Phosphodiesterase Inhibitor Research Articles

Repurposing drugs for highly prevalent diseases: pentoxifylline, an old drug and a new opportunity for diabetic kidney disease.

Diabetic kidney disease is one of the most frequent complications in patients with diabetes and constitutes a major cause of end-stage kidney disease. The prevalence of diabetic kidney disease continues to increase as a result of the growing epidemic of diabetes and obesity. Therefore, there is mounting urgency to design and optimize novel strategies and drugs that delay the progression of this pathology and contain this trend. The new approaches should go beyond the current therapy focussed on the control of traditional risk factors such as hyperglycaemia and hypertension. In this scenario, drug repurposing constitutes an economic and feasible approach based on the discovery of useful activities for old drugs. Pentoxifylline is a nonselective phosphodiesterase inhibitor currently indicated for peripheral artery disease. Clinical trials and meta-analyses have shown renoprotection secondary to anti-inflammatory and antifibrotic effects in diabetic patients treated with this old known drug, which makes pentoxifylline a candidate for repurposing in diabetic kidney disease.

Protective effects of phosphodiesterase 2 inhibitor against Aβ1-42 induced neuronal toxicity

Our previous study suggested that inhibition of Phosphodiesterase 2 ameliorates memory loss upon exposure to oxidative stress. While whether memory enhancing effects of PDE2 inhibition on Alzheimer's disease mouse model are involved in antioxidant defense and neuronal remodeling, are largely unexplored. The present study addressed whether and how PDE2 inhibitor Bay 60–7550 rescued Aβ oligomers (Aβo)-induced neuronal damage and memory impairment. The results suggested that exposure of primary cortical neurons to Aβo induced neuronal cells damage and increased PDE2 expression, which were paralleled to an increase in the oxidative parameter malondialdehyde (MDA) level and cellular apoptosis. However, this Aβo-induced oxidative damage was blocked by pre-treatment with protein kinase A or G (PKA or PKG) inhibitor, suggesting the involvement of cAMP/cGMP signaling. Moreover, microinjection of Aβo into the prefrontal cortex of mice increased the MDA level; while Bay 60–7550 reversed this effect and increased antioxidant and anti-apoptotic factors, i.e. increased trolox-equivalent-antioxidant capacity and Bcl-2/Bax ratio. Bay 60–7550 also rescued Aβo-induced synaptic atrophy and memory deficits, as evidenced by the increased synaptic proteins' levels and spine density in the prefrontal cortex, and improved cognitive behaviors by decreased working memory errors in the eight-arm maze and increased discrimination index in the novel object recognition test. These findings suggest that inhibition of PDE2 contributes to antioxidant defense and neuronal remodeling by regulation of cAMP/cGMP signaling, which provide a theoretical basis for the future use of PDE2 inhibitors as the anti-AD drugs.

Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

Fibroblast growth factor 21 (FGF21) functions as a polypeptide hormone to regulate glucose and lipid metabolism, and its expression is regulated by cellular metabolic stress. Pyruvate is an important intermediate metabolite that acts as a key hub for cellular fuel metabolism. However, the effect of pyruvate on hepatic FGF21 expression and secretion remains unknown. Herein, we examined the gene expression and protein levels of FGF21 in human hepatoma HepG2 cells and mouse AML12 hepatocytes in vitro, as well as in mice in vivo. In HepG2 and AML12 cells, pyruvate at concentrations above 0.1 mM significantly increased FGF21 expression and secretion. The increase in cellular cAMP levels by adenylyl cyclase activation, phosphodiesterase (PDE) inhibition and 8-Bromo-cAMP administration significantly restrained pyruvate-stimulated FGF21 expression. Pyruvate significantly increased PDE activities, reduced cAMP levels and decreased CREB phosphorylation. The inhibition of exchange protein directed activated by cAMP (Epac) and cAMP response element binding protein (CREB) upregulated FGF21 expression, upon which pyruvate no longer increased FGF21 expression. The increase in plasma pyruvate levels in mice induced by the intraperitoneal injection of pyruvate significantly increased FGF21 gene expression and PDE activity with a reduction in cAMP levels and CREB phosphorylation in the mouse liver compared with the control. In conclusion, pyruvate activates PDEs to reduce cAMP and then inhibits the cAMP–Epac–CREB signaling pathway to upregulate FGF21 expression in hepatocytes.

Roflumilast, a cyclic nucleotide phosphodiesterase 4 inhibitor, protects against cerebrovascular endothelial injury following cerebral ischemia/reperfusion by activating the Notch1/Hes1 pathway

The loss of tight junction (TJ) and adherens junction (AJ) proteins leads to the damage of the blood-brain barrier (BBB) during cerebral ischemia. Inhibition of cyclic nucleotide phosphodiesterase 4 (PDE4) by roflumilast (Roflu) protects against ischemic stroke-induced neuronal damage. However, the effects of Roflu on vascular endothelial injury and BBB integrity remain unknown. Here, we investigated whether and how Roflu protects against cerebrovascular endothelial injury caused by cerebral ischemia/reperfusion. We demonstrated that PDE4B knocking-down increased the expression of TJ and AJ proteins in human brain microvascular endothelial cells (HBMECs) subjected to oxygen-glucose deprivation reperfusion (OGD/R). Inhibition of PDE4 by Roflu (1.0 μM) showed similar effects as PDE4B knocking-down. We then found that Roflu activated Notch1/Hairy and enhancer of split 1 (Hes1) signaling. Consistently, the effects of Roflu on TJ and AJ proteins were reversed by the γ-secretase inhibitor DAPT or Hes1 knocking-down. Furthermore, Roflu (1.0 mg/kg) improved neurobehavioral outcomes and ameliorated BBB disruption in rats following ischemic stroke. Roflu also increased the levels of TJ proteins and AJ proteins in vivo. Collectively, these data suggest that Roflu is a promising compound for the prevention of BBB damage. The protective effects of Roflu are mediated through activation of the Notch1/Hes1 pathway.

Selective inhibition of phosphodiesterase 4D increases tau phosphorylation at Ser214 residue.

Tau is a protein that normally participates in the assembly and stability of microtubules. However, it can form intraneuronal hyperphosphorylated aggregates that are hallmarks of Alzheimer's disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP-dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU-S214), an event that was shown to reduce the pathological assembly of the protein. Given that the neuronal cAMP/PKA-activated cascade is involved in synaptic plasticity and memory, and that cAMP-enhancing strategies demonstrated promising therapeutic potential for the treatment of cognitive deficits, we investigated the impact of cAMP on pTAU-S214 in N2a cells and rat hippocampal slices. Our results confirm that the activation of adenylyl cyclase increases pTAU-S214 in both model systems and, more interestingly, this effect is mimicked by GEBR-7b, a phosphodiesterase 4D inhibitor with proven pro-cognitive efficacy in rodents.

E2027 Cardiac Safety Evaluation With Concentration-Response Modeling of ECG Data to Inform Dose Selection in Studies in Patients With Dementia With Lewy Bodies.

E2027 is a novel, highly selective and potent inhibitor of phosphodiesterase 9 in development for dementia with Lewy bodies. Cardiac safety assessments for emerging agents are essential to avoid drug-induced QT interval prolongation, which may predispose individuals to potentially fatal ventricular arrhythmias. To evaluate the cardiac safety of E2027 and to inform dose selection for the phase 2 study of E2027 in dementia with Lewy bodies, we evaluated concentration-response modeling of pooled electrocardiogram data. A post hoc concentration-QTc analysis evaluated potential QT effects using data from 2 randomized, double-blind studies in healthy subjects: a single ascending dose (SAD) study and a multiple ascending dose (MAD) study. Daily E2027 doses ranged from 5 to 1200 mg. A linear mixed-effects model was used to establish the relationship between plasma concentrations of E2027 and change from the baseline of QTcF (ΔQTcF). A significant but shallow relationship was observed in the estimated slope of the concentration-ΔQTcF: 0.002 ms/ng/mL (90% confidence interval: 0.0007-0.0031) with a small, nonsignificant treatment effect-specific intercept of -0.6 ms. Based on this pooled concentration-QTc analysis, an effect on the QTcF interval >10 ms can be excluded up to E2027 plasma concentrations of ∼3579 ng/mL, corresponding to a dose at least 4-fold larger than the 50 mg phase 2 dose. This pooled post hoc analysis evaluating cardiac safety of E2027 demonstrated that clinically concerning QTcF prolongation and related cardiac complications are highly unlikely with proposed E2027 doses planned for phase 2.

Effect of milrinone on the meiosis resumption and cytoplasm maturation of buffalo oocytes.

Buffalo has many excellent economic traits and it is one of the greatest potential livestock. Compared with cattle, buffalo has poorer reproductivity, it is of great significance to improve the development potential of oocytes. Buffalo oocyte in vitro maturation (IVM) has been widely used in production, but the poor development ability of bovine oocytes IVM limits the development of buffalo reproductivity. Milrinone as a phosphodiesterase inhibitor could affect the maturation of oocytes in goat and mice, but there have been few reported studies in water buffalo. To optimize buffalo oocyte in vitro maturation systems, the effects of phosphodiesterase inhibitor (milrinone) on pre-maturation culture of buffalo oocytes were investigated in this study. Buffalo cumulus-oocyte complexes (COCs) were cultured in medium with different concentrations (0, 12, 25, 50 and 100 mol/l) of milrinone for different times (0, 4, 8, 12, 16, 22 and 24 h). The results showed that the buffalo COCs nuclear maturation process could be inhibited by milrinone (25-100 mol/l) in a dose-dependent manner. The inhibitory effect of milrinone on in vitro maturation of buffalo oocytes did not decrease with the extension of time. This indicated that milrinone can be used as a nuclear maturation inhibitor during the maturation process in buffalo oocytes. In addition, milrinone can inhibit the effect of follicle stimulating hormone (FSH)-induced IVM of buffalo oocytes, but with time FSH partially eliminated the inhibition. Therefore, inhibition of milrinone on the nuclear maturation of buffalo oocytes was reversible, and buffalo oocytes can mature normally after the inhibition is lessened.

Neutrophils and Asthma.

Although eosinophilic inflammation is characteristic of asthma pathogenesis, neutrophilic inflammation is also marked, and eosinophils and neutrophils can coexist in some cases. Based on the proportion of sputum cell differentiation, asthma is classified into eosinophilic asthma, neutrophilic asthma, neutrophilic and eosinophilic asthma, and paucigranulocytic asthma. Classification by bronchoalveolar lavage is also performed. Eosinophilic asthma accounts for most severe asthma cases, but neutrophilic asthma or a mixture of the two types can also present a severe phenotype. Biomarkers for the diagnosis of neutrophilic asthma include sputum neutrophils, blood neutrophils, chitinase-3-like protein, and hydrogen sulfide in sputum and serum. Thymic stromal lymphoprotein (TSLP)/T-helper 17 pathways, bacterial colonization/microbiome, neutrophil extracellular traps, and activation of nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 pathways are involved in the pathophysiology of neutrophilic asthma and coexistence of obesity, gastroesophageal reflux disease, and habitual cigarette smoking have been associated with its pathogenesis. Thus, targeting neutrophilic asthma is important. Smoking cessation, neutrophil-targeting treatments, and biologics have been tested as treatments for severe asthma, but most clinical studies have not focused on neutrophilic asthma. Phosphodiesterase inhibitors, anti-TSLP antibodies, azithromycin, and anti-cholinergic agents are promising drugs for neutrophilic asthma. However, clinical research targeting neutrophilic inflammation is required to elucidate the optimal treatment.

Experimental assessment of the toxicity of a cardiac drug based on a phosphodiesterase-3 inhibitor and ethylmethylhydroxypyridine succinate

The study aimed to establish the parameters of chronic toxicity of the newly developed drug based on phosphodiesterase-3 inhibitor and ethylmethylhydroxypyridine succinate in experiments on laboratory animals. The analysis was performed on white sexually mature young male Wistar rats weighing 170–185 g. Four groups of white rats were formed. The first experimental group was administered Bendamine based on a phosphodiesterase-3 inhibitor and ethylmethylhydroxypyridine succinate at a therapeutic dose. Rats of the second experimental group were injected with the experimental drug in a 5-fold dose. Rats of the third experimental group were administered the drug in a 10-fold dose. The fourth group served as control. The study of chronic toxicity of Bendamine in white rats indicates that long-term 30-day administration in therapeutic doses or 5-fold dose does not cause clinical signs of poisoning, as evidenced by the physiological limits of fluctuations in the studied morphological and biochemical parameters of blood rats. Prolonged administration of Bendamine to rats in a 10-fold dose is accompanied by a slight suppression of the body's physiological state, as indicated by a decrease in total erythrocytes and hemoglobin by 10.1 % against an increase in white blood cells by 59.8% (P < 0.001). In addition, there was a decrease in the functional state of the liver, as evidenced by a probable reduction in total protein by 8.0% and urea – by 13.5 %, as well as an increase in ALT, AST, and alkaline phosphatase by 31.6 %, 7.4 %, and 53.9% respectively. Probable changes in the coefficients of liver and spleen mass have been established. When administered intramuscularly to rats with the drug Bendamine for 30 days, the macroscopic and microscopic structure of the studied internal organs is preserved in all groups of animals. The second experimental group revealed reversible moderate histostructural changes in the liver and kidneys. In rats treated with ten times the therapeutic dose of the drug, histologically found hemodynamic disorders and alterations in dystrophic nature, mainly of protein origin, with focal localization in the parenchyma of the liver, kidneys, and myocardium, which in most cases are reversible and result from the compensatory response. Macroorganism on the introduction of a high dose of the study drug.

Direct-to-consumer vs. traditional prescriptions of PDE5 inhibitors: A contemporary cost-analysis

ABSTRACT Introduction After a text screen and focused telehealth visit, patients can now have access to phosphodiesterase inhibitor (PDEi) prescriptions through online direct to consumer (DTC) healthcare companies. Prescription of PDEis has unknown costs relative to the traditional office visit, especially with the availability of online discounts. Objective This study seeks to quantify the cost of DTC PDEi treatment compared to a traditional physician visit and local pharmacy prescription. Methods Nine pharmacies were included, including two DTC companies, two compounding pharmacies with national reach, three online Canadian pharmacies licensed by the Canadian International Pharmacy Association, and sixteen American pharmacy chains, representing all locally available chains. National pharmacies which are subsidiaries were grouped together. Prices for chains were determined using their publicly available price on GoodRx® with coupons in Irvine, CA (92612), Cleveland, OH (44101), New York, NY (10001), Dallas, TX (75201), and Birmingham, AL (35201). Cost of physician visit was determined using 2020 CMS reimbursement for a level 3 new patient visit. Prices for DTC companies were acquired from direct communication with the company. Where offerings did not align with 90 dose regimens, price per pill was determined and price for 90 doses was calculated. Results For a 90-dose supply of sildenafil 20mg, a physician visit and local prescription cost a mean of $191.51 with a low of $125.45, compounding pharmacy prescription cost a mean of $218.28 with a low of $144.35, and Canadian pharmacy prescription cost a mean of $240.25 with a low of $169.34, compared to a mean of $220.00 and a low of $180.00 for online DTC. For sildenafil 100mg, a physician visit and local prescription had a mean cost of $524.11 with a low cost of $137.16, compounding pharmacy prescription a mean cost of $401.78 with a low cost of $289.35, and Canadian pharmacy prescription a mean cost of $262.63 with a low cost of $200.36, compared to a mean and low cost of $900.00 for DTC. For tadalafil 5mg, a physician visit and local prescription cost a mean of $205.79 with a low of $125.80, compounding pharmacy prescription cost a mean of $179.33 with a low of $169.35, and “Canadian” pharmacy prescription cost a mean of $207.59 with a low of $195.34, compared to an average and low of $720.00 for DTC. Finally, for tadalafil 20mg, a physician visit and local prescription cost a mean of $643.70 with a low of $161.00, compounding pharmacy prescription cost a mean of $359.33 with a low of $289.35, and Canadian pharmacy prescription cost a mean of $291.74 with a low of $229.00, compared to a mean of $3420.00 and a low of $2880.00 for DTC (Table 1). Conclusions Our findings suggest that local pharmacies, in conjunction with online coupons, provide a markedly less expensive option for fulfillment of PDEi prescriptions than online DTC services, compounding, or Canadian pharmacies, along with the established benefits of physician visits for screening. This is most pronounced if patients have flexibility in where they fill their prescriptions and can use the available local prices. Disclosure Work supported by industry: no. A consultant, employee (part time or full time) or shareholder is among the authors (FY: Coloplast (consultant), Cynosure (consultant) LJ: Hims (consultant)).

Ex vivo and In vivo Assays for Drug Discovery and Detection of Photoreceptor Survival Factors

Retinitis pigmentosa (RP) is one of the most common inherited diseases of the retina (retinopathies) that is estimated to affect 1 in 4,000 people in the United States and worldwide. This collection of genetic diseases leads to progressive photoreceptor cell degeneration and ultimately vision loss, with more than 60 genes implicated in molecular pathways towards photoreceptor cell death. Currently, there are no treatments available and, due to its vast diverse genetic background, a putative gene‐based therapy is challenging. Consequently, mutation‐independent strategies to protect photoreceptors against continued damage and progressive vision loss are appealing approaches. One of them is the use of survival, neurotrophic and neuroprotective factors that target common signaling pathways of cell death in the different types of RP. The aim of this study is to optimize methods to evaluate the efficacy of compounds to protect photoreceptor cells against death.We used PSVue® to optimize the cell death read‐out in cultured retinal explants and in retinas of a mouse model for a human inherited autosomal recessive RP, the rd10 mouse. Retinal degeneration in the rd10 mice is caused by a spontaneous mutation in the gene for the β‐subunit of phosphodiesterase. PSVue® is a robust probe for detecting phosphatidyl serine on the surface of cells undergoing death. We used retinal tissue explanted from wild type mouse eyes, and cultured it ex vivo. Induction of photoreceptor cell death was achieved with Zaprinast, an inhibitor of phosphodiesterase, to mimic the rd10 photoreceptor cell death. Low probe concentrations and short incubation times resulted in a reproducible detection of photoreceptor death in the retina by confocal super resolution microscopy. The PSVue® detection method yielded lower fluorescence background than the TUNEL detection method. Pigment epithelium‐derived factor (PEDF), a photoreceptor survival protein, was used as control to prevent photoreceptor cell death induced by Zaprinast. PEDF supplementation prior to Zaprinast treatment decreased cell death detection, as expected. We used the mouse model rd10 to evaluate photoreceptor death in vivo. PSVue® eye‐drops were administered to the left eye of rd10 mice of P17‐P25 postnatal ages, and fluorescence was detected by fundoscopy using MICRON III a day later. HBBS eye‐drops were administered to the right eye as a control reference. The fluorescence of the probe detected in the back of mouse eyes increased between ages P17 and P23 relative to that of HBBS‐treated eyes. The results revealed a peak of photoreceptor cell degeneration that occurred at P25 days of age in living rd10 mouse models.We have optimized methods to determine photoreceptor cell death in the ex vivo and in vivo experimental systems described here using a fluorescent probe. The methodologies are simple, fast, and complementary, and together they will prove to be suitable for screening factors and drugs against RP.

Dynamic Changes in ABCC4 Protein‐Protein Interactions during PKA Signaling: Role of the ABCC4 PDZ Motif

We recently demonstrated that a protein interaction between the PDZ motif of ABCC4 and a single PDZ‐domain‐containing protein, MPP1 had an important role in ABCC4 function in acute myeloid leukemia (Pitre et al. Nature Comm. 2017). To gain a better understanding into the role of the highly conserved ABCC4 PDZ‐motif, we performed live cell imaging using total internal reflectance fluorescence, TIRF, of ABCC4‐GFP expression constructs with or without the PDZ motif. The expression constructs were introduced into HEK293 cells where ABCC4 had been deleted by CRISPR‐Cas9 technology. ABCC4‐GFP was static at the membrane showing little movement, while deletion of the PDZ motif promoted rapid movement. Activation of PKA with forskolin and the phosphodiesterase inhibitor, iso‐butyl‐methyl xanthine increased intracellular cAMP and promoted membrane movement of ABCC4 harboring the PDZ motif. To get an unbiased understanding of the proteins interacting with ABCC4 both in cis‐ and trans‐, and under PKA activation conditions, we developed stable cell lines in the ABCC4‐null HEK293 cells. These stable cell lines harbored either an APEX2‐ABCC4 or an APEX2‐ABCC4 lacking a PDZ motif. APEX2‐derived ascorbate oxidase and biotin phenol, coupled with Avidin‐beads was used to interrogate the neighborhood of proteins within ~10 nm of ABCC4. We also developed stable cells harboring expression constructs for either Avi‐tagged ABCC4 or Avi‐ABCC4 without the PDZ motif to determine proteins that directly interacted with ABCC4 through the PDZ motif. Each Avi‐expression construct harbored a biotin ligase, thus enabling ABCC4 to be tagged by biotin and readily “pulled down” by avidin‐beads. The interacting proteins were identified by quantitative proteomics. Several PDZ specific interacting proteins were identified in these cell lines. By using analytical density gradient ultracentrifugation, we identified distinct PDZ specific ABCC4‐complexes that formed upon PKA activation. The actin‐binding cytoskeletal protein, Moesin (MSN) associated with ABCC4, requiring the PDZ‐motif. We used quantitative proteomics and APEX2 ABCC4 constructs to show that the neighborhood surrounding ABCC4 lacking the PDZ‐motif had fewer proteins, especially cytoskeletal proteins interacting with it. We propose that the ABCC4‐MSN interaction is important for tethering ABCC4 to the cytoskeletal actin network. Importantly, siRNA knockdown of MSN disrupted the association between ABCC4 and its interactors indicating the key role of MSN in the dynamic protein interactions occurring between ABCC4 and its interactors.

GPCR signaling measurement and drug profiling with an automated live‐cell microscopy system

Time‐lapse microscopy combined with genetically encoded fluorescent biosensors is a powerful tool for quantitatively measuring live cell signaling dynamics with high spatial and temporal resolution in single cells, providing rich signaling information beyond population‐based biochemical assays and plate reading measurements. One major limitation, however, is the low experimental throughput. To overcome this limitation, we created a highly customizable MATLAB‐based platform called FALCOscope (Flexible Automated Liquid‐handling Combined Microscope) that coordinates between an OpenTrons liquid handler and a fluorescent microscope to automate drug stimulations, fluorescence imaging and single cell analysis. We demonstrated the feasibility of our approach by quantifying GPCR‐stimulated Protein Kinase A (PKA) activity dynamics and by measuring cAMP dynamics stimulated by a panel of 14 GPCR agonists and antagonists. We then characterized the cAMP dynamics induced by GPR68, a proton sensing GPCR, in response to extracellular pH changes in the presence of its positive allosteric modulator ogerin. We found that GPR68 induced cAMP responses are more transient for acidic pH’s than neutral pH’s, suggesting receptor desensitization at acidic pHs. We further identified that ogerin, in addition to enhancing the cAMP response mostly strongly for pH 7.0, also sustains the cAMP response for acidic pH’s, demonstrating the novel findings captured by live‐cell dynamics. In addition, we show that 100 µM ogerin also has off‐target effects on cAMP/PKA signaling, likely as a phosphodiesterase inhibitor. This FALCOscope system enables single‐cell dynamic measurement in high throughput, providing a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells as well as drug profiling and screening.

Synthesis of Spiropyrazoles Under Organic and Nonorganic Catalysis

Abstract: Spiropyrazoles display many biological activities such as antitumor, vasodilation, analgesic, phosphodiesterase inhibitors, aldosterone antagonistic, anabolic, androgenic, antiinflammatory, progestational and salt-retaining activities, and they also exert neuroprotection in dopaminergic cell death. Many efforts have been made to obtain these derivatives with high yield and excellent regio-, diastereo- and enantioselectivities. Most of the spiroprazole synthesis methods were proceeded in good to excellent yield in the presence of organic catalysts, such as squaramide, NHC pre-catalyst, pyrrole derivatives, bis-oxazoline, etc. DMAP, DABCO, thiourea derivatives, DBU, acetic acid and quinoline catalysts. In addition, the inorganic and organometallic catalysts have been proven their efficiency in the synthesis of various types of spiro-pyrazoles in excellent yield. Thus, in this review, we have compiled all citations for the synthesis of spiropyrazoles in the presence of various types of catalysts such as organic, inorganic, and metalorganic catalysts in the range 2020 to 2012. This review article is a useful compilation for researchers interested in the synthesis of spiropyrazole derivatives and will assist them in selecting appropriate catalysts for the preparation of their spiropyrazoles.

Molecular and Functional Characterization of the Dual‐Specificity Phosphodiesterase Type 10A in Human White Adipocytes

The second messenger cyclic nucleotides cAMP and cGMP are key effectors of thermogenesis in adipocytes in response to a diverse array of cues. Their intracellular levels are normally regulated by the hydrolytic action of phosphodiesterases (PDEs), a conserved superfamily of enzymes composed of 12 families in humans (PDE1‐12) with distinct substrate specificities and tissue distribution profiles. Phosphodiesterase inhibitors are thus promising drugs for the treatment of metabolic disease by promoting cyclic nucleotide‐mediated adipose tissue thermogenesis, but so far mainly those that elevate either cAMP or cGMP signaling alone have been studied. We previously showed that inhibition of the dual‐specificity phosphodiesterase type 10A (PDE10A) with the small molecule drug MP‐10 sufficiently induces a thermogenic gene expression program in differentiated human white pre‐adipocytes (HWP‐ds) to a similar degree as combined cAMP and cGMP treatment. Here, we asked (1) which PDE10A isoforms are expressed in HWP‐ds, (2) the downstream signaling pathways PDE10A controls, and (3) how PDE10A expression is influenced by inflammatory stimuli in this cell type. Immunoblot analysis revealed that both the cytosolic (PDE10A1) and membrane‐bound (PDE10A2) isoforms of PDE10A are highly expressed in HWP‐ds, predominantly in the former subcellular compartment. This was confirmed by fractionation and immunofluorescence analyses. Inhibition of PDE10A with MP‐10 dose‐dependently increased PKA and PKG signaling as revealed by phosphorylation of cyclic response element binding protein (CREB) and vasodilator‐stimulated phosphoprotein (VASP) at S133 and S239, respectively. Consistently, the stimulation of lipolysis by MP‐10 was fully prevented by combined pre‐incubation of HWP‐ds with selective PKA and PKG inhibitors. Finally, mimicking the adipose tissue microenvironment in obesity by applying pro‐inflammatory cytokines onto HWP‐ds caused an early (4‐hour) and sustained (24‐hour) increase in PDE10A mRNA expression. These results provide new insight into the molecular character, function, and regulation of PDE10A in human adipocytes and further justify the use of selective inhibitors for the treatment of metabolic disease through stimulating adipose tissue thermogenesis in a more efficient and targeted manner.

BS-400-08 HETEROGENOUS CAMP SIGNALING IN THE INTACT HEART IS SEX DEPENDENT

cAMP is key for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. Yet, if cAMP signaling occurs heterogeneously throughout the intact heart, and how this translates into functional responses, has not been explored. To determine the spatiotemporal kinetics of cAMP signaling in the intact heart and the underlying mechanisms responsible. Male and female cardiac-specific CAMPER reporter mice that report cAMP binding by changes in FRET were used at 12 weeks. Hearts were excised and Langendorff-perfused for simultaneous cAMP and Vm imaging on a novel integrated whole heart optical imaging system. Following perfusion, tissue samples from base and apex were assessed for phosphodiesterase (PDE) activity. In male hearts, cAMP was uniformly activated in response to β-AR stimulation with bolus norepinephrine (NE, 1.5 μM). Conversely, in female hearts NE led to a greater change in cAMP activity in basal regions vs. the apex (n=7, p<0.05). Moreover, cAMP deactivation was slower in the base vs. apex, in female (n=6, p<0.01) but not male hearts (n=6). Apex-base differences were also evident following PDE inhibition with IBMX (100 μM), with a greater change in cAMP activity in the apex vs. base in both female (n=7, p<0.001) and male hearts (n=5, p<0.05). Likewise, PDE activity assays showed higher total PDE activity in apical regions (n=10, p<0.01), with more apical PDE activity in female vs. male hearts (n=5, p<0.05). In female hearts, faster apical cAMP deactivation following bolus NE was associated with a significant difference in action potential duration (APD80) between apex and base (n=3, p<0.05), but APD80 was not significantly different between regions in male hearts. Using novel whole heart imaging, we have shown female hearts display lower maximal cAMP activity and faster deactivation in the apex, in part, due to elevated PDE activity in this region. This heterogeneity was not observed in male hearts. These findings may have important implications for electrophysiological responses regulated by the cAMP pathway, particularly in heart failure, where PDE activity is altered.

Abstract 473: Cilostazol Decreases Venous Outflow Inflammation And Improves Avf Patency Inmurine Arteriovenous Fistula

Background: Arteriovenous fistula (AVF) have high failure rates, and no pharmacotherapeutics can improve AVF patency. The most common cause of AVF failure is inflammation-driven neointimal hyperplasia and thrombosis. Limited observational data suggests that cilostazol (CIL), an FDA-approved phosphodiesterase-3 inhibitor that inhibits platelet activation and smooth muscle cell proliferation, could help improve AVF maturation. Here, we investigated the effects of cilostazol on murine AVF patency, and further assessed the fibrinogenesis and anti-inflammatory effects using intravital fluorescence molecular imaging. Methods: One week prior to fistula creation, mice were randomized to CIL (40mg/kg/day) or PBS group (n=20 each). AVF was created using an end-to-side external jugular vein and carotid artery anastomosis. AVF blood flow was measured post-operatively (Transonic) and then every 7 days until AVF was occluded (&lt;0.1 ml/min). Macrophages and fibrin were imaged after 10mg/kg FMX-AF555 and/or 4.5nmol FTP11CyAm7 i.v. injection prior to imaging on day 7 or day 14, respectively. The target-to-background ratio (TBR) was calculated as mean signal intensity (MSI) of the fistula divided by the MSI of the contralateral vein. Results: Post-operative AVF blood flow was similar between CIL and PBS treated groups (p=0.3). Average AVF patency was significantly longer in CIL group compared to PBS group (42 vs 21 days, p&lt;0.01, figure 1A). Both day 7 and day 14 venous outflow fibrinogenesis signal assessed by IVM was significantly lower in CIL group compared to PBS group (ps&lt;0.05, Figure 1B). On day 14, CIL significantly lower venous outflow FMX signals compared to PBS group (p&lt;0.05). Conclusion: Oral cilostazol therapy prolongs AVF patency, and decreases venous outflow inflammation and fibrinogenesis in experimental AVF in vivo. The current data supports investigating cilostazol as a clinical pro-AVF patency pharmacotherapeutic strategy in dialysis patients.

Discovery of Novel Pain Therapies Without Side‐effects by Combining Targeted Mutagenesis and Phenotype‐based Drug Screening in Larval Zebrafish

IntroductionOpioid drugs provide effective pain relief but come with considerable side‐effects including addiction and respiratory depression which can be lethal with overdose. During the COVID19 pandemic, the opioid epidemic reached almost 100,000 deaths in the United States, highlighting the urgent need to identify pain killers with reduced liability so they can be safely prescribed. One approach to develop safe pain killers is to combine existing opioid analgesics with drugs reducing respiratory depression. Using our novel biotechnology platforms in larval zebrafish, we propose to identify new molecular targets and drugs with potent analgesic properties, reduced respiratory liability, and without addictive properties.MethodsCombining targeted mutagenesis and phenotype‐based drug screening approaches in larval zebrafish in vivo,we can quickly and effectively identify new molecular targets and screen new drug compounds to either target respiratory depression, analgesia (Zaig and Montandon, 2021, eLife), as well as addiction. We first screened customized drug libraries targeting voltage‐gated calcium channels and the adenylyl cyclase pathway, two molecular pathways involved in pain. We are also creating mutants using CRISPR‐cas9 in first generation zebrafish embryos to identify new molecular targets. Using our new models, we quantified respiratory depression by fentanyl in day post‐fertilization 7 larvae, as well as the nociceptive response to formalin.ResultsTo determine the role of calcium channels opioid‐mediated effects, we combined fentanyl and the calcium channel activators nefiracetam (1µM) or FLP‐64176 (1µM). Both nefiractem and FLP‐64176 prevented respiratory depression by fentanyl (1µM). To target the adenylyl cyclase pathway, we combined fentanyl and the phosphodiesterase inhibitors pentoxifylline (50µM), roflumilast (1µM), or dipyridamole (100µM). Pentaxifylline prevented respiratory depression by fentanyl but not roflumilast and dipyridamole. To perform targeted mutagenesis, we injected 4 single guide RNAs targeting the orpm1 gene, i.e. the gene coding for µ‐opioid receptors. Six days later, we measured respiratory depression by fentanyl in knockout larvae or controls. In oprm1‐/‐ knockout larvae, respiratory depression by fentanyl was substantially reduced compared to controls, therefore confirming the validity of our targeted mutagenesis approach. We are currently targeting the genes coding for calcium channels and phosphodiesterases to determine whether they can be molecular targets for respiratory depression and/or analgesia.DiscussionThe successful identification of new drug combinations to prevent respiratory depression while preserving analgesia suggests that our drug discovery platform in larval zebrafish will substantially accelerate the identification of new opioid drugs with reduced morbidity and mortality. Our platform is also being leveraged to discover non‐opioid pain killers with reduced side‐effects.

Phosphodiesterase 7B regulates prostanoid‐ but not ß‐adrenergic‐stimulated cAMP levels in primary human airway smooth muscle cells

Human airway smooth muscle (HASM) cells contain G‐protein coupled receptor (GPCR) signal transduction pathways that are organized in the plasma membrane and form cAMP signaling compartments. One such compartment contains ß2‐adrenergic receptors (ß2AR) coupled to adenylyl cyclase (AC)6 and is principally localized in lipid rafts. Another compartment expresses E prostanoid receptors 2 and 4 (EP2/4) coupled to AC2 and is restricted to non‐raft regions of the plasma membrane. Various phosphodiesterase (PDE) isozymes modulate cyclic AMP (cAMP) levels in response to these pathways. Non‐specific or PDE‐family specific PDE inhibitors have been used to treat asthma and chronic obstructive pulmonary disease with limited success, in part due to expression of these PDE isoforms throughout the body. Drugs that inhibit specific PDE isozymes might produce therapeutic effects in airways with fewer off‐target effects. RNA‐sequencing data in HASM cells indicates that PDE7B is the third most highly expressed PDE isoform and includes expression of both the PDE7B‐201 and PDE7B‐203 splice variants. We sought to characterize the role of PDE7B in the lipid raft and non‐raft cAMP signaling compartments. We used siRNA to knock down both PDE7B splice variants, confirmed protein reduction via Western Blot analysis, and then observed live cells’ real‐time cAMP levels in response to either isoproterenol (ßAR agonist) or PGE2 (EP agonist). The cADDis‐FMP biosensor, which localizes in lipid raft regions, did not report altered cAMP dynamics in response to ß2AR or EPR agonists following PDE7B knockdown. However, the cADDis‐FS15 biosensor, which reports cAMP levels in non‐raft plasma membrane regions, reported significant increases in cAMP levels stimulated by EPR agonists but no change in cAMP levels stimulated by ß2AR agonist. PDE7B may play a unique role in regulating non‐raft associated EP2/4 signaling without altering cAMP levels in other areas of the cell, thereby presenting a novel therapeutic target for pulmonary diseases.

Single doses of a highly selective inhibitor of phosphodiesterase 1 (lenrispodun) in healthy volunteers: a randomized pharmaco-fMRI clinical trial.

Lenrispodun is a potent and highly selective inhibitor of phosphodiesterase (PDE) type 1, which is thought to prolong intracellular second messenger signaling within cortical and subcortical dopaminergic brain regions. This is the first study of a PDE1 inhibitor in healthy volunteers using behavioral and neuroimaging approaches to examine its effects on neural targets and to provide a safety and tolerability assessment. The primary objectives were to determine whether lenrispodun induces changes in BOLD fMRI signals in the inferior frontal gyrus (IFG) during the stop signal task, and the dorsal anterior insula (dAI) during the extinction phase of a fear conditioning/extinction task. Using a double-blind, placebo-controlled, within-subjects design, 26 healthy individuals (22 completed all fMRI sessions) received in random order a single oral dose of placebo, lenrispodun 1.0 milligram (mg) or lenrispodun 10.0 mg and completed several tasks in the scanner including the stop signal (n = 24) and fear conditioning/extinction tasks (n = 22). Prespecified region-of-interest analyses for the IFG and dAI were computed using linear mixed models. Lenrispodun induced increases in IFG activity during the stop signal task at 1.0 mg (Cohen's d = 0.63) but not 10.0 mg (Cohen's d = 0.07) vs. placebo. Lenrispodun did not induce changes in dAI activity during fear extinction at either dose. Exploratory outcomes revealed changes in cardiac interoception. Lenrispodun administration was well-tolerated. These results provide evidence that 1.0 mg lenrispodun selectively improved neural inhibitory control without altering fear extinction processing. Future investigations should determine whether lenrispodun improves inhibitory control in target populations such as individuals with attention deficit hyperactivity disorder. Trial registration: ClinicalTrials.gov identifier: NCT03489772.