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.

The PDE4 inhibitor roflumilast improves memory in rodents at non-emetic doses.

Enhancement of central availability of the second messenger cAMP is a promising approach to improve cognitive function. Pharmacological inhibition of phosphodiesterase type 4 (PDE4), a group of cAMP hydrolyzing enzymes in the brain, has been shown to improve cognitive performances in rodents and monkeys. However, inhibition of PDE4 is generally associated with severe emetic side-effects. Roflumilast, an FDA-approved PDE4 inhibitor for treatment of chronic obstructive pulmonary disease (COPD), is yielding only mild emetic side effects. In the present study we investigate the potential of roflumilast as a cognition enhancer and to determine the potential coinciding emetic response in comparison to rolipram, a classic PDE4 inhibitor with pronounced emetic effects. Cognition enhancement was evaluated in mice and it was found that both roflumilast and rolipram enhanced memory in an object location task (0.03mg/kg), whereas only roflumilast was effective in a spatial Y-maze (0.1mg/kg). Emetic potential was measured using competition of PDE4 inhibition for α2-adrenergic receptor antagonism in which recovery from xylazine/ketamine-mediated anesthesia is used as a surrogate marker. While rolipram displayed emetic properties at a dose 10 times the memory-enhancing dose, roflumilast only showed increased emetic-like properties at a dose 100 times the memory-enhancing dose. Moreover, combining sub-efficacious doses of the approved cognition-enhancer donepezil and roflumilast, which did not improve memory when given alone, fully restored object recognition memory deficit in rats induced by the muscarinic receptor antagonist scopolamine. These findings suggest that roflumilast offers a more favorable window for treatment of cognitive deficits compared to rolipram.

Possible overlapping time frames of acquisition and consolidation phases in object memory processes: a pharmacological approach.

In previous studies, we have shown that acetylcholinesterase inhibitors and phosphodiesterase inhibitors (PDE-Is) are able to improve object memory by enhancing acquisition processes. On the other hand, only PDE-Is improve consolidation processes. Here we show that the cholinesterase inhibitor donepezil also improves memory performance when administered within 2 min after the acquisition trial. Likewise, both PDE5-I and PDE4-I reversed the scopolamine deficit model when administered within 2 min after the learning trial. PDE5-I was effective up to 45 min after the acquisition trial and PDE4-I was effective when administered between 3 and 5.5 h after the acquisition trial. Taken together, our study suggests that acetylcholine, cGMP, and cAMP are all involved in acquisition processes and that cGMP and cAMP are also involved in early and late consolidation processes, respectively. Most important, these pharmacological studies suggest that acquisition processes continue for some time after the learning trial where they share a short common time frame with early consolidation processes. Additional brain concentration measurements of the drugs suggest that these acquisition processes can continue up to 4-6 min after learning.

PDE5 inhibition improves object memory in standard housed rats but not in rats housed in an enriched environment: implications for memory models?

Drug effects are usually evaluated in animals housed under maximally standardized conditions. However, it is assumed that an enriched environment (EE) more closely resembles human conditions as compared to maximally standardized laboratory conditions. In the present study, we examined the acute cognition enhancing effects of vardenafil, a PDE5 inhibitor, which stimulates protein kinase G/CREB signaling in cells, in three different groups of male Wistar rats tested in an object recognition task (ORT). Rats were either housed solitarily (SOL) or socially (SOC) under standard conditions, or socially in an EE. Although EE animals remembered object information longer in the vehicle condition, vardenafil only improved object memory in SOL and SOC animals. While EE animals had a heavier dorsal hippocampus, we found no differences between experimental groups in total cell numbers in the dentate gyrus, CA2-3 or CA1. Neither were there any differences in markers for pre- and postsynaptic density. No changes in PDE5 mRNA- and protein expression levels were observed. Basal pCREB levels were increased in EE rats only, whereas ß-catenin was not affected, suggesting specific activation of the MAP kinase signaling pathway and not the AKT pathway. A possible explanation for the inefficacy of vardenafil could be that CREB signaling is already optimally stimulated in the hippocampus of EE rats. Since previous data has shown that acute PDE5 inhibition does not improve memory performance in humans, the use of EE animals could be considered as a more valid model for testing cognition enhancing drugs.

Object recognition testing: methodological considerations on exploration and discrimination measures.

The object recognition task (ORT) is a popular one-trial learning test for animals. In the current study, we investigated several methodological issues concerning the task. Data was pooled from 28 ORT studies, containing 731 male Wistar rats. We investigated the relationship between 3 common absolute- and relative discrimination measures, as well as their relation to exploratory activity. In this context, the effects of pre-experimental habituation, object familiarity, trial duration, retention interval and the amnesic drugs MK-801 and scopolamine were investigated. Our analyses showed that the ORT is very sensitive, capable of detecting subtle differences in memory (discrimination) and exploratory performance. As a consequence, it is susceptible to potential biases due to (injection) stress and side effects of drugs. Our data indicated that a minimum amount of exploration is required in the sample and test trial for stable significant discrimination performance. However, there was no relationship between the level of exploration in the sample trial and discrimination performance. In addition, the level of exploration in the test trial was positively related to the absolute discrimination measure, whereas this was not the case for relative discrimination measures, which correct for exploratory differences, making them more resistant to exploration biases. Animals appeared to remember object information over multiple test sessions. Therefore, when animals have encountered both objects in prior test sessions, the object preference observed in the test trial of 1h retention intervals is probably due to a relative difference in familiarity between the objects in the test trial, rather than true novelty per se. Taken together, our findings suggest to take into consideration pre-experimental exposure (familiarization) to objects, habituation to treatment procedures, and the use of relative discrimination measures when using the ORT.

Object recognition testing: statistical considerations.

The object recognition task is a commonly used test for the assessment of memory functions in rodents. In this paper different aspects concerning the analysis of object recognition data are discussed. Using a set of experimental studies and fictive data sets, it was shown that the absolute discrimination measure (d1) behaves different from the ratio measures (d2 and d3). Furthermore, it is suggested that, besides group differences, one should also examine whether each individual group actually discriminates between the novel and the familiar object. For this purpose, the use of a fictive group showing no discrimination is advisable. Furthermore, on basis of 48 object recognition task studies it is shown that discrimination performance does not fall significantly below zero. Therefore one-sided testing is allowed, provided that place- or object biases can be ruled out. Finally, it was shown that differences in exploration levels may affect the statistical evaluation of group differences. Several suggestions for statistical analysis are given.

Object recognition testing: rodent species, strains, housing conditions, and estrous cycle.

The object recognition task (ORT) allows assessing learning and memory processes in rodents. In this study, two areas in which knowledge about the ORT could be extended were addressed; i.e. generality to species and strains, and intervening variables including housing and estrous cycle. Regarding generality to species and strains, the ORT performance of golden hamsters was assessed. The hamsters showed sufficient exploration times, object recognition performance, and a retention-interval dependent decline similar to rats and mice. Subsequently, we tested three mouse strains which have not been described before in the ORT; i.e. OF1, NMRI, and SJL mice. OF1 and NMRI strains performed equally well, whereas the SJL strain showed low exploration times and no memory retention. Therefore, the SJL strain is unsuited for ORT experiments using a 1h retention interval and a fixed (3 min) trial duration. Furthermore, the sensitivity to a pharmacological memory deficit model (scopolamine) was tested in three rat strains. Each strain showed a dose dependent relationship, but the least effective dose of scopolamine differed among the three strains, the effect being greater in the order of Wistar, Long-Evans, Hooded Lister rats. Finally, to investigate potential intervening variables in the ORT, the effects of housing conditions and estrous cycle were investigated with rats. Single housing resulted in absolute higher performance than social housing. Furthermore, females in pro-estrus/estrus showed better performance compared to females in met-estrus/di-estrus. Taken together, object recognition appears to be a common ability of rodent species, but different strains have different memory capacities and sensitivities to scopolamine, individual housing leads to higher performance, and performance of females is dependent on the estrous cycle phase. Thus, rodent species, strain, housing, and estrous cycle should be taken into consideration in ORT studies.

Phosphodiesterase type 5 (PDE5) inhibition improves object recognition memory: indications for central and peripheral mechanisms.

A promising target for memory improvement is phosphodiesterase type 5 (PDE5), which selectively hydrolyzes cyclic guanosine monophosphate (cGMP). In rodents, PDE5 inhibitors (PDE5-Is) have been shown to improve memory performance in many behavioral paradigms. However, it is questioned whether the positive effects in animal studies result from PDE5 inhibition in the central nervous system or the periphery. Therefore, we studied the effects of PDE5 inhibition on memory and determined whether compound penetration of the blood-brain barrier (BBB) is required for this activity. Two selective PDE5-Is, vardenafil and UK-343,664, were tested in the object recognition task (ORT) in both a MK-801- and scopolamine-induced memory deficit model, and a time-delay model without pharmacological intervention. Compounds were dosed 30 min before the learning trial of the task. To determine if the PDE5-Is crossed the BBB, their concentrations were determined in plasma and brain tissue collected 30 min after oral administration. Vardenafil improved object recognition memory in all three variants of the ORT. UK-343,664 was ineffective at either preventing MK-801-induced memory disruption or time-dependent memory decay. However, UK-343,664 attenuated the memory impairment of scopolamine. Vardenafil crossed the BBB whereas UK-343,664 did not. Further, co-administration of UK-343,664 and scopolamine did not alter the brain partitioning of either molecule. This suggests that the positive effect of UK-343,664 on scopolamine-induced memory decay might arise from peripheral PDE5 inhibition. The results herein suggest that there may be multiple mechanisms that mediate the efficacy of PDE5 inhibition to improve memory performance in tasks such as the ORT and that these involve PDE5 located both within and outside of the brain. To further elucidate the underlying mechanisms, the cellular and subcellular localization of PDE5 needs to be determined.

Phosphodiesterase 2 and 5 inhibition attenuates the object memory deficit induced by acute tryptophan depletion.

The underlying mechanism of short-term memory improvement after inhibition of specific phosphodiesterases (PDEs) is still poorly understood. The present study aimed to reveal the ability of PDE5 and PDE2 inhibitors, that increase cyclic guanosine monophosphate (cGMP) and both cyclic adenosine monophosphate (cAMP) and cGMP, respectively, to reverse an object recognition deficit induced by acute tryptophan depletion. Acute tryptophan depletion is a pharmacological challenge tool to lower central serotonin (5-hydroxytryptamine; 5-HT) levels by depleting the availability of its dietary precursor tryptophan. Short-term object memory was tested in male Wistar rats by exposing them to the object recognition task. First, the effects of acute tryptophan depletion upon object recognition 2 h after administration of the nutritional mixture were established. Subsequently, acute tryptophan depletion was combined with the PDE5 inhibitor vardenafil (1, 3 and 10 mg/kg) or with the PDE2 inhibitor BAY 60-7550 (0.3, 1 and 3 mg/kg), 30 min prior to testing. Acute tryptophan depletion significantly lowered plasma tryptophan levels and impaired object recognition performance. Vardenafil (3 and 10 mg/kg) and BAY 60-7550 (3 mg/kg) were able to attenuate the acute tryptophan depletion induced object recognition impairment. Thus, both PDE5 and PDE2 inhibition improved short-term object recognition performance after an acute tryptophan depletion induced deficit. The underlying mechanisms, however, remain poorly understood and further studies are needed to determine whether the present findings can be explained by a direct effect of enhanced cAMP and cGMP levels upon 5-HT activity, or even other neurotransmitter systems, and possibly an interaction with synthesis of nitric oxide or effects upon cerebral blood flow function.