In vitro and in vivo profiling of CHF5022 and CHF5074

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In vitro and in vivo profiling of CHF5022 and CHF5074
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  Pharmacological Research 55 (2007) 318–328 In vitro and in vivo profiling of CHF5022 and CHF5074Two   -amyloid 1–42  lowering agents Bruno P. Imbimbo a , ∗ , Elda Del Giudice b , Valentina Cenacchi a , Roberta Volta a , Gino Villetti a ,Fabrizio Facchinetti a , Benedetta Riccardi a , Paola Puccini a , Nadia Moretto c ,Francesca Grassi c , Simone Ottonello c , Alberta Leon b a  Research & Development Department, Chiesi Farmaceutici, Via Palermo 26/A, 43100 Parma, Italy b  Research & Innovation, Via Svizzera 16, 35127 Padua, Italy c  Department of Biochemistry and Molecular Biology, University of Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy Accepted 18 December 2006 Abstract Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may delay or prevent the onset of Alzheimer’s disease (AD). A subsetof NSAIDs, including flurbiprofen, has been shown to selectively inhibit the production of    -amyloid 1–42  (A  42), independently from theircyclooxygenase (COX) inhibiting activity. We evaluated the in vitro and in vivo profiles of CHF5022 and CHF5074, two flurbiprofen analogues.The in vitro A   inhibiting activity was evaluated in a human neuroglioma cell line (H4) carrying the double Swedish mutation (K595N/M596L)of the human amyloid precursor protein (APPsw). The in vitro anti-COX activity was evaluated using human recombinant enzymes isolatedfrom transfected Sf-9 cells. The in vivo pharmacokinetic and pharmacodynamic profiles of the two compounds were evaluated in young APPswtransgenicmice(Tg2576)afteroral gavage (100or300mgkg − 1 day − 1 for4–5days)andaftermedicateddiet(375ppmfor4weeks).  R -Flurbiprofenwas used as comparator. In vitro, CHF5022 and CHF5074 were found to be 3- and 7-fold more potent than  R -flurbiprofen in inhibiting A  42secretion (IC 50 s of 92, 40 and 268  M, respectively). Differently from  R -flurbiprofen, CHF5022 and CHF5074 did not affect COX-1 (at 100  M)and COX-2 (at 300  M) activity. Similarly to  R -flurbiprofen, no significant alteration in the expression profile of a subset of Notch intracellulardomain-responsive genes was observed with either CHF5022 or CHF5074. In Tg2576 mice, CHF5022 was well tolerated when administered byoral  gavage  (100mgkg − 1 day − 1 for 5 days) or by medicated diet (56mg kg − 1 day − 1 for 4 weeks).  R -Flurbiprofen was poorly tolerated in the diet(32mgkg − 1 day − 1 ) with 55% of the animals dying during the first week of treatment. After 4–5 days of oral  gavage , CHF5022 and CHF5074plasma and brain levels at 3h were found to increase with the dose, leading to brain concentrations of about 10% and 5% of the correspondingplasma concentrations, respectively. In animals fed for 4 weeks with compound-supplemented diet, mean plasma (580  M) and brain (20  М )concentrations of CHF5022 were 8 and 15 times higher than those of   R -flurbiprofen. Plasma A  42 concentration was dose-dependently decreasedby CHF5022 and CHF5074. Brain A   levels (formic acid-extractable) were not significantly affected by either compound, although A  42 levelstended to inversely correlate ( P =0.105) with CHF5022 concentration in the brain. CHF5022 and CHF5074 thus appear to have a promising invitro and in vivo profile. This warrants further evaluation of their long-term effects on A   brain pathology.© 2007 Elsevier Ltd. All rights reserved. Keywords:   -Amyloid;   -Secretase modulators; Transgenic Tg2576 mouse 1. Introduction Long-term use of non-steroidal anti-inflammatory drugs(NSAIDs) may delay or prevent the onset of Alzheimer’sdisease (AD). A subset of NSAIDs, including flurbiprofen,has been shown to selectively inhibit the production of    - ∗ Corresponding author. Tel.: +39 0521 279278; fax: +39 0521 279579.  E-mail address: (B.P. Imbimbo). amyloid 1–42  (A  42), independently from their cyclooxygenase(COX) inhibiting activity [1–3] and to counteract the pro-gression of A   pathology in transgenic mouse models of AD[4–8]. The proposed mechanism for this activity is an allosteric modulation of presenilin-1, the major component of the   -secretase complex, that is responsible for the formation of A   [3,9–11]. These A  42 lowering NSAIDs differ from tra-ditional   -secretase inhibitors because they do not inhibit the  -secretase-mediatedcleavagesofeitheramyloidprecursorpro-tein (APP) at the alternative    site, or Notch-1 at the S3 1043-6618/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.phrs.2006.12.010   B.P. Imbimbo et al. / Pharmacological Research 55 (2007) 318–328  319Fig. 1. Structural formula of   R -flurbiprofen, CHF5022 and CHF5074. site [1,12]. These   -secretase-mediated cleavages generate twointracellular domains with transcription factor activity knownas Notch intracellular domain (NICD) and APP intracellulardomain (AICD), respectively. CHF5022 and CHF5074 are flur-biprofen analogues with A  42 lowering properties but devoidof anti-COX activity (Fig. 1) [13,14]. In rats, CHF5022 and CHF5074 are well absorbed after oral administration (absoluteoral bioavailability of 91% and 50%, respectively) and slowlyeliminated from plasma (elimination half-lives of 33 and 21h,respectively). Their high plasma protein binding (99%) regu-lates central nervous system penetration with concentrations incerebrospinal fluid around 2% of those in plasma [13]. In the presentstudywecomparedtheinvitroactivityofCHF5022andCHF5074 with that of   R- flurbiprofen, an A  42 lowering agentpresentlyinPhase3clinicaltrialsforAD[15].Thepharmacoki- netics and pharmacodynamics of the two new compounds werealso evaluated after multiple dosing in young transgenic mice(Tg2576) expressing the Swedish mutation of APP (APPsw).Short-term (4–5 days), dose–response (100 and 300mgkg − 1 day − 1 ) studies were initially carried out. Later, based on thehigher brain penetration, clearer dose-proportionality and aslightly better tolerability emerging from the short-term stud-ies, CHF5022 was selected for a 4-week study in Tg2576 mice.The overall objective was the proper design of long-term stud-ies aimed at evaluating the effects of long-term administrationof the compound on brain A   pathology. 2. Materials and methods 2.1. A β 42 and A β 40 assay A  42 and A  40 were measured in the culture medium of H4 cells, a human neuroglioma cell line expressing the doubleSwedish mutation (K595N/M596L) of human APP (APPsw).Cells were seeded onto 24-well plates (2 × 10 5 cell well − 1 )and allowed to grow to confluence for 24h, in 5% CO 2  /95%air in a humidified atmosphere. Increasing concentrations (from3 to 300–400  M) of the compounds were added to the cellsovernight in a final volume of 0.5ml.  R -Flurbiprofen was usedaspositivecontrol(3–1000  M).DMSO(1%)wasusedasnega-tive control. At the end of the incubation, 100  l of supernatantswere removed and treated with a biotinylated mouse mono-clonal antibody (4G8, Signet Laboratories Inc., Dedham, MA,USA), specifically recognizing the 17–24 amino acid regionof A   and two rabbit polyclonal antibodies (C-term 42 andC-term 40, BioSource International, Camarillo, CA, USA),specifically recognizing the C-terminus of A  42 and A  40,respectively.Antigen–antibodiescomplexeswererecognizedbyTAG-donkey anti-rabbit IgG (Jackson ImmunoResearch Lab-oratories, Soham, UK). Streptavidin coated magnetic beadscaptured the complexes and the signals were read by an electro-chemiluminescence instrument (Origen M8 Analyzer, BioVerisCorporation, Gaithersburg, MD, USA).The cytotoxicity potential of test compounds was assessedin the same cells of the A   assay (H4) with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)assay. MTT is a soluble pale yellow salt that is reduced by mito-chondrial succinate dehydrogenase to form an insoluble dark blue formazan product to which the cell membrane is imperme-able.TheabilityofcellstoreduceMTTprovidesanindicationof mitochondriaintegrityandactivityanditmaybeinterpretedasameasure of viability and/or cell number. After medium removalfor A  42 and A  40 determination, cells were incubated for 3hwith 500  l culture medium containing 0.5mgml − 1 MTT, at37 ◦ C, 5% CO 2  and saturated humidity. After removal of themedium, 500  l of 100% DMSO were added to each well. Theamount of formed formazan was determined reading the sam-ples at 570nm (background 630nm) using a microplater reader(model 450, Bio-Rad, Hercules, CA, USA). 2.2. COX-1 and COX-2 assay The inhibition of the cyclooxygenase activity was estimatedmeasuring PGE 2  production from arachidonic acid accordingto a modified version of the method described by Glaser etal. [16]. Recombinant human prostaglandin H 2  synthase-1(PGHS-1) and -2 (PGHS-2) were expressed in transfectedSpodoptera frugiperda (Sf-9) cells (Invitrogen, San Diego,CA, USA). The microsomal fractions were prepared from thetransfected cells and used to assay the enzymatic activities.Briefly, the enzymes (2  g) reconstituted in a buffer (100mMTris–HCl, pH 8.0) containing 2mM phenol, were preincubatedwith vehicle (DMSO) or test compounds in DMSO (1%DMSO in the final assay) for 20min at 22 ◦ C. The reactionmixture was completed with 1  M hematin. The reaction wasinitiated adding arachidonic acid (4 and 2  M for COX-1 andCOX-2, respectively) and the mixture was incubated for 5minat 22 ◦ C for COX-1 assay, or for 10min at 25 ◦ C for COX-2assay. For control measurements, arachidonic acid was omittedfrom the reaction mixture. The reactions were stopped by thesequential addition of 1M HCl and 1M Tris–HCl (pH 8.0),followed by cooling to 4 ◦ C. The amount of PGE 2  present in thereaction mixture was quantified using an enzyme-immunoassay  320  B.P. Imbimbo et al. / Pharmacological Research 55 (2007) 318–328 detection kit (Cayman Chemical, Ann Arbor, MI, USA) andthe measurements were made with a microplate reader. 2.3. Expression profiling of AICD- and NICD-responsivegenes Theinhibitionof   -secretasemediatedcleavageatthe  siteof APPand/orattheS3siteofNotchwasestimatedbygeneexpres-sionprofilingofasubsetofAICD-andNICD-responsivegenes.Oligonucleotides(60mer)matching8AICD( KTN1 , FST  , TIEG , CASP7  ,  CALB1 ,  KAI1 ,  FMR1 ,  FMR2 ) and 20 NICD (  HES1 ,  HES7  ,  HEY2 ,  HEY1 ,  HEYL ,  ASCL1 ,  NEUROG1 ,  GAA ,  CD4 ,  MYOG , CTNNB1 ,  NFKBIA , GSK3B , UTG1A9 ,  DF  , GCG , CCK  , SCT  ,  SST  ,  CDKN1A ) responsive genes [17–19] were designed with the OligoArray 1.0 program [20], chemically synthesized with a 5  -amino linker modification and spotted in duplicateonto charged nylon membranes (Eurogentec, Liege, Belgium).Four “housekeeping genes” ( GAPD ,  HPRT1 ,  ACTB ,  B2M  )were also spotted in duplicate, along with sonicated genomicDNA from human placenta (20 spots). The latter, togetherwith 20 empty spots, served as technical controls for datanormalization and analysis. A list of the genes and of the corre-spondingoligonucleotidesutilisedisprovidedinSupplementaryTable 1.H4 cells were incubated for 12h in the presence of either  R -Flurbiprofen (250  M), CHF5022 (100  M) or CHF5074(100  M). DMSO (1%, v/v) and DAPT (1  M) (  N  -[  N  -(3,5-difluorophenacetyl)- l -alanyl]- S  -phenylglycine  t  -butyl ester), atraditional   -secretase inhibitor, were used as negative andpositive controls, respectively. Total RNA was extracted withthe Trizol reagent (Invitrogen), reverse-transcribed (60min at50 ◦ C) and radioactively labelled as previously described [21].The resulting cDNAs were purified on Bio-Spin 6 columns(Bio-Rad Laboratories, Segrate, Italy) and quantified by scin-tillation counting. Filters were prehybridised (2h at 42 ◦ C) with5.0  g of denatured Cot-1 DNA, 5.0  g of poly-dA and 4mlof hybridisation solution (5 ×  SSC, 0.5% SDS, 5 ×  Denhardt’s),followed by the addition of the radiolabelled cDNAs and sub-sequent hybridisation for 18h at 42 ◦ C. After washing at 55 ◦ C(twowasheswith2 × SSC–0.05%SDSand1 × SSC–0.1%SDS;one wash with 0.1 ×  SSC–0.1% SDS; 20min each), membraneswere aligned on a phosphorimaging screen (Super ResolutionSR, Perkin-Elmer, Branchburg, NJ) and exposed for 24–72h atroom temperature. Target intensities, measured and visualizedwith a Cyclone phosphorimager (Perkin-Elmer) at a resolutionof 42  m per pixel, were digitized with the OptiQuant software.Three technical replicates were conducted for each treatment(“T”)andfortheDMSOcontrol(“C”)andthreeimagesofcom-parable overall intensity for each condition, as determined withthe Quantity One program (Bio-Rad), were subjected to furtheranalysisusingImaGene5.0(Biodiscovery,MarinaDelRey,CA)as described previously [20]. Signals from spots consistently tagged as “flag 0” in the three replicates as well as within indi-vidual duplicates from the same membrane were averaged, andnormalized signal intensity ratios (  I  T  /   I  C  and  I  C  /   I  T ) were calcu-lated.Geneswithan  I  T  /   I  C  oran  I  C  /   I  T  valuegreaterthanorequalto 1.5 were judged as upregulated or downregulated in treated(“T”; NSAIDs plus DAPT) versus control (“C”; DMSO) cells,respectively. 2.4. Studies in Tg2576 transgenic mice Young male and female transgenic mice (Tg2576) express-ing the human  APP  gene with the Swedish double mutation(K670N/M671L) under the transcriptional control of the ham-ster prion protein promoter [22] were used for the in vivostudies. Male animals were housed singly in individual cageswhile female animals were placed in groups of 3–5 animals percage. The experiments were performed in accordance with EECGuidelines (86/609/ECC) for the use of laboratory animals. 2.4.1. Study 1 Twenty-one male mice of 4–5 months of age were given byoral  gavage  vehicle (Kool-Aid 7.5mlkg − 1 ) or a suspension of CHF5022 (100 or 300mgkg − 1 day − 1 in Kool-Aid) once dailyfor 5 days. This vehicle was selected to replicate that reportedwith flurbiprofen in similar studies [1,3]. On Day 5, mice were givenafinaldoseof100or300mgkg − 1 orvehicleandsacrificed3h later, as described below. 2.4.2. Study 2 Seventeen female mice of 5–7 months of age were given byoral  gavage  vehicle (Kool-Aid 7.5mlkg − 1 ) or a suspension of CHF5074 (100 or 300mgkg − 1 day − 1 in Kool-Aid) once dailyfor 4 days. On Day 4, mice were given a final dose of 100 or300mgkg − 1 or vehicle and sacrificed 3h later, as describedbelow. 2.4.3. Study 3 Thirty-threemaleandfemalemiceof4–5monthsweregivenvehicle or CHF5022 or  R -flurbiprofen-supplemented chow adlibitum for 4 weeks. There were 11 animals in each treat-ment group.  R -Flurbiprofen (Sigma, St. Louis, MO, USA) andCHF5022 were formulated into standard, colour-coded, rodentdiet by Charles River (Calco, Italy) at a final drug concentrationof 375ppm. The concentration of the drugs in the diet was thesameasthatusedforibuprofeninpreviousstudies[4,5,7].Body weight and food consumption were monitored every 3–4 days. 2.5. Plasma and brain A β  measurements Twenty-four hours before starting treatment, one bloodsamplewascollectedbymeansofretro-bulbarpunctureformea-surement of baseline plasma, A  40 and A  42 concentrations.On the last day of treatment, mice were sacrificed by decapita-tion. Blood samples were collected in EDTA-coated tubes andcentrifuged at 800rpm for 20min to separate plasma. Plasmasamples were divided into two aliquots of approximately 100  leach and stored at  − 80 ◦ C until A   and drug assay. The brainswere quickly removed and placed on an ice-cold plate. Cor-tex and hippocampus were dissected and immediately frozenon dry ice and stored at  − 80 ◦ C for A   assay. The remain-ing brain was immediately frozen on dry ice and stored at − 80 ◦ C for drug level measurements. Plasma was diluted 1:4   B.P. Imbimbo et al. / Pharmacological Research 55 (2007) 318–328  321 for A  42 and 1:20 for A  40. For measurement of A  , braintissue samples were homogenized in 70% formic acid at 1:10(w/v). Homogenates were agitated at 4 ◦ C for 3h and thencentrifuged at 15,000 × g  for 25min at 4 ◦ C. The supernatantswere collected and neutralized with 1M Tris, pH 11 at 1:20(w/v) dilution with 3 × protease inhibitor mixture (BoehringerMannheim, Mannheim, Germany). Levels of A  40 and A  42in plasma and in brain homogenate supernatants were measuredwith commercial ELISA kits (The Genetics Company, Zurich,Switzerland). The micro-titre plates were coated with capturingpurified monoclonal antibodies specifically recognizing the C-terminus of human A  40 (clone G2-10, reactive to aminoacidresidues 31–40, isotype IgG2b, kappa) or A  42 (clone G2-13,reactive to aminoacid residues 33–42, isotype IgG1, kappa). Asdetection antibody, a monoclonal biotin conjugated antibodyrecognizing the N-terminus of human A   (clone W0-2, reac-tive to aminoacid residues 4–10, isotype IgG2a, kappa) wasused. The assay was linear in the range 25–500pgml − 1 andthe detection limit was 25pgml − 1 . 2.6. Plasma and brain drug measurements CHF5022 and CHF5074 levels in plasma and in brain sam-ples were measured by liquid chromatography as previouslydescribed[12].Briefly,sampleswerepreparedbyadding300  lacetonitrile and 40  l phosphoric acid 40% to 100  l plasmaor brain homogenate and placing the mixture in a vortexfor 5s. Plasma and brain samples were then centrifuged at14,000rpmfor5minandthesupernatants(15and50  l,respec-tively) were injected into the HPLC system. Equipment systemswith fluorescence (Waters 474, Waters, Guyancourt, France)or mass spectrometry (API 2000, Applied Biosystems, Fos-ter City, CA, USA) detectors were used. The chromatographicconditions were adapted to each compound to obtain goodpeak separation and detection sensitivity. A mixture of ammo-nium formate (20mM) buffer–acetonitrile–methanol was usedas mobile phase for the fluorescence detector. For CHF5022,the assay was linear in the range 20–4000ngg − 1 in the brainand 5–1000ngml − 1 in plasma with limits of quantitation of 20ngg − 1 in the brain and 5ngml − 1 in plasma. For CHF5074,the assay was linear between 400 and 20,000ngg − 1 in the brainand 100–8500ngml − 1 in plasma with limits of quantitation of 400ngg − 1 in the brain and 100ngml − 1 in plasma. 2.7. Statistical analysis The effects of test compounds on in vitro A   secretion andCOX activity were expressed as percent of controls. IC 50 s wereestimated by logistic regression analysis.Expression profilings of NICD- and AICD-responsive geneswere analysed with the Bayesian  t  -test implemented in theCyber-T software ( ).Intheinvivostudies,two-wayanalysisofvariance(ANOVA)for repeated measures was used to detect significant treatmentand time differences in plasma A  40 and A  42 concentrations.One-way ANOVA was used to test treatment differences inbrain A   levels. After a significant factor effect by ANOVA,individual group differences were analysed using Holm-Sidak’sprocedure for multiple comparisons versus control group (vehi-cle). In Study 3, using male and female animals, a two-wayANOVA was used to test treatment and gender differences inbrain A  levels. When appropriate, plasma and brain A  40 andA  42 levels were transformed (log or square root) to normalizedataandtoobtainhomoscedasticity.Exactprobabilities( P )werereportedformultiplecomparisons.CalculationsweredonewiththestatisticalsoftwareSigmaStat TM 2.0forWindows TM (SPSS,Chicago, IL, USA). Data are presented either as individual val-ues or as mean ± S.E.M. (standard error of mean) in the text. 3. Results 3.1. In vitro studies InH4cells,cytotoxicityofthetestedcompoundswasmodest(<15%) at concentrations up to 100  M. Significant toxic-ity (30–40%) was observed at 1000  M with  R -flurbiprofen(40.3 ± 3.0%) and at 300  M with CHF5074 (29.4 ± 5.9%) andCHF5022 (37.7 ± 1.3%).  R -Flurbiprofen concentration-dependently inhibited A  42secretion with an IC 50  of 268  M (Fig. 2). A  40 was not inhib-itedatconcentrationsupto300  M(103.5 ± 1.7%ofthecontrolvalues). At 500–1000  M, A  40 secretion was inhibited by20–40%, but these concentrations were also cytotoxic (Fig. 2).At 100 and 300  M,  R -flurbiprofen inhibited COX-1 activityby 50 ± 5% and 88 ± 3%, respectively (Table 1). At 300  M,it also displayed a significant inhibitory activity on COX-2( − 46 ± 6%). At 250  M,  R -flurbiprofen did not influence anyof the tested AICD- and NICD-responsive genes while DAPT(1  M) downregulated the expression of five genes (Table 2).CHF5074showedaclearconcentration-dependentinhibitoryactivity on A  42 secretion (IC 50  =40  M, Fig. 2). At non- cytotoxic concentrations, no inhibition of the production of A  40 was observed (100.4 ± 3.8% of the control value at100  M, Fig. 2). COX-1 activity was not inhibited at 100  M.At 300  M, an inhibitory effect was detected on COX-1( − 40 ± 13%)butnotonCOX-2(Table1).At100  M,CHF5074did not alter the expression levels of any of the tested AICD- orNICD-responsive genes (Table 2).CHF5022 was less potent than CHF5074 in inhibiting A  42secretion (IC 50  =92  M, Fig. 2) and retained a modest activ- ity on A  40 at non-cytotoxic concentrations ( − 22.2 ± 4.8% at Table 1Effectsof   R -flurbiprofen,CHF5022andCHF5074onhumanrecombinantCOX-1 and COX-2 activity from Sf-9 insect cellsCompound COX-1 (% of control) COX2 (% of control)100  M a 300  M a 300  M a  R -Flurbiprofen 50.0  ±  5.0 12.0  ±  2.6 54.0  ±  5.5CHF5022 99.3  ±  0.3 97.0  ±  10.8 129.4  ±  9.1CHF5074 99.5  ±  2.5 59.9  ±  12.5 110.7  ±  9.6Enzyme activity is expressed as percent of control values (mean ± S.E.M.,  n =3per experiment). a Compound concentration.  322  B.P. Imbimbo et al. / Pharmacological Research 55 (2007) 318–328 Fig. 2. Effects of   R -flurbiprofen, CHF5022 and CHF5074 on A  42 (upperpanel) and A  40 (lower panel) secretion in human neuroglioma cells (H4)expressing APPsw. A   concentrations are expressed as percent of controlvalues (mean ± S.E.M.,  n =3 for each concentration). Estimated IC 50 s of   R -flurbiprofen, CHF5022 and CHF5074 for A  42 were 268  M, 92  M and40  M, respectively. Reliable IC 50 s for A  40 were not computable. 100  M, Fig. 2). It did not inhibit COX-1 and COX-2 up to a concentration of 300  M. At 100  M, CHF5022 significantlydownregulated two AICD-responsive genes, without any effecton the tested NICD target genes (Table 2). 3.2. In vivo Study 1 One animal in the vehicle-treated group (at Day 2) andthree animals in the highest CHF5022 dose level-treated group Fig. 3. Individual plasma and brain levels of CHF5022 (100 or300mgkg − 1 day − 1 for 5 days, upper panel) and CHF5074 (100 or300mgkg − 1 day − 1 for 4 days, lower panel) in young Tg2576 mice. Sampleswere taken 3h after the last dose. (at Day 3, Day 4 and Day 5) died. At Day 5, mean bodyweight of the surviving animals was similar in the three groups(23.1 ± 0.7g, 21.4 ± 0.6g and 22.6 ± 0.8g in the vehicle, 100and 300mg/kg/day group, respectively). Mean liver and kidneyweights were similar, while spleen weight tended (  p =0.065)to decrease dose-dependently in the CHF5022-treated groupscompared to vehicle (89 ± 9mg, 71 ± 6mg and 63 ± 6mg invehicle, 100 and 300mgkg − 1 day − 1 group, respectively).At Day 5, 3h after the last dose, CHF5022 mean plasmaand brain levels were found to be dose-proportional (Fig. 3). Mean brain to plasma percent ratio was 11.0 ± 4.7% in the100mgkg − 1 day − 1 and 9.6 ± 1.0% in the 300mgkg − 1 day − 1 groups.At Day 5, plasma A  40 concentrations tended to decrease inboththe100mgkg − 1 day − 1 ( P =0.059)and300mgkg − 1 day − 1 ( P =0.063) groups compared to baseline (Fig. 4), while A  42 Table 2NICD- and AICD-responsive genes downregulated in H4 cells incubated overnight with DAPT, R-flurbiprofen, CHF5022 and CHF5074Gene (accession number) Gene description Regulator Fold change (  I  C  /   I  T )DAPT (1  M)  R -Flurbiprofen (250  M) CHF5022 (100  M) CHF5074 (100  M)  HEY1  (Q9Y5J3) Hairy enhancer-of-splitrelated with YRPW motif 1NICD 1.9 – – –  HES7   (AB049064) Hairy and enhancer of split 7 NICD 1.8 – – – FST   (M19481) Follistatin AICD 1.6 – – – KTN1  (Q86UP2) Kinectin 1 AICD 1.5 – 1.7 – TIEG  (U21847) Transforming growth factor-  inducible early growthresponseAICD 2.2 – 2.8 –Foldchangevaluesareexpressedasratiosbetweenaveragesignalintensitiesmeasuredinthreereplicates(eachonecontainingduplicatespots)fortheDMSOcontrol(  I  c ) and the test compounds (  I  T ). Only genes with a significant ( P <0.05) ≥ 1.5 fold-change are reported.
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