Assessing a theoretical risk of dolutegravir-induced developmental immunotoxicity in juvenile rats.

HIV-1 integrase inhibitors (INIs) are a promising class of antiretrovirals for the treatment of HIV in adults; there is interest in expanding their use into pediatric populations. A theoretical concern for developmental immunotoxicity was raised after a publication suggested that two HIV INI tool compounds inhibited in vitro cleavage activity of recombination activating genes 1 and 2 (RAG1/2) through the inhibition of their binding to recombination signal sequences. RAG1/2 are required for the development of mature B and T lymphocyte populations. The potential effects of the investigational INI dolutegravir on RAG1/2 were addressed by developing assays in juvenile rats to measure T cell receptor (TCR) Vß usage by flow cytometry as an indicator of TCR repertoire diversity and a T cell dependent antibody response (TDAR) as an indicator of immunosuppression. These endpoints were incorporated into a juvenile rat toxicity study, along with immunophenotyping, hematology, and histopathology of immunologic organs. Dose levels of 0, 0.5, 2, or 75mg/kg/day dolutegravir were given via oral gavage from postnatal day 4 through 66. At the highest dose, there was decreased body weight gain and two preweanling deaths; however, there were no treatment-related effects on developmental parameters. There were no effects on immunologic competence, as measured by TDAR, and no effects on lymphocyte subsets or CD4 and CD8 TCR Vß usage in peripheral blood. Histopathology of immunologic organs (spleen, thymus, lymph nodes) and hematology evaluation revealed no effects. The no observed adverse effect level for immunotoxicity endpoints was 75mg/kg/day.

Primary antibody response to keyhole limpet hemocyanin in rat as a model for immunotoxicity evaluation.

To address current regulatory expectations on immunotoxicity testing of new chemicals, we describe an animal model that measures the primary antibody response to the T-cell dependent antigen, keyhole limpet hemocyanin (KLH). Single immunization with KLH by either footpad (300microg/rat) or intravenous (300microg/kg) route in Sprague Dawley rats resulted in increased germinal center formation in the spleen and a robust anti-KLH IgM (70-388microg/ml) and IgG (230-470microg/ml) antibody response with peak detection on Days 5 and 14 post-immunization, respectively. Subcutaneous immunization with KLH (300microg/kg) resulted in a much weaker anti-KLH IgM and IgG (< or =20microg/ml) antibody response with no detectable increase in splenic germinal center formation. The utility of a rat KLH immunization model in detecting immunosuppression was evaluated with the known immunosuppressive drugs: cyclosporin, azathioprine and prednisolone. Rats, treated with drug at a maximum tolerated dose, were immunized with KLH by footpad or intravenous injection and serum samples were collected at various intervals up to 2 weeks post-immunization. Additional study parameters included terminal body weight, hematology and/or histopathology. All three drugs inhibited the IgM (60%) and IgG (> or =90%) antibody responses in the absence of overt toxicity based on evaluation of the standard toxicology parameters. In conclusion, measurement of a rat primary antibody response to KLH by ELISA is a reliable and readily standardized method for assessing immunotoxicity of pharmaceuticals.