Supplemental material to this article can be found at:
`http://jpet.aspetjournals.org/content/suppl/2013/05/24/jpet.113.203489.DC1
`
`1521-0103/346/2/219–228$25.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
`
`http://dx.doi.org/10.1124/jpet.113.203489
`J Pharmacol Exp Ther 346:219–228, August 2013
`
`Inhibition of Btk with CC-292 Provides Early Pharmacodynamic
`Assessment of Activity in Mice and Humans s
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`Erica K. Evans, Richland Tester, Sharon Aslanian, Russell Karp, Michael Sheets,
`Matthew T. Labenski, Steven R. Witowski, Heather Lounsbury, Prasoon Chaturvedi,
`Hormoz Mazdiyasni, Zhendong Zhu, Mariana Nacht, Martin I. Freed, Russell C. Petter,
`Alex Dubrovskiy, Juswinder Singh, and William F. Westlin
`Celgene Avilomics Research, Bedford, Massachusetts
`Received January 23, 2013; accepted May 13, 2013
`
`ABSTRACT
`Targeted therapies that suppress B cell receptor (BCR) signaling
`have emerged as promising agents in autoimmune disease and
`B cell malignancies. Bruton’s tyrosine kinase (Btk) plays a crucial
`role in B cell development and activation through the BCR
`signaling pathway and represents a new target for diseases
`characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-
`(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phe-
`nyl)acrylamide (CC-292) is a highly selective, covalent Btk
`inhibitor and a sensitive and quantitative assay that measures
`CC-292-Btk engagement has been developed. This translational
`pharmacodynamic assay has accompanied CC-292 through
`each step of drug discovery and development. These studies
`
`demonstrate the quantity of Btk bound by CC-292 correlates
`with the efficacy of CC-292 in vitro and in the collagen-induced
`arthritis model of autoimmune disease. Recently, CC-292 has
`entered human clinical trials with a trial design that has provided
`rapid insight into safety, pharmacokinetics, and pharmacody-
`namics. This first-in-human healthy volunteer trial has demon-
`strated that a single oral dose of 2 mg/kg CC-292 consistently
`engaged all circulating Btk protein and provides the basis for
`rational dose selection in future clinical trials. This targeted
`covalent drug design approach has enabled the discovery and
`early clinical development of CC-292 and has provided support
`for Btk as a valuable drug target for B-cell mediated disorders.
`
`Introduction
`Bruton’s tyrosine kinase (Btk) is a kinase expressed exclusively
`in B cells and myeloid cells and has a well characterized, vital
`role in B cells highlighted by the human primary immune
`deficiency disease, X-linked agammaglobulinemia (XLA),
`which results from mutation in the Btk gene (Smith et al.,
`1998). As a result of incomplete B cell differentiation, XLA
`patients have a near complete absence of mature B cells in the
`peripheral blood (Campana et al., 1990) and cannot produce
`immunoglobulins (Conley, 1985; Nonoyama et al., 1998). The
`human XLA phenotype is recapitulated, although less
`severely, in Btk knock-out mice (Khan et al., 1995) and in
`xid mice, which have a naturally occurring Btk mutation
`(Rawlings et al., 1993).
`Specifically, Btk plays an essential role in the B cell
`receptor (BCR) signaling pathway. Antigen binding to the
`BCR results in B cell receptor oligomerization, Syk and Lyn
`kinase activation (Gauld et al., 2002), followed by Btk kinase
`activation (Park et al., 1996; Rawlings et al., 1996; Baba et al.,
`
`dx.doi.org/10.1124/jpet.113.203489.
`s This article has supplemental material available at jpet.aspetjournals.org.
`
`2001). Once activated, Btk forms a signaling complex with
`proteins such as BLNK, Lyn, and Syk and phosphorylates
`phospholipase C (PLC)g2 (Baba et al., 2001; Tsukada et al.,
`2001). This leads to downstream release of intracellular Ca21
`stores and propagation of the BCR signaling pathway through
`extracellular signal-regulated kinase and nuclear factor-kB
`signaling, ultimately resulting in transcriptional changes to
`foster B cell survival, proliferation, and/or differentiation
`(Baba et al., 2001; Maas and Hendriks, 2001; Mohamed et al.,
`2009).
`While BCR signaling is essential in the normal develop-
`ment and function of B cells, several pathologies have been
`attributed to dysregulated BCR activity. These include diseases
`of autoreactivity, such as that observed in lupus, multiple
`sclerosis, and rheumatoid arthritis, in which B cells inappro-
`priately break self-tolerance to produce antibodies contributing
`to autoimmune disease (Edwards and Cambridge, 2005, 2006;
`Teng et al., 2007). BCR signaling also contributes to several B cell
`malignancies, such as chronic lymphocytic leukemia (CLL) (Chen
`et al., 2005; Hoellenriegel et al., 2011; Stevenson et al., 2011),
`mantle cell lymphoma, and subsets of diffuse large B cell
`leukemia (Chen et al., 2008; Lenz et al., 2008; Baran-Marszak
`et al., 2010; Davis et al., 2010; Suljagic et al., 2010; Pighi et al.,
`
`ABBREVIATIONS: BCR, B cell receptor; BSA, bovine serum albumin; Btk, Bruton’s tyrosine kinase; CC-292, N-(3-(5-fluoro-2-(4-(2-
`methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide; CIA, collagen-induced arthritis; CLL, chronic lymphocytic leukemia; CNX-500,
`N1-(3-(3-(4-(3-acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propyl)-N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-
`d]imidazol-4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide; CST, Cell Signaling Technology; ELISA, enzyme-linked immunosorbet assay;
`PBS, phosphate-buffered saline; PD, pharmacodynamic; PK, pharmacokinetic; PLC, phospholipase C; XLA, X-linked agammaglobulinemia.
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`Evans et al.
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`2011). However, until recently, therapies that target the B cell
`have resulted in depletion of the B cell repertoire, while
`therapeutic strategies that reduce BCR activity are rela-
`tively new for treatment of these diseases.
`Promising recent clinical data generated by inhibition of
`distinct BCR signaling components, including Syk, phospha-
`tidylinositide 3-kinase d, and Btk with fostamatinib, idelalisib
`(also known as GS-1101 or CAL-101), and ibrutinib (PCI-
`32765), respectively, have provided great excitement for this
`approach. Inhibition of Syk with fostamatinib has demon-
`strated efficacy in human clinical trials in rheumatoid
`arthritis as well as in B cell malignancies dependent on
`BCR signaling such as CLL (Braselmann et al., 2006; Chen
`et al., 2008; Podolanczuk et al., 2009; Friedberg et al., 2010;
`Genovese et al., 2011). Similarly, inhibition of phosphatidy-
`linositide 3-kinase d with GS-1101 has also shown promising
`results in CLL (Herman et al., 2010; Hoellenriegel et al., 2011;
`Lannutti et al., 2011). Btk, downstream of Syk in the BCR
`signaling pathway, also represents an attractive drug target
`in diseases characterized by aberrant B cell activity. More-
`over, owing to its highly restricted expression pattern in
`B cells and myeloid cells, Btk provides an opportunity for
`selective therapeutic targeting. Preclinically, small molecule
`inhibition of Btk with CGI1746 and ibrutinib demonstrated
`therapeutic activity in several models of autoimmune disease
`(Honigberg et al., 2010; Chang et al., 2011; Di Paolo et al.,
`2011). Ibrutinib has shown promising results in early clinical
`development
`for the treatment of B cell malignancies
`(Harrison, 2012; Advani et al., 2013) and is currently in phase
`III trials in CLL, providing evidence that Btk represents
`a viable and efficacious therapeutic target.
`We describe our work on N-(3-(5-fluoro-2-(4-(2-methoxyeth-
`oxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide
`(CC-292), which is a potent, highly selective, covalent in-
`hibitor of Btk that inhibits BCR signaling and has efficacy in
`a rheumatoid arthritis disease model. We also describe
`a pharmacodynamic (PD) assay that has been implemented
`throughout all stages of preclinical development to measure
`activity of CC-292 and correlate Btk inhibition with functional
`outcome both in vitro and in vivo. Finally, we report the
`substantial oral exposure to CC-292 in humans and use this
`PD assay to unequivocally and quantitatively demonstrate
`complete Btk engagement in a first-in-human setting. This
`work represents the first report of a selective Btk inhibitor
`appropriate for use in a human clinical setting of autoimmune
`disease and uses a powerful translational approach to confirm
`on-target activity in human B cells.
`
`Materials and Methods
`B-Lymphocyte Isolation for In Vitro Signaling, Prolifera-
`tion, and Activation. Human naïve, primary B cells (CD191, IgD1)
`were isolated from anticoagulated whole blood by density centrifuga-
`tion through Histopaque-1077 and peripheral blood mononuclear cell
`(PBMC) isolation. PBMCs were subject to red blood cell lysis using
`Red Blood Cell Lysis Buffer (Boston BioProducts, Ashland, MA) followed
`by incubation with MACS reagent (130-091-150) and negative selection
`over a MACS column to obtain naïve primary B cells with .85%
`purity.
`Immunoblot Analysis. Cells were incubated in serum-free RPMI
`media for 1–1.5 hours. Isolated human B cells were incubated with
`CC-292 at a final concentration of 0.001, 0.01, 0.1 and 1 mM. Ramos
`cells were incubated with 0.1 nM–3 mM CC-292. Cells were then
`
`incubated in the presence of compound for 1 hour at 37°C. Following
`incubation, cells were centrifuged and resuspended in 100 ml of
`serum-free RPMI and BCR was stimulated with addition of 5 mg/ml
`a-human IgM. Samples were centrifuged, washed in phosphate-
`buffered saline (PBS), and lysed in 100 ml of Cell Extraction Buffer
`[cat. no. FNN0011; Life Technologies (Invitrogen), Carlsbad, CA] plus
`1:10 (v/v) PhosSTOP Phosphatase Inhibitor (Roche, Basel, Switzerland)
`and 1:10 (v/v) Complete Protease Inhibitor (cat. no. 11836145001;
`Roche). Antibodies used for immunoblot analysis include P-PLCg2 [cat.
`no. 3872; Cell Signaling Technology, Beverly, MA (CST)], PLCg2 (3871;
`CST), Syk (2712; CST), P-Syk (2710; CST), Btk (cat. no. 611116; BD
`Biosciences, Franklin Lakes, NJ), P-Btk (cat. no. 2207-1; Epitomics,
`Berlingame, CA), and Tubulin (cat. no. T6199; Sigma-Aldrich, St. Louis,
`MO). Membranes were scanned on a Li-Cor Odyssey scanner using
`infrared fluorescence detection (Li-Cor Biosciences, Lincoln, NB).
`B-Lymphocyte Proliferation (3H-Thymidine Incorporation).
`A suspension of resting purified naïve human B cells isolated by
`negative selection (MACS reagent 130-091-150) in RPMI was pre-
`pared at 0.4–0.5  106 cells/ml. Cells were mixed together with a-human
`IgM (final concentration of 5 mg/ml in each well) and vehicle (dimethyl
`sulfoxide) or CC-292 (final concentrations of 0.01, 0.1, 1.0, 10.0, 100.0, or
`1000 nM per well) and seeded in a 96-well plate. Cells were incubated
`for 56 hours in a humidified incubator maintained at 37°C and 5%
`CO2. 3H-Thymidine was added (final concentration of 1 mCi in each well)
`and cells were incubated overnight, harvested, and measured for 3H
`incorporation. Experiments were performed in triplicate.
`Btk Target Site Occupancy Enzyme-Linked Immunosor-
`bent Assay. An enzyme-linked immunosorbent assay (ELISA)
`method for the detection of free uninhibited Btk in mouse, rat, dog,
`monkey, and human lysates was developed at Celgene Avilomics
`Research, and a validation of this method in human B cell lysate
`was performed by a federal Certified Laboratories Improvement
`Amendments-certified laboratory (Cambridge Biomedical Laborato-
`ries, Boston, MA) The parameters that were assessed included: accuracy,
`linearity, dilution, precision (intra- and interassay), stability, refer-
`ence range, freeze-thaw cycles, reportable range, specificity, sensitiv-
`ity, and carryover. All specifications for linearity, precision (intra- and
`interassay), accuracy, and carryover defined in the validation protocol
`were met. Samples were stable at –80°C for 5 weeks and the re-
`portable range of the Btk ELISA was 12–12,800 pg of free Btk. Cell
`lysates or spleen homogenates were incubated with N1-(3-(3-(4-(3-
`acrylamidophenylamino)-5-methylpyrimidin-2-ylamino)phenoxy)propyl)-
`N5-(15-oxo-19-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-
`4-yl)-4,7,10-trioxa-14-azanonadecyl)glutaramide (CNX-500) (Celgene
`Avilomics Research; final concentration 1 mM) in a PBS, 0.05% Tween-
`20, 1% bovine serum albumin (BSA) solution for 1 hour at room
`temperature. Standards and samples were transferred to a streptavi-
`din-coated 96-well ELISA plate and mixed while shaking for 1 hour at
`room temperature. The a-Btk antibody (BD 611116, 1:1000 dilution in
`PBS 1 0.05% Tween-20 1 0.5% BSA) was then incubated for 1 hour at
`room temperature. After wash, goat anti-mouse horseradish peroxi-
`dase (1:5000 dilution in PBS 1 0.05% Tween-20 1 0.5% BSA) was
`added and incubated for 1 hour at room temperature. The ELISA was
`developed with addition of tetramethyl benzidine followed by Stop
`Solution (CST) and read at optical density 450 nm. The standard
`curve (11.7–3000 pg/ml) was generated with human full-length
`recombinant Btk protein and plotted using a 4-parameter curve fit in
`Gen5 software. Uninhibited Btk detected from samples was normal-
`ized to mg total protein as determined by BCA protein analysis (cat.
`no. 23225; Pierce, Rockford, IL).
`Spleen Homogenization. Spleens were harvested from mice,
`frozen immediately in liquid nitrogen, and stored at –80°C. To
`generate spleen lysates, each spleen was sliced in half and lysed using
`a Precellys 24 Bead Homogenizer in 500 ml of Bio-Rad Bio-Plex Lysis
`Buffer plus protease inhibitors (Bio-Rad, Hercules, CA). Supernatant
`was transferred to a fresh microfuge tube and stored frozen at –80°C
`until analysis.
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`Btk Inhibition with the Covalent Inhibitor CC-292
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`potent, selective inhibitor of Btk. CC-292 was rationally
`designed to possess high affinity for the ATP binding pocket
`and to form a specific covalent bond with cysteine 481 in Btk,
`a poorly conserved amino acid among kinases. In biochemical
`assays, CC-292 is a potent inhibitor of Btk kinase activity
`(IC50apparent , 0.5 nM, kinact/KI 5 7.69  104 M–1s21) and is
`highly selective (Supplemental Tables 1 and 2; see Supple-
`mental Methods for more information). Because biochemical
`kinase assays may overestimate the potency of small molecule
`kinase inhibitors due to high ATP concentrations found in the
`cellular environment, cell activity for several of these closely
`related kinase family members was assessed. CC-292 dem-
`onstrated a high degree of selectivity against kinases with
`a cysteine in a homologous position as Cys481 in Btk (epidermal
`growth factor receptor, Itk, Janus kinase 3; Supplemental
`Tables 3 and 4). Full details of the in vitro properties of
`CC-292 as well as confirmation of the covalent mechanism of
`action by mass spectrometry are shown in Supplemental Figs.
`2–4 and Supplemental Tables 1–3. To demonstrate specific
`inhibition of Btk in cells, CC-292 was evaluated in Ramos
`cells, which express an intact BCR signaling pathway that is
`activated robustly by addition of anti-IgM. CC-292 potently
`inhibited Btk autophosphorylation on Tyr223 (EC50 5 8 nM;
`Fig. 2A; Supplemental Fig. 5), phosphorylation of the Btk
`substrate, PLCg2, as well as activation of the downstream
`kinase extracellular signal-regulated kinase, all previously
`shown to be sensitive to Btk inhibition (Honigberg et al., 2010;
`Di Paolo et al., 2011). It is noteworthy that while CC-292
`inhibited autophosphorylation of Btk, it had no effect on the
`phosphorylation of Btk on Tyr551, a site phosphorylated by
`Lyn and Syk and required for Btk activation (Afar et al.,
`1996). These data demonstrate CC-292 is selective for Btk and
`does not inhibit the Src-family kinases upstream of Btk in the
`BCR signaling pathway (Fig. 2A).
`Consistent with its covalent mechanism of action, CC-292
`provided prolonged inhibition of kinase activity hours after
`the drug was removed from cells. In contrast to reversible
`inhibition with the potent Btk inhibitor dasatinib (Hantschel
`et al., 2007), for which kinase activity had almost completely
`returned 6 hours after drug removal, recovery of Btk activity
`following a 1-hour exposure to CC-292 continued to be suppressed
`∼8 hours in drug-free media (Fig. 2B). This prolonged period
`of Btk inhibition correlated well with Btk protein turnover
`assayed in the presence of the protein synthesis inhibitor
`cyclohexamide. These experiments indicated that existing
`cellular Btk was degraded slowly (36% reduction of protein in
`8 hours and 63% reduction at 17 hours) (Supplemental Fig. 6).
`Since Btk exposed to CC-292 is irreversibly bound and inhibited,
`the return of Btk-dependent signaling relies on the appearance
`of new Btk protein as a result of protein synthesis in a CC-292–free
`environment.
`Quantitative Analysis of Btk Occupancy. The covalent
`mechanism of action of CC-292 has enabled design of a
`companion PD assay that directly quantifies covalent bonding
`to Btk protein after drug exposure. A probe (CNX-500) was
`developed consisting of a covalent Btk inhibitor chemically
`linked to biotin (Fig. 3A; Supplemental Fig. 7). This molecule
`retains inhibitory activity against Btk (IC50app 5 0.5 nM)
`as well as the ability to form a covalent bond with Btk
`(Supplemental Fig. 8) and has demonstrated selectivity
`against the structurally related kinase epidermal growth
`factor receptor (IC50app . 25 nM), and upstream Src-family
`
`Collagen-Induced Arthritis Model. Experiments were carried
`out at Bolder Biopath, Boulder, CO. All experiments were carried out
`in compliance with regulations of the Institutional Animal Care and
`Use Committee and were conducted in accordance with principles and
`procedures dictated by the highest standards of humane animal care.
`Dba1 mice were injected at the base of the tail with 150 ml of Freund’s
`Complete Adjuvant (Sigma-Aldrich) containing bovine type II
`collagen (Elastin Products, Owensville, MO) (2 mg/ml) on day 0 and
`again on day 21. On study days 25–27, onset of arthritis occurred, and
`mice were randomized into treatment groups (10 per treatment
`group, four per group for normal). Randomization into each group was
`done after swelling was obviously established in at least one paw, and
`attempts were made to assure approximately equal mean scores
`across the groups at the time of enrollment. Treatment was initiated
`after enrollment. Treatment continued daily (QD at 24-hour inter-
`vals) through arthritis day 14. Clinical scores were assessed for each
`of the paws on study arthritis days 1–15 using the following scoring
`system: 0 5 normal, 1 5 one hind or fore paw joint affected or minimal
`diffuse erythema and swelling, 2 5 two hind or fore paw joints affected
`or mild diffuse erythema and swelling, 3 5 3 hind or fore paw joints
`affected or moderate diffuse erythema and swelling, 4 5 marked
`diffuse erythema and swelling or 4 digit joints affected, 5 5 severe
`diffuse erythema and severe swelling entire paw, unable to flex digits.
`Spleens and plasma were harvested 2 or 24 hours after the last dose of
`CC-292 on arthritis day 14 and paws were removed and fixed in
`formalin for histopathological analysis.
`Clinical Study. A double-blind, placebo-controlled, ascending
`single-dose, randomized study in normal healthy human volunteers
`was conducted at a single clinical research unit in accordance with
`Declaration of Helsinki principles. Informed consent statements were
`obtained from all subjects prior to inclusion in the study. Subjects
`were admitted to the unit 1 day before dosing and discharged 96 hours
`after dosing. Six subjects were administered a single oral dose of
`2 mg/kg CC-292, monitored for safety and evaluated for drug action by
`pharmacokinetic (PK) and PD analysis.
`Isolation of Enriched B Lymphocyte Population from
`Human Healthy Volunteers. Human whole blood (21 ml) was
`collected from each subject at each time point into BD Vacutainer
`CPT Cell Preparation Tubes containing sodium heparin. RosetteSep
`Human B Cell Enrichment Cocktail (cat. no. 15024; StemCell Technol-
`ogies, Vancouver, BC, Canada) was added to each CPT tube and
`centrifuged for 25 minutes at 1800g at room temperature. Isolated
`cells were harvested into a clean 50-ml conical tube that was pooled by
`subject. Each enriched B cell suspension was centrifuged at 400g for
`15 minutes at room temperature. Cell suspensions were diluted in
`1 ml of red blood cell lysis buffer for 3 minutes at room temperature.
`Cell pellets were lysed with 150 ml Bio-Plex lysis buffer (cat. no.
`171-304012; Bio-Rad). The lysates were stored frozen at #–70°C until
`Btk target site occupancy analysis by ELISA.
`
`Results
`CC-292: A Potent, Highly Selective Btk Inhibitor. We
`have identified CC-292 (Fig. 1; Supplemental Fig. 1) as a
`
`Fig. 1. Chemical structure of CC-292 (N-(3-(5-fluoro-2-(4-(2-methoxye-
`thoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide).
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`Fig. 2. CC-292 demonstrates concentration-dependent silencing of Btk activity and prolonged duration of action after a-IgM stimulation of the B cell
`receptor in Ramos cells. (A) Ramos cells were treated with increasing concentrations of CC-292 (0.3–3000 nM) and then stimulated with 5 mg/ml of the
`BCR ligand a-IgM. Btk autophosphorylation as well as Btk substrate phosphorylation (P-Y1217-PLCg2) and downstream activation of extracellular
`signal-regulated kinase (Erk) were assayed by immunoblot. Quantitation of immunoblot demonstrated that CC-292 inhibits Btk autophosphorylation
`with EC50 = 8 nM (n = 4) (Supplemental Fig. 4). (B) Ramos cells were treated with compound for 1 hour. Cells were then resuspended in compound-free
`media and stimulated with 5 mg/ml a-IgM at 0, 4, 6, or 8 hours after compound removal. Btk substrate phosphorylation was measured by immunoblot.
`Btk remains inhibited up to 8 hours after treatment with the covalent modifier CC-292, whereas Btk activity returns quickly after treatment with the
`reversible inhibitor, dasatinib. Representative immunoblot of n = 3 experiments.
`
`kinases including Syk (IC50app . 1000 nM) and Lyn (IC50app .
`3500 nM). Moreover, the specificity of the Btk target
`occupancy ELISA derives from the use of a detection mono-
`clonal antibody that selectively recognizes Btk immobilized
`on the streptavidin substrate by the covalent probe and,
`therefore, this assay measures only Btk bound to the covalent
`probe. By building a standard curve with known amounts of
`recombinant Btk protein bound to CNX-500, the amount of
`Btk in any sample can be precisely quantitated. Used in
`a competition assay, this probe detected free, uninhibited Btk
`and was excluded from interaction with Btk previously
`bonded by CC-292 (Fig. 3B). Results from this analysis can
`be reported in absolute values, such as picograms of free Btk
`per microgram of total protein or in relative terms by
`normalization to control samples not exposed to inhibitor. In
`Ramos cells exposed to a range of CC-292 concentrations, the
`amount of Btk captured by the probe was compared with
`untreated samples and the extent of Btk bonded was
`demonstrated to be proportional to CC-292 drug concentra-
`tion (Supplemental Fig. 9). It is noteworthy that the degree of
`Btk covalently bonded by CC-292, herein referred to as Btk
`occupancy, correlated with inhibition of Btk kinase activity.
`Extensive analysis has revealed that the EC50 of Btk
`occupancy from a CC-292 dose-response in Ramos cells
`(EC50 5 6 nM) correlated directly with the cellular EC50 of
`Btk kinase inhibition with CC-292 (EC50 5 8 nM) (Supple-
`mental Figs. 5 and 9). Furthermore, the concentration at
`which CC-292 inhibited 90% of Btk activity in Ramos cells
`was 35 nM while the concentration of CC-292 required for
`90% occupancy of Btk was 39 nM, supporting a direct
`stoichiometric correlation between target occupancy and
`inhibition of Btk activity. This correlative relationship was
`also demonstrated in freshly isolated human primary naïve
`
`B cells ex vivo. In naïve human B cells, the kinase activity of
`Btk was inhibited 42% at 10 nM, a concentration that
`produced 37% Btk occupancy (Fig. 4A). It is noteworthy that
`kinase inhibition and occupancy also reflected efficacy in
`B cell functional assays such as B cell proliferation (EC50 5
`3 nM; Fig. 4B) and activation as determined by inhibition of
`upregulation of the activation marker, CD69, in response to
`stimulation by anti-IgM (Supplemental Table 5). These data
`demonstrate a strong quantitative relationship among CC-292
`concentration, extent of Btk enzyme inhibition, and level of
`Btk occupancy. Therefore, measurement of Btk occupancy
`can serve as a robust surrogate measurement of Btk kinase
`inhibition that correlates with inhibition of BCR signaling
`and its functional consequences.
`As described above, once covalently bound by CC-292, an
`individual Btk protein is permanently silenced. Therefore, the
`return of activity must depend on new Btk protein synthesis.
`Determination of Btk protein resynthesis rates in mice in vivo
`was enabled by maximally inhibiting Btk with a single dose of
`CC-292 and then monitoring the return of Btk in spleen
`lysates over time with the covalent probe. Mouse spleens were
`collected at several time points after a single oral dose of
`50 mg/kg CC-292, a dose level projected to achieve complete
`Btk engagement, and assayed with the covalent probe to track
`emergence of new Btk protein. New Btk protein was detected
`at low levels 8 hours after compound administration, and
`achieved 43% of predose Btk protein levels at 24 hours and
`71% of predose levels 48 hours after drug administration
`(Fig. 5). It is noteworthy that PK analysis of mouse plasma
`from this experiment indicated circulating CC-292 was absent
`in five of six animals by the 8-hour time point (unpublished
`data). Presently, the potential contribution of active metabo-
`lites of CC-292 cannot be excluded. These data provide precise
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`Fig. 3. Covalent probe CNX-500 allows direct assessment of Btk occupancy in vitro and in vivo. (A) Covalent probe CNX-500. (B) Covalent probe CNX-
`500 detects free, uninhibited Btk in lysates derived from tissue culture, animal tissues, or clinical samples. Samples treated with CC-292 are lysed and
`then incubated with 1 mM CNX-500. Uninhibited Btk in the lysate is captured by CNX-500 and quantitated by streptavidin (SA)-coated ELISA plate.
`Normalization to untreated control sample allows determination of the percentage of Btk occupancy.
`
`determination of the extent and duration of covalent in-
`hibition of Btk protein in mice.
`Relationship of Btk Occupancy and Efficacy of
`CC-292 in the Collagen-Induced Arthritis Model of
`Arthritis. The collagen-induced arthritis (CIA) model has
`been shown previously to respond to both B cell modulating
`therapies as well as direct Btk inhibition (Pine et al., 2007;
`Honigberg et al., 2010; Chang et al., 2011; Di Paolo et al.,
`2011; Liu et al., 2011b). Oral efficacy of CC-292 in an
`established CIA model in mice was measured. Dose-
`dependent inhibition of the clinical signs of inflammatory
`disease was observed during the in-life portion of the model,
`including reduction in joint and paw swelling and visible
`redness of the affected paws. Reduction of clinical signs of
`disease was measured at 95, 85, and 50% for 30, 10, and 3 mg/kg,
`respectively (Fig. 6A). Moreover, all three dose levels of CC-292
`prevented the loss in body weight typically associated with
`severity of disease observed in this model (Supplemental
`Fig. 10). It is noteworthy that CC-292 also demonstrated
`significant effects on the generation of inflammatory chemo-
`kines and cytokines in this model,
`including KC (mouse
`equivalent of interleukin-8), interleukin-6, and tumor necrosis
`factor a (Supplemental Table 6). The precise mechanism
`for this protective effect is currently under investigation but
`suggests direct or indirect modulation of effector cell function
`and may be independent of the role of Btk in B cells. To
`demonstrate the relationship between inhibition of inflam-
`matory activity and direct engagement of CC-292 with Btk,
`spleens collected either 2 or 24 hours after the last CC-292
`
`dose were assayed for Btk occupancy. Occupancy in spleen
`lysates tracked closely with inhibition of the clinical signs of
`disease: 34% occupancy at 3 mg/kg at 2 hours correlated
`with 50% inhibition of disease, Btk occupancy of 84% was
`detected 2 hours after dosing with 10 (85% inhibition of
`disease) or 30 mg/kg (97% occupancy, 95% inhibition of
`disease) of CC-292. Consistent with Btk resynthesis experi-
`ments described earlier, only 19% Btk occupancy remained 24
`hours after the 3-mg dose, whereas sustained occupancy of
`.40% at 24 hours was achieved with dose levels of 10 and 30
`mg/kg. This analysis demonstrated that once-a-day dosing at
`the higher doses resulted in continuous CC-292–Btk engage-
`ment at levels greater than 40% and that this was sufficient
`for .85% inhibition of disease with therapeutic dosing of CC-
`292 (Fig. 6B). Morphologic and histopathologic analysis of six
`affected joints (four paws, two knees) demonstrated a dose-
`dependent protection from joint damage, including pannus
`formation, cartilage degradation, and bone erosion. The
`disease-modifying activity of CC-292 correlated with both
`Btk occupancy and the pronounced inhibition of the clinical
`inflammation characteristic of arthritis in this model (Fig.
`6C). This correlation between Btk occupancy and inhibition
`of disease strongly suggests that selective inhibition of Btk
`provided the protective effect of CC-292 activity in this
`collagen-induced arthritis model.
`Human Clinical PK-PD Relationship with CC-292.
`CC-292 demonstrated covalent bonding, prolonged, selective
`inhibition of Btk in vitro, and efficacy in preclinical models in
`vivo. In addition, there was a strong correlation between the
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`Fig. 4. Btk occupancy with CC-292 in human primary B
`cells correlates with inhibition of Btk signaling and in-
`hibition of B cell proliferation. (A) Human naïve B cells were
`isolated by negative selection, treated with the indicated
`concentrations of CC-292 for 1 hour, stimulated with 5 mg/ml
`a-IgM for 10 minutes, and lysed. Lysates were split in two for
`immunoblot and occupancy analyses. Representative immu-
`noblot from three independent experiments shown and
`occupancy from three experiments plotted as mean 6 S.D.
`(B) Human naïve B cells (CD19+, IgD+) were purified from
`whole blood by negative selection with MACS purification.
`Purified naïve B cells were stimulated with 5 mg/ml a-IgM for
`72 hours. 3H-Thymidine was added to the media for the final
`16 hours and 3H incorporation measured. Experiments were
`done in triplicate. The mean 6 S.D. were plotted and the
`EC50 calculated from n = 3 separate experiments. CC-292
`inhibits B cell proliferation EC50 = 3 nM.
`
`paired traditional pharmacokinetic analysis of plasma
`drug levels with Btk occupancy analysis in a B cell–enriched
`fraction from freshly isolated human blood to determine the
`PK-PD relationship of CC-292 following single oral admin-
`istration in humans. After initial dose escalation, 2 mg/kg
`CC-292 was found to be optimal for analysis of this PK-PD
`relationship. Six healthy adult subjects were adminis-
`tered a single oral dose of CC-292 (2.0 mg/kg) and
`sequential blood samples were isolated over time to
`determine the relationship between the plasma concen-
`tration of CC-292 and Btk occupancy in an enriched B cell
`population.
`There was rapid absorption of 2 mg/kg CC-292 in all
`subjects, with peak plasma concentrations achieved within
`30–120 minutes after dose administration and a mean
`measured maximum plasma concentration of 542 ng/ml
`(Cmax) was attained. Plasma concentrations declined to near
`or below the lower limit of detection (0.1 ng/ml) within 24
`hours post-dose with a median terminal elimination half-life
`of 1.9 hours. Plasma concentrations of CC-292 at 48 hours
`post-dose were below the lower limit of quantification in all
`subjects (Fig. 7).
`Analysis of Btk occupancy was determined at each time
`point using the covalent probe ELISA. The absolute value of
`free Btk in lysates of enriched B