The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

Effect of Roflumilast, a Selective PDE4 Inhibitor, on Bone Phenotypes in ADO2 Mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We previously created mouse models of ADO2 (p.G213R) with one of the most common mutations (G215R) as found in humans and demonstrated that this mutation in mice phenocopies the human disease of ADO2. Previous studies have shown that roflumilast (RF), a selective phosphodiesterase 4 (PDE4) inhibitor that regulates the cAMP pathway, can increase osteoclast activity. We also observed that RF increased bone resorption in both wild-type and ADO2 heterozygous osteoclasts in vitro, suggesting it might rescue bone phenotypes in ADO2 mice. To test this hypothesis, we administered RF-treated diets (0, 20 and 100 mg/kg) to 8-week-old ADO2 mice for 6 months. We evaluated bone mineral density and bone micro-architecture using longitudinal in-vivo DXA and micro-CT at baseline, and 6-, 12-, 18-, and 24-week post-baseline time points. Additionally, we analyzed serum bone biomarkers (CTX, TRAP, and P1NP) at baseline, 12-, and 24-week post-baseline. Our findings revealed that RF treatment did not improve aBMD (whole body, femur, and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group treated with a normal diet. Furthermore, we did not observe any significant changes in serum levels of bone biomarkers due to RF treatment in these mice. Overall, our results indicate that RF does not rescue the osteopetrotic bone phenotypes in ADO2 heterozygous mice.

Equine metabolic investigation of the phosphodiesterase-4 inhibitor ibudilast as a potential performance enhancer.

RATIONALE: Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. METHODS: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. RESULTS: A total of 20 in vivo metabolites were identified under experimental conditions, including 12 Phase I and 8 Phase II conjugated metabolites. Phase I metabolites were predominantly formed through hydroxylation (mono-, di-, and tri-hydroxylation). Demethylated metabolites were also identified during this investigation. Additionally, the research detected Phase II metabolites conjugated with glucuronic and sulfonic acids. CONCLUSIONS: The data presented here can assist in detecting the PDE4 inhibitor ibudilast and uncover its illicit use in competitive sports.

PDE4 inhibitor eliminates breast cancer stem cells via noncanonical activation of mTOR.

Ineffective cancer treatment is implicated in metastasis, recurrence, resistance to chemotherapy and radiotherapy, and evasion of immune surveillance. All these failures occur due to the persistence of cancer stem cells (CSCs) even after rigorous therapy, thereby rendering them as essential targets for cancer management. Contrary to the quiescent nature of CSCs, a gene profiler array disclosed that phosphatidylinositol-3-kinase (PI3K), which is known to be crucial for cell proliferation, differentiation, and survival, was significantly upregulated in CSCs. Since PI3K is modulated by cyclic adenosine 3',5' monophosphate (cAMP), analyses of cAMP regulation revealed that breast CSCs expressed increased levels of phosphodiesterase 4 (PDE4) in contrast to normal stem cells. In accordance, the effects of rolipram, a PDE4 inhibitor, were evaluated on PI3K regulators and signaling. The efficacy of rolipram was compared with paclitaxel, an anticancer drug that is ineffective in obliterating breast CSCs. Analyses of downstream signaling components revealed a switch between cell survival and death, in response to rolipram, specifically of the CSCs. Rolipram-mediated downregulation of PDE4A levels in breast CSCs led to an increase in cAMP levels and protein kinase A (PKA) expression. Subsequently, PKA-mediated upregulation of phosphatase and tensin homolog antagonized the PI3K/AKT/mTOR pathway and led to cell cycle arrest. Interestingly, direct yet noncanonical activation of mTOR by PKA, circumventing the influence of PI3K and AKT, temporally shifted the fate of CSCs toward apoptosis. Rolipram in combination with paclitaxel indicated synergistic consequences, which effectively obliterated CSCs within a tumor, thereby suggesting combinatorial therapy as a sustainable and effective strategy to abrogate breast CSCs for better patient prognosis.

Drotaverine Inhibitor of PDE4: Reverses the Streptozotocin Induced Alzheimer's Disease in Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with decline in memory and cognitive impairments. Phosphodiesterase IV (PDE4) protein, an intracellular cAMP levels regulator, when inhibited act as potent neuroprotective agents by virtue of ceasing the activity of Pro-inflammatory mediators. The complexity of AD etiology has ever since compelled the researchers to discover multifunctional compounds to combat the AD and neurodegeneration. The aim of this study was to probe into role of drotaverine a PDE4 inhibitor in the management of AD. Albino mice were divided into seven groups (n = 10). Group 1 control group received carboxy methyl cellulose (CMC 1 mL/kg), group II diseased group treated with streptozotocin (STZ 3 mg/kg) by intracerebroventricular (ICV) route, group III administered standard drug Piracetam 200 mg/kg and groups IV-VII were given drotaverine (10, 20, 40, and 80 mg/kg i/p respectively). Groups II-VII were given STZ (3 mg/kg, ICV) on 1st and 3rd day of treatment to induce AD. All the groups were given their respective treatments for 23 days. Improvement in learning and memory was evaluated by using behavioral tests like open field test, elevated plus maze test, Morris water maze test and passive avoidance test. Furthermore, brain levels of biochemical markers of oxidative stress, neurotransmitters, ß-amyloid and tau protein were also measured. Drotaverine showed statistically significant dose dependent improvement in behavioral and biochemical markers of AD: the maximum response was achieved at a dose level of 80 mg/kg. The Study concluded that drotaverine ameliorates cognitive impairment and as well as exhibited modulated the brain levels of neurotransmitters.

Ultrasound assisted rapid synthesis of mefenamic acid based indole derivatives under ligand free Cu-catalysis: Their pharmacological evaluation.

An improved and rapid synthesis of mefenamic acid based indole derivatives has been achieved via the ligand free Cu-catalyzed coupling-cyclization method under ultrasound irradiation. This simple, straightforward and inexpensive one-pot method involved the reaction of a terminal alkyne derived from mefenamic acid with 2-iodosulfanilides in the presence of CuI and K2CO3 in PEG-400. The reaction proceeded via an initial CC bond formation (the coupling step) followed by CN bond formation (the intramolecular cyclization) to afford the mefenamic acid based indole derivatives in good to acceptable yields. Several of these compounds showed inhibition of PDE4 in vitro and the SAR (Structure Activity Relationship) within the series is discussed. The compound 3d has been identified as a promising and selective inhibitor of PDE4B (IC50 = 1.34 ± 0.46 µM) that showed TNF-α inhibition in vitro (IC50 = 5.81 ± 0.24 µM) and acceptable stability in the rat liver microsomes.

Roflumilast, a Phosphodiesterases-4 (PDE4) Inhibitor, Alleviates Sepsis­induced Acute Kidney Injury.

BACKGROUND Sepsis causes acute kidney injury (AKI) in critically ill patients. Roflumilast, a phosphodiesterases-4 (PDE4) inhibitor, has been shown to be therapeutically effective in sepsis-induced organ injury. However, the function of roflumilast in sepsis-induced AKI is not clearly understood. The present study aimed to explore the protective effect of roflumilast on sepsis-induced AKI in mice. MATERIAL AND METHODS A sepsis model was established by cecal ligation and puncture surgery. Roflumilast (1 mg/kg and 3 mg/kg) was used once daily for 7 consecutive days for treatment. Kidney tissues were pathologically examined by hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining. The levels of kidney injury markers including blood urea nitrogen (BUN), creatinine (Cre), kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL), and inflammatory cytokines including interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-1ß were detected by their corresponding test kits. The protein expression was measured using western blot and cell apoptosis of kidney tissue was determined by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. RESULTS Roflumilast was demonstrated to alleviate sepsis-induced AKI by reducing histopathological changes and decreasing the levels of kidney injury markers in a concentration-dependent way. The production of TNF-alpha, IL-6, and IL-1ß was significantly suppressed by roflumilast. Besides, roflumilast inhibited the activation of NLRP3 (nucleotide-binding domain (NOD)-like receptor protein 3) and NF-kappaB (nuclear factor kappa-light-chain-enhancer of activated B cells). Additionally, roflumilast inhibited cell apoptosis and changes in expression of apoptosis related proteins induced by sepsis. Finally, high concentration of roflumilast (3 mg/kg) did not have an adverse effect on liver, heart, lung, or spleen. CONCLUSIONS Our study indicated that roflumilast could ameliorate AKI induced by sepsis through restraining inflammatory response and apoptosis of the kidney, providing a molecular basis for a novel medical treatment of septic AKI.

Tetrahydroquinoline and tetrahydroisoquinoline derivatives as potential selective PDE4B inhibitors.

Tetrahydroquinoline and tetrahydroisoquinoline derivatives containing 2-phenyl-5-furan moiety were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. The bioassay results showed that title compounds showed good inhibitory activity against PDE4B and blockade of LPS (lipopolysaccharide) induced TNF-α release, which also exhibited considerable in vivo activity in animal models of asthma/COPD (chronic obstructive pulmonary disease) and sepsis induced by LPS. The bioactivity of compounds containing tetrahydroquinoline (series 4) was higher than that of tetrahydroisoquinoline derivatives (series 3). Compound 4 m with 4-methoxybenzene moiety exhibited the best potential selective activity against PDE4B. The primary structure-activity relationship study and docking results showed that the tetrahydroquinoline moiety of compound 4 m played a key role to form hydrogen bonds and π-π stacking interaction with PDE4B protein while the rest part of the molecule extended into the catalytic domain to block the access of cAMP and formed the foundation for inhibition of PDE4B. Based on LPS induced sepsis model for the measurement of TNF-α inhibition in Swiss Albino mice and neutrophilia inhibition for asthma and COPD in Sprague Dawley rats with the potential molecules, compound 4 m would be great promise as a hit inhibitor in the future study.

Stable C-N axial chirality in 1-aryluracil scaffold and differences in in vitro metabolic clearance between atropisomers of PDE4 inhibitor.

We report herein the stable C-N axial chirality in a 1-phenyl-6-aminouracil scaffold owing to the presence of various functional groups at the ortho-position of the N(1)-phenyl group. Racemic 1-phenyl-6-aminouracils were first separated by chiral HPLC or converting them to the corresponding diastereomers using a chiral resolving agent. We then determined the rotational barrier of each atropisomer by a thermal racemization method and found that these compounds have rotational barriers similar to other C-N axially chiral biaryls. In addition, there was a good correlation between the rotational barriers and van der Waals radii of an ortho-substituent of the N(1)-phenyl group. To explore the possibility of the chiral 1-phenyl-6-aminouracil scaffold as a drug lead, we synthesized both atropisomers as phosphodiesterase-4 inhibitors 10. The atropisomers showed significantly different metabolic stabilities while their PDE4 inhibitory activities were somewhat similar. This finding demonstrates the potential utility of stable C-N bond atropisomers in the development of chiral drugs.

Targeted Delivery of an Anti-inflammatory PDE4 Inhibitor to Immune Cells via an Antibody-drug Conjugate.

Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody-drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.

RECEPTOR AFFINITY AND PHOSPHODIESTERASES 4B AND 10A ACTIVITY OF OCTAHYDRO- AND 6,7-DIMETHOXY-3,4-DIHYDRO- ISOQUINOLIN-2(1H)-YL-ALKYL DERIVATIVES OF IMIDAZO- AND PYRIMIDINO[2,1-f]PURINES.

A series of octahydro- and 6,7-dimethoxy-3,4-dihydro- isoquinolin-2(1H)-yl-alkyl derivatives of imidazo- and pyrimidino[2,1-f]purines were synthesized and biologically evaluated in in vitro competition binding experiments for serotonin 5-HT(1A), 5-HT(6), 5-HT(7), and dopamine D2 receptors and inhibitory potencies for phosphodiesterases - PDE4B1 and PDE10A. The structure-activity relationships allowed to determine the structural features responsible for receptor and enzyme activity. Compound 5 (8-(4-(6,7-dimethoxy-3,4-dihydroiso- quinolin-2(1H)butyl)1,3-dimethyl-H-imidazo[2,1-f]purine-2,4(3H,8H)-dione) could be regarded as promising structure for further modification and detailed mechanistic study for obtained hybrid ligands.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A-D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4(+) T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 µmol/kg) and leukocyte infiltration (ED50 = 0.188 µmol/kg) with an efficacy comparable to a high dose of budesonide (1 µmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitor.

1. The metabolism of CHF 6001, a novel PDE4 inhibitor, was determined in vitro in mouse, rat, dog, monkey and human microsomes and hepatocytes and in vivo in plasma, urine, feces and bile of rats after intravenous and intratracheal administration. 2. The behavior of CHF 6001 in microsomes and hepatocytes changed across species. CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the metabolism of CHF 6001 in human liver microsomes. 3. In the rat, CHF 6001 was found extensively metabolized in urine, feces and bile, but not in plasma, where CHF 6001 was the main compound present. The metabolite profiles were different in the four biological matrices from both qualitative and quantitative point of view. 4. CHF 6001 was metabolized through hydrolysis with the formation of the alcohol CHF 5956, loss of a chlorine atom, loss of the N-oxide, hydroxylation, loss of the cyclopropylmethyl group in the alcohol moiety, conjugation with glucuronic acid, glutathione and cysteine-glycine. 5. The major metabolite present in the bile was isolated and characterized by nuclear magnetic resonance analysis. It derived from CHF 6001 through contraction of the pyridine-N-oxide ring to N-hydroxy pyrrole and conjugation with glucuronic acid.

Ischemic preconditioning inhibits mitochondrial permeability transition pore opening through the PTEN/PDE4 signaling pathway.

OBJECTIVES: Ischemic preconditioning (IPC) induces cardioprotection against ischemia-reperfusion (IR) injury by inhibiting the mitochondrial permeability transition pore (mPTP). Here, we tested the hypothesis that IPC-induced cardioprotection is mediated by the phosphatase PTEN and PDE4 (phosphodiesterase 4). METHODS: Isolated hearts from wild-type mice (WT, n = 110) and myocyte-specific PTEN-knockout mice (PKO, n = 94) were exposed to IPC or control conditions followed by IR. Subcellular fractionation was performed by sucrose gradient ultracentrifugation. RESULTS: IPC limited myocardial infarct size (IS) in WT mice. The PDE4 inhibitor rolipram abolished the protective effect of IPC. However, small IS was found in PKO hearts after IR, and IPC did not decrease IS but enlarged it in PKO hearts. IPC promoted PDE4D localization to caveolin-3-enriched fractions in WT mice by increasing Akt levels at the caveolae. In PKO hearts, basal PDE4D levels were elevated at the caveolae, and IPC decreased PDE4D levels. Consistent with the subcellular PDE4D protein levels and its activity, elevation in intracellular Ca(2+) levels in the ischemic heart and opening of mPTP after IR were inhibited by IPC in WT mice, but not by IPC in PKO mice. CONCLUSIONS: IPC inhibits mPTP opening by regulating the PTEN/PDE4 signaling pathway.

Functional rescue of CFTR in rectal organoids from patients carrying R334W variant by CFTR modulators and PDE4 inhibitor Roflumilast.

BACKGROUND: Many disease-causing variants in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene remain uncharacterized and untreated. Restoring the function of the impaired CFTR protein is the goal of personalized medicine, particularly in patients carrying rare CFTR variants. In this study, functional defects related to the rare R334W variant were evaluated after treatment with CFTR modulators or Roflumilast, a phosphodiesterase-4 inhibitor (PDE4i). METHODS: Rectal organoids from subjects with R334W/2184insA and R334W/2183AA > G genotypes were used to perform the Forskolin-induced swelling (FIS) assay. Organoids were left drug-untreated or treated with modulators VX-770 (I), VX-445 (E), and VX-661 (T) mixed, and their combination (ETI). Roflumilast (R) was used alone or as a combination of I + R. RESULTS: Our data show a significant increase in FIS rate following treatment with I alone. The combined use of modulators, such as ETI, did not increase further swelling than I alone, nor in protein maturation. Treatment with R shows an increase in FIS response similar to those of I, and the combination R + I significantly increases the rescue of CFTR activity. CONCLUSIONS: Equivalent I and ETI treatment efficacy was observed for both genotypes. Furthermore, significant organoid swelling was observed with combined I + R used that supports the recently published data describing a potentiating effect of only I in patients carrying the variant R334W and, at the same time, corroborating the role of strategies that include PDE4 inhibitors further to potentiate the effect of I for this variant.

A study of roflumilast treatment on functional and structural changes in hippocampus in depressed Adult male Wistar rats.

Depression is a common stress disability disorder that affects higher mental functions including emotion, cognition, and behavior. It may be mediated by inflammatory cytokines that interfere with neuroendocrine function, and synaptic plasticity. Therefore, reductions in inflammation might contribute to treatment response. The current study aims to evaluate the role of Protein Kinase (PKA)- cAMP response element-binding protein (CREB)- brain derived neurotropic factor (BDNF) signaling pathway in depression and the effects of roflumilast (PDE4 inhibitor) as potential antidepressant on the activity of the PKA-CREB-BDNF signaling pathway, histology, and pro-inflammatory cytokine production. Forty Adult male Wistar rats were divided into 4 groups: Control group, Positive Control group: similar to the controls but received Roflumilast (3 mg / kg / day) by oral gavage for the last 4 weeks of the experiment, Depressed group which were exposed to chronic stress for 6 weeks, and Roflumilast-treated group which were exposed to chronic stress for 6 weeks and treated by Roflumilast (3 mg / kg / day) by oral gavage for the last 4 weeks of the experiment. The depressed group showed significant increase in immobility time with significant decrease in swimming and struggling times, significant decrease in hippocampal PKA, CERB, BDNF, Dopamine, Cortisone, and Superoxide dismutase while hippocampal Phosphodiesterase-E4, Interleukin-6, and Malondialdhyde levels were significantly elevated. These findings were significantly reversed upon Roflumilast treatment. Therefore, it could be concluded that depression is a neurodegenerative inflammatory disease and oxidative stress plays a key role in depression. Roflumilast treatment attenuated the depression behavior in rats denoting its neuroprotective, and anti-inflammatory effects.

Rolipram attenuates bile duct ligation-induced liver injury in rats: a potential pathogenic role of PDE4.

Anti-inflammatory and antifibrotic effects of the broad spectrum phosphodiesterase (PDE) inhibitor pentoxifylline have suggested an important role for cyclic nucleotides in the pathogenesis of hepatic fibrosis; however, studies examining the role of specific PDEs are lacking. Endotoxemia and Toll-like receptor 4 (TLR4)-mediated inflammatory and profibrotic signaling play a major role in the development of hepatic fibrosis. Because cAMP-specific PDE4 critically regulates lipopolysaccharide (LPS)-TLR4-induced inflammatory cytokine expression, its pathogenic role in bile duct ligation-induced hepatic injury and fibrogenesis in Sprague-Dawley rats was examined. Initiation of cholestatic liver injury and fibrosis was accompanied by a significant induction of PDE4A, B, and D expression and activity. Treatment with the PDE4-specific inhibitor rolipram significantly decreased liver PDE4 activity, hepatic inflammatory and profibrotic cytokine expression, injury, and fibrosis. At the cellular level, in relevance to endotoxemia and inflammatory cytokine production, PDE4B was observed to play a major regulatory role in the LPS-inducible tumor necrosis factor (TNF) production by isolated Kupffer cells. Moreover, PDE4 expression was also involved in the in vitro activation and transdifferentiation of isolated hepatic stellate cells (HSCs). Particularly, PDE4A, B, and D upregulation preceded induction of the HSC activation marker α-smooth muscle actin (α-SMA). In vitro treatment of HSCs with rolipram effectively attenuated α-SMA, collagen expression, and accompanying morphologic changes. Overall, these data strongly suggest that upregulation of PDE4 expression during cholestatic liver injury plays a potential pathogenic role in the development of inflammation, injury, and fibrosis.

Monitoring phosphodiesterase-4 inhibitors using liquid chromatography/(tandem) mass spectrometry in sports drug testing.

RATIONALE: The recent discovery of resveratrol's capability to inhibit cAMP-specific phosphodiesterases (PDEs) and, as a consequence, to enhance particularly the activity of Sirt1 in animal models has reinforced the interest of preventive doping research organizations, especially in PDE4 inhibitors. Among these, the archetypical PDE4-inhibitor rolipram significantly increased the number of mitochondria in laboratory rodents, which further demonstrated a performance increase in a treadmill-test (time-to-exhaustion) of approximately 40%. Besides rolipram, a variety of new PDE4-inhibiting substances including cilomilast, roflumilast, and numerous additional new drug entities were described, with roflumilast being the first-in-class having received clinical approval for the treatment of chronic obstructive pulmonary disease (COPD). Due to the availability of these substances, and the fact that a misuse of such compounds in sport cannot be excluded, it deems relevant to probe for the prevalence of these compounds in sports drug testing programs. METHODS: Known urinary phase-I metabolites of rolipram, roflumilast, and cilomilast were generated by in vitro incubations employing human liver microsomal preparations. The metabolites obtained were studied by liquid chromatography with high-resolution/high-accuracy tandem mass spectrometry (LC/MS/MS) and the reference product ion mass spectra of established and most relevant metabolites were utilized to provide the information necessary for comprehensive doping controls. The analytical procedure was based on conventional routine doping control assays employing enzymatic hydrolysis followed by liquid-liquid extraction and subsequent LC/MS/MS measurement. RESULTS: Structures of diagnostic product ions and dissociation pathways of target analytes were elucidated, providing the information required for implementation into an existing test method for routine sports drug testing. The established method allowed for detection limits for the intact drugs of 1-5 ng/mL, and further assay characteristics (intraday precision 1.5-13.7%, interday precision 7.3-18.6%, recovery 20-100%, ion suppression/enhancement, and specificity) were determined. In addition, proof-of-concept analyses concerning roflumilast were conducted with a urine sample obtained from a COPD patient under roflumilast treatment.

Identification of the fused bicyclic 4-amino-2-phenylpyrimidine derivatives as novel and potent PDE4 inhibitors.

2-Phenyl-4-piperidinyl-6,7-dihydrothieno[3,4-d]pyrimidine derivative (2) was found to be a new PDE4 inhibitor with moderate PDE4B activity (IC50=150 nM). A number of derivatives with a variety of 4-amino substituents and fused bicyclic pyrimidines were synthesized. Among these, 5,5-dioxo-7,8-dihydro-6H-thiopyrano[3,2-d]pyrimidine derivative (18) showed potent PDE4B inhibitory activity (IC50=25 nM). Finally, N-propylacetamide derivative (31b) was determined as a potent inhibitor for both PDE4B (IC50=7.5 nM) and TNF-α production in mouse splenocytes (IC50=9.8 nM) and showed good in vivo anti-inflammatory activity in the LPS-induced lung inflammation model in mice (ID50=18 mg/kg). The binding mode of the new inhibitor (31e) in the catalytic site of PDE4B is presented based on an X-ray crystal structure of the ligand-enzyme complex.

Phosphodiesterase (PDE) 4 inhibition boosts Schwann cell myelination in a 3D regeneration model.

Phosphodiesterase 4 (PDE4) inhibitors have been extensively researched for their anti-inflammatory and neuroregenerative properties. Despite the known neuroplastic and myelin regenerative properties of nonselective PDE4 inhibitors on the central nervous system, the direct impact on peripheral remyelination and subsequent neuroregeneration has not yet been investigated. Therefore, to examine the possible therapeutic effect of PDE4 inhibition on peripheral glia, we assessed the differentiation of primary rat Schwann cells exposed in vitro to the PDE4 inhibitor roflumilast. To further investigate the differentiation promoting effects of roflumilast, we developed a 3D model of rat Schwann cell myelination that closely resembles the in vivo situation. Using these in vitro models, we demonstrated that pan-PDE4 inhibition using roflumilast significantly promoted differentiation of Schwann cells towards a myelinating phenotype, as indicated by the upregulation of myelin proteins, including MBP and MAG. Additionally, we created a unique regenerative model comprised of a 3D co-culture of rat Schwann cells and human iPSC-derived neurons. Schwann cells treated with roflumilast enhanced axonal outgrowth of iPSC-derived nociceptive neurons, which was accompanied by an accelerated myelination speed, thereby showing not only phenotypic but also functional changes of roflumilast-treated Schwann cells. Taken together, the PDE4 inhibitor roflumilast possesses a therapeutic benefit to stimulate Schwann cell differentiation and, subsequently myelination, as demonstrated in the biologically relevant in vitro platform used in this study. These results can aid in the development of novel PDE4 inhibition-based therapies in the advancement of peripheral regenerative medicine.