Recovery Animals in Toxicology Studies: An Innovation and Quality Consortium Perspective on Best Practices With Case Study Examples.

The inclusion of recovery animals in nonclinical safety studies that support clinical trials is undertaken with a wide diversity of approaches even while operating under harmonized regulatory guidance. While empirical evaluation of reversibility may enhance the overall nonclinical risk assessment, there are often overlooked opportunities to reduce recovery animal use by leveraging robust scientific and regulatory information. In the past, there were several attempts to benchmark recovery practices; however, recommendations have not been consistently applied across the pharmaceutical industry. A working group (WG) sponsored by the 3Rs Translational and Predictive Sciences Leadership Group of the IQ Consortium conducted a survey of current industry practice related to the evaluation of reversibility/recovery in repeat dose toxicity studies. Discussion among the WG representatives included member company strategies and case studies that highlight challenges and opportunities for continuous refinements in the use of recovery animals. The case studies presented in this paper demonstrate increasing alignment with the Society of Toxicologic Pathology recommendations (2013) towards (1) excluding recovery phase cohorts by default (include only when scientifically justified), (2) minimizing the number of recovery groups (e.g., control and one dose level), and (3) excluding controls in the recovery cohort by leveraging external and/or dosing phase data. Recovery group exclusion and decisions regarding the timing of reversibility evaluation may be driven by indication, modality, and/or other scientific or strategic factors using a weight of evidence approach. The results and recommendations discussed present opportunities to further decrease animal use without impacting the quality of human risk assessment.

Preclinical toxicity of a geldanamycin analog, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance.

PURPOSE: 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs. METHODS: Doses of 0, 2.4, 12 and 24 mg/m2 per day were administered to rats, while dogs received doses of 0, 8 or 16 mg/m2 per day. In both species, 17-DMAG was administered i.v. (slow bolus for rats; 1-h infusion for dogs) daily for 5 days. An additional cohort of dogs received 16 mg/m2 per day orally for 5 days. Clinical observations were noted, and standard hematology and clinical chemistry parameters were monitored. Selected tissues were evaluated microscopically for drug-related lesions. Tissue and plasma 17-DMAG concentrations were measured by HPLC/MS at selected time-points on days 1 and 5. RESULTS: Daily i.v. administration of 17-DMAG at doses of 24 mg/m2 per day in rats or 16 mg/m2 per day in dogs produced lethality on day 6, approximately 24 h following the last dose. Body weight loss was common in rats and dogs. Drug-related gastrointestinal, bone marrow and hepatic toxicities were also common in rats and dogs. Dogs also exhibited signs of renal and gallbladder toxicity. Plasma concentrations of 17-DMAG increased proportionately with dose in rats and disproportionately with dose in dogs. In rat tissues, however, only fourfold to sixfold increases in 17-DMAG concentrations were observed with a tenfold increase in dose. The highest concentrations of 17-DMAG were found in the liver of rats, with progressively lower concentrations in the spleen, lung, kidney and plasma. Regardless of the route of administration, higher drug concentrations were present in plasma (rat and dog) and tissue (rat) samples obtained on day 5 compared to those obtained on day 1. Although plasma concentrations decreased with time, 17-DMAG was still detected in dog plasma for at least 24 h after drug administration. CONCLUSIONS: With the recent approval of 17-DMAG for clinical use, the data generated from these preclinical studies will provide guidance to clinicians as they administer this drug to their patients. The MTD of 17-DMAG was 12 mg/m2 per day in rats and 8 mg/m2 per day in dogs; therefore, the recommended starting dose for phase I trial is 1.3 mg/m2 per day for 5 days. Gastrointestinal and bone marrow toxicity were dose-limiting in rats, and gastrointestinal, renal, gallbladder and bone marrow toxicity were dose-limiting in dogs. All adverse effects were fully reversible in surviving animals after treatment was complete.

Effect of preexposure to ultrafine carbon black on respiratory syncytial virus infection in mice.

Epidemiological studies have indicated that exposure to elevated levels of particulate matter exacerbates several pulmonary diseases, including asthma, bronchitis, and viral infections. Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in infants and may lead to the development of asthma in childhood. To determine whether particle exposure modulates the immune response to RSV, eight-week-old female BALB/c mice received an intratracheal (i.t.) instillation of either 40 micro g ultrafine carbon black (CB) particles or vehicle. The following day, mice were i.t. instilled with either 106 pfu RSV or uninfected media. End points were examined 1, 2, 4, 7, and 10 days during RSV infection. Compared with RSV alone, tumor necrosis factor-alpha (TNF-alpha) protein was reduced in the bronchoalveolar lavage fluid (BALF) on days 1 and 2 of infection; there was also a reduction in BALF lymphocyte numbers on day 4, which correlated with reductions in both IFN-gamma-inducible protein (IP-10), lymphotactin, and IFN-gamma mRNAs in the lungs of RSV + CB mice. Multiprobe ribonuclease protection assays of RSV + CB lung tissue showed no changes in the RSV-associated chemokines regulated upon activation, normal T cell expressed and secreted (RANTES), eotaxin, monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP)-1 alpha or MIP-1 beta. Viral titers in RSV + CB mice were lower than RSV on days 2-4 of infection. By day 7 of infection, however, neutrophil numbers, proinflammatory cytokine mRNA expression, and protein levels of TNF-alpha and the Th2 cytokine interleukin (IL)-13 were increased in the lungs of RSV + CB mice, indicating an exacerbation of infection. These data indicate that preexposure to ultrafine particles induces an inflammatory milieu promoting allergic immune responses rather than IFNgamma production necessary for microbial defense.

Ultrafine carbon black particles enhance respiratory syncytial virus-induced airway reactivity, pulmonary inflammation, and chemokine expression.

Exposure to particulate matter (PM) may exacerbate preexisting respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), bronchitis, and pneumonia. However, few experimental studies have addressed the effects of PM on lower respiratory tract (LRT) viral infection. Respiratory syncytial virus (RSV) is a major etiological agent for LRT infections in infants, the elderly, and the immunocompromised and may lead to chronic wheezing and the development of asthma in children. In this study, we examined the effects of carbon black (CB) on RSV-induced pulmonary inflammation, chemokine and cytokine expression, and airway hyperresponsiveness in a mouse model of RSV. Female BALB/c mice were instilled via the trachea (i.t.) with 1 x 106 plaque forming units (pfu) RSV or with uninfected culture media. On day 3 of infection, mice were i.t. instilled with either 40 micro g ultrafine CB particles or with saline. End points were examined on days 4, 5, 7, and 14 of RSV infection. Viral titer and clearance in the lung were unaffected by CB exposure. Neutrophil numbers were elevated on days 4 and 7, and lymphocyte numbers were higher on days 4 and 14 of infection in CB-exposed, RSV-infected mice. CB exposure also enhanced RSV-induced airway hyperresponsiveness to methacholine, bronchoalveolar lavage (BAL) total protein, and virus-associated chemokines monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1 alpha), and regulated upon activation, normal T cell expressed and secreted (RANTES). MIP-1 alpha mRNA expression was increased in the alveolar epithelium, where ultrafine particles deposit in the lung. These data demonstrate a synergistic effect of ultrafine CB particles on RSV infection, and suggest a potential mechanism for increased respiratory infections in human populations after PM exposure.