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Effect of the Treatment Period With Erythromycin on Cytochrome P450 3A Activity in Humans
Toshiaki Okudaira, Tsutomu Kotegawa, Hiromitsu Imai, Kimiko Tsutsumi, Shigeyuki Nakano and Kyoichi Ohashi
J. Clin. Pharmacol. 2007; 47; 871
DOI: 10.1177/0091270007302562

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Effect of the Treatment Period With
Erythromycin on Cytochrome
P450 3A Activity in Humans
Toshiaki Okudaira, MD, Tsutomu Kotegawa, MD, PhD, Hiromitsu Imai, MD,
Kimiko Tsutsumi, PhD, Shigeyuki Nakano, MD, PhD, and Kyoichi Ohashi, MD, PhD
The aim of the present study was to estimate the time
course change in cytochrome P450 3A (CYP3A) activity
during repeated doses of erythromycin. Twelve healthy
male volunteers participated in this randomized, 4 × 4 Latin
square design study. The pharmacokinetics of a single oral
dose of midazolam, a probe for CYP3A activity, were
assessed in 4 conditions: (1) midazolam (5 mg) without ery-
thromycin (EM0), (2) erythromycin 2 days + midazolam (2.5
mg) (EM2), (3) erythromycin 4 days + midazolam (2.5 mg)
(EM4), and (4) erythromycin 7 days + midazolam (2.5 mg)
(EM7). The dose of erythromycin was 800 mg/d. Erythromycin
produced a 2.3-, 3.4-, and 3.4-fold increase in dose-corrected
area under the curve of midazolam for EM2, EM4, and EM7,
respectively, as compared with EM0 (P <.05/6). A significant
E rythromycin, a macrolide antimicrobial agent, has
a potent inhibitory effect on cytochrome P450 3A
(CYP3A).1,2 Because of this property, erythromycin has
often been used as a representative CYP3A inhibitor in
drug interaction studies. The mechanism of CYP inhi-
bition by erythromycin is an inactivation of enzyme
attributable to irreversible binding of erythromycin
metabolite to CYP3A.3,4 A compound that elicits this
type of enzyme inactivation results in a time-dependent
decrease in the remaining intact enzyme,5,6 suggest-
ing that the magnitude of drug interactions can be
enhanced by a longer treatment period with inactiva-
tors including erythromycin.
Many studies have shown that pretreatment with
erythromycin produces significant increases in plasma
From the Department of Clinical Pharmacology and Therapeutics, Oita
University Faculty of Medicine, Oita, Japan. Address for correspondence:
Tsutomu Kotegawa, MD, PhD, Department of Clinical Pharmacology
and Therapeutics, Oita University Faculty of Medicine, 1-1 Idaigaoka,
Hasama-machi, Yufu-city, Oita, 879-5593, Japan.
DOI: 10.1177/0091270007302562
J Clin Pharmacol 2007;47:871-876
DRUG METABOLISM
prolongation of terminal half-life was observed in EM4
and EM7. The relationship between the duration of ery-
thromycin treatment and total clearance of midazolam
indicated that a plateau level of CYP3A inhibition can be
achieved by 4 days or more of erythromycin treatment. The
repeated treatment with erythromycin yields CYP3A inhibi-
tion in a duration-dependent manner. A 4-day course of
erythromycin treatment produces 90% or more of the max-
imal inhibition of CYP3A in humans.
Keywords: Erythromycin; cytochrome P450 3A; treat-
ment period; drug interaction; midazolam
Journal of Clinical Pharmacology, 2007;47:871-876
© 2007 the American College of Clinical Pharmacology
concentrations of clinically important drugs such as
immune modulators,7 HIV antivirals,8 theophylline,9,10
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase inhibitors,11 and benzodiazepines.12,13
However, these studies were performed with varied
treatment periods of 2 to 10 days of erythromycin,
which may affect the extent of drug interactions via
CYP3A inhibition. In clinical practice for mycoplasma
and chlamydial infection, a 14- to 21-day course of
erythromycin is usually prescribed.14,15 Data obtained
from interaction studies with a shorter treatment
period of erythromycin may underestimate the extent
of a drug interaction occurring in clinical practice.
Information on the time course change in CYP3A
activity after initiation of the repeated treatment with
erythromycin may help in estimating the magnitude
of interaction in various clinical situations.
When one is designing an interaction study, the
dosage of inhibitors should be carefully determined
to produce a sufficient inhibition of the enzyme.
However, an excessive dosage should be avoided to
minimize the risk of adverse reactions occurring in
871
 OKUDAIRA ET AL
volunteers. Data concerning the relationship between
the dosing period and enzyme activity, if available,
would provide information for designing interaction
studies with an adequate dosage.
Midazolam is an established in vivo probe for
human CYP3A activity and is not a substrate of p-
glycoprotein.16 In the present study, using midazolam,
we aimed to estimate the time course change in CYP3A
activity during repeated doses of erythromycin.
MATERIALS AND METHODS
Subjects
The Institutional Review Board of Oita University
Hospital reviewed and approved the protocol. Twelve
healthy male volunteers, aged 21 to 28 years and
weighing 54.3 to 73.4 kg (body mass index 18.3-25.9
kg/m2), participated in the present study after giving
written informed consent. Before entering the study,
each subject underwent a routine physical examina-
tion and laboratory investigations. None of the subjects
were taking other medications. None had a history
of either medical disease or smoking. The subjects
were asked not to consume grapefruit juice and herbal
dietary supplements including St John’s wort from 2
weeks before the first trial until the end of the last
trial. Alcoholic drinks and caffeine-containing food
and beverages were not allowed during study days.
Study Design
A randomized, open label, 4 × 4 Latin square design
was used. The 4-way conditions were (1) only mida-
zolam (EM0), (2) erythromycin treatment for 2 days +
midazolam (EM2), (3) erythromycin treatment for 4
days + midazolam (EM4), and (4) erythromycin treat-
ment for 7 days + midazolam (EM7). The interval of
each dosage of midazolam was at least 2 weeks.
Procedures
Subjects were administered erythromycin 200 mg
(Erythrocin; Dainippon Pharmaceutical Co, Osaka,
Japan) 4 times a day (08:00, 13:00, 18:30, 23:00) for
2, 4, or 7 days. On the study day (the last day of
erythromycin treatment) at 07:00, the subjects
entered the Clinical Pharmacology Center at Oita
University Faculty of Medicine. Subjects took a stan-
dardized light breakfast at 07:20. They ingested ery-
thromycin with 150 mL of water at 08:00, followed
by a single oral dose of midazolam 5 mg in EM0 or
872 • J Clin Pharmacol 2007;47:871-876
2.5 mg in EM2, EM4, and EM7 (Dormicum, injectable
solution; Astellas Pharma Inc, Tokyo, Japan) at 09:00.
The reduced dose of midazolam (2.5 mg) was to
minimize the risk of adverse reactions such as exces-
sive sedation and fall caused by the interaction of
midazolam and erythromycin. The doses of midazo-
lam were chosen with an assumption that the phar-
macokinetics are linear over this dose range. This
linearity of midazolam pharmacokinetics was shown
within a dose range of up to 15 mg although it
became nonlinear between doses of 15 and 30 mg.17
For additional administration of erythromycin on
the study day (13:00, 18:30, 23:00) and blood sam-
pling, subjects remained in the Clinical Pharmacology
Center for 25 hours. Blood samples were obtained
with a catheter placed in a forearm vein that was
kept patent with heparinized saline. Venous blood
samples (9 mL) were collected before and 0.33, 0.66,
1, 1.5, 2, 3, 5, 8, 14, and 22 hours after midazolam
administration. Samples were collected in heparinized
tubes and centrifuged at 3000 × g for 10 minutes.
The separated plasma was stored at –20°C until the
time of assay.
Drug Assay
Plasma concentrations of midazolam were assayed by
high-performance liquid chromatography. Plasma
was extracted as follows. One hundred microliters of
1N NaOH, 20 ng of flurazepam as internal standard
(IS), and 500 µL or 1 mL of plasma were applied to the
extraction column (Extrelut NT1, Merk, Darmstadt,
German). One milliliter of plasma was extracted at 5,
8, 14, and 22 hours after administration, at which
lower levels of midazolam were anticipated. Ten min-
utes later each sample was extracted twice with 4 mL
of extraction liquid (70% heptane, 30% chloroform).
The extracts were collected in a glass tube and evap-
orated to dryness. The residue was reconstructed in
200 µL of mobile phase, and a 100-µL aliquot was
injected into an analytical column (XterraTM RP18 5
µm, 4.6 ×150 mm, Waters, Milford, Mont). The mobile
phase consisted of 10 mM ammonium acetate buffer
(pH 8.2), acetonitrile, and methanol (65:26:9, v/v/v).
The flow rate was 1.5 mL/min. Peaks were quantified
by an ultraviolet absorbance detector (2487λUV/VIS
Detector, Waters, Milford, Mont) at 230 nm. The
retention times of midazolam and IS were 17 and 22
minutes, respectively. The standard curves for mida-
zolam were linear within the ranges of 1 to 100 ng/mL
in plasma. The correlation coefficients between the
peak height ratios (drug/IS) and the concentrations of
midazolam were >0.999. Coefficients of intra-assay
 CYTOCHROME P450 3A ACTIVITY IN HUMANS

Statistical Analysis
Plasma midazolam concentration

25
EM 0 Data were analyzed using a statistical program (SPSS
EM 2
20
EM 4
12.0J; SPSS Inc, Chicago, Ill). Pharmacokinetic para-
meters among 4 trials were compared by the
•2.5mg/dose (ng/mL)

EM 7
15
Wilcoxon signed rank test. Differences were regarded
as statistically significant if P values were below
10
.05/6 (Bonferroni correction).
5
RESULTS
0
0 5 10 15 20
Time after administration (h)
Pharmacokinetics

All 12 enrolled subjects completed the study. No

Figure 1. Plasma midazolam concentration–time curves (mean ±
SD). Open circles = control (EM0); solid triangles = 2 days of treat-
ment with erythromycin (EM2); open squares = 4 days of treatment
with erythromycin (EM4); solid squares = 7 days of treatment with
erythromycin (EM7).
and interassay validations at 1 ng/mL were 13.7%
and 8.5%, respectively. All samples were assayed in
duplicate.
Pharmacokinetics of Midazolam
The actual data were used for peak plasma concentra-
tion (Cmax) and time of peak concentration (tmax). The
elimination constant ke was calculated by log-linear
regression in the terminal elimination phase. The ter-
minal half-life (t1/2) was calculated by ln2/ke. The area
under the plasma concentration–time curve (AUC0-t)
was determined by the linear trapezoidal method.
This was extrapolated to infinity using ke (AUC0-∞).
Total clearance (CL/F) was calculated from the
dose/AUC0-∞ ratio.
adverse events were observed. Plasma midazolam
concentration–time curves and pharmacokinetic para-
meters of midazolam are shown in Figure 1 and Table I,
respectively. The time course changes in individual
CL/F are shown in Figure 2. Erythromycin signifi-
cantly increased Cmax, increased dose-corrected AUC0-∞,
and decreased CL/F. The increases in dose-corrected
AUC0-∞ were 2.3-, 3.4-, and 3.4-fold for EM2, EM4, and
EM7, respectively. A significant prolongation of t1/2
was observed in EM4 and EM7 but not in EM2.
DISCUSSION
The present study showed that CL/F of midazolam dur-
ing the repeated dosing of erythromycin progressively
decreased, indicating that the degree of inhibition of
CYP3A by erythromycin depends on the duration of
administration. Erythromycin is a well-known mecha-
nism-based inactivator.3,4 Erythromycin metabolite
binds to hem iron of CYP and forms metabolite-
intermediate (MI) complex, thereby irreversibly
inactivating the enzyme. Therefore, the inhibition

Table I Pharmacokinetic Parameters of Midazolam

Parameter EM0 EM2 EM4 EM7

Cmax, ng/mLa 11.0 ± 2.6 20.0 ± 5.9* 26.4 ± 8.7* 25.8 ± 9.9*
tmax, h 1.5 (0.67-2) 1 (0.33-2) 1 (0.67-2) 1 (0.33-2)
t1/2, h 2.3 ± 0.9 3.4 ± 1.9 5.1 ± 2.8* 6.9 ± 4.4*,**
AUC0-∞, ng ⋅ h/mLa 35.8 ± 7.8 83.1 ± 37.7* 118.7 ± 57.0* 119.2 ± 50.8*
AUC0-∞, ratio vs controla 1.0 2.32 ± 0.9 3.38 ± 1.6 3.38 ± 1.4
CL/F, mL/min/kg 19.3 ± 5.5 9.4 ± 4.2* 6.5 ± 2.5* 6.6 ± 3.0*
Values are mean ± SD or median (minimum-maximum), n = 12. EM0, control; EM2, 2 days of treatment with erythromycin; EM4, 4 days of treatment
with erythromycin; EM7, 7 days of treatment with erythromycin; Cmax, peak plasma concentration; tmax, time of peak concentration; t1/2, terminal half-
life; AUC, area under the curve; CL/F, total clearance.
a. Dose corrected value (Cmax and AUC values were divided by dose/2.5mg),
*Compared with EM0 P < .05/6.
**EM2 vs EM7 P < .05/6.

DRUG METABOLISM 873

 OKUDAIRA ET AL
of CYP3A by erythromycin is regulated not only by
the pharmacokinetics of erythromycin and its
metabolites but also by the formation rate of MI
complex and by the reproduction of CYP3A.
Takedomi et al18 reported that repeated treatment
with erythromycin duration-dependently increased
MI complex content in the liver of rat without affect-
ing the total content of CYP. In their study, a progres-
sive increase in AUC of midazolam with duration of
erythromycin treatment (up to 4 days) was also
observed. It is conceivable that the duration-
dependent inhibition of CYP3A by erythromycin
observed in this study is attributable to a progressive
reduction of the intact CYP3A remaining in the body.
A number of studies have reported the dosing
period–dependent effect of erythromycin on the phar-
macokinetics of CYP3A substrates. A 7-day course of
erythromycin significantly decreased clearance of
alfentanil, although 1 day of treatment with ery-
thromycin did not significantly affect the pharmacoki-
netics of alfentanil.19 For theophylline, a CYP1A2 and
3A4 substrate, 10 days of treatment with erythromycin
produced a significant prolongation of t1/2 and a
decrease of clearance, but 3 days of treatment with ery-
thromycin did not.9 This delayed onset of the effect
of erythromycin may be attributable not only to the
duration-dependent inhibition of CYP3A but also to
less contribution of CYP3A in the metabolism of theo-
phylline as compared with CYP1A2. Diltiazem is also
a mechanism-based inactivator of CYP3A.6 Ohashi
et al20 studied the effect of a single dose and multiple
doses (4 and 7 days of treatment) of diltiazem on the
pharmacokinetics of nifedipine. The study showed that
diltiazem increased AUC of nifedipine in a duration-
dependent manner. Together with the results in the
present study, it was confirmed that the dosing period
of mechanism-based inactivators affected the extent of
interaction in vivo.
In the present study, 4 and 7 days of treatments with
erythromycin produced the same magnitude (3.38-
fold) of increases in AUC of midazolam, suggesting
that a plateau level of CYP3A inhibition can be
achieved by 4 days or more of erythromycin treat-
ment. Concerning the magnitude of the change in the
pharmacokinetics of midazolam caused by CYP3A
inhibitors, Tsunoda et al21 found that 3 doses of 200
mg of oral ketoconazole, which is one of the most
potent inhibitors of CYP3A, increased AUC of mida-
zolam by 16-fold. On the other hand, the increase in
AUC of midazolam was 4.4-fold with a 7-day treat-
ment with erythromycin 1500 mg/d.1 These data sug-
gest that the magnitude of inhibition by a usual dosage
of erythromycin may be far less than that produced by
874 • J Clin Pharmacol 2007;47:871-876
ketoconazole. It is likely that erythromycin causes a
partial inhibition of midazolam clearance even at the
maximum inhibition with infinite time exposure to
erythromycin. Assuming that CL/F of midazolam
during repeated dosing of erythromycin (CL/F t)
decreases asymptotically toward CL/F at the maxi-
mum inhibition with infinite time following a first-
order process, the half-life of the decrease in CL/F was
estimated as 22.8 hours (Figure 2). Using the half-life
of the decrease in CL/F, 1 and 3 days of treatment with
erythromycin produces approximately 50% and 90%
of the maximal decrement of CL/F, respectively. These
estimations suggest that a sufficient inhibition of
CYP3A can be obtained by 3 days or longer of ery-
thromycin treatment in interaction studies. When
designing an interaction study, excessive dosage
should be avoided to minimize the risk of adverse
reactions occurring in volunteers. The results obtained
in the current study may contribute to a suitable
dosage regimen of erythromycin used for interaction
studies in humans. However, the extent of the change
in the pharmacokinetics of midazolam showed a large
interindividual difference. Greater SD values were
observed in AUC of midazolam after erythromycin
treatment (EM2, EM4, and EM7) compared with con-
trol (EM0) (Table I), indicating that the extent of inhi-
bition of CYP3A by erythromycin shows a large
interindividual difference. One of the factors explain-
ing this large interindividual difference is the genetic
polymorphism of CYP3A5, which was not examined
in this study. The polymorphic CYP3A5 expression in
humans is strongly dependent on the presence of the
CYP3A5*3 allele.22 An in vitro study showed that ver-
apamil, a mechanism-based inhibitor of CYP3A, and
its metabolite inhibited CYP3A5 to a lesser extent
compared with CYP3A4.23 This suggests that the pres-
ence of CYP3A5 affects the extent of CYP3A inhibi-
tion caused by mechanism-based inactivators. Further
studies are necessary to examine the influence of the
polymorphism of CYP3A5 on interactions of ery-
thromycin and CYP3A substrates in vivo.
Midazolam undergoes an extensive first-pass
metabolism with oral bioavailability less than 50%.24
Both intestinal CYP3A and hepatic CYP3A contribute
to the first-pass metabolism and are subject to the inhi-
bition by erythromycin. Therefore, the time course
change in midazolam pharmacokinetics observed in
the current study reflects the combined changes in
both hepatic and intestinal CYP3A activity. However,
the present study could not separate the effect sites
(the intestine or liver) of erythromycin because only
oral midazolam was administered. Further studies are
necessary concerning the difference in the time course
 CYTOCHROME P450 3A ACTIVITY IN HUMANS
35
30
25
CL/F (mL/min/kg)
20
half life 22.8 hr
15
10
5 azithromycin on the pharmacokinetics and pharmacodynamics of
0 3. Franklin MR. Cytochrome P450 metabolic intermediate com-
0 50 100 150
Erythromycin treatment periods (hr)
Figure 2. The relationship between the duration of ery-
thromycin treatment and total clearance of midazolam (CL/F).
Hairline = individual data; open square = mean values of CL/F;
thick line = a line fitted to mean values of CL/F.
change in CYP3A activity between organs.
Thus far, most interaction studies with erythromycin
have adopted a 3- to 7-day course of erythromycin treat-
ment. The results of the present study indicate that 4
days or longer of erythromycin treatment used by other
studies is sufficient to obtain a maximum inhibition of
CYP3A. However, the magnitude of inhibition depends
on the dose of inhibitor as well. The 7-day treatment
with erythromycin caused a 3.4-fold increase in mida-
zolam AUC in the present study. Olkkola et al reported
that the same duration (7 days) of erythromycin treat-
ment increased AUC of midazolam more than 4 times.1
This greater increase observed by Olkkola et al1 is attrib-
utable to the dose of erythromycin (800 vs 1500 mg/d).
In clinical practice, the treatment of mycoplasma or
chlamydial infection requires a 14-day or longer course
of erythromycin 2 g/d.14,15 It is likely that the greater
dose of erythromycin used in clinical practice produces
a more potent CYP3A inhibition compared with that
observed in the current study.
CONCLUSIONS
The repeated treatment with erythromycin yields
CYP3A inhibition in a duration-dependent manner. It
is likely that a 4-day course of erythromycin treatment
is sufficient to obtain 90% or more of the maximal
DRUG METABOLISM
inhibition of CYP3A in humans. Further information
on the dose of erythromycin and CYP3A5 polymor-
phism may allow a more accurate prediction of the
extent of interaction in vivo.
Financial disclosure: None declared.
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