Lumateperone Normalizes Pathological Levels of Acute Inflammation through Important Pathways Known to Be Involved in Mood Regulation.

Lumateperone is indicated for the treatment of schizophrenia in adults and for depressive episodes associated with bipolar I or II disorder (bipolar depression) in adults, as monotherapy and as adjunctive therapy with lithium or valproate (Calabrese et al., 2021). It is currently under evaluation for the treatment of major depressive disorder (www.ClinicalTrials.gov). Lumateperone acts by selectively modulating serotonin, dopamine, and glutamate neurotransmission in the brain. However, other mechanisms could be involved in the actions of lumateperone, and because of the connection between the immune system and psychiatric health, we hypothesized that lumateperone might improve symptoms of depression, at least in part, by normalizing pathologic inflammation. Here, we show that in male and female C57BL/6 mice subjected to an acute immune challenge, lumateperone reduced aberrantly elevated levels of key proinflammatory cytokines (e.g., IL-1ß, IL-6, and TNF-α) in both brain and serum; lumateperone also reduced proinflammatory cytokines in male mice under acute behavioral stress. Further, we demonstrate that lumateperone altered key genes/pathways involved in maintaining tissue integrity and supporting blood-brain barrier function, such as claudin-5 and intercellular adhesion molecule 1. In addition, in acutely stressed male Sprague Dawley rats, lumateperone conferred anxiolytic- and antianhedonic-like properties while enhancing activity in the mammalian target of rapamycin complex 1 pathway in the PFC. Together, our preclinical findings indicate that lumateperone, in addition to its ability to modulate multiple neurotransmitter systems, could also act by reducing the impact of acute inflammatory challenges.SIGNIFICANCE STATEMENT Lumateperone is indicated in adults to treat schizophrenia and depressive episodes associated with bipolar I or II disorder, as monotherapy and adjunctive therapy with lithium or valproate. Because aberrant immune system activity is associated with increased depressive symptoms, the relationship between lumateperone and immune function was studied. Here, lumateperone reduced the levels of proinflammatory cytokines that were increased following an immune challenge or stress in mice. Additionally, lumateperone altered genes and pathways that maintain blood-brain barrier integrity, restored an index of blood-brain barrier function, reduced anxiety-like behavior in rodents, and enhanced mammalian target of rapamycin complex 1 pathway signaling in the PFC. These results highlight the anti-inflammatory actions of lumateperone and describe how lumateperone may reduce immune pathophysiology, which is associated with depressive symptoms.

Selective Phosphodiesterase 1 Inhibition Ameliorates Vascular Function, Reduces Inflammatory Response, and Lowers Blood Pressure in Aging Animals.

Diminished nitric oxide-cGMP-mediated relaxation plays a crucial role in cardiovascular aging, leading to decreased vasodilation, vascular hypertrophy and stiffening, and ultimately, cardiovascular dysfunction. Aging is the time-related worsening of physiologic function due to complex cellular and molecular interactions, and it is at least partly driven by DNA damage. Genetic deletion of the DNA repair enzyme ERCC1 endonuclease in Ercc1Δ/- mice provides us an efficient tool to accelerate vascular aging, explore mechanisms, and test potential treatments. Previously, we identified the cGMP-degrading enzyme phosphodiesterase 1 as a potential treatment target in vascular aging. In the present study, we studied the effect of acute and chronic treatment with ITI-214, a selective phosphodiesterase 1 inhibitor on vascular aging features in Ercc1Δ/- mice. Compared with wild-type mice, Ercc1Δ/- mice at the age of 14 weeks showed decreased reactive hyperemia, diminished endothelium-dependent and -independent responses of arteries in organ baths, carotid wall hypertrophy, and elevated circulating levels of inflammatory cytokines. Acute ITI-214 treatment in organ baths restored the arterial endothelium-independent vasodilation in Ercc1Δ/- mice. An 8-week treatment with 100 mg/kg per day ITI-214 improved endothelium-independent relaxation in both aorta and coronary arteries, at least partly restored the diminished reactive hyperemia, lowered the systolic and diastolic blood pressure, normalized the carotid hypertrophy, and ameliorated inflammatory responses exclusively in Ercc1Δ/- mice. These findings suggest phosphodiesterase 1 inhibition would provide a powerful tool for nitric oxide-cGMP augmentation and have significant therapeutic potential to battle arteriopathy related to aging. SIGNIFICANCE STATEMENT: The findings implicate the key role of phosphodiesterase 1 in vascular function and might be of clinical importance for the prevention of mortalities and morbidities related to vascular complications during aging, as well as for patients with progeria that show a high risk of cardiovascular disease.

Inhibition of calcium-calmodulin-dependent phosphodiesterase (PDE1) suppresses inflammatory responses.

A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses. ITI-214 dose-dependently suppressed lipopolysaccharide (LPS)-induced gene expression of pro-inflammatory cytokines in an immortalized murine microglial cell line, BV2 cells. RNA profiling (RNA-Seq) was used to analyze the impact of ITI-214 on the BV2 cell transcriptome in the absence and the presence of LPS. ITI-214 was found to regulate classes of genes that are involved in inflammation and cell migration responses to LPS exposure. The gene expression changes seen with ITI-214 treatment were distinct from those elicited by inhibitors of other PDEs with anti-inflammatory activity (e.g., a PDE4 inhibitor), indicating a distinct mechanism of action for PDE1. Functionally, ITI-214 inhibited ADP-induced migration of BV2 cells through a P2Y12-receptor-dependent pathway, possibly due to increases in the extent of cAMP and VASP phosphorylation downstream of receptor activation. Importantly, this effect was recapitulated in P2 rat microglial cells in vitro, indicating that these pathways are active in native microglial cells. These studies are the first to demonstrate that inhibition of PDE1 exerts anti-inflammatory effects through effects on microglia signaling pathways. The ability of PDE1 inhibitors to prevent or dampen excessive inflammatory responses of BV2 cells and microglia provides a basis for exploring their therapeutic utility in the treatment of neurodegenerative diseases associated with increased inflammation and microglia proliferation such as Parkinson's disease and Alzheimer's disease.

Discovery of ITI-333, a Novel Orally Bioavailable Molecule Targeting Multiple Receptors for the Treatment of Pain and Other Disorders.

Development of more efficacious medications with improved safety profiles to manage and treat multiple forms of pain is a critical element of healthcare. To this end, we have designed and synthesized a novel class of tetracyclic pyridopyrroloquinoxalinone derivatives with analgesic properties. The receptor binding profiles and analgesic properties of these tetracyclic compounds were studied. Systematic optimizations of this novel scaffold culminated in the discovery of the clinical candidate, (6bR,10aS)-8-[3-(4-fluorophenoxy)propyl]-6b,7,8,9,10,10a-hexahydro-1H-pyrido[3',4':4,5]pyrrolo[1,2,3-de]quinoxalin-2(3H)-one (compound 5, ITI-333), which exhibited potent binding affinity to serotonin 5-HT2A (Ki = 8.3 nM) and µ-opioid receptors (MOR, Ki = 11 nM) and moderate affinity to adrenergic α1A (Ki = 28 nM) and dopamine D1 (Ki = 50 nM) receptors. ITI-333 acts as a 5-HT2A receptor antagonist, a MOR partial agonist, and an adrenergic α1A receptor antagonist. ITI-333 exhibited dose-dependent analgesic effects in rodent models of acute pain. Currently, this investigational new drug is in phase I clinical development.

Pharmacologic profile of ITI-333: a novel molecule for treatment of substance use disorders.

RATIONALE: Medications are urgently needed to treat symptoms of drug withdrawal and mitigate dysphoria and psychiatric comorbidities that drive opioid abuse and relapse. ITI-333 is a novel molecule in development for treatment of substance use disorders, psychiatric comorbidities, and pain. OBJECTIVE: Characterize the preclinical profile of ITI-333 using pharmacological, behavioral, and physiological assays. METHODS: Cell-based assays were used to measure receptor binding and intrinsic efficacy of ITI-333; animal models were employed to assess effects on opioid reinstatement, precipitated oxycodone withdrawal, and drug abuse liability. RESULTS: In vitro, ITI-333 is a potent 5-HT2A receptor antagonist (Ki = 8 nM) and a biased, partial agonist at µ-opioid (MOP) receptors (Ki = 11 nM; lacking ß-arrestin agonism) with lesser antagonist activity at adrenergic α1A (Ki = 28 nM) and dopamine D1 (Ki = 50 nM) receptors. In vivo, ITI-333 blocks 5-HT2A receptor-mediated head twitch and MOP receptor-mediated effects on motor hyperactivity in mice. ITI-333 alone is a naloxone-sensitive analgesic (mice) which suppresses somatic signs of naloxone-precipitated oxycodone withdrawal (mice) and heroin cue-induced reinstatement responding without apparent tolerance or physical dependence after chronic dosing (rats). ITI-333 did not acutely impair gastrointestinal or pulmonary function (rats) and was not intravenously self-administered by heroin-maintained rats or rhesus monkeys. CONCLUSIONS: ITI-333 acts as a potent 5-HT2A receptor antagonist, as well a biased MOP receptor partial agonist with low intrinsic efficacy. ITI-333 mitigates opioid withdrawal/reinstatement, supporting its potential utility as a treatment for OUD.

A review of the pharmacology and clinical profile of lumateperone for the treatment of schizophrenia.

Schizophrenia is associated with a tremendous individual and societal burden. The disease is characterized by a complex set of symptoms including psychosis, hallucinations, delusions and related positive symptoms combined with social function deficits, cognitive disturbances and, often, devastating mood disorder, such as comorbid depression. Management of the disease often requires lifelong pharmacotherapy. However, many pharmacotherapies do not improve all symptoms (e.g., social withdrawal, depression, cognitive deficits) and can be associated with intolerable side effects such as weight gain and metabolic disturbances, motor dysfunction and endocrine dysregulation. Lumateperone (ITI-007, CAPLYTA™) is a novel antipsychotic agent, discovered and developed by Intra-Cellular Therapies, Inc. (ITCI) and approved for treatment of schizophrenia in adults in December 2019. Lumateperone simultaneously modulates serotonin, dopamine and glutamate neurotransmission, three key neurotransmitters implicated in schizophrenia. It achieves efficacy with a favorable safety profile. The clinical development program included 20 clinical trials with over 1900 individuals exposed to lumateperone. The program demonstrated the efficacy for lumateperone in two positive well controlled trials in patients with schizophrenia. The unique pharmacology of lumateperone supports the observed benefits across a wide range of symptoms, including social function and depression, and supports its favorable safety profile. Here, we review the discovery of lumateperone's unique biological effects and its clinical actions in the treatment of schizophrenia.

The Effects of Acute and Chronic Selective Phosphodiesterase 1 Inhibition on Smooth Muscle Cell-Associated Aging Features.

Age-related cardiovascular diseases (CVDs) remain among the leading global causes of death, and vascular smooth muscle cell (VSMC) remodeling plays an essential role in its pathology. Reduced NO-cGMP pathway signaling is a major feature and pathogenic mechanism underlying vasodilator dysfunction. Recently, we identified phosphodiesterase (PDE) 1, an enzyme that hydrolyzes and inactivates the cyclic nucleotides cAMP and cGMP, and thereby provides a potential treatment target for restoring age-related vascular dysfunction due to aging of VSMC. Based on this hypothesis, we here tested the effects of PDE1 inhibition in a model of SMC-specific accelerated aging mice. SMC-KO and their WT littermates received either vehicle or the PDE1 inhibitor lenrispodun for 8 weeks. Vascular function was measured both in vivo (Laser Doppler technique) and ex vivo (organ bath). Moreover, we deployed UV irradiation in cell culture experiments to model accelerated aging in an in vitro situation. SMC-KO mice display a pronounced loss of vasodilator function in the isolated aorta, the cutaneous microvasculature, and mesenteric arteries. Ex vivo, in isolated vascular tissue, we found that PDE1 inhibition with lenrispodun improves vasodilation, while no improvement was observed in isolated aorta taken from mice after chronic treatment in vivo. However, during lenrispodun treatment in vivo, an enhanced microvascular response in association with upregulated cGMP levels was seen. Further, chronic lenrispodun treatment decreased TNF-α and IL-10 plasma levels while the elevated level of IL-6 in SMC-KO mice remained unchanged after treatment. PDE1 and senescence markers, p16 and p21, were increased in both SMC-KO aorta and cultured human VSMC in which DNA was damaged by ultraviolet irradiation. This increase was lowered by chronic lenrispodun. In contrast, lenrispodun increased the level of PDE1A in both situations. In conclusion, we demonstrated that PDE1 inhibition may be therapeutically useful in reversing aspects of age-related VSMC dysfunction by potentiating NO-cGMP signaling, preserving microvascular function, and decreasing senescence. Yet, after chronic treatment, the effects of PDE1 inhibition might be counteracted by the interplay between differential PDE1A and C expression. These results warrant further pharmacodynamic profiling of PDE enzyme regulation during chronic PDE1 inhibitor treatment.

Vascular Ageing Features Caused by Selective DNA Damage in Smooth Muscle Cell.

Persistently unrepaired DNA damage has been identified as a causative factor for vascular ageing. We have previously shown that a defect in the function or expression of the DNA repair endonuclease ERCC1 (excision repair cross complement 1) in mice leads to accelerated, nonatherosclerotic ageing of the vascular system from as early as 8 weeks after birth. Removal of ERCC1 from endothelial alone partly explains this ageing, as shown in endothelial-specific Ercc1 knockout mice. In this study, we determined vascular ageing due to DNA damage in vascular smooth muscle cells, as achieved by smooth muscle-selective genetic removal of ERCC1 DNA repair in mice (SMC-KO: SM22αCre+ Ercc1fl/-). Vascular ageing features in SMC-KO and their wild-type littermates (WT: SM22αCre+ Ercc1fl/+) were examined at the age of 14 weeks and 25 weeks. Both SMC-KO and WT mice were normotensive. Compared to WT, SMC-KO showed a reduced heart rate, fractional shortening, and cardiac output. SMC-KO showed progressive features of nonatherosclerotic vascular ageing as they aged from 14 to 25 weeks. Decreased subcutaneous microvascular dilatation and increased carotid artery stiffness were observed. Vasodilator responses measured in aortic rings in organ baths showed decreased endothelium-dependent and endothelium-independent responses, mostly due to decreased NO-cGMP signaling. NADPH oxidase 2 and phosphodiesterase 1 inhibition improved dilations. SMC-KO mice showed elevated levels of various cytokines that indicate a balance shift in pro- and anti-inflammatory pathways. In conclusion, SMC-KO mice showed a progressive vascular ageing phenotype in resistant and conduit arteries that is associated with cardiac remodeling and contractile dysfunction. The changes induced by DNA damage might be limited to VSMC but eventually affect EC-mediated responses. The fact that NADPH oxidase 2 as wells as phosphodiesterase 1 inhibition restores vasodilation suggests that both decreased NO bioavailability and cGMP degradation play a role in local vascular smooth muscle cell ageing induced by DNA damage.

Discovery of novel alpha7 nicotinic receptor antagonists.

Two distinct families of small molecules were discovered as novel alpha7 nicotinic acetylcholine receptor (nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selectivity for the neuronal alpha7 subtype over other nAChRs and good brain penetration. Neuroprotection was demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as antidote for organophosphorus nerve agent intoxication.

Advanced research on dopamine signaling to develop drugs for the treatment of mental disorders: biochemical and behavioral profiles of phosphodiesterase inhibition in dopaminergic neurotransmission.

Dopamine plays a central role in the regulation of psychomotor functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade and therefore controlled by phosphodiesterases (PDEs). Multiple PDEs with different substrate specificities and subcellular localization are expressed in the striatum, and the functional roles of PDE10A, PDE4, and PDE1B are extensively studied. Biochemical and behavioral profiles of PDE inhibition by selective inhibitors and/or genetic deletion related to dopaminergic neurotransmission are compared among those PDEs. The inhibition of PDE up-regulates cAMP/PKA signaling in three neuronal subtypes, resulting in the stimulation of dopamine synthesis at dopaminergic terminals, the inhibition of dopamine D(2)-receptor signaling in striatopallidal neurons, and the stimulation of dopamine D(1)-receptor signaling in striatonigral neurons. Predominant roles of PDE families or isoforms are implicated in each neuronal subtype: PDE4 at dopaminergic terminals, PDE10A and PDE4 in striatopallidal neurons, and PDE1B in striatonigral neurons. PDE10A and PDE4 inhibition may exhibit D(2) antagonist-like, antipsychotic effects, whereas PDE1B inhibition may exhibit D(1) agonist-like effects in the striatum. Development of PDE isoform-specific inhibitors is essential for better understanding of the function of each PDE isoform and treatment of neuropsychiatric disorders.

Distinct roles of PDE4 and PDE10A in the regulation of cAMP/PKA signaling in the striatum.

Phosphodiesterase (PDE) is a critical regulator of cAMP/protein kinase A (PKA) signaling in cells. Multiple PDEs with different substrate specificities and subcellular localization are expressed in neurons. Dopamine plays a central role in the regulation of motor and cognitive functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade, and therefore controlled by PDE activity. We used in vitro and in vivo biochemical techniques to dissect the roles of PDE4 and PDE10A in dopaminergic neurotransmission in mouse striatum by monitoring the ability of selective PDE inhibitors to regulate phosphorylation of presynaptic [e.g., tyrosine hydroxylase (TH)] and postsynaptic [e.g., dopamine- and cAMP-regulated phosphoprotein of M(r) 32 kDa (DARPP-32)] PKA substrates. The PDE4 inhibitor, rolipram, induced a large increase in TH Ser40 phosphorylation at dopaminergic terminals that was associated with a commensurate increase in dopamine synthesis and turnover in striatum in vivo. Rolipram induced a small increase in DARPP-32 Thr34 phosphorylation preferentially in striatopallidal neurons by activating adenosine A(2A) receptor signaling in striatum. In contrast, the PDE10A inhibitor, papaverine, had no effect on TH phosphorylation or dopamine turnover, but instead robustly increased DARPP-32 Thr34 and GluR1 Ser845 phosphorylation in striatal neurons. Inhibition of PDE10A by papaverine activated cAMP/PKA signaling in both striatonigral and striatopallidal neurons, resulting in potentiation of dopamine D(1) receptor signaling and inhibition of dopamine D(2) receptor signaling. These biochemical results are supported by immunohistochemical data demonstrating differential localization of PDE10A and PDE4 in striatum. These data underscore the importance of individual brain-enriched cyclic-nucleotide PDE isoforms as therapeutic targets for neuropsychiatric and neurodegenerative disorders affecting dopamine neurotransmission.

General anesthetics selectively modulate glutamatergic and dopaminergic signaling via site-specific phosphorylation in vivo.

Isoflurane, propofol and ketamine are representative general anesthetics with distinct molecular mechanisms of action that have neuroprotective properties in models of excitotoxic ischemic damage. We characterized the effects of these agents on neuronal glutamate and dopamine signaling by profiling drug-induced changes in brain intracellular protein phosphorylation in vivo to test the hypothesis that they affect common downstream effectors. Anesthetic-treated and control mice were killed instantly by focused microwave irradiation, frontal cortex and striatum were removed, and the phosphorylation profile of specific neuronal signaling proteins was analyzed by immunoblotting with a panel of phospho-specific antibodies. At anesthetic doses that produced loss of righting reflex, isoflurane, propofol, and ketamine all reduced phosphorylation of the activating residue T183 of ERK2 (but not of ERK1); S897 of the NR1 NMDA receptor subunit; and S831 (but not S845) of the GluR1 AMPA receptor subunit in cerebral cortex. At sub-anesthetic doses, these drugs only reduced phosphorylation of ERK2. Isoflurane and ketamine also reduced phosphorylation of spinophilin at S94, but oppositely regulated phosphorylation of presynaptic (tyrosine hydroxylase) and postsynaptic (DARPP-32) markers of dopaminergic neurotransmission in striatum. These data reveal both shared and agent-specific actions of CNS depressant drugs on critical intracellular protein phosphorylation signaling pathways that integrate multiple second messenger systems. Reduced phosphorylation of ionotropic glutamate receptors by all three anesthetics indicates depression of normal glutamatergic synaptic transmission and reduced potential excitotoxicity. This novel approach indicates a role for phosphorylation-mediated down-regulation of glutamatergic synaptic transmission by general anesthetics and identifies specific in vivo targets for focused evaluation of anesthetic mechanisms.

Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling.

Adenosine A(2A) receptors are predominantly expressed in the dendrites of enkephalin-positive gamma-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A(2A) receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A(2A) receptor signaling in the striatum. Specifically, the adenosine A(2A) receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A(2A) receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-d-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, M(r) 32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca(2+)/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A(2A) receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680, whereas presynaptic PKA substrates were unresponsive to this agent, consistent with the postsynaptic localization of adenosine A(2A) receptors. Finally, the phosphorylation state of these proteins was further assessed in vivo by systemic administration of caffeine.

PDE Inhibitors for the Treatment of Schizophrenia.

Schizophrenia is a pervasive neuropsychiatric disorder affecting over 1% of the world's population. Dopamine system dysfunction is strongly implicated in the etiology of schizophrenia. Data support the long-standing concept of schizophrenia as a disease characterized by hyperactivity within midbrain (striatal D2) dopamine systems. In addition, there is now considerable evidence that glutamate neurotransmission, mediated through NMDA-type receptors, is deficient in patients with schizophrenia and that hypoactivity in cortical dopamine and glutamate pathways is a key feature of this serious mental disorder. While current antipsychotic medications-with a common mechanism involving dopamine D2 receptor antagonism or pre-synaptic partial agonism-adequately address positive symptoms of the disease, such as the acute hallucinations and delusions, they fail to substantially improve negative features, such as social isolation, and can further compromise poor cognitive function associated with schizophrenia. In fact, cognitive impairment is a core feature of schizophrenia. The treatment of cognitive impairment and other residual symptoms associated with schizophrenia, therefore, remains a significant unmet medical need. With current cell-surface receptor-based pharmacology falling short of addressing these core cognitive symptoms, more recent approaches to treatment development have focused on processes within the cell. In this review, we discuss the importance of cyclic nucleotide (cNT) phosphodiestereases (PDEs)-intracellular enzymes that control the activity of key second messenger signaling pathways in the brain-which have been proposed as targets for new schizophrenia therapies. We also discuss the challenge facing those developing drugs to target specific PDE enzymes involved in psychopathology without involving other systems that produce concomitant side effects.

Dopamine Targeting Drugs for the Treatment of Schizophrenia: Past, Present and Future.

Schizophrenia is a chronic and debilitating neuropsychiatric disorder affecting approximately 1% of the world's population. This disease is associated with considerable morbidity placing a major financial burden on society. Antipsychotics have been the mainstay of the pharmacological treatment of schizophrenia for decades. The traditional typical and atypical antipsychotics demonstrate clinical efficacy in treating positive symptoms, such as hallucinations and delusions, while are largely ineffective and may worsen negative symptoms, such as blunted affect and social withdrawal, as well as cognitive function. The inability to treat these latter symptoms may contribute to social function impairment associated with schizophrenia. The dysfunction of multiple neurotransmitter systems in schizophrenia suggests that drugs selectively targeting one neurotransmission pathway are unlikely to meet all the therapeutic needs of this heterogeneous disorder. Often, however, the unintentional engagement of multiple pharmacological targets or even the excessive engagement of intended pharmacological targets can lead to undesired consequences and poor tolerability. In this article, we will review marketed typical and atypical antipsychotics and new therapeutic agents targeting dopamine receptors and other neurotransmitters for the treatment of schizophrenia. Representative typical and atypical antipsychotic drugs and new investigational drug candidates will be systematically reviewed and compared by reviewing structure-activity relationships, pharmacokinetic properties, drug metabolism and safety, pharmacological properties, preclinical data in animal models, clinical outcomes and associated side effects.

Preclinical profile of ITI-214, an inhibitor of phosphodiesterase 1, for enhancement of memory performance in rats.

RATIONALE: Therapeutic agents for memory enhancement in psychiatric disorders, such as schizophrenia, are urgently needed. OBJECTIVE: The aim of this study is to characterize the preclinical profile of ITI-214, a potent inhibitor of phosphodiesterase 1 (PDE1). METHODS: ITI-214 was assayed for inhibition of PDE1 versus other PDE enzyme families using recombinant human PDE enzymes and for off-target binding to 70 substrates (General SEP II diversity panel; Caliper Life Sciences). Effects of ITI-214 (0.1-10 mg/kg, po) on memory performance were assayed in rats using the novel object recognition (NOR) paradigm, with drug given at specified time points prior to or following exposure to objects in an open field. ITI-214 was evaluated for potential drug-drug interaction with risperidone in rats using conditioned avoidance response (CAR) and pharmacokinetic assessments. RESULTS: ITI-214 inhibited PDE1A (K i = 33 pmol) with >1000-fold selectivity for the nearest other PDE family (PDE4D) and displayed minimal off-target binding interactions in a 70-substrate selectivity profile. By using specific timing of oral ITI-214 administration, it was demonstrated in the NOR that ITI-214 is able to enhance acquisition, consolidation, and retrieval memory processes. All memory effects were in the absence of effects on exploratory behavior. ITI-214 did not disrupt the risperidone pharmacokinetic profile or effects in CAR. CONCLUSIONS: ITI-214 improved the memory processes of acquisition, consolidation, and retrieval across a broad dose range (0.1-10 mg/kg, po) without disrupting the antipsychotic-like activity of a clinical antipsychotic medication, specifically risperidone. Clinical development of ITI-214 is currently in progress.

Discovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric Diseases.

A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compounds as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochemical and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clinical candidate, (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate (ITI-214), which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.

Phosphodiesterase 1B knock-out mice exhibit exaggerated locomotor hyperactivity and DARPP-32 phosphorylation in response to dopamine agonists and display impaired spatial learning.

Using homologous recombination, we generated mice lacking phosphodiesterase-mediated (PDE1B) cyclic nucleotide-hydrolyzing activity. PDE1B(-/-) mice showed exaggerated hyperactivity after acute D-methamphetamine administration. Striatal slices from PDE1B(-/-) mice exhibited increased levels of phospho-Thr34 DARPP-32 and phospho-Ser845 GluR1 after dopamine D1 receptor agonist or forskolin stimulation. PDE1B(-/-) and PDE1B(+/-) mice demonstrated Morris maze spatial-learning deficits. These results indicate that enhancement of cyclic nucleotide signaling by inactivation of PDE1B-mediated cyclic nucleotide hydrolysis plays a significant role in dopaminergic function through the DARPP-32 and related transduction pathways.

Functional profile of a novel modulator of serotonin, dopamine, and glutamate neurotransmission.

RATIONALE: Schizophrenia remains among the most prevalent neuropsychiatric disorders, and current treatment options are accompanied by unwanted side effects. New treatments that better address core features of the disease with minimal side effects are needed. OBJECTIVES: As a new therapeutic approach, 1-(4-fluoro-phenyl)-4-((6bR, 10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3',4':4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-butan-1-one (ITI-007) is currently in human clinical trials for the treatment of schizophrenia. Here, we characterize the preclinical functional activity of ITI-007. RESULTS: ITI-007 is a potent 5-HT2A receptor ligand (K i = 0.5 nM) with strong affinity for dopamine (DA) D2 receptors (K i = 32 nM) and the serotonin transporter (SERT) (K i = 62 nM) but negligible binding to receptors (e.g., H1 histaminergic, 5-HT2C, and muscarinic) associated with cognitive and metabolic side effects of antipsychotic drugs. In vivo it is a 5-HT2A antagonist, blocking (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI)-induced headtwitch in mice with an inhibitory dose 50 (ID50) = 0.09 mg/kg, per oral (p.o.), and has dual properties at D2 receptors, acting as a postsynaptic D2 receptor antagonist to block D-amphetamine hydrochloride (D-AMPH) hyperlocomotion (ID50 = 0.95 mg/kg, p.o.), yet acting as a partial agonist at presynaptic striatal D2 receptors in assays measuring striatal DA neurotransmission. Further, in microdialysis studies, this compound significantly and preferentially enhances mesocortical DA release. At doses relevant for antipsychotic activity in rodents, ITI-007 has no demonstrable cataleptogenic activity. ITI-007 indirectly modulates glutamatergic neurotransmission by increasing phosphorylation of GluN2B-type N-methyl-D-aspartate (NMDA) receptors and preferentially increases phosphorylation of glycogen synthase kinase 3ß (GSK-3ß) in mesolimbic/mesocortical dopamine systems. CONCLUSION: The combination of in vitro and in vivo activities of this compound support its development for the treatment of schizophrenia and other psychiatric and neurologic disorders.

Regulation of AMPA receptor dephosphorylation by glutamate receptor agonists.

Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 at Ser(845) enhances AMPA channel activity. This study demonstrates that Ser(845) is rapidly dephosphorylated upon AMPA receptor activation in nucleus accumbens slices. AMPA-induced dephosphorylation at Ser(845) was blocked by CNQX, an AMPA receptor antagonist, by nifedipine, an L-type Ca(2+) channel antagonist, or by cyclosporin A, a calcineurin inhibitor. N-methyl-D-aspartate (NMDA) treatment also decreased phosphorylation of Ser(845), an effect that was blocked by MK-801, an NMDA receptor antagonist, but not by nifedipine. Accumbens neurons are enriched for dopamine- and cyclic AMP (cAMP)-regulated phosphoprotein, Mr 32,000 (DARPP-32), a potent inhibitor of protein phosphatase 1 (PP1) when phosphorylated by PKA (at Thr(34)). We tested the hypothesis that the AMPA/KA or NMDA-stimulated dephosphorylation of DARPP-32 via calcineurin, leading to increased PP1 activity and dephosphorylation of GluR1. AMPA or NMDA treatment decreased phospho-Thr(34)-DARPP-32 levels, effects that were blocked by receptor antagonists, or cyclosporin A. However, dephosphorylation of Ser(845) mediated by AMPA or NMDA receptors was unaffected in DARPP-32/inhibitor-1 knockout mice. These data suggest that AMPA- or NMDA-induced dephosphorylation of GluR1 at Ser(845) occurs by a mechanism that is independent of DARPP-32 and PP1, but involves activation of calcineurin. Thus, Ca(2+)-dependent dephosphorylation of GluR1 may serve as a negative feedback mechanism for the regulation of AMPA receptor activity in neurons.