Abstract
Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed an alternative technology for generating a long acting potent GHR antagonist through translational fusion of a mutated GH linked to GH binding protein and tested three candidate molecules. All molecules had the amino acid change (G120R), creating a competitive GHR antagonist and we tested the hypothesis that an amino acid change in the GH binding domain (W104A) would increase biological activity. All were antagonists in bioassays. In rats all antagonists had terminal half-lives >20 hours. After subcutaneous administration in rabbits one variant displayed a terminal half-life of 40.5 hours. A single subcutaneous injection of the same variant in rabbits resulted in a 14% fall in IGF-I over 7 days. In conclusion: we provide proof of concept that a fusion of GHR antagonist to its binding protein generates a long acting GHR antagonist and we confirmed that introducing the W104A amino acid change in the GH binding domain enhances antagonist activity.
Highlights
In addition to the Glyine-120 to lysine antagonistic substitution in site two, Pegvisomant has eight further amino acid changes in site 1
GHA3 showed the greatest inhibition of growth hormone (GH) activity and the order of greatest inhibition to least was GHA3 >GHA1 (IC50 = 44.4 ± 4.7 nM >GHA2 (IC50 = 198.4 ± 25.2 nM)
We have demonstrated that a GHR antagonist generated through an amino acid change in the site 2 binding site of GH for its receptor has delayed clearance when fused to GH binding protein
Summary
In addition to the Glyine-120 to lysine antagonistic substitution in site two, Pegvisomant has eight further amino acid changes in site 1 These result in a GH derivative that forms a high-affinity complex with one GHR molecule. We previously demonstrated that a chimeric molecule comprised of GH linked to GHBP is a potent and long-acting GHR agonist[14] The pharmacokinetic characteristics suggested this fusion molecule could provide GH agonist therapy requiring injection every 21–28 days compared to daily GH administration This technology clearly demonstrated that fusion to GH binding protein is an effective technology to delay clearance[15]. The results demonstrate that this approach generates a GH antagonist with delayed clearance and biological activity in vivo
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