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.
Okabe
M
M
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co, Ltd, Shizuoka, Japan.
Asano
M
M
Kuga
T
T
Komatsu
Y
Y
Yamasaki
M
M
Yokoo
Y
Y
Itoh
S
S
Morimoto
M
M
Oka
T
T
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