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This article reviews published evidence describing the enzymatic and nonenzymatic formation and the routes of metabolism of the hepoxilins. Also treated are the major approaches used for the chemical synthesis of these compounds and for some of their analogs.

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 degrees C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 degrees C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3 +/- 9.1 nM and 8.86 +/- 1.4 pmol/ml per 2 x 10(6) cells (+/- S.E.M.) respectively reflecting approx. 2.67 x 10(6) sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

Bleomycin has been suggested to incite plasma extravasation and influx of inflammatory cells leading to pulmonary fibrosis. We hypothesized that stable analogs of the 12-lipoxygenase product, hepoxilin, may attenuate these effects. We initially investigated the effects of the four hepoxilin analogs (PBT-1 to -4) coadministered intradermally with bleomycin and found that PBT-1 and -2 significantly opposed the vascular permeability effects of bleomycin in rat skin. We subsequently tested the hepoxilin analogs for their actions in opposing the intratracheal bleomycin-evoked acute inflammatory phase of lung fibrosis in the mouse, characterized by a marked accumulation of macrophages and an increase in the rate of collagen synthesis and deposition. We found that the bleomycin-evoked effects on macrophage influx were inhibited by all the hepoxilin analogs (PBT-1, -3, and -4 Ͼ PBT-2) administered i.p. for 8 days. Increased total lung collagen was completely abrogated by PBT-1 and -2, whereas PBT-3 and -4 had little effect. A doseresponse study with PBT-1 indicated that the effective dose for inhibition of bleomycin-induced inflammatory and histological changes was below 10 g/day. These studies demonstrate an in vivo action of stable analogs of hepoxilin and support an effect on inflammation and vascular permeability from these novel compounds, especially for PBT-1.Idiopathic pulmonary fibrosis is a devastating disorder that is poorly understood and resistant to treatment (Cooper, 2000). The observation that the antibiotic bleomycin sulfate (BL), a potent cancer chemotherapeutic agent (Adamson, 1984;Nici et al., 1998), may cause interstitial lung fibrosis in humans (Yagoda et al., 1972) led to the development of animal models in which a single dose of BL administered intratracheally induced changes resembling human idiopathic pulmonary fibrosis histopathologically (Kelley et al., 1980). The acute phase of this response is characterized by a marked accumulation of inflammatory cells and an increase in the rate of collagen synthesis and deposition (Cooper et al., 1988;Gurujeyalakshmi and Giri, 1995;Giri and Hollinger, 1996).Previous animal studies have demonstrated that BLevoked lung fibrosis is exacerbated with nordihydroguaiaretic acid, a lipoxygenase inhibitor, suggesting that a lipoxygenase product may be involved in endogenous mechanisms controlling lung fibrosis (Giri and Hollinger, 1996). The hepoxilins (HXs) may be candidates for the control of lung fibrosis as they are formed through the 12-lipoxygenase pathway of metabolism of arachidonic acid (Pace-Asciak et al., 1983;Pace-Asciak, 1984;Pace-Asciak and Martin, 1984). These compounds have previously been shown to have significant biological actions (Pace-Asciak, 1984;Dho et al., 1990;Laneuville et al., 1992;Pace-Asciak et al., 1995;Reynaud et al., 1996Reynaud et al., , 1999Sutherland et al., 2000). HXs raise free intracellular calcium in human neutrophils ex vivo through the release from stores and vascular tissue in vitro and block the intracellular calc...

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We have previously shown that the methyl ester of hepoxilin A Q causes a receptor-induced rise in intracellular calcium through the release from intracellular stores in suspended human neutrophils. The corresponding free acid was devoid of activity. We now report that the action of the free acid form of hepoxilin A Q is dependent on the type of vehicle used, i.e. it is active in releasing calcium when used in an ethanol vehicle but not in DMSO. The methyl ester is equally active in either vehicle. The pattern of calcium release between the free acid and the methyl ester is qualitatively different. Both compounds show a biphasic pattern, i.e. an initial rapid phase followed by a slow decline in calcium levels but never reaching pre-hepoxilin A Q baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl = 188 þ 14 S.D., free acid = 135 þ 11 S.D. nM, P 6 0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl = 88 þ 8 S.D., free acid = 107 þ 15 S.D. nM, not significant). Lanthanum chloride (an inhibitor of calcium influx) interfered with the second phase of the curve causing calcium levels to return to normal pre-hepoxilin levels for both compounds. Addition of lanthanum chloride prior to the hepoxilin addition or carrying out the experiments in calciumfree medium, eliminated the second phase completely, with the calcium peak returning rapidly to normal baseline levels, suggesting that the second phase is due to calcium influx. Again the methyl ester is more active than the free acid (methyl, 189 þ 12; free acid, 145 þ 6 S.D. nM, P 6 0.005). Additional experiments with tritium-labelled methyl ester of hepoxilin A Q demonstrated that the compound is hydrolyzed into the free acid intracellularly. These experiments demonstrate that DMSO interacts with hepoxilin free acid, interfering with its entry into the cell while ethanol does not. Once inside the cell, hepoxilin interacts with its own receptor to release calcium rapidly from stores, but it also causes a more prolonged influx of calcium from the extracellular milieu.z 1999 Federation of European Biochemical Societies.

We report herein a novel class of thromboxane receptor (TP receptor) antagonists modeled on unstable natural lipids that we identified several years ago, the hepoxilins. These antagonists have been rendered chemically and biologically more stable than the natural compounds through structural modification by chemical synthesis. We demonstrate that the analogs inhibit the aggregation of human platelets in vitro evoked by the thromboxane receptor agonists,-heptenoic acid) and U46619 (9,11-dideoxy-9␣,11␣-methanoepoxy-prosta-5Z,13E-dien-1-oic acid). The most potent of the analogs described, PBT-3 [10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester], has an IC 50 versus aggregation by I-BOP ϭ 0.6 ϫ 10 Ϫ7 M and versus U46619 ϭ 7 ϫ 10 Ϫ7 M, representing one of the most potent anti-aggregating substances so far described. PBT-3 also inhibits thromboxane formation and aggregation evoked by collagen with an IC 50 ϭ 8 ϫ 10 Ϫ7 M. Other PBT (hepoxilin cyclopropane) analogs so far tested were 5-to 10-fold less active, and the native hepoxilins were about 500-fold less active. Neither PBT-3 nor the other analogs inhibited 12-lipoxygenase, phospholipase A 2 , or cyclooxygenase 1 or 2, and weakly stimulated adenyl cyclase (threshold stimulation at 10 Ϫ7 M and little selectivity for each of the PBT compounds). TP antagonism by PBT-3 was further demonstrated in receptor binding studies through use of 125