RU2850269C2 - Pharmaceutical combinations for treatment of malignant tumours - Google Patents

Abstract

FIELD: chemistry; pharmaceuticals.

SUBSTANCE: invention relates to chemistry and pharmaceuticals, namely to the use of a therapeutically effective amount of a CDK inhibitor corresponding to formula Ib: or a pharmaceutically acceptable salt thereof, for the treatment of a malignant blood tumour in combination with a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof, wherein the malignant blood tumour is selected from acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), acute lymphocytic leukaemia (ALL) and chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, and primary central nervous system lymphoma.

EFFECT: to provide a synergistic effect of the combination of active agents in the treatment of malignant blood tumours.

9 cl, 33 dwg, 4 tbl, 16 ex

Description

CROSS-REFERENCE

[1] This application claims the benefit of U.S. Provisional Application Serial No. 62/314,356, filed March 28, 2016, the contents of which are incorporated herein by reference in their entirety.

LEVEL OF TECHNOLOGY

[2]In any At any given time, numerous cancer-targeted therapeutics are in Phase I or Phase II clinical trials and evaluation; however, most do not progress further. In fact, it is estimated that over 90% of cancer-targeted therapeutics fail in Phase I or II clinical trials. The failure rate in Phase III clinical trials is nearly 50%, and the cost of developing new drugs from discovery to Phase III trials ranges from $0.8 billion to $1.7 billion and takes eight to ten years.

[3] Furthermore, many patients do not respond even to standard drugs for which they have been shown to be effective. For reasons that are currently not entirely clear or that are not easy to assess, individual patients may not respond to therapy with a standard drug. In some cases, administering drug combinations may be more effective in treating a malignancy than drugs administered individually. These drug combinations may act synergistically, enhancing the activity of the drugs against the malignancy. In some cases, drugs that are not particularly effective may find new and unexpected uses when combined with additional drug therapies.

ESSENCE OF THE INVENTION

[4] In one aspect, the invention relates to a method of treating a malignant blood tumor, comprising administering to an individual in need thereof a therapeutically effective amount of a CDK inhibitor according to formula I:

or its pharmaceutically acceptable salt, where:

R ₁ is optionally substituted phenyl;

each of R ₂ and R ₃ is independently selected from hydroxy and OR ₈ , wherein R ₈ is optionally substituted C ₁ -C ₁₀ alkyl;

R ₄ is optionally substituted C ₁ -C ₄ alkyl; and

R₉is hydrogen or optionally substituted C₁-C₄-alkyl;

and a therapeutically effective amount of a BCL-2 inhibitor.

[5] In some aspects, the invention relates to a method of treating a malignant tumor comprising administering to an individual in need thereof a therapeutically effective amount of a CDK inhibitor according to formula I:

I,

or its pharmaceutically acceptable salt, where:

R ₁ is optionally substituted phenyl;

each of R ₂ and R ₃ is independently selected from hydroxy and OR ₈ , wherein R ₈ is optionally substituted C ₁ -C ₁₀ alkyl;

R ₄ is optionally substituted C ₁ -C ₄ alkyl; and

R₉is hydrogen or optionally substituted C₁-C₄-alkyl;

and a therapeutically effective amount of a proteasome inhibitor. In certain embodiments, the malignant tumor is selected from hematopoietic cancer and triple-negative breast cancer (TNBC).

[6] In certain embodiments, the compound of Formula I is a compound of Formula Ia:

Ia or its pharmaceutically acceptable salt.

[7] In certain embodiments, for a compound or salt of Formula I or Ia, R ₁ is optionally substituted with one or more substituents independently selected from hydroxy, cyano, halogen, amino, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, and nitro. In certain embodiments, R ₁ is substituted with one or more substituents independently selected from halogen and C ₁ -C ₄ haloalkyl. In certain embodiments, R ₁ is 2-chloro-4-trifluoromethylphenyl.

[8] In certain embodiments, for a compound of Formula I or Formula Ia, each of R ₂ and R ₃ is independently selected from hydroxy and OR ₈ , wherein R ₈ is C ₁ -C ₁₀ alkyl optionally substituted with one or more substituents independently selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, each of R ₂ and R ₃ is hydroxy.

[9] In certain embodiments, for a compound of Formula I or Formula Ia, R ₄ is C ₁ -C ₄ alkyl substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₄ is C ₁ -C ₄ alkyl substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₄ is 2-hydroxymethyl.

[10] In certain embodiments, for a compound of Formula I or Formula Ia, R ₉ is C ₁ -C ₄ alkyl optionally substituted with hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₉ is methyl. In certain embodiments, a compound of Formula I is a compound of Formula Ib:

Ib

or its pharmaceutically acceptable salt.

[11] In certain embodiments, a BCL-2 inhibitor according to the methods described herein is a BH3 mimetic. The BCL-2 inhibitor can specifically inhibit the Bcl-2 protein. The BCL-2 inhibitor can be selected from navitoclax, venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, S44563, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitory peptide V5, Bax inhibitory peptide P5, Bax channel blocker, and ARRY 520 trifluoroacetate. In certain embodiments, the BCL-2 inhibitor according to the methods described herein is selected from navitoclax and venetoclax, or a pharmaceutically acceptable salt of either.

[12] In certain embodiments, a hematologic malignancy according to the methods described herein is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic lymphoma (ALL) and chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphomas, extranodal marginal zone B-cell lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, and primary central nervous system lymphoma. The malignant blood tumor may be diffuse large B-cell lymphoma, acute myeloid leukemia, or chronic lymphocytic leukemia.

[13] For certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor can be administered simultaneously. For the methods described herein, the CDK inhibitor and the BCL-2 inhibitor can be administered sequentially within about 12 hours of each other, such as within about 5 hours of each other.

[14] For certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor may be co-formulated in a pharmaceutical composition.

[15] For certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor may be administered every day, every other day, or every third day.

[16] For certain methods described herein, the proteasome inhibitor is selected from bortezomib, marizomib, ixazomib, disulfiram, epigallocatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, MG132, and a pharmaceutically acceptable salt of any of them. In certain embodiments, the proteasome inhibitor is selected from bortezomib, marizomib, ixazomib, and a pharmaceutically acceptable salt of any of them.

[17] In certain methods described herein, the CDK inhibitor and the proteasome inhibitor are administered simultaneously. The CDK inhibitor and the proteasome inhibitor can be administered sequentially within about 12 hours of each other, such as within 5 hours of each other.

[18] In certain methods described herein, the CDK inhibitor and the proteasome inhibitor are formulated together in a pharmaceutical composition.

[19] In certain methods described herein, the CDK inhibitor and the BCL-2 inhibitor are administered every day, every other day, or every third day.

[20] In some aspects, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a CDK inhibitor according to formula I:

or its pharmaceutically acceptable salt, where:

R₁represents optionally substituted phenyl;

each of R ₂ and R ₃ is independently selected from hydroxy and OR ₈ , wherein R ₈ is optionally substituted C ₁ -C ₁₀ alkyl;

R ₄ is optionally substituted C ₁ -C ₄ alkyl; and

R₉is hydrogen or optionally substituted C₁-C₄-alkyl;

a therapeutically effective amount of a BCL-2 inhibitor or a proteasome inhibitor, and a pharmaceutically acceptable excipient.

[21] In certain embodiments, the compound or salt of Formula I corresponds to Formula Ia: Ia.

[22] For the compositions described herein for compounds of Formula I or Formula Ia, R ₁ may optionally be substituted with one or more substituents independently selected from hydroxy, cyano, halogen, amino, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, and nitro. In certain embodiments, R ₁ is substituted with one or more substituents independently selected from halogen and C ₁ -C ₄ haloalkyl. In certain embodiments, R ₁ is 2-chloro-4-trifluoromethylphenyl.

[23] For compositions described herein for compounds of Formula I or Formula Ia, each of R ₂ and R ₃ may be independently selected from hydroxy and OR ₈ , wherein R ₈ is C ₁ -C ₁₀ alkyl optionally substituted with one or more substituents independently selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, each of R ₂ and R ₃ is hydroxy.

[24] For compositions described herein for compounds of Formula I or Formula Ia, R ₄ is C ₁ -C ₄ alkyl substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₄ is C ₁ -C ₄ alkyl substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₄ is 2-hydroxymethyl.

[25] For compositions described herein for compounds of Formula I or Formula Ia, R ₉ can be C ₁ -C ₄ alkyl optionally substituted with hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₉ is methyl.

[26] For the compositions described herein, the compound of formula I may be a compound of formula Ib: Ib, or a pharmaceutically acceptable salt thereof.

[27] For the compositions described herein comprising a BCL-2 inhibitor, the BCL-2 inhibitor may be selected from navitoclax, venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, S44563, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitory peptide V5, Bax inhibitory peptide P5, Bax channel blocker, ARRY 520 trifluoroacetate, and a pharmaceutically acceptable salt of any of them. The BCL-2 inhibitor may be selected from navitoclax and venetoclax or a pharmaceutically acceptable salt of any of them. In certain embodiments, the BCL-2 inhibitor is venetoclax.

[28] For compositions described herein comprising a proteasome inhibitor, the proteasome inhibitor may be selected from bortezomib, marizomib, ixazomib, disulfiram, epigallocatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, MG132, and a pharmaceutically acceptable salt of any of them. In certain embodiments, the proteasome inhibitor is selected from bortezomib, marizomib, ixazomib, and a pharmaceutically acceptable salt of any of them.

INCLUSION BY REFERENCE

[29] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF DRAWINGS

[30] The novel features of the invention are set forth more particularly in the appended claims. A better understanding of the features and advantages of the present invention may be obtained from the following detailed description, which sets forth illustrative embodiments in which the principles of the invention are employed, and the accompanying drawings, in which:

[31] Figure 1 illustrates a comparison of the activity of voruciclib and flavopiradol against 38 kinases.

[32] Figure 2 illustrates the single-digit nM potency of voruciclib against cyclin-dependent kinases.

[33] Figures 3A-3D illustrate the synergistic effect of voruciclib in combination with venetoclax (ABT-199). Voruciclib inhibits the induction of myeloid leukemia cell differentiation protein-1 (MLC-1) by venetoclax in diffuse large B-cell lymphoma (DLBCL) NU-DHL-1 cells. MCL-1=red; DAPI=blue; fluorescent imaging marker (FTM)=green.

[34] Figures 4A-4D illustrate increased apoptosis in diffuse large B-cell lymphoma (DLBCL) NU-DHL-1 cells by combination treatment with voruciclib and venetoclax. Cleaved caspase-3 (CC3) = red; DAPI = blue; fluorescent imaging marker (FTM) = green.

[35] Figures 5A-5D illustrate the synergistic effect of voruciclib in combination with navitoclax (ABT-263). Treatment of the Burkitt lymphoma cell line Ramos with voruciclib and navitoclax induces apoptosis. Cleaved caspase-3 (CC3) = red; DAPI = blue; fluorescent imaging marker (FTM) = green.

[36] Figures 6A-6E illustrate the synergistic effect of voruciclib in combination with venetoclax in five models of diffuse large B-cell lymphoma.

[37] Figure 7 illustrates that inhibition of MCL-1 via CDK9 helps direct cells toward apoptosis.

[38] Figures 8A-8D illustrate that proteasome inhibition induces MCL-1 activation in triple-negative breast cancer (TNBC). Figure 8A illustrates the list of compounds screened in the HCC1187 TNBC xenograft model. Figure 8B illustrates the mean % change in MCL-1 induction by treatment with the various compounds. Figures 8C-8D illustrate cell staining for CC3 (shown in red) following treatment with vehicle and bortezomib.

[39] Figures 9A-9D illustrate the synergistic effect of voruciclib in combination with marizomib on NudHL1 DLBCL cells. Cleaved caspase-3 (CC3)=red; DAPI=blue; fluorescent imaging marker (FTM)=green.

[40] Figures 10A-10D illustrate the synergistic effect of voruciclib in combination with bortezomib on NudHL1 DLBCL cells. Cleaved caspase-3 (CC3)=red; DAPI=blue; fluorescent imaging marker (FTM)=green.

[41] Figures 11A-11D illustrate the synergistic effect of voruciclib in combination with bortezomib on triple-negative breast cancer cells. Cleaved caspase-3 (CC3)=red; DAPI=blue; fluorescent imaging marker (FTM)=green.

[42] Figures 12A-12E illustrate the synergistic effect of voruciclib in combination with bortezomib on HCC1187 triple-negative breast cancer cells. Figures 12A-12D illustrate reduced tumor volume in the HCC1187 TNBC mouse model treated with voruciclib and bortezomib. Figure 12E illustrates a Western blot showing reduced MCL-1 expression in HCC1187 TNBC cells treated with voruciclib and bortezomib or voruciclib and tunicamycin.

[43] Figure 13 illustrates the effect of voruciclide and bortezomib and the combination of voruciclib and bortezomib on body weight in the HCC1187 TNBC mouse model.

[44] Figure 14 illustrates the effect of bortezomib in combination with palbociclib, a CDK4/6 inhibitor, in the HCC1187 TNBC mouse model.

[45] Figures 15A-15B show that voruciclib reduces bortezomib-induced expression of MCL-1 and the E3 ubiquitin protein ligase XIAP. Figure 15A illustrates a putative model of voruciclib inhibition of CDK9. Figure 15B illustrates a Western blot showing that voruciclib reduces bortezomib-induced increase in MCL-1 and the E3 ubiquitin protein ligase XIAP expression.

[46] Figures 16A-16B illustrate cells resistant to bortezomib treatment. Figure 16A illustrates cells resistant to bortezomib treatment in an area otherwise cleared of cells by bortezomib treatment. Figure 16B illustrates that bortezomib-resistant cells express GRP78, a protein expressed as part of the ER stress response.

[47] Figure 17 illustrates three different pathways of response to ER stress.

[48] Figures 18A-18B illustrate that voruciclib can affect the IRE1α-dependent ER stress response cascade. Figure 18A illustrates the IRE1α-dependent ER stress response pathway. Figure 18B illustrates a Western blot showing that the ER stress inducer tunicamycin significantly upregulates X-box binding protein 1 (XBP1), a pro-survival (anti-tumor cell death) protein. This effect was significantly reduced by voruciclib. At 6 hours, only tunicamycin exhibited this effect, however, at 24 hours, both bortezomib and tunicamycin exhibited this effect.

[49] Figures 19A-19B illustrate suppression of bortezomib-induced XBP1 transcription by voruciclib. STF083010=Inhibitor of IRE1α endoribonuclease activity; Tm=tunicamycin.

[50] Figures 20A-20D illustrate the synergistic effect of voruciclib in combination with ixazomib. Cleaved caspase 3 (CC3) = red; DAPI = blue; fluorescent imaging marker (FTM) = green.

[51] Figures 21A-21B illustrate solid tumor sections treated with the CDK inhibitors voruciclib, palbociclib, dinaciclib, and flavopiradol in combination with ixazomib. Only voruciclib+ixazomib reproducibly produced significant tumor cell killing within 24 h.

[52] Figures 22A-B illustrate the synergy of voruciclib and venetoclax in the SU-DHL-4 model for diffuse large B-cell lymphoma (DLBCL).

[53] Figures 23A-23C illustrate the synergy of voruciclib and venetoclax in the SU-DHL-4 model, the OCI Ly10 model, and the U2932 model of diffuse large B-cell lymphoma (DLBCL).

[54] Figure 24 illustrates that voruciclib suppresses MCL1 expression in DLBCL xenograft models.

[55] Figures 25A-25B illustrate the synergistic effect of voruciclib and venetoclax in ABC-type DLBCL (RIVA) in mice.

[56] Figures 26A-26B illustrate the synergistic effect of voruciclib and venetoclax and the effect on body weight in the U2932 mouse model of DLBCL.

[57] Figures 27A-27B illustrate the synergistic effect of voruciclib and venetoclax in the NUDHL1 mouse model of DLBCL.

[58] Figure 28 illustrates the synergistic effect of voruciclib and venetoclax in the SUDHL4 model for GC DLBCL.

[59] Figure 29 illustrates that voruciclib restores p53 abrogated by venetoclax.

[60] Figures 30A-3C illustrate that voruciclib has activity in AML cell lines as a single agent.

[61] Figure 31 illustrates that the combination of voruciclib and venetoclax induces synergistic cell death in AML cell lines.

[62] Figure 32 illustrates the synergistic effect of the combination of voruciclib and venetoclax in inhibiting tumor growth in SKM1 AML xenografts.

[63] Figure 33 illustrates that voruciclib-induced apoptosis correlates with MCL suppression.

DETAILED DESCRIPTION

[64] The invention relates to combination therapies for the treatment of a malignant tumor. In particular, the invention relates to combination therapies of CDK inhibitors with other anti-malignant agents for the treatment of a malignant tumor. In one aspect, the invention relates to compositions and methods for treating a malignant tumor with a CDK inhibitor in combination with a BCL-2 inhibitor. Such a combination provides synergistic effects in the treatment of malignant tumors and, in particular, in the treatment of malignant blood tumors, such as leukemia and lymphoma.

[65] In another aspect, the invention relates to compositions and methods for treating a malignant tumor with a CDK inhibitor in combination with a proteasome inhibitor. Such a combination provides a synergistic effect in the treatment of a malignant tumor, and in particular in the treatment of hematopoietic tumors and triple-negative breast cancer.

[66] The basic terms used above and below preferably have the following meanings in the context of the present invention, unless otherwise indicated. Accordingly, the definitions of the basic terms as used in the context of the present invention are provided below in the present description:

[67] The singular form includes the plural unless the context clearly indicates otherwise.

[68] The term "about," as used herein, generally refers to an acceptable range of error for a particular quantity, as determined by one of skill in the art, which may depend in part on how the quantity is measured or determined. For example, "about" may mean within 1 standard deviation or more than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of the given quantity. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5 times, and within 2 times the value of the quantity.

[69] As used herein, the term "at least one" refers to one or more. For example, the term "at least one anti-cancer agent" means that the combination comprises one anti-cancer agent or more anti-cancer agents.

[70] The term "effective amount" or "therapeutically effective amount," as used herein, generally refers to an amount of a compound described herein that is sufficient to provide the intended, intended, or prescribed use, including, but not limited to, the treatment of a disease or condition. A therapeutically effective amount may vary depending on the use (e.g., in vitro or in vivo ), or the individual being treated and the disease, such as the individual's weight and age, the severity of the disease condition, and the route of administration. The term may also refer to a dose that induces a specific response in target cells, such as a decrease in proliferation or suppression of the activity of a target protein. A specific dose may vary depending on the specific compounds selected, the dosing regimen followed, administration in combination with other compounds, the time of administration, the tissue into which the administration is administered, and the physical delivery system in which it is administered.

[71]As used within the framework of the invention, the term "Pharmaceutically acceptable" means that the carrier, diluent, excipients, and/or salt must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. "Pharmaceutically acceptable" also means that the compositions or dosage forms, within the judgment of a healthcare professional, are suitable for use in animals or humans without undue toxicity, irritation, allergic response, or other problem or complication, and meet an acceptable benefit/risk ratio.

[72] As used herein, the term "combination" or "pharmaceutical combination" refers to the combined administration of anti-cancer agents. The combinations of the invention comprise a CDK inhibitor, such as a compound of formula I, Ia, or Ib, and at least one anti-cancer agent selected from a BCL-2 inhibitor and a proteasome inhibitor; and these anti-cancer agents can be administered to an individual in need thereof, such as simultaneously or sequentially.

[73] The term "synergistic" or "synergistic effect" or "synergism" as used herein generally refers to an effect in which one or more effects of a combination of compositions exceeds one or more effects of each component separately, or may exceed the sum of one or more effects of each component separately. A synergistic effect may be approximately 10%, 20%, 30%, 50%, 75%, 100%, 110%, 120%, 150%, 200%, 250%, 350%, or 500% or more greater than the effect on an individual of one of the components separately, or the sum of the effects of each of the components when administered individually. The effect may be any of the quantifiable effects described herein. Advantageously, such synergy of the agents in a combination may allow the use of lower doses of one or both agents, may provide higher efficacy at the same doses, and may prevent or delay the development of multiple drug resistance. To determine the synergistic, additive, or antagonistic effect of the agents used in combination, the additivity index (CI) method of Chou and Talalay can be used. When the CI value is less than 1, synergy exists between the compounds used in the combination; when the CI value is 1, an additive effect exists between the compounds used in the combination; and when the CI value exceeds 1, an antagonistic effect exists. A synergistic effect can be achieved by co-formulating the agents of a pharmaceutical combination. A synergistic effect can be achieved by administering two or more agents as separate formulations, administered simultaneously or sequentially.

[74]Cyclin-dependent kinases (CDKs) are a family of enzymes that are activated during specific phases of the cell cycle. CDKs consist of a catalytic subunit (the actual cyclin-dependent kinase or CDK) and a regulatory subunit (cyclin). There are at least nine CDKs (CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, etc.) and at least 15 different types of cyclins (cyclin A, B1, B2, D1, D2, D3, E, H, etc.). Each stage of the cell cycle is regulated by such CDK complexes: G1/S transition (CDK2/cyclin A, CDK4/cyclin D1-D3, CDK6/cyclin D3), S-phase (CDK2/cyclin A), G2-phase 30 (CDK1/cyclin A), G2 transition phase/M (CDK1/cyclin B).

[75] As used herein, the term "CDK inhibitor" refers to an agent capable of inhibiting one or more cyclin-dependent kinases (CDKs). Aberrant expression and overexpression of these kinases has been demonstrated in many diseases, such as cancer. In the context of the present invention, the CDK inhibitor of the pharmaceutical combination described herein may be a compound of Formula I, Ia, or Ib, or a pharmaceutically acceptable salt thereof. The compounds of the present invention may specifically inhibit one or more of CDK1/cyclin B, CDK2/cyclin E, CDK4/cyclin D, CDK4/cyclin D1, and CDK9/cyclin T1. In certain embodiments, a compound of the invention specifically inhibits CDK9/cyclin T1 or CDK9.

[76] The present disclosure describes combination therapies for the treatment of malignancy, such as leukemia, lymphoma, and cancer. The methods and compositions described herein may include a cyclin-dependent kinase (CDK) inhibitor, such as a compound of Formula I, Ia, or Ib, or a pharmaceutically acceptable salt thereof. In some cases, the combination therapy may include a CDK inhibitor in combination with a proteasome inhibitor. In other cases, the combination therapy may include a CDK inhibitor in combination with a BCL-2 inhibitor.

[77] In certain embodiments, a CDK inhibitor of the invention corresponds to a compound described in U.S. Patent Nos. 7,271,193; 7,915,301; 8,304,449; 7,884,127; 8,563,596, the entire contents of each of which are incorporated herein by reference. In certain embodiments, a CDK inhibitor of the invention is a compound of Formula I:

I,

or a pharmaceutically acceptable salt thereof, where:

R ₁ is optionally substituted phenyl;

each of R ₂ and R ₃ is independently selected from hydroxy and OR ₈ , wherein R ₈ is optionally substituted C ₁ -C ₁₀ alkyl;

R ₄ is optionally substituted C ₁ -C ₄ alkyl; and

R₉is hydrogen or optionally substituted C₁-C₄-alkyl.

[78] In certain embodiments, the compound or salt of Formula I corresponds to Formula Ia:

Ia.

[79] In certain embodiments, for a compound of Formula I or Formula Ia, R ₁ is optionally substituted with one or more substituents independently selected from hydroxy, cyano, halogen, amino, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₄ haloalkyl, and nitro. In certain embodiments, R ₁ is substituted with one or more substituents independently selected from hydroxy, cyano, halogen, C ₁ -C ₄ alkyl, and C ₁ -C ₄ haloalkyl. In certain embodiments, R ₁ is substituted with one or more substituents independently selected from halogen and C ₁ -C ₄ haloalkyl. In certain embodiments, R ₁ is 2-chloro-4-trifluoromethylphenyl.

[80] The term "alkyl" refers to a straight or branched hydrocarbon radical consisting solely of carbon and hydrogen atoms and having no unsaturation. In certain embodiments, alkyl contains from one to eight carbon atoms (i.e., C ₁ -C ₈ alkyl). In other embodiments, alkyl contains from one to five carbon atoms (i.e., C ₁ -C ₅ alkyl). In other embodiments, alkyl contains from one to four carbon atoms (i.e., C ₁ -C ₄ alkyl). In other embodiments, alkyl contains from one to three carbon atoms (i.e., C ₁ -C ₃ alkyl). In other embodiments, alkyl contains from one to two carbon atoms (i.e., C ₁ -C ₂ alkyl). In other embodiments, alkyl contains one carbon atom (i.e., C ₁ alkyl). In other embodiments, alkyl contains from five to eight carbon atoms (i.e., C ₅ -C ₈ alkyl). In other embodiments, the alkyl contains two to five carbon atoms (i.e., C ₂ -C ₅ alkyl). In other embodiments, the alkyl contains three to five carbon atoms (i.e., C ₃ -C ₅ alkyl). In certain embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (isopropyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is bonded to the remainder of the molecule by a single bond. Unless otherwise specifically indicated in the description, the alkyl group is optionally substituted with one or more substituents, such as the substituents described herein.

[81] The term "alkoxy" refers to a radical bonded through an oxygen atom of the formula -O-alkyl, where alkyl is an alkyl chain as defined above.

[82] The term "amino" refers to the group -NR'R", where R' and R" are independently selected from hydrogen; and alkyl, hydroxyl, aryl, cycloalkyl, heterocycloalkyl, and heteroaryl, any of which may be optionally substituted with one or more substituents such as hydroxy, cyano, halogen, amino, _C1 - _C4- alkyl, _C1 - _C4 -alkoxy, _C1 - _C4- hydroxyalkyl, _C1 - _C4 -haloalkyl, and nitro.

[83] The term "C _xy ", when used in conjunction with a chemical moiety such as alkyl, includes groups that contain from x to y carbon atoms in the chain. For example, the term "C _xy alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched alkyl groups, that contain from x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[84]The term "haloalkyl" refers to an alkyl group that is substituted by one or more halogen radicals, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-chloromethyl-2-fluoroethyl, and the like. In some embodiments, the alkyl portion of the haloalkyl is further optionally substituted as described herein.

[85]The term "hydroxyalkyl" refers to an alkyl group that is substituted by one or more hydroxy radicals, for example, hydroxymethyl, hydroxyethyl, dihydroxymethyl, etc. In some embodiments, the alkyl portion of the hydroxyalkyl is further optionally substituted as described herein.

[86] In certain embodiments, for a compound or formula of Formula I or Ia, each of R ₂ and R ₃ is independently selected from hydroxy and OR ₈ , wherein R ₈ is C ₁ -C ₁₀ alkyl optionally substituted with one or more substituents independently selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₈ at each occurrence is selected from optionally substituted C ₁ -C ₆ alkyl, such as optionally substituted C ₁ -C ₄ alkyl. In certain embodiments, each of R ₂ and R ₃ is independently hydroxy.

[87] In certain embodiments, for a compound or formula of Formula I or Ia, R ₄ is optionally substituted C ₁ -C ₄ alkyl, wherein R ₄ is optionally substituted with one or more substituents selected from hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ alkoxy, and nitro. In certain embodiments, R ₄ is optionally substituted C ₁ -C ₂ alkyl. In certain embodiments, R ₄ is hydroxyalkyl, such as 2-hydroxymethyl.

[88] In certain embodiments, for a compound or formula of Formula I or Ia, R ₉ is C ₁ -C ₄ -alkyl, optionally substituted with hydroxy, cyano, halogen, amino, =O, =S, C ₁ -C ₄ -alkoxy and nitro. In certain embodiments, R ₉ is an optionally substituted C ₁ -C ₂ alkyl. In certain embodiments, R ₉ is methyl. In certain embodiments, R ₉ is hydrogen.

[89] In certain embodiments, for a compound or formula of Formula I or Ia, the compound of Formula I is a compound or pharmaceutically acceptable salt selected from: (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-5,7-dimethoxy-chromen-4-one; (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-one; and (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methyl-pyrrolidin-3-yl)-chromen-4-one hydrochloride.

[90] In certain embodiments, the compound of formula I or Ia is a compound of formula Ib:

or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound of Formula I, Ia, or Ib is in the form of an acid addition salt, such as a hydrochloric acid salt.

[91] The term "substituted" refers to moieties having substituents replacing hydrogen on one or more carbon atoms or heteroatoms of the structure. It will be understood that "substitution" or "substituted by" includes the implied condition that such substitution is in accordance with the permissible valence of the substituted atom and substituent and also provides a stable compound that does not readily undergo transformation such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" includes all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. One or more permissible substituents may be present and they may be the same or different for the respective organic compounds. For the purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible organic substituents described herein that satisfy the valences of the heteroatoms.

[92] Substituents may include any substituents described herein, for example, halogen, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as a thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, carbocycle, heterocycle, cycloalkyl, heterocycloalkyl, aromatic, and heteroaromatic moiety. In some embodiments, the substituents may include any of the substituents described herein, for example: halogen, hydroxy, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO ₂ ), imino (=NH), oximo (=N-OH), hydrazino (=N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC(O)-R ^a , -R ^b -OC(O)-OR ^a , -R ^b -OC(O)-N(R ^a ) ₂ , -R ^b -N(R ^a ) ₂ , -R ^b -C(O)R ^a , -R ^b -C(O)OR ^a , -R ^b -C(O)N(R ^a ) ₂ , -R ^b -OR ^c -C(O)N(R ^a ) ₂ , -R ^b -N(R ^a )C(O)OR ^a , -R ^b -N(R ^a )C(O)R ^a , -R ^b -N(R ^a )S(O) _t R ^a (where t is 1 or 2), -R ^b -S(O) _t R ^a (where t is 1 or 2), -R ^b -S(O) _t OR ^a (where t is 1 or 2) and -R ^b -S(O) _t N(R ^a ) ₂ (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl and heteroarylalkyl, any of which may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxy, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO ₂ ), imino (=NH), oximo (=N-OH), hydrazine (=N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC(O)-R ^a , -R ^b -OC(O)-OR ^a , -R ^b -OC(O)-N(R ^a ) ₂ , -R ^b -N(R ^a ) ₂ , -R ^b -C(O)R ^a , -R ^b -C(O)OR ^a , -R ^b -C(O)N(R ^a ) ₂ , -R ^b -OR ^c -C(O)N(R ^a ) ₂ , -R ^b -N(R ^a )C(O)OR ^a , -R ^b -N(R ^a )C(O)R ^a , -R ^b -N(R ^a )S(O) _t R ^a (where t is equal to 1 or 2), -R ^b -S(O) _t R ^a (where t is equal to 1 or 2), -R ^b -S(O) _t OR ^a (where t is equal to 1 or 2) and -R ^b -S(O) _t N(R ^a ) ₂ (where t is equal to 1 or 2); wherein each R ^a is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R ^a , if valence permits, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO ₂ ), imino (=NH), oximo (=N-OH), hydrazine (=N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC(O)-R ^a , -R ^b -OC(O)-OR ^a , -R ^b -OC(O)-N(R ^a ) ₂ , -R ^b -N(R ^a ) ₂ , -R ^b -C(O)R ^a , -R ^b -C(O)OR ^a , -R ^b -C(O)N(R ^a ) ₂ , -R ^b -OR ^c -C(O)N(R ^a ) ₂ , -R ^b -N(R ^a )C(O)OR ^a , -R ^b -N(R ^a )C(O)R ^a , -R ^b -N(R ^a )S(O) _t R ^a (where t is 1 or 2), -R ^b -S(O) _t R ^a (where t is 1 or 2), -R ^b -S(O) _t OR ^a (where t is 1 or 2) and -R ^b -S(O) _t N(R ^a ) ₂ (where t is 1 or 2); and where each R ^b is independently selected from a direct bond or a straight or branched alkylene, alkenylene or alkynylene chain and each R ^c is a straight or branched alkylene, alkenylene or alkynylene chain.

[93] Methods for producing compounds of formula I, Ia and Ib or pharmaceutically acceptable salts thereof can be found in PCT Patent Publication No. WO2004004632 (corresponding to U.S. Patent 7,271,193) and PCT Patent Publication No. WO2007148158.

[94] The present invention relates to pharmaceutically acceptable salts of any of the compounds described herein, for example, a compound of formula I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors. Pharmaceutically acceptable salts include, for example, acid addition salts and base addition salts. The acid that is added to the compound to form an acid addition salt can be an organic acid or an inorganic acid. The base that is added to the compound to form a base addition salt can be an organic base or an inorganic base. In some cases, the pharmaceutically acceptable salt is a metal salt. In some cases, the pharmaceutically acceptable salt is an ammonium salt.

[95] Acid addition salts can result from the addition of an acid to a compound described herein. In some cases, the acid is organic. In some cases, the acid is inorganic. Non-limiting examples of suitable acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, phosphoric acid, nicotinic acid, isonicotinic acid, lactic acid, salicylic acid, 4-aminosalicylic acid, tartaric acid, ascorbic acid, gentisic acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, citric acid, oxalic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, Glycolic acid, malic acid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, phenylacetic acid, N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid, 3-phosphoglyceric acid, glucose-6-phosphoric acid and amino acid.

[96]Salts Metals can be formed by adding an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, an alkaline earth metal, or a main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[97] In some embodiments, the metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.

[98] Ammonium salts can be formed by adding ammonia or an organic amine to a compound described herein. Non-limiting examples of suitable organic amines include triethylamine, diisopropylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, piperazole, imidazole, pyrazine, piperazine, ethylenediamine, N,N'-dibenzylethylene diamine, procaine, chloroprocaine, choline, dicyclohexylamine, and N-methylglucamine.

[99] Non-limiting examples of suitable ammonium salts include triethylamine salt, diisopropylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, morpholine salt, N-methylmorpholine salt, piperidine salt, N-methylpiperidine salt, N-ethylpiperidine salt, dibenzylamine salt, piperazine salt, pyridine salt, pyrazole salt, piperazole salt, imidazole salt, pyrazine salt, piperazine salt, ethylenediamine salt, N,N'-dibenzylethylenediamine salt, procaine salt, chloroprocaine salt, choline salt, dicyclohexylamine salt, and N-methylglucamine salt.

[100] Non-limiting examples of suitable acid addition salts include hydrochloride, hydrobromide, hydroiodate, nitrate, nitrite, sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate, bicarbonate, nicotinate, isonicotinate, lactate, salicylate, 4-aminosalicylate, tartrate, ascorbate, gentisinate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, pantothenate, acetate, propionate, butyrate, fumarate, succinate, citrate, oxalate, maleate, hydroxymaleate, methyl maleate, glycolate, malate, cinnamate, mandelic acid, 2-phenoxybenzoate, 2-acetoxybenzoate, embonate, phenylacetate, N-cyclohexylsulfamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, ethane-1,2-disulfonate, 4-methylbenzenesulfonate, naphthalene-2-sulfonate, naphthalene-1,5-disulfonate, 2-phosphoglycerate, 3-phosphoglycerate, glucose-6-phosphate and amino acid salt.

[101] The compounds described herein, such as the compounds and salts of Formulas I, Ia, Ib, BCL-2 inhibitors, and proteasome inhibitors, may in some cases exist as diastereomers, enantiomers, or other stereoisomeric forms. The compounds described herein include all diastereomeric, enantiomeric, and epimeric forms, as well as corresponding mixtures thereof. Separation of stereoisomers can be accomplished by chromatography, or by forming diastereomers and separating by recrystallization or chromatography, or any combination thereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions," John Wiley And Sons, Inc., 1981, incorporated herein by reference.) Stereoisomers can also be prepared by stereoselective synthesis.

[102] The compounds described herein, for example, the compounds and salts of formulas I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors include the use of amorphous forms as well as crystalline forms (also known as polymorphs). The compounds described herein may be in the form of pharmaceutically acceptable salts. In addition, active metabolites of these compounds having the same type of activity are included within the scope of the present invention. In addition, the compounds described herein may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the compounds described herein are also considered to be disclosed herein.

[103] The compounds described herein, for example, the compounds and salts of formulas I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors, include compounds that exhibit their natural isotopic abundance and compounds wherein one or more atoms are artificially enriched in a particular isotope having the same atomic number but an atomic mass or mass number different from the atomic mass or atomic number primarily found in nature. The scope of the present invention encompasses all isotopic variations of the compounds of the present invention, both radioactive and non-radioactive. For example, hydrogen has three naturally occurring isotopes designated ¹ H (protium), ² H (deuterium) and ³ H (tritium). Protium is the most abundant isotope of hydrogen in nature. Increasing the deuterium content may provide certain therapeutic advantages, such as increased half-life and/or in vivo exposure, or may provide a compound suitable for studying drug elimination and metabolism pathways in vivo . Isotopically enriched compounds can be produced.

[104] The compounds described herein, for example, the compounds and salts of formulas I, Ia, Ib, BCL-2 inhibitors and proteasome inhibitors, wherein the compound has carbon-carbon double bonds or carbon-nitrogen double bonds, may exist, when feasible, in the Z- or E -form (or cis- or trans -form). Moreover, some chemical structures may exist in different tautomeric forms. Unless otherwise indicated, the chemical structures described herein also include all Z- , E- and tautomeric forms.

[105]In certain cases, a compound described herein may be a prodrug, for example, where the carboxylic acid present in the parent compound is in the form of an ester. The term "prodrug" encompasses compounds that are converted to pharmaceutical agents under physiological conditions, i.e., The parent compound of the present invention. One method for producing a prodrug is to include one or more selectable moieties that are hydrolyzed under physiological conditions to form the desired molecule. In certain embodiments, the prodrug is converted by enzymatic activity of the host animal, such as enzymatic activity in certain target cells of the host animal. For example, preferred prodrugs of the present invention are esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids).

[106] Prodrugs are often useful because they may be easier to administer than the parent drug in some situations. They may be, for example, orally bioavailable, whereas the parent compound is not. Prodrugs may provide enhanced penetration of compounds into cells relative to the parent drug. For example, a prodrug may have improved cellular permeability relative to the parent compound. A prodrug may also have increased solubility in pharmaceutical formulations relative to the parent drug. In some embodiments, the prodrug format increases the lipophilicity of the pharmaceutical. In some embodiments, the prodrug format increases the effective aqueous solubility.

[107] In certain embodiments, a cyclin-dependent kinase (CDK) inhibitor, such as a compound or salt of Formula I, Ia, or Ib, can be used in combination with an inhibitor of one or more BCL-2 family proteins. Inhibitors of the BCL-2 family of anti-apoptotic proteins alter at least the cell survival cascade. Activation of apoptosis can occur through an extrinsic pathway triggered by activation of death receptors on the cell surface, or an intrinsic pathway triggered by developmental signals and a variety of intracellular stresses. This intrinsic pathway, also known as the stress pathway or mitochondrial pathway, is primarily regulated by the BCL-2 family, a class of key regulators of caspase activation consisting of anti-apoptotic (pro-survival) proteins having BH1-BH4 domains (BCL-2, i.e., the BCL-2 protein, which is a member of the BCL-2 family of anti-apoptotic proteins), BCL-xL, BCL-w, A1, MCL-1, and BCL-B); and pro-apoptotic proteins having BH1, BH2, and BH3 domains (BAX, BAK, and BOK); and proapoptotic BH3-only proteins (BIK, BAD, BID, BIM, BMF, HRK, NOXA, and PUMA) (see, e.g. , Cory et al., Nature Reviews Cancer 2:647–56 (2002); Cory et al., Cancer Cell 8:5–6 (2005); Adams et al., Oncogene 26:1324–1337 (2007)). Activation of the antiapoptotic proteins BCL-2 blocks the activation of the proapoptotic multidomain proteins BAX and BAK (see, e.g., Adams et al., Oncogene 26:1324–37 (2007)).

[108] As used herein, the term "BCL-2 inhibitor" refers to an agent that is capable of inhibiting one or more proteins of the BCL-2 family of anti-apoptotic proteins, such as BCL-2, BCL-xL, and BCL-w. In certain embodiments, a BCL-2 inhibitor of the invention selectively inhibits one protein of the BCL-2 family, such as a BCL-2 inhibitor may selectively inhibit BCL-2 but not BCL-xl or BCL-w.

[109] A BCL-2 inhibitor described herein may inhibit one or more of BCL-2, BCL-xL, and BCL-w. In certain embodiments, an inhibitor of the BCL-2 anti-apoptotic protein family inhibits BCL-2. In certain embodiments, an inhibitor of the BCL-2 anti-apoptotic protein family inhibits BCL-2 and does not inhibit other members of the BCL-2 protein family, such as does not inhibit BCL-xL or BCL-w. In certain embodiments, the BCL-2 inhibitor is a BH3 mimetic.

[110] In certain embodiments, a BCL-2 inhibitor of the invention inhibits the function of BCL-xL. In addition to inhibiting BCL-xL, the inhibitor can also interact with and/or inhibit one or more functions of BCL-2, such as BCL-xL/BCL-2 inhibitors. In certain embodiments, a BCL-2 inhibitor of the invention inhibits each of BCL-xL and BCL-w. In certain embodiments, a BCL-2 inhibitor of the invention inhibits BCL-xL, BCL-2, and BCL-w.

[111] In certain embodiments, a BCL-2 inhibitor interferes with the interaction between a BCL-2 family member of anti-apoptotic proteins and one or more ligands or receptors that the BCL-2 family member of anti-apoptotic proteins would bind in the absence of the inhibitor. In other embodiments, an inhibitor of one or more BCL-2 family members of anti-apoptotic proteins, wherein the inhibitor specifically inhibits at least one BCL-2 protein, binds only to one or more of BCL-xL, BCL-2, BCL-w, but not to other Bcl-2 family members of anti-apoptotic proteins such as Mcl-1 and BCL2A1.

[112] The binding affinity of a BCL-2 inhibitor to BCL-2 family proteins can be quantified. As an example, the binding affinity of a BCL-xL inhibitor can be determined using a competitive fluorescence polarization assay in which a fluorescent BAK peptide with a BH3 domain is incubated with the BCL-xL protein (or another BCL-2 family protein) in the presence or absence of increasing concentrations of a BCL-xL inhibitor, as described previously (see, e.g., U.S. Patent Publication 20140005190; Park et al., Cancer Res . 73:5485-96 (2013); Wang et al., Proc. Natl. Acad. Sci USA 97:7124-9 (2000); Zhang et al., Anal. Biochem. 307:70-5 (2002); Bruncko et al., J. Med. Chem . 50:641-62 (2007)). Percent inhibition can be determined using the equation: 1 - [(mP value for well - negative control)/range)] x 100%. The inhibition constant (K _i ) is determined using the formula: K _i = [ I ] ₅₀ / ([L] ₅₀ / K _d + [ P ] ₀ / K _d +1), as described in Bruncko et al., J. Med. Chem . 50:641–62 (2007) (see also Wang, FEBS Lett . 360:111–114 (1995)).

[113] Examples of BCL-2 inhibitors include ABT-263 (4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide or IUPAC, (R)-4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide) (see, e.g. , Park et al., 2008, J. Med. Chem. 51:6902; Tse et al., Cancer Res., 2008, 68:3421; International Patent Application Publication No. WO 2009/155386; U.S. Patent Nos. 7,390,799, 7,709,467, 7,906,505, 8,624,027) and ABT-737 (4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide, benzamide, 4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]- or 4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide) (see, e.g. , Oltersdorf et al., Nature , 2005, 435:677; U.S. Patent No. 7,973,161; U.S. Patent No. 7,642,260).

[114]In other embodiments, the BCL-2 inhibitor is a quinazoline sulfonamide compound (see, e.g., Sleebs et al., 2011,J. Med. Chem.54:1914). In another embodiment, the BCL inhibitor is a small molecule compound as described by Zhou et al.,J. Med. Chem.,2012, 55:4664 (see, for example, compound 21 (R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-5-ethyl-1-methyl-1H-pyrrole-2-carboxylic acid) and Zhou et al., J. Med. Chem.,2012, 55:6149 (see, for example, compound 14 (R)-5-(4-chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-ethyl-2-methyl-1H-pyrrole-3-carboxylic acid; compound 15 (R)-5-(4-chlorophenyl)-4-(3-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic acid). In other embodiments, the BCL inhibitor is a BCL-2/BCL-xL inhibitor, such as BM-1074 (see, e.g., Aguilar et al., 2013,J. Med. Chem. 56:3048); BM-957 (see, e.g., Chen et al., 2012,J. Med. Chem. 55:8502); BM-1197 (see, for example, Bai et al., PLoS One2014 Jun 5;9(6):e99404. Doi: 10.1371/journal.pone. 009904); U.S. Patent Application No. 2014/0199234; N-acylsulfonamide compounds (see, e.g., International Patent Application Publication No. WO 2002/024636, International Patent Application Publication No. WO 2005/049593, International Patent Application Publication No. WO 2005/049594, U.S. Patent No. 7,767,684, U.S. Patent No. 7,906,505). In another embodiment, the BCL-2 inhibitor is a small molecule macrocyclic compound (see, e.g., International Patent Application Publication No. WO 2006/127364, U.S. Patent No. 7,777,076). In another embodiment, the BCL-2 inhibitor is an isoxazolidine compound (see, e.g., International Patent Application Publication No. WO 2008/060569, U.S. Patent No. 7,851,637, U.S. Patent No. 7,842,815). In another embodiment, the BCL-2 inhibitor is S44563 (see, e.g., Loriot et. al.,Cell Death and Disease, 2014, 5, e1423). In one embodiment, the BCL-2 inhibitor is (R)-3-((4'-chloro-[1,1'-biphenyl]-2-yl)methyl)-N-((4-(((R)-4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)sulfonyl)-2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoline-8-carboxamide. In another embodiment, the BCL-2 inhibitor is a small molecular weight heterocyclic compound (see, e.g., U.S. Patent No. 9,018,381).

[115] In certain cases, a BCL-2 inhibitor is used in combination with a compound or salt of Formula I, Ia, or Ib. Any BCL-2 inhibitor can be used and may exhibit a synergistic effect when used in combination with a compound or salt of Formula I, Ia, or Ib. An inhibitor of the BCL-2 family may inhibit one or more members of the BCL-2 family, including Bcl-2, Bcl-xL, Bcl-w, BAK1, BAX, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL2L10, BCL2L13, BCL2L14, BOK, and MCL1. In certain embodiments, a compound or salt of Formula I, Ia, or Ib is used in combination with any of the following: navitoclax, venetoclax, A-1155463, A-1331852, ABT-737, obatoclax, TW-37, A-1210477, AT101, HA14-1, BAM7, sabutoclax, UMI-77, gambogic acid, maritoclax, MIM1, methylprednisolone, iMAC2, Bax inhibitory peptide V5, Bax inhibitory peptide P5, Bax channel blocker, and ARRY 520 trifluoroacetate. In some examples, voruciclib is used in combination with navitoclax. In certain embodiments, voruciclib is used in combination with venetoclax.

[116] In some embodiments, a BCL-2 inhibitor is used in combination with a CDK inhibitor of the invention, such as a compound of Formula I, Ia, or Ib, to treat a hematopoietic tumor. In certain embodiments, the hematopoietic tumor is a leukemia, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic lymphoma (ALL), and chronic lymphocytic leukemia (CLL). In certain embodiments, the hematopoietic tumor is a non-Hodgkin's lymphoma, such as a B-cell or T-cell lymphoma. B-cell lymphomas include diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphocytosis (SLL), mantle cell lymphoma, marginal zone B-cell lymphomas, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, and primary central nervous system lymphoma. T-cell lymphomas include precursor T-cell lymphoblastic lymphoma, peripheral T-cell lymphomas, cutaneous T-cell lymphomas, adult T-cell lymphoma subtypes: indolent chronic, acute and lymphoma, angioimmunoblastic T-cell lymphoma, estranodal natural killer/T-cell lymphoma, nasal type, enteropathy-associated intestinal T-cell lymphoma (EATL) subtypes I and II, and anaplastic large cell lymphoma (ALCL). The combinations of the present invention, for example, combinations of CDK inhibitors and BCL-2 inhibitors described herein, can be used to treat the hematopoietic malignancy described herein.

[117] The terms "treat,""treatment," or "treating," as used herein, may include alleviating, mitigating, or mitigating the symptoms of a disease or condition, preventing additional symptoms, alleviating or preventing the underlying causes of symptoms, inhibiting the disease or condition, such as stopping the progression of a disease or condition, alleviating the disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or stopping the symptoms of a disease or condition either prophylactically and/or therapeutically.

[118] The invention relates to methods for preventing or reducing the likelihood of recurrence of a cancer in an individual in need thereof. In certain embodiments, the term "prevent" or "prevention" associated with a disease or disorder may refer to a compound or combination that, in a statistical sample, reduces the incidence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample. The method comprises administering the combination therapy described herein for the treatment of minimal residual disease, and/or as maintenance therapy, such as as prolonged or extended therapy after cessation of other cancer therapy. For example, the combination therapy may be administered after cessation of other cancer therapy, such as chemotherapy, radiation therapy, and/or surgery.

[119] In some aspects, a proteasome inhibitor can be combined or used in combination with a CDK inhibitor of the invention, such as a compound or salt of any of Formulas I, Ia, or Ib. In eukaryotic cells, the ubiquitin (Ub)-proteasome cascade (UPS) involves the modification of Ub and subsequent degradation of protein substrates. The UPS controls the levels of many cellular regulatory proteins, including transcription factors, cell cycle regulatory proteins, and factors involved in various cellular processes. A common feature of the UPS pathway is that highly conserved Ub covalently binds to target proteins through a series of enzymes, namely, the Ub-activating enzyme E1, the Ub-conjugating enzyme E2, and the Ub ligase E3. E1 first activates Ub and transfers it to E2. From the E2 enzyme, Ub is transferred directly to the target protein or indirectly via the Ub ligase E3. The polyubiquitinated protein is recognized and degraded by the 26S proteasome, a large complex with multiple proteolytic activities.

[120] As used herein, the term "proteasome inhibitor" refers to an agent that blocks the action of the proteasome. Inhibition of the proteasome may prevent the degradation of proapoptotic factors such as the p53 protein, allowing the activation of programmed cell death in neoplastic cells dependent on the suppression of proapoptotic cascades.

[121] Any proteasome inhibitor can be used and may exhibit a synergistic effect when used in combination with a CDK inhibitor, such as a compound or salt of Formula I, Ia, or Ib. Non-limiting examples of proteasome inhibitors may include: bortezomib, marizomib, ixazomib, disulfiram, epigallocatechin-3-gallate, salinosporamide A, carfilzomib, ONX 0912, CEP-18770, MLN9708, epoxomicin, and MG132.

[122] In some embodiments, a proteasome inhibitor is used in combination with a CDK inhibitor of the invention, such as a compound of Formula I, Ia, or Ib, to treat a hematopoietic cancer such as large B-cell lymphoma or triple-negative breast cancer.

[123] In some aspects, the combinations described herein, such as combinations of CDK inhibitors with BCL-2 inhibitors or proteasome inhibitors, can be used to treat a cancer. The combination therapy described herein can reduce the likelihood of metastasis in an individual in need thereof. In some embodiments, the metastasis is a solid tumor. In some embodiments, the metastasis is a liquid tumor. Cancers that are liquid tumors can be tumors that arise, for example, in the blood, bone marrow, and lymph nodes, and can include, for example, leukemia, myeloid leukemia, lymphocytic leukemia, lymphoma, Hodgkin's lymphoma, melanoma, and multiple myeloma. Leukemias include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and hairy cell leukemia. Malignant tumors, which are solid tumors, include, for example, prostate cancer, testicular cancer, breast cancer, brain cancer, pancreatic cancer, colon cancer, thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi's sarcoma, skin cancer, squamous cell carcinoma of the skin, kidney cancer, head and neck cancer, throat cancer, squamous cell carcinomas that form the moist mucous linings of the nose, mouth, throat, bladder cancer, osteosarcoma, cervical cancer, endometrial cancer, esophageal cancer, liver cancer, and kidney cancer. In some embodiments, the condition treated by the methods described herein is a metastasis of melanoma cells, prostate cancer cells, testicular cancer cells, breast cancer cells, brain cancer cells, pancreatic cancer cells, colon cancer cells, thyroid cancer cells, gastric cancer cells, lung cancer cells, ovarian cancer cells, Kaposi's sarcoma cells, skin cancer cells, kidney cancer cells, head and neck cancer cells, throat cancer cells, squamous cell carcinoma cells, bladder cancer cells, osteosarcoma cells, cervical cancer cells, endometrial cancer cells, esophageal cancer cells, liver cancer cells, or kidney cancer cells.

[124] The methods described herein can also be used to inhibit the progression of metastatic malignancies. Non-limiting examples of malignant tumors include adrenal carcinoma, childhood adrenal carcinoma, AIDS-related malignancies, anal cancer, appendiceal cancer, basal cell carcinoma, childhood basal cell carcinoma, bladder cancer, childhood bladder cancer, bone cancer, brain tumor, childhood astrocytomas, childhood brainstem glioma, childhood atypical teratoid/rhabdoid tumor of the central nervous system, childhood embryonal tumors of the central nervous system, childhood germ cell tumors of the central nervous system, childhood craniopharyngioma of the brain, childhood ependymoma of the brain, breast cancer, childhood bronchial tumors, carcinoid tumor, childhood carcinoid tumor, carcinoid tumor of the gastrointestinal tract, carcinoma of unknown origin, childhood carcinoma of unknown origin, childhood cardiac tumors, cervical cancer, Childhood cervical cancer, childhood chordoma, chronic myeloproliferative disorders, colon cancer, colorectal cancer, childhood colorectal cancer, extrahepatic bile duct cancer, bile duct carcinoma in situ (DCIS), endometrial cancer, esophageal cancer, childhood esophageal cancer, childhood esthesioneuroblastoma, malignant tumor of the eye, malignant fibrous histiocytoma of bone, gallbladder cancer, gastric cancer (stomach), childhood gastric cancer, gastrointestinal stromal tumors (GIST), childhood gastrointestinal stromal tumors (GIST), childhood extracranial germ cell tumor, extragonadal germ cell tumor, gestational trophoblastic tumor, glioma, head and neck cancer, childhood head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, kidney cancer, renal cancer, Wilms' tumor, childhood tumors kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (cml), hairy cell leukemia, lip cancer, liver cancer (primary), childhood liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system (CNS) lymphoma, melanoma, childhood melanoma, intraocular melanoma, Merkel cell carcinoma, malignant mesothelioma, childhood malignant mesothelioma, Metastatic squamous cell carcinoma of the neck with occult primary midline carcinoma involving the NUT gene, oral cancer, childhood multiple endocrine neoplasia syndromes, mycosis fungoides, myelodysplastic syndromes, myelodysplastic neoplasms, myeloproliferative neoplasms, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhood oral cancer, oropharyngeal cancer, ovarian cancer, childhood ovarian cancer, epithelial ovarian cancer, low-grade ovarian cancer, pancreatic cancer, childhood pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), childhood papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, childhood pleuropulmonary blastoma, prostate cancer, rectal cancer, transitional cell carcinoma of the renal pelvis, retinoblastoma, salivary gland cancer, childhood salivary gland cancer, Ewing sarcoma family of tumors, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, childhood skin cancer, nonmelanoma skin cancer, small bowel cancer, squamous cell carcinoma, childhood squamous cell carcinoma, testicular cancer, childhood testicular cancer, throat cancer, thymoma and thymic carcinoma, childhood thymoma and thymic carcinoma, thyroid cancer, childhood thyroid cancer, transitional cell carcinoma of the ureter, urethral cancer, endometrial cancer of the corpus endometriosis, vaginal cancer, vulvar cancer, and macroglobulinemia Waldenstrom.

[125] The combination therapies described herein may be used in conjunction with other therapies, such as radiation therapy. The chemotherapy and radiation therapy regimens may include a finite number of cycles of drug therapy followed by drug-free therapy, or may include a finite time frame in which chemotherapy or radiation therapy is administered. Protocols may be determined by clinical trials, drug labels, and clinical staff in consultation with the individual being treated. The number of cycles of chemotherapy or radiation therapy, or the total duration of the chemotherapy or radiation therapy regimen, may vary depending on the individual's response to cancer therapy. The pharmaceutical agent described herein may be administered after completion of the chemotherapy or radiation therapy regimen.

[126] In some aspects, the combinations described herein can be used to treat an individual in need thereof. In some cases, the individual treated with the methods and compositions described herein can be a human. The individual treated with the methods and compositions described herein can be a non-human animal. Non-limiting examples of non-human animals can include a non-human primate, livestock, a companion animal, and a laboratory animal.

[127] In certain embodiments, the combination therapy described herein may be administered as separate agents or may be combined into a single pharmaceutical composition. For example, a combination of a CDK inhibitor, such as a compound or salt of Formula I, Ia, or Ib, and a BCL-2 inhibitor, such as venetoclax or navitoclax, may be formulated as two separate pharmaceutical compositions or the two agents may be co-formulated as a single pharmaceutical composition.

[128] In certain embodiments, a CDK inhibitor, such as a compound or salt of Formula I, Ia, or Ib, is co-formulated with a BCL-2 inhibitor or a proteasome inhibitor. In some cases, a compound of Formula I, Ia, or Ib is co-formulated with any of navitoclax, venetoclax, bortezomib, marizomib, or ixazomib, or a combination thereof.

[129] In certain embodiments, the invention provides a pharmaceutical composition, e.g., for oral or parenteral administration, comprising a compound or salt of Formula I, Ia, or Ib. In some aspects, the pharmaceutical composition comprises a compound or salt of Formula I, Ia, or Ib in an amount of at least about 1 mg to about 1000 mg, about 100 mg to about 400 mg, about 100 mg to about 200 mg, about 200 mg to about 400 mg, or about 250 mg to about 350 mg. For example, a pharmaceutical composition of the invention may contain about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, or about 500 mg of a compound of Formula I, Ia, or Ib. For a compound described herein, for example, a compound of Formula Ib, formulated into a pharmaceutical composition in the form of a salt, the amount of the compound may reflect the weight of the free base rather than the weight of the salt form. In certain embodiments, the pharmaceutical composition of a compound or salt of Formula I, Ia, or Ib does not include an additional anticancer agent, such as a BCL-2 inhibitor or a proteasome inhibitor. In certain embodiments, the pharmaceutical composition includes an additional anticancer agent, such as a BCL-2 inhibitor or a proteasome inhibitor.

[130] A therapeutically effective amount of a compound of the invention, e.g., a compound or salt of Formula I, Ia, or Ib, can be expressed as mg of compound per kg of body weight of an individual. In some cases, the dosage of the therapeutically effective amount can be at least about 0.1 mg/kg to about 20 mg/kg, such as about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, or about 20 mg/kg. For a compound described herein, for example, a compound of Formula Ib, formulated into a pharmaceutical composition in the form of a salt, the therapeutically effective amount of the compound may reflect the mass of the free base rather than the mass of the salt form.

[131] In certain embodiments, the invention relates to a pharmaceutical composition, e.g., for oral or parenteral administration, comprising a BCL-2 inhibitor, e.g., venetoclax or navitoclax. The pharmaceutical composition may comprise a BCL-2 inhibitor in an amount of at least about 1 mg to about 1000 mg, about 100 mg to about 1000 mg, about 100 mg to about 800 mg, about 200 mg to about 800 mg, or about 300 mg to about 8000 mg. For example, a pharmaceutical composition of the invention may comprise about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, about 500 mg, about 520 mg, about 540 mg, about 560 mg, about 580 mg, about 600 mg, about 620 mg, about 640 mg, about 660 mg, about 680 mg, about 700 mg, about 720 mg, about 740 mg, about 760 mg, approximately 780 mg, approximately 800 mg, approximately 820 mg, approximately 840 mg, approximately 860 mg, approximately 880 mg, approximately 900 mg, approximately 920 mg, approximately 940 mg, approximately 960 mg, approximately 980 mg, or approximately 1000 mg of a BCL-2 inhibitor, such as venetoclax or navitoclax.

[132] In certain embodiments, the invention relates to a pharmaceutical composition, e.g., for oral or parenteral administration, comprising a proteasome inhibitor, e.g., bortezomib, marizomib, or ixazomib. The pharmaceutical composition may comprise a proteasome inhibitor in an amount of at least about 0.5 mg to about 50 mg, about 1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about 10 mg, or about 1 mg to about 5 mg. For example, a pharmaceutical composition of the invention may contain about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg of a proteasome inhibitor, such as bortezomib, marizomib, or ixazomib.

[133] In certain embodiments, the formulations of the invention comprise a compound or salt of Formula I, Ia, or Ib, a BCL-2 inhibitor, or a proteasome inhibitor, wherein the compound or salt is from about 70% to about 99.99%, from about 80% to about 99.9%, from about 85% to about 99%, from about 90% to about 99%, from about 95% to about 99%, from about 97% to about 99%, from about 98% to about 99%, from about 98% to about 99.9%, from about 99% to about 99.99%, from about 99.5% to about 99.99%, from about 99.6% to about 99.99%, from about 99.8% to about 99.99%, or from about 99.9% to about 99.99% free from impurities.

[134] In certain embodiments, a pharmaceutical composition of the invention comprises both a compound or salt of Formula I, Ia, or Ib and a BCL-2 inhibitor in the amounts described herein, such as a pharmaceutical composition with 100-400 mg of a compound or salt of Formula Ib and 200 to 800 mg of a BCL-2 inhibitor, such as venetoclax.

[135] In certain embodiments, a pharmaceutical composition of the invention comprises both a compound or salt of Formula I, Ia, or Ib and a proteasome inhibitor in amounts as described herein, such as a pharmaceutical composition with 100-400 mg of a compound or salt of Formula Ib and 1-10 mg of a proteasome inhibitor, such as ixazomib.

[136] The pharmaceutical compositions described herein may be in the form of a liquid formulation, a solid formulation, or a combination thereof. Non-limiting examples of formulations may include a tablet, capsule, pill, gel, paste, liquid solution, and cream. In some cases, a therapeutic agent, such as a compound or salt of Formula I, Ia, or Ib, a BCL-2 inhibitor, or a proteasome inhibitor, may be in crystallized form. In pharmaceutical compositions containing two or more therapeutic agents, each agent may be crystallized separately and then combined, or they may be crystallized together. The compositions may contain two or more therapeutic agents in one or more physical states. For example, the composition may be a tablet containing one therapeutic agent in a solid formulation and another therapeutic agent or drug in a gel formulation. In certain embodiments, the composition is a single pharmaceutical composition comprising a compound or salt of Formula I, Ia or Ib in a first physical state and a BCL-2 family inhibitor or proteasome inhibitor in a second physical state.

[137] The compositions of the present invention may further comprise an excipient or additive. Excipients may include any and all solvents, coatings, chelating agents, flavoring agents, coloring agents, lubricants, disintegrating agents, preservatives, sweeteners, antifoaming agents, buffering agents, polymers, antioxidants, binders, diluents, and fillers (or carriers). Typically, the excipient is compatible with the therapeutic compositions of the present invention.

[138] Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicles before use. Such liquid preparations can be prepared using conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.

[139] Furthermore, the present invention encompasses anhydrous compositions and dosage forms containing the active ingredient, since water can promote the degradation of some compounds. The anhydrous compositions and dosage forms of the present invention can be prepared using anhydrous or low-moisture ingredients and low-moisture or low-humidity conditions. Compositions and dosage forms of the present invention that contain lactose can be made anhydrous if significant contact with moisture and/or humidity is expected during manufacturing, packaging, and/or storage. The anhydrous composition can be prepared and stored in a manner that maintains its anhydrous nature. Thus, anhydrous compositions can be packaged using materials that impede the effects of water so that they can be included in suitable kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foil, unit dose containers, blister packs, and strip packs.

[140] The ingredient described herein can be combined in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier can have a variety of forms depending on the desired form of administration. In preparing compositions for an oral dosage form, any of the usual pharmaceutical media can be used as a carrier, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, in the case of oral liquid preparations (such as suspensions, solutions and elixirs) or aerosols; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without the use of lactose. For example, in the case of oral solid preparations, suitable carriers include powders, capsules and tablets. If desired, tablets can be coated by standard aqueous or non-aqueous methods.

[141] Some examples of materials that serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffered saline; and (21) other non-toxic compatible substances used in the field of pharmaceutical compounding.

[142] Binders suitable for use in dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as gum arabic, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.

[143] Examples of suitable excipients for use in the compositions and dosage forms described herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof.

[144] When aqueous suspensions and/or elixirs are desired for administration, the active ingredient therein may be combined with various sweetening or flavoring agents, dyes or pigments and, if desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerol and various combinations thereof.

[145] In one embodiment, the composition may include a solubilizer to ensure high solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This may be particularly important for compositions not for oral use, such as for injection compositions. The solubilizer may also be added to enhance the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or uniform solution or dispersion.

[146] The pharmaceutical compositions described herein may be suitable for administration to an individual in need thereof. In some cases, sustained release formulations for oral administration may be prepared to achieve controlled release of the active substance in contact with body fluids in the gastrointestinal tract and to provide a substantially constant and effective level of the active substance in the blood plasma. For this purpose, the crystalline form may be embedded in a polymer matrix of a biodegradable polymer, a water-soluble polymer, or a mixture thereof, and optionally suitable surfactants. In this context, embedding may mean incorporating microparticles into a polymer matrix. Controlled release formulations are also prepared by encapsulating dispersed microparticles or emulsified microdroplets by known dispersion or emulsification methods.

[147] In some embodiments, the compositions can be formulated as a food composition. For example, the compositions can be a beverage or other liquids, solid food products, semi-solid food products, with or without an edible carrier. For example, the compositions can include black tea with the addition of any of the compositions described herein. The composition can be a dairy product with the addition of any of the compositions described herein. In some embodiments, the compositions can be formulated as a food composition. For example, the compositions can comprise a beverage, a solid food product, a semi-solid food product, or an edible carrier.

[148] In certain embodiments, pharmaceutical formulations may be in a form suitable for parenteral injection, such as a sterile suspension, solution, or emulsion in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Pharmaceutical formulations for parenteral administration include, for example, aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared, for example, as oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, isopropyl palmitate, or medium chain triglycerides, or liposomes. In preferred embodiments, the formulation for parenteral administration is an aqueous suspension.

[149] A compound described herein may be present in a composition in a concentration range, wherein the range is defined by an upper and lower value selected from the concentrations described above. For example, a compound or salt of the invention may be present in a composition at a concentration of from about 1 nM to about 100 mM, from about 10 nM to about 10 mM, from about 100 nM to about 1 mM, from about 500 nM to about 1 mM, from about 1 mM to about 50 mM, from about 10 mM to about 40 mM, from about 20 mM to about 35 mM, or from about 20 mM to about 30 mM.

[150] Methods for preparing compositions containing the compounds described herein may include formulating the compounds with one or more inert pharmaceutically acceptable excipients. Liquid compositions include, for example, solutions in which the compound is dissolved, emulsions containing the compound, or a solution containing liposomes, micelles, or nanoparticles containing the compound as described herein. These compositions may also contain minor amounts of non-toxic excipients such as wetting agents or emulsifiers, pH buffering agents, and other pharmaceutically acceptable additives.

[151] Injectable formulations may be presented in unit dosage form, for example, in ampoules or multiple-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The compositions may be presented in single-dose or multiple-dose containers, for example, in sealed ampoules or vials, and may be stored in powder form or in the freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately before use. From sterile powders, granules, and tablets of the type described above, extemporaneous injectable solutions and suspensions can be prepared.

[152] Pharmaceutical formulations for parenteral administration include aqueous and non-aqueous (oleaginous) sterile injection solutions of the active compounds, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.

[153] A composition described herein, for example, a pharmaceutical composition of a compound or salt of Formula I, Ia, or Ib, or a BCL-2 inhibitor or proteasome inhibitor, or a co-formulation of a compound of Formula I, Ia, or Ib with a BCL-2 inhibitor or proteasome inhibitor, can be administered once or more than once per day. The composition can be administered periodically (e.g., every day without a break during the treatment regimen). In some cases, the treatment regimen can be less than a week, a week, two weeks, three weeks, a month, or more than a month. In some cases, the composition of the invention is administered for at least 12 weeks. In other cases, the composition is administered over a period of one day, at least two consecutive days, at least three consecutive days, at least four consecutive days, at least five consecutive days, at least six consecutive days, at least seven consecutive days, at least eight consecutive days, at least nine consecutive days, at least ten consecutive days, or at least more than ten consecutive days. In some cases, the therapeutically effective amount may be administered once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, 10 times a week, 11 times a week, 12 times a week, 13 times a week, 14 times a week, 15 times a week, 16 times a week, 17 times a week, 18 times a week, 19 times a week, 20 times a week, 25 times a week, 30 times a week, 35 times a week, 40 times a week, or more than 40 times a week. In some cases, the therapeutically effective amount may be administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, eight times a day, nine times a day, 10 times a day, or more than 10 times a day. In some cases, the composition is administered at least twice a day. In other cases, the composition is administered at least every hour, at least every two hours, at least every three hours, at least every four hours, at least every five hours, at least every six hours, at least every seven hours, at least every eight hours, at least every nine hours, at least every 10 hours, at least every 11 hours, at least every 12 hours, at least every 13 hours, at least every 14 hours, at least every 15 hours, at least every 16 hours, at least every 17 hours, at least every 18 hours, at least every 19 hours, at least every 20 hours, at least every 21 hours, at least every 22 hours, at least every 23 hours, or at least every day.

[154] The pharmaceutical compositions of the invention may be administered either short-term or long-term. The pharmaceutical compositions of the invention may be administered as a single treatment or as a course of treatment. Treatment may be administered once daily, twice daily, three times daily, in the morning, in the evening, before bedtime, or continuously throughout the day. Treatment may be administered every day, every other day, every third day, twice a week, once a week, once every two weeks, once a month, once every six weeks, once every two months, once every three months, once every six months, once a year, once every two years, once every 5 years, or as needed.

[155] In certain embodiments, the administered dose of a drug may be temporarily reduced or temporarily withheld for a certain period of time. In certain embodiments, a patient has a drug off-day where the patient does not receive the drug or receives a reduced amount of the drug for a period of time. The drug off-day may be, for example, from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, or more than 28 days. The drug off-day may be about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. The dose reduction during the drug withdrawal period may be, for example, 10%-100% of the initial administered dose, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%. For further examples, the dose reduction may be from 10% to 100%, from 20% to 80%, from 30% to 70%, from 50% to 90%, from 80% to 100%, or from 90% to 100%.

[156] Once the patient's condition has improved, a maintenance dose may be administered if necessary. The dosage or frequency of administration, or both, may then be reduced as symptoms dictate, to a level at which improvement in the disease, disorder, or condition can be maintained.

[157] Additional routes of administration of the formulations described herein include, for example, delivery by the enteral route, including oral, gastric or duodenal feeding tube, rectal suppository, rectal enema, parenteral route, injection, infusion, intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, intracameral, epidural, subcutaneous, inhalation, transdermal, transmucosal, sublingual, buccal, topical, cutaneous, dermal, ear drops, intranasal and vaginal administration. The compounds described herein can be administered locally to the area requiring treatment, for example, by local infusion during surgery, topical application such as creams or ointments, injection, catheter, or implant. Administration can also be accomplished by direct injection into the affected tissue or organ.

[158] The duration of administration and/or the amounts administered may be determined by a physician or other type of clinical specialist. The physician or clinical specialist may monitor the individual's response to the administered compositions and adjust the dosage based on the individual's performance. For example, the dosage for individuals who exhibit reduced effects on energy regulation may be increased to achieve the desired results.

[159] In some embodiments, the combination therapy described herein may be administered together at the same time by the same route, or may be administered separately. In some embodiments, the components in the compositions may be administered using the same or different routes of administration.

[160] In some embodiments, the invention also relates to methods for producing the compositions described herein. In some embodiments, producing a composition described herein involves mixing or combining two or more components.

[161] In some embodiments, the compositions may be combined or admixed with a pharmaceutically active or therapeutic agent, a carrier, and/or an excipient. Examples of such components are described herein. The combination compositions may be formulated into unit dosages such as tablets, capsules, gelatin capsules, sustained-release tablets, and the like.

[162] In some embodiments, the composition is prepared such that a solid composition is obtained that contains a substantially uniform mixture of one or more components, such that the one or more components are uniformly dispersed in the composition, so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules.

[163] The unit dose may be packaged in a container for transport to the user. The unit dose may be packaged in a test tube, jar, box, vial, bag, tray, can, bottle, syringe, or tin.

[164] Another aspect of the invention relates to achieving desired effects in one or more individuals after administering a combination composition described herein for a certain period of time. For example, the beneficial effects of the compositions described herein can be observed after administering the compositions to an individual for 1, 2, 3, 4, 6, 8, 10, 12, 24, or 52 weeks.

[165] In certain embodiments, the combination therapies described herein can be administered via a combination treatment regimen. A combination treatment regimen can encompass treatment regimens in which administration of a compound described herein or a pharmaceutically acceptable salt thereof is initiated before, during, or after administration of a second agent described herein and is continued until any point in treatment with the second agent or after completion of treatment with the second agent. The invention also encompasses treatment methods in which a compound described herein or a pharmaceutically acceptable salt thereof and a second agent used in combination are administered simultaneously or at different time points and/or at decreasing or increasing intervals during the treatment period. Combination treatment further includes intermittent treatment that is started and stopped at different time points to facilitate clinical control of the patient.

[166] In certain embodiments, combination therapy may provide a therapeutic advantage due to differential toxicity associated with the two treatments. For example, treatment with CDK inhibitors, such as the CDK inhibitors described herein, may result in particular toxicities that are not observed with an anti-cancer agent, such as a BCL-2 inhibitor or a proteasome inhibitor, and vice versa. As such, this differential toxicity may allow each treatment to be administered at a dose at which this toxicity is non-existent or minimal, such that the combination therapy provides a therapeutic dose while avoiding the toxicity of each component of the combination agents. Moreover, when the therapeutic effects achieved by the combination therapy are synergistic, the doses of each agent can be further reduced, thereby further reducing the toxicity associated with them.

[167] The compounds described herein, or pharmaceutically acceptable salts thereof, and combination therapies can be administered before, during, or after the onset of a disease or condition, and the timing of administration of compositions containing the compound varies. The compounds described herein can be used as a prophylactic agent and can be administered continuously to individuals predisposed to developing conditions or diseases to prevent the onset of the disease or condition. The compounds described herein and compositions thereof can be administered to an individual in the presence of, or as early as possible after the onset of, symptoms. A compound described herein can be administered as early as is practical after the onset of a disease or condition is detected or suspected, and for a period of time necessary to treat the disease.

EXAMPLES

[168] The following examples are provided to illustrate various embodiments of the invention and do not limit the present invention in any way. The given example, as well as the methods described in this description, reflect preferred embodiments, are illustrative and do not limit the scope of the invention. Changes thereto and other applications that are encompassed by the essence of the invention, as defined by the scope of the claims, will become clear to those skilled in the art.

[169] For all in vitro studies of the mechanism of action of drugs, the materials and methods described below were used.

[170] The cell lines presented in Table 1 , either attached or in suspension, were processed in 6-well dishes or T10 flasks, respectively, at a seeding density of one million per ml before processing in total volumes of 3-10 ml. All agents used for in vitro cell treatment were formulated in water/DMSO to achieve the required concentrations (as indicated in the respective figure legends), with DMSO concentrations not exceeding 0.01%. Drug sources are listed in the table under the column "CIVO microinjection studies". Cells were harvested at experimental time points: 2, 6, or 24 hours after treatment, as indicated in the respective figures, by first washing the cells twice with ice-cold PBS in 15-ml falcon tubes, centrifuging at 1000 rpm for 5 min, followed by lysis of the corresponding cell pellets as described in the section below. Note: Drug-resistant NUDHL1 Dox 1000 nM and Toledo Dox 1000 nM cell lines were generated at Presage by culturing the parental lines in medium with stepwise increasing concentrations of doxorubicin over several passages. These cells were used for xenograft generation when resistance to 1 μM Dox was achieved.

Table 1

DLBCL cell lines Name of cell line Supplier Cultivation conditions at 5% CO2/37°C RIVA (or RI-1) DSMZ, Germany RPMI 1640 (Thermofisher)+10% FBS SU-DHL-4 ATCC, USA RPMI 1640 (Thermofisher)+10% FBS U2932 DSMZ RPMI 1640 (Thermofisher)+10% FBS NUDHL1 DSMZ RPMI 1640 (Thermofisher)+10% FBS TOLEDO DSMZ RPMI 1640 (Thermofisher)+10% FBS OCI Ly10 UHN, Princess Margaret Hospital, Canada IMDM (Thermofisher) + 20% human serum (Valley biomedical) - fresh medium each passage TNBC cell line HCC1187 ATCC RPMI 1640 (Thermofisher)+10% FBS AML cell lines SKM1 DSMZ RPMI 1640 (Thermofisher)+10% FBS MV-4-11 ATCC IMDM (Thermofisher) + 20% human serum (Valley biomedical) OCI AML3 DSMZ RPMI 1640 (Thermofisher)+10% FBS

[171] For all studies using Western blot analysis, the materials and methods described below were used.

[172] Protein lysates were prepared by adding ice-cold 1X RIPA buffer (from 10X, Millipore) with 1X-3X Halt protease and phosphatase inhibitor cocktail (100X, Pierce) to cell pellets, followed by pulsed sonication (~3-5 s) three times. Lysates were cleared by centrifugation at 14,000 rpm for 15-20 min, and the supernatant (lysate) was collected for further analysis. To prepare lysates from cryopreserved tumor graft tissue, tissue was first homogenized using a hand-held tissue homogenizer (Cole Palmer) in the lysis buffer described above, followed by pulsed sonication and centrifugation as described above. The above steps were performed under ice-cold conditions. Protein samples were quantified using the Bradford assay (Biorad) at dilutions of 1:10 or higher using appropriate blank controls with 1x RIPA. 20-25 micrograms of protein from each sample were subjected to SDS-PAGE using 4-12% Bis-Tris BOLT gels (Thermofisher) and transferred to membranes with a pore size of either 0.45 or 0.2 micrometers (depending on the molecular size of the protein) and either nitrocellulose (NC) or pvdf (Thermofisher) using the manufacturer's protocol, blocked for one hour in 5% milk at room temperature, followed by the addition of primary antibodies (Table 2) at 4°C overnight and appropriate HRP-conjugated secondary antibodies (Jackson Immunoresearch, dilutions 1:10,000-1:20,000) for 1 hour at room temperature. After each antibody incubation, three washes with PBS/0.01% Tween-20 (5-10 min each) were performed. Chemiluminescent ECL substrate (Pierce or GE) and autoradiographic film (Thermofisher) were used to detect protein signals.

Table 2

Experiments with DLBCL and AML Primary antibody View Supplier/catalog number Dilution/thinner cPARP Rabbit CST 5625 1:1000 for NC (or 1:4000 for PVDF membranes) in 5% milk MCL1 Rabbit CST 39224; CST 5453 1:1000 for NC (or 1:4000 for PVDF membranes) in 5% milk cMYC Rabbit Ab32072 1:10000 (PVDF) in 5% milk P53 Rabbit CST 2527 and CST 9282 1:4000 (PVDF) in 5% milk phospho RNA polymerase II (Ser 2) Rat Active Motif 61083 1:1000 (PVDF) in 5% BSA/PBS Beta-actin (load control) Mouse CST 12262 1:40000 (PVDF) in 5% milk BCL2 Mouse Dako M0887 1:5000 (PVDF) in 5% milk Experiments with TNBC Primary antibody View Supplier Breeding XBP1s Rabbit CST 12782 1:1000 (NC) in 5% milk XIAP Rabbit CST 2042 1:1000 (NC) in 5% milk MCL1 Rabbit CST 94296 1:1000 (NC) in 5% milk Beta-actin (load control) Mouse Sigma A5441 1:10,000 (NC) in 5% milk phospho P38MAPK Rabbit CST 4631 1:1000 (NC) in 5% BSA phospho JNK Rabbit CST 9251 1:1000 (NC) in 5% BSA General p38 Rabbit CST 9212 1:1000 (NC) in 5% BSA General JNK Rabbit CST 9225 1:1000 (NC) in 5% BSA NOXA Mouse Millipore OP180 clone 1:500 (NC) in 5% milk BAK Rabbit Ab32371 1:1000 (NC) in 5% BSA pIRE1a Rabbit Novus NB100 2323 1:1000 (NC) in 5% BSA GADD34 Rabbit Protein Tech 1:1000 (NC) in 5% BSA

[173] For all in vivo studies, the materials and methods described below were used.

[174]All The work on mice was approved by the IACUC Board of Presage Biosciences, Seattle, WA. All procedures were performed under anesthesia, and every effort was made to minimize pain and suffering. None of the mice involved in the study became ill or died before the end of the experiment, and all mice treated with drugs as described below underwent routine health monitoring and were humanely euthanized at the end of the experiment. Subcutaneous flank xenografts were generated using the cell lines/mouse strains listed in the table below (Table 3). 200 μl of cell suspension at a 1:1 ratio with Matrigel (Corning) were injected into the right flank using 1 ml syringes (BD 309659) and 27G needles (BD 305109).

Table 3

DLBCL and AML xenografts cell line Source Number of inoculated cells per mouse Line of mice RIVA (or RI-1) DSMZ 5×10 ⁶ million NOD SCID (Envigo) SU-DHL-4 ATCC 5×10 ⁶ million SCID Beige (Charles River/Envigo) U2932 DSMZ 5×10 ⁶ million NOD SCID (Envigo) SKM1 (AML) DSMZ 5×10 ⁶ million NOD SCID (Envigo) NUDHL1 DSMZ 5×10 ⁶ million NOD SCID (Envigo) TOLEDO DSMZ 5×10 ⁶ million NOD SCID (Envigo) TNBC xenografts HCC1187 ATCC 5×10 ⁶ million Athymic nudes (Envigo)

[175] For all CIVO microinjection studies, the materials and methods described below were used.

[176] Mice were included in CIVO drug studies when tumor volume reached approximately 1000 mm ³ . Microinjection studies were performed using the CIVO device as described previously (Klinghoffer et al, Science Translational Medicine 2015). The device was equipped with 6 injection needles and set for an injection length of 6 mm and a total delivery volume of 3 µL. A fluorescent imaging marker (FTM) was added to each drug reservoir in the delivery vehicle along with each drug or drug combination. All microdoses were equivalent to or less than the doses that would be allowed under FDA guidance for Investigational New Drug (IND) testing and due to the solubility of the drug in the vehicle. The total amounts of drugs injected are listed in Table 4.

Table 4

Microinjections of DLBCL CIVO Medicine Quantity Source Compound Vorutsiklib 15.2 mcg Presage 5% DMSO/water Venetoclax (ABT-199) 26 ng Chemietek 5% DMSO/water Navitoclax (ABT-263) 29.2 ng Chemietek 5% DMSO/water TNBC CIVO microinjections Medicine Quantity Source Compound Ixazomib (screening) 15 mcg Selleck 20% HPbCD Doxorubicin (screening) 5.1 mcg Pfizer 0.9% saline solution Carboplatin (screening) 15 mcg Selleck Water 5-FU (screening) 2 mcg Selleck 5% DMSO/water Abraxane (screening) 14.25 Celgene 0.9% saline solution Olaparib (screening) 13.05 mcg Selleck 5% DMSO/water Trametinib (screening) 18.5 mcg Selleck 5% DMSO/water Everolimus (screening) 7.2 mcg Selleck 5% DMSO/water BEZ 235 (screening) 14.1 mcg Selleck 5% DMSO/water PU H71 (screening) 15.4 mcg Selleck Water Erlotinib (screening) 2.95 mcg Selleck 5% DMSO/water Sunitinib (screening) 3 mcg Selleck 5% DMSO/water Sorafenib (screening) 13.95 mcg Selleck 5% DMSO/water Dasatanib (screening) 14.6 mcg Selleck 5% DMSO/water Bortezomib (screening) 1.2 mcg Chemietek 5% DMSO/water Dinaciclib (screening) 11.88 mcg Selleck 5% DMSO/water Flavopiridol (screening) 12.1 mcg Sigma 5% DMSO/water Palbociclib (screening) 13.44 mcg Chemietek 5% DMSO/water Marizomib 94 ng Triphase 5% DMSO/0.9% saline

[177] For all studies of systemic drug efficacy, the materials and methods described below were used.

[178] Mice were included in the study when tumors reached a mean volume of 150-200 mm ³ . Tumor volume was calculated as V = length × width × height, all three dimensions were measured using digital thickness gauges simultaneously with body weight. The study was terminated when any of the three measured dimensions exceeded 2 cm, the volume exceeded 2500 mm ³ , ulceration was observed, or body weight decreased by more than 20%.

Tumor growth inhibition % (TGI) is defined as:

where sizes are averaged across tumors in the respective groups. The Wilcoxon rank-sum test or Mann-Whitney test were used as statistical tests to determine differences between groups.

[179]Doses of drugs, Routes of administration, administration regimens, and formulation protocols are described below. Instech feeding tubes, Ref: FTP-20-38, were used for oral tube feeding, and BD insulin syringes were used for intravenous feeding.

DLBCL Research

- RIVA : Voruciclib 200 mg/kg (po) 6 administrations/1 rest and ABT 199 (po) 1 mg/kg 2x/week for 4 weeks

- SUDHL4 : Voruciclib 200 mg/kg (po) 6 doses/1 rest and ABT 199 (po) 25 mg/kg 6 doses/1 rest for 4 weeks

- U2932: Voruciclib 200 mg/kg (po) 6 administrations/1 rest and ABT 199 (po) 10 mg/kg 2x/week for 4 weeks

AML research

- SKM1: Voruciclib 200 mg/kg (po) 6 administrations/ 1 rest + ABT 199 (po) 10 mg/kg 6 administrations/ 1 rest for 3 weeks

TNBC Research

- HCC1187: Voruciclib 200 mg/kg (po) 5th administration/2nd rest + ixazomib/MLN2238 (po) 3 mg/kg days 3, 6, 10, 13 for 2 weeks

- HCC1187: palbociclib 120 mg/kg (po) QD x 14 + bortezomib 0.42 mg/kg (iv), days 1, 4, 8, 11 for 2 weeks

- HCC1187: Voruciclib 200 mg/kg (po) + bortezomib 0.42 mg/kg (iv), days 1, 4, 8, 11 for 2 weeks

Example 1. Voruciclib is a potent inhibitor of cyclin-dependent kinase-9 (CDK9)

[180] Voruciclib is believed to be a potent inhibitor of CDK4/6. Voruciclib's activity against 38 different kinases was tested in comparison to flavopiradol, a known CDK9 inhibitor. Figure 1 shows the structures of voruciclib and flavopiridol, as well as a comparison of the inhibition profiles of voruciclib and flavopiradol. Figure 1 demonstrates that, similar to flavopiradol, voruciclib is a potent inhibitor of CDK9 (K _i < 10 nM). However, voruciclib is more specific for CDKs than flavopiradol. Figure 1 shows that flavopiradol is a potent inhibitor of the serine/threonine protein kinases MAK and ICK, while voruciclib does not exhibit potent inhibition of either MAK or ICK. MAK and ICK are associated with intestinal epithelial cells and may be responsible for severe diarrhea associated with flavopiradol treatment. Voruciclib, on the other hand, may exhibit fewer gastrointestinal side effects. DiscoveRx and Thermofisher Screening: Percentage inhibition of kinase activity for 468 kinase assays in the DiscoveRx ScanMax kinase panel and 414 kinase assays in the ThermoFisher SelectScreen kinase panel at 10 μM and 50 nM. Test compound is preweighed in Presage into silanized amber glass vials with screw caps and shipped to DiscoveRx and Reaction Biology. For ThermoFisher, the compound is weighed and dissolved at 10 mM in DMSO before shipping under ambient conditions. Upon receipt, vials are stored at -20°C until use. The powder is dissolved in DMSO at a concentration of 10 mM.

[181] Figure 2 shows the single-digit nM potency of voruciclib against CDKs 1, 4, 6, and 9, with CDK9 inhibition being most potent. Reaction Biology Profiling: The objective of this study was to rank-order the sensitivity of 48 kinases to voruciclib hydrochloride. Testing was performed at Reaction Biology Corp. Kinase activity was quantified using a filter binding assay with radioactive γ-33P-ATP as the phosphate donor. The ATP concentration was close to the Km for the respective kinase. For each kinase, the IC50 value was calculated from the 10-point concentration curve for the test compound and converted to Ki values. The 48 kinases tested in this example were identified in previous screening experiments as the most promising candidates.

Example 2. Voruciclib exhibits a synergistic effect in combination with the Bcl-2 family inhibitor venetoclax (ABT-199)

[182]Cells diffuse large B-cell lymphoma (DLBCL) NU-DHL-1 were treated with vehicle, voruciclib alone, venetoclax (ABT-199) alone, or a combination of voruciclib and venetoclax.Fig. 3A-3DNU-DHL-1 cells stained for the anti-apoptotic protein myeloid leukemia cell differentiation protein-induced protein Mcl-1 (MCL-1) (shown in red).Fig. 3ACells treated with vehicle only are shown.Fig.3B:voruciclib alone reduced MCL-1 expression, as shown by the increase in darker areas in the fragment301Venetoclax alone increased MCL-1 expression, as shown by the light areas in the fragment.302 Fig. 3CThe combination of voruciclib and venetoclax resulted in a significant reduction in MCL-1 expression, as shown inFig. 3D, shown by dark areas in the fragment303.

[183] Figures 4A-4D demonstrate the correlation between MCL-1 suppression and synergistic induction of apoptosis. Diffuse large B-cell lymphoma (DLBCL) NU-DHL-1 cells were treated with vehicle, voruciclib alone, venetoclax (ABT-199) alone, or a combination of voruciclib and venetoclax. Cells were stained for cleaved caspase-3 (CC3) (shown in red) as a marker of apoptosis. Figure 4A shows cells that were treated with vehicle alone. Voruciclib alone, as shown in Figure 4B , showed slightly increased CC3 expression, as shown by the open areas in Figure 4O1 . Similarly, venetoclax alone, as shown in Figure 4C , showed a slight increase in CC3 expression, as shown by the open areas in Figure 4O2 . The combination of voruciclib and venetoclax in Fig. 4D demonstrated a synergistic increase in CC3 expression, as indicated by the large bright region at fragment 403 , suggesting increased induction of apoptosis in treated cells.

[184] Figures 6A-6E demonstrate that the synergistic effect of voruciclib and venetoclax on apoptosis is reproducible across multiple DLBCL models. Briefly, RIVA, Toledo, Toledo/Dox1000, NUDHL, and NUDHL/Dox1000 cells were treated with voruciclib alone, venetoclax alone, or voruciclib and venetoclax in combination, as shown in Figure 6A, Figure 6B, Figure 6C, Figure 6D , and Figure 6E , respectively. Voruciclib alone and venetoclax alone had little effect on CC3 expression, however, the combination of voruciclib and venetoclax demonstrated synergistic induction of CC3 expression in all five DLBCL models.

[185] Figure 7 shows a proposed model for this synergistic effect. Voruciclib, through inhibition of MCL-1 via CDK9, can direct cells to apoptosis when combined with Bcl-2 inhibition.

Example 3. Voruciclib exhibits a synergistic effect in combination with the Bcl-2 family inhibitor navitoclax

[186]Cells Burkitt lymphoma Ramos were treated with vehicle, voruciclib alone, navitoclax (ABT-263) alone, or a combination of voruciclib and navitoclax.Fig. 5A-5DShown are Ramos Burkitt lymphoma cells stained for cleaved caspase-3 (CC3) (shown in red) as a marker of apoptosis.Fig. 5ACells treated with vehicle alone are shown. Voruciclib alone, as shown inFig. 5B, showed a slight increase in CC3 expression, as shown by the light areas in the fragment501. Similarly, venetoclax alone, as shown inFig. 5C, showed a slight increase in CC3 expression, as shown by the light areas in the fragment502The combination of voruciclib and navitoclax demonstrated a marked increase in CC3 expression, as shown by the large number of bright areas in the fragment.503 Fig. 5D, indicating increased induction of apoptosis in treated cells.

Example 4: Voruciclib exhibits a synergistic effect in combination with a proteasome inhibitor

[187] Drug screening was performed to test multiple drugs simultaneously. Figure 8A shows a list of the various compounds that were injected into the HCC1187 triple-negative breast cancer (TNBC) xenograft model. Figure 8B shows the mean % change in MCL-1 induction in the xenografts. The proteasome inhibitor bortezomib showed an increase in MCL-1 expression. Figure 8C shows vehicle-treated xenografts. Figure 8D shows bortezomib-treated xenografts stained for MCL-1 expression (shown in red). Bortezomib significantly increased MCL-1 expression in these cells, as shown by the increase in bright areas in the 801 region.

Example 5. Voruciclib exhibits a synergistic effect in combination with the proteasome inhibitor marizomib

[188] DLBCL NudHL1 xenografts were treated for 6 hours with vehicle, voruciclib alone, marizomib alone, or voruciclib and marizomib in combination. Figures 9A-9D show cells that were treated as described above and stained for cleaved caspase-3 (CC3) as a marker of apoptosis (shown in red). Figure 9A shows cells that were treated with vehicle alone. Cells treated with voruciclib alone, as shown in Figure 9B , had either little or no increase in CC3 expression, as indicated by the small number of bright areas in plot 901 . Similarly, cells treated with marizomib alone, as shown in Figure 9C , had either little or no increase in CC3 expression, as indicated by the small number of bright areas in plot 902 . Cells treated with the combination of voruciclib and marizomib showed a marked increase in CC3 expression, as indicated by the large number of bright regions in panel 903 of Fig. 9D , suggesting a synergistic effect on the induction of apoptosis in these cells.

Example 6. Voruciclib exhibits a synergistic effect in combination with the proteasome inhibitor bortezomib

[189] Briefly, DLBCL NudHL1 xenografts were treated for 6 hours with vehicle, voruciclib alone, bortezomib alone, or voruciclib and bortezomib in combination. Figures 10A-10D show cells treated as described above and stained for cleaved caspase-3 (CC3) as a marker of apoptosis (shown in red).

[190] Figure 10A shows cells that were treated with vehicle alone. Cells treated with voruciclib alone, as shown in Figure 10B , had either little or no increase in CC3 expression. Similarly, cells treated with bortezomib alone, as shown in Figure 10C , had either little or no increase in CC3 expression, as indicated by the small number of bright areas in plot 1001 . Cells treated with the combination of voruciclib and bortezomib showed a marked increase in CC3 expression, as indicated by the large number of bright areas in plot 1002 of Figure 10D , suggesting a synergistic effect on the induction of apoptosis in these cells.

[191] Figure 11 shows a similar experiment performed in the HCC1187 triple-negative breast cancer (TNBC) xenograft model. Briefly, cells were treated with vehicle, voruciclib alone, bortezomib alone, or voruciclib and bortezomib in combination. Cells were then stained for CC3 expression (shown in red) as a marker of apoptosis. Figure 11A shows cells that were treated with vehicle alone. Cells treated with voruciclib alone, as shown in Figure 11B , had either little or no increase in CC3 expression. Similarly, cells treated with bortezomib alone, as shown in Figure 11C , had either little or no increase in CC3 expression. Cells treated with the combination of voruciclib and bortezomib showed a marked increase in CC3 expression, as indicated by the large number of bright regions in panel 1101 of Fig. 11D , suggesting a synergistic effect on the induction of apoptosis in these cells.

[192] Triple-negative breast cancer (TNBC) xenograft-bearing mice model HCC1187 were administered vehicle, voruciclib (200 mg/kg), bortezomib (0.42 mg/kg), or voruciclib (200 mg/kg) and bortezomib (0.42 mg/kg) in combination. Tumor volume was measured in each mouse over time and the results are shown in Figures 12A-12D . Mice administered the combination of voruciclib and bortezomib ( Figure 12D ) showed a significant reduction in normalized tumor volume over time compared to mice administered voruciclib alone ( Figure 12B ) or bortezomib alone ( Figure 12C ). Figure 12E shows a Western blot for MLC-1 protein expression. Voruciclib-treated cells had decreased MCL-1 protein expression compared to untreated cells, while bortezomib-treated cells had increased MCL-1 expression compared to untreated cells. Furthermore, voruciclib reduced the bortezomib-induced increase in MCL-1 expression.

[193] Figure 13 shows that body weight in mice was not affected by administration of voruciclib alone, bortezomib alone, or voruciclib and bortezomib in combination. These data may indicate that combination therapy with voruciclib and bortezomib may exhibit little or no toxicity.

[194] Furthermore, voruciclib antagonizes bortezomib-induced activation of the pro-survival proteins MCL-1 and the E3 ubiquitin protein ligase XIAP. Figure 15A shows a putative model of CDK9 inhibition by voruciclib. Figure 15B shows MCL-1 and XIAP protein levels in cells treated with voruciclib alone, bortezomib alone, or voruciclib and bortezomib in combination. Western blot demonstrates that voruciclib reduces bortezomib-induced activation of both MCL-1 and XIAP protein in these cells.

Example 7. Palbociclib, a CDK 4/6 inhibitor, does not demonstrate a synergistic effect in combination with bortezomib

[195] Triple-negative breast cancer (TNBC) HCC1187 xenograft-bearing mice were administered vehicle, palbociclib (120 mg/kg), bortezomib (0.42 mg/kg), or palbociclib (120 mg/kg) and bortezomib (0.42 mg/kg) in combination. Tumor volume was measured in each mouse over time and the results are shown in Figure 14. Unlike voruciclib, palbociclib did not show a synergistic effect on tumor volume when combined with bortezomib. Palbociclib is a specific inhibitor of CDK4/6, while voruciclib targets CDK9. Thus, this difference may be CDK9 specific.

Example 8. The endoplasmic reticulum (ER) stress response cascade may play a role in the synergistic effects of voruciclib in combination with bortezomib

[196] Figure 16A shows a tissue section treated with bortezomib. Bortezomib induced apoptosis in some cells, while some cells were resistant to bortezomib treatment. Cells were stained for the ER chaperone protein GRP178/BiP. Figure 16B shows that cells resistant to apoptosis by bortezomib express GRP178/BiP.

[197] Next, HCC1187 triple-negative breast cancer cells were treated with voruciclib alone, bortezomib alone, tunicamycin alone (an ER stress inducer), or in combination. Tunicamycin alone strongly induced X-box binding protein 1 (XBP1), an ER stress protein from the IRE1α component of the ER stress cascade, at 6 h. Furthermore, both tunicamycin and bortezomib strongly induced XBP1 expression at 24 h (Figure 18B) . The combination of voruciclib with either bortezomib or tunicamycin reduced XBP1 activation. These results indicate that voruciclib can inhibit the IRE1α component of the ER stress pathway (Figure 18B) . Voruciclib also reduces bortezomib-induced XBP1 transcriptional induction (Figs. 19A–19B) . HCC1187 cells were treated with voruciclib (1.5 μM), bortezomib (10 nM), the IRE1a endoribonuclease inhibitor STF083010 (Millipore) (60 μM), or tunicamycin (Sigma) (100 nM) for 4 h. mRNA was collected using a Qiagen RNAeasy Kit and quantified using a Nanodrop spectrophotometer. 1 μg of mRNA from each condition was used to prepare cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the manufacturer's protocol. 100 ng of cDNA was used to perform PCR assays to amplify unspliced or spliced forms of XBP1 (Thermofisher PCR Supermix) - 94°C for 2 min, (94°C for 15 s, 55°C for 30 s, 72°C for 1 min) x 30 cycles, 72°C for 7 min, hold at 4°C. Spliced and unspliced forms of XBP1 were detected.

Example 9. Voruciclib exhibits a synergistic effect in combination with the proteasome inhibitor ixazomib

[198] Figures 20A-20D show the HCC1187 triple-negative breast cancer (TNBC) xenograft model. Briefly, cells were treated with vehicle, voruciclib alone, ixazomib alone, or voruciclib and ixazomib in combination. Cells were then stained for CC3 expression (shown in red) as a marker of apoptosis.

[199] Figure 20A shows cells that were treated with vehicle alone. Cells treated with voruciclib alone, as shown in Figure 20B , had either little or no increase in CC3 expression, as indicated by the small number of bright areas in plot 2001 . Similarly, cells treated with ixazomib alone, as shown in Figure 20C , had either little or no increase in CC3 expression, as indicated by the small number of bright areas in plot 2002 . Cells treated with the combination of voruciclib and ixazomib showed a marked increase in CC3 expression, as indicated by the large number of bright areas in plot 2003 of Figure 20D , suggesting a synergistic effect on the induction of apoptosis in these cells.

[200] Various CDK inhibitors were tested in combination with ixazomib, as shown in Figures 21A and 21B . Only voruciclib showed a synergistic effect on tumor cell apoptosis in combination with ixazomib, as shown by the bright areas in panels 2101 and 2102 . None of vehicle alone, ixazomib alone, palbociclib and ixazomib, dinaciclib and ixazomib, and flavopiradol and ixazomib showed the synergistic effect on tumor cell apoptosis that the combination of voruciclib and ixazomib did.

Example 10. Voruciclib exhibits a synergistic effect in combination with venetoclax

[201] Figures 22A-B illustrate the synergy of voruciclib and venetoclax in the DLBCL SU-DHL-4 model. Cleaved PARP (cPARP) can be used as a biomarker of apoptosis. To evaluate the efficacy of the two-agent combination of voruciclib and venetoclax, low doses of each (150 nM for voruciclib and 20 nM for venetoclax) were tested as single agents or in combination in the DLBCL SUDHL4 model. Cells were exposed to drug for 24 h before cell lysis and Western blotting using antibodies specific for the cleaved form of PARP, MCL-1, C-MYC, and beta-actin (as a protein loading control) in Figure 22A and BCL2 as an additional protein loading control in Figure 22B . Figure 22A illustrates that voruciclib suppresses MCL1 at 1.5 μM in the DLBCL model.

[202] Figure 22B shows the use of a 0.15 μM concentration of voruciclib, which was 1/10 the concentration shown to be effective in inducing apoptosis as a single agent. This level of voruciclib retains some efficacy in suppressing MCL-1 expression (see lanes 1 and 2 of Figure 22B ), while exposure of cells to venetoclax induces MCL-1 expression (see lanes 1 and 3 of Figure 22B ). Low exposure to voruciclib also suppresses venetoclax-induced MCL-1 (see lanes 3 and 4 of Figure 22B ).

[203] Figures 23A-C illustrate the synergy of voruciclib and venetoclax in the SU-DHL-4 model, the OCI Ly10 model, and the U2932 model of DLBCL. Cleaved PARP (cPARP) can be used as a biomarker of apoptosis. To evaluate the efficacy of the two-agent combination of voruciclib and venetoclax, low doses of each were tested as single agents or in combination in three DLBCL models. Cells were exposed to drug for 24 h before cell lysis and Western blotting using antibodies specific for the cleaved form of PARP and beta-actin (as a protein loading control).

[204] Figure 23A illustrates that the combination of 0.15 μM voruciclib and 20 nM venetoclax exhibits synergy in inducing apoptosis in the SUDHL4 DLBCL model, which is not induced by either compound alone. Figure 23B illustrates that the combination of 5 μM voruciclib and 50 nM venetoclax exhibits synergy in inducing apoptosis in the OCI Ly10 DLBCL model, where neither compound alone significantly induced apoptosis. Figure 23C illustrates that the combination of 5 μM voruciclib and 50 nM venetoclax exhibits synergy in inducing apoptosis in the U2932 DLBCL model, where the combination of the two compounds is significantly superior to the addition of each compound alone.

Example 11. Voruciclib suppresses MCL1 expression in a DLBCL xenograft mouse model

[205] Figure 24 illustrates that voruciclib downregulates MCL1 expression in mouse DLBCL tumor xenografts. Mice bearing DLBCL tumor xenografts (OCILY10) were administered voruciclib or vehicle control by gavage every day for 5 days, and tumors were harvested for MCL-1 expression analysis 4 h after the last dose. Tumor samples were cut in half, with one half solubilized in lysis buffer for Western blot analysis and the other half fixed in formalin and prepared for immunohistochemistry. Western blotting of tumor lysates was performed for 3 individual mice per treatment group using antibodies specific for MCL-1 or beta-actin (protein loading control). The graph shows that voruciclib as a single agent suppressed MCL1 expression in DLBCL xenograft tumors at 100 mg/kg.

Example 12. Voruciclib and venetoclax inhibit the growth of DLBCL tumor xenografts in mice

[206] Figures 25A-B illustrate the effect of the combination of voruciclib and venetoclax on inhibiting the growth of DLBCL tumor xenografts. Immunodeficient NOD SCID mice were implanted with RIVA (activated B cells or ABC), a model of DLBCL. The tumor xenografts were allowed to grow in the NOD SCID host until they reached 200 ^mm3 , after which the xenograft-bearing animals were randomly assigned to 4 treatment groups: 1. Vehicle control; 2. Voruciclib 200 mg/kg; 3. Venetoclax 1 mg/kg; and 4. The combination of voruciclib 200 mg/kg and venetoclax 1 mg/kg. Voruciclib was dosed via gavage every day for 6 days of the week, with one day of rest between courses. Venetoclax was dosed via gavage on days 1 and 4 of each weekly course. Treatment effects were assessed by a technician blinded to treatment status in each study group, measuring tumor volume twice weekly. N = 5-6 animals per treatment group.

[207] Figure 25A shows plots of mean tumor volume (mm ³ ) for each of the 4 study groups: vehicle, voruciclib alone, venetoclax alone, and the combination of voruciclib and venetoclax. Administration of the individual agents demonstrated a slower reduction in tumor volume across study days. Combination administration demonstrated a significantly greater reduction in tumor volume, with a more than 2-fold reduction in mean tumor volume after 3 weeks of the study.

[208] Figure 25B illustrates tumor images from animals in each of the 4 study groups: vehicle, voruciclib as a single agent, venetoclax as a single agent, and the combination of voruciclib and venetoclax. The tumor size in the vehicle group was the largest, as shown in panel 2501. The tumor in the voruciclib group as a single agent was smaller, as shown in panel 2502. The tumor in the venetoclax group as a single agent was also smaller than that in the vehicle group, as shown in panel 2503. The tumor in the combination therapy group, shown in panel 2504 , was significantly smaller than that in the vehicle group or either of the single agent therapies, indicating a synergistic effect.

[209] Figures 26A-B illustrate the effect of the combination of voruciclib and venetoclax on the growth of DLBCL tumor xenografts. Immunodeficient NOD SCID mice were implanted with U2932, a model of DLBCL. Tumor xenografts were allowed to grow in the NOD SCID host until they reached 200 mm ³ , after which the xenograft-bearing animals were randomly assigned to 4 treatment groups: 1. Vehicle control; 2. Voruciclib 200 mg/kg; 3. Venetoclax 10 mg/kg; and 4. The combination of voruciclib 200 mg/kg and venetoclax 10 mg/kg. Voruciclib was dosed via gavage every day for 6 days of the week, with one day of rest between courses. Venetoclax was dosed via gavage on days 1 and 4 of each weekly course. Treatment effects were assessed by a technician blinded to treatment status in each study group, measuring tumor volume twice weekly. N = 5-6 animals per treatment group.

[210] Figure 26A shows plots of mean tumor volume (mm ³ ) for each of the 4 study groups. Individual administration of the agents demonstrated a slower decrease in tumor volume across study days. Combined administration demonstrated virtually no tumor growth, with the mean tumor volume remaining constant throughout the 29-day study period.

[211] Figure 26B shows plots of mean body weight in grams (g) for individuals in each of the 4 study groups. All 4 study groups demonstrated stable and consistent body weight, which is a key indicator of health and safety.

[212] Figures 27A-B illustrate the effect of the combination of voruciclib and venetoclax on the growth of DLBCL tumor xenografts. Immunodeficient NOD SCID mice were implanted with NUDHL1, a model of DLBCL. Tumor xenografts were allowed to grow in the NOD SCID host until they reached 200 ^mm3 , after which the xenograft-bearing animals were randomly assigned to 4 treatment groups: 1. Vehicle control; 2. Voruciclib 200 mg/kg; 3. Venetoclax 50 mg/kg; and 4. The combination of voruciclib 200 mg/kg and venetoclax 50 mg/kg. Voruciclib was dosed by gavage every day for 6 days a week with one day of rest between treatments. Venetoclax was administered via gavage on days 1 and 4 of each weekly course. Drug effects were assessed by a technician blinded to treatment status in each study group, measuring tumor volume twice weekly. N = 5-6 animals per treatment group.

[213] Figure 27A shows plots of mean tumor volume (mm ³ ) for each of the 4 study groups. Individual administration of the agents demonstrated a slower reduction in tumor volume across study days. Combined administration demonstrated a significantly greater reduction in tumor volume, with a more than 2-fold reduction in mean tumor volume after 3 weeks of the study.

[214] Figure 27B illustrates tumor images from animals in each of the 4 study groups: vehicle, voruciclib as a single agent, venetoclax as a single agent, and the combination of voruciclib and venetoclax. The tumor size in the vehicle case was the largest, as shown in Figure 2701 . The tumor in the voruciclib single agent case is smaller, as shown in Figure 2702 . The tumor in the venetoclax single agent case is also smaller than the vehicle case, as shown in Figure 2703 . The tumor in the combination therapy case is shown in Figure 2704 and is significantly smaller than the vehicle case or either of the single agent therapies, indicating a synergistic effect.

[215] Figure 28 illustrates the effect of the combination of voruciclib and venetoclax on the growth of DLBCL tumor xenografts. Immunodeficient NOD SCID mice were implanted with SUDHL4, a model of DLBCL. Tumor xenografts were allowed to grow in the NOD SCID host until they reached 200 mm ³ , after which the xenograft-bearing animals were randomly assigned to 4 treatment groups: 1. Vehicle control; 2. Voruciclib 200 mg/kg; 3. Venetoclax 25 mg/kg; and 4. The combination of voruciclib 200 mg/kg and venetoclax 25 mg/kg. Voruciclib was dosed by gavage every day for 6 days a week with one day of rest between treatments. Venetoclax was dosed via gavage every day for 6 days of the week, with one day of rest between courses. Drug effects were assessed by a technician blinded to treatment status in each study group, measuring tumor volume twice weekly. N = 5-6 animals per treatment group. Figure 28 shows plots of normalized tumor volume (mm ³ ) for each of the 4 treatment groups. Single-agent therapy demonstrated an increase in tumor volume across study days. Combined administration demonstrated less tumor growth than single-agent and vehicle therapy.

[216] Each lane in Figure 29 represents an individual SUDHL4 xenograft. Collectively, Western blotting illustrates that voruciclib restores p53 abrogated by venetoclax. Tumors were harvested on day 26, 4 hours after administration. Each tumor was fragmented into two halves. One half was used for FFPE-based IHC analysis, and the other half was used for Western blotting analysis.

Example 13. Voruciclib inhibits MCL1 expression in AML cell lines

[217] Figures 30A-3C illustrate that voruciclib has single-agent activity in AML cell lines. AML cell lines (SKM-1, MV-4-11, and OCI-AML-3) were either left untreated or exposed to clinically achievable levels of voruciclib (1.5 and 5.0 μmol/L) for 2, 6, or 24 h. At these time points, cells were lysed and analyzed using Western blotting with antibodies that specifically recognize phosphorylated RNA polymerase II (pSer2), MCL-1, cleaved PARP, and beta-actin. In all three AML cell lines (SKM-1, MV-4-11, and OCI-AML-3), voruciclib suppresses MCL-1 and induces apoptosis, as shown in fragments 3001 , 3003 , and 3005. Cleaved PARP (cPARP) can be used as a biomarker of apoptosis. In all three AML cell lines (SKM-1, MV-4-11, and OCI-AML-3), voruciclib induces apoptosis, as shown in fragments 3002 , 3004 , and 3006 .

Example 14. The combination of voruciclib and venetoclax induces cell death in AML cell lines

[218] Figure 31 illustrates that the combination of voruciclib and venetoclax induces synergistic cell death in AML cell lines. AML cell lines (SKM-1, MV-4-11, and OCI-AML-3) were either left untreated or exposed to levels of voruciclib (1.5 μmol/L), venetoclax (100 nM) previously shown to be below the levels required to induce apoptosis by either agent alone, or the two drugs in combination at these subeffective concentrations for 24 h. At these time points, cells were lysed and analyzed using Western blotting with antibodies that specifically recognize phosphorylated RNA polymerase II (pSer2), MCL-1, cleaved PARP, and beta-actin. As shown in fragments 3101 and 3102 , the combination of voruciclib and venetoclax induces apoptosis (PARP), where neither agent alone induces apoptosis. Venetoclax was found to induce MCL-1 activation in all three cell lines. Voruciclib abolishes the MCL-1-mediated resistance mechanism in all three cell lines.

Example 15. The combination of voruciclib and venetoclax inhibits tumor growth in AML xenografts

[219] Figure 32 illustrates that the combination of voruciclib and venetoclax inhibits tumor growth in AML SKM1 xenografts. Single-agent venetoclax therapy has a normalized tumor volume similar to that of vehicle. Single-agent voruciclib demonstrates a slightly lower tumor volume, while the combination of voruciclib and venetoclax significantly inhibits tumor growth, with a final tumor volume approximately half that of vehicle at day 22 of the study.

Example 16. Voruciclib induces apoptosis as a single agent in DLBCL models

[220] Figure 33 illustrates that voruciclib-induced apoptosis correlates with MCL-1 suppression. Three DLBCL models were used: SUDHL4, RIvA, and U2932. Cleaved PARP (cPARP) can be used as a biomarker of apoptosis. Western blotting demonstrates that apoptosis increases with increasing voruciclib concentration. Similarly, voruciclib suppresses MCL-1 and induces apoptosis with increasing voruciclib concentration.

[221] Although preferred embodiments of the present invention have been shown and described in this specification, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, or substitutions will now be apparent to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein can be used in practicing the present invention. It will be understood that the following claims define the scope of the invention and that it covers methods and structures within the scope of these claims and their equivalents.

Claims (12)

1. Administration of a therapeutically effective amount of a CDK inhibitor corresponding to formula Ib: Ib, or a pharmaceutically acceptable salt thereof, for the treatment of malignant blood tumors in combination with a therapeutically effective amount of venetoclax or a pharmaceutically acceptable salt thereof, wherein the malignant blood tumor is selected from acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic lymphoma (ALL) and chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, intravascular large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma, marginal zone B-cell lymphomas, extranodal marginal zone B-cell lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, and primary central nervous system lymphoma. 2. The use according to claim 1, wherein the malignant blood tumor is diffuse large B-cell lymphoma, acute myeloid leukemia or chronic lymphocytic leukemia. 3. The use according to claim 1 or 2, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered simultaneously to an individual in need thereof. 4. The use according to claim 1 or 2, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered sequentially to an individual in need thereof. 5. The use according to claim 4, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered sequentially within 12 hours of each other to an individual in need thereof. 6. The use according to claim 4, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered sequentially within 5 hours of each other to an individual in need thereof. 7. The use according to claim 1 or 2, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are formulated together in a pharmaceutical composition. 8. The use according to any one of claims 1-7, wherein the CDK inhibitor and venetoclax or a pharmaceutically acceptable salt thereof are administered every day, every other day, or every third day to an individual in need thereof. 9. The use according to any one of claims 1 to 8, wherein the compound of formula Ib is (+)-trans-2-(2-chloro-4-trifluoromethylphenyl)-5,7-dihydroxy-8-(2-hydroxymethyl-1-methylpyrrolidin-3-yl)-chromen-4-one hydrochloride.