The Research About Silkworm Powder Helping The Aborbtion Of RhGM-CSF Through Oral Administration

The oral delivery of protein drug is a worldwide problem. In this study, we isolated a certainprotein from silkworm pupa powder. And as it to be drug loading material, we manufactured anew oral dosage form of rhGM-CSF (recombined human Gramulocyte/Macrophase colonystimulated factor）.In experiment, pupa powder was orally administered to Km mice as drug loading materialmixed rhGM-CSF drugs. Blood sample was collected at0,15,40,60,120min post oraladministration of the mixture, and the serum were used to detect the concentration of rhGM-CSFby using a rhGM-CSF ELISA Kit. The result indicated, the concentration of rhGM-CSF inbloodstream was highest of group, which was the mixture of pupa powder and rhGM-CSF drug,at40min post oral administration. And the concentration was twice of group, which was onlycomposed with rhGM-CSF. This showed that, as drug loading material, pupa powder was able tohelp the oral absorption of rhGM-CSF certainly.To find the active ingredient which helped rhGM-CSF oral absorption from the pupa powder,we divided pupa powder into soluble components and insoluble components by homogenate andcentrifugation. The result of ELISA maintained,40min post oral administration, theconcentration of rhGM-CSF in mixture group of supernatant and rhGM-CSF was four times ofmixture group of precipitant and rhGM-CSF. This was suggested, the active ingredient was in thesupernatant.Protein in supernatant was precipitated by ammonium sulfate precipitation and was dividedby2kDa,10kDa,30kDa,100kDa filters. The result of ELISA showed, the best promotingeffect of oral absorption of rhGM-CSF was the retentate of10kDa filter, the better was theretentate of100kDa filter, and the remaining is the worst. The result of different mixing ratio ofrhGM-CSF drugs and10kDa filter retentate lyophilized powder showed, the effect of10kDafilter retentate was relative to the mixing ratio, and the more of10kDa filter retentate lyophilizedpowders, the better of the promoting. It was positive correlation.The analysis of SDS-PAGE and Tricin-SDS-PAGE indicated that hardly any protein was in2kDa filter retentant，the main protein in10kDa filter retentat was10-20kDa and litter was30kDa, almost protein in30kDa filter retentat was30kDa, and the protein in100kDa filterretentat was30kDa and80kDa. So the active ingredient in10kDa filter retentat was10-20kDaproteins.The result of Co-IP suggested there was no interaction factor of rhGM-CSF in10kDa filtercassettes. The analysis of MALDI TOF MS and LTQ MS showed that the protein of10-20kDaproteins were ubiquitin, chymotrypsin inhibitor, Cationic peptide CP8precursor, Kazal-type proteinase inhibitor, RecName: Full=Chymotrypsin inhibitor SCI-II and RecName:Full=Chymotrypsin inhibitor SCI-I. In addition to ubiquitin, the rest of the proteins are serineprotease inhibitor, which can prevernt protease hydrolysis. This may be the reason why10-20kDa proteins can help the rhGM-CSF oral administration.