Phosphodiesterase 4 Inhibitor Research Articles

Identification of phosphodiesterase-4 as the therapeutic target of arctigenin in alleviating psoriatic skin inflammation

IntroductionArctigenin, derived from Arctium lappa L., has multiple pharmacological activities, including immunoregulatory, anti-diabetic, anti-tumor, and neuroprotective effects. Nevertheless, the potential therapeutic target of arctigenin in modulating inflammation remains undefined. ObjectivesIn the present study, we identified that arctigenin was a phosphodiesterase-4 (PDE4) selective inhibitor for the first time. Further investigations were performed to fully uncover the effects and mechanism of arctigenin on experimental murine psoriasis model. MethodsCrystal structure determination, PDEs enzyme assay, and isothermal titration calorimetry were included to illustrate the binding specialty, inhibitory effects, and selectivity of arctigenin on PDE4D. The anti-inflammatory effects were conducted in LPS-activated human peripheral blood mononuclear cells (PBMCs) and RAW264.7 cells. Imiquimod-induced murine psoriasis was performed to uncover the therapeutic effects and mechanism of arctigenin in vivo. ResultsArctigenin could bind to the catalytic domain of PDE4D via formation of hydrogen bonds as well as π-π stacking interactions between the dibenzyl butyrolactone of arctigenin and several residues of PDE4D. Accordingly, arctigenin showed prominent anti-inflammation in human PBMCs and murine RAW264.7 cells. PDE4 inhibition by arctigenin resulted in elevation of intracellular cyclic adenosine monophosphate (cAMP) and phosphorylation of cAMP-response element binding protein (CREB), which were largely blocked through intervention of protein kinase A (PKA) activity by H89 treatment or reduction of protein expression by siRNA transfection. Moreover, we first identified that a topical application of arctigenin ameliorated experimental psoriatic manifestations in imiquimod-induced murine psoriasis model by decreasing adhesion and chemotaxis of several inflammatory cells. Further proteomics analysis revealed that arctigenin could rectify the immune dysfunction and hyperactivation of keratinocytes in the inflamed skin microenvironments, which might be largely related to the expression of Keratins. ConclusionThe research provided credible clew that inhibition of PDE4 by arctigenin might function as the potential therapeutic approach for the treatment of psoriasis.

Pharmacological rescue in patient iPSC and mouse models with a rare DISC1 mutation

We previously identified a causal link between a rare patient mutation in DISC1 (disrupted-in-schizophrenia 1) and synaptic deficits in cortical neurons differentiated from isogenic patient-derived induced pluripotent stem cells (iPSCs). Here we find that transcripts related to phosphodiesterase 4 (PDE4) signaling are significantly elevated in human cortical neurons differentiated from iPSCs with the DISC1 mutation and that inhibition of PDE4 or activation of the cAMP signaling pathway functionally rescues synaptic deficits. We further generated a knock-in mouse line harboring the same patient mutation in the Disc1 gene. Heterozygous Disc1 mutant mice exhibit elevated levels of PDE4s and synaptic abnormalities in the brain, and social and cognitive behavioral deficits. Pharmacological inhibition of the PDE4 signaling pathway rescues these synaptic, social and cognitive behavioral abnormalities. Our study shows that patient-derived isogenic iPSC and humanized mouse disease models are integral and complementary for translational studies with a better understanding of underlying molecular mechanisms.

Phosphodiesterase-4 Inhibition in Psoriasis.

Psoriasis is a chronic immune-mediated inflammatory disorder. Phosphodiesterase-4 (PDE-4) is an enzyme that mediates inflammatory responses and plays a role in psoriasis pathogenesis. PDE-4 degrades its substrate cyclic adenosine monophosphate (cAMP) to adenosine monophosphate (AMP), which subsequently leads to the production of pro-inflammatory mediators. Inhibitors of PDE-4 work by blocking the degradation of cAMP, which leads to a reduction in inflammation. Apremilast is the only approved oral PDE-4 inhibitor for the treatment of psoriasis. While it is effective for some patients, it may be limited by adverse effects in others. A topical PDE-4 inhibitor, roflumilast, is being investigated in psoriasis and showing promising results. Crisaborole, a topical PDE-4 inhibitor approved for use in atopic dermatitis, has also been investigated in psoriasis. This is an updated comprehensive review to summarize the currently available evidence for the PDE-4 inhibitors apremilast, roflumilast and crisaborole in the treatment of psoriasis, with a focus on data from randomized clinical trials.

QSAR and docking studies of 3, 5-dimethylpyrazole as potent inhibitors of Phosphodiesterase-4

A quantitative structure-activity relationship (QSAR) study was performed to develop a model on a series of 3, 5-dimethylpyrazole containing furan moiety derivatives which exhibited considerable inhibitory activity against PDE4B. The obtained model has correlation coefficient (r) of 0.934, squared correlation coefficient (r2) of 0.872, and leave-one-out (LOO) cross-validation coefficient (Q2) value of 0.733. The predictive power of the developed model was confirmed by the external validation which has (r2) value of 0.812. These parameters confirm the stability and robustness of the model to predict the activity of a new designed set of 3,5-dimethyl-pyrazole derivatives (I-XV), results indicated that the compound III, V, XIII, and XV showed the strongest inhibition activity (IC50 = 0.2813, 0.5814, 0.6929, 0.6125μM, respectively) against PDE4B compared to the reference rolipram with (IC50=1.9μM). Molecular docking was performed on a new designed compound with PDE4B protein (3o0j). Docking results showed that compounds (X and IX) have high docking affinity of -36.2037 and -33.2888 kcal/mol respectively.
 Keywords: QSAR, molecular docking, pyrazole derivatives, PDE4 inhibitors, anti-inflammatory.

Roflumilast Ameliorates Isoflurane-Induced Inflammation in Astrocytes via the CREB/BDNF Signaling Pathway.

Background and purpose: Astrocyte-mediated neuroinflammation plays an important role in anesthetic isoflurane-induced cognitive impairment. Roflumilast, a selective inhibitor of phosphodiesterase-4 (PDE-4) used for the treatment of chronic obstructive pulmonary disease (COPD), has displayed a wide range of anti-inflammatory capacity in different types of cells and tissues. In the current study, we aimed to investigate whether roflumilast possesses a protective effect against isoflurane-induced insults in mouse primary astrocytes. Methods: Primary astrocytes were isolated from the cerebral cortices of immature rats. The production of NO was determined using DAF-FM DA staining assay. QRT-PCR and western blot were used to evaluate the expression levels of iNOS, COX-2, and BDNF in the astrocytes treated with different therapies. The gene expressions and concentrations of IL-6 and MCP-1 released by the astrocytes were detected using qRT-PCR and ELISA, respectively. The expression levels of phosphorylated CREB and PGE2 were determined using western blot and ELISA, respectively. H89 was introduced to evaluate the function of CREB. Recombinant human BDNF and ANA-12 were used to verify the role of BDNF. Results: The upregulated iNOS, excessive production of NO, IL-6, and MCP-1, and activated COX-2/PGE2 signaling pathways in the astrocytes induced by isoflurane were significantly reversed by the introduction of roflumilast, in a dose-dependent manner. Subsequently, we found that BDNF could be upregulated by roflumilast, which was verified to be related to the activation of CREB and blocked by H89 (a CREB inhibitor). In addition, the COX-2/PGE2 signaling pathway activated by isoflurane can be inactivated by recombinant human BDNF. Finally, the regulatory effect of roflumilast against the isoflurane-activated COX-2/PGE2 signaling pathway was significantly blocked by ANA-12, which is a BDNF inhibitor. Conclusion: Roflumilast might ameliorate isoflurane-induced inflammation in astrocytes via the CREB/BDNF signaling pathway.

Roflumilast: A Review of Chronic Obstructive Pulmonary Disease (COPD) Treatment

Abstract Chronic Obstructive Pulmonary Disease (COPD) is a chronic airway inflammation with resulting progressive airflow limitation that has a high incidence, morbidity, and mortality. Roflumilast is an oral phosphodiesterase-4 inhibitor as a therapy to decrease the risk of COPD exacerbations in patients with moderate-severe COPD with a history of chronic bronchitis. Roflumilast can be given orally once daily as a single or combination drug. It can be used as COPD moderate-severe but also more beneficial as COPD mild treatment. The efficacy of Roflumilast can prevent exacerbations of repetition and can fixed lung function both in terms of FEV1 and vital capacity to force. The incidence of side effects, which are diarrhea and digestive disorders. The cost-effectiveness showed that Quality of life patient with Roflumilast is better than the group without additional. This paper aimed to review systematically Roflumilast as COPD treatment for the clinical application.Keywords : COPD, Roflumilast, phosphodiesterase-4 inhibitorCorrespondence : fauna@staff.ubaya.ac.id

Roflumilast protects against spatial memory impairments and exerts anti-inflammatory effects after transient global cerebral ischemia.

Phosphodiesterase 4 (PDE4) inhibitors have been shown to present beneficial effects in cerebral ischemic injury because of their ability to improve cognition and target different phases and mechanisms of cerebral ischemia, including apoptosis, neurogenesis, angiogenesis, and inflammation. The present study investigated whether repeated treatment with the PDE4 inhibitor roflumilast rescued memory loss and attenuated neuroinflammation in rats following transient global cerebral ischemia (TGCI). TGCI caused memory impairments, neuronal loss (reflected by Neuronal nuclei (NeuN) immunoreactivity), and compensatory neurogenesis (reflected by doublecortin (DCX) immunoreactivity) in the hippocampus. Also, increases in the protein expression of the phosphorylated response element-binding protein (pCREB) and inflammatory markers such as the glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba-1), were detected in the hippocampus in TGCI rats. Repeated treatment with roflumilast (0.003 and 0.01mg/kg) prevented spatial memory deficits without promoting hippocampal protection in ischemic animals. Roflumilast increased the levels of pCREB, arginase-1, interleukin (IL) 4, and IL-10 in the hippocampus 21days after TGCI. These data suggest a protective effect of roflumilast against functional sequelae of cerebral ischemia, which might be related to its anti-inflammatory properties.

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.

Roflumilast Reduced the IL-18-Induced Inflammatory Response in Fibroblast-Like Synoviocytes (FLS).

Pro-inflammatory cytokines, such as the IL-18-induced inflammatory response and associated damage in fibroblast-like synoviocytes (FLS), play an important role in the pathogenesis of rheumatoid arthritis (RA). Roflumilast, an inhibitor of phosphodiesterase-4 (PDE-4), has been licensed for the treatment of chronic obstructive pulmonary disease (COPD). However, it is unknown whether roflumilast possesses a protective effect against the IL-18-induced inflammatory response in FLS. We found that roflumilast attenuated IL-18-induced oxidative stress by reducing the production of reactive oxygen species and malondialdehyde (MDA) in MH7A fibroblast-like synoviocytes (FLS). Additionally, roflumilast prevented IL-18-induced expressions and secretions of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. Importantly, we found that roflumilast inhibited IL-18-induced expressions of chemokines such as CCL5, CXCL9, and CXCL10. Further, roflumilast inhibited the expression of extracellular matrix degradative enzymes, such as matrix metalloproteinase-3 (MMP-3) and MMP-13. Mechanistically, we found that roflumilast suppressed the activation of the transcriptional factor AP-1 and NF-κB. Our results suggest that roflumilast might be a potential therapeutic agent for the treatment of RA.

Modulation of cAMP metabolism for CFTR potentiation in human airway epithelial cells

Cystic fibrosis (CF) is a genetic disease characterized by CF transmembrane regulator (CFTR) dysfunction. With over 2000 CFTR variants identified, in addition to known patient to patient variability, there is a need for personalized treatment. The discovery of CFTR modulators has shown efficacy in certain CF populations, however there are still CF populations without valid therapeutic options. With evidence suggesting that single drug therapeutics are insufficient for optimal management of CF disease, there has been an increased pursuit of combinatorial therapies. Our aim was to test cyclic AMP (cAMP) modulation, through ATP Binding Cassette Transporter C4 (ABCC4) and phosphodiesterase-4 (PDE-4) inhibition, as a potential add-on therapeutic to a clinically approved CFTR modulator, VX-770, as a method for increasing CFTR activity. Human airway epithelial cells (Calu-3) were used to test the efficacy of cAMP modulation by ABCC4 and PDE-4 inhibition through a series of concentration–response studies. Our results showed that cAMP modulation, in combination with VX-770, led to an increase in CFTR activity via an increase in sensitivity when compared to treatment of VX-770 alone. Our study suggests that cAMP modulation has potential to be pursued as an add-on therapy for the optimal management of CF disease.

A Perspective on Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against Neurodegeneration.

As life expectancy increases, dementia affects a growing number of people worldwide. Besides current treatments, phosphodiesterase 9 (PDE9) represents an alternative target for developing innovative small molecules to contrast neurodegeneration. PDE inhibition promotes neurotransmitter release, amelioration of microvascular dysfunction, and neuronal plasticity. This review will provide an update on natural and nature-inspired PDE9 inhibitors, with a focus on the structural features of PDE9 that encourage the development of isoform-selective ligands. The expression in the brain, the presence within its structure of a peculiar accessory pocket, the asymmetry between the two subunits composing the protein dimer, and the selectivity towards chiral species make PDE9 a suitable target to develop specific inhibitors. Additionally, the world of natural compounds is an ideal source for identifying novel, possibly asymmetric, scaffolds, and xanthines, flavonoids, neolignans, and their derivatives are currently being studied. In this review, the available literature data were interpreted and clarified, from a structural point of view, taking advantage of molecular modeling: 3D structures of ligand-target complexes were retrieved, or built, and discussed.

Combination of apremilast and narrowband ultraviolet B light in the treatment of generalized vitiligo in skin phototypes IV to VI: A randomized split-body pilot study

To the Editor: Apremilast is an oral phosphodiesterase-4 inhibitor that is approved for the treatment of psoriasis.1 It was shown to be effective in controlling progression of vitiligo,2 but another study found that apremilast does not bring any additional benefit to narrowband ultraviolet B light (NB-UVB) phototherapy.3 That study, however, included a low proportion of skin phototypes (SPTs) IV to VI and lacked the advantage of a bilateral comparison study design. Our study evaluated the efficacy of apremilast plus NB-UVB phototherapy compared with NB-UVB phototherapy alone in patients with SPTs IV to VI with generalized vitiligo (≥20% body surface area).

CRD-733, a Novel PDE9 (Phosphodiesterase 9) Inhibitor, Reverses Pressure Overload-Induced Heart Failure.

Augmentation of NP (natriuretic peptide) receptor and cyclic guanosine monophosphate (cGMP) signaling has emerged as a therapeutic strategy in heart failure (HF). cGMP-specific PDE9 (phosphodiesterase 9) inhibition increases cGMP signaling and attenuates stress-induced hypertrophic heart disease in preclinical studies. A novel cGMP-specific PDE9 inhibitor, CRD-733, is currently being advanced in human clinical studies. Here, we explore the effects of chronic PDE9 inhibition with CRD-733 in the mouse transverse aortic constriction pressure overload HF model. Adult male C57BL/6J mice were subjected to transverse aortic constriction and developed significant left ventricular (LV) hypertrophy after 7 days (P<0.001). Mice then received daily treatment with CRD-733 (600 mg/kg per day; n=10) or vehicle (n=17), alongside sham-operated controls (n=10). CRD-733 treatment reversed existing LV hypertrophy compared with vehicle (P<0.001), significantly improved LV ejection fraction (P=0.009), and attenuated left atrial dilation (P<0.001), as assessed by serial echocardiography. CRD-733 prevented elevations in LV end diastolic pressures (P=0.037) compared with vehicle, while lung weights, a surrogate for pulmonary edema, were reduced to sham levels. Chronic CRD-733 treatment increased plasma cGMP levels compared with vehicle (P<0.001), alongside increased phosphorylation of Ser273 of cardiac myosin binding protein-C, a cGMP-dependent protein kinase I phosphorylation site. The PDE9 inhibitor, CRD-733, improves key hallmarks of HF including LV hypertrophy, LV dysfunction, left atrial dilation, and pulmonary edema after pressure overload in the mouse transverse aortic constriction HF model. Additionally, elevated plasma cGMP may be used as a biomarker of target engagement. These findings support future investigation into the therapeutic potential of CRD-733 in human HF.

Apremilast mitigates interleukin (IL)-13-induced inflammatory response and mucin production in human nasal epithelial cells (hNECs)

ABSTRACT Interleukin (IL)-13-associated inflammatory response is important for the pathogenesis of allergic rhinitis (AR). Apremilast is a phosphodiesterase-4 (PDE4) inhibitor approved for psoriasis treatment. Here, we investigated the potential effects of Apremilast against IL-13-induced injury in human nasal epithelial cells (hNECs). Firstly, Apremilast ameliorated oxidative stress in IL-13-challenged cells by decreasing the levels of reactive oxygen species (ROS) and the production of malondialdehyde (MDA). Secondly, Apremilast inhibited the expressions of IL-6 and IL-8. Moreover, Apremilast inhibited the expressions of the chemokines colony-stimulating factor 2 (CSF2) and chemokine ligand 11 (CCL11). Interestingly, exposure to IL-13 increased the expressions of mucin 4 and mucin 5AC (MUC5AC), which was ameliorated by treatment with Apremilast. Interestingly, we found that Apremilast inhibited the phosphorylation of c-Jun-N-terminal kinase (JNK). Importantly, Apremilast reduced the levels of c-fos and c-Jun, the two AP-1 subfamilies. The luciferase reporter assay demonstrates that Apremilast reduced the transcriptional activity of activator protein 1 (AP-1). Lastly, we found that Apremilast prevented the activation of nuclear factor kappa-B (NF-κB) by decreasing the levels of nuclear NF-κB p65 and the luciferase activity of the NF-κB reporter. In summary, we conclude that Apremilast possesses a protective effect against IL-13-induced inflammatory response and mucin production in hNECs by inhibiting the activity of AP-1 and NF-κB.

Anti-inflammatory topical medication - newdevelopments in the treatment of atopicdermatitis.

Atopic dermatitis is a chronic inflammatory disease that arises from polygenic disposition, a dysfunction of the physicochemical epithelial barrier, a cutaneous dysbiosis, and a faulty neurosensory activity and shows a highly individual acuity due to epigenetic factors. An essential component of therapeutic management is the application of anti-inflammatory topical medication. Currently, topical glucocorticoids and topical calcineurin inhibitors are routinely used in reactive and proactive therapy. In recent years, the development of molecular medicine has identified several new therapeutic targets that have enabled the development of innovative therapeutic approaches. In addition to phosphodiesterase-4 inhibitors and aryl hydrocarbon receptor modulators, it is mainly Janus kinase inhibitors with different selectivity that are emerging as new effective and safe options for topical therapy. The current data suggests that in the coming months and years representatives of the above-mentioned substance classes will be approved for topical use.

Effectiveness of Apremilast in Real Life in Patients with Psoriasis: A Longitudinal Study.

Apremilast is an oral selective phosphodiesterase-4 inhibitor developed recently for psoriasis treatment. The aim of this study is to assess the real-life outcomes of use of apremilast in patients with psoriasis in everyday clinical practice. A total of 159 adult patients (90 males) with plaque psoriasis were included in the study. Fifty of the patients (31%) had psoriatic arthritis. All patients started apremilast at the time of enrolment. There was a marked improvement in Psoriasis Area and Severity Index, body surface area and Dermatology Life Quality Index scores across the follow-up period (12 months). The improvements in these scores were also consistent when the patients were stratified according to increasing body mass index. Only 10.6% of the patients discontinued apremilast, because of no response. In conclusion, apremilast is an effective and safe treatment in patients with psoriasis, and its effect is not influenced by body mass index.

Cytokine Levels of Skin Lesions in Moderate and Severe Psoriasis as Predictors for the Type 4 Fosphodiesterase Inhibitor (Apremilast) Therapy Effectivness

Background.Psoriasis is a widespread skin disorder characterized by cytokines as the main modulators of skin inflammation. A promising psoriasis therapy approach is targeted on cytokine networks by inhibiting the phosphodiesterase 4 (PDE-4). The efficacy and safety of the PDE-4 inhibitor apremilast in psoriasis confirmed in multicenter controlled studies, which, however, are accompanied by reports of unsuccessful therapy cases.Aims to determine early immunological predictors for PDE-4 inhibitor (apremilast) therapy efficacy based on skin lesions cytokines analyzes in patients with moderate to severe psoriasis.Methods.An open, uncontrolled prospective clinical study was performed. Efficacy of apremilast was analyzed by PASI, BSA, sPGA and DLQI. The outcomes at 26 therapy week were compared with the cytokine levels in skin biopsies selected before starting therapy and analyzed using xMAP technology. The multiparameter prognostic model for apremilast therapy effectiveness was developed based on a heterogeneous sequential recognition procedure.Results.The 34 patients with moderate to severe psoriasis were included in the study. According to the clinical outcomes, two comparison groups were formed: high (75% reduction in PASI score or more; n=14) and insufficient (50% reduction in PASI score or less; n=20) apremilast efficiency. Cytokines analyses showed an inverse relationship between the initial anti-inflammatory IL-10 level and the PASI reduction (r=0.37; p0.025). On the other hand, the relationships between pro-inflammatory cytokines IL-1 and IL-6 levels and PASI score at the 26th week of therapy were characterized by positive correlation: r=0.31 (p0.05) and r=0.37 (p0.025), respectively. Based on the data obtained, a predictive model for the apremilast therapy efficiency was developed. The expected predictive values of this model were 86% for a high therapy effectiveness and 60% for an insufficient effectiveness, respectively.Conclusions.The study results for the first time describe the cytokine profiles in the skin lesions in patients, who responded differently to PDE-4 inhibitor therapy. These dada allows to predict the treatment efficacy and give a chance to personalize apremilast usage for the moderate and severe psoriasis treatment.

Roflumilast prevents ischemic stroke-induced neuronal damage by restricting GSK3β-mediated oxidative stress and IRE1α/TRAF2/JNK pathway

Inhibition of phosphodiesterase 4 (PDE4) protects against neuronal apoptosis induced by cerebral ischemia. However, the exact mechanisms responsible for the protection of PDE4 inhibition have not been completely clarified. Roflumilast (Roflu) is an FDA-approved PDE4 inhibitor for the treatment of chronic obstructive pulmonary disease. The potential protective role of Roflu against ischemic stroke-associated neuronal injury remains unexplored. In this study, we investigated the effect and mechanism of Roflu against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) and in vivo rat middle cerebral artery occlusion (MCAO) models. We demonstrated that Roflu significantly reduced the apoptosis of HT-22 cells exposed to OGD/R, enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2), and reduced oxidative stress. Treatment with Roflu increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3β (GSK3β) but decreased the level of phosphorylated inositol requiring enzyme 1α (IRE1α). Interestingly, constitutively active GSK3β (S9A) mutation abolished the effects of Roflu on oxidative stress and IRE1α phosphorylation. Moreover, Roflu decreased the binding of IRE1α to tumor necrosis factor receptor-associated factor 2 (TRAF2) and attenuated the phosphorylation of c-Jun N-terminal kinase (JNK). We also found that PDE4B knockdown reduced the phosphorylation of both IRE1α and JNK, while overexpression of PDE4B antagonized the role of PDE4B knockdown on the activation of IRE1α and JNK. Besides, the inhibition of PDE4 by Roflu produced similar effects in primary cultured neurons. Finally, Roflu ameliorated MCAO-induced cerebral injury by decreasing infarct volume, restoring neurological score, and reducing the phosphorylation of IRE1α and JNK. Collectively, these data suggest that Roflu protects neurons from cerebral ischemia reperfusion-mediated injury via the activation of GSK3β/Nrf-2 signaling and suppression of the IRE1α/TRAF2/JNK pathway. Roflu has the potential as a protective drug for the treatment of cerebral ischemia.

In Vitro, In Vivo and In Silico Effectiveness of LASSBio-1386, an N-Acyl Hydrazone Derivative Phosphodiesterase-4 Inhibitor, Against Leishmania amazonensis.

Leishmaniasis are group of neglected diseases with worldwide distribution that affect about 12 million people. The current treatment is limited and may cause severe adverse effects, and thus, the search for new drugs more effective and less toxic is relevant. We have previously investigated the immunomodulatory effects of LASSBio-1386, an N-acylhydrazone derivative. Here we investigated the in vitro and in vivo activity of LASSBio-1386 against L. amazonensis. LASSBio-1386 inhibited the proliferation of promastigotes of L. amazonensis (EC50 = 2.4 ± 0.48 µM), while presenting low cytotoxicity to macrophages (CC50 = 74.1 ± 2.9 µM). In vitro incubation with LASSBio-1386 reduced the percentage of Leishmania-infected macrophages and the number of intracellular parasites (EC50 = 9.42 ± 0.64 µM). Also, in vivo treatment of BALB/c mice infected with L. amazonensis resulted in a decrease of lesion size, parasitic load and caused histopathological alterations, when compared to vehicle-treated control. Moreover, LASSBio-1386 caused ultrastructural changes, arrested cell cycle in G0/G1 phase and did not alter the membrane mitochondrial potential of L. amazonensis. Aiming to its possible molecular interactions, we performed docking and molecular dynamics studies on Leishmania phosphodiesterase B1 (PDB code: 2R8Q) and LASSBio-1386. The computational analyses suggest that LASSBio-1386 acts against Leishmania through the modulation of leishmanial PDE activity. In conclusion, our results indicate that LASSBio-1386 is a promising candidate for the development of new leishmaniasis treatment.

COPD patients with chronic bronchitis and higher sputum eosinophil counts show increased type‐2 and PDE4 gene expression in sputum

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase‐4‐inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32‐day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophilhigh ≥ 3%; eosinophillow < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty‐one genes were differentially expressed in primary samples (p‐adjusted for false discovery rate < 0.05); all up‐regulated in eosinophilhigh patients and functionally enriched for type‐2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophilhigh patients. These findings suggest that type‐2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials.