Study On N-terminal PEG/PEG-hexadecane Modification Of Human Growth Hormone And Growth Hormone Antagonist

Human growth hormone (hGH) has been used to treat children with short stature, renal failure and Turner's Syndrome. Growth hormone antagonist (GHA), an analog of growth hormone (GH), can inhibit GH action and treat acromegaly. However, clinical application of hGH and GHA suffers from its short plasma half-life and low bioavailability. PEGylation and albumin binding are two of the most effective approaches to prolong the plasma half-life of hGH. However, the steric shielding effects of polyethylene glycol (PEG) and albumin can drastically decrease the bioactivity of hGH, which is opposite to the increased pharmacokinetics (PK). In order to solve the problems, we establish the relationship of the PEG size and the PD property of GHA for the development of long-acting therapeutic GHA. On this basis, two N-terminally mono-PEGylated hGHs were prepared using aldehyde chemistry. Phenyl amide and ethyl moieties were used as the linkers between PEG and hGH, respectively. The present study was aimed to optimize the linker chemistry for improving the PK and PD of the N-terminally mono-PEGylated hGH. In order to further increase the pharmacological function, we designed and prepared by N-terminal PEG-hexadecane modification of hGH.The dissertation is divided into three parts. The first part focused on N-terminal mono-PEGylation of GHA with 20 kDa and 40 kDa PEG were used to look for a balance of the two competing factors. Sedimentation velocity analysis suggested that 40 kDa PEG can generate a stronger hydrated layer than 20 kDa PEG to surround GHA. As reflected by marginal suppression of insulin-like growth factor I (IGF-I), GHA conjugated with 40 kDa PEG can marginally inhibit hGH action. In contrast, GHA conjugated with 20 kDa PEG can apparently inhibit hGH action, as reflected by IGF-I suppression of 30?43%. Thus, our work demonstrated the effective therapeutic potency of N-terminally mono-PEGylated GHA.The second part investigated two N-terminally mono-PEGylated hGHs prepared using aldehyde chemistry. Phenyl amide and ethyl moieties were used as the linkers between PEG and hGH, respectively. The hydrodynamic volume, proteolytic sensitivity and immunogenicity of the PEGylated hGH with phenyl amide linker (hGH-phenyl-PEG) were lower than those of the one with propyl linker (hGH-prop-PEG). In addition, hGH-phenyl-PEG showed a higher in vitro bioactivity and better PK and PD than hGH-prop-PEG. The better PK of hGH-phenyl-PEG was mainly due to its lower proteolytic sensitivity and low immunogenicity. The better PD of hGH-phenyl-PEG was mainly due to its higher in vitro bioactivity. Thus, the phenyl amide linker can improve the overall pharmacological profiles of the PEGylated hGH.The third part designed and prepared by N-terminal PEG-hexadecane modification of hGH, using PEG (3.5 kDa or 10 kDa) as the linker. PEGylation and albumin binding with hexadecane can increase the hydrodynamic volume and decrease the immunogenicity of hGH, thereby markedly increasing the PK of hGH. Since N-terminus is far from the bioactive domain of hGH, N-terminal modification of hGH can minimize the steric shielding effects on the bioactive domain of hGH. Hexadecane-bound albumin can be slowly released from hGH during the in vivo circulation, which can slowly restore the bioactivity of hGH. Thus, the high bioactivity of PEG-hexadecane modified hGH (hGH-PEG-HD) was synergistically achieved by N-terminal PEG-hexadecane modification and slow-release of albumin.In conclusion, the novel method of optimizing the PEG chain, linker chemistry and using the noncvalent binding technology can improve the pharmacodynamics of hGH efficiently. Therefore, the present study can facilitate the development of a potent long-acting protein drug with high therapeutic efficacy.