NZ755485B2 - The use of inhibitors of Bruton's tyrosine kinase (Btk) - Google Patents

Abstract

Use of ibrutinib in the manufacture of a medicament for treating multiple myeloma (MM) in an individual in need thereof, wherein the medicament is formulated for administration at an ibrutinib dosage of about 420 to about 840 mg once per day in a solid oral dosage form. Preferably the medicament is in the form of capsules containing a once per day dosage of 420 mg, 560 mg or 840 mg. in the form of capsules containing a once per day dosage of 420 mg, 560 mg or 840 mg.

Description

THE USE OF INHIBITORS OF BRUTON'S TYROSINE KINASE (BTK) RELATED APPLICATIONS The present application claims the benefit of priority from U.S. ional Patent Application No. 61/351,130, filed June 3, 2010; U.S. Provisional Patent ation 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. ,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 herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION 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 cells types except T lymphocytes and natural killer cells. Btk plays an essential role in the B-cell signaling pathway g cell surface B-cell receptor (BCR) stimulation to downstream intracellular responses.

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 a number of other hematopoietic cell signaling ys, e.g., Toll like receptor (TLR) and cytokine receptor–mediated TNF-α tion in hages, IgE receptor (FcepsilonRI) signaling in Mast cells, inhibition of Fas/APO-1 apoptotic signaling in B-lineage lymphoid cells, and collagen-stimulated platelet aggregation.

See, e.g., C. A. Jeffries, et al., (2003), Journal of Biological Chemistry 278:26258-26264; N. J.

Horwood, et al., (2003), The Journal of Experimental Medicine 197:1603-1611; Iwaki et al. (2005), Journal of Biological try ):40261-40270; 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 [0003a] In a first aspect of the invention, there is provided use of ibrutinib in the manufacture of a medicament for treating multiple myeloma (MM) in an dual in need thereof, n the (26859011_1):AXG medicament is formulated for administration at an ibrutinib dosage of about 420 to about 840 mg once per day in a solid oral dosage form.

Disclosed herein, in certain embodiments, is a method for treating a hematological malignancy in an dual 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 ments, the amount of the irreversible Btk inhibitor is ient to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, the hematological malignancy is CLL. In some embodiments, the treating the hematological malignancy comprises managing the (26859011_1):AXG hematological malignancy. In some ments, the logical malignancy is a B-cell malignancy. In some embodiments, the hematological malignancy is a leukemia, lymphoproliferative disorder, or d. In some embodiments, the mobilized cells are myeloid cells or lymphoid cells. In some embodiments, analyzing the zed plurality of cells comprises measuring the peripheral blood tration of the mobilized plurality of cells. In some embodiments, the method r comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases as compared to the tration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a subsequent decrease in peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer ent regimen after the eral blood tration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method r comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as 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 number of mobilized ity of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises ing the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiirther comprises administering a second cancer ent 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 comprises ing a biomarker profile for a population of cells isolated from the plurality of cells, wherein the biomarker profile indicates the expression of a biomarker, the expression level of a biomarker, mutations in a biomarker, or the presence of a biomarker. In some ments, the biomarker is any cytogenetic, cell surface molecular or protein or RNA expression marker. In some embodiments, the biomarker is: ZAP70; t(14,18); [3-2 microglobulin; p53 mutational status; ATM mutational status; del(17)p; del(11)q; del(6)q; CD5; CD1 1c; CD19; CD20; CD22; CD25; CD38; CD103; CD138; secreted, e or asmic immunoglobulin expression; VH mutational status; or a combination thereof. In some embodiments, the method further ses providing a second cancer treatment regimen based on the biomarker profile. In some embodiments, the method fiarther comprises not administering based on the biomarker profile. In some embodiments, the method further comprises predicting the efficacy of a treatment regimen based on the biomarker . In some embodiments, the hematological malignancy is a chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, or a 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 high grade B cell lymphoma, or extranodal marginal zone B cell lymphoma. In some embodiments, the hematological malignancy is chronic myelogenous (or myeloid) leukemia, or acute lymphoblastic leukemia. In some embodiments, the hematological malignancy is relapsed or tory diffuse large B-cell ma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or tory CLL; relapsed or tory SLL; ed or refractory multiple myeloma. In some embodiments, the Btk inhibitor forms a covalent bond with a cysteine ain of a Bruton’s tyrosine kinase, a Bruton’s tyrosine kinase homolog, or a Btk tyrosine kinase cysteine homolog, In some ments, the irreversible Btk inhibitor is (R)(3-(4-amino(4-phenoxyphenyl)—1H—pyrazolo[3,4-d]pyrimidinyl)piperidinyl)prop- 2-enone. In some embodiments, the amount of the irreversible Btk inhibitor is from 300 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of the irreversible Btk inhibitor is from 420 mg/day up to, and including, 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 AUC0_24 of the Btk inhibitor is between about 150 and about 3500 ng*h/mL. In some embodiments, the 4 of the Btk inhibitor is between about 500 and about 1100 ng*h/mL. In some embodiments, the Btk inhibitor is stered orally. In some embodiments, the Btk tor is administered once per day, twice per day, or three times per day. In some embodiments, the Btk inhibitor is stered until disease progression, unacceptable toxicity, or individual . 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 e ssion, unacceptable toxicity, or individual choice. In some embodiments, the Btk inhibitor is a front line therapy, second line therapy, third line therapy, fourth line therapy, fifth line therapy, or sixth line therapy. In some ments, the Btk tor treats a refractory hematological malignancy. In some embodiments, the Btk inhibitor is a maintenance therapy. 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 ments, the second cancer treatment regimen comprises a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway tor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk tor, a PI3K inhibitor, a Blnk inhibitor, a PLCy inhibitor, a PKCB inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, a histone ylase tor, a protein kinase tor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jakl/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, ifosphamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, bine, fostamatinib, paclitaxel, docetaxel, umab, rituximab, dexamethasone, sone, CAL- lOl, ibritumomab, tositumomab, bortezomib, pentostatin, atin, 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 mab. In some embodiments, the second cancer treatment regimen ses hosphamide, 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 sidechain of a Bruton’s tyrosine kinase, a Bruton’s tyrosine kinase g, or a Btk tyrosine kinase cysteine homolog. In some embodiments, the inhibitor of Bruton’s tyrosine kinase has the structure of Formula (D): N \ \ lk / ’N N T Y\Z R6 R‘8 R7 Formula (D) wherein: La is CH2, 0, NH or S; Ar is a tuted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; Y is an optionally substituted group ed from among 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 l or 2; R7 and R8 are independently H; or R7 and R8 taken together form a bond; R6 is H; and pharmaceutically active metabolites, or pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs f. In some embodiments, the Bruton’s tyrosine kinase inhibitor is (R)-l-(3-(4-amino(4-phenoxyphenyl)— lH-pyrazolo[3 ,4-d]pyrimidin-l-yl)piperidiny1)propen-l-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)2. In some embodiments, each of R7 and R8 is H. In some embodiments, Y is a 4-, 5-, 6-, or 7-membered cycloalkyl ring; or Y is a 4-, 5-, 6-, or ered heterocycloalkyl ring.

Disclosed herein, in certain embodiments, is a method for treating relapsed or refractory dgkin’s lymphoma in an dual in need thereof, comprising: administering to the individual a eutically-effective amount of (R)(3-(4-amino(4-phenoxypheny1)-1H- pyrazolo[3,4-d]pyrimidinyl)piperidinyl)propenone. In some embodiments, the non- Hodgkin’s lymphoma is relapsed or refractory diffuse large B-cell ma (DLBCL), relapsed or refractory mantle cell lymphoma, or relapsed or refractory ular lymphoma. In some embodiments, the amount of (R)—l—(3—(4—amino—3—(4—phenoxyphenyl)—lH-pyrazolo[3,4- d]pyrimidin-l-yl)piperidin-l-yl)prop—2—en—1 —one is from 300 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of (R)—1—(3—(4-amino(4-phenoxyphenyl)—lH- pyrazolo[3,4-d]pyrimidin- l -yl)piperidinyl)prop—2—en— 1 -one is from 420 mg/day up to, and including, 840 mg/day. In some embodiments, the amount of (R)(3-(4-amino(4- phenoxyphenyl)— 1 H-pyrazolo [3 ,4-d]pyrimidiny1)piperidinyl)propenone is about 420 mg/day, about 560 mg/day, or about 840 . In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg/day. In some embodiments, the AUC0_24 of the Btk inhibitor is between about 150 and about 3500 ng*h/mL. In some embodiments, the AUC0_24 of the Btk inhibitor is between about 500 and about 1100 ng*h/mL. In some embodiments, (R)-l- (3 -(4-amino(4-phenoxypheny1)-1H—pyrazolo[3,4-d]pyrimidiny1)piperidin- l -yl)propen- 1-one is administered orally. In some embodiments, (R)(3-(4-amino(4-phenoxyphenyl)- azolo[3,4-d]pyrimidinyl)piperidinyl)propenone is administered once per day, twice per day, or three times per day. In some embodiments, (R)(3-(4-amino(4- phenoxyphenyl)— 1 H—pyrazolo [3 ,4—d]pyrimidin—1—yl)piperidinyl)propenone is administered until e progression, unacceptable toxicity, or individual choice. In some embodiments, (R)(3-(4-amino-3—(4—phenoxyphenyl)— 1 H-pyrazolo [3 yrimidin yl)piperidin-l-yl)propenone is administered until disease progression, unacceptable toxicity, or individual choice. In some embodiments, (R)(3-(4-amino(4-phenoxyphenyl)- 1H-pyrazolo [3 ,4-d]pyrimidinyl)piperidinyl)prop—2-enone is administered daily until disease progression, unacceptable toxicity, or individual choice. In some embodiments, (R) (3 -(4-amino(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidinyl)piperidinyl)propen- 1-one is administered every other day until disease progression, unacceptable toxicity, or individual choice. In some embodiments, (R)—1-(3-(4-amino(4-phenoxyphenyl)-1H- pyrazolo [3 ,4-d]pyrimidinyl)piperidin— 1 —yl)propen— 1 -one is a second line therapy, third line therapy, fourth line therapy, fifth line therapy, or sixth line therapy. In some embodiments, the Btk inhibitor is a nance therapy. In some embodiments, the method fiirther ses administering a second cancer treatment regimen. In some embodiments, the second cancer treatment n is administered after zation of a plurality of lymphoid cells from the non-Hodgkin’s lymphoma. In some embodiments, the second cancer treatment regimen is administered after lymphocytosis of a plurality of lymphoid cells from the dgkin’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 ments, the second cancer ent 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 tor, a PLCy inhibitor, a PKCB inhibitor, or a combination thereof In some embodiments, the second cancer treatment regimen comprises an dy, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jakl/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, ifosphamide, bicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, mab, dexamethasone, prednisone, CAL-101, momab, 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 mab. In some embodiments, the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and Iituximab. In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, Vincristine, and prednisone, and ally, 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 domide.

[0006] Disclosed herein, in certain embodiments, is a method for treating diffiJse large B-cell lymphoma, activated B cell-like subtype LBCL), in an individual in need thereof, comprising: administering to the individual an irreversible Btk inhibitor in an amount from 300 mg/day up to, and including, 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 the gene ce of one or more kers in a plurality of lymphoid cells isolated from the diffuse large B—cell lymphoma. In some embodiments, the irreversible Btk inhibitor is (3 -(4-amino(4-phenoxyphenyl)- 1 H-pyrazolo [3 ,4- d]pyrimidin-l-yl)piperidin-l-yl)propenone. In some ments, the ABC-DLBCL is characterized by a CD79B mutation. In some embodiments, the CD79B on is a mutation of the immunoreceptor tyrosine—based activation motif (ITAM) signaling module. In some ments, the CD79B mutation is a missense mutation of the first immunoreceptor tyrosine- based tion motif (ITAM) ne. In some embodiments, the CD79B mutation increases surface BCR expression and attenuates Lyn kinase activity. In some embodiments, the ABC- DLBCL is characterized by a CD79A on. In some embodiments, the CD79A on is in the immunoreceptor tyrosine-based activation motif (ITAM) signaling module. In some embodiments, the CD79A mutation is a splice-donor—site mutation of the immunoreceptor tyrosine-based activation motif (ITAM) signaling module. In some embodiments, the CD79A mutation s the immunoreceptor tyrosine-based activation motif (ITAM) signaling module.

In some ments, the ABC-DLBCL is characterized by a mutation in MyD88, A20, or a combination thereof In some embodiments, the MyD88 mutation is the amino acid substitution L265P in the MYD88 Toll/IL-l receptor (TIR) domain. In some embodiments, the amount of the irreversible Btk inhibitor is from 420 mg/day up to, and including, 840 mg/day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg/day, about 560 , or about 840 mg/day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg/day. In some embodiments, the AUC0_24 of the Btk inhibitor is n about 150 and about 3500 ng*h/mL. In some embodiments, the AUC0_24 of the Btk inhibitor is between about 500 and about 1100 L. In some 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 e progression, unacceptable toxicity, or individual choice. In some embodiments, the irreversible Btk tor is a front 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 logical malignancy. In some embodiments, the irreversible Btk inhibitor is a maintenance therapy. In some embodiments, the method fiarther 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, ion therapy, a targeted therapy, or a ation thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, B cell or signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a mmunotherapeutic, is a damaging agent, a proteosome 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.

Disclosed herein, in certain embodiments, is a method of determining a cancer treatment regimen for an dual with a logical malignancy, comprising: (a) administering to the dual an amount of an irreversible Btk inhibitor sufficient to mobilize a plurality of cells from the malignancy; (b) ing 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 PLCy inhibitor, a PKCB inhibitor, or a combination thereof. In some embodiments, the cancer treatment regimen comprises a B cell receptor pathway inhibitor.

In some ments, the cancer treatment regimen comprises a CD79A inhibitor, a CD79B tor, a CD19 inhibitor, a Lyn tor, a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCy inhibitor, a PKCB tor, or a combination thereof In some embodiments, the cancer treatment regimen comprises an antibody, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteosome inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jakl/2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof. In some embodiments, the cancer ent regimen comprises mbucil, ifosphamide, doxorubicin, mesalazine, omide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, umab, rituximab, thasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof. In some embodiments, the cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, stine, and prednisone, and optionally, rituximab. In some ments, the cancer treatment regimen comprises bendamustine, and mab. In some embodiments, the cancer treatment regimen comprises bine, cyclophosphamide, and rituximab. In some embodiments, the cancer treatment regimen comprises cyclophosphamide, Vincristine, and sone, and optionally, rituximab. In some ments, the cancer treatment regimen comprises etoposide, doxorubicin, tine, cyclophosphamide, prednisolone, and optionally, rituximab. In some embodiments, the cancer treatment regimen comprises dexamethasone and lenalidomide.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 depicts the role of Btk activity in a number of processes in a CLL cell that contribute to the pathogenesis of the disease Fig. 2 presents the absolute yte count during the course of ent with an irreversible Btk inhibitor for an individual with CLL.

Fig. 3 presents change in the sum of the product of the diameters of lymph node (LN) in patients with CLL and SLL who are treated with an irreversible Btk inhibitor.

Fig. 4 depicts LN response in patient suffering from CLL. Left panel depicts LN prior to ent with an irreversible Btk inhibitor and Right panel depicts LN post-treatment with an irreversible Btk inhibitor.

Fig. 5 depicts the effect of an rsible Btk inhibitor on LN disease burden and lymphocytosis over time in the patients suffering with CLL and/or SLL.

[0013] Fig. 6 depicts adverse effects in patients treated with an irreversible Btk inhibitor.

Grades 1-4 ent severity of effects with 1 representing very mild to 4 representing extreme discomfort.

Fig. 7 depicts the absolute cyte count (ALC)/109 L vs. Cycle Day after administering a Btk inhibitor to individuals with follicular lymphoma who achieved complete or partial response (ClVPR). The Y Axis shows the Absolute Lymphocyte Counts (ALC) at each time point by cycle number and day in the X axis. All Patients (except Pt 32009) were treated on schedule of 4 weeks on treatment followed by one week off. Thus, dayl of each cycle s one week off drug for these patients. Note the ses of ALC during most cycles of most patients, and the fall ofALC at the beginning of subsequent cycles. This pattern is often blunted in later cycles as patients ded to treatment. Patient 32009 received treatment without interruption and did not show this cyclic pattern, but did show an increase at Cycle 1, daylS, and gradual increases during Cycles 2 to 5.

Fig. 8 s the absolute lymphocyte count (ALC)/109 L vs. Cycle Day after administering a Btk inhibitor to duals with follicular lymphoma who had Stable Disease (SD) during treatment. The Y Axis shows the te Lymphocyte Counts (ALC) at each time point by cycle number and day in the X axis. All Patients were treated on schedule of 4 weeks on treatment followed by one week off. Thus, day] of each cycle follows one week off drug for these ts. Note the gradual increase of blood ALC mobilization of Patient 32004, who initially was stable but later had Progressive Disease (PD).

[0016] Fig. 9 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Day after administering a Btk inhibitor to PD individuals with follicular lymphoma. The Y Axis shows the Absolute Lymphocyte Counts (ALC) at each time point by cycle number and day in the X axis.

All Patients except 38010 were treated on schedule of 4 weeks on treatment followed by one week off. Thus, dayl of each cycle s one week off drug for these patients. Note lack of mobilization, especially patients 38010 and 3200]. t 323001 had limited treatment before being taken off study. The lymphocyte response suggests that this patient might had responded if it had been possible to stay on treatment longer.

Fig. 10 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Day after administering a Btk inhibitor to PR and SD individuals with DLBCL. The Y Axis shows the Absolute Lymphocyte Counts (ALC) at each time point by cycle number and day in the X axis.

Patient 38011 was d on le of 4 weeks on treatment followed by one week off. Thus, dayl of each cycle follows one week off drug for this patient. Patients 38008 and 324001 were treated with continuous daily doses.

Fig. 11 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Day after administering a Btk inhibitor to PD individuals with DLBCL. The Y Axis shows the Absolute Lymphocyte Counts (ALC) at each time point by cycle number and day in the X axis. All Patients were treated on schedule of 4 weeks on treatment followed by one week off. Thus, dayl of each cycle s one week off drug for these patients. Note lack of mobilization for 3 of the 4 patients. Patient 32002 received only one cycle of ent.

Fig. 12 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Day after stering a Btk inhibitor to individuals with mantle cell lymphoma. The Y Axis shows the Absolute Lymphocyte Counts (ALC) at each time point by cycle number and day in the X axis.

Patients 32006, 38003, and 38004 were treated on schedule of 4 weeks on treatment followed by one week off. Thus, dayl of each cycle follows one week off drug for these patients. The other patients were treated with continuous daily . Note that the patient with initial PD (32014) failed to show mobilization.

Fig. 13 depicts the absolute lymphocyte count (ALC)/109 L vs. Cycle Day for after administering a Btk inhibitor to the individuals with mantle cell lymphoma shown in Figure 12. The axis has been changed, as compared to Fig. 12, to demonstrate low amplitude fluctuations. Note that all responding patients showed some degree of mobilization.

Fig. 14 demonstrates that lymphocyte mobilization, specifically B Cell type, consistent with lymphoma cells, decreases as disease ds. Patient 32007, Cohort 4, had follicular lymphoma, grade 3, which gradually regressed from SD to CR. Although the changes ofALC in this case are not dramatic, the B cell on is undergoing characteristic cyclic increases in response to ent with a Btk inhibitor. Also note the decreasing cycle by cycle magnitude of shifts consistent with cumulative disease control.

Fig. 15 demonstrates that there is increased B Cell mobilization with disease progression. Patient 32004, Cohort 2, had follicular lymphoma, grade 1, which progressed from SD initially to PD following Cycle 6.

Fig. 16 depicts early mobilization and eventual decrease of a CD45DIM B cell subpopulation in responding mantle cell ma t 5. This subpopulation has a typical MCL immunophenotype (CD45DIM) and is different than that of normal lymphocytes.

Fig. 17 depicts abnormal high light scatter CD19+ cells mobilizing and then regressing in CR DLBCL Pt 324001. These CD45+ cells with light scatter (SSC-H) in the upper panels were gated upon and their CD3 vs CD19 staining displayed in the lower panels. Here the ve malignant cells were “hidden” in the large MNC window normally defining monocytes.

The sequence of mobilization followed by response is similar to other es.

Figure 18 presents the responses for a clinical trial involving administering a Btk inhibitor to elderly patients with CLL or SLL, who are naive for drug intervention. Individuals were administered 420 mg/day of a Btk inhibitor.

[0026] Figure 19 ts the responses for a al trial ing administering a Btk inhibitor to R/R patients with CLL or SLL. duals were administered 420 mg/day of a Btk inhibitor.

Figure 20 presents the responses for a clinical trial involving administering a Btk inhibitor to individuals with high risk CLL.

[0028] Figure 21 presents the se over time for a clinical trial involving administering a Btk inhibitor to individuals with CLL or SLL.

Figure 22 presents the best responses for all patients in a clinical trial ing administering a Btk tor to individuals with CLL or SLL.

Figure 23 presents the best responses for abstract patients in a clinical trial involving administering a Btk inhibitor to individuals with CLL or SLL.

Figure 24 presents the best response by prognostic factor in CLL or SLL patients involved in a clinical trial involving administering a Btk inhibitor.

Figure 25 presents initial (Cycle 2) response assessment and best response (420mg Cohorts) in CLL or SLL patients involved in a al trial involving administering a Btk inhibitor.

Figure 26 presents initial (Cycle 2) response assessment by dose in relapsed/refractory CLL or SLL patients involved in a clinical trial involving administering a Btk inhibitor.

Figure 27 presents improvements in hematological parameters in CLL or SLL patients involved in a clinical trial involving administering a Btk inhibitor.

Figure 28 present data showing the results of a combination of a Btk inhibitor and Carboplatin or Velcade in DoHH2 cells.

Figure 29 present data showing the results of a combination of a Btk inhibitor and Dexamethasone or Lenalidomide in DoHH2 cells.

Figure 30 present data showing the s of a ation of a Btk inhibitor and olimus or R406 in DoHH2 cells.

Figure 31 present data showing the s of a combination of a Btk inhibitor and abine or Doxorubicin in DoHH2 cells.

Figure 32 present data showing the results of a combination of a Btk inhibitor and Cal-101 in TMD8 cells.

Figure 33 present data showing the results of a combination of a Btk inhibitor and R406 in TMD8 cells.

[0041] Figure 34 present data showing the results of a combination of a Btk inhibitor and vincristine in TMD8 cells.

Figure 35 present data showing the results of a combination of a Btk inhibitor and doxorubicin in TMD8 cells.

Figure 36 present data showing the results of a combination of a Btk tor and lenolidomide in TMD8 cells.

Figure 37 present data showing the results of a combination of a Btk inhibitor and velcade in TMD8 cells.

Figure 38 present data showing the results of a combination of a Btk inhibitor and Fludarabine in TMD8 cells.

[0046] Figure 39 present data g the results of a combination of a Btk inhibitor and taxol in TMD8 cells.

DETAILED DESCRIPTION OF THE INVENTION There is currently a need for methods of treating (including, diagnosing) hematological malignancies, including relapsed and refractory B cell malignancies, and ABC-DLBCL. The present application is based, in part, on the unexpected ery that Btk inhibitors induce mobilization (or, in some cases, lymphocytosis) of id cells in solid hematological malignancies. Mobilization of the lymphoid cells increases their exposure to additional cancer treatment ns and their availability for biomarker screening. The inventors have also found that Btk inhibitors are useful for treating relapsed and refractory malignancies and ABC- DLBCL.

Disclosed herein, in n embodiments, is a method for ng a logical malignancy in an individual in need thereof, comprising: (a) stering 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. Disclosed herein, in certain embodiments, is a method for treating diffuse large B—cell lymphoma, activated B cell-like e (ABC-DLBCL), in an individual in need thereof, comprising: administering to the individual an irreversible Btk inhibitor in an amount from 300 mg/day up to, and ing, 1000 mg/day. Further sed , in certain embodiments, is a method for treating relapsed or refractory non-Hodgkin’s lymphoma in an dual in need f, comprising: administering to the individual a eutically—effective amount of (R)-l -(3-(4-amino(4- phenoxyphenyl)— l H-pyrazolo [3 yrimidin— l —yl)piperidin- l -yl)propenone.

Certain Terminology

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the d subject matter belongs. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the intemet can come and go, but equivalent information can be found by ing the intemet. Reference thereto evidences the availability and public dissemination of such information.

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 subject matter claimed. In this application, the use of the ar includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “ 33 “ a an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise.

Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, but not limited to, patents, patent applications, articles, books, manuals, and ses are hereby expressly incorporated by reference in their entirety for any purpose.

Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg CED ORGANIC CHEMISTRY 4TH ED.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional s of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed. Unless specific definitions are provided, the nomenclature employed in connection with, and the laboratory ures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. 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 e.g., using kits of manufacturer's specifications or as commonly accomplished in the art or as bed . The foregoing techniques and procedures can be generally performed of conventional methods well known in the art and as described in various general and more specific nces that are cited and discussed throughout the present specification.

It is to be understood that the methods and itions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be 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 will be limited only by the appended claims.

All publications and patents mentioned herein are incorporated herein by reference in their entirety for the e of describing and disclosing, for example, the constructs and methodologies that are described in the ations, which might be used in connection with the methods, compositions and compounds described herein. The publications discussed herein are provided solely for their sure prior to the filing date of the present application. g herein is to be construed as an ion that the inventors described herein are not entitled to te such disclosure by virtue of prior invention or for any other reason.

An “alkyl” group refers to an aliphatic arbon group. The alkyl moiety may be a “saturated alkyl” group, which means that it does not contain any alkene or alkyne moieties. The alkyl moiety may also be an “unsaturated alkyl” moiety, which means that it contains at least one alkene or alkyne moiety. An “alkene” moiety refers to a group that has at least one carbon- carbon double bond, and an e” moiety refers to a group that has at least one - carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be ed, ht chain, or cyclic. Depending on the structure, an alkyl group can be a monoradical or a diradical (i.e., an alkylene group). The alkyl group could also be a “lower alkyl” having 1 to 6 carbon atoms.

As used herein, C1-Cx includes C1-C2, C1-C3 . . C1-Cx, The “alkyl” moiety may have 1 to 10 carbon atoms (whenever it appears herein, a cal range such as “1 to 10” refers to each integer in the given range; e.g, “1 to 10 carbon atoms” means that the alkyl group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. to and including 10 carbon atoms, although the present definition also covers the , up occurrence of the term “alkyl” where no cal range is designated). The alkyl group of the compounds described herein may be designated as “C1-C4 alky ” or similar designations. By way of example only, “C1-C4 alkyl” indicates that there are one to four carbon atoms in the alkyl chain, z'.e., the alkyl chain is selected from among methyl, ethyl, propyl, iso-propyl, n-butyl, iso- butyl, sec-butyl, and t-butyl. Thus C1-C4 alkyl includes C1-C2 alkyl and C1-C3 alkyl. Alkyl groups can be substituted or unsubstituted. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0058] As used herein, the term “non-cyclic alkyl” refers to an alkyl that is not cyclic (i.e., a straight or branched chain containing at least one carbon atom). clic alkyls can be fillly saturated or can contain non-cyclic alkenes and/or alkynes. Non-cyclic alkyls can be optionally substituted.

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 atoms —C(R)=C(R)-R, wherein R refers to the ing portions of the alkenyl group, which may be the same or different. The alkenyl moiety may be branched, straight chain, or cyclic (in which case, it would also be known as a “cycloalkenyl” . Depending on the structure, an alkenyl group can be a monoradical or a diradical (i.e., an alkenylene group).

Alkenyl groups can be optionally substituted. miting examples of an alkenyl group include —CH=CH2, -C(CH3)=CH2, —CH=CHCH3, —C(CH3)=CHCH3. lene groups include, but are not limited to, —CH=CH—, —C(CH3)=CH—, —CH=CHCH2—, —CH=CHCH2CH2— and — C(CH3)=CHCH2—. Alkenyl groups could have 2 to 10 carbons. The l group could also be a “lower alkenyl” having 2 to 6 carbon atoms.

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 atoms —CEC-R, wherein R refers to the remaining portions of the alkynyl group, which may be the same or different. The “R” n of the alkynyl moiety may be branched, straight chain, or cyclic.

Depending on the structure, an l group can be a monoradical or a diradical (i.e., an alkynylene group). Alkynyl groups can be optionally substituted. Non-limiting examples of an alkynyl group include, but are not limited to, —CECH, 3, —CECCH2CH3, —CEC—, and — CECCH2—. Alkynyl groups can have 2 to 10 carbons. The alkynyl group could also be a “lower alkynyl” having 2 to 6 carbon atoms.

An “alkoxy” group refers to a (alkyl)O— group, where alkyl is as defined herein.

“Hydroxyalkyl” refers to an alkyl radical, as defined herein, substituted with at least one hydroxy group. Non-limiting examples of a hydroxyalkyl e, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3—hydroxypropyl, l-(hydroxymethyl)- 2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4—hydroxybutyl, 2,3-dihydroxypropyl, roxymethyl)hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2-(hydroxymethyl)-3 -hydroxypropyl. yalkyl” refers to an alkyl l, as defined herein, substituted with an alkoxy group, as defined herein.

An “alkenyloxy” group refers to a (alkenyl)O- group, where alkenyl is as defined herein.

The term “alkylamine” refers to the —N(alkyl)XHy group, where x and y are selected from among x=l , y=l and x=2, y=0. When x=2, the alkyl groups, taken together with the N atom to which they are attached, can optionally form a cyclic ring system.

[0066] “Alkylarninoalkyl” refers to an alkyl radical, as defined herein, substituted with an alkylamine, as defined herein.

An “amide” is a chemical moiety with the formula -C(O)NHR or )R, where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded h a ring carbon). An amide moiety may form a linkage between an amino acid or a peptide molecule and a compound bed herein, thereby forming a prodrug. Any amine, or carboxyl side chain on the compounds described herein can be amidified. The procedures and specific groups to make such amides are known to those of skill in the art and can y be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is orated herein by reference in its entirety.

The term “ester” refers to a chemical moiety with formula -COOR, where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl (bonded h a ring carbon) and heteroalicyclic (bonded through a ring carbon). Any hydroxy, or yl side chain on the nds described herein can be fied. The procedures and specific groups to make such esters are known to those of skill in the art and can readily be found in reference sources 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.

[0069] 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-aromatics (e. g., cycloalkyls and non-aromatic heterocycles). Rings can be optionally substituted. Rings can be monocyclic or polycyclic.

[0070] As used herein, the term “ring system” refers to one, or more than one ring.

The term “membered ring” can embrace any cyclic structure. The term “membered” is meant to denote the number of skeletal atoms that tute the ring. Thus, for example, exyl, pyridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, filran, and thiophene are 5-membered rings.

[0072] The term “fused” refers to structures in which two or more rings share one or more bonds.

The term “carbocyclic” or “carbocycle” refers to a ring n each of the atoms forming the ring is a carbon atom. Carbocycle includes aryl and cycloalkyl. The term thus distinguishes carbocycle from heterocycle (“heterocyclic”) in which the ring backbone contains at least one atom which is different from carbon (i.e a heteroatom). Heterocycle includes heteroaryl and heterocycloalkyl. Carbocycles and heterocycles can be optionally substituted.

The term “aromatic” refers to a planar ring having a lized n-electron system containing 4n+2 7: electrons, where n is an integer. Aromatic rings can be formed from five, six, seven, eight, nine, or more than nine atoms. Aromatics can be optionally substituted. The term “aromatic” es both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g., pyridine). The term includes clic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups.

As used herein, the term “aryl” refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl rings can be formed by five, six, seven, eight, nine, or more than nine carbon atoms. Aryl groups can be optionally substituted. Examples of aryl groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, fluorenyl, and indenyl. ing on the structure, an aryl group can be a monoradical or a diradical (i.e., an e group).

An xy” group refers to an (aryl)O— group, where aryl is as defined herein.

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

[0078] enyl” means an alkenyl radical, as defined herein, substituted with an aryl group, as defined herein.

The term “cycloalkyl” refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, partially rated, or fully unsaturated.

Cycloalkyl groups include groups having from 3 to 10 ring atoms. rative examples of cycloalkyl groups include the following moieties: PgfibfibKjQ’OQ DUO» O, OaOCO 9% =0 UOfifiQ lkyl group can be a monoradical or a diradical (e.g., an cycloalkylene group). The cycloalkyl group could also be a “lower cycloalkyl” having 3 to 8 carbon atoms.

“Cycloalkylalkyl” means an alkyl radical, as defined herein, substituted with a cycloalkyl group. Non-limiting cycloalkylalkyl groups include ropylmethyl, cyclobutylmethyl, entylmethyl, cyclohexylmethyl, and the like.

[0081] The term “heterocycle” refers to heteroaromatic and heteroalicyclic groups containing one to four heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent O or S atoms. Herein, whenever the number of carbon atoms in a heterocycle is indicated (e.g., C1-C6 heterocycle), at least one other atom (the heteroatom) must be present in the ring. Designations such as “C1-C6 heterocycle” refer only to the number of carbon atoms in the ring and do not refer to the total number of atoms in the ring. It is understood that the heterocylic ring can have additional heteroatoms in the ring. Designations such as “4-6 membered heterocycle” refer to the total number of atoms that are contained in the ring (i.e., a four, five, or six membered ring, in which at least one atom is a carbon atom, at least one atom is a heteroatom and the remaining two to four atoms are either carbon atoms or heteroatoms). In heterocycles that have two or more atoms, those two or more heteroatoms can be the same or different from one another. Heterocycles can be optionally substituted. Binding to a heterocycle can be at a heteroatom or via a carbon atom. Non-aromatic heterocyclic groups e groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring . The heterocyclic groups include benzo-fused ring systems. An example of a 4-membered cyclic group is azetidinyl (derived from ine). An example of a 5—membered cyclic group is lyl. An example of a 6-membered heterocyclic group is pyridyl, and an example of a lO-membered heterocyclic group is quinolinyl. Examples ofnon—aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofiiranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, ydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, l,2,3,6-tetrahydropyridinyl, olinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H- pyranyl, dioxanyl, l,3-dioxolanyl, pyrazolinyl, nyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, lidinyl, imidazolinyl, imidazolidinyl, 3- azabicyclo[3.l.0]hexanyl, 3-azabicyclo[4.l.0]heptanyl, 3H—indolyl and izinyl. es of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, lyl, pyrazinyl, olyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, nolinyl, indolyl, benzimidazolyl, benzofiaranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups, as derived from the groups listed above, may be C—attached or N—attached where such is possible. For instance, a group derived from pyrrole may be pyrrol—l—yl (N—attached) or pyrrolyl (C- attached). Further, a group derived from imidazole may be imidazol-l-yl or imidazol-3 -yl (both N-attached) or imidazolyl, imidazol-4—yl or imidazol—S-yl (all C-attached). The heterocyclic groups include benzo-fused ring s and ring systems tuted with one or two oxo (=0) moieties such as pyrrolidinone. Depending on the structure, a heterocycle group can be a monoradical or a diradical (i.e., a heterocyclene group).

The terms oaryl” or, alternatively, “heteroaromatic” refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfiJr. An N- containing “heteroaromatic” or “heteroaryl” moiety refers to an aromatic group in which at least one ofthe skeletal atoms of the ring is a en atom. Illustrative examples of heteroaryl groups include the following moieties: JrJOOUDO r? orb and the like. Depending on the structure, a heteroaryl group can be a monoradical or a diradical (i.e., a heteroarylene group).

As used herein, the term “non-aromatic cycle”, “heterocycloalkyl” or “heteroalicyclic” refers to a non-aromatic ring wherein one or more atoms forming the ring is a heteroatom. A romatic heterocycle” or “heterocycloalkyl” group refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen and sulfur. The radicals may be fused with an aryl or heteroaryl. Heterocycloalkyl rings can be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Heterocycloalkyl rings can be ally substituted. In certain embodiments, non—aromatic heterocycles contain one or more carbonyl or thiocarbonyl groups such as, for example, 0x0- and thio-containing groups.

Examples of cycloalkyls e, but are not limited to, lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin, oxane, 1,4-dioxin, 1,4-dioxane, zine, 1,3-oxathiane, 1,4- oxathiin, l,4—oxathiane, tetrahydro—l,4—thiazine, 2H—l,2—oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-l,3,5-triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3- dioxolane, l,3-dithiole, l,3-dithiolane, isoxazoline, isoxazolidine, ine, oxazolidine, oxazolidinone, thiazoline, thiazolidine, and 1,3-oxathiolane. Illustrative examples of heterocycloalkyl groups, also referred to as omatic heterocycles, e: Cab000C700 C7 C7 C7 D C700 [O mgrgufir) 00,17s H )OK 0 N~§= , or.)O the like. The term heteroalicyclic also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the accharides. Depending on the structure, a heterocycloalkyl group can be a monoradical or a diradical (i.e., a heterocycloalkylene group).

[0084] The term “halo” or, alternatively, “halogen” or “halide” means fluoro, chloro, bromo and iodo.

The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl” and “haloalkoxy” include alkyl, l, alkynyl and alkoxy structures in which at least one hydrogen is replaced with a halogen atom. In certain embodiments in which two or more en atoms are replaced with halogen atoms, the halogen atoms are all the same as one another. In other ments in which two or more hydrogen atoms are replaced with n atoms, the halogen atoms are not all the same as one another.

The term “fluoroalkyl,” as used herein, refers to alkyl group in which at least one hydrogen is replaced with a fluorine atom. Examples of fluoroalkyl groups include, but are not limited to, -CF3, —CH2CF3, —CF2CF3, —CH2CH2CF3 and the like.

As used herein, the terms “heteroalkyl” “heteroalkenyl” and “heteroalkynyl” include optionally substituted alkyl, alkenyl and l radicals in which one or more al chain atoms is a heteroatom, e.g, oxygen, nitrogen, sulfur, silicon, orus or combinations thereof. The heteroatom(s) 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)—CH3, -CH2-S-CH2- CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2—CH3, —CH=CH-O-CH3, 3)3, -CH2-CH=N- OCH3, and —CH=CH-N(CH3)-CH3. In addition, up to two heteroatoms may be consecutive, such as, by way of example, -CH2-NH-OCH3 and —CH2—O-Si(CH3)3.

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

The term “bond” or “single bond” refers to a chemical bond between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure.

[0090] An “isocyanato” group refers to a —NCO group.

An “isothiocyanato” group refers to a —NCS group.

The term “moiety” refers to a specific segment or functional group of a molecule.

Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

[0093] A “sulfrnyl” group refers to a -S(=O)-R.

A “sulfonyl” group refers to a -S(=O)2-R.

A “thioalkoxy” or “alkylthio” group refers to a —S-alkyl group.

A “alkylthioalkyl” group refers to an alkyl group substituted with a —S-alkyl group.

As used herein, the term “O-carboxy” or xy” refers to a group of formula O-.

“Carboxy” means a -C(O)OH l.

As used herein, the term “acetyl” refers to a group of formula -C(=O)CH3.

“Acyl” refers to the group —C(O)R.

As used herein, the term “trihalomethanesulfonyl” refers to a group of formula O)2- where X is a halogen.

As used herein, the term ” refers to a group of formula -CN.

“Cyanoalkyl” means an alkyl radical, as defined herein, substituted with at least one cyano group.

As used herein, the term “N-sulfonamido” or “sulfonylamino” refers to a group of formula RS(=O)2NH—.

As used herein, the term bamyl” refers to a group of formula - OC(=O)NR2.

As used herein, the term “N-carbamyl” refers to a group of formula ROC(=O)NH-.

As used herein, the term “O-thiocarbamyl” refers to a group of a - OC(=S)NR2.

As used herein, the term “N-thiocarbamyl” refers to a group of a ROC(=S)NH—.

As used herein, the term “C-amido” refers to a group of formula -C(=O)NR2.

“Aminocarbonyl” refers to a -CONH2 radical.

As used herein, the term “N-amido” refers to a group of formula RC(=O)NH-.

[00112] As used , the substituent “R” appearing by itself and without a number designation refers to a substituent selected from among from alkyl, cycloalkyl, aryl, heteroaryl (bonded h a ring carbon) and non—aromatic heterocycle (bonded through a ring carbon).

The term “optionally substituted” or “substituted” means that the referenced group may be substituted with one or more onal group(s) individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, , aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, acyl, nitro, haloalkyl, lkyl, amino, including mono— and di-substituted amino , and the protected derivatives thereof. By way of e an optional substituents may be LSRS, wherein each LS is independently selected from a bond, -O-, -C(=O)-, -S-, -S(=O)-, -S(=O)2-, -NH-, - NHC(O)—, -C(O)NH-, NH-, -NHS(=O)2, -OC(O)NH-, -NHC(O)O-, -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); and each RS is independently selected from H, (substituted or tituted C1-C4alkyl), (substituted or unsubstituted C3-C6cycloalkyl), heteroaryl, or heteroalkyl. The 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.

The term “Michael acceptor moiety” refers to a fianctional group that can participate in a l reaction, wherein a new covalent bond is formed between a portion of the Michael acceptor moiety and the donor moiety. The Michael acceptor moiety is an electrophile and the “donor moiety” is a nucleophile.

[00115] The term “nucleophile” or “nucleophilic” refers to an on rich compound, or moiety thereof. An example of a nucleophile includes, but in no way is limted to, a cysteine residue of a molecule, such as, for example Cys 481 of Btk.

The term “electrophile”, or “electrophilic” refers to an electron poor or on deficient molecule, or moiety thereof. Examples of ophiles include, but in no way are limited to, l acceptor moieties.

The term “acceptable” or “pharmaceutically acceptable”, with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated or does not abrogate the biological activity or ties of the nd, and is relatively nontoxic.

“B-cell lymphoproliferative ers (BCLD) biomarkers”, as used herein, refer to any biological molecule (found either in blood, other body fluids, or tissues) or any chromosomal abnormality that is a sign of a BCLD-related ion or disease.

"Tumor," as used , refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

"Neoplastic," as used , refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Thus, "neoplastic cells" include malignant and benign cells having dysregulated or unregulated cell growth.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. es of cancer include, but are not limited to, B-cell proliferative disorders (BCLDs), such as lymphoma and leukemia, and solid tumors. By "B cell-related cancer" or "cancer of B-cell lineage" is intended any type of cancer in which the dysregulated or unregulated cell growth is associated with B cells.

By "refractory" in the context of a cancer is intended the particular cancer is resistant to, or non-responsive to, therapy with a ular therapeutic agent. A cancer can be refractory to y with a particular therapeutic agent either from the onset of treatment with the particular eutic agent (i.e., non-responsive to initial exposure to the therapeutic agent), or as a result of developing resistance to the therapeutic agent, either over the course of a first treatment period with the therapeutic agent or during a subsequent treatment period with the therapeutic agent.

By "agonist activity" is intended that a substance functions as an agonist. An agonist combines with a receptor on a cell and initiates a reaction or activity that is similar to or the same as that initiated by the receptor's natural ligand.

By "antagonist activity" is intended that the substance functions as an antagonist.

An antagonist of Btk prevents or reduces ion of any of the responses meidated by Btk.

By "significan " agonist activity is intended an agonist activity of at least 30%, %, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than the agonist activity induced by a neutral substance or negative control as measured in an assay of a B cell " agonist activity is response. Preferably, "significan an agonist ty that is at least 2-fold greater or at least 3-fold greater than the agonist ty induced by a neutral substance or negative control as measured in an assay of a B cell response. Thus, for example, where the B cell response of interest is B cell proliferation, ficant" agonist activity would be induction of a level of B cell proliferation that is at least 2-fold greater or at least 3-fold greater than the level of B cell proliferation induced by a neutral substance or negative control.

] A substance "free of significant agonist activity" would t an agonist activity of not more than about 25% greater than the agonist activity induced by a neutral substance or negative control, preferably not more than about 20% greater, 15% greater, 10% greater, 5% greater, 1% greater, 0.5% greater, or even not more than about 0.1% greater than the agonist activity induced by a neutral substance or negative control as measured in an assay of a B cell response.

In some embodiments, the Btk inhibitor therapeutic agent is an antagonist anti- Btk antibody. Such antibodies are free of significant agonist activity as noted above when bound to a Btk antigen in a human cell. In one embodiment of the ion, the antagonist anti-Btk antibody is free of significant t ty in one ar response. In another ment of the invention, the antagonist anti-Btk antibody is free of significant agonist activity in assays of more than one cellular response (e.g., proliferation and differentiation, or proliferation, differentiation, and, for B cells, dy production).

By "Btk-mediated signaling" it is intended any of the biological activities that are dependent on, either directly or indirection, the activity of Btk. Examples of Btk-mediated ing are signals that lead to proliferation and survival of Btk-expressing cells, and stimulation of one or more Btk-signaling ys within Btk-expressing cells.

A Btk "signaling pathway" or "signal transduction pathway" is ed to mean at least one biochemical reaction, or a group of biochemical reactions, that results from the activity of Btk, and which generates a signal that, when transmitted through the signal pathway, leads to activation of one or more downstream molecules in the signaling cascade. Signal transduction pathways involve a number of signal uction les that lead to transmission of a signal from the cell—surface across the plasma ne of a cell, and through one or more in a series of signal transduction molecules, through the cytoplasm of the cell, and in some instances, into the cell's nucleus. Of ular interest to the present ion are Btk signal transduction pathways which tely regulate (either e or inhibit) the activation ofNF-KB Via the NF-KB signaling pathway.

The methods of the present invention are directed to s for treating cancer that, in certain embodiments, utilize antibodies for determining the expression or presence of certain BCLD biomarkers in these methods. The following terms and definitions apply to such dies.

Antibodies" and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. The terms are used synonymously. In some instances the antigen specificity of the immunoglobulin may be known.

The term "antibody" is used in the broadest sense and covers fully assembled antibodies, dy fragments that can bind antigen (e.g., Fab, F(ab’)2, FV, single chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, and the like), and recombinant peptides comprising the forgoing.

[00132] The terms "monoclonal antibody" and "mA " as used herein refer to an antibody obtained from a substantially neous population of antibodies, i.e., the individual antibodies comprising the tion are identical except for possible naturally occurring mutations that may be present in minor amounts.

Native antibodies" and "native globulins" are usually tetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) . Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide es varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain 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 chain variable s.

[00134] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies. Variable regions confer antigen-binding specificity. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions, both in the light chain and the heavy-chain variable domains.

The more highly conserved portions ofvariable domains are celled in the framework (FR) regions. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a B-pleated-sheet ration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the B—pleated—sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of dies (see, Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. 1, pages 647-669). The constant domains are not involved directly in binding an dy to an antigen, but exhibit various effector functions, such as Fe receptor (FcR) binding, participation of the dy in antibody-dependent cellular toxicity, initiation of complement dependent cytotoxicity, and mast cell degranulation.

The term "hypervariable region," when used herein, refers to the amino acid residues of an antibody that are responsible for antigen—binding. The hypervariable region comprises amino acid residues from a "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 ; Kabat et a1. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md.) and/or those residues from a "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 (13) in the heavy chain variable domain; Clothia and Lesk, (1987) J. Mol.

Biol., 196:901-917). "Framework“ or "FR" residues are those variable domain residues other than the hypervariable region residues, as herein deemed. ody fragments" se a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody nts e Fab, Fab, F(ab')2, and FV fragments; diabodies; linear antibodies (Zapata et a1. (1995) Protein Eng. 10: 1057-1062); single-chain dy molecules; and multispecific antibodies formed from dy fragments. Papain digestion of antibodies produces two identical n- g fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" nt, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen—combining sites and is still capable of cross- linking antigen.

“FV” is the minimum antibody fragment that contains a complete n 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. tively, the six CDRs confer n—binding specificity to the antibody. However, even a single le domain (or half of an FV comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first nt domain (CH1) of the heavy chain. 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 dy hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine e(s) of the constant domains bear a free thiol group. Fab' fragments are ed by reducing the F(ab')2 fragment‘s heavy chain disulfide bridge. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be ed to one of two clearly ct types, called kappa (K) and lambda (9»), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to ent classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be filrther divided into subclasses (isotypes), e. g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy- chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, n, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector fimctions. For example, human IgG1 and IgG3 isotypes have ADCC (antibody dependent cell-mediated xicity) activity.

The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

The term “acceptable” or “pharmaceutically acceptable”, with respect to a formulation, composition or ient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated or does not abrogate the biological activity or properties of the compound, and is relatively nontoxic.

As used herein, the term “agonist” refers to a nd, the presence of which results in a biological activity of a protein that is the same as the biological activity resulting from the presence of a lly occurring ligand for the protein, such as, for example, Btk.

As used herein, the term “partial agonist” refers to a compound the presence of which results in a biological activity of a n that is of the same type as that resulting from the presence of a naturally occurring ligand for the protein, but of a lower magnitude.

As used , the term “antagonist” refers to a compound, the presence of which results in a decrease in the magnitude of a biological activity of a protein. In certain embodiments, the ce of an antagonist s in complete inhibition of a biological activity of a protein, such as, for example, Btk. In certain ments, an antagonist is an inhibitor.

The term “Bruton’s tyrosine kinase (Btk),” as used herein, refers to Bruton’s tyrosine kinase from Homo sapiens, as disclosed in, e.g., US. Patent No. 6,326,469 (GenBank Accession No. NP_000052).

The term n’s tyrosine kinase homolog,” as used herein, refers to orthologs of Bruton’s tyrosine kinase, e.g., the orthologs from mouse nk Accession No.

AAB47246), dog (GenBank Accession No. XP_549139.), rat (GenBank Accession No.

NP_001007799), chicken (GenBank Accession No. 564), or zebra f1sh (GenBank Accession No. XP_6981 l7), and fusion proteins of any of the foregoing that exhibit kinase activity towards one or more substrates of Bruton’s tyrosine kinase (e.g. a peptide substrate haVing the amino acid sequence “AVLESEEELYSSARQ”).

The terms “co-administration” or nation therapy” and the like, as used herein, are meant to encompass administration of the selected therapeutic agents to a single patient, and are ed to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

The term “effective amount,” as used herein, refers to a sufficient amount of a Btk inhibitory agent or a Btk inhibitor compound being administered which will result in an increase or ance in the blood of a subpopulation of lymphocytes (e.g., pharmaceutical debulking). For example, an “effective amount” for diagnostic and/or prognostic uses is the amount of the composition ing a nd as disclosed herein required to provide a clinically significant decrease an increase or appearance in the blood of a subpopulation of cytes without undue adverse side effects. An riate “effective ” in any individual case may be determined using ques, such as a dose escalation study.

The term “therapeutically effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will e to some extent one or more of the symptoms s B-cell proliferative disorder (BCLD). The result can be 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 ” of a compound disclosed herein is an amount effective to achieve a desired pharmacologic effect or eutic improvement Without undue adverse side effects. It is understood that “an effect amount” or “a eutically effective amount” can vary from t to subject, due to variation in metabolism of the compound of any of Formula (A), Formula (B), Formula (C), or Formula (D), age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. By way of example only, therapeutically effective amounts may be ined by routine experimentation, including but not limited to a dose escalation clinical trial.

The terms “enhance” or “enhancing” means to increase or prolong either in y or duration a desired effect. By way of e, “enhancing” the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or ion. An “enhancing- effective amount,” as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, amounts ive 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.

] The term “homologous cysteine,” as used herein refers to a cysteine residue found with in a sequence position that is homologous to that of cysteine 481 of Bruton’s tyrosine kinase, as defined herein. For example, cysteine 482 is the gous cysteine of the rat ortholog of Bruton’s tyrosine kinase, cysteine 479 is the homologous cysteine of the chicken ortholog; and cysteine 481 is the gous cysteine in the zebra fish ortholog. In another example, the homologous cysteine ofTXK, a Tec kinase family member related to Bruton’s tyrosine, is Cys 350. See also the sequence alignments of tyrosine kinases (TK) published on the world wide web at kinase.com/human/kinome/phylogeny.html.

The term “identical,” as used herein, refers to two or more ces or subsequences which are the same. In addition, the term “substantially identical,” as used herein, refers to two or more ces which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using comparison thms or by manual alignment and visual inspection. By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% cal, or about 95% identical over a specified . Such percentages to describe the nt identity” of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across 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 identical when 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 specified region. The identity can exist over a region that is at least about 75-100 amino acids in , over a region that is about 50 amino acids in length, or, where not specified, across the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are “substantially identical” if the nucleic acid es are about 60% cal, 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 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, across the entire sequence of a polynucleotide sequence.

The terms “inhibits”, “inhibiting”, or itor” of a kinase, as used herein, refer to inhibition of enzymatic phosphotransferase activity.

The term “irreversible inhibitor,” as used herein, refers to a compound that, upon contact with a target protein (e.g., a kinase) causes the ion of a new covalent bond with or within the protein, whereby one or more of the target protein’s biological activities (e.g., phosphotransferase activity) is shed or abolished notwithstanding the subsequent presence or absence of the irreversible inhibitor.

The term “irreversible Btk inhibitor,” as used herein, refers to an inhibitor of Btk that can form a covalent bond with an amino acid residue of Btk. In one embodiment, the irreversible inhibitor of Btk can form a covalent bond with a Cys residue of Btk; in particular embodiments, the rsible tor can form a covalent bond with a Cys 481 residue (or a homolog thereof) of Btk or a cysteine residue in the gous corresponding position of another tyrosine .

The term “isolated,” as used herein, refers to separating and removing a component of interest from components not of interest. Isolated substances can be in either a dry or semi-dry state, or in solution, including but not limited to an aqueous solution. The isolated ent can be in a homogeneous state or the isolated component can be a part of a pharmaceutical ition that comprises additional pharmaceutically acceptable carriers and/or excipients. By way of example only, nucleic acids or proteins are “isolated” when such nucleic acids or proteins are free of at least some of the cellular components with which it is associated in the natural state, or that the c acid or protein has been trated to a level greater than the concentration of its in vivo or in vitro production. Also, by way of example, a gene is isolated when separated from open reading frames which flank the gene and encode a protein other than the gene of interest.

A “metabolite” of a compound disclosed herein is a derivative of that compound that is formed when the compound is metabolized. The term “active metabolite” refers to a biologically active derivative of a compound that is formed when the compound is metabolized.

The term “metabolized,” as used herein, refers to the sum of the ses (including, but not limited to, hydrolysis reactions and reactions catalyzed by enzymes, such as, oxidation reactions) by which a particular substance is d by an organism. Thus, enzymes may e specific ural alterations to a compound. For example, cytochrome P450 catalyzes a variety of oxidative and reductive reactions while uridine diphosphate glucuronyl transferases catalyze the transfer of an activated glucuronic—acid le to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines and free sulfliydryl groups. Further information on metabolism may be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of the compounds disclosed herein can be identified either by administration of compounds to a host and analysis of tissue s from the host, or by incubation of compounds with hepatic cells in vitro and analysis of the resulting compounds.

Both methods are well known in the art. In some embodiments, metabolites of a compound are formed by oxidative processes and correspond to the corresponding hydroxy-containing compound. In some embodiments, a compound is metabolized to pharmacologically active metabolites.

The term “modulate,” as used herein, means to interact with a target either directly or indirectly so as to alter the ty of the target, including, by way of example only, to enhance the activity of the target, to inhibit the activity of the target, to limit the activity of the target, or to extend the activity of the target.

] As used herein, the term ator” refers to a compound that alters an activity of a molecule. For example, a modulator can cause an increase or decrease in the magnitude of a certain activity of a molecule compared to the magnitude of the activity in the absence of the modulator. In n embodiments, a modulator is an inhibitor, which decreases the magnitude of one or more activities of a molecule. In certain embodiments, an inhibitor completely ts 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.

As used herein, the term “selective binding compound” refers to a compound that selectively binds to any n of one or more target proteins.

As used herein, the term “selectively binds” refers to the ability of a selective binding compound to bind to a target n, such as, for example, Btk, with greater 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 greater than the affinity for a non-target.

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, specific modulator refers to modulating a target activity at least 10, 50, 100, 250, 500, 1000 times more than a rget activity.

The term “substantially purified,” as used herein, refers to a ent of interest that may be substantially or ially free of other components which ly any or interact with the component of interest prior to purification. By way of example only, a ent of interest may be “substantially purified” when the 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 about 1% (by dry weight) of contaminating components. Thus, a “substantially purified” component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater.

The term “subject” as used herein, refers to an animal which is the object of treatment, observation or experiment. By way of example only, a subject may be, but is not d to, a mammal including, but not limited to, a human.

As used herein, the term “target activity” refers to a biological activity capable of being modulated by a selective modulator. Certain exemplary target activities include, but are not limited to, binding affinity, signal uction, tic activity, tumor growth, effects on particular biomarkers related to B—cell lymphoproliferative disorder ogy.

As used herein, the term “target protein” refers to a le or a portion of a protein capable of being bound by a selective binding compound. In certain embodiments, a target protein is Btk.

The terms “treat,” “treating” or “treatment”, as used herein, e alleviating, abating or rating a disease or condition, or symptoms thereof; managing a disease or condition, or symptoms thereof; preventing additional symptoms; ameliorating or preventing the underlying metabolic causes of ms; inhibiting the disease or condition, e.g., arresting the development of the disease or condition; relieving the disease or condition; causing regression of the disease or condition, relieving a condition caused by the disease or condition; or ng the symptoms of the e or condition. The terms “treat,” “treating” or ment”, include, but are not limited to, lactic and/or therapeutic treatments.

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

[00170] As used herein, ECso refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

Hematological Malignancies Disclosed herein, in certain embodiments, is a method for treating a hematological ancy 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 rsible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, the logical malignancy is CLL. In some embodiments, analyzing the mobilized ity of cells ses 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 tration of the mobilized plurality of cells ses as 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 peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an se in the peripheral blood concentration of the mobilized plurality of cells as ed to the concentration before administration of the Btk inhibitor. In some embodiments, the method r comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells has increased for a predetermined length of time. In some ments, analyzing the mobilized plurality of cells comprises counting the number of mobilized ity of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells ses measuring the duration of an increase in the number of mobilized plurality of cells in the eral blood as compared to the number before stration of the Btk tor. In some embodiments, the method further comprises 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, the hematological malignancy is a chronic lymphocytic leukemia (CLL), small lymphocytic ma (SLL), high risk CLL, or a 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 a, marginal zone lymphoma, Burkitt’s lymphoma, non- Burkitt high grade B cell ma, or extranodal marginal zone B cell lymphoma. In some embodiments, the hematological malignancy is acute or chronic myelogenous (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), ed or refractory mantle cell lymphoma, relapsed or refractory ular lymphoma, relapsed or refractory CLL; relapsed or refractory SLL; relapsed or refractory multiple myeloma. In some embodiments, the hematological malignancy is a hematological malignancy that is classified as high-risk. In some ments, the hematological malignancy is high risk CLL or high risk SLL.

B-cell lymphoproliferative disorders (BCLDs) are neoplasms of the blood and encompass, inter alia, non-Hodgkin ma, multiple myeloma, and ia. BCLDs can originate either in the lymphatic s (as in the case of lymphoma) or in the bone marrow (as in the case of leukemia and myeloma), and they all are involved with the uncontrolled growth of lymphocytes or white blood cells. There are many subtypes of BCLD, e. g., chronic lymphocytic leukemia (CLL) and non—Hodgkin lymphoma (NHL). The disease course and treatment of BCLD is dependent on the BCLD subtype; however, even within each subtype the clinical presentation, morphologic appearance, and response to therapy is heterogeneous.

Malignant lymphomas are stic transformations of cells that reside predominantly within lymphoid tissues. Two groups of malignant lymphomas are n's lymphoma and non-Hodgkin's ma (NHL). Both types of lymphomas infiltrate loendothelial tissues. However, they differ in the neoplastic cell of origin, site of disease, presence of ic symptoms, and response to treatment (Freedman et al., "Non-Hodgkin's Lymphomas" r 134, Cancer Medicine, (an approved publication of the an Cancer Society, B.C. Decker Inc., Hamilton, Ontario, 2003).

Non-Hodgkin ’s Lymghomas Disclosed herein, in certain embodiments, is a method for treating a non- Hodgkin’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 malignancy; and (b) analyzing the zed 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 n after the peripheral blood concentration of the mobilized plurality of cells increases as 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 peripheral blood tration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the on of an increase in the peripheral blood concentration of the mobilized ity of cells as compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method fithher comprises administering a second cancer treatment regimen after the peripheral blood concentration of the zed plurality of cells has increased for a ermined length of time. In some embodiments, analyzing the mobilized ity of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as compared to the concentration before administration of the Btk tor. In some embodiments, administering the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises 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.

Further sed herein, in certain ments, is a method for treating relapsed or refractory non-Hodgkin’s lymphoma in an individual in need thereof, sing: administering to the individual a therapeutically-effective amount of (R)-l -(3-(4-amino(4- phenoxyphenyl)— l zolo [3 ,4-d]pyrimidin- l -yl)piperidin- l open- I -one. In some embodiments, the dgkin’s lymphoma is relapsed or tory diffuse large B-cell lymphoma ), relapsed or refractory mantle cell lymphoma, or relapsed or refractory follicular lymphoma.

Non-Hodgkin lymphomas (NHL) are a diverse group of malignancies that are predominately of B-cell origin. NHL may develop in any organs associated with lymphatic system such as spleen, lymph nodes or tonsils and can occur at any age. NHL is often marked by enlarged lymph nodes, fever, and weight loss. NHL is classified as either B-cell or T-cell NHL.

Lymphomas related to lymphoproliferative disorders following bone marrow or stem cell transplantation are usually B-cell NHL. In the Working Formulation classification scheme, NHL has been divided into low-, intermediate—, and high—grade categories by virtue of their natural histories (see "The dgkin's Lymphoma Pathologic Classification Proj ect," Cancer 49(1982):2112-2135). The low-grade lymphomas are indolent, with a median survival of 5 to 10 years (Homing and erg (1984) N. Engl. J. Med. 311:1471-1475). Although chemotherapy can induce remissions in the majority of indolent lymphomas, cures are rare and most ts eventually relapse, requiring r therapy. The intermediate- and high-grade lymphomas are more aggressive tumors, but they have a greater chance for cure with herapy. However, a significant proportion of these patients will relapse and require r treatment.

A non-limiting list of the B-cell NHL includes Burkitt's lymphoma (e. g., Endemic Burkitt's Lymphoma and ic t's Lymphoma), Cutaneous B-Cell Lymphoma, Cutaneous Marginal Zone Lymphoma (MZL), Diffuse Large Cell Lymphoma (DLBCL), e Mixed Small and Large Cell Lympoma, e Small Cleaved Cell, Diffuse Small Lymphocytic Lymphoma, odal Marginal Zone B-cell lymphoma, follicular lymphoma, Follicular Small Cleaved Cell (Grade 1), Follicular Mixed Small Cleaved and Large Cell (Grade 2), ular Large Cell (Grade 3), Intravascular Large B-Cell Lymphoma, Intravascular Lymphomatosis, Large Cell Immunoblastic Lymphoma, Large Cell Lymphoma (LCL), Lymphoblastic Lymphoma, MALT ma, 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 zone B-cell ma—mucosa—associated lymphoid tissue (MALT) lymphoma, Mediastinal Large B-Cell Lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, primary mediastinal B-cell lymphoma, lymphoplasmocytic lymphoma, hairy cell leukemia, Waldenstrom's Macroglobulinemia, and primary l nervous system (CNS) lymphoma. Additional non-Hodgkin's lymphomas are contemplated within the scope of the present invention and apparent to those of ordinary skill in the art.

DLBCL Disclosed herein, in certain embodiments, is a method for treating a 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 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 ments, analyzing the mobilized ity 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 tration of the mobilized ity of cells increases as compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer ent regimen occurs after a uent decrease in peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method fiarther comprises administering a second cancer ent regimen after the peripheral blood concentration of the zed plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method r comprises administering a second cancer treatment regimen afier the number of mobilized ity of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the eral blood. In some ments, analyzing the mobilized plurality of cells comprises ing the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises 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.

As used herein, the term “Diffuse large B-cell ma (DLBCL)” refers to a neoplasm of the germinal center B lymphocytes with a diffuse grth pattern and a high- intermediate proliferation index. DLBCLS ent approximately 30% of all lymphomas and may present with several morphological variants including the centroblastic, immunoblastic, T- cell/histiocyte rich, anaplastic and plasmoblastic subtypes. Genetic tests have shown that there are ent subtypes of DLBCL. These subtypes seem to have different outlooks (prognoses) and responses to treatment. DLBCL can affect any age group but occurs mostly in older people (the average age is mid-60s).

Disclosed herein, in n 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 from 300 mg/day up to, and including, 1000 mg/day. The ABC subtype of diffuse large B-cell lymphoma LBCL) is thought to arise from post germinal center B cells that are arrested during plasmatic differentiation. The ABC subtype of DLBCL (ABC-DLBCL) ts for approximately 30% total DLBCL diagnoses. It is considered the least curable of the DLBCL molecular subtypes and, as such, patients sed with the ABC-DLBCL typically y significantly reduced survival rates compared with individuals with other types of DLCBL.

ABC-DLBCL is most commonly associated with chromosomal translocations deregulating the germinal center master regulator BCL6 and with mutations inactivating the PRDMl gene, which encodes a riptional repressor required for plasma cell differentiation.

] A particularly relevant signaling pathway in the pathogenesis DLBCL is the one mediated by the nuclear factor (NF)—KB transcription complex. The NF-KB family comprises 5 s (p50, p52, p65, c—rel and RelB) that form homo- and heterodimers and function as transcriptional factors to mediate a variety of eration, apoptosis, inflammatory and immune responses and are critical for normal B—cell development and survival. NF-KB is widely used by eukaryotic cells as a tor of genes that control cell proliferation and cell survival. As such, many different types of human tumors have misregulated NF-KB: that is, NF- KB is tutively active. Active NF-KB turns on the expression of genes that keep the cell proliferating and protect the cell from conditions that would otherwise cause it to die via apoptosis.

The dependence ofABC DLBCLs on NF-kB depends on a signaling pathway upstream of IkB kinase comprised of CARD] 1, BCL10 and MALTl (the CBM complex).

Interference with the CBM pathway extinguishes NF—kB signaling in ABC DLBCL cells and induces apoptosis. The molecular basis for constitutive ty of the NF-kB pathway is a subject of current investigation but some somatic alterations to the genome ofABC DLBCLs clearly invoke this pathway. For example, somatic mutations of the coiled—coil domain of CARDll in DLBCL render this signaling ld protein able to spontaneously nucleate protein-protein interaction with MALTl and BCL10, causing IKK activity and NF-kB activation. Constitutive activity of the B cell receptor signaling pathway has been implicated in the activation ofNF-kB in ABC DLBCLs with wild type CARD] 1, and this is ated with mutations within the cytoplasmic tails of the B cell receptor subunits CD79A and CD79B.

Oncogenic activating mutations in the signaling adapter MYD88 activate NF-kB and synergize with B cell receptor signaling in sustaining the survival ofABC DLBCL cells. In addition, inactivating mutations in a negative regulator of the NF-kB pathway, A20, occur almost exclusively in ABC DLBCL.

Indeed, c tions affecting multiple components of the NF-KB signaling pathway have been recently identified in more than 50% ofABC-DLBCL ts, where these s promote constitutive NF-KB activation, thereby contributing to lymphoma growth. These include mutations of CARDll (~10% of the cases), a lymphocyte—specific cytoplasmic scaffolding protein that—together with MALTl and BCLlO—forms the BCR signalosome, which relays signals from antigen ors to the downstream mediators ofNF-KB activation.

An even larger on of cases (~30%) carry biallelic genetic lesions inactivating the negative NF-KB regulator A20. Further, high levels of expression ofNF-KB target genes have been ed in ABC-DLBCL tumor samples. See, e.g., U. Klein et al., (2008), Nature s Immunology 8:22-23; R.E. Davis et al., (2001), Journal ofExperimental ne 194: 1861- 1874; G. Lentz et al., (2008), Science 319:1676—1679; M. no et al., (2009), Nature 459:712-721; and L. Srinivasan et al., (2009), Cell 139:573-586).

DLBCL cells of the ABC subtype, such as OCI-Ly10, have chronic active BCR signalling and are very sensitive to the Btk inhibitors described herein. The irreversible Btk inhibitors described herein potently and irreversibly inhibit the growth of 10 (EC50 continuous exposure = 10 nM, EC50 1 hour pulse = 50 nM). In addition, induction of apoptosis, as shown by capsase activation, Annexin-V flow cytometry and increase in sub-G0 fraction is observed in OCILy10. Both ive and resistant cells express Btk at similar , and the active site of Btk is fully occupied by the inhibitor in both as shown using a fluorescently labeled affinity probe. OCI-Ly10 cells are shown to have chronically active BCR signalling to NF-kB which is dose dependently inhibited by the Btk tors described herein. The activity of Btk inhibitors in the cell lines studied herein are also characterized by comparing signal transduction profiles (Btk, PLCy, ERK, NF—kB, AKT), cytokine secretion profiles and mRNA expression profiles, both with and without BCR stimulation, and observed significant differences in these profiles that lead to clinical biomarkers that identify the most sensitive patient populations to Btk inhibitor treatment. See US. Patent No. 7,711,492 and Staudt et al., Nature, Vol. 463, Jan. 7, 2010, pp. 88—92, the contents of which are incorporated by reference in their entirety.

Follicular Lymphoma sed herein, in certain embodiments, is a method for treating a follicular lymphoma in an individual in need f, 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 tor is sufficient to induce cytosis 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 ity of cells increases as compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment n occurs after a subsequent decrease in peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized ity of cells ses measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as ed to the concentration before administration of the Btk tor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood tration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiirther comprises 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.

] As used herein, the term “follicular lymphoma” refers to any of several types of non-Hodgkin's lymphoma in which the lymphomatous cells are clustered into nodules or les. The term follicular is used e the cells tend to grow in a circular, or nodular, n in lymph nodes. The average age for people with this lymphoma is about 60.

CLL/SLL Disclosed herein, in certain embodiments, is a method for treating a 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 malignancy; and (b) ing the mobilized plurality of cells. In some embodiments, the CLL or SLL is isk.

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 ses 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 as 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 peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method fithher ses stering a second cancer treatment regimen after the peripheral blood tration of the mobilized plurality of cells has increased for a predetermined length of time. In some ments, analyzing the zed plurality of cells comprises counting the number of mobilized ity of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment n occurs after a uent decrease in the number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells ses measuring the on of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood has sed for a ermined length of time.

[00188] Chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL) are commonly thought as the same disease with slightly different manifestations. Where the cancerous cells gather determines Whether it is called CLL or SLL. When the cancer cells are primarily found in the lymph nodes, lima bean shaped structures of the lymphatic system (a system primarily of tiny vessels found in the body), it is called SLL. SLL accounts for about 5% to 10% of all lymphomas. When most ofthe cancer cells are in the tream and the bone marrow, it is called CLL.

Both CLL and SLL are slow-growing diseases, gh CLL, which is much more common, tends to grow slower. CLL and SLL are treated the same way. They are usually not considered curable with standard treatments, but depending on the stage and growth rate of the disease, most patients live longer than 10 years. Occasionally over time, these slow-growing lymphomas may transform into a more aggressive type of lymphoma.

Chronic lymphoid leukemia (CLL) is the most common type of leukemia. It is estimated that 100,760 people in the United States are living with or are in remission from CLL.

Most (>75%) people newly diagnosed with CLL are over the age of 50. Currently CLL treatment focuses on controlling the disease and its ms rather than on an outright cure.

CLL is treated by herapy, radiation therapy, biological therapy, or bone marrow transplantation. Symptoms are mes treated surgically (splenectomy removal of enlarged spleen) or by radiation therapy ("de-bulking" swollen lymph nodes). Though CLL progresses slowly in most cases, it is considered generally ble. Certain CLLs are classified as high- risk. As used herein, “high risk CLL” means CLL terized by at least one of the following 1) l7pl3-; 2) llq22-; 3) unmutated IgVH together with ZAP-70+ and/or CD3 8+; or 4) trisomy

[00191] CLL treatment is typically stered when the 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.

Small lymphocytic leukemia (SLL) is very similar to CLL described supra, and is also a cancer of B-cells. In SLL the al lymphocytes mainly affect the lymph nodes.

However, in CLL the abnormal cells mainly affect the blood and the bone marrow. The spleen may be affected in both conditions. SLL accounts for about lin 25 of all cases of non-Hodgkin lymphoma. It can occur at any time from young adulthood to old age, but is rare under the age of 50. SLL is considered an nt lymphoma. This means that the disease progresses very slowly, and patients tend to live many years after diagnosis. However, most patients are diagnosed with ed disease, and although SLL responds well to a variety of herapy drugs, it is generally considered to be incurable. Although some cancers tend to occur more often in one gender or the other, cases and deaths due to SLL are evenly split between men and women. The average age at the time of diagnosis is 60 years.

Although SLL is indolent, it is tently progressive. The usual pattern of this disease is one of high response rates to radiation therapy and/or chemotherapy, with a period of disease remission. This is followed months or years later by an inevitable relapse. Re-treatment leads to a response again, but again the disease will relapse. This means that although the short- terrn prognosis of SLL is quite good, over time, many patients develop fatal complications of recurrent e. Considering the age of the duals typically diagnosed with CLL and SLL, there is a need in the art for a simple and effective treatment of the disease with minimum side- effects that do not impede on the t’s quality of life. The instant invention fulfills this long standing need in the art.

Mantle Cell Lymphoma Disclosed , in certain embodiments, is a method for treating a Mantle cell lymphoma in an dual in need thereof, comprising: (a) stering 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, analyzing the mobilized plurality of cells comprises ing the peripheral blood concentration of the mobilized plurality of cells. In some ments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases as compared to the tration before stration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a subsequent se in eral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some ments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized ity of cells in the peripheral blood increases as compared to the tration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized plurality of cells in the eral blood. In some embodiments, analyzing the zed plurality of cells ses measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiirther comprises administering a second cancer treatment regimen after the number ofmobilized ity of cells in the peripheral blood has increased for a predetermined length of time.

As used , the term, “Mantle cell lymphoma” refers to a subtype of B-cell lymphoma, due to CD5 positive antigen-naive pregerminal center B-cell within the mantle zone that surrounds normal germinal center follicles. MCL cells generally over-express cyclin D1 due to a t(l l :14) chromosomal ocation in the DNA. More specifically, the translocation is at t(l l;l4)(ql3;q32). Only about 5% of mas are of this type. The cells are small to medium in size. Men are affected most often. The average age of patients is in the early 60s. The lymphoma is usually widespread when it is diagnosed, involving lymph nodes, bone marrow, and, very often, the spleen. Mantle cell lymphoma is not a very fast growing lymphoma, but is difficult to treat.

Marginal Zone B-cell Lymphoma Disclosed herein, in certain embodiments, is a method for treating a marginal zone B-cell lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor ient to mobilize a plurality of cells from the malignancy; and (b) ing the mobilized ity 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 zed 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 as 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 peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises ing the duration of an increase in the peripheral blood concentration of the mobilized ity of cells as 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 the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of zed plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized ity of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiirther comprises 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.

As used herein, the term “marginal zone B—cell lymphoma” refers to a group of d B-cell neoplasms that involve the lymphoid tissues in the marginal zone, the patchy area e the follicular mantle zone. Marginal zone lymphomas account for about 5% to 10% of lymphomas. The cells in these lymphomas look small under the microscope. There are 3 main types of marginal zone lymphomas including extranodal marginal zone B-cell lymphomas, nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma.

MALT ] Disclosed herein, in n embodiments, is a method for treating a MALT in an individual in need f, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor ient 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 ity of cells. In some embodiments, the method further comprises administering a second cancer ent regimen after the peripheral blood concentration of the mobilized plurality of cells ses as ed to the concentration before administration of the Btk inhibitor. In some embodiments, stering the second cancer treatment regimen occurs after a subsequent decrease in peripheral blood tration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized ity of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method fithher ses administering a second cancer treatment regimen afier the peripheral blood concentration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the zed plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized ity of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment n after the number of mobilized plurality of cells in the peripheral blood has increased for a predetermined length of time.

The term “mucosa-associated lymphoid tissue (MALT) lymphoma”, as used herein, refers to extranodal manifestations ofmarginal-zone lymphomas. Most MALT lymphoma are a low grade, although a minority either manifest initially as intermediate-grade non-Hodgkin lymphoma (NHL) or evolve from the low-grade form. Most of the MALT lymphoma occur in the stomach, and roughly 70% of gastric MALT lymphoma are associated with bacter pylori infection. Several cytogenetic alities have been identified, the most common being trisomy 3 or t(11;18). Many of these other MALT lymphoma have also been linked to infections with bacteria or viruses. The average age of patients with MALT ma is about 60.

Nodal Marginal Zone B-Cell ma Disclosed , in certain embodiments, is a method for treating a nodal marginal zone B-cell lymphoma in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor ent to ze a plurality of cells from the malignancy; and (b) analyzing the mobilized plurality of cells. In some embodiments, the amount of the rsible Btk inhibitor is sufficient to induce lymphocytosis of a plurality of cells from the malignancy. In some ments, analyzing the mobilized plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method fiarther comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells increases as 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 peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before stration of the Btk inhibitor. In some ments, the method further comprises administering a second cancer treatment regimen after the eral blood concentration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized ity of cells comprises counting the number ofmobilized plurality of cells in the peripheral blood. In some ments, the method r comprises administering a second cancer treatment regimen after the number of zed plurality of cells in the peripheral blood increases as compared to the concentration before stration of the Btk inhibitor. In some embodiments, administering the second cancer treatment n occurs afier a subsequent decrease in the number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiarther comprises administering a second cancer treatment regimen afier the number of mobilized plurality of cells in the peripheral blood has increased for a predetermined length of time.

The term “nodal marginal zone B—cell lymphoma” refers to an indolent B-cell lymphoma that is found mostly in the lymph nodes. The disease is rare and only accounts for 1% of all dgkin’s Lymphomas (NHL). It is most ly diagnosed in older patients, with women more susceptible than men. The disease is classified as a marginal zone lymphoma because the on occurs in the marginal zone of the s. Due to its confinement in the lymph nodes, this disease is also classified as nodal.

Splem'c Marginal Zone B-Cell Lymphoma Disclosed herein, in certain embodiments, is a method for treating a splenic marginal zone B-cell lymphoma in an individual in need f, 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 ments, 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 zed plurality of cells comprises measuring the peripheral blood concentration of the mobilized plurality of cells. In some embodiments, the method fill'th61‘ comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells ses as compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a uent decrease in peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk tor. In some embodiments, the method further comprises administering a second cancer ent regimen after the peripheral blood concentration of the zed plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as compared to the tration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a uent decrease in the number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the zed plurality of cells comprises measuring the on of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiarther comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood has sed for a predetermined length of time.

[00203] The term “splenic marginal zone B—cell lymphoma” refers to specific low-grade small B-cell lymphoma that is incorporated in the World Health Organization classification.

Characteristic features are splenomegaly, moderate cytosis with Villous morphology, intrasinusoidal pattern of involvement of various organs, especially bone marrow, and relative indolent course. Tumor progression with increase of blastic forms and aggressive or are observed in a minority of patients. Molecular and cytogenetic studies have shown heterogeneous results ly because of the lack of rdized diagnostic criteria.

Burkz'tt Lymphoma sed , in certain embodiments, is a method for treating a Burkitt lymphoma in an individual in need f, comprising: (a) stering 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 tor is sufficient to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, analyzing the mobilized plurality of cells comprises ing 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 ses as 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 peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises ing the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as compared to the tration before stration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the peripheral blood tration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of zed plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiirther ses stering 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.

] The term “Burkitt lymphoma” refers to a type ofNon-Hodgkin Lymphoma (NHL) that commonly s children. It is a highly aggressive type of B-cell lymphoma that often starts and involves body parts other than lymph nodes. In spite of its fast-growing nature, Burkitt’s lymphoma is often curable with modern intensive ies. There are two broad types of Burkitt’s lymphoma — the sporadic and the endemic varieties: Endemic Burkitt’s lymphoma: The disease involves children much more than adults, and is related to Epstein Barr Virus (EBV) infection in 95% cases. It occurs primarily is equatorial Africa, where about half of all childhood s are t’s lymphoma. It characteristically has a high chance of involving the jawbone, a rather distinctive feature that is rare in sporadic Burkitt’s. It also commonly involves the abdomen. ic Burkitt’s ma: The type of Burkitt’s lymphoma that s the rest of the world, ing Europe and the Americas is the sporadic type. Here too, it's mainly a disease in children. The link between Epstein Barr Virus (EBV) is not as strong as with the c variety, though direct evidence of EBV infection is present in one out of five patients.

More than the involvement of lymph nodes, it is the abdomen that is notably affected in more than 90% of the children. Bone marrow involvement is more common than in the sporadic variety.

Waldenstrom Macroglobulz'nemz'a Disclosed , in certain embodiments, is a method for treating a Waldenstrom macroglobulinemia in an individual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk inhibitor ent to mobilize a ity of cells from the malignancy; and (b) ing the zed 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 zed 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 n after the peripheral blood concentration of the mobilized plurality of cells increases as 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 peripheral blood concentration of the mobilized ity of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as 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 the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer ent regimen occurs after a subsequent decrease in the number of zed plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an se in the number of mobilized plurality of cells in the eral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method fiirther comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood has sed for a predetermined length of time.

The term “Waldenstrom macroglobulinemia”, also known as lymphoplasmacytic lymphoma, is cancer involving a subtype of White blood cells called lymphocytes. It is characterized by an uncontrolled clonal proliferation of terminally entiated B lymphocytes.

It is also characterized by the lymphoma cells making an antibody called immunoglobulin M (IgM). The IgM antibodies circulate in the blood in large amounts, and cause the liquid part of the blood to thicken, like syrup. This can lead to decreased blood flow to many organs, which can cause problems with vision (because ofpoor circulation in blood vessels in the back of the eyes) and neurological ms (such as headache, dizziness, and confiasion) caused by poor blood flow within the brain. Other symptoms can include feeling tired and weak, and a tendency to bleed . The ying etiology is not fiilly understood but a number of risk factors have been identified, including the locus 6p21.3 on chromosome 6. There is a 2- to 3-fold risk increase of developing WM in people with a personal history of autoimmune diseases with autoantibodies and particularly ed risks associated with hepatitis, human immunodeficiency virus, and rickettsiosis.

Multigle Myeloma ] sed , in certain embodiments, is a method for treating a 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 malignancy; and (b) analyzing the zed 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 tration 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 as compared to the concentration before administration of the Btk inhibitor. In some ments, administering the second cancer treatment regimen occurs after a subsequent decrease in peripheral blood concentration of the zed plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the eral blood concentration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method fiarther ses administering a second cancer ent regimen after the peripheral blood concentration of the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen afier the number of zed plurality of cells in the eral blood increases as ed to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment n occurs after a subsequent decrease in the number of mobilized plurality of cells in the peripheral blood. In some embodiments, ing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk tor. In some embodiments, the method further comprises 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.

] Disclosed herein, in certain embodiments, is a method for treating a multiple myeloma in an individual in need thereof, sing: (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, 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 ses as compared to the concentration before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs afier a subsequent decrease in peripheral blood tration of the mobilized plurality of cells. In some embodiments, analyzing the zed plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the zed plurality of cells as 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 the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized ity of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as 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 number of mobilized plurality of cells in the peripheral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number ofmobilized plurality of cells in the peripheral blood has increased for a predetermined length of time.

Multiple myeloma, also known as MM, myeloma, plasma cell a, or as Kahler's disease (after Otto Kahler) is a cancer of the white blood cells known as plasma cells. A type of B cell, plasma cells are a crucial part of the immune system sible for the production of antibodies in humans and other vertebrates. They are produced in the bone marrow and are transported h the lymphatic system.

Leukemia

[00213] Disclosed herein, in n embodiments, is a method for treating a ia in an individual in need thereof, comprising: (a) administering to the dual 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 ments, the amount of the irreversible Btk inhibitor is ent to induce lymphocytosis of a plurality of cells from the ancy. 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 as 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 peripheral blood tration of the mobilized plurality of cells. In some embodiments, ing the mobilized plurality of cells comprises measuring the duration of an increase in the peripheral blood concentration of the mobilized plurality of cells as 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 the mobilized plurality of cells has increased for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of zed plurality of cells in the peripheral blood increases as compared to the concentration before administration of the Btk inhibitor. In some ments, administering the second cancer treatment regimen occurs after a subsequent se in the number of mobilized plurality of cells in the peripheral blood. In some ments, analyzing the mobilized ity of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as ed to the number before administration of the Btk inhibitor. In some embodiments, the method r comprises 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.

Leukemia is a cancer ofthe blood or bone marrow characterized by an abnormal increase of blood cells, y leukocytes (white blood cells). Leukemia is a broad term covering a spectrum of diseases. The first division is between its acute and chronic forms: (i) acute leukemia is characterized by the rapid increase of immature blood cells. This crowding makes the bone marrow unable to produce y blood cells. Immediate treatment is required in acute leukemia due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. Acute forms of leukemia are the most common forms of leukemia in children; (ii) chronic leukemia is guished by the excessive build up of relatively mature, but still abnormal, white blood cells. Typically taking months or years to progress, the cells are produced at a much higher rate than normal cells, resulting in many al white blood cells in the blood. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Additionally, the diseases are subdivided according to which kind of blood cell is affected. This split divides leukemias into blastic or lymphocytic ias and myeloid or myelogenous leukemias: (i) lymphoblastic or lymphocytic leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form lymphocytes, which are infection-fighting immune system cells; (ii) myeloid or myelogenous leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form red blood cells, some other types of white cells, and platelets.

Within these main categories, there are several subcategories including, but not limited to, Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML), Chronic myelogenous leukemia (CML), and Hairy cell leukemia (HCL).

Btk inhibitors Also presented herein are methods for treating a cancer such as by way of e only, a BCLD, in a subject wherein the subject has been treated with a dosing regimen of a Btk inhibitor. In the ing description of irreversible Btk compounds suitable for use in the methods bed herein, definitions of referred—to standard chemistry terms may be found in reference works (if not otherwise defined herein), including Carey and Sundberg “Advanced c Chemistry 4th Ed.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods ofmass spectroscopy, NMR, HPLC, n chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the ordinary skill of the art are employed. In addition, nucleic acid and amino acid sequences for Btk (e. g., human Btk) are known in the art as disclosed in, e.g., US. Patent No. 6,326,469. Unless specific ions are provided, the nomenclature employed in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and ry, and treatment of ts.

] The Btk inhibitor compounds described herein are selective for Btk and kinases having a ne residue in an amino acid sequence position of the tyrosine kinase that is homologous to the amino acid sequence position of cysteine 481 in Btk. Generally, an irreversible inhibitor compound of Btk used in the methods described herein is identified or characterized in an in vitro assay, e. g., an acellular mical assay or a cellular functional assay. Such assays are useful to determine an in vitro IC50 for an irreversible Btk inhibitor compound.

For example, an acellular kinase assay can be used to determine Btk activity after incubation of the kinase in the absence or presence of a range of concentrations of a candidate irreversible Btk inhibitor nd. If the candidate compound is in fact an irreversible Btk inhibitor, Btk kinase activity will not be recovered by repeat washing with inhibitor-free medium. See, e.g., J. B. Smaill, et al. (1999), J. Med. Chem, 42(10): 1803-1815. Further, covalent x formation between Btk and a candidate irreversible Btk inhibitor is a useful indicator of rsible inhibition of Btk that can be readily determined by a number of methods known in the art (e. g., mass spectrometry). For example, some rsible Btk-inhibitor compounds can form a covalent bond with Cys 481 of Btk (e. g., Via a l reaction).

] Cellular functional assays for Btk inhibition include measuring one or more cellular nts in response to stimulating a Btk—mediated pathway in a cell line (e.g., BCR activation in Ramos cells) in the absence or ce of a range of concentrations of a candidate irreversible Btk tor compound. Useful endpoints for determining a response to BCR tion include, e.g., autophosphorylation of Btk, phosphorylation of a Btk target protein (e.g., PLC-y), and cytoplasmic calcium flux.

] High throughput assays for many acellular biochemical assays (e.g., kinase assays) and cellular functional assays (e.g., calcium flux) are well known to those of ordinary skill in the art. In addition, high hput screening systems are 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 entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. Automated systems thereby allow the identification and characterization of a large number of irreversible Btk compounds t undue effort.

[00221] In some embodiments, the Btk inhibitor is selected from the group consisting of a small organic molecule, a macromolecule, a peptide or a non-peptide.

In some embodiments, the Btk inhibitor provided herein is a reversible or irreversible inhibitor. In certain embodiments, the Btk inhibitor is an rsible inhibitor.

] In some embodiments, the irreversible Btk inhibitor forms a covalent bond with a cysteine sidechain of a Bruton’s tyrosine kinase, a Bruton’s tyrosine kinase homolog, or a Btk tyrosine kinase cysteine g.

Irreversible Btk inhibitor compounds can use for the manufacture of a medicament for treating any of the foregoing conditions (e.g., autoimmune diseases, inflammatory diseases, allergy disorders, B—cell proliferative ers, or thromboembolic disorders).

In some embodiments, the irreversible Btk inhibitor compound used for the methods described herein inhibits Btk or a Btk homolog kinase activity with an in vitro IC50 of less than 10 uM. (e.g., less than 1 uM, less than 0.5 uM, less than 0.4 uM, less than 0.3 uM, less than 0.1, less than 0.08 uM, less than 0.06 uM, less than 0.05 uM, less than 0.04 uM, less than 0.03 uM, less than less than 0.02 uM, less than 0.01, less than 0.008 uM, less than 0.006 uM, less than 0.005 uM, less than 0.004 uM, less than 0.003 uM, less than less than 0.002 uM, less than 0.001, less than 0.00099 uM, less than 0.00098 uM, less than 0.00097 uM, less than 6 uM, less than 0.00095 uM, less than 0.00094 uM, less than 0.00093 uM, less than 0.00092, or less than 0.00090 uM).

In one embodiment, the irreversible Btk inhibitor compound selectively and irreversibly inhibits an activated form of its target tyrosine kinase (e.g., a phosphorylated form of the tyrosine kinase). For example, ted Btk is hosphorylated at tyrosine 551. Thus, in these embodiments the irreversible Btk inhibitor inhibits the target kinase in cells only once the target kinase is activated by the signaling events.

In other embodiments, the Btk inhibitor used in the methods describe herein has the structure of any of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F). Also described herein are pharmaceutically able salts, pharmaceutically able solvates, pharmaceutically active metabolites, and pharmaceutically acceptable prodrugs of such nds. Pharmaceutical compositions that include at least one such compound or a pharmaceutically acceptable salt, ceutically acceptable solvate, ceutically active metabolite or pharmaceutically acceptable prodrug of such compound, are provided. In some embodiments, when compounds disclosed herein contain an oxidizable nitrogen atom, the nitrogen atom can be converted to an N-oxide by methods well known in the art. In certain embodiments, isomers and chemically protected forms of compounds having a structure represented by any of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or Formula (F), are also provided. a (A) is as follows: R3\ ’R2 N R1 N \ \ m / A N N.

Formula (A) wherein: A is independently selected from N or CR5; R1 is H, L2-(substituted or unsubstituted alkyl), L2-(substituted or unsubstituted cycloalkyl), bstituted or unsubstituted alkenyl), L2-(substituted or unsubstituted cycloalkenyl), L2-(substituted or unsubstituted heterocycle), L2—(substituted or unsubstituted heteroaryl), or L2-(substituted or unsubstituted aryl), where L2 is a bond, 0, 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, wherein, L3 is optional, and when t is a bond, optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted cycloalkyl, optionally tuted or unsubstituted alkenyl, optionally substituted or unsubstituted alkynyl; X is optional, and when present is a bond, 0, , S, -S(=O), -S(=O)2, -NH, -NR9, - NHC(O), -C(O)NH, O), -C(O)NR9, —S(=O)2NH, -NHS(=O)2, -S(=O)2NR9-, - NR9S(=O)2, -OC(O)NH-, -NHC(O)O-, —OC(O)NR9-, -NR9C(O)O-, -CH=NO-, - ON=CH-, -NR10C(O)NR10-, heteroaryl, aryl, -NR10C(=NR11)NR10-, -NR10C(=NR11)- or -C(=NR11)O-; , -C(=NR11)NR10-, -OC(=NR11)—, L4 is al, and when t is a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, tuted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; or L3, X and L4 taken together form a nitrogen containing heterocyclic ring; 0 R6 R6 R6 (I? R6 0 OQS/IO 3 g ‘1 R7 EMR LLLL R7 '15 R7 R26 R7 G is R8 6 R8 R8 or R8 , , , , , wherein, R6, R7 and R8 are independently selected from among H, lower alkyl or tuted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower lkyl, and substituted or tituted lower cycloalkyl; 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- ituted or unsubstituted aryl), wherein L6 is a bond, 0, 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 among H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl; each R10 is independently H, tuted or unsubstituted lower alkyl, or substituted or unsubstituted lower cycloalkyl; or two R10 groups can together form a 5-, 6—, 7-, or 8-membered heterocyclic ring; or R9 and R10 can together form a 5-, 6-, 7-, or 8-membered cyclic ring; or each R11 is independently selected from H, —S(=O)2Rg, 2NH2, g, -CN, -N02, heteroaryl, or heteroalkyl; and pharmaceutically active metabolites, pharrnaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof.

In one aspect are compounds having the structure of Formula (Al): R3\ ’R2 N R1 N \ \ l / .A N N Formula (Al), wherein A is ndently selected from N or CR5; R1 is H, L2-(substituted or unsubstituted alkyl), bstituted or unsubstituted cycloalkyl), L2-(substituted or unsubstituted alkenyl), bstituted or unsubstituted cycloalkenyl), bstituted or unsubstituted heterocycle), L2—(substituted or unsubstituted aryl), or L2-(substituted or unsubstituted aryl), where L2 is a bond, 0, S, -S(=O), -S(=O)2, C(=O), tituted 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, wherein, L3 is optional, and when present is a bond, or an optionally substituted group selected from alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, eteroaryl, or alkylheterocycloalkyl; X is optional, and when present is a bond, 0, —C(=O), S, -S(=O), -S(=O)2, -NH, -NR9, - NHC(O), -C(O)NH, -NR9C(O), —C(O)NR9, —S(=O)2NH, -NHS(=O)2, -S(=O)2NR9-, - NR98(=O)2, -OC(O)NH-, -NHC(O)O—, —OC(O)NR9-, -NR9C(O)O-, -CH=NO-, - ON=CH-, -NR10C(O)NR10-, heteroaryl, aryl, -NR10C(=NR11)NR10-, -NR10C(=NR11)- or -C(=NR11)O-; , -C(=NR11)NR10-, -OC(=NR11)—, L4 is optional, and when present is a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, tuted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; or L3, X and L4 taken together form a nitrogen containing heterocyclic ring, or an optionally substituted group selected from alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl; GisWEMJ‘M9% JKNRa\SjR/RtéaO\SO// 8%/ Where Ra is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and either R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4alkyl, substituted or tituted C1- C4heteroalkyl, C1-Cgalkylaminoalkyl, ydroxyalkylaniinoalkyl, C1- Cgalkoxyalkylarninoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1—Cgalkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted aryl, C1-C4alkyl(aryl), C1-C4alkyl(heteroaryl), C1- Cgalkylethers, C1-Cgalkylamides, or C1-C4alkyl(C2—Cgheterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalkylaminoalkyl, C1-Cghydroxyalkylaminoalkyl, C1- xyalkylaminoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1—Cgalkle3—C6cycloalkyl, tuted or tituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1—C4alkyl(aryl), C1-C4alkyl(heteroaryl), C1- Cgalkylethers, C1-Cgalkylamides, or lkyl(C2-Cgheterocycloalkyl); or R6 and R8 taken together form a bond; R7 is H, substituted or unsubstituted C1-C4a1kyl, substituted or unsubstituted C1- C4heteroalkyl, C1-C8alkylaminoalkyl, C1-Cghydroxyalkylaminoalkyl, C1- xyalkylaminoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1—Cgalkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4alkyl(aryl), C1-C4alky1(heteroaryl), C1- Cgalkylethers, C1-C8alkylamides, or C1-C4a1ky1(C2-Cgheterocycloalkyl); or R5 is H, halogen, -L6-(substituted or unsubstituted C1-C3 alkyl), -L6-(substituted or unsubstituted C2-C4 alkenyl), ubstituted or unsubstituted heteroaryl), or —L6- (substituted or unsubstituted aryl), n L6 is a bond, 0, 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 among H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl; each R10 is independently H, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower cycloalkyl; or two R10 groups can together form a 5-, 6-, 7-, or 8—membered heterocyclic ring; or R9 and R10 can together form a 5-, 6-, 7-, or 8-membered heterocyclic ring; or each R11 is independently selected from H, —S(=O)2Rg, —S(=O)2NH2, -C(O)Rg, -CN, -N02, heteroaryl, or heteroalkyl; and pharmaceutically active metabolites, pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts of compounds of a (Al). By way of example only, are salts of an amino group formed with inorganic acids such as hloric acid, hydrobromic acid, phosphoric acid, ic acid and perchloric acid or with 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 an anion, such as adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, ate, lfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy—ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, e, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, e, palmitate, e, pectinate, fate, ylpropionate, ate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Further salts include those in which the counterion is an cation, such as sodium, lithium, potassium, calcium, magnesium, ammonium, and quaternary ammonium (substituted with at least one c moiety) cations.

In another embodiment are pharmaceutically acceptable esters of compounds of Formula (Al), including those in which the ester group is ed from a formate, acetate, propionate, butyrate, acrylate and ethylsuccinate.

In another embodiment are pharmaceutically acceptable carbamates of compounds of a (A1). In another embodiment are pharmaceutically acceptable N—acyl derivatives of compounds of Formula (A1). Examples yl groups include N—acetyl and N- ethoxycarbonyl groups.

] In a further embodiment, the nd of Formula (A) has the following structure of Formula (B): Formula (B) wherein: Y is alkyl or substituted alkyl, or a 4-, 5-, or 6—membered cycloalkyl ring; each Ra is independently H, n, —CF3, —CN, -N02, OH, NH2, -La-(substituted or unsubstituted alkyl), -La-(substituted or unsubstituted alkenyl), —La-(substituted or unsubstituted heteroaryl), or —La-(substituted or tituted aryl), wherein La is a bond, 0, S, -S(=O), -S(=O)2, NH, C(O), CH2, -NHC(O)O, -NHC(O), or -C(O)NH; 0 R6 O°s”o R6 R6 R5 0 (.8? 9 HMFG HMR § E R, E R7 R20, R7 G is R8 6 R8 R8 or R8 , , , , , wherein, R6, R7 and R8 are independently ed from among H, lower alkyl or substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower cycloalkyl, and substituted or unsubstituted lower heterocycloalkyl; R12 is H or lower alkyl; or Y and R12 taken together form a 4-, 5—, or 6—membered cyclic ring; and pharmaceutically acceptable active metabolites, pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof. )% 9W In filI'thCI‘ embodiments, G is selected from among O 0 ) l ) )WN/ )W )WO/ I £78“ 0 O O and 0/ \O a , , .

Y\N,R12 In further embodiments, ML» is ed from among 6NTJIY’Oqfi’ b 6?“ NH 6Nj / Rm in NTJIY’E >1, “LIV / 9 EQand ;' In a r embodiment, the compound of Formula (Al) has the following ure of Formula (Bl): Formula (B 1), wherein: Y is an optionally substituted group selected from among ne, heteroalkylene, arylene, heteroarylene, alkylenearylene, alkyleneheteroarylene, and alkyleneheterocycloalkylene; each Ra is independently H, halogen, -CF3, -CN, -N02, OH, NH2, -La-(substituted or unsubstituted alkyl), -La-(substituted or unsubstituted alkenyl), —La-(substituted or unsubstituted heteroaryl), or —La—(substituted or unsubstituted aryl), wherein La is a bond, 0, S, -S(=O), -S(=O)2, NH, C(O), CH2, -NHC(O)O, -NHC(O), or -C(O)NH; 0 R6 0 R6 0 R6 MR7%0 R6 ifi/kR7 J‘KNRMR7 G is Rs R8 R8 , , , ?R6 0 R6 I O\/ 3 / R7 fliNRa ka,S R8 or, R8 where R3 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and either R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalky1aminoalkyl, C1—Cghydroxyalkylarninoalkyl, C1— Cgalkoxyalkylaminoalkyl, tuted or unsubstituted C3-C6cycloalky1, substituted or unsubstituted C1-Cgalkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2—Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alkyl(aryl), C1—C4alkyl(heteroaryl), C1-Cgalky1ethers, C1-Cgalkylan1ides, or C1- C4alkyl(C2-Cgheterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1—C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalkylan1inoalkyl, C1—Cghydroxyalkylaniinoalkyl, C1- Cgalkoxyalkylaniinoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1-Cgalkle3-Cécycloalkyl, substituted or tituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or tituted aryl, C1- C4alkyl(aryl), lkyl(heteroaryl), C1-C8alkylethers, C1-Cgalkylan1ides, or C1- C4alkyl(C2-Cgheterocycloalkyl); or R6 and R8 taken together form a bond; R7 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-C8alkylarninoalkyl, C1-C8hydroxyalkylan1inoalkyl, C1- Cgalkoxyalkylarninoalkyl, substituted or unsubstituted ycloalkyl, substituted or unsubstituted C1-C8alkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alkyl(aryl), C1-C4alkyl(heteroaryl), lky1ethers, C 1 -Cga1ky1amides, or C1- C4alkyl(C2-Cgheterocycloalkyl); R12 is H or lower alkyl; or Y and R12 taken together form a 4-, 5-, or 6-membered heterocyclic ring; and pharmaceutically acceptable active metabolites, pharmaceutically acceptable es, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof.

In further embodiments, G is selected from among 0 O , , M“ M“ WI?!\ ,R wR W0\ / O R O and O where R is H, alkyl, alkylhydroxy, , , , ., cycloalkyl, heteroaryl, alkylalkoxy, alkylalkoxyalkyl.

Y\N,R12 In further embodiments,MWIW is selected from among (5:3 if ¢ 6\ Ox:f b\ H ”R | N\ N\ HN\ s; .3: WW rrrr m 9" ,and re: , , , In a further embodiment, the compound of Formula (B) has the following structure of Formula (C): N \ \ lN/ N R12‘N Formula (C) Y is alkyl or substituted alkyl, or a 4-, 5-, or ered cycloalkyl ring; R12 is H or lower alkyl; or Y and R12 taken together form a 4-, 5-, or 6-membered heterocyclic ring; ll/OR6 9 R6 9 R6 ”EMF” MR /8 g E R7 at/ WAR? I R7 G 1s. R8 6, R8 R8 . or R8 , , , whereln, R6, R7 and R8 are ndently selected from among H, lower alkyl or substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, tuted or unsubstituted lower cycloalkyl, and substituted or unsubstituted lower cycloalkyl; and pharmaceutically acceptable active metabolites, pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof.

In r embodiment, the compound of Formula (Bl) has the following structure of Formula (C l): R12‘N’ Formula (C 1), Y is an optionally substituted group selected from among alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, and alkylheterocycloalkyl; R12 is H or lower alkyl; or Y and R12 taken together form a 4-, 5-, or 6-membered heterocyclic ring; MRMKMRfMR : /R60R7 fNFE/QER R8 where Ra is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and either R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalkylarninoalkyl, C1-C8hydroxyalkylarninoalkyl, C1- Cgalkoxyalkylarninoalkyl, substituted or tituted ycloalkyl, substituted or unsubstituted C1-Cgalkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted eterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alky1(aryl), C1-C4alkyl(heteroaryl), lkylethers, C1-Cgalkylamides, or C1- C4alkyl(C2-Cgheterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted lkyl, substituted or unsubstituted C1- C4heteroalkyl, C1—Cgalkylaminoalkyl, C1—Cghydroxyalkylaminoalkyl, C1- Cgalkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1-Cgalkle3—C6cycloalkyl, substituted or unsubstituted aryl, tuted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alkyl(aryl), C1-C4alkyl(heteroaryl), C1-Cgalkylethers, C1-Cgalkylamides, or C1- C4alkyl(C2-Cgheterocycloalkyl); or R6 and R8 taken together form a bond; R7 is H, substituted or tituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalkylaminoalkyl, C1-Cghydroxyalkylaminoalkyl, C1- Cgalkoxyalkylarninoalkyl, substituted or unsubstituted C3—C6cycloalky1, substituted or unsubstituted C1-C8a1kle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4all<yl(aryl), C1-C4alkyl(heteroaryl), C1-Cgalkylethers, C1-Cgalky1amides, or C1- C4alkyl(C2—Cgheterocycloalkyl); and pharmaceutically acceptable active metabolites, ceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable gs thereof.

In a further or alternative embodiment, the “G” group of any of Formula (Al), Formula (B1), or Formula (Cl) is any group that is used to tailor the physical and biological properties of the molecule. Such tailoring/modifications are achieved using groups which modulate Michael acceptor chemical reactivity, acidity, basicity, lipophilicity, solubility and other physical properties of the le. The physical and biological properties modulated by such modifications to G include, by way of example only, enhancing al vity of Michael acceptor group, lity, in viva absorption, and in viva metabolism. In addition, in viva metabolism includes, by way of example only, controlling in viva PK properties, rget activities, ial toxicities associated with cpr450 interactions, drug-drug ctions, and the like. Further, modifications to G allow for the tailoring of the in viva efficacy of the compound h the modulation of, by way of example, specific and non-specific protein binding to plasma proteins and lipids and tissue distribution in viva.

In another ment, provided herein is a compound of Formula (D). Formula (D) is as follows: N \ \ lk / ’N N T \Z :R6 R8 R7 Formula (D) wherein: La is CH2, 0, NH or S; Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; Y is an optionally tuted group selected from among 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; R6, R7, and R8 are each independently selected from among H, tuted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4heteroalkyl, substituted or tituted ycloalkyl, substituted or unsubstituted C2-C6heterocycloalkyl, C1-C6alkoxyalkyl, C1—Cgalkylaminoalkyl, substituted or tituted C3- Cécycloalkyl, substituted or unsubstituted aryl, tuted or unsubstituted heteroaryl, substituted or unsubstituted C1—C4alkyl(aryl), substituted or unsubstituted C1-C4alkyl(heteroaryl), substituted or unsubstituted C1-C4alkyl(C3-Cgcycloalkyl), or tuted or unsubstituted C1-C4a1ky1(C2—Cgheterocycloalkyl); or R7 and R8 taken together form a bond; and pharmaceutically active metabolites, or pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof.

In one embodiment are compounds having the structure of Formula (D1): N \ \ lk / ’N N T \2 :R6 R3 R7 Formula (D1) wherein La is CH2, 0, NH or S; Ar is an optionally tuted aromatic carbocycle or an aromatic heterocycle; Y is an optionally tuted group selected from among alkylene, heteroalkylene, arylene, heteroarylene, alkylenearylene, alkyleneheteroarylene, and alkyleneheterocycloalkylene, or combination thereof; Z is C(=O), NHC(=O), NRaC(=O), NRaS(=O)X, where x is 1 or 2, and Ra is H, substituted or unsubstituted alkyl, substituted or unsubstituted lkyl; and either R7 and R8 are H; R6 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalkylaminoalkyl, C1-Cghydroxyalkylaminoalkyl, C1- Cgalkoxyalkylaminoalkyl, substituted or unsubstituted C3—C6cycloalkyl, substituted or unsubstituted C1-C8alkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alkyl(aryl), C1-C4alkyl(heteroaryl), lkylethers, C1-Cgalkylamides, or C1- l(C2—Cgheterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1—C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1-Cgalkylaminoalkyl, C1—Cghydroxyalkylaminoalkyl, C1- xyalkylaminoalkyl, substituted or tituted C3-C6cycloalkyl, substituted or unsubstituted C1-Cgalkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or tituted eterocycloalkyl, tuted or unsubstituted heteroaryl, C1- C4alkyl(aryl), C1-C4alkyl(heteroaryl), C1-Cgalkylethers, C1-Cgalkylamides, or C1- C4alkyl(C2-Cgheterocycloalkyl); or R6 and R8 taken er form a bond; R7 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, lkylaminoalkyl, C1-C8hydroxyalkylaminoalkyl, C1- Cgalkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6cycloalkyl, tuted or unsubstituted C1-Cgalkle3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alky1(aryl), C1-C4alkyl(heteroaryl), C1-Cgalky1ethers, C1-Cgalky1amides, or C1- C4a1kyl(C2-Cgheterocycloalkyl); or combinations thereof; and pharmaceutically active metabolites, or pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof.

In another embodiment are provided pharmaceutically acceptable salts of compounds of Formula (D1). By way of e only, are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with 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 rion is an anion, such as adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, lfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, e, 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. Further salts include those in Which the counterion is an cation, such as sodium, lithium, potassium, m, magnesium, ammonium, and quaternary ammonium (substituted With at least one organic moiety) cations.

In another embodiment are pharmaceutically acceptable esters of compounds of Formula (Dl), ing those in Which the ester group is selected from a formate, acetate, propionate, butyrate, acrylate and ethylsuccinate.

In another embodiment are pharmaceutically acceptable ates of compounds of Formula (Dl). In another embodiment are pharmaceutically acceptable N—acyl derivatives of compounds of Formula (D1). Examples yl groups include N—acetyl and N- ethoxycarbonyl .

] In a r embodiment, La is 0.

In a r embodiment, Ar is phenyl.

] In a further embodiment, Z is C(=O), NHC(=O), or NCH3C(=O).

In a further embodiment, each of R1, R2, and R3 is H.

In one embodiment is a compound of Formula (Dl) wherein R6, R7, and R8 are all H. In another embodiment, R6, R7, and R8 are not all H.

For any and all of the embodiments, tuents can be selected from among from a subset of the listed atives. For example, in some embodiments, La is CH2, 0, or NH.

In other embodiments, La is O or NH. In yet other embodiments, La is 0.

In some embodiments, Ar is a substituted or unsubstituted aryl. In yet other ments, Ar is a 6-membered aryl. In some other embodiments, Ar is phenyl.

In some embodiments, x is 2. In yet 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.

In some embodiments, R7 and R8 are independently selected from among H, unsubstituted C1-C4 alkyl, substituted lkyl, unsubstituted eteroalkyl, and substituted C1-C4heteroalkyl; or R7 and R8 taken together form a bond. In yet other embodiments, each of R7 and R8 is H; or R7 and R8 taken together form a bond.

In some embodiments, R6 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4heteroalkyl, C1-C6alkoxyalky1, C1-C2alkyl-N(C1-C3alky1)2, substituted or unsubstituted aryl, tuted or unsubstituted heteroaryl, C1-C4a1ky1(ary1), C1- C4alkyl(heteroaryl), C1-C4alkyl(C3-Cgcycloalkyl), or C1-C4alkyl(C2-Cgheterocycloalkyl). In some other embodiments, R6 is H, substituted or unsubstituted C1-C4alky1, substituted or unsubstituted C1—C4heteroalkyl, lkoxyalkyl, C1-C2alkyl-N(C1-C3alkyl)2, C1-C4alkyl(aryl), C1-C4alkyl(heteroaryl), C1—C4alkyl(C3—C8cycloalkyl), or C1—C4alkyl(C2-Cgheterocycloalkyl). In yet other embodiments, R6 is H, substituted or unsubstituted lkyl, -CH2-O-(C1-C3alkyl), - CH2-N(C1-C3alkyl)2, C1-C4alkyl(phenyl), or C1—C4alkyl(5— or ered heteroaryl). In some embodiments, R6 is H, substituted or unsubstituted C1-C4alkyl, -CH2-O-(C1-C3alkyl), -CH2- N(C1-C3alkyl)2, C1-C4alkyl(phenyl), or C1-C4alkyl(5— or 6-membered heteroaryl containing 1 or 2 N atoms), or C1-C4alkyl(5- or 6-membered heterocycloalkyl containing 1 or 2 N atoms).

In some embodiments, Y is an optionally substituted group ed from among alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl. In other embodiments, Y is an optionally tuted group ed from among C1-C6alkyl, C1-C6heteroalkyl, 4-, 5-, 6- or 7-membered cycloalkyl, and 4-, 5-, 6- or 7-membered heterocycloalkyl. In yet other embodiments, Y is an optionally substituted group selected from among C1-C6alkyl, C1-C6heteroalkyl, 5-, or 6- ed cycloalkyl, and 5-, or 6-membered heterocycloalkyl containing 1 or 2 N atoms. In some other embodiments, Y is a 5-, or 6-membered cycloalkyl, or a 5-, or 6-membered heterocycloalkyl containing 1 or 2 N atoms.

Any combination of the groups described above for the various variables is contemplated herein. It is understood that substituents and substitution patterns on the compounds provided herein can be selected by one of ordinary skill in the art to e compounds that are ally stable and that can be synthesized by techniques known in the art, as well as those set forth herein.

In one embodiment the irreversible inhibitor of a kinase has the structure of Formula (E): Formula (E) wherein: n is a moiety that binds to the active site of a kinase, including a tyrosine kinase, further including a Btk kinase cysteine homolog; Y is an optionally substituted group ed from among alkylene, heteroalkylene, arylene, heteroarylene, heterocycloalkylene, cycloalkylene, alkylenearylene, alkyleneheteroarylene, alkylenecycloalkylene, and alkyleneheterocycloalkylene; Z is C(=O), , NHC(=O), NCH3C(=O), C(=S), S(=O)x, OS(=O)X, NHS(=O)X, Where x is l or 2; R6, R7, and R8 are each independently selected from among H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4heteroalkyl, substituted or tituted C3-C6cycloalkyl, tuted or unsubstituted C2-C6heterocycloalkyl, C1-C6alkoxyalkyl, C1-C8alkylaminoalkyl, substituted or unsubstituted C3- C6cycloalkyl, substituted or unsubstituted aryl, tuted or unsubstituted heteroaryl, substituted or unsubstituted C1—C4alkyl(aryl), substituted or unsubstituted C1-C4alkyl(heteroaryl), substituted or unsubstituted C1-C4a1ky1(C3-Cgcycloalky1), or substituted or unsubstituted C1-C4alkyl(C2-Cgheterocycloalkyl); or R7 and R8 taken together form a bond; and pharmaceutically active metabolites, or pharmaceutically acceptable es, pharmaceutically able salts, or pharmaceutically acceptable prodrugs thereof.

In some embodiments, is a substituted fused biaryl moiety selected from / / / l \ l \ / \ N \ N \ /N\( | N | N / N N/ N/ \ N \ N / \ N / l N/ \N T ‘ \” N/ / k k ” \N N/ \ /N\( I N In one , provided herein are compounds of Formula (F). Formula (F) is as La/Ar l\ 5“ N/ 'i‘ Y\Z R6 R‘8 R7 Formula (F) wherein La is CH2, 0, NH or S; Ar is a substituted or unsubstituted aryl, or a tuted or unsubstituted heteroaryl; and either (a) Y is an optionally substituted group selected from among alkylene, heteroalkylene, arylene, heteroarylene, alkylenearylene, alkyleneheteroarylene, alkylenecycloalkylene and alkyleneheterocycloalkylene; Z is C(=O), NHC(=O), NRaC(=O), NRaS(=O)x, where x is l or 2, and Ra is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and either (i) R6, R7, and R8 are each independently selected from among H, substituted or unsubstituted lkyl, substituted or unsubstituted C1-C4heteroalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C2-C6heterocycloalkyl, C1- C6alkoxyalkyl, C1-Cgalkylarninoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1- C4alkyl(aryl), substituted or unsubstituted C1—C4alkyl(heteroaryl), substituted or tituted lkyl(C3-C8cycloalkyl), or substituted or unsubstituted C1-C4alkyl(C2-Cgheterocycloalkyl); (ii) R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1- C4heteroalkyl, C1—Cgalkylaminoalkyl, C1—C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1-Cgalkle3—C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1- C4alkyl(aryl), lkyl(heteroaryl), C1—Cgalkylethers, C1-Cgalkylan1ides, or C1- C4a1kyl(C2-Cgheterocycloalkyl); or (iii) R7 and R8 taken together form a bond; R6 is selected from among H, substituted or unsubstituted C1-C4alkyl, substituted or unsubstituted C1-C4heteroalkyl, substituted or unsubstituted ycloalkyl, substituted or unsubstituted C2-C6heterocycloalkyl, C1-C6alkoxyalkyl, C1-Cgalkylan1inoalkyl, tuted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4alkyl(aryl), substituted or unsubstituted C1—C4alky1(heteroaryl), substituted or tituted C1- C4alkyl(C3-Cgcycloalkyl), or substituted or unsubstituted C1-C4alkyl(C2- Cgheterocycloalkyl) or (b) Y is an optionally substituted group selected from cycloalkylene or cycloalkylene; Z is C(=O), NHC(=O), O), NRaS(=O)X, where x is l or 2, and Ra is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl; and either (i) R7 and R8 are H; R6 is substituted or unsubstituted C1-C4alkyl, tuted or unsubstituted C1-C4heteroalkyl, C1-Cgalkylaminoalkyl, C1—C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3—C6cycloalkyl, substituted or tituted C1-Cgalkle3- Cécycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2- Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4alkyl(aryl), C1- C4all<yl(heteroaryl), C1-Cgalkylethers, C1—Cgalkylamides, or lkyl(C2- Cgheterocycloalkyl); (ii) R6 and R8 are H; R7 is substituted or unsubstituted C1—C4alkyl, substituted or unsubstituted eteroalkyl, C1-Cgalkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1—C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C1-Cgalkle3- C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2- rocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4alky1(ary1), C1- yl(heteroaryl), C1-Cgalkylethers, C1-Cgalky1amides, or C1-C4a1kyl(C2- Cgheterocycloalkyl); or (iii) R7 and R8 taken er form a bond; R6 is substituted or unsubstituted C1—C4alkyl, substituted or unsubstituted eteroalkyl, C1-Cgalkylaminoalkyl, C1-Cghydroxyalkylaminoalkyl, C1-Cgalkoxyalkylaminoall<yl, substituted or unsubstituted C3-C60ycloalkyl, substituted or unsubstituted C1-Cgalkle3- C6cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2- Cgheterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4alkyl(aryl), C1- C4all<yl(heteroaryl), C1-Cgalkylethers, C1-Cgalkylamides, or C1-C4alkyl(C2- Cgheterocycloalkyl); and pharmaceutically active metabolites, or pharmaceutically acceptable solvates, pharmaceutically acceptable salts, or pharmaceutically acceptable prodrugs thereof Further embodiments of nds of Formula (A), Formula (B), Formula (C), a (D), include, but are not limited to, compounds selected from the group consisting of: NH2 NH2 NH2 NH2 NH2 N \ \ N \ \ \N N \ \ \N | N l / N l / N/ | N Nl \ / / / / N N N N 0 N CW O _ \ _ _ O O _ O O O O O 9 9 9 9 9 O/Q OQ O’Q OQ NH; NH2 NHZ NH2 NHz NI \ \N NI \ \N [\l \ \N Nt\ \N N \ \N N/ N/ N/ N/ / / N N O /= — — N-“s HN HN HN HN o o o o In still another embodiment, nds provided herein are selected from In one aspect, provided herein is a compound selected from among: l-(3 -(4- amino(4-phenoxyphenyl)- l H-pyrazolo[3,4—d]pyrimidin- l -yl)piperidin-l -yl)propenone (Compound 4); (E)- l -(3-(4-amino(4-phenoxyphenyl)- l H-pyrazolo [3 ,4-d]pyrimidin- l - yl)piperidin- l -yl)butenone (Compound 5); l-(3-(4-amino-3 -(4-phenoxyphenyl)— l H- pyrazolo [3 ,4-d]pyrimidin- l -yl)piperidin- l -y1)sulfonylethene (Compound 6); l-(3 ino-3 - noxyphenyl)- l H—pyrazolo [3 ,4—d]pyrimidinyl)piperidin- l —y1)propynone (Compound 8); 4-amino(4-phenoxyphenyl)—1 H-pyrazolo [3 ,4—d]pyrimidin— 1 - yl)piperidin-l-y1)propenone (Compound 9); N-((1s,4s)(4-amino(4-phenoxyphenyl)— lH-pyrazolo [3 ,4-d]pyrimidinyl)cyclohexyl)acrylamide (Compound 10); (4-amino(4-phenoxyphenyl)— l H—pyrazolo [3 ,4—d]pyrimidin—1—yl)pyrrolidin-l-yl)propen-l-one (Compound 1 1); l-((S)—3—(4—amino—3—(4—phenoxyphenyl)— l H-pyrazolo [3 ,4-d]pyrimidin- l - yl)pyrrolidin- l openone (Compound 12); —3-(4-amino(4-phenoxyphenyl)- lH-pyrazolo [3 ,4-d]pyrimidin- l -yl)piperidin— l —yl)prop—2—enone (Compound 13); l-((S)-3 -(4- amino(4-phenoxyphenyl)- l H-pyrazolo[3,4—d]pyrimidin- l -yl)piperidin-l -yl)propenone (Compound 14); and (E)— l -(3-(4-amino(4—phenoxyphenyl)- l H-pyrazolo [3 ,4-d]pyrimidin- l - yl)piperidin- l -yl)(dimethylamino)buten-l —one (Compound 1 5).

In some embodiments, the Btk inhibitor is (R)- l -(3-(4-amino-3 -(4- phenoxyphenyl)— l H-pyrazolo [3 ,4-d]pyrimidin- l -yl)piperidin- l -yl)propenone.

In one embodiment, the Btk inhibitor is (x-cyano-B-hydroxy-B- methyl-N—(2,5 - dibromophenyl)propenamide (LFM-Al 3), AVL- 1 01 , 4-tert-butyl-N-(3 -(8 - (phenylamino)imidazo[ l ,2-a]pyrazin-6—yl)pheny1)benzamide, 5 -(3-aminomethylphenyl)— l - methyl(4-(morpholinecarbonyl)pheny1amino)pyrazin-2( l H)-one, N-(2-methyl-3 -(4- methyl(4-(morpholinecarbonyl)phenylamino)—5-oxo-4,5-dihydropyrazin yl)phenyl)acetamide, 4-tert-butyl-N-(2-methy1—3-(4—methyl-6—(4—(morpholine yl)phenylamino)—5 -oxo-4,5-dihydropyrazinyl)pheny1)benzamide, 5 -(3 -(4-tert- butylbenzylamino)—2-methylphenyl)— 1 —methyl(4-(morpholine—4— carbonyl)phenylamino)pyrazin-2( 1 H)-one, 5-(3-(3-tert-butylbenzylamino)methylphenyl) methyl(4-(morpholinecarbonyl)phenylamino)pyrazin-2( 1 H)-one, 3-tert-butyl-N-(2-methyl(4-methyl(4—(morpholine—4—carbonyl)phenylamino)—5—oxo-4,5-dihydropyrazin nyl)benzamide, 6—tert—butyl—N—(2—methyl—3—(4—methyl(4-(morpholine carbonyl)phenylamino)-5 ,5—dihydropyrazin—2—yl)phenyl)nicotinamide, and terreic acid.

Throughout the cation, groups and substituents thereof can be chosen by one skilled in the field to e stable moieties and compounds.

] In certain embodiments, any of the Btk inhibitors and/or the second agent provided herein for the invention methods is included in a pharmaceutical composition comprising: i) a physiologically acceptable carrier, diluent, and/or excipient.

In some embodiments, the Btk inhibitor of the invention methods is administered at a dose of from about 125 mg/kg/day to about 12.5 day. In certain ments, the Btk inhibitor is administered at a dose selected from the group ting of about 1.25 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.

In some embodiments provide the kers in accordance with the practice of the present invention is selected from , CD5, t(14;18), CD38, [3-2 microglobulin, p53 mutational , ATM mutational status, chromosome 17p deletion, chromosome llq deletion, e or cytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22, CD1 1c, CD 103, chromosome 7q deletion, and VH mutational status.

In some ments, determining the expression or presence of one or more biomarkers from one or more ulation of cytes is of a combination of biomarkers.

In certain embodiments, the combination ofbiomarkers is CD19 and CD5 or CD20 and CD5.

In other embodiments, the second agent is administered at a dose of from about 1.25 mg/kg/day to about 12.5 mg/kg/day. In n 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 determined expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes. A person skilled in the art such as a physician can readily determine the suitable regimen (e.g. dosage of the second agent) based on the diagnostic results.

[00272] In other embodiments, the present invention provides methods for treating a cancer comprising determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes in a subject that has received a dose of a Btk inhibitor; and administering a second agent based on the determined expression profile.

In other embodiments, the present invention also provides methods for treating a cancer comprising administering a Btk inhibitor ent to result in an increase or appearance in the blood of a subpopulation of lymphocytes defined by immunophenotyping; and administering a second agent once the increase or appearance in the blood of the subpopulation of lymphocytes is determined.

In some embodiments, the subject is a human.

] In some embodiments, the Btk tors are orally administered.

In any of the aforementioned aspects are fithher embodiments in which administration is enteral, parenteral, or both, and wherein (a) the effective amount of the Btk inhibitor is systemically administered to the mammal; (b) the effective amount of the Btk inhibitor is administered orally to the mammal; (c) the effective amount of the Btk inhibitor is intravenously administered to the mammal; (d) the effective amount of the Btk inhibitor administered by inhalation; (e) the effective amount of the Btk tor is administered by nasal administration; or (f) the effective amount of the Btk inhibitor is stered by injection to the mammal; (g) the effective amount of the Btk inhibitor is administered topically l) to the mammal; (h) the effective amount of the Btk tor is administered by lmic administration; or (i) the effective amount of the Btk inhibitor is administered rectally to the mammal.

In any of the aforementioned aspects are further ments sing single administrations of the effective amount of the Btk inhibitor, including r embodiments in which (i) the Btk inhibitor is administered once; (ii) the Btk inhibitor is administered to the mammal le times over the span of one day; (iii) continually; or (iv) continuously.

In any of the aforementioned aspects are fiarther embodiments comprising multiple administrations of the effective amount of the Btk inhibitor, including fiarther embodiments in which (i) the Btk inhibitor is administered in a single dose; (ii) the time between multiple administrations is every 6 hours; (iii) the Btk inhibitor is administered to the mammal every 8 hours. In further or alternative embodiments, the method comprises a drug holiday, wherein the administration of the Btk inhibitor is temporarily suspended or the dose of the Btk inhibitor being administered is temporarily reduced; at the end of the drug holiday, dosing of the Btk inhibitor is d. The length of the drug holiday can vary from 2 days to 1 year.

In any of the aforementioned aspects are further embodiments in which administration is enteral, parenteral, or both, and wherein (a) the effective amount of the second agent is systemically administered to the mammal; (b) the effective amount of the second agent is administered orally to the mammal; (c) the effective amount of the second agent is intravenously administered to the mammal; (d) the effective amount of the second agent administered by inhalation; (e) the effective amount of the second agent is administered by nasal administration; or (f) the effective amount of the second agent is administered by ion to the mammal; (g) the ive amount ofthe second agent is administered topically (dermal) to the mammal; (h) the effective amount of the second agent is administered by ophthalmic administration; or (i) the effective amount of the second agent is administered rectally to the mammal.

In any of the aforementioned aspects are r embodiments comprising single administrations of the effective amount of 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 over the span of one day; (iii) continually; or (iv) continuously.

] In any of the aforementioned aspects are fithher embodiments comprising multiple administrations of the ive amount of the second agent, including fiarther embodiments in which (i) the second agent is administered in a single dose; (ii) the time n multiple administrations is every 6 hours; (iii) the second agent is administered to the mammal every 8 hours. In further or ative embodiments, the method comprises a drug holiday, wherein the administration of the second agent is temporarily suspended or the dose of the second agent being administered is temporarily reduced; at the end of the drug holiday, dosing of the second agent is resumed. The length of the drug holiday can vary from 2 days to 1 year.

] In any of the aforementioned aspects the second agent is ed from the group consisting of alemtuzumab, arsenic trioxide, asparaginase (pegylated or non-), bevacizumab, cetuximab, platinum-based compounds such as cisplatin, cladribine, daunorubicin/doxorubicin/idarubicin, irinotecan, fludarabine, S-fluorouracil, gemtuzumab, methotrexate, PaclitaxelTM, taxol, temozolomide, thioguanine, or classes of drugs including hormones (an antiestrogen, an antiandrogen, or gonadotropin releasing hormone analogues, interferons such as alpha interferon, nitrogen mustards such as busulfan or melphalan or mechlorethamine, retinoids such as tretinoin, topoisomerase inhibitors such as irinotecan or topotecan, tyrosine kinase inhibitors such as gefinitinib or imatinib, or agents to treat signs or symptoms induced by such therapy including allopurinol, filgrastim, granisetron/ondansetron/palonosetron, dronabinol.

Preparation ofCompounds Compounds of Formula D may be synthesized using standard tic techniques known to those of skill in the art or using s known in the art in combination with methods bed herein. In ons, solvents, atures and other reaction conditions presented herein may vary according to those of skill in the art. As a further guide the ing synthetic methods may also be utilized.

The reactions can be employed in a linear sequence to e the compounds described herein or they may be used to size fragments which are subsequently joined by the methods described herein and/or known in the art.

Farmatl'an 0fCovalent Linkages by Reaction ofan ophz'le with a Nucleophz'le The compounds described herein can be modified using various electrophiles or nucleophiles to form new functional groups or substituents. Table 1 entitled “Examples of Covalent Linkages and Precursors Thereof” lists selected examples of covalent linkages and precursor functional groups which yield and can be used as guidance toward the variety of electrophiles and nucleophiles combinations available. Precursor fianctional groups are shown as electrophilic groups and philic .

Table 1: Examples of Covalent Linkages and Precursors Thereof Electrophile Alkyl thiol Alkyl ethers Alkyl amines Alkyl thiol Vinyl sulfone Alkyl ethers Vinyl sulfone Alkyl amines Vinyl e Vinyl sulfide Use of Protecting Groups In the reactions described, it may be necessary to protect reactive fianctional , for example hydroxy, amino, imino, thio or y groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Protecting groups are used to block some or all reactive moieties and prevent such groups from participating in chemical reactions until the protective group is removed. In one embodiment, each protective group be removable by a different means. Protective groups that are d under totally disparate reaction conditions fulfill the requirement of differential removal. Protective groups can be removed by acid, base, and hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile and may be used to t carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile. Carboxylic acid and y reactive moieties may be blocked with base labile groups such as, but not limited to, methyl, ethyl, and acetyl in the ce of amines blocked with acid labile groups such as t-butyl ate or with carbamates that are both acid and base stable but hydrolytically removable.

[00287] Carboxylic acid and hydroxy reactive moieties may also be blocked with hydrolytically removable protective groups such as the benzyl group, while amine groups capable of hydrogen bonding with acids may be blocked with base labile groups such as Fmoc.

Carboxylic acid reactive moieties may be protected by conversion to simple ester compounds as exemplified herein, or they may be d with oxidatively-removable tive groups such as 2,4-dimethoxybenzyl, while co-existing amino groups may be blocked with fluoride labile silyl carbamates.

Allyl blocking groups are usefiil in then presence of acid- and base- protecting groups since the former are stable and can be uently removed by metal or pi-acid catalysts. For example, an allyl-blocked carboxylic acid can be deprotected with a talyzed reaction in the presence of acid labile t—butyl carbamate or base—labile acetate amine protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate may be attached. As long as the residue is ed to the resin, that functional group is blocked and cannot react. Once released from the resin, the fianctional group is available to react.

Typically ng/protecting groups may be selected from: H2 0 H CH32 C\ 0C\O)l\ E ’0 H2040\C/ \ H20/ 3 \fl/ H30/ H2 2 allyl Bn Cbz alloc Me H2 H3C\ [CH3 o c - .2 H30/ \ (H3C)3C/ (H30)3C Sl\, /S'\/\ A (CH3)3C 0 Et t-bUtyl TBDMS T900 H2 0k C\ o O HZC’ (CH3)3C/ \H/ (CeH5)3C~ H CL O 3 0.0 Bee PMB trityl acetyl Fmoc 00290 Other rotectin roups, plus adetailed description of techni ues aq pplicable to the creation of ting groups and their removal are described in Greene and Wuts, Protective Groups in Organic sis, 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 entirety.

Further Forms ofCompounds The compounds described herein may possess one or more stereocenters and each center may exist in the R or S configuration. The compounds presented herein include all diastereomeric, enantiomeric, and ic forms as well as the appropriate mixtures thereof.

Stereoisomers may be obtained, if desired, by methods known in the art as, for example, the separation of stereoisomers by chiral chromatographic columns.

Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known, for example, by chromatography and/or fractional crystallization. In one embodiment, enantiomers can be ted by chiral chromatographic columns. In other embodiments, enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and ting (e.g., hydrolyzing) the individual diastereomers to the ponding pure omers. All such isomers, ing diastereomers, enantiomers, and mixtures thereof are considered as part of the compositions described herein.

The methods and formulations described herein include the use of es, lline forms (also known as polymorphs), or pharmaceutically acceptable salts of compounds bed herein, as well as active metabolites of these compounds having the same type of activity. In some situations, compounds may exist as tautomers. All tautomers are ed within the scope of the compounds presented herein. In addition, the compounds described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The ed forms of the compounds presented herein are also considered to be disclosed herein. nds of Formula D in unoxidized form can be prepared from N-oxides of compounds of Formula D by treating with a reducing agent, such as, but not limited to, sulfiar, sulfur dioxide, triphenyl ine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like in a suitable inert organic solvent, such as, but not limited to, acetonitrile, ethanol, aqueous e, or the like at 0 to 80°C.

[00295] In some embodiments, compounds described herein are prepared as prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a compound described herein, which is administered as an ester (the “prodrug”) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is lically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is lized to reveal the active moiety. In n ments, upon in viva administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or eutically active form of the compound. To produce a prodrug, a pharmaceutically active compound is d such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side s or toxicity, to improve the flavor of a drug or to alter other characteristics or ties of a drug. By virtue of knowledge macodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound. (see, 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 , Academic Press, Inc., San Diego, pages 352-401, Saulnier et al., (1994), Bioorganic and nal Chemistry Letters, Vol. 4, p. 1985).

Prodrug forms of the herein described compounds, wherein the prodrug is metabolized in vivo to produce a derivative as set forth herein are included within the scope of the . In some cases, some of the herein-described compounds may be a g for another derivative or active compound.

Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for ce, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in ceutical compositions over the parent drug. Prodrugs may be designed as reversible drug derivatives, for use as modifiers to enhance drug transport to site—specific tissues. In some embodiments, the design of a prodrug increases the effective water solubility. See, e. g., Fedorak et al., Am. J. l., 269:G210-218 (1995); McLoed et al., Gastroenterol, 106:405-413 (1994); Hochhaus et al., Biomed. Chrom., 6:283-286 (1992); J. Larsen and H. Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987); 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 A.C.S. Symposium Series; and Edward B. Roche, ersible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, all incorporated herein in their entirety.

[00298] Sites on the aromatic ring portion of compounds of Formula D can be susceptible to various metabolic reactions, therefore incorporation of appropriate substituents on the aromatic ring structures, such as, by way of example only, halogens can reduce, minimize or eliminate this metabolic pathway.

Compounds bed herein include ically—labeled nds, which are identical to those d in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl, respectively.

Certain isotopically-labeled compounds described herein, for example those into which radioactive es such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Further, tution with es such as deuterium, i.e., 2H, can afford n therapeutic advantages resulting from r lic stability, for example increased in vivo half-life or reduced dosage requirements.

In additional or further ments, the compounds bed herein are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. nds described herein may be formed as, and/or used as, pharmaceutically acceptable salts. The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid on salts, formed ) by reacting the free base form of the compound with a pharmaceutically acceptable: inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid, and the like; or with an organic acid such as acetic acid, nic 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, c acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, esulfonic 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-l-carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxyene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, ic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion (e. g. lithium, sodium, ium), an alkaline earth ion (e. g. magnesium, or calcium), or an aluminum ion; or coordinates with an organic base. able organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, ylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

The corresponding counterions of the pharmaceutically acceptable salts may be analyzed and identified using various methods including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof.

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

It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms or crystal forms thereof, particularly es or polymorphs.

Solvates contain either stoichiometric or non—stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of 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. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the nds and methods provided herein.

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

Compounds described herein may be in various forms, including but not d to, amorphous forms, milled forms and nano—particulate forms. In addition, compounds described herein include lline forms, also known as polymorphs. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound.

Polymorphs usually have ent X—ray diffraction patterns, ed spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility.

Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single l form to dominate.

The screening and terization of the pharmaceutically acceptable salts, polymorphs and/or solvates may be accomplished using a y of techniques including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor on, and copy.

Thermal analysis methods s thermo chemical degradation or thermo physical ses including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, determine weight loss, to find the glass transition temperature, or for excipient compatibility studies. Such methods include, but are not limited to, Differential scanning calorimetry (DSC), Modulated Differential ng Calorimetry (MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric and Infrared analysis ). X-ray diffraction methods include, but are not limited to, single l and powder diffractometers and synchrotron sources. The s spectroscopic techniques used include, but are not limited to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not d to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR copy, and Raman microscopy.

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

Cancer Treatment Regimens Disclosed herein, in certain embodiments, is a method for ng 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 zed plurality of cells. In some embodiments, the amount of the irreversible Btk tor 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 n after the peripheral blood concentration of the mobilized plurality of cells increases as 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 peripheral blood concentration of the mobilized plurality of cells. In some embodiments, analyzing the mobilized plurality of cells comprises ing the duration of an increase in the peripheral blood tration of the mobilized plurality of cells as compared to the concentration before administration of the Btk inhibitor. In some ments, the method further comprises administering a second cancer treatment regimen after the peripheral blood concentration of the mobilized plurality of cells has sed for a predetermined length of time. In some embodiments, analyzing the mobilized plurality of cells comprises counting the number of mobilized plurality of cells in the peripheral blood. In some embodiments, the method further comprises administering a second cancer treatment regimen after the number of mobilized plurality of cells in the peripheral blood increases as compared to the number before administration of the Btk inhibitor. In some embodiments, administering the second cancer treatment regimen occurs after a subsequent decrease in the number of mobilized plurality of cells in the eral blood. In some embodiments, analyzing the mobilized plurality of cells comprises measuring the duration of an increase in the number of mobilized plurality of cells in the peripheral blood as compared to the number before administration of the Btk inhibitor. In some ments, the method r ses administering a second cancer treatment n after the number of mobilized plurality of cells in the peripheral blood has increased for a predetermined length of time.

In some embodiments, administering a Btk tor before a second cancer treatment regimen reduces immune—mediated reactions to the second cancer ent regimen.

In some embodiments, administering a Btk inhibitor before ofatumumab reduces immune- mediated reactions to ofatumumab.

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 tor, a Blnk inhibitor, a PLCy inhibitor, a PKCB inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises an antibody, B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapeutic, a DNA ng agent, a proteosome inhibitor, a histone deacytlase inhibitor, a protein kinase inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jakl/Z inhibitor, a protease tor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

In some embodiments, the second cancer treatment regimen comprises mbucil, ifosphamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, atinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL—101, ibritumomab, tositumomab, bortezomib, tatin, endostatin, or a combination f.

In some embodiments, the second cancer treatment regimen comprises hosphamide, hydroxydaunorubicin, stine, and prednisone, and optionally, rituximab.

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

In some embodiments, the second cancer ent regimen comprises fludarabine, cyclophosphamide, and rituximab.

In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, Vincristine, and sone, and ally, rituximab.

In some embodiments, the second cancer treatment n comprises etoposide, doxorubicin, vinristine, cyclophosphamide, prednisolone, and optionally, rituximab.

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

Additional cancer treatment regimens e Nitrogen Mustards such as for example, bendamustine, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, prednimustine, trofosfamide; Alkyl Sulfonates like busulfan, ulfan, treosulfan; Ethylene Imines like uone, thiotepa, triaziquone; Nitrosoureas like carmustine, fotemustine, lomustine, nimustine, ranimustine, semustine, streptozocin; Epoxides such as for e, etoglucid; Other Alkylating Agents such as for example dacarbazine, mitobronitol, pipobroman, temozolomide; Folic Acid Analogues such as for example methotrexate, permetrexed, pralatrexate, raltitrexed; Purine Analogs such as for e cladribine, clofarabine, fludarabine, mercaptopurine, nelarabine, tioguanine; dine Analogs such as for e azacitidine, capecitabine, carmofur, cytarabine, decitabine, fluorouracil, abine, tegafur; Vinca Alkaloids such as for example Vinblastine, Vincristine, Vindesine, vinflunine, vinorelbine; Podophyllotoxin Derivatives such as for example ide, teniposide; Colchicine derivatives such as for e demecolcine; Taxanes such as for example docetaxel, paclitaxel, paclitaxel umex; Other Plant Alkaloids and Natural Products such as for example trabectedin; Actinomycines such as for example dactinomycin; Antracyclines such as for example aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, zorubincin; Other Cytotoxic Antibiotics such as for e bleomycin, ixabepilone, mitomycin, ycin; Platinum Compounds such as for example carboplatin, cisplatin, oxaliplatin, satraplatin; Methylhydrazines such as for example procarbazine; Sensitizers such as for e aminolevulinic acid, efaproxiral, methyl aminolevulinate, porfimer sodium, temoporfin; Protein Kinase Inhibitors such as for example dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, nib, temsirolimus; Other Antineoplastic Agents such as for example alitretinoin, altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, estramustine, hydroxycarbamide, irinotecan, lonidamine, masoprocol, miltefosein, mitoguazone, mitotane, rsen, pegaspargase, pentostatin, romidepsin, sitimagene ceradenovec, tiazofurine, topotecan, tretinoin, vorinostat; Estrogens such as for example diethylstilbenol, ethinylestradiol, fosfestrol, polyestradiol ate; Progestogens such as for e gestonorone, medroxyprogesterone, megestrol; Gonadotropin Releasing Hormone Analogs such as for example buserelin, goserelin, leuprorelin, triptorelin; Anti—Estrogens such as for example fulvestrant, tamoxifen, toremifene; ndrogens such as for example bicalutamide, flutamide, nilutamide, , Enzyme Inhibitors, aminoglutethimide, anastrozole, exemestane, formestane, ole, vorozole; Other e nists such as for example abarelix, degarelix; Immunostimulants such as for e histamine dihydrochloride, mifamurtide, pidotimod, plerixafor, roquinimex, thymopentin; Immunosuppressants such as for example everolimus, gusperimus, leflunomide, mycophenolic acid, sirolimus; Calcineurin Inhibitors such as for e ciclosporin, tacrolimus; Other Immunosuppressants such as for example azathioprine, lenalidomide, methotrexate, thalidomide; and Radiopharmaceuticals such as for example, iobenguane.

Additional cancer treatment regimens include interferons, interleukins, Tumor Necrosis Factors, Growth Factors, or the like.

Additional cancer treatment regimens include Immunostimulants such as for example ancestim, filgrastim, lenograstim, molgramostim, pegfilgrastim, sargramostim; Interferons such as for example interferon alfa natural, interferon alfa-Za, interferon alfa-2b, interferon alfacon- l eron l interferon beta natural, eron beta-la, interferon , , beta-lb, interferon gamma, peginterferon alfa—2a, peginterferon alfa-Zb; Interleukins such as for example aldesleukin, oprelvekin; Other Immunostimulants such as for example BCG vaccine, glatiramer e, ine dihydrochloride, immunocyanin, an, melanoma vaccine, mifamurtide, pegademase, mod, afor, poly IzC, poly ICLC, roquinimex, tasonermin, thymopentin; Immunosuppressants such as for example abatacept, us, alefacept, antilymphocyte immunoglobulin (horse), ymocyte immunoglobulin (rabbit), eculizumab, efalizumab, everolimus, gusperimus, leflunomide, muromab-CD3, enolic acid, natalizumab, sirolimus; TNF alpha Inhibitors such as for example adalimumab, afelimomab, certolizumab pegol, cept, golimumab, infliximab; Interleukin Inhibitors such as for example anakinra, basiliximab, canakinumab, daclizumab, mepolizumab, rilonacept, tocilizumab, ustekinumab; Calcineurin Inhibitors such as for example ciclosporin, tacrolimus; Other Immunosuppressants such as for example azathioprine, lenalidomide, methotrexate, thalidomide.

Additional cancer treatment regimens e 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.

Additional cancer treatment regimens include Monoclonal Antibodies such as for example alemtuzumab, bevacizumab, catumaxomab, mab, edrecolomab, gemtuzumab, ofatumumab, mumab, rituximab, zumab, , Immunosuppressants, eculizumab, efalizumab, muromab-CD3, natalizumab; TNF alpha Inhibitors such as for example adalimumab, afelimomab, certolizumab pegol, golimumab, infliximab, Interleukin Inhibitors, basiliximab, canakinumab, daclizumab, mepolizumab, tocilizumab, ustekinumab, , Radiopharmaceuticals, ibritumomab tiuxetan, tositumomab; Others Monoclonal Antibodies such as for example abagovomab, adecatumumab, alemtuzumab, anti-CD30 monoclonal dy Xmab2513, anti-MET monoclonal antibody MetMab, apolizumab, apomab, arcitumomab, ximab, bispecific antibody 2B1, blinatumomab, brentuximab vedotin, capromab pendetide, cixutumumab, claudiximab, mumab, dacetuzumab, denosumab, eculizumab, epratuzumab, epratuzumab, ertumaxomab, etaracizumab, figitumumab, fresolimumab, galiximab, ganitumab, gemtuzumab icin, glembatumumab, momab, inotuzumab ozogamicin, ipilimumab, lexatumumab, lintuzumab, lintuzumab, lucatumumab, mapatumumab, matuzumab, zumab, monoclonal antibody CC49, mumab, nimotuzumab, ofatumumab, oregovomab, pertuzumab, ramacurimab, ranibizumab, siplizumab, izumab, mab, tositumomab, zumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, Visilizumab, volociximab, zalutumumab. onal cancer treatment ns include agents that affect the tumor micro- enviroment such as cellular signaling k (e.g. phosphatidylinositol 3-kinase (PI3K) signaling pathway, signaling from the B-cell receptor and the IgE or). 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 is a PKCy inhibitor such as by way of example only, enzastaurin.

Examples of agents that affect the tumor micro-environment include PI3K signaling inhibitor, syc kinase inhibitor, Protein Kinase Inhibitors such as for example dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, temsirolimus; Other Angiogenesis Inhibitors such as for example GT-111, JI-101, R1530; Other Kinase Inhibitors such as for example AC220, AC480, ACE-041, AMG 900, AP24534, Arry- 614, AT7519, AT9283, AV-951, axitinib, AZD1152, AZD7762, 5, AZD8931, bafetinib, BAY 73-4506, BGJ398, , BI 811283, B16727, BIBF 1120, BIBW 2992, EMS-690154, EMS-777607, EMS-863233, BSK-461364, CAL—101, CEP—l 1981, CYC116, DCC-2036, dinaciclib, dovitinib lactate, E7050, EMD 1214063, ENMD-2076, fostamatinib um, GSK2256098, GSK690693, INCB18424, INNO-406, JNJ-26483327, JX-594, KX2- 391, nib, LY2603618, MGCD265, MK-0457, MK1496, MLN8054, MLN8237, MP470, 16354, NMS—1286937, ON Na, OSI—027, OSI-930, Btk inhibitor, PF-00562271, PF-02341066, PF-03814735, PF—04217903, PF—04554878, PF-04691502, PF-3758309, PHA- , PLC3397, progenipoietin, R547, R763, ramucirumab, regorafenib, R05185426, SAR103168, 33CH 727965, SGI—1176, SGX523, SNS-314, TAK-593, TAK-901, TKI258, 2, TTP607, XL147, XL228, XL281R05126766, XL418, XL765.

[00324] Further examples of anti-cancer agents for use in combination with a Btk inhibitor compound include inhibitors ofmitogen—activated protein kinase signaling, e.g., U0126, PD98059, PD184352, PD0325901, 42886, SB239063, SP600125, BAY 43- 9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies (e.g.,

[00325] Other anti-cancer agents that can be employed in combination with a Btk inhibitor compound include Adriamycin, Dactinomycin, Bleomycin, stine, Cisplatin, aciVicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; mycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; tamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; nar ; bropirimine; busulfan; cactinomycin; calusterone; mide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; mbucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; icin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; bine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; done; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; icin hydrochloride; ifosfamide; iimofosine; interleukin 11 (including recombinant interleukin II; or r1L2), interferon alfa-Za; eron alfa-Zb; interferon alfa-nl; eron alfa-n3; interferon beta-1 a; interferon gamma-l b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; ine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; topurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; omin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; ycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; cin hydrochloride; pyrazofurin; ine; imide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; fene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglycinate e; Vinleurosine sulfate; Vinorelbine te; Vinrosidine sulfate; Vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.

[00326] Other anti-cancer agents that can be employed in combination with a Btk inhibitor nd include: -1, 25 dihydroxyvitamin D3; S-ethynyluracil; erone; aclarubicin; acylfulvene; adecypenol; adozelesin; eukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; lide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing genetic protein—1; antiandrogen, tic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara—CDP—DL—PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin l; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; stat; BCIVABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam tives; lethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine imine; calcipotriol; calphostin C; camptothecin tives; canarypox IL—2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; age derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanosperrnine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; rphyrin; cladribine; ene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; tol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-S -azacytidine; 9- ycin; yl spiromustine; nol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; flnasteride; flavopiridol; flezelastine; fluasterone; bine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-such as for example grth factor-l receptor tor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; j asplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; ycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; ole; linear ine ue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; lin; ninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; stim; mismatched double stranded RNA; mitoguazone; ctol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 —based therapy; mustard anticancer agent; mycaperoxide B; cterial cell wall extract; myriaporone; N—acetyldinaline; N—substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; atin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; ine; pentosan polysulfate ; pentostatin; pentrozole; perflubron; famide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; rpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; um-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl ridone; prostaglandin J2; proteasome inhibitors; n A-based immune modulator; n kinase C inhibitor; protein kinase C inhibitors, microalgal; n tyrosine phosphatase inhibitors; purine nucleoside phosphorylase tors; ins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII mide; rogletimide; rohitukine; romurtide; imex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal uction tors; single chain antigen-binding protein; sizofiran; xane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem- cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; tent stem cell factor; translation tors; tretinoin; triacetyluridine; ibine; trimetrexate; triptorelin; etron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC tors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresolg ne; verdins; verteporfin; Vinorelbine; Vinxaltine; Vitaxin; vorozole; zanoterone; atin; zilascorb; and zinostatin amer.

Yet other anticancer agents that can be employed in combination with a Btk inhibitor compound include alkylating , antimetabolites, natural products, or hormones, e. g., nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (e. g., an), nitrosoureas (e.g., carmustine, lomusitne, ete.), or triazenes (decarbazine, etc.). Examples of antimetabolites include but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine s (e.g., Cytarabine), purine analogs (e. g., topurine, thioguanine, pentostatin).

Examples of alkylating agents that can be employed in combination a Btk inhibitor compound include, but are not limited to, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin, etc.), or nes (decarbazine, ete.). Examples of antimetabolites include, but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e. g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.

Examples of anti-cancer agents which act by arresting cells in the GZ-M phases due to stabilized microtubules and which can be used in ation with a Btk tor compound include without limitation the following marketed drugs and drugs in pment: Erbulozole (also known as R-55104), Dolastatin 10 (also known as DLS-l 0 and NSC-376128), lin 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), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C), Spongistatins (such as Spongistatin l, 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 (such as Epothilone A, lone B, Epothilone C (also known as epothilone A or , Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothilone B ), Epothilone E, Epothilone F, Epothilone B N-oxide, lone A N- oxide, 16-aza-epothilone B, 21-arninoepothilone 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), Soblidotin (also known as TZT-1027), LSP (Pharmacia, also known as LS-4577), LS-4578 acia, also known as -P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-l82877 (Fujisawa, also known as WS-9885B), GS-l64 a), GS-198 a), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, also known as ILX- 651 and LU-223651 ), SAH-49960 /Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM—l32 (Armad), AM—l38 (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, CSL-Ser.HCI, and RPR-25 8062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known as NSC-106969), T-l38067 (Tularik, also known as T-67, TL-138067 and TI- ), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State sity), Oncocidin A1 (also known as BTO- 956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of ne, also known as MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), 72 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-191), TMPN na State University), Vanadocene acetylacetonate, T—138026 (Tularik), Monsatrol, lnanocine (also known as NSC-698666), 3-lAABE (Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, also known as T—900607), RPR- 115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, ondrin B, 1 (Asta Medica), D-68l44 (Asta Medica), Diazonarnide A, A-293620 (Abbott), NPI-2350 s), Taccalonolide A, TUB-245 (Aventis), 54 (Abbott), Diozostatin, (—)—Phenylahistin (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-3 183 15 (Abbott), HTI—286 (also known as SPA-110, trifluoroacetate salt) (Wyeth), D-823l7 (Zentaris), D—82318 (Zentaris), SC-12983 (NCI), Resverastatin ate sodium, BPR-OY—007 (National Health Research Institutes), and SSR- 250411 ).

Biomarkers sed herein, in certain embodiments, is a method for treating a hematological malignancy in an dual in need thereof, comprising: (a) administering to the individual an amount of an irreversible Btk tor sufficient to mobilize a plurality of cells from the malignancy; and (b) ing 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 preparing a ker profile for a population of cells isolated from the plurality of cells. In some embodiments, the biomarker expression profile is used to diagnose, determine a prognosis, or create a predictive profile of a hematological malignancy. In some embodiments, the biomarker profile indicates the expression of a biomarker, the expression level of a biomarker, mutations in a ker, or the presence of a biomarker. In some embodiments, the biomarker is any cytogenetic, cell surface molecular or protein or RNA expression marker. In some embodiments, the biomarker is: ZAP70; t(14,18); [3-2 microglobulin; p53 mutational status; ATM mutational 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; VH mutational status; or a combination f. In some embodiments, the method fiarther comprises providing a second cancer treatment regimen based on the biomarker profile. In some embodiments, the method further comprises not administering based on the biomarker profile. In some ments, the method further comprises predicting the efficacy of a treatment regimen based on the biomarker profile.

In certain embodiments, the methods comprise diagnosing, determining a prognosis, or ng a predictive profile of a hematological maligancy ncy based upon the expression or presence of certain biomarkers. In other embodiments, the methods fithher comprise stratefying patient populations based upon the sion or presence of certain biomarkers in the affected lymphocytes. In still other embodiments, the methods further se determining a eutic regimen for the subject based upon the expression or presence of certain biomarkers in the affected lymphocytes. In yet other embodiments, the s further comprise predicting a response to therapy in a subject based upon the sion or presence of certain biomarkers in the affected lymphocytes.

In certain aspects, provided herein are methods of sing, ining a prognosis, or creating a predictive profile of a hematological maligancy in a subject comprising: (a) stering a Btk inhibitor to the subject sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes; and (b) determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes; wherein the expression or presence of one or more biomarkers is used to diagnose the hematological maligancy, determine the prognosis of the hematological maligancy, or create a predictive profile of the hematological maligancy. In one embodiment, the increase or appearance in the blood of a subpopulation of lymphocytes is determined by immunophenotyping. In another embodiment, the increase or appearance in the blood of a subpopulation of lymphocytes is determined by cent activated cell sorting (FACS).

In other aspects, provided herein are methods of stratifying a patient population having a hematological maligancy comprising: (a) administering a Btk inhibitor to the subject sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes; and (b) determining the expression or ce of one or more biomarkers from one or more subpopulation of cytes; wherein the expression or presence of one or more biomarkers is used to stratify patients for treatment of the hematological maligancy. In one embodiment, the se or appearance in the blood of a subpopulation of lymphocytes is determined by phenotyping. In another embodiment, the increase or appearance in the blood of a subpopulation of lymphocytes is ined by fluorescent activated cell sorting (FAC S).

In still other aspects, provided herein are s of determining a eutic regimen in a subject having a hematological maligancy comprising: (a) administering a Btk inhibitor to the subject sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes; and (b) determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes; wherein the expression or presence of one or more biomarkers is used to determine the therapeutic regimen for the ent of the logical maligancy. In one embodiment, the increase or appearance in the blood of a ulation of lymphocytes is determined by immunophenotyping. In another embodiment, the increase or appearance in the blood of a ulation of lymphocytes is determined by fluorescent activated cell sorting (FACS).

In yet other aspects, ed herein are methods of predicting a response to therapy in a subject having a hematological maligancy comprising: (a) administering a Btk inhibitor to the subject sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes; and (b) determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes; wherein the expression or presence of one or more biomarkers is used to predict the subject’s response to therapy for the hematological maligancy. In one embodiment, the increase or appearance in the blood of a subpopulation of cytes is determined by immunophenotyping. In another embodiment, the increase or appearance in the blood of a subpopulation of lymphocytes is determined by fluorescent activated cell sorting (FACS).

In certain aspects, provided herein are methods of diagnosing, determining a prognosis, or creating a predictive profile of a hematological ncy in a subject comprising determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes in a subject that has received a dose of a Btk inhibitor wherein the expression or presence of one or more biomarkers is used to diagnose the hematological maligancy, determine the prognosis ofthe hematological maligancy, or create a predictive profile of the hematological maligancy. In one embodiment, the dose of Btk inhibitor is sufficient to result in an se or appearance in the blood of a ulation of lymphocytes defined by immunophenotyping. In another embodiment, the determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes further comprises isolating, detecting or measuring one or more type of lymphocyte. In still r embodiment, the Btk inhibitor is a ible or irreversible inhibitor.

] In other aspects, provided herein are methods of stratifying a patient tion having a hematological maligancy comprising determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes in a subject that has received a dose of a Btk inhibitor n the expression or presence of one or more biomarkers is used to stratify ts for treatment of the logical maligancy. In one embodiment, the dose of Btk inhibitor is sufficient to result in an increase or appearance in the blood of a ulation of lymphocytes defined by immunophenotyping. In another embodiment, the determining the sion or presence of one or more biomarkers from one or more ulation of lymphocytes further comprises isolating, detecting or measuring one or more type of cyte. In still another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

In still other aspects, provided herein are methods of determining the therapeutic n in a subject having a hematological maligancy comprising determining the sion or presence of one or more biomarkers from one or more subpopulation of lymphocytes in a subject that has received a dose of a Btk tor n the expression or presence of one or more biomarkers is used to ine the therapeutic regimen for the treatment of the hematological maligancy. In one embodiment, the dose of Btk inhibitor is sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes defined by immunophenotyping. In r embodiment, the determining the sion or presence of one or more biomarkers from one or more subpopulation of lymphocytes further comprises isolating, detecting or measuring one or more type of lymphocyte. In still another embodiment, the Btk inhibitor is a reversible or rsible inhibitor.

In yet other aspects, provided herein are methods of predicting a response to therapy in a subject having a hematological maligancy comprising determining the sion or presence of one or more biomarkers from one or more circulating lymphocytes in a subject that has received a dose of a Btk inhibitor wherein the expression or presence of one or more biomarkers is used to predict the subject’s response to therapy for the hematological maligancy.

In one embodiment, the dose of Btk inhibitor is sufficient to result in an increase or appearance in the blood of a subpopulation of lymphocytes defined by phenotyping. In another ment, the determining the expression or ce of one or more biomarkers from one or more ulation of lymphocytes further comprises isolating, detecting or measuring one or more type of lymphocyte. In still another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

] As plated herein, any biomarker related to hematological ncies are in some embodiments utilized in the present methods. These biomarkers include any biological le (found either in blood, other body fluids, or tissues) or any chromosomal abnormality that is a sign of a hematological maligancy. In certain embodiments, the biomarkers include, but are not limited to, TdT, CD5, CD11c, CD19, CD20, CD22, CD79a, CD15, CD30, CD38, CD13 8, CD103, CD25, ZAP-70, p53 mutational status, ATM mutational status, mutational status of IgVH, chromosome 17 deletions (del 17p), chromosome 6 deletions (del 6q), chromosome 7 deletions (del 7q), chromosome 11 deletions (del llq), trisomy 12, chromosome 13 deletions (del 13 q), t(1 1 : 14) chromosomal translocation, 8) chromosomal translocation, CD10, CD23, beta—2 microglobulin, bcl—2 expression, CD9, presence of Helicobacter pylori, CD154/CD40, Akt, NF—KB,WNT, Mtor, ERK, MAPK, and Src tyrosine kinase expression. In certain embodiments, the biomarkers include ZAP-70, CD5, t(14;18), CD38, 13-2 microglobulin, p53 mutational status, ATM mutational status, chromosome 17p on, chromosome 1 lq deletion, surface or cytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22, CD11c, CD 103, chromosome 7q deletion, VH onal status, or a combination thereof.

In certain embodiments, subpopulations of patients having a logical maligancy cancer or pre- that would benefit from a known treatment regimen are identified by screening candidate subjects for one or more clinically useful biomarkers known in the art. Any clinically useful prognostic marker known to those of skill in the art can be used. In some embodiments, the subpopulation includes patients having chronic cytic leukemia (CLL), and the clinically useful prognostic markers of particular interest e, but are not limited to, , CD38, .beta.2 microglobulin, and cytogenetic markers, for example, p53 mutational status, ATM mutational status, chromosome deletions, such as the chromosome 17p deletion and the chromosome llq deletion, all of which are clinically useful prognostic s for this disease.

ZAP-70 is a tyrosine kinase that associates with the zeta subunit of the T cell n receptor (TCR) and plays a pivotal role in T cell activation and development (Chan et al. (1992) Cell 71 :649-662). ZAP-70 undergoes ne phosphorylation and is essential in mediating signal transduction ing TCR stimulation. Overexpression or constitutive activation of tyrosine kinases has been demonstrated to be involved in a number of malignancies including leukemias and several types of solid tumors. For example, increased ZAP-70 RNA expression levels are a prognostic marker of 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 high levels in the B-cells of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) patients, and more particularly in the subset of CLL patients who tend to have the more aggressive al course that is found in CLL/SLL ts with unmutated Ig genes (Wiestner et al. (2003) Blood 101: 4944-4951; US. Patent ation Publication No. 20030203416).

Because of the correlation between ZAP-70 expression levels and lg gene mutation status, ZAP- 70 can be used as a prognostic indicator to identify those patients likely to have severe disease (high ZAP-70, unmutated Ig genes), and who are therefore ates for aggressive y.

CD38 is a signal transduction molecule as well as an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP ribose ). CD38 expression is present at high levels in bone marrow precursor B cells, is down-regulated in resting normal B cells, and then is re—expressed in terminally differentiated plasma cells (Campana et al. (2000) Chem. Immunol. 75: 169-188). CD38 is a reliable prognostic indicator in B-CLL, with the expression of CD38 generally indicating a less favorable outcome (D'Arena et al. (2001) Leuk. Lymphoma 42: 109; Del Poeta et al. (2001) Blood 3; Durig et a1. (2002) Leukemia 16:30; Ibrahim et al. (2001) Blood 98:181; Deaglio et al. (2003) Blood 102:2146-2155). The unfavorable clinical indications that CD38 expression has been associated with include an advanced stage of disease, poor responsiveness to chemotherapy, a shorter time before initial treatment is required, and a shorter survival time (Deaglio et al. (2003) Blood 102:2146—2155). Initially, a strong ation n CD38 expression and IgV gene mutation was observed, with patients having ted V genes displaying higher percentages of CD38.sup.+ B-CLL cells than those with mutated V genes (Damle et a1. (1999) Blood 94:1840-1847). However, subsequent studies have indicated that CD38 expression does not always correlate with the ngement of the IgV genes (Hamblin et al. (2002) Blood 99: 1023; Thunberg et al. (2001) Blood 97:1892). p53 is a nuclear phosphoprotein that acts as a tumor suppressor. Wild-type p53 is involved in regulating cell growth and division. p53 binds to DNA, stimulating the production of a protein (p21) that interacts with a cell division—stimulating protein (cdk2). When p21 is bound to cdk2, the cell is d from entering the next stage of cell division. Mutant p53 is incapable of binding DNA effectively, thus ting p21 from acting as the stop signal for cell division, resulting in uncontrolled cell division, and tumor formation. p53 also regulates the induction of programmed cell death (apoptosis) in response to DNA damage, cell stress or the aberrant sion of some oncogenes. Expression of wild type p53 in some cancer cell lines has been shown to e grth suppression control (Casey et a1. (1991) Oncogene 6: 1791-1797; shi 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. p53 mutations have been found to be associated with Burkitt's lymphoma, L3-type B-cell acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia (Gaidano et al. (1991) Proc. Natl.

Acad. Sci. USA. 88:5413-5417). p53 alities have also been found associated with B- cell prolymphocytic leukemia (Lens et al. (1997) Blood 89:2015-2023). The gene for p53 is located on the short arm of chromosome 17 at 17p13.105-p12.

] Bmicroglobulin is an extracellular protein that is noncovalently associated with the .alpha. chain of the class I major ompatibility complex (MHC). It is detectable in the serum, and is an adverse prognostic indicator in CLL (Keating et al. (1998) Blood 86:606a) and Hodgkin's lymphoma (Chronowski et a1. (2002) Cancer 95:2534-2538). It is clinically used for lymphoproliferative diseases including ia, ma, and multiple myeloma, where serum Bmicroglobulin levels are related to tumor cell load, prognosis, and disease activity (Bataille et a1. (1983) Br. J. ol. 55:439—447; Aviles et al. (1992) Rev. Invest. Clin. 44:215-220). P2 microglobulin is also usefiil in staging myeloma patients (Pasqualetti et al. (1991) Eur. J. Cancer 27: 1 123-1 126).

Cytogenetic tions may also be used as markers to create a predictive profile of a hematological maligancy. For example, chromosome abnormalities are found in a large percentage of CLL patients and are l in predicting the course of CLL. For example, a 17p deletion is tive of aggressive disease progression. In addition, CLL patients with a chromosome 17p deletion or mutation in p53, or both, are known to respond poorly to chemotherapeutics and rituximab. Allelic loss on chromosome 17p may be also be a useful prognostic marker in colorectal cancer, where patients with a 17p deletion are associated with an increased tendency of disease dissemination in colorectal cancer (Khine et a1. (1994) Cancer 73 :28-35).

Deletions of the long arm of some 11 (11q) are one of the most frequent structural chromosome aberrations in s types of lymphoproliferative disorders. CLL patients with chromosome 11q deletion and possibly ATM mutations have a poor survival compared to patients without either this defect or the 17p deletion. Furthermore, an 11q deletion is often accompanied by ive lymph node involvement (Dohner et a1. (1997) Blood 89:2516-2522). This deletion also identifies patients who are at high risk for disease persistence after high-dose therapy and autologous transplantation.

[00348] The ataxia telangiectasia mutated (ATA4) gene is a tumor suppressor gene that is involved in cell cycle , apoptosis, and repair ofDNA double-strand breaks. It is found on chromosome 11. ATMmutations are associated with increased risk for breast cancer among women with a family history of breast cancer (Chenevix-Trench et al. (2002) J. Natl. Cancer Inst. 942205-215; Thorstenson et al. (2003) Cancer Res. 63:3325-3333) and/or early-onset breast cancers (Izatt et al. (1999) Genes Chromosomes Cancer 26:286—294; a et a1. (2001) Cancer -487). There is also a high frequency of association of rhabdomyosarcoma with ATM gene mutation/deletion (Zhang et al. (2003) Cancer Biol. Ther. 1:87-91).

Methods for detecting chromosomal abnormalities in a patient are well known in the art (see, for example, Cuneo et al. (1999) Blood 93: 1372-1380; Dohner et a1. (1997) Blood 6-2522). Methods to e mutated proteins, such as ATM, are well known in the art (see, for example, Butch et al. (2004) Clin. Chem. 50: 2302-2308).

Thus, the biomarkers that are evaluated in the methods described herein include the cell survival and apoptotic ns described supra, and proteins involved in hematological maligancy-related signaling pathways. Determining the expression or presence can be at the protein or nucleic acid level. Thus, the biomarkers include these proteins and the genes encoding these proteins. Where detection is at the protein level, the biomarker protein comprises the full- length polypeptide or any detectable fragment thereof, and can include variants of these protein sequences. Similarly, where detection is at the nucleotide level, the biomarker nucleic acid includes DNA comprising the full-length coding sequence, a fragment of the full-length coding sequence, variants of these sequences, for example naturally occurring variants or splice- variants, or the complement of such a sequence. ker nucleic acids also include RNA, for example, mRNA, comprising the full-length sequence encoding the biomarker protein of interest, a fragment of the full-length RNA ce of interest, or variants of these sequences.

Biomarker proteins and biomarker nucleic acids also include variants of these sequences. By "fragment" is ed a portion of the polynucleotide or a portion of the amino acid sequence and hence protein d thereby. cleotides that are fragments of a biomarker nucleotide ce generally comprise 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 t in a fiill-length biomarker polynucleotide disclosed . A fragment of a biomarker cleotide will generally encode at least 15, 25, , 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 ed to mean substantially similar sequences. Generally, variants of a particular biomarker of the ion will have 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 determined by sequence alignment programs known in the art.

As provided above, any method known in the art can be used in the methods for determining the expresion or presence of ker described herein. Circulating levels of biomarkers in a blood sample ed from a candidate subject, can be measured, for example, by ELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Western blot, multiplexing technologies, or other similar methods. Cell e expression of biomarkers can be measured, for example, by flow cytometry, immunohistochemistry, Western Blot, precipitation, ic bead selection, and quantification of cells expressing either of these cell surface markers. ker RNA expression levels could be measured by RT-PCR, Qt-PCR, microarray, Northern blot, or other similar technologies.

As previously noted, determining the expression or presence of the biomarker of interest at the protein or nucleotide level can be accomplished using any detection method known to those of skill in the art. By ting expression" or "detecting the level of' is intended determining the expression level or presence of a biomarker protein or gene in the biological sample. Thus, "detecting expression" encompasses instances where a biomarker is determined not to be expressed, not to be ably expressed, expressed at a low level, expressed at a normal level, or overexpressed.

In certain s of the method provided herein, the one or more subpopulation of lymphocytes are isolated, detected or ed. In certain embodiments, the one or more subpopulation of lymphocytes are isolated, detected or ed using immunophenotyping techniques. In other embodiments, the one or more subpopulation of cytes are isolated, detected or measured using fluorescence activated cell sorting (FACS) techniques.

In certain embodiments of the methods provided herein, the one or more biomarkers comprises ZAP-70, CD5, t(l4;l8), CD38, [3—2 microglobulin, p53 mutational status, ATM mutational , chromosome 17p deletion, chromosome llq deletion, surface or cytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22, CD1 lc, CD 103, chromosome 7q deletion, VH mutational status, or a combination thereof.

In certain aspects, the methods described herein, the determining step requires determining the expression or presence of a combination of biomarkers. In certain embodiment, the combination of biomarkers is CD19 and CD5 or CD20 and CD5.

In certain aspects, the ssion 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, using, for example, immunohistochemistry techniques or nucleic acid-based techniques such as in situ ization and RT-PCR. In one embodiments, the sion or presence of one or more biomarkers is carried out by a means for nucleic acid cation, a means for nucleic acid sequencing, a means utilizing a nucleic acid microarray (DNA and RNA), or a means for in situ hybridization using cally labeled probes.

In other embodiments, the determining the expression or presence of one or more biomarkers is carried out through gel electrophoresis. In one embodiment, the determination is carried out h transfer to a membrane and hybridization with a specific probe.

] In other embodiments, the determining the expression or presence of one or more biomarkers d out by a diagnostic g technique.

In still other embodiments, the determining the expression or presence of one or more biomarkers carried out by a able solid substrate. In one embodiment, the detectable solid substrate is paramagnetic nanoparticles functionalized with antibodies.

] In another aspect, provided herein are methods for detecting or measuring residual ma following a course of treatment in order to guide continuing or discontinuing treatment or changing from one therapeutic regimen to another sing ining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes in a subject wherein the course of treatment is treatment with a Btk tor. s for ing expression of the biomarkers described herein, and optionally ne markers, within the test and control biological samples comprise any methods that determine the quantity or the presence of these markers either at the nucleic acid or protein level. Such methods are well known in the art and include but are not limited to western blots, northern blots, ELISA, immunoprecipitation, immunofluorescence, flow cytometry, histochemistry, c acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In ular embodiments, expression of a biomarker is detected on a protein level using, for example, antibodies that are directed against specific biomarker proteins. These antibodies can be used in various methods such as Western blot, ELISA, multiplexing technologies, immunoprecipitation, or immunohistochemistry techniques. In some embodiments, detection of cytokine markers is accomplished by electrochemiluminescence (ECL).

Any means for specifically identifying and quantifying a biomarker (for example, biomarker, a biomarker of cell survival or proliferation, a biomarker of apoptosis, a biomarker of a Btk-mediated signaling pathway) in the biological sample of a candidate subject is contemplated. Thus, in some embodiments, expression level of a ker n of st in a biological sample is detected by means of a binding protein capable of interacting specifically with that biomarker n or a biologically active variant thereof. Preferably, labeled antibodies, binding ns thereof, or other binding rs may be used. The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze al alteration of a substrate compound or composition that is detectable.

The antibodies for detection of a biomarker protein may be monoclonal or polyclonal in origin, or may be synthetically or recombinantly produced. The amount of complexed protein, for example, the amount of biomarker protein associated with the binding protein, for example, an antibody that specifically binds to the ker protein, is determined using standard protein detection methodologies known to those of skill in the art. A detailed review of immunological assay design, theory and protocols can be found in numerous texts 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) t Protocols in logy (John Wiley & Sons, Inc., New York, NY.) The choice of marker used to label the antibodies will vary depending upon the application. However, the choice of the marker is readily determinable to one d in the art.

These labeled antibodies may be used in immunoassays as well as in histological applications to detect the presence of any biomarker or protein of interest. The labeled antibodies may be polyclonal or monoclonal. Further, the dies for use in detecting a protein of interest may be labeled with a radioactive atom, an enzyme, a chromophoric or fluorescent moiety, or a colorimetric tag as bed elsewhere herein. The choice of tagging label also will depend on the detection limitations desired. Enzyme assays (ELISAs) typically allow detection of a colored product formed by interaction of the enzyme-tagged complex with an enzyme substrate.

Radionuclides that can serve as detectable labels include, for e, 1-131, 1-123, 1-125, Y-90, Re-188, , At-211, Cu-67, , and Pd—109. Examples of enzymes that can serve as detectable labels include, but are not d to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucosephosphate dehydrogenase. Chromophoric moieties include, but are not limited to, fluorescein and rhodamine. The antibodies may be conjugated to these labels by methods known in the art. For e, enzymes and chromophoric molecules may be conjugated to the antibodies by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Alternatively, conjugation may occur through a ligand-receptor pair.

Examples of suitable ligand-receptor pairs are biotin-avidin or biotin—streptavidin, and dy- antigen.

In certain embodiments, expression or presence of one or more biomarkers or other proteins of interest within a biological , for example, a sample of bodily fluid, is determined by radioimmunoassays or enzyme—linked immunoassays s), competitive g enzyme-linked immunoassays, dot blot (see, for example, Promega Protocols and Applications Guide (2Ild ed.; Promega Corporation (1991), Western blot (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Vol. 3, Chapter 18 (Cold Spring Harbor tory Press, Plainview, NY), chromatography, preferably high performance liquid chromatography (HPLC), or other assays known in the art. Thus, the detection assays can involve steps such as, but not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, or precipitation.

In n other embodiments, the methods of the ion are usefiil for fying and treating hematological maligancys, including those listed above, that are tory to (i.e., resistant to, or have become resistant to) first-line oncotherapeutic treatments.

The expression or presence of one or more of the biomarkers described herein may also be ined at the nucleic acid level. Nucleic acid-based techniques for assessing expression are well known in the art and include, for example, determining the level of biomarker mRNA in a biological sample. Many expression detection methods use isolated RNA.

Any RNA isolation technique that does not select against the isolation ofmRNA can be utilized for the purification ofRNA (see, e. g., Ausubel et al., ed. (1987-1999) Current Protocols in Molecular Biology (John Wiley & Sons, New York). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process disclosed in US. Pat. No. 4,843,155.

Thus, in some embodiments, the detection of a biomarker or other protein of interest is assayed at the nucleic acid level using nucleic acid probes. The term "nucleic acid probe" refers to any molecule that is capable of selectively binding to a specifically ed target nucleic acid molecule, for example, a tide transcript. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled, for example, with a radioactive label, a cent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or other labels or tags that are discussed above or that are known in the art. Examples ofmolecules that can be utilized as probes include, but are not d to, RNA and DNA.

[00369] For e, isolated mRNA can be used in hybridization or amplification assays that include, but are not d to, Southern or Northern analyses, polymerase chain reaction es and probe arrays. One method for the detection ofmRNA levels es ting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a biomarker, biomarker described herein above.

Hybridization of an mRNA with the probe indicates that the biomarker or other target protein of interest is being expressed.

] In one ment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by g the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a ne, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid e and the mRNA is contacted with the probe(s), for example, in a gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level ofmRNA ng the biomarkers or other proteins of interest.

[00371] An alternative method for determining the level of a mRNA of interest in a sample involves the process of c acid amplification, e. g., by RT-PCR (see, for example, US. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et a1. (1990) Proc. Natl. Acad. Sci.

USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad.

Sci. USA 86: 1 173-1 177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1 197), rolling circle replication (US. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low s. In particular aspects of the invention, biomarker expression is assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System).

Expression levels of an RNA of interest may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic . See US. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The detection of expression may also comprise using nucleic acid probes in solution.

] In one embodiment of the ion, microarrays are used to determine expression or presence of one or more biomarkers. Microarrays are particularly well suited for this purpose because of the reproducibility n different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes ed to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing ve gene expression levels. See, US. Pat.

Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample. ques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., US. Pat. No. 5,384,261, incorporated herein by reference in its entirety.

Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric es, fibers such as fiber optics, glass or any other appropriate substrate, see US. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each h is hereby orated in its entirety for all purposes. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an clusive device. See, for example, US.

Pat. Nos. 5,856,174 and 5,922,591, herein incorporated by reference.

Pharmaceutical Compositions/Formulations Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically able carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art. A summary of pharmaceutical itions described herein may be found, for example, in Remington: The Science and ce ofPharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington ’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug ry Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999), herein incorporated by reference in their entirety.

A pharmaceutical composition, as used herein, refers to a mixture of a nd described herein, such as, for example, compounds of Formula D or the second agent, with other chemical components, such as rs, stabilizers, diluents, dispersing agents, suspending agents, thickening , and/or excipients. The pharmaceutical composition facilitates stration of the compound to an organism. In practicing the s of ent or use provided herein, therapeutically effective s of compounds described herein are administered in a pharmaceutical composition to a mammal having a 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 y of the compound used and other factors. The compounds can be used singly or in combination with one or more therapeutic agents as components of mixtures.

In certain embodiments, compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium ide, sodium phosphate, sodium , sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as e/dextrose, sodium onate and um chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In other ments, compositions may also include one or more salts in an amount required to bring osmolality ofthe 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; suitable salts include sodium chloride, ium de, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

The term “pharmaceutical combination” as used herein, means a product that s from the mixing or combining of more than one active ingredient and es both fixed and non-fixed ations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g. a compound described herein and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g. a compound described herein and a co- agent, are administered to a t as separate entities either simultaneously, concurrently or tially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also s to cocktail therapy, e.g. the administration of three or more active ingredients.

The pharmaceutical formulations bed herein can be administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, aneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration 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 melt formulations, tablets, es, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. ceutical compositions including a compound described herein may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee—making, levigating, emulsifying, encapsulating, entrapping or compression processes.

“Antifoaming agents” reduce foaming during processing which can result in coagulation of aqueous dispersions, bubbles in the finished film, or generally impair processing. ary anti-foaming agents include silicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. In certain embodiments, antioxidants enhance al ity where ed.

In certain embodiments, compositions provided herein may also include one or more vatives to 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.

Formulations described herein may benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, 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 nine, (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) extrins, (1) pentosan polysulfate and other heparinoids, (m) divalent s such as magnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e. g., alginic acid and salts thereof; cellulose derivatives such as carboxymethylcellulose, methylcellulose (e. g., Methoce1®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e. g., Klucel®), ethylcellulose (e. g., Ethocel®), and microcrystalline cellulose (e.g., ®); microcrystalline se; amylose; magnesium aluminum te; ccharide acids; bentonites; gelatin; nylpyrrolidone/vinyl acetate copolymer; crosspovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, a sugar, such as sucrose (e.g., ), e, dextrose, molasses, mannitol, sorbitol, xylitol (e. g., Xylitab®), and lactose; a natural or tic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone (e. g., Polyvidone® CL, Kollidon® CL, Polyplasdone® XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

A “carrier” or “carrier materials” include any commonly used excipients in pharmaceutics and should be selected on the basis of ibility with compounds disclosed herein, such as, compounds of any of Formula D and the second agent, and the e profile properties of the desired dosage form. Exemplary carrier materials include, e. g., binders, suspending agents, disintegration , filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. “Pharmaceutically compatible carrier materials” may include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, ine, magnesium silicate, nylpyrrollidone (PVP), terol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium de, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e. g., Remington: The e and ce ofPharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., ton’s Pharmaceutical Sciences, Mack Publishing Co., Easton, lvania 1975; Liberman, HA. and Lachman, L., Eds, Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery s, Seventh Ed. (Lippincott Williams & Wilkins1999).

“Dispersing agents,” and/or “viscosity modulating agents” include materials that control the diffirsion and homogeneity of a drug through liquid media or a granulation method or blend method. In some embodiments, these agents also facilitate the effectiveness of a coating or eroding matrix. Exemplary diffusion facilitators/dispersing agents include, e.g., hydrophilic polymers, electrolytes, Tween ® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and the carbohydrate-based dispersing agents such as, for example, hydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC , carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), stalline cellulose, magnesium um te, triethanolamine, polyvinyl l (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)- phenol polymer with ethylene oxide and dehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68®, F8 8®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®, which is a tetrafiinctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, pany, N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (8-630), polyethylene glycol, e. g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, osics, such as, e. g., sodium carboxymethylcellulose, cellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, ers, polyvinyl alcohol (PVA), tes, chitosans and combinations thereof.

Plasticizcers such as cellulose or triethyl cellulose can also be used as dispersing agents.

Dispersing agents particularly useful in mal dispersions and self-emulsifying sions are dimyristoyl phosphatidyl choline, l phosphatidyl e from eggs, natural phosphatidyl glycerol from eggs, cholesterol and isopropyl myristate.

Combinations of one or more erosion facilitator with one or more ion facilitator can also be used in the present compositions.

] The term “diluent” refers to chemical compounds that are used to dilute the compound of interest prior to delivery. Diluents can also be used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain embodiments, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for nous blend for capsule filling. Such compounds include e.g., lactose, starch, mannitol, sorbitol, se, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di—Pac® (Amstar); mannitol, ypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner’s sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate rate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, m carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

The term “disintegrate” includes both the ution and dispersion of the dosage form when ted with gastrointestinal fluid. “Disintegration agents or disintegrants” facilitate the breakup or disintegration of a substance. Examples of egration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amij el®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e. g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcoce1®, Vivacel®, Ming Tia®, and Solka- Floc®, methylcellulose, croscarmellose, or a cross—linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac—Di—Sol®), cross—linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crosspovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in ation starch, and the like.

“Drug absorption” or “absorption” typically refers to the process of nt of drug from site of administration of a drug across a barrier into a blood vessel or the site of action, e.g., a drug moving from the intestinal tract into the portal vein or lymphatic system.

An “enteric coating” is a substance that remains substantially intact in the stomach but dissolves and es the drug in the small intestine or colon. Generally, the enteric coating ses a polymeric material that prevents release in the low pH environment of the stomach but that s at a higher pH, typically a pH of 6 to 7, and thus dissolves sufficiently in the small intestine or colon to release the active agent therein.

“Erosion facilitators” include als that control the erosion of a particular material in intestinal fluid. n facilitators are lly known to those of ordinary skill in the art. Exemplary erosion facilitators include, e.g., hydrophilic polymers, olytes, ns, peptides, and amino acids.

“Filling agents” include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Flavoring agents” and/or “sweeteners” US€fill in the formulations described herein, include, e. g., acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, l, cherry, cherry cream, chocolate, cinnamon, bubble gum, , citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, , glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, neotame, orange, pear, peach, peppermint, mint cream, Prosweet® Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, Spearmint, Spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, rin, aspartame, acesulfame potassium, mannitol, talin, sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, a, walnut, elon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, e.g., anise—menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream, a-mint, and mixtures thereof.

“Lubricants” and “glidants” are compounds that prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, m hydroxide, talc, sodium stearyl filmerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali- metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, ol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CarbowaxTM, sodium oleate, sodium benzoate, glyceryl behenate, hylene , magnesium or sodium lauryl sulfate, colloidal silica such as SyloidTM, Cab-O-Sil®, a starch such as corn starch, silicone oil, a surfactant, and the like.

A rable serum concentration” or “measurable plasma concentration” describes the blood serum or blood plasma concentration, lly measured in mg, g, or ng of therapeutic agent per ml, dl, or 1 of blood serum, absorbed into the bloodstream after administration. As used herein, measurable plasma trations are typically measured in ng/ml or g/ml.

] “Pharmacodynamics” refers to the factors which determine the biologic response observed relative to the concentration of drug at a site of action.

“Pharmacokinetics” refers to the factors which determine the attainment and maintenance of the appropriate concentration of drug at a site of action.

“Plasticizers” are compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. In some ments, plasticizers can also function as dispersing agents or wetting .

[00402] “Solubilizers” include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, n E TPGS, ylacetamide, N—methylpyrrolidone, N—hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n—butanol, isopropyl alcohol, cholesterol, bile salts, hylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.

“Stabilizers” include compounds such as any antioxidation , buffers, acids, preservatives and the like. y state,” as used herein, is when the amount of drug administered is equal to the amount of drug eliminated within one dosing interval resulting in a plateau or constant plasma drug exposure.

“Suspending agents” include nds such as polyvinylpyrrolidone, e. g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e. g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, ypropylmethylcellulose, hydroxymethylcellulose acetate stearate, rbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e. g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e. g., sodium carboxymethylcellulose, cellulose, sodium carboxymethylcellulose, ypropylmethylcellulose, yethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, an monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e. g., Pluronic® (BASF), and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol , octoxynol 40. In some embodiments, surfactants may be included to enhance physical stability or for other es.

“Viscosity enhancing ” include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, ypropylmethyl cellulose e stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof. ng agents” include compounds such as oleic acid, yl monostearate, sorbitan monooleate, sorbitan monolaurate, anolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan urate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

Dosage Forms The compositions described herein can be formulated for administration to a subject via any conventional means including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), buccal, intranasal, rectal or transdermal administration routes. As used herein, the term “subject” is used to mean an animal, preferably a mammal, including a human or non-human. The terms patient and subject may be used interchangeably.

[00410] Moreover, the pharmaceutical compositions described herein, which include a compound of any of Formula D or the second agent can be formulated into any suitable dosage form, including but not limited to, s oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a t to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent ations, lyophilized ations, tablets, powders, pills, s, es, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate ations, and mixed immediate release and controlled release formulations.

Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if d, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or ne) or calcium ate. If desired, disintegrating agents may be added, such as the cross-linked rmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are ed with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, hylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for fication or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of n, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium te and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid n, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral stration should be in dosages suitable for such administration.

In some ments, the solid dosage forms disclosed herein may be in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration , a rapid-disintegration tablet, an escent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder) a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, s, es, or an aerosol. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a , including but not limited to, a fast—melt . Additionally, pharmaceutical formulations described herein may be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation is administered in two, or three, or four, capsules or tablets.

In some embodiments, solid dosage forms, e. g., s, effervescent s, and capsules, are prepared by mixing particles of a compound of any of Formula (Al-A6), a (Bl-B6), Formula (C1-C6), or Formula (D1-D6), with one or more pharmaceutical excipients to form a bulk blend ition. When referring to these bulk blend compositions as homogeneous, it is meant that the particles of the nd of any of Formula (Al-A6), Formula (Bl-B6), Formula (Cl—C6), or Formula (D1—D6), are dispersed evenly throughout the composition so that the composition may be y subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The individual unit dosages may also include film gs, which disintegrate upon oral ingestion or upon contact with diluent. These formulations can be ctured by conventional pharmacological techniques.

Conventional pharmacological techniques include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous ation, (5) wet granulation, or (6) . See, e.g., Lachman et al., The Theory and Practice of Industrial Pharmacy (1986). Other methods include, e.g., spray drying, pan coating, melt granulation, ation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.

The pharmaceutical solid dosage forms described herein can include a compound described herein and one or more pharrnaceutically acceptable additives such as a compatible carrier, , filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration er, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more ation thereof. In still other aspects, using standard g procedures, such as those described in Remington '5 Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of the compound of any of a (Al-A6), Formula (Bl-B6), Formula (Cl-C6), or Formula (D1-D6). In one embodiment, some or all of the particles of the compound of any of a (Al-A6), Formula (Bl-B6), Formula (Cl-C6), or Formula (D1-D6), are coated. In another embodiment, some or all of the particles of the compound of any of Formula (Al-A6), Formula (Bl—B6), Formula (Cl-C6), or Formula ), are microencapsulated. In still another embodiment, the particles of the compound of any of Formula (Al-A6), Formula (Bl—B6), Formula (C1-C6), or Formula (D1-D6), are not microencapsulated and are uncoated.

Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, n, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium de, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose e stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

Suitable filling agents for use in the solid dosage forms described herein e, but are not limited to, e, calcium ate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, ypropylmethycellulose (HPMC), ypropylmethycellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

In order to release the compound of any of Formula (Al-A6), Formula (Bl-B6), Formula (C1-C6), or Formula (D1-D6), from a solid dosage form matrix as ently as le, disintegrants are often used in the formulation, especially when the dosage forms are compressed with binder. Disintegrants help rupturing the dosage form matrix by swelling or capillary action when moisture is absorbed into the dosage form. Suitable disintegrants for use in the solid dosage forms bed herein include, but are not limited to, natural starch such as corn starch or potato , a pregelatinized starch such as National 1551 or Amij el®, or sodium starch glycolate such as Promogel® or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked ose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross—linked carboxymethylcellulose, or cross-linked croscarmellose, a cross- linked starch such as sodium starch glycolate, a linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum® HV sium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug ion that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose (e. g.

Hypromellose USP Pharmacoat-603, ypropylmethylcellulose acetate stearate (Aqoate HS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e. g., Klucel®), ellulose (e. g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesium aluminum silicate, ccharide acids, bentonites, gelatin, polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac®), glucose, dextrose, molasses, ol, sorbitol, l (e. g., Xylitab®), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, nylpyrrolidone (e.g., ne® CL, Kollidon® CL, Polyplasdone® XL-lO, and Povidone® K-l2), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fillers which itself can act as moderate . Formulators skilled in art can determine the binder level for the ations, but binder usage level of up to 70% in tablet formulations is common.

Suitable lubricants or glidants for use in the solid dosage forms described herein e, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal and alkaline earth metal salts, such as aluminum, m, magnesium, zinc, stearic acid, sodium stearates, magnesium te, zinc stearate, waxes, Stearowet®, boric acid, sodium te, sodium acetate, sodium chloride, leucine, a hylene glycol or a methoxypolyethylene glycol such as CarbowaxTM, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrin), polyols (including mannitol, xylitol, and ol), cyclodextrins and the like.

The term “non water-soluble diluent” represents compounds typically used in the formulation of pharmaceuticals, such as calcium phosphate, m sulfate, starches, modified starches and microcrystalline cellulose, and microcellulose (e.g., having a density of about 0.45 g/cm3, e.g. , powdered cellulose), and talc.

Suitable wetting agents for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan urate, triethanolamine oleate, polyoxyethylene sorbitan eate, polyoxyethylene sorbitan monolaurate, quaternary um compounds (e.g., Polyquat 10®), sodium oleate, sodium lauryl sulfate, magnesium stearate, sodium docusate, triacetin, Vitamin E TPGS and the like.

] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene an monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, nylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, Vinyl pyrrolidone/Vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy- propylmethylcellulose, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e. g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e. g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan urate, povidone and the like.

Suitable idants for use in the solid dosage forms bed herein include, for example, e. g., butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol.

It should be appreciated that there is considerable overlap between additives used in the solid dosage forms described herein. Thus, the listed additives should be taken as merely exemplary, and not limiting, ofthe types of additives that can be included in solid dosage forms described herein. The amounts of such additives can be y ined by one skilled in the art, according to the particular properties desired.

In other embodiments, one or more layers of the ceutical formulation are plasticized. Illustratively, a cizer is generally a high boiling point solid or liquid. Suitable plasticizers can be added from about 0.01% to about 50% by weight (w/w) of the coating composition. Plasticizers include, but are not limited to, diethyl ate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, triacetin, polypropylene glycol, polyethylene glycol, triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, and castor oil.

[00432] Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In s embodiments, compressed tablets which are designed to dissolve in the mouth will include one or more flavoring agents. In other ments, the compressed tablets will include a film surrounding the final compressed tablet. In some ments, the film coating can provide a d release of the compound of of any of Formula D or the second agent, from the formulation. In other embodiments, the film coating aids in patient compliance (e.g., Opadry® coatings or sugar coating). Film coatings including Opadry® typically range from about 1% to about 3% of the tablet weight. In other embodiments, the compressed tablets e one or more excipients.

A capsule may be prepared, for example, by placing the bulk blend of the formulation of the compound of any of Formula D or the second agent, described above, inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other ments, the formulation is placed in a sprinkle capsule, wherein the e may be swallowed whole or the capsule may be opened and the contents sprinkled on food prior to eating. In some embodiments, the therapeutic dose is split into multiple (e.g., two, three, or four) capsules. In some embodiments, the entire dose of the formulation is delivered in a capsule form.

In various embodiments, the les of the compound of any of Formula D or the second agent, and one or more excipients are dry d and ssed into a mass, such as a tablet, having a hardness sufficient to provide a pharmaceutical composition that substantially disintegrates within less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 s, or less than about 60 minutes, after oral administration, thereby releasing the formulation into the gastrointestinal fluid.

In another aspect, dosage forms may include ncapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH rs, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending , disintegration agents, filling agents, tants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

Materials useful for the microencapsulation described herein include materials compatible With compounds of any of Formula D or the secodn agent, which sufficiently isolate the compound of any of Formula D or the secodn agent, from other non-compatible excipients.

Materials compatible With compounds of any of a D or the secodn agent, are those that delay the e of the compounds of of any of Formula D or the secodn agent, in viva.

Exemplary microencapsulation materials useful for delaying the release of the formulations including compounds described herein, include, but are not limited to, hydroxypropyl cellulose ethers (HPC) such as K1uce1® or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A, hydroxypropylmethylcellulose e stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel®, n®-EC, Surelease®, Polyvinyl alcohol (PVA) such as Opadry AMB, hydroxyethylcelluloses such as Natrosol®, ymethylcelluloses and salts of carboxymethylcelluloses (CMC) such as Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymers such as Kollicoat IR®, monoglycerides (Myverol), cerides (KLX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit® EPO, Eudragit® L30D- 55, Eudragit® FS 30D Eudragit® L100-55, Eudragit® L100, Eudragit® SlOO, Eudragit® RD100, Eudragit® E100, Eudragit® L125, Eudragit® S125, Eudragit® NE30D, and Eudragit® NE 40D, cellulose acetate phthalate, sepifilms such as mixtures ofHPMC and c acid, extrins, and mixtures of these materials.

In still other embodiments, cizers such as polyethylene glycols, e. g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, and triacetin are incorporated into the microencapsulation material. In other ments, the microencapsulating al useful for delaying the e of the pharmaceutical compositions is from the USP or the National Formulary (NF). In yet other embodiments, the ncapsulation material is Klucel. In still other embodiments, the microencapsulation material is methocel.

Microencapsulated compounds of any of Formula D or the secodn agent, may be formulated by methods known by one of ordinary skill in the art. Such known methods include, e. g., spray drying processes, spinning disk-solvent processes, hot melt processes, spray chilling methods, fluidized bed, electrostatic deposition, centrifiagal extrusion, rotational suspension separation, polymerization at liquid-gas or solid-gas interface, pressure extrusion, or ng solvent extraction bath. In addition to these, several chemical techniques, e.g., complex coacervation, solvent evaporation, polymer—polymer incompatibility, interfacial polymerization in liquid media, in situ polymerization, in—liquid drying, and desolvation in liquid media could also be used. Furthermore, other methods such as roller compaction, extrusion/spheronization, coacervation, or rticle coating may also be used.

[00440] In one embodiment, the particles of compounds of any of Formula D or the secodn agent, are microencapsulated prior to being formulated into one of the above forms. In still another embodiment, some or most ofthe particles are coated prior to being fiarther formulated by using standard coating procedures, such as those bed in Remington 's Pharmaceutical es, 20th Edition (2000).

[00441] In other embodiments, the solid dosage formulations of the compounds of any of Formula D or the second agent, are plasticized (coated) with one or more layers. Illustratively, a plasticizer is generally a high boiling point solid or liquid. Suitable plasticizers can be added from about 0.01% to about 50% by weight (w/w) of the coating composition. Plasticizers include, but are not limited to, diethyl phthalate, citrate esters, polyethylene , glycerol, acetylated glycerides, triacetin, polypropylene glycol, hylene , yl citrate, dibutyl sebacate, stearic acid, stearol, stearate, and castor oil.

In other embodiments, a powder including the formulations with a compound of any of a D or the secodn agent, described , may be formulated to e one or more pharmaceutical excipients and flavors. Such a powder may be prepared, for example, by mixing the formulation and optional pharmaceutical ents to form a bulk blend composition. Additional embodiments also include a suspending agent and/or a wetting agent.

This bulk blend is uniformly subdivided into unit dosage packaging or multi-dosage packaging units.

In still other embodiments, effervescent s are also prepared in accordance with the present disclosure. Effervescent salts have been used to disperse nes in water for oral administration. Effervescent salts are granules or coarse powders containing a medicinal agent in a dry mixture, usually ed of sodium bicarbonate, citric acid and/or tartaric acid.

When salts of the itions described herein are added to water, the acids and the base react to liberate carbon dioxide gas, thereby causing “effervescence.” Examples of effervescent salts include, e.g., the following ingredients: sodium bicarbonate or a mixture of sodium bicarbonate and sodium carbonate, citric acid and/or tartaric acid. Any acid-base combination that results in the liberation of carbon dioxide can be used in place of the combination of sodium bicarbonate and citric and tartaric acids, as long as the ingredients were suitable for pharmaceutical use and result in a pH of about 6.0 or .

In some embodiments, the solid dosage forms described herein can be formulated as enteric coated delayed release oral dosage forms, i.e., as an oral dosage form of a pharmaceutical composition as described herein which utilizes an c coating to affect release in the small intestine of the gastrointestinal tract. The enteric coated dosage form may be a compressed or molded or extruded tablet/mold (coated or ed) containing granules, , pellets, beads or particles of the active ingredient and/or other composition components, which are themselves coated or uncoated. The c coated oral dosage form may also be a capsule d or uncoated) containing pellets, beads or granules of the solid carrier or the ition, which are themselves coated or uncoated.

] The term “delayed release” as used herein refers to the delivery so that the release can be accomplished at some generally table location in the intestinal tract more distal to that which would have been accomplished if there had been no delayed e alterations. In some embodiments the method for delay ofrelease is coating. Any coatings should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile can be used as an enteric coating in the methods and compositions bed herein to achieve delivery to the lower gastrointestinal tract. In some embodiments the polymers described herein are anionic carboxylic polymers. In other embodiments, the polymers and compatible mixtures thereof, and some of their properties, include, but are not limited to: (a) Shellac, also called purified lac, a refined product obtained from the resinous secretion of an insect. This coating dissolves in media ofpH >7; (b) Acrylic polymers. The performance of acrylic polymers rily their solubility in biological fluids) can vary based on the degree and type of tution. Examples of suitable acrylic polymers e methacrylic acid copolymers and ammonium methacrylate copolymers. The Eudragit series E, L, S, RL, RS and NE (Rohm Pharma) are available as solubilized in organic solvent, aqueous dispersion, or dry powders. The Eudragit series RL, NE, and RS are insoluble in the gastrointestinal tract but are permeable and are used primarily for colonic ing. The it series E dissolve in the h. The Eudragit series L, L-30D and S are insoluble in stomach and ve in the intestine; (c) Cellulose Derivatives. es of suitable cellulose derivatives are: ethyl cellulose; reaction mixtures of partial acetate esters of cellulose with phthalic anhydride. The mance can vary based on the degree and type of substitution. Cellulose acetate phthalate (CAP) dissolves in pH >6. Aquateric (FMC) is an aqueous based system and is a spray dried CAP psuedolatex with particles <1 um. Other components in Aquateric can include ics, Tweens, and acetylated ycerides. Other suitable cellulose derivatives include: cellulose acetate trimellitate (Eastman); cellulose (Pharmacoat, Methocel); hydroxypropylmethyl cellulose phthalate (HPMCP); hydroxypropylmethyl cellulose succinate (HPMCS); and hydroxypropylmethylcellulose acetate succinate (e.g., AQOAT (Shin Etsu)). The performance can vary based on the degree and type of substitution. For example, HPMCP such as, HP-50, HP—55, HP-55S, HP-55F grades are suitable. The performance can vary based on the degree and type of substitution. For example, suitable grades of ypropylmethylcellulose acetate succinate e, but are not limited to, AS-LG (LF), which dissolves at pH 5, AS-MG (MF), which dissolves at pH 5.5, and AS-HG (HF), which dissolves at higher pH. These polymers are offered as granules, or as fine powders for aqueous dispersions; Poly Vinyl e Phthalate (PVAP). PVAP dissolves in pH >5, and it is much less permeable to water vapor and gastric fluids.

In some embodiments, the coating can, and usually does, contain a plasticizer and possibly other coating excipients such as colorants, talc, and/or magnesium stearate, which are well known in the art. Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400), l phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl ate. In particular, anionic carboxylic acrylic polymers usually will contain 10-25% by weight of a plasticizer, especially dibutyl phthalate, polyethylene glycol, yl e and triacetin. Conventional coating techniques such as spray or pan coating are employed to apply coatings. The coating thickness must be sufficient to ensure that the oral dosage form remains intact until the desired site of topical delivery in the inal tract is reached.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants (e. g., carnuba wax or PEG) may be added to the coatings besides plasticizers to solubilize or disperse the g material, and to improve coating mance and the coated product.

[00448] In other embodiments, the formulations described herein, which include compounds of Formula D or the secodn agent, are delivered using a pulsatile dosage form. A pulsatile dosage form is capable ofproviding one or more immediate release pulses at predetermined time points after a controlled lag time or at specific sites. Many other types of controlled release systems known to those of ordinary skill in the art and are suitable for use with the formulations described herein. Examples of such delivery systems include, e.g., polymer-based systems, such as polylactic and polyglycolic acid, plyanhydrides and polycaprolactone; porous matrices, nonpolymer-based systems that are lipids, including s, such as cholesterol, cholesterol esters and fatty acids, or neutral fats, such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings, bioerodible dosage forms, compressed tablets using conventional binders and the like. See, e.g., Liberman et al., Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214 (1990); Singh et al., Encyclopedia ofPharmaceutical Technology, 2Ild Ed., pp. 751-753 (2002); US. Pat. Nos. 725, 4,624,848, 4,968,509, 5,461,140, 923, 5,516,527, 5,622,721, 5,686,105, ,700,410, 5,977,175, 6,465,014 and 6,932,983, each of which is cally incorporated by reference.

In some embodiments, pharmaceutical formulations are provided that include particles of the compounds of any of a D or the secodn agent, described herein and at least one dispersing agent or ding agent for oral administration to a t. The formulations may be a powder and/or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group ing, but not limited to, ceutically acceptable aqueous oral dispersions, ons, solutions, elixirs, gels, and syrups. See, e. g., Singh et al., Encyclopedia ofPharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to the particles of compounds of Formula (Al—A6), the liquid dosage forms may include additives, such as: (a) egrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions can further include a lline inhibitor.

The aqueous suspensions and dispersions described herein can remain in a homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The homogeneity should be determined by a sampling method tent with regard to determining homogeneity of the entire composition. In one ment, an aqueous suspension can be pended into a homogenous suspension by physical agitation lasting less than 1 minute. In r embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by al agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a nous suspension by physical agitation g less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

Examples of disintegrating agents for use in the aqueous suspensions and dispersions include, but are not d to, a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amij el®, or sodium starch glycolate such as Promogel® or Explotab®; a cellulose such as a wood product, methylcrystalline cellulose, e. g., Avicel®, Avicel® PHlOl, Avicel® PH102, Avicel® PHlOS, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, and Solka—Floc®, methylcellulose, croscarmellose, or a cross- linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross- linked carboxymethylcellulose, or cross-linked croscarmellose; a cross-linked starch such as sodium starch glycolate; a cross-linked polymer such as vidone; a cross-linked polyvinylpyrrolidone; alginate such as alginic acid or a salt of c acid such as sodium alginate; a clay such as Veegum® HV sium aluminum silicate); a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth; sodium starch glycolate; ite; a natural sponge; a surfactant; a resin such as a cation-exchange resin; citrus pulp; sodium lauryl sulfate; sodium lauryl e in combination starch; and the like.

In some embodiments, the dispersing agents suitable for the aqueous suspensions and dispersions described herein are known in the art and include, for example, hydrophilic polymers, electrolytes, Tween ® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and the carbohydrate—based dispersing agents such as, for example, hydroxypropylcellulose and hydroxypropyl cellulose ethers (e. g., HPC, , and HPC-L), hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylmethyl—cellulose phthalate, hydroxypropylmethyl-cellulose acetate stearate; noncrystalline cellulose, magnesium um silicate, anolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone/Vinyl acetate copolymer (Plasdone®, e.g., 8-630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e. g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e. g., Tetronic 908®, also known as Poloxamine 908®, which is a unctional block copolymer derived from sequential addition ofpropylene oxide and ne oxide to ethylenediamine (BASF Corporation, Parsippany, N.J.)). In other embodiments, the dispersing agent is ed from a group not comprising one of the ing agents: hydrophilic polymers; electrolytes; Tween ® 60 or 80; PEG; polyvinylpyrrolidone (PVP); hydroxypropylcellulose and hydroxypropyl cellulose ethers (e. g., HPC, HPC-SL, and HPC-L); hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, HPMC K100M, and coat® USP 2910 (Shin-Etsu)); ymethylcellulose sodium; methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl- cellulose phthalate; hydroxypropylmethyl—cellulose acetate stearate; non-crystalline cellulose; magnesium aluminum te; triethanolamine; polyvinyl alcohol (PVA); ,3,3- tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde; mers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); or poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®).

[00454] Wetting agents suitable for the aqueous suspensions and dispersions bed herein are known in the art and include, but are not limited to, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fatty acid esters (e.g., the commercially available Tweens® such as e. g., Tween 20® and Tween 80® (ICI Specialty Chemicals», and polyethylene s (e. g., Carbowaxs 3350® and 1450®, and Carbopol 934® (Union Carbide», oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, yethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Vitamin E TPGS, sodium taurocholate, simethicone, phosphotidylcholine and the like Suitable preservatives for the aqueous suspensions or dispersions described herein include, for example, potassium sorbate, parabens (e.g., methylparaben and propylparaben), benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl alcohol or benzyl alcohol, phenolic compounds such as phenol, or nary compounds such as benzalkonium chloride. Preservatives, as used herein, are orated into the dosage form at a concentration sufficient to inhibit microbial growth.

Suitable viscosity enhancing agents for the aqueous suspensions or dispersions described herein include, but are not limited to, methyl cellulose, xanthan gum, carboxymethyl cellulose, ypropyl ose, ypropylmethyl cellulose, Plasdon® 8-630, er, polyvinyl alcohol, alginates, acacia, chitosans and ations thereof. The concentration of the viscosity enhancing agent will depend upon the agent selected and the viscosity desired.

Examples of sweetening agents suitable for the aqueous suspensions or dispersions bed herein include, for example, acacia syrup, acesulfame K, alitame, anise, apple, ame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola, cool , cool citrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, rin, aspartame, acesulfame potassium, mannitol, talin, ose, sorbitol, swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, e.g., anise-menthol, cherry-anise, cinnamon- orange, cherry-cinnamon, chocolate—mint, honey—lemon, lemon-lime, lemon—mint, menthol- eucalyptus, orange-cream, a-mint, and es thereof. In one embodiment, the aqueous liquid dispersion can comprise a sweetening agent or flavoring agent in a concentration g from about 0.001% to about 1.0% the volume of the aqueous dispersion. In another embodiment, the aqueous liquid dispersion can comprise a sweetening agent or ng agent in a tration ranging from about 0.005% to about 0.5% the volume of the s dispersion. In yet another embodiment, the aqueous liquid dispersion can comprise a sweetening agent or flavoring agent in a concentration ranging from about 0.01% to about 1.0% the volume of the aqueous dispersion.

In on to the additives listed above, the liquid formulations can also include inert diluents commonly used in the art, such as water or other ts, solubilizing agents, and emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl l, ethyl carbonate, ethyl acetate, benzyl l, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylforrnamide, sodium lauryl sulfate, sodium ate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.

In some embodiments, the pharmaceutical ations described herein can be self-emulsifying drug delivery systems ). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by Vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase can be added just prior to stration, which ensures stability of an unstable or hydrophobic active ingredient.

Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. SEDDS may provide improvements in the ilability of hydrophobic active ingredients. Methods ofproducing self-emulsifying dosage forms are known in the art and include, but are not limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563, each of which is specifically incorporated by reference.

It is to be appreciated that there is overlap between the above-listed additives used in the aqueous dispersions or sions described herein, since a given additive is often classified differently by ent practitioners in the field, or is commonly used for any of several different functions. Thus, the listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in ations described herein. The amounts of such additives can be readily determined by one skilled in the art, ing to the particular properties desired.

Intranasal Formulations Intranasal formulations are known in the art and are bed in, for example, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each of which is specifically incorporated by reference. Formulations that include a compound of any of Formula (A1 -A6), Formula (Bl-B6), Formula (C1-C6), or Formula (D1-D6), which are prepared according to these and other techniques well-known in the art are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. These ingredients are known to those skilled in the ation of nasal dosage forms and some of these can be found in TON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st n, 2005, a standard reference in the field. The choice of suitable carriers is highly dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient.

Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or ing and other stabilizing and solubilizing agents may also be present. The nasal dosage form should be isotonic with nasal secretions.

For administration by inhalation, the compounds of any of Formula D or the second agent, described herein may be in a form as an aerosol, a mist or a . ceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other le gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be ated containing a powder mix of the compound described herein and a suitable powder base such as e or starch.

Buccal Formulations Buccal formulations that include nds of any of Formula D or the second agent may be administered using a y of formulations known in the art. For example, such formulations include, but are not limited to, US. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136, each of which is specifically incorporated by reference. In addition, the buccal dosage forms bed herein can further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal . The buccal dosage form is fabricated so as to erode gradually over a ermined time period, wherein the delivery of the compound of any of Formula D or the second agent, is provided essentially hout. Buccal drug delivery, as will be iated by those skilled in the art, avoids the disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and/or first-pass inactivation in the liver. With regard to the bioerodible (hydrolysable) polymeric carrier, it will be appreciated that virtually any such carrier can be used, so long as the desired drug release profile is not compromised, and the carrier is compatible with the compound of any of Formula D or the second agent, and any other components that may be present in the buccal dosage unit. Generally, the ric carrier comprises hydrophilic -soluble and water-swellable) polymers that adhere to the wet e of the buccal mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers and co, e. g., those known as “carbomers” (Carbopol®, which may be obtained from BF. Goodrich, is one such polymer). Other components may also be incorporated into the buccal dosage forms described herein include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. For buccal or gual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a tional manner.

Transdermal Formulations Transdermal formulations described herein may be administered using a y of devices which have been described in the art. For example, such s e, but are not limited to, US. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636, 3,972,995, 3,993,072, 3,993,073, 934, 4,031,894, 4,060,084, 4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378, 5,837,280, ,869,090, 6,923,983, 6,929,801 and 6,946,144, each of which is specifically incorporated by reference in its entirety.

] The transdermal dosage forms bed herein may incorporate certain pharmaceutically acceptable excipients which are conventional in the art. In one embodiments, the transdermal formulations described herein include at least three ents: (1) a formulation of a compound of any of Formula D or the secodn agent; (2) a penetration enhancer; and (3) an aqueous adjuvant. In addition, transdermal ations can include additional components such as, but not limited to, gelling agents, creams and ointment bases, and the like.

In some ments, the transdermal formulation can further include a woven or non-woven backing material to enhance absorption and prevent the removal of the transdermal formulation from the skin. In other embodiments, the transdermal formulations described herein can maintain a saturated or supersaturated state to promote diffiasion into the skin.

Formulations suitable for transdermal administration of compounds described herein may employ transdermal delivery devices and transdermal ry patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery of the compounds bed herein can be accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches can provide controlled delivery of the compounds of any of a D or the second agent. The rate of tion can be slowed by using rate—controlling membranes or by trapping the compound within a polymer matrix or gel. Conversely, absorption enhancers can be used to increase absorption. An absorption enhancer or carrier can include absorbable pharmaceutically acceptable solvents to assist passage through the skin. For e, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, ally a rate controlling barrier to r the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

Injectable Formulations Formulations that include a compound of any of Formula D or the secodn agent, suitable for intramuscular, subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, sions, suspensions or emulsions, and sterile s for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, s (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and inj e organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. ations suitable for subcutaneous ion may also n additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by s antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium de, and the like. Prolonged absorption of the inj ectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum earate and gelatin.

For intravenous injections, compounds described herein may be formulated in aqueous solutions, preferably in physiologically compatible s such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be ted are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations may include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally known in the art.

[00469] eral injections may involve bolus injection or uous infusion.

Formulations for ion may be presented in unit dosage form, e.g, in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.

Suitable ilic solvents or vehicles include fatty oils such as sesame oil, or tic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. ally, the suspension may also contain le stabilizers or agents which se the solubility of the compounds to allow for the ation of highly trated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Other Formulations In certain ments, delivery systems for pharmaceutical compounds may be employed, such as, for example, liposomes and emulsions. In certain embodiments, compositions provided herein can also include an mucoadhesive polymer, selected from among, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl te mer, sodium alginate and dextran.

In some embodiments, the compounds described herein may be administered lly and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and vatives.

The nds described herein may also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, ning conventional itory bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted.

Dosing and Treatment Regimens Disclosed herein, in n 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) ing the mobilized plurality of cells. In some embodiments, the amount of the irreversible Btk inhibitor is ent to induce lymphocytosis of a plurality of cells from the malignancy. In some embodiments, the amount of the irreversible Btk inhibitor is from 300 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of the irreversible Btk inhibitor is from 420 mg/day up to, and including, 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 . In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg/day. In some embodiments, the AUC0_24 of the Btk inhibitor is between about 150 and about 3500 ng*h/mL. In some embodiments, the AUC0_24 of the Btk inhibitor is between about 500 and about 1100 ng*h/mL. In some embodiments, the Btk inhibitor is administered orally. In some embodiments, the Btk inhibitor is administered once per day, twice per day, or three times per day. In some embodiments, the Btk inhibitor is administered until disease ssion, ptable toxicity, or individual choice. In some ments, the Btk inhibitor is stered daily until disease progression, unacceptable ty, 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 a nance therapy.

The compounds described herein can be used in the preparation of medicaments for the inhibition of Btk or a homolog thereof, or for the treatment of diseases or conditions that would t, at least in part, from inhibition of Btk or a homolog thereof, including a patient and/or subject diagnosed with a hematological ancy. In addition, a method for treating any of the es or conditions described herein in a subject in need of such treatment, involves administration of pharmaceutical compositions ning at least one compound of any of Formula (A), Formula (B), Formula (C), or Formula (D), described herein, or a pharmaceutically acceptable salt, pharmaceutically acceptable N—oxide, ceutically active metabolite, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof, in therapeutically effective amounts to said subject.

] The compositions ning the compound(s) described herein can be stered for prophylactic, eutic, or maintenance treatment. In some embodiments, compositions containing the compounds described herein are administered for therapeutic applications (e. g., administered to a patient diagnosed with a hematological ancy). In some embodiments, compositions ning the compounds described herein are administered for therapeutic applications (e.g., dministered to a patient susceptible to or otherwise at risk of developing a hematological malignancy). In some embodiments, compositions containing the compounds described herein are administered to a patient who is in remission as a maintenance therapy. s of a compound disclosed herein will depend on the use (e. g., therapeutic, prophylactic, or maintnenace). Amounts of a compound disclosed herein will depend on severity and course of the disease or condition, previous therapy, the patients health status, weight, and response to the drugs, and the judgment of the ng physician. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (including, but not d to, a dose escalation clinical trial). In some ments, the amount of the irreversible Btk inhibitor is from 300 mg/day up to, and ing, 1000 mg/day. In some embodiments, the amount of the irreversible Btk inhibitor is from 420 mg/day up to, and including, 840 . In some embodiments, the amount of the Btk inhibitor is from 400 mg/day up to, and including, 860 mg/day. In some embodiments, the amount of the Btk inhibitor is about 360 mg/day. In some embodiments, the amount of the Btk inhibitor is about 420 mg/day. In some embodiments, the amount of the Btk inhibitor is about 560 mg/day. In some embodiments, the amount of the Btk inhibitor is about 840 mg/day. In some embodiments, the amount of the Btk inhibitor is from 2 mg/kg/day up to, and including, 13 mg/kg/day. In some embodiments, the amount of the Btk inhibitor is from 2.5 mg/kg/day up to, and including, 8 mg/kg/day. In some embodiments, the amount of the Btk inhibitor is from 2.5 mg/kg/day up to, and including, 6 mg/kg/day. In some embodiments, the amount of the Btk inhibitor is from 2.5 mg/kg/day up to, and including, 4 mg/kg/day. In some embodiments, the amount of the Btk inhibitor is about 2.5 mg/kg/day. In some embodiments, the amount of the Btk inhibitor is about 8 mg/kg/day.

In some embodiments, a Btk inhibitior disclosed herein is administered daily. In some embodiments, a Btk tor disclosed herein is administered every other day.

In some embodiments, a Btk inhibitior disclosed herein is administered once per day. In some embodiments, a Btk inhibitior disclosed herein is administered twice per day. In some ments, a Btk inhibitior disclosed herein is administered here times per day. In some embodiments, a Btk inhibitior disclosed herein is administered times per per day.

In some embodiments, the Btk inhibitor is administered until disease progression, unacceptable toxicity, or dual 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 stered every other day until disease progression, unacceptable toxicity, or individual choice.

In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the compounds may be given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday can vary between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose ion during a drug holiday may be from 10%-100%, including, by way of example only, %, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the t's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is ed. Patients can, however, require intermittent treatment on a long- term basis upon any recurrence of symptoms.

] The amount of a given agent that will correspond to such an amount will vary ing upon factors such as the particular compound, the severity of the disease, the ty (e.g., weight) of the subject or host in need of treatment, but can heless be routinely determined in a manner known in the art according to the particular stances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In general, however, doses employed for adult human treatment will typically be in the range of 002-5000 mg per day, or from about 1-1500 mg per day. The desired dose may iently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for e as two, three, four or more sub-doses per day.

The pharmaceutical composition described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compound. The unit dosage may be in the form of a package ning discrete quantities of the formulation. Nonlimiting examples are ed tablets or es, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single—dose non-reclosable containers.

Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to e a preservative in the composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative. In some embodiments, each unit dosage form comprises 210 mg of a compound disclosed herein. In some embodiments, an individual is administerd 1 unit dosage form per day. In some embodiments, an individual is administerd 2 unit dosage forms per day. In some embodiments, an individual is administerd 3 unit dosage forms per day. In some embodiments, an individual is administerd 4 unit dosage forms per day.

] The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages may be altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the nt of the practitioner.

Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by rd pharmaceutical ures in cell es or experimental animals, ing, but not limited to, the determination of the LD50 (the dose lethal to 50% of the tion) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds ting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

Kits/Articles of Manufacture The present invention also encompasses kits for carrying out the methods of the present invention. For example, the kit can comprise a d compound or agent capable of detecting a biomarker described herein, e.g., a biomarker of sis, cellular proliferation or al, or a Btk-mediated signaling pathway, either at the protein or nucleic acid level, in a ical sample and means for determining the amount of the biomarker in the sample (for example, an antibody or an ucleotide probe that binds to RNA encoding a biomarker of st) following incubation of the sample with a BCLD therapeutic agent of interest. Kits can be packaged to allow for detection iple biomarkers of interest by including individual d compounds or agents capable of detecting each dual biomarker of interest and means for determining the amount of each ker in the sample.

The particular choice of the second agent used will depend upon the diagnosis of the attending physicians and their judgment of the condition of the patient and the appropriate treatment protocol of the Btk inhibitors.

EXAMPLES The following specific and non—limiting examples are to be construed as merely illustrative, and do not limit the t disclosure in any way whatsoever. Without further elaboration, it is ed that one skilled in the art can, based on the description , utilize the present disclosure to its t extent. All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the intemet can come and go, but equivalent information can be found by searching the intemet. nce thereto evidences the availability and public dissemination of such information.

Example 1: Treatment of Non-Hodgkin Lymphoma by Administering a Btk inhibitor to Induce Pharmaceutical Debulking Two groups of patients with Non-Hodgkin Lymphoma (15 each) are treated with or without a Btk inhibitor followed by administering a second agent (Taxane). Group 1 is subject to the second agent treatment only (Taxane) and Group 2 is subject to a Btk inhibitor treatment for 2 days followed by administering the second agent based on the determined expression or presence of one or more B-cell lymphoproliferative disorder (BCLD) biomarkers from one or more subpopulation of lymphocytes.

Example 2. Determining the Expression or Presence of BCLD after Administering the Btk inhibitor for the Treatment of Non-Hodgkin Lymphoma Determining the expression or ce of BCLD after administering compound to a patient of Group 1 is used by the known procedures. e 3. Use of Taxane for the treatment of Non-Hodgkin Lymphoma Following determination of the expression or ce of one or more B-cell lymphoproliferative er (BCLD) kers from one or more subpopulation of lymphocytes in the patient, Taxane is used for Group 2 patients.

Example 4: Clinical Example of Determination of BCLDs Using a Btk inhibitor A patient with BCLD completes treatment with a Btk inhibitor or another treatment, and appears to be in complete remission. After this treatment is stopped, a short course of the Btk inhibitor is then given. If cells with markers of the malignant cells appear in the peripheral blood, in some embodiments it is an indication for ued treatment or for ng another treatment. One example ofthe cell subpopulation investigated for in the peripheral blood is cells bearing both the CD5 and CD20 markers, which is typical of CLL/SLL and Mantle Cell Lymphoma. These markers can be detectable by flow cytometry. A fiarther example of cell type is follicular lymphoma, which is terized by cells with t(l4;18) which in other embodiments are detectable by PCR or in situ hybridization in cells harvested from the eral blood.

Based on the markers ofthe malignant cells as ined in the peripheral blood, a suitable second treatment regimen is administered.

Example 5: Pharmaceutical Compositions:

[00494] The compositions described below are presented with a compound of Formula (D) for illustrative purposes; any of the nds of any of Formulas (A), (B), (C), or (D) can be used in such pharmaceutical compositions.

Example 5a: Parenteral Composition To prepare a parenteral pharmaceutical composition suitable for administration by injection, 100 mg of a water-soluble salt of a compound of a (D) is dissolved in DMSO and then mixed with 10 mL of 0.9% sterile saline. The mixture is incorporated into a dosage unit form suitable for administration by injection.

Example 5b: Oral Composition To prepare a pharmaceutical composition for oral delivery, 100 mg of a compound of a (D) is mixed with 750 mg of starch. The mixture is incorporated into an oral dosage unit for, such as a hard gelatin capsule, which is suitable for oral administration.

Example 5c: Sublingual (Hard Lozenge) Composition To prepare a pharmaceutical composition for buccal delivery, such as a hard lozenge, mix 100 mg of a compound of Formula (D), with 420 mg of powdered sugar mixed, with 1.6 mL of light corn syrup, 2.4 mL distilled water, and 0.42 mL mint extract. The mixture is gently blended and poured into a mold to form a lozenge suitable for buccal administration.

Example 5d: Inhalation Composition

[00498] To prepare a pharmaceutical composition for inhalation delivery, 20 mg of a compound of Formula (D) is mixed with 50 mg of anhydrous citric acid and 100 mL of 0.9% sodium de solution. The mixture is incorporated into an inhalation delivery unit, such as a nebulizer, which is suitable for inhalation administration.

Example 5e: Rectal Gel ition

[00499] To prepare a pharmaceutical composition for rectal delivery, 100 mg of a compound of a (D) is mixed with 2.5 g ofmethylcellulose (1500 mPa), 100 mg of methylparapen, 5 g of glycerin and 100 mL ofpurified water. The resulting gel mixture is then incorporated into rectal delivery units, such as syringes, which are suitable for rectal administration. e 5f: l Gel Composition To prepare a pharmaceutical topical gel composition, 100 mg of a compound of Formula (D) is mixed with 1.75 g of hydroxypropyl ose, 10 mL of propylene glycol, 10 mL of isopropyl myristate and 100 mL ofpurified alcohol USP. The resulting gel mixture is then orated into containers, such as tubes, which are suitable for topical administration.

Example 5g: Ophthalmic Solution Composition To prepare a pharmaceutical mic solution composition, 100 mg of a nd of Formula (D) is mixed with 0.9 g ofNaCl in 100 mL of purified water and filtered using a 0.2 micron filter. The resulting isotonic on is then orated into ophthalmic delivery units, such as eye drop containers, which are suitable for ophthalmic administration. e 6—Clinical Trial to Determine Efficacy of a Btk irreversible inhibitor in CLL and SLL patients Patients with CLL and/or SLL: The data provided herein is a pooled analysis of patients with CLL or SLL from two clinical trials of a Btk irreversible inhibitor. The initial trial (Study 04753) was a Phase 1A mu1ti-cohort, first-in-human, dose escalation trial of a Btk irreversible inhibitor in patients with ed or tory B-cell. 56 patients were enrolled between March 2009 and September 2010 and two doses were ted, namely oral once-daily dosing of a Btk irreversible inhibitor with a 28-day—on, 7-day-off le, and a continuous daily oral dosing schedule. Of the 56 patients ed, 16 CLL/SLL patients are included in this pooled analysis.

The second trial (Study 1102) is a Phase 1B/II trial of two once-daily oral doses of a Btk irreversible inhibitor in 2 populations of patients with CLL or SLL; a cohort containing patients with relapsed of refractory disease after at least 2 prior treatment regimens, and a second cohort of elderly patients with treatment—naive disease. This study began ment in May 2010, and has enrolled 56 patients to date. For the e of this pooled analysis, 38 patients, with a minimum of 28 days -up and 28 patients with on study tumor assessments are included in this analysis. In sum, 56 patients from the two studies are included in this analysis.

[00504] The baseline characteristics of patients enrolled to the two studies are summarized here. In study 04753, the median age was 66, there were 11 patients with CLL and ts with SLL. The median # of prior therapies was 3, with a range of 1-10. X% of patients had received prior nuc1eoside analogues, and x% had received prior D20 agents.

In study 1102, the median age was 68, 32 patients had CLL and 2 patients had SLL. Of the patients with CLL, 10 had del 17p. 15 patients had bulky disease, defined as a noda1 mass >5 cm diameter. In the relapsed/ tory cohort, the median # of prior ns was X.3 Per the eligibility requirements, all patients had received a nuc1eoside analogue-based regimen. 93% had received prior anti-CD20 agents, 9% alemtuzumab, and 19% bendamustine.

Objectives of the analysis

[00507] The ive of this pooled analysis is to characterize the nature and kinetics of the response to a Btk irreversible inhibitor in CLL. The Btk irreversible inhibitor compound is one of a new class of BCR signaling inhibitors, and, similar to other inhibitors of this pathway, the kinetics of response differ between the peripheral blood and the nodal compartments. The second objective was to ize the current status of the two studies with respect to best response, patient disposition, and time on treatment. The final objective was the ization of the adverse event profile of the Btk inhibitor on a larger and more diverse population of patients with CLL or SLL.

Response criteria Different response criteria were applied to patients with CLL and SLL respectively in these trials. Though considered biologically similar (or identical) diseases, given the phenotypic differences in presentation, the IW criteria for CLL are based on improvement in circulating cytes, nodal/ splenic/ marrow-based disease, and normalization of hematologic parameters. In st, the NHL criteria used to gauge the lymphomatous presentation of this disease (or SLL) are based only on improvement in lymphadenopathy and organomegaly. cyte count Figure 5 depicts the change with treatment in the cyte count for a 57 year- old patient with disease relapse following multiple prior therapies and the poor-risk cytogenetic feature dell lq began treatment with a Btk irreversible inhibitor nearly 6 months ago. Typical of the majority of CLL patients treated with a Btk irreversible tor, there was an initial, rapid, and prominent reduction in nodal disease and spleen size, with a corresponding rise in the circulating lymphocyte count, likely a consequence of the inhibitory effects of a Btk irreversible inhibitor on lymphocyte homing to the nodal and splenic compartments. Simultaneous with these changes, patients reported symptomatic improvement consistent with the tion of bulky e. Over time, the initial rise in lymphocytes returns to pre-treatment levels in spite of sustained reductions in adenopathy and megaly. Cases such as this seen with a Btk rsible inhibitor and similar , highlights the difficulty in ng standard response criteria to newer agents.

Effect of treatment on Lymph Node SPD As shown in Figure 6, patients treated with a Btk irreversible inhibitor had an immediate and marked nodal response to treatment. 85% of evaluable patients ed a partial response and even more had some LN shrinkage. 80% of ts with measurable LN disease achieved a 50% reduction in their SPD within 2 cycles of therapy. Figure 7 shows the remarkable shrinkage in Lymph node post-treatment for the 57 year-old patient described supra.

Change in Lymph Node and absolute lymphocyte count (ALC) Figure 8 depicts the effect of a Btk irreversible tor on LN disease burden and lymphocytosis over time in the patients from the Phase Ia trial. y statistics from the patients with an early lymphocytosis show a similar pattern in the median percent change over time in both ALC and in LN disease burden measured by the SPD. Immediately following treatment, patients develop an early lymphocytosis which decreases with time to pre-treatment or normal levels. There is a sustained decrease in disease burden shown by the LN sum of perpendicular diameters. Thus, with some variability in timing, many patients show a marked decrease in tumor burden in both peripheral blood and in LN disease with sustained treatment.

Adverse Effects Adverse effects seen as a side effect of the treatment were monitored as outlined in Figure 9. The effects were categorized by severity into grades 1—4. Grade 3 or greater events have been very uncommon. The vast majority of events have been mild in severity. Diarrhea, nausea, and fatigue have been the most commonly reported adverse , with most of the reports occurring early in treatment Thus, the oral Btk inhibitor has marked activity in patients with CLL and SLL including high-risk pts. It provides good disease control with longer follow-up commonly exceeds 6 months. There is no evidence of drug—related myelosuppression or cumulative toxicity.

Example 7 al Trial to ine Safety and y of Compounds of Formula (D)

[00514] The purpose of this clinical trial is to study the side effects and best dose of a nd of Formula (D) and to ine its efficacy in the treatment of patients diagnosed with recurrent B-cell lymphoma.

STUDY DESIGN Cohorts of 6 ts each receive a compound of Formula (D) at 1.25, 2.5, 5.0, 8.3, 12.5, 17.5 mg/kg/d until the MTD is established. In cases where MTD is not reached, dosing levels are increased beyond 17.5mg/kg/d by 33% increments. Patients e daily treatment for 28 days followed by a 7 day rest period (one cycle). Tests for Btk occupancy by the drug (“occupancy”) are med on Day 1, 2, 8, 15 and 29 during Cycle 1 and on Day 1 and 15 of Cycles 3, 5, 7, 9, and 11. If S 1 DLT (“dose-limiting toxicity”) is observed in the cohort during Cycle 1, escalation to the next cohort will proceed. Patients are enrolled in the next cohort if four of the six patients enrolled in the cohort completed Cycle 1 without experiencing a DLT, while the remaining two patients are completing evaluation. If 2 2 DLTs are observed during Cycle 1, dosing at that dose and higher is suspended and the MTD is established as the previous cohort. ts are allowed to continue dosing at the MTD. If 2 2 DLTs are seen at the 5.0 d cohort an additional cohort of 6 ts can be added at 3.75 mg/kg/d.

Upon ination of the MTD, a cohort of 6 patients is enrolled to receive a compound of Formula (D) at the MTD or “preferred occupying dose” continuously for 35 days with no rest period (one cycle).

STUDY POPULATION Up to 52 patients with recurrent surface immunoglobulin ve B cell non- Hodgkin’s lymphoma according to WHO classification (including small lymphocytic lymphoma /chronic lymphocytic leukemia) STUDY OBJECTIVES 1. Primary Objectives include: A. Determine pharmacokinetics (PK) of an orally administered compound of Formula (D).

B. Evaluate tumor response. Patients have screening (i.e., baseline) disease assessments within 30 days before beginning treatment. Patients o follow-up disease assessments following specified dosing cycles. Patients without evidence of e progression on treatment are followed for a maximum of 6 months off treatment for disease progression. At screening, a ed tomography (CT) (with contrast unless contraindicated) and positron- emission tomography (PET) or CT/PET scan of the chest, abdomen, and pelvis are ed. At other visits, a CT (with contrast unless contraindicated) scan of the chest, abdomen, and pelvis are ed. A CT/PET or PET is required to confirm a te response. Bone marrow biopsy is optional. In patients known to have positive bone marrow before treatment with study drug, a repeat biopsy should be done to confirm a complete se following treatment. All patients are evaluated for response based on International Working Group Revised Response Criteria for Malignant Lymphoma, ines for the diagnosis and treatment of c lymphocytic leukemial4, or m Response Criteria in Waldenstrom’s Macroglobulinemia.

C. e pharrnacodynamic (PD) ters to include drug ncy of Btk, the target enzyme, and effect on biological markers of B cell function. Specifically, this study examines the pharmacodynamics (PD) of the drug in peripheral blood mononuclear cells (PBMCs) using two PD assays. The first PD assay measures occupancy of the Btk active site by the drug using a specially designed fluorescent probe. The second PD assay measures inhibition of B cell activation by stimulating the PBMCs ex vivo at the BCR with anti-IgM/IgG, and then assaying cell surface expression of the tion marker CD69 by flow cytometry The PD biomarkers are measured in vitro from a blood sample removed from patients 4-6 hours following an oral dose of the drug. These assays determine what drug levels are required to achieve maximal occupancy of Btk and maximal inhibition ofBCR signaling. When possible, similar studies are conducted on circulating tumor cells isolated from blood of patients. 2. Secondary Objectives include: A. To analyze tumor biopsy samples (when possible) for apoptotic biomarker expression analysis.

Inclusion Criteria To be eligible to participate in this study, a patient must meet the ing Women and men 2 18 years of age Body weight 2 40 kg Recurrent surface immunoglobulin ve B cell non-Hodgkin’s lymphoma (NHL) according to WHO classification, including small cytic lymphoma/ chronic lymphocytic ia (SLL/CLL) and lymphoplasmacytic lymphoma, including Waldenstrom's Macroglobulinemia (WM) Measurable disease (for NHL, bidimensional disease 2 2 cm diameter in at least one dimension, for CLL Z 5000 leukemia cells/mm3, and for WM presence of immunoglobulin M paraprotein with a minimum IgM level 2 1000 mg/dL and infiltration of bone marrow by lymphoplasmacytic cells) Have failed 2 1 us treatment for lymphoma and no standard therapy is ble. Patients with diffuse large B cell lymphoma must have failed, refused or be ineligible for autologous stem cell transplant ECOG performance status ofS 1 Ability to swallow oral capsules without difficulty Willing and able to sign a written informed consent Exclusion Criteria A patient meeting any of the ing criteria will be excluded from this study: More than four prior systemic therapies (not counting maintenance rituximab), except for CLL patients. Salvage therapy/conditioning regimen g up to autologous bone marrow transplantation is considered to be one regimen Prior allogeneic bone marrow transplant Immunotherapy, chemotherapy, radiotherapy or experimental therapy within 4 weeks before first day of study drug dosing Major surgery within 4 weeks before first day of study drug dosing CNS involvement by lymphoma Active unistic infection or treatment for opportunistic infection within 4 weeks before first day of study drug dosing o Uncontrolled illness including but not limited to: ongoing or active infection, matic congestive heart failure (New York Heart Association Class III or IV heart failure), unstable angina pectoris, cardiac arrhythmia, and atric illness that would limit compliance with study requirements 0 History of myocardial infarction, acute coronary syndromes ding le angina), coronary angioplasty and/or stenting within the past 6 months 0 Known HIV infection 0 Hepatitis B sAg or Hepatitis C positive 0 Other medical or psychiatric illness or organ dysfianction which, in the opinion of the investigator, would either compromise the patient’s safety or interfere with the evaluation of the safety of the study agent 0 Pregnant or lactating women (female patients of child-bearing potential must have a negative serum pregnancy test within 14 days of first day of drug dosing, or, if positive, a pregnancy ruled out by ound) 0 History of prior cancer < 2 years ago, except for basal cell or squamous cell carcinoma of the skin, cervical cancer in situ or other in situ carcinomas Results: ] 29 pts (12 follicular, 7 CLL/SLL, 4 DLBCL, 4 mantle, 2 marginal) with a median of 3 prior therapies have been ed on cohorts 1-4. y was well tolerated with most adverse events < grade 2. One protocol defined DLT (dose delay > 7 d due to penia) was observed. 19/22 pts from cohorts 1-3 are evaluable. The ORR is 42%; 1 CR (SLL), 7 PR (4 CLL/SLL, 2 MCL and lFL). In cohort 2, PD demonstrate complete occupancy of Btk by a compound of Formula (D), with >95% enzyme occupancy 4 hours post dose in all pts. Basophil degranulation, a Btk-dependent cellular process, was completely inhibited up to 24 hrs. T-cell responses were not ed, and no significant depletion of peripheral blood B, T or NK cell counts was observed. Positive ation (R2 = 0.93) was found between Btk active-site occupancy in PBMCs (mean of Days 1 and 8) and a compound of Formula (D) plasma AUCO -° (Day 1) at the 1.25 mg/kg dose.

Example 8: Clinical Example of Diagnosis of BCLDs Using a Btk inhibitor

[00527] A patient with BCLD completes treatment with a Btk inhibitor or another treatment, and appears to be in te remission. After this treatment is stopped, a short course of the Btk inhibitor is then given. If cells with markers of the malignant cells appear in the peripheral blood, in some embodiments it is an indication for continued treatment or for starting another treatment. One example ofthe cell subpopulation investigated for in the peripheral blood is cells bearing both the CD5 and CD20 markers, which is typical of L and Mantle Cell Lymphoma. These markers can be able by flow cytometry. A fiarther example of cell type is follicular lymphoma, which is characterized by cells with t(l4;18) which in other embodiments are detectable by PCR or in situ ization in cells harvested from the peripheral blood.

For patients initially starting on treatment an increase of the malignant subpopulation can be an early predictive marker of se or duration of response.

For patients who have previously received treatment and are suspected of progressing based upon changes (for example in a scan) that are non-diagnostic, the BTK test for peripheral blood cell increases could add diagnostic information that enable earlier treatment of relapse. This would be valuable in determining whether to re-start treatment for BCLD or to watch or to pursue an alternative diagnosis.

The test could yield better diagnostic information for ts whose BCLD is suspected to be orming into a more aggressive cellular form. For example both CLL/SLL and lower grade follicular lymphoma can transform into a higher grade process which may resemble diffiise large B cell lymphoma, and require more aggressive treatment. e 9: Patient ion ] Patient selection screens are performed to identify an individual with the ABC subtype of DLBCL. Gene expression profiling is conducted using FFPE biopsy material, using RNA amplified with a Nugen kit and assayed on an Affymetrix U133Plus 2.0 arrays.

Samples are screened for recurrent somatic mutations. This is accomplished by conventional resequencing of candidate genes in the NF-kB and B cell receptor signaling pathways (e. g. CARDl l, CD79A, CD79B, MYD88, TNFAIP3) plus p53 by exon amplification and standard dideoxy automated DNA sequencing.

The patient selection screen also identifies patients with ABC DLBCL that are particularly sensitive or resistant to Btk inhibitors. A ve result for a CARDll mutation tes that the individual is resistant to Btk inhibitors e CARDll mutations te the NF-kB pathway at a step that is downstream of BTK.

] Genomic copy number analysis is also required to tely assess the activity of oncogenic pathways that may be relevant for the response to Btk inhibitors as well as to assess prognosis. In particular, ABC DLBCLs harbor genomic deletions of the TNFAIP3 locus, which encodes A20, a negative regulator ofNF—kB. Thus, a full assessment ofA20 status es both resequencing to look for somatic mutations and copy number analysis to look for deletions. In addition, patients are identified with DLBCL tumors that harbor genomic deletions in the INK4a/ARF locus or have trisomy of chromosome 3 because these genomic aberrations are associated with poor prognosis in ABC DLBCL. A single pass high throughput DNA sequencing is performed using the Illumina HiSeq2000 platform to assess genomic copy number globally.

Example 10: PK and Efficacy of a Btk inhibitor in Individuals with CLL or SLL A Btk inhibitor was administered to 33 individuals diagnosed with CLL or SLL.

Efficacy and PK was ined.

Day 8 Dose AUCO-24 IWG Resp 2c m_ Patient ID Grou Sex mL) C cle Mar 2011 420 073-203 Naive 420 217-107 R/R ___“ 420 217-202 Naive 420 032-110 R/R ___“ 420 217-104 R/R ___“ 420 032-201 Naive ___“ 420 217-103 R/R ___“ 420 032-104 R/R ___“ 420 217-102 R/R ___“ WWWNNNNNNNNNNHHHHHHHHHHNHOOOONQUIADJNHOQOQQQMLWNHOCOONOUI#WNH. 420 217-106 1VR-_—-I 420 032-109 IVR 420 217-110 IVR 420 038-101 M 420 217-111 M 420 217-109 M 420 032-107 M ___“ 420 1 Naive 420 032-105 R/R ___“ 420 217-101 R/R ___“ 420 1 R/R 420 217-105 R/R ___“ 420 032-101 R/R ___“ 420 2 Naive ___“ 420 217-112 RR 420 217-201 Naive ___“ 420 073-204 Naive 420 217-108 R/R ___“ 420 032-108 R/R 420 032-106 R/R ___“ 420 032-102 R/R 420 3 NR 420 032-202 Naive ___“ Day 8 AUCO-24 IWG Resp N0. 1 Patient ID Groun Sex (n_.h/mL) C cle Mar 2011 Example 11: Clinical Trial with Btk tor A phase Ib/H clinical trial was performed to study the effects of a Btk inhibitor on individuals with CLL.

Study Type: Interventional Allocation: Non-Randomized Endpoint Classification: Safety Study Intervention Model: Parallel ment g: Open Label Primary Purpose: Treatment Group I (elderly, naive, individuals) received 420 mg/day of the Btk inhibitor.

Group 11 (NR individuals, who had twice been treated With fludara) received 420 mg/day of the Btk inhibitor. Group 111 (MR individuals, who had twice been treated with fludara) ed 840 mg/day of the Btk inhibitor.

Patient Characteristics 231;. 22 {222; :22 {2226315 Prism-r therapy}.% Nucleaside analing 2} £323} 2"? {392922} 33219525} 22-32222 2:222 252223 322721: fillfflfilfigi *2 $9223 24(55%} 2'? {3322} 232222222122 229252 5 {2222} 3-2222} 222222222. 22222} 2 {222232 22 {32221 12122325 8 £952} 3 {323222} EB {32%}: 2212 2 1522-321 1. {222; 2 {2222} 12 (22.22} H222 «z: 11g{22 2 {3223} 2 {152;} 12 (5222} . .: . 3215 {32%} 132132 23152} 13,124 {2532}, . 2 222122}: 2;:2 £122} 2122 } 12,125 {222} 2 2312 (212} 23222222} ,5 Tumor assessment was performed every 2 treatment cycles. ives Describe the characteristics of the antitumor effect of a Btk inhibitor in duals with CLL/SLL, e.g., reduction in lymphadenopathy/splenornegaly, and kinetics of change in absolute lymphocyte count (ACL).

Summarize the safety profile of the Btk inhibitor. ion Criteria FOR ENT-NAIVE GROUP ONLY: Men and women 2 65 years of age with confirmed diagnosis of CLL/SLL, who require treatment per NCI or International Working Group inesl l- l 4 FOR RELAPSED/REFRACTORY GROUP ONLY: Men and women 2 18 years of age with a confirmed diagnosis of relapsed/refractory CLL/SLL unresponsive to therapy (ie, failed 2 2 previous ents for CLL/SLL and at least 1 n had to have had a purine analog [eg, fludarabine] for subjects with CLL) Body weight 2 40 kg ECOG performance status ofS 2 Agreement to use contraception during the study and for 30 days after the last dose of study drug if sexually active and able to bear children Willing and able to participate in all required evaluations and procedures in this study ol including swallowing capsules without difficulty Ability to understand the purpose and risks of the study and provide signed and dated informed consent and authorization to use protected health information (in accordance with al and local subject privacy regulations) Exclusion Criteria A life-threatening illness, medical ion or organ system dysfiinction which, in the investigator's opinion, could compromise the subject's safety, interfere with the absorption or metabolism of Btk inhibitor P0, or put the study outcomes at undue risk Any immunotherapy, chemotherapy, radiotherapy, or experimental therapy within 4 weeks before first dose of study drug (corticosteroids for disease-related symptoms allowed but require l-week washout before study drug administration) Central s system (CNS) involvement by lymphoma Major surgery within 4 weeks before first dose of study drug Creatinine > 1.5 x institutional upper limit of normal (ULN); total bilirubin > 1.5 x ULN (unless due to Gilbert's e); and aspartate aminotransferase (AST) or e aminotransferase (ALT) > 2.5 x ULN unless disease related Concomitant use ofmedicines known to cause QT prolongation or torsades de pointes Significant screening electrocardiogram (ECG) abnormalities including left bundle branch block, 2nd degree AV block type 11, 3rd degree block, bradycardia, and QTc > 470 msec Lactating or pregnant Response Criteria NHL IWG criterial were applied to SLL cases without modification ] The 2008 CLL IWG criteria were d to CLL cases with the following modifications: a. An isolated lymphocytosis, in the absence of other parameters meeting the criteria for PD, was not considered PD b. Patients experiencing a cytosis, but obtaining a PR by other measurable parameters, were classified as "nodal" response until there was a 50% reduction in ALC from baseline in which case they were categorized as PR. c. Patients with a normal ALC (<5K) at baseline with treatment-related lymphocytosis required normalization to <5K to be categorized as PR.

Results Subject Disposition Fnfiow-up Median si} 5.3 13 «3.5 Range 1.33- 912 DJ -» 9,5 {3,3 -- r55 Saw-eat Bismminued Best Response Best se by Risk Features ESE-1311a 8- 831% 3?% Results further summarized in Figs. 18 — 27. Figure 18 presents the responses for the naive, 420 mg/day group. Figure 19 presents the responses for the R/R, 420 mg/day group.

Figure 20 presents the responses by prognostic factors. Figure 21 presents responses over time.

Figure 22 ts the best responses for all patients. Figure 23 presents the best responses for abstract patients. Figure 24 presents the best response by prognostic factor. Figure 25 presents initial (Cycle 2) response assessment and best response (420mg Cohorts). Figure 26 ts initial (Cycle 2) response assessment by dose: relapsed/refractory. Figure 27 presents ements in hematological parameters.

Conclusions The interim Phase II data confirm that a Btk inhibitor is highly active in both treatment-naive and relapsed/ refractory CLL/ SLL patients Class-specific rapid lymph node reduction with concurrent lymphocytosis seen in the majority of patients 2008 CLL IWG objective responses (PR + CR) and nodal responses appear to be durable and independent of high risk genomic es A high proportion (85%) of relapsed or refractory patients are free-of—progression at 6 months (420mg ) Example 12: Long Term —Up Trial for Individuals Taking Btk inhibitor The purpose of this study is to determine the erm safety of a fixed-dose, daily regimen of Btk inhibitor P0 in subjects with B cell lymphoma or chronic cytic leukemia/small lymphocytic leukemia (CLL/SLL).

Study Type: Interventional Allocation: Non-Randomized Endpoint Classification: Safety Study Intervention Model: Single Group Assignment Masking: Open Label Primary Purpose: Treatment ] Intervention: 420 mg/day of a Btk inhibitor ] Applicable conditions: B-cell c cytic ia; Small Lymphocytic Lymphoma; Diffuse Well—Differentiated Lymphocytic Lymphoma; B Cell Lymphoma; Follicular Lymphoma; Mantle Cell Lymphoma; Non-Hodgkin's Lymphoma; Waldenstrom Macroglobulinemia; Burkitt Lymphoma; B-Cell Diffiase Lymphoma Primary Outcome Measures: Adverse / Safety Tolerability [Time Frame: 30 days after last dose of study drug] - frequency, severity, and dness of adverse events Secondary Outcome Measures: Tumor Response [Time Frame: frequency of tumor assessments done per standard of care] - tumor response will be assessed per established response criteria. This study will capture time to e ssion and duration of response.

Tumor Response [Time Frame: Time to disease progression] - Duration of response as measured by ished response criteria for B cell lymphoma and chronic lymphocytic leukemia Inclusion Criteria Men and women with B cell lymphoma or CLL/small lymphocytic lymphoma (SLL) who had stable disease or response to Btk inhibitor PO for at least 6 months on a prior Btk inhibitor study and want to continue study drug or who had disease progression on PCYC- 04753 and want to try a higher dose Eastern Cooperative Oncology Group (ECOG) performance status of S 2 Agreement to use contraception during the study and for 30 days after the last dose of study drug if sexually active and able to bear children Willing and able to participate in all required evaluations and procedures in this study protocol including swallowing capsules without difficulty Ability to tand the e and risks of the study and provide signed and dated informed consent and authorization to use protected health information (in ance with national and local subject privacy regulations) Exclusion Criteria ] A life-threatening illness, l condition or organ system dysfiinction which, in the investigator's opinion, could compromise the subject's safety, interfere with the absorption or metabolism of Btk inhibitor P0, or put the study outcomes at undue risk Concomitant immunotherapy, chemotherapy, radiotherapy, osteroids (at dosages equivalent to prednisone > 20 mg/day ), or experimental therapy Concomitant use ofmedicines known to cause QT prolongation or torsades de pointes Central nervous system (CNS) involvement by ma Creatinine > 1.5 x utional upper limit of normal (ULN); total bilirubin > 1.5 x ULN (unless due to Gilbert's disease); and aspartate aminotransferase (AST) or alanine aminotransferase (ALT) > 2.5 x ULN unless e related Lactating or pregnant Example 13: Phase 11 Study of Btk inhibitor in R/R MCL The purpose of this study is to: Evaluate the efficacy of Btk inhibitor in relapsed/refractory ts with MCL who have not had prior omib, and who have had prior bortezomib The secondary objective is to evaluate the safety of a fixed daily dosing regimen of Btk inhibitor capsules in this population.

Study Type: Interventional Allocation: Non-Randomized Endpoint fication: Safety/Efficacy Study Intervention Model: Parallel Assignment Masking: Open Label Primary Purpose: Treatment Intervention: 560 mg/day of a Btk inhibitor Primary Outcome Measures To Measure the Number of Participants with a Response to Study Drug [Time Frame: Participants will be followed until progression of disease or start of another anti-cancer treatment] Secondary Outcome Measures To Measure the Number of Participants with Adverse Events as a e of Safety and Tolerability [Time Frame: Participants will be followed until progression of disease or start of another anti-cancer treatment] To Measure the Number of Participants cokinetics to Assist in Determining How the Body Responds to the Study Drug [Time Frame: Procedure to be Performed During the First Month of Receiving Study Drug] Patient Reported es [Time Frame: Participants will be followed until progression of e or start of another anti-cancer treatment] To measure the number icipants reported outcomes in determining the health related quality of life.

Inclusion Criteria: Men and women 2 18 years of age ECOG performance status ofS 2 Pathologically confirmed MCL, with documentation of either overexpression of cyclin D1 or t(l 1,14), and measurable disease on cross nal imaging that is 2 2 cm in the longest diameter and measurable in 2 perpendicular dimensions Documented failure to e at least partial response (PR) with, or documented disease progression disease after, the most recent treatment regimen At least 1, but no more than 5, prior treatment regimens for MCL (Note: ts having received 22 cycles of prior treatment with bortezomib, either as a single agent or as part of a combination therapy regimen, will be considered to be bortezomib-exposed.) Willing and able to ipate in all required evaluations and procedures in this study ol including swallowing capsules without difficulty Ability to understand the e and risks of the study and e signed and dated informed consent and authorization to use protected health information (in accordance with national and local subject privacy regulations) Major exclusion criteria: ] Prior chemotherapy within 3 weeks, nitrosoureas within 6 weeks, therapeutic anticancer antibodies within 4 weeks, radio— or toxin-immunoconjugates within 10 weeks, radiation therapy within 3 weeks, or major surgery within 2 weeks of first dose of study drug Any life-threatening illness, medical condition or organ system dysfiinction which, in the investigator's opinion, could compromise the subj ect's safety, interfere with the absorption or metabolism of Btk inhibitor capsules, or put the study outcomes at undue risk ally significant cardiovascular disease such as uncontrolled or symptomatic arrhythmias, congestive heart failure, or myocardial infarction within 6 months of screening, or any Class 3 or 4 cardiac disease as defined by the New York Heart Association Functional Classification orption syndrome, e significantly affecting gastrointestinal fisnction, or ion of the stomach or small bowel or ulcerative colitis, symptomatic inflammatory bowel e, or l or complete bowel obstruction Any of the following laboratory abnormalities: a. Absolute neutrophil count (ANC) < 750 cells/mm3 (0.75 X 109/L) unless there is documented bone marrow ement b. Platelet count < 50,000 cells/mm3 (50 x 109/L) independent of transfusion support unless there is documented bone marrow involvement c. Serum aspartate transaminase (AST/SGOT) or alanine transaminase (ALT/SGPT) 2 3.0 X upper limit of normal (ULN) d. Creatinine > 2.0 x ULN Example 14: Phase 11 Study of Btk inhibitor + ofatumumab in R/R CLL The purpose of this study was to determine the efficacy and safety of a fixed- dose, daily n of orally administered Btk inhibitor combined with umab in subjects with relapsed/refractory CLL/SLL and related diseases Study Type: Interventional Allocation: ndomized Endpoint Classification: Safety Study Intervention Model: Single Group Assignment Masking: Open Label ] Primary Purpose: Treatment Intervention: 420 mg/day of a Btk tor, standard dose of ofatumumab Applicable conditions: B-cell Chronic Lymphocytic Leukemia; Small Lymphocytic Lymphoma; Diffuse Well—Differentiated Lymphocytic Lymphoma; Prolymphocyctic Leukemia; Richter‘s Transformation Primary e Measures: Response and safety of Btk inhibitor [Time Frame: At the end of cycles 1 and 3] Response rate as defined by recent guidelines in Chronic Lymphocytic Leukemia Secondary Outcome Measures: Pharmacokinetic/Pharmacodynamic assessments [Time Frame: during l-2 cycles] Pharmacodynamics of Btk inhibitor (ie, drug occupancy of Btk and effect on biological market 1/2) of Btk inhibitor.

Tumor Response [Time Frame: at the end of Cycles 2,4 and 6 (28 days for each Overall response rate as defined by recent guidelines on CLL Inclusion Criteria: Subjects with histologically confirmed chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), prolymphocytic leukemia (PLL) as defined by WHO classification of hematopoietic neoplasms, or Richter’s transformation arising out of CLL/SLL and satisfying 2 l of the following conditions: Progressive splenomegaly and/or lymphadenopathy fied by physical examination or radiographic studies Anemia (<11 g/dL) or thrombocytopenia (<100,000/uL) due to bone marrow involvement Presence of unintentional weight loss > 10% over the preceding 6 months NCI CTCAE Grade 2 or 3 fatigue Fevers > 100.5 degree or night sweats for > 2 weeks without evidence of infection Progressive lymphocytosis with an se of > 50% over a 2 month period or an pated ng time of < 6 months Need for cytoreduction prior to stem cell transplant Subjects must have failed 2 2 prior therapies for CLL including a nucleoside analog or Z 2 prior therapies not including nucleoside analog if there is a contraindication to such therapy > 10% expression of CD20 on tumor cells ECOG performance status S 2 Life expectancy 2 12 weeks ] Subjects must have organ and marrow function as defined below: Absolute phil count (ANC) Z 1000/uL in the e of bone marrow involvement Platelets Z /uL Total bin S 1.5 x utional upper limit of normal unless due to Gilbert's disease AST(SGOT) S 2.5 x institutional upper limit of normal unless due to infiltration of the liver Creatinine S 2.0 mg/dL OR creatinine clearance 2 50 mL/min No history of prior anaphylactic reaction to rituximab ] No history of prior exposure to ofatumumab Age 2 18 years Body weight 2 40 kg Able to w capsules without difficulty and no history of malabsorption syndrome, disease significantly affecting gastrointestinal fianction, or resection of the stomach or small bowel or ulcerative colitis, symptomatic inflammatory bowel disease, or partial or complete bowel obstruction Exclusion Criteria: A life-threatening illness, medical condition or organ system dysfianction which, in the investigator's opinion, could compromise the t's safety, interfere with the absorption or metabolism of Btk inhibitor P0, or put the study outcomes at undue risk ] Any anticancer immunotherapy, chemotherapy, radiotherapy, or experimental therapy within 4 weeks before first dose of study drug. Corticosteroids for disease-related symptoms are allowed provided 1 week washout occurs.

Active central nervous system (CNS) involvement by lymphoma Major surgery within 4 weeks before first dose of study drug Lactating or pregnant y of prior ancy, except for adequately d basal cell or squamous cell skin cancer, in situ cervical cancer, or other cancer from which the subject has been disease free for at least 2 years or which will not limit survival to < 2 years History of Grade 2 2 toxicity (other than alopecia) continuing from prior ncer therapy.

Results 6 Patients have been evaluated for DLT through end of cycle 2. 0 DLTs occurred in these patients. 4 patients have had end of cycle 3 scans and blood counts. 3 of 4 are responder per IWG criteria. Our response rate is 75% for these pts.

Example 15: Phase II Study of Btk inhibitor + BR or FCR in R/R CLL The purpose of this study is to establish the safety of orally administered Btk inhibitor in combination with bine/cyclophosphamide/rituximab (FCR) and bendamustine/rituximab (BR) in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic ma(SLL).

Study Type: Interventional Allocation: ndomized Endpoint Classification: Safety Study Intervention Model: Single Group Assignment Masking: Open Label Primary Purpose: Treatment ] Intervention: 420 mg/day of a Btk inhibitor, standard FCR or BR regimen Applicable conditions: B-cell Chronic Lymphocytic Leukemia; Small Lymphocytic Lymphoma; Diffuse Well-differentiated Lymphocytic Lymphoma Primary Outcome Measures: To measure the number of participants with prolonged hematologic toxicity [Time Frame: 8 weeks from first dose] Secondary Outcome Measures: To measure the number ofparticipants with adverse events as a measure of safety and tolerability [Time Frame: For 30 days after the last dose of Btk inhibitor] To measure the number ofpatients who respond to ent by measuring the increase or decrease of disease in the lymph nodes and/or blood test results [Time Frame: Patients may remain on study until the last subject enrolled completes a maximum of 12 cycles of Btk inhibitor. Any subjects still ing Btk inhibitor at that time may enroll in a long-term -up study to continue to receive Btk inhibitor capsules] Inclusion Criteria: Histologically confirmed CLL or SLL and satisfying at least 1 of the following criteria for requiring treatment: Progressive splenomegaly and/or lymphadenopathy identified by physical examination or radiographic s Anemia (<11 g/dL) or ocytopenia 000/uL) due to bone marrow involvement Presence of unintentional weight loss > 10% over the preceding 6 months NCI CTCAE Grade 2 or 3 fatigue Fevers > 100.50 or night sweats for > 2 weeks without ce of ion Progressive lymphocytosis with an increase of > 50% over a 2 month period or an anticipated doubling time of < 6 months 1 to 3 prior treatment regimens for CLL/SLL ECOG performance status ofS 1 2 18 years of age Willing and able to ipate in all required evaluations and procedures in this study protocol including swallowing capsules without difficulty Ability to understand the purpose and risks of the study and provide signed and dated informed consent and authorization to use protected health information (in accordance with national and local subject privacy regulations) Exclusion Criteria: Any chemotherapy, therapeutic antineoplastic antibodies (not ing radio- or toxin immunoconjugates), radiation therapy, or experimental antineoplastic therapy within 4 weeks of first dose of study drug Radio— or toxin—conjugated antibody therapy within 10 weeks of first dose of study drug Concomitant use cines known to cause QT prolongation or torsades de pointes ormed lymphoma or Richter's transformation Any life-threatening illness, medical condition or organ system dysfiinction which, in the igator's opinion, could mise the subj ect's safety, interfere with the absorption or metabolism of Btk inhibitor P0, or put the study outcomes at undue risk ] Any of the following laboratory abnormalities: a. Absolute neutrophil count (ANC) < 1000 cells/mm3 (1.0 x 109/L) b. Platelet count < 50,000/mm3 (50 x 109/L) 0. Serum aspartate minase (AST/SGOT) or alanine transaminase (ALT/SGPT) 2 3.0 X upper limit al (ULN) d. Creatinine > 2.0 X ULN or creatinine clearance < 40 mL/min Example 16: Phase 11 Study of Btk inhibitor in R/R DLBCL The purpose of this study is to evaluate the efficacy of Btk inhibitor in relapsed/refractory de novo activated B—cell (ABC) and germinal—cell B—Cell (GCB) Diffiase Large B-cell Lymphoma (DLBCL).

Study Type: Interventional Allocation: ndomized Endpoint Classification: Safety Study ] Intervention Model: Single Group Assignment Masking: Open Label Primary e: Treatment ] Intervention: 560 mg/day Btk inhibitor Primary Outcome Measures: To measure the number ofpatients with a response to study drug [Time Frame: 24 weeks from first dose] Participants will be followed until progression of disease or start of another anti- cancer treatment.

Secondary Outcome Measures: To measure the number of patients with adverse events as a measure of safety and tolerability. [Time Frame: For 30 days after the last dose of Btk inhibitor] Participants will be followed until progression of the disease or start of another anticancer treatment.

To measure the number ofparticipants pharmacokinetics to assist in determining how the body responses to the study drug. [Time Frame: Procedure will be performed during the first month of receiving study drug] Inclusion Criteria: Men and women 2 18 years of age. n Cooperative gy Group (ECOG) performance status of S 2.

Pathologically confirmed de novo DLBCL; ts must have available al tissue for central review to be eligible.

Relapsed or refractory disease, defined as either: 1) recurrence of disease after a complete remission (CR), or 2) partial response (PR), stable disease (SD), or progressive disease (PD) at completion of the treatment regimen preceding entry to the study (residual e):Subj ects must have previously ed an appropriate first-line treatment regimen.

Subjects with ted residual disease after the treatment regimen directly ing study enrollment must have biopsy demonstration of al DLBCL. Subjects who have not received high dose chemotherapy/autologous stem cell transplant (HDT/ASCT) must be ineligible for HDT/ASCT as defined by meeting any of the following criteria: Age 2 70 years ,Diffuse lung capacity for carbon de (DLCO) < 50% by pulmonary function test (PFT), Left ventricular ejection fraction (LVEF) < 50% by multiple gated acquisition(MUGA)/echocardiograph (ECHO), Other organ dysfunction or comorbidities precluding the use of HDT/ASCT on the basis of unacceptable risk of treatment-related ity, Subject refusal of HDT/ASCT.

Subjects must have 2 l measurable (> 2 cm in longest dimension) disease sites on computed tomography (CT) scan.

Exclusion Criteria: Transformed DLBCL or DLBCL with coexistent histologies (eg, follicular or -associated lymphoid tissue [MALT] lymphoma) ] Primary mediastinal (thymic) large B—cell lymphoma (PMBL) Known central nervous system (CNS) lymphoma Any chemotherapy, external beam radiation therapy, or ncer antibodies within 3 weeks of the first dose of study drug Radio— or toxin—immunoconjugates within 10 weeks of the first dose of study drug Major surgery within 2 weeks of first dose of study drug Any life-threatening illness, medical condition or organ system dysfunction which, in the investigator's opinion, could compromise the subj ect's , or put the study outcomes at undue risk Clinically significant cardiovascular disease such as uncontrolled or matic arrhythmias, congestive heart failure, or myocardial infarction within 6 months of screening, or any Class 3 or 4 cardiac disease as defined by the New York Heart ation Functional Classification Unable to swallow capsules or malabsorption syndrome, disease significantly affecting gastrointestinal fianction, or resection of the stomach or small bowel or tive colitis, symptomatic inflammatory bowel disease, or partial or complete bowel obstruction Any of the following laboratory abnormalities: a. Absolute neutrophil count (ANC) < 750 cells/mm3 (0.75 x lO9/L) unless there is documented bone marrow involvement b. et count < 50,000 cells/mm3 (50 x lO9/L) ndent of transfilsion t unless there is documented bone marrow involvement c. Serum aspartate transaminase (AST/SGOT) or alanine transaminase (ALT/SGPT) 2 3.0 upper limit of normal (ULN) d. Creatinine > 2.0 x ULN Example 17: Assay of Drug Combinations Combinations of a Btk tor and additional cancer treatment agents were assayed using DoHH2 cells.

] DOHH2 is a DLBCL (diffuse large B—cell lymphoma) cell line, from a transformed follicular lymphoma patient. It is moderately sensitive to a Btk inhibitor.

The Btk inhibitor was incubated with other cancer drugs for 2 days. Assay was an alamar blue assay.

The combinations were: a. Btk inhibitor and Gemicitabine; b. Btk tor and thasone; 9.0 Btk inhibitor and Lenalinomide; Btk inhibitor and R-406; o Btk inhibitor and Temsirolimus; Btk tor and Carboplatin; Btk inhibitor and Bortezomib; and Btk inhibitor and Doxorubicin.

Results are presented in figures 28—3 1.

Example 18: Assay of Drug Combinations ] Combinations of a Btk inhibitor and additional cancer treatment agents were assayed using TMD8 cells.

TMD8 is a NF-kB signalling-dependent ABC-DLBCL cell line. It is sensitive to BTK inhibitors alone at low nanomolar concentrations (G150 ~l-3 nM). A Btk inhibitor was incubated with other cancer drugs for 2 days. Assay was an alamar blue assay.

The combinations were: .99 Btk inhibitor and CAL-101; .57 Btk inhibitor and Lenalinomide; .0 Btk inhibitor and R—406; .9 Btk inhibitor and Bortezomib; Frqornro Btk inhibitor and Vincristine; Btk inhibitor and Taxol; Btk inhibitor and abine; and Btk inhibitor and Doxorubicin.

[00726] Results are presented in figures 32—39.

Example 19: Clinical Trial of Btk Inhibitor in Combination with BR A clinical trial was performed to determine the effects of combining a Btk inhibitor with BR (bendamustine and rituximab). The Btk inhibitor was administered. Following an increase in the concentration of lymphoid cells in the peripheral blood, BR was administered. l results indicated that the combination of the Btk inhibitor and BR ed in substantially no lymphoid cells in the peripheral blood.

Example 20: Clinical Trial of Btk Inhibitor in Combination with Ofatumumab A al trial was performed to determine the effects of combining a Btk inhibitor with Ofatumumab. The Btk inhibitor was administered. ing an increase in the concentration of lymphoid cells in the peripheral blood, Ofatumumab was administered. Initial results indicated that the combination of the Btk inhibitor and Ofatumumab resulted in a decrease in lymphoid cells in the peripheral blood.

Claims (13)

CLAIMS :

1. Use of ibrutinib in the manufacture of a medicament for treating multiple myeloma (MM) in an individual in need thereof, wherein the medicament is formulated for administration at an ibrutinib dosage of about 420 to about 840 mg once per day in a solid oral dosage form.

2. The use of claim 1, n the ibrutinib dosage is about 420 mg once per day.

3. The use of claim 1, wherein the ibrutinib dosage is about 560 mg once per day.

4. The use of claim 1, wherein the ibrutinib dosage is about 840 mg once per day.

5. The use of any one of claims 1-4, wherein the solid oral dosage form is a capsule.

6. The use of any one of claims 1-4, wherein the solid oral dosage form is a tablet.

7. The use of any one of claims 1-6, wherein following stration of the medicament, the individual achieves a stable disease, a partial response, or a complete response.

8. The use of claim 7, wherein following administration of the medicament, the individual achieves a stable disease.

9. The use of claim 7, wherein following administration of the medicament, the individual achieves a partial response.

10. The use of claim 7, wherein following stration of the medicament, the dual achieves a te response.

11. The use of any one of claims 1-10, wherein following administration of the medicament, the individual does not experience a progressive disease.

12. Use of claim 1, wherein (27092897_1):AXG the medicament is formulated for oral administration of the ibrutinib to the individual at a dose of 560 mg on a continuous once-daily regimen until progression of the multiple myeloma or unacceptable toxicity; and the individual achieves a complete se.

13. The use of claim 12, wherein lymphocytosis is not considered ssion of the multiple myeloma. (27092897_1):AXG