Selective Phosphodiesterase Research Articles

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

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

Difamilast, a selective phosphodiesterase 4 inhibitor, ointment in paediatric patients with atopic dermatitis: a phase III randomized double-blind, vehicle-controlled trial.

SummaryBackgroundIn atopic dermatitis (AD), phosphodiesterase 4 (PDE4) inhibition reduces proinflammatory mediators and cytokines. Difamilast is a new selective PDE4 inhibitor.ObjectivesTo demonstrate the superiority of topical difamilast to vehicle in Japanese paediatric patients with AD.MethodsThis was a phase III randomized, double‐blind, vehicle‐controlled trial. Patients aged 2–14 years with an Investigator Global Assessment (IGA) score of 2 or 3 received difamilast 0·3% (n = 83), difamilast 1% (n = 85) or vehicle (n = 83) ointment twice daily for 4 weeks.ResultsThe primary endpoint was the percentage of patients with an IGA score of 0 or 1 with improvement by at least two grades at week 4. The success rates in IGA score at week 4 were 44·6%, 47·1% and 18·1% in the difamilast 0·3%, difamilast 1% and vehicle groups, respectively. Both difamilast groups demonstrated significantly higher success rates in IGA score compared with vehicle at week 4 [difamilast 0·3% (P < 0·001); difamilast 1% (P < 0·001)]. Regarding secondary endpoints, improvements in Eczema Area and Severity Index (EASI; improvement of ≥ 50%, ≥ 75% and ≥ 90% in overall score) at week 4 were significantly higher in patients in the difamilast 0·3% and 1% groups than those in the vehicle group. EASI score in the difamilast 0·3% and 1% groups was significantly reduced compared with that of patients in the vehicle group at week 1. The significant difference between both the difamilast groups and the vehicle groups was maintained from week 1 through to week 4. Most treatment‐emergent adverse events were mild or moderate, and no serious events or deaths were reported.ConclusionsDifamilast 0·3% and 1% ointments are superior to vehicle and well tolerated in Japanese paediatric patients with AD.

Rectal roflumilast improves trinitrobenzenesulfonic acid-induced chronic colitis in rats.

Roflumilast, a highly selective oral phosphodiesterase IV inhibitor, exerts anti-inflammatory and anti-fibrotic effects. Oral roflumilast causes gastrointestinal side effects, especially vomiting, which could be reduced by administering roflumilast via off-label routes. Inhaled roflumilast reportedly improved inflammatory and histopathological changes in asthmatic mice. The current study investigated the effects of oral and rectal roflumilast on trinitrobenzenesulfonic acid (TNBS)-induced chronic colitis in rats, an experimental model resembling human Crohn's disease. Five groups of rats (n=8) were used: normal control, TNBS-induced colitis, and three TNBS-treated colitic groups, which received oral sulfasalazine (500 mg·kg-1·day-1), oral roflumilast (5 mg·kg-1·day-1), or rectal roflumilast (5 mg·kg-1·day-1) for 15 days after colitis induction. Then, the following were assessed: the colitis activity score, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and IL-6 serum levels, colonic length, and myeloperoxidase, malonaldehyde, and glutathione levels. Histological examinations employed H&E, Masson trichrome, and PAS stains in addition to immunostaining for KI-67 and TNF-α. The TNBS-induced colitis rats showed significant increases in disease activity scores, serum TNF-α, IL-2, and IL-6 levels, and colonic myeloperoxidase and malonaldehyde content. They also showed significant decreases in colonic length and glutathione levels in addition to histopathological and immunohistochemical changes. All the treatments significantly improved all these changes. Sulfasalazine provided the greatest improvement, followed by oral roflumilast, and then rectal roflumilast. In conclusion, both oral and rectal roflumilast partially improved TNBS-induced chronic colitis, suggesting the potential of roflumilast as an additional treatment for Crohn's disease.

Abstract 10955: The Effects of Acute and Chronic Selective Phosphodiesterase 1 Inhibition in Associated Smooth Muscle Cell-Specific Aging Features

Introduction: Aging-related vascular smooth muscle cell (VSMC) remodeling plays an essential role in cardiovascular diseases. Reduced NO-cGMP pathway signalling is a major feature and pathogenic mechanism underlying VSMC dysfunction. Recently, we found that phosphodiesterase (PDE) 1, an enzyme that hydrolyzes and inactivates the cyclic nucleotides cAMP and cGMP, is a potential treatment target for restoring age-related deficits in VSMC signalling. We here tested the effects of PDE1 inhibition in SMC-specific Ercc1 DNA repair mutant mice, a model of isolated, accelerated SMC aging. Design and Method: SM22α Cre+ Ercc1 fl/- (SMC-KO) and SM22α Cre+ Ercc1 fl/+ littermate (SMC-LM) mice receive either vehicle or the PDE1 inhibitor lenrispodun (dose 40mg/kg/day) in drinking water for 8 weeks. Reactive hyperemia was measured in-vivo at the age of 14 and 25 weeks. Mice were sacrificed at 26 weeks. The endothelium-dependent (ED) and -independent (EI) relaxation of the thoracic aorta was assessed by wire myography. Moreover, we investigated the effect of PDE1 inhibition on the expression of selected PDEs involved in the vasculature by qPCR. Results: Reactive hyperemia was dramatically reduced in vehicle-treated SMC-KO vs. SMC-LM at the age of 25 weeks (P=0.0002) but was preserved in lenrispodun-treated SMC-KO mice. In vehicle-treated SMC-KO mice, the ED and EI relaxations were greatly diminished compared to SMC-LM (ED: 23.92±4.34 vs. 68.04±4.86, P&lt;0.0001; EI: 57.93±5.31 vs. 87.43±4.01, P=0.0004). Acute administration of lenrispodun in the organ bath improved both ED and EI responses (ED: 38.61±4.04, P=0.001; EI 69.99±4.29 P=0.01) while the relaxations remained unchanged after chronic lenrispodun treatment. In addition, lenrispodun upregulated PDE1a mRNA expression in SMC-KO, while no changes were observed in other PDEs. Conclusions: PDE1 inhibition may therapeutically be useful in restoring age-related vascular dysfunction by preserving microvascular function and potentiating NO-cGMP signaling. Yet, its chronic effects might be counteracted by the upregulation of PDE1a in vasculature. These results warrant further pharmacodynamic profiling of PDE enzyme regulation during chronic PDE1 inhibitor treatment.

Treatment with IMR-687, a Highly Selective PDE9 Inhibitor, Increases HbF and Reduces VOCs in Adults with Sickle Cell Disease in a Long-Term, Phase 2a, Open-Label Extension Study

IntroductionIMR-687 (tovinontrine) is a highly selective phosphodiesterase 9 (PDE9) inhibitor being developed as an orally administered therapy for patients with sickle cell disease (SCD) and beta-thalassemia. IMR-687 increases intracellular cGMP levels and has been shown in preclinical studies to increase fetal hemoglobin (HbF) expression and reduce hemolysis and sickling of RBCs, which can lead to painful vaso-occlusive crisis (VOC). In a Phase 2a, randomized, double-blind, placebo-controlled study of adult patients with SCD (N=93) (NCT03401112), IMR-687 was generally well-tolerated as a monotherapy and in combination with hydroxyurea (HU) (European Hematology Association Annual Congress 2021, Abstract S263). IMR-687 treatment with 50-200 mg once daily (N=63) for up to 6 months, as compared with placebo (N=30), resulted in a 40% lower mean annualized rate of VOCs, 38% lower mean annualized rate of VOC-related hospitalizations, and increased median time to first VOC (169 days vs 87 days, respectively, p=0.029). Improvements were also observed in patient-reported outcomes regarding pain episode severity (ASCQ-Me) and percentage of RBCs containing HbF (F-cells).MethodsA Phase 2a open-label extension (OLE) study (NCT04053803) is ongoing to assess the long-term safety and benefit of IMR-687 administered to adult subjects with SCD for up to 4 years. Secondary long-term pharmacokinetic (PK) and pharmacodynamic (PD) parameters are also being examined. The use of background HU is permitted. The Safety Review Committee meets regularly and ad hoc as necessary.Subjects were eligible upon completion of the Phase 2a parent study (16 or 24 weeks depending on the assigned treatment arm and without early withdrawal) and if they met all inclusion and exclusion criteria. For subjects without treatment interruption, baseline values from the parent study were used, as appropriate. The dose of IMR-687 initially administered in the OLE study was 200 mg as a once daily oral dose. The dose of IMR-687 is planned to be increased to 300 or 400 mg daily, depending on the subject's weight.Subjects are evaluated every 4-6 months for safety, PK and PD parameters (including HbF, F-cells, hemoglobin [Hb] and other biomarkers) as well as incidence of VOCs.ResultsAs of a data cut-off of 12-May-2021, 24 subjects were enrolled in the OLE study, 17 of which had a treatment interruption between the parent and OLE studies, and 7 subjects had direct roll-over to the OLE study. Seventeen of the OLE subjects were treated with IMR-687 monotherapy and 7 subjects were treated with combination IMR-687 + HU.IMR-687 continued to be well-tolerated in the OLE study (N=24). There were no treatment-related serious adverse events (SAEs), and the most common (≥10%) AEs were headache (21% of subjects), back pain (17%) and nausea (13%). There were 3 (13%) subjects with VOC-related hospitalizations.Annualized VOC rate was analyzed for 18 subjects treated for at least 200 days in the OLE study. Subjects who were previously in the active treatment arm in the parent study (N=13) maintained a low mean annualized VOC rate while continuing IMR-687 in the OLE study (1.35/yr [parent] and 1.85/yr [OLE]); subjects previously treated with placebo (N=5) had a 39% reduction in VOC rate when switched to IMR-687 (4.71/yr [parent] vs 2.89/yr [OLE]) (Figure 1). Of 15 subjects with evaluable PD data at 8 months, 7 (47%) subjects had a ≥ 6% absolute increase in F-cells, and 4 of 11 (36%) subjects had a ≥ 3% absolute increase in HbF (Figure 2). Updated data for subjects completing 12 months in the OLE study will be presented.ConclusionsConsistent with the parent study, preliminary results from the ongoing Phase 2a OLE study demonstrate that daily dosing of 200 mg IMR-687 was well-tolerated with longer-term treatment as a monotherapy or in combination with HU. IMR-687 treatment reduced the annualized rate of VOCs and increased HbF (%) and F-cells (%). Based on these encouraging data, a Phase 2b study (NCT04474314) is ongoing to further explore IMR-687 at doses up to 400 mg daily as a disease-modifying therapy for SCD. [Display omitted] DisclosuresBarysauskas: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Yen: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Tang: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Ballal: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Attie: Imara Inc.: Current Employment, Current equity holder in publicly-traded company.

Inhibition of phosphodiesterase type 9 reduces obesity and cardiometabolic syndrome in mice.

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show that cyclic GMP-selective phosphodiesterase 9A inhibition (PDE9-I) in both male and ovariectomized female mice suppresses preestablished severe diet-induced obesity/CMS with or without superimposed mild cardiac pressure load. PDE9-I reduces total body, inguinal, hepatic, and myocardial fat; stimulates mitochondrial activity in brown and white fat; and improves CMS, without significantly altering activity or food intake. PDE9 localized at mitochondria, and its inhibition in vitro stimulated lipolysis in a PPARα-dependent manner and increased mitochondrial respiration in both adipocytes and myocytes. PPARα upregulation was required to achieve the lipolytic, antiobesity, and metabolic effects of PDE9-I. All these PDE9-I-induced changes were not observed in obese/CMS nonovariectomized females, indicating a strong sexual dimorphism. We found that PPARα chromatin binding was reoriented away from fat metabolism-regulating genes when stimulated in the presence of coactivated estrogen receptor-α, and this may underlie the dimorphism. These findings have translational relevance given that PDE9-I is already being studied in humans for indications including heart failure, and efficacy against obesity/CMS would enhance its therapeutic utility.

Difamilast ointment in adult patients with atopic dermatitis: A phase 3 randomized, double-blind, vehicle-controlled trial

Difamilast is a selective phosphodiesterase 4 inhibitor. Phosphodiesterase 4 is involved in cytokine production linked with inflammatory disorders, including atopic dermatitis. To demonstrate the superiority of difamilast ointment 1% to vehicle in adult Japanese patients with atopic dermatitis. In this phase 3, randomized, double-blind trial, patients aged 15-70years with an investigator global assessment score of 2 or 3 received topical difamilast ointment 1% (n=182) or a vehicle (n=182) twice daily for 4weeks. The success rate in investigator global assessment score at week 4 (primary endpoint)-the percentage of patients achieving an investigator global assessment score of 0 or 1 with ≥2-grade improvement-was significantly higher with 1% difamilast than with the vehicle (38.46% vs 12.64%, respectively, P<.0001).The success rates in ≥50%, ≥75%, and ≥90% improvement in overall eczema area and severity index score at week 4 followed the same trend. Difamilast at 1% provided significant mean percent improvement from baseline in overall eczema area and severity index score versus vehicle from week 1 to 4. Treatment-emergent adverse events were mostly mild or moderate and less frequent with difamilast. Study treatment was limited to 4weeks. Difamilast ointment 1% demonstrated superiority to the vehicle and favorable safety in adult Japanese patients with atopic dermatitis.

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

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

Dose dependent effect of cilostazol in induced testicular ischemia reperfusion via modulation of HIF/VEGF and cAMP/SIRT1 pathways

Twisting of the spermatic cord is a common dangerous health problem that may be accompanied with testicular necrosis and infertility. Cilostazol (CLZ) is a selective phosphodiesterase (PDE) 3A inhibitor used for treatment of intermittent claudication. It has a great role in myocardial, spinal cord and hepatic ischaemia/reperfusion. However, till now, there are no researches evaluating its role in testicular ischaemia/reperfusion (TIR). The current work studies its capability to improve TIR induced injury with more concentration on the mechanisms involved in such effect. Four groups of animals were included: sham, TIR induced group, TIR plus CLZ low dose (10 mg/kg), TIR plus CLZ high dose (30 mg/kg). Our results proved that TIR had significant decrease of the serum ELISA of testosterone, marked disturbances in oxidative stress evaluated parameters as malondialdehyde (MDA), reduced glutathione (GSH), total antioxidant capacity (TAC), ELISA measurement of tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL1β) inflammatory mediators, apoptotic marker (caspase3) using western blotting, immunohistochemistry of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). TIR reduced the protective agents as cyclic adenosine monophosphate (cAMP) and sirtuin-1 (SIRT1) by ELISA method with marked germinal cell apoptosis. The biochemical results were confirmed by the histopathological findings that showed marked decrease in both Johnsen's score and Cosentino's score. However, treatment with CLZ significantly reversed the profound TIR damaging effects, on the basis of its anti‐inflammatory, anti-oxidant, and anti‐apoptotic activities with recuperation of the testicular vascularity. Modulation of HIF/VEGF and cAMP/SIRT1 pathways showed a great role in mediating such effect.

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

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

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

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

Phosphodiesterase-4 Inhibitors for Non-COPD Respiratory Diseases.

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

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

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

Abstract 1243: Effects of ITI-214, a potent and selective phosphodiesterase type 1 inhibitor, on tumor myeloid cellular composition, tumor volume and survival in mouse models of colorectal cancer when combined with an anti-PD-1 checkpoint inhibitor

Abstract We investigated the effect of ITI-214, a selective phosphodiesterase type 1 (PDE-1) inhibitor [Li et al., J Med Chem, 2016, 1149-64] on immune cell composition of the tumor microenvironment, tumor volume and survival in syngeneic mouse model of colorectal cancer. The studies are based on our previous preclinical work showing that PDE-1 inhibitors modulate the function of immune cells (microglia, macrophages) by reducing immune cell motility and down-regulating key cytokines (CCL2 and TNFa). We hypothesize that ITI-214 will inhibit macrophage infiltration into the tumor microenvironment and reverse macrophage suppression of immune responses in immunogenic cancers, thereby promoting antitumor immunity and tumor killing. The effects of ITI-214, alone or in combination with sub-effective doses of programmed cell death-1 (PD-1) immune checkpoint inhibitor, were assessed on the growth of CT26 tumor in BALB/c mice. Tumor responses in tumor-bearing mice treated with isotype antibodies (i.e. control group) were compared with groups of mice treated with ITI-214 monotherapy (50mg/kg, i.p. qd, from day 7), a sub-effective dose of anti-PD-1 antibody (150 µg anti-PD-1 RPMI-14, days 7, 11, 14, 17), and a combination of each dose of ITI-214 plus a sub-effective dose of the anti PD-1 antibody. Results showed that only mice receiving the combination of ITI-214 with a sub-effective dose of anti-PD-1 displayed significantly reduced tumor growth (p=0.001). Flow cytometry analysis of tumor-infiltrating immune cells revealed that tumors from ITI-214 monotherapy-treated mice showed significantly decreased numbers of tumor (infiltrating) macrophages which were further decreased in mice receiving the combination treatment. Further, the combination therapy of ITI-214 and anti-PD-1 enhanced anti-tumor immunity by eliciting increased numbers of Natural Killer (NK) cells and TNFa-producing CD4 T-cells. The results suggest that ITI-214 alone reduces numbers of tumor macrophages, an effect which we believe unmasks the anti-tumor immune response induced by anti-PD-1 antibodies. In separate experiments we assessed the effect of combined anti-PD-1 (5mg/kg anti-PD-1 RPMI-14, biwk x 2) and ITI-214 treatment (25 or 50mg/kg, oral, qd, from day 7) on survival; the effect of combined anti-PD-1 and ITI-214 treatment produced a complete response in about 50% of treated mice, translating into a statistically significant effect on survival as compared to control (p=0.001). Together, these studies demonstrate a synergistic ability of ITI-214 and anti-PD-1 to alter the tumor environment and control tumor growth. Translating these findings to humans suggests that ITI-214 may provide a means to expand the utility of immune checkpoint inhibitors in immunogenic tumors. Citation Format: Gretchen L. Snyder, Peter A. John, Marc V. Pulanco, Xingxing Zang, Allen A. Fienberg, Robert E. Davis. Effects of ITI-214, a potent and selective phosphodiesterase type 1 inhibitor, on tumor myeloid cellular composition, tumor volume and survival in mouse models of colorectal cancer when combined with an anti-PD-1 checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1243.

Structure-based redesigning of pentoxifylline analogs against selective phosphodiesterases to modulate sperm functional competence for assisted reproductive technologies

Phosphodiesterase (PDE) inhibitors, such as pentoxifylline (PTX), are used as pharmacological agents to enhance sperm motility in assisted reproductive technology (ART), mainly to aid the selection of viable sperm in asthenozoospermic ejaculates and testicular spermatozoa, prior to intracytoplasmic sperm injection (ICSI). However, PTX is reported to induce premature acrosome reaction (AR) and, exert toxic effects on oocyte function and early embryo development. Additionally, in vitro binding studies as well as computational binding free energy (ΔGbind) suggest that PTX exhibits weak binding to sperm PDEs, indicating room for improvement. Aiming to reduce the adverse effects and to enhance the sperm motility, we designed and studied PTX analogues. Using structure-guided in silico approach and by considering the physico-chemical properties of the binding pocket of the PDEs, designed analogues of PTX. In silico assessments indicated that PTX analogues bind more tightly to PDEs and form stable complexes. Particularly, ex vivo evaluation of sperm treated with one of the PTX analogues (PTXm-1), showed comparable beneficial effect at much lower concentration—slower AR, higher DNA integrity and extended longevity of spermatozoa and superior embryo quality. PTXm-1 is proposed to be a better pharmacological agent for ART than PTX for sperm function enhancement.

Selective Phosphodiesterase 1 Inhibition Ameliorates Vascular Function, Reduces Inflammatory Response, and Lowers Blood Pressure in Aging Animals.

Diminished nitric oxide-cGMP-mediated relaxation plays a crucial role in cardiovascular aging, leading to decreased vasodilation, vascular hypertrophy and stiffening, and ultimately, cardiovascular dysfunction. Aging is the time-related worsening of physiologic function due to complex cellular and molecular interactions, and it is at least partly driven by DNA damage. Genetic deletion of the DNA repair enzyme ERCC1 endonuclease in Ercc1Δ/- mice provides us an efficient tool to accelerate vascular aging, explore mechanisms, and test potential treatments. Previously, we identified the cGMP-degrading enzyme phosphodiesterase 1 as a potential treatment target in vascular aging. In the present study, we studied the effect of acute and chronic treatment with ITI-214, a selective phosphodiesterase 1 inhibitor on vascular aging features in Ercc1Δ/- mice. Compared with wild-type mice, Ercc1Δ/- mice at the age of 14 weeks showed decreased reactive hyperemia, diminished endothelium-dependent and -independent responses of arteries in organ baths, carotid wall hypertrophy, and elevated circulating levels of inflammatory cytokines. Acute ITI-214 treatment in organ baths restored the arterial endothelium-independent vasodilation in Ercc1Δ/- mice. An 8-week treatment with 100 mg/kg per day ITI-214 improved endothelium-independent relaxation in both aorta and coronary arteries, at least partly restored the diminished reactive hyperemia, lowered the systolic and diastolic blood pressure, normalized the carotid hypertrophy, and ameliorated inflammatory responses exclusively in Ercc1Δ/- mice. These findings suggest phosphodiesterase 1 inhibition would provide a powerful tool for nitric oxide-cGMP augmentation and have significant therapeutic potential to battle arteriopathy related to aging. SIGNIFICANCE STATEMENT: The findings implicate the key role of phosphodiesterase 1 in vascular function and might be of clinical importance for the prevention of mortalities and morbidities related to vascular complications during aging, as well as for patients with progeria that show a high risk of cardiovascular disease.

Topical Delivery of Apremilast Loaded Nanostructured Lipid Carrier Based Hydrogel for Psoriasis Therapy

Background: Apremilast (APR) is an orally administered selective phosphodiesterase 4 inhibitor approved to treat plaque psoriasis and psoriatic arthritis and is available as an oral tablet formulation. However, its systemic side effects limit its application. The low solubility and permeability of apremilast make it difficult to administer it through the skin. Hence an attempt is made to incorporate apremilast into a suitable nanocarrier to facilitate its topical delivery.&#x0D; Aims: To formulate and characterize Apremilast loaded nanostructured lipid carriers for the management of psoriasis to reduce the systemic side effects.&#x0D; Methodology: Apremilast loaded Nanostructured Lipid carriers (NLC) were prepared by melt emulsification accompanied by probe sonication. The formulation was prepared using GMS, Sefsol 218, Tween 80 and Transcutol P as Solid Lipid, Liquid lipid, Surfactant and Penetration Enhancer. The NLC was incorporated into carbapol 934 dispersion to convert it into a gel. The NLC formulation was evaluated for size, Polydispersity Index, Zeta Potential, Entrapment efficiency, Transmission Electron Microscopy. After that, the NLC gel was examined for Spreadability, Extrudabilty, Viscosity, In vitro drug release, Ex vivo permeation, Skin deposition and In vivo studies.&#x0D; Results: The formulated Apremilast loaded showed particle size of less than 200 nm (i.e.170.32nm) with a narrow PDI of 0.267. Entrapment efficiency revealed that 89.26±01.22% of the drug was entrapped. Transmission electron microscopy images confirmed the spherical nature of the nanocarrier. The extended-release pattern of the formulated NLC for 24h was observed in the in vitro release studies and followed the Higuchi model(R2=0.9966). Ex vivo permeability showed a 6.14 fold increase in permeability and 74.05±0.25% deposition of apremilast loaded NLC gel compared to apremilast gel. The formulation was stable for three months without significant changes. In vivo skin studies showed that the prepared NLC did not have any skin irritation potential. The antipsoriatic activity demonstrated by the Apremilast loaded NLC gel in the imiquimod induced psoriasis model in mice was comparable to the standard treatment.&#x0D; Conclusion: Apremilast loaded NLC demonstrated enhanced permeation, improved skin retention and extended-release compared to conventional gel. The developed formulation can be an alternative for psoriasis therapy after clinical trials in the future.

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

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

Evaluation of Neutrophil Dynamics Change by Protective Effect of Tadalafil After Renal Ischemia/Reperfusion Using In Vivo Real-time Imaging.

Neutrophils play a major role in ischemia/reperfusion injury (IRI) in renal transplantation and acute kidney injury. However, it has been difficult to observe changes in neutrophil dynamics over time in living mice kidney. We investigate neutrophil dynamics in IRI in living mice using novel in vivo multiphoton microscope imaging techniques and characterize the renoprotective effects of a selective phosphodiesterase 5 inhibitor, tadalafil. Wild-type and endothelial nitric oxide synthase knockout mice, a model of endothelial dysfunction, were used to establish in vivo real-time imaging in living mouse kidneys. Neutrophils were labeled green with Ly-6G monoclonal antibody, and plasma flow was labeled red with BSA. Tadalafil was administered orally 1 h before surgery. Both kidney pedicles were reperfused after 37°C warm ischemia for 45 min. Our novel approach revealed that neutrophils were trapped in glomerulus within a few minutes after reperfusion. They gradually increased over time and infiltrated neutrophils were observed in the tubular lumen and peritubular capillary. The neutrophils were clearly visualized rolling on peritubular capillary plexus at 3 μm/min. The administration of tadalafil significantly reduced neutrophil influx into the glomerulus in both wild-type and endothelial nitric oxide synthase knockout mice. Reduced neutrophil infiltration in tadalafil groups, which was confirmed by flow cytometry, resulted in histopathologically decreased tubular injury. The expression of vascular cell adhesion molecule 1 and kidney injury molecule 1 was partially prevented by tadalafil. Use of a novel technique contributed to elucidation of neutrophil dynamics after reperfusion. Tadalafil has a potential for inhibiting neutrophil infiltration in renal IRI.

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

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