Use of Bruton's tyrosine kinase (Btk) inhibitors

Abstract

The present invention provides a pharmaceutical composition for use in the treatment of follicular lymphoma. [Solution] The pharmaceutical composition comprises 560 mg of a BTK inhibitor and is formulated for a continuous once-daily dosing regimen, and the BTK inhibitor is as follows: JPEG2026065005000042.jpg6042 A pharmaceutical composition having the following structure is provided.

Description

[Technical Field]

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

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

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

[0004] In certain embodiments, a method for treating a hematological malignancy in an individual requiring it is disclosed herein, comprising the steps of: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to pharmacokineticate multiple cells from the malignancy; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignancy. In some embodiments, the hematological malignancy is CLL. In some embodiments, the step of treating the hematological malignancy includes the step of managing the hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the hematological malignancy is leukemia, lymphoproliferative disorder, or myeloid. In some embodiments, the pharmacokinetic cells are myeloid cells or lymphoid cells. In some embodiments, the step of analyzing the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased compared to the concentration prior to administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after the concentration of pharmacokinetic cells subsequently decreases. In some embodiments, the step of analyzing the pharmacokinetic cells includes measuring the duration of the increase in the peripheral blood concentration of pharmacokinetic cells compared to the concentration prior to administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the step of analyzing the pharmacokinetic cells includes counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased compared to the concentration prior to administration of the Btk inhibitor. In some embodiments, the step of administering the second cancer treatment regimen occurs after the number of pharmacokinetic cells in the peripheral blood subsequently decreases.In some embodiments, the step of analyzing a plurality of pharmacokinetic cells includes measuring the period of increase in the number of pharmacokinetic cells in peripheral blood compared to the number before administration of a Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of pharmacokinetic cells in peripheral blood has increased over a predetermined period of time. In some embodiments, the step of analyzing a plurality of pharmacokinetic cells includes preparing biomarker properties relating to a population of cells isolated from the plurality of cells, where the biomarker properties indicate biomarker expression, biomarker expression level, changes in the biomarker, or the presence of the biomarker. In some embodiments, the biomarker is an expression marker of any cytogenetic, cell surface molecule, protein, or RNA. In some embodiments, the biomarkers are:ZAP70;t(14,18):β-2 microglobulin;mutation status of p53;mutation status of ATM;del(17)p;del(11)q;del(6)q;CD5;CD11c;CD19;CD20;CD22;CD25;CD38;CD103;CD138;secreted, surface or intracellular immunoglobulin expression;mutation status of VH; or a combination thereof. In some embodiments, the method further includes the step of providing a second cancer treatment regimen based on biomarker characteristics. In some embodiments, the method further includes the step of administering based on biomarker characteristics. In some embodiments, the method further includes the step of predicting the effect of a treatment regimen based on biomarker characteristics. In some embodiments, the hematological malignancies are chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, or non-CLL / SLL lymphoma. In some embodiments, hematological malignancies include follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Valdenström macroglobulinemia, multiple myeloma, marginal lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, or extranodular marginal B-cell lymphoma.In some embodiments, the hematological malignancy is chronic myeloid (or spinal) leukemia or acute lymphoblastic leukemia. In some embodiments, the hematological malignancy is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or refractory CLL; relapsed or refractory SLL; relapsed or refractory multiple myeloma. In some embodiments, the Btk inhibitor forms a covalent bond with the cysteine ​​side chain of Bruton's tyrosine kinase, a Bruton's tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog. In some embodiments, the irreversible Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one. In some embodiments, the amount of the irreversible Btk inhibitor is from 300 mg / day to 1000 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is from 420 mg / day to 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day, about 560 mg / day, or about 840 mg / day. In some embodiments, the amount of the irreversible Btk inhibitor is about 420 mg / day. In some embodiments, the AUC of the Btk inhibitor. 0-24 These are approximately 150 and 3500 ng * It is between h / mL. In some embodiments, the AUC of the Btk inhibitor 0-24 These are approximately 500 and 1100 ng *The concentration is between h / mL. In some embodiments, the Btk inhibitor is administered orally. In some embodiments, the Btk inhibitor is administered once daily, twice daily, or three times daily. In some embodiments, the Btk inhibitor is administered until disease progression, unacceptable toxicity, or individual selection. In some embodiments, the Btk inhibitor is administered daily until disease progression, unacceptable toxicity, or individual selection. In some embodiments, the Btk inhibitor is administered every other day until disease progression, unacceptable toxicity, or individual selection. In some embodiments, the Btk inhibitor is a first-line therapy, second-line therapy, third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy. In some embodiments, the Btk inhibitor treats refractory hematological malignancies. In some embodiments, the Btk inhibitor is maintenance therapy. In some embodiments, the second cancer treatment regimen includes chemotherapeutic agents, steroids, immunotherapy agents, targeted therapies, or combinations thereof. In some embodiments, the second cancer treatment regimen includes a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen includes antibodies, B cell receptor signaling inhibitors, PI3K inhibitors, IAP inhibitors, mTOR inhibitors, radioimmunotherapy agents (radioimmunotherapeutic), DNA damage agents, bioprecipitation inhibitors, histone deacetylase inhibitors, protein kinase inhibitors, hedgehog inhibitors, Hsp90 inhibitors, telomerase inhibitors, Jak1 / 2 inhibitors, protease inhibitors, PKC inhibitors, PARP inhibitors, or a combination thereof.In some embodiments, the second cancer treatment regimen includes chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof. In some embodiments, the second cancer treatment regimen includes cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen includes bendamustine and rituximab. In some embodiments, the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab. In some embodiments, the second cancer treatment regimen comprises cyclophosphamide, vincristine, and prednisone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises etoposide, doxorubicin, vincristine, cyclophosphamide, prednisolone, and optionally, rituximab. In some embodiments, the second cancer treatment regimen comprises dexamethasone and lenalidomide. In some embodiments, the Bruton's tyrosine kinase inhibitor is a reversible inhibitor. In some embodiments, the Bruton's tyrosine kinase inhibitor is an irreversible inhibitor. In some embodiments, the Bruton's tyrosine kinase inhibitor forms a covalent bond with the cysteine ​​side chain of Bruton's tyrosine kinase, a Bruton's tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog. In some embodiments, the Bruton's tyrosine kinase inhibitor has the structure of formula (D):

[0005] [ka]

[0006] Here: L ais CH2, O, NH or S; Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; Y is any substituent selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; Z is C(=O), OC(=O), NHC(=O), C(=S), S(=O) x OS (=O) x , NHS (=O) x And here, x is either 1 or 2; R7 and R8 are independently H; Alternatively, R7 and R8 together form a single bond; R6 is H; and the inhibitor of Bruton's tyrosine kinase has a pharmaceutically active metabolite, a pharmaceutically acceptable solvent compound, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug. In some embodiments, the Bruton's tyrosine kinase inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one. In some embodiments, La is O. In some embodiments, Ar is phenyl. In some embodiments, Z is C(=O), NHC(=O), or S(=O)2. In some embodiments, R7 and R8 are each H. In some embodiments, Y is a cycloalkyl group with a 4-membered, 5-membered, 6-membered, or 7-membered ring; or Y is a heterocycloalkyl group with a 4-membered, 5-membered, 6-membered, or 7-membered ring.

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

[0008] Disclosed herein is a method for treating diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) in individuals requiring treatment, comprising the step of administering an irreversible Btk inhibitor to the individual at doses ranging from 300 mg / day to 1000 mg / day. In some embodiments, the method further comprises the step of diagnosing the individual as having diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) by determining the gene sequences of one or more biomarkers in multiple lymphoid cells isolated from diffuse large B-cell lymphoma. In some embodiments, the irreversible Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one. In some embodiments, ABC-DLBCL is characterized by a CD79B mutation. In some embodiments, the CD79B mutation is a mutation in the immunoreceptor-activated tyrosine motif (ITAM) signaling module. In some embodiments, the CD79B mutation is a missense mutation of the initial immunoreceptor-activated tyrosine motif (ITAM) tyrosine. In some embodiments, the CD79B mutation increases surface BCR expression and decreases Lyn kinase activity. In some embodiments, ABC-DLBCL is characterized by the CD79A mutation. In some embodiments, the CD79A mutation is present in the immunoreceptor-activated tyrosine motif (ITAM) signaling module. In some embodiments, the CD79A mutation is a splice donor site mutation in the immunoreceptor-activated tyrosine motif (ITAM) signaling module. In some embodiments, the CD79A mutation removes the immunoreceptor-activated tyrosine motif (ITAM) signaling module. In some embodiments, ABC-DLBCL is characterized by MyD88, A20, or a combination thereof. In some embodiments, the MyD88 mutation is the amino acid substitution L265P in the MYD88 Toll / IL-1 receptor (TIR) ​​domain.In some embodiments, the amount of irreversible Btk inhibitor ranges from 420 mg / day to 840 mg / day. In some embodiments, the amount of irreversible Btk inhibitor is approximately 420 mg / day, approximately 560 mg / day, or approximately 840 mg / day. In some embodiments, the amount of irreversible Btk inhibitor is approximately 420 mg / day. In some embodiments, the AUC of the Btk inhibitor. 0-24 These are approximately 150 and 3500 ng * It is between h / mL. In some embodiments, the AUC of the Btk inhibitor 0-24 These are approximately 500 and 1100 ng * The concentration is between h / mL. In certain embodiments, the irreversible Btk inhibitor is administered orally. In some embodiments, the irreversible Btk inhibitor is administered daily until disease progression, unacceptable toxicity, or individual selection. In some embodiments, the irreversible Btk inhibitor is administered every other day until disease progression, unacceptable toxicity, or individual selection. In some embodiments, the irreversible Btk inhibitor is first-line, second-line, third-line, fourth-line, fifth-line, or sixth-line therapy. In some embodiments, the irreversible Btk inhibitor treats refractory hematological malignancies. In some embodiments, the irreversible Btk inhibitor is maintenance therapy. In some embodiments, the method further includes the step of administering at least one additional cancer treatment regimen. In some embodiments, the additional cancer treatment regimen includes chemotherapeutic agents, immunotherapy agents, steroids, radiotherapy, targeted therapy, or a combination thereof. In some embodiments, the second cancer treatment regimen comprises antibodies, B cell receptor signaling inhibitors, PI3K inhibitors, IAP inhibitors, mTOR inhibitors, radioimmunotherapeutic agents, bioprecipitation inhibitors, histone deacetylase inhibitors, protein kinase inhibitors, hedgehog inhibitors, Hsp90 inhibitors, telomerase inhibitors, Jak1 / 2 inhibitors, protease inhibitors, PKC inhibitors, PARP inhibitors, or a combination thereof.

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

[0010] [Figure 1] Figure 1 illustrates the role of Btk activity in many processes in CLL cells that contribute to the pathogenesis of the disease. [Figure 2] Figure 2 shows the absolute number of lymphocytes during treatment with irreversible Btk inhibitors for individuals with CLL. [Figure 3] Figure 3 shows the change in the sum of lymph node (LN) diameter products in patients with CLL and SLL treated with irreversible Btk inhibitors. [Figure 4] Figure 4 shows the LN response in patients with CLL. The left panel shows LN before treatment with an irreversible Btk inhibitor, and the right panel shows LN after treatment with an irreversible Btk inhibitor. [Figure 5] Figure 5 shows the effects of irreversible Btk inhibitors on the time course of LN disease burden and lymphocytosis in patients with CLL and / or SLL. [Figure 6] Figure 6 shows the side effects in patients treated with irreversible Btk inhibitors. Grades 1-4 represent the severity of the effect, with 1 representing very mild and 4 representing very unpleasant. [Figure 7] Figure 7 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle day after administration of a Btk inhibitor to individuals with follicular lymphoma who achieved complete or partial remission (CR / PR). The Y-axis shows the absolute lymphocyte count (ALC) at each point in time, based on the cycle number and day on the X-axis. All patients (except Pt32009) were treated on a 4-week schedule followed by a one-week treatment off period. Therefore, day 1 of each cycle represents a one-week drug off period for these patients. Note the increase in ALC during most cycles in most patients, and the decrease in ALC at the beginning of subsequent cycles. This pattern is often blunted in subsequent cycles as the patient responds to treatment. Patient 32009 received non-interfering treatment and did not exhibit this cyclical pattern, but showed an increase on day 15 of cycle 1 and a gradual increase during cycles 2-5. [Figure 8] Figure 8 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle day after administration of a Btk inhibitor to follicular lymphoma patients who had stable disease (SD) during treatment. The Y-axis shows the absolute lymphocyte count (ALC) at each point in time, based on the cycle number and day on the X-axis. All patients (except Pt32009) were treated on a 4-week schedule, followed by a 1-week rest period. Therefore, on day 1 of each cycle, drug administration to these patients was suspended for one week. Note the gradual increase in hematological ALC pharmacokinetics in patient 32004, who was initially stable but later developed progressive disease (PD). [Figure 9]Figure 9 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle day after administration of a Btk inhibitor to PD individuals with follicular lymphoma. The Y-axis shows the absolute lymphocyte count (ALC) at each time point, with the cycle number and day on the X-axis. All patients except 38010 were treated on a 4-week schedule, followed by a 1-week rest period. Therefore, day 1 of each cycle represents a 1-week rest period for these patients. Note the lack of pharmacokinetics, particularly in patients 38010 and 32001. Patient 323001 had limited treatment before being included in the study. The lymphocyte response suggests that this patient might have responded if they had been able to remain on longer-term treatment. [Figure 10] Figure 10 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle days after administration of Btk inhibitors to individuals with DLBCL in PR and SD. The Y-axis shows the absolute lymphocyte count (ALC) at each time point, with the cycle number and day on the X-axis. Patient 38011 was treated on a 4-week schedule, followed by a 1-week rest period. Therefore, day 1 of each cycle represents a 1-week rest period for these patients. Patients 38008 and 324001 were treated with a continuous daily dose. [Figure 11] Figure 11 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle days after administration of a Btk inhibitor to PD individuals with DLBCL. The Y-axis shows the absolute lymphocyte count (ALC) at each time point, with the cycle number and day on the X-axis. All patients were treated on a 4-week schedule, followed by a 1-week rest period. Therefore, day 1 of each cycle represents a 1-week rest period for these patients. Note the lack of pharmacokinetics in 3 out of 4 patients. Patient 32002 received only one cycle of treatment. [Figure 12]Figure 12 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle days after administration of a Btk inhibitor to individuals with mantle cell lymphoma. The Y-axis shows the absolute lymphocyte count (ALC) at each point in time, based on the cycle number and day on the X-axis. Patients 32006, 38003, and 38004 were treated on a 4-week schedule, followed by a 1-week rest period. Therefore, on day 1 of each cycle, drug administration to these patients was suspended for one week. Other patients were treated with continuous daily drug infusions. Note that patient 32014, who had early PD, did not show pharmacokinetics. [Figure 13] Figure 13 shows the absolute lymphocyte count (ALC) / 10⁹L versus cycle days after administration of a Btk inhibitor to individuals with mantle cell lymphoma shown in Figure 12. Compared to Figure 12, the axes have been modified to demonstrate lower amplitude fluctuations. Note that all responding patients showed some degree of pharmacokinetics. [Figure 14] Figure 14 demonstrates that lymphocyte dynamics, particularly the B-cell type, are consistent with lymphoma cells and decrease as the disease responds. Patient 32007, group 4, had follicular lymphoma (grade 3) that gradually regressed from SD to CR. In this case, the ALC changes were not dramatic, but the B-cell fraction showed a characteristic increase in ring structure in response to treatment with a Btk inhibitor. Note also the decrease in cycles due to the cycle scale of displacement, which is consistent with overlapping disease management. [Figure 15] Figure 15 demonstrates that B-cell dynamics increase with disease progression. Patient 32004, a peer group of 2, had follicular lymphoma (grade 1) that first progressed from stable disease (SD) to disease progression (PD) after cycle 6. [Figure 16] Figure 16 shows the initial pharmacokinetics and eventual decline of the CD45DIM B cell subgroup in response to patient 200-005 with mantle cell lymphoma. This subgroup has a typical MCL immunophenotype (CD45DIM) and differs from that of normal lymphocytes. [Figure 17]Figure 17 shows abnormal high-light-scatter CD19+ cells that kineticate and then regress in CR DLBCLPt324001. These CD45+ cells with light dispersion (SSC-H) are gated in the upper panel, and their CD3 vs. CD19 staining is shown in the lower panel. Here, putative malignant cells are "hidden" in a large MNC window that normally defines monocytes. The sequence of kinetication after the response is similar to other examples. [Figure 18] Figure 18 presents responses to a clinical trial involving the administration of a Btk inhibitor to elderly patients with CLL or SLL who were not receiving drug intervention. Individuals were administered 420 mg / day of a Btk inhibitor. [Figure 19] Figure 19 presents the response to a clinical trial involving the administration of a Btk inhibitor to R / R patients with CLL or SLL. Individuals were administered 420 mg / day of a Btk inhibitor. [Figure 20] Figure 20 shows the response to a clinical trial involving the administration of a Btk inhibitor to individuals with high-risk CLL. [Figure 21] Figure 21 shows the time course of a clinical trial involving the administration of a Btk inhibitor to individuals with CLL or SLL. [Figure 22] Figure 22 shows the best response for all patients in a clinical trial involving the administration of a Btk inhibitor to individuals with CLL or SLL. [Figure 23] Figure 23 shows the hypothetical best patient response in a clinical trial involving the administration of a Btk inhibitor to individuals with CLL or SLL. [Figure 24] Figure 24 shows the best response based on prognostic factors in patients with CLL or SLL involved in clinical trials regarding the administration of Btk inhibitors. [Figure 25] Figure 25 presents the initial (cycle 2) response evaluation and best response (420 mg same-age group) in CLL or SLL patients involved in clinical trials regarding the administration of Btk inhibitors. [Figure 26] Figure 26 presents the initial (cycle 2) response evaluation by dose in patients with relapsed / refractory CLL or SLL involved in clinical trials, relating to the administration of Btk inhibitors. [Figure 27] Figure 27 shows the improvement in blood parameters in patients with CLL or SLL involved in clinical trials related to the administration of Btk inhibitors. [Figure 28] Figure 28 presents data showing the results of Btk inhibitors in combination with carboplatin or Velcade in DoHH2 cells. [Figure 29] Figure 29 presents data showing the results of Btk inhibitors and combinations of dexamethasone or lenalidomide in DoHH2 cells. [Figure 30] Figure 30 presents data showing the results of Btk inhibitors in combination with temsirolimus or R406 in DoHH2 cells. [Figure 31] Figure 31 presents data showing the results of Btk inhibitors in combination with gemcitabine or doxorubicin in DoHH2 cells. [Figure 32] Figure 32 presents data showing the results of combining Btk inhibitors with Cal-101 ​​in TMD8 cells. [Figure 33] Figure 33 presents data showing the results of combining Btk inhibitors with R406 in TMD8 cells. [Figure 34] Figure 34 presents data showing the results of combining Btk inhibitors with vincristine in TMD8 cells. [Figure 35] Figure 35 presents data showing the results of combining Btk inhibitors with doxorubicin in TMD8 cells. [Figure 36] Figure 36 presents data showing the results of combining Btk inhibitors with lenolidomide in TMD8 cells. [Figure 37] Figure 37 presents data showing the results of combining a Btk inhibitor with Velcade TMD8 cells. [Figure 38] Figure 38 presents data showing the results of the combination of Btk inhibitors and fludarabine in TMD8 cells. [Figure 39] Figure 39 presents data showing the results of combining Btk inhibitors with Taxol in TMD8 cells. [Modes for carrying out the invention]

[0011] There is currently a need for methods (including diagnostic steps) to treat hematological malignancies, including relapsed and refractory B-cell malignancies, and ABC-DLBCL. This application is partly based on the unexpected discovery that Btk inhibitors induce pharmacokinetics (or, in some cases, lymphocytosis) of lymphoid cells in solid hematological malignancies. Phenokinetics of lymphoid cells increases their exposure to additional cancer treatment regimens and their availability for biomarker screening. The inventors have also found that Btk inhibitors are useful in treating relapsed and refractory malignancies, as well as ABC-DLBCL.

[0012] In certain embodiments, a method for treating hematological malignancies in an individual requiring the treatment thereof is disclosed herein, comprising the steps of: (a) administering to the individual an amount of irreversible Btk inhibitor sufficient to pharmacokineticate multiple cells from the malignancy; and (b) analyzing the pharmacokinetic multiple cells. In certain embodiments, a method for treating diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) in an individual requiring the treatment thereof is disclosed herein, comprising the steps of: administering to the individual an amount of irreversible Btk inhibitor ranging from 300 mg / day to 1000 mg / day. In certain embodiments, a method for treating relapsed or refractory non-Hodgkin lymphoma in an individual in need thereof is further disclosed herein, comprising the steps of: administering a therapeutically effective amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one to the individual.

[0013] <Specific technical terms> Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art, relating to the subject matter of the claims. Where there are multiple definitions of a term herein, the definition in this section shall prevail. Where references are made to URLs or other such identifiers or addresses, it is understood that such identifiers may change, and specific information on the Internet may appear or disappear, but equivalent information may be found by searching the Internet. Citations to these confirm the availability and general dissemination of such information.

[0014] It should be understood that the general descriptions above and the detailed descriptions below are illustrative and descriptive only and do not limit any subject matter to be claimed. In this application, the use of the singular includes the plural unless otherwise specified. As used in the specification and appended claims, the singular forms "a," "an," and "the" include multiple referents unless the context clearly indicates otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of other forms such as "include," "includes," and "included," as well as the term "including," is not limited.

[0015] The section headings used herein are for the organized purpose only and should not be construed as limiting the subject matter described herein. However, all documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, manuals, and professional works, are expressly incorporated herein by reference in whole for any purpose.

[0016] Definitions of standard chemical terms can be found in reference materials, including Carey and Sundberg, “ADVANCED ORGANIC CHEMISTRY 4TH ED.” Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPCL, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used within the techniques of the art. Unless specific definitions are provided, the technical terms and their corresponding tests and techniques used herein in relation to analytical chemistry, organic synthesis, and medicinal chemistry and pharmaceutical chemistry are known in the art. Standard techniques may be used in chemical synthesis, chemical analysis, preparation, formulation, and delivery of pharmaceuticals, and in patient treatment. Standard techniques may be used in recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Reaction and purification techniques may be performed, for example, using manufacturer's specification kits, or as commonly achieved in the art, or as described herein. The aforementioned techniques and procedures may be carried out in conventional methods well known to the techniques and as described in various general and more specific references cited and discussed throughout this specification.

[0017] It should be understood that the methods and compositions described herein are not limited to the specific methodologies, protocols, cell lines, components, and reagents described herein, and that such may be modified. Furthermore, it should be understood that the terms used herein are intended to describe only specific embodiments and are not intended to limit the scope of the methods and compositions described herein to those limited only by the appended claims.

[0018] All publications and patents referenced herein are incorporated herein by reference in their entirety, for example, to describe and disclose the structures and methodologies described in the publications, and may be used herein in relation to the methods, compositions and compounds described herein. The publications considered herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein shall be construed as an acknowledgment that the inventors described herein have no prior rights to such disclosures by prior art or for any other purpose.

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

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

[0021] The “alkyl” portion may have 1 to 10 carbon atoms (wherever it appears herein, the numerical range, such as “1 to 10,” refers to each integer within a given range; for example, “1 to 10 carbon atoms” means that the alkyl group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., including 10 carbon atoms; however, this definition also includes the appearance of the term “alkyl” without specifying a numerical range). The alkyl groups of the compounds described herein may be represented as “C1-C4 alkyl” or similar names. As just one example, “C1-C4 alkyl” indicates that 1 to 4 carbon atoms are present in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Thus, C1-C4 alkyl includes C1-C2 alkyl and C1-C3 alkyl. Alkyl groups may or may not be substituted. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0022] As used herein, the term “acyclic alkyl” refers to an alkyl that is not cyclic (i.e., a linear or branched alkyl containing at least one carbon atom). Acyclic alkyls may be fully saturated or may include acyclic alkenes and / or alkynes. Acyclic alkyls may be optionally substituted.

[0023] The term "alkenyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a double bond that is not part of the aromatic group. That is, an alkenyl group begins with the atom -C(R)=C(R)-R, where R refers to the rest of the same or different alkenyl group. The alkenyl moiety can be branched, linear, or cyclic (in which case it may also be known as a "cycloalkenyl" group). Depending on the structure, an alkenyl group can be a monoradical or a diradical (i.e., an alkenylene group). Alkenyl groups can be optionally substituted. Unrestricted examples of alkenyl groups include -CH=CH2, -C(CH3)=CH2, -CH=CHCH3, and -C(CH3)=CHCH3. Alkenylene groups include, but are not limited to, -CH=CH-, -C(CH3)=CH-, -CH=CHCH2-, -CH=CHCH2CH2-, and -C(CH3)=CHCH2-. An alkenyl group may have 2 to 10 carbon atoms. An alkenyl group may also be a "lower alkenyl" having 2 to 6 carbon atoms.

[0024] The term "alkynyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a triple bond. That is, an alkynyl group begins with the atom -C≡CR, where R refers to the rest of the alkynyl group, which may be the same or different. The "R" portion of the alkynyl moiety can be branched, linear, or cyclic. Depending on the structure, an alkynyl group can be a monoradical or a diradical (i.e., an alkynylene group). Alkynyl groups can be optionally substituted. Unrestricted examples of alkynyl groups include, but are not limited to, -C≡CH, -C≡CCH3, -C≡CCH2CH3, -C≡C-, and -C≡CCH2-. Alkynyl groups can have 2 to 10 carbon atoms. Alkynyl groups can also be "lower alkynyls" having 2 to 6 carbon atoms.

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

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

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

[0028] The term "alkylamine" is -N(alkyl) x H y This refers to a group, where x and y are selected from x=1, y=1 and x=2, y=0. In the case of x=2, the alkyl group obtained together with the attached N atom can optionally form a cyclic ring structure.

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

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

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

[0032] As used herein, the term “ring” refers to any covalently closed structure. Rings include, for example, carbocyclic rings (e.g., aryl and cycloalkyl), heterocyclic rings (e.g., heteroaryl and non-aromatic heterocyclic rings), aromatic compounds (e.g., aryl and heteroaryl), and non-aromatic compounds (e.g., cycloalkyl and non-aromatic heterocyclic rings). Rings may be optionally substituted. Rings may be monocyclic or polycyclic.

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

[0034] The term "membered ring" can encompass any cyclic structure. The term "member" is intended to indicate the number of skeletal atoms that make up the ring. For example, cyclohexyl, pyridine, pyran, and thiopyran are 6-membered rings, while cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

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

[0036] The term "carbocyclic formula" or "carbocyclic ring" refers to a ring in which each of the atoms forming the ring is a carbon atom. Carbocyclic rings include aryl and cycloalkyl groups. Therefore, the term distinguishes carbocyclic rings from heterocyclic rings ("heterocyclic formulas") in which the ring backbone contains at least one atom other than carbon (i.e., a heteroatom). Heterocyclic rings include heteroaryl and heterocycloalkyl groups. Carbocyclic rings and heterocyclic rings can be substituted as desired.

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

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

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

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

[0041] The term "cycloalkyl" refers to monocyclic or polycyclic radicals that contain only carbon and hydrogen atoms and can be saturated, partially unsaturated, or completely unsaturated. Cycloalkyl groups include groups with 3 to 10 cyclic atoms. Examples of cycloalkyl groups include the following:

[0042] [ka]

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

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

[0045] The term "heterocyclic" refers to heteroaromatic and heteroalicyclic groups containing 1 to 4 heteroatoms, each selected from O, S, and N, wherein each heterocyclic group has 4 to 10 atoms in its ring system, provided that the ring of the group does not contain two adjacent O or S atoms. In this specification, whenever the number of carbon atoms in a heterocyclic ring is indicated (e.g., C1-C6 heterocyclic ring), there must always be at least one other atom (heteroatom) present in the ring. Designations such as "C1-C6 heterocyclic ring" refer only to the number of carbon atoms in the ring, and not to the total number of atoms in the ring. It is understood that a heterocyclic ring may have additional heteroatoms within the ring. Designations such as "4- to 6-membered heterocyclic ring" refer to the total number of atoms contained in the ring (i.e., a 4-, 5-, or 6-membered ring where at least one atom is a carbon atom, at least one atom is a heteroatom, and the remaining 2 to 4 atoms are carbon atoms or heteroatoms). In heterocyclic compounds having two or more heteroatoms, these two or more heteroatoms may be the same or different from one another. Heterocyclic compounds can be optionally substituted. Bonding to heterocyclic compounds may occur via heteroatoms or via carbon atoms. Non-aromatic heterocyclic groups include groups having only four atoms in their ring system, while aromatic heterocyclic groups must have at least five atoms in their ring system. Heterocyclic groups include benzo-condensed ring systems. An example of a four-membered heterocyclic group is azetidinyl (derived from azetidine). An example of a five-membered heterocyclic group is thiazolyl. An example of a six-membered heterocyclic group is pyridyl, and an example of a ten-membered heterocyclic group is quinolinyl.Examples of non-aromatic heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiacepinyl, and 1,2,3,6-tetrahydropyranyl These include dinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, and quinolidinyl. Examples of aromatic heterocyclic groups include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, sinnolinyl, indazolyl, indolidinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, flazanyl, benzoflazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthylidinyl, and phlopyridinyl. The aforementioned groups, such as those derived from the above groups, may be C- or N- whenever possible. For example, a group derived from pyrrole may be pyrrole-1-yl (with N attached) or pyrrole-3-yl (with C attached). Furthermore, a group derived from imidazole may be imidazol-1-yl or imidazol-3-yl (both with N attached), or imidazol-2-yl, imidazol-4-yl, or imidazol-5-yl (all with C attached). Heterocyclic groups include benzo-condensed ring systems and ring systems substituted with one or two oxo (=O) moieties, such as pyrrolidine-2-one. Depending on the structure, heterocyclic groups may be monoradicals or diradicals (i.e., heterocyclene groups).

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

[0047] [ka]

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

[0049] As used herein, the terms “non-aromatic heterocyclic compound,” “heterocycloalkyl,” or “heteroalicyclic” refer to a non-aromatic ring, where one or more atoms forming the ring are heteroatoms. “Non-aromatic heterocyclic compound” or “heterocycloalkyl” group refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Radicals may be condensed with aryl or heteroaryl groups. Heterocycloalkyl rings may be formed by 3, 4, 5, 6, 7, 8, 9, or more than 9 atoms. Heterocycloalkyl rings are optionally substituted. In certain embodiments, non-aromatic heterocyclic compounds include one or more carbonyl or thiocarbonyl groups, such as an oxygen-containing group and a thio-containing group. Examples of heterocycloalkyls include, but are not limited to, lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, tetrahydrothiopyrans, 4H-pyrans, tetrahydropyrans, piperidines, 1,3-dioxins, 1,3-dioxanes, 1,4-dioxins, 1,4-dioxanes, piperazines, 1,3-oxatian, 1,4-oxathian, 1,4-oxatian, tetrahydro-1,4-thiazines, 2H-1,2-oxazines, maleimides, succinimides, barbiturates, thiobarbiturates, and dioxopiperazines. This includes hydantoin, dihydrouracil, morpholine, trioxane, hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazolin, pyrazolidine, imidazoline, imidazolidine, 1,3-dioxol, 1,3-dioxolane, 1,3-dithiol, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidinone, and 1,3-oxathiolane. Examples of heterocycloalkyl groups, also referred to as non-aromatic heterocycles, include:

[0050] [ka]

[0051] The term "heteroalicyclic" also includes, but is not limited to, all cyclic structures of carbohydrates, including monosaccharides, disaccharides, and oligosaccharides. Depending on the structure, heterocycloalkyl groups can be monoradicals or diradicals (i.e., heterocycloalkylene groups).

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

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

[0054] As used herein, the term “fluoroalkyl” refers to an alkyl group in which at least one hydrogen atom is substituted with a fluorine atom. Examples of fluoroalkyl groups include, but are not limited to, -CF3, -CH2CF3, -CF2CF3, -CH2CH2CF3, etc.

[0055] As used herein, the terms “heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” include optionally substituted alkyl, alkenyl, and alkynyl radicals in which one or more chain atoms of the backbone are heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, or combinations thereof). The heteroatoms may be located at any internal position of the heteroalkyl group or at the position where the heteroalkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-H3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Furthermore, up to two heteroatoms can be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

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

[0057] The term "bond" or "single bond" refers to a chemical bond between two atoms or parts, where the atoms linked by the bond are considered to be part of a larger underlying structure.

[0058] The "isocyanate" group refers to the -NCO group.

[0059] The "isothiocyanate" group refers to the -NCS group.

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

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

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

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

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

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

[0066] "Carboxylate" refers to the -C(O)OH group.

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

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

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

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

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

[0072] As used herein, the terms “N-sulfonamide” or “sulfonylamino” refer to the group of the formula RS(=O)2NH-.

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

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

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

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

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

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

[0079] As used herein, the term “N-amide” refers to the group of the formula RC(=O)NH-.

[0080] As used herein, a substituent "R" appearing alone and without a specified number refers to a substituent selected from alkyl, cycloalkyl, aryl, heteroaryl (bonded via ring carbon) and non-aromatic heterocyclic (bonded via ring carbon).

[0081] The term "optionally substituted" or "substituted" means that the reference group may be substituted by one or more additional groups individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, alkyl sulfoxide, aryl sulfoxide, alkyl sulfone, aryl sulfone, cyano, halo, acyl, nitro, haloalkyl, fluoroalkyl, amino, and monosubstituted and disubstituted amino groups, and their protected derivatives. As an example, the optional substituent is L s R s However, here each L is independently selected from a single bond, -O-, -C(=O)-, -S-, -S(=O)-, -S(=O)2-, -NH-, -NHC(O)-, -C(O)NH-, -S(=O)2NH-, -NHS(=O)2, -OC(O)NH-, -NHC(O)O-, -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); each R is independently selected from H, (substituted or unsubstituted C1-C4 alkyl), (substituted or unsubstituted C3-C6 cycloalkyl), heteroaryl, or heteroalkyl. Protecting groups that can form protected derivatives of the above substituents are known to those skilled in the art and can be found in the above references such as Greene and Wuts.

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

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

[0084] The term "electrophile" or "electrophilicity" refers to a molecule or part of a molecule that is electron-poor or lacking in electrons. Examples of electrophiles include, but are not limited to, the Michael acceptor.

[0085] As used herein, with respect to formulations, compositions, or components, the terms “acceptable” or “pharmaceutically acceptable” mean that they do not have any lasting adverse effects on the overall health of the subject being treated, or that they do not inhibit the biological activity or properties of the compound and are relatively non-toxic.

[0086] As used herein, “B-cell lymphoproliferative disorder (BCLD) biomarker” refers to any biomolecule (found in blood, other bodily fluids, or tissues) or any chromosomal abnormality that is a sign of a BCLD-related disease or disorder.

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

[0088] "Cancer" and "cancer" refer to or describe physiological diseases in mammals typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, B-cell lymphoproliferative disorders (BCLDs) such as lymphoma and leukemia, and solid tumors. "B-cell related cancer" or "B-cell lineage cancer" refers to any type of cancer in which dysregulated or uncontrolled cell proliferation is associated with B cells.

[0089] In the context of cancer, "refractory" means that a particular cancer is resistant to or does not respond to treatment with a specific drug. Cancer may be refractory to treatment with a specific drug throughout the entire period of drug treatment, or during subsequent periods of drug treatment, from the start of treatment with the specific drug (i.e., not responding to initial exposure to the drug), or as a result of progressive resistance to the drug.

[0090] "Agonist activity" means that a substance is intended to function as an agonist. An agonist binds to a receptor on a cell and initiates a reaction or activity similar to, or identical to, one initiated by the receptor's native ligand.

[0091] "Antagonist activity" means that a substance is intended to function as an antagonist. A Btk antagonist prevents or reduces the induction of any of the reactions mediated by Btk.

[0092] "Significant" agonist activity is intended to be at least 30%, 35%, 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 the B-cell response assay. Preferably, "significant" agonist activity is at least twice or at least three times greater than the agonist activity induced by a neutral substance or negative control, as measured in the B-cell response assay. For example, if the B-cell response of interest is B-cell proliferation, "significant" agonist activity is the induction of a level of B-cell proliferation at least twice or at least three times greater than the level of B-cell proliferation induced by a neutral substance or negative control.

[0093] Substances that "lack significant agonist activity" exhibit agonist activity that is at least about 25% greater than the agonist activity induced by a neutral substance or negative control, and preferably at least about 20% greater, 15% greater, 10% greater, 5% greater, 1% greater, 0.5% greater, or even more than about 0.1% greater than the agonist activity induced by a neutral substance or negative control, as measured in a B-cell reaction assay.

[0094] In some embodiments, the Btk inhibitor therapeutic agent is an antagonist anti-Btk antibody. Such an antibody, when bound to the Btk antigen in human cells, does not exhibit the significant agonist activity described above. In one embodiment of the present invention, the antagonist anti-Btk antibody does not exhibit significant agonist activity in the reaction of a single cell. In another embodiment of the present invention, the antagonist anti-Btk antibody does not exhibit significant agonist activity in assays of reactions of one or more cells (e.g., proliferation and differentiation, or, in the case of proliferation, differentiation, and B cells, antibody production).

[0095] "Btk-mediated signaling" refers to biological activity that is directly or indirectly dependent on Btk activity. Examples of Btk-mediated signaling include signals that cause proliferation and survival of Btk-expressing cells, as well as signals that stimulate one or more Btk signaling pathways within Btk-expressing cells.

[0096] The term Btk “signaling pathway” or “signaling pathway” is intended to mean at least one biochemical reaction, or group of biochemical reactions, that generate a signal resulting from Btk activity and, when transmitted through the signaling pathway, causes the activation of one or more downstream molecules in a signaling cascade. Signaling pathways include many signaling molecules that cause the transmission of signals from the cell surface, across the cell's plasma membrane and through one or more of a series of signaling molecules, and in some examples, to the cell's nucleus. Of particular interest to the present invention is the Btk signaling pathway that ultimately controls (enhances or inhibits) NF-κB activation via the NF-κB signaling pathway.

[0097] The present invention relates to a method for treating cancer that, in certain embodiments, utilizes an antibody to determine the expression or presence of a specific BCLD biomarker in a method for treating cancer. The following terms and definitions apply to such antibodies.

[0098] Antibodies and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. The terms are used synonymously. In several cases, the antigen specificity of immunoglobulins is known.

[0099] The term "antibody" is used in its broadest sense and includes fully combined antibodies, antibody fragments capable of binding antigens (e.g., Fab, F(ab')2, Fv, single-chain antibodies, bispecific antibodies (diabodies), antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, etc.), and recombinant peptides including the aforementioned.

[0100] The terms “monoclonal antibody” and “mAb” as used herein refer to antibodies obtained from a substantially homogeneous population of antibodies, i.e., individual antibodies within the population are identical except for the possibility of spontaneously generating mutations that may exist in small amounts.

[0101] "Natural antibodies" and "natural immunoglobulins" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced disulfide bridges within the chain. Each heavy chain has a variable domain (V) at one end, followed by many constant regions. H Each light chain has a variable domain (V) at one end. L The light chain has constant regions at the terminals and other ends; the light chain constant region is aligned with the first constant region of the heavy chain, and the light chain variable domain is aligned with the heavy chain variability domain. Certain amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains.

[0102] The term "variable" refers to the fact that specific portions of the variable domain differ widely in the sequence within an antibody. The variable region confers antigen-binding specificity. However, variability is not uniformly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light and heavy chain variability domains. The more highly conserved portions of the variable domain are called (celled) framework (FR) regions. Each of the natural heavy and light chain variability domains contains four FR regions, primarily employing a β-pleated sheet structure and connected by three CDRs that form loops connecting to the β-pleated sheet structure, and in some cases form parts of it. The CDRs in each chain are held together in close proximity by the FR region, and CDRs from other chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I, pages 647-669). The constant region is not directly involved in antibody-antigen binding, but exhibits various effector functions, such as Fc receptor (FcR) binding, antibody involvement in antibody-dependent cytotoxicity, initiation of complement-dependent cytotoxicity, and mast cell degranulation.

[0103] As used herein, the term "hypervariable region" refers to the amino acid residue of an antibody that is responsible for antigen binding. The hypervariable region includes amino acid residues from the "complementarily determining region" or "CDR" (i.e., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variability domain, and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variability domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md.), and / or residues from the "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variability domain, and (H1), 53-55 (H2), and 96-101 (13) in the heavy chain variability domain; Clothia and Lesk, (1987) J.Mol.Biol., 196:901-917). As used herein, “framework” or “FR” residues are residues of the variability domains other than the residues of the hypervariable regions.

[0104] An "antibody fragment" contains an intact antibody, preferably a portion of the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab, F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies (Zapata et al. (1995) Protein Eng. 10:1057-1062); single-chain antibody molecules; and multiselective antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, each called a "Fab" fragment, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and can further cross-link antigens.

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

[0106] The Fab fragment also includes a constant region of the light chain and a first constant region (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by adding a few residues at the carboxyl endpoint of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the herein designation for Fab', where the cysteine ​​residue in the constant region has a freed thiol group. The Fab' fragment is produced by reducing the heavy chain disulfide crosslinking of the F(ab')2 fragment. Other chemical conjugations of antibody fragments are also known.

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

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

[0109] As used herein, the word “label” refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody in order to produce a “labeled” antibody. A label may be detectable on its own (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical transformation of a detectable substrate compound or composition. As used herein, with respect to a formulation, composition, or component, the terms “acceptable” or “pharmaceutically acceptable” mean that it does not have a persistent adverse effect on the overall health of the subject being treated, or that it does not inhibit the biological activity or properties of the compound and is relatively non-toxic.

[0110] As used herein, the term “agonist” refers to a compound that is responsible for the biological activity of a protein, which is the same as the biological activity that results from the presence of a naturally occurring ligand for that protein, such as Btk.

[0111] As used herein, the term “semi-agonist” refers to a compound that is of the same type as, but lesser in nature, a result of the presence of a naturally occurring ligand for a protein, but which contributes to the bioactivity of the protein.

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

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

[0114] The term “Bruton’s tyrosine kinase homolog” means, as used herein, an orthologue of a Bruton’s tyrosine kinase, e.g., an orthologue from any of the aforementioned fusion proteins that exhibit kinase activity for one or more substrates of a Bruton’s tyrosine kinase (e.g., a peptide substrate having the amino acid sequence “AVLESEEELYSSARQ”), such as mouse (GenBank Accession No. AAB47246), dog (GenBank Accession No. XP_549139), rat (GenBank Accession No. NP_001007799), chicken (GenBank Accession No. NP_989564), or zebrafish (GenBank Accession No. XP_698117), or Bruton’s tyrosine kinase.

[0115] The terms “concurrent administration” or “combined administration,” as used herein, mean the administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which the therapeutic agents are administered by the same or different routes of administration or at the same or different times.

[0116] The term “effective dose” means, as used herein, a sufficient amount of a Btk inhibitor or Btk inhibitor compound administered that results in an increase or appearance of a subgroup of lymphocytes (e.g., a reduction in the dosage of a drug). For example, for diagnostic and / or prognostic use, “effective dose” is the amount of a composition containing a compound as disclosed herein that is required to clinically adequately reduce the increase or appearance of a subgroup of lymphocytes without excessive adverse side effects. An appropriate “effective dose” in any individual case may be determined using techniques such as dose escalation studies.

[0117] The term “therapeutically effective dose” means, as used herein, a sufficient amount of a drug or compound administered to alleviate to some extent one or more symptoms of B-cell lymphoproliferative disorder (BCLD). This may result in a reduction and / or mitigation of the signs, symptoms, or causes of BCLD, or any other desired change in the biological system. The term “therapeutically effective dose” includes, for example, a dose effective for prevention. The “effective dose” of any compound disclosed herein is the amount effective in achieving the desired pharmacological effect or therapeutic improvement without excessive adverse side effects. It is understood that the “effective dose” or “therapeutically effective dose” may vary from subject to subject due to changes in the metabolism of any compound of formula (A), formula (B), formula (C), or formula (D), the subject’s age, weight, general condition, the disease being treated, the severity of the disease being treated, and the prescribing physician’s judgment. As just one example, the therapeutically effective dose may be determined by routine experimentation, including, but not limited to, dose-increase clinical trials.

[0118] The terms “enhance” or “enhancing” mean increasing or extending a desired effect, either in terms of potency or duration. For example, “enhancing” the effect of a therapeutic agent refers to the ability to increase or extend the effect of the therapeutic agent during the treatment of a disease, disorder, or illness, either in terms of potency or duration. As used herein, the term “effective amount for enhancement” refers to an appropriate amount for enhancing the effect of a therapeutic agent in the treatment of a disease, disorder, or illness. When administered to a patient, the effective amount for this use will depend on the severity and course of the disease, disorder, or illness, previous treatments, the patient’s health condition and response to the drug, and the judgment of the treating physician.

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

[0120] As used herein, the term “identical” refers to two or more sequences or subsequences that are the same. In addition, as used herein, the term “substantially identical” refers to two or more sequences that have the same percentage of sequence units when compared and repositioned for maximum match across a comparison window, or when specifying a region measured using a comparison algorithm or by manual alignment and visual inspection. As just one example, two or more sequences are “substantially identical” if their consecutive units are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical over a specified region. The percentages are for describing the “percentage identity” of two or more sequences. Sequence identity may exist over a region of at least approximately 75–100 consecutive units, over a region of approximately 50 consecutive units, or, not specifically specified, over the entire sequence. This definition also refers to the complement of a test sequence. As just one example, when two or more polypeptide sequences are identical when they have the same amino acid residues, they are considered "substantially identical" if the amino acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical in a specified region. The identity may be over a region of at least 75–100 amino acids, over a region of about 50 amino acids, or, not specifically specified, over the entire polypeptide sequence. Furthermore, as just one example, when two or more polynucleotide sequences are identical when they have the same nucleic acid residues, they are considered "substantially identical" if the nucleic acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical in a specified region. Identity may exist in a region of at least approximately 75–100 nucleic acid lengths, in a region of approximately 50 nucleic acid lengths, or, not specifically, across the entire sequence of the polynucleotide.

[0121] As used herein, the terms “inhibit,” “suppress,” or “inhibitor” in relation to kinases refer to the inhibition of the phosphate transferase activity of an enzyme.

[0122] As used herein, the term “irreversible inhibitor” refers to a compound that, upon contact with a target protein (e.g., kinase), causes the formation of new covalent bonds in or within the protein, thereby reducing or neutralizing one or more of the target protein’s biological activities (e.g., phosphate transferase activity) despite the subsequent presence or absence of the irreversible inhibitor.

[0123] As used herein, the term “irreversible Btk inhibitor” refers to an inhibitor of Btk that can form a covalent bond with an amino acid residue of Btk. In one embodiment, an irreversible inhibitor of Btk can form a covalent bond with a cysteine ​​residue of Btk. In a particular embodiment, an irreversible inhibitor can form a covalent bond with the cysteine ​​481 residue of Btk (or its homologue) or with a cysteine ​​residue at a corresponding position in a homologue of another tyrosine kinase.

[0124] As used herein, the term “isolated” refers to the separation and removal of a component of interest from a component of no interest. The separated substance may exist in a dry, semi-dried state, or in a solution, including but not limited to aqueous solutions. The separated component may exist in a homogeneous state, or it may become part of a pharmaceutical composition including additional pharmaceutically acceptable carriers and / or excipients. As just one example, a nucleic acid or protein is “isolated” when it is not present in at least some of the cellular components that naturally accompany it, or when the nucleic acid or protein is concentrated to a level higher than its in vivo or in vitro production concentration. Also as just one example, a gene is isolated when it is separated from a read frame that protects (flanks) the gene and codes for proteins other than the gene of interest.

[0125] The “metabolites” of the compounds disclosed herein are derivatives of the compound formed when the compound is metabolized. The term “active metabolite” refers to a biologically active derivative of the compound formed when the compound is metabolized. As used herein, the term “metabolism” refers to the entire process by which an organism transforms a particular substance, including, but not limited to, enzyme-catalyzed reactions such as hydrolysis and oxidation. Enzymes can thus bring about specific structural changes in a compound. For example, cytochrome P450 catalyzes a variety of oxidation and reduction reactions, while uridine diphosphate glucuronyltransferase catalyzes the transfer of activated glucuronic acid molecules to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free sulfhydryl groups. Further information on metabolism can be found in The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of the compounds disclosed herein can be identified by either administering the compounds to a host and analyzing tissue samples from the host, or by in vitro incubation of the compounds by hepatocytes and analysis of the resulting compounds. Both methods are well known in the art. In some embodiments, metabolites of the compounds are formed by an oxidation process and correspond to the corresponding hydroxyl-containing compounds. In some embodiments, the compounds are metabolized to pharmacologically active metabolites.

[0126] The term "modulate" as used herein means interacting with a target, either directly or indirectly, in such a way as to alter the activity of the target, including, but not limited to, enhancing, inhibiting, limiting, or expanding the activity of the target.

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

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

[0129] As used herein, the term “selectively binding” refers to the ability of a selectively binding compound to bind to a target protein, such as Btk, with an affinity higher than that to a non-target protein. In certain embodiments, selective binding refers to binding to a target with an affinity at least 10, 50, 100, 250, 500, 1000 times higher or greater than the affinity to a non-target protein.

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

[0131] As used herein, the term “well purified” means a component of interest that is sufficiently or substantially free of other components that would normally be associated with or interact with the component of interest before purification. As just one example, a component of interest may be “well 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 1% of contaminants (relative to dry weight). Thus, a “well purified” component of interest may have a purification level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or higher.

[0132] As used herein, the term “subject” refers to an animal that is the subject of treatment, observation, or experimentation. For example, a subject may be a mammal, but is not limited to humans.

[0133] As used herein, the term “target activity” refers to a biological activity that has the ability to be regulated by a selective modulator. Specific examples of targeted activity include, but are not limited to, effects on specific biomarkers relating to binding affinity, signaling, enzyme activity, tumor growth, and the pathology of B-cell lymphocyte proliferative disorders.

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

[0135] The terms “to treat,” “to treat,” or “treatment” as used herein include reducing or improving a disease or illness or its symptoms; managing a disease or illness or its symptoms; preventing further symptoms; improving or preventing the underlying metabolic cause of symptoms; inhibiting a disease or illness, e.g., preventing its progression; alleviating a disease or illness; causing recovery from a disease or illness; alleviating a condition caused by a disease or illness; or stopping the symptoms of a disease or illness. The terms “to treat,” “to treat,” or “treatment” include, but are not limited to, preventive and / or therapeutic treatments.

[0136] As used herein, IC 50 This refers to the amount, concentration, or dose of a specific test compound that achieves 50% inhibition of the maximum reaction, such as Btk inhibition, in the assay for measuring the aforementioned reaction.

[0137] As used herein, EC 50 This refers to the dose, concentration, or amount of a specific test compound that elicits, triggers, or enhances a dose-dependent response at 50% of the maximum expression of a particular response.

[0138] <Hematological malignancies> Disclosed herein is a method for treating a hematological malignancy in an individual requiring it, comprising the steps of: (a) administering to the individual an amount of an irreversible Btk inhibitor sufficient to pharmacokineticate multiple cells from the malignancy; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignancy. In some embodiments, the hematological malignancy is CLL. In some embodiments, the step of analyzing the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic multiple cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after the concentration of the pharmacokinetic multiple cells has subsequently decreased. In some embodiments, the step of analyzing the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of multiple pharmacokinetic cells has increased over a predetermined period of time. In some embodiments, the step of analyzing the multiple pharmacokinetic cells includes counting the number of multiple pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of multiple pharmacokinetic cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the step of administering the second cancer treatment regimen occurs after the number of multiple pharmacokinetic cells in the peripheral blood has subsequently decreased. In some embodiments, the step of analyzing the multiple pharmacokinetic cells includes measuring the duration of the increase in the number of multiple pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of multiple pharmacokinetic cells in the peripheral blood has increased over a predetermined period of time.In some embodiments, the hematological malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, or non-CLL / SLL lymphoma. In some embodiments, the hematological malignancy is follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Valdenström macroglobulinemia, multiple myeloma, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, or extranodular marginal zone B-cell lymphoma. In some embodiments, the hematological malignancy is acute or chronic myeloid (or spinal) leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia. In some embodiments, hematological malignancies include relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or refractory CLL; relapsed or refractory SLL; and relapsed or refractory multiple myeloma. In some embodiments, hematological malignancies are hematological malignancies classified as high-risk. In some embodiments, hematological malignancies are high-risk CLL or high-risk SLL.

[0139] B-cell lymphoproliferative disorders (BCLDs) are newer hematological tumors, encompassing, among others, non-Hodgkin lymphoma, multiple myeloma, and leukemia. BCLDs can arise from lymphoid tissue (as in lymphomas) or bone marrow (as in leukemia and myeloma). They are all related to either unrestricted growth of lymphocytes or leukocytes. Many subtypes of BCLD exist (e.g., chronic lymphocytic leukemia (NHL), non-Hodgkin lymphoma (CLL)). Disease strategies and treatments for BCLD depend on the BCLD subtype; however, even within each subtype, clinical manifestations, morphological aspects, and responses to treatment are heterogeneous.

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

[0141] Non-Hodgkin lymphoma This specification describes methods for treating non-Hodgkin lymphoma in particular embodiments, depending on the needs, and comprising (a) administering to an individual a sufficient amount of an irreversible Btk inhibitor to phagocytosis of multiple cells from the malignant tumor; and (b) analyzing the phagocytosed multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor present is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, hematological malignancies are present with apogosipol. In some embodiments, the analysis of the phagocytosed multiple cells comprises measuring the peripheral blood concentration of the phagocytosed multiple cells. In some embodiments, the method further comprises administering a second cancer treatment regimen compared to the peripheral blood concentration of the phagocytosed multiple cells after administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a decrease in the peripheral blood concentration of the phagocytosed multiple cells. In some embodiments, the analysis of the phagocytosed multiple cells comprises measuring the duration of the increase in the peripheral blood concentration of the phagocytosed multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of pharmacokinetic cells has increased in the peripheral blood compared to the concentration before administration of a Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes measuring the period of increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of a Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0142] Furthermore, in certain embodiments disclosed herein are methods for treating relapsed or refractory non-Hodgkin lymphoma in individuals requiring such treatment, (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one; in some embodiments, non-Hodgkin lymphoma can be relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, or relapsed or refractory follicular lymphoma.

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

[0144] An unspecified list of B-cell NHLs includes Burkitt's lymphoma (e.g., endemic Burkitt's lymphoma and sporadic Burkitt's lymphoma), cutaneous B-cell lymphoma, cutaneous marginal zone lymphoma (MZL), diffuse large cell lymphoma (DLBCL), diffuse mixed small cell and large cell lymphoma, diffuse small cleaved cell lymphoma, diffuse small lymphocytic lymphoma, extranodal marginal zone B-cell lymphoma, follicular lymphoma, and follicular small cleaved cell follicular lymphoma. Cell) (Grade 1), Mixed type of small incised nuclear cell lymphoma and large cell follicular lymphoma (Grade 2), Large cell follicular lymphoma (Grade 3), Intravascular Large B-Cell Lymphoma, Intravascular Lymphomatosis, Large Immunoblastic Lymphoma, Large Cell Lymphoma (LCL), Lymphoblastic Lymphoma, MALT Lymphoma, Mantle Cell Lymphoma (MCL), Immunoblastic Large Cell Lymphoma, Progenitor B-Lymphoblastic Lymphoma, Mantle Cell Lymphoma, Chronic Lymphocytic Leukemia (CLL) / Small Lymphocyte Lymphoma (SLL), Extranodal Marginal B-Cell Lymphoma, Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma, Mediastinal Large B-Cell Lymphoma B-cell lymphoma, marginal B-cell lymphoma of the lymph nodes, marginal B-cell lymphoma of the spleen, B-cell lymphoma of the first mediastinal region, lymphoplasmacytic lymphoma, hairy cell leukemia, Valdenström macroglobulinemia, and first central nervous system (CNS) lymphoma. Additional non-Hodgkin lymphomas have been considered within the scope of this invention and are apparent and present to those skilled in the art. DLBCL

[0145] This specification discloses a method for treating DLCBL in an individual as required in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to pharmacokineticate multiple cells from a malignant tumor; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from a malignant tumor. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic multiple cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the period of increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0146] As used herein, the term “diffuse B-cell lymphoma (DLBCL)” refers to a tumor of germinal center B lymphocytes with a diffuse growth pattern and a high intermediate proliferation index. DLBCL accounts for approximately 30% of all lymphomas and can exhibit several morphological variations, including germinal center cells, immunoblasts, T cells / histiocytes, undifferentiated subtypes, and plasmablast subtypes. Genetic testing reveals the existence of different subtypes of DLBCL. These subtypes appear to have different appearances (prognoses) and responses to treatment. DLBCL can affect any age group, but it most commonly occurs in older individuals (average age is mid-60s).

[0147] This specification discloses a method for treating a subtype of diffuse large B-cell lymphoma, such as activated B-cell lymphoma (ABC-DLBCL), in individuals requiring it in certain embodiments, comprising the step of administering an irreversible Btk inhibitor to the individual in amounts ranging from 300 mg / day to 1000 mg / day (including 1000 mg / day). The ABC subtype of diffuse large B-cell lymphoma (ABC-DLBCL) is thought to arise from post-germinal center B cells that are inhibited during plasma differentiation. The ABC subtype of DLBCL (ABC-DLBCL) accounts for approximately 30% of DLBCL diagnoses in total. The DLBCL subtype and the least likely to cure patients diagnosed with ABC-DLBCL as such typically show significantly reduced survival rates compared to individuals with other types of DLBCL. ABC-DLBCL is generally associated with chromosomal translocations that de-regulate the germinal center master regulator BCL6, and mutations that inactivate the PRDM1 gene, which encodes a transcriptional repressor necessary for plasma cell differentiation.

[0148] The signaling pathway particularly relevant to the developmental mechanism of ABC-DLBCL is the signaling pathway mediated by nuclear factor (NF)-κB transcription complexes. The NF-κB family consists of five members (p50, p52, p65, c-rel, and RelB), which form homodimers and heterodimers and function as transcriptional factors mediating various proliferation, apoptosis, inflammatory, and immune responses, and are also of crucial importance for normal B cell development and survival. NF-κB is widely used by eukaryotic cells as a regulator of genes that control cell proliferation and cell survival. Therefore, various types of human tumors have misregulated NF-κB; that is, NF-κB is inherently active. Active NF-κB initiates the expression of genes that promote cell proliferation and protect cells from disease that would otherwise lead to apoptosis and death.

[0149] The dependence of ABC DLBCL on NF-κB depends on the upstream signaling pathway of IκB kinase, which is composed of CARD11, BCL10, and MALT1 (CBM complex). Interference with the CBM pathway eliminates NF-κB signaling in ABC DLBCL cells and induces apoptosis. While the molecular basis of constitutive activity of the NF-κB pathway is the subject of current investigation, several somatic mutations in the ABC DLBCL genome clearly activate this pathway. For example, somatic mutations in the multi-coil domain of CARD11 in DLBCL can render this signaling scaffold protein capable of spontaneously forming nuclei from protein-protein interactions with MALT1 and BCL10, resulting in IκB activity and NF-κB activation. Constitutive activity of the B cell receptor signaling pathway influences NF-κB activation in ABC DLBCL with wild-type CARD11, which is related to mutations inside the cytoplasmic tail of the B cell receptor subunits CD79A and CD79B. Tumor-activating mutations within the signaling adapter MYD88 activate NF-κB, synergistically influencing B cell receptor signaling in maintaining the survival of ABC DLBCL cells. In addition, mutations that inactivate the NF-κB pathway (A20) as a negative modulator occur almost exclusively in ABC DLBCL.

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

[0151] DLBCL cells of ABC subtypes, such as OCI-Ly10, possess chronic active BCR signaling and are highly sensitive to the Btk inhibitors described herein. The irreversible Btk inhibitors described herein inhibit the growth of OCI-Ly10 (continuous EC50 exposure = 10 nM, EC50 (1-hour pulse) = 50 nM). In addition, induction of apoptosis and caspase activity are observed in OCI-Ly10, as indicated by annexin-V flow cytometry and increase in the sub-G0 fraction. Both sensitive and resistant cells express Btk at similar levels, and the active site of Btk is fully occupied by the inhibitor, as indicated using a fluorescently labeled affinity probe. OCI-Ly10 cells are shown to possess chronically active BCR signaling to NF-κB present at doses that are dependently inhibited by the Btk inhibitors described herein. The activity of Btk inhibitors in the cell lines studied in this specification was characterized by a comparison of signaling characteristics (Btk, PLCγ, ERK, NF-κB, AKT), cytokine secretion characteristics, and mRNA expression characteristics in both BCR-stimulated and unstimulated cells. Significant differences were observed in these characteristics, and these significant differences lead to clinical biomarkers for identifying patients most sensitive to Btk inhibitor treatment. See U.S. Patent No. 7,711,492 and Staudt et al., Nature, Vol. 463, January 7, 2010, pp. 88-92. These contents are incorporated herein by full citation.

[0152] <Follicular lymphoma> This specification discloses a method for treating follicular lymphoma in an individual, which, in certain embodiments, requires, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from the malignant tumor; and (b) analyzing the phagocytised multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of the phagocytised multiple cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytised multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0153] As used herein, the term “follicular lymphoma” refers to one of several types of non-Hodgkin lymphoma in which the lymphoma cells are divided into clusters of nodules or vesicles. The term follicular is used because the cells tend to grow in an annular pattern within the lymph node, or in a pattern in which nodules are present. The average age of people with this lymphoma is about 60 years.

[0154] <CLL / SLL> This specification discloses methods for treating CLL or SLL in an individual as required in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to pharmacokineticate multiple cells from a malignant tumor; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, CLL or SLL is high-risk. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from a malignant tumor. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic multiple cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to a number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

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

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

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

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

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

[0160] SLL is painless, but it is constantly progressive. The usual pattern of this disease is one of high response rates to radiotherapy and / or chemotherapy during periods of disease remission, followed by months or years of inevitable relapse. Retreatment leads to another response, but again, the disease relapses. This means that while the short-term prognosis for SLL is quite good, over time, many patients develop the fatal complication of relapsing disease. Typically, given the age of individuals diagnosed with CLL and SLL, there is a need for a simple and effective treatment technique for the disease with minimal side effects that do not interfere with the patient's quality of life. This invention meets this long-term, ongoing need for such a technique.

[0161] <Mantle cell lymphoma> This specification discloses a method for treating mantle cell lymphoma in an individual in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to pharmacokineticate multiple cells from the malignant tumor; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to a pre-administration concentration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to a pre-administration concentration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the period of increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0162] The term “mantle cell lymphoma” as used herein refers to a subtype of B-cell lymphoma caused by CD5-positive antigen-naive pregarminal center B cells within the mantle zone surrounding normal germinal center follicles. MCL cells generally overexpress cyclin D1 due to a t(11:14) chromosomal translocation in their DNA. More specifically, the translocation is located at t(11;14)(q13;q32). Only about 5% of lymphomas are of this type. The cells are small in size in culture medium. Males are most often affected. The average age of patients is the early 1960s. Lymphoma is usually widespread when diagnosed, as it involves lymph nodes, bone marrow, and often the spleen. Mantle cell lymphoma is not a very rapidly growing lymphoma, but it is difficult to treat.

[0163] <Marginal B-cell lymphoma> This specification discloses a method for treating marginal B-cell lymphoma in an individual in which it is required in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to pharmacokineticate multiple cells from the malignant tumor; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic multiple cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0164] As used herein, the term “marginal B-cell lymphoma” refers to a group of related B-cell tumors, which include lymphoid tissue in the marginal region, a patchy area outside the mantle zone of the follicles. Marginal lymphomas account for approximately 5% to 10% of all lymphomas. The cells in these lymphomas appear small under a microscope. There are three main types of marginal lymphoma, including extranodal marginal B-cell lymphoma, lymph node marginal B-cell lymphoma, and splenic marginal lymphoma.

[0165] <Mucosal-associated lymphoid tissue (MALT)> This specification discloses a method for treating mucosal-associated lymphoid tissue of an individual, which, in certain embodiments, requires, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from a malignant tumor; and (b) analyzing the phagocytised multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from a malignant tumor. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0166] The term “mucosa-associated lymphoid tissue (MALT) lymphoma” as used herein refers to the extranodal manifestation of marginal lymphoma. Most MALT lymphomas are low-grade, although a small number initially present as intermediate-grade non-Hodgkin lymphoma (NHL) or develop from lower-grade forms. Most MALT lymphomas occur in the stomach. Approximately 70% of gastric MALT lymphomas are associated with Helicobacter pylori infection. Several cytogenetic abnormalities have been identified, the most common being trisomy 3 or t(11;18). Many of these other MALT lymphomas have also been associated with bacterial or viral infections. The average age of patients with MALT lymphoma is approximately 60.

[0167] <Marginal B-cell lymphoma> This specification discloses a method for treating marginal B-cell lymphoma in an individual, which, in certain embodiments, requires, comprising: (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from the malignant tumor; and (b) analyzing the phagocytised multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0168] The term "marginal B-cell lymphoma" refers to painless B-cell lymphoma, usually found in the lymph nodes. The disease is rare, accounting for only 1% of all non-Hodgkin lymphomas (NHL). It is most commonly diagnosed in older patients, particularly in women who are more susceptible than men. Because the mutation occurs at the periphery of the B cells, the disease is classified as a marginal lymphoma. Due to its limited presence within the lymph nodes, it is also classified as a nodular lymphoma.

[0169] <Splenic marginal B-cell lymphoma> This specification discloses a method for treating marginal B-cell lymphoma of the spleen in an individual, which, in certain embodiments, requires, comprising: (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from the malignant tumor; and (b) analyzing the phagocytised multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the phagocytised multiple cells compared to a pre-administration concentration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytised multiple cells compared to a pre-administration concentration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0170] The term "marginal B-cell lymphoma of the spleen" refers to a specific, low-grade B-cell lymphoma included in the World Health Organization classification. Its characteristics include moderate lymphocytosis of splenomegaly, a villous morphology, sinusoidal capillary patterns in various organs (particularly the bone marrow), and a relatively painless course. Tumor development and aggressive behavior, with increased blast morphology, are observed in a small number of patients. Molecular and cytogenetic studies have shown heterogeneous results, perhaps due to the lack of standardized diagnostic criteria.

[0171] Burkitt lymphoma This specification discloses a method for treating Burkitt lymphoma in an individual, as required in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from the malignant tumor; and (b) analyzing the phagocytized multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the phagocytized multiple cells includes measuring the peripheral blood concentration of the phagocytized multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration in the multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the phagocytized multiple cells. In some embodiments, the analysis of the phagocytized multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytized multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0172] The term "Burkitt lymphoma" generally refers to a type of non-Hodgkin lymphoma (NHL) that affects children. It is a highly aggressive form of B-cell lymphoma that frequently begins and includes parts of the body other than lymph nodes. Despite its rapidly growing nature, Burkitt lymphoma is often curable with modern intensive care. There are two broad forms of Burkitt lymphoma: a sporadic variant and an indigenous variant.

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

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

[0175] Waldenström macroglobulinemia This specification discloses a method for treating Waldenström macroglobulinemia in an individual as required in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from a malignant tumor; and (b) analyzing the phagocytised multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from a malignant tumor. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0176] The term "Waldenström macroglobulinemia," also known as lymphoplasmacytic lymphoma, is a cancer related to a subtype of white blood cell called lymphocytes. It is characterized by the unsuppressed proliferation of ultimately differentiated B lymphocytes. It is also characterized by lymphoma cells that produce antibodies called immunoglobulin M (IgM). IgM antibodies circulate in large quantities in the blood, thickening the fluid portion of the blood like syrup. This leads to reduced blood flow to many organs and can cause problems with vision (due to poor blood flow in the blood vessels behind the eyes) and neurological problems (such as headaches, dizziness, and confusion) caused by poor blood flow in the brain. Other symptoms may include feeling tired and weak, and a tendency to bleed easily. The underlying etiology is not fully understood, but many risk factors have been identified, including location 6p21.3 on chromosome 6. There is a 2-3 times increased risk of developing WM in individuals with autoantibodies related to hepatitis, human immunodeficiency virus, and rickettsial diseases, as well as a personal history of autoimmune diseases, particularly those at high risk.

[0177] Multiple myeloma This specification discloses a method for treating myeloma in an individual, which, in certain embodiments, requires, comprising: (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to phagocytose multiple cells from the malignant tumor; and (b) analyzing the phagocytised multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a decrease in the peripheral blood concentration of the phagocytised multiple cells. In some embodiments, the analysis of the phagocytised multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the phagocytised multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0178] This specification discloses a method for treating multiple myeloma in an individual in certain embodiments, comprising the steps of (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to pharmacokineticate multiple cells from the malignant tumor; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to a concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0179] Multiple myeloma, also known as MM, myeloma, plasma cell myeloma, or Karel's disease (post-Otto Köhler), is a cancer of white blood cells known as plasma cells. A type of B cell, plasma cells are a vital part of the immune system present in humans and other vertebrates, responsible for antibody production. They are produced in the bone marrow and transported by the lymphatic system.

[0180] <leukemia> This specification discloses a method for treating leukemia in an individual, as required in certain embodiments, comprising: (a) administering to the individual a sufficient amount of an irreversible Btk inhibitor to pharmacokineticate multiple cells from a malignant tumor; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from a malignant tumor. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after an increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the analysis of the pharmacokinetic multiple cells includes measuring the duration of the increase in the peripheral blood concentration of the pharmacokinetic multiple cells compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined length of time. In some embodiments, the analysis of pharmacokinetic cells includes the step of counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after an increase in the number of pharmacokinetic cells in the peripheral blood compared to the concentration of the Btk inhibitor before administration. In some embodiments, the administration of the second cancer treatment regimen occurs after a subsequent decrease in the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the analysis of pharmacokinetic cells includes the step of measuring the duration of the increase in the number of pharmacokinetic cells in the peripheral blood compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes the step of administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined length of time.

[0181] Leukemia is a cancer of the blood or bone marrow characterized by an abnormal increase in blood cells, usually white blood cells (leukocytes). Leukemia is a broad term covering a range of diseases. Between its acute and chronic forms lie: (i) Acute leukemia is characterized by a rapid increase in immature blood cells. This proliferation causes the bone marrow to produce healthy blood cells. Emergency treatment is required in acute leukemia due to the rapid progression and accumulation of malignant cells, which then enter the bloodstream and spread to other organs of the body. The acute form of leukemia is the most common form of leukemia in children; (ii) Chronic leukemia is identified by an excess of relatively mature but still abnormal white blood cells. Typically, it takes months or years to progress, and the cells are produced at a much faster rate than normal cells, resulting in a large number of abnormal white blood cells in the blood. Chronic leukemia usually occurs in older people, but theoretically it can occur in any age group. Furthermore, the disease is subdivided according to the type of blood cells affected. This division separates lymphoblastic or lymphocytic leukemia from myeloid or myeloid leukemia: (i) Lymphoblastic or lymphocytic leukemia, cancerous changes occur in a type of myeloid cell that normally forms lymphocytes, which fight against cells of the immune system; (ii) Myeloid or myeloid leukemia, cancerous changes occur in a type of myeloid cell that normally forms red blood cells, several other types of white blood cells and platelets.

[0182] Within these main categories, there are several subcategories that are not limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and hairy cell leukemia (HCL).

[0183] <Btk inhibitors> Furthermore, this specification presents, as an example, a method for treating cancer such as BCLD in subjects, in which subjects were treated with a regimen of oral Btk inhibitor drugs. In the following descriptions of reversible Btk compounds suitable for use in the methods described herein, definitions of standard chemical terms can be found in reference materials, including Carey and Sundberg, "Advanced Organic Chemistry 4th Ed. Vols. A (2000) and B (2001), Plenum Press, New York" (unless otherwise defined herein). Unless otherwise specified, conventional methods of mass spectrometry, NMR, HPCL, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used by those skilled in the art. Furthermore, nucleic acid and amino acid sequences for Btk (e.g., human Btk) are known by art, for example, as disclosed in U.S. Patent No. 6,326,469. Unless otherwise specified, the technical terms used herein in relation to analytical chemistry, organic synthesis chemistry, and medicinal chemistry and pharmaceutical chemistry, as well as the methods and techniques thereof, are known and exist in the art. Standard techniques may be used in chemical synthesis, chemical analysis, preparation, formulation, and delivery of pharmaceuticals, and in patient care.

[0184] The Btk inhibitor compounds described herein are selectively present for Btk and kinases that have a cysteine ​​residue in the amino acid sequence position of a tyrosine kinase homologous to the amino acid sequence position of cysteine ​​481 in Btk. Generally, irreversible inhibitory compounds of Btk used in the methods described herein are identified or characterized in an in vitro assay, such as a non-cellular biochemical assay or a cellular functional assay. The assay is an in vitro IC for irreversible Btk inhibitory compounds. 50 It is useful for measuring [something].

[0185] For example, cell-free kinase assays are used to measure Btk activity after incubation of kinases lacking or presenting various concentrations of candidate irreversible Btk inhibitor compounds. If the candidate compound is indeed an irreversible Btk inhibitor, Btk kinase activity will not be recovered by repeated washing with inhibitor-free media. See, for example, JBSmaill et al. (1999), J.Med.Chem. 42(10):1803-1815. Furthermore, the covalent complex composition between Btk and a candidate irreversible Btk inhibitor is a useful indicator of irreversible inhibition of Btk, and can be readily measured by many methods known in the art (e.g., mass spectrometry). For example, some irreversible Btk inhibitors form covalent bonds with the aforementioned cysteine ​​residues (e.g., via the Michael reaction).

[0186] Assays of cellular function for Btk inhibition involve measuring one or more cellular endpoints in response to the stimulation of Btk-mediated pathways in a cell line (e.g., BCR activation in Ramos cells) in the absence or presence of various concentrations of candidate irreversible Btk inhibitor compounds. Useful endpoints for measuring responses to BCR activation include, for example, Btk autophosphorylation, phosphorylation of Btk target proteins (e.g., PLC-γ), and cytoplasmic calcium flux.

[0187] High-productivity assays for numerous cell-free biochemical assays (e.g., kinase assays) and cellular functional assays (e.g., calcium efflux) are well known to those skilled in the art. Furthermore, high-throughput 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 the entire procedure, including pipetting of all samples and reagents in microplates with detectors appropriate for the assay, liquid preparation, timed incubation, and final numerical readings. Automated systems thereby enable the identification and characterization of numerous irreversible Btk compounds without excessive effort.

[0188] In some embodiments, the Btk inhibitor is selected from the group consisting of small organic molecules, macromolecules, peptides, or non-peptides.

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

[0190] In some embodiments, irreversible Btk inhibitors form covalent bonds having cysteine ​​side chains of Bruton-type tyrosine kinase, a Bruton-type tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog.

[0191] Irreversible Btk inhibitor compounds can be used in the manufacture of drugs to treat any of the aforementioned conditions (e.g., autoimmune diseases, inflammatory diseases, allergic disorders, B cell proliferation disorders, or thromboembolic disorders).

[0192] In some embodiments, the irreversible Btk inhibitor compound used for the methods described herein is Btk or less than 10 μM in vitro IC50. 50 It inhibits Btk homologous kinase activity (e.g., <1 μM, <0.5 μM, <0.4 μM, <0.3 μM, <0.1 μM, <0.08 μM, <0.06 μM, <0.05 μM, <0.04 μM, <0.03 μM, <0.02 μM, <0.01 μM, <0.008 μM, <0.006 μM, <0.005 μM, <0.004 μM, <0.003 μM, <0.002 μM, <0.001 μM, <0.00099 μM). (Less than 0.00098 μM, Less than 0.00097 μM, Less than 0.00096 μM, Less than 0.00095 μM, Less than 0.00094 μM, Less than 0.00093 μM, Less than 0.00092 μM, or Less than 0.00090 μM) In one embodiment, an irreversible Btk inhibitor selectively and irreversibly inhibits the activated form of its target tyrosine kinase (e.g., a phosphorylated form of the tyrosine kinase). For example, activated Btk is transphosphorylated at tyrosine 551. Thus, in these embodiments, once the target kinase is activated by a signaling event, the irreversible Btk inhibitor inhibits the target kinase within the cell.

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

[0194] Equation (A) is as follows:

[0195] [ka]

[0196] In the formula, A is selected independently of N or CR5; R1 is H, L2-(substituted or unsubstituted alkyl), L2-(substituted or unsubstituted cycloalkyl), L2-(substituted or unsubstituted alkenyl), L2-(substituted or unsubstituted cycloalkenyl), L2-(substituted or unsubstituted heterocyclic compound), L2-(substituted or unsubstituted heteroaryl), L2-(substituted or unsubstituted aryl), where L2 is a single bond, O, S, -S(=O), -S(=O)2, C(=O), -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); R2 and R3 are independently selected from H, lower alkyl, and substituted lower alkyl; R4 is L3-X-L4G, where L3 is optional and, if present, a single bond, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl; X is optional and, if present, can be a single bond, O, -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, -NR9S(=O)2, -OC(O)NH-, -NHC(O)O, -OC(O)NR9, -NR9C(O)O, -CH=NO-, -ON=CH-, -NR 10 C(O)NR 10 -, heteroaryl, aryl, -aryl 10 C(=NR11)NR 10 -, - NR 10 C(=NR 11 )-, -C(=NR 11 )NR 10 -, -OC(=NR 11 )-, or -C(=NR 11 )O- and; L4 is optional and, if present, is a single bond, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted biheterocyclic compound; Alternatively, the L3, X, and L4 obtained together form a nitrogen atom containing a heterocycle; G is

[0197] [ka]

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

[0199] In one embodiment, a compound having the structure of formula (A1) is disclosed, Equation (A1) is,

[0200] [ka]

[0201] And, A is selected independently of N or CR5; R1 is H, L2-(substituted or unsubstituted alkyl), L2-(substituted or unsubstituted cycloalkyl), L2-(substituted or unsubstituted alkenyl), L2-(substituted or unsubstituted cycloalkenyl), L2-(substituted or unsubstituted heterocyclic compound), L2-(substituted or unsubstituted heteroaryl), L2-(substituted or unsubstituted aryl), where L2 is a single bond, O, S, -S(=O), -S(=O)2, C(=O), -(substituted or unsubstituted C1-C6 alkyl), or -(substituted or unsubstituted C2-C6 alkenyl); R2 and R3 are independently selected from H, lower alkyl groups, and substituted lower alkyl groups; R4 is L3-X-L4-G, where L3 is optional and, if present, is an optional substituent selected from a single bond, alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl; X is optional and, if present, can be a single bond, O, -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, -NR9S(=O)2, -OC(O)NH-, -NHC(O)O, -OC(O)NR9, -NR9C(O)O, -CH=NO-, -ON=CH-, -NR 10 C(O)NR 10 -, heteroaryl, aryl, -aryl 10 C(=NR 11 )NR 10 -, -NR 10 C(=NR 11 )-, -C(=NR 11 )NR 10 -, -OC(=NR 11 )-, or -C(=NR 11 )O- and; L4 is optional and, if present, can be a single bond, an optionally substituted alkyl, an optionally substituted cycloalkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted aryl, an optionally substituted heteroaryl, or an optionally substituted heterocyclic compound; Alternatively, the resulting L3, X, and L4 may form any substituent selected from nitrogen, alkyl, heteroalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, or alkylheterocycloalkyl that encloses a heterocycle; G is

[0202] [ka]

[0203] And here, R a R7 and R8 are H, a substituted or unsubstituted alkyl, or a substituted or unsubstituted cycloalkyl; and R7 and R8 are also H; R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkyl C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, C1-C8 alkyl amide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or, R6 and R8 form a single bond; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkyl C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, C1-C8 alkyl amide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or, R5 is H, halogen, -L6-(substituted or unsubstituted C1-C3 alkyl), -L6-(substituted or unsubstituted C2-C4 alkenyl), -L6-(substituted or unsubstituted heteroaryl), or -L6-(substituted or unsubstituted aryl), where L6 is a single bond, O, S, -S(=O), S(=O)2, NH, C(O), -NHC(O)O, -OC(O)NH, -NHC(O) or -C(O)NH; Each R9 is independently selected from H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl; Each R 10is, independently, H, optionally substituted lower alkyl, or optionally substituted lower cycloalkyl; or, Two Rs 10 groups, together, can form a 5-, 6-, 7- or 8-membered heterocyclic ring; or, R9 and R 10 together, can form a 5-, 6-, 7- or 8-membered heterocyclic ring; or, Each R 11 is independently selected from H, -S(=O)2R8, -S(=O)2NH2, -C(O)R8, -CN, -NO2, heteroaryl or heteroalkyl; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of the foregoing constitution.

[0204] In another embodiment, pharmaceutically acceptable salts of the compound of formula (A1) are provided. As just one example, the salts exist as salts of the amino group made by inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Further salts are available if the counterion is one of the following anions, namely adipartate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formic acid, fumaric acid, glucoheptate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate This includes lactobionic acid, lactates, lauric acid, lauryl sulfate, malic acid, maleic acid, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrates, oleates, oxalates, palmitates, pamoate, pectinates, persulfates, phenyl 3-propionate, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonate, undecanoate, and valerates. Furthermore, the salts contain counterions of the following cations, namely sodium, lithium, potassium, calcium, magnesium, ammonium, and quaternary ammonium group (substituted with at least one organic moiety) cations.

[0205] In another embodiment, there exist pharmaceutically acceptable esters of compounds of formula (A1) in which the ester group is selected from formic acid esters, acetates, propionates, butyrates, acrylates, and ethyl succinates.

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

[0207] In further embodiments, there exists a compound of formula (A) having the structure of formula (B):

[0208] [ka]

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

[0210] [ka]

[0211] Here, R6, R7 and R8 are independently selected from H, lower alkyl or substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower cycloalkyl, and substituted or unsubstituted lower heterocycloalkyl; R 12 is H, or a lower alkyl group; or Y and R obtained together. 12forms a 4-, 5- or 6-membered heterocyclic ring; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvate, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of the foregoing constitution.

[0212] In a further embodiment, G is

[0213]

Chemical formula

[0214] selected from

[0215] In a further embodiment,

[0216]

Chemical formula

[0217] is

[0218]

Chemical formula

[0219] selected from

[0220] In a further embodiment, the compound of formula (A1) has the structure of the following formula (B1). That is,

[0221]

Chemical formula

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

[0223] [ka]

[0224] [ka]

[0225] And here, R a R7 and R8 are H, a substituted or unsubstituted alkyl, or a substituted or unsubstituted cycloalkyl; and R7 and R8 are also H; R6 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R6 and R8 are H; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); or, R6 and R8 form a single bond; R7 is H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, C1-C8 alkylaminoalkyl, C1-C8 hydroxyalkylaminoalkyl, C1-C8 alkoxyalkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C2-C8 heterocycloalkyl, substituted or unsubstituted heteroaryl, C1-C4 alkyl(aryl), C1-C4 alkyl(heteroaryl), C1-C8 alkyl ether, C1-C8 alkylamide, or C1-C4 alkyl(C2-C8 heterocycloalkyl); R 12 is H, or a lower alkyl group; or Y and R obtained together. 12 It forms a 4, 5, or 6-membered heterocycle; The compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvent compound, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug having the above configuration.

[0226] In a further embodiment, G is

[0227] [ka]

[0228] Selected from, where R is H, alkyl, alkylhydroxy, heterocycloalkyl, heteroaryl, alkylalkoxy, or alkylalkoxyalkyl.

[0229] In a further embodiment,

[0230] [ka]

[0231] teeth,

[0232] [ka]

[0233] Selected from.

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

[0235] [ka]

[0236] Y is an alkylene, a substituted alkylene, or a 4, 5, or 6-membered ring cycloalkylene; R 12 is H, or a lower alkyl group; Alternatively, Y and R obtained together 12 It forms a 4, 5, or 6-membered heterocycle; G is

[0237] [ka]

[0238] Herein, R6, R7 and R8 are independently selected from H, lower alkyl, substituted lower alkyl, lower heteroalkyl or substituted lower heteroalkyl, substituted or unsubstituted lower cycloalkyl, substituted or unsubstituted lower heterocycloalkyl; and the compound comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvent compound, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug of the above configuration.

[0239] In a further embodiment, the compound of formula (B1) has the structure of the following formula (C1). That is,

[0240] [ka]

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

[0242] [ka]

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

[0244] In further or alternative embodiments, the "G" group of any of formulas (A1), (B1), and (C1) is any group used to modulate the physical properties, biological characteristics, and other properties of the molecule. The modification is achieved using groups that modulate the Michael acceptor chemical reactivity, acidity, basicity, lipophilicity, solubility, and other physical properties of the molecule. The physical characteristics and biological characteristics modulated by the modification to G include, as just a few examples, enhancing the Michael acceptor group, solubility, in vivo absorption, and in vivo metabolic chemical reactivity. Furthermore, the in vivo metabolic modifications include, as just a few examples, controlling the in vivo PK properties, off-target activity, potential toxicity related to cypP450 interactions, drug-drug interactions, and so on. In addition, modifications to G allow for the modification of the in vivo efficacy of the compound, for example, by modulating specific and nonspecific protein binding to plasma proteins and lipids, and in vivo tissue distribution.

[0245] In another embodiment, the compound described herein is of formula (D), which is as follows:

[0246] [ka]

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

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

[0249] [ka]

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

[0251] In another embodiment, pharmaceutically acceptable salts of the compound of formula (D1) are provided. As just one example, the salt is a salt of an amino group formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or an organic acid 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 adipartate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formic acid, fumaric acid, glucoheptate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobion The salts include anions such as acids, lactates, lauric acid, lauryl sulfate, malic acid, maleic acid, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrates, oleates, oxalates, palmitates, pamoate, pectinates, persulfates, phenyl 3-propionate, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonate, undecanoate, and valerates. Further salts include those in which the counterion is a cation such as sodium, lithium, potassium, calcium, magnesium, ammonium, and quaternary ammonium (substituted with at least one organic moiety).

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

[0253] In another embodiment, there are pharmaceutically acceptable carbamates of the compound of formula (D1). In yet another embodiment, there are pharmaceutically acceptable N-acyl derivatives of the compound of formula (D1). Examples of N-acyl groups include N-acetyl groups and N-ethoxycarbonyl groups.

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

[0255] In further embodiments, Ar is phenyl.

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

[0257] In a further embodiment, each of R1, R2, and R3 is H.

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

[0259] In any and all embodiments, the substituent is selected from a subset of the alternatives listed. For example, in some embodiments, L a is CH2, O, or NH. In other embodiments, L a is O or NH. In yet another embodiment, L a It is O.

[0260] In some embodiments, Ar is a substituted or unsubstituted aryl compound. In yet another embodiment, Ar is a six-membered aryl compound. In several other embodiments, Ar is a phenyl compound.

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

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

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

[0264] In some embodiments, Y is an optional substituent selected from alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl groups. In other embodiments, Y is an optional substituent selected from C1-C6 alkyl, C1-C6 heteroalkyl, tetra-, quin-, hex- or seven-membered cycloalkyl, and tetra-, quin-, hex- or seven-membered heterocycloalkyl groups. In yet another embodiment, Y is an optional substituent selected from C1-C6 alkyl, C1-C6 heteroalkyl, quin- or six-membered cycloalkyl groups containing 1 or 2N atoms, and quin- or six-membered heterocycloalkyl groups containing 1 or 2N atoms. In some other embodiments, Y is a quin- or six-membered cycloalkyl group containing 1 or 2N atoms, or a quin- or six-membered heterocycloalkyl group containing 1 or 2N atoms.

[0265] Any combination of the above groups for various variations is considered herein. It will be understood that substituents and substitution patterns on the compounds provided herein may be selected by those skilled in the art to provide compounds that are chemically stable and can be synthesized by art known in the art, as are the compounds specified herein.

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

[0267] [ka]

[0268] During the ceremony, Here, the sphere in formula (E) is a region that binds to the active site of a kinase, which contains a tyrosine kinase and further contains a Btk kinase-cysteine ​​homolog; Y is an arbitrary substituent selected from alkylene, heteroalkylene, arylene, heteroarylene, heterocycloalkylene, cycloalkylene, alkylenearylene, alkylenehetearene, alkylenecycloalkylene, and alkylenehetearene; Z is C(=O), OC(=O), NHC(=O), NCH3C(=O), C(=S), S(=O) x OS (=O) x , NHS (=O) x And here, x is either 1 or 2; R6, R7, and R8 are H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4 alkyl(aryl), substituted or unsubstituted C1-C4 alkyl(heteroaryl), substituted or unsubstituted C1-C4 alkyl(C3-C8 cycloalkyl), or substituted or unsubstituted C1-C4 alkyl(C2-C8 heterocycloalkyl); or R7 and R8 together form a single bond, and the irreversible inhibitor has its pharmaceutically active metabolite, or a pharmaceutically acceptable solvent compound, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug.

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

[0270] [ka]

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

[0272] [ka]

[0273] During the ceremony, L a is CH2, O, NH or S; Ar is a substituted or unsubstituted aryl or a substituted or unsubstituted heteroaryl; (a) Y is an optional substituent selected from alkylene, heteroalkylene, arylene, heteroarylene, alkylene arylene, alkylene heteroarylene, alkylene cycloalkylene, and alkylene heterocycloalkylene; Z is C(=O), NHC(=O), NR a C(=O), NR a S(=O) x where x is or 2, and R a is H, a substituted or unsubstituted alkyl, or a substituted or unsubstituted cycloalkyl; and (i) R6, R7, R8 are H, a substituted or unsubstituted C1-C4 alkyl, a substituted or unsubstituted C1-C4 heteroalkyl, a substituted or unsubstituted C3-C6 cycloalkyl, a substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, a substituted or unsubstituted C3-C6 cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a substituted or unsubstituted C1-C4 alkyl(aryl), a substituted or unsubstituted C1-C4 alkyl(heteroaryl), a substituted or unsubstituted C1-C4 alkyl(C3-C8 cycloalkyl), or a substituted or unsubstituted C1-C4 alkyl(C2-C8 heterocycloalkyl); or (ii) R6 and R8 are H; R7 is H, a substituted or unsubstituted C1-C4 alkyl, a substituted or unsubstituted C1-C4 heteroalkyl, a C1-C8 alkylaminoalkyl, a C1-C8 hydroxyalkylaminoalkyl, a C1-C8 alkoxyalkylaminoalkyl, a substituted or unsubstituted C3-C6 cycloalkyl, a substituted or unsubstituted C1-C8 alkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted C2-C8 heterocycloalkyl, a substituted or unsubstituted heteroaryl, a C1-C4 alkyl(aryl), a C1-C4 alkyl(heteroaryl), a C1-C8 alkyl ether, a C1-C8 alkylamide, or a C1-C4 alkyl(C2-C8 heterocycloalkyl); or (iii) R7 and R8 form a single bond; R6 is selected from H, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C2-C6 heterocycloalkyl, C1-C6 alkoxyalkyl, C1-C8 alkylaminoalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C1-C4 alkyl (aryl), substituted or unsubstituted C1-C4 alkyl (heteroaryl), substituted or unsubstituted C1-C4 alkyl (C3-C8 cycloalkyl), or substituted or unsubstituted C1-C4 alkyl (C2-C8 heterocycloalkyl), or (b) Y is an optional substituent selected from cycloalkylene or heterocycloalkylene; Z is C(=O), NHC(=O), NR a C(=O) NR a S(=O) x And here, x is either 1 or 2, and R a is H, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl; and, (i) R7 and R8 are H; R6 is either a substituted or unsubstituted C1-C4 alkyl, a substituted or unsubstituted C1-C4 heteroalkyl, a C1-C8 alkylaminoalkyl, a C1-C8 hydroxyalkylaminoalkyl, a C1-C8 alkoxyalkylaminoalkyl, a substituted or unsubstituted C3-C6 cycloalkyl, a substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted C2-C8 heterocycloalkyl, a substituted or unsubstituted heteroaryl, a C1-C4 alkyl(aryl), a C1-C4 alkyl(heteroaryl), a C1-C8 alkyl ether, a C1-C8 alkylamide, or a C1-C4 alkyl(C2-C8 heterocycloalkyl); (ii) R6 and R8 are H; R7 is a substituted or unsubstituted C1-C4 alkyl, a substituted or unsubstituted C1-C4 heteroalkyl, a C1-C8 alkylaminoalkyl, a C1-C8 hydroxyalkylaminoalkyl, a C1-C8 alkoxyalkylaminoalkyl, a substituted or unsubstituted C3-C6 cycloalkyl, a substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted C2-C8 heterocycloalkyl, a substituted or unsubstituted heteroaryl, a C1-C4 alkyl(aryl), a C1-C4 alkyl(heteroaryl), a C1-C8 alkyl ether, a C1-C8 alkylamide, or a C1-C4 alkyl(C2-C8 heterocycloalkyl); or (iii) R7 and R8 both form a single bond; R6 is a substituted or unsubstituted C1-C4 alkyl, a substituted or unsubstituted C1-C4 heteroalkyl, a C1-C8 alkylaminoalkyl, a C1-C8 hydroxyalkylaminoalkyl, a C1-C8 alkoxyalkylaminoalkyl, a substituted or unsubstituted C3-C6 cycloalkyl, a substituted or unsubstituted C1-C8 alkylC3-C6 cycloalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted C2-C8 heterocycloalkyl, a substituted or unsubstituted heteroaryl, a C1-C4 alkyl(aryl), a C1-C4 alkyl(heteroaryl), a C1-C8 alkyl ether, a C1-C8 alkylamide, or a C1-C4 alkyl(C2-C8 heterocycloalkyl), and formula (F) is its pharmaceutically active metabolite, a pharmaceutically acceptable solvent compound, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug.

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

[0275] [ka]

[0276] [ka]

[0277] [ka]

[0278] In another embodiment, the compounds provided herein are selected from the following:

[0279] [ka]

[0280] In one embodiment, the compounds provided herein are selected from the following:That is, 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one (compound 4); (E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)but-2-en-1-one (compound 5); 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)sulfo Nylethene (compound 6); 1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-in-1-one (compound 8); 1-(4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one (compound 9); N-((1s,4s)-4-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl) Chlohexyl)acrylamide (compound 10); 1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)pyrrolidine-1-yl)prop-2-en-1-one (compound 11); 1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)pyrrolidine-1-yl)prop-2-en-1-one (compound 12); 1-((R)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3 ,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one (compound 13); 1-((S)-3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one (compound 14); and (E)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)-4-(dimethylamino)but-2-en-1-one (compound 15).

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

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

[0283] Throughout this specification, groups and their substituents may be selected by those skilled in the art to provide stable moieties and compounds.

[0284] In certain embodiments, any of the Btk inhibitors and / or the second agent provided herein with respect to the present invention comprises i) a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0285] In some embodiments, the Btk inhibitor of the present invention is administered in doses ranging from about 1.25 mg / kg / day to about 12.5 mg / kg / day. In specific embodiments, the Btk inhibitor is administered in doses selected from groups consisting of about 1.25 mg / kg / day, about 2.5 mg / kg / day, about 5 mg / kg / day, about 8.3 mg / kg / day, or about 12.5 mg / kg / day.

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

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

[0288] In other embodiments, the secondary agonist is administered at doses ranging from approximately 1.25 mg / kg / day to approximately 12.5 mg / kg / day. In specific embodiments, the secondary agonist is administered at doses selected from the group consisting of approximately 1.25 mg / kg / day, approximately 2.5 mg / kg / day, approximately 5 mg / kg / day, approximately 8.3 mg / kg / day, or approximately 12.5 mg / kg / day. The dosage of the secondary agonist is based on the measured expression or presence of one or more biomarkers from one or more lymphocyte subgroups. Persons skilled in the art, such as physicians, can readily determine an appropriate regimen (e.g., dosage of the secondary agonist) based on the diagnostic results.

[0289] In other embodiments, the present invention provides a method for treating cancer, comprising the steps of: measuring the expression or presence of one or more biomarkers in one or more lymphocyte subgroups of a subject who has received a dose of a Btk inhibitor; and administering a secondary agonist based on the measured expression characteristics.

[0290] In other embodiments, the present invention also provides a method for treating cancer, comprising the steps of: administering a Btk inhibitor in such a way that a lymphocyte subgroup defined by immunophenotyping results in an increase or appearance in the blood; and administering a secondary agent in which the increase or appearance of the lymphocyte subgroup in the blood is measured.

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

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

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

[0294] In any of the embodiments described above, there are further embodiments that include a single dose of an effective amount of the Btk inhibitor, which further embodiments include (i) a single dose of the Btk inhibitor, (ii) multiple doses of the Btk inhibitor per day administered to a mammal, (iii) intermittently, or (iv) continuously.

[0295] In any of the embodiments described above, there are further embodiments that include multiple doses of an effective amount of Btk inhibitor, the embodiments of which include (i) administering a single dose of the Btk inhibitor; (ii) administering the multiple doses at intervals of 6 hours; and (iii) administering the Btk inhibitor to a mammal every 8 hours. In further or alternative embodiments, the method includes a drug-free period, during which the administration of the Btk inhibitor is temporarily interrupted or the dose of the Btk inhibitor being administered is temporarily reduced, and at the end of the drug-free period, the administration of the Btk inhibitor is resumed. The length of the drug-free period may vary between 2 days and 1 year.

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

[0297] In any of the embodiments described above, there are further embodiments that include a single dose of an effective amount of the second agonist, the embodiments of which include (i) a single dose of the second agonist, (ii) administration of the second agonist to a mammal multiple times a day, (iii) intermittently, or (iv) continuously.

[0298] In any of the embodiments described above, there are further embodiments that include multiple doses of an effective amount of the secondary agonist, the embodiments of which include (i) administering the secondary agonist in a single dose, (ii) having intervals of 6 hours between the multiple doses, and (iii) administering the secondary agonist to a mammal every 8 hours. In further or alternative embodiments, the method includes a drug-free period, during which the administration of the secondary agonist is temporarily interrupted or the dose of the secondary agonist being administered is temporarily reduced, and at the end of the drug-free period, the administration of the secondary agonist is resumed. The length of the drug-free period may vary between 2 days and 1 year.

[0299] In any of the embodiments described above, the second agonist is alemtuzumab, arsenic trioxide, asparaginase (pegylated or non-pegylated), bevacizumab, cetuximab, platinum-based compounds such as cisplatin, cladribine, daunorubicin / doxorubicin / idarubicin, irinotecan, fludarabine, 5-fluorouracil, gemtuzumab, methotrexate, paclitaxel (trademark), taxol, temozolomide, thioguanine, or hormones (anti-estrogen, anti-androgen, or gonadotropin-releasing hormone analogs). The medications selected are those used to treat signs and symptoms induced by treatment, such as interferons including alpha-interferon, nitrogen mustards including busulfan, melphalan, or mechloretamine, retinoids including treynoin, topoisomerase inhibitors including irinotecan or topotecan, tyrosine kinase inhibitors including gefitinib or imatinib, or allopurinol, filgrastim, granisetron / ondansetron / palonosetron, or dronabinol.

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

[0301] Chemical reactions may be used in a linear sequence to provide the compounds described herein, or to synthesize fragments that are subsequently linked by methods described herein and / or known in the art.

[0302] <Formation of covalent bonds through the reaction of an electrophile with a nucleophile> The compounds described herein can be modified using various electrophilic and / or nucleophilic reagents to form new functional groups or substituents. Table 1, titled "Examples of Covalent Bonds and Their Precursors," lists examples of covalent chains and precursor functional group selections that produce a variety of available electrophilic and nucleophilic reagent combinations and can be used as guidance for electrophilic and nucleophilic reagent combinations. Precursor functional groups are shown as electrophilic and nucleophilic groups.

[0303] [Table 1-1]

[0304] [Table 1-2]

[0305] <Use of protective groups> In the reactions described, it may be necessary to protect reactive functional groups, such as hydroxy, amino, imino, thio, or carboxyl groups, that are desired in the final product, in order to avoid their unwanted involvement in the reaction. Protecting groups are used to block some or all of the reaction moieties and prevent such groups from participating in the chemical reaction until the protecting group is removed. In one embodiment, each protecting group can be removed by separate means. Protecting groups that are cleaved under collectively different reaction conditions satisfy the requirement of differential removal. Protecting groups can be removed by acid, base, and hydrolysis. Groups such as trityl, dimethoxytrityl, acetal, and t-butyldimethylsilyl are used to protect carboxyl and hydroxyl reactive moieties in the presence of an acid-dissociable, hydrolytically removable Cbz group, and a base-dissociable Fmoc group-protected amino group. The reactive moieties of carboxylic acids and hydroxyls can be blocked by acid-dissociating groups such as t-butylcarbamates, or by stable acid- or base-removable carbamates, in the presence of amines, with a base-dissociating group such as methyl, ethyl, and acetyl, but not limited to these.

[0306] The reactive carboxylic acid and hydroxyl moieties can also be blocked with hydrolytically removable protecting groups such as benzyl groups, while the acid-hydrogen-bonding amine group can be blocked with a base-dissociating group such as Fmoc. The carboxylic acid reaction moiety can be protected by conversion to a simple ester compound, as illustrated herein, or blocked with an oxidatively removable protecting group such as 2,4-dimethoxybenzyl, while the coexisting amino group can be blocked with a fluoride-degradable silylcarbamate.

[0307] Allyl blocking groups are useful in the presence of acid protecting groups and base protecting groups, because the former are stable and can be subsequently removed by metal or π-acid catalysts. For example, carboxylic acids blocked by allyls can be protected in the presence of acid-dissociable t-butylcarbamate or base-dissociable acetate amine protecting groups, such as Pd 0It can be deprotected by a catalyzed reaction. Another form of protecting group is a resin to which a compound or intermediate can be attached. As long as the residue is attached to the resin, its functional group is blocked and cannot react. Once released from the resin, the functional group can react.

[0308] Typical blocking / protecting groups can be selected from the following:

[0309] [ka]

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

[0311] <Further forms of the compound> The compounds described herein may have one or more stereocenters, each center may be in an R or S configuration. The compounds presented herein include all diastereomers, enantiomers, and epimers, as well as suitable mixtures thereof. Stereoisomers may be obtained, if desired, by methods known in the art, such as separation of stereoisomers by chiral chromatography column.

[0312] Diastereomer mixtures can be separated into individual diastereomers based on their physical and chemical differences by known methods, such as chromatography and / or fractional crystallization. In one embodiment, enantiomers can be separated by chiral chromatography column. In other embodiments, enantiomers can be separated by reaction with a suitable optically active compound (e.g., an alcohol) to convert the enantiomer mixture into a diastereomer mixture, separating the diastereomers, and converting the individual diastereomers into their corresponding pure enantiomers (e.g., by hydrolysis). All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered as part of the compositions described herein.

[0313] The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of the compounds described herein, as well as active metabolites of these compounds having similar activity. In some circumstances, the compounds may exist as tautomers. All tautomers fall within the range of compounds shown herein. Furthermore, the compounds described herein may exist in solvated and unsolvated forms using pharmaceutically acceptable solvents such as water and ethanol. The solvated forms of the compounds presented herein should also be disclosed herein.

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

[0315] In some embodiments, the compounds described herein are prepared as prodrugs. A “prodrug” refers to a drug that is converted to a parent drug in vivo. In some situations, prodrugs are often useful because they may be easier to administer than the parent drug. They may be bioavailable, for example, by oral administration, whereas the parent drug is not. Prodrugs may also have improved solubility in pharmaceutical compositions than the parent drug. An example of an unrestricted prodrug is a compound described herein, which, when administered as an ester (“prodrug”), facilitates translocation across cell membranes where solubility would negatively affect movement, but which is then metabolically hydrolyzed to a carboxylic acid, the active entity, once it is in a cell where water solubility is beneficial. Further examples of prodrugs may be short-chain peptides (polyamino acids) in which a peptide is metabolized and bound to an acid group that reveals the active site. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to a biological, pharmaceutically, or therapeutically active form of the compound. In certain embodiments, a prodrug is enzymatically metabolized to a biological, pharmaceutically, or therapeutically active form of a compound by one or more steps or processes. To produce a prodrug, a pharmaceutically active compound is modified so that the active compound is regenerated when administered in vivo. Prodrugs may be designed to alter the metabolic stability or transport properties of a drug, to shield against side effects or toxicity, to improve the flavor of a drug, or to alter other characteristics or properties of a drug. With knowledge of pharmacological processes and drug metabolism in vivo, those skilled in the art can design prodrugs of a compound once a pharmaceutically active compound is known.(For example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392; Silverman (1992), The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc., San Diego, pages 352-401, Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. (see 1985).

[0316] The prodrug form of the compounds described herein, which is metabolized in vivo to produce derivatives as specified herein, is included within the scope of the claims. In some cases, some of the compounds described herein may be prodrugs for other derivatives or active compounds.

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

[0318] The aromatic ring portion of the compound of formula (D) is susceptible to various metabolic reactions; therefore, the incorporation of appropriate substituents on the aromatic ring structure, such as halogens, can reduce, minimize, or eliminate this metabolic pathway.

[0319] The compounds described herein include isotopically labeled compounds, which are identical to those recited in the various chemical formulas and structures shown herein, except that one or more atoms are replaced by atoms 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 compounds of the present specification are isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Cl, respectively. Specific isotopically labeled compounds described herein, for example, 3 H and 14 C, etc., compounds incorporated with radioactive isotopes are useful for tissue distribution assays of drugs and / or substrates. Furthermore, substitution with isotopes such as deuterium, i.e., 2 H, can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or decreased required dosage.

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

[0321] The compounds described herein may be formed and / or used as pharmaceutically acceptable salts. The types of pharmaceutically acceptable salts include: (1) a free base of the compound with a pharmaceutically acceptable inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, metaphosphate; or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]octa-2-ene-1-carboxylic acid, glucoheptonic acid. (1) Includes acid addition salts formed by reaction with pharmaceutically acceptable organic acids such as (1) acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid; (2) Includes, but is not limited to, salts formed when an acidic proton present in the parent compound is substituted by a metal ion, such as an alkali metal ion (e.g., lithium, sodium, potassium), an alkaline earth ion (e.g., magnesium or calcium), or an aluminum ion, or coordinates with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.

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

[0323] The salt is recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent after filtration, evaporation of the solvent, or lyophilization in the case of an aqueous solution.

[0324] It should be understood that references to pharmaceutically acceptable salts include solvated forms or their crystalline forms, particularly solvates or polymorphs. Solvates may be formed during the crystallization process in a pharmaceutically acceptable solvent, such as water or ethanol, comprising either stoichiometric or non-stoichiometric amounts of solvent. Hydrates are formed when the solvent is water, or alkoxides are formed when the solvent is alcohol. Solvates of the compounds described herein may be conveniently prepared or formed during the processes described herein. Furthermore, the compounds provided herein may exist in non-solvated forms as well as solvated forms. Generally, solvated forms are considered equivalent to non-solvated forms for the purposes of the compounds and methods provided herein.

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

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

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

[0328] Throughout this specification, groups and their substituents may be selected by those skilled in the art to provide stable moieties and compounds.

[0329] <Cancer Treatment Regimen> In certain embodiments, a method for treating a hematological malignancy in a solid requiring it is described herein, comprising the steps of: (a) administering to the solid an amount of an irreversible Btk inhibitor sufficient to pharmacokineticate multiple cells from the malignancy; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignancy. In some embodiments, the step of analyzing the pharmacokinetic multiple cells includes measuring the peripheral blood concentration of the pharmacokinetic multiple cells. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic multiple cells has increased compared to the concentration before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after the peripheral blood concentration of the pharmacokinetic multiple cells has subsequently decreased. In some embodiments, analyzing the multiple pharmacokinetic multiple cells includes measuring the period over which the peripheral blood concentration of the pharmacokinetic multiple cells increases compared to the concentration before administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the peripheral blood concentration of pharmacokinetic cells has increased over a predetermined period of time. In some embodiments, the step of analyzing the pharmacokinetic cells includes counting the number of pharmacokinetic cells in the peripheral blood. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased compared to the number before administration of the Btk inhibitor. In some embodiments, the administration of the second cancer treatment regimen occurs after the number of pharmacokinetic cells in the peripheral blood has decreased. In some embodiments, the step of analyzing the pharmacokinetic cells includes measuring the period over which the number of pharmacokinetic cells in the peripheral blood increases compared to the number before administration of the Btk inhibitor. In some embodiments, the method further includes administering a second cancer treatment regimen after the number of pharmacokinetic cells in the peripheral blood has increased over a predetermined period of time.

[0330] In some embodiments, administering a Btk inhibitor prior to a second cancer treatment regimen reduces the immune-mediated response to the second cancer treatment regimen. In some embodiments, administering a Btk inhibitor prior to ofatumumab reduces the immune-mediated response to ofatumumab. In some embodiments, the second cancer treatment regimen includes chemotherapy agents, steroids, immunotherapy agents, targeted therapies, or combinations thereof. In some embodiments, the second cancer treatment regimen includes a B cell receptor pathway inhibitor. In some embodiments, the B cell receptor pathway inhibitor is a CD79A inhibitor, CD79B inhibitor, CD19 inhibitor, Lyn inhibitor, Syk inhibitor, PI3K inhibitor, Blnk inhibitor, PLCγ inhibitor, PKCβ inhibitor, or a combination thereof. In some embodiments, the second cancer treatment regimen includes antibodies, B cell receptor signaling pathway inhibitors, PI3K inhibitors, IAP inhibitors, mTOR inhibitors, radioimmunotherapy agents, DNA damage agents, proteosome inhibitors, histone deacylase inhibitors, protein kinase inhibitors, Hedgehog inhibitors, Hsp90 inhibitors, telomerase inhibitors, Jak1 / 2 inhibitors, protease inhibitors, PKC inhibitors, PARP inhibitors, or combinations thereof.

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

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

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

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

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

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

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

[0338] Additional cancer treatment regimens include, for example, nitrogen mustards such as bendamustine, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, melphalan, prednimastine, and trophosphamide; alkyl sulfonates such as busulfan, mannosulfan, and treosulfan; ethyleneimines such as carboquan, thiotepa, and triadiquan; nitrosoureas such as carmustine, fotemustine, lomustine, nimustine, ranimustine, semustine, and streptozocin; epoxides such as etogluside; other alkylating agents such as dacarbazine, mitobronitol, pipobromane, and temozolomide; and folic acid analogs such as methotrexate, permetrexed, pralatrexate, and larcitrexed. Analogues; for example, purine analogs such as cladribine, clofarabine, fludarabine, mercaptopurine, nerarabine, and thioguanine; pyrimidine analogs such as azacitidine, capecitabine, carmofur, cytarabine, decitabine, fluorouracil, gemcitabine, and tegafur; vinca alkaloids such as vinblastine, vincristine, vindesine, vinflunin, and vinorelbine; and podophyllotoxin derivatives such as etoposide and teniposide. Derivatives; for example, colchicine derivatives such as demecoltin; taxanes such as docetaxel, paclitaxel, and paclitaxel polygrumex; other plant alkaloids and natural products such as trabectedin; actinomycins such as dactinomycin; anthracyclines such as acralubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, barurubicin, and solubinin; other cytotoxic antibiotics such as bleomycin, ixabepyrone, mitomycin, and pricamycin; platinum compounds such as carboplatin, cisplatin, oxaliplatin, and satoraplatin; methylhydrazines such as procarbazine;For example, sensitizers such as aminolevulinic acid, efapoxial, methyl aminolevulinate, sodium porfimer, and temoporfin; for example, protein kinase inhibitors such as dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazonanib, sorafenib, sunitinib, and temsirolimus; for example, alitretinoin, altretamine, amzacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin difutitox, estramustine, and hydroxycarb Antineoplastic agents such as mid, irinotecan, ronidamin, masopropyl, miltefosein, mitogwazon, mitotane, oblimersen, pegaspargase, pentostatin, romidepsin, citrimazine, seradenovec, thiazophrine, topotecan, tretinoin, and vorinostat; estrogens such as diethylstilbenol, ethinylestradiol, phosphestrol, and polyestradiol phosphate; and gestolone. Progestogens such as medroxyprogesterone and megestrol; gonadotropin-releasing hormone analogs such as buserelin, goserelin, leuprorelin, and triptorelin; antiestrogens such as fulvestrant, tamoxifen, and toremifene; antiandrogens such as bicalutamide, flutamide, and nilutamide; enzyme inhibitors such as aminoglutethimide, anastrozole, exemestane, formestan, letrozole, and borozol; for example, abalelix and degarelix. Other hormone antagonists; for example, immunostimulants such as histamine hydrochloride, mifamultide, pidotimod, plerixafor, lokinimex, and timopentin; immunosuppressants such as everolimus, gusperimus, leflunomide, mycophenolic acid, and sirolimus; calcineurin inhibitors such as cyclosporine and tacrolimus; other immunosuppressants such as azathioprine, lenalidomide, methotrexate, and thalidomide; and radiopharmaceuticals such as iobenguan.

[0339] Additional cancer treatment regimens include interferon, interleukin, tumor necrosis factor, and growth factors.

[0340] Additional cancer treatment regimens include immunostimulants such as ancestim, filgrastim, lenograstim, morglamostim, pegfilgrastim, and salglamostim; for example, natural interferon alfa Interferons such as natural interferon, interferon alpha-2a, interferon alpha-2b, interferon alpha-con-1, interferon alpha-n1, natural interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma, pegylated interferon alpha-2a, pegylated interferon alpha-2b; interleukins such as aldethleukin, oprelbequin; other immunostimulants such as BCG vaccine, glatiramer acetate, histamine hydrochloride, immunocyanin, lentinan, melanoma vaccine, mifamultide, pegademase, pidotimod, prelixafor, polyI:C, polyICLC, lokinimex, tasonelmin, thymopentin; abatacept, Immunosuppressants such as abetimus, alefacept, anti-lymphocyte immunoglobulin (horse), anti-thymocyte immunoglobulin (rabbit), eculizumab, efalizumab, everolimus, gusperimus, leflunomide, muromanab-CD3, mycophenolate, natalizumab, sirolimus; for example, adalimumab, afelimomab, certolizumab pegol, etanercept, golimumab, This includes TNF-alpha inhibitors such as infliximab; interleukin inhibitors such as anakinra, basiliximab, canakinumab, daclizumab, mepolizumab, lilonacept, tocilizumab, and ustekinumab; calcineurin inhibitors such as cyclosporine and tacrolimus; and other immunosuppressants such as azathioprine, lenalidomide, methotrexate, and thalidomide.

[0341] Additional cancer treatment regimens include adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, infliximab, muromonab-CD3, natalizumab, panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, etc., or combinations thereof.

[0342] Additional cancer treatment regimens include, for example, monoclonal antibodies such as alemtuzumab, bevacizumab, catumakisomab, cetuximab, edrecolomab, gemtuzumab, ofatumumab, panitumumab, rituximab, and trastuzumab; immunosuppressants such as eculizumab, efalizumab, muromab-CD3, and natalizumab; TNF-alpha inhibitors such as adalimumab, aferimomab, certolizumab pegol, golimumab, and infliximab; and basiliximab and canakinumb. Interleukin inhibitors such as daclizumab, mepolizumab, tocilizumab, and ustekinumab; radiopharmaceuticals such as ibritumomab tiuxetan and tocitumomab; for example, abagovomab, adekatumumab, alemtuzumab, anti-CD30 monoclonal antibody Xmab2513, anti-MET monoclonal antibody MetMab, apolizumab, apomab, alsitumomab, basiliximab, bispecific antibody 2B1, blinatumomab, brentuximab vedotin, and capromab. Pendetid, ixtumumab, claudiximab, conatumumab, decatumumab, denosumab, eculizumab, epratuzumab, epratuzumab, ertumaxomab, etalacizumab, figtumumab, fresolimmab, galiximab, ganitumumab, gemtuzumab ozogamisin, grembatumumab, ibritumomab, inotuzumab ozogamisin, ipilimumab, lexatumumab, lintuzumab, lintuzumab, lucatumumab, mapatum This includes other monoclonal antibodies such as mab, matuzumab, milatuzumab, monoclonal antibody CC49, necitumumab, nimotuzumab, ofatumumab, oregobomab, pertuzumab, ramacurimab, ranibizumab, ciprizumab, sonepcizumab, tanezumab, tocitumomab, trastuzumab, tremelimumab, tucotzumab cermoleukin, bertuzumab, vizilizumab, boroxiximab, and zaltumumab.

[0343] Additional cancer treatment regimens may include agents that affect the tumor microenvironment, such as cellular signaling networks (e.g., the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which signals from B cell receptors and IgE receptors). In some embodiments, the secondary agonist is a PI3K signaling inhibitor or a SYC kinase inhibitor. In one embodiment, the SYC inhibitor is R788. In another embodiment, just as an example, there may be a PKCγ inhibitor such as Enzastaurin.

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

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

[0346] Other anticancer agents that can be used in combination with Btk inhibitor compounds include Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, Asibicin; Acralubicin; Acodazole hydrochloride; Acronin; Adzelesin; Aldesleukin; Altretamine; Ambomycin; Amethantrone acetate; Aminoglutethimide; Amsacrin; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetape; Azotomycin; Batymast; Benzodepa; Bicalutamide; Bisantren hydrochloride; Nimesil Bisnafide acid; Bizeresin; Bleomycin sulfate; Braquenal sodium; Bropyrimine; Busulfan; Cactinomycin; Carsterone; Calasemide; Carvetimer; Carboplatin; Carmustine; Carbicin hydrochloride; Carzeresin; Sedefingol; Chlorambucil; Cyloremycin; Cladribine; Crisnator mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Daunorubicin hydrochloride; Decitabine; Dexolomaplatin; Desaguanine; Desaguamine mesylate; Diadiquan; Doxorubicin; Doxorubicin hydrochloride; Doroxif Droloxyfene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; erusamitrusine; enloplatin; empromate; epipropidine; epirubicin hydrochloride; erbrozol; esorbicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etopurine; fadrozol hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosskido N; Fostriesin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Iimofosine; Interleukin II (including recombinant interleukin II or rlL2), Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-n1; Interferon alpha-n3; Interferon beta-la; Interferon gamma-lb; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide acetate;Rialosol hydrochloride; lometelexol sodium; lomustine; rosoxantrone hydrochloride; masopropyl; meitansine; mechloretamine hydrochloride; megestrol acetate; melenegestrol acetate; melphalan; menogalyl; mercaptopurine; methotrexate; methotrexate sodium; metoprin; metsuredepa; mitindomide; mitocalcin; mitochromin; mitogillin; mitomarcine; mitomycin; mitospel; mitotane; mitoxantrone hydrochloride Mycophenolic acid; Nocodazoie; Nogaramycin; Ormaplatin; Oxislan; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perphosphamide; Pipobroman; Piposulfan; Pyroxantrone hydrochloride; Plicamycin; Promestan; Porfimer sodium; Porphyromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofulin; Ribopurine; Logretimide; Saf Saffingol; Safingol hydrochloride; Semustine; Simtrazene; Sparphosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan sodium; Tegafur; Trexatron hydrochloride; Temoporfin; Teniposide; Teroxylone; Testolactone; Thiamipurine; Thioguanine; Thiotepa; Thiazofulin; Tirapazamine; Tretol citrate Mifene; Trestron acetate; Trisibilin phosphate; Trimethrexate; Trimethrexate glucuronate; Triptorelin; Tubrozol hydrochloride; Uracil mustard; Uredepa; Bupreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vingricinate sulfate; Vinolervine sulfate; Vinoresine tartrate; Vinrosidine sulfate; Vinzolidinedione sulfate; Borozol; Zeniplatin; Dinostatin; Solbicine hydrochloride.

[0347] Other anticancer agents that may be used in combination with Btk inhibitors include: 20-epi-1,25 dihydroxyvitamin D3; 5-ethinyluracil; abiraterone; acralubicin; akifluven; adesipeno; adzelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amiphostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographicolide; angiogenesis inhibitors; antagonist D; antagonist G; Antarelix; anti-dorsalizing morphogenetic proteins. protein)-1; anti-androgen prostate cancer; anti-estrogen; anti-neoplaston; antisense oligonucleotide; aphydicolin glycinate; apoptosis gene modulator; apoptosis regulator; aprinic acid; ara-CDP-DL-PTBA; arginine deaminase; asraculin; atamestan; atrimustin; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivative; varanol; batimastat; BCR / ABL antar Gonist; Benzochlorine; Benzoylstaurosporine; Beta-lactam derivatives; Beta-aretin; Beta-cramycin B; Betulinic acid; bFGF inhibitors; Bicalutamide; Bisantren; Bisaziridinylspermine; Bisnafide; Bistratin A; Bizeresin; Breflate; Bropyrimine; Budotitan; Butionine sulfoximine; Calcipotriol; Carphostin C; Camptothecin derivatives; Canarypox IL-2; Capecitabine; Carboxamide-amino-triazole; Carboxamide triazole; CaRest M3; CARN-700; cartilage-derived inhibitors; carzeresin; casein kinase inhibitors (ICOS); castanospermine; cecropine B; cetrorelix; chlorine; chloroquinoxalinesulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; colismycin A; colismycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; cristrinatol; cryptophycin 8;Cryptophycin A derivative; Clacin A; Cyclopentatranquinone; Cycloplatam; Cypemycin; Cytarabine ocphosphonate; Cytolytic factor; Cytostatin; Dacliximab; Decitabine; Dehydrodydemnin B; Deslorerin; Dexamethasone; Dexphosphamide; Dexrazoxane; Dexverapamil; Diadiquan; Didemnin B; Zidox; Diethylnorspermine; Dihydro-5-azacitidine; 9-Dioxamycin; Diphenylspiromustine; Docosanol; Dracetron; Doxyfluridine; Doroxifen; Doro Nabinol; Zuocarmycin SA; Ebselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Elemen; Emitefur; Epirubicin; Epristeride; Estramustine analog; Estrogen agonist, estrogen antagonist; Etanidazole; Etoposide phosphate; Exemestane; Fadrozol; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopyridol; Frezelastine; Fluasterone; Fludarabine; Fluorodaunornicin hydrochloride; Holphenimex; Formestan; Ho Striesin; Fotemustine; Gadolinium texaphylline; Gallium nitrate; Gallocitabine; Ganirelix; Gelatinase inhibitors; Gemcitabine; Glutathione inhibitors; Hapsulfame; Heregulin; Hexamethylene bisacetamide; Hypericin; Ibandronic acid; Idarubicin; Idoxifen; Idramanton; Irmofosin; Ilomast; Imidazoacridone; Imiquimod; Immunostimulant peptides; For example, insulin such as growth factor-1 receptor inhibitors; Interferon agonists, interferons; Interleukins; Yobeng Ann; Iododoxorubicin; Ipomeanol, 4-; Iroproct; Irsogladin; Isobengazole; Isohomohalichondrin B; Itasetron; Jasplakinol; Kahalalid F; Lamelalin-N Triacetate; Lanreotide; Reinamycin; Renograstim; Lentinan Sulfate; Leptolstatin; Letrozole; Leukemia Suppressant; Leukocyte Alpha Interferon; Leuprolide + Estrogen + Progesterone; Leuprorelin; Lebamisol; Rialozol; Linear Polyamine Analogues; Lipid-soluble Disaccharide Peptides; Lipid-soluble Platinum Compounds;Lissoclinamide 7; Lovaplatin; Lombrisin; Lometrexol; Ronidamin; Loxoxantrone; Lovastatin; Loxoribine; Lurtotecan; Lutetium Texaphylline; Lisophilin; Soluble peptide; Mytansine; Mannostatin A; Marimast; Masopropyl; Maspin; Matrilysine inhibitor; Matrix metalloproteinase (metalloproteinase) inhibitors; menogalyl; melbaron; meterelin; methioninase; metoclopramide; MIF inhibitors; mifepristone; miltefosine; mirimostim; inappropriate double-stranded RNA; mitogwazone; mitractol; mitomycin analogs; mitonafide; mitotoxin fibroblast growth factor saporin; mitoxantrone; mophalotene; moglamostim; monoclonal antibodies, human chorionic gonadotropins Monophosphoryl lipid A + myobacterium cell wall sk; mopidamole; multidrug resistance gene inhibitor; treatment based on extrinsic tumor suppressor 1; mustard anticancer agent; micaperoxide B; mycobacterial cell wall extract; myriapolon; N-acetyldinaline; N-substituted benzamide; nafarelin; nagrestip; naloxone + pentazocine; napavin; naphterpine; naltograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulator; nitroxide antioxidant; nitrulline; O6-benzylguanine; octreotide ;Oxenon;Oligoligonucleotides;Onapristone;Ondansetron;Ondansetron;Oracin;Oral cytokine inducer;Ormaplatin;Osateron;Oxaliplatin;Oxaunomycin;Paraamine;Palmitoyl rhizoxin;Pamidronic acid;Panaxytriol;Panomifene;Parabactin;Pazeliptin;Pegaspargase;Perdecine;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron;Perphosphamide;Periryl alcohol;Penadinomycin;Phenyl acetate;Phosphatase inhibitors;Picibanil;Pilocarpine hydrochloride;Pirarubicin;Pyritrexime;Placetin A;Placetin B; Plasminogen activator inhibitors; Platinum complexes; Platinum compounds; Platinum-triamine complexes; Porfimer sodium; Porphyromycin; Prednisone; Propylbis-acridone; Prostaglandin J2, proteasome inhibitors; Protein A-based immunomodulatory components; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae; Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; Purpurin; Pyrazoloacridine; Pyridoxylated hemoglobin, polyoxyethylene (poly oxyethylerie) conjugated; RAF antagonist; Lalcitrexed; Ramosetron; RAS farnesyl protein transferase inhibitor; RAS inhibitor; RAS-GAP inhibitor; Demethylated retelliptin; Rhenium Re186 etidronate; Rhizoxin; Ribozyme; RII retinamide; Logretimide; Rohitzkin; Romulutide; Lokinimex; Rubidinone B1; Ruboxyl; Safingol; Saintpine; SarCNU; Sarcophytol A; Sarglamostim; Sdi 1 Mimetic; Semustine; Aging-derived inhibitor 1; Sense oligonucleotide; Signal transduction inhibitor; Signal transduction modulator; Single-chain antigen-binding protein; Schizophyllan; Sobuzoxane; Borocaptate; Sodium phenylacetate; Sorbelol; Somatomedin-binding protein; Sonelmin; Sparphosic acid; Spicamycin D; Spiromustine; Suprenopentin; Spongestatin 1; Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipiamid; Stromelicin inhibitor; Sulfinosine; Superactive vasoactive small intestinal peptide Thyroid antagonist; Salazista; Suramin; Swinesonin; Synthetic glycosaminoglycan; Talimustin; Tamoxifen methiozide; Tauromustine; Tazarotene; Tecogalan sodium; Tegafur; Telrapyrilium; Telomerase inhibitor; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Salibrasticin; Thiocorallin; Thrombopoietin; Thrombopoietin mimetic; Thymalfacin; Thymopoietin receptor agonist; Thymotrinan; Thyroid-stimulating hormone; Ethylethioproprine tin; Tirapazamine;Titanocene dichloride; Topsentin; Toremifene; Pluripotent stem cell factor; Translation inhibitors; Tretinoin; Triacetyluridine; Trisilibine; Trimethrexate; Triptorelin; Tropicetron; Turosteride; Tyrosine kinase inhibitors; Chilphostine; UBC inhibitors; Ubenimex; Urogenital sinus-derived growth inhibitors; Urokinase receptor antagonists; Vapreotide; Variolin B; Vector erythrocyte gene therapy; Veraresol; Veramine; Verzin; Verteporfin; Vinorelbine; Vinxaltin; Vitaxin; Borozol; Zanoterone; Zeniplatin; Zirascorb; and Zinostatin stimalamer.

[0348] Other anticancer agents used in combination with Btk inhibitors include alkylating agents, antimetabolites, natural products, or hormones, such as nitrogen mustards (e.g., mechloretamine, cyclophosphamide, chlorambucil), alkyl sulfons (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine), or triazenes (e.g., decarbazine). Examples of antimetabolites include, but are not limited to, folate analogs (e.g., methotrexate), pyrimidine analogs (e.g., cytarabine), and purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).

[0349] Examples of alkylating agents used in combination with Btk inhibitors include, but are not limited to, nitrogen mustards (e.g., mechloroetamine, cyclophosphamide, chlorambucil, melphalan), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfons (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin), or triazenes (e.g., decarbazine). Examples of antimetabolites include, but are not limited to, folate analogs (e.g., methotrexate), pyrimidine analogs (e.g., fluorouracil, phloxouridine, cytarabine), and purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).

[0350] Examples of anticancer agents that act by stabilizing microtubules to arrest cells in the G2-M phase and can be used in combination with Btk inhibitor compounds include, without limitation, the following commercially available and investigational drugs: elbrozol (also known as R-55104), dorastatin 10 (also known as DLS-10 and NSC-376128), mybobrin isethionate (also known as CI-980), vincristine, NSC-639829, discodermorid (also known as NVP-XX-A-296), ABT-751 (also known as Abbott, E-7010), altriltine (A Altrilutinin A and Altrilutinin C, etc., Sponge statins (Sponge statin 1, Sponge statin 2, Sponge statin 3, Sponge statin 4, Sponge statin 5, Sponge statin 6, Sponge statin 7, Sponge statin 8 and Sponge statin 9, etc.), Semadotin hydrochloride (also known as LU-103793 and NSC-D-669356), Epotilon (Epotilon A, Epotilon B, Epotilon C, also known as Desoxyepotilon A or dEpoA, Epotilon D (KOS-862, also referring to dEpoB and Desoxyepotilon B), Epotilon E, Epotilon F, Epotilon B N-oxide, Epotilon A N-oxide, 16-aza-epotylon B, 21-aminoepotylon B (also known as BMS-310705), 21-hydroxyepotylon D (also known as desoxyepotylon F and dEpoF), 26-fluoroepotylon), auristatin PE (also known as NSC-654663), sobridotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4577) ), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian)Academy of Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651), SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739 (Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl and RPR-258062A), Bitilevamide, Tubricin A, Canadensol, Centaureidine (also known as NSC-106969), T-138067 (Tularik, also known as T-67, TL-138067, TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas (Arizona State University), Oncosidine A1 (also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Physianolide B, Laurimalid, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, also known as MF-569) and TI-138067, Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine, also known as MF-191), TMPN (Arizona StateUniversity), vanadcetin acetylacetonate, T-138026 (Tularik), monsatrol, inakosin (also known as NSC-698666), 3-1AABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607, RPR (Tuiarik, also known as T-900607), RPR-115781 (Aventis), eryuterobin (desmethyleryuterobin, desaetyleleutherobin, isoeryuterobin A, and Z-eryuterobin, etc.), caribaeoside, caribaeolin, halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccaronolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistine (also known as NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseberin B, D-43411 (Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (SPA-110, trifluoroacetate) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi).

[0351] <Biomarkers> In certain embodiments, a method for treating a hematological malignancy in an individual requiring it is disclosed herein, comprising the steps of: (a) administering to the individual an amount sufficient to pharmacokinetically induce multiple cells from the malignancy; and (b) analyzing the pharmacokinetic multiple cells. In some embodiments, the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignancy. In some embodiments, the step of analyzing the pharmacokinetic multiple cells includes preparing a biomarker profile of a population isolated from the multiple cells. In some embodiments, the biomarker expression profile is used to diagnose, determine prognosis, or create a predictive profile for hematological malignancies. In some embodiments, the biomarker profile indicates biomarker expression, biomarker expression levels, biomarker mutations, or the presence of a biomarker. In some embodiments, the biomarker is any cytogenetic, cell surface molecule or protein, or RNA expression marker.

[0352] In some embodiments, the biomarkers are: ZAP70; t(14,18)β-2 microglobulin; mutational states of p53; mutational states of ATM; del(17)p; del(11)q; del(6)q; CD5; CD11c; CD19; CD20; CD22; CD25; CD38; CD103; CD138; expression of secreted, surface, or intracytoplasmic immunoglobulins; V H Mutational states of; or combinations thereof. In some embodiments, the method further includes the step of providing a second cancer treatment regimen based on a biomarker profile. In some embodiments, the method does not further include the step of administering based on a biomarker profile. In some embodiments, the method further includes the step of predicting the effect of a treatment regimen based on a biomarker profile.

[0353] In certain embodiments, the method includes steps of diagnosing, determining prognosis, and creating a predictive profile of hematological malignancies based on the expression or presence of specific biomarkers. In other embodiments, the method further includes steps of strategizing a patient population based on the expression or presence of specific biomarkers in affected lymphocytes. In yet another embodiment, the method further includes steps of determining a treatment regimen for a subject based on the expression or presence of specific biomarkers in affected lymphocytes. In yet another embodiment, the method further includes steps of predicting a subject's response to treatment based on the expression or presence of specific biomarkers in affected lymphocytes.

[0354] In a particular embodiment, the following steps are provided herein: (a) administering to a subject a Btk inhibitor sufficient to increase or increase lymphocyte subgroups in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups, wherein the expression or presence of one or more biomarkers is used to diagnose a hematological malignancy, determine the prognosis of a hematological malignancy, or create a predictive profile of a hematological malignancy. In one embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by immunophenotyping. In another embodiment, the increase or expression in lymphocyte subgroups in the blood is measured by fluorescence-activated cell sorting (FACS).

[0355] In another embodiment, a method for stratifying a patient population of malignant tumors is described herein, comprising the steps of: (a) administering a subject a Btk inhibitor sufficient to increase or increase lymphocyte subgroups in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups, thereby stratifying patients for treatment of hematological malignancies. In one embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by an immunophenotyping assay. In another embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by a fluorescence-activated cell sorter (FACS).

[0356] In another embodiment, a method for determining a treatment regime for a subject having a hematological malignancy is provided herein, comprising the steps of: (a) administering to the subject a Btk inhibitor sufficient to increase or increase lymphocyte subgroups in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups; thereby determining a treatment regime for the treatment of the hematological malignancy using the expression or presence of one or more biomarkers. In one embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by an immunophenotyping assay. In another embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by a fluorescence-activated cell sorter (FACS).

[0357] In another embodiment, a method for predicting the response of a subject with a hematological malignancy to treatment is provided herein, comprising the steps of: (a) administering to the subject a Btk inhibitor sufficient to increase or increase lymphocyte subgroups in the blood; and (b) measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups, thereby using the expression or presence of one or more biomarkers to predict the subject's response to treatment of a hematological malignancy. In one embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by an immunophenotyping assay. In another embodiment, the increase or increase in lymphocyte subgroups in the blood is measured by a fluorescence-activated cell sorter (FACS).

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

[0359] In another embodiment, a method for stratifying a population of subjects having hematological malignancies is described herein, the method comprising the step of measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups in subjects who have been administered a Btk inhibitor, thereby stratifying patients for treatment of hematological malignancies using the expression or presence of one or more biomarkers. In one embodiment, the dose of the Btk inhibitor is sufficient to increase or express lymphocyte subgroups defined by an immunophenotyping method in the blood. In another embodiment, the step of measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups further comprises the steps of isolating, detecting, or measuring one or more types of lymphocytes. In yet another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

[0360] In another embodiment, a method for determining a treatment regimen in a subject having a hematological malignancy is disclosed herein, comprising the step of measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups in a subject who has been administered a Btk inhibitor, wherein the expression or presence of one or more biomarkers is used to determine a treatment regimen for the hematological malignancy. In one embodiment, the dose of the Btk inhibitor is sufficient to increase or express lymphocyte subgroups defined by an immunophenotyping method in the blood. In another embodiment, the step of measuring the expression or presence of one or more biomarkers from one or more lymphocyte subgroups further comprises the steps of isolating, detecting, and measuring one or more types of lymphocytes. In yet another embodiment, the Btk inhibitor is a reversible or irreversible inhibitor.

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

[0362] As discussed herein, any biomarkers relating to hematological malignancies are found in some embodiments utilized by the methods of the present invention. These biomarkers include any biomolecule (found in blood, other bodily fluids, or tissues) or any chromosomal abnormalities that are indicative of hematological malignancies. In certain embodiments, the biomarkers include TdT, CD5, CD11c, CD19, CD20, CD22, CD79a, CD15, CD30, CD38, CD138, CD103, CD25, ZAP-70, mutational states of p53, mutational states of ATM, and IgV HThis includes, but is not limited to, mutational states, deletion of chromosome 17 (del 17p), deletion of chromosome 6 (del 6q), deletion of chromosome 7 (del 7q), deletion of chromosome 11 (del 11q), trisomy 12, deletion of chromosome 13 (del 13q), t(11:14) chromosomal translocation, t(14:18) chromosomal translocation, expression of CD10, CD23, beta-2 microglobulin, bcl-2, CD9, presence of Helicobacter pylori, expression of CD154 / CD40, Akt, NF-κB, WNT, Mtor, ERK, MAPK, and Src tyrosine kinase. In certain embodiments, the biomarkers include ZAP-70, CD5, t(14;18), CD38, β-2 microglobulin, mutational states of p53, mutational states of ATM, deletion of chromosome 17p, deletion of chromosome 11q, surface or intracytoplasmic immunoglobulins, CD138, CD25, deletion of 6q, CD19, CD20, CD22, CD11c, CD103, deletion of chromosome 7q, V Hの This includes mutated states or combinations thereof.

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

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

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

[0366] p53 is a nuclear phosphoprotein that acts as a tumor suppressor. Wild-type p53 is involved in the regulation of cell proliferation and division. p53 binds to DNA and stimulates the production of a protein (p21) that interacts with a cell division-stimulating protein (cdk2). When p21 is bound to cdk2, the cell is inhibited from entering the next stage of cell division. Mutated p53 cannot effectively bind to DNA and therefore prevents p21 from acting as a cell division arrest signal, resulting in uncontrolled cell division and tumorigenesis. p53 also regulates the induction of apoptosis (programmed cell death) in response to DNA damage, cellular stress, or abnormal expression of several oncogenes. Expression of wild-type p53 in several cancer cell lines has been shown to restore growth suppression control (Casey et al. (1991) Oncogene 6:1791-1797; Takahashi et al. (1992) Cancer Res. 52:734-736). p53 mutations 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 lymphoma, L3 type B-cell acute lymphoblastic leukemia, and B-cell chronic lymphocytic leukemia (Gaidano et al. (1991) Proc. Natl. Acad. Sci. USA 88:5413-5417). Abnormalities in p53 have also been found to be associated with B-cell prelymphoblastic leukemia (Lens et al. (1997) Blood 89:2015-2023). The gene for p53 is located in region 17p13.105-p12 on the short arm of chromosome 17.

[0367] Beta-2-microglobulin is an extracellular protein that non-covalently binds to the α chain of the major histocompatibility complex (MHC) of class I. Beta-2-microglobulin is detectable in serum and is an indicator of adverse prognosis in chronic lymphoma (CLL) (Keating et al. (1998) Blood 86:606a) and Hodgekin's Lymphoma (Chronowski et al. (2002) Cancer 95:2534-2538). Beta-2-microglobulin is used clinically in lymphoproliferative disorders, including leukemia, lymphoma, and multiple myeloma, where serum beta-2-microglobulin levels are associated with tumor cell volume, prognosis, and disease activity (Bataille et al. (1983) Br.J.Haematol. 55:439-447; Aviles et al. (1992) Rev.Invest.Clin. 44:215-220). Additionally, p2-microglobulin is useful in staging myeloma patients (Pasqualetti et al. (1991) Eur.J.Cancer 27:1123-1126).

[0368] Furthermore, cytogenetic abnormalities can also be used as markers to create predictive profiles for hematological malignancies. For example, chromosomal abnormalities are found in a large proportion of CLL patients and are useful in predicting the course of CLL. For instance, deletion of chromosome 17p suggests aggressive disease progression. In addition, CLL patients with deletion of chromosome 17p, mutations in p53, or both are known to have a poor response to chemotherapy and rituximab. Allele deletions on chromosome 17p may be a useful prognostic marker in colorectal cancer, and patients with 17p deletion are associated with an increased tendency for disease metastasis in colorectal cancer (Khine et al. (1994) Cancer 73:28-35).

[0369] Deletion of the long arm of chromosome 11 (11q) is one of the most common chromosomal structural abnormalities in various types of lymphoproliferative disorders. Patients with CLL who have a deletion of chromosome 11q and a possible ATM mutation have poorer survival compared to patients who do not have either this deletion or a deletion of 17p. Furthermore, the deletion of 11q is often accompanied by extensive lymph node infiltration (Dohner et al. (1997) Blood 89:2516-2522). This deletion also identifies patients at high risk of disease persistence after high-dose therapy and autologous transplantation.

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

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

[0372] Therefore, biomarkers evaluated by the methods described herein include proteins involved in cell survival and the apoptotic proteins mentioned above, as well as proteins related to hematological malignancies and associated signaling pathways. Measurement of expression or presence may be at the protein or nucleate level. Thus, biomarkers include these proteins and the genes encoding these proteins. When detection is performed at the protein level, biomarker proteins include full-length polypeptides or any detectable fragments thereof, and may include variants of these protein sequences. Similarly, when detection is performed at the nucleotide level, biomarker nucleates include DNA containing full-length encoding sequences, fragments of full-length encoding sequences, variants of these sequences (e.g., spontaneous variants or splice variants), or complements of such sequences. Biomarker nucleates also include RNA such as mRNA containing a full-length sequence encoding a biomarker protein of interest, fragments of a full-length RNA sequence of interest, or variants of these sequences. Biomarker proteins and biomarker nucleates also include variants of these sequences. "Fragment" refers to a portion of a polynucleotide or a portion of an amino acid sequence, and therefore also to the proteins encoded by them. A polynucleotide, which is a fragment of a biomarker nucleotide sequence, generally contains 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 adjacent nucleotides, or generally contains up to the number of nucleotides present in the full-length biomarker polynucleotide disclosed herein. Generally, a fragment of a biomarker polynucleotide will encode at least 15, 25, 30, 50, 100, 150, 200, or 250 adjacent amino acids, or up to the total number of amino acids present in the full-length biomarker protein of the present invention. "Variant" is intended to refer to a substantially similar sequence.Generally, variants of the specific biomarkers of the present invention 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 the biomarker as measured by sequence alignment programs known in the art.

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

[0374] As previously stated, the measurement of the expression and presence of a biomarker of interest can be achieved using any detection method known to those skilled in the art. The “step of detecting expression” or “step of detecting the level of ~” refers to measuring the expression level or presence of a biomarker protein or gene in a biological sample. Thus, the “step of detecting expression” includes examples where the biomarker is measured as not expressed, not detected, expressed at a low level, expressed at a normal level, or overexpressed.

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

[0376] In certain embodiments provided herein, one or more biomarkers include ZAP-70, CD5, t(14;18), CD38, β-2-microglobulin, a mutation in p53, a mutation in ATM, chromosome 17p deletion, chromosome 11q deletion, surface or intracytoplasmic immunoglobulin, CD138, CD25, 6q deletion, CD19, CD20, CD22, CD11c, CD103, chromosome 7q deletion, a mutation in VH, or a combination thereof.

[0377] In a particular embodiment, the method described herein comprises a step of measuring the expression or presence of a combination of biomarkers. In a particular embodiment, the combination of biomarkers is CD19 and CD5, or CD20 and CD5.

[0378] In certain embodiments, the expression or presence of these various biomarkers and any clinically useful prognostic markers in a biological sample may be detected at the protein or nucleate level using, for example, immunohistochemistry techniques or nucleate-based techniques such as insight hybridization or RT-PCR. In one embodiment, the expression or presence of one or more biomarkers may be performed by methods of amplifying nucleates, methods of distributing nucleates, methods utilizing nucleate microarrays (DNA and RNA), or methods for insight hybridization using specifically labeled probes.

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

[0380] In other embodiments, the step of measuring the expression or presence of one or more biomarkers is performed by imaging techniques.

[0381] In yet another embodiment, the expression or presence of one or more biomarkers is measured using a detectable solid substrate. In one embodiment, the detectable solid substrate is paramagnetic nanoparticles functionalized with an antibody.

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

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

[0384] Any method for specifically identifying and measuring biomarkers (e.g., biomarkers, biomarkers of cell survival or proliferation, biomarkers of apoptosis, biomarkers of Btk-mediated signaling pathways) in candidate biological samples was explored. Thus, in some embodiments, the expression level of a biomarker protein of interest in a biological sample is detected using a binding protein or a biologically active variant thereof that can specifically interact with that biomarker protein. Preferably, a labeled antibody, its binding moiety, or other binding partner may be used. As used herein, the word “label” refers to a detectable compound or composition that is directly or indirectly altered to produce a “labeled” antibody. A label may be detectable on its own (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical transformation of a detectable substrate compound or composition.

[0385] Antibodies for detecting biomarker proteins may be monoclonal or polyclonal in origin, or they may be produced by synthesis or recombination techniques. The amount of protein complexes has been measured using standard protein detection methodologies known to those skilled in the art. A protein complex is, for example, a combination of a binding protein and a biomarker, and a binding protein is, for example, an antibody that specifically binds to a biomarker. Detailed reviews of immunological assay design, theory, and protocols can be found in numerous books in the art (see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology) (Greene Publishing and Wiley-Interscience, NY); Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, NY)).

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

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

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

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

[0390] Therefore, in some embodiments, nucleic acid probes are used to assay at the nucleic acid level for the detection of biomarkers or other proteins of interest. The term “nucleic acid probe” refers to any molecule that can selectively bind to a clearly intended target nucleic acid molecule, such as a nucleotide transcript. Probes can be synthesized by those skilled in the art or obtained from a suitable biological preparation. Probes may be specifically designed to be labeled, for example, with radioactive labels, fluorescent labels, enzymes, chemiluminescent tags, colorimetric tags or other labels, or tags or labels known above or in the art. Examples of molecules that can be used as probes include, but are not limited to, RNA and DNA.

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

[0392] In one embodiment, for example, mRNA is immobilized on a solid surface, and the separated mRNA is flowed over, for example, an agarose gel, and the mRNA is transferred from the gel to a membrane, such as nitrocellulose, to contact the probe. In an alternative embodiment, the probe is immobilized on a solid surface, and the mRNA is also in contact with the probe, for example, on a gene tip array. Those skilled in the art can easily adapt known mRNA detection methods to detect mRNA levels encoding biomarkers or other proteins of interest.

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

[0394] The expression levels of RNA of interest may be monitored using membrane blots (such as those used in hybridization analyses like Northern or dot blotting), microwells, sample tubes, gels, beads, or fibers (or any solid carrier containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, incorporated herein by reference. Detection of expression may also include the use of nucleic acid probes in solution.

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

[0396] Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent No. 5,384,261, which are incorporated entirely by reference herein. While planar array surfaces are preferred, arrays may be assembled on multiple surfaces, as well as virtually any shape of surface. Arrays may be beads, gels, polymer surfaces, fibers such as optical fibers, glass, or peptides or nucleic acids on other suitable substrates, each of which is incorporated entirely by reference herein for all purposes, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5,800,992. Arrays may be packaged to enable diagnostic or other operations of a device, including all of the above. See, for example, U.S. Patents Nos. 5,856,174 and 5,922,591, which are incorporated entirely by reference herein.

[0397] <Pharmaceutical Compositions / Formulations> Pharmaceutical compositions may be prepared in conventional ways using one or more physiologically acceptable carriers, which include excipients and adjuvants that facilitate the processing of active compounds into pharmaceutically usable formulations. The appropriate formulation depends on the chosen route of administration. Any known techniques, carriers, and excipients may be used as appropriate and as understood in the art. Summary of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), the entirety of which is incorporated herein by reference.

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

[0399] In certain embodiments, the pharmaceutical composition optionally includes one or more pH adjusters or buffers to modulate the buffer, such as: acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; alkalis such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trihydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amou...

Claims

1. A method for treating hematological malignancies in individuals requiring it, the method being: a. A step of administering to an individual an irreversible Btk inhibitor in an amount sufficient to pharmacokinetically activate multiple cells from a malignant tumor; a. A method characterized by comprising the step of analyzing multiple kinetically activated cells.

2. The method according to claim 1, characterized in that the amount of the irreversible Btk inhibitor is sufficient to induce lymphocytosis of multiple cells from the malignant tumor.

3. The method according to claim 2, characterized in that the malignant tumor is apogosipol.

4. The method according to claim 1, characterized in that the step of treating the hematological malignancy includes the step of managing the hematological malignancy.

5. The method according to claim 1, characterized in that the hematological malignancy is a B-cell malignancy.

6. The method according to claim 1, characterized in that the hematological malignancy is leukemia, lymphoproliferative disorder, or myeloid.

7. The method according to claim 1, characterized in that the kineticated cells are myeloid cells or lymphocyte-like cells.

8. The method according to claim 1, characterized in that the step of analyzing the plurality of kineticated cells includes the step of measuring the peripheral blood concentration of the plurality of kineticated cells.

9. The method according to claim 8, further comprising the step of administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic plurality of cells has increased compared to the concentration of the Btk inhibitor before administration.

10. The method according to claim 8, characterized in that the step of administering the second cancer treatment regimen occurs after a subsequent decrease in the peripheral blood concentration of the pharmacokinetic plurality of cells.

11. The method according to claim 1, characterized in that the step of analyzing the plurality of pharmacokinetic cells includes measuring the period of increase in the peripheral blood concentration of the plurality of pharmacokinetic cells compared to the concentration before administration of the Btk inhibitor.

12. The method according to claim 11, further comprising the step of administering a second cancer treatment regimen after the peripheral blood concentration of the pharmacokinetic plurality of cells has increased over a predetermined period of time.

13. The method according to claim 1, characterized in that the step of analyzing the plurality of kineticated cells includes the step of counting the number of the plurality of kineticated cells in the peripheral blood.

14. The method according to claim 13, further comprising the step of administering a second cancer treatment regimen after the number of the kineticated plurality of cells in the peripheral blood has increased compared to the number before administration of the Btk inhibitor.

15. The method according to claim 14, characterized in that the step of administering the second cancer treatment regimen occurs after a subsequent decrease in the number of the kineticated plurality of cells in the peripheral blood.

16. The method according to claim 1, characterized in that the step of analyzing the plurality of pharmacokinetic cells includes measuring the period of increase in the number of the plurality of pharmacokinetic cells in peripheral blood compared to the number before administration of the Btk inhibitor.

17. The method according to claim 16, further comprising the step of administering a second cancer treatment regimen after the number of the kineticated plurality of cells in the peripheral blood has increased over a predetermined period of time.

18. The method according to claim 1, wherein the step of analyzing the plurality of dynamoized cells includes the step of preparing biomarker properties for a population of cells isolated from the plurality of cells, wherein the biomarker properties indicate biomarker expression, biomarker expression level, mutation in the biomarker, or presence of the biomarker.

19. The method according to claim 18, characterized in that the biomarker is an expression marker for any cytogenetic cell surface molecule, protein, or RNA.

20. The method according to claim 18, characterized in that the biomarker is ZAP70; t(14,18):β-2 microglobulin; a mutation of p53; a mutation of ATM; del(17)p; del(11)q; del(6)q; CD5; CD11c; CD19; CD20; CD22; CD25; CD38; CD103; CD138; secreted surface or intracellular immunoglobulin expression; a mutation of VH; or a combination thereof.

21. The method according to claim 18, further comprising the step of providing a second cancer treatment regimen based on the biomarker characteristics.

22. The method according to claim 18, characterized in that it does not include the step of administering based on the biomarker characteristics.

23. The method according to claim 18, further comprising the step of predicting the effect of a second cancer treatment regimen based on the biomarker characteristics.

24. The method according to claim 1, characterized in that the hematological malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, or non-CLL / SLL lymphoma.

25. The method according to claim 1, characterized in that the hematological malignancy is follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Valdenström macroglobulinemia, multiple myeloma, marginal zone lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, or extranodular marginal zone B-cell lymphoma.

26. The method according to claim 1, characterized in that the hematological malignancy is acute or chronic myeloid (or spinal) leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia.

27. The method according to claim 1, characterized in that the hematological malignancy is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, relapsed or refractory follicular lymphoma, relapsed or refractory CLL; relapsed or refractory SLL; or relapsed or refractory multiple myeloma.

28. The method according to claim 1, characterized in that the Btk inhibitor forms a covalent bond with the cysteine ​​side chain of a Bruton's tyrosine kinase, a Bruton's tyrosine kinase homolog, or a Btk tyrosine kinase cysteine ​​homolog.

29. The method according to claim 1, characterized in that the irreversible Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one.

30. The method according to claim 1, characterized in that the amount of the irreversible Btk inhibitor is from 300 mg / day to 1000 mg / day.

31. The method according to claim 1, characterized in that the amount of the irreversible Btk inhibitor is from 420 mg / day to 840 mg / day.

32. The method according to claim 1, characterized in that the amount of the irreversible Btk inhibitor is about 420 mg / day, about 560 mg / day, or about 840 mg / day.

33. The method according to claim 1, characterized in that the amount of the irreversible Btk inhibitor is approximately 420 mg / day.

34. AUC of the aforementioned Btk inhibitor ０－２４ However, approximately 150 and approximately 3500 ng ＊ The method according to claim 1, characterized in that it is between h / mL.

35. AUC of the aforementioned Btk inhibitor ０－２４ However, approximately 500 and approximately 1100 ng ＊ The method according to claim 1, characterized in that it is between h / mL.

36. The method according to claim 1, characterized in that the Btk inhibitor is administered orally.

37. The method according to claim 1, characterized in that the Btk inhibitor is administered once a day, twice a day, or three times a day.

38. The method according to claim 1, characterized in that the Btk inhibitor is administered until disease progression, unacceptable toxicity occurs, or individual selection occurs.

39. The method according to claim 1, characterized in that the Btk inhibitor is administered daily until disease progression, unacceptable toxicity occurs, or individual selection occurs.

40. The method according to claim 1, characterized in that the Btk inhibitor is administered every other day until disease progression, unacceptable toxicity occurs, or individual selection occurs.

41. The method according to claim 1, characterized in that the Btk inhibitor is a first-line therapy, second-line therapy, third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy.

42. The method according to claim 1, characterized in that the Btk inhibitor is a refractory hematological malignancy.

43. The method according to claim 1, characterized in that the Btk inhibitor is used as maintenance therapy.

44. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises a chemotherapeutic agent, a steroid, an immunotherapy agent, a targeted therapy, or a combination thereof.

45. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen includes a B-cell receptor pathway inhibitor.

46. The method according to claim 45, characterized in that the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof.

47. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises an antibody, a B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapy agent, a DNA damage agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a Hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

48. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen includes chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

49. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally rituximab.

50. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises bendamustine and rituximab.

51. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab.

52. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises cyclophosphamide, vincristine, and prednisone, and optionally rituximab.

53. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises etoposide, doxorubicin, vincristine, cyclophosphamide, prednisolone, and optionally rituximab.

54. The method according to claim 9, 10, 12, 14, 15, 17, or 21, characterized in that the second cancer treatment regimen comprises dexamethasone and lenalidomide.

55. The aforementioned irreversible Btk inhibitor comprises the following structure: 【Chemistry 1】 Here: L ａ CH ２ , O, NH or S; Ar is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; Y is an optional substituent selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; Z is C(=O), OC(=O), NHC(=O), C(=S), S(=O) ｘ , OS(=O) ｘ , NHS(=O) ｘ where x is 1 or 2; R ６ , R ７ , and R ８ H, substituted or unsubstituted C １ -C ４ Alkyl, substituted, or unsubstituted C １ -C ４ Heteroalkyl, substituted, or unsubstituted C ３ -C ６ Cycloalkyl, substituted or unsubstituted C ２ -C ６ Heterocycloalkyl, C １ -C ６ Alkoxyalkyl, C １ -C ８ Alkylaminoalkyl, substituted or unsubstituted C ３ -C ６ Cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C １ -C ４ Alkyl (aryl), substituted or unsubstituted C １ -C ４ Alkyl (heteroaryl), substituted or unsubstituted C １ -C ４ Alkyl (C ３ -C ８ Cycloalkyl, or substituted or unsubstituted C １ -C ４ Alkyl (C ２ -C ８ Each is independently selected from among heterocycloalkyl groups; or R ７ and R ８ They form a single bond together; The method according to claim 1, characterized in that the irreversible Btk inhibitor comprises a pharmaceutically active metabolite, a pharmaceutically acceptable solvent compound, a pharmaceutically acceptable salt, or a pharmaceutically acceptable prodrug thereof.

56. The method according to claim 55, characterized in that La is O.

57. The method according to claim 55, characterized in that Ar is phenyl.

58. Z is C (=O), NHC (=O), or S (=O) ２ The method according to claim 55, characterized in that...

59. Each R ７ and R ８ The method according to claim 55, characterized in that is H.

60. Y is a cycloalkyl ring with four, five, six, or seven members; The method according to claim 55, characterized in that Y is a four-membered, five-membered, six-membered, or seven-membered heterocycloalkyl ring.

61. A method for treating relapsed or refractory non-Hodgkin lymphoma in an individual in need thereof, the method comprising the step of administering to the individual a therapeutically effective amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one.

62. The method according to claim 61, characterized in that the non-Hodgkin lymphoma is relapsed or refractory diffuse large B-cell lymphoma (DLBCL), relapsed or refractory mantle cell lymphoma, or relapsed or refractory follicular lymphoma.

63. The method according to claim 61, characterized in that the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is from 300 mg / day to 1000 mg / day.

64. The method according to claim 61, characterized in that the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is from 420 mg / day to 840 mg / day.

65. The method according to claim 61, characterized in that the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is about 420 mg / day, about 560 mg / day, or about 840 mg / day.

66. The method according to claim 61, characterized in that the amount of (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is about 420 mg / day.

67. (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one AUC ０－２４ However, approximately 150 and approximately 3500 ng ＊ The method according to claim 61, characterized in that it is between h / mL.

68. AUC of the aforementioned Btk inhibitor ０－２４ However, approximately 500 and approximately 1100 ng ＊ The method according to claim 67, characterized in that it is between h / mL.

69. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is administered orally.

70. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is administered once, twice, or three times a day.

71. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is administered until disease progression, unacceptable toxicity, or individual selection.

72. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is administered until disease progression, unacceptable toxicity, or individual selection.

73. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is administered daily until disease progression, unacceptable toxicity, or individual selection.

74. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is administered every other day until disease progression, unacceptable toxicity, or individual selection.

75. The method according to claim 61, characterized in that (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one is a second-line therapy, third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy.

76. The method according to claim 61, characterized in that the Btk inhibitor is used for maintenance therapy.

77. The method according to claim 61, further comprising the step of performing a second cancer treatment regimen.

78. The method according to claim 77, characterized in that the second cancer treatment regimen is administered after the pharmacokinetics of multiple lymphoid cells from non-Hodgkin lymphoma.

79. The method according to claim 77, characterized in that the second cancer treatment regimen is administered after lymphocytosis of multiple lymphoid cells from non-Hodgkin lymphoma.

80. The method according to claim 77, characterized in that the second cancer treatment regimen comprises a chemotherapeutic agent, a steroid, an immunotherapy agent, a targeted therapy, or a combination thereof.

81. The method according to claim 77, characterized in that the second cancer treatment regimen includes a B cell receptor pathway inhibitor.

82. The method according to claim 81, characterized in that the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof.

83. The method according to claim 77, characterized in that the second cancer treatment regimen includes an antibody, a B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapy agent, a DNA damage agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a Hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

84. The method according to claim 77, characterized in that the second cancer treatment regimen includes chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

85. The method according to claim 77, characterized in that the second cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally rituximab.

86. The method according to claim 77, characterized in that the second cancer treatment regimen comprises bendamustine and rituximab.

87. The method according to claim 77, characterized in that the second cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab.

88. The method according to claim 77, characterized in that the second cancer treatment regimen comprises cyclophosphamide, vincristine, and prednisone, and optionally rituximab.

89. The method according to claim 77, characterized in that the second cancer treatment regimen comprises etoposide, doxorubicin, vincristine, cyclophosphamide, prednisolone, and optionally rituximab.

90. The method according to claim 77, characterized in that the second cancer treatment regimen comprises dexamethasone and lenalidomide.

91. A method for treating diffuse large B-cell lymphoma, activated B-cell-like subtype (ABC-DLBCL) in an individual requiring such treatment, the method comprising the step of administering an irreversible Btk inhibitor to the individual in an amount ranging from 300 mg / day to 1000 mg / day.

92. The method according to claim 91, further comprising the step of diagnosing an individual having diffuse large B-cell lymphoma or an activated B-cell-like subtype (ABC-DLBCL) by determining the gene sequence of one or more biomarkers of multiple lymphoid cells isolated from diffuse large B-cell lymphoma.

93. The method according to claim 91, characterized in that the irreversible Btk inhibitor is (R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine-1-yl)piperidine-1-yl)prop-2-en-1-one.

94. The method according to claim 91, characterized in that the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by a CD79B mutation.

95. The method according to claim 94, characterized in that the CD79B mutation is a mutation in the tyrosine-based activation motif (ITAM) signaling module of an immune receptor.

96. The method according to claim 94, characterized in that the CD79B mutation is a missense mutation in the tyrosine of the tyrosine-based activation motif (ITAM) of the first immune receptor.

97. The method according to claim 94, characterized in that the CD79B mutation increases surface BCR expression and reduces lyn kinase activity.

98. The method according to claim 91, characterized in that the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by a CD79A mutation.

99. The method according to claim 98, characterized in that the CD79A mutation is located in the tyrosine-based activation motif (ITAM) signaling module of an immune receptor.

100. The method according to claim 98, characterized in that the CD79A mutation is a splice donor site mutation in the tyrosine-based activation motif (ITAM) signaling module of an immune receptor.

101. The method according to claim 98, characterized in that the CD79A mutation removes the tyrosine-based activation motif (ITAM) signaling module of an immune receptor.

102. The method according to claim 91, characterized in that the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by mutations in MyD88, A20, or a combination thereof.

103. The method according to claim 102, characterized in that the MyD88 mutation is an amino acid substitution L265P in the MYD88 Toll / IL-1 receptor (TIR) ​​domain.

104. The method according to claim 91, characterized in that the amount of irreversible Btk inhibitor is from 420 mg / day to 840 mg / day.

105. The method according to claim 91, characterized in that the amount of irreversible Btk inhibitor is about 420 mg / day, about 560 mg / day, or about 840 mg / day.

106. The method according to claim 91, characterized in that the amount of irreversible Btk inhibitor is approximately 420 mg / day.

107. AUC of the aforementioned Btk inhibitor ０－２４ However, approximately 150 and approximately 3500 ng ＊ The method according to claim 91, characterized in that it is between h / mL.

108. AUC of the aforementioned Btk inhibitor ０－２４ However, approximately 500 and approximately 1100 ng ＊ The method according to claim 107, characterized in that it is between h / mL.

109. The method according to claim 91, characterized in that the irreversible Btk inhibitor is administered orally.

110. The method according to claim 91, characterized in that the irreversible Btk inhibitor is administered daily until disease progression, unacceptable toxicity occurs, or individual selection occurs.

111. The method according to claim 91, characterized in that the irreversible Btk inhibitor is administered every other day until disease progression, unacceptable toxicity, or individual selection occurs.

112. The method according to claim 91, characterized in that the irreversible Btk inhibitor is a first-line therapy, second-line therapy, third-line therapy, fourth-line therapy, fifth-line therapy, or sixth-line therapy.

113. The method according to claim 91, characterized in that the irreversible Btk inhibitor treats refractory hematological malignancies.

114. The method according to claim 91, characterized in that the irreversible Btk inhibitor is used as maintenance therapy.

115. The method according to claim 91, further comprising the step of administering at least one additional cancer treatment regimen.

116. The method according to claim 115, characterized in that the additional cancer treatment regimen includes a chemotherapeutic agent, an immunotherapy agent, a steroid, radiotherapy, targeted therapy, or a combination thereof.

117. The method according to claim 116, characterized in that the second cancer treatment regimen includes an antibody, a B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapy agent, a DNA damage agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a Hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

118. A method for determining a cancer treatment regimen for an individual with a hematological malignancy, the method being: a. A step of administering to an individual an irreversible Btk inhibitor in an amount sufficient to pharmacokinetically activate multiple cells from a malignant tumor; b. The process of analyzing multiple kinetically activated cells; c. A method characterized by including the step of selecting a cancer treatment regimen.

119. The method according to claim 118, characterized in that the cancer treatment regimen includes a chemotherapeutic agent, a steroid, an immunotherapy agent, a targeted therapy, or a combination thereof.

120. The method according to claim 118, characterized in that the cancer treatment regimen includes a B cell receptor pathway inhibitor.

121. The method according to claim 118, characterized in that the cancer treatment regimen includes a CD79A inhibitor, a CD79B inhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3K inhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, or a combination thereof.

122. The method according to claim 118, characterized in that the cancer treatment regimen includes an antibody, a B cell receptor signaling inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTOR inhibitor, a radioimmunotherapy agent, a DNA damage agent, a bioprecipitation inhibitor, a histone deacetylase inhibitor, a protein kinase inhibitor, a Hedgehog inhibitor, an Hsp90 inhibitor, a telomerase inhibitor, a Jak1 / 2 inhibitor, a protease inhibitor, a PKC inhibitor, a PARP inhibitor, or a combination thereof.

123. The method according to claim 118, characterized in that the cancer treatment regimen includes chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

124. The method according to claim 118, characterized in that the cancer treatment regimen comprises cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, and optionally rituximab.

125. The method according to claim 118, characterized in that the cancer treatment regimen comprises bendamustine and rituximab.

126. The method according to claim 118, characterized in that the cancer treatment regimen comprises fludarabine, cyclophosphamide, and rituximab.

127. The method according to claim 118, characterized in that the cancer treatment regimen comprises cyclophosphamide, vincristine, and prednisone, and optionally rituximab.

128. The method according to claim 118, characterized in that the cancer treatment regimen comprises etoposide, doxorubicin, vincristine, cyclophosphamide, prednisolone, and optionally rituximab.

129. The method according to claim 118, characterized in that the cancer treatment regimen comprises dexamethasone and lenalidomide.

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