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

Abstract

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.

Plasma Polyethylene Glycol (PEG) Levels Reach Steady State Following Repeated Treatment with N8-GP (Turoctocog Alfa Pegol; Esperoct®)

“…The rate of decline was in line with the plasma halflife of PEG observed in the single-dose rat study [10]. As expected, human plasma PEG levels were several fold lower (five-to eightfold) than those reported at the no-observedadverse-effect-level in the rat toxicity study [17]. PEG exposure in children was slightly higher than in adults and adolescents, which is likely due to the higher N8-GP (and therefore PEG) dose combined with a shorter dose interval (60 IU/kg twice weekly in the paediatric trial vs. 50 IU/kg Q4D in the adult/adolescent trial).…”

“…Adolescents and adults (pathfinder2) were treated with 50 IU/kg N8-GP Q4D (open squares) [4,6]. Mean PEG exposure during the 52-week rat toxicity study [17] is indicated by the upper dotted line, and the LLoQ is shown by the lower dotted line. Predicted mean PEG exposure in children, adolescents and adults (grey solid line) is shown as a reference.…”

“…Unused plasma samples from an antidrug antibody analysis performed during a chronic 52-week toxicity study in Rowett nude rats were selected [17]. A total of 159 plasma samples were analysed from individual male rats receiving 0, 50, 150, 500, or 1200 IU/kg of N8-GP Q4D.…”

“…The consequences of different steady-state PEG levels can be assessed in repeat-dose toxicology studies. For N8-GP, a 52-week repeat-dose toxicology study in Rowett nude rats was conducted with intravenous administration at a dose range of 50-1200 IU/kg every 4th day (Q4D) [17]. No treatment-related adverse reactions or treatmentrelated changes in any organ were reported in this study, even at doses far beyond the clinical dose (maximum dose cohort: 1200 IU/kg Q4D).…”

PEGylated biologics in haemophilia treatment: Current understanding of their long‐term safety

“…7,8 In such studies, possible long-term effects of the PEGylated product can be investigated without interference by the immune system, and thus, a more relevant risk assessment for chronic effects can be performed. In order to overcome this situation, immunodeficient athymic rats have been used to evaluate PEGylated biologics in addition to existing toxicology programmes.…”