Mark G. Obukowicz

Novel, Selective Δ6 or Δ5 Fatty Acid Desaturase Inhibitors as Antiinflammatory Agents in Mice

Decreased synthesis of arachidonic acid by inhibition of the Δ6 or Δ5 desaturase was evaluated as a means to mitigate inflammation. Using quantitative in vitro and in vivo radioassays, novel compounds representing five classes of Δ5 desaturase inhibitors and one class of Δ6 desaturase inhibitor were identified. The Δ6 desaturase inhibitor, SC-26196, had pharmacokinetic and pharmacodynamic profiles in mice that allowed for the evaluation of the pharmacological effects of chronic inhibition of desaturase activity. SC-26196 decreased edema to the same extent as indomethacin or essential fatty acid deficiency in the carrageenan paw edema model in the mouse. The antiinflammatory properties of SC-26196 were consistent with its mechanism of action as a Δ6 desaturase inhibitor: 1) A correlation existed between inhibition of liver Δ6 desaturase activity and decreases in edema. 2) The onset of the decrease in edema was time dependent. 3) Selective reduction of arachidonic acid occurred dose dependently in liver, plasma and peritoneal cells. 4) In the presence of SC-26196, controlled refeeding of arachidonic acid, but not oleic acid, reversed the changes resulting from desaturase inhibition. The Δ6 desaturase may be a target for development of antiinflammatory drugs whose mechanism of action is unique.

Identification and Characterization of a Novel Δ6/Δ5 Fatty Acid Desaturase Inhibitor As a Potential Anti-Inflammatory Agent

Abstract The anti-inflammatory properties of essential fatty acid deficiency or n-3 polyunsaturated fatty acid supplementation have been attributed to a reduced content of arachidonic acid (AA; 20:4 n-6). An alternative, logical approach to depleting AA would be to decrease endogenous synthesis of AA by selectively inhibiting the Δ5 and/or the Δ6 fatty acid desaturase. High-throughput radioassays were developed for quantifying Δ5, Δ6, and Δ9 desaturase activities in vitro and in vivo . CP-24879 ( p -isopentoxyaniline), an aniline derivative, was identified as a mixed Δ5/Δ6 desaturase inhibitor during the screening of chemical and natural product libraries. In mouse mastocytoma ABMC-7 cells cultured chronically with CP-24879, there was a concentration-dependent inhibition of desaturase activity that correlated with the degree of depletion of AA and decreased production of leukotriene C 4 (LTC 4 ). Production of LTC 4 was restored by stimulating the cells in the presence of exogenous AA, indicating that endogenous AA was limiting as substrate. In the livers of mice treated chronically with the maximally tolerated dose of CP-24879 (3 mg/kg, t.i.d.), combined Δ5/Δ6 desaturase activities were inhibited approximately 80% and AA was depleted nearly 50%. These results suggest that Δ5 and/or Δ6 desaturase inhibitors have the potential to manifest an anti-inflammatory response by decreasing the level of AA and the ensuing production of eicosanoids.

Fish oil inhibits Δ6 desaturase activity in vivo: Utility in a dietary paradigm to obtain mice depleted of arachidonic acid

Abstract In mice that were alternately fasted and then refed an essential fatty acid-deficient (EFAD) diet, there was a rapid and substantial decline in tissue n-3 and n-6 polyunsaturated fatty acids (PUFAs) and a corresponding increase in n-9 fatty acids. Combined in vivo activities of Δ6 + Δ5 desaturases were quantified directly by measuring the conversion of 14C-linoleic acid (intraperitoneal injection) to 14C-arachidonic acid in liver lipids. Δ5 desaturase activity was quantified by measuring the conversion of 14C-dihomo-γ-linolenic acid (intraperitoneal injection) to 14C-arachidonic acid in liver lipids. The combined Δ6 + Δ5 desaturase activities in EFAD mice was very similar to that in chow-fed control mice (35% vs. 33% conversion of 14C-linoleic acid to 14C-arachidonic acid, respectively). Subsequent refeeding of EFAD mice with an EFAD diet supplemented with corn oil restored tissue n-6 PUFA levels, but did not alter Δ6 + Δ5 desaturase activities (33%). In contrast, subsequent refeeding of EFAD mice with a fish oil-supplemented diet markedly inhibited Δ6 + Δ5 desaturase activities (7%). Fatty acid analysis of the livers from the fish oil-fed mice showed that there was a depletion of the n-6 PUFAs, linoleic acid, and arachidonic acid, and an increase in the n-3 PUFAs, eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3). The inhibition of Δ6 + Δ5 desaturase activities was also maintained in EFAD mice fed a 1:1 mixture of fish oil:corn oil. As a consequence, a unique fatty acid composition in liver and plasma was obtained in which arachidonic acid was selectively depleted, whereas linoleic acid and n-3 PUFAs were increased. Δ5 desaturase activity was not affected by any of the fasting/refeeding paradigms. The data demonstrate that dietary n-3 PUFAs negatively regulate the in vivo synthesis of n-6 PUFAs at the level of the Δ6 desaturase. The inhibition of Δ6 desaturase activity by n-3 PUFAs provides a basis for a unique dietary route to selectively reduce tissue arachidonic acid, while providing sufficient linoleic acid, an essential fatty acid, to support normal cellular metabolism. This dietary paradigm may be effective in attenuating diseases characterized by excessive production of arachidonic acid-derived eicosanoids.

Dietary EPA reduces tumor load in ApcMin/+ mice by altering arachidonic acid metabolism, but conjugated linoleic acid, gamma--and alpha-linolenic acids have no effect.

Colorectal cancer is the second leading cause of cancer deaths in the United States and several lines of evidence implicate environmental factors, specifically dietary components, as major variables influencing colorectal cancer incidence. Among these, the amount and type of dietary fat consumed is of particular importance. Studies in humans and in animal models overwhelmingly indicate a protective effect of the (n-3) polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA, 20:5 (n-3)) and docosahexaenoic acid (DHA, 22:6 (n-3)) and the mechanism is largely thought to be related to interference with biosynthesis of 2-series prostaglandins from arachidonic acid (AA, 20:4 (n-6)). Compared to EPA and DHA, a-linolenic acid (ALA, 18:3 (n-3)), the parent fatty acid of the (n-3) family, conjugated linoleic acid (cLA, 18:2 (n-6) and (n-7))

Modulation of adjuvant-induced arthritis by dietary arachidonic acid in essential fatty acid-deficient rats.

Controlled feeding of linoleic acid (LA) or arachidonic acid (AA) to essential fatty acid-deficient (EFAD) rats was used to define the relationship between dietary AA and the inflammatory response evoked during adjuvant-induced arthritis. Based on energy percentage, EFAD rats were fed AA at the human daily equivalent (1×; 5.5 mg/day) or 10 times that amount (10×; 55 mg/day) or, alternatively, 0.5× of LA (273 mg/day). Feeding of 0.5×LA restored the plasma level of AA to that in chow-fed controls. In contrast, feeding of 1×AA only partially restored the plasma level of AA; 10×AA was required to fully replete AA. In parallel to the degree of repletion of AA in plasma, there were accompanying decreases in the levels of palmitoleic acid, oleic acid, and Mead acid. Compared to rats fed the standard laboratory chow diet (Control), edema in the primary hind footpads was decreased by 87% in EFAD, 71% in EFAD+1×AA, 45% in EFAD+10×AA, and 30% in EFAD+0.5×LA. The decrease in edema in the footpads of EFAD rats was nearly identical to the decrease in edema in the footpads of Control rats dosed with indomethacin. Hind footpad edema correlated with the final AA plasma level and eicosanoid levels extracted from hind footpad tissue, but not with neutrophil infiltration. The data showed that 0.5×LA and 10×AA, but not 1×AA, could quickly replete AA, accompanied by the synthesis of AA-derived eicosanoids and restoration of edema. These results suggest that in humans consumption of the average daily amount of AA without concurrent ingestion of LA would not alleviate an EFAD state.

Mitigation of arthritis by high-dose administration of a COX-2 inhibitor in the collagen-induced arthritis model in the mouse.

Non-steroidal anti-inflammatory drugs (NSAIDs), including selective cyclooxygenase (COX)-2 inhibitors, improve many parameters of arthritis in the adjuvant-induced arthritis model in the rat (Anderson et al., 1996). However, results with NSAIDs in the collagen-induced arthritis model in the mouse (Wooley, 1988) have been equivocal, possibly due to a mechanism that is prostaglandin-independent and because the level of dosing is limited due to gastrointestinal (GI) toxicity (Griswold et al., 1988; Phadke et al., 1985; Smith et al., 1990). With the advent of selective COX-2 inhibitors, it was possible to evaluate the efficacy of high-level dosing of a COX-2 inhibitor in the context of a GI-sparing background. The specific objectives of this study were to evaluate whether SC-046, a selective COX-2 inhibitor, mitigates the incidence and/or severity of arthritis and, possibly, is disease-modifying in the collagen-induced arthritis model in the mouse.