BDBM50103593 Methoin Mephenytoin Mesantoin
(5S)-5-ethyl-3-methyl-5-phenylimidazolidine-2,4-dione Mephenytoin, D- S-mephentoin S-Mephenytoin BDBM21361 (5S)-5-Ethyl-3-methyl-5-phenyl-2,4-imidazolidinedione
- ChEMBL_690430 (CHEMBL1635167) Inhibition of CYP2C19 using S-mephenytoin as probe
- ChEMBL_857034 (CHEMBL2162420) Reversible inhibition of CYP2C19 using mephenytoin as substrate
- ChEMBL_2265487 Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate
- ChEMBL_2291425 Inhibition of CYP2C19 in pooled human liver microsomes using S-mephenytoin as substrate assessed as reduction in OH-S-mephenytoin metabolite formation by LC-MS/MS analysis
- ChEBML_1683155 Inhibition of CYP2C19 in human liver microsomes using S-Mephenytoin as substrate
- ChEMBL_1729688 (CHEMBL4144966) Inhibition of CYP2C19 (unknown origin) using S-mephenytoin as substrate
- ChEMBL_1898404 (CHEMBL4400439) Inhibition of CYP2C19 (unknown origin) using S-Mephenytoin as substrate
- ChEMBL_2151597 (CHEMBL5036059) Inhibition of CYP2C19 in human liver microsome using mephenytoin as substrate
- ChEMBL_2234459 (CHEMBL5148231) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate
- ChEMBL_1487659 (CHEMBL3532645) Reversible inhibition of human CYP2C19 S-mephenytoin 4'-hydroxylase activity
- ChEMBL_1667015 (CHEMBL4016811) Inhibition of human liver microsomes CYP2C19 using S-mephenytoin as substrate
- ChEMBL_2103953 (CHEMBL4812456) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate
- ChEMBL_630552 (CHEMBL1108266) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate
- ChEMBL_765952 (CHEMBL1827968) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as probe
- ChEMBL_789831 (CHEMBL1925092) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate
- ChEMBL_875799 (CHEMBL2186745) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate
- ChEMBL_1656441 (CHEMBL4005911) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate in presence of NADPH
- ChEMBL_2100791 (CHEMBL4809187) Direct inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate
- ChEMBL_2240604 (CHEMBL5154500) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate in presence of NADPH
- ChEMBL_1362980 (CHEMBL3292548) Inhibition of CYP2C19 in human liver microsomes assessed as S-mephenytoin conversion to 4'-hydroxy mephenytoin preincubated for 10 mins followed by NADPH addition measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1661023 (CHEMBL4010635) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate by HPLC analysis
- ChEMBL_1838865 (CHEMBL4338998) Inhibition of human CYP2C19 using S-mephenytoin as substrate by LC/MS/MS analysis
- ChEMBL_2265896 Inhibition of CYP2C19 in human liver microsomes using S mephenytoin as substrate by LC-MS analysis
- ChEMBL_52552 (CHEMBL666251) Inhibition of human cytochrome P450 2C19 as S-mephenytoin-4-hydroxylation (100 uM)
- ChEMBL_1576884 (CHEMBL3806593) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate by LC-MS/MS analysis
- ChEMBL_2252751 (CHEMBL5166961) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate in presence or absence of NADPH
- ChEMBL_2272997 Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate by LC-MS/MS analysis
- ChEMBL_788300 (CHEMBL1919563) Inhibition of CYP2C19-mediated (S)-mephenytoin 4'-hydroxylation in human liver microsomes by LCMS analysis
- ChEMBL_874591 (CHEMBL2186227) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 10 mins
- ChEMBL_883347 (CHEMBL2214083) Inhibition of human liver microsome CYP2C19 using mephenytoin as a substrate by LC-MS-MS analysis
- ChEMBL_1501738 (CHEMBL3587718) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate by LC-MS/MS analysis
- ChEMBL_1653834 (CHEMBL4003200) Inhibition of CYP2C19 in human liver microsomes using S-(+)-mephenytoin as substrate by HPLC-MS/MS method
- ChEMBL_2110157 (CHEMBL4818832) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate by LC-MS/MS analysis
- ChEMBL_2149010 (CHEMBL5033408) Inhibition of human CYP2C19 using S-mephenytoin as substrate in presence of NADPH incubated for 15 to 45 mins
- ChEMBL_2156134 (CHEMBL5040794) Inhibition of CYP2C19 in human liver microsomes using (s)-Mephenytoin as substrate by LC-MS/MS analysis
- ChEMBL_690427 (CHEMBL1635164) Inhibition of CYP2C19 in human liver microsomes assessed as S-mephenytoin 4'-hydroxylation after 60 mins
- ChEMBL_850902 (CHEMBL2156885) Inhibition of human CYP2C19 using mephenytoin as substrate after 45 mins by LC/MS/MS analysis
- ChEMBL_962445 (CHEMBL2390191) Reversible inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate by LC/MS/MS analysis
- ChEMBL_977557 (CHEMBL2423174) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate by LC-MS/MS analysis
- ChEBML_1682568 Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate after 30 mins by LC-MS/MS analysis
- ChEMBL_1296350 (CHEMBL3129760) Reversible inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate by LC-MS/MS analysis
- ChEMBL_1624784 (CHEMBL3867196) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1659465 (CHEMBL4009077) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1858696 (CHEMBL4359425) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin substrate incubated for 10 mins in presence of NADPH
- ChEMBL_1869044 (CHEMBL4370110) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 10 mins in presence of NADPH
- ChEMBL_1880000 (CHEMBL4381394) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin substrate in presence of NADPH incubated for 10 mins
- ChEMBL_2100801 (CHEMBL4809197) Time-dependent inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 30 mins
- ChEMBL_2205590 (CHEMBL5118298) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate and measured by LC-MS/MS analysis
- ChEMBL_2246144 (CHEMBL5160354) Inhibition of human liver CYP2C19 expressed in baculovirus-infected insect cell supersomes assessed as mephenytoin 4-hydroxylation
- ChEMBL_2296119 Inhibition of CYP2C19 in human liver microsomes using s-mephenytoin as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_809874 (CHEMBL2014811) Inhibition of human CYP2C19 using S-mephenytoin as substrate after 45 mins by LC-MS/MS analysis
- ChEBML_1695900 Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate after 10 mins by LC-MS/MS analysis
- ChEMBL_1366895 (CHEMBL3297536) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate after 15 mins by LC/MS/MS analysis
- ChEMBL_1526172 (CHEMBL3636697) Reversible inhibition of CYP2C19 (S)-Mephenytoin 4'-hydroxylase activity in human liver microsomes by LC-MS/MS analysis
- ChEMBL_1553513 (CHEMBL3768880) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate incubated for 5 mins by LC-MS analysis
- ChEMBL_1578013 (CHEMBL3806762) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate after 30 mins by LC/MS/MS analysis
- ChEMBL_1584739 (CHEMBL3821541) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate after 40 mins by LC-MS analysis
- ChEMBL_1616179 (CHEMBL3858248) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate in presence of NADPH by LC/MS/MS analysis
- ChEMBL_1712698 (CHEMBL4122747) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2066472 (CHEMBL4721725) Inhibition of human liver microsome CYP2C19 using mephenytoin as substrate incubated for 20 mins by LC-MS/MS analysis
- ChEMBL_2160242 (CHEMBL5044992) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 15 to 45 mins in presence of NADPH
- ChEMBL_2222746 (CHEMBL5136080) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 15 to 45 mins in presence of NADPH
- ChEMBL_2226859 (CHEMBL5140372) Inhibition of CYP2C19 in human liver microsomes using s-mephenytoin as substrate incubated for 15 to 40 mins in presence of NADPH
- ChEMBL_800478 (CHEMBL1948598) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate after 60 mins by LC-MS/MS analysis
- ChEMBL_977239 (CHEMBL2416910) Inhibition of CYP2C19 in human liver microsomes assessed as S-mephenytoin 4'-hydroxylation after 20 mins by LCMS analysis
- ChEMBL_1486162 (CHEMBL3533281) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 5 mins by LC-MS/MS analysis
- ChEMBL_1487307 (CHEMBL3534774) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 8 mins by LC-MS/MS analysis
- ChEMBL_1616314 (CHEMBL3858383) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 15 mins by LC/MS/MS analysis
- ChEMBL_1703502 (CHEMBL4054735) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 15 mins by LC-MS/MS analysis
- ChEMBL_1803979 (CHEMBL4276271) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate after 10 to 30 mins by LC/MS analysis
- ChEMBL_1933000 (CHEMBL4478652) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 10 mins by LC/MS/MS analysis
- ChEMBL_2030245 (CHEMBL4684403) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 30 mins by LC-MS/MS analysis
- ChEMBL_2035471 (CHEMBL4689629) Inhibition of CYP2C19 in human liver microsomes using (S)-(+)-mephenytoin as substrate incubated for 30 mins by LC-MS/MS analysis
- ChEMBL_2101775 (CHEMBL4810171) Inhibition of CYP2C19 in human liver microsomes using s-mephenytoin as substrate substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2106515 (CHEMBL4815190) Inhibition of CYP2C19 in human liver microsomes using Mephenytoin as substrate measured after 20 mins by LC-MS/MS analysis
- ChEMBL_2150739 (CHEMBL5035201) Inhibition of CYP2C19 in human pooled liver microsomes using S-mephenytoin as substrate in presence of NADPH by LC-MS/MS analysis
- ChEMBL_690378 (CHEMBL1634995) Inhibition of CYP2C19 in human liver microsomes assessed as S-mephenytoin 4'-hydroxylation after 60 mins by Dixon plot analysis
- ChEMBL_790070 (CHEMBL1925460) Inhibition of human B-lymphoblastoid cell microsomal CYP2C19 assessed as (S)-mephenytoin 4'-hydroxylation preincubated for 5 mins with substrate
- ChEMBL_990820 (CHEMBL2444387) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate after 5 to 60 mins by LC-MS/MS analysis
- ChEMBL_1281753 (CHEMBL3100522) Inhibition of CYP2C19 in human liver microsomes assessed as S-mephenytoin 4'-hydroxylation after 20 mins by LC-MS/MS analysis
- ChEMBL_1487041 (CHEMBL3531867) Inhibition of CYP2C19 in human liver microsomes using (S)-Mephenytoin as substrate after 5 to 30 mins by LC-MS/MS analysis
- ChEMBL_1492466 (CHEMBL3528841) Inhibition of CYP2C19 in human liver microsomes assessed as (S)-mephenytoin 4'-hydroxylation after 3 mins by LC-MS/MS analysis
- ChEMBL_1492476 (CHEMBL3528851) Inhibition of CYP2C19 in human liver microsomes assessed as (S)-mephenytoin 4'-hydroxylation preincubated for 15 mins by LC-MS/MS analysis
- ChEMBL_1556411 (CHEMBL3772285) Inhibition of CYP2C19 (unknown origin) using mephenytoin as substrate preincubated for 10 mins followed by NADPH addition by LC/MS/MS analysis
- ChEMBL_1660770 (CHEMBL4010382) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate after 60 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1903443 (CHEMBL4405665) Inhibition of human liver microsome CYP2C19 using S-mephenytoin as substrate incubated for 5 to 20 mins by LC-MS/MS analysis
- ChEMBL_1904189 (CHEMBL4406411) Inhibition of CYP2C19 in human pooled liver microsomes using (S)-mephenytoin as substrate after 10 mins by UPLC-MS/MS analysis
- ChEMBL_1904560 (CHEMBL4406782) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate incubated for 40 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1973674 (CHEMBL4606492) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1992480 (CHEMBL4626215) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate incubated for 20 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_2069796 (CHEMBL4725049) Inhibition of human liver microsome CYP2C19 using S-mephenytoin as substrate incubated for 20 mins by LC-MS/MS with HPLC analysis
- ChEMBL_2073303 (CHEMBL4728837) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2209735 (CHEMBL5122684) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate preincubated with compound followed by NADPH addition by LC-MS/MS analysis
- ChEMBL_2254473 (CHEMBL5168683) Inhibition of CYP2C19 in human liver microsomes at 0.01 to 30 uM using mephenytoin as substrate in presence of NADPH by UPLC-MS/MS analysis
- ChEMBL_1586834 (CHEMBL3826666) Inhibition of CYP2C19 in human liver microsomes in presence of NADPH using mephenytoin as substrate measured within 2.5 mins by LC-MS/MS analysis
- ChEMBL_1632392 (CHEMBL3875098) Inhibition of CYP2C19 in human liver microsomes assessed as reduction in S-mephenytoin-4-hydroxylation at 1 to 25 uM by UPLC-MS/MS analysis
- ChEMBL_1713393 (CHEMBL4123442) Inhibition of CYP2C19 in human liver microsomes assessed as (S)-mephenytoin 4'-hydroxylation after 4 to 40 mins in presence of NADPH by LCMS analysis
- ChEMBL_1742660 (CHEMBL4158410) Inhibition of of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 20 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1760219 (CHEMBL4195227) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 15 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_1869496 (CHEMBL4370562) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate after 20 mins in presence of NADP by LC-MS/MS analysis
- ChEMBL_1913289 (CHEMBL4415872) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 10 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1924298 (CHEMBL4427254) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate after 10 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_1996554 (CHEMBL4630449) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 20 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2027340 (CHEMBL4681498) Inhibition of CYP2C19 in pooled human liver microsomes using mephenytoin as substrate in presence of NADPH incubated for 10 mins by LC-MS/MS analysis
- ChEMBL_2075864 (CHEMBL4731398) Inhibition of CYP2C19 in human pooled liver microsomes using S-mephenytoin as substrate measured up to 20 mins by UHPLC-MS/MS analysis
- ChEMBL_2101713 (CHEMBL4810109) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate preincubated for 5 mins followed by NADPH addition by LC-MS/MS analysis
- ChEMBL_2103388 (CHEMBL4811891) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 15 mins in presence of NADPH by LC/MS/MS analysis
- ChEMBL_2123615 (CHEMBL4832848) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate in presence of NADPH incubated for 10 mins by LC-MS/MS analysis
- ChEMBL_2132132 (CHEMBL4841647) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 10 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2253557 (CHEMBL5167767) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 5 to 45 mins in presence of NADPH by LC/MS analysis
- ChEMBL_2280223 Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate measured after 30 mins in the presence of NADPH by LC/MS/MS analysis
- ChEMBL_1493570 (CHEMBL3529384) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 20 mins with substrate prior to initiation of reaction with NADPH by HPLC analysis
- ChEMBL_1658367 (CHEMBL4007979) Inhibition of microsomal CYP2C19 (unknown origin) using S-mephenytoin as substrate preincubated for 5 mins followed by NADPH addition by LC-MS/MS analysis
- ChEMBL_1661009 (CHEMBL4010621) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition measured after 40 mins
- ChEMBL_1668233 (CHEMBL4018121) Inhibition of CYP2C19 in liver microsomes (unknown origin) assessed as formation of 4-hydroxymephenytoin from S-mephenytoin in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1760538 (CHEMBL4195546) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate measured after 10 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1859910 (CHEMBL4360766) Inhibition of CYP2C19 in human liver microsomes using s-mephenytoin as substrate measured after 10 mins in presence of NADPH by UHPLC-MS/MS analysis
- ChEMBL_1889155 (CHEMBL4390909) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured by LC-MS/MS analysis
- ChEMBL_2016609 (CHEMBL4670187) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate incubated for 5 to 20 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2022784 (CHEMBL4676597) Inhibition of CYP2C19 in human liver microsomes using S-Mephenytoin as substrate measured after 45 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2046446 (CHEMBL4701145) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate measured after 30 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2076128 (CHEMBL4731662) Inhibition of CYP2C19 (unknown origin) using S-mephenytoin as substrate measured after 5 to 20 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_2124313 (CHEMBL4833546) Inhibition of CYP2C19 in human liver microsomes assessed as mephenytoin 4-hydroxylation reaction incubated for 30 mins in presence of NADP by LC-MS/MS analysis
- ChEMBL_2277115 Inhibition of CYP2D6 (unknown origin) using (S)-mephenytoin as substrate incubated for 10 to 20 mins in presence of NADPH-generating system by LC-MS/MS analysis
- ChEMBL_306771 (CHEMBL831868) Inhibitory activity against cytochrome P450 determined using human liver microsome upon 15 min incubation with probes substrates s-mephenytoin (2C19)
- ChEMBL_872612 (CHEMBL2183771) Inhibition of CYP2C19 in human liver microsomes assessed as decrease in formation of 4-hydroxymephenytoin from mephenytoin substrate after 60 mins by LC-MS/MS
- ChEMBL_1677532 (CHEMBL4027675) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate upto 10 uM after 45 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1972621 (CHEMBL4605439) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate measured for 10 to 30 mins in presence of NADPH regenerating system by UPLC-MS analysis
- ChEMBL_2118303 (CHEMBL4827369) Reversible inhibition of CYP2C19 in human liver microsomes using (S)-Mephenytoin as substrate preincubated for 40 mins followed by NADPH addition by LC/MS/MS analysis
- ChEMBL_1502035 (CHEMBL3588594) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as susbtrate incubated for 30 mins prior to substrate addition for 10 mins by LC-MS/MS analysis
- ChEMBL_1661003 (CHEMBL4010615) Inhibition of human CYP2C19 using (S)-mephenytoin as substrate incubated for 5 mins followed by NADPH addition measured after 30 mins by LC-MS/MS analysis
- ChEMBL_1817311 (CHEMBL4316971) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured for 15 mins by LC-MS/MS analysis
- ChEMBL_2246768 (CHEMBL5160978) Inhibition of CYP2C19 in human liver microsomes using as S-mephenytoin substrate incubated for 10 min followed by NADPH addition for 10 mins by LC-MS/MS analysis
- ChEBML_1688703 Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate preincubated for 15 mins measured after 8 mins in presence or absence of NADPH by LC/MS/MS analysis
- ChEMBL_1493638 (CHEMBL3530054) Metabolism-dependent inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 20 mins with NADPH prior to initiation of reaction with probe substrate by HPLC analysis
- ChEMBL_1561113 (CHEMBL3776837) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated with substrate for 5 mins followed by NADPH addition measured after 20 mins by HPLC analysis
- ChEMBL_1764607 (CHEMBL4199854) Inhibition of CYP2C19 in human liver microsomes using S-Mephenytoin as substrate pretreated for 5 mins followed by NADPH addition and measured after 45 mins by LC-MS analysis
- ChEMBL_2124738 (CHEMBL4833971) Inhibition of CYP2C19 in pooled human liver microsomes using (S)-(+)-mephenytoin as substrate preincubated for 30 mins in presence of NADPH followed by substrate addition by LC-MS/MS analysis
- ChEMBL_2235523 (CHEMBL5149295) Inhibition of CYP2C19 in human liver microsome using mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2273351 Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1349494 (CHEMBL3271177) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 5 mins prior to substrate addition measured after 10 mins by LC/MS/MS analysis
- ChEMBL_1632086 (CHEMBL3874792) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition measured after 10 mins by LC/MS/MS method
- ChEMBL_1678332 (CHEMBL4028475) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by substrate addition measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1709452 (CHEMBL4119501) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition measured after 10 mins by LC/MS/MS analysis
- ChEMBL_1734943 (CHEMBL4150479) Inhibition of CYP2C19 in human liver microsomes assessed as reduction in 4-Hydroxymephenytoinn formation using mephenytoin as substrate after 10 to 20 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1739972 (CHEMBL4155722) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1750150 (CHEMBL4184910) Inhibition of CYP2C19 in human liver microsomes using mephenytoin as substrate preincubated for 10 mins with substrate followed by NADPH addition measured after 15 mins by LC/MS/MS analysis
- ChEMBL_1772289 (CHEMBL4224401) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition measured after 10 mins by LC-MS/MS analysis
- ChEMBL_1807805 (CHEMBL4307164) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate incubated for 10 mins followed by substrate addition further incubated for 10 mins by LC-MS/MS analysis
- ChEMBL_1845028 (CHEMBL4345455) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate preincubated for 5 mins followed by NADPH addition and measured for 10 to 30 mins by LC-MS/MS analysis
- ChEMBL_1849382 (CHEMBL4349923) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate preincubated for 5 mins followed by NADPH addition measured after 40 mins by LC-MS/MS analysis
- ChEMBL_1902238 (CHEMBL4404460) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 5 mins followed by NADPH addition and measured for 10 to 30 mins by LC-MS/MS analysis
- ChEMBL_1902873 (CHEMBL4405095) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_1931352 (CHEMBL4434603) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin pre-incubated for 10 mins before NADPH addition and measured after 10 mins by LC/MS/MS analysis
- ChEMBL_1988421 (CHEMBL4621968) Inhibition of CYP2C19 in human liver microsomes using s-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured after 10 mins by LC-MS/MS analysis
- ChEMBL_2014364 (CHEMBL4667942) Inhibition of recombinant human CYP2C19 expressed in baculovirus infected insect cells using s-mephenytoin as substrate incubated for 30 mins in presence of NADPH-regenerating system by fluorescence based assay
- ChEMBL_2212608 (CHEMBL5125557) Inhibition of CYP2C19 in human liver microsomes using (s)-mephenytoin as substrate preincubated for 5 mins followed by NADPH addition measured after 20 mins by LC-MS/MS analysis
- ChEMBL_2226335 (CHEMBL5139848) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured after 15 mins by LC-MS/MS analysis
- ChEMBL_2248275 (CHEMBL5162485) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and measured after 20 mins by LC/MS/MS analysis
- ChEMBL_2296576 Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate assessed as reduction in substrate hydroxylation incubated for 5 mins in presence of beta-NADPH measured by LC-MS/MS analysis
- ChEMBL_2296586 Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate assessed as reduction in substrate hydroxylation incubated for 30 mins in presence of beta-NADPH measured by LC-MS/MS analysis
- ChEMBL_1870801 (CHEMBL4371968) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate preincubated for 5 mins followed by NADPH cofactor addition and measured after 40 mins by LC-MS/MS analysis
- ChEMBL_2025119 (CHEMBL4678932) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 5 mins followed by NADPH generating system addition and measured after 120 mins by LC-MS/MS
- ChEMBL_2075215 (CHEMBL4730749) Inhibition of human liver microsome CYP2C19 using S-mephenytoin as substrate preincubated for 10 mins followed by substrate and NADPH addition and measured after 10 mins by LC-MS/MS analysis
- ChEMBL_2121113 (CHEMBL4830260) Inhibition of CYP2C19 in human liver Microsome using S-mephenytoin as substrate preincubated for 10 mins followed by NADPH addition and further incubated for 10 mins as substrate by LC-MS/MS analysis
- ChEMBL_2241097 (CHEMBL5155307) Time dependent inhibition of CYP2C19 in human liver microsomes at 40 uM using (S)-mephenytoin as substrate measured after 5 to 20 mins in presence of NADPH by LC/MS/MS analysis
- Cytochrome P450 2C9 Inhibition Assay (Mephenytoin) The inhibition of cytochrome P450 2C19-isoenzyme catalysed hydroxylation of Mephenytoin by the test compound is assayed at 37° C. with human liver microsomes. All assays are carried out on a robotic system in 96 well plates. The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), human liver microsomes (0.5 mg/ml), (S)-Mephenytoin (70 uM) and the test compound at five different concentrations or no compound (high control) in duplicate (e.g. highest concentration 10-50 uM with subsequent serial 1:4 dilutions). Following a short preincubation period, reactions are started with the cofactor (NADPH, 1 mM) and stopped by cooling the incubation down to 8° C. and subsequently by addition of one volume of acetonitrile.
- ChEMBL_1487322 (CHEMBL3535114) Inhibition of CYP2J2 in human liver microsomes using 7 probe cocktail containing phenacetin, paclitaxel, diclofenac, S-mephenytoin, dextromethorphan, astemizole and midazolam after 8 mins by LC-MS/MS analysis
- ChEMBL_1660977 (CHEMBL4010589) Inhibition of CYP2C19 in human liver microsomes using (S)-mephenytoin as substrate preincubated for 20 mins followed by substrate addition measured after 30 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1667892 (CHEMBL4017780) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by substrate addition measured after 10 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1668431 (CHEMBL4018319) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate pretreated for 10 mins followed by substrate addition measured after 10 mins in presence of NADPH by LC-MS/MS method
- ChEMBL_1745610 (CHEMBL4180120) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins followed by substrate addition measured after 10 mins in presence of NADPH by LC-MS/MS analysis
- ChEMBL_1904235 (CHEMBL4406457) Inhibition of CYP2C19 in human pooled liver microsomes using (S)-mephenytoin as substrate preincubated with NADPH for 30 mins followed by substrate addition and measured after 10 mins by UPLC-MS/MS analysis
- ChEMBL_2248036 (CHEMBL5162246) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as cocktail probe substrate preincubated for 5 mins followed by NADPH addition and measured after 30 mins by HPLC-MS/MS analysis
- ChEMBL_2033574 (CHEMBL4687732) Mixed type inhibition of human recombinant CYP2C19 using (S)-mephenytoin as substrate preincubated for 5 mins followed by NADPH-generating system addition measured after 40 mins by Lineweaver-Burk plot analysis
- ChEMBL_2080681 (CHEMBL4736472) Inhibition of CYP2C19 in human liver microsomes using S-mephenytoin as substrate preincubated for 10 mins in presence of NADPH generating system followed by substrate addition and measured after 20 mins by LC-MS/MS analysis
- ChEMBL_1660914 (CHEMBL4010526) Inhibition of CYP2C19 in human liver microsomes assessed as enzyme-mediated metabolite formation using (S)-mephenytoin as substrate incubated for 5 mins followed by NADPH addition measured after 30 mins by LC-MS/MS analysis
- Inhibition Assay Six test compound concentrations (0.1, 0.25, 1, 2.5, 10, 25 μM in DMSO; final DMSO concentration=0.25%) are incubated with human liver microsomes (0.5 mg/mL) and NADPH (1 mM) in the presence of the probe substrate mephenytoin (25 μM) for 60 min at 37° C. The selective CYP2C19 inhibitor, tranylcypromine, is screened alongside the test compounds as a positive control.
- Inhibition of Cytochrome P450 Oxidase The human liver microsomes (0.253 mg/mL protein) containing cytochrome P450, test compounds (0.05-50 μM), CYPs substrates (10 μM p-acetaminophen, 5 μM diclofenac, 30 μM mephenytoin, 5 μM dextromethorphan hydrobromide, 2 μM midazolam), 1.0 mM NADP were incubated at 37° C. for 10 minutes. Naflavone, sulfafenpyrazole, N-3-benzylnivan, quinidine, and ketoconazole were used as reference inhibitors. The results are shown in Table 3. The IC50 of the test compounds are all greater than 50 μM.
- Inhibition Assays The final incubation volume contains TRIS buffer (0.1 M), MgCl2 (5 mM), a certain concentration of human liver microsomes dependent on the P450 isoenzyme measured (P450 2C9, P450 3A4: 0.1 mg/ml; P450 2D6: 0.2 mg/ml; P450 2C19: 0.5 mg/ml; P450 2C8: 0.05 mg/ml) and a certain concentration of the individual substrate for each isoenzyme (P450 2C9: Diclofenac 10 μM; P450 3A4: Midazolam 5 μM; P450 2D6: Dextromethorphan 5 μM; P450 2C19: S-Mephenytoin 70 μM; P450 2C8: Amodiaquine 1 μM).
- Inhibition Assay Each five kinds of substrates, human hepatic microsome, and a test drug in 50 mmol/L Hepes buffer as a reaction solution was added to a 96-well plate as the composition ad described above, NADPH, as a coenzyme was added to initiate metabolism reactions as markers and, after the incubation at 37° C. for 15 minutes, a methanol/acetonitrile=1/1 (V/V) solution was added to stop the reaction. After the centrifugation at 3000 rpm for 15 minutes, resorufin (CYP1A2 metabolite) in the supernatant was quantified by a fluorescent multilabel counter and tolbutamide hydroxide (CYP2C9 metabolite), mephenytoin 4′ hydroxide (CYP2C19 metabolite), dextrorphan (CYP2D6 metabolite), and terfenadine alcohol (CYP3A4 metabolite) were quantified by LC/MS/MS.
- Cyp Inhibition For Cyp inhibition, human liver microsomes from BD Gentest were incubated with Compound 028 or Compound 032 (10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01 uM) and substrate (CYP1A2: Phenacetin at 30 uM; CYP2C9: Diclofenac at 10 uM; CYP2C19: S-Mephenytoin at 35 uM; CYP3A4: Midazolam at 5 uM and Testosterone at 80 uM; CYP2D6: Bufuralol at 10 uM) for the following incubation times: CYP1A2, 2C9, 2D6: 10 minutes, 37° C.; CYP2C19: 45 minutes, 37° C.; CYP3A4: 5 minutes, 37° C. Substrate conversion was measured by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Inhibition was calculated by curve fitting in Graph Pad Prism.
- Cyp inhibition For Cyp inhibition, human liver microsomes from BD Gentest were incubated with Compound 028 or Compound 032 (10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01 μM) and substrate (CYP1A2: Phenacetin at 30 μM; CYP2C9: Diclofenac at 10 μM; CYP2C19: S-Mephenytoin at 35 μM; CYP3A4: Midazolam at 5 μM and Testosterone at 80 μM; CYP2D6: Bufuralol at 10 μM) for the following incubation times: CYP1A2, 2C9, 2D6: 10 minutes, 37° C.; CYP2C19: 45 minutes, 37° C.; CYP3A4: 5 minutes, 37° C. Substrate conversion was measured by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Inhibition was calculated by curve fitting in Graph Pad Prism.
- CYP Enzyme Inhibition Assay Human liver microsomes (HLMs) (0.3 mg/mL in 0.1-M potassium phosphate buffer, pH 7.4) are incubated with CYP (cytochromes P450) isozyme-selective substrates (phenacetin for CYP1A2, amodiaquine for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, Dextromethorphan for CYP2D6, and midalozam for CYP3A4/5), and multiple concentrations of Compound (I-1) or Ko143 (0, 0.041, 0.12, 0.37, 1.11, 3.33, 10, and 30 uM) in 96-well plates. Reactions are initiated by the addition of β-NADPH (2 mM) and MgCl2 (3 mM) in 0.1-M potassium phosphate buffer, pH 7.4. Reactions are incubated for 12 minutes at 37° C., and then terminated by the addition of an equal volume of ACN containing 1-uM carbutamide (IS). The plates are refrigerated at approximately 4° C. for 15 minutes and then centrifuged in order to pellet the precipitated proteins.
- CYP Activity Assay Inhibition of hepatic cytochromes P450 was assessed in human liver microsomes using a substrate-specific approach of monitoring metabolites formed by each specific CYP enzyme. Metabolic reactions included phenacetin-O-deethylation (1A2), tolbutamide methylhydroxylation (2C9), S-mephenytoin 4'-hydroxylation (2C19), dextrome-thorphan-O-deethylation (2D6), and testosterone-6-β-hydroxylation (3A4). Each substrate was added at a concentration less than or equal to the Km for themetabolic pathway, and microsomal protein concentrations and assay incubation times were optimized for each reaction to ensure linear metabolite formation based on initial rates (Table 1). Microsomes were suspended in phosphate buffer and incubated at 37 °C in the presence of probe substrates. Reactions were initiated by the addition of an NADPH-regenerating buffer system and were quenched at the appropriate times using acetonitrile. Samples were centrifuged and concentrations of metabolites assessed by LC/MS.
- CYP P450 Enzyme Inhibition Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 μM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms.
- CYP P450 Enzyme Inhibition Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 uM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms.
- Enzyme Inhibition Assay CYP P450: Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 μM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms. The inhibition ratio is calculated as:(M t −M 0)/M water×100%in which Mt and M0 represent the concentrations of the specific probe substrate metabolite, which was formed by individual CYP450 isoform, at the beginning and end of the reaction in the presence of test compound; while Mwater represents the concentration of the specific metabolite at the end of the reaction in the absence of test compound.
- Enzyme Inhibition Assay Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 1 μM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms. The inhibition ratio is calculated as:(M t −M 0)/M water×100%in which Mt and M0 represent the concentrations of the specific probe substrate metabolite, which was formed by individual CYP450 isoform, at the beginning and end of the reaction in the presence of test compound; while Mwater represents the concentration of the specific metabolite at the end of the reaction in the absence of test compound.
- CYP P450 Enzyme Inhibition Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 μM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms. The inhibition ratio is calculated as: (Mt−M0)/Mwater×100% in which Mt and M0 represent the concentrations of the specific probe substrate metabolite, which was formed by individual CYP450 isoform, at the beginning and end of the reaction in the presence of test compound; while Mwater represents the concentration of the specific metabolite at the end of the reaction in the absence of test compound. Test compound concentration-dependent response data experiments performed in triplicate. Mean CYP2D6 IC50 values were derived from non-linear, least-squares fitting of dose-dependent response data to a standard logistic equation (Prism, GraphPad Software, Inc) to generate the CYP2D6 IC50 results.
- CYP P450 Enzyme Inhibition Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 μM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms. The inhibition ratio is calculated as:(Mt−M0)/Mwater×100%in which Mt and M0 represent the concentrations of the specific probe substrate metabolite, which was formed by individual CYP450 isoform, at the beginning and end of the reaction in the presence of test compound; while Mwater represents the concentration of the specific metabolite at the end of the reaction in the absence of test compound. Test compound concentration-dependent response data experiments performed in triplicate. Mean CYP2D6 IC50 values were derived from non-linear, least-squares fitting of dose-dependent response data to a standard logistic equation (Prism, GraphPad Software, Inc) to generate the CYP2D6 IC50.
- CYP P450 Enzyme Inhibition Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM) and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. For experiment, a reaction mixture of test compound at 10 uM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6 and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms. The inhibition ratio is calculated as:(M t −M 0)/M water×100%in which Mt and M0 represent the concentrations of the specific probe substrate metabolite, which was formed by individual CYP450 isoform, at the beginning and end of the reaction in the presence of test compound; while Mwater represents the concentration of the specific metabolite at the end of the reaction in the absence of test compound. Test compound concentration-dependent response data experiments performed in triplicate. Mean CYP2D6 IC50 values were derived from non-linear, least-squares fitting of dose-dependent response data to a standard logistic equation (Prism, GraphPad Software, Inc)
- CYP P450 Enzyme Inhibition Inhibitory activities of test compounds on 5 major isoforms of CYP P450 were evaluated by using pooled human liver microsome (HLM, purchased from BD Gentest) and selective substrates for those isoforms. Those CYP isoforms and their corresponding probe substrates are as follows: CYP1A2 (phenacetin, 30 μM), CYP2C9 (tolutamide, 100 μM), CYP2C19 (S-mephenytoin, 40 μM), CYP2D6 (dextromethorphan, 5 μM), and CYP3A4 (midazolam, 1 μM). All probe substrates were used at concentrations near or below their Kms. A reaction mixture of test compound at 10 μM or in serial dilution, CYP probe substrate described above and 0.2 mg/mL pooled HLM in phosphate buffer, pH 7.4 in a final volume of 200 μL was pre-incubated at 37° C. for 10 minutes in triplicate. The reaction was initiated by addition of NADPH at final concentration of 1 mM. The reaction was terminated after 10 minutes (CYP1A2, CYP2D6, and CYP3A4) or 30 minutes (CYP2C9 and CYP2C19) by addition of 100 μL ice-cold acetonitrile with internal standard (IS). The samples were then centrifuged at 13,000 rpm and the supernatants were injected to an LC-MS/MS (Agilent Technologies) to quantify the concentration of the specific metabolites of the probe substrates formed by individual CYP450 isoforms. The inhibition ratio is calculated as:(M t −M 0)/M water×100%in which Mt and M0 represent the concentrations of the specific probe substrate metabolite, which was formed by individual CYP450 isoform, at the beginning and end of the reaction in the presence of test compound; while Mwater represents the concentration of the specific metabolite at the end of the reaction in the absence of test compound. Test compound concentration-dependent response studies were performed in triplicate. Mean CYP2D6 IC50 values were derived from non-linear, least-squares fitting of dose-dependent response data to a standard logistic equation (Prism, GraphPad Software, Inc.) to generate the CYP2D6 IC50