Peter Demin

Hepoxilins: A review on their enzymatic formation, metabolism and chemical synthesis

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.

Hepoxilin signaling in intact human neutrophils: biphasic elevation of intracellular calcium by unesterified hepoxilin A3

We have previously shown that the methyl ester of hepoxilin A3 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 A3 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 A3 baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl=188±14 S.D., free acid=135±11 S.D. nM, P<0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl=88±8 S.D., free acid=107±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 calcium‐free 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±12; free acid, 145±6 S.D. nM, P<0.005). Additional experiments with tritium‐labelled methyl ester of hepoxilin A3 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.

Synthesis of racemic 11,12-cyclopropyl analogs of hepoxilins A3 and B3

Abstract Stable biologically active racemic 11,12-cyclopropyl analogs of hepoxilins A 3 and B 3 were prepared via polyacetylenic intermediates.

Hepoxilins raise circulating insulin levels in vivo.

We have demonstrated over a decade ago that hepoxilins cause the release of insulin from isolated pancreatic islets of Langerhans in vitro. However, no studies are available so far to indicate whether these compounds are active in vivo. The present study is the first to our knowledge which demonstrates that hepoxilins administered intra‐arterially in the anaesthetized rat cause the release of insulin in the circulation. This release is dependent on the glucose status of the rat. Hence, animals fasted overnight do not respond to hepoxilin administration, while animals that have had free access to food respond to hepoxilins with a rise in insulin concentrations in blood. The hepoxilin effect is rapid and varies with different hepoxilins, the most potent of which is hepoxilin A3 (HxA3) (both the 8S and the 8R enantiomers). Administration of 100 μg HxA3 produces a rise in blood insulin equivalent to that caused by the administration of 5 mg glucose. In view of earlier evidence showing that these compounds cause a rise in intracellular calcium levels in vitro at a <1 μg/ml concentration through a receptor‐mediated mechanism, we speculate that the actions of hepoxilins in causing the release of insulin from the pancreas may be due to alterations in calcium levels within the β‐cell. We believe that hepoxilins may represent new lead compounds as therapeutics in type II diabetes mellitus.

FORMATION OF 14,15-HEPOXILINS OF THE A3 AND B3 SERIES THROUGH A 15-LIPOXYGENASE AND HYDROPEROXIDE ISOMERASE PRESENT IN GARLIC ROOTS

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the “A”-type and the “B”-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S),15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5–10-fold less active than 11(S),12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.

Chemical synthesis and actions of 11,12-thiirano-hepoxilin A3.

A novel analog of hepoxilin A3 has been chemically synthesized in which the 11,12-epoxide group has been altered to a thiirano group. This has been accomplished through allylic rearrangement of unnatural (11 R, 12 R)-hepoxilin B3 under Mitsunobu conditions, first into unnatural (11 R, 12 R)-hepoxilin A3, followed by conversion of this compound with inversion of the epoxide centers into the thiirano-hepoxilin A3 having the natural 11 S, 12 S configuration. We also report herein evidence showing that thiirano-hepoxilin A3 raises intracellular calcium concentrations in intact human neutrophils.

Epimer-specific actions of hepoxilins A3 and B3 on PAF- and bradykinin-evoked vascular permeability in the rat skin in vivo.

The two epimers of hepoxilins A3 (8R and 8S) and B3 (l0R and l0S) were tested for effects on vascular permeability in the rat skin assay, a model for acute inflammation. Results showed epimer-specific effects, the R isomer being active, the S isomer being essentially inactive. While the hepoxilins on their own, at a dose of 20–40 ng/injection intradermally were essentially inactive, they potentiated the actions of bradykinin (both HxA3 and B3 were active) and PAF (only 10R was active) coinjected with the hepoxilins. These results suggest that hepoxilins may mediate (amplify) the actions of inflammatory mediators.

High-performance liquid chromatographic separation of fluorescent esters of hepoxilin enantiomers on a chiral stationary phase

Fluorescent anthryl (ADAM) derivatives of hepoxilins have been shown to possess good chromatographic properties affording good sensitivity for the high-performance liquid chromatographic analysis and detection of these compounds and related eicosanoids (12-hydroxyeicosatetraenoic acid) in biological samples. We report herein the separation of all possible stereoisomers of hepoxilins A3 and B3 as their methyl esters as well as their ADAM ester and acetate derivatives on a cellulose trisdimethyphenylcarbamate chiral stationary phase (Chiracel OD) in the normal-phase mode. This methodology is important to address the mechanistic route of biosynthesis of these products.

Hepoxilin analogs, potential new therapeutics in disease.

We have chemically synthesized several stable analogs of the naturally occurring hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

Stereoselective actions of hepoxilins A3 and B3 and their cyclopropane analogs (HxΔA3 and HxΔB3) on bradykinin and PAF-evoked potentiation of vascular leakage in rat skin

Native hepoxilins (Hx) A3 and B3 as well as their synthetic cyclopropane analogs, HxdeltaA3 and HxdeltaB3 are inactive on their own in causing changes in vascular permeability in rat skin measured by leakage of plasma-bound Evans Blue dye. Several of these compounds, however, were observed to potentiate the leakage of dye evoked by bradykinin (BK) and platelet-activating factor (PAF). The syn epimer of HxA3 was effective in potentiating dye leakage evoked by BK but not by PAF. The syn epimer of HxB3, on the other hand, was capable of potentiating both BK- and PAF-evoked plasma protein leakage. The anti epimer of both hepoxilins was inactive. In contrast, the anti epimer of the cyclopropane analog HxdeltaA3 potentiated only the BK-evoked changes, whereas the anti epimer of HxdeltaB3 potentiated only the PAF-evoked changes in dye leakage. The corresponding other epimer of each compound was inactive. Our findings indicate that the hepoxilin cyclopropane analogs appear to mimic the actions of the native compounds.