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.

Reduction of arthrofibrosis utilizing a collagen membrane drug-eluting scaffold with celecoxib and subcutaneous injections with ketotifen.

The dense formation of abnormal scar tissue after total knee arthroplasty results in arthrofibrosis, an unfortunate sequela of inflammation. The purpose of this study was to use a validated rabbit model to assess the effects on surgically-induced knee joint contractures of two combined pharmacological interventions: celecoxib (CXB) loaded on an implanted collagen membrane, and subcutaneously (SQ) injected ketotifen. Thirty rabbits were randomly divided into five groups. The first group received no intervention after the index surgery. The remaining four groups underwent intra-articular implantation of collagen membranes loaded with or without CXB at the time of the index surgery; two of which were also treated with SQ ketotifen. Biomechanical joint contracture data were collected at 8, 10, 16, and 24 weeks. At the time of necropsy (24 weeks), posterior capsule tissue was collected for messenger RNA and histopathologic analyses. At 24 weeks, there was a statistically significant increase in passive extension among rabbits in all groups treated with CXB and/or ketotifen compared to those in the contracture control group. There was a statistically significant decrease in COL3A1, COL6A1, and ACTA2 gene expression in the treatment groups compared to the contracture control group (P < .001). Histopathologic data also demonstrated a trend towards decreased fibrous tissue density in the CXB membrane group compared to the vehicle membrane group. The present data suggest that intra-articular placement of a treated collagen membrane blunts the severity of contracture development in a rabbit model of arthrofibrosis, and that ketotifen and CXB may independently contribute to the prevention of arthrofibrosis. Statement of clinical significance: Current literature has demonstrated that arthrofibrosis may affect up to 5% of primary total knee arthroplasty patients. For that reason, novel pharmacologic prophylaxis and treatment modalities are critical to mitigating reoperations and revisions while improving the quality of life for patients with this debilitating condition.

New world origin of canine distemper: Interdisciplinary insights.

OBJECTIVE: Canine distemper virus (CDV), human measles virus (HMV), and rinderpest virus (RPV) of cattle are morbilliviruses that have caused devastating outbreaks for centuries. This paper seeks to reconstruct the evolutionary history of CDV. MATERIALS AND METHODS: An interdisciplinary approach is adopted, synthesizing paleopathological analysis of 96 Pre-Columbian dogs (750-1470 CE) from the Weyanoke Old Town, Virginia site, with historical reports, molecular analysis and morbilliviral epidemiology. RESULTS: Both measles (c.900CE) and rinderpest (c. 376 BCE) were first reported in Eurasia, while canine distemper was initially described in South America much later (1735 CE); there are no paleopathological indications of CDV in Weyanoke Old Town dogs. Molecularly, CDV is closely related to HMV, while viral codon usage indicates CDV may have previously infected humans; South American measles epidemics occurred prior to the emergence of canine distemper and would have facilitated HMV transmission and adaptation to dogs. CONCLUSIONS: The measles epidemics that decimated indigenous South American populations in the 1500-1700 s likely facilitated the establishment of CDV as a canine pathogen, which eventually spread to Europe and beyond. SIGNIFICANCE: Understanding the historical and environmental conditions that have driven morbilliviral evolution provides important insights into potential future threats of animal/human cross-species infections. LIMITATIONS: Interpreting historical disease descriptions is difficult and the archaeological specimens are limited. Molecular sequence data and codon usage analyses rely on modern viruses. SUGGESTIONS FOR FURTHER RESEARCH: Interdisciplinary approaches are increasingly needed to understand diseases of the past and present, as critical information and knowledge is scattered in different disciplines.

Scientific and Regulatory Policy Committee Points to Consider: Histopathologic Evaluation in Safety Assessment Studies for PEGylated Pharmaceutical Products.

Colorless, intracytoplasmic vacuoles occur in multiple tissues in animals following repeated administration of polyethylene glycol (PEG)-conjugated molecules. The extent of vacuolation depends on physical characteristics and molecular backbone of the PEG and the dose, product, drug target/pharmacology, and duration of exposure. The collective experience gathered from multiple nonclinical toxicology studies of PEGylated biopharmaceuticals indicates that in general, PEG-related vacuolation is not associated with demonstrable cell and tissue damage or dysfunction and is reversible with sufficient duration of drug-free periods. Existing data are insufficient to predict whether nonclinical animal species differ in their sensitivity to develop PEG-associated vacuoles; however, recent data suggest that there may be species differences. Recent comprehensive reviews have addressed the basic challenges in developing PEGylated pharmaceutical products, including general reference to and description of PEG-associated tissue findings. These manuscripts have identified gaps in our current understanding of PEG-associated vacuolation, including the lack of a widely accepted standardized histological terminology and criteria to record and grade the severity of vacuolation as well as insufficient knowledge regarding the nature of the contents of these vacuoles. The goal of this article is to help address some of the gaps identified above by providing points to consider, including a pictorial review of PEG-associated microscopic findings, when evaluating and reporting the extent, severity, and significance (adversity or lack of adversity) of PEG-associated cytoplasmic vacuolation in safety assessment studies. [Box: see text].

Toxicity associated with ingestion of a polyacrylic acid hydrogel dog pad.

Superabsorbent sodium polyacrylate polymeric hydrogels that retain large amounts of liquids are used in disposable diapers, sanitary napkins, and other applications. These polymers are generally considered "nontoxic" with acute oral median lethal doses (LD50) >5 g/kg. Despite this favorable toxicity profile, we identified a novel toxic syndrome in dogs and rats following the ingestion of a commercial dog pad composed primarily of a polyacrylic acid hydrogel. Inappropriate mentation, cerebellar ataxia, vomiting, and intention tremors were observed within 24 h after the ingestion of up to 15.7 g/kg of the hydrogel by an adult, castrated male Australian Shepherd mix. These observations prompted an experimental study in rats to further characterize the toxicity of the hydrogel. Adult, female Sprague Dawley rats ( n = 9) were assessed before and after hydrogel ingestion (2.6-19.2 g/kg over 4 h) using a functional observation battery and spontaneous motor activity. Clinical signs consistent with neurotoxicity emerged in rats as early as 2 h after the end of hydrogel exposure, including decreased activity in an open field, hunched posture, gait changes, reduced reaction to handling, decreased muscle tone, and abnormal surface righting. Hydrogel-exposed rats also had reduced motor activity when compared with pre-exposure baseline data. Rats that ingested the hydrogel did not develop nervous system lesions. These findings support the conclusion that some pet pad hydrogel products can induce acute neurotoxicity in animals under high-dose exposure conditions.

Therapeutic potential of the translation inhibitor silvestrol in hepatocellular cancer.

BACKGROUND & AIMS: Although hepatocellular cancers (HCC) frequently arise in the setting of fibrosis and a hepatic regenerative response requiring new cell growth, therapeutic strategies for these cancers have not targeted protein synthesis. Silvestrol, a rocaglate isolated from Aglaiafoveolata, can inhibit protein synthesis by modulating the initiation of translation through the eukaryotic initiation factor 4A. In this study, we evaluated the therapeutic efficacy of silvestrol for HCC. METHODS: The efficacy of silvestrol was examined using human HCC cells in vitro using an orthotopic tumor cell xenograft model in a fibrotic liver. The impact of silvestrol on the liver was assessed in vivo in wild-type mice. RESULTS: Silvestrol inhibited cell growth with an IC50 of 12.5-86 nM in four different HCC cell lines. In vitro, silvestrol increased apoptosis and caspase 3/7 activity accompanied by loss of mitochondrial membrane potential and decreased expression of Mcl-1 and Bcl-xL. A synergistic effect was observed when silvestrol was combined with other therapeutic agents, with a dose-reduction index of 3.42-fold with sorafenib and 1.75-fold with rapamycin at a fractional effect of 0.5. In vivo, an antitumor effect was observed with 0.4 mg/kg silvestrol compared to controls after one week, and survival of tumor-bearing mice was improved with a median survival time of 42 and 28 days in the silvestrol and control groups, respectively. The effect on survival was not observed in orthotopic xenografts in non-fibrotic livers. Silvestrol treatment in vivo did not alter liver structure. CONCLUSIONS: These data identify silvestrol as a novel, structurally unique drug with potent anticancer activity for HCC and support the potential value of targeting initiation of translation in the treatment of HCC.

Rodent preclinical models for developing novel antiarthritic molecules: comparative biology and preferred methods for evaluating efficacy.

Rodent models of immune-mediated arthritis (RMIA) are the conventional approach to evaluating mechanisms of inflammatory joint disease and the comparative efficacy of antiarthritic agents. Rat adjuvant-induced (AIA), collagen-induced (CIA), and streptococcal cell wall-induced (SCW) arthritides are preferred models of the joint pathology that occurs in human rheumatoid arthritis (RA). Lesions of AIA are most severe and consistent; structural and immunological changes of CIA best resemble RA. Lesion extent and severity in RMIA depends on experimental methodology (inciting agent, adjuvant, etc.) and individual physiologic parameters (age, genetics, hormonal status, etc.). The effectiveness of antiarthritic molecules varies with the agent, therapeutic regimen, and choice of RMIA. All RMIA are driven by overactivity of proinflammatory pathways, but the dominant molecules differ among the models. Hence, as with the human clinical experience, the efficacy of various antiarthritic molecules differs among RMIA, especially when the agent is a specific cytokine inhibitor.

Recommendations for pathology peer review.

Pathology peer review verifies and improves the accuracy and quality of pathology diagnoses and interpretations. Pathology peer review is recommended when important risk assessment or business decisions are based on nonclinical studies. For pathology peer review conducted before study completion, the peer-review pathologist reviews sufficient slides and pathology data to assist the study pathologist in refining pathology diagnoses and interpretations. Materials to be reviewed are selected by the peer-review pathologist. Consultations with additional experts or a formal (documented) pathology working group may be used to resolve discrepancies. The study pathologist is solely responsible for the content of the final pathology data and report, makes changes resulting from peer-review discussions, initiates the audit trail for microscopic observations after all changes resulting from peer-review have been made, and signs the final pathologist's report. The peer-review pathologist creates a signed peer-review memo describing the peer-review process and confirming that the study pathologist's report accurately and appropriately reflects the pathology data. The study pathologist also may sign a statement of consensus. It is not necessary to archive working notes created during the peer-review process.

RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFalpha or anti-IL-1 therapies.

INTRODUCTION: Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFalpha, IL-1beta, and receptor activator of NF-kappaB ligand (RANKL). Anti-IL-1 or anti-TNFalpha therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared. METHODS: Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFalpha inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (alpha1AGP), multiple cytokines) were measured in serum (day 14 post onset). RESULTS: Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFalpha reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFalpha and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFalpha and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum alpha1AGP, IL-1beta, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFalpha and IL-17. Anti-TNFalpha reduced systemic alpha1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic alpha1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model. CONCLUSIONS: Anti-TNFalpha or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.

RANKL is a marker and mediator of local and systemic bone loss in two rat models of inflammatory arthritis.

UNLABELLED: RANKL is an essential mediator of bone erosions, but the role of RANKL in systemic bone loss had not been studied in arthritis. RANKL protein was increased in rat joint extracts and serum at the earliest stages of arthritis. Osteoprotegerin (OPG) treatment reversed local and systemic bone loss, suggesting that RANKL is both a marker and mediator of bone loss in arthritis. INTRODUCTION: RANKL is well established as an essential mediator of bone erosions in inflammatory arthritis, but the role of RANKL in systemic bone loss in arthritis had not been studied. We hypothesized that serum RANKL could serve as both a mediator and as a novel biomarker for local and systemic bone loss in arthritis. We challenged this hypothesis in two established rat models of inflammatory arthritis. We sought to determine whether serum RANKL was elevated early in disease progression and whether RANKL suppression could prevent both local and systemic bone loss in these models. MATERIALS AND METHODS: Detailed time-course studies were conducted in animals with collagen-induced (CIA) or adjuvant-induced (AIA) arthritis to evaluate the onset and progression of inflammation (paw swelling), bone erosions, osteoclast numbers, and RANKL protein levels in arthritic joints and in serum. Additional CIA and AIA rats (n=8/group) received placebo (PBS) or recombinant OPG (3 mg/kg three times weekly) for 10 days beginning 4 days after disease onset (first macroscopic evidence of hind paw erythema and edema) to assess the role of RANKL in local and systemic bone loss. RESULTS: RANKL protein was significantly elevated in the joints and serum of CIA and AIA rats within 1-2 days of disease onset. Increased RANKL levels were associated with local (hind paw) and systemic (vertebral) osteopenia in both models. The RANKL inhibitor OPG prevented local and systemic osteopenia in both models of established disease. CONCLUSIONS: RANKL protein is significantly increased both locally and systemically during the earliest stages of inflammatory arthritis in rats, suggesting that serum RANKL might have prognostic value for bone erosions and systemic osteopenia in this condition. RANKL inhibition through OPG prevented local and systemic bone loss in these arthritis models, suggesting that RANKL inhibition is a promising new approach for treating bone loss in arthritis.

Kinetics of bone protection by recombinant osteoprotegerin therapy in Lewis rats with adjuvant arthritis.

OBJECTIVE: To assess the effect of different dosages and treatment schedules of osteoprotegerin (OPG) on joint preservation in an experimental model of adjuvant-induced arthritis (AIA). METHODS: Male Lewis rats with AIA (6-8 per group) were treated with a subcutaneous bolus of recombinant human OPG according to one of the following schedules: daily OPG (an efficacious regimen) starting at disease onset (days 9-15), early intervention (days 9-11), delayed intervention (days 13-15), and extended therapy (days 9-22). Inflammation (hind paw swelling) was quantified throughout the clinical course; osteoporosis (bone mineral density [BMD], by quantitative dual x-ray absorptiometry) and morphologic appraisals of inflammation, bone damage, intralesional osteoclasts (by semiquantitative histopathologic scoring), and integrity of the articular cartilage matrix (by retention of toluidine blue stain) were determined in histology sections of arthritic hind paws. RESULTS: OPG provided dose- and schedule-dependent preservation of BMD and periarticular bone while essentially eliminating intralesional osteoclasts. Dosages > or = 2.5 mg/kg/day preserved or enhanced BMD and prevented essentially all erosions. A dosage of 4 mg/kg/day protected joint integrity to a comparable degree when given for 7 (days 9-15) or 14 (days 9-22) consecutive days. At this dosage, early intervention (days 9-11) was twice as effective as delayed intervention (days 13-15) at preventing joint dissolution. Erosions and osteoclast scores were greatly decreased for 26 days (measured from the first treatment) after 7 or 14 daily doses of OPG (4 mg/kg/day). OPG treatment also prevented loss of cartilage matrix proteoglycans, an indirect consequence of protecting the subchondral bone. No OPG dosage or regimen alleviated weight loss, inflammation, or periosteal osteophyte production. CONCLUSION: These data indicate that OPG preserves articular bone and (indirectly) articular cartilage in arthritic joints in a dose- and schedule-dependent manner, halts bone erosion when given at any point during the course of arthritis, produces sustained antierosive activity after a short course, and is most effective when initiated early in the disease.