Enhancement of antinociception by coadministration of nonsteroidal anti-inflammatory drugs and soluble epoxide hydrolase inhibitors

Abstract

Combination therapies have long been used to treat inflammation while reducing side effects. The present study was designed to evaluate the therapeutic potential of combination treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and previously undescribed soluble epoxide hydrolase inhibitors (sEHIs) in lipopolysaccharide (LPS)-challenged mice. NSAIDs inhibit cyclooxygenase (COX) enzymes and thereby decrease production of metabolites that lead to pain and inflammation. The sEHIs, such as 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), stabilize anti-inflammatory epoxy-eicosatrienoic acids, which indirectly reduce the expression of COX-2 protein. Here we demonstrate that the combination therapy of NSAIDs and sEHIs produces significantly beneficial effects that are additive for alleviating pain and enhanced effects in reducing COX-2 protein expression and shifting oxylipin metabolomic profiles. When administered alone, AUDA-BE decreased protein expression of COX-2 to 73 +/- 6% of control mice treated with LPS only without altering COX-1 expression and decreased PGE(2) levels to 52 +/- 8% compared with LPS-treated mice not receiving any therapeutic intervention. When AUDA-BE was used in combination with low doses of indomethacin, celecoxib, or rofecoxib, PGE(2) concentrations dropped to 51 +/- 7, 84 +/- 9, and 91 +/- 8%, respectively, versus LPS control, without disrupting prostacyclin and thromboxane levels. These data suggest that these drug combinations (NSAIDs and sEHIs) produce a valuable beneficial analgesic and anti-inflammatory effect while prospectively decreasing side effects such as cardiovascular toxicity.

Fig. 1.

Dose–response curves in a thermal hindpaw withdrawal latency model after pretreatment with various concentrations of COX inhibitors (rofecoxib, black; celecoxib, white; indomethacin, gray). The inhibitors reduce LPS-induced thermal hyperalgesia in a dose-dependent manner, indicated by an increase in withdrawal latency toward baseline. Thermal withdrawal latencies were assessed 6 h after LPS exposure. Data represent the average latency ± SD (n = 4) to paw withdrawal from a thermal stimulus. Mean latency values are normalized as percent of control mice receiving vehicle before LPS challenge. ∗, Significantly different from vehicle (P < 0.05) as determined by ANOVA followed by Dunnett's test. The dose is expressed in milligrams per kilogram in all figures.

Fig. 2.

Additive antinociception. (a) Coadministration of AUDA-BE and NSAIDs produces an additive effect in counteracting LPS-induced decreases in hindpaw withdrawal latency. Hindpaw withdrawal latency was assessed 6 h after LPS exposure. Data represent the latency ± SD (n = 4) to paw withdrawal from a thermal stimulus. The data are depicted as percentage of control mice receiving vehicle without LPS. Individual inhibitors alone are shown as dark gray bars. Coadministration of AUDA-BE with various COX-2 inhibitors are shown as light gray bars. ∗, Significantly different from NSAID alone (P < 0.05) as determined by ANOVA followed by Tukey's test. (b) Structures of the sEH and COX inhibitors. Names and synonyms are provided in the text.

Fig. 3.

Coadministration of AUDA-BE and NSAIDs produces a synergistic decrease in PGD₂ (gray bars) and PGE₂ (black bars) 6 h after LPS exposure. As expected, all inhibitors individually decreased PGs in a dose-related manner. The data indicate that using a prophylactic dose of AUDA-BE with a nonoptimum therapeutic dose of COX inhibitor can further reduce the proinflammatory PGD₂ and PGE₂. The data represent average ± SD (n = 4) and are depicted as percentage of control mice receiving vehicle without LPS. Control values are PGD₂, 1.1 (method detection limit), and PGE₂, 2.6 ± 0.3 nM. ∗, Significantly different from NSAID alone (P < 0.05) as determined by ANOVA followed by Tukey's test.

Fig. 4.

A prophylactic dose of AUDA-BE reduces hepatic COX-2 protein expression 6 h after LPS exposure relative to mice receiving vehicle without LPS. Results from individual inhibitors at various doses are shown in dark gray bars. Coadministration of the sEHI and NSAID is depicted as a light gray bar, indicating that a prophylactic dose of AUDA-BE used in conjunction with a nonoptimum therapeutic dose of celecoxib (50 mg/kg) can further decrease COX-2 induction. Data represent the COX-2 protein levels ± SD (n = 3) in murine liver after exposure to LPS as determined by independent Western blots. ∗, Significantly different from vehicle (P < 0.05) as determined by ANOVA followed by Tukey's test. #, Significantly different from AUDA-BE alone (P < 0.05) as determined by ANOVA followed by Tukey's test. In addition, celecoxib at any dose did not statistically alter hepatic COX-1 or sEH protein levels compared with LPS-challenged mice (P < 0.05).

Fig. 5.

The COX-2 inhibitors increase the flow of AA through the P450 pathway increasing EpETrEs [Σ (8(9), 11(12) and 14(15) EETs; black bars) and DiHTrEs (Σ 5,6; 8,9; 11,12; and 14,15 DHETs; gray bars) in murine plasma after exposure to LPS. The 5(6) EET data are excluded because of lactone formation during sample preparation. Although the regioisomers were measured separately, the data are presented with the regioisomers combined to simplify the tables. There is no statistical difference in the ratio of regioisomers of EETs or DHETs (P < 0.05). The data indicate that coadministration of AUDA-BE and COX inhibitors can further increase the anti-inflammatory EETs. The data are average ± SD (n = 4). ∗, Significantly different from NSAID alone (P < 0.05) as determined by ANOVA followed by Tukey's test.

Fig. 6.

Coadministration of AUDA-BE and COX-2 inhibitors does not appear to create an imbalance in the PGI₂ (stable metabolite 6-keto-PGF_1α, black bars) and TXA₂ (stable metabolite TXB₂, gray bars) concentrations 6 h after LPS exposure. High doses of COX-2 inhibitors alone generate this disparity, leading to increased risk of thrombotic events. The data are average ± SD (n = 4) and are depicted as percentage of control mice receiving vehicle without LPS. Control values are 6-keto-PGF_1α, 7.6 ± 0.4, and TXB₂, 4.1 ± 0.4 nM. ∗, Significantly different from vehicle (P < 0.05) as determined by ANOVA followed by Tukey's test.