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Reproductive Toxicology 28 (2009) 220–225

Contents lists available at ScienceDirect

Reproductive Toxicology

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evelopmental toxicity testing of monoclonal antibodies:n enhanced pre- and postnatal study design option

ane Stewart ∗

afety Assessment, AstraZeneca Pharmaceuticals, Macclesfield, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

r t i c l e i n f o

rticle history:eceived 12 March 2009eceived in revised form 7 April 2009ccepted 9 April 2009

a b s t r a c t

For many monoclonal antibodies (mAb), the preferred species for general and reproductive safety testingis often the cynomolgus monkey. This article describes the rationale for combining the traditional “seg-mented” embryofetal development study with the pre- and postnatal development (PPND) study into

vailable online 18 April 2009

eywords:onoclonal antibodies

ynomolgus monkeyevelopmental toxicity study designs

a single “enhanced” PPND study design in the cynomolgus monkey where a single cohort of animals isexposed throughout gestation and allowed to give birth naturally. The proposed “enhanced” PPND studydesign evaluates all the stages of the traditional two study design using fewer animals. It also assessesthe functional consequences of mid to late gestational exposure. This is of particular relevance to therisk assessment of monoclonal antibodies where fetal exposure to maternal IgG increases as pregnancyprogresses and where morphologic examination of a pre-term fetus may not be adequate to reveal the

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presence of adverse effec

. Introduction

The International Conference for Harmonisation (ICH) S5 guide-ine for the “Detection of toxicity to reproduction for medicinalroducts” [1] describes the most probable options for assessmentf medicines as a three study option focussing on (i) fertility andarly embryonic development, (ii) pre- and postnatal developmentPPND, including maternal function) and (iii) embryofetal devel-pment (EFD). This guideline codifies the traditional “segmented”pproach to reproductive toxicity testing which has been prevalentor small molecule testing of pharmaceuticals for nearly 40 years.

Inherent in using the three study option is the repetition ofaternal exposure to the test agent during organogenesis. Firstly,

he effects of maternal exposure during organogenesis on embry-fetal development are assessed directly by caesarean section athe end of gestation in an EFD study. This is considered sufficiento cover the type of inadvertent exposure that could occur during alinical trial of a small molecule where test article clearance occursapidly in hours or days. Effects on fetal development are then re-xamined in the pre- and postnatal development (PPND) study,here test article is administered from implantation throughout

estation (and lactation) and the overall effects on infant viability,rowth and function are assessed postnatally.

For many monoclonal antibodies (mAb), the preferred speciesor safety testing is often the cynomolgus monkey. The principles

∗ Tel.: +44 1625 513209.E-mail address: jane.stewart@astrazeneca.com.

890-6238/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.reprotox.2009.04.002

functional development of key target organs.© 2009 Elsevier Inc. All rights reserved.

guiding species selection for safety testing are described elsewhere[2–4]. Notwithstanding the technical issues regards undertakingstudies in pregnant monkeys, in general, the criteria requiring useof the cynomolgus for repeat dose toxicity tests (e.g. relevance ofprimary pharmacologic effect, avoidance of neutralising antibod-ies, etc.) will also apply to developmental and reproductive toxicitytests. For both ethical and financial reasons, where the cynomolgusmonkey has been chosen for reproductive safety testing, it is impor-tant to use the minimum number of animals to adequately assessthe effects on embryofetal development, pregnancy outcome andpostnatal development.

This article describes the rationale and means of combining thetraditional “segmented” EFD study with the PPND study into a sin-gle “enhanced” PPND study design.

2. Traditional embryofetal development studies—generallimitations

The traditional EFD study as described by ICHS5, requires dosingfrom implantation through to closure of the hard palate. Unfor-tunately, due to the low pregnancy rate in cynomolgus monkeys[5,6], animals are generally selected by ultrasound scanning at ges-tation day (GD) 18–20 to start on study and so there is no exposureat implantation or in the earliest stages of embryogenesis. Dosing

would then continue to the end of organogenesis at GD 50.

The critical endpoints in the traditional EFD study are the assess-ment of fetal viability, fetal growth (using fetal weight measuredat caesarean section) and morphologic assessment of the fetus toobserve malformations and anomalies.

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The difficulty of using the cynomolgus monkey as means ofcreening for dysmorphogenesis becomes apparent when con-idering that historically the typical starting group size for anmbryofetal development study is 12–16. Although these animalsill have been selected as pregnant at start of dosing, the cynomol-

us monkey has a high spontaneous abortion rate [5,6] and producesingleton fetus. Accordingly, there are typically only 10–12 fetuseser group available at caesarean section for fetal morphology exam-

nation. Given that the background incidence of malformation isstimated to be generally less than 1% [6,7], a test agent would haveo increase the incidence rate of any malformation substantivelyefore the typical study design is able to detect that dysmor-hogenic hazard.

To place the sensitivity to detect dysmorphogenesis of aynomolgus EFD study with approximately 12 evaluable fetuses perose group into context with another species with predominantlyingleton pregnancies, the new EMEA pregnancy labelling guideline8], effective from January 2009, suggests that sponsors will needata from at least 300 first trimester exposed human pregnanciesefore being able to conclude the treatment is not responsible for10 fold or more increase in the overall incidence of malforma-

ions. The EMEA based their guidance on humans having an overallncidence of malformations of 3%. In theory, given that the over-ll incidence of birth defects in cynomolgus is somewhat lower,t would require an even greater number of exposed cynomolgusregnancies to provide the equivalent statistical confidence as forumans.

Given all of the above, it is not surprising that the value of theraditional EFD study design for the risk assessment of any test agents questioned in ICH in that the guidance acknowledges that theumbers of primates used are often too low for the detection ofisk, and that primates are best used to characterise a relativelyertain toxicant rather than detect a hazard.

. Embryofetal exposure to monoclonal antibodies (mAb)

Placental transfer of antibodies in humans and nonhuman pri-ates is an active process which requires the presence of an Fc

omain on the antibody and is via placental FcRn receptors [9]. Theequirement for the Fc domain means that some antibody classese.g. IgA) or antibody fragments which do not have the FC region,re not transported across the placenta. However, the majority ofhe monoclonal antibodies (mAb) developed for therapeutic pur-oses are IgG, and in particular IgG1. The efficiency of placental IgGransport in humans varies with subclass and has been shown toe IgG1 > IgG4 > IgG3 > IgG2 [10].

Embryofetal exposure to maternal antibodies and exogenouslydministered monoclonal antibodies is not constant during gesta-ion. Prior to implantation and during the early perimplantationeriod, the actively erosive syncytiotrophoblast cells of the blas-ocyst invade the endometrial stroma creating lacunae filled with

aternal blood. At this very early stage, before either the yolkac placenta or chorioallantoic placenta is formed, it is not clearhether the inner cell mass of the embryo is exposed to maternal

ntibodies.A few weeks later in gestation, it has been shown that embryofe-

al exposure to maternal immunoglobulins during organogenesis isimited but measurable. For example, IgG is measurable in coelomicuid samples from 6 weeks of gestation in humans whereas IgM isot. This suggests that the placental transfer of IgG starts, at a low

evel, early in pregnancy [11]. In primates, after this limited expo-

ure in first trimester [12], the immunoglobulin transport into theetus increases during gestation, being greatest in third trimester13,14]. Overall this means fetal exposure to exogenously adminis-ered monoclonal antibodies is predicted to increase as pregnancyrogresses [15, submitted for publication].

logy 28 (2009) 220–225 221

4. Impact of mAb embryofetal exposure pattern on studydesign options

Monoclonal antibodies often have a prolonged half-life inhumans, allowing weekly or even monthly therapeutic administra-tion. The FcRn receptor, as well as mediating placental transport,also plays an important role in regulating antibody serum persis-tence in the serum in both cynomolgus monkeys and in humans.The FcRn appears to protect IgG from degradation, facilitating thelong half-life of this class of antibody in the serum [9]. This pro-longed half-life means that if there is inadvertent administrationto a woman in early pregnancy (perhaps before she is aware ofher pregnancy), the mAb may still be at pharmacologically relevantconcentrations in the woman after organogenesis, during the fetalgrowth and maturation phase of gestation when placental trans-port of maternal immunoglobulins into the fetus is increasing. Thissituation has been shown in nonhuman primates: high fetal expo-sure to an IgG mAb has been demonstrated at gestational day (GD)100 of pregnancy in a cynomolgus study where the administrationperiod stopped at GD 50 [16].

For small molecules with a short half-life, it is generally acceptedthat a traditional EFD study, with administration limited to theperiod of organogenesis, is sufficient to provide the basis of the riskassessment for inadvertent exposure in early pregnancy. Hence itis permitted under ICH regulations [17] to proceed into large-scaleclinical trials with women of child-bearing potential – where thereis statistical likelihood of an inadvertent pregnancy exposure – onthe basis of EFD data alone, without additional information from aPPND study. The PPND study is generally deferred to support mar-keting. However this approach of deferring the PPND study maynot be optimal for all long half-life mAbs because the informationregards the risk of causing fetal harm after organogenesis may becritical for the clinical management of the inadvertently exposedpregnant woman in large-scale Phase III trials. It is thereforebeholden on companies to consider the need for PPND informa-tion before there is long duration, large-scale exposure of fertilewomen to a mAb, whether that be in a clinical trial situation orpost-marketing. This need will vary with the disease condition.

Given that first trimester fetal exposure to mAb is likely tobe limited, dysmorphogenesis due to direct fetal exposure dur-ing organogenesis is of lesser concern than with small molecules.However, even if direct embryofetal exposure is limited, the mAbin the mother may still adversely affect the pregnancy, resultingin abortions. For example, the anti EGFR mAb panitumamab, aswell as causing the expected menstrual cycle irregularities in non-pregnant monkeys, also caused abortions (see VectibixTM productlabel). Given the existing literature information on the role of EGFRin ovarian physiology [18] and embryofetal development [19–21],the primate studies with the anti-EGFR mAb panitumamab couldbe classed as helping to characterise a relatively certain toxicant.

Where there is evidence of ovarian dysfunction, either fromthe literature or from menstrual cycle irregularities detected ina repeat dose toxicity study in primates, it would be helpful tobe able to assess the influence of mAb exposure on conception,pre-implantation and peri-implantation embryonic development.However, unfortunately, due to the low pregnancy rate in cynomol-gus monkeys, animals are not generally exposed to test materialbefore or immediately after mating. This means it is not realistic toassess the effects of an mAb on fertilisation or implantation. Instead,monkeys are selected by ultrasound scanning at GD 18–20 to starton studies which assess embryofetal development rather than the

fertility of the mother.

After organogenesis, placental IgG transport into the primatefetus is increasing. Therefore, there is concern that a mAb couldadversely affect those organ systems that are still in critical stagesof their development post-organogenesis, such as the immune

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ystem, or indeed there is concern for the effects on the functionalaturation of all the major organ systems. The “organogenesis

nly” exposure of a traditional EFD study is therefore not optimisedither for the exposure pattern that could occur in clinical use noror the developmental concerns regarding target organ growthnd maturation during the post-organogenesis period. Someompanies responded to this design dilemma by altering the EFDtudy design to prolong the administration of the mAb into theetal period and to delay the caesarean section from GD 100 to GD50 (i.e. approximately 10 days before term).

The pre- and postnatal study design described in ICHS5 designequires that the mother is dosed from implantation through lacta-ion. This means the offspring may be exposed in utero and throughuckling. However, it is acknowledged that in humans, the pre-ominant Ig class present in milk is secretory IgA and that the IgGomponent in milk is minimal [22]. Although the exact amount ofaternal IgG which is absorbed from the infant gut is not known,

verall it is accepted that in utero exposure accounts for the vastajority of the maternal IgG present in the neonate (illustrated in

uman case study of maternal thyrotropin receptor blocking anti-odies [23]). This is believed to be true of the cynomolgus also [12]hich means that there is little scientific rationale for extending

he dosing of an IgG mAb to the mother into the lactation phase asmeans of detecting direct adverse effects of the mAb on the devel-pment of the infant, as opposed to detection of effects on maternalunction.

. “Enhanced” pre- and postnatal study design

The ICH S5a guideline emphasises that alternate strategies coulde more valid according to circumstances, and the key factor is tollow direct or indirect evaluation of all stages of the reproductiverocess. The differences in maternal clearance and timing of fetalxposure to antibodies compared to small molecules provide thecientific rationale for examining alternatives to traditional EFD andPND designs.

The anti-TNF� mAb golimumab has been assessed in a seg-ented manner where the first study exposed cynomolgus fromD 20 to GD 50 and the second study (a peri- and postnatal study)xposed from GD 50 through birth to lactation Day 33 [16]. This isot the exact replicate of the ICH S5 small molecule testing regime

n rodents in that the PPND design described in ICH expects toave exposure throughout gestation, including organogenesis. Theexpected” ICH exposure regime for cynomolgus PPND study woulde from GD 20 through to lactation.

In the golimumab example [16], there were 12–14 cynomolgusonkeys per group in the EFD study and a further 12 monkeys per

roup in the peri- and postnatal study phase. In the EFD study, thereas no effect on pregnancy maintenance and at cesarean sectionn GD 100–103, there were no morphologic abnormalities detectedn the fetuses. In the peri- and postnatal phase there were a total ofstillbirths, none of which had morphologic abnormalities. Indeed

he critical purpose of the peri- and postnatal experiment was noto assess morphologic effects but to assess the effect of in utero andostnatal exposure to golimumab on T-cell and B-cell populationsnd immune response to antigen challenge in the infant.

Building on the success of the golimumab experiments, whichhowed that exposure throughout gestation did not affect matu-ation of the key target organ of concern (the immune system) inhe infant, an obvious adaptation would be to expose a single set of

nimals throughout gestation from GD 20, allow them to give birthaturally, and then assess infant viability, growth and morphol-gy postnatally. The maturation of key target organs, could then bessessed in the monkey infants using in vivo functional endpointsnd terminal morphologic/histologic endpoints.

logy 28 (2009) 220–225

Instead of running separate EFD and PPND studies, which require2 sets of pregnant animals to be exposed to test mAb, the mor-phology and viability endpoints of the EFD study can be assessedthrough a combination of methods within an “enhanced” PPND(ePPND) study. Initially this enhancement would be through ultra-sound examination to assess fetal viability and growth at specificpoints during gestation, then through detailed external visualinspection of the infant post birth, including basic morphometricmeasurements of limb length, head and chest circumference. Thiswould be supplemented by quantitative measures of infant growthduring the postnatal phase plus detailed skeletal inspection usingradiographic assessment performed either at termination or insedated infants at a pre-determined time postnatally. Examinationof visceral organ morphology would be performed at terminationof the infant.

Many laboratories performing EFD studies in cynomolgus mon-keys would routinely perform ultrasound examinations duringgestation to ascertain that the pregnancy is ongoing. At some facil-ities these would be carried out every 2 weeks and the monkeywould be sedated for each pregnancy scan. In addition to ascer-taining pregnancy status, just like in humans, it is possible to takequantitative measures of fetal growth using ultrasonography [24].However, obtaining the precise fetal images required to quantitatefetal growth takes longer than a simple scan to ascertain the pres-ence of a fetus with a heartbeat and so the procedure under sedationwill take longer which adds to the experimental burden on the ani-mal. On the basis that the duration and frequency of sedation shouldbe minimised in gestation to minimise the potential for disruptionto the pregnant animal, sponsors should consider the true scientificneed for frequent measurements of fetal growth in the monkey. Inutero growth is not assessed in any other species used for develop-mental toxicity testing; it is accepted that either the birth weightor the fetal weight at caesarean section is an adequate indicator ofsignificant prior changes in fetal growth. Based on the acceptabilityof the designs for other species, it may be reasonable to limit thequantitative measures of fetal growth by ultrasonography to 2 or 3occasions during gestation (e.g. D50, D90 and D130) with the actualbirth weights following at about D160.

This ePPND design allows visualisation of the postnatal morpho-logic consequences of gestational exposure in conjunction with thefunctional consequence in the same individual animal. So althougha caesarean section is not performed to assess fetal morphology,every F1 animal, including late stage abortions and stillbirths couldstill be assessed for morphologic abnormalities. This is possiblebecause, unlike rodents, cynomolgus monkeys do not eat theirstillborn offspring, leaving the cadaver available for morphologicexamination.

The live born infants would enter the postnatal phase of thestudy. The duration of the postnatal phase would vary dependingon the issues of particular concern for the project. Where overallinfant viability and growth is the primary concern, the postnatalphase may be only 1–2 months, covering the critical 1st postna-tal week when infant mortality is at its highest [6]. However, someadditional “functional” tests, tailored to meet the specific needs ofthe project, may require extension of the postnatal phase to the ear-liest time feasible to assess that specific functionality. This includesimmunophenotyping and functional assessment of the immunesystem (depending on the endpoint, starting from approximately 4months) or assessment of infant behaviour, learning and memory(unlikely to start before 6 months of age). These design features areillustrated in Fig. 1. It is anticipated that the majority of the studies

will involve the termination of the infants allowing full assessmentof skeletal morphology by radiography, visceral morphology, as wellas weights and histopathology of organs of interest.

Successful adaptation of the postnatal phase endpoints of aPPND study to investigate target organs of concern is well illus-

J. Stewart / Reproductive Toxicology 28 (2009) 220–225 223

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ig. 1. Enhanced pre- and postnatal development study design for the cynomolgusrgan concerns, e.g. ontogeny of immune system, CNS development, etc. Items inDAR, T-cell dependent antibody response; NK, natural killer.

rated in the cynomolgus study performed to assess the oxytocineceptor antagonist barusiban [25]. Barusiban is not a mAb but aynthetic peptide being developed for the treatment of pre-termabour. However the approach is of interest regards the under-ying rationale for the postnatal phase design. Given the likelyxposure in pre- and postnatal human infants, the antagonist wasdministered from GD 85 right through gestation to mimic theongest potential human exposure. There were concerns about theossible effects of oxytocin receptor antagonists on maternal and

nfant behaviour plus development of various organ systems suchs the kidney. This was addressed firstly by allowing the animalso give birth naturally and assess infant viability, and then byaving an extended 9 month postnatal phase in which there weretandard endpoints (such as clinical chemistry, ophthalmology andrgan histopathology) plus specific endpoints added in to addresspecific concerns, e.g. urinalysis, immune function endpoints andehaviour assessment. Because of specific concerns regards the

nfluence of oxytocin receptor antagonists on maternal behaviour,he postnatal assessment included a detailed study involving notnly assessment of the infant, but also video recording of theother–infant interaction on postnatal days 90–100.When using an ePPND design, exposure monitoring in the infant

t delivery then intermittently during the postnatal phase is rec-mmended: mAb exposure in infants whose mothers have beenosed in late gestation can be substantial and prolonged [16]. Thisxposure monitoring of the infant, coupled to “traditional” generaloxicity study endpoints such as clinical chemistry, haematologynd major organ histopathology in the infant, permits a more robustisk assessment for gestational and neonatal exposure than theriginal rodent PPND study designs which tended to focus on mea-uring concentrations of test agent in milk rather than measuringhe resultant exposure in the suckling infant. Measuring exposure inhe monkey infant may be very useful for those indications whereediatric use is anticipated in human infants. The data from thePPND study can then help provide a bridge to possible futuretudies with direct dosing of juvenile monkeys.

With most cynomolgus PPND designs for small molecules, its standard practice to determine the concentration of test agent

n the mothers milk. The excretion of immunoglobulins in human

ilk and the absorption in the infant are governed by the classf Ig and the age of the infant. It is therefore possible to predictilk excretion and pattern of absorption from the gastrointesti-

al tract with a certainty that is not possible with small molecules

ey. Postnatal phase duration and endpoints are designed to address specific targetare those endpoints traditionally assessed in an embryofetal development study.

[22,26]. There is limited excretion of IgG in human milk and lim-ited absorption of IgG from the infant human GI tract. SecretoryIgA is the predominant immunoglobulin excreted in human milkwhere it is believed to have a major role in protecting infants frominfection by pathogens having a mucosal portal of entry [22]. Incases where maternal IgG has been detected in the human neonateand associated with an adverse effect, it has been concluded thatthe majority of the exposure was from placental exposure and notfrom milk [23]. In this example, maternal thyrotrophin receptor-blocking antibodies caused a transient congenital hypothyroidismand delayed thyroid development in the human infant. Althoughthis human example is of endogenous IgG and not an exogenousmAB, it is a good example of a postnatal functional consequence inthe infant arising from exposure to maternal IgG which started inutero but persisted postnatally.

In the cynomolgus, there does not appear to be much datadirectly quantitating IgG secretion in milk and its subsequentuptake from the gastrointestinal tract in the suckling infant using anexperimental design that excludes possibility of previous in uteroexposure. Older studies [27,28] describing the timecourse and con-centrations of different Ig subtypes in primate infants conclude thatprimates are like humans in that most IgG in the primate neonate isfrom in utero exposure. However, these experiments do not defini-tively quantitate the relative amounts of IgG from in utero versuscolostral exposure. Where sponsors have measured therapeutic IgGmAb in cynomolgus milk, the concentration may be several logorders lower than in serum: golimumab concentration in milk wasapproximately one thousandth that in serum [16]. Given the low IgGconcentrations in milk and the low uptake by the suckling infant,it could be argued that it is scientifically unnecessary to continueadministration of the mAb to the mother beyond parturition andunnecessary to analyze milk for its mAb titre. Removal of maternaldosing procedures and milk collection procedures from the post-natal phase of the ePPND design would be a helpful simplificationthat reduces the experimental burden on the mother and allowsthe experiment to focus primarily on the assessment of postnataldevelopment of the monkey infant.

6. Future considerations regards acceptability of theenhanced pre- and postnatal development study design

A previous criticism of developmental and reproductive toxicitytesting in cynomolgus in monkeys is that the group size employed

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ay not be sufficient to adequately determine if there has beenn effect on pregnancy outcome. Adequacy of group size will bef critical importance for the success of the ePPND study designn that the design will have to accommodate two known peri-ds of F1 losses: firstly the early abortions (before GD 50) andecondly the periparturient losses and stillbirths [6]. There is anngoing collaboration between AstraZeneca Pharmaceuticals andovance Laboratories examining the background data on pregnancyutcome in the cynomolgus monkey with the aim of providing aetailed statistical exploration of the influence of starting group sizen the power of experiments to detect treatment related pregnancyosses above the background level.

It is likely that this statistical examination, which will takeccount of the variability in a large database of control monkeyregnancies, will recommend larger group sizes than are currentlysed for a standard EFD. However, an ePPND design would be theole pivotal study and would obviate the need for a standaloneFD study and so is expected to reduce rather than increase over-ll animal numbers. It is noteworthy that the golimumab exampleescribed previously [16] used in excess of 24 monkeys per groupcross the two separate studies. PPND groups of a similar size to theombined total for golimumab may be more acceptable to spon-ors if it is possible to reduce the number of groups on test. Whereotent mAb are being studied which produce measurable and mon-

torable pharmacodynamic effects in the adult cynomolgus monkeynd in humans, is it really essential to explore a dose response usingultiple dose groups, or would a single test group and one con-

rol group suffice? Is it essential to determine a no effect level oran the ePPND study serve purely to identify adverse effects onregnancy outcome related to the (pharmacologic) response to theelective mAb? By selecting a single relatively high dose, which ade-uately exceeds all likely therapeutic exposures, it may be possibleo confirm the presence or absence of adverse effects on pregnancyutcome using a single treated group of monkeys. If fewer doseroups were used, the overall number of monkeys required for anPPND design is unlikely to exceed that used for a traditional EFDulti-group design.There are concerns that the ePPND will not be acceptable

rom a regulatory standpoint because there is no morphologicxamination of fetuses. The ICHS5 guidance describes a combinedFD/PPPND option but stipulates that a cohort of pregnant animalshould be available for caesarean section and fetal examination.owever, this text was written for rodent studies where stillbornr non-viable offspring are likely to be cannibalised and lost toetailed morphologic examination. This would not be the case forynomolgus stillborns. Ths ICH S5 guideline also clearly allowsponsors to adapt designs to meet the scientific needs of the testaterial. The pattern of fetal exposure to immunoglobulin is strong

ustification for modification of the traditional designs.The barusiban study [25], cited in the previous section as a

uccessful adaptation of a PPND type study, also included bloodressure measurement and ECG recordings in the infants. The sci-ntific rationale for this detailed cardiovascular assessment is notxplained in that publication. While not doubting there was a soundcientific rationale for undertaking the cardiovascular investiga-ions in this study, their inclusion serves as a reminder that forhese PPND designs to gain in general acceptance among stakehold-rs, it is important not to overcomplicate the postnatal phase withndpoints which create a procedural burden on the animals with-ut significantly adding to the risk assessment. This will requireesigning the in vivo postnatal phase to include sufficient but not

xcessive provision of baseline data (e.g. infant growth parameters,imited timepoint exposure monitoring, hematology and clinicalhemistry) plus focussed assessment of identified key target organs.ust because a particular endpoint can be feasibly assessed duringhe in vivo phase in the monkey infant does not in itself justify its

logy 28 (2009) 220–225

inclusion in the design. Unlike rodent studies, the number of infantsavailable in the postnatal phase of a typical NHP study does notpermit division of the F1 population into different cohorts to allowassessment of different postnatal endpoints. So every procedure inan ePPND study is likely to be a procedural burden on every motherand infant within the experiment. Given that infant exposure to themAb is likely to be highest in the first couple of months postnatally,the assessments made during the postnatal in vivo phase need tobe sufficient to ascertain there has not been covert but serious dys-function in major body organ systems. But the most important invivo endpoints remains the growth and well being of the primateinfant: building in invasive monitoring or stressful procedures intothe postnatal phase of the design to ascertain normality in organsystems that have not been specifically identified as target organs ofconcern should be scientifically justified to avoid overcomplicatingthe design and overburdening the infant.

Finally, some sponsors are concerned that the ePPND studydesign is too long in duration for standard drug development time-lines where the information may be needed prior to recruitingWOCBP onto early clinical trials. The traditional EFD study wouldhave cesarean section at approximately GD 100 giving readoutof fetal weight and viability on the day of necropsy. The subse-quent detailed fetal morphometry data would accrue over the nextfew weeks and months as the specimens are examined. With theenhanced ePPND study, the critical data, which broadly equates tothe EFD data, is the infant birth weight, infant external appear-ance and the infant survival up to day 7 postpartum. With anaverage gestational length of approximately 160 days [6], this databecomes available 2 months after the traditional EFD study design.For those companies that have already moved to using the modifiedEFD study design where mAb is administered throughout gestationwith caesarean section on GD 150, the difference in time taken toacquire the critical data (e.g. fetal viability on GD 150 gestation ver-sus infant survival on day 7 postpartum) is only a few weeks. Thistimeline difference is small in comparison to the scientific and ethi-cal advantage of the design. With prior agreement of the monitoringauthority, it may be acceptable to proceed into small-scale clinicaltrials based on an interim report including the critical data up toand including the infant birth weight plus Day 7 viability and thencomplete the entire study in time for larger, long duration clinicaltrials or as a marketing commitment.

7. Conclusion

The ePPND study design evaluates all the stages of the tradi-tional two study design using fewer animals. It also assesses thefunctional consequences of mid to late gestational exposure whichis of particular relevance to the risk assessment of monoclonal anti-bodies.

Conflict of interest statement

I work for AstraZeneca Pharmaceuticals. AstraZeneca owns asubsidiary which develops monoclonal antibodies and undertakesdevelopmental toxicity studies.

Acknowledgements

The author is grateful to Jennifer Sims, Gerhard Weinbauer andDerek Newall for helpful discussions on primate developmentaltoxicity study designs.

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