WO2015171647A1

WO2015171647A1 - Treatment of cancer

Info

Publication number: WO2015171647A1
Application number: PCT/US2015/029297
Authority: WO
Inventors: Charles P. Hart; Jeffrey B. LANDAU; Jessica D. SUN; Stewart Kroll
Original assignee: Threshold Pharmaceuticals, Inc.
Priority date: 2014-05-05
Filing date: 2015-05-05
Publication date: 2015-11-12

Abstract

Administration of a hypoxia-activated prodrug, such as, e.g., TH-281, TH-302, and TH-308, in combination with carfilzomib or pomalidomide is useful for the treatment of cancers such as blood cancers, such as multiple myeloma.

Description

TREATMENT OF CANCER

[0001] The present invention relates to methods of treating of cancer, such as a blood cancer, by the administration of a combination of a hypoxia-activated prodrug, such as TH- 302, and carfilzomib, pomalidomide or bortezomib to a patient in need of treatment and so relates to the field of biology, medicine, toxicology, and pharmacology.

BACKGROUND OF THE INVENTION

[0002] Blood cancer refers to a class of cancers that attack the blood, bone marrow, and/or lymphatic system. This class of cancers includes multiple myeloma, which can be life- threatening disease for which new and more efficacious treatments are needed.

[0003] The relation between cancer cells and the tumor microenvironment affects the growth and survival of cancer cells (see Hiruma et al., Blood. 2009; 113(20): 4894-4902, and Podar et al., Leukemia. 2009; 23(1): 10-24, each of which is incorporated herein by reference). Hypoxia, or low oxygen level, is a characteristic of the microenvironment of many solid tumors and results from the poor vascularization that characterizes many solid tumors. High metastatic potential and poor prognosis correlate highly with hypoxia in solid tumors.

[0004] Clinical research has attempted to target therapies to the hypoxic regions of solid tumors for many years without notable success. Recently, however, a promising new class of hypoxia-activated prodrugs has emerged (see U.S. Patent No. 7,550,496, incorporated herein by reference), and the most promising compound in that class, called TH-302 (see PCT Pub. Nos. WO 2007/002931; WO 2008/083101; and WO 2010/048330, each of which is incorporated herein by reference), is now in advanced clinical testing.

[0005] Clinical testing of TH-302 has shown that it has potential for treatment of blood cancers as well (see PCT Pub. No. WO 2012/006032, incorporated herein by reference). Because of the dire prognosis for patients with blood cancers, further advancements in pharmaceutical management of the condition are needed. The present invention meets these needs. BRIEF SUMMARY OF THE INVENTION

[0006] This invention provides medicines and technology for treating cancer: in particular, the invention provides methods and pharmaceutical formulations for treating cancer by administration of a combination of a hypoxia-activated prodrug, such as TH-302 and carfilzomib, pomalidomide or bortezomib. When used together, the two drug combination can be more effective than any of the drugs used alone, without materially altering tolerability.

[0007] Thus, one aspect of this invention relates to methods and pharmaceutical formulations for treating cancer in which a combination of pharmaceutical agents comprising a hypoxia-activated prodrug such as TH-302 and carfilzomib, pomalidomide or bortezomib is administered to a cancer patient. The drug combination may be used for simultaneous or sequential use in the treatment of cancer, although in many embodiments, the TH-302 will be administered first (see PCT Pub. No. WO 2010/048330). Another aspect of the invention is a method of treating cancer by administering an effective combination of pharmaceutical agents comprising a hypoxia-activated prodrug such as TH-302 and carfilzomib, pomalidomide or bortezomib. A further aspect of the invention is the use of a hypoxia- activated prodrug such as TH-302 and carfilzomib, pomalidomide or bortezomib in the manufacture of a medicament or medicament combination for treatment of cancer.

[0008] Provided herein are methods for treating various blood cancers, such as multiple myeloma, comprising administering a therapeutically effective amount of a hypoxia-activated prodrug. Suitable hypoxia activated prodrugs include a compound of formula (I):

(I) wherein Y₂ is O, S, NR₆, NCOR₆, or NS0₂R6 wherein Re is (Ci- C₆) alkyl, Ci-C₆ heteroalkyl, aryl, or heteroaryl; R₃ and R4 are independently selected from the group consisting of 2- haloalkyl, 2-alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-heteroalkylsulfonyloxyalkyl; Ri has the formula L-Z₃; L is C(Z₁)₂; each Zi independently is hydrogen, halogen, Ci-C₆ alkyl, Ci-C₆ heteroalkyl, aryl, heteroaryl, C₃-Cg cycloalkyl, heterocyclyl, Ci-C₆ acyl, Ci- C₆ heteroacyl, aroyl, or heteroaroyl; or L is:

Z₃ is a bioreductive group having a formula selected from the group consisting of:

wherein each Xi is independently N or CR₈; X₂ is NR₇, S, or O; each R₇ is independently Ci- C₆ alkyl, Ci-C₆ heteroalkyl, C₃-Cg cycloalkyl, heterocyclyl, aryl or heteroaryl; and Rg is independently hydrogen, halogen, cyano, CHF₂, CF₃, C0₂H, amino, Ci-C₆ alkyl, Ci-C₆ heteroalkyl, Ci-C₆ cycloalkyl, Ci-C₆ alkoxy, Ci-C₆ alkylamino, Ci-C₆ dialkylamino, aryl, CON(R₇)₂, Ci-C₆ acyl, Ci-C₆ heteroacyl, aroyl or heteroaroyl; or a pharmaceutically acceptable salt thereof. In various embodiments of the invention, the hypoxia-activated prodrug utilized in this invention is a compound of formula I that is TH-281, TH-302, or TH- 308 (structures provided below).

[0009] In one embodiment, the other chemotherapeutic agent is carfilzomib. In another embodiment, the other chemotherapeutic agent is pomalidomide. In another embodiment, the other chemotherapeutic agent is bortezomib. Carfilzomib (PR-171), has the following chemical structure and name.

(S)-4-methyl-N~((S)- 1 -(((S)-4-methyl- 1 -((R)-2-methyloxiran-2-yl)- 1 -oxopen- tan-2- yl)amino)- 1 -oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido- )-4- phenylbutanamido)pentanamide .

[0010] Pomalidomide is known as INN, CC-4047, 3 -amino-thalidomide, and the trade name Pomalyst. Pomalidomide has the following chemical structure and IUPAC name 4- amino-2-(2,6-dioxopiperidin-3-yl)isoindole-l,3-dione.

[0011] Bortezomib is known as BAN, INN, USAN and PS-341, and the trade name Velcade. Bortezomib has the following chemical structure and IUPAC name [(lR)-3-methyl- l-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid.

[0012] In one embodiment, TH-302 or another compound of formula I is administered as combination therapy with another chemotherapeutic agent to treat a blood cancer such as Acute Lymphoblastic Leukemia (ALL), Acute Myeloid (Myelogenous) Leukemia (AML), Chronic Lymphocytic (or Lymphoid) Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Myelodysplasia Syndrome (MDS), Myelofibrosis (MF), and Multiple Myeloma (MM), including relapsed and refractory forms of these cancers. In one embodiment, the blood cancer is multiple myeloma, including relapsed and refractory forms of MM.

[0013] The chemotherapeutic may be administered after the hypoxia-activated prodrug. The hypoxia-activated prodrug, and the chemotherapeutic may be administered in a plurality of cycles. By way of illustration, each cycle may comprise consecutively administering one or more of said drugs one after another on the same day, once a week for two consecutive weeks, followed by one week in which none of said drugs is administered.

[0014] The hypoxia-activated prodrug may be administered at a dose of about 150 to 1000 mg/m². In one embodiment, TH-302 or another compound of formula I is administered on specific days of a 28 day cycle, and the dose is between 170 and 670 mg/m²/day. Suitable administration schedules for doses of TH-302 include the following (TH-302 is administered intravenously):

days one and eight of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib);

days one and fifteen of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib);

days one, eight, and fifteen of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib);

days eight, fifteen and twenty-two of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and

bortezomib);

days one, two, eight, nine, fifteen, and sixteen on a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for for carfilzomib, pomalidomide and bortezomib); days one, four, eight, eleven, fifteen, and eighteen on a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib,

pomalidomide and bortezomib); and

days one to five, eight to twelve and fifteen to nineteen on a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib).

[0015] Suitable administration schedules and doses of TH-302 in non-human animal models include the following (TH-302 is administered intravenously):

days one to five, eight to twelve, and fifteen to nineteen on a twenty-one day cycle (second cycle begins at day 22) at a dose of between about 12. 5 mg/kg (mpk), to about

25 mpk to about 50 mpk, to about 100 mpk (this regimen is suitable for carfilzomib, pomalidomide and bortezomib); and

days one, four, eight, eleven, fifteen, and eighteen on a twenty-one day cycle

(second cycle begins at day 22) at a dose of between about 50 mpk and 100 mpk (this regimen is suitable for carfilzomib, pomalidomide and bortezomib).

[0016] Each of the above schedules can be considered a "cycle" of therapy. Patients will generally receive more than one cycle of therapy, although there may be breaks of at least a day, and more generally a week or longer, between each cycle of therapy. There may be variations up to a day when patients are administered one of the drugs. Other compounds of Formula I are generally dosed in accordance with the above schedules and amounts, with the amount adjusted to reflect how active the compound is relative to TH-302. In some embodiments, TH-302 or another compound of formula 1 is administered as an intravenous infusion over 30 minutes. In one embodiment, the other chemotherapeutic agent is carfilzomib. In one embodiment, the other chemotherapeutic agent is pomalidomide. In one embodiment, the other chemotherapeutic agent is bortezomib. Treatment cycles are continued until cure or evidence of progressive disease or intolerable toxicity.

[0017] The carfilzomib may be administered at its commerically supplied or any FDA (or other regulatory authority) approved dose. In one embodiment, carfilzomib is administered intravenously at a daily dose of 10 to 60 mg/m²/day, including but not limited to 20 mg/m²/day, 27 mg/m²/day, and 56 mg/m²/day, which is typically administered on days 1, 2, 8, 9, 15 and 16 of a 28 day cycle (second cycle starts on day 29). In one embodiment, carfilzomib can be administered to non-human animals intravenously at a daily dose of about 1.5 to about 2, to about 5 mpk, which is typically administered on days 1, 2, 8, 9, 15 and 16 of a 21 day cycle (second cycle starts on day 22).

[0018] The pomalidomide may be administered at its commerically supplied or any FDA (or other regulatory authority) approved dose. In one embodiment, pomalidomide is administered orally at a dose of either 2 mg/day, 3 mg/day, or 4 mg/day on days one to twenty-one of a twenty-eight day cycle (second cycle begins on day 29). In another embodiment, pomalidomide is administered orally at a dose of 2 mg/day on days one to twenty-eight of a twenty-eight day cycle (second cycle begins on day 29). In one embodiment, pomalidomide can be administered to non-human animals intravenously at a daily dose of about 50 mpk, which is typically administered on days 1-5, 8-12 and 15-19 of a 21 day cycle (second cycle starts on day 22).

[0019] The bortezomib may be administered at its commerically supplied or any FDA (or other regulatory authority) approved dose. In one embodiment, bortezomib is administered intravenously at a daily dose of less than 1.0, for example 0.6, 0.7, 0.8, or 0.9 mg/m2/day, which is typically administered on days 1, 4, 8 and 11 of a 21 day cycle (second cycle starts on day 22). In one embodiment, bortezomib can be administered to non-human animals intravenously at a daily dose of about 0.6, 0.7, 0.8, or 0.9 mg/m2/day, which is typically administered on days 2, 5, 9, 12, 16 and 19 of a 21 day cycle (second cycle starts on day 22). [0020] In various embodiments, for those days in an administration cycle in which the TH- 302 or another compound of formula I is administered on the same day as the carfilzomib, pomalidomide or bortezomib, the TH-302 or other compound of formula I is administered at least 30 minutes to 4 hours, i.e., 1 or 2 hours, before the carfilzomib, pomalidomide or bortezomib. [0021] In one embodiment, TH-302 or another compound of formula I is administered as a combination therapy with another chemotherapeutic agent to treat multiple myeloma, including relapsed or refractory forms of this disease, including but not limited to patients who have failed bortezomib and/or lenalidomide (or thalidomide) therapy.

[0022] Subjects that undergo treatment with known therapeutic agents may experience resistance to treatment, and the present invention offers new therapies for these patients. For example, although bortezomib was approved by the FDA for relapsed/refractory, relapsed, and newly diagnosed MM, some patients do not respond and others acquire resistance to bortezomib, and the present invention offers new therapies for these patients. The present invention also can be used to treat patients that resistant to an immunomodulatory drug (IMiD), such as thalidomide (Thalomid, marketed by Celgene), lenalidomide (Revlimid, marketed by Celgene, or pomelidamide (Pomalyst, marketed by Celgene). In one embodiment, the treatment methods described herein are employed in combination with bortezomib, thalidomide, or lenalidomide therapy in MM patients. Thus, in some embodiments, the methods described herein are used to overcome resistance to proteasome inhibitor or IMiD treatment.

[0023] In one embodiment, TH-302 or another compound of formula I is provided as a concentrate in a sterile liquid formulation containing 70% ethanol anhydrous, 25% dimethylacetamide, and 5% polysorbate 80. It is supplied in a 10-mL glass vial with a rubber stopper and flip-off seal. Each single use vial contains a nominal fill volume of 6.5 mL of TH-302 drug product for a nominal total of 650 mg of TH-302 (corresponds to 100 mg/rnL) and is diluted prior to administration as described in the Examples, below. [0024] In various embodiments of the invention, a biomarker of hypoxia is used to select patients for treatment and/or to identify patients that are responding to therapy. See, e.g., PCT Pub. Nos. WO 2013/116385 and WO 2008/124651, incorporated herein by reference, for suitable biomarkers and methods for use in this embodiment.

[0025] These and other aspects and embodiments of the invention are described in additional detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will now be described in more detail, with reference to the accompanying Figures, in which:

[0027] Figures 1A and IB show a comparison of body weight change after administration of carfilzomib, as a monotherapy or in combination with TH-302, in C57BL/6 mice;

[0028] Figures 2A and 2B show a comparison of M-spike levels after administration of carfilzomib, bortezomib and TH-302 alone and in combination in Vk*MYC mice;

[0029] Figures 3A-F shows a comparison of body, spleen and liver weight change, MM percentage and M-spike levels after administration of carfilzomib, bortezomib and TH-302, alone and in combination, in 5T33MM mice.

[0030] Figures 4A-D shows a comparison of body weight change and white blood cell, neutrophil and lymphocyte counts after administration of pomalidomide in combination with TH-302 in different doses and regimens in CD-I mice.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The practice of the present invention includes the use of conventional techniques of biochemistry, medicinal chemistry, pharmacology and immunology, which are within one of skill in the art.

Definitions

[0032] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the meanings below. All numerical designations, e.g., pH, temperature, time, concentration, and weight, including ranges, are approximations that typically may be varied (+) or (-) by increments of 0.1, 1.0, or 10.0, as appropriate. All numerical designations may be understood as preceded by the term "about". Reagents described herein are exemplary and equivalents of such may be known in the art.

[0033] The singular form "a", "an", and "the" includes plural references unless the context clearly dictates otherwise.

[0034] The term "comprising" means any recited elements are necessarily included and other elements may optionally be included. "Consisting essentially of means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. "Consisting of means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention. [0035] Certain terms related to formula I are defined below.

[0036] "Acyl" refers to -CO- alkyl, wherein alkyl is as defined here.

[0037] "Aroyl" refers to -CO-aryl, wherein aryl is as defined here.

[0038] "Alkoxy" refers to -O-alkyl, wherein alkyl is as defined here.

[0039] "Alkenyl" refers to a linear monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double bond, but no more than three double bonds. For example, (C2-C₆)alkenyl includes, ethenyl, propenyl, 1 ,3-butadienyl and the like. Alkenyl can be optionally substituted with substituents, including for example, deuterium ("D"), hydroxyl, amino, mono or di(CiC₆)alkyl amino, halo, C₂-C₆ alkenyl ether, cyano, nitro, ethynyl, Ci-C₆ alkoxy, Ci-C₆ alkylthio, -COOH, -CONH₂, mono- or di(Ci-C₆)alkylcarboxamido, -S0₂NH2, -OS0₂-(Ci-C6)alkyl, mono or di(Ci-C₆) alkylsulfonamido, aryl, heteroaryl, alkyl or heteroalkylsulfonyloxy, and aryl or heteroarylsulfonyloxy.

[0040] "Alkyl" refers to a linear saturated monovalent hydrocarbon radical or a branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix. As used in this disclosure, the prefixes (Ci-C_qq), Ci-_qq, or Ci-C_qq, wherein qq is an integer from 2-20, have the same meaning. For example, (C_rC₆)alkyl, Ci-₆ alkyl, or Ci C₆ alkyl includes methyl, ethyl, n-propyl, 2-propyl, n- butyl, 2-butyl, tert-butyl, pentyl, and the like. For each of the definitions herein (e.g., alkyl, alkenyl, alkoxy, etc.), when a prefix is not included to indicate the number of main chain carbon atoms in an alkyl portion, the radical or portion thereof will have six or fewer main chain carbon atoms. (Ci-C₆)alkyl can be optionally substituted with substituents, including for example, deuterium ("D"), hydroxyl, amino, mono or di(Ci- C₆) alkyl amino, halo, C₂-C6 alkenyl ether, cyano, nitro, ethenyl, ethynyl, Ci-C₆ alkoxy, Ci-C₆ alkylthio, -COOH, -CONH₂, mono- or di(Ci- C6)alkylcarboxamido, - S0₂NH₂, -OS0₂-(Ci-C6)alkyl, mono or di(Ci-C₆) alkylsulfonamido, aryl, heteroaryl, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy or heteroarylsulfonyloxy. [0041] "Alkylamino" or mono-alkylamino refers to -NH-alkyl, wherein alkyl is as defined here.

[0042] "Alkynyl" refers to a linear monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one triple bond, but no more than two triple bonds. For example, (C₂- C₆)alkynyl includes, ethynyl, propynyl, and the like. Alkynyl can be optionally substituted with substituents, including for example, deuterium ("D"), hydroxyl, amino, mono or di(Ci - C₆)alkyl amino, halo, C₂-C₆ alkenyl ether, cyano, nitro, ethenyl, d-C₆ alkoxy, d-C₆ alkylthio, - COOH, -CONH₂, mono- or di(Ci-C₆)alkylcarboxamido, -S0₂NH₂, -OS0₂-(Ci-C₆)alkyl, mono or di(Ci- C6)alkylsulfonamido, aryl, heteroaryl, alkyl or heteroalkylsulfonyloxy, and aryl or heteroarylsulfonyloxy.

[0043] "Aryl" refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms which is substituted independently with one to eight substituents, preferably one, two, three, four of five substituents selected from deuterium ("D"), alkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, acylamino, mono- alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), -(CR'R")ⁿ-COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl) or -(CR'R")_n-CONRxRy (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and Rx and Ry are independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl). In one embodiment, R_x and R^y together is cycloalkyl or heterocyclyl. More specifically the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2- naphthyl, and the substituted forms thereof. [0044] "Cycloalkyl" refers to a monovalent cyclic hydrocarbon radical of three to seven ring carbons. The cycloalkyl group can have one or more double bonds and can also be optionally substituted independently with one, two, three or four substituents selected from alkyl, optionally substituted phenyl, or -C(0)R^z (where R^z is hydrogen, alkyl, haloalkyl, amino, mono-alkylamino, di-alkylamino, hydroxyl, alkoxy, or optionally substituted phenyl). More specifically, the term cycloalkyl includes, for example, cyclopropyl, cyclohexyl, cyclohexenyl, phenylcyclohexyl, 4- carboxycyclohexyl, 2-carboxamidocyclohexenyl, 2- dimethylaminocarbonyl- cyclohexyl, and the like. [0045] "Dialkylamino" or di-alkylamino refers to -N(alkyl)², wherein alkyl is as defined here.

[0046] "Heteroalkyl" refers to an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, -OR^w, -NR^xR^y, and -S(0)_pR^z (where p is an integer from 0 to 2), with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom of the heteroalkyl radical. R^w is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, aralkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl. R^x is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl or araalkyl. R^y is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl. R^z is hydrogen (provided that n is 0), alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, amino, mono- alkylamino, di-alkylamino, or hydroxyalkyl. Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and 2-methylsulfonyl-ethyl. For each of the above, R^w, R^x, R^y, and R^z can be further substituted by amino, halo, fluoro, alkylamino, di-alkylamino, OH or alkoxy. Additionally, the prefix indicating the number of carbon atoms (e.g., Ci-Cio) refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, -OR^w, -NR^xR^y, or - S(0)_pR^z portions. In one embodiment, R^x and R^y together is cycloalkyl or heterocyclyl.

[0047] "Heteroaryl" refers to a monovalent monocyclic, bicyclic or tricyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring. The heteroaryl ring is optionally substituted independently with one to eight substituents, preferably one, two, three or four substituents, selected from alkyl, cycloalkyl, cycloalkyl-alkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, -COR (where R is hydrogen, alkyl, phenyl or phenylalkyl, - (CR'R")n-COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cydoalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), or - (CR'R")n-CONR^xR^y (where n is an integer from 0 to 5, R and R" are independently hydrogen or alkyl, and R^x and R^y are, independently of each other, hydrogen, alkyl, cydoalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl). In one embodiment, R^x and R^y together is cydoalkyl or heterocyclyl. More specifically the term heteroaryl includes, but is not limited to, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyridazinyl, pyrimidinyl, benzofuranyl, tetrahydrobenzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyl, quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl, benzisoxazolyl or benzothienyl, indazolyl, pyrrolopyrymidinyl, indolizinyl, pyrazolopyridinyl, triazolopyridinyl, pyrazolopyrimidinyl, triazolopyrimidinyl, pyrrolotriazinyl, pyrazolotriazinyl, triazolotriazinyl, pyrazolotetrazinyl, hexaaza-indenly, and heptaaza- indenyl and the derivatives thereof. Unless indicated otherwise, the arrangement of the hetero atoms within the ring can be any arrangement allowed by the bonding characteristics of the constituent ring atoms.

[0048] "Heterocyclyl" or "cycloheteroalkyl" refers to a saturated or unsaturated non- aromatic cyclic radical of 3 to 8 ring atoms in which one to four ring atoms are heteroatoms selected from O, NR (where R is hydrogen, alkyl, cydoalkyl, cycloalkylalkyl, phenyl or phenylalkyl), P(=0)OR^w, or S(0)_p (where p is an integer from 0 to 2), the remaining ring atoms being C, wherein one or two C atoms can optionally be replaced by a carbonyl group. The heterocyclyl ring can be optionally substituted independently with one, two, three or four substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cydoalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, mono-alkylamino, di- alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cydoalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R")_n-COOR (n is an integer from 0 to 5, R and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cydoalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R")_n-CONR^xR^y (where n is an integer from 0 to 5, R and R" are independently hydrogen or alkyl, R^x and R^y are, independently of each other, hydrogen, alkyl, cydoalkyl, cycloalkylalkyl, phenyl or phenylalkyl). More specifically the term heterocyclyl includes, but is not limited to, pyridyl, tetrahydropyranyl, N-methylpiperidin-3- yl, N-methylpyrrolidin-3-yl, 2-pyrrolidon-l-yl, furyl, quinolyl, thienyl, benzothienyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1 , 1 -dioxo-hexahydro- lA⁶-thiopyran-4-yl, tetrahydroimidazo[4,5-c]pyridinyl, imidazolinyl, piperazinyl, and piperidin-2-only and the derivatives thereof. The prefix indicating the number of carbon atoms (e.g., C₃ - C₁₀) refers to the total number of carbon atoms in the portion of the cycloheteroalkyl or heterocyclyl group exclusive of the number of heteroatoms. [0049] "Heteroacyl" refers to -CO-heteroalkyl, wherein heteroalkyl is as defined here.

[0050] "Heteroaroyl" refers to -CO-heteroayl, wherein heteroaryl is as defined here. [0051] "Rsui sulfonyloxy" refers to R_sui-S(=0)2-0- and includes alkylsulfonyloxy, heteroakylsulfonyloxy, cycloalkylsulfonyloxy, heterocyclylsulfonyloxy, arylsulfonyloxy and heteroarylsulfonyloxy wherein R_sui is alkyl, heteroakyl, cycloalkyl, heterocyclyl, aryl and heteroaryl respectively, and wherein alkyl, heteroakyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are as defined here. Examples of alkylsulfonyloxy include Me-S(=0)2-0-, Et- S(=0)2-0-, CF₃-S(=0)2-0- and the like, and examples of arylsulfonyloxy include:

wherein R_ar is H, methyl, or bromo.

[0052] "Substituents" refers to, along with substituents particularly described in the definition of each of the groups above, those selected from: deuterieum, -halogen, - OR', -

NR'R", -SR^*,

-SiRR"R"^*,-OC(0)R^*, -C(0)R^*, -C0₂R, -CONR'R", -OC(0)NR^*R", -NR"C(0)R, -NR^*- C(0)NR"R"^*, -NR"C(0)₂R, -NH-C(NH₂)=NH, -NR'C(NH)=NH, -NH- C(NH₂)=NR, - S(0)R, -S(0)₂R, -S(0)₂NRR", -NR^*S(0)₂R", -CN, -N0₂, -R, -N₃, perfluoro(C i-C₄)alkoxy, and perfluoro(C i-C₄)alkyl, in a number ranging from zero to the total number of open valences on the radical; and where R, R" and R" are independently selected from hydrogen, Ci_8 alkyl, C₃_₆ cycloalkyl, C2-8 alkenyl, C2-8 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-Ci_₄ alkyl, and unsubstituted aryloxy-Ci_₄ alkyl, aryl substituted with 1-3 halogens, unsubstituted Ci_g alkyl, Ci.galkoxy or Ci.gthioalkoxy groups, or unsubstituted aryl- Ci_₄ alkyl groups. When R and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, - NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of from 1-4 carbon atoms. Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T² - C(O)— (CH₂)_q-U³-, wherein T² and U³ are independently -NH-, -0-, -CH₂- or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)_rB-, wherein A and B are independently CH₂-, -0-, -NH-, -S-, -S(O)-, -S(0)₂ -, -S(0)₂NR^*- or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH₂)_s-X⁵-(CH₂)t -, wherein s and t are independently integers of from 0 to 3, and X⁵ is -0-, -NR^*-, -S-, -S(O)-, -S(0)₂-, or -S(0)₂NR^*-. The substituent R' in -NR^*- and - S(0)₂NR- is selected from hydrogen or unsubstituted Ci_₆ alkyl. [0053] Certain compounds utilized in the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example, and without limitation, tritium (³H), iodine- 125 (¹²⁵1) or carbon- 14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. [0054] Other terms related to this invention are defined below.

[0055] An "active agent" or "pharmaceutical" is a compound with a desired pharmacological effect. It includes all pharmaceutically acceptable forms of the active agent described. Unless explicitly stated otherwise, all embodiments of the invention may be practiced with any one or more different isomers, stereoisomers, and pharmaceutical salts of each of the active ingredients that has the desired effect. Reference to any drug or active agent in this disclosure includes any and all isomers, stereoisomers, pharmaceutically compatible salts, solvates, and pharmaceutical compositions thereof that retain at least some of the physiological or chemotherapeutic effects of the drug itself, unless such isomers, salts, solvates, and/or compositions are explicitly excluded. Any such compound may be used as an alternative to the drug itself to improve efficacy, tolerability, delivery, or pharmacokinetics, or simply by choice within the good judgment of the manufacturer, distributor, pharmacist, clinician, or end user.

[0056] "Acute" in the context of blood cancers, refers to the relatively short time course in which these cancers can become extremely serious and even lead to the death of a patient (e.g., they can be fatal in as little as a few weeks if left untreated) and differentiates them from "chronic" blood cancers, which may not have extremely debilitating effects on or lead to the death of a patient for many years. "Acute leukemias" refer to ALL, AML, and the like. "Chronic leukemias" refer to CLL, CML, myelofibrosis, and the like.

[0057] "Acute Lymphoblastic Leukemia (ALL)" refers to a blood cancer, particularly a cancer affecting the white blood cells, and is characterized by hyperproliferation of lymphoblasts. In ALL, malignant, immature white blood cells continuously multiply and are overproduced in the bone marrow. ALL cells crowd out normal cells in the bone marrow and may metastasize to other organs. ALL is also known as acute lymphocytic leukemia and acute childhood leukemia.

[0058] "Acute Myeloid (Myelogenous) Leukemia (AML)" refers to a blood cancer in which white blood cells known as "myeloid cells" become cancerous. In AML, the bone marrow produces abnormal blood cells called "myeloblasts," leading to the replacement of normal blood cells with abnormal cells and disrupting the normal function of the bone marrow. With the abnormal production of "blast" cells, the production of normal blood marrow cells is inhibited, causing a deficiency of red blood cells, normal white blood cells, and platelets, leading to deleterious effects such as anemia, vulnerability to bruising and bleeding, and increased risk of infection.

[0059] "Administering" or "administration of a drug to a patient" (and grammatical equivalents of this phrase) refer both to direct administration, which may be administration to a patient by a medical professional or may be self-administration, as well as to indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.

[0060] "Blood cancer" refers to a hematological malignancy involving abnormal hyperproliferation or malignant growth and/or metastasis of a blood cell. Blood cancers include, without limitation, acute leukemias (AML and ALL), chronic leukemias (CML and CLL), idiopathic myelofibrosis (MF, also known as agnogenic myeloid metaplasia or AMM), lymphoma, myelodysplasia syndrome (MDS), and multiple myeloma (MM).

[0061] "Bone marrow stem cell transplant" refers to replacing a patient's bone marrow with new bone marrow. In these transplants, chemotherapy drugs are used to kill the stem cells in the bone marrow (including those creating diseased lymphocytes), and then, healthy adult blood stem cells from a donor (allogenic transplant) or from the patient's own peripheral blood (or bone marrow) (each, an autologus transplant) are infused into the blood, wherein they travel to the bone marrow and begin making healthy blood cells.

[0062] "Chronic lymphocytic (or lymphoid) leukemia (CLL)" refers to a blood cancer affecting B cell lymphocytes. B cells originate in the bone marrow and develop in the lymph nodes. In CLL, the B cells grow in an uncontrolled manner and accumulate in the bone marrow and blood, wherein they crowd out healthy blood cells. As the disease advances, CLL results in swollen lymph nodes, spleen, and liver.

[0063] "Chronic myelogenous leukemia (CML)" refers to a blood cancer in which the bone marrow produces granulocytes, some of which never mature into white blood cells. The "immature" white blood cells are called "blasts." Over time, the granulocytes and blasts grow out of control and result in a platelet and red blood cell deficiency in the bone marrow. CML patients may have a gene mutation called the "Philadelphia chromosome." This chromosome causes the bone marrow to make certain tyrosine kinases that result in the development of granulocytes or blasts. Some CML patients have a form of the disease resistant to treatment with tyrosine kinase inhibitors. CML includes, without limitation, chronic myelogenous leukemia, chronic myeloid leukemia, chronic myelocytic leukemia, and chronic granulocytic leukemia (CGL).

[0064] A "chemotherapeutic agent" is a pharmaceutical compound that is given to a cancer patient primarily to eradicate, diminish, stabilize, or decrease the growth rate or metabolism of one or more malignant tumors in the patient. Included are agents such as carfilzomib, pomalidomide or bortezomib. The more general term "therapeutic agent" includes chemotherapeutics and radiation therapy.

[0065] "Combination therapy" refers to the use of two or more drugs in therapy, i.e., use of a hypoxia-activated prodrug as described herein together with conventional drugs used to treat blood cancer is a combination therapy. Administration in "combination" refers to the administration of two agents (e.g., a hypoxia-activated prodrug and an agent known for treating a blood cancer) in any manner in which the pharmacological effects of both manifest in the patient at the same time. Thus, administration in combination does not require that a single pharmaceutical composition, the same dosage form, or even the same route of administration be used for administration of both agents or that the two agents be administered at precisely the same time. For example, and without limitation, it is contemplated that carfilzomib, pomalidomide or bortezomib can be administered in combination with a hypoxia-activated prodrug in accordance with the present invention.

[0066] The terms "dose" and "dosage" refer to a specific amount of active or therapeutic agent(s) for administration at one time. A "dosage form" is a physically discrete unit that has been packaged or provided as unitary dosages for subjects being treated. It contains a predetermined quantity of active agent calculated to produce the desired onset, tolerability, and therapeutic effect.

[0067] "Hypoxia-activated prodrug" refers to a drug that is less active or inactive under normoxia than under hypoxia or anoxia. Hypoxia-activated prodrugs include drugs that are activated by a variety of reducing agents and reductase enzymes, including without limitation single electron transferring enzymes (such as cytochrome P450 reductases) and two electron transferring (or hydride transferring) enzymes (see US Pat. App. Pub. Nos. 2005/0256191, 2007/0032455, and 2009/0136521, and PCT Pub. Nos. WO 2000/064864, WO 2004/087075, and WO 2007/002931, each of which is incorporated herein by reference). The hypoxia- activated prodrugs useful in the methods of the present invention are compounds of formula I, including but not limited to compounds where Z₃, as defined by that formula, is a 2-nitroimidazole moiety. Examples of particular hypoxia-activated prodrugs useful in the methods of the invention include without limitation TH-281, TH-302, and TH-308. Methods of synthesizing and formulating TH-302 and other compounds of formula I are described in PCT Pub. Nos. WO 2007/002931 and WO 2008/083101, each of which is incorporated herein by reference.

[0068] "Multiple myeloma (MM)" refers to a blood cancer having clonal B cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. There are several types of multiple myeloma, including smoldering multiple myeloma (SMM), plasma cell leukemia, nonsecretory myeloma, osteosclerotic myeloma (POEMS syndrome), solitary plasmacytoma (also called solitary myeloma of the bone), and extramedullary plasmacytoma.

[0069] "Myelodysplastic syndrome (MDS)" refers to a blood cancer that occurs when the bone marrow stops producing healthy blood cells and instead produces immature blood cells that function poorly. This results in the production of too many defective blood cells and not enough healthy blood cells. In people with MDS, the disorder begins when a defect occurs in a stem cell in the bone marrow. That stem cell, in turn, produces blood cells that carry the same defect. These defective cells grow to outnumber healthy blood cells and live longer. These defective cells may also kill other stem cells too early, resulting in low blood counts. The abnormal cells also crowd out the healthy cells. MDS can progress over time into acute myelogenous leukemia.

[0070] "Myelofibrosis (MF)" refers to a type of chronic leukemia that disrupts the body's normal production of blood cells. Myelofibrosis can occur on its own (primary myelofibrosis) or it can occur as a result of another bone marrow disorder (secondary myelofibrosis). Advanced myelofibrosis gets progressively worse and can eventually develop into a more serious form of leukemia.

[0071] "Patient" or "subject" refers to mammals, particularly humans, but also to animals such as simians, cattle, horses, dogs, cats, and rodents suffering from blood cancer.

[0072] A "prodrug" is a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active agent with respect to at least one beneficial property or effect.

[0073] "Relapsed or refractory" refers to a type of blood cancer that is resistant to treatment with an agent, or responds to treatment with an agent but comes back without being resistant to that agent, or responds to treatment with an agent but comes back resistant to that agent.

[0074] "Standard chemotherapy" refers to treatment with drugs in accordance with FDA labeling instructions and/or good clinical practice. Standard chemotherapy is well known to those of skill in the medical arts. [0075] "TH-281 " refers to the compound of formula:

[0076] TH-302" refers to the compound of formula:

[0077] "TH-308" refers to the compound of formula:

[0078] "Therapeutically effective amount" of a drug or an agent refers to an amount of drug or agent that, when administered to a patient with blood cancer, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of the blood cancer in the patient. A therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of therapeutically effective doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0079] "Treating" or "treatment of a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this technology, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of blood cancer; diminishment of extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state; or other beneficial results.

[0080] This invention is based in part on the discovery that a hypoxia-activated prodrug such as TH-302 and carfilzomib, pomalidomide or bortezomib work especially well together in treating malignant conditions such as blood cancers, including but not limited to multiple myeloma. The two drug combinations of this invention (when administered in doses and on schedules adjusted for the particular animal model employed, relative to the human doses and schedules described herein) substantially inhibit cancer cell proliferation and increase survival in animal models of cancer and are expected to demonstrate similar benefit in human therapy. The benefit provided by the drug combinations of the invention will be, for many patients, more than that provided by any of the drugs alone and beyond what could be predicted. The present invention represents a significant advance in the treatment of this deadly disease. [0081] In some patients, this two-drug combination provided by the invention may also be more tolerable to some patients than monotherapy. The use of the drug combinations described herein represents an important advance in cancer management and treatment.

Hypoxia- activated prodrugs

[0082] A hypoxia-activated prodrug is inert or has less activity than the active form and converts to the active form in vivo at or around a tumor site that is hypoxic, relative to normal tissues with physiological oxygenation. These drugs typically contain one or more bioreducible groups. The preparation and use of illustrative hypoxia-activated prodrugs is described in PCT Pub. Nos. WO 04/087075, WO 00/064864, WO 07/002931 , and WO 08/083101 , and US Pub. Nos. 2005/0256191 , US 2007/0032455, and US 2009/0136521 , each of which is incorporated herein by reference.

[0083] This invention may be conducted with hypoxia-activated prodrugs in the same class as bromo-isophosphoramide mustard (Br-IPM), having DNA bis-alkylator activity. Such compounds may have the structure shown in Formula I:

(I) wherein Y₂ is O, S, NRe, NCORe, or NS0₂R₆ wherein Re is Ci-C₆ alkyl, Ci-C₆ heteroalkyl, aryl, or heteroaryl; R₃ and R₄ are independently selected from the group consisting of 2-haloalkyl, 2-alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-heteroalkylsulfonyloxyalkyl; Ri has the formula L-Z₃; L is C(Zi)₂; each Zi independently is hydrogen, halogen, Ci-C₆ alkyl, Ci-C₆ heteroalkyl, aryl, heteroaryl, C₃-Cg cycloalkyl, heterocyclyl, Ci-C₆ acyl, Ci-C₆ heteroacyl, aroyl, or heteroaroyl; or L is:

wherein each Xi is independently N or CR₈; X₂ is NR₇, S, or O; each R₇ is independently Ci-C₆ alkyl, Ci-C₆ heteroalkyl, C₃-Cg cycloalkyl, heterocyclyl, aryl or heteroaryl; and Rg is independently hydrogen, halogen, cyano, CHF₂, CF₃, C0₂H, amino, Ci-C₆ alkyl, Ci-C₆ heteroalkyl, Ci-C₆ cycloalkyl, Ci-C₆ alkoxy, Ci-C₆ alkylamino, Ci-C₆ dialkylamino, aryl, CON(R₇)₂, Ci-C₆ acyl, Ci-C₆ heteroacyl, aroyl or heteroaroyl; or a pharmaceutically acceptable salt thereof. [0084] Exemplary are TH-281, TH-302, and TH-308 which respectively have the following structures:

TH-302, TH-281, and TH-308 convert to a cytotoxic agent selectively under hypoxic conditions in vivo at or around hypoxic areas. [0085] When used herein, reference to the compounds TH-302, TH-281, and TH-308 are for illustrative purposes for the general class of compounds having the structure shown in formula I. Unless expressly limited to a particular compound, the various aspects of the invention discussed in reference to TH-302, TH-281, and TH-308 may be put into practice using TH-302, TH-281, and TH-308 interchangeably, or using other hypoxia-activated prodrugs having the structure of Formula I, at the user's discretion.

[0086] In various embodiments, the hypoxia-activated prodrug is TH-302, which is administered at a daily dose of about 170 mg/m2 to about 670 mg/m2. Suitable doses of TH- 302 in this range include 240 mg/m2, 300 mg/m2, 340 mg/m2, 480 mg/m2, and 575 mg/m2. Doses are typically administered intravenously (iv). Suitable iv administration schedules for doses of TH-302 in this range include the following:

days one and fifteen of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib); days one, eight, and fifteen of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib);

bortezomib);

days one, two, eight, nine, fifteen, and sixteen on a twenty-eight day cycle

(second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for for carfilzomib, pomalidomide and bortezomib);

days one, four, eight, eleven, fifteen, and eighteen on a twenty-eight day cycle

(second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g.

300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib,

pomalidomide and bortezomib); and

[0087] For an adult human, a dose of 1 mg/m² of an active agent (drug) = about 1.7 mg of that agent or drug per patient (i.e., the prototypical adult human has 1.7 m² of surface area).

Therefore, for example, 100 mg/m² of a drug = about 170 mg of that drug per patient. The dose of TH-302 administered in accordance with the present invention is converted to a dose in mg using the above conversion.

[0088] Suitable administration schedules and doses of TH-302 in non-human animal models include the following (TH-302 is administered intravenously):

days one, four, eight, eleven, fifteen, and eighteen on a twenty-one day cycle

(second cycle begins at day 22) at a dose of between about 50 mpk and 100 mpk (this regimen is suitable for carfilzomib, pomalidomide and bortezomib). [0089] A therapeutically effective dose determined for a non-human animal can be converted to the corresponding human equivalent dose (HED) as described in the table below:

To convert animal dose in mg/kg to_HED (assumes a 60 kg human) in mg/kg, divide animal dose by HED conversion factor. For species not listed or for weights outside the standard ranges, human equivalent dose (HED) can be calculated from the formula:

HED = animal dose in mg/kg x (animal weight in kg/human weight in kg) 0.33. b For example, cynomolgus, rhesus, or stumptail.

[0090] A dose in mg/m² can be converted to a mg/kg dose in adult humans by dividing the mg/m² dose by a factor of 37; in children the corresponding dividing factor is 25.

[0091] In various embodiments, carfilzomib is administered in a daily dose of about 10 mg/m² to about 60 mg/m². Suitable doses of carfilzomib in this range include 20 mg/mg², 27 mg/m², and 56 mg/m². Doses are typically administered intravenously (iv). Suitable iv administration schedules for doses of carfilzomib in this range include the following: twice a week; and days one, two, eight, nine, fifteen, and sixteen of a 28 day cycle. An oral formulation of a carfilzomib analog (ONX 0912) is currently in clinical development. ONX 0912 can be administered orally in accordance with the invention. In one embodiment, the ONX 0912 is administered on days 1 through 5 of a 14-day cycle at a dose of 30 mg per day or at any higher dose shown to be safe and tolerable.

[0092] In various embodiments, pomalidomide is administered in a daily dose of about 2-4 mg. Doses are typically administered orally (po). A suitable administration schedule for pomalidomide in this range is daily for 21 days of a 28 day cycle. [0093] In various embodiments, bortezomib is administered in a daily dose of about 0.6 to 1.3 mg. Doses are typically administered intravenously (iv). A suitable administration schedule for bortezomib in this range is daily dose of less than about 1.0, e.g. 0.6, 0.7, 0.8 to 0.9 mg/m², which is typically administered on days 1, 4, 8 and 11 of a 21 day cycle (second cycle starts on day 22).

[0094] Each of the above schedules can be considered a "cycle" of therapy. Patients will generally receive more than one cycle of therapy, although there may breaks of at least a day, and more generally a week or longer, between each cycle of therapy. Other compounds of Formula I are generally dosed in accordance with the above schedules and amounts, with the amount adjusted to reflect how active the compound is relative to TH-302.

Chemotherapeutic agents

[0095] Carfilzomib is a tetrapeptide epoxyketone and a selective proteasome inhibitor and is marketed under the tradename Kyprolis^®. The principal mechanism of action is thought to be as a selective proteasome inhibitor. Carfilzomib has the chemical formula:

[0096] The U.S. Food and Drug Administration (FDA) has approved carfilzomib for use in patients with multiple myeloma who have received at least two prior therapies, including treatment with bortezomib and an immunomodulatory therapy and have demonstrated disease progression on or within 60 days of completion of the last therapy. [0097] While carfilzomib can be used at any FDA approved dose in accordance with the methods of the invention, in various embodiments, carfilzomib is administered at a daily dose ranging from 10 mg/m 2 to 60 mg/m 2 (e.g., 20 mg/m 2 , 27 mg/m 2 or 56 mg/m 2 ) as an iv infusion over 30 minutes on days 1, 2, 8, 9, 15, and 16 of a 28-day cycle.

[0098] Dexamethasone is frequently co-administered with other anti-cancer drugs and may be included, in accordance with the invention, in any of the combination therapies described herein. While dexamethasone can be used at any FDA approved dose in accordance with the methods of the invention, in various embodiments, dexamethasone is administered at a dose of about 4 mg/m² orally on at least those days in the cycle when at least one of the other two drugs (TH-302 and carfilzomib or TH-302 and pomelidamide, for example, are administered). Dexamethasone is frequently given 30 minutes to 4 hours before carfilzomib is administered and may be similarly administered in accordance with the invention. [0099] Pomalidomide is an analog of thalidomide is marketed under the tradename Pomalyst^®. The principal mechanism of action is thought to be as an immunomodulator (IMiD). Pomalidomide has the chemical formula:

[0100] The U.S. Food and Drug Administration (FDA) has approved pomalidomide as a treatment in patients with relapsed and refractory multiple myeloma, i.e., patients with multiple myeloma who received at least two prior therapies, including lenalidomide (Revlimid®) and bortezomib (Velcade®), and whose disease progressed within 60 days of completing the last therapy.

[0101] While pomalidomide can be used at any FDA approved dose in accordance with the methods of the invention, in various embodiments, pomalidomide is administered at a dose ranging from 2-4 mg orally on days 1-21 of a 28-day cycle. Dexamethasone may be administered, as described above, prior to or contemporaneous with the dose of pomalidomide.

[0102] Bortezomib is marketed under the tradename Velcade®. The principal mechanism of action is thought to be as an proteosome inhibitor. Bortezomib has the chemical formula:

[0103] The U.S. Food and Drug Administration (FDA) has approved bortezomib as a treatment in patients with relapsed multiple myeloma and mantle cell lymphoma. [0104] While Bortezomib can be used at any FDA approved dose in accordance with the methods of the invention, in various embodiments, Bortezomib is administered at a dose ranging from 0.6 to 0.7 to 0.8 to 0.9 mg iv on days 1, 4, 8 and 11 of a 21 -day cycle.

Formulations

[0105] A "medicament combination" as used herein refers to two or more medications that are used in combination and may be co-formulated (admixed together) or separately formulated (not admixed or otherwise combined together in a single unit dose form).

[0106] Formulations of TH-302, TH-308, and TH-281 suitable for iv injection and methods for administering them in the treatment of cancer that are suitable for use in practice of the present invention are described in PCT Pub. Nos. WO 07/002931, WO 08/083101, and WO 10/048330, incorporated herein by reference. Suitable formulations of these drugs for oral administration are described in WO 12/142520, incorporated herein by reference.

[0107] While the various methods of the invention are illustrated specifically with TH-302, carfilzomib, pomalidomide and bortezomib, other particular compounds of Formula I, as well as other formulations, and dosing schedules of the invention, can be assessed for safety and efficacy in preclinical models and clinical trials. In such studies, one uses a compound, formulation, or drug combination of the invention in a preclinical animal model, for example, a homograft or allograft model using tumor cell lines derived from the same species, or a xenograft of human tumor cells in an immune-compromised animal, or in a human clinical trial approved by the appropriate regulatory authority. Using such systems and models, the investigator may determine, for example, the maximum tolerable dose and the dose required for a significant beneficial therapeutic effect using such models.

[0108] Depending on efficacy and side effect profile, a hypoxia-activated prodrug, carfilzomib, pomalidomide and bortezomib may be distributed and administered separately in a treatment of a particular disease or condition. Thus, in accordance with the invention, a hypoxia-activated prodrug may be combined with carfilzomib, pomalidomide or bortezomib for administration together; or the drugs may be separately formulated and administered.

[0109] The invention also encompasses various combinations of agents for marketing or distribution together. Such combinations are optionally marketed and distributed in kit form. The combinations or kits may comprise separate packs of an effective amount of a hypoxia- activated prodrug, exemplified by Formula I, such as TH-302; and carfilzomib, pomalidomide or bortezomib. The combination or kit will be suitably packaged and may also contain or be marketed in combination with written instructions that direct the clinician on the use of the combination or elements of the kit for chemotherapy in accordance with the invention.

Treatment Methods and Uses

[0110] In one aspect, the present invention provides a method of treating a blood cancer comprising administering a therapeutically effective amount of a hypoxia-activated prodrug of formula I in combination with carfilzomib, pomalidomide or bortezomib. In various embodiments, the hypoxia-activated prodrug is selected from the group consisting of TH- 281, TH-302, and TH-308. In one important embodiment, the prodrug is TH-302. The hypoxia-activated prodrug is administered in a therapeutically effective amount to a patient in need of such treatment, thereby treating the blood cancer. Illustrative blood cancers amenable to treatment include multiple myeloma, including relapsed or refractory forms of MM.

[0111] In one embodiment, the hypoxia-activated prodrug administered is TH-302. In one embodiment, TH-302 or another compound of formula I is administered as a 30 minute intravenous infusion daily on days one and eight every 21 days. In one embodiment, TH-302 or another compound of formula I is administered as a 30 minute intravenous infusion daily on one of the following admistration schedules: days one and eight of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib);

bortezomib);

days one, two, eight, nine, fifteen, and sixteen on a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for for carfilzomib, pomalidomide and bortezomib);

days one, four, eight, eleven, fifteen, and eighteen on a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib,

pomalidomide and bortezomib); and

[0112] Suitable administration schedules and doses of TH-302 in non-human animal models include the following (TH-302 is administered intravenously):

days one to five, eight to twelve, and fifteen to nineteen on a twenty-one day cycle (second cycle begins at day 22) at a dose of between about 12. 5 mg/kg (mpk), to about 25 mpk to about 50 mpk, to about 100 mpk (this regimen is suitable for carfilzomib, pomalidomide and bortezomib); and

days one, four, eight, eleven, fifteen, and eighteen on a twenty-one day cycle (second cycle begins at day 22) at a dose of between about 50 mpk and 100 mpk (this regimen is suitable for carfilzomib, pomalidomide and bortezomib).

[0113] In one embodiment, TH-302 or another compound of formula I is administered for 3 or more such cycles, including but not limited to 6 cycles and 9 cycles. In various embodiments, TH-302 or another compound of formula I is administered for up to about 25 or up to about 50 such cycles.

[0114] In suitable embodiments, combinations of suitable for carfilzomib, pomalidomide or bortezomib and hypoxia-activated prodrug are administered to a patient, separately, sequentially, or simultaneously, for the treatment of the blood cancer. When administered in combination with carfilzomib, pomalidomide or bortezomib in one embodiment, TH-302 or another compound of formula I is administered before administering carfilzomib, pomalidomide or bortezomib. For example, TH-302 may be administered the day before carfilzomib, pomalidomide or bortezomib is administered or, if the two drugs are administered on the same day, then TH-302 or another compound of formula I may be administered from at least 30 minutes to up to 1 hour or 2 hours or 4 hours or 6 hours or even 8 hours before carfilzomib, pomalidomide or bortezomib. See e.g. PCT Pub. No. WO 2008/083101, incorporated herein by reference. In other embodiments, TH-302 or another compound of formula I is administered after administering carfilzomib, pomalidomide or bortezomib. For example, pomalidomide may be administered before TH-302 is administered.

[0115] In various embodiments, a method of the invention is employed as a first, second, third or later line of treatment. As used herein, a "first line" or "second line" or "third line" of treatment refers to a place in the order of treatment with different medications or other therapies received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. Therefore, first line therapy is "the first treatment for a disease or condition." In patients with cancer, first line therapy, sometimes referred to as "primary therapy" or "primary treatment", can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. Typically, a patient is given a subsequent chemotherapy regimen (second or third line therapy), either because the patient did not show a positive clinical or showed only a sub-clinical response to a first or second line therapy or showed a positive clinical response but later experienced a relapse, sometimes with disease now resistant to the earlier therapy that elicited the earlier positive response.

[0116] Methods of preparation of and pharmaceutical compositions of hypoxia-activated prodrugs, and other methods of treating cancer by administering various hypoxia-activated prodrugs of formula I are described in Duan et al., J. Med. Chem. 2008, 51, 2412-2420 PCT Pub. Nos. WO 2007/002931, WO 2008/083101, and WO 2010/048330, each of which is incorporated herein by reference. Other methods of treating blood cancers, which may be used in combination with the methods of the present invention, are known to one of skilled in the art, and are described, for example, in the product descriptions found in the 2010 or more current edition of the Physician's Desk Reference, Medical Economics Company, Inc., Oradell, NJ; Goodman and Gilman's The pharmacological basis of therapeutics., Eds. Hardman et al., McGraw-Hill. New York. (US) 2011, 12th Ed., and in publications of the U.S. Food and Drug Administration and the NCCN Guidelines (National Comprehensive Cancer Network). Such described and known methods can be appropriately modified by one of skill in the art, in view of this disclosure, to practice the treatment methods of the present technology.

[0117] In one embodiment, TH-302 or another compound of formula I is provided as a concentrate in a sterile liquid formulation containing 70% ethanol anhydrous, 25% dimethlyacetamide, and 5% polysorbate 80. It is supplied in a 10-mL glass vial with a rubber stopper and flip-off seal. Each single use vial contains a nominal fill volume of 6.5 mL of TH-302 drug product for a nominal total of 650 mg of TH-302 (corresponds to 100 mg/rnL) and is diluted prior to administration as described in Example 2, below.

[0118] The methods of the invention are now described in the context of treating multiple myeloma with carfilzomib, pomalidomide or bortezomib.

Treatment of Multiple Myeloma with a hypoxia activated prodrug and carfilzomib, pomalidomide or bortezomib

[0119] In one embodiment, the blood cancer treated in accordance with the methods of the invention is multiple myeloma (MM). In one embodiment, the MM is refractory or relapsed MM, including but not limited to lenalidomide or thalidomide refractory MM or bortezomib refractory MM. For treating MM and other blood cancers, TH-302 or another compound of formula I is administered at a frequency and in amounts described herein in combination with carfilzomib or pomalidomide. [0120] In one embodiment of the methods of the invention to treat MM, TH-302 or another compound of formula I is administered intravenously over about 30 to about 60 minutes on an administration schedules as follows (TH-302 is administered intravenously):

days one, eight, and fifteen of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and bortezomib); days eight, fifteen and twenty-two of a twenty-eight day cycle (second cycle begins at day 29) at a dose between 170 mg/m²/day and 670 mg/m²/day, e.g. 300 mg/m², 340 mg/m², or 480 mg/m² (this regimen is suitable for carfilzomib, pomalidomide and

bortezomib);

days one, two, eight, nine, fifteen, and sixteen on a twenty-eight day cycle

pomalidomide and bortezomib); and

[0121] Suitable administration schedules and doses of TH-302 in non-human animal models include the following (TH-302 is administered intravenously):

[0122] In various embodiments, TH-302 or another hypoxia-activated prodrug of formula I and carfilzomib or pomalidomide are administered in combination with other drugs, including other anti cancer drug(s). In the combination therapies of the invention, the other drug(s) are administered, in some embodiments, in frequencies and amounts, and via routes, substantially similar if not identical to those conventionally employed.

[0123] In one embodiment, TH-302 or another hypoxia-activated prodrug of formula I is administered in combination with is carfilzomib, pomalidomide or bortezomib and/or non- drug therapy conventionally used to treat MM. An example of a suitable non-drug therapy includes, without limitation, radiation and/or bone marrow stem cell transplantation

[0124] In one embodiment, TH-302 or another compound of formula I is administered as a combination therapy with carfilzomib, pomalidomide or bortezomib to treat MM, including relapsed or refractory forms of this disease, including but not limited to patients who have failed prior bortezomib and/or lenalidomide (or thalidomide) therapy. In one embodiment, TH-302 is administered on a schedule described above at least 30 minutes to 8 hours, i.e., up to 1 hour or 2 hours or 4 hours or 6 hours, before the carfilzomib, pomalidomide or bortezomib, on the days when both drugs are administered. Accordingly the day that carfilzomib, pomalidomide or bortezomib is scheduled to be administered may be up to a day later than the administration schedules disclosed herein.

[0125] In one embodiment, TH-302 is administered as a combination therapy with pomalidomide to treat multiple myeloma, including relapsed or refractory forms of this disease, including but not limited to patients who have failed bortezomib and/or lenalidomide (or thalidomide) therapy. In one embodiment, the TH-302 or another compound of formula I is administered on days and at doses as described above. In this embodiment, the pomalidomide is administered as commercially supplied at a dose of 2-4 mg on days 1 to 21 of a 28 day cycle.

Hypoxic Markers

[0126] In various embodiments of the invention, a marker of hypoxia is used to select patients for treatment and/or to identify patients that are responding (or not responding) to therapy. Hypoxia markers have been developed in the course of studies showing that hypoxia promotes more aggressive solid tumor phenotypes and associates with resistance to radiation and many chemotherapies, as well as likelihood of tumor invasion and poor patient survival. In particular, cells at p0₂ <10 mm Hg resist the ionizing effect of radiotherapy and cytotoxic effect of chemotherapy. Thus, a variety of methods have been devised to assess degree of hypoxia in xenografts and patient tumors, and, in accordance with the invention, these methods, suitably modified and practiced as described herein, are used in certain embodiments of the methods of the invention to select patients and assess response to therapy. In general, the invention provides methods for identifying patients suitable for therapy with a hypoxia-activated prodrug in which a marker of hypoxia is used to identify that a patient's cancer is hypoxic and then the patient is treated with a hypoxia-activated prodrug, i.e., the higher the degree of hypoxia, the more likely the patient will respond to therapy with a hypoxia-activated prodrug. Those of skill in the art will appreciate, in view of this disclosure, that these methods are useful in all cancers, not just blood cancers.

[0127] Traditionally, the gold standard for measuring hypoxia has been the use of a polarographic oxygen-sensitive probe, which provides direct measurement of tissue oxygen tension. However, this method has limitations, such as its inability to differentiate between viable and necrotic foci, the inaccessibility of many tumor tissues, including those associated with hematologic malignancies of the bone marrow, and the lack of a practical means to apply the technique in large scale.

[0128] Other hypoxic markers that have been identified that are suitable for use in accordance with the methods of the invention include GLUT-1, HIF-la, CA-IX (see PCT Pub. No. WO 13/116385, incorporated herein by referenc), LDH-A, VEGF, osteopontin, and microRNA markers, including but not limited to miR-210. Each of these proteins or microRNAs is up-regulated in hypoxia, and they can be detected by tumor biopsy. More conveniently, however, some of these markers, i.e., CA-IX LDH-A, VEGF, osteopontin, and microRNA markers, including but not limited to miR-210, will be detectable in the blood, serum, or plasma of a patient, allowing a simple blood test, instead of a tumor biopsy, to be used to select patients for hypoxia-activated prodrug therapy. [0129] In addition, studies have examined the spatial relationship between tumor hypoxia assessed by immunohistochemistry and PET tracers, such as [18FJ-EF5, [18FJ-FAZA, [18F]- FMISO, and [18F]-HX4 (see PCT Pub. No. WO 2008/124651, incorporated herein by reference), can be employed in accordance with the methods of the invention. In addition to autoradiography and PET imaging, MRI imaging of hypoxia, in particular dynamic contrast- enhanced MRI (DCE-MRI), can be used to identify hypoxic cancers and thus identify patients ideal for treatment with hypoxia-activated prodrugs.

[0130] Pimonidazole and EF5, both 2-nitroimidazole compounds, are hypoxia markers that, via immunohistochemical identification of pimonidazole or EF5 protein adducts, can give a reliable estimate of hypoxia. This method reliably identifies viable hypoxic cells specifically (necrotic cells cannot metabolize pimonidazole or EF5). Hypoxyprobe®-l (pimonidazole hydrochloride, marketed by Hypoxyprobe, Inc.) when administered, either IV or orally, is distributed to all tissues in the body including the brain but only forms adducts with proteins in those cells that have an oxygen concentration less than 14 micromolar (equivalent to a p0₂ of 10 mm Hg at 37 degrees Celsius). Hypoxyprobe- lMAbl is a mouse lgGl monoclonal antibody that detects protein adducts of Hypoxyprobe- 1 in hypoxic cells. This reagent is typically added to each tissue sample. Chromogenic or fluorescent secondary antibody reagents are then used in accordance with the invention to reveal where Hypoxyprobe- 1 adducts have formed in the hypoxic tissue. In one embodiment, hypoxia status can be checked using about 500 mg/m² pimonidazole hydrochloride (hypoxyprobe- 1) administered IV.

[0131] In addition to these markers of hypoxia, there are other markers that can be used to select patients for hypoxia-activated prodrug therapy. The hypoxia-activated prodrugs of the invention are activated by reductases, so biopsies or blood tests that show a patient has higher levels of an activating reductase, such as POR (P450 oxido-reductase), MTRR (methionine synthase reductase), and/or NOS (nitric oxide synthase), demonstrate that a patient is more likely to respond to hypoxia-activated prodrug therapy. Furthermore, the DNA damage induced by these hypoxia-activated prodrugs is repaired by the HDR (also known as HR) system, and the lower the levels of the proteins in this system, including but not limited to BRCA, FANC, XPF (also known as ERCC4), XRCC2 and/or XRCC₃, in the blood or tumor biopsy of a patient, the more likely the patient will respond to hypoxia-activated prodrug therapy. [0132] Thus, the methods of the invention include methods for determining whether a patient is suitable for or is responding to a therapeutic method of the invention.

[0133] The present invention, having been described in summary and in detail, is illustrated and not limited by the following examples.

EXAMPLES

[0134] The following examples are intended for illustration only and should not be construed to limit the scope of the claimed invention. The principal agents used are TH-302, as a representative species of hypoxia-activated drugs, and carfilzomib, pomalidomide or bortezomib.

Example 1. In vivo administration of TH-302 in combination with carfilzomib for the treatment of multiple myeloma

[0135] The combination of TH-302 and carfilzomib (CFZ) was administered in a C57BL mouse model (see Chesi et al, Blood 120: 376-385 (2012); Cancer Cell, 13: 167-180 (2008), incorporated herein by reference).

[0136] In these studies, CFZ was dosed at 2 or 4 mg/kg (mpk) iv; and TH-302 at 100 mpk ip. In the studies, the CFZ and TH-302 were formulated in one of the following formulations: (a) lOmM sodium citrate (pH 3.5) in 10% captisol; or

(b) 10% captisol. The frequency of administration is as follows:

(a) CFZ: days 1, 2 and 8, 9 in a two week test (for three week test, also dose on days 15,16); and TH-302: days 1 and 8 in a two week test (for three week test, also dose on day 15). [0137] On the days where CFZ and TH-302 are both dosed on the same day, there was a 4 hour delay between the TH-302 dose (dosed first) and the CFZ dose. The treatment of TH- 302 in combination with CFZ did not increase the toxicity, compared to CFZ-alone in C57BL mice see Figures 1 and 2. C57BL mice were well tolerated with 2 mg/kg CFZ in combination with TH-302 (100 mpk, weekly ip). C57BL mice were well tolerated with 4 mg/kg CFZ as a monotherapy. Example 2. In vivo administration of TH-302 in combination with carfilzomib or bortezomib or the treatment of multiple myeloma

[0138] The combination of TH-302 and carfilzomib (CFZ) or bortezomib (BTZ) were administered in a Vk*myc transgenic mice (see Chesi et al, Blood 120: 376-385 (2012); Cancer Cell, 13: 167-180 (2008), incorporated herein by reference). In these studies, CFZ is dosed at 2 mg/kg (mpk) iv; TH-302 at 100 or 50 mpk ip; and BTZ is dosed at 0.8 mg/kg (mpk) ip. The frequency of administration is as follows:

(a) CFZ: days 2, 3 and 9, 10 in a two week test (for three week test, also dose on days 16,17); and TH-302: days 2 and 9 in a two week test (for three week test, also dose on day 16);

(b) CFZ: days 2, 3 and 9, 10 in a two week test (for three week test, also dose on days 16,17); and TH-302: days 1-5 and 8-12 in a two week test (for three week test, also dose on days 15-19);

(c) BTZ: days 2, 5 and 9, 12 in a two week test (for three week test, also dose on days 16, 19); and TH-302: days 2 and 9 in a two week test (for three week test, also dose on day 16); and

(d) BTZ: days 2, 5 and 9, 12 in a two week test (for three week test, also dose on days 16,19); and TH-302: days 1-5 and 8-12 in a two week test (for three week test, also dose on days 15-19). [0139] On the days where CFZ or BTZ and TH-302 are both dosed on the same day, there was a 1 hour delay between the TH-302 dose (dosed first) and the CFZ or BTZ dose. The in vivo combination treatment showed additive effects when TH-302 (both 50 mpk daily and 100 mpk 2x/wk) was used in combination with BTZ (see Figs. 3 and 4). Additive effects were also observed when TH-302 (50 mpk daily) was used in combination with CFZ. There was no evident body weight loss in the treated groups during the study period.

Example 3. In vivo administration of TH-302 in combination with carfilzomib for the treatment of multiple myeloma

[0140] The combinations of TH-302 and carfilzomib (CFZ) and TH-302 are administered in a 5T33MMw mouse model (see Hu et al. Blood, 116(9): 1524-1527, 2010; Disease Models and Mechanisms, 5:763-771, 2012; and Mol. Cancer Ther., 12(9): 1763-1773 (2013), incorporated herein by reference). In these studies, CFZ is dosed at 4 mg/kg (mpk) iv; TH- 302 at 100 mpk ip; and BTZ at 0.6 mpk ip. The frequency of administration is as follows:

(a) CFZ: days 2, 3 and 9, 10 in a two week test (for three week test, also dose on days 16,17); and TH-302: days 2 and 9 in a two week test (for three week test, also dose on day 16).

(b) BTZ: days 2, 5 and 9, 12 in a two week test (for three week test, also dose on days 16 and 19).

[0141] On the days where CFZ and TH-302 are both dosed on the same day, TH-302 is dosed first, before the CFZ dose.

The in vivo combination treatment shows no severe toxicity, see Fig. 4.

Example 4. In vivo administration of TH-302 in combination with pomalidomide for the treatment of multiple myeloma

[0142] The combination of TH-302 and pomalidomide (PM or POM) were administered in a CD-I mouse model (see Chia et al., Nat Genet. 37: 1181-1186 (2005); incorporated herein by reference).

[0143] In these studies, TH-302 was dosed at 50, 75 or 100 mpk ip; and PM at 10 mpk ip. The frequency of administration is as follows:

(a) PM: days 1-5 and 8-12 in a two week test (for three week test, also dose on days 15-19); and TH-302: days 1 and 4 and 8 and 11 in a two week test (for three week test, also dose on days 15 and 18); and

(b) PM and TH-302 both dosed on days 1-5 and 8-12 in a two week test (for three week test, also dose on days 15-19).

[0144] On the days where PM or CFZ and TH-302 are both dosed on the same day, there was a 2 hour delay between the TH-302 dose (dosed first) and the PM dose. Animals were euthanized 4 days after the last treatment for hematologic analysis.

[0145] The in vivo combination treatment shows no severe toxicity when TH-302 was used in combination with pomalidomide (see Fig. 5). Example 5. In vivo administration of TH-302 in combination with carfilzomib or pomalidomide for the treatment of multiple myeloma

[0146] The combinations of TH-302 and carfilzomib (CFZ) and TH-302 and pomalidomide (PM) are administered in a 5T33MMvv mouse model (see Hu et al. Blood, 116(9):1524- 1527, 2010; Disease Models and Mechanisms, 5:763-771, 2012; and Mol. Cancer Ther., 12(9): 1763-1773 (2013); incorporated herein by reference) and in a Vk*MYC mouse model (see Chesi et al, Blood 120: 376-385 (2012); Cancer Cell, 13: 167-180 (2008), incorporated herein by reference). The in vivo combination treatment shows improvements in one or more disease parameters, such as decreased cancer cell burden, reduced paraprotein secretion, and reduced microvessel density (MVD), compared to TH-302-alone or CFZ-alone or PM-alone treated 5T33MMw mice (p <0.01) (see Hu et al. Blood, 116(9): 1524-1527, 2010; Disease Models and Mechanisms, 5:763-771, 2012; and Mol. Cancer Ther., 12(9): 1763-1773 (2013)) or TH-302-alone or CFZ-alone or PM-alone treated Vk*MYC mice (p <0.01) (see e.g. Chesi et al, Blood 120: 376-385 (2012); Cancer Cell, 13: 167-180 (2008)).

[0147] In these studies, CFZ is dosed at 2 mg/kg (mpk) iv; TH-302 at 100 mpk ip; and PM at 50 mpk ip. In some studies, dexamethasone is also employed at 10 mpk iv. The frequency of administration is as follows:

(a) CFZ: days 1, 2 and 8, 9 in a two week test (for three week test, also dose on days 15,16); and TH-302: days 1 and 8 in a two week test (for three week test, also dose on day

15);

(b) CFZ: days 1, 2 and 8, 9 in a two week test (for three week test, also dose on days 15,16); and TH-302: days 1 and 4 and 8 and 11 in a two week test (for three week test, also dose on days 15 and 18); (c) PM: days 1-5 and 8-12 in a two week test (for three week test, also dose on days

15-19); and TH-302: days 1 and 8 in a two week test (for three week test, also dose on day 15);

(d) PM: days 1-5 and 8-12 in a two week test (for three week test, also dose on days 15-19); and TH-302: days 1 and 4 and 8 and 11 in a two week test (for three week test, also dose on days 15 and 18); and (e) PM and TH-302 both dosed on days 1-5 and 8-12 in a two week test (for three week test, also dose on days 15-19).

[0148] On the days where PM or CFZ and TH-302 are both dosed on the same day, there is a 2 hour delay between the TH-302 dose (dosed first) and the PM or CFZ dose.

Example 6: Demonstration of Efficacy in Humans

[0149] A human clinical trial is conducted to demonstrate the safety and tolerability, define the maximum tolerated dose (MTD), and demonstrate the efficacy of the combination of TH-302 with carfilzomib, pomalidomide or bortezomib. Suitable patients for such a trial include previously treated or untreated subjects with MM. A human clinical trial can include analysis of any of a series of biomarkers, as described above.

[0150] The starting dose of TH-302 is 170 mg/m² or higher. The protocol described here foresees to manage toxicities by using a conservative dose escalation, dose modification, and by the use of growth factors if necessary. Prophylactic and therapeutic treatments against skin and mucosal toxicity and therapeutic recommendations in case of injection site reactions can be used as needed.

[0151] The trial can be conducted as an open-label, phase 1, multicenter, dose escalation trial of TH-302 combined with carfilzomib, pomalidomide or bortezomib in previously untreated subjects with MM or in MM patients who have progressed despite prior therapy (i.e., relapsed or refractory MM patients). The trial investigates safety and tolerability, and defines the MTD of TH-302 combined with carfilzomib, pomalidomide or bortezomib. In the trial, TH-302 is administered 30 minutes to 4 hours prior to administration of carfilzomib, pomalidomide or bortezomib.

[0152] The trial is conducted in 3 phases: screening, treatment, and follow-up. Screening assessments are conducted within 21 days prior to Cycle 1 Day 1. Study drugs are administered in successive 28-day cycles (any of the 28-day cycles of administration described herein may be used) until there is evidence of progressive disease according to IMWG, intolerable toxicity, or the subject discontinues the study drugs for other reasons. Upon discontinuation from the study drugs, an end of treatment visit will be conducted followed by a post-treatment safety visit either 30 days (± 3 days) after the last administration of study drugs or immediately before the administration of a new cancer therapy. [0153] Consented subjects who meet the eligibility criteria will be assigned to a TH-302 dose escalation cohort. All screening evaluations are completed within 21 days prior to the first TH-302 administration. After the subject gives written, informed consent the following evaluations will be performed: complete physical examination, assessment of weight, vital signs measurements (blood pressure, heart rate (HR), respiratory rate, temperature), demographic data recording, 12-lead ECG, Eastern Cooperative Oncology Group (ECOG) performance status, medical history recording, cancer history recording, medication history recording, standard laboratory tests (hematology, biochemistry, urinalysis), pregnancy test in women of childbearing potential, sample collection for serum and plasma hypoxia markers, and assessment of eligibility.

[0154] The treatment phase will consist of 28-day cycles. On Days 1, 8, and 15 of each cycle, TH-302 is administered iv at least 30 minutes, i.e., 2 to 4 hours, prior to the iv administration of carfilzomib at 20 mg/m² or 27 mg/m² or 56 mg/m² or pomalidomide at 2, 3, or 4 mg or bortezomib at 0.6 mg/m² to 0.7 mg/m² to 0.8 mg/m² to 0.9 mg/m² (carfilzomib, pomalidomide or bortezomib are dosed in amounts and at frequencies approved by the FDA). In addition, doses of carfilzomib, pomalidomide or bortezomib, and TH-302 doses can be delayed or modified or both for hematological and non-hematological toxicity. The combination treatment will continue until disease progression, intolerable toxicity, or subject discontinuation of study drug for other reasons. [0155] The evaluations that may be performed at specified time points are: limited physical examination, update of medical history, assessment of weight, vital signs measurements (blood pressure, HR, respiratory rate, temperature), 12-lead ECGs, ECOG performance status, standard laboratory tests (hematology, biochemistry, urinalysis), PK sampling, sample collection for serum and plasma hypoxia markers, pregnancy test in women of childbearing potential, sample collection for pharmacogenomics testing, concomitant medication recording, and AE assessment.

[0156] Safety and tolerability endpoints will consist of TEAEs graded according CTCAE version 4.03, SAEs, and deaths. In addition, drug exposure and standard laboratory tests (hematology, biochemistry, urinalysis, and pregnancy test in women of childbearing potential), 12-lead ECGs, physical examinations, and assessment of weight and vital signs may also be performed. [0157] All subjects are eligible to receive best supportive care defined as any standard supportive measure that is not considered primary treatment of the disease under study. Use of growth factors for the treatment of myelosuppression is according to the American Society of Clinical Oncology (ASCO) guidelines should they prove necessary. The combination treatment will continue until disease progression, intolerable toxicity, or subject discontinuation (e.g., withdrawal of consent) occurs.

[0158] TH-302 (concentrate for solution for administration) drug product for use in the trial is a sterile liquid formulation of TH-302. TH-302 is formulated with 70% ethanol anhydrous, 25% dimethlyacetamide, and 5% polysorbate 80. It will be supplied by the sponsor in a 10-mL glass vial with a rubber stopper and flip-off seal. TH-302 drug product is a clear, colorless to light yellow solution, essentially free of visible particulates. Each single use vial contains a nominal fill volume of 6.5 mL of TH-302 drug product for a nominal total of 650 mg of TH-302 (corresponds to 100 mg/mL) and will be labeled clearly, disclosing the lot number, route of administration, required storage conditions, sponsor's name, and appropriate precautionary labeling as required by applicable regulations. Dilution prior to administration is required per the pharmacy manual.

[0159] TH-302 drug product is supplied in a 10 mL glass vial and will be diluted prior to administration with commercially available 5% dextrose in water to a total volume of 500 mL (1000 mL for total dose of > 1000 mg) per administration to obtain the desired final concentration. Each dose of TH-302 will be prepared in a non di(2 ethylhexyl)phthalate (non DEHP), containing 5% dextrose in water, and administered IV via a non DEHP containing IV administration set. The starting dose of TH-302 will be 170 mg/m². Two additional dose levels are planned (240 and 340 mg/m²). Additional intermediate dose levels may be investigated to manage emerging toxicities. [0160] The body surface area (BSA) should be recalculated and the dose adjusted on each dosing occasion. Body surface area should be calculated based on a standard formula, such as the Mosteller formula: BSA (m²) = ([height(cm) x weight(kg)] / 3600 )^½, e.g., BSA = square root((cm*kg)/3600) or in inches and pounds: BSA (m²) = ([height(in) x weight(lbs)] / 3131)^½. [0161] Each TH-302 dose is administered as a 500 mL (1000 mL for total dose of > 1000 mg) volume that should be given IV over 30 minutes. Longer durations of administration are permitted based on the investigator's judgment of the time required to administer the dose. [0162] Supra venous erythema and hyperpigmentation have been reported at the injection site; severe cellulitis, vessication, and tissue necrosis may occur if extravasation of TH-302 occurs during administration. Care in the administration of TH-302 will reduce the chance of perivenous infiltration. If any signs or symptoms of extravasation occur, the administration of TH 302 should be immediately terminated and restarted in another vein. TH-302 should always be administered via a freely flowing IV line, preferably, where feasible, through a central venous catheter. Administration through small veins, particularly on the hands and feet is discouraged. Because of the progressive nature of extravasation reactions, close observation and plastic surgery consultation is recommended. [0163] Prophylaxis against nausea and vomiting should be implemented using a regimen intended for moderately emetogenic chemotherapy.

[0164] TH-302 administration reactions have been observed. These reactions have been characterized by lip swelling and urticaria that responded to steroid and antihistamine treatment. It is recommended that a steroid such as dexamethasone (or equivalent) be included in the antiemetic regimen prior to administration. Symptoms and signs of hypersensitivity include fever, myalgia, headache, rash, pruritus, urticaria, angioedema, chest discomfort, dyspnea, coughing, cyanosis, and hypotension. If the nature and the severity of the reaction require termination of treatment, it should be determined if the reaction may or may not be an immunoglobulin E mediated process. If there are symptoms such as upper airway obstruction or hypotension that suggest anaphylaxis or an anaphylactoid reaction, then treatment with an antihistamine (e.g., diphenhydramine 25 to 50 mg oral, intramuscular, or slow IV, or equivalent) and low dose steroid (e.g., hydrocortisone, 100 mg IV or equivalent) should be considered by the investigator as appropriate. If the event is clearly anaphylaxis, then epinephrine (1/1000, 0.3 to 0.5 mL given subcutaneously or equivalent) should be considered as well as standard treatment approaches. In the case of bronchospasm, inhaled β- agonist should be considered. Idiosyncratic reaction may also be treated with an antihistamine and low dose steroids depending on their severity. Reactions to the administration of TH-302 should be assessed and treated in a similar manner. For all reactions to TH-302, the investigator should consult with the Medical Monitor to determine the appropriate course of action for future treatment.

[0165] Any medications (other than those excluded by the protocol) that are considered necessary for the subjects' welfare and will not interfere with the study drugs may be given at the investigator's discretion. Female subjects who have been on hormone replacement therapy for menopausal symptoms for a period of at least 2 months will not be excluded from the trial provided the hormone replacement therapy regimen remains unchanged during the conduct of the trial. Prophylactic hematopoietic colony-stimulating factors may be implemented in subsequent cycles if neutropenia results in dose reduction or dose delay at prior doses. Therapeutic use of hematopoietic colony-stimulating factors is permitted following ASCO guidelines.

[0166] Prophylaxis against nausea and vomiting should be implemented using a regimen intended for moderately emetogenic chemotherapy. Inclusion of dexamethasone (or equivalent) in the antiemetic regimen is recommended. Prophylactic and therapeutic recommendations against skin and mucosal toxicity and therapeutic recommendations in case of injection site reactions may be taken as described above.

[0167] Palliative radiotherapy to non-target lesions is permitted if it becomes necessary while the subjects are in the trial. Prophylactic and therapeutic steps against skin and mucosal toxicity and therapeutic treatment in case of injection site reactions is permissible.

[0168] The follow-up phase will consist of 2 visits, an end of treatment visit and a safety visit. The end of treatment visit will be conducted within 1 week after discontinuation of the study drug treatment or immediately before initiation of any other anti-cancer therapy, whichever occurs first. The safety visit will be conducted either 30 (± 3) days after the last administration of study drug or immediately before initiation of any other anti-cancer therapy. Upon discontinuation from the treatments (end of treatment visit), the subject will have a complete physical examination, assessment of weight, ECOG performance status, vital signs measurements (blood pressure, HR, respiratory rate, temperature), 12-lead ECG, standard laboratory tests (hematology, biochemistry, urinalysis), sample collection for serum and plasma hypoxia markers, pregnancy test for women of childbearing potential, tumor imaging (CT or MRI, only required if not performed within the past 8 weeks and if clinically appropriate), and concomitant medication recording, and AEs assessment.

[0169] A safety visit will be conducted either 30 days (± 3 days) after the last administration of study drugs or immediately before initiation of any other cancer therapy. Information on further lines of therapy will be collected. The evaluations performed at the safety visit are: complete physical examination, vital signs assessments (blood pressure, HR, respiratory rate, temperature), standard laboratory tests (hematology, biochemistry, urinalysis), and pregnancy test for women of childbearing potential. Subjects will be contacted for AEs and subsequent cancer therapy information every 3 months for a minimum of 12 months.

[0170] The results show that the methods of the invention are effective in treating blood cancers.

[0171] For all purposes in the United States of America, each and every publication and patent document cited herein is incorporated herein by reference for all purposes as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

[0172] While the invention has been described with reference to the specific embodiments, changes can be made and equivalents can be substituted to adapt to a particular context or intended use, thereby achieving benefits of the invention without departing from the scope of the claims that follow. [0173] While certain embodiments have been illustrated and described in the foregoing examples, it will be understood that changes and modifications can be made in the foregoing methods to practice the present technology in accordance with ordinary skill in the art without departing from the present technology in its broader aspects as defined in the following claims. [0174] The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [0175] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED IS: 1. A combination of a hypoxia-activated prodrug selected from the group consisting of TH-281, TH-302, and TH-308 and another chemotherapeutic agent selected from the group consisting of carfilzomib and pomalidomide for simultaneous or sequential use in the treatment of a blood cancer.

2. A method of treating a blood cancer comprising administering a therapeutically effective amount of a prodrug selected from the group consisting of TH-281, TH-302, and TH-308 in combination with another chemotherapeutic agent selected from the group consisting of carfilzomib and pomalidomide to a patient in need of such treatment thereby treating the blood cancer.

3. The combination or method of claim 1 or 2, wherein the blood cancer treated is a multiple myeloma.

4. The combination or method of claim 3, wherein the multiple myeloma is a relapsed or refractory multiple myeloma.

5. The combination or method of any one of claims 1 to 3, wherein the patient has demonstrated disease progression on a previous therapy.

6. The combination or method of any one of claims 1 to 5, wherein the patient is unsuitable for standard chemotherapy.

7. The combination or method of any one of claims 1 to 6, wherein carfilzomib or pomalidomide is administered at least about 30 minutes to about 8 hours after treatment with the prodrug on the days when both drugs are administered on the same day.

8. The combination or method of any one of claims 1 to 7, wherein the compound is TH-302.

9. The combination or method of any one of claims 1 to 8, wherein the chemotherapeutic is carfilzomib.

10. The combination or method of any one of claims 1 to 8, wherein the chemotherapeutic is pomalidomide.

11. The combination or method of any one of claims 1 to 10, wherein TH- 302 is administered intravenously at a dose in a range from about 170 mg/m²/day to 670 mg/m²/day.

12. The combination or method of claim 11, wherein TH-302 is administered as a combination therapy with carfilzomib or pomalidomide to treat multiple myeloma, in a plurality of cycles of 28 days.

13. The combination or method of claim 12, wherein TH-302 is administered as a combination therapy with carfilzomib or pomalidomide on days one, eight, and fifteen of the twenty-eight day cycle at a dose between 170 mg/m²/day and 670 mg/m²/day.

14. The combination or method of claim 12, wherein TH-302 is administered as a combination therapy with carfilzomib or pomalidomide on days one, two, eight, nine, fifteen, and sixteen on a twenty-eight day cycle at a dose between 170 mg/m²/day and 670 mg/m²/day.

15. The combination or method of claim 12, wherein TH-302 is administered as a combination therapy with carfilzomib or pomalidomide on days one and eight of the twenty-eight day cycle at a dose between 170 mg/m²/day and 670 mg/m²/day.

16. The combination or method of claim 12, wherein TH-302 is administered as a combination therapy with carfilzomib or pomalidomide on days one, four, eight, eleven, fifteen, and eighteen on a twenty-eight day cycle at a dose between 170 mg/m²/day and 670 mg/m²/day.

17. The combination or method of any of claims 12 to 16, wherein TH-302 is administered as a combination therapy further comprising carfilzomib or pomalidomide in conjunction with surgery or radiation therapy.