Research Of Oral Drugs Of Human Erythropoietin(rhEPO) Expressed In Bombyx Mori

In order to study the pharmacological effect and safety evaluation of recombinant humanerythropoietin (rhEPO) expressed in silkworm pupae in the treatment of renal anemia by oraladministration, we used Bac-to-Bac system to construct a recombinant human erythropoietin B.mori baculovirus (vBm-rhEPO)，virus titer of whose was determined by real-time fluorescentquantitative PCR. By the identification of SDS-PAGE and Western blotting, rhEPO protein(BmrhEPO) was successfully expressed with a molecular size of~25kDa in BmN cell, silkwormlarvae and pupa infected by vBm-rhEPO, respectively. The silkworm pupa infected by vBm-rhEPOwere made into lyophilized powder by homogenate and lyophilization, and quantification ofrecombinant rhEPO protein (BmrhEPO) showed that the expression of BmrhEPO reached the levelof approximately8382IU/g pupae lyophilized powder. The in vitro biological activity of BmrhEPOdetermined by evaluating its ability to enhance the proliferation of erythroleukemic TF-1cell usingMTT method was similar to that of rhEPO expressed by CHO cell. Under the same concentration,the in vitro biological activity of BmrhEPO was much higher than that of rhEPO expressed by CHOcell. And the half effective concentration EC50of BmrhEPO was0.36IU/mL, which was slightlyless than that of rhEPO expressed by CHO cell. Acute toxicity experiment of oral administration ofBmrhEPO pupae lyophilized powder in mice showed that it was non-toxic for mice, and themaximum tolerated dose of BmrhEPO was more than147882IU/kg. Renal anemia mice model wasconstructed using continuous gavage of adenine, and identified by the detection of renal functionand blood routine. The pharmacological effects of BmrhEPO after oral administration in renalanemia mice, which were gavaged BmrhEPO once a day at the dose of73958.8,24652.9and14791.8IU/kg, respectively, were evaluated by circulating reticulocyte counts and percentage ofcirculating reticulocytes of erythrocytes, respectively, in comparison to those for subcutaneousadministration of positive drug Recormon at the dose of200IU/kg. The results showed that thereticulocyte number and percentage of three oral groups were increased significantly compared withnegative control group, and positively correlated with oral dose. It suggests that it was effective tooral BmrhEPO in the treatment of renal anemia in mice. This research provides experimental basisfor the development of recombinant human erythropoietin oral drugs.