HMG-CoA reductase inhibitors and P-glycoprotein modulation

Article abstract of DOI:10.1038/sj.bjp.0703920

1. Five 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability to reverse P-glycoprotein (P-gp) mediated rhodamine 123 (R123) transport in a murine monocytic leukaemia cell line that over-expresses the multi-drug resistance protein la/b (mdrla/lb). 2. P-gp modulation was studied by a fluorimetric assay and confocal microscopy by means of R123 efflux and uptake experiments, respectively. 3. Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited R123 transport in a concentration-dependent manner. Lovastatin acid, simvastatin acid, fluvastatin and pravastatin did not show a significant inhibition of the R123 transport in our cell system. Atorvastatin methyl ester and lactone showed the highest affinities for P-gp and results were comparable for both methods. 4. In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to fluorimetric assay is a sensitive tool to study P-gp affinity in drug screening that is especially useful for early phases of drug development.

Full text of DOI:10.1038/sj.bjp.0703920

British Journal of Pharmacology (2001) 132, 1183 ± 1192
ã
2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00
www.nature.com/bjp
HMG-CoA reductase inhibitors and P-glycoprotein modulation
1
1
2
1Katrijn Bogman, Anne-Kathrin Peyer, Michael Torok, Ernst Kusters & *,1Jurgen Drewe
2
1Department of Clinical Pharmacology and Toxicology, University Hospital/Kantonsspital, Basel, Switzerland and Department
of Chemical and Analytical Development, Novartis Pharma AG, Basel, Switzerland
1
Five 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), (e.g.
atorvastatin, ¯uvastatin, lovastatin, pravastatin and simvastatin), were investigated for their ability
to reverse P-glycoprotein (P-gp) mediated rhodamine 123 (R123) transport in a murine monocytic
leukaemia cell line that over-expresses the multi-drug resistance protein 1a/b (mdr1a/1b).
2
P-gp modulation was studied by a ¯uorimetric assay and confocal microscopy by means of R123
e‚ux and uptake experiments, respectively.
Atorvastatin acid, methyl ester and lactone, lovastatin lactone and simvastatin lactone inhibited
3
R123 transport in a concentration-dependent manner. Lovastatin acid, simvastatin acid, ¯uvastatin
and pravastatin did not show a signi®cant inhibition of the R123 transport in our cell system.
Atorvastatin methyl ester and lactone showed the highest anities for P-gp and results were
comparable for both methods.
4
In conclusion, monitoring of R123 transport in living cells by confocal microscopy in addition to
¯uorimetric assay is a sensitive tool to study P-gp anity in drug screening that is especially useful
for early phases of drug development.
British Journal of Pharmacology (2001) 132, 1183 ± 1192
Keywords: Statins; P-glycoprotein; rhodamine; in vitro; confocal microscopy; drug interactions; HMG-CoA reductase
inhibitors
Abbreviations: ATO, atorvastatin; cMOAT, canalicular multispeci®c organic anion transporter; CYP450 3A4, cytochrome
P450 3A4; DMSO, dimethyl sulfoxide; FLU, ¯uvastatin; HBSS, Hank's balanced salt solution; HMG-CoA,
3-hydroxy-3-methylglutaryl coenzyme A; IP, inhibitory potency; LOV, lovastatin; Log P, log of octanol/
water partition coecient; MDR, multi-drug resistance; OATP, organic anion transporter; P-gp, P-
glycoprotein; PI, propidium iodide; PRA, pravastatin; R123, rhodamine 123; SIM, simvastatin; t50, time
needed to reach the half-maximal e€ect
Introduction
P-glycoprotein (P-gp, mdr1-gene product), a member of the
superfamily of ATP-binding cassette (ABC) transporters, has
been intensively examined because of its multi-drug resistance
(MDR) phenotype in oncology. This multi-drug transporter
prevents the intracellular accumulation of anti-cancer drugs
by actively removing them from the cell membrane before
they reach their intracellular targets.
and as a result increase oral absorption, decrease hepatic/
renal excretion and/or alter distribution into tissues where
there is expression of this protein.
It is of interest to note that the cytochrome P450 system,
especially the isoform P450 3A, has an overlapping substrate
speci®city to that of P-gp (Kim et al., 1999; Wacher et al.,
1995).
In the body, P-gp is widely expressed on the luminal
surface of capillary endothelial cells in brain and testis,
adrenal cortex, brush border membrane of the proximal renal
tubule epithelium, canalicular membranes of hepatocytes,
mucosa of the small and large intestine, pancreatic ductules,
endometrium of gravid uterus and placenta (Ambudkar et al.,
1999). Strong morphological and genetic evidence exist
demonstrating a role of P-gp in absorption, distribution
and excretion of certain hydrophobic, amphiphatic drugs and
xenobiotics in mice and probably in humans (Schinkel, 1997).
This may also lead to pharmacokinetic drug ± drug interac-
tions in human as described by di€erent authors (Boyd et al.,
2000; Hunter & Hirst, 1997; Yu, 1999). Thus two drugs that
are transported by P-gp could compete for the transporter
The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase inhibitors (statins) represent an established class of
drugs for the treatment of hypercholesterolaemia, with
potentially fatal adverse events (such as rhabdomyolysis).
The lipophilic drugs lovastatin, simvastatin, atorvastatin and
¯uvastatin are metabolized via the cytochrome P450 system
in the liver and the gut, making them subject to potential
interactions with concomitantly administered drugs that are
competing for metabolism via this system (Christians et al.,
1998). Pravastatin is water-soluble and does not undergo
metabolism via CYP450 to any signi®cant extent. Both
lovastatin and simvastatin are inactive lactone pro-drugs
and are converted by hydrolytic enzymes in plasma and liver
to the active acid form (Tang & Kalow, 1995) whereas
atorvastatin, ¯uvastatin and pravastatin are administrated in
their active acid form (Transon et al., 1996). All statins
included in this study, except pravastatin, show a high
hepatic extraction ratio. Furthermore it is known that
¯uvastatin (Lennernas & Fager, 1997; Lindahl et al., 1996),
*Author for correspondence at: Department of Clinical
Pharmacology and Toxicology, University Hospital/Kantonsspital,
Petersgraben 4, CH 4031 Basel, Switzerland;
E-mail: Juergen.Drewe@unibas.ch

1184
K. Bogman et al
In vitro screening of P-glycoprotein substrates
lovastatin (Halpin et al., 1993) and simvastatin metabolites
(Cheng et al., 1994) are excreted into bile, and that
pravastatin has an important glomerular ®ltration and
tubular secretion.
Membrane transport of these compounds into di€erent
tissues can occur by passive and/or carrier-mediated
processes. Implication of the latter processes for liver
selectivity or elimination has been described or hypothesized
earlier for some statins (Boyd et al., 2000; Christians et al.,
1998; Hsiang et al., 1999; Lindahl et al., 1996; Tamai et al.,
1997; Wu et al., 2000; Yamazaki et al., 1997).
inactivated foetal calf serum, 1 mM sodium pyruvate, 10 mM
HEPES bu€er, 0.02 mg ml71 asparagine, 16minimum essen-
tial medium with non-essential amino acids, 16minimum
essential medium with vitamins, 0.05 mM b-mercaptoethanol,
50 mg ml71 gentamycin. Culture conditions were 5% CO2/
95% air atmosphere at 378C.
The P388/MDR cells were continuously grown in the
presence of 0.25 mg ml71 doxorubicin. One day before
each experiment, cells were grown in doxorubicin-free
medium.
Numerous factors contributing to the risk for adverse drug
interactions with statins have been reported recently
(Bottor€, 1999; Christians et al., 1998; Corsini et al., 1999),
and should be considered when patients are receiving
additional drugs. Knowledge about the di€erences in the
adverse drug interaction pro®le of statins is an important
determinant of safety in long-term therapy of hypercholester-
olaemia.
In vitro screening methods for detection of P-gp anity are
numerous and include primary cell culture systems (Drewe et
al., 1999), fresh tissue (Fricker et al., 1999), over-expressing
cell lines (Pourtier-Manzanedo et al., 1992), membrane
vesicles (Shapiro et al., 1997), puri®ed P-gp on h.p.l.c.
columns (Zhang et al., 2000).
In the present study, we developed an in vitro ¯uorimetric
assay and a confocal microscopy method to study the ability
of ®ve statins and their derivates for modulation of transport
of a ¯uorescent P-gp substrate rhodamine 123 (R 123). As
cell system, a murine mdr1a/1b over-expressing monocytic
leukaemia cell line and its parental analogue (Boesch et al.,
1991) was used.
Isolation of atorvastatin
Twenty-nine Sortis1 tablets (corresponding to a total of
8.4 g of atorvastatin acid) were suspended in a mixture
of 151 ml methylene chloride : methanol : hexane : acetic acid
(50 : 50 : 50 : 1) (v v71 v71 v71) and stirred for 2 h. The
insoluble excipients were ®ltered o€ and the solution was
evaporated to dryness at 508C under vacuum, yielding
800 mg evaporation residue. Final puri®cation was achieved
with preparative high-pressure liquid chromatography
(h.p.l.c.) using a preparative column ®lled with 100 g silica
gel (15 mm, Merck no. 11635) as stationary phase. Of the
above mentioned evaporation residue 500 mg were dissolved
in 20 ml methylene chloride : methanol (1 : 1) (v v71), poured
onto the column and eluted with methylene chloride : tert.-
butylmethylether : methanol (2 : 2 : 1) (v v71 v71) as mobile
phase with a ¯ow rate of 10 ml min71. After 2 h, the mobile
phase was changed to methylene chloride : tert.-butylmethyl-
ether : methanol (1 : 1 : 2) (v v71 v71) and atorvastatin was
eluted immediately within 50 min. The solvents were removed
at 508C under vacuum, yielding 301 mg of drug substance
with a purity of 98.3 % (h.p.l.c.). The product was ®nally
1
characterized with mass spectrometry and H-NMR-spectro-
scopy.
Methods
Materials
Isolation of atorvastatin lactone and atorvastatin
methyl ester
Lovastatin lactone and simvastatin lactone were kind gifts
from Merck Sharp & Dohme Ltd (Rahway, NJ, U.S.A.).
Fluvastatin sodium salt and SDZ-PSC833 was from
Novartis Pharma AG (Basel, Switzerland), and pravastatin
sodium salt from Bristol-Meyers Squibb (Sankyo; Japan).
Sixty-six mg of atorvastatin drug substance (isolated as
described above) dissolved in 6 ml acetonitrile and 100 ml
phosphoric acid (85%) were heated for 3 h at 808C. After
cooling down to room temperature the sample was
evaporated to dryness and the evaporation residue was
again dissolved in 3 ml methylene chloride : methanol (1 : 1)
(v v71). The solution was poured on three preparative thin
layer chromatography plates (Merck no. 5715) and the
compounds were subsequently eluted with methylene chlor-
ide : tert.-butylmethylether : methanol (90 : 5 : 5) (v v71 v71).
Using 254 nm UV-detection, two zones became visible.
Atorvastatin acid was isolated from
a
commercially
available drug. Rhodamine 123 was purchased from
Molecular Probes (Eugene, OR, U.S.A.). All other
chemicals used were of best quality available and were
purchased from Merck (Darmstadt, Germany), Sigma or
Fluka (Buchs, Switzerland).
Foetal calf serum and all culture medium contents were
from Gibco (Paisley, UK), 96-well microtiter plates were
from Costar (Cambridge, MA), OptiplateTM-96 were from
Packard (Zurich, Switzerland) and the Lab-Tek chambered
coverglass system from Nunc (Napperville, IL, U.S.A.).
Atorvastatin lactone was concentrated in zone
1 with
rf=0.60, whereas atorvastatin methyl ester was concentrated
in zone 2 with rf=0.81. After removal of the separated
zones from the plates the combined lactone and methyl ester
samples were washed with 3 ml hexane and eluted with 3 ml
methylene chloride : methanol (1 : 1) (v v71 v71). After
evaporation to dryness the compounds were lyophilized in
6 ml acetonitrile : water (1 : 1) (v v71) each, yielding 15.2 mg
atorvastatin lactone and 6.9 mg atorvastatin methyl ester.
The purity for both compounds was 498% and the
structures were con®rmed with mass spectrometry and 1H-
NMR-spectroscopy.
Cell lines
The murine monocytic leukaemia cell lines P388/P and the
doxorubicin-resistant subline P388/MDR were obtained from
Dr C. Geroni (Pharmacia & Upjohn, Centro Ricerche e
Sviluppo, Milano, Italy). Both cell lines were cultured in
RPMI glutamax 1640 supplemented with 10% (v v71) heat-
British Journal of Pharmacology vol 132 (6)

K. Bogman et al
In vitro screening of P-glycoprotein substrates
1185
Preparation of simvastatin acid and lovastatin acid
(PI) a 650 nm long-pass ®lter was used. Objectives used were
a Zeiss Plan-Neo¯uar 636oil immersion objective with a
numerical aperture of 1.25. Optical sections of 1.1 mm
thickness were scanned through the z-plane of the sample.
Auto¯uorescence of the cells was not detected. After a
10 min incubation phase in 0.15 mM R123 in HBSS at room
temperature, test drug was added and time-dependent R123
accumulation in the cells was recorded. Dead cells were
stained with PI. Final DMSO and ethanol concentrations
were below 0.6 and 1%, respectively. For data analysis,
twenty cells in one z-section of each experiment were
randomly chosen, with exclusion of death cells, and the pixel
intensity was measured using the public domain NIH Image
1.61 program. Data are reported as an average measured
pixel intensity.
Simvastatin acid and lovastatin acid were reconstituted as
described earlier (Tsuji et al., 1993), respectively from
simvastatin and lovastatin by hydrolysis in 0.05 N NaOH
solution with stirring at 208C for 30 min. The hydrolysed
solutions were adjusted to pH 7.4 with 0.2 N HCl and stored
at 48C until used.
Stock solutions
Stock solutions of test compounds were prepared using
dimethyl sulfoxide (DMSO). The rhodamine 123 stock
solution was prepared in ethanol (99.8%).
Qualitative HPLC method
For kinetic analysis, the R123 accumulation was measured
10 min after addition of statins and 30 min after addition of
verapamil and SDZ-PSC833.
Stability in Hank's balanced salt solution (HBSS) was tested
for all statins using h.p.l.c. Samples were analysed by
chromatography on
a
heated (608C) RP18 endecapped
Statistical analysis
Superspher1 100 (Lichrochart1 125-4) column, with isocratic
elution and a water : acetonitrile : tert.-butylmethylether:pho-
sphoric acid (85%) (600 : 300 : 70 : 0.2) (v v71 v71 v71) mobile
phase with a constant ¯ow rate of 1 ml min71. The e‚uent
was monitored using 236 nm UV-detection and diode array.
For estimation of the kinetic parameters,
a non-linear
regression program was used (Microcal Origin, version 6.0,
Origin Microcal Software, Inc., Northampton, MA, U.S.A.).
Data are given as means+s.e.mean. Comparison tests were
performed by ANOVA with Bonferroni's adjustment for post
hoc pairwise comparisons.
Fluorimetry
Uptake assay In order to measure net R123 uptake, 26106
cells ml71 were pre-incubated at 378C for 10 min in the
presence or absence of test drugs, followed by incubation
with 10 mM R123. After 10 min, uptake was stopped by
transferring samples on ice, cells were washed twice in the
presence or absence of test drugs at 48C and lysed in 0.8%
Triton X-100, aliquots were transferred into OptiplateTM-96
plates and ¯uorescence of the lysate was analysed with a HTS
7000 Plus Bio Assay Reader (Perkin Elmer Ltd., Buck-
inghamshire, UK) with 485 nm excitation and 535 nm
emission ®lters.
Calculation of kinetic parameters
IC50, or the concentration of test compound that exerts a
half-maximal inhibition of the R123 transport was calculated
from a saturable relationship. R123 concentrations used were
1 mM for the microtiter plate based ¯uorimetric assay and
0.15 mM for the confocal microscopy based assay.
The inhibitory potency (IPx), or the capacity of a test
compound, at concentration x mM, to inhibit the R123
transport was systematically related to the IP100 and IP10 of
verapamil in the same assay for ¯uorimetric and confocal
assays, respectively.
E‚ux assay To load R123 into P388/MDR cells prior to
e‚ux, cells were incubated at 378C in 1 mM R123 in the
presence of 100 mM verapamil in order to block P-gp. After
10 min, incubation was stopped by transferring samples on
ice; cells were dispersed into a 96-well microtiter plate
(Costar 3799) and washed twice with or without test drugs
at 48C. E‚ux was initiated by resuspending the cell pellet in
R123-free HBSS with or without test drug (e‚ux bu€er) at
room temperature. After 6 min e‚ux, 200 ml samples of the
e‚ux bu€er were analysed for R123 ¯uorescence in the HTS
7000 Plus Bio Assay Reader (Perkin Elmer Ltd., Buck-
inghamshire, U.K.) with 485 nm excitation and 535 nm
emission ®lters.
The resulting IPrel was calculated with formula 1.
IPrel ˆÁFtest compound at conc: x=ÁFverapamil
;
ꢀ1†
x
where DF is the di€erence in R123 ¯uorescence in absence
and presence of P-gp modulator.
Results
Method validation
In order to characterize P-glycoprotein (P-gp) mediated
transport of rhodamine 123 (R123) in our cell system,
parental (P388/P) and P-gp over-expressing (P388/MDR)
cells were compared for their R123 speci®c ¯uorescence levels
after incubation in 10 mM R123. The absence of P-gp
expression in P388/P cells and the high amount in P388/
MDR cells were demonstrated by Western blot earlier in our
laboratory (Drewe et al., 1999). Figure 1 shows the in¯uence
of a representative P-gp inhibitor (verapamil) and energy
depletion on the R123 accumulation in P388/MDR cells. In
the absence of P-gp modulator, the P388/MDR cells
Laser scanning microscopy
The Lab-Tek chambered coverglass containing 0.56106 living
cells ml71 in HBSS, supplemented with 1 mM pyruvate for
energy supply, was mounted on the stage of a Zeiss LSM 510
inverted laser scanning microscope. An Ar 488 nm laser line
and 505 ± 530 nm band-pass ®lter were used for R123
detection, whereas for the detection of propidium iodide
British Journal of Pharmacology vol 132 (6)

1186
K. Bogman et al
In vitro screening of P-glycoprotein substrates
displayed low ¯uorescence (26.0%) in comparison to their
P388/P cell controls. R123 accumulation up to 75.6% and
62.0% of P388/P cell controls was obtained by P-gp
inhibition using 100 mM verapamil and ATP-depletion with
NaN3, NaF respectively. This showed an energy-dependent,
P-gp mediated active extrusion of the rhodamine dye in P388/
MDR cells.
To analyse a test substance for its P-gp modulating
properties, we designed two complementary assays.
A
microtiter plate based ¯uorimetric e‚ux assay with moderate
throughput and a more sensitive confocal microscopy based
uptake assay with live monitoring of single cells.
The inter-assay variability was normalized by the inhibi-
tory potency of verapamil. For the microtiter plate based
assay the coecients of variation for intra- and inter-assay
variability were 3.7 and 9.5% respectively, the confocal
microscopy based assay had coecients of variation of 6.0
and 30.0% in between assays and in between cells
respectively.
In order to compare the two above described methods,
verapamil and SDZ ± PSC833, well-characterized P-gp in-
hibitors (Pourtier-Manzanedo et al., 1992), were analysed.
Figure 2 shows the concentration-dependent inhibition of
R123 e‚ux in P388/MDR cells, pre-incubated with 1 mM
R123, by SDZ ± PSC833 and verapamil. IC50 values were
2.7 mM and 6.5 mM respectively.
Figure 2 Microtiter plate based ¯uorimetric assay. E€ect of SDZ-
PSC833 and verapamil on R123 e‚ux out of P388/MDR cells, cells
were preloaded in 1 mM R123. Data are expressed as percentage of
R123 e‚ux in absence of inhibitor. Values represent means+s.e.-
mean for n=5.
The concentration-dependent R123 accumulation in P388/
MDR cells by SDZ ± PSC833 and verapamil were also
assessed by confocal microscopy and resulted in IC50 values
of 0.4 mM and 1.3 mM respectively (Figure 3).
In both methods, SDZ ± PSC833 was the more potent
inhibitor of P-gp, but IC50 values calculated from confocal
analysis were 6 ± 5-times lower for SDZ ± PSC833 and
verapamil, respectively, and a 12% higher di€erence of the
inhibitory potencies was measured, when compared to the
¯uorimetric assay.
Figure 3 Confocal microscopy based assay. Concentration depen-
dent e€ect of SDZ-PSC833 and verapamil on intracellular R123
accumulation in P388/MDR cells, pre-incubated with 0.15 mM R123.
Data are expressed as blank corrected mean optical densities per cell.
Values represent mean+s.e.mean for 20 cells.
Applications
To study the P-gp modulatory e€ect of a series of HMG-
CoA reductase inhibitors (e.g. atorvastatin, ¯uvastatin,
pravastatin, lovastatin and simvastatin), a combination of
both assays was used.
Prior to P-gp anity studies, possible quenching of R123
¯uorescence induced by the test compound was determined
by analysing the decrease in ¯uorescence of a ®xed R123
concentration (25 nM) upon addition of increasing concen-
trations of test compound dissolved in HBSS. Only
¯uvastatin showed a signi®cant quenching and could reduce
the R123 ¯uorescence up to 55% of its control value.
Figure 1 R123 uptake in P388/P and P388/MDR cells and in¯uence
of ATP depletion; (NaN3 10 mM, NaF 10 mM) and a representative
P-gp inhibitor (verapamil 100 mM) on R123; restoration in P388/
MDR cells. Data are given as percentage of R123 ¯uorescence in
P388/P cells. Values represent means+s.e.mean for n=5, P-values
are determined by ANOVA with Bonferroni's adjustment for post
hoc pairwise comparisons.
British Journal of Pharmacology vol 132 (6)

K. Bogman et al
In vitro screening of P-glycoprotein substrates
1187
Five to six concentrations of statins were tested in n=5
replicates starting always with the control (0 mM) and 3 mM as
the lowest concentration up to at maximum 200 mM of test
substance. This range was supposed to cover pharmacological
concentrations and never achieved toxic concentrations in the
assays. The latter was veri®ed by measuring lactate
dehydrogenase activity of the medium and by PI staining
(data not shown). None of the drugs showed signs of
cytotoxicity above 50 mM concentrations except simvastatin
lactone and lovastatin lactone.
pravastatin did show any signi®cant modulating e€ect
(P40.05). The mentioned quenching confounded ¯uvastatin
e‚ux data and therefore interpretation of the corrected
values was dicult. Estimates of the kinetic parameters
resulted in similar values for atorvastatin acid, simvastatin
and lovastatin lactone with IC50 values of 30.1, 38.2 and
45.8 mM respectively, whereas atorvastatin lactone and
methyl ester were more potent inhibitors (IC50=5.2 mM
and 3.2 mM respectively). Spontaneous and fast lactone
hydrolysis in aqueous medium led to variability in e‚ux
data for low simvastatin lactone and lovastatin lactone
concentrations. Probably due to interference with the more
hydrophilic acid form which does not show P-gp anity.
H.p.l.c. analysis of simvastatin and lovastatin hydrolysis
products showed that in aqueous medium, 30% of lactone
was transformed to the acid form within 2 h (data not
shown), this was consistent with previous reported data
(Masters et al., 1995).
Microtiter plate based fluorimetric assay
The microtiter plate based ¯uorimetric e‚ux assay (Figure
4 and Table 1), showed that atorvastatin lactone, methyl
ester and acid, simvastatin lactone and lovastatin lactone
induced a concentration-dependent inhibition of the R123
e‚ux but not simvastatin acid neither lovastatin acid nor
Figure 4 Microtiter plate based ¯uorimetric assay. Concentration dependent e€ect of P-gp modulators on R123 e‚ux out of P388/
MDR cells, cells were preloaded in 1 mM R123. (A) Simvastatin lactone and simvastatin acid; (B) lovastatin lactone and lovastatin
acid; (C) atorvastatin lactone, atorvastatin methyl ester and atorvastatin acid; (D) pravastatin and ¯uvastatin (¯uvastatin values
were corrected for quenching). Data are expressed as percentage of R123 e‚ux in absence of inhibitor. Values represent
means+s.e.mean for n=5.
British Journal of Pharmacology vol 132 (6)

1188
K. Bogman et al
In vitro screening of P-glycoprotein substrates
Table 1 Ranking of a series of P-gp modulators according to their capacity to modulate R123 transport. Comparison of a microtiter
plate based ¯uorimetric e‚ux assay with a confocal microscopy based accumulation assay and relation to lipophilicity expressed as
log P values
ATO
methyl
ester
ATO
lactone
ATO
acid
SIM
lactone
SIM
acid
LOV
lactone
LOV
acid
PRA
FLU
PSC833 Verapamil
Microtiter based fluorimetric assay
a
IC50 (mM)
3.2+0.4
5.2+0.7 30.1+12.1 38.2+24.8
ND
45.8+14.7 ND
ND
ND
2.7+2.5
6.5+1.5
b
IPrel (% of 110.3+1.1 110.0+2.0 53.1+3.5 76.9+3.8 72.9+2.8 90.7+1.8 5.8+3.4 711.6+5.4 17.6+3.1 127.7+1.5 100+2.2
IPverapamil
Confocal microscopy based assay
)
a
IC50 (mM)
t50 (min)
1.8+1.0
21.9
3.1+0.4 21.5+2.2 24.5+67.6
ND
ND
22.54+7.1
13.4
ND
ND
ND
ND
ND
ND
0.4+0.04 1.3+0.1
29.3
c
5.2
27.1
5.3
9.6
Compound properties
Log P
6.64d
(clog P)
6.04d
(clog P)
4.06e
4.7f
2.1f
4.3f
1.7f
70.23f
3.80d
3.24e
ND
3.79g
(clog P)
aConcentration of test compound needed to reach half-maximal P-gp inhibition. R123 concentration was 1 mM in the microtiter plate
based assay and 0.15 mMin the confocal microscopy based assay. Values are calculated with a saturable relationship. bInhibitory
potency, calculated with formula (1) using experimental values for R123 e‚ux, at test compound and verapamil concentrations 50 mM
(5 mM for PSC833) and 100 mM respectively. cTime needed to reach half-maximal R123 restoration after addition of test compound.
e
f
g
dSRC, KowWin public domain; (Corsini et al., 1999), (Lennernas & Fager, 1997), SRC Physprop Database, public domain; ND=not
determined.
a
b
c
d
Figure 6 Confocal microscopy based assay. Concentration depen-
dent e€ect of simvastatin lactone, lovastatin lactone, atorvastatin
acid, atorvastatin methyl ester, atorvastatin lactone on intracellular
R123 restoration in P388/MDR cells, pre-incubated with 0.15 mM
R123. Data are expressed as blank corrected mean optical densities
per cell. Values represent mean+s.e.mean for 20 cells.
Figure 5 Representative confocal microscopy pictures of P388/P (a),
P388/MDR (b) cells after incubation in 0.15 mM R123 and P388/
MDR after subsequent addition of verapamil 10 mM (c) and
atorvastatin methyl ester 10 mM (d). Addition of test compounds
that did not inhibit P-gp, resulted in pictures as shown in (b). R123
stains mitochondria in living cells. Dead cells (pointed by the arrow)
show a delocalized cytosolic dye staining. Bar=20 mM.
R123 0.15 mM and subsequent treatment with test com-
pounds.
P388/P cells accumulated within minutes a high amount of
R123 that is preferentially staining mitochondria. In contrast,
the P388/MDR cells did not accumulate the dye for at least
2 h. When cells were naturally energy depleted, a small
fraction of the dye could enter the cytosol; adding sodium
pyruvate could reverse this reaction. Upon addition of P-gp
inhibitors, P388/MDR cells started accumulating R123
resulting in an intracellular staining.
Confocal microscopy based assay
The confocal microscope based assay was applied in parallel
to the above-described method, whereby the statins were
analysed for their capacity to induce a R123 accumulation in
P388/MDR cells.
Figure 5 shows representative confocal micrographs of
P388/P and P388/MDR cells after 10 min incubation in
Results obtained by quantitative analysis of intracellular
R123 ¯uorescence upon addition of di€erent concentrations
of test compound are represented in Figure 6 and are
British Journal of Pharmacology vol 132 (6)

K. Bogman et al
In vitro screening of P-glycoprotein substrates
1189
consistent with data provided by the microtiter plate based
¯uorimetric assay. The procedure of image capture and
subsequent quanti®cation of intracellular dye ¯uorescence
with the analysis program NIH Image has been validated
before (Miller & Pritchard, 1991). The observed ranking of P-
gp modulators based on apparent IC50 was atorvastatin
methyl ester 5 atorvastatin lactone 4 lovastatin lactone %
e€ect measured by the ¯uorimetric based assay. They could
be explained by the di€erent experimental designs, e‚ux
versus uptake assays. During the e‚ux phase after loading of
the cells, the direction of R123 passive di€usion and P-gp
mediated extrusion was the same and sink conditions were
maintained. Whereas in the uptake assay the two ¯uxes were
in opposite directions. The same observations were reported
in ¯ow cytometry assays (Wang et al., 2000). Also an
intracellular trapping of dye had to be considered, a fraction
that would not be available anymore for active extrusion.
Furthermore, e‚ux was stopped after 5 min, where uptake
was analysed at least for 10 ± 30 min after addition of
modulator. In addition, a 6.7-times lower R123 concentration
was used in confocal microscopy.
simvastatin lactone
% atorvastin acid (Table 1). No
signi®cant (P40.05) inhibitory e€ect was seen for pravastatin
neither ¯uvastatin, nor for simvastatin and lovastatin in their
acid forms.
The time needed to reach half-maximal R123 accumulation
(t50) was calculated with a saturable relationship and resulted
in
a higher accumulation rate for simvastatin lactone,
atorvastatin lactone and lovastatin lactone than for atorvas-
tatin methyl ester and atorvastatin acid (Table 1).
In confocal microscopy, calculated IC50 values for the
statins may be over-estimated, due to the small number of
samples in the low concentration range of the dose-response
curve. The latter method showed an unexpectedly high
IPrel750 value for simvastatin (268%) and lovastatin (215%)
in their lactone forms that can not be explained yet.
Nevertheless, the overall ranking from strong over weak to
none P-gp inhibition and anity was: SDZ-PSC833 4
atorvastatin methyl ester 5 atorvastatin lactone 4 verapamil
4 simvastatin lactone % lovastatin lactone % atorvastatin
Discussion
The aim of the present work was to study P-gp modulation
by a series of HMG-CoA reductase inhibitors. Recently,
atorvastatin has been identi®ed as P-gp substrate (Boyd et al.,
2000; Wu et al., 2000), for some of the other statins P-gp
interaction was assumed but not yet proven (Christians et al.,
1998; Dimitroulakos & Yeger, 1996; Lindahl et al., 1999).
The high hepatic extraction (Corsini et al., 1999; Lennernas
& Fager, 1997) of these compounds gives evidence for
transporter-mediated excretion into the bile by P-gp and
MRP2 (cMOAT), which are located at the canalicular plasma
membrane (Muller & Jansen, 1997).
acid
4 pravastatin % ¯uvastatin. This ranking was
comparable for both methods and consistent with results of
other groups for known inhibitors (Pourtier-Manzanedo et
al., 1992).
Lipophilicity
Because of its hydrophilicity, pravastatin's passive di€usion
into hepatocytes is limited and transport across the sinusoidal
membrane is facilitated by the sodium dependent organic
anion transporter, hOATP2; this process can be inhibited by
atorvastatin, simvastatin and lovastatin, suggesting that these
statins possibly compete for this transport (Hsiang et al.,
1999). Bile excretion of pravastatin is facilitated by MRP2
(Yamazaki et al., 1997).
When drugs compete for a common transporter, drug
interactions are likely to occur. Until now, e€ects on
metabolism explained most clinical interactions with HMG-
CoA reductase inhibitors. Pravastatin is not metabolized by
the CYP450 3A4 system, therefore it is likely that the
increased pravastatin plasma concentration after co-adminis-
tration with cyclosporin A is caused by interaction on one or
more transporter proteins.
We established two in vitro assay systems that can be used
complementary to assess drug mediated P-gp modulation.
The confocal microscopy based assay can be used as a ®rst-
line highly sensitive method to analyse new substances for
their e€ect on R123 accumulation properties. The need for
only small amounts of compounds and cells and the real-time
process monitoring makes it an appropriate method for early
preclinical drug screening. Potential cellular toxicity and
changes in the dye accumulation pattern is readily detectable.
The microtiter plate based ¯uorimetric assay performs a fast
further screening of extended concentrations and saturation
kinetics. The latter assay allows a higher throughput of
samples.
In previous structure ± activity ± relationship studies on mod-
ulators of P-glycoprotein (Ecker & Chiba, 1995; Khan et al.,
1998; Zamora et al., 1988), it has been shown that a
correlation exists between the log
P values and the
chemosensitizing activity of a compound. In the present
study, high anity (low IC50) correlates with rather high log
P values (Table 1) with the exception of ¯uvastatin, which
has log P higher than 3. Our ®ndings suggest that ¯uvastatin
does not interact with P-gp, but the exact mechanism of
interaction with cell membranes has to be elucidated.
Its ability to decrease the surface tension at the membrane
surface and postulated interaction with lipids that could
result in an altered membrane ¯uidity has been reported
(Lindahl et al., 1999). Involvement of other transporters such
as cMOAT and OATP, both anion transporters, cannot be
excluded and will be investigated in future experiments.
Looking at the R123 accumulation rate initiated by a
modulator, fast and slow processes can be distinguished. In
addition, solubility of the modulator, transport rate through
membrane bilayer and anity for the transporter can
in¯uence P-gp interaction. The reported lower trans-mem-
brane movement rate for SDZ-PSC833 compared to the one
for verapamil (Ambudkar et al., 1999; Eytan & Kuchel, 1999;
Smith et al., 1998), is well re¯ected in its high t50 value of
29.3 min in comparison to that of verapamil (t50=9.6 min).
Furthermore, the fast onset of P-gp inhibition initiated by
verapamil was proven in intestinal perfusion studies with
talinolol in healthy volunteers (Grammate & Oertel, 1999).
Our data suggested that the more lipophilic HMG-CoA
reductase inhibitors had a higher R123 uptake rate (lower t50)
(Table 1).
The di€erences in IC50 values between the two assays
(Table 1) were most likely under-estimations of the inhibitory
British Journal of Pharmacology vol 132 (6)

1190
K. Bogman et al
In vitro screening of P-glycoprotein substrates
Table 2 Reported drug interactions with HMG-CoA reductase inhibitors
Compound
Interaction site Manifestation
P-gp Fold : AUCdigoxin
Atorvastatin
Simvastatin
Lovastatin
Pravastatin Fluvastatin
f
Digoxin
15%a
+0.3 ng/mlf
?
NSf
NS; renal CI
of digoxin:b
1.9e
Cyclosporin A P-gp+CYP3A4 Fold : AUCstatin
Diltiazem
Rhabdomyolysisc
NR
6d
3.6 (lactone)
3.7 (acid)h
20l
20e
5-23e
NSh
P-gp+CYP3A4 Fold : plasma
concentrationstatin
4.3h
NR
Itraconazole
Mibefradil
P-gp+CYP3A4 Fold : AUCstatin
P-gp+CYP3A4 Fold : AUCstatin
IC50 of mibefradil (mM)
for inhibition of statin
metabolism (in vitro)
2l
15l
NR
0.35i
NSl
1.2j
NSl
1.1k
NSi
3.4j
0.4i
7k
0.6i
NRi
Gemfibrozil
CYP3A4
Rhabdomyolysisf Rhabdomyolysisf Rhabdomyolysis,
creatine kinase
NS
: AUC of
NSf
20-30x:f
metabolitesf
AUC=area under the plasma concentration ± time curve; NR=not reported; NS=no signi®cant change; ?=no clinically signi®cant
c
interaction, information on plasma concentration not given. a(Boyd et al., 2000), b(Deslypere, 1994), (Maltz et al., 1999), d(Lennernas
f
i
& Fager, 1997), e(Corsini et al., 1999), (Stockley, 1999), g(Mousa et al., 2000), h(Azie et al., 1998), (Prueksaritanont et al., 1999),
l
j(Donahue et al., 1999), k(Muck, 2000), (Bottor€, 1999).
Drug ± drug interactions
be needed to prove the hypothesis. A variety of other factors
such as genetic polymorphism and interindividual variation
(Ho€meyer et al., 2000; Lown et al., 1997) or susceptibility
to adverse e€ects are further determinants of clinical
outcome.
In conclusion, the present study demonstrated that the
described methods are able to analyse P-gp modulating
properties of test compounds in vitro. We have shown that
atorvastatin acid, atorvastatin methyl ester and lactone,
simvastatin lactone and lovastatin lactone are P-gp mod-
ulators. No e€ect was seen with simvastatin acid, lovastatin
acid, ¯uvastatin and pravastatin. Clinical and in vitro data
suggest that P-gp modulating statins have a general trend to
high probability for drug interactions.
What is the value of this information to predict potential
pharmacokinetic drug interactions? Recently, it was demon-
strated that mibefradil is an inhibitor of CYP450 3A4
(Prueksaritanont et al., 1999). Mibefradil is also shown to
be a P-gp inhibitor (Wandel et al., 2000).
Itraconazole, a P-gp inhibitor and CYP450 3A4 substrate,
has a clinical interaction pattern with statins, that could be
explained by both P-gp and CYP450 3A4 e€ects. Among the
reported drug interactions (Table 2), the increase in digoxin
(a P-gp substrate which is not metabolized by CYP450 3A4)
plasma levels under concomitant administration of atorvas-
tatin and simvastatin can be explained by P-gp inhibition
alone.
Increased statin levels after co-administration with cyclos-
porin A can be explained both by P-gp and CYP450 3A4
inhibition. However the 5 ± 23-fold increased pravastatin
bioavailability is more likely caused by cMOAT inhibition
rather than an e€ect on P-gp and CYP450 3A4. In addition,
statins that showed P-gp modulating properties in our assays,
are presumably also transported by the latter transporter.
Our in vitro cell system can be used to screen potential
drug interactions, but well-designed clinical studies will still
We thank U. Behrens and A. Templeton for excellent technical
assistance and critically reviewing of the manuscript. We thank
Merck Sharp & Dohme Ltd (Rahway, NJ, U.S.A.), Novartis
Pharma AG (Basel, Switzerland) and Bristol-Meyers Squibb
(Sankyo Japan) for providing the test substances. This study was
supported by grants from Bristol-Meyers Squibb, Ho€mann-La
Roche Research Foundation and Swiss National Fonds Founda-
tion (Grant No. 32-52918.97).
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