1521-0103/360/1/226–238$25.00 http://dx.doi.org/10.1124/jpet.116.236224THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 360:226–238, January 2017Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Bruton’s Tyrosine Kinase Small Molecule Inhibitors Induce aDistinct Pancreatic Toxicity in Ratss

Rebecca I. Erickson, Leah K. Schutt, Jacqueline M. Tarrant, Michelle McDowell, Lichuan Liu,Adam R. Johnson, Sock-Cheng Lewin-Koh, Maj Hedehus, Jed Ross, Richard A. D. Carano,Karin Staflin, Fiona Zhong, James J. Crawford, Shelly Zhong, Karin Reif, Arna Katewa,Harvey Wong, Wendy B. Young, Donna M. Dambach, and Dinah L. MisnerGenentech, Inc., South San Francisco, California (R.I.E., L.K.S., J.M.T., M.M., L.L., A.R.J., S.-C.L.-K., M.H., J.R., R.A.D.C., K.S.,F.Z., J.J.C., S.Z., K.R., A.K., W.B.Y., D.M.D., D.L.M.); and University of British Columbia, Vancouver, British Columbia (H.W.);Primary Laboratory of Origin: Genentech, Inc., 1 DNA Way, MS59, South San Francisco, CA 94080

Received July 18, 2016; accepted October 31, 2016

ABSTRACTBruton’s tyrosine kinase (BTK) is a member of the Tec family ofcytoplasmic tyrosine kinases involved in B-cell and myeloid cellsignaling. Small molecule inhibitors of BTK are being investigatedfor treatment of several hematologic cancers and autoimmunediseases. GDC-0853 ((S)-2-(39-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,49-bipyridin]-29-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selectiveand reversible oral small-molecule BTK inhibitor in developmentfor the treatment of rheumatoid arthritis and systemic lupus erythe-matosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days orlonger caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-ladenmacrophages with adjacent lobular exocrine acinar cell atro-phy, degeneration, and inflammation. Similar findings were not

observed in mice or dogs at much higher exposures. Hemorrhagein the peri-islet vasculature emerged between four and seven dailydoses of GDC-0853 and was histologically similar to spontane-ously occurring changes in aging SD rats. This suggests thatGDC-0853 could exacerbate a background finding in youngeranimals. Glucose homeostasis was dysregulated following aglucose challenge; however, this occurred only after 28 days ofadministration and was not directly associated with onsetor severity of pancreatic lesions. There were no changesin other common serum biomarkers assessing endocrine andexocrine pancreatic function. Additionally, these lesions werenot readily detectable via Doppler ultrasound, computed tomog-raphy, or magnetic resonance imaging. Our results indicatethat pancreatic lesions in rats are likely a class effect of BTKinhibitors, which may exacerbate an islet-centered pathologythat is unlikely to be relevant to humans.

IntroductionBruton’s tyrosine kinase (BTK) is a cytoplasmic tyrosine

kinase in the Tec kinase family (Bmx, Btk, Itk, Rlk, Tec) ex-pressed primarily in hematopoetic cell lineages that has essen-tial functions in B cells and myeloid cells. Antagonism of BTKenzymatic activity in B cells leads to inhibition of B-cell receptor(BCR)–dependent signaling. This results in the disruption ofchemotactic signals important for B-cell survival, migration, andproliferation, and a reduction in inflammatory cytokine pro-duction from myeloid cells by preventing signaling through the

FCgRIII receptor. Small-molecule BTK inhibitors are cur-rently being developed for treatment of several hematologiccancers and autoimmune diseases.Ibrutinib (Imbruvica; Pharmacyclics, Janssen Biotech, Inc.,

Sunnyvale, CA) is a highly effective BTK inhibitor for thetreatment ofB-cellmalignancies that is generallywell toleratedand largely devoid of leukopenia and hypogammaglobulinemia.Lymphocytosis is observed clinically with ibrutinib treatmentand is thought to be related to inhibition of BCR signalingand efflux of cells from lymphoid tissues into the systemic cir-culation (Herman et al., 2014). Common adverse events foribrutinib in patients with chronic lymphocytic leukemia includenausea, vomiting, diarrhea, constipation, petechiae, contusions,atrial fibrillation, and hypertension (Lipsky et al., 2015; Molica,

This work was funded by Genentech, Inc.dx.doi.org/10.1124/jpet.116.236224.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: AUC0-24, area under the concentration-time curve from time 0 to 24 hours; BCR, B-cell receptor; BTK, Bruton’s tyrosine kinase;CRL, Charles River Laboratories; CT, computed tomography; DAB, diaminobenzidine; DMSO, dimethylsulfoxide; ELISA, enzyme-linked immunosorbentassay; F-344, Fischer-344; GDC-0853, (S)-2-(39-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,49-bipyridin]-29-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one; GNE-309, 2-(39-(hydroxymethyl)-5-((5-(2-methoxyethyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)amino)-1-methyl-6-oxo-1,6-dihydro-[3,49-bipyridin]-29-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one; HRP, horseradish peroxidase; IVGTT, intravenous glucose tolerance test; KO, knockout; MRI,magnetic resonance imaging; OGTT, oral glucose tolerance test; pBTK, phospho-BTK; PE, phycoerythrin; SD, Sprague-Dawley; TE, echo time; US,ultrasound; WH, Wistar-Han; XLA, X-linked agammaglobulinemia.

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2015). It is unclear whether any of these findings are directlyrelated to inhibition of BTK activity. Ibrutinib inhibits BTKcovalently and irreversibly by targeting a cysteine residue(Cys481) conserved in 10 other kinases within the kinome.Consequently, ibrutinib potently inhibits several “off-target”kinases at therapeutic doses, which may also contribute to theadverse events observed (Pan et al., 2007; Evans et al., 2013).For example, EGFR, TEC, and PI3K-AKT pathway inhibitionhave been associated with diarrhea, bleeding, and atrialfibrillation, respectively (McMullen et al., 2014; Stephens andSpurgeon, 2015; Byrd et al., 2016).Loss of BTK function through genetic mutation in humans or

knockout (KO) in mice suggests that selective BTK inhibitorsshould cause suppression of antibody-mediated immune re-sponses while being generally well tolerated in patients. Inhumans, loss-of-function mutations in the gene encoding BTKresult in X-linked agammaglobulinemia (XLA). Due to a blockin B-cell development between the pro- and pre-B-cell stage,patients with XLA have a profound reduction in serum Igconcentration of all classes, and fail to mount effective humoralimmune responses (Conley et al., 2000; Bao et al., 2012).Patients with XLA are susceptible to recurrent bacterial andenteroviral infections; however, with the advent of Ig infusiontherapy, most of them live well into adulthood (Howard et al.,2006). Btk KO and X-linked immunodeficient mice have a lesssevere immunologic phenotype than humans due to only apartial block in B-cell development and some compensationby other BCR signaling components, including TEC kinase(Rawlings et al., 1993; Kerner et al., 1995; Ellmeier et al., 2000;Lindvall et al., 2005). Serum IgG and IgM concentrations in KOmice are reduced but not absent (Satterthwaite et al., 1997).Conversely, serum IgE concentration is increased, which hasbeen attributed to an increase in class-switched B cells in thespleen (Iyer et al., 2011). Moreover, to the extent that BTK-deficient humans andmice have been evaluated, few clinical orhistopathological abnormalities external to the hematopoeticsystem have been reported.GDC-0853 ((S)-2-(39-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-

4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,49-bipyridin]-29-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a potent,selective, reversible, small-molecule BTK inhibitor in develop-ment for the treatment of autoimmune diseases (Young andCrawford, 2016). In preclinical assessments to characterize itstoxicity profile, GDC-0853 was well tolerated when orally ad-ministered daily to Sprague-Dawley (SD) rats for up to 4 weeks.Findings that were considered related to pharmacologic activ-ity of GDC-0853 included mild increases in blood lymphocytecount, minimal B-cell depletion in lymphoid organs, and changesin serum Ig concentrations. Unexpectedly, themajor finding waspancreatic toxicity, characterized microscopically by multifocalislet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cellatrophy, degeneration, and inflammation. We conducted a com-prehensive characterization of the distinct pancreatic lesionsassociated with GDC-0853 administration in SD rats. Ourresults suggest that inhibition of BTK enzymatic activity isinvolved in the pathogenesis of these lesions. The most sensi-tive strain was the SD rat; lesions in Fischer-344 (F-344) andWistar-Han (WH) rats were of a lesser severity and/or requireda longer treatment duration to develop. The observed lesionswere histologically similar to spontaneously occurring changes

in aging SD rats. Pancreatic lesions were subclinical, with fewchanges in standard clinical pathology parameters in exocrineor endocrine pancreatic function, and were not detectable byimaging techniques. Our results indicate that pancreaticlesions in rats are likely a class effect of BTK inhibitors, whichmay exacerbate an islet-centered pathology that is unlikely tobe relevant to humans.

Materials and MethodsTest Articles. All test articles, including GDC-0853, GNE-309

(2-(39-(hydroxymethyl)-5-((5-(2-methoxyethyl)-4,5,6,7-tetrahydropyr-azolo[1,5-a]pyrazin-2-yl)amino)-1-methyl-6-oxo-1,6-dihydro-[3,49-bipyr-idin]-29-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one), ibrutinib, and spebrutinib, were synthesizedat Genentech, Inc. (South San Francisco, CA) and formulated for oraladministration in the same vehicle: 1.0% (w/v) 4000 cps hydroxypro-pylmethylcellulose (Fagron, Rotterdam, Netherlands), 0.2% (v/v)Tween 80 (Avantor, Center Valley, PA), and 100 mM citrate buffer(pH 3.0 6 0.1; Spectrum Laboratory Products, Inc., New Brunswick,NJ), prepared in reverse osmosis water. The same vehicle served asthe control article in the in vivo studies.

Animal Care and Use. All in vivo experiments were performed instrict accordance with the guidelines in the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals. Allprotocols were approved by the Institutional Animal Care and UseCommittees at Genentech or Covance, Inc., and appropriate effortswere made to reduce animal suffering. Three strains of rats were usedin the in vivo studies: Sprague-Dawley [Crl:SD(SD)], Wistar-Han[CRL:Crl:WI(Han)], and Fischer-344 (F344 Fischer). Sprague-Dawleyrats were obtained from Charles River Laboratories (CRL; Hollister,CA or Portage, MI). Wistar-Han rats were obtained from CRL(Hollister, CA or Margate, UK). Fischer-344 rats were obtained fromCRL (Kingston, NY). At the start of the studies, animals were6–12 weeks old. Dose formulations (described earlier) were adminis-tered once daily by oral gavage at a dose volume of 5 or 10 ml/kg.Clinical observations (daily) and body weights (at least twice weekly)were collected for all animals.

Following the initial identification of pancreatic pathologic changesin rats administered GDC-0853 in 7- and 28-day toxicity studies, andthe absence of similar findings in dogs administered GDC-0853 in a28-day toxicity study, a series of experiments were conducted in rats tobetter characterize the pancreas lesions, to determine any strain differ-ences in susceptibility, and to determine whether the developing lesioncould bemonitored by glucose tolerance testing and/or imagingmethods.

Toxicokinetics. Concentrations of test articles in plasma werequantified using the liquid chromatography–tandem mass spectrom-etry method. Toxicokinetic parameters [Cmax, time to maximal con-centration (Tmax), and area under the concentration-time curve fromtime 0 to 24 hours (AUC0-24)] were determined by noncompartmentalmethods using the extravascular input model (Phoenix WinNonlin,version 6.3.0; CertaraUSA Inc., Princeton, NJ). Concentrations of testarticles were also evaluated in pancreas and liver tissue for compar-ison with concentrations in plasma at the same time point.

Clinical Pathology. Rats were fasted overnight prior to collectionof blood for all clinical pathology measurements. Blood samples forhematology analyses were collected from the retro-orbital plexusunder isoflurane-induced anesthesia into EDTA-containing tubesand analyzed on a Sysmex XT 2000iV (Sysmex America, Inc.,Mundelein, IL). For measurement of IgE and IgG concentrations,blood was collected into serum separator tubes from the abdominalaorta under isoflurane-induced anesthesia and ketamine. Concentra-tion of serum IgE was measured by enzyme-linked immunosorbentassay (ELISA) using mouse anti-rat IgE for capture and detection(Clone B41-1 and Clone B41-3, respectively; BDBiosciences, San Jose,CA) and streptavidin–horseradish peroxidase (HRP;GELife Sciences,Marlborough, MA). Purified rat IgE isotype control (BD Biosciences)

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was used as a standard. Concentration of serum IgGwasmeasured byELISA with goat anti-rat IgG-Fc and HRP-conjugated goat anti-ratIgG-Fc as capture and detection antibodies, respectively (BethylLaboratories, Montgomery, TX). Rat reference serum (14 mg/ml ofrat IgG; Bethyl Laboratories) was used as a standard. Serum amylase,lipase, glucose, and fructosamine were analyzed in blood collectedfrom the jugular vein in conscious rats into serum separator tubes andmeasured on a Roche Modular Analytics (Roche Diagnostics Corpo-ration, Indianapolis, IN) or on a Beckman Coulter AU680 (BeckmanCoulter Inc., West Sacramento, CA) for fructosamine using themanufacturer’s applications. Serum insulin was measured by ELISA(Mesoscale Discovery, Gaithersburg, MD).

Histopathology. In all in vivo studies, a necropsy was performedthe day following administration of the last dose of test articles. Theexceptions were studies that included a 4-week recovery-phase cohort.Pancreas was collected and processed for histopathological evalua-tion. The tissue was preserved in formalin, embedded in paraffin, andslideswere prepared from 5-mm-thick sections that were subsequentlystained with hematoxylin and eosin. Any macroscopic observationswere recorded.

Histochemistry and Immunohistochemistry. Formalin-fixed,paraffin-embedded sections of pancreas from a subset of control,unaffected, and affected animals administered 30 mg/kg/day GDC-0853 were cut at 4 mm for staining with Masson’s trichrome stain orimmunolabeling with antibodies against CD68, clone ED1 (Serotec,Kidlington, UK); cytokeratin 19 (Lifespan Biosciences, Seattle, WA);or smooth muscle actin, clone E184 (Epitomics, Burlingame, CA).

For CD68, sections were deparaffinized and pretreated for antigenretrieval using Target Retrieval Solution (Dako, Carpinteria, CA). Theslides were subsequently blocked for endogenous peroxidase activityusing 3%H2O2 and for avidin/biotin using an avidin/biotin blocking kit(Vector Laboratories, Burlingame, CA). Sections were preblocked fornonspecific binding sites with blocking buffer. They were then in-cubated in mouse anti-CD68 at 10 mg/ml, followed by biotinylatedhorse anti-mouse IgG or biotinylated goat anti-rabbit IgG, respec-tively (Vector Laboratories). The sections were incubated in Vectas-tain ABC Elite reagent (Vector Laboratories), followed by MetalEnhanced Diaminobenzidine (DAB; Thermo Scientific, Rockford, IL).

For cytokeratin 19, after deparaffinization, the sections underwentantigen retrieval in Lab Vision EDTA (pH 8.0; Thermo FisherScientific, Waltham, MA), blocking, and then incubation in rabbitanti–cytokeratin 19 at 5 mg/ml. The primary antibody was detectedwith PowerVision Poly anti-Rabbit HRP (Leica, Newcastle, UK),followed by DAB.

For smooth muscle actin, all steps were performed on the VentanaDiscovery XT Platform (Ventana Medical Systems, Tucson, AZ).Sectionswere deparaffinized using EZ Prep (VentanaMedical Systems,Tuscon, AZ), and pretreatment was accomplished with Cell Condition-ing 1 (VentanaMedical Systems,Tucson, AZ) using standard incubationtime. Sections were then incubated with rabbit anti–smooth muscleactin at 0.075 mg/ml. The sections were subsequently incubated withanti-rabbit OmniMap-HRP reagent (Ventana Medical Systems), fol-lowed by DAB. All slides were counterstained with hematoxylin,dehydrated, cleared in xylene, and coverslipped.

Glucose Tolerance Testing. Animals were fasted overnight andadministered vehicle or GDC-0853 2 hours prior to oral glucose toler-ance test (OGTT) or intravenous glucose tolerance test (IVGTT). For theOGTT studies (days 4, 14, and 32), animals were administered a 50%dextrose solution via oral gavage at a volume of 5 ml/kg (total dose of2500mg/kg dextrose). Blood (approximately 0.25ml)was collected priorto administration of GDC-0853 and dextrose and at 15, 30, 60, and120 minutes after administration of dextrose. Blood samples werecollected via the tail vein (days 4 and 14) or jugular vein (day 32) onto aglucometer (days 4 and 14: Nova StatStrip; NovaBiomedical,Waltham,MA; day 32: AlphaTRAK2; VWR, Radnor, PA) for analysis of bloodglucose level. The remaining blood was processed to plasma for anal-ysis of insulin levels. For the IVGTT study (days 4, 14, and 28), ratswere cannulated in the femoral artery. Animals were intravenously

administered a 50%dextrose solution via tail vein at a volume of 5ml/kg(total dose of 2500 mg/kg dextrose). Whole blood (0.1 ml) was collectedprior to and at 2, 5, 10, 15, 30, and 60 minutes after administration ofdextrose. Blood samples were collected via the tail vein onto a NovaStatStrip glucometer for analysis of blood glucose level. The remainingblood was processed to plasma for analysis of insulin levels.

Ultrasound Imaging Study. Micro-ultrasound imaging wasperformed with the Vevo 2100 microimaging system (VisualSonics,Toronto, Ontario, Canada) after 7 and 14 days of administration ofvehicle, GNE-309, or GDC-0853. Pancreatic images were obtainedwith an MS400 256-element array transducer (30-MHz center fre-quency, VisualSonics, Toronto, ON, Canada), 12-mm field-of-view,axial resolution of 0.04 mm, and lateral resolution of 0.1 mm. Three-dimensional pancreatic images were acquired with a motorized drivemechanism that traverses the z-axis while acquiring x-y axis imagesat regular spatial intervals of 0.05 mm. Both b-mode and powerDoppler images were acquired. Average b-mode intensity (in arbitraryunits) for the pancreas was obtained from a single imaging slice thatcontains the largest transverse region of pancreas. Power Dopplerimages for the same slicewere used to estimate percentage vascularity[(power Doppler vascular area/total pancreatic area) � 100].

Computed Tomography Imaging Study. An eXplore Ultrasmall-animal computed tomography (CT) scanner (GE Healthcare,Little Chalfont, Buckinghamshire, United Kingdom) was used to scanthe pancreas at baseline (day 21) and after 7 and 14 days ofadministration of vehicle or GDC-0853. To attain dynamic contrastenhancement, a bolus of 0.6 ml of iohexol (Omnipaque-300, GEHealthcare, Little Chalfont, Buckinghamshire, United Kingdom)was administered via lateral tail vein at a rate of 7.2 ml/s, with adelay of 5 seconds before beginning the scan. Regions of interest weregenerated for the head, body, and tail of pancreas as well as for theabdominal aorta, liver, and portal vein to calculate the followingparameters:

• Time-attenuation curve• Pancreatic enhanced and unenhanced phases• Hepatic enhanced and unenhanced phases• Functional perfusion data: blood volume (ml), blood flow

(ml/min), mean transit time (minutes), and permeability(ml/100 g of tissue/min).

The ratio of the late to early enhancement obtained from the hepaticand pancreatic phases (L:E) was quantified. For a healthy humanwithno pancreatic disease, the L:E ratio is #1 (Hashimoto et al., 2011).

Magnetic Resonance Imaging Study. Magnetic resonance im-aging was conducted after 7 days (4 males from each group on day 8,and 4 males from each group on day 9) and 14 days (day 15 or 16) ofadministration of vehicle or GNE-309. Experiments were performedon a 4.7T horizontal imaging system (Agilent Technologies, SantaClara, CA) using a 63-mm quadrature volume coil. The transverserelaxation time (T2) wasmeasured using a two-dimensional multislicefast spin echo experiment with field of view of 60� 60mm2; 16 coronalslices of 1.0-mm thickness; matrix size of 128 � 128 (zero-filled to256 � 256); echo train length of 16; four echo times (TEs) of 7, 16, 26,and 36ms; and four averages. Data acquisition was synchronizedwithrespiration by triggering with the respiration signal produced by theSA Instruments (Stony Brook, NY) equipment, which resulted in arepetition time of approximately 2000 ms. For each image pixel, T2

and S0 were determined by fitting to the equation:

SðTEÞ5S0exp�2TET2

�

�2

TET2

�2TET2

T2, or transverse relaxation time, is the decay constant for thecomponent of magnetization that is perpendicular to the staticmagnetic field of the magnet, S0. The pancreas was manually outlined

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on the S0 image. The volume and average T2 of the pancreas wascalculated. Pancreatic lesions could lead to an increase in volume or T2

due to neoplasm, inflammation, or edema, or a decrease in T2 due tohyperdense tissue (hyperplasia).

Kinase Panel Selectivity Testing. GDC-0853, GNE-309, andibrutinib were tested at a concentration of 1 mM against a panel of221 recombinant human kinase biochemical assays, including cyto-plasmic and receptor tyrosine kinases, serine/threonine kinases, andlipid kinases (Invitrogen SelectScreen Kinase Profiling Services,Thermo Fisher Scientific). For kinase activity assays, the ATPconcentrations used were within 2-fold of the apparent Michaelisconstant (Km) for each kinase, whereas for ATP-site competitionbinding assays, the active-site competitive probe concentration usedwas approximately equal to its dissociation constant (Kd).

Human Ex Vivo Whole-Blood CD69 Assay. Heparinized wholeblood (100 ml) from healthy human volunteers in 96-well squaretop/tapered V-bottom deep-well plates (Analytical Sales, PomptonPlains, NJ) was incubated for 1 hour at 37°C with a titration of BTKinhibitor or vehicle in duplicate [0.38% dimethylsulfoxide (DMSO)].Bloodwas then stimulatedwith 50mg/ml goat anti-human IgMF(ab9)2(Southern Biotech, Birmingham, AL). After an 18-hour incubation,cells in the blood were stained with 45 ml per well of a cocktailcontaining anti-mouse CD19-PerCP-Cy5.5, clone SJ25C1 (1.1 mg/ml;BD Biosciences); anti-mouse CD27-fluorescein isothiocyanate, cloneL128 (0.18 mg/ml; BD Biosciences), and anti-mouse CD69-PE (CD69-phycoerythrin), clone FN50 (7.5 ml per 45 ml of cocktail; BD Pharmin-gen, San Jose, CA) or PEmouse IgG1, k isotype control (10 ml per 45 mlcocktail; BD Pharmingen) for 30 minutes at room temperature. Thiswas followed by lysis of red blood cells using 1�BD Pharm lysis buffer(BD Biosciences). Cells were washed with fluorescence-activated cellsorting buffer and fixed with 2% paraformaldehyde. Samples wereacquired and analyzed on a BDLSRII flow cytometer (BDBiosciences,San Jose, CA). Activation of B cells (CD191CD27–) was assessed basedon CD69 PE mean fluorescence intensity using FACSDiva software(BD Biosciences). The CD69 PE mean fluorescence intensity wasplotted against the log10 of the BTK inhibitor concentration and fit bynonlinear regression using Prism version 5.0 (GraphPad Software, LaJolla, CA) to the variable-slope four-parameter sigmoidal inhibitionequation to determine the half-maximal inhibitory concentration(IC50). Inhibitors were tested against blood from at least threedifferent donors, and the mean IC50 (6SD) was calculated.

Human Ex Vivo Whole-Blood Phospho-BTK Assay.Heparinized whole blood (304 ml) from healthy human volunteers in96-well square top/tapered V-bottom deep-well plates was incubatedfor 6 hours at 37°C with a titration of BTK inhibitor or vehicle induplicate (8% DMSO) in a water reservoir within a tissue cultureincubator (0.4%DMSO). After the incubation, the plate was put on ice,and the blood was dilutedwith 320 ml of 2� lysis buffer (Cell SignalingTechnology, Danvers, MA) containing complete protease inhibitors(Roche Diagnostics), 2� phosphatase inhibitor cocktails 2 and3 (Sigma-Aldrich, St. Louis, MO), and 200 mM sodium orthovanadate(New England Biolabs, Ipswich, MA). The diluted blood was thor-oughly mixed, and the plate was sealed and stored at –80°C. Forphospho-BTK (pBTK)(Y223) detection, High Bind 96-well plates(Mesoscale Discovery, Gaithersburg, MD) were spotted with 10 mlper well of 10 mg/ml mouse anti-BTK monoclonal antibody (BDBiosciences) and then allowed to dry before use. Phospho-BTK from250 ml of thawed lysate was detected using rabbit anti-human pBTK(Y223) monoclonal antibody (13.2 mg/ml; Epitomics) and SULFO-TAGconjugated goat anti-rabbit IgG antibody (1 mg/ml; Mesoscale Discov-ery). The electrochemiluminescence signal was recorded with aSECTOR Imager 6000 MSD reader (Mesoscale Discovery). The pBTKelectrochemiluminescence signal was plotted against the log10 of theinhibitor concentration and fit by nonlinear regression using Prismversion 5.0 (GraphPad Software) to the variable slope four-parametersigmoidal inhibition equation to determine the IC50. Inhibitors weretested against blood from at least three different donors, and themeanIC50 (6SD) was calculated.

Expression of Btk in Pancreas. Laser capture microdissec-tion was conducted on exocrine and endocrine tissue from SD ratpancreatic tissue (three samples) using Zeiss Palm (Zeiss, Thornwood,NY). RNA was isolated using the Arcturus PicoPure kit (AppliedBiosystems, Carlsbad, CA) and DNase treated on-column beforebioanalyzer quantification and qualification (RNA 6000 Pico Kit;Agilent, Santa Clara, CA). Reverse transcription to cDNA (Super-Script VILO Master Mix; Thermo Fisher Scientific) was subsequentlyperformed on the laser capture microdissection samples: human isletRNA (four anonymous samples, sex unknown; Prodo Laboratories,Irvine, CA), universal RNA (rat/human; Clontech, Mountain View,CA), and total RNA from pooled isolated rat islet preparations(AllCells, Alameda, CA). This was followed by preamplificationutilizing PreAmp Master Mix (Fluidigm, South San Francisco, CA).Subsequent polymerase chain reaction for relative gene expressionused Taqman Fast Universal PCR Master Mix (Applied Biosystems)and the 96.96 Dynamic Array Integrated Fluidic Circuit (Fluidigm,South San Francisco, CA) on the Biomark HD System (Fluidigm). Alltarget and housekeeping gene assays used for preamplification andrelative gene expression were species-specific Taqman Assays pur-chased from Life Technologies. Nonblank values in all samples wereall detectable with a good dynamic range, and there were no falsepositives in any assay blanks. Standard curves from diluted islet sampleswere within the linear range for robust assay detection. Finally, geneexpression was normalized against the ribosomal endogenous controlgene, 18S. Data are represented as delta CTs, relative to the highlyexpressed 18S, where negative numbers are lower than 18S and positivenumbers are greater than 18S.

Statistical Analyses. Data are expressed as themean6 standarddeviation. Repeated-measures models were fitted to the longitudinalblood glucose and insulin data with treatment, time points, and theirinteraction as fixed effects and animal as a random effect. Effect oftreatment on change from baseline was evaluated at each time pointusing contrast t test with Bonferroni correction for multiple compar-isons. Log transformation was applied prior to analysis to addressheteroskedasticity where appropriate.

Levene’s test was used to test for heteroskedasticity in b-modeintensity and percentage vascularity for ultrasound (US) and mag-netic resonance imaging (MRI) T2 data. If Levene’s test was signifi-cant, treatment effect was evaluated usingWelch’s t test; otherwise, atwo-sample t test assuming equal variances was conducted.

For all comparisons, statistical analyses were evaluated at a 5%significance level, and statistical analysis of data were performedusing SAS 9.2 (SAS Institute, Cary, NC).

For the CT study, differences between animals administered GDC-0853 or vehicle were tested using a t test with P , 0.05 in thestatistical package.

For the MRI study, differences in volume or overall pancreatic T2

between animals administered GNE-309 or vehicle were tested usinga t test with P , 0.05 in the statistical package JMP (SAS Institute).

ResultsBTK Inhibitors Induce Distinct Pancreatic Lesions

in Sprague-Dawley Rats. GDC-0853, a potent, selective,reversible, small-molecule BTK inhibitor, was orally admin-istered to SD rats for 1–28 consecutive days at doses up to100 mg/kg/day (Table 1). Exposure to GDC-0853 (assessed byCmax and AUC0-24) increased over the dose range tested.Generally, increase in exposure was dose-proportional up to30 mg/kg/day and greater than dose-proportional between30 and 100 mg/kg/day. No toxicologically significant accumu-lation or sex-dependent differences in exposure were observed(Supplemental Fig. 1; Table 2). GDC-0853 was generally welltolerated. Occasional hypoactivity, reduced mean body weightgain, and reduced food consumption (approximately 80% of

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control means) were observed only after 28 days at the highestdose tested (100 mg/kg/day).GDC-0853–related changes in clinical pathology parame-

ters included mildly increased blood lymphocyte counts (up to50% over control mean) and elevated serum IgE concentration(up to 5-fold over control mean) with no clear change in IgGconcentration (Supplemental Fig. 2). These changes wereconsistent with reported effects of BTK inhibition followingadministration of ibrutinib or through loss of function in KOmice (Satterthwaite et al., 1997; Iyer et al., 2011; Hermanet al., 2014).GDC-0853–related microscopic findings in the pancreas

were observed at doses $5 mg/kg/day after dosing durationsof 7–28 days. Multifocal pancreatic lesions, often islet-centered but sparing islet cells, were characterized by varying

degrees (minimal to moderate) of islet/peri-islet hemorrhage,fibrosis, mixed cell infiltrates, and pigment-laden macro-phages. Additionally, significant involvement of adjacentexocrine acinar tissue was noted in some animals, consistingof lobular acinar cell atrophy with acinar cell degeneration/apoptosis, interstitial mixed-cell (predominantly macro-phages with fewer lymphocytes and neutrophils) infiltrates,and/or interlobular hemorrhage and edema. Despite substan-tial involvement of islet tissue, there was little microscopicevidence of islet cell degeneration. Pancreatic lesions were notobserved in rats administered 30mg/kg/dayGDC-0853 for 1 or3 days, suggesting the onset of the lesion was between 4 and7 days of dosing. Representative photomicrographs depictingthe variety of pancreatic findings present are shown in Fig. 1.Themost subtle microscopic change wasminimal hemorrhage

TABLE 1In vivo study design

Purpose Duration (days) Test Article Rat Strain Number (N) of Animals Dose Levels EndpointsEvaluated

mg/kg

Pilot toxicity 7 GDC-0853 SD N = 4/sex/group 0, 5, 15, 30 TKt, CP, PHPGeneral toxicity 28–32 GDC-0853 SD N = 15/sex/group, with N =

5/sex/group undergoing a28-day recovery period

0, 0.5, 10, 30, 100 TKs, CP, OGTT,PHP

Characterization of pancreaspathology

21 GDC-0853 SD N = 6 males/group 0, 0.5, 30 TKt, CP, PHP,IHC

Oral glucose metabolism 14 GDC-0853 SD N = 6 males/group 0, 10, 100 TKt, OGTT,PHP

Intravenous glucosemetabolism

28 GDC-0853 SD N = 10 males/group 0, 30 TKt, IVGTT,PHP

Strain sensitivity 14 GDC-0853 SD, WH, F-344 N = 6 males/group 0, 30 TKt, PHPStrain sensitivity 14 GNE-309 SD, WH, F-344 N = 6 males/group 0, 100 TKt, PHPDoppler ultrasound imaging 14 GNE-309 SD N = 8 males/group 0, 100 TKt, US, PHPDoppler ultrasound imaging 14 GDC-0853 SD N = 16 males/group 0, 30 TKt, US, PHPComputed tomography

imaging14 GDC-0853 SD N = 16 males/group 0, 30 TKt, CT, PHP

Magnetic resonance imaging 14 GNE-309 SD N = 8 males/group 0, 100 TKt, MRI, PHP

CP, standard clinical pathology; IHC, immunohistochemistry of pancreatic tissue; PHP, standard pancreatic histopathology (hematoxylin and eosin); TKs, toxicokinetics insatellite animals; TKt, toxicokinetics in toxicity animals.

TABLE 2Comparison of BTK inhibitors and results in Sprague-Dawley rats: BTK potency and selectivity, toxicokinetics, and incidence of pancreatic lesions

BTKInhibitor

Whole-Blood Potencya

Kinases Inhibited at 1 mMb StudyDuration Dose Cmax

c AUC0-24c Incidence of Pancreatic

LesionsdpBTK IC50 CD69 IC50

nM nM days mg/kg/day mM hour×mM

GDC-0853 11 6 4 8 6 6 BTK (.80%), SRC, FGR, BMX (.50%) 7 5 1.52 7.55 1/4 M, 0/4 F10 4.29 23.9 2/4 M, 0/4 F30 15.1 149 2/4 M, 0/4 F

GNE-309 15 6 2 13 6 3 BTK (.80%), SRC, FGR (.50%) 7 30 4.17 17.4 0/4 M, 1/4 F100 17.7 183 2/4 M, 2/4 F300 54.8 566 2/4 M, 3/4 F

Ibrutinib 7 6 2 12 6 1 BTK, SRC, FGR, BMX, BLK, BRK,TEC, YES, RIPK2, HCK, CSK,LCK, LYN, TXK, ERBB4, ERBB2,EGFR, FRK, RET, SRM, TNK2,FLT3, ITK, JAK3 (.80%), FLT4,KDR, B-RAF, TIE2, CSF1R,FGF1R, PDGFRa (.50%)

14 5 0.349 0.608 0/6 M, 1/6 F25 2.20 5.56 1/6 M, 1/6 F

Spebrutinib 186 6 214 392 6 146 BTK (.80%), SRC (.50%), BMX,STK16, TXK, TEC, JAK3 (.80%),ERBB4, ARK5, JNK1, RET, FLT3,AuroraA, JNK3, AuroraB, DRAK1,WEE1, JAK2, FLT4, IRAK1 (.50%)

14 30 1.00 2.66 3/6 M100 2.26 10.5 2/6 M300 6.64 36.3 1/6 M

F = female; M = male.aThe mean IC50 6 standard deviation from three to six donors is presented.bBTK inhibitors were tested against a panel of 221 active recombinant human kinases.cToxicokinetics performed on the last day of dosing: group mean Cmax and AUC0-24 (male and female combined where relevant due to no appreciable sex differences).dNot observed in any control animal treated with vehicle.

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Fig. 1. Representative photomicrographs of pancreatic histopathology observed in Sprague-Dawley rats following daily oral administration ofGDC-0853 for 21 or 28 days are presented. (A) Minimal peri-islet hemorrhage, day 29 (bar = 50 mm). (B) Islet/peri-islet hemorrhage and fibrinwith mixed-cell infiltrates and exocrine degeneration/atrophy, day 29 (bar = 100 mm). (C) Islet/peri-islet fibroplasia/fibrosis with adjacent exocrineatrophy, day 22 (bar = 100 mm). (D) Multifocal lesions at different stages present concurrently, day 29 (bar = 500 mm). (E) Masson’s trichromestain highlighting collagen (blue) in peri-islet fibrosis, day 22 (bar = 100 mm). (F) Immunohistochemical labeling of smooth muscle actin, day22 (bar = 100 mm). (G) Immunohistochemical labeling of CD68, day 22 (bar = 100 mm). (H) Immunohistochemical labeling of cytokeratin 19, day22 (bar = 100 mm).

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in the small blood vessels of the peri-islet vasculature at theinterface of the islet and exocrine tissue (Fig. 1A). In moreseverely affected islets, the hemorrhage was more extensive,and islet/peri-islet lesions had the appearance of “explodingislets,”with clusters or individual islet cells floating in lakes ofhemorrhage, fibrin and secondary degeneration of adjacentexocrine acinar cells, and varying amounts of mixed-cellinflammatory infiltrates and pigment-laden macrophages(Fig. 1B). Other islets were surrounded and/or dissected bylarge areas of dense fibroplasia and fibrosis (Fig. 1C). Atrophyof adjacent lobules of exocrine pancreas was suggestive ofimpaired blood flow through the affected peri-islet vascula-ture. Multifocal lesions at different stages of developmentwere often present in the same animal, suggesting an ongoinginsult (Fig. 1D). Pancreatic lesions were not observed in anyanimal treated with vehicle or 0.5 mg/kg/day GDC-0853 for upto 28 days. Thus, 0.5 mg/kg GDC-0853 was considered to bethe no-observed-adverse-effect level for this finding. Therewas considerable resolution of the pancreatic pathology at theend of the 28-day recovery period, with only small amounts ofmature fibrous connective tissue dissecting islet/peri-isletareas and pigment-laden macrophages in the interstitial andinterlobular connective tissue remaining.Immunohistochemical and histochemical evaluation of the

pancreatic lesion was performed to further characterize thepathologic changes observed microscopically by hematoxylinand eosin staining. Histochemical staining of the affectedpancreatic sections with Masson’s trichrome stain demon-strated an increase in blue-staining collagen fibers consistentwith fibrosis within affected islet/peri-islet and interstitialareas (Fig. 1E). Increased smoothmuscle actin labeling within

areas of pancreatic islet/peri-islet fibrosis could originate fromseveral different cell types, including blood vessel–associatedsmoothmuscle pericytes or a localized activation of pancreaticstellate cells (Fig. 1F). Increased CD68 labeling confirmed amacrophage infiltrate (Fig. 1G). Increased numbers of weaklycytokeratin 19–positive tubular structures were present insurrounding areas of islet/peri-islet fibrosis and within atro-phic exocrine lobules. This was consistent with pancreaticductular proliferation and/or formation of duct-like tubularcomplexes by atrophic acinar cells (Fig. 1H). These findingsconfirmed the presence of fibrosis and a CD68-positive mac-rophage infiltrate and suggested that blood vessel–associatedpericytes or activated pancreatic stellate cells may be involvedin the production of the fibroplastic reaction.To help understand whether inhibition of BTK enzyme

activity was driving the SD rat pancreatic pathologic changes,we evaluated three additional BTK inhibitors in SD rats:GNE-309, ibrutinib, and spebrutinib (Fig. 2). GNE-309 is apotent and highly selective BTK inhibitor that is structurallyrelated to GDC-0853. Ibrutinib and spebrutinib are structur-ally distinct, potent, and less-selective BTK inhibitors. Incontrast to GDC-0853, which reversibly inhibits BTK at theATP binding site, ibrutinib and spebrutinib inhibit BTKcovalently and irreversibly by targeting a cysteine residue(Cys481) near the ATP binding site that is conserved in10 other kinases within the kinome (Pan et al., 2007; Evanset al., 2013). As a consequence, covalent-binding BTK inhib-itors potently inhibit many of these “off-target” kinases.The on-target potency of the four molecules was compared

using two human ex vivo whole-blood assays to evaluate BTK-pathway inhibition. Additionally, the selectivity was assessed

Fig. 2. Molecular structures of BTK inhibitors GDC-0853,GNE-309, ibrutinib, and spebrutinib.

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using a panel of 221 in vitro kinase assays, includingcytoplasmic and receptor tyrosine kinases, serine/threoninekinases, and lipid kinases. This single point testing at 1 mMshowed that, relative to ibrutinib and spebrutinib, GDC-0853and GNE-309 were much more selective for BTK over otherkinases (Table 2). GDC-0853 and GNE-309 were also evalu-ated in a broad panel of biochemical radioligand-binding andenzyme assays, including 42 targets of major classes ofbiogenic amine receptors, neuropeptide receptors, ion channelbinding, and neurotransmitter transporter. The results ofthese assays did not reveal any significant off-target binding(data not shown).Doses of GNE-309, ibrutinib, and spebrutinib were selected

at levels that would result in clinically relevant plasmaexposures (Pharmacyclics, Inc., 2015; Brown et al., 2016).When administered to SD rats for 7–14 consecutive days,exposure to each of the molecules (Cmax and AUC0-24) in-creased with the dose over the dose range tested (Table 2). Allmolecules were well tolerated with no clinical signs or bodyweight changes attributed to treatment. Microscopic pancre-atic lesions similar to those observed in SD rats administeredGDC-0853 were present with GNE-309, ibrutinib, and spe-brutinib at all dose levels examined, but not in animalstreated with vehicle (Table 2).Taken together with the highly selective properties of GDC-

0853, where no off-target kinase activity is expected at thelow-observed-adverse-effect level for pancreatic lesions in SDrats (7 days at 5 mg/kg/day), these data indicate that in-hibition of BTK enzymatic activity in SD rats is likely involvedin the development of the pancreatic lesions (Fig. 3).Sprague-Dawley Rat Was the Most Sensitive Strain

Tested. During the histopathologic characterization of theGDC-0853–related pancreatic lesions, it was noted that themore mildly affected pancreatic islets in young (10- to12-week-old) SD rats treated with GDC-0853 closely resem-bled the microscopic changes of the age-related spontaneouspancreatic islet hemorrhage and fibrosis in SD rats (Reavenand Reaven, 1981; Dillberger, 1994; Imaoka et al., 2007). Thissimilarity suggested that BTK inhibitors might exacerbate abackground pancreatic change in younger SD rats, and thatthe SD strain may be highly sensitive to the pancreatic effects

of GDC-0853. To test this hypothesis, we investigated theeffects of BTK inhibitors on the pancreas of two othercommonly used laboratory rat strains, F-344 and WH. Fol-lowing administration of GDC-0853 (30 mg/kg/day) or GNE-309 (100 mg/kg/day) for 14 consecutive days, exposure wasconfirmed in all treated animals. Plasma concentrations ofboth GDC-0853 and GNE-309 were similar across strains at3 hours postdose, near the anticipated Tmax, on day 14 (Fig. 4).With both molecules, the same incidence and severity of BTKinhibitor–related pancreatic lesions was observed across thestrains: in 5 of 6 SD (moderate), 1 of 6 F-344 (minimal), and 0 of6 WH rats. Thus, SD rats were particularly sensitive to theeffects of BTK inhibitors on the pancreas (Fig. 4). The reducedsensitivity of WH rats was further confirmed in longer-termstudies with GDC-0853. No GDC-0853–related pancreaticlesions were observed in WH rats administered GDC-0853at doses up to 30 mg/kg/day for 28 days. However, following6 months of daily dosing up to 30 mg/kg/day in WH rats,minimal-to-mild pancreatic islet/peri-islet fibrosis andpigment-laden macrophages were observed in malesadministered $2 mg/kg/day GDC-0853 (data not shown).These pancreatic changes were similar in extent and severityto the islet changes occurring spontaneously in aging SD rats,but did not occur in any vehicle-treated animals, and thereforewere considered related to GDC-0853 administration.BTK Is Expressed at Low Levels in the Rat and

Human Pancreas. BTK is predominantly expressed inmosthematopoietic cells and tissues harboring these cell types. Todate, there is no reported expression or function of BTK in theendocrine or exocrine pancreas. To better understand whetherBTK enzyme inhibition in the pancreas itself could be causingthe lesions in rats, we evaluated local drug concentrations ofGDC-0853 and Btk expression in pancreatic tissue. Following21 days of dosing at 30 mg/kg in SD rats, GDC-0853 concen-tration near Tmax (3 hours postdose) in plasma (8.12 mM) wasgreater than in pancreas (0.319 mM) or liver (2.44 mM). Thisconfirmed local exposure but demonstrated no preferentialdrug accumulation in pancreas and liver. Transcriptionalprofiling of Btk was performed on laser-captured microdis-sected endocrine and exocrine tissue from SD rats andpurchased human islet samples. Compared with lymph node

Fig. 3. Exposure to GDC-0853 in Sprague-Dawley rats relative to BTK (on-target) and off-target (BMX, FGR, SRC) kinase half-maximalinhibitory concentrations (IC50) is presented.Pharmacokinetic profiles were plotted for theno-observed-adverse-effect level (NOAEL;0.5 mg/kg/day) and low-observed-adverse-effectlevel (LOAEL; 5 mg/kg/day) doses identified in28- and 7-day studies, respectively. Kinase IC50values were determined in in vitro biochemicalactivity assays at Genentech (for BTK) orInvitrogen (for BMX, FGR, SRC) using ATPconcentrations equal to the apparent Michaelisconstant (Km) for each kinase.

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or universal RNA samples, Btk was expressed at significantlylower levels across islet/endocrine and exocrine tissue fromboth rats and humans (Fig. 5). It is not known whether theseBtk transcripts would translate to protein expression.BTK Inhibition Has Mild Effects on Glucose

Metabolism. After administration of GDC-0853 to SD ratsat doses up to 100 mg/kg/day for 28 consecutive days, despitethe presence of pancreatic lesions, there were no changes inserum biomarkers of exocrine and endocrine pancreatic func-tion, including amylase, lipase, insulin, or fructosamine (Fig.6). There was a mild increase in blood glucose (10–30%, P ,0.05) in fasted rats administered 100 mg/kg/day GDC-0853after 12 and 28 days of dosing (Fig. 7A); however, given thatpancreatic lesions of a similar incidence and severity wereobserved in animals administered $10 mg/kg/day in thisstudy, the relationship between fasted blood glucose levelsand pancreatic lesions was uncertain. An OGTT was per-formed as amore sensitive test of subclinical effects on glucosehomeostasis. At 28 days, there was an increased and pro-longed peak in blood glucose (without corresponding changesin insulin) relative to animals administered vehicle at $10and $30 mg/kg/day GDC-0853, respectively (Fig. 7, B and C).To evaluate the effectiveness of the OGTT as a premonitoryendpoint in predicting the onset of pancreatic changes, asubsequent study evaluated the effects of 10 and 100 mg/kg/day GDC-0853 after 4 and 14 days of administration. At these

earlier time points, there were no differences in the OGTTresponse of blood glucose or serum insulin between animalstreated with vehicle and GDC-0853, despite the presence ofpancreatic lesions in the majority of animals administeredGDC-0853 (9 of 12) at 14 days. An IVGTT in SD rats admin-istered 30 mg/kg/day GDC-0853 evaluated at 4, 14, and28 days of dosing showed minor changes only after 28 days(Supplemental Fig. 3). Collectively, these findings show thatGDC-0853 administration results in mild glucose dysregula-tion after prolonged treatment in rats. This effect is not clearlyrelated to the pancreatic lesions; therefore, fasted glucose orglucose challenge tests are not biomarkers of BTK-relatedpancreatic toxicity in SD rats.Doppler Ultrasound, Computed Tomography, and

Magnetic Resonance Imaging Were Not AdequatelySensitive to Identify BTK Inhibitor–Related PancreaticPathologic Changes In Vivo. Histopathologic characteriza-tion of the BTK inhibitor–related pancreatic changes in SDrats identified hemorrhage, edema, and fibrosis as the majorstructural alterations. We used several clinically relevantimaging techniques to determine whether the pancreaticchanges in SD rats could be visualized in vivo to evaluate theirpotential application as a clinical monitoring tool.In studies evaluating US, GDC-0853 (30 mg/kg/day) or

GNE-309 (100 mg/kg/day) was orally administered to maleSD rats for 14 days, and imaging was performed on days 7 and

Fig. 5. Relative Btk transcript expression (dCT;delta Cycle Threshold) in pancreatic tissue fromhumans and Sprague-Dawley rats is presented.BTK is expressed at low levels in endocrine andexocrine tissue from rat and human pancreas.Laser capture microscopy was performed onexocrine and endocrine pancreas from rats.Transcriptional expression was analyzed byFluidigm in human islets (four samples), ratexocrine and islet tissues (three samples each),and rat lymph nodes (three samples). The resultsare normalized to 18S/baseline. Values arepresented as the mean 6 standard deviation.

Fig. 4. Strain sensitivity to BTK inhibitor–induced pan-creatic lesions is presented. GDC-0853 (30 mg/kg/day) andGNE-309 (100 mg/kg/day) were administered orally for14 consecutive days to SD, F-344, and WH rats (n = 6 malesper group per strain). With each test article, pancreaticlesions were observed in 5, 1, and 0 SD, F-344, andWH rats,respectively. All lesions in SD rats were moderate inseverity, whereas those in F-344 rats were minimal inseverity. Plasma concentrations (mM) of GDC-0853 andGNE-309 at 3 hours postdose are presented as the mean 6standard deviation.

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14. Histopathologic evaluations at the day 15 necropsy con-firmed pancreatic lesions in seven of eight and eight of eightanimals administered GDC-0853 and GNE-309, respectively.There were no significant differences detected by b-modeintensity or percentage vascularity parameters after 7 daysof treatment with either molecule. After 14 days of treatment,b-mode intensity and percentage vascularity were signifi-cantly elevated over the respective controls for animals treat-ed with GNE-309, but not GDC-0853 (Fig. 8). Together withthe lack of association between individual severity of pancre-atic findings and these parameters, a consistent relationshipwith the histologic changes could not be supported.CT and MRI evaluations were conducted following admin-

istration of GDC-0853 or GNE-309 for 14 or 17 days, re-spectively. Pancreatic lesions were confirmed in all treatedanimals. The CT imaging demonstrated no clear GDC-0853–related effects detected by time-attenuation curve andL:E ratio, pancreatic enhanced and unenhanced phases, orfunctional perfusion (Supplemental Fig. 4). Similarly, therewere no clear treatment-related changes in MRI T2 maps(Supplemental Fig. 5).

DiscussionIn these experiments, we showed that administration of a

selective BTK small molecule inhibitor, GDC-0853, to SD ratsresults in distinct microscopic pancreatic lesions character-ized by multifocal islet/peri-islet hemorrhage, inflammation,fibrosis, and pigmented macrophages with adjacent lobularexocrine acinar cell atrophy, degeneration, and inflammation.These lesions demonstrated significant reversibility following

a 4-week recovery period, with only mature islet/peri-isletfibrosis and pigmented macrophages remaining. AdditionalBTK inhibitors—two structurally distinct, irreversible inhib-itors (ibrutinib and spebrutinib) and a second highly selective,reversible inhibitor (GNE-309)—caused the same pancreaticlesions when administered to SD rats under the sameconditions. For the selective BTK inhibitors, the pancreaticlesions were observed at doses where no significant off-targetactivity was expected. Collectively, these data strongly sug-gest that inhibition of BTK enzymatic activity is involved inthe pathogenesis of these lesions, which may be considered aclass effect of BTK inhibitors in rats.As evidence of species specificity, no similar pancreatic

findings were observed in CD-1 mice or beagle dogs adminis-tered GDC-0853 or other potent BTK inhibitors for up to9 months, despite achieving exposures up to 24 times theestablished low-observed-adverse-effect level in SD rats (Sup-plemental Fig. 6). In addition, there are no reports of pancre-atic changes in mice with BTK mutations (knockout orX-linked immunodeficient) or pancreatic disease or dysfunc-tion in male patients who lack functional BTK enzyme (XLA)in at least six published clinical series and/or registries in-cluding more than 400 patients (Lederman and Winkelstein,1985; Hermaszewski and Webster, 1993; Plebani et al., 2002;Moin et al., 2004; Aghamohammadi et al., 2006; Winkelsteinet al., 2006). Also of note, ibrutinib has been administered tothousands of patients and is not associated with pancreatictoxicity. Although SD rats were the most sensitive strain testedin the current studies, F-344 and WH rats developed BTKinhibitor–related pancreatic changes of a lesser severity after2 weeks or 6 months, respectively. Taken together, these

Fig. 6. Amylase, lipase, insulin, and fructosamine levels in Sprague-Dawley rats (n # 15 per sex per group) following administration of GDC-0853 arepresented. GDC-0853 was administered orally at doses of 0.5–100 mg/kg/day for 28 days. Values are presented as the mean 6 standard deviation.

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results support the hypothesis that biologic differences pre-dispose rats to BTK inhibitor–induced pancreatic changes,with the SD rat being an exceptionally sensitive strain.Furthermore, the likelihood of clinical translatability is low.Spontaneous pancreatic islet hemorrhage and fibrosis in SD

rats is well described (Reaven and Reaven, 1981; Dillberger,1994; Imaoka et al., 2007). The changes are characterized byan initial extravasation of red blood cells from the peri-isletcapillaries and hemosiderin (pigment) deposition in the centeror periphery of the islet. The earliest observations arerecorded in 12-week-old animals, but the incidence andseverity of the changes increase considerably by 26 weeks ofage (Imaoka et al., 2007). These spontaneous islet changes aremore commonly observed in males, and a similar sex pre-dilection for males was observed in the GDC-0853–relatedpancreatic findings. The relative sex- and strain-susceptibilitydifferences may be related to body weight gain and overallmetabolic status. Male SD rats have a significantly fastergrowth rate compared with male WH rats, owing to higherdaily food consumption. At 12 weeks of age, male SD rats arealready 30–70% larger than male WH rats (Hayakawa et al.,2013; Charles River Laboratories International, Inc.). Re-ducing body weight by exercise and/or caloric restriction (from800 to 500 g at 12 months of age) significantly reduced theincidence of spontaneous pancreatic islet pathologic changeswhen compared with sedentary SD rats fed ad libitum(Reaven and Reaven, 1981). SD rats have higher rates ofhyperglycemia and a higher predisposition to streptozotocin-induced diabetes. These findings may be associated withstrain differences in the developing islet (Ojiro et al., 1993).Pancreatic islet pathology observed in rat strains with a

genetic predisposition to obesity/diabetes suggests that ratsare sensitive to developing islet hemorrhage in response toincreased metabolic demands. Spontaneous pancreatic islet

Fig. 8. Ultrasound imaging of the pancreas in Sprague-Dawley ratsfollowing administration of GNE-309 (n = 8 males per group) or GDC-0853(n = 16 males per group) is presented. GNE-309 and GDC-0853 wereadministered orally at doses of 100 and 30 mg/kg/day, respectively, for14 days. B-mode intensity and percentage vascularity were measured onday 14. Values are presented as the mean 6 standard deviation.Significance was defined at P , 0.05. Veh, vehicle.

Fig. 7. Glucose and insulin levels in Sprague-Dawley rats following administration of GDC-0853 are presented. GDC-0853 was administered orally atdoses of 0.5–100mg/kg/day for up to 32 days. (A) Fasted serum glucose in rats (n# 15 per sex per group) wasmeasured on days 13 and 28. An oral glucosetolerance test in rats (n = 5 per sex per group) on day 32 included blood glucose (B) and serum insulin (C) measurements. Values are presented as themean 6 standard deviation. Significance was defined at P , 0.05. Veh, vehicle.

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changes in the OLETF,WY/Kob, ZDF, and Goto-Kakizaki rats(Lacraz et al., 2009; Jones et al., 2010; Katsuda et al., 2014)appear to be related to increased insulin demand and hyper-insulinemia. They commonly include early islet hypertrophy/hyperplasia and degranulation of beta cells, followed byhemorrhage, hemosiderin deposition, inflammation, and/orfibrosis. Evidence of vascular injury in response to increasedmetabolic demand, including hemorrhage and hemosiderindeposition, appears to be unique to rats. In mouse models oftype 2 diabetes and in human patients with type 2 diabetes,islet changes have been observed that include beta-cellhyperplasia followed by decreased beta-cell mass, amyloiddeposition, and inflammation; notably, however, hemorrhagehas not been reported as a feature characterizing the pancre-atic pathology (Junger et al., 2002; Hull et al., 2005; Iizukaet al., 2005; Bonner-Weir and O’Brien, 2008; Donath et al.,2008; Talchai et al., 2009). Compared with mice and othermammals, including dogs and primates, rats have uniqueanatomic features within the microvasculature perfusing theendocrine and exocrine tissue (Greaves, 2012), which may beassociated with increased susceptibility to injury.The spontaneous pancreatic islet lesions observed in SD

rats are thought to be preceded by alterations in glucosemetabolism. In contrast, GDC-0853–treated SD rats do nothave significantly altered glucose metabolism at the earlystages of development of lesions, indicating that the BTKinhibitor–induced pancreatic changes likely involve a distinctpathogenicmechanism. In the acute stage, occurring within asfew as 7 days of administration of GDC-0853, pancreaticpathology was characterized by islet/peri-islet hemorrhage,suggesting drug-induced microvascular injury to the thin-walled capillaries within this region. A similar hypothesis wassuggested by Brenneman et al. (2014), who described a verysimilar drug-related pancreatic finding in SD rats for whichthe drug target was not disclosed. In their investigations, anincrease in immunohistochemical markers of endothelialcytotoxicity in the isletmicrovasculature between 1 and 5 daysof administration was identified. This was suggestive of testarticle–induced vascular injury, although it was acknowl-edged that a primary versus secondary drug effect could notbe determined. In light of the histopathologic characteristics ofthe islet-centered changes seen with GDC-0853 involvingcompromised vascular integrity with resulting local acutehemorrhage and edema and chronic fibrosis, we attempted toimage the rat pancreatic lesions using several modalities. ByUS, there were no significant differences between GDC-0853–treated and control animals in either b-mode or per-centage vascularity after 14 days. Furthermore, there were nodifferences in MRI or CT imaging parameters, the latterhaving shown promise in detecting fibrotic changes in patientsexperiencing pancreatic anastomotic failure following pan-creatoduodenectomy (Hashimoto et al., 2011).Despite the presence of significant pancreatic pathology, SD

rats administered GDC-0853 demonstrated good overalltolerability with no related clinical signs. Additionally, nosignificant changes in standard clinical pathology parametersassociated with exocrine or endocrine function were observedin studies up to 4 weeks in duration. There were no significantchanges in serum amylase, lipase, insulin, or fructosamine(glycated serum albumin used to assess glycemic controlduring the preceding approximately 2 weeks). Fasted bloodglucose was mildly increased in rats administered the highest

dose of GDC-0853. However, given that pancreatic lesions of asimilar incidence and severity were observed in animalsreceiving lower doses in this study with no apparent changesin glucose levels, the relationship between the minor increasein glucose levels and pancreatic lesions is uncertain. Elevatedrelative glucose levels were observed following OGTT at alldoses of GDC-0853, where pancreatic lesions were observed,but these effectswere detected only after 28 days (not after 4 or14 days). Thus, there appear to be GDC-0853–related changesin glucose homeostasis, but the relationship to pancreaticfindings has not been definitively established and may beconsidered either secondary to the development of the lesionor even a separate effect of BTK inhibitors in rats. Moreover,the relative timing and mild magnitude of the changes inglucose regulation in rats suggest that similar tests would beof questionable clinical utility for monitoring the onset of anypancreatic changes in a heterogeneous patient population.In summary, our results suggest that BTK inhibitors, as a

class, cause pancreatic lesions in rats that may be due toexacerbation of a unique susceptibility of rats (especially inthe SD strain) to develop peri-islet hemorrhage and sub-sequent inflammation and fibrosis. These pancreatic changesare subclinical, with few changes in standard clinical pathol-ogy parameters associated with exocrine or endocrine pancre-atic function, suggesting significant functional reserve. Theselesions were not detected by three imaging methods, makingmonitoring of development of the lesions difficult. Nonethe-less, the absence of similar changes in other nonclinicalspecies administered BTK inhibitors and in BTK mutantmouse models, and the lack of clinical reports of pancreaticdysfunction in patients with XLA and patients treated withibrutinib argue that this type of injury is very unlikely to occurwith BTK inhibitor therapeutics in humans.

Acknowledgments

The authors thank Laura de Forge, Arna Katewa, Joseph Lubach,Michael Sweeney, and Covance Laboratories, Inc., for assistance inthe conduct of the in vivo and ex vivo experiments.

Authorship Contributions

Participated in research design: Erickson, Schutt, Tarrant,McDowell, Lewin-Koh, Hedehus, Ross, Carano, Staflin, Craw-ford, S. Zhong, Reif, Wong, Young, Dambach, Misner.

Conducted experiments: McDowell, Hedehus, Ross, Staflin,Katewa.

Contributed new reagents or analytic tools: McDowell, Hedehus,Ross, Carano, Crawford.

Performed data analysis: Erickson, Schutt, Tarrant, McDowell,Liu, Johnson, Lewin-Koh, Hedehus, Ross, Carano, Staflin, F. Zhong,Katewa, Wong, Misner.

Wrote or contributed to the writing of the manuscript: Erickson,Schutt, Tarrant, Johnson, Carano, Staflin, Wong, Young, Dambach,Misner.

References

Aghamohammadi A, Fiorini M, Moin M, Parvaneh N, Teimourian S, Yeganeh M,Goffi F, Kanegane H, Amirzargar AA, Pourpak Z, et al. (2006) Clinical, immuno-logical and molecular characteristics of 37 Iranian patients with X-linked agam-maglobulinemia. Int Arch Allergy Immunol 141:408–414.

Bao Y, Zheng J, Han C, Jin J, Han H, Liu Y, Lau Y-L, Tu W, and Cao X (2012)Tyrosine kinase Btk is required for NK cell activation. J Biol Chem 287:23769–23778.

Bonner-Weir S and O’Brien TD (2008) Islets in type 2 diabetes: in honor of Dr. RobertC. Turner. Diabetes 57:2899–2904.

Brenneman KA, Ramaiah SK, Rohde CM, Messing DM, O’Neil SP, Gauthier LM,Stewart ZS, Mantena SR, Shevlin KM, Leonard CG, et al. (2014) Mechanisticinvestigations of test article-induced pancreatic toxicity at the endocrine-exocrineinterface in the rat. Toxicol Pathol 42:229–242.

BTK Inhibitors Induce Pancreatic Toxicity in Rats 237

at ASPE

T Journals on A

pril 15, 2022jpet.aspetjournals.org

Dow

nloaded from

Brown JR, Harb WA, Hill BT, Gabrilove J, Sharman JP, Schreeder MT, Barr PM,Foran JM, Miller TP, Burger JA, et al. (2016) Phase I study of single-agent CC-292,a highly selective Bruton’s tyrosine kinase inhibitor, in relapsed/refractory chroniclymphocytic leukemia. Haematologica 101:e295–e298.

Byrd JC, Harrington B, O’Brien S, Jones JA, Schuh A, Devereux S, Chaves J, WierdaWG, Awan FT, Brown JR, et al. (2016) Acalabrutinib (ACP-196) in RelapsedChronic Lymphocytic Leukemia. N Engl J Med 374:323–332.

Conley ME, Rohrer J, and Minegishi Y (2000) X-linked agammaglobulinemia. ClinRev Allergy Immunol 19:183–204.

Dillberger JE (1994) Age-related pancreatic islet changes in Sprague-Dawley rats.Toxicol Pathol 22:48–55.

Donath MY, Schumann DM, Faulenbach M, Ellingsgaard H, Perren A, and Ehses JA(2008) Islet inflammation in type 2 diabetes: from metabolic stress to therapy.Diabetes Care 31 (Suppl 2):S161–S164.

Ellmeier W, Jung S, Sunshine MJ, Hatam F, Xu Y, Baltimore D, Mano H,and Littman DR (2000) Severe B cell deficiency in mice lacking the tec kinasefamily members Tec and Btk. J Exp Med 192:1611–1624.

Evans EK, Tester R, Aslanian S, Karp R, Sheets M, Labenski MT, Witowski SR,Lounsbury H, Chaturvedi P, Mazdiyasni H, et al. (2013) Inhibition of Btk withCC-292 provides early pharmacodynamic assessment of activity in mice and hu-mans. J Pharmacol Exp Ther 346:219–228.

Greaves P (2012) Exocrine pancreas/endocrine pancreas, in Histopathology of Pre-clinical Toxicity Studies: Interpretation and Relevance in Drug Safety Evaluation,pp 489–510, Academic Press, Amsterdam, Netherlands.

Hashimoto Y, Sclabas GM, Takahashi N, Kirihara Y, Smyrk TC, Huebner M,and Farnell MB (2011) Dual-phase computed tomography for assessment of pan-creatic fibrosis and anastomotic failure risk following pancreatoduodenectomy. JGastrointest Surg 15:2193–2204.

Hayakawa K, Mimura Y, Tachibana S, Furuya M, Kodama T, Aoki T, Hosokawa S,Fukui M, Shibata S, Yoshida M, et al. (2013) Study for collecting background dataon Wistar Hannover [Crl:WI(Han)] rats in general toxicity studies–comparativedata to Sprague Dawley rats. J Toxicol Sci 38:855–873.

Herman SEM, Mustafa RZ, Gyamfi JA, Pittaluga S, Chang S, Chang B, Farooqui M,and Wiestner A (2014) Ibrutinib inhibits BCR and NF-kB signaling and reducestumor proliferation in tissue-resident cells of patients with CLL. Blood 123:3286–3295.

Hermaszewski RA and Webster AD (1993) Primary hypogammaglobulinaemia: asurvey of clinical manifestations and complications. Q J Med 86:31–42.

Howard V, Greene JM, Pahwa S, Winkelstein JA, Boyle JM, Kocak M, and ConleyME (2006) The health status and quality of life of adults with X-linked agamma-globulinemia. Clin Immunol 118:201–208.

Hull RL, Shen Z-P, Watts MR, Kodama K, Carr DB, Utzschneider KM, Zraika S,Wang F, and Kahn SE (2005) Long-term treatment with rosiglitazone andmetformin reduces the extent of, but does not prevent, islet amyloid deposition inmice expressing the gene for human islet amyloid polypeptide. Diabetes 54:2235–2244.

Iizuka S, Suzuki W, Tabuchi M, Nagata M, Imamura S, Kobayashi Y, Kanitani M,Yanagisawa T, Kase Y, Takeda S, et al. (2005) Diabetic complications in a newanimal model (TSOD mouse) of spontaneous NIDDM with obesity. Exp Anim 54:71–83.

Imaoka M, Satoh H, and Furuhama K (2007) Age- and sex-related differences inspontaneous hemorrhage and fibrosis of the pancreatic islets in Sprague-Dawleyrats. Toxicol Pathol 35:388–394.

Iyer AS, Morales JL, Huang W, Ojo F, Ning G, Wills E, Baines JD, and August A(2011) Absence of Tec family kinases interleukin-2 inducible T cell kinase (Itk) andBruton’s tyrosine kinase (Btk) severely impairs Fc epsilonRI-dependent mast cellresponses. J Biol Chem 286:9503–9513.

Jones HB, Nugent D, and Jenkins R (2010) Variation in characteristics of islets ofLangerhans in insulin-resistant, diabetic and non-diabetic-rat strains. Int J ExpPathol 91:288–301.

Junger E, Herberg L, Jeruschke K, and Leiter EH (2002) The diabetes-prone NZO/Hlstrain. II. Pancreatic immunopathology. Lab Invest 82:843–853.

Katsuda Y, Ohta T, Miyajima K, Kemmochi Y, Sasase T, Tong B, Shinohara M,and Yamada T (2014) Diabetic complications in obese type 2 diabetic rat models.Exp Anim 63:121–132.

Kerner JD, Appleby MW, Mohr RN, Chien S, Rawlings DJ, Maliszewski CR, WitteON, and Perlmutter RM (1995) Impaired expansion of mouse B cell progenitorslacking Btk. Immunity 3:301–312.

Lacraz G, Giroix M-H, Kassis N, Coulaud J, Galinier A, Noll C, Cornut M, SchmidlinF, Paul J-L, Janel N, et al. (2009) Islet endothelial activation and oxidative stressgene expression is reduced by IL-1Ra treatment in the type 2 diabetic GK rat.PLoS One 4:e6963.

Lederman HM and Winkelstein JA (1985) X-linked agammaglobulinemia: an anal-ysis of 96 patients. Medicine (Baltimore) 64:145–156.

Lindvall JM, Blomberg KEM, Väliaho J, Vargas L, Heinonen JE, Berglöf A,Mohamed AJ, Nore BF, Vihinen M, and Smith CIE (2005) Bruton’s tyrosine kinase:cell biology, sequence conservation, mutation spectrum, siRNA modifications, andexpression profiling. Immunol Rev 203:200–215.

Lipsky AH, Farooqui MZ, Tian X, Martyr S, Cullinane AM, Nghiem K, Sun C, ValdezJ, Niemann CU, Herman SE, et al. (2015) Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated withibrutinib. Haematologica 100:1571–1578.

McMullen JR, Boey EJH, Ooi JYY, Seymour JF, Keating MJ, and Tam CS (2014)Ibrutinib increases the risk of atrial fibrillation, potentially through inhibition ofcardiac PI3K-Akt signaling. Blood 124:3829–3830.

Moin M, Aghamohammadi A, Farhoudi A, Pourpak Z, Rezaei N, Movahedi M,Gharagozlou M, Ghazi BMS, Zahed A, Abolmaali K, et al. (2004) X-linked agam-maglobulinemia: a survey of 33 Iranian patients. Immunol Invest 33:81–93.

Molica S (2015) The clinical safety of ibrutinib in chronic lymphocytic leukemia.Expert Opin Drug Saf 14:1621–1629.

Ojiro K, Kitamura H, Shimada T, and Nakamura M (1993) A morphometrical studyof the postnatal development of rat pancreatic islets, with special regard to thedifferences between Wistar and Sprague-Dawley strains. Kaibogaku Zasshi 68:190–203.

Pan Z, Scheerens H, Li SJ, Schultz BE, Sprengeler PA, Burrill LC, Mendonca RV,Sweeney MD, Scott KC, Grothaus PG, et al. (2007) Discovery of selective irre-versible inhibitors for Bruton’s tyrosine kinase. ChemMedChem 2:58–61.

Pharmacyclics, Inc. (2015) Imbruvica: Highlights of prescribing information.Retrieved from http://www.accessdata.fda.gov/drugsatfda_docs/label/2015/205552s002lbl.pdf

Plebani A, Soresina A, Rondelli R, Amato GM, Azzari C, Cardinale F, Cazzola G,Consolini R, De Mattia D, Dell’Erba G, et al.; Italian Pediatric Group for XLA-AIEOP (2002) Clinical, immunological, and molecular analysis in a large cohort ofpatients with X-linked agammaglobulinemia: an Italian multicenter study. ClinImmunol 104:221–230.

Rawlings DJ, Saffran DC, Tsukada S, Largaespada DA, Grimaldi JC, Cohen L, MohrRN, Bazan JF, Howard M, Copeland NG, et al. (1993) Mutation of unique region ofBruton’s tyrosine kinase in immunodeficient XID mice. Science 261:358–361.

Reaven EP and Reaven GM (1981) Structure and function changes in the endocrinepancreas of aging rats with reference to the modulating effects of exercise andcaloric restriction. J Clin Invest 68:75–84.

Satterthwaite AB, Cheroutre H, Khan WN, Sideras P, and Witte ON (1997) Btkdosage determines sensitivity to B cell antigen receptor cross-linking. Proc NatlAcad Sci USA 94:13152–13157.

Stephens DM and Spurgeon SE (2015) Ibrutinib in mantle cell lymphoma patients:glass half full? Evidence and opinion. Ther Adv Hematol 6:242–252.

Talchai C, Lin HV, Kitamura T, and Accili D (2009) Genetic and biochemical path-ways of beta-cell failure in type 2 diabetes. Diabetes Obes Metab 11 (Suppl 4):38–45.

Winkelstein JA, Marino MC, Lederman HM, Jones SM, Sullivan K, Burks AW,Conley ME, Cunningham-Rundles C, and Ochs HD (2006) X-linked agammaglob-ulinemia: report on a United States registry of 201 patients. Medicine (Baltimore)85:193–202.

Young W and Crawford J (2016) Discovery of GDC-0853: A Highly Potent, Selectiveand Non-Covalent BTK Inhibitor, American Chemistry Society, San Diego, CA.

Address correspondence to: Leah K. Schutt, 1 DNA Way, MS59, South SanFrancisco, CA 94080. E-mail: schutt.leah@gene.com

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