4/1/2015 Targeting Btk in Lymphoma: PCI-32765 Inhibits Tumor Growth in Mouse Lymphoma Models and a Fluorescent Analog of PCI-32765 Is an Active-Site Probe …
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`Blood (ASH Annual Meeting Abstracts) 2007 110: Abstract 1592
`© 2007 American Society of Hematology
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`Poster Session
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`Molecular Pharmacology: Novel Therapies
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`Molecular Pharmacology, Drug Resistance
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`Targeting Btk in Lymphoma: PCI-32765 Inhibits Tumor Growth in
`Mouse Lymphoma Models and a Fluorescent Analog of PCI-32765 Is an
`Active-Site Probe That Enables Assessment of Btk Inhibition In Vivo.
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`Lee A. Honigberg, PhD1,*, Ashley M. Smith, B.S.1,*, Jun Chen, Ph.D.1,*, Patti Thiemann, B.S.1,* and
`Erik Verner, Ph.D.1,*
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`(Intr. by Markus F. Renschler) 1 Pharmacyclics, Inc., Sunnyvale, CA, USA.
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`There is increasing evidence indicating that B-cell-receptor (BCR) signaling is required for survival of non-
`Hodgkin’s lymphoma (NHL) cells. Bruton’s tyrosine kinase (Btk) is required for BCR signaling and mutations that
`inactivate human Btk cause X-linked-agammaglobulinemia, a B-cell immunodeficiency. Although Btk functions
`selectively in B cells, the Btk active site is structurally similar to the active site in several Src and Abl kinases and as a result, there have been few highly
`selective small molecule inhibitors of Btk. We have developed a series of covalent Btk inhibitors that target Cys-481 in Btk and this approach results in
`increased potency and selectivity over related kinases that lack a Cys residue at this position (ChemMedChem 15:58). PCI-32765 is a Cys-481 targeting
`Btk inhibitor that has been optimized for potency, selectivity and pharmacokinetics. In cellular assays, PCI-32765 inhibits BCR-stimulus induced
`phosphorylation of Phospholipase-C-gamma, a Btk substrate, as well as downstream phosphorylation of Erk (IC50 < 100 nM). In addition, PCI-32765
`induces apoptosis and inhibits proliferation in a subset of NHL cell lines including DHL-4, DHL-6, WSU-DLCL2, OCI-Ly10 and DOHH2 (IC50s = 0.6–
`1.6uM). We have used RNAi knockdown in DOHH2 cells as an independent method to confirm that Btk is required for lymphoma cell proliferation. In
`vivo, orally dosed PCI-32765 (50mg/kg) inhibits growth of DOHH2 and WSU-DLCL2 xenografts. PCI-32765 also prevents disease progression in a mouse
`collagen-induced arthritis model (12.5mg/kg PO), indicating that other B cell lineage diseases are sensitive to Btk inhibition. In order to further
`characterize the selectivity and in vivo potency of PCI-32765, we have developed PCI-33380, an active-site probe consisting of a covalent Btk inhibitor
`linked to the fluorophore Bodipy-FL. PCI-33380 binds to Btk and can be detected by flow cytometry or by denaturing gel electrophoresis of cell lysates. In
`cell lysates, the probe labels a single predominant band of the same molecular weight as Btk and this band is absent in cells from xid mice. Labeling of this
`band is inhibited (IC50=10nM) by a brief pre-treatment of cells with PCI-32765, indicating that the probe can be used to assess occupancy of Btk by a
`covalent inhibitor. We have used the probe to quantitate the inhibition of Btk by PCI-32765 in vivo. A single oral dose of PCI-32765 (10mg/kg) delivered
`to mice leads to rapid and complete inhibition of Btk in spleen. In addition, a single oral dose of PCI-32765 fully inhibits Btk in xenograft tumors and
`peripheral blood cells and this inhibition is maintained for up to 24hr. The Btk probe provides pharmacodynamic measurements that may allow
`optimization of dosing and schedule for in vivo studies and we are currently adapting the probe assays for use in monitoring the inhibition of Btk in human
`clinical trials.
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`Footnotes
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`Disclosure: Employment: All authors are employees of Pharmacyclics, Inc. Ownership Interests:; All authors are shareholders in Pharmacyclics, Inc.
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`4/1/2015 Targeting Btk in Lymphoma: PCI-32765 Inhibits Tumor Growth in Mouse Lymphoma Models and a Fluorescent Analog of PCI-32765 Is an Active-Site Probe …
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`http://abstracts.hematologylibrary.org/cgi/content/abstract/110/11/1592?maxtoshow=&hits=10&RESULTFORMAT=&fuff lltext=Honigberg&searchid=1&FIRSTIND… 2/2
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