Sridhar Natesan

Dissociation between In Vivo Occupancy and Functional Antagonism of Dopamine D2 Receptors: Comparing Aripiprazole to Other Antipsychotics in Animal Models

The novel antipsychotic aripiprazole requires high (>90%) striatal D2 receptor occupancy (D2RO) to be clinically active, but despite its high D2RO it does not show extrapyramidal symptoms. While most antipsychotics are active at nearly 65% D2RO, they show motor side effects when D2RO exceeds 80%. We investigated this discrepancy between D2RO, 5HT2 receptor occupancy (5-HT2RO) and in vivo functional activity of aripiprazole in comparison to haloperidol (typical) and risperidone (atypical) in animal models. All three drugs showed dose-dependent D2RO. While risperidone clearly showed higher 5-HT2RO than D2RO, aripiprazole and haloperidol showed higher D2RO than 5-HT2RO at all doses. Haloperidol and risperidone induced catalepsy at doses producing >80% D2RO, while aripiprazole despite higher D2RO (>90%) induced no catalepsy. Haloperidol and risperidones ED50 values for inhibition of conditioned avoidance response (CAR) and amphetamine-induced locomotor activity (AIL) corresponded to ∼60% D2RO. In contrast, aripiprazole showed a significant dissociation; while it blocked AIL at similar D2RO, a 23-fold higher dose (86% D2RO) was required to inhibit CAR. FOS expression in shell region of the nucleus accumbens was significant for all drugs at D2ROs that were effective in CAR. However, in the core region of the nucleus accumbens and dorsolateral striatum, aripiprazole differed from the others in that despite high D2RO it induced low FOS. Haloperidol and risperidone showed dose/occupancy-dependent prolactin elevations, while aripiprazole did not. Across models, haloperidol and risperidone show similar occupancy-functional antagonism of the D2 system, while aripiprazole shows a clear dissociation. Partial agonism of aripiprazole offers a good explanation for this dissociation and provides a framework for understanding occupancy-functional relationships of partial D2 agonist antipsychotics.

Effect of Chronic Antipsychotic Treatment on Brain Structure: A Serial Magnetic Resonance Imaging Study with Ex Vivo and Postmortem Confirmation

BACKGROUND There is increasing evidence that antipsychotic (APD) may affect brain structure directly. To examine this, we developed a rodent model that uses clinically relevant doses and serial magnetic resonance imaging (MRI), followed by postmortem histopathological analysis to study the effects of APD on brain structures. METHODS Antipsychotic , haloperidol, and olanzapine were continuously administered to rats via osmotic minipumps to maintain clinic-like steady state levels for 8 weeks. Longitudinal in vivo MRI scanning (T₂-weighted) was carried out at baseline, 4 weeks, and 8 weeks, after which animals were perfused and their brains preserved for ex vivo MRI scanning. Region of interest analyses were performed on magnetic resonance images (both in vivo as well as ex vivo) along with postmortem stereology using the Cavalieri estimator probe. RESULTS Chronic (8 weeks) exposure to both haloperidol and olanzapine resulted in significant decreases in whole-brain volume (6% to 8%) compared with vehicle-treated control subjects, driven mainly by a decrease in frontal cerebral cortex volume (8% to 12%). Hippocampal, corpus striatum, lateral ventricles, and corpus callosum volumes were not significantly different from control subjects, suggesting a differential effect of APD on the cortex. These results were corroborated by ex vivo MRI scans and decreased cortical volume was confirmed postmortem by stereology. CONCLUSIONS This is the first systematic whole-brain MRI study of the effects of APD, which highlights significant effects on the cortex. Although caution needs to be exerted when extrapolating results from animals to patients, the approach provides a tractable method for linking in vivo MRI findings to their histopathological origins.

Contrasting Effects of Haloperidol and Lithium on Rodent Brain Structure: A Magnetic Resonance Imaging Study with Postmortem Confirmation

BACKGROUND Magnetic resonance imaging (MRI) studies suggest that antipsychotic -treated patients with schizophrenia show a decrease in gray-matter volumes, whereas lithium-treated patients with bipolar disorder show marginal increases in gray-matter volumes. Although these clinical data are confounded by illness, chronicity, and other medications, they do suggest that typical antipsychotic drugs and lithium have contrasting effects on brain volume. METHODS Rodent models offer a tractable system to test this hypothesis, and we therefore examined the effect of chronic treatment (8 weeks) and subsequent withdrawal (8 weeks) with clinically relevant dosing of an antipsychotic (haloperidol, HAL) or lithium (Li) on brain volume using longitudinal in vivo structural MRI and confirmed the findings postmortem using unbiased stereology. RESULTS Chronic HAL treatment induced decreases in whole brain volume (-4%) and cortical gray matter (-6%), accompanied by hypertrophy of the corpus striatum (+14%). In contrast, chronic Li treatment induced increases in whole-brain volume (+5%) and cortical gray matter (+3%) without a significant effect on striatal volume. Following 8 weeks of drug withdrawal, HAL-induced changes in brain volumes normalized, whereas Li-treated animals retained significantly greater total brain volumes, as confirmed postmortem. However, the distribution of these contrasting changes was topographically distinct: with the haloperidol decreases more prominent rostral, the lithium increases were more prominent caudal. CONCLUSIONS The implications of these findings for the clinic, potential mitigation strategies, and further drug development are discussed.

The Dopamine Stabilizers (S)-(-)-(3-Methanesulfonyl-phenyl)-1-propyl-piperidine [(-)-OSU6162] and 4-(3-Methanesulfonylphenyl)-1-propyl-piperidine (ACR16) Show High in Vivo D2 Receptor Occupancy, Antipsychotic-Like Efficacy, and Low Potential for Motor Side Effects in the Rat

“Dopamine stabilizers” are a new class of compounds that have the ability to reverse both hypo- as well as hyperdopaminergia in vivo. This class, exemplified by the phenylpiperidines (S)-(-)-3-(3-methanesulfonyl-phenyl)-1-propyl-piperidine [(-)-OSU6162] and 4-(3-methanesulfonyl-phenyl)-1-propyl)-piperidine [ACR16] although lacking high in vitro binding affinity for dopamine D2 receptor [(-)-OSU6162, Ki = 447 nM; ACR16, Ki > 1 μM], shows functional actions, suggestive of their interaction. Hence, we evaluated in vivo D2 occupancy of these agents in rats and correlated it to observed effects in a series of behavioral, neurochemical, and endocrine models relevant to the dopamine system and antipsychotic effect. Both (-)-OSU6162 and ACR16 showed robust dose-dependent striatal D2 occupancy with ED50 values of 5.27 and 18.99 mg/kg s.c., respectively, and functional assays showed no partial agonism. Over an occupancy range of 37 to 87% (3-60 mg/kg) for (-)-OSU6162 and 35 to 74% (10-60 mg/kg) for ACR16, we observed both inhibitory (amphetamine-induced locomotor activity) and stimulatory effects (in habituated rats). Haloperidol, over a similar occupancy range (33-78%), potently inhibited psychostimulant activity and induced catalepsy, but it failed to activate habituated animals. In the conditioned avoidance response assay, ACR16 was clearly more efficacious than (-)-OSU6162. In addition, both these compounds demonstrated significant preferential Fos induction in the nucleus accumbens compared with the dorsolateral striatum, a strong predictor of atypical antipsychotic efficacy. The results suggest that dopamine stabilizers exhibit locomotor stabilizing as well as antipsychotic-like effects, with low motor side effect liability, in a dose range that corresponds to high D2 in vivo occupancy.

Loss of phosphodiesterase 10A expression is associated with progression and severity in Parkinson’s disease

The mechanisms underlying neurodegeneration and loss of dopaminergic signalling in Parkinsons disease are still only partially understood. Phosphodiesterase 10A (PDE10A) is a basal ganglia expressed dual substrate enzyme, which regulates cAMP and cGMP signalling cascades, thus having a key role in the regulation of dopaminergic signalling in striatal pathways, and in promoting neuronal survival. This study aimed to assess in vivo the availability of PDE10A in patients with Parkinsons disease using positron emission tomography molecular imaging with (11)C-IMA107, a highly selective PDE10A radioligand. We studied 24 patients with levodopa-treated, moderate to advanced Parkinsons disease. Their positron emission tomography imaging data were compared to those from a group of 12 healthy controls. Parametric images of (11)C-IMA107 binding potential relative to non-displaceable binding (BPND) were generated from the dynamic (11)C-IMA107 scans using the simplified reference tissue model with the cerebellum as the reference tissue. Corresponding region of interest analysis showed lower mean (11)C-IMA107 BPND in the caudate (P < 0.001), putamen (P < 0.001) and globus pallidus (P = 0.025) in patients with Parkinsons disease compared to healthy controls, which was confirmed with voxel-based analysis. Longer Parkinsons duration correlated with lower (11)C-IMA107 BPND in the caudate (r = -0.65; P = 0.005), putamen (r = -0.51; P = 0.025), and globus pallidus (r = -0.47; P = 0.030). Higher Unified Parkinsons Disease Rating Scale part-III motor scores correlated with lower (11)C-IMA107 BPND in the caudate (r = -0.54; P = 0.011), putamen (r = -0.48; P = 0.022), and globus pallidus (r = -0.70; P < 0.001). Higher Unified Dyskinesia Rating Scale scores in those Parkinsons disease with levodopa-induced dyskinesias (n = 12), correlated with lower (11)C-IMA107 BPND in the caudate (r = -0.73; P = 0.031) and putamen (r = -0.74; P = 0.031). Our findings demonstrate striatal and pallidal loss of PDE10A expression, which is associated with Parkinsons duration and severity of motor symptoms and complications. PDE10A is an enzyme that could be targeted with novel pharmacotherapy, and this may help improve dopaminergic signalling and striatal output, and therefore alleviate symptoms and complications of Parkinsons disease.

Reduced Cortical Volume and Elevated Astrocyte Density in Rats Chronically Treated with Antipsychotic Drugs—Linking Magnetic Resonance Imaging Findings to Cellular Pathology

BACKGROUND Increasing evidence suggests that antipsychotic drugs (APD) might affect brain structure directly, particularly the cerebral cortex. However, the precise anatomical loci of these effects and their underlying cellular basis remain unclear. METHODS With ex vivo magnetic resonance imaging in rats treated chronically with APDs, we used automated analysis techniques to map the regions that show maximal impact of chronic (8 weeks) treatment with either haloperidol or olanzapine on the rat cortex. Guided by these imaging findings, we undertook a focused postmortem investigation with stereology. RESULTS We identified decreases in the volume and thickness of the anterior cingulate cortex (ACC) after chronic APD treatment, regardless of the APD administered. Postmortem analysis confirmed these volumetric findings and demonstrated that chronic APD treatment had no effect on the total number of neurons or S100β+ astrocytes in the ACC. In contrast, an increase in the density of these cells was observed. CONCLUSIONS This study demonstrates region-specific structural effects of chronic APD treatment on the rat cortex, primarily but not exclusively localized to the ACC. At least in the rat, these changes are not due to a loss of either neurons or astrocytes and are likely to reflect a loss of neuropil. Although caution needs to be exerted when extrapolating results from animals to patients, this study highlights the power of this approach to link magnetic resonance imaging findings to their histopathological origins.

Altered PDE10A expression detectable early before symptomatic onset in Huntington’s disease

There is an urgent need for early biomarkers and novel disease-modifying therapies in Huntingtons disease. Huntingtons disease pathology involves the toxic effect of mutant huntingtin primarily in striatal medium spiny neurons, which highly express phosphodiesterase 10A (PDE10A). PDE10A hydrolyses cAMP/cGMP signalling cascades, thus having a key role in the regulation of striatal output, and in promoting neuronal survival. PDE10A could be a key therapeutic target in Huntingtons disease. Here, we used combined positron emission tomography (PET) and multimodal magnetic resonance imaging to assess PDE10A expression in vivo in a unique cohort of 12 early premanifest Huntingtons disease gene carriers with a mean estimated 90% probability of 25 years before the predicted onset of clinical symptoms. We show bidirectional changes in PDE10A expression in premanifest Huntingtons disease gene carriers, which are associated with the probability of symptomatic onset. PDE10A expression in early premanifest Huntingtons disease was decreased in striatum and pallidum and increased in motor thalamic nuclei, compared to a group of matched healthy controls. Connectivity-based analysis revealed prominent PDE10A decreases confined in the sensorimotor-striatum and in striatonigral and striatopallidal projecting segments. The ratio between higher PDE10A expression in motor thalamic nuclei and lower PDE10A expression in striatopallidal projecting striatum was the strongest correlate with higher probability of symptomatic conversion in early premanifest Huntingtons disease gene carriers. Our findings demonstrate in vivo, a novel and earliest pathophysiological mechanism underlying Huntingtons disease with direct implications for the development of new pharmacological treatments, which can promote neuronal survival and improve outcome in Huntingtons disease gene carriers.

Evaluation of N -Desmethylclozapine as a Potential Antipsychotic—Preclinical Studies

There is growing interest in N-desmethylclozapine (NDMC), the major metabolite of clozapine, as a unique antipsychotic because it acts in vitro as a 5-HT2 antagonist and as a partial agonist to dopamine D2 and muscarinic receptors. To explore this, we compared NDMC to a typical (haloperidol), atypical (clozapine), and partial-agonist atypical (aripiprazole) antipsychotic in preclinical models. The comparison was carried out using: brain D2 and 5-HT2 receptor occupancy; animal models predictive of antipsychotic efficacy (amphetamine-induced hyperlocomotion (AIL) and conditioned avoidance response (CAR) models); measures predictive of side effects (catalepsy and prolactin elevation); and molecular markers predictive of antipsychotic action (striatal Fos induction). NDMC (10–60 mg/kg/s.c.) showed high 5-HT2 (64–79%), but minimal D2 occupancy (<15% at 60 mg/kg) 1 h after administration. In contrast to other antipsychotics, NDMC was not very effective in reducing AIL or CAR and showed minimal induction of Fos in the nucleus accumbens. However, like atypical antipsychotics, it showed no catalepsy, prolactin elevation, and minimal Fos in the dorsolateral striatum. It seems unlikely that NDMC would show efficacy as a stand-alone antipsychotic, however, its freedom from catalepsy and prolactin elevation, and its unique pharmacological profile (muscarinic agonism) may make it feasible to use this drug as an adjunctive treatment to existing antipsychotic regimens.

Phosphodiesterase 10A PET Radioligand Development Program: From Pig to Human

Four novel phosphodiesterase 10A (PDE10A) PET tracers have been synthesized, characterized in preclinical studies, and compared with the previously reported 11C-MP-10. Methods: On the basis of in vitro data, IMA102, IMA104, IMA107, and IMA106 were identified as potential PDE10A radioligand candidates and labeled with either 11C via N-methylation or with 18F through an SN2 reaction, in the case of IMA102. These candidates were compared with 11C-MP-10 in pilot in vivo studies in the pig brain. On the basis of these data, 11C-IMA106 and 11C-IMA107 were taken into further evaluation and comparison with 11C-MP-10 in the primate brain. Finally, the most promising radioligand candidate was progressed into human evaluation. Results: All 5 tracers were produced with good radiochemical yield and specific activity. All candidates readily entered the brain and demonstrated a heterogeneous distribution consistent with the known expression of PDE10A. Baseline PET studies in the pig and baboon showed that 11C-IMA107 and 11C-MP-10 displayed the most favorable tissue kinetics and imaging properties. The administration of selective PDE10A inhibitors reduced the binding of 11C-IMA107 and 11C-MP-10 in the PDE10A-rich brain regions, in a dose-dependent manner. In the nonhuman primate brain, the tissue kinetics of 11C-IMA107 and 11C-MP-10 were well described by a 2-tissue-compartment model, allowing robust estimates of the regional total volume of distribution. Blockade with unlabeled MP-10 confirmed the suitability of the cerebellum as a reference tissue and enabled the estimation of regional binding potential as the outcome measure of specific binding. Conclusion: 11C-IMA107 was identified as the ligand with the highest binding potential while still possessing reversible kinetics. The first human administration of 11C-IMA107 has demonstrated the expected regional distribution and suitably fast kinetics, indicating that 11C-IMA107 will be a useful tool for the investigation of PDE10A status in the living human brain.

Microglial activation in the rat brain following chronic antipsychotic treatment at clinically relevant doses.

Neuroinflammation is increasingly implicated in the pathogenesis of Schizophrenia (SCZ). In addition, there is increasing evidence for a relationship between the dose and duration of antipsychotic drug (APD) treatment and reductions in grey matter volume. The potential contribution of microglia to these phenomena is however not yet defined. Adult rats were treated with a common vehicle, haloperidol (HAL, 2 mg/kg/day) or olanzapine (OLZ, 10 mg/kg/day) for 8 weeks via an osmotic mini-pump implanted subcutaneously. Microglial cells, identified by their Iba-1 immunoreactivity, were quantified in four regions of interest chosen based on previous neuroimaging data: the hippocampus, anterior cingulate cortex, corpus striatum, and secondary somatosensory cortex. Those cells were also analysed according to their morphology, providing an index of their activation state. Chronic APD treatment resulted in increased density of total microglia in the hippocampus, striatum, and somatosensory cortex, but not in the ACC. Importantly, in all brain regions studied, both APD tested led to a dramatic shift towards an amoeboid, reactive, microglial morphology after chronic treatment compared to vehicle-treated controls. These data provide the first in vivo evidence that chronic APD treatment at clinically relevant doses leads to microglial proliferation and morphological changes indicative of activated microglia in the naïve rat brain. Although caution needs to be exerted when extrapolating results from animals to patients, these data suggest a potential contribution of antipsychotic medication to markers of brain inflammation. Further investigation of the links between antipsychotic treatment and the immune system are warranted.