Species selection for nonclinical safety assessment of drug candidates: Examples of current industry practice.

In drug development, nonclinical safety assessment is pivotal for human risk assessment and support of clinical development. Selecting the relevant/appropriate animal species for toxicity testing increases the likelihood of detecting potential effects in humans, and although recent regulatory guidelines state the need to justify or dis-qualify animal species for toxicity testing, individual companies have developed decision-processes most appropriate for their molecules, experience and 3Rs policies. These generally revolve around similarity of metabolic profiles between toxicology species/humans and relevant pharmacological activity in at least one species for New Chemical Entities (NCEs), whilst for large molecules (biologics) the key aspect is similarity/presence of the intended human target epitope. To explore current industry practice, a questionnaire was developed to capture relevant information around process, documentation and tools/factors used for species selection. Collated results from 14 companies (Contract Research Organisations and pharmaceutical companies) are presented, along with some case-examples or over-riding principles from individual companies. As the process and justification of species selection is expected to be a topic for continued emphasis, this information could be adapted towards a harmonized approach or best practice for industry consideration.

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.

An innovative investigative approach to characterize the effects observed in a combined fertility study in male and female rats.

This paper describes the characterization of male- and female-mediated effects in a standard ICH rat fertility and early embryonic development study with a discontinued clinical small molecule. In the standard study, the test item had no effect on the number of treated females becoming pregnant, but litter sizes were reduced at the high dose level. In the treated male rats, increased incidences of abnormal sperm, decreases in average sperm path and straight line velocities, and minimal retention of mature sperm in the seminiferous tubules were observed at all dose-levels tested. These findings were unexpected in view of a lack of histopathological changes in the reproductive organs of either gender in 4-week repeat dose studies in rats and monkeys. A follow-up fertility study was conducted using an innovative flexible study design and a single high-dose level. In the first instance, treated male rats were mated with untreated females, followed by necropsy of a subset of males. The intention was then to re-mate the males after an 8-week wash-out period with either treated or untreated females depending on the outcome of the first mating. On this occasion, litter sizes were not affected, but the testicular effects were reproduced. A second mating with treated females reproduced the reduced litter sizes due to increased pre- and post-implantation loss, demonstrating that the effect on fecundity was female-mediated. The testicular changes in males were shown to be reversible after a 12-week recovery period.

Consideration of the Development of the Gastrointestinal Tract in the Choice of Species for Regulatory Juvenile Studies.

Although the rat is close to being the automatic choice for regulatory juvenile toxicity studies, there are several shortcomings in this model. Choosing a species should take into serious consideration the importance of gastrointestinal tract development in the period before weaning as this may have a major effect on exposure. A comparison of the anatomical changes in the stomach and small intestine of mini-pig and rat show that the mini-pig is a far closer model for man than is the rat, although there are a few unusual aspects of small intestinal development in the mini-pig. Considerations of the development of the gastrointestinal tract and choice of species have the potential to affect safety assessment and should be a prime consideration when planning an investigation. Birth Defects Research 110:56-62, 2018. © 2017 Wiley Periodicals, Inc.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

Anti-Folate Receptor-α IgE but not IgG Recruits Macrophages to Attack Tumors via TNFα/MCP-1 Signaling.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen-specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127-41. ©2017 AACR.

Vehicle Systems and Excipients Used in Minipig Drug Development Studies.

Minipigs have been used for dermal drug development studies for decades, and they are currently more frequently considered as the second nonrodent species for pivotal nonclinical studies, in lieu of the dog or nonhuman primate, for compounds delivered via standard systemic routes of administration. Little is known about the tolerability of different excipients in minipigs; sharing knowledge of excipient tolerability and compositions previously used in nonclinical studies may avoid testing of inadequate formulations, thereby contributing to reduced animal usage. This article reviews vehicles employed in the Göttingen(®)minipig based on the combined experience from a number of pharmaceutical companies and contract research organizations. The review includes vehicles tolerated for single or multiple dosing by the Göttingen minipig, some of which are not appropriate for administration to other common nonrodent species (e.g., dogs). By presenting these data for dermal, oral, subcutaneous, and intravenous routes of administration, studies to qualify these vehicles in minipigs can be minimized or avoided. Additionally, investigators may more frequently consider using the minipig in place of higher species if the tolerability of a vehicle in the minipig is known.

Organ data from the developing Göttingen minipig: first steps towards a juvenile PBPK model.

The Göttingen minipig is the most commonly used pig breed in preclinical drug development in Europe and has recently also been explored for physiologically based pharmacokinetic modelling. To develop such a model, not only physiological data from adult animals but also data from juvenile animals are required, especially when using this model for paediatric drug development. Therefore, the aim of our study was to document body and organ weights (brain, heart, lungs, liver, gastrointestinal tract, spleen and kidney), lengths of the small and large intestines and pH values of the gastrointestinal tract in Göttingen minipigs from the foetal stage until the age of 5 months. Postnatal organ and body weights were fitted to regression models to find suitable equations that could be used to estimate organ weights as a function of body weight in the neonatal and juvenile Göttingen minipig. Most organs followed a non-linear growth curve during the first 5 months of life. In general, relative organ weights were the highest during the first week of life, during which the gastric pH was more alkaline than at 28 days of age.

Regulatory Forum Opinion Piece: Carcinogen Risk Assessment: The Move from Screens to Science.

Throughout the last 50 years, the paradigm for carcinogenicity assessment has depended on lifetime bioassays in rodents. Since 1997, the International Conference on Harmonisation (ICH) S1B has permitted the use of a 2-year rodent bioassay (usually in the rat) and an alternative, genetically modified mouse model to support cancer risk assessment of pharmaceuticals. Since its introduction, it has become apparent that many of the stated advantages of the 6-month Tg mouse bioassay have, in actual fact, not been realized, and the concern exists that an albeit imperfect, 2-year mouse bioassay has been replaced by a similarly imperfect 6-month equivalent. This essay argues strongly that model systems, using cancer as the end point, should be discontinued, and that the recent initiatives, from the Organization for Economic Cooperation and Development and Institute of Peace and Conflict Studies, on "mode of action," "adverse outcome pathways," and "human relevance framework" should be embraced as being risk assessments based upon the available science. The recent suggested revisions to the ICH S1 guidelines, utilizing carcinogenicity assessment documents, go some way to developing a science-based risk assessment that does not depend almost entirely on a single, imperfect, cancer-based end point in nonrelevant animal species.

Vigabatrin-induced CNS changes in juvenile rats: Induction, progression and recovery of myelin-related changes.

The purpose of this study was to expand on the knowledge previously published on the central nervous system effects of Vigabatrin in juvenile animals. By employing extended sectioning of the brain and by using four different tissue staining techniques it is demonstrated that oral administration of Vigabatrin to juvenile rats (treatment periods of post-natal day (PND) 4-7, 7-14 or 14-30) will cause histological CNS changes at dose levels of 15 and 50mg/kg/day, but not at a dose level of 5mg/kg/day. No evidence of neuronal degeneration or gliosis was seen at any stage of treatment. Consistent with previous reports microvacuolation, as well as effects on myelination and on oligodendrocytes were recorded. The present study expands on these findings and demonstrates that the variation in the location of the vigabatrin-induced lesions in the juvenile rat brain (both neuropil vacuolation and reduction of myelin) appears to be consistent with the process of myelination: In the youngest animals (PND 4-7) myelination occurs mainly in the hind brain (medulla oblongata and pons) where neuropil vacuolations is recorded. In animals dosed during PNDs 7-14 or during PNDs 14-30, the first changes were found in the thalamus. It seems likely that the earlier stages of myelination are more vulnerable to treatment related effects and the swollen oligodendrocytes seen as the initial change in the thalamus in animals treated during PNDs 4-7 and 7-14 represents an early stage in the development of the myelin lesion which is seen later as neuropil vacuolation.

The development of myelin in the brain of the juvenile rat.

The development process of myelination varies between region and species. Fully myelinated fibers are required if mammalian neural circuits are to function normally. Histology samples at staggered time points throughout the study were examined at days 4, 5, 7, 8, 10, 14, 17, 24, 37, and 44. We suggest that the development of myelin in the juvenile rodent brain can be conveniently separated into 3 phases. Evaluation of myelin basic protein-stained sections of the areas of brain that contain the elements of the developing limbic system over the sensitive period from postnatal day (PND) 14 to 34 may provide an insight into possible toxicity that may lead to cognition and learning issues in adults. We will hope to develop this notion further in the future. The precise chronology of the development of the blood-brain barrier in rats has yet to be established; thus, there is potential for significant exposure of the juvenile brain to chemicals that do not cross the blood-brain barrier in the adult. Thus, it is suggested that evaluation of myelin development should probably be extended to all new chemical entities intended for pediatric use, and not just those that are intended for central nervous system use.

Juvenile toxicity: are we asking the right questions?

The recent increase in numbers of juvenile toxicity studies over the past few years has doubtless added to our knowledge of the development of organ systems in the young rodent, but it is perhaps rather more difficult to answer the questions as to whether this body of work has served us well in terms of assessing risk in the pediatric population. If there are shortcomings, are they the result of poor study design or poor sensitivity of the model, or are the wrong questions being posed? This article provides a superficial overview of the progress made thus far and considers which aspects of study design and model choice currently fail to adequately address the major issues surrounding pediatric toxicology.

Revisiting alternative approaches to the assessment of carcinogenic risk.

The rodent bioassay has been a mainstay of carcinogenic risk assessment for close to half a century; not that it has been without criticism in that time. As early as the 1990s, there were clearly established shortcomings with regard to relevance and extrapolation, but despite these concerns, this test continues relatively unchanged. As our understanding of the mechanisms of carcinogenesis and the availability of investigative techniques improve, there seems less reason than ever to persist with this exercise that provides little meaningful scientific data at great cost in terms of animal usage and pathologist time. This article highlights possible ways to gather and present data pertinent to carcinogenesis in man and suggests that it is the toxicological pathology community that should take the lead here in persuading regulators that there is great room for improvement in this particular aspect of regulatory pathology.