In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor.
About 100 derivatives of human recombinant granulocyte colony-stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell-proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co, Ltd, Shizuoka, Japan.
M
M
Okabe
M
M
Asano
T
T
Kuga
Y
Y
Komatsu
M
M
Yamasaki
Y
Y
Yokoo
S
S
Itoh
M
M
Morimoto
T
T
Oka
D000818:Animals; D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000097:blood / Q000235:genetics / Q000494:pharmacology*; D016179:Granulocyte Colony-Stimulating Factor; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009154:Mutation; D011994:Recombinant Proteins / Q000097:blood / Q000494:pharmacology
D003115:Colony-Stimulating Factors; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor