Long-Term Safety of PEGylated Coagulation Factor VIII in the Immune-Deficient Rowett Nude Rat.

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals.

Local tolerance and systemic toxicity of single and repeated intramuscular administrations of two different formulations of the RTS,S malaria candidate vaccine in rabbits.

RTS,S malaria antigen is weakly immunogenic as such and needs to be formulated with an adjuvant to improve the magnitude and duration of the immune responses to RTS,S. Two Adjuvant Systems, AS01 and AS02 were evaluated during the development of the RTS,S vaccine. The evaluation included non-clinical studies in rabbits to evaluate the local intramuscular tolerance following administration on a single occasion, and the local and systemic effects following repeated administrations of RTS,S/AS01 or RTS,S/AS02 formulations. In the first study, rabbits were injected on one occasion with RTS,S/AS01, RTS,S/AS02 or controls, and the local intramuscular tolerance was evaluated up to 3 days after injection. In the second study, the different formulations were injected on Days 0, 14, 28 and 42. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Macroscopic and microscopic evaluations were made after termination of the study. No sign of toxicity was detected following single or repeated administrations of the adjuvanted RTS,S formulations. Changes in haematology or clinical chemistry parameters were indicative of a developing immune response in the groups receiving either RTS,S formulation. All examined parameters returned to normal within 28 days after the last injection. The absence of toxicological effects following the injection of RTS,S/AS01 or RTS,S/AS02 in rabbits was supportive of further clinical evaluation of these two formulations.

The haemotoxicity of mitomycin in a repeat dose study in the female CD-1 mouse.

Mitomycin (MMC), like many antineoplastic drugs, induces a predictable, dose-related, bone marrow depression in man and laboratory animals; this change is generally reversible. However, there is evidence that MMC may also cause a late-stage or residual bone marrow injury. The present study in female CD-1 mice investigated the haematological and bone marrow changes induced by MMC in a repeat dose study lasting 50 days. Control and MMC-treated mice were dosed intraperitoneally on eight occasions over 18 days with vehicle, or MMC at 2.5 mg/kg, autopsied (n = 6-12) at 1, 7, 14, 28, 42 and 50 days after the final dose and haematological changes investigated. Femoral nucleated bone marrow cell counts and levels of apoptosis were also evaluated and clonogenic assays carried out; serum levels of FLT3 ligand (FL) were assessed. At day 1 post-dosing, MMC induced significant reductions in RBC, Hb and haematocrit (HCT) values, and there were decreases in reticulocyte, platelet, and femoral nucleated cell counts (FNCC); neutrophil, lymphocyte and monocyte values were also significantly reduced. On days 7 and 14 post-dosing, all haematological parameters showed evidence of a return towards normal values, but at these times, and at day 28, values for RBC and FNCC remained significantly reduced in comparison with controls. At days 42 and 50 post-dosing, many haematological parameters in MMC-treated mice had returned to control levels; however, there remained evidence of late-stage effects on RBC, Hb and HCT values, and FNCC also continued to be significantly decreased. Results for granulocyte-macrophage colony-forming units and erythroid colonies showed a profound decrease immediately post-dosing, but a return to normal values was evident at day 50. Serum FL concentrations demonstrated very significant increases in the immediate post-dosing period, but a return to normal was seen at day 50 post-dosing; a relatively similar pattern was seen in the number of apoptotic femoral marrow nucleated cells. The histopathological examination of kidney tissues from MMC animals at day 42 and 50 post-dosing showed evidence of hydronephrosis with cortical glomerular/tubular atrophy and degeneration. It is therefore concluded that MMC administered on eight occasions over 18 days to female CD-1 mice at 2.5 mg/kg induced profound changes in haematological and bone marrow parameters in the immediate post-dosing period with a return to normal levels at day 50 post-dosing; however, there was evidence of mild but significant late-stage/residual effects on RBC and FNCC, and on cells of the erythroid lineage in the bone marrow.