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Cellular uptake of the atypical antipsychotic clozapine is a
carrier-mediated process
David Dickens1^, Steffen Rädisch1^, George N. Chiduza1, Athina Giannoudis2, Michael J.
Cross1, Hassan Malik3, Elke Schaeffeler4,5, Rowena L. Sison-Young1, Emma L. Wilkinson1,
Christopher E. Goldring1, Matthias Schwab4,6,7, Munir Pirmohamed1, Anne T. Nies4,5
^ =equal contribution
1Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool,
UK 2Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool,
UK3Liverpool Hepatobiliary Unit, University Hospital Aintree, Liverpool, UK4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany 5University Tübingen, Tübingen, Germany 6Department of Clinical Pharmacology, University Hospital Tübingen, Tübingen, Germany7Department of Pharmacy and Biochemistry, University Tübingen, Tübingen, Germany
Authors for Correspondence:
Dr. David Dickens, Department of Molecular and Clinical Pharmacology, Wolfson Centre for
Personalised Medicine, University of Liverpool, Block A: Waterhouse Building, 1-5
Brownlow Street, Liverpool, L69 3GL, United Kingdom.
Email: [email protected]
OR
Dr. Anne Nies, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology,
Auerbachstrasse 112, 70376 Stuttgart, Germany.
Email: [email protected]
1
Graphical Abstract
Neutrophil Hepatocyte
Bile
Brain endothelial cell
Blood
Clozapine
2
Abstract
The weak base antipsychotic clozapine is the most effective medication for treating refractory
schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity
indicating transporter-mediated uptake, which has been insufficiently studied. This is
important because it could have significant impact on clozapine’s efficacy, drug-drug
interaction and safety profile. A major limitation of clozapine’s use is the risk of clozapine-
induced agranulocytosis/ granulocytopenia (CIAG), which is a rare but severe hematological
adverse drug reaction.
We firstly studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3).
Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-
dependent and saturable (Vmax=3299 pmol/million cells/min, Km=35.9 µM). The chemical
inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline and
chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the
in vivo interactions observed in rodents or humans for these compounds. An extensive set of
studies utilising transporter-overexpressing cell lines and siRNA-mediated transporter
knockdown in hCMEC/D3 cells, showed that clozapine was not a substrate of OCT1
(SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1
(SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide
analysis the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3)
were identified as additional candidate transporters. We therefore also investigated clozapine
transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor
an inhibitor of OATP1B1 and OATP1B3.
In summary, we have identified a carrier-mediated process for clozapine uptake into brain,
which may be partly responsible for clozapine’s high unbound accumulation in the brain and
its drug-drug interaction profile. Cellular clozapine uptake is independent from currently
known drug transporters and thus, molecular identification of the clozapine transporter will
help to understand clozapine’s efficacy and safety profile.
3
Keywords
Agranulocytosis, blood-brain barrier, clozapine, drug transport, organic anion transporter,
organic cation transporter, schizophrenia, SLC transporters
4
Introduction
Schizophrenia is a severe psychiatric illness affecting about 24 million people worldwide. It
is characterised by symptoms of altered perception, thought, affect and behaviour.
Schizophrenia can have a devastating effect on a patient’s life, and is associated with
increased mortality with life expectancy reduced by 12-15 years compared to that of the
general population.1 Pharmacological treatment is of fundamental importance for coping with
the symptoms of schizophrenia. However, about one third of all patients do not respond
adequately to standard treatment options and remain refractory.1 In the UK, a patient is
classified as refractory if treatment with at least two antipsychotic drugs, including one non-
clozapine, second-generation antipsychotic drug has failed.2 Clozapine is approved for the
treatment of schizophrenia in otherwise refractory patients and has demonstrated clear
superiority to typical antipsychotics with a response rate of 30% versus 4%. Despite being
effective, clozapine is only licensed as a treatment option for refractory patients because of
the substantially increased risk of agranulocytosis.1
Clozapine’s site of action is the brain; however, the blood-brain barrier (BBB) can restrict
drug penetration into the brain. The BBB is an active cellular barrier that provides both a
physical obstruction via tight junctions that reduce the paracellular route and by the
expression of drug transporters, a dynamic mechanism for affecting drug permeability into
the brain.3,4 The BBB comprises specialised brain endothelial cells that form the walls of
micro-capillaries and can regulate the passage of endogenous substances and xenobiotics into
the brain, which maintains brain homeostasis and neuronal signalling. The barrier is not static
but is a biologically active interface with regulatory functions involving transport, secretory
and enzymatic roles.3
Cellular uptake of clozapine consistent with a carrier-mediated process has been observed in
leukaemia cells5 and in in vivo rodent models. A study in rats following clozapine dosing has
5
shown that the total drug concentration (free and bound) was 24-fold higher in the brain
compared to the serum with a peak at 30 min following intraperitoneal administration.6
Moreover, clozapine, determined both in brain homogenate and by microdialysis, has an
unbound free drug concentration in the brain compared to the blood (Kpuu brain) greater than 2
in rodents, suggesting that carrier-mediated transport of clozapine is responsible for this
above unity Kpuu brain.7 Finally a human study with 11C-clozapine as a PET tracer found that
clozapine and maybe also clozapine metabolites preferentially accumulate in the liver and
brain, compared to other tissues.8 However, the specific type of transporter was not identified
and no transporters were specifically investigated for carriage of clozapine in any of these
studies.
The totality of evidence suggests that transporter-mediated uptake at the BBB is involved in
modulating clozapine entry into the brain, thus generating the high unbound concentration in
the brain, which could be important with respect to the efficacy of clozapine but also in
relation to drug-drug interactions (DDIs). Transporter-mediated clozapine uptake may also be
relevant for the serious adverse reactions of clozapine-induced agranulocytosis/
granulocytopenia (CIAG), which is a severe haematological adverse drug reaction occurring
in about 1% of treated patients and limits the use of clozapine.9 The bioactivation of
clozapine or its major metabolites N-desmethylclozapine (DM-CLZ) and clozapine N-oxide
(CLZ-NO) to reactive nitrenium ions by the neutrophils has been considered a potential
mechanism of clozapine-induced cytotoxicity.10 Although the aetiology of CIAG is unknown,
genetic causes may contribute. A genome-wide association (GWA) study identified certain
human leukocyte antigen alleles associated with the risk of CIAG.11 Recently, in a subsequent
GWA study, an association between the genetic variant rs149104283 and CIAG was
identified.12 This variant is located within a genomic region on chromosome 12 covering the
genes SLCO1B3, SLCO1B7 and SLCO1B1. The liver-specific organic anion transporter
6
polypeptides OATP1B3 and OATP1B1, encoded by SLCO1B3 and SLCO1B1, respectively,
mediate the hepatocellular uptake of organic anions across the sinusoidal hepatocyte
membrane.13 In contrast, OATP1B7, also known as LST-3TM12 and encoded by SLCO1B7,
is located in the endoplasmic reticulum of hepatocytes.14 Therefore, only OATP1B1 and
OATP1B3 may contribute causally to CIAG by mediating the hepatocellular uptake of
clozapine with pharmacokinetic consequences as suggested by Legge et al.12 This is
supported by the fact that neutrophils do not express the SLCO1B3/SLCO1B7/SLCO1B1
genomic cluster.15 In interpreting the data published by Legge et al.12 it is important to
determine whether clozapine is in fact a substrate for OATP1B1 and/or OATP1B3, which
was not investigated by Legge et al.12
The objective of this study was therefore to investigate clozapine transport in an in vitro
model of the BBB and to assess if it is a substrate of hepatic OAT1B1 and OATP1B3
transporters.
7
Experimental Section
Materials
Unless otherwise stated, reagents were obtained from Sigma-Aldrich (Gillingham, UK). [N-
methyl-3H]-clozapine (1 mCi/ml, specific activity 80 Ci/mmol), [3H]-lamotrigine (1 mCi/ml,
specific activity 5 Ci/mmol), [3H]-L-carnitine (1 mCi/ml, specific activity 60 Ci/mmol), [3H]-
adenosine (1 mCi/ml, specific activity 40 Ci/mmol) and [14C]-TEA+ (0.1 mCi/ml, specific
activity 55 mCi/mmol) were obtained from American Radiolabeled Chemicals Inc. (St.
Louis, MO, USA). [3H]Uridine (1 mCi/ml, specific activity 25.5 Ci/mmol) and
[14C]metformin (specific activity 107 mCi/mmol) were from PerkinElmer (Waltham, MA,
USA) and Moravek Inc. (Brea, CA, USA), respectively. [3H]-labelled CLZ-NO (0.5 mCi/ml,
specific activity 80.0 Ci/mml) and [3H]-labelled DM-CLZ (0.5 mCi/ml, specific activity 21
Ci/mmol) were purchased from Novandi Chemistry (Södertälje, Sweden). [Estradiol-6,7-
3H(N)]-17β-glucuronide with specific activity of 52.9 Ci/mmol was purchased from
PerkinElmer (Boston, MA).
Cell culture
The human chronic myeloid leukaemia cell line (KCL22) was previously stably transfected
with an empty plasmid (control) or a plasmid encoding for SLC22A1 (OCT1) or SLC22A4
(OCTN1) by means of electroporation and single cell cloning.16,17 Cells were cultured in
RPMI-1640 medium supplemented with 10 % FBS (v/v) and 1 % penicillin-streptomycin
(v/v) at 37 °C and 5% CO2. hCMEC/D3 is an immortalised cell clone derived from primary
brain endothelial cells and was a kind gift of Professor Pierre-Olivier Couraud (INSERM,
Paris, France) and cultured as previously described.18 HEK293 cells were cultivated in
DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin
(Lonza, Basel, Switzerland) at 37 °C and 5% CO2.
8
Cloning of human SLCO1B1, SLCO1B3, SLC29A2 and SLC29A4 and generation of
stably-transfected cell lines
The full-length cDNA encoding human OATP1B1 was amplified from human liver and
cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA). The following primers
were used for amplification: SLCO1B1_for1: 5'-TTTCAATCATGGACCAAAATCAAC-3’
and SLCO1B1_rev1: 5'-TTAACAATGTGTTTCACTATCTGC-3’. The SLCO1B1 cDNA
was then excised with NotI and cloned into NotI-digested pcDNA3.1(+) expression vector
(ThermoFisher Scientific, Waltham, MA, USA). Finally, SLCO1B1 cDNA was excised with
KpnI/ApaI and cloned into KpnI/ApaI-digested pcDNA5/FRT FlpIn vector (ThermoFisher
Scientific). The coding sequence was identical to the reference sequence NM_006446.4
except for two T>C nucleotide exchanges at position 675 and 1568. Both substitutions were
synonymous so that the amino acid sequence remained identical to the published reference
protein sequence of OATP1B1 (NP_006437.3). Stable transfection of the HEK293 human
embryonic kidney cell line (FlpIn, ThermoFisher Scientific) with SLCO1B1 was carried out
as described previously.19 FlpIn HEK cells stably transfected with the empty pcDNA5/FRT
FlpIn vector served as controls.
In the human SLCO1B3 gene, the two common genetic variants rs4149117 (c.334T>G,
p.Ser112Ala) and rs7311358 (c.699G>A, p.Met233Ile) are linked.20–24 The two variant alleles
form a haplotype designated as haplotype 1 (c.334G, c.699A) having a frequency of 80-88%
in Caucasians, Mexicans and Han Chinese.23 Haplotype 2 with the two alleles c.334T and
c.699G is designated as the reference sequence (NM_019844.3) and has a frequency of 12-
17% in Caucasians, Mexicans and Han Chinese.23 Culturing of HEK293 cells expressing full-
length cDNA encoding OATP1B3 refseq (NP_062818.1) has been described previously.25
For cloning of OATP1B3 haplotype 1, the full-length cDNA encoding human OATP1B3
haplotype 1 in vector pCMV-XL4 was purchased from Origene (Rockville, MD, USA;
9
cat.no. SC113235), excised with NotI/XbaI and cloned into NotI/XbaI-digested
pcDNA3.1(+) expression vector (ThermoFisher Scientific). The presence of the variants
c.334G and c.699A was verified by sequencing. Stable transfection of HEK293 cells (CRL-
1573; American Type Culture Collection, Manassas, VA) with the vector encoding SLCO1B3
haplotype 1 was carried out as described previously.26 Cells were transfected using
Metafectene Pro (Biontex, München, Germany) and grown for 2 – 3 weeks in the presence of
800 μg/ml G418. Cell clones stably expressing OATP1B3 haplotype 1 were selected by
immunofluorescence analysis. HEK293 cells stably transfected with the empty pcDNA3.1(+)
vector served as controls for experiments with both OATP1B3 haplotypes. Cell lines were
incubated with 5 mM butyrate 24 h before use to increase protein levels of the recombinant
transporters.25
The full-length cDNA encoding human ENT2 was amplified from Huh7 cells and cloned into
the pcDNA3.1 V5 His Topo Vector (Thermo Fisher Scientific). The following primers were
used for amplification: SLC29A2_for: 5'-GCGGCCATGGCGCGAGGAGACG-3’ and
SLC29A2_rev: 5'-TCAGAGCAGCGCCTTGAAGAGGAAGGAGAGG-3’. The SLC29A2
cDNA was then excised with KpnI/NotI and cloned into KpnI/NotI-digested pcDNA5/FRT
FlpIn vector (ThermoFisher Scientific). The coding sequence was identical to the reference
sequence NM_001532.2 except for one G>A nucleotide exchange at position 1060, which is
synonymous, so that the amino acid sequence is identical to the published reference protein
sequence of ENT2 (NP_001523.2).
For cloning of PMAT (ENT4), the full-length cDNA of human SLC29A4 (NM_
NM_153247) in vector pCMV-XL6 was purchased from Origene (Rockville; cat.no.
SC101059), excised with NotI and cloned into NotI-digested pcDNA5/FRT FlpIn vector
(ThermoFisher Scientific).
10
Stable transfection of the HEK293 human embryonic kidney cell line (FlpIn, ThermoFisher
Scientific) with SLC29A2 and SLC29A4 was carried out as described above.
Immunofluorescence microscopy of OATP transfectants
Immunofluorescence staining of OATP transfectants was carried out as previously
described.26 Transfected cells were grown on PCA chamber slides (Sarstedt, Nümbrecht,
Germany) for 2 days and fixed with methanol at -20 °C for 10 min as described previously.26
Fixed cells were then incubated with the primary antibodies, followed by incubation with the
Alexa488-conjugated goat anti-rabbit secondary antibody (1:300, ThermoFisher Scientific)
for 1 hour as described.26 Primary antibodies were diluted in PBS as follows: ESL antiserum
against OATP1B127 1:100; HPA004943 antibody (Sigma-Aldrich, Taufkirchen, Germany)
against OATP1B3 1:100. Nuclei were stained with TO-PRO®-3 (1 µM final concentration,
ThermoFisher Scientific). Images were taken with a confocal laser scanning microscope
(TCS SP8, Leica Microsystems, Wetzlar, Germany).
Primary cell extraction and culture
Primary human hepatocytes were derived from patients that underwent surgery for resectable
primary hepatocellular carcinoma or colorectal liver metastases. All patients gave their
written informed consent to the use of their resected tissue for experimental purposes and the
Liverpool Central Research Ethics Committee gave ethical approval. Excess healthy liver
parenchyma was resected as part of the regular procedure and immediately stored in ice-cold
HEPES buffer (10 mM HEPES, 136 mM NaCl, 5 mM KCl, 0.5 % glucose, pH 7.6) on ice.
The extraction process for primary human hepatocytes was initiated and is described in detail
by Heslop et al.28 In brief, perfusion was carried out with collagenase to digest the connective
tissue. The digested tissue was then carefully disassociated and cells poured through a nylon
11
mesh and centrifuged. The resulting cell pellet was resuspended in full William´s medium E
(supplemented with 1 % penicillin-streptomycin (v/v), 2 mM L-glutamine, 1 % of 100x
insulin-transferrin-selenium (ITS) liquid media supplement (v/v), 100 nM dexamethasone).
Initially, 500,000 cells were seeded into each well of ready to use collagen-coated (collagen I
from rat tail) 24-well plates (Life Technologies Ltd., Paisley, UK) and incubated at 37 °C and
5 % CO2 for 3 hours. The medium was removed, cells washed once with WiIlliam´s medium
E, and hepatocytes finally incubated for 24 hours at 37 °C and 5 % CO2 in full William´s
medium E (supplemented with 1 % penicillin-streptomycin (v/v), 2 mM L-glutamine, 1 % of
100x ITS liquid media supplement (v/v), 100 nM dexamethasone).
Primary human cardiac microvascular endothelial cells (HCMECs, lot-3011401 (#1), lot-
9090701.2 (#2)) and primary human brain microvascular endothelial cells (#1, HBMECs, lot-
1111603.7) were purchased from PromoCell (Heidelberg, Germany), cultured and RNA was
extracted as previously described.29 Total RNA from primary human brain endothelial cells
(#2) was acquired from ScienCell (San Diego, California).
Drug uptake assay
Uptake measurements into hCMEC/D3 cells, and KCL22 transfected cells were conducted at
37 °C as previously outlined.30 Transport buffer consisted of Hanks balanced salt solution
(HBSS), 25 mM HEPES at pH 7.4 with a tracer concentration of [3H]-labelled drug (0.05
µCi/ml or 0.1 µCi/ml), non-labelled drug to give a final concentration ranging from 0.1 – 300
µM, and 0.1 % BSA (w/v). The vehicle concentration did not exceed 0.2% per reaction
(DMSO). Uptake assays for adherent cells were performed in 6 or 12 well plates with cells
equilibrated in transport buffer. The reaction was initiated by the addition of transport buffer
containing radiolabelled compound for the indicated time points. To stop uptake, ice cold
HBSS was added and cells washed three times followed by lysis in 5% SDS solution. The
12
lysed cells were added to scintillation cocktail and radioactivity determined with a
scintillation counter (1500 Tri Carb LS Counter; Packard, Meriden, CT 06450, USA). Results
were normalised to pmoles per million cells for drug uptake. Non-adherent cells (KCL22)
were attached to 6 well plates by poly-L-lysine coating and assayed as above for drug uptake.
Uptake measurements into HEK transfectants were carried out as described previously at 37
°C.26 Uptake of the prototypic substrate estradiol 17β-glucuronide was measured as described
previously at a concentration of 5 µM including a tracer amount of 20 nM [estradiol-6,7-
3H(N)]-17β-glucuronide. For inhibition studies, uptake of estradiol 17β-glucuronide was
carried out in the presence of different clozapine concentrations or in the presence of 50 µM
rifampicin (positive control inhibitor)31 and terminated after 5 min. Uptake of clozapine was
measured at various concentrations. The uptake buffer with clozapine included a tracer
amount of 12.5 nM [N-methyl-3H] clozapine. Considering therapeutically achievable Cmax
concentrations of 4 µM clozapine32 and a plasma unbound fraction of 0.05533 the
concentration of 0.2 µM thereby reflects the systemic in vivo situation. An unbound
concentration of 0.95 µM can be potentially reached at the inlet to the liver (calculation see
Supporting Information). Uptake of DM-CLZ and CLZ-NO was measured at a concentration
of 2 µM including a tracer amount of 6.25 and 10 nM, respectively, of radiolabelled
compound.
Uptake of 5 µM metformin into PMAT-expressing cells was carried out as described
previously.34 Uptake of 1 µM uridine into ENT2-expressing cells was carried out in the same
way.
Uptake was stopped after different time points and cells were lysed with 0.2% SDS as
described previously.26 Intracellular radioactivity was determined by liquid scintillation
counting (Hidex 300SL TDCR liquid scintillation counter, Turku, Finland) and protein
content of lysed cells using the bicinchoninic acid assay.26
13
Confirmation of functional OATP1B expression in transfected cell lines
All three OATP1B- transfected cell lines expressed OATP1B1 or OATP1B3 in the plasma
membrane as evidenced by positive immunofluorescence staining (Supporting Figure 1A-C).
The OATPs were functionally active and showed significant OATP1B1- and OATP1B3-
mediated uptake of the positive control substrate estradiol 17β-glucuronide comparable to
previously published data (Supporting Figure 1D-F).25,27
Calculation of clozapine charge at pH 7.4
The “isoelectric point” plugin of MarvinSketch 15.9.14 was used to calculate the pH-
dependent net charge distribution. At the physiological pH 7.4, clozapine was calculated to
carry a net charge of +1.7, consistent with the data provided by Drugbank Version 5.0
(https://www.drugbank.ca/).35
Chemical similarity
Chemical similarity to clozapine was quantified using the Tanimoto coefficient as calculated
using Molecular ACCess System (MACCS) structural fingerprints as described.36 The
Tanimoto coefficient varies from 0 to 1, with 1 being the highest degree of similarity.
RNA extraction and real time PCR
RNA was extracted with Tri reagent according to the manufactures recommendation as
previously described.18 Following RNA extraction, reverse transcription utilising TaqMan
reverse transcription reagents (ThermoFisher, Paisley, UK) was performed. TaqMan gene
expression assays were supplied by ThermoFisher Scientific; FAM; SLC22A1
(Hs00427554_m1), SLC22A2 (Hs00533907_m1), SLC22A3 (Hs01009568_m1), SLC22A4
(Hs00268200_ml), SLC22A5 (Hs00929869_m1), SLC29A1 (Hs01085704_g1), SLC29A2
14
(Hs00155426_m1), SLC29A3 (Hs00217911_m1), SLC29A4 (Hs00928283_m1), SLCO1B1
(Hs00272374_m1), SLCO1B3 (Hs00251986_m1), SLCO1B7 (Hs00991170_m1),
β–actin/ACTB (Hs99999903_m1), GAPDH (Hs03929097_g1) and GAPDH VIC (4310884E).
The expression data were normalised to GAPDH expression using the comparative Ct
method to determine relative expression. For the primary cells of different origins, the
normalized ΔCt was calculated as Ctgene of interest – Ctcontrol where the control Ct was the
geometric mean of the Cts of β – actin and GAPDH for that given sample.37
siRNA transfections
siRNA transfections were carried out on the hCMEC/D3 cells with lipofectamine RNAi max
(ThermoFisher) as previously described18 or with dharmafect (GE Dharmacon, Lafayette,
CO, USA) following manufacturer’s instructions. siRNA oligos used were human
siGENOME SMARTpool siRNAs (GE Dharmacon): SLC22A5 (M-007456-02-0005),
SLC29A1 (M-003709-01-0005), and non-targeting siRNA pool #2 (D-001206‐14‐05).
Statistical tests
A one-way ANOVA followed by a Dunnett’s post-hoc test or a one sample t-test was carried
out for statistical analysis for experiments with multiple comparisons as appropriate. Single
comparisons were analysed by an independent two-tailed t-test. Significant results are
indicated with * for p<0.05, ** for p<0.01, and *** for p<0.001. Analysis was performed
with SPSS Statistics version 20 (IBM United Kingdom Ltd., Hampshire, UK) or Prism 5 or 6
(GraphPad Software Inc., La Jolla, CA, USA). Kinetic parameters were calculated with Prism
6 by fitting a non-linear Michaelis-Menten regression curve to the data. The inhibitor
concentration that achieved half-maximum inhibition (IC50) of substrate accumulation was
15
determined by fitting a non-linear regression curve with variable slope to the data using Prism
5 or 6. All data are presented as means ± SD.
16
Results
Carrier-mediated uptake of clozapine into brain endothelial cells
To investigate the potential of carrier-mediated clozapine uptake into hCMEC/D3 cells, a
time-course assay was carried out. A clozapine concentration as low as 1 µM (range from 0.3
– 2.4 µM) has been reported as the average peak plasma concentration at steady-state
following twice-daily doses of 100 mg.38 This concentration was chosen for the majority of
the uptake assays presented in this work. Clozapine uptake into hCMEC/D3 cells exhibited
typical transporter-mediated uptake kinetics with a linear phase of uptake (about two
minutes) that was saturable (Figure 1A) and with significantly decreased uptake at 4 °C
(Figure 1B).
Chemical inhibitor screening was applied as a first step to identify the transporter class
potentially responsible for clozapine uptake into the hCMEC/D3 cells. The selected
compounds are listed in Supporting Table 1 with corresponding typical and important drug
transporter classes affected. The accumulation of 1 µM clozapine was determined separately
in the presence of all selected chemical inhibitors (Figure 1C) and at a 1 min time point for
verapamil (Figure 1D). Prazosin and verapamil were the most potent inhibitors, resulting in
reduced clozapine accumulation of 94 % and 83 %, respectively. The presence of lamotrigine
resulted in reduced clozapine accumulation by 26 % compared to the vehicle control. As up
to 94% of clozapine uptake could be chemically inhibited, this suggests the major route of
uptake of this drug into the hCMEC/D3 cells is carrier-mediated and not by passive diffusion.
To further investigate this carrier-mediated process, the kinetics were determined at 37 °C in
the linear phase of uptake at a fixed time-point with increasing clozapine concentrations
ranging from 0.1 – 300 µM (Figure 1E) as saturation is hallmark of a carrier-mediated
transport process. Clozapine uptake followed Michaelis-Menten kinetics and curve fitting
yielded a Vmax of 3299 pmol/million cells/min, and Km of 35.9 µM (Figure 1E). Considering
17
that average clozapine peak plasma concentrations are around 1 µM, a Km of 35.9 µM
indicates a high-capacity clozapine uptake transporter at therapeutically relevant
concentrations. Taken together, the observed chemical inhibitor sensitivity and saturable
kinetics of clozapine uptake are consistent with the involvement of a transporter in the influx
of the compound into brain endothelial cells.
To determine if the uptake process of clozapine into the hCMEC/D3 cells was through a
process similar to that described for clozapine uptake into promyelocytic leukaemia cells,5
indatraline was utilised (Figure 1F). In the presence of 10 µM indatraline, a significant
decrease of clozapine uptake was observed (35%) consistent with that observed by Henning
et al. who observed 62% inhibition in the presence of indatraline.5
Interaction of clozapine with a herbal medicine and psychotropic drugs in brain
endothelial cells
An interaction between the anthraquinone rhein and clozapine has recently been reported to
occur by an unknown mechanism in the rat in vivo that reduces the Kpuu of clozapine.39 We
therefore investigated whether rhein could affect the uptake of clozapine into the hCMEC/D3
cells. We used concentrations of 20 µM and 100 µM rhein, which are achieved in humans
after single oral doses of 100 and 300 mg rhein, respectively.40,41 We found that rhein could
significantly reduce the clozapine uptake into the hCMEC/D3 cells (Figure 1G) implicating
uptake inhibition as a possible cause of the drug-herbal interaction observed in vivo.
To test if psychotropic drugs that have either a possible DDI with clozapine (quetiapine,
carbamazepine)42,43 or high unbound drug concentration in the brain compared to plasma
(chlorpromazine, olanzapine)7 interact with the clozapine transporter, we tested these
compounds at two different concentrations and compared this to the untreated clozapine
uptake in the hCMEC/D3 cells (Figure 1H). In the presence of quetiapine, chlorpromazine
18
and olanzapine, we observed a significant decrease in clozapine uptake suggesting that these
compounds can interact with the clozapine transporter.
The chemical compounds that affect the uptake of clozapine were ranked by their chemical
similarity to clozapine, providing a basis for predicting which of these compounds are likely
to be a substrate as well as an inhibitor due to their chemical structure being similar to
clozapine’s (Table 1).
Interaction at transport level between clozapine and lamotrigine
Clozapine exhibits typical SLC22A1 (OCT1) inhibitor characteristics namely, a positive net
charge at physiological pH and high lipophilicity. In addition, clozapine has been reported to
inhibit the OCT1-mediated uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium
(ASP+) by 47.5 %.44 Due to the effect of 100 µM lamotrigine on the uptake of clozapine in
the hCMEC/D3 cells, and the combined clinical use of clozapine and lamotrigine in man with
the potential for DDI,43 we investigated this in vitro observation in more detail. To test the
effect of clozapine on lamotrigine transport, KCL22 cells, either transfected with empty
vector or SLC22A1, were incubated with 5 µM lamotrigine in the presence of increasing
clozapine concentrations ranging from 0.1 – 100 µM (Figure 2A). The same experiment was
performed with 2.73 µM TEA+ as an OCT1 model substrate (Figure 2B). A dose-response
was observed for increasing clozapine concentrations with both OCT1 substrates. Curve-
fitting yielded IC50 values for clozapine of 1.8 µM (lamotrigine as a substrate) and 5.7 µM
(TEA+ as a substrate), respectively, for the inhibition of OCT1-mediated transport. The
analysis was extended to the hCMEC/D3 cell line, again with 5 µM lamotrigine as a substrate
and increasing clozapine concentrations ranging from 0.1 – 100 µM (Figure 2C). As before, a
dose-response was observed and curve-fitting resulted in a similar IC50 of 2.0 µM. These in
vitro results indicate that clozapine can potently inhibit OCT1-mediated lamotrigine transport
19
at therapeutically relevant concentrations. To see if the reverse was applicable, we utilised the
hCMEC/D3 cell line with 1 µM clozapine in the presence of increasing lamotrigine
concentrations ranging from 1 – 1000 µM after 30 min (Figure 2D). An inhibitory effect was
observed with lamotrigine concentrations ≥ 100 µM, but not within the therapeutic range.
The manufacturer's product monograph specifies that lamotrigine peak plasma levels range
between 2 – 18 µM following single lamotrigine doses of 50 – 400 mg. A peak plasma
concentration up to 47 µM has been reported in a study that assessed individual therapeutic
thresholds in epilepsy patients with doses up to 1,200 mg.45 This value represents an extreme
and is indicated in Figure 2D by a dotted line as maximum relevant lamotrigine
concentration.
Assessment of clozapine uptake by organic cation transporters
Due to clozapine’s ability to be an OCT1 inhibitor and the results from the chemical inhibitor
screening, we investigated clozapine as a potential substrate for OCT1 in a time course assay
utilising SLC22A1-transfected and empty vector-transfected KCL22 cell lines. This is a
validated and widely recognized method to study whether a drug is a substrate for an uptake
transporter.46 According to these recommendations a compound should display at least a
twofold higher accumulation in transporter-expressing than in control cells.46 A time-course
accumulation assay over 30 min did not show any significant difference between empty
vector-transfected and SLC22A1-transfected KCL22 cell lines (Figure 3A). A positive control
utilising 5 µM lamotrigine as an OCT1 substrate (30 min time point) confirmed the
functionality of the assay (Figure 3B).17 These results demonstrate that clozapine, at clinically
relevant concentrations, is not a substrate for OCT1 in vitro.
Other well-characterised OCT transporter candidates from within the SLC22A family, which
are also expressed in the hCMEC/D3 cells (Supporting Figure 2), are SLC22A4 (OCTN1)
20
and SLC22A5 (OCTN2). A KCL22 cell line transfected with empty vector or SLC22A4 was
available and clozapine investigated as a potential substrate for this particular transporter
after 1, 5 and 30 min, respectively. The model substrate TEA+ was utilised as a positive
control.18 No differences in clozapine uptake could be observed between control and
SLC22A4-transfected cells (Figure 3C). TEA+ accumulation was significantly increased in
SLC22A4-transfected cells, showing functionality of the assay (Figure 3D). These results
suggest that clozapine is not an OCTN1 substrate.
SLC22A2 (OCT2) mRNA levels are not detectable in the hCMEC/D3 cell line (Supporting
Figure 2), but given that we still witnessed transport into this cell line, this would suggest that
OCT2 is not relevant. There are detectable SLC22A3 mRNA levels encoding for the
extraneuronal monoamine transporter OCT3.17,47 Monoamines are a class of molecules that
include important neurotransmitters such as dopamine, serotonin, and norepinephrine. Two
monoamine transporter systems are distinguished in the literature and referred to as uptake1
and uptake2. Uptake1 consists of high-affinity neuronal monoamine transporters, mainly
SLC6A2-4 (NET, DAT, and SERT), while uptake2 consists of high-capacity extraneuronal
monoamine transporters, particularly SLC22A3 (OCT3) and SLC29A4 (ENT4 or PMAT).48
At this stage, OCT3 was considered the most promising transporter candidate for clozapine
and the observed high-capacity uptake. Additional screening was thus applied with several
known OCT3 inhibitors. The selected inhibitors are listed in Supporting Table 1 and
verapamil was included as a positive control. While verapamil again potently inhibited
clozapine uptake, none of the other inhibitors had an effect after 30 min (Figure 3E)
suggesting that OCT3 is not involved in clozapine transport.
The remaining, well-characterised transporter candidate from within the SLC22A family is
SLC22A5 (OCTN2). We have previously used siRNA-mediated knockdown of transporters
of interest to identify the specific transporter for gabapentin uptake into brain endothelial
21
cells.18 Here we used siRNA targeting the OCTN2 transporter and achieved sufficient
knockdown at the mRNA level (Figure 3F) and reduced uptake of a model substrate, L-
carnitine (Figure 3G). However the uptake of clozapine in the SLC22A5 siRNA-mediated
knockdown at both the 1 and 30 min time points were not altered compared to the scrambled
control siRNA transfected cells (Figure 3H) suggesting that OCTN2 is not the clozapine
transporter.
Involvement of SLC29A subfamily members in clozapine uptake
Given that we had not identified a transporter for clozapine in the SLC22A family, we
extended our screening approach to the monoamine transporters SLC29A1-4 (ENT1-4),
which are also expressed in the hCMEC/D3 cells (Supporting Figure 2). Because SLC29A3
(ENT3) is located intracellularly in lysosomes and mitochondria and therefore not involved in
cellular uptake,49 we did not further investigate its role in clozapine transport. In contrast,
SLC29A4 (ENT4/PMAT) is of particular interest because, together with OCT3, it is
recognised as a high-capacity monoamine neurotransmitter transporter with expression being
detected in various brain regions.50 Interestingly, PMAT has also been demonstrated to
exhibit similar, but distinct, substrate and inhibitor characteristics as organic cation
transporters from the SLC22 family, particularly OCT3.48,50,51 Moreover, clozapine has been
found to act as a potent inhibitor of PMAT-mediated MPP+ transport.52 Corticosterone up to a
concentration of 100 µM, in contrast, had no inhibitory effect on the uptake of serotonin into
stably SLC29A4-transfected MDCK cells,50 fitting with the data obtained for clozapine in
hCMEC/D3 cells (Figure 3E).
SLC29A1 siRNA treatment of the hCMEC/D3 cells resulted in a significantly reduced
relative SLC29A1 gene expression of 26.0 ± 3.7% (P<0.001) compared to negative control
siRNA. Successful knockdown was also functionally observed by a reduced accumulation of
22
adenosine, a typical substrate of ENT149 (Fig. 4A). However, clozapine accumulation was not
different between SLC29A1- and negative control siRNA-treated cells for both 1 and 30 min
time points (Fig. 4B). For assessing involvement of ENT2 and PMAT in clozapine
accumulation, we generated HEK transfected cells expressing either transporter.
Accumulation of typical substrates of ENT2 (uridine)49 and of PMAT (metformin)53 was
significantly higher in the transporter-expressing cells than in vector-transfected cells
(Fig.4C, E). However, clozapine did not accumulate twofold higher in transporter-expressing
than in vector-transfected control cells (Fig. 4D, F) indicating that ENT2 and PMAT do not
mediate cellular uptake of clozapine.
SLCO1B gene expression in endothelial cells and primary hepatocytes
The recent publication by Legge et al. suggested a potential involvement of OATP1B
transporters in cellular uptake of clozapine. The results from the chemical inhibitor screen in
the brain endothelial cells is not consistent with this as methotrexate and MK571 can inhibit
both OATP1B1 and OATP1B3 but had no effect on clozapine uptake (Figure 1C). OAT1B7
is localized in the endoplasmic reticulum of hepatocytes and therefore not involved in cellular
uptake of compounds.14 We also investigated the expression of these transporters in the
hCMEC/D3 cells, primary endothelial cells of different organ origins and primary
hepatocytes as SLCO1B1 and SLCO1B3 are known to be highly expressed in hepatocytes.
The gene expression results show that the three transporters are expressed in two differently
derived primary hepatocytes preparations as expected26 but in none of the primary or
immortalised endothelial cell types (Figure 5A-C). We can conclude from this negative
expression data that the three OATP1B transporters are not involved in the uptake of
clozapine into the brain endothelial cells.
23
Assessment of clozapine and its metabolites as transported substrates and inhibitors of
OATP1B1 and OATP1B3
In order to determine whether OATP1B1 and OATP1B3 are involved in hepatocellular
uptake of clozapine or its metabolites, we analysed clozapine uptake into OATP1B-
expressing versus vector-transfected control cells. We used concentrations of 0.2 µM, 2 µM
and 20 µM clozapine and of 2 µM DM-CLZ or CLZ-NO and incubation times of 2, 10 and
30 min (Supporting Figure 3). None of these conditions yielded a twofold higher
accumulation of clozapine into the OATP1B transfectants than into the control cells
indicating that neither clozapine nor the clozapine metabolites are transported by OATP1B1
or OATP1B3.
Next, we investigated clozapine and its major metabolites as potential inhibitors of
OATP1B1 or OATP1B3 (Figure 6). There was no inhibition of OATP1B-mediated estradiol
17β-glucuronide uptake by 0.2 µM, 2 µM or 20 µM clozapine, DM-CLZ or CLZ-NO. As
expected, OATP1B-mediated estradiol 17β-glucuronide uptake was almost completely
abolished using the positive control inhibitor rifampicin confirming functionality of the
assay.31
24
Discussion
Clozapine is a lipophilic drug with an experimentally derived logD (pH 7.4) of 2.754 that is
within the range of lipophilicity described as optimal for “passive diffusion” across the
BBB.55 Additionally the “Overton rule” states that lipophilicity is the driving force for uptake
through the plasma membrane.56 Therefore, characterisation of the uptake process of
clozapine has not received much attention. However, there are exceptions to the dogma of the
“Overton rule”, for example YM155 is a highly lipophilic compound (predicted LogD pH 7.4
of 4.89) but its uptake into cells is governed by the SLC35F2 transporter57 and the OCT1 and
OCT2 transporters.58 Furthermore it has been suggested that diffusion of all drugs across the
lipid bilayer is negligible irrespective of their lipophilicity and that passage across the
membrane is mediated by transporters.59
In this study we have therefore determined that clozapine uptake into brain endothelial cells
is a carrier-mediated process consistent with the criteria defined by Sugano et al.60: we
demonstrated time-dependent saturation, saturation with increasing concentrations of
substrate, and inhibition with chemical inhibitors that share structural similarity to the
substrate. Furthermore, inhibition by up to 95% shows the major involvement of a carrier-
mediated process rather than “passive diffusion”. Our results are consistent with an uptake
process that was previously described for clozapine in a leukaemia cell line,5 with indatraline
inhibiting clozapine uptake in both cell types.
Summerfield et al. determined for a number of tricyclic compounds, including clozapine,
chlorpromazine and olanzapine, a Kpuu brain greater than unity, suggestive of a transporter-
mediated uptake process at the BBB.7 Our findings correlate with this and we also show that
both chlorpromazine and olanzapine inhibited the carrier-mediated uptake of clozapine. It is
possible that these 3 compounds share the same uptake transporter because of their chemical
similarity. A number of recent studies have investigated the Kpuu brain for clozapine in rodents
and obtained variable numbers.7,61,62 These differences could be due to the lag observed in the
25
brain concentration relative to the plasma concentration resulting in a Kpuu brain of 0.04 at 30
min to 1.71 at 240 min.63 However, irrespective of the precise ratio, a recent study found an
increase in the blood concentration and decrease in the unbound brain concentration of
clozapine with a corresponding decreased pharmacodynamic effect following treatment with
rhein in rodents.39 Our finding that rhein can at least in part inhibit the uptake of clozapine
provides a possible mechanism for this in vivo interaction.
Transporters can be a site for DDI if two or more compounds share or inhibit the same
transporter. Clozapine increases the serum concentration to dose ratio for quetiapine by 82%
in patients,42 but the mechanism is unknown. Our in vitro observation that quetiapine can
inhibit the clozapine transporter leads us to tentatively suggest that inhibition of transport
could be involved in the change of drug concentrations detected in patients. Clozapine can
potently inhibit OCT1-mediated lamotrigine transport at therapeutically relevant
concentrations. These in vitro observations might have consequences for the safety and/or
effectiveness of lamotrigine when co-administered with clozapine. It should be noted that no
effect was observed on clozapine plasma levels following addition of lamotrigine in a small
clinical trial.64 However a recent review that summarises case reports for DDI relating to
clozapine, proposes that lamotrigine is likely to increase clozapine plasma concentrations.43
The results of the chemical inhibitor screen reported here, lead to us to hypothesise that an
organic cation transporter was responsible for clozapine transport. However, we followed this
up with extensive molecular studies, and not only ruled out OCT1 as the clozapine
transporter in agreement with a previous report,65 but also excluded other structurally and
functionally similar transporters including OCTN1, OCTN2, ENT1, ENT2, and PMAT
transporters. These are important negative results as these transporters are relevant for
transport of other drugs and for endogenous substrate uptake.66,67 Up to 10% of the genome
encodes for transporters, despite this they are one of the least characterised group of proteins,
so it remains challenging to identify the specific transporter for a given compound,
26
particularly given that limited information is available in the literature on the substrate
specificities of transporters.68 Additionally, there is bias in the knowledge base which has
largely focused on the well-known and early identified transporter classes.68,69 Furthermore, at
least one of the compounds that reduced clozapine uptake, verapamil, has recently been
linked to a transport process by an unknown uptake transporter at both the BBB and blood–
retinal barrier.70 In addition to transporter-mediated uptake, clozapine might also accumulate
via receptor internalisation. Clozapine has been shown to induce the internalization of
serotonin receptor 2A (5-HT2A) after short-term stimulation of 15-30 minutes.71,72 Together
with the observation that 5-HT2A is expressed in hCMEC/D3 cells73 clozapine-induced
5-HT2A internalization might therefore be an additional route of clozapine accumulation in
hCMEC/D3 cells.
Variability in the plasma concentrations of clozapine has been observed between different
patients following the same oral dose.32 A recent study found an association between CYP1A2
genetic variants and clinical (heavy smoking) parameters with plasma concentrations of
clozapine but these two parameters only accounted for 16% of the variability observed in
those patients.74 Once a specific clozapine transporter is identified it could become an
additional variable to model the variation observed in plasma concentrations. This might then
provide an improved model that can predict the variability in the plasma concentrations
between different patients allowing more precise definition of doses in individual patients.
The reason why clozapine is only licensed as a reserve treatment option is the substantially
increased risk of CIAG. Given our findings, the role of transporter-mediated cellular uptake
of clozapine into neutrophils or their precursors would be worth studying. Indeed, this is
consistent with the fact that: (a) there is transporter-mediated clozapine uptake into the
promyelocytic leukemia cell line HL-605 and (b) eight times higher clozapine concentrations
were detected in leukocytes from a patient with CIAG compared with ten patients without
agranulocytosis.75 A recent study has found that variants in the SLCO1B1, SLCO1B3 and
27
SLCO1B7 genes are correlated with neutropenia.12 However, these genes are neither
expressed in brain endothelial cells nor in neutrophils15 suggesting that they are not
responsible for clozapine uptake into brain or neutrophils.
Nevertheless, OATP1B1 and OATP1B3 might be involved in hepatocellular uptake of
clozapine as well as its major metabolites with pharmacokinetic consequences as suggested.12
However, the lack of clozapine or clozapine metabolite transport itself and the lack of
inhibition of OATP1B-mediated estradiol 17β-glucuronide uptake by clozapine and the
metabolites indicate that intracellular hepatic clozapine levels do not depend on hepatic
uptake by OATP1B1 or OATP1B3. This observation is supported by the fact that clozapine is
an organic cation whereas all currently identified substrates of OATP1B1 and OATP1B3 are
amphipathic organic anions or uncharged molecules.76 The direct involvement of OATP1B1
and OATP1B3 in the absorption or disposition of clozapine with consequences on CIAG is
highly debatable given that clozapine is not transported by OATP1B1 or OATP1B3 in vitro.
It is a well-recognized major limitation of GWA studies that an identified genetic variant
often does not correlate with the trait of interest but is rather a surrogate for the true causal
variant.77 We therefore propose, as one possible explanation for the significant association of
rs149104283 with CIAG reported by Legge et al.,12 that there is a linkage of rs149104283 to
other genes in the human genome relevant for CIAG, which needs further elucidation.
In conclusion, a potent clozapine uptake process has been identified and characterised in the
hCMEC/D3 cell line, an in vitro model of the BBB. This process may critically affect
clozapine treatment efficacy and is exemplified by the fact that in humans, clozapine and
maybe partly also its metabolites accumulate in the brain.8 Theoretically, genetic variants or
differences in expression of this unknown clozapine transporter might predict the
effectiveness (e.g. uptake into the brain) and/or adverse effects (e.g. uptake into leukocytes,
adipocytes, or brain) of clozapine. Furthermore, our findings also provide some mechanistic
insights into DDIs associated with clozapine. Taken together, our findings indicate that
28
carrier-mediated uptake of clozapine is important, and identification of the clozapine
transporter will shed further light on variability in efficacy and safety associated with
clozapine use, and the potential for DDIs.
29
Acknowledgments
This work was funded by a Tenure Track Fellowship to D.D., an EU Marie-Curie Early
Stage Researcher award to S.R. within the ‘Fighting Drug Failure’ network (grant agreement
number 238132) and the Robert-Bosch Foundation, Stuttgart, Germany to E.S., M.S., A.T.N..
M.P. is a NIHR Senior Investigator, and thanks the MRC Centre for Drug Safety Science for
providing infrastructure support. D.D. thanks Douglas Kell for a useful discussion. We thank
Silvia Hübner for her expert technical assistance.
Supporting information
Supporting Method:
Calculation of the pharmacological relevant clozapine concentration at the inlet to the liver
Supporting Table 1:
Selected drug transporter inhibitors for chemical screening
Supporting Figure 1:
Characterization of OATP1B-transfected cells
Supporting Figure 2:
Gene expression of SLC22A1-5 and SLC29A1‐4 in the hCMEC/D3 cell line
Supporting Figure 3:
Assessment of clozapine and clozapine metabolites as transported substrates of OATP1B1
and OATP1B3
30
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36
Table 1. Summary of compounds that reduced the uptake of clozapine
The chemical structure of the compounds that reduce clozapine uptake into brain endothelial
cells are ranked from high to low in terms of chemical similarity to clozapine. Chemical
similarity was derived from Tanimoto similarity using MACCS structural fingerprints. This
provides a numerical measure of molecular similarity of 0 to 1 with the higher the number the
more chemical similarity the compound has to clozapine.
Compound Chemical structure Chemical similarity to clozapine
Clozapine
1
Olanzapine
0.83
Chlorpromazine
0.58
Quetiapine
0.54
Prazosin
0.48
Lamotrigine0.39
Indatraline
0.37
37
Compound Chemical structure Chemical similarity to clozapine
Verapamil
0.33
Rhein0.11
38
Figure legends
Figure 1. Inhibitable and saturable uptake of clozapine into human brain endothelial cells.
(A) Time-course uptake assay of 1 µM [3H]-clozapine in hCMEC/D3 cells. (B) Accumulation
of [3H]-clozapine in hCMEC/D3 cells after 30 min at 37°C and 4°C, respectively. (C) Uptake
of [3H]-clozapine in the hCMEC/D3 cell line after 30 min in the presence or absence of
various drug transporter inhibitors. (D) Uptake of [3H]-clozapine in the hCMEC/D3 cell line
after 1 min in the presence or absence of 100 µM verapamil. (E) Clozapine uptake into
hCMEC/D3 cells was determined after 1 min with clozapine concentrations ranging from 0.1
- 300 µM. A Michaelis-Menten regression curve was fitted to the data and kinetic parameters
Vmax and Km were calculated. (F) Uptake of [3H]-clozapine in the hCMEC/D3 cell line in the
presence or absence of 10 µM indatraline. (G) Uptake of clozapine into hCMEC/D3 cells
after 30 min in the absence or presence of rhein. (H) Uptake of clozapine into the hCMEC/D3
cell line after 30 min in the absence or presence of several psychotropic compounds. Data are
expressed as means ± standard deviation (n = 3 independent experiments in triplicate). *
p<0.05, ** p<0.01, *** p<0.001 vs. control.
Figure 2. Clozapine as an inhibitor of OCT1-mediated transport. Uptake of 5 µM [3H]-
lamotrigine (A) and 2.73 µM [14C]-TEA+ (B) into KCL22 cells transfected with empty vector
or OCT1, determined after 30 min in the presence of increasing clozapine concentrations (0.1
– 100 µM). OCT1-mediated uptake was calculated by subtracting the data derived from
empty vector-transfected from OCT1-transfected KCL22 cells. (C) Accumulation of
lamotrigine in the hCMEC/D3 cell line after 30 min in the presence of increasing clozapine
concentrations (0.1 – 100 µM). (D) Accumulation of clozapine in the hCMEC/D3 cell line
after 30 min in the presence of increasing lamotrigine concentrations (1 – 1000 µM). The
therapeutically relevant maximum lamotrigine concentration of 47 µM is indicated by a
39
dotted line. Data are expressed as means ± standard deviation (n = 3 independent experiments
in triplicate).
Figure 3. Assessment of clozapine uptake by organic cation transporters (OCT1, OCT3,
OCTN1, OCTN2). (A) Time-course of clozapine uptake into KCL22 cells transfected with
empty vector or OCT1. (B) Positive control with uptake of lamotrigine into KCL22 cells
transfected with empty vector or OCT1 after 30 min at 37 °C. (C) Clozapine and (D) TEA+
uptake into empty vector-transfected and OCTN1-transfected KCL22 cells was determined
after 1, 5 and 30 min, respectively. (E) Second chemical inhibitor screening to investigate
OCT3 as a potential clozapine transporter. Accumulation of clozapine in the hCMEC/D3 cell
line in the presence of different drug transporter inhibitors after 30 min. (F) The knockdown
efficiency of the OCTN2 siRNA is plotted as the remaining relative gene expression in % as
compared to negative control siRNA transfected cells. Accumulation of L-carnitine (G) at 3
min or clozapine after 1 or 30 min (H) in hCMEC/D3 cells utilising a siRNA knockdown
approach. Cells were transfected either with negative control siRNAs or OCTN2 targeting
siRNAs. Data are expressed as means ± standard deviation (n = 3 independent experiments in
triplicate). * p<0.05, ** p<0.01, *** p<0.001 vs. control.
Figure 4. Assessment of the involvement of SLC29A subfamily members in clozapine
uptake. Accumulation of adenosine at 3 min (A) or clozapine at 1 or 30 min (B) in
hCMEC/D3 cells after utilising a siRNA screening approach for SLC29A1. Cells were
transfected either with negative control siRNAs or SLC29A1 targeting siRNA. Accumulation
of uridine at 2 min (C) or clozapine at 1 or 30 min (D) in ENT2-overexpressing HEK cells.
Accumulation of metformin at 5 min (E) or clozapine at 1 or 30 min (F) in ENT2-
40
overexpressing HEK cells. Data are expressed as means ± standard deviation (n= 3
independent experiments). * p<0.05, ** p<0.01, *** p<0.001 vs. control.
Figure 5. Gene expression profile of SLCO1B1, SLCO1B3 and SLCO1B7 transporters in
primary hepatocytes and a number of endothelial cells of different origins. The relative
mRNA expression of human SLCO1B1 (A), SLCO1B3 (B) and SLCO1B7 (C) was
determined. Primary cells from different patient samples were obtained from different tissue
or cell type and includes hepatocytes, cardiac endothelial cells, brain endothelial cells and
hCMEC/D3 cells.
Figure 6. Assessment of clozapine and clozapine metabolites as inhibitors of OATP1B1 and
OATP1B3. OATP1B-mediated estradiol 17β-glucuronide transport was measured in the
presence of different concentrations of (A) clozapine (CLZ), (B) N-desmethyl clozapine
(DM-CLZ) and (C) clozapine N-oxide (CLZ-NO) or the positive control inhibitor rifampicin
(RIF). Data are expressed as means ± standard deviations (n=3 independent experiments in
triplicate). * p<0.05, ** p<0.01, *** p<0.001 vs. control (no compound).
41
1 µM Clozapine (hCMEC/D3)
Contro
l
M Inda
tralin
e
+1
0
0
100
200
300
400
500
pmol
/milli
on c
ells *
1 µM Clozapine (hCMEC/D3)
Contro
l
M Que
tiapin
e
+20
M Que
tiapin
e
+100
M C
hlorpr
omaz
ine
+20
M Chlo
rprom
azine
+100
M Olan
zapin
e
+2
0 M O
lanza
pine
+100
M Carb
amaz
epine
+20
M Carb
amaz
epine
+100
0
200
400
600
pmol
/milli
on c
ells
* ** * * *
F
H
1 µM Clozapine (hCMEC/D3)
Contro
l
M Praz
osin
+100
M Vera
pamil
+100
M Lamotr
igine
+100
M Tar
iquida
r
+1
M P
SC-833
+1
0 M K
o143
+1
M MK57
1
+50
M Indo
methac
in
+100
M M
ethotr
exate
+100
050
100150200250300350400450500
pmol
/milli
on c
ells
******
****
1 µM Clozapine (hCMEC/D3)
0 1 2 3 4 5 6 7 8 9 10 15 20 25 300
200
400
600
800
Time (min)
pmol
/milli
on c
ells
Figure 1
A B
C D
0 50 100 150 200 250 3000
1000
2000
3000
4000
Clozapine (µM)
Vel
ocity
((pm
ol/m
illion
cel
ls)/m
in)
E
1 µM Clozapine (hCMEC/D3)
Contro
l
M Rhe
in
+2
0 M R
hein
+100
0
200
400
600
pmol
/milli
on c
ells
*
G
1 µM Clozapine (hCMEC/D3)
Contro
l
M Vera
pamil
+100
0
50
100
150
200
250
pmol
/milli
on c
ells
*
1 µM Clozapine (hCMEC/D3)
37 °C 4 °C0
200
400
600
pmol
/milli
on c
ells
*
42
1 µM CLZ (hCMEC/D3)
10 - 1 10 0 10 1 10 2 10 3 10 40
100
200
300
400
500
Lamotrigine (µM)
CLZ
acc
umul
atio
n(p
mol
/milli
on c
ells
)IC50 = 630.8 µM
5 µM LTG (OCT1-mediated)
10 - 2 10 - 1 10 0 10 1 10 2 1030
20
40
Clozapine (µM)
LTG
acc
umul
atio
n(p
mol
/milli
on c
ells
)
IC50 = 1.8 µM
Figure 2
A 2.73 µM TEA+ (OCT1-mediated)
10 - 2 10 - 1 10 0 10 1 10 2 10 30
5
10
15
Clozapine (µM)
TEA
+ acc
umul
atio
n(p
mol
/mill
ion
cells
)
IC50 = 5.7 µM
B
5 µM LTG (hCMEC/D3)
10 - 2 10 -1 100 101 10 2 1030
20
40
60
Clozapine (µM)
LTG
acc
umul
atio
n(p
mol
/milli
on c
ells
)
IC50 = 2.0 µM
C D
43
1 µM CLZ (KCL22)
0 5 10 15 20 25 300
25
50
75
100
125
Time (min)
CLZ
acc
umul
atio
n(p
mol
/milli
on c
ells
)
SLC22A1 (OCT1)Control
Figure 3
A B
C D
5 µM LTG (KCL22)
0
20
40
60
80
LTG
acc
umul
atio
n(p
mol
/milli
on c
ells
)
**
E F
G H
1 µM CLZ (KCL22)
1 min 5 min 30 min0
10
20
30
40
50
CLZ
acc
umul
atio
n(p
mol
/milli
on c
ells
)
Control
SLC22A4(OCTN1)
171 µM TEA+ (KCL22)
5 min 30 min0
10
20
30
40
TEA
+ acc
umul
atio
n(p
mol
/mill
ion
cells
)
Control
SLC22A4(OCTN1)
*
***
1 µM CLZ (hCMEC/D3)
Contro
l
M Vera
pamil
+100
M A
baca
vir
+1
0 M Phe
nytoi
n
+1
0 M Cort
icoste
rone
+1
0 M C
ortico
steron
e
+100
050
100150200250300350400450500
CLZ
acc
umul
atio
n(p
mol
/milli
on c
ells
)
***
0
10
20
30
40
50
37 nM L-Carnitine (hCMEC/D3)
Car
nitin
e ac
cum
ulat
ion
(fmol
/milli
on c
ells
)
*
hCMEC/D3Knockdown efficiency
SLC22A5 (OCTN2)0
25
50
75
100
Rel
ativ
e ge
ne e
xpre
ssio
n (%
)
1 µM CLZ (hCMEC/D3)
1 min 30 min0
200
400
600
800
CLZ
acc
umul
atio
n(p
mol
/mill
ion
cells
)
Control
SLC22A5(OCTN2)siRNA
1 µM CLZ (hCMEC/D3)
44
0
100
200
300
2 nM Adenosine (hCMEC/D3)
Ade
nosi
ne a
ccum
ulat
ion
(fmol
/mill
ion
cells
)
**
0
20
40
60
80
100
1 µM Uridine (HEK)
Urid
ine
accu
mul
atio
n(p
mol
/mg
prot
ein)
**
A
Figure 4
B
1 µM CLZ (hCMEC/D3)
1 min 30 min0
200
400
600
800
CLZ
acc
umul
atio
n(p
mol
/mill
ion
cells
)
Control
SLC29A1(ENT1)siRNA
1 µM CLZ (hCMEC)
C D 1 µM CLZ (HEK)
1 µM CLZ (HEK)
1 min 30 min0
100
200
300
CLZ
acc
umul
atio
n(p
mol
/mg
prot
ein) Control
SLC29A2(ENT2)
**
F 1 µM CLZ (HEK)
1 µM CLZ (HEK)
1 min 30 min0
100
200
300
400
CLZ
acc
umul
atio
n(p
mol
/mg
prot
ein) Control
SLC29A4(PMAT)
*
E
0
10
20
30
40
5 µM Metformin (HEK)
Met
form
in a
ccum
ulat
ion
(pm
ol/m
g pr
otei
n)
***
45
Figure 5R
elat
ive
SLC
O1B
1ex
pres
sion
hepato
cytes
1
hepato
cytes
2
card
iac en
dothelial
cells
1
card
iac en
dothelial
cells
2
brain en
dothelial
cells
1
brain en
dothelial
cells
2
hCMEC/D3 cell
s0.0
0.5
1.0
1.5
A B
Rel
ativ
e SL
CO
1B3
exp
ress
ion
hepato
cytes
1
hepato
cytes
2
cardiac
endotheli
al ce
lls 1
cardiac
endotheli
al ce
lls 2
brain en
dothelial
cells
1
brain en
dothelial
cells
2
hCMEC/D3 cell
s0.0
0.5
1.0
1.5
C
Rel
ativ
e SL
CO
1B7
expr
essi
on
hepato
cytes
1
hepato
cytes
2
card
iac en
dothelial
cells
1
card
iac en
dothelial
cells
2
brain en
dothelial
cells
1
brain en
dothelial
cells
2
hCMEC/D3 cell
s0.0
0.5
1.0
1.5
2.0
2.5
46
47
OATP1B1
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
CLZ (µM) 0 0.2 2 20 0RIF (µM) 0 0 0 0 50
OATP1B3 refseq
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
CLZ (µM) 0 0.2 2 20 0RIF (µM) 0 0 0 0 50
OATP1B3 haplo1
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
CLZ (µM) 0 0.2 2 20 0RIF (µM) 0 0 0 0 50
OATP1B1
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
DM-CLZ (µM) 0 0.2 2 20 0RIF (µM) 0 0 0 0 50
OATP1B3 refseq
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
RIF (µM) 0 0 0 0 50DM-CLZ (µM) 0 0.2 2 20 0
OATP1B3 haplo1
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
RIF (µM) 0 0 0 0 50DM-CLZ (µM) 0 0.2 2 20 0
OATP1B1
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
CLZ-NO (µM) 0 0.2 2 20 0RIF (µM) 0 0 0 0 50
OATP1B3 refseq
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
RIF (µM) 0 0 0 0 50CLZ-NO (µM) 0 0.2 2 20 0
OATP1B3 haplo1
Est
radi
ol 1
7-g
lucu
roni
deup
take
(% u
ninh
ibite
d)
0
50
100
150
RIF (µM) 0 0 0 0 50CLZ-NO (µM) 0 0.2 2 20 0
********
*******
********
Inhibition by clozapine N-oxideC
Inhibition by N-desmethyl clozapineB
Inhibition by clozapineA
Figure 6
