Ronald G. Rossetti

Oxidative metabolism of anandamide

In addition to the well studied hydrolytic metabolism of anandamide, a number of oxidative processes are also possible. Several routes somewhat analogous to the metabolism of free arachidonic acid have been reported. These involve mediation by various lipoxygenases and COX-2 and lead to ethanolamide analogs of the prostaglandins and HETES. The physiological significance of these products is not well understood at this time. There are also preliminary data suggesting a pathway involving oxidation of the hydroxy group of anandamide to a putative metabolite, N-arachidonyl glycine (AA-gly). This molecule displays activities in experimental models that suggest that it may play a role in some of the activities attributed to its precursor, anandamide.

Dimethylheptyl‐THC‐11 OIC acid: A nonpsychoactive antiinflammatory agent with a cannabinoid template structure

OBJECTIVE To assess the antiinflammatory activity of dimethylheptyl-THC-11 oic acid (DMH-11C), a nonpsychoactive synthetic derivative of tetrahydrocannabinol. METHODS Acute inflammation was induced by injection of interleukin-1beta and tumor necrosis factor alpha into subcutaneous air pouches formed on the backs of mice. Inflammation was quantified 6 hours later by pouch fluid leukocyte counts. Adjuvant-induced polyarthritis in rats was used as a model of chronic inflammation and joint tissue injury. Animals were either untreated, treated with safflower oil, or treated with DMH-11C in safflower oil. Arthritis was assessed by clinical observation and by histomorphologic evaluation of tibiotarsal joints. RESULTS Oral administration of DMH-11C reduced the accumulation of pouch fluid leukocytes and significantly reduced the severity of adjuvant-induced polyarthritis. Histopathologic studies of tibiotarsal joints showed that DMH-11C treatment attenuated pannus formation and joint tissue injury. CONCLUSION DMH-11C suppresses acute inflammation in the subcutaneous air pouch in mice and chronic joint inflammation characteristic of adjuvant disease in rats. These results demonstrate the potential use of this nonpsychoactive cannabinoid as an antiinflammatory agent.

Regulation of anandamide tissue levels by N-arachidonylglycine

N-arachidonylglycine (NAGly), the carboxylic analog of the endocannabinoid anandamide, occurs in rat and bovine brain as well as in peripheral sites and shows activity against tonic, formalin-induced pain. It was also observed, using cell membrane preparations, that it inhibits the hydrolytic activity of fatty acid amide hydrolase (FAAH) on anandamide (N-arachidonylethanolamide). These data suggested that it may serve as an endogenous regulator of tissue anandamide concentrations. In this report, we show findings derived from mass spectrometric analyses, indicating that blood levels of anandamide in rats given 10 mg/kg p.o. of NAGly were increased significantly by more than 9-fold when compared with vehicle-treated controls. In vitro evidence in RAW 264.7 cells using a deuterium-labeled NAGly demonstrated that it was not a precursor or source of arachidonic acid for the observed 50% rise in anandamide levels, suggesting that the increase was due to some effect other than increased biosynthesis of anandamide. Moreover, the findings presented here suggest that NAGly can serve as a model for the design of agents to provide pharmacological control of tissue anandamide concentrations.

ORAL ADMINISTRATION OF UNSATURATED FATTY ACIDS : EFFECTS ON HUMAN PERIPHERAL BLOOD T LYMPHOCYTE PROLIFERATION

Oils enriched in certain polyunsaturated fatty acids suppress joint pain and swelling in rheumatoid arthritis (RA) patients. Because T lymphocyte activation is important to propagation of joint tissue injury in patients with RA, we examined the effects of fatty acids administered by mouth in vivo on proliferation of human lymphocytes activated through the T cell receptor complex. T cell proliferation was reduced after oral administration of 2.4 g gammalinolenic acid in capsules of borage seed oil. Oral administration of oils enriched in linoleic acid, the parent n‐6 fatty acid, and alpha linolenic acid, the parent n‐3 fatty acid, did not influence growth of stimulated cells. Fatty acid analyses indicated that suppression of lymphocyte proliferation after gammalinolenic acid administration was associated with increased plasma and peripheral blood mononuclear cell concentrations of gammalinolenic acid and dihomogammalinolenic acid. J. Leukoc. Biol. 62: 438–443; 1997.

Gammalinolenic acid, an unsaturated fatty acid with anti-inflammatory properties, blocks amplification of IL-1 beta production by human monocytes.

Administration of gammalinolenic acid (GLA), an unsaturated fatty acid, reduces joint inflammation in patients with rheumatoid arthritis. Addition of GLA in vitro suppresses release of IL-1β from human monocytes stimulated with LPS. LPS-induced IL-1β release is followed by IL-1-induced IL-1β release, an amplification process termed autoinduction. We show here with peripheral blood monocytes from normal volunteers and from patients with rheumatoid arthritis by using IL-1R antagonist to block autoinduction and IL-1α stimulation to simulate autoinduction that ∼40% of IL-1β released from LPS-stimulated cells is attributable to autoinduction and that GLA reduces autoinduction of IL-1β while leaving the initial IL-1β response to LPS intact. Experiments with cells in which transcription and protein synthesis were blocked suggest that GLA induces a protein that reduces pro-IL-1β mRNA stability. IL-1β is important to host defense, but the amplification mechanism may be excessive in genetically predisposed patients. Thus, reduction of IL-1β autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.

Suppression of human monocyte interleukin-1β production by ajulemic acid, a nonpsychoactive cannabinoid

Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, reduces joint tissue damage in rats with adjuvant arthritis. Because interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) are central to the progression of inflammation and joint tissue injury in patients with rheumatoid arthritis, we investigated human monocyte IL-1beta and TNFalpha responses after the addition of AjA to cells in vitro. Peripheral blood and synovial fluid monocytes (PBM and SFM) were isolated from healthy subjects and patients with inflammatory arthritis, respectively, treated with AjA (0-30 microM) in vitro, and then stimulated with lipopolysaccharide. Cells were harvested for mRNA, and supernatants were collected for cytokine assay. Addition of AjA to PBM and SFM in vitro reduced both steady-state levels of IL-1beta mRNA and secretion of IL-1beta in a concentration-dependent manner. Suppression was maximal (50.4%) at 10 microM AjA (P<0.05 vs untreated controls, N=7). AjA did not influence TNFalpha gene expression in or secretion from PBM. Reduction of IL-1beta by AjA may help explain the therapeutic effects of AjA in the animal model of arthritis. Development of nonpsychoactive therapeutically useful synthetic analogs of Cannabis constituents, such as AjA, may help resolve the ongoing debate about the use of marijuana as medicine.

Suppression of fibroblast metalloproteinases by ajulemic acid, a nonpsychoactive cannabinoid acid

Production of matrix metalloproteinases (MMP) in joint tissue of patients with inflammatory arthritis facilitates cartilage degradation and bone erosion, and leads to joint deformities and crippling. Thus, MMPs are important targets for agents designed to treat inflammatory arthritis. Oral administration of ajulemic acid (AjA), a synthetic, nonpsychoactive cannabinoid acid, prevents joint tissue injury in rats with adjuvant arthritis. AjA binds to and activates PPARγ directly. Therefore, we investigated the influence of AjA on MMP production in human fibroblast‐like synovial cells (FLS), and examined the role of PPARγ in the mechanism of action of AjA. FLS, treated or not with a PPARγ antagonist, were treated with AjA then stimulated with TNFα or IL‐1α. Release of MMPs‐1, 3, and 9 was measured by ELISA. The influence of AjA on MMP‐3 release from stimulated PPARγ positive (PPAR+/−) and PPARγ null (PPAR−/−) mouse embryonic fibroblasts (MEF) was also examined. Addition of AjA to FLS suppressed production of MMPs whether or not PPARγ activation was blocked. Secretion of MMP‐3 was also suppressed by AjA in both TNFα‐ and IL‐1α‐stimulated PPARγ+/− and PPARγ−/− MEF. Suppression of MMP secretion from FLS by AjA appears to be PPARγ independent. Prevention by AjA of joint tissue injury and crippling in the rat adjuvant arthritis model may be explained in large part by inhibition of MMPs. These results suggest that AjA may be useful for treatment of patients with rheumatoid arthritis and osteoarthritis. J. Cell. Biochem. 100: 184–190, 2007.

Ajulemic acid, a nonpsychoactive cannabinoid acid, induces apoptosis in human T lymphocytes.

Oral administration of ajulemic acid (AjA), a synthetic nonpsychoactive cannabinoid acid, prevents joint cartilage and bone damage in an experimental model of arthritis in rats. Joint tissue injury in patients with rheumatoid arthritis (RA) is due in part to activation of T lymphocytes in the synovium, and T lymphocytes in synovium of RA patients are resistant to apoptosis. Thus, a potential mechanism whereby AjA prevents joint tissue injury in the animal model might be enhanced apoptosis of T lymphocytes. Apoptosis of human T cells in vitro was assessed by Annexin V expression, caspase-3 activity, DNA fragmentation, and microscopy. AjA induced apoptosis of T cells in a dose- and time-dependent manner. Apoptosis preceded loss of cell viability by trypan blue dye exclusion, confirming that cell loss was due to programmed cell death rather than necrosis. A nontoxic compound such as AjA may be a useful therapeutic agent for patients with diseases such as RA which are characterized by T-cell-driven chronic inflammation and tissue injury.

Human peripheral blood T lymphocyte proliferation after activation of the T cell receptor: Effects of unsaturated fatty acids

Oils enriched in certain polyunsaturated fatty acids suppress joint pain and swelling in rheumatoid arthritis patients with active synovitis. Because T lymphocyte activation is important for propagation of joint tissue injury in patients with rheumatoid arthritis, we examined the effects of fatty acids added in vitro on proliferation of human T lymphocytes stimulated with monoclonal antibodies to CD3 and CD4. Unsaturated fatty acids reduced T cell proliferation in a dose dependent manner (dihomogammalinolenic acid > gammalinolenic acid > eicosapentaenoic acid > arachidonic acid). Removal of fatty acids from cultures before cell stimulation did not change the effects, but addition of fatty acids after cell stimulation failed to reduce T cell responses. The saturated palmitic acid did not influence T cell growth. These studies indicate that small changes in cellular fatty acids can have profound effects on early events in T cell signaling and on T cell function.

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

Ajulemic acid (AjA), a synthetic nonpsychoactive cannabinoid, and lipoxin A4 (LXA4), 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 LXA4, a known antiinflammatory and proresolving mediator. Addition of AjA (0‐30 p, M) in vitro to human blood and synovial cells increased production of LXA4 (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 LXA4 identified by mass spectrometry. Blockade of 12/15 LOX, which leads to LXA4 synthesis via 15‐HETE production, reduced (>90%) the ability of AjA to enhance production of LXA4 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.— Zurier, R. B., Sun, Y.‐P., George, K. L., Stebu‐lis, J. A., Rossetti, R. G., Skulas, A., Judge, E., Serhan, C. N. Ajulemic acid, a synthetic cannabinoid, increases formation of the endogenous proresolving and antiinflammatory eicosanoid, lipoxin A4. FASEB J. 23, 1503–1509 (2009)