Journal of Pharmaceutical Sciences xxx (2016) 1e11

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Journal of Pharmaceutical Sciences

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Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Mechanism of Drug-Drug Interactions Between Warfarin and Statins

Abdul Naveed Shaik 1, 2, *, Tonika Bohnert 2, David A. Williams 1, Lawrence L. Gan 3,
Barbara W. LeDuc 1
1
Department of Pharmaceutical Sciences, MCPHS University, 179 Longwood Avenue, Boston, Massachusetts 02115
2
Department of Drug Metabolism and Pharmacokinetics, Biogen, 14 Cambridge Center, Cambridge, Massachusetts 02140
3
Development Center for Biotechnology, Taipei 221, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: The anticoagulant drug warfarin and the lipid-lowering statin drugs are commonly co-administered to
Received 5 February 2016 patients with cardiovascular diseases. Clinically significant drug-drug interactions (DDIs) between these
Revised 9 March 2016 drugs have been recognized through case studies for many years, but the biochemical mechanisms
Accepted 10 March 2016
causing these interactions have not been explained fully. Previous theories include kinetic alterations in
cytochrome P-450emediated drug metabolism or disturbances of drug-protein binding, leading to
anticoagulant activity of warfarin; however, neither the enantioselective effects on warfarin metabolism
Keywords:
nor the potential disruption of drug transporter function have been well investigated. This study
cytochrome P450
CYP enzymes
investigated the etiology of the DDIs between warfarin and statins. Liquid chromatographyemass
organic anion transporters spectrometry methods were developed and validated to quantify racemic warfarin, 6 of its hydroxyl-
organic anion-transporting polypeptide ated metabolites, and pure enantiomers of warfarin; these methods were applied to study the role of
transporters different absorption, distribution, metabolism, and excretion properties, leading to DDIs. Plasma protein
P-glycoprotein binding displacement of warfarin was performed in the presence of statins using equilibrium dialysis
protein binding method. Substrate kinetics of warfarin and pure enantiomers were performed with human liver mi-
drug interactions crosomes to determine the kinetic parameters (Km and Vmax) for the formation of all 6 hydroxywarfarin
LC-MS
metabolites, inhibition of warfarin metabolism in the presence of statins, was determined. Uptake
enzyme kinetics
transport studies of warfarin were performed using overexpressing HEK cell lines and efflux transport
inhibition
using human adenocarcinoma colonic cell line cells. Fluvastatin significantly displaced plasma protein
binding of warfarin and pure enantiomers; no other statin resulted in significant displacement of
warfarin. All the statins that inhibited the formation of 10-hydroxywarfarin, atorvastatin, pitavastatin,
and simvastatin were highly potent compared to other statins; in contrast, only fluvastatin was found to
be a potent inhibitor of formation of 7-hydroxy warfarin. Uptake and efflux drug transporters do not play
any role in these DDIs. The results showed that DDIs between warfarin and statins are primarily caused
by cytochrome P-450 inhibition.
© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction

Abbreviations used: 17b DG, estradiol 17-b-D Glucuronide; Caco-2, colon
adenocarcinoma; CPDPX, 8-cyclopentyl-1,3-dipropyl xanthine; CYP P450, cyto-
chrome P450 enzymes; DDI, drug-drug interaction; DMSO, dimethyl sulfoxide; E3S,
estrone-3-sulfate; HEK, human embryonic kidney; HLM, human liver microsomes;
INR, international normalized ratio; LC-MS/MS, liquid chromatography-mass
spectrometry; LSC, liquid scintillation counter; OAT, organic anion transporters;
OATP, organic anion transporting polypeptides; P-gp, P-glycoprotein; PPB, plasma
protein binding; QC, quality control.
This article contains supplementary material available from the authors by request
or via the Internet at http://dx.doi.org/10.1016/j.xphs.2016.03.011.
* Correspondence to: Abdul Naveed Shaik (Telephone: þ1-407-313-7009;
Fax: þ1-407-313-7030).
E-mail address: naveed.shaik@my.mcphs.edu (A.N. Shaik).
Warfarin is an anticoagulant used in the treatment and pre-
vention of thrombosis and thromboembolism; it acts by inhibiting
the enzyme vitamin K epoxide reductase which is essential for
functioning of factor VII and IX during anticoagulation.1,2 Warfarin
is a narrow therapeutic index drug, and it is the third most common
drug causing hospital admissions due to adverse drug effects.3
Warfarin is known to cause interactions with foods, herbs, and
different classes of drugs.4 Changes in warfarin plasma concentra-
tion due to interfering compounds, over-dosage, or under-dosage
will cause difficulty in maintaining INR (international normalized
ratio) levels, which is an indication of anticoagulation range.5 The

http://dx.doi.org/10.1016/j.xphs.2016.03.011
0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11
predominant adverse effect of warfarin is bleeding, which may be
fatal. Even though warfarin possesses a high risk of adverse drug
reactions, which limits its usage, it is the most prescribed antico-
agulant. Warfarin is preferred over other oral anticoagulants like
factor Xa inhibitors, rivaroxaban (Xarelto™), apixaban (Eliquis™),
and edoxaban (Lixiana™); and oral thrombin inhibitor dabigatran
(Pradaxa™), due to its cost effectiveness, and more importantly as
overdose effects of warfarin can easily be reversed by either
infusing fresh plasma or by dosing reduced vitamin K.6,7
Warfarin is a racemic mixture of R and S warfarin; S-warfarin is
approximately 3-5 times more potent than the R-enantiomer and is
more rapidly cleared from the body.1 Warfarin shows low hepatic
clearance (CL), and is metabolized by multiple cytochromes (CYPs).
Both pure enantiomers of warfarin show CYP-mediated meta-
bolism to form 6 different hydroxylated metabolites: 30 -hydroxy,
40 -hydroxy, 6-hydroxy, 7-hydroxy, 8-hydroxy, and 10-hydorxy
warfarin. The pure enantiomers are stereoselectively metabolized
by the CYP system; S-warfarin undergoes 7-hydroxylation by
CYP2C9, while R-warfarin forms 10-hydroxy warfarin via CYP3A4.8
In elderly patients, warfarin is often co-administered with
multiple drugs such as non-steroidal anti-inflammatory drugs,
statins, barbiturates, and antifungalsdstatins being the fourth
major co-administered class with warfarin.9 Statins are plasma
lipid lowering drugs, and are widely prescribed for reducing the
risk of cardiovascular disease. Statins act by inhibiting the enzyme
3-hydroxy-3-methylglutaryl-coenzyme A reductase which cata-
lyzes the rate-limiting step of cholesterol biosynthesis, thereby
reducing plasma low density lipoproteins and cholesterol.10 Many
of the statins are extensively metabolized by multiple CYPs and
have been reported to inhibit the CYP enzymes.11
Drug-drug interactions (DDIs) occur whenever the effect of one
drug is modified by the presence of another drug, either leading to
therapeutic failure, toxicity, or serious complications; DDIs are one
of the leading causes for withdrawal of drugs from the market or
issuance of black box warnings.12 DDIs between warfarin and sta-
tins have been reported in clinical studies and case reports since the
early 1990s.13 The co-administration of statins with warfarin leads
to serious complications resulting in elevation of INR in patients, an
indication of the increased risk of bleeding.13,14 Case studies have
shown that co-administration of atorvastatin, fluvastatin, rosu-
vastatin, and simvastatin with warfarin has led to an increase in INR
values, which returned to normal after discontinuation of the sta-
tins or changing the dose; these reports predict the mechanism of
DDIs by alteration of one of the absorption, distribution, meta-
bolism, and excretion properties, especially alteration of CYP-
mediated metabolism as the predominant cause for the observed
DDIs leading to bleeding or thromboembolism.15-18 However,
neither change in PK (pharmacokinetic) profile of warfarin nor the
exact mechanisms and the magnitude by which these interactions
occur is studied yet.15,19 There is no substantial in vitro data which
explore the inhibition of warfarin metabolism by specific statins to
elucidate the role of CYP-mediated metabolism in DDIs. Warfarin is
highly plasma protein bound, as are many statins, warfarin, which
is approximately 99% protein bound; the 1% free fraction is
considered to bring pharmacological action, and any increase in
this free fraction at the target may lead to adverse events including
bleeding.1,20,21 PPB (plasma protein binding) displacement of
warfarin by statins is one of the suggested reasons for DDIs.22,23
One other suggested cause of DDIs between warfarin and statins
is through interference of drug transport of warfarin; both efflux as
well as uptake transporters are suggested to play a role. Warfarin
and statins are known substrates of P-glycoprotein (P-gp)20; addi-
tionally, warfarin might be a substrate for OATP1B1, OATP1B3
(organic anion transporting polypeptides), OAT1, and OAT3
(organic anion transporters) as reported by some of the clinical
findings.24,25 All of the statins are reported to be substrates of up-
take transporters OATP1B1 and OATP1B3,20 and an interaction at
these uptake transporters might cause DDIs. Therefore, this study
used in vitro methods to elucidate the biochemical mechanisms
underlying DDIs between warfarin and the statins, thus helping to
predict safer clinical dosing in patients taking warfarin, decreasing
the incidence of serious injury and hospitalization.
Materials and Methods
Materials
Racemic warfarin, 8-cyclopentyl-1,3-dipropyl xanthine (CPDPX),
and HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid,
N-(2-Hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid)) was ob-
tained from Sigma-Aldrich (St. Louis, MO). R-(þ)-warfarin, S-()-
warfarin, 30 -hydroxywarfarin, 40 -hydroxywarfarin, 6-hydroxywarfarin,
7-hydroxywarfarin, 8-hydroxywarfarin, 10-hydroxywarfarin, ator-
vastatin, cerivastatin, fluvastatin, lovastatin, pravastatin, simva-
statin, simvastatin acid, and rosuvastatin were obtained from
Toronto Research Chemicals, Inc. (Ontario, Canada). Pitavastatin was
obtained from TLC Pharmachem, Inc. (Ontario, Canada). 3H (R,S)
warfarin phenyl-4-3H, 3H R-warfarin phenyl-4-3H, and 3H
S-warfarin phenyl-4-3H were obtained from Moravek Biochemicals,
(Brea, CA). [3H]-digoxin, mannitol D-[1-14C], [N-methyl-3H]-verap-
amil hydrochloride, P-[glycyl-1-14C]-aminohippuric acid, estrone
sulfate, ammonium salt-, [6,7-3H(N)], propranolol, L-[4-3H], estra-
diol 17 b-D-glucuronide, and [estradiol-6,7-3H(N)] were obtained
from Perkin Elmer (Waltham, MA). Liquid chromatographyemass
spectrometry (LC-MS) grade acetonitrile and methanol were ob-
tained from JT Baker Chemicals (Center Valley, PA) and all the other
chemicals used were of highest quality available. The pooled human
liver microsome (HLM) preparation was obtained from Xenotech
LLC (Kansas City, KS). Human plasma was obtained from Bio-
reclamation LLC (Westbury, NY). Caco-2 cells (human adenocarci-
noma colonic cell line) were purchased from ATCC. Human
embryonic kidney (HEK) cell lines, either mock transfected or
transfected with OATP1B1, OATP1B3, OAT1, or OAT3, were obtained
from the University of California South San Francisco.
LC-MS/MS Method Development and Validation
An LC-MS/MS method was developed for separation and
quantitation of racemic warfarin and metabolites using API-5500
QTrap mass spectrometer (AB Sciex, Framingham, MA); sepa-
rately a chiral method using a chiral column was developed for
separation and quantitation of warfarin enantiomers using API
5500 QTrap; these 2 methods were validated using FDA bio-
analytical method validation guidelines. In brief, a Shimadzu
Prominence LC system (Shimadzu Corporation, Kyoto, Japan) was
used to inject 20 mL aliquots of the processed samples onto a Zobrax
eclipse XDB® C18 column (2.1  150 mm, 5 mm, Agilent Technolo-
gies, Waldbronn, Germany) which was heated in a column oven to
27 C. A binary gradient mobile phase consisting of (A) 25 mM
ammonium acetate pH 4.85 (prepared using Milli-Q water: Milli-
pore, Milford, MA) and (B) acetonitrile was delivered at 0.80 mL/
min with a gradient flow for 22 min; the compounds were sepa-
rated on the column, and the eluent plus the separated compounds
were delivered into the mass spectrometer's electrospray ioniza-
tion chamber. Quantitation was achieved by MS-MS detection in
positive ion mode for all the metabolites of warfarin, warfarin, and
the internal standard (IS-CPDPX), using an API-5500 QTrap equip-
ped with a Turboionspray™ interface at 500 C. Furthermore, a
chiral method was developed for analyzing of R- and S-warfarin
using a chiral column (Chiracel OD-RH 2.1  150 mm, 5 mm), a
 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11 3

binary gradient mobile phase consisting of (A) 10 mM ammonium Metabolism Studies

acetate at pH 4.4, and (B) 100% acetonitrile. These 2 methods were
validated to meet the acceptance criteria of FDA guidance for bio- Drug metabolism studies were performed with racemic
analytical method validation guidelines; 6 replicates were run each warfarin or pure enantiomers to determine the metabolites formed
time and on different days to determine intra-day and inter-day from each enantiomer. Followed by initial experiments to deter-
variability.26 Detection of all the ions was performed in the Multi- mine the linear conditions, HLM concentration at 1 mg/mL, NADPH
ple Reaction Monitoring mode; the transition pair settings and at 5 mM, and time of incubation was fixed at 30 min. Substrate
retention times for 30 , 40 , 6, 7, 8, and 10 hydroxylated warfarin curves were run for racemic warfarin, R-warfarin, and S-warfarin to
metabolites, racemic warfarin, and pure enantiomers of warfarin generate their kinetic parameters, Km, and Vmax. In-house experi-
along with instrument-specific parameters were previously mental conditions were followed and were modified based on
reported by our group.27 The analytical data were processed by Ghosal et al.42 The final assay parameters were as follows: HLM at
Multiquant® software version 2.1.1 (AB Sciex, Framingham, MA). 1.0 mg/mL, cofactor NADPH (5 mM), and assay buffer was potas-
sium phosphate 100 mM containing 3 mM MgCl2 (pH 7.4); the
Plasma Protein Binding reaction volume was 200 mL. Drugs were incubated for 30 min in a
humidified incubator set at 37 C containing an orbital shaker set at
PPB was performed by equilibrium dialysis; the dialysis 60 RPM to mimic the in vivo conditions. Warfarin or the pure en-
assembly (Spectrum Labs, Rancho Dominguez, CA) consists of 2 antiomers R-warfarin and S-warfarin were incubated individually
chambers which are separated by a semipermeable cellulose in a 96-well plate at the following concentrations: 0.01, 0.1, 0.2, 0.5,
membrane. In one of the chambers, 1 mL of plasma containing 1.0, 5.0, 10.0, 50.0, and 100.0 mM, and each of the concentrations
drug was pipetted while in the other chamber 1 mL of buffer was was incubated in triplicate. After a 30-min incubation, the reaction
added. The separating membrane had a molecular weight cutoff of was terminated by adding 200 mL of acetonitrile containing 10 ng/
6-8 kDa (Spectra Por 1, size 1; Spectrum Labs). The dialysis mL of analytical internal standard, followed by the sample prepa-
assembly was then transferred to a dialyzer, and then the dialyzer ration procedure as described in the PPB section. Similarly, cali-
with dialysis assembly was placed in an incubator set at 37 C. At bration curves were prepared for all the 6 hydroxylated
the end of the incubation time, the dialysis assembly was metabolites: 30 -OH, 40 -OH, 6-OH, 7-OH, 8-OH, and 10-OH warfarin.
removed, and plasma and buffer were collected separately with a
1-mL Hamilton syringe to determine the magnitude of the volume
Inhibition Studies
shift as described by Tozer et al.28-30 The collected plasma was
weighed and compared with the original weight of 1 mL of
CYP inhibition studies were performed similarly to substrate
plasma.28-30 The volumes of plasma and concentrations of drug
kinetic studies, briefly, in a total incubation volume of 200 mL
obtained at each time point were used to calculate the % bound
consisting of potassium phosphate buffer 100 mM containing
drug. For sample analysis, an aliquot of 50 mL of plasma from the
MgCl2 (3 mM) pH 7.4, HLMs at 1 mg/mL, and cofactor
plasma compartment was mixed with 50 mL of blank buffer, and
NADPH (5 mM). The experiment was initiated with addition of the
50 mL of buffer from the buffer compartment was mixed with 50
inhibitor to pre-incubate for 10 min and the rest of the experi-
mL of blank plasma. To the samples 200 mL of acetonitrile con-
mental conditions described above were followed. The concentra-
taining 10 ng/mL of IS-CPDPX was added. The contents were
tion of warfarin and pure enantiomers were used at Km generated
transferred into a 96-well 1-mL plate (Millipore); the plate was
in-house, and the inhibitor (statins) concentration varied from 0.01
centrifuged at 4000  g for 10 min in a tabletop centrifuge
to 100 mM. Midazolam (2.5 mM) and tolbutamide (150 mM) were
(Eppendorf-5810 R) and 100 mL of the supernatant was transferred
into a 96-well deep well analysis plate with 2-mL volume capacity
(Millipore). To the supernatant, 200 mL of diluent containing a
mixture of acetonitrile-Milli-Q-formic acid in the ratio 80:20:0.1
was added and loaded on to LC-MS/MS for analysis. The stability of
the drug was calculated by taking an aliquot of drug in plasma and
maintained at the incubation conditions in a separate vial, and
recovery was calculated using plasma and buffer collected from
the dialyzer chamber at the end of incubation period. Similarly,
calibration curves were prepared for racemic warfarin, R-warfarin,
and S-warfarin in 1:1 vol/vol plasma-buffer with a concentration
range of 1-2000 nM. The % bound and the stability of the drug
were calculated from the concentrations obtained from plasma
and buffer samples. Followed by initial experiment to determine
the linear condition and equilibrium times of warfarin, pure en-
antiomers, and statins, each of the drug's Cmax was used to design
the PPB displacement experiment, each experiment was con-
ducted in 4 replicates. The concentration of warfarin and enan-
tiomers was fixed at 2 mM and the statins were incubated at
clinical molar ratios obtained from the clinical Cmax values from
literature. Warfarin or pure enantiomers were incubated at 2
mM,31 atorvastatin at 221 mM,32-34 cerivastatin at 102 mM,35 flu-
vastatin 5426 mM,20 however, PPB displacement of fluvastatin was
performed at 2000 mM due to limited solubility, lovastatin at 95
mM,20,36 pitavastatin at 781 mM,33 pravastatin at 540 mM,33,37 Figure 1. PPB displacement of racemic warfarin in the presence of statins at clinical
rosuvastatin at 205 mM,38,39 and simvastatin was incubated at molar ratios. Data represent the mean and standard error of mean of a minimum of 4
265 mM.40,41 independent experiments.
4 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11

Figure 2. PPB displacement of (a) S-warfarin and (b) R-warfarin in the presence of statins. Data represent the mean and standard error of mean of a minimum of 4 independent
experiments.

used as positive controls as these are preferred substrates as sug-
gested by FDA guidelines,43 and ketoconazole (0.002-5.0 mM) and
sulfaphenazole (0.003-20 mM) were used as inhibitors for CYP2C9
and CYP3A4, respectively.
Efflux Studies
Caco-2 cells were cultured in T-75 flasks (Corning [75 cm2]) with
approximately 1.5  105 cells. The cells were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% heat-
inactivated fetal bovine serum (DMEM/FBS) and 1% non-essential
amino acid at 37 C in a 5% CO2 atmosphere with 95% relative hu-
midity. The cell culture medium was replaced 3 times weekly. The
cells were split on achieving approximately 80% confluency by
trypsinization. The cells were then seeded onto a 24-well cluster
plate (Corning Falcon™ HTS 24-Multiwell, 1 mm pore) at 4  104
cells/cm2 with 400 mL of cell suspension added to each apical
chamber. The inserts were then transferred into the feeder plates
(Corning FalconTM) containing 1 mL/well of culture medium and
placed in a CO2 incubator set at 37 C, 95% relative humidity, and 5%
CO2 throughout the culturing period. The medium was changed
every other day until 21 days. The cell monolayers were monitored
using transepithelial electrical resistance (TEER) (EVOM® Epithelial
Volt Ohmmeter, World Precision Instruments, Sarasota, FL) values.
Wells having TEER values greater than 1000 U$cm2 were used for
the experiment.
Permeability experiments across monolayers were performed
on fully differentiated Caco-2 cells at the end of 21 days. Before the
start of the experiment, each transwell insert was washed twice
with the assay buffer (HBSS buffer containing 10 mM HEPES, pH
7.4) and TEER values were measured before and after the washing
steps to ensure the integrity of the monolayers. Unidirectional flux
of warfarin was performed by adding warfarin to either the apical
or to the basolateral side of the cell layer and analyzing the
samples from both the basolateral and apical sides. Permeability
(Papp) was calculated according to the equation shown below
in apical to basolateral (A-B) and basolateral to apical (B-A)
directions.44
dQ=dt
Papp ½cm=sec ¼
Co$A
where dQ/dt is the rate of permeation, “t” is the incubation time in
s; C0 is the initial concentration of warfarin in mM of the donor
compartment, and A is the surface area of the cell monolayer (cm2).
The efflux ratio was obtained by using the following formula: efflux
ratio ¼ Papp B-A/Papp A-B (Papp obtained from B to A divided by the
Papp obtained from A to B).45
Transport of warfarin was studied along with the positive con-
trol digoxin (5 mM) for P-gp, the low permeability marker mannitol
(3 mM), and the high permeability marker propranolol (3 mM).
Compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM
and diluted into transport assay buffer; the final concentration of
DMSO was below 1%. Before the start of the experiment, cells were
washed and equilibrated for 30 min with assay buffer (HBSS con-
taining 10 mM HEPES, pH 7.4). Dosing solutions were prepared by
diluting non-radiolabeled and radiolabeled stock solutions of 3H-
warfarin, 3H-digoxin, 3H-mannitol, or 3H-propranolol into the
transport assay buffer. Transport was initiated by adding the
compounds to the donor chamber followed by incubation at 37 C in

Table 1
Summary of Kinetic Parameters (Km and Vmax) of 6 Hydroxylated Metabolites of Warfarin Formed From Racemic Warfarin, R-Warfarin, and S-Warfarin Using MME

Compound Kinetic Parameters 30 -OH War 40 -OH War 6-OH War 7-OH War 8-OH War 10-OH War

Racemic-warfarin Km m M a
NA 7.09 ± 1.4 8.44 ± 0.8 3.9 ± 0.7 24.3 ± 2.0 14.9 ± 2.9
Vmax (pmol/min/mg) NA 54.7 ± 2.8 127 ± 3.5 202 ± 8.7 38.1 ± 1.1 268 ± 15.7
CLint (mL/min/g) 7.72 15 51.8 1.57 17.9
R-warfarin Km mMa NA 19.6 ± 5.7 236 ± 33.6 41.3 ± 12.2 237 ± 33.5 30.9 ± 3.2
Vmax (pmol/min/mg) NA 807 ± 75 5386 ± 568 466 ± 56.0 1937 ± 506 8351 ± 314
CLint (mL/min/g) 41.1 22.8 11.2 8.17 270
S-warfarin Km mMa NA 16.2 ± 2.2 5.18 ± 0.3 3.65 ± 0.4 12.7 ± 0.9 43.1 ± 4.3
Vmax (pmol/min/mg) NA 754 ± 32.2 964 ± 16.5 207 ± 59.7 116 ± 2.5 582 ± 24.3
CLint (mL/min/g) 46.5 186 56.7 9.13 13.5
a
Apparent Km.
 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11
Figure 3. Inhibition of 7-OH warfarin formation from racemic warfarin in the presence
of (a) atorvastatin, (b) fluvastatin, and (c) pitavastatin.
a CO2 incubator with 90% relative humidity and 5% CO2. The incu-
bation plate was placed on an orbital shaker set at 90 rpm
throughout the duration of the experiment. At the end of the in-
cubation time (2 h), samples from both the donor and receiver
compartments were removed and transferred to scintillation vials.
To these samples 5 mL of scintillation fluid (Microscint 40, Perkin
Elmer) was added. These vials were then transferred to liquid
scintillation counter (Beckman LS6500 scintillation counter, Beck-
man Coulter, Fullerton, CA) for counting of radioactivity. The
5
experiments were performed in triplicate with 3 independent
studies. The cell monolayer integrity of each monolayer was
measured pre- and post-experiment by TEER values. TEER values of
1000 U$cm2 or greater, which indicated acceptable monolayer
integrity, were used for calculation of permeability (Papp). At the
end of the experiment, contents from both the donor and receiver
compartments were analyzed for mass balance.
Uptake Studies
An in-house standardized assay protocol which was modified
based on Van de Steeg46 and Ishiguro et al.47 was used to study
warfarin uptake by transporters. The appropriate HEK cells were
seeded in 24-well plates pre-coated with Poly-D-Lysine (BD Bio-
sciences) at 0.3  106 cells/well for OATP1B1 and OATP1B3, 0.4 
106 cells/well for OAT1 and OAT3 in DMEM with 10% FBS, penicillin,
streptomycin, hygromycin (65 mg/mL), and 5 mM sodium butyrate.
The cells were grown in the plates for 18 h at 37 C in an incubator
with 5% CO2 with 95% humidity. During the experiment, the cells
were washed with HBSS 4 times to remove the residual media. A
primary stock solution of cold warfarin (100 mM) was prepared in
DMSO; secondary stocks were prepared in HBSS and an equal
amount of radioactivity was added to each of the secondary stock
solutions. The cells were incubated with warfarin for 3 min at 37 C
in an incubator shaker. After incubation, 1 mL of stop solution (ice-
cold HBSS) was added to each well, the cells were dried, and 200 mL
of 1 N NaOH was added to the dried cells for cell digestion. The
plates were allowed to shake overnight at 600  g. After the
overnight shaking from each well containing 200 mL cell lysate, an
aliquot of 10 mL of cell lysate was transferred into a 96-well plate for
protein estimation. Protein was determined using the BCA assay
(Pierce, Thermo Scientific). After protein determination, 100 mL of
cell lysate was transferred into a scintillation vial and 5 mL of
scintillation fluid (Microscint 40, Perkin Elmer) was added. The
samples were analyzed using a liquid scintillation counter
(LSC6500, Beckman Coutler). Uptake was calculated from the dis-
integrations per minute. The following concentrations of warfarin
were used for uptake studies: 10, 50, 75, 100, 200, 500, 1000, and
1500 mM. The total radioactivity in each concentration was 0.025
mM with the remaining concentration being composed of unlabeled
compound. In separate plates HEK cells transfected with OATP1B1,
OATP1B3, OAT1, or OAT3 were seeded and uptake of assay positive
control, estradiol-17-b-D-glucuronide (17BDG), and para-amino
hippuric acid was performed to determine uptake mediated by
OATP1B1, OATP1B3, OAT1, and OAT3, respectively.
Data Analysis
Kinetic parameters Km and Vmax were generated by non-linear
regression analysis of data by fitting Michealis-Menten Equation
(MME), and the in vitro IC50 values were evaluated by fitting the
data using Prism Software version 6 (GraphPad Software, Inc., San
Diego, CA). The statistical significance of differences between
experimental groups for PPB was analyzed using one-way analysis
of variance followed by Tukey post hoc analysis (Prism software).
All data are presented as the mean of each treatment group ±
standard error of mean as indicated in the figure legend.
Results
LC-MS/MS Method Validation
All the 6 hydroxylated metabolites of warfarin and racemic
warfarin were separated on column for achiral method and the R-
and S-enantiomers of warfarin were separated using a chiral
6 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11

Figure 4. Inhibition of 10-OH warfarin formation from racemic warfarin in the presence of (a) atorvastatin, (b) cerivastatin, (c) lovastatin, (d) pitavastatin, (e) pravastatin, (f)
rosuvastatin, and (g) simvastatin.

column. Both the achiral and chiral methods were validated to meet
the acceptance criteria of FDA guidance for bioanalytical method
validation.26 Six replicates were run each time and on different days
to determine intra-day and inter-day variability, accuracy, precision,
and recovery. The calibration curves were generated and all the
standards were within linear range with a deviation of ±20% for
lower concentrations and ±15% for higher concentrations
as described in the FDA guidelines for bioanalysis; the linear re-
gressions using a weighting factor of 1/X2 were 0.99 or greater. The
validation parameters, accuracy, and precision were calculated using
6 replicates at low, mid, and high quality control samples; inter-day
and intra-day validation parameters are shown in Supplemental
Tables 1 and 2, respectively for achiral and chiral methods and
were reported earlier by our group.27
PPB Displacement
The time to reach PPB equilibrium by racemic warfarin, R-
and S-warfarin was 2 h; S-warfarin showed tighter binding than
R-warfarin, 0.8% and 1.0% for S- and R-warfarin, respectively, up
to 24 h (Supplemental Fig. 1). The time to reach equilibrium for
atorvastatin was 4 h; for cerivastatin 2 h; for fluvastatin 2 h;
 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11 7

Figure 5. Inhibition of 10-OH warfarin formation from R-warfarin in the presence of (a) atorvastatin, (b) cerivastatin, (c) fluvastatin, (d) lovastatin, (e) pitavastatin, (f) pravastatin, (g)
rosuvastatin, and (h) simvastatin.

for lovastatin 2 h; for pitavastatin 4 h; for pravastatin 2 h;
for rosuvastatin 4 h; and for simvastatin 2 h (data not shown);
the PPB of statins was comparable with literature values.48 Once
the time to reach equilibrium for warfarin, R-, S-warfarin, and
the statins was determined, PPB displacement studies were
performed, wherein warfarin or R- or S-warfarin was equili-
brated in plasma for the requisite time, and then the statin was
added and allowed to equilibrate, after which the plasma
was injected into a dialyzing unit to determine any displace-
ment. PPB displacement studies of racemic warfarin in the
presence of statins at clinical molar concentrations resulted in
significant PPB displacement of racemic warfarin by fluvastatin
and pitavastatin as shown in Figure 1; statistical analysis was
done by one-way analysis of variance followed by Tukey's test.
The unbound fraction of racemic warfarin doubled (2.19-fold) in
the presence of fluvastatin and was increased by 1.2-fold by
pitavastatin. No other statin showed significant PPB displace-
ment of racemic warfarin, whereas studies with S-warfarin in
the presence of statins resulted in significant PPB displacement
of S-warfarin by fluvastatin and simvastatin (Fig. 2a). The un-
bound fraction of S-warfarin increased by 2.18-fold in the
presence of fluvastatin and it was increased by 1.7-fold by
simvastatin. No other statin showed significant PPB displace-
ment of S-warfarin. R-warfarin showed significant displacement
8 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11

Figure 6. Inhibition of 7-OH warfarin formation from S-warfarin in the presence of (a) fluvastatin and (b) pitavastatin.

by fluvastatin only, where the unbound fraction of R-warfarin (Fig. 6b). The IC50 of all statins are summarized in Table 2. Assay
increased to 2.2-fold in the presence of fluvastatin (Fig. 2b). controls were performed with the inhibition experiments. Tolbu-
None of the other statins showed significant PPB displacement tamide was used as a positive control for CYP2C9-mediated
of R-warfarin (data not shown). metabolism, whereas midazolam was used as a positive control
for CYP3A4-mediated metabolism. Inhibition of 7-OH warfarin and
4-OH tolbutamide (data not shown) formation was monitored in
Metabolism Studies
the presence and absence of sulfaphenazole, a known inhibitor of
CYP2C9-mediated metabolism. For CYP3A4, inhibition of 10-OH
The kinetic parameters of 6 hydroxylated metabolites of
warfarin and 1-OH midazolam (data not shown) was monitored
warfarin determined using MME (Supplemental Figs. 2 and 3) are
in the presence and absence of ketoconazole, a known inhibitor of
summarized in Table 1; as the metabolism is contributed by mul-
CYP3A4-mediated metabolism.
tiple CYPs, the Km value is given as apparent Km. Intrinsic CL was
calculated using the formula CLint ¼ Vmax/Km.49 The intrinsic CL is
expressed as mL/min/g.49,50 Km and Vmax values were generated for Efflux Studies
formation of all the hydroxylated metabolites from racemic, R, and
S warfarin. The kinetic parameter values generated for racemic The warfarin efflux experiment was performed at different
warfarin, and the Km and Vmax values for 7-OH and 10-OH warfarin concentrations ranging from 0.1 to 100 mM. The Papp of warfarin for
generated using R- and S-warfarin were in accordance with litera- apical to basolateral (A-B) was approximately 30  106 cm/s,
ture values.51-54 The 30 -OH warfarin concentration was below low
limit of quantification for all the incubations. The kinetic parame-
Table 2
ters were generated using MME after ruling out atypical kinetics IC50 Values of Statins Toward Formation of 7-OH Warfarin and 10-OH Warfarin
(Supplemental Fig. 4) as reported earlier by our group,27 the Km and Metabolism From Racemic Warfarin
Vmax generated from these experiments were used subsequently
Drug Co-Administered Drug Reaction IC50 (mM)
for CYP inhibition studies testing statins as inhibitors. The substrate
79.7 ± 1.9
concentration chosen for inhibition studies was fixed at 3 mM for R- Racemic warfarin Atorvastatin 7-Hydroxylation
10-Hydroxylation 8.65 ± 1.6
and S-warfarin enantiomers and 10 mM for racemic warfarin based Cerivastatin 7-Hydroxylation >100
on the results obtained from substrate kinetic studies. 10-Hydroxylation 28.1 ± 1.1
Fluvastatin 7-Hydroxylation 5.86 ± 0.9
10-Hydroxylation 55.6 ± 4.1
Inhibition Studies Lovastatin 7-Hydroxylation >100
10-Hydroxylation 24.8 ± 3.5
Inhibition of major metabolic pathways of warfarin leading to Pitavastatin 7-Hydroxylation 64.0 ± 3.1
10-Hydroxylation 14.9 ± 1.4
the formation of 7-OH warfarin and 10-OH warfarin from racemic Pravastatin 7-Hydroxylation >100
(Figs. 3 and 4), R- (Fig. 5), and S-warfarin (Fig. 6) in the presence of 10-Hydroxylation 27.8 ± 1.2
statins are summarized in Table 2. 7-OH warfarin formation from Rosuvastatin 7-Hydroxylation >100
racemic warfarin was completely inhibited by fluvastatin alone 10-Hydroxylation 67.4 ± 3.3
Simvastatin 7-Hydroxylation >100
(Fig. 3b); atorvastatin (Fig. 3a) and pitavastatin (Fig. 3c) showed
10-Hydroxylation 15.6 ± 2.7
maximum inhibition of 77% and 60%, respectively, at the highest R-Warfarin Atorvastatin 10-Hydroxylation 8.56 ± 1.5
tested concentration, whereas atorvastatin, cerivastatin, lovastatin, Cerivastatin 10-Hydroxylation 10.5 ± 1.2
pitavastatin, pravastatin, and simvastatin inhibited 10-OH warfarin Fluvastatin 10-Hydroxylation 34.2 ± 4.4
formation from racemic warfarin as summarized in Table 2. Inhi- Lovastatin 10-Hydroxylation 7.43 ± 1.5
Pitavastatin 10-Hydroxylation 8.21 ± 1.1
bition of 10-OH warfarin formation from R-warfarin was quantified Pravastatin 10-Hydroxylation 13.3 ± 0.9
in the presence of statins; all statins that inhibited 10-OH warfarin Rosuvastatin 10-Hydroxylation 33.1 ± 3.9
formation with different IC50 values are summarized in Table 2 and Simvastatin 10-Hydroxylation 10.5 ± 0.6
rosuvastatin showed a maximum inhibition of 75% at the highest S-Warfarin Atorvastatin 7-Hydroxylation >100
Cerivastatin 7-Hydroxylation >100
tested concentration (Fig. 5g). Inhibition of 7-OH warfarin forma-
Fluvastatin 7-Hydroxylation 3.68 ± 0.6
tion from S-warfarin was quantified in the presence of statins; only Lovastatin 7-Hydroxylation >100
fluvastatin showed potent inhibition of 7-OH warfarin formation Pitavastatin 7-Hydroxylation 46 ± 1.9
from S-warfarin with an IC50 of 3.68mM. Pitavastatin inhibited 7-OH Pravastatin 7-Hydroxylation >100
warfarin formation with an IC50 of 46 mM; however, a maximum of Rosuvastatin 7-Hydroxylation >100
Simvastatin 7-Hydroxylation >100
70% inhibition was observed at the highest tested concentration
 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11 9

Table 3 was chosen so as to quantitate 0.1-1.0% free drug, which would

Papp Values and Efflux Ratio of Warfarin Permeability Across Fully Confluent Caco-2 correspond to 2-20 nM. Initial experiments with 2 mM statin con-
Monolayers
centration resulted in significant PPB displacement by fluvastatin
Drug Papp (10-6 cm/s) B-A/A-B only (Supplemental Fig. 5), but to appropriately reflect clinical sit-
A-B B-A Ratio uations, the statins concentration for the PPB displacement studies
were maintained at clinical molar ratios obtained from literature.
Warfarin 0.1 mM 30.1 26.8 0.9
Warfarin 0.5 mM 29.6 29.1 1 Atorvastatin, cerivastatin, lovastatin, pravastatin, and rosuvastatin
Warfarin 1 mM 30.7 28.3 0.9 did not displace warfarin or R- or S-warfarin. Fluvastatin signifi-
Warfarin 5 mM 31.8 31.8 1 cantly displaced PPB of racemic warfarin (p < 0.0001), R-warfarin
Warfarin 10 mM 30.6 30.8 1
(p ¼ 0.0085), and S-warfarin (p < 0.0001), whereas pitavastatin
Warfarin 50 mM 30.6 30.3 1
Warfarin 100 mM 32.2 28.3 0.9 significantly displaced PPB of racemic warfarin (p ¼ 0.0022) but did
Digoxin 0.6 16.7 26.2 not displace PPB of either R- or S-warfarin. Simvastatin significantly
displaced PPB of S-warfarin (p ¼ 0.004), but did not show any effect
on PPB of racemic warfarin or R-warfarin; these results are novel
whereas the Papp for basolateral to apical (B-A) was 26-30  106 findings. There is, however, caution warranted for the interpreta-
cm/s. The efflux ratio [B-A/A-B] was 0.9-1.0 as shown in Table 3. The tion of these new results. The drug concentrations used were well
efflux of warfarin was determined across Caco-2 cell monolayers; above the concentrations encountered clinically. Warfarin shows
the efflux ratio was found to be around 1 for all the concentrations low hepatic extraction where the CL is independent of hepatic
tested ranging from 0.1 to 100 mM. The apparent permeability of blood flow, and is determined by the intrinsic metabolizing ca-
high permeability marker propranolol, low permeability marker pacity (CLHep ¼ CLint  fu). Any increase in the free fraction of the
mannitol, and efflux marker digoxin were similar to the literature drug will have effect on metabolism of the drug and will signifi-
values. cantly alter area under the curve and CL which will warrant dose
adjustment,21 but studies using a systematic approach and a
pharmacokinetic-pharmacodynamic equilibration time approach
Uptake Studies
have shown that PPB displacement of warfarin which shows long
pharmacokinetic-pharmacodynamic equilibration time (14 h) does
The uptake of warfarin in OATP1B1-, OATP1B3-, OAT1-, or OAT3-
not result in clinically significant interactions,55 as increased free
transfected cells was determined. Initial experiments of radio-
drug concentration would lead to rapid elimination of the drug
labeled warfarin uptake to determine the time-dependent uptake
until steady state is obtained.21 The findings from this study need to
up to 3 min with time points at 10, 20, 30, 45, 60, 90, 120, and 180 s
be understood in conjunction with CYP inhibition, as well as the
resulted in either equal or higher values of uptake of warfarin in
higher experimental concentrations than observed clinically.
mock-transfected cells than in OATP1B1-, OATP1B3-, OAT1-, or OAT3-
Currently, CYP inhibition is considered to be the major cause of
transfected cells (Fig. 7). The experiments were repeated 4 times.
DDIs between warfarin and the statins. The formation of 2 major
Concentration-dependent uptake of warfarin up to 30 min showed
metabolites, 7-OH warfarin and 10-OH warfarin, formed by
equal or higher uptake of warfarin in mock cells than in OATP1B1-,
CYP2C9 and CYP3A4, respectively, were monitored. These 2 me-
OATP1B3-, OAT1-, or OAT3-transfected cells.
tabolites are known to be the major metabolites, and they
significantly contribute to the hepatic CL of warfarin. Furthermore,
Discussion warfarin metabolism is inhibited by its own metabolites, inhibi-
tion of warfarin metabolism by its hydroxylated metabolites, and
The objective of this study was to investigate the mechanisms presence of 2 subsets of enzymes will lead to atypical kinetics;
underlying DDIs between warfarin and the statins, including however, studies have shown that warfarin metabolism follows
displacement of warfarin PPB, role of different CYPs, and drug Michaelis-Menten Kinetics.56 It has further shown that for highly
transporters. The PPB displacement of warfarin and R- and S- protein-bound drugs, the in vivo CL by atypical kinetics is negli-
warfarin by statins has never been explored previously. Warfarin gible57 and typical kinetics give good in vitro and in vivo correla-
and R- and S-warfarin were incubated at 2 mM; this concentration tion for which typical MME was used to generate the kinetic

Figure 7. Time-dependent uptake of racemic warfarin in HEK transfected cells with (a) mock or OATP1B-1 transfected cells and (b) mock or OATP1B-3 transfected cells.
10 A.N. Shaik et al. / Journal of Pharmaceutical Sciences xxx (2016) 1e11
parameters. The kinetic parameters Km and Vmax generated were
in accordance with the literature values for 7-OH and 10-OH
warfarin.58,59 The kinetic parameters generated for 30 , 40 , 6, and
8-OH warfarin metabolites from racemic warfarin, especially from
R- and S-enantiomers, are novel findings. Fluvastatin which shows
high PPB displacement and potent inhibition of 7-OH warfarin
formation from racemic warfarin and S-warfarin will affect the PK
parameters of warfarin.13 When pure S-warfarin is dosed with
fluvastatin it may lead to more severe DDIs than compared to a
dose of racemic warfarin with fluvastatin.60 Atorvastatin, pit-
avastatin, and simvastatin potently inhibit the formation of 10-OH
warfarin from racemic warfarin as well as R-warfarin. These re-
sults further confirm the hypothesis that CYP3A4 inhibition is the
cause of DDIs between warfarin and atorvastatin, leading to
elevated INR levels,16,17 between warfarin and simvastatin,15,17 and
inhibition of CYP2C9 as the cause of DDIs between warfarin and
fluvastatin.18 Our group findings further confirm the clinical ob-
servations, where co-administering simvastatin in an 82-year-old
patient lead to increase in INR levels from 2.0-3.5 to 8.0; this
elevation ultimately lead to fatal cerebral hemorrhage, and potent
inhibition of 10 hydroxylation of warfarin by simvastatin with an
IC50 10.5 mM could lead to increase in INR.15 Similarly, potent in-
hibition of 7-hydroxylation of warfarin by fluvastatin with an IC50
of 5.8 mM could be the cause for elevation of INR levels when
warfarin and fluvastatin are co-administered, which leads to
either change in fluvastatin dose or replacing fluvastatin with
atorvastatin as reported by Andrus.13 Furthermore, Simonson et
al.14 have reported that patients on warfarin therapy and co-
administration of rosuvastatin at 40 mg/kg showed INR levels of
2-3. When the rosuvastatin dose was increased to 80 mg/kg it
resulted in the increase in INR levels above 3, which might be a
result of moderate inhibition of 10-OH warfarin formation by
rosuvastatin.14 Inhibition of 10-OH warfarin by lovastatin results in
increased INR levels as observed by Iliadis and Konwinski.61 Based
on our results and clinical observations reported in the literature,
we suggest that inhibition of CYP-mediated metabolism of
warfarin by statins is the primary cause of DDIs observed and
should be considered when co-administering with warfarin.
The in vitro uptake and efflux transport of warfarin has not
been reported previously. This study explored warfarin as a
possible substrate for uptake transporters (OATP1B1, OATP1B3,
OAT-1, or OAT-3) as well as the efflux transporter P-gp, known to
be the predominant efflux transporter expressed in Caco-2 cells. In
the experiments conducted, warfarin was not shown to be a
substrate for any of the transporters tested. The uptake of warfarin
was similar in transfected as well as mock cells, and the uptake
was further ruled out by performing uptake of verapamil and
salicylic acid (Supplemental Fig. 6), which are highly permeable,
non-substrates of OATP1B1, OATP1B3, OAT1, or OAT3; both the
compounds showed profiles similar to warfarin which ruled out
any role of these transporters in uptake of warfarin. Although
previously reported studies suggested that warfarin might be a
substrate for P-gp, we could not calculate the efflux ratio of
warfarin in the Caco-2 system due to its high permeability. The
Papp of warfarin was approximately 30  106 cm/s in both A to B
and B to A directions for all of the tested concentrations ranging
from 0.1 to 100 mM.
One of the limitations of this study was high concentration of
warfarin used for PPB displacement studies due to analytical
detection limits. Although CYP inhibition studies were performed
with great detail, however mechanism of inhibition was not
evaluated. Further studies of PPB displacement of warfarin in the
presence of statins at clinical concentration, CYP inhibition studies
to understand the mechanism of inhibition, or a clinical DDI would
broaden our understanding of DDIs between warfarin and statins.
Conclusions
In summary, CYP inhibitions are the significant cause of DDIs
between warfarin and statins; inhibition profile (IC50 values) of
warfarin and pure enantiomers in the presence of statins will
broaden our understanding of DDIs between warfarin and statins
and will help in dose adjustment of warfarin when co-administered
with any of the statins. PPB displacement of warfarin and enan-
tiomers by different statins will not lead to clinically significant
DDIs; uptake or efflux drug transporters do not play any role in
DDIs involving warfarin and statins.
Acknowledgments
The authors thank the Department of Drug Metabolism &
Pharmacokinetics at Biogen for sponsoring this project. We thank
Dr. Chakri Lagishetty and Dr. Raja Venkatasubramanian of CPSP,
University of Florida, for the critical review of this manuscript.
Authorship Contributions.
Participated in research design: Abdul Naveed Shaik, Tonika
Bohnert, and Barbara LeDuc.
Conducted experiments: Abdul Naveed Shaik.
Contributed new reagents or analytic tools: Abdul Naveed Shaik.
Performed data analysis: Abdul Naveed Shaik, Tonika Bohnert.
Wrote or contributed to the writing of the manuscript: Abdul
Naveed Shaik, Tonika Bohnert, Barbara LeDuc, David A. Williams,
and Lawrence Gan.
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