Roflumilast inhibits the release of chemokines and TNF-α from human lung macrophages stimulated with lipopolysaccharide

BACKGROUND AND PURPOSE

Lung macrophages are critically involved in respiratory diseases. This study assessed the effects of the PDE4 inhibitor roflumilast and its active metabolite, roflumilast N-oxide on the release of a range of chemokines (CCL2, 3, 4, CXCL1, 8, 10) and of TNF-α, from human lung macrophages, stimulated with bacterial lipopolysaccharide LPS.

EXPERIMENTAL APPROACH

Lung macrophages isolated from resected human lungs were incubated with roflumilast, roflumilast N-oxide, PGE₂, the COX inhibitor indomethacin, the COX-2 inhibitor NS-398 or vehicle and stimulated with LPS (24 h). Chemokines, TNF-α, PGE₂ and 6-keto PGF_1α were measured in culture supernatants by immunoassay. COX-2 mRNA expression was assessed with RT-qPCR. PDE activities were determined in macrophage homogenates.

KEY RESULTS

Expression of PDE4 in lung macrophages was increased after incubation with LPS. Roflumilast and roflumilast N-oxide concentration-dependently reduced the LPS-stimulated release of CCL2, CCL3, CCL4, CXCL10 and TNF-α from human lung macrophages, whereas that of CXCL1 or CXCL8 was not altered. This reduction by the PDE4 inhibitors was further accentuated by exogenous PGE₂ (10 nM) but abolished in the presence of indomethacin or NS-398. Conversely, addition of PGE₂ (10 nM), in the presence of indomethacin restored inhibition by roflumilast. LPS also increased PGE₂ and 6-keto PGF_1α release from lung macrophages which was associated with an up-regulation of COX-2 mRNA.

CONCLUSIONS AND IMPLICATIONS

Roflumilast and roflumilast N-oxide reduced LPS-induced release of CCL2, 3, 4, CXCL10 and TNF-α in human lung macrophages.     

Defects in mouse nephrogenesis induced by selective and non-selective cyclooxygenase-2 inhibitors

BACKGROUND AND PURPOSE

Deletion of the cyclooxygenase-2 (COX-2) gene causes impairment of kidney development, but the effect of selective inhibitors of COX-2 (coxibs) or the non-selective inhibitors of COX (the classical non-steroidal anti-inflammatory drugs; NSAIDs) on kidney development was less well described.

EXPERIMENTAL APPROACH

We assessed the effects of equipotent analgesic doses of celecoxib, rofecoxib, valdecoxib, etoricoxib and lumiracoxib and of the NSAIDs, diclofenac and naproxen, on postpartum kidney development in mice, from postnatal day 1 (P1) to P21.

KEY RESULTS

All the COX inhibitors, at the doses used, blocked COX-2 activity by more than 80% as assayed by PGE₂ synthesis in lipopolysaccharide-stimulated mouse blood samples. Rofecoxib, etoricoxib and lumiracoxib exerted the most marked impairment of postpartum kidney development, demonstrated by attenuation of kidney growth, reduction in size of glomeruli, increase in immature superficial glomeruli, thinning of subcapsular cortical mass and reduction in size of juxtamedullary glomeruli. These defects were less severe than those in kidneys from COX-2^−/− mice. Administration of diclofenac and naproxen revealed renal defects similar to those after coxib treatment, but both NSAIDs induced greater arrest of immature superficial glomeruli in the outer cortex and increased the number of undifferentiated proliferating cell nuclear antigen-positive cells. Treatment with celecoxib or valdecoxib caused only minimal changes in renal morphology.

CONCLUSIONS AND IMPLICATIONS

Classical NSAIDs cause similar or even stronger nephrodysgenesis than the coxibs. Also, the ranking of coxibs regarding adverse effects on renal development, using equi-analgesic doses, is rofecoxib = etoricoxib = lumiracoxib > valdecoxib > celecoxib.     

A crucial role for TNF-α in mediating neutrophil influx induced by endogenously generated or exogenous chemokines,KC/CXCL1 and LIX/CXCL5

Background and purpose:

Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice.

Experimental approach:

Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-α. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy.

Key results:

Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-α, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-α antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-α production, which were inhibited by reparixin or anti-TNF-α treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-α upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-α or anti-LIX/CXCL5.

Conclusion and implications:

Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-α, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.     

Lipoxin A4 attenuates zymosan-induced arthritis by modulating endothelin-1 and its effects

BACKGROUND AND PURPOSE

Lipoxin A₄ (LXA₄) is a lipid mediator involved in the resolution of inflammation. Increased levels of LXA₄ in synovial fluid and enhanced expression of the formyl peptide receptor 2/lipoxin A₄ receptor (FPR2/ALX) in the synovial tissues of rheumatoid arthritis patients have been reported. Endothelins (ETs) play a pivotal pro-inflammatory role in acute articular inflammatory responses. Here, we evaluated the anti-inflammatory role of LXA₄, during the acute phase of zymosan-induced arthritis, focusing on the modulation of ET-1 expression and its effects.

EXPERIMENTAL APPROACH

The anti-inflammatory effects of LXA₄, BML-111 (agonist of FPR2/ALX receptors) and acetylsalicylic acid (ASA) pre- and post-treatments were investigated in a murine model of zymosan-induced arthritis. Articular inflammation was assessed by examining knee joint oedema; neutrophil accumulation in synovial cavities; and levels of prepro-ET-1 mRNA, leukotriene (LT)B₄, tumour necrosis factor (TNF)-α and the chemokine KC/CXCL1, after stimulation. The direct effect of LXA₄ on ET-1-induced neutrophil activation and chemotaxis was evaluated by shape change and Boyden chamber assays respectively.

KEY RESULTS

LXA₄, BML-111 and ASA administered as pre- or post-treatment inhibited oedema and neutrophil influx induced by zymosan stimulation. Zymosan-induced preproET-1 mRNA, KC/CXCL1, LTB₄ and TNF-α levels were also decreased after LXA₄ pretreatment. In vitro, ET-1-induced neutrophil chemotaxis was inhibited by LXA₄ pretreatment. LXA₄ treatment also inhibited ET-1-induced oedema formation and neutrophil influx into mouse knee joints.

CONCLUSION AND IMPLICATION

LXA₄ exerted anti-inflammatory effects on articular inflammation through a mechanism that involved the inhibition of ET-1 expression and its effects.     

Prostaglandin E2 couples through EP4 prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines

Background and purpose:

Prostaglandin (PG) E₂ and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE₂ signals principally through prostanoid receptors of the EP₂ and/or EP₄ subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP₂ and EP₄ receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE₂-induced IL-8 production.

Experimental approach:

To identify the causative receptor, we knocked-down and over-expressed EP₂ and EP₄ receptor subtypes in colonic epithelial cells and studied the effect of several selective EP₂/EP₄ receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE₂ and Bmax values for the EP₂ and EP₄ receptor on colonic epithelial cells were determined by radioligand-binding assays with [³H]PGE₂.

Key results:

PGE₂ had the highest affinity for the EP₄ receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP₄ receptor agonist, ONO-AE1-329, and abolished by silencing the EP₄ receptor gene by using siRNA techniques, a selective EP₄ receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase.

Conclusions and implications:

These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE₂ acting via the EP₄ receptor subtype.     

Delayed apoptosis of human monocytes exposed to immune complexes is reversed by oxaprozin: role of the Akt/I��B kinase/nuclear factor ��B pathway

Background and purpose

Monocytes-macrophages play a key role in the initiation and persistence of inflammatory reactions. Consequently, these cells represent an attractive therapeutic target for switching off overwhelming inflammatory responses. Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most common drugs for the symptomatic treatment of rheumatic diseases. Their effects have been explained on the basis of cyclooxygenase (COX) inhibition. However, some of the actions of these drugs are not related to inhibition of prostaglandin synthesis.

Experimental approach

We examined the effect of oxaprozin on apoptosis of immune complex-activated monocytes in comparison with drugs of the same class, and the signalling pathway that leads activated monocytes exposed to oxaprozin to apoptosis. In particular, we studied the activity of caspase-3, the involvement of IκB kinase (IKK)-nuclear factor κB (NF-κB) system and the activity of X-linked mammalian inhibitor of apoptosis protein (XIAP), Akt and mitogen-activated protein kinase (MAPK) in activated monocytes in the presence of oxaprozin.

Key results

Immune complexes caused the inhibition of monocyte apoptosis. Oxaprozin reversed in a dose-dependent manner immune complex-induced survival of monocytes, without affecting the apoptosis of resting cells. Other NSAIDs are ineffective. The activity of oxaprozin was related to inhibition of Akt activation that, in turn, prevented p38 MAPK, IKK and NF-κB activation. Consistently, the inhibition of NF-κB activation reduced the production of the anti-apoptotic molecule XIAP, leading to uncontrolled activity of caspase 3.

Conclusions and implications

These results suggest that oxaprozin exerts its anti-inflammatory activity also through COX-independent pathways. It is likely that oxaprozin-mediated inhibition of the Akt/IKK/NF-κB pathway contributes to its anti-inflammatory properties.     

TNF-α induces CXCL1 chemokine expression and release in human vascular endothelial cells in vitro via two distinct signaling pathways

Aim： Chemokines usually direct the movement of circulating leukocytes to sites of inflammation or injury. CXCL1/GRO-a has been shown to be upregulated in atherosclerotic lesions and various cancers. The aim of this study was to investigate the mechanisms underlying the TNF-α-induced release of CXCL1 from human vascular endothelial cells in vitro. Methods： Human umbilical vein endothelial cells （HUVECs） were treated with different proinflam-matory mediators and growth factors. CXCL1 expression and secretion were determined using RT-PCR and ELISA, respectively. TNF-a-induced cell signaling was assayed with Western blotting. Cell viability/growth was determined using MTTassay. Monocyte migration was measured with transwell migration assay. Results： Among the 17 mediators and growth factors tested, TNF-α, LPS and thrombin induced marked increase in CXCL1 release from HUVEC cells. TNF-α （2, 5 ng/mL） induced CXCL1 release and mRNA expression in the cells in concentration- and time-dependent manners. TNF-α （5 ng/mL） caused activation of JNK, p38 MAPK, PI3K and Akt, whereas pretreatment with JNK inhibitor （SP600125）, p38 MAPK inhibitor （SB202190） or PI-3K inhibitor （LY294002） significantly suppressed TNF-a-induced CXCL1 release from the cells. But only SP600125 significantly reduced TNF-a-induced CXCL1 mRNA expression in the cells. Moreover, dexamethasone （up to 500 nmol/L） failed to affect TNF-a-induced CXCL1 release from the cells. In functional studies, recombinant CXCL1 enhanced HUVEC proliferation, and both recombinant CXCL1 and TNF-a-induced CXCL1 from HUVECs attracted human monocyte migration. Conclusion： TNF-a stimulates CXCL1 release from human ECs through JNK-mediated CXCL1 mRNA expression and p38 MAPK- and PI-3K-mediated CXCL1 secretory processes.     

The role of PKA and PKCε pathways in prostaglandin E2-mediated hypernociception

Background and purpose:

Protein kinase (PK) A and the ε isoform of PKC (PKCε) are involved in the development of hypernociception (increased sensitivity to noxious or innocuous stimuli) in several animal models of acute and persistent inflammatory pain. The present study evaluated the contribution of PKA and PKCε to the development of prostaglandin E₂ (PGE₂)-induced mechanical hypernociception.

Experimental approach:

Prostaglandin E₂-induced mechanical hypernociception was assessed by constant pressure rat paw test. The activation of PKA or PKCε was evaluated by radioactive enzymic assay in the dorsal root ganglia (DRG) of sensory neurons from the hind paws.

Key results:

Hypernociception induced by PGE₂ (100 ng) by intraplantar (i.pl.) injection, was reduced by i.pl. treatment with inhibitors of PKA [A-kinase-anchoring protein St-Ht31 inhibitor peptide (AKAPI)], PKCε (PKCεI) or adenylyl cyclase. PKA activity was essential in the early phase of the induction of hypernociception, whereas PKC activity was involved in the maintenance of the later phase of hypernociception. In the DRG (L4-L5), activity of PKA increased at 30 min after injection of PGE₂ but PKC activity increased only after 180 min. Moreover, i.pl. injection of the catalytic subunit of PKA induced hypernociception which was markedly reduced by pretreatment with an inhibitor of PKCε, while the hypernociception induced by paw injection of PKCε agonist was not affected by an inhibitor of PKA (AKAPI).

Conclusions and implications:

Taken together, these findings are consistent with the suggestion that PKA activates PKCε, which is a novel mechanism of interaction between these kinases during the development of PGE₂-induced mechanical hypernociception.     

Therapeutic inhibition of CXCR2 by Reparixin attenuates acute lung injury in mice

Background and purpose:

Acute lung injury (ALI) remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of ALI. The main chemokine receptor on neutrophils is CXCR2, which regulates neutrophil recruitment and vascular permeability, but no small molecule CXCR2 inhibitor has been demonstrated to be effective in ALI or animal models of ALI. To investigate the functional relevance of the CXCR2 inhibitor Reparixin in vivo, we determined its effects in two models of ALI, induced by either lipopolysaccharide (LPS) inhalation or acid instillation.

Experimental approach:

In two ALI models in mice, we measured vascular permeability by Evans blue and evaluated neutrophil recruitment into the lung vasculature, interstitium and airspace by flow cytometry.

Key results:

Pharmacological inhibition of CXCR2 by Reparixin reduced CXCL1-induced leukocyte arrest in the microcirculation of the cremaster muscle, but did not influence arrest in response to leukotriene B₄ (LTB₄) demonstrating specificity. Reparixin (15 μg g⁻¹) reduced neutrophil recruitment in the lung by approximately 50% in a model of LPS-induced ALI. A higher dose did not provide additional reduction of neutrophil recruitment. This dose also reduced accumulation of neutrophils in the interstitial compartment and vascular permeability in LPS-induced ALI. Furthermore, both prophylactic and therapeutic application of Reparixin improved gas exchange, and reduced neutrophil recruitment and vascular permeability in a clinically relevant model of acid-induced ALI.

Conclusions and implications:

Reparixin, a non-competitive allosteric CXCR2 inhibitor attenuates ALI by reducing neutrophil recruitment and vascular permeability.     

The interaction of ibuprofen and diclofenac with aspirin in healthy volunteers

Background and purpose:

Aspirin reduces the risk of myocardial infarction and stroke by inhibiting thromboxane production in platelets. This inhibition can be competitively antagonized by some non-steroidal anti-inflammatory drugs (NSAIDs).

Experimental approach:

By measuring thromboxane B₂ production in healthy volunteers, we investigated whether ibuprofen (800 mg three times daily for 7 days) or diclofenac (50 mg three times daily for 7 days) taken concurrently with aspirin 80 mg (once daily for 7 days) influenced the inhibitory effect of aspirin. The effects were compared with aspirin 30 mg (once daily for 7 days), which is the lowest dose of aspirin with a proven thromboprophylactic effect.

Key results:

The median percentage inhibition of thromboxane B₂ levels by 30 mg or 80 mg aspirin was 90.3% (range 83.1–96.0%) and 98.0% (range 96.8–99.2%) respectively. The inhibition by concurrent administration of slow release diclofenac and 80 mg aspirin was 98.1% (range 97.2–98.9%), indicating no interference between aspirin and diclofenac. The inhibition decreased significantly by concurrent administration of immediate release ibuprofen and 80 mg aspirin (86.6%; range 77.6–95.1%) to a level less than 30 mg aspirin.

Conclusions and implications:

As alternatives are easily available, NSAIDs such as diclofenac should be preferred to ibuprofen for combined use with aspirin.     

Reply to ‘Indication bias or protopathic bias?’

Aim

The aim of the study was to characterize the extent of indication bias resulting from the excessive use of NSAIDs on the days preceding a spontaneous abortion to relieve pain.

Methods

We used data from a retrospective cohort study assessing the risk for spontaneous abortions following exposure to NSAIDs. Three definitions of exposure for cases of spontaneous abortions were compared, from the first day of pregnancy until the day of spontaneous abortion and until 3 and 2 days before a spontaneous abortion. Statistical analysis was performed using multivariate time programmed Cox regression.

Results

A sharp increase was observed in the dispensation of indomethacin, diclofenac and naproxen, and a milder increase was found in the use of ibuprofen during the week before a spontaneous abortion. Non- selective COX inhibitors in general and specifically diclofenac and indomethacin were found to be associated with spontaneous abortions when the exposure period was defined until the day of spontaneous abortion (hazard ratio (HR) 1.15, 95% confidence interval (CI) 1.04, 1.28; HR 1.31, 95% CI 1.08, 1.59 and HR 3.33, 95% CI 2.09, 5.29, respectively). The effect disappears by excluding exposures occurring on the day before the spontaneous abortion for non-selective COX inhibitors and on the last week before the spontaneous abortion for indomethacin. In general, decreasing HRs were found with the exclusion of exposures occurring on the days immediately before the spontaneous abortion.

Conclusions

The increased use of NSAIDs during the last few days that preceded a spontaneous abortion to relieve pain associated with the miscarriage could bias studies assessing the association between exposure to NSAIDs and spontaneous abortions.     

Prostaglandin E2 induces spontaneous rhythmic activity in mouse urinary bladder independently of efferent nerves

BACKGROUND AND PURPOSE

The acute effects of PGE₂ on bladder smooth muscle and nerves were examined to determine the origin of PGE₂-induced spontaneous rhythmic contractions.

EXPERIMENTAL APPROACH

Contraction studies, confocal Ca²⁺ imaging and electrophysiological recordings in strips of mouse urinary bladder were used to differentiate the effects of PGE₂ on bladder smooth muscle and efferent nerves.

KEY RESULTS

PGE₂ (50 µM) increased the tone and caused phasic contractions of detrusor smooth muscle strips. Confocal Ca²⁺ imaging showed that PGE₂ increased the frequency of whole-cell Ca²⁺ transients (WCTs) (72 ± 5%) and intracellular recordings showed it increased the frequency of spontaneous depolarizations, from 0.31·s⁻¹ to 0.90·s⁻¹. Non-selective inhibition of EP receptors using SC-51322 and AH-6809 (10 µM), or the L-type Ca²⁺ channel blocker nifedipine (1 µM), prevented these phasic contractions and WCTs, and reduced the tone (by 45 ± 7% and 59 ± 6%, respectively). Blocking P2X1 receptors with NF449 (10 µM) caused a small but significant reduction in the frequency of PGE₂-induced phasic contractions (24 ± 9%) and WCTs (28 ± 17%) but had no significant effect on spontaneous depolarizations or tone. Inhibiting muscarinic receptors with cyclopentolate (1 µM) had no significant effect on these measures. Spontaneous WCTs became synchronous in PGE₂, implying enhanced functional coupling between neighbouring cells. However, the electrical input resistance was unchanged.

CONCLUSIONS AND IMPLICATIONS

It was concluded that depolarization alone is sufficient to explain a functional increase in intercellular coupling and the ability of PGE₂ to increase detrusor spontaneous rhythmic activity does not require parasympathetic nerves.     

Pharmacokinetic-pharmacodynamic modeling of diclofenac in normal and Freund's complete adjuvant-induced arthritic rats

Aim:

To characterize pharmacokinetic-pharmacodynamic modeling of diclofenac in Freund''s complete adjuvant (FCA)-induced arthritic rats using prostaglandin E₂ (PGE₂) as a biomarker.

Methods:

The pharmacokinetics of diclofenac was investigated using 20-day-old arthritic rats. PGE₂ level in the rats was measured using an enzyme immunoassay. A pharmacokinetic-pharmacodynamic (PK-PD) model was developed to illustrate the relationship between the plasma concentration of diclofenac and the inhibition of PGE₂ production. The inhibition of diclofenac on lipopolysaccharide (LPS)-induced PGE₂ production in blood cells was investigated in vitro.

Results:

Similar pharmacokinetic behavior of diclofenac was found both in normal and FCA-induced arthritic rats. Diclofenac significantly decreased the plasma levels of PGE₂ in both normal and arthritic rats. The inhibitory effect on PGE₂ levels in the plasma was in proportion to the plasma concentration of diclofenac. No delay in the onset of inhibition was observed, suggesting that the effect compartment was located in the central compartment. An inhibitory effect sigmoid I_max model was selected to characterize the relationship between the plasma concentration of diclofenac and the inhibition of PGE₂ production in vivo. The I_max model was also used to illustrate the inhibition of diclofenac on LPS-induced PGE₂ production in blood cells in vitro.

Conclusion:

Arthritis induced by FCA does not alter the pharmacokinetic behaviors of diclofenac in rats, but the pharmacodynamics of diclofenac is slightly affected. A PK-PD model characterizing an inhibitory effect sigmoid I_max can be used to fit the relationship between the plasma PGE₂ and diclofenac levels in both normal rats and FCA-induced arthritic rats.     

The antipyretic effect of dipyrone is unrelated to inhibition of PGE(2) synthesis in the hypothalamus

BACKGROUND AND PURPOSE

Bacterial lipopolysaccharide (LPS) induces fever through two parallel pathways; one, prostaglandin (PG)-dependent and the other, PG-independent and involving endothelin-1 (ET-1). For a better understanding of the mechanisms by which dipyrone exerts antipyresis, we have investigated its effects on fever and changes in PGE₂ content in plasma, CSF and hypothalamus induced by either LPS or ET-1.

EXPERIMENTAL APPROACH

Rats were given (i.p.) dipyrone (120 mg·kg⁻¹) or indomethacin (2 mg·kg⁻¹) 30 min before injection of LPS (5 µg·kg⁻¹, i.v.) or ET-1 (1 pmol, i.c.v.). Rectal temperature was measured by tele-thermometry. PGE₂ levels were determined in the plasma, CSF and hypothalamus by elisa.

KEY RESULTS

LPS or ET-1 induced fever and increased CSF and hypothalamic PGE₂ levels. Two hours after LPS, indomethacin reduced CSF and hypothalamic PGE₂ but did not inhibit fever, while at 3 h it reduced all three parameters. Three hours after ET-1, indomethacin inhibited the increase in CSF and hypothalamic PGE₂ levels but did not affect fever. Dipyrone abolished both the fever and the increased CSF PGE₂ levels induced by LPS or ET-1 but did not affect the increased hypothalamic PGE₂ levels. Dipyrone also reduced the increase in the venous plasma PGE₂ concentration induced by LPS.

CONCLUSIONS AND IMPLICATIONS

These findings confirm that PGE₂ does not play a relevant role in ET-1-induced fever. They also demonstrate for the first time that the antipyretic effect of dipyrone was not mechanistically linked to the inhibition of hypothalamic PGE₂ synthesis.     

Protective effects of prostaglandin E1 on human umbilical vein endothelial cell injury induced by hydrogen peroxide

Aim:

To investigate the protective effects of prostaglandin E₁ (PGE₁) against H₂O₂-induced oxidative damage on human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were pretreated with PGE₁ (0.25, 0.50, and 1.00 μmol/L) for 24 h and exposed to H₂O₂ (200 μmol/L) for 12 h, and cell viability was measured by the MTT assay. LDH, NO, SOD, GSH-Px, MDA, ROS, and apoptotic percentage were determined. eNOS expression was measured by Western blotting and real-time PCR.

Results:

PGE₁ (0.25−1.00 μmol/L) was able to markedly restore the viability of HUVECs under oxidative stress, and scavenged intracellular reactive oxygen species induced by H₂O₂. PGE₁ also suppressed the production of lipid peroxides, such as MDA, restored the activities of endogenous antioxidants including SOD and GSH-Px, and inhibited cell apoptosis. In addition, PGE₁ significantly increased NO content, eNOS protein, and mRNA expression.

Conclusion:

PGE₁ effectively protected endothelial cells against oxidative stress induced by H₂O₂, an activity that might depend on the up-regulation of NO expression.     

Regioselective oxidation of phospho-NSAIDs by human cytochrome P450 and flavin monooxygenase isoforms: implications for their pharmacokinetic properties and safety

BACKGROUND AND PURPOSE

Phospho-ibuprofen (MDC-917) and phospho-sulindac (OXT-328) are highly effective in cancer and arthritis treatment in preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin monooxygenases (FMOs).

EXPERIMENTAL APPROACH

The CYP/FMO-catalysed metabolism of phospho-ibuprofen and phospho-sulindac was studied by using in silico prediction modelling and a direct experimental approach.

KEY RESULTS

The CYP isoforms catalyse the oxidation of non-steroidal anti-inflammatory drugs (NSAIDs) and phospho-NSAIDs, with distinct activity and regioselectivity. CYP1A2, 2C19, 2D6 and 3A4 oxidize phospho-ibuprofen, but not ibuprofen; whereas CYP2C9 oxidizes ibuprofen, but not phospho-ibuprofen. All CYPs tested oxidize phospho-sulindac, but not sulindac. Among the five CYPs evaluated, CYP3A4 and 2D6 are the most active in the oxidation of phospho-ibuprofen and phospho-sulindac respectively. FMOs oxidized phospho-sulindac and sulindac, but not phospho-ibuprofen or ibuprofen. FMOs were more active towards phospho-sulindac than sulindac, indicating that phospho-sulindac is a preferred substrate of FMOs. The susceptibility of phospho-NSAIDs to CYP/FMO-mediated metabolism was also reflected in their rapid oxidation by human and mouse liver microsomes, which contain a full complement of CYPs and FMOs. Compared with conventional NSAIDs, the higher activity of CYPs towards phospho-ibuprofen and phospho-sulindac may be due to their greater lipophilicity, a key parameter for CYP binding.

CONCLUSIONS AND IMPLICATIONS

CYPs and FMOs play an important role in the metabolism of phospho-NSAIDs, resulting in differential pharmacokinetic profiles between phospho-NSAIDs and NSAIDs in vivo. The consequently more rapid detoxification of phospho-NSAIDs is likely to contribute to their greater safety.     

Selectivity profiling of the novel EP2 receptor antagonist,PF-04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

BACKGROUND AND PURPOSE

Understanding the role of the EP₂ receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP₂ receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP₂ receptor over other EP receptors using a range of isolated tissue systems.

EXPERIMENTAL APPROACH

PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP₁); ONO-AE1-259 and PGE₂- induced relaxation of mouse and guinea pig trachea (EP₂); PGE₂-induced depolarization of guinea pig isolated vagus (EP₃); PGE₂-induced relaxation of human and rat trachea (EP₄). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays.

KEY RESULTS

In bioassay systems, where assessment of potency/selectivity is made against the ‘native’ receptor, PF-04418948 only acted as an antagonist of EP₂ receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE₂ respectively. However, the affinity of PF-04418948 was not equal in the two preparations.

CONCLUSIONS AND IMPLICATIONS

Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP₂-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP₂ receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE₂ and the role of EP₂ receptors in health and disease.     

Spinal glial TLR4-mediated nociception and production of prostaglandin E2 and TNF

Background and purpose:

Toll-like receptor 4 (TLR4) expressed on spinal microglia and astrocytes has been suggested to play an important role in the regulation of pain signalling. The purpose of the present work was to examine the links between TLR4, glial activation and spinal release of prostaglandin E₂ (PGE₂) and tumour necrosis factor (TNF), and the role these factors play in TLR4-induced tactile allodynia.

Experimental approach:

Toll-like receptor 4 was activated by intrathecal (i.t.) injection of lipopolysaccharide (LPS) and KDO₂-Lipid A (KDO₂) to rats. Tactile allodynia was assessed using von Frey filaments and cerebrospinal fluid collected through spinal dialysis and lumbar puncture. PGE₂ and TNF levels were measured by mass spectometry and elisa. Minocycline and pentoxifylline (glia inhibitors), etanercept (TNF-blocker) and ketorolac (COX-inhibitor) were given i.t. prior to injection of the TLR4-agonists, in order to determine if these agents alter TLR4-mediated nociception and the spinal release of PGE₂ and TNF.

Key results:

Spinal administration of LPS and KDO₂ produced a dose-dependent tactile allodynia, which was attenuated by pentoxifylline, minocycline and etanercept but not ketorolac. Both TLR4 agonists induced the spinal release of PGE₂ and TNF. Intrathecal pentoxifylline blunted PGE₂ and TNF release, while i.t. minocycline only prevented the spinal release of TNF. The release of PGE₂ induced by LPS and KDO₂ was attenuated by i.t. administration of ketorolac.

Conclusions and implications:

Activation of TLR4 induces tactile allodynia, which is probably mediated by TNF released by activated spinal glia.     

An exploratory microdialysis study investigating the effect of repeated application of a diclofenac epolamine medicated plaster on prostaglandin concentrations in skeletal muscle after standardized physical exercise

Aim

Muscle injuries and extensive exercise are associated with cyclo-oxygenase dependent formation of inflammatory prostaglandins. Since the effect of topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) on local cyclo-oxygenase is unknown, the present exploratory, open label, non-randomized study set out to measure exercise induced release of prostaglandins before and after epicutaneous administration of diclofenac.

Methods

Microdialysis was used to determine the local interstitial concentration of PGE₂ and 8-iso-PGF_2α as well as diclofenac concentrations in the vastus lateralis under rest, dynamic exercise and during recovery in 12 healthy subjects at baseline and after a treatment phase applying a total of seven plasters medicated with 180 mg of diclofenac epolamine over 4 days.

Results

At baseline PGE₂ concentrations were 1169 ± 780 pg ml⁻¹ at rest and 1287 ± 459 pg ml⁻¹ during dynamic exercise and increased to 2005 ± 1126 pg ml⁻¹ during recovery. After treatment average PGE₂ concentrations were 997 ± 588 pg ml⁻¹ at rest and 1339 ± 892 pg ml⁻¹ during exercise. In contrast with the baseline phase no increase in PGE₂ concentrations was recorded during the recovery period after treatment (PGE₂ 1134 ± 874 pg ml⁻¹). 8-iso-PGF_2α was neither affected by exercise nor by treatment with diclofenac. Local and systemic concentrations of diclofenac were highly variable but comparable with previous clinical pharmacokinetic studies.

Conclusions

We can hypothesize an effect of topical diclofenac epolamine plaster on limiting the increase of local concentrations of the pro-inflammatory prostaglandin PGE₂ induced in the muscle of healthy human subjects following standardized physical exercise. No effect of diclofenac treatment on 8-iso-PGF_2α concentrations was observed, mainly since isoprostane is produced by a free radical-catalyzed lipid peroxidation mechanism independent of cyclo-oxygenases.