Study On The Radioprotective Effect Of Heparin-Superoxide Dismutase Conjugates

With the rapid development of medical therapy technology, the advanced radiotherapy with increasing accuracy has been playing an important role in the tumor therapeutic field. However, the unsolved problem associated with the surrounding normal tissue injury impedes its further application. In order to develop a new radioprotector with high efficiency and low toxicity, the mechanism by which radiation injury occurs is arousing more and more attention. At present, the most widely accepted explaination is the hypothesis of oxidative response injury caused by the excess reactive oxygen species (ROS) which are produced by the reaction between the radiation and the related in vivo photosensitizers. The ROS family which can targetedly damage bio-macromolecules such as DNA, lipid and protein is composed of different members among which superoxide free radical (O2) is the first one being produced and then can stimulate the generation of the others. In this sense, as the unique enzyme responsible for scavenging O2, superoxide dismutase (SOD) is believed in its potency of ameliorating the ROS-mediated normal tissue injury. Nonetheless, like most of the other protein pharmaceuticals, the direct application of SOD is restricted for the common shortcomings such as its vulnerability to proteinase, poor stability, short half-life and so on. To overcome the above problems, chemical modification is one of the widely adopted measures. In this study, the functional mucopolysaccharide, heparin, was selected to modify SOD for improving the above-mentioned disadvantages as well as integrating and enlarging its biological functions. Based on the early researches, first of all. the porcine erythroeyte-derived SOD was purified and then the Hep-SOD conjugates were prepared. By means of SDS-PAGE, Native-PAGE, SEC-HPLC, gel filtration chromatography and multi-angle laser scattering methods, the purity and the weight-average molecular weight (Mw) of SOD and Hep-SOD were examined. In vitro, the radioprotective effect of Hep-SOD on the murine lung fibroblast cell L929was investigated using MTT assay. In the pharmacokinetic experiment, the foundational pharmacokinetic parameters of Hep-SOD were examined by detecting the enzymatic activity in the mice peripheral blood at different time points after peritoneal injection using SOD kit. At last, the in vivo systematic evaluation of the radioprotective effect of Hep-SOD on the irradiated mice was implemented according to the guiding principle of pre-clinic testing for anti-radiation drugs.1. Purification of the porcine erythrocyte-derived SODWith references to the early studies, crude SOD was purified by DEAE-Sepharose Fast Flow chromatography. Then, the purity of its purified products was determined qualitatively and quantitatively with the alliance of SDS-PAGE and SEC-HPLC. It was shown that the purity of SOD increased from87%to almost100%after purification. Meanwhile, enzymatic activity of the purified SOD was raised to4614u/mg prot with a50%increment compared with the crude SOD.2. Preparation of Hep-SOD and the analysis for its purity and molecular weightAlso based on the early studies, Hep-SOD conjugates were prepared through heparin activation, chemical modification and isolation by Q-Sepharose Fast Flow chromatography. The average modification rate of the free amino-group of SOD was32%detected by TNBS method. Then, two kinds of conjugates with different modification rates named as Hep-SOD1and Hep-SOD2were examined for their enzymatic activity. It was revealed that, compared with the crude SOD, Hep-SOD1remained68%of the original enzymatic activity while Hep-SOD2remained40%of that. By using gel filtration chromatography and multi-angle laser scattering method, the Mw of Hep-SOD1and Hep-SOD2was shown to be49.69kDa and61.07kDa respectively.3. In vitro evaluation for the radioprotective effect of Hep-SOD on L929cellsCell-seeded concentration, sensitive radiation dosage and detecting time after radiation were screened with MTT assay. It was exhibited that L929at the concentration of2000cells per well showed remarkable radiosensitivity to4Gy X rays48hours after the radiation which was evidenced by the significantly lowered related cell viability compared with the normal control group (P<0.01). Hep-SOD1showed no cytotoxicity in the examined dose range (1000～0μ.g/ml,990～0μ/ml) while Hep-SOD2was nontoxic below the concentration of500μg/ml (112.5U/ml) which might be due to its higher modification rate to some extent.112.5U/ml was selected as the final concentration for the two conjugates for further study on their radioprotective effects. Indicated by the indifference from the normal control group, both of Hep-SOD1and Hep-SOD2exhibited notably radioprotective effect on L929cells treated pre-radiation while no effect was observed when treated post-radiation.4. Determination of the foundational pharmacokinetic parameters of Hep-SODThe foundational pharmacokinetic parameters of Hep-SOD were examined by detecting the enzymatic activity in the mice peripheral blood at different time points after peritoneal injection. Analysed by the related software, in vivo pharmacokinetic action of Hep-SOD was shown to be in accordance with the two-compartment model. After chemical modification, half-life of SOD was lengthened remarkably. Mean retention time (MRT) of Hep-SOD2was shown to be (22.86±3.37)h compared with (10.82±0.98)h of SOD. Overall, Hep-SOD2exhibited better bioavailability.5. In vivo systematic evaluation of the radioprotective effect of Hep-SODBased on the aforementioned in vitro evaluation and detection of the foundational pharmacokinetic parameters of both conjugates, Hep-SOD2(named as Hep-SOD in the following) was preferred in this chapter for its definitely radioprotective effects in vitro and better bioavailability in vivo. By consulting to the guiding principle of pre-clinic testing for anti-radiation drugs, systematic study was developed on the irradiated mice using Hep-SOD. The results showed that Hep-SOD could ameliorate the radiation-induced myclotoxicity, reduce the oxidative response injury especially in lung, down-regulate both of GPT and GOT contents in the peripheral blood, suppress the related histopathological changes.However. agarose electrophoresis for the DNA extracted from the mouse thymus showed that the pre-administration of Hep-SOD to the irradiated mice was likely to worsen DNA injury. This phenomenon posed the necessity for further exploring the radiation injury and the concrete action mechanism of Hep-SOD in detail.In summary, we achieved several results and conclusions as follows in this study:(1) The weight-average molecular weight of Hep-SOD was detected for the first time by gel filtration chromatography and multi-angle laser scattering method.(2) Hep-SOD showed significantly radioprotective effects on the murine lung fibroblast cell L929when treated pre-radiation.(3) Through heparin modification, half-life of SOD was lengthened remarkably and bioavailability was increased significantly. Moreover, with the increasing modification rate, Hep-SOD exhibited better in vivo pharmacokinetic action property.(4) For the radiation-injured mice, Hep-SOD could effectively ameliorate radiation-induced myelotoxicity, reduce oxygen response injury in the tissue level, suppress the related histopathological changes. However, it was also exhibited that Hep-SOD administration pre-radiation was tended to worsen DNA injury which posed the necessity for comprehensive interpretation of the radiation injury and the concrete action mechanism of Hep-SOD in the subsequent researches.