Exploring Greater Flexibility for Chronic Toxicity Study Designs to Support Human Safety Assessment While Balancing 3Rs Considerations.

Chronic repeated-dose toxicity studies are required to support long-term dosing in late-stage clinical trials, providing data to adequately characterize adverse effects of potential concern for human safety. Different regulatory guidances for the design and duration of chronic toxicity studies are available, with flexibility in approaches often adopted for specific drug modalities. These guidances may provide opportunities to reduce time, cost, compound requirement and animal use within drug development programs if applied more broadly and considered outside their current scopes of use. This article summarizes presentations from a workshop at the 43rd Annual Meeting of the American College of Toxicology (ACT) in November 2022, discussing different approaches for chronic toxicity studies. A recent industry collaboration between the Netherlands Medicines Evaluation Board (MEB) and UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) illustrated current practices and the value of chronic toxicity studies for monoclonal antibodies (mAbs) and evaluated a weight of evidence (WOE) model where a 3-month study rather than a 6-month study might be adequate. Other topics included potential opportunities for single-species chronic toxicity studies for small molecules, peptides and oligonucleotides and whether a 6-month duration non-rodent study can be used more routinely than a 9-month study (similar to ICH S6(R1) for biological products). Also addressed were opportunities to optimize recovery animal use if warranted and whether restriction to one study only (if at all) can be applied more widely within and outside ICH S6(R1).

Carcinogenicity testing in drug development: Getting it right.

For a pharmaceutical drug, carcinogenicity testing occurs in rodents to identify its tumorigenic potential to allow assessment of the risk from its use in humans. Testing takes the form of 2-year studies in mice and rats and/or more recently, a 6-month study in transgenic mice. This paper examines the process of regulatory interaction regarding carcinogenicity testing, notably through the United States (US) Food and Drug Administration (FDA) Special Protocol Assessment (SPA) process to seek Executive Carcinogenicity Assessment Committee (ECAC) approval. The content of 37 submissions to CAC were examined. The paper also examines the outcome from such agency engagement, notably around study dose level selection as well as looking at the design of proposed carcinogenicity study protocols used in submissions (including numbers of animals, control group aspects and toxicokinetic [TK] evaluation). Overall, it was shown that the current process of regulatory interaction allows for studies acceptable to support eventual drug approval and marketing. However, it was established that areas exist to improve the content of submission documents and study design aspects.

Adeno-associated virus (AAV)-based gene therapy products: What are toxicity studies in non-human primates showing us?

A number of adeno-associated virus (AAV)-based gene therapy products have entered clinical development, with a few also reaching marketing approval. However, as our knowledge of them grows from nonclinical and clinical testing, it has become apparent that various actual and theoretical safety issues can arise from their use. This review of 19 Good Laboratory Practice (GLP)-compliant toxicity studies in non-human primates (NHPs) with AAV-based gene therapy products via a variety of different dose routes in the period 2017-2021 showed results ranging from no study findings different from controls, or findings considered to be non-adverse, to actual toxicity, with changes highlighting careful monitoring in the clinic. Similar findings were found from a review of a number of published toxicity studies in NHPs. It was confirmed that studies have a role in evaluating for dorsal root ganglion (DRG) and/or peripheral nerve toxicity, hepatotoxicity, adverse immunogenicity and, to a lesser degree, insertional mutagenesis as well as other potential unacceptable findings such as adverse inflammation for ocular therapy candidates. Overall, it was demonstrated that toxicity (and biodistribution) studies in NHPs are a vital part of the safety assessment of AAV-based gene therapy products prior to clinical entry.

Nonclinical Testing Evaluation of Liposomes as Drug Delivery Systems.

Various marketed drugs, as well as many in-development, have utilized liposomes, vesicles composed of one or more phospholipid bilayers, as a drug delivery system, often with the statement that they are "non-toxic" materials. This paper examined safety testing considerations and reviewed nonclinical packages used to support the safe clinical use and marketing of drugs using a liposomal drug delivery system, including liposome-only study findings. It was found that most experience has come from use of an established drug (especially in the oncology field) in a liposome formulation with known excipients. From this knowledge, it is proposed that the minimal package of studies (using an oncology indication as an example) needed to support clinical entry should include in vivo pharmacology in selected mouse xenograft models, pharmacokinetic characterization showing enhanced kinetics or disposition and including tumor exposure evaluation along with repeat-dose toxicity testing in one species. It was also found that the liposomes used in drug delivery systems are not truly "non-toxic" materials. However, the majority of findings in toxicity testing relate to macrophage processing of large amounts of lipid material, with no human known safety consequence. Of note, however, are cases of hypersensitivity for some PEGylated liposome forms which translate to the clinic.

Development of COVID-19 therapies: Nonclinical testing considerations.

Therapies have been developed in the last couple of years to allow vaccination against, or treatment of patients with, COVID-19 using pathways such as Emergency Use Authorization (EUA) in the USA and Conditional Marketing Authorization (CMA) in the EU and UK. However, nonclinical studies were performed to allow such authorization and these were reviewed for 6 vaccines, 7 biological (monoclonal antibodies [mAbs]) and 4 small molecule therapies to examine whether the number and types of studies normally needed for regulatory agency authorization have been reduced. Results showed that the short answer is generally no. Thus, a battery of immunogenicity/efficacy or related pharmacology/biological activity studies showing utility against SARS-CoV-2 were performed as well as general toxicity studies across all 3 compound classes along with pharmacokinetic studies for mAbs and small molecules and, reproduction toxicity testing for vaccines and small molecules; additionally, genotoxicity testing occurred for small molecules. What was different from conventional, lengthy drug development, was that for vaccines and small molecules, leverage to existing platform technology or data available for other development programs, respectively, occurred. Recognition that mAbs can target the spike protein leading to neutralization allowed rapid development into clinical candidates.

Successful regulatory agency interaction - A nonclinical regulatory strategist's perspective.

Regulatory agency interaction occurs from before a candidate drug enters clinical development and all the way to marketing approval and beyond. This paper presents ways to enable successful interaction by avoiding issues, with an emphasis on nonclinical testing aspects. Strategic thinking as to whether an early regulatory agency meeting should occur is discussed and if yes, how to make it a success by generating relevant questions with proper preparation including a robust Briefing Document. Examples of unfavourable regulatory agency feedback during meetings is given which may have been avoided. Similarly, ways for successful regulatory submission in the form of a Clinical Trials Application (CTA) in Europe or an Investigational New Drug (IND) application in the US are considered with examples of comments that can be received from regulatory agencies. At marketing application stage with submission of a Marketing Authorisation Application (MAA) in Europe and a New Drug Application (NDA) or a Biologic License Application (BLA) in the US, a key document is the Nonclinical Overview and suggested content and potential deficiencies are presented to allow avoidance of adverse regulatory agency responses and time delay. Successful regulatory agency interaction involves robust scientific thinking, proper planning and well-written documentation.

Nonclinical & clinical interface - extrapolation of nonclinical data to support Phase I clinical studies.

A review of the Investigator's Brochure and Clinical Study Reports for 58 non-oncology small molecule and biopharmaceutical drug candidates tested in a healthy volunteer subject population was conducted. Key findings were (1) a vital role for nonclinical pharmacology and toxicology testing was confirmed to allow setting of clinical starting dose and supporting use of highest dose based on No Observed Adverse Effect Levels (NOAELs), Pharmacologically Active Doses (PADs) and other approaches, (2) for clinical starting dose calculation, reference to the NOAEL was key, whether in calculation of a Maximum Recommended Starting Dose (MRSD), or by supporting PAD approaches (small molecules); or, through pharmacokinetic/pharmacodynamic (PK/PD) data modelling (biopharmaceuticals), (3) starting dose for small molecules was very conservative with human exposure >100- to 100-fold (46%) lower or between 10- and 100-fold (41%) lower than that seen at the NOAEL; high margins over exposure seen at NOAELs were also seen for biopharmaceuticals, (4) at the highest doses used, about 25% of studies for small molecules and 12% of studies for biopharmaceuticals showed exposure greater than that seen at the NOAEL and (5) adverse event evaluation showed that our current paradigm of moving from nonclinical testing into SAD/MAD Phase I testing is remarkably safe.

Nonclinical safety testing of imaging agents, contrast agents and radiopharmaceuticals.

Drug development includes imaging agents, contrast agents and radiopharmaceuticals; these materials differ from therapeutic drugs in that they are largely used to diagnose and/or monitor diseases and not treat them. Consequently, nonclinical safety testing needs are different. An examination of testing packages supporting clinical entry and/or marketing of these materials has shown a common approach to some study types (eg, imaging, biodistribution and toxicity testing). Recent regulatory guidelines to support development are the United States Food and Drug Administration (FDA)'s "Guidance for Industry Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations" and the European Medicines Agency (EMA)'s "Guideline on the Non-Clinical Requirements for Radiopharmaceuticals" (currently draft). It is hoped that these documents will allow developers to only perform nonclinical studies that are necessary to support functionality, follow distribution of the material and examine general safety/toxicity. However, as they are mainly focused on radiopharmaceuticals, companies are likely to apply knowledge of established testing packages to other new imaging agents and/or follow principles given in older regulatory guidelines, namely FDA's "Guidance for Industry Developing Medical Imaging Drug and Biological Products Part I Conducting Safety Assessments". Thus, in some cases, the need for regulatory agency interaction is still vital to avoid development surprises and delays due to an incomplete or badly performed testing package.

Opportunities for use of one species for longer-term toxicology testing during drug development: A cross-industry evaluation.

An international expert working group representing 37 organisations (pharmaceutical/biotechnology companies, contract research organisations, academic institutions and regulatory bodies) collaborated in a data sharing exercise to evaluate the utility of two species within regulatory general toxicology studies. Anonymised data on 172 drug candidates (92 small molecules, 46 monoclonal antibodies, 15 recombinant proteins, 13 synthetic peptides and 6 antibody-drug conjugates) were submitted by 18 organisations. The use of one or two species across molecule types, the frequency for reduction to a single species within the package of general toxicology studies, and a comparison of target organ toxicities identified in each species in both short and longer-term studies were determined. Reduction to a single species for longer-term toxicity studies, as used for the development of biologicals (ICHS6(R1) guideline) was only applied for 8/133 drug candidates, but might have been possible for more, regardless of drug modality, as similar target organ toxicity profiles were identified in the short-term studies. However, definition and harmonisation around the criteria for similarity of toxicity profiles is needed to enable wider consideration of these principles. Analysis of a more robust dataset would be required to provide clear, evidence-based recommendations for expansion of these principles to small molecules or other modalities where two species toxicity testing is currently recommended.

Toxicology Paradise: Sorting Out Adverse and Non-adverse Findings in Animal Toxicity Studies.

A challenge for all toxicologists is defining what study findings are actually adverse versus non-adverse in animal toxicity studies, and which ones are relevant for generating a no observed adverse effect level (NOAEL) to assess human risk. This article presents views on this challenge presented by toxicologists, toxicologic pathologists, and regulatory reviewers at the 2019 annual meeting of the American College of Toxicology during a workshop entitled "Toxicology Paradise: Sorting Out Adverse and Non-adverse Findings." The speakers noted that setting a NOAEL is not always straightforward, not only for small molecules but also for biopharmaceuticals, and that a "weight of evidence" approach often is more useful than a rigid threshold-setting algorithm. Regulators from the US Food and Drug Administration and European Union told how assessment of adverse nonclinical findings is undertaken to allow clinical studies to commence and drug marketing approvals to succeed, along with the process that allows successful dialogs with regulators. Nonclinical case studies of findings judged to be adverse versus non-adverse were presented in relation to the many factors that might halt or delay clinical development. The process of defining adverse findings and the NOAEL in final study reports was discussed, as well as who should be involved in the process.

Safety Evaluation of PQ Birch Allergy Immunotherapy to Support Product Development.

PQ Birch represents an allergen-specific immunotherapy for the treatment of birch pollinosis. It consists of native birch pollen extract chemically modified with glutaldehyde adsorbed to L-tyrosine in its microcrystalline form with addition of the adjuvant Monophosphoryl Lipid A (MPL®). A nonclinical safety testing strategy was designed based upon interpretation of current legislation and regulatory intelligence and comprised genotoxicity studies (bacterial reverse mutation and Chinese hamster ovary micronucleus assays), a rat repeat dose toxicology study and a rabbit local tolerance study. No safety findings of concern were found. Thus, no evidence of genotoxicity was found. Relatively minor, immunostimulatory effects were seen following repeated subcutaneous dosing (once every 2 weeks for 13 weeks) as reversible increased white cell count (notably neutrophils), increased globulin level (resulting in decreased albumin/globulin [A/G] ratio) and increased fibrinogen, as well as minor dose site reaction in the form of inflammatory cell infiltrate. These findings are likely due to the immunostimulatory nature of MPL® and/or the presence of L-tyrosine within the adjuvanted vaccine. Similar dose site inflammatory changes to the injected formulation were also noted in the rabbit local tolerance study.

New toxicity testing of PQ grass allergy immunotherapy to support product development.

PQ Grass represents an allergen-specific immunotherapy for pre-seasonal treatment of patients with seasonal allergic rhinitis (or rhinoconjunctivitis) with or without mild-to-moderate bronchial asthma. It consists of a native pollen extract for 13 grass species, chemically modified with glutaraldehyde, and adsorbed to l-tyrosine in a microcrystalline form with addition of the adjuvant Monophosphoryl Lipid A (MPL® ). Previous non-clinical safety testing, including rat repeat dose toxicity in adult and juvenile animals, rat reproductive toxicity and rabbit local tolerance studies showed no safety findings of concern. A new Good Laboratory Practice compliant rat subcutaneous repeat dose toxicity study to evaluate a higher clinical dose and modified posology (once every 2 weeks for 13 weeks) showed no signs of toxicity. As seen in previous studies, relatively minor, immunostimulatory effects were seen such as reversible increased white cell count (notably neutrophils), increased globulin level (resulting in decreased A/G ratio) and increased fibrinogen as well as minor dose site reaction in the form of inflammatory cell infiltrate. These findings are likely due to the immunostimulatory nature of MPL and/or the presence of l-tyrosine within the adjuvanted vaccine. This new toxicity study with PQ Grass therefore supports longer posology with higher dose levels.

Getting a molecule into the clinic: Nonclinical testing and starting dose considerations.

Examination of content of 35 Investigator Brochures (IBs) for small molecules (including some for oncology) used to support First-In-Human studies over a 2 year period (2014-2016) showed that a mean of 37 nonclinical studies were performed per molecule with pharmacology, ADME and toxicology testing contributing 43%, 32% and 24% of the studies, respectively. Examination of 11 IBs for biopharmaceuticals (monoclonal antibodies) over the same time frame showed that the mean number of nonclinical studies was 17 studies per molecule with pharmacology, ADME and toxicology testing contributing 82%, 6% and 12% of the studies, respectively. For both types of molecule, similar numbers of pharmacology studies were performed but the approximately 50% fewer studies for biopharmaceuticals was due to considerably limited ADME and toxicology testing. Despite available regulatory guidance to allow calculation of a safe clinical starting dose, examination of how this occurred in the examined IBs showed that a variety of approaches are in practice, although reference to the NOAEL in toxicology testing is still key, whether in calculation of a Maximum Recommended Starting Dose (small molecules), or after use of pharmacology and/or PK data (especially for biopharmaceuticals) to show acceptable safety margins over doses used/exposure seen in toxicology studies.

Pharmacokinetic and toxicology comparator testing of biosimilar drugs - Assessing need.

A key element in the development of a biosimilar molecule is the comparability of the biological activity/nonclinical similarity to the innovator drug. Although some regulatory guidelines are encouraging little or no in vivo testing, currently a common practice is to perform at least one toxicology and/or one pharmacokinetic (PK) study to assess if any different findings occur for in-life, clinical pathology and histopathological parameters or in exposure. An exercise was performed in which the results of such testing were evaluated. It was found that 10 PK comparison studies in the cynomolgus monkey across 4 monoclonal (Mab) classes showed similar exposure in all cases. In 17 toxicology comparison studies with 5 Mab classes performed in the same species and in 7 toxicology comparison studies with non-Mab biosimilars in the rat, no new/unexpected findings were seen and drug exposure measurement gave comparable values in all cases. Overall, although this work does not rule out possible utility of some in vivo testing (notably in the form of stand-alone PK testing) to confirm similar exposure between the 2 molecules tested, it is unclear what benefit can be gained from toxicology testing, especially if comparability has been demonstrated from physiochemical and in vitro characterisation.

Dose site reactions and related findings after vaccine administration in safety studies.

Potential new human vaccines undergo toxicology testing to evaluate local reactogenicity and systemic toxicity. A review of 30 recently published and in-house repeat dose toxicity studies with a variety of vaccines was performed. Species tested were generally rat or rabbit, usually by intramuscular (although occasionally subcutaneous) injection. Results showed no unexpected findings indicating vaccine toxicity, but classic signs of enhanced acute and/or chronic inflammation at the dose site compared with that seen in injected control animals, often accompanied by changes in draining lymph nodes and the spleen (lymphoid hyperplasia and/or increased weight). Other associated signs of a response to vaccine dosing were altered clinical pathology parameters (commonly raised blood neutrophil count and altered globulin level). No obvious difference in dose site or systemic reaction was seen across vaccine, species or the dose route used. A non-dose recovery period of 2 to 4 weeks was sufficient to show evidence of reversibility of dose site effects. Injection site, lymphoid tissue and clinical pathological changes can be interpreted as related to an expected reaction after vaccine dosing, with generation of an immune response largely as a result of the presence of adjuvant, although direct vaccine antigen involvement was also occasionally demonstrated by the presence of a slightly increased inflammatory response seen over adjuvant treatment only. Overall, the need for toxicity testing of vaccines is in line with current regulatory guideline requirements and has proven to be a valuable part of the safety evaluation process prior to human use. Copyright © 2016 John Wiley & Sons, Ltd.

Utility and importance of animal data in drug product labels.

Information on the use and safety of medicines to assist prescription by healthcare professionals occurs in drug labels (Summary of Product Characteristics in Europe and Package Insert in the USA). Animal data (notably genotoxicity, reproduction toxicity and carcinogenicity and/or repeat dose toxicity testing) comprise an important component of the information (having a vital role in giving assurance that an extensive safety assessment for the medicinal product has occurred) and regulatory guidance is available to help inform on its input into drug labels. However, an evaluation of animal data for the 27 new drugs approved in the USA in 2013 (and the same drugs if available in Europe) shows great variability in detail and level of information presented within and across regions and/or the possibility of confusion on interpretation of some of the presented animal study findings. It is concluded that it may be time to revisit what animal data are presented in drug product labels (although bearing in mind current regional regulatory guidance requirements), not only to allow within and across region consistency on information given but to present it in a way that fully assists healthcare professions when prescribing a medicine.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Association of Inhalation Toxicologists' (AIT) review of regulatory aspects for inhalation toxicology studies.

Regulatory guidelines are intended to provide recommendations on ways to achieve greater harmonization in the interpretation and application of technical procedures and requirements for product registration in order to reduce or obviate replication of the testing carried out during the research and development of new products. The objectives of such harmonization are more economical use of human, animal and material resources; the elimination of unnecessary delay in the global development and availability of new products while maintaining safeguards on quality, safety and efficacy; and the fulfillment of regulatory obligations to protect public health.

The evolution of juvenile animal testing for small and large molecules.

Recent formalised regulatory requirements for ensuring safe use of new drugs in children has increased the requirement, when considered relevant, to perform juvenile animal testing before commencing paediatric clinical trials. A key goal of this work is to identify or examine for a developmental or toxicity finding not seen in other toxicology testing. With our current knowledge, this paper examines what types of testing are occurring, what novel findings are being seen and their relevance in the safety evaluation process. Furthermore, trends for now and the future in the type of juvenile animal testing will be described including a need for more focused study designs and more published data on modern cross-species postnatal development.

The design of chronic toxicology studies of monoclonal antibodies: implications for the reduction in use of non-human primates.

The changing environment of monoclonal antibody (mAb) development is impacting on the cost of drug development and the use of experimental animals, particularly non-human primates (NHPs). The drive to reduce these costs is huge and involves rethinking and improving nonclinical studies to make them more efficient and more predictive of man. While NHP use might be unavoidable in many cases because of the exquisite specificity and consequent species selectivity of mAbs, our increasing knowledge base can be used to improve drug development and maximise the output of experimental data. Data on GLP regulatory toxicology studies for 58mAbs were obtained from 10 companies across a wide range of therapeutic indications. These data have been used to investigate current practice and identify study designs that minimise NHP use. Our analysis shows that there is variation in the number of animals used for similar studies. This information has been used to develop practical guidance and make recommendations on the use of science-based rationale to design studies using fewer animals taking into account the current regulatory guidance. There are eight recommendations intended to highlight areas for consideration. They include guidance on the main group size, the inclusion of recovery groups and the number of dose groups used in short and long term chronic toxicology studies.