Juvenile toxicity study of PF-07256472/recifercept, a recombinant human soluble fibroblast growth factor receptor 3, in 2-3-month-old cynomolgus monkeys.

Achondroplasia is an autosomal disorder caused by point mutation in the gene encoding fibroblast growth factor receptor 3 (FGFR3) and resulting in gain of function. Recifercept is a potential disease modifying treatment for achondroplasia and functions as a decoy protein that competes for ligands of the mutated FGFR3. Recifercept is intended to restore normal bone growth by preventing the mutated FGFR3 from negative inhibitory signaling in pediatric patients with achondroplasia. Here we evaluated the potential effects of twice weekly administration of recifercept to juvenile cynomolgus monkeys (approximately 3-months of age at the initiation of dosing) for 6-months. No adverse effects were noted in this study, identifying the high dose as the no-observed-adverse-effect-level and supporting the use of recifercept in pediatric patients from birth. Considering that juvenile toxicity studies in nonhuman primates are not frequently conducted, and when they are conducted they typically utilize animals ≥9 months of age, this study demonstrates the feasibility of executing a juvenile toxicity study in very young monkeys prior to weaning.

Embryo-fetal exposure and developmental outcome of lenalidomide following oral administration to pregnant cynomolgus monkeys.

Lenalidomide is an immunomodulatory drug and is very effective in the management of a number of malignancies, including multiple myeloma. Like thalidomide, lenalidomide interacts with the cereblon E3 ligase complex, which results in targeted destruction of proteins. This study was conducted to study the teratogenic potential of lenalidomide when administered to pregnant cynomolgus monkeys. Lenalidomide was administered orally on gestation days 20-50 at dosages of 0 (vehicle control), 0.5, 1, 2 and 4 mg/kg/day. Thalidomide was used as a positive control and was administered orally at 15 mg/kg/day on gestation days 26-28. Each group consisted of 5 pregnant monkeys. Pregnancy was terminated on gestation day 100 ± 1 by cesarean section and fetuses examined for external, internal and skeletal changes. Intrauterine loss was 40% in the thalidomide group and 20 % in each of the lenalidomide 2 and 4 mg/kg/day groups. Treatment with lenalidomide and thalidomide resulted in no effects on placental weights, fetal body weights and body measurements. External fetal examination revealed malformations in fetuses of all lenalidomide-treated groups, including malformations of upper and lower extremities. These external malformations had correlated skeletal findings and were similar to those seen in the thalidomide-treated group, where two of three fetuses showed the classic thalidomide syndrome of malformed upper and lower extremities. A no-observed-adverse-effect level was not identified in this study, and the mean maternal exposures at the lowest dosage, where fetal malformations were observed, were 5-folder lower than the exposures observed in the MM patients treated with 25 mg of lenalidomide.

Enhanced normograms and pregnancy outcome analysis in nonhuman primate developmental toxicity studies.

The incidence of spontaneous pregnancy/infant losses is highly variable in long-tailed macaques (cynomolgus monkey), making it potentially difficult to ascertain test item-related effects in developmental toxicity studies. Therefore, pregnancy normograms had been developed by Jarvis et al. [1] to aid in the distinction of normal (e.g. test facility background) versus non-normal pregnancy outcomes. These normograms were mostly derived from embryo-fetal development studies and from PPND studies with a postnatal phase limited to seven days. However, the enhanced pre- and postnatal developmental (ePPND) study paradigm has essentially replaced these former study types. This work aims at providing enhanced normograms (e-normograms) in the context of regulatory ePPND studies. Survival functions for the prenatal phase (286 control pregnancies) and the postnatal phase (222 live infants) were estimated using the Kaplan-Meier estimator. Normograms were generated from survival curves and pseudo-study simulations. Data were available from two test facilities with comparable EU-compliant animal husbandry. Pregnancy duration/outcome as well as survival functions did not differ significantly between test facilities indicating that this husbandry system yields comparable developmental observations across different test facilities, at least in this NHP species. These novel e-normograms were developed for pregnant long-tailed macaques and provide an extended postnatal period up to three months, a new concept of separate normograms for the prenatal and the postnatal period, specific information on the perinatal phase events, a prediction of expected number of live infants for group size management, and the option to evaluate effects on pregnancy duration through distinction of live births and infant losses.

Group size experiences with enhanced pre- and postnatal development studies in the long-tailed macaque (Macaca fascicularis).

Enhanced pre- and postnatal development (ePPND) studies have become the default developmental toxicity test for biopharmaceuticals if nonhuman primates represent the relevant species. Spontaneous pregnancy losses and infant deaths can be significant in macaques such as long-tailed macaques. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline S6(R1) states that pregnancy outcome can be judged also by the normogram-based variability of reference data according to a publication by Jarvis et al. (2010) defining a study as valid with six to eight live infants in the control group on postnatal day 7 (PND7). Since the release of ICH S6(R1) (2011), ePPND studies for biologics have replaced the former separate embryo-fetal and PPND study types. This work provides a retrospective analysis of pregnancy outcomes from 21 ePPND studies and group sizes of 14-24 animals per group. All studies reached the goal of at least six to eight infants on PND7, with overall losses ranging between 5 % and 45 %. Consistently, a group size of 14-24 maternal animals yielded more than six to eight infants on PND7. Therefore, it is suggested to reduce ePPND study group sizes from 20 to 14, yielding an animal number reduction of approx. 30 %.

Effects of SA237, a humanized anti-interleukin-6 receptor monoclonal antibody, on pre- and postnatal development in cynomolgus monkey.

BACKGROUND: SA237 is a humanized anti-interleukin-6 receptor (IL-6R) monoclonal antibody in which the constant and variable regions have been engineered for a longer plasma half-life. According to literature, blocking of IL-6 related functions could have an influence on pregnancy sustainment, development of the immune system, and brain growth. METHODS: SA237 effects on dams, embryo-fetal development, parturition and postnatal development were investigated in an enhanced pre- and postnatal development study, in which SA237 was subcutaneously administered to pregnant cynomolgus monkeys at dose levels of 2 or 50 mg/kg once weekly from gestation day 20 until parturition. Infant development, including immune function and learning ability tests, was comprehensively assessed at multiple examinations until approximately 10 months after birth. RESULTS: SA237 plasma concentrations were almost equivalent between dams and their infants and dropped throughout the postnatal period, pharmacologically relevant exposure was maintained for 147 days after birth at 50 mg/kg. Because the binding of SA237 to IL-6R inhibited IL-6R-mediated clearance of IL-6, serum IL-6 increased in dams and infants. However, there were no SA237-related adverse effects on dams, embryos, fetuses, or infants. SA237 pharmacological effects contributed to the suppression of plasma cell differentiation and antibody production by inhibiting IL-6 signaling, and T cell-dependent antibody reaction was minimally suppressed in infants, but physiological immunoglobulin class switching and general antibody production against a T cell-dependent antigen were maintained. CONCLUSION: The exposure to SA237 did not adversely affect dams, embryo-fetal development, parturition, and postnatal development, including immune function and neuronal development. Birth Defects Research 109:843-856, 2017. © 2017 Wiley Periodicals, Inc.

Continuing harmonization of terminology and innovations for methodologies in developmental toxicology: Report of the 8th Berlin Workshop on Developmental Toxicity, 14-16 May 2014.

This article is a report of the 8th Berlin Workshop on Developmental Toxicity held in May 2014. The main aim of the workshop was the continuing harmonization of terminology and innovations for methodologies used in the assessment of embryo- and fetotoxic findings. The following main topics were discussed: harmonized categorization of external, skeletal, visceral and materno-fetal findings into malformations, variations and grey zone anomalies, aspects of developmental anomalies in humans and laboratory animals, and innovations for new methodologies in developmental toxicology. The application of Version 2 terminology in the DevTox database was considered as a useful improvement in the categorization of developmental anomalies. Participants concluded that initiation of a project for comparative assessments of developmental anomalies in humans and laboratory animals could support regulatory risk assessment and university-based training. Improvement of new methodological approaches for alternatives to animal testing should be triggered for a better understanding of developmental outcomes.

Visual evoked potential-based acuity assessment: overestimation in amblyopia.

BACKGROUND/AIMS: When visual acuity (VA) is assessed with spatially repetitive stimuli (e.g., gratings) in amblyopes, VA can be markedly overestimated. We evaluated to what extent this also applies to VEP-based objective acuity assessment, which typically uses gratings or checkerboards. METHODS: Seventeen subjects with amblyopia (anisometropic and strabismic) participated in the study; decimal VA range of their amblyopic eye covered 0.03-1.0 (1.5-0.0 logMAR). Using the Freiburg Acuity VEP (FrAVEP) method, checkerboard stimuli with six check sizes covering 0.02°-0.4° were presented in brief-onset mode (40 ms on, 93 ms off) at 7.5 Hz. All VEPs were recorded with a Laplacian montage. Fourier analysis yielded the amplitude and significance at the stimulus frequency. Psychophysical VA was assessed with the Landolt-C-based automated Freiburg Visual Acuity Test (FrACT). RESULTS: Test-retest limits of agreement for both FrACT and FrAVEP were ±0.20 logMAR. In all but two dominant eyes and high-acuity amblyopic eyes (VA<0.3 logMAR), FrACT and FrAVEP agreed within the expected limits of ±0.3 logMAR. However, the VEP-based acuity procedure overestimated single Landolt-C acuity by more than 0.3 logMAR in 9 of 17 (53%) of the amblyopic eyes, up to 1 logMAR. While all subjects had a psychophysical acuity difference>0.2 logMAR between the dominant and amblyopic eye, only three of them showed such difference with the FrAVEP. CONCLUSION: Both measurements of visual acuity with the VEP and FrACT were highly reproducible. However, as expected, in amblyopia, acuity can be markedly overestimated using the VEP. We attribute this to the use of repetitive stimulus patterns (checkerboards), which also lead to overestimation in psychophysical measures. The VEP-based objective assessment never underestimated visual acuity, but needs to be interpreted with appropriate caution in amblyopia.

The enhanced pre- and postnatal development study for monoclonal antibodies.

The enhanced pre- and postnatal (ePPND) study design has been developed in response to new scientific knowledge and subsequent guideline changes [ICH M3(R2) and ICH S6(R1)]. The changes in study design were basically driven by the experiences obtained during preclinical development of biopharmaceuticals. The standard ePPND concept does not apply to conventional small molecule pharmaceuticals. In essence, the ePPND design is a pre- and postnatal development (PPND) study in which key elements of an embryo-fetal development study are investigated in newborns and infants rather than in the fetus. The cynomolgus monkey is the current relevant nonhuman primate model. The ICH S6(R1) guideline reached step 5 in June 2011 and provides detailed recommendations on various parameters and the conduct of an ePPND study. This chapter provides working guidance for monitoring menstrual cycles to generate pregnant animals, ultrasound monitoring of pregnancy, morphometric measurements of fetuses and newborns, in vivo skeletal examination, various protocols for evaluation of infants (e.g., neurobehavioral assessment, learning and memory test, grip strength, immune system evaluation) and a comprehensive list of additional infant evaluation parameters for the cynomolgus monkey.

Embryo fetal development studies in nonhuman primates.

Embryo fetal development (EFD) studies in nonhuman primates are frequently conducted in macaques with Macaca fascicularis (cynomolgus monkey, long-tailed macaque, crab-eating macaque) being the most accepted model. EFD studies are also feasible in the marmoset. Due to recent guideline changes (ICH M3(R2) and S6(R1)), EFD studies are largely confined to conventional pharmaceutical compounds rather than biopharmaceuticals. This chapter describes basic study designs and provides working protocols for collecting, processing, and staining fetuses, including collection of amniotic fluid and umbilical cord blood. The chapter also covers the examination and terminology for external, visceral, and skeletal examinations of fetuses. The species covered in this chapter are cynomolgus monkey (long-tailed macaque) and marmoset monkey.

Harmonization of description and classification of fetal observations: achievements and problems still unresolved: report of the 7th Workshop on the Terminology in Developmental Toxicology Berlin, 4-6 May 2011.

This article summarizes the 7th Workshop on the Terminology in Developmental Toxicology held in Berlin, May 4-6, 2011. The series of Berlin Workshops has been mainly concerned with the harmonization of terminology and classification of fetal anomalies in developmental toxicity studies. The main topics of the 7th Workshop were knowledge on the fate of anomalies after birth, use of Version 2 terminology for maternal-fetal observations and non-routinely used species, reclassification of "grey zone" anomalies and categorization of fetal observations for human health risk assessment. The paucity of data on health consequences of the postnatal permanence of fetal anomalies is relevant and further studies are needed. The Version 2 terminology is an important step forward and the terms listed in this glossary are considered also to be appropriate for most observations in non-routinely used species. Continuation of the Berlin Workshops was recommended. Topics suggested for the next Workshop were grouping of fetal observations for reporting and statistical analysis.

The enhanced pre- and postnatal study for nonhuman primates: update and perspectives.

The enhanced pre- and postnatal (ePPND) study design has been developed in response to new scientific knowledge and subsequent guideline changes, that is, ICH M3(R2) and ICH S6(R1). The design changes were basically driven by the experiences obtained during preclinical development of biopharmaceuticals. The ePPND concept typically does not apply to pharmaceuticals. In essence, the ePPND design is a PPND study in which key elements of an embryofetal development (EFD) study are being investigated in newborns and infants rather than in the fetus. The current relevant nonhuman primate model is the cynomolgus monkey. The ICH S6(R1) has reached step 4 during June 2011 and provides detailed recommendations on various parameters and the conduct of an ePPND study. By the time this article is written, it appears that for monoclonal antibodies, the ePPND study is the preferred approach although ICH S6(R1) also leaves options for modified EFD and PPND study concepts. Our data also demonstrate that social housing is feasible for developmental toxicity studies in the cynomolgus monkey model.

Postnatal development in cynomolgus monkeys following prenatal exposure to natalizumab, an alpha4 integrin inhibitor.

BACKGROUND: Natalizumab is a humanized monoclonal IgG4 antibody to human alpha4 integrin that blocks the interaction of alpha4beta1 and alpha4beta7 integrins with their ligands, including fibronectin, vascular cell adhesion molecule-1, and mucosal addressin cellular adhesion molecule-1. Because alpha4 integrins and their ligands are widely involved in mammalian development, lymphopoeisis, and hematopoiesis, natalizumab may interfere with these processes. METHODS: The effects of prenatal exposure to natalizumab on postnatal development were assessed in cynomolgus monkeys at doses of 0 and 30 mg/kg administered intravenously every other day from gestational day (GD) 20 to 70 or GD 20 to term. Infants were delivered by natural birth and evaluated for general health, survival, development, and immunological structure and function at 12 or 18 months. RESULTS: An increase in abortions was seen in the first cohort of natalizumab-treated dams (39.3 vs. 7.1% in the controls) but not in the second cohort (33.3, 37.5%). Infants in the term treatment group had elevated lymphocyte ( approximately 150%) and nucleated red blood cell counts ( approximately 400%), consistent with the pharmacological effect of natalizumab, and reductions in platelet counts ( approximately 28%), which were reversible following clearance of natalizumab. No anemia was observed. Infants in the term treatment group had significantly increased spleen weights at 12 months but not at 18 months. All other experimental observations in infants from natalizumab-treated dams were comparable with those of controls. CONCLUSION: Natalizumab had no adverse effects on the general health, survival, development, or immunological structure and function of infants born to dams treated with natalizumab during pregnancy.

Electrophysiological evaluation of retinal photoreceptor function after repeated bevacizumab injections.

Bevacizumab (Avastin, Genentech) was one of the first anti-VEGF substances used to treat macular edema or choroidal neovascularization in patients with vascular ocular pathologies. However, only few studies evaluate the safety of intravitreal bevacizumab injections in regard to retinal photoreceptor function. We evaluated retinal function after repeated (2-3) monthly injections of bevacizumab in a prospective case series of 10 patients with various retinal diseases. Study endpoints were visual acuity (VA) using ETDRS charts and 3 full-field electroretinography sessions with a flash intensity range of 0.0005-2 cds/m(2). V-log-I b-wave amplitudes were fitted by a Naka-Rushton model. No significant changes in scotopic or photopic ERG measures were observed between baseline ERG and last follow-up ERG. Individual patients showed transient alterations of ERG measures on the first follow-up visit. Mean visual acuity was stable over the time course of the study (logMAR = 0.42 at baseline and logMAR = 0.48 at last follow-up). In conclusion, three monthly repeated injections of bevacizumab do not affect mid-term electrophysiological retinal function. Transient alterations in ERG readings of individual patients 1 week after intravitreal bevacizumab injection may be attributed to short-term disruption of the retinal equilibrium through the trauma of injection. Evaluation of patients receiving more than three injections of anti-VEGF substances should be the focus of future studies.

Physiology and Endocrinology of the Ovarian Cycle in Macaques.

Macaques provide excellent models for preclinical testing and safety assessment of female reproductive toxicants. Currently, cynomolgus monkeys are the predominant species for (reproductive) toxicity testing. Marmosets and rhesus monkeys are being used occasionally. The authors provide a brief review on physiology and endocrinology of the cynomolgus monkey ovarian cycle, practical guidance on assessment and monitoring of ovarian cyclicity, and new data on effects of social housing on ovarian cyclicity in toxicological studies. In macaques, cycle monitoring is achieved using daily vaginal smears for menstruation combined with cycle-timed frequent sampling for steroid and peptide hormone analysis. Owing to requirements of frequent and timed blood sampling, it is not recommended to incorporate these special evaluations into a general toxicity study design. Marmosets lack external signs of ovarian cyclicity, and cycle monitoring is done by regular determinations of progesterone. Cynomolgus and marmoset monkeys do not exhibit seasonal variations in ovarian activity, whereas such annual rhythm is pronounced in rhesus monkeys. Studies on pair- and group-housed cynomolgus monkeys revealed transient alterations in the duration and endocrinology of the ovarian cycle followed by return to normal cyclicity after approximately six months. This effect is avoided if the animals had contact with each other prior to mingling. These experiments also demonstrated that synchronization of ovarian cycles did not occur.