Nonclinical Safety Research Articles

The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain

Assessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs.

Points to consider for non-clinical safety evaluation of <i>in vivo</i> gene therapy products

Advances in gene therapy have led to a wide range of gene therapy products. Hence, it is challenging to use standard non-clinical safety tests, which are typically used to evaluate chemically synthesized products. Therefore, a case-by-case safety evaluation based on the unique characteristics of each product is necessary. In Japan, the fundamental considerations for evaluating the non-clinical safety of gene therapy products are outlined in “Ensuring the Quality and Safety of Gene Therapy Products”. Notably, when designing non-clinical safety studies, it is essential to consider factors such as the efficiency of vector transduction, tissue/cell tropism of the vector, and species differences in the expression level and biological activity of transgene products in the target tissue/organs. However, it is also crucial to understand the limitations of animal studies that arise due to species differences and to assess the safety of expression vectors and expressed genes based on a thorough literature review of their biological properties. The aims of this review are to present non-clinical safety evaluation approaches for gene therapy products as outlined in the Japanese guidelines and key considerations for appropriate non-clinical safety evaluation of in vivo gene therapy products.

Marketing Regulatory Oversight of Advanced Therapy Medicinal Products in Europe.

Advanced therapy medicinal products (ATMP) in the European Union (EU) are regulated by Regulation 1394/2007 and comprise gene and cell therapy and tissue-engineered products. Under this framework, ATMP are authorised by the centralised procedure, coordinated by the European Medicines Agency (EMA), whereas clinical trial authorisations remain at the remit of each National Competent Authority. The Committee for Advanced Therapies is responsible for the scientific evaluation of the marketing authorisation applications and for generating a draft opinion that goes to the Committee for Human Medicinal Products for a final opinion. For every application, data and information relating to manufacturing processes and quality control of the active substance and final product have to be submitted for assessment together with data from non-clinical and clinical safety and efficacy studies. Technical requirements for ATMP are defined in the legislation, and guidance for different products is available through several EMA/CAT guidelines.Due to the diverse and complex nature of ATMP, a need for some regulatory flexibility was recognised. Thus, a risk-based approach was introduced in Regulation 1394/2007 allowing adapted regulatory requirements. This has led, for instance, to the development of good manufacturing practice (GMP) guidelines specific for ATMP. This, together with enhanced regulatory support, has allowed an increasing number of successful marketing authorisation applications resulting in 25 licensed ATMP in the EU, mainly gene therapy medicinal products. The promise of messenger RNA and genome editing technologies as therapeutic tools make the future for these innovative medicinal products look even brighter.This chapter reviews the regulatory landscape together with some of the support initiatives developed for ATMP in the EU.

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment_suppl

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment_suppl

The first nonclinical pharmacokinetic and safety report of IVL3004, a novel long-acting injectable naltrexone formulation

The first nonclinical pharmacokinetic and safety report of IVL3004, a novel long-acting injectable naltrexone formulation

Preclinical safety assessment of JNJ-10450232 (NTM-006), a structural analog of acetaminophen, that does not cause hepatotoxicity at supratherapeutic doses

JNJ-10450232 (NTM-006) is a new molecular entity that is structurally related to acetaminophen. A comprehensive non-clinical safety program was conducted to support first-in-human and clinical efficacy studies based on preclinical data suggesting that the compound has comparable or enhanced antinociceptive and antipyretic efficacy without causing hepatotoxicity at supratherapeutic doses. No hepatic toxicity was noted in a mouse model sensitive to acetaminophen hepatotoxicity or in rats, dogs, and non-human primates in 28-day repeat dose toxicity studies at and above doses/exposures at which acetaminophen is known to cause hepatotoxicity. In the 28-day toxicity studies, all treatment-related findings were monitorable and reversible. Methemoglobinemia, which was observed in dogs and to a lesser extent in rats, is also observed with acetaminophen. This finding is considered not relevant to humans due to species differences in metabolism. Thyroid hypertrophy and hyperplasia were also observed in dogs and were shown to be a consequence of a species-specific UGT induction also demonstrated with increased thyroid hormone metabolism. Indirect bilirubin elevation was observed in rats as a result of UGT1A1 Inhibition. JNJ-10450232 (NTM-006) had no toxicologically relevant findings in safety pharmacology or genotoxicity studies. Together, these data supported progressing into safety and efficacy studies in humans.

Toward implementing virtual control groups in nonclinical safety studies.

Historical data from control groups in animal toxicity studies are currently mainly used for comparative purposes to assess validity and robustness of study results. Due to the highly controlled environment in which the studies are performed and the homogeneity of the animal collectives it has been proposed to use the historical data to build so-called virtual control groups, which could partly or entirely replace the concurrent control group. This would constitute a substantial contribution to the reduction of animal use in safety studies. Before the concept can be implemented, the prerequisites regarding data collection, curation, and statistical evaluation together with a validation strategy need to be identified to avoid any impairment of the study outcome and subsequent consequences for human risk assessment. To further assess and develop the concept of virtual control groups, the transatlantic think tank for toxicology (t4) sponsored a workshop with stakeholders from the phar­maceutical and chemical industry, academia, FDA, contract research organizations (CROs), and non-governmental organizations in Washington, which took place in March 2023. This report sum­marizes the current efforts of a European initiative to share, collect, and curate animal control data in a centralized database and the first approaches to identify optimal matching criteria between virtual controls and the treatment arms of a study as well as first reflections about strategies for a qualifi­cation procedure and potential pitfalls of the concept.

Effects of intravenous infusion, vest wearing and repeated intravenous blood collection on clinical pathology parameters in cynomolgus monkeys (Macaca fascicularis).

Nonhuman primates are used extensively in a variety of nonclinical safety evaluation studies of new drugs. In those studies, intravenous infusion is a common treatment method, a noninvasive telemetry system is usually used for cardiovascular safety and pharmacology evaluation, and blood samples are repeatedly collected for various analysis. Intravenous infusion, vest wearing, and repeated intravenous blood collection can caused a stress response in cynomolgus monkeys, which may lead to changes in clinical pathology parameters in them. Here, we aimed to test the effects of the above operations on clinical pathology parameters in cynomolgus monkeys. Twenty monkeys (10 male/10 female) were administered 0.9% sodium chloride injections via intravenous infusions on Days 1 and 10. Each animal wore a vest before each dosing, and the vest was removed at 24 h after each dosing. Blood samples were collected before the first dose and at 2 min, 24 h, 48 h, 72 h, and 168 h after each dosing. As compared to values before the first dose (D-1) increases in reticulocytes (percentage and absolute count) and decreases in erythrocytes (red blood cells, hemoglobin, and hematocrit) were noted after dosing. The decrease in erythrocytes and increase in reticulocytes were considered to the related to the repeated intravenous blood collection. Increases in leukocytes (white blood cells and absolute count and percentage of neutrophils) and platelets (mean platelet volume and platelet distribution width) were noted at 2 min or 24 h post dose. Increases in aspartate aminotransferase, direct bilirubin, creatine kinase, C-reactive protein, and human cardiac troponin I and decreases in inorganic phosphate were noted at 2 min to 72 h post dose.

Toxicologic Pathology Forum Opinion: Interpretation of Gliosis in the Brain and Spinal Cord Observed During Nonclinical Safety Studies.

Gliosis, defined as a nonneoplastic reaction (hypertrophy and/or proliferation) of astrocytes and/or microglial cells, is a frequent finding in the central nervous system (CNS [brain and/or spinal cord]) in nonclinical safety studies. Gliosis in rodents and nonrodents occurs at low incidence as a spontaneous finding and is induced by various test articles (e.g., biomolecules, cell and gene therapies, small molecules) delivered centrally (i.e., by injection or infusion into cerebrospinal fluid or neural tissue) or systemically. Several CNS gliosis patterns occur in nonclinical species. First, gliosis may accompany degeneration and/or necrosis of cells (mainly neurons) or neural parenchyma (neuron processes and myelin). Second, gliosis often follows inflammation (i.e., leukocyte accumulation causing parenchymal damage) or neoplasm formation. Third, gliosis may appear as variably sized, randomly scattered foci of reactive glial cells in the absence of visible parenchymal damage or inflammation. In interpreting test article-related CNS gliosis, adversity is indicated by parenchymal injury (e.g., degeneration, necrosis, or inflammation) and not the mere existence of a glial reaction. In the absence of clear structural damage to the parenchyma, gliosis as a standalone CNS finding should be interpreted as a nonadverse reaction to regional alterations in microenvironmental conditions rather than as evidence of a glial reaction associated with neurotoxicity.

FDA and industry collaboration: Identifying opportunities to further reduce reliance on nonhuman primates for nonclinical safety evaluations

The nonhuman primate (NHP) has always been a limited resource for pharmaceutical research with ongoing efforts to conserve. This is due to their inherent biological properties, the growth in biotherapeutics and other modalities, and their use in small molecule drug development. The SARS-CoV-2 pandemic has significantly impacted the availability of NHPs due to the immediate need for NHPs to develop COVID-19 vaccines and treatments and the China NHP export ban; thus, accelerating the need to further replace, reduce and refine (3Rs) NHP use. The impact of the NHP shortage on drug development led DruSafe, BioSafe, and the United States (U.S.) Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) to discuss this issue at their 2021 annual meeting. This meeting identified areas to further the 3Rs in NHP use within the current nonclinical safety evaluation regulatory framework and highlighted the need to continue advancing alternative methods towards the aspirational goal to replace use of NHPs in the long term. Alignment across global health authorities is necessary for implementation of approaches that fall outside existing guidelines. This article captures the proceedings from this meeting highlighting current best practices and areas for 3Rs in NHP use.

Reference intervals and method sensitivity for electrocardiology, hemodynamics, and body temperature parameters in healthy cynomolgus monkeys

In nonclinical studies, electrocardiograms (ECG) of cynomolgus monkey are recorded intermittently by external leads in manually restrained animals (snapshot recording) or continuously by jacketed external telemetry (JET) or implanted radiotelemetry transmitter in freely moving animals. With the implanted device, blood pressure and core body temperature can be monitored simultaneously. Despite the frequent use of cynomolgus monkeys in nonclinical safety pharmacology testing, few reference data are available for this species, comparisons of the ECG recording methods are limited, and power analyses are seldom conducted. In this study, pretreatment data were recorded from 406, 663, and 131 healthy experimentally naïve monkeys using the snapshot, JET, and implantable method, respectively, from 2019 to 2021. Reference intervals were determined for ECG, blood pressure, and body temperature parameters. Diurnal effects were observed in these parameters, with the exception of QRS and pulse pressure. The QRS, QT, and heart rate-corrected QTc intervals, as well as blood pressure, had a weak positive relationship with age and/or body weight. There were no sex differences in these parameters, and the country of origin only had minimal influences. Compared to telemetry, snapshot ECG data had shorter RR, PR, and QT intervals and longer QRS interval. The JET and implanted telemetry ECG data were comparable. Effect size analysis was conducted to estimate the method sensitivity for each parameter in common non-clinical study design scenarios. Snapshot recording, JET, and implanted telemetry were sensitive to detect 7–15 milliseconds of changes in QTc intervals in standard study designs, indicating these are powerful methods for assessment of QT prolongation in vivo.

Nonclinical safety assessments of a novel synthetic toll-like receptor 4 agonist and saponin based adjuvant

A full nonclinical safety package was performed to support the clinical use of SPA14, a novel liposome-based vaccine adjuvant containing the synthetic toll-like receptor 4 agonist E6020 and saponin QS21.E6020 and QS21 were tested negative for their potential genotoxic effects in Ames, micronucleus, or mouse-lymphoma TK (thymidine kinase) assay. To evaluate the potential local and systemic effects of SPA14, two toxicity studies were performed in rabbits. In the first dose range finding toxicity study, rabbits received two intramuscular injections of SPA14 at increasing doses of E6020 combined with two antigens, a control (saline), the two antigens alone, or the antigens adjuvanted with a liposome-based adjuvant AS01B. No systemic toxicity was detected, supporting the dose of 5 μg of E6020 for the subsequent pivotal study. In the second repeated dose toxicity study, rabbits received four intramuscular injections of SPA14 alone, a control (saline), SPA14 combined with two antigens, the two antigens alone, or the antigens combined with AF03 adjuvant, which is a squalene-based emulsion. SPA14 alone or in combination with the antigens was well tolerated and did not cause any systemic toxicity. Finally, two safety pharmacology studies were conducted to assess potential cardiovascular and respiratory effects of E6020 and SPA14 in conscious telemetered cynomolgus monkeys and beagle dogs, respectively. One subcutaneous injection of E6020 in monkeys and one intramuscular injection of SPA14 in dogs had no consequences on respiratory and cardiovascular functions. Altogether these results support the clinical development of SPA14.

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format. The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Nonclinical Safety Assessment of Lipid Nanoparticle-and Emulsion-Based Self-Amplifying mRNA Vaccines in Rats.

Vaccines containing mRNA with the capacity to self-amplify represent an alternative to the mRNA vaccines that came to prominence during the COVID-19 pandemic. To gain further insights on the safety profile of self-amplifying mRNA- (SAM-) vaccines, this preclinical toxicology study in rats evaluated the effect of (i) the type of delivery system (lipid nanoparticle [LNP] vs cationic nano-emulsion [CNE]); (ii) antigen-encoding sequence (rabies glycoprotein G vs SARS-CoV-2 Spike); and (iii) RNA amplification. Further analyses also evaluated gene expression in peripheral blood after vaccination, and the biodistribution of vaccine RNA. The SAM vaccines administered as two doses 2-weeks apart had acceptable safety profiles in rats, with respect to clinical signs, blood biochemistry, and macroscopic and microscopic pathology. A transient increase in ALT/AST ratio occurred only in female rats and in the absence of muscle and liver damage was dependent on RNA amplification and appeared related to the greater quantities of vaccine RNA in the muscle and livers of female rats vs male rats. The RNA and delivery-vehicle components, but not the nature of the antigen-coding sequence or the requirement for RNA amplification, affected aspects of the stimulation of innate-immune activity, which was consistent with the transient activation of type I and type II interferon signaling. The delivery vehicle, LNP, differed from CNE as vaccine RNA in CNE compositions appeared independently to stimulate innate-immune activity at 4 hours after vaccination. Our analysis supports further studies to assess whether these differences in innate-immune activity affect safety and efficacy of the SAM vaccine.

Toxicology studies for compound preparations of traditional Chinese medicine in ancient classic prescriptions

According to Provisions for Drug Registration and Categories and Requirements of Application Dossiers for Traditional Chinese Medicine(TCM), non-clinical safety research/toxicology research should be conducted for classical compound Chinese medicine prescriptions(Categories 3.1 and 3.2), and the data should be submitted at the application for the marketing authorization. Based on the requirements of toxicology research in Categories and Requirements of Application Dossiers for Traditional Chinese Medicine, this study further refined and clarified the requirements of toxicology research on classical compound Chinese medicine preparations(Categories 3.1 and 3.2).

Preclinical development of a small molecule inhibitor of tau self‐association for Alzheimer’s disease and related dementias

AbstractBackgroundThe approach for targeting toxic tau aggregates was to develop a small molecule inhibitor of tau self‐association that is agnostic to the types of aggregates formed. Pharmacodynamic studies of the lead compound in mouse models of tauopathy were performed. Therapeutic treatment of aged P301L tau JNPL3 mice showed reduced tau aggregation and improved motor behavior. Preventive treatment studies in young JNPL3 and htau mice with the lead also showed reduced tau aggregation. These results supported preclinical development of the lead to evaluate the potential of the compound for an Investigational New Drug Application (IND) submission to enable a first‐in‐human study.MethodProcess development and manufacturing of the compound was performed for the nonclinical safety studies (non‐GLP) and for drug substance (GLP) for clinical studies. To enable nonclinical studies, formulation work for oral administration of the compound was performed for pharmacokinetic studies to ensure sufficient oral bioavailibility for the toxicity studies. Dose range finding studies included a maximum tolerated dose (MTD) phase and a 14‐day repeat oral dosing phase in rat and dog (non‐GLP).ResultManufacture of the compound has been scaled to a 3 Kg batch size with 100% purity by HPLC (% area) as a single polymorph. Manufacture of capsule drug product for clinical studies was completed. Both the drug substance and drug product have shown good stability at room temperature storage. Oral bioavailibility was about 70% in rats and 40‐60% in dogs. Oral doses of 100, 300 and 1000 mg/kg/day in rats, and doses of 50 and 150 mg/kg/day in dogs for 14 days were well tolerated clinically, but the 500 mg/kg/day dose in dogs caused slight generalized tremors 1‐2 hours post‐dosing on multiple days. Hepatic effects were seen in both species, mostly at the higher doses in rats.ConclusionThe results of the 14‐day studies enabled the selection of doses for the 28‐day rat and dog toxicity studies (GLP). The results of these studies in combination with the array of studies required for the IND application have been completed. The results of these studies were generally favorable, and the application is being prepared for submission.

Nonclinical safety evaluation of two vaccine candidates for herpes simplex virus type 2 to support combined administration in humans.

Herpes simplex virus type 2 (HSV-2) is the most common cause of genital disease worldwide. The development of an effective HSV-2 vaccine would significantly impact global health based on the psychological distress caused by genital herpes for some individuals, the risk transmitting the infection from mother to infant, and the elevated risk of acquiring HIV-1. Five nonclinical safety studies were conducted with the replication defective HSV529 vaccine, alone or adjuvanted with GLA-SE, and the G103 subunit vaccine containing GLA-SE. A biodistribution study was conducted in guinea pigs to evaluate distribution, persistence, and shedding of HSV529. A preliminary immunogenicity study was conducted in rabbits to demonstrate HSV529-specific humoral response and its enhancement by GLA-SE. Three repeated-dose toxicity studies, one in guinea pigs and two in rabbits, were conducted to assess systemic toxicity and local tolerance of HSV529, alone or adjuvanted with GLA-SE, or G103 containing GLA-SE. Data from these studies show that both vaccines are safe and well tolerated and support the ongoing HSV-2 clinical trial in which the two vaccine candidates will be given either sequentially or concomitantly to explore their potential synergistic and incremental effects.

A mini-review on limitations associated with UV filters

Ultraviolet light from the sun can produce long-term skin damage and cancer. The use of sunscreen products containing one or more UV filters is encouraged by health professionals for preventing the damaging effects resulting from sun exposure. However, recently there have been increasing concerns about the use of sunscreens and their safety for both humans and the environment. The sunscreen manufacturers should take the initiative in testing of the products for possible short-term skin toxicity and long-term health effects that might occur due to the absorption of UV filters through the skin. Published studies have shed light on this topic by investigating the harmful effects of UV filters such as oxybenzone on the hormone system of aquatic animals and humans. Currently, in vitro and in vivo animal models are being used to determine the mechanistic and cellular effects these products produce. With growing awareness of adverse effects posed by UV filters on the environment and exposed organisms, several jurisdictions are prohibiting their use in sunscreens. To our knowledge, very few reviews summarized the potential toxicities associated with UV filters. Therefore, the current reported findings are rather controversial due to the lack of nonclinical safety assessment data to determine the clinical significance of such exposure.

Nonclinical safety evaluation of food colorant lac dye via systematic toxicity profiling with assessment of in vivo antigenic potential.

Lac dye is a natural colorant derived mainly from the insect Kerria lacca (Kerr) and has been used in food and beverage as a red-coloring additive. Despite its increasing use for human consumption as an alternative for allergy-associated cochineal, its toxicity profile remained incomplete to sufficiently assess its safety for the intended use. In this study, we evaluated systemic and genetic toxicity by performing acute and subacute oral toxicity studies in Sprague-Dawley (SD) rats using highly purified lac dye (LD) formulated in water and a battery of genotoxicity tests, respectively. To assess antigenic potentials, we carried out an in vivo passive cutaneous anaphylaxis test. A single dose of LD did not cause mortality at 5000mg/kg body weight (BW), setting oral LD50 of >5000mg/kg BW in SD rats. In the 90-day study, transient salivation without accompanying histopathological lesions in the salivary glands in 200 and 500mg/kg BW groups and red-purple pigmentation on the surface of femora and skulls in 500mg/kg groups were observed as nonadverse effects associated with LD, and no adverse effect was detected in all of the parameters examined, establishing a 500mg/kg BW as no-observed-adverse-effect-level (NOAEL). Furthermore, LD was not mutagenic nor clastogenic in the genotoxicity tests. When tested for antigenicity, LD did not induce anaphylactic skin responses as opposed to the positive reaction by ovalbumin, suggesting a lack of antigenicity. Taken together, these findings provide extended toxicity information on LD with direct evidence supporting the lack of antigenicity, providing essential guidance for its safe use in humans.

Biology and postnatal development of organ systems of cynomolgus monkeys (Macaca fascicularis).

The cynomolgus macaque has become the most used non-human primate species in nonclinical safety assessment during the past decades. This review summarizes the biological data and organ system development milestones of the cynomolgus macaque available in the literature. The cynomolgus macaque is born precocious relative to humans in some organ systems (e.g., nervous, skeletal, respiratory, and gastrointestinal). Organ systems develop, refine, and expand at different rates after birth. In general, the respiratory, gastrointestinal, renal, and hematopoietic systems mature at approximately 3 years of age. The female reproductive, cardiovascular and hepatobiliary systems mature at approximately 4 years of age. The central nervous, skeletal, immune, male reproductive, and endocrine systems complete their development at approximately 5 to 9 years of age. The cynomolgus macaque has no meaningful developmental differences in critical organ systems between 2 and 3 years of age for use in nonclinical safety assessment.