Deborah Ann Rathjen

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

A novel long chain polyunsaturated fatty acid, β-oxa 21:3 n -3, inhibits T lymphocyte proliferation, cytokine production, delayed-type hypersensitivity, and carrageenan-induced paw reaction and selectively targets intracellular signals

A novel polyunsaturated fatty acid (PUFA), β-oxa 21:3n-3, containing an oxygen atom in the β position, was chemically synthesized, and found to have more selective biological activity than the n-3 PUFA, docosahexaenoic acid (22:6n-3) on cells of the immune system. Although β-oxa 21:3n-3 was very poor compared with 22:6n-3 at stimulating oxygen radical production in neutrophils, it was more effective at inhibiting human T lymphocyte proliferation (IC50 of 1.9 vs 5.2 μM, respectively). β-Oxa 21:3n-3 also inhibited the production of TNF-β, IFN-γ, and IL-2 by purified human T lymphocytes stimulated with PHA plus PMA, anti-CD3 plus anti-CD28 mAbs, or PMA plus A23187. Metabolism of β-oxa 21:3n-3 via the cyclooxygenase and lipoxygenase pathways was not required for its inhibitory effects. Consistent with its ability to suppress T lymphocyte function, β-oxa 21:3n-3 significantly inhibited the delayed-type hypersensitivity response and carrageenan-induced paw edema in mice. In T lymphocytes, β-oxa 21:3n-3 inhibited the agonist-stimulated translocation of protein kinase C-βI and -ε, but not -α, -βII, or -θ to a particulate fraction, and also inhibited the activation of the extracellular signal-regulated protein kinase, but not c-Jun NH2-terminal kinase and p38. In contrast, 22:6n-3 had no effects on these protein kinase C isozymes. The increase in antiinflammatory activity and loss of unwanted bioaction through the generation of a novel synthetic 22:6n-3 analogue provides evidence for a novel strategy in the development of anti-inflammatory agents by chemically engineering PUFA.

A Novel β-Oxa Polyunsaturated Fatty Acid Downregulates the Activation of the IκB Kinase/Nuclear Factor κB Pathway, Inhibits Expression of Endothelial Cell Adhesion Molecules, and Depresses Inflammation

Several novel polyunsaturated fatty acids (PUFAs) that contain either an oxygen or sulfur atom in the β-position were found to exhibit more selective antiinflammatory properties than their natural PUFA counterparts. One of these, β-oxa-23:4n-6, unlike natural PUFAs, lacked ability to stimulate oxygen radical production in neutrophils but caused marked inhibition of agonist-induced upregulation of leukocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC) and E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression. In addition, β-oxa-23:4n-6 inhibited acute and chronic inflammatory responses in mice as well as the upregulation of adhesion molecule expression in arterial endothelium. This action of β-oxa-23:4n-6 required a functional 12- but not 5-lipoxygenase or cyclooxygenases, consistent with its metabolism via the 12-lipoxygenase pathway. Whereas β-oxa-23:4n-6 did not affect the activation of mitogen-activated protein kinases by tumor necrosis factor, activation of the I&kgr;B kinase/nuclear factor &kgr;B pathway was selectively inhibited. These novel PUFAs could form the basis for a potential new class of pharmaceuticals for treating inflammatory diseases, including atherosclerosis.

Inhibition of Neutrophil Leukotriene B 4 Production by a Novel Synthetic N -3 Polyunsaturated Fatty Acid Analogue, β-Oxa 21:3 n -3

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.

Inhibitory effects of arachidonic acid (20:4,n-6) and its monohydroperoxy- and hydroxy-metabolites on procoagulant activity in endothelial cells

The procoagulant response of endothelium to pathophysiological agents such as tumour necrosis factor alpha (TNF alpha) and phorbol myristate acetate (PMA) alters the expression of proteins such as tissue factor. The modulation of such procoagulant activity (PCA) by the polyunsaturated fatty acid arachidonic acid (20:4,n-6) and its 15-hydroperoxy (15-HPETE) and 15-hydroxy (15-HETE) metabolites was examined since this may have important implications in cardiovascular disease and atherosclerosis. Treatment of human umbilical vein endothelial cells (HUVEC) for 30 min with 20:4, 15-HPETE or 15-HETE before induction of PCA with TNF alpha (100 U) or PMA (10(-7) M) caused a significant inhibition of PCA. This inhibition was seen at 2-5 microM fatty acids. Dose response curves with TNF alpha indicated that the inhibition was greatest at higher concentrations of TNF alpha (> or = 250U TNF alpha/ml). The mode of administration of the fatty acid was not critical as fatty acids presented as DPC-fatty acid micelles or solubilised in ethanol gave similar inhibitions of PCA. 20:4, 15-HPETE or 15-HETE did not alter the binding of I125-labelled TNF alpha to its surface receptors on HUVEC, suggesting that the effect of these fatty acids was not mediated by events at the cell surface receptor level. In support of this, these fatty acids were found to inhibit PCA induced by PMA which bypasses cell surface receptors to activate protein kinase C directly.(ABSTRACT TRUNCATED AT 250 WORDS)

SYNTHESIS OF HYDROPEROXIDE AND PERKETAL DERIVATIVES OF POLYUNSATURATED FATTY ACIDS AS POTENTIAL ANTIMALARIAL AGENTS

Hydroperoxide derivatives of beta-oxa-substituted polyunsaturated fatty acids were prepared by 15-lipoxygenase catalysed oxidation and perketal derivatives of fatty acid hydroperoxides were synthesized. The perketals are more stable than their parent fatty acid hydroperoxides, but less active as antimalarial agents in the in vitro growth inhibition of Plasmodium falciparum

Synthesis of polyunsaturated β-thia and γ-thia fatty acids from naturally derived polyunsaturated fatty alcohols and in vitro evaluation of their susceptibility to β-oxidation

Abstract A range of polyunsaturated β- and γ-thia fatty acids are conveniently prepared in two steps from naturally derived polyunsaturated fatty alcohols. In vitro β-oxidation studies using acyl Co-A oxidase from Arthrobacter species indicate that (all-Z)-(eicosa-5,8,11,14-tetraenyloxy)acetic acid, synthesised as reported earlier, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid and (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid are not susceptible to oxidation, whereas 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid is oxidised with V0 appproximately 40% of that of arachidonic acid. In competitive experiments, none of these novel polyunsaturated fatty acids effect the β-oxidation of arachidonic acid.

Effects of beta-oxa and beta-thia polyunsaturated fatty acids on agonist-induced increase in endothelial cell adhesion molecules

Fish oils contain an abundance of n-3 fatty acids derived from linolenic acid (18:3n-3), in particular eicosapentaenoic (20:5n-3) and docosahexaenoic acids (22:6n-3). Both of these fatty acids are generally found in low concentrations in nonmarine animals although 22:6n-3 is found in high concentrations in certain specialized tissues such as retina and spermatozoa. There is increasing evidence that the beneficial effects of fish diets are related to the presence of n-3 fatty acids. Thus the ingestion of these fatty acids is thought to be responsible for the lower incidence of cardiovascular disease in certain human populations (1). It has been suggested that they may exert their antiinflammatory effects by inhibiting the 5lipoxygenase pathway in neutrophils and monocytes. However, some fatty acids, notably cis monounsaturated (e.g., oleic acid) and n-6 fatty acids (arachidonic acid), stimulate superoxide production in these cells and could therefore be expected to increase the inflammatory response and cardiovascular homeostasis, resulting in myocardial injury. Recent findings from our laboratory showed that, unlike the n-3 and n-6 polyunsaturated fatty acids (PUFA), the 15-hydroperoxy derivatives such as HPETE were much more active than the parent fatty acids in depressing tumor necrosis factor (TNF) c~-induced increase in the expression of adhesion molecules in endothelial cells (2). This is interesting and of potential therapeutic importance in the treatment of intravascular inflammation since HPETE lacked leukocyte-activating properties, i.e., ability to stimulate neutrophils and macrophages to adhere to the endothelium, release oxygen radicals, and release tysosomal enzymes (3). As such these are devoid of some of the proinflammatory activities of the free fatty acids such as 20:4n-6, 20:5n-3, and 22:6n-3. However the instability of HPETE would preclude its use in any form of therapy. We have now been able to achieve the synthesis of some compounds (4,5) which have the advantage in that they are