Use of inhibitors of Bruton's tyrosine kinase (Btk)

Abstract

To provide pharmaceutical compositions for use in treatment of relapsed and refractory hematological malignancies, such as follicular lymphoma.SOLUTION: The invention provides a pharmaceutical composition for use in treating follicular lymphoma which contains 560 mg of a BTK inhibitor having a structure in the figure and is formulated for a continuous once-daily dosing regimen.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority from U.S. Provisional Patent Application No. 61 / 351,130, filed June 3, 2010; U.S. Provisional Patent Application No. 61 / 351,655, filed June 4, 2010; U.S. Provisional Patent Application No. 61 / 351,793, filed June 4, 2010; U.S. Provisional Patent Application No. 61 / 351,762, filed June 4, 2010; U.S. Provisional Patent Application No. 61 / 419,764, filed December 3, 2010; and U.S. Provisional Patent Application No. 61 / 472,138, filed April 5, 2011; all of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Bruton's tyrosine kinase (Btk), a member of the Tec family of non-receptor tyrosine kinases, is a key signaling enzyme expressed in all hematopoietic cell types except T lymphocytes and natural killer cells. Btk plays an essential role in the B cell signaling pathway, linking cell surface B cell receptor (BCR) stimulation to downstream intracellular responses.

[0003] Btk is a key regulator of B cell development, activation, signaling, and survival (Kurosaki, Curr Op Imm, 2000, 276-281; ​​Schaeffer and Schwartzberg, Curr Op Imm 2000, 282-288). In addition, Btk plays a role in many other hematopoietic cell signaling pathways, such as Toll-like receptor (TLR) and cytokine receptor-mediated TNF-α production in macrophages, IgE receptor (FcepsilonRI) signaling in mast cells, inhibition of Fas / APO-1 apoptotic signaling in B-lineage lymphoid cells, and collagen-stimulated platelet aggregation. For example, CA Jeffries, et al., (2003), Journal of Biological Chemistry 278:26258-26264; NJ Horwood, et al., (2003), The Journal of Experimental Medicine 197:1603-1611; Iwaki et al. (2005), Journal of Biological Chemistry 280(48):40261-40270; see Vassilev et al. (1999), Journal of Biological Chemistry 274(3):1646-1656, and Quek et al. (1998), Current Biology 8(20):1137-1140. Summary of the Invention [Problem to be solved by the invention]

[0004] Disclosed herein, in certain embodiments, is a method of treating a hematological malignancy in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of the plurality of cells from the malignancy. In some embodiments, the hematological malignancy is CLL. In some embodiments, treating the hematological malignancy comprises managing the hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the hematological malignancy is leukemia, lymphoproliferative disorder, or myeloid. In some embodiments, the mobilized cells are myeloid cells or lymphoid cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after the number of mobilized cells has subsequently decreased. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the peripheral blood concentration of mobilized cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined period of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after the number of mobilized cells in the peripheral blood has subsequently decreased.In some embodiments, analyzing the mobilized plurality of cells includes measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells includes preparing a biomarker signature for a population of cells separated from the plurality of cells, wherein the biomarker signature indicates the expression, expression level, change in biomarker, or presence of a biomarker. In some embodiments, the biomarker is any cytogenetic, cell surface molecular, protein, or RNA expression marker. In some embodiments, the biomarkers are: ZAP70; t(14,18):beta-2 microglobulin; p53 mutation status; ATM mutation status; del(17)p; del(11)q; del(6)q; CD5; CD11c; CD19; CD20; CD22; CD25; CD38; CD103; CD138; secreted, surface, or intracellular immunoglobulin expression; VH mutation status; or a combination thereof. In some embodiments, the method further comprises providing a second cancer treatment regimen based on the biomarker profile. In some embodiments, the method does not further comprise administering based on the biomarker profile. In some embodiments, the method further comprises predicting the efficacy of the treatment regimen based on the biomarker profile. In some embodiments, the hematological malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, or non-CLL / SLL lymphoma. In some embodiments, the hematological malignancy is follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high grade B cell lymphoma, or extranodal marginal zone B-cell lymphoma.In some embodiments, the hematological malignancy is chronic myeloid (or myeloid) leukemia or acute lymphoblastic leukemia. In some embodiments, the hematological malignancy is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or refractory CLL; relapsed or refractory SLL; or relapsed or refractory multiple myeloma. In some embodiments, the Btk inhibitor forms a covalent bond with a cysteine ​​side chain of Bruton's tyrosine kinase, a Bruton's tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog. In some embodiments, the irreversible Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one. In some embodiments, the amount of the irreversible Btk inhibitor is 300 mg / day to 1000 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is 420 mg / day to 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day, about 560 mg / day, or about 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day. In some embodiments, the AUC of the Btk inhibitor. 0-24 is about 150 and about 3500ng * h / mL. In some embodiments, the AUC of the Btk inhibitor 0-24 is about 500 and about 1100ng *h / mL. In some embodiments, the Btk inhibitor is administered orally. In some embodiments, the Btk inhibitor is administered once daily, twice daily, or three times daily. In some embodiments, the Btk inhibitor is administered until disease progression, unacceptable toxicity, or individual choice. In some embodiments, the Btk inhibitor is administered daily until disease progression, unacceptable toxicity, or individual choice. In some embodiments, the Btk inhibitor is administered every other day until disease progression, unacceptable toxicity, or individual choice. In some embodiments, the Btk inhibitor is first-line therapy, second-line therapy, third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy. In some embodiments, the Btk inhibitor treats refractory hematological malignancies. In some embodiments, the Btk inhibitor is maintenance therapy. In some embodiments, the second cancer treatment regimen includes a chemotherapeutic agent, a steroid, an immunotherapeutic agent, a targeted therapy, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, a B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.In some embodiments, the second cancer treatment regimen comprises chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises bendamustine and rituximab. In some embodiments, the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab. In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises etoposide, doxorubicin, vinristine, cyclophosphamide, prednisolone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises dexamethasone and lenalidomide. In some embodiments, the inhibitor of Bruton's tyrosine kinase is a reversible inhibitor. In some embodiments, the inhibitor of Bruton's tyrosine kinase is an irreversible inhibitor. In some embodiments, the inhibitor of Bruton's tyrosine kinase forms a covalent bond with a cysteine ​​side chain of Bruton's tyrosine kinase, a Bruton's tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog. In some embodiments, the inhibitor of Bruton's tyrosine kinase has the structure of Formula (D):

[0005] [ka]

[0006] where: L ais CH, O, NH or S; Ar is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; Y is an optional substituent selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; Z is C(=O), OC(=O), NHC(=O), C(=S), S(=O) x , OS(=O) x , NHS(=O) x where x is 1 or 2; R7 and R8 are independently H; or R7 and R8 together form a single bond; R6 is H; and the inhibitor of Bruton's tyrosine kinase has a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug thereof. In some embodiments, the Bruton's tyrosine kinase inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one. In some embodiments, La is O. In some embodiments, Ar is phenyl. In some embodiments, Z is C(=O), NHC(=O), or S(=O). In some embodiments, each of R7 and R8 is H. In some embodiments, Y is a 4-, 5-, 6-, or 7-membered cycloalkyl; or Y is a 4-, 5-, 6-, or 7-membered heterocycloalkyl.

[0007] In certain embodiments, disclosed herein are methods of treating relapsed or refractory non-Hodgkin's lymphoma in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one. In some embodiments, the non-Hodgkin's lymphoma is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, or relapsed or refractory follicular lymphoma. In some embodiments, the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is 300 mg / day to 1000 mg / day. In some embodiments, the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is 420 mg / day to 840 mg / day. In some embodiments, the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is about 420 mg / day, about 560 mg / day, or about 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day. In some embodiments, the AUC 0-24 is about 150 and about 3500ng * h / mL. In some embodiments, the AUC of the Btk inhibitor 0-24 is about 500 and about 1100ng *h / mL. In some embodiments, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is administered orally. In some embodiments, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is administered once daily, twice daily, or three times daily. In some embodiments, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is administered until disease progression, unacceptable toxicity, or individual choice. In some embodiments, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is administered until disease progression, unacceptable toxicity, or individual choice. In some embodiments, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is administered daily until disease progression, unacceptable toxicity, or individual choice. In some embodiments, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is administered every other day until disease progression, unacceptable toxicity, or individual choice. In some embodiments, the (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one is a second line therapy, a third line therapy, a fourth line therapy, a fifth line therapy, or a sixth line therapy. In some embodiments, the Btk inhibitor is a maintenance therapy. In some embodiments, the method further comprises administering a second cancer treatment regimen.In some embodiments, the second cancer treatment regimen is administered after mobilization of multiple lymphoid cells from non-Hodgkin's lymphoma. In some embodiments, the second cancer treatment regimen is administered after lymphocytosis of multiple lymphoid cells from non-Hodgkin's lymphoma. In some embodiments, the second cancer treatment regimen comprises a chemotherapeutic agent, a steroid, an immunotherapeutic agent, a targeted therapy, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises a B-cell receptor pathway inhibitor. In some embodiments, the B-cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, a B-cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a biotin inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises bendamustine and rituximab. In some embodiments, the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab.In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises etoposide, doxorubicin, vinristine, cyclophosphamide, prednisolone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises dexamethasone and lenalidomide.

[0008] Disclosed herein are methods for treating diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) in an individual in need thereof, comprising administering to the individual an irreversible Btk inhibitor in an amount of 300 mg / day to 1000 mg / day. In some embodiments, the method further comprises diagnosing the individual with diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) by determining gene sequencing of one or more biomarkers in lymphoid cells isolated from the diffuse large B-cell lymphoma. In some embodiments, the irreversible Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one. In some embodiments, the ABC-DLBCL is characterized by a CD79B mutation. In some embodiments, the CD79B mutation is a mutation in the immunoreceptor tyrosine-based activation motif (ITAM) signaling module. In some embodiments, the CD79B mutation is a missense mutation of the first immunoreceptor tyrosine-based activation motif (ITAM) tyrosine. In some embodiments, the CD79B mutation increases surface BCR expression and reduces Lyn kinase activity. In some embodiments, the ABC-DLBCL is characterized by a CD79A mutation. In some embodiments, the CD79A mutation is in the immunoreceptor tyrosine-based activation motif (ITAM) signaling module. In some embodiments, the CD79A mutation is a splice donor site mutation in the immunoreceptor tyrosine-based activation motif (ITAM) signaling module. In some embodiments, the CD79A mutation removes the immunoreceptor tyrosine-based activation motif (ITAM) signaling module. In some embodiments, the ABC-DLBCL is characterized by MyD88, A20, or a combination thereof. In some embodiments, the MyD88 mutation is the amino acid substitution L265P in the MYD88 Toll / IL-1 receptor (TIR) ​​domain.In some embodiments, the amount of the irreversible Btk inhibitor is 420 mg / day to 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day, about 560 mg / day, or about 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day. In some embodiments, the AUC of the Btk inhibitor. 0-24 is about 150 and about 3500ng * h / mL. In some embodiments, the AUC of the Btk inhibitor 0-24 is about 500 and about 1100ng * h / mL. In certain embodiments, the irreversible Btk inhibitor is administered orally. In some embodiments, the irreversible Btk inhibitor is administered daily until disease progression, unacceptable toxicity, or individual choice. In some embodiments, the irreversible Btk inhibitor is administered every other day until disease progression, unacceptable toxicity, or individual choice. In some embodiments, the irreversible Btk inhibitor is a first-line therapy, second-line therapy, third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy. In some embodiments, the irreversible Btk inhibitor treats a refractory hematological malignancy. In some embodiments, the irreversible Btk inhibitor is a maintenance therapy. In some embodiments, the method further comprises administering at least one additional cancer treatment regimen. In some embodiments, the additional cancer treatment regimen comprises a chemotherapeutic agent, an immunotherapeutic agent, a steroid, radiation therapy, a targeted therapy, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, a B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a damaging agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

[0009] Disclosed herein, in certain embodiments, is a method for determining a cancer treatment regimen for an individual with a hematological malignancy, comprising the steps of: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignancy; (b) analyzing the mobilized plurality of cells; and (c) selecting a cancer treatment regimen. In some embodiments, the cancer treatment regimen comprises a chemotherapeutic agent, a steroid, an immunotherapeutic agent, a targeted therapy, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the cancer treatment regimen comprises a B cell receptor pathway inhibitor. In some embodiments, the cancer treatment regimen comprises a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the cancer treatment regimen comprises an antibody, a B-cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, the cancer treatment regimen comprises chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.In some embodiments, the cancer treatment regimen includes cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the cancer treatment regimen includes bendamustine and rituximab. In some embodiments, the cancer treatment regimen includes fludarabine, cyclophosphamide, and rituximab. In some embodiments, the cancer treatment regimen includes cyclophosphamide, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the cancer treatment regimen includes etoposide, doxorubicin, vinristine, cyclophosphamide, prednisolone, and optionally, rituximab. In some embodiments, the cancer treatment regimen includes dexamethasone and lenalidomide. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 depicts the role of Btk activity in many processes in CLL cells that contribute to disease pathogenesis. [Figure 2] FIG. 2 presents absolute lymphocyte counts during treatment with irreversible Btk inhibitors for individuals with CLL. [Figure 3] Figure 3 presents the change in the sum of lymph node (LN) diameter products in patients with CLL and SLL treated with irreversible Btk inhibitors. [Figure 4] Figure 4 shows LN responses in patients with CLL. The left panel shows LNs before treatment with an irreversible Btk inhibitor, and the right panel shows LNs after treatment with an irreversible Btk inhibitor. [Figure 5] FIG. 5 depicts the effect of irreversible Btk inhibitors on LN disease burden and lymphocytosis over time in patients with CLL and / or SLL. [Figure 6] Figure 6 depicts side effects in patients treated with irreversible Btk inhibitors. Grades 1-4 represent the severity of the effect, with 1 representing very mild and 4 representing very unpleasant. [Figure 7] Figure 7 shows absolute lymphocyte count (ALC) / 109L versus cycle day after administration of a Btk inhibitor to individuals with follicular lymphoma who achieved complete or partial remission (CR / PR). The Y-axis shows absolute lymphocyte count (ALC) at each time point, with cycle number and day on the X-axis. All patients (except Pt32009) were treated on a 4-week schedule followed by one week off treatment. Therefore, day 1 of each cycle represents one week off drug for these patients. Note the increase in ALC during most cycles for most patients, and the decrease in ALC at the beginning of subsequent cycles. This pattern often blunts in subsequent cycles as patients respond to treatment. Patient 32009 received treatment without interference and did not show this cycle pattern, but showed an increase on day 15 of cycle 1 and a gradual increase during cycles 2–5. [Figure 8] Figure 8 shows absolute lymphocyte counts (ALC) / 109L versus cycle day after administration of a Btk inhibitor to individuals with follicular lymphoma who had stable disease (SD) during treatment. The Y-axis shows absolute lymphocyte counts (ALC) at each time point, with cycle number and day on the X-axis. All patients (except Pt32009) were treated on a 4-week schedule followed by a 1-week break from treatment. Therefore, on day 1 of each cycle, these patients received a 1-week break from drug administration. Note the gradual increase in blood ALC kinetics in patient 32004, who initially had stable disease but later developed progressive disease (PD). [Figure 9]Figure 9 shows absolute lymphocyte counts (ALC) / 109L versus cycle day after administration of a Btk inhibitor to PD individuals with follicular lymphoma. The Y-axis shows absolute lymphocyte counts (ALC) at each time point, with cycle number and day on the X-axis. All patients except 38010 were treated on a 4-week schedule followed by a 1-week treatment break. Therefore, on day 1 of each cycle, these patients received a 1-week break from drug administration. Note the lack of mobilization, particularly for patients 38010 and 32001. Patient 323001 had limited treatment before being withdrawn from the study. The lymphocyte response suggests that this patient may have responded if they had been able to remain on treatment longer. [Figure 10] Figure 10 shows absolute lymphocyte counts (ALC) / 109L versus cycle day after administration of a Btk inhibitor to individuals with PR and SD DLBCL. The Y-axis shows absolute lymphocyte counts (ALC) at each time point, with cycle number and day on the X-axis. Patient 38011 was treated on a 4-week schedule followed by a 1-week break from treatment. Thus, day 1 of each cycle marked a 1-week break from drug administration to these patients. Patients 38008 and 324001 were treated with continuous daily doses. [Figure 11] Figure 11 shows absolute lymphocyte count (ALC) / 109L versus cycle day after administration of a Btk inhibitor to individuals with PD and DLBCL. The Y-axis shows absolute lymphocyte count (ALC) at each time point, with cycle number and day on the X-axis. All patients were treated on a 4-week schedule followed by a 1-week break from treatment. Therefore, on day 1 of each cycle, these patients received a 1-week break from drug administration. Note the lack of mobilization in 3 of the 4 patients. Patient 32002 received only one cycle of treatment. [Figure 12]Figure 12 shows absolute lymphocyte count (ALC) / 109L versus cycle day after administration of a Btk inhibitor to individuals with mantle cell lymphoma. The Y-axis shows absolute lymphocyte count (ALC) at each time point, with cycle number and day on the X-axis. Patients 32006, 38003, and 38004 were treated on a 4-week schedule followed by a 1-week break from treatment. Therefore, on day 1 of each cycle, these patients received a 1-week break from drug administration. Other patients were treated with continuous daily infusions. Note that the patient with early PD (32014) did not demonstrate mobilization. [Figure 13] Figure 13 depicts absolute lymphocyte count (ALC) / 109L versus cycle day after administration of a Btk inhibitor to individuals with mantle cell lymphoma shown in Figure 12. Compared to Figure 12, the axes have been shifted to demonstrate lower amplitude fluctuations. Note that all responding patients showed some degree of mobilization. [Figure 14] Figure 14 demonstrates that lymphocyte mobilization, particularly B-cell types, coincides with lymphoma cells and decreases with disease response. Patient 32007, group 4, had follicular lymphoma (grade 3) that gradually regressed from SD to CR. While the changes in ALC in this case are not dramatic, the B-cell fraction undergoes a characteristic cyclic increase in response to treatment with a Btk inhibitor. Also note the decreasing cycles with increasing cycle magnitude, consistent with progressive disease control. [Figure 15] Figure 15 demonstrates increased B cell mobilization with disease progression. Patient 32004, cohort 2, had follicular lymphoma (grade 1) that initially progressed from SD to PD after cycle 6. [Figure 16] Figure 16 depicts the early mobilization and eventual decline of the CD45DIM B cell subpopulation in responding mantle cell lymphoma patient 200-005. This subpopulation has a typical MCL immunophenotype (CD45DIM) and differs from that of normal lymphocytes. [Figure 17]Figure 17 shows abnormal high light scatter CD19+ cells mobilizing and then regressing in CR DLBCLPt324001. These CD45+ cells with light scatter (SSC-H) were gated in the upper panel, and their CD3 vs. CD19 staining is displayed in the lower panel. Here, the putative malignant cells "hide" in the large MNC window that normally defines monocytes. The sequence of mobilization following the reaction is similar to other examples. [Figure 18] Figure 18 presents the response to a clinical trial involving administering a Btk inhibitor to elderly patients with CLL or SLL who were not receiving drug intervention. Individuals were administered 420 mg / day of the Btk inhibitor. [Figure 19] Figure 19 presents the response to a clinical trial involving administering a Btk inhibitor to patients in R / R with CLL or SLL. Individuals received 420 mg / day of the Btk inhibitor. [Figure 20] FIG. 20 presents the response to a clinical trial involving administering a Btk inhibitor to individuals with high-risk CLL. [Figure 21] FIG. 21 presents the response over time for a clinical trial involving administering a Btk inhibitor to individuals with CLL or SLL. [Figure 22] FIG. 22 presents the best overall patient response in a clinical trial of administering a Btk inhibitor to individuals with CLL or SLL. [Figure 23] FIG. 23 presents the best response for notional patients in clinical trials involving administering Btk inhibitors to individuals with CLL or SLL. [Figure 24] FIG. 24 presents best response by prognostic factors in patients with CLL or SLL involved in clinical trials for administering a Btk inhibitor. [Figure 25] FIG. 25 presents the initial (Cycle 2) response assessment and best response (420 mg cohort) in patients with CLL or SLL involved in a clinical trial administering a Btk inhibitor. [Figure 26] FIG. 26 presents the first (Cycle 2) response assessment by dose in patients with relapsed / refractory CLL or SLL involved in a clinical trial of administering a Btk inhibitor. [Figure 27] FIG. 27 presents the improvement of blood parameters in CLL or SLL patients involved in a clinical trial with administration of a Btk inhibitor. [Figure 28] FIG. 28 presents data showing the results of combining a Btk inhibitor with carboplatin or velcade in DoHH2 cells. [Figure 29] FIG. 29 presents data showing the results of combining a Btk inhibitor with dexamethasone or lenalidomide in DoHH2 cells. [Figure 30] FIG. 30 presents data showing the results of combining Btk inhibitors with temsirolimus or R406 in DoHH2 cells. [Figure 31] FIG. 31 presents data showing the results of combining a Btk inhibitor with gemcitabine or doxorubicin in DoHH2 cells. [Figure 32] Figure 32 presents data showing the results of combining a Btk inhibitor with Cal-101 ​​in TMD8 cells. [Figure 33] Figure 33 presents data showing the results of combining a Btk inhibitor with R406 in TMD8 cells. [Figure 34] FIG. 34 presents data showing the results of combining a Btk inhibitor with vincristine in TMD8 cells. [Figure 35] Figure 35 presents data showing the results of combining a Btk inhibitor with doxorubicin in TMD8 cells. [Figure 36] Figure 36 presents data showing the results of combining a Btk inhibitor with lenolidomide in TMD8 cells. [Figure 37] FIG. 37 presents data showing the results of combining a Btk inhibitor with Velcade in TMD8 cells. [Figure 38] Figure 38 presents data showing the results of combining a Btk inhibitor with fludarabine in TMD8 cells. [Figure 39] Figure 39 presents data showing the results of combining a Btk inhibitor with taxol in TMD8 cells. DETAILED DESCRIPTION OF THE INVENTION

[0011] There is currently a need for methods of treating (including diagnosing) hematologic malignancies, including relapsed and refractory B-cell malignancies, and ABC-DLBCL. This application is based, in part, on the surprising discovery that Btk inhibitors induce lymphoid cell mobilization (or, in some cases, lymphocytosis) in solid hematologic malignancies. Mobilization of lymphoid cells increases their exposure to additional cancer treatment regimens and their availability for biomarker screening. The inventors have also discovered that Btk inhibitors are useful for treating relapsed and refractory malignancies, as well as ABC-DLBCL.

[0012] In certain embodiments, disclosed herein is a method of treating a hematological malignancy in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In certain embodiments, disclosed herein is a method of treating diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) in an individual in need thereof, comprising: administering to the individual an irreversible Btk inhibitor in an amount of from 300 mg / day to 1000 mg / day. Further disclosed herein, in certain embodiments, is a method of treating relapsed or refractory non-Hodgkin's lymphoma in an individual in need thereof, comprising the step of administering a therapeutically effective amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one to the individual.

[0013] 〈Specific technical terms〉 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In the event that there are multiple definitions for terms herein, those in this section prevail. When reference is made to a URL or other such identifier or address, it is understood that such identifiers may change and particular information on the Internet may come and go, but that equivalent information may be found by searching the Internet. Reference thereto confirms the availability and general dissemination of such information.

[0014] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of other forms such as "include," "includes," and "included," as well as the term "including," is open-ended.

[0015] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, papers, books, manuals, and treatises, are expressly incorporated herein by reference in their entirety for any purpose.

[0016] Definitions of standard chemical terms can be found in reference sources, including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED." Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used, within the skill of the art. Unless specific definitions are provided, the terms used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, and their laboratory procedures and techniques, described herein, are those known in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Reactions and purification techniques can be performed, for example, using kits according to manufacturer's specifications, or as commonly accomplished in the art, or as described herein. The foregoing techniques and procedures may be carried out in a conventional manner well known in the art and as described in various general and more specific references cited and discussed throughout this specification.

[0017] It is understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, as such may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which are limited only by the appended claims.

[0018] All publications and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in the publications, which may be used in connection with the methods, compositions, and compounds described herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other purpose.

[0019] An "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl moiety may be a "saturated alkyl" group, meaning that the alkyl moiety does not contain any alkene or alkyne moieties. The alkyl moiety may also be an "unsaturated alkyl" moiety, meaning that the alkyl moiety contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group having at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group having at least one carbon-carbon triple bond. The alkyl moiety may be saturated or unsaturated and may be branched, straight-chain, or cyclic. Depending on the structure, an alkyl group may be a monoradical or a diradical (i.e., an alkylene group). An alkyl group may also be a "lower alkyl" having 1 to 6 carbon atoms.

[0020] As used herein, C1-C x are C1-C2, C1-C3..C1-C x Includes:

[0021] An "alkyl" moiety can have 1 to 10 carbon atoms (whenever it appears herein, a numerical range such as "1 to 10" refers to each integer in the given range; for example, "1 to 10 carbon atoms" means that the alkyl group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, but this definition also includes appearances of the term "alkyl" without a numerical range specified). The alkyl group of the compounds described herein can be designated as "C1-C4 alkyl" or similar designation. By way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from among methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Thus, C1-C4 alkyl includes C1-C2 alkyl and C1-C3 alkyl. The alkyl group can be substituted or unsubstituted. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0022] As used herein, the term "acyclic alkyl" refers to an alkyl that is not cyclic (i.e., a straight or branched chain containing at least one carbon atom). An acyclic alkyl can be fully saturated or can contain acyclic alkenes and / or alkynes. An acyclic alkyl can be optionally substituted.

[0023] The term "alkenyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a double bond that is not part of an aromatic group. That is, an alkenyl group begins with the atom -C(R)=C(R)-R, where R refers to the remainder of the alkenyl group, which may be the same or different. The alkenyl portion can be branched, straight-chain, or cyclic (in which case it would also be known as a "cycloalkenyl" group). Depending on the structure, an alkenyl group can be a monoradical or a diradical (i.e., an alkenylene group). An alkenyl group can be optionally substituted. Non-limiting examples of alkenyl groups include -CH=CH, -C(CH)=CH, -CH=CHCH, and -C(CH)=CHCH. Alkenylene groups include, but are not limited to, -CH=CH-, -C(CH)=CH-, -CH=CHCH-, -CH=CHCHCH-, -CH=CHCHCH-, and -C(CH)=CHCH-. An alkenyl group can have 2 to 10 carbons. An alkenyl group can also be a "lower alkenyl" having 2 to 6 carbon atoms.

[0024] The term "alkynyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a triple bond. That is, an alkynyl group begins with the atom -C≡CR, where R refers to the remainder of the alkynyl group, which can be the same or different. The "R" portion of the alkynyl moiety can be branched, straight-chain, or cyclic. Depending on the structure, an alkynyl group can be a monoradical or a diradical (i.e., an alkynylene group). An alkynyl group can be optionally substituted. Non-limiting examples of alkynyl groups include, but are not limited to, -C≡CH, -C≡CCH, -C≡CCHCH, -C≡C-, and -C≡CCH-. An alkynyl group can have 2 to 10 carbons. An alkynyl group can also be a "lower alkynyl" having 2 to 6 carbon atoms.

[0025] An "alkoxy" group refers to a (alkyl)O- group, where alkyl is as defined herein.

[0026] "Hydroxyalkyl" refers to an alkyl radical, as defined herein, substituted with at least one hydroxy group. Non-limiting examples of hydroxyalkyl include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2-(hydroxymethyl)-3-hydroxypropyl.

[0027] "Alkoxyalkyl" refers to an alkyl radical, as defined herein, substituted by an alkoxy group. An "alkenyloxy" group refers to a (alkyl)O- group, where alkenyl is as defined herein.

[0028] The term "alkylamine" refers to an -N(alkyl) x H y refers to the group wherein x and y are selected from among x=1, y=1 and x=2, y=0. When x=2, the alkyl group taken together with the N atom to which it is attached can optionally form a cyclic ring structure.

[0029] "Alkylaminoalkyl" refers to an alkyl radical, as defined herein, substituted by an alkylamine, as defined herein.

[0030] An "amide" is a chemical moiety with the formula -C(O)NHR or -NHC(O)R, where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon). The amide moiety forms a linkage between an amino acid or peptide molecule and a compound described herein, thereby forming a prodrug. Any amine or carboxyl side chain on the compounds described herein can be aminated. Procedures and specific groups for making such amides are known to those of skill in the art and can be readily found in references such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0031] The term "ester" refers to a chemical moiety with the formula -COOR, where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon). Any hydroxy or carboxyl side chain on the compounds described herein can be esterified. Procedures and specific groups for making such esters are known to those of skill in the art and can be readily found in references such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety.

[0032] As used herein, the term "ring" refers to any covalently closed structure. Rings include, for example, carbocycles (e.g., aryls and cycloalkyls), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics (e.g., aryls and heteroaryls), and non-aromatic (e.g., cycloalkyls and non-aromatic heterocycles). Rings can be optionally substituted. Rings can be monocyclic or polycyclic.

[0033] As used herein, the term "ring system" refers to one or more rings.

[0034] The term "membered ring" can encompass any cyclic structure. The term "membered" is intended to indicate the number of skeletal atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are six-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are five-membered rings.

[0035] The term "fused" refers to a structure in which two or more rings share one or more bonds.

[0036] The term "carbocyclic" or "carbocycle" refers to a ring in which each of the atoms forming the ring is a carbon atom. Carbocycle includes aryl and cycloalkyl. Thus, the term distinguishes carbocycle from heterocycle ("heterocyclic"), in which the ring backbone includes at least one atom other than carbon (i.e., a heteroatom). Heterocycle includes heteroaryl and heterocycloalkyl. Carbocycles and heterocycles can be optionally substituted.

[0037] The term "aromatic" refers to a planar ring having a delocalized π-electron system containing 4n+2π electrons, where n is an integer. An aromatic ring can be formed from 5, 6, 7, 8, 9, or more than 9 atoms. An aromatic can be optionally substituted. The term "aromatic" includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) groups.

[0038] As used herein, the term "aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. The aryl ring can be formed by 5, 6, 7, 8, 9, or more than 9 carbon atoms. The aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. Depending on the structure, the aryl group can be a monoradical or a diradical (i.e., an arylene group). An "aryloxy" group refers to an (aryl)O- group, where aryl is as defined herein.

[0039] "Aralkyl" means an alkyl radical, as defined herein, substituted with an aryl group. Non-limiting aralkyl groups include benzyl, phenethyl, and the like.

[0040] "Aralkenyl" means an alkenyl radical, as defined herein, substituted by an aryl group, as defined herein.

[0041] The term "cycloalkyl" refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen and is saturated, partially unsaturated, or fully unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include the following moieties:

[0042] [ka]

[0043] Depending on the structure, a cycloalkyl group can be a monoradical or a diradical (i.e., a cycloalkylene group). A cycloalkyl group can also be a "lower cycloalkyl" having from 3 to 8 carbon atoms.

[0044] "Cycloalkylalkyl" means an alkyl radical, as defined herein, substituted with a cycloalkyl group. Non-limiting cycloalkylalkyl groups include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like.

[0045] The term "heterocycle" refers to heteroaromatic and heteroalicyclic groups containing 1 to 4 heteroatoms, each selected from O, S, and N, where each heterocyclic group has 4 to 10 atoms in its ring system, with the proviso that the ring of the group does not contain two adjacent O or S atoms. Whenever the number of carbon atoms in a heterocycle is indicated herein (e.g., C-C heterocycle), at least one other atom (heteroatom) must also be present in the ring. Designations such as "C-C heterocycle" refer only to the number of carbon atoms in the ring, not the total number of atoms in the ring. It is understood that heterocyclic rings can have additional heteroatoms in the ring. Designations such as "4- to 6-membered heterocycle" refer to the total number of atoms contained in the ring (i.e., a 4-, 5-, or 6-membered ring in which at least one atom is a carbon atom, at least one atom is a heteroatom, and the remaining 2 to 4 atoms are carbon atoms or heteroatoms). In heterocyclic compounds having two or more heteroatoms, the two or more heteroatoms can be the same or different. Heterocyclic compounds can be optionally substituted. Attachment to the heterocyclic compound can be at a heteroatom or through a carbon atom. Non-aromatic heterocyclic groups include groups with only four atoms in their ring system, while aromatic heterocyclic groups must have at least five atoms in their ring system. Heterocyclic groups include benzo-fused ring systems. An example of a four-membered heterocyclic group is azetidinyl (derived from azetidine). An example of a five-membered heterocyclic group is thiazolyl. An example of a six-membered heterocyclic group is pyridyl, and an example of a ten-membered heterocyclic group is quinolinyl.Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyranyl, and 1,2,3,6-tetrahydropyranyl. dioxanyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The aforementioned groups, as derived from the above groups, may be C- or N-attached where possible. For example, a group derived from pyrrole can be (N-attached) pyrrol-1-yl or (C-attached) pyrrol-3-yl. Furthermore, a group derived from imidazole can be (both N-attached) imidazol-1-yl or imidazol-3-yl, or (all C-attached) imidazol-2-yl, imidazol-4-yl, or imidazol-5-yl. Heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or two oxo (=O) moieties, such as pyrrolidin-2-one. Depending on the structure, heterocyclic groups can be monoradicals or diradicals (i.e., heterocyclene groups).

[0046] The term "heteroaryl" or alternatively "heteroaromatic" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. An N-containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. Illustrative examples of heteroaryl groups include the following moieties:

[0047] [ka]

[0048] Depending on the structure, a heteroaryl group can be a monoradical or a diradical (i.e., a heteroarylene group).

[0049] As used herein, the terms "non-aromatic heterocyclic compound", "heterocycloalkyl", or "heteroalicyclic" refer to non-aromatic rings, where one or more of the atoms forming the ring are heteroatoms. A "non-aromatic heterocyclic compound" or "heterocycloalkyl" group refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The radical can be condensed with an aryl or heteroaryl. The heterocycloalkyl ring can be formed by 3, 4, 5, 6, 7, 8, 9, or more than 9 atoms. The heterocycloalkyl ring is optionally substituted. In certain embodiments, the non-aromatic heterocyclic compound includes one or more carbonyl or thiocarbonyl groups, such as oxygen-containing groups and thio-containing groups. Examples of heterocycloalkyl include, but are not limited to, lactam, lactone, cyclic imide, cyclic thioimide, cyclic carbamate, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane, 1,4-oxathiin, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1,3-dioxol, 1,3-dioxolane, 1,3-dithiol, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, and 1,3-oxathiolane. Examples of heterocycloalkyl groups, also referred to as non-aromatic heterocycles, include the following:

[0050] [Chemical formula]

[0051] The term "heteroalicyclic" also includes all ring forms of carbohydrates, including, but not limited to, monosaccharides, disaccharides, and oligosaccharides. Depending on the structure, a heterocycloalkyl group can be a monoradical or a diradical (i.e., a heterocycloalkylene group).

[0052] The term "halo", or alternatively "halogen", or "halide", refers to fluoro, chloro, bromo and iodo.

[0053] The terms "haloalkyl," "haloalkenyl," "haloalkynyl," and "haloalkoxy" include alkyl, alkenyl, alkynyl, and alkoxy structures in which at least one hydrogen is replaced by a halogen atom. In certain embodiments in which two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are all the same as each other. In other embodiments in which two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are not all the same as each other.

[0054] As used herein, the term "fluoroalkyl" refers to an alkyl group in which at least one hydrogen is replaced by a fluorine atom. Examples of fluoroalkyl groups include, but are not limited to, -CF, -CHCF, -CFCF, -CHCHCF, and the like.

[0055] As used herein, the terms "heteroalkyl," "heteroalkenyl," and "heteroalkynyl" include optionally substituted alkyl, alkenyl, and alkynyl radicals in which one or more skeletal chain atoms is a heteroatom (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, or combinations thereof). The heteroatom may be placed at any interior position of the heteroalkyl group or at the position at which the heteroalkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-H3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Additionally, up to two heteroatoms may be consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0056] The term "heteroatom" refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from among oxygen, sulfur, nitrogen, silicon, and phosphorus, but are not limited to these atoms. In embodiments in which two or more heteroatoms are present, the two or more heteroatoms can all be the same as one another, or some or all of the two or more heteroatoms can be different from each other.

[0057] The term "bond" or "single bond" refers to a chemical bond between two atoms or two moieties when the atoms connected by the bond are considered to be part of a larger substructure.

[0058] An "isocyanato" group refers to a --NCO group.

[0059] An "isothiocyanato" group refers to a -NCS group.

[0060] The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often recognized as a chemical entity embedded in or attached to a molecule.

[0061] A "sulfinyl" group refers to a -S(=O)-R.

[0062] A "sulfonyl" group refers to a -S(=O)2-R.

[0063] A "thioalkoxy" or "alkylthio" group refers to an --S-alkyl group.

[0064] An "alkylthioalkyl" group refers to an alkyl group substituted with an --S-alkyl group.

[0065] As used herein, the term "O-carboxy" refers to a group of formula RC(=O)O-.

[0066] "Carboxy" refers to the radical --C(O)OH.

[0067] As used herein, the term "acetyl" refers to a group of formula -C(=O)CH3.

[0068] "Acyl" refers to the group -C(O)R.

[0069] As used herein, the term "trihalomethanesulfonyl" refers to a group of formula X3CS(=O)2- where X is a halogen.

[0070] As used herein, the term "cyano" refers to a group of formula -CN.

[0071] "Cyanoalkyl" means an alkyl radical, as defined herein, substituted with at least one cyano group.

[0072] As used herein, the term "N-sulfonamido" or "sulfonylamino" refers to a group of formula RS(=O)2NH-.

[0073] As used herein, the term "O-carbamyl" refers to a group of formula -OC(=O)NR2.

[0074] As used herein, the term "N-carbamyl" refers to a group of formula ROC(=O)NH-.

[0075] As used herein, the term "O-thiocarbamyl" refers to a group of formula -OC(=S)NR2.

[0076] As used herein, the term "N-thiocarbamyl" refers to a group of formula ROC(=S)NH-.

[0077] As used herein, the term "C-amido" refers to a group of formula -C(=O)NR2.

[0078] "Aminocarbonyl" refers to the -CONH2 radical.

[0079] As used herein, the term "N-amido" refers to a group of formula RC(=O)NH-.

[0080] As used herein, the substituent "R" occurring alone and without a number designation refers to a substituent selected from among alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and non-aromatic heterocycle (bonded through a ring carbon).

[0081] The term "optionally substituted" or "substituted" means that the referenced group can be substituted with one or more additional groups individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, acyl, nitro, haloalkyl, fluoroalkyl, amino, including mono- and di-substituted amino groups, and protected derivatives thereof. As an example, an optional substituent is L s R s where each L is independently selected from a single bond, -O-, -C(=O)-, -S-, -S(=O)-, -S(=O)-, -NH-, -NHC(O)-, -C(O)NH-, -S(=O)NH-, -NHS(=O)-, -OC(O)NH-, -NHC(O)O-, -(substituted or unsubstituted C-C alkyl), or -(substituted or unsubstituted C-C alkenyl); and each R is independently selected from H, (substituted or unsubstituted C-C alkyl), (substituted or unsubstituted C-C cycloalkyl), heteroaryl, or heteroalkyl. Protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, above.

[0082] The term "Michael acceptor moiety" refers to a functional group capable of participating in a Michael reaction, in which a new covalent bond is formed between a portion of the Michael acceptor moiety and a donor moiety. The Michael acceptor moiety is an electrophile, and the "donor moiety" is a nucleophile.

[0083] The term "nucleophile" or "nucleophilic" refers to an electron-rich compound or portion thereof. Examples of nucleophiles include, but are not limited to, cysteine ​​residues in molecules such as Cys481 of Btk.

[0084] The terms "electrophile" or "electrophilic" refer to an electron-poor or electron-deficient molecule or moiety. Examples of electrophiles include, but are not limited to, Michael acceptor moieties.

[0085] As used herein, the term "acceptable" or "pharmaceutically acceptable" with respect to a formulation, composition, or ingredient means that it has no lasting adverse effects on the general health of the subject receiving treatment or abrogates the biological activity or properties of the compound, and is relatively non-toxic.

[0086] As used herein, "B-cell lymphoproliferative disorder (BCLD) biomarker" refers to any biological molecule (found either in blood, other body fluids, or tissues) or any chromosomal abnormality that is indicative of a BCLD-related disease or disorder.

[0087] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, whether malignant or benign. As used herein, "neoplastic" refers to any form of dysregulated or uncontrolled cell proliferation, resulting in abnormal tissue growth, whether malignant or benign. Thus, "tumor cells" include malignant and benign cells with dysregulated or uncontrolled cell proliferation.

[0088] "Cancer" and "cancerous" refer to or describe a physiological disease in mammals that is typically characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, B-cell lymphoproliferative disorders (BCLDs), such as lymphomas and leukemias, and solid tumors. By "B-cell-associated cancer" or "cancer of the B-cell lineage" is intended any type of cancer in which dysregulated or unregulated cell proliferation is associated with B cells.

[0089] By "refractory" in the context of cancer, it is intended that a particular cancer is resistant to or does not respond to treatment with a particular therapeutic agent. A cancer may be refractory to treatment with a particular therapeutic agent from the initiation of treatment with the particular therapeutic agent (i.e., failure to respond to initial exposure to the therapeutic agent), throughout the entire treatment period with the therapeutic agent, or during subsequent treatment periods with the therapeutic agent, or as a result of developing resistance to the therapeutic agent.

[0090] By "agonist activity" is meant that a substance functions as an agonist, which binds to a receptor on a cell and initiates a response or activity similar to or the same as that initiated by the receptor's natural ligand.

[0091] By "antagonist activity" is meant that the substance functions as an antagonist. An antagonist of Btk prevents or reduces the induction of any of the responses mediated by Btk.

[0092] By "significant" agonist activity is intended at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater agonist activity than the agonist activity induced by a neutral substance or a negative control as measured in an assay of B cell responses. Preferably, "significant" agonist activity is at least two-fold greater or at least three-fold greater than the agonist activity induced by a neutral substance or a negative control as measured in an assay of B cell responses. Thus, for example, if the B cell response of interest is B cell proliferation, "significant" agonist activity would be induction of a level of B cell proliferation that is at least two-fold greater or at least three-fold greater than the level of B cell proliferation induced by a neutral substance or a negative control.

[0093] A substance that is "free of significant agonist activity" exhibits agonist activity that is at most about 25% greater than the agonist activity induced by a neutral substance or a negative control, and preferably at most about 20% greater, 15% greater, 10% greater, 5% greater, 1% greater, 0.5% greater, or even at most about 0.1% greater than the agonist activity induced by a neutral substance or a negative control as measured in an assay of B cell response.

[0094] In some embodiments, the Btk inhibitor therapeutic agent is an antagonist anti-Btk antibody. Such an antibody, when bound to the Btk antigen on human cells, lacks significant agonist activity as described above. In one embodiment of the invention, the antagonist anti-Btk antibody lacks significant agonist activity in an assay of one or more cellular responses (e.g., proliferation and differentiation, or proliferation, differentiation, and, for B cells, antibody production).

[0095] By "Btk-mediated signaling" is intended any biological activity that depends, directly or indirectly, on the activity of Btk. Examples of Btk-mediated signaling are signals that are responsible for the proliferation and survival of Btk-expressing cells, and stimulation of one or more Btk signaling pathways in Btk-expressing cells.

[0096] A Btk "signal pathway" or "signal transduction pathway" is intended to mean at least one biochemical reaction, or group of biochemical reactions, which results from the activity of Btk and generates a signal that, when propagated through the signal pathway, causes activation of one or more downstream molecules in a signal transduction cascade. A signal transduction pathway includes many signaling molecules that are responsible for transmitting a signal from the cell surface, across the cell's plasma membrane, and through one or more in a series of signaling molecules, through the cell's cytoplasm, and in some instances, to the cell's nucleus. Of particular interest to the present invention is the Btk signal transduction pathway, which ultimately controls (enhances or inhibits) activation of NF-κB via the NF-κB signaling pathway.

[0097] The methods of the present invention are, in certain embodiments, directed to methods of treating cancer that utilize antibodies to determine the expression or presence of particular BCLD biomarkers in methods of treating cancer. The following terms and definitions apply to such antibodies.

[0098] "Antibodies" and "immunoglobulins" (Ig) are glycoproteins with the same structural characteristics. The terms are used interchangeably. In some instances, the antigen specificity of an immunoglobulin is known.

[0099] The term "antibody" is used in the broadest sense and includes intact antibodies, antibody fragments capable of binding antigen (e.g., Fab, F(ab'), Fv, single chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, etc.), and recombinant peptides containing the foregoing.

[0100] The terms "monoclonal antibody" and "mAb," as used herein, refer to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts.

[0101] "Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V) followed by a number of constant regions. H Each light chain has a variable domain (V L ) and a constant region at its other end; the light-chain constant region is aligned with the first constant region of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains.

[0102] The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies. The variable regions confer antigen-binding specificity. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework (FR) regions. Natural heavy-chain and light-chain variable domains each contain four FR regions, primarily adopting a β-pleated sheet configuration, and connected by three CDRs that form, in some cases, loops connecting the β-pleated sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al. (1991) NIH Publication No. 91-3242, Vol. I, pages 647-669). The constant regions are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as Fc receptor (FcR) binding, participation of the antibody in antibody-dependent cellular toxicity, initiation of complement-dependent cytotoxicity, and mast cell degranulation.

[0103] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the "complementarily determining region" or "CDR" (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain, and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and / or residues from the "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chain variable domain, and (H1), 53-55 (H2), and 96-101 (H3) in the heavy-chain variable domain; Clothia and Lesk, (1987) J. Mol. Biol., 196:901-917. As considered herein, "framework" or "FR" residues are those variable domain residues other than the hypervariable region residues.

[0104] An "antibody fragment" comprises an intact antibody, preferably a portion of the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab, F(ab'), and Fv fragments; bispecific antibodies; linear antibodies (Zapata et al. (1995) Protein Eng. 10:1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, each called an "Fab" fragment, with a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects the ability to readily crystallize. Pepsin treatment yields an F(ab') fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0105] An "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0106] Fab fragments also contain the light chain constant region and the first heavy chain constant region (CH1). Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab', in which the cysteine ​​residues of the constant regions bear a free thiol group. Fab' fragments are generated by reducing the heavy chain disulfide bridges of the F(ab')2 fragment. Other chemical conjugations of antibody fragments are also known.

[0107] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0108] Depending on the amino acid sequence of their heavy chain constant region, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgG1 and IgG3 isotypes have ADCC (antibody-dependent cellular cytotoxicity) activity.

[0109] The word "label," as used herein, refers to a detectable compound or composition that is attached directly or indirectly to an antibody to produce a "labeled" antibody. The label may be detectable alone (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze the chemical alteration of a substrate compound or composition that is detectable. As used herein, the terms "acceptable" or "pharmaceutically acceptable," with respect to a formulation, composition, or ingredient, means that it has no lasting deleterious effects on the general health of the subject receiving treatment or does not abrogate the biological activity or properties of the compound, and is relatively non-toxic.

[0110] As used herein, the term "agonist" refers to a compound that is an entity that produces a biological activity of a protein that is the same as the biological activity that results from the presence of a naturally occurring ligand for the protein, e.g., Btk.

[0111] As used herein, the term "partial agonist" refers to a compound that is an entity that produces the same type of biological activity of a protein as would result from the presence of a naturally occurring ligand for the protein, but to a lesser extent.

[0112] As used herein, the term "antagonist" refers to a compound, the presence of which results in a decrease in the degree of biological activity of a protein. In certain embodiments, the presence of an antagonist results in complete suppression of the biological activity of a protein, such as Btk. In certain embodiments, an antagonist is an inhibitor.

[0113] As used herein, the term "Bruton's tyrosine kinase (Btk)" refers to Bruton's tyrosine kinase from humans, as disclosed, for example, in U.S. Pat. No. 6,326,469 (GenBank Accession No. NP_000052).

[0114] The term "Bruton's tyrosine kinase homolog," as used herein, refers to an ortholog of Bruton's tyrosine kinase, e.g., an ortholog from mouse (GenBank Accession No. AAB47246), dog (GenBank Accession No. XP_549139), rat (GenBank Accession No. NP_001007799), chicken (GenBank Accession No. NP_989564), or zebrafish (GenBank Accession No. XP_698117), and fusion proteins of any of the foregoing that exhibit kinase activity toward one or more substrates of Bruton's tyrosine kinase (e.g., a peptide substrate having the amino acid sequence "AVLESEEELYSSARQ").

[0115] The terms "co-administration" or "co-administration" and the like, as used herein, are meant to encompass the administration of selected therapeutic agents to one patient and are intended to include treatment regimens in which the therapeutic agents are administered by the same or different routes of administration or at the same or different times.

[0116] The term "effective amount," as used herein, refers to a sufficient amount of a Btk inhibitor or Btk-inhibiting compound being administered to result in an increase or appearance of a lymphocyte subpopulation in the blood (e.g., a reduction in the dosage of a pharmaceutical agent). For example, an "effective amount" for diagnostic and / or prognostic uses is the amount of a composition comprising a compound as disclosed herein required to clinically sufficiently reduce the increase or appearance of a lymphocyte subpopulation in the blood without undue adverse side effects. An appropriate "effective amount" in any individual case can be determined using techniques such as dose escalation studies.

[0117] The term "therapeutically effective amount," as used herein, refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more symptoms of B-cell lymphoproliferative disorder (BCLD). This may result in a reduction and / or alleviation of the signs, symptoms, or causes of BCLD, or any other desired alteration of a biological system. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a compound disclosed herein is an amount effective to achieve a desired pharmacological effect or therapeutic improvement without undue adverse side effects. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject due to variations in the metabolism of the compound of any of Formula (A), Formula (B), Formula (C), or Formula (D), the subject's age, weight, general condition, the disease being treated, the severity of the disease being treated, and the judgment of the prescribing physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including, but not limited to, a dose-escalation clinical trial.

[0118] The terms "enhance" or "enhancing" mean to increase or prolong, either in potency or duration, a desired effect. By way of example, "enhancing" the effect of a therapeutic agent refers to the ability to increase or prolong, either in potency or duration, the effect of the therapeutic agent during treatment of a disease, disorder, or condition. As used herein, the term "enhancing-effective amount" refers to an amount adequate to enhance the effect of the therapeutic agent in treating a disease, disorder, or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0119] As used herein, the term "cognate cysteine" refers to a cysteine ​​residue found at a position in the sequence that is homologous to that of cysteine ​​481 of Bruton's tyrosine kinase, as defined herein. For example, cysteine ​​482 is the cognate cysteine ​​in the rat ortholog of Bruton's tyrosine kinase. Cysteine ​​479 is the cognate cysteine ​​in the chicken ortholog. And cysteine ​​481 is the cognate cysteine ​​in the zebrafish ortholog. In another example, the cognate cysteine ​​in TXK, the Tec kinase family related to Bruton's tyrosine kinase, is cysteine ​​350. See also the sequence alignment of tyrosine kinases (TKs) published on the World Wide Web at kinase.com / human / kinome / phylogeny.html.

[0120] As used herein, the term "identical" refers to two or more sequences or subsequences that are the same. Additionally, as used herein, the term "substantially identical" refers to two or more sequences that have a percentage of sequence units that are the same when compared and aligned for maximum correspondence over a comparison window, or when specifying a region as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences are "substantially identical" if contiguous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The percentages are intended to describe the "percent identity" of two or more sequences. Sequence identity can exist over a region that is at least about 75-100 contiguous units in length, over a region that is about 50 contiguous units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are "substantially identical" if the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a designated region. Identity can exist over a region that is at least about 75-100 amino acids in length, over a region that is about 50 amino acids in length, or, where not specified, over the entire sequence of the polypeptide sequence. Further, by way of example only, two or more polynucleotide sequences are the same when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a designated region. The identity can exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or, where not specified, over the entire sequence of the polynucleotide sequence.

[0121] As used herein, the terms "inhibit," "inhibiting," or "inhibitor" of a kinase refer to the inhibition of the phosphotransferase activity of the enzyme.

[0122] As used herein, the term "irreversible inhibitor" refers to a compound that, upon contact with a target protein (e.g., a kinase), causes the formation of a new covalent bond at or within the protein, such that one or more of the biological activities of the target protein (e.g., phosphotransferase activity) is reduced or abolished despite the subsequent presence or absence of the irreversible inhibitor.

[0123] As used herein, the term "irreversible Btk inhibitor" refers to an inhibitor of Btk that can form a covalent bond to an amino acid residue of Btk. In one embodiment, the irreversible inhibitor of Btk can form a covalent bond to a cysteine ​​residue of Btk. In certain embodiments, the irreversible inhibitor can form a covalent bond to the cysteine ​​481 residue of Btk (or a homolog thereof) or a cysteine ​​residue at the corresponding position in a homolog of another tyrosine kinase.

[0124] As used herein, the term "isolated" refers to the separation and removal of a component of interest from components that are not of interest. An isolated material may exist in a dry state, a semi-dry state, or in a solution, including, but not limited to, an aqueous solution. An isolated component may exist in a homogeneous state, or the isolated component may be part of a pharmaceutical composition that includes additional pharmaceutically acceptable carriers and / or excipients. By way of example only, a nucleic acid or protein is "isolated" when it is free from at least some of the cellular components that naturally accompany it, or when the nucleic acid or protein is concentrated to a level higher than its in vivo or in vitro production concentration. Also, by way of example, a gene is isolated when it is separated from open reading frames that flank the gene and encode proteins other than the gene of interest.

[0125] A "metabolite" of a compound disclosed herein is a derivative of that compound formed when the compound is metabolized. The term "active metabolite" refers to a biologically active derivative of a compound formed when the compound is metabolized. As used herein, the term "metabolism" refers to the totality of processes by which a particular substance is transformed by an organism, including, but not limited to, hydrolysis and enzyme-catalyzed reactions such as oxidation. Thus, enzymes can cause specific structural changes to a compound. For example, cytochrome P450 catalyzes various oxidation and reduction reactions, while uridine diphosphate glucuronyltransferase catalyzes the transfer of activated glucuronic acid molecules to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free sulfhydryl groups. Further information regarding metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of the compounds disclosed herein can be identified by either administering the compound to a host and analyzing tissue samples from the host, or by incubating the compound with hepatocytes in vitro and analyzing the resulting compound. Both methods are well known in the art. In some embodiments, metabolites of the compounds are formed by an oxidation process and correspond to the corresponding hydroxy-containing compound. In some embodiments, the compounds are metabolized to pharmacologically active metabolites.

[0126] The term "modulate," as used herein, means to interact, either directly or indirectly, with a target so as to alter the activity of the target, including, by way of example only, enhancing the activity of the target, inhibiting the activity of the target, limiting the activity of the target, or extending the activity of the target.

[0127] As used herein, the term "modulator" refers to a compound that alters the activity of a molecule. For example, a modulator can cause an increase or decrease in the magnitude of a particular activity of a molecule compared to the magnitude of the activity in the absence of the modulator. In certain embodiments, a modulator is an inhibitor, which decreases the magnitude of one or more activities of a molecule. In certain embodiments, an inhibitor completely prevents one or more activities of a molecule. In certain embodiments, a modulator is an activator, which increases the magnitude of at least one activity of a molecule. In certain embodiments, the presence of a modulator results in an activity that does not occur in the absence of the modulator.

[0128] As used herein, the term "selective binding compound" refers to a compound that selectively binds to any portion of one or more target proteins.

[0129] As used herein, the term "selectively binds" refers to the ability of a selective binding compound to bind to a target protein, e.g., Btk, with a higher affinity than it binds to a non-target protein. In certain embodiments, specific binding refers to binding to a target with an affinity that is at least 10, 50, 100, 250, 500, 1000, or more times higher than the affinity for a non-target.

[0130] As used herein, the term "selective modulator" refers to a compound that selectively modulates a target activity relative to a non-target activity. In certain embodiments, a specific modulator refers to modulating a target activity at least 10, 50, 100, 250, 500, 1000 or more times over a non-target activity.

[0131] As used herein, the term "substantially purified" refers to a component of interest that is sufficiently or substantially free of other components that normally accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" when a preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest can have a purification level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater.

[0132] As used herein, the term "subject" refers to an animal that has been the object of treatment, observation, or experiment. By way of example only, a subject may be a mammal, including, but not limited to, a human.

[0133] As used herein, the term "target activity" refers to a biological activity capable of being modulated by a selective modulator. Specific exemplary target activities include, but are not limited to, binding affinity, signal transduction, enzyme activity, tumor growth, and effects on specific biomarkers related to the pathology of B-cell lymphoproliferative disorders.

[0134] As used herein, the term "target protein" refers to a molecule or a portion of a molecule that has the ability to be bound by a selective binding compound. In certain embodiments, the target protein is Btk.

[0135] The terms "treat", "treating" or "treatment", as used herein, include alleviating or ameliorating a disease or disorder, or a symptom thereof; managing a disease or disorder, or a symptom thereof; preventing further symptoms; ameliorating or preventing the underlying metabolic cause of a symptom; inhibiting a disease or disorder, e.g., preventing the progression of a disease or disorder; relieving a disease or disorder; causing remission of a disease or disorder, relieving a condition caused by a disease or disorder; or arresting the symptoms of a disease or disorder. The terms "treat", "treating" or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatments.

[0136] As used herein, IC 50 refers to the amount, concentration, or dosage of a particular test compound that achieves 50% inhibition of a maximal response, such as inhibition of Btk, in an assay that measures said response.

[0137] As used herein, EC 50 refers to the dose, concentration, or amount of a particular test compound that induces a dose-dependent response at 50% of the maximal expression of a particular response induced, caused, or potentiated by the particular test compound.

[0138] <Hematologic malignancies> Disclosed herein, in certain embodiments, is a method for treating a hematological malignancy in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, the hematological malignancy is CLL. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after the degree of the mobilized plurality of cells has subsequently decreased. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after the number of mobilized cells in the peripheral blood has subsequently decreased. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.In some embodiments, the hematological malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, or non-CLL / SLL lymphoma. In some embodiments, the hematological malignancy is follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Waldenstrom's macroglobulinemia, multiple myeloma, marginal zone lymphoma, Burkitt's lymphoma, non-Burkitt's high-grade B-cell lymphoma, or extranodal marginal zone B-cell lymphoma. In some embodiments, the hematological malignancy is acute or chronic myeloid (or myeloid) leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia. In some embodiments, the hematological malignancy is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or refractory CLL; relapsed or refractory SLL; relapsed or refractory multiple myeloma. In some embodiments, the hematological malignancy is a hematological malignancy classified as high-risk. In some embodiments, the hematological malignancy is high-risk CLL or high-risk SLL.

[0139] B-cell lymphoproliferative disorders (BCLDs) are hematologic neoplasms, including non-Hodgkin's lymphoma, multiple myeloma, and leukemia, among others. BCLDs can arise from lymphoid tissue (as in lymphomas) or bone marrow (as in leukemia and myeloma), and all involve either unrestrained growth of lymphocytes or leukocytes. Many subtypes of BCLD exist (e.g., chronic lymphocytic leukemia (NHL) and non-Hodgkin's lymphoma (CLL)). The course and treatment of BCLD depend on the BCLD subtype; however, even within each subtype, there is heterogeneity in clinical manifestations, morphological features, and response to treatment.

[0140] Malignant lymphomas result from the malignant transformation of cells that reside primarily within lymphoid tissue. Two types of malignant lymphoma exist: Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL). Both types of lymphoma invade tissues of the reticuloendothelial system. However, they differ in the tumor cell of origin, the site of disease, the presence of systemic symptoms, and response to treatment (Freedman et al., "Non-Hodgkin's Lymphomas," Chapter 134, (Cancer Medicine), (a 2003 publication approved by the American Cancer Society), B.C. Decker Inc., Hamilton, Ontario).

[0141] <Non-Hodgkin's lymphoma> In certain embodiments, described herein are methods for treating non-Hodgkin's lymphoma in need thereof, comprising: (a) administering to an individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, the hematological malignancy is treated with apogossypol. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells increases compared to the concentration in the peripheral blood before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0142] Further disclosed herein, in certain embodiments, is a method of treating relapsed or refractory non-Hodgkin's lymphoma in an individual in need thereof, comprising administering (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one; in some embodiments, the non-Hodgkin's lymphoma is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, or relapsed or refractory follicular lymphoma.

[0143] Non-Hodgkin's lymphoma (NHL) represents a diverse group of malignant tumors predominantly of B-cell origin. NHL can develop in any organ associated with the lymphatic system, such as the spleen, lymph nodes, or tonsils, and can occur at any age. NHL is often characterized by lymphadenopathy, fever, and weight loss. NHL is classified as either B-cell or T-cell NHL. Lymphomas associated with lymphoproliferative disorders following bone marrow or stem cell transplantation usually present in B-cell NHL. The Working Formulation classification system divides NHL into low-grade, intermediate-grade, and high-grade NHLs based on the value of their natural history (see "Non-Hodgkin's-Lymphoma Pathologic Classification Project") (Cancer, 49 (1982): 2112-2135). Low-grade lymphomas remain indolent, with an intermediate median survival of 5 to 10 years (Horning and Rosenberg (1984) N. Engl. J. Med. 311:1471-1475). Chemotherapy can induce remission in the majority of indolent lymphomas, but cures are rare, and most patients eventually relapse, requiring further treatment. Intermediate-grade and high-grade malignant lymphomas are more aggressive tumors, but they have a greater chance of cure with chemotherapy. However, a significant proportion of these patients relapse and require further treatment.

[0144] A non-limiting list of B-cell NHLs includes Burkitt's lymphoma (e.g., Endemic Burkitt's Lymphoma and Sporadic Burkitt's Lymphoma), Cutaneous B-Cell-Lymphoma, Cutaneous Marginal Zone Lymphoma (MZL), Diffuse Large Cell Lymphoma (DLBCL), Diffuse Mixed Small and Large Cell Lymphoma Diffuse Small Cleaved Cell, Diffuse Small Lymphocytic Lymphoma, Extranodal Marginal Zone B-Cell Lymphoma, Follicular Lymphoma, Follicular Small Cleaved Cell Follicular Lymphoma Cell) (Grade 1), Mixed Small Cleavage Cell Lymphoma and Large Cell Follicular Lymphoma (Grade 2), Large Cell Follicular Lymphoma (Grade 3), Intravascular Large B-Cell Lymphoma, Intravascular Lymphomatosis, Large Immunoblastic Lymphoma, Large Cell Lymphoma (LCL), Lymphoblastic Lymphoma, MALT Lymphoma, Mantle Cell Lymphoma (MCL), Immunoblastic Large Cell Lymphoma, Precursor B-Lymphoblastic Lymphoma, Mantle Cell Lymphoma, Chronic Lymphocytic Leukemia (CLL) / Small Lymphocytic Lymphoma (SLL), Extranodal Marginal B-Cell Lymphoma, Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma, Mediastinal Large B-Cell Lymphoma Non-Hodgkin's lymphomas include lymph node marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, Waldenstrom's macroglobulinemia, and primary central nervous system (CNS) lymphoma. Additional non-Hodgkin's lymphomas are contemplated within the scope of the present invention and will be apparent to those skilled in the art. DLBCL

[0145] Disclosed herein, in certain embodiments, is a method for treating DLCBL in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from a malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from a malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0146] As used herein, the term "diffuse B-cell lymphoma (DLBCL)" refers to a tumor of germinal center B lymphocytes with a diffuse growth pattern and a high-intermediate proliferation index. DLBCL accounts for approximately 30% of all lymphomas and can exhibit several morphological variants, including germinal center, immunoblastic, T-cell / histiocytic, anaplastic, and plasmablastic subtypes. Genetic testing indicates that different subtypes of DLBCL exist. These subtypes appear to have different appearances (prognoses) and responses to treatment. DLBCL can affect any age group, but most often occurs in older people (average age mid-60s).

[0147] Disclosed herein, in certain embodiments, is a method for treating diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) in an individual in need thereof, comprising administering to the individual an irreversible Btk inhibitor in an amount ranging from 300 mg / day to 1000 mg / day (including 1000 mg / day). The ABC subtype of diffuse large B-cell lymphoma (ABC-DLBCL) is thought to arise from post-germinal center B cells that are arrested during plasma differentiation. The ABC subtype of DLBCL (ABC-DLBCL) accounts for approximately 30% of DLBCL diagnoses in total. This molecular subtype of DLBCL, and as such, patients diagnosed with ABC-DLBCL have the lowest chance of cure, typically exhibiting significantly reduced survival rates compared to individuals with other types of DLBCL. ABC-DLBCL is generally associated with chromosomal translocations that deregulate the germinal center master regulator BCL6 and mutations that inactivate the PRDM1 gene, which encodes a transcriptional repressor required for plasma cell differentiation.

[0148] A signaling pathway particularly relevant to the pathogenesis of ABC-DLBCL is that mediated by the nuclear factor (NF)-κB transcription complex. The NF-κB family contains five members (p50, p52, p65, c-rel, and RelB), which form homodimers and heterodimers and function as transcription factors mediating various proliferation, apoptosis, inflammatory, and immune responses, and are critical for normal B cell development and survival. NF-κB is widely used by eukaryotic cells as a regulator of genes that control cell proliferation and cell survival. Therefore, various types of human tumors misregulate NF-κB; i.e., NF-κB is constitutively active. Active NF-κB initiates the expression of genes that promote cell proliferation and protect cells from diseases that would otherwise lead to death by apoptosis.

[0149] The dependence of ABC DLBCLs on NF-kB depends on the IkB kinase upstream signaling pathway, which is composed of CARD11, BCL10, and MALT1 (the CBM complex). Interference with the CBM pathway abolishes NF-kB signaling in ABC DLBCL cells and induces apoptosis. While the molecular basis for constitutive activity of the NF-kB pathway is currently under investigation, several somatic alterations to the genome of ABC DLBCLs clearly activate this pathway. For example, somatic mutations in the coiled-coil domain of CARD11 in DLBCLs render this signaling scaffold protein capable of spontaneously nucleating from protein-protein interactions with MALT1 and BCL10, resulting in IKK activity and NF-kB activation. Constitutive activity of the B cell receptor signaling pathway influences NF-kB activation in ABC DLBCLs with wild-type CARD11, which is associated with mutations within the cytoplasmic tails of the B cell receptor subunits CD79A and CD79B. Oncogenic activating mutations in the signaling adaptor MYD88 activate NF-kB and synergize with B cell receptor signaling in maintaining ABC DLBCL cell survival. In addition, inactivating mutations in a negative regulator of the NF-kB pathway (A20) occur almost exclusively in ABC DLBCL.

[0150] Indeed, genetic alterations affecting multiple components of the NF-κB signaling pathway have recently been identified in over 50% of ABC-DLBCL patients. These lesions promote component NF-κB activation, thereby contributing to lymphoma growth. These include mutations in CARD11 (up to 10% of cases), a lymphocyte-specific cytoplasmic scaffolding protein that, together with MALT1 and BCL10, forms the BCR signalosome, relaying signals from antigen receptors to downstream mediators of NF-κB activation. An even larger fraction (up to 30%) carries biallelic genetic lesions that inactivate the negative NF-κB regulator A20. Furthermore, high levels of expression of NF-κB target genes have been observed in ABC-DLBCL tumor samples. For example, U. Klein et al. (2008) Nature Reviews Immunology 8:22-23; REDavis et al. (2001) Journal of Experimental Medicine 194:1861-1874; G. Lentz et al. (2008) Science 319:1676-1679; M. See Compagno et al. (2009), Science 459:712-721; and L. Srinivasan et al. (2009), Cell 139:573-586.

[0151] DLBCL cells of the ABC subtype, such as OCI-Ly10, have chronically active BCR signaling and are highly sensitive to the Btk inhibitors described herein. The irreversible Btk inhibitors described herein inhibit OCI-Ly10 growth (EC50 continuous exposure = 10 nM, EC50 (1-hour pulse) = 50 nM). In addition, induction of apoptosis and caspase activity are observed in OCI-Ly10, as demonstrated by Annexin-V flow cytometry and an increase in the sub-G fraction. Both sensitive and resistant cells express Btk at similar levels, and the active site of Btk is fully occupied by the inhibitor, as demonstrated using a fluorescently labeled affinity probe. OCI-Ly10 cells are shown to have a chronically active BCR signaling to NF-kB, which is present at doses that are subsequently inhibited by the Btk inhibitors described herein. The activity of Btk inhibitors in the cell lines studied herein was characterized by comparing signaling (Btk, PLCγ, ERK, NF-kB, AKT), cytokine secretion, and mRNA expression profiles both with and without BCR stimulation, and significant differences in these profiles were observed, leading to clinical biomarkers that identify patients most sensitive to Btk inhibitor treatment. See U.S. Patent No. 7,711,492 and Staudt et al., Nature 463, January 7, 2010, pp. 88-92, the contents of which are incorporated herein by reference in their entireties.

[0152] <Follicular lymphoma> Disclosed herein, in certain embodiments, is a method for treating follicular lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after the number of mobilized cells in the peripheral blood has decreased. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0153] As used herein, the term "follicular lymphoma" refers to any of several types of non-Hodgkin's lymphoma in which the lymphoma cells are clustered into small nodules or follicles. The term follicular is used because the cells tend to grow in a circular, or nodular, pattern in the lymph nodes. The average age of people with this lymphoma is about 60 years old.

[0154] CLL / SLL Disclosed herein, in certain embodiments, are methods for treating CLL or SLL in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the CLL or SLL is high-risk. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after an increase in the number of mobilized cells in the peripheral blood compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0155] Chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL / SLL) are generally considered the same disease with slightly different manifestations. Where the cancer cells collect determines whether it is called CLL or SLL. When cancer cells are found primarily in lymph nodes, the lima-bean-shaped structures of the lymphatic system (a system of small vessels found primarily in the body), it is called SLL. SLL accounts for about 5% to 10% of all lymphomas. When most of the cancer cells are present in the bloodstream and bone marrow, it is called CLL.

[0156] Although CLL (which is more common) tends to grow slowly, both CLL and SLL are slow-growing diseases. CLL and SLL are treated in the same way. They are usually not curable with standard treatments, but depending on the stage and growth rate of the disease, most patients survive longer than 10 years. Sometimes, over time, these slow-growing lymphomas can transform into more aggressive forms of lymphoma.

[0157] Chronic lymphocytic leukemia (CLL) is the most common type of leukemia. It is estimated that 100,760 people in the United States are living with or in remission from CLL. Most people (>75%) newly diagnosed with CLL are over the age of 50. Currently, CLL treatment focuses on controlling the disease and its symptoms rather than focusing on a complete cure. CLL is treated with chemotherapy, radiation therapy, biologic therapy, or bone marrow transplant. Symptoms are sometimes treated surgically (splenectomy for splenomegaly) or with radiation therapy ("debulking" for lymphadenopathy). CLL is generally considered incurable, although it progresses slowly in most cases. Certain CLLs are classified as high-risk. As used herein, "high-risk CLL" refers to at least one of the following: 1) 17p13-; 2) 11q22-; 3) unmutated IgVH with ZAP-70+ and / or CD38+; or 4) trisomy 12.

[0158] CLL treatment is typically administered when a patient's clinical symptoms or blood counts indicate that the disease has progressed to a point where it may affect the patient's quality of life.

[0159] Small lymphocytic leukemia (SLL) is very similar to CLL, described above, and is a cancer of B cells. In SLL, abnormal lymphocytes primarily affect lymph nodes. However, in CLL, abnormal cells primarily affect the blood and bone marrow. The spleen may be affected in both conditions. SLL accounts for approximately 1 in 25 of all cases of non-Hodgkin's lymphoma. It can occur at any time, from adolescence to old age, but is rare in people under 50 years of age. SLL is considered an indolent lymphoma, meaning the disease progresses very slowly. Patients tend to live many years after diagnosis. However, most patients are diagnosed with advanced disease. SLL responds well to various chemotherapy drugs, but it is generally considered incurable. While some cancers tend to occur more frequently in one gender or the other, cases and deaths from SLL are evenly divided between men and women. The average age at diagnosis is 60 years.

[0160] Although SLL is indolent, it is relentlessly progressive. The usual pattern of the disease is one of high response rates to radiation therapy and / or chemotherapy, with periods of disease remission, followed by months or years of inevitable relapse. Retreatment again leads to response, but the disease recurs again. This means that while the short-term prognosis for SLL is quite good, over time, many patients suffer from fatal complications of recurrent disease. Given the age of individuals typically diagnosed with CLL and SLL, there is a need in the art for simple, effective treatments of the disease with minimal side effects that do not interfere with the patient's quality of life. The present invention fulfills this long-standing and continuing need in the art.

[0161] Mantle cell lymphoma Disclosed herein, in certain embodiments, are methods for treating mantle cell lymphoma in an individual, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after the number of mobilized cells in the peripheral blood has decreased. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0162] As used herein, the term "mantle cell lymphoma" refers to a subtype of B-cell lymphoma characterized by CD5-positive antigen-naive pregerminal B cells in the mantle zone surrounding normal germinal center follicles. MCL cells typically overexpress cyclin D1 due to a t(11:14) chromosomal translocation in DNA. More specifically, the translocation is t(11;14)(q13;q32). Only approximately 5% of lymphomas exhibit this subtype. The cells are medium-to-small in size. Males are most commonly affected. The average age of patients is in the early 60s. At diagnosis, lymphoma is usually widespread, involving lymph nodes, bone marrow, and often the spleen. While mantle cell lymphoma is not a very fast-growing lymphoma, it is difficult to treat.

[0163] Marginal B-cell lymphoma Disclosed herein, in certain embodiments, is a method for treating marginal B-cell lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0164] As used herein, the term "marginal zone B-cell lymphoma" refers to a group of related B-cell tumors that involve lymphoid tissue in the marginal zone, a patchy area outside the mantle zone of the follicle. Marginal zone lymphomas account for approximately 5% to 10% of lymphomas. The cells in these lymphomas are small under a microscope. There are three major types of marginal zone lymphoma, including extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma.

[0165] Mucosa-associated lymphoid tissue (MALT) Disclosed herein, in certain embodiments, is a method for treating mucosa-associated lymphoid tissue in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from a malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from a malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0166] As used herein, the term "mucosa-associated lymphoid tissue (MALT) lymphoma" refers to the extranodal manifestation of marginal lymphoma. Although a minority initially present as intermediate-grade non-Hodgkin's lymphoma (NHL) or evolve from a lower-grade form, most MALT lymphomas are low-grade. Most MALT lymphomas arise in the stomach. Approximately 70% of gastric MALT lymphomas are associated with Helicobacter pylori infection. Several cytogenetic abnormalities have been identified, the most common being trisomy 3 or t(11;18). Many of these other MALT lymphomas have also been associated with bacterial or viral infections. The average age of patients with MALT lymphoma is approximately 60.

[0167] <Marginal zone B-cell lymphoma> Disclosed herein, in certain embodiments, is a method for treating lymph node marginal zone B-cell lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0168] The term "nodal marginal zone B-cell lymphoma" refers to an indolent B-cell lymphoma that is mostly found in lymph nodes. The disease is rare, accounting for only 1% of all non-Hodgkin's lymphomas (NHL). It is most commonly diagnosed in older patients, with women more susceptible than men. Because mutations occur in the marginal zone of B cells, the disease is classified as marginal zone lymphoma. Due to its restriction in lymph nodes, the disease is also classified as nodal.

[0169] <Splenic marginal zone B-cell lymphoma> Disclosed herein, in certain embodiments, is a method for treating splenic marginal B-cell lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0170] The term "splenic marginal zone B-cell lymphoma" refers to a specific low-grade B-cell lymphoma incorporated into the World Health Organization classification. It is characterized by moderate lymphocytosis with splenomegaly, villous morphology, intrasinusoidal pattern in various organs (especially the bone marrow), and a relatively indolent course. Tumor progression with increased blast morphology and aggressive behavior is observed in a minority of patients. Molecular and cytogenetic studies have shown heterogeneous results, likely due to the lack of standardized diagnostic criteria.

[0171] <Burkitt lymphoma> Disclosed herein, in certain embodiments, is a method of treating Burkitt's lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0172] The term "Burkitt lymphoma" refers to a type of non-Hodgkin's lymphoma (NHL) that commonly affects children. It is a highly aggressive form of B-cell lymphoma that frequently begins and involves body parts other than lymph nodes. Despite its fast-growing nature, Burkitt lymphoma is often curable with modern intensive treatment. There are two widespread types of Burkitt lymphoma: the sporadic variety and the endemic variety.

[0173] Endemic Burkitt lymphoma: The disease affects children far more than adults and is associated with Epstein-Barr virus (EBV) infection in 95% of cases. It occurs primarily in equatorial Africa, where approximately half of all childhood cancers are Burkitt lymphoma. It is more likely to involve the jawbone, a rare suggestive feature of sporadic Burkitt lymphoma. It also commonly involves the abdomen.

[0174] Sporadic Burkitt lymphoma: The type of Burkitt lymphoma that affects the rest of the world, including Europe and the Americas, is the sporadic type. Here, too, it is primarily a disease of children. Direct evidence of Epstein-Barr virus infection is present in one in five patients, but the link between Epstein-Barr virus (EBV) is not as strong as with the endemic variant. Beyond lymph node involvement, the abdomen is significantly affected in over 90% of children. Bone marrow involvement is more common than with the sporadic variant.

[0175] <Waldenström's macroglobulinemia> Disclosed herein, in certain embodiments, is a method for treating Waldenstrom's macroglobulinemia in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from a malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from a malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0176] Waldenström's macroglobulinemia, also known as lymphoplasmacytic lymphoma, is a cancer involving a subtype of white blood cells called lymphocytes. It is characterized by the uncontrolled clonal proliferation of differentiated B lymphocytes. It is also characterized by lymphoma cells producing an antibody called immunoglobulin M (IgM). IgM antibodies circulate in the bloodstream, causing the liquid portion of the blood to thicken like syrup. This leads to reduced blood flow to many organs, which can cause problems with vision (due to poor circulation in the blood vessels behind the eyes) and neurological problems (such as headaches, dizziness, and confusion) caused by poor blood flow in the brain. Other symptoms may include feeling tired and weak and a tendency to bleed easily. Although the underlying etiology is not fully understood, many risk factors have been identified, including the location 6p21.3 on chromosome 6. There is a two- to three-fold increased risk of developing WM in people with a personal history of autoimmune diseases, with autoantibodies related to hepatitis, human immunodeficiency virus, and rickettsiosis being particularly high risk.

[0177] <Multiple myeloma> Disclosed herein, in certain embodiments, is a method for treating myeloma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0178] Disclosed herein, in certain embodiments, is a method for treating multiple myeloma in an individual, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after an increase in the number of mobilized cells in the peripheral blood compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0179] Multiple myeloma, also known as MM, myeloma, plasma cell myeloma, or Kahler's disease (after Otto Kohler), is a cancer of white blood cells known as plasma cells. Plasma cells, a type of B cell, are a vital part of the immune system responsible for producing antibodies in humans and other vertebrates. They are produced in the bone marrow and transported by the lymphatic system.

[0180] <leukemia> Disclosed herein, in certain embodiments, are methods for treating leukemia in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignant tumor; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignant tumor. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of the increase in the peripheral blood concentration of the mobilized plurality of cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized cells in the peripheral blood. In some embodiments, analyzing the mobilized cells comprises measuring the duration of the increase in the number of mobilized cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined length of time.

[0181] Leukemia is a cancer of the blood or bone marrow characterized by an abnormal increase in blood cells, usually white blood cells (leukocytes). Leukemia is a broad term covering a range of diseases. Primary division exists between its acute and chronic forms: (i) acute leukemia is characterized by a rapid increase in immature blood cells. This overcrowding forces the bone marrow to produce healthy blood cells. Urgent treatment is required in acute leukemia due to the rapid progression and accumulation of malignant cells, which then migrate into the bloodstream and spread to other organs of the body. The acute form of leukemia is the most common form in children; (ii) chronic leukemia is distinguished by an excessive composition of relatively mature, yet still abnormal, white blood cells. It typically takes months or years to progress, with cells being produced at a much faster rate than normal cells, resulting in a large number of abnormal white blood cells in the blood. Chronic leukemia most often occurs in older people, but theoretically can occur in any age group. The disease is further subdivided according to the type of blood cells affected. This division separates lymphoblastic, or lymphocytic, leukemia from myeloid, or myelogenous, leukemia: (i) lymphoblastic, or lymphocytic, leukemia, in which cancerous changes occur in a type of bone marrow cell that then normally forms lymphocytes, which are fighting cells of the immune system; (ii) myeloid, or myelogenous, leukemia, in which cancerous changes occur in a type of bone marrow cell that then normally forms red blood cells, several other types of white blood cells, and platelets.

[0182] Within these major categories, several subcategories exist, including, but not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and hairy cell leukemia (HCL).

[0183] <Btk inhibitor> Also presented herein are methods for treating cancer, such as BCLD, in a subject, by way of example only, where the subject is treated with a Btk inhibitor drug regimen. In the following description of reversible Btk compounds suitable for use in the methods described herein, definitions referring to standard chemical terms can be found in references, including Carey and Sundberg, Advanced Organic Chemistry, 4th Ed. Vols. A (2000) and B (2001), Plenum Press, New York (unless otherwise defined herein). Unless otherwise indicated, those skilled in the art will employ conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology. Furthermore, nucleic acid and amino acid sequences for Btk (e.g., human Btk) are known in the art, for example, as disclosed in U.S. Pat. No. 6,326,469. Unless specific definitions are provided, the terminology used in connection with, and the laboratory methods and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0184] The Btk inhibitor compounds described herein are selective for Btk and kinases that have a cysteine ​​residue in the amino acid sequence position of a tyrosine kinase homologous to the amino acid sequence position of cysteine ​​481 in Btk. Generally, irreversible inhibitors of Btk used in the methods described herein are identified or characterized in an in vitro assay, e.g., a non-cellular biochemical assay or a cellular functional assay. The assay may be used to determine an in vitro IC for an irreversible Btk inhibitor compound. 50 It is useful for measuring

[0185] For example, cell-free kinase assays can be used to measure Btk activity after incubation of the kinase in the absence or presence of various concentrations of a candidate irreversible Btk inhibitor compound. If the candidate compound is indeed an irreversible Btk inhibitor, Btk kinase activity will not be restored by repeated washing with inhibitor-free medium. See, e.g., J.B. Smaill et al. (1999), J. Med. Chem. 42(10):1803-1815. Furthermore, the formation of covalent complexes between Btk and a candidate irreversible Btk inhibitor is a useful indicator of irreversible inhibition of Btk, which can be easily measured by numerous methods known in the art (e.g., mass spectrometry). For example, some irreversible Btk inhibitor compounds form covalent bonds with the cysteine ​​residues described above (e.g., via a Michael reaction).

[0186] Cellular functional assays for Btk inhibition involve measuring one or more cellular endpoints in response to stimulating a Btk-mediated pathway in a cell line (e.g., BCR activation in Ramos cells) in the absence or presence of varying concentrations of a candidate irreversible Btk inhibitor compound. Useful endpoints for measuring the response to BCR activation include, for example, Btk autophosphorylation, phosphorylation of Btk target proteins (e.g., PLC-γ), and cytosolic calcium flux.

[0187] Many high-throughput assays for acellular biochemical assays (e.g., kinase assays) and cellular functional assays (e.g., calcium flux) are well known to those skilled in the art. Furthermore, high-throughput screening systems exist commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc., Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate the entire procedure, including pipetting of all samples and reagents in a microplate, timed incubation, and final readout in a detector appropriate for the assay. Automated systems thereby enable the identification and characterization of a large number of irreversible Btk compounds without undue effort.

[0188] In some embodiments, the Btk inhibitor is selected from the group consisting of a small organic molecule, a polymer, a peptide, or a non-peptide.

[0189] In some embodiments, the Btk inhibitors provided herein are reversible or irreversible inhibitors. In certain embodiments, the Btk inhibitor is an irreversible inhibitor.

[0190] In some embodiments, the irreversible Btk inhibitor forms a covalent bond with a cysteine ​​side chain of Bruton's tyrosine kinase, a Bruton's tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog.

[0191] The irreversible Btk inhibitor compounds can be used in the manufacture of a medicament for treating any of the aforementioned conditions (e.g., an autoimmune disease, an inflammatory disease, an allergic disorder, a disorder of B cell proliferation, or a thromboembolic disorder).

[0192] In some embodiments, the irreversible Btk inhibitor compounds used for the methods described herein inhibit Btk or have an in vitro IC of less than 10 μM. 50 (e.g., less than 1 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.1 μM, less than 0.08 μM, less than 0.06 μM, less than 0.05 μM, less than 0.04 μM, less than 0.03 μM, less than 0.02 μM, less than 0.01 μM, less than 0.008 μM, less than 0.006 μM, less than 0.005 μM, less than 0.004 μM, less than 0.003 μM, less than 0.002 μM, less than 0.001 μM, less than 0.00099 μM) In one embodiment, an irreversible Btk inhibitor compound selectively and irreversibly inhibits the activated form of its target tyrosine kinase (e.g., a phosphorylated form of a tyrosine kinase). For example, activated Btk is transphosphorylated at tyrosine 551. Thus, in these embodiments, the irreversible Btk inhibitor inhibits the target kinase in a cell once the target kinase has been activated by a signaling event.

[0193] In other embodiments, the Btk inhibitors used in the methods described herein have the structure of any of Formula (A), (B), (C), (D), (E), or (F). Also described herein are pharmaceutically acceptable salts, pharmaceutically acceptable solvates, pharmaceutically active metabolites, and pharmaceutically acceptable prodrugs of the compounds. Pharmaceutical compositions containing at least one such compound, or a pharmaceutically acceptable salt, pharmaceutically acceptable solvate, pharmaceutically active metabolite, or pharmaceutically acceptable prodrug of such a compound, are provided. In some embodiments, when a compound disclosed herein contains an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods known in the art. In certain embodiments, isomers and chemically protected forms of compounds having a structure represented by any of Formula (A), (B), (C), (D), (E), or (F) are provided.

[0194] Formula (A) is as follows:

[0195] [ka]

[0196] wherein A is independently selected from N or CR5; R1 is H, L2-(substituted or unsubstituted alkyl), L2-(substituted or unsubstituted cycloalkyl), L2-(substituted or unsubstituted alkenyl), L2-(substituted or unsubstituted cycloalkenyl), L2-(substituted or unsubstituted heterocycle), L2-(substituted or unsubstituted heteroaryl), L2-(substituted or unsubstituted aryl), where L2 is a single bond, O, S, -S(=O), -S(=O)2, C(=O), -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); R2 and R3 are independently selected from H, lower alkyl, and substituted lower alkyl; R4 is L3-X-L4G, where L3 is optional and, if present, is a single bond, optionally optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl; X is optional and, when present, may be a single bond, O, -C(=O), S, -S(=O), -S(=O), -NH, -NR, -NHC(O), -C(O)NH, -NRC(O), -C(O)NR, -S(=O)NH, -NHS(=O), -S(=O)NR, -NRS(=O), -OC(O)NH-, -NHC(O)O, -OC(O)NR, -NRC(O)O, -CH=NO-, -ON=CH-, -NR 10 C(O)NR 10 -, heteroaryl, aryl, -NR 10 C(=NR11)NR 10 -,-NR 10 C(=NR 11 )-, -C(=NR 11 )NR 10 -, -OC(=NR 11 )-, or -C(=NR 11 )O-; L4 is optional and, when present, is a single bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted biheterocyclic compound; or L3, X and L4 taken together form a nitrogen containing heterocycle; G is

[0197] [ka]

[0198] wherein R6, R7, and R8 are independently selected from H, lower alkyl or substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower cycloalkyl, and substituted or unsubstituted lower heterocycloalkyl; R5 is H, halogen, -L6-(substituted or unsubstituted C1-C3 alkyl), -L6-(substituted or unsubstituted C2-C4 alkenyl), -L6-(substituted or unsubstituted heteroaryl), or -L6-(substituted or unsubstituted aryl), where L6 is a single bond, O, S, -S(=O), S(=O)2, NH, C(O), -NHC(O)O, -OC(O)NH, -NHC(O), or -C(O)NH; each R9 is independently selected from H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl; Each R 10 are independently H, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower cycloalkyl; or The Two R's 10 The groups together can form a 5-, 6-, 7-, or 8-membered heterocycle; or R9 and R 10 can together form a 5-, 6-, 7- or 8-membered heterocycle; or Each R 11 are independently selected from H, —S(═O)2R8, —S(═O)2NH2, —C(O)R8, —CN, —NO2, heteroaryl, or heteroalkyl; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of said constituents.

[0199] In one aspect, a compound having the structure of formula (A1) is disclosed: Formula (A1) is

[0200] [ka]

[0201] and A is independently selected from N or CR5; R1 is H, L2-(substituted or unsubstituted alkyl), L2-(substituted or unsubstituted cycloalkyl), L2-(substituted or unsubstituted alkenyl), L2-(substituted or unsubstituted cycloalkenyl), L2-(substituted or unsubstituted heterocycle), L2-(substituted or unsubstituted heteroaryl), L2-(substituted or unsubstituted aryl), where L2 is a single bond, O, S, -S(=O), -S(=O)2, C(=O), -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); R2 and R3 are independently selected from H, lower alkyl, and substituted lower alkyl; R4 is L3-X-L4-G, where L3 is optional and, when present, is an optional substituent selected from a single bond, alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl; X is optional and, when present, may be a single bond, O, -C(=O), S, -S(=O), -S(=O), -NH, -NR, -NHC(O), -C(O)NH, -NRC(O), -C(O)NR, -S(=O)NH, -NHS(=O), -S(=O)NR, -NRS(=O), -OC(O)NH-, -NHC(O)O, -OC(O)NR, -NRC(O)O, -CH=NO-, -ON=CH-, -NR 10 C(O)NR 10 -, heteroaryl, aryl, -NR 10 C(=NR 11 )NR 10 -, -NR 10 C(=NR 11 )-, -C(=NR 11 )NR 10 -, -OC(=NR 11 )-, or -C(=NR 11 )O-; L4 is optional and, when present, is a single bond, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic compound; or L3, X and L4 taken together form an optional substituent selected from nitrogen, alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl containing heterocycle; G is

[0202] [ka]

[0203] where R a is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or R6 and R8 form a single bond; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or R5 is H, halogen, -L6-(substituted or unsubstituted C1-C3 alkyl), -L6-(substituted or unsubstituted C2-C4 alkenyl), -L6-(substituted or unsubstituted heteroaryl), or -L6-(substituted or unsubstituted aryl), where L6 is a single bond, O, S, -S(=O), S(=O)2, NH, C(O), -NHC(O)O, -OC(O)NH, -NHC(O) or -C(O)NH; each R9 is independently selected from H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl; Each R 10are independently H, optionally substituted lower alkyl, or optionally substituted lower cycloalkyl; or The Two R's 10 The groups together can form a 5-, 6-, 7-, or 8-membered heterocycle; or R9 and R 10 can together form a 5-, 6-, 7- or 8-membered heterocycle; or Each R 11 are independently selected from H, —S(═O)2R8, —S(═O)2NH2, —C(O)R8, —CN, —NO2, heteroaryl, or heteroalkyl; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of said constituents.

[0204] In another embodiment, pharmaceutically acceptable salts of the compound of formula (A1) are provided. By way of example only, the salts may be salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Further salts may be formed where the counterion is one of the following anions: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, and the like. Salts include, but are not limited to, esters of hydroxypropyl methyl esters such as hydroxypropyl methyl esters, ...

[0205] In another embodiment, there are pharmaceutically acceptable esters of the compound of formula (A1), including those in which the ester group is selected from formate, acetate, propionate, butyrate, acrylate, and ethylsuccinate.

[0206] In another embodiment, there are pharmaceutically acceptable carbamates of the compound of formula (A1). In another embodiment, there are pharmaceutically acceptable N-acyl derivatives of the compound of formula (A1). Examples of N-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

[0207] In a further embodiment, there is a compound of Formula (A) having the structure of Formula (B):

[0208] [ka]

[0209] wherein Y is alkylene, substituted alkylene, or 4-, 5-, or 6-membered cycloalkylene; Each R a are independently H, halogen, —CF3, —CN, —NO2, OH, NH2, -La-(substituted or unsubstituted alkyl), -La-(substituted or unsubstituted alkenyl), -La-(substituted or unsubstituted heteroaryl), or -La-(substituted or unsubstituted aryl), where La is a single bond, O, S, —S(═O), —S(═O)2, NH, C(O), CH2, —NHC(O)O, —NHC(O) or —C(O)NH; G is

[0210] [ka]

[0211] wherein R6, R7, and R8 are independently selected from H, lower alkyl or substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower cycloalkyl, substituted or unsubstituted lower heterocycloalkyl; R 12 is H, or lower alkyl; or Y and R taken together 12form a 4-, 5-, or 6-membered heterocycle; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of said structure.

[0212] In a further embodiment, G is

[0213] [ka]

[0214] is selected from.

[0215] In a further embodiment,

[0216] [ka]

[0217] teeth,

[0218] [ka]

[0219] is selected from.

[0220] In a further embodiment, the compound of formula (A1) has the structure of formula (B1):

[0221] [ka]

[0222] wherein Y is an optional substituent selected from alkylene, heteroalkylene, arylene, heteroarylene, alkylenearylene, alkyleneheteroarylene, alkyleneheterocycloalkylene; Each R aare independently H, halogen, —CF3, —CN, —NO2, OH, NH2, -La-(substituted or unsubstituted alkyl), -La-(substituted or unsubstituted alkenyl), -La-(substituted or unsubstituted heteroaryl), or -La-(substituted or unsubstituted aryl), where La is a single bond, O, S, —S(═O), —S(═O)2, NH, C(O), CH2, —NHC(O)O, —NHC(O) or —C(O)NH; G is

[0223] [ka]

[0224] [ka]

[0225] where R a is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or R6 and R8 form a single bond; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R 12 is H, or lower alkyl; or Y and R taken together 12 forms a 4-, 5- or 6-membered heterocycle; The compound may comprise a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of the compound.

[0226] In a further embodiment, G is

[0227] [ka]

[0228] wherein R is H, alkyl, alkylhydroxy, heterocycloalkyl, heteroaryl, alkylalkoxy, alkylalkoxyalkyl.

[0229] In a further embodiment,

[0230] [ka]

[0231] teeth,

[0232] [ka]

[0233] is selected from.

[0234] In a further embodiment, the compound of formula (B) has the structure of formula (C):

[0235] [ka]

[0236] Y is alkylene, substituted alkylene, or 4-, 5-, or 6-membered cycloalkylene; R 12 is H, or lower alkyl; Or, Y and R taken together 12 forms a 4-, 5- or 6-membered heterocycle; G is

[0237] [ka]

[0238] wherein R, R, and R are independently selected from H, lower alkyl, substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower cycloalkyl, substituted or unsubstituted lower heterocycloalkyl; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of said compound.

[0239] In a further embodiment, the compound of formula (B1) has the structure of formula (C1):

[0240] [ka]

[0241] Y is an optional substituent selected from alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, alkylheterocycloalkyl; R 12 is H, or lower alkyl; or Y and R obtained together 12 forms a 4-, 5- or 6-membered heterocycle; G is

[0242] [ka]

[0243] where R a is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or R6 and R8 form a single bond; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, or C1-C4 alkyl(C2-C8 heterocycloalkyl); and the compound comprises a pharmaceutically active metabolite, pharmaceutically acceptable solvate, pharmaceutically acceptable salt, or pharmaceutically acceptable prodrug of said composition.

[0244] In further or alternative embodiments, the "G" group in any of Formula (A1), Formula (B1), and Formula (C1) is an optional group used to adjust the physical and biological properties of a molecule. Such adjustment / modification can be achieved using groups that adjust the Michael acceptor chemical reactivity, acidity, basicity, lipophilicity, solubility, and other physical properties of the molecule. The physical and biological properties adjusted by such modifications to G include, by way of example only, enhancing the chemical reactivity of Michael acceptor groups, solubility, in vivo absorption, and in vivo metabolism. Furthermore, in vivo metabolism can include, by way of example only, controlling in vivo PK properties, off-target activity, potential toxicity associated with CYP P450 interactions, drug-drug interactions, and the like. Furthermore, modifications to G allow for the tailoring of the in vivo efficacy of a compound by, by way of example only, modulating specific and nonspecific protein binding to plasma proteins and lipids and in vivo tissue distribution.

[0245] In another embodiment, described herein is a compound of formula (D), wherein formula (D) is:

[0246] [ka]

[0247] Here, L a is CH2, O, NH or S; Ar is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; Y is an optional substituent selected from alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, and heteroarylene; Z is C(=O), OC(=O), NHC(=O), C(=S), S(=O)x, OS(=O)x, NHS(=O)x, where x is 1 or 2; R6, R7, and R8 are each independently selected from H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 cycloalkyl, substituted or unsubstituted alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4 alkyl(aryl), substituted or unsubstituted C1-C4 alkyl(heteroaryl), substituted or unsubstituted C1-C4 alkyl(C3-C8 cycloalkyl), or substituted or unsubstituted C1-C4 alkyl(C2-C8 heterocycloalkyl); or R7 and R8 form a single bond; and the pharmaceutical formulation comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of the compound.

[0248] In some embodiments, the compound has the structure of formula (D1):

[0249] [ka]

[0250] L ais CH2, O, NH or S; Ar is an optionally substituted aromatic carbocyclic or aromatic heterocyclic compound; Y is an optional substituent selected from alkylene, heteroalkylene, arylene, heteroarylene, alkylenearylene, alkyleneheteroarylene, alkyleneheterocycloalkylene, or combinations thereof; Z is C(=O), NHC(=O), NR a C(=O)NR a S(=O)x, where x is 1 or 2, and R a is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, substituted or unsubstituted alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido or C1-C4 alkyl(C2-C8 heterocycloalkyl); R6 and R8 are H; R7 is H, optionally substituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 alkoxyaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or R6 and R8 form a single bond; R7 is H, optionally substituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 alkoxyaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or a combination thereof; and the pharmaceutical formulation comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of the compound.

[0251] In another embodiment, pharmaceutically acceptable salts of the compound of formula (D1) are provided. By way of example only, the salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Further salts include those in which the counterion is adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, or the like. Included are salts in which the counterion is an anion such as acid, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Additional salts include those in which the counterion is a cation such as sodium, lithium, potassium, calcium, magnesium, ammonium, and quaternary ammonium (substituted by at least one organic moiety).

[0252] In another embodiment are pharmaceutically acceptable esters of the compounds of formula (DI), including those in which the ester group is selected from formate, acetate, propionate, butyrate, acrylate, and ethylsuccinate.

[0253] In another embodiment, there is a pharmaceutically acceptable carbamate of the compound of formula (D1). In another embodiment, there is a pharmaceutically acceptable N-acyl derivative of the compound of formula (D1). Examples of N-acyl groups include N-acetyl and N-ethoxycarbonyl groups.

[0254] In a further embodiment, L a is O.

[0255] In a further embodiment, Ar is phenyl.

[0256] In further embodiments, Z is C(=O), NHC(=O) or NCH3C(=O).

[0257] In further embodiments, each of R 1 , R 2 and R 3 is H.

[0258] In one embodiment, there is a compound of formula (D1) wherein R6, R7, and R8 are all H. In another embodiment, R6, R7, and R8 are not all H.

[0259] For any and all embodiments, substituents are selected from among a subset of the listed alternatives. For example, in some embodiments, L a is CH, O, or NH. In other embodiments, L a is O or NH. In yet other embodiments, L a is O.

[0260] In some embodiments, Ar is substituted or unsubstituted aryl. In still other embodiments, Ar is 6-membered aryl. In some other embodiments, Ar is phenyl.

[0261] In some embodiments, x is 2. In still other embodiments, Z is C(=O), OC(=O), NHC(=O), S(=O) x , OS(=O) x , or NHS(=O) x In some other embodiments, Z is C(=O), NHC(=O), or S(=O)2.

[0262] In some embodiments, R7 and R8 are independently selected from H, unsubstituted C1-C4 alkyl, substituted C1-C4, unsubstituted C1-C4 heteroalkyl, and substituted C1-C4 heteroalkyl; or R7 and R8 together form a single bond. In other embodiments, each of R7 and R8 is H; or R7 and R8 together form a single bond.

[0263] In some embodiments, R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C6 alkoxyalkyl, C1-C2 alkyl-N(C1-C3 alkyl)2, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4(heteroaryl), C1-C4 alkyl(C3-C8 cycloalkyl), or C1-C4 alkyl(C2-C8 heterocycloalkyl). In some other embodiments, R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C6 alkoxyalkyl, C1-C2 alkyl-N(C1-C3 alkyl), C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C4 alkyl(C3-C8 cycloalkyl), or C1-C4 alkyl(C2-C8 heterocycloalkyl). In still other embodiments, R6 is H, substituted or unsubstituted C1-C4 alkyl, —CH2—O—(C1-C3 alkyl), —CH2—N(C1-C3 alkyl), C1-C4 alkyl(phenyl), or C1-C4 alkyl(five- or six-membered heteroaryl). In some embodiments, R6 is H, substituted or unsubstituted C1-C4 alkyl, -CH2-O-(C1-C3 alkyl), -CH2-N(C1-C3 alkyl)2, C1-C4 alkyl(phenyl), or C1-C4 alkyl(five- or six-membered heteroaryl containing 1 or 2 N atoms), or C1-C4 alkyl(five- or six-membered heterocycloalkyl containing 1 or 2 N atoms).

[0264] In some embodiments, Y is an optional substituent selected from alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl. In other embodiments, Y is an optional substituent selected from C1-C6 alkyl, C1-C6 heteroalkyl, 4-, 5-, 6-, or 7-membered cycloalkyl, and 4-, 5-, 6-, or 7-membered heterocycloalkyl. In still other embodiments, Y is an optional substituent selected from C1-C6 alkyl, C1-C6 heteroalkyl, 5- or 6-membered cycloalkyl containing 1 or 2N atoms, and 5- or 6-membered heterocycloalkyl containing 1 or 2N atoms. In some other embodiments, Y is 5- or 6-membered cycloalkyl containing 1 or 2N atoms, or 5- or 6-membered heterocycloalkyl containing 1 or 2N atoms.

[0265] Any combination of the above groups for various variables is contemplated herein. It is understood that the substituents and substitution patterns on the compounds provided herein can be selected by one skilled in the art to provide compounds that are chemically stable and can be synthesized by techniques known in the art, similar to the compounds specified herein.

[0266] In one embodiment, the irreversible inhibitor of a kinase has the structure of Formula (E):

[0267] [ka]

[0268] During the ceremony, wherein the sphere in formula (E) is a moiety that binds to the active site of a kinase, including tyrosine kinases, and further including Btk kinase cysteine ​​homologs; Y is an optional substituent selected from alkylene, heteroalkylene, arylene, heteroarylene, heterocycloalkylene, cycloalkylene, alkylenearylene, alkyleneheteroarylene, alkylenecycloalkylene, and alkyleneheterocycloalkylene; Z is C(=O), OC(=O), NHC(=O), NCH3C(=O), C(=S), S(=O) x , OS(=O) x , NHS(=O) x where x is 1 or 2; R6, R7, and R8 are H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4 alkyl(aryl), substituted or unsubstituted C1-C4 alkyl(heteroaryl), substituted or unsubstituted C1-C4 alkyl(C3-C8 cycloalkyl), or substituted or unsubstituted C1-C4 alkyl(C2-C8 heterocycloalkyl); or R7 and R8 together form a single bond, and the irreversible inhibitor has a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug thereof.

[0269] In some embodiments, the sphere in formula (E) is a substituted fused biaryl moiety selected from:

[0270] [ka]

[0271] In one embodiment, provided herein is a compound of formula (F):

[0272] [ka]

[0273] During the ceremony, L a is CH2, O, NH or S; Ar is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; (a) Y is an optional substituent selected from among alkylene, heteroalkylene, arylene, heteroarylene, alkylenearylene, alkyleneheteroarylene, alkylenecycloalkylene, and alkyleneheterocycloalkylene; Z is C(=O), NHC(=O), NR a C(=O), NR a S(=O) x where x is 2 or 2 and R a is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and (i) R6, R7, R8 are H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4 alkyl(aryl), substituted or unsubstituted C1-C4 alkyl(heteroaryl), substituted or unsubstituted C1-C4 alkyl(C3-C8 cycloalkyl), or substituted or unsubstituted C1-C4 alkyl(C2-C8 heterocycloalkyl); (ii) R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkylC1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or (iii) R7 and R8 form a single bond; R6 is selected from H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4 alkyl(aryl), substituted or unsubstituted C1-C4 alkyl(heteroaryl), substituted or unsubstituted C1-C4 alkyl(C3-C8 cycloalkyl), or substituted or unsubstituted C1-C4 alkyl(C2-C8 heterocycloalkyl); (b) Y is an optional substituent selected from cycloalkylene or heterocycloalkylene; and Z is C(=O), NHC(=O), NR a C(=O)NR a S(=O) x where x is 1 or 2, and R a is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and (i) R7 and R8 are H; R6 is substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido, or C1-C4 alkyl(C2-C8 heterocycloalkyl); (ii) R6 and R8 are H; R7 is substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamido, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or (iii) R7 and R8 together form a single bond; R6 is or is a substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkylether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl), and Formula (F) is a pharmaceutically active metabolite, pharmaceutically acceptable solvate, pharmaceutically acceptable salt, or pharmaceutically acceptable prodrug thereof.

[0274] Further embodiments of compounds of Formula (A), Formula (B), Formula (C), and Formula (D) include, but are not limited to, compounds selected from the group consisting of:

[0275] [ka]

[0276] [ka]

[0277] [ka]

[0278] In yet another embodiment, the compound provided herein is selected from the following:

[0279] [ka]

[0280] In one aspect, provided herein is a compound selected from the following:That is, 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (compound 4); (E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)but-2-en-1-one (compound 5); 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)sulfo N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-yn-1-one (Compound 8); 1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 9); N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)silicate cyclohexyl)acrylamide (Compound 10); 1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one (Compound 11); 1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1-one (Compound 12); 1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3 ,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 13); 1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (Compound 14); and (E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-(dimethylamino)but-2-en-1-one (Compound 15).

[0281] In some embodiments, the Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one.

[0282] In one embodiment, the Btk inhibitor is α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)propenamide (LFM-A13), AVL-101, 4-tert-butyl-N-(3-(8-(phenylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)benzamide, 5-(3-amino-2-methylphenyl)-1-methyl-3-(4-(morpholine-4-carbonyl)phenyl)propen ... phenylamino)pyrazin-2(1H)-one, N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)acetamide, 4-tert-butyl-N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl ) benzamide, 5-(3-(4-tert-butylbenzylamino)-2-methylphenyl)-1-methyl-3-(4-(morpholine-4-carbonyl)phenylamino)pyrazin-2(1H)-one, 5-(3-(3-tert-butylbenzylamino)-2-methylphenyl)-1-methyl-3-(4-(morpholine-4-carbonyl)phenylamino)pyrazin-2(1H)-one, 3-tert-butyl-N -(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide, 6-tert-butyl-N-(2-methyl-3-(4-methyl-6-(4-(morpholine-4-carbonyl)phenylamino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)nicotinamide, and tereic acid.

[0283] Throughout the specification, groups and substituents thereof can be chosen by one skilled in the art to provide stable moieties and compounds.

[0284] In certain embodiments, any of the Btk inhibitors and / or second agents provided herein in connection with the present invention are included in a pharmaceutical compound that includes: i) a physiologically acceptable carrier, diluent, and / or excipient.

[0285] In some embodiments, the Btk inhibitor of the methods of the present invention is administered at a dose of about 1.25 mg / kg / day to about 12.5 mg / kg / day. In certain embodiments, the Btk inhibitor is administered at a dose selected from the group consisting of about 1.25 mg / kg / day, about 2.5 mg / kg / day, about 5 mg / kg / day, about 8.3 mg / kg / day, or about 12.5 mg / kg / day.

[0286] In some embodiments, biomarkers provided in accordance with the practice of the invention include ZAP-70, CD5, t(14;18), CD38, beta-2 microglobulin, p53 mutation status, ATM mutation status, chromosome 17p deletion, chromosome 11q deletion, surface or intracytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22, CD11c, CD103, chromosome 7q deletion, and V H Selected from mutation status.

[0287] In some embodiments, measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations is a combination of biomarkers, hi certain embodiments, the combination of biomarkers is CD19 and CD5, or CD20 and CD5.

[0288] In other embodiments, the second agent is administered at a dose of about 1.25 mg / kg / day to about 12.5 mg / kg / day. In certain embodiments, the second agent is administered at a dose selected from the group consisting of about 1.25 mg / kg / day, about 2.5 mg / kg / day, about 5 mg / kg / day, about 8.3 mg / kg / day, or about 12.5 mg / kg / day. The dosage of the second agent is based on the measured expression or presence of one or more biomarkers from one or more lymphocyte subpopulations. A person skilled in the art, such as a physician, can easily determine an appropriate regimen (e.g., dosage of the second agent) based on the diagnostic results.

[0289] In another embodiment, the invention provides a method for treating cancer, comprising determining the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations in a subject who has received a dose of a Btk inhibitor; and administering a second agent based on the measured expression profile.

[0290] In another embodiment, the present invention also provides a method for treating cancer, comprising administering a Btk inhibitor sufficient to result in an increase or appearance in the blood of lymphocyte subpopulations defined by immunophenotyping; and administering a second agent once, where the increase or appearance in the blood of the lymphocyte subpopulation is measured.

[0291] In some embodiments, the subject is a human.

[0292] In some embodiments, the Btk inhibitor is administered orally.

[0293] In any of the foregoing aspects, there are further embodiments in which administration is enteral, parenteral, or both, wherein (a) an effective amount of a Btk inhibitor is administered systemically to a mammal, (b) an effective amount of a Btk inhibitor is administered orally to a mammal, (c) an effective amount of a Btk inhibitor is administered intravenously to a mammal, (d) an effective amount of a Btk inhibitor is administered by inhalation, (e) an effective amount of a Btk inhibitor is administered intranasally, and (f) an effective amount of a Btk inhibitor is administered by injection to a mammal, (g) an effective amount of a Btk inhibitor is administered topically (transdermally) to a mammal, (h) an effective amount of a Btk inhibitor is administered by eye drops, or (i) an effective amount of a Btk inhibitor is administered rectally to a mammal.

[0294] In any of the foregoing aspects, there are further embodiments comprising a single administration of an effective amount of the Btk inhibitor, including further embodiments in which (i) the Btk inhibitor is administered once, (ii) the Btk inhibitor is administered to the mammal multiple times per day, (iii) intermittently, or (iv) continuously.

[0295] In any of the foregoing aspects, there are further embodiments comprising multiple administrations of an effective amount of a Btk inhibitor, including further embodiments in which (i) the Btk inhibitor is administered in a single dose; (ii) the multiple administrations are spaced apart every 6 hours; and (iii) the Btk inhibitor is administered to the mammal every 8 hours. In further or alternative embodiments, the method includes a drug holiday, in which administration of the Btk inhibitor is temporarily suspended or the administered dose of the Btk inhibitor is temporarily reduced, and at the end of the drug holiday, dosing of the Btk inhibitor is resumed. The length of the drug holiday can vary from two days to one year.

[0296] In any of the foregoing aspects, there are further embodiments in which administration is enteral, parenteral, or both, wherein (a) an effective amount of the second agent is administered systemically to the mammal; (b) an effective amount of the second agent is administered orally to the mammal; (c) an effective amount of the second agent is administered intravenously to the mammal; (d) an effective amount of the second agent is administered by inhalation; (e) an effective amount of the second agent is administered intranasally; or (f) an effective amount of the second agent is administered by injection to the mammal; (g) an effective amount of the second agent is administered topically (transdermally) to the mammal; (h) an effective amount of the second agent is administered ophthalmically; or (i) an effective amount of the second agent is administered rectally to the mammal.

[0297] In any of the foregoing aspects, there are further embodiments comprising a single administration of an effective amount of the second agent, including further embodiments in which (i) the second agent is administered once, (ii) the second agent is administered to the mammal multiple times daily, (iii) intermittently, or (iv) continuously.

[0298] In any of the foregoing aspects, there are further embodiments comprising multiple administrations of an effective amount of the second agent, including further embodiments wherein (i) the second agent is administered in a single dose, (ii) the multiple administrations are spaced apart every 6 hours, and (iii) the second agent is administered to the mammal every 8 hours. In further or alternative embodiments, the method includes a drug holiday, in which administration of the second agent is temporarily suspended or the dose of the administered second agent is temporarily reduced, and at the end of the drug holiday, dosing of the second agent is resumed. The length of the drug holiday can vary from two days to one year.

[0299] In any of the foregoing embodiments, the second agent is selected from the group consisting of alemtuzumab, arsenic trioxide, asparaginase (pegylated or non-pegylated), bevacizumab, cetuximab, platinum-based compounds such as cisplatin, cladribine, daunorubicin / doxorubicin / idarubicin, irinotecan, fludarabine, 5-fluorouracil, gemtuzumab, methotrexate, paclitaxel™, taxol, temozolomide, thioguanine, or hormones (anti-estrogen, anti-androgen, or gonadotropin-releasing hormone analogs). , interferons such as alpha interferon, nitrogen mustards such as busulfan or melphalan or mechlorethamine, retinoids such as trenoin, topoisomerase inhibitors such as irinotecan or topotecan, tyrosine kinase inhibitors such as gefitinib or imatinib, or agents for treating signs and symptoms induced by such treatments, including allopurinol, filgrastim, granisetron / ondansetron / palonosetron, dronabinol, and the like.

[0300] Preparation of Compounds Compounds of formula (D) can be synthesized using standard synthetic techniques known to those skilled in the art, or in combination with the methods described herein. Additionally, the solvents, temperatures, and other reaction conditions shown herein can be varied by those skilled in the art. The following synthetic methods can also be used as further guidance.

[0301] The chemical reactions can be used in a linear sequence to provide the compounds described herein, or can be utilized to synthesize fragments that are then joined by methods described herein and / or known in the art.

[0302] Formation of a Covalent Bond by Reaction of an Electrophile with a Nucleophile The compounds described herein can be modified using a variety of electrophiles and / or nucleophiles to form new functional groups or substituents. Table 1, entitled "Examples of Covalent Linkages and Their Precursors," lists selected examples of covalent linkages and precursor functional groups that can be used as guidance for combining electrophiles and nucleophiles, producing a wide variety of available electrophile and nucleophile combinations. The precursor functional groups are shown as electrophile and nucleophile groups.

[0303] [Table 1-1]

[0304] [Table 1-2]

[0305] Use of Protecting Groups In the reactions described, it may be necessary to protect reactive functional groups, such as hydroxy, amino, imino, thio, or carboxy groups, desired in the final product to prevent unwanted participation in the reaction. Protecting groups are used to block some or all reactive moieties and prevent such groups from participating in chemical reactions until the protecting group is removed. In one embodiment, each protecting group is removable by a different means. Protecting groups that are cleaved under totally different reaction conditions meet the requirement of differential removal. Protecting groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal, and t-butyldimethylsilyl are used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with the Cbz group, which is acid-labile and removable by hydrogenolysis, and the Fmoc group, which is base-labile. Carboxylic acid and hydroxy reactive moieties can be blocked with acid-labile groups such as t-butyl carbamate, or base-labile groups such as, but not limited to, methyl, ethyl, and acetyl in the presence of amines that are both acid- or base-stable but blocked with hydrolytically removable carbamates.

[0306] Carboxylic acid and hydroxy reactive moieties can also be blocked with hydrolytically removable protecting groups such as benzyl groups, while amine groups capable of hydrogen bonding with acids can be blocked with base-labile groups such as Fmoc. Carboxylic acid reactive moieties can be protected by conversion to simple ester compounds, as exemplified herein, or alternatively, blocked with oxidatively removable protecting groups such as 2,4-dimethoxybenzyl, while coexisting amino groups can be blocked with fluoride-decomposable silyl carbamates.

[0307] Allyl blocking groups are useful in the presence of acid- and base-protecting groups because the former are stable and can be subsequently removed by metal or pi-acid catalysts. For example, allyl-blocked carboxylic acids can be reacted with Pd 0Another form of protecting group is a resin to which a compound or intermediate can be attached. As long as the residue is attached to the resin, the functional group is blocked and cannot react. Once released from the resin, the functional group can react.

[0308] Typical blocking / protecting groups may be selected from:

[0309] [ka]

[0310] Detailed descriptions of other protecting groups, as well as techniques applicable to the creation and removal of protecting groups, are provided in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference in their entireties.

[0311] Further forms of the compound The compounds described herein may have one or more stereocenters, and each center may exist in the R or S configuration. The compounds presented herein include all diastereomeric, enantiomeric, and epimeric forms, as well as the appropriate mixtures thereof. If desired, stereoisomers can be obtained by methods known in the art, such as, for example, separation of stereoisomers by chiral chromatography columns.

[0312] Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by known methods, such as, for example, chromatography and / or fractional crystallization. In one embodiment, enantiomers can be separated by chiral chromatography columns. In another embodiment, enantiomers can be separated by converting the enantiomeric mixture to a diastereomeric mixture by reaction with a suitable optically active compound (e.g., alcohol), separating the diastereomers, and converting the individual diastereomers to their corresponding pure enantiomers (e.g., by hydrolysis). All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered part of the compositions described herein.

[0313] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of the compounds described herein, as well as active metabolites of these compounds with similar activity. In some situations, compounds may exist as tautomers. All tautomers are included within the scope of the compounds described herein. Furthermore, the compounds described herein may exist in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, as well as in unsolvated forms. Solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0314] The unoxidized form of the compound of formula (D) can be prepared from the N-oxide of the compound of formula (D) by treatment with a reducing agent such as, but not limited to, sulfur, sulfur dioxide, triphenylphosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, in a suitable inert organic solvent such as, but not limited to, acetonitrile, ethanol, aqueous dioxane, at 0-80°C.

[0315] In some embodiments, the compounds described herein are prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. In some situations, prodrugs are often useful because they may be easier to administer than the parent drug. They may be, for example, bioavailable by oral administration, whereas the parent drug is not. A prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A non-limiting example of a prodrug is a compound described herein that is administered as an ester ("prodrug") to facilitate transport across cell membranes where solubility adversely affects transport, but is then metabolically hydrolyzed to the active carboxylic acid once inside the cell, where water solubility is beneficial. A further example of a prodrug may be a short-chain peptide (polyamino acid) linked to an acid group, where the peptide is metabolized to reveal the active moiety. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to the biologically, pharmaceutically, or therapeutically active form of the compound. In certain embodiments, prodrugs are enzymatically metabolized by one or more steps or processes into the biologically, pharmaceutically, or therapeutically active form of the compound. To generate a prodrug, a pharmaceutically active compound is modified so that the active compound is regenerated upon in vivo administration. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, mask side effects or toxicity, improve the flavor of a drug, or modify other characteristics or properties of a drug. With knowledge of pharmacological processes and in vivo drug metabolism, one skilled in the art can design a prodrug of a compound once a pharmaceutically active compound is known.(For example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392; Silverman (1992), The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc., San Diego, pages 352-401, Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. (see 1985).

[0316] Prodrug forms of the compounds described herein, where the prodrug is metabolized in vivo to produce a derivative as specified herein, are included within the scope of the claims. In some cases, some of the compounds described herein may be prodrugs for another derivative or active compound.

[0317] Prodrugs are often useful because in some situations they may be easier to administer than the parent drug. They may, for example, be bioavailable by oral administration, whereas the parent drug is not. Prodrugs may also have improved solubility in pharmaceutical compositions over the parent drug. Prodrugs can be designed as reversible drug derivatives for use as modifiers to enhance drug transport to site-specific tissues. In some embodiments, the design of the prodrug increases effective solubility. See, e.g., Fedorak et al., Am. J. Physiol., 269:G210-218 (1995); McLoed et al., Gastroenterol, 106:405-413 (1994); Hochhaus et al., Biomed. Chrom., 6:283-286 (1992); J. Larsen et al., Int. J. Pharmaceutics, 47, 103 (1988); Sinkula et al., J. Pharm. Sci., 64:181-210 (1975); T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series; and Edward B. Roche, Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, all of which are incorporated herein by reference in their entireties.

[0318] The aromatic ring moiety of compounds of formula (D) is susceptible to a variety of metabolic reactions, and therefore, incorporation of appropriate substituents on the aromatic ring structure, such as, by way of example only, halogens, may reduce, minimize or eliminate this metabolic pathway.

[0319] The compounds described herein include isotopically labeled compounds, which are identical to those listed in the various chemical formulas and structures shown herein, except for the fact that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those usually found in nature. Examples of isotopes that can be incorporated into the compounds herein include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 35 S, 18 F, 36 Certain isotopically labeled compounds described herein, e.g., 3 H and 14 Compounds incorporating radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. Additionally, deuterium, i.e., 2 Substitution with isotopes such as H may afford certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

[0320] In additional or further embodiments, the compounds described herein are metabolized upon administration to an organism in need thereof to produce metabolites that are then used to produce a desired effect, including a desired therapeutic effect.

[0321] The compounds described herein can be formed and / or used as pharmaceutically acceptable salts. Types of pharmaceutically acceptable salts include: (1) salts of the free base of the compound with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, metaphosphoric acid, and the like; or salts of acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, and the like. (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth ion (e.g., magnesium, or calcium), or an aluminum ion, or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0322] The corresponding counter ions of pharmaceutically acceptable salts can be analyzed and identified using a variety of methods, including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectrometry, mass spectrometry, or any combination thereof.

[0323] The salts are recovered by using at least one of the following techniques: filtration, filtration followed by precipitation with a non-solvent, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[0324] It should be understood that the reference to a pharmaceutically acceptable salt includes solvent addition forms or crystalline forms thereof, particularly solvates or polymorphs.Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and can be formed during the crystallization process with a pharmaceutically acceptable solvent such as water, ethanol, etc.Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol.Solvates of the compounds described herein can be conveniently prepared or formed during the processes described herein.In addition, the compounds provided herein can exist in unsolvated as well as solvated forms.Generally, solvated forms are considered equivalent to unsolvated forms for the purposes of the compounds and methods provided herein.

[0325] It should be understood that reference to a salt includes solvent addition forms or its crystalline forms, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystalline packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors, such as the recrystallization solvent, the rate of crystallization, and the storage temperature, can cause a single crystalline form to dominate.

[0326] The compounds described herein may be in various forms, including, but not limited to, amorphous, pulverized, and nanoparticle forms. Furthermore, the compounds described herein include crystalline forms, also known as polymorphs. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystalline shapes, optical and electrical properties, stability, and solubility. Various factors, such as recrystallization solvents, crystallization speeds, and storage temperatures, can cause a single crystalline form to dominate.

[0327] Screening and characterization of pharmaceutically acceptable salts, polymorphs, and / or solvates can be accomplished using a variety of techniques, including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermochemical decomposition or thermophysical processes, including, but not limited to, polymorphic transformations. Such methods are used to analyze relationships between polymorphic forms, measure weight loss to determine glass transition temperatures, or for excipient compatibility studies. Such methods include, but are not limited to, differential scanning calorimetry (DSC), modulated differential scanning calorimetry (MDSC), thermogravimetric analysis (TGA), and thermogravimetric and infrared analysis (TG / IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron radiation sources. Various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX), environmental scanning electron microscopy with EDX (in a gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

[0328] Throughout the specification, groups and substituents thereof can be chosen by one skilled in the art to provide stable moieties and compounds.

[0329] Cancer Treatment Regimen In certain embodiments, described herein are methods for treating a hematological malignancy in an individual in need thereof, comprising the steps of: (a) administering to the individual an irreversible Btk inhibitor in an amount sufficient to mobilize a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after the peripheral blood concentration of the mobilized plurality of cells has subsequently decreased. In some embodiments, analyzing the plurality of mobilized cells comprises measuring the period during which the peripheral blood concentration of the mobilized plurality of cells increases compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of mobilized cells has increased for a predetermined period of time. In some embodiments, analyzing the mobilized cells comprises counting the number of mobilized cells in the peripheral blood. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased compared to the number before administration of the Btk inhibitor. In some embodiments, administration of the second cancer treatment regimen occurs after the number of mobilized cells in the peripheral blood has decreased. In some embodiments, analyzing the mobilized cells comprises measuring the period during which the number of mobilized cells in the peripheral blood increases compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering the second cancer treatment regimen after the number of mobilized cells in the peripheral blood has increased for a predetermined period of time.

[0330] In some embodiments, administering a Btk inhibitor before a second cancer treatment regimen reduces an immune-mediated response to the second cancer treatment regimen. In some embodiments, administering a Btk inhibitor before ofatumumab reduces an immune-mediated response to ofatumumab. In some embodiments, the second cancer treatment regimen comprises a chemotherapy agent, a steroid, an immunotherapy agent, a targeted therapy, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, a B cell receptor signaling pathway inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapy, a DNA damaging agent, a proteosome inhibitor, a histone deacylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

[0331] In some embodiments, the second cancer treatment regimen comprises chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

[0332] In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, vincristine and prednisone, and optionally rituximab.

[0333] In some embodiments, the second cancer treatment regimen comprises bendamustine and rituximab.

[0334] In some embodiments, the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab.

[0335] In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, vincristine and prednisone, and optionally rituximab.

[0336] In some embodiments, the second cancer treatment regimen comprises etoposide, doxorubicin, vinristine, cyclophosphamide, prednisolone, and optionally rituximab.

[0337] In some embodiments, the second cancer treatment regimen comprises dexamethasone and lenalidomide.

[0338] Additional cancer treatment regimens include, for example, nitrogen mustards such as bendamustine, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, prednimustine, trofosfamide; alkyl sulfonates such as busulfan, mannosulfan, treosulfan; ethyleneimines such as carboquone, thiotepa, triaziquone; nitrosoureas such as carmustine, fotemustine, lomustine, nimustine, ranimustine, semustine, streptozocin; epoxides such as etoglucide; other alkylating agents such as dacarbazine, mitobronitol, pipobroman, temozolomide; folic acid analogs such as methotrexate, permetrexed, pralatrexate, raltitrexed. Analogues; for example, purine analogues such as cladribine, clofarabine, fludarabine, mercaptopurine, nelarabine, and thioguanine; pyrimidine analogues such as azacitidine, capecitabine, carmofur, cytarabine, decitabine, fluorouracil, gemcitabine, and tegafur; vinca alkaloids such as vinblastine, vincristine, vindesine, vinflunine, and vinorelbine; podophyllotoxin derivatives such as etoposide and teniposide Derivatives); colchicine derivatives, such as demecolcine; taxanes, such as docetaxel, paclitaxel, and paclitaxel poliglumex; other plant alkaloids and natural products, such as trabectedin; actinomycins, such as dactinomycin; anthracyclines, such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, and zorubicin; other cytotoxic antibiotics, such as bleomycin, ixabepilone, mitomycin, and plicamycin; platinum compounds, such as carboplatin, cisplatin, oxaliplatin, and satraplatin; methylhydrazines, such as procarbazine;Sensitizing agents such as aminolevulinic acid, efaproxiral, methyl aminolevulinate, porfimer sodium, and temoporfin; protein kinase inhibitors such as dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, and temsirolimus; and anti-inflammatory drugs such as alitretinoin, altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, estramustine, and hydroxycarbazone. Antineoplastic agents such as fluticasone, irinotecan, lonidamine, masoprocol, miltefosine, mitoguazone, mitotane, oblimersen, pegaspargase, pentostatin, romidepsin, sitimagine seradenovec, tiazofurin, topotecan, tretinoin, and vorinostat; estrogens such as diethylstilbenol, ethinylestradiol, fosfestrol, and polyestradiol phosphate; and estronolone. progestogens such as medroxyprogesterone, megestrol; gonadotropin-releasing hormone analogues such as buserelin, goserelin, leuprorelin, and triptorelin; antiestrogens such as fulvestrant, tamoxifen, and toremifene; antiandrogens such as bicalutamide, flutamide, and nilutamide; enzyme inhibitors such as aminoglutethimide, anastrozole, exemestane, formestane, letrozole, and vorozole; and enzyme inhibitors such as abarelix and degarelix. immunostimulants such as histamine dihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex, and thymopentin; immunosuppressants such as everolimus, gusperimus, leflunomide, mycophenolate, and sirolimus; calcineurin inhibitors such as cyclosporine and tacrolimus; other immunosuppressants such as azathioprine, lenalidomide, methotrexate, and thalidomide; and radiopharmaceuticals such as iobenguane.

[0339] Additional cancer treatment regimens include interferons, interleukins, tumor necrosis factors, growth factors, and the like.

[0340] Additional cancer treatment regimens include, for example, immune stimulants such as ancestim, filgrastim, lenograstim, molgramostim, pegfilgrastim, sargramostim; interferons such as interferon alfa-2a, interferon alfa-2b, interferon alfacon-1, interferon alfa-n1, natural interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, peginterferon alfa-2a, peginterferon alfa-2b; interleukins such as aldesleukin and oprelvekin; other immune stimulants such as BCG vaccine, glatiramer acetate, histamine dihydrochloride, immunocyanin, lentinan, melanoma vaccine, mifamurtide, pegademase, pidotimod, plerixafor, poly I:C, poly ICLC, roquinimex, tasonermin, and thymopentin; Immunosuppressants such as avetimus, alefacept, antilymphocyte immunoglobulin (horse), antithymocyte immunoglobulin (rabbit), eculizumab, efalizumab, everolimus, gusperimus, leflunomide, muromonab-CD3, mycophenolate, natalizumab, and sirolimus; for example, adalimumab, afelimomab, certolizumab pegol, etanercept, and golimumab. TNF-alpha inhibitors such as infliximab; interleukin inhibitors such as anakinra, basiliximab, canakinumab, daclizumab, mepolizumab, rilonacept, tocilizumab, and ustekinumab; calcineurin inhibitors such as cyclosporine and tacrolimus; and other immunosuppressants such as azathioprine, lenalidomide, methotrexate, and thalidomide.

[0341] The additional cancer treatment regimen is adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, infliximab, muromonab-CD3, natalizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, or the like, or a combination thereof.

[0342] Additional cancer treatment regimens include, for example, monoclonal antibodies such as alemtuzumab, bevacizumab, catumaxomab, cetuximab, edrecolomab, gemtuzumab, ofatumumab, panitumumab, rituximab, and trastuzumab; immunosuppressants such as eculizumab, efalizumab, muromab-CD3, and natalizumab; TNF-alpha inhibitors such as adalimumab, afelimomab, certolizumab pegol, golimumab, and infliximab; and anti-cancer drugs such as basiliximab, canakinumab, and rituximab. Interleukin inhibitors such as daclizumab, mepolizumab, tocilizumab, and ustekinumab; radiopharmaceuticals such as ibritumomab tiuxetan and tositumomab; and radiopharmaceuticals such as abagovomab, adecatumumab, alemtuzumab, anti-CD30 monoclonal antibody Xmab2513, anti-MET monoclonal antibody MetMab, apolizumab, apomab, arcitumomab, basiliximab, bispecific antibody 2B1, blinatumomab, brentuximab vedotin, and capromab. Pendetide, cixutumumab, claudiximab, conatumumab, decatumumab, denosumab, eculizumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab, galiximab, ganitumab, gemtuzumab ozogamicin, glembatumumab, ibritumomab, inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lucatumumab, mapatum Other monoclonal antibodies include mab, matuzumab, milatuzumab, monoclonal antibody CC49, nesutumumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab, ramacurimab, ranibizumab, siplizumab, sonepcizumab, tanezumab, tositumomab, trastuzumab, tremelimumab, tucotuzumab-celmoleukin, veltuzumab, visilizumab, volociximab, and zalutumumab.

[0343] Additional cancer treatment regimens include agents that affect the tumor microenvironment, such as cellular signaling networks (e.g., the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which signals from B cell receptors and IgE receptors). In some embodiments, the second agent is a PI3K signaling inhibitor or a syc kinase inhibitor. In one embodiment, the syk inhibitor is R788. In another embodiment, there is a PKCγ inhibitor, such as, by way of example only, enzastaurin.

[0344] Examples of drugs that affect the tumor microenvironment include PI3K signaling inhibitors, syc kinase inhibitors, protein kinase inhibitors such as dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pasonanib, sorafenib, sunitinib, and temsirolimus; and other vascular inhibitors such as GT-111, JI-101, and R1530. Formation inhibitors; for example, AC220, AC480, ACE-041, AMG900, AP24534, Arry-614, AT7519, AT9283, AV-951, axitinib, AZD1152, AZD7762, AZD8055, AZD8931, bafetinib, BAY73-4506, BGJ398, BGT226, BI811283, BI6727, BIB F1120, BIBW2992, BMS-690154, BMS-777607, BMS-863233, BSK-461364, CAL-101, CEP-11981, CYC116, DCC-2036, dinaciclib, dovitinib lactate, E7050, EMD1214063, ENMD-2076, fostamatinib b) Disodium, GSK2256098, GSK690693, INCB18424, INNO-406, JNJ-26483327, JX-594, KX2-391, linifanib, LY2603618, MGCD265, MK-0457, MK1496, MLN8054, MLN8237, MP470, NMS-1116354, NMS-1286937, ON 01919.Na, OSI-027, OSI-930 Btk inhibitor, PF-00562271, PF-02341066, PF-03814735, PF-04217903, PF-04554878, PF-04691502, PF-3758309, PHA-739358, PLC3397, progenipoietin, R547, R763, ramucirumab, regorafenib, RO5185426, SAR103168, S3333333CH Other kinase inhibitors include 727965, SGI-1176, SGX523, SNS-314, TAK-593, TAK-901, TKI258, TLN-232, TTP607, XL147, XL228, XL281RO5126766, XL418, and XL765.

[0345] Further examples of anti-cancer agents for use in combination with the Btk inhibitor compounds include inhibitors of mitogen-activated protein kinase signaling, such as U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY43-9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies (e.g., Rituxan).

[0346] Other anticancer drugs that may be used in combination with Btk inhibitor compounds include adriamycin, dactinomycin, bleomycin, vinblastine, cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; nimesil Visafide; Bizelesin; Bleomycin sulfate; Brequinar sodium; Bropirimine; Busulfan; Cactinomycin; Calsterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cilolemycin; Cladribine; Crisnatol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Daunorubicin hydrochloride; Decitabine; Dextromaplatin; Dezaguanine; Dezaguamine mesylate; Diaziquone; Doxorubicin; Doxorubicin hydrochloride; Droloxifene phen; droloxifene citrate; dromostanolone propionate; zuazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; elbrozole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etopurine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluroocitabine; foskid fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin II (including recombinant interleukin II, or rlL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-la; interferon gamma-lb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;Liarozole hydrochloride; Lometerexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindomide; Mitocalcin; Mitochromine; Mitogillin; Mitomarcin; Mitomycin; Mitospel; Mitotane; Mitoxantrone hydrochloride ;Mycophenolic acid;Nocodazoie;Nogalamycin;Ormaplatin;Oxislan;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate;Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride;Plicamycin;Promestane;Porfimer sodium;Porfiromycin;Prednimustine;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pirazofurin;Riboprin;Rogletimide;Safu Fingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Tallysomycin; Tecogalan sodium; Tegafur; Trexatron hydrochloride; Temoporfin; Teniposide; Teloxylon; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Trecitrate Miphen; trestron acetate; tricibirine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tuburozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglicinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrocidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride;

[0347] Other anticancer agents that may be used in combination with Btk inhibitor compounds include: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; akylfulvene; adecipenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein (anti ... protein)-1;Antiandrogen prostate cancer;Antineoestrogen;Antineoplaston;Antisense oligonucleotide;Aphidicolin glycinate;Apoptosis gene modulator;Apoptosis regulator;Apurinic acid;ara-CDP-DL-PTBA;Arginine deaminase;Asulaculin;Atamestane;Atrimustine;Axinastatin 1;Axinastatin 2;Axinastatin 3;Azasetron;Azatoxin;Azatyrosine;Baccatin III derivative;Balanol;Batimastat;BCR / ABL anta Agonists; Benzochlorins; Benzoylstaurosporines; Beta-lactam derivatives; Beta-arretin; Betaclomicin B; Betulinic acid; bFGF inhibitors; Bicalutamide; Bisantrene; Visazilidinyl spermine; Visafide; Bistraten A; Bizelesin; Breflate; Bropirimine; Budotitanium; Buthionine sulfoximine; Calcipotriol; Calphostin C; Camptothecin derivatives; Canarypox IL-2; Capecitabine; Carboxamido-amino-triazoles; Carboxamidotriazoles; CaRest M3; CARN-700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorin; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; colismycin A; colismycin B; combretastatin A4; combretastatin analogs; conagenin; clambescidin 816; crisnatol; cryptophycin 8;Cryptophycin A derivatives; Curacin A; Cyclopentanthraquinone; Cycloplatam; Sipemycin; Cytarabine ocfosfate; Cytolytic factors; Cytostatin; Dacliximab; Decitabine; Dehydrodidemnin B; Deslorelin; Dexamethasone; Dexphosphamide; Dexrazoxane; Dexverapamil; Diaziquone; Didemnin B; Didox; Diethylnorspermine; Dihydro-5-azacytidine; 9-dioxamycin; Diphenylspiromustine; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Dorothymidine Navinol; Duocarmycin SA; Ebselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Elemene; Emitefur; Epirubicin; Epristeride; Estramustine analogs; Estrogen agonists, estrogen antagonists; Etanidazole; Etoposide phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Flezelastine; Fluasterone; Fludarabine; Fluorodaunornithine hydrochloride; Forfenimex; Formestane; Ho Striesin; fotemustine; gadolinium texaphyrin; gallium nitrate; gallocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hapsulfame; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridone; imiquimod; immune-stimulating peptides; insulin, such as growth factor-1 receptor inhibitors; interferon agonists, interferons; interleukins; iovenous Ann; Iododoxorubicin; Ipomeanol, 4-; Ilopract; Irsogladine; Isobengazole; Isohomohalichondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprolide + estrogen + progesterone; Leuprorelin; Levamisole; Liarozole; Linear polyamine analogs; Lipid-soluble disaccharide peptides; Lipid-soluble platinum compounds;Lissoclinamide; lobaplatin; lombricin; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lisofylline; lytic peptides; maytansine; mannostatin A; marimastat; massoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors (metalloproteinase) inhibitors; menogaril; melbarone; meterelin; methioninase; metoclopramide; MIF inhibitors; mifepristone; miltefosine; millimostim; mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogs; mitonafide; mitotoxin; fibroblast growth factor; saporin; mitoxantrone; mofalotene; molgramostim; monoclonal antibodies, human chorionic gonadotropin ;Monophosphoryl lipid A + myobacterial cell wall SK;Mopidamol;Multidrug resistance gene inhibitors;Exogenous tumor suppressor 1-based therapy;Mustard anticancer drugs;Mycaperoxide B;Mycobacterial cell wall extract;Myriaporone;N-acetyldinaline;N-substituted benzamides;Nafarelin;Nagrestipine;Naloxone + pentazocine;Napavine;Naphterpine;Nartograstim;Nedaplatin;Nemorubicin;Neridronic acid;Neutral endopeptidase;Nilutamide;Nisamycin;Nitric oxide modulators;Nitroxide antioxidants;Nitrulline;O6-benzylguanine;Octreotide ;Oxenon;Oligonucleotides;Onapristone;Ondansetron;Ondansetron;Oracin;Oral cytokine inducers;Ormaplatin;Osateron;Oxaliplatin;Oxaunomycin;Palauamine;Palmitoylrhizoxin;Pamidronic acid;Panaxytriol;Panomifene;Parabactin;Pazeliptin;Pegaspargase;Perdecin;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron;Perfosfamide;Perillyl alcohol;Phenazinomycin;Phenylacetate;Phosphatase inhibitors;Picibanil;Pilocarpine hydrochloride;Pirarubicin;Piritrexim;Pracetin A;Plassetin B; Plasminogen activator inhibitors; Platinum complexes; Platinum compounds; Platinum-triamine complexes; Porfimer sodium; Porfiromycin; Prednisone; Propylbis-acridone; Prostaglandin J2, proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae; Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; Purpurin; Pyrazoloacridine; Pyridoxylated hemoglobin; Polyoxyethylene (poly Oxyethylerie) conjugates; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated leteriptin; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukin; romurtide; roquinimex; rubidinone B1; ruboxyl; safingol; saintpin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetic; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; single-chain antigen-binding protein; sizofiran; sobuzoxane; borocaptate; sodium phenylacetate; sorberol; somatomedin-binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal peptide Tide antagonists; Saradista; Suramin; Swainsonine; Synthetic glycosaminoglycans; Talimustine; Tamoxifen methiodide; Tauromustine; Tazarotene; Tecogalan sodium; Tegafur; Terlapyrylium; Telomerase inhibitors; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Saliblastine; Thiocoraline; Thrombopoietin; Thrombopoietin mimetics; Thymalfasin; Thymopoietin receptor agonists; Thymotrin; Thyroid-stimulating hormone; Ethyl etiopropionine tin; Tirapazamine;Titanocene dichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector-based erythrocyte gene therapy; veraresol; veramine; verudin; verteporfin; vinorelbine; vinxartin; vitaxin; vorozole; zanoteron; zeniplatin; zilascorub; and zinostatin stimalamer.

[0348] Other anti-cancer agents that may be used in combination with Btk inhibitor compounds include alkylating agents, antimetabolites, natural products, or hormones, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, etc.), or triazenes (e.g., dacarbazine, etc.). Examples of antimetabolites include, but are not limited to, folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).

[0349] Examples of alkylating agents that can be used in combination with Btk inhibitor compounds include, but are not limited to, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, melphalan, etc.), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, etc.), or triazenes (e.g., dacarbazine). Examples of antimetabolites include, but are not limited to, folic acid analogs (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).

[0350] Examples of anti-cancer drugs that act by arresting cells in the G2-M phase through stabilized microtubules and that may be used in combination with Btk inhibitor compounds include, without limitation, the following marketed drugs and drugs in development: elbrozole (also known as R-55104), dolastatin 10 (also known as DLS-10 and NSC-376128), mibobrin isethionate (also known as CI-980), vincristine, NSC-639829, discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), altritolutin (Abbott, also known as E-7010), and thiazolinone (Abbott, also known as thiazolinone). Altrilutin A and Altrilutin C), spongistatins (spongistatin 1, spongistatin 2, spongistatin 3, spongistatin 4, spongistatin 5, spongistatin 6, spongistatin 7, spongistatin 8 and spongistatin 9), cemadotin hydrochloride (also known as LU-103793 and NSC-D-669356), epothilones (epothilone A, epothilone B, epothilone C, also known as desoxyepothilone A or dEpoA, epothilone D (KOS-862, also referred to as dEpoB and desoxyepothilone B), epothilone E, epothilone F, epothilone B N-oxide, epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705), 21-hydroxyepothilone D (also known as desoxyepothilone F and dEpoF), 26-fluoroepothilone), auristatin PE (also known as NSC-654663), sobridotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4577) (Pharmacia), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (HungarianAcademy of Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651), SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), cryptophycin 52 (also known as LY-355703), AC-7739 (Ajinomoto, also known as AVE-8063A and CS-39.HCI), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCI and RPR-258062A), vitilebuamide, tublysin A, canadensol, centaureydin (also known as NSC-106969), T-138067 (Tularik, also known as T-67, TL-138067, TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute), physianolide B, laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, also known as MF-569) and TI-138067, narcosine (also known as NSC-5366), nascapine, D-24851 (Asta Medica), A-105972 (Abbott), hemiasterlin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine, also known as MF-191), TMPN (Arizona StateUniversity), vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, inacosin (also known as NSC-698666), 3-lAABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607, RPR (Tuiarik, also known as T-900607), RPR-115781 (Aventis), eleutherobin (such as desmethyleleutherobin, desacetyleleutherobin, isoeleutherobin A, and Z-eleutherobin), carbaeoside, carbaeolin, halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), diozostatin, (-)-phenylahistine (also known as NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), myoseverin B, D-43411 (Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (also known as SPA-110, trifluoroacetate) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), resbelastatin sodium phosphate, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi).

[0351] Biomarkers Disclosed herein, in certain embodiments, is a method for treating a hematological malignancy in an individual in need thereof, comprising: (a) administering to the individual an irreversible Btk inhibitor in an amount sufficient to mobilize a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of irreversible Btk inhibitor is sufficient to induce lymphocytosis of the plurality of cells from the malignancy. In some embodiments, analyzing the mobilized plurality of cells comprises preparing a biomarker profile of a population separated from the plurality of cells. In some embodiments, the biomarker expression profile is used to diagnose, prognose, or generate a predictive profile of the hematological malignancy. In some embodiments, the biomarker profile indicates the expression, expression level, mutation, or presence of a biomarker. In some embodiments, the biomarker is any cytogenetic, cell surface molecule or protein, or RNA expression marker.

[0352] In some embodiments, the biomarkers are: ZAP70; t(14,18) beta-2 microglobulin; p53 mutation status; ATM mutation status; del(17)p; del(11)q; del(6)q; CD5; CD11c; CD19; CD20; CD22; CD25; CD38; CD103; CD138; secreted, surface, or cytoplasmic immunoglobulin expression; V H or a combination thereof. In some embodiments, the method further comprises providing a second cancer treatment regimen based on the biomarker profile. In some embodiments, the method does not further comprise administering based on the biomarker profile. In some embodiments, the method further comprises predicting the efficacy of the treatment regimen based on the biomarker profile.

[0353] In certain embodiments, the methods include diagnosing, determining a prognosis, or creating a predictive profile of a hematological malignancy based on the expression or presence of a particular biomarker. In other embodiments, the methods further include stratifying a patient population based on the expression or presence of a particular biomarker in affected lymphocytes. In yet other embodiments, the methods further include determining a treatment regimen for a subject based on the expression or presence of a particular biomarker in affected lymphocytes. In yet other embodiments, the methods further include predicting a subject's response to treatment based on the expression or presence of a particular biomarker in affected lymphocytes.

[0354] In certain embodiments, provided herein are methods for diagnosing, determining prognosis, or creating a predictive profile for a malignancy in a subject, comprising the steps of: (a) administering to the subject a Btk inhibitor sufficient to increase or cause lymphocyte subpopulations to appear in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations, wherein the expression or presence of the one or more biomarkers is used to diagnose, determine the prognosis for, or create a predictive profile for a hematological malignancy. In one embodiment, the increase or appearance of lymphocyte subpopulations in the blood is measured by immunophenotyping. In another embodiment, the increase or expression of lymphocyte subpopulations in the blood is measured by fluorescence-activated cell sorting (FACS).

[0355] In another aspect, described herein is a method for stratifying a patient population for a malignant tumor, comprising: (a) administering to a subject a Btk inhibitor sufficient to increase or increase the presence of lymphocyte subpopulations in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations; wherein the expression or presence of the one or more biomarkers is used to stratify the patient for treatment of the hematological malignancy. In one embodiment, the increase or increase in the presence of lymphocyte subpopulations in the blood is measured by immunophenotyping. In another embodiment, the increase or increase in the presence of lymphocyte subpopulations in the blood is measured by fluorescence-activated cell sorting (FACS).

[0356] In yet another aspect, provided herein is a method for determining a therapeutic regimen for a subject with a hematological malignancy, comprising the steps of: (a) administering to the subject a Btk inhibitor sufficient to increase or appear in the blood of lymphocyte subpopulations; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations; wherein the expression or presence of the one or more biomarkers is used to determine a therapeutic regimen for treating the hematological malignancy. In one embodiment, the increase or appearance in the blood of lymphocyte subpopulations is measured by immunophenotyping. In another embodiment, the increase or appearance in the blood of lymphocyte subpopulations is measured by fluorescence-activated cell sorting (FACS).

[0357] In yet another aspect, provided herein is a method for predicting a response to treatment of a subject with a hematological malignancy, comprising: (a) administering to the subject a Btk inhibitor sufficient to increase or represent a lymphocyte subpopulation in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations; wherein the expression or presence of the one or more biomarkers is used to predict the subject's response to treatment of the hematological malignancy. In one embodiment, the increase or representation of lymphocyte subpopulations in the blood is measured by immunophenotyping. In another embodiment, the increase or representation of lymphocyte subpopulations in the blood is measured by fluorescence-activated cell sorting (FACS).

[0358] In certain aspects, provided herein are methods for diagnosing, determining prognosis, and creating a predictive profile of a patient's hematological malignancy, comprising measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations in a subject administered a Btk inhibitor, wherein the expression or presence of the one or more biomarkers is used to diagnose, determine the prognosis of, or create a predictive profile of the hematological malignancy. In one embodiment, the dose of the Btk inhibitor is sufficient to increase or express lymphocyte subpopulations defined by immunophenotyping in the blood. In another embodiment, measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations further comprises isolating, detecting, or measuring one or more types of lymphocytes. In yet another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

[0359] In another aspect, described herein is a method for stratifying a population of subjects with a hematological malignancy, comprising measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations in a subject administered a Btk inhibitor, wherein the expression or presence of the one or more biomarkers is used to stratify the patient for treatment of the hematological malignancy. In one embodiment, the dose of the Btk inhibitor is sufficient to increase or express lymphocyte subpopulations defined by immunophenotyping in the blood. In another embodiment, measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations further comprises isolating, detecting, or measuring one or more types of lymphocytes. In yet another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

[0360] In yet another aspect, a method for determining a therapeutic regimen in a subject with a hematological malignancy is disclosed herein, comprising measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations in the subject administered a Btk inhibitor, wherein the expression or presence of the one or more biomarkers is used to determine a therapeutic regimen for treating the hematological malignancy. In one embodiment, the dose of the Btk inhibitor is sufficient to increase or express lymphocyte subpopulations defined by immunophenotyping in the blood. In another embodiment, measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations further comprises isolating, detecting, or measuring one or more types of lymphocytes. In yet another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

[0361] In yet another aspect, described herein is a method for predicting response to treatment in a subject with a hematological malignancy, the method comprising measuring the expression or presence of one or more biomarkers from one or more circulating lymphocytes in the subject administered a Btk inhibitor, wherein the expression or presence of the one or more biomarkers is used to predict the subject's response to treatment for the hematological malignancy. In one embodiment, the dose of the Btk inhibitor is sufficient to increase or express lymphocyte subpopulations defined by immunophenotyping in the blood. In another embodiment, measuring the expression or presence of one or more biomarkers from one or more lymphocyte subpopulations further comprises isolating, detecting, and measuring one or more types of lymphocytes. In yet another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

[0362] As contemplated herein, any biomarker for hematological malignancies may be utilized in some embodiments in the methods of the present invention. These biomarkers include any biomolecule (found in either blood, other body fluids, or tissues) or any chromosomal abnormality that is indicative of a hematological malignancy. In certain embodiments, the biomarkers include TdT, CD5, CD11c, CD19, CD20, CD22, CD79a, CD15, CD30, CD38, CD138, CD103, CD25, ZAP-70, p53 mutation status, ATM mutation status, IgV HThese include, but are not limited to, mutation status of chromosome 17 (del 17p), chromosome 6 (del 6q), chromosome 7 (del 7q), chromosome 11 (del 11q), trisomy 12, chromosome 13 (del 13q), t(11:14) chromosomal translocation, t(14:18) chromosomal translocation, expression of CD10, CD23, beta-2 microglobulin, bcl-2, CD9, presence of Helicobacter pylori, expression of CD154 / CD40, Akt, NF-κB, WNT, Mtor, ERK, MAPK, and Src tyrosine kinase. In certain embodiments, the biomarkers are ZAP-70, CD5, t(14;18), CD38, beta-2 microglobulin, p53 mutation status, ATM mutation status, chromosome 17p loss, chromosome 11q loss, surface or intracytoplasmic immunoglobulin, CD138, CD25, 6q loss, CD19, CD20, CD22, CD11c, CD103, chromosome 7q loss, V Hの mutational state, or a combination thereof.

[0363] In certain embodiments, a subpopulation of subjects with hematological malignancies or pre-hematological malignancies who will benefit from a known treatment regimen is identified by screening candidate subjects for one or more clinically useful biomarkers known in the art. Any clinically useful prognostic marker known to those skilled in the art can be used. In some embodiments, the subpopulation includes patients with chronic lymphocytic leukemia (CLL), and clinically useful prognostic markers of particular interest include, but are not limited to, ZAP-70, CD38, beta-2 microglobulin, and cytogenetic markers such as p53 mutation status, ATM mutation status, and chromosomal defects (such as chromosome 17p deletion and chromosome 11q deletion), all of which are clinically useful prognostic markers for this disease.

[0364] ZAP-70 is a tyrosine kinase that binds to the zeta subunit of the T cell antigen receptor (TCR) and plays a crucial role in T cell activation and development (Chan et al. (1992) Cell 71:649-662). ZAP-70 tyrosine phosphorylates and is essential for mediating signal transduction following TCR stimulation. Overexpression or constitutive activation of tyrosine kinases has been demonstrated to be associated with many malignancies, including leukemia and several types of solid tumors. For example, increased ZAP-70 RNA expression levels are a prognostic marker for chronic lymphocytic leukemia (CLL) (Rosenwald et al. (2001) J. Exp. Med. 194:1639-1647). ZAP-70 is expressed in T cells and natural killer cells, but is not known to be expressed in normal B cells. However, ZAP-70 is expressed at elevated levels in B cells of chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL) patients and, more particularly, in a subset of CLL patients who tend to have a more aggressive clinical course, particularly those with unmutated Ig genes (Wiestner et al., (2003) Blood 101:4944-4951; U.S. Patent Application Publication No. 20030203416). Because there is a correlation between ZAP-70 expression levels and the mutational status of Ig genes, ZAP-70 can be used as a prognostic indicator to identify patients who may have severe disease (high ZAP-70 and unmutated Ig genes) and are therefore candidates for aggressive treatment.

[0365] CD38 is a surface enzyme that catalyzes the synthesis and degradation of cyclic ADP-ribose (cADPR) and is also a signaling molecule. CD38 expression is expressed at high levels on bone marrow precursor B cells, down-regulated on resting normal B cells, and re-expressed on differentiated plasma cells (Campana et al. (2000) Chem. Immunol. 75:169-188). CD38 is a reliable prognostic indicator in B-CLL, and its expression generally indicates a less favorable outcome (D'Arena et al. (2001) Leuk. Lymphoma 42:109; Del Poeta et al. (2001) Blood 98:2633; Durig et al. (2002) Leukemia 16:30; Ibrahim et al. (2001) Blood 98:181; Deaglio et al. (2003) Blood 102:2146-2155). Unfavorable clinical indicators associated with CD38 expression include advanced disease, poor response to chemotherapy, shorter time before initial treatment is required, and shorter survival time (Deaglio et al. (2003) Blood 102:2146-2155). Initially, a strong correlation between CD38 expression and IgV gene mutations was observed, with patients with unmutated V genes exhibiting a higher percentage of CD38.sup.+ B-CLL cells than patients with mutated V genes (Damle et al. (1999) Blood 94:1840-1847). However, later studies showed that CD38 expression does not necessarily correlate with IgV gene rearrangements (Hamblin et al. (2002) Blood 99:1023; Thunberg et al. (2001) Blood 97:1892).

[0366] p53 is a nuclear phosphoprotein that acts as a tumor suppressor. Wild-type p53 is involved in the control of cell growth and division. p53 binds to DNA and stimulates the production of a protein (p21) that interacts with a protein (cdk2) that stimulates cell division. When p21 is bound to cdk2, the cell is prevented from entering the next stage of cell division. Mutant p53 cannot bind DNA effectively, thus preventing p21 from acting as a cell division stop signal, resulting in uncontrolled cell division and tumor formation. p53 also controls the induction of cell apoptosis in response to DNA damage, cellular stress, or aberrant expression of several oncogenic genes. Expression of wild-type p53 in several cancer cell lines has been shown to restore growth suppression control (Casey et al. (1991) Oncogene 6:1791-1797; Takahashi et al. (1992) Cancer Res. 52:734-736). Mutations in p53 are found in most tumor types, including tumors of the colon, breast, lung, ovary, bladder, and many other organs. Mutations in p53 have been found to be associated with Burkitt's lymphoma, L3 B-cell acute lymphoblastic leukemia, and B-cell chronic lymphocytic leukemia (Gaidano et al. (1991) Proc. Natl. Acad. Sci. USA 88:5413-5417). Abnormalities in p53 have also been found to be associated with B-cell prolymphocytic leukemia (Lens et al. (1997) Blood 89:2015-2023). The p53 gene is located in the region 17p13.105-p12 of the short arm of chromosome 17.

[0367] B2-microglobulin is an extracellular protein that noncovalently binds to the α chain of the class I major histocompatibility complex (MHC). B2-microglobulin is detectable in serum and is an adverse prognostic indicator in CLL (Keating et al. (1998) Blood 86:606a) and Hodgekin's lymphoma (Chronowski et al. (2002) Cancer 95:2534-2538). B-2-microglobulin is used clinically in lymphoproliferative disorders, including leukemia, lymphoma, and multiple myeloma, where serum levels correlate with tumor volume, prognosis, and disease activity (Bataille et al. (1983) Br. J. Haematol. 55:439-447; Aviles et al. (1992) Rev. Invest. Clin. 44:215-220). P2-microglobulin is also useful for staging myeloma patients (Pasqualetti et al. (1991) Eur. J. Cancer 27:1123-1126).

[0368] Cytogenetic abnormalities can also be used as markers to create predictive profiles of hematologic malignancies. For example, chromosomal abnormalities are found in a large proportion of CLL patients and are useful in predicting the course of CLL. For example, loss of 17p indicates aggressive disease progression. Furthermore, CLL patients with loss of chromosome 17p, mutations in p53, or both are known to respond poorly to chemotherapy and rituximab. Allelic loss on chromosome 17p can be a useful prognostic marker in colorectal cancer, and patients with loss of 17p are associated with an increased propensity for disease metastasis in colorectal cancer (Khine et al. (1994) Cancer 73:28-35).

[0369] Deletion of the long arm of chromosome 11 (11q) is one of the most common chromosomal structural abnormalities in various types of lymphoproliferative disorders. CLL patients with deletion of chromosome 11q and possible ATM mutation have poorer survival rates than patients without either this defect or deletion of 17p. Furthermore, deletion of 11q is often accompanied by extensive lymph node involvement (Dohner et al. (1997) Blood 89:2516-2522). This deletion also identifies patients at high risk of persistent disease after high-dose therapy and autologous transplantation.

[0370] The ataxia telangiectasia mutated (ATA4) gene is a tumor suppressor gene involved in cell cycle arrest, apoptosis, and DNA double-strand break repair. It is found on chromosome 11. Mutations in ATM are associated with an increased risk of breast cancer in women with a family history of breast cancer (Chenevix-Trench et al. (2002) J. Natl. Cancer Inst 94:205-215; Thorstenson et al. (2003) Cancer Res 63:3325-3333) and / or early-onset breast cancer (Izatt et al. (1999) Genes Chromosomes Cancer 26:286-294; Teraoka et al. (2001) Cancer 92:479-487). Furthermore, rhabdomyosarcoma is frequently associated with mutations / deletions in the ATM gene (Zhang et al. (2003) Cancer Biol. Ther. 1:87-91).

[0371] Methods for detecting chromosomal abnormalities in patients are well known in the art (see, e.g., Cuneo et al. (1999) Blood 93:1372-1380; Dohner et al. (1997) Blood 89:2516-2522). Methods for measuring mutant proteins such as ATM are well known in the art (see, e.g., Butch et al. (2004) Clin. Chem. 50:2302-2308).

[0372] Thus, biomarkers assessed by the methods described herein include cell survival and apoptosis proteins, as well as proteins involved in hematologic malignancy-related signaling pathways. Measurement of expression or presence can be at the protein or nucleic acid level. Thus, biomarkers include these proteins and the genes encoding them. When detection is performed at the protein level, biomarker proteins include full-length polypeptides or any detectable fragments thereof, and may include variants of these protein sequences. Similarly, when detection is performed at the nucleotide level, biomarker nucleic acids include DNA containing full-length coding sequences, fragments of full-length coding sequences, variants of these sequences (e.g., naturally occurring variants or splice variants), or the complements of such sequences. Biomarker nucleic acids also include RNA, such as mRNA, containing the full-length sequence encoding the biomarker protein of interest, fragments of the full-length RNA sequence of interest, or variants of these sequences. Biomarker proteins and biomarker nucleic acids also include variants of these sequences. The term "fragment" refers to a portion of a polynucleotide or amino acid sequence, and thus to the protein encoded thereby. Polynucleotides that are fragments of a biomarker nucleotide sequence generally contain at least 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400 contiguous nucleotides, or up to the number of nucleotides present in a full-length biomarker polynucleotide disclosed herein. Generally, a fragment of a biomarker polynucleotide will encode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids, or up to the total number of amino acids present in a full-length biomarker protein of the invention. "Variant" is intended to refer to a substantially similar sequence.Generally, a variant of a specific biomarker of the present invention has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that biomarker as measured by sequence alignment programs known in the art.

[0373] As provided above, any method known in the art can be used in the methods for measuring the expression or presence of biomarkers described herein. Circulating levels of biomarkers in blood samples obtained from candidate subjects can be measured, for example, by ELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Western blotting, multiplexing techniques, or other similar methods. Cell surface expression of biomarkers can be measured, for example, by flow cytometry, immunohistochemistry, Western blotting, immunoprecipitation, magnetic bead selection, and quantifying cells expressing any of these cell surface markers. Biomarker RNA expression levels can be measured by RT-PCR, Qt-PCR, microarrays, Northern blotting, or other similar techniques.

[0374] As previously mentioned, measuring the expression and presence of a biomarker of interest can be accomplished using any detection method known to those of skill in the art. "Detecting expression" or "detecting the level of" refers to measuring the expression level or presence of a biomarker protein or gene in a biological sample. Thus, "detecting expression" includes instances where a biomarker is measured as not expressed, not detectably expressed, expressed at low levels, expressed at normal levels, or overexpressed.

[0375] In certain aspects of the methods provided herein, one or more lymphocyte subpopulations are isolated, detected, or measured. In certain embodiments, one or more lymphocyte subpopulations are isolated, detected, or measured using immunophenotyping. In other embodiments, one or more lymphocyte subpopulations are isolated, detected, or measured using fluorescence-activated cell sorting (FACS) technology.

[0376] In certain embodiments provided herein, the one or more biomarkers comprise ZAP-70, CD5, t(14;18), CD38, beta-2-microglobulin, p53 mutation status, ATM mutation status, chromosome 17p loss, chromosome 11q loss, surface or intracytoplasmic immunoglobulin, CD138, CD25, 6q loss, CD19, CD20, CD22, CD11c, CD 103, chromosome 7q loss, VH mutation status, or a combination thereof.

[0377] In certain aspects, the method described herein, wherein the measuring step involves measuring the expression or presence of a combination of biomarkers, hi certain embodiments, the combination of biomarkers is CD19 and CD5, or CD20 and CD5.

[0378] In certain aspects, the expression or presence of these various biomarkers, and any clinically useful prognostic markers, in a biological sample can be detected at the protein or nucleic acid level, for example, using immunohistochemistry or nucleic acid-based techniques such as in situ hybridization or RT-PCR. In one embodiment, the expression or presence of one or more biomarkers is determined by nucleic acid amplification methods, nucleic acid array methods, methods utilizing nucleic acid microarrays (DNA and RNA), or methods for in situ hybridization using specifically labeled probes.

[0379] In other embodiments, measuring the expression or presence of one or more biomarkers is performed through gel electrophoresis. In one embodiment, the measurement is performed through transfer to a membrane and hybridization using a specific probe.

[0380] In other embodiments, determining the expression or presence of one or more biomarkers is performed by an imaging technique.

[0381] In yet other embodiments, the expression or presence of one or more biomarkers is determined by a detectable solid substrate, hi one embodiment, the detectable solid substrate is an antibody-functionalized paramagnetic nanoparticle.

[0382] In another aspect, provided herein are methods for detecting or measuring residual lymphoma, the methods following a course of treatment that induces continuous or non-continuous treatment or changes from one therapeutic regimen to another, the methods comprising determining the expression or presence of one or more biomarkers in one or more subpopulations of lymphocytes in a subject, wherein the treatment is treatment with a Btk inhibitor.

[0383] Methods for detecting the expression of the biomarkers described herein, optionally cytokine markers, in a test or control biological sample include any method for determining the amount or presence of these markers at either the nucleic acid or protein level. Such methods are well known in the art and include, but are not limited to, Western blot, Northern blot, ELISA, immunoprecipitation, immunofluorescence, flow cytometry, immunohistochemistry, nucleic acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In certain embodiments, biomarker expression is detected at the protein level, for example, using antibodies directed against specific biomarker proteins. These antibodies can be used in a variety of methods, including Western blot, ELISA, multiplexing techniques, immunoprecipitation, or immunohistochemistry. In some embodiments, detection of cytokine markers is achieved by electrochemiluminescence (ECL).

[0384] Any method for specifically identifying or measuring a biomarker (e.g., a biomarker, a biomarker for cell survival or proliferation, or a biomarker for apoptosis, a biomarker for a Btk-mediated signaling pathway) in a candidate biological sample is contemplated. Thus, in some embodiments, the expression level of a biomarker protein of interest in a biological sample is detected using a binding protein or a biologically active variant thereof that can specifically interact with the biomarker protein. Preferably, a labeled antibody, binding portion thereof, or other binding partner may be used. As used herein, the word "label" refers to a detectable compound or composition that is modified, directly or indirectly, with an antibody to generate a "labeled" antibody. The label may be detectable alone (e.g., a radioisotope label or fluorescent label) or, in the case of an enzymatic label, may catalyze the chemical conversion of a substrate compound or composition that is detectable.

[0385] Antibodies for detecting biomarker proteins can be monoclonal or polyclonal in origin, or they may be synthetically or recombinantly produced. Standard protein detection methodologies known to those of skill in the art are used to measure the amount of protein complexes. A protein complex can be, for example, a binding protein bound to a biomarker, and a binding protein can be, for example, an antibody that specifically binds to a biomarker. Detailed reviews of immunoassay design, theory, and protocols can be found in numerous publications in the art (see, for example, Ausubel et al., eds. (1995) Current Protocols in Molecular Biology (Greene Publishing and Wiley-Interscience, NY); Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY)).

[0386] The choice of marker used to label the antibody varies depending on the application. However, the choice of marker can be easily determined by one of ordinary skill in the art. These labeled antibodies may be used in histological applications or immunoassays to detect the presence of any biomarker or protein of interest. Labeled antibodies may be polyclonal or monoclonal. Furthermore, antibodies used to detect proteins of interest may be labeled with radioactive atoms, enzymes, chromogenic or fluorescent moieties, or colorimetric tags as described elsewhere herein. The choice of whether to tag the label also depends on the desired limit of detection. Enzyme assays (ELISAs) typically allow for the detection of colored products formed by the interaction of an enzyme-tagged complex with an enzyme substrate. Radionuclides useful as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. Examples of enzymes that serve as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase. Chromogenic moieties include, but are not limited to, fluorescein and tiotropium. Antibodies may be modified with these labels by methods known in the art. For example, enzymes and chromophore molecules may be attached to antibodies via coupling agents such as dialdehydes, carbodiimides, and dimaleimides. Alternatively, binding may occur via a ligand-receptor pair. Examples of suitable ligand-receptor pairs are biotin-avidin or biotin-streptavidin, and antibody-antigen.

[0387] In certain embodiments, the expression or presence of one or more biomarkers or other proteins of interest in a biological sample (e.g., a sample of a body fluid) is determined by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA), competitive-binding enzyme-linked immunosorbent assay, dot blot (see, e.g., Promega Protocols and Applications Guide (2nd ed.; Promega Corporation (1991)), Western blot (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Vol. 3, Chapter 18 (Cold Spring Harbor Laboratory Press, Plainview, NY)), chromatography, preferably high-performance liquid chromatography (HPLC), or other assays known in the art. Thus, detection assays can include, but are not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, or immunoprecipitation steps.

[0388] In certain other embodiments, the methods of the present invention are useful for identifying and treating hematological malignancies, including those mentioned above, that are resistant to first-line cancer therapies.

[0389] The expression or presence of one or more of the biomarkers described herein may be measured at the nucleic acid level. Nucleic acid-based techniques for assessing expression are well known in the art and include, for example, measuring the level of biomarker mRNA in a biological sample. Many expression detection methods use isolated RNA. Any RNA isolation technique that does not select as opposed to mRNA isolation can be utilized for RNA purification (see, e.g., Ausubel et al., ed. (1987-1999) Current Protocols in Molecular Biology (John Wiley & Sons, New York)). Furthermore, many tissue samples can be readily processed using techniques well known to those of skill in the art, such as the single-step RNA isolation process disclosed in U.S. Patent 4,843,155.

[0390] Thus, in some embodiments, detection of biomarkers or other proteins of interest is assayed at the nucleic acid level using nucleic acid probes. The term "nucleic acid probe" refers to any molecule capable of selectively binding to a specifically intended target nucleic acid molecule, e.g., a nucleotide transcript. Probes can be synthesized by one skilled in the art or obtained from an appropriate biological preparation. Probes can be specifically designed for labeling, e.g., with a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or other label, or tags or labels described above or known in the art. Examples of molecules that can be used as probes include, but are not limited to, RNA and DNA.

[0391] For example, the isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction analysis, and probe arrays. One method for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the detected gene. The nucleic acid probe can be, for example, a full-length cDNA or a portion thereof consisting of at least 7, 15, 30, 50, 100, 250, or 500 oligonucleotides, sufficient to specifically hybridize under stringent conditions to mRNA or genomic DNA encoding a biomarker, such as the biomarkers described herein. Hybridization of the mRNA with the probe indicates that the biomarker or other target protein of interest is expressed.

[0392] In one embodiment, for example, mRNA is immobilized on a solid surface and contacted with a probe, for example, by running the separated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in a gene chip array. One of skill in the art can readily adapt known mRNA detection methods to detect levels of mRNA encoding biomarkers or other proteins of interest.

[0393] Alternative methods for measuring the level of mRNA for a protein of interest in a sample include, for example, RT-PCR (see, e.g., U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Qβ replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (US Pat. No. The method includes a process of nucleic acid amplification using PCR (e.g., 5,854,033), or any other nucleic acid amplification method, followed by methods for detecting the amplified molecules that are well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low numbers. In certain embodiments of the present invention, biomarker expression is assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® system).

[0394] The expression level of an RNA of interest may be monitored using membrane blots (such as those used in hybridization assays such as Northerns and dots), microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Detection of expression may also involve the use of nucleic acid probes in solution.

[0395] In one embodiment of the present invention, microarrays are used to measure the expression or presence of one or more biomarkers. Microarrays are particularly suitable for this purpose because of their reproducibility between different experiments. DNA microarrays provide one method for simultaneously measuring the expression levels of many genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. The hybridization intensity of each probe on the array is measured and converted to a quantitative value corresponding to the relative gene expression level. See U.S. Patent Nos. 6,040,138, 5,800,992, 6,020,135, 6,033,860, and 6,344,316, all of which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for ascertaining gene expression profiles for many RNAs in a sample.

[0396] Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent 5,384,261, which is incorporated herein by reference in its entirety. While planar array surfaces are preferred, arrays may be fabricated on surfaces of virtually any shape, or even multiple surfaces. Arrays may be peptide or nucleic acid arrays on beads, gels, polymeric surfaces, fibers such as fiber optics, glass, or other suitable substrates; see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5,800,992, each of which is incorporated herein in its entirety for all purposes. Arrays may also be packaged to enable diagnostic or other operations of the all-inclusive device. See, for example, U.S. Patent Nos. 5,856,174 and 5,922,591, which are incorporated herein by reference in their entirety.

[0397] Pharmaceutical Compositions / Preparations Pharmaceutical compositions can be prepared by using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate the processing of active compounds into pharmaceutical preparations.Suitable preparations depend on the route of administration selected.All well-known techniques, carriers and excipients can be used as suitable and as understood in the art. Summaries of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference in their entireties.

[0398] As used herein, a pharmaceutical composition refers to a mixture of a compound described herein, such as a compound of Formula D or a second agent, with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents, and / or excipients. A pharmaceutical composition facilitates administration of a compound to a living organism. In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a compound described herein is administered in a pharmaceutical composition to a mammal suffering from the disease, disorder, or condition to be treated. Preferably, the mammal is a human. A therapeutically effective amount can vary widely, depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. The compound can be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0399] In certain embodiments, pharmaceutical compositions optionally include one or more pH adjusting or buffering agents, such as: acids, such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; alkalis, such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trihydroxymethylaminomethane; and buffers, such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

[0400] In other embodiments, the composition also contains one or more salts in an amount needed to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0401] As used herein, the term "pharmaceutical combination" refers to a product resulting from the mixing or combination of one or more active ingredients, including both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that both active ingredients, such as a compound described herein and an auxiliary agent, are administered to a patient simultaneously in the form of a single entity or single dose. The term "unfixed combination" means that the active ingredients, such as a compound described herein and an auxiliary agent, are administered to a patient simultaneously, in parallel, or sequentially separately, without any specific time limit between them, such that such administration provides an effective level of the two compounds to the patient's body. The latter also applies to cocktail therapy, for example, the administration of three or more active ingredients.

[0402] The pharmaceutical formulations described herein can be administered to a subject by multiple routes of administration, including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), nasal, buccal, topical, rectal, or transdermal routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolve formulations, tablets, capsules, tablets, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and immediate mix and controlled release formulations.

[0403] Pharmaceutical compositions containing the compounds described herein can be manufactured in a conventional manner, by means of, by way of example only, conventional mixing, dissolving, granulating, dragee-making, micronizing, emulsifying, encapsulating, entrapping, or compressing processes.

[0404] "Antifoaming agents" reduce foaming during processing, which can lead to coagulation of the aqueous dispersion, resulting in bubbles in the finished coating, or generally hindering processing. Typical antifoaming agents include silicone emulsions or sorbitan sesquoleate.

[0405] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium bisulfite, and tocopherol. In certain embodiments, antioxidants enhance chemical stability when needed.

[0406] In certain embodiments, the compositions provided herein also contain one or more preservatives that inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0407] The formulations described herein may benefit from antioxidants, metal chelators, thiol-containing compounds, and other general stabilizers. Examples of these stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0408] "Binders" impart cohesive properties and include, for example, alginic acid and its salts; cellulose derivatives such as carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®); microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crospovidone; povidone; starch; alpha-starch; sugars such as tragacanth, dextrin, sucrose (e.g., Dipac®), glucose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), and lactose; natural or synthetic gums such as acacia, tragacanth, ghatti gum, and isapol bark. husk mucus, polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone® XL-10), larch arabogalactan, Veegum®, polyethylene glycol, wax, sodium alginate...

Claims

1. A pharmaceutical composition for use in treating mantle cell lymphoma in an individual, said pharmaceutical composition comprising 560 mg of a BTK inhibitor and formulated for a continuous once-daily dosing regimen, and said BTK inhibitor having the following structure: 【Chemistry 1】

2. 10. The pharmaceutical composition of claim 1, wherein the individual has previously received at least one treatment for the mantle cell lymphoma.

3. The pharmaceutical composition described in claim 1 or 2, wherein the mantle cell lymphoma is recurrent or refractory mantle cell lymphoma.

4. A pharmaceutical composition described in any one of claims 1-3, wherein the pharmaceutical composition is for administration with an additional cancer treatment.

5. 5. The pharmaceutical composition of claim 4, wherein the additional cancer treatment is bendamustine, bortezomib, CAL-101, chlorambucil, cyclophosphamide, dexamethasone, docetaxel, doxorubicin, endostatin, everolimus, etoposide, fludarabine, fostamatinib, hydroxydaunorubicin, ibritumomab, ifosfamide, lenalidomide, mesalazine, ofatumumab, paclitaxel, pentostatin, prednisone, rituximab, temsirolimus, thalidomide, tositumomab, vincristine, or a combination thereof.

6. A pharmaceutical composition described in any one of claims 1-5, wherein the pharmaceutical composition is in the form of a solid oral dosage form.

7. 7. The pharmaceutical composition of claim 6, wherein the solid oral dosage form is a capsule.

8. 7. The pharmaceutical composition of claim 6, wherein the solid oral dosage form is a tablet.

9. 9. The pharmaceutical composition of any one of claims 1-8, wherein after administration of the BTK inhibitor, the individual achieves stable disease, partial remission, or complete remission.

10. 9. The pharmaceutical composition of any one of claims 1-8, wherein the individual achieves a partial or complete remission after administration of the BTK inhibitor.

11. 9. The pharmaceutical composition of any one of claims 1-8, wherein the individual achieves complete remission after administration of the BTK inhibitor.

12. 1. A pharmaceutical composition for use in treating mantle cell lymphoma in an individual who has previously received at least one therapy for mantle cell lymphoma, the pharmaceutical composition comprising 560 mg of a BTK inhibitor and formulated for a continuous once-daily dosing regimen until progression of the mantle cell lymphoma or unacceptable toxicity is observed, wherein lymphocytosis is not considered progression of the mantle cell lymphoma, and the BTK inhibitor has the structure: 【Chemistry 1】 and the individual achieves a complete remission.

13. Administration of the BTK inhibitor results in > about 100 ng ＊ AUC in h / mL （０－２４） The pharmaceutical composition of claim 12, wherein

14. The pharmaceutical composition described in claim 12 or 13, wherein administration of the BTK inhibitor results in >90% of the BTK active sites in peripheral blood mononuclear cells of the individual being occupied by the BTK inhibitor 24 hours after administration.

15. A pharmaceutical composition described in any one of claims 12-14, wherein the once-daily administration regimen is continued for at least 6 months.

16. 1. A pharmaceutical composition for use in the treatment of mantle cell lymphoma, the pharmaceutical composition comprising 560 mg of a BTK inhibitor and formulated for a continuous once-daily dosing regimen until progression of the mantle cell lymphoma or unacceptable toxicity is observed, wherein lymphocytosis is not considered progression of the mantle cell lymphoma, and the BTK inhibitor has the structure: 【Chemistry 1】 and the individual achieves a complete remission.

17. The pharmaceutical composition of claim 16 , wherein the individual is treatment-naive.

18. Administration of the BTK inhibitor results in > about 100 ng ＊ AUC in h / mL （０－２４） The pharmaceutical composition according to claim 16 or 17, wherein

19. A pharmaceutical composition described in any one of claims 16-18, wherein administration of the BTK inhibitor results in >90% of the BTK active sites in peripheral blood mononuclear cells of the individual being occupied by the BTK inhibitor 24 hours after administration.

20. A pharmaceutical composition described in any one of claims 16-19, wherein the once-daily administration regimen is continued for at least 6 months.

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