Human cytochromes p450 mediating phenacetin 0-deethylation in vitro: Validation of the high affinity component as an index of CYP1A2 activity

Article abstract of DOI:10.1021/js980255z

Phenacetin 0-deethylation, widely used as an index reaction for cytochrome P450 1A2 (CYP1A2) activity, displays biphasic kinetics in human liver microsomes. CYP1A2 has been identified as contributing to the high affinity component, but is not verified as the sole contributor to the high affinity phase. In addition, the human CYP isoforms accounting for the low affinity phase have not been identified. We have used heterologously expressed human CYP isoforms to identify, kinetically characterize, and predict the relative contribution of the major human liver CYP isoforms mediating phenacetin 0-deethylation. CYP1A2 (Km 31 /J.M) is the only high affinity phenacetin 0-deethylase in human liver microsomes, while CYPs 2A6 (K_m 4098 pM), 2C9 (K_m 566 μM), 2C19 (K_m 656 μM), 2D6 (K_m 1021 μM), and 2E1 (K_m 1257 μM) all contribute to the low affinity phase of the reaction. Considering the relative abundance of the various CYPs in human liver, CYP1A2 accounts for 86% of net reaction velocity at a substrate concentration of 100 fM, while CYP2C9 becomes the primary phenacetin 0-deethylase at substrate concentrations of 865 /M and higher and accounts for 31% of the net Vmsst of the reaction. Predictions from kinetic studies on heterologously expressed CYPs are consistent with chemical inhibition studies on human liver microsomes with sulfaphenazole and a-naphthoflavone that suggest a greater role for CYP2C9, and a smaller role for CYP1A2, at higher substrate concentrations. Thus CYP1A2 is the only high affinity human liver phenacetin 0-deethylase, thereby validating the use of the high affinity component as an index of CYP1A2 activity in human liver microsomes.

Full text of DOI:10.1021/js980255z

Human Cytochromes P450 Mediating Phenacetin O-Deethylation in
Vitro: Validation of the High Affinity Component as an Index of
CYP1A2 Activity
KARTHIK VENKATAKRISHNAN, LISA L. VON MOLTKE, AND DAVID J. GREENBLATT*
Contribution from Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine,
Boston, Massachusetts 02111 and the Division of Clinical Pharmacology, New England Medical Center Hospital,
Boston, Massachusetts 02111.
Received June 19, 1998.
Final revised manuscript received September 15, 1998.
Accepted for publication September 17, 1998.
deethylase activity;7-9 and (c) the ability of heterologously
expressed CYP1A2 to catalyze phenacetin O-deethylation
with a Km comparable to that of the high affinity component
in human liver microsomes.2,10,11 However, CYP1A2 has
not been conclusively established as the only high affinity
isoform mediating phenacetin O-deethylation. Existence
of additional high affinity (low abundance and/or capacity)
phenacetin O-deethylases in human liver is a possibility
that cannot be excluded based on chemical inhibition or
correlation studies alone. This is important information
since such alternate high affinity enzymes, if inducible, can
theoretically assume importance even at low substrate
concentrations, especially in livers containing relatively low
levels of CYP1A2. This in turn could compromise the use
of phenacetin as an index substrate for measuring CYP1A2
activity. In addition, the CYP isoform(s) responsible for
the low affinity component of human liver microsomal
phenacetin O-deethylation have not been identified.
The objectives of the present study were thus to identify
and kinetically characterize the major human liver mi-
crosomal CYP isoforms that catalyze phenacetin O-deethy-
lation using heterologously expressed CYPs. This infor-
mation, in conjunction with the relative abundance of the
various CYPs in human liver, has then been applied in
simulating the relative contribution of each CYP isoform
to phenacetin O-deethylation rate as a function of substrate
concentration. This study identifies CYP1A2 as the only
enzyme responsible for the high affinity component of
phenacetin O-deethylation by human liver microsomes,
whereas the low affinity component is accounted for by at
least six different CYP isoforms.
Abstract 0 Phenacetin O-deethylation, widely used as an index
reaction for cytochrome P450 1A2 (CYP1A2) activity, displays biphasic
kinetics in human liver microsomes. CYP1A2 has been identified as
contributing to the high affinity component, but is not verified as the
sole contributor to the high affinity phase. In addition, the human
CYP isoforms accounting for the low affinity phase have not been
identified. We have used heterologously expressed human CYP
isoforms to identify, kinetically characterize, and predict the relative
contribution of the major human liver CYP isoforms mediating
phenacetin O-deethylation. CYP1A2 (K 31 µM) is the only high
m
affinity phenacetin O-deethylase in human liver microsomes, while
CYPs 2A6 (K 4098 µM), 2C9 (K 566 µM), 2C19 (K 656 µM),
m
m
m
2D6 (K 1021 µM), and 2E1 (K 1257 µM) all contribute to the low
m
m
affinity phase of the reaction. Considering the relative abundance of
the various CYPs in human liver, CYP1A2 accounts for 86% of net
reaction velocity at a substrate concentration of 100 µM, while CYP2C9
becomes the primary phenacetin O-deethylase at substrate concentra-
tions of 865 µM and higher and accounts for 31% of the net Vmax of
the reaction. Predictions from kinetic studies on heterologously
expressed CYPs are consistent with chemical inhibition studies on
human liver microsomes with sulfaphenazole and R-naphthoflavone
that suggest a greater role for CYP2C9, and a smaller role for CYP1A2,
at higher substrate concentrations. Thus CYP1A2 is the only high
affinity human liver phenacetin O-deethylase, thereby validating the
use of the high affinity component as an index of CYP1A2 activity in
human liver microsomes.
Introduction
Materials and Methods
Phenacetin O-deethylation is widely used as an index
reaction to reflect human liver microsomal cytochrome
P450 1A2 (CYP1A2) activity. Although this reaction is
reported to be highly specific for CYP1A2 activity at low
concentrations of substrate, human liver microsomal kinet-
ics of phenacetin O-deethylation display biphasic kinetics.1-3
This suggests that the pathway is mediated by at least two
different enzymes with distinct affinities, although the
identity of the low affinity component has not been
established. Indirect evidence suggesting the role of
CYP1A2 as the primary human liver microsomal high
affinity phenacetin O-deethylase includes (a) sensitivity of
the high affinity component to specific chemical inhibitors
of CYP1A2 such as furafylline and R-naphthoflavone;1,3-6
(b) significant correlations between immunoquantified
levels of this isoform and high affinity phenacetin O-
Ma t er ia lssPhenacetin, acetaminophen, R-naphthoflavone,
sulfaphenazole, and omeprazole were purchased from the Sigma
Chemical Co. (St. Louis, MO), and 2-acetamidophenol from Aldrich
Chemical Co. (Milwaukee, WI). The cofactors NADP+, (()-isocitric
acid, MgCl2, and isocitrate dehydrogenase were from Sigma, as
was the KH2PO4 salt used in buffer solution.
Microsomes from cDNA transfected human lymphoblastoid cells
expressing CYP 1A2, 2A6, 2B6, 2C9-Arg144, 2C19, 2D6, 2E1, or
12
3A4
were obtained from Gentest Corporation (Woburn, MA),
aliquoted and stored at -80 °C, and thawed on ice before use.
Microsomal protein concentrations (determined by the Lowry
protein assay) and CYP content (determined by carbon monoxide
difference spectrophotometry) were provided by the manufacturer.
Liver samples, obtained from the International Institute for the
Advancement of Medicine (Exton, PA) or the Liver Tissue Procure-
ment and Distribution Service (University of Minnesota) were from
12 different transplant donors with no known liver disease. The
donor population (median age 27 years; range 3-50 years) was
balanced with respect to sex, race, smoking habits, and alcohol
* Corresponding author. Phone: 617-636-6997, Fax: 617-636-6738.
1502 / Journal of Pharmaceutical Sciences
10.1021/js980255z CCC: $15.00
Published on Web 10/24/1998
© 1998, American Chemical Society and
Vol. 87, No. 12, December 1998
American Pharmaceutical Association

Figure 1sPhenacetin O-deethylation by heterologously expressed human CYPs 1A2 (panel A), 2A6 (panel B), 2C9 (panel C), 2C19 (panel D), 2D6 (panel E),
and 2E1 (panel F). Solid dots are experimental data points, and the lines are fitted functions. A Michaelis−Menten model (eq 1) was used for CYPs 1A2, 2A6,
and 2E1. A Hill enzyme model (eq 2) was used for CYPs 2C9, 2C19, and 2D6. See Table 1 for kinetic parameters.
consumption. The tissue was partitioned and kept at -80 °C until
the time of microsome preparation as described previously.13,14
Meth od ssIncubations of phenacetin with microsomes from
human lymphoblastoid cells expressing CYP 1A2, 2A6, 2B6, 2C9-
Arg144, 2C19, 2D6, 2E1, or 3A4 were performed in polypropylene
tubes in a volume of 0.25 mL at 37 °C using a protein concentration
of 800 µg/mL and a reaction time of 20 min. The incubation
method was identical to that described earlier for human liver
microsomes,3 except that incubations were performed without
agitation, but with only mild shaking, as recommended by the
supplier of the cDNA-expressed enzymes. Reactions were stopped
by the addition of 100 µL of acetonitrile, and acetaminophen
assayed in incubates using a HPLC method described previously.3
The kinetics of phenacetin O-deethylation by cDNA-expressed
CYPs were characterized using the following ranges of substrate
concentrations: 5-2000 µM (CYP1A2), 250-10000 µM (CYP2A6,
2C19 and 2D6), 250-5000 µM (CYP2C9), and 50-10000 µM
(CYP2E1). CYP2B6 and 3A4 displayed no detectable activity at
2000 µM phenacetin concentration. Control incubations were
performed with microsomes from cells transfected with expression
vector alone and showed no detectable activity.
Km is the substrate concentration at which the reaction velocity
is 50% of Vmax, the maximal reaction velocity. A is the Hill
coefficient for cooperative substrate binding.
Nonlinear regression was performed using the SigmaPlot
software (J andel Scientific). The choice of model was based on
examination of Michaelis-Menten plots, Eadie-Hofstee plots,
visual inspection of nonlinear regression fits, and by application
of the Akaike’s Information criterion (AIC).15
Chemical inhibition studies with R-naphthoflavone (1 µM),
sulfaphenazole (10 µM), and omeprazole (10 µM) were performed
by coincubating phenacetin (50 or 2000 µM) and inhibitor with
human liver microsomes for 20 min at a protein concentration of
250 µg/mL. This has been shown previously to be in the linear
range of time and protein concentration for this reaction.3 The
specificity of R-naphthoflavone (1 µM) toward CYP1A2 was
confirmed by studying the effect of the inhibitor on phenacetin
O-deethylation catalyzed by cDNA-expressed CYPs 1A2, 2A6, 2C9,
2C19, 2D6, and 2E1 at a substrate concentration of 1000 µM. The
concentrations of sulfaphenazole5 and omeprazole16 were chosen
to ensure relative specificity versus CYP 2C9 and 2C19, respec-
tively. Inhibition studies were performed on four human liver
microsomal preparations, and the differences in inhibition at 50
and 2000 µM phenacetin were analyzed by a two-tailed paired
t-test. Phenacetin O-deethylation rates were further measured
at two different substrate concentrations (40 µM and 1000 µM) in
12 liver microsomal preparations to test the predictability of liver
microsomal rates from kinetic studies on cDNA-expressed CYP
isoforms.
The kinetics of phenacetin O-deethylation by cDNA-expressed
CYPs were described by one of the following models:
A one enzyme Michaelis-Menten model:
V
maxS
V )
(1)
(2)
Km + S
A Hill enzyme model:
Results
V
maxSA
KAm + SA
V )
An initial screen of cDNA-expressed CYPs 1A2, 2A6,
2B6, 2C9-Arg144, 2C19, 2D6, 2E1, and 3A4 for phenacetin
Journal of Pharmaceutical Sciences / 1503
Vol. 87, No. 12, December 1998

Figure 2sEadie−Hofstee plots of data presented in Figure 1. Note that substrate activation (Hill kinetics) is evident for CYPs 2C9, 2C19, and 2D6.
Table 1sKinetic Parameters of Phenacetin O-Deethylation by
Heterologously Expressed Human CYP Isoforms
thoflavone caused a 90% inhibition of phenacetin O-
deethylation by cDNA-expressed CYP1A2 (IC50 0.16 µM;
Figure 3B), whereas the activities of cDNA-expressed CYPs
2A6, 2C9, 2C19, 2D6, and 2E1 were not significantly
altered (93-110% of control activity). This justified the
use of this concentration of R-naphthoflavone as a CYP1A2
isoform-specific chemical inhibitor in human liver microso-
mal inhibition studies. Human liver microsomal phenace-
tin O-deethylation was inhibited by 1 µM R-naphthoflavone
at both 50 and 2000 µM phenacetin, although the percent-
age inhibition at 50 µM (83 ( 6%; n ) 4 human livers)
was significantly higher (p < 0.0001) than that at 2000 µM
phenacetin (30 ( 8%; n ) 4 human livers). Sulfaphenazole
(10 µM) produced a 5 ( 0.6% inhibition of human liver
microsomal phenacetin O-deethylation rate at a 50 µM
concentration of substrate, which was significantly lower
(p < 0.005) than the 14 ( 0.7% inhibition observed at a
substrate concentration of 2000 µM. However, this con-
centration of sulfaphenazole (10 µM) produced a 89%
inhibition of phenacetin O-deethylation catalyzed by cDNA-
expressed CYP2C9 at a substrate concentration of 2000
µM. Omeprazole (10 µM) caused a 13 ( 0.9% inhibition
at a substrate concentration of 50 µM phenacetin, while
no inhibition was observed at 2000 µM phenacetin.
abundance-
a
CYP
V
adjusted
max
isoform (pmol/min/pmol CYP)
V
max
b (%)
K a (µM)
A
m
CYP1A2c
CYP2A6c
CYP2C9d
CYP2C19d
CYP2D6d
CYP2E1c
2.38 ± 0.05
2.65 ± 0.12
2.77 ± 0.11
4.55 ± 0.08
6.38 ± 0.25
3.71 ± 0.06
22.99
8.07
30.96
12.11
7.27
30.9 ± 2.8
4098 ± 428
566 ± 40
656 ± 27
1021 ± 84
1257 ± 62
2.2 ± 0.3
1.75 ± 0.1
1.68 ± 0.2
18.59
a Table entries are mean ± SE of parameter estimates. b Abundance values
used are 12.7% (1A2), 4% (2A6), 14.7% (2C9), 3.5% (2C19), 1.5% (2D6),
and 6.6% (2E1) of total human hepatic CYP content. c A Michaelis−Menten
model (eq 1) was used. d A Hill enzyme model (eq 2) was used.
O-deethylase activity at a substrate concentration of 2000
µM showed that all CYPs except CYP2B6 and 3A4 dis-
played activity. The kinetic parameters for the CYP
isoforms are shown in Table 1. Primary plots and Eadie-
Hofstee transformed plots are shown in Figures 1 and 2,
respectively. While CYPs 1A2, 2A6, and 2E1 displayed
Michaelis-Menten kinetics (eq 1; Figures 1A, 1B, 1F, 2A,
2B, and 2F), CYPs 2C9, 2C19, and 2D6 showed Hill enzyme
kinetics (eq 2; Figures 1C, 1D, 1E, 2C, 2D, and 2E) as
evident from the Eadie-Hofstee plots shown in Figure 2.
Calculation of AIC values indicated that the Hill model was
preferred over the Michaelis-Menten model for CYPs 2C9,
2C19, and 2D6.
Discussion
Phenacetin O-deethylation is a widely used index reac-
tion for the measurement of CYP1A2 activity. Despite the
recognition of biphasic kinetics of this reaction in human
liver microsomes, the identity of the CYP isoform(s) that
contribute to the low affinity component has not been
The results of chemical inhibition studies of phenacetin
O-deethylation by human liver microsomes are presented
in Figure 3A. At the concentration used (1 µM), R-naph-
1504 / Journal of Pharmaceutical Sciences
Vol. 87, No. 12, December 1998

CYP1A2 activity in human liver microsomes. On the other
hand, six distinct CYP isoforms contribute to the low
affinity component of the reaction. Heterologously ex-
pressed CYPs 2C9, 2C19, and 2D6 displayed Hill enzyme
kinetics (Figure 2, Table 1) suggestive of substrate activa-
tion and allosteric binding. A possible artifact that may
result in Hill kinetics is substrate consumption at low
substrate concentrations. This possibility was ruled out,
since the reactions were performed under conditions that
ensured linearity with respect to time and protein concen-
tration, and substrate consumption was not significant
even at the lowest concentrations. Substrate activation is
a characteristic feature of many CYP3A-mediated reac-
tions. In addition, it has been shown that CYP2C9-
mediated amitriptyline N-demethylation17 and dapsone
metabolism18 demonstrate Hill kinetics. However, there
are no reports to our knowledge about CYP2C19- and 2D6-
mediated reactions demonstrating substrate activation.
These observations suggest that Hill enzyme kinetics are
not restricted to CYP3A-mediated reactions.
The relative contribution of each CYP isoform to net
human liver microsomal reaction rate (fi) was predicted as
a function of substrate concentration (eq 3; Figure 4), using
the relative abundance (Ai) of each CYP isoform in human
liver, and the substrate concentration-velocity functions
[vi(s)] for each CYP isoform determined from kinetic studies
on heterologously expressed enzymes.12,19
Aivi(s)
fi(%) )
× 100
(3)
6
Aivi(s)
∑
i)1
We have used the average immunoquantified levels of
CYPs 1A2, 2A6, 2D6, and 2E1 (12.7%, 4%, 1.5%, and 6.6%
8
of total hepatic CYP) in human liver microsomes
as
scaling factors in this simulation. CYP2C enzymes account
for 18.2% of total human hepatic CYP.8 The ratio of
immunoquantified levels of CYP2C9 and 2C19 in human
liver microsomes was recently reported as 20:1,20 but we
determined the ratio of the relative activity factors for
CYP2C9 and 2C19 to be 4.26 ( 1.55 (mean ( S.E, n ) 10
human livers) using cDNA-expressed CYP2C9 and 2C19,
and human liver microsomes from our liver bank, with
tolbutamide methylhydroxylation and S-mephenytoin 4′-
hydroxylation as measures of CYP2C9 and 2C19 activity,
respectively.19 We used this ratio of relative activity factors
(4.26:1) in our analysis, yielding an estimate that CYP2C9
and 2C19 should account for 14.7% and 3.5% of total
hepatic microsomal CYP. The simulation shown in Figure
4 suggests that while CYP1A2 is the primary isoform
mediating phenacetin O-deethylation at low substrate
concentrations such as those typically used in chemical
inhibition studies, CYP2C9 becomes the primary phenace-
tin O-deethylase at phenacetin concentrations greater than
865 µM. When the Vmax values of the CYP isoforms are
multiplied by their respective abundances, CYP2C9 ac-
counts for 31% of the Vmax while CYP1A2 contributes to
23% of the Vmax. Typically, phenacetin concentrations of
100 µM are used in studies evaluating the inhibitory
potential of new drugs toward CYP1A2. Figure 4 suggests
that CYP1A2 should account for 86% of phenacetin O-
deethylation rate at 100 µM phenacetin, a prediction that
is consistent with an approximately 80% inhibition by 1
µM R-naphthoflavone observed at this substrate concentra-
tion.21 To test the role of CYP2C9 at high phenacetin
concentrations as predicted by Figure 4, we performed
chemical inhibition studies of phenacetin O-deethylation
by human liver microsomes with 10 µM sulfaphenazole at
substrate concentrations of 50 and 2000 µM. Inhibition
Figure 3s(A) Effects of R-naphthoflavone (ΑNF), sulfaphenazole (SPA), and
omeprazole (OMEP) on phenacetin O-deethylation by human liver microsomes.
Mean percent inhibition ± S.E (n ) 4 human livers) is presented. The black
and gray bars indicate substrate concentrations of 50 and 2000 µM,
respectively. (B) Inhibition by R-naphthoflavone of phenacetin O-deethylation
catalyzed by cDNA-expressed CYP1A2 at a substrate concentration of 1000
µM.
established, nor has it been verified that CYP1A2 is the
sole contributor to the high affinity component.
Using heterologously expressed human CYP isoforms,
we have demonstrated that CYPs 1A2, 2A6, 2C9, 2C19,
2D6, and 2E1 can all catalyze phenacetin O-deethylation,
although with very different affinities (Table 1, Figure 1).
We have not evaluated the kinetics of phenacetin O-
deethylation by CYP1A1 due to the minimal constitutive
expression of this isoform in human liver. Our conclusions
are thus restricted to uninduced human liver and cannot
be extended to other tissues or to induced states. The Km
for CYP1A2 determined in this study (31 µM) is within the
range of Km values for this isoform (25-95 µM) determined
in earlier studies,2,10,11 as well as Km values for the high
affinity component of phenacetin O-deethylation by human
liver microsomes (35-152 µM) as reported previously.3
Lower Km values of 5-10 µM for the high affinity phase
have been reported in other studies1,2 that used a different
range of substrate concentrations. Since the Km for CYP1A2
(30.9 µM) is more than 1 order of magnitude smaller than
the next lowest Km (566 µM for CYP2C9), it can be
concluded that CYP1A2 is the only CYP that contributes
importantly to the high affinity component of phenacetin
O-deethylation in human liver microsomes. This validates
the use of the high affinity component as an index of
Journal of Pharmaceutical Sciences / 1505
Vol. 87, No. 12, December 1998

Figure 4sA simulation of the relative contribution of CYPs 1A2, 2A6, 2C9, 2C19, 2D6 and 2E1 to overall phenacetin O-deethylation rate as a function of
substrate concentration (eq 3).
by sulfaphenazole at 2000 µM phenacetin was significantly
higher than that at 50 µM, while the reverse was true for
R-naphthoflavone (Figure 3A). These findings are quali-
tatively in agreement with the predictions from Figure 4.
Although Figure 4 predicts a 36% contribution of CYP2C9
to phenacetin O-deethylation rate at a concentration of
2000 µM, only 14% inhibition was observed with 10 µM
sulfaphenazole. This may be related to the fact that the
cDNA-expressed CYP2C9 used in this study is the 2C9-
Arg144 allelic form, while human liver microsomes contain
several allelic forms of this isoform. The various alleles
may have reduced affinity and/or capacity as phenacetin
O-deethylases, as has been shown for CYP2C9 index
substrates.22 The possibility that CYP2C9-mediated phen-
acetin O-deethylation may be resistant to inhibition by
sulfaphenazole was ruled out, since 89% inhibition of the
reaction catalyzed by cDNA-expressed CYP2C9 was ob-
served in the presence of 10 µM sulfaphenazole at 2000
µM phenacetin.
CYP2C19 has been proposed as a low affinity human
liver microsomal phenacetin O-deethylase based on non-
linear mixed effect modeling.23 However, Figure 4 predicts
CYP2C9 to be the primary phenacetin O-deethylase at high
substrate concentrations. In addition, omeprazole, a rela-
tively specific CYP2C19 inhibitor at 10 µM,16 did not inhibit
phenacetin O-deethylation at a substrate concentration of
2000 µM. Thus, our results suggest that CYP2C19 does
not have a quantitatively important role in human liver
microsomal phenacetin O-deethylation, even at high con-
centrations. The reason for the 13% inhibition produced
by omeprazole at 50 µM phenacetin concentration is not
clear and may reflect a relative rather than absolute
specificity of this inhibitor toward CYP2C19.
pathway of interest is mediated by multiple CYP isoforms,
as in this case. Although the use of cDNA-expressed
enzymes circumvents this problem, the validity of in vitro-
in vitro scaling of liver microsomal kinetics from kinetic
studies on cDNA-expressed enzymes is not established. The
scaling factors that can be reliably used to predict rates of
reactions catalyzed by liver microsomal preparations from
kinetic studies on cDNA expressed enzymes are yet to be
determined. Relative activity factors (RAFs) have been
proposed as suitable scaling factors for this purpose,12
although the validity of the RAF approach remains to be
established. We have used the immunoquantified levels
of individual CYP isoforms as the scaling factors in
predicting the relative contribution of the various CYP
isoforms to net phenacetin O-deethylation rate as a func-
tion of substrate concentration. While the ratio of RAFs
for the major drug-metabolizing human CYP isoforms 1A2,
2D6, and 3A4 (determined using 7-ethoxyresorufin O-
deethylation, high affinity component of nortriptyline 10-
hydroxylation, and diazepam 3-hydroxylation as index
reactions, respectively, in a panel of 12 human livers)
(Venkatakrishnan, K. et al.: unpublished data) are similar
to the ratio of their immunoquantified levels reported
previously,8 this is not true for CYP2C9 and 2C19,19 as
described earlier. On the basis of existing data, we have
assumed that the ratio of immunoquantified levels together
with the necessary correction for CYP2C9:CYP2C19 RAF
ratio can be used in the simulation analysis (eq 3) for
predicting relative contributions. This assumption only
allows the scaling approach to predict relative contribu-
tions, but not absolute liver microsomal reaction rates. This
is because, although the ratio of RAFs for the major CYP
isoforms is similar to that of immunoquantified levels
(corrected for 2C9:2C19 ratio), the absolute magnitude of
the RAFs is higher, probably suggestive of lower turnover
numbers of cDNA-expressed enzymes when compared to
their counterparts in human liver microsomes. This
limitation of the scaling approach became evident when
the kinetic data from cDNA-expressed enzyme studies was
used in conjunction with previously published values of
average concentrations of CYP isoforms in human liver
(corrected for CYP2C9:2C19 ratio)8,19 to predict average
rates of phenacetin O-deethylation at two fixed substrate
concentrations: 40 µM and 1000 µM. The predicted liver
microsomal rates are 60 and 296 pmoles/min/mg protein,
respectively. However, in a panel of 12 human liver
Kinetic studies on cDNA-expressed human CYP iso-
forms, and chemical inhibition studies, both have been used
in the present study to predict the relative contribution of
human liver microsomal CYP isoforms to net phenacetin
O-deethylation rate as a function of substrate concentra-
tion. Both approaches have intrinsic limitations. Chemical
inhibitors are not absolutely specific and frequently inhibit
multiple CYP isoforms, requiring a careful selection of
inhibitor concentration. Despite a prudent choice of inhibi-
tor concentrations, inhibitors such as omeprazole (2C19)
and diethyldithiocarbamic acid (2E1) are only relatively
selective, and results of inhibition studies with these agents
have to be interpreted with caution, especially when the
1506 / Journal of Pharmaceutical Sciences
Vol. 87, No. 12, December 1998

9. Shimada, T.; Mimura, M.; Inoue, K.; Nakamura, S.; Oda, H.;
Ohmori, S.; Yamazaki, H. Cytochrome P450-dependent drug
oxidation activities in liver microsomes of various animal
species including rats, guinea pigs, dogs, monkeys, and
humans. Arch. Toxicol. 1997, 71, 401-408.
10. J ensen, K. G.; Poulsen, H. E.; Doehmer, J .; Loft, S. Kinetics
and inhibition by fluvoxamine of phenacetin O-deethylation
in V79 cells expressing human CYP1A2. Pharmacol. Toxicol.
1995, 76, 286-288.
11. Rodrigues, A. D.; Surber, B. W.; Yao, Y.; Wong, S. L.; Roberts,
E. M. [O-ethyl14C]Phenacetin O-deethylase activity in human
liver microsomes. Drug Metab. Dispos. 1997, 25(9), 1097-
1100.
12. Crespi, C. L. Xenobiotic metabolizing human cells as tools
for pharmacological and toxicological research. Adv. in Drug
Res. 1995, 26, 179-235.
microsomes, the observed reaction rates were 540 ( 62 and
1718 ( 125 pmoles/min/mg protein (mean ( SEM) at 40
µM and 1000 µM phenacetin, respectively. This suggests
that immunoquantified CYP isoform levels may not be
suitable scaling factors to predict absolute liver microsomal
rates of drug biotransformations from kinetic studies on
cDNA-expressed CYP isoforms. Their use in simulating
the relative contribution of CYP isoforms to net reaction
rate as a function of substrate concentration (such as the
simulation in Figure 4) is contingent upon the requirement
that the ratio of the immunoquantified levels is similar to
that of the true scaling factors, the identity of which
remains to be determined.
A limitation of the scaling approach used here is that it
does not account for the interindividual variability in
human liver microsomal levels of CYP isoforms. The levels
of expression of CYP1A2 as well as the other isoforms
investigated here are highly variable. The simulation
presented in Figure 4 is thus only an average picture of
the relative contributions of the various CYP isoforms to
phenacetin O-deethylation rate as a function of substrate
concentration. Variable inhibition from liver to liver by
isoform-selective chemical inhibitors is also expected due
to interindividual variability in the levels of expression of
the CYP isoforms when multiple enzymes are involved.
In conclusion, phenacetin O-deethylation is catalyzed by
six distinct human CYP isoforms. CYP1A2 is the only high
affinity isoform in uninduced human liver and accounts for
greater than 80% of the reaction rate at low substrate
concentrations (50-100 µM phenacetin). It can be pre-
dicted from kinetic studies on heterologously expressed
CYPs, and chemical inhibition studies on human liver
microsomes, that CYP2C9 may contribute significantly to
the reaction at high concentrations of substrate, with
additional minor contributions of CYP2A6, 2C19, 2D6, and
2E1.
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Acknowledgments
This work was supported by Grants MH-34223, DA-05258, MH-
19924, and RR-00054 from the Department of Health and Human
Services. Dr. von Moltke is the recipient of a Scientist Development
Award (K21-MH-01237) from the National Institutes of Mental
Health.
J S980255Z
Journal of Pharmaceutical Sciences / 1507
Vol. 87, No. 12, December 1998