PDE4 Subtypes Research Articles

Amelioration of functional and histopathological consequences after spinal cord injury through phosphodiesterase 4D (PDE4D) inhibition

Spinal cord injury (SCI) is a life-changing event that severely impacts the patient's quality of life. Modulating neuroinflammation, which exacerbates the primary injury, and stimulating neuro-regenerative repair mechanisms are key strategies to improve functional recovery. Cyclic adenosine monophosphate (cAMP) is a second messenger crucially involved in both processes. Following SCI, intracellular levels of cAMP are known to decrease over time. Therefore, preventing cAMP degradation represents a promising strategy to suppress inflammation while stimulating regeneration. Intracellular cAMP levels are controlled by its hydrolyzing enzymes phosphodiesterases (PDEs). The PDE4 family is most abundantly expressed in the central nervous system (CNS) and its inhibition has been shown to be therapeutically relevant for managing SCI pathology. Unfortunately, the use of full PDE4 inhibitors at therapeutic doses is associated with severe emetic side effects, hampering their translation toward clinical applications. Therefore, in this study, we evaluated the effect of inhibiting specific PDE4 subtypes (PDE4B and PDE4D) on inflammatory and regenerative processes following SCI, as inhibitors selective for these subtypes have been demonstrated to be well-tolerated. We reveal that administration of the PDE4D inhibitor Gebr32a, even when starting 2 dpi, but not the PDE4B inhibitor A33, improved functional as well as histopathological outcomes after SCI, comparable to results obtained with the full PDE4 inhibitor roflumilast. Furthermore, using a luminescent human iPSC-derived neurospheroid model, we show that PDE4D inhibition stabilizes neural viability by preventing apoptosis and stimulating neuronal differentiation. These findings strongly suggest that specific PDE4D inhibition offers a novel therapeutic approach for SCI.

Discovery of 3-trifluoromethyl-substituted pyrazoles as selective phosphodiesterase 10A inhibitors for orally attenuating isoprenaline-induced cardiac hypertrophy

Recently, MP-10, a previous drug candidate with potent inhibition of phosphodiesterase 10A (PDE10A) in clinical phase II trials for schizophrenia or Alzheimer's disease, has shown significant potential in preventing and treating cardiovascular diseases. However, its poor metabolic stability and high permeability across the blood-brain barrier (BBB) make it unsuitable for preventing and treating peripheral cardiovascular diseases. Herein, the hit-to-lead optimization was performed to discover novel 3-trifluoromethyl-substituted pyrazole derivatives as potent and selective PDE10A inhibitors. The structure-activity relationships, biological characterization, molecular mechanism, and drug-like evaluation were discussed to identify compound C7 which showed potent inhibition against PDE10A (half maximal inhibitory concentration, IC50 = 11.9 nmol/L), more than 840-fold selectivity over other PDE subtypes, enhanced liver microsomes stability (T1/2 = 239 min) compared to MP-10 and low BBB permeability. Importantly, oral pretreatment with C7·3HCl at a dose of 5.0 mg/kg significantly attenuated the pathological and functional changes induced by isoprenaline (ISO)-induced pathological cardiac hypertrophy in mice, particularly suppressing increase of cardiac weight, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) hypertrophic markers along with cardiac fibrosis. These findings further support that targeting PDE10A provides an innovative therapeutic approach for preventing and treating cardiac diseases.

Elevated PDE4C level serves as a candidate diagnostic biomarker and correlates with poor survival in thyroid carcinoma

Thyroid carcinoma (THCA) is the most common endocrine cancer. Phosphodiesterase (PDE) 4 enzyme family, as specific regulator of cyclic adenosine monophosphate, may play a important role in THCA. However, few studies on PDE4 enzyme family in THCA have been reported yet. Therefore, this study aimed to systematically analyze the changes of PDE4 enzyme family in THCA, and look for potential target for THCA therapy. We systematically analyzed the expression differences, prognostic value, genetic alteration, methylation modification, and the correlation with tumor immune microenvironment of PDE4 family in THCA using several public databases, including TCGA, GEO, GSCA, TNMplot, cBioPortal, DiseaseMeth and TIMER. Besides, functional enrichment analysis and protein–protein interaction (PPI) network of PDE4 family was investigated using Metascape and STRING databases. The expression levels of PDE4A, PDE4B and PDE4D were down-regulated in THCA patients at different cancer stages, while the expression level of PDE4C was significantly up-regulated. Moreover, THCA patients with higher PDE4C expression had shorter progress free survival compared with those with lower PDE4C expression. The low genomic alteration frequencies and mildly increased methylation levels of PDE4 family were found in THCA patients. Except for PDE4A, the expression levels of PDE4B, PDE4C and PDE4D could affect many immune cells infiltration during THCA progression. Four PDE4 subtypes were all enriched in cAMP catabolic process. Nevertheless, PDE4C was not enriched in the cAMP binding signal pathway, and PDE4B was not enriched in the G alphas signaling events. Notably, PDE4C participated in cAMP metabolic process by regulating adenylate cyclases (ADCYs), which involved ADCY1, ADCY5, ADCY6, ADCY8 and ADCY9. The findings of this study provide a partial basis for the role of PDE4 family in the occurrence and development of THCA. In addition, this study also suggested that PDE4C might be a potential prognostic marker of THCA, which could serve as a reference for future basic and clinical research.

Uncovering the selectivity mechanism of phosphodiesterase 7A/8A inhibitors through computational studies.

The expression of phosphodiesterase 7A (PDE7A) and phosphodiesterase 8A (PDE8) genes is integral to human signaling pathways, and the inhibition of PDE7A has been associated with the onset of various diseases, including effects on the immune system and nervous system. The development of PDE7 selective inhibitors can promote research on immune and nervous system diseases, such as multiple sclerosis, chronic inflammation, and autoimmune responses. PDE8A is expressed alongside PDE8B, and its inhibitory mechanism is still unclear. Studying the mechanisms of selective inhibitors against different PDE subtypes is crucial to prevent potential side effects, such as nausea and cardiac toxicity, and the sequence similarity of the two protein subtypes was 55.9%. Therefore, it is necessary to investigate the differences of both subtypes' ligand binding sites. Selective inhibitors of two proteins were chosen to summarize the reason for their selectivity through molecular docking, molecular dynamics simulation, alanine scanning mutagenesis, and MM-GBSA calculation. We found that Phe384PDE7A, Leu401PDE7A, Gln413PDE7A, Tyr419PDE7A, and Phe416PDE7A in the active site positively contribute to the selectivity towards PDE7A. Additionally, Asn729PDE8A, Phe767PDE8A, Gln778PDE8A, and Phe781PDE8A positively contribute to the selectivity towards PDE8A.

Natural Phosphodiesterase-4 Inhibitors with Potential Anti-Inflammatory Activities from Millettia dielsiana.

The results of in silico screening of the 50 isolated compounds from Millettia dielsiana against the target proteins PDE4 (PDE4A, PDE4B, and PDE4D) showed binding affinity ranges from -5.81 to -11.56, -5.27 to -13.01, and -5.80 to -12.12 kcal mol-1, respectively, with median values of -8.83, -8.84, and -8.645 kcal mol-1, respectively. Among these compounds, Millesianin F was identified as the most promising PDE4A inhibitor due to its strongest binding affinity with the target protein PDE4A. (-11.56 kcal mol-1). This was followed by the compound 5,7,4'-trihydroxyisoflavone 7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside (D50) with the binding affinity value of -11.35 kcal mol-1. For the target protein PDE4B, compound D50 exhibited the strongest binding affinity value of -13.01 kcal mol-1, while showing poorer inhibition ability for PDE4D. The 100 ns MD simulation examination (radius of gyration, Solvent Accessible Surface Area (SASA), Root-Mean-Square Deviation (RMSD), Root-Mean-Square Fluctuation (RMSF), and hydrogen bonding) was carried out to examine the overall stability and binding efficiency of the protein-ligand complex between compounds (Millesianin F, Millesianin G, Claclrastin-7-O-β-d-glucopyranoside, 7-hydroxy-4',6 dimethoxyisoflavone-7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside, 7-hydroxy-4',8-dimethoxyisoflavone 7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside, Odoratin-7-O-β-d-glucopyranoside, and 5,7,4'-trihydroxyisoflavone 7-O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside) and PDE4 (A, B) subtype proteins. Compound D50 has shown strong anti-inflammatory activity, as evidenced by experimental results. It effectively inhibits PDE4B and PDE4D, with IC50 values of 6.56 ± 0.7 µM and 11.74 ± 1.3 µM, respectively. Additionally, it reduces NO production, with an IC50 value of 5.40 ± 0.9 µM. Based on these findings, it is promising and considered a potential novel anti-inflammatory drug for future development.

Phosphodiesterase and psychiatric disorders: a two-sample Mendelian randomization study

BackgroundPhosphodiesterases (PDEs) have been associated with psychiatric disorders in observational studies; however, the causality of associations remains unestablished.MethodsSpecifically, cyclic nucleotide PDEs were collected from genome-wide association studies (GWASs), including PDEs obtained by hydrolyzing both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) (PDE1A, PDE2A, and PDE3A), specific to cGMP (PDE5A, PDE6D, and PDE9A) and cAMP (PDE4D and PDE7A). We performed a bidirectional two-sample Mendelian randomization (MR) analysis to investigate the relationship between PDEs and nine psychiatric disorders. The inverse-variance-weighted (IVW) method, MR-Egger, and weighted median were used to estimate causal effects. The Cochran’s Q test, MR-Egger intercept test, MR Steiger test, leave-one-out analyses, funnel plot, and MR pleiotropy residual sum and outlier (MR-PRESSO) were used for sensitivity analyses.ResultsThe PDEs specific to cAMP were associated with higher-odds psychiatric disorders. For example, PDE4D and schizophrenia (SCZ) (odds ratios (OR) = 1.0531, PIVW = 0.0414), as well as major depressive disorder (MDD) (OR = 1.0329, PIVW = 0.0011). Similarly, PDE7A was associated with higher odds of attention-deficit/hyperactivity disorder (ADHD) (OR = 1.0861, PIVW = 0.0038). Exploring specific PDE subtypes and increase intracellular cAMP levels can inform the development of targeted interventions. We also observed PDEs (which hydrolyzes both cAMP and cGMP) was associated with psychiatric disorders [OR of PDE1A was 1.0836 for autism spectrum disorder; OR of PDE2A was 0.8968 for Tourette syndrome (TS) and 0.9449 for SCZ; and OR of PDE3A was 0.9796 for MDD; P < 0.05]. Furthermore, psychiatric disorders also had some causal effects on PDEs [obsessive–compulsive disorder on increased PDE6D and decreased PDE2A and PDE4D; anorexia nervosa on decreased PDE9A]. The results of MR were found to be robust using multiple sensitivity analysis.ConclusionsIn this study, potential causal relationships between plasma PDE proteins and psychiatric disorders were established. Exploring other PDE subtypes not included in this study could provide a more comprehensive understanding of the role of PDEs in psychiatric disorders. The development of specific medications targeting PDE subtypes may be a promising therapeutic approach for treating psychiatric disorders.

Phosphodiesterase-4 Inhibition in Parkinson's Disease: Molecular Insights and Therapeutic Potential.

Clinicians and researchers are exploring safer and novel treatment strategies for treating the ever-prevalent Parkinson's disease (PD) across the globe. Several therapeutic strategies are used clinically for PD, including dopamine replacement therapy, DA agonists, MAO-B blockers, COMT blockers, and anticholinergics. Surgical interventions such as pallidotomy, particularly deep brain stimulation (DBS), are also employed. However, they only provide temporal and symptomatic relief. Cyclic adenosine monophosphate (cAMP) is one of the secondary messengers involved in dopaminergic neurotransmission. Phosphodiesterase (PDE) regulates cAMP and cGMP intracellular levels. PDE enzymes are subdivided into families and subtypes which are expressed throughout the human body. PDE4 isoenzyme- PDE4B subtype is overexpressed in thesubstantia nigraof the brain. Various studies have implicated multiple cAMP-mediated signaling cascades in PD, and PDE4 is a common link that can emerge as a neuroprotective and/or disease-modifying target. Furthermore, a mechanistic understanding of the PDE4 subtypes has provided perceptivity into the molecular mechanisms underlying the adverse effects of phosphodiesterase-4 inhibitors (PDE4Is). The repositioning and development of efficacious PDE4Is for PD have gained much attention. This review critically assesses the existing literature on PDE4 and its expression. Specifically, this review provides insights into the interrelated neurological cAMP-mediated signaling cascades involving PDE4s and the potential role of PDE4Is in PD. In addition, we discuss existing challenges and possible strategies for overcoming them.

Selective PDE4B and PDE4D inhibitors produce distinct behavioral responses to ethanol and GABAergic drugs in mice

Apremilast is a phosphodiesterase (PDE) type 4 inhibitor that is nonselective at subtypes PDE4A-D. It modulates ethanol and GABAergic responses via protein kinase A (PKA) phosphorylation of specific GABAA receptor subunits and has opposite effects on ethanol-induced ataxia in wild-type and GABAA β3-S408/409A knock-in mice. We hypothesized that these different effects are due to preferential actions at different PDE4 subtypes. To test this hypothesis, we compared effects of selective PDE4 inhibitors on responses to ethanol and GABAergic drugs in male and female C57BL/6J mice. The PDE4B inhibitor A33 accelerated recovery from ataxia induced by ethanol and diazepam but did not alter ataxia induced by propofol. The PDE4D inhibitor D159687 accelerated recovery from diazepam-induced ataxia but prolonged recovery from ethanol- and propofol-induced ataxia. A33 shortened, while D159687 prolonged, the sedative-hypnotic effects of ethanol. Both drugs shortened diazepam's sedative-hypnotic effects. The modulatory effects of A33 and D159687 were completely prevented by the PKA inhibitor H89. Only D159687 prevented development of acute functional tolerance to ethanol-induced ataxia. D159687 transiently reduced two-bottle choice drinking in male and female mice that had consumed ethanol for 3 weeks and transiently reduced two-bottle choice, every-other-day drinking in male mice. A33 did not alter ethanol drinking in either procedure. Neither drug altered binge-like ethanol consumption or blood ethanol clearance. Thus, D159687 produced behavioral effects similar to apremilast, although it produced a more transient and smaller reduction in drinking. These results indicate that PDE4D inhibition contributes to apremilast's ability to reduce ethanol drinking, whereas PDE4B inhibition is not involved.

576 Difamilast, a new topical PDE4 inhibitor, ameliorates not only chronic allergic dermatitis but also idiopathic dermatitis induced by psychological stress in mice

Difamilast is a novel PDE4 inhibitor with the strongest inhibitory activity against PDE4B among the four PDE4 subtypes. Moizerto® ointment (0.3%, 1% difamilast) has been recently approved for treatment of atopic dermatitis (AD) in Japan. Exogenous antigen stimulation or psychological stress can trigger or exacerbate AD symptoms. In this study, we evaluated the therapeutic effects of difamilast, tacrolimus, and betamethasone valerate on two different mice models of dermatitis: one was chronic contact hypersensitivity (CCH) induced by repeated application of hapten every 2 days for 52 days, the other was idiopathic dermatitis due to persistent self-scratching induced by application of hapten three times a week for 16 weeks, followed by individual housing (social isolation) for 6 months. Efficacy was assessed by repeated topical application of each drug for 4 or 6 weeks after each dermatitis established. In the CCH model, difamilast ointment group significantly improved skin hyperplasia from 0.03% to 3% dose-responsibly with inhibition of inflammatory cell infiltration such as T cells, eosinophils, and neutrophils. Betamethasone valerate ointment (0.12%) improved skin hyperplasia more strongly than 3% difamilast ointment. Tacrolimus ointment (0.1%) temporarily improved skin symptoms, but eventually had no effect. In the idiopathic dermatitis model, difamilast (1%, 3%) significantly improved skin symptoms with a decrease in MIP-1 alpha and MIP-2 content. Tacrolimus (0.1%) slightly improved skin symptoms, while betamethasone valerate (0.1%) exacerbated it. Notably, difamilast ameliorates both CCH and psychological stress-induced idiopathic dermatitis. Betamethasone valerate worsened the idiopathic dermatitis probably due to skin atrophy as steroid’s side effect and tacrolimus had weak effects in both models. Therefore, difamilast is expected to be a possible treatment for AD symptoms such as chronic allergic dermatitis and psychological stress-exacerbated idiopathic dermatitis.

Pharmacologic or Genetic PDE4 Inactivation Reduces Obesity and Improves Glucose Handling in Mice

PurposeType 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a group of four isoenzymes (PDE4A to D) that hydrolyze the second messenger cAMP. Non/PAN‐selective PDE4 inhibitors exert potent anti‐inflammatory properties and are approved for the treatment of COPD and psoriasis, but are also associated with significant side effects, including nausea, emesis and weight loss. Here, we explored the idea that the weight loss associated with PAN‐PDE4 inhibitor use may be pursued as a desirable therapeutic outcome in the treatment of obesity‐related metabolic syndromes. As a first step, we tested whether treatment of mice with a PAN‐PDE4 inhibitor would replicate clinical data and reduce ageing‐ and/or high‐fat diet‐induced obesity in the animals. As each of the four PDE4 subtypes plays unique physiological roles, targeting individual PDE4s is a promising approach to improve the tolerability of PDE4 inhibitor therapy. To this end, we determined the metabolic phenotypes of individual PDE4 subtypes in mice.MethodsBody and tissue weights, food and water consumption, glucose and insulin tolerance, serum insulin and glucagon levels, and locomotor activity and exercise capacity were assessed in aged mice, as well as in young mice fed a high‐fat diet. To delineate the role of PDE4s, the phenotypes of mice genetically deficient (KO) in each of the four individual PDE4 subtypes, PDE4A to PDE4D, were compared to their wildtype littermates, and mice treated with the PAN‐PDE4 inhibitor Roflumilast were compared to solvent controls.ResultsTreatment with the PAN‐PDE4 inhibitor Roflumilast reduced high‐fat diet‐induced obesity in mice, as reflected by reduced body weight and white fat pads, without lowering food consumption or increasing physical activity (unchanged locomotor activity and exercise capacity) suggesting that the weight‐loss effect of PDE4 inhibition observed in clinical trials is replicated in the mouse. Genetic ablation of PDE4B or PDE4D in mice replicated the effects of PAN‐PDE4 inhibition by reducing body‐ and adipose tissue weights in mice, whereas ablation of PDE4A or PDE4C had no effect. Reduced adiposity as a result of PDE4 inactivation was associated with improved glucose handling and insulin sensitivity, while serum levels of insulin and glucagon were unchanged. These data suggest that PDE4 inactivation does not act via pancreatic hormone release but acts in downstream target tissues to enhance glucose utilization.ConclusionsInactivation of PDE4s represents a promising approach to tackle obesity and associated metabolic abnormalities such as elevated blood glucose levels. Targeting PDE4B and/or PDE4D with subtype‐selective PDE4 inhibitors appears sufficient to mediate these therapeutic benefits and may be free of the adverse effects associated with the PAN‐PDE4 inhibitors available to date.

Ablation of PDE4A Exacerbates Pseudomonas aeruginosa‐Induced Acute Lung Injury in Mice

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a family of four isoenzymes, PDE4A to D, that hydrolyze the second messenger cAMP. Three of these enzymes, PDE4A, PDE4B and PDE4D, are widely expressed throughout pulmonary and immune cell types, and their concurrent inhibition with non/PAN‐selective PDE4 inhibitors is well‐known to exert potent anti‐inflammatory benefits. To determine if PDE4 inhibition may represent a therapeutic approach for acute lung injury resulting from severe pneumonia, we explored the effect of genetic ablation of individual PDE4 subtypes, or their concurrent inhibition with PAN‐PDE4 inhibitors, in a model of acute intra‐tracheal Pseudomonas aeruginosa (PA) infection in mice. We have shown recently that selective ablation of PDE4B is sufficient to protect mice from lung injury in this model by reducing pulmonary and systemic levels of pro‐inflammatory cytokines, as well as pulmonary vascular leakage and mortality. With the present study we explored whether selective inactivation of PDE4A or treatment with PAN‐PDE4 inhibitors may provide additional therapeutic benefits.MethodsMice deficient in PDE4A (PDE4A‐KO) and their wildtype (WT) littermate controls were infected intratracheally with the PA lab strain PA01 and subjected 16 h later to bronchoalveolar lavage (BAL), cardiac blood draws, and tissue extractions. Alternatively, vascular/alveolar leakage was assessed by measuring Evans Blue Dye extravasation. To test the effect of PAN‐PDE4 inhibition, mice were treated with the PDE4 inhibitor Piclamilast (5 mg/kg, i.p.) at 2 h prior to PA01 infection.ResultsCompared to WT controls, PDE4A‐KO mice displayed a hyper‐inflammatory phenotype characterized by increased levels of pro‐inflammatory cytokines, immune cells (neutrophils) in BAL fluid, and vascular leakage. Despite these hyperinflammatory responses, the bacterial load in lungs was also increased in PDE4A‐KO mice compared to WT controls. Treatment with a PAN‐PDE4 inhibitor produced incongruent effects, with an increased bacterial load mirroring the effect of PDE4A ablation, whereas reduced levels of inflammatory cytokines such as TNFα mimicked the effect of PDE4B ablation.ConclusionsAblation of PDE4A produces detrimental effects in this model of acute PA‐lung infection. Current studies aim to determine whether an impaired bacterial killing, and the resulting increase in bacterial load, are responsible for the hyperinflammation, or whether these are independent effects of PDE4A ablation. PAN‐PDE4 inhibition mimics some of the beneficial effects of selective PDE4B ablation, but also some of the detrimental effects of PDE4A ablation. These findings suggest that selective inactivation of PDE4B may be a more effective therapeutic approach, compared to PAN‐PDE4 inhibition, in settings of acute PA‐lung infection.

Assessment of PDE4 Inhibitor-Induced Hypothermia as a Correlate of Nausea in Mice

Simple SummaryType 4 cAMP-phosphodiesterases (PDE4s) comprise a family of four isoenzymes, PDE4A to D, that hydrolyze and inactivate the second messenger cAMP. Non/PAN-selective PDE4 inhibitors, which inhibit all four PDE4 subtypes simultaneously, produce many promising therapeutic benefits, such as anti-inflammatory or cognition- and memory-enhancing effects. However, unwanted side effects, principally, nausea, diarrhea, and emesis, have long hampered their clinical and commercial success. Targeting individual PDE4 subtypes has been proposed for developing drugs with an improved safety profile, but which PDE4 subtype(s) is/are actually responsible for nausea and emesis remains ill-defined. Based on the observation that nausea is often accompanied by hypothermia in humans and other mammals, we used the measurement of core body temperatures of mice as a potential correlate of nausea induced by PDE4 inhibitors in humans. We find that selective inactivation of any of the four PDE4 subtypes did not change the body temperature of mice, suggesting that PAN-PDE4 inhibitor-induced hypothermia (and hence nausea in humans) requires the simultaneous inhibition of multiple PDE4 subtypes. This finding contrasts with prior reports that proposed PDE4D as the subtype mediating these side effects of PDE4 inhibitors and suggests that subtype-selective inhibitors that target any individual PDE4 subtype, including PDE4D, may not cause nausea.Treatment with PAN-PDE4 inhibitors has been shown to produce hypothermia in multiple species. Given the growing body of evidence that links nausea and emesis to disturbances in thermoregulation in mammals, we explored PDE4 inhibitor-induced hypothermia as a novel correlate of nausea in mice. Using knockout mice for each of the four PDE4 subtypes, we show that selective inactivation of individual PDE4 subtypes per se does not produce hypothermia, which must instead require the concurrent inactivation of multiple (at least two) PDE4 subtypes. These findings contrast with the role of PDE4s in shortening the duration of α2-adrenoceptor-dependent anesthesia, a behavioral surrogate previously used to assess the emetic potential of PDE4 inhibitors, which is exclusively affected by inactivation of PDE4D. These different outcomes are rooted in the distinct molecular mechanisms that drive these two paradigms; acting as a physiologic α2-adrenoceptor antagonist produces the effect of PDE4/PDE4D inactivation on the duration of α2-adrenoceptor-dependent anesthesia, but does not mediate the effect of PDE4 inhibitors on body temperature in mice. Taken together, our findings suggest that selective inhibition of any individual PDE4 subtype, including inhibition of PDE4D, may be free of nausea and emesis.

Baicalein Ameliorates Aβ-Induced Memory Deficits and Neuronal Atrophy via Inhibition of PDE2 and PDE4.

Inhibition of phosphodiesterase 2 and 4 (PDE2A and PDE4) increases the intracellular cAMP and/or cGMP levels, which may prevent Amyloid β 42 oligomers (Aβ) related cognitive impairment and dementias. Baicalein, one of natural flavones found in the root of Scutellaria baicalensis Georgi, has a wide range of pharmacological activities including antioxidant and anti-inflammatory effects. However, no studies suggest whether baicalein mediated anti-Alzheimer’s disease (AD) events involve PDEs subtypes-mediated neuroprotective pathways. The present study examined whether memory enhancing effects of baicalein on Aβ- induced cognitive impairment are related to regulating neuroplasticity via PDE2 and PDE4 subtypes dependent cAMP/cGMP neuroprotective pathway. The results suggested that microinjected of Aβ into CA1 of hippocampus induced cognitive and memory impairment in mice, as evidenced by decreased recognition index in the novel object recognition (NOR) task, impaired memory acquisition, retention and retrieval in the Morris water maze (MWM) and shuttle box tests. These effects were reversed by treatment with baicalein for 14 days. Moreover, Aβ-induced neuronal atrophy and decreased expression of two synaptic proteins, synaptophysin and PSD 95, were prevented by baicalein. The increased expression of PDE2A and PDE4 subtypes (PDE4A, PDE4B and PDE4D), and decreased levels of cAMP/cGMP, pCREB/CREB and BDNF induced by Aβ were also blocked by chronic treatment of baicalein for 14 days. These findings suggest that baicalein’s reversal of Aβ-induced memory and cognitive disorder may involve the regulation of neuronal remodeling via regulation of PDE2/PDE4 subtypes related cAMP/cGMP -pCREB-BDNF pathway.

Rolipram plays an anti-fibrotic effect in ligamentum flavum fibroblasts by inhibiting the activation of ERK1/2

BackgroundFibrosis is an important factor and process of ligamentum flavum hypertrophy. The expression of phosphodiesterase family (PDE) is related to inflammation and fibrosis. This article studied the expression of PDE in hypertrophic ligamentum flavum fibroblasts and investigated whether inhibition of PDE4 activity can play an anti-fibrotic effect.MethodsSamples of clinical hypertrophic ligamentum flavum were collected and patients with lumbar disc herniations as a control group. The collagenase digestion method is used to separate fibroblasts. qPCR is used to detect the expression of PDE subtypes, type I collagen (Col I), type III collagen (Col III), fibronectin (FN1) and transforming growth factor β1 (TGF-β1). Recombinant TGF-β1 was used to stimulate fibroblasts to make a fibrotic cell model and treated with Rolipram. The morphology of the cells treated with drugs was observed by Sirius Red staining. Scratch the cells to observe their migration and proliferation. WB detects the expression of the above-mentioned multiple fibrotic proteins after drug treatment. Finally, combined with a variety of signaling pathway drugs, the signaling mechanism was studied.ResultsMultiple PDE subtypes were expressed in ligamentum flavum fibroblasts. The expression of PDE4A and 4B was significantly up-regulated in the hypertrophic group. Using Rolipram to inhibit PDE4 activity, the expression of Col I and TGF-β1 in the hypertrophic group was inhibited. Col I recovered to the level of the control group. TGF-β1 was significantly inhibited, which was lower than the control group. Recombinant TGF-β1 stimulated fibroblasts to increase the expression of Col I/III, FN1 and TGF-β1, which was blocked by Rolipram. Rolipram restored the increased expression of p-ERK1/2 stimulated by TGF-β1.ConclusionThe expressions of PDE4A and 4B in the hypertrophic ligamentum flavum are increased, suggesting that it is related to the hypertrophy of the ligamentum flavum. Rolipram has a good anti-fibrosis effect after inhibiting the activity of PDE4. This is related to blocking the function of TGF-β1, specifically by restoring normal ERK1/2 signal.

The cAMP-phosphodiesterase 4 (PDE4) controls β-adrenoceptor- and CFTR-dependent saliva secretion in mice

Saliva, while often taken for granted, is indispensable for oral health and overall well-being, as inferred from the significant impairments suffered by patients with salivary gland dysfunction. Here, we show that treatment with several structurally distinct PAN-PDE4 inhibitors, but not a PDE3 inhibitor, induces saliva secretion in mice, indicating it is a class-effect of PDE4 inhibitors. In anesthetized mice, while neuronal regulations are suppressed, PDE4 inhibition potentiates a β-adrenoceptor-induced salivation, that is ablated by the β-blocker Propranolol and is absent from homozygous ΔF508-CFTR mice lacking functional CFTR. These data suggest that PDE4 acts within salivary glands to gate saliva secretion that is contingent upon the cAMP/PKA-dependent activation of CFTR. Indeed, PDE4 contributes the majority of total cAMP-hydrolytic capacity in submandibular-, sublingual-, and parotid glands, the three major salivary glands of the mouse. In awake mice, PDE4 inhibitor-induced salivation is reduced by CFTR deficiency or β-blockers, but also by the muscarinic blocker Atropine, suggesting an additional, central/neuronal mechanism of PDE4 inhibitor action. The PDE4 family comprises four subtypes, PDE4A-D. Ablation of PDE4D, but not PDE4A-C, produced a minor effect on saliva secretion, implying that while PDE4D may play a predominant role, PDE4 inhibitor-induced salivation results from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data reveal a critical role for PDE4/PDE4D in controlling CFTR function in an in vivo model and in inducing salivation, hinting at a therapeutic potential of PDE4 inhibition for cystic fibrosis and conditions associated with xerostomia.

Inhibition of cAMP‐Phosphodiesterase 4 (PDE4) Induces Salivation in Mice

While exploring potential anti-inflammatory benefits of PDE4 inhibition in a mouse model of bacterial lung infection, we noticed that treatment with the PDE4 inhibitor Piclamilast visibly induced salivation in the animals. Given the predominant role of autonomic nervous system regulation in the control of salivation, and the potential impact of altered autonomics triggered by PDE4 inhibition on our infection model, we have further explored this observation. InKetamine/Xylazine-anesthetized mice, in which saliva production was tracked by the weight increase of thin filter paper strips placed into the mouths of the animals, treatment with PDE4 inhibitors did not induce saliva production per se, but synergized with low doses of the β-adrenoceptor agonist Isoprenaline to induce substantial salivation that was ablated by treatment with the β-blocker Propranolol, but not the mACh-blocker Atropine, and was absent in CFTRΔF508/ΔF508 mice that lack functional CFTR. Conversely, in awake mice, visual scoring (elevated/abnormal salivation=1; no elevated/abnormal salivation=0) indicated that treatment with PDE4 inhibitors was by itself sufficient to induce substantial salivation, which was ablated by treatment with either Propranolol or Atropine, and was also absent in ΔF508-CFTR-mutant mice. Our findings suggest that in anesthetized mice, in which autonomic nervous system regulations are suppressed, inhibition of cAMP degradation by PDE4 within salivary gland cells potentiates the effects of increased cAMP production resulting from β-adrenoceptor activation, and thereby promote the cAMP/PKA-dependent activation of CFTR that promotes saliva secretion. However, in awake mice, PDE4 inhibition acts via an additional mechanism on the autonomic, muscarinic regulation of salivation, likely by affecting neurotransmitter release. In both awake and anesthetized mice, salivation was induced by several structurally distinct PDE4 inhibitors, but not by a PDE3 inhibitor, suggesting that salivation is a class effect of PAN-PDE4 inhibitors. The PDE4 family comprises four PDE4 subtypes, PDE4A to D. Genetic ablation of any individual PDE4 subtype in the respective KO mouse strains did not induce salivation in either awake or anesthetized mice, indicating that salivation must result from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data identify PDE4 as a critical regulator of CFTR function in an in vivo model and suggests a therapeutic potential of PDE4 inhibition to alleviate CFTR hypofunction in cystic fibrosis. In addition, our data reveal a therapeutic potential for PDE4 inhibition in the treatment of dry mouth, a condition frequently associated with drug-, radiation-, or ageing-induced salivary gland dysfunction.

Inhibition of PDE4 Potentiates the Anesthetic Effects of Isoflurane in Mice

Despite major advances, there remains a need for novel anesthetic drugs or drug combinations with improved efficacy and safety profiles. Here, we show that inhibition of cAMP-phosphodiesterase 4 (PDE4), while not inducing anesthesia by itself, potently enhances the anesthetic effects of Isoflurane in mice. Treatment with several distinct PAN-PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast and RS25344, significantly delayed the time-to-righting of mice after removal from Isoflurane anesthesia. Conversely, treatment with the PDE3 inhibitor Cilostamide, or treatment with the potent, but non-brain-penetrant PDE4 inhibitor YM976, had no effect. These findings suggest that potentiation of Isoflurane hypnosis is a class effect of brain-penetrant PDE4 inhibitors, and that they act by synergizing with Isoflurane in the inhibition of neuronal activity. The PDE4 family comprises four PDE4 subtypes, PDE4A to PDE4D. Genetic deletion of any of the four PDE4 subtypes in mice did not affect Isoflurane anesthesia per se. However, PDE4D knock-out mice are largely protected from the effect of pharmacologic PDE4 inhibition, suggesting that PDE4D is the predominant, but not the sole PDE4 subtype involved in potentiating Isoflurane anesthesia. Pretreatment with Naloxone or Propranolol alleviated the potentiating effect of PDE4 inhibition, implicating opioid- and β-adrenoceptor signaling in mediating PDE4 inhibitor-induced augmentation of Isoflurane anesthesia. Conversely, stimulation or blockade of α1-adrenergic, α2-adrenergic, or 5-HT3-serotonergic signaling did not affect the potentiation of Isoflurane hypnosis by PDE4 inhibition. In a separate line of study, we show that pretreatment with a PDE4 inhibitor boosts the delivery of bacteria into the lungs of mice, if the bacteria are delivered via intranasal infection under Isoflurane, thus providing a first example that PDE4 inhibitor-induced potentiation of Isoflurane anesthesia can critically impact animal models and must be considered as a factor in experimental design. Our findings suggest that PDE4 inhibition may serve as a tool to delineate the exact molecular mechanisms of Isoflurane anesthesia, which remain poorly understood, and may potentially be exploited to reduce the clinical doses of Isoflurane required to maintain hypnosis.

Inhibition of cAMP-phosphodiesterase 4 (PDE4) potentiates the anesthetic effects of Isoflurane in mice

Despite major advances, there remains a need for novel anesthetic drugs or drug combinations with improved efficacy and safety profiles. Here, we show that inhibition of cAMP-phosphodiesterase 4 (PDE4), while not inducing anesthesia by itself, potently enhances the anesthetic effects of Isoflurane in mice. Treatment with several distinct PAN-PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast, and RS25344, significantly delayed the time-to-righting after Isoflurane anesthesia. Conversely, treatment with a PDE3 inhibitor, Cilostamide, or treatment with the potent, but non-brain-penetrant PDE4 inhibitor YM976, had no effect. These findings suggest that potentiation of Isoflurane hypnosis is a class effect of brain-penetrant PDE4 inhibitors, and that they act by synergizing with Isoflurane in inhibiting neuronal activity. The PDE4 family comprises four PDE4 subtypes, PDE4A to PDE4D. Genetic deletion of any of the four PDE4 subtypes in mice did not affect Isoflurane anesthesia per se. However, PDE4D knockout mice are largely protected from the effect of pharmacologic PDE4 inhibition, suggesting that PDE4D is the predominant, but not the sole PDE4 subtype involved in potentiating Isoflurane anesthesia. Pretreatment with Naloxone or Propranolol alleviated the potentiating effect of PDE4 inhibition, implicating opioid- and β-adrenoceptor signaling in mediating PDE4 inhibitor-induced augmentation of Isoflurane anesthesia. Conversely, stimulation or blockade of α1-adrenergic, α2-adrenergic or serotonergic signaling did not affect the potentiation of Isoflurane hypnosis by PDE4 inhibition. We further show that pretreatment with a PDE4 inhibitor boosts the delivery of bacteria into the lungs of mice after intranasal infection under Isoflurane, thus providing a first example that PDE4 inhibitor-induced potentiation of Isoflurane anesthesia can critically impact animal models and must be considered as a factor in experimental design. Our findings suggest that PDE4/PDE4D inhibition may serve as a tool to delineate the exact molecular mechanisms of Isoflurane anesthesia, which remain poorly understood, and may potentially be exploited to reduce the clinical doses of Isoflurane required to maintain hypnosis.

Imperatorin Alleviates Psoriasiform Dermatitis by Blocking Neutrophil Respiratory Burst, Adhesion, and Chemotaxis Through Selective Phosphodiesterase 4 Inhibition.

Aim: Neutrophil infiltration and increased oxidative stress are involved in the pathogenesis and severity of psoriasis. Although the therapy of psoriasis remains elusive, targeting treatment to reduce oxidative stress is considered a potential option. Our study demonstrates the anti-inflammatory effects of a natural furocoumarin, imperatorin, on activated human neutrophils and psoriasiform dermatitis in mice. Results: Imperatorin inhibited superoxide anion generation, neutrophil adhesion, and migration in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-stimulated human neutrophils. Further studies showed that imperatorin induced a decrease in cAMP-specific phosphodiesterase (PDE) activity, and increased intracellular cAMP levels and protein kinase A (PKA) activity in human neutrophils. The enzyme activities of PDE4 subtypes, but not PDE3 and PDE7, were inhibited by imperatorin. Furthermore, imperatorin inhibited the phosphorylation of protein kinase B (Akt), extracellular regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), as well as Ca2+ mobilization in fMLF-stimulated neutrophils. These suppressive effects of imperatorin on cell responses and signaling were reversed by PKA inhibitor, suggesting that cAMP/PKA is involved in the anti-inflammatory effects of imperatorin. In vivo studies of imiquimod- and interleukin-23-induced mouse psoriasiform dermatitis demonstrated that imperatorin alleviated skin desquamation, epidermal thickening, keratinocyte hyperproliferation, and neutrophil infiltration. Innovation and Conclusion: Our results demonstrate that imperatorin inhibits human neutrophil respiratory burst, adhesion, and migration through the elevation of cAMP/PKA to inhibit Akt, ERK, JNK, and Ca2+ mobilization. Imperatorin is a natural inhibitor of PDE4A/B/C and may serve as a lead for developing new therapeutics to treat neutrophilic psoriasis. Antioxid. Redox Signal. 35, 885-903.

PAN-selective inhibition of cAMP-phosphodiesterase 4 (PDE4) induces gastroparesis in mice.

Inhibitors of cAMP-phosphodiesterase 4 (PDE4) exert a number of promising therapeutic benefits, but adverse effects, in particular emesis and nausea, have curbed their clinical utility. Here, we show that PAN-selective inhibition of PDE4, but not inhibition of PDE3, causes a time- and dose-dependent accumulation of chow in the stomachs of mice fed ad libitum without changing the animals' food intake or the weight of their intestines, suggesting that PDE4 inhibition impairs gastric emptying. Indeed, PDE4 inhibition induced gastric retention in an acute model of gastric motility that traces the passage of a food bolus through the stomach over a 30minutes time period. In humans, abnormal gastric retention of food is known as gastroparesis, a syndrome predominated by nausea (>90% of cases) and vomiting (>80% of cases). We thus explored the abnormal gastric retention induced by PDE4 inhibition in mice under the premise that it may represent a useful correlate of emesis and nausea. Delayed gastric emptying was produced by structurally distinct PAN-PDE4 inhibitors including Rolipram, Piclamilast, Roflumilast, and RS25344, suggesting that it is a class effect. PDE4 inhibitors induced gastric retention at similar or below doses commonly used to induce therapeutic benefits (e.g., 0.04mg/kg Rolipram), thus mirroring the narrow therapeutic window of PDE4 inhibitors in humans. YM976, a PAN-PDE4 inhibitor that does not efficiently cross the blood-brain barrier, induced gastroparesis only at significantly higher doses (≥1mg/kg). This suggests that PDE4 inhibition may act in part through effects on the autonomic nervous system regulation of gastric emptying and that PDE4 inhibitors that are not brain-penetrant may have an improved safety profile. The PDE4 family comprises four subtypes, PDE4A, B, C, and D. Selective ablation of any of these subtypes in mice did not induce gastroparesis per se, nor did it protect from PAN-PDE4 inhibitor-induced gastroparesis, indicating that gastric retention may result from the concurrent inhibition of multiple PDE4s. Thus, potentially, any of the four PDE4 subtypes may be targeted individually for therapeutic benefits without inducing nausea or emesis. Acute gastric retention induced by PDE4 inhibition is alleviated by treatment with the widely used prokinetic Metoclopramide, suggesting a potential of this drug to alleviate the side effects of PDE4 inhibitors. Finally, given that the cause of gastroparesis remains largely idiopathic, our findings open the possibility that a physiologic or pathophysiologic downregulation of PDE4 activity/expression may be causative in a subset of patients.