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.

Genotoxicity test battery - An assessment of its utility in early drug development.

Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.

Core battery safety pharmacology testing - An assessment of its utility in early drug development.

Requirements for safety pharmacology testing have been in place since the issue of initial regulatory guidance over 20 years ago. An evaluation of such testing, supporting first clinical entry of 105 small molecule drug candidates over the last decade, showed that a "core battery" of in vitro electrophysiological (hERG), conscious non-rodent telemetry cardiovascular, rodent central nervous system (CNS) (modified Irwin's or functional observational battery [FOB] test) and respiratory function (plethysmography) studies was performed. Routine use of the latter 2 studies appears to have limited utility, with only 21% and 28% of studies, respectively, giving findings of which none were identified as of obvious concern to moving the affected drugs into the clinic. The use of a stand-alone hERG assay does not appear to be particular sensitive in predicting proarrythmic risk as a tool by itself. Telemetry study testing had utility especially for identifying effects on QTc interval (about 10% of studies), resulting on some occasions in a lower clinical starting dose and/or increased awareness for potential effects on the cardiovascular system in the Phase I study. Overall, this investigation provides information supporting an overhaul of the current "box ticking" core battery approach used for safety pharmacology testing. However, in order to achieve a more focused examination to investigate potential undesirable pharmacodynamic effects of a new candidate drug and also support 3Rs (Replacement, Reduction and Refinement) thinking in performing unnecessary studies, there will not only need to be a sea change by drug developers but also a change in current regulatory guidance.

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.

Nonclinical Immunotoxicity Testing in the Pharmaceutical World: The Past, Present, and Future.

An examination for potential direct or indirect adverse effects on the immune system (immunotoxicity) is an established component of nonclinical testing to support safe use of new drugs. Testing recommendations occur in various regulatory guidance documents, especially ICH S8, and these will be presented. Key evaluation usually occurs in toxicology studies with further investigative work a consideration if a positive signal is seen. Expectations around whether findings may occur are related to the type of compound being developed, including a chemically synthesized small molecule, a small molecule oncology drug, a biopharmaceutical, an oligonucleotide, a gene therapy/stem cell product, a vaccine, or reformulation of drugs in liposomes or depots. Examples of immunotoxicity/immunogenicity findings will be discussed for all of these types of compound. Overall, it can be concluded that our main tool for evaluation of potential immunotoxicity/immunogenicity for a new drug still remains standard toxicology study testing with key assessment for effects on clinical pathology and lymphoid organs/tissues (weights and cellularity). Additional evaluation from studies using a T cell-dependent antibody response (TDAR) and lymphocyte phenotyping is also valuable, if needed. Thus, using the tools from the past, it is the role of toxicologists to work with clinical teams now and in the future, to interpret findings from nonclinical testing to possible adverse findings in humans.

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.

Use of "Big Data" to Evaluate Responses to Changes in Regulatory Guidelines: Trends in Genotoxicity Testing Packages for New Pharmaceutical Products.

BACKGROUND: In 2006, a concept paper (ICH S2(R1)) describing the need for revision of the ICH guidelines on genotoxicity testing for new "small molecule" pharmaceuticals (then ICH S2A and ICH S2B) was finalised. As a result, testing strategy has changed, and flexibility has been introduced in the form of two "equally suitable" options for completing the battery of genotoxicity studies required to support clinical development and marketing of new products. METHODS: The TIBCO Spotfire® platform was used to create a specific view of available in-house data on genotoxicity studies conducted to support pharmaceutical product development over a period of approximately 12 years. The available in-house dataset comprised all reportable non-clinical data from Good Laboratory Practice (GLP) compliant and non-GLP/screening studies on regulated products (including pharmaceuticals, industrial chemicals, agrochemicals, and medical devices) across multiple testing sites, in different geographical locations. RESULTS: The analysis showed clear trends in the numbers and types of genotoxicity studies conducted on small molecule pharmaceutical products during development, which correlated with the changes in the available regulatory guidance over time. More interestingly, where two "equally suitable" options for genotoxicity testing are described in the international guidance, there is a clear preference for ICH S2(R1) Option 1 (70-80% of testing) compared to Option 2 (20-30%). CONCLUSION: Use of 'Big Data' identified trends in the newer approach to genotoxicity testing by industry in the light of the updated regulatory guidance.

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.

Nonclinical Immunotoxicity Testing in the Pharmaceutical World: The Past, Present, and Future.

An examination for potential direct or indirect adverse effects on the immune system (immunotoxicity) is an established component of nonclinical testing to support safe use of new drugs. Testing recommendations occur in various regulatory guidance documents, especially ICH S8, and these will be presented. Key evaluation usually occurs in toxicology studies with further investigative work a consideration if a positive signal is seen. Expectations around whether findings may occur are related to the type of compound being developed, including a chemically synthesized small molecule, a small molecule oncology drug, a biopharmaceutical, an oligonucleotide, a gene therapy/stem cell product, a vaccine, or reformulation of drugs in liposomes or depots. Examples of immunotoxicity/immunogenicity findings will be discussed for all of these types of compound. Overall, it can be concluded that our main tool for evaluation of potential immunotoxicity/immunogenicity for a new drug still remains standard toxicology study testing with key assessment for effects on clinical pathology and lymphoid organs/tissues (weights and cellularity). Additional evaluation from studies using a T cell-dependent antibody response (TDAR) and lymphocyte phenotyping is also valuable, if needed. Thus, using the tools from the past, it is the role of toxicologists to work with clinical teams now and in the future, to interpret findings from nonclinical testing to possible adverse findings in humans.

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.

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.

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.

Biocompatibility assessments for medical devices - evolving regulatory considerations.

INTRODUCTION: Biocompatibility assessment provides key data supporting medical device development and marketing. Although regional and international guidance is available, differences in proposed biocompatibility assessments or test methods lead to confusion and inefficiencies in generating the package of supporting nonclinical data. Areas covered: Modifications to available guidance for biological safety testing of medical devices, as described by the International Organisation for Standardisation (ISO) and the US Food and Drug Administration (FDA), have, over time, sometimes increased and sometimes decreased the level of harmonisation in testing requirements. These requirements continue to evolve, as shown by refinements and supplements to existing ISO 10,993 standards, new ISO standards under development and new finalised guidance from the FDA - which shows a shift away from routine testing-based approaches and much greater emphasis on characterisation, with use of existing literature or demonstration of equivalence to established comparator products, where possible. Expert commentary: This article examines the impact of recent changes in guidance for biocompatibility assessment of new medical devices and shows that, although a high level of consistency now occurs in ISO and FDA requirements, there are still areas where a 'standard approach' is not possible, allowing hurdles for global development of medical devices to persist.