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Inhibition of NF-B and AP-1 activation by R- and S-flurbiprofen^{1 ,2}

Zentrum der Pharmakologie, Johann Wolfgang Goethe-Universität, Frankfurt, 60590 Frankfurt am Main, Germany; and
^* Institut für Experimentelle Pharmakologie and Toxikologie, Universität Erlangen, Fahrstr.19, 91054 Erlangen, Germany

³Correspondence: Center of Pharmacology, Johann Wolfgang Goethe-University of Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany. E-mail. geisslinger{at}em.uni-frankfurt.de

SPECIFIC AIMS

Flurbiprofenbelongs to the 2-aryl-propionic acid group of nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs consist of two optical isomers. R-flurbiprofen is considered to be the ‘inactive’ isomer because it does not inhibit cyclooxygenase (COX) activity. However, previous studies have revealed that it has antinociceptive and anti-tumor effects. Since R-flurbiprofen is not epimerized to the S-enantiomer in rats and humans, these effects are not mediated through the COX-inhibiting S-isomer. The aim of the present study was to evaluate the potential mechanisms that might explain the observed effects of R-flurbiprofen and to find out whether R-flurbiprofen was also able to reduce inflammation.

PRINCIPAL FINDINGS

1. In the zymosan-induced paw inflammation model in rats, R-flurbiprofen reduced inflammation as effectively as dexamethasone and was even more effective than the cyclooxygenase inhibiting S-isomer
Since the anti-inflammatory efficacy of NSAIDs is considered to be closely related to the COX inhibitory properties, a lack of anti-inflammatory efficacy for R-flurbiprofen was hypothesized. To address this hypothesis, we assessed its effects in the zymosan-induced hind paw inflammation model in rats. R- and S-flurbiprofen (1, 3, and 9 mg/kg) and dexamethasone (0.5 mg/kg, positive control) were injected intraperitoneally (i.p.) 15 min before the intraplantar injection of 0.625 mg zymosan. The paw volume as indicator of the inflammatory response was measured using a plethysmometer. The time course is shown in Fig. 1 . Surprisingly, R-flurbiprofen reduced the paw edema at least as effectively as S-flurbiprofen. At 3 and 9 mg/kg, effects of R-flurbiprofen were indistinguishable from those of 0.5 mg/kg dexamethasone. For statistical comparisons, the area under the ‘paw volume increase’ vs. ‘time’ curves were calculated. R-flurbiprofen significantly ( < 0.05) reduced the paw edema at 1, 3, and 9 mg/kg. Effects of S-flurbiprofen were statistically significant only between 4 and 8 h.

View larger version (26K): [in this window] [in a new window]   Figure 1. Time course of the anti-inflammatory effects of R- (A) and S-flurbiprofen (B) in zymosan-induced hind-paw inflammation in rats. Rats (6 per group) received an intraperitoneal injection of 1 (), 3 (), and 9 () mg/kg R- or S-flurbiprofen or 0.5 mg/kg dexamethasone (•) dissolved in 1 ml of phosphate buffer. Control animals received 1 ml of vehicle (). Fifteen minutes after drug administration, 0.625 mg zymosan suspended in 100 µl phosphate buffer was injected into the plantar surface of the right hind paw. The paw volume was determined by plethysmometry at the times indicated, and the absolute increase (PW) as compared to the volume measured before zymosan injection is shown. Each curve represents the mean ± standard deviation.

To evaluate the concentration range and to check the rate of epimerization, plasma concentrations of R- and S-flurbiprofen were determined after i.p. administration of 9 mg/kg R-flurbiprofen. Maximum plasma concentrations of R-and S-flurbiprofen were 520.9 ± 126.9 µmol/l and 3.89 ± 0.64 µmol/l, respectively. Based on AUC calculations, 1.61 ± 0.21% of R-flurbiprofen was found to be epimerized to S-flurbiprofen.

2. R- and S-flurbiprofen inhibited the zymosan-induced COX-2 mRNA and protein expression in the inflamed paw and spinal cord, which was associated with a significant reduction of prostaglandin release
To evaluate the underlying mechanisms, we first assessed the effects of R- and S-flurbiprofen on prostaglandin E₂ (PGE₂) release in the inflamed paw and spinal cord. As expected because of its COX inhibitory properties, S-flurbiprofen significantly reduced PGE₂ release at both sites. Unexpectedly, R-flurbiprofen was almost as effective as S-flurbiprofen. Since R-flurbiprofen does not inhibit COX enzymatically, we hypothesized that it might affect its expression. To address this hypothesis COX-2 mRNA and protein expression was assessed by means of quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively.

In the noninflamed paw, only minimal COX-2 mRNA was measurable. After zymosan injection, however, it was increased about 15-fold. This increase was significantly reduced by 9 mg/kg R- and S-flurbiprofen. In the spinal cord both COX isoforms were expressed in control rats i.e. constitutively. After zymosan injection into the paw, COX-2 mRNA in the spinal cord increased about sevenfold and this increase was dose dependently reduced by R- and S-flurbiprofen and by dexamethasone.

COX-2 protein expression was similarly affected. Lumbar spinal cord was excised 6 h after zymosan injection and protein extracts of lumbar spinal cord homogenates were subjected to Western blot analysis. An obvious reduction of zymosan-induced COX-2 protein expression was observed with 9 mg/kg R- and S-flurbiprofen and 0.5 mg/kg dexamethasone.

3. R- and S-flurbiprofen inhibited lipopolysaccharide (LPS) -induced activation of the transcription factor NF- B in RAW 264.7 macrophages by blocking its nuclear translocation. R-flurbiprofen also inhibited activator protein 1 (AP-1)
The transcription of the COX-2 gene is regulated among others by the transcription factor NF-B. Therefore, we were interested in whether the observed inhibition of COX-2 expression might be mediated through inhibition of NF-B activation. RAW 264.7 mouse macrophages were used to study NF- B activation in vitro. DNA binding activity was assessed by electrophoretic mobility shift assays. Cells were stimulated with 10 µg/ml LPS for 1 h in the absence or presence of R- and S-flurbiprofen (Fig. 2) . Nuclear extracts were incubated with end ³² P-labeled NF-B consensus oligonucleotide. NF-B DNA binding was minimal in control cells but strongly increased after LPS stimulation. This increase of DNA binding activity was dose dependently reduced by R-flurbiprofen and less so by S-flurbiprofen (Fig. 2) . Immunocytochemistry using an antibody directed against the p65 subunit of NF-B revealed that R-flurbiprofen inhibited the nuclear translocation of NF-B. S-flurbiprofen had similar effects but was less potent.

View larger version (42K): [in this window] [in a new window]   Figure 2. Electrophoretic mobility shift assay showing the effects of R- and S-flurbiprofen on DNA binding activity of NF-B proteins in LPS-stimulated murine macrophages. A representative EMSA of 3 repeat experiments is shown. RAW 264.7 cells were left untreated or incubated with R- and S-flurbiprofen at the doses indicated, followed by stimulation with 10 µg/ml LPS for 30 min.

In addition to NF-B, the transcription factor AP-1 is substantially involved in the regulation of the inflammatory response. Therefore, we assessed whether flurbiprofen also affected AP-1 activation. Specific AP-1 binding activity was markedly increased in LPS-stimulated cells as compared to unstimulated control cells. R-flurbiprofen (10–1000 µM) inhibited AP-1 DNA binding in a dose-dependent manner. At 1000 µM, it was completely suppressed. With S-flurbiprofen, a reduction was observed only at 1000 µM.

4. Inhibition of NF-B activation by R- and S-flurbiprofen was independent of I-B phosphorylation and degradation
In unstimulated cells, NF-B is inactive because it is retained in the cytoplasm by its inhibitor I-B. Upon stimulation, I-B is phosphorylated by I-B kinases and subsequently degraded. This releases NF-B, which is then translocated to the nucleus. To evaluate the stage at which R-flurbiprofen blocked NF-B activation we assessed whether I-B breakdown could take place in its presence. Cytosolic extracts were subjected to Western blot analysis using an antibody directed against I-B. The I-B signal was rapidly reduced after LPS stimulation (maximum at 30 min). Its degradation was affected by neither R- nor S-flurbiprofen.

CONCLUSIONS AND SIGNIFICANCE

It has been shown before that the R-enantiomer of flurbiprofen is a potent antinociceptive drug although it does not inhibit cyclooxygenase activity at therapeutically relevant concentrations. The present study demonstrates that R-flurbiprofen also reduces inflammation nearly as effectively as dexamethasone in zymosan-induced paw inflammation in rats. Different criteria suggest that the observed anti-inflammatory effects are mediated at least in part by inhibition of NF-B and AP-1 activation: R-flurbiprofen inhibited 1) NF-B and AP-1 DNA binding activity, 2) LPS-induced nuclear translocation of NF-B, and 3) NF-B-dependent gene transcription. The inhibitory effects on NF-B appear to be independent of I-B degradation. Concerning NF-B and AP-1 activity and paw inflammation, S-flurbiprofen was less effective. In vitro, NF-B and AP-1 inhibition occurred at concentrations of 100 to 1000 µM. Similar concentrations were found in plasma of rats treated with 9 mg/kg R-flurbiprofen. Thus, it may be assumed that the mechanisms observed in vitro are relevant for the in vivo effects of the drugs.

Recently, high NF-B and AP-1 DNA binding activity have been found in the synovium of patients with rheumatoid arthritis and osteoarthritis. In collagen-induced arthritis in mice, NF-B and AP-1 activation preceded both clinical arthritis and gene transcription of collagenases (metalloproteinases), which play an important role in the degradation of cartilage and bone. These findings suggest that activation of NF-B and AP-1 may be involved in the pathogenesis of rheumatoid arthritis and explain the beneficial effects of glucocorticoids, which inhibit activation of NF-B. Since R-flurbiprofen reduced inflammation nearly as effectively as dexamethasone in the present study and appears to act through a similar mechanism, it might likewise be able to reduce the clinical symptoms and joint destruction in rheumatoid arthritis.

In addition to the previously reported antinociceptive and the here demonstrated anti-inflammatory activity, R-flurbiprofen has been shown to inhibit tumor formation and growth in multiple intestinal neoplasia mice, with up to 90% inhibition of the total tumor number at a dose of 20 mg/(kg · day). The mechanism of this anticarcinogenic effect is not known. However, considering the anti-apoptotic role of NF-B in several human cancer cells, it may be assumed that NF-B inhibition might be the mechanism underlying this chemopreventive efficacy.

Since it has been first recognized that glucocorticoids and acetylsalicylic acid inhibit the transactivation of the transcription factor NK-B, much attention has been focused on NF-B as a potential target in the treatment of inflammatory diseases. Treatment with glucocorticoids and acetylsalicylic acid, however, is limited by their potential toxicities. That R-flurbiprofen inhibits NF-B and AP-1 at much lower concentrations than acetylsalicylic acid and exerts anti-inflammatory effects similar to those of dexamethasone suggests that R-flurbiprofen might be a useful ‘new’ drug in the treatment of RA.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0130fje To cite this article, use (November 14, 2000) FASEB J. 10.1096/fj.00-0130fje

² This work was supported by Deutsche Forschungs-gesellschaft (SFB 553 C6).

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This Article Full Text (PDF) All Versions of this Article: 15/3/595 15/1/2 00-0130fjev1 00-0130fjev2    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by TEGEDER, I. Articles by GEISSLINGER, G. Search for Related Content PubMed PubMed Citation Articles by TEGEDER, I. Articles by GEISSLINGER, G.