘GCAGGCAGCC CGTGGAGTTG H ‘TTCAGCTGCT CCAGCAAGGA G iAATTTAGGGT TCTTCCTGGA G IGCTGGGCGGA GTTGGGGGCC T The ODNs employed may be modified to increase the ity of the ODN in Vivo. For example, the ODNs may be employed as phosphorothioate derivatives (replacement of a non—bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. MOE (2—methoxyethyl)) modification (ISIS ne) is also effective. The construction of such modified ODNs is bed in detail in U.S. Patent No. 6,900,187 B2, the contents of which are incorporated by reference. In some embodiments, the ODN is custirsen.

As used herein, “custirsen” refers to an antisense oligonucleotide that reduces clusterin expression having the sequence CAGCAGCAGAGTCTTCATCAT (SEQ ID NO: 3), wherein the anti-clusterin oligonucleotide has a phosphorothioate backbone throughout, has sugar es of nucleotides 1—4 and 18-21 bearing 2’—O—methoxyethyl modifications, has nucleotides 5~l7 which are 2’deoxynucleotides, and has 5—methylcytosines at nucleotides 1, 4, and 19. Custirsen is also known as TV—lOll, OGX—Oll, ISIS 112989 and Custirsen Sodium.

As used herein, “RNA inducing molecule” refers to a molecule capable of ng RNA interference or “RNAi” of rin expression. RNAi es mRNA degradation, but many of the biochemical mechanisms underlying this interference are unknown. The use of RNAi has been described in Fire et al., 1998, Carthew et al., 2001, and Elbashir et al., 2001, the contents of which are incorporated herein by reference.

Isolated RNA molecules can mediate RNAi. That is, the isolated RNA les of the present invention mediate degradation or block expression of mRNA that is the transcriptional product of the gene, W0 2012!123823 which is also referred to as a target gene. For convenience, such mRNA may also be referred to herein as mRNA to be ed. The terms RNA, RNA molecule(s), RNA segment(s) and RNA fragment(s) may be used interchangeably to refer to RNA that mediates RNA U) interference. These terms include double—stranded RNA, small interfering RNA (siRNA), hairpin RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from lly occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non—nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). Nucleotides in the RNA molecules of the t invention can also comprise nonstandard nucleotides, including non—naturally occurring nucleotides or deoxyribonucleotides. Collectively, all such altered RNAi les are referred to as analogs or s of naturally—occurring RNA.

RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi.

As used herein the phrase "mediate RNAi" refers to and indicates the ability to distinguish which mRNA molecules are to be affected by the RNAi machinery or process. RNA that mediates RNAi interacts with the RNAi machinery such that it s the machinery to degrade particular mRNAs or to otherwise reduce the sion of the target n. In one embodiment, the present invention relates to RNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi inhibition by cleavage or blocking expression of the target mRNA.

As noted above, the RNA molecules of the t invention in general comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion. The total number of nucleotides in the RNA molecule is suitably less than in order to be effective mediators of RNAi. In preferred. RNA. molecules, the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21—23. Suitable ces are set forth in the U] present application as SEQ ID NOszl9 to 42 (Table 2).

Table 2. Sequence Identification Numbers for RNA Interference Inducing Molecules SEQ ID NO: Sequence 1 l9 CCAGAGCUCG CCCUUCUACT T GUAGAAGGGC GAGCUCUGGT T 21 GAUGCUCAAC ACCUCCUCCT T GGUG UUGAGCAUCT T UAAUUCAACA AAACUGUTT [\J .1}. GACAGUUUUA UUGAAUUAGT T (\D (.D UAAUUCAACA UTT N O\ ACAGUUUUGU UGAAUUATT 27 AUGAUGAAGA CUCUGCUGCT T 28 GCAGCAGAGU CUUCAUCAUT T 29 UGAAUGAAGG GACUAACCUG TT CAGGUUAGUC CCUUCAUUCA TT w H CAGAAAUAGA CAAAGUGGGG TT (A) N UUUG UCUAUUUCUG TT U.) (A) ACAGAGACUA AGGGACCAGA TT OJ .1}. ACAGAGACUA AGGGACCAGA TT w U1 CCAGAGCUCG CCCUUCUACT T (JJ OX GUAGAAGGGC GAGCUCUGGT T U) \] CAUC GUCCGCAGCT T LL) OD GCUGCGGACG GACT T >b>J>UJ HOKO yb 2 The siRNA les of the invention are used in therapy to treat patients, including human patients, that have cancers or other diseases of a type where a eutic benefit is obtained by the inhibition of expression of the targeted protein. siRNA molecules U] of the invention are stered to patients by one or more daily injections (intravenous, subcutaneous or intrathecal) or by continuous intravenous or intrathecal administration for one or more treatment cycles to reach plasma and tissue concentrations le for the regulation of the targeted mRNA and protein.

As used herein, a “mammalian subject affected by prostate cancer” means a mammalian subject who was been atively diagnosed to have prostate cancer.

As used herein, “androgen~independent prostate ” encompasses cells and. tumors containing' cells that are not androgen—dependent (not androgen sensitive); often such cells progress from being androgen—dependent to being androgen-independent. In some embodiments, androgen independent prostate cancer has progressed since the administration of hormone ablation therapy and/or hormone de therapy. In some embodiments, there is increased AR expression in the androgen—independent prostate cancer compared to prostate cancer that is not androgen~independent.

As used herein, “castration—resistant prostate ” asses any en—independent prostate cancer that is resistant to hormone ablation therapy and/or hormone blockade therapy. In some embodiments, castration-resistant prostate cancer has progressed since the administration of hormone on or hormone blockade therapy. In some embodiments, there is increased AR expression in the castration—resistant prostate cancer ed to prostate cancer that is not castration resistant.

As used herein, “Hsp90 inhibitor” refers to an agent that perturbs or reduces a function of Hsp90, including inhibiting' a Hsp90-protein interaction, Hsp90 signaling, or Hsp90 protein expression. Hsp90 inhibitors e but are not d to Hsp90—specific monoclonal antibodies, oligonucleotides that target Hsp90 expression (such as Hsp9O targeting antisense oligonucleotides or RNA inducing molecules), peptide agents specific for Hsp90, and small molecule inhibitors specific for Hsp90. Non—limiting examples of Hsp90 tors are Hsp90i—l, Hsp90i—2, Hsp90i—2—PRO and —2—PRO2. ~l is a Hsp90 inhibitor. Hsp90i—l is also known as 17— allylamino—l7—demethoxygeldanamycin (17*AAG), Telatinib, Tanespimycin, NSC—330507, CNF—lOl, KOS—953, GLD—36, and CP 127374.

The CAS Registry Number of Hsp90i—1 is 75747—14—7. The Hsp90i—1 used for the experiments described herein is also referred to as l7—AAG and was obtained from the National Institutes of Health (Bethesda, Maryland, USA). 17—AAG has been discussed in Egorin et al., 1998, and. Koga et al., 2009, and is also available for purchase from gen (San Diego, California, USA). —2 is a Hsp90 inhibitor. -2 is also known as PF— 04928473, and SNX—2112, and (4—(6,6—Dimethyl-4~0xo—3—trifluoromethyl— 4,5,6,7-tetrahydro—indazol—1—yl)—2-(4—hydroxy—cyclohexylamino)- benzamide). The CAS Registry No. for Hsp90i-2 is 908112—43—6. Hsp90i- 2 has the following structure: Hsp90i—2 is discussed in Lamoureux et al., 2011, the entire contents of which are incorporated herein by reference.

WO 23823 PCT/132012/000696 —2—PRO is a Hsp90 inhibitor. Hsp90i—2-PRO is the prodrug of —2. The CAS Registry No. for Hsp90i—2—PRO is 908115—27—5.

Hsp90i—2—PRO is also known as SNX—5422 and PF—04929ll3. Hsp90i—2—PRO has the following structure: Hsp90i—2—PRO is discussed in Lamoureux et al., 2011, the entire contents of which are incorporated herein by reference.

Hsp90i—2—PR02 is another prodrug of Hsp90i—2. Hsp90i—2—PRO2 is discussed in Chandarlapaty et al., 2008, the entire contents of which are incorporated herein by reference. Hsp90i—2—PR02 is also known as SNX—5542.

Methods of synthesis for Hsp90i—2, Hsp90i—2—PRO and Hsp90i—2—PRO2 are bed in Huang et al., J) Med Chem. 52:4288—4305 (2009), and U.S.

Patent No. 7,928,135, the entire contents of which are orated herein by reference. Alternatively, —Z, Hsp90i—2~PRO and Hsp90i—2—PR02 are available from Pfizer Inc. (New York, New York, USA) and Serenex Inc. (Durham, North Carolina, USA).

Those having ry skill in the art of organic synthesis will appreciate that modifications to the general procedures shown in the synthesis schemes of this ation can be made to yield structurally diverse compounds. For example, where aryl rings are present, all positional isomers are contemplated and may be synthesized using standard aromatic substitution chemistry. The number and types of substituents may also vary around the aryl rings.

Furthermore, where alkyl groups are present, the chain length may be ed using methods well known to those of ordinary skill in the art. Where ester formation is contemplated, lactones may' be used wherein the e ring is opened by reaction with a nucleophile, such as an ether—containing moiety described hereinabove. Suitable organic transformations are described. in March’s Advanced Organic Chemistry: Reactions, Mechanisms, and ure (Wiley—Interscience; 6th edition, 2007), the content of which is hereby incorporated by nce.

Compounds of the subject invention can be converted to prodrugs to optimize absorption and bioavailability. Formation of a prodrug include, but is not limited to, reaction of a free hydroxyl group with a carboxylic acid to form an ester, reaction of a free hydroxyl group with an phosphorus oxychloride followed by hydrolysis to form a ate, or reaction of a free hydroxyl group with an amino acid to form an amino acid ester, the process of which has been described previously by Chandran in . The substituents are chosen and ing analogs are evaluated according to principles well known in the art of medicinal and pharmaceutical chemistry, such as quantification of structure—activity relationships, optimization of biological activity and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties.

Except where otherwise specified, when the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or 8 configuration. It is to be understood ingly that the isomers arising from such try (e.g., all enantiomers and diastereomers) are included. within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation ques and by stereochemically controlled synthesis, such as those described in "Enantiomers, tes and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general e and without limitation, isotopes of hydrogen include m and deuterium. Isotopes of carbon include C—13 and C—l4.

It will be noted that any notation of a carbon in structures throughout this application, when used. without r notation, are intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein.

It will also be noted that any on of a hydrogen in structures throughout this application, when used. without further notation, are intended to represent all isotopes of en, such as 1H, 2H, or 3H. Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.

Isotopically—labeled compounds can generally be prepared by tional techniques known. to those skilled. in the art using appropriate isotopically—labeled reagents in place of the non— labeled reagents employed.

As used herein, "alkyl" es both branched and straight—chain ted aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted. Thus, Cl—Cn as in “Cl—Chalkyl" is defined to include groups having 1, 2, ...U -48— WO 23823 PCT/IBZO12/000696 n-l or n carbons in a linear or branched arrangement. For example, cl_C€-I as in ”Cy—C6alkyl" is defined to e groups having 1, 2, 3/ 4i 5/ or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n—propyl, isopropyl, n-butyl, t—butyl, pentyl, hexyl, and octyl.

As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or clic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be tituted or substituted. Examples of such aryl elements include phenyl, p- toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, phenanthryl, anthryl or hthyl. In cases where the aryl substituent is bicyclic and one ring is non—aromatic, it is understood that attachment is via the aromatic ring.

The term “heteroaryl”, as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S.

Bicyclic ic heteroaryl groups include but are not d to phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused. to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a ered aromatic urated) heterocyclic ring having one atom selected from O, N or S.

Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, inyl, furanyl, indolinyl, indolyi, zinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, W0 2012/123823 PCT/B20121000696 pyridazinyl, pyridopyridinyl, pyridazinyl, l, pyrimidyl, yl, olinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, azolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4—dioxanyl, hexahydroazepinyl, UI dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, ofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, ooxazolyl, opyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, oquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetra— hydroquinoline. In cases where the heteroaryl substituent is bicyclic and one ring is non—aromatic or contains no heteroatoms, it is understood that ment is via the aromatic ring or via the heteroatom. containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N~ oxides thereof are also encompassed by this definition.

The alkyl, aryl, and heteroaryl substituents may be substituted or unsubstituted, unless specifically defined otherwise.

The compounds of the instant invention may be in a salt form. As used herein, a “salt” is a salt of the instant nds which has been modified by making‘ acid. or base salts of the compounds. In some embodiments, the salt is ceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or nic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, s, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, , potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non—toxic, inorganic and c acid or base on salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the nds of the invention, or by separately reacting' a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. entative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, e, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, eptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

The compositions of this invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the e in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered. independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or e, for delivering the instant compounds to the animal or human. The carrier W0 2012/123823 PCTXIB2012/000696 may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a ceutically acceptable carrier.

The dosage of the compounds administered; in treatment will vary depending upon s such as the pharmacodynamic teristics of a specific herapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds may comprise a single compound or mixtures f with additional anticancer agents. The compounds can be administered in oral dosage forms as tablets, es, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), eritoneal, aneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other' methods, all using‘ dosage forms well known to those of ordinary skill in the pharmaceutical arts.

The compounds can be stered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referredt to herein as a pharmaceutically acceptable carrier) suitably selected. with respect to the intended, formv of administration and, as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, enous or direct injection or parenteral stration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co—administered in the fornl of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers WO 23823 include e, sucrose, n and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain le binders, lubricants, diluents, disintegrating agents, U) coloring agents, flavoring agents, flow—inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other c solvents, including esters, ons, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted. from non—effervescent granules and. escent preparations reconstituted. from. effervescent granules. Such liquid dosage forms may n, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms ally contain flavorants and. coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Techniques and compositions for making dosage forms useful in the present invention are bed in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in ceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical logy; J. G. Hardy, S. S. Davis, Clive G. Wilson, W0 2012/123823 Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical es, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned ations are incorporated by nce herein.

U] Tablets may contain suitable s, lubricants, disintegrating agents, coloring agents, flavoring agents, flow—inducing agents, and melting . For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such. as lactose, gelatin, agar, starch, e, glucose, methyl cellulose, magnesium. stearate, dicalciunt phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as e or actose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium te, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compounds can also be administered. in the form. of liposome delivery systems, such as small unilamellar vesicles, large unilamallar es, and Hmltilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The nds may be administered as components of tissue—targeted emulsions.

The compounds may also be coupled to soluble polymers as able 3O drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide—phenol, polyhydroxyethylasparta- midephenol, or polyethyleneoxide—polylysine substituted with oyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of ctic and ycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, Polyacetals, polydihydropyrans, polycyanoacylates, and inked or amphipathic U] block copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, ose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as ned release products to e for continuous release of medication over a period. of hours. Compressed. tablets can be sugar . or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration. in the gastrointestinal tract.

For oral administration in liquid dosage form, the oral drug components are combined with any oral, xic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. es of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, ing , emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted. from. non—effervescent granules and. effervescent preparations reconstituted. from. effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, ners, and melting agents.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous se (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for W0 2012/123823 parenteral administration preferably contain a water e salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing' agents such as sodiunl bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing . Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium. chloride, — or propyl— n, and chlorobutanol. Suitable pharmaceutical carriers are described. in Remington‘s Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

The compounds of the instant invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using' those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

Parenteral and enous forms may also include minerals and other materials to make them. compatible with the type of injection or delivery system chosen.

The inhibition of clusterin expression may be transient, and. may occur in combination with a Hsp90 tor. In humans with prostate , this means that inhibition of expression should be effective starting within a day or two of Hsp90 inhibition or administration of an Hsp90 inhibitor, and extending for about 3 to 6 months thereafter.

This may require multiple doses to accomplish. It will be appreciated, however, that the period of time may be more prolonged, ng Hsp90 inhibition and extending for substantial time afterwards without departing from the scope of the invention. ~56- Aspects of the ion can be applied to the treatment of en— independent prostate , or to prevent prostate cancer from ng en—independent.

Aspects of the invention can be applied to the treatment of castration—resistant prostate cancer, or to prevent te cancer from becoming castration—resistant.

“Combination” means either at the same time and ncy, or more usually, at different times and. frequencies as an oligonucleotide targeting clusterin expression, as part of a single treatment plan.

Aspects of the ion include the administration of the oligonucleotide before, after, and/or during the administration of a Hsp90 inhibitor. A Hsp90 inhibitor may therefore be used, in combination with an oligonucleotide according to the ion, but yet be administered at different times, different dosages, and at a different frequency, than the oligonucleotide.

As used herein, an “amount” or “dose” of an oligonucleotide ed in milligrams refers to the milligrams of oligonucleotide present in a drug product, regardless of the form of the drug product.

As used herein, “effective” when referring to an amount of oligonucleotide which reduces clusterin expression, a Hsp90 inhibitor, or‘ any combination thereof refers to the quantity of oligonucleotide, Hsp90 inhibitor, or any combination thereof that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with. a reasonable benefit/risk ratio when used in the manner of this invention.

As used herein, “treating” encompasses, e.g., inhibition, regression, or stasis of the progression of prostate .

Treating also encompasses the prevention or amelioration of any symptom or symptoms of prostate cancer. _57- As used herein, “inhibition” of disease progression or e complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.

As used herein, a “symptom” associated with prostate cancer includes any clinical or laboratory manifestation ated with prostate cancer, and is not limited to what the subject can feel or observe.

As used herein, “pharmaceutically able carrier” refers to a carrier or excipient that is suitable for use with humans and/or animals t undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be a pharmaceutically able solvent, suspending agent or vehicle, for delivering the instant nds and/or combinations to the t.

The following abbreviations are used herein PCa prostate cancer CRPC castrate resistant prostate cancer HSP heat shock proteins CLU clusterin PSA prostate specific antigen 17—AAG 17~allylamino—17—demethoxygeldanamycin Aso antisense oligonucleotide Dosage Units Administration of an oligonucleotide that targets clusterin expression can be carried out using the various mechanisms known in the art, including naked administration and administration in pharmaceutically acceptable lipid carriers. For example, lipid carriers for antisense delivery are disclosed in U.S. Patent Nos. ,855,911 and 5,417,978, which are orated herein by nce.

In general, the oligonucleotide is administered by intravenous ~58- (i.v.), intraperitoneal (i.p.), subcutaneous , or oral routes, or direct local tumor injection. In preferred embodiments, an oligonucleotide targeting clusterin expression is administered by i.v. injection. In some embodiments, the amount of oligonucleotide administered is 640mg.

The amount of antisense oligonucleotide administered is one effective to inhibit the expression of clusterin in prostate cells.

It will be appreciated that this amount will vary both with the effectiveness of the antisense oligonucleotide employed, and. with the nature of any carrier used.

The amount of antisense oligonucleotide targeting clusterin sion administered may be from 40 to 640 mg, or 300—640 mg.

Administration of the antisense oligonucleotide may be once in a seven day period, 3 times a week, or more specifically on days 1, 3 and 5, or 3, 5 and 7 of a seven day period. In some embodiments administration of the antisense oligonucleotide is less frequent than once in a seven day period. Dosages may be calculated by patient weight, and therefore a dose range of about 1—20 mg/kg, or about 2—10 mg/kg, or about 3-7 mg/kg, or about 3—4 mg/kg could be used. This dosage is repeated at intervals as needed. One clinical concept is dosing once per week with 3 loading doses during week one of treatment. The amount of antisense oligonucleotide administered is one that has been demonstrated to be effective in human ts to t the expression of clusterin in cancer cells.

In some embodiments of the invention, the amount of oligonucleotide targeting the expression of rin required for treatment of prostate cancer is less in combination with a Hsp90 inhibitor, than would be required with oligonucleotide monotherapy.

Custirsen may be ated at a tration of 20 mg/mL as an isotonic, phosphate—buffered saline on for IV administration _59_ W0 2012/123823 and can be supplied as an 8 mL solution containing 160 mg custirsen sodium in a single Vial.

Custirsen may be added to 250 mL 0.9% sodium chloride (normal saline). The dose may be administered using either a peripheral or central indwelling catheter intravenously as an infusion over 2 hours. Additionally, an infusion pump may be used.

Administration of an Hsp90 inhibitor may be oral, nasal, pulmonary, parenteral, i.v., i.p., intra—articular, ermal, intradermal, s.c., topical, uscular, rectal, intrathecal, intraocular, and buccal. One of skill in the art will recognize that higher doses may be required for oral administration than for i.v. injection.

The dose of Hsp90 inhibitor may be 60mg/kg, 55mg/kg, 45mg/kg, 40mg/kg, 35mg/kg, g, 20mg/kg, lSmg/kg, lOmg/kg, 5mg/kg or less.

A dosage unit of the oligonucleotide which reduces clusterin expression and an Hsp90 inhibitor may comprise one of each singly or mixtures thereof. A combination of an oligonucleotide which reduces clusterin sion and an Hsp9O inhibitor can be administered in oral dosage forms as tablets, capsules, pills, s, granules, elixirs, tinctures, sions, syrups, and emulsions. An oligonucleotide which reduces clusterin expression and/or Hsp9O inhibitor may also be administered in intravenous (bolus or infusion), intraperitoneal, aneous, or intramuscular form, or introduced directly, e.g. by injection or other methods, into or onto a te cancer lesion, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

An oligonucleotide which reduces clusterin expression and/or Hsp9O tor can be stered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers ctively referred to herein as a pharmaceutically acceptable carrier) suitably selected. with respect to the intended form of stration and. as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral stration. The oligonucleotide and/or Hsp90 inhibitor can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can. be a solid. or liquid, and. the type of r is generally chosen based on the type of administration being used. Capsule or tablets can be easily formulated and can be made easy to w or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow~ inducing agents, and. melting’ agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or sions reconstituted from non- effervescent granules and effervescent preparations reconstituted from escent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying , suspending agents, diluents, sweeteners, ners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

An oligonucleotide which reduces clusterin expression and/or Hsp90 inhibitor can also be stered in the form of liposome delivery s, such as small ellar vesicles, large unilamallar vesicles, and multilamellar es. Liposomes can be formed from a y of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue—targeted emulsions.

For oral administration in liquid dosage form, an Hsp90 inhibitor may be combined‘ with any oral, non-toxic, pharmaceutically acceptable ~61— inert carrier such as ethanol, glycerol, water, and the like.

Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically able fats and oils, ls or other organic solvents, including esters, emulsions, U] syrups or elixirs, sions, solutions and/or suspensions tituted‘ from. fervescent granules and effervescent preparations reconstitutedt from: effervescent granules. Such liquid dosage forms may n, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting .

In some embodiments of the invention, the amount of Hsp90 inhibitor required for treatment of prostate cancer is less in combination with an oligonucleotide targeting the expression of clusterin, than would be required with Hsp90 monotherapy.

A dosage unit may comprise a single compound or mixtures of compounds. A dosage unit can be prepared for oral or injection dosage forms.

According to an aspect of the invention, there is provided an ucleotide which reduces rin expression—containing pharmaceutical composition packaged in dosage unit form, wherein the amount of the oligonucleotide in each dosage unit is 640mg or less.

Said pharmaceutical composition may include an Hsp90 inhibitor, and may be in an injectable solution or suspension, which may further contain sodium ions.

According to another aspect of the invention, there is provided the 3O use of an oligonucleotide targeting clusterin expression and a Hsp90 inhibitor in the manufacture of a medicament for the treatment of , where the medicament is formulated to deliver a dosage of 640mg or less of oligonucleotide to a patient. The medicament may contain sodium ions, and/or be in the form of an injectable solution.

General ques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: s (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd n (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, , Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James ty, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the ceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. , Eds.); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol. 40 (Gilbert S. , Christopher T. Rhodes, Eds.). These references in their entireties are hereby incorporated by reference into this application.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

W0 2012/123823 mental Details Example 1. Hsp90 inhibitors induce expression of HSPs in prostate cancer (PCa) cells in vitro and in viva.

Dose— and. time~dependent effects of Hsp90i—l or Hsp90i—2 on the expression of CLU, Hsp90, Hsp70 and Akt protein and mRNA levels was evaluated in LNCaP and PC-B cells. Both Hsp90i—l and Hsp90i~2 increased Hsp70 and CLU protein levels up to 3 fold in a dose— and time—dependent manner (Fig. 1A, B and C). Hsp90 inhibition induced a dose— and a time dependent decline of Akt sion as previously reported (Lamoureux et al., 2011). mRNA levels of CLU, Hsp70 and. Hsp90 also increased. after Hsp90 inhibitor treatment (Fig. 1D).

Next the s of —2 treatment on CLU expression were assessed in vivo in CRPC LNCaP xenografts using immunohistochemistry and western blot (Fig. 2). CLU expression increased 4—fold after treatment with Hsp90i—2—PRO (***, p<0.001) 2O compared with vehicle treated tumor (Fig. 2A, B). Similarly, Hsp70, which is considered a pharmacodynamic measure of Hsp90 inhibition (Solit et al., 2003; Eccles et al., 2008), increased 2.3—fold after treatment with Hsp90i—2—PRO (***, p<0.001) (Fig. 2A).

Example 2. Ekeatment-induced feed forward loop involving CLU and HSF-l activity.

Since HSF—l is the pre—dominant regulator of the heat shock response ji et al., 2008; Workman et al., 2007), the effect of Hsp90 inhibition on HSF—l—activity and expression of HSPs was evaluated. Hsp90i—l or Hsp90i—2 icantly induced CLU (Fig. l) as well as HSF—l activity in a dose—dependent manner (***, pS0.00l; Fig. 3A). CLU overexpression protected PC—3 tumor cells from Hsp90i—2—induced apoptosis (**, pS0.0l; Fig. 8A). Moreover, HSF~1 own using siRNA decreases CLU sion, sensitizing tumor cells to apoptosis—induced by Hsp90i-2 (Fig. 8B), confirming that the tive effect of CLU is mediated by HSF—l. Surprisingly, overexpression of CLU also increased HSF—l activity (***, pS0.00l, Fig. 3B), while CLU knockdown using siRNA or custirsen significantly decreased HSF—l ty (*, pS0.05; ***, pSO.DOl; Fig. 3C), identifying novel feed—forward. tion of HSF—l by U‘I CLU. Indeed, silencing of CLU ted HSF—l transcriptional activityninduced by Hsp90i~l or Hsp90i—2 (Fig. 3C), as well as REF— 1 regulated genes such as Hsp27 and Hsp70 (Fig. 3D). This effect can be explained by the ability of CLU knockdown to sequester HSF—l in the cytoplasm (Fig. 8B).

Example 3. Increased. potency of combination ent, comprising custirsen and Hsp90 tor in increasing apoptosis in prostate tumor cell lines compared to monotherapy.

Since Hsp90 inhibitors induce up—regulation of CLU and functions as a mediator in treatment resistance (Zoubeidi et al. 2010; Gleave et al., 2005; Zellweger et al., 2003), it was next evaluated if CLU own combined with Hsp90 inhibition increased treatment effectiveness. LNCaP cells were d with custirsen and subsequently treated with indicated concentrations of Hsp90i—l or Hsp90i—2. The combination. had. significantly enhanced Hsp90i—l or Hsp90i—2 effectiveness, reducing cell viability by 20% at lOOnM and lOOOnM (*, p<0.05) compared with cells treated with l Sch A80 and Hsp90 inhibitors (Fig. 4A). To determine whether this effect was additive or a combination effect, the dose—dependent effects with constant ratio design and the combination index (CI) values was performed and calculated. ing' to the Chou and. y median effect principal (Chou et al., 1984). Figure 4B shows the dose response curve (combination treatment, custirsen or HSP90i—2 monotherapy) and the combination index plots, indicating that custirsen and HSP90i—2 had enhanced ed potency on tumor cell growth compared to custirsen or Hsp90i—2 inhibitor monotherapy.

Moreover, OGX—Oll potentiates the effect of Hsp90 inhibitor to induce apoptosis (Fig. 4C and D). Flow cytometric analysis shows that apoptotic rates (subGl fraction) increased icantly (p<0.00l) when custirsen is combined with Hsp90i—1 (53%) or Hsp90i— 2(65.4%), compared to control Sch ASO (4.2%), custirsen alone (17.4%), control Sch ASO+Hsp90i—l ) or control Sch ASO+Hsp90i~2 (24.8%; Fig. 4C). Moreover, the combination custirsen with —l or Hsp90i~2 induced more caspase—dependent apoptosis compared to Hsp90 inhibitor— or custirsen monotherapy, as shown by cleaved PARP and. caspase—3 expression. (Fig 4C). The icant increase of caspase—3 activity confirms that custirsen sensitizes cells to Hsp90 inhibition with increased apoptotic rates (Fig. 4D).

Reduced cell viability from combined CLU plus Hsp90 tion results, in part, from decreases in p—Akt levels in both PC—3 and LNCaP cells, as well as AR (and. PSA) expression in LNCaP cells (Fig. 4C).

Example 4. Potent combination therapy of custirsen and Hsp90i—l in PC~3 afts in vivo.

The effects of combining custirsen with Hsp90i—1 was evaluated in PC—3 tumors in vivo. Male nude mice bearing PC—3 xenograft swere randomly selected. for treatment (custirsen —+ Hsp90i—1 vs control Sch + Hsp90i—1; n=7). Custirsen + Hsp90i—1 had icantly enhanced the mor effects compared to of Sch + Hsp90i—1 in Vivo, ng the mean tumor volume from 2935.3 mm3 to 1176.9 mm3 after 68 days (**; pS0.0l), compared to control Sch (Fig. 5A).

Cancer specific survival was significantly prolonged with combined custirsen + Hsp90i~1 compared with controls (71.4% vs 14.3% at day 72, respectively; *; pS0.05; Fig. 5B. Immunohistochemical analysis s decreased CLU, K167, and Akt, expression after treatment with custirsen + Hsp90i—1 compared to other groups (Fig 5C).

Additionally, custirsen + Hsp90i—l d tumors had higher apoptosis as shown by increased TUNEL staining compared with other groups (Fig 5C).

Example 5. Potent combination therapy of sen and —2—PRO in LNCaP CRPC xenografts in vivo.

Next the effects of combined ent with custirsen and Hsp90i—2— PRO was assessed in castrate resistant LNCaP tumors. Mice bearing LNCaP tumors were ted when PSA values exceeded SOng/ml. Once PSA levels relapsed above pre-castration levels mice were randomly assigned to vehicle control, Hsp90i—2—PRO alone, Hsp90i—2—PRO + control Sch, or Hsp90i—2—PRO + custirsen (n=lO in each. group).

Mice treated with Hsp90i—2—PRO + sen had significant delays in tumor growth compared. with all other groups (Fig 6A) (at l0 days, respectively 265.3 mm3, and 892.7 mm3 for control, 646.4mm3 for Hsp90i—2—PRO alone and 551.56 mm? for Hsp90i—2—PRO+control Sch). By 7 weeks post ent, all mice in the control had been euthanized; tumor volume in the Hsp90i—2—PRO + custirsen group was r517.4 mm3 compared to 2483.6mm3 for HSP90i~2~PRO alone and 2176.4 mm? for Hsp90i-2—PRO+control Sch; ***, l; Fig. 6A).

Serunl PSA levels were also significantly lower (~4—fold) in the mice receiving custirsen + Hsp90i—2—PRO compared with other groups (***, p<0.001; Fig~ 6B). The combination custirsen ~+ Hsp90i—2—PRO group had a mean PSA level of lZOng/ml after 42 days compared to 418.7ng/Hd in e group, 527ng/ml in Hsp90i~2—PRO alone, or 480.3ng/ml in sch + Hsp90i~2~PRO groups. The combination custirsen+ Hsp90i—2—PRO group had a significantly increased. PSA ng time (33.6 weeks; *, p<0.05) and decreased PSA velocity (l3.78ng/mL/week; *, p<0.05) compared with other groups (PSA doubling time: ~2.4weeks; velocity: ~85ng/mL/week; Fig. 6C).

Overall survival was also icantly longer in mice treated with combined custirsen + Hsp90i—2—PRO (Fig’ 6D). By day 57, all mice died or were euthanized. due to high tumor burden in l, Hsp90i—2—PRO alone, or control Sch + Hsp90i—2—PRO groups compared with the combined custirsen + Hsp90i—2-PRO group, where all mice were still alive (p<0.001) after 62 days. These data demonstrate that targeting CLU using custirsen in combination with HSP90i—2—PRO ~67- inhibits tumor growth and prolongs survival in human CRPC xenograft model significantly more than monotherapy.

Consistent with in vitro findings, immunohistochemical analysis U} revealed decreased CLU, Ki67, Akt, and AR expression after treatment with combined custirsen + Hsp90i—2—~PRO compared with other groups (Fig 7A). The staining results were orated by western blotting (Fig 7B). Additionally, tumors treated with combination custirsen + Hsp90i—2—PRO had higher apoptosis rates compared with other groups as shown by increased TUNEL staining (Fig 7A). These data suggest that decreases in tumor progression custirsen -F Hsp90i-2—PRO treated tumors result from both reduced proliferation rates as well as sed apoptosis rates.

Example 6. Materials and Methods for Examples 1—5.

Tumor cell lines and reagents: The human PCa cell line PC—3 was sed from the American Type Culture Collection (2008, ATCC-authentication by isoenzymes analysis) and maintained in DMEM (Invitrogen-Life Technologies, Inc.) mented with 5% fetal bovine serum and 2mmol/L Lglutamine. LNCaP cells were kindly provided. by Dr. Leland. W.K.

Chung' (1992, MDACC, n Tx) and. tested. and. authenticated. by whole—genome and whole—transcriptome sequencing on na Genome Analyzer IIx platfornt in July 2009. LNCaP cells were maintained RPMI 1640 (Invitrogen Life Technologies, Inc.) supplemented with 5% fetal bovine serun1 and. 2mmol/l; L-glutamine. All cell lines were cultured i11 a humidified 5% (Kb/air atmosphere at 37°C. All cell lines were passaged. for less than 3 months after resurrection.

Western blotting and/or real time PCR was performed for AR and PSA each time when LNCaP cells were resurrected.

Therapeutic : Hsp90 inhibitor, HSP90i—2 (4—(6,6—Dimethyl—4—oxo-3—trifluoromethyl— 4,5,6,7—tetrahydro—indazol—l—yl)—2—(4-hydroxy—cyclohexylamino)— ide) and its prodrug HSP90i—2~PRO were used respectively for W0 2012/123823 in vitro and in vivo studies. These compounds are novel synthetic small molecular weight inhibitors that bind the N—terminal ine triphosphate binding site of Hsp90 and HSP90i—2—PRO is orally bioavailable. For the in vitro studies, —2 was dissolved in dimethyl ide (DMSO) at lOmM stock solutions and stored at ~2OOC. For the in vivo studies, HSP90i—2—PRO was dissolved in PBS 1% carboxymethylcellulose and 0.5% Tween 80 (Invitrogen—Life Technologies, Inc.) at lSmg/ml and stored at 4°C.

HSP90i—l lylamino—l7—demethoxygeldanamycin (l7—AAG)) was used for in vitro and in vivo studies. For the studies, l7—AAG was dissolved in dimethyl sulfoxide (DMSO) at lOmM stock solutions and stored at ~200C.

Clusterin siRNA and Antisense ucleotides siRNAs were purchased from Dharmacon Research, Inc. (Lafayette, CO) using the siRNA sequence corresponding to the human CLU initiation site in exon 2 and a scramble control as previously bed (Sowery et al., 2008). Second—generation antisense (custirsen) and scrambled (Sch) oligonucleotides with a 2'—O—(2—methoxy)ethyl modification were supplied by OncoGenex Pharmaceuticals (Vancouver, British Columbia, Canada). Custirsen sequence (5'— CAGCAGCAGAGTCTTCATCAT—3'), SEQ ID NO:3 corresponds to the initiation site in exon II of human CLU. The Sch l sequence was 5’-CAGCGCTGACAACAGTTTCAT~3' (SEQ ID NO: 44). Prostate cells were d with siRNA or oligonucleotides using protocols described previously (Sowery et al., 2008).

Cell proliferation and apoptosis : Prostate cells lines were plated. in appropriate media (DMEM or RPMI) with 5% FBS and treated. with Hsp90i~2~PRO or Hsp90i—l at indicated concentration and time and cell growth was measured using the crystal violet assay as described. previously (Leung et al., 2000). Detection and quantitation of apoptotic cells were done by WC 23823 flow—cytometry (described below) and western ng analysis.

Each assay was repeated in triplicate.

The combination index (Cl) was evaluated using CalcuSyn dose effect is software (Biosoft, dge, UK). This method, based on the multiple drug effect equation of Chou—Talalay (Chou et al., 1984), is suitable for caluculating combined drug activity over a wide range of growth inhibition: CI =1, additivity; C: >1, antagonism; CI <1, combination effect. CI was calculated at EDW and ED75 .

Caspase-3 activity was assessed 3 days after treatment using the kit CaspACE Assay System, Fluorometric (Promega, Madison, WI, USA).

Fifty pg of total cell lysate were incubated with caspase—3 substrate AC-DEVD—AMC at room temperature for 4h and caspase-3 activity was quantified in a fluorometer with tion at 360nm and emission 460nm.

Cell cycle analysis: Prostate cancer cell lines were incubated. in the absence or the presence of 1pM Hsp90i—2 or Hsp90i—l for 72h, trypsinized, washed twice and incubated in PBS containing 0.12% Triton X-100, 0.12mM EDTA and lOOug/ml ribonuclease A; SOug/ml propidium iodide was then added to each sample for 20min at 4°C. Cell cycle distribution was ed by flow cytometry (Beckman Coulter Epics Elite, Beckman, Inc., Miamai, FL), based on 2N and 4N DNA content. Each assay was done in triplicate. western blotting analysis: Samples containing equal s of protein (depending on the antibody, ) from lysates of cultured tumor prostate cell lines underwent electrophoresis on SDS—polyacrylamide gel and were transferred to nitrocellulose filters. The filters were blocked in Odyssey Blocking Buffer (LI-COR ences) at room ature for lh and blots were probed overnight at 4°C with primary antibodies to detect proteins of interests. After incubation, the filters were washed. 3 times with washing buffer (PBS containing 0.1% Tween) for 5min. Filters were then incubated for 1h with 1:5,000 diluted Alexa Fluor secondary antibodies (Invitrogen) at room temperature. Specific proteins were detected using ODYSSEY IR imaging system (LI—COR Biosciences) after washing.

Quantitative Reverse Transcription—PCB: Total RNA was extracted from cultured cells after 48h of treatment using TRIzol reagent (Invitrogen Life Technologies, Inc.). Two ug of total RNA was ed transcribed using the Transcriptor First Strand CDNA Synthesis Kit (Roche Applied Science). Real~time monitoring‘ of PCR amplification of complementary DNA (cDNA) was med using DNA primers emental table 81) on ABI PRISM 7900 HT ce Detection System (applied Biosystems) with SYBR PCR. Master Mix (Applied Biosystems). Target gene expression was normalized tt> GAPDH levels in tive samples as an internal standard, and the comparative cycle threshold (Ct) method was used to calculated relative quantification of target mRNAs. Each assay 2O was performed in cate.

Luciferase assay: LNCaP and C4—2 cells (2.5x105) were plated on six—plates and transfected using lipofectin (6uL per well; Invitrogen Life Technologies, Inc.). The total amount HSE ds DNA used were normalized to lug per well by the addition of a control plasmid.

One uM -2 or Hsp90i~l was added 4h after the transfection and for 48h. HSE—luciferase activity was measured using uciferase Reporter Assay System (Promega) with the aid of a microplate luminometer (EG&G Berthold). All experiments were carried out in triplicate wells and repeated 3 times using different preparations of plasmids. fluorescence: Tumor cells were grown on coverslips and d. with different concentration of Hsp90i—2 or Hsp90i-l for 48h. After treatment, cells were fixed in ice‘cold methanol completed with 3% acetone for 10min at —20°C. Cells were the washed thrice with PBS and ted with 0.2% Triton/PBS for 10min, followed by washing and 30min blocking in 3% nonfat milk before the addition of antibody overnight to detect HSF—l (1:250). ns were visualized using anti—mouse antibody coupled with FITC (1:500; 30 min).

Photomicrographs were taken at 20X magnification using Zeiss Axioplan II fluorescence microscope, followed by analysis with g re (Northern Eclipse, Empix g, Inc.).

Animal Treatment: Male athymic nude mice (Harlan Sprague—Dawley, lnc.) were injected s.c. with 2x106 LNCaP cells (suspended in O.lmL Matrigel; BD Biosciences). The mice were ted once tumors reach between 300 and 500mm3 or the PSA level increased above SOng/mL. Once tumors progressed to castrate resistance, mice were randomly assigned to vehicle, Hsp90iPRO alone, Hsp90i-2—PRO + Sch ASO or Hsp90i—2— PRO + custirsen. —2—PRO (Prodrug, 25mg/kg; formulation in 0.5'0P CMC+0.5% Tween—80) is orally administered three times per week and custirsen or Sch ASO (lSmg/kg) was injected intra—peritoneally once daily for the first week and then three times per week. Each experimental group consisted of 10 mice. Tumor volume was measured twice weekly (length x width x depthv x 0.5432). Serum. PSA was determined weekly by enzymatic assay (Abbott IMX, Montreal, Quebec, Canada). PSA doubling time (PSAdt) and velocity were calculated by the log~slope method (PSAt PSAinitial x emt). Data points were expressed as average tumor volume r SEM or average PSA concentration i SEM.

To establish PC—3 tumors, 2 x 106 PC—3 cells were inoculated s.c. in the flank region of 6—8 week—old male athymic mice (Harlan Sprague— Dawley, Inc.). When tumors reached lOOmm3, usually 3—4 weeks after injection, mice were randomly selected for treatment with HspBOi-l (25mg/kg) + control Sch ASO (lSmg/kg) or Hsp90i—1 + custirsen (15mg/kg). Hsp90i—l was injected. i.p. three times per week, and custirsen or Sch were injected i.p. once/day for the first week and then three times per week. For each experimental group consisted of 7 mice. Tumor volume was measured twice weekly. Data points were expressed as average tumor volume i SEM.

When tumor volume reached 210% of body , mice were sacrificed and tumors harvested for evaluation of protein expression by western blotting analyses and immunohistochemistry. All animal procedures were performed according to the guidelines of the Canadian Council on Animal Care and appropriate institutional certification.

Immunohistochemistry: Immunohistochemical stains were performed on formalin—fixed and paraffin—embedded 4pm sections of tumor samples using te primary antibody, and the a autostainer Discover XT (Ventana Medical System) with enzyme labeled biotin streptavidin system and solvent resistant 3,3’sdiaminobenyidine Map kit. All comparisons of staining intensities were made at 200x ications.

Statistical analysis: All in vitro data were assessed using the t t test and Mann— Whitney test. Tumor volumes of mice were compared using Kruskal— Wallis test. Overall survival was analyzed using Kaplan—Meier curves and statistical significance between the groups was assessed with the log—rank test (Graphpad. Prism). Levels of tical 3O significance were set at P<0.05.

Antibodies used for western ng: PARP (1/1000) Caspase 3 (1/1000), Akt (1/1000), p-Akt (1/500), are from cell signaling. Cyclin D1 (1/1000), HSP9O 0), HSP70 (l/lOOO), clusterin (l/lOOO), AR (l/lOOO), PSA (l/lOOO) W0 2012/123823 HSF-l (1/1000) are from Santa Cruz. HSP27 (1/5000) is from Assays Designs.

Table 3: Primers used for quantitative ime PCT ce Sequence 5 ’ to 3' Sequence 5 ’ to 3’ name forward reverse Clusterin ‘GAGCAGCTGAACGAGCAGTTT (SEQ CTTCGCCTTGCGTGAGGT ID NO: 45) (SEQ ID NO: 46) Hsp7O TGCCCTATCCAGATCCTGCTA GAGCCATCAGACTGAGGAGTGA (SEQ (SEQ ID NO: 47) ID NO: 48) -Hsp90 TTCAGGCCCTTCCCGAAT TCACTCCTTCCTTGGCAACAT (SEQ ID NO: 49) (SEQ ID NO: 50) Discussion Prostate cancer responds initially to anti-androgen therapies, however, progression of castration ant e frequently U] occurs. Small molecule inhibitors of Hsp90 show promise in the treatment of castration—resistant prostate cancer (CRPC) and other cancers, however these inhibitors trigger a heat shock response that attenuates drug iveness. In prostate cancer, treatment resistance emerges early due to compensatory isms involving activation of heat shock factor 1 (HSFnl). Once released from Hsp90, HSF—l translocates to the nucleus, binds to heat shock elements (HSE) of Hsp genes and increases Hsp transcription activity (Whitesell et al., 2005). Therefore, Hsp90 inhibition induces a heat shock response with increased. expression. of several Hsps including Hsp90, Hsp70, Hsp27 and. clusterin (CLU), which enhance tumor cell survival and treatment resistance. The up-regulation of these molecular chaperones has been reported to play a role in cellular recovery from stress by restoring n homeostasis, ing thermotolerance, cell survival, and treatment resistance (Takayama et al., 2003; Zoubeidi 2O et al., 2010). The data herein show that preventing CLU induction in this response would enhance Hsp90 inhibitor—induced CRPC cell death in vitro and in Vivo. As disclosed herein, CRPC was treated with Hsp90 inhibitor HSP90i—2—PRO or HSP90i—l in the absence or presence of custirsen, an antisense drug‘ that targets CLU. Treatment with either Hsp90 inhibitor alone increased. nuclear translocation and transcriptional activity of the heat shock factor HSF—l, which stimulated dose— and ependent increases in heat shock protein sion, including especially CLU‘ sion. Treatment—induced increases in CLU were blocked by custirsen, such that the ation of custirsen and either Hsp90 inhibitor had enhanced. inhibition activity on CRPC cell growth and apoptosis compared to custirsen or Hsp90 tor monotherapy. Accompanying these effects was a decrease in HSF—l transcriptional activity as well as expression of HSPs, Akt, PSA and androgen receptor. In vivo evaluation of the Hsp90 inhibitors with sen in xenograft models of human CRPC demonstrated that custirsen markedly potentiated anti-tumor efficacy, W0 2012/123823 leading to an 80% inhibition of tumor growth with prolonged survival compared to Hsp90 inhibitor monotherapy. Together, the findings herein indicate that Hsp9O inhibitor-induced activation of the heat shock response and CLU is attenuated by custirsen, with combination U] therapy having increased potency on delaying CRPC progression.

Development of treatment resistance is a common feature of most ancies and the underlying basis for most cancer deaths.

Treatment resistance evolves, in part, from selective pressures of treatment that collectively increase the tic rheostat of cancer cells. Survival proteins up—regulated after treatment stress include anti—apoptotic members of the bcl—2 protein family, survivin, and molecular chaperones like CLU and other HSPs (Zellweger et al. 2003). lar chaperones help cells cope with stress—induced. protein ation, and play prominent roles in cell signaling and transcriptional regulatory networks. Chaperones act as genetic buffers izing the phenotype of various cells and organisms at times of environmental stress, and enhance Darwinian fitness of cells during cancer progression and treatment resistance (Whitesell et al., 2005). Heat shock ones are key components of the heat shock response, a highly conserved stress—activated protective mechanism also associated with oncogenic transformation and thermo- tolerance (Dai et al., 2007). Chaperones are particularly important in ting misfolded protein and endoplasmic reticular (ER) stress responses, an emerging area of interest in ent stress and resistance. A g enthusiasm for therapeutic tion of this proteostasis network highlights Hsp's and CLU as rational targets because of their multifunctional roles in signaling' and transcriptional networks associated. with cancer ssion and treatment resistance. Cancer cells express higher levels of molecular chaperones and pirate the protective ons of HSFl to support their transformation (Dai et al., 2007). Indeed, inhibitors of Hsp90, Hsp70, Hsp27 or CLU‘ have all been reported. to induce 2012/000696 cancer cell death and. ize chemotherapy (Lamoureux et al., 2011; Guo et al., 2005).

Increased expression of clusterin (CLU) has been associated with U) chemoresistance, radioresistance, and hormone resistance (Zellweger et al., 2003; July et al., 2004). CLU is a stress—induced cytoprotective chaperone that inhibits protein ation in a manner analogous to small HSPs, and its promoter contains a 14—bp element recognized by the transcription factor HSF—l (Humphreys et al., 1999). In human PCa, CLU levels are low in Gleason grade 3 untreated hormone—naive tissues, but increase with higher Gleason score (Steinberg et al., 1997) and. within weeks after androgen deprivation (July et al., 2002). CLU expression. correlates with loss of the tumor suppressor gene ka3.1 during the l stages of‘ prostate tumorigenesis in ka3.1 knockout mice (Song* et al., 2009). Experimental and clinical studies associate CLU with development of ent resistance, where CLU sses treatment-induced cell death in response to androgen withdrawal, chemotherapy or radiation (Miyake et al., 2000a; July et al., 2002; Miyake et al., 2000b; Miyake et al., 2000c). Over—expression of CLU in human prostate LNCaP cells accelerates progression after hormone— or chemo—therapy (Miyake et al., 2000a; Miyake et al., 2000c), identifying CLU as an anti—apoptotic gene up—regulated by treatment stress that confers therapeutic resistance. sen is a second—generation phosphorothioate antisense oligonucleotide tly in late stage clinical development that potently ts CLU expression and enhances the efficacy of ncer therapies in various human cancers including PCa (Zoubeidi et al., 2010, Gleave et al., 2005). While targeting CLU enhances the cytotoxic effects of chemotherapy and delays tumor growth in various human cancers including PCa (Miyake et al., 2005), a role for CLU has not been characterized. in the context of Hsp90 inhibitor treatment and resistance. As shown herein, Hsp90 inhibition induces a heat shock response with increased HSF—l activity and CLU expression, which functions to inhibit treatment-induced apoptosis and enhance emergence of treatment ance. Knockdown of CLU using custirsen potentiates the effect of Hsp90 inhibitors in CRPC.

Aspects of the present invention relate to the unexpected ery that an oligonucleotide targeting rin expression such as custirsen, together with a Hsp90 inhibitor is a potent combination for treatment of prostate cancer. The discovery that an ant— clusterin therapy combined with Hsp90 is so potent is particularly surprising~ because Hsp90 is known to increase the expression. of multiple otective proteins.

Several Hsp90 inhibitors including HSP90i—2 have potent anti—tumor activity in various preclinical models (Lamoureux et al., 2001; Chandarlapaty et al., 2008; Okawa et al., 2009) and are in clinical trials (Lamoureux et al., 2011; Sydor‘ et al., 2006). Consistent with prior reports (Lamoureux et al., 2011; Cervantes-Gomez et al., 2009), the data herein show that Hsp90 inhibitors induce a stress response with activation of the ription factor HSF—l and subsequent increased levels of Hsp90 itself, Hsp70 and CLU. This heat shock response likely enhance emergence of treatment resistance, as inhibition of transcription using Actinomycin D attenuates -1—mediated Hsp70 and Hsp27 expression and iates the effect of HSP90i-l in vitro (Cervantes-Gomez et al., 2009). Additionally, inhibition of the stress response by silencing‘ HSF—l also increases the ty of Hsp90 inhibitors (Bagatell et al., 2000). The experiments disclosed herein evaluated the role of CLU in this heat shock response since CLU is dramatically induced. by Hsp90 inhibitor treatment and CLU inhibitors are in late stage clinical pment.

CLU is associated. with many varied. patho—physiological processes including reproduction, lipid transport, complement regulation and apoptosis (Zoubeidi et a1. 2010; Rosenberg et al., 1995). CLU expression is rapidly upregulated in various tissues undergoing apoptosis, including normal and ant prostate and breast PCT/IBZOIZIOOO696 tissues following e withdrawal (Kyprianou et a1., 1990; Kyprianou et a1., 1991). Previous studies have also linked CLU expression with induction and progression of many cancers, including CRPC (Zoubeidi et a1., 2010). Furthermore, CLU up— U) regulation following' androgen ablation in xenograft. tumor‘ models accelerates progression. to castrate resistance and. renders cells resistant to other apoptotic stimuli, including taxane chemotherapy (Miyake et a1., 2000; Miyake et al., 2001). Consistent with these accumulated findings (Miyake et a1., 2001), inhibition of CLU using custirsen istically' es conventional as well as molecular targeted therapies in PCa preclinical models (Sowery et a1., 2008). Indeed, custirsen is now in Phase III trials as Phase II studies reported >90% inhibition of CLU in human prostate cancer tissues (Chi et a1., 2005), and 7 months prolonged survival when OGX—Oll is combined with docetaxel in CRPC (Chi et a1., 2008; Chi et a1., 2010).

The data herein show that Hsp90 inhibitors increase CLU levels both in Vitro and in vivo, while clusterin inhibits HSP90i—2 or —l induced CLU. As expected (Cervantes—Gomez et a1., 2009; Bagatell et a1., 2000), —2 or HSP90i-1 induces HSF—l transcriptional activity leading to up—regulation of HSPs expression. Surprisingly, the experiments described herein found that CLU silencing abrogates, while CLU overexpression enhances, Hsp90 inhibitor— induced HSF—l ription activity, identifying a role for CLU in the regulation of HSF—l and the heat shock response itself. CLU knockdown blocks the translocation to HSF—l to the nucleus following treatment with Hsp90 inhibitors. This effect of CLU on HSF-l activity is biologically relevant since CLU’ overexpression ts, while CLU ing enhances, cytotoxicity of Hsp90 inhibitors. Consistent with these in vitrc results, istic effects were also observed in vivo in PC—3 and LNCaP models when custirsen was combined with Hsp90 inhibitors. Combination custirsen plus Hsp90 inhibitor significantly delay CRPC tumor growth and prolonged al in PC—3 and LNCaP . Increased apoptotic rates with combined Hsp90 and CLU inhibition suggests that d tumor progression resulted from enhanced treatment—induced apoptosis. Systemic administration of an ucleotide which reduces clusterin expression plus a Hsp90 inhibitor decreases tumor growth compared with control Sch ASO plus an Hsp90 inhibitor in PC—3 model and LNCaP castration—resistant prostate cancer, respectively. This inhibition of tumor ssion is accompanied withv a prolongation of survival in both prostate cancer models.

Detection of increased apoptosis after ed rin plus Hsp90 inhibition by detection of TUNEL using immunohistochemistry suggests that delayed tumor progression after combined y results from enhanced Hsp90 inhibitor—induced apoptosis.

Collectively, these results highlight, for the first time, a ically relevant feed—forward tion loop of CLU on HSF—l and the heat shock response.

The effect of an oligonucleotide which reduces clusterin expression in combination with an Hsp90 inhibitor on PSA level was examined in the LNCaP castration—resistant prostate cancer model as disclosed herein above. As shown , targeting CLU using siRNA or the antisense drug, custirsen, suppressed treatment—induced CLU induction and enhanced Hsp90 inhibitor—induced cell death in prostate cancer cells. Serum PSA level is an established and useful biomarker regulated by androgen receptor (AR) in the presence of androgens (Magklara et al., 2002), and a le tool in the follow—up of patients to assess the efficacy of chemotherapy. In addition to the effects of CLU inhibition on the heat shock response, observations in the castrate—sensitive, AR—positive LNCaP model highlight another possible benefit of combined CLU and Hsp90 suppression involving* AR. activity. Hsp90 inhibition is known to destabilize and e the AR with decreased PSA expression (Solit et al., 2002; Georget et al., 2002). In vivo, serum PSA levels as well as PSA doubling time and velocity, were significantly reduced with combination OGX—Oll therapy compared with PF—04929ll3 monotherapy. Serum PSA level is an established and useful AR— WO 23823 PCT/IBZOIZIOOO696 regulated biomarker (Kim et al., 2004) and a valuable tool in assessing efficacy of chemotherapy. Interestingly, at the low doses of Hsp90 tor used in this in vivo study, no effect on serum PSA level was nt. Lower PSA levels with combination therapy correlated with lower AR levels. This ation between CLU inhibition and lower AR levels may involve the regulation loop of CLU’ on ESP-l and. the role of HSF—l in regulating" expression of other AR ones (eg. Hsp27, Hsp70, Hsp90, FKBP5.2) and we are actively exploring the molecular basis in ongoing experiments.

While CLU is known to be transcriptionally activated by HSF—l (Zoubeidi et al., 2010), the data herein also show that CLU exerts a feed forward loop that in turn activates HSF—l. CLU knockdown decreases HSF~l riptional activity and abrogates its r translocation, which subsequently leads to decreased Hsp27, Hsp70 and Hsp90 expression, similar to that observed after HSF—l knockdown (Rossi et al., 2006). Consequently, AR stability is reduced because of lowered chaperone levels.

In addition to increased y of anti—tumor activity, 2O combination therapy may also allow dose reduction strategies to reduce toxicity. For example, HSPSOi—l induced. hepatotoxicity as monotherapy at 60mg/kg/day (Glaze et al., 2005), while HSP90i—2—PRO caused body weight loss at 50mg/kg/day. In a previous study, 50mg/kg HSP90i—2—PRO as monotherapy ted LNCaP CRPC tumor progression (Lamoureux et al., 2011). At sub—therapeutic doses of ng/kg/day used in the present study, HSP90i—2—PRO monotherapy showed marginal, non—significant decreases in tumor volume and no effect on serum PSA levels; however, significant delays in tumor progression were seen at this lower dose when HSP90i—2—PRO was combined with custirsen, with no toxicity observed.

The data disclosed herein help define how stress induced by Hsp90 inhibitors regulates CLU by induction of HSF—l activity and, in turn, how CLU regulates HSF—l ty, cell survival, and treatment resistance. As demonstrated herein, for the first time, -81— W0 2012/123823 that CLU inhibition abrogates the heat shock response induced Hsp90 inhibitors. These observations are clinically relevant since CLU inhibitors are in phase III clinical trials, and provide a ork for building new drug combinations based on mechanism- based interventions to overcome drug resistance. The present ion relates to the development of targeted strategies ing custirsen in combination with Hsp9O inhibitors to improve patient outcome in CRPC.

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Claims (13)

What is claimed

1. Use of i) an antisense or RNAi oligonucleotide that is complementary to the sequence of clusterin of a mammalian subject, and that reduces clusterin expression; and ii) a Heat Shock n 90 (Hsp90) inhibitor comprising 4— (6,6—Dimethyl—4—oxo—3-trifluoromethyl—4,5,6,7-tetrahydro— indazol—l—yl)—2—(4~hydroxy—cyclohexylamino)—benzamide, or a pharmaceutically acceptable salt thereof, or a prodrug that is metabolized to release 4—(6,6—Dimethyl—4—oxo~3~ trifluoromethyl~4,5,6,7—tetrahydro—indazol—l—yl)—2—(4—hydroxy— cyclohexylamino)~benzamide in the manufacture of a medicament for treating prostate cancer in the mammalian subject.

2. The use of claim 1, wherein the cancer is androgen—independent prostate cancer.

3. The use of claim 1 or 2, wherein the ian subject is human.

4. The use of any one of claims 1—4, wherein the oligonucleotide is an antisense oligonucleotide.

5. The use of claim 5, wherein the antisense ucleotide comprises one of Seq ID NOs: 1—11.

6. The use of claim 5, wherein the antisense oligonucleotide comprises SEQ ID NO: 3.

7. The use of claim 6, wherein the antisense ucleotide is ed to enhance in vivo stability relative to an unmodified oligonucleotide of the same sequence.

8. The use of claim 7, wherein the oligonucleotide is custirsen.

The use of any one of claims 1—4, n the oligonucleotide is an RNAi oligonucleotide.

10. The use of claim 9, wherein the RNAi oligonucleotide comprises one Of SEQ ID NOS: 19-42.

ll. The use of any one of claims 1—10, wherein the combination comprises Hsp90i—2—PRO.

12. The use of any one of claims 1—11, wherein the combination of the oligonucleotide and the Hsp90 inhibitor is effective to inhibit the proliferation of prostate cancer cells.

13. The use of claim 1, ntially as herein described with reference to any one of the Examples and/or