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`3 7°C with 5% CO2 . After 72 hours of culture, cell viability was determined using an (3-( 4,5-
`
`dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4-sulfophenyl)-2H-tetrazolium) (MTS)
`
`assay (Cell Titer 96, Promega). Viability data were used to generate cell viability curves for each
`
`drug alone and in combination for each sample. The potential synergy of the combination of the
`
`BTK inhibitor of Formula XVIII and the PBK-8 inhibitor of Formula IX at a given equimolar
`
`concentration was determined using the median effect model as described in Chou and Talalay,
`
`Adv Enzyme Regul. 1984, 22, 27-55. The statistical modeling was run in R using a script that
`
`utilizes the median effect model as described in Lee et al., J. Biopharm. Stat. 2007, 17, 461-80.
`
`A value of 1, less than 1, and greater than 1 using R defines an additive interaction, synergistic
`
`and antagonistic, respectively. The Lee et al. method calculates a 95% confidence interval for
`
`each data point. For each viability curve, to be considered synergistic, a data point must have an
`
`interaction index below 1 and the upper confidence interval must also be below 1. In order to
`
`summarize and demonstrate collective synergy results, an interaction dot blot was generated for
`
`the primary patient samples.
`
`[00297] A similar approach was utilized to study diffuse large B cell lymphoma (DLBCL)
`
`(TMD8) and MCL (MINO) cell Lines. Cells were treated with each drug alone and with six
`
`equimolar concentrations of the BTK inhibitor of Formula XVIII and the PBK-8 inhibitor of
`
`Formula IX ranging from 0.003 nM to 1.0 µM (for TMD8) or 0.03 nM to 10 µM (for MINO) on
`
`96-well plates in triplicate. Plated cells were then cultured in standard conditioned media plus
`
`FBS at 37°C with 5% CO2 . After 72 hours of culture, viability was determined using an MTS
`
`assay (Cell Titer 96, Promega). Viability data were used to generate cell viability curves for
`
`each drug alone and in combination for each sample. The results of the experiments described in
`
`this example are shown in FIG. 1, FIG. 2, FIG. 3, and FIG. 4.
`
`Example 2 - Synergistic Combination of a BTK inhibitor and a PBK-o inhibitor
`
`[00298] Combination experiments were performed to determine the synergistic, additive, or
`
`antagonistic behavior of drug combinations using the Chou/Talalay method/algorithm by
`
`defining combination indexes for drug combinations. Information about experimental design for
`
`evaluation of synergy is described in e.g. Chou and Talalay, Adv. Enzyme Regul. 1984, 22, 27-55
`
`and more generally in e.g. Greco et al., Pharrnacol. Rev. 1995, 47, 331-385. The study was
`
`performed using the BTK inhibitor of Formula (XVIII) and the PBK-8 inhibitor of Formula
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`(IX). Single agent activities were first determined in the various cell lines and subsequently, the
`
`combination indexes were established using equimolar ratios taking the single agent drug EC50s
`
`into consideration. For individual agents that displayed no single agent activity, equimolar ratios
`
`were used at fixed concentrations to establish combination indexes. The readout from 72 hour
`
`proliferation assays using Cell TiterGlo (ATP content of remaining cells) determined the fraction
`
`of cells that were effected as compared to untreated cells (Fa = fraction affected =
`
`( 1- ( ( cells +
`
`inhibitor)- background signal)/ ((cells+ DMSO)- background signal)).
`
`[00299] The combination index obtained was ranked according to Table 1.
`
`TABLE 1. Combination Index (Cl) Ranking Scheme
`
`Ran2e of CT
`<0.1
`0.1-0.3
`0.3-0.7
`0.7-0.85
`0.85-0.9
`0.9-1.1
`1.1-1.2
`1.2-1.45
`1.45-3.3
`3.3-10
`>10
`
`Descriution
`Very strong synergism
`Strong synenrism
`Synergism
`Moderate synergism
`Slight synergism
`Nearly additive
`Slight antagonism
`Moderate antagonism
`Antagonism
`Strong antagonism
`Very strong antagonism
`
`[00300] The detailed results of the cell line studies for the BTK inhibitor of Formula (XVIII)
`
`and the PBK-8 inhibitor of Formula (IX) are given in FIG. 5 to FIG. 37. The results of the cell
`
`line studies are summarized in Table 2.
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`TABLE 2. Summary of results of the combination of a BTK inhibitor with a PBK-8 inhibitor (S
`
`= synergistic, A= additive, X = no effect).
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`Attorney Docket No. 055112-5002-WO
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`Cell Line
`Raii
`Ramos
`Daudi
`Mino
`Pfeiffer
`DOHH
`REC-I
`U937
`K562
`SU-DHL-1
`SU-DHL-2
`HBL-1
`TMD8
`LY19
`LY7
`LYI
`SU-DHL-6
`SupB15
`CCRF
`
`Indication
`Burkitt's
`Burkitt's
`Burkitt's
`MCL
`iNHL
`iNHL
`iNHL
`Myeloid
`CML
`ABC
`ABC
`ABC
`ABC
`GCB
`GCB
`GCB
`GCB
`B-ALL
`B-ALL
`
`ED25
`s
`X
`s
`s
`s
`s
`s
`s
`X
`s
`s
`s
`s
`X
`s
`X
`s
`s
`s
`
`EDS0
`s
`X
`s
`s
`s
`s
`s
`s
`X
`A
`s
`s
`s
`X
`s
`X
`s
`s
`A/S
`
`ED75
`s
`X
`s
`s
`s
`s
`A
`s
`X
`X
`s
`s
`s
`X
`s
`X
`s
`s
`X
`
`ED90
`s
`X
`s
`s
`s
`s
`A
`s
`X
`X
`s
`s
`s
`X
`s
`X
`s
`s
`X
`
`Example 3 - BTK Inhibitory Effects on Solid Tumor Microenvironment m an Orthotopic
`
`Pancreatic Cancer Model
`
`[00301] An orthotopic pancreatic cancer model was used to investigate the therapeutic efficacy
`
`of the combination of the BTK inhibitor of Formula (XVTTT) and the PBK-8 inhibitor of Formula
`
`(IX) through treatment of the solid tumor microcnvironmcnt. Mice were dosed orally with 15
`
`mg/kg of Formula (XVIII), 15 mg/kg of Formula (IX), or a combination of 15 mg/kg of both
`
`drugs.
`
`[00302] Cell
`
`line derived
`
`from KrasG12D;Trp53R172H;Pdxl-Cre
`
`(KPC) mice were
`
`orthotopically implanted into the head of the pancreas after 35 passages. Based on the mice
`background from where the cell lines were generated, 1 x 106 cells were injected in C57BL/6
`
`mice. Throughout the experiment, animals were provided with food and water ad libitum and
`
`subjected to a 12-h dark/light cycle. Animal studies were performed in accordance with the U.S.
`
`Public Health Service "Guidelines for the Care and Use of Laboratory Animals" (IACUC). After
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`euthanization, pancreatic tumors were dissected out, weighed and single cell suspensions were
`
`prepared for flow cytometry analysis.
`
`[00303] Results of the experiments are shmvn in FIG. 38, which illustrates tumor growth
`
`suppression in the orthotopic pancreatic cancer model. The statistical p-value (presumption
`
`against null hypothesis) is shown for each tested single agent and for the combination against the
`
`vehicle. The results show that all three treatments provide statistically significant reductions in
`
`tumor volume in the pancreatic cancer model.
`
`[00304] Additional
`
`results of the experiments
`
`relating
`
`to
`
`treatment of the
`
`tumor
`
`microenvironment are shown in FTG. 39 to FTG. 41. FTG. 39 shows the effects of oral dosing
`
`with 15 mg/kg of the BTK inhibitor of Formula (XVIII), 15 mg/kg of the PI3K inhibitor of
`
`Formula (IX), or a combination of both drugs on myeloid tumor-associated macrophages
`
`(TAMs) in pancreatic tumor-bearing mice. FIG. 40 illustrates the effects of oral dosing with 15
`
`mg/kg of the BTK inhibitor of Formula (XVIII), 15 mg/kg of the PI3K inhibitor of Formula (IX),
`
`or a combination of both inhibitors on myeloid-derived suppressor cells (MDSCs) in pancreatic
`
`tumor-bearing mice. FIG. 41 illustrates the effects of oral dosing with 15 mg/kg of the BTK
`
`inhibitor of Formula (XVIII), 15 mg/kg of the PI3K inhibitor of Formula (IX), or a combination
`
`of both inhibitors on regulatory T cells (Tregs) in pancreatic tumor-bearing mice. The results
`
`shown in FIG. 39 to FIG. 41 demonstrate that administration of the BTK inhibitor of Formula
`
`(XVIII) and the combination of the BTK inhibitor of Formula (XVIII) and the PBK inhibitor of
`
`Formula (IX) reduce immunosuppressive tumor associated myeloid cells and Tregs in pancreatic
`
`tumor-bearing mice. Overall, BTK inhibition with Formula (XVIII) or a combination of
`
`Formula (XVIII) and Formula (IX) significantly reduced tumor burden in an aggressive
`
`orthotopic PDA model, decreased immature myeloid infiltrate, reduced the number of tumor
`
`associated macrophages, and reduced the number of immunosuppressive Tregs, demonstrating a
`
`strong effect on the tumor microenvironment.
`
`Example 4 - BTK Inhibitory Effects on Solid Tumor Microenvironment in an Ovarian Cancer
`
`Model
`
`[00157] The ID8 syngeneic orthotropic ovarian cancer murine model was used to investigate the
`
`therapeutic efficacy of the BTK inhibitor of Formula (XVIII) through treatment of the solid
`
`tumor microenvironment. Human ovarian cancer models, including the ID8 syngeneic
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`orthotropic ovarian cancer model and other animal models, are described in Fong and Kakar, J.
`
`Ovarian Res. 2009, 2, 12; Greenaway et al., Gynecol. Oneal. 2008, 108, 385-94; Urzua et al.,
`
`Tumour Biol. 2005, 26, 236-44; Janat-Amsbury et al., Anticancer Res. 2006, 26, 3223-28; Janat(cid:173)
`
`Amsbury et al., Anticancer Res. 2006, 26, 2785-89. Animals were treated with vehicle or
`
`Formula (XVIII), 15 mg/kg/BID given orally. The results of the study are shown in FIG. 42,
`
`FIG. 43, FIG. 44, FIG. 45, FIG. 46, FIG. 47, FIG. 48, and FIG.49.
`
`[00158] FIG. 42 and FIG. 43 demonstrate that the BTK inhibitor of Formula (XVIII) impairs
`
`ID8 ovarian cancer growth in the ID8 syngeneic murine model. FIG. 44 shows that tumor
`
`response to treatment with the BTK inhibitor of Formula (XVIII) correlates with a significant
`
`reduction in immunosuppressive tumor-associated lymphocytes in tumor-bearing mice. FIG. 45
`
`shows treatment with the BTK inhibitor of Formula (XVIII) impairs ID8 ovarian cancer growth
`
`(through reduction in tumor volume) in the syngeneic murine model. FIG. 46 and FIG. 47 show
`
`that the tumor response induced by treatment with the BTK inhibitor of Formula (XVlll)
`
`correlates with a significant reduction in immunosuppressive B cells in tumor-bearing mice.
`
`FIG. 48 and FIG. 49 show that the tumor response induced by treatment with the BTK inhibitor
`
`of Formula (XVIII) correlates with a significant reduction in immunosuppressive tumor
`
`associated Tregs and an increase in CDS+ T cells.
`
`[00159] The results shown in FIG. 42 to FIG. 49 illustrate the surprising efficacy of the BTK
`
`inhibitor of Formula (XVIII) in modulating tumor microenvironment in a model predictive of
`
`efficacy as a treatment for ovarian cancer in humans.
`
`Example 5 - BTK Inhibitory Effects on Solid Tumor Microenvironment Through Modulation of
`
`Tumor-Infiltrating MDSCs and T AMs
`
`[00160] A study was performed to observe potential reduction in tumor burden through
`
`modulation of tumor infiltrating MDSCs and TAMs using the BTK inhibitor of Formula (XVIII)
`
`and/or gemcitabine ('-Gem"). In this study, KPC derived mouse pancreatic cancer cells
`
`(KrasG12D;Trp53Rl 72H;Pdxl-Cre) were injected into the pancreases. Animals were treated
`
`with (1) vehicle; (2) Formula (XVIII), 15 mg/kg/BID given orally; (3) gemcitabine 15 mg/kg
`
`intravenous (IV) administered every 4 days for 3 injections; or (4) Formula (XVIII), 15
`
`mg/kg/BID given orally with together with gemcitabine, 15 mg/kg IV administered every 4 days
`
`for 3 injections.
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`[00161] Single cell suspensions from tumor samples. Mouse tumor tissue was collected and
`
`stored in PBS/0.1 % soybean trypsin inhibitor prior to enzymatic dissociation. Samples were
`
`finely minced with a scissors and mouse tissue was transferred into DMEM containing 1.0
`
`mg/ml collagenase IV (Gibco), 0.1% soybean trypsin inhibitor, and 50 U/ml DNase (Roche) and
`
`incubated at 37C for 30 min. with constant stirring while human tissue was digested in 2.0 mg/ml
`
`collagenase IV, 1.0 mg/ml hyluronidase, 0.1% soybean trypsin inhibitor, and 50 U/ml DNase for
`
`45 minutes. Suspensions were filtered through a l 00 micron filter and washed with F ACS buffer
`
`(PBS/0.5% BSA/2.0 mM EDTA) prior to staining. Two million total cells were stained with
`
`antibodies as indicated. Intracellular detection of FoxP3 was achieved following
`
`pernieabilization with BD Perni Buffer TH (BD Biosciences) and eBioscience Fix/Perni
`
`respectively. Following surface staining, samples were acquired on a BD Fortessa and analyzed
`
`using FlowJo (Treestar) software.
`
`[00162] ln FIG. 50, the reduction in tumor size upon treatment is shown. The effects on
`
`particular cell subsets are shown in the flow cytometry data presented in FIG. 51, FIG. 52, FIG.
`
`53, and FIG. 54.
`
`[00163] The results shown in FIG. 50 to FIG. 54 illustrate reduction in tumor burden by
`
`modulating the tumor infiltrating MDSCs and TAMs, which affects Treg and CD8- T cell levels,
`
`through inhibition ofBTK using Formula (XVIII).
`
`Example 6- Effects ofBTK Inhibitors on Thrombosis
`
`[00164] Clinical studies have shown that targeting the BCR signaling pathway by inhibiting
`
`BTK produces significant clinical benefit (Byrd, et al., N. Engl. J. Afed. 2013, 369(1), 32-42,
`
`Wang, et al., N. Engl. J. Afed. 2013, 369(6), 507-16). However, in these studies, bleeding has
`
`been reported in up to 50% of ibrutinib-treated patients. Most bleeding events were of grade 1-2
`
`(spontaneous bruising or petechiae) but, in 5% of patients, they were of grade 3 or higher after
`
`trauma. These results are reflected in the prescribing information for ibrutinib, where bleeding
`
`events of any grade, including bruising and petechiae, were reported in approximately half of
`
`patients treated with ibrutinib (IMBRUVICA package insert and prescribing information, revised
`
`July 2014, U.S. Food and Drug Administration).
`
`[00165] Constitutive or aberrant activation of the BCR signaling cascade has been implicated in
`
`the propagation and maintenance of a variety of B cell malignancies. Small molecule inhibitors
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`ofBTK, a protein early in this cascade and specifically expressed in B cells, have emerged as a
`
`new class of targeted agents. There are several BTK inhibitors, including Formula XXVII (CC-
`
`292), and Formula XX-A (PCI-32765, ibmtinib), in clinical development. Importantly, early
`
`stage clinical trials have found ibrutinib to be particularly active in chronic lymphocytic
`
`leukemia (CLL) and mantle cell lymphoma (MCL), suggesting that this class of inhibitors may
`
`play a significant role in various types of cancers (Aalipour and Advani, Br. J. Haematol. 2013,
`
`163, 436-43). However, their effects are not limited to leukemia or lymphomas as platelets also
`
`rely on the T ec kinases family members BTK and T ec for signal transduction in response to
`
`various thrombogenic stimuli (Oda, et al., Blood 2000, 95(5), 1663-70; Atkinson, et al. Blood
`
`2003, 102(/0), 3592-99). In fact, both Tee and BTK play an important role in the regulation of
`
`phospholipase Cy2 (PLCy2) downstream of the collagen receptor glycoprotein VI (GPVI) in
`
`human platelets. In addition, BTK is activated and undergoes tyrosine phosphorylation upon
`
`challenge of the platelet thrombin receptor, which requires the engagement of allbf33 integrin
`
`and PI3K activity (Laffargue, et al., FEBS Lett. 1999, 443(1), 66-70). It has also been implicated
`
`in GPTba-dependent thrombus stability at sites of vascular injury (Liu, et al., Blood 2006, 108(8),
`
`2596-603). Thus, BTK and Tee arc involved in several processes important in supporting the
`
`formation of a stable hemostatic plug, which is critical for preventing significant blood loss in
`
`response to vascular injury. Hence, the effects of the BTK inhibitor of Formula (XVIII) and
`
`ibmtinib were evaluated on human platelet-mediated thrombosis by utilizing the in vivo human
`
`thrombus formation in the VWF HAI mice model described in Chen et al. Nat. Biotechnol. 2008,
`
`26(1), 114-19.
`
`[00166] Administration of anesthesia, insertion of venous and arterial catheters, fluorescent
`labeling and administration of human platelets (5 x 108/ml), and surgical preparation of the
`cremaster muscle in mice have been previously described (Chen et al. Nat Biotechnol. 2008,
`
`26(1), 114-19). Injury to the vessel wall of arterioles (~40-65 mm diameter) was performed
`
`using a pulsed nitrogen dye laser ( 440 nm, Photonic Instruments) applied through a 20x water(cid:173)
`
`immersion Olympus objective (LUMPlanFl, 0.5 numerical aperature (NA)) of a Zeiss Axiotech
`
`vario microscope. Human platelet and wall interactions were visualized by fluorescence
`
`microscopy using a system equipped with a Yokogawa CSU-22 spinning disk confocal scanner,
`
`iXON EM camera, and 488 nm and 561 nm laser lines to detect BCECF-labeled and rhodamine(cid:173)
`
`labe1ed platelets, respectively (Revolution XD, Andor Technology). The extent of thrombus
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`formation was assessed for 2 min after injury and the area (µm 2
`
`) of coverage determined (Image
`
`IQ, Andor Technology). For the Formula (XVIII), Formula (XXVII) (CC-292), and Formula
`
`(XX-A) (ibrntinib) inhibition studies, the BTK inhibitors were were added to purified human
`
`platelets for 30 min before administration.
`
`[00167] The in vivo throbus effects of the BTK inhibitors, Formula (XVIII), Formula (XXVII)
`
`(CC-292), and Formula (X-X-A) (ibrutinib ), were evaluated on human platelet-mediated
`
`thrombosis by utilizing the in vivo human thrombus formation in the VWF HAI mice model,
`
`which has been previously described (Chen et al. Nat Biotechnol. 2008, 26( 1), 114-19). Purified
`
`human platelets were preincubated with various concentrations of the BTK inhibitors (0.1 µM,
`
`0.5 µM, or 1 µM) or DMSO and then administered to VWF HAI mice, followed by laser(cid:173)
`
`induced thrombus formation. The BTK inhibitor-treated human platelets were fluorescently
`
`labeled and infused continuously through a catheter inserted into the femoral artery. Their
`
`behavior in response to laser-induced vascular injury was monitored in real time using two(cid:173)
`
`channel confocal intravital microscopy (Furie and Furie, J. Clin. Invest. 2005, 115 (12), 2255-62).
`
`Upon induction of arteriole injury untreated platelets rapidly formed thrombi with an average
`thrombus size of 6,450 ± 292 mm2 (mean± s.e.m.), as shown in FIG. 55 and FIG. 56. Similarly,
`
`Formula (XVIII) (1 µM) treated platelets formed a slightly smaller but not significantly different
`thrombi with an average thrombus size of 5733 ± 393 mm2 (mean± s.e.m.). In contrast, a
`
`dramatic reduction in thrombus size occured in platelets pretreated with 1 µM of Formula XX-A
`(ibrntinib), 2600 ± 246 mm2 (mean± s.e.m.), resulting in a reduction in maximal thrombus size
`
`by approximately 61% cornpared with control (P > 0.001) (FIG. 55 and 57), Similar results were
`
`obtained with platelets pretreated with 500 nM of Formula (XVIII) or ibrutinib: thrombus size of
`5946 ± 283 mm2
`, and 2710 ± 325 mm2 respectively. These initial results may provide some
`
`mechanic background and explaination on the reported 44c% bleeding related adverse event rates
`
`in the Phase III RESONATE™ study comparing ibrutinib with ofatumumab. The results
`
`obtained for Formula XXVII (CC-292) were similar to that for Formula XX-A (ibrutinib ), as
`
`shown in FIG. 55, 56, and 57. The effect of the BTK inhibitor concentration is shown in FIG.
`
`58. These results demonstrate the surprising advantage of the BTK inhibitor of Formula (XVIII),
`
`which does not interfere with thrombus formation, while the BTK inhibitors of Formula XXVII
`
`(CC-292) and Formula XX-A (ibrutinib) interfere with thrombus formation.
`
`[00168] The objective of this study was to evaluate in vivo thrombus formation in the presence
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`ofBTK inhibitors. In vivo testing of novel antiplatelet agents requires informative biomarkers.
`
`By utilizing a genetic modified mouse von Willebrand factor (VWFR1326H) model that
`
`supports human but not mouse platelet-mediated thrombosis, we evaluated the effects of
`
`Formula (XVIII), Formula XXVII (CC-292), and Formula XX-A (ibrutinib) on thrombus
`
`formation. These results show that Formula (XVIII) had no significant effect on human platelet(cid:173)
`
`mediated thrombus formation while Formula XX-A (ibrutinib) was able to limit this process,
`
`resulting in a reduction in maximal thrombus size by 61 % compared with control. Formula
`
`XXVII (CC-292) showed an effect similar to Formula XX-A (ibrutinib). These results, which
`
`show reduced thrombus formation for ibrutinib at physiologically relevant concentrations, may
`
`provide some mechanistic background for the Grade 2:: 3 bleeding events (eg, subdural
`
`hematoma, gastrointestinal bleeding, hematuria and postprocedural hemorrhage) that have been
`
`reported in~ 6% of patients treated with Formula XX-A (ibrutinib).
`
`[00169] GPVl platelet aggregation was measured for Formula (XVlll) and Formula XX-A
`
`(ibrutinib ). Blood was obtained from untreated humans, and platelets were purified from
`
`plasma-rich protein by centrifugation. Cells were resuspended to a final concentration of
`
`350,000/~tL in buffer containing 145 mmol/L NaCl, 10 mmol/L HEPES, 0.5 mmol/L Na2HPO4,
`
`5 mmol/L KCl, 2 mmol/L MgCh, 1 mmol/L CaCh, and 0.1% glucose, at pH 7.4. Stock
`
`solutions of Convulxin (CVX) GPVI were prepared on the day of experimentation and added to
`
`platelet suspensions 5 minutes (37 °C, 1200 rpm) before the induction of aggregation.
`
`Aggregation was assessed with a Chronolog Lumi-Aggregometer (model 540 VS; Chronolog,
`
`Havertown, PA) and permitted to proceed for 6 minutes after the addition of agonist. The results
`
`are reported as maximum percent change in light transmittance from baseline with platelet buffer
`
`used as a reference. The results are shown in FIG. 59.
`
`[00170] In FIG. 60, the results of CVX-induced (250 ng/mL) human platelet aggregation results
`
`before and 15 min after administration of the BTK inhibitors to 6 healthy individuals are shown.
`
`[00171] The results depicted in FIG. 59 and FIG. 60 indicate that the BTK inhibitor of Formula
`
`XX-A (ibrutinib) significantly inhibits GPVI platelet aggregation, while the BTK inhibitor of
`
`Formula (XVIII) does not, further illustrating the surprising benefits of the latter compound.
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`Example 7 - Study of a BTK Inhibitor and a Combination of a BTK Inhibitor and a PI3K
`
`Inhibitor in Canine Lymphoma
`
`[00172] Canine B cell lymphoma exists as a pathological entity that is characterized by large
`
`anaplastic, centroblastic or immunoblastic lymphocytes with high proliferative grade, significant
`
`peripheral lymphadenopathy and an aggressive clinical course. While some dogs respond
`
`initially to prednisone, most canine lymphomas progress quickly and must be treated with
`
`combination therapies, including cyclophosphamide, vincristine, doxorubicin, and prednisone
`
`(CHOP), or other cytotoxic agents. In their histopathologic features, clinical course, and high
`
`relapse rate after initial treatment, canine B cell lymphomas resemble diffuse large B cell
`
`lymphoma (DLBCL) in humans. Thus, responses of canine B cell lymphomas to experimental
`
`treatments are considered to provide proof of concept for therapeutic candidates in DLBCL.
`
`[00173] In this example, companion dogs with newly diagnosed or relapsed/refractory LSA
`
`were enrolled on a veterinary clinical trial of the BTK inhibitor of Formula (XVIII) ("Arm l ") or
`
`the BTK inhibitor of Formula (XVIII) and the PBK-8 inhibitor of Formula (IX) ("Arm 2").
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`Enrollment has completed for Arm 1 and is ongoing for Arm 2. With approximately 1/3 of Arm
`
`2 subjects treated, the preliminary results show that combined treatment with the BTK inhibitor
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`of Formula (XVIII) and the PBK-8 inhibitor of Formula (IX) may have greater efficacy than
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`treatment with the BTK inhibitor of Formula (XVIII) alone in aggressive lymphoma.
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`[00174] Twenty-one dogs were treated in Arm 1 with the BTK inhibitor of Formula (XVIII) at
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`dosages of 2.5 mg/kg once daily to 20 mg/kg twice daily. Intra-subject dose escalation was
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`allowed. Six of the 11 dogs that initiated at 2.5 or 5 mg/kg once daily were escalated and
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`completed the study with dosages of 10 mg/kg twice daily. Among all the dose cohorts, 8 dogs
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`had shrinkage of target lesions >20%; the best tumor responses were between 45-49% reduction
`
`in the sum of target lesions in two dogs. Complete responses ("CR", disappearance of all
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`evidence of disease per evaluator judgment; and absence of new lesions) were not observed in
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`Arm 1.
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`[00175] In the combination phase of the study (Arm 2), 7 dogs have been treated with 10 mg/kg
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`the BTK inhibitor of Formula (XVIII) and the PBK-8 inhibitor of Formula (IX) at 2.5 or 3.5
`
`mg/kg, on a twice daily schedule. To date, 4 dogs had shrinkage of target lesions > 20%; and the
`
`best tumor responses were between 58-65% reduction in the sum of target lesions, with one
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`sustained CR observed. Initial reductions in the sum of target lesions were observed to deepen
`
`during the course of therapy in 4 of the 7 dogs. A summary of the results is presented in Table 5.
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`TABLE 5. Summary of the results of the canine lymphoma study.
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`Response Metric
`
`Formula (XVIII) and
`Formula IX a
`4/7
`
`Formula (XVIII)
`monothera
`8/21 38.1 %)
`6/21 28.6 %
`0/21
`
`a Arm 2 is still recruiting subjects
`bLD, longest diameter ofup to 5 target lesion
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`[00305] These preliminary data suggest that in companion dogs with naturally occurring B cell
`
`lymphomas, treatment with the combination of the BTK inhibitor of Formula (XVIII) and the
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`PBK-8 inhibitor of Formula (IX) may provide increased biological activity (tumor shrinkage and
`
`stable disease) and may possibly lead to deeper responses than treatment with the BTK inhibitor
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`of Formula (XVIII) alone. Although the available data represent only 1/3 of the planned Arm 2
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`population, the extended response time (median time to best response) and observation of a CR
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`among the few dogs treated to date may be evidence of synergy between Formula (XVIII) and
`
`Formula (IX) in this highly aggressive disease.
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`We claim:
`
`CLAIMS
`
`1. A method of treating a hyperproliferative disorder in a subject, comprising co(cid:173)
`
`administering to a subject in need thereof a therapeutically effective amount of a
`
`phosphoinositide 3-kinase (PBK) inhibitor, or of a pharmaceutically acceptable salt
`
`thereof, in combination with a Bruton's tyrosine kinase (BTK) inhibitor, or of a
`
`pharmaceutically acceptable salt thereof.
`
`2. The method of Claim 1, wherein the PBK inhibitor is selected from the group consisting
`
`of a PBK-y inhibitor, a PBK-8 inhibitor, and a PBK-y,8 inhibitor.
`
`3. The method of claim 2, wherein the PBK inhibitor is a PBK-y,8 inhibitor.
`
`4. The method of any one of claims 1 to 3, wherein the hyperproiferative disorder is
`
`leukemia, lymphoma, or a solid tumor cancer.
`
`5. The method of any one of Claims 1 to 4, wherein the solid tumor cancer is selected from
`
`the group consisting of breast, lung, colorectal, thyroid, bone sarcoma and stomach
`
`cancers.
`
`6. The method of any one of Claims 1 to 5, wherein the combination of the PBK inhibitor
`
`with the BTK inhibitor is administered by oral, intravenous, intramuscular,
`
`intraperitoneal, subcutaneous or transdermal means.
`
`7. The method of any one of Claims 1 to 6, wherein the PBK inhibitor and/or BTK inhibitor
`
`is in the form of a pharmaceutically acceptable salt.
`
`8. The method of any one of Claims 1 to 7, wherein the PBK inhibitor is administered to
`
`the subject before administration of the BTK inhibitor.
`
`9. The method of any one of Claims 1 to 7, wherein the PB K inhibitor is administered
`
`concurrently with the administration of the BTK inhibitor.
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`10. The method of any one of Claims 1 to 7, wherein the PI3K inhibitor is administered to
`
`the subject after administration of the BTK inhibitor.
`
`11. The method of any one of Claims 1 to 10, wherein the PB K inhibitor is:
`
`or a pharmaceutically-acceptable salt thereof, wherein:
`X 1 is C(R9
`) or N;
`X 2 is C(R10) or N;
`Y is N(R11
`), 0 or S;
`Z is CR8 orN;
`
`n is 0, 1, 2 or 3;
`R 1 is a direct-bonded or oxygen -linked saturated, partially saturated or unsaturated 5-, 6- or 7-
`
`membered monocyclic ring containing 0, 1, 2, 3 or 4 atoms selected from N, 0 and S, but
`
`containing no more than one O or S, wherein the available carbon atoms of the ring are
`substituted by 0, 1 or 2 oxo or thioxo groups, wherein the ring is substituted by O or 1 R 2
`substituents, and the ring is additionally substituted by 0, 1, 2 or 3 substituents independently
`
`selected from halo, nitro, cyano, C1_4alkyl, OC1_4alkyl, OC1_4haloalkyl, NHCi-4, N(C1-
`
`4alkyl)C1_4alkyl and C1-4haloalkyl;
`R 2 is selected from halo, C 1-4haloalkyl, cyano, nitro, -C( O)R\-C(=O)ORa, -C(=O)NRaRa,
`-C( NRa)NRaRa, -OR\-OC(=O)Ra, -OC(=O)NRaRa. -OC(=O)N(Ra)S(=O)zR\ -
`
`OC2-6alkylNRaRa, -OC2--6alkylOR\ -SR\ OS(=O)R\ -S(=O)2R\ -S(-O)2NRaR\ -
`
`S(=O)2N(Ra)C(=O)R\-S( O)zN(Ra)C(=O)OR\-S(=0)2N(Ra)C(=O)NRaR\-NRaRa,
`
`-N(Ra)C(=O)Ra, -N(Ra)C(=O)OR\ -N(Ra)C(-O)NRaRa, -N(Ra)C( NRa)NRaRa, -
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`N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaRa, -NRaC2-6 alkylNRaRa and-NRaC2-6alkylORa; or
`R2 is selected from C 1_6alkyl, phenyl, benzyl, heteroaryl, heterocycle, -(C1_3alkyl)heteroaryl,
`-(C1_3alkyl)heterocycle, -O(C1_3alkyl)heteroaryl, -O(C1_3alkyl)heterocycle, -NRa(C1_
`3alkyl)heteromyl, -NR \ C 1_3alkyl)heterocycle, - ( C 1_3alkyl)phenyl, -0( C1_3alkyl)phenyl
`
`and -NRa(C1_3alkyl)phenyl all of which are substituted by 0, 1, 2 or 3 substituents selected
`from C1_4haloalkyl, OC1_4alkyl, Br, Cl, F, I and C 1-4alkyl;
`R3 is selected from H, halo, C 1-4haloalkyl, cyano, nitro, -C(=O)R\ -C(=O)R\ -
`C( O)NRaRa, -C( NRa)NRaRa, -OR a, -OC( O)R\ -OC(=O)NRaRa, -
`OC(=O)N(Ra)S( O)2R2, -OC2-6alkylNRaR\ -OC2-6alkylOR\-SR\-S( O)Ra, -
`S(=O)2R\-S(=O)2NRaRa, -S(=0)2N(Ra)C( O)RU, -S(=O)2N(Ra)C(=O)ORU, -
`
`S(=O)2N(Ra)C(=O)NRaRa, -NRaRU,-N(Ra)C(=O)RU, -N(Ra)C(=O)ORa, -
`
`N(Ra)C(=O)NRaR\ -N(Ra)C( NRa)NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)S(=O)2NRaNRaRa,
`
`-NRaC2_6alkylORU, C 1_6alkyl, phenyl, benzyl, heteroaryl and heterocycle, wherein the C1_
`6alkyl, phenyl, benzyl, heteroaryl and heterocycle are additionally substituted by 0, 1, 2 or 3
`substituents selected from C1--6haloalkyl, OC1_6alkyl, Br, Cl, F, I and C 1_6alkyl;
`R 4 is, independently, in each instance, halo, nitro, cyano, C 1-4alkyl, OC 1-4alkyl, OC 1_4haloalkyl,
`NHC1_4alkyl, N(C1_4alkyl)C1_4alkyl or C1-4haloalkyl;
`R 5 is, independently, in each instance, H, halo, C 1_6alkyl, C 1-4haloalkyl, or C 1_6alkyl substituted
`by 1, 2 or 3 substituents selected from halo, cyano, OH, OC 1_4alkyl, C 1_4alkyl, C 1_3haloalkyl,
`OC1_4alkyl, NH2, NHC1_4alkyl, N(C1_4alkyl)C1_4alkyl; or both R 5 groups together form a C3_
`6spiroalkyl substituted by 0, 1, 2 or 3 substituents selected from halo, cyano, OH, OC 1-4alkyl,
`C1_4alkyl, C1_3haloalkyl, OC1_4alkyl, NH2, NHC1_4alkyl, N(C1_4alkyl)C1_4alkyl;
`R 6 is selected from H, halo, C 1_6 alkyl, C 1_4haloalkyl, cyano, nitro, -C(=O)R\-C( O)OR\(cid:173)
`C(=O)NRaR\-C( NRa)NRaRa, -S(=O)R\-S(=OhR\-S(=O)2NRaRa, -
`
`S(=O)2N(Ra)C(=O)R\ -S( O)2N(Ra)C( O)ORU, -S(=0)2N(Ra)C(=O)NRaR\
`R 7 is selected from H, halo, C 1_6alkyl, C 1_4haloalkyl, cyano, nitro, -C(=O)R\ -C(-O)ORU, -
`C( O)NRaRU,-C( NRa)NRaRa, -S(=O)R\-S(=OhR\-S(=O)2NRaRa, -
`
`S(=O)2N(Ra)C(=O)R\ -S( OhN(Ra)C( O)ORU, -S(=0)2N(Ra)C(=O)NRaR\
`R 8 is selected from H, C1-6haloalkyl, Br, Cl, F, I, OR\ NRaRa, C1-6alkyl, phenyl, benzyl,
`
`heteroaryl and heterocycle, wherein the C1-6alkyl, phenyl, benzyl, heteroaryl and heterocycle
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`are additionally substituted by 0, 1, 2 or 3 substituents selected from C 1_6haloalkyl, OC1_
`6alkyl, Br, Cl, F, I and C1-6alkyl;
`R 9 is selected from H, halo, C 1_4haloalkyl, cyano, nitro, -C(=O)R\ -C(=O)O