Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034

CI-1034, an endothelin-A receptor antagonist was being developed for pulmonary hypertension. Drug-drug interaction studies using human hepatic microsomes were conducted to assess CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 inhibition potential; CYP3A4 induction potential was evaluated using primary human hepatocytes. CI-1034 moderately inhibited CYP2C9 (IC(50) 39.6 microM) and CYP3A4 activity (IC(50) 21.6 microM); CYP3A4 inhibition was metabolism-dependent. In human hepatocytes, no increase in CYP3A4 activity was observed in vitro, while mRNA was induced 15-fold, similar to rifampin, indicating that CI-1034 is both an inhibitor and inducer of CYP3A4. A 2-week clinical study was conducted to assess pharmacokinetics, pharmacodynamics and safety. No significant changes were observed in [formula: see text] between days 1 and 14. However, reversible elevations of serum liver enzymes were observed with a 50mg BID dose and the program was terminated. To further understand the interactions of CI-1034 in the liver and possible mechanisms of the observed hepatotoxicity, we evaluated the effect of CI-1034 on bile acid transport and previously reported that CI-1034 inhibited biliary efflux of taurocholate by 60%, in vitro. This indicated that inhibition of major hepatic transporters could be involved in the observed hepatotoxicity. We next evaluated the in vitro inhibition potential of CI-1034 with the major hepatic transporters OATP1B1, OATP1B3, OATP2B1, MDR1, MRP2 and OCT. CI-1034 inhibited OATP1B1 (K(i) 2 microM), OATP1B3 (K(i) 1.8 microM) and OATP2B1 activity (K(i) 3.3 microM) but not OCT, MDR1 or MRP2 mediated transport. Our data indicates that CI-1034 is an inhibitor of major hepatic transporters and inhibition of bile efflux may have contributed to the observed clinical hepatotoxicity. We recommend that in vitro drug-drug interaction panels include inhibition and induction studies with transporters and drug metabolizing enzymes, to more completely assess potential in vivo interactions or toxicity.     

Influence of lipophilicity on the interactions of hydroxy stilbenes with cytochrome P450 3A4

Resveratrol, a polyphenol found in red wine, was recently suggested to act as an irreversible, mechanism-based inactivator of cytochrome P450 3A4 (CYP3A4). We found a significant inhibition of human CYP3A4-dependent transformation of cyclosporine by resveratrol, with IC50 = 4.5 microM. We studied the kinetics parameters of CYP3A4 transformation of resveratrol and structurally related, naturally occurring stilbenes. Resveratrol, piceid, resveratroloside, 5,4'-dihydroxy-3-O-methoxystilbene, and 5,3-dihydroxy-4'-O-methoxystilbene were all shown to inhibit hydroxylation of testosterone by CYP3A4. Both methoxy-stilbenes had lower IC50 values, ranging from 0.43 to 0.47 microM, suggesting that lipophilicity rather than number or positions of free hydroxyls (3,5 or 5,4') determines the CYP3A4 inhibition capacity of polyphenols. In line with these findings, both glucosyl-stilbenes were found to be weak inhibitors of CYP3A4. The affinity of the enzyme towards methoxy-stilbenes, expressed as apparent Km, was indeed higher than those for the parent resveratrol and its glucosides, in CYP3A4 reaction mixtures. Vmax values were similar, except for piceid. These results support the role of lipophilicity in the interaction of polyphenols with CYP3A4. It is suggested that selective structural modifications of substrates add significantly to knowledge acquired through molecular modifications of the enzyme.     

Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction

The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metabolism, whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large number of chemicals in the drug discovery phase for CYP3A4 inhibitory and inducing potential.     

Cree antidiabetic plant extracts display mechanism-based inactivation of CYP3A4

Seventeen Cree antidiabetic medicinal plants were studied to determine their potential to inhibit cytochrome P450 3A4 (CYP3A4) through mechanism-based inactivation (MBI). The ethanolic extracts of the medicinal plants were studied for their inhibition of CYP3A4 using the substrates testosterone and dibenzylfluorescein (DBF) in high pressure liquid chromatography (HPLC) and microtiter fluorometric assays, respectively. Using testosterone as a substrate, extracts of Alnus incana, Sarracenia purpurea, and Lycopodium clavatum were identified as potent CYP3A4 MBIs, while those from Abies balsamea, Picea mariana, Pinus banksiana, Rhododendron tomentosum, Kalmia angustifolia, and Picea glauca were identified as less potent inactivators. Not unexpectedly, the other substrate, DBF, showed a different profile of inhibition. Only A. balsamea was identified as a CYP3A4 MBI using DBF. Abies balsamea displayed both NADPH- and time-dependence of CYP3A4 inhibition using both substrates. Overall, several of the medicinal plants may markedly deplete CYP3A4 through MBI and, consequently, decrease the metabolism of CYP3A4 substrates including numerous medications used by diabetics.     

Mechanism-based inhibition of rat liver microsomal diazepam C3-hydroxylase by mifepristone associated with loss of spectrally detectable cytochrome P450

Since initial studies with the steroids norethindrone and ethynylestradiol, reported by White and Muller-Eberhard in 1977 (Biochem. J. 166, 57-64), there has been continuing interest in xenobiotics that bear terminal or sub-terminal acetylenic groups which can cause catalysis-dependent inhibition of CYP monooxygenases associated either with loss of prosthetic group heme or protein adduct formation. Mifepristone is a synthetic steroid bearing a propyne substitution on carbon 17 and this suggested to us that it may act as a mechanism-based inhibitor of the CYP isoforms responsible for its metabolism. In human and rat liver, CYP3A isoforms have been implicated in mifepristone clearance and mifepristone administration to rats has also been shown to induce CYP3A enzymes and the associated diazepam C3-hydroxylase activity (Cheesman, Mason and Reilly, J. Steroid Biochem. Mol. Biol., 58, 1996, 447-454). With microsomes prepared from the livers of untreated female rats and others in which diazepam C3-hydroxylase has been induced, we show here that mifepristone can cause catalysis-dependent inhibition of this monooxygenase. In addition, incubation of microsomes with mifepristone in the presence, but not in the absence, of NADPH caused loss of spectrally detectable cytochrome P450. These results suggest that heme adduct formation may result from mifepristone metabolism by CYP3A monooxygenases which undergo self-catalysed irreversible inactivation with this drug as substrate. Since mifepristone administration in vivo is able also to cause induction of the synthesis of hepatic CYP3A apoprotein, mifepristone may have the potential in human medicine for complex interactions with other co-administered drugs which are also substrates for CYP3A monooxygenases.     

Inhibitory effects of the essential oil of chamomile (Matricaria recutita L.) and its major constituents on human cytochrome P450 enzymes

Chamomile extracts and tea are widely used herbal preparations for the treatment of minor illnesses (e.g. indigestion, inflammation). In this study the inhibitory effect of chamomile essential oil and its major constituents on four selected human cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) was investigated. Increasing concentrations of the test compounds were incubated with individual, recombinant CYP isoforms and their effect on the conversion of surrogate substances was measured fluorometrically in 96-well plates; enzyme inhibition was expressed as IC50 and Ki value in relation to positive controls. Crude essential oil demonstrated inhibition of each of the enzymes, with CYP1A2 being more sensitive than the other isoforms. Three constituents of the oil, namely chamazulene (IC50 = 4.41 microM), cis-spiroether (IC50 = 2.01 microM) and trans-spiroether (IC50 = 0.47 microM) showed to be potent inhibitors of this enzyme, also being active towards CYP3A4. CYP2C9 and CYP2D6 were less inhibited, only chamazulene (IC50 = 1.06 microM) and alpha-bisabolol (IC50 = 2.18 microM) revealed a significant inhibition of the latter. As indicated by these in vitro data, chamomile preparations contain constituents inhibiting the activities of major human drug metabolizing enzymes; interactions with drugs whose route of elimination is mainly via cytochromes (especially CYP1A2) are therefore possible.     

CYP3A4 inhibitory activity of new bisalkaloids,dipiperamides D and E,and cognates from white pepper

Two new bisalkaloids, dipiperamides D and E, were isolated as inhibitors of a drug metabolizing enzyme cytochrome P450 (CYP) 3A4 from the white pepper, Piper nigrum. Their structures were elucidated by spectroscopic methods. Dipiperamides D and E showed potent CYP3A4 inhibition with IC(50) values of 0.79 and 0.12 microM, respectively, and other metabolites from the pepper were moderately active or inactive.     

Effective cytochrome P450 (CYP) inhibitors isolated from tarragon (Artemisia dracunculus)

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC50 values of 10.0 ± 1.3 μM for compound 1 and 3.3 ± 0.2 μM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market.     

Identification of the human cytochrome P450 enzymes involved in the in vitro biotransformation of lynestrenol and norethindrone

This study examined the cytochrome P450 (CYP) enzyme selectivity of in vitro bioactivation of lynestrenol to norethindrone and the further metabolism of norethindrone. Screening with well-established chemical inhibitors showed that the formation of norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC(50)=0.02 microM) and with CYP2C9 inhibitor sulphaphenazole (IC(50)=2.13 microM); the further biotransformation of norethindrone was strongly inhibited by ketoconazole (IC(50)=0.09 microM). Fluconazole modestly inhibited both lynestrenol bioactivation and norethindrone biotransformation. Lynestrenol bioactivation was mainly catalysed by recombinant human CYP2C9, CYP2C19 and CYP3A4; rCYP3A4 was responsible for the hydroxylation of norethindrone. A significant correlation was observed between norethindrone formation and tolbutamide hydroxylation, a CYP2C9-selective activity (r=0.63; p=0.01). Norethindrone hydroxylation correlated significantly with model reactions of CYP2C19 and CYP3A4. The greatest immunoinhibition of lynestrenol bioactivation was seen in incubations with CYP2C-Ab. The CYP3A4-Ab reduced norethindrone hydroxylation by 96%. Both lynestrenol and norethindrone were weak inhibitors of CYP2C9 (IC(50) of 32 microM and 46 microM for tolbutamide hydroxylation, respectively). In conclusion, CYP2C9, CYP2C19 and CYP3A4 are the primary cytochromes in the bioactivation of lynestrenol in vitro, while CYP3A4 catalyses the further metabolism of norethindrone.     

Reduction of toxic metabolite formation of acetaminophen

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.     

Reversible and Irreversible Inhibition of High-Affinity Choline Transport Caused by Ethylcholine Aziridinium Ion

The effect of ethylcholine aziridinium ion (AF64A) on choline transport in hippocampal, striatal, and cerebrocortical synaptosomes was studied. Synaptosomes prepared from these three brain regions were equally sensitive to AF64A. Low concentrations of AF64A produced a reversible inhibition (IC50 values = 1.35-2.25 microM), whereas higher concentrations produced an irreversible inhibition (IC50 values = 25-30 microM), which started as competitive. The irreversible component of the inhibition was independent of extracellular Na+ concentration, a finding suggesting that the choline transporter is alkylated at its outward position. The kinetics of the inhibition were rapid and similar in the three brain regions examined. The high-affinity choline transport was more sensitive to the toxin than the low-affinity choline transport. Based on these results, we propose a kinetic model that explains the reversible and the irreversible inhibitions induced by AF64A. The possible relationships between the concentrations that in vitro produce reversible and irreversible inhibition and those that in vivo produce selective and nonselective cholinergic hypofunction are discussed.     

Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors

Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found to show extremely poor oral bioavailability. Compound 1 is one of the first reported non-amidine type FXa inhibitors. To discover novel and orally active FXa inhibitors, we investigated flexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyridin-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC(50) of 0.05 microM, comparable with that of 1. Further modification to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active 2-methylpyridine analogue 2s (FXa IC(50) of 0.061 microM), for which the liability of CYP3A4 inhibition was significantly weakened compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.     

Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes

Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.     

Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents

The inhibition by azole antifungals of human cytochrome CYP3A4, the major form of drug metabolising enzyme within the liver, was compared with their inhibitory activity against their target enzyme, Candida albicans sterol 14alpha-demethylase (CYP51), following heterologous expression in Saccharomyces cerevisiae. IC(50) values for ketoconazole and itraconazole CYP3A4 inhibition were 0.25 and 0. 2 microM. These values compared with much lower doses required for the complete inhibition of C. albicans CYP51, where IC(50) values of 0.008 and 0.0076 microM were observed for ketoconazole and itraconazole, respectively. Additionally, stereoselective inhibition of CYP3A4 and CYP51 was observed with enantiomers of the azole antifungal compounds diclobutrazol and SCH39304. In both instances, the RR(+) configuration at their asymmetric carbon centres was most active. Interestingly, the SS(-) enantiomeric form of SCH39304 was inactive and failed to bind CYP3A4, as demonstrable by Type II binding spectra.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

Novel retinoic acid 4-hydroxylase (CYP26) inhibitors based on a 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl scaffold

Retinoic acid (RA), the biologically active metabolite of vitamin A, is used medicinally for the treatment of hyperproliferative diseases including dermatological conditions and cancer. The antiproliferative effects of RA have been well documented as well as the limitations owing to toxicity and the development of resistance to RA therapy. RA metabolism inhibitors (RAMBAs or CYP26 inhibitors) are attracting increasing interest as an alternative method for enhancing endogenous levels of retinoic acid in the treatment of hyperproliferative disease. Here the synthesis and inhibitory activity of novel 3-(1H-imidazol- and triazol-1-yl)-2,2-dimethyl-3-(4-(phenylamino)phenyl)propyl derivatives in a MCF-7 CYP26A1 microsomal assay are described. The most promising inhibitor methyl 2,2-dimethyl-3-(4-(phenylamino)phenyl)-3-(1H-1,2,4-triazol-1-yl)propanoate (6) exhibited an IC(50) of 13 nM (compared with standards Liarozole IC(50) 540 nM and R116010 IC(50) 10 nM) and was further evaluated for CYP selectivity using a panel of CYP with >100-fold selectivity for CYP26 compared with CYP1A2, 2C9 and 2D6 observed and 15-fold selectivity compared with CYP3A4. The results demonstrate the potential for further development of these potent inhibitors.     

Inhibition of cytochrome P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors

p-Nitrophenol hydroxylation is widely used as a probe for microsomal CYP2E1. Several drugs are known as CYP2E1 inhibitors because of their capability to inhibit p-nitrophenol hydroxylation. Our results suggest further participation of CYP2A6 and CYP2C19 enzymes in p-nitrophenol hydroxylation. Moreover, CYP2A6 and CYP2C19 may be considered as the primary catalysts, whereas CYP2E1 can also contribute to the hydroxylation of p-nitrophenol. Further aim of our study was to evaluate the selectivity of p-nitrophenol hydroxylase inhibitors towards cytochrome P450 enzymes. The effects of antifungals: bifonazole, econazole, clotrimazole, ketoconazole, miconazole; CNS-active drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1. Strong inhibition was observed for CYP3A4 by antifungals with IC(50) values in submicromolar range. However, ketoconazole was the only imidazole derivative that could be considered as a selective inhibitor of CYP3A4. The CNS-active drugs investigated were found to be weak inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac efficiently inhibited CYP2C9 and to a less extent CYP3A4 enzyme.     

Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietary flavonoids

CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study we have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC(50) in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.     

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.