Effects of the phosphodiesterase 2 inhibitor BI 474121 on central nervous system cyclic guanosine monophosphate concentrations: Translational studies.

AIMS: Phosphodiesterase 2 (PDE2) regulates intracellular cyclic adenosine monophosphate and guanosine monophosphate (cAMP/cGMP) levels, which contribute to processes crucial for learning and memory. BI 474121, a potent and selective PDE2 inhibitor, is in development for treating cognitive impairment associated with schizophrenia. METHODS: The effects of BI 474121 on cGMP concentrations were first assessed in rat cerebrospinal fluid (CSF) to demonstrate central nervous system (CNS) and functional target engagement. Next, a Phase I study in healthy participants assessed the pharmacokinetics of BI 474121 in CSF vs. plasma, the pharmacodynamics of BI 474121 by measuring cGMP concentrations in the CSF, and the safety of BI 474121. RESULTS: In rats, BI 474121 was associated with a dose-dependent increase (71% at the highest dose tested [3.0 mg kg-1]) in cGMP levels in the CSF relative to vehicle (P < 0.001). In healthy participants, the maximum-measured concentration CSF-to-plasma ratio for BI 474121 exposure was similar following single oral doses of BI 474121 2.5, 10, 20 and 40 mg (dose-adjusted geometric mean: 8.96% overall). BI 474121 2.5-40 mg administration in healthy participants also increased cGMP levels in CSF (maximum exposure-related change from baseline ratio, BI 474121: 1.44-2.20 vs. placebo: 1.26). The most common treatment-emergent adverse event (AE) was mild-to-moderate post-lumbar puncture syndrome, which resolved with standard treatment. No AEs of special interest were observed. CONCLUSIONS: BI 474121 crosses the blood-brain barrier to inhibit PDE2, supporting cGMP as a translational marker to monitor CNS target engagement. These findings promote further clinical development of BI 474121. CLINICALTRIALS: gov number (NCT04672954).

Development of the novel GlyT1 inhibitor, iclepertin (BI 425809), for the treatment of cognitive impairment associated with schizophrenia.

Schizophrenia is a psychiatric disorder characterised by symptoms in three domains: positive (e.g. delusions, hallucinations), negative (e.g. social withdrawal, lack of motivation) and cognitive (e.g. working memory and executive function impairment). Cognitive impairment associated with schizophrenia (CIAS) is a major burden for patients and negatively impacts many aspects of a patient's life. Antipsychotics are the standard-of-care treatment for schizophrenia but only address positive symptoms. So far there are no approved pharmacotherapies for the treatment of CIAS. Iclepertin (BI 425809) is a novel, potent and selective glycine transporter 1 (GlyT1) inhibitor, under development by Boehringer Ingelheim for the treatment of CIAS. Phase I studies have shown it to be safe and well tolerated in healthy volunteers, and central target engagement (inhibition of GlyT1) was achieved in a dose-dependent manner from 5 to 50 mg in healthy volunteers. A Phase II study has demonstrated that iclepertin is safe and well tolerated in patients with schizophrenia and improves cognition at doses of 10 mg and 25 mg. Phase III studies are ongoing to confirm these initial positive safety and efficacy findings with the 10 mg dose, and if successful, iclepertin could become the first approved pharmacotherapy used to treat CIAS.

Effects of the Glycine Transporter-1 Inhibitor Iclepertin (BI 425809) on Sensory Processing, Neural Network Function, and Cognition in Animal Models Related to Schizophrenia.

N-methyl-D-aspartate (NMDA) receptor hypofunction leading to neural network dysfunction is thought to play an important role in the pathophysiology of cognitive impairment associated with schizophrenia (CIAS). Increasing extracellular concentrations of the NMDA receptor co-agonist glycine through inhibition of glycine transporter-1 (GlyT1) has the potential to treat CIAS by improving cortical network function through enhanced glutamatergic signaling. Indeed, the novel GlyT1 inhibitor iclepertin (BI 425809) improved cognition in a recent clinical study in patients with schizophrenia. The present study tested the ability of iclepertin to reverse deficits in auditory sensory processing and cortical network function induced by the uncompetetive NMDA receptor antagonist, MK-801, using electroencephalography (EEG) to measure auditory event-related potentials (AERPs) and 40 Hz auditory steady-state response (ASSR). In addition, improvements in memory performance with iclepertin were evaluated using the T-maze spontaneous alternation test in MK-801-treated mice and the social recognition test in naïve rats. Iclepertin reversed MK-801-induced deficits in the AERP readouts N1 amplitude and N1 gating, as well as reversing deficits in 40 Hz ASSR power and intertrial coherence. Additionally, iclepertin significantly attenuated an MK-801-induced increase in basal gamma power. Furthermore, iclepertin reversed MK-801-induced working memory deficits in mice and improved social recognition memory performance in rats. Overall, this study demonstrates that inhibition of GlyT1 is sufficient to attenuate MK-801-induced deficits in translatable EEG parameters relevant to schizophrenia. Moreover, iclepertin showed memory-enhancing effects in rodent cognition tasks, further demonstrating the potential for GlyT1 inhibition to treat CIAS. SIGNIFICANCE STATEMENT: Despite the significant patient burden caused by cognitive impairment associated with schizophrenia, there are currently no approved pharmacotherapies. In this preclinical study, the novel glycine transporter inhibitor iclepertin (BI 425809) reversed sensory processing deficits and neural network dysfunction evoked by inhibition of N-methyl-D-aspartate receptors and enhanced working memory performance and social recognition in rodents. These findings support previous clinical evidence for the procognitive effects of iclepertin.

Preclinical validation of the micropipette-guided drug administration (MDA) method in the maternal immune activation model of neurodevelopmental disorders.

Pharmacological treatments in laboratory rodents remain a cornerstone of preclinical psychopharmacological research and drug development. There are numerous ways in which acute or chronic pharmacological treatments can be implemented, with each method having certain advantages and drawbacks. Here, we describe and validate a novel treatment method in mice, which we refer to as the micropipette-guided drug administration (MDA) procedure. This administration method is based on a sweetened condensed milk solution as a vehicle for pharmacological substances, which motivates the animals to consume vehicle and/or drug solutions voluntarily in the presence of the experimenter. In a proof-of-concept study, we show that the pharmacokinetic profiles of the atypical antipsychotic drug, risperidone, were similar whether administered via the MDA procedure or via the conventional oral gavage method. Unlike the latter, however, MDA did not induce the stress hormone, corticosterone. Furthermore, we assessed the suitability and validity of the MDA method in a mouse model of maternal immune activation, which is frequently used as a model of immune-mediated neurodevelopmental disorders. Using this model, we found that chronic treatment (>4 weeks, once per day) with risperidone via MDA led to a dose-dependent mitigation of MIA-induced social interaction deficits and amphetamine hypersensitivity. Taken together, the MDA procedure described herein represents a novel pharmacological administration method for per os treatments in mice that is easy to implement, cost effective, non-invasive, and less stressful for the animals than conventional oral gavage methods.

The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

Overexpression of Protein Kinase Mζ in the Hippocampus Enhances Long-Term Potentiation and Long-Term Contextual But Not Cued Fear Memory in Rats.

The persistently active protein kinase Mζ (PKMζ) has been found to be involved in the formation and maintenance of long-term memory. Most of the studies investigating PKMζ, however, have used either putatively unselective inhibitors or conventional knock-out animal models in which compensatory mechanisms may occur. Here, we overexpressed an active form of PKMζ in rat hippocampus, a structure highly involved in memory formation, and embedded in several neural networks. We investigated PKMζ's influence on synaptic plasticity using electrophysiological recordings of basal transmission, paired pulse facilitation, and LTP and combined this with behavioral cognitive experiments addressing formation and retention of both contextual memory during aversive conditioning and spatial memory during spontaneous exploration. We demonstrate that hippocampal slices overexpressing PKMζ show enhanced basal transmission, suggesting a potential role of PKMζ in postsynaptic AMPAR trafficking. Moreover, the PKMζ-overexpressing slices augmented LTP and this effect was not abolished by protein-synthesis blockers, indicating that PKMζ induces enhanced LTP formation in a protein-synthesis-independent manner. In addition, we found selectively enhanced long-term memory for contextual but not cued fear memory, underlining the theory of the hippocampus' involvement in the contextual aspect of aversive reinforced tasks. Memory for spatial orientation during spontaneous exploration remained unaltered, suggesting that PKMζ may not affect the neural circuits underlying spontaneous tasks that are different from aversive tasks. In this study, using an overexpression strategy as opposed to an inhibitor-based approach, we demonstrate an important modulatory role of PKMζ in synaptic plasticity and selective memory processing. SIGNIFICANCE STATEMENT: Most of the literature investigating protein kinase Mζ (PKMζ) used inhibitors with selectivity that has been called into question or conventional knock-out animal models in which compensatory mechanisms may occur. To avoid these issues, some studies have been done using viral overexpression of PKMζ in different brain structures to show cognitive enhancement. However, electrophysiological experiments were exclusively done in knock-out models or inhibitory studies to show depletion of LTP. There was no study showing the effect of PKMζ overexpression in the hippocampus on behavior and LTP experiments. To our knowledge, this is the first study to combine these aspects with the result of enhanced memory for contextual fear memory and to show enhanced LTP in hippocampal slices overexpressing PKMζ.

Pharmaceutical Characterization of Tropomyosin Receptor Kinase B-Agonistic Antibodies on Human Induced Pluripotent Stem (hiPS) Cell-Derived Neurons.

Brain-derived neurotrophic factor (BDNF) is a central modulator of neuronal development and synaptic plasticity in the central nervous system. This renders the BDNF-modulated tropomyosin receptor kinase B (TrkB) a promising drug target to treat synaptic dysfunctions. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the so-called GRINCH neurons were derived from human-induced pluripotent stem cells. These GRINCH neurons were used as model cells for pharmacologic profiling of two TrkB-agonistic antibodies, hereafter referred to as AB2 and AB20 In next-generation sequencing studies, AB2 and AB20 stimulated transcriptional changes, which extensively overlapped with BDNF-driven transcriptional modulation. In regard to TrkB phosphorylation, both AB2 and AB20 were only about half as efficacious as BDNF; however, with respect to the TrkB downstream signaling, AB2 and AB20 displayed increased efficacy values, providing a stimulation at least comparable to BDNF in respect to VGF transcription, as well as of AKT and cAMP response element-binding protein phosphorylation. In a complex structure of the TrkB-d5 domain with AB20, determined by X-ray crystallography, the AB20 binding site was found to be allosteric in regard to the BDNF binding site, whereas AB2 was known to act orthosterically with BDNF. In agreement with this finding, AB2 and AB20 acted synergistically at greater concentrations to drive TrkB phosphorylation. Although TrkB downstream signaling declined faster after pulse stimulation with AB20 than with AB2, AB20 restimulated TrkB phosphorylation more efficiently than AB2. In conclusion, both antibodies displayed some limitations and some benefits in regard to future applications as therapeutic agents.

Age-related synaptic dysfunction in Tg2576 mice starts as a failure in early long-term potentiation which develops into a full abolishment of late long-term potentiation.

Tg2576 mice are widely used to study amyloid-dependent synaptic dysfunction related to Alzheimer's disease. However, conflicting data have been reported for these mice with regard to basal transmission as well as the in vitro correlate of memory, long-term potentiation (LTP). Some studies show clear impairments, whereas others report no deficiency. The present study uses hippocampal slices from 3-, 10-, and 15-month-old wild-type (WT) and Tg2576 mice to evaluate synaptic function in each group, including experiments to investigate basal synaptic transmission, short- and long-term plasticity by inducing paired-pulse facilitation, and both early and late LTP. We show that synaptic function remains intact in hippocampal slices from Tg2576 mice at 3 months of age. However, both early and late LTP decline progressively during aging in these mice. This deterioration of synaptic plasticity starts affecting early LTP, ultimately leading to the abolishment of both forms of LTP in 15-month-old animals. In comparison, WT littermates display normal synaptic parameters during aging. Additional pharmacological investigation into the involvement of NMDA receptors and L-type voltage-gated calcium channels in LTP suggests a distinct mechanism of induction among age groups, demonstrating that both early and late LTP are differentially affected by these channels in Tg2576 mice during aging.

Touchscreen tasks in mice to demonstrate differences between hippocampal and striatal functions.

In mammals, hippocampal and striatal regions are engaged in separable cognitive processes usually assessed through species-specific paradigms. To reconcile cognitive testing among species, translational advantages of the touchscreen-based automated method have been recently promoted. However, it remains undetermined whether similar neural substrates would be involved in such behavioral tasks both in humans and rodents. To address this question, the effects of hippocampal or dorso-striatal fiber-sparing lesions were first assessed in mice through a battery of tasks (experiment A) comprising the acquisition of two touchscreen paradigms, the Paired Associates Learning (dPAL) and Visuo-Motor Conditional Learning (VMCL) tasks, and a more classical T-maze alternation task. Additionally, we sought to determine whether post-acquisition hippocampal lesions would alter memory retrieval in the dPAL task (experiment B). Pre-training lesions of dorsal striatum caused major impairments in all paradigms. In contrast, pre-training hippocampal lesions disrupted the performance of animals trained in the T-maze assay, but spared the acquisition in touchscreen tasks. Nonetheless, post-training hippocampal lesions severely impacted the recall of the previously learned dPAL task. Altogether, our data show that, after having demonstrated their potential in genetically modified mice, touchscreens also reveal perfectly adapted to taxing functional implications of brain structures in mice by means of lesion approaches. Unlike its human counterpart requiring an intact hippocampus, the acquisition of the dPAL task requires the integrity of the dorsal striatum in mice. The hippocampus only later intervenes, when acquired information needs to be retrieved. Touchscreen assays may therefore be suited to study striatal- or hippocampal-dependent forms of learnings in mice.

Inhibition of PDE2A, but not PDE9A, modulates presynaptic short-term plasticity measured by paired-pulse facilitation in the CA1 region of the hippocampus.

Phosphodiesterase (PDE) inhibitors are currently considered promising therapeutic targets for treatment of cognitive impairment in diseases such as Schizophrenia and Alzheimer's disease. Inhibitors of PDE2A and PDE9A have emerged as potential candidates shown to improve synaptic plasticity and memory function in animals. However, the functional relevance of their putative different localization in the neuron is not understood. Thus, this study aims at elucidating potential presynaptic effects of PDE2A inhibition in comparison to the inhibition of PDE9A. For this purpose, we used paired-pulse facilitation (PPF), a model of short-term synaptic plasticity related to presynaptic function. First, we performed a series of experiments to validate the model in acute rat hippocampal slices using several reference substances including calcium channel blockers, glutamatergic receptor antagonists, and GPCR agonists. Second, we analysed the effect of PDE2A and PDE9A inhibition and their role regulating the influence that the second messengers cAMP and cGMP exert on basal transmission. Our results show that the interplay between the adenylyl cyclase activator forskolin, the soluble guanylyl cyclase activator BAY 41-8543 and the PDE2A inhibitor PF-999 reveals a primarily presynaptic mechanism of action of PDE2A inhibition. On the contrary, inhibition of PDE9A did not alter PPF under similar conditions. In conclusion, these data provide new evidence supporting a role of PDE2A modulating short-term synaptic plasticity. Moreover, this function of PDE2A is suggested to rely on an active modulation of the cAMP hydrolysis as a response to changes in cGMP levels at the presynaptic level.

Neuroimaging Biomarkers for Drug Discovery and Development in Schizophrenia.

Schizophrenia is a chronic mental illness that affects up to 1% of the population. While efficacious therapies are available for positive symptoms, effective treatment of cognitive and negative symptoms remains an unmet need after decades of research. New developments in the field of neuroimaging are accelerating our knowledge gain regarding the underlying pathophysiology of symptoms in schizophrenia and psychosis spectrum disorders, inspiring new targets for drug development. However, no validated and qualified biomarkers are currently available to support the development of new therapeutics. This review summarizes the current use of neuroimaging technology in clinical drug development for psychotic disorders. As exemplified by drug development programs that target NMDA receptor hypofunction, neuroimaging results play a critical role in target discovery and establishing target engagement and dose selection. Furthermore, pharmacological neuroimaging may provide response biomarkers that allow for early decision making in proof-of-concept studies that leverage pharmacological challenge models in healthy volunteers. That said, while response and predictive biomarkers are starting to be evaluated in patient populations, they continue to play a limited role. Novel approaches to neuroimaging data acquisition and analysis may aid the establishment of biomarkers that are predictive at the individual level in the future. Nevertheless, various gaps in knowledge need to be addressed and biomarkers need to be validated to establish them as "fit for purpose" in drug development.

Neural effects of dopaminergic compounds revealed by multi-site electrophysiology and interpretable machine-learning.

Background: Neuropsychopharmacological compounds may exert complex brain-wide effects due to an anatomically and genetically broad expression of their molecular targets and indirect effects via interconnected brain circuits. Electrophysiological measurements in multiple brain regions using electroencephalography (EEG) or local field potential (LFP) depth-electrodes may record fingerprints of such pharmacologically-induced changes in local activity and interregional connectivity (pEEG/pLFP). However, in order to reveal such patterns comprehensively and potentially derive mechanisms of therapeutic pharmacological effects, both activity and connectivity have to be estimated for many brain regions. This entails the problem that hundreds of electrophysiological parameters are derived from a typically small number of subjects, making frequentist statistics ill-suited for their analysis. Methods: We here present an optimized interpretable machine-learning (ML) approach which relies on predictive power in individual recording sequences to extract and quantify the robustness of compound-induced neural changes from multi-site recordings using Shapley additive explanations (SHAP) values. To evaluate this approach, we recorded LFPs in mediodorsal thalamus (MD), prefrontal cortex (PFC), dorsal hippocampus (CA1 and CA3), and ventral hippocampus (vHC) of mice after application of amphetamine or of the dopaminergic antagonists clozapine, raclopride, or SCH23390, for which effects on directed neural communication between those brain structures were so far unknown. Results: Our approach identified complex patterns of neurophysiological changes induced by each of these compounds, which were reproducible across time intervals, doses (where tested), and ML algorithms. We found, for example, that the action of clozapine in the analysed cortico-thalamo-hippocampal network entails a larger share of D1-as opposed to D2-receptor induced effects, and that the D2-antagonist raclopride reconfigures connectivity in the delta-frequency band. Furthermore, the effects of amphetamine and clozapine were surprisingly similar in terms of decreasing thalamic input to PFC and vHC, and vHC activity, whereas an increase of dorsal-hippocampal communication and of thalamic activity distinguished amphetamine from all tested anti-dopaminergic drugs. Conclusion: Our study suggests that communication from the dorsal hippocampus scales proportionally with dopamine receptor activation and demonstrates, more generally, the high complexity of neuropharmacological effects on the circuit level. We envision that the presented approach can aid in the standardization and improved data extraction in pEEG/pLFP-studies.

Globular and protofibrillar aß aggregates impair neurotransmission by different mechanisms.

In Alzheimer's disease, substantial evidence indicates the causative role of soluble amyloid ß (Aß) aggregates. Although a variety of Aß assemblies have been described, the debate about their individual relevance is still ongoing. One critical issue hampering this debate is the use of different methods for the characterization of endogenous and synthetic peptide and their intrinsic limitations for distinguishing Aß aggregates. Here, we used different protocols for the establishment of prefibrillar Aß assemblies with varying morphologies and sizes and compared them in a head-to-head fashion. Aggregation was characterized via the monomeric peptide over time until spheroidal, protofibrillar, or fibrillar Aß aggregates were predominant. It could be shown that a change in the ionic environment induced a structural rearrangement, which consequently confounds the delineation of a measured neurotoxicity toward a distinct Aß assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aß assemblies, based on the stability of the applied Aß aggregates in these settings. In contrast to monomeric or fibrillar Aß, different prefibrillar Aß aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aß aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aß aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aß assemblies resulted in a time-dependent suppression of basal neurotransmission, indicative of a mechanistic switch in synaptic impairment. Thus, we emphasize the importance of addressing the metastability of prefacto characterized Aß aggregates in assigning a biological effect.

Effects of the mGlu2/3 receptor agonist LY379268 on two models of disturbed auditory evoked brain oscillations in mice.

Cognitive impairment is a core feature of schizophrenia and is poorly addressed by currently available medication. This is partly because the underlying circuits are insufficiently understood, and available animal models for brain dysfunction do not adequately mimic human pathology. To improve the translatability of animal studies and complement behavioral data, EEG measurements are being increasingly used in preclinical research. Brain oscillations are similar across species and can be impaired via several means. In this study, we used two approaches to impair early sensory processing and cortical oscillations in mice: a pharmacological model targeting NMDA receptor function in the whole brain via systemic MK-801 application and an optogenetic model targeting parvalbumin-positive (PV+) interneurons locally in the medial prefrontal cortex (mPFC). We evoked brain activity using auditory stimulation, a tool with high translatability from mouse to human. We then investigated the effect of LY379268, an agonist of mGlu2/3 receptors, a potential therapeutic target for schizophrenia, on single neuron and EEG responses. LY379268 was able to rescue MK-801-induced deficits for a variety of clinically relevant early sensory EEG biomarkers. Single neuron recordings revealed a strong effect of LY379268 on the signal-to-noise ratio during auditory stimulation and optogenetic inhibition of PV+ interneurons. Our results contribute to a better understanding of how group II metabotropic glutamate receptors modulate neuronal population and network activity under sensory stimulation while challenged pharmacologically or optogenetically.

Quantitative electroencephalography parameters as neurophysiological biomarkers of schizophrenia-related deficits: A Phase II substudy of patients treated with iclepertin (BI 425809).

Patients with schizophrenia experience cognitive impairment related to neural network dysfunction and deficits in sensory processing. These deficits are thought to be caused by N-methyl-D-aspartate receptor hypofunction and can be assessed in patient populations using electroencephalography (EEG). This substudy from a Phase II, randomized, double-blind, placebo-controlled, parallel-group study investigating the safety and efficacy of the novel glycine transporter-1 inhibitor, iclepertin (BI 425809), assessed the potential of EEG parameters as clinically relevant biomarkers of schizophrenia and response to iclepertin treatment. Eligible patients were randomized to once-daily add-on iclepertin (2, 5, 10, or 25 mg), or placebo (1:1:1:1:2 ratio) for 12 weeks. EEG data were recorded from a subgroup of patients (n = 79) at baseline and end of treatment (EoT). EEG parameters of interest were mismatch negativity (MMN), auditory steady-state response (ASSR), and resting state gamma power, and their correlations with clinical assessments. At baseline, MMN and ASSR exhibited consistent correlations with clinical assessments, indicating their potential value as neurophysiological biomarkers of schizophrenia-related deficits. ASSR measures were positively correlated to the MATRICS Consensus Cognitive Battery overall and neurocognitive composite scores; MMN amplitude was positively correlated with Positive and Negative Syndrome Scale scores. However, correlations between change from baseline (CfB) at EoT in clinical assessments, and baseline or CfB at EoT for EEG parameters were modest and inconsistent between dose groups, which might indicate low potential of these EEG parameters as predictive and treatment response biomarkers. Further methodological refinement is needed to establish EEG parameters as useful drug development tools for schizophrenia.

Pro-cognitive effects of the GlyT1 inhibitor Bitopertin in rodents.

N-methyl-D-aspartate-receptor (NMDAR) hypofunction contributes to cognitive impairments in neuropsychiatric disorders such as schizophrenia. Reduced NMDAR signalling can be enhanced by increasing extracellular levels of the NMDAR co-agonist glycine through inhibition of its transporter (GlyT1). This may be one option to improve cognitive deficits or negative symptoms of schizophrenia. In this preclinical study, we aimed at investigating effects of the GlyT1-inhibitor Bitopertin on cognition, social function and motivation. Central target engagement was assessed by Bitopertin-induced changes in glycine levels in rats' cerebrospinal fluid (CSF) and prefrontal cortex (PFC). Behavioural effects of Bitopertin on recognition memory were evaluated using a social-recognition test in rats, while its effects on working memory were tested in a spontaneous alternation task in mice pre-treated with the NMDAR antagonist MK-801. Bitopertin was further investigated using a social interaction test in rats pre-treated with the NMDAR antagonist phencyclidine, and the effects on effortful motivation were explored in progressive ratio tasks in rats. Results show that Bitopertin increased glycine levels in CSF and PFC. Moreover, it enhanced recognition memory and reduced MK-801-induced working memory deficits. By contrast, Bitopertin had no significant effects on PCP-induced social interaction deficits, and it did not alter effort-related responding. Collectively, our data demonstrate that GlyT1 inhibition by Bitopertin increased CSF and extracellular glycine levels and advocated for pro-cognitive effects of GlyT1 inhibition both in intact and NMDAR antagonists-pre-treated rodents. Together, these findings support the use of GlyT1-inhibitors for the treatment of cognitive symptoms in pathologies characterized by NMDR hypofunction, such as schizophrenia.

Increasing the Excitatory Drive Rescues Excitatory/Inhibitory Imbalance and Mismatch Negativity Deficit Caused by Parvalbumin Specific GluA1 Deletion.

Disturbance in synaptic excitatory and inhibitory (E/I) transmission in the prefrontal cortex is considered a critical factor for cognitive dysfunction, a core symptom in schizophrenia. However, the cortical network pathophysiology induced by E/I imbalance is not well characterized, and an effective therapeutic strategy is lacking. In this study, we simulated imbalanced cortical network by using mice with parvalbumin neuron (PV) specific knockout of GluA1 (AMPA receptor subunit 1) (Gria1-PV KO) as an experimental model. Applying high-content confocal imaging and electrophysiological recordings in the medial prefrontal cortex (mPFC), we found structural and functional alterations in the local network of Gria1-PV KO mice. Additionally, we applied electroencephalography (EEG) to assess potential deficits in mismatch negativity (MMN), the standard readout in the clinic for measuring deviance detection and sensory information processing. Gria1-PV KO animals exhibited abnormal theta oscillation and MMN, which is consistent with clinical findings in cognitively impaired patients. Remarkably, we demonstrated that the glycine transporter 1 (GlyT1) inhibitor, Bitopertin, ameliorates E/I imbalance, hyperexcitability, and sensory processing malfunction in Gria1-PV KO mice. Our results suggest that PV-specific deletion of GluA1 might be an experimental approach for back translating the E/I imbalance observed in schizophrenic patients. Our work offers a systematic workflow to understand the effect of GlyT1 inhibition in restoring cortical network activity from single cells to local brain circuitry. This study highlights that selectively boosting NMDA receptor-mediated excitatory drive to enhance the network inhibitory transmission from interneurons to pyramidal neurons (PYs) is a potential therapeutic strategy for restoring E/I imbalance-associated cognitive-related abnormality.

Effects of Cytochrome P450 3A4 Induction and Inhibition on the Pharmacokinetics of BI 425809, a Novel Glycine Transporter 1 Inhibitor.

BACKGROUND AND OBJECTIVE: Increased glycine availability at the synaptic cleft may enhance N-methyl-D-aspartate receptor signalling and provide a promising therapeutic strategy for cognitive impairment associated with schizophrenia. These studies aimed to assess the pharmacokinetics of BI 425809, a potent glycine-transporter-1 inhibitor, when co-administered with a strong cytochrome P450 3A4 (CYP3A4) inhibitor (itraconazole) and inducer (rifampicin). METHODS: In vitro studies using recombinant CYPs, human liver microsomes, and human hepatocytes were conducted to determine the CYP isoforms responsible for BI 425809 metabolism. In addition, two open-label, fixed-treatment period, phase I studies in healthy male volunteers are described. Period 1: participants received oral BI 425809 25 mg (single dose) on day 1; period 2: participants received multiple doses, across 10 days, of oral itraconazole or rifampicin combined with a single dose of oral BI 425809 25 mg on day 4/7 of the itraconazole/rifampicin treatment, respectively. Pharmacokinetic and safety endpoints were assessed in the absence/presence of itraconazole/rifampicin and included area under the concentration-time curve (AUC) over the time interval 0-167 h (AUC0‒167; itraconazole), 0-168 h (AUC0‒168; rifampicin), or 0-infinity (AUC0-∞; rifampicin and itraconazole), maximum measured concentration (Cmax) of BI 425809, and adverse events. RESULTS: In vitro results suggested that CYP3A4 accounted for ≥ 90% of the metabolism of BI 425809. BI 425809 exposure (adjusted geometric mean ratio [%]) was higher in the presence of itraconazole (AUC0‒167: 265.3; AUC0-∞: 597.0; Cmax: 116.1) and lower in the presence of rifampicin (AUC0‒168: 10.3; AUC0-∞: 9.8; Cmax: 37.4) compared with BI 425809 alone. Investigational treatments were well tolerated. CONCLUSIONS: Systemic exposure of BI 425809 was altered in the presence of strong CYP3A4 modulators, corroborating in vitro results that CYP3A4 mediates a major metabolic pathway for BI 425809. TRIAL REGISTRATION NUMBER: NCT02342717 (registered on 15 January 2015) and NCT03082183 (registered on 10 March 2017).

Symptomatic and preventive effects of the novel phosphodiesterase-9 inhibitor BI 409306 in an immune-mediated model of neurodevelopmental disorders.

BI 409306, a phosphodiesterase-9 inhibitor under development for treatment of schizophrenia and attenuated psychosis syndrome (APS), promotes synaptic plasticity and cognition. Here, we explored the effects of BI 409306 treatment in the polyriboinosinic-polyribocytidilic acid (poly[I:C])-based mouse model of maternal immune activation (MIA), which is relevant to schizophrenia and APS. In Study 1, adult offspring received BI 409306 0.2, 0.5, or 1 mg/kg or vehicle to establish an active dose. In Study 2, adult offspring received BI 409306 1 mg/kg and/or risperidone 0.025 mg/kg, risperidone 0.05 mg/kg, or vehicle, to evaluate BI 409306 as add-on to standard therapy for schizophrenia. In Study 3, offspring received BI 409306 1 mg/kg during adolescence only, or continually into adulthood to evaluate preventive effects of BI 409306. We found that BI 409306 significantly mitigated MIA-induced social interaction deficits and amphetamine-induced hyperlocomotion, but not prepulse inhibition impairments, in a dose-dependent manner (Study 1). Furthermore, BI 409306 1 mg/kg alone or in combination with risperidone 0.025 mg/kg significantly reversed social interaction deficits and attenuated amphetamine-induced hyperlocomotion in MIA offspring (Study 2). Finally, we revealed that BI 409306 1 mg/kg treatment restricted to adolescence prevented adult deficits in social interaction, whereas continued treatment into adulthood also significantly reduced amphetamine-induced hyperlocomotion (Study 3). Taken together, our findings suggest that symptomatic treatment with BI 409306 can restore social interaction deficits and dopaminergic dysfunctions in a MIA model of neurodevelopmental disruption, lending preclinical support to current clinical trials of BI 409306 in patients with schizophrenia. Moreover, BI 409306 given during adolescence has preventive effects on adult social interaction deficits in this model, supporting its use in people with APS.

A randomized Phase II trial evaluating efficacy, safety, and tolerability of oral BI 409306 in attenuated psychosis syndrome: Design and rationale.

AIM: Attenuated psychosis syndrome (APS), a condition for further study in the Diagnostic and Statistical Manual of Mental Disorders-5, comprises psychotic symptoms that are qualitatively similar to those observed in schizophrenia but are less severe. Patients with APS are at high risk of converting to first-episode psychosis (FEP). As evidence for effective pharmacological interventions in APS is limited, novel treatments may provide symptomatic relief and delay/prevent psychotic conversion. This trial aims to investigate the efficacy, safety, and tolerability of BI 409306, a potent and selective phosphodiesterase-9 inhibitor, versus placebo in APS. Novel biomarkers of psychosis are being investigated. METHODS: In this Phase II, multinational, double-blind, parallel-group trial, randomized (1:1) patients will receive BI 409306 50 mg or placebo twice daily for 52 weeks. Patients (n = 300) will be enrolled to determine time to remission of APS, time to FEP, change in everyday functional capacity (Schizophrenia Cognition Rating Scale), and change from baseline in Brief Assessment of Cognition composite score and Positive and Negative Syndrome Scale scores. Potential biomarkers of psychosis under investigation include functional measures of brain activity and automated speech analyses. Safety is being assessed throughout. CONCLUSIONS: This trial will determine whether BI 409306 is superior to placebo in achieving sustainable remission of APS and improvements in cognition and functional capacity. These advances may provide evidence-based treatment options for symptomatic relief in APS. Furthermore, the study will assess the effect of BI 409306 on psychotic conversion and explore the identification of patients at risk for conversion using novel biomarkers.