Cannabinoids, inflammation, and fibrosis.

Cannabinoids apparently act on inflammation through mechanisms different from those of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs). As a class, the cannabinoids are generally free from the adverse effects associated with NSAIDs. Their clinical development thus provides a new approach to treatment of diseases characterized by acute and chronic inflammation and fibrosis. A concise survey of the anti-inflammatory actions of the phytocannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol, cannabichromene, and cannabinol is presented. Mention is also made of the noncannabinoid plant components and pyrolysis products, followed by a discussion of 3 synthetic preparations-Cesamet (nabilone; Meda Pharmaceuticals, Somerset, NJ, USA), Marinol (dronabinol; THC; AbbVie, Inc., North Chicago, IL, USA), and Sativex (Cannabis extract; GW Pharmaceuticals, Cambridge United Kingdom)-that have anti-inflammatory effects. A fourth synthetic cannabinoid, ajulemic acid (AJA; CT-3; Resunab; Corbus Pharmaceuticals, Norwood, MA, USA), is discussed in greater detail because it represents the most recent advance in this area and is currently undergoing 3 phase 2 clinical trials by Corbus Pharmaceuticals. The endogenous cannabinoids, including the closely related lipoamino acids, are then discussed. The review concludes with a presentation of a possible mechanism for the anti-inflammatory and antifibrotic actions of these substances. Thus, several cannabinoids may be considered candidates for development as anti-inflammatory and antifibrotic agents. Of special interest is their possible use for treatment of chronic inflammation, a major unmet medical need.-Zurier, R. B., Burstein, S. H. Cannabinoids, inflammation, and fibrosis.

Ultrapure ajulemic acid has improved CB2 selectivity with reduced CB1 activity.

Ajulemic acid, a side-chain analog of Δ(8)-THC-11-oic acid, was designed as a potent therapeutic agent free of the psychotropic adverse effects typical of most cannabinoids. Subsequent studies of ajulemic acid have yielded widely divergent findings on the occurrence of these adverse effects. To help resolve these discrepancies, we have prepared highly purified ajulemic acid using a different synthetic method than previously reported in the literature and compared its cannabinoid receptor binding constants with those obtained using several other preparations from different sources. Whereas CB2 binding did not vary greatly among all of the samples, the CB1 binding showed a wide range of affinities. The highly purified product (JBT-101) reported here had the weakest affinity for CB1 while the original preparation (HU-239) showed the strongest affinity for CB1. The CB1/CB2 ratio of affinities was 12.3 for JBT-101 whereas that for HU-239 was 0.19, a 65-fold difference. Functional responses such as catalepsy and hypothermia using JBT-101 versus HU-239 displayed reduced CB1 activity in keeping with the receptor binding data. Thus, earlier conclusions on the limited therapeutic index for ajulemic acid need to be reconsidered in the light of the data now obtained using JBT-101.

Treatment of rheumatoid arthritis with marine and botanical oils: influence on serum lipids.

The gap in mortality between patients with rheumatoid arthritis (RA) and the general population (1.5-3.0 fold risk) is increasing. This disparity is attributable mainly to cardiovascular disease (CVD), as the CVD risk is comparable to patients with diabetes mellitus. The purpose of this study is to determine whether borage seed oil rich in gamma-linolenic acid, fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), or the combination of both oils are useful treatments for dyslipidemia in patients with RA. We randomized patients into a double blind, 18 month trial. Mixed effects models were used to compare trends over time in serum lipids. No significant differences were observed between the three groups: All three treatment groups exhibited similar meaningful improvement in the lipid profile at 9 and 18 months. When all groups were combined, these treatments significantly reduced total and LDL-cholesterol and triglycerides, increased HDL-cholesterol, and improved the atherogenic index. All improvements observed at 9 months persisted at 18 months (P < 0.001 verses baseline). Conclusion. Marine and botanical oils may be useful treatment for rheumatoid arthritis patients who are at increased risk for cardiovascular disease compared to the general population.

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 nonpsychoactive cannabinoid acid, suppresses osteoclastogenesis in mononuclear precursor cells and induces apoptosis in mature osteoclast-like cells.

Oral administration of ajulemic acid (AjA), a cannabinoid acid devoid of psychoactivity, prevents joint tissue injury in rats with adjuvant induced arthritis. Because activation of osteoclasts is central to the pathogenesis of bone erosion in patients with rheumatoid arthritis (RA), we investigated the influence of AjA on osteoclast differentiation and survival. Osteoclast cultures were established by stimulation of RAW264.7 cells and primary mouse bone marrow cultures with receptor activator of NF-kappaB ligand (RANKL). Simultaneous addition of AjA (15 and 30 microM) and RANKL to both culture systems significantly suppressed development of multinucleated osteoclasts (osteoclastogenesis) in a dose dependent manner, as determined by quantification of multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive cells. AjA impaired growth of RAW264.7 monocytes and prevented further osteoclast formation in cultures in which osteoclastogenesis had already begun. Reduction by AjA of both monocyte growth and osteoclast formation was associated with apoptosis, assayed by annexin V and propidium iodide staining, and caspase activity. The anti-osteoclastogenic effects of AjA did not require the continuous presence of AjA in the cell cultures. Based on these findings, we propose that AjA or other nonpsychoactive synthetic analogs of Cannabis constituents may be useful therapy for diseases such as RA and osteoporosis in which bone resorption is a central feature.

CD4+CD25high T cell numbers are enriched in the peripheral blood of patients with rheumatoid arthritis.

Accumulating evidences support that CD4(+)CD25(high) T regulatory (Treg) cells play an essential role in controlling and preventing autoimmunity. Paradoxically, RA patients have elevated numbers of circulating CD4(+)CD25(high) T cells, however, the inflammation is still ongoing. Further identification of these CD4(+)CD25(high) T cells may contribute to a better understanding of underlying mechanisms. We show here that these CD4(+)CD25(high) T cells were composed of CD4(+)CD25(high)FoxP3(+) Treg cells and activated CD4(+)CD25(high)FoxP3(-) effector cells. Moreover, there were significantly more Treg cells and effector T cells expressing GITR, and more monocytes expressing GITR-L. Thus, although RA patients have elevated numbers of CD4(+)CD25(high) T cells, the suppressive function is not increased, because of the increased number of activated effector T cells. In addition, the GITR-GITR-L system was activated in RA patients, which might lead to diminish suppressive activity of Treg cells and/or lead to resistance of activated effector T cells to suppression by Treg cells, thus, contributing to the ongoing inflammation in RA patients.

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.

Analysis of the thiol status of peripheral blood leukocytes in rheumatoid arthritis patients.

Although the exact etiology of rheumatoid arthritis (RA) remains unknown, there is increasing evidence that reactive oxygen species and a pro-oxidant/antioxidant imbalance are an important part of the pathogenesis of joint tissue injury. Flow cytometry was used to evaluate the thiol status [surface-thiols and intracellular glutathione (iGSH)] of leukocytes from RA patients and controls. Levels of surface-thiols and iGSH of leukocytes from RA patients were significantly lower than of leukocytes from controls. CD53, a glycoprotein of the tetraspanin superfamily, which coprecipitates with the GSH recycling enzyme gamma-glutamyl transpeptidase, was elevated significantly on leukocytes from RA patients compared with leukocytes from controls. Surface-thiols and GSH play important roles in redox buffering of cells, providing protection from oxidative stress. The chronic inflammation of RA has been associated with oxidative stress, which is shown to cause a decline in the levels of cellular antioxidant sulfhydryls (R-SH). As antioxidant-protective levels also decline with age, the problem is compounded in older RA patients, who did have fewer R-SH. Chronic stress can also have an effect on telomere lengths, determining cell senescence and longevity. Although telomeres shorten with increasing age, our flow cytometry studies indicate that accelerated shortening in telomere lengths occurs with increasing age of RA patients, suggesting premature cellular aging. The paradox is that lymphocytes from RA patients are believed to resist apoptosis, and we suggest that the elevated expression of CD53, which results from the increased oxidative stress, may protect against apoptosis.

Liver abnormalities in rheumatic diseases.

Abnormalities of LFTs and liver function occur not infrequently in patients with rheumatic conditions, and many diagnostic possibilities exist. Systemic inflammation that is related to uncontrolled rheumatic disease and periods of disease remission have been described as a cause for fluctuations in levels of serum aminotransferases. Although these benign extra-articular manifestations of rheumatic disease are the most common manifestations, more serious hepatic involvement, including vasculitis, nodular regenerative hyperplasia, and primary biliary cirrhosis, have been observed in specific rheumatic diseases. The cause of rheumatic disease is unclear. Occult HCV infection and associated cryoglobulinemia can mimic rheumatic disease. HCV infection should be suspected routinely in patients with mixed cryoglobulinemia, especially because antiviral therapy may be beneficial. The medical management of rheumatic disease involves medications that are often hepatotoxic. Routine laboratory monitoring, imaging studies, and, if necessary, biopsy examination in situations in which serum aminotransferases remain abnormal, are recommended.

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.

Ajulemic acid: A novel cannabinoid produces analgesia without a "high".

A long-standing goal in cannabinoid research has been the discovery of potent synthetic analogs of the natural substances that might be developed as clinically useful drugs. This requires, among other things, that they be free of the psychotropic effects that characterize the recreational use of Cannabis. An important driving force for this goal is the long history of the use of Cannabis as a medicinal agent especially in the treatment of pain and inflammation. While few compounds appear to have these properties, ajulemic acid (AJA), also known as CT-3 and IP-751, is a potential candidate that could achieve this goal. Its chemical structure was derived from that of the major metabolite of Delta9-THC, the principal psychotropic constituent of Cannabis. In preclinical studies it displayed many of the properties of non-steroidal anti-inflammatory drugs (NSAIDs); however, it seems to be free of undesirable side effects. The initial short-term trials in healthy human subjects, as well as in patients with chronic neuropathic pain, demonstrated a complete absence of psychotropic actions. Moreover, it proved to be more effective than placebo in reducing this type of pain as measured by the visual analog scale. Unlike the narcotic analgesics, signs of dependency were not observed after withdrawal of the drug at the end of the one-week treatment period. Data on its mechanism of action are not yet complete; however, the activation of PPAR-gamma, and regulation of eicosanoid and cytokine production, appear to be important for its potential therapeutic effects.

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.

Regulation of Macrophage-Independent T-Cell Activation by Prostaglandins and Nonsteroidal Anti-Inflammatory Drugs.

We examined the hypothesis that both misoprostol (miso), a prostaglandin E(1) (PGE(1)) analog, and nonsteroidal anti-inflammatory drugs (NSAIDs) have significant and largely inhibitory effects on T-cell activation and, consequently, influence T-cell function. Studies were done using a macrophage-independent system for T-cell activation by mitogenic combinations of immobilized anti-T-cell monoclonal antibodies (mabs), including combinations of anti-CD3 with anti-CD4 or anti-CD6. The results indicate that misoprostol, like other prostaglandins, can inhibit T-cell proliferation and the expression of mRNAs for cytokines critical in T-cell growth and immunoregulation. Somewhat higher concentrations of misoprostol are required than of PGE(1) to achieve comparable effects. However, even very low doses of misoprostol (0.01 &mgr;g ml(minus sign1)) may sometimes enhance T-cell activation. The effects on cytokine mRNA are not uniform; for a given dose and agent, one cytokine mRNA may be enhanced while another is unchanged or diminished. Whether a response is increased or decreased depends on several factors, including the dose of misoprostol or other PG tested, the strength of the response on which the agent is acting, and the level of preexisting activation or cytokine gene expression. Similar findings can be seen with treatment in vitro with NSAIDs, including sodium salicylate. When PGs (including misoprostol) and NSAIDs are combined, effects are often additive and sometimes appear synergistic, but paradoxical results can also occur, whereby each agent separately may be modestly inhibitory, but together they are stimulatory. The in vitro response of T cells to both PGs and NSAIDs (either separately or together) is, thus, highly complex. These data support the notion that these agents may have significant effects on the regulation of T-cell activation and function.

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.

Cannabinoids, endocannabinoids, and related analogs in inflammation.

This review covers reports published in the last 5 years on the anti-inflammatory activities of all classes of cannabinoids, including phytocannabinoids such as tetrahydrocannabinol and cannabidiol, synthetic analogs such as ajulemic acid and nabilone, the endogenous cannabinoids anandamide and related compounds, namely, the elmiric acids, and finally, noncannabinoid components of Cannabis that show anti-inflammatory action. It is intended to be an update on the topic of the involvement of cannabinoids in the process of inflammation. A possible mechanism for these actions is suggested involving increased production of eicosanoids that promote the resolution of inflammation. This differentiates these cannabinoids from cyclooxygenase-2 inhibitors that suppress the synthesis of eicosanoids that promote the induction of the inflammatory process.

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.

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.

Ajulemic acid, a synthetic nonpsychoactive cannabinoid acid, bound to the ligand binding domain of the human peroxisome proliferator-activated receptor gamma.

Ajulemic acid (AJA) is a synthetic analog of THC-11-oic acid, a metabolite of tetrahydrocannabinol (THC), the major active ingredient of the recreational drug marijuana derived from the plant Cannabis sativa. AJA has potent analgesic and anti-inflammatory activity in vivo, but without the psychotropic action of THC. However, its precise mechanism of action remains unknown. Biochemical studies indicate that AJA binds directly and selectively to the isotype gamma of the peroxisome proliferator-activated receptor (PPARgamma) suggesting that this may be a pharmacologically relevant receptor for this compound and a potential target for drug development in the treatment of pain and inflammation. Here, we report the crystal structure of the ligand binding domain of the gamma isotype of human PPAR in complex with ajulemic acid, determined at 2.8-A resolution. Our results show a binding mode that is compatible with other known partial agonists of PPAR, explaining their moderate activation of the receptor, as well as the structural basis for isotype selectivity, as observed previously in vitro. The structure also provides clues to the understanding of partial agonism itself, suggesting a rational approach to the design of molecules capable of activating the receptor at levels that avoid undesirable side effects.