The atypical antipsychotic, clozapine, exerts superior efficacy in therapy-resistant schizophrenia, but unfortunately induces agranulocytosis with an incidence of 0.8 - 1 %. In this study, we investigated the cellular uptake of clozapine into human promyelocytic leukaemia HL-60 cells using HPLC with electrochemical detection. On incubation with 1.25 to 40 µM clozapine for 30 min, a saturable, energy- and temperature-dependent uptake process takes place (Km = 18.8 µM, kcat = 1.36 nmol/5 min/mg protein at 37° C). This suggests membrane passage of clozapine by a carrier mechanism. 10 µM Indatraline, an inhibitor of dopamine, noradrenaline and serotonin (5-HT) reuptake, but not the selective 5-HT reuptake inhibitor fluvoxamine, markedly reduced the transport of clozapine by 62 %, whereas addition of 10 mM glucose to the incubation medium increased intracellular clozapine concentrations by 28 %. Since cyclosporine A, vinblastine or verapamil up to a final concentration of 10 µM did not alter the intracellular accumulation of clozapine, an involvement of P-glycoprotein seems to be unlikely. In summary, clozapine uptake into HL-60 cells meets criteria of an active unidirectional transport. Its molecular correlates remain to be established.
References
-
1
Amar A, Segman R H, Shtrussberg S, Sherman L, Safirman C, Lerer B, Brautbar C.
An association between clozapine-induced agranulocytosis in schizophrenics and HLA-DQB1*0201.
Int J Neuropsychopharmacol.
1998;
1
(1)
41-44
-
2
den-Boer J A, Westenberg H G, De-Leeuw A S, van-Vliet I M.
Biological dissection of anxiety disorders: the clinical role of selective serotonin reuptake inhibitors with particular reference to fluvoxamine.
Int Clin Psychopharmacol.
1995;
4
47-52
-
3
Bradford M M.
A rapid and sensitive method for the quantification of milligram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem.
1976;
252
1990-1997
-
4
Cardarelli C O, Aksentijevich I, Pastan I, Gottesman M M.
Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters.
Cancer Res.
1995;
55
(5)
1086-1091
-
5
Claghorn J, Honigfeld G, Abuzzahab F S.
The risks and benefits of clozapine versus chlorpromazine.
J Clin Psychopharmacol.
1987;
7
377-384
-
6
Dettling M, Sachse C, Müller-Oerlinghausen B, Roots I, Brockmöller J, Rolfs A, Cascorbi I.
Clozapine-induced agranulocytosis and hereditary polymorphisms of clozapine metabolizing enzymes: no association with myeloperoxidase and cytochrome P4502D6.
Pharmacopsychiatry.
2000;
33
(6)
218-220
-
7
Gardner I, Zahid N, MacCrimmon D, Uetrecht J P.
A comparison of the oxidation of clozapine and olanzapine to reactive metabolites and the toxicity of these metabolites to human leukocytes.
Mol Pharmacol.
1998;
53
991-998
-
8
Gasparini L, Racchi M, Benussi L, Curti D, Binetti G, Bianchetti A, Trabucchi M, Govoni S.
Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells. Neurosci.
Lett..
1997;
231
(2)
113-117
-
9
Guest L, Sokoluk B, MacCrimmon J, Uetrecht J P.
Examination of possible toxic and immune mechanisms of clozapine-induced agranulocytosis.
Toxicology.
1998;
131
53-65
-
10
Guitton C, Kinowski J M, Aznar R, Bressolle F.
Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection.
J Chromatogr B Biomed Sci Appl.
1997;
690
(1-2)
211-222
-
11
Hyttel J, Larsen J J.
Neurochemical profile of LU 19-005, a potent inhibitor of uptake of dopamine, noradrenaline and serotonin.
J Neurochem.
1985;
44
1615-1622
-
12
Kane J, Honigfeld G, Singer J, Meltzer H.
Clozapine for the treatment-resistant schizophrenic. A double-blind comparison with chlorpromazine.
Arch Gen Psychiatry.
1988;
45
789-796
-
13
Klimke A, Klieser E.
The atypical neuroleptic clozapine (Leponex) - current knowledge and recent clinical aspects.
Fortschr Neurol Psychiatr.
1995;
63
173-193
-
14 Klimke A, Löffler S, Fehsel K, Henning U. In Naber D, Müller-Spahn F, editors Neue Forschungsergebnisse zur Clozapin-induzierten Agranulozytose. Springer, Berlin, Heidelberg, New York 2001: 21-29
-
15 Krieger K. Untersuchungen zum Transport von [2,5,6-3H]-Dopamin und l-[4,6-propyl-3H]-Dihydroalprenolol an nativen Lymphozyten und immortalisierten Zellen. Heinrich-Heine-Universität Düsseldorf (Dissertation) 1999
-
16
Lesch K P, Jatzke S, Meyer J, Stober G, Okladnova O, Mossner R, Riederer P.
Mosaicism for a serotonin transporter gene promoter-associated deletion: decreased recombination in depression.
J Neural Transm.
1999;
106
(11-12)
1223-1230
-
17
Liu Z C, Uetrecht J P.
Clozapine is oxidized by activated human neutrophils to a reactive nitrenium ion that irreversibly binds to the cells.
J Pharmacol Exp Ther.
1995;
275
1476-1483
-
18
Löffler S, Fehsel K, Klimke A, Henning U.
Nitric oxide is not involved in apoptosis induction by psychoactive drugs.
XI World Congress of Psychiatry.
1999;
II
251
-
19
Meltzer H Y.
Multiple-outcome criteria in schizophrenia: an overview of outcome with clozapine.
Eur Psychiatry.
1995;
10
(1)
19S-26S
-
20
Mechetner E B, Roninson I B.
Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal antibody.
Proc Natl Acad Sci USA.
1992;
89
(13)
5824-5828
-
21
Musgrave I F, Seifert R.
Human neutrophils and HL-60 cells do not possess a2-adrenoceptors.
Biochem Pharmacol.
1994;
47
(2)
233-239
-
22
Nordenberg J, Fenig E, Landau M, Weizman R, Weizman A.
Effects of psychotropic drugs on cell proliferation and differentiation.
Biochem Pharmacol.
1999;
58
(8)
1229-1236
-
23
Ohnishi T, Tamai I, Sakanaka K, Sakata A, Yamashima T, Yamashita J, Tsuji A.
In vivo and in vitro evidence for ATP-dependency of P-glycoprotein-mediated efflux of doxorubicin at the blood-brain barrier.
Biochem Pharmacol.
1995;
49
(10)
1541-1544
-
24
Peroutka S J, Snyder S H.
Relationship of neuroleptic drug effects at brain dopamine, serotonin, α-adrenergic and histamine receptors to clinical potency.
Am J Psychiatry.
1980;
137
1518-1522
-
25
Pisciotta A V, Konings S A.
[51Cr] release assay of clozapine-induced cytotoxicity: evidence for immunogenic mechanism.
J Clin Psychiatry.
1994;
55
(B)
143-148
-
26
Rochat B, Baumann P, Audus K L.
Transport mechanisms for the antidepressant citalopram in brain microvessel endothelium.
Brain Res.
1999;
831
(1-2)
229-236
-
27
Safa A R, Agresti M, Bryk D, Tamai I.
N-(p-azido-3-[125I]iodophenethyl)-spiperone binds to specific regions of P-glycoprotein and another multidrug binding protein, spiperophilin, in human neuroblastoma cells.
Biochemistry.
1994;
33
(1)
256-265
-
28
Seifert R, Schulz G.
The superoxide-forming NADPH oxidase of phagocytes. An enzyme system regulated by multiple mechanisms.
Rev Physiol Biochem Pharmacol.
1991;
117
1-338
-
29
Syed S K, Christopherson R I, Roufogalis B D.
Reversal of vinblastine transport by chlorpromazine in membrane vesicles from multidrug-resistant human CCRF-CEM leukaemia cells.
Br J Cancer.
1998;
78
(3)
321-327
-
30
Uetrecht J P, Zahid N, Tehim A, Fu J M, Rakhit S.
Structural features associated with reactive metabolite formation in clozapine analogues.
Chem Biol Interact.
1997;
104
(2-3)
117-129
-
31
Williams D P, Pirmohamed M, Naisbitt D J, Uetrecht J P, Park B K.
Induction of metabolism-dependent and -independent neutrophil apoptosis by clozapine.
Mol Pharmacol.
2000;
58
(1)
207-216
Dr. Uwe Henning
Neurobiochemical Research Unit Department of Psychiatry
Heinrich-Heine-University Düsseldorf
Bergische Landstr. 2
40629 Düsseldorf
Germany
Email: KN27120@mail.lvr.de
