RESUMO
Double antibody sandwich-type ELISA was used to detect rhG-CSF in serum to study the pharmacokinetics of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF in mice and to confirm that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF. Pharmacokinetic parameters were calculated with 3P87 software. T1/2 s of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF are 2. 1 , 14.2 and 10. 6 h, respectively. T1/2 s of PEG- rhG-CSF and rHSA-hG-CSF are 7, 5 times than T1/2 s of rhG-CSF, respectively. Tpeak s of PEG-rhG-CSF and rHSA-hG-CSF are 15, 13 times than Tpeak of rhG-CSF, respectively. The result of ELISA indicates that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacocinética , Polietilenoglicóis/química , Proteínas Recombinantes de Fusão/farmacocinética , Albumina Sérica/química , Animais , Área Sob a Curva , Ensaio de Imunoadsorção Enzimática/métodos , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/química , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/química , Proteínas RecombinantesRESUMO
Poor pharmacokinetics and resistance within some tumor cell lines have been the major obstacles during the preclinical or clinical application of TRAIL (tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand). The half-life of TRAIL114-281 (114 to 281 amino acids) was revealed to be no more than 30 minutes across species. Therefore maleimido activated PEG (polyethylene glycol) and MMAE (Monomethyl Auristatin E) were applied to site-specifically conjugate with the mutated cysteines from different monomers of TRAIL successively, taking advantage of steric effects involved within TRAIL mutant conjugations. As a result, TRAIL trimer was hetero-modified for different purposes. And the resulting PEG-TRAIL-vcMMAE conjugate exhibited dramatically improved half-life (11.54 h), favourable in vivo targeting capability and antitumor activities while no sign of toxicity in xenograft models, suggesting it's a viable therapeutic and drug delivery strategy.
Assuntos
Antineoplásicos/química , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Pulmonares/tratamento farmacológico , Oligopeptídeos/química , Polietilenoglicóis/química , Ligante Indutor de Apoptose Relacionado a TNF/química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Meia-Vida , Humanos , Células K562 , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células MCF-7 , Masculino , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Ligante Indutor de Apoptose Relacionado a TNF/farmacocinética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent that specifically induces apoptosis in broad-spectrum tumor cell lines, meanwhile leaving normal cells unaffected. Unfortunately, the clinical development of TRAIL was hampered, and could be attributed to its instability, bioavailability or poor delivery. Although N-terminal specific PEGylation provides a means to improve the pharmacokinetic and stability of TRAIL, it took a bit longer time to accomplish the PEGylation process than expected. We therefore designed another PEGylation approach, mutated Cys-SH site-specific PEGylation, to conjugate methoxypoly(ethylene glycol) maleimide (mPEG-MAL) with TRAIL (95-281) mutant N109C. Asn-109 was chosen as the PEGylated site for it is a potential N-linked glycosylation site. It was shown that ~90% TRAIL mutant N109C could be PEGylated by mPEG-MAL within 40 min. And mPEG(MAL)-N109C was revealed to possess superior in vitro stability and antitumor activity than N-terminal specifically PEGylated TRAIL (114-281) (mPEG(ALD)-TRAIL(114-281)). What's more, mPEG(MAL)-N109C exhibited more therapeutic potentials than mPEG(ALD)-TRAIL(114-281) in tumor xenograft model, benefitting from better drug delivery and bioavailability. These results have demonstrated mutated Cys-SH specific PEGylation is an alternative to site-specifically PEGylate TRAIL efficiently and effectively other than N-terminal specific PEGylation.