Efficacy-study-protocol-in-human-primary-tumor-mod

ProtocolofefficacystudyofcompoundsinhumanprimarytumorxenograftmodelPurpose:Totestanti-tumoreffectsofcompoundsinectopicmodelofhumanprimarytumormodelAnimals:MaleorfemaleSCID/nudemiceaged5~6weeks,withbodyweightabout18~20g,willbeorderedfromvendor,andwillbehousedinspecificpathogen-free(SPF)conditionsatChemPartner.Animalswillbeheldforaminimumofthreedaysforacclimationpriortothebeginningofstudy.Procedure:1.Takeasepticallythetumorsthataregreaterthan300mgbutnomorethan1000mg.2.Removenecrotictissue,cutthetumorsintoabout10-15mgpiecesandmixthetumorpiecesinHBSSplusantibiotic/antimycoticonice.3.Themixedtumorpieceswillbeimplanted(singleflank)intoSCIDmice/nudemiceaftermixedwith20-50µLMatrigel,3-6piecesabout50-90mgpermouse,theaddingnumberoftumorpiecesandvolumeofmatrigelwillbedependedontheprofileofspecialcase.4.Bodyweightswillberecordedonceortwiceperweekbeforegrouping.5.Tumorvolume(length×width)willbemeasuredonceortwiceperweekbyusingDigitalcalipersandtumorvolumeswillbecalculatedbasedonthefollowingformula:Tumorvolume＝(length×width2)/2.6.Groupingtumorbearingmice(randomlydividedbasedonthetumorvolume),whentheirtumorsvolumehasreachedappropriatesize(100~300mm3).Thenumberofgroupswillbebasedonhowmanyanimalsareeligible.Andtheotheranimalswillbeeuthanized.Theanticipatedgroupswillbeasfollow:Group1:N=10,vehiclecontrolfortestcompoundsGroup2:N=10,positivecompoundcontrolGroup3:N=10,highdoseoftestcompoundsGroup4:N=10,middledoseoftestcompoundsGroup5:N=10,lowdoseoftestcompounds7.Compoundadministration:Theformulationofcompoundswillbepreparedaccordingtoclient’srequest.Dosingvolume:animalswereadministeredat0.1ml/10gbasedonthelatestanimalbodyweight(onthedayofdosing).8.Daysoftreatmentwillbedeterminedaccordingtotheresultsandafterdiscussionwithclient.9.Thebodyweightofmicewillbemeasuredeverydaybeforedosing,andcalculatedrelativechangeofbodyweightofeachmouseaccordingtothefollowingformula:RCBW(%)=(BWi–BW0)/BW0×100;BWiisthebodyweight;BW0isthebodyweightonthefirstdayoftreatment.ThetumorvolumeofeachmousewasmeasuredtwiceperweekandcalculatedTGIofeachgroupaccordingtothefollowingformula:TGI(%)=[1-(Ti-T0)/(Vi-V0)]×100;Tiisthetumorvolumeoftreatmentgroup,T0isthetumorvolumeofthetreatmentgrouponthefirstdayoftreatment;Viisthetumorvolumeofvehiclecontrolgroup,V0isthetumorvolumeofthevehiclegrouponthefirstdayoftreatment.Theexperimentalresultswillberecordedonthelaboratorynotebook.10.Monitoredclinicalconditioncarefullyforanyindicationthattheanimalsareunwell(e.g.bodyweightlosses,subduedbehavior,pilo-erection,hunched,skintenting,laboredrespiration,paleextremities,abnormalgait,diarrhoea,vocalization).Anyevidenceofdeviationsfromnormalityshouldeitherresultinananimalbeinghumanelyterminatedorresultincarefulsubsequentobservationthroughoutthedaytocheckforanydeteriorationinanimalcondition.Ifsymptomsworsen,animalsshouldbehumanelyculled.11.Allanimalswouldbeeuthanizedamaximumof6weeksafterinitiationofcompoundsadministration.Theexperimentwillbeterminatedaccordingtotheresultsandafterdiscussionwithclient.12.Thefinalsampleswillbecollectedaccordingtotheclient’srequestandafterdiscussionwithclient.ThesnapfrozentissuesusedforPKandproteinexpressionanalysiswillbeusedwithdryiceandthenkeptin-80℃,andthesnapfrozentissuesusedforRNAanalysiswillbeusedwithRNAlater@(Ambion,Cat.No.:Am7021).Materials:NameSourceCat.NoOtherHBSSInvitrogen14170Storeat2-8℃Antibiotic-AntimycoticInvitrogen15240Storeat-20℃Centrifugaltube50mlCostar430828DishCorningCLS430167-500EA10cmindiameterOphthalmicforceps66Vision-TechOphthalmicscissors66Vision-TechCancerimplantneedlesPopper&Sons,inc7927DigimaticcallipersMitsutoyo500-171-20HumanprimarytumorfragmentChemPartnerMatrigelBD354234Storeat-20℃SorafenibLCLABORATORIESS-8502Theformulation:CremophorEL/ethanol(50:50).The4-folderstocksolutionwillbepreparedfreshlyeverythreedays.The1solutionwillbepreparedfreshforimmediatedosingbydilutingthe4stocksolutionwithsterilizeddistilledwater.LowerdoseswillbepreparedbydilutionwithCremophor/ethanol/water12.5/12.5/75(1vehicle).GEMZAR@LillyFranceNo.7501theeffectivecontentofgemcitabineis46.51%,preparedfreshlybeforedosing,dissolvedinsaline