School of Psychology and Counselling, Queensland University of Technology, Brisbane, Australia, and Alcohol Research Center, Neuropsychiatric Institute, University of California, Los Angeles, California, USA
Division of Mental Health, Royal Brisbane Hospital, Brisbane, Queensland, Australia, and Alcohol Research Center, Neuropsychiatric Institute, University of California, Los Angeles, California, USA
Division of Mental Health, Royal Brisbane Hospital, Brisbane, Australia
Alcohol Research Center, Neuropsychiatric Institute, University of California, Los Angeles, California, USA
Division of Mental Health, Royal Brisbane Hospital, Brisbane, Australia
Alcohol Research Center, Neuropsychiatric Institute, University of California, Los Angeles, California, USA
Correspondence: Professor Ernest P. Noble, Alcohol Research Center, Neuropsychiatric Institute, University of California, Los Angeles, CA 90024-1759, USA. Tel: +1 310 825 1891; fax: +1 310 206 7309; e-mail: epnoble{at}ucla.edu
Declaration of interest Funding from the Risperidal Foundation, Janssen-Cilag.
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ABSTRACT |
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Aims To examine the role of DRD2 polymorphism on prolactin levels in patients treated with antipsychotic medication.
Method Antipsychotic drugs with different degrees of D2 receptor binding were given to 144 patients with schizophrenia. Serum prolactin levels were obtained and Taq1A DRD2 alleles were determined.
Results Prolactin levels increased across medication groups reflecting increasingly tight D2 receptor binding (clozapine, olanzapine, typical antipsychotics and risperidone). In the combined medication group, patients with the DRD2*A1allele had 40% higher prolactin levels than patients without this allele. In patients treated with clozapine (the loosest D2 receptor binding agent), patients with the DRD2*A1allele had prolactin levels twice those of patients without this allele.
Conclusions Patients with the DRD2A1 allele receiving antipsychotic medications had higher prolactin levels and were overrepresented among those with hyperprolactinaemia, suggesting greater functional D2 receptor binding in this group.
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INTRODUCTION |
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METHOD |
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Assessments
A total of 144 unrelated White patients (123 men, 21 women), attending
various psychiatric units for the treatment of their schizophrenia, were
enrolled in the study. Their average age was 36.4 years (s.d.= 12.0); 61
participants were in-patients and 83 were out-patients. A clinical history was
taken by either a psychiatrist (S.B., B.L., M.B., W.W.) or a clinical
psychologist (R.Y.). Demographic details including ethnic background data were
obtained. The Positive and Negative Syndrome Scale (PANSS;
Kay, 1990) was used to assess
psychotic symptoms. All raters were trained to a criterion of 90% agreement
using PANSS training videos. Interrater reliability was obtained through
random checks of the PANSS by independent raters of the same patient. These
reliabilities were sound.
Medications
All participants received their prescribed antipsychotic medication for at
least 1 month at a stable dosage. Thirty-one patients were prescribed
clozapine, 31 olanzapine, 33 typical antipsychotics (12 flupentixol, 2
fluphenazine decanoate, 13 zuclopenthixol, 3 haloperidol decanoate, 1
thioridazine, 1 thiothixene and 1 trifluoperazine) and 49 risperidone.
Antipsychotic dosage was transformed to chlorpromazine equivalents per
kilogram. The mean chlorpromazine equivalent dosages in the four medication
groups were clozapine 5.14 mg/kg (s.d.=2.94), olanzapine 5.37 mg/kg
(s.d.=2.62), typicals 5.60 mg/kg (s.d.=3.70) and risperidone 4.82 mg/kg
(s.d.=2.00). There was no significant difference in dosage among the four drug
groups (F(3,128)=0.51, P=0.67). Adherence by the in-patients
was sound as all medication was administered by nursing staff; outpatient
adherence was estimated by self-report and assessment by the treating
psychiatrist. Thirty of the 33 patients receiving typical medication were
treated with nurse-administered depot preparations.
Prolactin levels and DRD2 alleles
A 10 ml blood sample was drawn from each participant for DNA extraction and
prolactin determination. The DNA was sent to the University of California Los
Angeles for genotyping, and prolactin determination was conducted at the Royal
Brisbane Hospital. Serum prolactin level was determined by a heterogeneous
sandwich magnetic separation assay (Immuno 1 system; Bayer Diagnostics,
Newbury, Berkshire, UK) which was standardised against the World Health
Organization 3rd IRP 84/500 Reference Manual.
DNA was extracted from leucocytes using standard techniques and subsequently used as a template for determination of Taq1A DRD2 alleles by the polymerase chain reaction (Grandy et al, 1993). As previously described (Noble et al, 1994), the amplification of DNA was carried out using a Perkin Elmer GeneAmp 9600 thermocycler (Perkin Elmer, Boston, MA, USA). Approximately 500 ng of amplified DNA was then digested with 5 units of Taq1 restriction enzyme (Gibco/BRL, Grand Island, NY, USA) at 65°C overnight. The resulting products were analysed by electrophoresis in a 2.5% agarose gel containing ethidium bromide and visualised under ultraviolet light. The A1/A2 genotype is revealed by three fragments (310 bp, 180 bp and 130 bp) and the A2/A2 genotype by two fragments (180 bp and 130 bp); the A1/A1 genotype is shown by the uncleaved 310 bp fragment. Participants with A1/A1 and A1/A2 genotypes were considered to have A1+allelic status and those with the A2/A2 genotype were considered to have A1- allelic status.
Data analysis
Information coded from interview proformas was entered into a computer
database along with prolactin results. The Taq1A DRD2
allelic data were entered last. Chi-squared tests (Yates' corrected) were
employed to compare differences in categorical variables between
A1+ and A1- allelic groups. Analysis
of variance (ANOVA) was used to compare differences in prolactin levels among
the various drug groups. Similarly, one-way ANOVA was employed to examine
differences in prolactin levels between the A1+ and
A1- allelic groups. A P value of 0.05 was
considered to be statistically significant.
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RESULTS |
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Analysis of variance revealed significant differences in prolactin levels among the four medication groups (F(3,140)=19.0, P<0.0001). Figure 1 shows prolactin levels in each of the treatment groups. Post hoc pairwise comparisons were undertaken to test differences between groups. Olanzapine compared with clozapine treatment resulted in significantly higher prolactin levels (F(1,60)=4.76, P=0.033). Patients treated with typical antipsychotics had significantly higher prolactin levels than their olanzapine-treated counterparts (F(1,62)=7.60, P=0.007). Finally, significantly higher levels of prolactin were evident in patients treated with risperidone compared with those treated with typical antipsychotics (F(1,80)=8.97, P=0.004).
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The genotypes of the 144 patients were as follows: A1/A1 (n=7), A1/A2
(n=55) and A2/A2 (n=82). The mean age of the
A1+ group (35.5 years, s.d.=12.7) was not significantly
different from that of the A1- group (37.1 years,
s.d.=11.5) (F(1,141)=0.66, P=0.42). There was no significant
difference between the A1+ and A17
groups according to gender (2(1)=0.048, P=0.83),
in-patient or outpatient status (
2(1)=0.00,
P>0.99), family history of schizophrenia
(
2(1)=0.00, P>0.99), criminality
(
2(1)=0.02, P=0.90), binge drinking
(
2(1)=0.08, P=0.78) or suicide attempts
(
2(1)=2.03, P=0.16). There was also no significant
difference in markers of psychosis severity, as measured by number of
admissions (F(1,140)=1.45, P=0.23), PANSS positive symptoms
(F(1,140)=0.06, P=0.81) and PANSS negative symptoms
(F(1,139)=2.60, P=0.11). There was no difference in
antipsychotic chlorpromazine equivalent dosage between A1+
and A1- patients taking clozapine (F(1,128)=2.36,
P=0.14), olanzapine (F(1,27)=1.0, P=0.33), typical
antipsychotics (F(1,24)=0.33, P=0.57) and risperidone
(F(1,45)=0.47, P=0.50).
Table 1 shows the serum prolactin levels of A1+ and A1- group patients treated with antipsychotic medications. Analysis of variance of the total sample of patients indicated that those carrying the A1 allele had about a 40% higher prolactin level than patients without this allele (F(1,142)=4.50, P=0.04).
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Clozapine, the loosest-binding antipsychotic drug, showed a significant difference across allelic groups, with a prolactin level that was twice as high in A1+ patients compared with A1- patients (F(1,29)=4.63, P=0.04). When the analysis was conducted for male patients only (there were too few women patients for analysis), significance remained (F(1,26)4.58, P=0.04). There was no significant difference in prolactin levels between A1+ and A1- patients in the other antipsychotic drug groups.
Hyperprolactinaemia is defined using community sample cut-off levels set at
a 95% reference range: 430 mU/l in men and
560 mU/l in women
(Vanderpump et al,
1998). In total, 64 patients, 44% of the sample, exceeded these
levels. Only 7 of these patients were prescribed lower-binding agents
(clozapine and olanzapine) confirming that these medications are rarely
associated with hyperprolactinaemia (5% of the sample). Forty of the patients
on risperidone exceeded these prolactin levels, indicating that 81% of
patients on this medication were in the hyperprolactinaemic range. A
Yates'-corrected
2 analysis conducted to compare allelic
status in the group with prolactin levels in the normal range and those with
hyperprolactinaemia was significant (
2(1)=5.52,
P=0.02). Those with A1+ allelic status were
significantly overrepresented in the group of patients with clinical
hyperprolactinaemia.
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DISCUSSION |
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Individuals with the A1 allele and higher prolactin levels when treated with antipsychotic medication and were overrepresented among patients with clinical hyperprolactinaemia. The A1+ participants also had significantly higher prolactin levels than A1- participants when treated with the loosely binding agent clozapine. The greater prolactin response to antipsychotics observed in A1+ patients with schizophrenia in this and other studies (Mihara et al, 2000, 2001) may be the result of A1+ individuals having fewer unbound dopamine receptors at any given antipsychotic dose. Our results indicate that in addition to the D2 receptor binding dissociation constant of antipsychotic medications, individual D2 receptor density is also important in determining prolactin response to antipsychotic agents.
Individuals with the A1 allele have a reduced density of brain D2 receptors (Noble et al, 1991; Thompson et al, 1997; Phjalainen et al, 1998; Jonsson et al, 1999). An early brain autopsy study (Noble et al, 1991) found a significant reduction of approximately 30% in the number of D2 dopamine receptors (Bmax) in the caudate nucleus of A1+ compared with A1- subjects; there was no difference in D2 binding affinity (Kd) between the two allelic groups. Thompson et al (1997) also reported a 30-40% reduction in D2 receptor density in the striatum of A1+ compared with A1- individuals. An in vivo study of healthy Finnish volunteers (Pohjalainen et al, 1998) showed significantly decreased D2 receptor density in the striatum of A1+ compared with A1- individuals, with no difference in Kd between the two groups. In another positron emission tomography study of healthy humans using [11C]-labelled raclopride (Jonsson et al, 1999), a significant association of the A1 allele was found with low D2 receptor density. Taq1A DRD2 variants are now known to be in linkage disequilibrium with C957T, a synonymous mutation in the human DRD2 (Duan et al, 2003). Furthermore, C957T affects messenger ribonucleic acid (mRNA) folding, leading to a decrease in mRNA stability and a 50% decrease in D2 dopamine receptor proteins. These effects dramatically diminish dopamine-induced upregulation of D2 receptors. As a result of fewer D2 receptors at any dose of antipsychotic medication, A1+ individuals may have a lower density of free, unbound D2 receptors, and consequently an enhanced prolactin response.
Dopamine receptor drug occupancy and consequent receptor blockade are necessary for both clinical antipsychotic action (Kapur & Remington, 2001) and a variety of other effects. Studies with conventional antipsychotic drugs report that approximately 70% occupancy results in maximal therapeutic efficacy (Nordstrom et al, 1993). A trend towards improved efficacy in patients with treatment-resistant schizophrenia was found when doses of olanzapine were increased to an average of 30.4 mg (Volavka et al, 2002). Preliminary investigations have been undertaken to increase the efficacy of clozapine, an agent with a high D2 receptor dissociation constant or loose binding, by adding haloperidol, an agent with a low dissociation constant (Kapur et al, 2001). Individuals lacking the A1 allele may be more likely to benefit from these approaches to improve drug D2 receptor occupancy given that they may have relatively more free, unbound, D2 receptors. Conversely, A1+ individuals are unlikely to derive as much improvement from this approach, with optimal therapeutic effect being likely at lower dosages in these patients. Patients lacking the A1 allele may require higher doses for maximal antipsychotic effect, particularly when prescribed a loosely binding antipsychotic such as clozapine or quetiapine.
According to the rapid dissociation model (Kapur & Seeman, 2001), loose-binding atypical agents are hypothesised to have an antipsychotic action without causing other effects of dopamine blockade such as raised prolactin levels or extrapyramidal side-effects (Kapur & Seeman, 2001). Our data are not consistent with this, because clozapine has definite effects on serum prolactin levels, with A1+ individuals having significantly raised prolactin levels compared with A1- patients. The D2 blockade effect of clozapine in A1+ patients is not limited to an antipsychotic effect alone.
Other clinical parameters influenced by D2 receptors require investigation. For example, D2 receptor occupancy correlates with liability to extrapyramidal adverse effects in patients treated with risperidone (Yamada et al, 2002) and a variety of antipsychotic drugs, including clozapine (Broich et al, 1998), haloperidol (Kapur et al, 2000) and olanzapine (Jauss et al, 1998). Individuals with the A1 allele treated with antipsychotic medication may experience extrapyramidal adverse effects at lower dose than A1- patients, as these patients have decreased nigrostriatal D2 receptor density (Thompson et al, 1997).
Limitations of the study
Although the total number of patients investigated is adequate, one of the
limitations of this study is the relatively small number of patients in each
medication subgroup. Further research involving larger numbers of patients
with each individual medication is indicated in order to ascertain whether or
not this association occurs with specific antipsychotic agents. Prospective
studies examining the changes in prolactin levels over time are also
recommended.
Implications of the study
Our study implicates the D2 receptor dissociation constant of
the antipsychotic agent as well as DRD2 variants
as important determinants of D2 receptor blockade induced by
antipsychotic medications. Patients carrying the A1 allele generally
display higher prolactin levels, probably as a result of lower density of
free, unbound, D2 receptors, and may be at increased risk of
adverse effects associated with hyperprolactinaemia. The results demonstrate
that this association is most evident with the loose D2 receptor
binding antipsychotic agent, clozapine. Future research should employ this
pharmacogenetic approach to investigate clinical parameters other than
prolactin response. This may result in clinicians being able to optimise
antipsychotic treatment with regard to drug selection, dose and possible
adverse effects.
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Clinical Implications and Limitations |
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LIMITATIONS
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ACKNOWLEDGMENTS |
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Received for publication October 6, 2003. Revision received March 5, 2004. Accepted for publication March 6, 2004.
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