Relative contribution of rat cytochrome P450 isoforms to the metabolism of caffeine: The pathway and concentration dependence

Article abstract of DOI:10.1016/j.bcp.2007.12.017

The aim of the present study was to estimate the relative contribution of rat P450 isoforms to the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance for estimating the activity of P450 in rat liver and its potential for

Full text of DOI:10.1016/j.bcp.2007.12.017

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biochempharm
Relative contribution of rat cytochrome P450 isoforms
to the metabolism of caffeine: The pathway and
concentration dependence
*
Marta Kot, Władysława A. Daniel
Department of Pharmacokinetics and Drug Metabolism, Institute of Pharmacology, Polish Academy of Sciences,
Sme˛tna 12, PL 31-343 Krako´w, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of the present study was to estimate the relative contribution of rat P450 isoforms to
the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance
for estimating the activity of P450 in rat liver and its potential for pharmacokinetic inter-
actions in pharmacological experiments. The results obtained using rat cDNA-expressed
P450s indicated that 8-hydroxylation was the main oxidation pathway of caffeine (70%) in
the rat. CYP1A2 was found to be a key enzyme catalyzing 8-hydroxylation (72%) and
substantially contributing to 3-N-demethylation (47%) and 1-N-demethylation (37.5%) at
a caffeine concentration of 0.1 mM (relevant to ‘‘the maximum therapeutic concentration in
humans’’). Furthermore, CYP2C11 considerably contributed to 3-N-demethylation (31%).
The CYP2C subfamily (66%) – mainly CYP2C6 (27%) and CYP2C11 (29%) – played a major role
in catalyzing 7-N-demethylation. At higher substrate concentrations, the contribution of
CYP1A2 to the metabolism of caffeine decreased in favor of CYP2C11 (N-demethylations)
and CYP3A2 (mainly 8-hydroxylation). The obtained results were confirmed with liver
microsomes (inhibition and correlation studies). Therefore, caffeine may be used as a
marker substance for assessing the activity of CYP1A2 in rats, using 8-hydroxylation (but
not 3-N-demethylation—like in humans); moreover, caffeine may also be used to simulta-
neously, preliminarily estimate the activity of CYP2C using 7-N-demethylation as a marker
reaction. Hence caffeine pharmacokinetics in rats may be changed by drugs affecting the
activity of CYP1A2 and/or CYP2C, e.g. by some antidepressants.
Received 8 November 2007
Received in revised form
19 December 2007
Accepted 21 December 2007
Keywords:
Caffeine metabolism
Rat cytochrome P450
cDNA-expressed isoforms
Liver microsomes
Cytochrome P450 inhibitors
Correlation analysis
# 2008 Elsevier Inc. All rights reserved.
1.
Introduction
caffeine increases the release of various neurotransmitters,
and at higher concentrations inhibits phosphodiesterase and
Caffeine (1,3,7-trimethylxanthine), a purine alkaloid, is the
most universally used psychoactive substance as a compo-
nent of coffee, tea and numerous drugs. It has multiple
pharmacological effects. As an adenosine receptor antagonist,
GABA receptors [1]. Due to its ability to interact with
neurotransmission in different regions of the brain, caffeine
displays psychomotor stimulant properties, promoting beha-
vioral functions such as vigilance, attention, mood and
* Corresponding author. Tel.: +48 12 6374022; fax: +48 12 6374500.
E-mail address: nfdaniel@cyf-kr.edu.pl (W.A. Daniel).
Abbreviations: CYP; P450, cytochrome P450; FMO, flavin-containing monooxygenase; HPLC, high performance liquid chromatography;
aNF, a-naphthoflavone; SULF, sulfaphenazole; CIM, cimetidine; QUIN, quinine; trans-DCE, trans-1,2-dichloroethylene; ERY, erythromycin;
Km, the Michaelis constant; Vmax, maximum velocity of the reaction.
0006-2952/$ – see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2007.12.017

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
1539
arousal [2]. In recent years caffeine and newly synthesized
xanthine derivatives have been investigated as adenosine
receptor antagonists and neuroprotective drugs in animal
models of neurodegenerative diseases in vivo [3–6]. Since the
disposition of caffeine in vivo depends on cytochrome P450,
mutual drug interactions between caffeine and other centrally
acting drugs at both the pharmacodynamic and pharmaco-
kinetic level are feasible [7].
in rats [15]. Our results concerning the influence of psycho-
tropic drugs on the metabolism of caffeine also indicate that
more than one enzyme contributes to the oxidative metabo-
lism of caffeine in the rat [16-17]; moreover, recent studies
with selective P450 inducers suggest that CYP2C11 may be
engaged in the 7-N-demethylation of caffeine in this species
[18].
Although a number of studies have indicated that phase I of
caffeine metabolism in the liver may proceed via a few
cytochrome P450 isoforms, their identification and relative
quantitative contribution to the specific metabolic pathways
of the test drug has not been precisely estimated as yet,
especially in laboratory rodents. So far, most studies into the
involvement of P450s in the metabolism of caffeine in rats
have been carried out using high (1–10 mM) substrate
concentrations, predominantly unspecific P450 inducers or
inhibitors [11,19], often not including all P450 isoforms [12,20]
or all caffeine metabolic reactions [19]. Therefore it was
difficult to identify all the P450 isoforms involved in caffeine
metabolism; furthermore, quantitative estimation of the
contribution of individual P450s to the four oxidative meta-
bolic reactions of caffeine at concentrations relevant to those
found in humans was practically impossible. According to
the literature, the concentration value of ca. 100 mM seems to
be the highest encountered in the clinic [21,22] and may be
assumed as ‘‘the maximum therapeutic concentration in
humans’’. Pharmacokinetic studies showed that caffeine
concentrations found in the blood plasma of coffee drinkers
were below 100 mM (e.g. 10–31 mM after consumption of 2–6
cups of coffee [23,24].
On the other hand, due to its natural character, caffeine is
used as a marker substance for estimating phenotypes with
regard to the activity of CYP1A2 in man. 3-N-Demethylation
to paraxanthine is regarded as
a reaction specifically
catalyzed by CYP1A2 in humans [8–10]. However, it remains
to be ascertained whether this reaction may also be used for
testing the activity of CYP1A2 in laboratory rodents, since no
detailed study on the contribution of individual P450
isoforms to the metabolism of caffeine has been carried
out to date.
Interestingly, caffeine is oxidized in a few positions of its
structure—apart from its 3-N-demethylation to paraxanthine
catalyzed by CYP1A2, the compound undergoes 1-N-demethy-
lation, 7-N-demethylation and 8-hydroxylation (to theobro-
mine, theophylline and 1,3,7-trimethyluric acid, respectively),
(Fig. 1). While in man 3-N-demethylation is quantitatively the
main oxidation pathway, in the rat 8-hydroxylation seems to
be a dominant metabolic reaction [8,11,12]. Some literature
data suggest that other oxidation pathways of caffeine may be
mediated by P450 isoforms different to CYP1A2, both in
humans [9,13,14] and rats [8,11,12,15]. It seems that the CYP3A
subfamily is the main isoenzyme catalyzing 8-hydroxylation
to 1,3,7-trimethyluric acid [9,12]; however, some other data
also suggest the contribution of CYP2B1 and CYP2E1 to the
catalyzing of this reaction in the rat [11]. It has also been
suggested that 1-N-demethylation and mainly 7-N-demethy-
lation, which lead to the formation of theobromine and
theophylline, respectively, are likely to engage CYP2B isoforms
Since the latest achievements in biotechnology have
facilitated the obtaining and availability of cDNA-expressed
P450s, which at present constitute a modern model for testing
drug metabolism, it was possible to carry out systematic
studies into caffeine metabolism, including all individual rat
P450s and the four oxidative metabolic reactions of caffeine.
Fig. 1 – The main metabolic pathways of caffeine.

1540
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
The aim of the present study was to qualitatively and
untreated and trans-DCE-treated rats at the substrate con-
centration of 100 mM.
quantitatively estimate the contribution of P450 isoforms to
the metabolism of caffeine at different drug concentrations in
rat liver and, on the basis of the obtained results, to assess the
usefulness of caffeine as a marker substance for estimating
the activity of P450 and its potential for pharmacokinetic
interactions in pharmacological experiments with rats. To this
aim, rat cDNA-expressed P450s were used, and the obtained
results were confirmed with liver microsomes (inhibition and
correlation studies).
Incubations were carried out in a system containing the
liver microsomes (ca. 1 mg of protein/ml), a phosphate buffer
(0.15 M, pH 7.4), MgCl2Á6H2O (6 mM), NADP (1.2 mM), DL-
isocitric acid (6 mM) and isocitric dehydrogenase (1.2 U/ml).
The final incubation volume was 1 ml. After a 3-min (with a-
naphthoflavone), a 15-min (with cimetidine) or a 20-min (with
erythromycin) preincubation at 37 8C, the reaction was
initiated by adding caffeine (the other inhibitors were not
preincubated). After a 50-min incubation, the reaction was
terminated by adding 700 ml of a 2% ZnSO4 and 50 ml of 2 M HCl.
To verify possible involvement of flavin-containing mono-
oxygenase (FMO) in caffeine metabolism, thermal inactivation
of FMO was applied [26,27]. To this end, control liver micro-
somes were preincubated with or without NADPH at 50 8C for
1.5 min before incubating the complete reaction mixture
containing NADPH and caffeine (100 mM) at 37 8C. Caffeine
and its metaboliteswere analyzedbya high-performance liquid
chromatography method (HPLC) described below.
2.
Materials and methods
2.1.
Drugs and chemicals
Caffeine and its metabolites—theobromine, paraxanthine,
theophylline and 1,3,7-trimethyluric acid, phenacetin, as well
as a-naphthoflavone, sulfaphenazole, cimetidine, quinine,
trans-1,2-dichloroethylene, erythromycin, NADP, DL-isocitric
acid (trisodium salt), isocitric dehydrogenase, NADPH were
purchased from Sigma (St. Louis, USA). Acetamidophenol,
MgCl2Á6H2O and all organic solvents with HPLC purity were
supplied by Merck (Darmstadt, Germany).
2.4.
Correlation analysis of the data
Due to the dose-dependent contribution of the P450 isoforms
studied to the oxidative metabolism of caffeine, a correlation
study was carried out at a caffeine concentration of 100 mM
(relevant to ‘‘the maximum therapeutic concentration in
humans’’) and 800 mM (relevant to the Km value for 3-N-
demethylation in rat liver microsomes). The rates of caffeine
1-N-, 3-N- and 7-N-demethylation and 8-hydroxylation were
correlated with the activity of P450 isoforms in liver micro-
somes.
2.2.
Animal procedures
All the experiments with animals were performed in accor-
dance with the Polish governmental regulations (Animals
Protection Act, DZ.U. 97.111.724, 1997). The experiments were
carried out on male Wistar rats (230–260 g) kept under
standard laboratory conditions. Liver microsomes were pre-
pared by differential centrifugation in 20 mM Tris/KCl buffer
(pH 7.4), including washing with 0.15 M KCl according to a
conventional method.
The activity of CYP2C6 was studied by measuring the rate of
warfarin 7-hydroxylation at the substrate concentration of
60 mM as described previously [28]. The concentrations of
warfarin and its metabolite 7-hydroxywarfarin formed in liver
microsomes were assessed by the HPLC method of Lang and
Bocker [29]. The activities of CYP2A2, CYP2B, CYP2C11 and
CYP3A2 were studied by measuring the rate of P450-specific
reactions: the 7a-, 16b-, 2a- and 2b-hydroxylation of testoster-
one (respectively) at the substrate concentration of 100 mM as
described previously [30]. The concentrations of testosterone
and its metabolites formed in liver microsomes were assessed
by the HPLC method of Sonderfan et al. [31]. The activity of
CYP1A2 was studied by measuring the rate of phenacetin O-
deethylation to acetamidophenol according to the method of
Eagling et al. [32] at the substrate concentration of 10 mM [33].
The CYP2E1 inhibitor trans-1,2-dichloroethylene (trans-
DCE) was administered intraperitoneally in
a dose of
100 mg/kg to rats [25]. Control animals were injected with
the same volume of water. After 5 h, untreated and trans-DCE-
treated rats were sacrificed, and their livers were removed and
homogenized. Liver microsomes were prepared as described
above.
2.3.
Caffeine metabolism in liver microsomes
Studies into caffeine metabolism in liver microsomes were
carried out at the linear dependence of product formation on
time, protein and substrate concentration. For determining
enzyme kinetics (Eadie–Hofstee plots), the caffeine concen-
trations used ranged from 0.1 to 2.8 mM. For inhibition studies,
the concentrations of caffeine were from 50 to 400 mM.
Caffeine was incubated with liver microsomes in the absence
or presence of one of the selective inhibitors added in vitro:
0.025–0.1 mM a-naphthoflavone (a CYP1A2 inhibitor), 0.5–
5.0 mM sulfaphenazole (a CYP2C6 inhibitor), 25–100 mM cime-
tidine (a CYP2C6 + CYP2C11 inhibitor), 1.25–10 mM quinine (a
CYP2D inhibitor), 50–400 mM erythromycin (a CYP3A2 inhibi-
tor). The CYP2E1 inhibitor trans-1,2-dichloroethylene (trans-
DCE) was administered intraperitoneally as described above.
Caffeine metabolism was studied in the liver microsomes of
Each pair of data was compared by
a simple linear
regression analysis using the statistical Prism 2.01 program
(GraphPad Software Inc., San Diego, CA).
2.5.
cDNA-expressed rat P450s
Microsomes from baculovirus-infected insect cells expressing
CYP1A1,CYP1A2, CYP2A2, CYP2B1, CYP2C6, CYP2C11,CYP2C13,
CYP2D1, CYP2D2, CYP2E1 and CYP3A2 co-expressed with
NADPH P450 oxidoreductase (Supersomes) were obtained from
Gentest Co. (Woburn, MA, USA). CYP2A2, CYP2B1, CYP2C6,
CYP2C13, CYP2E1 and CYP3A2 were also co-expressed with b5.
Microsomal protein expressing NADPH P450 oxidoreductase

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
1541
and cytochrome b5 was used as a control. Studies into caffeine
metabolism in the Supersomes were carried out at the linear
dependence of product formation on time, the amount of P450
isoform and substrate concentration. Caffeine metabolism was
studied under experimental conditions similar to those
described for liver microsomes, using 100, 200, 400, 800 and
1600 mM caffeine, except for the factthat the finalconcentration
and incubation time of P450s was 100 pmol/ml and 50 min for
CYP1A1, 50 pmol/ml and 30 min for CYP1A2, 60 pmol/ml and
30 min for CYP2A2, 70 pmol/ml and 30 min for CYP2B1,
50 pmol/ml and 60 min for CYP2C6, 60 pmol/ml and 30 min
for CYP2C11, 70 pmol/ml and 30 min for CYP2C13, 60 pmol/ml
and 20 min for CYP2D1, 60 pmol/ml and 45 min for CYP2D2,
100 pmol/ml and 60 min for CYP2E1, 50 pmol/ml and 15 min for
CYP3A2.
7000 System Manager was used. The analytical column
(Supelcosil LC-18, 15 cm  4.6 mm, 5 mm) was from Supelco
(Bellefonte, USA). The mobile phase consisted of 0.01 M
acetate buffer (pH 3.5) and methanol (91:9, v/v). The flow rate
was 1 ml/min (0–26.5 min), followed by 3 ml/min (26.6–
35 min). The column temperature was maintained at 30 8C.
The absorbance of caffeine and its metabolites was measured
at a wavelength of 254 nm. The compounds were eluted in the
following order: theobromine (9.7 min), paraxanthine
(15.8 min), theophylline (16.9 min), 1,3,7-trimethyluric acid
(23.4 min), caffeine (30.5 min). The sensitivity of the method
allowed for quantification of theobromine as low as
0.001 nmol, paraxanthine as low as 0.004 nmol, theophylline
as low as 0.005 nmol, 1,3,7-trimethyluric acid as low as
0.01 nmol and caffeine as low as 0.005 nmol in one sample.
The accurracy of the method amounted to 1.2% (para-
xanthine), 1.3% (theophylline), 2.1% (theobromine), 2.3%
(1,3,7-trimethyluric acid) and 2.9% (caffeine). The intra- and
inter-assay coefficients of variance were below 4 and 6%,
respectively.
2.6.
Determination of caffeine and its metabolites
Caffeine and its four primary metabolites were assessed using
the HPLC method based on Rasmussen et al. [34] as previously
described [16]. Briefly, after incubation, samples were cen-
trifuged and the water phase containing caffeine and its
metabolites was extracted with 6 ml of an organic mixture
consisting of ethyl acetate and 2-propanol (8:1, v/v). The
residue obtained after evaporation of the microsomal extract
was dissolved in 100 ml of the mobile phase described below.
An aliquot of 20 ml was injected into the HPLC system. The
Merck-Hitachi chromatograph, ‘‘LaChrom’’ (Darmstadt, Ger-
many), equipped with a L-7100 pump, an UV detector and a D-
3.
Results
3.1.
Caffeine metabolism in rat liver microsomes
Fig. 2A–D shows the Eadie–Hofstee plots for caffeine oxidation
processes in the pooled liver microsomes of five rats. All the
plots were non-linear indicating for the multiple-enzyme
Fig. 2 – Eadie–Hofstee plots for caffeine 1-N-demethylation (A), 3-N-demethylation (B), 7-N-demethylation (C), and
8-hydroxylation (D) in rat liver microsomes. Pooled liver microsomes of five control rat were incubated in a 0.15 M
phosphate buffer (pH 7.4) with caffeine (0.1À2.8 mM), 6 mM MgCl2Á6H2O and the NADPH generating system (1.2 mM NADP,
6 mM DL-isocitric acid, 1.2 U/ml isocitric dehydrogenase) for 50 min.

1542
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
Table 1 – Kinetic parameters of caffeine metabolism in cDNA-expressed rat CYPs (Supersomes)
P450
1-N-Demethylation
3-N-demethylation
7-N-Demethylation
C-8-hydroxylation
(theobromine)
(paraxanthine)
(theophylline)
(1,3,7-trimethyluric acid)
aKm
bVmax Vmax/Km aKm
bVmax Vmax/Km
aKm
bVmax Vmax/Km aKm
bVmax Vmax/Km
1A1 (not constitutive)
0.214
0.121
0.137
0.080
0.547
18.735
0.333
1.184
1.230
0.291
0.484
0.117
0.804
0.163
0.072
0.102
1.828
0.129
0.207
0.068
0.187
0.198
0.547
6.645
1.190
0.900
0.186
0.097
0.387
0.175
0.055
0.643
0.409
0.171
0.074
0.837
0.675
0.470
2.559
0.919
1.506
0.277
5.405
5.555
0.288
0.791
0.146
0.082
0.062
1.038
0.075
0.315
0.048
0.596
1.519
1.684
10.689
0.174
0.121
0.132
0.406
0.082
0.209
0.173
0.110
0.273
5.029
0.429
1.433
6.521
1.558
10.260
1.160
1.397
0.592
5.305
4.050
0.423
0.675
0.669
1.100
1.438
7.725
0.310
0.740
0.389
0.987
2.120
0.084
1.573
0.467
0.169
0.923
0.753
0.267
0.530
0.657
0.186
0.523
0.158
0.091
0.806
0.338
0.751
1.189
0.720
0.096
0.174
5.750
5.800
1.094
9.427
0.751
0.154
0.934
0.301
0.052
0.281
0.265
0.625
52.100
6.924
103.593
0.932
1A2
2A2
2B1
0.456
2C6
2C11
2C13
2D1
2D2
2E1
1.244
0.253
0.072
2.930
1.523
0.109
3A2
8.983
aKm (mM), bVmax (pmol/pmol P450 isoform/min). The highest intrinsic clearances are shown in bold. Kinetic parameters were derived following
non-linear analysis (program SigmaPlot 8.0; Enzyme Kinetics).
catalysis of both N-demethylations and 8-hydroxylation.
There was no significant difference in the rate of caffeine
metabolism (100 mM) between liver microsomes preincubated
with and without NADPH at 50 8C (data not shown).
(Program Sigma Plot 8.0; Enzyme Kinetics), are presented in
Table 1. These kinetic parameters indicate distinct inter-
isoform differences, which is consistent with the multi-
enzyme Eadie–Hofstee plots derived from liver microsomes
(Fig. 2A–D). CYP1A2 showed the highest intrinsic clearance
(Vmax/Km) towards caffeine metabolism, its highest value
being reached for 8-hydroxylation. The intrinsic clearance of
CYP1A2 was also relatively high in the case of 3-N-demethyla-
tion and 1-N-demethylation. In the case of 7-N-demethylation,
3.2.
A study with rat cDNA-expressed P450s
Kinetic parameters showing the caffeine metabolism in rat
cDNA-expresssed P450s, obtained using non-linear analysis
Fig. 3 – The biotransformation of caffeine via 1-N-demethylation (A), 3-N-demethylation (B), 7-N-demethylation (C), and
C-8-hydroxylation (D) by rat cDNA-expressed P450s (Supersomes). Caffeine (100 mM) was incubated with Supersomes
(50–100 pmol P450/ml), 6 mM MgCl2Á6H2O and the NADPH generating system (1.2 mM NADP, 6 mM DL-isocitric acid, 1.2 U/ml
isocitric dehydrogenase) for 15–60 min, depending on optimum conditions for a P450 isoform. Each bar represents the
mean value of two independent analyses.

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
1543
the highest intrinsic clearance was found for CYP1A2, CYP2C6
and CYP2C11.
CYP1A2 is a chief enzyme catalyzing 8-hydroxylation (72%)
and substantially contributing to 3-N-demethylation (47%) and
1-N-demethylation (37.5%). Moreover, 8-hydroxylation was
also visibly mediated by CYP3A2 (15%), while 3-N-demethyla-
tion – by CYP2C11 (31%) and CYP3A2 (7%), and 1-N-demethyla-
tion – by CYP3A2 (17%) and CYP2C13 (14%). The catalysis of
7-N-demethylation was predominantly governed by CYP2C11
(29%) and CYP2C6 (27%), and to lower extent – by CYP1A2
(14%), CYP2C13 and CYP3A2 (10% each). At a higher concen-
tration of the substrate, CYP1A2 contribution to the metabo-
lism of caffeine decreased – mostly in favor of CYP2C11 (in the
case of N-demethylations) and/or CYP3A2 (mainly in the case
of 8-hydroxylation) (Fig. 4).
The ability of rat cDNA-expressed P450s to metabolize
caffeine at its therapeutic concentration (100 mM) is shown in
Fig. 3A–D. According to the calculated intrinsic clearance
values, the preference of P450 enzymes for catalyzing caffeine
metabolism was as follows (pmol of product/pmol of P450 iso-
form/min):
CYP1A2 > CYP2A2 > CYP2B1 > CYP2E1 > CYP1A1 > CYP3A2 >
CYP2C13 > CYP2C6 > CYP2D1 > CYP2C11 > CYP2D2 for 1-N-
demethylation, CYP1A2 > CYP1A1 > CYP2C11 > CYP3A2 >
CYP2D1 > CYP2A2 > CYP2D2 > CYP2C6 > CYP2B1 > CYP2E1 >
CYP2C13 for 3-N-demethylation, CYP1A2 > CYP2C6 >
CYP2C11 > CYP2D2 > CYP2D1 > CYP3A2 > CYP2A2 > CYP2-
CYP2C13 > CYP2E1 > CYP2B1 > CYP1A1 for 7-N-demethyla-
tion, CYP1A2 > CYP3A2 > CYP1A1 > CYP2D1 > CYP2D2 >
CYP2C6 > CYP2A2 > CYP2B1 > CYP2C11 > CYP2E1 > CYP2C13
for 8-hydroxylation.
3.4.
Inhibition of caffeine metabolism by specific P450
inhibitors in rat liver microsomes
a-Naphtoflavone (a CYP1A2 inhibitor) exerted
a strong
inhibitory effect on the rate of the four oxidative caffeine
pathways, being most effective towards 8-hydroxylation (to
34% of the control value; Ki = 0.048 mM), and the least active
towards 7-N-demethylation (to 48% of the control value;
Ki = 0.136 mM) (Fig. 5 and Table 3).
3.3.
Quantitative estimation of the contribution of P450
enzymes to the specific metabolic pathways of caffeine
We roughly estimated the contribution of the P450 isoforms
studied to caffeine phase I oxidation pathways on the basis of
the rate of those reactions in Supersomes and the contribution
of each isoform to the total P450 content in the liver (Table 2).
CYP2B1, the only available CYP2B isoform, was assumed to
be a representative of CYP2B1/2 isoforms, regarding their
similar structure (a 97% amino acid sequence similarity); [35]
and catalytic function [36,37]. The calculations done at a
caffeine concentration of 100 mM (relevant to ‘‘the maximum
therapeutic concentration in humans’’) indicated that CYP1A2
was the main isoform responsible for caffeine metabolism.
Sulfaphenazole (a CYP2C6 inhibitor) significantly
decreased the rate of 7-N-demethylation (to 71% of the control
value; Ki = 2.8 mM), not affecting other reactions. Cimetidine (a
CYP2C6 + CYP2C11 inhibitor) reduced the rate of 7-N-
demethylation (to 57% of the control value; Ki = 35.2 mM),
and to a smaller extent that of 3-N-demethylation (to 76% of
the control value; Ki = 138 mM). Erythromycin (a CYP3A
inhibitor) significantly diminished the rate of 1-N-demethyla-
tion and 8-hydroxylation (to 69 and 86%, respectively, of the
control value; Ki = 490 and 858 mM, respectively). Neither
Table 2 – Estimation of the contribution of P450 isoforms to the particular metabolic pathways of caffeine on the basis of
the rates of these reaction in Supersomes and average P450 contents in the liver
P450
Relative contribution
of the iso-form to the
total P450 contents in liver
microsomes (fraction)a
Relative contribution of the isoforms to caffeine
metabolism in liver microsomes (percentage)b
Caffeine1-N-
Caffeine 3-N-
Caffeine 7-N-
Caffeine C-8-
hydroxylation
demethylation
demethylation
demethylation
Caffeine
Caffeine
Caffeine
Caffeine
100 mM
800 mM
100 mM
800 mM
100 mM
800 mM
100 mM
800 mM
CYP1A2
CYP2A2
CYP2B1
CYP2C6
CYP2C11
CYP2C13
CYP2D1
CYP2D2
CYP2E1
CYP3A2
0.050a
0.035a
0.010a
0.200a
0.400a
0.230a
0.025c
0.025c
0.100b
0.200b
37.5
6.9
22.6
3.5
46.9
1.6
19.2
1.2
14.1
3.1
5.4
1.7
71.9
30.4
1.0
0.1
0.6
0.02
6.1
2.8
0.4
0.4
0.3
0.7
1.6
0.6
0.2
0.2
0.2
0.3
6.6
6.7
5.2
2.8
26.8
29.3
10.0
2.0
24.5
41.3
6.9
6.5
6.4
13.8
1.0
23.8
14.3
1.1
31.3
4.2
50.8
3.9
2.7
0.7
1.0
1.5
1.5
1.1
0.2
0.4
0.7
0.4
1.4
0.6
0.6
9.2
9.5
2.2
4.1
3.0
3.1
0.3
16.8
17.4
6.6
15.9
10.1
14.6
15.3
58.1
a
Data according to aLewis [38]; bShimojo et al. [39]; cLarrey et al. [40].
b
Relative contribution of P450s to the particular metabolic pathways of caffeine was calculated as percentage of the sum of predicted
velocities in liver microsomes. The predicted velocity in liver microsomes was calculated by multiplying the velocity in Supersomes (see Fig. 3)
by the relative contribution of isoform to the total P450 content in liver microsomes. For details of calculations, see Wo´jcikowski et al. [41].
Essential differences between 100 and 800 mM concentration of caffeine are shown in bold.

1544
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
Fig. 4 – The concentration-dependent contribution of P450 isoforms to caffeine metabolism in rat liver (based on the rate of
caffeine metabolism in Supersomes and the mean of P450s in the liver). For further explanations see Fig. 3.
quinine (a CYP2D inhibitor) nor in vivo administration of trans-
DCE (a CYP2E1 inhibitor) significantly affected caffeine
metabolism in vitro.
strong as those for 3-N- or 1-N-demethylation at 800 mM
caffeine (Table 4B). Moreover, the rate of 3-N-demethylation
positively correlated with the activities of CYP2B and CYP2C11,
while the rate of 7-N-demethylation potently correlated with
the activity of CYP2C11, as well as with the activities of CYP2B
and CYP2C6. As observed previously for 100 mM caffeine, 8-
hydroxylation in the case of 800 mM caffeine, was also strongly
correlated with the activity of CYP3A2.
3.5.
Correlation study
It was shown that the 8-hydroxylation of caffeine was a major
metabolic reaction (ꢀ70%). An approximately sixfold inter-
individual variability of the rates of caffeine metabolic
reactions both at 100 and 800 mM concentrations of caffeine
was found (data not shown). The rate of formation of caffeine
metabolites by different preparations of rat liver microsomes,
determined for two concentrations of caffeine (100 and
800 mM), was compared with the monooxygenase activity
assessed in our laboratory for each liver preparation. The
results of those analyses are shown in Table 4A (for 100 mM
caffeine) and B (for 800 mM caffeine), where the correlation
coefficient (r) and the p value are given for each pair of data.
At a caffeine concentration of 100 mM, the rates of all the
caffeine metabolic reactions strongly correlated with the
activity of CYP1A2. Moreover, the rate of 3-N-demethylation
significantly correlated with the activity of CYP2B, the rate of
1-N-demethylation correlated with the activity of CYP2C6,
whereas the rate of 8-hydroxylation strongly correlated with
the activity of CYP3A2 (Table 4A).
4.
Discussion
The data presented above are the first comprehensive report on
the qualitative and quantitative contribution of individual P450
isoforms to the four oxidation pathways of caffeine metabolism
in the rat. The obtained results indicate that the main oxidation
pathway of caffeine in the rat is 8-hydroxylation (70%), which is
specifically catalyzed by CYP1A2 (72%) at a concentration of
100 mM (relevant to ‘‘the maximum therapeutic concentration
in humans’’), and may be used as a marker reaction for this
enzyme in the above-mentioned species. The above results
showing the importance of 8-hydroxylation for caffeine
metabolism are consistent with those obtained by Berthou
et al. [8] with rats, but disagree with those pertaining to humans
which show that 3-N-demethylation is the main metabolic
pathway of caffeine [8,9]. Since both these main reactions in the
two species (8-hydroxylation in rats and 3-N-demethylation in
humans) are specifically catalyzed by CYP1A2, they may imply
species differences in CYP1A2 catalytic competence and
kinetics, as well as in caffeine metabolism.
At a caffeine concentration of 800 mM, the rates of all the
caffeine metabolic reactions also significantly correlated with
the activity of CYP1A2 (Table 4B), however, the correlations
concerning 8-hydroxylation and in particular 7-N-demethyla-
tion were weaker than at 100 mM caffeine (Table 4A) and not as

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
1545
tion). The above conclusions are based on our results of the
Eadie–Hofstee analysis (non-linear plots suggesting a multi-
ple-enzyme catalysis) and the proven ability of cDNA-
expressed P450s to metabolize caffeine; they are also
confirmed by some further data derived from correlation
and inhibition studies with liver microsomes.
The use of cDNA-expressed P450s revealed that CYP1A2
had the biggest intrinsic clearance (Vmax/Km
) towards
caffeine metabolism, which reached its highest value for
8-hydroxylation. Although lower than in the case of 8-
hydroxylation, the clearance by CYP1A2 was also the highest
in the case of 1-N- and 3-N-demethylation, compared to
other P450s. Accordingly, the theoretically calculated con-
tribution of P450 isoforms to caffeine oxidation pathways
(on the basis of the rate of these reactions in Supersomes
and the mean of P450s in the liver) indicates that CYP1A2 is a
key enzyme that catalyzes 8-hydroxylation (72%) and
substantially contributes to 3-N-demethylation (47%) and
1-N-demethylation (37.5%) at a caffeine concentration of
100 mM, corresponding to ‘‘the maximum therapeutic con-
centration in humans’’. Furthermore, also CYP2C11 con-
siderably contributes to 3-N-demethylation (31%), while
CYP3A2 facilitates 1-N-demethylation and 8-hydroxylation
(17 and 15%, respectively).
Fig. 5 – The effect of P450-specific inhibitors on the rate of
caffeine 1-N-demethylation (A), 3-N-demethylation (B), 7-
N-demethylation (C), and 8-hydroxylation (D) in rat liver
microsomes. Microsomes were incubated with 100 mM
caffeine in the absence (control) or presence of P450-
specific inhibitors: 0.1 mM a-naphthoflavone (aNF), 5 mM
sulfaphenazole (SULF), 50 mM cimetidine (CIM), 10 mM
quinine (QUIN) or 400 mM erythromycin (ERY); trans-1,2-
dichloroethylene (trans-DCE) was administered in vivo in a
dose of 100 mg/kg i.p. Absolute control values were
0.0026 W 0.0001 nmol of theobromine/mg of protein/min,
0.0036 W 0.0001 nmol of paraxanthine/mg of protein/min,
0.0021 W 0.0001 nmol of theophylline/mg of protein/min,
0.0144 W 0.0001 nmol of 1,3,7-trimethyluric acid/mg of
protein/min. Mean values W S.E.M. (n = 5) are presented.
Statistical significance was assessed using Student’s t test
and indicated with ***, p < 0.001; **, p < 0.01; *, p < 0.05. For
further explanation, see Fig. 2.
The above results are in line with the observed inhibition of
the four metabolic pathways of caffeine by the CYP1A2
inhibitor a-naphtoflavone (the strongest effect on 8-hydro-
xylation, and the weakest one on 7-N-demethylation) and
with a significant correlation of the rate of these reactions with
the activity of CYP1A2 in liver microsomes. The results
obtained using cDNA-expressed P450s are also in line with
the inhibition of 3-N-demethylation by cimetidine (a
CYP2C6 + CYP2C11 inhibitor), and with the inhibition of 1-N-
demethylation and 8-hydroxylation by erythromycin (a CYP3A
inhibitor) in liver microsomes. It is noteworthy that the rate of
3-N-demethylation correlates with the activity of CYP2C11 (at
800 mM caffeine). Moreover, the relatively high clearance of
CYP3A2 and its theoretically calculated contribution to
caffeine 8-hydroxylation in rat liver correspond to the
significant correlation of the rate of this reaction with the
activity of CYP3A2 in liver microsomes. The contribution of
CYP3A2 to 8-hydroxylation markedly increases at the higher
substrate concentration of 100À800 mM (from 15 to 58%) at the
expense of CYP1A2 (a decrease from 72 to 30%).
1-N- and 3-N-demethylations are also preferentially cata-
lyzed by CYP1A2, to a lesser degree than 8-hydroxylation,
though, while 7-N-demethylation is mainly mediated by
isoforms of the subfamily CYP2C (66%). At higher substrate
concentrations, the contribution of CYP1A2 to the metabolism
of caffeine decreases in favor of the other P450s: CYP2C11 (N-
demethylation reactions) and CYP3A2 (mainly 8-hydroxyla-
Table 3 – The influence of P450 inhibitors on the metabolism of caffeine in rat liver microsomes
P450 Inhibitors
Caffeine 1-N-
demethylation
(theobromine)
Ki (mM)
Caffeine 3-N-
demethylation
(paraxanthine)
Ki (mM)
Caffeine 7-N-
demethylation
(theophylline)
Ki (mM)
Caffeine C-8-
hydroxylation)
(1,3,7-trimethyluric
acid) Ki (mM)
a-Naphthoflavone (1A2)
Sulfaphenazole (2C6)
Cimetidine (2C6 + 2C11)
Quinine (2D)
0.083
0.085
0.136
0.048
No effect
No effect
No effect
No effect
490
No effect
137.8
2.8
No effect
158
35.2
No effect
No effect
No effect
No effect
No effect
No effect
No effect
No effect
858
trans-DCE (2E1)
Erythromycin (3A)
The presented inhibition constants (Ki) for inhibition of particular metabolic pathways were calculated using Dixon analysis. Trans-DCE, trans-
1,2-dichloroethylene. For further explanation, see Fig. 5.

1546
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
In contrast to 8-hydroxylation, the CYP1A2 clearance
related to 7-N-demethylation is relatively low and comparable
to that of CYP2C6 and CYP2C11. However, the amount of the
metabolite formed by an individual P450 isoform in the liver
depends not only on enzyme kinetics (intrinsic clearance), but
also on the contribution of an isoform to the total P450
content. The roughly estimated contribution of P450 isoforms
to the 7-N-demethylation of caffeine indicates that the CYP2C
isoforms, mainly CYP2C6 (27%) and CYP2C11 (29%), play a
major role in catalyzing this reaction. The contribution of
CYP2C11 to 7-N-demethylation increases from 29 to 41% at a
higher substrate concentration, while this of CYP1A2 shows a
decreasing tendency (from 14 to 5%). The theoretically
calculated contribution of the CYP2C isoforms to the 7-N-
demethylation of caffeine can be supported by the results
obtained with liver microsomes, namely those concerning
inhibition of the rate of this reaction by sulfaphenazole
(CYP2C6 inhibitor) and cimetidine (CYP2C6 + CYP2C11 inhi-
bitor), as well as the positive correlation between the rate of 7-
N-demethylation of caffeine (800 mM) and the activity of the
two above-mentioned CYP2C isoforms. On the other hand, the
correlation between the rate of 7-N-demethylation of caffeine
(800 mM) and the activity of CYP1A2 is relatively weak
compared to that concerning CYP2C isoforms. The substantial
contribution of CYP2C11 to 7-N-demethylation and – as has
been mentioned elsewhere – to 3-N-demethylation is in line
with the results of our previous study obtained using P450
inducers [18] and with the findings of Bienvenu et al. [42] who
observed a decrease in the rate of these reactions after a
continuous infusion of the growth hormone to hypophysec-
tomized male rats.
The correlation between the rate of 7-N-demethylation and
the activity of CYP2B appears to be accidental due to the ability
of CYP2C11 (constituting ꢀ40% of the total P450) to catalyze the
16b-hydroxylation of testosterone, which is a marker reaction
of CYP2B activity (our unpublished results). An analogous
miscorrelation seems to occur between the rate of 3-N-
demethylation and the activity of CYP2B. As has been
mentioned elsewhere, the contribution of CYP2C11 to 3-N-
demethylation reaches 31 and 50% at caffeine concentrations
of 100 and 800 mM, respectively.
The above results are only partly in line with the
conclusions drawn by other authors. One of the causes may
be that caffeine metabolism was studied in different experi-
mental models and/or that conclusions were drawn from one
experimental model only [20,43,44]. Other possible causes are
the use of unspecific inducers (or inhibitors) and relating the
rates of caffeine reactions to the activity of only one (mainly an
induced one) P450 isoform, as well as the application of high
(1–10 mM) caffeine concentrations and not taking account of
all P450 isoforms in both experimentation and data inter-
pretation [11,12,15,19].
The obtained data only partly agree with the conclusions
drawn by Morita et al. [12] which indicate that CYP3A2 is
responsible for the 8-hydroxylation of caffeine. The latter
authors concentrated exclusively on CYP3A2 and performed
their studies on liver microsomes at a high (10 mM) caffeine
concentration. As shown in our present work, CYP1A2 is the
major enzyme catalyzing caffeine 8-hydroxylation at
a
concentration of 100 mM (relevant to ‘‘the maximum

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 5 3 8 – 1 5 4 9
1547
therapeutic concentration in humans’’), but the contribution
of CYP3A2 to 8-hydroxylation markedly rises at higher
substrate concentrations at the expense of CYP1A2. Our
results do not closely correspond, either, to the findings of
Chung et al. [11,19] who proposed – on the basis of unspecific
P450 and FMO inducers/inhibitors [45] and using the very
high 10 mM concentration of caffeine – that caffeine 8-
hydroxylation was catalyzed mainly by CYP2B1, CYP3A1,
CYP2E1, while N-demethylation reactions by CYP1A2 and
FMO. Our present study indicates that FMO does not
significantly contribute to caffeine metabolism measured
in vitro at the substrate concentration of 100 mM, as shown by
the thermal inactivation of FMO. On the other hand, our
experiment with cDNA-expressed P450s shows that CYP2B1
and CYP2E1 only insignificantly contribute to the 8-hydro-
xylation and total metabolism of caffeine, which is in line
with the conclusions of our recent study with selective P450
inducers [18].
governed by P450s of the CYP2C subfamily (and not by CYP2B
as suggested previously); (3) 8-hydroxylation, the main
oxidation pathway of caffeine in the rat, is specifically
mediated by CYP1A2 (and not by CYP3A as suggested
previously) at a concentration of 100 mM, corresponding to
‘‘the maximum therapeutic concentration in humans’’. At
higher substrate concentrations, the contribution of CYP1A2
to the metabolism of caffeine decreases in favor of CYP2C11
(N-demethylations) and CYP3A2 (mainly 8-hydroxylation).
Therefore, caffeine may be applied as a marker substance
for assessing the activity of CYP1A2 in the rat, using 8-
hydroxylation (but not 3-N-demethylation—like in humans) as
a specific reaction. Furthermore, caffeine can be used to
preliminarily and simultaneously estimate CYP2C activity
using 7-N-demethylation as a marker reaction. The above-
presented results indicate species-differences in caffeine
metabolism and show for the first time the contribution of
the CYP2C subfamily to this metabolism. Moreover, they
suggest that caffeine pharmacokinetics in rats may be
changed by drugs affecting the activity of CYP1A2 and/or
CYP2C, e.g. by some antidepressants.
Our results correspond to the hypothesis of Bienvenu et al.
[44] that the N-demethylation reactions of caffeine are
catalyzed by polycyclic hydrocarbon-inducible P450s; on the
other hand, however, they do not support the hypothesis put
forward by Berthou et al. [15] who – on the basis of the auto-
induction of caffeine metabolism accompanied with increases
in CYP1A and CYP2B protein levels and activities and in the
formation of theophylline – inferred that the 7-N-demethyla-
tion of caffeine (1 mM) was predominantly mediated by
CYP2B. In all the above-cited studies the authors did not take
account of P450 isoforms of the CYP2C subfamily, which are
abundantly expressed in rat liver and constitute about 80% of
the total P450.
Acknowledgments
This study was supported by Grant no. 4 PO5F 024 26 from the
State Committee for Scientific Research (KBN), Warszawa, and
as part of the statutory activity of the Institute of Pharmacol-
ogy of the Polish Academy of Sciences in Krako´w.
r e f e r e n c e s
Strain differences may influence the relative contribution
of P450 isoforms to caffeine metabolism in the case of Dark
Agouti rats with an increased CYP3A level [12,46,47]. However,
researches use mainly Wistar or Sprague–Dawley rats
[11,12,15] and differences between studies have been observed
within the same strain (e.g. Wistar in Morita et al. [12], and our
study). Therefore it seems that different substrate concentra-
tions (0.1–10 mM) and unspecific tests used by the authors are
chiefly responsible for the observed discrepancies in the
involvement of individual P450 isoforms in caffeine metabo-
lism in rats.
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