Inhibition of Neutrophil Leukotriene B4 Production by a Novel Synthetic N-3 Polyunsaturated Fatty Acid Analogue, β-Oxa 21:3n-3

Abstract

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the β-position, β-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the β-oxa-PUFA substantially depresses the production of leukotriene B4 (LTB4) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, β-oxa 21:3 n-6, is also a strong inhibitor of LTB4 production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB4. The parent β-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB4 formation. β-Oxa-21:3n-3 inhibits the conversion of [3H]20:4n-6 to [3H]5-hydroxyeicosatetraenoic acid and [3H]LTB4 by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. β-Oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of β-oxa-21:3n-3 to a 16-monohydroxy-β-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, β-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, β-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.

RBx 7796: A novel inhibitor of 5-lipoxygenase

“…Some of these molecules were effective in acute infl ammation models but failed to offer protection in chronic models of infl ammation in experimental animals and in clinic [16,26,30,31]. Reports exist to suggest that long chain fatty acids [32,33] and molecules like α-Tocopherol [34] with long carbon chain can inhibit 5-lipoxygenase, albeit, with weak II-potency. Through extensive medicinal chemistry efforts, we have discovered RBx 7796 (Clafrinast) -a distinct chemical entity from N-hydroxyurea, that inhibits 5-lipoxygenase enzyme in a competitive manner and exhibits effi cacy in several experimental animal models of airway infl ammation.…”

The Immunomodulatory Effects of Novel β-Oxa, β-Thia, and γ-Thia Polyunsaturated Fatty Acids on Human T Lymphocyte Proliferation, Cytokine Production, and Activation of Protein Kinase C and MAPKs

“…When long chain fatty acids are added to cells, they become esterified in different lipid pools, including membrane phospholipids (17). The incorporation and composition of phospholipids may vary with time.…”

“…The oxa fatty acid is UV active, so on a TLC plate sprayed with the dichlorofluorescein and viewed under UV light, it quenches the fluorescence of the dichlorofluorescein to show up as a dark spot on a green background. The samples were transesterified, and the liberated fatty acid methyl esters were quantitated by GLC, as described above (17). The positional distribution of ␤-oxa-21: 3n-3 in purified phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol was assessed by degrading the compounds with phospholipase A 2 and measuring the amount of the ␤-oxa-PUFA associated with the reaction products (unesterified fatty acids and lysophospholipids) after isolation by TLC (20).…”

“…Incorporation of the ␤-oxa 21:3n-3 into membrane phospholipids was performed, as described previously (17). Briefly, T lymphocytes (4 ϫ 10 7 ) were incubated with or without 20 M ␤-oxa-21:3n-3 in 4 ml of HBSS at 37°C for 30 min.…”

Mechanisms by which fatty acids regulate leucocyte function

“…This compound significantly inhibited T-lymphocyte proliferation and cytokine production in vitro when compared with DHA [46]. Other chemically engineered FAs, with either an oxygen atom in the β position (β-oxa) or a sulfur atom in the β (β-thia) or γ (γ -thia) position, have also been synthesized and have specific effects on neutrophil and lymphocyte function [85,86]. These novel FAs may be useful as anti-inflammatory agents, as they exhibit increased biological activity compared with the natural long-chain PUFAs.…”