title: Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae.
abstract: Recent studies have demonstrated that recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) produced by the silkworm pupae bioreactor is absorbed into blood through oral administration and functions as an active cytokine. The aim of this study was to further examine and identify synergetic protein factors in silkworm pupae that improve rhGM-CSF absorption via an oral route. The concentrations of rhGM-CSF in serum were evaluated in mice after oral administration of rhGM-CSF using different chemical compositions of silkworm pupae as pharmaceutical excipients. The experimental data revealed that the supernatant lyophilized powder (SLP) of a homogenized slurry of silkworm pupae caused a significant increase in the rhGM-CSF level in blood when rhGM-CSF was orally administered with SLP, suggesting that synergetic protein factors that improve the oral absorption of rhGM-CSF primarily exist in SLP. As shown by scanning electron microscopy, microspheres were formed when rhGM-CSF was coated with SLP. Animal experimental data showed that the absorption of orally administered rhGM-CSF through the gastrointestinal (GI) tract primarily resulted from protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration. Surface plasmon resonance spectroscopy analysis demonstrated that several protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration were bound to rhGM-CSF. Proteins bound to rhGM-CSF by liquid chromatography-mass spectrometry were identified as chymotrypsin inhibitor SCI-II precursor, cationic peptide CP8 precursor, Kazal-type proteinase inhibitor, and chymotrypsin inhibitor SCI-I. These findings indicate that these proteinase inhibitors play an important role in improving rhGM-CSF absorption in the GI tract.
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Fang
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Jianhong
authors.initials: J
authors.lastname: Shu
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Si
authors.initials: S
authors.lastname: Li
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Jin
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Zuoming
authors.initials: Z
authors.lastname: Nie
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Zhengbing
authors.initials: Z
authors.lastname: Lv
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
authors.forename: Yaozhou
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000284:Administration, Oral; D000818:Animals; D019149:Bioreactors; D012831:Bombyx / Q000378:metabolism*; D002853:Chromatography, Liquid; D005260:Female; D041981:Gastrointestinal Tract / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D011679:Pupa / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D014462:Ultrafiltration
chemical_list: D016178:Granulocyte-Macrophage Colony-Stimulating Factor
keywords: oral administration; proteinase inhibitor; rhGM-CSF; silkworm pupae; synergetic protein factors