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Pharmacogenetics is the use of genetic information to determine who will (or will not) respond to a given treatment. In order to have a pharmacogenetic basis, there should be variability in the treatment response. Although there are data on the variability of the treatment response to all classes of medications that are used in the treatment of asthma- i.e., glucocorticoids, teophyllin, β2 agonists and leukotriene modifiers- pharmacogenetic associations have been described only for β2 agonists and leukotriene modifiers [1].

The leukotrienes (cysLTs) are lipid mediators derived from the ubiquitous membrane component arachidonic acid. The plethora of biological activities suggested that they activate different receptors. A number of elegant genetic and biochemical studies have been performed to elucidate the nature of these receptors. Results from classical pharmacological and molecular studies indicate that there are two main classes of leukotriene receptors. One based on the biological activities of leukotriene B4 and related hydroxyacids, referred to as BLT receptors, and a second class identified by the cysteinyl- leukotrienes (cysLTs). Activation of the BLT receptors initially was shown to produce potent chemotactic activities on leukocytes whereas the latter class (CysLT receptors) stimulated smooth muscle as well as other cells. Furthermore, there is now sufficient information from classical pharmacological and biochemical assays as well as molecular investigations to divide both categories into receptor sub-classes, namely, BLT1 and BLT2 as well as CysLT1 and CysLT2 (Table 1).

One of the major reasons for pursuing the chemical structure of the biological material known as slow-reacting substance of anaphylaxis (SRS-A) was that this material was known to be a potent bronchoconstrictor substance in guinea pigs [1] and in isolated human airways [2]. Thirty years ago, the simple concept was that SRS-A was released from sensitized cells following antigen sensitization and challenge, and that the released material transduced a signal at an as yet to be identified receptor leading to smooth muscle constriction and therefore the manifestations of human asthmA. It has been almost 25 years since the elucidation of the structure of SRS-A as a mixture of the cysteinyl leukotrienes (LT) [3] and just over 5 years since agents that act on leukotriene pathway have been available as asthma treatments [4,5]. What have we learned about asthma from the use of these agents and what is the role of these agents in the treatment of asthma?

The compounds inhibiting the leukotrienes (LTs) biosynthesis and/or antagonizing their biological functions can be utilized in the antiastmatic therapy1,2. We synthesized3 the series of 2,4-dihydroxyacetophenone derivatives 1 and 2 bearing carboxyl and their antileukotrienic activities have been determined. The distances D between carboxyl and lipophilic part of molecule were calculated for energetically optimized conformations using CHEM-X, Windows 95 programme. The initial geometry with all trans torsions in connecting chain between aromatic nuclei was confirmed in a solid state by the X-ray analysis of two selected derivatives 1. Lipophilicity of compounds was measured by the use of partitioning tic and log P were calculated from log P – RM relationships. The analysis of quantitative relationships was performed using Statgraphic Programme vers. 4.2.

The three well recognized lipoxygenases in humans are best known for their occurrence in different types of blood cells (1). And these three enzymes appear to have distinct biological roles. The leukocyte 5S-lipoxygenase clearly functions in the initiation of a metabolic pathway; the leukotriene products have distinct cell receptors and mediate proin-flammatory activities. The platelet 12S lipoxygenase synthesizes 12S-HpETE, and following reduction by cellular peroxidase, the hydroperoxide is converted efficiently and almost exclusively to the hydroxy derivative, 12S-HETE. The 12S-HETE may be a signaling molecule involved in cell-cell communication. Overall, however, there is no strong consensus on the biological role of this highly specific 12S-lipoxygenase product.

The cysteinyl leukotrienes are powerful mediators of vaso- and bronchoconstriction, edema formation, and mucus secretion1. They appear to play a key role in asthma and may be involved in cardiac and renal disease as well1–8. The parent compound, leukotriene C4 (LTC4), is formed by the conjugation of leukotriene A4 with glutathione (GSH). Thus it may be expected that the metabolism of LTC4 resembles GSH conjugates formed with carcinogens, toxins, and xenobiotics9. Until recently, the only enzyme known to metabolize this class of compounds as well as GSH itself was γ-glutamyl transpeptidase (GGT)10–12. GSH conjugates including LTC4 are metabolized to their cysteinylglycine derivatives. In the case of LTC4 the resulting leukotriene is LTD4. This compound is the most potent of the cysteinyl leukotrienes and has been found to be a consistently more effective agonist than LTC4 and 10 to 100 times more effective than LTE4, a metabolite of LTD4 13–16. LTC4 is synthesized in the liver and is secreted into the bile15. It is also produced in peripheral tissues1,2,16. Conversion of LTC4 to LTD4 is not well understood although it is believed to occur extracellularly because GGT is an ectoenzyme10–17. We became interested in cysteinyl leukotriene metabolism when we developed mice deficient in GGT18. Our subsequent studies unexpectedly showed that GGT-deficient mice are competent to metabolize LTC4.

Glutathione S-transferases (GSTs) comprise a family of ubiquitous enzymes mainly responsible for xenobiotic metabolism, drug biotransformation, and protection against peroxidative damage via the catalysis of reduced glutathione (GSH) with hydrophobic electrophiles1. Predominantly cytosolic2, four known membrane-bound forms of GSTs have been documented, now part of a novel supergene family designated MAPEG (Membrane Associated Proteins in Eicosanoid and glutathione Metabolism)3. A member of this class of enzymes is leukotriene (LT) C4 synthase and unlike all other GSTs, has minimal activity for conventional GST substrates, and is committed to the biosynthesis of cysteinyl leukotrienes (LTC4, LTD4, LTE4) via the conjugation of GSH to LTA4 4.

This study was undertaken to correlate the follicular levels of interleukin (IL)-1 alpha, IL-2, tumor necrosis factor-alpha (TNF-alpha), and leukotriene (LT) B4 with oocyte maturity, fertilization, and achievement of pregnancy.The material was obtained from 22 women undergoing IVF, 8 of whom became pregnant and 14 of whom did not.All of the studied cytokines and LT B4 were found in follicular fluids, but there were no significant differences according to oocyte maturity, fertilization, embryo quality, and achievement of pregnancy. On the other hand, a significant positive correlation was found between IL-1 alpha and TNF-alpha, IL-1 alpha, and LT B4 as well as between TNF-alpha and LT B4 in follicular fluids with subsequently fertilized oocytes.It seems that IL-1 alpha, TNF-alpha and LT B4 may take part in the process of follicle wall degradation, and their follicular correlations may suggest more optimal follicular and oocyte development and maturation.

Sulfidopeptide leukotrienes (sLT) are arachidonic acid (AA) metabolites involved in the inflammatory response (Dahlen, 1981). LT production from the unstable metabolic intermediate LTA4 is cell-type dependent, as polymorphonuclear leukocytes (PMNL) generate predominantly LTB4 while LTC4 is the main product synthesized by eosinophils and mast cells; in addition, cooperation between PMNL (donor cells for LTA4) and endothelial cells (EC, acceptor cells) leads to formation of LTC4 by “transcellular biosynthesis” (Feinmark, 1986; Maclouf, 1989).

Some Recent Advances in Leukotriene Research B. Samuelsson. Practical Aspects of Prostaglandin E1 before and after Solid Organ Transplantation H. Muller et al. Prostaglandins in Liver Transplantation R.M. Merion. Prostaglandins in Heart Transplantation F. Iberer et al. Eicosanoids in Peridontal Diseases: Potential for Systemic Involvement S.M. Damareet al. Mitogen-Activated Protein Kinases and Endothelial Prostacyclin Secretion J.H. Grose et al. Crosstalk between Elevation of [Ca2+I, Reactive Oxygen Species Generation and Phospholipase A2 Stimulation in Human Keratinocyte Cell Line R. Goldman et al. Cloning Tissue-Specific Expression and Regulation of the Bovine Thromboxane A2 Receptor S. Muck, K. Schror. Weak Inhibitors of Cyclooxygenases May Exert their Antinociceptive Effect by Modulation of Transcription Factors N. Scheuren et al. New Group of Lipid Mediators Containing )-Hydroxyarachidonic Acid (20-HETE) K. Mao et al. Biosynthesis of Novel Divinyl Ether Oxylipins by Enzyme from Garlic (Allium Sativum L.) Bulbs A.N. Grechkin, M. Hamberg. Catalytic and Spectroscopic Properties of Linoleate Diol Synthase of the Fungus Gaumannomyces Graminis E.H. Oliw, C. Su. 87 Additional Articles. Index.

In recent years a number of leukotriene modulators have shown promise in the treatment of asthma.1, 2 Most of these have been Cys-LT1 antagonists.2 Inhibitors of both cysteinyl leukotrienes and LTB4 however are likely to be more effective given the pro-inflammatory characteristics of LTB4. In addition, these inhibitors would also inhibit the formation of the mediators formed from double lipoxygenation such as lipoxins.3 Thus far, most bioavailable inhibitors of 5-lipoxygenase have been related to zileuton or ZD-2138.4 An alternative to 5-lipoxygenase inhibition is the antagonism of the 5-lipoxygenase activating protein (FLAP).5 Three compounds with this mechanism have reached the clinic but all three have apparently been withdrawn.4 We have synthesized a number of leukotriene inhibitors that are not direct inhibitors of 5-lipoxygenase with optimized pharmacokinetic and pharmacological characteristics. The best of these is A-93178.

Leukotriene (LT) A4 hydrolase catalyses the final step in the biosynthesis of the lipid mediator LTB4 (5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), which is a potent leukocyte chemotactic and aggregating agent. In the biosynthesis of leukotrienes arachidonic acid is converted by 5-lipoxygenase into the unstable epoxide intermediate LTA4 (5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatctraenoic acid), the common precursor for all leukotrienes1.

Leukotriene (LT) A4 hydrolase catalyzes hydrolysis of LTA4 (5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) into the proinflammatory compound LTB4 (5(S), 12 (R)-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid, in a reaction without any cofactor requirement. The substrate LTA4 is in turn derived from arachidonic acid by two consecutive reactions catalyzed by 5-lipoxygenase1.

The leukotrienes are formed by transformation of arachidonic acid into an unstable epoxide intermediate, leukotriene A4 (LTA4), which can be converted enzymatically by hydration to LTB4, and by addition of glutathione to LTC4. This last compound is metabolized to LTD4 and LTE4 by successive elimination of a γ-glutanyl residue and glycine1, 2.

Platelet-activating factor (PAF) has been suggested to be an important chemical mediator in bronchial asthma1). When given by inhalation, PAF produces short-term bronchoconstriction in both normal2) and asthmatic subjects2,3), increases airway reactivity in normal volunteers4,5), stimulates leukotriene and thromboxane A2 production in asthmatics6), and also worsens gas exchange in mild asthma7). In aminal models, it has been reported that exogenous PAF causes bronchoconstriction, microvascular leakage, mucous hypersecretion, an increase in bronchial responsiveness, and eosinophil recruitment into the airways of guinea pigs1). Thus, PAF induces various pathophysiologic changes relevant to bronchial asthma.

The identities and roles of arachidonic acid (AA) metabolites in human diseases are often elusive. For example, allergens cause tissues to secrete agents that attract eosinophils (Eo) and induce these cells to release granule enzymes, Superoxide anion, and bioactive molecules that lead to the organ dysfunctions seen in allergy1. Zileuten, an anti-lipoxygenase drug, relaxes the airways of asthmatics, perhaps by blocking the synthesis of an Eo-targeting eicosanoid2-4. The drug clearly inhibits allergen-induced Eo fluxes to lung as well as bronchospasm in primates and lower mammals5,6. Thus, an eicosatetraenoate (ETE) may link Eo to allergy. Among the eicosanoids occupying human allergen-reactive sites, 15(S)-hydroxy-ETE (15-HETE), prostaglandins, and thromboxanes are not chemotactic for Eo; leukotriene (LT)B4 attracts Eo but is more active on polymorphonuclear neutrophils (PMN); and peptido-LTs, lipoxins, and 5(S)-hydroxy-ETE (5-HETE) act weakly on both cell types1,7-10. These agents are unlikely to mediate Eo-based lesions in vivo. However, a newly defined 5-HETE analog, 5-oxoETE, has in vitro actions suggesting that it could be such a mediator.

In line with the original report on allergen-induced release of slow reacting substance of anaphylaxis (SRS-A) in human lung tissue [1], it was soon after the discovery of leukotrienes (LT) [reviewed in 2] possible to establish that allergen provocation of lung tissue from asthmatics indeed promoted release of the three leukotriene constituents of SRS-A, namely the cysteinyl-containing LTC4, LTD4 and LTE4 [3]. Furthermore, the contraction response to allergen challenge of bronchi from atopics was substantially inhibited when leukotriene release was blocked [3], supporting the hypothesis [4] that SRS-A was an important mediator of allergen-induced bronchoconstriction in man.

Leukotriene (LT)A4 is formed from arachidonic acid in two consecutive reactions, both catalyzed by 5-lipoxygenase (1). This unstable epoxide intermediate is hydrolyzed into the proinflamrnatory compound LTB4, by the enzyme LTA4 hydrolase (review in 2). From sequence comparison with certain zinc containing aminopeptidases and proteases, typified by thermolysin, a potential zinc binding site was found in the sequence of LTA4 hydrolase (3, 4). This finding prompted us to investigate the possible zinc content and peptidase activity of LTA4 hydrolase.

The cytokine interleukin-8 (IL-8) has direct effects on neutrophils such as increasing the expression of CD1lb adhesion molecules or chemotactic peptide receptors and also enhances agonist-mediated responses such as the generation of superoxide. The metabolism of endogenous 3H-arachidonate can be estimated by the release of radioactive metabolites from the cell. Incubation of purified neutrophils with IL-8 (10-500 ng/ml) for up to 30 min. did not directly stimulate this activity, however IL-8 at 10 and 100 ng/ml enhanced the activity of cells stimulated with calcium ionophore (1 µM) by 126 ± 16% (n = 5) and 144 ± 11% (n = 12), respectively. To examine the role of leukotriene metabolites such as 5-HETE or LTB4 in the signal transduction pathways of IL-8, we employed two lipoxygenase inhibitors, piriprost (1) at 87 µM and MK886 (2) at 100 nM, concentrations that inhibited the release of radioactivity from ionophore-stimulated cells by 94 ± 1% (n = 5) and 89 ± 2% (n = 11), respectively. The inhibitory activity of these compounds on leukotriene synthesis under identical conditions was confirmed by reverse phase HPLC. MK886 inhibited the release of leukotrienes from IL-8-primed cells stimulated with ionophore by 96 ± 1% (n = 4), but did not inhibit the upregulation of CD 1 lb stimulated by IL-8, nor did it inhibit the priming of fMLP-stimulated superoxide production by IL-8. Similar data were obtained with piriprost. We conclude therefore that leukotriene synthesis does not mediate the direct activation of phagocytes by IL-8, nor the priming of the fMLPstimulated respiratory burst.

Products of the arachidonic acid cascade have been implied to play a role in growth and in the clinical manifestations of malignant brain tumors. These indications are based on the findings of elevated levels of prostaglandins and leukotrienes in operative specimen (5, 2, 9) and on the observation of antitumoral effects of prostaglandin and leukotriene inhibitors in experimental models (11). Besides the potentially modulatory effects on growth, the common findings of peritumoral edema, in many cases the direct cause of death in brain tumor patients, might be related to release of arachidonic acid metabolites. Thus, intracranial infusion of leukotrienes do induce edema (3), and the amount of peritumoral edema was observed to relate to amounts of leukotrienes in the tumor tissue (2). Finally, the first hand treatment of brain tumors is the administration of corticosteroids. This is necessary for the control of the brain edema and could potentially mediate its effects by inhibiting phospholipase A2 and thereby inhibit the release of arachidonic acid.

The expression of the 5-lipoxygenase and the 5-lipoxygenase activating protein (FLAP) genes in human tonsillar B cells and in a lymphoblastoid B cell line was demonstrated at the transcriptional level by the polymerase chain reaction (PCR) technique. Intact B cells produced very low amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid in comparison to those formed by sonicates of these cells. Incubation of intact lymphoblastoid B cells with the glutathione depleting agent, azodicarboxylic acid bis[dimethylamidel] (diamide), prior to the addition of the calcium ionophore A23187 and arachidonic acid, led to the formation of similar amounts of LTB4 as those produced by sonicated cells. These results indicate that the glutathione status is of importance for the activity of 5-lipoxygenase in B lymphocytes.

Gallstone disease is a significant health problem; an estimated 15 million adults are affected by this biliary tract disease in North America. Cholesterol gallstone formation is accompanied by impaired GB motility. A later stage is cholecystitis- an inflammatory event which may increase smooth muscle tone, resulting in pain1. In animal models of cholecystitis, significant increases in the levels of arachidonic acid metabolites in GB perfusates has been demonstrated2. These metabolites, comprised of prostanoids (PS) and leukotrienes (LT), have traditionally been associated with many inflammatory processes3. The role of PS in the etiology of cholecystitis and gallstone formation has been well defined. However, to date, little is known regarding the role assumed by the LT’s in these disease states.

Harkavy1 was the first to report that an alcohol-soluble extract of sputum from allergic asthmatic patients contained an agent which provoked spasms of cat and rabbit intestines in vitro. Few years later, von Euler2 and Goldblatt3 showed that extracts from human prostate gland and seminal vesicles decreased the blood pressure and stimulated the smooth muscles of the uterus. The compounds were designated as prostaglandins2. A new area of research on arachidonic acid metabolism started by the characterization of prostaglandins E and F by Bergstrom and Sjovall4,5. The final structures of PGE1 PGF1α and PGF1β were elucidated later6. The thromboxanes were first described as the rabbit aorta contracting substance (RCS) by Piper and Vane7. Hamberg et al.8 showed that RCS corresponded to the unstable thromboxane A2. Another line of investigations focused on the nature of substances causing a slowly developing and long-lasting contraction of guinea pig jejunum in vitro9. This substance called slow reacting substance (SRS) was shown to be released during the antigen-antibody reaction in guinea pig anaphylaxis and later designated as slow reacting substances of anaphylaxis (SRS-A). SRS-A were purified and characterized for the first time by Morris et al.10. The first product of the SRS-A has been identified as leukotriene C by Murphy et al.11 and Morris et al.12. The two other components of SRS-A were demonstrated to be the leukotriene D4 (LTD4) formed through cleavage of the γ-glutamyl moiety of the gluthathione side chain of LTC4 13,15 and the leukotriene E4 (LTE4) formed through the cleavage of the glycine residue from the peptide chain of LTD4 14,16. Leukotriene B4 (LTB4) was originally isolated by Borgeat and Samuelsson, in incubates of rabbit PMNL17 or human PMNL18 stimulated with arachidonic acid and calcium ionophore.

The enzymatic steps involved in the synthesis of prostaglandins, thromboxanes, and leukotrienes have been elucidated in substantial detail in numerous biochemical studies (1,2). In most cases, these studies involved the addition of radiolabeled arachidonic acid to homogenous cell suspensions with characterization of the resultant radiolabeled metabolites. Thus, most of the studies during the 1960s and 1970s were designed to establish those oxidative pathways of arachidonic acid within a single cell type. As a result, the metabolic patterns of eicosanoids generated by a given cell type has been fairly well established, for example, those cells which are found in the blood have been studied in great detail and their primary cyclooxygenase and lipoxygenase metabolites are known (Table 1). While most cells in the blood do metabolize arachidonic acid by one or more cascades, there are some cells which do not participate in the oxidative conversion of arachidonic acid into reactive intermediates. Table 1 indicates that the red blood cell and the lymphocyte (3) cannot metabolize arachidonic acid by either the cyclooxygenase or lipoxygenase pathways. Normally, one considers most cells in the blood to have a Table 1 Arachidonic Acid Metabolites Generated by Stimulation of Isolated Blood Cells Cells Eicosanoid Oxidase Intermediate Secondary Enzyme Platelet TxB2 C.O. PGH2 Thromboxane Synthase HHT C.O. PGH2 Thromboxane Synthase 12-HETE 12-LO 12-HPETE --- Polymorphonuclear Leukocyte LTB4 5-LO LTA4 LTA4-Hydrolase Eosinophil LTC4 5-LO LTA4 LTC4-Synthase 5-HETE 15-LO 15-HPETE --- Basophil LTC4 5-LO LTA4 LTC4-Synthase Monocyte Macrophage TxA2 C.O. PGH2 Thromboxane Synthase LTB4 5-LO LTA4 LTA4-Hydrolase LTC4 5-LO LTA4 LTC4-Synthase Lymphocyte None None --- --- RBC None None --- --- substantial capacity for the production of active eicosanoids. For example, platelets are known to produce thromboxane A2, a pro-aggregatory substance in response to stimulation and that this eicosanoid plays an important role in thrombus formation (4). The neutrophil on the other hand appears only to have a 5-lipoxygenase pathway for metabolism of arachidonic acid and stimulation of this cell leads to the formation of LTB4, a chemotactic factor (5). The eosinophil and basophil also metabolize arachidonate by the 5-lipoxygenase pathway and convert the reactive LTA4 into the sulfidopeptide leukotriene LTC4, the potent smooth muscle contracting agent and substance which enhances capillary permeability (6). The studies in this manner have greatly expanded our understanding of the complexity of arachidonic acid metabolism and the role which the eicosanoid metabolites may play in health and disease. However, it appears that the synthesis of eicosanoids may be more complex than only occurring within one cell and may involve cell-cell interactions.

Recently, we reported formation of lipoxins from endogenous substrate, via platelet-dependent lipoxygenation of granulocyte-derived leukotriene (LT)A4 (1). Furthermore, human platelet suspensions transformed synthetic LTA4 to lipoxins. The platelets also efficiently converted LTA4 to cysteinyl-containing leukotrienes (2).

Leukotriene (LT) A4 hydrolase converts the unstable epoxide intermediate LTA4 into the potent proinflammatory compound LTB4. The formation of LTA4 is catalyzed by the enzyme 5-lipoxygenase and involves the dioxygenation of arachidonic acid with subsequent epoxide formation (1).

The lipoxins are a series of biologically active eicosanoids which contain a conjugated tetraene structure as a characteristic feature (1). The two main compounds which carry biological activities are positional isomers: one designated lipoxin A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) and the other lipoxin B4 (5S,14R,15S, trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid). Multiple pathways have been documented in vitro which can lead to the formation of lipoxins (1–4). It appears that the biosynthetic pathways utilized are species-, cell type- and substratespecific. One route, documented with results from both isotopic oxygen studies as well as the identification of alcohol trapping products, involves the transformation of 15-HETE to a 5(6)epoxytetraene by leukocytes (1). When this intermediate (15S-hydroxy-5,6-epoxy-7,9,13-trans-11-ciseicosatetraenoic acid) was synthesized and incubated with purified cytosolic epoxide hydrolase, it was quantitatively converted into LXA4 (5). During the formation of lipoxins from exogenous 15-HETE by human neutrophils (an event which may occur in cell-cell interactions), an inverse relationship is observed between leukotriene and lipoxin production (6). Thus, the generation of epoxide-containing intermediates by human PMN appears to play a pivotal role in the biosynthesis of both leukotrienes and lipoxins.

Leukotrienes D4 (1a) and E4 (1b) are potent contractile substances which have been implicated in the ethiology of human asthma. These leukotrienes exert their effects through interaction with specific receptors on pulmonary smooth muscle and other tissues and thus the design of specific antagonists of these receptors offers the potential for the discovery of novel anti-asthma drugs. We have undertaken to characterize and map the LTD4 receptor via a variety of means. These include (a) analysis of structure-activity of the leukotrienes and of known LTD4 antagonists; (b) physical characterization of the receptor using radioactive labelled agonists and antagonists and (c) NMR and molecular modelling conformational analysis studies on leukotriene agonists and antagonists. The initial studies allowed us to propose a hypothetical model of the LTD4 receptor which was instrumental in the design of MK-571 (2), a LTD4 receptor antagonist which has undergone clinical testing. We have subsequently attempted to further refine this model using MK-571 and novel synthetic agonists at the LTD4 receptor as tools for such studies.

Polymorphonuclear leukocytes (PMNL) are known to play a major role in the inflammatory process in part through their ability to produce and respond to chemotactic factors. Leukotriene (LT) B4, a metabolite of arachidonic acid derived from the 5-lipoxygenase pathway, is produced by phagocytes and has potent chemotactic and chemokinetic effects on these cells1. PMNL stimulated with the ionophore A23187 synthesize large amounts of LTB4 2, while receptor-mediated activation of PMNL and monocytes-macrophages by agonists such as the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP), the complement fragment C5a, platelet-activating factor (paf-acether) or by phagocytosis also leads to LTB4 synthesis3–7. However LTB4 synthesis induced by natural agonists is of lower magnitude as it is often not detectable by HPLC procedures8. Evidence has accumulated during the last decade, supporting that platelet/leukocyte interactions occur in several pathophysiological situations9, and in particular that platelets might modulate inflammation. Indeed, activated platelets release arachidonic acid metabolites, pafacether, platelet-derived growth factor (PDGF), platelet factor 4 (PF4), serotonin and adenine nucleotides which could affect PMNL functions such as migration, degranulation, adherence and production of superoxide anion10–13.

The leukotrienes are established mediators of inflammation and anaphylaxis (1,2) but have also been indicated to modulate e.g. lymphocyte function (3,4) and influence hematopoietic progenitor replication (5,6). Furthermore, a growth factor for these progenitor cells (GM-CSF) has been reported to stimulate the production of LTC4 in human eosinophils (7). The purpose of the present investigation was to compare the leukotriene producing capacity of peripheral blood leukocytes from patients with CML to that of leukocytes from patients with another myeloproliferative disorder (polycythemia vera), non-malignant leukocytosis patients, and healthy donors. The results indicate an increased LTC4 formation in CML, but not in PV or non-malignant inflammatory disease (8).

For approximately 50 years the mediator termed slow reacting substance of anaphylaxis (SRS-A) was suspected to play an important role in human allergic reactions, prolonged bronchoconstriction and asthma yet its chemical structure remained elusive (1, 2). Details concerning the biosynthetic origin of this molecule as well as the regulatory mechanisms involved in controlling production and degradation of SRS-A were unknown. In 1979, the structure of SRS-A was elucidated (3) as a family of three novel compounds, having both a lipid portion derived from arachidonic acid and a peptide portion derived from glutathione (4). These molecules are now termed sulfidopeptide leukotrienes (leukotriene C4, D4, E4), which differ in the number of amino acid residues resident in the peptide portion as either gamma-glutamylcysteinylglycine, cysteinylglycine, or cysteine respectively. During the past decade, a great deal of information has been obtained describing the biosynthesis of these molecules, the activation of phospholipase A2 in liberating free arachidonic acid esterified to storage phospholipids (5), the importance of 5-lipoxygenase in generating the reactive intermediate leukotriene A4 (6) and LTC4 synthase which catalyzes the condensation of glutathione with leukotriene A4 yielding LTC4 (7). Furthermore, it is now recognized that sulfidopeptide leukotrienes can be synthesized in a variety of cells including mast cells (8), eosinophils (9), macrophages (10), and basophils (11). Interest in these molecules continues because of the potent biological activities which they possess including bronchoconstriction (12), vasoconstriction (13), and increased vascular permeability (14). Metabolism of LTC4 is known to take place rapidly and includes sequential peptide cleavage reactions (leading to the sulfidopeptide leukotriene described above) as well as ω- and β-oxidation with ultimate elimination of metabolites into the urine (15).

Leukotriene (LT)A4, the unstable intracellular intermediate in leukotriene biosynthesis, may be released to the extracellular space by activated leukocytes (1). As a consequence, the metabolism of LTA4 is not restricted to cells with 5-lipoxygenase activity, but can also be exerted by other cell types equipped with LTA4-metabolizing enzymes. Thus, LTA4, released by 5-lipoxygenase expressing cells, may be converted to LTB4 by surrounding erythrocytes (2), endothelial cells (3) or lymphocytes (4), all possessing LTA4 hydrolase activity. Similarly, mast cells (1), endothelial cells (3, 5) and smooth muscle cells (6) have been demonstrated to convert LTA4 to cysteinyl-containing leukotrienes. The present chapter describes some of our recent data regarding the metabolism of synthetic or granulocyte-derived LTA4 in human platelets and respiratory tissue leading to formation of cysteinyl-containing leukotrienes and lipoxins (LX).

The 5-lipoxygenase from leukocytes catalyzes the oxidation of arachidonic acid to 5hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 (LTA4) as the first two steps of the leukotriene biosynthesis pathway. The reaction catalyzed by 5-lipoxygenase is similar to that of other mammalian and plant lipoxygenases, showing activation by the hydroperoxide product, kinetic lag phases and turnover-dependent inactivation, but with additional requirements for ATP, Ca2+1 and for a leukocyte protein (FLAP) presumably involved in the translocation of the enzyme to the membrane during cellular leukotriene production2.

Leukotrienes are a family of compounds originally termed the slow-reacting substances of anaphylaxis and are recognized as important mediators of anaphylaxis (1). In particular, leukotriene C4 (LTC4 ) and leukotriene D4 (LTD4) are potent spasmogens in a variety of smooth muscle tissues including trachea, lung, ileum and the vasculature (2.4). The mechanism by which contraction is induced by the leukotrienes (LTs) is not yet known. However, in some experimental systems, but not all, a cyclooxygenase product of arachidonic acid metabolism, possibly thromboxane B2 (TxB2 ), may be involved in mediating leukotrieneinduced effects (3,4). This is based on the observation that inhibitors of cyclooxygenase, such as indomethacin and meclofenamic acid, can block leukotriene-induced contraction of the lung parenchyma, vasculature and the ileum (2.4). In other tissues, most notably guinea pig trachea, the cyclooxygenase products appear to be relatively unimportant mediators of leukotriene effects (5). Furthermore, in the tissues in which cyclooxygenase products appear to be important, thromboxane synthesis may be crucial for the leukotriene responses (4). Although TxB2 is usually assumed to be of platelet origin, the cellular source of LT-induced thromboxane synthesis is not known.

The oxygenated derivatives of arachidonic acid that are biologically active are defined as eicosanoids (Needleman et al., 1986). Among the eicosanoids are prostaglandins, including prostacyclin, thromboxanes, leukotrienes, and various hydroxy acids (Fig. 1). The eicosanoid precursor, arachidonic acid, is esterified in the 2-position of several phospholipids, and it must be hydrolyzed before the eicosanoids can be synthesized (Fig. 1). The liberated arachidonic acid can be enzymatically oxygenated by a membrane-bound cyclooxygenase, a microsomal cytochrome p450 or a cytosolic lipoxygenase with the formation of unstable intermediate products (Fig.1). These intermediate products include endoperoxides for prostaglandin production and epoxides and hydroperoxides for leukotriene and hydroxy acid formation (Fig 1). The type of arachidonate oxygenation is characteristic of the enzymes that each cell contains.

Until recently there has been very little known about the ultimate metabolitc fate of the sulfidopeptide leukotrienes. A basic principle of the pharmacology of mediator substances requires that mechanisms exist for biological inactivation of these very potent molecules. One such mechanism might be simple uptake of leukotrienes by the cells which make or respond to them. Studies showing that LTC4, produced in the isolated perfused lung in response to the calcium ionophore A23187, was largely retained by the lung over a span of 10 minutes, and that very little conversion to LTD4/LTE4 took place, suggesting the possibility that a reuptake mechanism for LTC4 in the lung existed (1). Other eicosanoids (LTB4, 6-keto-PGF1, thromboxane B2 and PGE2) were largely released into the perfusate. It is also possible that LTC4 was retained through binding to tissue proteins with high affinity and not taken up into cells. Since direct experiments have now shown that leukotrienes are not stored in cells from which they are released, and, while the retention of LTC4 by the isolated perfused rat lung has not been fully explained, simple reuptake of LTC4 in the lung seems unlikely. At present it is thought that metabolic biotransformation is the primary mechanism of inactivation of the leukotrienes.

Leukotrienes (LTs) are the most important vasoactive eicosanoids formed by the lipoxygenase pathway of arachidonic acid metabolism. The leukotrienes constitute an exciting chapter in modern pathophysiological research. They have been studied for over half a century but were only chemically identified in 1979 by Samuelsson and colleagues (1). Leukotriene A4 is the parent compound of the leukotriene pathway. Leukotriene A4 acts as a precursor for the synthesis of LTB4, the potent chemotactic eicosanoid by one branch of the pathway, and forms LTC4, the initial peptide leukotriene by another branch of the pathway (2). LTC4 is converted to LTD4. which in turn is converted to LTE4 All three are peptide leukotrienes and have important biological activity. Collectively LTC4, LTD4 and LTE4 are known as the “slow reacting substance of anaphylaxis” or SRS-A (3).

Various arachidonic acid metabolites are released from the lungs during hypersensitivity reactions such as anaphylaxis or asthma. Thromboxane A2 and leukotrienes were shown to play a key role in the bronchoconstriction associated with these conditions. In our experiments, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and Enzyme Immunoassay (EIA) techniques were use (a) to study the profile of cyclooxygenase and lipoxygenase products released during guinea pig anaphylaxis, (b) to characterize their time-course of release, and (c) to investigate the complex interactions regulating their synthesis. The lungs of guinea pigs sensitized to ovalbumin (100 mg intraperitonealy and 100 mg subcutaneously) were perfused with Krebs solution. Anaphylaxis was induced by specific challenge (ovalbumin, 100 ug/ml) and the effluent was collected at 1 min intervals for the measure of prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotrienes B4 (LTB4) and D4 (LTD4). In a set of experiments, the 12-hydroxyheptadecatrienoic acid (HHT) and 12-keto heptadecatrienoic acid (12-keto-HT) were analysed. Our results showed that the time-course of release of all the arachidonic acid metabolites were maximal approximately 5–6 min following the onset of the challenge. Leukotriene D4 and to a higher extent LTB4 were the major lipoxygenase products detected during anaphylaxis. Perfusion of the lungs with aspirin and indomethacin decreased the formation of PGE2 and TxB2 but did not modify the release of leukotrienes. On the contrary, BW755C and eicosatetraynoic acid (ETYA) reduced the release of all icosanoids from the lungs. FPL-55712, a selective leukotriene antagonist, significantly reduced the release of PGE2, TxB2, LTB4 and LTD4 at the high concentration (20 uM). In summary, this study made use of novel techniques to quantify arachidonic acid metabolites in the effluent of perfused lungs. New metabolites were described. Our study also stresses the possible significance of LTB4 as a mediator of anaphylaxis and asthma.

The formation of leukotrienes (LTs) from arachidonic acid derived from phospholipids of the cell membrane is initially catalysed by 5-lipoxygenasel. Metabolism of the unstable epoxide LTA4 leads to the formation of LTB4 and the cysteinyl-containing LTs C4, D4 and E4. All these LTs have potent, although different, biological activities. LTB4 is a powerful chemotactic agent for leukocytes whereas LTs C4, D4 and E4 have potent smooth muscle stimulating actions and account for the biological activity of the allergic mediator previously known as slow-reacting substance of anaphylaxis (SRS-A)2. Leukotriene B4 has pro-inflammatory actions but little smooth muscle stimulating activity of its own whereas cysteinyl-containing LTs have potent actions in the cardiovascular system and in the airways in vitro and in vivo (see3,4).

Most of the studies conducted in the seventies as well as in the early eighties, have been designed to establish the oxidative pathways of arachidonic acid by specific cell types resulting in the determination of the structure of eicosanoids by a given cell. Such an approach has delineated specific enzymatic patterns by certain cells in the blood and vascular system. Such findings are summarized in Table I. Platelets produce mainly 12-hydroxyeicosatetraenoic acid, 12-hydroxy heptadecatrienoic acid and thromboxane A2. Endothelial cells from human umbilical cord or vascular smooth muscle synthesize mainly prostaglandin I2 (prostacyclin) and prostaglandins E2 and F2α whereas this production is different for endothelial cells from the microvasculature. Polymorphonuclear granulocytes synthesize nearly exclusively LTB4 and eosinophils generate LTC4. Monocytes, depending upon their origin or their maturation, seem to produce both leukotrienes. In contrast, red blood cells and lymphocytes lack the specific oxygenases to form eicosanoids.

Glutathione transferases constitute a group of enzymes that catalyze several reactions involving glutathione (Mannervik, 1985). Their main function, according to current concepts, is to detoxify and accelerate the excretion of certain xenobiotic compounds (Chasseaud, 1979) by catalyzing the conjugation of glutathione with these electro-philic substrates. In addition, glutathione transferases also catalyze other reactions (e. g., peroxidase and isomerase reactions). We have recently reported (Mannervik et al., 1984) that six basic glutathione transferases from rat liver cytosol (Mannervik and Jensson, 1982) catalyze the conversion of LTA4 or its methyl ester to LTC4. One of the enzymes was a considerably more efficient catalyst of the reaction than the remaining five.

The 5-lipoxygenase pathway for oxidative metabolism of unsaturated fatty acids was first recognized less than 10 years ago with the definition of 5S-hydroxy-eicosatetraenoic acid (5-HETE) as a product, l and its potential biological relevance to inflammation was defined solely by the modest chemotactic activity of 5-HETE.2 However, major interest in this pathway did not occur until 5 years ago when leukotriene B4 (LTB4), 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid was first described3 and the elusive “slow reacting substance of anaphylaxis (SRS-A),” was chemically defined as three additional leukotriene products of this pathway: LTC4, 5S-hydroxy-6R-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acido; LTD4, 5S-hydroxy-6R-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid5-7; and LTE4, 5S-hydroxy-6R-S-cysteinyl-7,9-trans-11,14-cis-eicosatetraenoic acid.8 That efforts to decrease the generation of the leukotriene compounds could have a significant effect in down-regulating a variety of inflammatory events has been strongly suggested by several types of data developed over the past 5 years, with regard to the breadth of proinflammatory effects manifested by these compounds, indications that a variety are mediated via specific receptors that do not recognize other naturally-occurring compounds, an expanding knowledge of the inflammatory cell types which serve as sources for the leukotrienes, and the demonstration that leukotrienes are recoverable from complex biological fluids in both in vivo models of inflammation and human disease. Although there is potential for antagonizing the biological effects of each leukotriene at the end-organ receptor level, the present discussion will focus mainly on regulation of leukotriene biosynthesis as an anti-inflammatory therapeutic approach. Furthermore, the potential of dietary alteration as an adjunct to developing pharmacotherapeutic inhibitors of the 5-lipoxygenase pathway will be specifically considered.

The oxidative metabolism of arachidonic acid by 5-lipoxygenase to form 5-hydro-peroxy-6-trans8-cis-eicosatetraenoic acid (5-HPETE) is followed by enzymatic conversion of 5-HPETE to 5,6-oxido-7,9-trans-ll,14-cis-eicosatetraenoic acid (LTA4). An epoxide hydrolase converts LTA4 to 5S,12R-dmydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), whereas a glutathione-S-transferase adducts glu-tathione to yield 5S-hydroxy-6R-S-gluta.thionyl-7,9-trans-11.14-cis-eicossitrae-noic acid (LTC4). Sequential cleavages by γ-glutamyltranspeptidase of glutamic acid and by a dipeptidase of glycine form 5S-hydroxy-6R-S-cysteinylglycyl-7,9-trans-11 1,14-cis-eicosatetraenoic acid (LTD4) and 5S-hydroxy-6R-S-cysteinyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTE4), respectively. Leukotrienes C4, D4, and E4 are generically described as sulfidopeptide leukotrienes and constitute the activity previously termed slow reacting substance of anaphylaxis (Samuelsson, 1983; Lewis and Austen, 1984). The evidence indicating that the sulfidopeptide leukotrienes exert their physiological effects through interaction with several distinct receptors includes their differential functional activities on different tissues, the differential effects of pharmacological inhibitors on the agonist effects of the three sulfidopeptide leukotrienes, different receptor characteristics defined by radioligand binding studies, and apparent differences in the subcellular distribution of sulfi-dopeptide leukotriene binding sites.

Research carried out in numerous laboratories has led to the hypothesis that metabolites of the ∆5-lipoxygenase (5∆-L0) pathway (e.g., leukotrienes and 5-HETE) may play an important role in mediating a number of inflammatory diseases including asthma, inflammatory bowel disease, and diseases associated with cutaneous inflammation. The purpose of this chapter is to briefly review the rationale supporting this hypothesis and to present the results of some experimental studies with promising ∆5-L0 inhibitors and leukotriene antagonists which would support the clinical evaluation of these types of drugs in the three disease states.