CA2265708C - Lipoxin compounds and their use in treating cell proliferative disorders - Google Patents

Abstract

Compounds having the active site of natural lipoxins, but a longer tissue ha lf- life are disclosed. In particular, 15-epi-lipoxins and their use in ameliorating undesired cell proliferation, which characterizes diseases such as cancer, are also disclosed.

Description

101520253035W0 98/1 10491CA 02265708 l999-03- 10PCT/US97/16342LIPOXIN COMPOUNDS AND THEIR USE IN TREATING CELLPROLIFERATIVE DISORDERSBackgroundLipoxins are a group of biologically active mediators derived fromarachidonic acid through the action of lipoxygenase (LO) enzyme systems. (Serhan,C.N. and Samuelsson, B. (1984) Proc. Natl. Acad. Sci. USA 81 :5335). Formation inhuman cell types is initiated by 5-LO or l5—LO. (Serhan, CN. (1991) J. Bioenerg.Biomembr. 23:105). Single-cell types generate lipoxins at nanogram levels duringhuman neutrophil-platelet and eosinophil transcellular biosynthesis of eicosanoids.(Serhan, C.N. and Sheppard, K.-A. (1990) J. Clin. Invest. 852772). LXs are conjugatedtetraene-containing eicosanoids that modulate cellular events in several organ systems.Lipoxin A4 (LXA4) and lipoxin B4 ( LXB4) are the two major lipoxins.Each enhances protein kinase C (PKC) activity in nuclei of erythroleukemia cells at 10nM (Beckman, B.S. et al. (1992) Proc. Soc. Exp. Biol. Med. 201 :l69). Each elicitsprompt vasodilation at nM levels (Busija, D.W. et al. ( 1989) Am. J. Physiol. 256:H468;Katoh, T. et al. (1992) Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32):F436).The vasodilatory effects of lipoxins are well-documented. For example, administrationof LXA4 in micromolar amounts via inhalation blocks bronchoconstriction in asthmaticpatients. (Christie, P.E. et al. (1992) Am. Rev. Respir. Dis. l45:1281).In the 10'”) M range, LXA4 also stimulates cell proliferation incombination with suboptimal concentrations of granulocyte-macrophage colonystimulating factor (GM—CSF) to induce myeloid bone marrow colony formation(Stenke, L. et al. (1991) Biochem. Biophys. Res. Commun. l80:255). LXA4 also .stimulates human mononuclear cell colony formation (Popov, G.K. et al. (1989) Bull.Exp. Biol. Med. 107293).LXA4 inhibits chemotaxis of polymorphonuclear leukocytes (Lee, T.H. etal. (1991) Biochem. Biophys. Res. Commun. 180: 1416). An equimolar combination oflipoxins has been found to modulate the polymorphonuclear neutrophil-mesangial cellinteraction in glomerular inflammation. (Brady, H.R. et al (1990) Am. J. Physiol. 809).Activation of the polymorphonuclear neutrophils (PMN) includes the release ofmediators of structural and functional abnormalities associated with the early stages ofglomerular inflammation. (Wilson, C.B. and Dixon, F.J. (1986) In: The Kidney, editedby B.M. Brenner and F.C. Rector. Philadelphia, PA: Saunders, p. 800-891).Lipoxins act as antagonists to leukotrienes (LT), which are mediators ofinflammation. LXA4 modulates LTC4-induced obstruction of airways in asthmaticIO1520253035CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342patients. (Christie, P.E. et al. (1992) Am. Rev. Respir. Dis. 145:128l). LXA4 inhibitsLTD4- and LTB4-mediated inflammation in animal in vivo models. (Badr, K.F. et al(1989) Proc. Natl. Acad. Sci. 86:3438; Hedqvist, P. et al. (1989) Acta Physiol. Scand.137:571). Prior exposure to LXA4 (nM) blocks renal vasoconstrictor actions of LTD4(Katoh, T. et al. (1992) Am. J .Physiol. 263 (Renal Fluid Electrolyte Physiol. 32) F436).Leukotriene-induced inflammation occurs, for example, in arthritis, asthma, varioustypes of shock, hypertension, renal diseases, allergic reactions, and circulatory diseasesincluding myocardial infarction.Although lipoxins are potent small molecules that could be administeredin vivo to treat a number of diseases and conditions, these molecules are short-lived invivo. Compounds having the same bio-activities as natural lipoxins, but a longer in vivohalf-life would be valuable pharmaceuticals.Summary of the InventionThis invention features substantially purified 15-epi—lipoxin compounds.In one embodiment, the 15-epi—lipoxin compound is l5R—5,6,l 5-trihydroxy-7,9,13-trans-1 1-cis-eicosatetraenoic acid and in another embodiment, this acid has a 5S,6R,configuration (15—epi-LXA4). In other embodiments, the 15-epi—lipoxin compound is1SR-5,14,15—trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid, and this acid has a5S,l4R configuration (15-epi—LXB4). In still other embodiments, the 15-epi—lipoxincompound is 15-hydroxyeicosatetraenoic acid (15-HETE), and this acid has a 15Rconfiguration.This invention also features lipoxin analogs, which have an active regionthat is the same or similar to natural lipoxin, but a metabolic transformation regionwhich is more resistant to in vivo catabolism. The instant disclosed lipoxin analogstherefore have the biological activity of natural lipoxins, but a longer metabolic half—life.Certain of the instant disclosed lipoxin analogs may additionally have an increased inviva potency, higher binding affinity to lipoxin receptors or enhanced bio-activity ascompared to natural lipoxins.Like natural lipoxins, the instant disclosed small molecules are highlypotent and biocompatible (i.e. non-toxic). However, unlike natural lipoxins, lipoxinsanalogs inhibit, resist, or more slowly undergo metabolism and therefore have a longerpharmacological activity. Further, the instant disclosed compounds are more lipophilicthan natural lipoxins and therefore are more readily taken up by biological membranes.In addition, the invention features methods of ameliorating an undesiredproliferation of certain cells based on contacting the cells with an effective amount of a101520253035CA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342substantially purified 15-epi—lipoxin compound. In preferred embodiments, the cells areundergoing cancerous or tumorous growth. Also in preferred embodiments, the cells areselected from the group consisting of: an epithelial cell, a leukocyte, an endothelial cell,and/or a fibroblast. In certain preferred embodiments of the invention, cells arecontacted in vivo. In another embodiment, cells are contacted ex viva.The invention also features methods for ameliorating a cell proliferativedisorder in a subject by administering an effective amount of a substantially purified 15-epi-lipoxin compound.In another aspect, the invention features pharmaceutical compositionshaving the substantially purified 15—epi-lipoxin compound of the present invention and apharmaceutically acceptable carrier. In a preferred embodiment, the 15-epi-lipoxincompound is in an amount effective to prevent an undesired proliferation of cells in asubject. In another embodiment, the pharmaceutical composition includes an effectiveamount of acetylsalicylic acid (ASA).The invention further relates to diagnostic and research uses of thelipoxin compounds. Additional features and advantages of the invention will becomemore apparent from the following detailed description and claims.Brief Description of the DrawingsFIG. IA is a graph showing prostaglandin endoperoxide synthase (PGHS) andlipoxygenase (LO) expression in human tumor cell line (A549 cells) alveolar type IIepithelial cells. Cells were grown for 24 h at 37 °C in T-75 cmz flasks in the presenceor absence of Interleukin-13 (IL-1 5) (1 ng/ml). Extracted total RNA (1 ug) was takenfor Reverse Transcription (RT) and PCR using specific oligonucleotides for PGHS-1and -2, 15-, 12- and 5-LO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Radioactive bands were quantified directly by phosphorimager analysis, normalized tothe expression of GAPDH and expressed as fold increase in mRNA levels after exposureto IL-1 B. The inset of FIG. 1A shows 15-LO mRNA expression in human lung tissueand peripheral blood monocytes (PBM) with ND meaning 15—LO mRNA expression notdetected.FIG. 1B is a graph showing a RP—HPLC profile of [3H]-labeled mono-hydroxyeicosatetraenoic acids (HETES) from permeabilized IL-1 B-treated A549 cells(1 .5x1 06 cells/ml) exposed to [3H]-arachidonic acid (20pM) for 20 min at 37 °C.Products were extracted and chromatographed using a linear gradient ofmethanol:H20:acetic acid (65:35:0.0l; v/v/v) and methanolzacetic acid (99.9:0.1, v/v) ata flow rate of 1.0 ml/min. Arrows denote co—chromatography of synthetic standards.101520253035W0 98/1 1049CA 02265708 l999-03- 10PCTIUS97l16342FIG. 2A is a graph showing the generation of l5-HETE. A549 cells (6x106cells/flask) were treated with IL-1 5 (lng/ml) for 24 h, subjected to freeze-thaw (twocycles), exposed for 20 min to either vehicle (0.1% vol/vol ethanol (EtOH)),acetylsalicylic acid (ASA), the cytochrome P450 inhibitor (17-octadecaynoic acid (17-ODYA), 5 p.M) or the 5-LO inhibitor (Rev-5901 isomer, 5 uM) and incubated witharachidonic acid (20 uM) for 20 min at 37°C. In some experiments, cells were heat-denatured (100 °C, 60 min) before incubation. Incubations were stopped with additionof methanol (2v), and products were extracted for reversed phase (RP) —high-pressureliquid chromatography (HPLC). Data are means :l: SEM from four to six separate flasks.*, P<0.05 and **, P<0.0l for treatments versus control are shown.FIG. 2B is a graph showing the time course of 15-HETE formation fromendogenous sources. A549 cells (1.5 x 106 cells per ml) were grown for 48 h in theabsence or presence of IL-1 5 (1 ng/ml) and incubated (30 min at 37 °C) in 4 ml HBSSwith or without A231g7 (5 uM). 15-HETE levels were determined by RIA. Resultsrepresent the mean dz SEM of three different experiments determined by duplicate. *,P<0.05 for treatments versus vehicle are shown.FIG. 3 is a graph showing the relative chiralities of 15-HETE triggered by ASA.A549 cells (107 cells per flask) were exposed to IL-1 [3 (1 ng/ml) for 24 h, treated withvehicle (0.1% vol/vol ethanol) (El) or ASA (I) for 20 min and then incubated (30 min,37 °C) in HBSS containing arachidonic acid (20 uM) and A231g7 (5 uM). Productswere chromatographed by RP-HPLC (as in FIG. 1B) and the region containing 15-HETE was collected, extracted with chloroform and treated with diazomethane. Chiralanalysis was performed with a Bakerbond DNBPG (see Methods for details). Resultsare representative of two separate experiments showing similar results. The inset ofFIG. 3 shows the ratio between A549-derived 15R and 15S-HETE in the absence orpresence (filled bars) of ASA.FIG. 4A is a graph showing a RP-HPLC chromatogram of products fromepithelial cell—polymorphonuclear neutrophils (PMN) costimulation. Confluent A549cells were exposed to IL-13 (1 ng/ml) for 24 h, treated with ASA (20 min) andarachidonic acid (20 uM, 60 s) and each incubated with freshly isolated PMN (A549cell:PMN cell ratio of 1:8) followed by stimulation with ionophore A3187 (5 uM) in 4ml of Hank's balanced salt solution (HBSS) for 30 min at 37 °C. Products wereextracted and taken to RP-HPLC as described in the Methods section of Example 5. Thechromatogram was plotted at 300 nm and is representative of n=6 experiments.FIG. 4B is a graph showing on-line ultra-violet (UV) spectra of products fromthe epithelial cell-PMN costimulation described in FIG. 4A. Material eluting beneathpeak B was identified as predominantly 15-epi—LXB4.1015202530354‘CA 02265708 l999-03- 10W0 98/11049 PCTIUS97/16342FIG. 4C is a graph showing on-line UV spectra of products from the epithelialcell-PMN constimulation described in FIG. 4A. Material eluting beneath the illustratedpeaks was identified as predominantly 15-epi-LXA4,FIG. 5A is a graph showing ASA modulating the formation of tetraene-containing lipoxins (lipoxins plus I5-epi-lipoxins) during epithelial cell-PMNcostimulation. A549 cells were exposed to IL-1 [3 (lng/ml, 24 h) and treated (20 min, 37°C) with either vehicle (0.1% vol/vol) or ASA, before the addition of arachidonic acid(20 }JM, 1 min) and freshly isolated PMN (A549/PMN cell ratio of 1:5). Costimulationswere carried out as in FIG. 4A. Results represent the mean i SEM from 3-5 separatedonors. The inset of FIG. 5A shows the effect of cell ratio on generation of tetraene-containing lipoxins (lipoxins plus 15-epi-lipoxins) during co—incubations of A549 cellswith PMN in the absence (0) or presence (I) of ASA.FIG. 5B is a graph showing ASA modulating the formation ofpeptidoleukotrienes (LTC4 plus LTD4) during epithelial cell-PMN costimulationaccording to the conditions outlined in FIG. 5A. The inset of FIG. 5B shows the effectof cell ratio on generation of peptidoleukotrienes (LTC4 plus LTD4) during co-incubations of A549 cells with PMN in the absence (0) or presence (I) of ASA.FIG. 6A is a graph showing the effect of Lipoxin A4 (LXA4), Lipoxin B4(LXB4), Dexamethasone (DEX) and vehicle alone treatment on A549 cell number overtime. A549 cells in 96-well plates were treated with either vehicle (0.15% EtOH) orequimolar concentrations (10'6 M) of LXA4, LXB4 or DEX for up to 96 hours at 37 °C.At the indicated intervals, cells were harvested for the 3,(4,5-dimethylthiazoyl-2-yl) 2,5(diphenyl-tetrazolium bromide) MTT assay. Data are means i SEM of 3-7 experimentsperformed in quadruplicate. *, P<0.05 and **, P<0.005 for compounds versus vehicleare shown.FIG. 6B is a graph showing the effect of LXA4, LXB4, and DEX treatment onthe percent inhibition of A549 cell proliferation at varying A549 cell concentrations.A549 cells were exposed to LXA4, LXB4 or DEX at the indicated concentrations for 72hours at 37 °C. Results are means i SEM of 5-8 experiments performed inquadruplicate. Results are expressed as the percent inhibition of proliferation relative tovehicle. *, P<0.05, **, P<0.025 and ***, P<0.005 for compounds versus vehicle areshown.FIG. 7A is a graph showing the effect of LXA4, LXB4 and DEX on A549 cellDNA synthesis, as indicated by 3H-thymidine incorporation, where A549 cells weregrown for 72 hours in the presence of LXA4, LXB4 and DEX at varying concentrationsranging between 5nM to 500 nM. Twenty-four hours before the assay, methyl-[3H]thymidine (2 pCi/ml) was added to each well. Cells were subsequently washed four1015CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342times with DPBS-2+ (4°C), lysed with 0.25 N Sodium Hydroxide (NaOH), andradioactivity incorporation was monitored. Values represent mean :t SEM of 3 differentexperiments performed in quadruplicate. Results are expressed as the percent of[3H]thymidine incorporation relative to vehicle alone. *, P<0.05 and **, P<0.005 forcompounds versus vehicle are shown.FIG. 7B is a graph showing the effect of LXA4, LXB4 and DEX on theinhibition of A549 cells, where A 549 cells were seeded in 12-well culture plates in thepresence of LXA4, LXB4 and DEX (1 ;,LM) and cell counts were obtained at 72 hoursby enumerating the trypan-excluding cells. Values represent mean i SEM of 3 differentexperiments. Results are expressed as the percent inhibition of proliferation relative tobuffer. *, P<0.05 for compounds versus vehicle are shown.FIG. 8 is a diagram showing the proposed biochemical pathway for generating15-epi-lipoxins. ASA—acetylated PGHS-2 and/or P450 activities contribute to 15R-HETE. Epithelial 15R-HETE undergoes transcellular conversion by Leukocyte 5-LO toa 15-epi-5(6)-epoxytetraene intermediate, which is common to both 15-epi-LXA4 and15-epi-LXB4.101520253035W0 98/1 1049iCA 02265708 l999-03- 10PCT/US97/ 16342Detailed Description of the InventionAs used herein, the following phrases and terms are defined as follows:A "lipoxin compound" shall mean a natural lipoxin compound (lipoxinA4 or lipoxin B4) and/or a lipoxin analog.A "lipoxin analog" shall mean a compound which has an "active region"that functions like the active region of a "natural lipoxin", but which has a "metabolictransformation region" that differs from natural lipoxin. Lipoxin analogs includecompounds which are structurally similar to a natural lipoxin, compounds which sharethe same receptor recognition site, compounds which share the same or similar lipoxinmetabolic transformation region as lipoxin, and compounds which are art-recognized asbeing analogs of lipoxin. Lipoxin analogs include lipoxin analog metabolites. Thecompounds disclosed herein may contain one or more centers of asymmetry. Whereasymmetric carbon atoms are present, more than one stereoisomer is possible, and allpossible isomeric forms are intended to be included within the structural representationsshown. Optically active (R) and (S) isomers may be resolved using conventionaltechniques known to the skilled artisan. The present invention is intended to include thepossible diastereisomers as well as the racemic and optically resolved isomers.A preferred lipoxin compound for use in the subject invention is a "15-epi-lipoxin compound". As used herein, "15—epi-lipoxin compound" is a lipoxincompound in which the absolute configuration at the 15 Carbon is R.The term "15-epi-lipoxin compound" is intended to encompassprecursors. The term "precursor" is intended to refer to chemical intermediates that canbe converted in vivo, ex vivo and/or in vitro to form the 15-epi-lipoxin compounds of theinvention. The term "precursor" also contemplates prodrugs which are converted in vivoto the 15-epi-lipoxin compounds of the invention (see, e.g., R.B. Silverrnan, 1992, "TheOrganic Chemistry of Drug Design and Drug Action", Academic Press, Chp. 8).Examples of such prodrugs include, but are not limited to esters of hydroxyls and/orcarboxyl groups and/or compounds which can be hydrolyzed or otherwise converted invivo or, ex vivo and/or in vitro into the 15-epi-lipoxin compounds of the presentinvention.The terms "corresponding lipoxin" and "natural lipoxin" refer to anaturally-occurring lipoxin or lipoxin metabolite. Where an analog has activity for alipoxin-specific receptor, the corresponding or natural lipoxin is the normal ligand forthat receptor. For example, where an analog is a LXA4 analog having specific activityfor a LXA4 specific receptor on differentiated HL-60 cells, the corresponding lipoxin isLXA4. Where an analog has activity as an antagonist to another compound (such as a101520253035W0 98/11049CA 02265708 l999-03- 10PCT/US97/16342leukotriene), which is antagonized by a naturally-occurring lipoxin, that natural lipoxinis the corresponding lipoxin.The term "active region" shall mean the region of a natural lipoxin orlipoxin analog, which is associated with in viva cellular interactions. The active regionmay bind the "recognition site" of a cellular lipoxin receptor or a macromolecule orcomplex of macromolecules, including an enzyme and its cofactor. Preferred lipoxin A4analogs have an active region comprising C5-C15 of natural lipoxin A4. Preferredlipoxin B4 analogs have an active region comprising C5-C14 of natural lipoxin B4.The term "recognition site" or receptor is art-recognized and is intendedto refer generally to a functional macromolecule or complex of macromolecules withwhich certain groups of cellular messengers, such as hormones, leukotrienes, andlipoxins, must first interact before the biochemical and physiological responses to thosemessengers are initiated. As used in this application, a receptor may be isolated, on anintact or perrneabilized cell, or in tissue, including an organ. A receptor may be from orin a living subject, or it may be cloned. A receptor may normally exist or it may beinduced by a disease state, by an injury, or by artificial means. A compound of thisinvention may bind reversibly, irreversibly, competitively, noncompetitively, oruncompetitively with respect to the natural substrate of a recognition site.The term "metabolic transformation region" is intended to refer generallyto that portion of a lipoxin, a lipoxin metabolite, or lipoxin analog including a lipoxinanalog metabolite, upon which an enzyme or an enzyme and its cofactor attempts toperform one or more metabolic transformations which that enzyme or enzyme andcofactor normally transform on lipoxins. The metabolic transformation region may ormay not be susceptible to the transformation. A nonlimiting example of a metabolictransformation region of a lipoxin is a portion of LXA4 that includes the C-13,14 doublebond or the C-15 hydroxyl group, or both.The term "detectable label molecule" is meant to include fluorescent,phosphorescent, and radiolabeled molecules used to trace, track, or identify thecompound or receptor recognition site to which the detectable label molecule is bound.The label molecule may be detected by any of the several methods known in the art.The term " labeled lipoxin analog" is further understood to encompasscompounds which are labeled with radioactive isotopes, such as but not limited totritium ( 3H), deuterium ( 2H), carbon ( 14C), or otherwise labeled (e.g. fluorescently).The compounds of this invention may be labeled or derivatized, for example, for kineticbinding experiments, for further elucidating metabolic pathways and enzymaticmechanisms, or for characterization by methods known in the art of analyticalchemistry.101520253035W0 98/ 1 10491CA 02265708 l999-03- 10PCT/U S97/ 16342The term "inhibits metabolism" means the blocking or reduction ofactivity of an enzyme which metabolizes a native lipoxin. The blockage or reductionmay occur by covalent bonding, by irreversible binding, by reversible binding which hasa practical effect of irreversible binding, or by any other means which prevents theenzyme from operating in its usual manner on another lipoxin analog, including alipoxin analog metabolite, a lipoxin, or a lipoxin metabolite.The term "resists metabolism" is meant to include failing to undergo oneor more of the metabolic degradative transformations by at least one of the enzymeswhich metabolize lipoxins. Two nonlimiting examples of LXA4 analog that resistsmetabolism are 1) a structure which can not be oxidized to the 15-oxo form, and 2) astructure which may be oxidized to the 15—oxo form, but is not susceptible to enzymaticreduction to the 13, 14-dihydro form.The term "more slowly undergoes metabolism" means having slowerreaction kinetics, or requiring more time for the completion of the series of metabolictransformations by one or more of the enzymes which metabolize lipoxin. Anonlimiting example of a LXA4 analog which more slowly undergoes metabolism is astructure which has a higher transition state energy for C-15 dehydrogenation than doesLXA4 because the analog is sterically hindered at the C-16.The term "tissue" is intended to ‘include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smooth muscles, andorgans.The term "halogen" is meant to include fluorine, chlorine, bromine andiodine, or fluoro, chloro, bromo, and iodo.The term "pharmaceutically acceptable salt" is intended to include art-recognized pharmaceutically acceptable salts. These non—toxic salts are usuallyhydrolyzed under physiological conditions, and include organic and inorganic bases.Examples of salts include sodium, potassium, calcium, ammonium, copper, andaluminum as well as primary, secondaiy, and tertiary amines, basic ion exchange resins,purines, piperazine, and the like. The term is further intended to include esters of lowerhydrocarbon groups, such as methyl, ethyl, and propyl.The term "pharmaceutical composition" comprises one or more lipoxinanalogs as active ingredient(s), or a pharmaceutically acceptable salt(s) thereof, and mayalso contain a pharmaceutically acceptable carrier and optionally other therapeuticingredients. The compositions include compositions suitable for oral, rectal,ophthalmic, pulmonary, nasal, dermal, topical, parenteral (including subcutaneous,intramuscular and intravenous) or inhalation administration. The most suitable route inany particular case will depend on the nature and severity of the conditions being treated101520253035W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342_ -and the nature of the active ingredient(s). The compositions may be presented in unitdosage form and prepared by any of the methods well—known in the art of pharmacy.Dosage regimes may be adjusted for the purpose to improving the therapeutic response.For example, several divided dosages may be administered daily or the dose may beproportionally reduced over time. A person skilled in the art normally may determinethe effective dosage amount and the appropriate regime. A lipoxin analog pharmaceuticcomposition can also refer to a combination comprising lipoxins, lipoxin analogs, and/orlipoxin metabolites, including metabolites of lipoxin analogs. A nonlimiting example ofa combination is a mixture comprising a lipoxin analog x which inhibits one enzymewhich metabolizes lipoxins and which optionally has specific activity with a lipoxinreceptor recognition site, and a second lipoxin analog y which has specific activity witha lipoxin receptor recognition site and which optionally inhibits or resists lipoxinmetabolism. This combination results in a longer tissue half-life for at least y since xinhibits one of the enzymes which metabolize lipoxins. Thus, the lipoxin actionmediated or antagonized by y is enhanced.The term "substantially pure or purified" lipoxin compounds are definedas encompassing natural or synthetic compounds of lipoxins having less than about 20%(by dry weight) of other biological macromolecules , and preferably having less thanabout 5% other biological macromolecules (but water, buffers, and other smallmolecules, especially moleculess having a molecular weight of less than 5000, can bepresent. The term "purified" as used herein preferably means at least 80% by dryweight, more preferably in the range of 95-99% by weight, and most preferably at least99.8% by weight, of biological macromolecules of the same type present (but water,buffers, and other small molecules, especially molecules having a molecular weight ofless than 5000, can be present). The term "pure" as used herein preferably has the samenumerical limits as "purified" immediately above. "Isolated" and "purified" do notencompass either natural materials in their native state or natural materials that havebeen separated into components (e.g., in an acrylamide gel) but not obtained either aspure (e.g. lacking contaminating proteins, or chromatography reagents such asdenaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.The term "subject" is intended to include living organisms susceptible toconditions or diseases caused or contributed to by inflammation, inflammatoryresponses, vasoconstriction, myeloid suppression and/or undesired cell proliferation.Examples of subjects include humans, dogs, cats, cows, goats, and mice. The termsubject is further intended to include transgenic species.The term "cell proliferative disorder" includes disorders involving theundesired proliferation of a cell. Non-limiting examples of such disorders include101520253035W0 98/1 10491CA 02265708 l999-03- 10PCT/U S97/ 16342_ 1 1 -tumors, (e.g., brain, lung (small cell and non-small cell), ovary, prostate, breast or colon)or other carcinomas or sarcomas (e.g., leukemia, lymphoma).The term "ameliorated" in intended to include treatment for, preventionof, limiting of and/or inhibition of undesired cell proliferation and/or a cell proliferativedisorder.Lipoxin CompoundsThe instant invention is based on the surprising finding that substantially pure15-epi-lipoxin compounds ameliorate undesired cell proliferation in a subject. The 15-epi—lipoxin compounds of the present invention include 15R-5,6,15-trihydroxy-7,9,l3-trans-11-cis-eicosatetraenoic acid, in a 5S,6R configuration (15-epi-LXA4). The 15-epi-lipoxin compounds of the present invention also include 15R-5,14,! 5-trihydroxy-6,10,12-trans—8-cis—eicosatetraenoic acid, in a 5S,14R configuration (15-epi-LXB4).The 15-epi-lipoxins of the present invention further include 15-hydroxyeicosatetraenoicacid, particularly in a 15R configuration.The instant invention is based on the surprising finding that lipoxins arerapidly metabolized in a unique fashion by certain cells in vivo. Although other LO-derived products (e.g. leukotrienes) are metabolized by co-oxidation followed by [3-oxidation ( Huwyler et al., (1992) Eur.J. Biochem. 206, 869-879), the instant inventionis based on the unexpected finding that lipoxins are metabolized by a series of oxidationand reduction reactions acting on certain sites of the lipoxin molecule. For example,LXA4 metabolism has been found to occur, at least in part, via oxidation of the C-15hydroxyl to generate 15-oxo-LXA4, reduction of the C-13,14 double bond to yield 13,14—dihydro-15-oxo—LXA4 and further reduction to yield 13,14-dihydro-LXA4. In LXB4and its natural isomers the analogous oxidation occurs at the C-5 hydroxyl and reductionoccurs at the C-6,7 double bond.Thus, the instant invention features lipoxin analogs having lipoxinactivity, but which are chemically modified to prevent dehydrogenation and thereforesubsequent degradation in vivo. In these analogs, the C-1 to C-13 portion of the naturallipoxin may or may not be conserved. Variations of the C-1 to C-13 portion includedifferent cis or trans geometry as well as substitutions. The disclosed compounds isrepresented below by a structural genus, which is further divided into subgenuses.Subgenuses included in each of the following two R groups is denoted by a Romannumeral on the left of the page.CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97ll6342The instant lipoxins comprising an "active region" and a "metabolictransformation region" as both terms are defined herein are generally of the followingstructure:wherein R] can beQl\1,11, III,IV Vfl/R XHO Q3H R2R4 R3Y1 R2 0Y2-»..,_.M /U\V (CH2)n OR:Rsa R3bVI,VII,VIII, M1x,x \ OR0 J\X‘ (CH2 n ORa10 R33 R3bICA 02265708 l999-03- 10W0 98/11049 PCT/US97l16342- 13 _and R2 can beCA 02265708 l999-03- 10W0 98/ 1 1049 PCT/US97/16342- 14 -In one embodiment, the lipoxin analogs of this invention have thefollowing structural formula I: wherein X is R], OR], or SR1;wherein R, is(i) hydrogen;10 (ii) alkyl of 1 to 8 carbons atoms, inclusive,which may be straight chain or branched;(iii) cycloalkyl of 3 to 10 carbon atoms,inclusive;(iv) aralkyl of 7 to 12 carbon atoms;15 (v) phenyl;(vi) substituted phenylZi ZilZiiiZv Ziv20 wherein Z i , Z m_, and Zv are eachindependently selected from the groupconsisting of -N02, -CN, -C(=O)-R1,-SO3H, and hydrogen;wherein Zii and Z“, are each25 independently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(vii) detectable label molecule; or101520253035W0 98/110491CA 02265708 l999-03- 10PCT/US97/ 16342_ 15 -(viii) straight or branched chain alkenyl of 2 to8 carbon atoms, inclusive;wherein Q] is (C=O), S02 or (CN);wherein Q3 is O, S or NH;wherein one of R2 and R3 is hydrogen and the other is(a) H;(b) alkyl of 1 to 8 carbon atoms, inclusive, which maybe straight chain or branched;(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;((1) alkenyl of 2 to 8 carbon atoms, inclusive, whichmay be straight chain or branched; or(C) RaQ2Rbwherein Q2 is -0- or -S—;wherein Ra is alkylene of 0 to 6 carbons atoms,inclusive, which may be straight chain orbranched; and wherein Rb is alkyl of 0 to 8carbon atoms, inclusive, which may be straightchain or branched;wherein R4 is(a) H;(b) alkyl of 1 to 6 carbon atoms, inclusive, which maybe straight chain or branched;wherein Y, or Y2 is -OH , methyl, or -SH and wherein theother is(a) H(b) CHaZbwherea+b=3,a=0to 3,b=0to3;andZ is cyano, nitro, or halogen;(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched; or(d) alkoxy of 1 to 4 carbon atoms, inclusive;or Y1 and Y2 taken together are(a) =N; or(b) =0;wherein R5 is(a) alkyl of 1 to 9 carbon atoms which may be straightchain or branched;CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342_ 15 _(b) -(CH2)n 'Riwherein n = O to 4 and Ri is(i) cycloalkyl of 3 to 10 carbon atoms,inclusive;(ii) phenyl; or(iii) substituted phenylZi ZiiZii iZv Zivwherein Z i , Z in’, and ZV are eachindependently selected from the groupconsisting of hydrogen, -N02, -CN,-C(=O)-R], methoxy, and -SO3H; andwherein Z“ and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(C) - aQaRbwherein Q, = -0- or -S-;wherein R, is alkylene of 0 to 6 carbonsatoms, inclusive, which may be straight chainor branched;wherein Rb is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain orbranched;(<1) -C(Riii)(Riv)'Riwherein Rm and RV are selected independentlyfrom the group consisting of(i) H;(ii) CHaZbwherea+b=3,a=0to 3,b=0+3, and wherein any Z is independently selectedfrom the group consisting of halogen;(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 toICA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342_ 17 _6 halogen atoms, inclusive, straight chain or branched;andwherein R6 is(a) H;5 (b) alkyl from 1 to 4 carbon atoms, inclusive, straightchain or branched;(c) halogen; but excluding the C-1 position amides, C-1 position alkanoates, and pharmaceutically acceptableC-1 position salts of (SS, 6R, 15S)-trihydroxy-7E, 9E,10 11Z, 13E-eicosatetraenoic acid (LXA4); and excludingC-5, C-6, and C-15 position alkanoates of LXA4.In one embodiment of this invention, the lipoxin analogs have thefollowing structure II:15 II. wherein X is R], OR], or SR];wherein R, is20 (i) hydrogen;(ii) alkyl of 1 to 8 carbons atoms, inclusive,which may be straight chain or branched;(iii) cycloalkyl of 3 to 10 carbon atoms,inclusive;25 (iv) aralkyl of 7 to 12 carbon atoms;(v) phenyl;(vi) substituted phenyl1015202530W0 98/1 1049CA 02265708 l999-03- 10PCT/U S97/16342- 13 -ZiiiZv Zivwherein Z i , Z iii’, and ZV are eachindependently selected from the groupconsisting of -N02, -CN, -C(=O)-R, ,hydrogen, and —SO3H;wherein Zii and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(vii) detectable label molecule, such as but notlimited to fluorescent labels; or(viii) alkenyl of 2 to 8 carbon atoms, inclusive,straight chain or branched;wherein Q, is (C=O), S02 or (C=N);wherein Q3 is O, S or NH;wherein one of R2 and R3 is hydrogen and the other is(a) H;(b) alkyl of 1 to 8 carbon atoms, inclusive, which maybe straight chain or branched;(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;(d) alkenyl of 2 to 8 carbon atoms, inclusive, whichmay be straight chain or branched; or(9) RaQ2Rbwherein Q2 is -0- or -S-;wherein Ra is alkylene of 0 to 6 carbons atoms,inclusive, which may be straight chain orbranched;wherein Rb is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain orbranched;wherein R4 is(a) H;ICA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342- 19 -(b) alkyl of 1 to 6 carbon atoms, inclusive, which maybe straight chain or branched;wherein Y, or Y2 is -OH , methyl, -H or -SH and wherein theother is5 (a) H;(b) CHaZbwherea+b=3,a=Oto3,b=0to3Z is cyano, nitro, or halogen including F, Cl, Br, I;(c) alkyl of 2 to 4 carbon atoms, inclusive, straight10 chain or branched;(d) alkoxy of 1 to 4 carbon atoms, inclusive;or Y, and Y2 taken together are(a) =N; or(b) =0;15 wherein R5 is(a) alkyl of 1 to 9 carbon atoms which may be straightchain or branched;(b) '(CH2)n ‘Rswherein n = O to 4 and Ri is20 (i) cycloalkyl of 3 to 10 carbon atoms,inclusive;(ii) phenyl; or(iii) substituted phenylZi ZiiZiii2 5 ZV Zi Vwherein Z i , Z iii‘, and ZV are eachindependently selected from the groupconsisting of hydrogen, -N02, -CN,30 -C(=O)-RI, methoxy, and -SO3H;wherein Zii and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,CA 02265708 l999-03- 10W0 98/ l 1049 PCT/U S97! 16342- 20 _hydrogen, andhydroxyl;(C) 'RaQ¢1Rbwherein Qa = -0- or -S-; and5 wherein Ra is alkylene of 0 to 6 carbonsatoms, inclusive, which may be straight chainor branched;wherein Rb is alkyl of O to 8 carbon atoms,inclusive, which may be straight chain or10 branched;(d) 'C(Riii)(Riv)‘Riwherein Rm and Riv are selected independentlyfrom the group consisting of(i) H; and15 (ii)CHaZbwherea+b=3,a=0t03,b=0+3wherein any Z is selected from the groupconsisting of halogen.(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and l to6 halogen atoms, inclusive, straight chain or branched.20 In one embodiment of this invention, the lipoxin analogs have thefollowing structure III:III. 25wherein X is R1, OR,, or SR,;wherein R, is(i) hydrogen;1015202530W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97/16342_ -(ii) alkyl of 1 to 8 carbons atoms, inclusive,which may be straight chain or branched;(iii) cycloalkyl of 3 to 10 carbon atoms,inclusive;(iv) aralkyl of 7 to 12 carbon atoms;(v) phenyl;(vi) substituted phenylZi ZiiZi i iZv Zivwherein Z i , Z in’, and ZV are eachindependently selected from the groupconsisting of -N02, -CN, -C(=O)-R],hydrogen, and -SO3H;wherein Zii and Z“, are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, andhydroxyl;(vii) detectable label molecule; or(viii) alkenyl of 2 to 8 carbon atoms, inclusive,straight chain or branched;wherein Q, is (C=O), S02 or (C==N);wherein Q3 is O, S or NH;wherein one of R2 and R3 is hydrogen and the other is(a) H;(b) alkyl of 1 to 8 carbon atoms, inclusive, which maybe straight chain or branched;(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;((1) alkenyl of 2 to 8 carbon atoms, inclusive, whichmay be straight chain or branched; or(3) RaQ2Rbwherein Q2 is -O- or -S-;CA 02265708 l999-03- 10WO 98111049 PCT/US97/16342- 22 _wherein Ra is alkylene of O to 6 carbons atoms,inclusive, which may be straight chain orbranched;wherein Rb is alkyl of 0 to 8 carbon atoms,5 inclusive, which may be straight chain orbranched;wherein R4 is(a) H; or(b) alkyl of 1 to 6 carbon atoms, inclusive, which may10 be straight chain or branched;wherein Y, or Y2 is hydroxyl , methyl, hydrogen or thiol andwherein the other is(a) H;(b) CHaZb15 wherea+b=3,a=0to3,b=0to3Z is cyano, nitro, or halogen [including F, Cl, Br,I];(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;20 ((1) alkoxy of 1 to 4 carbon atoms, inclusive;or Y, and Y2 taken together are(a) =N; or(b) =0; andwherein R5 is25 (a) alkyl of 1 to 9 carbon atoms which may be straightchain or branched;(b) -(CH2)n ‘Riwherein n = 0 to 4 and Ri is(i) cycloalkyl of 3 to 10 carbon atoms,30 V inclusive;(ii) phenyl;(iii) substituted phenyl10152025W0 98/ 1 1049ICA 02265708 l999-03- 10PCT/US97I16342_ 23 -wherein Z i , Z “L, and Z‘, are eachindependently selected from the groupconsisting of hydrogen, —NO2, -CN,-C(=O)-R], methoxy, and -SO3H;wherein Zii and Z“, are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(C) ‘RaQaRbwherein Q3 = -0- or -S-;wherein Ra is alkylene of 0 to 6 carbonsatoms, inclusive, which may be straight chainor branched;wherein Rb is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain orbranched; or(C1) "C(Riii)(Riv)’Riwherein Rm and Riv are selected independentlyfrom the group consisting of(i) H;(ii)CHaZbwherea+b=3,a=0to3,b=0+3wherein any Z is selected from the groupconsisting of halogen.In another embodiment of this invention, lipoxin analogs have the10152025CA 02265708 l999-03- 10W0 98lll049 PCTlUS97I16342-24-following structural formula IV:IV. wherein X is R], OR1, or SR];wherein R] is(i) hydrogen;(ii) alkyl of l to 8 carbons atoms, inclusive,which may be straight chain or branched;(iii) cycloalkyl of 3 to 10 carbon atoms,inclusive;(iv) aralkyl of 7 to 12 carbon atoms;(V) phenyl;(vi) substituted phenylZi ZiiZiiiZv Zivwherein Z i , Z iii’, and Zv are eachindependently selected from the groupconsisting of -N02, -CN, -C(=O)-R],methoxy, hydrogen, and -SO3H;wherein Z“ and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(vii) detectable label molecule; or(viii) alkenyl of 2 to 8 carbon atoms, inclusive,101520253035CA 02265708 l999-03- 10W0 98/ 1 1049 PCT/US97/16342-25-straight chain or branched;wherein Q1 is (C=O), S02 or (CN);wherein Q3 is O, S or NH;wherein one of R2 and R3 is hydrogen and the other is(a) H;(b) alkyl of 1 to 8 carbon atoms, inclusive, which maybe straight chain or branched;(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;(d) alkenyl of 2 to 8 carbon atoms, inclusive, whichmay be straight chain or branched; or(C) RaQ2Rbwherein Q2 is -0- or —S-;wherein R, is alkylene of 0 to 6 carbons atoms,inclusive, which may be straight chain orbranched;wherein Rb is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain orbranched;wherein R4 is(a) H; or(b) alkyl of 1 to 6 carbon atoms, inclusive, which maybe straight chain or branched;wherein Y, or Y2 is —OH , methyl, or -SH and wherein theother is(a) H;(b) CHaZbwherea+b=3,a=Oto3,b=0to3,Z is cyano, nitro, or halogen;(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched; or(d) alkoxy of 1 to 4 carbon atoms, inclusive;or Y, and Y2 taken together are(a) =N; or(b) =0;wherein R5 is(a) alkyl of 1 to 9 carbon atoms which may be straightchain or branched;1015202530W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342_ 25 -(b) -(CH2)n 'Riwherein n = 0 to 4 and Ri is(i) cycloalkyl of 3 to 10 carbon atoms,inclusive;(ii) phenyl; or(iii) substituted phenylZi ZiiZi i lZv Zivwherein Z i , Z iii’, and Z‘, are eachindependently selected from the groupconsisting of hydrogen, -N02, -CN,-C(=O)-R1, methoxy, and -SO3H;wherein Z“ and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(C) RaQaRbwherein Qa = -0- or -S-;wherein Ra is alkylene of O to 6 carbonsatoms, inclusive, which may be straight chainor branched;wherein Rb is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain orbranched;(d) ‘C(Riii)(Riv)'Riwherein Rm and Riv are selected independentlyfrom the group consisting of(i) H; or(ii)CHaZbwherea+b=3,a=Oto3,b=0+3, andwherein any Z is selected from the groupICA 02265708 l999-03- 10W0 98/ 11049 PCT/U S97/ 16342-27-consisting of halogen; or(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and l to6 halogen atoms, inclusive, straight chain or branched;and5 wherein R6 is(a) H;(b) alkyl from 1 to 4 carbon atoms, inclusive, straightchain or branched; or(c) halogen.10 In another embodiment of this invention, lipoxin analogs have thefollowing structural formula V:V. 15wherein R, is(i) hydrogen;(ii) alkyl of 1 to 8 carbons atoms, inclusive,which may be straight chain or branched;20 (iii) cycloalkyl of 3 to 10 carbon atoms,inclusive;(iv) aralkyl of 7 to 12 carbon atoms;(V)Ph€nyh(vi) substituted phenyl25101520253035CA 02265708 l999-03- 10W0 98/11049 PCT/U S97/ 16342-23-wherein Z i , Z iii’, and Zv are eachindependently selected from the groupconsisting of -N02, —CN, -C(=O)-R,,hydrogen, and —SO3H;wherein Z“ and Z“, are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, and hydroxyl;(vii) detectable label molecule; or(viii) alkenyl of 2 to 8 carbon atoms, inclusive,straight chain or branched;wherein n = 1 to 10, inclusive;wherein R2, R33, and R31, are independently selected from(a) H;(b) alkyl of 1 to 8 carbon atoms, inclusive, which maybe straight chain or branched;(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;(d) alkenyl of 2 to 8 carbon atoms, inclusive, whichmay be straight chain or branched; or(9) RaQ2Rbwherein Q2 is -0- or -S-;wherein Ra is alkylene of 0 to 6 carbons atoms,inclusive, which may be straight chain orbranched; and wherein Rb is alkyl of 0 to 8carbon atoms, inclusive, which may be straightchain or branched;wherein Y, or Y2 is -OH , methyl, hydrogen, or -SH andwherein the other is(a) H;(b) CHaZbwherea+b=3,a=0to 3,b=0to 3, andZ is cyano, nitro, or halogen;(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;(d) alkoxy of 1 to 4 carbon atoms, inclusive, straightchain or branched;or Y] and Y2 taken together are101520253035CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342-29-(a) =N; or(b) =0;wherein Y3 or Y4 is -OH , methyl, hydrogen, or -SH andwherein the other is(a) H;(b) CHaZbwherein a + b =3, a= 0 to 3, b = 0 to 3,and any Z is cyano, nitro, or halogen;(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;(d) alkoxy of l to 4 carbon atoms, inclusive, straightchain or branched;or Y3 and Y4 taken together are(a) =N; or(b) =0;wherein Y5 or Y6 is -OH , methyl, hydrogen, or -SH andwherein the other is(a) H;(b) CHaZbwherea+b=3,a=0to 3,b=0to3Z is cyano, nitro, or halogen;(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;(d) alkoxy of 1 to 4 carbon atoms, inclusive, straightchain or branched;or Y5 and Y6 taken together are(a) =N; or(b) =0;wherein R5 is(a) alkyl of l to 9 carbon atoms which may be straightchain or branched;('3) -(CH2).. ‘R1wherein n = 0 to 4 and Ri is(i) cycloalkyl of 3 to 10 carbon atoms,inclusive;(ii) phenyl; or(iii) substituted phenyl1015202530W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342- 30 _wherein Z i , Z “L, and Zv are eachindependently selected from the groupconsisting of hydrogen, -N02, -CN,-C(=O)-R1, and -SO3H;wherein Zn and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, methoxy, and hydroxyl;(C) 'RaQaRbwherein Q, = -0- or -S-; andwherein Ra is alkylene of 0 to 6 carbonsatoms, inclusive, which may be straight chainor branched;wherein Rb is either alkyl of 0 to 8 carbonatoms, inclusive, which may be straight chainor branched or substituted phenyl;(d) -C(Riii)(Riv)-Riwherein Rm and Riv are selected independentlyfrom the group consisting of(i) H; or(ii) CHaZbwherea+b=3,a=Oto 3,b=0+3, andwherein any Z is selected from the groupconsisting of halogen; or(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to6 halogen atoms, inclusive, straight chain or branched;but excluding the C-1 position amides, C-1 positionalkanoates, and pharmaceutically acceptable C-1position salts of (SS, 14R, 15S)-trihydroxy-6E, 8Z,'10E, 12E-eicosatetraenoic acid (LX134); C-5, C-6, and1CA 02265708 l999-03- 10W0 98/1 1049 PCT/U S97/ 16342- 3 1 -C-5 position alkanoates of LXB4 _In another embodiment of this invention, lipoxin analogs have thestructural formula VI:5 VI.HO OH O2 RbOHwherein Ra is selected from the group(a) H; or10 (b) alkyl of 1 to 8 carbon atoms;wherein Rb selected from the group consisting of:Zii.__CH2Q Ziii ; -—-CH2O OCH3Ziv15In another preferred embodiment of this invention, lipoxin analogs havethe following structural formula VII:VII.20 wherein Ra is selected from the group(a) H; or(b) alkyl of 1 to 8 carbon atoms;25 wherein Rb and Re are independently selected from the groupCA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342- -(a) H;(b) hydroxyl, or thiol;(c) methyl or halomethyls including -CF3 and -CH2F;(d) halogen;5 (e) alkoxy of 1 to 3 carbon atoms, including methoxy;wherein Rd and Re are selected independently from the group(a) H;(b) hydroxyl, or thiol;(c) methyl or halomethyl including -CF3 and -CHZF;10 (d) halogen;(e) alkoxy of 1 to 3 carbon atoms, inclusive, includingmethoxy; or(0 alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive,which may be straight chain or branched; but excluding15 the C-1 position amides, C-1 position alkanoates, andpharmaceutically acceptable C-1 position salts of (SS,6R, 15S)-trihydroxy-7E, 9E, 11Z, 13E-eicosatetraenoicacid (LXA4); C-5, C-6, and C-15 position alkanoates of LXA4.20In another preferred embodiment of this invention, the lipoxinanalogs have the structural formula VIII:VIII.25wherein R3 is selected from the group(a) H; or(b) alkyl of I to 8 carbon atoms;30 wherein Rb and RC are independently selected from the group(a) H;lCA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342- 33 _(b) hydroxyl or thiol;(c) halomethyl, including CF3;(d) halogen;(6) alkyl of 1 to 3 carbon atoms, inclusive, straight5 chain or branched; or(f) alkoxy of 1 to 3 carbon atoms, inclusive;wherein Rd and Re are selected independently from the group(a) H;(b) hydroxyl, or thiol;10 (c) methyl or halomethyl including -CF3 and —CH2F;(d) halogen;(e) alkoxy of 1 to 3 carbon atoms, inclusive, includingmethoxy; or(0 alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive,15 which may be straight chain or branched.In another preferred embodiment of this invention, the lipoxinanalogs have the structural formula IX:IX. 20wherein R3 is selected from the group(a) H; or(b) alkyl of 1 to 8 carbon atoms;25 wherein Rb and Re are independently selected from the group(a) H;(b) hydroxyl or thiol;(c) halomethyl, including CF3 and CH2F;(d) halogen;CA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342-34-(e) alkyl of l to 3 carbon atoms, inclusive, straightchain or branched;(0 alkoxy of l to 3 carbon atoms, inclusive; andwherein R5 is5 (a) alkyl of 1 to 9 carbon atoms which may be straightchain or branched;(b) -(CH2)n -R1wherein n = 0 to 4 and Ri is(i) cycloalkyl of 3 to 10 carbon atoms,10 inclusive;(ii) phenyl; or(iii) substituted phenylZi ZiiZiiiZv Ziv15wherein Z i , Z M’, and ZV are eachindependently selected from the groupconsisting of hydrogen, -N02, -CN,-C(=O)-R], and -SO3H;20 wherein Z“ and Ziv are eachindependently selected from the groupconsisting of halogen, methyl,hydrogen, methoxy, and hydroxyl;(C) 'RaQaRb25 wherein Q,, = -0- or -S—;wherein R3 is alkylene of 0 to 6 carbonsatoms, inclusive, which may be straight chainor branched;wherein Rb is either alkyl of 0 to 8 carbon30 atoms, inclusive, which may be straight chainor branched or substituted phenyl;(<1) -C(Rm)(Rav)-Rawherein Rm and Riv are selected independently101520251CA 02265708 l999-03- 10W0 98/1 1049 PCT/U S97/ 16342-35-from the group consisting of(i) H; or(ii) CHaZbwherea+b=3,a=0to 3,b=0+3wherein any Z is selected from the groupconsisting of halogen; or(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to6 halogen atoms, inclusive, straight chain or branched.In another preferred embodiment, the compounds have thestructural formula X:X.OH OHO Rb "I;/,RCwherein R3 is selected from the group(a) H; or(b) alkyl of 1 to 8 carbon atoms, inclusive, straightchain or branched; andwherein Rb and RC are independently selected from the group(a) H;(b) hydroxyl or thiol;(c) halomethyl, including, for example, CF3;(d) halogen;(e) alkyl of 1 to 3 carbon atoms, inclusive, straightchain or branched;(0 alkoxy of 1 to 3 carbon atoms, inclusive, includingmethoxy.1015202530CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342-36-In another preferred embodiment, the compounds have thestructural formula X1:X1.0Ra wherein Ra is(i) hydrogen;(ii) alkyl of 1 to 8 carbons atoms, inclusive,which may be straight chain or branched; or(iii) detectable label molecule;wherein n = l to 10, inclusive;wherein Y2, R33, and R3b are independently selected from(a) H;(b) alkyl of 1 to 8 carbon atoms, inclusive, which maybe straight chain or branched;(c) cycloalkyl of 3 to 6 carbon atoms, inclusive;(d) alkenyl of 2 to 8 carbon atoms, inclusive, whichmay be straight chain or branched; or(C) RaQ2Rbwherein Q2 is -O- or -S-;wherein R8 is alkylene of 0 to 6 carbons atoms,inclusive, which may be straight chain orbranched; and wherein Rb is alkyl of 0 to 8carbon atoms, inclusive, which may be straightchain or branched;wherein Y] is -OH , methyl, or -SH ;wherein Y2 is(a) H;(b) CHaZbwherea+b=3,a=0to3,b=0to3Z is halogen; or1015202530W0 98/1 10491CA 02265708 l999-03- 10PCT/US97/16342-37-(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;wherein Y3 and Y5 are independently selected from the groupconsisting of :(8) H;(b) CHaZbwhereina+b=3,a=0to3,b=0to3and any Z is cyano, nitro, or halogen; or(c) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;wherein Y4 and Y6 are independently selected from the groupconsisting of:(w H;(b) alkyl of 2 to 4 carbon atoms, inclusive, straightchain or branched;(c) alkoxy of 1 to 4 carbon atoms, inclusive, straightchain or branched; or((1) hydroxyl or thiol; andwherein R5 is(a) alkyl of 1 to 9 carbon atoms which may be straightchain or branched;(b) -(CH2)n 'Riwherein n = 0 to 3 and R, is(i) cycloalkyl of 3 to 10 carbon atoms,inclusive;(ii) phenyl;(iii) substituted phenylZi ZiiZiiiZv Zivwherein Zii and ZN are eachindependently selected from the groupconsisting of halogen, methyl,CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342- 38 -hydrogen, methoxy, and hydroxyl;(C) ‘RaQaRbwherein Q3 = -0- or -S-;wherein Ra is alkylene of 0 to 6 carbons5 atoms, inclusive, which may be straight chainor branched;wherein Rb is_._CH2Q Ziii ; :CH2O OCH3Ziv1 0(d) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6halogen atoms, inclusive, straight chain or branched;orbut excluding the C-1 position amides, C-1 positionalkanoates, and pharmaceutically acceptable C-1 position salts15 of (SS, 14R, 15S)-trihydroxy-6E, 8Z, 10E, 12E-eicosatetraenoic acid (LXB4);C-5, C-6, and C-5 position alkanoates (acetates) of LXB4 _In the most preferred embodiment of this invention, the compounds of this invention20 have the following structural formulas:ICA 02265708 l999-03- 10W0 98/ 1 1049 PCT/U S97/16342-39-OH OHCA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342-40- OR.CH3OR'CH3OIIIIIIIIECOH O OR.OH 5Hwhere R’ is H or CH3;and where the substituents at C* are in the R configuration.In other preferred embodiments of this invention, the compounds of thisinvention have the following structural formulas:CA 02265708 l999-03- 10W0 98/11049 PCT/U S97l 16342\ \ OH/or OH 10152025W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342- 42 - OH\\\//A\\v/,cooH\HO OHwhere the substituents at the C* are in the R configuration.Method for Making Lipoxin CompoundsPreferred compounds can be made as specifically described in thefollowing Example 1. Other compounds of this invention can be made by combinedstrategies for lipoxin (LX) and prostaglandin analog synthesis using standard chemicalmethods such as selective hydrogenation, Pd(O)-Cu(I) coupling, Wittig-type coupling,Sharpless epoxidation, and asymmetric reductions following coupling of the majorintermediates described below and in the literature to generate the stable LX analogs ofthis invention.(Webber, S.E. et al. (1988) Adv. Exp. Med. Biol. 229261; Radiichel, B.and Vorbriiggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263;and Nicolaou, K.C. et al. (1991) Angew Chem. Int. Ed. Engl. 30:1100). Geometricalvariations can be accomplished e.g. as described in U.S. Patent No. 4,576,758 andNicolaou, K.C. (1989) J. Org. Chem. 54: 5527.As shown below, LX analog compounds comprising subgenus 1 inScheme I can be prepared as three major fragments (A, 1;, and Q), which can then becombined to form the total molecule.ICA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342_ -Scheme Il-—- 5.2.o R2Q1 PhHO \/R310 Synthesis of the epoxy-alcohol for precursor fragment Q can be generatedwith substitutents R2, R3 and R4 selected from hydrogen, phenyl, halogen or methyl.Each of these respective epoxy-alcohols may be transformed into phenyl urethanederivatives as 3.15 Q0 R4 R2Q1 PhPmm)ko \/R3with PhNCO, pyrimidine and CH2Cl2 followed by Lewis acid catalysis by SN2 openingto give a 1,2 cyclic carbonate that contains the vicinal diol at20 C-6 in the (R) configuration required for binding and C-5 in the (S) configuration alsoestablished for bioactivity and binding at a recognition site. These alcohols are nextprotected to generate the precursor A fragment as 5.10152025W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/ 16342-44-H3 These A fragments can now be coupled to the fragment _I_3_ intermediate, aphosphonium bromide Q as in Webber, S.E. et al. (1988) Adv. Exp. Med. Biol. 229:6] ingram quantities to generate the combined A + 3 fragment products Q.: P(Ph)3Me3Si9A + B OH Q3H R2QMe3Si R4 R3Fragment _C_ intermediates from Scheme I are generated in parallel topreparation of A—B couplings. In these _C_ fragments, substitutions at Y1 and/or Y2 aremethyl, methoxy, hydrogen, cyano, nitro, or halogen; see specific example 3. Thus,carrying l5-methyl and/or, for example, 16-methyl or 16—phenoxy-derivatives permitsthese substituted-LXA4 analogs to be not susceptible to dehydrogenation.Thus, the _C_ fragments carrying the preferred resistance to enzymaticoxidation and/or dehydrogenation may be converted by protection of key sites, followedby bromination to give vinyl bromide products of fragment Q such as Q that is coupledto Q by using catalytic amounts of P(Ph3)4 and CuI to generate the complete backbonestructure of the LXA4 analogs of genus formula I. This scheme is further illustrated bythe following Examples.ICA 02265708 l999-03- 10W0 98/11049 PCT/U S97/ 16342_ 45 _ 5Scheme II.‘\‘~\‘A R2B :3’ Q1" (3‘)“~~ R4 R3I C RY1 Y2 5The compounds of this invention within subgenus formulas II and III10 may be made synthesized in a similar manner.Compounds in genuses II and III are generated by first individuallypreparing substituted compounds of fragment A that are each coupled as in Scheme I toindividually prepared fragment 1; to generate 1 or A1 + l_3_1 fragments possessingindividual substitutions at X, Q1, R2, R3, and R4 as indicated._ — HO Q3H R2\ \ Q‘\xMe3Si R4 R3101520CA 02265708 l999-03- 10 W0 98/11049 PCT/US97/ 16342_ 45 -§\\BThe C1 fragment § carrying the acetylenic group C-14,15 and the co-C-20end substitutions will each be generated as shown above for structure 6 prostaglandinanalogs and converted to their corresponding vinyl bromide products as in (KCN JAC1985, Webber) to yield brominated products of each individual substituted fragment C1or § species that are suitable for coupling to 2_0 using catalytic amounts of P(Ph3)4 andCuI to generate the combined products of the acetylenic-LXA4 analog class. Each of thefinal products may then be subject to gradient RP-HPLC using rapid diode arraydetection (as in Serhan, C.N., Methods in Enzymology) for purification. The presenceof the modification at C-15 thru C-20 of LXA4 can alter metabolism by dehydrogenasesand oxidases by providing steric hindrance, stable prostaglandin analogs carrying C-15to u)-end substitutions have been prepared and are not metabolized by dehydrogenases(Radiichel, B. and Vorbriiggen, H. (1985) Adv. Prostaglandin Thromboxane LeukotrieneRes. 142263 and Vorbriiggen, H. et al. In: Chemistry, Biochemistry, andPharmacological Activity of Prostanoids (Roberts, S.M., Scheinmarm, F. eds.). Oxford:Pergamon Press).The cyclo-LXA4 compounds of this invention within genus formula IVmay be made in the following manner.Scheme III.|\O 10152025W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97/16342-47-The parent compound of this class is also subject to a similar totalsynthesis strategy and is assigned three main fragments A, B, and Q in structure ffl. Aprecursor for fragment A may be prepared by routes used in Nicolaou, K.C. (1989) J.Org. Chem. 54:5527 to prepare _1__Q in the synthesis of 7-cis,1 1-trans—LXA4 methyl ester.E OHR6Fragment B in 30 can be obtained via the precursor 11 or saligenin-([0-hydroxybenzylalcohol) as generated in (Vorbrtiggen et al., p. 353). The benzyl alcohol1_1_ is reacted with _1_Q (1:1) in the presence of NaH in DMF to give 12. This keyintermediate is silylated in BSTFA followed by coupling with individual fragmentsdesigned for C precursors.2HO Q3H R2\ Q1 KOH R3R6Q can then be coupled to vinyl brominate fragment C of given individualdesign by treating the bromite precursor with 4.0 equivalents of AgNO3, then 7.0 equiv.of KCN, EIOH/THF/H20 (1 :1 :1), 0 :> 25°C, 2-4 h. the individual products are thensubject to Lindlar cat. for selective catalytic mild hydrogenation in CH2Cl2 2-3 h to giveindividual compounds belonging to the genus IV. Each can be saponified in LiOH/THFto give corresponding free acids after isolation by RP-HPLC.CA 02265708 l999-03- 10W0 98/ 1 1049 PCTIUS97/16342- 43 _.12R0 Q3H R2Q1\\XSiMe3 35 The invention of genus IV compounds is illustrated further below, thesynthesis of l5(i)methyl-cyclo-LXA4 methyl ester and corresponding free acid.The compounds of this invention within genus formula V may be madein the following manner. Several systems studied with LXB4 indicate that several siteswithin the natural compound are required for bioactivity (Serhan, C.N. (1991) J.10 Bioenerg. and Biomembr. 232105). These sites include the C-14 alcohol in the (R)configuration and the double bond at 08,9 of the tetraene in the cis configuration. Inaddition, based on metabolic studies resulting in the instant invention, several keyaddition sites have been identified as being necessary to preserve LXB4 bioactivity.These include preserving the C-15 alcohol from dehydrogenase activity (i.e., block 5-15 oxo—LXB4 formation); maintaining both the A8 bond and 14(R) alcohol; and preventingreduction of A6-7 double bond and [3/co -oxidations of the resultant compounds.Scheme IV20_1_fi Thus, genus V (fl) maintains the regions of LXB4 which are necessaryfor its bioactivity, but modifies the regions available to metabolic degradation. Again, a25 retrosynthetic analysis gives priority to three key fragments A, B and C designated in l_4.Coupling of key intermediates to generate members of the LXB4 analog class usesstandard techniques as outlined for LXA4 and its analogs namely, selective101520W0 98/1 10491CA 02265708 l999-03- 10PCT/US97/16342-49-hydrogenation (to generate 8-cis geometry); Pd(O)—Cn(I) coupling to join A & 3fragments carrying unique substitutions; Wittig-type coupling to join C fragments thatcarry the required substitutions and Sharpless epoxidation to yield the l4(R) vicinalalcohol (see ref. Nicolaou, K.C. et al. ( 1991) Angew. Chem. Int. Ed. Engl. 30:1 100.),ref.; Weber, S.E. er al. (1988) Adv. Exp. Med. Biol. 229161 Ed. Wong, P.K. and Serhan,C.N.) and those cited within). Thus by using a similar strategy to LXA4 analogs and theconstruction of native LXB4 the specific analogs can be obtained.1.5. The A fragments of _lj that carry substitutions at R2, R6, R7 that can be(H, CH3, OCH3, phenyl, halo-substituted phenyl are generated by standard methodsfrom, for example _l_6 where= CH2 of increasing chain length._1_6O R.Q1\Cl (CH2)n XRHO Rl /Q1% (CH2)n \XMe3Si RJ5OHRQ:% (CH2)r1/ \X....t -_.._......._.—..——-.................w......,._.,.~.. ,10152025W0 98/1 1049CA 02265708 l999-03- 10PCT/U S97! 16342_ 50 _Compound _1_6 is converted to the vinyl bromide _l_5 as in (N icolaou K.C. et al. (1991)Angew. Chem. Int. Ed. Engl. 30: 1100-16.) via a trimethylsilyl acetylenic intermediatel_7_ that is reduced by pinanyl—9BBN then n- BuN4NF in THF to yield _l_8 than is nowsubmitted for bromination afier protecting essential moieties such the alcohol to give 1;(fragment A).The fragment _C_I in E is generated from compound 12 with the R5substitutions as indicated.ER522HO“ R52_1CH RTBDMS 5The acetylenic alcohol 1_2 is then reduced in LAH followed by Sharplessasymmetric epoxidation to generate 2_0 that is isolated by RP-HPLC to yield the (+)isomer that is used to generate the required C-14 alcohol of LXB4 analogs in the (R)configuration compound go is transformed to the corresponding aldehyde 2_l_ afterprotection of the substituted groups carried at R4 and R5 as well as the alcohol usingPCC in methylene chloride. (Nicolaou, K.C. et al. (1991) Angew. Chem. Int. Ed. Engl.3021100).22.Br’IJ;(Ph)3lCA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342_ 5] -The phosphonium salt Q can be prepared as in Ref.; (Weber, S.E. et al.(1988) Adv. Exp. Med. Biol. 229:6l, Ed. Wong, P.K. and Serhan, C.N.) and used hereto generate the B-_C fragment coupling via a Wittig-type coupling to give Q carrying thedesignated substitutions in the group of 2; where R4 and R5 carry substitutions. This cis5 double bond of _2_; can be is isomerized to give the trans isomer using 12 as a catalyst togive the parent precursor form of _1_f1_ as 23. 2-;1024"" TBDMSR% \ \ R5Me3SiE15 TBDMS RThen coupling of 23 to 1_5_ is accomplished by Pd(O) - Cu(I) coupling togive the acetylenic precursor of 13 designated 2;. Following selective Lindlar catalytichydrogenation the individual LXB4 analogs can be further purified via RP-HPLC used101520253035W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342- 52 -the tetraene skeleton as a convenient means to isolate individual products employingrapid diode array detection (Serhan, C.N. (1990) Meth. Enzymol. 187: 167).UtilitiesThe compounds of this invention have the biological activity of naturalLXs, but are more resistant to degradation or alternatively inhibit the degradation ofnatural LXs. The disclosed compounds therefore have utility as pharmaceuticals fortreating or preventing a number of diseases or conditions associated with inadequate orinappropriate LX mediated cellular response in a subject.Based on the anti-proliferative effect of the disclosed 15-epi-lipoxincompounds, the invention provides methods for ameliorating undesired cell proliferationby contacting cells with a pharmaceutical composition including an effective amount ofthe substantially purified 15-epi-LX compound and a pharmaceutically acceptablecarrier. The cell can be contacted in vivo and/or in vitro. Alternatively, cells can beremoved from a subject; contacted with the substantially purified l5—epi-lipoxincompound of the present invention ex vivo and implanted in the subject. The inventionalso provides a method for ameliorating a cell proliferative disorder in a subjectincluding administering an effective amount of a substantially purified 15-epi-LXcompound.The effective amount is ordinarily the amount which is required to assuresufficient exposure to a target cell population. Such an amount will ordinarily dependupon the nature of the 15-epi-LX compound, the mode of administration, the severity ofthe undesired cell proliferation or cell proliferative disorder and other factors consideredby a person of ordinary skill when determining a dosage regimen.Target cells to be contacted can be undergoing cancerous and/ortumorous growth. Alternatively, target cells can be undergoing abnormal cellproliferation in response to a stimulus, such as restenosis; and/or target cells can consistof transformed cells having a genetic makeup altered from that of the original cells.Preferred target cells include epithelial cells, leukocytes, endothelial cells,and/or a fibroblasts.Based on the stimulatory action of LXs on selected cells, the inventionalso provides methods for treating a subject with a myeloid suppressive disorder byadministering to the subject an effective amount of a pharmaceutical compositioncomprising a LX analog. The effective amount is ordinarily that amount which isrequired to assure sufficient exposure to the target cell population. Such an amount willordinarily depend upon the nature of the analog, the mode of administration, the severity101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/U S97! 16342_ 53 -of the myeloid suppression, and other factors considered by a person of ordinary skillwhen determining a dosage regimen.Therapeutic use of a cell proliferative LX analog also includes removingcells from a subject, stimulating cell growth in vitro, and reintroducing the enhanced cellpreparation, in whole or in part, into the subject. Additional therapeutic agents (e.g.cytokines such as GM—CSF) may be optionally used in conjunction with the LX duringstimulation or in conjunction with the introduction of the cell preparation.In another embodiment, the compounds of this invention are used to treator prevent inflammation or an inflammatory response. LXA4inhibits the activation of leukocytes which are mediators of inflammation. The LXA4 -induced effect includes inhibition of leukocyte migration, generation of reactive oxygenspecies, and the formation of pro-inflammatory mediators involved in tissue swelling.(Raud, J. et al. (1991) Adv. Exp. Med. Biol. 314:185. Cel1—Cel1 Interactions in theRelease of Inflammation Mediators vol. 314) LXB4 exhibits radioprotective actions,such as preventing diarrhea and ataxia, in an in vivo assay with mouse hematopoieticstem cells. (Walken, T.L. Jr.,(l988) J . Radiat. Res. 292255)The leukocyte-mediated inflammation or inflammatory responses causeor contribute to a wide variety of diseases and conditions including various forms ofasthma and arthritis. Included within the present invention are inflammatory responsesto physical injury, such as physical trauma, radiation exposure, and otherwise.In another embodiment, the compounds of this invention are used to treator prevent inflammation by antagonizing the action of leukotrienes. LXA4 inhibitsLTB4—induced inflammation, blocking both plasma leakage and leukocyte migration inan in vivo assay of the hamster cheek pouch. (Hedqvist, P. et al. (1989) Acta Physiol.Scand. 137 : 571. ) Plasma leakage and leukocyte migration are key events in bothwound healing and inflammation. LXA4 also antagonizes LTD4-induced renalhemodynamic actions and blocks the binding of LTD4 to mesangial cellswhich are responsible, in part, for regulating hemodynamics in the kidney . (Badr. K.F.et al. (1989) Proc. Natl. Acad. Sci. USA 86: 438. )The compounds of this invention may be administered to antagonize theaction of sulfidopeptide leukotrienes, such as LTD4, LTC4, and LTB4. Leukotriene-mediated vasoconstrictive responses are associated with diseases such as: asthma,anaphylactic reactions, allergic reactions, shock, inflammation, rheumatoid arthritis,gout, psoriasis, allergic rhinitis, adult respiratory distress syndrome, Crohn's disease,endotoxin shock, traumatic shock, hemmorrhagic shock, bowl ischemic shock, renalglomerular disease, benign prostatic hypertrophy, inflammatory bowl disease,101520253035W0 98/11049CA 02265708 l999-03- 10PCT/US97l16342- -myocardial ischemia, myocardial infarction, circulatory shock, brain injury, systemiclupus erythematosus, chronic renal disease, cardiovascular disease, and hypertension.In another embodiment, the compounds of this invention are used to treator prevent a vasocontractive response or condition. LXs induce endothelium-dependentvasodilation (LXA4) (Lefer, A.M. et al (1988) Proc. Natl. Acad. Sci. USA 8518340) anddilation of cerebral arterioles in new born pigs in vivo (LXA4 and LXB4) ( Busija, D.W.et al. (1989) Am . J. Physiol. 256:468. ). Furthermore, LXA4 induces rapid arteriolardilation in hamster cheek pouch in vivo (Dahlen, S.-E. et al. (1987) Acta Physiol.Scand. 1302643 ) and in the renal hemodynamics of the rat. ( Badr, K.F. et al. (1987)Biochem. Biophys. Res. Commun. 145: 408).Vasocontractive responses or conditions cause, contribute, or areassociated with diseases and conditions such as renal hemodynamic diseases, includingglomerular diseases, cardiovascular diseases including hypertension, myocardialinfarction, myocardial ischemia, and vascular diseases and gastrointestinal diseases.Also encompassed by this invention is a method of screening LX analogsor other compounds to identify those having a longer tissue half-life than thecorresponding natural LX. This method can be used to determine whether thecompound inhibits, resists, or more slowly undergoes metabolism compared to thenatural LX. This method is performed by preparing at least one enzyme whichmetabolizes LXs, contacting the compound with the enzyme preparation, anddetermining whether the compound inhibits, resists, or more slowly undergoesmetabolism by the enzyme. Cells having a LX recognition site, such aspolymorphonuclear neutrophils, peripheral blood monocytes, and differentiated HL-60cells are among the appropriate sources for the enzyme preparation. The LX recognitionsite may exist naturally, or be induced artificially, by a disease state, or by an injury. Anon-limiting example of artificially-induced LXA4 recognition sites is the induction ofsuch sites in differentiated HL-60 cells.In one embodiment, preparation of the enzymes comprised harvestingcells and performing freeze-thaw lysis three times, followed by ultracentrifugation toyield a 100,000g supernatant. A cell-free 100,000g pellet may also be used. In addition,an enzyme preparation may comprise any enzymes that do not participate in natural LXmetabolism, but perform transformations upon LXs similar or equivalent to thosetransformations performed by the enzyme or enzymes which naturally metabolize LXS.Nonlimiting examples of appropriate enzymes are 15-hydroxyprostaglandindehydrogenase, cytochrome P—450 monogenases from human leukocytes, and rat andhuman liver microsomes.101520253035W0 98/11049ICA 02265708 l999-03- 10PCT/US97/16342_ 55 _Characterization of LX metabolites included standard techniques such asextraction, chromatography, and quantitative I-IPLC followed by trimethyl silylderivatization, O-methoxime derivatization and gas chromatography/mass spectroscopyanalysis. The experimental details of this embodiment are described below in Example1.LX analogs can also be screened for binding activity with a LX receptorrecognition site, for example by contacting the compound with a receptor recognitionsite and determining whether and to what degree the compound binds. Examples ofkinetic binding assays include homologous displacement, competitive binding, isotherm, and equilibrium binding assays.The receptor recognition site may normally exist or it may be induced bya disease state, by an injury, or by artificial means. For example, retinoic acid, PMA, orDMSO may be used to induce differentiation in HL-60 cells. Differentiated HL-60 cellsexpress LXA4-specific receptor recognition sites. Examples of other cells which may bescreened for LX specificity include PMN, epithelial cells, and peripheral bloodmonocytes.Selection of competitive ligands will depend upon the nature of therecognition site, the structure of the natural substrate, any structural or functionalanalogs of the natural substrate known in the art, and other factors considered by askilled artisan in making such a determination. Such ligands also include knownreceptor antagonists. The compounds of this invention may be radiolabelled withisotopes including 2H, 3H, '3C, and '4C by standard techniques known in the art ofradiochemistry or synthetic chemistry.In one embodiment of this method, the structural specificity of inducedLXA4 recognition sites was assessed with LXB4, LTC4, LTB4 and trihydroxyheptanoicmethyl ester. The experimental details of this embodiment are described below inExample 2.In addition, the compounds of this invention may be used to exert certainactions on specific cell types as developmental models for inflammation and injury. Forexample, LXA4 stimulates the mobilization of intra—cellular Ca“, lipid remodeling, andchemotaxis without aggregation in human PMN (Palmblad, J . et al. Biochem. Biophys.Res. Commun. (1987) 145: 168; Lee, T.H. et al. Clin.Sci. (1989) 771195; Nigam, S. etal. J.Cell. Physiol. (1990) 143:5l2; Luscinskas, F.W. et al. ( 1990) Biochem. Pharmacol.39:355). LXA4 also blocks both LTB4 and FMLP-induced responses, such as IP3generation. LXB4 also stimulates lipid remodeling. LXA4 activates isolated PKC, andis specific for the y-subspecies of PKC which is found in the brain and spinal cord.(Hansson, A. et al. Biochem. Biophys. Res. Commun. (1986) 134: 1215; Shearman,CA 02265708 2002-05-14i\"l.S. er al. FEBS Lett. (1989) 245: 167)_The present invention is further illustrated by the following exampleswhich should in no way be construed as being further limiting.ICA 02265708 l999-03- 10W0 98/ 11049 PCT/U S97/ 16342- 57 -ExamplesExample 1 Synthesis of Lipoxin Analog Compounds1 CAo+A»L.,.Me1 2HO OH O HO OH O\) II V\ IC °“OH H3 4HO OH O HO OH 0Preparation of the methvl ester precursor of compound 1:To a solution of 3-methyl-3-trimethy1si1oxy-1-bromo—1 -octene (130 mg. 0.44mmol) in benzene (1.5 mL) was added n-propylamine (0.05 mL, 0.61 mmol) and10 Pd(PPh3)4 (20 mg. 0.02 mmol) and the solution was protected from light. It was thendegassed by the freeze-thaw method and stirred at rt for 45 min. (7E, 9E, SS, 6R)Methyl 5,6-di(tert-butyldimethylsiloxy)-dodeca—7,9-diene-1 1—ynoate (183 mg. 0.44101520253035W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342- 58 -mmol) (compound 12) and copper iodide (14 mg. 0.07 mmol) were added and thesolution was one more time degassed by the freeze-thaw method. The mixture wasstirred for 3 h at rt and quenched with saturated aqueous solution of NH4C1 andextracted with ether. It was then washed with brine and dried over MgSO4 and thesolvent was evaporated. Flash column chromatography (silica, 3% ether hexanes)afforded pure compound as a colorless liquid (171 mg. 57% yield).To a solution of the compound (171 mg. 0.25 mmol) in THF (0.5 mL) was addedn-BuN4F(0.9 mL. 0.90 mmol) and the mixture was stirred at rt. The reaction wascompleted in 2 h at which time it was poured into water and extracted with ether. Theether extracts were washed with brine, dried over Na2SO4 and the solvent wasevaporated. Flash column chromatography (silica 4% MeOH/CH2Cl2) afforded themethyl ester (24 mg.) together with some of the corresponding lactone. HPLC retentiontime: 9:39 min (microsorb reverse phase, 4.6mm X 25 cm, C-18 column, MeOH/H2070:30 flow rate 1 ml/ min, UV detector at 300nm). UV in MeOH: ?tmax283, 294, 311nm. 1H NMR (500 MHz CDC13) 56.53 (dd. 15.2 10.9 Hz, 1 H), 6.32 (dd, J = 15.1,11.0 Hz, 1 H), 6.17 (d, J=15.9 Hz,1 H) 5.83 (dd. J= 17.5, 2.1 Hz, 1 H), 5.80 (dd. J =15.2, 6.7 Hz, 1 H), 5.72 (dd. J = 17.0, 2.1 Hz, 1 H), 4.14 (m, 1 H), 3.68-3.64 (m, 4H),2.35-2.31 (m, 2 H), 1.51-1.48 (m, 1 H), 1.43-1.42 (m, 2 H), 1.30-1.23 (m, 15 H) 0.85 (t,3 H). 13 C NMR (126 MHZ, CDC13) 5150.01, 140.18, 132.95, 132.26, 112.43, 107.50,75.23, 73.76, 42.49, 33.67, 32.17, 31.36, 27.96, 23.56, 22.58, 21.03, 14.03.Preparation of the methyl ester precursor of compound 2:A solution of the methyl ester precursor of compound 1 (3 mg. in CH2C12 (1ml) was mixed with Lindlar's catalyst (1 mg.) and placed under a hydrogen atmosphere.The mixture was stirred at rt in the dark followed by HPLC until about 80% conversion(1 h). Filtration over celite evaporation of the solvent and separation by HPLC gave apure methyl ester. HPLC retention time: 10:02 min (microsorb reverse phase. 10 mmX 25cm C-18 column, MeOH/H20 70:30 flow rate 4 ml/min. UV detector at 300nm).UV in MeOH: nmax 287, 301, 315 nm.Preparation of the methyl ester precursor of compound 3:This compound was prepared similarly to the preparation of the methyl esterprecursor of compound 1 (from 3-cyclohexyl-3-trimethylsiloxy-1-bromo-1-octene).Desilylation of this compound was also performed in a similar manner to afford themethyl ester. HPLC retention time 8:02 min (microsorb reverse phase, 4.6mm X 25cm.1015202530CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97l16342-59-C-18 column, MeOH/H20 70:30, flow rate 1 ml/min, UV detector at 300nm). UV inMeOH: kmax 282, 293, 311 nm. 1H NMR (360 MHz, CDC13) 56.56 (dd, 15.4, 10.9Hz, 1 H), 6.33 (dd, J =15.2, 10.9 Hz, 1 H), 6.13 (dd, J = 15.8, 6.5 Hz, 1 H), 5.81 (dd, J =15.2, 6.4 Hz, 1 H), 5.80 (d, J = 15.6 Hz, 1 H), 5.73 (dd, J = 15.4, 2.1 Hz, 1 H), 4.15 (br,1 H), 3.93-3.90 (m, 1 H), 3.67 (br, 1 H), 3.65 (s, 3 H), 2.34 (t, 2 H), 1.82-1.65 (m, 10 H),1.46-1.38 (m, 3 H), 1.26-1.01 (m, 5 H).Preparation of the methyl ester precursor of compound 4:Selective hydrogenation of the methyl ester precursor of compound 3, followedby HPLC purification gave the methyl ester precursor of compound 4. HPLC retentiontime: 9.72 min (microsorb reverse phase, 10 mm X 25cm C-18 column, MeOH/H2070:30 flow rate 4 ml./min. UV detector at 300nm), UV in MeOH: lmax 288, 301, 315nm. 1H NMR (250 MHZ, C6D5) 5 6.66-6.89 (m, 2 H), 5.95-6.24 (m, 4 H), 5.55-5.66(m, 2 H), 3.82 (m, 1 H), 3.73 (m, 1 H), 3.41 (m, 1 H), 3.31 (s, 3H, OCH3), 2.08 (t, 2 H,CH2COO), 1.00-1.81 (m, 18 H).The methylesters can be converted to corresponding alcohols usingstandard techniques.Synthesis of 15(R)-15-methyl-LXA& and l5(i)methyl-LXA4_;Approximately 1 gm acetylenic ketone 2_1 is prepared using Friedel-Craftsacylation of bis(trimethylsilyl) acetylene with hexanoyl chloride and is reduced using (-)-pinayl-9-BBN to give the (S) alcohol in CH3N2 as in Webber, S.E. et al. (1988) Adv.Exp. Med. Biol. 229261; Nicolaou, K.C. et al. (1991) Angew. Chem. Int. Ed. Engl.30:1 100; and Vorbriiggen, H. et al.: In: Chemistry, Biochemistry, and PharmacologicalActivity of Prostanoids (Roberts, S.M., Scheinmann, F. eds.). Oxford: Pergamon Press,to generate the methyl at C-15.120%M63Alternatively, the keto group can be treated with CH3MgBr (60:>70°C)as in Vorbrtiggen, H. et al.: In: Chemistry, Biochemistry, and Pharmacological Activity10152025W0 98/11049CA 02265708 l999-03- 10PCT/US97/ 16342_ 50 _of Prostanoids (Roberts, S.M., Scheinmann, F. eds.). Oxford: Pergamon Press to yieldthe 15(:t:)methyl of L (2-5 g) in dry CH2Cl_7_ (~20 ml) at 0°C with sequential additions of2,6-lutidine (5.2 ml) and tert-butyldimethylsilyl triflate (6.9 ml). This reaction is mixedfor 1 h and then diluted with 100 ml ether for aqueous extraction and drying withMgSO4.D M63The product 9 is then coupled with Q10ID.OMeM6351that is generated as in Nicolaou, K.C. et al. (1991) Angew Chem. Int. Ed. Engl. 30:1 100;Nicolaou, K.C. et al. (1989) J. Org. Chem. 5425527 and Webber, S.E. et al. (1988) Adv.Exp. Med. Biol. 229:6l. Structure Q from fragment A in Scheme I is suspended in 4.0equiv. of AgNO3, then 7.0 equiv. of KCN, containing EtOI-I:THF:H20 (l:l:1), 0—25°Cfor 2 h to generate the C-methyl ester protected 15-methyl-LXA4 analog that isconcentrated and saponified in THF with LiOH (2 drops, 0.1 M) at 4°C 12-24 h to givethe corresponding free acid.ICA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342Synthesis of 16-dimethvl-LXA54_ L"‘oH5 This compound is generated using the similar strategy by coupling g_above with e_: vide supra, or L to generate the 15-phenyl-LXA4 analog, or g to generatethe 17-m-chlorophenoxy—LXA4 analogs.IO10 OHfBrOH“ :15 gB\/\/0 Cl6HhB\/\/O ziiZiiiZiv20101520CA 02265708 l999-03- 10W0 98/ 1 1049 PCT/US97/ 16342-52-The appropriate _C_) fragments in Scheme I (i.e. g. f, g, h, ) are each prepared as reviewedin Radfichel, B. and Vorbriiggen, H. (1985) Adv. Prostaglandin ThromboxaneLeukotriene Res. l4:263 for the known corresponding prostaglandin analogues. In 1;,R=H; Cl, methoxy or halogen .Synthesis of 13.14-acetvlenic-LXA£ and halogen-containing analogs. Using the A2B2 generated fragment from Scheme 11, the correspondingC2 fragments are prepared for coupling.Structuresj and g are generated as in Nicolaou, K.C. et al. (1989) J. Org. Chem. 54:5527and methylated as in Radfichel, B. and Vorbriiggen, H. (1985) Adv. ProstaglandinThromboxane Leukotriene Res. 14:263 are coupled to Z to yield these LX analogues.The materials may be subject to RP-HPLC for purification vide supra.I5?‘ 1020W0 98/1 10491CA 02265708 l999-03- 10PCT/US97/16342-63..Synthesis of 14,15-acetylenic—LXA_4_. The designated combined AZB2 fragment can be prepared from couplings of fragmentsA1 and B1, illustrated in Route II to carry the structure of Z or 51 vide supra forcoupling to fragment Q2. The precursor for the Q2 fragment 1 can be prepared as inRadfichel, B. and Vorbriiggen, H. (1985) Adv. Prostaglandin Thromboxane LeukotrieneRes. 142263 for a prostaglandin analog.g-_.MCZA OSitBuMe2ISOMe é)Si‘_LBuMe2 0Precursor m as prepared previously (Nicolaou, K.C. (1989) J. Org.Chem. 54:5527) is added at 1.2 equiv. to 0.05 equiv. of Pd(PPh3)4, 0.16 equiv. of Cul,n-PrNH2, in benzene with Me2Al—carrying 1, 2-3 h RT to yield Q.10152025W0 98/1 1049CA 02265708 l999-03- 10PCT/U S97! 16342-64-I13OMe The alcohol protecting groups TBDMS=R are removed with 10 equiv. ofHF-pyr, THF, 0-25°C (4 h) followed by exposure to 3.0 equivalents of Et3N, MeOH,25°C 15 min to open acid-induced 8-lactones that usually form between C-1—carboxyand C-5 alcohol in the LXs (Serhan, C.N. (1990) Meth. Enzymol.l87:l67 and Nicolaou,K.C. (1989) J. Org. Chem. 5425527). After mild treatment with Lindlar cat. 5% byweight, the extracted material may be subjected to LiOH saponification in THF togenerate the free acid of the target molecule that can be subject to further purification byRP-HPLC gradient mobile phase as in (Serhan, C.N. et al. (1990) Meth. Enzymol.l87:167).Synthesis of l5(:l:)methvl-cvclo—LXA£IOHO OH 0/§ OMe Compound 9 as the SiMe3 derivative can be placed (~ 1 gm) in a roundbottom 100 ml flask under an atmosphere enriched with argon in degassed benzene (20ml). To this add 3.0 equivalents of a vinyl bromide fragment vide infra. This couplingreaction is carried out in catalytic amounts of Pd (PPh3)4 and CuI and can be monitoredby injected aliquots of this suspension into RP-HPLC monitored by UV abundance witha rapid scanning diode. The progression line course 1-3 h at 23°C after which thematerial is extracted with ethyl acetate: H20 4:1 v/v) and concentrated byICA 02265708 l999-03- 10W0 98/11049 PCT/U S97/ 16342- 55 _rotoevaporation. The methyl ester can be saponified in LiOH/THF to give quantitativeyields of the free carboxylic acid. Other derivatives can be prepared as above usingfragment A with different fragment 3 moieties that have been substituted to give forexample a dimethyl or other derivative. This can be obtained by taking the readily5 available ketone 12 and treating it with CH3MgBr (60°C) to generate g that can also becoupled to fragment A as above using conventional techniques such as Pd(O)-Cu(I)coupling. Increased chain length from C-15 can also be obtained.2O1 0 C1 9H0 CH3x <cH2>n/\15 Synthesis of 5-Methyl-LXB,3 and 4.4-Dimethvl-LXBE.The 5—methyl -LXB4 hinders or retards 5-oxo-LXB4 formation. Usingthe general scheme outlined above, the A fragment can be constructed to carry the 5-methyl in a vinyl bromide r precursor that is coupled to a joined B + Q fragment by20 Pd(0)—Cu(I) coupling. O (TBDMS) O2 5 OMC1015202530W0 98lll049 CA 02265708 l999-03- 10PCT/US97/16342- 55 -TBDMSO TBDMS6 To (TBDMS) M633‘The vinyl bromide ; can be obtained from the § that contains eitherdimethyl or hydrogen substituents at its C-4 position. The protected precursortcontaining fragments B; + Q is generated as reported in reference (Nicolaou K.C. et al.(1991) Angew. Chem. Int. Ed. Engl. 30: 1100-16.). Compound 1 is converted to § or Qby coupling with the indicated vinyl bromide. Thus the target molecule can begenerated by adding r_ at 1.0 equv. (z 1 gm) to a round bottom flask degassed containingEt2NH as solvent with t_ injected in Et2NH at 1.2 equiv. Pd(Ph3P)4 is added at 0.02equiv. to give the 8(9)-containing acetylenic precursor methyl ester of §.The material is extracted and subj ect to rotoevaporation suspended inquinoline (0.5 eq) in CH2Cl2 and subject to hydrogenation using (10%; 25°C) Lindlarcatalyst and a stream of H2 gas to selectively reduce the acetylenic double bond atposition 8. The formation of the tetraene component of the methylester of 5-methyl-LXB4 or 4—dimethyl—LXB4 methyl ester can be monitored by RP-HPLC to assesscompletion of the reduction (i.e., 1-3h). The methylfiesters are next saponified to theircorresponding free acids by treating the products with LiOH in THF 25 ul H20 added at0=>24°, 8 — 24h.Example 2 LeukemiaCells and Monocytes : Half—life AssayLipoxin A4 Metabolism by Human PromyelocyticHL-60 cells were purchased from American Type Culture Collection(Rockville, MD), and other cell culture reagents were from GIBCO (Grand Island, NY).Versene (EDTA) was from Whittaker Bioproducts (Walkersville, MD). Synthetic11,12-acetylenic LXA4 methyl ester and LXs were from Cascade Biochemical (Reading,U.K.). 15(S)-15-m-PGEI, PGEl and 5-HETE were from Cayman Chemical Co. (Ann1020253035CA 02265708 2002-05-14_ 57 _Arbor. MI). [1 1.12-3H]LXA4 was prepared from 11,12-acetylenic LXA4 using Lindlarcatalyst as a custom tritiation (NET—259. lot 0 2793275, New England Nuclear, Boston,MA). Tritiated products were isolated using RP—HPLC (Flore er al. (1992) J. Biol.Chem. 267216168; Serhan, C.N. (1990)Meth. Enzymol. 187:167). Methoxyamine and NAD were from Sigma ChemicalCompany (St. Louis, MO). Manganese dioxide and Adams reagent were from AldrichChemical Co. (Milwaukee, WI).Human PMN were obtained from healthy volunteers by gradientcentrifugation of heparinized fresh venous blood (Biiyum, A. (1986) Scand. J. Clin. Lab.Invest. 21 :77). HL-60 cells were seeded in RPMI supplemented with penicillin (100U/ml), streptomycin (100 it/ml), fetal bovine serum (10%) (Hyclone, Logan, UT) andincubated (3 7°C with 5% CO2 atmosphere) in plastic 250 ml flasks. Individual flaskscontaining 5 x 107 HL-60 cells/ml were incubated in the presence or absence of phorbol12—myristate 13-acetate (PMA) (10 or 16 nM, 24-27 h) and adherence was monitored forinduction of macrophage-like phenotype as in Collins, SJ. (1987) Blood 70:1233.Peripheral blood monocytes were obtained (Goldyne, M.E. er al. (1984) J. Biol. Chem.259:8815 after plating fresh mononuclear cells onto plastic petri dishes containing PBSwith glucose (1 mg/ml) for l h at 37°C. Non-adherent cells were removed and adherentmononuclear cells; were gently resuspended using Versene (7 ml/plate) and washed inPBS. PMN (>98%), adherent monocytes (>95%) and HL~60 cells were enumerated bylight microscopy, suspending in PBS for incubations, and <2—3% in each case werepenneable to trypan blue. For some experiments, cell—free supematants were preparedfrom HL-60 cells treated with PMA (16 nM) for 24 - 72 h. After harvesting, thedifferentiated cells were washed, then subject to freeze-thaw lysis (repeated 3 times) andultracentrifugation (100,000 g, 1 h).' lncubations with eicosanoids were stopped with cold methanol containingeither PGB2 or 5-HETE as internal standards (5-HETE was used when 15—oxo-ETE wasquantified). Products were extracted using Sep-pak C18 and routinely chromatographedas in Serhan , C.N. (1990) Meth. Enzymol. 187:167. RP HPLC system consisted of anLKB gradient dual pump equipped with an Altex Ultraspher:ODS (4.6 mm x 25 cm)column, flow rate 1 ml/min eluted (0-20 min) with methanol/H20/acetic acid(65:35:0.01) and methanol/acetic acid (99.99/0.1) in a linear gradient (20-45 min) thatwas used to quantitate the co-metabolites of LTB4 (i.e. 20-COOH and 20-OI-I-LTB4) aswell as LXA4. Recovery of internal standards was 82-2 7.9, mean S.D. (n=13).Compounds I-IV were separated using an Altex UltrasphereX;ODS column (10 mm x 25cm) eluted at a flow rate of 3.0 ml/min with methanol/H20/acetic acid (60:40:0.0l ,v/v/v). Formation of 15-oxo-ETE by 100,000 g supematants (cf. Agins, A.P. et al."‘Tradc-mark101520253035CA 02265708 2002-05-14-63-(1987) Agents Actions 212397; Xun. C-Q. er al. (1991) Biochem. J. 2792553; and Sok,D—E. er al. (1988) Biochem. Biophys. Res. Commun. 156:524) was quantified afterRP—HPLC using an ODS column (4.6 mm X 25 cm) eluted with methanol/H20/aceticacid (70:30:0.0l , v/v/v) monitored at 280 rim with a flow rate of 1 ml/min.Monocyte—derived products were also chromatographed using a Hypersil-xicolumn (5 IL,4 mm x 300 mm) eluted with methanol/H20/acetic acid (60.40:0.0l, v/v/v) and a flowrate of 1 ml/min. On-linc spectra were recorded using a diode array detector(Hewlett-Packard 1040M series 11) equipped with HPLC313 ChemStation software (DOSseries). Spectra were acquired using step 4 nm, Bw = 10 nm, range = 235-360 nm with asampling interval of 1.28 sec.GC/MS was performed with a Hewlett-Packard 5971A mass selectivedetector quadrupole equipped with a HPG1030A workstation and GC 5890. The columnwas a HPUltra 2 (cross-linked 5°/o phenyl methyl silicone gum phase; 25 m x 0.2 mm x0.33 um) and injections were made in the splitless mode in bis(TMS)trifluoroacetamide(BSTFA). The temperature program was initiated at 150 °C and reached 250°C at 10min and 325° at 20 min. Standard saturated fatty acid methyl esters C16—C26 gave thefollowing retention times (minzsec; mean of n=6). C16, 8.03; C13, 9.77; C20, 12.22; C22,16.11; C24, 20.72; C26, 23.62 that were used to calculate respective C values ofLX-derived metabolites as in Serhan, C.N. (1990) Meth. Enzymol. 1871167.Diazomethane was prepared and the methyl ester products were treated with BSTFA(Pierce Chemical Co., Rockford, IL) to obtain Me3Si derivatives. Methyl ester0-methoxime derivatives were prepared as in Kelly,R.W. and Abel, M.H. (1983) Biomed. Mass Spectrom. 10:276. Catalytichydrogenations were performed in methanol (1 ml) with Adams reagent (Aldrich,Milwaukee, WI) by saturating the platinum IV oxide (1-2 mg) with a stream of bubblinghydrogen (20 min, RT). After extraction, materials were treated with diazomethanefollowed by BSTFA (overnight; RT).RESULTSMetabolism of LXA4: Intact neutrophils from peripheral blood of healthydonors did not significantly metabolize exogenous LXA4 while cells from the samedonors rapidly transformed LXA4 via (o—oxidation. In contrast, PMA-treated HL-60 cellsthat displayed monocyte/macrophage-like characteristics rapidly transformed LXA4.Within the first 60 s of exposure, > 70% of LXA4 was metabolized. In the absence ofPMA treatment, neither intact HL-60 cells (undifferentiated) nor their cell-freesupernatants (100,000 x g) fi=orm LXA4 (n=3).*Trade-mark101520253035CA 02265708 l999-03- 10W0 98/11049 PCT/US97/16342-69-Differentiated HL-60 cells incubated with LXA4 converted thiseicosanoid to several products. Labeled LXA4 was transformed to four main productsthat carried tritium (denoted compounds I-IV), which were collected for further analysis.Structures of compounds I-IV To obtain quantities of these compoundsenabling structural studies, their retention times in RP-HPLC were established using the3H-label elution profile to mark boundaries, and unlabeled samples pooled from severalincubations were chromatographed and individually collected from within these regionsfor GC/MS analysis. Selected ion monitoring of the products obtained after treatmentwith diazomethane and BSTFA revealed that compounds I-IV each displayed prominentions at m/z 203 [—CH(OSiMe3)-(CH2)3-COOCH3] indicating that carbons 1 through 5 ofLXA4 (carboxylic carbon is number 1) were not modified, although each product gave adifferent retention time than LXA4. The methyl ester, trimethylsilyl derivative of LXA4displayed prominent ions in its electron impact spectrum at m/z 203 (base peak) and173, with its molecular ion at 582 (M+4). Other ions of diagnostic value in thisderivative of LXA4 are observed at m/z 171 (203-32), 409 (M-173), 379 (M-203), 482(M-100) and 492 (M-90) (Serhan, C.N. et al. (1984) Proc. Natl. Acad. Sci. USA8l:5335; and Serhan, C.N. (1990) Meth. Enzymol 1872167. It is noteworthy that theLXs in general are known to give extremely weak molecular ion peaks (Serhan, C.N. etal. (1984) Proc. Natl. Acad. Sci. USA 81 :5335). Nevertheless, compounds labeled I & IIalso possess prominent ions at m/z 173 (Me3SiO+=CH—(CH2)4-CH3) indicating that thecarbon 15-20 fragment of these LXA4-derived products was intact, while the ion at rn/2173 was not evident in compounds III and IV. Thus, the conclusion that compounds I-IVare metabolites of LXA4 was based upon: their physical properties (HPLC and GC/MS),the finding that they carry tritium label, as well as the absence of these products inincubations with HL-60 cells not treated with PMA.Next, compounds III and IV were focused on since it appeared that theyrepresent metabolites with structural modifications in the carbon 15 through 20 fragmentof LXA4. Since co—oxidation (hydroxylation at carbon 20) was a possibility, ions thatcould result from the respective 20-OH and 20-COOH forms of LXA4 afterderivatization, namely m/z 261 and 217 (Me3SiO+=CH-(CH2)4-CH2OSiMe3 andMe3SiO+=CH-(CH2)4-CO2Me), were scanned in the acquired GC—MS data profiles.Neither III nor IV displayed prominent ions at either m/z 261 or 217 indicating thatthese products were not likely the result of a)—oxidation.]The mass spectrum (C value 24.3) of the Me3 Si derivative, methyl esterof compound III was obtained. Prominent ions in its spectrum were observed at m/z 203(base peak, CH(OSiMe3)-(CH2)3-COOCH3), 171 (203—32; elimination of CH3OH), 215[(M-203)-90, elimination of trimethylsilanol (Me3SiOH)] and 99 (O=C—(CH2)4—CI-I3)..,.-.-.............—....-.—..................,........._. ._. ..,.,..,.. . , ._101520253035W0 98/11049CA 02265708 l999-03- 10PCT/US97/ 16342- 70 -Ions of lower intensity were a m/z 508 (M"') and 418 (M—90; loss of Me3SiOH). Thepresence of these ions suggested that the material that coeluted with 3H-labeledcompound III was the 15-oxo-derivative of LXA4. This is supported by several lines ofevidence, namely the virtual loss of the prominent ion at m/z 173(Me3SiO+=CH-(CH2)4-CH3), presence of m/z 99 (O=C-(CH2)4-CH3), the absence of atetraene chromophore and appearance of a new chromophore at UV lmax at 335-340 nm.The tetraenone chromophore was confirmed by treating LXA4 with MnO2 in chloroformas used for prostaglandin conversion (Anggard, E. and Samuelsson, B. (1964) J. Biol.Chem. 23924097). Also, the mass spectrum of the catalytic hydrogenation product gavea C value of 25.1 with prominent ions at m/z 203 (base peak), m/z 99 (66%), m/z 313(M-203 or M—CH(OSiMe3)-(CH2)3-COOCH3; 35%) and m/z 171 (36%) with noprominent ion at m/z 173. Less intense ions were at m/z 516 (M+) and m/z 426 (M-90).Thus, the upward shift of 8 amu and framentation of this saturated derivative wereconsistent with the generation of the corresponding 15-oxo-derivative.To examine this LXA4-derived product further, an aliquot of the materialeluting beneath the peak labeled III was treated with diazomethane followed bymethoximation (as in Bergholte, J .M. et al. (1987) Arch. Biochem. Biophys. 257:444)and treatment with BSTFA. Its spectrum, C value 25.4, showed prominent ions at m/z203 (base peak), 171 (203-32; loss of CH3OH) and 229 [M-128 orCH3O-N=C-(CH2)4CH3-(2x90)]. Ions of lower intensity were at rn/2 537 (M+), 466(M-71, the ott—cleavage ion M-CH2(CH2)3CH3), 481 (M—56 or M-CH2=CH-CH2-CH3, aMcLafferty rearrangement ion), 431 [M-106 (possibly loss of C-,H5N+)], 401 [M-136(elimination of Me3SiOH + CH3 + - OCH3) and 460 (M—77, loss of NOCH3 plusMeOH). Again, an ion at m/z 173 that would have originated from an alcohol-containingC;15 fragment (Me3SiO+-CH-(CH2)4-CH3) was virtually absent in its spectrum. Thus,the ions present are consistent with the methyl ester, O-methoxime derivative generatedfrom the 15-oxo-containing derivative of LXA4. Together the prominent ions observedwith these different derivatives suggest that material eluting beneath the peak labeled IIIwas the 15-oxo- product of LXA4 (i.e. 15-oxo-LXA4).The mass spectrum of the methyl ester Me3 Si derivative (C value 26.0) ofcompound IV showed. prominent ions at m/z 203 (base peak, CH(OSiMe3)-(CH2)3-COOCH3), 171 (203-32; loss of CH3OH), 99 (O=C-(CH2)4-CH3) and 307 (M-203 or M-CH(OSiMe3)—(CH2)3-COOCH3). Ions of lower intensity were at m/z 510 (M‘‘”), 420(M—90, loss of trimethylsilanol) and 208 (M—(99+203)). Its UV spectrum showed atriplet of absorbance with maxima at 259, 269 and 280 nm, consistent for a conjugatedtriene chromophore. The presence of these ions and UV spectrum suggest that IV was adihydro-15-oxo-metabolite of LXA4. This basic structure was supported by the presence101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97/ 16342_ 71 -of the ion at m/z 99 that is consistent with a keto group at position carbon 15, and thepresence of m/z 203 as base peak revealed that the alcohol groups at carbons 5 and 6remain intact. In addition, the absence of a trienone chromophore (K cal = 310 nm)indicates that loss of a double bond was at A13—l4 position to give the observed trienechromophore. Together these results indicate that compound IV was13,14-dihydro-15-oxo-LXA4.The methyl ester, Me3SiO derivative of compound II (C value - 25.4)gave ions at m/z 203 (base peak; CH(OSiMe3)-(CH2)3COOCH3), 173(Me3SiO+=CH—(CH2)4-CH3), 171 (203-32) and 584 (M+). Its molecular ion was twomass units higher than the LXA4 derivative. These ions and a triplet band of absorbance)\.flMCOH 259 nm, 269 and 282 nm suggest that compound II was a dihydro-derivativeof LXA4. The methyl ester, Me3SiO derivative of compound I from HL—6O cells gavetwo products in GC. The major one (C value = 25.0) gave similar ions in its massspectrum as LXA4, but instead its molecular ion was at m/z 586 with ions also present atm/z 555 (M-31) and 496 (M—90), indicating that two of the four double bonds werereduced (not shown). However, identical products were not observedwith peripheral blood monocytes (vide infra), and thus the HL—6O cell-derived materialsfrom peak I were not further characterized in the present experiments. The structures ofI-IV indicate that LXA4 is not metabolized by 0)-oxidation by intact leukocytes butinstead is both dehydrogenated at carbon 15 alcohol and also transformed from aconj ugated tetraene to triene structures. Taken together these observations suggested thatLXA4 may be attacked by NAD—dependent 15-prostaglandin dehydrogenase (5-PGDH),an enzyme known to carry out similar reactions with prostanoids as substrate (Anggard,E. and Samuelsson, B. (1964) J. Biol. Chem. 239:4097, and reviewed in Hansen, H.S.(1976) Prostaglandins 122647).15-PGDH activity was recently shown to be induced in HL-60 cells(Xun, C-Q. et al. (1991) Biochem. J. 2792553), and it apparently utilizes 15-HETE assubstrate with 92% efficiency compared to PGE2 (Agins, A.P. et al. (1987) AgentsActions 21 :397). Indeed, 100,000 g supematants prepared from PMA-treated HL-60cells converted 15-HETE to 15-oxo-ETE indicating the presence of a dehydrogenaseactivity after differentiation. LXA4 competed for catalysis of 15—HETE giving a Ki = 8.22!: 2.6 p.M (S.E.M., n=6) calculated from Lineweaver-Burke plots. At equimolarconcentrations of LXA4 and 15-HETE, LXA4 blocked 15-oxo-ETE formation by 260%.The relative conversion for LX compared to PGE, by 100,000 g supematants indicatedthat LXA4, 11-trans-LXA4 as well as LXB4 but not 15-methyl—PGE1 were converted.Together, these results suggest that LXA4 > 1 1-trans—LXA4 > LXB4 are substrates for15- PGDH or an equivalent enzyme system.101520253035W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342- 72 -Since PMA induces differentiation to monocyte-macrophage-like lineageof HL-60 cells (Collins, S.J. (1987) Blood 7021233), peripheral blood monocytes wereincubated to determine if they metabolize LX. LXss display potent actions withmonocytes (Stenke, L. et al. (l99lb) Biochem. Biophys. Res. Commun. 180:255) andthese cells do not co-oxidize eicosanoids (Goldyne, M.E. et al. (1984) J. Biol. Chem.25928815). When suspensions of both intact monocytes (n=5) and permeabilized cells(freeze-thaw or saponin-treated, n=5) were exposed to LXA4, it was converted to15-oxo-LXA4 and conjugated triene-containing products, 13,14-dihydro-LXA4 and13,14-dihydro-15-oxo-LXA4. As with differentiated HL-60 cells, monocytes rapidlyconverted LXA4 (> 60%) within 30 s. The temporal relationships for formation of thesemetabolites in both intact and permeabilized monocytes were similar and suggest that15-oxo-LXA4 metabolite is a transient intermediate. Also, in each monocyte suspensionincubated with 3H-LXA4 (d=33), 13,14-dihydro-l 5-oxo-LXA4 and 13,14-dihydro—LXA4were major products carrying radiolabel. It is noteworthy that a product eluting before13,14-dihydro-LXA4 at 15.5-17 min was observed that also displayed a trienechromophore and was likely the ll-trans isomer of 13,14—dihydro-LXA4 that resultsfrom cis—trans isomerization encountered during work-up, The ll-cis double bond ofnative LXA4 is labile and readily isomerizes to all-trans during extraction and isolation(Romano, M. and Serhan, C.N. (1992) Biochemistry 31:8269).Example 3 Binding Affinity to Lipoxin Receptors AnalogsHuman promyelocytic leukemia cells (HL—60) were purchased from theAmerican Type Culture Collection (Rockville, MD). RPMI medium and cell culturereagents were from GIBCO (Grand Island, NY). Synthetic LXA4, trihydroxyheptanoicacid (methyl ester), LXB4, LTD4, LTC4 and LTB4 were from Biomol (PlymouthMeeting, PA), and SKF 104353 was from Smith Kline and French Laboratories. ONO4057 was from ONO Pharmaceutical Co., Ltd. (Osaka, Japan). [14,l5-3H]LTB5_ (32.8mCi/mmole), [1-‘4C]arachidonic acid (50.2 mCi/mmole), 32PyATP (3,000 Ci/mmole),[9,10‘3H(N)]palmitic acid (30.0 mCi/mmole), and [9,10-3H(N)]myristic acid (30.7Ci/mmole) were purchased from New England Nuclear (DuPont Co., Boston, MA).11,12-acetylenic LXA4 was from Cascade Biochemicals (Oxford, UK). Microcentrifugetube filters (0.45 pm cellulose acetate) were purchased from PGC Scientific(Gaithersburg, MD) and silicon oils were from Harwick Chemical Corp. (Akron, OH)(d=1.05) and Thomas Scientific (Swedesboro, NJ) (d=0.963), respectively. Nitrobluetetrazolium, PMA, DMSO, proteases, retinoic acid and Actinomycin D were purchasedfrom Sigma (St. Louis, MO). Islet activating protein (IAP) was from LIST Biological101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97/ 16342- 73 _Lab., Inc. (Campbell, CA). Plasticware, Whatman LK6D TLC plates and solvent(HPLC grade) were from Fisher (Springfield, NJ).Preparation of | 1 1,12-.3.H|LXA_4. Tritiation of 1l,l2—acetylenic LXA4methyl ester was carried out under a custom tritiation service (NET-259:92-2326) byNew England Nuclear (Boston, MA). Briefly, 1 1,12-acetylene methyl ester wascharacterized by UV absorbance and reverse-phase HPLC as in (Nicolaou, K.C. et al.(1985) J. Am. Chem. Soc. 107:7515) and exposed to tritium atmosphere in methylenechloride at room temperature. This incubation was stirred in the presence of Lindlarcatalyst (1.0 mg from Fluka Chemicals) for ~ 1 h. The resulting mixture was stored inmethanol and isolated using RP-HPLC. Tritiated products were chromatographed asmethyl esters utilizing a gradient HPLC system equipped with a photodiode array rapidspectral detector (Serhan, C.N., Methods in Enzymology: Arachidonate Related LipidMediators, in Murphy R.C., Fitzpatrick, F. (eds.), vol. 187. Orlando, FL, Academic,(1990), p. 167). This mixture contained both [1 1,12-3H]LXA4 and [11,12-3H]—1 1-trans~LXA4 methyl esters (~ 1:3 ratio) as determined by coelution with synthetic standards.After RP-HPLC, fractions containing [1 1,12-3H]LXA4 were collected, and extractedinto ethyl acetate. The free acid was prepared by LiOH saponification (Fiore, S. et al.(1992) J. Biol. Chem. 267:l6l68). Material from these fractions, when injected intoUV-electrochemical detection HPLC, gave greater than 90% of radioactivity associatedwith a tetraene-containing product that coeluted with synthetic LXA4. Materials thateluted with the retention time of authentic LXA4 in two HPLC systems were taken forbinding experiments. The specific activity calculated for [11,12-3H]LXA4 was 40.5Ci/mmole.Cell cultures and differentiation. HL-60 cells were seeded in RPMImedium supplemented with 100 U/ml penicillin, 100 ug/ml streptomycin, and 10% fetalcalf serum (Hyclone, Logan, UT), incubated at 37°C with 5% CO2 atmosphere in 250ml flasks. Individual flasks containing ~ 50 x 106 cells/ml next received dimethylsulfoxide (DMSO) (1.12% v/v, 120 h) or retinoic acid (RA) (1 uM, 120 h) or phorbolmyristate acetate (PMA) (20 nM, 48 h). Before performing binding assays, cells werewashed twice in phosphate buffered saline (PBS), Ca3+- and Mg2+- free, enumerated andsuspended at 20 x 106 cells/ml in Tris buffer (10 mM), pH 7.4 (Fiore, S. et al. (1992) J.Biol. Chem. 267:l6l68). Nitro blue tetrazolium reduction was performed to monitorinduction of polymorphonuclear phenotype as in (Imaizumi, M. and Breitman, T.R.(1986) Blood 67: 1273), and cell adherence was determined for induction ofmacrophage—like phenotype (Collins, S.J. (1987) Blood 70: 1273). Human umbilicalvein endothelial cells (HUVEC) maintained in culture third passage were obtained fromDr. M. Gimbrone (Brigham and Women's Hospital Department of Pathology).101520253035CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342-74-PMN. platelet and RBC isolation from peripheral blood. Human PMNwere obtained by the modified Boyum method (Boyum, A. (1968) Scan. J. Clin. Lab.Invest. 21 :77) from fresh heparinized blood after venipuncture of healthy normalvolunteers. Suspensions in PBS were monitored for cell number and viability by theirability to exclude trypan blue both exceeding 98%. Red blood cells were obtained from10 ml of heparinized blood after three centrifugations in PBS (2,500 rpm, 10 min at 21°C). Blood drawn in acidic citrate dextrose (9:1, v/v) was used to isolate platelets aspreviously described (Serhan, C.N. and Sheppard, K.-A. (1990) J. Clin. Invest. 85:772).Ligand binding. 3H-LXA4 and 3H-LTB4 binding was performedessentially as in Fiore, S. et al. (1992) J. Biol. Chem. 267216168. After suspending cellsin Tris buffer 10 mM (pH 7.4, Ca2+ 2.5 mM, Mg2+ 1.2 mM), aliquots (0.5 ml) wereincubated (20 min at 4°C) with 3H- ligands alone (0.3 nM) or in the presence ofincreasing concentrations of homoligands or other compounds (3-300 nM). Theincubations were rapidly centrifuged (60 sec, 12,000 g) on silicon oil (d=l .028), andcell-associated radioactivity was determined by liquid scintillation counting (Wallac1409, Pharmacia, Piscataway, NJ). Binding experiments with HUVEC cells wereperformed in 12 well plates with 3.5 x 105 cells/well. After 10 min, wells were washedtwice with PBS and cell-associated label was recovered by adding glacial acetic acid(0.5 ml). Results obtained from these assays were submitted for further analysis usingthe Ligand program (Elsevier-Biosoft, Cambridge, UK).PLD activity. Human PMN and HL-60 cells (50 x 105 cells/ml) preparedas above were incubated with 3H—myristic acid or 3H-palmitic acid (8 uCi/50 x 106cells) for 40-60 min at 37°C in PBS. Cell uptake ranged between 60-80% of addedlabel, 7.1 :I: 4.2% (n=l0; mean d: S.D.) and 32.6 :1: 10.3% (n=6; mean i S.D.) into totalphospholipid classes of PMN and HL-60 cells, respectively. Incubations were carriedout at 37°C (10 x 105 cells/ ml PBS). Agonists were added in 50 uL PBS or with 1:10(v/v) EtOH:PBS for phosphatidylethanol (PEt) formation as in Billah, M.M. et al.(1989) J. Biol. Chem. 264:17069. At indicated times, incubations were stopped byadding 3.5 ml of ice cold CHCI3/MeOH (2/5, v/v) containing 1-'4C—arachidonic acid(5,000 cpm) used here as internal standard to quantitate extraction recoveries. Sampleswere extracted using a modified Bligh and Dyer extraction as in (Serhan, C.N. andSheppard, K.-A. (1990) J. Clin. Invest. 85:772). Organic phases, concentrated in 50 i.tLof CHCI3/MeOH (8/2, v/v), were spotted onto linear K6D TLC plates developed withthe organic phase of ethyl acetate/isooctane/acetic acid/water(110/50/20/100, v/v/v/v)for 50 min (Billah, M.M. et al. (1989) J. Biol. Chem. 264: 1 7069). In this system,phosphatidic acid (PA) gave an Rf: 0.1 i 0.04 and PEt gave an Rf: 0.56 i 0.04; n = 38:i: S.D. that were clearly separated from other phospholipids (that remained at the origin)101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/U S97/ 16342_ 75 -or neutral lipids (R{= 0.75-0.90). Lipids were visualized with iodine vapor andidentified by co—elution with authentic standards that were also spotted andchromatographed in each TLC plate. Regions corresponding to PA, PEt, and internalstandards were scraped and quantified by liquid scintillation counting. In addition to3H-myristate or 3H-palmitate labeling, both [1-‘4C]arachidonate (0.25 p.Ci/30 x 105PMN) and 32PyATP (20 uCi/50 x 105 PMN) labeled cells were used to monitor LXA4_stimulated formation of PA and PEt generation. In these experiments, PA was resolvedusing ethyl acetate/isooctane/acetic acid (45/15/10, v/v/v) as solvent system (Bocckino,S.B. et al. (1989) Anal. Biochem. 180224) and gave an Rf: 0.46 :1: 0.03 (n = 15). Allvalues reported for PEt formation in Tables 4 and 5 were calculated by subtracting thedpm obtained in the presence of agonist(s) alone minus those measured in the presenceof agonist(s) and 0.5% EtOH.Impact of IAP and staurosporine on LXA£-induced PLD activity. PLDactivity assays, performed as described (vide supra), were preceded by cell exposure toeither IAP or staurosporine. IAP treatment of PMN was performed as previouslydescribed (Nigam, S. et al. (1990) J. Cell Physiol. 143:5l2), and HL-60 cells wereincubated in the presence or absence of IAP (300 ng/ml) for 2 h at 37°C (Kanaho, Y. etal. (1992) J. Biol. Chem. 267:23554). Aliquots (107 cells/0.5 ml) were added to 0.4 mlbuffer. Next, incubated cells were exposed to either 100 pl of vehicle (PBS, EtOH0.04%) or LXA4 (IO'7 M and IO'9 M), in the presence or absence of 0.5% EtOH.Staurosporine (1-00 nM) was added to cell suspensions for 5 min at 37°C before additionof LXA4.RESULTSAfter preparation of synthetic [1 1,12-3H]LXA4, its specific binding topromyelocytic cells (HL-60) was characterized and direct comparisons with specificbinding of [14,15—3H]LTB4 were performed. When routine phenotypic markers weremonitored, untreated HL-60 cells displayed a low level of specific binding for both 3H-LXA4 and 3H-LTB4 ligands. Differentiation induced by exposure to either DMSO(1.12%) or retinoic acid (1 uM) for 5 days was accompanied with a three to fivefoldincrease in specific binding for both radioligands. PMA-treated cells (20 nM, 48 h)displaying characteristics of macrophagic-like phenotype, i.e. NBT negative cells thatadhere to, plastic (see Imaizumi, M. and Breitman, T.R. (1986) Blood 67: 1273; Collins,SJ. (1987) Blood 70:1233), also led to the appearance of specific binding with both 3H-LXA4 and 3H-LTB4. Equilibrium binding with 3H-LXA4 at 4°C was reached at 10 minthat remained virtually unchanged for the next 20 min.101520253035W0 98/1 1049CA 02265708 l999-03- 10PCT/US97I16342_ 76 _To assess whether induction of specific binding for both 3H— ligandsrequired de novo synthesis, Actinomycin D (2 pg/ml) was added with PMA incubations.Actinomycin D blocked the PMA-induced increment in specific binding for both labeledeicosanoids, suggesting inhibition of de novo protein biosynthesis also blockedappearance of specific binding sites. The impact of protease and glycosidase treatmentswas assessed with both differentiated HL-60 cells and human PMN for 3H-LXA4specific binding. Protease treatment reduced specific binding and provided additionalevidence in support of a protein component of LXA4 specific binding sites.Results from isotherm binding assays (4°C, 10 min) with differentiatedHL-60 cells and [1 1,12-3H]LXA4 (0.1-30 nM) showed that [1 1,12-3H]LXA4 specificbinding sites gave a Kd = 0.6 i 0.3 nM. A nonlinear portion of the Scatchard plot wasobserved for concentrations observed with LXA4 specific binding with human PMN(Fiore, S. et al. (1992) J. Biol. Chem. 267: 16168). Results obtained here with LTB4specific binding with HL-60 cells, Kd = 0.12 nM, are essentially in agreement withvalues recently reported by Harada (Xie, M. et al. (1991) J. Clin. Invest. 88:45), namelyKd = 0.23 nM.To further characterize the interactions of 3H-LXA4 with its specificbinding sites, competition binding experiments were performed with differentiated HL-60 cells. LXA4, LXB4, LTB4, LTC4 and the leukotriene receptor antagonists SKF104353 (LTD4 antagonist; Harada, Y. (1990) Hiroshima J. Med. Sci. 39:89) and ONO-4057 (LTB4 antagonist; Gleason, J .G. et al. (1987) J. Med. Chem. 30:959) were assessedas potential competing ligands. Neither LXB4, LTB4, nor trihydroxyheptanoic acid(methyl ester) (300 nM) were able to displace 3H-LXA4 specific binding withdifferentiated HL-60 cells while LTC4 caused ~30% decrease of specific binding whenadded in 3 log molar excess. The finding that LXA4 (300 nM) was unable to competewith 3H—LTB4 (0.3 nM) binding to differentiated HL-60 cells suggests that LXA4 andLTB4 interact with separate classes of specific binding sites. Leukotriene receptorantagonists SKF 1043 53 and ONO-4057 did not displace 3H-LXA4 binding withdifferentiated HL-60 cells, but SKF 1043 53 and LTD4 were effective in competing thespecific 31-I-LXA4 binding with HUVEC. HUVEC displayed a Kd of 11.0 :1: 2.6 nM anda By! of 2.5 x 10-10 M for 3H-LXA4, and virtually identical values were calculated forLTD4 competition. In the case of 3H—LTB4, HUVEC did not specifically bind LTB4, butnon-specific cell association was evident with this EH-ligand (n=3; not shown). Specificassociation of 3H—LXA4 was not evident among several other cell types surveyed. Here,neither washed platelets, RBCS, the B-cell (Raji), nor T-cell (Jurkat) cultured cell linesdisplayed specific binding for 3H-LXA4. Taken together, these results indicate thatLXA4 interacts with unique binding sites in differentiated HL-60 cells that is not101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/U S97/ 16342- 77 ..sensitive to either leukotriene receptor antagonist (SKF 104353 or ONO-4057). InHUVEC, 3H-LXA4 specific binding was sensitive to both LTD4 and SKF 104353 butnot ONO-4057, suggesting that 3H-LXA4 specific binding in this cell type may reflectits interaction with putative LTD4 receptors.LXA4 rapidly stimulates phosphatidic acid formation in humanneutrophils (Nigam, S. et al. (1990) J. Cell Physiol. 1432512). To determine if 3H-LXA4binding confers PLD activation, PEt and PA were monitored in both PMN and HL-60cells. Results indicated that LXA4 stimulates PLD activity in these cell types withsimilar temporal responses. PMN exposed to LXA4 (10''0 M) rapidly generated theethanol trapping product PEt within 60 s that declined to baseline levels by 5 min. Inthe absence of added EtOH, PEt was not formed at statistically significant levels. Abiphasic concentration dependence was obtained for PEt formation in both PMN anddifferentiated HL-60 cells. An apparent maximal response was noted with theconcentration range of ~1O‘9-1O“° M LXA4, and a second peak of activity was observedat 10‘7 M LXA4. Below IO‘3 M, both the chemotactic peptide FMLP and LXA4 gaveresults of similar magnitude with F MLP appearing to be slightly more potent with PMNfrom some donors. To evaluate the potential contribution of other biosyntheticpathways, LXA4—induced PA formation was also examined in both 32PyATP and [1-‘4C]—arachidonate-labeled PMN. 32P-labeled PA was evident in statistically significantlevels only after 30 min of exposure to LXA4 (10'7 M). Similar results were obtainedwith 14C-labeled PA formation derived from ‘4C-arachidonate—labeled precursors.These findings indicated that LXA4 can also stimulate other routes of PA formation inPMN but only after 30 min of exposure.Only differentiated HL-60 cells (expressing specific binding sites for 3H-LXA4) incubated with LXA4 rapidly generated PEt that was evident within 30 sec.Undifferentiated HL-60 cells incubated with LXA4 (1O'9 M) did not rapidly generatePEt. The concentration dependence with these cells also gave a biphasic response withLXA4 and gave an apparent maximum at 10‘9 M. To investigate possible signaltransduction events involved in LXA4-mediated PLD activation, PMN and HL-60 cellswere next exposed to either IAP or staurosporine. Results indicate that the LXA4-mediated PLD activity evoked within the lower concentration range (10‘9—1 0'10 M) wassensitive to IAP treatment in both cell types and, similarly, the PLD activity stimulatedat higher concentrations of LXA4 (1O'7 M) was inhibited by staurosporine. Thus, inboth cellular systems at concentrations below 10‘3 M, LXA4 rapidly interacts withspecific binding sites that trigger PLD activity and hence confers a functional response,while within submicromolar concentrations of LXA4, it may stimulate additionalprocesses that can also lead to activation of PLD.‘101520253035W0 98/1 1049CA 02265708 l999-03- 10PCT/U S97/ 16342- 73 -Example 4: Lipoxin Bioactivity AssaysSeveral of the preferred LX analogs (shown structurally as compounds 1through 8 above) were prepared by total synthesis as described in Example 1. Followingpreparation and isolation of these compounds via HPLC, compounds were first assessedto determine whether they retain biological activity using the neutrophil adhesion assayand epithelial cell transmigration assays (as described in Nash, S et al., (1987) J. Clin.Invest. 80:1 104-1113; Nash, S et al., (1991) J. Clin. Invest. 87: 1474-1477; Parkos, C.A.et al., (1991) J. Clin. Invest. 88:l605-1612; Parkos, C.A. et al. (1992) J. Cell. Biol.ll7:757-764; Madara J.L. er al., (1992) J. Tiss. Cult. Meth. 14:209-216).Compounds 1 through 8 (l0'7 - 10'10M) were found to inhibit neutrophiladhesion to endothelial cells and their transmigration on epithelial cells. The acetylenicprecursors ( compound 1, 3, 5 and 7) were found to be physically more stable than theirtetraene counterparts. Compound 7, which did not have an alcohol group in the C15position or other modifications in the series, showed no biological activity in the assays.It would therefore appear that a substituent in the C15 position of LX is necessary forthe biological activity of at least LXA4 analogs. 15-methyl-LX A4 (compound 2) alsoproved to inhibit polymorphonuclear (PMN) adhesion triggered by leukotriene B4(LTB4) to human endothelial cells with an IC50 of ~ 1 nM. LX analogs 1 through 8were found to block migration at potencies greater than or equal to synthetic LXA4.Compound 7 was found to be essentially inactive within the concentration range forinhibition induced by LXA4 or other analogs. The results in these neutrophil-containingbioassays indicate that LXA4 analogs with modifications in C15-C20 positions retaintheir biological action and can inhibit PMN transmigration and adhesion events.The "bio-half-life" of compounds 1-8 was assessed using phorbol ester-treated human promyelocytic leukemia (HL-60) cells as described in Example 2. Thesecells converted more than 95% of LX A4 within five minutes of its addition to the cellincubation. LXA4 in this system was rapidly transformed to 15-oxo-LXA4. However,in the same assay, 15-methyl-LXA4 ( compound 2) and cyclohexyl-LXA4( compound4) werequantitatively recovered in the incubation medium at times up to two hours.These results illustrate that modification in the carbon 20 through the carbon 15positions prevents the further metabolism of LXA4 by leukocytes.In addition, the stability of the acetylenic methyl ester LXA4 ( compound1) was recovered essentially intact after 60 minutes of incubation in whole blood (3 7°C) , as assessed after extraction and reverse phase HPLC. When taken together,these results indicate that LX analogs retain biological action and are resistant to furthermetabolism in vitro.101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97/16342- 79 _Example 5: Effect of 15-epi-Lipoxins' on Cell ProliferationMATERIALS and METHODSSynthetic (5S,6R,1 5R)-trihydroxy-7,9,1 3 -trans-l 1-cis—eicosatetraenoate:carboxymethyl ester (15-epi-LXA4-methyl ester) was prepared by total organicsynthesis and was a gift of Prof. N. A. Petasis (Department of Chemistry, University ofSouthern California). 15-epi-LXA4 free acid was obtained by saponification of 15-epi-LXA4~methyl ester in tetrahydrofuran with LiOH (0.1 M) at 4°C for 24 h. Syntheticeicosanoid reference samples were from Cascade Biochem Limited (Reading,Berkshire, England). Inhibitors of 5—LO (Rev 5901 isomer) and cytochrome P450 (17-octadecaynoic acid, l7—ODYA) activities were from Biomol (Plymouth Meeting, PA).Radiolabeled ([32P]) dCTP and ([3H]) arachidonic acid and methyl-thymidine werefrom Dupont NEN (Boston, MA). Ionophore (A231g7), ASA, 3,(4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyl-tetrazolium bromide) (MTT) and guanidinium isothyocyanate werepurchased from Sigma Chemical Co (St. Louis, MO). Recombinant human interleukin 1B (IL-13) was obtained from R&D systems (Minneapolis, MN). Du1becco's phosphate-buffered saline containing both CaCl2 (0.6 mM) and MgCl2 (1.0 mM) (pH 7.4)(DPBS2+), fetal bovine serum (FBS), penicillin and streptomycin were from BioWhittaker (Walkersville, MD). Hank's balanced salt solution (HBSS) and F-12K nutrientmixture were from Gibco Laboratories (Grand Island, NY). High-pressure liquidchromatography (HPLC) grade solvents and cesium chloride were purchased from JTBaker (Phillipsburg, NJ), methyl formate was from Eastman Kodak Co (Rochester, NY)and Sep-Pak C18 cartridges were from Waters Associates (Milford, MA). Diazomethanewas prepared from N-methyl—N'-nitro-N-nitroguanidine purchased from AldrichChemical Company (Milwaukee, WI). N,0-bis (trimethylsilyl) trifluoroacetamide(BSTFA) was from Pierce (Rockford, IL). First strand cDNA synthesis kit and othermolecular biology reagents were from Promega (Madison, WI). Oligonucleotide primerswere purchased to Integrated DNA Technologies (Coralville, IA).Cell isolation and culture:Human type II epithelial A549 cells from human lung carcinoma and normalhuman skin fibroblast (breast) were obtained from the American Type CultureCollection (Rockville, MD). The A549 cell line originated from a human alveolar cellcarcinoma and was a useful cell line because it was easily assessable and could bemaintained in culture without contaminating tissue macrophages. (Lieber, M., et al.101520253035W0 98/11049CA 02265708 l999-03- 10PCT /U S97] 16342_ go -(1976) A continuous tumor-cell line from a human lung carcinoma with properties oftype II alveolar epithelial cells. Int. J. Cancer Fibroblasts were utilized within theirlimited window of viability and their passage numbers were recorded (results arereported for cells from 3-5 passages). Epithelial A549 cells were seeded into T-75 cm2tissue culture flasks and maintained in F-12K medium supplemented with 10% heat-inactivated FBS, penicillin (50 U/mL) and streptomycin (50 (g/mL). Human PMN fromhealthy donors who had not taken ASA or other medications for at least two weeks wereobtained by Ficoll-Hypaque gradient centrifugation and dextran sedimentation, (BoyumA. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolationof mononuclear cells by one centrifugation, and of granulocytes by combiningcentrifugation and sedimentation at 1 g. Scand. J Clin. Lab. Invest. 21 (Suppl. 97): 77-89.), and suspended in DPBS+ at pH 7.4. Viability of A549 cells and PMN wasdetermined by their ability to exclude trypan blue and were 95 i 2 and 97 :i: 1%,respectively. These values were not significantly altered during the reported incubations.Incubation Conditions:In incubations involving permeabilized A549 cells (prepared by a rapid freeze-thaw cycle), IL-1 B-treated (1 ng/ml, 24 h) A549 cells (1.5x106 cells/ml) were pretreatedfor 20 min with either vehicle (0.1% EtOH), ASA (500 (M; used throughout), 5 (M 17-ODYA, an inhibitor of cytochrome P450, (Muerhoff A.S., et al. (1989) Prostaglandinand fatty acid 0) and (0)-1)-oxidation in rabbit lung: acetylenic fatty acid mechanism-based inactivators as specific inhibitors. .1. Biol. Chem. 244: 749-756.), or 5 mM Rev5901 isomer, a 5-LO inhibitor, subjected to two cycles of rapid freezing in a dry ice-acetone bath and thawing to room temperature (full cycle <20 min), and incubated witharachidonic acid (20 (M) for 20 min at 37°C in 4 ml of DPBS2+. In experimentsinvolving radiolabeled arachidonic acid, incubations were initiated with the addition of[3H]-arachidonic acid (0.25 (Ci/ml) plus unlabeled arachidonic acid (20 (M) for 20 minat 37 °C.For time-course experiments involving the generation of l5-HETE fromendogenous sources (see FIG. 2B), intact A549 cells were exposed to IL-1 [5 (1 ng/ml)for up to 48 h and then treated with vehicle (containing 0.1% EtOH) or ASA for 20 minfollowed by addition of ionophore A3187 (5 uM) in 4 ml of HBSS for 30 min at 37°C.In coincubation experiments, confluent A549 cells were exposed to IL-1 3 (1ng/ml, 24 h), washed in HBSS and treated with vehicle alone or ASA for 20 min andarachidonic acid (20 (M) for 60 seconds at 37°C. Coincubations were performed by101520253035CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342.8].adding PMN to A549 cell monolayers followed by costimulation with ionophoreA23187 (5 LLM) in 4 ml 0fHBSS for 30 min at 37°C.Analysis of eicosanoids:Incubations were stopped with 2 volumes cold MeOH containing prostaglandinB2 (200 ng) and products were extracted using Sep-Pak C18 cartridges. Materials whicheluted in the methyl formate fractions were concentrated under a stream of N2 andscanned for ultraviolet-absorbing material (in methanol) with a model 8452spectrophotometer (Hewlett Packard Co, Palo Alto, CA) prior to injection into areversed phase (RP)-HPLC system. This system consisted of a dual pump gradient(LKB, Bromma, Sweden), a diode array detector (Hewlett-Packard 1040M series II) anda HPLC3D ChemStation software. The collected UV data were recalled at 300 nm tomonitor conjugated tetraenes, at 270 nm for trienes and 234 for monoHETEs. All UVspectra were acquired using step = 4 nm, Bw = 10 nm, and range = 220-360 nm with asampling interval of 0.96 s.The monohydroxy eicosanoids (i. e. 5-, 12- and 15-HETE) from A549 cells wereanalyzed using a Ultrasphere~ODS column (5 nm, 4.6 mm x 25 cm) (BeckmanInstruments, Fullerton, CA) was eluted with MeOH/H20/acetic acid (65:35:0.01; v/v/v)as phase one (10 -20 min), and a linear gradient with MeOH/acetic acid (99.9:0.1, v/v) asphase two (20-45 min) at a flow rate of 1.0 ml/min. The R- and S-enantiomers of 15-HETE were resolved and identified using a chiral HPLC system similar to that reportedby Hawkins et al. (1988). (Hawkins et al. (1988) Resolution of enantiomers ofhydroxyeicosatetraenoate derivatives by chiral phase high-pressure liquidchromatography. Anal. Biochem. 173: 456-462.) Briefly, after RP—HPLC materialeluting beneath 15-HETE peak was extracted with chloroform and converted into methylester by ethereal diazomethane treatment, chiral analysis was performed with aBakerbond DNBPG (covalent) chiral column (5 nm, 4.6 mm x 25 cm) (JT Baker,Phillipsburg, NJ) eluted with n-hexane/2-propanol (l00:O.4; v/v) at a flow rate of 0.8ml/min. When indicated, generation of 15-HETE from endogenous sources wasmonitored by radioimmunoassay (RIA). The antibody was raised against 15S-HETEwith 0.1% crossreactivity at 50% B/B0 for 5-HETE (PerSeptive Diagnostics,Cambridge, MA).For analysis of LXs (including LXs and 15-epi-LXs) from A549 cell-PMN,coincubations were carried out using either a Waters (Bondapak C18 (3.9 x 300 nm)column eluted with an isocratic mobile phase MeOH/H20/acetic acid (60:40:0.0l;v/v/v) with a flow rate of 0.6 ml/min or an Altex Ultrasphere ODS column (5 um, 10202530CA 02265708 2002-11-05-33-mm x 25 cm) eluted with McOH/l-I20/acetic acid (65:35:0.0l; v/v/v) at a flow of3ml/min. Peptidoleukotrienes (LTC4 and LTD4) eluted in the MeOH fractions from Sep-Pak cartridge extractions were resolved with a Beckman Ultrasphere-ODS columneluted with MeOH/H20/acetic acid (65:35:0.01; v/v/v), pH 5.7, at 1 ml/min. Incubationsof PMN with l5R-HETE were stopped with MeOH and methyl formate fractions of theSep—Pal< C18 extracted products were injected into an Altex Ultrasphere ODS column(5 pm, l0 mm x 25 cm) eluted with MeOH/H20/acetic acid (65:35:0.0l; v/v/v) using aflow of 3 ml/min. The material beneath peaks absorbing at 300 nm were individuallycollected after RP-HPLC and analyzed by gas chromatography-mass spectrometry (GC-MS) employing a Hewlett-Packard 5890 GC series II equipped with a 597lA mass-selective quadrapole detector as in.Claria, J. and Serhan, C.N. (1995) Aspirin triggerspreviously undescribed bioactive eicosanoids by human endothelial cell-leukocyteinteraction. Proc. Natl Acad. Sci. USA 92:9475-9479.Reverse Transcription (RT) and PCR:Total RNA was obtained from A549 cells by the guanidinium isothiocyanate-cesium chloride method and cDNA was produced by RT. Oligonucleotide primers wereconstructed from published sequences of PGHS—l and PGHS-2 ((5'-TGC CCA GCTCCT GGC CCG CCG CTT-3' (sense), 5'-GTG CAT CAA CAC AGG CGC CTC TTC-3' (antisense)), and (5'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3' (sense) and5'-AGA TCA TCT CTG CCT GAG TAT CTT-3' (antisense)), respectively) , 15-LO (5'-ATG GGT CTC TAC CGC ATC CGC GTG TCC ACT—3’ (sense) and 5'-CAC CCAGCG GTA ACA AGG GAA CCT GAC CTC-3' (antisense)), 12-LO, (Funk, C.D. andFitzGerald, GSA. (1991) Eicosanoid forming enzyme mRNA in human tissues. J. Biol.Chem. 266: 12508-12513), (5'-AGT TCC TCA ATG GTG CCA AC—3' (sense) and 5'-ACA GTG TTG GGG TTG GAG AG-3' (antisense)) and 5-LO (5'-GAA GAC CTGATG TTI‘ GGC TACC-3' (sense) and 5'-AGG GTT CTC ATC TCC CGG-3'(antisense)). Amplification of the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) primers was performed with 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3' (sense) and 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (antisense). PGHS-l,PGHS-2 and GAPDH samples were amplified for 25 cycles of denaturation at 94 °C for1 min, annealing at 58°C for 2 min and extension at 72 °C for 3 min. 5-, 12- and 15-LOwere amplified at 94°C (1 min), 55°C (2 min) and 72°C (2.5 min) for 35 cycles. PCRproducts were analyzed by electrophoresis in 2% agarose gel and their identity wasmonitored by restriction enzyme analysis. For detection of specific PCR-amplified101520253035CA 02265708 l999-03- 10W0 98/11049 PCTIU S97/ 16342-83-targets, 0.5 (Ci of [32P]dCTP (3000 Ci/mmol) was added to the PCR mixture and theproducts were quantified by phosphorimager using Image-Quant programming(Molecular Dynamics, San Lorenzo, CA).Cell Proliferation:A microculture 3,(4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyl-tetrazolium bromide)(MTT) assay, (Marshall, N.J., et al. (1995) A critical assessment of the use ofmicroculture tetrazolium assays to measure cell growth and function. Growth Regul. 5:69-84.), was used to examine the actions of LXs and other eicosanoids on cellproliferation. A549 cells and fibroblasts from exponential-phase maintenance cultureswere counted and dispensed within replicate 96-well culture plates in 100 pl volumes ofmedium (~2000 cells/well). Following 24 hours at 37 °C, the culture medium wasremoved, and fresh medium (100 pl) containing either the compounds (5-1000 nM) orvehicle (medium plus 0.15% ethanol) was added to 4 replicates for each conditionstudied and the culture plates were then incubated for up to 96 hours at 37 °C in a 5%CO2 atmosphere. At the end of this period, 25 pl of freshly prepared MTT in HBSS (5mg/ml) was added to the wells and the plates were incubated for 4 hours at 37 °C. Dyesolution was aspirated, wells were washed once with HBSS and dye taken up by thecells was extracted in 100 pl of isopropyl alcoholzl N HCl (9624, v/v) and quantitated at570 nm using a microplate reader (Molecular Devices, Menlo Park, CA). In someexperiments, cells were grown in 12-well culture plates, treated as above andenumerated using a Neubauer hemocytometer. Viability was assessed routinely usingtrypan blue exclusion assay. For the A549 cells and fibroblasts, a linear relation wasestablished for the MTT values and cell number within the range of the experimentsshown (r = 0.995, P < 0.005). A549 cells grown for 72 hours in the presence of thecompounds (5—1000 nM) for vehicle (0.15% EtOH) were lysed with 0.25 N NaOH andthe cellular protein content was determined by applying the Bio-Rad (Richmond, CA)microassay method using bovine serum albumin as standard. The mean cellular proteincontent in resting A549 cells was 46.6 ;t 1.5 pg/cell.Thymidine incorporation and DNA synthesis:A549 cells (-2 x 104 cells/ml) were seeded in 96-well plates, allowed to settlefor 24 hours and grown for an additional 72 hours in the presence of compounds (5-1000 nM) or vehicle (medium plus 0.15% ethanol). Twenty-four hours before the assay,2 (Ci/ml of methyl-[3H]thymidine (specific activity 6.7 Ci/mmol) were added to each101520253035W0 98/11049CA 02265708 l999-03- 10PCT/US97/ 16342_ 84 _well. (See Cybulsky et al. (1992) Eicosanoids enhance epidermal growth factor receptoractivation and proliferation in glomerular epithelial cells. Am. J. Physiol. 262 (RenalFluid Electrolyte Physi0l.3l): F 639-F646.) After pulse-labeling, each well was washedfour times with cold DPBS2+, and the cells were lysed with NaOH (0.25 N) andradioactivity measured.The Student's t—test was used for statistical analysis and differences were consideredsignificant at a P value ( 0.05).RESULTSEicosanoids are formed by initial oxygenation of arachidonic acid by PGHS orL0 enzymatic pathways. (Samuelsson B., et al. (1987) Leukotrienes and Lipoxins:structures, biosynthesis, and biological effects. Science 237: 1171-1176.) To assesswhich of the eicosanoid-generating enzymes are present and/or regulated by cytokines inA459 cells, mRNA levels of PGHS-1 and -2 and 5-, 12- and 15-LO from A549 cellsgrown in the presence or absence of IL-1 B were monitored by RT-PCR followed byphosphorimager analysis. As illustrated in FIG. 1A, mRNA levels for PGHS-2 weresignificantly increased (~ twofold) after stimulation of A549 cells with IL-15. Incontrast, mRNA levels for PGHS-l and 5-LO were not significantly altered afterexposure of the cells to cytokine (FIG. 1A). A549 cells failed to show either 15- or 12-LO expression before or after cytokine induction (FIG. IA). The absence of 15-LOmRNA in A549 cells, was further confirmed by performing the RT-PCR in parallel withhuman lung tissue and peripheral blood monocyte RNA, which are known positive andnegative sources, (cf Funk C.D. and FitzGera1d G.A. (1991) Eicosanoid formingenzyme mRNA in human tissues. J. Biol. Chem. 266: 12508-12513), of 15-LO mRNA,respectively (see FIG. 1A, inset).To characterize the profile of monohydroxy products produced by airwayepithelial cells, IL-1 B-stimulated A549 cells (1 .5x106 cells/ml) were perrneabilized andincubated with arachidonic acid, and the products formed were extracted and analyzedby RP-HPLC. The chromatographic profile revealed the presence of a major productwith strong UV absorbance at 234 nm which coeluted with synthetic 15-HETE. Also,when [3H]-arachidonic acid was added to IL-1 B-treated A549 cells, radiolabeledmaterial was recovered beneath the peak coeluting with 15-HETE (FIG. 1B). In theseexperiments, the formation of either 5- or 12-HETE was not consistently observed. ASAtreatment of A549 cells led to a marked increase in the formation of 15-HETE, whileincubation of permeabilized A549 cells with 17-ODYA, a reported inhibitor of P450eicosanoid metabolism, (Muerhoff, A.S. et al. (1989) Prostaglandin and fatty acid to and101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97l16342.. 8 5 -(co-1)—oxidation in rabbit lung: acetylenic fatty acid mechanism-based inactivators asspecif1cinhibitors,J. Biol. Chem. 244:749—756), resulted in ~50% reduction in 15-HETE(FIG. 2A). l7-ODYA is a potent inhibitor of P450 eicosanoid metabolism and does notselectively inhibit either cyclooxygenase or L0 activity. (See Muerhoff et al. andsupplier's supporting materials.) The 5-LO inhibitor (Rev-5901 isomer) did not alter theamount of 15-HETE produced by A549 cells in a statistically significant fashion. Heat-denatured A549 cells reduced the quantities of 15-HETE by ~90%_, suggesting anenzymatic component in its formation. Production of 15-HETE from endogenoussources of arachidonate was also obtained from intact A549 cells (25.0 :1: 10.0 ng/107cells) treated with IL-1 5 (1 ng/ml) for 24 hours. Taken together these results indicatethat 15-HETE is the main monohydroxy product generated by A549 cells and suggestthat acetylated PGHS-2 and cytochrome P450 each contributes to its biosynthesis.To investigate the time-course for generation of 15-HETE from endogenoussources of arachidonate, intact A549 cells were exposed to IL-1 B (1 ng/ml) for up to 48h and the amount of immunoreactive 15-HETE present in the cell supernatant wasmonitored by means of a specific RIA. In resting conditions, A549 cells producedsignificant levels of immunoreactive 15-HETE (19.1 d: 10.5 ng/107 cells, n = 3, d = 2).These values were unchanged by addition of ionophore A3187 (5 (M) in the absence ofIL-1 [3 (FIG. 2B). Also, in the absence of ionophore stimulation, addition of IL-1 5 toA549 cells for up to 48 h did not result in an augmented generation of 15-HETE (FIG.2B). In sharp contrast, addition of IL-1 3 plus A23137 stimulation of A549 cells led to amarked increase in the production of 15-HETE (FIG. 2B). The maximal levels werefound at 24 h of exposure to the cytokine with the levels of 15-HETE decliningthereafter.Because the stereochemistry of the alcohol in 15-HETE produced by A549 cellswas of interest, the relative amounts of individual R and S enantiomers of 15-HETEgenerated by IL-1 B-treated A549 cells were examined using a chiral phase HPLCanalysis. (See Methods.) PGHS-2 as well as cytochrome P450 are enzymes other than15-LO that can each convert arachidonic acid to 15-HETE. ASA-acetylated PGHS-2-derived 15-HETE carries its carbon (C)—l 5 alcohol group mainly in the R configuration.(Holtzman, M.J., et al., (1992) Identification of a pharrnacologically distinctprostaglandin H synthase in cultured epithelial cells, J. Biol. Chem. 267221438-21445).Here, 15-HETE produced by IL-1 3-primed A549 cells was converted to its methyl esterand subjected to SP-HPLC chiral analysis. The 15-HETE from activated A549 cells was65% in the R and 35% in the S configuration (FIG. 3). Pretreatment of A549 cells withASA (20 min, 37 °C) resulted in a 3-fold increase in the amounts of 15R-HETE whereasformation of 15S—HETE remained unaltered (FIG. 3). In the presence of ASA, 15R-101520253035CA 02265708 l999-03- 10W0 98/1 1049 PCT/US97/16342-86-HETE accounted for 85% of the total amount of 15-HETE produced by A549 cells.These results indicate that the majority of 15-HETE generated by IL-1 B-primed A549cells in presence of ASA was in the R configuration.Transcellular eicosanoid biosynthesis is an important means of amplifying lipidmediators as well as generating new mediators. (Marcus, A.J. (1995) Aspirin asprophylaxis against colorectal cancer, N Engl. J. Med. 333:656-658.) Costimulation ofhuman endothelial cells and PMN after ASA treatment results in the formation of a newclass of bioactive eicosanoids. (Claria, J. and Serhan, C.N. (1995) Aspirin triggerspreviously undescribed bioactive eicosanoids by human endothelial cell-leukocyteinteractions, Proc. Natl. Acad. Sci. USA 92:9475-9479.) These novel eicosanoids wereidentified as l5-epi—LXs and their biosynthesis involve leukocyte transformation ofASA-triggered endothelial-derived 15R-HETE. In view of these results, it is possiblethat formation of new eicosanoids by transcellular biosynthesis also occurs duringepithelial cell-PMN interactions. To test this hypothesis, confluent A549 cells wereexposed to IL-13 (1 ng/ml, 24 h), treated with ASA and costimulated with PMN. FIG.4A shows a representative HPLC profile of material obtained from stimulated cellsexposed to ASA, which revealed the presence of four major products with strong UVabsorbance when plotted at 300 nm. On-line spectral analysis of these products showedthat they each displayed a triplet of absorbing bands characteristic of conjugatedtetraene-containing chromophores indicative of the LX basic structure (maxima at 301nm and shoulders at 288 and 316 i 2 nm) (FIGS. 4B and 4C).LXA4 and 15-epi-LXA4 were identified in the chromatographic profiles on thebasis of coelution with synthetic standards and the presence of the characteristicchromophore. In these coincubations, 15-epi-LXA4 accounted for ~88% of the totalamount of LXA4 detected which is in agreement with the observation that the majorityof 15-HETE generated by IL-1 [3-primed A549 cells exposed to ASA is predominantly inthe R configuration. (Cf FIGS. 3 and 4A, 4B and 4C). In this RP-HPLC system, l5-epi-l 1-trans-LXA4 and LXB4 coeluted, as did 11-trans-LXA4 and 15-epi-LXB4 (notshown). These LX isomers were not further resolved by HPLC and are denoted in theprofile beneath peaks labeled as peaks A and B, respectively (FIG. 4A). The compoundsbeneath peaks A and B did resolve as OTMS, methyl ester derivatives in GC-MS (videinfra). The products were present in ~8:2 ratio in favor of their 15R epimers (n=3).Compound C (FIG. 4A) did not coelute with any of the previously identified LXs andwas also present in the RP-HPLC profile from activated PMN incubated with 15R-HETE (data not shown, 11 = 5). Material eluting beneath Compound C matched thephysical properties of Compound III that was recently isolated from endothelial cell-PMN interactions. (Cf Claria, J. and Serhan, C.N. (1995) Aspirin triggers previously101520253035W0 98/1 1049ICA 02265708 l999-03- 10PCT/US97/16342_ g7 _undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions,Proc. Natl. Acad. Sci. USA 92:9475-9479.) Although the complete stereochemistry ofcompound C remains to be determined, the UV spectral data and chromatographicmobilities suggest that it may be the 15-epimer form of 7-cis-1 l-trans-LXA4.(Nicolaou, K.C., et al. (1989) Identification of a novel 7-cis-11-translipoxin A4,generated by human neutrophils: total synthesis, spasmogenic activities and comparisonwith other geometric isomers of LXs A4 and B4, Biochim. Biophys. Acta 1003244-53.)Thus, the LXs generated during epithelial (A549 cell)—PMN costimulation after ASAtreatment were predominantly I5-epi—LX (FIG. 4A).In addition to the ability to produce LXS, coincubations of activated PMN withA549 cells also generate significant amounts (~ 8 times more than tetraene-containingLXs) of peptidoleukotrienes (pLTs; LTC4, and LTD4) in the absence of ASA (FIGS. 5Aand 5B). The amounts of both LXs and pLTs produced in these coincubations weredependent upon individual cell ratios (FIG. 5A and 5B, insets). Exposure of airwayepithelial A549 cells to ASA (20 min) before addition of PMN led to an increase in theformation of LXs and a decrease in pLTs (F IGs. 5A and SB). Neither PMN nor A549cells incubated separately, in the absence or presence of ASA, generate detectable levelsof LXs or pLTs (FlGs. 5A and 5B and data not shown). Taken together, these resultsindicate that, during A549 cell-PMN interactions, both LXs and pLTs originate fromtranscellular routes.LXS are vasodilators and potent regulators of leukocyte responses, such asinhibition of chemotaxis, adhesion to endothelial cells and transmigration acrossepithelium. (See Serhan, C.N. (l 994) Lipoxin biosynthesis and its impact ininflammatory and vascular events. biochim. Biophys. Acta 1212:1-25.) In contrast,pLTs possess both vasoconstrictor and proinflammatory actions as well as stimulate thegrowth of several cell types including fibroblasts, smooth-muscle and glomerularepithelial cells. (Baud, L., et al. (I 985) Leukotriene C4 binds to human glomerularepithelial cells and promotes their proliferation in vitro. J. Clin. Invest. 76:374-377.)LXs reverse the vasoconstrictor action of LTD4 in rat renal hemodynamics and blockLTC4-stimulated hematopoiesis. (Serhan, C.N. (1994) Lipoxin biosynthesis and itsimpact in inflammatory and vascular events. Biochim. Biophys. Acta 1212:1-25.)Because ASA enhances l5—epi-LX formation and inhibits pLT biosynthesis (FIGS. 5Aand 5B), these eicosanoids may play counterregulatory actions on cell proliferation andcontribute to ASA's protective mechanisms in human cancer. To this end, the effect ofthese LO products on epithelial cell proliferation (FIGS. 6A and 6B) was tested and theiractions was compared to that of dexamethasone, a well-established inhibitor, employinga soluble microculture tetrazolium (MTT) assay. (Alley, M.C., et al. (1988) Feasibility1015202530W0 98/11049CA 02265708 l999-03- 10PCT/US97/16342_ 33 -of drug screening with panels of human tumor cell lines using a microculturetetrazolium assay. Cancer Res. 48:589-601.) These experiments were performed withsynthetic LXA4 and LXB4, which were available in sufficient quantities for bioassay,rather than the 15-epi-LX, which are the major LX produced by these cells. As shown inFIGs. 6A and 6B, LXA4 and LXB4 inhibited A549 cell proliferation in a time- (A) anddose-dependent (B) fashion. LXA4 and LXB4 as well as dexamethasone [1 uM]inhibited A549 cell proliferation after 72 and 96 hours of treatment (FIG. 6A). After 72hours, LXA4 shared the anti-proliferative properties (FIG. 6A) observed fordexamethasone with these cells. (Cf Croxtall, J.D., and Flower R.J. (1992) Lipocortin lmediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cellline. Proc. Natl. Sci. USA 89:357l-3575.) The half maximum inhibition (IC50) forLXA4 was ~80 nM compared to that of dexamethasone, which was ~7 nM. LXB4 atconcentrations of 0.5 and 1 uM was (3 times more active than either LXA4 ordexamethasone (FIG. 6B). Of interest, both LXA4 and LXB4 showed essentially equalpotency for blocking A549 cell growth when each was added repeatedly (i.e., 24-hourintervals) to the cells for 3 consecutive days (data not shown, n = 3, d = 4), suggestingthat LX may be inactivated by these epithelial cells. Results from additional experimentsemploying direct cell enumeration (FIG. 7B) and measurement of total cellular proteincontent (data not shown, n = 3, d=4) paralleled those obtained with MTT assay, thusconfirming the anti-proliferative actions of LXs in A549 cells. Furthermore, a blockageof DNA synthesis, as determined by 3H—thymidine incorporation, occurred when A549cells were exposed for 72 hours to concentrations of 50 nM or higher of LXB4 ordexamethasone (FIG. 7A). Afier incubation of A549 cells with the compounds, theviability of the cells, as determined by trypan blue exclusion assay, was found to be~98%, indicating that these compounds were not cytotoxic within the range ofconcentrations used in these experiments.The l5-hydroxy epimeric forms of LXA4 and LXB4 (15-epi-LXA4 and l5-epi-LXB4, respectively), which were the dominant forms of LX isolated from these cells,proved to also be potent inhibitors of epithelial cell proliferation, as shown in thefollowing Table 1.10152025W0 98/11049ICA 02265708 l999-03- 10PCT/US97/ 16342- 39 _Table 1: LX and 15-epi-LX actions on cell proliferationCompound [10-7 % inhibition P value*Vehicle 1.6 i 10.3 NSLXA4 16.3 J; 5.2 NS15-epi-LXA4 20.1 2». 1.9 <0.025LXB4 30.0 : 8.5 <0.00515-epi-LXB4 79.3 i 0.3 <0.001&Dexamethasone 42.1 i 5.4 <0.005Cells (2000 A549 cells/well) were grown in 96-well plates and exposed to vehicle(0. 1 5% vol/vol in EtOH/F-12K media) or equimolar concentrations (1O‘7 M) of LXA4,15-epi-LXA4, LXB4, 15-epi-LXB4, or dexamethasone for 72 hours at 37 °C. Valuesrepresent i SEM from 3 to 7 experiments performed in quadruplicate and are expressedas percent inhibition of cell growth. *P values denot statical differences as compared tocells alone. & P<0.001 for 15-epi-LXB4.At equimolar levels (100 nM), 15-epi-LXA4 inhibited A549 cell growth to a similarextent as LXA4. On the other hand, 15—epi-LXB4 isolated from conversion of 15R-HETE by activated PMN and added back to the A549 cells gave a more potent anti-proliferative activity than LXB4 (~80% vs. ~34% inhibition proliferation, P<0.001;Table 1). This compound was characterized by UV, HPLC and GC-MS (C value: 23.3).Diagnostic ions for the OTMS methyl ester were m/z 173 (base peak), 203, 289, 379 andless prominent ions at 482 (M+-100) [It was not possible to obtain its molecular ionbecause of its low abundance]. Thus, the predominant material beneath the peak labeledB in FIG. 4B was consistent with that of 15-epi-LXB4, which gave a shorter C value andseparated from LXB4 as OTMS, methyl ester derivative in GC-MS analysis. The massspectra of 15-epi-LXB4 and LXB4 were essentially identical (not shown) but their Cvalues were distinct. Material eluting beneath the peak denoted as C (isolated fromactivated PMN incubated with 15R-HETE) also showed a mild inhibitory action onepithelial cell growth (18 d: 2% inhibition proliferation, n = 3, d = 4). In sharp contrast,15-epi-trans-LXA4, 11-trans—LXB4, 8,9-acetylenic-LXB4, peptidoleukotrienes (LTC4and LTD4) and the LX precursors (15S— and 15R-HETE) each tested at 10'6 — 10‘9 Mwere not able to significantly inhibit A549 cell proliferation (data not shown, n = 3-5, (1= 4). These results indicate that LX and 15-epi-LX gave a stereoselective action inblocking cell proliferation in A549 cells. LXA4 and LXB4 was tested with human skinfibroblasts to determine if they were antiproliferative for this cell type. At 100 nM, bothLXA4 and LXB4 inhibited proliferation of fibroblasts. LXB4 gave 38.0 ( 7.5%101520W0 98/1 1049CA 02265708 l999-03- 10PCT/US97/16342- 90 _inhibition and LXA4 10.7 ( 1.8% compared to dexamethasone (29.7 ( 0.4%) as positivecontrol (n=3).The presence of an active cytochrome P450 enzyme system in human airwayA549 cells, (Vogel, et al. (1994) Transforming growth factor-Bl inhibits TCDD-inducedcytochrome P45OIA1 expressions in human lung cancer A549 cells. Arch. Toxicol. 68:303-307.), together with the results that inhibition of P450 as well as heat denaturingblocks 15-HETE generation in these cells (FIG. 2A) suggests that this enzyme system inepithelial cells also contributes to l5—HETE biosynthesis and the generation of l5-epi-lipoxin by transcellular routes (FIG. 8). Taken together, these observations (FIGS. 1-4)establish the existence of two separate enzymatic pathways (i.e. ASA-acetylated PGHS-2 and cytochrome P450), which can initiate the formation of 15—epi-lipoxin duringairway epithelial cell-PMN interactions (FIG. 8). Also, it should be noted that, in viewof ASA's ability to induce P450 enzymes, Pankow, D. et al. (1994) Acetylsalicylic acid -inducer of cytochrome P-450 2E1? Arch. T oxicol. 68: 261-265, it is possible that thesetwo independent routes may act in concert to generate l5-epi-lipoxin.EguivalentsThose skilled in the art will recognize, or be able to ascertain using nomore than routine experimentation, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scope of thisinvention and are covered by the following claims.CA 02265708 1999-09-09-91-SEQUENCE LISTINGGENERAL INFORMATION:APPLICANT:NAME: BRIGHAM AND WOMEN'S HOSPITALSTREET: 75 FRANCIS STREETCITY: BOSTONSTATE: MASSACHUSETTSCOUNTRY: USPOSTAL CODE (ZIP): 02115TELEPHONE:TELEFAX:TITLE OF INVENTION: LIPOXIN COMPOUNDS AND THEIR USE INTREATING CELL PROLIPERATIVE DISORDERSNUMBER OF SEQUENCES: 12CORRESPONDENCE ADDRESS:ADDRESSEE: RICHES, MCKENZIE 8 HRBERTSTREET: 2 BLOOR STREET EAST, SUITE 2900CITY: TORONTOSTATE: ONTARIOCOUNTRY: CANADAPOSTAL CODE: MIW 3J5CGMUTER READABLE FORM:COMPUTER: IBM PC compatibleOPERATING SYSTEM: PC-DOS/MS-DOSSOFTWARE: ASCII TEXTCURRENT APPLICATION DATA:APPLICATION NUMBER: 2,265,708FILING DATE: 15-SEPTEMBER-1997cLAssIrIcATION:cO7c 59/42, AGIK 31/32, co7c 69/732, CO7F 7/O8PRIOR APPLICATION DATA:APPLICATION NUMBER: Us O8/712,610FILING DATE: 13-SEPTEMER-1996PATENT AGENT INFORMATION:NAME: RICHES, MOKENZIE 8 HERBERTREFERENCE 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Claims (18)

1. A substantially purified 15-epi-lipoxin compound in which the absolute configuration at the 15 carbon is R.

2. The 15-epi-lipoxin compound of claim 1, wherein the 15 epi-lipoxin compound is 15R-5, 6, 15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid.

3. The 15-epi-lipoxin compound of claim 2, wherein the 15R-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid has a 5S,6R
configuration.

4. The lipoxin compound of claim 1, wherein the 15-epi-lipoxin compound is 15R-5,14,15-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid.

5. The 15-epi-lipoxin compound of claim 4, wherein the 15R-5,14,15-trihydroxy-6,10,12-traps-8-cis-eicosatetraenoic acid has a 5S, 14R
configuration.

6. The 15-epi-lipoxin compound of claim 1, wherein the 15-epi-lipoxin compound is a 15-hydroxyeicosatetraenoic acid.

7. A pharmaceutical composition comprising the 15-epi-lipoxin compound of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein the 15-epi-lipoxin compound is in an amount effective to prevent an undesired proliferation of cells in a subject.

9. The pharmaceutical composition of claim 8, wherein the cells are epithelial cells.

10. The pharmaceutical composition of claim 8, wherein the cells are leukocytes.

11. The pharmaceutical composition of claim 8, wherein the cells are endothelial cells.

12. The pharmaceutical composition of claim 8, wherein the cells are fibroblasts.

13. The pharmaceutical composition of claim 8, wherein the cells are undergoing cancerous growth.

14. The pharmaceutical composition of any one of claims 7 to 13, further comprising an effective amount of acetylsalicylic acid.

15. Use of an effective amount of a substantially purified 15-epi-lipoxin compound for inhibiting or preventing an undesired proliferation of a cell.

16. Use of claim 15, wherein the cell is in vivo.

17. Use of claim 15, wherein the cell is ex vivo.

18. Use of an effective amount of a substantially purified 15-epi-lipoxin compound for treating or preventing a cell proliferative disorder.