Dopamine D2 Receptors as Treatment Targets in Schizophrenia

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1 Translational Medicine Dopamine D2 Receptors as Treatment Targets in Schizophrenia Philip Seeman 1 Abstract The antipsychotic effectiveness of chlorpromazine and haloperidol started a search for their therapeutic targets. The antipsychotic receptor target turned out to be a dopamine receptor, now cloned as the dopamine D2 receptor. The D2 receptor is the common target for antipsychotics. Antipsychotic clinical doses correlate with their affinities for this receptor. Therapeutic doses of antipsychotics occupy 60 to 80% of brain D2 receptors in patients, but aripiprazole occupies up to 90%. While antipsychotics may take up to six hours to occupy D2 receptors, much clinical improvement occurs within a few days. The receptor has high- and low-affinity states. The D2High state is functional for dopaminelike agonists such as aripiprazole. Most individuals with schizophrenia are supersensitive to dopamine. Animal models of psychosis show that a variety of risk factors, genetic and nongenetic, are associated with behavioral supersensitivity to dopamine, reflected in elevated levels of dopamine D2High receptors. Although antipsychotics such as haloperidol alleviate psychosis and reverse the elevation of D2High receptors, long-term use of traditional antipsychotics can further enhance dopamine supersensitivity in patients. Therefore, switching from a traditional antipsychotic to an agonist antipsychotic such as aripiprazole can result in the emergence of psychotic signs and symptoms. Clozapine and quetiapine do not elicit parkinsonism and rarely result in tardive dyskinesia because they are released from D2 within 12 to 24 hours. Traditional antipsychotics remain attached to D2 receptors for days, preventing relapse, but allowing accumulation that can lead to tardive dyskinesia. Future goals include imaging D2High receptors and desensitizing them in early-stage psychosis. Key Words: Schizophrenia, Dopamine D2 Receptor, D2High, Dopamine Supersensitivity, D2 Polymorphisms, D3, Antipsychotics, Parkinsonism Introduction This review is an update to an earlier perspective on the role of dopamine receptors in the psychotic process (1). This review suggests that current knowledge about dopamine receptors supports the dopamine hypothesis of schizophrenia; the final section of the review addresses criticisms and limitations of the hypothesis. 1 Pharmacology Department, Faculty of Medicine, University of Toronto Address for correspondence: Philip Seeman, MD, PhD, 260 Heath St., West, Suite 605, Toronto, Ontario, M5P 3L6, Canada Phone: ; Philip.Seeman@utoronto.ca Submitted: August 14, 2009; Revised: September 30, 2009; Accepted: November 17, 2009 The history of dopamine receptors is intertwined with the history of psychosis and antipsychotics. The research path starts with the development of antihistamines after World War II, especially with H. Laborit using these compounds to enhance analgesia during surgery and obstetrics (2). When patients received one of these medications, compound 4560, Laborit observed a euphoric quietude; the patients were calm and somnolent, with a relaxed and detached expression (2). Compound 4560, now known as chlorpromazine, was tested by many French physicians for a variety of medical illnesses. The 1952 report by Delay et al. (2) showed that, within three days, chlorpromazine stopped internal voices in eight patients, a dramatic finding in terms of both efficacy and speed of action (see also 3-5; cf. 6). 56 Clinical Schizophrenia & Related Psychoses April 2010

2 Philip Seeman Figure 1 Competition between dopamine at dopamine D1 receptors (using [3H]SCH23390), and between dopamine and [3H]domperidone at dopamine D2 receptors in homogenates of rat striatum. There is a clear demarcation between competition at the high-affinity states of the receptors, D1High and D2High, and the low-affinity states of the receptors, D1Low and D2Low. The proportion of D2 receptors in the high-affinity state is generally between 15 and 20% in the rat striatum. The average dissociation constants (Ki values; n=4 5 independent experiments) were calculated by the Cheng-Prusoff equation (36) from the concentrations that 50% inhibited the high-affinity component and the low-affinity component. Nonspecific binding defined by the presence of 1 µm (+)-butaclamol for D1, and 10 µm S-sulpiride for D2 (from [39]; with permission of Wiley-Liss, Inc.). With the antipsychotic action of chlorpromazine capturing the attention of the psychiatric community, the discovery of the specific target of action for chlorpromazine became a goal for basic science. The working assumption then, as now, was that finding such a target would open the pathway to uncovering a biochemical cause of psychosis in general and schizophrenia in particular. Prior to 1967, antipsychotics were known to affect metabolism and turnover of adrenaline, noradrenaline, and serotonin, but no selective action on these or any other neurotransmission systems had been shown, nor were neurotransmitter receptors then directly detectable. After studies on the amphetamine-blocking actions of chlorpromazine and haloperidol (7, 8), Van Rossum advanced the hypothesis that antipsychotic drugs (neuroleptics) might be selective blockers of dopamine receptors (9, 10). Van Rossum stated: The hypothesis that neuroleptic drugs may act by blocking dopamine receptors in the brain has been substantiated by preliminary experiments with a few selective and potent neuroleptic drugs. There is an urgent need for a simple isolated tissue that selectively responds to dopamine so that less specific neuroleptic drugs can also be studied and the hypothesis further tested (10). Van Rossum s 1967 need for a simple isolated tissue that selectively responds to dopamine ultimately materialized with the advent of a radioreceptor assay, using [3H]haloperidol, on homogenized tissue (11-15) and later on cloned pure receptors (16). For the first time, this work provided direct evidence that all antipsychotics selectively blocked dopamine receptors with clinical potencies that correlated with their affinity for a dopamine receptor in vitro (12, 13). Based on these early findings, the target for the antipsychotic drugs was named the antipsychotic/dopamine receptor (13), but was later renamed the dopamine D2 receptor (17, 18). The definitions and terminology for dopamine receptors are in the next section. Rather than attempt to review the 37,000 known publications on dopamine receptors, this review will focus on selected aspects of dopamine D2 receptors, particularly the basic properties of dopamine D2 receptors that are relevant to schizophrenia. Nomenclature of Dopamine Receptors A dopamine receptor is defined as a target or site that responds to dopamine with a potency greater than either noradrenaline, adrenaline, or serotonin or any other endog- Clinical Schizophrenia & Related Psychoses April

3 Dopamine D2 Receptors and Schizophrenia enous neurotransmitter. This rank order of potency holds for all dopamine receptors. Although exogenous compounds were not critical in defining dopamine receptors, they were helpful insofar as most dopamine receptors had the following rank order of potency: bromocriptine>apomorphine>(+) 6,7-dihydroxy-2-aminotetralin>dopamine>noradrenaline> adrenaline>serotonin. The D1 Receptor The name for the dopamine D1 receptor has stood the test of time and is associated with the stimulation of dopamine-stimulated adenylate cyclase (18). The DNA of this receptor has been cloned (19). The D2 Receptor As noted above, the antipsychotic/dopamine receptor (12, 13), as labeled by [3H]haloperidol, was renamed the D2 receptor (17, 18). The D2 receptor is associated with an inhibition of adenylate cyclase (20-22). However, before the D2 receptor DNA was cloned (16), it had acquired different names in different laboratories, including D4 (23), D0 (24), or D2Low, which was the low-affinity state of the D2 receptor (25, 26). Now that the D2 receptor DNA has been cloned, these three terms are no longer in use. Current Nomenclature for the Dopamine Receptors The dopamine receptors are now all identified by their DNA and amino acid sequences. (Note: The D1 receptor has 446 amino acids [19]; the D2Short receptor has 414 amino acids [27]; the D2Long receptor has 443 amino acids [28]; the D2Longer receptor has 445 amino acids [29]; the D3 receptor has 400 amino acids [30]; the D4.4 receptor has 419 amino acids [31-33]; the D5 receptor has 477 amino acids [34].) Dopamine D1 Receptors With the advent of radioreceptor assays in the 1980 s, the discovery of SCH23390 as a selective antagonist for the D1 receptor enabled the preparation and use of [3H]SCH23390 as a radioreceptor ligand for D1 receptors. To this day, [3H] SCH23390 is a reliable and commonly used ligand to label D1 receptors. There are two D1-like receptors, D1 and D5. Examples of the competition between dopamine and the ligands used to label D1 and D2 receptors are shown in Figure 1. A biphasic pattern of inhibition is readily seen. The component of inhibition that occurs at low concentration of dopamine is generally referred to as the high-affinity component or high-affinity state of the receptor for dopamine, D1High, or for D2High. The high-affinity component is absent in the presence of high concentrations of a guanine nucleotide (37). The component of inhibition that occurs at Table 1 Dopamine Dopamine Adrenaline Adrenaline Endogenous Agonist Dissociation Constants, nm, at Dopamine D1 and D2 Receptors (rat striata) KiHigh KiLow KiHigh KiLow Noradrenaline KiHigh Noradrenaline KiLow Serotonin Serotonin KiHigh KiLow D1 [3H]Sch23390 Kd=0.35 nm* 30 5, , ,300 9,690 9,690 * Reference (39); References (39; this laboratory.) high concentrations of dopamine is referred to as the lowaffinity component or low-affinity state of the receptor for dopamine, D1Low, or D2Low (see Figure 1). Using data of the sort shown in Figure 1, the dissociation constants or inhibition constants (Ki values) of many drugs have been reported (37, 38). Dopamine-like agonists that have biphasic patterns of ligand inhibition yield dissociation constants (Ki values) for D1High and D1Low, as shown in Figure 1, and listed in Table 1. The data in Table 1 show that the dissociation constants of dopamine, noradrenaline and serotonin have the appropriate rank order in meeting the main criterion for defining a dopamine receptor. This holds true when considering the dissociation constants (Ki values) for either the high-affinity or the low-affinity states. Dopamine D2 Receptors D2 [3H]Domperidone Kd=0.48 nm 2.4 3, , >20 >2,000 Dopamine D2 Antagonists The dopamine D2 receptor was originally defined as a dopamine receptor that did not stimulate adenylate cyclase (18). Subsequent work showed that D2 receptors actually inhibited adenylate cyclase (21, 22). There are three D2-like dopamine receptors: D2, D3, and D4, all of which are associated with the inhibition of adenylate cyclase. Since the early reports and summaries of the potencies of various antipsychotics on the dopamine D2 receptor, using either [3H]haloperidol, [3H]spiperone (40), [3H] domperidone (41, 42) or other ligands (12, 43, 44), there has been a vast number of publications listing thousands of dissociation constants of new and old antipsychotics, and various congeners, on D2 receptors (an earlier summary is in [45]). 58 Clinical Schizophrenia & Related Psychoses April 2010

4 Philip Seeman Table 2 Antipsychotic Dissociation Constants, Ki, at Dopamine Receptors Human Clone: D1 D2 D2 D2 D3 D4 nm nm nm nm nm nm [3H]Ligand Used: Sch Raclo Spip Nem Raclo Spip Kd of Ligand, nm: Amisulpride-( )-S * Amoxapine Aripiprazole 1.8 Benperidol Bifeprunox 3.8 Butaclamol-(+) Chlorpromazine Chlorprothixene Clozapine , 83* 22 Clozapine-iso [3H]Domperidone Droperidol Epidepride Flupentixol-cis Fluphenazine Haloperidol , 27* 2 Iloperidone (HP873) Loxapine Melperone Metoclopramide 16 Molindone Moperone Nemonapride Norclozapine Olanzapine Perlapine Perphenazine Pimozide * Prochlorperazine Quetiapine Raclopride , 1.8* 2400 Remoxipride Risperidone , 6.7* 4.4 Risperidone-9-OH 1.6 Sertindole , 1.6* 11 Spiperone Sulpiride-S , 8* 1000 Thioridazine , 2.3* 11 Thiothixene-cis Trifluperazine Trifluperidol Ziprasidone Ki values (3 14 replicates) measured at ligand concentrations of 2 x Kd of ligand (50, 51). Ki values calculated by Cheng-Prusoff relation (36). D2=D2Long receptor. *Reference (52). Dashes indicate not done. Sch=Schering 23390; Raclo=Raclopride; Spip=Spiperone; Nem=Nemonapride. However, the dissociation constant of a particular antipsychotic often differs among laboratories. There are many reasons for this. One reason is that the final concentration of tissue is different in different laboratories (46). Another reason is that a drug consistently shows a higher dissociation constant when competing versus a highly fatsoluble ligand, as compared to its competition versus a more water-soluble ligand (47). This is shown in Table 2, where the antipsychotic dissociation constants are progressively higher when measured with [3H]raclopride, [3H]spiperone and [3H]nemonapride. For example, haloperidol has a dissociation constant of 0.74 nm with [3H]raclopride, 2.7 nm with [3H]spiperone, and 8.4 nm with [3H]nemonapride. This latter principle is important when different hospital groups use different radioligands to measure the D2 occupancy by antipsychotics when treating patients with schizophrenia. In addition, the principle of competition in vitro Clinical Schizophrenia & Related Psychoses April

5 Dopamine D2 Receptors and Schizophrenia Table 3 Clinical Doses of Antipsychotics Figure 2 Spiperone Benperidol Trifluperidol Cis-flupenthixol Pimozide Fluphenazine Risperidone Haloperidol Sertindole Aripiprazole Olanzapine Raclopride Thiothixene Ziprasidone Bifeprunox Trifluperazine Perphenazine LY Prochlorperazine Molindone Loxapine Ziprasidone Amoxapine Thioridazine Clozapine Quetiapine Remoxipride S-Sulpiride Chlorpromazine Promazine Average Dose Mg/Day Oral Range Mg/Day Oral Ref # 0.2 to to to 5 2 to to 8 3 to 9 5 to to to to to to to to to to to to to to to 1500 (13) (13) (13) (54) (13) (13) (55) (13, 53, 56) (55, 56) (55, 56) (55) (55) (13, 53) (55) (55) (13, 53) (53) (57) (13) (13, 53) (53) (56) (55) (13, 53, 55) (13) (55, 56) (54) (54) (13, 53) (13) Clinically Used As Racemate Partial agonist Partial agonist Glutamate/ dopamine agonist No longer used Racemate between dopamine and a D2 ligand is used to examine abnormalities in schizophrenia and other neurological disorders (48, 49). Clinical Correlate of D2 Antagonist Potencies In order to relate antipsychotic potencies to clinical doses, it is best to use a set of antipsychotic dissociation constants that have all been obtained under similar conditions, such as those listed in Table 2 for [3H]raclopride on the human cloned D2 receptor. Despite the worldwide variation in antipsychotic doses, Table 3 gives a list of the most commonly used doses and their range in clinical practice. Using the data in Tables 2 and 3, the correlation between the antipsychotic doses and the dissociation constants is shown in Figure 2. Although the doses for chlorpromazine and thioridazine are off the correlation, most of these two drugs are bound to plasma proteins in the patient. When allowance is made for this high binding to plasma The clinical doses of antipsychotic medications are related to their affinities for the dopamine D2 receptor. The antipsychotic dissociation constants at D2, obtained using [3H]raclopride, are shown on the ordinate. The glutamate agonist LY (57) has an affinity for the dopamine D2High receptor with a dissociation constant of 10 nm at D2High (using [3H]domperidone, because [3H]raclopride does not readily reveal D2High receptors [58]). This value of 10 nm predicts a clinical dose of approximately mg/day, in general agreement with the dose of 80 mg/day used by Patil et al. (57). Because of the very high binding (exceeding 98%) of chlorpromazine and thioridazine to plasma proteins (54), these antipsychotics require high daily doses. However, the final concentrations of all the antipsychotics (including chlorpromazine and thioridazine) in the plasma water in treated patients are almost identical to their dissociation constants (54, 59). (Adapted from [59]; with permission from Scholarpedia.) proteins, it turns out that the correlation between all the antipsychotic dissociation constants and their unbound (or free) concentration of the antipsychotics in the patients correlate very well (51, 54). Also included in the correlation in Figure 2 is the glutamate agonist LY This compound has recently been tested and found to have an antipsychotic effect (57). Using the D2-selective ligand [3H]domperidone, however, LY has a clear affinity for the dopamine D2High receptor with a dissociation constant of about 10 nm (59, 60), a concentration that would predict a clinical daily dose of approximately mg per day for psychosis (see also [55, 61]); Patil et al. (57) used a daily dose of 80 mg. D2 Antagonists, Parkinsonism, Transient Antipsychotic Action Therapeutic doses of antipsychotics generally occupy 60 to 80% of the brain D2 receptors in vivo (62, 63), with the 60 Clinical Schizophrenia & Related Psychoses April 2010

6 Philip Seeman Table 4A Drug Potencies at Serotonin 5HT2A Receptors, Relative to Ki Values at the D2 Receptor (human clones) Table 4B Drug Potencies at Serotonin 5HT1A Receptors, Relative to Ki Values at the D2 Receptor Ki at 5HT2A/Ki at D2 Parkinsonism Amoxapine 0.03 Low Iloperidone 0.04 Low Clozapine 0.05 None Sertindole 0.15 Low Clozapine-iso 0.15 Catalepsy Perlapine 0.16 Low Risperidone 0.18 Moderate Loxapine 0.19 Yes Olanzapine 0.46 Moderate Quetiapine 0.96 None Ziprasidone 1.1 Moderate Melperone 1.2 Low Thioridazine 1.2 Low Chlorpromazine 1.7 Yes Trifluperazine 6.3 Yes Fluphenazine 6.9 Yes Spiperone 32 Yes Remoxipride 99 None Haloperidol 100 Yes Molindone 1061 Yes Raclopride 2750 Yes exception of the dopamine partial agonist aripiprazole which probably internalizes D2 receptors into the cytoplasm, as do other D2 agonists, and which occupies D2 receptors in excess of 90%. Aside from aripiprazole, occupation of brain D2 receptors at a percentage higher than 80% generally results in parkinsonism (tremor, akinesia, and rigidity) in humans. Some antipsychotics such as clozapine and quetiapine, however, elicit negligible parkinsonism, even at relatively high doses. It has often been suggested that the blockade of serotonin-2 receptors or stimulation of serotonin-1 receptors alleviates the parkinsonism of D2 blockade (64). However, when one calculates the ratio of the Ki values for D2 receptors (in Table 2) divided by the Ki values for serotonin- 2A receptors, as shown in Table 4A, there does not appear to be any obvious relation between parkinsonism and the ratio of the Ki values. Furthermore, there is no clear relation between the parkinsonism-inducing tendencies of antipsychotics and the ratio of the D2/serotonin-1A Ki values (see Table 4B). There does not appear to be any all-encompassing explanation of why some antipsychotics such as clozapine and quetiapine do not induce parkinsonism while others do. However, an important factor may be the speed of dissociation of antipsychotics from the D2 receptor. For example, all eight antipsychotics that result in low or negligible parkinsonism (remoxipride, clozapine, quetiapine, norclozapine, perlapine, S-( )-amisulpride, aripiprazole, and amoxapine) (C50% for GTP-γ-S)/ Ki at D2 Parkinsonism Perlapine 0.05 Low Quetiapine 0.16 None Clozapine 1.7 None Iloperidone (HP873) 2 Low Melperone 3.9 Low Clozapine-iso 5.2 Catalepsy Loxapine 9.8 Yes Risperidone 13.8 Moderate Ziprasidone 14 Moderate Remoxipride 19.4 None Thioridazine 27 Low Trifluperazine 32 Yes Olanzapine 39 Moderate Amoxapine 71 Low Sertindole 316 Low Spiperone Yes Chlorpromazine High Yes Fluphenazine High Yes Haloperidol High Yes Molindone High Yes Raclopride High Yes Human cloned 5HT2A in HEK293 cells (2 8 replicates), using 0.5 nm [3H]ketanserin (Kd=0.55 nm) (50, 51, 65). Human cloned 5HT1A in CHO cells (3 replicates). GTPγS method (66). dissociate rapidly from the D2 receptor, as summarized in Table 5 (51, 67, 68). The attachment of clozapine, quetiapine, or amisulpiride to D2 receptors is transient in the sense that these drugs quickly dissociate from D2 and no longer occupy the D2 receptor after 12 to 24 hours. The patient s signs and symptoms, however, remain abated, perhaps because these transiently acting antipsychotics reset a disturbed electricalchemical pattern. This is speculation; the actual process is not known. Another factor related to parkinsonism are the cholinergic potencies of some antipsychotics, particularly clozapine, whose dissociation constant is equal to that of benztropine (51). While an anticholinergic action may account for the relatively low level of parkinsonism seen with clozapine and olanzapine, it does not explain the low or absent parkinsonism seen with remoxipride (Table 6). D2 Agonists There is considerable evidence that enzymes and hormone receptors can have a high-affinity state and a lowaffinity state (69-72). Although the dopamine D2High state of the receptor has not yet been visualized, there is every reason to think that the same principles hold as with other receptors (69-72) Clinical Schizophrenia & Related Psychoses April

7 Dopamine D2 Receptors and Schizophrenia and that imaging this state will prove useful in the diagnosis and treatment of schizophrenia in the future. Clinical Correlates of D2 Agonist Potencies Figure 3 shows that the concentrations of dopaminelike drugs to inhibit the release of prolactin from rat anterior pituitary cells (in primary culture) are similar or identical to the concentrations or dissociation constants that inhibit the binding of [3H]spiperone to the high-affinity state of the dopamine D2 receptor, D2High, in porcine homogenized anterior pituitary. Dopamine itself, for example, has identical values of about 9 or 10 nm for these two actions. The dissociation constants of the dopamine-like drugs at the lowaffinity state, D2Low, are approximately two or three orders of magnitude higher. Because the molarities of these dopamine agonists in Figure 3 are so similar on D2High and on the functional inhibition of prolactin release, the data suggest that the highaffinity state is physiologically functional. In the central nervous system, however, it has been difficult to obtain data relating in vivo function with either D2High or D2Low. The data in Figure 4 show a correlation Table 5 Time for Drugs to Dissociate by 50% from D2 Cloned Receptor 50% off D2 at Parkinsonism Remoxipride* 13 sec None Clozapine* 16 sec None Quetiapine* 16 sec None Norclozapine 18 sec None Perlapine 24 sec Low Amisulpride-( )-S* sec Low Aripiprazole 52 sec Low Amoxapine 66 sec Low Molindone 3 min Moderate Clozapine-iso 3.6 min Catalepsy Iloperidone 4.2 min Low Ziprasidone 11 min Moderate Loxapine 16.5 min Yes Olanzapine* 17 min Moderate Sertindole* min Low Sulpiride-S* min Yes Raclopride* 24 min Yes Risperidone 27 min Yes Haloperidol* min Yes Chlorpromazine* 31 min Yes Method 1: 100 µm raclopride was added at t=0 to displace bound [3H]antipsychotic (concentration at Ki); D2Long. Time measured for 50% displacement. Method 2: Antipsychotic (at concentration=ki at D2) was bound to D2Long; was diluted with 10 ml buffer at t=0; aliquots filtered at different times, filter rinsed, 5 nm [3H]raclopride added; time for 50% re-occupation of D2Long by [3H]raclopride measured. *Data are average of results using methods 1 and 2. (References in [51].) between the antiparkinson drug concentrations of dopamine agonists and their dissociation constants at the D2High receptor (39); these data support the principle that the D2High state of the D2 receptor is the functional state. Do D2High States Exist In Vivo? Despite considerable reproducible data from many laboratories showing that high-affinity and low-affinity states of the D2 receptor can be detected in homogenized tissues, as well as the clear regulation of D2High by guanine nucleotides, the existence of D2High states in vivo is less well established. For example, Sibley et al. (74) and Skinbjerg et al. (75) could not detect D2High sites in intact cells, although such sites could be detected in intact cells by others (76). In particular, Skinbjerg et al. (75) found that dopamine inhibited the binding of [3H]sulpiride at a single binding site (Ki of 389 nm) in intact tissue culture cells, but did detect D2High (KiHigh of 51 nm) in homogenized cells with [3H]methylspiperone. The same was found for two other dopamine-like agonists, NPA and 2-methoxy-NPA. In such experiments, however, it remains to be examined whether [3H]sulpiride can reveal D2High sites in homogenized tissue, because it has been found that apomorphine competition with [3H] sulpiride on homogenized striata yields two sites (20 nm and 131 nm) which are not significantly different than a single site (77). A search to detect D2High states in vivo by means of positron emission tomography is encountering difficulties and inconsistencies. Several studies show that amphetamine injected intravenously (before the ligand is injected) inhibits the radioactive agonist more than the radioactive antagonist. Table 6 Dissociation Constants, Ki, at Muscarinic M1 Receptors, Relative to Ki Values at Human Cloned D2 Receptors Ki at M1/ Ki at D2 Parkinsonism Clozapine 0.13 None Olanzapine 0.28 Moderate Clozapine-iso 0.93 Catalepsy Quetiapine 0.98 None Amoxapine 2.3 Low Chlorpromazine 2.8 Yes Loxapine 12 Yes Iloperidone 20 Low Ziprasidone 98 Moderate Sertindole 210 Low Pimozide 336 Moderate Haloperidol 1100 Yes Remoxipride High None Risperidone High Moderate Ki at M1 measured with [3H]quinuclidinylbenzilate. Ki at D2Long measured with [3H]raclopride. (References [50, 51].) 62 Clinical Schizophrenia & Related Psychoses April 2010

8 Philip Seeman Figure 3 Figure 4 The concentrations of dopamine-like drugs to inhibit the release of prolactin from rat anterior pituitary cells (in primary culture) are similar or identical to the concentrations or dissociation constants that inhibit the binding of [3H] spiperone to the high-affinity state of the dopamine D2 receptor, D2High, in porcine homogenized anterior pituitary. The dissociation constants of the dopamine-like drugs at the low-affinity state, D2Low, are approximately 2 or 3 orders of magnitude higher. 1, (+)-epinephrine; 2, ( )-epinephrine; 3, ( )-norepinephrine; 4,(+)-3-N-propyl-3-(3-hydroxyphenyl)-piperidine; 5, ( )-3-N-propyl-3-(3-hydroxyphenyl)-piperidine; 6, dopamine; 7, RU-24213; 8, RU-24926; 9, (±)-6,7-dihydroxy-2-aminotetralin; 10, ( )-apomorphine; 11, bromocriptine; 14, pergolide; 15, (±)-N-propyl-norapomorphine (NPA) IC50% value for prolactin inhibition, 84 nm, was taken as twice that for ( )-NPA, 42 nm; 31, (±)-5,6-dihydroxy-2-aminotetralin; 33, (+)-norepinephrine; 34, ( )-Npropyl-norapomorphine; 35, (+)-N-propyl-norapomorphine; 36, RU-29717; 43, 8-aminoergoline; 46, (±)-5-OH-N,N-dipropyl-2-aminotetralin; 47, N,Ndipropyl-2-aminotetralin. (Re-drawn from data of [73].) Correlation between the antiparkinson drug concentrations of dopamine agonists and their dissociation constants (KHigh values) at the dopamine D2High receptor. The therapeutic concentrations were derived from the known concentrations in the plasma, corrected for the fraction bound to plasma proteins. Although no good correlation exists for D1High, D1Low or D2Low, the concentrations of bromocriptine, apomorphine, and pramipexole in human plasma water are similar to the low-affinity dissociation constants of these drugs at D2. (Adapted from [39]; with permission of Wiley-Liss, Inc.) Intravenous amphetamine, for example, inhibited the binding of radioactive methoxy-npa or radioactive (+)PHNO more than it inhibited the binding of radioactive raclopride (78, 79). Such results might support the principle that tracer radioactive concentrations bind preferentially to D2High and are more readily inhibited by endogenous dopamine released by amphetamine. However, Finnema et al. (80) found that apomorphine injected intravenously was about equally effective in displacing the agonist [11C]methoxy-NPA and the antagonist [11C]raclopride, data that suggest that there is no distinction between the binding of agonist and antagonist to D2 and that there may be no detectable D2High state in vivo. McCormick et al. (81) made a similar finding. However, both Finnema et al. (80) and McCormick et al. (81) injected the cold agonist (apomorphine or NPA) intravenously many minutes before the intravenous injection of the radioligands. This procedure may result in an apparently identical pattern of inhibition of the radioactive agonist and the radioactive antagonist. In contrast, Ross and Jackson (82) simultaneously co-injected a dopamine agonist and the radioligand intravenously to measure D2 receptors in vivo. By this method, Ross and Jackson successfully identified high-affinity and low-affinity D2 receptors occupied by the ligand but displaced by the agonist (such as pergolide), similarly to the pattern obtained in vitro. Using this co-injection method, therefore, it was found that an intravenous injection of the agonist NPA inhibited the binding of [3H](+)PHNO significantly more than the D2 antagonist [3H]raclopride (83), as measured ex vivo. This finding is compatible with the idea that the sites for the binding of agonist and antagonist differ and that D2High sites exist in vivo. Overall, the greater inhibition of [3H](+) PHNO than [3H]raclopride by NPA suggests that the additional inhibition may reflect competition at D2High receptors. In other words, the additional difference of 17% inhibition (63% 46%) by NPA may reflect D2High receptors (83). This would agree with in vitro work that shows that between 10 and 20% of the D2 population exists in the D2High state (84, 85). Moreover, NPA inhibited 19% of the specific binding of [3H](+)PHNO at the dose of 10 µg/kg, which is a dose that would not inhibit the binding of [3H]raclopride (83). In fact, the data of Finnema et al. (80) show that their lowest Clinical Schizophrenia & Related Psychoses April

9 Dopamine D2 Receptors and Schizophrenia dose of 10 µg/kg apomorphine inhibited up to 15% more radioactive agonist than radioactive antagonist. This amount of 15% matches the proportion of D2 receptors that are normally in the D2High state. The major biological difference between the preinjection method (80, 81) and the co-injection method (82) is that the co-injection method ensures that the arrival of the ligand and the NPA agonist occurs at precisely the same moment. Considering that a receptor agonist can rapidly desensitize a receptor or internalize receptors within seconds or minutes (86, 87), the arrival of NPA prior to that of [3H](+) PHNO would reduce the amount of D2High receptors available to [3H](+)PHNO. Moreover, the protocol of Seneca et al. (79) had amphetamine injected twenty minutes before the radioligand; amphetamine-released dopamine may here contribute to desensitization or receptor internalization with consequent fewer D2High states. In addition, the protocol of Finnema et al. (80) may also lead to a rapid desensitization or receptor internalization by apomorphine with a reduction in D2High states in the three minutes before the agonist radioligand arrives. Because the high-affinity state of beta-adrenoceptors has been successfully detected in intact cells by Toews et al. (70), it is highly likely that the high-affinity state of the D2 receptor will be identified and labeled in patients. The intracellular levels of GTP-like compounds (88) may make this difficult. Finally, the injection of a G-protein inhibitor directly into the striatum to block the G proteins that mediate dopamine-inhibited cyclase attenuates apomorphineinduced stereotyped behavior (89). This classical experiment may be one of the few experiments to demonstrate that the high-affinity state of D2 may be the functional state in the nervous system. Although this finding is compatible with the situation in the anterior pituitary showing that D2High is the functional state (see Figure 3), the pituitary cells do not have presynaptic neurons, and, therefore, may be more analogous to denervated neurons. Clinical Relevance of D2High States While the detection of D2High sites in vivo requires additional investigation, the in vitro measurement of D2High sites, especially as measured by [3H]domperidone, has been helpful in examining the basis of dopamine supersensitivity in animals. Dopamine supersensitivity to dopamine-like drugs occurs in a majority of individuals with schizophrenia, whether or not they are taking antipsychotic medications (90, 91). In fact, a wide variety of factors known to increase the risk for psychosis or schizophrenia cause dopamine supersensitivity in animals. Such factors include street drugs (cocaine, phencyclidine, amphetamine; [92, 93]), social isolation, birth injury with anoxia, brain injury, gene mutations, and high-dose steroids. For example, knockouts of the RGS9 gene result in mice that are dopamine supersensitive and a marked increase in the proportion of D2 receptors that are in the D2High state. The control proportion of D2 receptors in the highaffinity state is usually 18 20%, but the RGS9 knockout striata had a D2High proportion of 60%. In contrast, animals with knockouts of the adenosine-a2a receptor are hypoactive and subsensitive to dopamine agonists. Correspondingly, the D2High proportions were much reduced. A summary of all the animal models tested for the relation between dopamine supersensitivity and the proportion of D2High receptors is given in Figure 5. Details are provided in the legend to Figure 5. Many knockouts, including those for GSKbeta (glycogen synthase kinase), the metabotropic glutamate receptor-5, the D3 receptor, the D1 receptor and histamine receptors, did not change the animal s sensitivity to dopamine-like drugs and did not elevate the proportion of D2High receptors. These findings in Figure 5 are compatible with many other observations that dopamine supersensitivity is often not accompanied by any change in the density of dopamine D2 receptors (108). In fact, all or almost all the D2High elevations in Figure 5 are not accompanied by any change in the total density of D2 receptors (see also [ ] for effect of antipsychotics on D2 and on D2High). Schizophrenia and the Molecular Biology of D2 Receptors The origin of the dopamine hypothesis of schizophrenia is from Van Rossum (10), who based his proposal on the possible mode of action of neuroleptics. A history of Van Rossum s original hypothesis is provided by Baumeister and Francis (113), who describe that only indirect data and nonselective biochemical effects of antipsychotics existed before 1967: When the hypothesis of dopamine blockade by neuroleptic agents can be further substantiated it may have fargoing consequences for the pathophysiology of schizophrenia. Overstimulation of dopamine receptors could then be part of the aetiology. Obviously such an overstimulation might be caused by overproduction of dopamine, production of substances with dopamine actions., abnormal susceptibility of the receptors, etc. (10). The previous section, illustrating the abnormal susceptibility of elevated D2High receptors and its relation to dopamine supersensitivity in animal models of schizophrenia, supports the Van Rossum hypothesis. In addition, there 64 Clinical Schizophrenia & Related Psychoses April 2010

10 Philip Seeman Figure 5 Animals that are supersensitive to dopamine-like drugs (e.g., apomorphine, cocaine, methylphenidate, amphetamine) reveal elevated proportions of dopamine D2 receptors that are in the high-affinity state for dopamine, D2High (left). The data summarized here were obtained on striata from animals found to be dopamine supersensitive under the following conditions (listed from the top down; unless otherwise specified, details are found in [84, 85, 94]): mature rats with neonatal lesion of the hippocampus (95); rats sensitized by long-term treatment with amphetamine; rats socially isolated after weaning (96); knockouts of GABA B1(-/-) receptors in mice (B. Bettler and P. Seeman, unpublished); knockouts of metabotropic glutamate mglu3 receptors in mice (97); mglu2 metabotropic glutamate receptor knockouts in mice (97); five days of 10 mg/kg corticosterone treatment to rats; knockouts of the DBH (dopamine-beta hydroxylase) gene in mice; long-term ethanol treatment of rats (98); rats sensitized to phencyclidine; rats treated for 14 days with cannabinoid HU210 at 20 µg/kg (Moreno et al., 2005; F.J. Bermudez Silva, F. Rodriguez de Fonseca, J. Suarez, and P. Seeman, unpublished); knockouts of trace amine-1 receptors in mice (99); rats sensitized by long-term treatment with methamphetamine (100); rats sensitized and addicted by long-term self treatment with cocaine (101); knockouts of the RGS9-2 (regulator of G protein signaling-9) gene in mice; knockouts of the dopamine D4 receptor gene in mice; knockouts of the GPRK6 (G protein-coupled receptor kinase) gene in mice; cholinergic lesion in the cerebral cortex of rats; long-term high-dose treatment of rats with caffeine (102); mice made dopamine-deficient by tyrosine hydroxylase knockouts; knockouts of alpha-1b-adrenoceptors in mice (103); rats with entorhinal lesions of the hippocampus (104); knockouts of PSD95 (post-synaptic density 95) gene in mice (M. Beaulieu, M. Caron, and P. Seeman, unpublished); rats born by Caesarian section with anoxia; rats treated with reserpine (5 mg/kg for 3 days; 2 days no drug); mice with COMT (catechol-o-methyl transferase) gene knockouts; rats with neonatal lesion of the hippocampus (105); rats treated with cannabinoid WIN 55,212-2 (4 mg/day for 14 days; F.J. Bermudez Silva, F. Rodriguez de Fonseca, J. Suarez, and P. Seeman, unpublished); rats spontaneously active and explorative (no treatment; 106); mice with knockouts of dopamine transporter DAT or vesicle monoamine transporter-2 VMAT-2 (107); rats sensitized to quinpirole; mice with knockouts of RIIbeta protein kinase A. The right side (84, 85) shows either the lack of elevation, or a minor elevation, or an actual fall in the proportion of D2High receptors in mice with knockouts in the genes for glycogen synthase kinase (GSK3beta), metabotropic glutamate receptor mglur5, dopamine D1 or D3 receptors, histamine H1, H2 or H3 receptors, and adenosine A2A receptors; nine days of ketanserin treatment also had no effect on D2High receptors. (Adapted and extended from [85]; with permission from Wiley & Sons, Inc.) Clinical Schizophrenia & Related Psychoses April

11 Dopamine D2 Receptors and Schizophrenia Figure 6 The human dopamine D2Long receptor has 443 amino acids. The receptor is embedded within the cell membrane s bimolecular layers (of phospholipids and cholesterol) and extends from the extracellular surface (top) to the cytoplasmic surface (bottom). The hydroxyl groups of dopamine are considered to attach to the two serine amino acids 194 and 197 of transmembrane 5 (114; as shown here), while the amino group of dopamine is considered to attach to the aspartic acid 114 of transmembrane 3 (115). The amino nitrogen atom of all antipsychotic drugs is considered to attach to the aspartic acid 114. The variants of the D2Long receptor are: the D2Short receptor, where a segment of 29 amino acids has been spliced out during development of the tissue; the D2Longer receptor, where a dipeptide (VQ or valine-glutamine) is retained during splicing; variant V96A, where alanine replaces valine at position 96 in about 1% of the population; variant S311C, where cysteine replaces serine in about 4% of people; and variant P310S that occurs in about 0.4% of people. Amino acid 351 has a T351A polymorphism. The proline at position 319 is coded by either cytosine-cytosine-cytosine (CCC) or by cytosine-cytosine-thymidine (CCT), where the thymidine base occurs at base position 957. A rare polymorphism was found at V154I in a family with myoclonus dystonia (116), but other families with this illness did not reveal such a mutation (117, 118). The amino acids are: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine. (Adapted from Seeman et al., 2000, with permission from Elsevier Ltd and Copyright Clearance Center.) are features in the molecular biology of D2 receptors in schizophrenia that further support the dopamine hypothesis of schizophrenia. The molecular biology variants of the dopamine D2 receptor are shown in Figure 6. As indicated in the legend to Figure 6, these variants include D2Short, D2Long, D2Longer (29, 119, 120) and polymorphisms at amino acid positions 96, 154, 310, 311, 319, and 351. Of the various polymorphisms in D2, the variation at position 311 in the D2 receptor (see Figure 7) was found highly significantly associated with schizophrenia in a metaanalysis of 3,707 individuals (122). A further meta-analysis by Allen et al. (122a) found the silent polymorphism at amino acid position Proline319Proline (nucleotide 957; see Figure 7) to be highly significantly associated with schizophrenia (p< ). Furthermore, the genetic variants of D2 (but not the genes of D3 or D4 receptors) are associated with rapid onset and poor prognosis for methamphetamine psychosis (126). While these gene findings do not show that D2 is directly associated with schizophrenia, the findings supplement the principle that D2 is the main target for antipsychotic 66 Clinical Schizophrenia & Related Psychoses April 2010

12 Philip Seeman Figure 7 D2 receptor variants in schizophrenia. The dopamine D2 receptor has a variant at position 311 where cysteine replaces serine in approximately 7% of individuals with schizophrenia, in contrast to 3.6% of control individuals (121). This S311C variant was found to be highly statistically significantly associated with the illness in a meta-analysis of 3,707 patients with schizophrenia (122, 123). A thymidine nucleotide at base position 957 (within the codon for proline 319) is associated with a significantly higher prevalence, 56%, in individuals with schizophrenia, as compared to controls, 46% (124, 125). Figure 8 Functional link in vivo between D1 and D2 receptors. Reversal of methamphetamine-induced elevation of D2High receptors by a D1 receptor agonist. Left panel: Using the competition of dopamine with [3H]domperidone to measure dopamine D2High receptors, the level of dopamine D2High receptors in the striata of control rats was 22% of the D2 population. Middle panel: Repeated administration of methamphetamine (1 mg/ kg/day, i.p., for 5 days) sensitized the animals to become dopamine supersensitive and increased the proportion of D2High receptors to 58%. Right panel: Following sensitization by methamphetamine, rats were treated with a dopamine D1 agonist (SKF 38393; 3 mg/kg/day, i.p., for 7 days). The behavioral dopamine supersensitivity of these rats was reversed and the proportion of D2High receptors was also reversed to 31%. (Redrawn from original data; [100].) treatment (127) and are compatible with the additional principle that all psychotic roads lead to an elevation in D2High receptors (111). Overactivity of dopamine D2 receptors can be the basis of positive symptoms of schizophrenia such as hallucinations and delusions, and it can also be the basis of cognitive difficulties (128). Further support for the dopamine hypothesis is that the majority of individuals with schizophrenia are supersensitive to the dopamine-like actions of amphetamine or methylphenidate (90; see also supersensitivity psychosis in 129, 130). Additionally, healthy identical twins of individuals with schizophrenia reveal significantly more D2 receptors in the caudate nucleus (131). Finally, untreated individuals at risk for schizophrenia and who later developed schizophrenia showed a significant increase of 14% in D2 receptors before the onset of the illness (132). Clinical Schizophrenia & Related Psychoses April

13 Dopamine D2 Receptors and Schizophrenia Interactions between D1 and D2 Receptors There is an extensive literature on the interactions between dopamine D1 and D2 receptors (references in 133; 134, 135). Such interactions are not unexpected, considering the co-localization of D1 and D2 in the same neuron in many cases (136). Some of these interactions are synergistic (137, 138) and some are opposing, such as the effect on adenylate cyclase (18) or behavior (139). A practical example of a D1- D2 interaction that may have a clinical application is from Shuto et al. (100), where a D1 agonist (SKF38393) reversed the behavioral dopamine supersensitivity and reversed the elevated D2High receptors in methamphetamine-sensitized rats, as shown in Figure 8. The D2-Like Receptors: D3 and D4 Although the affinities of various antipsychotics are similar for the D2 and D3 receptors, D3 is significantly less sensitive to haloperidol, molindone, olanzapine, quetiapine, risperidone and especially remoxipride by more than tenfold (see Table 2). While there are many good studies researching the possible association of the D3 receptor with schizophrenia (using the Serine9Glycine polymorphism), there are a number of negative studies ( ). Furthermore, although many, but not all, of the antipsychotics have similar potencies on D2 and D3 (see Table 2), it is only the D2 receptor that is universally occupied to a therapeutic level of 60 80% by the antipsychotics (51). The D3 receptors are not occupied by therapeutic doses of antipsychotics (143). This overall observation suggests that D3 may not be a major treatment target in schizophrenia. In fact, BP8947, a partial agonist with a Ki of 0.9 nm for D3 that inhibits cocaine-seeking behavior (144), was not effective in treating schizophrenia (10 mg/day for 10 days; [145]). D4 belongs to the D2-like group of dopamine receptors (D2, D3, D4), all of which are associated with dopamineinhibited adenylate cyclase. As shown in Table 2 (and comparing data for [3H]spiperone only), many antipsychotic drugs have approximately similar potencies on D2 and D4 receptors, except for clozapine, which is about ten times more potent on D4. Much weaker on D4, compared to D2, are raclopride, remoxipride, sulpiride, flupentixol and fluphenazine. Overall, no association has been found between D4 polymorphisms and schizophrenia. Strengths and Weaknesses of the Dopamine Hypothesis of Schizophrenia A. While the present review of dopamine receptors in schizophrenia is naturally incomplete, one conclusion is that the dopamine D2 receptor is the main target for the treatment of schizophrenia because antipsychotic clinical doses correlate with their affinities for the dopamine D2 receptor. B. Despite the correlation between antipsychotic clinical doses and drug affinities for D2, and despite the relatively rapid access of antipsychotics into the brain, why is it often mentioned that patients with schizophrenia require two to three weeks before their psychosis is alleviated? Such a disparate time course between drug localization and patient improvement could question the clinical relevance of dopamine D2 receptors. However, as noted above, Delay et al. (2) observed that it only required approximately three days for chlorpromazine to attenuate the signs and symptoms of acute psychosis. The recent reviews by Agid et al. (3, 4) and Kapur et al. (5) have more rigorously demonstrated that antipsychotic clinical effects have their onset within the first twenty-four hours of administration, and reach a peak of improvement within the first few days or first week of treatment. These reports reinforce the results of Leucht et al. (146) showing an early onset of antipsychotic drug action (within seven days) in 1,708 patients with amisulpiride, an early onset of antipsychotic drug action in an animal model of psychosis (147), and further clinical early onsets of antipsychotic action within less than three days, sometimes within hours ( ). While it is true that continued improvement over additional weeks and months occurs, the most rapid rate of improvement occurs within the first week of antipsychotic treatment (152, 153). The high rate of improvement within the first few days of antipsychotic treatment strongly suggests that the blockade of D2 receptors is responsible for the improvement of the clinical signs and symptoms of psychosis. The many antipsychotics all have different profiles of receptor blockade, but they all share a common action in blocking D2 receptors at the predicted concentrations in plasma water. Moreover, antipsychotics such as remoxipride and amisulpiride are highly selective for D2 receptors, largely precluding the contribution of other receptors to clinical effects. C. D2 receptors are about ten times more numerous in the basal ganglia (putamen and caudate nucleus) than in the cerebral cortex. Since the basal ganglia are usually thought to be mainly associated with motor control, this finding appears not to support the dopamine hypothesis of schizophrenia. However, the paradox is that recent work shows that antipsychotic clinical effects are clearly related to the occupancy of dopamine D2 receptors in the striatal region and not in the extrastriatal regions (154), even though it has 68 Clinical Schizophrenia & Related Psychoses April 2010