Evaluation of the Toxicity and Neurological Effects of Fulranumab in Adult Cynomolgus Monkeys.

Fulranumab, an anti-human nerve growth factor antibody, was evaluated in a series of nonclinical toxicology studies. No treatment effects were observed in adolescent cynomolgus monkeys in standard design, repeat-dose toxicology studies of up to 6 months. Adverse effects on the developing nervous system were observed in offspring of pregnant cynomolgus monkeys treated with fulranumab. Subsequent studies including detailed morphologic investigations of the nervous system did reveal fulranumab-related changes in adult cynomolgus monkeys; this article is focused on those findings. A single dose of ≥1 mg/kg fulranumab administered subcutaneously (SC) caused a decrease in neuron and sympathetic ganglion size (superior cervical ganglion), observed morphologically and stereologically, with a resulting appearance of increased glial cell density. Similar results were observed in repeat-dose (15 to 52 weeks) toxicity studies at ≤50 mg/kg/wk fulranumab SC. These effects recovered after a 3-month treatment-free period. Fulranumab did not cause any neuronal death, necrosis, apoptosis, or any apparent decrease in function of sympathetic neurons/ganglia at any time point examined. A no observed effect level (NOEL) was established at 0.25 mg/kg fulranumab SC every 4 weeks for 28 weeks.

Applying a weight of evidence approach to the evaluation of developmental toxicity of biopharmaceuticals.

Toxicity studies in pregnant animals are not always necessary for assessing the human risk of developmental toxicity of biopharmaceuticals. The growing experience and information on target biology and molecule-specific pharmacokinetics present a powerful approach to accurately anticipate effects of target engagement by biopharmaceuticals using a weight of evidence approach. The weight of evidence assessment should include all available data including target biology, pharmacokinetics, class effects, genetically modified animals, human mutations, and a thorough literature review. When assimilated, this weight of evidence evaluation may be sufficient to inform risk for specific clinical indications and patient populations. While under current guidance this approach is only applicable for drugs and biologics for oncology, the authors would like to suggest that this approach may also be appropriate for other disease indications. When there is an unacceptable level of uncertainty and a toxicity study in pregnant animals could impact human risk assessment, then such studies should be considered. Determination of appropriate nonclinical species for developmental toxicity studies to inform human risk should consider species-specific limitations, reproductive physiology, and pharmacology of the biopharmaceutical. This paper will provide considerations and examples of the weight of evidence approach to evaluating the human risk of developmental toxicity of biopharmaceuticals.

ILSI/HESI maternal toxicity workshop summary: maternal toxicity and its impact on study design and data interpretation.

Workshops on maternal toxicity were held at the annual Society of Toxicology, Teratology Society, and European Teratology Society meetings in 2009. Speakers presented background information prior to a general discussion on this topic. The following recommendations/options are based on the outcome of the discussions at the workshops: 1. A comprehensive evaluation of all available data from general toxicity studies, range-finding Developmental and Reproductive Toxicology (DART) studies, class effects, structure-activity relationships, exposure studies, etc. is essential for appropriate dose selection for definitive DART studies. The intent is to avoid marked maternal toxicity leading to mortality or decreased body weight gains of greater than 20% for prolonged periods. (a) Evaluate alternative endpoints for dose selection and data interpretation (e.g., target tissue effects and pharmacology) for biotherapeutics. (B) Evaluate additional maternal parameters based on effects and/or target organs observed in short-term (e.g., 2- or 4-week) general toxicity studies. 2. Evaluate all available data to determine a cause-effect relationship for developmental toxicity. (a) Conduct a pair-feeding/pair-watering study as a follow-up. (b) Evaluate individual data demonstrating maternal toxicity in the mother with adverse embryo-fetal outcomes in the litter associated with the affected mother. (c) Conduct single-dose studies at increasing doses as a complement to conventional embryo-fetal toxicity studies for certain classes of compounds that affect the hERG channel. 3. Support statements that embryo-fetal effects are caused by maternal toxicity and/or exaggerated pharmacology, especially for malformations. (a) Provide mechanistic or other supporting data. (b) Establish the relevance of the DART findings in animals for human exposures. Birth Defects Res (Part B) 92:36-51, 2010. © 2011 Wiley-Liss, Inc.

Considerations in assessing the developmental and reproductive toxicity potential of biopharmaceuticals.

This report discusses the principles of developmental and reproductive toxicity (DART) testing for biopharmaceuticals. Biopharmaceuticals are large-molecular-weight proteins or peptides produced by modern biotechnology techniques incorporating genetic engineering and hybridoma technologies. The principles of DART testing for biopharmaceuticals are similar to those for small-molecule pharmaceuticals and in general follow the regulatory guidance outlined in International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) document S5(R2). However, because many biopharmaceuticals are species-specific, alternate approaches may be needed to evaluate DART potential as outlined in ICH S6. For molecules that show species-specific cross-reactivity restricted to non-human primates (NHP), some aspects of DART may require NHP testing. For biopharmaceuticals that are uniquely specific and only active on intended human targets or human and chimpanzee targets, surrogate molecules that cross-react with the more traditional rodent species may need to be developed and used for DART testing. Alternatively, genetically modified transgenic animals may also need to be considered. Surrogate molecules and transgenic animals may also be considered for DART testing even if the biopharmaceutical is active in NHPs in order to reduce the use of NHPs. Because of the unique properties of biopharmaceuticals, a case-by-case approach is needed for DART and general toxicity evaluation, which requires consideration of specific product attributes including biochemical and biophysical characteristics, pharmacological activity, and intended clinical indication.

Effects of natalizumab, an alpha4 integrin inhibitor, on fertility in male and female guinea pigs.

BACKGROUND: Natalizumab is a humanized monoclonal immunoglobulin G4 antibody directed against the human alpha4 integrin subunit disrupting interaction with its ligands. As alpha4 integrins and/or their ligands appear to be involved in reproductive function, the effects of natalizumab on fertility in male and female guinea pigs were investigated. METHODS: Natalizumab was administered by bolus intravenous injection every other day at doses of 0, 3, 10, and 30 mg/kg. Males began treatment at least 28 days prior to mating until necropsy (approximately 3 to 5 days after mating). Dosing in females was done from gestational day (GD) of an existing pregnancy to GD 30 of a second pregnancy. RESULTS: In male guinea pigs, natalizumab treatment had no effect on sperm parameters, reproductive organ weights, organ-weight ratios, or histology of the testis or epididymis. Natalizumab did not affect the ability of treated males to produce pregnancies in untreated females. In female guinea pigs, no treatment-related changes were seen in uterine weights or ovary weights. Pregnancy rates were reduced in females treated with 30 mg/kg natalizumab, but not those treated with 3 or 10 mg/kg. Pregnancy rates were 63.3, 66.7, 66.7, and 29.6% for groups treated with 0, 3, 10, and 30 mg/kg, respectively. Effects observed at 30 mg/kg were at exposures 36-fold those observed in humans. CONCLUSIONS: Natalizumab had no effects on male fertility, but did result in a reduction in pregnancy rates in females treated with the high dose of 30 mg/kg.

The guinea pig as an animal model for developmental and reproductive toxicology studies.

BACKGROUND: Regulatory guidelines for developmental and reproductive toxicology (DART) studies require selection of "relevant" animal models as determined by kinetic, pharmacological, and toxicological data. Traditionally, rats, mice, and rabbits are the preferred animal models for these studies. However, for test articles that are pharmacologically inactive in the traditional animal models, the guinea pig may be a viable option. This choice should not be made lightly, as guinea pigs have many disadvantages compared to the traditional species, including limited historical control data, variability in pregnancy rates, small and variable litter size, long gestation, relative maturity at birth, and difficulty in dosing and breeding. METHODS: This report describes methods for using guinea pigs in DART studies and provides results of positive and negative controls. Standard study designs and animal husbandry methods were modified to allow mating on the postpartum estrus in fertility studies and were used for producing cohorts of pregnant females for developmental studies. RESULTS: A positive control study with the pregnancy-disrupting agent mifepristone resulted in the anticipated failure of embryo implantation and supported the use of the guinea pig model. Control data for reproductive endpoints collected from 5 studies are presented. CONCLUSION: In cases where the traditional animal models are not relevant, the guinea pig can be used successfully for DART studies.

Effects of natalizumab, an alpha4 integrin inhibitor, on the development of Hartley guinea pigs.

BACKGROUND: Natalizumab is a humanized monoclonal immunoglobulin G4 antibody directed against the human alpha4 integrin subunit, disrupting interaction with its ligands. Natalizumab inhibits the interaction of alpha4 integrins with fibronectin, vascular cell adhesion molecule-1, and mucosal addressin cellular adhesion molecule-1, which are of potential importance in development. Two studies were undertaken to evaluate the effects of natalizumab on embryo/fetal development in guinea pigs. METHODS: In the first study, pregnant guinea pigs were treated with intravenous injections of 3, 10, or 30 mg/kg natalizumab or vehicle every other day from gestational day (GD) 4 to 30. In the second study, females were treated on alternate days starting at least 28 days prior to mating through GD 30. Fetal examinations and histopathologic examination of the liver, heart, thymus, spleen, and intestinal tract were performed following maternal euthanasia on GD 59-62. RESULTS: Natalizumab had no significant effect on embryo/fetal development in either study. Exposure to natalizumab during organogenesis did not result in treatment-related external, visceral, or skeletal variations or malformations or histopathologic changes. CONCLUSION: No fetotoxicity or teratogenic effects were attributable to natalizumab in these studies.

Embryo/fetal development studies with hydroxypropyl methylcellulose acetate succinate (HPMCAS) in rats and rabbits.

BACKGROUND: Hoshi et al. [Hoshi et al. J Toxicol Sci 10(Suppl):187-255, 1985a,b,c,d] evaluated the potential for hydroxypropyl methylcellulose acetate succinate (HPMCAS) to produce developmental and reproductive toxicity in a series of studies that included rat and rabbit teratology studies, a rat fertility study, and a rat peri- and postnatal study. The authors concluded that there were no compound-related findings. In the cesarean-section phase of the rat teratology study, however, clubfoot was reported for 0.8, 2.1, 5.5, and 4.1% of fetuses in the control, 625, 1250, and 2500 mg/kg groups, respectively. There were no significant increases in external anomalies, but the apparent dose-related increase in clubfoot was not specifically addressed. In the rabbit teratology study, the number of litters evaluated (12-13 per group) was not consistent with current regulatory guidelines. Therefore, to definitively establish the potential of HPMCAS to produce developmental toxicity, embryo/fetal development studies were carried out in rats and rabbits. METHODS: Groups of 20 pregnant Sprague-Dawley rats and New Zealand White rabbits were dosed with 0, 50, 150, 625, or 2500 mg/kg HPMCAS from gestational day (GD) 6-17 or GD 7-19 for rats and rabbits, respectively. Fetuses were collected by cesarean section and examined for external, visceral and skeletal development. RESULTS: No developmental toxicity was observed as a result of HPMCAS exposure demonstrating that maternal HPMCAS exposure during gestation does not induce developmental anomalies. There were no findings of clubfoot or other limb anomalies in these studies at dose levels equivalent to those that were previously associated with a possible increase in clubfoot. CONCLUSIONS: The conclusion of the earlier study indicating that treatment with HPMCAS at doses up to and including 2500 mg/kg did not produce developmental toxicity was confirmed with these studies. It is likely that the clubfoot noted in the earlier rat teratology study was a misdiagnosis or artifact.

Performing a testicular spermatid head count.

Procedure of counting spermatid heads using hemacytometer.