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The European Journal of Psychiatry

versión impresa ISSN 0213-6163

Eur. J. Psychiat. vol.23 no.3 Zaragoza jul./sep. 2009




Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes



Lalit K. Srivastava*,**, Neelakanta P.V. Nair*,**, Samarthji Lal*,**,***

* Douglas Mental Health University Institute, Montreal, Quebec
** Department of Psychiatry, McGill University
*** Department of Psychiatry, Montreal General Hospital, Montreal, Quebec. CANADA





Background and Objectives: Clozapine (CLOZ) and levomepromazine (LMP) improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ), a neuroleptic analog of LMP, to human recombinant dopamine (rDA) receptor subtypes.
Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells.
Results: (i) LMP has a high affinity (Ki, nM) for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9); (ii) LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6); (iii) CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7); (iv) CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v) CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ.
Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

Key words: Clozapine; Cyamemazine; Dopamine receptor subtypes; Levomepromazine; Schizophrenia.


Background and Objectives

The dopamine (DA) hypothesis of schizophrenia implicates an enhancement of DA function in the pathophysiology of schizophrenia1-3, at least with respect to positive symptoms4. All typical and atypical neuroleptics impair DA neurotransmission5,6. Subtypes of DA receptors have been identified, namely, D1-like (D1, D5) and D2-like (D2, D3, D4)7. Aside from reserpine, which depletes presynaptic DA, neuroleptics impair DA neurotransmission by blocking DA D2 receptors and do so in direct relation to their clinical antipsychotic potencies7. Neuroleptics show differences in their binding affinity for the various DA receptor subtypes7. Various authors have pointed to specific DA receptor subtypes as mediating the symptoms of schizophrenia and being the principle site of action of neuroleptics, namely, the D48, D39,10 or D1-like subtypes11. The superior efficacy of clozapine (CLOZ) in treating schizophrenia, especially in treatment-resistant schizophrenia12 has been attributed to antagonism at the D4 receptor8.

Following two open studies13,14, it has recently been shown that levomepromazine (LMP) also improves treatment-resistant schizophrenia15. LMP, a phenothiazine neuroleptic which is structurally similar to chlorpromazine (CPZ), has a methoxy group at carbon 2 of the phenothiazine ring instead of a chlorine atom and a methyl group at carbon 2 of the aliphatic side chain (Fig. 1). Cyamemazine (CMZ), also a clinically effective neuroleptic16,17, is an analog of LMP which differs from LMP in having a cyano group at carbon 2 of the phenothiazine ring instead of a methoxy group (Fig. 1). Neither LMP nor CMZ are marketed as antipsychotics in the United States. In Canada LMP is available for the treatment of schizophrenia but when used is usually prescribed as an adjunctive agent for its sedative-hypnotic effects. In France CMZ is the most frequently prescribed neuroleptic in the treatment of schizophrenia18.




Dopamine (DA) receptor binding studies with LMP19-21 or CMZ22-24 have been few and none have looked at the full spectrum of DA receptor subtypes. In view of current theories implicating specific subtypes of DA receptors in the pathophysiology of schizophrenia and the demonstration that both CLOZ and LMP improve treatment-resistant schizophrenia, we have investigated the binding of LMP and CMZ to human recombinant DA receptor subtypes and compared the binding affinities of LMP and CMZ to those of CLOZ.



Frozen membrane suspensions of recombinant human dopamine receptor subtypes expressed in Sf9 cells were purchased from Bio-Signal, Montreal. Radioligands for D2 receptor [3H] spiperone (15-30 Ci/mmol) and D1 receptor 3H-SCH23390 (70-80 Ci/mmol) were purchased from NEN Dupont. CMZ and LMP were gifts from Rhône Poulenc Pharma. Other drugs and chemicals were obtained from RBI.

The competition assay for drug binding to receptor radioligands was done in triplicate in 1.0 ml assay buffer (50 mM Tris-EDTA, pH 7.4, 154 mM NaCl) containing 0. 22 nM 3H-spiperone (for the D2L, D2S, D3 and D4.2) or 1.6 nM [3H] SCH23390 (for the D1 and D5), different concentrations of neuroleptics (0.1 nM-10µM) and 1 unit of membrane preparations. Non-specific binding was assayed in parallel in the presence of 10 µM (+) butaclamol. Incubation was carried out at 25o C for 60 min (D2) or 90 min (D1) at the end of which the contents were filtered on Whatman GF/B filters. The filters were washed four times with 5 ml of ice-cold Tris-EDTA buffer. Radioactivity bound to the filters was determined by placing the filters in scintillation cocktail (Packard) and counting, after an overnight equilibration, in a Beckman Scintillation counter. Competition data were analyzed by Prism software to obtain Ki (inhibitor constant, nM) values for drugs.

Data were analyzed by the two-tailed Student's t-test.



Results are shown in the Table. Noteworthy are (i) high affinities of LMP for rD2L, rD2S, rD3 and rD4.2 receptors; (ii) LMP and CLOZ have a similar order of magnitude of affinity for the rD1 receptor (54.3 vs 34.6) (p = NS); (iii) CMZ has high affinities for the rD2L, rD2S, rD3, rD4.2, rD1 and rD5 receptors; (iv) CMZ is nine times more potent than CLOZ at the rD1 receptor (p < 0.001) and five times more potent than CLOZ at the rD4.2 receptor (p < 0.001); (v) CMZ has high affinities for the rD1 and rD5 receptors compared with LMP and CLOZ (p < 0.001 for all four comparisons).





In binding studies in autopsied human brain frontal cortex, the binding affinity of LMP and CPZ to D2-like receptors (using 3H-spiperone) was considerably greater than for D1-like receptors (using 3H-SCH-23390), namely 23.5 and 30.7 times greater, respectively, whereas for CLOZ it was only 1.2 times greater19. In the present study using human rDA receptor subtypes the binding affinities of LMP for the rD2L, rD2S, rD3 and rD4.2 receptors, respectively, were 6.3, 12.6, 6.5 and 6.8 times greater than the affinity for D1 receptors and 5.6, 11.2, 5.8 and 6.1 times greater, respectively, than affinity for the rD5 receptor. For CLOZ the binding affinity to the rD1 receptor was considerably greater than for the affinity for the rD2L, rD2S, and rD3 and approximately equipotent in binding to the D4.2 receptor. The binding affinity of CLOZ to the rD5 receptor was approximately equipotent to the binding affinity to the rD2L and rD2S, and 4.2 times greater for the rD3 receptor. The binding affinity of CLOZ to the rD4.2 receptor was 6.9 times greater than for the rD5 receptor.

The superior efficacy of CLOZ in treating schizophrenia, especially in treatment-resistant schizophrenia12 has been attributed to antagonism at the D4 receptor as well as other neurotransmitter receptor targets. D4 receptors are increased 6-fold compared with only a 10% increase in D2 and D3 receptors in schizophrenia8 and cloned D4 receptors are blocked at CLOZ concentrations that are found in the spinal fluid from CLOZ treated patients7. In the present study both LMP and CMZ showed a greater affinity for the D4 receptor than CLOZ. However, the affinity of haloperidol, which is ineffective in treatment-resistant schizophrenia12,15 has a much greater binding affinity for the D4 receptor than CLOZ7. Further, the selective D4 antagonist, sonepiprazole, was without clinical benefit in patients with schizophrenia25 (in the doses used).

Both CLOZ12 and LMP15 improve treatment-resistant schizophrenia. The only similarity in DA receptor binding in our study is the affinity for the rD1 receptor. Compared with reported Ki values, CLOZ has a 2.6 and 2.3 times greater affinity for the rD1 receptor than CPZ and HAL, respectively7. Thus, the Ki values for CLOZ, LMP, CPZ and HAL for the rD1 receptor are of a similar order of magnitude. This may indicate that D1 receptors are important targets for antipsychotic activity. In this regard, CLOZ binds preferentially to cortical D1-like DA receptors in primate brain11. Further, chronic treatment with CLOZ and other antipsychotic agents down regulates D1 (and D5) receptors in primate prefrontal cortex26,27.

Hameg et al.23 investigated CMZ binding to human rDA receptors expressed in CHO cell lines but did not distinguish rD2L from rD2S or rD1 from rD5. Our findings are of a similar order of magnitude to those of Hameg et al.23. CMZ has a high affinity for the rD1 and rD4 receptors. The affinity of CMZ for the rD1 and the rD4 receptors is nine times and five times, respectively, more than that of CLOZ. If D1 and D4 receptors are important in the neuroleptic activity of CLOZ and other antipsychotic agents, then CMZ may have an important role in the management of treatment-resistant schizophrenia. This is of particular importance as CLOZ treatment is associated with a two percent cumulative incidence of agranulocytosis after 52 weeks of treatment28. Unfortunately, well controlled studies comparing CMZ to other neuroleptics have not been undertaken. Nurowska17 in a parallel design study comparing CMZ with LMP showed 88% of patients improved on CMZ and 86% on LMP. CMZ was better tolerated. The investigation, however, was an open study. Both CMZ and CLOZ affect many receptor types aside from DA so that a contribution of non-DA receptors, namely, serotonin receptor subtypes29, muscarinic M1 and glutamate receptors to the antipsychotic efficacy of these agents cannot be excluded. However, the DA hypothesis remains viable and given the focus on antagonism at the D1 and D4 receptors in the pathophysiology of schizophrenia, the finding that CMZ has a considerably greater affinity than CLOZ for D1 and D4 receptor sites points to the need for studies comparing the efficacy of CMZ to CLOZ, especially in treatment-resistant schizophrenia.



This work was supported by the Medical Research Council (Canada), now called Canadian Institutes of Heath Research (CIHR), and former Rhône Poulenc Pharma. We thank Nadia Zajac for secretarial assistance.



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S. Lal, MD, FRCP(C)
Douglas Mental Health University Institute
6875 LaSalle Boulveard
Montreal, Quebec H4H 1R3
Telephone: 1-514-934-1934 ext. 42362
Fax: 1-514-934-8471

Received 8 September 2008
Revised 10 March 2009
Accepted 7 April 2009

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