Faculty Bibliography

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

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

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

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

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

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

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

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