Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier-Mediated Process.

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 a 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 first 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 utilizing 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.

[1]  K. Dietz,et al.  Pharmacokinetics of clozapine and its metabolites in psychiatric patients: plasma protein binding and renal clearance. , 1998, British journal of clinical pharmacology.

[2]  Emma L. Wilkinson,et al.  Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability , 2016, Biology Open.

[3]  Joanne Wang,et al.  Selective Transport of Monoamine Neurotransmitters by Human Plasma Membrane Monoamine Transporter and Organic Cation Transporter 3 , 2010, Journal of Pharmacology and Experimental Therapeutics.

[4]  Stephen A Baldwin,et al.  The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. , 2013, Molecular aspects of medicine.

[5]  J. Wang,et al.  The plasma membrane monoamine transporter (PMAT): Structure, function, and role in organic cation disposition , 2016, Clinical pharmacology and therapeutics.

[6]  P. Couraud,et al.  ABC AND SLC TRANSPORTER EXPRESSION AND POT SUBSTRATE CHRACTERIZATION ACROSS THE HUMAN CMEC/ D3 BLOOD-BRAIN BARRIER CELL LINE , 2010 .

[7]  R. Cecchelli,et al.  A Simple Method for Assessing Free Brain/Free Plasma Ratios Using an In Vitro Model of the Blood Brain Barrier , 2013, PloS one.

[8]  S. Kim,et al.  In Vivo Tissue Pharmacokinetics of Carbon-11-Labeled Clozapine in Healthy Volunteers: A Positron Emission Tomography Study , 2015, CPT: pharmacometrics & systems pharmacology.

[9]  B. Jansson,et al.  In-depth neuropharmacokinetic analysis of antipsychotics based on a novel approach to estimate unbound target-site concentration in CNS regions: link to spatial receptor occupancy , 2016, Molecular Psychiatry.

[10]  Christoph Hiemke,et al.  How Does the Benzamide Antipsychotic Amisulpride get into the Brain?—An In Vitro Approach Comparing Amisulpride with Clozapine , 2003, Neuropsychopharmacology.

[11]  G. Superti-Furga,et al.  A Call for Systematic Research on Solute Carriers , 2015, Cell.

[12]  W. Pardridge,et al.  Drug Transport across the Blood–Brain Barrier , 2012, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[13]  Mikko Niemi,et al.  Genetics is a major determinant of expression of the human hepatic uptake transporter OATP1B1, but not of OATP1B3 and OATP2B1 , 2013, Genome Medicine.

[14]  Christel A. S. Bergström,et al.  Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1. , 2008, Journal of medicinal chemistry.

[15]  Stanley E. Lazic,et al.  Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models , 2012, PloS one.

[16]  M. Pirmohamed,et al.  Lamotrigine is a substrate for OCT1 in brain endothelial cells. , 2012, Biochemical pharmacology.

[17]  K. Mizuguchi,et al.  Identification of novel functional organic anion-transporting polypeptide 1B3 polymorphisms and assessment of substrate specificity , 2011, Pharmacogenetics and genomics.

[18]  S. Summerfield,et al.  Examining the Uptake of Central Nervous System Drugs and Candidates across the Blood-Brain Barrier , 2016, The Journal of Pharmacology and Experimental Therapeutics.

[19]  Guangji Wang,et al.  Prediction of human pharmacokinetics from preclinical information of rhein, an antidiabetic nephropathy drug, using a physiologically based pharmacokinetic model. , 2014, Basic & clinical pharmacology & toxicology.

[20]  K. Brouwer,et al.  In Vitro Methods to Support Transporter Evaluation in Drug Discovery and Development , 2013, Clinical pharmacology and therapeutics.

[21]  M. Pirmohamed,et al.  Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol , 2017, Scientific Reports.

[22]  L. Bertilsson,et al.  Genetic and Clinical Factors Affecting Plasma Clozapine Concentration. , 2015, The primary care companion for CNS disorders.

[23]  Lie-Chwen Lin,et al.  The Drug-Drug Effects of Rhein on the Pharmacokinetics and Pharmacodynamics of Clozapine in Rat Brain Extracellular Fluid by In Vivo Microdialysis , 2015, The Journal of Pharmacology and Experimental Therapeutics.

[24]  M. Panicker,et al.  Functional Selectivity in Serotonin Receptor 2A (5-HT2A) Endocytosis, Recycling, and Phosphorylation , 2013, Molecular Pharmacology.

[25]  Li Di,et al.  Coexistence of passive and carrier-mediated processes in drug transport , 2010, Nature Reviews Drug Discovery.

[26]  Bruno Stieger,et al.  Organic anion-transporting polypeptides. , 2014, Current topics in membranes.

[27]  D. Keppler,et al.  Localization and Genomic Organization of a New Hepatocellular Organic Anion Transporting Polypeptide* , 2000, The Journal of Biological Chemistry.

[28]  E. Perucca,et al.  Effect of Adjunctive Lamotrigine Treatment on the Plasma Concentrations of Clozapine, Risperidone and Olanzapine in Patients With Schizophrenia or Bipolar Disorder , 2006, Therapeutic drug monitoring.

[29]  H. Kamimura,et al.  Characterization of Human Organic Cation Transporter 1 (OCT1/SLC22A1)- and OCT2 (SLC22A2)-Mediated Transport of 1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)- 4,9-dihydro-1H-naphtho[2,3-d]imidazolium Bromide (YM155 Monobromide), a Novel Small Molecule Survivin Suppressant , 2010, Drug Metabolism and Disposition.

[30]  Y. Sawada,et al.  Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese subjects. , 2006, Drug metabolism and pharmacokinetics.

[31]  W. Dubin,et al.  Drug interactions affecting clozapine levels , 2015 .

[32]  David J. Begley,et al.  Structure and function of the blood–brain barrier , 2010, Neurobiology of Disease.

[33]  Douglas B. Kell,et al.  A ‘rule of 0.5’ for the metabolite-likeness of approved pharmaceutical drugs , 2015 .

[34]  M. Rapacz,et al.  Reference genes in real-time PCR , 2013, Journal of Applied Genetics.

[35]  J. Volavka,et al.  Multiple-Dose Pharmacokinetics of Clozapine in Patients , 1987, Pharmaceutical Research.

[36]  Joanne Wang,et al.  Interaction of Organic Cations with a Newly Identified Plasma Membrane Monoamine Transporter , 2005, Molecular Pharmacology.

[37]  M. Daly,et al.  Clozapine-induced agranulocytosis is associated with rare HLA-DQB1 and HLA-B alleles , 2014, Nature Communications.

[38]  M Pirmohamed,et al.  Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine. , 2000, Molecular pharmacology.

[39]  J R Chretien,et al.  Estimation of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors. , 1998, Journal of drug targeting.

[40]  Q. Al-Awqati.  One hundred years of membrane permeability: does Overton still rule? , 1999, Nature Cell Biology.

[41]  D. Keppler,et al.  A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. , 2000, American journal of physiology. Gastrointestinal and liver physiology.

[42]  P. Wolf,et al.  Lamotrigine therapeutic thresholds , 2008, Seizure.

[43]  A. Klimke,et al.  Uptake of Clozapine into HL-60 Promyelocytic Leukaemia Cells , 2002, Pharmacopsychiatry.

[44]  B. Roth,et al.  Clozapine and other 5-hydroxytryptamine-2A receptor antagonists alter the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro and in vivo , 1999, Neuroscience.

[45]  M. Wiciński,et al.  Clozapine-induced agranulocytosis/granulocytopenia: mechanisms and monitoring , 2018, Current opinion in hematology.

[46]  Amy E. Chadwick,et al.  Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile , 2016, Archives of Toxicology.

[47]  B. Pollock,et al.  Prediction of brain clozapine and norclozapine concentrations in humans from a scaled pharmacokinetic model for rat brain and plasma pharmacokinetics , 2014, Journal of Translational Medicine.

[48]  P. Visscher,et al.  Five years of GWAS discovery. , 2012, American journal of human genetics.

[49]  L. Gustavsson,et al.  Identification of novel substrates and structure-activity relationship of cellular uptake mediated by human organic cation transporters 1 and 2. , 2013, Journal of medicinal chemistry.

[50]  Douglas B Kell,et al.  What would be the observable consequences if phospholipid bilayer diffusion of drugs into cells is negligible? , 2015, Trends in pharmacological sciences.

[51]  Doris Chen,et al.  The solute carrier SLC35F2 enables YM155-mediated DNA damage toxicity. , 2014, Nature chemical biology.

[52]  S. Vavricka,et al.  Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver , 2002, Hepatology.

[53]  Joanne Wang,et al.  Identification and Characterization of a Novel Monoamine Transporter in the Human Brain* , 2004, Journal of Biological Chemistry.

[54]  H. E. Meyer zu Schwabedissen,et al.  LST‐3TM12 is a member of the OATP1B family and a functional transporter , 2018, Biochemical pharmacology.

[55]  David S Wishart,et al.  Using DrugBank for In Silico Drug Exploration and Discovery , 2016, Current protocols in bioinformatics.

[56]  A. Sekine,et al.  Catalog of 258 single-nucleotide polymorphisms (SNPs) in genes encoding three organic anion transporters, three organic anion-transporting polypeptides, and three NADH:ubiquinone oxidoreductase flavoproteins , 2001, Journal of Human Genetics.

[57]  D. Kell,et al.  Carrier-mediated cellular uptake of pharmaceutical drugs: an exception or the rule? , 2008, Nature Reviews Drug Discovery.

[58]  Joanne Wang,et al.  Metformin Transport by a Newly Cloned Proton-Stimulated Organic Cation Transporter (Plasma Membrane Monoamine Transporter) Expressed in Human Intestine , 2007, Drug Metabolism and Disposition.

[59]  O. Spigset,et al.  Quetiapine and drug interactions: evidence from a routine therapeutic drug monitoring service. , 2007, The Journal of clinical psychiatry.

[60]  B. Cohen,et al.  Tissue Concentrations of Clozapine and its Metabolites in the Rat , 1993, Neuropsychopharmacology.

[61]  M. Pirmohamed,et al.  Transport of gabapentin by LAT1 (SLC7A5). , 2013, Biochemical pharmacology.

[62]  M. Pirmohamed,et al.  Effective dasatinib uptake may occur without human organic cation transporter 1 (hOCT1): implications for the treatment of imatinib-resistant chronic myeloid leukemia. , 2008, Blood.

[63]  H. Bönisch,et al.  Interaction of the human plasma membrane monoamine transporter (hPMAT) with antidepressants and antipsychotics , 2009, Naunyn-Schmiedeberg's Archives of Pharmacology.

[64]  Ming Yang,et al.  Quantitative determination of rhein in human plasma by liquid chromatography-negative electrospray ionization tandem mass/mass spectrometry and the application in a pharmacokinetic study. , 2012, Journal of pharmaceutical and biomedical analysis.

[65]  J. Kopitz,et al.  High clozapine concentrations in leukocytes in a patient who developed leukocytopenia , 2007, Progress in Neuro-Psychopharmacology and Biological Psychiatry.

[66]  H. McLeod,et al.  Variants in the SLCO1B3 Gene: Interethnic Distribution and Association with Paclitaxel Pharmacokinetics , 2007, Clinical pharmacology and therapeutics.

[67]  Joanne Wang,et al.  Polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics. , 2016, Pharmacological research.

[68]  Ute Hofmann,et al.  Proton Pump Inhibitors Inhibit Metformin Uptake by Organic Cation Transporters (OCTs) , 2011, PloS one.

[69]  Bruno Stieger,et al.  The SLCO (former SLC21) superfamily of transporters. , 2013, Molecular aspects of medicine.

[70]  F. Behar-Cohen,et al.  Blood–brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P‐glycoprotein on a novel verapamil influx carrier , 2016, British journal of pharmacology.

[71]  D. Keppler,et al.  Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8). , 2004, Pharmacogenetics.

[72]  M. Schwab,et al.  Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors , 2015, The Pharmacogenomics Journal.

[73]  Euan J. Rodger,et al.  Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation , 2015, Scientific Reports.