Selective PDE4 Inhibitor Research Articles

Novel Celecoxib Derivative, RF26, Blocks Colon Cancer Cell Growth by Inhibiting PDE5, Activating cGMP/PKG Signaling, and Suppressing β-catenin-dependent Transcription.

Previous studies have reported that the cGMP-specific PDE5 isozyme is overexpressed in colon adenomas and adenocarcinomas and essential for colon cancer cell proliferation, while PDE5 selective inhibitors (e.g., sildenafil) have been reported to have cancer chemopreventive activity. This study aimed to determine the anticancer activity of a novel PDE5 inhibitor, RF26, using colorectal cancer (CRC) cells and the role of PDE5 in CRC tumor growth in vivo. The objective of this study was to characterize the anticancer activity of a novel celecoxib derivative, RF26, in CRC cells previously reported to lack COX-2 inhibition but have potent PDE5 inhibitory activity. Anticancer activity of RF26 was studied using human CRC cell lines. Its effects on intracellular cGMP levels, cGMP-dependent protein kinase (PKG) activity, β-catenin levels, TCF/LEF transcriptional activity, cell cycle distribution, and apoptosis were measured. CRISPR/cas9 PDE5 knockout techniques were used to determine if PDE5 mediates the anticancer activity of RF26 and validate PDE5 as a cancer target. RF26 was appreciably more potent than celecoxib and sildenafil to suppress CRC cell growth and was effective at concentrations that increased intracellular cGMP levels and activated PKG signaling. RF26 suppressed β-catenin levels and TCF/LEF transcriptional activity and induced G1 cell cycle arrest and apoptosis within the same concentration range. CRISPR/cas9 PDE5 knockout CRC cells displayed reduced sensitivity to RF26, proliferated slower than parental cells, and failed to establish tumors in mice. Further evaluation of RF26 for the prevention or treatment of cancer and studying the role of PDE5 in tumorigenesis are warranted.

Structural optimization of Moracin M as novel selective phosphodiesterase 4 inhibitors for the treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and high mortality lung disease. Although the antifibrotic drugs pirfenidone and nintedanib could slow the rate of lung function decline, the usual course of the condition is inexorably to respiratory failure and death. Therefore, new approaches and novel therapeutic drugs for the treatment of IPF are urgently needed. And the selective PDE4 inhibitor has in vivo and in vitro anti-fibrotic effects in IPF models. But the clinical application of most PDE4 inhibitors are limited by their unexpected and severe side effects such as nausea, vomiting, and diarrhea. Herein, structure-based optimizations of the natural product Moracin M resulted in a novel a novel series of 2-arylbenzofurans as potent PDE4 inhibitors. The most potent inhibitor L13 has an IC50 of 36 ± 7 nM with remarkable selectivity across the PDE families and administration of L13·citrate (10.0 mg/kg) exhibited comparable anti-pulmonary fibrosis effects to pirfenidone (300 mg/kg) in a bleomycin-induced IPF mice model, indicate that L13 is a potential lead for the treatment of IPF.

Age-related pharmacokinetics differences were observed between young and elderly populations of a novel PDE5 inhibitor, youkenafil, and its metabolite M459

PurposeYoukenafil is a novel oral selective PDE5 inhibitor for treating Erectile Dysfunction. This investigation assessed pharmacokinetics (PK), safety, and tolerability of youkenafil and its main metabolite (M459) after taking 100 mg youkenafil hydrochloride tablets in elderly and young subjects. MethodsThis Phase I, single-center, open-label, parallel-group, single-dose study was conducted on 24 individuals (12 elders and 12 youngsters). Each subject received a single oral 100 mg youkenafil hydrochloride tablets. Blood samples were collected before medication and up to 48 h after medication for PK analysis. Safety and tolerability were also assessed, including treatment-emergent adverse events (TEAEs), laboratory tests, 12-lead ECG, vital sign inspections, color vision examinations, and physical examinations. ResultsPlasma concentrations of youkenafil and M459 were quantified. PK parameters were determined by non-compartmental analysis. Median Tmax of elderly and young groups were both 0.733 h. However, Cmax, AUC0-t, and AUC0-∞ of youkenafil were separately 16.8 %, 37.2 %, and 37.5 % higher in elders and t1/2 of youkenafil was 2.1 h longer in elders. More great differences were observed for M459. T1/2 values were 4.05 h longer in elders, with Cmax, AUC0-t and AUC0-∞ 73.7 %, 81.1 %, and 81.4 % higher in elders. Two (8.3 %) elderly subjects reported TEAEs (all grade Ⅰ in severity) and both recovered without any treatment. No serious adverse reactions (SAEs) or serious unexpected suspected adverse reactions (SUSARs) occurred in this study. ConclusionsThis was the first PK research of youkenafil and M459 in elderly men. PK parameters differences between youkenafil and M459 were comparable between elderly and young groups. Moreover, safety and tolerability of youkenafil were favorable in both groups.

A Long-Acting Lyotropic Liquid Crystalline Implant Promotes the Drainage of Macromolecules by Brain-Related Lymphatic System in Treating Aged Alzheimer's Disease.

Numerous evidence has demonstrated that the brain is not an immune-privileged organ but possesses a whole set of lymphatic transport system, which facilitates the drainage of harmful waste from brains to maintain cerebral homeostasis. However, as individuals age, the shrinkage and dysfunction of meningeal and deep cervical lymphatic networks lead to reduced waste outflow and elevated neurotoxic molecules deposition, further inducing aging-associated cognitive decline, which act as one of the pathological mechanisms of Alzheimer's disease. Consequently, recovering the function of meningeal and deep cervical lymph node (dCLNs) networks (as an important part of the brain waste removal system (BWRS)) of aged brains might be a feasible strategy. Herein we showed that the drug brain-entering efficiency was highly related to administration routes (oral, subcutaneous, or dCLN delivery). Besides, by injecting a long-acting lyotropic liquid crystalline implant encapsulating cilostazol (an FDA-approved selective PDE-3 inhibitor) and donepezil hydrochloride (a commonly used symptomatic relief agent to inhibit acetylcholinesterase for Alzheimer's disease) near the deep cervical lymph nodes of aged mice (about 20 months), an increase of lymphatic vessel coverage in the nodes and meninges was observed, along with accelerated drainage of macromolecules from brains. Compared with daily oral delivery of cilostazol and donepezil hydrochloride, a single administered dual drugs-loaded long-acting implants releasing for more than one month not only elevated drug concentrations in brains, improved the clearing efficiency of brain macromolecules, reduced Aβ accumulation, enhanced cognitive functions of the aged mice, but improved patient compliance as well, which provided a clinically accessible therapeutic strategy toward aged Alzheimer's diseases.

IgG immune complex-induced acute lung injury is ameliorated by cAMP via down-regulation of C/EBP- and AP-1-mediated transcriptions

BackgroundAcute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are life threatening pulmonary diseases, and we are now lack of effective therapeutic methods. Inflammatory responses are essential for initiating ALI/ARDS. Thus, ameliorating inflammatory reaction might be beneficial for treatment of the disease. There are increasing data that phosphodiesterase-4 (PDE4)-selective inhibitors, which may elevate cellular cyclic adenosine 3′, 5′-monophosphate (cAMP) level, could suppress inflammation. However, whether they could be used to treat IgG immune complex (IgG-IC)-associated ALI has not been determined.MethodsALI is induced by treating mice with airway deposition of IgG immune complexes. Cellular cAMP concentrations are elevated by treating mice or macrophages with Rolipram/Roflumilast. The degree of pulmonary injury is reflected by lung permeability, leukocyte accumulation, histological change and expressions of pro-inflammatory mediators. 6-Bnz-cAMP and H-89 are used to regulate protein kinase A (PKA) activity, and 8-pCPT-2′-O-Me-cAMP is applied to activate exchange proteins directly activated by cAMP (Epac). Gene expressions are analyzed by real-time PCR, ELISA or Western blot. CCAAT/enhancer binding protein (C/EBP) and activation protein 1 (AP-1) transcription activities are estimated by measuring the luciferase productions.ResultsIgG-IC-induced ALI is attenuated by the PDE4-selective inhibitor, which is due to reduced expressions of cytokine and chemokines. Interestingly, we find that cAMP downstream effector molecule PKA but not Epac is involved in negative regulation of IgG-IC-mediated pro-inflammatory mediators’ productions. Mechanistically, activation of cAMP-PKA signal axis leads to inactivation of MAPK pathway, resulting in a decrease in C/EBP- and AP-1-mediated transcriptions of pro-inflammatory mediators.ConclusionsOur data demonstrate, for the first time, that cAMP-PKA signal is involved in down-regulation of IgG-IC-associated inflammatory responses via down-regulating MAPK activation, which is critical for transcriptional activities of C/EBP and AP-1. Collectively, our experiments provide theoretical base for the potential application of PDE4-selective inhibitor to clinic for treatment of IgG-IC-related acute lung injury.

The effects of a Cilostazol, a selective phosphodiesterase III inhibitor, on liver ischemic-reperfusion injury and liver regeneration; In vitro experimental study

Objective: Hepatectomy and transplantation cause liver damage through ischemic reperfusion and oxidative stress. There is no treatment available to improve liver regeneration and reduce ischemic-reperfusion injury. The present study aimed to investigate whether a selective phosphodiesterase III inhibitor, Cilostazol, improves ischemic reperfusion injury and liver regeneration following extended hepatectomy. Material and Methods: Wistar albino rats (n=40) were randomized and divided into 4 equal groups. All rats underwent 60% hepatectomy, and Cilostazol (5 mg/kg per day) was administered to the experimental group. The subjects were sacrificed on the 4th and 7th days following the resection. Blood samples were taken to evaluate liver enzymes (ALT, AST) and liver tissue samples were taken to analyze morphology. Biochemical, morphological, and histopathological parameters were compared between Groups. Results: No statistically significant differences were detected in ALT, AST values , and relative liver weights in rats treated with Cilostazol compared to the control group without Cilostazol. Although not statistically significant, a significant increase was detected in relative liver weight and a decrease in AST value in rats treated with Cilostazol. SOD activity was found to be significantly higher and GSH levels, MPO and AOPPs levels were significantly lower in Cilostazol applied Groups. It is seen in these findings that selective inhibition of PDE3 by Cilostazol improves hepatic circulation. It was also found that ischemic reperfusion injury decreased and regeneration markers such as mitosis index, even nucleus, and proliferating cell nuclear antigen ratio increased in rats treated with Cilostazol. Conclusion: The present study found that selective PDE3 inhibitor Cilostazol positively affected the histopathological parameters following extended liver resection and significantly increased hepatocellular proliferation.

Drug-Excipient Compatibility Studies In Binary Mixtures of Tadalafil by Using DSC, TGA and FTIR

Objective: During drug preformulation studies, thermal analysis and spectroscopic techniques are used to detect physical or chemical incompatibilities between the active compound and the excipients in the formulation, and to demonstrate the safety and/or efficacy of the final product. It has revolutionized erectile dysfunction with the development of a selective cyclic guanosine monophosphate-specific PDE-5 inhibitors. Tadalafil is one of these inhibitors. Excipients are included in dosage forms to assist in manufacture, absorption or application. Although considered to be pharmacologically inert, the drug active compound may impair effectiveness. Material and Method: DSC, TGA, and FTIR, were used in the work. Ascorbic acid, butylated hydroxyanisole, calcium phosphate dibasic, cellulose, magnesium stearate, mannitol, sodium carboxymethyl cellulose, sucrose, talc, starch, primojel, and citric acid exhibit interaction with Tadalafil. Binary mixtures of drug:excipient have been analyzed. Results: Based on spectroscopic and thermal results; Tadalafil is incompatible with magnesium stearate, mannitol, sucrose, and ascorbic acid. Conclusion: Studies to investigate drug-excipient compatibility are an important step in drug development studies. Thermal and spectroscopic techniques are widely used in such studies.

CRISPR/Cas9 Knock-Out in Primary Neonatal and Adult Cardiomyocytes Reveals Distinct cAMP Dynamics Regulation by Various PDE2A and PDE3A Isoforms.

Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.

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

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

Application of Box-Behnken Design in the Preparation, Optimization, and In-Vivo Pharmacokinetic Evaluation of Oral Tadalafil-Loaded Niosomal Film.

Benign prostatic hyperplasia (BPH) affects about 90% of men whose ages are over 65. Tadalafil, a selective PDE-5 inhibitor, was approved by FDA for BPH, however, its poor aqueous solubility and bioavailability are considered major drawbacks. This work intended to develop and evaluate oral fast dissolving film containing tadalafil-loaded niosomes for those who cannot receive the oral dosage form. Niosomes were statistically optimized by Box-Behnken experimental design and loaded into a polymeric oral film. Niosomes were assessed for their vesicular size, uniformity, and zeta potential. The thickness, content uniformity, folding endurance, tensile strength, disintegration time, and surface morphology were evaluated for the prepared polymeric film. The optimized niosomes revealed high entrapment efficiency (99.78 ± 2.132%) and the film was smooth with good flexibility and convenient thickness (110 ± 10 µm). A fast release of tadalafil was achieved within 5 min significantly faster than the niosomes-free drug film. The in-vivo bioavailability in rats established that the optimized niosomal film enhanced tadalafil systemic absorption, with higher peak concentration (Cmax = 0.63 ± 0.03 µg/mL), shorter Tmax value (0.66-fold), and relative bioavailability of 118.4% compared to the marketed tablet. These results propose that the oral film of tadalafil-loaded niosomes is a suitable therapeutic application that can be passed with ease to geriatric patients who suffer from BPH.

Essential role of the mitochondrial Na+/Ca2+ exchanger NCLX in mediating PDE2-dependent neuronal survival and learning.

Impaired phosphodiesterase (PDE) function and mitochondrial Ca2+ (i.e., [Ca2+]m) lead to multiple health syndromes by an unknown pathway. Here, we fluorescently monitor robust [Ca2+]m efflux mediated by the mitochondrial Na+/Ca2+ exchanger NCLX in hippocampal neurons sequentially evoked by caffeine and depolarization. Surprisingly, neuronal depolarization-induced Ca2+ transients alone fail to evoke strong [Ca2+]m efflux in wild-type (WT) neurons. However, pre-treatment with the selective PDE2 inhibitor Bay 60-7550 effectively rescues [Ca2+]m efflux similarly to caffeine. Moreover, PDE2 acts by diminishing mitochondrial cAMP, thus promoting NCLX phosphorylation at its PKA site. We find that the protection of neurons against excitotoxic insults, conferred by PDE2 inhibition in WT neurons, is NCLX dependent. Finally, the administration of Bay 60-7550 enhances new object recognition in WT, but not in NCLX knockout (KO), mice. Our results identify a link between PDE and [Ca2+]m signaling that may provide effective therapy for cognitive and ischemic syndromes.

Forebrain-specific conditional calcineurin deficiency induces dentate gyrus immaturity and hyper-dopaminergic signaling in mice

Calcineurin (Cn), a phosphatase important for synaptic plasticity and neuronal development, has been implicated in the etiology and pathophysiology of neuropsychiatric disorders, including schizophrenia, intellectual disability, autism spectrum disorders, epilepsy, and Alzheimer’s disease. Forebrain-specific conditional Cn knockout mice have been known to exhibit multiple behavioral phenotypes related to these disorders. In this study, we investigated whether Cn mutant mice show pseudo-immaturity of the dentate gyrus (iDG) in the hippocampus, which we have proposed as an endophenotype shared by these disorders. Expression of calbindin and GluA1, typical markers for mature DG granule cells (GCs), was decreased and that of doublecortin, calretinin, phospho-CREB, and dopamine D1 receptor (Drd1), markers for immature GC, was increased in Cn mutants. Phosphorylation of cAMP-dependent protein kinase (PKA) substrates (GluA1, ERK2, DARPP-32, PDE4) was increased and showed higher sensitivity to SKF81297, a Drd1-like agonist, in Cn mutants than in controls. While cAMP/PKA signaling is increased in the iDG of Cn mutants, chronic treatment with rolipram, a selective PDE4 inhibitor that increases intracellular cAMP, ameliorated the iDG phenotype significantly and nesting behavior deficits with nominal significance. Chronic rolipram administration also decreased the phosphorylation of CREB, but not the other four PKA substrates examined, in Cn mutants. These results suggest that Cn deficiency induces pseudo-immaturity of GCs and that cAMP signaling increases to compensate for this maturation abnormality. This study further supports the idea that iDG is an endophenotype shared by certain neuropsychiatric disorders.

Abstract 9578: Chronic Phosphodiesterase Type 5 Inhibition by Sildenafil Causes Regression of Heart Failure: Insights into Molecular and Cellular Mechanisms

Background: Sildenafil (SIL), a selective inhibitor of PDE5 has been shown to exert profound beneficial effects in heart failure (HF). However, whether and by what mechanisms SIL can stop the progression of HF, or maybe even reverse the clinical route remain unknown. We tested the hypothesis that chronic SIL would improve intrinsic myocyte function and [Ca 2+ ] i regulation and reverse HF-induced detrimental alterations on cardiac SERCA2a, β-adrenergic receptors (AR) and nitric oxide synthase (NOS) isoforms, thus playing a salutary role in HF. Methods: We evaluated LV and myocyte function and the protein levels of myocyte β 1 - and β 3 - AR, SR Ca 2+ -ATPase (SERCA2a), phospholamban (PLB), and 3 NOS in 3 groups of male rats (8/group) for a period of 3 months (M): 1) HF , 3 M after receiving isoproterenol (ISO) (170 mg/kg sq for 2 days); 2) HF/SIL , 2 M after ISO, then SIL (70 μg/kg/day sq via mini pump) was initiated and given for 1 M; and 3) Controls . Results: Versus C, ISO-treated rats progressed to severe HF at 3 M with significantly reduced LV contractility (E ES , 0.7 vs 1.1 mmHg/μl), and slowed LV relaxation. LV myocyte contraction (dL/dt max , 77 vs 136 μm/sec), relaxation (dR/dt max , 62 vs 104 μm/sec) and [Ca 2+ ] iT (0.15 vs 0.24) were significantly decreased accompanied by a diminished myocyte inotropic response to ISO (10 -8 M) superfusion. These abnormalities were associated with concomitant significant decreases in myocyte protein levels of β 1 -AR (0.23 vs 0.64), SERCA2a (0.46 vs 0.80), PLB Ser16 /PLB ratio, and eNOS (0.28 vs 0.46), but increased protein levels of β 3 -AR (0.23 vs 0.10) and iNOS (0.18 vs 0.08). Chronic SIL treatment prevented HF-caused reduced E ES (1.3 mmHg/μl) and EF (62%) and retained normal dL/dt max (131μm/s), dR/dt max (106 μm/s), and [Ca 2+ ] iT (0.24), and restored normal myocyte contractile response to ISO stimulation. With SIL, protein levels of myocyte β 1 - and β 3 -AR, SERCA2a were normalized to control values, but eNOS was significantly elevated (0.77). Conclusions: Chronic SIL prevents HF-caused downregulation of cardiac β 1 -AR and reverses contrast changes between iNOS and β 3 -AR with SERCA2a and eNOS expression, restores normal myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve, and leads to regression of LV dysfunction in a rat model of progressive HF.

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 (1mg/kg) and pentoxifylline (100mg/kg) and also combination of rolipram (0.5mg/kg) and pentoxifylline (50mg/kg), orally for five weeks in rats that became diabetic by STZ (55mg/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.

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

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

Enhanced bioaccesibility of selective PDE4 inhibitor N-(3,4-dihydro-2h-1,5-benzodioxepin-7-yl) pyridine-4-carboxamide using polyamidoamine dendrimer

The aim of this study was to improve the aqueous solubility of highly hydrophobic but selective PDE4 inhibitor N-(3,4-dihydro-2h-1,5-benzodioxepin-7-yl) pyridine-4-carboxamide by associating it with polyamidoamine dendrimer. The PAMAM dendrimer restraining ethylenediamine core synthesized by a divergent approach was utilized for encapsulation. The solubility of conjugates was evaluated on the basis of concentration and generation of the dendrimer, pH of the solution, and temperature. The phase solubility diagram confirmed an increase in aqueous solubility of drug with increase in dendrimer concentration with respect to pH in the order 9.0 > 7.0 > 4.0. Moreover, values of thermodynamic parameters such as negative value of ΔHo, positive value of ΔSo and ΔGo reflects an exothermic complexation, presence of number of particles and spontaneity of the complexation. Overall, investigations validate the enhancement in solubility of drug after complexation with polyamidoamine dendrimer which further confirmed promising bioactivity of the drug-dendrimer conjugates.

Abstract 11076: Cardiac AKAP12 Overexpression Reduces Cyclic Amp Accumulation in the Vicinity of β 2 Ar

Introduction: In Heart Failure (HF), signaling downstream the β 2- Adrenergic Receptor (β 2 AR) is critical. Sympathetic stimulation of β 2 AR triggers Protein Kinase A (PKA) dependent phosphorylation of proteins that regulate cardiac function. Cyclic adenosine monophosphate (cAMP) accumulation in the vicinity of β 2 AR is crucial for PKA stimulation. cAMP levels are regulated in part by phosphodiesterases (PDEs). A Kinase Anchoring Protein 12 (AKAP12) has been reported to directly bind β 2 AR, PKA, and PDE4. Previously, we reported that cardiac overexpression of AKAP12 (oxAKAP12) in both male and female mice reduced left ventricular ejection fraction (EF) and fractional shortening (FS) significantly after 14-days of chronic Isoproterenol treatment when compared to control groups. Hypothesis: AKAP12 overexpression progresses HF development by reducing local cAMP levels in the vicinity of β 2 AR. Methods: AC16 cells (Human ventricular cell lines) were grouped into [1] control group or [2] cells stably transfected to overexpress human AKAP12 (oxAKAP12). cAMP accumulation in real-time downstream the β 2 AR was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) post-treatment with 10μM Epinephrine (EPI). Additionally, cells were pre-treated for 2 hours with either 10μM Rolipram (selective PDE4 inhibitor) or 0.1mM IBMX (non-selective PDE inhibitor) followed by 10μM EPI treatment. Results: oxAKAP12 cells (expressing AKAP12 protein 3 folds higher than controls) had significantly lower maximum cAMP accumulation post-EPI treatment downstream the β 2 AR (2.06 ± 0.32) compared to controls (9.04 ± 0.73), [p=0.0001]. Rolipram pretreatment increased cAMP levels in the oxAKAP12 group to (3.75± 0.72), however, the control group was still significantly higher (9.63 ± 0.95), [p=0.0032]. IBMX pretreatment increased cAMP levels in the oxAKAP12 group to (6.94 ± 1.55) which wasn’t significantly different from the control group (9.75 ± 2.52), [p=0.4152]. Conclusions: Cardiac overexpression of AKAP12 significantly reduces cAMP levels downstream of the β 2 AR. Potentially through scaffolding other PDEs aside from PDE4.

Piclamilast mitigates 1,2-dimethylhydrazine induced colon cancer in rats through modulation of Ras/PI3K/Akt/mTOR and NF-κβ signaling

Colorectal cancer (CRC) is the third leading type of adult cancer in both genders with high morbidity and mortality worldwide. Even though the discovery of many antineoplastic drugs for CRC, the current therapy is not adequately efficient.This study was designed to investigate the effect and mechanism of Piclamilast (PIC), a selective PDE4 inhibitor, on a DMH-induced colorectal cancer (CRC) rat model. The rats were grouped (n = 10) into group 1 (control), group 2 (PIC 3 mg/kg, p.o.), groups 3–5 received DMH (20 mg/kg/week, S.C.), and groups 4 and 5 received PIC (1 and 3 mg/kg/day, p.o.) for 15 weeks. The DMH treatment increased aberrant crypt foci (ACF), Proliferating cell nuclear antigen (PCNA), and TBARS levels, along with decreased antioxidant defenses (GSH, GSH-Px, and catalase). Increased NF-κβ expression and inflammatory cytokines were also evident. PIC dose-dependently reduced ACF and restored oxidative stress and inflammatory markers favorably. Moreover, PIC in its large, tested dose only significantly increased the intracellular level of cAMP and suppressed the activation of Ras and PI3K and its downstream Akt/mTOR signaling. Furthermore, PIC promoted CRC apoptosis, and increased the gene expression of the apoptotic factors, caspase-3 and Bax, and decreased the anti-apoptotic factor Bcl-2. The results of this study show that PIC may be a promising therapeutic agent for the treatment of CRC. PIC might inhibit the proliferation of CRC cells and induce apoptosis via multiple mechanisms that involve its antioxidant effect and inhibition of NF-κβ and Ras/PI3K/Akt/mTOR signaling.

Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis.

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, −3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-β2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.