Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier

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Cellular uptake of the atypical antipsychotic clozapine is a carrier-mediated process David Dickens, Steffen Rädisch, George N. Chiduza, Athina Giannoudis, Michael J. Cross, Hassan Malik, Elke Schaeffeler, Rowena L. Sison-Young, Emma L. Wilkinson, Christopher E. Goldring, Matthias Schwab, Munir Pirmohamed, and Anne T. Nies Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00547 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Molecular Pharmaceutics

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

1

Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool,

UK 2

Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool,

UK 3

Liverpool Hepatobiliary Unit, University Hospital Aintree, Liverpool, UK

4

Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany

5

University Tübingen, Tübingen, Germany

6

Department of Clinical Pharmacology, University Hospital Tübingen, Tübingen, Germany

7

Department 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 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphical Abstract


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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 clozapineinduced 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 timedependent 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.



Keywords Agranulocytosis, blood-brain barrier, clozapine, drug transport, organic anion transporter, organic cation transporter, schizophrenia, SLC transporters


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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 nonclozapine, 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



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


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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.



Experimental Section Materials Unless otherwise stated, reagents were obtained from Sigma-Aldrich (Gillingham, UK). [Nmethyl-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-6,73

H(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.


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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 1217% in Caucasians, Mexicans and Han Chinese.23 Culturing of HEK293 cells expressing fulllength 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;



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 (CRL1573; 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).


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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



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), lot9090701.2 (#2)) and primary human brain microvascular endothelial cells (#1, HBMECs, lot1111603.7) 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


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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-6,73

H(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



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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 OATP1B3mediated 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 pHdependent 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


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(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 ∆Ct 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

Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier

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