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. 2004 Feb 18;24(7):1637-45.
doi: 10.1523/JNEUROSCI.3118-03.2004.

Specific Inhibition of IkappaB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in rats

Irmgard Tegeder  1 , Ellen Niederberger, Ronald Schmidt, Susanne Kunz, Hans Gühring, Olaf Ritzeler, Martin Michaelis, Gerd Geisslinger
Affiliations

Specific Inhibition of IkappaB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in rats

Irmgard Tegeder et al. J Neurosci. .

Abstract

Phosphorylation of IkappaB through IkappaB kinase (IKK) is the first step in nuclear factor kappaB (NF-kappaB) activation and upregulation of NF-kappaB-responsive genes. Hence, inhibition of IKK activity may be expected to prevent injury-, infection-, or stress-induced upregulation of various proinflammatory genes and may thereby reduce hyperalgesia and inflammation. In the present study, we tested this hypothesis using a specific and potent IKK inhibitor (S1627). In an IKK assay, S1627 inhibited IKK activity with an IC50 value of 10.0 +/- 1.2 nm. In cell culture experiments, S1627 inhibited interleukin (IL)-1beta-stimulated nuclear translocation and DNA-binding of NF-kappaB. Plasma concentration time courses after intraperitoneal injection revealed a short half-life of 2.8 hr in rats. Repeated intraperitoneal injections were, therefore, chosen as the dosing regimen. S1627 reversed thermal and mechanical hyperalgesia at 3x 30 mg/kg in the zymosan-induced paw inflammation model and reduced the inflammatory paw edema at 3x 40 mg/kg. S1627 also significantly reduced tactile and cold allodynia in the chronic constriction injury model of neuropathic pain at 30 mg/kg once daily. The drug had no effect on acute inflammatory nociception in the formalin test and did not affect responses to heat and tactile stimuli in naive animals. As hypothesized, S1627 prevented the zymosan-induced nuclear translocation of NF-kappaB in the spinal cord and the upregulation of NF-kappaB-responsive genes including cyclooxygenase-2, tumor necrosis factor-alpha, and IL-1beta. Our data indicate that IKK may prove an interesting novel drug target in the treatment of pathological pain and inflammation.

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Figures

Figure 1.
Figure 1.
Concentration-dependent inhibition of IKK activity with S1627 assessed in an in vitro kinase assay. The IC50 value (10.0 ± 1.2 nm; mean ± SE) was calculated with a standard sigmoidal Emax model.
Figure 2.
Figure 2.
A, Western blot analysis showing the concentration-dependent inhibition of IL-1β-stimulated nuclear translocation of NF-κB (p65 subunit) in human umbilical vein endothelial cells. Cells were preincubated with S1627 or vehicle for 30 min, stimulated for 60 min with 1 nm IL-1β in the presence or absence of S1627, and then harvested for preparation of nuclear and cytosolic extracts. Nuclear p65 was detected with a specific antibody. Erk-2 was used as loading control. B, Concentration-dependent inhibition of IL-1β-stimulated nuclear translocation and DNA binding of NF-κB in human umbilical vein endothelial cells (mean ± SE) assessed using an ELISA that uses an oligonucleotide-coated plate and detection of protein binding with a p65-specific antibody. Results of four repeated experiments are shown.
Figure 3.
Figure 3.
Plasma concentration time course of S1627 (A) and its primary metabolite S658 (B) after intraperitoneal injection of one, two, or three doses of S1627 at the indicated times (4 rats in each group). The inset in A shows CSF concentrations after a single dose of 30 mg/kg (6 rats per time). Plasma and CSF concentrations were determined with liquid chromatography coupled with tandem mass spectrometry. The data represent the mean ± SE.
Figure 4.
Figure 4.
Effects of S1627 in naive animals on thermal PWL (A) and paw withdrawal thresholds (B) to von Frey stimulation. Three doses of 30 mg/kg S1627 (n = 6; ▵) or vehicle (n = 6; •) were injected intraperitoneally at the indicated times. Data represent the mean ± SE.
Figure 5.
Figure 5.
A, Effects of S1627 in the formalin assay. Two doses of 30 mg/kg were injected intraperitoneally 90 and 30 min before injection of formalin into the hind paw. The total number of flinches did not differ between groups. The data represent the mean ± SE of five rats in each group. B, Dose-dependent reduction of thermal hyperalgesia (Hargreaves test) in the zymosan-induced paw inflammation model. Either 10 mg/kg S1627 (n = 6; □) or 30 mg/kg (n = 6; ▵) was injected intraperitoneally at the indicated times. Controls (n = 12; •) received the appropriate volume of vehicle (1:1, vol/vol; polyethyleneglycol:water). The data represent the mean ± SE. ANOVA comparing the AUCs revealed significant effects for both doses (p < 0.05). C, Dose-dependent reduction of tactile hyperalgesia assessed with von Frey hairs in the zymosan-induced paw inflammation model. Either 10 mg/kg S1627 (n = 6; □) or 30 mg/kg (n = 6; ▵) was injected intraperitoneally at the indicated times (controls, n = 12; •). The data represent the mean ± SE. Antinociceptive effects of 3× 30 mg/kg (comparison of AUCs) were statistically significant with p < 0.05. 5. D, Reduction of pressure hyperalgesia (Randall Selitto test) in the zymosan-induced paw inflammation model. Three doses of 40 mg/kg (n = 4) were injected intraperitoneally 0.5 hr before and 2.25 and 5 hr after zymosan injection. The data represent the mean ± SE. The pressure pain threshold significantly differed between groups with p < 0.05 (indicated with the asterisk). E, Reduction of the zymosan-induced inflammatory paw edema with S1627. Two doses of 30 mg/kg or three doses of 40 mg/kg were injected intraperitoneally at the indicated times (n = 4 in each group). Controls received an equal volume of vehicle (n = 8). The data represent the mean ± SE. The reduction of paw swelling was statistically significant (comparison of AUCs) with the higher dose of 3× 40 mg/kg at p < 0.05.
Figure 6.
Figure 6.
A, Tactile allodynia after chronic constriction of the sciatic nerve (CCI model). Rats (n = 12 in each group) received 30 mg/kg S1627 or vehicle once daily for 5 d starting on day 4 after surgery. The nociceptive threshold was assessed daily with von Frey hairs before injection of the daily dose. The data represent the mean ± SE. The differences between the AUCs of control and S1627-treated rats was statistically significant at p < 0.05. B, Cold allodynia after chronic constriction of the sciatic nerve (CCI model). Rats (n = 12 in each group) received 30 mg/kg S1627 or vehicle once daily for 5 d starting at day 4 after surgery. Cold allodynia was assessed daily before drug injection by application of a drop of acetone onto the plantar surface of the hind paw. The time the rats spent licking, shaking, or lifting the paw after acetone application was measured with a stopwatch during an observation time of 2 min. The data represent the mean ± SE. The differences between the AUCs of control and S1627-treated rats was statistically significant at p < 0.05.
Figure 7.
Figure 7.
A, Western blot analysis of nuclear p65 (NF-κB subunit) using nuclear extracts from lumbar spinal cord tissue (L3-L5). Rats received a single dose of 100 mg/kg or vehicle 30 min before injection of 6 mg of zymosan into the hindpaw. The spinal cord was dissected 1 hr after zymosan injection. B, Western blot analysis of COX-2 in lumbar spinal cord tissue (L3-L5). Rats were treated as indicated, and the spinal cord was dissected 24 hr after zymosan injection. The times of drug or vehicle injection were 0.5 hr before zymosan and 2.25 and 5.5 hr after zymosan injection. C, D, TNF-α and IL-1β levels in lumbar spinal cord homogenates (L3-L5) assessed with commercially available ELISA assays. Rats were treated as indicated, and the spinal cord was dissected 48 hr after zymosan injection (6 mg). The times of drug or vehicle injection were 0.5 hr before zymosan and 2.25 and 5.5 hr after zymosan injection. Data are the mean ± SE. Zymosan-induced upregulation of TNF-α and IL-1β was significantly inhibited with 3× 30 mg/kg S1627, indicated with the asterisk (p < 0.05).
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