Vesicle-Encapsulated Rolipram (PDE4 Inhibitor) and Its Anticancer Activity.

Vesicular carriers of drugs are popular for specific targeting and delivery. The most popular vesicles among these are liposomes. However, they suffer from some inherent limitations. In this work, alternative vesicles with enhanced stability, i.e., niosomes and bilosomes have been prepared, characterized, and their delivery efficiency studied. Bilosomes have the additional advantage of being able to withstand the harsh environment of the gastrointestinal tract (GIT). The taurine-derived bile salt (NaTC) was incorporated into the bilosome bilayer. The inspiration behind NaTC insertion is the recent reports on antiaging action and immune function of taurine. Fluorescence probing was used to study the vesicle environment. The entrapment and subsequent release of the important cAMP-specific PDE4 inhibitor/drug Rolipram, which has antibreast cancer properties, was assessed on the breast cancer cell line MCF-7. Rolipram has important therapeutic applications, one of the most significant in recent times being the treatment of Covid-19-triggered pneumonia and cytokine storms. As for cancer chemotherapy, the localization of drug, targeted delivery, and sustained release are extremely important issues, and it seemed worthwhile to explore the potential of the bilosomes and niosomes to entrap and release Rolipram. The important finding is that niosomes perform much better than bilosomes in the hormone-responsive breast cancer mileau MCF-7. Moreover, there was a 4-fold decrease in the IC50 of Rolipram encapsulated in niosomes compared to Rolipram alone. On the other hand, bilosome-encapsulated Rolipram shows higher IC50 value. The results can be further understood by molecular docking studies.

Rolipram-loaded PgP nanoparticle reduces secondary injury and enhances motor function recovery in a rat moderate contusion SCI model.

Spinal cord injury (SCI) results in immediate axonal damage and cell death, as well as a prolonged secondary injury consist of a cascade of pathophysiological processes. One important aspect of secondary injury is activation of phosphodiesterase 4 (PDE4) that leads to reduce cAMP levels in the injured spinal cord. We have developed an amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that can deliver Rolipram, the PDE4 inhibitor. The objective of this work was to investigate the effect of rolipram loaded PgP (Rm-PgP) on secondary injury and motor functional recovery in a rat moderate contusion SCI model. We observed that Rm-PgP can increase cAMP level at the lesion site, and reduce secondary injury such as the inflammatory response by macrophages/microglia, astrogliosis by activated astrocytes and apoptosis as well as improve neuronal survival at 4 weeks post-injury (WPI). We also observed that Rm-PgP can improve motor functional recovery after SCI over 4 WPI.

Inhibition of autophagy and induction of glioblastoma cell death by NEO214, a perillyl alcohol-rolipram conjugate.

Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma.Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide.

Rolipram and pentoxifylline combination ameliorates the morphological abnormalities of dorsal root ganglion neurons in experimental diabetic neuropathy by reducing mitochondrial dysfunction and apoptosis.

Diabetic neuropathy (DN) is the most prevalent complication of diabetes. Pharmacological treatments for DN are often limited in efficacy, so the development of new agents to alleviate DN is essential. The aim of this study was to evaluate the effects of rolipram, a selective phosphodiesterase-4 inhibitor (PDE-4I), and pentoxifylline, a general PDE inhibitor, using a rat model of DN. In this study, a diabetic rat model was established by i.p. injection of STZ (55 mg/kg). Rats were treated with rolipram (1 mg/kg), pentoxifylline (100 mg/kg), and combination of rolipram (0.5 mg/kg) and pentoxifylline (50 mg/kg), orally for 5 weeks. After treatments, sensory function was assessed by hot plate test. Then rats were anesthetized and dorsal root ganglion (DRG) neurons isolated. Cyclic adenosine monophosphate (cAMP), adenosine triphosphate (ATP, adenosine diphosphate and mitochondrial membrane potential (MMP) levels, Cytochrome c release, Bax, Bcl-2, caspase-3 proteins expression in DRG neurons were assessed by biochemical and ELISA methods, and western blot analysis. DRG neurons were histologically examined using hematoxylin and eosin (H&E) staining method. Rolipram and/or pentoxifylline significantly attenuated sensory dysfunction by modulating nociceptive threshold. Rolipram and/or pentoxifylline treatment dramatically increased the cAMP level, prevented mitochondrial dysfunction, apoptosis and degeneration of DRG neurons, which appears to be mediated by inducing ATP and MMP, improving cytochrome c release, as well as regulating the expression of Bax, Bcl-2, and caspase-3 proteins, and improving morphological abnormalities of DRG neurons. We found maximum effectiveness with rolipram and pentoxifylline combination on mentioned factors. These findings encourage the use of rolipram and pentoxifylline combination as a novel experimental evidence for further clinical investigations in the treatment of DN.

Therapeutic treatment with phosphodiesterase-4 inhibitors alleviates kidney injury and renal fibrosis by increasing MMP-9 in a doxorubicin-induced nephrotoxicity mouse model.

Nephrotic syndrome (NS) is associated with kidney dysfunction and is an important cause of morbidity and mortality in industrialized countries. Here, we evaluated the effects of the phosphodiesterase-4 (PDE-4) inhibitors rolipram and roflumilast on a doxorubicin-induced NS model. Early-stage rolipram treatment preserved glomerular filtration barrier function, as indicated by reduced serum protein and albumin loss and the prevention of hypercholesterolemia. These effects were associated with reduced glomerular and tubular lesions and abrogated renal cell apoptosis. In addition, rolipram treatment reduced inflammation, which was characterized by a decrease in macrophage accumulation and reduced levels of CCL2 and TNF in the kidneys. Rolipram also reduced renal fibrosis, which was associated with decreased α-smooth muscle actin (α-SMA) area and increased metalloproteinase 9 (MMP9) activity in renal tissue. Late-stage rolipram or roflumilast treatment preserved glomerular filtration barrier function, as characterized by reduced serum albumin loss, decreased proteinuria, and the prevention of hypercholesterolemia. Importantly, only roflumilast treatment was associated with a reduction in glomerular and tubular lesions at this time point. In addition, both rolipram and roflumilast reduced renal tissue fibrosis and MMP9 activity in renal tissue.

Rolipram and pentoxifylline combination ameliorates experimental diabetic neuropathy through inhibition of oxidative stress and inflammatory pathways in the dorsal root ganglion neurons.

Diabetic neuropathy (DN) is the most challenging microvascular complication of diabetes and there is no suitable treatment for it, so the development of new agents to relieve DN is urgently needed. Since oxidative stress and inflammation play an essential role in the development of DN, clearance of these factors are good strategies for the treatment of this disease. According to key role of cyclic adenosine monophosphate (cAMP) in the regulation of oxidative stress and inflammatory pathways, it seems that phosphodiesterase inhibitors (PDEIs) can be as novel drug targets for improving DN through enhancement of cAMP level. The aim of this study was to evaluate the effects of rolipram, a selective PDE4 inhibitor, and pentoxifylline, a general PDE inhibitor on experimental model of DN and also to determine the possible mechanisms involved in the effectiveness of these agents. We investigated the effects of rolipram (1 mg/kg) and pentoxifylline (100 mg/kg) and also combination of rolipram (0.5 mg/kg) and pentoxifylline (50 mg/kg), orally for five weeks in rats that became diabetic by STZ (55 mg/kg, i.p.). After treatments, motor function was evaluated by open-field test, then rats were anesthetized and dorsal root ganglion (DRG) neurons isolated. Next, oxidative stress biomarkers and inflammatory factors were assessed by biochemical and ELISA methods, and RT-PCR analysis in DRG neurons. Rolipram and/or pentoxifylline treatment significantly attenuated DN - induced motor function deficiency by modulating distance moved and velocity. Rolipram and/or pentoxifylline treatment dramatically increased the cAMP level, as well as suppressed DN - induced oxidative stress which was associated with decrease in LPO and ROS and increase in TAC, total thiol, CAT and SOD in DRG neurons. On the other hand, the level of inflammatory factors (TNF-α, NF-kB and COX2) significantly decreased following rolipram and/or pentoxifylline administration. The maximum effectiveness was with rolipram and/or pentoxifylline combination on mentioned factors. These findings provide novel experimental evidence for further clinical investigations on rolipram and pentoxifylline combination for the treatment of DN.

Novel Liposomal Rolipram Formulation for Clinical Application to Reduce Emesis.

Introduction: The phosphodiesterase 4 (PDE4) inhibitor, rolipram, has beneficial effects on tissue inflammation, injury and fibrosis, including in the liver. Since rolipram elicits significant CNS side-effects in humans (ie, nausea and emesis), our group developed a fusogenic lipid vesicle (FLV) drug delivery system that targets the liver to avoid adverse events. We evaluated whether this novel liposomal rolipram formulation reduces emesis. Methods: C57Bl/6J male mice were used to compare the effect of three doses of free and FLV-delivered (FLVs-Rol) rolipram in a behavioral correlate model of rolipram-induced emesis. Tissue rolipram and rolipram metabolite levels were measured using LC-MS/MS. The effect of FLVs-Rol on brain and liver PDE4 activities was evaluated. Results: Low and moderate doses of free rolipram significantly reduced anesthesia duration, while the same doses of FLVs-Rol had no effect. However, the onset and duration of adverse effects (shortening of anesthesia period) elicited by a high dose of rolipram was not ameliorated by FLVs-Rol. Post-mortem analysis of brain and liver tissues demonstrated that FLVs affected the rate of rolipram uptake by liver and brain. Lastly, administration of a moderate dose of FLVs-Rol attenuated endotoxin induced PDE4 activity in the liver with negligible effect on the brain. Discussion: The findings that the low and moderate doses of FLVs-Rol did not shorten the anesthesia duration time suggest that FLV delivery prevented critical levels of drug from crossing the blood-brain barrier (BBB) to elicit CNS side-effects. However, the inability of high dose FLVs-Rol to prevent CNS side-effects indicates that there was sufficient unencapsulated rolipram to cross the BBB and shorten anesthesia duration. Notably, a moderate dose of FLVs-Rol was able to decrease PDE4 activity in the liver without affecting the brain. Taken together, FLVs-Rol has a strong potential for clinical application for the treatment of liver disease without side effects.

Inhibition of phosphodiesterase-4 in the spinal dorsal horn ameliorates neuropathic pain via cAMP-cytokine-Cx43 signaling in mice.

BACKGROUND: The spinal phosphodiesterase-4 (PDE4) plays an important role in chronic pain. Inhibition of PDE4, an enzyme catalyzing the hydrolysis of cyclic adenosine monophosphate AMP (cAMP), produces potent antinociceptive activity. However, the antinociceptive mechanism remains largely unknown. Connexin43 (Cx43), a gap junction protein, has been shown to be involved in controlling pain transduction at the spinal level; restoration of Cx43 expression in spinal astrocytes to the normal levels reduces nerve injury-induced pain. Here, we evaluate the novel mechanisms involving spinal cAMP-Cx43 signaling by which PDE4 inhibitors produce antinociceptive activity. METHODS: First, we determined the effect of PDE4 inhibitors rolipram and roflumilast on partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity. Next, we observed the role of cAMP-Cx43 signaling in the effect of PDE4 inhibitors on PSNL-induced mechanical hypersensitivity. RESULTS: Single or repeated, intraperitoneal or intrathecal administration of rolipram or roflumilast significantly reduced mechanical hypersensitivity in mice following PSNL. In addition, repeated intrathecal treatment with either of PDE4 inhibitors reduced PSNL-induced downregulation of cAMP and Cx43, and upregulation of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1ß. Furthermore, the antinociceptive effects of PDE4 inhibitors were attenuated by the protein kinase A (PKA) inhibitor H89, TNF-α, or Cx43 antagonist carbenoxolone. Finally, PSNL-induced upregulation of PDE4B and PDE4D, especially the PDE4B subtype, was reduced by treatment with either of the PDE4 inhibitors. CONCLUSIONS: The results suggest that the antinociceptive effect of PDE4 inhibitors is contributed by increasing Cx43 expression via cAMP-PKA-cytokine signaling in the spinal dorsal horn.

Rolipram Rescues Memory Consolidation Deficits Caused by Sleep Deprivation: Implication of the cAMP/PKA and cAMP/Epac Pathways.

BACKGROUND: Over the last few years, the number of people suffering from sleeping disorders has increased significantly despite negative effects on cognition and an association with brain inflammation. OBJECTIVES: We assessed memory deficits caused by Sleep Deprivation (SD) to determine the therapeutic effect of phosphodiesterase 4 (PDE4) inhibitors on SD-induced memory deficits and to investigate whether the modulation of memory deficits by PDE4 inhibitors is mediated by a protein kinase A (PKA)-independent pathway in conjunction with a PKA-dependent pathway. METHODS: Adult male mice were divided into four groups. Three SD groups were deprived of Rapid Eye Movement (REM) sleep for 12 h a day for six consecutive days. They were tested daily in the Morris water maze to evaluate learning and memory. One of the SD groups was injected with a PDE4 inhibitor, rolipram (1 mg/kg ip), whereas another had rolipram co-administered with chlorogenic acid (CHA, 20 mg/kg ip), an inhibitor of PKA. After 6 days, the mice were sacrificed, and the hippocampi were evaluated for cyclic AMP (cAMP) and nuclear factor Nrf-2 levels. The hippocampal expression of PKA, phosphorylated cAMP Response Element-Binding Protein (CREB), and phosphorylated glycogen synthase 3ß (Ser389) were also evaluated. RESULTS: SD caused a significant decrease in cAMP levels in the brain and had a detrimental effect on learning and memory. The administration of rolipram or rolipram+CHA resulted in an improvement in cognitive function. CONCLUSION: The present study provides evidence that restoration of memory with PDE4 inhibitors occurs through a dual mechanism involving the PKA and Epac pathways.

The protective effect of the PDE-4 inhibitor rolipram on intracerebral haemorrhage is associated with the cAMP/AMPK/SIRT1 pathway.

Rolipram specifically inhibits phosphodiesterase (PDE) 4, thereby preventing inactivation of the intracellular second messenger cyclic adenosine monophosphate (cAMP). Rolipram has been shown to play a neuroprotective role in some central nervous system (CNS) diseases. However, the role of PDE4 and the potential protective effect of rolipram on the pathophysiological process of intracerebral haemorrhage (ICH) are still not entirely clear. In this study, a mouse model of ICH was established by the collagenase method. Rolipram reduced brain oedema, blood-brain barrier (BBB) leakage, neuronal apoptosis and inflammatory cytokine release and improved neurological function in our mouse model of ICH. Moreover, rolipram increased the levels of cAMP and silent information regulator 1 (SIRT1) and upregulated the phosphorylation of AMP-activated protein kinase (AMPK). Furthermore, these effects of rolipram could be reversed by the SIRT1 inhibitor sirtinol. In conclusion, rolipram can play a neuroprotective role in the pathological process of ICH by activating the cAMP/AMPK/SIRT1 pathway.

Harnessing inflammation resolving-based therapeutic agents to treat pulmonary viral infections: What can the future offer to COVID-19?

Inflammation is generally accepted as a component of the host defence system and a protective response in the context of infectious diseases. However, altered inflammatory responses can contribute to disease in infected individuals. Many endogenous mediators that drive the resolution of inflammation are now known. Overall, mediators of resolution tend to decrease inflammatory responses and provide normal or greater ability of the host to deal with infection. In the lung, it seems that pro-resolution molecules, or strategies that promote their increase, tend to suppress inflammation and lung injury and facilitate control of bacterial or viral burden. Here, we argue that the demonstrated anti-inflammatory, pro-resolving, anti-thrombogenic and anti-microbial effects of such endogenous mediators of resolution may be useful in the treatment of the late stages of the disease in patients with COVID-19.

Discovery and Optimization of α-Mangostin Derivatives as Novel PDE4 Inhibitors for the Treatment of Vascular Dementia.

To validate PDE4 inhibitors as novel therapeutic agents against vascular dementia (VaD), 25 derivatives were discovered from the natural inhibitor α-mangostin (IC50 = 1.31 µM). Hit-to-lead optimization identified a novel and selective PDE4 inhibitor 4e (IC50 = 17 nM), which adopted a different binding pattern from PDE4 inhibitors roflumilast and rolipram. Oral administration of 4e at a dose of 10 mg/kg exhibited remarkable therapeutic effects in a VaD model and did not cause emesis to beagle dogs, indicating its potential as a novel anti-VaD agent.

Selective Modulation of A1 Astrocytes by Drug-Loaded Nano-Structured Gel in Spinal Cord Injury.

Astrogliosis has a very dynamic response during the progression of spinal cord injury, with beneficial or detrimental effects on recovery. It is therefore important to develop strategies to target activated astrocytes and their harmful molecular mechanisms so as to promote a protective environment to counteract the progression of the secondary injury. The challenge is to formulate an effective therapy with maximum protective effects, but reduced side effects. In this study, a functionalized nanogel-based nanovector was selectively internalized in activated mouse or human astrocytes. Rolipram, an anti-inflammatory drug, when administered by these nanovectors limited the inflammatory response in A1 astrocytes, reducing iNOS and Lcn2, which in turn reverses the toxic effect of proinflammatory astrocytes on motor neurons in vitro, showing advantages over conventionally administered anti-inflammatory therapy. When tested acutely in a spinal cord injury mouse model, it improved motor performance, but only in the early stage after injury, reducing the astrocytosis and preserving neuronal cells.

Effect of co-administration of two different phosphodiesterase inhibitors and a ß3-adrenoceptor agonist in an experimental model of detrusor overactivity.

The purpose of this study was to evaluate in vitro the effect of the combination of BRL 37344 (ß3-adrenoceptor agonist) with tadalafil (phosphodiesterase type 5 inhibitor) or rolipram (phosphodiesterase type 4 inhibitor) in an experimental model of detrusor overactivity. The experiments were carried out in two phases using bladder strips of mice. In the first phase, on the top of 40 mM potassium-induced contraction, strips isolated from control mice were exposed to increasing concentrations of each study drug. In another series of experiments, prior to contraction, strips were incubated with either tadalafil or rolipram, followed by the addition of increasing concentrations of BRL 37344. In the second phase, the same protocols were performed with animals previously treated with L-NAME for 30 days. Chronic L-NAME administration leads to detrusor overactivity due to nitric oxide synthase inhibition. In phase one, preincubation with tadalafil enhanced relaxation response to BRL 37344 at two concentrations. Pretreatment with rolipram had no effect on BRL 37344-induced relaxation. In L-NAME-treated mice, rolipram induced more relaxation than the other drugs, enhancing relaxation response to BRL 37344 at almost all concentrations, but no synergistic effect with tadalafil was observed. The relaxant effect of BRL 37344 was enhanced by rolipram but not by tadalafil, suggesting that PDE4 inhibition, especially when associated with ß3-adrenoceptor stimulation, could represent a potential treatment for overactive bladder.

Molecular Bases of PDE4D Inhibition by Memory-Enhancing GEBR Library Compounds.

Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.

Genetic Understanding of Stroke Treatment: Potential Role for Phosphodiesterase Inhibitors.

Phosphodiesterase (PDE) gene family is a large family having at least 21 genes and multiple versions (isoforms) of the phosphodiesterase enzymes. These enzymes catalyze the inactivation of intracellular mediators of signal transduction such as cAMP and cGMP and therefore, play a pivotal role in various cellular functions. PDE inhibitors (PDEI) are drugs that block one or more of the five subtypes of the PDE family and thereby prevent inactivation of the intracellular cAMP and cGMP by the respective PDE-subtypes. The first clinical use of PDEI was reported almost three decades ago. Studies later found the ability of these compounds to increase the levels of ubiquitous secondary messenger molecules that can cause changes in vascular tone, cardiac function and other cellular events and thus these findings paved the way for their use in various medical emergencies. PDEs are found to be distributed in many tissues including brain. Therefore, new therapeutic agents in the form of PDEI are being explored in neurodegenerative diseases including stroke. Although studies have revealed their use in cerebral infarction prevention, their full-fledged application in times of neurological emergency or stroke in specific has been very limited so far. Nevertheless, recent investigations suggest PDE4 and PDE5 inhibitors to play a vital role in mitigating stroke symptoms by modulating signaling mechanisms in PDE pathway. Further, extensive research in terms of their pharmacological properties like dosing, drug specific activities, use of simultaneous medications, ancillary properties of these compounds and studies on adverse drug reactions needs to be carried out to set them as standard drugs of use in stroke.

Rational design of conformationally constrained oxazolidinone-fused 1,2,3,4-tetrahydroisoquinoline derivatives as potential PDE4 inhibitors.

Improvement of subtype selectivity of an inhibitor's binding activity using the conformational restriction approach has become an effective strategy in drug discovery. In this study, we applied this approach to PDE4 inhibitors and designed a series of novel oxazolidinone-fused 1,2,3,4-tetrahydroisoquinoline derivatives as conformationally restricted analogues of rolipram. The bioassay results demonstrated the oxazolidinone-fused tetrahydroisoquinoline derivatives exhibited moderate to good inhibitory activity against PDE4B and high selectivity for PDE4B/PDE4D. Among these derivatives, compound 12 showed both the strongest inhibition activity (IC50=0.60µM) as well as good selectivity against PDE4B and good in vivo activity in animal models of asthma/COPD and sepsis induced by LPS. The primary SAR study showed that restricting the conformation of the catechol moiety in rolipram with the scaffold of oxazolidinone-fused tetrahydroisoquinoline could lead to an increase in selectivity for PDE4B over PDE4D, which was consistent with the observed docking simulation.

Attenuation of ethanol abstinence-induced anxiety- and depressive-like behavior by the phosphodiesterase-4 inhibitor rolipram in rodents.

RATIONALE: Withdrawal symptoms stand as a core feature of alcohol dependence. Our previous results have shown that inhibition of phosphodiesterase-4 (PDE4) decreased ethanol seeking and drinking in alcohol-preferring rodents. However, little is known about whether PDE4 is involved in ethanol abstinence-related behavior. OBJECTIVE: The objective of this study was to characterize the role of PDE4 in the development of anxiety- and depressive-like behavior induced by abstinence from ethanol exposure in different animal models. METHODS: Using three rodent models of ethanol abstinence, we examined the effects of rolipram, a prototypical, selective PDE4 inhibitor, on (1) anxiety-like behavior induced by repeated ethanol abstinence in the elevated plus maze test in fawn-hooded (FH/Wjd) rats, (2) anxiety-like behavior in the open-field test and light-dark transition test following acute ethanol abstinence in C57BL/6J mice, and (3) anxiety- and depressive-like behavior induced by protracted ethanol abstinence in the elevated plus maze, forced-swim, and tail-suspension tests in C57BL/6J mice. RESULTS: Pretreatment with rolipram (0.1 or 0.2 mg/kg) significantly increased entries and time spent in the open arms of the elevated plus maze test in rats with repeated ethanol abstinence. Similarly, in mice with acute ethanol abstinence, administration of rolipram (0.25 or 0.5 mg/kg) dose-dependently increased the crossings in the central zone of the open-field test and duration and transitions on the light side of the light-dark transition test, suggesting anxiolytic-like effects of rolipram. Consistent with these, chronic treatment with rolipram (0.1, 0.3, or 1.0 mg/kg) increased entries in the open arms of the elevated plus maze test; it also reduced the increased duration of immobility in both the forced-swim and tail-suspension tests in mice after protracted ethanol abstinence, suggesting antidepressant-like effects of rolipram. CONCLUSIONS: These results provide the first demonstration for that PDE4 plays a role in modulating the development of negative emotional reactions associated with ethanol abstinence, including anxiety and depression. PDE4 inhibitors may be a novel class of drugs for treatment of alcoholism.

The resolution of acute inflammation induced by cyclic AMP is dependent on annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.

The antiepileptogenic effect of low-frequency stimulation on perforant path kindling involves changes in regulators of G-protein signaling in rat.

G-protein coupled receptors may have a role in mediating the antiepileptogenic effect of low-frequency stimulation (LFS) on kindling acquisition. This effect is accompanied by changes at the intracellular level of cAMP. In the present study, the effect of rolipram as a phosphodiesterase inhibitor on the antiepileptogenic effect of LFS was investigated. Meanwhile, the expression of αs- and αi-subunit of G proteins and regulators of G-protein signaling (RGS) proteins following LFS application was measured. Male Wistar rats were kindled by perforant path stimulation in a semi-rapid kindling manner (12 stimulations per day) during a period of 6days. Application of LFS (0.1ms pulse duration at 1Hz, 200 pulses, 50-150µA, 5min after termination of daily kindling stimulations) to the perforant path retarded the kindling development and prevented the kindling-induced potentiation and kindling-induced changes in paired pulse indices in the dentate gyrus. Intra-cerebroventricular microinjection of rolipram (0.25µM) partially prevented these LFS effects. Twenty-four hours after the last kindling stimulation, the dentate gyrus was removed and changes in protein expression were measured by Western blotting. There was no significant difference in the expression of α-subunit of Gs and Gi/o proteins in different experimental groups. However, application of LFS during the kindling procedure decreased the expression RGS4 and RGS10 proteins (that reduce the activity of Gi/o) and prevented the kindling-induced decrease of RGS2 protein (which reduces the Gs activity). Therefore, it can be postulated that the Gi/o protein signaling pathways may be involved in antiepileptogenetic effect of LFS, and this is why decreasing the cAMP metabolism by rolipram attenuates this effect of LFS.