Case analysis and key considerations of toxicology experiments on young animals

Dan Xiaolei, Huang Fanghua *, Jiang Kaidi

(Drug Evaluation Center of the National Medical Products Administration, Beijing 100022)



In recent years, pediatric medication has become a hot topic in drug development, and clinical trials for pediatric populations have gradually increased. When there is a lack of non clinical and clinical data to support the safety of conducting pediatric clinical trials, it is necessary to consider conducting juvenile animal trials aimed at addressing safety issues that cannot be fully addressed in other non clinical or pediatric clinical trials, including potential long-term safety impacts. This article is based on the non clinical safety guidelines for pediatric drug development supported by ICH S11 released in 2020, and combines practical cases to explain the key considerations for the design and implementation of juvenile animal experiments, in order to provide reference for pediatric drug development in China.

In recent years, China has continuously encouraged and promoted the research and innovation of children's medication, and the number of applications and evaluations for children's medication has shown an increasing trend year by year. The development of drugs for children, including products already used in adults and products considered for first-time use in children, should consider conducting juvenile animal toxicology studies (JAS) based on specific circumstances. The design and implementation of JAS are complex and difficult. This article will use case studies to elaborate and analyze some key considerations of JAS, providing reference for the development of pediatric medication.

No.1

Guidelines for Non Clinical Safety Evaluation of Pediatric Drug Development

Due to the fact that children are in the stage of growth and development, their organ systems may not be mature or mature during drug treatment, which may have an impact on the pharmacokinetics, pharmacodynamics, and off target effects of drugs, leading to differences in drug safety and efficacy between children and adults. Therefore, it is necessary to consider whether JAS is necessary to address safety issues that cannot be fully addressed in other non clinical trials or pediatric clinical trials [1].

Since the 21st century, countries have successively issued non clinical safety research guidelines to support the development of drugs for children. The FDA released the "Guidance for Industry: Non clinical Safety Evaluation of Pediatric Drug Products" in 2006; EMA released the "Guidelines on the Need for Non Clinical Testing in Juvenile Animals on Human Pharmaceuticals for Pediatric Indicators" in 2008, and the Japanese Ministry of Health Labour and Welfare (MHLW) released the "Guidelines on Non Clinical Safety Studies in Juvenile Animals for Pediatric Drug Development" in 2012. In order to promote regional coordination and consistency, the International Council on Technical Requirements for Registration of Medicines for Human Use (ICH) established the S11 Expert Working Group in 2014 to internationally coordinate non clinical safety research content supporting pediatric drug development. On April 14, 2020, ICH S11 "Guiding Principles for Non Clinical Safety Evaluation Supporting Pediatric Drug Development" (hereinafter referred to as S11) was released. The National Medical Products Administration (NMPA) of China participated in the coordination of ICH S11 issues as a member of ICH and issued the S11 Implementation Announcement (No. 15 of 2021) on January 21, 2021.

S11 recommends internationally coordinated standards for non clinical safety evaluation to support the development of drugs for children, applicable to small molecule drugs and biologics intended for use in the pediatric population, and highlights the necessity of JAS and specific requirements for JAS design. Due to the necessity of JAS and the need for specific analysis of its experimental design, it has considerable flexibility. Therefore, this article combines case analysis to explore and analyze some key considerations for young animal experiments, in order to provide reference for the development of drugs for children in China.

No.2

Consideration of additional JAS

ICH M3 and S11 propose [1,5] that additional non clinical trials should only be conducted when existing non clinical and clinical research data are deemed insufficient to support pediatric research. The additional non clinical trials generally refer to JAS. S11 proposed the weight of evidence (WoE) method for determining whether additional non clinical safety studies are necessary. The key factors to be considered in WoE evaluation include but are not limited to the minimum age of the intended patient, adverse effects on developing organs, the quantity and type of existing data (including clinical and non clinical data), the effect of drug targets on organ development, drug selectivity and specificity, and clinical administration duration. Among them, the minimum age of the proposed patient and the adverse effects on the developing organ system are the most important (i.e. the highest weight). The WoE method requires simultaneous evaluation of multiple factors, taking into account the importance of each factor, in order to ultimately determine whether existing data can fully address safety concerns in the applied child population, or whether additional JAS can address these concerns [1].

Lacosamide (trade name Vimpat) is an antiepileptic drug developed by USB Corporation in the United States. It was first approved by the FDA in October 2008 for use in patients aged 17 and above [6], and requires clinical trials in children aged 1 month to 17 years after market launch [7]. Based on multiple pediatric clinical trial data conducted after the launch of Lacosamide, the FDA currently approves Lacosamide for the treatment of partial seizures in patients over 1 month of age and as an adjuvant therapy for primary generalized tonic clonic seizures in patients 4 years of age and older [8].

Lacosamide acts on the central nervous system (CNS), and the minimum age for clinical use is 1 month. Existing data is not sufficient to demonstrate the safety of the drug for children, and both target and toxicological tests suggest potential effects on the developing CNS. Therefore, in addition to the general toxicology data package (including single and repeated dose toxicity tests in rats and dogs, genetic toxicity, reproductive toxicity, and carcinogenicity tests), the non clinical safety studies conducted on Lacosamide also included repeated dose toxicity tests in young rats to support clinical trials in pediatric populations. In addition, a GLP test was conducted to explore the dose range of repeated dose toxicity in young dogs before applying for marketing [9]. In addition, this breed underwent a 33 week repeated administration toxicity test on young dogs.



In the JAS of Lacosamide rats, young rats were orally administered Lacosamide (30, 90, 180 mg/kg per day) starting from postnatal day (PND) 7 for 42 consecutive days. This resulted in weight loss during lactation, delayed female sexual maturation, decreased absolute and relative brain weight, and long-term neurobehavioral changes (such as changes in open field behavior, learning and memory deficits) [6,9]. Moreover, the exposure to developmental neurotoxicity in rats was lower than the maximum recommended dose of 400 mg per day for humans. In addition, in vitro experiments have shown that lacosamide interferes with the activity of CRMP-2, a brain failure response regulatory protein involved in neuronal differentiation and controlling axonal outward growth [8]. Therefore, it cannot be ruled out that lacosamide may have potential adverse reactions related to CNS development.



In summary, as a CNS drug, the minimum age for clinical use of lacosamide is 1 month old. According to existing research results, potential concerns about the drug's impact on developing CNS cannot be ruled out. Prior to the pediatric clinical trial, rat JAS was conducted, and it was found that lacosamide has adverse effects on CNS development in rats. It is worth noting that it is generally believed that in terms of brain development, the early postnatal period in rats corresponds to late pregnancy in humans. Therefore, the FDA has included the above risk warning information in the "Pregnancy" and "Pediatric Medications" sections of the "Special Population Medication" instructions to provide safety information for clinical use [8].

No.3

Key points of experimental design
3.1 Selection of animal species

On the basis of existing toxicology data packages that support adult clinical trials, when JAS is required, in most cases, single animal species testing is sufficient. In principle, the same species used in repeated dose toxicity testing in adult animals should be the first species considered by JAS, with rodents being the preferred species. In all cases, it should be demonstrated that the selected species is reasonable and belongs to the relevant animal species. Only when used in pediatrics or when there are multiple special concerns about postnatal development that cannot be addressed by a single species, is JAS for two genera necessary.

Rats are a commonly used species for repeated drug toxicity testing in adult animals, with rich historical control data. Their short developmental time allows for the inclusion of a large number of endpoint indicators in the experiment, observing the development of multiple organ systems after birth. Therefore, rats (when they are related animal species) are the animals commonly used in JAS. For biological products, in most cases, non-human primates (NHPs) are pharmacology related species. However, due to scientific and practical reasons, performing JAS on pre weaning NHPs poses great challenges (such as reproduction, transportation, and handling of mother/infant pairs), and the maturity of organ systems in post weaning NHPs (approximately 6 months old) often exceeds many pediatric related ages. The added value of JAS in post weaning NHPs is limited, and only in a few cases, JAS in pre weaning NHPs has reasonable value, such as in intended use for newborns and enhanced pre - and post natal development. Due to insufficient exposure in developmental, ePPND experiments, S11 encourages the use of alternative methods (such as in vitro experiments, transgenic animals, alternative molecules) [1].

The usual clinical strategy for pediatric or pediatric specific drugs is to conduct the first human (FIH) trial in healthy adult volunteers before pediatric trials. However, in some cases where medication is administered to pediatric patients without data from adult patients or healthy volunteers (such as life-threatening or debilitating diseases that only exist in children, or when the medication cannot be safely administered to adult volunteers), FIH trials will be conducted in pediatric patients, and non clinical plans typically include one rodent and one non rodent JAS, as well as the same safety pharmacology and genotoxicity tests required for adult medication [1].

Nusinersen sodium (trade name Spinraza) is an antisense oligonucleotide (ASO) drug developed by Biogen Idec in the United States. It can increase the inclusion of exon 7 in the mRNA transcript of survival motor neuron 2 (SMN2) and the production of full-length SMN protein. It is administered intrathecally via lumbar puncture and is used to treat spinal muscular atrophy (SMA), a serious autosomal recessive genetic disease that causes infant and child mortality due to SMN protein deficiency caused by chromosome 5q mutations. Nordenafil Sodium was approved for marketing by the US FDA in December 2016, and is suitable for use in newborns to 17 year olds [10].

The non clinical safety studies completed before the market launch of Norsen Sodium include: safety pharmacology tests for intrathecal administration in rats, genetic toxicity tests [bacterial recovery mutation test, in vitro Chinese hamster ovary cell (CHO cell) chromosome aberration test, mouse in vivo bone marrow micronucleus test], reproductive toxicity tests (mouse fertility and embryonic development test, rabbit embryo fetal development toxicity test), and toxicity tests in young monkeys and mice [11]. The FDA requires a 2-year carcinogenicity test for subcutaneous administration in mice and a perinatal toxicity test for rodents after listing [12].

The following describes a case study of toxicology of a pediatric drug using two young animal species.

In terms of repeated administration toxicity tests, sodium noshenate was subjected to JAS in two animals, including 14 week and 53 week repeated administration toxicity tests in young crab eating monkeys, and 13 week repeated administration toxicity tests in young mice. Among them, crab eating monkeys are more representative in terms of tissue distribution, cell uptake, metabolism, and toxicity sensitivity of ASO, so the crab eating monkey test is the core experiment. Young crab eating macaques were given intrathecal injections of 0.3, 1, and 3 mg of sodium noshenate each time for 14 consecutive weeks (the starting age of monkey administration was 9-10 weeks old, with a loading dose phase of once a week for a total of 5 times, followed by a maintenance phase of once every 2 weeks or once a week) or 0.3, 1, and 4 mg each time for 53 consecutive weeks (the starting age of monkey administration was 9-11 months old, with a loading dose phase of once a week for a total of 5 times, and then a maintenance phase of once every 6 weeks). In two experiments, pathological changes in brain tissue were observed in the high and medium dose groups (hippocampal neuronal vacuolization and necrosis, as well as cell debris), while acute and transient low-level spinal reflex defects were observed in the high dose group. In addition, in the 53 week monkey trial, potential neurobehavioral deficits were observed in the learning and memory tests of the high-dose group. After annual calculation and correction for differences in cerebrospinal fluid volume between different species, the pathological dose of monkey nerve tissue without effect (0.3 mg each time) is approximately equivalent to the human dose [10-11]. In addition, a 13 week repeated administration toxicity test was conducted on young CD-1 mice, administered between 4-95 days after birth, and showed good tolerance at a maximum dose of 50 mg/kg per dose, indicating oligonucleotide related changes [11].

In summary, as the world's first approved ASO drug for SMA treatment, sodium nosine is a pediatric drug intended for use in newborns. Clinical trials were conducted directly on children, and non clinical safety studies were conducted on young mice at 3 months, as well as on young crab eating macaques via intrathecal administration for 4 and 53 weeks. CNS assessments were also conducted, indicating adverse effects on the nervous system. This risk warning information is included in the instruction manual.

3.2 Age at which animals start receiving medication

The age at which animals start administering medication is a key element in JAS design, and the minimum age corresponding to the intended patient's development should be selected, taking into account the toxicological concerns of comparing organ system development stages between humans and animals. Due to the inconsistent correlation between organ systems of different species and organs, priority should be given to any potential target organs/systems of concern, or developing systems that are particularly vulnerable in the intended patient population [1]. S11 provides a detailed overview of the comparative development of organ systems in different animal species in the appendix, which can help select appropriate animal species and ages to fully address the safety concerns of the applied pediatric population.



In a survey published in 2011 on non clinical safety studies of pediatric drugs from 24 pharmaceutical companies in the United States, Europe, and Japan [13], based on the limited number of trials provided in the survey at the age of onset of administration, it appears that there was a trend in the late 1990s and early 2000s to start administration at the earliest age of administration, rather than at the age representing the developmental stage. Among the 118 rat JAS experiments, 94 were administered before weaning, but only 23 were intended for newborns, while the rest were for children over 2 years old. This trend began to shift towards a more representative age group in early 2006, which corresponds to the guidelines issued by the FDA in 2006. Domestic JAS started relatively late and practical experience is not yet mature. It is difficult to conduct experiments using pre weaning animals, so there are often cases where JAS experimental animals start to be administered at an older age and cannot support clinical trials using the minimum age of patients.

In a JAS study of 15 targeted CNS drugs published in 2019 (all sourced from electronic documents of the EMA Pediatric Committee), [14] described a case of animals starting treatment at an older age. Epi6 (code, product name not listed for commercial protection reasons) is used for the treatment of partial epileptic seizures. The intended patients include premature newborns. In the critical JAS trial, the age of administration to rats was PND28. From the perspective of CNS development stage, PND28 was older than the minimum age of the intended patients. In addition, compared with PND28, the toxicity severity of rats administered from PND21 was significantly increased (based on the occurrence of severe clinical symptoms at lower doses in younger rats leading to terminal euthanasia), indicating that rats at younger ages are more sensitive to toxicity. Therefore, starting from PND28, rats missed the sensitive critical window period of brain development and were unable to sensitively detect adverse effects related to the test substance.

3.3 Dose Exploration Experiment
The difficulty of conducting JAS experiments is high, and multiple factors can affect the success or failure of the experiments. Therefore, dose range fitting (DRF) experiments should be considered before formal experiments. S11 recommends conducting DRF experiments on young animals in other groups to evaluate tolerance related to exposure and age. This is particularly valuable for formal JAS that is administered before weaning, as it can avoid most accidental deaths or excessive toxicity caused by unrelated exposure levels. DRF should be administered at the minimum age planned for animals in the formal JAS to evaluate tolerance and exposure differences during the most critical period. In formal JAS, the dosing regimen should ensure that the relevant exposure levels can be achieved and maintained during the developmental period of concern. If the maturation of the animal absorption, distribution, metabolism, and excretion (ADME) system leads to significant changes in system exposure, dose adjustments (increase or decrease) should be considered during the formal JAS process to maintain a certain degree of consistency and clinical relevance of exposure. Normally, dose adjustments are not expected to exceed once during JAS [1].

POSOBIEC et al. described a case study of dose adjustment based on DRF results in formal JAS [15]. In this case, a rat JAS administered at PND7-45 is planned to support medication in the population of patients aged 3 months to adults. Due to the observation of ovarian toxicity (0.02 mg/kg per day) that may be related to pharmacological effects in adult rat toxicity tests, the administration period of JAS was continued until the expected sexual maturity of female rats to coincide with the administration period of adult animal toxicity tests, and the estrous cycle of sexually mature animals was evaluated in JAS. Firstly, an exploratory experiment was conducted on the administration of PND7-21 to rats, and severe toxicity was observed at doses ≥ 0.1 mg/kg; Subsequently, a preliminary dose exploration experiment was conducted for PND7-35 administration in rats, with doses of 0.01, 0.02, and 0.05 mg/kg. Toxicokinetic testing was performed at PND13, 21, and 35, and it was observed that the drug exposure sharply decreased during administration. At the final time point (PND35), the exposure levels at the three doses were similar and lower than the expected clinical exposure levels. Next, a dose range experiment was conducted, with rats divided into two groups and given 0.05 mg/kg at PND7-21. To explore how to better maintain consistent exposure levels in the later stages of development, the dose was increased from PND22, with one group increasing to 0.08 mg/kg and the other group increasing to 0.17 mg/kg. After adjustment, the exposure levels of the two groups showed a significant difference. Finally, based on the results of the dose exploration experiment, in the formal JAS, rats were administered doses of 0.0125, 0.025, and 0.05 mg/kg at PND7-21, and the doses were increased to 0.08, 0.17, and 0.35 mg/kg starting from PND22. Through this dose adjustment design, the exposure level overlaps with the high dose of adult animals (0.08 mg/kg for females and 0.2 mg/kg for males), and the same group of rats have relatively consistent exposure to the drug during development. The exposure levels of the three dose groups can be distinguished, allowing for observation of dose-response correlations, and the exposure levels of the medium and high dose groups also cover the expected clinical exposure level of 370 ng · h-1 · ml-1.

3.4 Selection of endpoint indicators
The JAS endpoint indicators include core endpoint indicators and additional endpoint indicators. In addition to general toxicology indicators, the core endpoint indicators also include growth (body weight and long bone length) and sexual development (such as vaginal opening time and male foreskin separation time in female rodents). In addition, to elaborate on the identified concerns, additional endpoint indicators should be included based on the type and intensity of concerns identified in the WoE evaluation, including other growth endpoints, bone assessment, clinical pathology, anatomical pathology, ophthalmic examination, CNS assessment, reproductive assessment, and immune assessment. Due to the high safety risk of neurodevelopmental toxicity (DNT), which is difficult to detect in clinical trials or post marketing monitoring, CNS assessment is a frequently added endpoint indicator in JAS research, especially for drugs with CNS activity.



A study summarized and analyzed 44 JAS trials of 32 drugs with CNS activity in FDA reviewed NDA applications between 2009 and 2014 [16]. Most drugs (20/32) underwent a rat JAS, some drugs (11/32) underwent JAS in rats and dogs, and only one drug (1/32) underwent a dog JAS. In all JAS, standard general toxicological parameters and growth and development indicators are included, and extended neurohistopathological evaluations, bone density measurements, reproductive evaluations, and neurobehavioral/CNS functional evaluations are typically conducted. Due to their CNS activity, over half of the trials (25/44) included extended neuropathological evaluations, while the remaining trials (19/44) underwent routine CNS histopathological examinations. Due to drug exposure occurring during the dynamic period of bone formation and growth in pediatric patients, bone development is also a focus of JAS. There were 23 experiments with specific bone development endpoints (excluding histopathology), among which 14 experiments (including 8 rat and 6 dog experiments) evaluated bone length and bone density (dual energy X-ray absorptiometry), and 9 experiments (rat experiments) only included bone length. In terms of reproductive assessment, mating and pregnancy outcome indicators were evaluated in all rat experiments (32 items), reproductive organ histopathology was evaluated in all rat and dog experiments (44 items), and sperm analysis was performed in 16 rat and dog experiments.

In terms of neurobehavioral/CNS functional assessment, various neurological and neurobehavioral tests were used, with detection indicators varying by species, mainly including neurological examination or functional observation combination (FOB) in dogs, as well as more specific and quantitative assessments in rats, such as motor activity, auditory startle behavior, and learning and memory detection. All 32 rat experiments were conducted using automated systems for one or more learning and memory tests, and most of the experiments measured motor activity and auditory startle behavior. In most rat JAS, complex water mazes were used to assess whether they caused long-term cognitive impairment, including Morris water maze (11 items), Biel water maze (8 items), and Cincinnati water maze (8 items).

These JAS provide safety information for pediatric medication and are included in the instructions (78%) to indicate risks, with the most common CNS effects being neurobehavioral abnormalities, including changes in motor activity, auditory startle reflex, and learning and memory. Among the neurobehavioral effects included in the instructions, 54% of the experiments found that these effects were persistent, indicating neurodevelopmental toxicity.


No.4
summary
The non clinical safety evaluation to support the development of pediatric drugs poses challenges for both applicants and trial researchers. On the one hand, this is due to the difficulty in designing and implementing non clinical/clinical trials in pediatric drug development, and on the other hand, it requires a comprehensive evaluation ability of existing research information. JAS can provide important reference information for risk identification and characterization of pediatric populations, and can elucidate safety concerns related to drug exposure risks during specific developmental stages. It is an important non clinical safety evaluation method for pediatric drug development. Based on the weight analysis of evidence (WoE), specific problems should be analyzed, and JAS should be designed comprehensively, scientifically and reasonably according to the various information obtained in the early stage, while ensuring the good implementation of the experimental process, in order to obtain valuable toxicological data to support the conduct of clinical trials in children and ensure the safety of pediatric subjects.


Content source: Chinese Journal of Pharmaceuticals 2022, 53 (11)