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Cytochrome P450 (CYP) inhibition screening: Comparison of three tests
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Cytochrome P450 (CYP) inhibition screening: Comparison of three tests Miia Turpeinen a,∗ , Laura E. Korhonen b , Ari Tolonen c , Jouko Uusitalo c , Risto Juvonen b , Hannu Raunio b , Olavi Pelkonen a a b c
Department of Pharmacology and Toxicology, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland Novamass Analytical Ltd., Oulu, Finland
a r t i c l e
i n f o
a b s t r a c t
Article history:
There are several different experimental systems for screening of in vitro inhibitory potency
Received 1 January 2006
of drugs under development. In this study we compared three different types of cytochrome
Received in revised form 9 May 2006
P450 (CYP) inhibition tests: the traditional single substrate assays, the fluorescent probe
Accepted 20 June 2006
method with recombinant human CYPs, and a novel n-in-one technique. All major hep-
Published on line 27 June 2006
atic drug-metabolizing CYPs were included (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4). Six compounds (sotalol, propranolol, citalopram, fluoxetine, oxazepam and diazepam) were
Keywords:
selected for detailed comparisons. The IC50 values of each of these compounds were mea-
Cytochrome P450 (CYP)
sured using the three assay types. The inhibitory potencies of these model drugs were
Inhibition
generally within the same order of magnitude and followed similar inhibition profiles in
Human liver microsomes
all the assay types. Clinically observed inhibitory interactions, or lack thereof, were pre-
Recombinant enzymes
dictable with all three assays. Comparison of potencies of ‘diagnostic’ inhibitors revealed
In vitro cocktail
also some notable differences between the assays, especially regarding CYP2E1. The potency
Fluorescent probes
of inhibitors towards CYP3A4 was dependent on the substrate and reaction measured. Gen-
Sotalol
erally all three assays gave reasonably comparable results, although some unexplained
Propranolol
differences were also noted
Citalopram
© 2006 Elsevier B.V. All rights reserved.
Fluoxetine Oxazepam Diazepam
1.
Introduction
Early knowledge of the metabolic properties of a new drug candidate is pivotal during drug development. Lead compounds need to be studied for metabolic characteristics to avoid drugs being withdrawn from clinical studies or the market due to major kinetic problems, such as strong interaction potential due to metabolism. This is the overpower-
∗
Corresponding author. Tel.: +358 8 5375255; fax: +358 8 5375247. E-mail address: miia.turpeinen@oulu.fi (M. Turpeinen).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.06.005
ing rationale for in vitro methods to assess drug metabolism and metabolic interactions at early phases of drug discovery and development (Pelkonen et al., 2005). Such an approach requires a high throughput assay infrastructure (Zlokarnik et al., 2005). Metabolism tests most suitable for high throughput screening usually include metabolic stability and drug interaction (CYP inhibition) assays (Li, 2001; White, 2000), and also more
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elaborate test systems are available. However, none of the in vitro systems currently available for drug metabolism is yet formally validated as to their performance in predicting in vivo interactions and comprehensive controlled data bases are rare or include only a modest number of drugs (Pelkonen and Raunio, 2005; Pelkonen et al., 2005; Hutzler et al., 2005). The aim of this work was to systematically compare the performance of three basic types of in vitro metabolism inhibition tests. These tests are (1) a conventional single substrate assay, (2) fluorescence based technology and (3) a newer, nin-one technique, all developed for measuring the inhibition of major drug-metabolizing cytochrome P450 (CYP) activities. Six compounds (sotalol, propranolol, citalopram, fluoxetine, oxazepam and diazepam) from three different therapy classes (-adrenergic receptor antagonists, selective serotonin reuptake inhibitors and benzodiazepines) were selected on the basis of previous literature reports on their CYP inhibition potential.
2.
Materials and methods
2.1.
Chemicals
Fluoxetine was a kind gift from Solvay Pharma (Brussels, Belgium). Sotalol, propranolol, citalopram, oxazepam and diazepam were obtained from Sigma Chemical Company (St. Louis, MO). Bupropion and hydroxybupropion were generous donations from GlaxoSmithKline (Research Triangle, NC), midazolam and ␣-hydroxymidazolam from Hoffmann-La Roche (Basel, Switzerland) and omeprazole, omeprazole sulphone and 5-hydroxyomeprazole from ¨ AstraZeneca (Molndal, Sweden). The metabolite standards dextrorphan, 6-hydroxychlorzoxazone, hydroxytolbutamide and 6-hydroxytestosterone were purchased from Ultrafine Chemical Company (Manchester, U.K.). Formic acid, LichroSolv GG methanol and acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). All other chemicals used were from Sigma–Aldrich (St. Louis, MO) and were of the highest purity available. Water was in-house freshly prepared with Simplicity 185 (Millipore S.A., Molsheim, France) water purification system and was UP grade (ultra pure, 18.2 M).
2.2.
Biological material
2.2.1.
Human liver microsomes
Human liver samples used in this study were obtained from the University Hospital of Oulu as a surplus from transplantation donors. The collection of surplus tissue was approved by the Ethics Committee of the Medical Faculty of the University of Oulu, Finland. The livers were transferred to ice immediately after surgical excision, cut into pieces, snapfrozen in liquid nitrogen and stored at −80 ◦ C until the microsomes were prepared by standard differential ultracentrifugation. A weight-balanced microsomal pool of ten liver microsomal preparations which have been extensively characterized for primary screening (sufficient model activities, no known polymorphisms, expected effects of model inhibitors, quantification of CYPs by Western blotting) was employed. The final microsomal pellet was suspended in 100 mM phosphate
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buffer pH 7.4. Accurate protein content was determined by the method of Bradford (1976).
2.2.2.
cDNA expressed human P450s
Baculovirus-insect cell expressed human CYP2B6 was purchased from BD Biosciences Discovery Labware (Bedford, MA) and used according to manufacturer’s instructions.
2.3.
Metabolic assays
2.3.1.
Single substrate assays
The incubation conditions and instrumentation used to assess the enzyme activities of CYP1A1/2 (ethoxyresorufin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide hydroxylation), CYP2D6 (dextromethorphan Odemethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4/5 (midazolam ␣-hydroxylation) have been described previously in detail in Taavitsainen et al. (2001) and the assay for CYP2B6 (bupropion hydroxylation) in Turpeinen et al. (2004). Each reaction mixture was preincubated for 2 min at 37 ◦ C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany). The reaction was started by adding NADPH and terminated by adding equal volume of ice-cold acetonitrile and subsequently cooled in an ice bath to precipitate the proteins. All incubates were done in duplicates. The mixture was vortex mixed and spun at 10,000 × g for 15 min. Supernatants were collected and stored at −20 ◦ C until analyzed. The amodiaquine N-deethylation assay for CYP2C8 was a modification from Li et al. (2002): incubation mixtures contained 0.5 mg protein/ml and 30 M amodiaquine and the incubation time was 20 min. Omeprazole 5-hydroxylation and sulphoxidation assays for CYP2C19 and CYP3A4, respectively, ¨ were adapted from Abel o¨ et al. (2000): incubation mixtures contained 0.5 mg microsomal protein/ml and 40 M omeprazole and incubation period was 20 min. Otherwise the incubations contained buffer and NADPH and were performed as described above.
2.3.2.
n-in-one assay
Conduction of the n-in-one assay was described previously in detail in Turpeinen et al. (2005). Shortly, each incubation mixture contained 0.5 mg microsomal protein/ml, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH and all 10 probe substrates. Substrates and their concentrations for the incubations were: melatonin (0.4 mM), coumarin (0.2 mM), bupropion (0.1 mM), amodiaquine (0.2 mM), tolbutamide (0.4 mM), omeprazole (0.2 mM), dextromethorphan (0.02 mM), chlorzoxazone (0.6 mM), midazolam (0.04 mM) and testosterone (0.1 mM). Reaction mixture, in a final volume of 200 l, was preincubated for 2 min at +37 ◦ C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany) before the reaction was initiated by addition of NADPH. Each reaction was terminated after 30 min by adding 100 l of ice-cold acetonitrile containing warfarin (1.5 M) as an internal standard (ISTD). Samples were subsequently cooled in an ice bath to precipitate the proteins. Supernatants were collected into clean tubes and stored at −20 ◦ C until analyzed. For analysis all incubation samples were thawed at room temperature, shaken and centrifuged for 10 min at 10,000 × g. The
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Table 1 – Experimental conditions of fluorescent probe assays in recombinant human CYP enzymes CYP isoform 1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4
Substrate and concentration (M)
Fluorescent metabolite
Incubation time (min)
Enzyme (pmol)
7-Ethoxyresorufin (1) Coumarin (3) EFC (2.5) DBF (1) MFC (75) BFC (25) MAMC (7.5) MFC (100) BFC (50)
Resorufin 7-Hydroxycoumarin HFC Fluorescein HFC HFC HAMC HFC HFC
20 15 30 30 45 45 60 45 30
0.5 0.75 0.75 3 0.75 2.5 1.5 1.5 0.75
EFC: 7-ethoxy-4-(trifluoromethyl)coumarin; DBF: dibenzyl fluorescein; MFC: 7-methoxy-4-(trifluoromethyl)coumarin; BFC: 7-benzyloxy-4(trifluoromethyl)coumarin; MAMC: 7-methoxy-4-(aminomethyl)coumarin; HFC: 7-hydroxy-4-(trifluoromethyl)coumarin; HAMC: 7-hydroxy-4(aminomethyl)coumarin.
supernatants were transferred to a Waters Total Recovery vial (Waters Corporation, Milford, MA, USA) to await LC/TOF-MS analysis.
2.3.3.
Fluorescent probe assays
Incubations were performed in 96-well plates. The experimental conditions are summarized in Table 1. In each well, a 150-l incubation volume contained 100 mM Tris–HCl buffer (pH 7.4), 4.2 mM MgCl2 , 1–100 M substrate and cDNA expressed CYP. The reaction was initiated by adding the NADPH-generating system (50 l) after 10 min preincubation at 37 ◦ C. After a given incubation time the reactions were terminated by the addition of 110 l, 80% acetonitrile/20% 0.5 M Tris Base. For CYP2A6 in each well, a 100-l incubation volume contained 75 l of 50 mM Tris–HCl buffer (pH 7.4) and 5.0 mM MgCl2 . The reaction was initiated by addition of NADPH (25 l) and terminated by adding 60 l, 10% TCA. Immediately before the measurement 140 l, 1.6 M glycine–NaOH buffer (pH 10.4) was added. For CYP2C8 the reaction was terminated by 2 M NaOH. The formed fluorescence was measured with a Victor2 plate counter (Perkin-Elmer Life Sciences Wallac, Turku, Finland). For assays using DBF, the signal to noise ratio was greatly increased when the plate was incubated (37 ◦ C) approximately 2 h after the addition of NaOH. Several control incubations were carried out to determine the effect of quenching by the inhibitors and other interfering factors. All IC50 values were determined in duplicate (http://www.gentest.com; Korhonen et al., 2005; Rahnasto et al., 2005).
2.4.
HPLC conditions
A Waters Alliance 2695 chromatographic system with autosampler, vacuum degasser and column oven was used. The analytical column was a Waters XTerra MS C18, 2.1 mm × 50 mm, 3.5 m with Phenomenex Luna C18(2) precolumn, 4.0 mm × 2.0 mm, 3.0 m (Phenomenex, Torrance, California, USA). The flow rate was 0.3 ml/min and the column oven temperature was 30 ◦ C. The eluents used were (A) aqueous 0.1% formic acid (pH 2.75) and (B) methanol. Gradient elution from 15% B to 60% B in 10 min was used. The flow was split postcolumn with Acurate Post-Column Stream Splitter (LC Packings, Amsterdam, The Netherlands). The split ratio was 1:4 to MS and waste, respectively.
2.5.
LC/ESI/MS conditions
LC/MS data were recorded with a Micromass LCT time-of-flight high resolution mass spectrometer (Micromass Ltd., Manchester, England) equipped with Z-Spray electrospray ionization source. A generic positive electrospray ionization method was used for all substrates and metabolites including internal standard. Basic parameter conditions were capillary, sample cone and extraction cone voltages 3000, 20 and 3 V, desolvation and nebulization N2 flows 600 and 70 l/h, and desolvation and source temperatures 220 and 150 ◦ C, respectively. The mass spectrometer and HPLC system were operated under Micromass MassLynx 3.4 software, which also was used to process the measured LC/MS data. Chromatographic traces of protonated metabolites of CYP specific substrates were extracted from total ion chromatograms or traces of appropriate molecular ions (protonated and/or Na+ or K+ adducts) and/or in-source fragments were combined. Mass chromatograms were then integrated and peak areas were normalized to ISTD. No quantification was performed. The peak areas of metabolites in different incubations were compared as such after internal standard corrections were made. The standard deviations of ISTD peak areas were typically below 10%.
2.6.
In vitro inhibition and reference inhibitors
For determinations of the IC50 values the study compounds were added into the incubation mixture in five different concentrations (0.01, 0.1, 1, 10 and 100 M) in a small volume of an appropriate solvent. The solvents used were water (sotalol and citalopram), methanol (diazepam) or DMSO (propranolol, fluoxetine and oxazepam). The performance of traditional CYP-selective model inhibitors (for references see Turpeinen et al., 2005) was characterized with fluorescent probe assay also as described above. Montelukast was used for inhibition of CYP2C8 activity (Walsky et al., 2005) in all assay types. The IC50 values for inhibitors were determined graphically of the logarithmic plot of inhibitor concentration versus percentage of activity remaining after inhibition using Microcal Origin, Version 6.0 (Microcal Software Inc., Northampton, MA). All data points represent the average of duplicate incubations. The enzyme activities in the presence of inhibitors were compared
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Table 2 – IC50 values (M) of sotalol and propranolol as measured by traditional single substrate assay, fluorescent probe assay and n-in-one approach CYP isoform
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 ␣-OH-MDZ 6-TES SO-OME 3-OH-OME
Sotalol
Propranolol
Single
Fluorescent
n-in-one
Single
Fluorescent
n-in-one
>100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100
30.7 >100 >100 >100 >100 88.3 5.8 >100
47.1 >100 >100 >100 >100 40.0 2.65 >100 >100
26.1 >100 41.1 >100 >100 60.8 2.5 >100
>100 >100 >100 >100
>100 >100 >100 n.d.
>100 >100 >100 n.d.
>100 >100 >100 >100
n.d.: not determined.
with the control incubations (incubations with the solvent but without an inhibitor).
3.
Results
3.2.
The results obtained with the test compounds are summarized in Table 2 (sotalol and propranolol), Table 3 (citalopram and fluoxetine) and Table 4 (oxazepam and diazepam).
3.1.
were observed with all three assay types. In addition, inhibition of CYP2B6 activity with an IC50 value of 41.1 M was observed when the n-in-one assay was used (Table 2).
Sotalol and propranolol
With sotalol, no inhibition was observed in any model activity in any of the three assays even at the highest concentration used (100 M). With propranolol, a strong inhibition of CYP2D6 with IC50 values of 5.8, 2.65 and 2.5 M for single substrate, fluorescent probe and n-in-one assays were observed, respectively. Moderate inhibition of CYP1A2 with IC50 values ranging from 26.1 to 47.1 M and CYP2C19 (IC50 values 40.0–88.3 M)
Citalopram and fluoxetine
Citalopram was found to inhibit CYP2D6 with IC50 values of 12.1, 3.3 and 4.6 M in single substrate, fluorescent probe and n-in-one assays, respectively (Table 3). The CYP3A4 associated model reactions testosterone 6-hydroxylation and omeprazole 3-hydroxylation showed inhibition with IC50 values of 31.6 and 10.0 M, respectively, for citalopram when the nin-one test was used. Fluoxetine inhibited CYP2D6 equally well in all three assays, with IC50 values of 0.3, 0.25 and 0.15 M in single substrate, fluorescent probe and n-in-one assay, respectively. Fluoxetine was a relatively potent inhibitor towards CYP2C19 and CYP2C9, with IC50 values of 0.33 and 3.61 M in the fluorescent assay, respectively. The IC50 values were considerably higher in single substrate and n-in-
Table 3 – IC50 values (M) of citalopram and fluoxetine as measured by traditional single substrate assay, fluorescent probe assay and n-in-one approach CYP isoform
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 ␣-OH-MDZ 6-TES SO-OME 3-OH-OME n.d.: not determined.
Citalopram
Fluoxetine
Single
Fluorescent
n-in-one
Single
Fluorescent
n-in-one
>100 >100 >100 >100 >100 >100 12.1 >100
>100 >100 >100 >100 >100 >100 3.3 >100 >100
>100 >100 >100 >100 >100 >100 4.6 >100
>100 >100 62.0 >100 48.6 33.1 0.3 >100
>100 >100 84.9 16.4 3.61 0.33 0.25 >100 17.0
>100 >100 21.9 >100 12.3 22.0 0.15 >100
>100 31.6 >100 10.0
78.8 9.9 52.3 n.d.
>100 >100 >100 n.d.
64.8 7.4 24.4 20.0
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Table 4 – IC50 values (M) of oxazepam and diazepam as measured by traditional single substrate assay, fluorescent probe assay and n-in-one approach CYP isoform
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 ␣-OH-MDZ 6-TES SO-OME 3-OH-OME
Oxazepam Single
Fluorescent
>100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
Diazepam n-in-one
>100 >100 >100 n.d.
Single
Fluorescent
n-in-one
31.6 >100 >100 >100 >100 >100 92.4 >100
>100 >100 >100 >100 >100 39.5 97.0 >100
>100 >100 >100 >100 >100 45.9 82.2 >100 >100
>100 >100 57.8 >100 >100 41.1 39.6 >100
>100 >100 >100 >100
>100 >100 >100 n.d.
>100 >100 >100 >100
n.d.: not determined.
one assays, with IC50 values of 33.1 and 22.0 M (CYP2C19) and 48.6 and 12.3 M (CYP2C9), respectively. Fluoxetine inhibited CYP3A4 moderately, but the IC50 value was dependent on the assay type and the model reaction used. The values obtained with the single substrate assay ranged from 9.9 M (testosterone 6-hydroxylation) to 78.8 M (midazolam ␣-hydroxylation) and with the n-in-one assay from 7.4 M (testosterone 6-hydroxylation) to 64.8 M (midazolam ␣hydroxylation). The fluorescence probe assay gave a value of 17.0 M. CYP2B6 was inhibited by fluoxetine in all three assays (IC50 values between 21.9 and 84.9 M). CYP2C8 was inhibited only in the fluorescence probe assay, with an IC50 value of 16.4 M.
3.3.
Oxazepam and diazepam
With oxazepam, moderate inhibition of CYP1A2 (IC50 = 31.6 M) and CYP2D6 (IC50 = 92.4 M) was seen with the n-in-one assay (Table 4). Other assay types showed no
inhibition. Diazepam was found to inhibit CYP2D6 with IC50 values of 97.0, 82.2 and 39.6 M in single substrate, fluorescent probe and n-in-one assays, respectively. Moderate inhibition of CYP2C19 by diazepam was seen in all assay types with IC50 values ranging from 39.5 to 45.9 M. In addition, in the n-in-one assay diazepam inhibited CYP2B6 activity with an IC50 value of 57.8 M.
3.4. Effect of CYP-specific reference inhibitors in the fluorescent probe assay To further compare the three types of tests with each other, the effects of well established reference inhibitors were evaluated in the single substrate, fluorescent probe and n-in-one assays. The effects of well established reference inhibitors were measured in the fluorescent probe assay and compared with the results obtained from our previous study with the single substrate and n-in-one assays (Turpeinen et al., 2005). The results are summarized in Table 5. Each diagnostic inhibitor
Table 5 – IC50 values (M) of CYP-specific diagnostic inhibitors in traditional single substrate assay, n-in-one technique and fluorescent probe assay CYP isoform 1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4
Inhibitor Fluvoxamine Tranylcypromine Ticlopidine Montelukast Sulphaphenazole Fluconazole Quinidine Pyridine Ketoconazole
Single 0.08 0.38 0.31 0.4 0.35 23.8 0.08 2.75 0.065c , 1.8d
Fluorescent
n-in-one
0.1 0.25 0.4 0.16 0.3 8.2 0.004 6.2 0.01
0.08 1.0 0.1 0.9 0.2 5.7a , 6.4b 0.035 36.6 1.8c , 0.07d , 0.08e , 0.13f
Data for single and n-in-one assays excluding montelukast is adapted from Turpeinen et al. (2005). a b c d e f
IC50 value for omeprazole demethylation. IC50 value for omeprazole 5-hydroxylation. C50 value for midazolam ␣-hydroxylation. IC50 value for testosterone 6-hydroxylation. IC50 value for omeprazole 3-hydroxylation. IC50 value for omeprazole sulphoxidation.
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was found to inhibit the model activities in a very similar fashion. There were, however, some notable differences. Ketoconazole was at least eight-fold more potent in the fluorescence probe assay than in the single substrate or n-in-one assays. In the latter two assays, there were large differences in the IC50 values between different CYP3A4 substrates. CYP2E1 model inhibitor pyridine displayed IC50 values of 2.7, 6.2 and 36.6 M for single substrate, fluorescent probe and n-in-one assay, respectively.
4.
Discussion
In this study, three different types of tests were developed and adapted for determining the inhibition capacities of chemical compound towards human drug-metabolizing CYP enzymes. Nine distinct CYP forms were included in each of the assays, including CYP2B6 and CYP2C8, two forms that have recently been identified as significant contributors to drug metabolism (Ekins and Wrighton, 1999; Totah and Rettie, 2005). The performance of the three assays was first assessed using well established in vitro inhibitors of CYPs. To cross-validate the assays, the inhibitory capacities of six selected drugs in clinical use were analyzed. The results show that the three assay types yield remarkably similar results with the majority of tested CYP forms. Of the two -receptor antagonists studied, sotalol has negligible first-pass metabolism and the majority of oral dose is excreted unchanged in urine (Sotaniemi et al., 1979). No inhibition of any hepatic CYP form by sotalol has been reported previously. A clear lack of inhibition was observed also in this study in all the three assay types. Propranolol is known to be a potent CYP2D6 inhibitor (Rowland et al., 1994) with moderate inhibition affinity also against CYP1A2 (Gillam and Reilly, 1988). In the present study, all three assays predicted these inhibitions and their magnitude correctly. Also moderate inhibition of CYP2C19 ranging from 40 to 88.3 M was observed in all the assays. The n-in-one technique suggested a moderate inhibition of CYP2B6 by propranolol with IC50 value of approximately 40 M, but this was not observed in the other two assay types. The two serotonin selective antidepressants studied, fluoxetine and citalopram, were selected because they have widely different CYP inhibition profiles. Citalopram is considered to be one of the compounds least prone to metabolic interactions among serotonin selective antidepressants. After continuous treatment with citalopram, the in vivo activity of CYP2D6 has been found to be slightly reduced (Gram et al., 1993; Baumann and Rochat, 1995), but inhibition of other CYP forms has not been reported. In agreement with these data, citalopram showed an inhibition of CYP2D6 which was observed in all three tests, while no inhibition of the other investigated CYP isoforms was observed. Fluoxetine is known to undergo extensive hepatic biotransformation mainly to the metabolite norfluoxetine (Fuller et al., 1991), with both having a strong inhibition capacity against CYP2D6 (Stevens and Wrighton, 1993), and notably low IC50 values for CYP3A4 (Greenblatt et al., 1996), CYP2C9 (Shader et al., 1994) and CYP2B6 (Hesse et al., 2000). In the current study, the magnitude of inhibition towards CYP forms by flu-
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oxetine was consistent with previous literature results. The assay utilizing the fluorescent probe technique predicted also an inhibition against CYP2C8, which was not observed in the other tests. To our knowledge, there are no literature reports about inhibition of CYP2C8 by fluoxetine. With fluoxetine, the IC50 values concerning CYP2C19 varied from approximately 0.5–33 M depending on the assay used. This particular discrepancy merits further investigations. The benzodiazepine oxazepam and the parent compound diazepam were selected because of close structural similarity (Fig. 1) but drastically different metabolic profiles. Oxazepam belongs structurally to 3-hydroxybenzodiazepines, which undergo direct conjugation by phase II enzymes and excretion to urine, while diazepam undergoes hepatic metabolism (Chouinard et al., 1999). In the present study, the results with both oxazepam and diazepam were in a fair agreement with literature information. Diazepam inhibited relatively modestly CYP2C19 and CYP2D6 and the inhibition was evident in all assay systems. A 2–2.5-fold difference in the CYP2D6 IC50 value between single substrate, fluorescent probe and n-in-one assay seems not too significant. False (?) positive results from the n-in-one assay regarding CYP2B6, CYP1A2, and CYP2D6 may be due to the fact that for the sake of more sensitive analytical technique lower substrate concentrations can be used in the n-in-one assay, which results in smaller IC50 values with competitive inhibitors. This same explanation is probably behind false (?) positives in the fluorescent probe assay. Reference inhibitors were found to perform mainly as expected and the IC50 values were within the same order of magnitude with all the three screening tests. The largest discrepancy was observed with the CYP2E1 inhibitor pyridine, which yielded IC50 values ranging from 2.8 to 36.6 M in the single substrate and n-in-one assays, respectively. The reason for this remains open and is discussed previously in detail in a paper by Turpeinen et al. (2005). Neither cross-reactivity of other cocktail components nor the effect of organic solvents was found to be the reason for this difference. Inhibition of CYP3A4 was tested with ketoconazole using multiple probe substrates. It was not unexpected that the inhibition potency of ketoconazole varied depending on the specific reaction measured. Previously, inhibition of CYP3A4 has been shown to be substrate-dependent (Harlow and Halpert, 1998). A drug molecule can bind to an allosteric site of CYP3A4 and change the affinity of the substrate-binding site (Wang et al., 2000). Molecular modeling and metabolism studies suggest that the active site of CYP3A4 has the capacity to accommodate large molecules and more than one substrate at the same time (Shou et al., 1994; Lewis et al., 1996). Because of the unique properties of CYP3A4, the reactions catalyzed do not always follow typical enzyme kinetics. Thus, CYP3A4 enzyme kinetics is unpredictable and depends on many different factors such as the substrates and inhibitors used (Galetin et al., 2005). A further complication is that with “CYP3A4-selective” probe drugs, it is not easy to differentiate between three human CYP3A enzymes, CYP3A4, CYP3A5, and CYP3A7, except that the last one is principally a fetal form (see Turpeinen et al., 2005). In this study this effect was observed especially with citalopram, which showed a moderate CYP3A4 inhibition in
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Fig. 1 – Chemical structures of study compounds: (a) sotalol, (b) propranolol, (c) citalopram, (d) fluoxetine, (e) oxazepam and (f) diazepam.
both the single substrate and n-in-one assays when testosterone 6-hydroxylation and omeprazole 3-hydroxylation were measured, but no inhibition occurred when midazolam ␣-hydroxylation or omeprazole sulphoxidation assays were carried out. The use of multiple CYP3A4 substrates is therefore recommended when screening the CYP3A4 inhibition potential of new chemical entities.
We estimated the percentage of inhibition in vivo for the test drugs and those CYPs most consistently inhibited (Table 6). Rather high inhibition percentages were predicted for propranolol and fluoxetine. Several clinically significant CYP-based interactions have been reported for these drugs (Stockley, 2004). Citalopram and diazepam, and expectedly sotalol and oxazepam, gave rather low expected inhibition
Table 6 – Calculated in vivo inhibition (%) of study compounds on selected CYP enzymes Study compound Sotalol Propranolol Fluoxetine Citalopram Oxazepam Diazepam
CYP isoform
Cmax (M)a
IC50 (M)b
Inhibition in vivo (%)c
Reference
NA 1A2 2D6 2D6 2D6 NA 2D6 2C19
2.8 1.2 1.2 1 0.4 1.4 1.1
>100 26.1–47.1 2.5–5.8 0.15–0.3 4.6–12.1 >100 39.6–97.0 39.5–45.9
NA 4–2 32–17 87–77 8–3 NA 3–1 3–2
Kimura et al. (1996) Pelkonen et al. (1998)
1.1
NA: not applicable. a b c
Maximum plasma concentrations (Cmax ) of study compounds after average oral dose. See Tables 2–4. The percentage of inhibition was calculated according to the equation: I/(I + IC50 ) × 100 (Pelkonen et al., 1998).
Pelkonen et al. (1998) Pelkonen et al. (1998) Sonne et al. (1988) Friedman et al. (1992)
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percentages. This is consistent with the lack of clinically significant CYP-based metabolic interactions caused by these drugs. In conclusion, two in vitro assays with higher throughput, the n-in-one technique and the fluorescent probe assay, were found to be useful and suitable for measuring the inhibition of major drug-metabolizing CYP activities compared to a conventional single substrate assay. The rationale behind using more efficient and robust methods in early stage of drug development is the cost-effectiveness of screening methods and the aim to rank hit/lead candidates for selection purposes. These tests provide tentative inhibition values and point out the CYP forms potentially inhibited. If detailed and more accurate information is wanted, the traditional single substrate technique would still be the recommended choice.
Acknowledgements The contribution of Dr. Larissa Kankainen and Dr. Petri Reponen and the valuable technical assistance of Mrs. Ritva Tau¨ riainen, Mrs. Paivi Tyni, Ms. Hannele Jaatinen and Ms. Virpi Koponen are greatly acknowledged. This study belongs to the Research Programme “Drug 2000” (subsection “screening methods”) of the Finnish Technological Research Agency (TEKES). This work was funded by the grants from TEKES and supported by the grants of Finnish Cultural Foundation, Finnish Medical Society Duodecim, Juliana von Wendt Foundation and Farmos Research and Science Foundation to M.T.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Cytochrome P450 (CYP) inhibition screening: Comparison of three tests Miia Turpeinen a,∗ , Laura E. Korhonen b , Ari Tolonen c , Jouko Uusitalo c , Risto Juvonen b , Hannu Raunio b , Olavi Pelkonen a a b c
Department of Pharmacology and Toxicology, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland Novamass Analytical Ltd., Oulu, Finland
a r t i c l e
i n f o
a b s t r a c t
Article history:
There are several different experimental systems for screening of in vitro inhibitory potency
Received 1 January 2006
of drugs under development. In this study we compared three different types of cytochrome
Received in revised form 9 May 2006
P450 (CYP) inhibition tests: the traditional single substrate assays, the fluorescent probe
Accepted 20 June 2006
method with recombinant human CYPs, and a novel n-in-one technique. All major hep-
Published on line 27 June 2006
atic drug-metabolizing CYPs were included (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4). Six compounds (sotalol, propranolol, citalopram, fluoxetine, oxazepam and diazepam) were
Keywords:
selected for detailed comparisons. The IC50 values of each of these compounds were mea-
Cytochrome P450 (CYP)
sured using the three assay types. The inhibitory potencies of these model drugs were
Inhibition
generally within the same order of magnitude and followed similar inhibition profiles in
Human liver microsomes
all the assay types. Clinically observed inhibitory interactions, or lack thereof, were pre-
Recombinant enzymes
dictable with all three assays. Comparison of potencies of ‘diagnostic’ inhibitors revealed
In vitro cocktail
also some notable differences between the assays, especially regarding CYP2E1. The potency
Fluorescent probes
of inhibitors towards CYP3A4 was dependent on the substrate and reaction measured. Gen-
Sotalol
erally all three assays gave reasonably comparable results, although some unexplained
Propranolol
differences were also noted
Citalopram
© 2006 Elsevier B.V. All rights reserved.
Fluoxetine Oxazepam Diazepam
1.
Introduction
Early knowledge of the metabolic properties of a new drug candidate is pivotal during drug development. Lead compounds need to be studied for metabolic characteristics to avoid drugs being withdrawn from clinical studies or the market due to major kinetic problems, such as strong interaction potential due to metabolism. This is the overpower-
∗
Corresponding author. Tel.: +358 8 5375255; fax: +358 8 5375247. E-mail address: miia.turpeinen@oulu.fi (M. Turpeinen).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.06.005
ing rationale for in vitro methods to assess drug metabolism and metabolic interactions at early phases of drug discovery and development (Pelkonen et al., 2005). Such an approach requires a high throughput assay infrastructure (Zlokarnik et al., 2005). Metabolism tests most suitable for high throughput screening usually include metabolic stability and drug interaction (CYP inhibition) assays (Li, 2001; White, 2000), and also more
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 9 ( 2 0 0 6 ) 130–138
elaborate test systems are available. However, none of the in vitro systems currently available for drug metabolism is yet formally validated as to their performance in predicting in vivo interactions and comprehensive controlled data bases are rare or include only a modest number of drugs (Pelkonen and Raunio, 2005; Pelkonen et al., 2005; Hutzler et al., 2005). The aim of this work was to systematically compare the performance of three basic types of in vitro metabolism inhibition tests. These tests are (1) a conventional single substrate assay, (2) fluorescence based technology and (3) a newer, nin-one technique, all developed for measuring the inhibition of major drug-metabolizing cytochrome P450 (CYP) activities. Six compounds (sotalol, propranolol, citalopram, fluoxetine, oxazepam and diazepam) from three different therapy classes (-adrenergic receptor antagonists, selective serotonin reuptake inhibitors and benzodiazepines) were selected on the basis of previous literature reports on their CYP inhibition potential.
2.
Materials and methods
2.1.
Chemicals
Fluoxetine was a kind gift from Solvay Pharma (Brussels, Belgium). Sotalol, propranolol, citalopram, oxazepam and diazepam were obtained from Sigma Chemical Company (St. Louis, MO). Bupropion and hydroxybupropion were generous donations from GlaxoSmithKline (Research Triangle, NC), midazolam and ␣-hydroxymidazolam from Hoffmann-La Roche (Basel, Switzerland) and omeprazole, omeprazole sulphone and 5-hydroxyomeprazole from ¨ AstraZeneca (Molndal, Sweden). The metabolite standards dextrorphan, 6-hydroxychlorzoxazone, hydroxytolbutamide and 6-hydroxytestosterone were purchased from Ultrafine Chemical Company (Manchester, U.K.). Formic acid, LichroSolv GG methanol and acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). All other chemicals used were from Sigma–Aldrich (St. Louis, MO) and were of the highest purity available. Water was in-house freshly prepared with Simplicity 185 (Millipore S.A., Molsheim, France) water purification system and was UP grade (ultra pure, 18.2 M).
2.2.
Biological material
2.2.1.
Human liver microsomes
Human liver samples used in this study were obtained from the University Hospital of Oulu as a surplus from transplantation donors. The collection of surplus tissue was approved by the Ethics Committee of the Medical Faculty of the University of Oulu, Finland. The livers were transferred to ice immediately after surgical excision, cut into pieces, snapfrozen in liquid nitrogen and stored at −80 ◦ C until the microsomes were prepared by standard differential ultracentrifugation. A weight-balanced microsomal pool of ten liver microsomal preparations which have been extensively characterized for primary screening (sufficient model activities, no known polymorphisms, expected effects of model inhibitors, quantification of CYPs by Western blotting) was employed. The final microsomal pellet was suspended in 100 mM phosphate
131
buffer pH 7.4. Accurate protein content was determined by the method of Bradford (1976).
2.2.2.
cDNA expressed human P450s
Baculovirus-insect cell expressed human CYP2B6 was purchased from BD Biosciences Discovery Labware (Bedford, MA) and used according to manufacturer’s instructions.
2.3.
Metabolic assays
2.3.1.
Single substrate assays
The incubation conditions and instrumentation used to assess the enzyme activities of CYP1A1/2 (ethoxyresorufin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide hydroxylation), CYP2D6 (dextromethorphan Odemethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), CYP3A4/5 (midazolam ␣-hydroxylation) have been described previously in detail in Taavitsainen et al. (2001) and the assay for CYP2B6 (bupropion hydroxylation) in Turpeinen et al. (2004). Each reaction mixture was preincubated for 2 min at 37 ◦ C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany). The reaction was started by adding NADPH and terminated by adding equal volume of ice-cold acetonitrile and subsequently cooled in an ice bath to precipitate the proteins. All incubates were done in duplicates. The mixture was vortex mixed and spun at 10,000 × g for 15 min. Supernatants were collected and stored at −20 ◦ C until analyzed. The amodiaquine N-deethylation assay for CYP2C8 was a modification from Li et al. (2002): incubation mixtures contained 0.5 mg protein/ml and 30 M amodiaquine and the incubation time was 20 min. Omeprazole 5-hydroxylation and sulphoxidation assays for CYP2C19 and CYP3A4, respectively, ¨ were adapted from Abel o¨ et al. (2000): incubation mixtures contained 0.5 mg microsomal protein/ml and 40 M omeprazole and incubation period was 20 min. Otherwise the incubations contained buffer and NADPH and were performed as described above.
2.3.2.
n-in-one assay
Conduction of the n-in-one assay was described previously in detail in Turpeinen et al. (2005). Shortly, each incubation mixture contained 0.5 mg microsomal protein/ml, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH and all 10 probe substrates. Substrates and their concentrations for the incubations were: melatonin (0.4 mM), coumarin (0.2 mM), bupropion (0.1 mM), amodiaquine (0.2 mM), tolbutamide (0.4 mM), omeprazole (0.2 mM), dextromethorphan (0.02 mM), chlorzoxazone (0.6 mM), midazolam (0.04 mM) and testosterone (0.1 mM). Reaction mixture, in a final volume of 200 l, was preincubated for 2 min at +37 ◦ C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany) before the reaction was initiated by addition of NADPH. Each reaction was terminated after 30 min by adding 100 l of ice-cold acetonitrile containing warfarin (1.5 M) as an internal standard (ISTD). Samples were subsequently cooled in an ice bath to precipitate the proteins. Supernatants were collected into clean tubes and stored at −20 ◦ C until analyzed. For analysis all incubation samples were thawed at room temperature, shaken and centrifuged for 10 min at 10,000 × g. The
132
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Table 1 – Experimental conditions of fluorescent probe assays in recombinant human CYP enzymes CYP isoform 1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4
Substrate and concentration (M)
Fluorescent metabolite
Incubation time (min)
Enzyme (pmol)
7-Ethoxyresorufin (1) Coumarin (3) EFC (2.5) DBF (1) MFC (75) BFC (25) MAMC (7.5) MFC (100) BFC (50)
Resorufin 7-Hydroxycoumarin HFC Fluorescein HFC HFC HAMC HFC HFC
20 15 30 30 45 45 60 45 30
0.5 0.75 0.75 3 0.75 2.5 1.5 1.5 0.75
EFC: 7-ethoxy-4-(trifluoromethyl)coumarin; DBF: dibenzyl fluorescein; MFC: 7-methoxy-4-(trifluoromethyl)coumarin; BFC: 7-benzyloxy-4(trifluoromethyl)coumarin; MAMC: 7-methoxy-4-(aminomethyl)coumarin; HFC: 7-hydroxy-4-(trifluoromethyl)coumarin; HAMC: 7-hydroxy-4(aminomethyl)coumarin.
supernatants were transferred to a Waters Total Recovery vial (Waters Corporation, Milford, MA, USA) to await LC/TOF-MS analysis.
2.3.3.
Fluorescent probe assays
Incubations were performed in 96-well plates. The experimental conditions are summarized in Table 1. In each well, a 150-l incubation volume contained 100 mM Tris–HCl buffer (pH 7.4), 4.2 mM MgCl2 , 1–100 M substrate and cDNA expressed CYP. The reaction was initiated by adding the NADPH-generating system (50 l) after 10 min preincubation at 37 ◦ C. After a given incubation time the reactions were terminated by the addition of 110 l, 80% acetonitrile/20% 0.5 M Tris Base. For CYP2A6 in each well, a 100-l incubation volume contained 75 l of 50 mM Tris–HCl buffer (pH 7.4) and 5.0 mM MgCl2 . The reaction was initiated by addition of NADPH (25 l) and terminated by adding 60 l, 10% TCA. Immediately before the measurement 140 l, 1.6 M glycine–NaOH buffer (pH 10.4) was added. For CYP2C8 the reaction was terminated by 2 M NaOH. The formed fluorescence was measured with a Victor2 plate counter (Perkin-Elmer Life Sciences Wallac, Turku, Finland). For assays using DBF, the signal to noise ratio was greatly increased when the plate was incubated (37 ◦ C) approximately 2 h after the addition of NaOH. Several control incubations were carried out to determine the effect of quenching by the inhibitors and other interfering factors. All IC50 values were determined in duplicate (http://www.gentest.com; Korhonen et al., 2005; Rahnasto et al., 2005).
2.4.
HPLC conditions
A Waters Alliance 2695 chromatographic system with autosampler, vacuum degasser and column oven was used. The analytical column was a Waters XTerra MS C18, 2.1 mm × 50 mm, 3.5 m with Phenomenex Luna C18(2) precolumn, 4.0 mm × 2.0 mm, 3.0 m (Phenomenex, Torrance, California, USA). The flow rate was 0.3 ml/min and the column oven temperature was 30 ◦ C. The eluents used were (A) aqueous 0.1% formic acid (pH 2.75) and (B) methanol. Gradient elution from 15% B to 60% B in 10 min was used. The flow was split postcolumn with Acurate Post-Column Stream Splitter (LC Packings, Amsterdam, The Netherlands). The split ratio was 1:4 to MS and waste, respectively.
2.5.
LC/ESI/MS conditions
LC/MS data were recorded with a Micromass LCT time-of-flight high resolution mass spectrometer (Micromass Ltd., Manchester, England) equipped with Z-Spray electrospray ionization source. A generic positive electrospray ionization method was used for all substrates and metabolites including internal standard. Basic parameter conditions were capillary, sample cone and extraction cone voltages 3000, 20 and 3 V, desolvation and nebulization N2 flows 600 and 70 l/h, and desolvation and source temperatures 220 and 150 ◦ C, respectively. The mass spectrometer and HPLC system were operated under Micromass MassLynx 3.4 software, which also was used to process the measured LC/MS data. Chromatographic traces of protonated metabolites of CYP specific substrates were extracted from total ion chromatograms or traces of appropriate molecular ions (protonated and/or Na+ or K+ adducts) and/or in-source fragments were combined. Mass chromatograms were then integrated and peak areas were normalized to ISTD. No quantification was performed. The peak areas of metabolites in different incubations were compared as such after internal standard corrections were made. The standard deviations of ISTD peak areas were typically below 10%.
2.6.
In vitro inhibition and reference inhibitors
For determinations of the IC50 values the study compounds were added into the incubation mixture in five different concentrations (0.01, 0.1, 1, 10 and 100 M) in a small volume of an appropriate solvent. The solvents used were water (sotalol and citalopram), methanol (diazepam) or DMSO (propranolol, fluoxetine and oxazepam). The performance of traditional CYP-selective model inhibitors (for references see Turpeinen et al., 2005) was characterized with fluorescent probe assay also as described above. Montelukast was used for inhibition of CYP2C8 activity (Walsky et al., 2005) in all assay types. The IC50 values for inhibitors were determined graphically of the logarithmic plot of inhibitor concentration versus percentage of activity remaining after inhibition using Microcal Origin, Version 6.0 (Microcal Software Inc., Northampton, MA). All data points represent the average of duplicate incubations. The enzyme activities in the presence of inhibitors were compared
133
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Table 2 – IC50 values (M) of sotalol and propranolol as measured by traditional single substrate assay, fluorescent probe assay and n-in-one approach CYP isoform
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 ␣-OH-MDZ 6-TES SO-OME 3-OH-OME
Sotalol
Propranolol
Single
Fluorescent
n-in-one
Single
Fluorescent
n-in-one
>100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100
30.7 >100 >100 >100 >100 88.3 5.8 >100
47.1 >100 >100 >100 >100 40.0 2.65 >100 >100
26.1 >100 41.1 >100 >100 60.8 2.5 >100
>100 >100 >100 >100
>100 >100 >100 n.d.
>100 >100 >100 n.d.
>100 >100 >100 >100
n.d.: not determined.
with the control incubations (incubations with the solvent but without an inhibitor).
3.
Results
3.2.
The results obtained with the test compounds are summarized in Table 2 (sotalol and propranolol), Table 3 (citalopram and fluoxetine) and Table 4 (oxazepam and diazepam).
3.1.
were observed with all three assay types. In addition, inhibition of CYP2B6 activity with an IC50 value of 41.1 M was observed when the n-in-one assay was used (Table 2).
Sotalol and propranolol
With sotalol, no inhibition was observed in any model activity in any of the three assays even at the highest concentration used (100 M). With propranolol, a strong inhibition of CYP2D6 with IC50 values of 5.8, 2.65 and 2.5 M for single substrate, fluorescent probe and n-in-one assays were observed, respectively. Moderate inhibition of CYP1A2 with IC50 values ranging from 26.1 to 47.1 M and CYP2C19 (IC50 values 40.0–88.3 M)
Citalopram and fluoxetine
Citalopram was found to inhibit CYP2D6 with IC50 values of 12.1, 3.3 and 4.6 M in single substrate, fluorescent probe and n-in-one assays, respectively (Table 3). The CYP3A4 associated model reactions testosterone 6-hydroxylation and omeprazole 3-hydroxylation showed inhibition with IC50 values of 31.6 and 10.0 M, respectively, for citalopram when the nin-one test was used. Fluoxetine inhibited CYP2D6 equally well in all three assays, with IC50 values of 0.3, 0.25 and 0.15 M in single substrate, fluorescent probe and n-in-one assay, respectively. Fluoxetine was a relatively potent inhibitor towards CYP2C19 and CYP2C9, with IC50 values of 0.33 and 3.61 M in the fluorescent assay, respectively. The IC50 values were considerably higher in single substrate and n-in-
Table 3 – IC50 values (M) of citalopram and fluoxetine as measured by traditional single substrate assay, fluorescent probe assay and n-in-one approach CYP isoform
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 ␣-OH-MDZ 6-TES SO-OME 3-OH-OME n.d.: not determined.
Citalopram
Fluoxetine
Single
Fluorescent
n-in-one
Single
Fluorescent
n-in-one
>100 >100 >100 >100 >100 >100 12.1 >100
>100 >100 >100 >100 >100 >100 3.3 >100 >100
>100 >100 >100 >100 >100 >100 4.6 >100
>100 >100 62.0 >100 48.6 33.1 0.3 >100
>100 >100 84.9 16.4 3.61 0.33 0.25 >100 17.0
>100 >100 21.9 >100 12.3 22.0 0.15 >100
>100 31.6 >100 10.0
78.8 9.9 52.3 n.d.
>100 >100 >100 n.d.
64.8 7.4 24.4 20.0
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Table 4 – IC50 values (M) of oxazepam and diazepam as measured by traditional single substrate assay, fluorescent probe assay and n-in-one approach CYP isoform
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4 ␣-OH-MDZ 6-TES SO-OME 3-OH-OME
Oxazepam Single
Fluorescent
>100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
Diazepam n-in-one
>100 >100 >100 n.d.
Single
Fluorescent
n-in-one
31.6 >100 >100 >100 >100 >100 92.4 >100
>100 >100 >100 >100 >100 39.5 97.0 >100
>100 >100 >100 >100 >100 45.9 82.2 >100 >100
>100 >100 57.8 >100 >100 41.1 39.6 >100
>100 >100 >100 >100
>100 >100 >100 n.d.
>100 >100 >100 >100
n.d.: not determined.
one assays, with IC50 values of 33.1 and 22.0 M (CYP2C19) and 48.6 and 12.3 M (CYP2C9), respectively. Fluoxetine inhibited CYP3A4 moderately, but the IC50 value was dependent on the assay type and the model reaction used. The values obtained with the single substrate assay ranged from 9.9 M (testosterone 6-hydroxylation) to 78.8 M (midazolam ␣-hydroxylation) and with the n-in-one assay from 7.4 M (testosterone 6-hydroxylation) to 64.8 M (midazolam ␣hydroxylation). The fluorescence probe assay gave a value of 17.0 M. CYP2B6 was inhibited by fluoxetine in all three assays (IC50 values between 21.9 and 84.9 M). CYP2C8 was inhibited only in the fluorescence probe assay, with an IC50 value of 16.4 M.
3.3.
Oxazepam and diazepam
With oxazepam, moderate inhibition of CYP1A2 (IC50 = 31.6 M) and CYP2D6 (IC50 = 92.4 M) was seen with the n-in-one assay (Table 4). Other assay types showed no
inhibition. Diazepam was found to inhibit CYP2D6 with IC50 values of 97.0, 82.2 and 39.6 M in single substrate, fluorescent probe and n-in-one assays, respectively. Moderate inhibition of CYP2C19 by diazepam was seen in all assay types with IC50 values ranging from 39.5 to 45.9 M. In addition, in the n-in-one assay diazepam inhibited CYP2B6 activity with an IC50 value of 57.8 M.
3.4. Effect of CYP-specific reference inhibitors in the fluorescent probe assay To further compare the three types of tests with each other, the effects of well established reference inhibitors were evaluated in the single substrate, fluorescent probe and n-in-one assays. The effects of well established reference inhibitors were measured in the fluorescent probe assay and compared with the results obtained from our previous study with the single substrate and n-in-one assays (Turpeinen et al., 2005). The results are summarized in Table 5. Each diagnostic inhibitor
Table 5 – IC50 values (M) of CYP-specific diagnostic inhibitors in traditional single substrate assay, n-in-one technique and fluorescent probe assay CYP isoform 1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4
Inhibitor Fluvoxamine Tranylcypromine Ticlopidine Montelukast Sulphaphenazole Fluconazole Quinidine Pyridine Ketoconazole
Single 0.08 0.38 0.31 0.4 0.35 23.8 0.08 2.75 0.065c , 1.8d
Fluorescent
n-in-one
0.1 0.25 0.4 0.16 0.3 8.2 0.004 6.2 0.01
0.08 1.0 0.1 0.9 0.2 5.7a , 6.4b 0.035 36.6 1.8c , 0.07d , 0.08e , 0.13f
Data for single and n-in-one assays excluding montelukast is adapted from Turpeinen et al. (2005). a b c d e f
IC50 value for omeprazole demethylation. IC50 value for omeprazole 5-hydroxylation. C50 value for midazolam ␣-hydroxylation. IC50 value for testosterone 6-hydroxylation. IC50 value for omeprazole 3-hydroxylation. IC50 value for omeprazole sulphoxidation.
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was found to inhibit the model activities in a very similar fashion. There were, however, some notable differences. Ketoconazole was at least eight-fold more potent in the fluorescence probe assay than in the single substrate or n-in-one assays. In the latter two assays, there were large differences in the IC50 values between different CYP3A4 substrates. CYP2E1 model inhibitor pyridine displayed IC50 values of 2.7, 6.2 and 36.6 M for single substrate, fluorescent probe and n-in-one assay, respectively.
4.
Discussion
In this study, three different types of tests were developed and adapted for determining the inhibition capacities of chemical compound towards human drug-metabolizing CYP enzymes. Nine distinct CYP forms were included in each of the assays, including CYP2B6 and CYP2C8, two forms that have recently been identified as significant contributors to drug metabolism (Ekins and Wrighton, 1999; Totah and Rettie, 2005). The performance of the three assays was first assessed using well established in vitro inhibitors of CYPs. To cross-validate the assays, the inhibitory capacities of six selected drugs in clinical use were analyzed. The results show that the three assay types yield remarkably similar results with the majority of tested CYP forms. Of the two -receptor antagonists studied, sotalol has negligible first-pass metabolism and the majority of oral dose is excreted unchanged in urine (Sotaniemi et al., 1979). No inhibition of any hepatic CYP form by sotalol has been reported previously. A clear lack of inhibition was observed also in this study in all the three assay types. Propranolol is known to be a potent CYP2D6 inhibitor (Rowland et al., 1994) with moderate inhibition affinity also against CYP1A2 (Gillam and Reilly, 1988). In the present study, all three assays predicted these inhibitions and their magnitude correctly. Also moderate inhibition of CYP2C19 ranging from 40 to 88.3 M was observed in all the assays. The n-in-one technique suggested a moderate inhibition of CYP2B6 by propranolol with IC50 value of approximately 40 M, but this was not observed in the other two assay types. The two serotonin selective antidepressants studied, fluoxetine and citalopram, were selected because they have widely different CYP inhibition profiles. Citalopram is considered to be one of the compounds least prone to metabolic interactions among serotonin selective antidepressants. After continuous treatment with citalopram, the in vivo activity of CYP2D6 has been found to be slightly reduced (Gram et al., 1993; Baumann and Rochat, 1995), but inhibition of other CYP forms has not been reported. In agreement with these data, citalopram showed an inhibition of CYP2D6 which was observed in all three tests, while no inhibition of the other investigated CYP isoforms was observed. Fluoxetine is known to undergo extensive hepatic biotransformation mainly to the metabolite norfluoxetine (Fuller et al., 1991), with both having a strong inhibition capacity against CYP2D6 (Stevens and Wrighton, 1993), and notably low IC50 values for CYP3A4 (Greenblatt et al., 1996), CYP2C9 (Shader et al., 1994) and CYP2B6 (Hesse et al., 2000). In the current study, the magnitude of inhibition towards CYP forms by flu-
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oxetine was consistent with previous literature results. The assay utilizing the fluorescent probe technique predicted also an inhibition against CYP2C8, which was not observed in the other tests. To our knowledge, there are no literature reports about inhibition of CYP2C8 by fluoxetine. With fluoxetine, the IC50 values concerning CYP2C19 varied from approximately 0.5–33 M depending on the assay used. This particular discrepancy merits further investigations. The benzodiazepine oxazepam and the parent compound diazepam were selected because of close structural similarity (Fig. 1) but drastically different metabolic profiles. Oxazepam belongs structurally to 3-hydroxybenzodiazepines, which undergo direct conjugation by phase II enzymes and excretion to urine, while diazepam undergoes hepatic metabolism (Chouinard et al., 1999). In the present study, the results with both oxazepam and diazepam were in a fair agreement with literature information. Diazepam inhibited relatively modestly CYP2C19 and CYP2D6 and the inhibition was evident in all assay systems. A 2–2.5-fold difference in the CYP2D6 IC50 value between single substrate, fluorescent probe and n-in-one assay seems not too significant. False (?) positive results from the n-in-one assay regarding CYP2B6, CYP1A2, and CYP2D6 may be due to the fact that for the sake of more sensitive analytical technique lower substrate concentrations can be used in the n-in-one assay, which results in smaller IC50 values with competitive inhibitors. This same explanation is probably behind false (?) positives in the fluorescent probe assay. Reference inhibitors were found to perform mainly as expected and the IC50 values were within the same order of magnitude with all the three screening tests. The largest discrepancy was observed with the CYP2E1 inhibitor pyridine, which yielded IC50 values ranging from 2.8 to 36.6 M in the single substrate and n-in-one assays, respectively. The reason for this remains open and is discussed previously in detail in a paper by Turpeinen et al. (2005). Neither cross-reactivity of other cocktail components nor the effect of organic solvents was found to be the reason for this difference. Inhibition of CYP3A4 was tested with ketoconazole using multiple probe substrates. It was not unexpected that the inhibition potency of ketoconazole varied depending on the specific reaction measured. Previously, inhibition of CYP3A4 has been shown to be substrate-dependent (Harlow and Halpert, 1998). A drug molecule can bind to an allosteric site of CYP3A4 and change the affinity of the substrate-binding site (Wang et al., 2000). Molecular modeling and metabolism studies suggest that the active site of CYP3A4 has the capacity to accommodate large molecules and more than one substrate at the same time (Shou et al., 1994; Lewis et al., 1996). Because of the unique properties of CYP3A4, the reactions catalyzed do not always follow typical enzyme kinetics. Thus, CYP3A4 enzyme kinetics is unpredictable and depends on many different factors such as the substrates and inhibitors used (Galetin et al., 2005). A further complication is that with “CYP3A4-selective” probe drugs, it is not easy to differentiate between three human CYP3A enzymes, CYP3A4, CYP3A5, and CYP3A7, except that the last one is principally a fetal form (see Turpeinen et al., 2005). In this study this effect was observed especially with citalopram, which showed a moderate CYP3A4 inhibition in
136
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Fig. 1 – Chemical structures of study compounds: (a) sotalol, (b) propranolol, (c) citalopram, (d) fluoxetine, (e) oxazepam and (f) diazepam.
both the single substrate and n-in-one assays when testosterone 6-hydroxylation and omeprazole 3-hydroxylation were measured, but no inhibition occurred when midazolam ␣-hydroxylation or omeprazole sulphoxidation assays were carried out. The use of multiple CYP3A4 substrates is therefore recommended when screening the CYP3A4 inhibition potential of new chemical entities.
We estimated the percentage of inhibition in vivo for the test drugs and those CYPs most consistently inhibited (Table 6). Rather high inhibition percentages were predicted for propranolol and fluoxetine. Several clinically significant CYP-based interactions have been reported for these drugs (Stockley, 2004). Citalopram and diazepam, and expectedly sotalol and oxazepam, gave rather low expected inhibition
Table 6 – Calculated in vivo inhibition (%) of study compounds on selected CYP enzymes Study compound Sotalol Propranolol Fluoxetine Citalopram Oxazepam Diazepam
CYP isoform
Cmax (M)a
IC50 (M)b
Inhibition in vivo (%)c
Reference
NA 1A2 2D6 2D6 2D6 NA 2D6 2C19
2.8 1.2 1.2 1 0.4 1.4 1.1
>100 26.1–47.1 2.5–5.8 0.15–0.3 4.6–12.1 >100 39.6–97.0 39.5–45.9
NA 4–2 32–17 87–77 8–3 NA 3–1 3–2
Kimura et al. (1996) Pelkonen et al. (1998)
1.1
NA: not applicable. a b c
Maximum plasma concentrations (Cmax ) of study compounds after average oral dose. See Tables 2–4. The percentage of inhibition was calculated according to the equation: I/(I + IC50 ) × 100 (Pelkonen et al., 1998).
Pelkonen et al. (1998) Pelkonen et al. (1998) Sonne et al. (1988) Friedman et al. (1992)
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percentages. This is consistent with the lack of clinically significant CYP-based metabolic interactions caused by these drugs. In conclusion, two in vitro assays with higher throughput, the n-in-one technique and the fluorescent probe assay, were found to be useful and suitable for measuring the inhibition of major drug-metabolizing CYP activities compared to a conventional single substrate assay. The rationale behind using more efficient and robust methods in early stage of drug development is the cost-effectiveness of screening methods and the aim to rank hit/lead candidates for selection purposes. These tests provide tentative inhibition values and point out the CYP forms potentially inhibited. If detailed and more accurate information is wanted, the traditional single substrate technique would still be the recommended choice.
Acknowledgements The contribution of Dr. Larissa Kankainen and Dr. Petri Reponen and the valuable technical assistance of Mrs. Ritva Tau¨ riainen, Mrs. Paivi Tyni, Ms. Hannele Jaatinen and Ms. Virpi Koponen are greatly acknowledged. This study belongs to the Research Programme “Drug 2000” (subsection “screening methods”) of the Finnish Technological Research Agency (TEKES). This work was funded by the grants from TEKES and supported by the grants of Finnish Cultural Foundation, Finnish Medical Society Duodecim, Juliana von Wendt Foundation and Farmos Research and Science Foundation to M.T.
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