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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.114.221952
J Pharmacol Exp Ther 353:351–359, May 2015
Pilocarpine-Induced Convulsive Activity Is Limited by Multidrug
Transporters at the Rodent Blood-Brain Barrier
K. Römermann, J. P. Bankstahl,1 W. Löscher, and M. Bankstahl
Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, and Center for Systems
Neuroscience, Hannover, Germany
Received December 11, 2014; accepted March 6, 2015
Introduction
The pilocarpine-induced status epilepticus (SE) model is
the most widely used rodent model of both SE itself and
chronic temporal lobe epilepsy (TLE) developing as a longterm consequence of SE. In humans, SE is a life-threatening
emergency, often refractory to anticonvulsive drug treatment
(Treiman et al., 1998; Mayer et al., 2002; Young, 2006),
resulting in a mortality rate of 40% or higher (Young, 2006),
and is often followed by severe neuropathological consequences. Chronic TLE, the most frequent type of epilepsy, is
associated with pharmacoresistance in more than two-thirds
of cases (Semah et al., 1998). Post-SE animal models of TLE,
such as the pilocarpine model, provide insight into pathologic
mechanisms of epileptogenesis and pharmacoresistant epilepsy (Löscher and Brandt, 2010). The pilocarpine model is
extensively applied in rats and, more recently with increasing
frequency, in mice (Schauwecker, 2012). In this model, SE
This work was supported by a grant from the German Research Foundation
(DFG) [Grant Lo 274/10-2]. The authors have no conflicts of interest.
K.R. and J.P.B. contributed equally to this work.
1
Current affiliation: Department of Nuclear Medicine, Preclinical Molecular
Imaging, Hannover Medical School, Hannover, Germany.
dx.doi.org/10.1124/jpet.114.221952.
lithium chloride or tariquidar. Concentration equilibrium transport
assays (CETA) were performed using cells overexpressing murine
PGP or BCRP. Lower pilocarpine doses were necessary for SE
induction in PGP-deficient mice. Brain-plasma ratios were higher
in mice lacking PGP or PGP and BCRP, which was also observed
after pretreatment with tariquidar in mice and in rats. Lithium
chloride did not change brain penetration of pilocarpine. CETA
confirmed transport of pilocarpine by PGP and BCRP. Pilocarpine
is a substrate of PGP and BCRP at the rodent blood-brain barrier,
which restricts its convulsive action. Future studies to reveal
whether strain differences in pilocarpine sensitivity derive from
differences in multidrug transporter expression levels are
warranted.
induction via systemic or intracerebral administration of the
cholinergic muscarinic agonist pilocarpine serves as an initial
insult for provoking epileptogenesis. Importantly, the model
reflects many features of human TLE in terms of seizure
characteristics and histopathological findings (Curia et al.,
2008). Furthermore, the pilocarpine model is highly valuable for
studying consequences of seizures and SE induced by cholinergic agents in chemical weapons and for developing counteractions (Tetz et al., 2006; de Araujo Furtado et al., 2012).
Pilocarpine is also clinically relevant as a topical treatment of
glaucoma (Lee and Higginbotham, 2005), and local or systemic
treatment of dry mouth (xerostomia; Visvanathan and Nix,
2010).
Since the first description of the model by Turski et al. (1984),
different protocols have been developed for systemic SE
induction by pilocarpine. In many laboratories, SE is induced
via injection of a single high pilocarpine dose. This approach is
often accompanied by a high mortality rate and does not always
lead to SE induction in a satisfactory proportion of animals
(Turski et al., 1989; Glien et al., 2001; Buckmaster and Haney,
2012; Mazzuferi et al., 2012). Using a fractionated protocol with
repetitive injection of smaller pilocarpine doses can improve
the outcome in terms of SE induction rate and low mortality in
rats and in mice (Glien et al., 2001; Gröticke et al., 2007;
ABBREVIATIONS: BCRP, breast cancer resistance protein; CETA, concentration equilibrium transport assays; HPLC, high-performance liquid
chromatography; Ko143 hydrate, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-( 2-methylpropyl)-1,4-dioxopyrazino[19,29:1,6]pyrido[3,4-b]
indole-3-propanoic acid 1,1-dimethylethyl ester hydrate; LLC cells, Lilly Laboratories cells; MDCK-II cells, Madin-Darby canine kidney type II cells; PGP,
P-glycoprotein; PSC833, (3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-6,9,18,24-tetraisobutyl-3,21,30-triisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl33-[(2R,4E)-2-methyl-4-hexenoyl]-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone; SE, status epilepticus;
TLE, temporal lobe epilepsy; WT, wild-type.
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ABSTRACT
As a result of the growing availability of genetically engineered
mouse lines, the pilocarpine post–status epilepticus (SE) model of
temporal lobe epilepsy is increasingly used in mice. A discrepancy
in pilocarpine sensitivity in FVB/N wild-type versus P-glycoprotein
(PGP)–deficient mice precipitated the investigation of the interaction between pilocarpine and two major multidrug transporters at the blood-brain barrier. Doses of pilocarpine necessary
for SE induction were determined in male and female wild-type
and PGP-deficient mice. Brain and plasma concentrations were
measured following low (30–50 mg×kg21 i.p.) and/or high (200
mg×kg21 i.p.) doses of pilocarpine in wild-type mice, and mice
lacking PGP, breast cancer resistance protein (BCRP), or both
transporters, as well as in rats with or without pretreatment with
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Römermann et al.
Materials and Methods
Animals. For the present study, 187 adult mice of both sexes [n 5
98 FVB/N WT mice, n 5 77 Mdr1a/b(2/2) mice, n 5 6 Bcrp1(2/2) mice,
n 5 6 Mdr1a/b(2/2)Bcrp1(2/2) mice], and 18 adult male SpragueDawley rats (Harlan, Horst, The Netherlands) were used. Breeding
pairs of the four mouse genotypes were kindly provided by the group of
Dr. A. Schinkel (The Netherlands Cancer Institute, Amsterdam, The
Netherlands), and offspring were used in this study. Twenty-one of the
WT mice were obtained from Charles River (Sulzfeld, Germany) and
used for the pharmacokinetic experiment. Mice and rats were housed
in groups separated by sex under controlled environmental conditions
(22 6 1°C, 40–70% humidity) with a 12-hour light/dark cycle (lights on
at 6:00 AM) and ad libitum access to food and water. All experiments
were performed in accordance with the European Communities Council
Directive of November 24, 1986 (86/609/EEC) and the German Law on
Animal Protection (“Tierschutzgesetz”). Ethical approval for the study
was granted by an ethical committee (according to x15 of the
Tierschutzgesetz) and the government agency (Lower Saxony State
Office for Consumer Protection and Food Safety; LAVES) responsible
for approval of animal experiments in Lower Saxony. All efforts were
made to minimize pain or discomfort in the animals used.
Chemicals and Cell Lines. Pilocarpine hydrochloride, lithium
chloride (LiCl), and methylscopolamine bromide were purchased from
Sigma-Aldrich (Taufkirchen, Germany) and dissolved in injectible
water. The PGP/BCRP inhibitor tariquidar dimesylate was kindly
provided by Xenova Ltd. (Slough, UK) and freshly dissolved prior to each
administration in 5% (w/v) aqueous dextrose solution. Administration
volume was 10 mg×kg21 for all compounds in mice, and 2 ml×kg21 (LiCl,
tariquidar) or 3 ml×kg21 (pilocarpine) in rats. For the in vitro experiments,
tariquidar, the BCRP inhibitor Ko143 hydrate [(3S,6S,12aS)-1,2,3,4,6,7,12,12aoctahydro-9-methoxy-6-( 2-methylpropyl)-1,4-dioxopyrazino[19,29:1,6]
pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester hydrate]
(Sigma-Aldrich) and unlabeled digoxin (Sigma-Aldrich), as a reference
substrate for PGP, were dissolved in dimethyl sulfoxide (,0.1% DMSO in
final solution). The PGP inhibitor PSC833 [(3S,6S,9S,12R,15S,18S,21S,
24S,30S,33S)-6,9,18,24-tetraisobutyl-3,21,30-triisopropyl-1,4,7,10,
12,15,19,25,28-nonamethyl-33-[(2R,4E)-2-methyl-4-hexenoyl]1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontane-2,5,8,11,14,
17,20,23,26,29,32-undecone] (kindly provided by Novartis Pharma
GmbH, Nürnberg, Germany) was dissolved in ethanol/Tween 80 (9:1)
and then diluted with double-distilled water (1:2). [14C]Mannitol and
[3H]digoxin (specific activity: 1.85–2.22 kBq/mmol and 0.555–1.48
GBq/mmol, respectively) were obtained from Hartmann Analytic
GmbH (Braunschweig, Germany).
For in vitro transport studies, Lilly Laboratories cell (LLC)-PK1 cells
transduced with murine Mdr1a and respective WT LLC cells were
used. Madin-Darby canine kidney type II (MDCK-II) cells transduced
with murine Bcrp1 and respective WT MDCK-II cells were also used.
Culturing of the cells was performed as described previously (Baltes
et al., 2007). Both cell lines are widely used and were recommended by
the US Food and Drug Administration for identifying PGP substrates
(Food and Drug Administration, 2006). The sole use of these cell lines as
a model system does not reflect all properties of the complex
composition of a blood-brain barrier, and therefore, does not allow
direct translation to the in vivo situation. In this study, cell assays were
used to confirm the in vivo data.
SE Induction by Pilocarpine in Mice. In total, 53 (21 males, 32
females) WT mice and 65 (25 males, 40 females) Mdr1a/b(2/2) mice
were used for SE induction experiments. To reduce peripheral side
effects of pilocarpine, mice were pretreated with 1 mg×kg21 i.p.
methylscopolamine 30 minutes before the first injection of pilocarpine.
Based on a prior fractionated protocol (Gröticke et al., 2007), the
pilocarpine dose necessary to induce self-sustaining SE was adapted to
the mouse genotypes by intraperitoneal administration of an initial
bolus (100–200 mg×kg21) and up to nine subsequent injections of
50–150 mg×kg21 every 20 or 30 minutes. Animals were continuously
monitored, and time of occurrence as well as number and severity
[referred to Racine’s scale (Racine, 1972)] of each single seizure prior to
SE was recorded. SE onset was defined as continuous ongoing seizure
activity following occurrence of one or two generalized tonic-clonic
seizures [stage 4 or 5 on the Racine scale (Racine, 1972)]. SE was
characterized by head nodding in an upright (sitting) body position,
Straub tail, and slight to moderate convulsions of forelimbs, sometimes
interrupted by further generalized tonic-clonic seizures (see Bankstahl
et al., 2012). Behavioral seizure activity and SE duration was confirmed
by individual EEG recordings in a subset of animals (for methodological
details, see Klein et al., 2015). For EEG recordings, animals were
implanted with bipolar electrodes in the dorsal hippocampus [anteroposterior (AP) 1.8 mm, laterolateral (LL) 1.6 mm, and dorsoventral
(DV) 2.0 mm relative to bregma] as described elsewhere (Gröticke et al.,
2008).
Pharmacokinetic Profile of Pilocarpine in Mice. Pilocarpine
was administered intraperitoneally at a subconvulsive dose of 50 mg×kg21
to WT mice, which were closely observed for potential occurrence of
seizures. After 5, 10, 15, 20, 30, 60, and 120 minutes, mice were deeply
anesthetized with carbon dioxide (n 5 3 per time point) and blood was
sampled by retro-orbital puncture into vials preloaded with 5 mM
EDTA. After cervical dislocation, brains were immediately removed,
weighed, and homogenized after addition of double distilled water
(∼1 ml per 100 mg tissue), and frozen in aliquots of 200 ml. Blood
samples were centrifuged (12,000 rpm, 3 minutes; room temperature)
and the supernatant plasma was frozen. All samples were stored at
–30°C until analysis. Plasma elimination half-life (t1/2) was estimated
by PK Solutions 2.0 (Summit Research Services, Montrose, CO).
Animals were not perfused before taking brain samples, as this can lead
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Schauwecker, 2012). Additionally, lithium chloride is often
administered the evening before the pilocarpine experiment,
which drastically reduces the required pilocarpine dose for SE
induction, thereby increasing the proportion of animals
surviving SE (Clifford et al., 1987; Bersudsky et al., 1994;
Shaldubina et al., 2007).
Unfortunately, SE induction and survival rates can dramatically differ not only between rodent strains (Chen et al.,
2005; Winawer et al., 2007a,b), but also between animals of
the same strain bred by different vendors and even between
inbred mice originating from different breeding rooms of the
same vendor (Borges et al., 2003; Bankstahl et al., 2009, 2012;
Müller et al., 2009). The mechanisms underlying this variable
efficacy of pilocarpine are unknown. In our laboratory, we
assessed the SE induction protocol in a batch of rodents from
new strains or vendors before starting the main experiment.
By this means, we found evidence that mice lacking the
multidrug transporter P-glycoprotein [PGP; Mdr1a/b(2/2)
mice] required lower doses of pilocarpine for SE induction
than wild-type (WT; FVB/N) mice. Multidrug transporters are
expressed at tissue barriers throughout the body, such as the
blood-brain barrier, and serve as protective mechanisms,
transporting a wide spectrum of xenobiotic compounds
(Löscher and Potschka, 2005b). Since the substrate spectrum
of the most widely investigated multidrug transporter PGP
considerably overlaps with that of breast cancer resistance
protein (BCRP) (Löscher and Potschka, 2005a), we hypothesized that pilocarpine is transported by each of these transporters at the blood-brain barrier in mice and rats, thereby
influencing the uptake of pilocarpine by the brain, and,
consequently, its convulsant action. In vivo experiments with
genetic knockout or pharmacological inhibition of multidrug
transporters were performed and validated by in vitro
transport assays using cells overexpressing murine PGP or
BCRP.
Pilocarpine and Multidrug Transporters
counter (MicroBeta Trilux; Wallac/PerkinElmer LAS GmbH, Rodgau,
Germany).
Determination of Pilocarpine in Brain, Plasma, and Transport Assay Samples. Concentrations of pilocarpine were determined via HPLC with UV detection. The chromatographic system was
composed of a LC-6A pump (Shimadzu, Duisburg, Germany) and
a UV detector (SPD-6A; Shimadzu) set at 213 nm. Separations were
obtained on a C18 column (Refill Nucleosil 120–5 C18; Macherey and
Nagel, Düren, Germany) preceded by a guard column (Nucleosil
120–5 C18; Knauer, Berlin, Germany). The mobile phase consisted of
potassium dihydrogen phosphate buffer (0.05 M) and acetonitrile (70:
40, v/v), and 0.1% triethylamine (final pH of 7.5). The flow rate was
1 ml×min21, and the injections were carried out via a 50-ml loop. Data
processing was handled by CBM-20A software (Shimadzu). For
sample preparation, plasma (50 ml) or cell medium (40 ml) were
mixed with 150 ml (plasma) or 60 ml (cell medium) of acetonitrile,
followed by 1 minute of agitation and 5 minutes centrifugation (13,200
rpm, room temperature). The upper layer was removed and stored at
–30°C until measurement. Homogenized brain samples (200-ml aliquots)
were mixed with 200 ml of acetonitrile, followed by 5 minutes of agitation and 15 minutes centrifugation (15,000 rpm, 4°C). The supernatant
was centrifuged in 0.2-mm filters (6000 rpm, 4°C) and the lower layer
was used for injection into the HPLC apparatus. Mean recovery for
pilocarpine was 99.8% in plasma, 100.7% in brain, and 102.8% in cell
medium. The quantification limit was 200 ng/ml for plasma, 1.1 mg×g21
for brain, and 625 ng×ml21 for cell medium. Retention times under
these conditions were 6.0 minutes in plasma samples and 6.5 minutes
in brain and cell medium samples.
Statistical Analysis. Statistical analysis was performed using
Prism 5 software (GraphPad Software Inc., La Jolla, CA). For
comparison of two groups, Student’s t test was used. In the case of
more than two groups, a one-way analysis of variance followed by
Bonferroni’s multiple comparison test was performed. Fisher’s exact
test was used to assess sex differences in SE induction and mortality
rates, respectively. CETA data were statistically analyzed by two-way
analysis of variance followed by Bonferroni test to compare replicate
means. All tests were used two-tailed, and a P value ,0.05 was
considered statistically significant. Unless stated otherwise, all
values are given as mean 6 S.E.M.
Results
SE Induction in Mdr1a/b(2/2) Mice Requires Lower
Pilocarpine Doses Than in WT Mice. SE induction in mice
was performed by fractionated pilocarpine injection. As described previously for C57BL/6 and NMRI mice (Gröticke et al.,
2007; Bankstahl et al., 2012), SE development was preceded by
immobility, head nodding, Straub tail, tremor, chewing, and at
least one generalized clonic-tonic seizure (type 4 or 5 on the
Racine scale). SE itself was characterized by continuous limbic
seizure activity (head nodding, forelimb convulsions, infrequent
generalized tonic-clonic seizures). Convulsive activity characteristics and SE-associated EEG patterns were comparable in
mouse genotypes. In 40% of EEG-monitored mice, SE was selflimiting before diazepam administration. In animals with selflimiting SE, EEG analysis revealed that SE duration was
shorter in WT mice than in Mdr1a/b(2/2) mice (53.0 6 11.2
minutes, n 5 4, versus 83.5 6 3.0 minutes, n 5 4; P 5 0.039).
Both in female and male WT mice, the pilocarpine dose necessary to induce SE was higher compared with Mdr1a/b(2/2)
mice (female, 465 6 32 mg×kg21 versus 295 6 12 mg×kg21, P ,
0.0001; male, 340 6 22 mg×kg21 versus 271 6 15 mg×kg21, P 5
0.013; Fig. 1). Interestingly, female WT mice required higher
pilocarpine doses compared with male WT mice (P 5 0.030),
whereas this sex difference was not found in PGP-deficient
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to a washout effect and falsely low brain concentrations (unpublished
data). Thus, we cannot exclude that brain concentrations are overestimated owing to remaining pilocarpine in the brain vasculature, but
as all animals in this study were treated identically, group comparisons
are expected to be reliable.
Brain-Plasma Ratio of Pilocarpine in Mice and Rats with
and without Tariquidar and LiCl Pretreatment. Fifty milligrams per kilogram pilocarpine were administered intraperitoneally to
all four mouse genotypes (n 5 6 per group). A separate group of WT
mice (n 5 6) received an injection of 15 mg×kg21 tariquidar intravenously 1 hour before pilocarpine for complete inhibition of PGP
and partial inhibition of BCRP (Bankstahl et al., 2013). A further group
(n 5 6) were pretreated with 10 mEq×kg21 (423.3 mg×kg21) LiCl
intraperitoneally 13 hours before pilocarpine (Bersudsky et al., 1994) to
evaluate whether LiCl increases brain levels of pilocarpine as suggested
previously (Marchi et al., 2009). Interestingly, the potency of LiCl to
lower convulsive doses of pilocarpine is much higher in rats than in
mice (Turski et al., 1989). Therefore, in the present study, we used LiCl
doses known to successfully potentiate pilocarpine effects (10 mEq×kg21
in mice and 3 mEq×kg21 in rats). Further WT (n 5 6) and Mdr1a/b(2/2)
mice (n 5 6) were treated with a convulsive dose of pilocarpine
(200 mg×kg21). All mice were closely observed for occurrence of seizures.
Twenty minutes following pilocarpine injection, blood samples were
taken and brains excised and stored as described above.
Three subsets of rats (n 5 6 per group) were treated intraperitoneally
with 30 mg×kg21 pilocarpine and various pretreatments: untreated,
pretreated with 15 mg×kg21 tariquidar intravenously 1 hour before
pilocarpine to achieve complete PGP inhibition (Kuntner et al., 2010),
or pretreated with 3 mEq×kg21 (127 mg×kg21) LiCl by mouth 13 hours
before pilocarpine (Glien et al., 2001). Rats were closely observed for
occurrence of seizures. Thirty minutes following pilocarpine injection,
blood and brains were sampled and stored as described above.
Concentration Equilibrium Transport Assays. We employed
concentration equilibrium transport assays (CETA), in which the
influence of passive diffusion is minimized by adding the drug at equal
concentrations to the apical and the basolateral compartments of the
Transwell system (Luna-Tortós et al., 2008; Löscher et al., 2011). CETA
were performed in triplicate as described previously (Luna-Tortós et al.,
2008). LLC cells were seeded with a density of 0.3 106 cells cm22 on
transparent polyester membrane filters (Transwell, six-well, 24-mm
diameter, 0.4-mm pore size; Corning Costar Corporation, Cambridge,
MA). MDCK-II cells were seeded with a density of 0.4 106 cells cm22
on translucent polyester membrane filters (ThinCert, six-well, 24.85-mm
diameter, 0.4-mm pore size; Greiner Bio-One, Frickenhausen,
Germany). Transport experiments were performed 5–7 days after
the cells reached 100% confluence. At 1 hour preincubation, culture
medium was replaced by serum-free Opti-MEM with or without
inhibitor (10 mM PSC833 for PGP inhibition or 0.1 mM Ko143 for
BCRP inhibition). For experiments with LLC cells, Ko143 (0.1 mM)
was added to inhibit endogenous BCRP, and for experiments with
MDCK-II cells, tariquidar (0.2 mM) was added to inhibit endogenous
PGP (Kühnle et al., 2009). The subsequent incubation was performed
with pilocarpine in fresh Opti-MEM on the apical and basolateral
sides of the monolayer at a concentration of 25 mM. After an initial
distribution phase, samples were taken from both compartments after
60, 120, 240, 360, 480, and 600 minutes and the amount of pilocarpine
was quantified by high-performance liquid chromatography (HPLC)
analysis. The results were calculated as percentage of initial concentration. The calculated values after the distribution phase may be associated
with a loss of compound, which may relate to residual adherence to the
walls or membrane of the inserts (Luna-Tortós et al., 2008). As control
experiment, CETA were performed with [3H]digoxin (diluted with
unlabeled digoxin to result in an activity of 1.85 kBq/ml and a final
concentration of 10 nM) as a reference substrate. The membrane integrity of monolayers was determined by [14C]mannitol diffusion and
transepithelial electrical resistance, following exclusion criteria described
earlier (Luna-Tortós et al., 2008). [3H]Digoxin and [14C]mannitol were
measured in disintegrations per minute (DPM) with a b-scintillation
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Römermann et al.
Fig. 1. Dose of pilocarpine necessary to induce a status epilepticus in
FVB/N wild-type and Mdr1a/b(2/2) mice of both sexes. Pilocarpine was
administered intraperitoneally by a fractionated dosing protocol as
described in Materials and Methods. Data are shown as means 6 S.E.M.
[n = 23, female WT; n = 31, female Mdr1a/b(2/2); n = 15, male WT; n = 19,
male Mdr1a/b(2/2)]. Asterisk indicates statistically significant difference
versus WT mice of the same sex; hash mark indicates statistically
significant difference between female and male WT mice (P , 0.05).
Fig. 2. Pilocarpine concentration in brain and plasma and brain-plasma
ratio at 5, 10, 15, 20, 30, 60, and 120 minutes after intraperitoneal
injection of a subconvulsive pilocarpine dose (50 mg×kg21) in FVB/N wildtype mice (n = 3 per time point). Data are shown as means 6 S.E.M.
Unexpectedly, after administration of a subconvulsive dose of
50 mg×kg21 pilocarpine, brain concentrations in WT and
Mdr1a/b(2/2) were not statistically different. Only a tendency
toward higher concentration in Mdr1a/b(2/2) compared with
WT mice was identified (Fig. 3A; P 5 0.0843, Student’s t test).
However, following administration of 200 mg×kg21 pilocarpine, an increase in brain-plasma ratio of Mdr1a/b(2/2) mice
was statistically significant compared with WT mice (Fig. 3B).
An unexpected decrease in brain-plasma ratio was found in
mice lacking only BCRP compared with WT and tripleknockout mice (Fig. 3A), suggesting compensatory overexpression of other transporters in this mouse genotype. In
Mdr1a/b(2/2)Bcrp1(2/2) mice, a statistically significant increase in brain-plasma ratio of pilocarpine was observed
compared with WT mice that was higher than the increase in
the PGP-deficient mice. For comparison, we added a group of
WT mice pretreated with the dual PGP and BCRP inhibitor
tariquidar and found a similar ratio to the triple knockout
mice (Fig. 3C). By contrast, LiCl pretreatment did not alter
pilocarpine brain uptake (Fig. 3C).
For species comparison, we administered 30 mg×kg21
pilocarpine in rats. Consistent with our mouse data, increased
brain-plasma ratio of tariquidar pretreated rats was statistically significant compared with control animals, whereas LiCl
pretreatment had no effect on pilocarpine brain uptake (Fig.
3D).
Pilocarpine Is Transported by Murine PGP and
BCRP In Vitro. To confirm the in vivo findings, in vitro
CETA using Mdr1a- or Bcrp1-transduced cells were performed.
CETA allow evaluation of active transport almost independently of the passive permeability component (Luna-Tortós
et al., 2008; Löscher et al., 2011). Since endogenous PGP or
BCRP of LLC or MDCK-II cells may influence pilocarpine
transport, we inhibited function of these transporters by
adding tariquidar [at a concentration (0.2 mM) that is reported
to selectively inhibit PGP (Kühnle et al., 2009)], to Bcrp1overexpressing cells; and a BCRP inhibitor (Ko143) to Mdr1aoverexpressing cells. As we did not test whether this
concentration inhibits only PGP in our assays, we cannot
exclude that murine BCRP was partially inhibited by 0.2 mM
tariquidar. However, as BCRP is the predominantly expressed
transporter in the transduced MDCK-II cells and transport is
clearly present, any crossinhibition appears to be negligible. In
Mdr1a-overexpressing cells, transport of pilocarpine from the
basolateral to the apical compartment was detected (Fig. 4A).
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mice. SE induction rates were comparable between female
and male WT (female, 71.9% versus male, 71.4%, P 5 1.00) as
well as female and male Mdr1a/b(2/2) mice (female, 77.5%
versus male, 76.0%, P 5 1.00). No differences were found
between mouse genotypes in the number of stage 3–to–stage
5 seizures prior to reaching SE [stage 3, 0.39 6 0.12 versus
0.16 6 0.066; stage 4, 1.7 6 0.24 versus 1.5 6 0.18; stage 5,
1.1 6 0.18 versus 1.2 6 0.19, WT versus Mdr1a/b(2/2),
respectively]. Mortality rate in WT mice was not statistically
different between sexes (female, 21.4% versus male, 6.7%;
P 5 0.11). However, in PGP-deficient mice, mortality was
higher in male compared with female mice (female, 26.1%
versus male, 47.2%; P 5 0.025). This mortality in PGPdeficient male mice was also higher than in male WT mice
[Mdr1a/b(2/2), 47.2% versus WT, 6.7%; P 5 0.020], whereas
mortality in female mice did not differ between mouse
genotypes (P 5 0.321).
Pharmacokinetic Properties of Pilocarpine in WT
Mice. To gain information on blood-brain barrier penetration
of pilocarpine in WT mice, we determined pilocarpine concentrations in brain and plasma over a sampling period of 2 hours
following intraperitoneal administration of a single subconvulsive dose of pilocarpine (50 mg×kg21). Maximum plasma
concentration (29.20 6 0.86 mg×ml21) was measured 10
minutes following administration (Fig. 2). Estimated plasma
elimination half-life (t1/2) was 35 minutes. Brain concentrations peaked between 10–30 minutes after injection
(about 22 mg×g21; Fig. 2). On the basis of these results, the
time point of 20 minutes postinjection was used to compare
pilocarpine brain uptake in different mouse genotypes and
following pharmacological treatment (see below). Brainplasma ratio was approximately 1 at 30 minutes after injection
and remained constant thereafter.
Brain Accumulation of Pilocarpine Is Limited by
PGP and BCRP. To evaluate the influence of PGP on
pilocarpine brain uptake, we compared brain-plasma ratios in
WT and Mdr1a/b(2/2) mice. As PGP and BCRP are
characterized by an overlapping substrate spectrum, we
included Bcrp1(2/2) mice as well as triple knockout mice
[Mdr1a/b(2/2)Bcrp1(2/2) mice] in this part of the study.
Pilocarpine and Multidrug Transporters
355
Transport was eliminated in the presence of PGP inhibitor
PSC833 (Fig. 4B). Unexpectedly, in LLC WT cells, pilocarpine
transport was observed as well, regardless of PSC833 presence
(Fig. 4, C and D), suggesting pilocarpine transport by another
noninhibited transporter expressed to higher extent in WT cells
than in PGP-overexpressing cells. In Bcrp1-overexpressing
cells, distinct transport of pilocarpine into the apical compartment
was observed, which was not detected in WT cells (Fig. 4, E and
G). Following inhibition of murine BCRP by Ko143, pilocarpine
transport was ablated (Fig. 4F). As a routine positive control
experiment, CETA with reference substrate digoxin was
performed in LLC cells overexpressing Mdr1a. Distinct transport of digoxin was observed (Fig. 4H), which could be
completely inhibited by PSC833 (not shown).
Fig. 4. CETA with pilocarpine (25 mM) without and with respective inhibitors (10 mM PSC833 for PGP and 0.1 mM Ko143 for BCRP) in (A and B) LLC
cells overexpressing murine Mdr1a, (C and D) LLC wild-type (WT) cells, (E and F) MDCK-II cells overexpressing murine Bcrp1, and (G) MDCK-II
WT cells. In all experiments with LLC cells, 0.1 mM Ko143 was added to inhibit endogenous BCRP. In all experiments with MDCK-II cells, 0.2 mM
tariquidar was added to inhibit endogenous PGP. (H) CETA with digoxin (10 nM) as positive control (n = 1) in LLC cells overexpressing Mdr1a. Digoxin
transport was inhibited by PSC833 (not shown). Data are expressed as percentage initial pilocarpine or digoxin concentration in the apical and
basolateral chamber. Experiments in A–G were performed in replicates (n = 3–6). Data are shown as means 6 S.E.M. Asterisk indicates statistically
significant difference between basolateral and apical compartments (P , 0.05).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 3. Brain-plasma ratios 20 minutes after intraperitoneal administration of (A) a subconvulsive
pilocarpine dose (50 mg×kg21) in wild-type (WT) and
transporter knockout mice, (B) 200 mg×kg21 pilocarpine in WT and Mdr1a/b(2/2) mice, and (C)
a subconvulsive pilocarpine dose (50 mg×kg21) in
WT mice pretreated either intravenously with
15 mg×kg21 tariquidar 1 hour before pilocarpine,
or intraperitoneally with 10 mEq×kg21 LiCl 13 hours
before pilocarpine injection. (D) Brain-plasma ratios
30 minutes after administration of 30 mg×kg21
pilocarpine in control rats and rats pretreated either
intravenously with 15 mg×kg21 TQD 1 hour before
pilocarpine, or intraperitoneally with 3 mEq×kg21 LiCl
13 hours before pilocarpine injection. Data are shown as
means 6 S.E.M. (n = 6 per group). Asterisk indicates
statistically significant difference versus WT mice
or control rats; hash mark indicates statistically
significant difference versus Mdr1a/b(2/2)Bcrp1(2/2)
mice (P , 0.05).
356
Römermann et al.
Discussion
TABLE 1
Comparison of brain and plasma concentrations after single administration of pilocarpine in rats and mice
Species, Strain
Pilocarpine
Dose
Time Point
Route of
Brain
of Sampling
Administration Postadministrationa Concentration
mg×kg–1
Rat, Wistar Unilever
mg×g–1
Rat, Sprague-Dawley
300
400
350
i.p.
Rat, Sprague-Dawley
Rat, Sprague-Dawley
30
0.3 [14C]pilocarpine
i.p.
p.o.
Mouse, NMRI
300
i.p.
Mouse, FVB/N
50
i.p.
200
50
i.p.
Mouse, Mdr1a/b(2/2)
Mouse, Bcrp1(2/2)
Mouse, Mdr1a/b(2/2)Bcrp1(2/2)
i.p.
p.a., postadministration.
a
Only time points up to 4 hours postadministration are considered.
b
Calculated from values given in the paper.
c
For further time points, see Fig. 2.
15–20 min
15–20 min
At onset of stage
4 seizures
(∼20–30 min p.a.)
30 min
30 min
1h
4h
10 min
3 h (mice with
SE)
10 min
20 minc
20 min
20 min
Plasma
Concentration
BrainPlasma
Ratio
Reference
mg×ml–1
138.4
193.7
41.6b
78.10
1.772 Zgrajka et al. (2010)
168.0
1.153
b
291.2 (serum) 0.143b Marchi et al. (2007)
12.74
0.058
0.028
0.004
173.9
20.7
12.14
0.123
0.062
0.008
317
21.3
1.055
0.472
0.452
0.500
0.549
0.972
Present study
Omori et al. (2004)
21.19
21.43
100.8
25.85
19.28
28.57
29.20
25.80
98.26
27.79
28.18
25.49
0.728
0.827
1.021
0.923
0.676
1.122
Present study
Mazzuferi et al. (2012)
Present study
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
The major findings of our study are that higher doses of
pilocarpine are necessary to induce a self-sustaining SE in
mice expressing PGP as compared with mice in which the
PGP-encoding gene is knocked out. This phenomenon is
reflected by increased pilocarpine brain uptake in PGP- and
PGP/BCRP-deficient mice, as well as in WT mice and rats
following pharmacological inhibition of PGP and BCRP. The
transport of pilocarpine by PGP and BCRP is further
substantiated by in vitro transport assays.
SE Induction by Pilocarpine in FVB/N WT and
Mdr1a/b(2/2) Mice. The mouse pilocarpine model of TLE
has recently gained increasing relevance in preclinical
epilepsy research (Buckmaster and Haney, 2012). In this
study, we used a fractionated pilocarpine injection protocol for
SE induction in the inbred mouse strain FVB/N, which is
a common background strain for generation of genetically
modified mice (Taketo et al., 1991). Here, this protocol showed
superior outcome compared with a recently published work in
FVB mice by Buckmaster and Haney (2012). Although the
reported bolus protocol led to an almost identical SE induction
rate of 69.2% versus 71.7% in our study, mortality was much
higher (51.5% versus 21.1%). This supports the advantage of
individual dosing in the fractionated pilocarpine model.
The finding that Mdr1a/b(2/2) mice were more susceptible
to pilocarpine’s convulsive effects than FVB/N WT mice was
somewhat surprising, as there is no evidence from the
literature for pilocarpine transport by PGP. According to the
“rule of fours,” compounds with a molecular mass ,400 g/mol,
sum of N and O atoms #4, and base pKa ,8 are probably not
PGP substrates (Didziapetris et al., 2003). Thus, it was not
self-evident to expect pilocarpine (molecular mass, 208 g/mol,
sum of N and O atoms 5 4, pKa 5 6.78) to be transported by
PGP. However, considering the presence of phytotoxins
during evolution, it is plausible that pilocarpine, which is
contained in the leaves of a tropical plant, would be among
substrates of multidrug transporters that have protective
function at tissue barriers throughout the body. Interestingly,
we also found that higher pilocarpine doses are needed for SE
induction in female than in male WT mice. This sex difference
was not observed in PGP-deficient mice and therefore could be
explained by a PGP-dependent mechanism. Similar sex differences in SE induction rate have previously been described in
́
mice (Buckmaster and Haney, 2012) and rats (Mejıas-Aponte
et al., 2002), and are probably not based on sex differences in
pharmacokinetics of pilocarpine (Omori et al., 2004). Rather,
they may be attributed to sex-dependent sexual hormone levels
occurring in the estrus cycle, which have been shown to
influence expression levels of PGP (Schiengold et al., 2006).
Indeed, evidence suggests that the degree of multidrug
transporter–mediated efflux activity at the blood-brain barrier
might be sex-related (Morris et al., 2003). For example, brain
uptake of the PGP substrate verapamil was lower in PGPcompetent female compared with male mice, which may indicate
an increased functional PGP activity in female mice (Dagenais
et al., 2001).
Transport of Pilocarpine by PGP and BCRP. Pilocarpine has an oil-water coefficient (log P) of 0.95–1.15 and a low
molecular mass (208.26 g×mol21). Additionally, low plasma
protein binding (,5%) has been reported (Omori et al., 2004).
Surprisingly, although the pilocarpine model has been used in
epilepsy research for decades, only limited information is
available concerning brain penetration in rodents (Table 1). It
was recently hypothesized that pilocarpine-mediated secondary effects like peripheral inflammation or elevated potassium levels in the brain are required for SE induction (Marchi
et al., 2007, 2009). Furthermore, it was suggested that
pilocarpine bears only modest blood-brain barrier permeability following systemic administration in rats (Table 1) (Marchi
et al., 2007). By contrast, our data suggest good penetration of
pilocarpine across the rat blood-brain barrier, demonstrated
by a brain-plasma ratio of about 1 at 30 minutes postadministration. This is consistent with data from Zgrajka et al.
Pilocarpine and Multidrug Transporters
was present in this species. Consistent with the mouse data,
pharmacological transporter inhibition with tariquidar led to
comparably increased pilocarpine brain uptake in rats,
supporting its classification as a multidrug transporter
substrate across species (Fig. 3D).
In vitro CETA clearly confirmed transport of pilocarpine by
overexpressed murine PGP and BCRP, which could be
blocked by respective specific inhibitors of both transporters
(Fig. 4). Nevertheless, transport of pilocarpine was less
distinct than transport of digoxin, which is frequently used
as a model PGP substrate (Schwab et al., 2003). Unexpectedly, pilocarpine transport was also present in LLC WT cells
without PGP overexpression (Fig. 4, C and D). This finding
cannot be interpreted as pilocarpine transport mediated by
endogenous PGP or BCRP, as both were blocked in WT cells
by respective inhibitors. Rather, persistent transport despite
PGP/BCRP inhibition suggests transport mediated by other
endogenous transporters expressed to a higher degree in LLC
WT cells than in PGP-overexpressing cells.
Effect of Pretreatment with LiCl on Pilocarpine
Brain Uptake. It is well known that pretreatment with LiCl
potentiates the SE-inducing effect of pilocarpine in rats as well
as in mice (Jope et al., 1986; Clifford et al., 1987; Bersudsky
et al., 1994; Chaudhary et al., 1999; Shaldubina et al., 2007).
The underlying mechanism is not completely understood. One
hypothesis suggests that LiCl-mediated proinflammatory processes affect blood-brain barrier integrity (Marchi et al., 2009),
subsequently resulting in increased brain uptake of pilocarpine. This concept is supported by the findings of Marchi et al.
(2007) that pretreatment with LiCl leads to activation of white
blood cells, increased blood interleukin-1b levels, and, importantly, increased blood-brain barrier permeability of fluorescein isothiocyanate–labeled albumin. Therefore, we evaluated
whether LiCl pretreatment would lead to higher pilocarpine
brain levels compared with vehicle-treated animals as a consequence of blood-brain barrier disturbance. However, our results
do not support a conclusion that potentiation of pilocarpine
convulsant activity can be attributed to LiCl-mediated bloodbrain barrier impairment, as a subsequent increase in brain
concentration of pilocarpine does not occur.
Potential Implication for Clinical Use of Pilocarpine.
Awareness of pilocarpine as a transporter substrate is of
relevance for clinical use of this compound, especially relating
to drug interactions. Although we have not directly investigated transport of pilocarpine by multidrug transporters
in the periphery, it is probable that transporters also play
a role in peripheral pharmacokinetics of pilocarpine. For
example, the combination of clinically-used, transporterinhibiting compounds, such as verapamil or cyclosporine,
with pilocarpine in patients with xerostomia may lead to
increased side effects, or even toxic effects, of pilocarpine
owing to increased tissue concentrations.
Conclusion. In conclusion, our findings demonstrate that
pilocarpine has good brain penetration in mice and rats that is
not further increased by pretreatment with LiCl. However,
cerebral uptake is limited by at least two major multidrug
transporters at the blood-brain barrier, leading to increased
convulsant potency of pilocarpine in transporter knockout mice.
Differences in multidrug transporter expression levels at the
blood-brain barrier may therefore explain differences in pilocarpine susceptibility. Besides genetic modification or mutation,
alterations in transporter expression can be disease-associated,
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
(2010), who report equal or even higher brain-plasma ratios
(Table 1). In Naval Medical Research Insitute (NMRI) mice,
Mazzuferi and colleagues (2012) report brain-plasma ratios
comparable to the present study (Table 1).
For comparison of pilocarpine brain uptake in the different
mouse genotypes, we initially administered a subconvulsive
dose to exclude influence of a seizure-induced blood-brain
barrier disruption (Sahin et al., 2003; van Vliet et al., 2007;
Li et al., 2013). Unexpectedly, no increase in brain levels was
observed in PGP-deficient mice. However, increasing the dose
to levels used for SE induction revealed a statistically
significant increase in brain uptake among PGP-deficient
mice (Fig. 3B). It has been suggested that in the absence of
a multidrug transporter, compensatory changes in expression
of other multidrug transporters may occur (Cisternino et al.,
2004; Hoffmann and Löscher, 2007). For example, Cisternino
and colleagues (2004) showed that in mutant Mdr1a-deficient
mice, mRNA expression of Bcrp1 is increased ∼3-fold. We
consider it possible that such a compensatory increase in
transporter expression in Mdr1a/b(2/2) mice may have
prevented increased uptake of the subconvulsive pilocarpine
dose, but that this potential compensatory transport was at
least partially saturated by the convulsive dose. However,
a direct translation of mRNA data to the protein level is not
possible, and conclusions should be drawn with caution. In
combined PGP/BCRP-deficient mice, the increase of pilocarpine brain uptake was higher than in either PGP or BCRP
knockout mice, suggesting an additive function of PGP and
BCRP. Similar observations have been made for other substrates of PGP and BCRP (de Vries et al., 2007; Chen et al.,
2009; Polli et al., 2009). Furthermore, pretreatment of WT mice
with 15 mg×kg21 of the dual PGP and BCRP inhibitor
tariquidar (Bankstahl et al., 2013) resulted in increased
pilocarpine brain uptake to a degree comparable with PGP/
BCRP-deficient mice, underscoring that pilocarpine is indeed
transported by both multidrug transporters. Interestingly, in
Bcrp1(2/2) mice we did not observe increased pilocarpine brain
uptake, although in vitro results clearly show BCRP-mediated
transport. Again, this finding may be explained by a compensatory overexpression of other transporters in Bcrp1(2/2) mice.
In addition, many compounds interacting with multidrug
transporters are described to behave dose dependently as both
substrate and inhibitor (Bankstahl et al., 2013). Further
studies are needed to determine whether this concept may be
applicable for pilocarpine.
Published studies suggest differences between mouse strains
with respect to their susceptibility to pilocarpine-induced
seizures/SE and resulting neuropathology (Bankstahl et al.,
2012; Schauwecker, 2012). FVB/N mice appear to exhibit high
vulnerability compared with other inbred (C57BL/6) or outbred
strains (CD1, NMRI) (Chen et al., 2005). Conversely, C57BL/6
mice are highly resistant to pilocarpine in terms of SE
induction (Borges et al., 2003; Müller et al., 2009; Bankstahl
et al., 2012). In view of the present results, these strain
differences might be explained in part by different transporter
expression levels at the blood-brain barrier, leading to mouse
strain–dependent brain uptake of pilocarpine. Therefore, it
would be of interest to compare brain-plasma ratios of
pilocarpine in commonly used outbred and inbred mouse
strains for epilepsy research in future studies.
As the pilocarpine model is commonly used in rats, we
investigated whether similar efflux transport of pilocarpine
357
358
Römermann et al.
e.g., in mouse models of Alzheimer’s disease (Cirrito et al., 2005;
Hartz et al., 2010). Therefore, the efflux-transporter-substrate
properties of pilocarpine should be considered when applied for
SE induction in rodents with altered multidrug transporter
expression or function.
Acknowledgments
The authors thank Piet Borst and Alfred Schinkel (The Netherlands
Cancer Institute) for providing the cell lines and breeding pairs of
transporter knockout mice used in this study. The skillful technical
assistance of Martina Gramer and Maria Hausknecht with HPLC
analysis is gratefully acknowledged. The authors thank Manfred
Kietzmann for help with pharmacokinetic analysis. The authors are
grateful to James Thakeray for carefully revising the manuscript.
Authorship Contributions
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Address correspondence to: Dr. Marion Bankstahl, Department of
Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine
Hannover, Bünteweg 17, 30559 Hannover, Germany. E-mail: marion.bankstahl@
tiho-hannover.de
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