assessment of fetal exposure risk following seminal excretion of a therapeutic igg4 t igg4 monoclonal antibody using a rabbit model

Dose mg/kg Hours post-dose Intravaginal:IV Dose mg/kg Hours post-dose Intravenous route Serum T-IgG4 concentration ␮g/mL Serum T-IgG4 ␮g/mL per mg/kg IV dose dose normalized d a T-IgG4,

Reproductive Toxicology

jo u r n al hom e p ag e :w w w e l s e v i e r c o m / l o c a t e / r e p r o t o x

Assessment of fetal exposure risk following seminal excretion of a

therapeutic IgG4 (T-IgG4) monoclonal antibody using a rabbit model

William J Breslin∗, Kim G Hilbish, Todd J Page, David E Coutant

Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, USA

a r t i c l e i n f o

Article history:

Received 24 March 2014

Received in revised form 28 April 2014

Accepted 5 May 2014

Available online xxx

Keywords:

IgG

Monoclonal antibodies

Placental transfer

Seminal transfer

Vaginal absorption

a b s t r a c t

partner

Thepotentialfordrugsandotherchemicalstobeexcretedinto

semenatconcentrationssufficienttoadverselyaffectsperm

func-tionordevelopmentoftheembryoorfetushasbeenatopicdebated

inthescientificliteratureoverthepastfewdecades.Two

excel-lentreviewsonthissubjectarepresentedbyPichinietal.[1]and

KlemmtandScialli[2].ThereviewbyPichiniet al.[1]coversa

varietyofimportanttopicsthatincludesthepharmacokineticsof

smallmolecularweightdrugexcretionintoseminalfluidacrossa

broadspectrumoftherapeuticdrugclasses.Althoughmostofthe

smallmolecularweightdrugswerereportedtobepresentinsemen

atconcentrationslessthanthatobservedinplasma,someofthe

antimicrobialswerefoundtoconcentrateinsemen.Clindamycin

forexamplewasreportedinhumanseminalfluidatconcentrations

approximately11-foldhigherthanplasma[3].Thedemonstrated

concentrationofsomedrugsinsemenincreasestheconcernfor

potentialadverse reproductiveand developmental effects from

eitherdirectdamagetospermorfromtheseminaltransferofdrug

fromtreated malesto untreated femalepartners Althoughthe

∗ Corresponding author at: Lilly Research Laboratories, Lilly Corporate Center

DC0730, Indianapolis, IN 46285, USA Tel.: +1 317 433 3601; fax: +1 317 260 7040.

E-mail address: wjbreslin@lilly.com (W.J Breslin).

effectofdirectdrugexposureonmalefertilityandearlyembryonic developmentcanbeassessedthroughstandardnonclinicalfertility studyprotocols,suchasthat describedformalefertility assess-mentinICHS5(R2)[4],thedevelopmentaleffects fromseminal drugtransfertotheuntreatedfemaleisnotroutinelyevaluated

Toaddressthepotentialexposureriskfromseminaltransfer

of chemicalstotheconceptus, Klemmtand Scialli[2] provided

afemale/conceptus exposureassessment basedona worst-case scenariousingmeasuredclindamycinseminalconcentrationsof 0.125mg/mL,asemenvolumeof5mL,100%vaginalabsorption, anda volumeofdistributionof1.1L/kgfora 60kgwoman.The bloodconcentrationofclindamycininawomanexposedthrough semenwascalculatedtobethreeordersofmagnitudelowerthan thebloodexposureofthetreatedmanwhoproducedthesemen Theresultingbloodconcentrationinthefemalewasthoughtnotto haveclinicalrelevance

Despite thefindings reportedbyKlemmtand Scialli[2]that predictalowexposurerisk,thereremainsafetyconcernsforthe transferofdrugsthroughsemen.Relativelyrecently,theUnited Kingdom[5]issuedguidanceontheuseofcontraceptioninhuman clinicaltrialsinwhichtheystate,“Wherethereisariskofdrug secretionthroughtheejaculate,malesubjects(includingmenwho havehad vasectomies)whosepartnersarepregnantshoulduse condomsforthedurationofthestudyandforasuitabletime after-wards(e.g.fivehalf-lives).Thebarrierprotectionrequirementsfor http://dx.doi.org/10.1016/j.reprotox.2014.05.004

0890-6238/© 2014 The Authors Published by Elsevier Inc This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/3.0/ ).

investiga-tionalmedicalproductthroughseminaltransferandsubsequent

vaginalabsorption.”Inaddition,theliteraturesurrounding

semi-nalexcretionandvaginalabsorptionofdrugsprimarilyaddresses

smallmoleculesanddoesnotcovertheriskofbiopharmaceuticals

presentinseminalfluid[1,2].Thelackofdataonseminaltransfer

andvaginalabsorptionofbiopharmaceuticalsrepresenta

signifi-cantdatagapgiventhehighproportionofdrugsthatfallintothis

category

Inordertoassessthepotentialfetalexposureriskduetothe

seminaltransferofatherapeuticIgG4antibodydrug(T-IgG4),a

seriesofstudieswereconductedtoassessseminalexcretion,

vagi-nalvs.intravenousabsorption,andplacentaltransferofaT-IgG4

antibody

2.1 Generalmethodology

2.1.1 Animalcare

NewZealandWhite (NZW)rabbitswereobtainedfrom

Cov-anceResearchProducts,Incorporated(Kalamazoo,MIorDenver,

PA).Eachrabbitwasuniquelyidentifiedbynumberusingaplastic

eartag.Therabbitswereindividuallyhousedincleansuspended

stainlesssteelcagesinanenvironmentallycontrolledroom

dur-ingthestudy.Theroomtemperatureandhumiditycontrolswere

settomaintainenvironmentalconditionsof66±5◦F(19±3◦C)

and50±20%relativehumidity.Temperatureandrelative

humid-ityweremonitoredcontinuously.Fluorescentlightingcontrolled

bylighttimersprovidedilluminationfora12-hlight/dark

pho-toperiod.Theventilationratewassetataminimumof10roomair

changesperhour.Reverseosmosis-purifiedwaterwasavailablead

libitum.PMINutritionInternational,LLCCertifiedRabbitLabDiet®

5322wasofferedadlibitumthroughouttheacclimationandstudy

period.Periodicanalysesofthecertifiedfeedwereperformedby

themanufacturertoensurethatheavymetalsandpesticideswere

notpresent at concentrations thatwould beexpected toaffect

theoutcomeofthestudy.Intheeventofinappetence,kalewas

providedasasupplement.Clinicalsignsweremonitoredpriorto

and1-hpost-doseanddailythereafter.ConcentrationsofT-IgG4

inserumandseminalplasmaweredeterminedbyenzyme-linked

immunosorbentassays(ELISA)usingavalidatedmethod

2.1.2 BioanalyticalmethodologyforthemeasurementofT-IgG4

ELISA methods were validated that involved the capture of

humanT-IgG4frommatrix(eitherrabbitserumorrabbit

semi-nalplasma)ontohumanT-IgG4-coatedplates.Mouseanti-human

IgG-HRPconjugatewasusedtodetecttheboundT-IgG4.Forthe

rabbitserum assay,therange ofquantification wasfrom0.015

to0.96␮g/mL.Fortherabbitseminalplasmaassay,therangeof

quantitationwas0.012–0.256␮g/mL.Sampleswithconcentrations

abovethevalidationrangeweremeasuredfollowingdilution.In

rabbitserumandinrabbitseminalplasma,stabilitywas

demon-stratedforuptothreefreeze–thawcyclesandinthawedsamples

foratleast17hatambienttemperature.Long-termfreezer

stabil-itywasestablishedatapproximately−70◦Cfor372daysinrabbit

serumandfor298daysinrabbitseminalplasma.Allsampleswere

analyzedwithin thevalidatedlimits For both assays,intra-run

precision(%relative standard deviation,or%RSD) and accuracy

(%relativeerror,or%RE)was≤19%,andinter-runprecisionand

accuracywas≤17%.Datawereevaluatedandarepresentedusing

descriptivestatisticsthatincludemean,standarddeviation(S.D.)

orstandarderror(S.E.), and thenumber ofanimals(N)usedto

calculatethemean.Nocomparativestatisticswereconducted

2.2 SeminalexcretionofT-IgG4inmalerabbits FifteenmaleNZWrabbits,approximately15monthsoldand weighing approximately 3.2–4.0kg were randomly assigned to threegroupsoffiverabbits;0(vehiclecontrol),6,and100mg/kg T-IgG4 TheT-IgG4dosingsolutionswereprepared inan aque-ous diluent containing 10mM sodium citrate, 150mM sodium chloride,and0.02%polysorbate80(pH6.0)toobtaindosing con-centrationsof 0 (vehiclecontrol), 1.2mg/mL (6mg/kgdose), or

20mg/mL(100mg/kgdose).Dosesolutionswereshowntobe sta-bleatconcentrationsrangingfrom0.2to56.5mg/mLwhenstored

inpolypropylenecontainersat5◦Cforupto8daysandupto24hat roomtemperature.Thedosingformulationsusedinthesestudies werestored refrigeratedand allowed toequilibrate to approx-imately room temperature prior to dosing.Body weights were recordedonthedayofdosingonlyfordosevolumecalculations

5mL/kgofthedosingsolutionwereadministeredintravenouslyvia slowpushinjectionintothemarginalearveinoverapproximately

2min.Blood(approximately1mL)wascollectedfromthemarginal earveinortheauriculararteryandsemenwascollectedviauseof

anartificialvaginaat8,24,48,and72hafterdosing.Blood sam-pleswereallowedtoclotforapproximately30min,centrifuged

atapproximately2700rpmfor10minandserumharvestedand storedat−80◦Cuntilanalysis.Thevolumeofeachsemensample, minustheplug,wasrecordedpriortocentrifugationtoobtain semi-nalplasma.Semensampleswerevortexedforapproximately30s andthencentrifuged(approximately2700rpm)forapproximately

10minatroomtemperaturetosettleparticulatematter.The semi-nalplasmawaswithdrawnandstoredat−80◦Cuntilanalysis.The amountofT-IgG4presentinsemensamplesateachcollectiontime pointwascalculated astheseminalplasmaconcentration mul-tipliedbythesemenvolume.Followingthelastcollectiontime, rabbitswereeuthanizedbyintravenousinjectionofsodium pen-tobarbital

Toxicokineticanalysisconsistedofdeterminationofserumand seminalplasmaconcentrations ofT-IgG4, theratioofT-IgG4in serumtoseminalplasma,thetotalseminalplasmaT-IgG4from eachsemensample,thetotalseminalplasmaT-IgG4fromallfour semen samples, and the proportion of IV administered T-IgG4 recoveredinseminal plasmafromthefoursemensamples col-lectedoverthe72hpost-dosingperiod.Formulasforthecalculated endpointsarelistedbelow

Serum:seminal plasma ratio=concentration of T-IgG4 in serum/concentrationofT-IgG4inseminalplasma

TotalseminalplasmaT-IgG4ineachsemensample=seminalplasma concentrationofT-IgG4ineachejaculatesample×semenvolume Total seminal plasma T-IgG4 from all four semen sam-ples=summation of the total seminal plasma T-IgG4 from eachofthefourejaculatesamplescollectedover72h

ProportionofIVadministereddoseofT-IgG4recoveredinseminal plasma=totalseminalplasmaT-IgG4recoveredover72h/mg/kg

IVdose×kgbodyweight

2.3 Intravaginalvs.intravenousabsorptionofT-IgG4female rabbits

Eight nonpregnant female NZW rabbits, approximately 6 monthsofageweighingapproximately2.9–3.6kg,wererandomly assignedtotwo groupsof fourrabbits(6or 100mg/kg)forthe intravaginalexposureassessment.Eightadditionalnonpregnant femaleNZWrabbitsofsimilarageandweightfromacombined fertilityandembryo-fetaldevelopmentaltoxicitystudywere ran-domlyassignedtotwogroupsoffourrabbits(6or100mg/kg)for theintravenousexposureassessment.Bodyweightswereobtained

onDays0and 2fortheintravaginaldosedanimalsandDays0

solu-tionwasprepared atappropriate concentrations ina matrix of

10mMsodiumcitrate,150mMNaCl,and0.02%Tween®80atapH

ofapproximately6.0.Forintravaginaladministration,theT-IgG4

solutionwassubsequently mixedwitha vehiclegel(0.30%

car-bopol974P,1.00%methylcellulose(4000cps),5.00%glycerin;0.10%

methylparaben;0.05%propylparaben;and93.55%purifiedwater

forinjection)ataratioofapproximately55:45(T-IgG4solution:gel)

toachievetheappropriateconcentrationnecessarytodeliverthe

targeteddoseona mg/kgmeanbody weightbasis usinga dose

volumeof4mL/rabbit.Thegelmatrix wasemployedtoslightly

increasetheviscosityofthedosingsolutioninordertoprevent

lossordrainagefromthevaginafollowingdosing.Theratioof

T-IgG4solutiontogelremainedconstantacrossthetwodosegroups

ThefinalT-IgG4dosingformulationremainedasasolutionandwas

administeredintravaginallyasasingledoseusingarubbercatheter

atavolumeof4mLperrabbit.Fortheintravenousgroup,thedosing

solutionwasadministeredasasingledoseatavolumeof5mL/kg

viathemarginalearveinusinga2minpush

Bloodsamples(approximately1mL)werecollectedfromthe

marginalearveinorauriculararteryat8,24,and48hafterdosing

Additionalbloodsamplesweretakenfromtheintravenouslydosed

rabbitsforextendeddurationtoxicokineticsbutarenotreported

here.Bloodsampleswereprocessedandanalyzedaspreviously

described.Followingthelastbloodcollection,allrabbitsfromthe

intravaginaldosegroupswereeuthanizedwithoutnecropsy.The

rabbitsfromtheintravenousdosegroupsremainedonstudyfor

subsequentevaluation.ToxicokineticparametersforT-IgG4were

calculatedfromtheindividualrabbitserumconcentrationvalues

ateachdosagelevelandinterval

2.4 MaternalandfetalT-IgG4exposurefollowingIV

administration

Thismaternaland fetal exposurestudy waspartof a larger

combinedNZW rabbitfertility andembryo-fetaldevelopmental

toxicity study Nonpregnantrabbits approximately7–8months

of age and weighing approximately 2.9–3.9kg were randomly

assigned to each treatment group For the main fertility and

embryo-fetaldevelopmentalstudy,groupsof25rabbits/treatment

were administered 0 (vehicle control), 6, or 100mg/kg T-IgG4

weeklystarting14dayspriortomatingandcontinuingthrough

gestationday21.Anadditionalsatellitegroupof6femaleswere

administered100mg/kg T-IgG4weeklyat thesameintervalsas

themainstudyanimalsfor maternalandfetalexposure

assess-mentonGD21.AqueousdosingsolutionsofT-IgG4wereprepared

in a matrix of 10mM citrate, 150mM NaCl, and 0.02% Tween

80;pHapproximately6.0.Becausethisstudywaspartofadrug

registrationpackageconductedunderGoodLaboratoryPractice,

doseformulationswereanalyzedfor proteincontentfor

confir-mationthat thetargeteddoseformulation concentrationswere

achieved Initialdoses were administered via the marginal ear

vein at a volume of 5mL/kg via a slow push injection over a

period of approximately 2min Due to adverse reactions and

mortality in some rabbits from the rate of infusion, the dose

administrationtimewaschangedtoapproximately15minusing

aninfusionpump.Following2weeksof treatment,thefemales

werematedwithmales.Forthemainstudyphasefemales,blood

sampleswere collected prior tonecropsy on gestationday 29,

whiletheexposuresatellitefemaleshadbloodsamplescollected

from three females each at approximately 8 or 24h following

doseadministration on gestation day 21 Within 1h of

collec-tionofmaternalbloodforbothmainstudyandexposuresatellite

females,fetalbloodwascollectedfromeachviablefetusper

lit-ter Theblood volume collectedfrom alladult females at each

time point, as well as from all fetal litters wasapproximately

1.0mL Blood was collected via a marginal ear vein for adult females andfromtheumbilical veinsof thefetuses intoserum separatortubescontainingnoanticoagulant;bloodcollectedfrom the fetuses was pooled by litter All animals were euthanized viaanintravenous (adults) orsubscapular (fetuses)injectionof sodiumpentobarbitalaftercompletionoftheirrespectivestudy periods

3.1 SeminalexcretionofT-IgG4inmalerabbits DatafromtheseminalexcretionstudyarepresentedinTable1 Allrabbitssurvivedtoscheduledterminationandtherewereno abnormalclinicalfindingsnotedatthedailyexaminationsinany group.Meanbodyweightsweresimilaracrossallgroups

No measurableconcentrationsof T-IgG4(all <0.0075␮g/mL) were foundin anyof theserum samplesfrom controlanimals andthereforethecontroldatawerenottabulated Themajority (17of 20)oftheseminalplasmasamplesfromcontrolanimals had concentrations of T-IgG4 that were below the quantita-tion limit (<0.012␮g/mL) One sample had insufficientvolume

to assay, and two seminal plasma samples had concentrations less than 0.050␮g/mL Overall, thevery low T-IgG4 concentra-tions in two of the control (0mg/kg) seminal plasma samples werenotconsideredtohaveadverselyimpactedthetoxicokinetic evaluation

LowlevelsofT-IgG4wereexcretedintorabbitseminalplasma followingIVadministration.Meanseminalplasmaconcentrations

ofT-IgG4rangedfrom0.3to1.8␮g/mLinthe6mg/kggroupand from1.9to12.2␮g/mLinthe100mg/kggroup.Themeantimeto maximumconcentration(Tmax)forT-IgG4inseminalplasmawas

24hpost-doseforboththe6and100mg/kgdosegroups

Mean serum concentrations of T-IgG4 ranged from 115 to

239␮g/mLin the6mg/kg groupandfrom1746to2979␮g/mL

inthe100mg/kggroup.ThemeanTmaxforT-IgG4inserumwas

8hpost-doseforboththe6and100mg/kgdosegroups, consis-tentwiththeexpectedserumkineticsforanantibodyfollowingIV administration

Following single dose intravenous administration of 6 or

100mg/kg T-IgG4, both serum and seminal plasma exposure increasedinaslightlylessthanproportionalmannerwithdose.The meanmaximumconcentration(Cmax)ofT-IgG4inseminalplasma increasedseven-foldandserumCmaxincreased12-foldbetween thedosesof6and100mg/kg

At8hpost-dose in the6mg/kg dosegroup,seminalplasma concentrations ofT-IgG4wereeither lowor belowthe limitof quantitation(<0.012␮g/mL).At100mg/kg,the8-hpost-doseratio

of mean serum to mean seminal plasma T-IgG4 concentration was 1599 to 1 From 24 to 72h post-dose in both the 6 and

100mg/kggroups,meanseminalplasmaconcentrationsofT-IgG4 werefound tobe101-to372-fold less than meanserum con-centrations.Thehighserumtoseminalplasmaratiosatthe8-h post-dosingperiodindicatedthattherewasalagtimeinthe dis-tributionofT-IgG4intotheseminalvesiclesfollowingintravenous administration

TheT-IgG4dosedeliveredthroughsemenwascalculatedby multiplying the seminal plasma concentration by the match-ing semen volume at each time point (Table 1) The total dose delivered in semen from the four seminal plasma sam-plescollectedovera72hperiodwascalculatedtobe1.92␮gin malesadministered6mg/kgand15.04␮ginmalesadministered

100mg/kg.ThetotalT-IgG4inthefourseminalplasmasamples was4–4.5ordersofmagnitudelessthantheIVadministereddose (Table1)

a in

Rabbit weight

b (kg)

b (␮

b (mL)

a to

f Range

3.2 Intravaginalvs.intravenousabsorptionofT-IgG4female rabbits

Allrabbitssurvivedtoscheduledtermination.Therewereno clinicalfindingsnotedatthedailyexaminationsinanygroupand meanbodyweightsweresimilaracrossallgroups(datanot pre-sented)

Serum T-IgG4 exposure data following intravaginal and IV administration arepresented in Table2.Followingintravaginal administration,T-IgG4waspoorlyabsorbed.At6mg/kg intravagi-nal,serumT-IgG4wasquantifiable(0.023␮g/mL)inonlyoneof fouranimalsatasingletimepoint(24h)post-dose.Theremaining serumsamplesfromthe6mg/kgintravaginalgroupwerebelow thelimitofquantitation(0.015␮g/mL).At100mg/kgintravaginal, serumT-IgG4wasquantifiableatallthreetimepointsinoneof fouranimalsandat24and48hpost-doseinasecondanimal.The remainingsamplesfromthe100mg/kgintravaginalgroupwere belowthelimit ofquantitation Themeanserumconcentration

ofT-IgG4inthe100mg/kgintravaginalrabbitthathaddetectable concentrationsatallthreetimepointswas0.577␮g/mL.TheCmax

inthevaginallydosedrabbitsoccurred24hpost-dosing

FollowingIVadministration,allanimalsgiven6or100mg/kg haddetectableconcentrationsofT-IgG4atalltimepoints evalu-ated.ThemeanconcentrationofT-IgG4overthe48hevaluation periodintheIVdosedrabbitswas110␮g/mLatthe6mg/kgdose and 1803␮g/mL atthe 100mg/kg dose and increased approxi-matelyproportional todose Whendosenormalized, themean serumconcentrationwas18␮g/mLpermg/kgT-IgG4administered forboththe6and100mg/kggroups

TheratioofserumT-IgG4concentrationsbetweenthe intravagi-nalandIVroutesofadministrationwas≤0.00016forthe6mg/kg dosegroupand≤0.00032forthe100mg/kgdosegroup(Table2) BecausemostoftheT-IgGserumconcentrationswerebelowthe limitofquantificationfollowingintravaginaldosing,theseratios werecalculatedusingthehighestavailablemeanindividual ani-maldatafortheintravaginallydosedanimals,vs.themeanoffour animalsfollowingIVdosing.Giventhatmostoftheserumsamples fromtheintravaginalexposureswerebelowthelimitof quantita-tion,theseratiosrepresenta“worstcase”calculationandindicate systemicexposureviathevaginalrouteisatleast3.5ordersof magnitudelessthanthatobtainedfollowingintravenousexposure 3.3 MaternalandfetalT-IgG4exposurefollowingIV

administration TheT-IgG4concentrationofeachsampleddosingsolutiondid notdeviatesubstantiallyfromitstheoreticalvalue.Meanassayed concentrationsofT-IgG4inthedosingsolutionwere100%of theo-reticalvalues.FormulationsassessedforT-IgG4concentrationhad RSDvaluesof≤0.7%andwereconsideredhomogenous

Compound-relatedmortalitywasnotedinthe100mg/kggroup duringthe treatmentperiod In themain study group,2 of 25 femalesdiedduringthedosingprocessorshortlythereafter(3min post-dose),oneonGD7andoneonGD14.Intheexposure satel-litegroup,twoofsixfemalesgiven100mg/kgwerefounddead, oneonGD14andoneonGD21.Thecausesofthesedeathswere notdeterminedbutwereattributedtotreatmentwithT-IgG4.Only minorclinicalsignsofanogenitalsoilinganddecreaseddefecation werenotedinsomeanimalsgiven100mg/kg.Bodyweightswere unaffectedbytreatment(datanotpresented)

3.3.1 Gestationalday21exposureassessment

Ofthesixfemales in the100mg/kg GD21satelliteexposure assessment(threewereassignedtothe8-hpost-dosinginterval andthree wereassigned tothe24-hpost-dosinginterval),only twofemales,oneateachsamplingtime-point,wereavailablefor

Table 2

Serum T-IgG4 a concentrations following intravaginal and intravenous exposure in nonpregnant rabbits.

Dose

(mg/kg)

Hours

post-dose

Intravaginal:IV

Dose

(mg/kg)

Hours

post-dose

Intravenous route Serum T-IgG4 concentration (␮g/mL) Serum T-IgG4 (␮g/mL)

per mg/kg IV dose (dose normalized) d

a T-IgG4, experimental therapeutic humanized IgG4 monoclonal antibody; BQL, below the limit of quantitation (0.015 ␮g/mL); IV, intravenous; NA, not applicable; SD, standard deviation.

b Intravaginal:intravenous T-IgG4 serum concentration ratios were calculated using the highest intravaginal serum values and the mean IV values.

c Mean intravaginal serum T-IgG4 for animal 66259 was calculated using values of 0.015 ␮g/mL (limit of quantification) for time points with values BLQ.

d Dose normalized = concentration of T-IgG4 in serum (␮g/mL) divided by the dose administered in mg/kg.

exposureassessment(Table3).Theotherfourrabbitsdied(two

ofsix)orwerenotgravid(twoofsix).Maternalexposureswere

1236␮g/mL8-hafterdosing(Table3)and0.262␮g/mL24-hafter

dosing(datanottabulated).Therewasnoquantifiableexposure

toT-IgG4inthefetusesat8or24hpost-dosingongestationday

21inthetwoanimalsevaluated,suggestingplacentaltransferwas

eitherlimitedatthisstageofgestationortherewasinterference

fromanti-drugantibodies(ADA).Inapreviouspilotstudyitwas

determinedthatrabbitsdevelopADAsfollowingrepeateddosing

withT-IgG4(unpublisheddata)

3.3.2 Mainstudygestationday29exposureassessment

T-IgG4concentrationswereBLQinallmaternalandfetal

sam-plescollectedfromthemainstudyphaseanimalsat6mg/kgon

gestationday29,approximately192hafterthelastdose,withthe

exception ofone animal thathad a maternalserum concentra-tionof10.7␮g/mLandameanfetalconcentrationof16.1␮g/mL (Table3).The lowexposureatthe192hpost-dose timeperiod

isconsistentwithanADAresponse,albeitADAswerenot specif-icallymeasuredinthisstudy.ThemeanconcentrationofT-IgG4

in thefetuses wasapproximately 1.5× therespectivematernal concentration

At100mg/kg,serum T-IgG4wasquantifiablein15 of19 lit-ters The meanserum concentrations of T-IgG4on GD29 were

268␮g/mLin maternalanimalsand 413␮g/mL in fetuses Con-sistentwiththe6mg/kgdata,themeanconcentrationofT-IgG4

in thefetuses wasapproximately 1.5× therespectivematernal concentrations indicating that T-IgG4 readily crosses the pla-centawithslightaccumulationinthefetusestowardtheendof gestation

Placental transfer: T-IgG4 a concentrations in maternal and fetal serum.

Dose

(mg/kg)

ratio

a T-IgG4, experimental therapeutic humanized IgG4 monoclonal antibody; BLQ, below the quantification limit of 0.015 ␮g/mL.

b Only one of three females was confirmed to be gravid and survived to gestation day 21.

c Following weekly intravenous dosing, several rabbits had low or absent exposure to T-IgG4 consistent with an anti-drug antibody (ADA) response, albeit ADAs were not specifically measured.

d Of the surviving animals on gestation day 29, 16 of 17 dams and 16 of 17 matched pooled fetal samples were BQL (<0.015 ␮g/mL) or had insufficient sample.

e Mean ± standard deviation.

Table 4

Estimated fetal exposure following seminal excretion, vaginal absorption and placental transfer of a T-IgG4 a antibody in rabbits.

Vaginal:IV a route exposure ratio c

Serum T-IgG4 (␮g/mL) per mg/kg

IV dose (dose normalized) d

GD a 29 fetal:maternal T-IgG4 serum ratio e

Estimated fetal exposure from seminal transfer on

GD 29 f

Fetal exposure ratio (seminal transfer/maternal IV) g

a T-IgG4, experimental therapeutic humanized IgG4 monoclonal antibody; IV, intravenous; NA, not applicable; SD, standard deviation; GD, gestation day.

b The percent of dose excreted in semen = total seminal plasma T-IgG4 recovered in four ejaculates over 72 h/mg/kg IV dose × kg body weight of rabbit.

c Vaginal:IV exposure ratio = serum T-IgG4 concentration following a 100 mg/kg intra-vaginal dose/serum T-IgG4 concentration following 100 mg/kg IV dose.

d The dose normalized serum T-IgG4 concentration converts the dose administered on a mg/kg basis to ␮g/mL of T-IgG4 in serum This value was obtained by dividing the measured serum T-IgG4 concentration by the mg/kg dose following IV administration of T-IgG4.

e Fetal:maternal T-IgG4 serum ratio = fetal serum T-IgG4 concentration/maternal serum T-IgG4 concentration on gestation day 29 following a 100 mg/kg IV dose.

f Estimated fetal exposure from seminal transfer = 100 mg/kg IV dose to male × proportion of dose excreted in semen (0.000041) × vaginal:IV route exposure ratio (0.00032) × dose normalization factor (18) which converts IV dose equivalent to serum exposure × fetal:maternal T-IgG4 serum ratio (1.5).

g Seminal transfer:maternal IV fetal exposure ratio = estimated fetal exposure from seminal transfer on GD29 (3.5 × 10 −5 ␮g/mL) following a 100 mg/kg IV dose to the male/(maternal exposure on GD29 (1803 ␮g/mL) following a 100 mg/kg IV dose to the female × GD29 fetal:maternal T-IgG4 serum ratio (1.5)).

3.4 Estimationofpotentialfetalexposurefromexcretionof

T-IgG4insemen

Combiningthedatafromtheseminaltransfer,vaginal

absorp-tion,andplacentaltransferstudies,anestimateoffetalexposure

fromT-IgG4excretedintothesemenandtransferredtoapregnant

femalerabbitviavaginaldepositioncanbecalculated(Table4)

Malerabbitsdosedat100mg/kgIVweregiven370mgT-IgG4

basedonanaveragebodyweightof3.7kg(Table1)

100mg/kg·3.7kg=370mgor370000␮g

Theproportionofa100mg/kgIVdoseof T-IgG4excretedin

foursemensamplescollectedovera72hperiodwas4.08×10−5

(Table1)

15.09␮gT-IgG4collectedinfoursemensamples/370000␮gIV

dose=4.08×10−5or

4.08×10−3%ofT-IgG4doseeliminatedinsemenover72hperiod

TheproportionofT-IgG4absorbedfollowingvaginalexposure

wasestimatedbycalculatingthevaginalroute:IVrouteserum

con-centrationratiofollowingdosesof100mg/kg(Table2)

0.577␮g/mLvaginalroute/1803␮g/mLIVroute=3.2×10−4

Theestimateddoseabsorbedsystemicallyinthefemale

follow-ingvaginal administrationisthedoseadministeredtothemale

timesthe proportionof theadministered dosetransferred into

sementimestheproportionofdrugthatissystemicallyavailable

infemalesaftervaginalabsorption=1.31×10−6mg/kg

100mg/kg·4.08×10−5·3.2×10−4=1.31×10−6mg/kg

Becausea 1.31×10−6mg/kg systemic doseof T-IgG4 would

result in serum concentrations below the limit of detection

(0.015␮g/mL),adosenormalizationfactorwasusedtocalculate

theresultingserumexposure.Thedosenormalizationfactor

con-vertsIVdoseadministeredonamg/kgbasistoserumexposureon

a␮g/mLbasis(Table2).Foreverymg/kgdoseadministeredbyIV

administration,theserumconcentrationofT-IgG4wasestimated

tobe18␮g/mL.ThisdosenormalizedT-IgG4serumconcentration

isbasedontheassumptionthatT-IgG4concentrationsremained

fairlyconstant up to48h postdose The averageT-IgG4 serum

exposureovera48hintervalresultingfroma1.31×10−6mg/kg

intravaginaldoseofT-IgG4wascalculatedtobe2.36×10−5␮g/mL

1.31×10−6mg/kg·18␮g/mL/mg/kg=2.36×10−5␮g/mL

Thefinalfactorinestimatingfetalexposurefromseminal

trans-fer is the calculation of the fetal:maternal T-IgG4 serum ratio

(Table3).Thefetal:maternalT-IgG4serumratioonGD29at192h postdosewas1.5.Forcalculationpurposes,wehaveassumedthat theratioofmaternal/fetalsystemicexposuretoT-IgG4doesnot changefrom0to192hpost-dose.Thisisasimplification,butallows foraconservativeestimateoffetalexposurefollowingmaternal dosingofT-IgG4

Puttingit alltogether, theestimateof fetal serum exposure resultingfroma100mg/kgdoseofT-IgG4tothemale(excretion

ofT-IgG4intosemen,vaginalabsorptionofT-IgG4,andplacental transfertothefetus)iscalculatedtobe3.5×10−5␮g/mL(Table4)

100mg/kg·4.08×10−5·3.2×10−4·18␮g/mL per mg/kg dose·1.5=3.5×10−5␮g/mL

ThefetalexposureratiofollowingseminaltransferofT-IgG4 rel-ativetodirectIVdosingofthepregnantfemalewasestimatedtobe 1.3×10−8(Table4).Thisvaluewasdeterminedbydividingthe esti-matedfetalserumT-IgG4concentrationfromseminaltransferby theproductofthemeasuredmaternalGD29serumT-IgG4 concen-trationandtheGD29fetal:maternalserumT-IgG4concentration ratio

3.5×10−5␮g/mL/(1803␮g/mL·1.5)=1.3×10−8

AsreviewedbyKlemmtandScialli[2],therearethreepotential mechanismsforexposuretotheconceptusfromdrugsinsemen These transport mechanisms include: (1) absorption from the vaginaintothematernalcirculationfollowedbyplacental trans-fertotheconceptus,(2)directexposuretotheconceptusfollowing physicaltransferthroughthecervixandintotheuterinecavity,and (3)directdeliverytotheeggviaadsorptionorbindingtosperm AlthoughlargemolecularweightIgGs(150kDa)arenotthoughtto readilydiffusethroughcellularmembranesasobservedwithsmall molecularweight(<1000Da)organicmolecules,IgGsareknownto crosscellularmembranesthroughatranscytotic,FcRn-mediated transportmechanism[6,7].Lietal.[8]demonstratedthatFcRngene expressionwaspresentinepithelialcellswithinthefemalegenital tract(cervical,uterine,andvaginal)ofhumansandmiceandthat FcRnboundIgGinthesetissues.Theauthorsfurtherdemonstrated FcRnmediatedbidirectionalIgGtransportacrosshuman endome-trialmonolayersandprimaryhumangenitalepithelialcellsinvitro WhenIgGwasintroducedvaginallytowildtype(WT)and FcRn-knockout(FcRn-KO)mice,IgGwasreadilydetectedintheseraof

WTmicebutnotintheseraofFcRn-KOmice,supportingthe posi-tionthatIgGabsorptionfromthegenitaltractisFcRn-mediated andapotentialpathwayforconceptusexposurefollowing semi-naltransfer.Conversely,exposureoftheconceptustoIgGdrugs

is not consideredto representa significantconcernas cervical

mucusfiltersoutseminalplasmafromspermatozoaandactsasa

barriertolargemolecules[9,10].Furthermore,thequantityofIgG

drugpotentiallyadhering/bindingtothesurfaceofthe

spermato-zoaisexpectedtobesmallornonexistentgiventhehighlyselective

bindingcharacteristicsofmostantibodydrugs,theextremelylow

expressionofFcRninthetestes[11],andthereforeonthesurfaceof

spermatozoa,andtherelativelysmallsizeandlimitedIgGcarrying

capacityofthespermatozoa

Toassesspotentialexposurerisktotheconceptusfrom

excre-tionofaT-IgG4antibodyintosemen,systemicabsorptionfrom

thevagina,andplacentaltransfertothefetus,datafromaseries

of studies using the rabbit as a model were analyzed In the

firststudy,blood(serum)andseminalplasmasamplesforT-IgG4

concentration determination were collected from male rabbits

followingasingle6or100mg/kgIVdoseofT-IgG4.The

concen-trationsofT-IgG4inseminalplasmarangedfrom0.3(6mg/kg)

to 12.2 (100mg/kg)␮g/mL from 24 to 72h post-dosing The

serum:seminalT-IgG4ratiorangedfromapproximately100–370,

indicatingrelativelylowlevelsofT-IgG4weresecretedintosemen

Theconcentrations of T-IgG4in rabbitseminal plasma andthe

rabbitT-IgG4serum:seminalplasmaratiosweresimilartothose

reportedforendogenousIgG4inhealthyhumanvolunteers.Raux

etal.[12]reportedtotalIgG4serumandseminalsecretion

con-centrationsof 517␮g/mL and 1.6␮g/mL, respectively, resulting

inaserum:seminalsecretionsratioof323.Rauxetal.[12]also

reportedsimilarhumanserum:seminalconcentrationsratiosfor

IgG1(317:1)andIgG2(310:1),butahigherratioforIgG3(1425:1),

indicatingthatallIgGsubtypesareexcretedatrelativelylow

lev-els in semen.Similarly, other investigators have reported total

endogenousIgGserumandsemenconcentrationsthatproduceda

serum:semenratioofapproximately115innormospermaticmen

[13].TheconsistencybetweentherabbitT-IgG4andthehuman

endogenousIgG4data,alongwiththehumantotalIgGdata,

sug-gestthatthereislikelynodifferencebetweentheseminalexcretion

oftherapeuticandendogenousIgGantibodiesandthattherabbit

isausefulmodelforpredictingexcretionofT-IgGantibodiesinto

humansemen

ThetotalamountofT-IgG4excretedinfoursemensamples

col-lectedover 72hin therabbit,based onT-IgG4seminalplasma

concentrationsmultipliedbythesemenvolumes,wasestimated

tobe0.004–0.009%oftheadministereddose.Giventhe

congru-encebetweenexcretionofIgG4intorabbitand humanseminal

plasma,andtherelativelysmallvolume(1–5mL)ofsemen

ejac-ulatedinhumans,thetransferofT-IgG4drugsthroughsemenin

humanswouldalsobeexpectedtoberelativelysmall,

approxi-matelyfour-ordersofmagnitudelessthanthedoseadministered

tothemale

In the second experiment, a T-IgG4 solution was vaginally

administered to nonpregnant female rabbitsat doses of 6 and

100mg/kg Inorder tomaximizethetime theT-IgG4remained

in contactwiththe vaginalepithelium, theformulation forthe

vaginallyadministeredT-IgG4wasmodified toslightly increase

viscosityand preventitfrom quicklydrainingfrom thevagina

Followingdosing,visualobservationsweremadethatconfirmed

littletonodosingsolutionwaslost todrainage.The prolonged

retentionofthedosingsolutionrepresentsaworstcasescenarioas

anecdotalevidenceindicateshumansemenisnotreadilyretained

intravaginally.AlthoughLietal.[8]demonstratedthattherewere

FcRn-mediatedtransport mechanismsin thevagina,theresults

fromourvaginalabsorptionstudyindicatethatminimal

absorp-tionofT-IgG4occurredfollowingvaginaldosing,asmeanserum

T-IgG4concentrationsmeasuredovera48hperiodfollowing

vagi-nalabsorptionwereapproximately0.032%ofserumconcentrations

followingIVinjection.Itisacknowledgedthatthevaginallydosed

rabbitswerenonpregnantandpotentialdifferencesinvaginalFcRn concentrationsbetweenthenonpregnantandpregnantstatecould

beasourceofvariabilityforvaginalabsorption

Inthefinalplacentaltransferstudy,rabbitsdemonstratedlow placental transfer ofT-IgG4 onGD21 (belowthequantification limit),buthightransferonGD29,withGD29fetalserumT-IgG4 concentrations1.5-foldhigherthanmaternalserumT-IgG4 con-centrations.Thesmallsamplesize(onelitter)andtheinterference fromantidrugantibodiesmayhaveconfoundedtheGD21 placen-taltransferdata.Currently,itisnotknownwhethermaternalADAs affectthematernal:fetalIgG4serumconcentrationratiosinrabbits However,lowerratesofIgGplacentaltransferareknowntooccur

in rabbitsand primatesearlier in gestation.It isalso unknown whethermaternalADAimpactthematernal:fetalIgGratios Bow-manetal.[14]reportedthatrabbitfetalserumIgGconcentrations rose600-foldfromGD19toGD28,reachingorexceedingmaternal serumconcentrationsonGD28.Similarly,asreviewedbyPentsuk andvanderLaan[15],increasingratesofplacentaltransferofIgG antibodieswerereportedasgestationprogressedinhumans,with

noorverylowlevelsofIgGintheembryonic/fetalcompartment duringtheearlysecondtrimester.Asobservedwithrabbits,human latefetalandinfantIgGconcentrationsreachedorexceeded mater-nalconcentrations[15].Basedonthesimilaritiesbetweenrabbit andhumanplacentaltransferofIgGantibodiesduringthe mid-late(secondandthirdtrimesterequivalent)gestationalperiods,the rabbitappearstobeagoodmodelforassessingplacentaltransfer riskinhumansduringthefetalperiod.Thereiscurrently insuffi-cientinformationinbothrabbitsandhumanstoassesstheutility

oftherabbitmodelinpredictingearlyembryonic(firsttrimester equivalent)placentaltransferofIgGantibodiesinhumans[14] Combiningthedatafromtheseminaltransfer,vaginal absorp-tion,andplacentaltransferstudies,theestimatedexposuretothe conceptusfromtheseminaltransferofT-IgG4drugisextremely lowand T-IgG4isunlikelytobepresentintheembryo/fetusat concentrationssufficienttoproduceabiologicalresponse.Using therabbitasamodel,theamountofdrugdeliveredinsemenfrom multipleejaculatesovera72hperiodisapproximately0.00408%of thedoseadministeredtothemale.TheamountofT-IgG4absorbed fromthevaginaisalsoextremelylow,approximately0.032%ofthe vaginaldosedelivered.Usingthisdata,alongwiththedose nor-malizationfactorof18toconvertdoseadministeredonamg/kg basistobloodexposureona␮g/mL basis,andadjustingforthe fetal:maternal GD29 serum concentrationratio of 1.5-fold, the estimatedGD29fetalT-IgG4serumconcentrationresultingfrom seminaltransferfollowing a100mg/kgdosetothemalewould

beapproximately3.5×10−5␮g/mL,andapproximately1.3×10−8 timeslessthantheexposurewhenthepregnantfemalewasdosed viaIVinjection(Table4).Theseexposuremultipleswouldbe signif-icantlybelowanytherapeuticorbiologicallyactivelevelsofT-IgG4 (seediscussionbelow)

To support the position of low embryo/fetal exposure risk fromseminaltransferofT-IgG4drug,humandatawereapplied

tothemodel.Usingthemaximumdoseadministeredinclinical trials with T-IgG4, the highest serum exposures (Cmax) were

≤50␮g/mL (unpublisheddata).GiventheT-IgG4serum:seminal plasma concentration ratios of 100–370 in the rabbit, and the similarfindings withendogenousIgG/IgG4 in humans[12], the human semen concentration can be conservatively estimated

to be approximately 0.50␮g/mL (50␮g/mL×1/100) Using a maximumsemenvolumeofapproximately5mLforman[16],the dose delivered vaginally is 2.5␮g (0.5␮g/mL×5mL) Applying thevaginalabsorptionfactorof0.00032obtainedinrabbits,the dose absorbed by the female is 0.0008␮g (0.00032×2.5␮g) The 0.0008␮g vaginally absorbed dose is equivalent to a 1.14×10−8mg/kg dose using a 70kg female (0.0008␮g/70kg body weight×1mg/1000␮g) The human fetal exposure

from the maternal dose is estimated to be 3.07×10−7␮g/mL

by converting maternal systemic dose into plasma

concen-tration and adjusting for the embryo:maternal serum ratio

(1.14×10−8mg/kg×18␮g/mL/mg/kg×1.5 fetal:maternal serum

ratio) Forthis T-IgG4, theeffective humanconcentrations

pro-ducing a 20% (EC20) or 80% (EC80) inhibition of the target is

approximately 0.8␮g/mL and 12.4␮g/mL, respectively

(unpub-lished data) The dissociation constant (Kd) for the T-IgG4 and

itshumanligandis5.9×10−4␮g/mL.Theestimatedhumanfetal

serumconcentrationof3.07×10−7␮g/mLresultingfromseminal

transferof T-IgG4, when administered at thehighest proposed

clinicaldose,isatleast1000-foldlowerthantheKd andatleast

sevenordersofmagnitudelowerthatthehumanEC20.Therefore,

the estimated human fetal exposure to T-IgG4 from seminal

transferisinsufficienttoproduceabiologicresponseandwould

notrepresentameaningful humanrisk.It shouldalsobenoted

thatthis exposureestimaterepresentsaworst-casescenarioas

themostconservativevalueswereappliedintheassessmentand

placentaltransferofIgGduringthecriticalembryonic periodof

developmentis consideredtobesignificantlylessthan thelate

gestationalplacentaltransfervaluesutilizedinthecalculations

RelativelylowlevelsofT-IgG4antibodywereexcretedinto

rab-bitsemen,withconcentrationsrangingfrom100-to370-foldlower

thantheconcentrationsobservedinserum.Theserum:semen

con-centrationsratiosforT-IgG4inrabbitweresimilartoendogenous

IgG4andtotalIgGserum:semenconcentrationratiosreportedfor

humans;therefore,therabbitappearstobeagoodpredictivemodel

forassessingthepotentialfor excretionofIgG4antibodydrugs

intohumansemen.ThelowconcentrationofT-IgG4excretedinto

semencombinedwiththesmallvolumeofsemenejaculatedresults

inalowdelivereddosetothefemale,approximately4–4.5orders

ofmagnitudelowerthanthetherapeuticdoseadministeredtothe

malebyIVinjection.VaginalabsorptionofT-IgG4wasalsolow,

approximately3.5ordersofmagnitudelowerthanIV

administra-tion.TherabbitGD29fetalT-IgG4exposurefromexcretionofdrug

intothesemenofamaledosed(IV)at100mg/kgwasestimated

tobeextremelylow(3.5×10−5␮g/mL)andapproximatelyeight

ordersofmagnitudebelowthefetalexposurefollowingadirect

100mg/kgIVdosetoapregnantfemalerabbit.Applyinghuman

serumT-IgG4exposuredatatothemodel,usingthehighestapplied

humandose,theestimatedhumanfetalserumconcentrationfrom

seminaltransferofT-IgG4was3.07×10−7␮g/mL,alevelatleast

1000-foldlowerthantheKdandatleastsevenordersofmagnitude

lowerthatthehumaninvivoEC20.Thesedataindicatethat

excre-tionofthisT-IgG4intosemenwouldnotresultinanybiologically

meaningfulexposurerisktotheconceptusofanuntreatedpartner

TheauthorsareemployeesofEliLillyandCompany

Acknowledgments

TheauthorsacknowledgeWILResearchforconductthestudy and internal Eli Lilly and Company staff for review of the manuscript.ThisworkwasfundedbyEliLillyandCompany

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