Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes.
1. Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions. 2. The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10-200 mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates. 3. The Km (or S50) and Vmax values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis-Menten or Hill equation, greatly depended on the buffer components and their concentration. 4. The CLint values in phosphate buffer were 1.2-3.0-fold (paclitaxel) or 3.1-6.4-fold (triazolam) higher than in Tris-HCl buffer at 50-100 mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100 mM which are both commonly used in drug metabolism studies. 5. These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro-in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism.
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Toshiyuki
T
Kudo
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Yuya
Y
Ozaki
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Tomomi
T
Kusano
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Eri
E
Hotta
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Yuka
Y
Oya
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Seina
S
Komatsu
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Hitomi
H
Goda
a Research Institute of Pharmaceutical Sciences, Musashino University , Shinmachi, Nishitokyo-shi , Tokyo , Japan.
Kiyomi
K
Ito
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D065727:Cytochrome P-450 CYP2C8 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D017239:Paclitaxel / Q000493:pharmacokinetics*; D014229:Triazolam / Q000493:pharmacokinetics*
D007527:Isoenzymes; D014229:Triazolam; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D017239:Paclitaxel
CYP2C8; CYP3A4; Phosphate buffer; Tris-HCl buffer; human liver microsomes; paclitaxel; triazolam