Treatment of rheumatoid arthritis with marine and botanical oils: an 18-month, randomized, and double-blind trial.

Objective. To determine whether a combination of borage seed oil rich in gamma linolenic acid (GLA) and fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is superior to either oil alone for treatment of rheumatoid arthritis (RA). Methods. Patients were randomized into a double-blind, 18-month trial. Mixed effects models compared trends over time in disease activity measures. Results. No significant differences were observed in changes in disease activity among the three randomized groups. Each group exhibited significant reductions in disease activity (DAS28) at 9 months (fish: -1.56[-2.16, -0.96], borage: -1.33[-1.83, -0.84], combined: -1.18[-1.83, -0.54]) and in CDAI (fish: -16.95[-19.91, -13.98], borage: -11.20[-14.21, -8.19], and combined: -10.31[-13.61, -7.01]). There were no significant differences in change of RA medications among the three groups. Reduced disease activity in study patients was similar to matched patients from an RA registry, and reduction in DMARD use was greater (P < 0.03) in study patients. Conclusion. All 3 treatment groups exhibited similar meaningful clinical responses after 9 months, improvements which persisted for 18 months, and a response similar to matched patients from an RA registry. Study patients were able to reduce DMARD therapy given in combination with TNF antagonists to a greater extent than registry patients. This paper is dedicated to the memory of Dr. John T. Sharp, M.D., a pioneer and innovator in the field of musculoskeletal radiology.

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 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.

Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells.

AIMS: To better understand mechanisms whereby Ajulemic acid (AjA), a synthetic antiinflammatory cannabinoid, promotes resolution of acute and chronic inflammation in animal models, we investigated its influence on cyclooxygenase 2 (COX2) expression and eicosanoid production in human fibroblast-like synovial cells (FLS). MAIN METHODS: FLS isolated from tissue obtained at joint replacement surgery or cultured from synovial fluid were treated for 60 min with AjA (10-30 microM), then stimulated with tumor necrosis factor alpha (TNFalpha). COX2 mRNA was measured by hybridization/colorimetric assay of whole cell lysates collected 4 h after stimulation. To determine effects on arachidonic acid release, FLS were incubated with (14)C-arachidonic acid for 20 h then treated with AjA (8-32 microM). Arachidonic acid release was measured by scintillation counting. Prostaglandins (PG) were measured by enzyme linked immunosorbent assay (ELISA) in cell supernatants collected 4 and 24 h after stimulation. KEY FINDINGS: AjA increased the steady state levels of COX2 mRNA in and arachidonic acid release from FLS. Treatment of FLS with AjA increased 15-deoxy-delta(12,14)-PGJ(2) (15d-PGJ(2)) production in a concentration dependent manner, but did not affect PGE(2) production significantly. SIGNIFICANCE: The capacity of AjA to increase selectively and markedly 15d-PGJ(2), an eicosanoid which facilitates resolution of inflammation, suggests that AjA may have value as a therapeutic agent for the treatment of rheumatoid arthritis (RA) and other diseases characterized by acute and chronic inflammation.

Suppression of human macrophage interleukin-6 by a nonpsychoactive cannabinoid acid.

Interleukin-6 (IL-6) is a multifunctional cytokine which contributes to inflammation and tissue injury in several diseases. Thus, inhibition of IL-6 production may be a useful strategy for treatment of patients with diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). A synthetic nonpsychoactive cannabinoid, ajulemic acid (AjA), prevents joint damage in experimental arthritis. Results of experiments presented here indicate that addition of AjA (3-30 microM) to human monocyte derived macrophages in vitro reduces steady state levels of IL-6 mRNA and the subsequent secretion of IL-6 from LPS stimulated cells. Although AjA binds to and activates PPARgamma, its anti IL-6 effects are PPARgamma independent. These studies provide evidence to support the view that AjA may prove to be an effective, safe antiinflammatory agent.

Potential anti-inflammatory actions of the elmiric (lipoamino) acids.

A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and evaluated for activity as potential anti-inflammatory agents. The compounds were tested in vitro for their effects on cell proliferation and prostaglandin production, and compared with their effects on in vivo models of inflammation. LPS stimulated RAW 267.4 mouse macrophage cells were the in vitro model and phorbol ester-induced mouse ear edema served as the principal in vivo model. The prostaglandin responses were found to be strongly dependent on the nature of the fatty acid part of the molecule. Polyunsaturated acid conjugates produced a marked increase in media levels of i15-deoxy-PGJ(2) with minimal effects on PGE production. It is reported in the literature that prostaglandin ratios in which the J series predominates over the E series promote the resolution of inflammatory conditions. Several of the elmiric acids tested here produced such favorable ratios suggesting that their potential anti-inflammatory activity occurs via a novel mechanism of action. The ear edema assay results were generally in agreement with the prostaglandin assay findings indicating a connection between them.

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 PPARgamma directly. Therefore, we investigated the influence of AjA on MMP production in human fibroblast-like synovial cells (FLS), and examined the role of PPARgamma in the mechanism of action of AjA. FLS, treated or not with a PPARgamma antagonist, were treated with AjA then stimulated with TNFalpha or IL-1alpha. Release of MMPs-1, 3, and 9 was measured by ELISA. The influence of AjA on MMP-3 release from stimulated PPARgamma positive (PPAR+/-) and PPARgamma null (PPAR-/-) mouse embryonic fibroblasts (MEF) was also examined. Addition of AjA to FLS suppressed production of MMPs whether or not PPARgamma activation was blocked. Secretion of MMP-3 was also suppressed by AjA in both TNFalpha- and IL-1alpha-stimulated PPARgamma+/- and PPARgamma-/- MEF. Suppression of MMP secretion from FLS by AjA appears to be PPARgamma 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.

Fibroblast-like synovial cells derived from synovial fluid.

OBJECTIVE: To obtain fibroblast-like synovial cells (FLS) from synovial fluid (SF). METHODS: SF aspirated from joints of patients with rheumatoid arthritis (RA), other types of inflammatory arthritis, and osteoarthritis (OA) was centrifuged and the resulting cell pellet resuspended in growth medium. After 2 days, nonadherent cells were removed. FLS were also cultured from surgical specimens of synovial tissue (td-FLS). Phenotype characterization of fluid derived FLS (fd-FLS) was accomplished by flow cytometry and immunohistochemistry staining. Tumor necrosis factor-alpha (TNF-alpha) induced interleukin 6 (IL-6), IL-8, and cyclooxygenase 2 (COX-2) mRNA levels were assessed. RESULTS: Second and later passage fd-FLS exhibited uniform fibroblast-like morphology. Fd-FLS and td-FLS expressed a similar profile of cell surface antigens including the fibroblast marker Thy-1. Less than 2% of either cell type expressed surface markers characteristic of dendritic cells, phagocytic cells, T cells, or leukocytes. Immunohistochemistry staining revealed the presence of fibroblast products prolyl-4 hydroxylase, procollagen I, and procollagen III in both culture types. TNF-a induced increases in IL-6, IL-8, and COX-2 mRNA were suppressed by dexamethasone in both fd-FLS and td-FLS. CONCLUSION: FLS can be cultured from SF. The fibroblast phenotype was confirmed by analysis of surface antigens and intracellular proteins. Inflammatory mediators produced after stimulation of both fd-FLS and td-FLS were suppressed by dexamethasone. In addition to providing a more accessible source of FLS, fd-FLS may also facilitate study of synovial cells in early RA when tissue specimens are not readily available.

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.

Suppression of human monocyte interleukin-1beta 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.

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 gammalinolenic acid, an unsaturated fatty acid with anti-inflammatory properties, modulates interleukin-1beta 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 interleukin-1beta (IL-1beta) from human monocytes stimulated with lipopolysaccharide (LPS). LPS-induced IL-1beta release is followed by IL-1-induced IL-1beta release, an amplification process termed "autoinduction." We show here, using IL-1alpha stimulation to simulate autoinduction, that administration of GLA to healthy volunteers and to patients with inflammatory arthritis reduces LPS-induced IL-1beta secretion mainly by reducing autoinduction of IL-1beta. GLA reduces LPS-induced pro-IL-1beta mRNA modestly and IL-la-induced pro-IL-1beta gene expression markedly. In addition to reducing amplification of IL-1beta, GLA increases the amount of IL-1 receptor antagonist (IL-1Ra) secreted from stimulated cells, thereby facilitating an increase in the secreted IL-1Ra/IL-1beta ratio. IL-1beta is important to host defense, but the amplification mechanism may be excessive in genetically predisposed individuals. Thus, reduction of IL-1beta autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.

Effect of Misoprostol on Acute and Chronic Inflammation.

Prostaglandins and their fatty acid precursors are important to regulation of cell function, and immune and inflammatory responses. Prostaglandin E compounds in particular have been shown to reduce inflammation and tissue injury. We examined the ability of misoprostol, the orally active analog of Prostaglandin E1, to influence inflammation in two animal models. In the subcutaneous air pouch model, acute inflammation was induced by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Misoprostol reduced pouch leukocyte counts in a dose dependent manner (33--49% v control), but did not alter fluid accumulation. The non-steroidal antiinflammatory agent, diclofenac, also reduced leukocyte counts in a dose dependent manner (28--66%) without affecting pouch fluid volume. Low dose misoprostol and low dose diclofenac used together reduced leukocyte counts by 39%, suggesting an advantageous snyergy in suppression of acute inflammation. Collagen induced arthritis (CIA) is a model of chronic inflammation. Misoprostol had no effect on the severity or course of CIA. Diclofenac reduced significantly all indices of inflammation tested, including joint swelling, number of affected joints and ability to walk. Misoprostol interfered with the antiinflammatory effect of diclofenac when the two compounds were administered together in the CIA model. These studies suggest that Misoprostol suppresses neutrophil mediated acute inflammation.