Characterization of six in vitro reactions mediated by human cytochrome P450: application to the testing of cytochrome P450-directed antibodies.
The identification of cytochrome P450 (CYP) isozymes mediating metabolic pathways of drugs has become increasingly important in anticipating pharmacokinetic drug interactions. The activity of individual CYPs can be monitored in vitro with human liver microsomes by means by relatively specific metabolic reactions: for CYP1A1/2, phenacetin O-deethylation; for CYP2C9, phenytoin 4-hydroxylation; for CYP2C19, S-mephenytoin 4'-hydroxylation; for CYP2D6, dextromethorphan O-demethylation; for CYP3A3/4, alprazolam 4-hydroxylation, and for CYP2E1, chlorzoxazone 6-hydroxylation. We determined the kinetic parameters (Vmax, Km) of these reactions and utilized them to test a monoclonal rat liver CYP1A1 antibody, a monoclonal rat liver CYP2E1 antibody, a polyclonal rabbit anti-rat liver CYP3A1 antibody, and a polyclonal goat anti-rat liver CYP2C11 antibody for their specificity and inhibitory capacity. The CYP1A1 monoclonal antibody (MAb), the CYP2E1 MAb, and the CYP3A1 polyclonal antibody (PAb) inhibited only their respective index reactions. The CYP2C11 PAb inhibited both phenytoin 4-hydroxylation and S-mephenytoin 4'-hydroxylation. At a microsomal versus antibody protein mass ratio of 1:15, 4-hydroxyalprazolam formation was reduced by 73.4% of control with the CYP3A1 PAb; 4'-hydroxymephenytoin formation decreased by 66.3% and 4-hydroxyphenytoin formation by 43.4% with the CYP2C11 PAb; phenacetin O-deethylation was reduced by 39.7% with the CYP1A1 MAb, and 6-hydroxychlorzoxazone formation decreased by 30.0% with the CYP2E1 MAb. Thus, all antibodies tested are at least CYP subfamily specific. The PAbs exhibited greater than 60% inhibition versus the CYP3A3/4- and the CYP2C19-mediated reactions, whereas the MAbs produced less than 50% inhibition for their respective index reactions. Because of their limited inhibitory capacity, MAbs may be correspondingly limited as tools for identification of human CYP enzymes via immunoinhibition studies.
Schmider
J
J
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA.
Greenblatt
D J
DJ
von Moltke
L L
LL
Harmatz
J S
JS
Duan
S X
SX
Karsov
D
D
Shader
R I
RI
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D004364:Pharmaceutical Preparations / Q000378:metabolism; D051381:Rats
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A