Application of liquid chromatography/mass spectrometry in accelerating the identification of human liver cytochrome P450 isoforms involved in the metabolism of iloperidone.
Iloperidone, [1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]eth anone, 1, is currently undergoing clinical trials as a potential antipsychotic agent. The metabolism of iloperidone was studied in human liver microsomes to define the metabolic pathways and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of major iloperidone metabolites. Iloperidone was extensively metabolized in vitro via hydroxylation, reduction and O-demethylation to produce 1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxyphenyl]-2- hydrox yethanone, 4; 4-[3-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]propoxy]-3-methoxy-alpha-met hylben zene methanol, 3, and 1-[4-[3-[4-(6-fluoro-1, 2-benzisoxazol3-yl)-1-piperidinyl]propoxy]-3-hydroxyphenyl]etha none, 2, respectively, in decreasing order of abundance. The major in vitro metabolite, 4, present in trace quantities in urine, was postulated to be either eliminated in bile as a conjugate or further metabolized to a phenol, 4-[3-[4-(6-fluoro-1, 2-benzoisoxazol-3-yl)-piperidin1-yl]propoxy]-3-methoxyphenol , 5. The formation of the three major in vitro metabolites 2, 3 and 4 was NADPH dependent. The major circulating and urinary metabolite in humans dosed with 1 was metabolite 3. The mean apparent Km and Vmax for formation of 2 by human liver microsomes was 7.4 +/- 3.0 microM and 0.0343 +/- 0.0134 nmol min-1 mg-1, respectively. The mean apparent Km and Vmax for 3 was 101.2 +/- 34.7 microM and 0.1414 +/- 0.0346 nmol min-1 mg-1, respectively. The mean apparent Km and Vmax for 4 was 39.7 +/- 10.8 microM and 0.1372 +/- 0.056 nmol min-1 mg-1, respectively. The CYP isoenzymes responsible for the formation of metabolites 2, 3 and 4 were determined by using selective chemical inhibitors and by correlation studies. Metabolites 2 and 4 were formed by CYP3A4 and by the polymorphic CYP2D6 respectively. Metabolite 3 is postulated to be produced mainly by a cytosolic enzyme(s), although CYP3A, CYP1A2 and CYP2E1 isozymes were shown to be involved in its formation as well. The power of liquid chromatography/mass spectrometry in greatly accelerating the process of identifying the human liver CYP isoforms involved in the metabolism of iloperidone was demonstrated in this study. Liquid chromatography/mass spectrometry was used in the initial studies to confirm the identities of the metabolites. This was followed by accurate and reliable quantitation of individual metabolites present in biological extracts by operating the mass spectrometer in the selected ion monitoring mode.
Mutlib
A E
AE
Department of Chemical Research, Neuroscience Therapeutic Area, Hoechst Marion Roussel Inc., Bridgewater, New Jersey, USA.
Klein
J T
JT
D014150:Antipsychotic Agents / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000502:physiology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology*; D007555:Isoxazoles / Q000378:metabolism*; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000502:physiology; D010880:Piperidines / Q000378:metabolism*
D014150:Antipsychotic Agents; D007527:Isoenzymes; D007555:Isoxazoles; D010880:Piperidines; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C081732:iloperidone