Assessment of PDE4 Inhibitor-Induced Hypothermia as a Correlate of Nausea in Mice

Treatment with PAN-PDE4 inhibitors has been shown to produce hypothermia in multiple species. Given the growing body of evidence that links nausea and emesis to disturbances in thermoregulation in mammals, we explored PDE4 inhibitor-induced hypothermia as a novel correlate of nausea in mice. Using knockout mice for each of the four PDE4 subtypes, we show that selective inactivation of individual PDE4 subtypes per se does not produce hypothermia, which must instead require the concurrent inactivation of multiple (at least two) PDE4 subtypes. These findings contrast with the role of PDE4s in shortening the duration of α2-adrenoceptor-dependent anesthesia, a behavioral surrogate previously used to assess the emetic potential of PDE4 inhibitors, which is exclusively affected by inactivation of PDE4D. These different outcomes are rooted in the distinct molecular mechanisms that drive these two paradigms; acting as a physiologic α2-adrenoceptor antagonist produces the effect of PDE4/PDE4D inactivation on the duration of α2-adrenoceptor-dependent anesthesia, but does not mediate the effect of PDE4 inhibitors on body temperature in mice. Taken together, our findings suggest that selective inhibition of any individual PDE4 subtype, including inhibition of PDE4D, may be free of nausea and emesis.

ROSTMEMA: A Double-Blind Randomized Placebo-Controlled Trial with the PDE4 Inhibitor Roflumilast in Patients Suffering from Long-term Cognitive Sequela After Stroke

Background : The aim of this study is to examine whether treatment with roflumilast improves cognition in patients suffering from cognitive sequelae after stroke. The results may provide a proof of concept on the potential of roflumilast (or other phosphodiesterase-4 (PDE-4) inhibitors) as pharmacotherapeutic treatment to enhance cognition, and will further increase our knowledge on the role of PDE4 in human cognition in general. Cognitive processes, in particular memory, will be assessed. Methods : 100 female and male patients (41-70 years old) suffering from cognitive complaints 1 year after stroke will be recruited via advertisements via social media and via local caretaking organizations. The first phase of the study will be conducted according to a double-blind, randomized placebo-controlled, between-subjects design. After a baseline measurement, participants will be tested for acute (1 hour after drug intake) and subsequent chronic (1.5 and 3 months after start treatment) treatment effects. In a second phase, the placebo group (50 people) will be given the opportunity to receive roflumilast. This is an open label design. The roflumilast group will be tested for long-term treatment effects at three months after the end of treatment. Discussion : Strengths of the current study are the open-label design as well as the assessment of long-term effects. The findings of the current study will demonstrate if the mechanism of PDE4 inhibition is a relevant target to improve cognitive functions in patients with brain damage after a stroke. If positive effects are found in this patient group, this treatment could also be relevant for other brain disorders (e.g. head trauma, mild cognitive impairment, Fragile X syndrome, schizophrenia) in which enhanced neuronal plasticity is required to improve cognition. Trial registration : The Medical Ethics Committee of Maastricht University Medical Center+ granted ethics approval of the fourth version of the protocol on February 25, 2021. The trial was registered at the European Drug Regulatory Affairs Clinical Trials (EudraCT) register on July 29, 2020, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2020-003768-16 and Clinicaltrials.gov (NCT04854811) at April 21, 2021, https://clinicaltrials.gov/ct2/show/NCT04854811. The Central Committee on Research Involving Human Subjects (CCMO) granted approval on May 4, 2021.

Inhibition of PDE4 by Roflumilast ameliorates sleep deprivation-induced cognitive dysfunction in C57BL/6J mice

Sleep deprivation interferes with long-term memory and cognitive functions by over-activation of phosphodiesterase (PDE) enzymes. PDE4 is a non-redundant regulator of the cyclic nucleotides (cAMP), is densely expressed in the hippocampus, and is involved in learning and memory processes. In the present study, we investigated the effects of Roflumilast (ROF), a PDE4 inhibitor, on sleep deprivation induced cognitive dysfunction in a mouse model. Memory assessment was performed using a novel object recognition task and the cAMP level was estimated by ELISA. The alterations in the expressions of PDE4B, amyloid beta, CREB, BDNF, and synaptic proteins (Synapsin I, SAP 97, PSD 95) were assessed to gain insights on the possible mechanisms of action of ROF using the western blot technique. Results show that ROF reverse SD induced cognitive decline in mice. ROF down-regulated PDE4B and amyloid beta expressions. Additionally, ROF improved cAMP levels and the expressions of synapsin I, SAP 97, and PSD 95 in the hippocampal region of SD mice. Taken together, these results suggest that ROF can suppress the deleterious effects of SD-induced cognitive dysfunction via PDE4-mediated cAMP/CREB/BDNF cascade.

Phosphodiesterase-4 Inhibition Reduces Cutaneous Inflammation and IL-1β Expression in a Psoriasiform Mouse Model but Does Not Inhibit Inflammasome Activation

Apremilast (Otezla®) is an oral small molecule phosphodiesterase 4 (PDE4) inhibitor approved for the treatment of psoriasis, psoriatic arthritis, and oral ulcers associated with Behçet’s disease. While PDE4 inhibition overall is mechanistically understood, the effect of apremilast on the innate immune response, particularly inflammasome activation, remains unknown. Here, we assessed the effect of apremilast in a psoriasis mouse model and primary human cells. Psoriatic lesion development in vivo was studied in K5.Stat3C transgenic mice treated with apremilast for 2 weeks, resulting in a moderate (2 mg/kg/day) to significant (6 mg/kg/day) resolution of inflamed plaques after 2-week treatment. Concomitantly, epidermal thickness dramatically decreased, the cutaneous immune cell infiltrate was reduced, and proinflammatory cytokines were significantly downregulated. Additionally, apremilast significantly inhibited lipopolysaccharide- or anti-CD3-induced expression of proinflammatory cytokines in peripheral mononuclear cells (PBMCs). Notably, inflammasome activation and secretion of IL-1β were not inhibited by apremilast in PBMCs and in human primary keratinocytes. Collectively, apremilast effectively alleviated the psoriatic phenotype of K5.Stat3 transgenic mice, further substantiating PDE4 inhibitor-efficiency in targeting key clinical, histopathological and inflammatory features of psoriasis. Despite lacking direct effect on inflammasome activation, reduced priming of inflammasome components upon apremilast treatment reflected the indirect benefit of PDE4 inhibition in reducing inflammation.

Tanimilast, A Novel Inhaled Pde4 Inhibitor for the Treatment of Asthma and Chronic Obstructive Pulmonary Disease

Chronic respiratory diseases are the third leading cause of death, behind cardiovascular diseases and cancer, affecting approximately 550 million of people all over the world. Most of the chronic respiratory diseases are attributable to asthma and chronic obstructive pulmonary disease (COPD) with this latter being the major cause of deaths. Despite differences in etiology and symptoms, a common feature of asthma and COPD is an underlying degree of airways inflammation. The nature and severity of this inflammation might differ between and within different respiratory conditions and pharmacological anti-inflammatory treatments are unlikely to be effective in all patients. A precision medicine approach is needed to selectively target patients to increase the chance of therapeutic success. Inhibitors of the phosphodiesterase 4 (PDE4) enzyme like the oral PDE4 inhibitor roflumilast have shown a potential to reduce inflammatory-mediated processes and the frequency of exacerbations in certain groups of COPD patients with a chronic bronchitis phenotype. However, roflumilast use is dampened by class related side effects as nausea, diarrhea, weight loss and abdominal pain, resulting in both substantial treatment discontinuation in clinical practice and withdrawal from clinical trials. This has prompted the search for PDE4 inhibitors to be given by inhalation to reduce the systemic exposure (and thus optimize the systemic safety) and maximize the therapeutic effect in the lung. Tanimilast (international non-proprietary name of CHF6001) is a novel highly potent and selective inhaled PDE4 inhibitor with proven anti-inflammatory properties in various inflammatory cells, including leukocytes derived from asthma and COPD patients, as well as in experimental rodent models of pulmonary inflammation. Inhaled tanimilast has reached phase III clinical development by showing promising pharmacodynamic results associated with a good tolerability and safety profile, with no evidence of PDE4 inhibitors class-related side effects. In this review we will discuss the main outcomes of preclinical and clinical studies conducted during tanimilast development, with particular emphasis on the characterization of the pharmacodynamic profile that led to the identification of target populations with increased therapeutic potential in inflammatory respiratory diseases.

Anticancer Activity of Continentalic Acid in B-Cell Lymphoma

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant’s diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.

A European pharmacotherapeutic agent Roflumilast exploring integrated preclinical and clinical evidences for SARS CoV-2 mediated inflammation to organ damage

COVID-19 has spread globally, affecting almost 160 million individuals. Elderly and pre-existing patients (such as diabetes, heart disease and asthma), seems more susceptible to serious illness with COVID-19. Roflumilast was licensed for usage in the European Union in July 2010 as a phosphodiesterase-4 (PDE4) inhibitor. Roflumilast has been shown to decrease bleomycin-induced lung fibrosis, lung hydroxyproline, right heart thickning in animal prophylactic. The current study reviewed existing data that the PDE-4 inhibitor protects not just renal tissues but also other major organ systems after COVID-19 infection by decreasing immune cell infiltration. These immune-balancing effects of roflumilast were related with a decrease in oxidative and inflammatory burden, caspase-3 suppression, and increased PKA/cAMP levels in renal and other organ tissue.

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.

Abstract MP241: Roflumilast-mediated Phosphodiesterase 4d Inhibition Reverses Diabetes-associated Cardiac Dysfunction And Remodeling: Effects Beyond Glucose Lowering

Introduction: Patients with type 2 diabetes (T2DM) have a substantial risk of developing cardiovascular disease. Our recent studies reveal that hyperinsulinemia attenuates cardiac contractility by inducing expression of phosphodiesterase 4D (PDE4D) that increases cAMP degradation. Furthermore, there is growing evidence that PDE4 dysregulation is of pathophysiological importance in metabolic disorders. Hypothesis: We propose that inhibition of PDE4D might ameliorate diabetes-associated cardiac dysfunction, in addition to lowering glucose. Methods: Male C57BL/6J mice fed with high-fat diet (HFD) were treated with PDE4 inhibitor roflumilast (currently used to treat chronic obstructive pulmonary disease, COPD). Myocardial structure, contractile function and remodeling were evaluated. For mechanistic studies, specific silencing of cardiac PDE4D and overexpression of miR-1 were administrated in mice undergoing high-fat feeding. These studies were complemented by in vitro analysis in primary cultured rat cardiomyocytes and cardiac fibroblasts. Results: Mice on HFD developed greater body weight compared to normal chow diet-fed mice, manifesting hyperglycemia, hyperinsulinemia, cardiac contractile dysfunction and remodeling, and cardiac PDE4D5 upregulation by 24 weeks. The expression of PDE4D5 was also elevated in human hearts with diabetes. In comparison with vehicle-treated HFD controls, PDE4 inhibitor roflumilast (1mg/kg/day) can prevent and even reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of cardiac PDE4D expression. In addition, cardiac miR-1 level was reduced in HFD mice, which was restored by PDE4 inhibitor roflumilast treatment. Either cardiac specific PDE4D5 knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD-mice, despite persistence of hyperglycemia and glucose intolerance. Gain- and loss-of-function studies of PDE4D in cardiomyocytes implicated that suppression of PDE4D protected cardiac hypertrophy via SERCA2a-mediated miR-1 restoration. Moreover, inhibition of PDE4D prevented insulin-activated TGF-β1 signaling which promotes miR-1 reduction in cardiac fibroblasts and subsequent fibrosis. Conclusions: These studies elucidate a novel mechanism by which PDE4D contributes to HFD-induced heart failure through reducing miR-1 expression in both cardiomyocytes and cardiac fibroblasts and suggest a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction associated with diabetes.