VX-765isanorallyactiveIL-convertingenzyme/caspase-1inhibitor,inhibitsIL-1βreleasewithsimilarpotencyinPBMCsfromFCAS(IC₅₀=0.99±0.29μM)andcontrol(IC₅₀=1.1±0.61μM)subjects.

Caspase-1^[1]

VX-765inhibitsthereleaseofLPS-inducedIL-1βandIL-18byhumanPBMCswithanIC₅₀of~0.7μMandreducesinflammatoryresponseinmurinemodelsofinflammatorydisease^[1].VX-765isapotent,specificinhibitorofthecaspase-1subfamily^[2].

VX-765doses50,100,and200mg/kgsignificantly(p<0.05) reduces="" serum="" il-1β="" levels="" by="" as="" much="" as="" 60%,="" whereas="" 25="" mg/kg="" has="" a="" smaller="" effect="" (~35%="" inhibition)="" that="" is="" not="" statistically="" significant.="" it="" is="" noteworthy="" that="" the="" effect="" of="" vx-765="" on="" the="" release="" of="" il-1β="" induced="" by="" lps="" reached="" a="" plateau="" at="" 100="" mg/kg.="" vx-765="" (25,="" 50,="" and="" 100="" mg/kg="" ×="" 2)="" significantly="" reduces="" ear="" edema.="" vx-765="" also="" dose-dependently="" reduces="" the="" concentrations="" of="" cytokines,="" chemokines,="" and="" inflammatory="" mediators="" in="" the="" ear="" biopsy="">^[2].VX-765atdosesof25,50,and200mg/kgsignificantlydelaysthetimetoseizureonsetby1.5-totwofold(p<0.01), reduces="" the="" number="" of="" seizures="" by="" 40%=""><0.01) and="" the="" total="" time="" spent="" in="" eeg="" seizure="" activity="" by="" 30="" to="" 50%=""><>^[3].

• [1].StackJH,etal.IL-convertingenzyme/caspase-1inhibitorVX-765blocksthehypersensitiveresponsetoaninflammatorystimulusinmonocytesfromfamilialcoldautoinflammatorysyndromepatients.JImmunol.2005Aug15;175(4):2630-4.

  [2].WannamakerW,etal.(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylicacid((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765),anorallyavailableselectiveinterleukin(IL)-converting

  [3].RavizzaT,etal.Inactivationofcaspase-1inrodentbrain:anovelanticonvulsivestrategy.Epilepsia.2006Jul;47(7):1160-8.

  [4].XuY,etal.NLRP3inflammasomeactivationmediatesestrogendeficiency-induceddepression-andanxiety-likebehaviorandhippocampalinflammationinmice.BrainBehavImmun.2016Aug;56:175-86.

  [5].ZhuangJ,etal.TDP-43upregulationmediatedbytheNLRP3inflammasomeinducescognitiveimpairmentin22",4,4"-tetrabromodiphenylether(BDE-47)-treatedmice.BrainBehavImmun.2017Oct;65:99-110.

Enzymeinhibitionisassayedbytrackingoftherateofhydrolysisofanappropriatesubstratelabeledwitheitherp-nitroanilineoraminomethylcoumarin(AMC)asfollows:ICE/caspase-1,suc-YVAD-p-nitroanilide;caspase-4,Ac-WEHD-AMC;caspase-6,Ac-VEID-AMC;caspase-3,-7,-8,and-9,Ac-DEVD-AMC;andgranzymeB,Ac-IEPD-AMC.Enzymesandsubstratesareincubatedinareactionbuffer[10mMTris,pH7.5,0.1%(w/v)CHAPS,1mMdithiothreitol,and5%(v/v)DMSO]for10minat37°C.Glycerolisaddedtothebufferat8%(v/v)forcaspase-3,-6,and-9andgranzymeBtoimprovestABIlityofenzymes.Therateofsubstratehydrolysisismonitoredusingafluorometer.AssaysforcathepsinBandtrypsinareperformed^[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

VX-765issolubilizedinDMSOandstored,andthendilutedwithRPMI1640completemedium(DMSO0.2%)beforeuse^[1].

Atotalof2×10⁵cells/well(100μLcellsUSPension)isdistributedintriplicateinflat-bottom96-wellplates.Either50μLofVX-765(40μMinRPMI1640completemediumcontaining0.2%DMSO)orvehiclecontrolisaddedtoappropriatewells.Followinga30-minincubationat37°C,50μLofLPSdilutedinRPMI1640completemediumisaddedatfinalconcentrationsvaryingfrom0.001to10ng/mL.Cellsarereturnedtoa37°Cincubator.At4hafterLPSaddition,75μLofsupernatantisremovedfromwells,clearedbycentrifugationfor5minat1500rpm,andstoredat4°Cuntilassayed.Cellsarereturnedtoa37°Cincubatoruntil24hafterLPSaddition,atwhichtime100μLofsupernatantisremoved,clearedbycentrifugation,andstoredat4°C.SupernatantsaretestedusingELISAkitsforIL-1β,IL-6,IL-18,andIL-1α^[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

VX-765ispreparedin25%CremophorELinwater(Mice)^[2].
VX-765ispreparedin20%Cremophor(Rat)^[3].

Mice^[2]
SingledosesofVX-765(10,21,43,and84mg/kg)invehicle(25%CremophorELinwater)areadministeredviaoralgavage.Bloodsamples(approximately0.25-0.3mL)arecollectedbeforedoseadministrationand0.167,0.25,0.5,1,1.5,2,3,4,6,8,and24hafterdosingviatheretroorbitalsinusandprocessedforplasma.Ahigh-performanceliquidchromatography/massspectrometrymethodologyisusedtodeterminetheconcentrationofVX-765andVRT-043198inplasmasamples.NoncompartmentalanalysisiscarriedoutusingWinNonlinPro,version4.0.1.
Rat^[3]
MaleSprague-Dawleyrats(250-280g)areused.VX-765(25,50,200mg/kg)isdissolvedin20%Cremophorandinjectedipinratsonceadayfor3consecutivedays.Onthefourthday,ratsreceivedVX-765,45minand10minbeforeintrahippocampalinjectionofkainicacid.Respectivecontrolsaresimilarlyinjectedwithvehiclebeforekainicacid.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

• [1].StackJH,etal.IL-convertingenzyme/caspase-1inhibitorVX-765blocksthehypersensitiveresponsetoaninflammatorystimulusinmonocytesfromfamilialcoldautoinflammatorysyndromepatients.JImmunol.2005Aug15;175(4):2630-4.

  [2].WannamakerW,etal.(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylicacid((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765),anorallyavailableselectiveinterleukin(IL)-converting

  [3].RavizzaT,etal.Inactivationofcaspase-1inrodentbrain:anovelanticonvulsivestrategy.Epilepsia.2006Jul;47(7):1160-8.

  [4].XuY,etal.NLRP3inflammasomeactivationmediatesestrogendeficiency-induceddepression-andanxiety-likebehaviorandhippocampalinflammationinmice.BrainBehavImmun.2016Aug;56:175-86.

  [5].ZhuangJ,etal.TDP-43upregulationmediatedbytheNLRP3inflammasomeinducescognitiveimpairmentin22",4,4"-tetrabromodiphenylether(BDE-47)-treatedmice.BrainBehavImmun.2017Oct;65:99-110.

509.0

C₂₄H₃₃ClN₄O₆

273404-37-8

RoomtemperatureincontinentalUS;mayvaryelsewhere

DMSO:≥27mg/mL

VX-765isdissolvedinDMSOasastockandthendilutedbysesameoil^[4].
VX-765isdissolvedin20%cremophor^[5].

*"<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

• [1].StackJH,etal.IL-convertingenzyme/caspase-1inhibitorVX-765blocksthehypersensitiveresponsetoaninflammatorystimulusinmonocytesfromfamilialcoldautoinflammatorysyndromepatients.JImmunol.2005Aug15;175(4):2630-4.

  [2].WannamakerW,etal.(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylicacid((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide(VX-765),anorallyavailableselectiveinterleukin(IL)-converting

  [3].RavizzaT,etal.Inactivationofcaspase-1inrodentbrain:anovelanticonvulsivestrategy.Epilepsia.2006Jul;47(7):1160-8.

  [4].XuY,etal.NLRP3inflammasomeactivationmediatesestrogendeficiency-induceddepression-andanxiety-likebehaviorandhippocampalinflammationinmice.BrainBehavImmun.2016Aug;56:175-86.

  [5].ZhuangJ,etal.TDP-43upregulationmediatedbytheNLRP3inflammasomeinducescognitiveimpairmentin22",4,4"-tetrabromodiphenylether(BDE-47)-treatedmice.BrainBehavImmun.2017Oct;65:99-110.