Evaluation of cAMS for 14C microtracer ADME studies

21 Pages·2017·1.24 MB·English

ETH Library
Evaluation of cAMS for 14C
microtracer ADME studies:
opportunities to change the current
drug development paradigm
Journal Article
Author(s):
Lozac'h F.; Fahrni, Simon; De Maria, Daniele; Welte, Caroline; Bourquin, Joël; Synal, Hans-Arno; Pearson, David; Walles, Markus;
Camenisch, Gian
Publication date:
2018-03
Permanent link:
https://doi.org/10.3929/ethz-b-000252746
Rights / license:
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Originally published in:
Bioanalysis 10(5), https://doi.org/10.4155/bio-2017-0216
This page was generated automatically upon download from the ETH Zurich Research Collection.
For more information, please consult the Terms of use.
Methodology
Forreprintorders,pleasecontact:[email protected]
14
Evaluation of cAMS for C microtracer
ADME studies: opportunities to change the
current drug development paradigm
Fre´de´ricLozac’h*,1, SimonFahrni2, DanieleDeMaria3, CarolineWelte3, Joe¨lBourquin2,
Hans-ArnoSynal3, DavidPearson1, MarkusWalles1&GianCamenisch1
1NovartisPharmaAG,NovartisInstitutesforBiomedicalResearch,PKSciences,Fabrikstrasse14,CH-4002Basel,Switzerland
2IonplusAG,Dietikon,Switzerland
3Eidgeno¨ssischeTechnischeHochschuleZu¨rich,DepartmentPhysik,Zu¨rich,Switzerland
*Authorforcorrespondence:Tel.:[phone];Fax:[phone];[email protected]
Aim:Althoughregulatoryguidancesrequirehumanmetabolisminformationofdrugcandidatesearlyin
thedevelopmentprocess,thehumanmassbalancestudy(orhADMEstudy),isperformedrelativelylate.
hADMEstudiestypicallyinvolvetheadministrationofa14C-radiolabelleddrugwherebiologicalsamples
aremeasuredbyconventionalscintillationcountinganalysis.Anotherapproachistheadministrationof
therapeuticdosescontaininga14C-microtracerfollowedbyacceleratormassspectrometry(AMS)analysis,
enablinghADMEstudiescompletionmuchearlier.Consequently,thereisanopportunitytochangethe
currentdrugdevelopmentparadigm.Materials&methods:ToevaluatetheapplicabilityoftheMICADAS–
cAMSmethod,wesuccessfullyperformed:thevalidationofMICADAS–cAMSforradioactivityquantifica-
tioninbiomatricesand,aratADMEstudy,wheretheconventionalmethodologywasassessedagainsta
microtracerMICADAS–cAMSapproach.Results&discussion:CombustionAMS(cAMS)technologyisap-
plicabletomicrotracerstudies.AfavorableopinionfromEMAtocompletethehADMEinaPhaseIsetting
wasreceived,openingthepossibilitiestochangedrugdevelopment.
Firstdraftsubmitted: 2October2017;Acceptedforpublication: 12December2017;Publishedonline:
16February2018
Keywords: 14C•cAMS•clearance•combustionacceleratormassspectrometry•humanADME•Leniolisib•mass
balance•metabolism•MICADAS•microtracer
In the scope of drug development, regulatory guidances encourage the early identification of relevant human
metabolites[1–3].AhumanADMEstudythatusedradiolabeleddrugsallowstheidentificationofmetabolitesand
theelucidationofkeybiotransformationpathwaysandclearancemechanismsinhumans.However,duetothehigh
study cost, the needs for a dosimetry assessment (which include a rat distribution investigation) and for a GMP
manufacturingaswellasthehighattritionratesencounteredindrugdevelopment,theconventionalhADMEis
generallyperformedlateindrugdevelopment(PhaseII,seeSupplementaryFigure1).Theadministereddoseina
conventionalhADMEstudycancontainupto3.7mBqofradioactivitywhichisanalysedbyscintillationcounting.
To address the regulatory demands earlier, a first investigation of pharmacokinetics (PK) and excretion routes is
typicallyperformedwitharadiolabeleddruginrodents.Subsequently,exploratoryanalysisofpotentiallyrelevant
metabolites is investigated in early clinical samples (Phase I) without the use of radiolabel [4,5] (Supplementary
Figure 1). However, the applied LC–MS methods can fail to identify unknown metabolites. If human-specific
metabolitesaregeneratedinearlyclinicalsamples,thesemetaboliteswillnotbedetectedinpreclinicalspeciesusing
radiolabeleddrug,andwillnotbediscoveredinhumansuntiltheradiolabeledhumanmassbalanceADMEstudy
iscompleted.Toaddressthisgap,weproposetodoseanormal,therapeuticallyrelevantdosespikedwithverylow
amounts of a radiotracer to healthy volunteers or patients in a Phase I setting. Thereafter, the mass balance, the
excretionroutesandlevelsofcirculatingmetabolitesinhumansaredeterminedbyacceleratormassspectrometry
(AMS)assuggestedbyLappinandGarner[6,7].
AMSisahighlysensitivemethodforthedetectionofradioactive14C,andthistechnologyallowshumanclinical
studies tobe performed atradiochemical exposuresthat donotdiffer substantially fromthe naturalbackground
10.4155/bio-2017-0216(cid:2)C 2018Fre´de´ricLozac’h Bioanalysis(2018)10(5),321–339 ISSN1757-6180 321
Methodology Lozac’h,Fahrni,DeMariaetal.
ofradioactivity[8,9].Ingeneral,AMSusinggraphitizationofsamplecarbon(gAMS)hasbeenacceptedbyhealth
authoritiesasamethodtoprovidedataforhumanADMEstudies[10,11].Inapreviouswhitepaper,theapplication
of gAMS for the measurement of parent compound and/or metabolites circulating in plasma was described [12].
However,theconversionofsamplecarbontographitemakesgAMSslow,resourcedemandingandexpensiveand,
therefore,isunsuitablefortheanalysisoflargenumbersofsamplesinanearlyclinicalsetting.Thedevelopment
ofcombustionAMS(cAMS)formicrogramlevelsamples[13]hasmadetheAMStechnologymoreapplicableto
ADMEstudies.Forinstance,theanalyteofinterestincAMSisconvertedtogaseousCO anddirectlymeasured.
2
ThisdirectmeasurementsignificantlysimplifiesthesamplepreparationforcAMSandgreatlyincreasesthesample
throughput [14,15]. Futhermore, large, expensive and resource-demanding high-voltage AMS systems have been
miniaturized and simplified substantially. Recent technology improvements such as the MIni CArbon DAting
System(MICADAS)haveledtolow-voltagecAMSinstrumentswithasmallfootprintandreducedcostscompared
withstandardAMSsystems[16,17].
Even though the transition to smaller AMS instruments positively affects the affordability and practicality,
thesenewlydevelopedsystemshaveonlybeentestedfortheirbiomedicalapplicabilityusinggraphitizationAMS
(gAMS)[18,19].However,thepotentialofthemethodcouldnotbereachedduetolimitationsofthegraphitization
process. This process is slow and also requires significant amounts of carrier carbon addition (on the order of
hundredsofmicrograms)toallowforsamplehandling.Inthepresentstudy,weevaluatethedirectmeasurement
ofsampleCO (cAMS)combinedwithMICADAS(MICADAS–cAMS)forADMEstudies.Wheneverpossible,
2
ourinvestigationsfollowpreviousrecommendations[8,12,20]oncriticalbioanalysisparametersneededtoperform
adequateradioactivityquantificationinthescopeofanADMEstudy.
InADMEstudiesusingconventionalradiotracerandanalyticalmethodsthebiomatrices(blood/plasma,urine
and feces) are usually analyzed directly for radioactivity to determine the entirety of the drug-related material.
Fromthetotalradioactivedoseandthetotalradioactivityinurineandfeces(atspecifiedtimeintervals),themass
balance can be derived. Metabolic profiles are generated by sample preparation and chromatographic separation
ofthedifferentdrug-relatedcomponents.ThemicrotracerapproachusingMICADAS–cAMSanalyticalmethods
canbeappliedtoADMEstudiesinthesameway:directcAMSanalyzesoftotal14C(directcAMS),whichinvolves
verylimitedsamplepreparation,thatis,preparingthesampleforcombustion.IndirectcAMSanalyzesofspecific
14C-relatedcomponentsinavarietyofmatricesfollowingmethodsofextraction,chromatographicseparationand
isolation(indirectAMS).
InordertovalidatetheMICADAS–cAMStechnologyforuseinADMEstudieswith14Casaradiotracer,the
dynamicrange,sensitivity,accuracyandprecisionofMICADAS–cAMSwereevaluatedbyspikingbiomatriceswith
a labeled test compound (LDE225, Sonidegib). This compound has been previously used successfully in gAMS
investigations[10].
To demonstrate the applicability for ADME studies with MICADAS–cAMS, a head-to-head comparison of
both the conventional and MICADAS–cAMS methodology was performed in a rat ADME study of CDZ173
(Leniolisib). CDZ173 is an inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) which is currently in clinical
development for autoimmune diseases. The study contained two arms: a conventional radiotracer with classical
radiodetection(liquidscintillationcounting[LSC]andTopCount)and,amicrotracerwithAMSmassspectrometric
atom-countingincombinationwithhighsensitivityradiodetection(MICADAS–cAMSincombinationwithlow
levelliquidscintillationcounting[LLLSC]).
Materials&methods
Chemicals&samplepreparation
Chemicals
Ammoniumformate,formicacid,sodiumbenzoate andphthalicanhydride ofthehighestgradewerepurchased
fromSigma-Aldrich(Steinheim,Germany).Water,acetonitrileandmethanolwereofthehighestgradeavailable
and were obtained from Thermo Fisher Scientific (Loughborough, UK). Acetanilide was acquired from Merck
(Darmstadt,Germany).
Biomatriceslineardilutionsamplepreparation
Fourbiomatrices(blood,plasma,urineandfeceshomogenate[containing1%carboxymethylcellulose])werespiked
withaknownamountof[14C]-LDE225.ThisamountwasinitiallydeterminedineachmatrixbyLLLSC.Subse-
quently,alineardilution(weight/weight(w/w))withbiomatrixwasappliedtostaywellwithintheMICADAS–
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EvaluationofcAMSfor14CmicrotracerADMEstudies:opportunitiestochangethecurrentdrugdevelopmentparadigm Methodology
cAMS dynamic range and below the activities tolerable for a natural abundance laboratory (samples typically
correspondingto0.5–1000mBq/g).Thetotal14CcontentwasdeterminedthroughdirectanalysisbyMICADAS–
cAMS.Quantificationofthetestcompoundoverthedynamicrangewasperformedintriplicate(15μlaliquotsof
biomatrix).
InvivoADMEsamplepreparation
[14C]-CDZ173 was synthesized by the isotope laboratory of Novartis, Basel, Switzerland, and purified by Phar-
maron,Cardiff,UK.[14C]-CDZ173wasdilutedwithunlabeledCDZ173toachievethedesiredspecificactivities
fortheconventionalandmicrotracerstudies.
Animaldosing
TheanimalstudieswereapprovedbyAnimalCareandUseCommitteesoftheKantonBasel,Switzerland.Hannover
Wistarrats(CharlesRiverFrance,299–326g,8–10weeks)receivedanoraldose(p.o.)of10mg/kg[14C]CDZ173
solutioncontaining0.5%(w/w)methylcelluloseand0.5%(w/w)Tween-80asvehicle.Theradioactivedoseswere
20.8 MBq/kg and 564 Bq/kg (mean) across the three rats of the conventional (Group 1) and the three rats of
themicroradiotracer(Group2)studies,respectively(SupplementaryTable1).Afterdosing,bloodwascollected
sublingually in anesthetized rats at 0.25, 0.5, 1, 4, 8, 24 and 48 h postdose. From each time-point, blood was
processedimmediatelytoplasmabycentrifugationat3000×gfor10minat4◦C.ForGroup1,asmallaliquot
◦
wastakenfortotalradioactivitydetection andtheremainder materialwasstoredat-80 Cuntilfurtheranalysis.
◦
ForGroup2,allmaterialwasstoredat-80 Cuntilfurtheranalysis.
Animalswerehousedinmetaboliccagesforurineandfecescollection.Urineandfeceswerecollectedquanti-
tativelyfrom0-to48-htime-intervalpostdose.Fecesweredirectlyhomogenizedafteradditionofa1%aqueous
carboxymethylcellulosesolution(aboutten-timesofthefecesweight).
Preparationofaplasmapool
Individualtime-pointsperratweremeasuredbyLSCfortotal14Cdeterminationinplasmaoftheconventional
study, and then identical volumes were pooled per collection time-point for metabolite profiling by microplate
scintillationcountingviaTopCount.Forthemicrotracerarm,identicalplasmavolumeswerepooledpercollection
timepoint,andtotal14CcontentaswellasmetaboliteprofilingweredeterminedbyMICADAS–cAMS.
Preparationofaurinepool
Urinesamplesbetween0and48hwerepooledacrossthethreeanimalsofthesamedosinggroupbycombining
identical percentage (by weight) of individual urine fractions. Samples from the conventional arm were assayed
bymicroplatescintillationcountingviaTopCountformetaboliteprofiling,whereassamplesfromthemicrotracer
groupweremeasuredbyMICADAS–cAMS.
Preparationofafecespool
Feces homogenates were pooled over 0–48-h time interval of the three animals of the same dosing group. The
pools were prepared by combining identical percentages of the amounts of the individual feces homogenates.
Radioactivitiesweremeasuredusingthesameapproachasforurinepools.
Samplepreparationofmatricesformetaboliteprofiling
A subfraction from the above plasma and feces pools were prepared by protein precipitation/extraction with
acetonitrile (fourfold volume addition). Following vortexing, sonication and centrifugation, the supernatant was
collected and the resulting pellet was extracted two more times with a mixture of water/acetonitrile/methanol
(4/4/2,v/v/v)andacetonitrile.Allsupernatantswerecombinedandconcentratedunderastreamofnitrogen.The
residueswerereconstitutedwithamixtureofwater/acetonitrile(9/1,v/v).Urinepoolswerepreparedbyvortex
andcentrifugation.Analiquotofthereconstitutedsampleswasinjectedformetaboliteprofiling.
Analyticalmethods
Ultra-performanceliquidchromatography&metaboliteprofiling
Ultra-performance liquid chromatography (UHPLC) was performed on an Acquity chromatographic system
(Waters, MA, USA) using a HSS T3 column (2.1 × 150 mm, 1.8 μm particles; Waters), protected with a
precolumn(2.1×5mm)ofthesamestationaryphase.Theprecolumnandcolumntemperaturesweresetat50◦C.
The components were eluted with a gradient of mobile phase A (10 mM ammonium formate, pH 3 [adjusted
futuresciencegroup www.future-science.com 323
Methodology Lozac’h,Fahrni,DeMariaetal.
with formic acid]) versus mobile phase B (acetonitrile). Mobile phase B was initially set up at 2% for the first
2min,increasedto12%at2.1min,to30%at20minandto98%at24min,thenmaintainedatthisleveluntil
25.5min.Thetotalflowratewas0.5ml/min.Thecolumneffluentwascollectedin6sfractionswithaGX271
fraction collector (Gilson AG, Mettmenstetten, Switzerland) for metabolite profiling. UHPLC instrumentations
weremanagedunderUNIFI™(Waters,version1.8).
Metabolitestructurecharacterization
MetabolitestructureswerecharacterizedbyUHPLCcombinedwithmassspectrometryanalysisoftheremaining
aliquotspreparedformetaboliteprofilingpurpose,withthesameUHPLCinstrumentationcoupledwithaXevo
QtoF(accurate-massquadrupoletime-of-flight)(Waters)conductedunderUNIFI™(version1.8).AftertheTUV
(tunable,dual-wavelengthultraviolet/visible)detection(setat254nm)thecollumneffluentwassplitin∼1:5ratio.
Onlytheminorportionwasdirectedintotheelectrosprayinterfaceofthemassspectrometer(operatedinpositive
ionmode).Themajorportionwasdirectedtothewaste.Argonwasusedforcollisionalactivation.Asolutionof
leucineenkephalinwasinfusedthoughtheLockSpray™interfacetogeneratealock-massthroughouttheanalysis
andobtainaccuratemassspectrometrydata.
Nomenclature&assignmentofthemetabolites
MetaboliteswithanassignedstructurebasedonfullchromatographicandMS/MSdatawerelabeledwithan‘M’
number,forexampleM1.MetaboliteswithlimitedstructuralinformationwerenamedasPx(wherexrepresents
theapproximateretentiontime).
Set-upformetaboliteidentification&profiling
A two-injections strategy, which was successfully applied in previous AMS studies [10], was performed. The
configuration is illustrated in Supplementary Figure 2. First, one small aliquot (15 μl) is injected on UHPLC
instrumentation,andfractionsarecollectedin96-wellplates,containingsolidscintillator(LumaPlate96deepwell
plate, PerkinElmer; Group 1), or precleaned tin cups (tin capsules for solids or liquids, PerkinElmer; Group 2).
The 96-well plates content was assayed by TopCount (PerkinElmer, WA, USA) or measured on a MICADAS
instrument at ETH Zurich (Ionplus AG, Switzerland) for Group 1 and Group 2, respectively. Sodium benzoate
(anonvolatile salt)wasusedasacarboncarrierinMICADAS–cAMSmeasurements.Asolutionofthebenzoate
carrier was manually pipetted in tin cups and dried prior to the fraction collection. After the first injection, a
secondaliquot(largervolume,upto150μl)isinjectedontheUHPLCsystemcoupleddirectlytohighresolution
mass spectrometer for metabolite identification. With the help of the reconstructed radiochromatogram profiles
generatedbyMICADAS–cAMSorTopCount,themetabolitemassesundertherelevantpeakswereidentified.
Quantificationstrategy
Radioactivity in the different matrices was quantified by MICADAS–cAMS, LLLSC or LSC. Total activities in
plasma, urine and feces were measured by the appropriate quantification technology based on sensitivity and
dynamic range. Radioactivity determinations from the conventional study (plasma, urine, feces and carcass) and
inothersamples(doseformulation,cagewash)weremeasuredbyLSC.The14Ccontentinbiologicalsamplesof
the microtracer study was determined by MICADAS–cAMS technology (plasma, urine) or LLLSC (urine, feces
and dose formulation). All sample preparation techniques (e.g., homogenization, quenching etc.) followed the
guidelinesfromSwartetal.([21]anditssupplementaldata).Alldeterminationsofradioactivitywereconductedin
weighedsamples,andvolumeswereadjustedaccordingtotheanalyticaltechniqueused(e.g.,∼0.1gforLSCand
∼0.015gforcAMS).Measurementofradioactivityforplasmawasconvertedintoconcentrations(molequivalents
pervolumeorgram)consideringthespecificradioactivityandassumingamatrixdensityof1g/ml.
MICADAS–cAMSsetup
All low-level 14C measurements were performed on a MICADAS–cAMS setup at the Laboratory of Ion Beam
PhysicsatETHZurich,Switzerland(SupplementaryFigure3).Inthissetup,alltheprocessesaresoftware-controlled
andfullyautomated.Anelementalanalyzer(EA,Elementar,Germany)withasample-feedingwheelfor80samples
wasusedasacombustionunitforsampleswrappedincylindricaltincapsules(Elementar).Thegasesarisingfrom
the samples combustion were separated thermochromatographically into N , CO and H O and quantified by
2 2 2
meansofathermalconductivitydetectordownstreamfromthethermochromatographycolumn.Thesamplegases
324 Bioanalysis(2018)10(5) futuresciencegroup
EvaluationofcAMSfor14CmicrotracerADMEstudies:opportunitiestochangethecurrentdrugdevelopmentparadigm Methodology
arethenhandledwithadedicatedgasinterfacesystem(GIS,IonplusAG,Switzerland,SupplementaryFigures3
& 4), allowing to trap the CO peak using a molecular sieve and to regulate its injection into the ion source of
2
MICADAStoensureaconstantcarbonmassflow.ThehybridionsourceofMICADASallowsbothmeasurements
ofgraphitizedandgaseousCO2samplesandisequippedwitharandom-accesssamplechangerfor40cathodes[16].
Adynamicrangeofmorethanfourordersofmagnitudecanbereachedwiththissetupandthecurrenttechnology.
MICADAS–cAMSanalysis
Linearbiomatricesstudieswereperformedinshortmeasurementcampaignsofonlyafewdays,whereasADME
measurementswereperformedon15workdaysoveraperiodof3weeks(SupplementaryFigure5).Mostsamples
weremeasuredwithacountingtimeof5minandatotalsample-to-sampletimeofjustunder8min.Inaddition
to samples, AMS standards (oxalic acid 2, NIST SRM 4990C, OX-2) and background (fossil, radiocarbon-free,
phthalic anhydride, Sigma-Aldrich, Germany) samples, were measured regularly to ensure stable measurement
conditions and for quality control. Furthermore, solid acetanilide (K37102211, Merck, Germany) was used
for carbon mass calibration of the elemental analyzer as precise mass determination was necessary for precise
determinationofthesampleactivity.
TheMICADAS–cAMSmeasurementwasrunindailybatchesofca.80–140samples,foratotalof1755samples
(1608 samples, 74 background/acetanilide and 73 standard samples) measured within 15 consecutive workdays
(Supplementary Figure 5). A quick retuning of the AMS instrument and the analysis of two standards and two
backgroundsamples,forqualitycontrol,normalizationandblanksubtraction,wereperformedatthebeginningof
eachmeasurement’sday.Inparallel,theadsorptiontrapwascleanedwithaseriesofcombustionswithoutsamples.
Dataanalysis
Biomatriceslineardilutionanalysis
Standarddeviation(SD)andrelativedeviation(RD,%)fromthemeasurementswerecalculatedaswellasthedevi-
ation(D,%)betweentheknownamount(calculatedfromthelineardilution[w/w])andthecAMSquantification
(meanofquantifiedaliquots).Thevalidationcriteriaintargetmatrix(performedonlyintriplicateinthisstudy)
were following the recommendation of the European Bioanalysis Forum [12], whenever possible, and applied to
determinethedynamicrange,limitofdetection[LOD]andlowerlimitofquantification[LLOQ]inbiomatrices.
DataanalysisforAMS
AMSdatawereevaluatedaccordingtostandardproceduresandconventionswiththeBATSsoftware[22].Unlike
the conventional radiometric methods, AMS does not measure radioactivity in samples but rather the isotopic
ratios(12C/14C)onafractionofthesamplecarbon.Therefore,notonlythesamplemassanditsactivityhaveto
beknownforactivitydetermination,butratherthesamplemass,thecarbonmassofthatsampleanditsisotopic
ratio.Inthepresentstudy,thesamplemassforAMSanalysisofbiomatriceswastypicallysetto∼15mgandcarbon
amounts were determined by the EA. For HPLC eluates, carrier carbon on the order of 40 μg C was added to
fractionsasdilutionfor14Candforsamplehandlingandquantification.Inthiscase,onlytheisotopicratioand
thecarbonadditionhadtobequantified.
ADMEanalysis
Massbalancecalculations
Themeancumulativeexcretionofradioactivitywascalculatedfromthesumoftheindividualpercentagesofdose
excretedduringthe24-hcollectionintervalsinurineandfeces.
PKanalysis
PKparametersoftotalradioactivityinplasmawereanalyzedusingBioBookIDBSelectronicnotebook(template
version10.0.1createdforNovartis).TheAUC werecalculatedusingthelineartrapezoidalrule.Formetabolite
last
profiles, the peaks in the radiochromatograms were integrated using the RadioStar software (version 5.0) from
BertholdTechnologies(BadWildbad,Germany).Relativepeakareas,andconcentrationsofindividualradiolabeled
components in plasma and the amounts of individual radiolabeled components in excreta were calculated from
thetotal14C-labeledcomponentsintherespectiveoriginalsamplepools.Absorption,expressedasapercentageof
dose,wasdeterminedbasedonradioactivityinurineandfeces.
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Methodology Lozac’h,Fahrni,DeMariaetal.
Results&discussion
InstrumentqualificationfordirectMICADAS–cAMSanalysis
SummaryofMICADAS–cAMSstudies,LOD&LLOQ
Atotalof19blood,23plasma,27urineand22fecessampleswereanalyzedoveradynamicrangefrom∼0.5to
∼1000mBq/g.Ingeneral,SDandRDacrosstriplicatemeasurementsofthesameconcentration,aswellasDwere
excellent. In blood, plasma, urine and feces the averaged RD and D values were all under 15% (Supplementary
Tables 2–5). Linearity, reproducibility and accuracy were demonstrated within a dynamic range in all matrices.
Althoughtheanalyseswereperformedonlyintriplicate,theresultswereinaccordancewithrequiredbioanalysis
criteria for ADME studies (RD: ± 20% within dynamic range, ± 25% at LLOQ, [12]). The observed LLOQ
(∼1, 0.5, ∼5 and ∼10 mBq/g for blood, plasma, urine and feces, respectively) were in line with the needs of
microtracerADMEstudiesandwithLLOQfromothercAMSinstruments(SupplementaryTable6).Itshouldbe
notedhere,thattheLLOQinthecurrentAMSstudyaswellasinsimilarAMSstudiesispredominantlylimited
bysamplepreparation(duetothepartialcontaminationofsamplesfromhigheractivitysources(e.g.,throughout
thesamplepreparation,administration,collection,storageetc.)andtheresultingvariabilityofbackgroundsamples
ratherthanthesamplecombustionormeasurementbycAMS.ThebackgroundoftheMICADAS–cAMSmethod
determined during the present ADME study (n = 62) corresponded to 0.014 mBq/g in a 15 mg aliquot with a
background variation of ca. 0.008 mBq/g. These samples had not been processed along with the other samples
at Novartis but through pipetting of aliquots at ETH Zurich. With this background, an LLOQ (background
value + 10 × background variation) on the order of 0.1 mBq/g should be achievable by the MICADAS–cAMS
technology.ThiscorrespondstoanLODof0.04mBq/g(backgroundvalue+3×backgroundvariation).However,
a prestudy analysis with 14C-free sodium benzoate from Novartis (which was also used in the ADME study as
carriercarbon)revealedthattheLLOQwasdominatedbythevariabilityofcontaminationwith14Cduringsample
preparationandcorrespondedto∼1.5mBq/g(backgroundof∼0.16mBq/gwithavariabilityof∼0.13mBq/g).
A large variation of values such as the ones observed for the lowest concentrations in Supplementary Table 4 is,
therefore,alsolikelycausedbythelargevariabilitycomingfromthesamplepreparation(e.g.,aftercentrifugation,
drying,handlingwithtweezersetc.)ratherthancAMSmeasurementsdirectly.Wethereforethinkthatthesomewhat
elevatedLLOQsfoundinurineandfecesareaconsequenceofeitherpotentialcontaminationwithextraneous14C
and/orhomogeneityissuesinthecaseoffeces.Theoverallcross-talkorcarry-overofsamplesprocessedwiththe
MICADAS–cAMSmethodhasbeendeterminedto0.5–1%oftheactivityoftheprevioussample.Intheanalysis
ofchromatograms,thiscross-talkisusuallymaskedbythebroadnessofchromatographicpeakswhichisnotsharp
toasinglefractionbutusuallyspanstwoormorefractions.
ULOQ&dynamicrange
ThecAMSupperLOQ(ULOQ)dependsonthecarboncontentofthesample.Itisontheorderof400mBq/g
for a 15 mg urine sample with a low carbon content of 0.4% or 4 Bq/g for a 15 mg plasma sample with 4%
carboncontentwhennocarbondilutionisused.Hence,thetheoreticaldynamicrangeoftheMICADAS–cAMS
instrumentationspans4ormoreordersofmagnitudefromLLOQtoULOQ.Additionofdilutingcarbonatthe
finalstepofthesamplepreparationcanextendtheULOQevenfurtherastheULOQismainlygivenbythehighest
14C/12CratiothatcanbemeasuredinAMS(i.e.,thehighestcountrate).Inordertoavoidanyinfluenceofthe
diluting carbon on the sample preparation, this carbon is introduced at the last step of the sample preparation
for a cAMS measurement, which is just before drying down the collected sample for combustion or even as a
separate tin capsule added during combustion in the elemental analyzer. The mentioned ULOQ values between
400mBq/gand4Bq/garewellwithinthedynamicrangeofLLLSCasdemonstratedbySwartetal.[21].Another
technique,whichcouldbeconsidered,istodilutesampleshigherthantheULOQwithblankmatrices.However,
adilutionofsamplescouldintroducetheriskofanalytes(e.g.,drug)adsorbingtosurfacesorprecipitatingduring
the dilution steps. The linear overlap of the dynamic ranges of cAMS with those of less sensitive radioactivity
quantificationtechnologies,namelyLSCandLLLSC,allowmeasurementofsamplesusingacombinationofthe
different technologies based on the absolute radioactivity content as these technologies have been determined to
giveidenticalresultswithintheirrespectivedynamicranges[21].
326 Bioanalysis(2018)10(5) futuresciencegroup
EvaluationofcAMSfor14CmicrotracerADMEstudies:opportunitiestochangethecurrentdrugdevelopmentparadigm Methodology
Table1.Concentrationoftotalradiolabeledcomponentsandpharmacokineticparametersinplasma(Group1and
Group2).
Time(h) MeanfromGroup1(μM)† MeanfromGroup2(μM)†
0.25 5.02 5.33
0.5 3.24 4.05
1 2.76 4.27
4 1.83 2.38
8 0.671 1.17
24 0.105 0.613
48 0.0475 0.422
Tmax(h) 0.25 0.25
Dose(mg/kg) 9.77 11.0
Cmax(μM) 5.02 5.33
T1/2(h)(0.25–8h) 3.1 3.8
T1/2(h)(8–48h) 11 28
AUClast(h·μM) 23.1 47.7
Concentrationoftotalradiolabeledcomponents(μM)andcorrespondingpharmacokineticparametersinplasmaofmaleratsfollowingasingleadministrationat∼10mg/kgp.o.
†Obtainedfromspecificactivitiesofeachdosingsolution.
Headtoheadcomparisonofconventional&microtracermethodologiesinthescopeofaratADME
Althougha40,000-foldlowerradioactivitywasusedinthemicrotracerarm(Group2)thanintheconventional
study (Group 1), similar results were obtained from the MICADAS–cAMS quantification (Group 2) and LSC
quantification(Group1),providingevidencefortheutilityofMICADAS–cAMSforADMEstudies.
PKofradioactivity
The plasma samples of Group 2 were directly measured by MICADAS–cAMS without an LLLSC precheck as
14C-compound concentrations were expected to be within the dynamic range of MICADAS–cAMS and the
restrictions of the natural abundance 14C laboratory at ETH Zurich. The concentrations of total radiolabeled
componentsinplasmaweremeasureableupto48hafteradministrationsinbothGroup1and2(Supplementary
Tables7&8).TheMICADAS–cAMSdatainplasmaarelistedinSupplementaryTable8.Theprecisionwasgood
withtherelativedeviationof<10.5%foralltriplicatemeasurements.Allamountswereabovethepredetermined
MICADAS–cAMS LLOQ in plasma (Supplementary Table 3). The derived PK data of both arms are listed in
Table1.Theconcentration-timecurvesplotsaredepictedinFigure1andSupplementaryFigure6.
Followingadministrationofasingledoseof14C-CDZ173(Leniolisib)inrats,themedianT (timetoreach
max
the maximum concentration) and C (maximum concentration) values of radioactivity in plasma were similar
max
(T :0.25hforbothgroups,C :5.02and5.33μMforGroup1andGroup2,respectively).
max max
For both groups, the initial half-lives were similar with 3.1 and 3.8 h for Groups 1 and 2, respectively, but a
differencewasobservedfortheterminalhalf-lives,whichwere11(Group1)and28h(Group2),respectively.The
concentrationsmeasuredinGroup2byMICADAS–cAMStechnologywereslightlyhigherthantheconcentrations
measuredinGroup1byLSC,especiallyforthelatertimepoints.Thereasonforthisobservationisnotclearatthis
point and it may be due to the low-level contamination in sample preparation as mentioned in the ‘Instrument
qualification for direct MICADAS–cAMS analysis’ section. Once these sample preparation issues have been
addressed,MICADAS–cAMSquantificationatthelatertimepointsmightbemoreaccurateduetothesuperior
sensitivity(atomcounting14C)forAMSversusdecaycountingforliquidscintillationtechnologies[20,23]andbetter
precisionofMICADAS–cAMSoverLSC[24].Thiswouldleadtoabetterestimationoftheterminalhalf-lifeofthe
compoundintheGroup2(Figure1).
TheRDcomparisonacrosstheintraindividualtriplicatemeasurementsfromGroup1(byLSC,Supplementary
Table7)andGroup2(byMICADAS–cAMS,SupplementaryTables8&9)alsoemphasizethebetterprecisionof
MICADAS–cAMStechnologyversusLSC.TheAUC betweenthegroupsalsodifferedandwerecalculatedtobe
last
23.1μM·hand47.7μM·h(Groups1&2).Thedifferencebetweenthegroupsisinlinewithapreviousstudy,in
whichca.twofoldvariabilityinAUC valueswasobservedbetweentwogroupsofratshousedindifferentcages,
last
despitelowintraindividualvariabilitywithineachgroup.
futuresciencegroup www.future-science.com 327
Methodology Lozac’h,Fahrni,DeMariaetal.
10.0
Group 1 (LSC technology)
M) Group 2 (MICADAS technology)
µ
n (
o
ati 1.0
r
nt
e
c
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c
a
m
s
a
Pl 0.1
0.0
0 12 24 36 48
Time (h)
Figure1. Pharmacokineticplots(logarithmicscale)oftotalradioactivityinplasmaafterdosing[14C]CDZ173torats
withconventionalradiolabeleddose(Group1)andmicrotracedose(Group2).
Massbalanceofradioactivity
To stay within 14C activities of the ULOQ of MICADAS–cAMS and due to the limitations imposed by the
restrictions of a natural abundance laboratory, urine and feces were initially measured by LLLSC, followed by
repeatmeasurementofurinebyMICADAS–cAMSwhenconcentrationswerewithintherequiredrange.
Supplementary Tables 10 and 11 show the CDZ173 (Leniolisib) radioactivity values recovered in urine from
LLLSCandMICADAS–cAMSdata,respectively.Themeasuredconcentrationsinthe0–24-htimeintervalwere
inthedynamicrangeofquantificationbybothLLLSCandcAMS(∼875mBq/gurine),andvaluesobtainedwith
bothmethodswerecomparable(SupplementaryTables10&11).Forthe24–48-haliquots,LLLSCmeasurements
(SupplementaryTable10)gaveahighrelativedeviationandthevalueswerebelowtheLLOQofLLLSCaccording
toSwartetal.[21].TheMICADAS–cAMSrelativedeviationforthesesampleswas<9%acrossallmeasurements
(SupplementaryTable11),andthereforeonlycAMSdatawereconsideredforfinalassessment.
Infeces(SupplementaryTable12),theradioactivityvaluesmeasuredbyLLLSCwereacceptableandabovethe
definedLLOQforLLLSCof100dpm/g[21],andwerethereforenotreassessedbycAMSinordertoavoidpotential
contaminationoftheinstrument.TherelativedeviationfromLLLSCmeasurementswasacceptablefornearlyall
collected intervals. Only for the 24–48-h sample interval, the LLLSC value was calculated to be slightly high at
21.7%.However,thepreparationoffecesdidnotresultinsufficienthomogenizationwhichcouldcontributeto
theobservedhighrelativedeviation.
The quantitative excretion profiles of both groups are shown in Table 2, and are very similar. Within 48 h
afteroraladministrationofGroup1,5%oftheadministeredradioactivitywasexcretedinurineand90%infeces
(Table2)withthemajorportionrapidlyexcretedwithin24h.InGroup2(Table2),theurinaryandfecalexcretion
amountedto6and96%ofdose,respectively,after48h.Attheendoftheexperiments(at48-hpostdose),only
2%ofdosewasmeasuredinthecarcassesfromGroup1.Ingeneral,agoodrecoveryofdrugrelatedmaterial in
rangeof97.3–102%(mean,n=3)wasobtainedforbothgroups.
Metabolism
Metabolitestructures
The structural characterization of metabolites in plasma and excreta was carried out by LC-MSe analysis. The
productionspectrum(SupplementaryFigures7&8)showedthe[M+H]+ ionatmassovercharge(m/z)451,
328 Bioanalysis(2018)10(5) futuresciencegroup
EvaluationofcAMSfor14CmicrotracerADMEstudies:opportunitiestochangethecurrentdrugdevelopmentparadigm Methodology
3
7
ml) Z1
m/ CD
p
d
y (
vit
cti
adioa M1 M13 M21M5 M6 M7M22
R
0 5 10 15 20 25
Retention time (min)
3
ml) Z17
m/ CD
p
d
y (
vit
cti
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Radio M1 M13 M19 M10M21M5 M6M7 M22 M25 M9
0 5 10 15 20 25
Retention time (min)
1
1 M2 M6 M7
ml) M
m/ 5
Radioactivity (dp Front peak P3.2 M28 M18 M20 M14M16 M23M24M26M4P17.6P17.8 MP19.8P19.9 CDZ173 M25M9
0 5 10 15 20 25
Retention time (min)
ml) M1
m/
Radioactivity (dp Front peak M28 M18M20P8.1M14P8.4M16 M23M24M26M19M4P17.7M10M21 M5 M6M7 M22 CDZ173
0 5 10 15 20 25
Retention time (min)
Figure2. Metaboliteprofilesinplasma,urineandfecesafterasingleoraldoseof10mg/kg14C-CDZ173inrat
(Group1,TopCounttechnology).Plasmapoolfromallthreeratsat0.25h(A),plasmapoolfromallthreeratsat8h
(B),urinepoolfromallthreeratsacrossthe0–48-hinterval(C)andfecespoolfromallthreeratsacrossthe0–48-h
interval(D).Theunitofradioactivityonthey-axisisdisintegrationpermin(dpm)permlofcolumneffluent(dpm/ml).