Improvement Of The Safety Profile And Cytoprotective Efficacy Of Amifostine By PEGylation Strategy

Amifostine,an organic thiophosphate prodrug,has been clinically utilized for selective protection of normal tissues with high expression of alkaline phosphatase from oxidative damage elicited by chemotherapy or radiotherapy.Amifostine can be dephosphorylated quickly by alkaline phosphatase for conversion into its active metabolite with sulfhydryls which can scavenge oxygen-derived free radicals and prevent DNA damage.Preclinical and clinical investigations have suggested that the cytoprotective selectivity of amifostine is mostly caused by the variations in alkaline phosphatase expression and extracellular acidity between normal tissues and tumors.For a majority of neoplastic tissues,hypoxia,weaklyacidic interstitial p H,and reduced expression of alkaline phosphatase in the plasma membrane of tumor cells all limit generation of the active metabolite,and therefore,amifostine is typically incompetent to protect these tumors from oxidative damages.In contrast,amifostine can be efficiently hydrolyzed into its active metabolite in most normal tissues with high expression of alkaline phosphatase and physiological environment,leading to selective protection of these normal tissues from the cytotoxicity of radiotherapy and chemotherapy.In clinical practice,amifostine is intravenously administered at the dosage of 740 mg/m2 which usually reaches the saturate drug concentration in blood.Since the active metabolite of amifostine shows very limited stability in vivo due to the rapid formation of disulfide from free sulfhydryls,such a high dosage is required to achieve desirable protective effects.However,the extremely high drug concentration in blood results in inevitable adverse effects such as hypotension,nausea,and vomiting,which has significantly restricted the extensive application of amifostine.Strategies for improvement of the protective efficacy and toxicity profile of amifostine are urgently required.PEGylation,covalently conjugating drug molecules with polyethylene glycol(PEG)chains,has shown great potential in improving the pharmacokinetics,pharmacodynamics and safety profiles of parent drugs.Since PEG is commercially available and has been approved by FDA for clinical use,PEGylation has been widely applied for the development of therapeutic agents including protein and peptide drugs,small molecule drugs,and drug delivery systems.PEGylation has the capability to change the unfavorable properties of drugs in solubility,biodistribution,immunogenicity,enzymatic degradation,renal filtration and reticuloendothelial system phagocytosis.Therefore,the circulatory half-life and bioavailability of drugs can be elevated,while drug toxicity can be reduced.This prompted us to consider whether PEGylation of amifostine can improve drug stability and efficacy,thereby allowing low injection dosage for a favorable safety profile.So far,modification of amifostine by PEG has never been reported in the literature.Considering the loading efficiency of small molecules to PEG polymers,we chose the 4-arm PEG with functional terminal for conjugation to amifostine.Here we constructed a PEGylated amifostine(PEG-amifostine)by a mild one-step reaction.The physiochemical properties,capacity of hydrolysis by alkaline phosphatase,stability,in vitro and in vivo toxicity and biological activities of PEGamifostine were evaluated.Successful conjugation of amifostine to 4-arm PEG was verified by various analyses,one 4-arm PEG was found conjugated with three amifostine molecules on average.The relatively large PEG-amifostine molecules clustered into spherical nanoparticles due to the hydrogen bonds and electrostatic interaction,resulting in distinct hydrolysis properties,cell uptake profile and antioxidative activity compared with the free small molecules.Amifostine was quickly hydrolyzed by alkaline phosphatase,releasing the active IX sulfhydryl groups.Whereas PEGylation prolonged the hydrolysis time of amifostine,which was likely attributed to the formation of clusters,providing sustained transformation to its functional metabolites.PEG-amifostine could be internalized into cells and translocated to acidic organelles in a time-dependent manner,a pattern distinct from the diffusion behavior of the small molecule,amifostine.The prolonged contact between PEG-amifostine and alkaline phosphatases on the membrane during the endocytosis guarantee the sufficient release of active sulfhydryl groups from immobilized amifostine.The in vitro safety profile of amifostine showed acute cytotoxicity.Testified by the inhibition of alkaline phosphatase,the severe cytotoxicity of amifostine was likely induced by the rapid formation of sulfhydryl groups.Interestingly,the intrinsic cytotoxicity of amifostine,which is related to the reductive reactivity of its metabolites and their ability to diffuse readily,was attenuated after PEGylation.This modification impeded the interaction between free sulfhydryl and functional biomolecules,providing PEG-amifostine with an improved safety profile in vitro.We established oxidative damage cell model by simulating cells with hydrogen peroxide.PEG-amifostine exhibited a long-term antioxidative activity with a slowly accumulative profile in 24 h without showing any toxicity.On the contrary,treatment of amifostine exhibited a shorter protection time and sensitized cells to H2O2 stimulation with 24 hours incubation,leading to a higher ROS level.Similar cytoprotective effects of amifostine and PEG-amifostine were observed in the prevention of cisplatin-induced cytotoxicity.Moreover,PEGylation did not change the selective protection of amifostine and is expected to enhance the protective effects of amifostine against chemotherapy at a higher dose.PEGylated form of amifostine significantly improved the efficacy and decreased the adverse effects of this antioxidant in vitro.Therefore,In vivo study to confirm and redeem the cytoprotective effects of the PEG-amifostine was carried out.Because of the increment of molecular weight by PEGylation,the safe dose of PEG-amifostine for in vivo experiments was validated first,the effective components in which equaled only one tenth of applied dose of free amifostine.Subsequently,Amino-fluorescein was conjugated to the 4-arm PEG for simulation of PEG-amifostine.Detection of the biodistribution of PEG-fluorescein at a safe dose found retention in kidney.Ultimately,in vivo cytoprotective effects of the PEG-amifostine were evaluated in mice with injection of cisplatin or receiving the ? radiation.The nephrotoxicity by cisplatin was prevented by PEG-amifostine through the measurement of the lipid peroxidation degree in kidney.Unexpectedly,amifostine amplified the kidney impairment instead of protective effects.In irradiated mice,both amifostine and PEG-amifostine mildly increased the percentage of live lymphocyte in bone marrow,whereas the cell injury could be reversed completely by neither of them.Overall,our study for the first time developed a PEGylated form of amifostine which significantly improved the efficacy and decreased the adverse effects of this antioxidant with great promise for clinical translation.