Selective Phosphodiesterase Inhibitors Research Articles

Use of a Fission Yeast Platform to Identify and Characterize Small Molecule PDE Inhibitors

Cyclic nucleotide phosphodiesterases (PDEs) have been proven to be targets for which highly selective and potent drugs can be developed. Mammalian genomes possess 21 genes whose products are pharmacologically grouped into 11 families; however related genes from pathogenic organisms display sufficient divergence from the mammalian homologs such that PDE inhibitors to these enzymes could be used to treat parasitic infections without acting on the related human PDEs. We have developed a platform for expressing cloned PDEs in the fission yeast Schizosaccharomyces pombe, allowing for inexpensive, but robust screening for small molecule inhibitors that are cell permeable. Such compounds typically display the expected biological activity when tested in cell culture, including anti-inflammatory properties for PDE4 and PDE7 inhibitors. The genetic pliability of S. pombe also allows for molecular genetic screens to identify mutations in target PDE genes that confer some resistance to these inhibitors as a way of investigating the PDE-inhibitor interaction. This screening method is readily accessible to academic laboratories as it does not require the purification of large quantities of a target protein. This allows for the discovery and profiling of PDE inhibitors to treat inflammation or of inhibitors of targets such as pathogen PDEs for which there may not be a sufficient financial motivation for pharmaceutical companies to identify selective PDE inhibitors using more traditional in vitro enzyme-based screening methods.

Phosphodiesterase‑4 inhibitors: a review of current developments (2013–2021)

ABSTRACT Introduction Cyclic nucleotide phosphodiesterase 4 (PDE4) is responsible for the hydrolysis of cAMP, which has become an attractive therapeutic target for lung, skin, and severe neurological diseases. Here, we review the current status of development of PDE4 inhibitors since 2013 and discuss the applicability of novel medicinal-chemistry strategies for identifying more efficient and safer inhibitors. Areas covered This review summarizes the clinical development of PDE4 inhibitors from 2013 to 2021, focused on their pharmacophores, the strategies to reduce the side effects of PDE4 inhibitors and the development of subfamily selective PDE4 inhibitors. Expert opinion To date, great efforts have been made in the development of PDE4 inhibitors, and researchers have established a comprehensive preclinical database and collected some promising data from clinical trials. Although four small-molecule PDE4 inhibitors have been approved by FDA for the treatment of human diseases up to now, further development of other reported PDE4 inhibitors with strong potency has been hampered due to the occurrence of severe side effects. There are currently three main strategies for overcoming the dose limitation and systemic side effects, which provide new opportunities for the clinical development of new PDE4 inhibitors.

Cilostazol for Aneurysmal Subarachnoid Hemorrhage: An Updated Systematic Review and Meta-Analysis

Background and Purpose: Delayed cerebral ischemia is a major cause of morbidity and mortality in patients with aneurysmal subarachnoid hemorrhage (aSAH). Cilostazol, a selective inhibitor of phosphodiesterase 3, was reported to reduce cerebral vasospasm and improve outcomes. We aimed to conduct an updated systematic review and meta-analysis of the efficacy and safety of cilostazol in aSAH. Methods: We systematically searched PubMed, Embase, MEDLINE, and the Cochrane Library for articles published in English with the latest publishing time in August 2020. Articles reporting favorable outcome as the primary outcome and reporting severe angiographic vasospasm (aVS), symptomatic vasospasm (sVS), new cerebral infarction, or mortality as the secondary outcome were included in this review. Furthermore, we examined whether clinical outcomes were associated with the dosage of cilostazol (300 mg/day vs. 100–200 mg/day). Results: Data from 405 patients in 4 randomized controlled trials (RCTs) and 461 patients in 4 observational studies (OSs) were included. In RCT studies, cilostazol was associated with significant favorable outcomes at discharge or 1 month (risk ratio [RR] 1.41, 95% confidence interval [CI] 1.01–1.97, p = 0.04) or 3 or 6 months (RR 1.16, 95% CI 1.05–1.28, p = 0.002). However, in OSs, no significant difference was indicated in favorable outcomes at discharge or 1 month (RR 1.22, 95% CI 0.94–1.60, p = 0.14) nor 3 or 6 months (RR 1.29, 95% CI 0.92–1.81, p = 0.14). The analyses found that cilostazol significantly reduced the incidences of severe aVS (RCT: RR 0.64, 95% CI 0.41–1.01, p = 0.05; OS: RR 0.61, 95% CI 0.43–0.88, p = 0.007), sVS (RCT: RR 0.46, 95% CI 0.31–0.70, p = 0.0002; OS: RR 0.38, 95% CI 0.21–0.68, p = 0.001), and new cerebral infarction (RCT: RR 0.40, 95% CI 0.24–0.67, p = 0.0005; OS: RR 0.38, 95% CI 0.23–0.64, p = 0.0002). However, no significant difference in mortality (RCT: RR 0.86, 95% CI 0.23–3.21, p = 0.82; OS: RR 0.16, 95% CI 0.02–1.24, p = 0.08) was found. In 3 OSs which reported different doses of cilostazol (300 mg/day vs. 100–200 mg/day) for aSAH, the 300-mg/day cilostazol groups showed decreased delayed cerebral infarction (RR 0.27, 95% CI 0.09–0.81, p = 0.02) but no significant difference in shunt-dependent hydrocephalus (RR 0.92, 95% CI 0.33–2.60, p = 0.88) or functional outcomes (RR 1.14, 95% CI 0.74–1.75, p = 0.56) compared with the 100–200 mg/day cilostazol groups. Conclusions: The meta-analyses suggest the credible efficacy and safety of cilostazol in treating aSAH. Furthermore, 300-mg/day cilostazol treatment appeared to be more effective than 100–200 mg/day treatment.

Roflupram attenuates α-synuclein-induced cytotoxicity and promotes the mitochondrial translocation of Parkin in SH-SY5Y cells overexpressing A53T mutant α-synuclein

We have previously shown that inhibition of cAMP-specific 3′,5′-cyclic phosphodiesterase 4 (PDE4) protects against cellular toxicity in neuronal cells. Since α-synuclein (α-syn) toxicity contributes to the neurodegeneration of Parkinson's disease (PD). The aim of this study was to explore the effects and mechanisms of PDE4 on α-syn-induced neuronal toxicity. Using mutant human A53T α-syn overexpressed SH-SY5Y cells, we found that PDE4B knockdown reduced cellular apoptosis. Roflupram (ROF, 20 μM), a selective PDE4 inhibitor, produced similar protective effects and restored the morphological alterations of mitochondria. Mechanistic studies identified that α-syn enhanced the phosphorylation of Parkin at Ser131, followed by the decreased mitochondrial translocation of Parkin. Whereas both PDE4B knockdown and PDE4 inhibition by ROF blocked the effects of α-syn on Parkin phosphorylation and mitochondrial translocation. Moreover, PDE4 inhibition reversed the increase in the phosphorylation of p38 mitogen-activated protein kinase (MAPK) induced by α-syn. ROF treatment also reduced the binding of p38 MAPK to Parkin. Consistently, overexpression of PDE4B blocked the roles of ROF on p38 MAPK phosphorylation, Parkin phosphorylation, and the subsequent mitochondrial translocation of parkin. Furthermore, PDE4B overexpression attenuated the protective role of ROF, as evidenced by reduced mitochondria membrane potential and increased cellular apoptosis. Interestingly, ROF failed to suppress α-syn-induced cytotoxicity in the presence of a protein kinase A (PKA) inhibitor H-89. Our findings indicate that PDE4 facilitates α-syn-induced cytotoxicity via the PKA/p38 MAPK/Parkin pathway in SH-SY5Y cells overexpressing A53T mutant α-synuclein. PDE4 inhibition by ROF is a promising strategy for the prevention and treatment of α-syn-induced neurodegeneration.

Clinical Pharmacology of Sildenafil in Infants and Children

Sildenafil is a competitive and selective inhibitor of phosphodiesterase 5. Sildenafil is cleared by hepatic CYP3A (major route) and CYP2C9 (minor route) and concomitant administration of potent CYP3A inducers (e.g., bosentan) causes decreases in plasma levels of sildenafil. CYP3A4 inhibitors (erythromycin and cimetidine) inhibit sildenafil metabolism prolonging the half-life and elevating blood levels of sildenafil. Sildenafil is a pulmonary arterial vasodilator and it has been used in the treatment of persistent pulmonary hypertension. The initial oral dose is 250 to 500 µg/kg 4 times-daily in infants and the oral dose is 10 to 20 mg thrice-daily in children with a body-weight up to 20 kg or > 20 kg, respectively. Sildenafil has been found efficacy and safe in infants and children but it may induce adverse-effects. Following an oral dosing, the absorption rate constant is 0.343 h-1, and the elimination half-life is 2.41 hours in children suggesting that sildenafil is rapidly absorbed and eliminated. The interaction of sildenafil with drugs and the metabolism of sildenafil have been extensively studied. The principal routes of sildenafil metabolism are: N-demethylation, oxidation, and aliphatic dihydroxylation, and the major metabolite is N-desmethyl sildenafil. The treatment of infants and children with sildenafil has been extensively studied. Sildenafil citrate and sildenafil cross the human placenta and sildenafil migrates into the breast-milk in significant amounts. The aim of this study is to review the sildenafil dosing, efficacy and safety, effects, adverse-effects, pharmacokinetics, interaction with drugs, metabolism, treatments, and sildenafil placental transfer and migration into the breast-milk.

Sildenafil-Mediated Neuroprotection from Adult to Neonatal Brain Injury: Evidence, Mechanisms, and Future Translation.

Cerebral stroke, traumatic brain injury, and hypoxic ischemic encephalopathy are among the most frequently occurring brain injuries. A complex pathogenesis, characterized by a synergistic interaction between alterations of the cerebrovascular system, cell death, and inflammation, is at the basis of the brain damage that leads to behavioral and neurodevelopmental disabilities in affected subjects. Sildenafil is a selective inhibitor of the enzyme phosphodiesterase 5 (PDE5) that is able to cross the blood–brain barrier. Preclinical data suggest that sildenafil may be a good candidate for the prevention or repair of brain injury in both adults and neonates. The aim of this review is to summarize the evidence supporting the neuroprotective action of sildenafil and discuss the possible benefits of the association of sildenafil with current therapeutic strategies.

Hybrid NiO nanostructured/sulfanilamide polymeric film for studying possible pharmacokinetic interaction between avanafil and nimodipine in real human serum by their simultaneous determination using square-wave voltammetry

Avanafil (AVN) is a new selective phosphodiesterase-5 (PDE-5) inhibitor, which is used as an oral treatment of erectile dysfunction. Coadministration of avanafil (AVN) with some antihypertensive drugs especially in elderly patients is a very common case. Nimodipine (NIM) is one of these hypotensive drugs that are commonly coadministered. Synergistic hypotension effect as a result of AVN and NIM coadministration is expected; therefore, it is mandatory to study the possible pharmacokinetic interaction between them. The main aim of the current work is to develop a simple, sensitive, and selective electrochemical method for simultaneous determination of AVN and NIM in human serum samples. Nickel oxide nanoparticles/poly(sulfanilamide) film was used as a new electrochemical modifier, which is electrodeposited on the surface of pencil graphite electrode (PGE) to improve its electrochemical properties. The best resolution between the two studied drugs was achieved by using Britton-Robinson buffer (BRB) at pH 3.0 producing two peaks at 1.45 V and 0.80 V for AVN and NIM, respectively. Furthermore, cyclic voltammetry was utilized for the first time to study the oxidation behavior of AVN and NIM and a plausible oxidation mechanism was suggested for both of them. The proposed square wave voltammetric method was successfully applied for trace quantification of AVN and NIM in real human serum samples with detection and quantitation limits of 0.037 and 0.10 μmol L−1 for AVN and 0.32 and 0.98 μmol L−1 for NIM, respectively. Moreover, the suggested approach was effectively implemented to investigate the possible pharmacokinetic interaction between AVN and NIM in serum samples of heathy human male volunteers. It was found from the pharmacokinetic parameters calculated for AVN when administered alone or in presence of NIM that there is a significant increase of serum concentration of AVN, when it is coadminstered with NIM, which stresses the importance of dose adjustment of AVN when coadministered with NIM.

Upregulation of Phosphodiesterase 2A Augments T Cell Activation by Changing cGMP/cAMP Cross-Talk.

3′,5′-cyclic adenosine monophosphate (cAMP) is well-known for its diverse immunomodulatory properties, primarily inhibitory effects during T cell activation, proliferation, and production of pro-inflammatory cytokines. A decrease in cAMP levels, due to the hydrolyzing activity of phosphodiesterases (PDE), is favoring inflammatory responses. This can be prevented by selective PDE inhibitors, which makes PDEs important therapeutic targets for autoimmune disorders. In this study, we investigated the specific roles of PDE2A and PDE3B in the regulation of intracellular cAMP levels in different mouse T cell subsets. Unexpectedly, T cell receptor (TCR) activation led to a selective upregulation of PDE2A at the protein level in conventional T cells (Tcon), whereas no changes were detected in regulatory T cells (Treg). In contrast, protein expression of PDE3B was significantly higher in both non-activated and activated Tcon subsets as compared to Treg, with no changes upon TCR engagement. Live-cell imaging of T cells expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, has enabled cAMP measurements in real time and revealed stronger responses to the PDE2A inhibitors in activated vs non-activated Tcon. Importantly, stimulation of intracellular cGMP levels with natriuretic peptides led to an increase of cAMP in non-activated and a decrease of cAMP in activated Tcon, suggesting that TCR activation changes the PDE3B-dependent positive to PDE2A-dependent negative cGMP/cAMP cross-talk. Functionally, this switch induced higher expression of early activation markers CD25 and CD69. This constitutes a potentially interesting feed-forward mechanism during autoimmune and inflammatory responses that may be exploited therapeutically.

27874 Correlation of itch response to roflumilast cream with disease severity and patient-reported outcomes in patients with chronic plaque psoriasis

Roflumilast cream is a nonsteroidal, selective phosphodiesterase-4 inhibitor in development for plaque psoriasis (PsO). A Phase 2b, double-blinded trial randomized adults with PsO (2-20% body surface area) to once daily roflumilast 0.3%, roflumilast 0.15%, or vehicle for 12 weeks (NCT03638258). Throughout the trial, itch and its impact were evaluated via patient reported outcomes (PROs): Worst Itch Numeric Rating Scale (WI–NRS), Itch related Sleep Loss (IRSL), and Dermatology Life Quality Index (DLQI).

Phosphodiesterase-4 Inhibitors for Non-COPD Respiratory Diseases.

Selective phosphodiesterase (PDE) inhibitors are a class of nonsteroid anti-inflammatory drugs for treating chronic inflammatory diseases. Modulation of systemic and airway inflammation is their pivotal mechanism of action. Furthermore, PDE inhibitors modulate cough reflex and inhibit airway mucus secretion. Roflumilast, a selective PDE4 inhibitor, has been extensively studied for the efficacy and safety in chronic obstructive pulmonary disease (COPD) patients. According to the mechanisms of action, the potential roles of PDE inhibitors in treating chronic respiratory diseases including severe asthma, asthma-COPD overlap (ACO), noncystic fibrosis bronchiectasis, and chronic cough are discussed. Since roflumilast inhibits airway eosinophilia and neutrophilia in COPD patients, it reduces COPD exacerbations in the presence of chronic bronchitis in addition to baseline therapies. The clinical studies in asthma patients have shown the comparable efficacy of roflumilast to inhaled corticosteroids for improving lung function. However, the clinical trials of roflumilast in severe asthma have been limited. Although ACO is common and is also associated with poor outcomes, there is no clinical trial regarding its efficacy in patients with ACO despite a promising role in reducing COPD exacerbation. Since mucus hypersecretion is a result of neutrophil secretagogue in patients with chronic bronchitis, experimental studies have shown that PDE4s are regulators of the cystic fibrosis transmembrane conductance regulator (CFTR) in human airway epithelial cells. Besides, goblet cell hyperplasia is associated with an increased expression of PDE. Bronchiectasis and chronic bronchitis are considered neutrophilic airway diseases presenting with mucus hypersecretion. They commonly coexist and thus lead to severe disease. The role of roflumilast in noncystic fibrosis bronchiectasis is under investigation in clinical trials. Lastly, PDE inhibitors have been shown modulating cough from bronchodilation, suppressing transient receptors potential (TRP), and anti-inflammatory properties. Hence, there is the potential role of the drug in the management of unexplained cough. However, clinical trials for examining its antitussive efficacy are pivotal. In conclusion, selective PDE4 inhibitors may be potential treatment options for chronic respiratory diseases apart from COPD due to their promising mechanisms of action.

Apremilast downregulates interleukin-17 production and induces splenic regulatory B cells and regulatory T cells in imiquimod-induced psoriasiform dermatitis

BackgroundApremilast, a selective inhibitor of the enzyme phosphodiesterase 4, is efficacious for psoriasis. However, detailed in vivo effects of apremilast on psoriasis remain to be elucidated. ObjectiveTo examine the in vivo effects of apremilast on psoriasis. MethodsPsoriasiform dermatitis was induced by applying imiquimod (IMQ) on the murine shaved back skin for six days. Mice were treated with apremilast or vehicle intraperitoneally daily. ResultsApremilast alleviated IMQ-induced psoriasiform dermatitis clinically and pathologically on days 3–6 by reducing infiltration of antigen-presenting cells and interleukin (IL)-17A-positive cells and increasing infiltration of Foxp3-postive cells into the skin on day 6, although a significant increase in IL-10 mRNA level was not observed on day 2. In addition, mRNA expression of IL-17A, IL-17F, and IL-22 was lower in the skin of IMQ-applied mice treated with apremilast than in those without apremilast on day 2, and apremilast inhibited infiltration of IL-17A-producing γδ T cells into the dermis on day 6. Furthermore, apremilast induced regulatory T cells and regulatory B cells in the spleen but not in the draining lymph nodes. ConclusionApremilast downregulated IL-17 production and induced splenic regulatory B cells and regulatory T cells in an IMQ-induced psoriasiform dermatitis mouse model.

Positive effects of roflumilast on behavior, neuroinflammation, and white matter injury in mice with global cerebral ischemia.

Inhibition of phosphodiesterase 4 (PDE4) is a promising pharmacological strategy for the treatment of cerebral ischemic conditions. To increase the relevance and increase the translational value of preclinical studies, it is important to conduct experiments using different animal species and strains, different animal models, and to evaluate long-term functional outcomes after cerebral ischemia. In the present study, the effects of the selective PDE4 inhibitor roflumilast were evaluated in vivo and in vitro. Balb/c mice were subjected to bilateral common carotid artery occlusion (BCCAO) and tested during 21 days in multiple behavioral tasks to investigate the long-term effects of roflumilast on functional recovery. The effects of roflumilast were also investigated on hippocampal cell loss, white matter injury, and expression of neuroinflammatory markers. Roflumilast prevented cognitive and emotional deficits induced by BCCAO in mice. Roflumilast also prevented neurodegeneration and reduced the white matter damage in the brain of ischemic animals. Besides, roflumilast decreased Iba-1 (microglia marker) levels and increased Arginase-1 (Arg-1; microglia M2 phenotype marker) levels in the hippocampus of these mice. Likewise, roflumilast suppressed inducible nitric oxide synthase (microglia M1 phenotype marker) expression and increased Arg-1 levels in a primary mouse microglia culture. These findings support evidence that PDE4 inhibition by roflumilast might be beneficial in cerebral ischemic conditions. The neuroprotective effects of roflumilast appear to be mediated by a decrease in neuroinflammation.

PDE3 Inhibition by Cilostazol Ameliorated Behavioral and Biochemical Deficits in Prenatal Alcohol Induced Experimental ADHD

Background: Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder with complex aetiology and phenotypes. Maternal consumption of alcohol is known to produce deleterious effects in the progeny, generating ADHD-related phenotypes. Phosphodiesterase-3 (PDE3) has been shown to provide benefits in various brain conditions. Objective: To investigate the role of a selective PDE3 inhibitor, effects of cilostazol administration on core phenotypes of Prenatal Alcohol Exposure (PAE) model of ADHD were assessed. Methods: PAE was established by exposing animals to 6/4 g.kg-1 (weekdays/weekends) ethyl alcohol from gestational days 8-20 and cilostazol (30/60 mg.kg-1 p.o.) was administered to the offspring (PND21- 48) of females exposed to ethyl alcohol. To identify probable mechanisms involved, the effects on protein markers of neuronal function such as, neuronal survival-BDNF, neuronal transcription factor-pCREB, brain inflammation (IL-6, IL-10 and TNF-α) and brain oxidative stress (TBARS and GSH) were studied in frontal cortex, cerebellum, and striatum. Also, effects on behaviour such as hyperactivity, inattention and anxiety were assessed. Result: PAE induced hyper-locomotion, inattention, and anxiety in tested animals. Administration of cilostazol to PAE group of animals resulted in amelioration of behavioural deficits. Also, cilostazol resulted in a significant increase in the levels of BDNF, pCREB, IL-10 and GSH along with a significant decrease in TNF-α, IL-6 and TBARS in different brain areas of PAE group. Conclusion: Cilostazol, a selective PDE3 inhibitor rectified behavioural phenotypes associated with ADHD, possibly by altering protein markers associated with neuronal survival, neuronal transcription factor, brain inflammation, and brain oxidative stress.

Roflumilast Suppresses Adipogenic Differentiationvia AMPK Mediated Pathway.

Obesity and related disorders have increasingly become global health problems over the years. In recent years, obesity has been recognized as the most important risk factor for a variety of diseases including cardiovascular diseases, type 2 diabetes, steatohepatitis, and cancer. The medical anti-obesity treatment is to intervene in the metabolic process of adipocytes by suppressing adipogenesis and promoting lipolysis. The Phosphodiesterase-4 (PDE4) pathway is involved in fat mass control and metabolic regulation. The present study aimed to investigate the effects of Roflumilast, a selective PDE4 inhibitor, on the differentiation of 3T3-L1 cells and the high fat diet-induced obesity in mice. We showed that treatment with Roflumilast inhibited lipid accumulation and triglycerides storage in mature 3T3-L1 cells, suggesting that Roflumilast suppressed adipogenesis. Mechanistically, we found that Roflumilast decreased the differentiation-induced expression of the adipogenesis genes including SREBP1C, FABP4, and Glut4, as well as their regulators including PPAR-γ and C/EBPα. Moreover, we proved that the effect of Roflumilast was dependent on the activation of the metabolic regulator AMPKα. The treatment with Roflumilast remarkably decreased the animals’ body weight, visceral adipose tissue weight, and adipocyte size in high fat diet-induced obese mice. In conclusion, our study revealed that Roflumilast suppressed adipogenesis and promoted lipolysis in cell culture and mice models via AMPK-mediated inhibition of PPAR-γ and C/EBPα. These findings imply roflumilast could have therapeutic potential in obesity-related diseases.

Selective Inhibition of Phosphodiesterase 7 Enzymes Reduces Motivation for Nicotine Use through Modulation of Mesolimbic Dopaminergic Transmission.

Approximately 5 million people die from diseases related to nicotine addiction and tobacco use each year. The nicotine-induced increase of corticomesolimbic dopaminergic (DAergic) transmission and hypodopaminergic conditions occurring during abstinence are important for maintaining drug-use habits. We examined the notion of reequilibrating DAergic transmission by inhibiting phosphodiesterase 7 (PDE7), an intracellular enzyme highly expressed in the corticomesolimbic circuitry and responsible for the degradation of cyclic adenosine monophosphate (cAMP), the main second messenger modulated by DA receptor activation. Using selective PDE7 inhibitors, we demonstrated in male rats that systemic PDE7 enzyme inhibition reduced nicotine self-administration and prevented reinstatement to nicotine seeking evoked by cues or by the pharmacological stressor yohimbine. The effect was also observed by direct application of the PDE7 inhibitors into the nucleus accumbens (NAc) shell but not into the core. Inhibition of PDE7 resulted in increased DA- and cAMP-regulated neuronal phosphoprotein and cAMP response element-binding protein and their phosphorylated forms in the NAc. It also enhanced the DA D1 receptor agonism-mediated effects, indicating potentiation of protein kinase A–dependent transmission downstream of D1 receptor activation. In electrophysiological recordings from DA neurons in the lateral posterior ventral tegmental area, the PDE7 inhibitors attenuated the spontaneous activity of DA neurons. This effect was exerted through the potentiation of D1 receptor signaling and the subsequent facilitation of γ-aminobutyric acid transmission. The PDE7 inhibitors did not elicit conditioned place preference and did not induce intravenous self-administration, indicating lack of reinforcing properties. Thus, PDE7 inhibitors have the potential to treat nicotine abuse.SIGNIFICANCE STATEMENT The World Health Organization estimates that there are 1.25 billion smokers worldwide, representing one-third of the global population over the age of 15. Nicotine-induced increase of corticomesolimbic DAergic transmission and hypodopaminergic conditions occurring during abstinence are critical for maintaining drug-use habits. Here, we demonstrate that nicotine consumption and relapse to nicotine seeking are attenuated by reequilibrating DAergic transmission through inhibition of PDE7, an intracellular enzyme responsible for the degradation of cAMP, the main second messenger modulated by DA receptor activation. PDE7 inhibition may represent a novel treatment approach to aid smoking cessation.

Role of Roflumilast Combined with ESHAP Chemotherapy in Relapsed/Refractory Patients with Diffuse Large B-Cell Lymphoma.

PurposeThere are unmet needs associated with the current treatment strategies for relapsed/refractory diffuse large B-cell lymphoma (DLBCL) due to the poor treatment outcomes of these strategies. Roflumilast, a selective phosphodiesterase-4 inhibitor used for treating chronic obstructive pulmonary disease, is effective against B-cell malignancy via phosphoinositide 3-kinase (PI3K)–activity suppression. We analyzed the effects of roflumilast combined with ESHAP (etoposide, cisplatin, methylprednisolone, and cytarabine) chemotherapy in experimental and clinical settings.Materials and MethodsAn in vitro study using lymphoma cell lines and a pilot study on relapsed/refractory DLBCL patients were conducted to investigate the effects and mechanism of the combination of roflumilast and chemotherapy. The complete response (CR), overall response rate (ORR), and 1-year progression-free survival (PFS) were analyzed.ResultsWe found that roflumilast is efficient when combined with other chemotherapy drugs, especially cytarabine. Synergistic effects between these two drugs influence the translation of mammalian target of rapamycin and myeloid cell leukemia 1, resulting in apoptosis and inhibition of B-cell lymphoma proliferation. In clinical setting, the roflumilast group showed better rates of CR (46.2% vs. 34.6%), ORR (76.9% vs. 53.8%), and 1-year PFS (50.0% vs. 25.9%) compared with the control group, though not statistically significant. The roflumilast group showed a higher incidence of asthenia and gastrointestinal adverse events. However, grade 3 or 4 adverse events were similar in both groups.ConclusionWe found that roflumilast, when combined with ESHAP chemotherapy, for relapsed/refractory DLBCL was clinically active and well tolerated. This combined treatment was able to suppress PI3K activity, which is correlated with the degree of clinical response.

467 Novel selective phosphodiesterase inhibitors promote the adipogenic function of dermal fibroblasts: Implication to treat hair loss

467 Novel selective phosphodiesterase inhibitors promote the adipogenic function of dermal fibroblasts: Implication to treat hair loss

Phosphodiesterase Inhibitors in Acute Lung Injury: What Are the Perspectives?

Despite progress in understanding the pathophysiology of acute lung damage, currently approved treatment possibilities are limited to lung-protective ventilation, prone positioning, and supportive interventions. Various pharmacological approaches have also been tested, with neuromuscular blockers and corticosteroids considered as the most promising. However, inhibitors of phosphodiesterases (PDEs) also exert a broad spectrum of favorable effects potentially beneficial in acute lung damage. This article reviews pharmacological action and therapeutical potential of nonselective and selective PDE inhibitors and summarizes the results from available studies focused on the use of PDE inhibitors in animal models and clinical studies, including their adverse effects. The data suggest that xanthines as representatives of nonselective PDE inhibitors may reduce acute lung damage, and decrease mortality and length of hospital stay. Various (selective) PDE3, PDE4, and PDE5 inhibitors have also demonstrated stabilization of the pulmonary epithelial–endothelial barrier and reduction the sepsis- and inflammation-increased microvascular permeability, and suppression of the production of inflammatory mediators, which finally resulted in improved oxygenation and ventilatory parameters. However, the current lack of sufficient clinical evidence limits their recommendation for a broader use. A separate chapter focuses on involvement of cyclic adenosine monophosphate (cAMP) and PDE-related changes in its metabolism in association with coronavirus disease 2019 (COVID-19). The chapter illuminates perspectives of the use of PDE inhibitors as an add-on treatment based on actual experimental and clinical trials with preliminary data suggesting their potential benefit.

Phosphoproteomic Analysis as an Approach for Understanding Molecular Mechanisms of cAMP-Dependent Actions.

In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cAMP. Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This minireview describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors' laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone-producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate-limiting step likely regulate these and, by analogy, many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. SIGNIFICANCE STATEMENT: Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment, providing many possible regulatory targets but also highlighting questions about which phosphosites are functionally important. These data imply that multistep processes are regulated by phosphorylation at not one but rather many sites. Most previous studies assumed a single step or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed substoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed.

Cilomilast Ameliorates Renal Tubulointerstitial Fibrosis by Inhibiting the TGF-β1-Smad2/3 Signaling Pathway.

Background: Renal tubulointerstitial fibrosis is the key pathological feature in chronic kidney diseases (CKDs) with no satisfactory therapies in clinic. Cilomilast is a second-generation, selective phosphodiesterase-4 inhibitor, but its role in renal tubulointerstitial fibrosis in CKD remains unclear.Material and Methods: Cilomilast was applied to the mice with unilateral ureteric obstruction (UUO) and renal fibroblast cells (NRK-49F) stimulated by TGF-β1. Renal tubulointerstitial fibrosis and inflammation after UUO or TGF-β1 stimulation were examined by histology, Western blotting, real-time PCR and immunohistochemistry. KIM-1 and NGAL were detected to evaluate tubular injury in UUO mice.Results: In vivo, immunohistochemistry and western blot data demonstrated that cilomilast treatment inhibited extracellular matrix deposition, profibrotic gene expression, and the inflammatory response. Furthermore, cilomilast prevented tubular injury in UUO mice, as manifested by reduced expression of KIM-1 and NGAL in the kidney. In vitro, cilomilast attenuated the activation of fibroblast cells stimulated by TGF-β1, as shown by the reduced expression of fibronectin, α-SMA, collagen I, and collagen III. Cilomilast also inhibited the activation of TGF-β1-Smad2/3 signaling in TGF-β1-treated fibroblast cells.Conclusion: The findings of this study suggest that cilomilast is protective against renal tubulointerstitial fibrosis in CKD, possibly through the inhibition of TGF-β1-Smad2/3 signaling, indicating the translational potential of this drug in treating CKD.