Investigation of the effect of mutations of rat albumin on the binding affinity to the alpha(4)beta(1) integrin antagonist, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), using recombinant rat albumins.
The authors reported previously rat strain differences in plasma protein binding to alpha(4)beta(1) antagonist D01-4582, resulting in a great strain difference in its pharmacokinetics (19-fold differences in the AUC). The previous study suggested that amino acid changes of V238L and/or T293I in albumin reduced the binding affinity. In order to elucidate the relative significance of these mutations, an expression system was developed to obtain recombinant rat albumins (rRSA) using Pichia pastoris, followed by a binding analysis of four rRSAs by the ultracentrifugation method. The equilibrium dissociation constant (K(d)) of wild-type rRSA was 210 nM, while K(d) of rRSA that carried both V238L and T293I mutations was 974 nM. K(d) of artificial rRSA that carried only V238L was 426 nM, and K(d) of artificial rRSA that carried only T293I was 191 nM. These results suggested that V238L would be more important in the alteration of K(d). However, since none of the single mutations were sufficient to explain the reduction of affinity, the possibility was also suggested that T293I interacted cooperatively to reduce the binding affinity of rat albumin to D01-4582. Further investigation is required to elucidate the mechanism of the possible cooperative interaction.
Ito
Takashi
T
Drug Metabolism & Pharmacokinetics Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., Tokyo, Japan. ito.takashi.uh@daiichisankyo.co.jp
Takahashi
Masayuki
M
Okazaki
Osamu
O
Sugiyama
Yuichi
Y
D000418:Albumins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002853:Chromatography, Liquid; D016023:Integrins / Q000037:antagonists & inhibitors*; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010671:Phenylurea Compounds / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011759:Pyrrolidines / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
C516971:4-(1-(3-chloro-4-(N-(2-methylphenyl)ureido)phenylacetyl)-4-fluoropyrrolidine-2-yl)methoxybenzoic acid; D000418:Albumins; D016023:Integrins; D010671:Phenylurea Compounds; D011759:Pyrrolidines; D011994:Recombinant Proteins