Pegvisomant is a specific GH antagonist developed for the treatment of acromegaly. Pegvisomant is a GH antagonist molecule with an amino acid substitution that blocks the conformational change necessary for signal transduction and polyethylene glycol (PEG) moieties to improve clinical efficacy. Pegvisomant has a long plasma half-life and its mode of clearance has not been established. We hypothesised that GHR mediated internalisation of Pegvisomant might be one mechanism for it clearance. To study internalisation, 293 cells expressing GHR were exposed for 30 minutes to GH or Pegvisomant at concentrations similar to those found in the treatment of acromegaly (200and 5000 ng per ml respectively). The ligands were detected by immunofluorescence. GH showed little labelling at the cell surface and the majority of GH was internalised. In cells exposed to Pegvisomant, clear plasma membrane binding and internalised ligand was detected. The percentage of total intracellular fluorescence was 91.4.0 plus or minus 2.1% for GH and 58.3 plus or minus 6.0% for Pegvisomant (p<0.001 for Pegvisomant vs GH). Pegvisomant concentrations led to a dose-dependent increase in intracellular mean cell fluorescence. In order to follow the passage of ligand within the cell, 293GHR cells were exposed to a 15 minutes pulse of either GH or Pegvisomant, in the presence of alexa-594 transferrin. Intracellular fluorescent vesicles were detectable in both GH and Pegvisomant treated cells. Their intracellular localisation was similar, showing co-localisation with transferrin .
Our studies demonstrate that, despite its size, Pegvisomant is internalised by cells expressing the GHR. As Pegvisomant doesn't activate intracellular signal transduction systems, our results support the concept that the conformational changes required for GHR signaling are not essential for the intracellular trafficking of the ligand and establish one mechanism for Pegvisomant clearance.
22 - 24 Mar 2004
British Endocrine Societies