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

PLoS ONE

Although many clinical pathological states are now detectable using imaging and biochemical analyses, neuropsychological tests are often considered as valuable complementary approaches to confirm diagnosis, especially for disorders like Alzheimer's or Parkinson's disease, and schizophrenia. The touchscreen-based automated test battery, which was introduced two decades ago in humans to assess cognitive functions, has recently been successfully back-translated in monkeys and rodents. We focused on optimizing the protocol of three distinct behavioral paradigms in mice: two variants of the Paired Associates Learning (PAL) and the Visuo-Motor Conditional Learning (VMCL) tasks. Acquisition of these tasks was assessed in naive versus pre-trained mice. In naive mice, we managed to define testing conditions allowing significant improvements of learning performances over time in the three aforementioned tasks. In pre-trained mice, we observed differential acquisition rates after specific task combinations. Particularly, we identified that animals previously trained in the VMCL paradigm subsequently poorly learned the sPAL rule. Together with previous findings, these data confirm the feasibility of using such behavioral assays to evaluate the power of different models of cognitive dysfunction in mice. They also highlight the risk of interactions between tasks when rodents are run through a battery of different cognitive touchscreen paradigms

Acquisition curves in naive mice trained under various conditions in the VMCL task.

<p>*p<0.05 group 4 vs group 1 and p<0.01 group 4 vs group 3.</p

Acquisition curves in naive mice trained either in the sPAL (similar objects) or the dPAL (different objects) tasks.

<p>* p<0.05 and **p<0.01 vs the dPAL group.</p

Effect of a previous training experience in touchscreen boxes on the acquisition of a new task.

<p>Comparison of the acquisition curves of naive vs trained mice in the dPAL (left panel), the VMCL (central panel) or the sPAL task (right panel). *p<0.05 and ***p<0.001 vs the sPAL (task 1).</p

Additional parameters measured in touchscreen boxes during experiments A, B and C.

<p>CT: Correction Trials; CTL: Correct Touch Latency; ITL: Incorrect Touch Latency; ML: Magazine Latency.</p><p>All parameters are expressed as mean values ± SEM.</p><p>*p<0.05,</p><p>**p<0.01 <i>vs</i> sPAL group (task 1);</p>##<p>p<0.01 <i>vs</i> VMCL conditions 4 group.</p

Global repartition of the correct responses of all animals assessed in the PAL tasks (left panel) or in the VMCL task (right panel).

<p>In both PAL paradigms, there are six possible object-place combinations. In the VMCL task, mice can only respond to the left or the right part of the screen after the first central nose-poke.</p

Global design of touchscreen experiments.

<p>In experiments A and B, naive mice were trained in the dPAL, sPAL or VMCL tasks according to specific learning conditions (groups 1–4, from top to bottom). In experiment C, most of the mice previously trained in a first touchscreen paradigm were assessed in a different task; because of their experience, early stages of pokey training were purposely skipped. For animals trained in stages with defined limited holding times (LHT), corresponding values are given in white. FD: Food Deprivation; IT: „Initial Touch“; MT: „Must Touch“; MI: „Must Initiate“; PI: „Punish Incorrect“; PT: „Pre-Training“.</p