Ajulemic acid, a synthetic cannabinoid, increases formation of the endogenous proresolving and anti-inflammatory eicosanoid, lipoxin A4

Abstract

Ajulemic acid (AjA), a synthetic nonpsychoactive cannabinoid, and lipoxin A(4) (LXA(4)), an eicosanoid formed from sequential actions of 5- and 15-lipoxygenases (LOX), facilitate resolution of inflammation. The purpose of this study was to determine whether the ability of AjA to limit the progress of inflammation might relate to an increase in LXA(4), a known anti-inflammatory and proresolving mediator. Addition of AjA (0-30 microM) in vitro to human blood and synovial cells increased production of LXA(4) (ELISA) 2- to 5-fold. Administration of AjA to mice with peritonitis resulted in a 25-75% reduction of cells invading the peritoneum, and a 7-fold increase in LXA(4) identified by mass spectrometry. Blockade of 12/15 LOX, which leads to LXA(4) synthesis via 15-HETE production, reduced (>90%) the ability of AjA to enhance production of LXA(4) in vitro. These results suggest that AjA and other agents that increase endogenous compounds that facilitate resolution of inflammation may be useful for conditions characterized by inflammation and tissue injury.

Figure 1.

Cannabinoid structures. A) Δ9-THC (tetrahydrocannabinol) is the component of Cannabis responsible for psychoactivity. B) THC-11-oic acid is a nonpsychoactive metabolite of Δ9 THC. C) AjA (1′,1′-dimethylheptyl THC-11-oic acid) is a nonpsychoactive synthetic analog of THC-11-oic acid.

Figure 2.

AjA stimulates LXA₄ production by human cells. A) PMNs (3×10⁶) incubated with vehicle alone (0 AjA) or treated 1 h with AjA and stimulated 4 h with 10 ng/ml rhTNFα. Supernatant LXA₄ determined by ELISA. Values are means of 6 replicates; error bars represent sd. *P < 0.04 vs. 0 AjA; Student’s t test (n=6). B) Peripheral blood incubated without or with AjA (3, 10, 30 μM) for 5 h. Supernatant LXA₄ concentration measured by ELISA. Values are means of 5 experiments with blood from 5 separate donors; error bars are sd. *P < 0.05 vs. 0 AjA; ANCOVA (3 and 10 μM, n=4; baseline and 30 μM, n=5).

Figure 3.

AjA stimulates LXA₄ production by human FLSs. FLSs were incubated with vehicle (0 AjA) or AjA (10, 20, 30 μM) for 1 h, then stimulated with 1 ng/ml TNFα for 18 h. Supernatant LXA₄ determined by ELISA. Values are means of 4 experiments; error bars are sd. *P < 0.05 vs. 0 AjA; ANCOVA (n=4).

Figure 4.

Treatment of murine peritonitis with oral administration of AjA. Male CD-1 mice were treated by mouth with 10 mg/kg/day AjA or with safflower oil (vehicle control) for 3 days before i.p. injection of 1 mg zymosan A. Peritoneal fluid obtained 3 h after zymosan installation. Values are means of total cell counts determined by light microscopy; error bars are sd. *P = 0.03 vs. untreated controls; ANCOVA, two separate experiments, 3 mice/group (n=6). NS, no stimulation.

Figure 5.

AjA stimulation of endogenous LXA₄ generation and reduction of peritoneal infiltration of PMNs in zymosan-A-induced peritonitis. AjA (1.5 mg/kg/100 μl sterile saline) was injected into tail vein of 6- to 8-wk-old male FVB mice, followed by i.p. injection of 1 mg zymosan A. Peritoneal lavages were collected, volumes were measured, and 2 vol of methanol was added. Following solid-phase extraction, samples were then analyzed using LC/MS/MS. Aliquots of lavages were assessed for PMN concentration as described in Materials and Methods. A) MS/MS spectrum of AjA-treated samples. Chromatogram spectra are representative of n = 4; a–d and c′ denote prominent diagnostic ions used for identification (see inset). a, ([M-H]) = m/z 351; b, ([M-H]–CO₂) = m/z 307; c, ([M-H]–CHO(CH₂)3COOH) = m/z 235; c′, (CHO(CH2)3COOH) = m/z 115; d, ([M-H]—CHO(CH2)4CH3) = m/z 215. B) LXA₄ generation quantified in AjA-treated samples vs. control samples. Values represent LXA₄ generation per mouse. Samples were combined before quantitation to allow for adequate detection. C) PMN concentrations quantified in peritoneal lavages from mice treated with AjA vs. untreated controls. Values are means; error bars are sd. *P < 0.05 vs. untreated controls; Student’s t test (n=4).

Figure 6.

Blockade of AjA enhancement of LXA_4. Human blood was incubated with AjA for 1 h, and LXA₄ was assessed as described in Materials and Methods. AjA (25 μM)-induced production of LXA₄ was reduced significantly when the 12/15 LOX inhibitor baicalein was added to cells before incubation with AjA. Values are means; error bars represent sd. *P < 0.05 vs. AjA alone; Student’s t test (n=3).