Multitarget Approach for the Treatment of Alzheimer's Disease: Inhibition of Phosphodiesterase 9 (PDE9) and Histone Deacetylases (HDACs) Covering Diverse Selectivity Profiles.

Here, we present a series of dual-target phosphodiesterase 9 (PDE9) and histone deacetylase (HDAC) inhibitors devised as pharmacological tool compounds for assessing the implications of these two targets in Alzheimer's disease (AD). These novel inhibitors were designed taking into account the key pharmacophoric features of known selective PDE9 inhibitors as well as privileged chemical structures, bearing zinc binding groups (hydroxamic acids and ortho-amino anilides) that hit HDAC targets. These substituents were selected according to rational criteria and previous knowledge from our group to explore diverse HDAC selectivity profiles (pan-HDAC, HDAC6 selective, and class I selective) that were confirmed in biochemical screens. Their functional response in inducing acetylation of histone and tubulin and phosphorylation of cAMP response element binding (CREB) was measured as a requisite for further progression into complete in vitro absorption, distribution, metabolism and excretion (ADME) and in vivo brain penetration profiling. Compound 31b, a selective HDAC6 inhibitor with acceptable brain permeability, was chosen for assessing in vivo efficacy of these first-in-class inhibitors, as well as studying their mode of action (MoA).

Taking Advantage of the Selectivity of Histone Deacetylases and Phosphodiesterase Inhibitors to Design Better Therapeutic Strategies to Treat Alzheimer's Disease.

The discouraging results with therapies for Alzheimer's disease (AD) in clinical trials, highlights the urgent need to adopt new approaches. Like other complex diseases, it is becoming clear that AD therapies should focus on the simultaneous modulation of several targets implicated in the disease. Recently, using reference compounds and the first-in class CM-414, we demonstrated that the simultaneous inhibition of histone deacetylases [class I histone deacetylases (HDACs) and HDAC6] and phosphodiesterase 5 (PDE5) has a synergistic therapeutic effect in AD models. To identify the best inhibitory balance of HDAC isoforms and PDEs that provides a safe and efficient therapy to combat AD, we tested the compound CM-695 in the Tg2576 mouse model of this disease. CM-695 selectively inhibits HDAC6 over class I HDAC isoforms, which largely overcomes the toxicity associated with HDAC class 1 inhibition. Furthermore, CM-695 inhibits PDE9, which is expressed strongly in the brain and has been proposed as a therapeutic target for AD. Chronic treatment of aged Tg2576 mice with CM-695 ameliorates memory impairment and diminishes brain Aß, although its therapeutic effect was no longer apparent 4 weeks after the treatment was interrupted. An increase in the presence of 78-KDa glucose regulated protein (GRP78) and heat shock protein 70 (Hsp70) chaperones may underlie the therapeutic effect of CM-695. In summary, chronic treatment with CM-695 appears to reverse the AD phenotype in a safe and effective manner. Taking into account that AD is a multifactorial disorder, the multimodal action of these compounds and the different events they affect may open new avenues to combat AD.

Discovery of in Vivo Chemical Probes for Treating Alzheimer's Disease: Dual Phosphodiesterase 5 (PDE5) and Class I Histone Deacetylase Selective Inhibitors.

In order to determine the contributions of histone deacetylase (HDAC) isoforms to the beneficial effects of dual phosphodiesterase 5 (PDE5) and pan-HDAC inhibitors on in vivo models of Alzheimer's disease (AD), we have designed, synthesized, and tested novel chemical probes with the desired target compound profile of PDE5 and class I HDAC selective inhibitors. Compared to previous hydroxamate-based series, these molecules exhibit longer residence times on HDACs. In this scenario, shorter or longer preincubation times may have a significant impact on the IC50 values of these compounds and therefore on their corresponding selectivity profiles on the different HDAC isoforms. On the other hand, different chemical series have been explored and, as expected, some pairwise comparisons show a clear impact of the scaffold on biological responses (e.g., 35a vs 40a). The lead identification process led to compound 29a, which shows an adequate ADME-Tox profile and in vivo target engagement (histone acetylation and cAMP/cGMP response element-binding (CREB) phosphorylation) in the central nervous system (CNS), suggesting that this compound represents an optimized chemical probe; thus, 29a has been assayed in a mouse model of AD (Tg2576).

Design, synthesis, biological evaluation and in vivo testing of dual phosphodiesterase 5 (PDE5) and histone deacetylase 6 (HDAC6)-selective inhibitors for the treatment of Alzheimer's disease.

We have identified chemical probes that act as dual phosphodiesterase 5 (PDE5) and histone deacetylase 6 (HDAC6)-selective inhibitors (>1 log unit difference versus class I HDACs) to decipher the contribution of HDAC isoforms to the positive impact of dual-acting PDE5 and HDAC inhibitors on mouse models of Alzheimer's disease (AD) and fine-tune this systems therapeutics approach. Structure- and knowledge-based approaches led to the design of first-in-class molecules with the desired target compound profile: dual PDE5 and HDAC6-selective inhibitors. Compound 44b, which fulfilled the biochemical, functional and ADME-Tox profiling requirements and exhibited adequate pharmacokinetic properties, was selected as pharmacological tool compound and tested in a mouse model of AD (Tg2576) in vivo.

A First-in-Class Small-Molecule that Acts as a Dual Inhibitor of HDAC and PDE5 and that Rescues Hippocampal Synaptic Impairment in Alzheimer's Disease Mice.

The targeting of two independent but synergistic enzymatic activities, histone deacetylases (HDACs, class I and HDAC6) and phosphodiesterase 5 (PDE5), has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). Here we report the discovery of a new first-in-class small-molecule (CM-414) that acts as a dual inhibitor of PDE5 and HDACs. We have used this compound as a chemical probe to validate this systems therapeutics strategy, where an increase in the activation of cAMP/cGMP-responsive element-binding protein (CREB) induced by PDE5 inhibition, combined with moderate HDAC class I inhibition, leads to efficient histone acetylation. This molecule rescued the impaired long-term potentiation evident in hippocampal slices from APP/PS1 mice. Chronic treatment of Tg2576 mice with CM-414 diminished brain Aß and tau phosphorylation (pTau) levels, increased the inactive form of GSK3ß, reverted the decrease in dendritic spine density on hippocampal neurons, and reversed their cognitive deficits, at least in part by inducing the expression of genes related to synaptic transmission. Thus, CM-414 may serve as the starting point to discover balanced dual inhibitors with an optimal efficacy and safety profile for clinical testing on AD patients.

Design, Synthesis, and Biological Evaluation of First-in-Class Dual Acting Histone Deacetylases (HDACs) and Phosphodiesterase 5 (PDE5) Inhibitors for the Treatment of Alzheimer's Disease.

Simultaneous inhibition of phosphodiesterase 5 (PDE5) and histone deacetylases (HDAC) has recently been validated as a potentially novel therapeutic approach for Alzheimer's disease (AD). To further extend this concept, we designed and synthesized the first chemical series of dual acting PDE5 and HDAC inhibitors, and we validated this systems therapeutics approach. Following the implementation of structure- and knowledge-based approaches, initial hits were designed and were shown to validate our hypothesis of dual in vitro inhibition. Then, an optimization strategy was pursued to obtain a proper tool compound for in vivo testing in AD models. Initial hits were translated into molecules with adequate cellular functional responses (histone acetylation and cAMP/cGMP response element-binding (CREB) phosphorylation in the nanomolar range), an acceptable therapeutic window (>1 log unit), and the ability to cross the blood-brain barrier, leading to the identification of 7 as a candidate for in vivo proof-of-concept testing ( Cuadrado-Tejedor, M.; Garcia-Barroso, C.; Sánchez-Arias, J. A.; Rabal, O.; Mederos, S.; Ugarte, A.; Franco, R.; Segura, V.; Perea, G.; Oyarzabal, J.; Garcia-Osta, A. Neuropsychopharmacology 2016 , in press, doi: 10.1038/npp.2016.163 ).

Pharmacokinetic investigation of sildenafil using positron emission tomography and determination of its effect on cerebrospinal fluid cGMP levels.

Sildenafil (Viagra) is a selective inhibitor of phosphodiesterase type 5 (PDE5), which degrades cyclic guanosine monophosphate to the linear nucleotide. Sildenafil is acutely used in erectile dysfunction and chronically in pulmonary hypertension. Evidence in the last decade shows that sildenafil may have potential as a therapeutic option for Alzheimer's disease or other neurodegenerative disorders. The purpose of this work was to explore whether sildenafil crosses the blood-brain barrier. Pharmacokinetic properties of sildenafil in rodents were investigated using (11) C-radiolabeling followed by in vivo positron emission tomography (PET) and ex vivo tissue dissection and gamma counting. PET results in rats suggest penetration into the central nervous system. Ex vivo data in perfused animals suggest that trapping of [(11) C]sildenafil within the cerebral vascular endothelium limits accumulation in the central nervous system parenchyma. Peroral sildenafil administration to Macaca fascicularis and subsequent chemical analysis of plasma and cerebrospinal fluid (CSF) using liquid chromatography coupled with tandem mass spectrometry showed that drug content in the CSF was high enough to achieve PDE5 inhibition, which was also demonstrated by the significant increases in CSF cyclic guanosine monophosphate levels. Central actions of sildenafil include both relaxation of the cerebral vasculature and inhibition of PDE5 in neurons and glia. This central action of sildenafil may underlie its efficacy in neuroprotection models, and may justify the continued search for a PDE5 ligand suitable for PET imaging. Sildenafil interacts with phosphodiesterase type 5 (PDE5) expressed in the endothelium and/or smooth muscle cells of brain vessels and also crosses the blood-brain barrier to interact with PDE5 expressed in brain cells. At therapeutic doses, the concentration of sildenafil in the cerebrospinal fluid (CSF) is high enough to inhibit PDE5 in the neural cells (neurons and glia). In turn, the concentration of cGMP likely increases in parenchymal cells and, as shown in this report, in the CSF. Read the Editorial Highlight for this article on page 220. Cover Image for this issue: doi: 10.1111/jnc.13302.

Decreased levels of guanosine 3', 5'-monophosphate (cGMP) in cerebrospinal fluid (CSF) are associated with cognitive decline and amyloid pathology in Alzheimer's disease.

AIMS: Levels of the cyclic nucleotides guanosine 3', 5'-monophosphate (cGMP) or adenosine 3', 5'-monophosphate (cAMP) that play important roles in memory processes are not characterized in Alzheimer's disease (AD). The aim of this study was to analyse the levels of these nucleotides in cerebrospinal fluid (CSF) samples from patients diagnosed with clinical and prodromal stages of AD and study the expression level of the enzymes that hydrolyzed them [phosphodiesterases (PDEs)] in the brain of AD patients vs. METHODS: For cGMP and cAMP CSF analysis, the cohort (n = 79) included cognitively normal participants (subjective cognitive impairment), individuals with stable mild cognitive impairment or AD converters (sMCI and cMCI), and mild AD patients. A high throughput liquid chromatography-tandem mass spectrometry method was used. Interactions between CSF cGMP or cAMP with mini-mental state examination (MMSE) score, CSF Aß(1-42) and CSF p-tau were analysed. For PDE4, 5, 9 and 10 expression analysis, brains of AD patients vs. controls (n = 7 and n = 8) were used. RESULTS: cGMP, and not cAMP levels, were significantly lower in the CSF of patients diagnosed with mild AD when compared with nondemented controls. CSF levels of cGMP showed a significant association with MMSE-diagnosed clinical dementia and with CSF biomarker Aß42 in AD patients. Significant increase in PDE5 expression was detected in temporal cortex of AD patients compared with that of age-matched healthy control subjects. No changes in the expression of others PDEs were detected. CONCLUSIONS: These results support the potential involvement of cGMP in the pathological and clinical development of AD. The cGMP reduction in early stages of AD might participate in the aggravation of amyloid pathology and cognitive decline.

Phosphodiesterase inhibition in cognitive decline.

Understanding the cellular and molecular processes involved in learning and memory will help in the development of safe and effective cognitive enhancers. The cAMP response element-binding (CREB) may be a universal modulator of processes required for memory formation, and increasing the levels of second messengers like cAMP and cGMP could ultimately lead to CREB activation. Phosphodiesterase (PDE) inhibitors regulate signaling pathways by elevating cAMP and/or cGMP levels, and they have been demonstrated to improve learning and memory in a number of rodent models of impaired cognition. The aim of this review is to summarize the outstanding progress that has been made in the application of PDE inhibitors for memory dysfunction. In addition, we have introduced some recent data we generated demonstrating that tadalafil could be considered as an optimal candidate for drug re-positioning and as a good candidate to enhance cognition.

Insulin-like growth factor 2 reverses memory and synaptic deficits in APP transgenic mice.

Insulin-like growth factor 2 (IGF2) was recently found to play a critical role in memory consolidation in rats and mice, and hippocampal or systemic administration of recombinant IGF2 enhances memory. Here, using a gene therapy-based approach with adeno-associated virus (AAV), we show that IGF2 overexpression in the hippocampus of aged wild-type mice enhances memory and promotes dendritic spine formation. Furthermore, we report that IGF2 expression decreases in the hippocampus of patients with Alzheimer's disease, and this leads us to hypothesize that increased IGF2 levels may be beneficial for treating the disease. Thus, we used the AAV system to deliver IGF2 or IGF1 into the hippocampus of the APP mouse model Tg2576 and demonstrate that IGF2 and insulin-like growth factor 1 (IGF1) rescue behavioural deficits, promote dendritic spine formation and restore normal hippocampal excitatory synaptic transmission. The brains of Tg2576 mice that overexpress IGF2 but not IGF1 also show a significant reduction in amyloid levels. This reduction probably occurs through an interaction with the IGF2 receptor (IGF2R). Hence, IGF2 and, to a lesser extent, IGF1 may be effective treatments for Alzheimer's disease.

Tadalafil crosses the blood-brain barrier and reverses cognitive dysfunction in a mouse model of AD.

Previous studies have demonstrated that cognitive function can be restored in mouse models of Alzheimer's disease (AD) following administration of sildenafil, a specific PDE5 inhibitor (Puzzo et al., 2009; Cuadrado-Tejedor et al.). Another very potent PDE5 inhibitor with a longer half-life and safe in chronic treatments, tadalafil, may represent a better alternative candidate for AD therapy. However, tadalafil was proven unable to achieve similar benefits than those of sildenafil in AD animal models (Puzzo et al., 2009). The lack of efficacy was attributed to inability to cross the blood-brain barrier (BBB). In this paper we first measured the blood and brain levels of tadalafil to prove that the compound crosses BBB and that chronic treatment leads to accumulation in the brain of the J20 transgenic mouse model of AD. We demonstrated the presence of PDE5 mRNA in the brain of the mice and also in the human brain. After a 10 week treatment with either of these PDE5 inhibitors, the performance of the J20 mice in the Morris water maze test improved when compared with the transgenic mice that received vehicle. Biochemical analysis revealed that neither sildenafil nor tadalafil altered the amyloid burden, although both compounds reduced Tau phosphorylation in the mouse hippocampus. This study provides evidence of the potential benefits of a chronic tadalafil treatment in AD therapy. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Phosphodiesterases as therapeutic targets for Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia among the elderly. In AD patients, memory loss is accompanied by the formation of beta-amyloid plaques and the appearance of tau in a pathological form. Given the lack of effective treatments for AD, the development of new management strategies for these patients is critical. The continued failure to find effective therapies using molecules aimed at addressing the anti-beta amyloid pathology has led researchers to focus on other non-amyloid-based approaches to restore memory function. Promising non-amyloid related candidate targets include phosphosdiesterases (PDEs), and indeed, Rolipram, a specific PDE4 inhibitor, was the first compound found to effectively restore cognitive deficits in animal models of AD. More recently, PDE5 inhibitors have also been shown to effectively restore memory function. Accordingly, inhibitors of other members of the PDE family may also improve memory performance in AD and non-AD animal models. Hence, in this review, we will summarize the data supporting the use of PDE inhibitors as cognitive enhancers and we will discuss the possible mechanisms of action underlying these effects. We shall also adopt a medicinal chemistry perspective that leads us to propose the most promising PDE candidates on the basis of inhibitor selectivity, brain distribution, and mechanism of action.