| Interferons (IFNs), with the functions of anti-viral, antiproliferative. and immunomodulatory activities, are a family of inducible proteins secreted by cells. They are known as ones of the most effective drugs against virus clinically. But during clinical therapies, IFNs showed some disadvantages, such as, unstable properties, immunogenicity and a relatively short half-life in circulation. Modification of IFNs by covalent attachment of soluble and nonimmunogenic polymers (for example polyethylene glycol, PEG) results in species with dramatically increased plasma half lives and low immunogenicity. Consequently, PEGylation might improve IFNs'therapeutic benefits. The investigation intended to study characterizations of PEG-IFNα2b and screen a stable protective additive for its freez-drying formulation, so as to laid the basic and preclinical research of PEG-IFNα2b. PEG-IFNα2b was analyzed for the biochemical characterizations. Its concentration was 0.66mg/ml determined by Lowry Method; Ultraviolet spectrophotometry of IFNα2b and PEG-IFNα2b showed no perceptable difference in spectra, about 280nm. PEG had no effect on the ultraviolet spectrophotometry of IFNα2b; Relative molecular weight 62.6KD of PEG-IFNα2b was acquired by MALDI-TOF-MOS. The data showed that relative molecular weight of modified IFNα2b was increased for two-fold. Peptide mappings of PEG-IFNα2b and IFNα2b had showed that an absorbance peak of IFNα2b was altered by the modification as evident by its disappearance from the map. Isoelectric focusing (pI) range of PEG-IFNα2b can be estimated between 5.2-6.56. As a control, the pI of IFNα2b was determined in a manner similar to that employed for the PEG-IFNα2b, and was found to possess a pI of 4.0-6.7, in agreement with the established value. Purifity and characterizations of PEG-IFNα2b was tested by RP-HPLC, in which retention time is 10.2min, purifity is 98.9%, while retention time of IFNα2b is 4.042. The anti-viral activity of PEG-IFNα2b was analyzed by VSV-Wish system. Its ability to protect wish cells had decreased for 12.2-fold, and its anti-viral activity remained 8.2% of unmodified IFNα2b. Trypsin degradation of PEG-IFNα2b and IFNα2b had been done. The results showed that anti-viral activity of IFNα2b decreased from 8.8×107IU/ml to 939IU/ml, however PEG-IFNα2b's from 4.8 ×106IU/ml to 6 ×105IU/ml on the effect of trypsin at 37℃for 1h. And PEG-IFNα2b had a much slower decrement trend on antiviral activity than that of IFNα2b in the trypsin degradation reaction at first 30min. PEG-IFNα2b and IFNα2b were put at 56℃to assay their heat resistance. Their antiviral activity had no obvious changes until 12 hours. At the 36th hour, antiviral activity of IFNα2b had decreased to below 105IU/ml, and that of PEG-IFNα2b remained above 106IU/ml. The results indicated that PEG-IFNα2b had greater resistance to heat than that of IFNα2b. HM mice were injected i.m. with the same antiviral activity (IU/kg) of IFNα2b and PEG-IFNα2b. Serum concentrations of PEG-IFNα2b were 105IU/ml after treatment for 72h. We could not assay serum concentrations of IFNα2b at 24h postdosing. Main pharmacokinetic parameters were calculated from the i.m. data.. Pegylation resulted in approximately a 20-fold increase in half-life in mice. And the...
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