Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes Kinetics and sensitivity to cytochrome P450 2D inhibitors Annalise Di Marco1, Dan Yao2and Ralph L
Trang 1Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes
Kinetics and sensitivity to cytochrome P450 2D inhibitors
Annalise Di Marco1, Dan Yao2and Ralph Laufer1
1 Department of Pharmacology, Istituto di Ricerche di Biologia Molecolare P Angeletti (IRBM), Merck Sharp and Dohme Research Laboratories, Rome, Italy;2Labeled Compound Synthesis, Department of Drug Metabolism,
Merck Research Laboratories, Rahway, NJ, USA
Liver microsomal preparations are routinely used to predict
drug interactions that can occur in vivo as a result of
inhi-bition of cytochrome P450 (CYP)-mediated metabolism
However, the concentration of free drug (substrate and
inhibitor)at its intrahepatic site of action, a variable that
cannot be directly measured, may be significantly different
from that in microsomal incubation systems Intact cells
more closely reflect the environment to which CYP
sub-strates and inhibitors are exposed in the liver, and it may
therefore be desirable to assess the potential of a drug to
cause CYP inhibition in isolated hepatocytes The objective
of this study was to compare the inhibitory potencies of a
series of CYP2D inhibitors in rat liver microsomes and
hepatocytes For this, we developed an assay suitable for
rapid analysis of CYP-mediated drug interactions in
both systems, using radiolabelled dextromethorphan, a
well-characterized probe substrate for enzymes of the
CYP2D family Dextromethorphan demethylation
exhib-ited saturable kinetics in rat microsomes and hepatocytes,
with apparent Km and Vmax values of 2.1 vs 2.8 lM
and 0.74 nmolÆmin)1 per mg microsomal protein vs
0.11 nmolÆmin)1 per mg cellular protein, respectively Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50values in the low micromolar range Some of these compounds exhibited biphasic inhibition kinetics, indicative
of interaction with more than one CYP2D isoform Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2–3-fold lower IC50 in hepatocytes than in microsomes The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracel-lular medium, suggesting that intracelextracel-lular accumulation may potentiate the effect of these compounds Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport
Keywords: CYP2D; cytochrome P450; hepatocytes; micro-somes
The pharmacokinetic and toxicokinetic properties of
phar-maceuticals depend in great part on their biotransformation
by drug-metabolizing enzymes The main
drug-metaboli-zing system in mammals is cytochrome P450 (CYP), a
family of microsomal isozymes present predominantly in
the liver Multiple CYPs catalyze the oxidation of chemicals
of endogenous and exogenous origin, including drugs,
steroids, prostanoids, eicosanoids, fatty acids, and
environ-mental toxins [1] If a drug that is metabolized by a
particular CYP isozyme is coadministered with an inhibitor
of that same enzyme, changes in its pharmacokinetics can
occur, which can give rise to adverse effects [2–5] It is
therefore important to predict and prevent the occurrence of
clearance changes caused by metabolic inhibition During the drug discovery process, it has become routine practice
in the pharmaceutical industry to assess CYP inhibition potential of drug candidates in order to exclude potent inhibitors from further development [6–8]
The extent of metabolic interaction between two drugs depends on their relative Kmand Kivalues and concentra-tions at the site of metabolism [3] In recent years, substantial progress has been made in the development
of in vitro screening methods to quantitatively determine kinetic parameters of CYP inhibition Using either recom-binant CYP proteins or liver microsomes, together with appropriate probe substrates, these assays can be used to measure Kivalues for competitive CYP inhibitors [7,9,10] It
is not entirely clear, however, whether these systems accurately and quantitatively reflect drug interactions that occur in vivo One possible drawback of recombinant enzymes is that inhibitory potency may depend on inter-actions with multiple CYPs present in the microsomal, but not recombinant, systems The intracellular concentration
of drugs (substrates and inhibitors)that is available for interacting with a particular CYP may also depend on
Correspondence to R Laufer, IRBM P Angeletti, Via Pontina km
30,600, 00040 Pomezia (Roma), Italy.
Fax: + 39 0691093 654, Tel.: + 39 0691093 440,
E-mail: ralph_laufer@merck.com
Abbreviation: CYP, cytochrome P450.
(Received 5 June 2003, revised 11 July 2003,
accepted 22 July 2003)
Trang 2processes lacking in microsomes, such as drug transport
across the plasma membrane, metabolism by cytosolic
enzymes, and binding to intracellular proteins Intact cells
more closely reflect the environment to which CYP
substrates and inhibitors are exposed in the liver, and it
may therefore be desirable to assess the potential of a drug
to cause CYP inhibition in isolated hepatocytes Isolated
hepatocytes have been used extensively to study drug
metabolism, cytotoxicity, and induction of
drug-metaboli-zing enzymes [11–15] However, there are few reports of
CYP inhibition studies using this system (see for example
[13,16–18]), probably because of the technical challenge
posed by the lower specific activity of CYP in cultured cells
relative to microsomal preparations
The objective of this study was to compare the inhibitory
potencies of CYP inhibitors in microsomes and hepatocytes
We developed an assay suitable for rapid analysis of
CYP-mediated drug interactions in both systems, using
radiolabelled dextromethorphan, a well-characterized probe
substrate for enzymes of the CYP2D family
Materials and methods
Materials
[O-methyl-14C]Dextromethorphan (61 mCiÆmmol)1)was
synthesized at Merck Research Laboratories, Rahway,
NJ, USA [3H]Quinine and [3H]quinidine were purchased
from American Radiolabeled Chemicals [3H]Taurocholic
acid was from Perkin–Elmer Life Sciences, and [14
C]for-maldehyde and [14C]formic acid were from Amersham
Biosciences Cell culture media were purchased from
Gibco-BRL, and chemicals from Sigma 96-well OasisTM HLB
extraction plates and vacuum mannifold were purchased
from Waters
Preparation of rat liver microsomes
Liver microsomes were prepared from male Sprague–
Dawley rats Livers were homogenized in 1.15% (w/v)
KCl, and the homogenate was centrifuged at 9000 g for
30 min The S-9 supernatant was centrifuged at 130 000 g
for 1 h The microsomal pellet was washed, resuspended in
0.15M Tris/HCl, pH 7.4, at a protein concentration of
10 mgÆmL)1and kept at)80 C
Isolation of rat hepatocytes
All animal care and experimental procedures were in
accordance with national and company guidelines Male
Sprague–Dawley rats weighing 250 g were subjected to
terminal anaesthesia using sodium pentobarbital Rat
hepatocytes were isolated by a two-step collagenase
per-fusion method [19] Cells were frozen in L15 medium
containing 10% fetal calf serum and 15% dimethyl sulfoxide
following the protocol described by Guyomard et al [20]
and kept in liquid nitrogen until use After quick thawing at
37C, cells were loaded on L15 medium containing 0.75M
glucose [21] and centrifuged for 1 min at 300 g Viable
hepatocytes were separated by centrifugation over 30%
Percoll solution for 3 min at 350 g Cell viability was
determined by Trypan Blue exclusion before freezing and
after thawing and was consistently greater than 90% The cells were resuspended in William’s Medium E containing GlutaMAXTM (Ala-Glu), 5 lgÆmL)1 insulin, 1 lM dexa-methasone, and penicillin/streptomycin, and seeded on collagen-precoated 24-well culture plates at a density of
100 000 cells per well Cultures were maintained at 37C in
a humidified atmosphere of 5% CO2 Four hours after plating, the medium was changed as described below Separation of [O-methyl-14
C]dextromethorphan from CYP2D-mediated demethylation products The CYP2D assay described in this study is based on a modification of procedures described previously for deter-mining the activity of various CYP isozymes, including CYP2D6, in hepatic microsomes [22,23] CYP-mediated demethylation of substrates which have the leaving methyl group radiolabelled with14C, yields [14C]formaldehyde as product, which can be isolated using reversed-phase (C8) extraction cartridges [24] We adapted this method to 96-well format, and modified the solid-phase matrix using Oasis extraction plates Solid-phase extraction was per-formed using a vacuum mannifold according to the instructions of the manufacturer When the radiolabelled substrate [O-methyl-14C]dextromethorphan, dissolved in either microsomal assay buffer or cell incubation medium, was applied to 96-well Oasis plates, over 99.7% of radioactivity was retained on the extraction plate, and could be recovered by elution with methanol In contrast, [14C]formaldehyde and [14C]formic acid, the products of CYP-mediated oxidation of [O-methyl-14 C]dextromethor-phan, were quantitatively recovered in the combined void volume and aqueous washing of Oasis extraction plates Microsomal CYP2D assays
Microsomal incubations were performed in 96-well conical plates (Corning) They contained, in a final volume of
100 lL, 0.1Mpotassium phosphate buffer, pH 7.4, 1 lM
[O-methyl-14C]dextromethorphan ( 15 000 d.p.m per assay), rat liver microsomes (3 lg), and NADPH-regenerating system (1 mM NADP, 5 mM glucose-6-phosphate, 3 mM MgCl2, 4 UÆmL)1 glucose-6-phosphate dehydrogenase) After preincubation for 10 min at 37C in the presence or absence of test compounds, reactions were started by the addition of the NADPH-regenerating system After 15 min, reactions were stopped by the addition of
10 lL 1MHCl Plates were centrifuged at 1100 g for 5 min using a microplate rotor, and supernatants loaded on 30-mg 96-well Waters Oasis extraction plates The flow-through was collected and plates were washed twice with 200 lL water Aliquots of the combined aqueous eluates were counted in a Packard TopCount scintillation counter using 24-well scintillation plates Product formation was totally dependent on the presence of NADPH and was linear with time for up to 20 min, and with microsomal protein concentration up to 1 mgÆmL)1(data not shown)
Hepatocyte CYP2D assays CYP2D assays in hepatocytes were performed at 37C in
a humidified atmosphere of 5% CO in 24-well culture
Trang 3plates containing 100 000 cells per well, unless indicated
otherwise Four hours after plating, cells were incubated
in 500 lL cell incubation medium {hepatocyte culture
medium (HCM [25]), supplemented with ITS +
(Colla-borative Research, Bedford, MA, USA)and 10 mM
sodium formate, which suppresses the formation of
14CO2 from [14C]formate in rat hepatocytes [26]} Plates
were preincubated for 10 min with CYP inhibitors or
vehicle [0.5% (v/v)dimethyl sulfoxide], before addition of
1 lM [O-methyl-14C]dextromethorphan ( 80 000 d.p.m
per assay) Reactions were stopped after 15 min by
addition of 50 lL 1M HCl, and cell lysates were
centrifuged in a tabletop centrifuge at high speed for
10 min The supernatants were loaded on 30-mg 96-well
Waters Oasis extraction plates and processed as described
above for the microsomal assays, except that extraction
plates were washed three times with 250 lL water
Uptake of drugs into rat hepatocytes
Uptake of radiolabelled quinine, quinidine, and taurocholic
acid into rat hepatocytes was determined at 37C in 250 lL
per well of a solution containing 116 mM NaCl, 5.3 mM
KCl, 1.1 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2,
10 mM glucose, and 10 mM Hepes, pH 7.4 Some
experi-ments were performed in sodium-free buffer containing
choline chloride instead of NaCl Incubations with 5 lM
[3H]quinine or [3H]quinidine were carried out for 1, 2, 3, 5,
and 10 min in the presence or absence of 2 lM carbonyl
cyanide p-trifluoromethoxyphenylhydrazone Incubations
with 1 lM[3H]taurocholic acid were performed for 20, 40,
60, 120, and 300 s in the presence or absence of extracellular
Na+ Plates were then washed 3 times with 1 mL ice-cold
buffer, cells were lysed with 0.1MNaOH, and radioactivity
was determined by scintillation counting Cell-associated
radioactivity for [3H]quinine and [3H]quinidine reached
steady-state levels after 10 min (data not shown) Results
were corrected for radioactivity associated with cells at time
zero, and expressed as cell/medium concentration ratio
(C/M)at steady state, using an estimated intracellular
volume of 4 lLÆ(106cells))1[27] [3H]Taurocholate uptake
was linear for up to 2 min (data not shown) Uptake
clearance was calculated by dividing the initial
uptake velocity by the substrate concentration
Determination of drug binding to hepatic proteins
For the determination of the liver tissue binding of
[3H]quinine and [3H]quinidine, rat liver was homogenized
in 0.1Mpotassium phosphate buffer and dialyzed against
the same buffer for 12 h at 4C to remove coenzymes The
compounds were mixed with tissue homogenates (10, 20
and 30%, w/v)or rat liver microsomes (0.03 mgÆmL)1)at
concentrations of 1 or 10 lM, and incubated at 37C for
30 min Reaction tubes were then centrifuged in a tabletop
centrifuge for 20 min at high speed, and the supernatants
were loaded on Centrifree ultrafiltration devices (Millipore)
to separate the unbound fractions Non-specific adsorption
of [3H]quinine to the filters was prevented by precoating
using unlabelled quinine (1 mM) The fraction not bound to
liver proteins (fu)was calculated according to the following
equation [28]:
fu ¼ Cf=½Cfþ ð100=n CbÞ ð1Þ where Cfis unbound drug in ultrafiltrate, Cbis bound drug, and n is the percentage of liver homogenate Biochemical assays
Protein was determined by the Bradford assay (Bio-Rad) using BSA as standard Lactate dehydrogenase activity was determined in hepatocyte cell suspensions before plating, and in monolayers 4 h after plating, using a colorimetric method (Cytotoxicity detection kit; Roche Diagnostics) ATP content of cell monolayers was determined after cell extraction with 1.7% (w/v)trichloroacetic acid using luciferase/luciferin reagent (Sigma)and luminescent pro-duct detection The intracellular concentration of ATP was calculated considering an intracellular volume of
4 lLÆ(106cells))1[27]
Statistical methods Curve fitting was performed by nonlinear regression according to the Levenberg-Marquardt algorithm, using
KALEIDAGRAPHTM 3.52 (Synergy Software, Reading, PA, USA) Statistical significance was assessed using a two-tailed Student’s t test
Results
Viability, metabolic and transport activities
of cryopreserved rat hepatocytes
To assess the metabolic state of hepatocytes used in this study,
we determined cell-attachment efficiency, ATP content, and
Na+-dependent taurocholate transport, a typical differenti-ated hepatocyte function medidifferenti-ated by the sodium taurocho-late cotransporting polypeptide (NTCP)[29] The efficiency
of cell attachment, determined by measuring cellular lactate dehydrogenase activities before and after plating, was
70 ± 6% (n¼ 2) Intracellular ATP concentrations were 2.3 ± 0.4 mM (mean ± SEM, n¼ 3), which is in close agreement with previously reported values (2.4 mM [30]) Cells transported [14C]taurocholate with an uptake clearance
of 24 ± 2 lLÆmin)1per mg cellular protein (n¼ 2) In the absence of extracellular Na+, uptake clearance was reduced sevenfold These values are similar to those previously reported for Na+–taurocholate cotransport in rat hepato-cytes (Vmax/Km¼ 17.5 lLÆmin)1Æmg)1[29])
Dextromethorphan O-demethylation in rat hepatocytes and microsomes
When [O-methyl-14C]dextromethorphan was incubated with rat hepatocytes, radiolabelled reaction product(s)were produced in a time-dependent and cell-concentration-dependent manner (Fig 1) The reaction products were not retained by OasisTMpolymeric reversed-phase sorbent, similarly to standard [14C]formaldehyde and [14C]formate (and unlike the substrate [O-methyl-14 C]dextromethor-phan) Metabolite formation from [O-methyl-14 C]dextro-methorphan in rat hepatocytes increased with substrate concentration in a saturable manner (Fig 2A) The reaction
Trang 4rate as a function of substrate concentration was fitted to
the Hill equation:
v¼Vmax S
n
Sn
where v and Vmaxare the observed and maximal rates of
metabolism, S50 is the substrate concentration at
half Vmax, and n is the Hill coefficient The values
obtained were S50¼ 2.80 ± 0.01 lM, Vmax ¼ 0.11 ±
0.01 nmolÆmin)1 per mg cellular protein, and
n¼ 0.82 ± 0.01 An Eadie–Hofstee plot of these data
was monotonous, with slight deviation from linearity
(Fig 2A, inset)
For comparison, we also determined the kinetics of dextromethorphan O-demethylation in rat microsomes (Fig 2B) Fitted kinetic constants were S50¼ 2.10 ± 0.01 lM, Vmax¼ 0.74 ± 0.01 nmolÆmin)1 per mg micro-somal protein, and n¼ 0.88 ± 0.01 Also in this case, the Eadie–Hofstee plot of these data was monotonous, with slight deviation from linearity (Fig 2B, inset)
We next examined the effect of isoform-specific CYP inhibitors on dextromethorphan O-demethylation As shown in Fig 3, the reaction in rat hepatocytes was inhibited by quinine, which is a known inhibitor of rat CYP2D [31–33], but not by a-naphthoflavone (inhibitor of
Fig 1 Time-dependent and cell-concentration-dependent demethylation
of [O-methyl- 14 C]dextromethorphan in rat hepatocytes Substrate was
incubated with 100 000 cells (circles)or 300 000 cells (squares)and
product formation was determined at the indicated times Results are
mean ± deviation from duplicate experiments.
Fig 2 Kinetics of [O-methyl-14C]dextromethorphan demethylation in rat hepatocytes (A) and rat liver microsomes (B) Data were fitted to the Hill equation as described in Results Each point is the mean ± deviation from duplicate experiments Insets: Eadie–Hofstee plots of the data.
Fig 3 Effect of CYP inhibitors on [O-methyl-14C]dextromethorphan demethylase activity in rat hepatocytes Results are expressed as per-centage enzymatic activity relative to that of the vehicle control Inhibitors used were: 1 l M a-naphthoflavone (ANF), 10 l M sulfa-phenazole (SPZ), 10 l M quinine (QUIN), and 10 l M troleandomycin (TAO) Results are mean ± deviation from duplicate experiments.
Trang 5rat CYP1A1/2 [34]), sulfaphenazole (rat CYP2C11 [35]),
and troleandomycin (rat CYP3A [36]) The selected
inhi-bitor concentrations were based on the above literature
references
Effect of quinine and quinidine on dextromethorphan
O-demethylation
A characteristic feature of rat CYP2D enzymes is that, in
contrast with the human enzyme, they are inhibited by
quinine more potently than by quinidine [17,31,37] As
shown in Fig 4A, quinine was a more potent inhibitor than
quinidine of [O-methyl-14C]dextromethorphan
O-demethy-lation in rat hepatocytes Inhibition curves were fitted to a
four-parameter logistic model:
1þ ðx=IC50Þn ð3Þ where Y is the fraction of enzyme activity relative to
no-inhibitor controls, X is the concentration of
inhi-bitor, IC50 the concentration for half-maximal inhibi-tion, and n the slope factor The results of the fitting are summarized in Table 1 Quinine and quinidine had IC50
values of 0.9 and 4.7 lM, respectively The slope factors were 0.57 and 0.64, respectively, suggesting interaction with more than one enzyme or binding site
Inhibition curves were also fitted to a two-site inhibition model (Fig 4):
1þ ðx=IC50 1Þþ
1 A
1þ ðx=IC50 2Þ ð4Þ where Y is the fraction of enzyme activity relative to no-inhibitor controls, A is the fraction of enzymes with
IC50-1, and 1) A the fraction of enzymes with IC50-2 As shown in Table 1, correlation coefficients (r)for the nonlinear regression curve fits using the two-enzyme model were slightly higher than those for the logistic fits Approximately 40% of the enzymatic activity in rat hepatocytes was inhibited by quinine and quinidine with high affinity (IC50-10.06 and 0.51 lM, respectively), and
Fig 4 Effect of quinine and quinidine on [O-methyl- 14 C]dextromethorphan demethylase activity (A)Rat hepatocytes; (B)rat liver microsomes Enzymatic activity was determined in the presence of quinine (circles)or quinidine (squares), and results were expressed as percentage of control activity in the absence of inhibitor Data represent mean ± SEM from three to five separate experiments Curves were fitted to a two-site inhibition model as described in Results.
Table 1 Kinetic parameters for inhibition of [O-methyl-14C]dextromethorphan demethylation by quinine and quinidine in rat liver microsomes and rat hepatocytes Inhibition data (Fig 4)were fitted to a four-parameter logistic model or a two-site inhibition model as described in the text n, slope factor; A, fraction of high-affinity sites; IC 50 , concentration that produces 50% inhibition; IC 50-1 , IC 50 for high-affinity sites; IC 50-2 , IC 50 for low-affinity sites; r, correlation coefficient of the nonlinear regression curve fit Results are parameter values (± SEM), as calculated by the curve-fitting software.
Inhibitor
Enzyme
source
Fit type 4-parameter logistic 2 enzymes
Quinine Hepatocytes 0.9912 0.9 ± 0.16 0.57 ± 0.05 0.9981 0.40 ± 0.04 0.06 ± 0.02 5.0 ± 1.0 Quinidine Hepatocytes 0.9956 4.7 ± 0.51 0.64 ± 0.04 0.9980 0.41 ± 0.07 0.51 ± 0.18 19.0 ± 4.7 Quinine Microsomes 0.9954 1.7 ± 0.21 0.53 ± 0.03 0.9986 0.45 ± 0.03 0.13 ± 0.03 12.6 ± 2.1 Quinidine Microsomes 0.9980 15.0 ± 0.9 0.72 ± 0.03 0.9976 0.45 ± 0.14 3.3 ± 1.5 48.9 ± 20.4
Trang 6about 60% with lower affinity (IC50-2 5 and 19 lM,
respectively) Also in rat liver microsomes quinine had a
lower IC50than quinidine, and both compounds
exhi-bited slope factors smaller than unity (Fig 4B and
Table 1) The IC50values for quinine and quinidine in
rat liver microsomes were twofold and threefold higher
than in hepatocytes, but this difference was statistically
significant only for quinidine (P < 0.01) When data
were fitted to a two-site inhibition model, relative ratios
of high-affinity and low-affinity binding sites in rat liver
microsomes were similar to those in hepatocytes Also in
this case, correlation coefficients for the two-enzyme
model curve fits were slightly better than those for the
logistic fits (Table 1) IC50 values of quinine and
quinidine for both high-affinity and low-affinity binding
sites in rat liver microsomes were between twofold and
threefold higher than the corresponding values in rat
hepatocytes (Table 1), but these differences were not
statistically significant (P > 0.05)
Even though the differences in IC50 values between
microsomes and hepatocytes were small and for the most
part not significant, there appeared to be a trend towards
lower IC50values in intact hepatocytes This may be due to
differences between the concentrations of free drug available
for enzyme inhibition in the two systems To test this
hypothesis, we measured the total concentration of quinine
and quinidine in rat hepatocytes, as well as their free
(non-protein-bound)fractions in both microsomes and hepatic
tissue Both quinine and quinidine accumulated in rat
hepatocytes and reached steady-state concentrations that
were 64-fold and 75-fold higher than their extracellular
concentrations, respectively (Table 2) More than 50% of
the accumulation of quinine and quinidine was inhibited by
ATP depletion using 2 lM carbonyl cyanide
p-trifluoro-methoxyphenylhydrazone, suggesting that it was mediated
by active drug transport into the hepatocytes (data not
shown) Cell-associated drugs can bind to tissue proteins,
and only the free fraction may be available for interaction
with microsomal CYP2D Radiolabelled quinine and
quinidine bound extensively to proteins in rat liver
homo-genates, with free fractions between 0.03 and 0.06 (Table 2)
In contrast, free fractions of both compounds were close to
unity in the rat liver microsome incubation system,
consis-tent with the very low concentration of microsomal protein
(30 lgÆmL)1)used in the assay (data not shown) Thus, free concentrations of quinine and quinidine inside rat hepato-cytes may not equal those in the extracellular medium, and
IC50should be corrected by a factor that takes into account cellular accumulation and protein binding The ratio between the intracellular concentration of free drug ([I]cell, free)and that of total drug added to the hepatocyte culture medium ([I]medium)is given by:
½Icell;free
½Imedium¼ fu;cellxC=M ð5Þ However, fu,cell, the free fraction of drug within the hepatocyte cytoplasm, cannot be measured experiment-ally If this value were similar to the free fraction in liver homogenate (i.e fu,cell¼ fu,tissue), then free drug concentrations inside hepatocytes would be 2–3-fold higher than that added to the culture medium
Inhibition of CYP2D activity
We next investigated the effects of several drugs on [O-methyl-14C]dextromethorphan O-demethylation in rat microsomes and hepatocytes As depicted in Fig 5A and summarized in Table 3, pyrilamine, propafenone, terfena-dine, verapamil and ketoconazole inhibited the reaction in intact hepatocytes with IC50values in the micromolar range Slope factors (determined by fitting the data to a four-parameter logistic equation)ranged from 0.5 (pyrilamine)
to 1.1 (propafenone) In rat liver microsomes, IC50values for pyrilamine, propafenone and verapamil were 2–3-fold higher than in hepatocytes, ketoconazole had comparable
IC50 values, and terfenadine a slightly lower IC50than in hepatocytes (Fig 5B and Table 3) This difference between microsomes and hepatocytes was statistically significant only for verapamil (P < 0.01) Slope factors for all compounds were very similar to those obtained in hepatocytes (Table 3) It was not possible to resolve the curves for these compounds into two distinct components using a two-site inhibition model (data not shown)
Discussion
Even though hepatic microsomes represent the most widely used in vitro system for the study of potential drug interactions, it has been reported that concentrative uptake
of some CYP inhibitors into the liver can cause drug interactions in vivo that are more pronounced than those predicted by inhibitory potency in a microsomal system [5,28,38,39] Freshly isolated and cryopreserved hepatocytes are an important experimental tool for the evaluation of drug metabolism, hepatotoxicity and induction of drug-metabolizing enzymes [11–15] The purpose of the present study was to use this system to determine the inhibitory potencies of a series of CYP2D inhibitors and to compare the results with those obtained in liver microsomes To this end, we developed a sensitive assay method suitable for rapidly assessing the potential of chemical compounds to inhibit CYP2D enzymes in both systems
CYP-catalyzed demethylation of substrates which had the leaving methyl group radiolabelled with 14C, yielding [14C]formaldehyde as product, has been previously used
Table 2 Accumulation in rat hepatocytes and hepatic protein binding
of quinine and quinidine Accumulation of quinine and quinidine (5 l M )
in rat hepatocytes is expressed as the steady state ratio (C/M)between
cell associated and extracellular drug concentrations Binding to rat
hepatic proteins was determined at two drug concentrations, 1
and 10 l M , and results were expressed as fraction of free drug, f u
To calculate f u · C/M, the f u for the two drug concentrations was
averaged.
Compound C/M
f u
f u · C/M
1 l M 10 l M
Quinine 64 0.037 0.056 3.0
Quinidine 75 0.028 0.031 2.2
Trang 7to assay the activity of various CYP isoforms in liver
microsomes [23,24,40,41] A related method is used to
determine CYP3A4 activity in human subjects in vivo The
so-called erythromycin breath test measures the disposition
in the breath of14CO2formed from further oxidation of
[14C]formaldehyde, the product of CYP3A4-catalyzed
N-demethylation of [N-methyl-14C]erythromycin [42] Even
though formation of 14C-labelled formaldehyde, formate
and CO2from CYP-mediated N-demethylation of
amino-pyrine in isolated hepatocytes was described over 25 years
ago [43], aminopyrine is not suitable as a substrate for
assaying the activity of specific CYPs, because its
N-demethylation is mediated by multiple CYP isozymes
[44] In contrast, [O-methyl-14C]dextromethorphan can be
used to specifically determine CYP2D activities in rat
hepatocytes The present experiments using
isoform-select-ive CYP inhibitors show that the demethylation of
[O-methyl-14C]dextromethorphan in rat hepatocytes was
mediated by enzymes of the CYP2D family The reaction
was inhibited by the CYP2D inhibitor quinine but not
by specific inhibitors of rat CYP1A1/2, CYP2C11 and
CYP3A In addition, the more potent inhibition by quinine relative to quinidine is a characteristic feature of rat CYP2D enzymes
Except for CYP1A and CYP2B, for which cell-based fluorimetric assays have been reported [45,46], non-HPLC assays suitable for determining CYP inhibition in intact hepatocytes have not been described to date The present method can be used for relatively high throughput screening
of CYP2D inhibitors, because of the possibility of carrying out reactions using as few as 100 000 cells attached to the wells of 24-well culture plates and processing the reaction products in 96-well solid-phase extraction plates Compared with conventional methods for measuring CYP activity in intact hepatocytes, which entail preparation of microsomes and HPLC separation of reaction products, the new CYP2D assay procedure described here has the advantage
of greatly improved simplicity, speed and sensitivity The latter factor is particularly important for assessing CYP inhibition, because competitive inhibition assays should be performed using substrate concentrations that are not much higher than the K The concentration of dextromethorphan
Table 3 Kinetic parameters for inhibition of [O-methyl- 14 C]dextromethorphan demethylation by CYP2D inhibitors in rat liver microsomes and rat hepatocytes Inhibition data (Fig 5)were fitted to a four-parameter logistic model n, slope factor; IC 50 , concentration that produces 50% inhibition Results are parameter values (± SEM), as calculated by the curve-fitting software.
Inhibitor
Hepatocytes Microsomes Hepatocytes Microsomes
Ketoconazole 0.7 ± 0.1 0.6 ± 0.1 0.69 ± 0.05 0.83 ± 0.09
Fig 5 Effect of CYP2D inhibitors on [O-methyl-14C]dextromethorphan demethylase activity (A)Rat hepatocytes; (B)rat liver microsomes Enzymatic activity was determined in the presence of pyrilamine (d), propafenone (j) , verapamil (m), ketoconazole (s), or terfenadine (h) Results were expressed as percentage of control activity in the absence of inhibitor Data represent mean ± SEM from three to four experiments Curves were fitted to a four-parameter logistic inhibition model as described in Results.
Trang 8(1 lM)used in the present hepatocyte assay fulfils this
requirement Even though we validated the assay for rat
CYP2D only, the general method of measuring the
radiolabelled products of CYP-mediated dealkylation
reac-tions should be easily adaptable to other CYP isoforms and
hepatocytes of other species, including humans, using
appropriate probe substrates, such as [O-ethyl-14
C]phenace-tin [40], [O-methyl-14C]naproxen [24], [N-methyl-14
C]eryth-romycin [23,41] and [N-methyl-14C]diazepam [24]
Dextromethorphan O-demethylation in rat liver
micro-somes and hepatocytes has previously been studied using
nonradiometric methods, and it was reported that this
reaction is mediated by multiple enzyme systems In rat liver
microsomes, O-demethylation of unlabelled
dextromethor-phan is mediated by high-affinity and low-affinity enzyme
systems, with apparent Kmvalues of 1–3 lMand 43–158 lM,
respectively [18,37] In rat hepatocytes, O-demethylation of
unlabelled dextromethorphan was reported to display
sigmoidal kinetics, with an S50value of 13 lMand a Hill
coefficient of 2.4 The rat CYP2D family comprises six
members, denominated CYP2D1-5 and CYP2D18 [47,48]
Dextromethorphan O-demethylation is catalyzed by
cDNA-expressed rat CYP2D2 but not CYP2D1 [49]
Indirect evidence suggests that other CYP2D isoforms can
catalyze this reaction Dextromethorphan interacts with
multiple CYP2D isoforms, as it was shown to inhibit the
metabolism of 7-methoxy-4-(aminomethyl)coumarin by
CYP2D1, CYP2D2, CYP2D3 and CYP2D4 with IC50
values of 264, 5.6, 18.6, and 136 lM, respectively [50] These
results suggest that the high-affinity component of the
reaction is mediated by CYP2D2 with a possible
contribu-tion of CYP2D3, while the low-affinity component may be
mediated by CYP2D1 and/or CYP2D4 In this study,
[O-methyl-14C]dextromethorphan demethylation in rat liver
microsomes and hepatocytes exhibited apparent Kmvalues
of 2.1 and 2.8 lM, and Vmaxvalues of 0.74 nmolÆmin)1per
mg microsomal protein vs 0.11 nmolÆmin)1per mg cellular
protein, respectively, with Hill coefficients close to unity and
Eadie–Hofstee plots that deviated only slightly from
linearity The apparent microsomal Km and Vmax values
are comparable to those previously reported [18,37] for the
high-affinity component of dextromethorphan
O-demethy-lation in rat liver microsomes (Km¼ 1.1–2.5 lM;
Vmax¼ 0.42–0.85 nmolÆmin)1Æmg)1) It is likely that
mark-edly biphasic kinetics were not observed in the present
experiments because the [O-methyl-14C]dextromethorphan
concentrations used did not exceed 25 lM(microsomes)and
100 lM(hepatocytes), which is close to the apparent Kmof
the low-affinity component reported in rat liver microsomes
In contrast, kinetic studies with unlabelled
dextromethor-phan were performed using substrate concentrations up to
500–600 lM [18,37] Thus, under the present conditions,
[O-methyl-14C]dextromethorphan O-demethylation was
pri-marily mediated by CYP2D isoforms with high substrate
affinity
The hypothesis that [O-methyl-14C]dextromethorphan
O-demethylation in rat liver microsomes and hepatocytes
is mediated by high-affinity CYP2D isoforms including
CYP2D2 and possibly CYP2D3 is supported by the
inhibition profile of quinine and quinidine In both
micro-somes and hepatocytes, these compounds inhibited the
reaction in a biphasic manner, suggesting interaction with at
least two enzyme systems Curve fitting to a logistic model
or to a two-site model produced excellent fits with correlation coefficients close to unity We preferred to analyze the data according to the two-site model for the following reasons Slope factors for the logistic fits were significantly smaller than 1, suggesting interaction with multiple enzyme systems, or allosteric behaviour Individual rat CYP2D isoforms display Michaelis–Menten kinetics with the substrate 7-methoxy-4-(aminomethyl)coumarin [50], and to our knowledge, allosteric kinetics has not been reported for other ligands On the other hand, it is well known that dextromethorphan can interact with multiple CYP2D isoforms [50], and the observed biphasic inhibition kinetics most likely reflect this property The higher-affinity component for quinine displayed IC50 values of 0.13 lM
(microsomes)and 0.06 lM (hepatocytes), whereas the lower-affinity component had IC50 values of 12.6 lM
(microsomes)and 5.0 lM (hepatocytes) These values are close to the reported IC50values of quinine for inhibition of CYP2D2-mediated and CYP2D3-mediated dealkylation of 7-methoxy-4-(aminomethyl)coumarin, 0.09 and 12.0 lM, respectively [50] The high-affinity and low-affinity compo-nents of quinidine inhibition of [O-methyl-14 C]dextrometh-orphan O-demethylation displayed IC50 values of 3.3 lM
(microsomes)and 0.51 lM (hepatocytes), vs 48.9 lM
(microsomes)and 19.0 lM (hepatocytes), respectively Again, these values are similar to the reported IC50values for inhibition of CYP2D2-mediated and CYP2D3-medi-ated dealkylation of 7-methoxy-4-(aminomethyl)coumarin, 2.8 and 26.9 lM, respectively
The effect of several additional drugs on [O-methyl-14C]dextromethorphan demethylase activity was assessed in both rat liver microsomes and hepatocytes Pyrilamine [51], propafenone [37] and terfenadine [52] are known to be potent rat and/or human CYP2D inhibitors, whereas verapamil was reported to be a weak (IC5060 lM) inhibitor of human CYP2D6 [53] Even though ketocon-azole has not been reported to inhibit CYP2D isoforms, it is known to be a nonspecific inhibitor of various rat CYPs, including CYP1A, CYP2C, CYP2E and CYP3A [54] Even though some of these compounds inhibited the reaction with slope factors significantly lower than 1, suggesting inter-action with more than one enzyme, the relative contribu-tions of distinct enzymatic systems could not be resolved by curve fitting Additional studies, using cDNA-expressed rat CYP2Ds will be needed to determine the interactions of these compounds with specific isoforms In general, there was reasonable agreement between IC50values determined
in microsomes vs hepatocytes Some inhibitors, including quinine and quinidine, displayed 2–3-fold lower IC50values
in hepatocytes than in microsomes, but this difference was statistically significant only for quinidine and verapamil One possible explanation for this trend is that some of the drugs accumulate to a moderate extent in hepatocytes We found that cell-associated concentrations of quinine and quinidine were about 70-fold higher than extracellular concentrations However, part of the cell-associated drug
is probably bound to intracellular proteins and may thus not
be available for interaction with CYP2D Both compounds were found to bind extensively to proteins in homogenates from rat liver Even though intracellular protein binding may be different from that observed in tissue homogenates,
Trang 9it is interesting to note that the cell/medium concentration
ratio, corrected for tissue protein binding, is between 2 and
3, i.e strikingly similar to some of the observed ratios
between IC50 values in microsomes vs hepatocytes In
conclusion, for the CYP inhibitors investigated in this study,
only slight differences in inhibitory potencies were observed
between intact hepatocytes and liver microsomes Even
though some drugs can reach high intrahepatic
concentra-tions [5], this effect may be partially offset by binding to
intracellular proteins Further studies are required to
determine whether, for compounds with important liver
uptake and low hepatic protein binding, hepatocyte IC50
values may provide more accurate predictions of in vivo drug
interactions than data obtained in microsomes
Acknowledgements
We thank Isabelle Gloaguen and Laura Rehak for technical assistance,
and Dr Ashok Chaudhary (Drug Metabolism, Merck Research
Laboratories, Rahway, NJ, USA)for his assistance in the preparation
of radiolabelled dextromethorphan.
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