CLINICAL SCIENCE

Lopinavir/Ritonavir Induces the Hepatic Activity of

Cytochrome P450 Enzymes CYP2C9, CYP2C19, and
CYP1A2 But Inhibits the Hepatic and Intestinal Activity
of CYP3A as Measured by a Phenotyping Drug Cocktail
in Healthy Volunteers
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Rosa F. Yeh, PharmD,* Vincent E. Gaver, PharmD,* Kristine B. Patterson, MD,Þ Naser L. Rezk, MS,*
Faustina Baxter-Meheux, BS,* Michael J. Blake, MD, PhD,þ Joseph J. Eron, Jr, MD,Þ
Cheri E. Klein, PhD,§ John C. Rublein, PharmD,§ and Angela D.M. Kashuba, PharmD*

ratios. Hepatic and intestinal + hepatic CYP3A activities were

Objective: The effect of lopinavir/ritonavir (LPV/r) administration
quantified by IV (CL) and PO (CL/F) MDZ clearance, respectively.
on cytochrome P450 (CYP) enzyme activity was quantified using a
Results: After LPV/r therapy, CYP2C9, CYP2C19, and CYP1A2 ac-
phenotyping biomarker cocktail. Changes in CYP2C9, CYP2C19,
tivity increased by 29%, 100%, and 43% (P = 0.001, 0.046, and 0.001),
CYP3A, CYP1A2, N-acetyltransferase-2 (NAT-2), and xanthine
respectively. No changes were seen in NAT-2 or XO activity. Hepatic
oxidase (XO) activities were evaluated using warfarin (WARF) +
and intestinal + hepatic CYP3A activity decreased by 77% (P G 0.001)
vitamin K, omeprazole (OMP), intravenous (IV) and oral (PO)
and 92% (P = 0.001), respectively.
midazolam (MDZ), and caffeine (CAF).
Conclusion: LPV/r therapy results in modest induction of
Design: Open-label, multiple-dose, pharmacokinetic study in
CYP1A2 and CYP2C9 and potent induction of CYP2C19 activity.
healthy volunteers.
Increasing doses of concomitant medications metabolized by these
Methods: Subjects (n = 14) simultaneously received PO WARF
enzymes may be necessary. LPV/r inhibited intestinal CYP3A
10 mg, vitamin K 10 mg, OMP 40 mg, CAF 2 mg/kg, and IV
to a greater extent than hepatic CYP3A activity. Doses of
MDZ 0.025 mg/kg on days (D) 1 and 14, and PO MDZ 5 mg on
concomitant CYP3A substrates should be reduced when combined
D2 and D15. LPV/r (400/100 mg twice daily) was administered on
with LPV/r, although intravenously administered compounds may
D4-17. CYP2C9 and CYP2C19 activities were quantified by S-WARF
require less of a relative dose reduction than orally administered
AUC0-inf and OMP/5-hydroxy OMP ratio, respectively. CYP1A2,
compounds.
NAT-2, and XO activities were quantified by urinary CAF metabolite
Key Words: lopinavir, ritonavir, pharmacokinetics, protease
inhibitors, cytochrome P450 enzyme system, induction, inhibition
Received for publication November 13, 2005; accepted February 1, 2006.
From the *School of Pharmacy, and †School of Medicine, University of
(J Acquir Immune Defic Syndr 2006;42:52Y60)
North Carolina at Chapel Hill, NC; ‡The Division of Pediatric
Pharmacology and Medical Toxicology, Pediatric Pharmacology Re-
search Unit, Children’s Mercy Hospital and Clinics, Kansas City, MO;
and §Abbott Laboratories, Clinical Pharmacokinetics and Antiviral
Global Team, Abbott Park, IL.
I n the treatment of HIV infection, drug interactions can be
detrimental to effective antiretroviral therapy as well as to
the treatment of other disease processes. HIV-infected
Funding source: Research grant support was provided by Abbott Laborato-
ries, the UNC Center for AIDS Research (P30A150410), the UNC patients usually take at least 3 antiretroviral agents and may
General Clinical Research Center (RR00046), the UNC Clinical Research receive other medications for supportive care, opportunistic
Nutrition Research Center (DK56350), AI54980 (A.D.M.K.), and the infections, and immunomodulation, in addition to using
Pediatric Pharmacology Research Unit, Children’s Mercy Hospitals and nutraceutical compounds for general well-being. Due to the
Clinics (grant 5 U10 HD01313-12, National Institute of Child Health and
Human Development, M.J.B.).
multiple effects of the nonnucleoside reverse transcriptase
Prior presentation: This research has been presented in part at the 5th inhibitors and protease inhibitors (PIs) on the cytochrome
International Workshop on Clinical Pharmacology of HIV Therapy, Rome, P450 (CYP) enzyme system, drug interactions are often
Italy, on April 1Y3, 2004, and the 6th International Workshop on Clinical unavoidable.1 The fixed-dose formulation of lopinavir (LPV)
Pharmacology of HIV Therapy, Quebec, Canada, on April 28Y30, 2005. and ritonavir (RTV) utilizes RTV’s potent inhibition of
Conflict of interest statement: Dr Eron is an ad hoc consultant to Abbott
Laboratories and receives support for conduct of clinical trials sponsored by CYP3A activity to enhance the systemic exposure of LPV.2
Abbott Laboratories through the University of North Carolina. Drs Klein CYP3A is the major enzyme subfamily responsible for the
and Rublein are employed by Abbott Laboratories. The remaining authors metabolism of most PIs and nonnucleoside reverse transcrip-
do not have conflicts of interest related to this research or manuscript. tase inhibitors. It is clear that lopinavir/ritonavir (LPV/r)
Reprints: Angela D.M. Kashuba, PharmD, 3318 Kerr Hall, CB#7360, School of
Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC
affects CYP3A enzyme activity, as many drug interactions
27599 (e-mail: akashuba@unc.edu). have been noted between LPV/r and other CYP3A sub-
Copyright * 2006 by Lippincott Williams & Wilkins strates.3Y5 However, very little data are available on the effect

52 J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006

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J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006
of LPV/r on other CYP isoforms. In vitro experiments suggest
no significant inhibitory effects on other CYP isoenzymes,6
but inductive effects have not been carefully studied. A
number of clinical reports have shown decreased exposure to
phenytoin,7 warfarin,8,9 methadone,10Y13 and ethinyl estradiol,10
but the mechanisms for these are unclear.
The primary objective of this study was to quantify the
influence of 10 days of LPV/r administration on the activity of
the 4 CYP isozymes responsible for metabolizing the majority
of drugs currently available. To accomplish this, we adminis-
tered a modified BCooperstown 5 + 1 cocktail^ consisting of
caffeine (CAF, a substrate for CYP1A2, N-acetyltransferase-2
[NAT-2], and xanthine oxidase [XO]), warfarin (WARF, a
substrate for CYP2C9), omeprazole (OMP, a substrate for
CYP2C19), and intravenous (IV) and oral (PO) midazolam
(MDZ, a substrate for CYP3A, but not P-glycoprotein).14,15
These data were generated to further understand currently
documented drug interactions with LPV/r and to predict the
extent of as-yet-undocumented drug interactions.
METHODS
Subjects
Sixteen volunteers were screened and assessed to be
healthy by physical examination, medical history, and
laboratory evaluations. Subjects weighed at least 50 kg,
were 18 to 45 years of age, and were HIV-1 seronegative.
Subjects were excluded if they had significant medical
conditions or laboratory abnormalities; had a history of
allergy to any study medication; were 20% more or less than
their ideal body weight; used any prescription or over-the-
counter medications, vitamin, or herbal supplements on a
regular basis; had a history of alcohol or drug abuse; were
unable to abstain from alcohol or grapefruit juice for the
duration of the study; used tobacco products within 2 months
before the first study visit; or had a baseline international
normalized ratio (INR) greater than 1.2. Sexually active
women were required to have a negative urine pregnancy
test and to use a barrier form of birth control for the duration
of the study. The University of North Carolina (UNC)
institutional review board approved the study protocol, and
all subjects provided written informed consent before any
study procedures.
Materials
LPV/r (Kaletra) soft gelatin capsules were obtained from
Abbott Laboratories (Abbott Park, IL). CAF 2-mg/mL PO
solution (formulated from CAF citrate purified powder,
Mallinckrodt, Phillipsburg, NJ), WARF 10-mg tablets (war-
farin, Dupont Pharmaceuticals, Newark, DE), vitamin K 10-mg
tablets (Mephyton, Merck and Co, Inc, Whitehouse Station,
NJ), OMP 40-mg capsules (Prilosec, AstraZeneca, Wilming-
ton, DE), MDZ 5-mg tablets (MDZ, Bedford Laboratories,
Bedford, OH), and MDZ 1-mg/mL IV solution (MDZ 2-mL
vials, Bedford Laboratories, Bedford, OH) were dispensed
from the UNC Hospitals Investigational Drug Service
pharmacy. Vitamin K was administered to prevent the phar-
macologic effect of WARF without altering the pharmacoki-
netics of the WARF stereoisomers.16
* 2006 Lippincott Williams & Wilkins
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Lopinavir/Ritonavir and Cytochrome P450 Activity
Study Design
This was an open-label, pharmacokinetic (PK) study
consisting of two 48-hour inpatient visits at the Verne S.
Caviness General Clinical Research Center (GCRC) at UNC
Hospitals and 2 follow-up outpatient visits (days 4 and 17).
The duration of the study was 17 days. As previous
investigations have not noted significant menstrual cycle
effects on phenotyping biomarker data, this study did not
control for menstrual cycle.17Y22
On days 1 and 14, all subjects were admitted to the
GCRC. After a peripheral IV line was placed for blood
collection, patients emptied their bladder and were dosed
with the phenotyping medications. On the first day of each PK
visit (days 1 and 14), each subject received simultaneous
administration of 4 PO medications (CAF 2 mg/kg, WARF
10 mg, vitamin K 10 mg, and OMP 40 mg) and 1 IV medication
(MDZ 0.025 mg/kg). CAF metabolites were continuously
collected in urine over 12 hours. Blood samples for WARF
were collected just before WARF administration and then at 3,
6, 9, 12, 24, 48, and 72 hours afterward. Blood samples for OMP
and 5-hydroxy metabolite (5-OH OMP) were collected 2 hours
after OMP administration. Blood samples for IV MDZ for the
first PK visit were collected just before MDZ administration
and at 5, 15, and 30 minutes and 1, 1.5, 2, 3, 4, 6, 8, 12, and
24 hours afterward.
During inpatient visits, standardized research meals
were served consisting of approximately 15% protein, 30%
fat, and 55% carbohydrate for a total daily intake of 2000 to
2500 calories. Meals were finished within 30 minutes and
given at least 2 hours after administration of phenotyping
medications. Nutrient intake analysis was performed by a
dietitian after each meal. No additional CAF was permitted.
Twenty-four hours after the first phenotyping medica-
tions were administered, on days 2 and 15, each subject
received an PO dose of MDZ 5 mg. Blood samples for PO
MDZ were collected just before MDZ administration and at
5, 15, and 30 minutes and 1, 1.5, 2, 3, 4, 6, 8, 12, 18, and
24 hours afterward.
Subjects were discharged from the GCRC approxi-
mately 24 hours after the PO MDZ dose, on days 3 and 16.
Subjects returned 24 hours later, on days 4 and 17, for an
outpatient visit to obtain a 72-hour WARF blood sample. At
day 4 outpatient visit, subjects took their first dose of LPV/r
soft gelatin capsules, administered orally at the usual clinical
dose of 400 LPV/100 mg RTV twice daily. Subjects were
advised to take LPV/r with food. Medication adherence for
this 10-day period was assessed by pill counts performed by
the study coordinator on day 15 and dosing administration
cards completed daily by the study subject. These data were
reviewed by the study coordinator before initiation of the
second inpatient study visit (day 14). Subjects with less than
95% adherence were removed from the study. In addition, to
further assess adherence, a blood sample for LPV and RTV
concentrations was drawn upon GCRC admission, before
administration of the phenotyping medications. LPV/r was
continued through day 17. During the second PK visit (PK 2,
days 14Y17), phenotyping procedures were performed in the
same manner as during days 1 to 4 (PK 1). During PK 2,
LPV/r administration was directly observed and given with
53
Yeh et al
food, at least 2 hours after administration of the phenotyping
medications.
Safety assessment at each PK visit included blood
laboratory testing (complete blood count with differential,
prothrombin time/INR, chemistry panel, liver function tests, and
urine pregnancy test), physical examination by study physician,
and adverse event assessment by the study coordinator. With
MDZ, respiratory rate, oxygenation, and mean arterial pressure
(MAP) were monitored every 15 minutes for 60 minutes after IV
administration and every 15 minutes for 120 minutes after PO
administration. Flumazenil was available at the bedside during
the entire inpatient stay. Adverse events were assessed using the
Adult AIDS Clinical Trials Group grading scale23 at initiation
and conclusion of both inpatient visits and outpatient visits.
Subjects were contacted by the study coordinator during the first
week of LPV/r administration to ensure medication tolerability
and to assess any early adverse event occurrences.
Analytical Methods
Blood samples were collected in vacutainers containing
8.55 mg K3EDTA (15% additive solution) as an anticoagulant
and kept on ice after collection for a maximum of 15 minutes.
Blood plasma was separated by centrifugation at 2800 rpm for
15 minutes at 4-C. During the study visit, plasma was stored at
the GCRC at j20-C. At the end of the study visit, frozen
plasma samples were transferred to a temperature-monitored
freezer, j80-C, for storage until analysis.
Urine containers were kept refrigerated at 4-C during the
12-hour urine collection. After collection, total urine volume
was measured, and 15-mL aliquots of urine were combined
with additional ascorbic acid to maintain a pH of less than 4.
These samples were frozen at j80-C until analysis.
Caffeine
Urinary concentrations of 5-acetylamino-6-formyl-
amino-3-methyluracil (AFMU), 1-methylxanthine (1X),
1-methyluric acid (1U), and 1,7-dimethyluric acid (17U)
were determined by reverse-phase high-performance
liquid chromatography with diode-array ultraviolet detec-
tion (Agilent 1100, Agilent Technologies, Palo Alto,
CA).7,24,25 Urine was filtered through a 0.22-Km nylon
centrifuge tube filter (Spin-X, Costar, Acton, MA) before
analysis. Samples (20 KL) were injected onto the HPLC
system with analytes of interest eluted on a C18 column
(4.6  250 mm, 5 Km; Aqua-C18, Phenomenex, Torrance,
CA) using a mobile phase of methanol/0.1% acetic acid
(85:15) at a flow rate of 1 mL/min. Eluates were monitored
by UV detection at 275 and 290 nm with CAF and
metabolite concentrations determined against calibration
standards. A 7-point standard curve using peak heights was
used to assess analyte concentrations. Standard curves for
all metabolites were linear over a range of 6 to 200 mmol/L
(r2 9 0.99). The coefficient of variation for all standards was
less than 10% except for the lower limit of quantitation
which was consistently less than 20%.
Warfarin
Concentrations of warfarin R- and S-isomers were
determined from blood plasma samples using a modified,
54
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J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006
validated reverse-phase HPLC method with ultraviolet
detection.26 Briefly, 300 KL of plasma was combined with
3 KL of 100 Kg/mL racemic ethyl-warfarin internal
standard in nanopure water. Samples were extracted using
BOND ELUT C18 solid-phase extraction column (100 mg,
10 mL; Varian, Harbor City, CA) and evaporated under
nitrogen at 45-C. Samples were reconstituted in 150 KL of
a 50% acetonitrile/50% glacial acetic acid (0.5%) solution
before injection onto a Agilent 1100 HPLC system. Isomer
separation was accomplished using a (R,R) Whelk-01 chiral
column (5.0 Km, 250  4.6 mm; Regis Technologies,
Morton Grove, IL) with a 5/100 Nitrile Guard Cartridge
(Regis Technologies) using gradient elution with a flow rate
of 1.0 mL/min. Calibration standard curves ranged from 25
to 3000 ng/mL. Intraday and interday coefficients of
variation were less than 10%.
Omeprazole
Concentrations of OMP and its CYP2C19-mediated
5-OH OMP were determined from blood plasma samples
using a modified, validated reverse-phase HPLC method
with ultraviolet detection.27 Briefly, 500 KL of plasma was
combined with 500 KL of 0.25 Kg/mL phenactin internal
standard in 100 mmol/L ammonium acetate buffer, pH 7.0.
Samples were extracted using BOND ELUT-C8 columns
(100 mg, 1.0 mL; Varian). Samples were evaporated using a
Turbo Vap LV Evaporator (Zymark Corp, Hopkinton, MA)
at 30-C for 30 minutes and reconstituted with 100 KL of
15% acetonitrile/85% HPLC-grade water (pH = 12) before
injection onto an Agilent 1100 HPLC system. Samples were
run on a XTerra MS-C8 column (3.5 Km, 3.9  100 mm;
Waters Scientific, Franklin, MA) with a XTerra MS-C8 Guard
column (3.5 Km, 3  20 mm; Waters Scienific) using gradient
elution with a flow rate of 800 KL/min. Calibration standard
curves ranged from 5 to 1000 ng/mL. Intraday and interday
coefficients of variation across the range of concentrations
were less than 5% for both OMP and 5-OH OMP.
Midazolam
Concentrations of MDZ were determined using a
modified, validated reverse-phase HPLC method with mass
spectroscopy detection.28 Briefly, 300 KL of plasma was added
to 100 KL of 0.01 Kg/mL alprazolam internal standard in
methanol. Samples were extracted with 1.5 mL tert-butyl
ether, evaporated to dryness using a Turbo Vap LV Evaporator
at 30-C for 10 minutes, and reconstituted with 100 KL of an
acetonitrile/water/acetic acid mixture before injection onto an
Agilent 1100 LCMSD system. Samples were run on a Luna C8
column (3.0 Km, 2  100 mm; Phenomenex, Torrance, CA)
using a gradient elution with a flow rate of 200 KL/min.
Calibration standard curves ranged from 0.1 to 500 ng/mL.
Intraday and interday coefficients of variation across the range
of concentrations were less than 8%.
Lopinavir and Ritonavir
LPV and RTV concentrations were measured individually
using plasma obtained from the second inpatient visit using a
validated HPLC method with ultraviolet detection.25,29 Briefly,
550 KL of plasma was combined with 550 KL of 1.0 mg/mL
* 2006 Lippincott Williams & Wilkins
J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006 Lopinavir/Ritonavir and Cytochrome P450 Activity

TABLE 1. CYP Phenotyping Results Before and After LPV/r Administration

Results*
Enzyme Phenotyping Measure Before LPV/r (PK 1) After LPV/r (PK 2) GMR (95% CI)
j1
CYP2C9 S-warfarin AUC (Kg I h I mL ) 14.97 (11.22Y22.13) 10.65 (8.66Y14.58) 0.71 (0.65Y0.79)*
CYP2C19 (PM included) OMP/5-OH OMP 1.55 (0.50Y4.19) 0.57 (0.27Y1.22) 0.15 (0.17Y1.03)*
CYP2C19 (NO PM) OMP/5-OH OMP 1.31 (0.78Y2.38) 0.57 (0.31Y1.32) 0.47 (0.20Y0.98)*
CYP3A (hepatic) IV MDZ CL (mL I minj1 I kgj1) 3.66 (3.15Y4.51) 0.83 (0.71Y1.03) 0.23 (0.18Y0.31)*
CYP3A (intestinal and hepatic) PO MDZ CL/F (mL I minj1 I kgj1) 13.73 (10.94Y19.64) 1.10 (0.96Y1.31) 0.08 (0.07Y0.11)*
CYP1A2 (1X + 1U + AFMU)/17U 4.64 (4.06Y5.53) 6.61 (5.71Y8.12) 1.43 (1.34Y1.54)*
NAT-2 (PM included) AFMU/(1X + 1U + AFMU) 0.10 (0.13Y0.36) 0.09 (0.12Y0.34) 0.95 (0.09Y2.90)
NAT-2 (NO PM) AFMU/(1X + 1U + AFMU) 0.20 (0.15Y0.38) 0.19 (0.14Y0.36) 0.96 (0.002Y3.06)
XO 1U/(1X + 1U) 0.70 (0.68Y0.72) 0.72 (0.69Y0.76) 1.03 (0.98Y1.09)
Results are expressed as geometric mean (95% CI). PK indicates pharmacokinetic visit.
*P G 0.05.

MDZ internal standard. Samples were extracted using BOND
ELUT-C18 columns (1.0 mL, 100 mg; Varian), evaporated to
dryness using a Turbo Vap LV Evaporator at 40-C, and
reconstituted in 100 KL of mobile phase before injection onto an
Agilent 1100 HPLC system. Samples were separated on a
Zorbax C18 analytical column (3.5 Km, 150  4.6 mm; Agilent,
Wilmington, DE) with a Zorbax C18 (3.5 Km, 12.5  4.6 mm;
Agilent) guard column and facilitated via a gradient elution.
Calibration standard curves ranged from 25 to 5000 ng/mL.
Intraday and interday coefficients of variation were less than 6%
for all analytes.
Data Analysis and Statistical Methods
CYP1A2, NAT-2, and XO activities were defined by
the urinary ratios of (1X + 1U + AFMU)/17U, AFMU/(1X +
1U + AFMU), and 1U/(1X + 1U), respectively. CYP2C9
activity was quantified by S-WARF AUC 0-inf , and
CYP2C19 activity was quantified by the OMP/5-OH OMP
ratio 2 hours postdose. Pharmacokinetic parameters for
MDZ and WARF were derived from plasma concentrations
using noncompartmental methods in WinNonLinPro V4.0.1
(Pharsight Corp, Mountain View, CA). Hepatic CYP3A
activity was quantified by IV MDZ clearance (CL) and
intestinal + hepatic CYP3A activity by the MDZ apparent
oral clearance (CL/F).
A priori sample size calculations determined that 13
evaluable study subjects would provide 80% power or more
to detect a 30% change in intraindividual phenotyping
measures, assuming a type I error of 0.05. Statistical
calculations were performed using SAS JMP 5.0 (SAS,
Cary, NC) by either the paired Student t test or Wilcoxon
rank sum test, depending on the distribution of the data.
Geometric mean ratios (GMRs) were calculated to compare
PK parameter changes within subjects between the 2 study
periods (PK 2/PK 1). Predictors of both baseline CYP
activity and the magnitude of change in CYP activity with
LPV/r exposure were evaluated using multivariate linear
regression and age, sex, race, body mass index, and LPV
and RTV concentrations as covariates. A P value of less
than 0.05 was considered significant. All data are presented
as the geometric mean (95% confidence interval [CI]),
unless otherwise stated.
RESULTS
Baseline Characteristics
Twenty-six subjects were screened, and 16 subjects
were enrolled into the study. One subject was withdrawn from
the study after 10 days of LPV/r due to the development of a
grade 2 rash. This resolved upon discontinuation of LPV/r
and administration of diphenhydramine and prednisone. An
additional subject was withdrawn due to poor LPV/r
adherence. Therefore, 14 subjects were used in the analysis
of CYP1A2, NAT-2, XO, CYP2C9, and CYP3A activities.
Of these subjects, 3 were white males, 4 were white females,
3 were African American males, and 4 were African
American females. Average age, weight, and height were 26.5
(23.9Y29.9) years, 70.3 (63.3Y80.0) kg, and 173.8 (168.1Y
180.1) cm, respectively. Because 3 subjects had no detectable
serum OMP or 5-OH OMP at either the baseline PK visit or
the treatment PK visit, 11 subjects were used to quantify
CYP2C19 activity.
All subjects were extensive metabolizers by phenotype
for CYP1A2,17,30,31 XO,17,30,31 and CYP2C9.32 One white
female was a CYP2C19 poor metabolizer (PM) by phenotype
(having an OMP/5-OH OMP ratio 96),33,34 and 1 African-
American female was an NAT-2 PM by phenotype [having an
AFMU/(1X + 1U + AFMU) ratio 90.5].17,30,31
Pharmacokinetic Analysis
A summary of the phenotyping measures for each drug-
metabolizing enzyme before and after LPV/r administration
and the GMRs are shown in Table 1. Figures 1A to C and
Figures 2A and B illustrate individual subject changes in
these markers upon exposure to LPV/r.
Upon LPV/r exposure, CYP1A2 metabolic ratios (Fig. 1A)
increased significantly by 43% (34%Y54%, P = 0.001), from a
ratio of 4.64 (4.06Y5.53) to a ratio of 6.61 (5.71Y8.12). NAT-2
and XO metabolic ratios did not change. NAT-2 metabolic

* 2006 Lippincott Williams & Wilkins 55

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Yeh et al
ratios were 0.20 (0.15Y0.38) before LPV/r and 0.19 (0.14Y0.36)
after LPV/r therapy (P = 0.34). XO metabolic ratios were 0.70
(0.68Y0.72) before LPV/r and 0.72 (0.69Y0.76) after LPV/r
therapy (P = 0.20).
CYP2C9 activity was measured by S-WARF expo-
sure (Fig. 1B). After LPV/r therapy, S-WARF AUC0-inf
significantly declined by 29% (17%Y34%, P = 0.001), from
14.97 (11.22Y22.13) Kg I h I mLj1 to 10.65 (8.66Y14.58)
Kg I h I mLj1. This represents a moderate increase in
CYP2C9 activity. Although not used as a CYP biomarker,
R-WARF AUC0-inf was also calculated for each study visit.
R-WARF exposure significantly decreased by 37% (23%Y46%,
P = 0.001), from 29.31 (22.17Y44.14) Kg I h I mLj1 to 18.39
(14.54Y27.86) Kg I h I mLj1.
CYP2C19 activity was estimated using an OMP
metabolic ratio (Fig. 1C). Among CYP2C19 extensive
metabolizers, the OMP metabolic ratio decreased signifi-
cantly by 53% (12%Y80%, P = 0.046), from 1.31 (0.78Y2.38)
to 0.57 (0.31Y1.32). This represents a 100% increase in
CYP2C19 activity. In the CYP2C19 PM, the OMP ratio also
decreased from 10.0 to less than 1. These data suggest a
significant increase in CYP2C19 activity, regardless of
phenotype.
56
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J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006
FIGURE 1. A, CAF metabolic ratio (CYP1A2 activity): PK 1 vs.
PK 2. Individual volunteer data are represented by the dotted
lines, and geometric mean data are represented by the solid
line. B, S-WARF AUC0-inf (CYP2C9 activity): PK 1 vs. PK 2.
Individual volunteer data are represented by the dotted lines,
and geometric mean data are represented by the solid line.
C, OMP metabolic ratio: PK 1 vs. PK 2 (CYP2C19 activity).
Individual volunteer data are represented by the dotted lines,
and geometric mean data are represented by the solid line.
Geometric mean line does not include PM. PK indicates
pharmacokinetic visit.
IV MDZ CL was used as a surrogate for hepatic
CYP3A activity, and the apparent oral clearance of MDZ
(CL/F) was used as a measure of combined intestinal and
hepatic CYP3A activity. Upon exposure to LPV/r, IV MDZ
CL and PO MDZ CL/F both decreased significantly,
suggesting a potent inhibitory effect on CYP3A activity in
both organs. IV MDZ CL declined by 77% (69%Y82%, P G
0.001), from 3.66 (3.15Y4.51) mL I minj1 I kgj1 to 0.83
(0.71Y1.03) mL I minj1 I kgj1 (Fig. 2A). PO MDZ CL/F
declined by 92% (89%Y93%, P = 0.001), from 13.73 (10.94Y
19.64) mL I minj1 I kgj1 to 1.10 (0.96Y1.31) mL I minj1 I kgj1
(Fig. 2B). As seen in Figure 3, significant correlations were
noted between baseline hepatic CYP3A activity and the
magnitude of LPV/r-induced hepatic inhibition (r2 = 0.44, P =
0.01) as well as between baseline intestinal + hepatic CYP3A
activity and the magnitude of LPV/r-induced inhibition (r2 =
0.59, P = 0.001).
Medication Adherence
Based on dose administration cards and pill counts,
medication adherence ranged from 95% to 100%. LPV
and RTV concentrations were measured using serum
samples collected upon admission for the PK 2 visit.
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J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006 Lopinavir/Ritonavir and Cytochrome P450 Activity

(86%) of 14 subjects; most by day 5 of LPV/r therapy. Two

subjects reported diarrhea throughout the study, but did not
report any diarrhea during their PK visits.
Both before and after LPV/r administration, no adverse
effects were associated with OMP, WARF, or vitamin K
administration. With MDZ administration, subjects displayed
either no or mild sedation. Respiratory rate decreased by a mean
of 2 (range, j6 to 0) breaths/min after the IV dose, and a mean
of 2 (range, j4 to 0) breaths/min after the PO dose. MAP
decreased by a mean of 4 (range, j20 to +5) mm Hg and
8 (range, j29 to 0) mm Hg after the IV and PO doses,
respectively. Subjects whose MAP decreased by 20 and 29 mm
Hg were otherwise asymptomatic of MDZ adverse events, and
the extent of MAP decrease was not dependent on LPV/r
administration. Flumazenil was not administered to any subject.

DISCUSSION
Treatment of HIV infection involves the use of multiple
antiretroviral medications. The number of drug interactions in
this population is unprecedented because many of these
compounds are metabolized by, and influence the activity of,
drug-metabolizing enzymes and transporters. Patients with
HIV infection often have concomitant disease states requiring
additional medication therapy which may also interact with
antiretroviral medications. Interactions are of particular con-
cern with drugs having narrow therapeutic indices, such as
anticonvulsants or anticoagulants. CYP phenotyping is a useful
tool for understanding and predicting these drug interactions.
Using phenotyping methods in healthy volunteers, we
found that 10 days of LPV/r therapy resulted in a 43% increase
in CYP1A2 activity, a 29% increase in CYP2C9 activity, and
an 100% increase in CYP2C19 activity. The degree of
CYP2C19 and CYP2C9 induction seen in this investigation is
consistent with in vitro data using prototypical inducers such
FIGURE 2. A, IV MDZ CL: PK 1 vs. PK 2 (hepatic CYP3A
activity). Individual volunteer data are represented by the
dotted lines, and geometric mean data are represented by the
solid line. B, PO MDZ CL/F: PK 1 vs. PK 2 (hepatic + intestinal
CYP3A activity). Individual volunteer data are represented by
the dotted lines, and geometric mean data are represented by
the solid line. PK indicates pharmacokinetic visit.

Subjects had samples obtained 8.4 T 0.6 hours (mean T

SD) after a self-recorded, nonobserved dose. LPV con-
centrations were 7.26 (5.86Y10.02) Kg/mL, and RTV
concentrations were 0.49 (0.36Y0.98) Kg/mL. These were
within the expected concentration range, as seen in
historical cohorts after observed dosing.4,7

Adverse Events
All study medications were generally well tolerated,
and all adverse effects were grade 2 or less, consistent with
the adverse reaction profile of LPV/r.4 The most common
adverse event associated with LPV/r was diarrhea. All 14
subjects experienced diarrhea, classified as grade 1 in 11
subjects (79%) and as grade 2 in 3 subjects (21%).
Loperamide was required for symptom relief in 4 of 11
subjects with grade 1 diarrhea and 3 of 3 subjects with grade 2
diarrhea. Diarrhea resolved before the second PK visit in 12
FIGURE 3. Correlation between baseline MDZ clearance and
the magnitude of change after 10 days of LPV/r. IV MDZ CL
(CYP3A hepatic activity) is represented by the open circles
(y = 0.48 j 0.06x, r = 0.66, P = 0.01 [solid line]), and PO MDZ
CL/F (CYP3A hepatic + intestinal activity) is represented by the
closed circles (y = 0.15 j 0.004x, r = 0.77, P = 0.001 [dotted
line]). PK indicates pharmacokinetic visit.

* 2006 Lippincott Williams & Wilkins 57

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Yeh et al
as rifampicin and phenobarbital, which show greater increases
in CYP2C19 activity than increases in CYP2C9 activity.35
Conversely, hepatic CYP3A activity declined by 77%, and
intestinal + hepatic CYP3A activity decreased by 92%. No
effects were noted on NAT-2 or XO activities. Although there
were no apparent differences in response associated with
weight, sex, race, age, and LPV or RTV concentrations (data
not shown), our sample size was not sufficient to conduct a
robust analysis.
To our knowledge, this is the first clinical study to show
induction of CYP1A2 by LPV/r. Commonly used CYP1A2
substrates include theophylline, antipsychotics (eg, clozapine,
haloperidol, and olanzapine), and tricyclic antidepressants
(eg, amitriptyline). RTV at a dose of 500 mg twice daily has
been shown to decrease exposure to theophylline36 by 43%
and olanzapine37 by 53%. Although the RTV dose in these
previous studies was greater than what was used in the current
study (and in current clinical practice), the magnitude of
induction was similar. Antidepressant and antipsychotic
agents are commonly used in HIV-infected patients, and
induction of CYP1A2 by PI combinations which include
RTV may result in decreased efficacy of these agents.
As determined by S-WARF exposure, LPV/r modestly
induced CYP2C9 activity. In vitro data from human liver
microsomes suggest that LPV/r is a very weak inhibitor of
CYP2C9 (Ki = 13.7Y23.0 Kmol/L [21.8Y36.6 Kg/mL]).
However, the microsome system lacks the cellular machinery
necessary to evaluate induction potential.6 S-WARF accounts
for approximately 70% of the overall anticoagulation response
with warfarin,38 and subtherapeutic INR values have been
reported in patients taking warfarin and at least 400 mg of RTV
twice daily.8,9 The clinical effect on INR could not be
evaluated in this study, as vitamin K was given with WARF.
However, based on the changes in S- and R-WARF exposure,
it appears that low doses of RTV in combination with LPV
may also cause a reduction in INR response.
The results of this study are also helpful in understanding
other unexpected drug interactions with LPV/r, such as that
seen with phenytoin or methadone. We previously demonstrat-
ed that LPV/r unexpectedly decreased phenytoin (PHT)
exposure by 31%.7 It has been reported that, at therapeutic
concentrations, PHT likely saturates CYP2C9, resulting in a
shift of PHT metabolism to the CYP2C19 pathway.39 It has
also been reported that LPV/r has significant binding affinity
for CYP2C9 (IC50 = 13.7 T 4.0 Kmol/L [8.6 T 2.5 Kg/mL] at a
LPV/RTV concentration ratio of 3:1).6,40 Our data suggest that
LPV/r likely increased PHT metabolism via induction of both
CYP2C9 and CYP2C19, although the specific contribution of
each isozyme cannot be determined from this study. Because
both PHT and LPV are highly protein bound (87%Y93%41 and
98%Y99%,4 respectively), protein-binding interactions cannot
be discounted in contributing to altered PHT concentrations.
Total methadone exposures have been shown to decrease
by 36% to 47% with concomitant LPV/r administration in both
HIV-infected and noninfected subjects,42,43 with inconsistent
reports of opioid withdrawal symptoms.10Y13 In 1 study, low-
dose RTV (100 mg twice daily) was not associated with
changes in total methadone exposure; therefore, the reduction
in methadone exposure was attributed to LPV.12 Although
58
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J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006
some in vitro data show that methadone is primarily
metabolized by CYP3A4 and, to a lesser extent, CYP2D6,44
other studies surmise that the decreased exposures may be due
to induction of CYP2B6, CYP2C9, CYP2C19, and/or glucur-
onidation pathways.13,42,43,45 Our data suggest that methadone
unlikely utilizes CYP3A4 as the primary pathway of metab-
olism (as LPV/r was found to be an overall inhibitor of
CYP3A4) and provide support for the role of CYP2C induction
in decreasing methadone exposures. However, as with
phenytoin, protein-binding interactions or stereoselective
effects may also be involved.43,46
The results of this investigation also suggest that
concomitant LPV/r therapy may increase the risk of failure
during treatment with drugs metabolized primarily by
CYP2C19. The increase in CYP2C19 activity observed in the
present study is consistent with other clinical data, such as the
LPV/r-mediated increase in AUC of the CYP2C19-generated
nelfinavir M8 metabolite.4,47 Other commonly used CYP2C19
substrates include diazepam,48 proton pump inhibitors (esome-
prazole, lansoprazole, and pantoprazole),49Y51 and antidepres-
sants (citalopram, sertraline, and amitriptyline).52,53 Interestingly,
the one subject phenotyped as a PM of CYP2C19 also exhibited a
large degree of CYP2C19 induction. This finding is unusual, as
PMs of other CYP isozymes are purported to have nonfunctional
alleles.54 However, 2 recent studies have found similar induction
potential in CYP2C19 PMs given the herbal product Ginkgo
biloba.55,56 This phenomenon needs further investigation, but
some hypotheses include induction of a non-CYP2C19 hydrox-
ylase or the presence of inducible, low-level CYP2C19 activity.56
Consistent with in vitro and in vivo reports, LPV/r
inhibited both hepatic and intestinal + hepatic CYP3A
activity, increasing exposure to IV and PO MDZ. A greater
degree of intestinal inhibition was seen in our study, likely
due to increased local concentrations of LPV and RTV during
absorption, and significant contribution of intestinal CYP3A
activity to MDZ metabolism.57Y59 Although MDZ coadmin-
istration with LPV/r is currently contraindicated,4 these data
may support the cautious use of IV MDZ, initiated at 25% of
the dose and given at 3 times the regular dosing interval.
Furthermore, for clinical studies which show overall induc-
tion of CYP3A substrates by LPV/r,10Y13 the mechanism of
induction is unlikely through CYP3A.
In vitro, LPV/r inhibits CYP3A with an IC50 of 1.1 KM
(0.69 Kg/mL).6 The metabolism of drugs that are primarily
CYP3A substrates such as atorvastatin,60 rifabutin,10 and
ketoconazole,61 when coadministered with LPV/r, is inhibited
in vivo to a similar extent as that seen with MDZ. However,
compounds that are also P-glycoprotein substrates (unlike MDZ)
may be affected to a greater extent.62 For example, tacrolimus is
both a CYP3A and P-glycoprotein substrate which requires a
28- to 140-fold decrease in dose upon the addition of LPV/r.63
Significant correlations were observed in this study
between the magnitude of decrease in CYP3A hepatic and
hepatic + intestinal clearances and initial CYP3A clearance.
Subjects with the highest initial MDZ clearances (and,
presumably, the highest CYP3A activity) exhibited the greatest
decline in activity with LPV/r treatment. This relationship has
been noted for other CYP3A inhibitorYsubstrate interactions,
such as MDZ-ketoconazole,57 grapefruit juiceYfelodipine,58 and
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J Acquir Immune Defic Syndr & Volume 42, Number 1, May 2006
MDZ-clarithromycin.59 This correlation is consistent with the
mechanism-based inhibition of CYP3A by RTV64 and, possibly,
LPV,65 where CYP3A is inactivated by the formation of a
metabolic intermediate complex. Further characterization of the
mechanism of the effects of LPV/r on CYP may improve in vivo
prediction models.
Because this study was performed in healthy volunteers,
it is possible that the magnitude of drug interactions between
LPV/r and other CYP substrates may differ. However, LPV
and RTV exposures are similar in HIV-negative and HIV-
positive individuals.4,7,66,67 Moreover, the magnitude of
interactions which have been performed in both HIV-negative
and HIV-positive persons using LPV/r in combination with
other drugs, such as efavirenz68,69 and saquinavir,70,71 is
similar. At minimum, these data provide a signal to clinicians
for drug interaction potential with LPV/r and other CYP
substrates, although the multiple medications taken by HIV-
infected subjects could impact the magnitude of interaction.
In conclusion, our data have important implications for
the appropriate use of combination drug therapy with LPV/r.
Failure to consider CYP enzyme system pathways in
treatment regimens that include LPV/r may result in
subtherapeutic or supratherapeutic drug concentrations; the
consequences of which might be treatment failure, the
progression of underlying disease processes, or drug toxicity.
Phenotyping cocktails are an efficient and useful way to
clinically evaluate drug interaction potential and target select
compounds for further evaluation.
ACKNOWLEDGMENTS
The authors thank Richard Bertz for scientific guidance
and support of this protocol, Nicole White for analytical
assistance, Chris Zurich for assistance with grants and
contracts, the research volunteers, and the nurses and staff of
the UNC GCRC.
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