M

M.M.L. In addition, combination treatment of main CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 like a restorative agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed. Intro Spleen tyrosine kinase (SYK) is definitely a 72-kDa cytoplasmic tyrosine kinase primarily indicated in hematopoietic cells including B-cells. In B-cells, transmission transduction is initiated by BCR activation via Src family kinase Lyn mediated phosphorylation of immune-receptor tyrosine-based activation motifs (ITAMs). This prospects to the recruitment, autophosphorylation, and sustained activity of SYK and activation of a number of downstream effectors (Mcsai et al., 2010). Recent evidence suggests KU 59403 that B-cell malignancies including non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) can be driven by aberrant activity of cellular signaling pathways and by extrinsic factors from your microenvironment that interact with the BCR (Caligaris-Cappio and Chiorazzi, 2010). Improved SYK manifestation and/or activity has been implicated in a number of NHL histologies (Rinaldi et al., 2006; Tavolaro et al., 2010 Chen et al., 2008; Davis et al., 2010). In CLL, constitutive SYK activity, as well as activation after BCR cross-linking, has been explained (Baudot et al., 2009; Gobessi et al., 2009). Improved manifestation of BCR connected kinases including SYK is definitely associated with a shorter treatment-free interval (Rodriguez et al., 2007), and SYK inhibition results in apoptosis (Baudot et al., 2009; Hoellenriegel et al., 2012) and disruption of chemokine activity (Rodriguez, et al., 2007; Hoellenriegel et al., 2012). Focusing on SYK has been explored using Fostamatinib disodium (R788), the pro-drug of R406. R788/406 is definitely a SYK inhibitor (IC50 = 41 nM) found to have activity inside a phase 2 study in NHL and CLL (Friedberg et al., 2010). However, R788/406 is known to possess significant off target effects, including FMS-related tyrosine kinase 3 (FLT-3), Lck, Janus kinase 1 and 3, and c-kit (Braselmann et al., 2006), which may be responsible for some of its activity (Davis et al., 2010). No further development of Fostamatinib or of another selective SYK inhibitor has been reported in lymphoid cancers. Consequently, the characterization of novel SYK inhibitors is definitely warranted. Given the restorative potential of SYK inhibition and the need to develop SYK inhibitors without off target effects, we evaluated P505-15, a novel and highly selective (IC50 = 1 nM) SYK inhibitor with anti-SYK activity that is 80-fold greater than its affinity for additional kinases. P505-15 offers been shown to potently inhibit SYK- and BCR-dependent activation of normal B-cells (Coffey, et al., 2012) and offers been shown to decrease CLL cell viability (Hoellenriegel et al., 2012). We targeted to further define the preclinical properties of P505-15 in NHL and CLL and its activity as a single agent or in combination with fludarabine Rabbit Polyclonal to A20A1 in main CLL samples, including those from individuals with poor risk biologic features. Materials and Methods Chemical Structure and Kinase Profiling. Synthesis and characterization of P505-15 [(4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino)pyrimidine-5-carboxamide acetate] (PRT062607) as well as its potency and selectivity for SYK have been reported (Coffey et al., 2012). Cell Lines and Antibodies. The human being non-Hodgkin lymphoma B-cell lines SUDHL-4 (#ACC495), SUDHL-6 (#ACC572), and Karpas-422 (#ACC32) were from DSMZ (Braunschweig, Germany); Toledo (#CRL-2631) and Ramos (#CRL-1596) were from the American Type Tradition Collection (ATCC; Manassas, VA). All cells were managed in RPMI press (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (ATCC) and penicillin/streptomycin (Invitrogen), and managed inside a 37C humidified cells tradition incubator. Antibodies included polyclonal goat F(ab)2 anti-human IgG and IgM (LifeTechnologies, Grand Island, NY); rabbit anti-human SYK, anti-human phospho-SYK (Y525/526), and alexafluor 488-conjugated anti-human phospho-ERK (Y204) (Cell Signaling Systems, Inc., Danvers, MA); phycoerythrin-conjugated anti-human phospho-AKT (S473), fluorescein isothiocyanate-conjugated anti-human CD19, allophycocyanin-conjugated anti-human CD5, phycoerythrin-conjugated anti-human phospho-SYK (Y352), fluorescein isothiocyanate-conjugated anti-mouse CD80 and CD86, and allophycocyanin-conjugated anti-mouse CD45R/B220 (BD Biosciences, San Jose, CA). SYK Autophosphorylation. Ramos cells.Good examples from mice treated with control goat serum (Fig. splenomegaly and significantly inhibited NHL tumor growth inside a xenograft model. In addition, combination treatment of main CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 like a restorative agent for B-cell malignancies. A dose finding research in healthful volunteers continues to be completed. Launch Spleen tyrosine kinase (SYK) is certainly a 72-kDa cytoplasmic tyrosine kinase mainly portrayed in hematopoietic cells including B-cells. In B-cells, sign transduction is set up by BCR activation via Src family members kinase Lyn mediated phosphorylation of immune-receptor tyrosine-based activation motifs (ITAMs). This qualified prospects to the recruitment, autophosphorylation, and suffered activity of SYK and activation of several downstream effectors (Mcsai et al., 2010). Latest evidence shows that B-cell malignancies including non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) could be powered by aberrant activity of mobile signaling pathways and by extrinsic elements through the microenvironment that connect to the BCR (Caligaris-Cappio and Chiorazzi, 2010). Elevated SYK appearance and/or activity continues to be implicated in several NHL histologies (Rinaldi et al., 2006; Tavolaro et al., 2010 Chen et al., 2008; Davis et al., 2010). In CLL, constitutive SYK activity, aswell as activation after BCR cross-linking, continues to be referred to (Baudot et al., 2009; Gobessi et al., 2009). Elevated appearance of BCR linked kinases including SYK is certainly connected with a shorter treatment-free period (Rodriguez et al., 2007), and SYK inhibition leads to apoptosis (Baudot et al., 2009; Hoellenriegel et al., 2012) and disruption of chemokine activity (Rodriguez, et al., 2007; Hoellenriegel et al., 2012). Concentrating on SYK continues to be explored using Fostamatinib disodium (R788), the pro-drug of R406. R788/406 is certainly a SYK inhibitor (IC50 = 41 nM) discovered to possess activity within a stage 2 research in NHL and CLL (Friedberg et al., 2010). Nevertheless, R788/406 may have got significant off focus on results, including FMS-related tyrosine kinase 3 (FLT-3), Lck, Janus kinase 1 and 3, and c-kit (Braselmann et al., 2006), which might be responsible for a few of its activity (Davis et al., 2010). No more advancement of Fostamatinib or of another selective SYK inhibitor continues to be reported in lymphoid malignancies. As a result, the characterization of book SYK inhibitors is certainly warranted. Provided the healing potential of SYK inhibition and the necessity to develop SYK inhibitors without off focus on effects, we examined P505-15, a book and extremely selective (IC50 = 1 nM) SYK inhibitor with anti-SYK activity that’s 80-fold higher than its affinity for various other kinases. P505-15 provides been proven to potently inhibit SYK- and BCR-dependent activation of regular B-cells (Coffey, et al., 2012) and provides been proven to diminish CLL cell viability (Hoellenriegel et al., 2012). We directed to help expand define the preclinical properties of P505-15 in NHL and CLL and its own activity as an individual agent or in conjunction with fludarabine in major CLL examples, including those extracted from sufferers with poor risk biologic features. Components and Methods Chemical substance Framework and Kinase Profiling. Synthesis and characterization of P505-15 [(4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino)pyrimidine-5-carboxamide acetate] (PRT062607) aswell as its strength and selectivity for SYK have already been reported (Coffey et al., 2012). Cell Lines and Antibodies. The individual non-Hodgkin lymphoma B-cell lines SUDHL-4 (#ACC495), SUDHL-6 (#ACC572), and Karpas-422 (#ACC32) had been extracted from DSMZ (Braunschweig, Germany); Toledo (#CRL-2631) and Ramos (#CRL-1596) had been extracted from the American Type Lifestyle Collection (ATCC; Manassas, VA). All cells had been taken care of in RPMI mass media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal leg serum (ATCC) and penicillin/streptomycin (Invitrogen), and taken care of within a 37C humidified tissues lifestyle incubator. Antibodies included polyclonal goat F(ab)2 anti-human IgG and IgM (LifeTechnologies, Grand Isle, NY); rabbit anti-human SYK, anti-human phospho-SYK (Y525/526), and alexafluor 488-conjugated anti-human phospho-ERK (Y204) (Cell Signaling Technology, Inc., Danvers, MA); phycoerythrin-conjugated anti-human phospho-AKT (S473), fluorescein isothiocyanate-conjugated anti-human Compact disc19, allophycocyanin-conjugated anti-human Compact disc5, phycoerythrin-conjugated anti-human phospho-SYK (Y352), fluorescein isothiocyanate-conjugated.This qualified prospects to the recruitment, autophosphorylation, and sustained activity of SYK and activation of several downstream effectors (Mcsai et al., 2010). Spleen tyrosine kinase (SYK) is certainly a 72-kDa cytoplasmic tyrosine kinase mainly portrayed in hematopoietic cells including B-cells. In B-cells, sign transduction is set up by BCR activation via Src family members kinase Lyn mediated phosphorylation of immune-receptor tyrosine-based activation motifs (ITAMs). This qualified prospects to the recruitment, autophosphorylation, and suffered activity of SYK and activation of several downstream effectors (Mcsai et al., 2010). Latest evidence shows that B-cell malignancies including non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) could be powered by aberrant activity of mobile signaling pathways and by extrinsic elements through the microenvironment that connect to the BCR (Caligaris-Cappio and Chiorazzi, 2010). Elevated SYK appearance and/or activity continues to be implicated in several NHL histologies (Rinaldi et al., 2006; Tavolaro et al., 2010 Chen et al., 2008; Davis et al., 2010). In CLL, constitutive SYK activity, aswell as activation after BCR cross-linking, continues to be referred to (Baudot et al., 2009; Gobessi et al., 2009). Elevated appearance of BCR linked kinases including SYK is certainly connected with a shorter treatment-free period (Rodriguez et al., 2007), and SYK inhibition leads to apoptosis (Baudot et al., 2009; Hoellenriegel et al., 2012) and disruption of chemokine activity (Rodriguez, et al., 2007; Hoellenriegel et al., 2012). Concentrating on SYK continues to be explored using Fostamatinib disodium (R788), the pro-drug of R406. R788/406 is certainly a SYK inhibitor (IC50 = 41 nM) discovered to possess activity within a stage 2 research in NHL and CLL (Friedberg et al., 2010). Nevertheless, R788/406 may have got significant off focus on results, including FMS-related tyrosine kinase 3 (FLT-3), Lck, Janus kinase 1 and 3, and c-kit (Braselmann et al., 2006), which might be responsible for a few of its activity (Davis et al., 2010). No more advancement of Fostamatinib or of another selective SYK inhibitor continues to be reported in lymphoid malignancies. As a result, the characterization of book SYK inhibitors is certainly warranted. Provided the healing potential of SYK inhibition and the necessity to develop SYK inhibitors without off focus on effects, we examined P505-15, a book and extremely selective (IC50 = 1 nM) SYK inhibitor with anti-SYK activity that’s 80-fold higher than its affinity for various other kinases. P505-15 provides been proven to potently inhibit SYK- and BCR-dependent activation of regular B-cells (Coffey, et al., 2012) and provides been proven to diminish CLL cell viability (Hoellenriegel et al., 2012). We directed to help expand define the preclinical properties of P505-15 in NHL and CLL and its own activity as an individual agent or in conjunction with fludarabine in major CLL examples, including those extracted from sufferers with poor risk biologic features. Materials and Methods Chemical Structure and Kinase Profiling. Synthesis and characterization of P505-15 [(4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino)pyrimidine-5-carboxamide acetate] (PRT062607) as well as its potency and selectivity for SYK have been reported (Coffey et al., 2012). Cell Lines and Antibodies. The human non-Hodgkin lymphoma B-cell lines SUDHL-4 (#ACC495), SUDHL-6 (#ACC572), and Karpas-422 (#ACC32) were obtained from DSMZ (Braunschweig, Germany); Toledo (#CRL-2631) and Ramos (#CRL-1596) were obtained from the American Type Culture Collection (ATCC; Manassas, VA). All cells were maintained in RPMI media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (ATCC) and penicillin/streptomycin (Invitrogen), and maintained in a 37C humidified tissue culture incubator. Antibodies included polyclonal goat F(ab)2 anti-human IgG and IgM (LifeTechnologies, Grand Island, NY); rabbit anti-human SYK, anti-human phospho-SYK (Y525/526), and alexafluor 488-conjugated anti-human phospho-ERK (Y204) (Cell Signaling Technologies, Inc., Danvers, MA); phycoerythrin-conjugated anti-human phospho-AKT (S473), fluorescein isothiocyanate-conjugated anti-human CD19, allophycocyanin-conjugated anti-human CD5, phycoerythrin-conjugated anti-human phospho-SYK (Y352), fluorescein isothiocyanate-conjugated anti-mouse CD80 and CD86, and allophycocyanin-conjugated anti-mouse CD45R/B220 (BD Biosciences, San Jose, CA). SYK Autophosphorylation. Ramos cells (107 cells per experiment) were preincubated for 30 minutes with or without P505-15 and then stimulated (30 minutes at 37C) with 1 = 7) were thawed at 37C, washed in 10 ml RPMI media plus 10% fetal calf serum by centrifugation, and resuspended at 106 cells/ml in the same media. Aliquots (200 = 5 per group) received daily oral BID doses of vehicle (0.5% methylcellulose in water) or P505-15 (15 mg/kg) for a total of 5 days. On study day 1, 1 hour after the first oral dose, mice received a single 200-= 15) and dosed twice daily by oral gavage with vehicle (0.5% methylcellulose in water) or 10, 15, or 20 mg/kg P505-15. Body weights were obtained at least once per week, and.Therefore, the characterization of novel SYK inhibitors is warranted. dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed. Introduction Spleen tyrosine kinase (SYK) is a 72-kDa cytoplasmic tyrosine kinase primarily expressed in hematopoietic cells including B-cells. In B-cells, signal transduction is initiated by BCR activation via Src family kinase Lyn mediated phosphorylation of immune-receptor tyrosine-based activation motifs (ITAMs). This leads to the recruitment, autophosphorylation, and sustained activity of SYK and activation of a number of downstream effectors (Mcsai et al., 2010). Recent evidence suggests that B-cell malignancies including non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) can be driven by aberrant activity of cellular signaling pathways and by extrinsic factors from the microenvironment that interact with the BCR (Caligaris-Cappio and Chiorazzi, 2010). Increased SYK expression and/or activity has been implicated in a number of NHL histologies (Rinaldi et al., 2006; Tavolaro et al., 2010 Chen et al., 2008; Davis et al., 2010). In CLL, constitutive SYK activity, as well as activation after BCR cross-linking, has been described (Baudot et al., 2009; Gobessi et al., 2009). Increased expression of BCR associated kinases including SYK is associated with a shorter treatment-free interval (Rodriguez et al., 2007), and SYK inhibition results in apoptosis (Baudot et al., 2009; Hoellenriegel et al., 2012) and disruption of chemokine activity (Rodriguez, et al., 2007; Hoellenriegel et al., 2012). Targeting SYK has been explored using Fostamatinib disodium (R788), the pro-drug of R406. R788/406 is a SYK inhibitor (IC50 = 41 nM) found to have activity in a phase 2 study in NHL and CLL (Friedberg et al., 2010). However, R788/406 is known to have significant off target effects, including FMS-related tyrosine kinase 3 (FLT-3), Lck, Janus kinase 1 and 3, and c-kit (Braselmann et al., 2006), which may be responsible for some of its activity (Davis et al., 2010). No further development of Fostamatinib or of another selective SYK inhibitor has been reported in lymphoid cancers. Therefore, the characterization of novel SYK inhibitors is warranted. Given the therapeutic potential of SYK inhibition and the need to develop SYK inhibitors without off target effects, we evaluated P505-15, a novel and highly selective (IC50 = 1 nM) SYK inhibitor with anti-SYK activity that is 80-fold greater than its affinity for other kinases. P505-15 has been shown to potently inhibit SYK- and BCR-dependent activation of normal B-cells (Coffey, et al., 2012) and has been shown to decrease CLL cell viability (Hoellenriegel et al., 2012). We aimed to further define the preclinical properties of P505-15 in NHL and CLL and its activity as a single agent or in combination with fludarabine in primary CLL samples, including those obtained from patients with poor risk biologic features. Materials and Methods Chemical Structure and Kinase Profiling. Synthesis and characterization of P505-15 [(4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino)pyrimidine-5-carboxamide acetate] (PRT062607) as well as its strength and selectivity for SYK have already been reported (Coffey et al., 2012). Cell Lines and Antibodies. The individual non-Hodgkin lymphoma B-cell lines SUDHL-4 (#ACC495), SUDHL-6 (#ACC572), and Karpas-422 (#ACC32) had been extracted from DSMZ (Braunschweig, Germany); Toledo (#CRL-2631) and Ramos (#CRL-1596) had been extracted from the American Type Lifestyle Collection (ATCC; Manassas, VA). All cells had been preserved in RPMI mass media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal leg serum (ATCC) and penicillin/streptomycin (Invitrogen), and preserved within a 37C humidified tissues lifestyle incubator. Antibodies included polyclonal goat F(ab)2 anti-human IgG and IgM (LifeTechnologies, Grand Isle, NY); rabbit anti-human SYK, anti-human phospho-SYK (Y525/526), and alexafluor 488-conjugated anti-human phospho-ERK (Y204) (Cell Signaling Technology, Inc., Danvers, MA); phycoerythrin-conjugated anti-human phospho-AKT (S473), fluorescein isothiocyanate-conjugated anti-human Compact disc19, allophycocyanin-conjugated anti-human Compact disc5, phycoerythrin-conjugated anti-human phospho-SYK (Y352), fluorescein isothiocyanate-conjugated anti-mouse Compact disc80 and Compact disc86, and allophycocyanin-conjugated anti-mouse Compact disc45R/B220 (BD Biosciences, San Jose, CA). SYK Autophosphorylation. Ramos cells (107 cells per test) had been preincubated for thirty minutes with or without P505-15 and stimulated (thirty minutes at 37C) with 1 = 7) had been thawed at 37C, cleaned in 10 ml RPMI mass media plus 10% fetal leg serum by centrifugation, and resuspended at 106 cells/ml in the same mass media. Aliquots (200 = 5 per group) received daily dental BID dosages of automobile (0.5% methylcellulose in water) or P505-15 (15 mg/kg) for a complete of 5 times. On study time 1, one hour after the initial oral dosage, mice received an individual 200-= 15) and dosed double daily by dental gavage with automobile (0.5% methylcellulose in water) or 10, 15, or 20 mg/kg P505-15. Body weights had been obtained at least one time per.Elevated expression of BCR linked kinases including SYK is normally connected with a shorter treatment-free interval (Rodriguez et al., 2007), and SYK inhibition leads to apoptosis (Baudot et al., 2009; Hoellenriegel et al., 2012) and disruption of chemokine activity (Rodriguez, et al., 2007; Hoellenriegel et al., 2012). Targeting SYK continues to be explored using Fostamatinib disodium (R788), the pro-drug of R406. results support the ongoing advancement of P505-15 being a healing agent for B-cell malignancies. A dosage finding research in healthful volunteers continues to be completed. Launch Spleen tyrosine kinase (SYK) is normally a 72-kDa cytoplasmic tyrosine kinase mainly portrayed in hematopoietic cells including B-cells. In B-cells, indication transduction is set up by BCR activation via Src family members kinase Lyn mediated phosphorylation of immune-receptor tyrosine-based activation motifs (ITAMs). This network marketing leads to the recruitment, autophosphorylation, and suffered activity of SYK and activation of several downstream effectors (Mcsai et al., 2010). Latest evidence shows that B-cell malignancies including non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) could be powered by aberrant activity of mobile signaling pathways and by extrinsic elements in the microenvironment that connect to the BCR (Caligaris-Cappio and Chiorazzi, 2010). Elevated SYK appearance and/or activity continues to be implicated in several NHL histologies (Rinaldi et al., 2006; Tavolaro et al., 2010 Chen et al., 2008; Davis et al., 2010). In CLL, constitutive SYK activity, aswell as activation after BCR KU 59403 cross-linking, continues to be defined (Baudot et al., 2009; Gobessi et al., 2009). Elevated appearance of BCR linked kinases including SYK is normally connected with a shorter treatment-free period (Rodriguez et al., 2007), and SYK inhibition leads to apoptosis (Baudot et al., KU 59403 2009; Hoellenriegel et al., 2012) and disruption of chemokine activity (Rodriguez, et al., 2007; Hoellenriegel et al., 2012). Concentrating on SYK continues to be explored using Fostamatinib disodium (R788), the pro-drug of R406. R788/406 is normally a SYK inhibitor (IC50 = 41 nM) discovered to possess activity within a stage 2 research in NHL and CLL (Friedberg et al., 2010). Nevertheless, R788/406 may have got significant off focus on results, including FMS-related tyrosine kinase 3 (FLT-3), Lck, Janus kinase 1 and 3, and c-kit (Braselmann et al., 2006), which might be responsible for a few of its activity (Davis et al., 2010). No more advancement of Fostamatinib or of another selective SYK inhibitor continues to be reported in lymphoid malignancies. As a result, the characterization of book SYK inhibitors is normally warranted. Provided the healing potential of SYK inhibition and the necessity to develop SYK inhibitors without off focus on effects, we evaluated P505-15, a novel and highly selective (IC50 = 1 nM) SYK inhibitor with anti-SYK activity that is 80-fold greater than its affinity for other kinases. P505-15 has been shown to potently inhibit SYK- and BCR-dependent activation of normal B-cells (Coffey, et al., 2012) and has been shown to decrease CLL cell viability (Hoellenriegel et al., 2012). We aimed to further define the preclinical properties of P505-15 in NHL and CLL and its activity as a single agent or in combination with fludarabine in main CLL samples, including those obtained from patients with poor risk biologic features. Materials and Methods Chemical Structure and Kinase Profiling. Synthesis and characterization of P505-15 [(4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino)pyrimidine-5-carboxamide acetate] (PRT062607) as well as its potency and selectivity for SYK have been reported (Coffey et al., 2012). Cell Lines and Antibodies. The human non-Hodgkin lymphoma B-cell lines SUDHL-4 (#ACC495), SUDHL-6 (#ACC572), and Karpas-422 (#ACC32) were obtained from DSMZ (Braunschweig, Germany); Toledo (#CRL-2631) and Ramos (#CRL-1596) were obtained from the American Type Culture Collection (ATCC; Manassas, VA). All cells were managed in RPMI media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (ATCC) and penicillin/streptomycin (Invitrogen), and managed in a 37C humidified tissue culture incubator. Antibodies included polyclonal goat F(ab)2 anti-human IgG and IgM (LifeTechnologies, Grand Island, NY); rabbit anti-human SYK, anti-human phospho-SYK (Y525/526), and alexafluor 488-conjugated anti-human phospho-ERK (Y204) (Cell Signaling Technologies, Inc., Danvers, MA); phycoerythrin-conjugated anti-human phospho-AKT (S473), fluorescein isothiocyanate-conjugated anti-human CD19, allophycocyanin-conjugated anti-human CD5, phycoerythrin-conjugated anti-human phospho-SYK (Y352), fluorescein isothiocyanate-conjugated anti-mouse CD80 and CD86, and allophycocyanin-conjugated anti-mouse CD45R/B220 (BD Biosciences, San Jose, CA). SYK Autophosphorylation. Ramos cells (107 cells per experiment) were preincubated for 30 minutes with or without P505-15 and then stimulated (30 minutes at 37C) with 1 = 7) were thawed at 37C, washed in 10 ml RPMI media plus 10% fetal calf serum by centrifugation, and resuspended at 106 cells/ml in the same media. Aliquots (200 = 5 per group) received daily oral BID doses of vehicle (0.5% methylcellulose in water) or P505-15 (15 mg/kg) for a total of 5.