Review

NUCLEIC ACID THERAPEUTICS Volume 24, Number 5, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/nat.2014.0490

Considerations for Assessment of Reproductive and Developmental Toxicity of Oligonucleotide-Based Therapeutics Joy Cavagnaro,1 Cindy Berman,2 Doug Kornbrust,3 Tacey White,4 Sarah Campion,5 and Scott Henry 6

This white paper summarizes the current consensus of the Reproductive Subcommittee of the Oligonucleotide Safety Working Group on strategies to assess potential reproductive and/or developmental toxicities of therapeutic oligonucleotides (ONs). The unique product characteristics of ONs require considerations when planning developmental and reproductive toxicology studies, including (a) chemical characteristics, (b) assessment of intended and unintended mechanism of action, and (c) the optimal exposure, including dosing regimen. Because experience across the various classes of ONs as defined by their chemical backbone is relatively limited, best practices cannot be defined. Rather, points to consider are provided to help in the design of science-based reproductive safety evaluation programs based upon product attributes.

Introduction

T

he basic principles of toxicology are applicable across all pharmacotherapeutic product classes; however, the rational basis for development of a specific toxicology program strategy is dependent on the specific attributes of the product. Specific product attributes are inherent in chemically synthesized small molecules or new chemical entities (NCEs) (drugs; pharmaceuticals) and biologically derived large molecules or new biological entities (NBEs) (biologics; biopharmaceuticals) (Table 1). The difference between pharmaceuticals and biopharmaceuticals can be viewed as a product continuum based upon size and complexity of molecular structure. Oligonucleotide-based therapeutics have attributes of both NCEs and NBEs [1] (Table 1). The safety assessment of oligonucleotides (ONs) has historically adhered to the nonclinical safety guidelines for small molecules because they are chemically synthesized and typically contain chemical moieties not naturally occurring in biological systems [2]. However, certain ON product attributes are more similar to those of biologics, including species specificity, structural similarities to endogenous molecules (i.e., DNA and/or RNA), and longer half-lives and/or pharmacodynamic effects. Therefore, the reproductive safety assessment considerations for ONs outlined in this document incorporate

concepts provided in the current guidelines for the assessment of both pharmaceuticals and biopharmaceuticals, based on the unique product attributes of ONs. The focus on product attributes has been the key principle supporting the ‘‘case-bycase’’ approach to the preclinical safety assessment of biopharmaceuticals [1,3,4] and is the recommended approach to the assessment of developmental and reproductive toxicology (DART) for ONs [5,6]. To date, the experience with the reproductive assessment has been obtained primarily with single-stranded phosphorothioate (PS)-containing ONs ranging from 18 to 22 nucleotides, with some containing alkoxy modifications on the 2¢ position of the ribose backbone, in simple saline solutions. Hence, the findings can be attributed to the ON structure. However, the principles discussed herein should apply equally to ON drug products containing delivery formulations or conjugate/linkers. In all cases, testing of the drug product is recommended. General Design of Reproductive Toxicity Studies

Reproductive and developmental toxicity studies are conducted to support enrollment of women of childbearing potential (WOCBP) into clinical trials, to support marketing applications, and to provide guidance to clinicians when prescribing to and counselling WOCBP and pregnant women

1

Access BIO, Boyce, Virginia. Berman Consulting, Wayland, Massachusetts. 3 Preclinisight, Reno, Nevada. 4 x E ponent, Inc., Philadelphia, Pennsylvania. 5 Pfizer, Inc., Groton, Connecticut. 6 Isis Pharmaceuticals, Inc., Carlsbad, California. 2

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Table 1. Comparison of Product Attributes Across Product Classes

Molecular weight Manufacture Structure Tissue distribution PK/ADME Species specificity Off-target toxicity

NCEs

ONs

NBEs

Low ( < 1kD) Chemical synthesis Single entity - Intra- and extracellular - Wide distribution - Species-specific metabolites - Short half life Less likely Often

Mid ( > 6 kD)a Chemical synthesis Single entity - Intra- and extracellular - Selected distribution - Catabolized to nucleotides - Long acting More likely Sometimes

High ( > 30 kDal) Biologically derived Heterogeneous - Largely extracellular - Limited distribution - Catabolized to amino acids - Long half life Often Rarely

Adapted from Schubert et al. 2012 [46]. NBEs, new biological entities; NCEs, new chemical entities; ONs, oligonucleotides.

(pre-and post-marketing). Studies to understand risks in case of unanticipated exposures during pregnancy are conducted either during product development or after approval. Studies are also conducted to assess potential effects on male and female fertility. The current guidelines for the evaluation of reproductive and developmental toxicity of pharmaceuticals are described in the International Conference on Harmonisation (ICH) S5 (R2) Guideline [7]. This guidance recommends that all aspects of the reproductive cycle be evaluated from gamete development through sexual maturation of the offspring. The complete cycle is divided into six subdivisions, and considerable flexibility is given for how testing should be accomplished (Fig. 1). The fertility and early embryonic development (F/EED) study (section 4.1.1, or segment 1 of ICH Guidline S5 [R2])

(Fig. 1) is typically conducted in rodents and is designed to evaluate reproductive function and early development of the embryo through implantation [8]. The drug is administered to adult males and females prior to conception and during the mating period, and to females through implantation of the embryo. Effects of the drug on estrous cycling are evaluated prior to mating as a measure of hormonal function, and the embryos are removed by caesarean section at mid gestation to assess ovulation, pregnancy rate, and implantation of fertilized eggs into the uterus. Females are exposed for 2 weeks prior to conception, whereas males are generally used for mating after 4 weeks of treatment. While in most cases males are dosed for only 4 weeks prior to mating, dosing continues through mating and through a full spermatogenic cycle of 9– 10 weeks, so that sperm assessments and/or histopathology can be conducted at the end of the exposure period. This

FIG. 1. Overview of reproduction toxicity evaluations described in ICH Guideline S5(R2): Detection of Toxicity to Reproduction for Medicinal Products and Toxicity to Male Fertility. A–F denote the stages in the complete reproductive life cycle from conception in one generation through conception in the following generation, as described in the guideline. The study types shown are standard study types described in the guideline as ‘‘the most probable option’’ for ensuring that all phases of the life cycle are evaluated and include studies for fertility and early embryonic development (section 4.1.1), embryo-fetal development (section 4.1.3) and pre- and postnatal development, including maternal function (section 4.1.2). Species listed are the typical species for each study type. Nonhuman primate (NHP) is shown in parentheses as one of several acceptable alternative species.

OSWG DART CONSIDERATIONS

study can be considered the functional complement to the evaluation of male and female reproductive tract in general repeat-dose toxicity studies. The embryo-fetal development (EFD) study (4.1.3 or segment 2) (Fig. 1) is traditionally conducted in one rodent species and one non-rodent species (typically the rabbit). The drug is administered to pregnant females from the day of implantation through the period of major organogenesis (equivalent to the first trimester in primates), and Caesarean sections are conducted near the end of gestation. The main endpoints are survival, growth, and morphology of the fetuses, as assessed by examination of their external, visceral, and skeletal features. Additionally, the study is intended to assess the maternal maintenance of pregnancy. Wise et al. [9] provide a more detailed description of these study designs. The pre- and postnatal development (PPND) study (4.1.2 or segment 3) (Fig. 1) is generally conducted in rodents and is designed to evaluate the postnatal functional consequences of in utero and lactational exposure [10].This study also evaluates the ability of the mother to carry a pregnancy to term, give birth, and properly rear offspring to weaning. The drug is administered to pregnant females from the time of implantation of the embryo, through parturition, and throughout the lactational period. A subset of pups from each litter is selected to continue into adulthood without further drug exposure and is tested for effects on learning and memory, reflex development, motor activity, and reproductive performance. As outlined in ICH S6(R1) [11] and highlighted in Table 1, the toxicity of biopharmaceuticals is assumed to be related to exaggerated pharmacology rather than chemical structure, so it is important to test for reproductive toxicity in a pharmacologically active species. Due to species specificity limitations, this requirement sometimes necessitates that studies be conducted in the nonhuman primate (NHP) [12]. Both stand-alone EFD and PPND studies can be conducted, but these separate studies require the use of a large number of primates. To optimize NHP usage, a combined enhanced pre- and postnatal development study (ePPND) has been recommended for biopharmaceuticals [13]. This study does not include fertility assessment, which is impractical in NHPs; therefore, if needed, assessment of reproductive toxicity potential is typically evaluated by histopathologic examination of reproductive organs as part of the general toxicity studies of at least 3 months’ duration. Publications [13–16] provide a more detailed discussion of the evaluation of reproductive toxicity in NHPs. General Guidance on the Timing of Studies to Support Clinical Development

The timing of DART studies relative to clinical development is dependent upon the design of the specific program and the patient population. General guidelines for the timing of DART testing as described in ICH M3(R2) [17] are applicable to ONs. EFD and fertility and early embryo development (F/EED) studies have traditionally been conducted to support enrollment of WOCBP into early clinical trials, whereas PPN studies are expected to be completed to support the marketing application. Sometimes small numbers of WOCBP are used in initial phase 1 first in human trials. Recent changes as outlined in ICH M3(R2) [17] allow

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greater flexibility to enroll up to *150 WOCBP into phase 2 clinical trials of up to 3 months’ duration based on preliminary developmental toxicity data (generally dose rangefinding) and general toxicity information on reproductive organs in animals, provided that pregnancy in patients is carefully controlled. Under this provision, definitive EFD and FEED studies would be conducted to support larger clinical trials with more WOCBP and longer duration. If assessment of reproductive risk needs to be conducted in NHPs, the timing of studies is generally more flexible given the length and complexity of these studies. For biopharmaceuticals meeting these conditions (e.g., monoclonal antibodies), ePPND studies can be conducted in parallel with phase 3 clinical trials, provided that appropriate precautions are included in the protocol to control for pregnancy (typically two forms of birth control are required) and no particular concerns exist for reproductive or developmental effects based on the mechanism of action [ICH S6(R1)] [11]. If these requirements cannot be met, submission of an EFD NHP study or an interim report from the ePPND study might be necessary prior to enrollment of WOCBP into phase 3 clinical trials. For ONs that are anticancer therapeutics for treatment of patients with advanced disease or no other therapeutic options, the extent and timing of reproductive toxicity testing is outlined in ICH S9 [18]. This guideline is focused on therapeutics for imminently life-threatening conditions and not for therapies that will be given prophylactically or as a maintenance dose. Most cancer therapeutics are assumed to be overtly toxic to dividing cells, so the risk of developmental toxicity is considered high. Therefore, the expectation is that cancer patients will be under careful control of pregnancy, and that informed consent will be given that takes into account the specific developmental toxicity risks of the molecule based on its mode of action or assumes worst case for describing potential risk. Because developmental toxicity is expected with many anticancer therapeutics, only a single confirmatory EFD study in one species is required (generally the rodent). A dose range-finding study in a single species can also be used to justify not conducting any additional reproductive or developmental toxicity studies. An EFD study in a second species (generally rabbit) would only be required if developmental toxicity is not observed in the first species. FEED and PPND studies are not required for anticancer therapeutics. Considerations Based upon Product Attributes of Oligonucleotide Drugs

For ONs, a number of distinct structural subclasses and different biological mechanisms exist that may require specific adaptation of reproductive study designs. Structural subclasses include platforms based on single-stranded DNA and double-stranded RNA (dsRNA) with varying degrees of PS, 2¢-O-alkyl, 2¢-fluoro, or other modifications [19]. Modifications can be made to the ribose sugar, such as with synthetic morpholinos, or to the interunit linkages by substitution with PS or phosphorodiamidate moieties. These differences in chemical structure can have significant impact on the pharmacokinetics (PK), metabolic stability, nonspecific interactions, and the possible need for sophisticated formulations. The primary mechanisms of action that rely on

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interaction with nucleic acid targets range from antisense inhibitors (involving Watson–Crick pairing with an mRNA target and subsequent degradation of the double-stranded target, usually mediated by RNaseH), RNA interference, which also relies on hybridization of a target mRNA after incorporation into the RNA-induced silencing complex (RISC), modulation of splicing of pre-RNA, or regulation of the transcriptional activity of multiple mRNAs through microRNA pathways. Alternatively, ON drugs [e.g., —C— phosphate—G—(CpG)-containing immunostimulatory ONs and aptamers] can exert their pharmacologic effect through interaction with specific proteins. An example of where the considerations will be unique to specific applications include ONs with CpG motifs that are intended to stimulate an immune response as compared with other chemical modifications of these molecules that are intended to minimize the immune response. Thus, similar to biopharmaceuticals, a single study design is not necessarily appropriate for all ON subclasses. Instead, the product attributes of each individual subclass of ONs will dictate the scientifically valid approach. The specific role of the pharmacologic target as it potentially relates to reproductive function and the potential for exaggerated pharmacologic effects on pregnancy or fetal development should be considered carefully in the design of the reproductive toxicity assessment [12]. For most ONs, this assessment would generally focus on the impact of decreased expression or function of the targeted protein. For microRNAs, the focus might be on the impact of increased expression of a family of RNAs. Therefore, if the pharmacological target for the ON is not well conserved between human and the standard species used for reproductive testing, assessment can prove challenging. It is helpful if the function of the specific target is known and there is literature on reproductive functions in related knockout or transgenic mouse models, as well as in human populations deficient in or over expressing the target. If such assessments are available, then the implication of altering function both systemically and in reproductive tissues, as well as during various stages of development, can be considered. If such information is unavailable, then other options should be considered (see section on animal-active analogues). The expression profile for the specific target should also be known in order to assess the level of concern. Typically, exposure of the testes to ONs has been low [20]. The opportunity for a direct effect on the target in the embryo or fetus may be limited if exposure of the fetus to ONs is low. For example, placental transfer of PS ONs has been shown to be very limited [21–23]; however, in some studies with PS ONs, quantifiable ON concentrations have been detected [20], as has modulation of target expression in the fetus [24,25]. Additionally, although it is not entirely clear whether ONs reach the fetus to an extent that could exert any direct fetal effects or at the proper time during development to exert an effect, pharmacology-related changes to maternal physiology could affect not only implantation of the embryo and maintenance of pregnancy, but also have the potential to cause fetal abnormalities (e.g., angiogenesis inhibitors or compounds whose pharmacologic effect leads to hypoglycaemia). Use of a pharmacologically relevant model is especially important when there are expected effects on reproduction or development.

CAVAGNARO ET AL. Selection of Animal Species

Ideally, the animal model(s) should be representative of humans with regard to PK, metabolism, sensitivity to toxicity, and pharmacologic effects. Aptamers and CpG-containing ONs that are designed to function through direct interaction with proteins typically exhibit robust activity across species and, therefore, are likely to be active in at least one of the species commonly used for EFD studies (rat and/or rabbit). In contrast, human antisense ONs are often not active in rodents and rabbits due to species differences in mRNA target sequence. In such cases, the use of an animal active analogue in reproductive toxicity studies may be warranted. Selection of the animal species should first consider whether the clinical candidate has pharmacologic activity in rodents and/or rabbits. For clinical candidates that are pharmacologically active in rodents and/or rabbits, or have targets that are not endogenous to any animal species (e.g., antimicrobial ONs), standard rodent and rabbit studies can be conducted, as is generally done for small molecules. Consistent with the recommendation of the Exaggerated Pharmacology Subcommittee of the Oligonucleotide Safety Working Group put forth for general toxicity studies [5], the use of one pharmacologically relevant species for reproductive toxicity testing should be sufficient. In those cases where the clinical candidate is active in only one of the standard species used for EFD studies (rat or rabbit), the EFD study in the second species is still warranted to thoroughly assess effects related to ON chemistry. If the clinical candidate is pharmacologically inactive in both the rodent and rabbit, studies in these standard species are still considered of value for clinical candidates whose chemical structures have not been tested previously in reproductive toxicity studies. However, when the clinical candidate lacks activity in either rats or rabbits, developers should consider other options to assess reproductive and developmental effects stemming from pharmacology, as discussed in the following paragraph and in the next section. In general, the use of NHPs for DART studies should be considered in the assessment of reproductive toxicity only for unique cases where there is a clear cause for concern and where the clinical candidate is only active in NHPs. This approach would enable the assessment of effects related to the sequence, chemistry, and pharmacodynamics (PD) of the clinical candidate. However, differences in target sequence may still limit absolute pharmacologic cross-reactivity. In addition, studies in NHPs often have limitations because animal numbers are too low for detection of risk. DART studies in NHPs are best used when the objective of the study is to characterize a relatively certain reproductive toxicant [11,26]. For example, they may be warranted in the context of an RNA target with specific reproductive concern that could not be addressed in rodents or based on a unique observation in general toxicity studies. Design and Use of Animal-Active Analogues

Use of an animal-active analogue is an option for addressing reproductive effects related to the intended pharmacology if the clinical candidate has an endogenous target but lacks homology and pharmacologic activity in either rodent or rabbit. This approach would enable the conduct of standard DART studies to evaluate the impact of the expected pharmacology on pregnancy and embryo-fetal development

OSWG DART CONSIDERATIONS

[27]. Typically, this approach follows the same expectations and strategy as for general toxicity studies, where pharmacological assessment is conducted in at least one species [5]. Most often, this assessment would be conducted in a rodent species because a mouse or rat analogue would likely have been used in the pharmacology and general toxicity studies. An advantage of the use of the rodent analogue is that all aspects of the reproductive cycle, including fertility and postnatal development, can be evaluated. In the selection of the analogue, the structure of the clinical candidate and the analogue should be as close as possible in both the chemical composition (length and modifications) and chemical class effects to facilitate interpretation of the data [5]. The use of an analogue is an acceptable strategy for ONs because it allows evaluation of effects related to both the chemical structure and the pharmacologic activity of the ON in larger numbers of animals, allowing a better characterization of risk than a corresponding smaller study in NHPs. Although animal-active analogues have the same chemical backbone as the clinical candidate, they may sometimes produce a different pattern of toxicity unrelated to target pharmacology5. Thus, when using analogues, the toxicity and toxicokinetic (TK) profile of the surrogate should be characterized and shown to be similar to the clinical candidate. The most common strategy for conducting reproductive toxicity assessments with rodent analogues has been to perform a full dose-response assessment with the clinical candidate to establish effect and no-effect dose levels related to the chemical structure, combined with a parallel satellite group that is treated with the analogue at a dose equivalent to one of the dose levels of the clinical candidate. The EFD study in the second species would only evaluate the clinical candidate. Ideally, the dose level selected for the analogue in rodents should be at a level that causes the expected PD effect or exaggerated PD effect. If the pharmacologically active ON has no effects on reproductive or developmental endpoints, then the evaluation is complete for the rodent study. If an effect on reproductive function is seen with evaluation of only a single dose level of the analogue in the rodent, more work may be needed to address the mechanism and dose response. Therefore, depending on the level of concern for any given target with regard to reproductive toxicity, a sufficient number of groups to characterize the dose-response for pharmacology-related reproductive effects with the analogue might be warranted to ensure that the PD is well characterized and to establish a no-adverse-effect level. In this case, a targeted assessment of the clinical candidate at just the highest dose level(s) might be possible. This strategy could be especially good for candidates from one of the more well characterized structural classes for which the reproductive toxicity potential of the chemical structure has already been thoroughly evaluated. Justification of Dosing Regimen

The route of administration for developmental toxicity studies generally mimics either the clinical regimen or that used in the nonclinical toxicity studies. For DART studies, the dosing regimen should be selected to achieve both a maximum tolerated maternal dose and to ensure adequate systemic exposure throughout the period of organogenesis. The latter objective is to provide an opportunity for fetal

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exposure to both the test article itself as well as its pharmacologic and toxicologic effects on the dam. For NCEs, the dosing regimen is typically once daily, which is generally consistent with their PK and PD half-lives as well as the clinical dosing frequency. However, for many ONs, achieving all objectives simultaneously can be challenging. Plasma half-lives are typically short, whereas tissue half-lives and PD half-lives tend to be quite long, and clinical dosing may be infrequent. Dose-limiting toxicities are more often related to tissue concentration than plasma concentration. On the other hand, the test species may not be pharmacologically responsive to the test article (this is often true for the rabbit) and the degree to which the test article crosses the placental barrier may be limited or unknown. Therefore, the dosing regimen for DART studies of ONs is often selected to achieve a balance between the objectives of maternal toxicity and exposure. Typical maternal toxicity includes adverse clinical observations, and changes in body weight, body weight gain, and food consumption. The goal is to balance excessive tissue accumulation with the need to dose regularly throughout organogenesis. The design should strive for plasma exposure to the chemical structure has frequently as possible especially when the chemical structure is not well characterized. For ONs whose PD requires a once-daily clinical dosing regimen, daily dosing should be used for the DART studies as well. Daily dosing should also be considered for ONs whose chemical structure is not well understood and ONs that show relatively low toxicity with repeated administration, regardless of the clinical dosing regimen. For ONs that are of a structural class that is metabolically stable and has a long tissue half-life and extended PD effects (e.g., PS ONs), where infrequent (e.g., weekly) dosing is used in the general toxicity and clinical studies, a dose range-finding study is recommended to evaluate whether daily dosing can be achieved in DART studies without eliciting excessive maternal toxicity. In cases where no toxicity occurs upon daily dosing, the highest dose could be a multiple of the highest anticipated human dose as is the case for biologics. One possibility for balancing accumulation with the need to dose regularly throughout organogenesis is to administer the same weekly dose as in the general toxicity studies but as smaller doses given on a more frequent basis (daily or several times per week) to ensure repeated exposure in the plasma compartment as frequently as possible throughout organogenesis. Additional work, such as measurement of target organ tissue concentrations, might be needed to ensure that the dosing regimen provides a similar weekly maternal tissue exposure as the single weekly dose. One drawback to a fractionated dosing scheme is that it could result in a lower maternal plasma maximum concentration (Cmax), relative to the same dose given on a weekly basis; hence, understanding the relationship between known or predicted toxicities and plasma Cmax versus tissue exposure is important. For ONs whose PD and/or toxicity is related to plasma concentration, a dosing regimen involving dose fractionation may not be advisable. In that case, alternative study designs could be considered. For example, use of multiple cohorts with staggered dosing at dose levels used in the repeateddose toxicity studies may be more appropriate; that is, each cohort receives the full dose at the clinical dosing interval to achieve the Cmax, and different cohorts are dosed on different gestation days or ranges of days to ensure dosing at each point

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during organogenesis. However, this study design is complicated and resource intensive, so this approach should be taken only if clearly warranted. Regardless of the type of ON, the objective is to achieve adequate exposure to both the pharmacodynamic effects and the chemical structure at each stage of organogenesis, and the approach should be justified. Exposure Assessment

Assessment of exposure in the reproductive toxicity studies may be warranted to enable a correlation with general toxicity studies and document the expected exposure in the study, but the optimal measure of exposure may differ for specific structural classes. In the case of stable single-stranded PS ONs, measurement of (maternal or paternal) target organ concentration (liver and/or kidney) might be the most relevant means of assessing this exposure. Within this class, the PK and tissue distribution properties are quite consistent from one sequence to another. A detailed assessment of area under the plasma concentration vs. time curve (AUC) may not be necessary if data already exist from other studies in the same species. The measurement of target organ concentration is also a useful way to compare the total exposure in the DART study to the general toxicity studies if the dose regimen used for this study was different. For other classes of ONs (such as polyethylene glycol modified, PEGylated aptamers) or those using sophisticated delivery formulations [such as liposome-formulated small interfering RNAs (siRNAs)], the exposure measure should be tailored to the specific PK properties. Depending on the nature of the ON product, the concentration of ON in placenta or fetal tissues could also be assessed. Concentrations were determined for several of the early single-stranded PS ONs because the transplacental transfer properties were not known. These studies revealed poor placental transfer with generally low concentrations measured in the placenta and less than detectable exposure to the fetus [21,22]. Therefore, pharmacological effects in these tissues are expected to be low to nonexistent for similar structural and chemical subclasses. Because of the consistent sequence-independent PK properties, such measurements may not be needed for all classes or may be determined initially for a specific chemical class. Class-Specific Considerations

Study design considerations may be specific based upon the ON subclass. For example, if a sponsor is developing several ONs with the same chemical backbone structure, information gained from testing one of the ONs in a full DART package using three dose levels in two species might be used to support the development of other chemically related ONs. In such circumstances, DART studies with subsequent closely related ONs may be limited to studies in pharmacologically active species only (or for which an active analogue is available). The ability to leverage data is particularly pertinent in the context of antisense ONs that are targeting a family of specific genetic mutations for orphan disease populations. Class-specific considerations related to single-stranded DNA antisense ONs

This class of ONs is designed to enter cells, bind mRNA, and decrease the level of protein expression through degra-

CAVAGNARO ET AL.

dation of the mRNA. Several development projects using PS ONs or 2¢-alkyl-modified ONs have advanced to phase 2 and phase 3 clinical trials and have required assessment of reproductive toxicity (see Table 2). To date, segment 1 and segment 2 studies have been conducted in mice and rats with human-specific and rodent-specific antisense ONs (12 total). segment 2 studies have also been conducted in rabbits with human-specific (11 total) and/or rabbit-specific antisense ONs on occasion. Two segment 3 studies—one for a PS ON and one for a 2¢-O-methoxy ethyl (2¢-MOE) antisense ON— have been conducted in rodents. The more proinflammatory ONs have been associated with increased rates of abortion, especially in rabbits, but generally no effects on fertility or fetal development have been observed. These studies have collectively examined the full spectrum of DART endpoints. The design of these studies has typically followed the standard protocols with some slight modifications to accommodate the drug properties of antisense ONs. Those special design features are typically related to dose regimen and the assessment of targeted pharmacology. The dose regimen often used in the general toxicity studies of the 2¢-MOE antisense ON class is once weekly. As discussed above, this dosing interval can be an issue for developmental toxicity studies in rodents and rabbits because of the short dosing period (12 to 13 days) during organogenesis, and the need to ensure exposure throughout all development windows. Therefore, weekly doses are sometimes divided for administration three times per week or every other day to ensure exposure in the blood compartment throughout the period of organogenesis. This divided dose regimen results in lower Cmax and AUC at each dose, but produces the same weekly AUC and maternal tissue concentrations. The other area that is typically adapted to suit the properties of antisense ONs is the strategy to assess the effects of targeted pharmacology. Although some mRNA targets have sequence homology between human and standard laboratory animal species, most do not. As discussed previously, in order to accommodate assessment of pharmacologic effects, studies have included testing of a surrogate antisense ON that is active in the test species (typically either mouse or rat). Surrogates have been used in a number of ways, but most often, studies are designed with a full dose-response of the human antisense ON and a satellite group treated with the active antisense ON surrogate at a dose that is associated with reduction of mRNA and pharmacologic activity in the animal model. Across the cumulative experience generated in the mouse and rabbit DART studies, no apparent impairment of fertility or pregnancy parameters and no teratogenic effects have been observed [21,22]. Also, antisense ONs or analogues have typically had no apparent effect on the offspring or postnatal endpoints evaluated. The lack of developmental toxicity is in part consistent with the observations that antisense ONs have not been shown to cross the placenta (at least in rodents and at later stages of gestation). However, there may still be risks for molecules with targets in the placenta. Increased rates of abortion may be seen with ONs of proinflammatory potential. The one common effect observed in rabbit teratology studies is a decrease in fetal body weight or premature delivery in the context of decreases in maternal body weight and food consumption. Although maternal effects are likely related to inflammatory effects at the high dose, the changes in

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Table 2. Examples of Study Designs for Antisense Oligonucleotides Chemical class PS ASO

ASO no. (target) 2302c (ICAM)

2105 (HPV) 2922 (CMV) 14803 (HCV) 3521 (PKC-a) MOE ASO

104838 (TNF-a)

Study type/ species

Dose (mg/kg/ dose) / route

Seg1/2 /Mouse 0, 3, 6, 12 / IV

Seg2/Rabbit Seg3/Mouse

0, 1, 3, 9 / IV 0, 3, 6, 12 / IV

Seg1/Rat Seg2/Rabbit Seg2/Rat Seg2/Rabbit Seg1/2 /Mouse Seg1/2 /Mouse Seg2/Rabbit Seg1/2 /Mouse Seg2/Rabbit

NA / ID NA / ID NA / ID 0, 1, 3, 9 / IV 0, 1, 3, 9 / IV 0, 3, 6, 15 / IV 0, 2, 8, 20 / IV 0, 2, 10, 20 / IV 0, 1, 3, 10 / IV

Seg1/2 /Mouse 0, 2, 8, 25 / IV

Regimen

Daily 15 days prior ISIS 3082 (3, 6, to cohabitation 12 mg/kg/day) and through GD 17 Daily GD 6–18 No Daily GD 6–22 ISIS 3082 (3, 6, 12 mg/kg/day) NA NA NA NA NA NA Daily GD 6–18 No QOD GD 0–18 No QOD GD 1–17 No QOD GD 6–18 No QOD GD 1–17 ISIS 4189 (10 mg/kg) QOD GD 7–19 No

Seg2/Rabbit Seg1/2 Mouse Seg2/Rabbit Seg3/Rat 388626a,b (SGLT2) Seg1/2 Mouse Seg2/Rabbit 329993a (CRP) Seg1/2/Rat Seg2/Rabbit 304801a (ApoCIII) Seg1/2/Mouse

0, 0, 0, 0, 0, 0, 0, 0, 0,

1, 4, 15 / IV 3, 10, 25 / SC 2.5, 5, 15 / SC 2, 10, 20 / SC 0.6, 3, 15 / SC 2.5, 5, 15 / SC 3, 10, 40 / IV 2.5, 5, 15 / SC 3, 10, 25 / SC

Every 4th day GD 0–14 QOD GD 6–18 QOD GD 0–16 QOD GD 6–18 QOD GD 6–21 QOD GD 0–15 QOD GD 6–18 QOD GD 5–19 QOD GD 6–18 QOD GD 0–15

Seg2/Rabbit Seg1/2 /Rat Seg2/Rabbit Seg1/2 /Mouse

0, 0, 0, 0,

3, 1, 1, 3,

QOD GD 6–18 QOD GD 0–19 Daily GD 6–18 QOD GD 0–15

301012 (ApoB)

113715a (PTP1b) 416858a (FXI)

6, 15 / SC 3, 8.5 / SC 2.9, 8.6 / SC 10, 25 / SC

Included rodent-specific ASO

ISIS 25302 (8 mg/kg) No ISIS 147764 ISIS 233183 ISIS 147768 No No ISIS 421985 No ISIS 440670 25 mg/kg)

(25 mg/kg) (15 mg/kg) (70 mg/kg/wk) (40 mg/kg) (3, 10,

ISIS 141925 (8.5 mg/kg) No ISIS 404171 (10, 25 mg/kg)

a

Active in monkey. Cross-species active including human, NHP, mouse, rat and rabbit. c Henry et al. 2004 [21,22]. ApoB, apolipoprotein B; ApoC, apolipoprotein C; ASO, antisense oligonucleotide; CMV, cytomegalo virus; CRP, C-reactive protein; FXI, factor XI; GD, gestation day; HCV, hepatitis C virus; HPV, human papillomavirus; ICAM, intercellular adhesion molecule; ID, intradermal; IV, intravenous; MOE, O-methoxy ethyl; PKC-a, protein kinase c-alpha; PS, phosphorothioate; PTP1b, protein tyrosine phosphatase 1B; QOD, every other day; SC, subcutaneous; Seg, segment; SGLT2, sodium/glucose transporter 2; TNF-a, tumor necrosis factor-alpha. b

reproductive parameters have been generally attributed to the decreased maternal food consumption. Class-specific considerations related to CpG ONs and immunostimulation

For CpG ONs, the mechanism of action raises concerns about potential effects on reproduction and development. CpG ONs stimulate the immune system through activation of toll-like receptor (TLR)-9 and subsequent induction of cytokines and chemokines [28]. In humans and NHPs, TLR-9 is expressed primarily in the endosomes of B cells and in plasmacytoid dendritic cells (pDC). Rodents express TLR-9 in B cells and pDCs, as well as in myeloid dendritic cells (mDCs) and monocytes. Activation of TLR-9 on pDCs leads to a rapid and massive production of interferon (IFN) by these cells, followed by differentiation to DCs and subsequent activation of T cells to produce interleukin (IL)-12, IFN-g, IL-4, IL-5 and IL-13 [29]. B cells activated through TLR9 produce IL-6 and IL-10, pDCs produce IFN-a, mDCs secrete

IL-12, and monocytes produce tumor necrosis factor (TNF)-a, IL-6, and IL-12. As a result of the broader cellular distribution of TLR9, rodents exhibit a broader spectrum of cytokines than seen in humans and NHP, which may result in over estimating the risk associated with CpG ONs. Rabbits do not show a clear pattern of CpG-oligodeoxynucleotide–dependent TLR9 activation, although they may respond to DNA nonspecifically as a polyanion. For example, incubation of rabbit B cells in vitro does not result in CpG-specific induction of cytokine expression. Because rabbits can exhibit quantitatively or quantitatively different responses to immunostimulatory (IS) ONs, as compared to rodents, performing an advance screen on rabbits may be prudent to ensure a relevant pharmacologic response to the IS ONs prior to selecting this species for use in the reproductive toxicity studies. Regardless of species, cytokine release if induced is transient, and levels return to baseline within approximately 12 hours. Unfortunately, data on CpG ONs are insufficient to determine whether they have direct effects on the fetus or

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whether they cross the placenta. However, due to their intended immunostimulatory effects, CpG ONs have been associated with adverse effects on both fertility and embryo-fetal development. Although the number of therapeutic CpG ONs evaluated for DART effects has been limited, academic research in mice has provided insight into the level of concern related to CpG ONs and TLR9 activation. Upon administration of a CpG ON (300 mg/dam) on gestational days 10 to 14, fetal resorption and/or preterm birth were induced in wild-type mice [30,31]. The absence of changes in TLR9 - / mice suggests that the effects are due to the pharmacological activity, rather than the ‘‘off-target’’ specific chemical modifications of the ON [31]. Administration of a CpG ON on gestation day (GD) 6 (300 or 400 mg), or GD 14 (300 mg) was associated with cranial and distal limb malformations [30,32]. A control ON and lower dose levels ( £ 250 mg) of the CpG ON had no adverse effect [30,32,33]. These findings of dose-related fetal loss and morphologic defects are consistent with the results of EFD studies of therapeutic CpG ONs of which the Subcommittee has knowledge. As previously mentioned, cellular distribution of TLR9 is broader in rodents than humans, and neonatal rodents appear to respond well to CpG ONs [34–38]. A plausible explanation for the reproductive effects of CpG ONs in animals is the large increase in IFN production that occurs with activation of TLR-9. High levels of IFN are known to interfere with the maintenance of pregnancy, as exemplified by the abortifacient activity reported for marketed IFN biopharmaceuticals in several species including primates [see prescribing information for Betaseron (IFN b1b), Actimmune (IFNg), Intron A (IFNa) and Avonex (IFN b1a)]. Additionally, developmental effects have been noted for Aldara, which is a pro-inflammatory adjuvant that stimulates pDCs to produce IFNs through activation of TLR7. A small number of studies have also linked IFN treatment with adverse pregnancy outcomes in clinical populations [39,40]. Some researchers have hypothesized that maternal Th1 cytokine induction could also play a role in reproductive effects with CpG ONs [30]. The innate immune system of human neonates is biased toward Th2 responses rather than the Th1 responses required for effective cell-mediated immunity. Indeed, the response of neonatal monocytes and DCs to TLR agonists differs significantly from that of adults. Specifically, CpG-induced IFN-a secretion is deficient in blood from 4-day-old neonates and the umbilical cord, as compared with adults [41]. Using cord blood and blood from infants of 3, 6, 9, and 12 months of age, Nyugen [42] showed that CpG-induced production of IFN-a–dependent chemokines increased with age but at 1 year of age was still significantly lower than in the adult controls. These findings suggest that CpG ONs will have reduced immunostimulatory effects in human neonates and, thus, carry reduced risk of adverse effects associated with pharmacological activity. However, the cellular distribution of TLR9 is broader in rodents than in humans, and neonatal rodents appear to respond well to CpG ONs [34–38]. Therefore, postnatal studies in rodents may over predict the risk associated with CpG ONs. The importance of study design for EFD studies of CpG ONs is highlighted by the dose response, the hypothesized involvement of maternal IFN and Th1 cytokine induction, the apparent species-specific sensitivity based on TLR activity and the transient nature of CpG-induced cytokine elevations.

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Sponsors should select dose levels and a dosing frequency to ensure PD effects throughout the period of major organogenesis. Infrequent dosing (as might be proposed for clinical administration) or dose fractionation (which lowers the Cmax and may decrease PD effect) may under-predict the risk of adverse effects. Therefore, separate cohorts with different dosing regimens may be needed to ensure adequate exposure at each stage of organogenesis. Although studies in rodents may over-predict risks of TLR-related reproductive or developmental toxicity, the findings with CpG ONs raise the question about potential effects related to immunostimulation via unintended TLR activation, as seen with certain proinflammatory antisense ONs. Less information is available on the potential for adverse effects on reproduction and development via unintentional activation of TLR-3, 7, and 8; however, similar considerations are recommended during the development of RNA ONs. Class-specific considerations related to siRNAs and microRNA mimetics/antagonists

At the time of the writing of this document, the Subcommittee was unaware of any reproductive toxicity assessments for these classes of ONs. These potential therapeutic ONs are similar in that they engage the cytoplasmic RISC complex responsible for processing of mRNA, but include a relatively broad array of chemical structural modifications and complex formulations used for tissue delivery. Potential similarities and differences in the approach to reproductive toxicity assessment relative to those previously discussed herein are outlined below. For microRNA antagonists, the mechanism of action is to hybridize with endogenous microRNAs and inhibit their function. These compounds are often structurally similar to traditional antisense ONs. Specifically, they are generally 12 to 20 nucleotides in length, fully modified on the 2¢-position and have PS linkages. Therefore, the PK, tissue distribution, and toxicity profile are generally similar to classes of PS ONs that have been well studied. Consequently, the principles for reproductive testing outlined for single-stranded PS ONs will apply. The sequence homology for microRNA is typically very well conserved across species, such that the human sequence would be expected to cross-react with at least one of the standard species used for embryo-fetal developmental toxicity studies. Similar considerations apply to microRNA mimetics. These ONs differ structurally from antagonists with respect to their double-stranded composition and the types of modifications employed. They are not intended to reduce gene expression but rather modulate a broad array of targets linked to the specific microRNA. However, like the antagonists, the microRNA mimetics usually exhibit robust cross-species activity and generally would not present challenges to assessing reproductive effects related to exaggerated pharmacology with the use of the standard species employed for such studies. siRNAs are also double-stranded and exert their effects via reduction in gene expression. Some of the siRNAs in development are delivered as the free ON (unformulated), in which case they may be chemically modified. These applications are typically limited to local delivery (e.g., intraocular

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or intrapulmonary) and would have limited distribution to reproductive tissues. However, in the majority of the current siRNA development programs, particularly those involving systemic delivery, the siRNA is delivered via a complex formulation, and the ON chemistry is generally natural (unmodified). Toxicity of these formulated siRNAs has been observed and is believed to be largely attributable to the formulation excipients (i.e., cationic lipids in the formulation). Although some examples of pharmacology-based toxicity exist, the profile is dominated mainly by the excipient effects. In addition, the lead siRNA candidates chosen for these programs often exhibit activity in either rat or rabbit (or both), and hence, reproductive effects related to pharmacology can potentially be assessed in one or both species. Therefore, the main challenge to proper conduct of reproductive toxicity studies with such products is to determine the appropriate dosing frequency, as is the case for other subclasses of ONs that are not administered daily. Although experience to date is minimal, the highest dose levels that can be tested in reproductive toxicity studies with formulated siRNAs are predicted to be limited by the excipient-related toxicity, and large clinical multiple-dose levels may not be achievable. However, despite this limitation, the Subcommittee does not recommend testing the free siRNA in reproductive studies, as the blood PK and tissue distribution will be dramatically different from those for the formulated siRNA (e.g., the plasma half-life may be several minutes for unformulated siRNAs vs. hours for the formulated product). Thus, reproductive toxicity evaluation of siRNAs or microRNA mimetics/antagonists should be planned with regard to the principles outlined for antisense ONs and other familiar subclasses, with the objectives of (1) identifying appropriate species for evaluating effects related to intended pharmacology and to chemistry (both ON and formulation excipient chemistry, if applicable); and (2) achieving appropriate exposure for reproductive toxicity assessment in terms of dosing regimen and other study design aspects. In addition, consideration should be given to the potential effects related to immunostimulation via unintentional activation of TLR-3, 7, and 8. Lin et al. [43] demonstrated that dsRNA can increase the resorption rate in mice due to TLR3 stimulation; similar results are obtained with a synthetic dsRNA, polyinosinicpolycytidylic acid [poly(I:C)] [44,45]. Treatment of pregnant mice with TLR3, 7, or 8 agonists can cause pregnancydependent hypertension and maternal changes similar to preeclampsia [46]. Furthermore, studies on polyI:C [45] suggest that TLR3 activation during pregnancy may affect the behavior of offspring. Together, these studies demonstrate that the timing of maternal exposure to the ON has a significant impact on the outcome; therefore, the dosing regimen of DART studies of RNA ONs is a critical aspect of study design. Class-specific considerations related to aptamer ONs

Aptamers are a unique subclass of ONs with respect to their mechanism of interaction (direct binding to a target protein) and structure (they are often PEGylated). Some aptamers are being developed as single-dose agents (e.g., those that target clotting proteins) or as intermittent local therapies (e.g., intraocular administration on a monthly basis), and such programs may not warrant reproductive toxicity studies. Those aptamers intended for repeated systemic administra-

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tion in WOCBP should be evaluated for reproductive effects. If the aptamer has been shown to exhibit robust cross-species pharmacologic activity, conducting the studies in the standard species should suffice, as per the guidelines described in previous sections. Consistent with the recommendations of the Exaggerated Pharmacology Subcommittee [5], relevant pharmacologic activity in just one of the standard species used for reproductive toxicity testing should be sufficient to address effects related to pharmacology. However, if the aptamer has activity only in primate species, consideration should be given to conducting the DART studies in NHPs. As a possible alternative strategy, when a surrogate is available, it may be acceptable to conduct studies with one or more dose levels of the surrogate, in addition to the standard three dose levels of the clinical candidate, to address potential reproductive and developmental effects related to pharmacology. However, the surrogate often has a different chemical structure from the clinical candidate and/or has not been optimized for pharmacologic activity. In such cases, the use of a surrogate to assess reproductive effects related to pharmacology may not be as scientifically sound as the testing of the clinical candidate in NHPs. These judgments should be made on a case-by-case basis with regard to the biology of the aptamer target and the likelihood of exposure of gonadal or embryo/fetal tissues versus the purported benefit for the patient population, among other considerations. Case Studies

The following section provides case studies of a variety of ONs to illustrate different approaches. Case study 1: Antisense ON administered via subcutaneous route

This section describes a case study for an antisense inhibitor intended for a cardiovascular indication (Table 2; ISIS 301012). Reproductive toxicity studies were conducted in mice (segment 1/2) and rabbits (segment 2). The drug candidate is a MOE ON that is not active in mouse or NHP. The general toxicity studies in mice used a top-level dose of 90 mg/kg administered as a once-weekly subcutaneous (SC) injection, and included a mouse-active surrogate. For the combined segment 1/2 study in mice, the highest doses administered by SC injection were up to *90 mg/kg/week, with three treatment groups of 25 male and 25 female mice. A mouse surrogate was also used in the study at a dose level of *90 mg/kg/week because the clinical candidate was not pharmacologically active in the mouse. The dose regimen used for female mice was every other day (QOD) for 2 weeks premating, during the mating period, and through GD15 to ensure repeated exposure during pregnancy and fetal development. Males were dosed weekly for a 10-week period encompassing pre-mating, mating, and post-mating phases of the study. The start of dosing for the males was 4 weeks prior to mating, and the last dose was administered on day 69, with necropsy on day 71 (approximately 48 hours after the last dose). Additionally, four groups of six animals per sex per group served as TK animals and received the control or test article in the same manner as the main study groups at the same dose levels and volume. A segment 2 study was conducted in rabbits with the human-specific antisense ON only. The dose regimen was

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QOD SC from GD 6 to 18 at dose levels of up to *55 mg/kg/ week (15 mg/kg/dose). On GD 29, each surviving study animal was euthanized and subjected to a complete necropsy for evaluation. Additionally, TK animals (three pregnant animals/group) were included for each dose group. No effects on fertility or fetal development were observed in either the segment 1/2 study in mice or in the segment 2 study in rabbits. Little or no accumulation of ON in the fetus or the placenta was seen after administration QOD during the gestation period in mice or rabbits. In addition, no effects on fetal development or fertility were observed in mice given a mouse-specific surrogate, which indicates no developmental toxicity associated with the target inhibition. In rabbits, maternal toxicity was evident as reductions in body weight and food consumption at the highest dose level. Furthermore, three pregnancies were aborted, and one female delivered early at the *55 mg/kg/week dose level. Decreases in fetal body weight observed and the abortions at *55 mg/kg/week in rabbits occurred in the presence of maternal toxicity, and may have been secondary to decreased food consumption and maternal body weight, and not a direct effect of the antisense ON on fetal development. This particular program is representative of other DART study programs for related compounds in this chemical class. The studies evaluated both the drug candidate and the potential effects related to exaggerated pharmacology. There was no apparent effect of fertility or fetal development independent of maternal toxicity. The exposure assessment documented dose-dependent tissue concentrations that were comparable to the general toxicity studies, but little to no measurable drug concentrations were seen in placenta or fetus from either species. Case study 2: Antisense ON administered via oral route

In another development program aimed at treating patients with inflammatory bowel disease, a disease that typically includes women of child-bearing potential, an antisense ON (Table 2: ISIS 104838), targeting an inflammation pathway, was evaluated. The ON has well documented activity in mice and NHPs, but not in rats. The clinical product consists of a specialized ‘‘gastro-protected’’ formulation (capsule) intended to deliver the ON to the lower gastrointestinal tract, thus minimizing systemic absorption. Very low systemic exposure was documented in general toxicity studies at highclinical-multiple oral dose levels using the clinically relevant formulation. Reproductive toxicity evaluation focused on EFD studies, and waivers were requested for the fertility and late gestation/early-lactation studies. Intravenous (IV) dosing was selected for the EFD studies to ensure adequate systemic exposure. In consideration of the documented pharmacologic activity in mice (as well as the previous experience with this species in general toxicity studies), the mouse was chosen (in lieu of rats) as the primary pharmacologically relevant species. Although the pharmacologic relevance was uncertain in rabbits (considered unlikely and not investigated), the rabbit was employed as the second species for the EFD studies to test the effects of chemical structure on reproduction and development. A study in NHPs, a pharmacologically relevant species, was considered unwarranted because, in general

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toxicity studies conducted in mice and NHPs, all observed toxicities reflected the familiar ‘class effects’ of PS ONs, with no suggestion of any adverse effect related to pharmacology. Doses were given daily to mimic the clinical regimen; the ON was a first-generation PS structure, and tissue stability was limited. Conventional range-finding EFD studies were conducted in both species, and maternal toxicity was well characterized based on clinical signs, body weight gain decrement, decreased food consumption, and gross necropsy (C-section) findings. Toxicokinetic (TK) sampling was not included in the range-finding studies, but was incorporated into the mouse EFD study (using satellite animals), and a dedicated PK study in pregnant rabbits was subsequently conducted to obtain exposure data in the second species. Sample analysis in the rabbit was limited to plasma from animals at the no-observed-adverse-effectlevel (NOAEL). This analysis demonstrated a very large exposure multiple with the IV route, as compared to human PK data from orally dosed patients (virtually undetectable with a sensitive hybridization-type assay). Adverse developmental effects were limited to the mouse in the form of increased post-implantation loss (embryo-fetal death) and decreases in fetal weight, which were also accompanied by substantial maternal toxicity. Case study 3: Aptamer administered via intravitreal route

Macugen (Pegaptanib) is a PEGylated modified ON that binds to VEGF and is approved for the treatment of wet agerelated macular degeneration (AMD). This ON has activity in mouse, rat, rabbit, dogs, and NHPs. Clinically, Macugen is administered by intravitreal (IVT) injection. IVT administration in nonclinical studies demonstrated a slow exit from the vitreous humor into the systemic circulation, with minimal systemic exposure. Nonclinical PK and general toxicity studies with Macugen were conducted using the SC, IV, and IVT routes. The safety of IVT- and IV-administered Macugen was evaluated in mice, rats, rabbits, dogs, and NHPs in acute, subchronic, and chronic studies. Macugen demonstrated very high tolerability following both systemic and IVT administration, and a maximum tolerated dose was not established. For the AMD indication, a limited reproductive toxicology program was conducted (range-finding EFD studies in the rabbit and mouse and a definitive EFD study in the mouse by IV administrations) due to the advanced age (median age is over 70 years old) and limited reproductive potential of the patient population. In addition to the initial indication of AMD, diabetic macular edema (DME) was later pursued as an indication, for which a younger patient population would be treated. The reproductive toxicology program was discussed with the U.S. Food and Drug Administration in light of both possible indications (AMD and DME), and it was agreed that fertility, reproduction, teratology, and PPND studies could be delayed until after the start of phase 3 for the DME indication. In addition, it was discussed that depending on the results of the EFD (segment 2) studies, a waiver for the second species could be justified. Based on discussions with other health authorities, IV mouse range-finding and definitive EFD and fertility studies, as well as a rabbit IVT rangefinding EFD study, were conducted prior to phase 3.

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In the mouse EFD study, IV administration was used to maximize systemic exposure, compared to the clinical route of administration, IVT. Macugen was administered daily from GD 6 to15; no teratogenicity, fetal mortality, or maternal toxicity was evident up to 40 mg/kg/day, the highest dose tested. Findings were limited to reduced fetal body weight (5%) and delayed ossification of forepaw phalanges, an effect that occurred at exposure levels of over 300-fold greater than the expected clinical exposure (based on AUC). Additionally, Macugen concentrations in the amniotic fluid were 0.05% of maternal plasma levels in the 40 mg/kg/day group. In the IVT range-finding EFD study in rabbits, no teratogenicity or other signs of embryo or fetal toxicity were seen at doses as high as 2 mg/eye given on GD 6, 13, and 19. In the fertility study in mice at IV dose levels up to 40 mg/kg/ day, no effects on fertility and reproduction were observed. The NOAEL (40 mg/kg) is approximately 1,300-fold (AUC) greater than the clinical exposure at the recommended dose. Although systemic exposure following IVT administration is very low, and placental transfer is probably poor, Europe European Medicines Agency and Japan Pharmaceuticals and Medical Devices Agency requested a definitive EFD rabbit study prior to the start of phase 3 (using the IVT route), and a mouse PPND study prior to registration (with IV dosing), due to concerns based on the role of VEGF in development. In the definitive rabbit study, IVT doses of up to 2 mg per eye were given on GD 7 and 13, and the only finding was an increase in the common skeletal variation of angulated hyoid alae in the highest dose group, which occurred at a 70-fold exposure safety margin (based on AUC). In the mouse PPND study, which was conducted at IV doses of 0.02, 0.15 and 1 mg/kg/day, maternal body weight gain was reduced at 1 mg/kg/day, but no effects on reproduction, viability, and growth of the offspring were observed. The highest dose tested of 1 mg/kg/day represents a 50-fold exposure safety margin (based on AUC at the same doses in other studies). Conclusions

ONs have attributes that are similar to NCEs and NBEs. Both sets of guidelines can be useful when planning reproductive toxicity studies. The approach to DART testing should take into account both the potential effects of chemical structure as well as the intended pharmacology of the ON. Standard reproductive toxicity species (rodent/rabbit) can often be used because most ON toxicity is related to chemical structure. Animal-active surrogates can be useful to assess potential reproductive effects related to pharmacology in addition to effects related to the chemical backbone. The choice of the relevant animal model, the dosing regimen, and whether to use the clinical candidate or an animal surrogate will all need to be considered carefully based on the specific product attributes of the ON. Information from general toxicity studies and previous experience with similar ONs can also help to inform these decisions. For various reasons outlined above, NHP studies should be used only as needed to answer specific questions or when surrogates are not practical. Dosing regimens should be tailored to ensure adequate exposure throughout the period of organogenesis without compromising the ability to assess PD- and PK-related effects.

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The lack of historical precedent in the assessment of these novel products allows for creativity and scientific expertise to play a major role in the design and interpretation of studies. Although ICH guidelines provide a common international basis for the basic reproductive toxicity protocol development process, they also allow for the necessary flexibility required in order to evaluate ONs in a scientifically valid manner. Ultimately, the successful development of ONs will depend on the use of an appropriate experimental model in each case to enable the predictive value of the preclinical safety evaluation, thus providing optimal safety information for the patients. Acknowledgments

The authors would like to thank Jeff Tepper, Stephen Shrewsbury, and Christopher Bowman for their critical review of the manuscript. Author Disclosure Statement

The submitted manuscript is a committee position document that does not promote any particular product(s), and no competing financial interests exist for any of the authors. References

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Address correspondence to: Joy Cavagnaro, PhD Access BIO PO Box 240 Boyce, VA 22620 E-mail: [email protected] Received for publication April 23, 2014; accepted after revision July 21, 2014.