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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 Warfarin is an anticoagulant used in the treatment and pre-
adenocarcinoma; CPDPX, 8-cyclopentyl-1,3-dipropyl xanthine; CYP P450, cyto- vention of thrombosis and thromboembolism; it acts by inhibiting
chrome P450 enzymes; DDI, drug-drug interaction; DMSO, dimethyl sulfoxide; E3S, the enzyme vitamin K epoxide reductase which is essential for
estrone-3-sulfate; HEK, human embryonic kidney; HLM, human liver microsomes;
functioning of factor VII and IX during anticoagulation.1,2 Warfarin
INR, international normalized ratio; LC-MS/MS, liquid chromatography-mass
spectrometry; LSC, liquid scintillation counter; OAT, organic anion transporters; is a narrow therapeutic index drug, and it is the third most common
OATP, organic anion transporting polypeptides; P-gp, P-glycoprotein; PPB, plasma drug causing hospital admissions due to adverse drug effects.3
protein binding; QC, quality control. Warfarin is known to cause interactions with foods, herbs, and
This article contains supplementary material available from the authors by request different classes of drugs.4 Changes in warfarin plasma concentra-
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;
tion due to interfering compounds, over-dosage, or under-dosage
Fax: þ1-407-313-7030). will cause difficulty in maintaining INR (international normalized
E-mail address: naveed.shaik@my.mcphs.edu (A.N. Shaik). 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 findings.24,25 All of the statins are reported to be substrates of up-
fatal. Even though warfarin possesses a high risk of adverse drug take transporters OATP1B1 and OATP1B3,20 and an interaction at
reactions, which limits its usage, it is the most prescribed antico- these uptake transporters might cause DDIs. Therefore, this study
agulant. Warfarin is preferred over other oral anticoagulants like used in vitro methods to elucidate the biochemical mechanisms
factor Xa inhibitors, rivaroxaban (Xarelto™), apixaban (Eliquis™), underlying DDIs between warfarin and the statins, thus helping to
and edoxaban (Lixiana™); and oral thrombin inhibitor dabigatran predict safer clinical dosing in patients taking warfarin, decreasing
(Pradaxa™), due to its cost effectiveness, and more importantly as the incidence of serious injury and hospitalization.
overdose effects of warfarin can easily be reversed by either
infusing fresh plasma or by dosing reduced vitamin K.6,7 Materials and Methods
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 Materials
more rapidly cleared from the body.1 Warfarin shows low hepatic
clearance (CL), and is metabolized by multiple cytochromes (CYPs). Racemic warfarin, 8-cyclopentyl-1,3-dipropyl xanthine (CPDPX),
Both pure enantiomers of warfarin show CYP-mediated meta- and HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid,
bolism to form 6 different hydroxylated metabolites: 30 -hydroxy, N-(2-Hydroxyethyl)piperazine-N0 -(2-ethanesulfonic acid)) was ob-
40 -hydroxy, 6-hydroxy, 7-hydroxy, 8-hydroxy, and 10-hydorxy tained from Sigma-Aldrich (St. Louis, MO). R-(þ)-warfarin, S-()-
warfarin. The pure enantiomers are stereoselectively metabolized warfarin, 30 -hydroxywarfarin, 40 -hydroxywarfarin, 6-hydroxywarfarin,
by the CYP system; S-warfarin undergoes 7-hydroxylation by 7-hydroxywarfarin, 8-hydroxywarfarin, 10-hydroxywarfarin, ator-
CYP2C9, while R-warfarin forms 10-hydroxy warfarin via CYP3A4.8 vastatin, cerivastatin, fluvastatin, lovastatin, pravastatin, simva-
In elderly patients, warfarin is often co-administered with statin, simvastatin acid, and rosuvastatin were obtained from
multiple drugs such as non-steroidal anti-inflammatory drugs, Toronto Research Chemicals, Inc. (Ontario, Canada). Pitavastatin was
statins, barbiturates, and antifungalsdstatins being the fourth obtained from TLC Pharmachem, Inc. (Ontario, Canada). 3H (R,S)
major co-administered class with warfarin.9 Statins are plasma warfarin phenyl-4-3H, 3H R-warfarin phenyl-4-3H, and 3H
lipid lowering drugs, and are widely prescribed for reducing the S-warfarin phenyl-4-3H were obtained from Moravek Biochemicals,
risk of cardiovascular disease. Statins act by inhibiting the enzyme (Brea, CA). [3H]-digoxin, mannitol D-[1-14C], [N-methyl-3H]-verap-
3-hydroxy-3-methylglutaryl-coenzyme A reductase which cata- amil hydrochloride, P-[glycyl-1-14C]-aminohippuric acid, estrone
lyzes the rate-limiting step of cholesterol biosynthesis, thereby sulfate, ammonium salt-, [6,7-3H(N)], propranolol, L-[4-3H], estra-
reducing plasma low density lipoproteins and cholesterol.10 Many diol 17 b-D-glucuronide, and [estradiol-6,7-3H(N)] were obtained
of the statins are extensively metabolized by multiple CYPs and from Perkin Elmer (Waltham, MA). Liquid chromatographyemass
have been reported to inhibit the CYP enzymes.11 spectrometry (LC-MS) grade acetonitrile and methanol were ob-
Drug-drug interactions (DDIs) occur whenever the effect of one tained from JT Baker Chemicals (Center Valley, PA) and all the other
drug is modified by the presence of another drug, either leading to chemicals used were of highest quality available. The pooled human
therapeutic failure, toxicity, or serious complications; DDIs are one liver microsome (HLM) preparation was obtained from Xenotech
of the leading causes for withdrawal of drugs from the market or LLC (Kansas City, KS). Human plasma was obtained from Bio-
issuance of black box warnings.12 DDIs between warfarin and sta- reclamation LLC (Westbury, NY). Caco-2 cells (human adenocarci-
tins have been reported in clinical studies and case reports since the noma colonic cell line) were purchased from ATCC. Human
early 1990s.13 The co-administration of statins with warfarin leads embryonic kidney (HEK) cell lines, either mock transfected or
to serious complications resulting in elevation of INR in patients, an transfected with OATP1B1, OATP1B3, OAT1, or OAT3, were obtained
indication of the increased risk of bleeding.13,14 Case studies have from the University of California South San Francisco.
shown that co-administration of atorvastatin, fluvastatin, rosu-
vastatin, and simvastatin with warfarin has led to an increase in INR LC-MS/MS Method Development and Validation
values, which returned to normal after discontinuation of the sta-
tins or changing the dose; these reports predict the mechanism of An LC-MS/MS method was developed for separation and
DDIs by alteration of one of the absorption, distribution, meta- quantitation of racemic warfarin and metabolites using API-5500
bolism, and excretion properties, especially alteration of CYP- QTrap mass spectrometer (AB Sciex, Framingham, MA); sepa-
mediated metabolism as the predominant cause for the observed rately a chiral method using a chiral column was developed for
DDIs leading to bleeding or thromboembolism.15-18 However, separation and quantitation of warfarin enantiomers using API
neither change in PK (pharmacokinetic) profile of warfarin nor the 5500 QTrap; these 2 methods were validated using FDA bio-
exact mechanisms and the magnitude by which these interactions analytical method validation guidelines. In brief, a Shimadzu
occur is studied yet.15,19 There is no substantial in vitro data which Prominence LC system (Shimadzu Corporation, Kyoto, Japan) was
explore the inhibition of warfarin metabolism by specific statins to used to inject 20 mL aliquots of the processed samples onto a Zobrax
elucidate the role of CYP-mediated metabolism in DDIs. Warfarin is eclipse XDB® C18 column (2.1 150 mm, 5 mm, Agilent Technolo-
highly plasma protein bound, as are many statins, warfarin, which gies, Waldbronn, Germany) which was heated in a column oven to
is approximately 99% protein bound; the 1% free fraction is 27 C. A binary gradient mobile phase consisting of (A) 25 mM
considered to bring pharmacological action, and any increase in ammonium acetate pH 4.85 (prepared using Milli-Q water: Milli-
this free fraction at the target may lead to adverse events including pore, Milford, MA) and (B) acetonitrile was delivered at 0.80 mL/
bleeding.1,20,21 PPB (plasma protein binding) displacement of min with a gradient flow for 22 min; the compounds were sepa-
warfarin by statins is one of the suggested reasons for DDIs.22,23 rated on the column, and the eluent plus the separated compounds
One other suggested cause of DDIs between warfarin and statins were delivered into the mass spectrometer's electrospray ioniza-
is through interference of drug transport of warfarin; both efflux as tion chamber. Quantitation was achieved by MS-MS detection in
well as uptake transporters are suggested to play a role. Warfarin positive ion mode for all the metabolites of warfarin, warfarin, and
and statins are known substrates of P-glycoprotein (P-gp)20; addi- the internal standard (IS-CPDPX), using an API-5500 QTrap equip-
tionally, warfarin might be a substrate for OATP1B1, OATP1B3 ped with a Turboionspray™ interface at 500 C. Furthermore, a
(organic anion transporting polypeptides), OAT1, and OAT3 chiral method was developed for analyzing of R- and S-warfarin
(organic anion transporters) as reported by some of the clinical 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
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- 7.4) and TEER values were measured before and after the washing
gested by FDA guidelines,43 and ketoconazole (0.002-5.0 mM) and steps to ensure the integrity of the monolayers. Unidirectional flux
sulfaphenazole (0.003-20 mM) were used as inhibitors for CYP2C9 of warfarin was performed by adding warfarin to either the apical
and CYP3A4, respectively. or to the basolateral side of the cell layer and analyzing the
samples from both the basolateral and apical sides. Permeability
Efflux Studies (Papp) was calculated according to the equation shown below
in apical to basolateral (A-B) and basolateral to apical (B-A)
Caco-2 cells were cultured in T-75 flasks (Corning [75 cm2]) with directions.44
approximately 1.5 105 cells. The cells were grown in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% heat- dQ=dt
inactivated fetal bovine serum (DMEM/FBS) and 1% non-essential Papp ½cm=sec ¼
Co$A
amino acid at 37 C in a 5% CO2 atmosphere with 95% relative hu-
midity. The cell culture medium was replaced 3 times weekly. The where dQ/dt is the rate of permeation, “t” is the incubation time in
cells were split on achieving approximately 80% confluency by s; C0 is the initial concentration of warfarin in mM of the donor
trypsinization. The cells were then seeded onto a 24-well cluster compartment, and A is the surface area of the cell monolayer (cm2).
plate (Corning Falcon™ HTS 24-Multiwell, 1 mm pore) at 4 104 The efflux ratio was obtained by using the following formula: efflux
cells/cm2 with 400 mL of cell suspension added to each apical ratio ¼ Papp B-A/Papp A-B (Papp obtained from B to A divided by the
chamber. The inserts were then transferred into the feeder plates Papp obtained from A to B).45
(Corning FalconTM) containing 1 mL/well of culture medium and Transport of warfarin was studied along with the positive con-
placed in a CO2 incubator set at 37 C, 95% relative humidity, and 5% trol digoxin (5 mM) for P-gp, the low permeability marker mannitol
CO2 throughout the culturing period. The medium was changed (3 mM), and the high permeability marker propranolol (3 mM).
every other day until 21 days. The cell monolayers were monitored Compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM
using transepithelial electrical resistance (TEER) (EVOM® Epithelial and diluted into transport assay buffer; the final concentration of
Volt Ohmmeter, World Precision Instruments, Sarasota, FL) values. DMSO was below 1%. Before the start of the experiment, cells were
Wells having TEER values greater than 1000 U$cm2 were used for washed and equilibrated for 30 min with assay buffer (HBSS con-
the experiment. taining 10 mM HEPES, pH 7.4). Dosing solutions were prepared by
Permeability experiments across monolayers were performed diluting non-radiolabeled and radiolabeled stock solutions of 3H-
on fully differentiated Caco-2 cells at the end of 21 days. Before the warfarin, 3H-digoxin, 3H-mannitol, or 3H-propranolol into the
start of the experiment, each transwell insert was washed twice transport assay buffer. Transport was initiated by adding the
with the assay buffer (HBSS buffer containing 10 mM HEPES, pH 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 5
Uptake Studies
Data Analysis
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 and intra-day validation parameters are shown in Supplemental
the acceptance criteria of FDA guidance for bioanalytical method Tables 1 and 2, respectively for achiral and chiral methods and
validation.26 Six replicates were run each time and on different days were reported earlier by our group.27
to determine intra-day and inter-day variability, accuracy, precision,
and recovery. The calibration curves were generated and all the PPB Displacement
standards were within linear range with a deviation of ±20% for
lower concentrations and ±15% for higher concentrations The time to reach PPB equilibrium by racemic warfarin, R-
as described in the FDA guidelines for bioanalysis; the linear re- and S-warfarin was 2 h; S-warfarin showed tighter binding than
gressions using a weighting factor of 1/X2 were 0.99 or greater. The R-warfarin, 0.8% and 1.0% for S- and R-warfarin, respectively, up
validation parameters, accuracy, and precision were calculated using to 24 h (Supplemental Fig. 1). The time to reach equilibrium for
6 replicates at low, mid, and high quality control samples; inter-day 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; and pitavastatin as shown in Figure 1; statistical analysis was
for rosuvastatin 4 h; and for simvastatin 2 h (data not shown); done by one-way analysis of variance followed by Tukey's test.
the PPB of statins was comparable with literature values.48 Once The unbound fraction of racemic warfarin doubled (2.19-fold) in
the time to reach equilibrium for warfarin, R-, S-warfarin, and the presence of fluvastatin and was increased by 1.2-fold by
the statins was determined, PPB displacement studies were pitavastatin. No other statin showed significant PPB displace-
performed, wherein warfarin or R- or S-warfarin was equili- ment of racemic warfarin, whereas studies with S-warfarin in
brated in plasma for the requisite time, and then the statin was the presence of statins resulted in significant PPB displacement
added and allowed to equilibrate, after which the plasma of S-warfarin by fluvastatin and simvastatin (Fig. 2a). The un-
was injected into a dialyzing unit to determine any displace- bound fraction of S-warfarin increased by 2.18-fold in the
ment. PPB displacement studies of racemic warfarin in the presence of fluvastatin and it was increased by 1.7-fold by
presence of statins at clinical molar concentrations resulted in simvastatin. No other statin showed significant PPB displace-
significant PPB displacement of racemic warfarin by fluvastatin 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
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
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