interference. The fluorescent HTS comes with great sensitivity, simple
operation, fast detection, lower expenditure, and quick turnaround time
(Crespi et al., 2000; Donato et al., 2004; Kariv et al., 2001; Sakai-Kato et al.,
1998; Trubetskoy et al., 2005). However, the fluorescence nature of the test
compounds and/or NADPH would interfere with the fluorescence detection. In
addition, most fluoroprobes are lacked of selectivity to individual CYPs and
are not thein vivoprobe substrates for CYPs. Poor correlations of fluorescence
HTS with LC/MS/MS assays using substrates selective to individual human
CYPs have been reported (Bjornsson et al., 2003; Cohen et al., 2003; Crespi
and Stresser, 2000; Donato et al., 2004; Stresser et al., 2002). It has been
suggested that the fluorescence results are not recommended for regulatory
submission (Bjornsson et al., 2003).
TABLE 16.5 Fluoroprobes and fluorescent metabolites for individual CYPs.
Substrates!products CYPs involved
Fluorescent detection:
excitation (nm)/emission (nm)AMMC!AHMC 2D6 390/460
BFC!HFC 3A4, 3A5, 3A7 409/530
BOMFC!HFC 3A4, 3A5, 2B6 400/502
BOMCC!CHC 3A4, 3A5, 2B6, 2C9 409/460
BOMF!fluorescein 2C9 485/530
BOMR!resorufin 3A4 530/585
7BQ!7-HQ 3A4 409/530
CEC!CHC 1A2, 2C19 409/460
BzRes!resorufin 1A1, 1B1, 3A4 530/590
Coumarin!7-HC 2A6 390/460
DBF!fluorescein 2C8, 2C9, 2C19, 3A4, 19 485/538
DBOMF!fluorescein 3A4, 3A5 485/530
EFC!HFC 2B6 409/530
EOMCC!CHC 2D6, 1A2, 2C19, 2E1 409/460
7-MFC!HFC 2C9, 2E1 409/530
MAMC!HAMC 2D6 390/460
MOBFC!HFC 2D6 400/502
OMF!fluorescein 2C19 485/538
OOMR!resorufin 2C9 530/585
AMMC, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin; AHMC, 3-[2-(N,N-
diethylamino)ethyl]-7-hydroxy-4-methylcoumarin hydrochloride; BFC, 7-benzyloxy-4-(trifluoro-
methyl)-coumarin; BOMFC, 7-benzyloxymethyloxy-4-trifluoromethyl coumarian; BOMCC,
7-benzyloxymethyloxy-3-cyanocoumarian; BOMF, benzyloxymethylfluorescein; BOMR, benzylox-
ymethylresorufin; 7BQ, 7-benzyloxyquinoline; BzRes, resorufin benzyl ether. CEC, 3-cyano-
7-ethoxycoumarin; CHC, 3-cyano-7-hydroxycoumarin; DBF, dibenzylfluorescein; DBOMF,
dibenzyloxcymethylfluorescein EFC, 7-ethoxy-4-trifluoromethylcoumarin; EOMCC, ethyloxymethy-
loxy-3-cyanocoumarian; 7-HQ, 7-hydroxyquinoline; HFC, 7-hydroxy-4-trifluoromethylcoumarin;
7-HC, 7-hydroxycoumarin; HAMC, 7-hydroxy-4-(aminomethyl)coumarin; 7-MFC, 7-methoxy-4-
trifluoromethylcoumarin; MAMC, 7-methoxy-4-(aminomethyl)coumarin; MOBFC, 7-(p-methoxy-
benzyloxy-4-trifluorocoumarin; OMF, 3-O-methylfluorescein; OOMR,n-Octyloxymethylresorufin.
FLUORESCENT ASSAY 533