Assessing systemic, developmental, and reproductive toxicity and estrogenicity of Korean red ginseng extract G1899 in juvenile Sprague-Dawley Rats.

Background: Korean red ginseng (KRG) is a product from ginseng roots, which is enriched with ginsenosides and has been utilized for a long time as an adaptogen to alleviate various physiological or disease conditions. While KRG is generally considered safe, conducting a thorough toxicological assessment of the spray-dried powder G1899 during the juvenile period is essential to establish its safety profile. This study aimed to assess the safety of G1899 during the juvenile period using Sprague-Dawley rats. Methods: Two studies were conducted separately: a juvenile toxicity study and a uterotrophic bioassay. To assess the potential toxicity at systemic, postnatal developmental, and reproductive levels, G1899 was orally gavaged once a day in post-weaning juvenile Sprague-Dawley (SD) rats at 0, 1250, 2500, or 5000 mg/kg/day. Estrogenicity was assessed by orally gavaging G1899 in immature female SD rats at 0, 2500, or 5000 mg/kg/day on postnatal days (PND) 19-21, followed by a uterotrophic bioassay. These studies were conducted in accordance with the Good Laboratory Practice (GLP) regulations and regulatory test guidelines. Results: Regarding juvenile toxicity, no abnormalities related to the G1899 treatment were observed in any group during the experiment. Moreover, no uterotrophic responses were observed in the dosed female group. Based on these results, the no observed adverse effect level (NOAEL) of G1899 was determined to be at least 5000 mg/kg/day for general systemic function, developmental/reproductive function, and estrogenic activity. Conclusion: Our results suggest that G1899 is not toxic to juveniles at doses of up to 5000 mg/kg/day.

Evaluation of gadolinium-based contrast agents in juvenile CD-1 mice including behavioral evaluations.

INTRODUCTION: Seven gadolinium-based contrast agents (GBCAs), four linear and three macrocyclic, were evaluated for potential effects on development, including behavior of juvenile CD-1 mice. METHODS: The GBCAs were administered via intravenous injection once daily on postnatal day (PND) 9, 12, 15, 18, and 21 (PND 1 was the day of delivery) at doses up to twice the human equivalent clinical dose (i.e., 0.63 mmol Gd/kg for gadoxetate disodium and 2.5 mmol Gd/kg for the other GBCAs). Mice were bled for evaluation of exposure (plasma) to gadolinium (Gd) on PND 9, 12, and 70. At scheduled euthanasia, the liver, spleen, brain, skin (dorsal surface), bone (left femur), and kidneys were excised from up to six mice/sex/group on PND 10, 22, or 70 for the determination of Gd levels and histopathological analysis. All mice were monitored for toxicity, growth and survival, sexual maturation, and behavior. CONCLUSION: Gd was quantifiable in the brain tissues with levels declining over time. There was no long-term effect on the growth and development for mice exposed to any of the GBCAs. There was no impact on neurodevelopment as assessed by brain histology and validated neurobehavioral tests, including a functional observational battery, motor activity, and learning and memory as evaluated in the Morris water maze. For all GBCAs, the highest dose tested represented the no-observable-adverse-effect level in juvenile mice.

Microdialysis as a safe and feasible method to study target-site piperacillin-tazobactam disposition in septic piglets and children.

OBJECTIVES: Knowledge on the tissue penetration of piperacillin-tazobactam in children with sepsis is lacking. In this study, the feasibility and performance of microdialysis experiments were explored in septic piglets and children as part of a translational research project. METHODS: Multiple-day microdialysis investigations were performed in muscle tissue of 22 piglets (of which 11 were septic) and 6 children with sepsis. An in vitro experiment preceded the (pre)clinical trials to derive optimal experimental settings and calibration technique. Linear mixed-effects models quantified the impact of sepsis on relative recovery (RR) and intercatheter, interindividual, interoccasion, and residual variability. RESULTS: In vivo microdialysis was well tolerated in piglets and children, with no significant adverse events reported. Using identical experimental settings, lower RR values were recorded in healthy and septic piglets (range: piperacillin, 17.2-29.1% and tazobactam, 23.5-29.1%) compared with the in vitro experiment (piperacillin, 43.3% and tazobactam, 55.3%), and there were unacceptably low values in children with sepsis (<10%). As a result, methodological changes were made in the pediatric trial. Realistic tissue concentration-time curves were derived in piglets and children. In piglets, sepsis reduced the RR. The greatest contributors to RR variability were residual (>40%) and interoccasion (>30%) variability. The internal standard method was the preferred calibration technique in both piglets and children. CONCLUSIONS: Microdialysis is a safe and applicable method for the measurement of tissue drug concentrations in piglets and children. This study demonstrated the impact of experimental settings, sepsis, and target population on individual RR.

PDGFRα monoclonal antibody: Assessment of toxicity in juvenile mice administered a murine surrogate antibody of olaratumab.

BACKGROUND: Olaratumab (Lartruvo™) is a recombinant human IgG1 monoclonal antibody that specifically binds PDGFRα. In order to support use of Lartruvo in pediatric patients, a definitive juvenile animal study in neonatal mice was conducted with a human anti-mouse PDGFRα antibody analog of olaratumab (LSN3338786). METHODS: A pilot study was used to set doses for the definitive juvenile mouse study. In the definitive study, juvenile mice were administered vehicle, 50, 100, or 150 mg/kg LSN3338786 by subcutaneous (SC) injection every 3 days between postnatal days (PND) 1 and 49, for a total of 17 doses. Blood samples were collected on PND 49 for antibody analysis and toxicokinetic evaluation. Tissues were collected on PND 52 for histopathologic examination. RESULTS: Results of the pilot study indicated that dosing neonatal mice starting on PND 1 via SC administration every 3 days was logistically feasible, produced exposures consistent with prior animal studies, and the selected dose levels were well tolerated by juvenile mice. In the definitive juvenile study, there were no LSN3338786-related deaths, clinical findings, and no effects on mean body weights, body weight gains, or food consumption. Additionally, there were no adverse LSN3338786-related hematology findings, and no macroscopic, organ weight, or microscopic findings of note. The highest dose evaluated, 150 mg/kg, was considered the NOAEL for juvenile toxicity. CONCLUSIONS: In conclusion, the juvenile animal studies did not identify any new toxicities or increased sensitivities for the intended pediatric patient population. The use of the surrogate antibody approach in a standard rodent model enabled the de-risking of theoretical concerns for toxicity in pediatric patients due to disruption of the PDGFRα pathway during early human development, such as pulmonary development.

Age-related enhanced degeneration of bioprosthetic valves due to leaflet calcification, tissue crosslinking, and structural changes.

AIMS: Bioprosthetic heart valves (BHVs), made from glutaraldehyde-fixed heterograft materials, are subject to more rapid structural valve degeneration (SVD) in paediatric and young adult patients. Differences in blood biochemistries and propensity for disease accelerate SVD in these patients, which results in multiple re-operations with compounding risks. The goal of this study is to investigate the mechanisms of BHV biomaterial degeneration and present models for studying SVD in young patients and juvenile animal models. METHODS AND RESULTS: We studied SVD in clinical BHV explants from paediatric and young adult patients, juvenile sheep implantation model, rat subcutaneous implants, and an ex vivo serum incubation model. BHV biomaterials were analysed for calcification, collagen microstructure (alignment and crimp), and crosslinking density. Serum markers of calcification and tissue crosslinking were compared between young and adult subjects. We demonstrated that immature subjects were more susceptible to calcification, microstructural changes, and advanced glycation end products formation. In vivo and ex vivo studies comparing immature and mature subjects mirrored SVD in clinical observations. The interaction between host serum and BHV biomaterials leads to significant structural and biochemical changes which impact their functions. CONCLUSIONS: There is an increased risk for accelerated SVD in younger subjects, both experimental animals and patients. Increased calcification, altered collagen microstructure with loss of alignment and increased crimp periods, and increased crosslinking are three main characteristics in BHV explants from young subjects leading to SVD. Together, our studies establish a basis for assessing the increased susceptibility of BHV biomaterials to accelerated SVD in young patients.

Neuropathology Evaluation in Juvenile Toxicity Studies in Rodents: Comparison of Developmental Neurotoxicity Studies for Chemicals With Juvenile Animal Studies for Pediatric Pharmaceuticals.

The developmental neuropathology examination in juvenile toxicity studies depends on the nature of the product candidate, its intended use, and the exposure scenario (eg, dose, duration, and route). Expectations for sampling, processing, and evaluating neural tissues differ for developmental neurotoxicity studies (DNTS) for chemicals and juvenile animal studies (JAS) for pediatric pharmaceuticals. Juvenile toxicity studies typically include macroscopic observations, brain weights, and light microscopic evaluation of routine hematoxylin and eosin (H&E)-stained sections from major neural tissues (brain, spinal cord, and sciatic nerve) as neuropathology endpoints. The DNTS is a focused evaluation of the nervous system, so the study design incorporates perfusion fixation, plastic embedding of at least one nerve, quantitative analysis of selected brain regions, and sometimes special neurohistological stains. In contrast, the JAS examines multiple systems, so neural tissues undergo conventional tissue processing (eg, immersion fixation, paraffin embedding, H&E staining only). An "expanded neurohistopathology" (or "expanded neuropathology") approach may be performed for JAS if warranted, typically by light microscopic evaluation of more neural tissues (usually additional sections of brain, ganglia, and/or more nerves) or/and special neurohistological stains, to investigate specific questions (eg, a more detailed exploration of a potential neuroactive effect) or to fulfill regulatory requests.

Regulatory Perspectives on Juvenile Animal Toxicologic Pathology.

The Society of Toxicologic Pathology's Annual Virtual Symposium (2021) included a session on "Regulatory Perspectives on Juvenile Animal Toxicologic Pathology." The following narrative summarizes the key concepts from the four talks included in this symposium session chaired by Drs Deepa Rao and Alan Hoberman. These encompass an overview of various global regulations impacting the conduct of juvenile animal studies in pharmaceutical drug development and chemical toxicity assessments in a talk by Dr Alan Hoberman. Given the numerous regulatory guidances and legal statutes that have covered the conduct of juvenile animal studies and the recent harmonization of these guidances for pharmaceuticals, Dr Paul Brown provided an update on the harmonization of these guidances for pharmaceuticals, in the recently finalized version of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S11 guidance document, "Nonclinical Safety Testing in Support of Development of Pediatric Medicines." The first two talks on regulations were followed by two talks focused on an evaluation of the postnatal development of two major organ systems relevant in juvenile animals. Dr Aurore Varela covered study design and endpoints impacting the skeletal system (bone), while Dr Brad Bolon presented a talk on the study design and conduct of neuropathology evaluations for the developing nervous system.

Juvenile Animal Testing: Assessing Need and Use in the Drug Product Label.

BACKGROUND: Juvenile animal testing has become an established part of drug development to support safe clinical use in the human pediatric population and for eventual drug product label use. METHODS AND RESULTS: A review of European Paediatric Investigation Plan decisions showed that from 2007 to mid-2017, 229 drugs had juvenile animal work requested, almost exclusively incorporating general toxicology study designs, in rat (57.5%), dog (8%), mouse (4.5%), monkey (4%), pig (2%), sheep (1%), rabbit (1%), hamster (0.5%), and species not specified (21.5%). A range of therapeutic areas were found, but the most common areas were infectious diseases (15%), endocrinology (13.5%), oncology (13%), neurology (11%), and cardiovascular diseases (10%). Examination of major clinical indications within these therapeutic areas showed some level of consistency in the species of choice for testing and the pediatric age that required support. Examination of juvenile animal study findings presented in product labels raises questions around how useful the data are to allow prescribing the drug to a child. CONCLUSION: It is hopeful that the new ICH S11 guideline "Nonclinical Safety Testing in Support of Development of Pediatric Medicines" currently in preparation will aid drug developers in clarifying the need for juvenile animal studies as well as in promoting a move away from toxicology studies with a conventional design. This would permit more focused testing to examine identified areas of toxicity or safety concerns and clarify the presentation/interpretation of juvenile animal study findings for proper risk assessment by a drug prescriber.

Lack of value of juvenile animal toxicity studies for supporting the safety of pediatric oncology phase I trials.

Toxicity studies in juvenile animals (JAS) are sometimes performed to support clinical trials in pediatric oncology patients, and there are differing conclusions on the value of JAS for pediatric drug development. This manuscript provides a review of the pediatric clinical data for 25 molecularly-targeted and 4 biologic anticancer therapeutics. Other publications that evaluated the value of JAS in pediatric drug development focus on differences in toxicity between juvenile animals and adult animals. The present paper examines pediatric-specific clinical findings to focus on dose setting in pediatric oncology patients and safety monitoring in terms of the potential value of JAS. Our assessment demonstrates that pediatric starting doses were safe for all 29 therapeutics examined in that no life-threatening toxicities occurred in the first cohort, and overall the ratio of the pediatric maximum tolerated dose (MTD) to the recommended adult dose was close to 1. In addition, the 4 serious adverse events (SAEs) that weren't detectable with standard monitoring plans for pediatric oncology trials would not have been detectable in a standard JAS. This review demonstrates that safe starting doses in pediatric oncology patients for these therapeutics could have been solely based on adult doses without any knowledge of findings in JAS.

The great barrier belief: The blood-brain barrier and considerations for juvenile toxicity studies.

Juvenile animal studies can be warranted to support the development of pediatric medicines. Drugs acting on the CNS or those which penetrate into the brain merit particular attention. The blood-brain barrier is functionally mature at birth, but undergoes functional postnatal modulation to provide a suitable microenvironment for the developing brain. In the past, dosing in rat juvenile studies has often commenced at 4 or 7days of age. However, rodents are very neurologically immature at birth compared with humans. We suggest that dosing of rat pups below two weeks of age is generally not warranted for the assessment of pediatric drugs. In the rare circumstances where exposure of younger rats is required to address a particular concern (e.g., an indication in preterm babies), consideration should be given to likely misleading signals of toxicity arising from high brain penetration of the drug, which may not be predictive for the human.

Nonclinical Safety Considerations for the Development of Pediatric-First Drugs: An Industry View.

This paper provides considerations on approaches to the development of medicines initially developed for pediatric use (ie, "pediatric-first" or "pediatric-only" drugs). The most common development approach for these types of medicines involves a first-in-human (FIH) clinical trial with healthy adult volunteers to assess safety and tolerability. This approach generally requires nonclinical repeat-dose studies in adult animals; safety pharmacology and in vivo genetic toxicology studies in adult animals are also performed for small-molecule drugs. Additional studies in juvenile animals may be required prior to clinical trials in pediatric patients, on a case-by-case basis. In this paradigm, the starting dose for pediatric patients is primarily driven by modeling from the adult pharmacokinetic assessment and pharmacology data. A second development approach is where the FIH clinical trial is conducted in pediatric patients. This approach is generally supported by repeat-dose studies in juvenile animals, with the onset of dosing at ages that developmentally correlate to the age of the pediatric patients. Safety pharmacology and in vivo genetic toxicology studies are generally performed in adult animals for small-molecule drugs. To define a safe yet minimally efficacious starting dose for pediatric patients, various complementing approaches can be used, including human equivalent dose, minimal anticipated biological effect level, and physiologically based pharmacokinetic modeling. Case examples for pediatric-first drug candidates show how both drug development approaches (ie, entry into human first in adults or directly in pediatric populations) are used in the pharmaceutical industry.

Drug safety evaluation in pediatric population:research advances / 国际药学研究杂志

The safety and risk assessment of pediatric pharmaceuticals has become a hot spot on drug development and man?agement in recent years. The shortage and unreasonable use of pediatric medicines and lack of preclinical juvenile safety evaluation of marketed pharmaceuticals have caused safety problems,a serious threat to the health and safety of pediatric population. Therefore,gov?ernments have formulated relevant regulative and administrative regulations and policies. In preclinical research ,due to the similarity of the young animals and children in the development process,a series of juvenile animal studies were carried out and the respective guidelines were gradually improved in Europe and the US. Therefore,the toxic effects of pharmaceuticals on children can be effective?ly predicted. In this paper,a detailed analysis and explanation is provided on the difficulties confronted with on R& D of pediatric pharmaceuticals,physiological differences between children and adults,and particularly,the importance and focus of preclinical safe?ty evaluation of pediatric pharmaceuticals,in order to offer a reference for the safety evaluation of pediatric drugs in China.

Determination of parameters of developmental toxicity study on pediatric drugs and focus of attention for safety evaluation of traditional Chinese medicine / 中国药理学与毒理学杂志

There is an increasing demand for neonatal and juvenile animal toxicity studies during the research and development of new drugs. In this paper,we discussed general evaluation parameters of pediatric non-clinical safety with pediatric drugs,such as growth and development and food intake,and paramenters of other organs and systems, such as the central nervous system,reproductive system, behavior evaluation in combination with our own experience. In addition,the characteristics of non-clin?ical safety evaluation of new traditional Chinese medicine materia medica used for juvenile animals were analyzed. This paper is intended reference for non-clinical safety evaluation of pediatric drugs and to gain some experience related to formulation of new guidelines.

Key principles of nonclinical safety evaluation for pediatric drugs and characteristics of traditional Chinese materia medica evaluation / 中国药理学与毒理学杂志

In current research and development of new drugs,the demand for toxicological study using neonatal and juvenile animals is becoming increasingly urgent. In this paper,we discussed the characteristics,importance and necessity of nonclinical safety evaluation for pediatric drugs,considerations for research design,selection of animal species and age,route and duration of drug administration and evaluation indexes. In addition,the characteristics of nonclinical safety evaluation of new traditional Chinese materia medica used for children were analyzed. It is hoped that these studies will not only provide support and reference for nonclinical safety evaluation of pediatric drugs but help accumulate material in formulating relevant guidelines.

Drug safety evaluation in pediatric population: Research advances / 国际药学研究杂志

The safety and risk assessment of pediatric pharmaceuticals has become a hot spot on drug development and management in recent years. The shortage and unreasonable use of pediatric medicines and lack of preclinical juvenile safety evaluation of marketed pharmaceuticals have caused safety problems, a serious threat to the health and safety of pediatric population. Therefore,governments have formulated relevant regulative and administrative regulations and policies. In preclinical research, due to the similarity of the young animals and children in the development process, a series of juvenile animal studies were carried out and the respective guidelines were gradually improved in Europe and the US. Therefore, the toxic effects of pharmaceuticals on children can be effectively predicted. In this paper, a detailed analysis and explanation is provided on the difficulties confronted with on R&D of pediatric pharmaceuticals, physiological differences between children and adults, and particularly, the importance and focus of preclinical safety evaluation of pediatric pharmaceuticals, in order to offer a reference for the safety evaluation of pediatric drugs in China.

Development of hematological and immunological characteristics in neonatal rats.

As major immunological and hematological parameters evolve during the early period of life, laboratory data must be interpreted in relation to developmental changes. Wistar (WU) rats were sacrificed on PND2, 4, 7, 10, 14, 17 and 21. Peripheral blood, bone marrow, thymus samples and spleen cells were collected and a bronchoalveolar lavage (BAL) performed. Parameters of blood counts changed considerably between time points. IgM and IgG levels steadily increased. Spontaneous spleen cell proliferation was low before PND21, although mitogens had stimulatory effects above baseline. In the spleen, T-lymphocyte counts tripled by PND17 (mainly attributed to CD8(+) cytotoxic T-cells and CD4(+) T-helper cells). In peripheral blood an increase in B-lymphocytes to about 60% of the cell number was observed. In BAL fluid, macrophages represented 95-98% of the cells. In thymus architecture, lymphoblast migration was seen and epithelial structures appeared. The data presented will help to distinguish between maturational changes and treatment-related effects.

An update of juvenile animal studies in the European Union: What do the numbers say?

Concern has been raised about the number of juvenile animal studies (JAS) being conducted in the European Union (EU) and the number of species required. The question of whether there has been an increase in the number of JAS requested by European Medicines Agency (EMA) and the question of single or multiple species studies are the focus of this manuscript. All source data on Pediatric Investigation Plans (PIP) were obtained from EMA pediatrics website and evaluated by year as obtained and adjusted to the date of the original PIP agreement rather than the newly assigned date of the PIP modification. The analysis of the data originally suggested an increase in the number of juvenile animal studies per year post 2010. However, a thorough examination of the adjusted PIP data from the EMA from December 2007 through 2013 support that the number of JAS being included in PIPs per year is not increasing but is in fact fairly stable at 15-30% per year. The data also, as expected, support that the JAS are mostly proposed with a single species and that species is predominately the rat and that multi-species JAS for an agreed PIP are infrequent.

Use of juvenile animal studies to support oncology medicine development in children.

Childhood cancer has remained a challenge because of long-term effects in children. The need to extend access of children into new cancer therapies requires early prediction of specific safety aspects and juvenile animal studies (JAS) are being conducted to screen for age-related toxicities and differences occurring during postnatal development. This paper investigates oncology approved medicines in the EU (1995-2014) and PIP (Paediatric Investigation Plans - 2007-2014), regarding the usefulness of JAS in their non-clinical development by evaluating information on the medicines labelling. The retrospective review from medicines and PIPs revealed a steady use of JAS to better characterize safety: Approximately 1 in 3 oncology medicine or PIP has conducted JAS. For 6 of the cancer medicines with JAS the toxicity profile in adult and juvenile animals showed some differences in study findings. The discussion of these cases is illustrative of the potential significance that JAS have provided in oncology medicines.

Safety of Excipients in Pediatric Formulations-A Call for Toxicity Studies in Juvenile Animals?

The development of drug products for pediatric use often requires age-appropriate formulations which can be more complex and may involve a broader range of excipients than adult dosage forms. Excipients established for adult use are not always appropriate for use in children because they can affect children differently than adults. Therefore, a comprehensive safety assessment of the excipients in a pediatric formulation is essential before use, referring to existing safety data from adult human and animals as well as safety data from pediatric use and juvenile toxicity studies, when available. The overall risk assessment needs to consider the safety risk from the excipients and the extent to which the risk from the disease as such will be ameliorated by the drug formulation. Non-clinical safety studies in juvenile animals are used to assess for specific toxicities or sensitivities of excipients and for establishing safe exposures in pediatric age groups. As for any active ingredient, non-clinical safety studies in juvenile animals should only be performed for excipients if important for clinical risk assessment and labelling. Pharmaceutical companies should be critical of excessive demands for juvenile animal testing, particularly of excipients when critically needed for significant therapeutic benefit.