*Heli Tuppurainen, MD, Department of Forensic Psychiatry, University of Kuopio, Niuvanniemi Hospital, FI-70240 Kuopio, Finland. Email: email@example.com
Aims: Aberrant dopamine transmission in extrastriatal brain regions has been repeatedly illustrated among patients with schizophrenia. Differences between typical and second-generation antipsychotics in dopamine D2 receptor modulation within various brain areas remain a topic for debate. The aim of the present study was therefore to investigate dopamine D2/3 receptor apparent binding potential (BPapp) and occupancy in midbrain and temporal cortex among clozapine-, olanzapine- and haloperidol-treated schizophrenia patients.
Methods: Dopamine D2/3 binding was studied on single-photon emission computed tomography ligand [123I]epidepride in 13 schizophrenia patients treated with medication (two with haloperidol, four with olanzapine and seven with clozapine), six drug-naïve patients and seven healthy controls.
Results: Statistically significant differences in midbrain dopamine D2/3 receptor BPapp (P = 0.015) and occupancy (P = 0.016) were observed between the clozapine, olanzapine and haloperidol groups. The lowest occupancy was found in clozapine-treated patients (5%), followed by olanzapine-treated patients (28%), compared to haloperidol-treated patients (40%). No significant differences were observed in the temporal poles. Occupancy changed substantially depending on the comparison group used (either drug-naïve vs healthy controls) in the examined brain areas (P = 0.001), showing an overestimation with all antipsychotics when the healthy control group was used.
Conclusion: Both typical and second-generation antipsychotics occupy cortical dopamine D2/3 receptors, thus mediating therapeutic efficacy. Observed differences in midbrain dopamine D2/3 occupancy between classical antipsychotics and second-generation antipsychotics may have clinical relevance by modulating altered nigrostriatal dopamine neurotransmission during the acute phase of schizophrenia.
THE ASSOCIATION BETWEEN drug potency to block subcortical dopamine D2 receptors and efficacy in the treatment of acute schizophrenia symptoms has been demonstrated repeatedly.1–3 Since the discovery of the first antipsychotic compound half a century ago the modulation of post-synaptic dopamine D2 receptors has remained as the single essential feature for antipsychotic drug action.4–6 More recently, neuroimaging and pharmacological evidence has supported the view that limbic cortical dopamine D2 receptors may be the main target of antipsychotic drug treatment.7
The relevance of striatal dopaminergic neurotransmission to the pathophysiology of schizophrenia continues to sustain the suggestion that an exaggerated phasic dopamine release in the basal ganglia occurs during the acute illness.8,9 The regulatory dopamine (DA) D2 receptors, which contain a feedback loop that projects from the cerebral cortex to the midbrain structures, are suggested to connect the aberrant dopamine function in schizophrenia patients, both in the striatum and in the extrastriatal regions.10–12
Studies on extrastriatal drug occupancy have provided conflicting results, because some studies have reported equally high D2 occupancy levels by both second-generation and traditional antipsychotics (90% vs 96%),13,14 while others found lower extrastriatal occupancy with second-generation compared to conventional neuroleptics.15,16 Some research groups have also discovered regional or limbic selectivity by second-generation drugs, in that significantly higher levels of D2 occupancy were found in extrastriatal areas when compared to striatum.13,17–20 One published study with monkeys did not find a divergence between different brain regions for D2 receptor occupancy, similar to the findings for clozapine-, risperidone- and haloperidol-treated animals when using the high-affinity ligand F-fallypride.18,21 This result was replicated using the same radiotracer with olanzapine- and haloperidol-treated schizophrenia patients, in whom D2 occupancy reached up to 68–78% in striatal and extrastriatal areas, except for substantia nigra/ventral tegmental area (VTA) structures, where a significantly lower occupancy level was observed with olanzapine compared to haloperidol (40% vs 59%).22
The majority of occupancy studies so far have made the assumption that dopamine D2 receptor densities do not differ between patients with schizophrenia and healthy controls in any brain regions. This may be relevant for the basal ganglia, although the actual distribution in D2 receptor binding may be larger among patients, because some patients have substantially elevated dopamine D2 levels.23–25 The most recent in vivo neuroimaging studies found a significant reduction of the dopamine D2/3 receptor density in several extrastriatal brain regions; that is, the temporal and anterior cingulate cortices, the thalamus and the midbrain among never-medicated patients with schizophrenia, compared to healthy controls.26–31
The aim of the present single-photon emission computed tomography (SPECT) study was to compare dopamine D2/3 apparent binding potential (BPapp) and drug occupancy in extrastriatal regions between clozapine-, olanzapine- and haloperidol-treated patients with schizophrenia using the high-affinity radioligand [123I]epidepride. The receptor occupancy during drug treatment was determined by using mean values obtained from drug-naïve schizophrenia patients, and healthy controls as reference values in the comparison.
Ethical approval for this study was obtained from the Kuopio University Hospital ethics committee. After a full explanation of the study protocol, both written and informed consent was received from each participant. Patients who met the ICD-10 criteria for either schizophrenia or schizophreniform disorder were included. Two patients treated with haloperidol (age 51 ± 8 years, F/M 1/1), four with olanzapine (age 35 ± 7 years, F/M 1/3) and seven with clozapine (age 35 ± 13 years, F/M 0/7) were recruited from psychiatric hospitals and outpatient units in Kuopio, Finland. The comparison groups consisted of six drug-naïve patients (age 33 ± 14 years, F/M 4/2) and seven healthy subjects (age 31 ± 9 years, F/M 3/4). Study design and data collection regarding drug-naïve patients and healthy controls has already been published in detail.29,30
A trained psychiatrist certified the diagnoses of all patients with the Structured Clinical Interview for the DSM III-R.32 Exclusion criteria were any neurological condition (ruled out on clinical examination and magnetic resonance imaging [MRI]) and substance misuse. All study individuals were right-handed. The antipsychotic-treated patients had been on the same, single medication after being drug free or drug naïve for at least 1 month, and the required washout period of previous medication was 3 months for oral and 6 months for depot antipsychotics prior the study. At the time of entry into the study the patients were treated within ordinary settings when drug dosages were clinically optimized and freely titrated for control of symptoms by the psychiatrists attending the patients. The mean daily dosage for patients receiving haloperidol was 6 mg (range 2.4–10 mg), 19 mg for patients receiving olanzapine (range 15–20 mg) and 593 mg for those receiving clozapine (range 400–800 mg), respectively. The mean dosages of each drug were within the recommended dose range (for haloperidol 5–20 mg/day, olanzapine 10–30 mg/day and clozapine 150–600 mg), but one haloperidol-treated patient a received lower daily dose (haloperidol depot injection 50 mg every 3 weeks), and the drug dose of three clozapine-treated patients was >600 mg/day.33 Chlorpromazine-equivalent mean dose for haloperidol was 300 mg/day, for olanzapine 400 mg/day and for clozapine 1200 mg/day.34,35 One haloperidol patient and all clozapine-treated patients received the last drug dose 2–3 h, and olanzapine-treated patients 14–15 h before the first SPET scan, and the subject receiving depot haloperidol was scanned 2 weeks following the last injection. Patients did not receive other medications that might interfere with D2 receptor binding during the present study (such as antidepressants, beta-blockers or anti-epileptic drugs). Psychopathological symptoms were assessed on the Positive and Negative Syndrome Scale (PANSS; Table 1).36
Table 1. Clinical scores (mean ± SD) of antipsychotic-treated patients
After an i.v. bolus injection of [123I]epidepride (185 MBq; supplied by MAP Medical Technologies, Tikkakoski, Finland) into the right antecubital vein, the first SPET scan was initiated after 30 min and the second scan 3 h after injection of the tracer. Both scans lasted approximately 30 min, using a dedicated MultiSPECT 3 gamma camera with fan-beam collimators (Siemens Medical Systems, Hoffman Estates, IL, USA). The energy window was centered on the photo peak of [123I] (i.e. 148–170 keV). During 360° rotation (120° per camera head), 40 view/head scans were acquired in a 128 × 128 matrix (with a pixel size of 2.8 mm). The radius of rotation was 13 cm. The imaging resolution was 9–10 mm and a soft filter (Butterworth, cut-off frequency 0.4 cm−1 and order 5) was used in reconstruction. Additional details of this SPET imaging procedure have already been described previously.37 After the scans, transaxial slices (6 mm thick) were reconstructed and corrected for attenuation but not for scatter and partial volume effect.
Regions of interest (ROI) in the reconstructed images were manually drawn onto the cerebellum (reference region: free + non-specific binding), the temporal pole and the midbrain (free + non-specific + specific binding). There was no significant difference in ROI area between control and patient groups in any of the regions studied. In all cases the operator was blind to the medication status of the participant. [123I]epidepride is a specific and high-affinity D2/3 receptor ligand that is optimal for imaging low density regions. In high-density areas (i.e. striatum) the use of [123I]epidepride is inadvisable because the tracer binding does not reach equilibrium.
The BPapp of [123I]epidepride in the ROI was calculated according to Eqn 1, as the ratio between radioactivity in the ROI (Cr) and in the cerebellum (Cc) minus 1 (Fig. 1):
This area ratio can be obtained by integrating time activity curves for the time interval of 0–210 min after tracer injection.
Several post-mortem and neuroreceptor-specific imaging studies have estimated the dopamine D2/3 receptor decline to be approximately 1% per year in striatal and extrastriatal brain regions in healthy subjects.38–40 Some previous neuroimaging studies on drug-naïve patients with schizophrenia have reported an age effect in striatum and also in some extrastriatal areas, that is, the frontal and temporal cortices.23,28 The age-related reduction in striatal D2/3 receptor density among schizophrenia patients has been found not to differ from that observed in healthy subjects.41 Age-related alterations in dopamine D2/3 binding were taken into account using Eqn 2 to exclude the possible influence of the different mean ages seen in the present study:
where BPapp-a is the age-adjusted apparent binding potential and BPapp-p is the apparent binding potential for each patient.
For the calculation of drug occupancy, we used the age-adjusted mean D2/3 receptor density of the drug-naïve schizophrenia patients because recent in vivo neuroimaging studies found a significantly reduced amount of dopamine D2/3 receptors in the extrastriatal brain regions of these patients, particularly in the temporal and anterior cingulate cortices, thalamus and midbrain regions compared to healthy controls.27,28,31 We also cross-checked the drug occupancy levels using the age-adjusted mean values from healthy volunteers. The D2/3 receptor occupancy, O, was calculated according to Eqn 3:
where BPapp-ap is the age-adjusted apparent binding potential for drug-treated patients and BPapp-ad/ac is the age-adjusted mean apparent binding potential of the drug-naïve group or the healthy control group.
Patients with schizophrenia underwent a tilted T1-weighted MRI with a 1.5-T Siemens Vision camera (Erlangen, Germany) using a standard head coil and coronal 3-D gradient echo sequence (repetition time [TR] 10 ms, echo time [TE] 4 ms, inversion time [TI] 250 ms, flip angle 12°, field of view 250 mm, matrix 256 × 192, one acquisition). MRI was used to exclude the structural abnormalities.
Statistical analysis was performed using appropriate non-parametric tests to compare group differences in age-adjusted dopamine D2/3 receptor binding values (Kruskal–Wallis test for all test groups and Mann–Whitney test to compare olanzapine and clozapine groups), receptor occupancies and clinical scores (Kruskal–Wallis test), and for right–left hemispheric differences of dopamine D2/3 binding within medicated patients (Wilcoxon rank–sum test). Spearman's two-tailed correlation was used for assessing the significance of relationships between D2/3 receptor occupancy (age-adjusted) and various dimensions of PANSS scores.
The clinical characteristics of the medicated groups are reported in Table 1. Mean scores of various PANSS items did not differ statistically between the study groups (P = 0.23–0.27, Kruskal–Wallis test).
The free + non-specific tracer uptake (area under the cerebellar time activity curve) did not significantly differ between the control and patient groups. The distributions of dopamine D2/3 receptor BPapp in both sides of the temporal pole and in the midbrain for the medicated patients are shown in Fig. 2. There was a significant difference in midbrain age-adjusted binding values (milliliter/milliliter; mean ± SD) between the medicated groups (1.00 ± 0.16 for haloperidol, 1.22 ± 0.10 for olanzapine and 1.59 ± 0.29 for clozapine, χ2 = 8.44, d.f. = 2, P = 0.015, Kruskal–Wallis test). In a re-analysis of the results between olanzapine- and clozapine-treated patients, a statistical significance was further observed (Z = −2.27, P = 0.023, Mann–Whitney test).
We found no statistically significant differences in age-adjusted dopamine D2/3 BPapp between medicated groups in the temporal pole (right side: haloperidol, 0.45 ± 0.03; olanzapine, 0.63 ± 0.09; clozapine, 0.67 ± 0.24; χ2 = 4.00, d.f. = 2, P = 0.136; left side: haloperidol, 0.41 ± 0.08; olanzapine, 0.66 ± 0.08; clozapine, 0.76 ± 0.27; χ2 = 4.71, d.f. = 2, P = 0.095, Kruskal–Wallis test).
We observed no general lateralization effect in binding values in the temporal pole (P = 0.18–0.72), except for clozapine-treated patients (Z = −2.20, P = 0.03, Wilcoxon rank–sum test). There were no statistical differences in BPapp between male and female participants in either the temporal poles or in the midbrain (P = 0.11–0.43, Kruskal–Wallis test). The midbrain ROI were similar among patients and controls.
The D2/3 receptor occupancy values in the midbrain and temporal pole for antipsychotics, relative to the drug-naïve state are shown in Table 2. Kruskal–Wallis test indicated statistically significant group differences in age-adjusted D2/3 receptor occupancy in relation to antipsychotic-naïve schizophrenia patients in the midbrain between the medicated groups. We were not able to detect statistically significant differences in temporal lobe occupancy between the medicated groups.
Table 2. Mean (range) age-corrected D2/3 percentage occupancy (O%)†
†Calculated in comparison with drug-naïve patients' mean apparent binding potential.
MB, midbrain; TP, temporal pole.
When compared to healthy subjects, drug occupancy levels (mean ± SD; age-adjusted) were prominently altered in the temporal pole (right side: haloperidol, 67 ± 2%; olanzapine, 53 ± 7%; clozapine, 49 ± 18%; left side: haloperidol, 71 ± 6%; olanzapine, 53 ± 5%; clozapine, 45 ± 19%), and in the midbrain (haloperidol, 53 ± 6%; olanzapine, 44 ± 4%; clozapine, 27 ± 13%), thus illustrating substantially overestimated values for all antipsychotics in all brain regions. There was a statistically significant difference between occupancy values (age-adjusted) depending on the comparison group used in all examined brain areas (right temporal pole, Z = −3.20, P = 0.001; left temporal pole, Z = −3.19, P = 0.001; midbrain, Z = −3.19, P = 0.001, Wilcoxon rank–sum test).
We found no correlations between age-adjusted dopamine D2/3 receptor occupancy and PANSS items in any brain regions (Table 3).
Table 3. Spearman's correlations (r) between age-adjusted dopamine D2/3 receptor occupancy and PANSS scores
P = 0.20–0.84 (NS).
MB, midbrain; PANSS, Positive and Negative Syndrome Scale; ROI, region of interest; TP, temporal pole.
The main finding of the present study was the statistically significant difference in midbrain dopamine D2/3 receptor apparent binding potential and occupancy between different antipsychotic compounds, in spite of the small sample size. The lowest occupancy was observed in the clozapine-treated patients (5%), followed by the olanzapine-treated patients (28%), when compared with haloperidol-treated patients (40%). This is the first study to demonstrate a statistically significant difference in midbrain dopamine D2/3 receptor binding between two second-generation antipsychotics, clozapine and olanzapine. The present results are in agreement with those of two separate F-fallypride positron emission tomography studies that produced the same rank order in substantia nigra/VTA occupancies for clozapine-, olanzapine- and haloperidol-treated patients.22,42 The group differences did not reach statistical significance in the temporal cortex in the present study, due to the low number of participants. The second-generation antipsychotics clozapine and olanzapine had similar mean D2/3 receptor occupancy (42% vs 46% on the right and 37% vs 45% on the left) in the temporal poles (although variance in occupancy values was larger within the clozapine group), while haloperidol had the higher occupancy (61% on the right and 66% on the left) in the present study.
In light of the most recent imaging studies, the reduction observed in the extrastriatal dopamine D2/3 receptor density among drug-naïve schizophrenia patients questions the relevance of using of healthy controls in a calculation of drug occupancy. As we expected, occupancy values changed substantially when compared to healthy controls, and more so than for medication-free patients. In the midbrain the D2/3 receptor occupancy was 53% in haloperidol-treated, 44% in olanzapine-treated and 27% in clozapine-treated patients. The occupancy values increased to 67–71% for haloperidol, 53% for olanzapine and 49–45% for clozapine in temporal lobes.
We presume that the midbrain signal mostly emanates from the substantia nigra, according to autoradiography studies.43,44 A partial volume effect for these imaging results, especially in small and low receptor density structures, cannot be totally ignored. Until now no volumetric data have been presented to cover the midbrain or substantia nigra of schizophrenia patients. There are arguments for the competitive effect of endogenous dopamine under dopamine depletion on [123I]epidepride binding in striatum and extrastriatal regions, which has been found to be transient.45,46 Amphetamine-challenged dopamine overflow, for example, has not demonstrated epidepride displacement in either experimental animal or human studies.47,48 Nevertheless, the putative effect of endogenous dopamine on D2/3 binding and occupancy results cannot completely be excluded.
Because there is evidence for age-dependent decline in striatal and extrastriatal dopamine D2/3 receptor density among healthy subjects, as well as among patients with schizophrenia, we adjusted the present results on apparent binding and occupancy for age.23,28,38–41 Confounding factors related to age correction may arise from possible regional differences in ROI size and increasing cortical atrophy during normal aging.49 None of the present study subjects suffered from brain atrophy, and ROI areas measured from SPET images were not different between any of the study groups.
The overall level of the present occupancy results in the examined brain areas is moderate or low compared to that determined in former studies. Such discrepancies in drug occupancy findings between research groups, however, may be caused by many factors. The radioligand and acquisition time used, in addition to drug dosage and physicochemical properties, for example, may substantially affect results.50 The molecular interplay between endogenous dopamine, antipsychotic agent and radio-tracer in different brain areas in vivo is not fully understood. We used a time interval of 0–210 min in the analyses of each ROI, which increases the reliability of the present findings in extrastriatal regions.
The present study subjects were treated within ordinary clinical settings, and medication was optimized by their own doctors. Chlorpromazine-equivalent doses for haloperidol and olanzapine were at a congruent level while clozapine dose was three–fourfold compared to that in other medication groups. Drug dosage has previously been demonstrated to influence the drug occupancy in a dose-dependent manner.4,51 The observed discrepancy in chlorpromazine-equivalent mean doses between different medication groups does not explain the present results in apparent binding potential or occupancy.
Despite the fact that the depot haloperidol-treated patient had a low daily drug dose (2.4 mg) his occupancy values (59–61% in temporal poles and 46% in substantia nigra) were nearly the same as those of the other patient who received a typical dose of 10 mg/day haloperidol (63–71% in temporal poles and 33% in substantia nigra). Olanzapine-patients treated with typical therapeutic doses of 15–20 mg/day had a D2/3 receptor occupancy range of 35–53% in the temporal lobes and 19–33% in the midbrain. The clozapine dose range was to some extent elevated (400–800 mg) according to treatment guidelines, but it resulted in a significantly lowest occupancy in the substantia nigra.33
Some of the newer antipsychotic drugs dissociate rapidly from dopamine receptors, particularly in areas of high receptor density with high levels of endogenous dopamine, but their mechanism of action in extrastriatal areas was unclear.50 Electrophysiological and experimental animal studies found substantial differences in modulation and overall level of dopaminergic transmission between mesocortical and nigrostriatal dopamine tracks.52,53 In the cortex, lower extracellular dopamine levels enable low-affinity drugs to block dopamine D2/3 receptors at a transiently high or moderate level.10 No regional selectivity was observed in the present study regarding the high-affinity drug haloperidol. In contrast, clozapine and olanzapine demonstrated preferential occupancy for the temporal cortex compared to the substantia nigra.
The present data emphasize the importance of cortical drug occupancy for both typical and second-generation antipsychotics, which is in line with the assumption that extrastriatal dopamine receptors are the primary target of antipsychotic drugs.7,54 Nigrostriatal dopamine transmission also plays a role in the pathophysiology of schizophrenia. We previously reported a reduced number of nigral dopamine D2/3 receptors (suggested to serve as autoreceptors that regulate dopamine transmission in the striatum) in schizophrenia patients, compared to healthy controls.30 Differences in the blockade of nigral dopamine D2/3 autoreceptors during elevated nigrostriatal dopamine transmission (observed in acute phase of schizophrenia) may partly explain superiority of clozapine in clinical efficacy compared to typical antipsychotics.55,56
According to animal studies dopamine D2/3 receptors in the substantia nigra play a major role in the modulation of motor functions.57 Extrapyramidal signs have been shown to be associated with regulation of nigrostriatal dopamine transmission.58 Differences in nigral dopamine D2/3 receptor occupancy may account for diverse risk between antipsychotic compounds to produce extrapyramidal symptoms.
We conclude that these observed differences in midbrain/substantia nigra occupancy between different antipsychotic agents may contribute to the differences in clinical profile of therapeutic efficacy, in addition to motor side-effects. Due to the methodological limitations discussed here, and the small sample size, the present results warrant further investigation.
The authors have no conflict of interest or financial involvement, which might have biased the present work. This study was supported by the Research Council for Health of the Finnish Academy, an EVO grant from Kuopio University Hospital and Annual EVO Financing from Niuvanniemi Hospital.