Allosteric inhibition of phosphodiesterase 4D induces biphasic memory-enhancing effects associated with learning-activated signaling pathways.

RATIONALE: Phosphodiesterase 4D negative allosteric modulators (PDE4D NAMs) enhance memory and cognitive function in animal models without emetic-like side effects. However, the relationship between increased cyclic adenosine monophosphate (cAMP) signaling and the effects of PDE4D NAM remains elusive. OBJECTIVE: To investigate the roles of hippocampal cAMP metabolism and synaptic activation in the effects of D159687, a PDE4D NAM, under baseline and learning-stimulated conditions. RESULTS: At 3 mg/kg, D159687 enhanced memory formation and consolidation in contextual fear conditioning; however, neither lower (0.3 mg/kg) nor higher (30 mg/kg) doses induced memory-enhancing effects. A biphasic (bell-shaped) dose-response effect was also observed in a scopolamine-induced model of amnesia in the Y-maze, whereas D159687 dose-dependently caused an emetic-like effect in the xylazine/ketamine anesthesia test. At 3 mg/kg, D159687 increased cAMP levels in the hippocampal CA1 region after conditioning in the fear conditioning test, but not in the home-cage or conditioning cage (i.e., context only). By contrast, 30 mg/kg of D159687 increased hippocampal cAMP levels under all conditions. Although both 3 and 30 mg/kg of D159687 upregulated learning-induced Fos expression in the hippocampal CA1 30 min after conditioning, 3 mg/kg, but not 30 mg/kg, of D159687 induced phosphorylation of synaptic plasticity-related proteins such as cAMP-responsive element-binding protein, synaptosomal-associated protein 25 kDa, and the N-methyl-D-aspartate receptor subunit NR2A. CONCLUSIONS: Our findings suggest that learning-stimulated conditions can alter the effects of a PDE4D NAM on hippocampal cAMP levels and imply that a PDE4D NAM exerts biphasic memory-enhancing effects associated with synaptic plasticity-related signaling activation.

PDE4 inhibition by difamilast regulates filaggrin and loricrin expression via keratinocyte proline-rich protein in human keratinocytes.

BACKGROUND: Difamilast, a topical phosphodiesterase 4 (PDE4) inhibitor, has been shown to be effective for treating atopic dermatitis (AD), but the molecular mechanism involved is unclear. Since skin barrier dysfunction including reduced expression of filaggrin (FLG) and loricrin (LOR) contributes to AD development, difamilast treatment may be able to improve this dysfunction. PDE4 inhibition increases transcriptional activity of cAMP-responsive element binding protein (CREB). Therefore, we hypothesized that difamilast may affect FLG and LOR expression via CREB in human keratinocytes. OBJECTIVE: To elucidate the mechanism by which difamilast regulates FLG and LOR expression via CREB in human keratinocytes. METHODS: We analyzed normal human epidermal keratinocytes (NHEKs) treated with difamilast. RESULTS: We observed increases of intracellular cAMP levels and CREB phosphorylation in difamilast (5 µM)-treated NHEKs. Next, we found that difamilast treatment increased mRNA and protein levels of FLG and LOR in NHEKs. Since reduced expression of keratinocyte proline-rich protein (KPRP) is reported to be involved in skin barrier dysfunction in AD, we examined KPRP expression in difamilast-treated NHEKs. We found that difamilast treatment increased mRNA and protein levels of KPRP in NHEKs. Furthermore, KPRP knockdown using siRNA transfection abolished the upregulation of FLG and LOR in difamilast-treated NHEKs. Finally, CREB knockdown canceled the upregulation of FLG, LOR, and KPRP in difamilast-treated NHEKs, indicating that PDE4 inhibition by difamilast treatment positively regulates FLG and LOR expression via the CREB-KPRP axis in NHEKs. CONCLUSION: These findings may provide further guidance for therapeutic strategies in the treatment of AD using difamilast.

Recent advances of Phosphodiesterase 4B in cancer.

INTRODUCTION: Phosphodiesterase 4B (PDE4B) is a crucial enzyme in the phosphodiesterases (PDEs), acting as a regulator of cyclic adenosine monophosphate (cAMP). It is involved in cancer process through PDE4B/cAMP signaling pathway. Cancer occurs and develops with the regulation of PDE4B in the body, suggesting that PDE4B is a promising therapeutic target. AREAS COVERED: This review covereed the function and mechanism of PDE4B in cancer. We summarized the possible clinical applications of PDE4B, and highlighted the possible ways to develop clinical applications of PDE4B inhibitors. We also discussed some common PDEs inhibitors, and expected the development of combined targeting PDE4B and other PDEs drugs in the future. EXPERT OPINION: The existing research and clinical data can strongly prove the role of PDE4B in cancer. PDE4B inhibition can effectively increase cell apoptosis, inhibit cell proliferation, transformation, migration, etc., indicating that PDE4B inhibition can effectively inhibit the development of cancer. Other PDEs may antagonize or coordinate this effect. As for the further study on the relationship between PDE4B and other PDEs in cancer, it is still a challenge to develop multi-targeted PDEs inhibitors.

Phosphodiesterase 4B inhibition: a potential novel strategy for treating pulmonary fibrosis.

Patients with interstitial lung disease can develop a progressive fibrosing phenotype characterised by an irreversible, progressive decline in lung function despite treatment. Current therapies slow, but do not reverse or stop, disease progression and are associated with side-effects that can cause treatment delay or discontinuation. Most crucially, mortality remains high. There is an unmet need for more efficacious and better-tolerated and -targeted treatments for pulmonary fibrosis. Pan-phosphodiesterase 4 (PDE4) inhibitors have been investigated in respiratory conditions. However, the use of oral inhibitors can be complicated due to class-related systemic adverse events, including diarrhoea and headaches. The PDE4B subtype, which has an important role in inflammation and fibrosis, has been identified in the lungs. Preferentially targeting PDE4B has the potential to drive anti-inflammatory and antifibrotic effects via a subsequent increase in cAMP, but with improved tolerability. Phase I and II trials of a novel PDE4B inhibitor in patients with idiopathic pulmonary fibrosis have shown promising results, stabilising pulmonary function measured by change in forced vital capacity from baseline, while maintaining an acceptable safety profile. Further research into the efficacy and safety of PDE4B inhibitors in larger patient populations and for a longer treatment period is needed.

BI 1015550: an investigational phosphodiesterase 4B (PDE4B) inhibitor for lung function decline in idiopathic pulmonary fibrosis (IPF).

INTRODUCTION: The two available therapies for idiopathic pulmonary fibrosis (IPF), pirfenidone and nintedanib, slow down but do not halt IPF progression. Therefore, several agents with specific molecular targets have been recently investigated to find a cure for IPF. Phosphodiesterase 4 (PDE4) inhibition is known for its anti-inflammatory and antifibrotic properties. BI 1015550, an oral preferential inhibitor of the isoform PDE4B, could express complementary activity to current therapies in IPF and other forms of progressive pulmonary fibrosis. AREAS COVERED: In this review, we first provide an overview toof the current IPF treatment market, followed by the description of pharmacokinetics and pharmacodynamics of BI 1015550. The main preclinical and early clinical evidence on BI 1015550 is then described, as well as its potential as an IPF treatment. EXPERT OPINION: Oral treatment with BI 1015550 was shown to stabilize lung function as compared to placebo over 12 weeks, both among patients with and without background antifibrotic use, with an acceptable safety profile in a phase 2 trial, and a phase 3 trial has been initiated. To date, this represents to date the largest effect size for an IPF investigational drug tested in a phase 2 trial with the shortest duration.

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS.

Phosphodiesterase 4 mRNA Level Suppression is Important for Extract of Black Carrot to Protect Against Hepatic Injury Induced by Ethanol.

Excessive alcohol use often results in alcoholic liver disease (ALD). An early change in the liver due to excessive drinking is hepatic steatosis, which may ultimately progress to hepatitis, liver fibrosis, cirrhosis, and liver cancer. Among these debilitating processes, hepatic steatosis is reversible with the appropriate treatment. Therefore, it is important to find treatments and foods that reverse hepatic steatosis. Black carrot has antioxidant and anti-inflammatory effects. In this study, we examined the effectiveness of black carrot extract (BCE) on hepatic steatosis in in vivo and in vitro ethanol-induced liver injury models. For the in vivo experiments, serum aminotransferase activities enhanced by ethanol- and carbon tetrachloride were significantly suppressed by the BCE diet. Furthermore, morphological changes in the liver hepatic steatosis and fibrosis were observed in the in vivo ethanol-induced liver injury model, however, BCE feeding resulted in the recovery to an almost normal liver morphology. In the in vitro experiments, ethanol treatment induced reactive oxygen species (ROS) levels in hepatocytes at 9 h. Conversely, ROS production was suppressed to control levels and hepatic steatosis was suppressed when hepatocyte culture with ethanol were treated with BCE. Furthermore, we investigated enzyme activities, enzyme protein levels, and messenger RNA levels of alcohol dehydrogenase (ADH), cytochrome p450 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH) using enzyme assays, western blot, and quantitative reverse transcription-polymerase chain reaction analyses. We found that the activities of ADH, CYP2E1, and ALDH were regulated through the cAMP-PKA pathway at different levels, namely, translational, posttranslational, and transcriptional levels, respectively. The most interesting finding of this study is that BCE increases cAMP levels by suppressing the Pde4b mRNA and PDE4b protein levels in ethanol-treated hepatocytes, suggesting that BCE may prevent ALD.

Apremilast exerts protective effects on stroke outcomes and blood-brain barrier (BBB) dysfunction through regulating Rho-associated protein kinase 2 expression.

AIMS: Stroke is a devastating event and a huge public health concern worldwide. Apremilast (APR) is a selective inhibitor of phosphodiesterase-4 involved in various neurological diseases, including stroke. However, the protective effects of APR on stroke have not been investigated. Here, we explored the effects of APR on stroke outcomes and blood-brain barrier (BBB) dysfunction using a middle cerebral artery occlusion (MCAO) stroke mice model. RESULTS: The results show that APR attenuated neurological injury in MCAO mice with decreased neurological deficit scores and infarct size, as well as increased hanging grip time. The increased BBB permeability and decreased expression of the tight junction protein Claudin-5 in MCAO mice were attenuated by APR treatment. APR treatment also mitigated neuroinflammation in MCAO mice, as shown by the decreased levels of inflammatory cytokines. In vitro assays also proved that APR ameliorated the oxygen/glucose deprivation/reoxygenation (OGD/R)-induced increase in endothelial permeability and restored the expression of Claudin-5 in bEnd.3 brain endothelial cells. Moreover, overexpression of ROCK2 in bEnd.3 cells abolished the protective effects of APR on endothelial permeability against OGD/R induction. CONCLUSION: Taken together, our results demonstrate that APR showed significant efficacy on ischemic stroke outcomes by alleviating enhanced BBB permeability and neuroinflammation by inhibiting ROCK2. These findings suggest a novel therapeutic window for ischemic stroke.

ZL-n-91, a specific Phosphodiesterase-4 inhibitor, suppresses the growth of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that frequently develops resistance to chemotherapy. A new approach to treating TNBC is required to improve patient survival. Phosphodiesterase-4 (PDE4) is an enzyme that is predominantly involved in the modulation of intracellular signaling mediated by cAMP. Although the efficacy of PDE4 inhibitors in several human inflammatory diseases is well documented, their clinical utility has been limited by side effects, including nausea and emesis. Recently, PDE4 has been used as a potential therapeutic target for different cancer types. In the present study, we investigated the anticancer effects of a novel PDE4 inhibitor ZL-n-91 on TNBC and the underlying mechanism. We showed that ZL-n-91 inhibited the proliferation of TNBC cells, induced cell apoptosis, and caused cell cycle arrest. Western blot analysis showed that ZL-n-91 increased Bax level and reduced Bcl-2 expression. Furthermore, downregulation of the cell cycle-related proteins, such as CDK2, CDK4, cyclin D1, PCNA, p-RB, and ZL-n-91, significantly inhibited the transcription of DNA repair genes and triggered an intracellular DNA damage response. Moreover, ZL-n-91 prevented the growth of the transplanted MDA-MB-231 tumor xenograft in nude mice and increased the γ-H2AX expression. These data demonstrate the anticancer effects of ZL-n-91 on TNBC cells and suggest its potential use in anticancer therapy.

Suppression of phosphodiesterase IV enzyme by roflumilast ameliorates cognitive dysfunction in aged rats after sevoflurane anaesthesia via PKA-CREB and MEK/ERK pathways.

Postoperative cognitive dysfunction (POCD) is a prevalent disorder after anaesthesia in the elderly patients. Roflumilast (RF), a phosphodiesterase 4 (PDE-4) inhibitor, could improve cognition with no side effects. Here, we sought to explore the efficacy of RF in the improvement of cognitive dysfunction caused by sevoflurane (Sev). Sprague-Dawley rats were anaesthetized, and the hippocampal neurons were treated with Sev to develop in vivo and in vitro POCD models, followed by RF administration. The mechanism of the PKA-CREB and MEK/ERK pathways in the pathogenesis of POCD was explored. Sev impaired the cognitive functions of rats, significantly reduced cyclic adenosine monophosphate (cAMP) concentrations and blocked the PKA-CREB and MEK/ERK pathways. Moreover, the Sev-treated rats and neurons exhibited enhanced apoptosis and reactive oxygen species (ROS). After treatment with RF, rats had better learning and memory function, and the activity of neurons in hippocampus and cortex was improved. Loss-of-function assay indicated that PKA-CREB and MEK/ERK signalling impairment reduced cAMP levels and promoted apoptosis and ROS in rat hippocampus and neurons. Generally, RF promotes neuronal activity in rats after Sev treatment by maintaining cAMP levels and sustaining the activation of PKA-CREB and MEK/ERK pathways. This might offer novel sights for POCD therapy.

CircAXL Knockdown Alleviates Aß1-42-Induced Neurotoxicity in Alzheimer's Disease via Repressing PDE4A by Releasing miR-1306-5p.

The development of Alzheimer's disease (AD) is implicated with the dysregulation of numerous circular RNAs (circRNAs). However, the function of several circRNAs remains unclear. The aim of this study was to investigate the role of circular AXL receptor tyrosine kinase (circAXL) in AD. Cell models of AD were constructed by treating SK-N-SH cells with amyloid-ß (Aß1-42). The expression of circAXL, miR-1306-5p and phosphodiesterase 4A (PDE4A) mRNA was detected by quantitative real-time PCR (qPCR). Cell viability was checked by CCK-8 assay. The production of inflammatory factors was monitored by ELISA. Cell apoptosis was checked by flow cytometry assay. Oxidative stress was assessed by ROS level, MDA level and SOD activity using commercial kits. Endoplasmic reticulum (ER) stress was assessed by ER-related protein markers using western blotting. The relationship between miR-1306-5p and circAXL or PDE4A was validated by RIP assay and dual-luciferase reporter assay. Serum exosomes were isolated by centrifugation to assess the diagnostic value of exosomal circAXL, miR-1306-5p and PDE4A. CircAXL was overexpressed in Aß1-42-treated SK-N-SH cells. CircAXL knockdown alleviated Aß1-42-induced cell cytotoxicity, cell apoptosis, inflammation, oxidative stress and endoplasmic reticulum (ER) stress in SK-N-SH cells. MiR-1306-5p was screened as a target of circAXL, and miR-1306-5p inhibition abolished the effects of circAXL knockdown. MiR-1306-5p inhibited the expression of PDE4A, and circAXL regulated PDE4A expression by targeting miR-1306-5p. MiR-1306-5p restoration also alleviated Aß1-42-induced cell injuries, while PDE4A reintroduction abolished the effects of miR-1306-5p restoration. Exosomal circAXL and exosomal miR-1306-5p had diagnostic values for AD. CircAXL knockdown alleviates Aß1-42-induced neurotoxicity in AD pathology via repressing PDE4A by releasing miR-1306-5p.

Phosphodiesterase 4 mediates interleukin-8-induced heterologous desensitization of the ß2 -adrenergic receptor.

Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.

New Hybrid Pyrazole and Imidazopyrazole Antinflammatory Agents Able to Reduce ROS Production in Different Biological Targets.

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure-activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets.

NPP4 is a procoagulant enzyme on the surface of vascular endothelium.

Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.

ß-Adrenergic receptor stimulation increases surface NKCC2 expression in rat thick ascending limbs in a process inhibited by phosphodiesterase 4.

The thick ascending limb of the loop of Henle (THAL) reabsorbs ∼30% of the filtered NaCl in a process mediated by the apical Na-K-2Cl cotransporter NKCC2. Stimulation of ß-adrenergic receptors in the THAL enhances NaCl reabsorption and increases intracellular cAMP. We found that intracellular cAMP stimulates NKCC2 trafficking to the apical membrane via protein kinase A (PKA). Several cAMP-specific phosphodiesterases (PDE) have been identified in rat THALs, and PDE4 decreases cAMP generated by ß-adrenergic stimulation in other cells. However, it is not known whether ß-adrenergic receptors activation stimulates NKCC2 trafficking. Thus we hypothesized that ß-adrenergic receptor stimulation enhances THAL apical membrane NKCC2 expression via the PKA pathway and PDE4 blunts this effect. THAL suspensions were obtained from Sprague-Dawley rats, and surface NKCC2 expression was measured by surface biotinylation and Western blot. Incubation of THALs with the ß-adrenergic receptor agonist isoproterenol at 0.5 and 1.0 µM increased surface NKCC2 by 17 ± 1 and 29 ± 5% respectively (P < 0.05). Preventing cAMP degradation with 3-isobutyl-methylxanthine (IBMX; a nonselective phosphodiesterase inhibitor) enhanced isoproterenol-stimulated surface NKCC2 expression to 51 ± 7% (P < 0.05 vs. isoproterenol). The ß-adrenergic receptor antagonist propranolol or the PKA inhibitor H-89 completely blocked isoproterenol + IBMX-induced increase on surface NKCC2, while propranolol or H-89 alone had no effect. Selective inhibition of PDE4 with rolipram (20 µM) potentiated the effect of isoproterenol on surface NKCC2 and increased cAMP levels. We concluded that ß-adrenergic receptor stimulation enhances surface NKCC2 expression in the THALs via PKA and PDE4 blunts this effect.

Antiproliferative effects of CC-8062 and CC-8075 in pancreatic cancer cells.

OBJECTIVES: Pancreatic cancer is one of the leading causes of cancer related deaths in the western world. It is also resistant to most chemotherapeutic modalities. Phosphodiesterase-4 inhibitors (PDE4is) have found applications in the treatment of respiratory diseases. The aim of this study is to investigate the cytotoxic effect of 2 novel PDE4is, the CC-8075 and CC-8062 compounds in pancreatic cancer cells. METHODS: Cell proliferation was measured using the sulforhodamine B protein dye. Induction of apoptosis was detected using enzyme-linked immunosorbent assay. Regulation of proteins and posttranslational modifications were determined using immunoblotting. RESULTS: Treatment of pancreatic cancer cells with CC-8075 and CC-8062 reduces their proliferation and increases apoptosis that is caspase dependent in T3M4 cells. Furthermore, PDE4is increase phosphorylation of p38MAPK, mitogen-activated protein kinase (MAPK) kinase 3/6,MAPKYactivated protein kinase 2, Atf2, and Hsp27. The use of thep38MAPK-specific inhibitors SB202190 and SB203580 results in a modest reduction in PDE4i-induced apoptosis in T3M4 cells. Also, retinoids enhance apoptosis induced by CC-8075 and CC-8062 in GER cells. CONCLUSIONS: These results highlight the antiproliferative effects of the phosphodiesterase inhibitors CC-8075 and CC-8062 in pancreatic cancer cells and suggest that activation of p38MAPK signaling pathway may be associated with this process.

Prophylactic effect of irsogladine maleate against indomethacin-induced small intestinal lesions in rats.

The effect of irsogladine maleate, a widely used antiulcer drug in Japan, on indomethacin-induced small intestinal lesions was examined in rats. Animals without fasting were given indomethacin (10 mg/kg, s.c.) and sacrificed 24 h later. Irsogladine (1-10 mg/kg) or 16,16-dimethyl prostaglandin E2 (dmPGE2 0.03 mg/kg) was given p.o. twice, 0.5 before and 6 h after indomethacin, while ampicillin (800 mg/kg) was given twice, 18 and 0.5 h before. Indomethacin caused severe lesions in the small intestine, mainly the jejunum and ileum, accompanied by intestinal hypermotility, the up-regulation of inducible nitric oxide synthase (iNOS) expression, and an increase of myeloperoxidase (MPO) activity as well as enterobacterial invasion in the mucosa. These events were all prevented by both dmPGE2 and ampicillin, except the intestinal hypermotility which was only prevented by dmPGE2. Likewise, irsogladine also significantly and dose-dependently prevented these lesions at > 1 mg/kg. This agent alone increased mucus secretion and significantly suppressed the decreased mucus response to indomethacin, resulting in a suppression of the bacterial invasion as well as the increase in MPO activity and iNOS expression. The protective effect of irsogladine was mimicked by isobutylmethylxanthine, a nonselective inhibitor of phosphodiesterase (PDE), as well as rolipram, a selective PDE4 inhibitor. These results suggest that irsogladine protects the small intestine against indomethacin-induced lesions, and this effect may be associated with the increased mucus secretion, probably due to the inhibitory actions of PDE, resulting in suppression of enterobacterial invasion and iNOS expression.