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Carbamazepine is not a substrate for P-glycoprotein
Andrew Owen, Munir Pirmohamed, Justice N. Tettey, Paul Morgan,1 David Chadwick2 & B. Kevin Park
Department of Pharmacology and Therapeutics, The University of Liverpool, Ashton Street, Liverpool, L69 3GE, U.K, 1Department of Drug Metabolism,
P®zer Global Research and Development, Sandwich, Kent, CT13 9NJ and 2Department of Neurological Sciences, The University of Liverpool,
Walton Centre for Neurology and Neurosurgery, Lower Lane, Liverpool, L9 7AE
Aims To determine whether the anticonvulsant carbamazepine (CBZ), a known
CYP3A4 substrate, is also a substrate for the multidrug ef¯ux transporter
P-glycoprotein (Pgp).
Methods The role of Pgp in the transport of CBZ was assessed in three systems: (a)
in mdr1a/1b(x/x) and wild-type mice after administration of 2 mg kgx1 and
20 mg kgx1, which served as a model for brain penetration; (b) in Caco-2 cells, an in
vitro model of the intestinal epithelium that is known to express high Pgp levels; and (c)
by ¯ow cytometry in lymphocytes using rhodamine 123, a ¯uorescent substrate for
PgP.
Results Brain penetration of both doses of CBZ at 1 h and 4 h was comparable in
wild-type and mdr1a/1b(x/x) mice. Transport across the Caco-2 cell monolayer was
Pgp-independent, and was not affected by the Pgp inhibitor PSC-833. CBZ had no
effect on rhodamine 123 ef¯ux from lymphocytes, in contrast to verapamil, which
increased ¯uorescence intensity ®vefold.
Conclusion CBZ is not a substrate for Pgp. Its ef®cacy is unlikely to be affected by Pgp
over-expression in the brain. Furthermore, the interaction of CBZ with drugs that
modulate both CYP3A4 and Pgp function such as verapamil is probably due to
inhibition of CYP3A4 and not Pgp.
Keywords: carbamazepine, epilepsy, P-glycoprotein
Introduction
Epilepsy is a common condition affecting 1% of the UK
population [1]. Approximately 30% of epileptics have
inadequate control of seizures with drugs [2]. The
mechanisms underlying such drug resistance are poorly
understood. It has been suggested that over-expression of
the multidrug transporter P-glycoprotein (Pgp) in the
blood±brain barrier may increase drug ef¯ux and limit
access to the epileptic focus [3]. Clearly, this would only be
important for drugs that are substrates for Pgp.
Carbamazepine (CBZ), a widely used anticonvulsant, is
regarded as ®rst line therapy for partial seizures [4]. It is also
commonly used in combination with other antiepileptic
drugs in the treatment of refractory epilepsy [2]. CBZ
undergoes extensive metabolism, with the initial oxidative
pathways being catalysed by CYP3A4 and CYP2C8 [5].
There is a well-known overlap between substrates for
CYP3A4 and Pgp [6], but it is not known whether CBZ
is also a substrate for Pgp. It is also interesting to note
that CBZ neurotoxicity can be precipitated by con-
comitant administration of verapamil, erythromycin or
grapefruit juice [7]. Although this has been ascribed to
inhibition of CBZ metabolism by CYP3A4, these
compounds are also known inhibitors of Pgp [8]. Thus,
it is possible that the interaction with CBZ may be due to
inhibition not only of CYP3A4, but also of Pgp.
In view of these concerns, in this study we have utilized
several model systems to investigate whether CBZ is
a substrate for Pgp.
Methods
Materials
Minimal Eagle's Medium (MEM), RPMI-1640, fetal calf
serum, trypsin EDTA and Hanks balanced salt solution
Correspondence: Dr M. Pirmohamed, Department of Pharmacology and
Therapeutics, The University of Liverpool, Ashton Street, Liverpool, L69
3GE, U.K. Tel.: 0151 7945549; Fax: 0151 7945540; E-mail: [email protected]
Received 28 June 2000, accepted 6 December 2000.
f 2001 Blackwell Science Ltd Br J Clin Pharmacol, 51, 345±349 345
(HBSS) were purchased from Gibco BRL (Life technol-
ogies, Paisley, UK). Rhodamine 123, imipramine,
verapamil and CBZ were obtained from Sigma (Poole,
Dorset, UK). Ethyl acetate and acetonitrile were
purchased from Fisher Scienti®c (Loughborough, UK).
Dosing of wild-type and transgenic knockout mice
Wild-type (fvb1) and mdr1a/1b(x/x) knockout mice
(Taconic Farms, Germantown, USA) were administered
CBZ (2 mg kgx1 or 20 mg kgx1 in 60% polyethylene
glycol:H2O; i.p.) 1 and 4 h before being sacri®ced (n=4
in each group). The doses used approximate to those used
clinically, at the start of therapy, and in those on
maintenance CBZ therapy. Blood (1.5 ml) was removed
by cardiac puncture prior to removal of brain. The samples
were frozen at x20uC until analysed by h.p.l.c.
Determination of drug transport in Caco-2 cells
Caco-2 cells (1r106), cultured in MEM supplemented
with 20% (v/v) fetal calf serum were placed in 35 mm,
six well-transwell (0.4 mm ®lters) culture plates (Costar,
Bucks, UK) for approximately 2 weeks prior to transport
experiments to allow the cells to adhere and reach
con¯uence. Fresh medium was added to both apical
(1.5 ml) and basolateral (2.5 ml) compartments every
2±3 days until the cells were 100% con¯uent. Cell
monolayer integrity was assessed by measuring the
transepithelial electrical resistance (TEER) using a Milli-
cell-ERS (electrical resistance system). For the transport
experiments, the cell monolayers were equilibriated in
warm (37uC) HBSS, following which the medium from
all apical and basolateral compartments was removed, and
replaced with CBZ (10 mM in HBSS) or HBSS alone.
Transport was also assessed in the presence of the Pgp
inhibitor PSC-833 (100 mM; a gift from Novartis, Basle),
following a 10 min preincubation period. Incubations
were performed for 1 h, after which the apical and
basolateral compartments were sampled for analysis.
Apparent permeability (Papp) values were then calculated
for both apical to basolateral (PappAtoB), and basolateral to
apical (PappBtoA) movement of compound [9]. A PappBtoA
to PappAtoB ratio of greater than 1.0 was indicative of
active ef¯ux transport of compound by an apical transport
protein such as P-gp.
Papp (cm s)=(Concentration in target compartment)/
(Concentration in origin compartment
rmembrane surface area (cm)rtime (s))
The Caco-2 cells used were shown to express high levels
of Pgp mRNA and protein by RT-PCR and Western
blotting, respectively (data not shown). Furthermore, the
same batch of cells had also been shown to have functional
Pgp activity in our laboratories [9].
Development of an h.p.l.c. method for quanti®cation ofcarbamazepine
Owing to lack of availability of radiolabelled CBZ, an
h.p.l.c. method was developed to allow quanti®cation in
the animal and tissue culture studies. All experiments
were carried out on whole brains, which were weighed
following removal, and then homogenized, prior to
extraction with ethyl acetate (3 mlr3). In initial experi-
ments, CBZ (0.8 mg mlx1) was recovered quantitatively
from spiked samples of brain and whole blood following
the addition of an internal standard (imipramine) and
extraction with ethyl acetate (3 ml, three times) with an
ef®ciency of 96.4t5.9% (n=3). A rectilinear relationship
was obtained between detector responses and CBZ
recovered from spiked samples over the concentration
range 0±1.6 mg mlx1 (r2>0.995, n=3). CBZ and imipra-
mine were separated from endogenous material at ambient
temperature on a Lichrospher1 60 RP-select B column
(Merck, Darmstadt, Germany, 125r4 mm, 5 m). The
mobile phase was composed of acetonitrile and ammo-
nium acetate buffer (25 mM, pH 4.0) and was delivered
at 1 ml minx1 with a Kontron-325 gradient pump
(Kontron Instruments, Massachusetts, USA) equipped
with a Kontron-360 autosampler and Kontron-332 u.v.
detector set at 285 nm. A gradient of 40%-80%
acetonitrile over 15 min was used and CBZ and imipra-
mine eluted at 3.5 and 8.5 min, respectively. Chromato-
grams were acquired and analysed using a Kontron PC
integration package. The limits of detection and quanti-
®cation were 30 ng mlx1 and 75 ng mlx1, respectively,
with intra- and interday assay precision of 97.9% and
98.0%, respectively (n=4). The data presented were all
above the limit of quanti®cation. The amount of CBZ
in whole brain was calculated as:
(YrX/A) ng gx1 of brain tissue
where Y=Total volume of reconstituted extract (ml).
X=Concentration of CBZ in reconstituted extract
(ng mlx1).
A=Weight of murine brain (g).
Effect of carbamazepine on ef¯ux of the ¯uorescent dyerhodamine 123
Lymphocytes were isolated from freshly drawn venous
blood as described previously [10]. Rhodamine 123 ef¯ux
studies were carried out by the method of Pro®t et al. [9].
Brie¯y, lymphocytes were loaded with rhodamine 123
(1.5 mg mlx1; 25min; 4uC) before being washed twice
with RPMI-1640 (1 ml; 1uC). The cells were then
A. Owen et al.
346 f 2001 Blackwell Science Ltd Br J Clin Pharmacol, 51, 345±349
incubated at 37uC or 4uC for 3 h in 1 ml of dye-free
media to allow dye ef¯ux, while parallel experiments
were performed at 37uC in the presence of either the
positive control, verapamil (30 mM), or CBZ (10 and
100 mM). The cells were then harvested, washed twice in
RPMI-1640, and ®xed before ¯ow cytometric analysis on
an EPICS-XL ¯ow cytometer (Beckman Coulter, Bucks,
UK). The lymphocyte population was electronically gated
to exclude debris. At least 5000 events were collected for
each sample, with cellular rhodamine 123 ¯uorescence
being plotted against the number of events. Data
acquisition was performed using the computer program
EXPO analysis software to determine median FL1
¯uorescence values.
Statistical analysis
All results are presented as meants.d. with 95%
con®dence intervals for the difference between the
means, where appropriate. Statistical analysis was per-
formed by using the unpaired t-test, after con®rming that
the data were normally distributed. A two-tailed P value of
<0.05 was accepted as being signi®cant.
Results
Studies in wild-type and mdr1a/1b(x/x) mice showed
that there were no differences in the brain concentrations
of CBZ after administration of low dose (2 mg kgx1) at
1 h (95% CI for the difference x657±236), and high dose
(20 mg kgx1) at both 1 (95% CI x6104±5004) and 4 h
(95% CI x357±282) (Figure 1).
Within whole blood, CBZ concentrations in wild-type
and knockout mice again did not show any differ-
ence and were as follows: 0.14t0.08 mg mlx1 and
0.40t0.29 mg mlx1 (95% CI for the difference
x0.1±0.6) 1 h after administration of 2 mg kgx1 CBZ
in wild-type and knockout mice, respectively; and after
administration of 20 mg kgx1 CBZ, the concentra-
tions were 7.61t1.35 mg mlx1 and 7.78t3.05 mg mlx1
(95% CIx3.9±4.3) at 1 h, and 0.56t0.18 mg mlx1
and 0.55t0.16 mg mlx1 (95% CI x0.3±0.3) at 4 h in
wild-type and knockout mice, respectively.
After low dose CBZ administration, the whole blood
and brain concentrations at 4 h were below the limit of
sensitivity of the assay.
For studies with Caco-2 cells, all monolayers had
a TEER greater than 200 V cmx2, indicating con¯uence
of the monolayer. The ratio of PappBtoA to PappAtoB
was 0.78t0.18 (n=4 experiments in triplicate), indicat-
ing that CBZ was not actively transported from the
basolateral to apical compartments. This was con®rmed
by the use of the Pgp inhibitor PSC-833, which failed to
alter the ratio (0.87t0.32; 95% CI for the difference
x0.5±0.4).
The effect of CBZ on rhodamine 123 ef¯ux in
lymphocytes was determined by ¯ow cytometry, and
was compared with the effect of verapamil (30 mM)
(Figure 2). In the presence of verapamil, the median
¯uorescence increased ®vefold (P<0.005). In contrast,
CBZ had no effect on ¯uorescence intensity at either
10 mM or 100 mM (Figure 2).
1h
Dose (mg kg–1)WT2
KO2
CB
Z c
once
ntra
tion
(ng
g–1 o
f bra
in)
14000
12000
10000
8000
6000
4000
2000
0
Time 1h 1h 1h 4h 4h
WT20
KO20
WT20
KO20
Figure 1 Brain concentrations of carbamazepine (CBZ) in
wild-type (WT) and mdr1a/1b(x/x) knockout (KO) mice at
1 and 4 h after administration of either low (2 mg kgx1) or high
(20 mg kgx1) doses of CBZ. Results represent mean t s.d. of
determinations in four mice within each group. No signi®cant
differences were found between WT and KO mice at the same
doses and times (Student's t-test).
Control Verapamil(30 µM)
Rat
io F
L1 r
elat
ive
to c
ontr
ol
16
14
12
10
8
6
4
0
2
CBZ(10 µM)
CBZ(100 µM)
*
Figure 2 Ratios of median ¯uorescence intensity (FL1) of
human lymphocytes in the presence of either verapamil or
carbamazepine (CBZ) relative to control lymphocytes. Data are
expressed as mean t s.d. of four separate experiments (performed
in duplicate). Statistical analysis performed by t-test: *P<0.005
(when compared with control).
Short report
f 2001 Blackwell Science Ltd Br J Clin Pharmacol, 51, 345±349 347
Discussion
P-glycoprotein (Pgp), an ATP-dependent membrane-
bound drug ef¯ux pump that is ubiquitously distributed,
transports a large number of therapeutically and structu-
rally disparate drugs [11]. It ®rst came to prominence as
a mechanism for conferring resistance to cancer che-
motherapy [12]. More recently, it has been suggested that
Pgp may also confer drug resistance in other diseases,
including HIV [13] and epilepsy [3].
Approximately 30% of epileptics become refractory
to drug treatment [2]. The ®nding of high levels of
Pgp expression in surgically resected temporal lobe speci-
mens has led to speculation of its role in the pathogenesis
of refractory epilepsy [3]. It is therefore important to
determine which of the anticonvulsants are Pgp substrates,
as such knowledge would be of obvious therapeutic value.
In this study, we have therefore investigated whether CBZ
is a Pgp substrate using three different model systems that
serve as surrogates for Pgp expression in different tissues.
Transgenic mdr1a/1b(x/x) mice are particularly
bene®cial when looking at brain penetration of drugs.
These knockout mice have provided valuable information
on the role of Pgp in drug disposition in vivo [11]. Our
results showed that there were no differences in brain and
whole blood concentrations of CBZ at both 1 and 4 h
after administration of either high or low doses. This is an
important observation since in patients with refractory
epilepsy where Pgp has been shown to be over-expressed
[3], the use of CBZ should lead to adequate brain
concentrations, and hence no impairment of ef®cacy.
Furthermore, many patients with refractory epilepsy are
already treated with CBZ, and yet are still not adequately
controlled [2]; this by itself indicates that factors other than
(or as well as) Pgp expression are also determinants of
treatment resistance in epilepsy.
The Caco-2 cell monolayer serves as a useful model to
investigate the role of Pgp in determining drug absorption
from the intestine. In accordance with the results obtained
with the mdr1a/1b(x/x) knockout mice, no active
transport of CBZ from the basolateral to apical membranes
was demonstrated indicating that CBZ is not a substrate for
intestinal Pgp. This was further con®rmed by the use
of PSC-833, a potent Pgp inhibitor [14], which failed
to change the directional transport. This observation is of
interest for two main reasons: ®rst, the interaction of CBZ
with verapamil and erythromycin [7] is therefore largely
due to inhibition of CYP3A4, and not Pgp. Taken
together with the fact that CBZ entry into the brain is not
in¯uenced by Pgp, it can be concluded that the interaction
is occurring at the level of the intestinal wall and liver, and
not brain. Second, it provides further evidence that not all
CYP3A4 substrates are also Pgp substrates, and is in
accordance with a recent study, which concluded that the
overlap in substrate speci®cities of CYP3A and Pgp was
nothing more than coincidental [6].
The third method used to assess Pgp-mediated transport
of CBZ was with ¯ow cytometry using the ¯uorescent dye
rhodamine 123, which allowed assessment of function
at the level of the individual cell [9]. This method gives an
indication whether a compound is a Pgp substrate, but
cannot by itself be used to conclusively prove that
a compound is not a substrate since there are multiple
binding sites within the Pgp molecule [15]. Furthermore,
rhodamine 123 ef¯ux may also partly be due to other
transporters such as MRP1 [16]. In accordance with
previous studies, verapamil, a known Pgp inhibitor [8],
interfered with rhodamine 123 ef¯ux from lymphocytes.
However, CBZ had no effect on rhodamine 123 ef¯ux
indicating that it did not compete for transport by Pgp,
or indeed by MRP1.
In conclusion, using three different systems, we have
shown that CBZ is not a substrate for the ef¯ux transporter
Pgp. Thus, its ef®cacy is unlikely to be inadvertently
affected by Pgp over-expression in the blood±brain
barrier. Furthermore, the interaction of verapamil and
other CYP3A4 substrates with CBZ is probably due to
inhibition of CYP3A4 and not Pgp.
AO is in receipt of a PhD studentship from P®zer Global Research
and Development. BKP is a Wellcome Principal Fellow. JNT is
a Glaxo Wellcome Postdoctoral fellow. The authors wish to thank
Novartis for supplying PSC-833.
References
1 Shorvon SD. Epidemiology, classi®cation, natural history, and
genetics of epilepsy. Lancet 1990; 336: 93±96.
2 Kwan P, Brodie MJ. Early identi®cation of refractory epilepsy.
N Engl J Med 2000; 342: 314±319.
3 Tishler DM, Weinberg KI, Hinton DR, Barbaro N, Annett
GM, Raffel C. Mdr1 gene-expression in brain of patients with
medically intractable epilepsy. Epilepsia 1995; 36: 1±6.
4 Chadwick D. Safety and ef®cacy of vigabatrin and
carbamazepine in newly diagnosed epilepsy: a multicentre
randomised double-blind study. Vigabatrin European
Monotherapy Study Group. Lancet 1999; 354: 13±19.
5 Kerr BM, Thummel KE, Wurden CJ, et al. Human liver
carbamazepine metabolism ± role of CYP3A4 and CYP2C8 in
10,11-epoxide formation. Biochem Pharmacol 1994; 47:
1969±1979.
6 Kim RB, Wandel C, Leake B et al. Interrelationship between
substrates and inhibitors of human CYP3A and P-glycoprotein.
Pharm Res 1999; 16: 408±414.
7 Dresser GK, Spence JD, Bailey DG.
Pharmacokinetic-pharmacodynamic consequences and clinical
relevance of cytochrome P450 3A4 inhibition. Clin
Pharmacokinet 2000; 38: 41±57.
8 Wandel C, Kim RB, Kajiji S, Guengerich P, Wilkinson GR,
Wood AJ. P-glycoprotein and cytochrome P-450 3A
inhibition: dissociation of inhibitory potencies. Cancer Res
1999; 59: 3944±3948.
A. Owen et al.
348 f 2001 Blackwell Science Ltd Br J Clin Pharmacol, 51, 345±349
9 Pro®t L, Eagling VA, Back DJ. Modulation of P-glycoprotein
function in human lymphocytes and Caco-2 cell monolayers
by HIV-1 protease inhibitors. AIDS 1999; 13: 1623±1627.
10 Pirmohamed M, Graham A, Roberts P, et al. Carbamazepine
hypersensitivity: assessment of clinical and in vitro chemical
cross-reactivity with phenytoin and oxcarbazepine. Br J Clin
Pharmacol 1991; 32: 741±749.
11 Schinkel AH, Wagenaar E, Mol CAAM, Vandeemter L.
P-glycoprotein in the blood±brain-barrier of mice in¯uences
the brain penetration and pharmacological activity of many
drugs. J Clin Invest 1996; 97: 2517±2524.
12 Germann UA. P-glycoprotein ± a mediator of
multidrug-resistance in tumor-cells. Eur J Cancer 1996;
32A: 927±944.
13 Kim RB, Fromm MF, Wandel C, et al. The drug
transporter P-glycoprotein limits oral absorption and brain
entry of HIV-1 protease inhibitors. J Clin Invest 1998; 101:
289±294.
14 Atadja P, Watanabe T, Xu H, Cohen D. PSC-833, a frontier in
modulation of P-glycoprotein mediated multidrug resistance.
Cancer Metastasis Rev 1998; 17: 163±168.
15 Buxbaum E. Co-operative binding sites for transported
substrates in the multiple drug resistance transporter Mdr1. Eur
J Biochem 1999; 265: 64±70.
16 Davey MW, Hargrave RM, Davey RA. Comparison of drug
accumulation in P-glycoprotein-expressing and
MRP-expressing human leukaemia cells. Leuk Res 1996;
8: 657±664.
Short report
f 2001 Blackwell Science Ltd Br J Clin Pharmacol, 51, 345±349 349
