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.

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.

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.

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.

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.

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.

Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges.

Biosimilars are biological medicinal products that contain a version of the active substance of an already authorised original biological medicinal product (the innovator or reference product). The first approved biosimilar medicines were small proteins, and more recently biosimilar versions of innovator monoclonal antibody (mAb) drugs have entered development as patents on these more complex proteins expire. In September 2013, the first biosimilar mAb, infliximab, was authorised in Europe. In March 2015, the first biosimilar (Zarxio™, filgrastim-sndz, Sandoz) was approved by the US Food and Drug Administration; however, to date no mAb biosimilars have been approved in the US. There are currently major differences between how biosimilars are regulated in different parts of the world, leading to substantial variability in the amount of in vivo nonclinical toxicity testing required to support clinical development and marketing of biosimilars. There are approximately 30 national and international guidelines on biosimilar development and this number is growing. The European Union's guidance describes an approach that enables biosimilars to enter clinical trials based on robust in vitro data alone; in contrast, the World Health Organization's guidance is interpreted globally to mean in vivo toxicity studies are mandatory. We reviewed our own experience working in the global regulatory environment, surveyed current practice, determined drivers for nonclinical in vivo studies with biosimilar mAbs and shared data on practice and study design for 25 marketed and as yet unmarketed biosimilar mAbs that have been in development in the past 5y. These data showed a variety of nonclinical in vivo approaches, and also demonstrated the practical challenges faced in obtaining regulatory approval for clinical trials based on in vitro data alone. The majority of reasons for carrying out nonclinical in vivo studies were not based on scientific rationale, and therefore the authors have made recommendations for a data-driven approach to the toxicological assessment of mAb biosimilars that minimises unnecessary use of animals and can be used across all regions of the world.

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.

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.

A battery of genotoxicity studies with an allergy vaccine adjuvanted with monophosphoryl lipid A (MPL®) for the treatment of grass pollen allergy.

The allergy vaccine Pollinex® Quattro Grass, developed for the prevention/relief of allergic symptoms caused by grass pollen in adults and children, contains extracts of 12 grass pollens and rye cereal (all chemically modified by glutaraldehyde), adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant monophosphoryl lipid A (MPL®). This paper represents the first publication on genetic toxicology data for such a vaccine. An Ames test using five strains of Salmonella typhimurium at concentrations up to 2000 standardized units (SU) per plate in the absence (-) and presence (+) of metabolic activation (rat liver S9) showed negative results, as did treatment - S9 in the mouse lymphoma assay (MLA). However, a reproducible positive response was noted in the MLA + S9, which could not be attributed to extreme culture conditions, but may have been an artefact of the exogenous metabolizing system. Up to 60% false positive results are reported for the MLA with noncarcinogens. Hence, in vivo genotoxicity studies were conducted. These further assessments comprised a rat bone marrow micronucleus test following subcutaneous (s.c.) doses of 10 000, 20 000 or 40 000 SU kg⁻¹ per day for 2 days, and a comet assay in liver and kidney cells from rats treated with Pollinex® Quattro Grass at 20 000 or 40 000 SU kg⁻¹ per day s.c. for 2 days. Plasma analysis showed evidence of systemic antibodies to Pollinex® Quattro Grass immunogens, but the exposure levels could not be quantified. No evidence of genotoxicity was observed in either of the in vivo studies. Overall, the genotoxicity test results supported safe clinical use of Pollinex® Quattro Grass.

Reproduction and juvenile animal toxicology studies in the rat with a new allergy vaccine adjuvanted with monophosphoryl lipid A (MPL(®)) for the treatment of grass pollen allergy.

Pollinex(®) Quattro Grass has been developed for the prevention or relief of allergic symptoms caused by pollen in both adults and children. Reproduction and juvenile animal toxicology studies have been performed. Subcutaneous injection on Day 14 prior to pairing and on Days 6 and 13 of gestation to pregnant rats at 2000SU/0.5 mL elicited no signs of maternal or embryo-foetal toxicity. Mating, fertility, fecundity and pup parameters were all unaffected by treatment. Once-weekly subcutaneous administration at ascending doses of 300, 800, 2000 and 2000SU/0.5 mL followed by a 4 week non-dose period to juvenile rats from 3 weeks of age showed no signs of obvious toxicity. As in a previously performed adult animal toxicology study with the vaccine, not unexpected, but relatively minor, immuno-stimulatory effects were seen in this study along with injection site reaction which can largely be attributed to the presence of tyrosine in the formulation.

Safety evaluation of biological drugs: what are toxicology studies in primates telling us?

A total of 26 toxicology studies performed with biological drugs (monoclonal antibodies and related immunoglobulins as well as recombinant human proteins) in the primate have been evaluated to give an insight into the types of study designs used and results. The evaluation involved examination of pivotal repeat dose studies which ranged from 2 to 13 weeks in duration, used to support early clinical investigation. Either no findings were seen or restricted to those which could largely be explained by the drug's pharmacology. Based on these results and supporting findings from a literature review of other similar drugs in development or approved for marketed use, a case has been made to revisit aspects of the standard design approach of toxicology studies for biological drugs. Thus, consideration should be given to a move away from the use of three drug-treated and numerous non-dose recovery groups currently used to support initial clinical entry to a robust toxicological assessment that could be achieved in many cases with one control and one or two drug-treated groups and with non-dose recovery groups only for the control and highest level used. An argument for not routinely measuring for anti-drug antibodies unless there is a specific drug-related reason is also made.

Juvenile animal testing in drug development--is it useful?

In pharmaceutical drug development, there has been increased interest in the need to perform juvenile animal studies to support the safety of use of new medicines in the pediatric population. Although such studies are not new, the increased interest has been "formalized" in recent regulatory guidelines. As a result, companies are now performing many more studies in juvenile animals, even when there is a lack of robust knowledge of cross-species functional and kinetic differences among juveniles that means extrapolation of any toxicology study finding to an immature human may not be easy or even relevant, especially if performed in the wrong species at the wrong time. It will be shown by presentation of some basic considerations needed in order to perform such testing, that juvenile animal studies are indeed feasible. However, it will also be highlighted that (based on available knowledge) there are currently not enough clear-cut examples to answer the question of whether juvenile animal toxicology studies to support pediatric development (by affecting the performance or design of a pediatric clinical trial or identifying a potential different-from-adult safety risk in clinical use) are truly useful or necessary.

Preclinical development of monoclonal antibodies: considerations for the use of non-human primates.

The development of mAbs remains high on the therapeutic agenda for the majority of pharmaceutical and biotechnology companies. Often, the only relevant species for preclinical safety assessment of mAbs are non-human primates (NHPs), and this raises important scientific, ethical and economic issues. To investigate evidence-based opportunities to minimize the use of NHPs, an expert working group with representatives from leading pharmaceutical and biotechnology companies, contract research organizations and institutes from Europe and the USA, has shared and analyzed data on mAbs for a range of therapeutic areas. This information has been applied to hypothetical examples to recommend scientifically appropriate development pathways and study designs for a variety of potential mAbs. The addendum of ICHS6 provides a timely opportunity for the scientific and regulatory community to embrace strategies which minimize primate use and increase efficiency of mAb development.

Safety evaluation to support First-In-Man investigations I: kinetic and safety pharmacology studies.

Kinetic (in vitro, pharmacokinetic, mass balance and toxicokinetic studies) and safety pharmacology data available in 34 Investigator's Brochures used to support First-In-Man clinical trials over a 10 year period have been evaluated to give an insight into the types of study designs used and how these have changed over the period analysed (1997-2006). For kinetic evaluation, study packages often had single dose pharmacokinetic studies in the rodent and non-rodent by oral and intravenous dose routes as well as protein binding measurement accompanied more recently by rodent mass balance studies and an examination of in vitro metabolism across species and/or enzyme activity with hepatocytes, liver microsomes or recombinantly expressed enzymes. Toxicokinetic data were examined from key repeat dose toxicity studies in rodents and non-rodents with the recent inclusion of sampling in control animals to check for drug contamination. For safety pharmacology evaluation, study packages included "core battery" rodent central nervous system as well as anaesthetised non-rodent cardiovascular and respiratory function studies. More recently, cardiovascular assessment occurred in conscious non-rodents, necessitating stand-alone rodent respiratory function studies although conscious models assessing both cardiovascular and respiratory function were also seen in some of the most recent packages. Recent packages also had an in vitro electrophysiology study, usually the hERG assay. Considerations for kinetic and safety pharmacology study designs are discussed.

Safety evaluation to support First-In-Man investigations II: toxicology studies.

Toxicology (single dose, range-finding, repeat dose and genotoxicity) data available in 34 Investigator's Brochures used to support First-In-Man clinical trials over a 10 year period have been evaluated to give an insight into the types of study designs used and how these have changed over the period analysed (1997-2006). Study packages had single dose toxicity studies in the rodent (although there has been a recent trend to reduce the number of these studies), range-finding toxicity studies in the rodent and non-rodent (with only small numbers of the latter used) and key 2- 4 week repeat dose toxicity studies in rodent (usually rat) and non-rodent (both dog and monkey). The majority of the latter studies established No Observed Adverse Effect Levels, showed the rodent to be generally less sensitive to target organ toxicity than the non-rodent and showed the liver and then the kidney to be the most common target organs. Genotoxicity assessment included 2 in vitro assays (a reverse mutation bacteria and either a chromosome aberration or mouse lymphoma assay) and commonly, an in vivo rodent bone marrow micronucleus test. Considerations for general toxicology and genotoxicity study designs are discussed along with the use of appropriate information to help set the clinical starting dose.

Pollinex Quattro Ragweed: safety evaluation of a new allergy vaccine adjuvanted with monophosphoryl lipid A (MPL) for the treatment of ragweed pollen allergy.

A novel allergy vaccine (Pollinex Quattro Ragweed) has been developed for the prevention or relief of allergic symptoms caused by pollen from Ambrosia spp. (ragweed). An extract from the pollen (chemically modified by glutaraldehyde) is adsorbed onto l-tyrosine with addition of the immunostimulatory adjuvant, monophosphoryl lipid A (MPL). A specific preclinical safety testing strategy was developed to support clinical use and comprised reference to preclinical data available for the marketed non-MPL adjuvanted form of the ragweed vaccine (Pollinex R) and a new repeat dose toxicity study in the rat. Studies with Pollinex R comprised single dose subcutaneous toxicity studies in mice and rats, repeat dose (10 injections over 20 days) parenteral toxicity studies in rats and dogs, an in vitro gene mutation assay along with single and multiple injection local tolerance studies in rats and dogs. The repeat dose subcutaneous toxicity study with Pollinex Quattro Ragweed involved seven injections over 3 weeks (which was more aggressive than the four weekly doses used in the clinic) with dose levels of up to 0.5 ml per animal used. Overall, the product showed no toxicological findings of significance at levels greatly in excess of those proposed for clinical use. As is a feature with vaccination, some dose site irritation was seen.

Safety evaluation of a new allergy vaccine containing the adjuvant monophosphoryl lipid A (MPL) for the treatment of grass pollen allergy.

A novel allergy vaccine (Pollinex Quattro) has been developed for the prevention or relief of allergic symptoms caused by a variety of pollens. Within this range, the grass pollen allergy vaccine contains extracts of 12 grass pollens and rye cereal (all chemically modified by glutaraldehyde) that are adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant monophosphoryl lipid A (MPL). A specific preclinical safety testing strategy was developed to support clinical use, comprising single-dose toxicity, repeat-dose toxicity and local tolerance studies. Dose levels of up to 0.5 ml per animal (mouse) and up to 1.0 ml per animal (rat and rabbit) were used with vaccines containing 2000 or 12 000 standardized units (SU) ml(-1) of grass pollen allergoids, 50 micro g ml(-1) of MPL adjuvant and 20 mg ml(-1) of tyrosine. Overall, the product showed no toxicological findings of significance at levels greatly in excess of those proposed for clinical use. A not unexpected, but relatively minor, immunostimulatory effect was seen following repeated dosing (once weekly for 3 weeks) at 1.0 ml per rat.