The benefits, limitations and opportunities of preclinical models for neonatal drug development.

Increased research to improve preclinical models to inform the development of therapeutics for neonatal diseases is an area of great need. This article reviews five common neonatal diseases - bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, perinatal hypoxic-ischemic encephalopathy and neonatal sepsis - and the available in vivo, in vitro and in silico preclinical models for studying these diseases. Better understanding of the strengths and weaknesses of specialized neonatal disease models will help to improve their utility, may add to the understanding of the mode of action and efficacy of a therapeutic, and/or may improve the understanding of the disease pathology to aid in identification of new therapeutic targets. Although the diseases covered in this article are diverse and require specific approaches, several high-level, overarching key lessons can be learned by evaluating the strengths, weaknesses and gaps in the available models. This Review is intended to help guide current and future researchers toward successful development of therapeutics in these areas of high unmet medical need.

Folic acid and primary prevention of neural tube defects: A review.

With 4 mg folic acid daily, it may take 20 weeks to reach red-blood-cell folate levels between 1050 and 1340 nmol/L, optimal for reduction of the neural tube defect risk. Therefore, folic acid supplementation should be started 5-6 months before conception. The residual risk with optimal red-blood-cell folate levels is reportedly 4.5 per 10,000 total births. The residual risk in pooled data from countries with mandatory folic acid fortification is 7.5 per 10,000 pregnancies, regardless of pre-fortification rates. European monitoring of folate intake with questionnaires should be replaced by periodic measurements of red-blood-cell folate. The risk of folate intake >1 mg/day does not outweigh the benefits of folic acid fortification, provided un-metabolized folic acid, RBC folate and vitamin B12 are monitored periodically. A European monitoring system, based on U.S. National Health and Nutrition Examination Surveys, should reside with the European Centre for Disease Prevention and Control.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

A database of embryo-fetal developmental toxicity (EFDT) studies of 379 pharmaceutical compounds in rat and rabbit was analyzed for species differences based on toxicokinetic parameters of area under the curve (AUC) and maximum concentration (Cmax) at the developmental lowest adverse effect level (dLOAEL). For the vast majority of cases (83% based on AUC of n = 283), dLOAELs in rats and rabbits were within the same order of magnitude (less than 10-fold different) when compared based on available data on AUC and Cmax exposures. For 13.5% of the compounds the rabbit was more sensitive and for 3.5% of compounds the rat was more sensitive when compared based on AUC exposures. For 12% of the compounds the rabbit was more sensitive and for 1.3% of compounds the rat was more sensitive based on Cmax exposures. When evaluated based on human equivalent dose (HED) conversion using standard factors, the rat and rabbit were equally sensitive. The relative extent of embryo-fetal toxicity in the presence of maternal toxicity was not different between species. Overall effect severity incidences were distributed similarly in rat and rabbit studies. Individual rat and rabbit strains did not show a different general distribution of systemic exposure LOAELs as compared to all strains combined for each species. There were no apparent species differences in the occurrence of embryo-fetal variations. Based on power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies.

Comparison of rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on the nature and severity of developmental effects.

Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo-fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo-fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo-fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.

Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

Juvenile animal testing of hydroxypropyl-ß-cyclodextrin in support of pediatric drug development.

Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is being explored as excipient for administration of poorly soluble NCE's in pediatrics. In support of pharmaceutical development, non-clinical studies were performed to investigate whether oral and intravenous administration of HP-ß-CD showed a different response in juvenile rats versus adult rats. Juvenile rats received HP-ß-CD via the intravenous route at dose levels of 50, 200 and 400mg/kg/day from postnatal day 16 to 44, or via oral gavage at 500, 1000 and 2000mg/kg/day from postnatal day 4 to 46. In addition to in vivo parameters, toxicokinetics and post-mortem evaluations were conducted. The main findings were related to the renal excretion of intact HP-ß-CD and were regarded as non-adverse transient adaptive responses. The pathogenesis of the osmotic nephrosis-like changes are discussed. With increasing age a more effective renal clearance of HP-ß-CD is present in line with the postnatal functional maturation of the kidney. In addition, following oral administration an increase in soft stools was seen which was related to osmotic water retention in the large intestine. The findings in the juvenile studies are very similar to those observed in previously performed adult rat studies at similar dose levels, same routes and similar or longer dose duration. No novel toxicity was seen in the juvenile studies.

In vivo longitudinal micro-CT study of bent long limb bones in rat offspring.

Micro-computed X-ray tomography (micro-CT) has been reported as a reliable method to assess ex vivo rat and rabbit fetal skeletons in embryo-fetal developmental toxicity studies. Since micro-CT is a non-invasive imaging modality it has the potential for longitudinal, in vivo investigation of postnatal skeletal development. This is the first paper using micro-CT to assess the reversibility of drug-induced bent long bones in a longitudinal study from birth to early adulthood in rat offspring. Analysis of the scans obtained on postnatal Day 0, 7, 21 and 80 showed complete recovery or repair of the bent long limb bones (including the scapula) within the first 3 weeks. When assessing risk the ability to demonstrate recovery is highly advantageous when interpreting such transient skeletal change. In summary, in vivo micro-CT of small laboratory animals can aid in non-clinical safety assessment, particularly for specific mechanistic purposes or to address a particular concern in developmental biology.

Fluctuating asymmetry as risk marker for stress and structural defects in a toxicologic experiment.

Fluctuating asymmetry (the directionally random asymmetry of bilateral structures, FA) is commonly used as a measure of developmental instability, and may increase with stress. As several studies reported a relation between FA and developmental abnormalities, we investigate whether FA could be an additional perhaps more sensitive marker of developmental toxicity. The aim of this work is analyzing patterns of FA in multiple traits in a large dataset of rabbit fetuses, which were prenatally exposed to a toxic compound and sacrificed just before natural delivery. Gravid females were exposed to three doses of this compound, inducing abnormalities in the fetuses at the high dose only. The average FA, however, was already higher than control in rabbit fetuses of the low-dose group but did not further increase with higher concentrations. Moreover, the increase in FA differed between traits, with the hindlimbs showing the strongest response. In addition, we did not find any association between FA and the presence of fetal abnormalities at the individual level. Although these results suggest that FA may act as "an early warning system," we did not find a dose-response relationship with increasing stress and effects were trait-specific. Further testing is needed before FA may be considered as a sensitive marker in developmental toxicity studies.

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

The zebrafish embryotoxicity/teratogenicity assay is described as a useful alternative screening model to evaluate the effect of drugs on embryofoetal development. Fertilized eggs were exposed to different concentrations of 15 compounds with teratogenic (8) and non-teratogenic (7) potential until 96h post-fertilization when 28 morphological endpoints and the level of compound uptake was assessed. The majority of drugs testing positive in mammals was also positive in zebrafish (75% sensitivity), while a relative high number of false positives were noted (43% specificity). Compound uptake determination appears useful for clarifying classifications as teratogenic or potential overdose although assay sensitivity could be improved to 71% if the exposure threshold, previously suggested as ∼50ng/larvae, is reconsidered. The zebrafish assay shows some potential, though limited in its current form, as a screening tool for developmental toxicity within Janssen drug development. Further assay refinement with respect to endpoints and body burden threshold is required.

Value of juvenile animal studies.

The Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute has undertaken a project to address the impact of juvenile animal studies on pediatric drug development. A workshop, sponsored and organized by the Health and Environmental Sciences Institute Developmental and Reproductive Toxicity Technical Committee, was held on May 5-6, 2010, in Washington, DC, to discuss the outcome of a global survey and the value of juvenile animal studies in the development of drugs intended for use in pediatric patients. During this workshop, summary data from the 2009-2010 survey were presented, and breakout sessions were used to discuss specific case studies to try to assess the impact of juvenile animal studies performed to support specific pediatric drug development. The objectives of the Workshop on The Value of Juvenile Animal Studies were to (1) provide a forum for scientists representing industry, academia, and regulatory agencies to discuss the impact of juvenile animal studies on pediatric drug development, (2) evaluate summary data from the survey to understand how the juvenile study data are being used and their impact in labeling and risk assessment, (3) discuss selected case studies from the survey to highlight key findings, and (4) identify the areas of improvement for the designs of juvenile animal studies. The take home message that resonated from the workshop discussions was that well-designed juvenile animal studies have demonstrated value in support of certain pediatric drug development programs. However, it was also clear that a juvenile animal study is not always warranted.

Modeling spatial learning in rats based on Morris water maze experiments.

The Morris water maze, developed by Morris (J Neurosci Methods 1984: 11: 47-60), is a behavioral experiment designed to test the spatial memory. When repeating the experiment several times, the changes in time (latency) and distance (path) taken to reach the platform are indicators for the learning and memory abilities of the rat. In juvenile toxicity studies, it is of interest to test whether dosing juvenile rats with some compound of interest has an effect on its learning ability. The traditional analysis uses non-parametric tests to check for a possible dose-effect. However, due to the many tests performed, this approach lacks power. Here, an alternative method is proposed, accounting for the longitudinal design of the study, the right-censoring of observations when animals did not find the platform and the correlation between the time and distance taken to reach the platform.

The ontogeny of drug metabolizing enzymes and transporters in the rat.

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals. The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1-3 and 6, Bsep and Bcrp, Oct1-2, Oat1-3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague-Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR. There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression. Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood-brain barrier. Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.

Real life juvenile toxicity case studies: the good, the bad and the ugly.

With the growing experience in the conduct of juvenile toxicity studies for multiple classes of compound, the 'case-by-case' approach has become under much more pressure. Instead, a general screen or 'standard design' is now commonly expected by regulatory authorities with more routine inclusion of neurological and reproductive assessments. Minor modifications or additions can be made to the design to address specific questions according to the class of drug or intended clinical use. This drift from a 'case-by-case' approach to a 'standard design' approach is present within certain reviewing divisions of the FDA, often requesting by default a rodent and non-rodent juvenile animal study. However, juvenile animal studies should be designed thoughtfully to fulfil a purpose based on scientific rationale, with each endpoint carefully considered in terms of practicality and interpretability of data generated. Only when using the appropriate strategy and design may juvenile studies add value by (1) identifying potential safety or pharmacokinetic issues for drugs intended for paediatric use, (2) suggesting additional clinical endpoints and (3) adding new information to the product label. As the knowledge from juvenile animal studies in various species grows, a better understanding of the significance/relevance of findings will be achieved.