Contribution of diacylglycerol lipase β to pain after surgery

Abstract

Background: Metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) yields arachidonic acid (AA), the precursor to proalgesic eicosanoids including prostaglandin E2 (PGE₂). Diacylglycerol lipase β (DAGLβ) is an enzyme that synthesizes 2-AG and its inhibition reduces eicosanoid levels and produces antinociceptive effects in models of inflammatory pain. Here we test whether inhibition of DAGLβ produces antinociceptive effects in a model of postoperative pain.

Methods: Rats were administered the selective DAGLβ inhibitor KT109 or vehicle and underwent plantar incision. Postsurgical pain/disability was examined using evoked (mechanical hyperalgesia), functional (incapacitance/weight bearing), and functional/spontaneous (locomotion) modalities.

Results: Activity-based protein profiling confirmed that KT109 inhibited DAGLβ in the lumbar spinal cord (LSC) and brain, confirming that it is a systemically active DAGLβ inhibitor. Treatment with KT109 reduced basal 2-AG, AA, and PGE₂ levels in the liver but not the brain, indicating that DAGLβ activity does not significantly contribute to basal PGE₂ production within the central nervous system. Plantar incision elevated the levels of 2-AG and PGE₂ in the LSC. Although KT109 did not alter postsurgical 2-AG levels in the LSC, it slightly reduced PGE₂ levels. In contrast, the clinically efficacious cyclooxygenase inhibitor ketoprofen completely suppressed PGE₂ levels in the LSC. Similarly, KT109 had no significant effect upon postsurgical 2-AG, AA, or PGE₂ levels at the incision site, while ketoprofen abolished PGE₂ production at this location. KT109 and ketoprofen reversed the weight bearing imbalance induced by plantar incision, yet neither KT109 nor ketoprofen had any significant effect on mechanical hyperalgesia. Treatment with ketoprofen partially but significantly rescued the locomotor deficit induced by incision while KT109 was without effect.

Conclusion: DAGLβ is not the principal enzyme that controls 2-AG derived AA and PGE₂ production after surgery, and inhibitors targeting this enzyme are unlikely to be efficacious analgesics superior to those already approved to treat acute postoperative pain.

Keywords: 2-AG; endocannabinoid; incision; pain; surgery.

Figure 1

KT109 inhibits rat DAGLβ in vitro and in vivo. Notes: (A) HEK293T cells expressing DAGLβ were incubated with KT109 or DMSO for 60 min at 25°C and subsequently probed with 1 µM HT-01. The first lane represents empty vector transfected HEK293T cells, while the next four lanes show cells expressing DAGLβ. The HT-01–labeled proteins in the gels were visualized by in-gel fluorescence. (B) Rats were injected with KT109 (30 mg/kg, IP) or vehicle 1 h prior to plantar incision. Brains and LSCs were harvested 4 h later and subjected to ABPP using HT-01. The bands corresponding to DAGLβ are indicated. *Indicates the known DAGLβ degradation product(s). Abbreviations: ABHD6, alpha/beta-hydrolase domain containing 6; ABPP, activity-based protein profiling; DAGLβ, diacylglycerol lipase β; DMSO, dimethylsulfoxide; IP, intraperitoneal; LSCs, lumbar spinal cords.

Figure 2

KT109 reduces the levels of 2-AG and downstream metabolites in the liver, but not brain. Notes: Levels of 2-AG, AA, and PGE2 in (A) liver and (B) brain 4 h after KT109 (30 mg/kg, IP) or vehicle administration. **p<0.01; ***p<0.001. Abbreviations: 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; IP, intraperitoneal; PGE2, prostaglandin E2.

Figure 3

Effects of KT109 and ketoprofen upon postsurgical PGE₂ levels. Notes: (A) Levels of 2-AG, AA, and PGE₂ in the heels of rats 4 h after plantar incision after treatment with 30 mg/kg KT109, 10 mg/kg ketoprofen, or vehicle (denoted as incision heel). Sham rats were injected with vehicle; they underwent a sham incision and their heels were harvested 4 h later. (B) 2-AG, AA, and PGE₂ levels in LSC 4 h after surgery in vehicle-, KT109-, or ketoprofen-treated rats. *p<0.05; **p<0.01; ***p<0.001. Abbreviations: 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; LSC, lumbar spinal cord; PGE2, prostaglandin E2.

Figure 4

Mechanical hyperalgesia and incapacitance in rats after plantar incision. Notes: (A) Mechanical thresholds (g) in the heel and plantar aspect of the paw at baseline and 4 h after plantar incision in rats treated with vehicle, 30 mg/kg KT109, or 10 mg/kg ketoprofen. (B) Hind limb weight bearing (differences in g) at baseline and 4 h after plantar incision in rats treated with vehicle, 30 mg/kg KT109, or 10 mg/kg ketoprofen. *p<0.05; **p<0.01; ***p<0.001.

Figure 5

Home cage locomotor activity and rearing before and after plantar incision. Notes: (A) Locomotor activity and (B) rearing over 12 h at baseline and after plantar incision in rats treated with vehicle, 30 mg/kg KT109, or 10 mg/kg ketoprofen. (C) Locomotion and (D) rearing over the first 6 h after plantar incision or at baseline in rats treated with vehicle, 30 mg/kg KT109, or 10 mg/kg ketoprofen. In each case, the left panel represents the baseline condition while the right panel represents incision. *p<0.05.

Scheme 1

Synthesis of KT109. Abbreviations: DIPEA: N,N-Diisopropylethylamine; THF, tetrahydrofuran; DMAP, 4-Dimethylaminopyridine; PdCl2 (dppf)2: (1,1′-Bis(diphenylphosphino)ferrocene) palladium(II) dichloride; K2CO3: potassium carbonate