Types of studies considered appropriate for biologicals

ICH S6 addresses the types of studies considered appropriate for biologicals and clearly defines the types of studies that are not generally considered applicable to biologicals. These studies are discussed below. In many cases, the guidance provided in ICH S6 is intentionally general to allow for the flexibility needed to address the challenges associated with the safety assessment of biologicals. Other ICH documents are included in the discussion as appropriate.

6.3.1 SAfETY Pharmacology Studies

Safety pharmacology studies are defined as "those studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure in the therapeutic range and above" (ICH S7A, 2001).34 The ICH S7A guidance document defines the general principles and recommendations for safety pharmacology studies. The guidance is applicable to small-molecular-weight molecules and to biologicals, but the guidance states that in the case of highly targeted biologicals, safety pharmacology endpoints can be included as endpoints in general toxicology studies, which reduces or eliminates the need for safety pharmacology studies for these products. However, ICH S7A recommends that a more extensive safety pharmacology battery be considered for a novel class of biologicals or for those biologicals that do not have a high degree of targeting. The guidance provided in ICH S6 allows for safety pharmacology indices to be addressed in independent studies or incorporated into toxicology studies. Regardless of the approach taken, safety pharmacology indices should be assessed in a pharmacologically relevant animal model.

The ICH S7A-defined core battery for safety pharmacology consists of functional assessments of organ systems critical for life and includes the central nervous system (CNS), cardiovascular system, and respiratory system. The extent to which these areas of concern can be assessed for biologicals is influenced by which animal model or models are identified as pharmacologically relevant. As stated previously, in many cases the only relevant animal model for safety evaluation of biologicals is a nonhuman primate. Cardiovascular and respiratory endpoints can be readily assessed in these animals. Laboratories that conduct studies in nonhuman primates have procedures for assessing CNS function, but these are more subjective in nonhuman primates than in other species, and well-established or validated methods in nonhuman primates are not available.

6.3.2 Exposure Assessment

The ICH S6 document addresses three aspects of exposure assessment: pharma-cokinetics and toxicokinetics (pharmacokinetics data obtained during the course of toxicology studies), assays, and metabolism. According to ICH S6, single- and multiple-dose pharmacokinetics, toxicokinetics, and tissue distribution studies in relevant species are useful, but studies intended to address mass balance are not useful. In practice, including toxicokinetic evaluations in toxicology studies is critical to the interpretation of toxicology data because it is the only way to confirm that exposure to the biological is maintained throughout the duration of the study. Because biologi-cals undergo proteolytic degradation, which can result in amino acids being incorporated into proteins/peptides not related to the biological drug, studies conducted with radiolabeled biologicals can be difficult to interpret. Validated assays should be used for measuring the amount of the biological present in serum samples collected during pharmacokinetics, pharmacology, and toxicology studies. Whenever possible, the assay method(s) used for laboratory animals should be same as that used for humans. The influence of antibodies to the biological product on assay performance should be determined.

Because they are proteins, biologicals undergo proteolytic degradation to small peptides and individual amino acids. Therefore, classical biotransformation studies, such as those performed for small-molecular-weight molecules, are not needed for biologicals.

6.3.3 Single-Dose Toxicity Studies

Data generated in these studies can be used to define dose-response relationships and to establish doses for repeated dose toxicity studies. Including safety pharmacology endpoints in these studies should be considered. Single-dose toxicity studies should be conducted in pharmacologically relevant models using the route of administration intended for the clinic.

6.3.4 Repeated-Dose Toxicity Studies

As is the case with all studies conducted with biologicals, these studies should be conducted in pharmacologically relevant models. As discussed previously, the route and frequency of administration should be appropriate for the intended clinical use. Generally speaking, the duration of treatment used for toxicology studies conducted with biologicals should be at least equal to the intended duration of treatment, with ICH S6 identifying six months as being generally appropriate for chronic indications such as psoriasis and rheumatoid arthritis. However, the ultimate duration of treatment used for each product is influenced by a number of factors, including clinical indication, toxicity profile of the product, and immunogenicity.18 In the case of serious, life-threatening diseases, such as cancer, patients can be treated for durations exceeding that used in toxicology studies, assuming that the clinical trials are designed to adequately monitor for adverse events.

An important determining factor of the duration of toxicology studies is immu-nogenicity, which refers to the animal developing antibodies to the biological. As discussed below, antibodies can neutralize the activity of biologicals or increase their rate of elimination to an extent that the animals are not being sufficiently exposed to the drug product. The occurrence of such antibodies can limit the duration of toxicology studies. For example, the formation of neutralizing antibodies by monkeys limited the duration of toxicology studies conducted with pegylated interferon-a 2b to four weeks,47 even though the approved duration of treatment with the product for patients with hepatitis C is one year.48

A recovery period should be included at the end of these toxicology studies to assess the reversal or potential worsening of pharmacological/toxicological effects. The length of the recovery period should be sufficient to allow for complete reversal of effects. In addition, the recovery period is important to allow for clearance of the drug in order to be able to monitor/measure antibody levels to the drug (as high drug concentrations generally interfere with the conduct of the antibody assay).

6.3.5 Immunotoxicity Studies

As shown in Table 6.5, many biologicals are intended to stimulate or suppress the immune system. The intended effects of these and other products on the immune


Approved Immunomodulatory Biologicals




Enbrel® (etanercept)

Humira® (adalimumab)

Amevive® (alefacept)

Raptiva® (efalizumab)

Interferes with lymphocyte activation by binding to lymphocyte antigen CD2 Binds to tumor necrosis factor (TNF) and blocks its interaction with cell surface TNF receptors Binds to TNF-alpha and blocks its binding to cell surface TNF receptors Inhibits adhesion of leukocytes to other cell types by binding to CD11a on the surface of leukocytes


Rheumatoid arthritis; polyarticular-course juvenile rheumatoid arthritis; psoriatic arthritis; psoriasis; ankylosing spondylitis Rheumatoid arthritis

Psoriasis system can be classified as immunopharmacology or as immunomodulatory effects. Adverse events can result from the intended immunomodulatory mechanism of action. For example, excessive down-regulation of the immune system can result in recrudescence of a previously inactive virus. Immunotoxicity, on the other hand, refers to adverse immune effects that occur with products that are not targeting the immune system or have unintended effects on the immune system. These effects include inflammatory reaction at the injection site and autoimmunity due to altered expression of surface antigens.

Although immunogenicity is an immune response of the animal to a foreign protein, it is not viewed as immunotoxicity per se. ICH S6 does not provide detailed guidance on immunotoxicity testing. It states that immunotoxicologic testing strategies may require screening studies followed by mechanistic studies, and it states that routine tiered testing approaches or standard testing batteries are not recommended for biologicals. As discussed below, there is an ongoing effort to establish better methods to assess intended and unintended effects of biologicals on the immune system of nonhuman primates. These efforts should lead to better understanding of the effects of biologicals on immune function.

FDA/CDER has published an immunotoxicity guidance document (Guidance for Industry, Immunotoxicology Evaluation of Investigational New Drugs, 2002).49 However, the guidance specifically states that it does not apply to biologicals. Additionally, an ICH document, ICH S8: Immunotoxicology Studies for Human Pharmaceuticals,36 has been developed. Similar to the FDA/CDER document, this document is not intended to be applied to biologicals. However, both documents contain useful information on approaches to assess immunotoxicity and can serve as a useful general reference to those developing biologicals.

6.3.6 Reproductive Performance and Developmental Toxicity Studies

ICH S6 contains two general recommendations regarding reproductive and developmental toxicity studies. First, the need for these studies is dependent upon the product, clinical indication, and intended patient population. Second, the specific study design and dosing schedule may be modified based on issues related to species specificity, immunogenicity, pharmacological activity, and a long elimination half-life. For example, concerns regarding developmental immunotoxicity can be addressed in studies designed to assess neonatal immune function.

More detailed information on reproductive toxicology studies than that presented in ICH S6 is found in ICH S5A (Detection of Toxicity to Reproduction for Medicinal Products, 1994)31 and ICH S5B(M) (Toxicity to Male Fertility, An Addendum to the ICH Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal Products)?2 These documents provide guidance on evaluating adult male and female reproductive function, embryo/fetal development, and postnatal development. They provide guidance on the specific phases of reproduction to be assessed, the selection of species, and the types of endpoints to be included in the studies. In general, the range of studies defined in ICH S5 is most applicable to products that are being tested in rats and rabbits — the primary species used for reproductive toxicology testing. If a biological is pharmacologically active in rats and rabbits, then ICH S5-recommended studies can be conducted unless immunogenicity limits the duration of testing. In many cases, however, biologicals are active only in humans and nonhuman primates. Conducting reproductive toxicity studies in nonhuman primates is associated with a number of challenges, which are discussed below.

6.3.7 Genotoxicity Studies

ICH S6 specifically states that the range and type of genotoxicity studies routinely conducted for small-molecular-weight drugs are not applicable to biologicals or for process contaminants that result during the manufacture of biologicals. Biologi-cals are not expected to interact directly with DNA or other chromosomal material, and they undergo proteolytic degradation to amino acids or peptides, which are not thought to have genotoxic potential. Furthermore, the manufacturing process of biologicals involves the use of physical methods of extraction and separation, as opposed to organic chemicals, eliminating the concern for potentially genotoxic organic impurities in final product. ICH S6 identifies the presence of an organic linker as the case in which biologicals should be evaluated in the genotoxicity tests typically reserved for small-molecular-weight drugs. An organic linker is a chemically synthesized small-molecular-weight molecule linking a radionuclide or an immunotoxin to a biological, typically a monoclonal antibody or antibody fragment. The types of genotoxicity studies considered appropriate for chemically synthesized small-molecular-weight products are defined in ICH S2B (Genotoxicity: A Standard Battery for Genotoxicity Testing for Pharmaceuticals).27

6.3.8 Carcinogenicity Studies

Carcinogenicity studies are conducted as part of the safety evaluation of small-molecule drugs if they are to be used continuously for at least six months or may be expected to be used repeatedly in an intermittent manner for a chronic or recurrent condition (e.g., allergic rhinitis, depression, and anxiety). Carcinogenicity studies are not needed if these products are to be administered infrequently or for short durations unless there is cause for concern. Causes for concern include carcinogenic potential in the class that is relevant to humans; structure-activity relationship suggesting carcinogenic risk; evidence of preneoplastic lesions in repeated-dose toxicity studies; and long-term retention of parent compound or metabolite(s) resulting in local tissue reactions or other pathophysiological responses (ICH S1).23 The carcinogenic potential of small-molecular-weight molecules is typically assessed in the rat and the mouse, with the study in rats being a two-year bioassay and the study in mice being the same or a shorter-term assay.

ICH S6 specifically states that the standard carcinogenicity bioassays conducted in rodents are "generally inappropriate" for biologicals. As stated previously, many biologicals are pharmacologically active only in nonhuman primates and it is not possible to conduct carcinogenicity studies in these species. In carcinogenicity studies, animals undergo lifelong treatment with the compound. The life span of monkeys would make carcinogenicity studies prohibitively long and require the use of excessive amounts of product. Additionally, the number of nonhuman primates needed for such a study would be equally prohibitive. If a human product is pharmacologically active in rodents, the ability to conduct carcinogenicity studies is potentially affected by immunogenicity, which can limit the feasible duration of treatment to considerably less than the two years needed for a rodent bioassay.

Because of these limitations, ICH S6 proposes an alternative approach to assessing the carcinogenic potential of biologicals that might have the potential to support or induce proliferation of transformed cells and clonal expansion, potentially leading to neoplasia. Such products should be evaluated in appropriate in vitro systems for their ability to stimulate growth. Appropriately designed in vivo studies might be needed if in vitro studies identify cause for concern. To date, concerns regarding carcinogenicity that might be associated with immunosuppressive products have not been routinely addressed by conducting a rodent bioassay, primarily due to a lack of pharmacological activity of the biological product in rodents. The potential for carcinogenicity is included in the approved package inserts for these products. For example, the approved package insert of Amevive® (alefacept; Biogen Idec, Inc., Cambridge, MA) addresses the concern for malignancies and states that caution should be exercised when considering the use of Amevive® in patients at high risk for malignancy.50

6.3.9 Local Tolerance Studies

As mentioned previously, virtually all biologicals are administered by an injection, which necessitates an assessment of the injection site for adverse effects. Assessment is made using visual observation and histopathological evaluation. These studies should be conducted with the formulation intended for the clinical candidate. It is possible to assess local tolerance as part of either single-dose or repeated-dose toxicity studies.

6.3.10 Tissue Cross-Reactivity Studies foR Monoclonal Antibodies

Tissue cross-reactivity studies define the binding of monoclonal antibodies to target and nontarget tissues using immunohistochemistry. Because binding to nontarget tissues can result in toxicity, these studies are an integral part of the safety assessment of monoclonal antibodies. Tissue cross-reactivity studies are conducted using cryosections of human tissues obtained during surgery or autopsy. They are also conducted using animal tissues to ensure that the animal model selected for toxicology studies exhibits a staining pattern similar to humans. Detailed guidance on the conduct of tissue cross-reactivity studies can be found in the FDA document entitled Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use.51


Many biological therapeutics are human proteins or specifically target human receptors, and thus have restricted species cross-reactivity. Because of the species-specific nature of biologicals, these drugs are often not pharmacologically active in nonprimate animal species (e.g., rodents or dogs) commonly used in toxicology studies conducted for traditional small-molecule drugs. Because nonhuman primates are phylogenetically closer to human, for many biological therapeutics they are the only relevant animal species for safety assessment studies.

The nonhuman primate has played an important role in the development of biotechnology products by facilitating general safety assessment and the evaluation of these products in specific diseases. For example, aging primates are used to study geriatric diseases, osteoporosis, and many ocular indications. Nonhuman primate models are also being developed to evaluate the effects of drugs on the reproductive system and the immune system in order to better understand effects that may be seen in humans.

Cynomolgus monkeys (Macaca fascilularis) are the principal nonhuman primates used for assessing the toxicity of biological therapeutics, although rhesus monkeys (Macaca mulatta) are also sometimes used. The main reason for choosing cynomolgus monkeys over rhesus monkeys is because cynomolgus monkeys are more appropriate for reproductive toxicity testing; rhesus monkeys are seasonal breeders, which makes reproductive toxicity testing especially difficult, and cynomolgus monkeys are not. Reproductive toxicity must be conducted in monkeys whenever the test compound binds only to the receptor in nonhuman primates.

A large historical database exists for endpoints measured in repeated-dose toxicology studies for both rhesus and cynomolgus monkeys, and many contract research organizations (CROs) have experience with both species so the use of either is a viable option for programs that do not require reproductive toxicity tests in monkeys. Another advantage of conducting toxicity studies in cynomolgus monkeys is their smaller size compared to rhesus monkeys, which requires less test material. Some advantages of using rhesus or cynomolgus macaques are that blood volume is not as limited as in rodents and many of the blood/serum-based markers of toxicity in nonhuman primates can then be used in clinical trials, allowing for direct comparison of the toxic effects of the drug in the preclinical studies with the effects seen in human patients.

Marmosets (Callithrix jacchus) may also be used, and their small size (350-500 g) is both an advantage and a disadvantage. The advantages can include the small amount of test material needed and the relatively small amount of space required for suitable housing. The main disadvantage of small size is the low volume of blood that may be obtained relative to that obtained from other species of nonhuman primates. In addition, marmosets are very sensitive to environmental stimuli and changes and are susceptible to stress factors. Also, many biological therapeutics crossreact with cynomolgus or rhesus targets, but not with marmoset. Fewer CROs have experience with the marmoset and the historical database is more limited; however, certain CROs do have considerable experience with toxicity testing in marmosets.52

In certain cases, the biological product is so species-specific that it will only cross-react with humans and chimpanzees (Pan troglodytes). Although safety studies can be conducted in chimpanzees, many limitations exist: no histopathology can be conducted since these are a highly protected species and are not euthanized at the termination of the study; only small animal numbers can be used; a limited number of CROs can conduct the studies; limited historical control data exist; obtaining protein-naive animals is difficult; and dosing parameters (frequency and dose level) are limited. Therefore, toxicity studies conducted with chimpanzees provide only limited data. In cases where the chimpanzee is the only relevant nonhuman species, alternative strategies should include testing a surrogate molecule (i.e., monoclonal antibodies or other proteins that are specific for the epitope or receptor in rodent or other animal species), or using transgenic or knock-out mice that overexpress or have a deletion of the targeted protein. Each of these approaches has issues that must be considered and will be discussed later in this chapter.

The age and size of the monkey are important considerations in the toxicity testing of biological therapeutics. Generally, cynomolgus monkeys should not be smaller than 2 kg, as the use of smaller animals limits the blood volume available for sampling. Younger animals are also more vulnerable to stress associated with various procedures encountered during the study and may be more prone to develop diarrhea and be more sensitive to the secondary effects (e.g., dehydration), leading to confounding toxicities unrelated to the test article. In addition, smaller animals are likely sexually immature and may respond to the drug differently from adult animals. The appropriate age of the animals may also depend on the biological activity of the compound and the age of the expected patient population. Most CROs have historical data ranges for clinical pathology parameters from animals of various age ranges as well as from various sources.

Several important factors should be considered when evaluating toxicology data from nonhuman primate studies. Differences can be seen among animals from different countries of origin (Chinese, Indonesian, Vietnamese, Mauritian) in clinical pathology parameters as well as other standard endpoints. In addition, nonhuman primate data should be reviewed on an animal-by-animal basis because of intra-animal heterogeneity and the small number of animals used. Statistics are therefore of limited utility in evaluating data from nonhuman primate studies. Maintaining the same strain and source of animals throughout the drug development program, and not switching because of animal availability, is very important.

Neonatal or juvenile monkey studies are difficult to conduct but may be necessary, depending on the intended patient population. If a juvenile monkey study is to be conducted to support use of the therapeutic in pediatric patients, it is important to carefully consider the appropriate age in the cynomolgus monkey so that it closely matches the intended patient population. The appropriate age may be difficult to define since this may vary depending on the target organ of the therapeutic. For example, the age of a monkey that is appropriate to mimic neurological development parameters in humans may be different from that which mimics immunologi-cal development or reproductive development and growth. Neonatal studies can only be conducted by CROs that have breeding capabilities on-site, as it is difficult to ship animals less than six months of age because of the stress of shipping. Unfortunately, few CROs have a large enough population of young animals of the same age to use for a toxicology study.

For the development of new therapies for geriatric diseases (prostate disease, ocular pathology, osteoporosis, diabetes, Alzheimer disease, etc.), it is important to understand how age-related disease and pathology develop. The cynomolgus monkey has been used for this type of testing and some CROs have special groups of older animals (generally 13 years of age and older). Ovariectomized cynomolgus monkeys are the most well established model for osteoporosis,53 and ocular toxicity testing is also well established in nonhuman primates.54-57

6.4.1 Immunotoxicity Testing in Nonhuman Primates

Immunotoxicity testing guidelines exist for small molecules for which the toxicology is largely unpredictable, and rodent species are typically used. For human biological therapeutics, the immune system is often the intended target of the therapy and the immunotoxicity observed is often exaggerated pharmacology. In this case, nonhuman primates are generally used and the immune tests need to be selected based on the known immunomodulatory properties of the drug. These assays can also be used as pharmacodynamic markers of drug activity or efficacy for these immune modulators. It is important to distinguish between immunopharmacology (where the immune system is the target organ of the therapeutic effect), immunotox-icity (where nontarget immune effects such as autoimmunity or immunosuppression may be observed), and immunogenicity (which represents an immune response to the drug, and not a toxicity per se).

Several important factors should be considered when including immunotoxicity testing into standard GLP toxicology studies. These include whether the assays have been validated; the use of main study animals or a satellite group; and the timing of these tests within the context of the GLP toxicology study. The advantages of using the main study animals for immunotoxicity testing are reduced animal use and the correlation of any immunotoxicity findings with other toxicities seen in those same animals. The disadvantage of using main study animals is that the additional manipulations for immune testing (e.g., injection of an antigen for determining antibody response) may influence the toxicity or immunogenicity of the therapeutic agent. Immunotoxicity testing is generally included in the one-month nonhuman primate toxicology studies. It is very important to include several baseline measurements because of the variability seen between animals and even in the same animal over time. Because of the small number of nonhuman primates in each group, it is important to reduce the variability in the assays as much as possible with regard to antigen source, technique, etc.

The FDA/CDER and ICH S8 immunotoxicology guidance documents do not apply to biologicals, but some of the recommendations in these documents can be applied to immunotoxicity testing of these products in nonhuman primates and in other species (e.g., if the biological cross-reacts in rodents). This guideline recommends that standard toxicity studies be used as the initial screen to detect immuno-toxicity, since standard hematology and immunopathology are generally sufficient to detect immune system alterations.58,59 Immunopathology includes total and differential white blood cell counts as well as evaluation of the histopathology of lym-phoid organs such as the thymus, spleen, lymph nodes, gut-associated lymphoid tissue (GALT), and the bone marrow. In addition, more detailed measurements of any change in size and cellularity of immune cells, germinal center development, cortex:medulla ratio of the thymus, and immunohistochemistry of the lymphoid organs should be included.

Flow cytometry can be included in a GLP toxicology study to evaluate changes in lymphocyte subsets, including T cells (CD4+, CD8+), B cells (CD20+), NK cells (CD16+), and monocytes (CD14+). These assays are typically conducted using peripheral blood, which allows for repeated sampling over time within the same animal. Immunophenotyping can also be conducted on tissues to determine whether lymphocyte trafficking is affected, although time points are limited to study termination (i.e., rodents); however, serial biopsies (i.e., on lymph nodes) can be performed in nonhuman primates. Serial biopsies may be difficult because they cannot be performed by all laboratories, and potential infections or other effects on the animals can affect data interpretation. Flow cytometry can also be used for more functional endpoints of immune competence, including lymphocyte activation, cytokine release, phagocytosis, apoptosis, oxidative burst, natural killer (NK) cell activity, etc. These can be added if the mechanism of action of the drug suggests involvement of a particular function or type of immune cells.

In nonhuman primates, the assay most commonly used to assess the ability to mount a T-cell-dependent antibody response (TDAR) is immunization with keyhole limpet hemocyanin (KLH) or tetanus toxoid (TT), and measurement of circulating antigen-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) methods. One method that can be applied for evaluating TDAR is immunization with KLH or TT before drug treatment to assess the effects on the secondary antibody response (i.e., first immunization given subcutaneously on Day -7 and second immunization 14 days later), and the other antigen can be injected after two weeks of treatment to determine the effect on the primary immune response 7 to 10 days later. This immunization regimen allows for the assessment of both the primary and the secondary T-cell-dependent antibody response within the one-month GLP toxicology study. For studies of longer duration, a booster immunization can be given at a later time point to assess the affect on the memory response, or to see whether an altered response returns to normal during the recovery period.

Other immune parameters can be measured in the nonhuman primate, including cytokine measurements and delayed type hypersensitivity measurements, although these are less well characterized. Many human ELISA kits for cytokines can be used to measure cytokines in the nonhuman primate, although it is very important to determine whether the reagents in these kits do truly cross-react with nonhuman primate cytokines. Many of the human reagents do crossreact, but exceptions exist and these need to be tested if they are used on a toxicology study.

Although immunomodulation can be assessed in the nonhuman primate, the assays are less well characterized than those used in the rodent. One issue is the lack of consistent protocols, and the timing of incorporating these assays into standard GLP toxicology studies varies. More historical control data are needed, and many assays have not been tested with an immunomodulatory control to confirm the level of sensitivity of the assay for detecting a mild/moderate immune modulator (both immunoenhancing and immunosuppressive activity).

Inherently, greater variability is seen in nonhuman primates than in inbred rodents, and the animal number per group is generally much smaller than in rodent studies. Finding ways of reducing the variability in the assay to allow for more meaningful data interpretation is critical. These can include decreasing the interanimal variability (using animals from the same source and of similar ages, decreasing stress during the study, increasing the number of baseline samples, etc.) and decreasing assay variability (standardizing the antigen source, assay technique, timing, etc.).

Currently, assays of immunomodulation can be conducted in nonhuman primates, but sufficient data are lacking regarding which assays are the most useful in predicting immunomodulatory effects in humans. Assay methods need to be standardized so that data can truly be compared to make that determination. Comparing data from the nonhuman primate with the immunotoxicity data in rodents would be useful to evaluate whether the nonhuman primate is more predictive of the human response. Additionally, regulatory agencies should continue to treat the immunotoxicity testing of biological therapeutics on a case-by-case basis. However, immune testing in nonhuman primates for biologicals goes beyond the estimation of immunotoxicity and can be very valuable for understanding the pharmacology of an immune modulator and can help to establish pharmacodynamic markers that can then be used in clinical trials. Combining all of the available data in nonhuman primates will allow for an improvement in the models and a better understanding of the value of these data. In addition, differences have been seen in immune parameters (especially immunophenotyping) among cynomolgus monkeys from different geographical locations. It is therefore very important to keep the same source of animals for toxicology studies throughout the drug development program.

6.4.2 Reproductive Testing in Nonhuman Primates

For biological therapeutics, the need for reproductive toxicity testing is dependent on the product, the clinical indication, and the patient population. Reproductive studies, including embryo-fetal development and male and female fertility, can be assessed in nonhuman primates. Although a traditional peri/postnatal development study (as conducted in rodents) would not be performed in nonhuman primates, a modified developmental ("late gestation") study could be conducted in nonhuman primates to assess placental transfer, excretion into milk, and evaluation of neonates (i.e., behavioral observations) up to six or nine months of age. As mentioned above, these studies are best conducted in cynomolgus monkeys because they are not seasonal breeders and are fertile throughout the year, unlike the rhesus monkey.

TABLe 6.6

Comparison across species: Approximate Gestation period (Days) of Embryonic and Fetal Development species

Mouse Rat pre-Implantation organogenesis Fetal Maturation

18 5G 57

19 21 29 155 27G


Cynomolgus monkey Human

Conducting reproductive toxicity testing in nonhuman primates offers many advantages. The endocrinology and duration of the menstrual cycle and early pregnancy are similar in humans and nonhuman primates. Other similarities include placental morphology and physiology, timing of implantation and subsequent rates of embryonic development, response to known teratogens, spermatogenesis, and placental transfer of IgG.60,61 A cross-species comparison of the time period of embryonic and fetal development is shown in Table 6.6.

Conducting reproductive toxicity testing in nonhuman primates also has several disadvantages compared to the use of other species. These include the small number of animals used and smaller number of offspring to evaluate (generally one fetus per dam); the cost and much longer duration of studies (~150-day gestation period); potential difficulty in obtaining sexually mature animals; low conception rate; high abortion rate; limited number of CROs with the ability to perform the studies; and the limited historical database. As a result, the timing of these studies in the development program may be later than for small-molecule therapeutics (ICH M3).37 This is especially an issue for development of biological therapeutics in Japan, where female fertility studies (which can take approximately nine months) and embryo-fetal development studies (which also take approximately nine months) are required prior to Phase 1 clinical trials that include women (ICH M3).37 These studies require a significant commitment in cost and time for a therapeutic that may well fail in Phase 1 or 2 trials.

Male fertility can be more easily assessed in cynomolgus monkeys by evaluating testicular volume and weight, sperm parameters (ejaculate weight, sperm count, morphology, motility), hormone analysis [testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin], and histopathologic evaluation of testicular biopsies with regard to spermatogenesis.62 In addition, flow cytometry techniques can be used to assess changes in cell types (Sertoli cells, Leydig cells, germ cells, etc.) and are very powerful for the detection of alterations in spermatid numbers and chromatin maturation.63 These parameters can be assessed in a separate male fertility study or they can be added to a subchronic or chronic repeated-dose toxic-ity study. The treatment phase would then cover approximately two spermatogenic cycles. A recovery period of at least six weeks (one spermatogenic cycle) should follow to evaluate the reversibility of effects.

The question remains whether it is necessary to assess mating in these studies. Successful mating as an endpoint in fertility investigations is a rather weak endpoint because mating may still be successful even if the reproductive system is severely impaired by administration of a test compound.62 Mating behavior can also be difficult to assess because each male is paired with a single female, and other compatibility issues may arise unrelated to effects from the test article. In addition, the female monkeys would then need to be followed until gestation Day 20 to determine whether they actually were pregnant. Considering the potentially low conception rate in nonhuman primates, any effects on this endpoint might be difficult to differentiate from the concurrent and historical control values. Because the number of animals is small, mating is unlikely to detect a test article-related effect that would not have been detected in the other parameters mentioned above. Using successful mating as an endpoint in nonhuman primate studies is further complicated by low conception and high abortion rates.

Female fertility studies generally consist of three pretreatment observation cycles, three treatment cycles, and one or more recovery cycles. Changes in menstrual cycling are measured, as well as cycle-related hormone analysis (FSH, LH, progesterone, estradiol). Again, mating is not generally needed at the end of a fertility study as the difficulties are similar to those mentioned above.

For an embryo-fetal study, confirmed pregnant animals are treated with the test article on gestation Days 20 through 50 (period of organogenesis). Animals then undergo Cesarean section on gestation Day 100, and fetal examinations are made. In addition, a modified developmental ("late gestation") study will examine the effects of test article treatment from gestation Day 100 until delivery on delivery parameters, neonatal effects, transfer of the test article across the placenta, and excretion of the test article into the milk. These two studies can be combined into one pre/ postnatal study with treatment from gestation Day 20 to parturition, with a cohort of animals undergoing Cesarean section on gestation Day 100 and a second cohort of animals allowed to deliver naturally.61

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