AU4271097A

Lipoxin compounds and their use in treating cell proliferative disorders

Abstract

This record has no abstract on file.

AU4271097A, drawing sheet 1 — Sheet 1 of 85

Term

Term ended

Projected expiry passed 15 September 2017, 9 years ago.

18 claims: 2 independent, 16 dependent

1
I claim, 1. A substantially purified 15-epi-lipoxin compound.
16
A method of inhibiting or preventing an undesired proliferation of a cell, which comprises contacting the cell with an effective amount of a substantially purified 15-epi 10 lipoxin compound.

Independent claims2

685 paragraphs in 167 sections, as filed

The present invention is intended to include the possible diastereisomers as well as the racemic and optically resolved isomers.

A preferred lipoxin compound for use in the subject invention is a 15epi-lipoxin compound. As used herein, 15-epi-lipoxin compound is a lipoxin compound in which the absolute configuration at the 15 Carbon is R.

The term 15-epi-lipoxin compound is intended to encompass precursors. The term precursor is intended to refer to chemical intermediates that can be converted in vivo, ex vivo and/or in vitro to form the 15-epi-lipoxin compounds of the invention. The term precursor also contemplates prodrugs which are converted in vivo to the 15-epi-lipoxin compounds of the invention (see, e.g., R.B. Silverman, 1992, The Organic 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/or carboxyl groups and/or compounds which can be hydrolyzed or otherwise converted in vivo or, ex vivo and/or in vitro into the 15-epi-lipoxin compounds of the present invention.

The terms corresponding lipoxin and natural lipoxin refer to a naturally-occurring lipoxin or lipoxin metabolite. Where an analog has activity for a lipoxin-specific receptor, the corresponding or natural lipoxin is the normal ligand for that receptor. For example, where an analog is a LXA4 analog having specific activity for a LXA4 specific receptor on differentiated HL-60 cells, the corresponding lipoxin is LXA4. Where an analog has activity as an antagonist to another compound (such as a

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-8leukolriene), which is antagonized by a naturally-occurring lipoxin, that natural lipoxin is the corresponding lipoxin.

The term '“active region shall mean the region of a natural lipoxin or lipoxin analog, which is associated with in vivo cellular interactions. The active region may bind the recognition site of a cellular lipoxin receptor or a macromolecule or complex of macromolecules, including an enzyme and its cofactor. Preferred lipoxin A4 analogs have an active region comprising C5-C15 of natural lipoxin A4. Preferred lipoxin B4 analogs have an active region comprising C5-C14 of natural lipoxin B4.

The term recognition site or receptor is art-recognized and is intended to refer generally to a functional macromolecule or complex of macromolecules with which certain groups of cellular messengers, such as hormones, leukotrienes, and lipoxins, must first interact before the biochemical and physiological responses to those messengers are initiated. As used in this application, a receptor may be isolated, on an intact orpermeabilized cell, or in tissue, including an organ. A receptor may be from or in a living subject, or it may be cloned. A receptor may normally exist or it may be induced by a disease state, by an injury, or by artificial means. A compound of this invention may bind reversibly, irreversibly, competitively, noncompetitively, or uncompetitively with respect to the natural substrate of a recognition site.

The term metabolic transformation region is intended to refer generally to that portion of a lipoxin, a lipoxin metabolite, or lipoxin analog including a lipoxin analog metabolite, upon which an enzyme or an enzyme and its cofactor attempts to perform one or more metabolic transformations which that enzyme or enzyme and cofactor normally transform on lipoxins. The metabolic transformation region may or may not be susceptible to the transformation. A nonlimiting example of a metabolic transformation region of a lipoxin is a portion of LXA4 that includes the C-13,14 double bond 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 the compound 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 encompass compounds which are labeled with radioactive isotopes, such as but not limited to tritium (3H), deuterium (2H), carbon ( l4C), or otherwise labeled (e.g. fluorescently).

The compounds of this invention may be labeled or derivatized, for example, for kinetic binding experiments, for further elucidating metabolic pathways and enzymatic mechanisms, or for characterization by methods known in the art of analytical chemistiy.

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The term inhibits metabolism means the blocking or reduction of activity of an enzyme which metabolizes a native lipoxin. The blockage or reduction may occur by covalent bonding, by irreversible binding, by reversible binding which has a practical effect of irreversible binding, or by any other means which prevents the enzyme from operating in its usual manner on another lipoxin analog, including a lipoxin analog metabolite, a lipoxin, or a lipoxin metabolite.

The term resists metabolism is meant to include failing to undergo one or more of the metabolic degradative transformations by at least one of the enzymes which metabolize lipoxins. Two nonlimiting examples of LXA4 analog that resists metabolism are 1) a structure which can not be oxidized to the 15-oxo form, and 2) a structure which may be oxidized to the 15-oxo form, but is not susceptible to enzymatic reduction to the 13,14-dihydro form.

The term more slowly undergoes metabolism means having slower reaction kinetics, or requiring more time for the completion of the series of metabolic transformations by one or more of the enzymes which metabolize lipoxin. A nonlimiting example of a LXA4 analog which more slowly undergoes metabolism is a structure which has a higher transition state energy for C-15 dehydrogenation than does LXA4 because the analog is sterically hindered at the C-16.

The term tissue is intended to include intact cells, blood, blood preparations such as plasma and serum, bones, joints, muscles, smooth muscles, and organs.

The term halogen is meant to include fluorine, chlorine, bromine and iodine, or fluoro, chloro, bromo, and iodo.

The term pharmaceutically acceptable salt is intended to include artrecognized pharmaceutically acceptable salts. These non-toxic salts are usually hydrolyzed under physiological conditions, and include organic and inorganic bases. Examples of salts include sodium, potassium, calcium, ammonium, copper, and aluminum as well as primary, secondary, and tertiary amines, basic ion exchange resins, purines, piperazine, and the like. The term is further intended to include esters of lower hydrocarbon groups, such as methyl, ethyl, and propyl.

The term pharmaceutical composition comprises one or more lipoxin analogs as active ingredients), or a pharmaceutically acceptable salt(s) thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. 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 in any particular case will depend on the nature and severity of the conditions being treated

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-10PCT/US97/16342 and the nature of the active ingredient(s). The compositions may be presented in unit dosage 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 be proportionally reduced over time. A person skilled in the art normally may determine the effective dosage amount and the appropriate regime. A lipoxin analog pharmaceutic composition can also refer to a combination comprising lipoxins, lipoxin analogs, and/or lipoxin metabolites, including metabolites of iipoxin analogs. A nonlimiting example of a combination is a mixture comprising a lipoxin analog x which inhibits one enzyme which metabolizes lipoxins and which optionally has specific activity with a lipoxin receptor recognition site, and a second lipoxin analog y which has specific activity with a lipoxin receptor recognition site and which optionally inhibits or resists lipoxin metabolism. This combination results in a longer tissue half-life for at least y since x inhibits one of the enzymes which metabolize lipoxins. Thus, the lipoxin action mediated or antagonized byy is enhanced.

The term substantially pure or purified lipoxin compounds are defined as encompassing natural or synthetic compounds of lipoxins having less than about 20% (by dry weight) of other biological macromolecules , and preferably having less than about 5% other biological macromolecules (but water, buffers, and other small molecules, especially moleculess having a molecular weight of less than 5000, can be present. The term purified as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term pure as used herein preferably has the same numerical limits as purified immediately above. Isolated and purified do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.

The term subject is intended to include living organisms susceptible to conditions or diseases caused or contributed to by inflammation, inflammatory responses, vasoconstriction, myeloid suppression and/or undesired cell proliferation. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species.

The term cell proliferative disorder includes disorders involving the undesired proliferation of a cell. Non-limiting examples of such disorders include

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PCT/US97/16342 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, prevention of, limiting of and/or inhibition of undesired cell proliferation and/or a cell proliferative disorder.

Lipoxin Compounds

The instant invention is based on the surprising finding that substantially pure 15-epi-lipoxin compounds ameliorate undesired cell proliferation in a subject. The 15epi-lipoxin compounds of the present invention include 157?-5,6,15-trihydroxy-7,9,13//ww-ll-cw-eicosatetraenoic acid, in a 55,67? configuration (15-epi-LXA4). The 15-epilipoxin compounds of the present invention also include 157?-5,14,15-trihydroxy6,10,12-/ra;?5-8-cfs-eicosatetraenoic acid, in a 55,147? configuration (15-epi-LXB4).

The 15-epi-lipoxins of the present invention further include 15-hydroxyeicosatetraenoic acid, particularly in a 157? configuration.

The instant invention is based on the surprising finding that lipoxins are rapidly metabolized in a unique fashion by certain ceils in vivo. Although other LOderived products (e.g. leukotrienes) are metabolized by ω-oxidation followed by βoxidation (Huwyler et al., (1992) Eur.J. Biochem. 206, 869-879), the instant invention is based on the unexpected finding that lipoxins are metabolized by a series of oxidation and 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-15 hydroxyl to generate I5-0X0-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 LXB4 and its natural isomers the analogous oxidation occurs at the C-5 hydroxyl and reduction occurs at the C-6,7 double bond.

Thus, the instant invention features lipoxin analogs having lipoxin activity, but which are chemically modified to prevent dehydrogenation and therefore subsequent degradation in vivo. In these analogs, the C-l to C-13 portion of the natural lipoxin may or may not be conserved. Variations of the C-l to C-13 portion include different cis or irons geometry as well as substitutions. The disclosed compounds is represented 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 Roman numeral on the left of the page.

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-12The instant lipoxins comprising an active region and a metabolic transformation region as both terms are defined herein are generally of the following structure:

Y> £2 o γ2· r ϊ j &3a R3b

VI, VII, VIII, IX, X

XI

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-13 and R-2 can be

Rd He

OH

VI

In one embodiment, the lipoxin analogs of this invention have the following structural formula I:

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- 14PCT/US97/16342

wherein X is R]5 OR], or SRj; wherein R| is (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;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z;, Zand Zv are each independently selected from the group consisting of -NO2, -CN, -C(-O)-Rj, -SO3H, and hydrogen;

wherein Zj; and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule; or

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-15PCT/US97/K5342 (viii) straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;

wherein Qj is (C=O), SO2 or (CN); wherein Q3 is 0, 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 may be straight chain or branched;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (e) RaQ2R|j wherein Q2 is -O- or -S-;

wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may he straight chain or branched;

wherein R4 is (a) H;

(b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chain or branched;

wherein Y| or Y2 is -OH, methyl, or -SH and wherein the other is (a) H (b) CHaZb where a + b =3, a= 0 to 3, b = 0 to 3; and Z is cyano, nitro, or halogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; or (d) alkoxy of 1 to 4 carbon atoms, inclusive; or Y! and Y2 taken together are (a) =N; or (b) =O; wherein R5 is (a) alkyl of 1 to 9 carbon atoms which may be straight chai'· or branched;

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-16(b)-(CH2)n-Ri wherein n = 0 to 4 and Rj is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or (iii) substituted phenyl

wherein Z;, Z and Zv are each independently selected from the group consisting of hydrogen, -NO2, -CN,

-C(=O)-Rj, methoxy, and -SO3H; and wherein Zb and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, and hydroxyl;

(c) -RaQaRb wherein Qa = -O- or -S-; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

(d) -C^XR^-Ri wherein R,-;; and Riv are selected independently from the group consisting of (i) H;

(ii) CHaZb where a + b = 3, a = 0to3, b = 0 + 3, and wherein any Z is independently selected from the group consisting of halogen;

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to

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-17PCT/US97/16342 halogen atoms, inclusive, straight chain or branched; and wherein Rg is (a)H;

(b) alkyl from I to 4 carbon atoms, inclusive, straight chain or branched;

(c) halogen; but excluding the C-l position amides, C1 position alkanoates, and pharmaceutically acceptable C-l position salts of (5S, 6R, 15S)-trihydroxy-7E, 9E,

11Z, 13E-eicosatetraenoic acid (LXA4); and excluding

C-5, C-6, and C-l 5 position alkanoates of LXA4.

In one embodiment of this invention, the lipoxin analogs have the following structure II:

II.

wherein X is Rj, ORt, or SR|; wherein Rj is (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;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

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wherein Ζ;, Z ii{ and Zv are each independently selected from the group consisting of -NO2, -CN, -C(=O)-Rj, hydrogen, and -SO3H;

wherein Zq and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule, such as but not limited to fluorescent labels; or (viii) alkenyl of 2 to 8 carbon atoms, inclusive, straight chain or branched;

wherein Qj is (C=O), SO2 or (C=N);

wherein Q3 is 0, 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 may be straight chain or branched;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (e) ^aQz^b wherein Q2 is-O-or-S-;

wherein fta is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may he straight chain or branched;

wherein R4 is (a)H;

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-19PCT/US97/16342 (b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chain or branched;

wherein Yj or Y2 is -OH, methyl, -H or -SH and wherein the other is (a) H;

(b) CHaZb where a + b =3, a= 0 to 3, b = 0 to 3 Z is cyano, nitro, or halogen including F, CI, Br, I;

(d) alkoxy of 1 to 4 carbon atoms, inclusive; or Yj and Y2 taken together are (a) =N; or (b) =0; wherein R5 is (a) alkyl of 1 to 9 carbon atoms which may be straight chain or branched;

(b) -(CH2)n-Ri wherein n = 0 to 4 and Rj is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or (iii) substituted phenyl

wherein Z;, Z jjj, and Zv are each independently selected from the group consisting of hydrogen, -NO2, -CN,

-C(=O)-R], methoxy, and -SO3H; wherein Z,j and Ziv are each independently selected from the group consisting of halogen, methyl,

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-20hydrogen, and hydroxyl;

(C) -RaQaRb wherein Qa = -0- or -S-; and wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

(d) -C(Rin)(Riv)-Ri wherein and R;v are selected independently from the group consisting of (i) H; and (ii) CHaZb where a + b = 3, a = 0 to 3, b = 0 + 3 wherein any Z is selected from the group consisting of halogen.

(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.

In one embodiment of this invention, the lipoxin analogs have the following structure III:

III.

wherein X is Rj, OR], or SR]; wherein R] is (i) hydrogen;

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-21(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 phenyl

wherein Z [, Z ϋ,· , and Zv are each independently selected from the group consisting of-NO2,-CN, -C(=O)-Rj, hydrogen, and -SO3jH;

wherein Zjj and Ziv are each independently selected from the group consisting 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 Q, is (C=O), SO2 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 may be straight chain or branched;

(d) alkenyl Of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (e) RaQ2^b wherein Q2 is -O- or -S-;

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-22PCT/US97/16342 wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

wherein R4 is (a) H; or (b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chain or branched;

wherein Y] or Y2is hydroxyl, methyl, hydrogen or thiol and wherein the other is (a) H;

(b) CHaZb where a + b =3, a= 0 to 3, b = 0 to 3 Z is cyano, nitro, or halogen [including F, CI, Br,

Φ (c) . alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(d) alkoxy of 1 to 4 carbon atoms, inclusive; or Y ] and Y2 taken together are (a) =N;or (b) =0; and wherein R5 is (a) alley 1 of 1 to 9 carbon atoms which may be straight chain or branched;

(bJ-iCH^-R, wherein n = 0 to 4 and Rj is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl;

(iii) substituted phenyl

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wherein Z j, Z ji5, and Zv are each independently selected from the group consisting of hydrogen, -NO2, -CN,

-C(=O)-Rj, methoxy, and -SO3H; wherein Zsi and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, and hydroxyl;

(c) ^aQa^b wherein Qa = -0- or -S-;

wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (d) -C(Rjjj)(Riv)-Rj wherein Rib and Riv are selected independently from the group consisting of (i) H;

(ii) CHaZb where a + b = 3, a = 0to3, b = 0 + 3 wherein any Z is selected from the group consisting of halogen.

In another embodiment of this invention, lipoxin analogs have the

X

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-24following structural formula IV:

IV.

OH Q3H R2 \ r I

wherein X is Rj, ORj, or SRj; wherein Rj is (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;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

wherein Z j, Z jy, and Zv are each independently selected from the group consisting of -NO2, -CN, -C(=O)-R]s methoxy, hydrogen, and -SO3H;

wherein Z;,· and Z-IV are each independently selected from the group consisting of halogen, methyl, hydrogen, and hydroxyl;

(vii) detectable label molecule; or (viii) alkenyl of 2 to 8 carbon atoms, inclusive,

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-25PCT/US97/J6342 straight chain or branched; wherein Qj is (C=O), SO2 or (CN); wherein Q3 is 0, S orNH;

wherein one of R2 and R3 is hydrogen and the other is (a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (e) RaQ2Rb wherein Q2 is -O- or -S-;

wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

wherein R4 is (a) H;or (b) alkyl of 1 to 6 carbon atoms, inclusive, which may be straight chain or branched;

wherein Yj or Y2 is -OH, methyl, or -SH and wherein the other is (a) H;

(b) CHaZb where a + b =3, a= 0 to 3, b = 0 to 3,

Z is cyano, nitro, or halogen;

(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; or (d) alkoxy of 1 to 4 carbon atoms, inclusive; or Y i and Y2 taken together are (a) =N; or (b) =O; wherein R5 is (a) alkyl of 1 to 9 carbon atoms which may be straight chain or branched;

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-26PCT/US97/16342 (b) -(CH2)„ -Ri wherein n = 0 to 4 and R; is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or (iii) substituted phenyl wherein Z j, Z ίΊί, and Zv are each independently selected from the group consisting of hydrogen, -NO2, -CN,

-C(=O)-R|, methoxy, and -SO3H; wherein Zb and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, and hydroxyl;

(c)RaQaRb wherein Qa = -O- or -S-;

wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

(dJ-CfRiiiXR^-Ri wherein RjH and Riv are selected independently from the group consisting of (i) H;or (ii) CHaZb where a + b = 3, a = 0to3,b = 0 + 3, and wherein any Z is selected from the group

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-27consisting of halogen; or (e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched; and wherein R6 is (a) H;

(b) alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; or (c) halogen.

In another embodiment of this invention, lipoxin analogs have the following structural formula V:

wherein Rj is (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;

(iv) aralkyl of 7 to 12 carbon atoms;

(v) phenyl;

(vi) substituted phenyl

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-28PCT/US97/16342 wherein Z j, Z , and Zv are each independently selected from the group consisting of-NO2, -CN, -C(=O)-RIs hydrogen, and -SO3H;

wherein Zb and Ziv are each independently selected from the group consisting 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, R3a, and R3b independently selected from (a) H;

(b) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (e) RaQjR-b wherein Q2 is -O- or -S-;

wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may he straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

wherein Y| or Y2 is -OH, methyl, hydrogen, or -Sh and wherein the other is (a) H;

(b) CHaZb where a + b =3, a= 0 to 3, b = 0 to 3, and Z is cyano, nitro, or halogen;

(d) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched;

or Y, and Y2 taken together are

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<td></td><td> (a) -N; or (b) =0; wherein Y3 or Y4 is -OH, methyl, hydrogen, or -SH and</td>

<td> '· 5 •</td><td> wherein the other is (a) H; (b) CHaZb wherein a + b =3, a= 0 to 3, b = 0 to 3,</td>

<td> 10</td><td> and any Z is cyano, nitro, or halogen; (c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; (d) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched;</td>

<td> 15</td><td> or Y3 and Y4 taken together are (a) or (b) =O; wherein Y5 or Y6 is -OH, methyl, hydrogen, or -SH and wherein the other is</td>

<td> 20</td><td> (a) H; (b) CHaZb where a + b =3, a= 0 to 3, b — 0 to 3</td>

<td> 25</td><td> Z is cyano, nitro, or halogen; (c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; (d) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched;</td>

<td> 30</td><td> or Y5 and Y6 taken together are (a) -N;or (b) =O; wherein R5 is (a) alkyl of 1 to 9 carbon atoms which may be straight chain or branched;</td>

<td> »</td><td> to)-'CH2)n-R{ wherein n = 0 to 4 and R,· is (i) cycloalkyl of 3 to 10 carbon atoms,</td>

<td> 35</td><td> inclusive; (ii) phenyl; or (iii) substituted phenyl</td>

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wherein Z;, Z ί5ι·, and Zv are each independently selected from the group consisting of hydrogen, -ΝΟ2, -CN,

-C(=O)-Rh and -SO3H; wherein Z;j and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, methoxy, and hydroxyl;

(c) -RaQaRb wherein Qa = -O- or -S-; and Wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is either alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched or substituted phenyl;

(d) -C(Rjjj)(Rjv)-Rj wherein Rii{ and R;v are selected independently from the group consisting of (i) H; or (ii) CHaZb where a + b = 3, a = 0 to 3, b - 0 + 3, and wherein any Z is selected from the group consisting of halogen; or (e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to halogen atoms, inclusive, straight chain or branched; but excluding the C-l position amides, C-l position alkanoates, and pharmaceutically acceptable C-l position salts of (5S, 14R, 15S)-trihydroxy-6E, 8Z,’ 10E, I2E-eicosatetraenoic acid (LXB4); C-5, C-6, and &

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-31- ·

C-5 position alkanoates of LXB4 ,

In another embodiment of this invention, lipoxin analogs have the structural formula VI;

VI.

Wherein Ra is selected from the group (a)H;or (b) alkyl of 1 to 8 carbon atoms;

wherein Rb selected from the group consisting of:

= In another preferred embodiment of this invention, lipoxin analogs have the following structural formula VII:

wherein Ra is selected from the group (a) H;or (b) alkyl of 1 to 8 carbon atoms;

wherein Rb and Rc are independently selected from the group

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-32(a) H;

(b) hydroxyl, or thiol;

(d) halogen;

(e) alkoxy of 1 to 3 carbon atoms, including methoxy; wherein and Re are selected independently from the group (a) H;

(b) hydroxyi, or thiol;

(d) halogen;

(e) alkoxy of 1 to 3 carbon atoms, inclusive, including methoxy; or (Q alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which may be straight chain or branched; but excluding the C-l position amides, C-l position alkanoates, and pharmaceutically acceptable C-l position salts of (5S,

6R, l5S)-trihydroxy-7E, 9E, 11Z, 13E-eicosatetraenoic acid (LXA4); C-5, C-6, and C-l 5 position alkanoates ofLXAzp

In another preferred embodiment of this invention, the lipoxin analogs have the structural formula VIII:

VIII.

wherein Ra is selected from the group (a) H;or (b) alkyl of 1 to 8 carbon atoms;

wherein Rfo and Rc are independently selected from the group (a)H;

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-33(b) hydroxyl or thiol;

(d) halogen;

(e) alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched; or .

(f) alkoxy of 1 to 3 carbon atoms, inclusive; wherein Rj and Re are selected independently from the group (a) H;

(b) hydroxyl, or thiol;

(d) halogen;

(e) alkoxy of 1 to 3 carbon atoms, inclusive, including methoxy; or (f) alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which may be straight chain or branched.

In another preferred embodiment of this invention, the lipoxin analogs have the structural formula IX:

IX.

wherein Ra is selected from the group (a) H; or (b) alkyl of 1 to 8 carbon atoms;

wherein Rfr and Rc are independently selected from the group (a) H;

(b) hydroxyl or thiol;

(d) halogen;

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-3410 (e) alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;

(f) alkoxy of 1 to 3 carbon atoms, inclusive; and wherein R5 is (a) alkyl of 1 to 9 carbon atoms wb;ch may be straight chain or branched;

(b) -(CH2)n -Rj wherein n = 0 to 4 and Rj is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl; or (iii) substituted phenyl

wherein Z ,·, Z and Zv are each independently selected from the group consisting of hydrogen, -NO2, -CN,

-C(=O)-R,, and-SO3H; wherein Zj; and Zjv are each independently selected from the group consisting of halogen, methyl, hydrogen, methoxy, and hydroxyl;

(c)-RaQaRb wherein Qa =-O-or-S-; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched;

wherein Rb is either alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched or substituted phenyl;

(dj-CCRfijXR^-Rj wherein Rjjj and Riv are selected independently

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-35PCT/US97/16342 from the group consisting of (i) H;or (ii) CHaZb where a + b = 3, a = 0 to 3, b = 0 + 3 wherein any Z is selected from the group consisting of halogen; or (e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.

In another preferred embodiment, the compounds have the structural formula X:

wherein Rg is selected from the group 15 (a)H;or (b) alkyl of 1 to 8 carbon atoms, inclusive, straight chain or branched; and wherein Rb and Rc are independently selected from the group (a)H;

(b) hydroxyl or thiol;

(d) halogen;

(e) alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;

(f) alkoxy of 1 to 3 carbon atoms, inclusive, including . methoxy.

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-36In another preferred embodiment, the compounds have the structural formula XI:

XI.

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= 1 to 10, inclusive;

wherein Y2, R3a, and R3j, are independently selected from (h) alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;

(d) alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or (e) RaQjRb wherein Q2 is -O- or -S-;

wherein Ra is alkylene of 0 to 6 carbons atoms, • inclusive, which may be straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched;

wherein Yj is -OH, methyl, or -SH; wherein Y2 is (a) H;

(b) CHaZb where a + b ~3, a=0to3, b*=0to3 Z is halogen; or

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-37(c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

wherein Y3 and Y5 are independently selected from the group consisting of:

(a) H;

(b) CHaZb wherein a + b =3, a= 0 to 3, b = 0 to 3 and any Z is cyano, nitro, or halogen; or (c) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

wherein Y4 and Y6 are independently selected from the group consisting of:

(a) H;

(b) alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;

(c) alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or (d) hydroxyl or thiol; and wherein R5 is (a) alkyl of 1 to 9 carbon atoms which may be straight chain or branched;

wherein n = 0 to 3 and Rj is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;

(ii) phenyl;

(iii) substituted phenyl

wherein Zjj and Ziv are each independently selected from the group consisting of halogen, methyl, hydrogen, methoxy, and hydroxyl;

(c) '-R-aQa^b wherein Qa = -O- or -S-;

wherein R, is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched; wherein Rb is

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(d) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched;or but excluding the C-l position amides, C-l position alkanoates, and pharmaceutically acceptable C-l position salts of (5S, I4R, 15S)-trihydroxy-6E, 8Z, 10E, 12Eeicosatetraenoic 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 invention 20 have the following structural formulas:

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-39FCT/tJS97/16342

OH

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where R' is H or CH3;

and where the substituents at G are in the R configuration.

In other preferred embodiments of this invention, the compounds of this invention have the following structural formulas:

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-41if

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-42PCT/US97/16342

where the substituents at the C* are in the R configuration.

Method for Making Lipoxin Compounds

Preferred compounds can be made as specifically described in the following Example 1, Other compounds of this invention can be made by combined strategies for lipoxin (LX) and prostaglandin analog synthesis using standard chemical methods such as selective hydrogenation, Pd(0)-Cu(I) coupling, Wittig-type coupling, Sharpless epoxidation, and asymmetric reductions following coupling of the major intermediates described below and in the literature to generate the stable LX analogs of this invention.(Webber, S.E. et al. (1988) Adv. Exp. Med. Biol. 229:61; Raduchel, 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). Geometrical variations can be accomplished e.g. as described in U.S. Patent No. 4,576,758 and Nicolaou, K.C. (1989) J. Org. Chem. 54:5527.

As shown below, LX analog compounds comprising subgenus 1 in

Scheme I can be prepared as three major fragments (A, B, and C), which can then be combined to form the total molecule.

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-43Scheme I

Synthesis of the epoxy-alcohol for precursor fragment 2 can be generated with substitutents R-2, R3 and R4 selected from hydrogen, phenyl, halogen or methyl. Each of these respective epoxy-alcohols may be transformed into phenyl urethane derivatives as 3.

with PhNCO, pyrimidine and CH2CI2 followed by Lewis acid catalysis by SN2 opening to give a 1,2 cyclic carbonate that contains the vicinal diol at

C-6 in the (R) configuration required for binding and C-5 in the (S) configuration also established for bioactivity and binding at a recognition site. These alcohols are next protected to generate the precursor A fragment as 4.

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These A fragments can now be coupled to the fragment B intermediate, a 5 phosphonium bromide 5 as in Webber, S.E. et al. (1988) Adv. Exp. Med. Biol. 229:61 in gram quantities to generate the combined A + B fragment products 6.

MejSi

Fragment C intermediates from Scheme I are generated in parallel to 15 preparation of A-B co<mlings. In these C fragments, substitutions at Y] and/or Y2 are methyl, methoxy, hydrogen, cyano, nitro, or halogen; see specific example 3. Thus, carrying I5-methyl and/or, for example, 16-methyl or 16-phenoxy-derivatives permits these substituted-LXA4 aiialogs to be not susceptible to dehydrogenation.

Thus, the C fragments carrying the preferred resistance to enzymatic 20 oxidation and/or dehydrogenation may be converted by protection of key sites, followed by bromination to give vinyl bromide products of fragment C such as 6b that is coupled to 6 by using catalytic amounts of P(Ph3)4 and Cul to generate the complete backbone structure of the LXA4 analogs of genus formula I. This scheme is further illustrated by the following Examples.

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The compounds of this invention within subgenus formulas II and III may be made synthesized in a similar manner.

Compounds in genuses II and III are generated by first individually preparing substituted compounds of fragment A that are each coupled as in Scheme I to individv,'1ly prepared fragment B to generate 7 or A j + B j fragments possessing individuax substitutions at X, Qj, R-2, R3, and R4 as indicated.

MejSi

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The Cj fragment 8 carrying the acetylenic group C-14,15 and the co-C-20 5 end substitutions will each be generated as shown above for structure 6 prostaglandin analogs and converted to their corresponding vinyl bromide products as in (KCN JAC 1985, Webber) to yield brominated products of each individual substituted fragment Cj or 8 species that are suitable for coupling to 20 using catalytic amounts of P(Ph3)4 and Cul to generate the combined products of the acetylenic-LXA4 analog class. Each of the final products may then be subject to gradient RP-HPLC using rapid diode array detection (as in Serhan, C.N., Methods in Enzymology) for purification. The presence of the modification at C-15 thru C-20 of LXA4 can alter metabolism by dehydrogenases and oxidases by providing steric hindrance, stable prostaglandin analogs carrying C-15 to ω-end substitutions have been prepared and are not metabolized by dehydrogenases (Raduchel, B. and Vorbriiggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 and Vorbriiggen, H. et al. In: Chemistry, Biochemistry, and Pharmacological Activity of Prostanoids (Roberts, S.M., Scheinmann, F. eds.). Oxford: Pergamon Press).

The cyclo-LXA4 compounds of this invention within genus formula IV may be made in the following manner.

Scheme III.

C

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-47- The parent compound of this class is also subject to a similar total synthesis strategy and Is assigned three main fragments A, B, and C in structure 30. A precursor for fragment A may be prepared by routes used in Nicolaou, K.C. (1989) 3. Org. Chem. 54:5527 to prepare 10 in the synthesis of 7-cisJl-trans-LXAq. methyl ester.

II

'X

Fragment B in 30 can be obtained via the precursor H or saligenin-([Ohydroxybenzylalcohol) as generated in (Vorbruggen et al, p. 353). The benzyl alcohol TI is reacted with JO (1:1) in the presence of NaH in DMF to give 12. This key intermediate is silylated in BSTFA followed by coupling with individual fragments designed for C precursors.

pp can then be coupled to vinyl brominate fragment C of given indi vidual design by treating the bromite precursor with 4.0 equivalents of AgNC>3, then 7.0 equiv. of KCN, EtOH/THF/H2O (11:1), 0 => 25°C, 2-4 h. the individual products are then Subject to Lindlar cat for selective catalytic mild hydrogenation in CH2C12 2-3 h to give individual compounds belonging to the genus IV. Each can be saponified in LiOH/THF to give corresponding free acids after isolation by RP-HPLC.

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The invention of genus IV compounds is illustrated further below, the synthesis of 15(±)methyl-cyclo-LXA4 methyl ester and corresponding free acid.

The compounds of this invention within genus formula V may be made in the following manner. Several systems studied with LXB4 indicate that several sites within the natural compound are required for bioactivity (Serhan, C.N. (1991) J.

Bioenerg. and Biomembr. 23:105). These sites include the C-14 alcohol in the (R) configuration and the double bond at C-8,9 of the tetraene in the cis configuration. In addition, based on metabolic studies resulting in the instant invention, several key addition sites have been identified as being necessary to preserve LXB4 bioactivity. These include preserving the C-15 alcohol from dehydrogenase activity (i.e., block 515 oxo-LXB4 formation); maintaining both the Δ8 bond and 14(R) alcohol; and preventing reduction of Δ6-7 double bond and β/ω -oxidations of the resultant compounds.

Scheme IV

Thus, genus V (14) maintains the regions of LXB4 which are necessary for its bioactivity, but modifies the regions available to metabolic degradation. Again, a retrosynthetic analysis gives priority to three key fragments A, B and C designated in J4. Coupling of key intermediates to generate members of the LXB4 analog class uses standard techniques as outlined for LXA4 and its analogs namely, selective

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-49hydrogenation (to generate 8-cis geometry); Pd(0)-Cn(I) coupling to join A & B fragments carrying unique substitutions; Wittig-type coupling to join C fragments that cany the required substitutions and Shaipless epoxidation to yield the 14(R) vicinal alcohol (seeref Nicolaou,K.C. etal. (1991) Angew. Chem. Int. Ed. Engl. 30:1100.), ref.; Weber, S.E. et al. (1988) Adv. Exp. Med. Biol. 229:61 Ed. Wong, P.K. and Serhan, C.N.) and those cited within). Thus by using a similar strategy to LXA4 analogs and the construction of native LXB4 the specific analogs can be obtained.

The A fragments of 15 that carry substitutions at R2, Rg, R7 that can be (H, CH3, OCH3, phenyl, halo-substituted phenyl are generated by standard methods from, for example 16 where= CH2 of increasing chain length.

Compound 16 is converted to the vinyl bromide 15 as in (Nicolaou K.C. et al. (1991)

Angew. Chem. Int. Ed. Engl. 30: 1100-16.) via a trimethylsilyl acetylenic intermediate

Γ7 that is reduced by pinanyl-9BBN then n- BuN4NF in THF to yield J8 than is now submitted for bromination after protecting essential moieties such the alcohol to give Γ5 (fragment A).

The fragment C in 14 is generated from compound 19 with the R5 substitutions as indicated.

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The acetylenic alcohol 19 is then reduced in LAH followed by Sharpless asymmetric epoxidation to generate 20 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 20 is transformed to the corresponding aldehyde 21 after protection of the substituted groups carried at R4 and R5 as well as the alcohol using PCC in methylene chloride. (Nicolaou, K.C. et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100).

Br'

P(Ph)3

H’

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-51 The phosphonium salt 22 can be prepared as in Ref.; (Weber, S.E. et al. (1988) Adv. Exp. Med. Biol. 229:61, Ed. Wong, P.K. and Serhan, C.N.) and used here to generate the B-C fragment coupling via a Wittig-type coupling to give 23 carrying the designated substitutions in the group of 23 where R4 and R5 carry substitutions. This cis double bond of 23 can be is isomerized to give the trans isomer using I2 as a catalyst to give the parent precursor form of 14 as 24.

Then coupling of 24 to 15 is accomplished by Pd(O) - Cu(I) coupling to give the acetylenic precursor of 14 designated 25. Following selective Lindlar catalytic hydrogenation the individual LXB4 analogs can be further purified via RP-HPLC used

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-52the tetraene skeleton as a convenient means to isolate individual products employing rapid diode array detection (Serhan, C.N. (1990) Meth. Enzymol. 187:167).

Utilities

The compounds of this invention have the biological activity of natural

LXs, but are more resistant to degradation or alternatively inhibit the degradation of natural LXs. The disclosed compounds therefore have utility as pharmaceuticals for treating or preventing a number of diseases or conditions associated with inadequate or inappropriate LX mediated cellular response in a subject.

Based on the anti-proliferative effect of the disclosed 15-epi-lipoxin compounds, the invention provides methods for ameliorating undesired cell proliferation by contacting cells with a pharmaceutical composition including an effective amount of the substantially purified 15-epi-LX compound and a pharmaceutically acceptable carrier. The cell can be contacted in vivo and/or in vitro. Alternatively, cells can be removed from a subject; contacted with the substantially purified 15-epi-lipoxin compound of the present invention ex vivo and implanted in the subject. The invention also provides a method for ameliorating a cell proliferative disorder in a subject including administering an effective amount of a substantially purified 15-epi-LX compound.

The effective amount is ordinarily the amount which is required to assure sufficient exposure to a target cell population. Such an amount will ordinarily depend upon the nature of the 15-epi-LX compound, the mode of administration, the severity of the undesired cell proliferation or cell proliferative disorder and other factors considered by a person of ordinary skill when determining a dosage regimen.

Target cells to be contacted can be undergoing cancerous and/or tumorous growth. Alternatively, target cells can be undergoing abnormal cell proliferation in response to a stimulus, such as restenosis; and/or target cells can consist of 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 invention also provides methods for treating a subject with a myeloid suppressive disorder by administering to the subject an effective amount of a pharmaceutical composition comprising a LX analog. The effective amount is ordinarily that amount which is required to assure sufficient exposure to the target cell population. Such an amount will ordinarily depend upon the nature of the analog, the mode of administration, the severity

of the myeloid suppression, and Other factors considered by a person of ordinary skill when determining a dosage regimen.

Therapeutic use of a cell proliferative LX analog also includes removing cells from a subject, stimulating cell growth in vitro, and reintroducing the enhanced cell preparation, 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 during stimulation or in conjunction with the introduction of the cell preparation.

In another embodiment, the compounds of this invention are used to treat or prevent inflammation or an inflammatory response. LXA4 inhibits the activation of leukocytes which are mediators of inflammation. The LXA4 induced effect includes inhibition of leukocyte migration, generation of reactive oxygen species, and the formation of pro-inflammatory mediators involved in tissue swelling. (Raud, J. et al. (1991) Adv. Exp. Med. Biol. 314:1 '85. Cell-Cell Interactions in the Release of Inflammation Mediators vol. 314) LXB4 exhibits radioprotective actions, such as preventing diarrhea and ataxia, in an in vivo assay with mouse hematopoietic stem cells. (Walken, T.L. Jr.,(1988) J, Radiat. Res. 29:255)

The leukocyte-mediated inflammation or inflammatory responses cause or contribute to a wide variety of diseases and conditions including various forms of asthma and arthritis. Included within die present invention are inflammatory responses to physical injury, such as physical trauma, radiation exposure, and otherwise.

In another embodiment, the compounds of this invention are used to treat or prevent inflammation by antagonizing the action of leukotrienes. LXA4 inhibits LTB4-induced inflammation, blocking both plasma leakage and leukocyte migration in an 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 both wound healing and inflammation. LXA4 also antagonizes LTD4-induced renal hemodynamic actions and blocks the binding of LTD4 to mesangial cells which 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 the action of sulfidopeptide leukotrienes, such as LTD4, LTC4, and LTB4. Leukotrienemediated 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, renal glomerular disease, benign prostatic hypertrophy, inflammatory bowl disease,

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-54myocardial ischemia, myocardial infarction, circulatory shock, brain injury, systemic lupus erythematosus, chronic renal disease, cardiovascular disease, and hypertension.

In another embodiment, the compounds of this invention are used to treat or prevent a vasocontractive response or condition. LXs induce endothelium-dependent vasodilation (LXA4) (Lefer, A.M. et al (1988) Proc. Natl. Acad. Sci. USA 85:8340) and dilation of cerebral arterioles in new bom pigs in vivo (LXA4 and LXB4) (Busija, D.W. et al. (1989) Am. J. Physiol. 256:468. ). Furthermore, LXA4 induces rapid arteriolar dilation in hamster cheek pouch in vivo (Dahlen, S.-E. et al. (1987) Acta Physiol.

Scand. 130:643 ) 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 are associated with diseases and conditions such as renal hemodynamic diseases, including glomerular diseases, cardiovascular diseases including hypertension, myocardial infarction, myocardial ischemia, and vascular diseases and gastrointestinal diseases.

Also encompassed by this invention is a method of screening LX analogs or other compounds to identify those having a longer tissue half-life than the corresponding natural LX This method can be used to determine whether the compound inhibits, resists, or more slowly undergoes metabolism compared to the natural LX. This method is performed by preparing at least one enzyme which metabolizes LXs, contacting the compound with the enzyme preparation, and determining whether the compound inhibits, resists, or more slowly undergoes metabolism by the enzyme. Cells having a LX recognition site, such as polymorphonuclear neutrophils, peripheral blood monocytes, and differentiated HL-60 cells are among the appropriate sources for the enzyme preparation. The LX recognition site may exist naturally, or be induced artificially, by a disease state, or by an injury. A non-limiting example of artificially-induced LXA4 recognition sites is the induction of such sites in differentiated HL-60 cells.

In one embodiment, preparation of the enzymes comprised harvesting cells and performing freeze-thaw lysis three times, followed by ultracentrifugation to yield 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 LX metabolism, but perform transformations upon LXs similar or equivalent to those transformations performed by the enzyme or enzymes which naturally metabolize LXs. Nonlimiting examples of appropriate enzymes are 15-hydroxyprostaglandin dehydrogenase, cytochrome P-450 monogenases from human leukocytes, and rat and human liver microsomes.

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-55Characterization of LX metabolites included standard techniques such as extraction, chromatography, and quantitative HPLC followed by trimethyl silyl derivatization, O-methoxime derivatization and gas chromatography/mass spectroscopy analysis. The experimental details of this embodiment are described below in Example

LX analogs can also be screened for binding activity with a LX receptor recognition site, for example by contacting tne compound with a receptor recognition site and determining whether and to what degree the compound binds. Examples of kinetic binding assays include homologous displacement, competitive binding, isotherm , and equilibrium binding assays.

The receptor recognition site may normally exist or it may be induced by a disease state, by an injury, or by artificial means. For example, retinoic acid, PMA, or DMSO may be used to induce differentiation in HL-60 cells. Differentiated FIL-60 cells express LXA4-specific receptor recognition sites. Examples of other cells which may be screened for LX specificity include PMN, epithelial cells, and peripheral blood monocytes.

Selection of competitive ligands will depend upon the nature of the recognition site, the structure of the natural substrate, any structural or functional analogs of the natural substrate known in the art, and other factors considered by a skilled artisan in making such a determination. Such ligands also include known receptor antagonists. The compounds of this invention may be radiolabelled with isotopes including 2H, 3H, 13C, and 14C by standard techniques known in the art of radiochemistry or synthetic chemistry.

In one embodiment of this method, the structural specificity of induced

LXA4 recognition sites was assessed with LXB4, LTC4, LTB4 and trihydroxyheptanoic methyl ester. The experimental details of this embodiment are described below in Example 2.

In. addition, the compounds of this invention may be used to exert certain actions on specific cell types as developmental models for inflammation and injury. For example, LXA4 stimulates the mobilization of intra-cellular Ca2+, lipid remodeling, and chemotaxis without aggregation in human PMN (Palmblad, J. et al. Biochem. Biophys. Res. Commun. (1987) lz5; 168; Lee, T.H. et al. Clin.Sci. (1989) 77:195; Nigam, S. et al. J.Cell. Physiol. (1990) 143:512; Luscinskas, F.W. et al. ( 1990) Biochem. Pharmacol. 39:355). LXA4 also blocks both LTB4 and FMLP-induced responses, such as IP3 generation. LXB4 also stimulates lipid remodeling. LXA4 activates isolated PKC, and is specific for the γ-subspecies of PKC which is found in the brain and spinal cord. (Hansson, A. et al. Biochem. Biophys. Res. Commun. (1986) 134: 1215; Shearman,

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-56M.S. et at. FEBS Lett. (1989) 245: 167); The publication Nicolaou, K.C. et al. Angew. Chem. Int. Ed. Engl. (1991) 30 :1100 and references cited within are expressly incorporated here by reference.

The present invention is further illustrated by the following examples 5 which should in no way be construed as being further limiting. The contents of all references and issued patents cited throughout all portions of this application including the background are expressly incorporated by reference.

Examples

Example 1 Synthesis of Lipoxin Analog Compounds

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7 8

Preparation of the methyl ester precursor of compound 1:

To a solution of 3-methyl-3-trimethylsiloxy-l-bromo-l-octene (130 mg. 0.44 mmol) in benzene (1,5 mL) was added n-propylamine (0.05 mL, 0.61 mmol) and

Pd(PPh3)4 (2θ mg· θ*θ2 mmol) and the solution was protected from light. It was then degassed by the freeze-thaw method and stirred at rt for 45 min. (7E, 9E, 5S, 6R)

Methyl 5,6-di(tert-butyldimethyIsiIoxy)-dodeca-7,9-diene-l 1-ynoate (183 mg. 0.44

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-58PCT/US97/16342 mmol) (compound 12) and copper iodide (14 mg. 0.07 mmol) were added and the solution was one more time degassed by the freeze-thaw method. The mixture was stirred for 3 h at rt and quenched with saturated aqueous solution of NH4CI and extracted with ether. It was then washed with brine and dried over MgSOq and the solvent 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 added n-BuN4F(0.9 mL. 0.90 mmol) and the mixture was stirred at rt. The reaction was completed in 2 h at which time it was poured into water and extracted with ether. The ether extracts were washed With brine, dried over NajSC^ and the solvent was evaporated. Flash column chromatography (silica 4% MeOH/CH^C^) afforded the methyl ester (24 mg.) together with some of the corresponding lactone. HPLC retention time: 9:39 min (microsorb reveise phase, 4.6mm X 25 cm, C-l 8 column, MeOH/H2O 70:30 flow rate 1 ml/ min, UV detector at 300nm). UV in MeOH: Xmax283,294,311 nm. JH NMR (500 MHz CDCI3) 66.53 (dd. 15.2 10.9 Hz, 1 H), 6.32 (dd, J = 15.1,

11.0 Hz, 1 H), 6.17 (d, >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 Η), 1.51-1.48 (m, 1 H), 1.43-1.42 (m, 2 H), 1.30-1.23 (m, 15 H) 0.85 (t,

H). 13 C NMR (126 MHz, CDCI3) 8150.01,140.18,132.95,132.26,112.43,107.50, 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 CH2CI2 (1 ml) 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 ceiite evaporation of the solvent and separation by HPLC gave a pure methyl ester. HPLC retention time: 10:02 min (microsorb reverse phase. 10 mm X 25cm C-l 8 column, MeOIT/H^O 70:30 flow rate 4 ml/min, UV detector at 300nm).

UV in MeOH: pmax 287, 301, 315 nm.

Preparation of the methyl ester precursor of compound 3:

This compound was prepared similarly to the preparation of the methyl ester precursor of compound 1 (from 3-cyclohexyl-3-trimethylsiloxy-l-bromo-l-octene).

Desilylation of this compound was also performed in a similar manner to afford the methyl ester. HPLC retention time 8:02 min (microsorb reverse phase, 4.6mm X 25cm.

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-59C-l o column, ΜεΟΗ/Η2Ο 70:30, flow rate 1 ml/min, UV detector at 300nm). UV in MeOH: Lmax 282, 293, 311 nm. NMR (360 MHz, CDCI3) 56.56 (dd, 15.4, 10.9 Hz, 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,

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 tlie methyl ester precursor of compound 3, followed by HPLC purification gave the methyl ester precursor of compound 4. HPLC retention time: 9.72 min (microsorb reverse phase, 10 mm X 25cm C-l8 column, MeOKTFjO 70:30 flow rate 4 ml./min. UV detector at 300nm), UV in MeOH: Lmax 288, 301,315 nm. IH NMR (250 MHz, C6D6) δ 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 using standard techniques.

Synthesis of 15fR)-15-methvl-LXA4 and 156L)methyl-LXA4

Approximately 1 gm acetylenic ketone a is prepared using Friedel-Crafts acylation 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. 229:61; Nicolaou, K.C. et al. (1991) Angew. Chem. Int. Ed. Engl.

30:1100; and Vorbriiggen, H. et ah In: Chemistry, Biochemistry, and Pharmacological Activity of Prostanoids (Roberts, S.M., Scheinmann, F. eds.). Oxford: Pergamon Press, to generate the methyl at C-l5.

Alternatively, the keto group can be treated with CI^MgBr (60=>70°C) as in Vorbriiggen, H. et al.: In: Chemistry, Biochemistry, and Pharmacological Activity of Prostanoids (Roberts, S.M., Scheinmann, F. eds.). Oxford: Pergamon Press to yield the 15(±)methyl of b (2-o g) in dry CH2CI2 (~20 ml) at 0°C with sequential additions of

2,6-lutidine (5.2 ml) and tert-butyldimethylsilyl triflate (6.9 ml). This reaction is mixed for 1 h and then diluted with 100 ml ether for aqueous extraction and drying with

MgSO4b

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The product c is then coupled with d

that is generated as in Nicolaou, K.C. et al. (1991) Angew Chem. Int. Ed, Engl. 30:1100; Nicolaou, K.C. et at (1989) J. Org. Chem. 54:5527 and Webber, S.E, et al. (1988) Adv.

Exp. Med. Biol. 229:61. Structure d from fragment A in Scheme I is suspended in 4.0 equiv. of ΑβΝΟβ, then 7.0 equiv. of KCN, containing EtOH:THF:H20 (1:1:1), 0-25°C for 2 h to generate the C-methyl ester protected 15-methyl-LXA4 analog that is concentrated and saponified in THF with LiOH (2 drops, 0,1 M) at 4°C 12-24 h to give the corresponding free acid.

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-61 Synthesis of 16-dimethvl-LXAzj

This compound is generated using the similar strategy by coupling d above with e vide supra, or f to generate the 15-phenyl-LXA4 analog, or g to generate the 17-/«-chlorophenoxy-LXA4 analogs.

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-62- .

The appropriate C fragments in Scheme I (i.e. e, £ g, h,) are each prepared as reviewed in RaduChel, B. and Vorbriiggen, H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 for the known corresponding prostaglandin analogues. In h, R=H; Cl, methoxy or halogen.

Using the A2B2 generated fragment from Scheme II, the corresponding

C2 fragments are prepared for coupling.

Structures^ and k are generated asin Nicolaou, K.C. et al. (1989) J. Org. Chem. 54:5527 and methylated as in Radiichel, B. and Vorbriiggen^ H. (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 are coupled to 7 to yield these LX analogues.

The materials may be subject to RP-HPLC for purification vide supra.

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The designated combined A2B2 fragment can be prepared from couplings of fragments A] and B}, illustrated in Route II to carry the structure of 7 or 4 vide supra for coupling to fragment O?. The precursor for the £2 fragment 1 can be prepared as in Raduchel, B. and Vorbruggen, H, (1985) Adv. Prostaglandin Thromboxane Leukotriene Res. 14:263 for a prostaglandin analog.

Me2Al---

Precursor 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, «-PrNfl·?, in benzene with Me2Al-carrying I,2-3 h RT to yield n.

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The alcohol protecting groups TBDMS=R are removed with 10 equiv, of 5 HF-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 δ-lactones that usually form between C-1 -carboxy and C-5 alcohol inthe LXs (Serhan, C.N. (1990) Meth. Enzymol. 187:167 and Nicolaou, K.C. (1989) J. Org. Chem. 54:5527). After mild treatment with Lindlar cat. 5% by weight, the extracted material may be subjected to LiOH saponification in THF to generate the free acid of the target molecule that can be subject to further purification by RP-HPLC gradient mobile phase as in (Serhan, C.N. et al. (1990) Meth. Enzymol. 187:167).

Synthesis of 15(±)methvl-cyclo-LXA^

Compound o as the SiMe3 derivative can be placed (~ 1 gm) in a round 20 bottom 100 ml flask under an atmosphere enriched with argon in degassed benzene (20 ml). To this add 3.0 equivalents of a vinyl bromide fragment vide infra. This coupling reaction is carried out in catalytic amounts of Pd (P?h3)4 and Cul and can be monitored by injected aliquots of this suspension into RP-HPLC monitored by UV abundance with a rapid scanning diode. The progression line course 1-3 h at 23°C after which the material is extracted with ethyl acetate; H2O 4:1 v/v) and concentrated by

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-65rotoevaporatiort. The methyl ester can be saponified in LiOH/THF to give quantitative yields of the free carboxylic acid. Other derivatives can be prepared as above using fragment A with different fragment B moieties that have been substituted to give for example a dimethyl or other derivative. This can be obtained by taking the readily available ketone p and treating it with CH3MgBr (60°C) to generate q that can also be coupled 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.

G

Synthesis of 5-Methvl-LXB^ and 4.4-Dimethvl-LXB^.

The 5-methyl -LXB4 hinders or retards 5-oxo-LXB4 formation. Using the general scheme outlined above, the A fragment can be constructed to carry the 5methyl in a vinyl bromide r precursor that is coupled to a joined B + C fragment by

Pd(0)-Cu(I) coupling.

O (TBDMS) q

OMe

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The vinyl bromide r can be obtained from the s that contains either dimethyl or hydrogen substituents at its C-4 position. The protected precursor t containing fragments B + C is generated as reported in reference (Nicolaou K.C. et al.

(1991) Angew. Chem. Int.. Ed. Engl. 30: 1100-16.). Compound t is converted to s or 28 by coupling with the indicated vinyl bromide. Thus the target molecule can be generated by adding rat 1.0 equv. (« 1 gm) to a round bottom flask degassed containing Et2NH as solvent with tinjected in Et2NH at 1.2 equiv. Pd(Ph3P)4 is added at 0.02 equiv. to give the 8(9)-containing acetylenic precursor methyl ester of s.

The material is extracted and subject to rotoevaporation suspended in quinoline (0.5 eq) in CH2C12 and subject to hydrogenation using (10%; 25°C) Lindlar catalyst and a stream of H2 gas to selectively reduce the acetylenic double bond at position 8. The formation of the tetraene component of the methylester of 5-methylLXB4 or 4-dimethyl-LXB4 methyl ester can be monitored by RP-HPLC to assess completion of the reduction (i.e., 1 -3h). The methy Westers are next saponified to their corresponding free acids by treating the products with LiOH in THF 25μ1 H2O added at 0=>24°,8-24h.

Example 2 Lipoxin A4 Metabolism by Human Pro myelocytic Leukemia

Cells and Monocytes : Half-life Assay

HL-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). Synthetic ll,12-acetylenic LXA4 methyl ester and LXs were from Cascade Biochemical (Reading, tLK.). 15(S)-l5-/w-PGE], PGE] and 5-HETE were from Cayman Chemical Co. (Ann

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TeLrijV -///TT .67- · /// .//

Arbor, MI). [11,12-3HJLXA4 was prepared from ! 1,12-acetyIenic LXA4 using Lindlar catalyst as a custom tritiation (NET-259, loi 0 2793-275, New England Nuclear Boston, MA). Tritiated products were isolated using RP-HPLC (Fiore et al. (1992) J. Biol.

4 Chem. 267.16168; Serhan, C.N. (1990)

Meth. Enzymol.T87:167). Methoxyamine and NAD were from Sigma Chemical » Company (St Louis, MO). Manganese dioxide and Adams reagent were from Aldrich

Chemical Co. (Milwaukee, WI). ' . \ : ' ' : '

Human PMN were obtained from healthy volunteers by gradient centrifugation of heparinized fresh venous blood (Boyum, A. (1986) Scand. J. Clin. Lab.

Invest. 21:77). HL-60 cells were seeded in RPMI supplemented with penicillin (100 U/ml), streptomycin (100 μ/ml), fetal bovine serum (10%) (Hyclone, Logan, UT) and incubated (37°C with 5% CO2 atmosphere) in plastic 250 ml flasks. Individual flasks containing 5x1 O'7 HL-60 cells/ml were incubated in the presence or absence of phorbol 12-myristate 13-acetate (PMA) (10 or 16 nM, 24-27 h) and adherence was monitored for induction of macrophage-like phenotype as in Collins, S.J. (1987) Blood 70:1233. Peripheral blood monocytes were obtained (Goldyne, M.E. et al. (1984) J. Biol. Chem. 259:8815 after plating fresh mononuclear cells onto plastic petri dishes containing PBS with glucose (1 mg/ml) for 1 h at 37°C. Non-adherent cells were removed and adherent mononuclear cells; were gently resuspended using Versene (7 ml/plate) and washed in

PBS. PMN (>98%), adherent monocytes (>95%) and HL-60 cells were enumerated by light microscopy, suspending in PBS for incubations, and <2-3% in each case were permeable to trypan blue. For some experiments, cell-free supernatants were prepared from HL-60 cells treated with PM A (16nM) for 24 - 72 h. After harvesting, the differentiated cells were washed, then subject to freeze-thaw lysis (repeated 3 times) and ultracentrifugation(100,000 g, 1 h).

Incubations with eicosanoids were stopped with cold methanol containing either PGB2 or 5-HETR as internal standards (5-HETE was used when 15-oxo-ETE was quantified). Products were extracted using Sep-pak Cl 8 and routinely chromatographed as in Serhan, C.N, (1990) Meth. Enzymol. 187:167. RP HPLC system consisted of an

LKB gradient dual pump equipped with ah Altex Ultrasphere-ODS (4.6 mm x 25 cm) column, flow rate 1 ml/min eluted (0-20 min) with methanoI/H2O/acetic acid (65:35:0.01) and methanol/acetic acid (99.99/0.1) in a linear gradient (20-45 min) that was used to quantitate the io-metabolites of LTB4 (i.e. 20-COOH and 20-OH-LTB4) as well as LXA4. Recovery of internal standards was 82.2 7.9, mean S.D. (n-13).

Compounds I-IV were separated using aft Altex Ultrasphere-ODS column (10 mm x 25 cm) eluted at a flow rate of 3.0 ml/min with methanol/B2O/acetic acid (60:40:0.01, v/v/v). Formation of 15-oxo-ETE by 100,000 g supernatants (cf. Agins, A.P. et al.

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-68(1987) Agents Actions 21:397; Xun, C-Q. et al. (1991) Biochem. J. 279:553; and Sok, D-E, et al. (1988) Biochem. Biophys. Res, Commun. 156:524) was quantified after RP-HPLC using an ODS column (4.6 mm x 25 cm) eluted With methanol/H2O/acetic acid (70:30:0.01, v/v/v) monitored at 280 nm with a flow rate of l ml/min.

Monocyte-derived products were also chromatographed using a Hypersil column (5 IL, mm x 300 mm) eluted with methanol-1’ ,0/acetic acid (60.40:0.01, v/v/v) and a flow rate of 1 ml/min. On-line spectra were i ·· _ded using a diode array detector (Hewlett-Packard 1040M series II) equipped with HPLC3D ChemStation software (DOS series), Spectra were acquired using step 4 nm, Bw = 10 nm, range = 235-360 nm with a sampling interval of 1.28 sec.

GC/MS was performed with a Hewlett-Packard 5971A mass selective detector quadrupoie equipped with a HPG1030A workstation and GC 5890. The column was a HPUItra 2 (cross-linked 5% phenyl methyl silicone gum phase; 25 m x 0.2 mm x 0.33 pm) and injections were made in the splitless mode in bis(TMS)trifluorOacetamide (BSTFA), The temperature program was initiated at 150 °C and reached 25Q°C at 10 min and 325° at 20 min. Standard saturated fatty acid methyl esters C16-C26 gave the following retention times (min:sec; mean of n=6). Cjg, 8.03; C18, 9.77; C20, 12.22; C22, 16.11; C24,20.72; C26,23.62 that were used to calculate respective C values of LX-derived metabolites as in Serhan, C.N. (1990) Meth. Enzymol. 187:167.

Diazomethane was prepared and the methyl ester products were treated with BSTFA (Pierce Chemical Co., Rockford, IL) to obtain Me3Si derivatives. Methyl ester O-methoxime derivatives were prepared as in Kelly,

R.W. and Abel, M.H. (1983) Biomed. Mass Spectrom. 10:276. Catalytic hydrogenations 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 bubbling hydrogen (20 min, RT). After extraction, materials were treated with diazomethane followed by BSTFA (overnight; RT).

RESULTS so ' ? WΪ / < e/'

Metabolism of LX A4: Intact neutrophils from peripheral blood of healthy donors did not significantly metabolize exogenous LXA4 while cells from the same donors rapidly transformed LXA4 via co-oxidation. In contrast, PMA-treated HL-60 cells that displayed monocyte/macrophage-like characteristics rapidly transformed LXA4.

Within the first 60 s of exposure, > 70% of LXA4 was metabolized. In the absence of PMA treatment, neither intact HL-60 cells (undifferentiated) nor their cell-fre e supernatants (100,000 x g) ft=orm LXA4 (n=3).

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Λ >. ; + 7+ j. -69- / ·

Differentiated HL-60 cells incubated with LXA4 converted this eicosanoid to several products. Labeled LXA4 was transformed to four main products that carried tritium (denoted compounds I-IV), which were collected for further analysis.

* Structures of compounds I-IV To obtain quantities of these compounds enabling structural studies, their retention times in RP-HPLC were established using the » 3H-label elution profile to mark boundaries, and unlabeled samples pooled from several incubations were chromatographed and individually collected from within these regions for GC/MS analysis. Selected ion monitoring of the products obtained after treatment with diazomethane and BSTFA revealed that compounds I-IV each displayed prominent ions atm/z203 f-CH(OSiMe3)-(CH2)3-COOCH3] indicating that carbons 1 through5 of

LXA4 (carboxylic carbon is number 1) were not modified, although each product gave a different retention time thanLXA4. The methyl ester, trimethylsilyl derivative of LXA4 displayed prominent ions in its electron impact spectrum at m/z 203 (base peak) and 173, with its molecular ion at 582 ,(M+4). Other ions of diagnostic value in this derivative of LXA4 are observed at m/z 171 (203-32), 409 (M-173), 379 (M-203), 482 (M-l 00) and 492 (M-90) (Serhan3 C,N. ei al. (1984) Proc. Natl. Acad. Sci. USA 81:5335; and Serhan, C,N. (1990) Meth. Enzymol 187:167· It is noteworthy that the LXs in general are known to give extremely weak molecular ion peaks (Serhan, C.N. er al. (1984) Proc. Natl, Acad, Sci, USA 81:5335). Nevertheless, compounds labeled 1 & II also possess prominent ions at m/z 173 (Me3SiO+=CH-(C 2H3) indicating that the carbon 15-20 fragment of these LXA4-derived products was .ntact, while the ion at m/z 173 was not evident in compounds III and IV. Thus, the conclusion that compounds I-IV are 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 in incubations with HL-60 cells not treated with PMA,

Next, compounds III and IV were focused on since it appeared that they represent metabolites with structural modifications in the carbon 15 through 20 fragment of LXA4. Since ώ-oxidation (hydroxylation at carbon 20) was a possibility, ions that could result from the respective 20-ΘΗ and 20-C0OH forms of LXA4 after derivatization, namely m/z 261 and 217 (Me3SiO+=CH-(CH2)4-CH2OSiMe3 and Me3SiOf=:CH-(CH2)4-CO2Me), were scanned in the acquired GC-MS data profiles. Neither Illnor IV displayed prominent ions at ei.ner m/z 261 ot 217 indicating that * these products were not likely the result of co-oxidation.]

The mass spectrum (C value 24.3) of the Me3 Si derivative, methyl ester of 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 CH30H), 215 [(M-203)-90, elimination of trimethylsilanol (Me3SiOII)] and 99 (O=C-(CH2)4-CH3).

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Ions of lower intensity were a m/z 508 (M+) and 418 (M-90; loss of Me3SiOH). The presence of these ions suggested that the material that coeluted with 3H-labeled compound III was the 15-oxo-derivative of LXA4. This is supported by several lines of evidence, 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 a tetraene chromophore and appearance of a new chromophore at UV Xmax at 335-340 nm. The tetraenone chromophore was confirmed by treating LXA4 with MnO2 in chloroform as used for prostaglandin conversion (Anggard, E. and Samuelsson, B. (1964) J. Biol. Chem. 239:4097). Also, the mass spectrum of the catalytic hydrogenation product gave a 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-COQCFI3; 35%) and m/z 171 (36%) with no prominent 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 were consistent with the generation of the corresponding 15-oxo-derivative.

To examine this LXA4-derived product further, an aliquot of the material eluting beneath the peak labeled ill was treated with diazomethane followed by methoximation (as in Bergholte, J.M. et ai (1987) Arch. Biochem. Biophys. 257:444) and treatment with BSTFA. Its spectrum, C value 25.4, showed prominent ions at m/z 203 (base peak), 171 (203-32; loss of CH3OH) and 229 [M-128 or CH3O-N=C-(CH2)4CH3-(2x90)]. Ions of lower intensity were at m/z 537 (M+), 466 (M-71, the at-cleavage ionM-CH2(CH2)3CH3), 481 (M-<6 orM-CH2=CH-CH2-CH3, a McLafferty rearrangement ion), 431 [M-106 (possibly loss of C7H5N+)], 401 [M-136 (elimination of Me3SiOH + CH3 + OCH3) and 460 (M-77, loss of NOCH3 plus MeOH). Again, an ion at m/z 173 that would have originated from an alcohol-containing C-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 generated from the15-oxo-containing derivative of LXA4. Together the prominent ions observed with these different derivatives suggest that material eluting beneath the peak labeled ΙΠ was the 15-oxo-product of LXA4 (i.e, 15-oxo-LXA4).

The mass spectrum of the methyl ester Me3Si derivative (C value 26.0) of compound IV showed, prominent ions at m/z 203 (base peak, CH(OSiMe3)-(CH2)3COOCH3), 171 (203-32; loss of CH3OH), 99 (O=C-(CH2)4-CH3) and 307 (M-203 or MCH(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 a triplet of absorbance with maxima at 259,269 and 280 nm, consistent for a conjugated triene chromophore. The presence of these ions and UV spectrum suggest that IV was a dihydro-15-oxo-metabolite of LXA4. This basic structure was supported by the presence

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-71 PCT/US97/16342 of the ion at m/z 99 that is consistent with a keto group at position carbon 15, and the presence of m/z 203 as base peak revealed that the alcohol groups at carbons 5 and 6 remain intact. In addition, the absence of atrienone chromophore (λ cal = 310 nm) indicates that loss of a double bond was at Δ13-14 position to give the observed triene chromophore. Together these results indicate that compound IV was

13,14-dihydro-l 5-oxo-LXA4.

The methyl e^ter, 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 two mass units higher than the LXA4 derivative. These ions and a triplet band of absorbance ZmaxMeOH 259 nm, 269 and 282 nm suggest that compound II was a dihydro-derivative of LXA4. The methyl ester, Me3SiO derivative of compound I from HL-60 cells gave two products in GC. The major one (C value = 25.0) gave similar ions in its mass spectrum as LXA4, but instead its molecular ion was at m/z 586 with ions also present at m/z 555 (M-31) and 496 (M-90), indicating that two of the four double bonds were reduced (not shown). However, identical products were not observed with peripheral blood monocytes (vide infra), and thus the HL-60 cell-derived materials from peak I were not further characterized in the present experiments. The structures of I-IV indicate that LXA4 is not metabolized by ω-oxidation by intact leukocytes but instead is both dehydrogenated at carbon 15 alcohol and also transformed from a conjugated tetraene to triene structures. Taken together these observations suggested that LXA4 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 12:647).

15-PGDH activity was recently shown to be induced in HL-60 cells (Xun, C-Q. eta!. (1991) Biochem. J. 279:553), and it apparently utilizes 15-HETE as substrate with 92% efficiency compared to PGE2 (Agins, A.P. et al. (1987) Agents Actions 21:397). Indeed, 100,000 g supernatants prepared from PMA-treated HL-60 cells converted 15-HETE to 15-oxo-ETE indicating the presence of a dehydrogenase activity after differentiation. LXA4 competed for catalysis of 15-HETE giving aK; = 8.2 ± 2,6 μΜ (S.E.M., n=6) calculated from Lineweaver-Burke plots. At equimolar concentrations of LXA4 and 15-HETE, LXA4 blocked 15-oxo-ETE formation by ^50%. The relative conversion for LX compared to PGE; by 100,000 g supernatants indicated that LXA4,1 l-/ra/«-LXA4 as well as LXB4 but not 15-methyl-PGEj were converted. Together, these results suggest that LXA4 > 11 -trans-LXA^ > LXB4 are substrates for 15- PGDH or an equivalent enzyme system.

Since PMA induces differentiation to monocyte-macrophage-like lineage of HL-60 cells (Collins, S.J. (1987) Blood 70:1233), peripheral blood monocytes were incubated to determine if they metabolize LX, LXss display potent actions with monocytes (Stenke,L. <?/a/(1991b) Biochem. Biophys. Res. Commun. 180:255) and these cells do not ω-oxidize eicosanoids (Goldyne, M.E. et al. (1984) J. Biol. Chem. 259:8815), 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 to 15-oxo-LXA4ailri conjugated triene-containing products, 13,14-dihydro-LXA4 and 13,14-dihydro--] 5-oxo-LXA4. As with differentiated HL-60 cells, monocytes Tapidly converted LXA4 (> 60%) within 30 s. The temporal relationships for formation of these metabolites in both intact and permeabilized monocytes were similar and suggest that 15-oxo-LXA4 metabolite is a transient intermediate. Also, in each monocyte suspension incubated with 3H-LXA4 (d=33), 13,14-dihydro-15-oxo-LXA4 and 13,14-dihydro-LXA4 were major products carrying radiolabel. It is noteworthy that a product eluting before

13,l4-dihydro-LXA4 at 15.5-17 min was observed that also displayed a triene chromophore and was likely the 11-trans isomer of 13,14-dihydro-LXA4 that results from cis-trans isomerization encountered during work-up, The 11 -cis double bond of native LXA4 is labile and readily isomerizes to all-trans during extraction and isolation (Romano, M. and Serhan, G.N. (1992) Biochemistry 31:8269'

Example 3 Binding Affinity to Lipoxin Receptors Analogs

Human promyelocytic leukemia cells (HL-60) were purchased from the American Type Culture Collection (Rockville, MD). RPMI medium and cell culture reagents were from GIBCO (Grand Island, NY). Synthetic LXA4, trihydroxyheptanoic acid (methyl ester), LXB4, LTD4, LTC4 and LTB4 were from Biomol (Plymouth Meeting, PA), and SKF104353 was from Smith Kline and French Laboratories. ONO 4057 was from ONO Pharmaceutical Co., Ltd. (Osaka, Japan). [14,15-3H]LTB4 (32.8 mCi/mmole), [l-I4C]arachidonic acid (50.2 mCi/mmole), 32ΡγΑΤΡ (3,000 Ci/mmole), [9,10'3H(N)3palmitic acid (30.0 mCi/mmole), and [9,10-3H(N)Jmyristic acid (30.7

Ci/mmole) were purchased from New England Nuclear (DuPont Co., Boston, MA). ll,12-acetylenicLXA4 was from Cascade Biochemicals (Oxford, UK), Microcentrifuge tube filters (0.45 pm cellulose acetate) were purchased from PGC Scientific (Gaithersburg, MD) and silicon oils were from Harwick Chemical Corp. (Akron, OH) (d=l.05) and Thomas Scientific (Swedesboro, NJ) (d-0.963), respectively. Nitroblue tetrazolium, PMA, DMSO, proteases, retinoic acid and Actinomycin D were purchased from Sigma (St. Louis, MO). Islet activating protein (IAP) was from LIST Biological

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Lab., Inc. (Campbell, CA). Plasticware, Whatman LK6D TLC plates and solvent (HPLC grade) were from Fisher (Springfield, NJ).

Preparation of Γ11.12-3H]LXA4. Tritiation of 11,12-acetyienic LXA4 methyl ester was earned out under a custom tritiation service (NET-259:92-2326) by New England Nuc)ear (Boston, MA). Briefly, 11,12-acetylene methyl ester was characterized by UV absorbance and reverse-phase HPLC as in (Nicolaou, K.C. et ah (1985) J. Am. Chem. Soc. 107:7515) and exposed to tritium atmosphere in methylene chloride at room temperature. This incubation was stirred in the presence of Lindlar catalyst (1.0 mg from Fluka Chemicals) for ~ 1 h. The resulting mixture was stored in methanol and isolated using RP-HPLC. Tritiated products were chromatographed as methyl esters utilizing a gradient HPLC system equipped with a photodiode array rapid spectral detector (Serhan, C.N., Methods in Enzymology: Arachidonate Related Lipid Mediators, in Murphy R.C., Fitzpatrick, F, (eds.), vol. 187. Orlando, FL, Academic, (1990), p. 167). This mixture contained both [11,12-3H]LXA4 and [ll,12-3H]-ll-/ra«.yLXA4 methyl esters (~ 1:3 ratio) as determined by coelution with synthetic standards, After RP-HPLC, fractions containing [ll,12-3H]LXA4 were collected, and extracted into ethyl acetate. The free acid was prepared by LiOFI saponification (Fiore, S. et al. (1992) J. Biol. Chem. 267:16168). Material from these fractions, when injected into UV-eleetrochemical detection HPLC, gave greater than 90% of radioactivity associated with a tetraene-containing product that coeluted with synthetic LXA4. Materials that eluted with the retention time of authentic LXA4 in two HPLC systems were taken for binding experiments. The specific activity calculated for [11,12-3H]LXA4 was 40.5 Ci/mmole.

Cell cultures and differentiation. HL-60 cells Were seeded in RPMI medium supplemented with 100 U/ml penicillin, 100 pg/ml streptomycin, and 10% fetal calf serum (Hycione, Logan, UT), incubated at 37°C with 5% CO2 atmosphere in 250 ml flasks. Individual flasks containing ~ 50 x 106 cells/ml next received dimethyl sulfoxide (DMSO) (1.12% v/v, 120 h) or retinoic acid (RA) (1 μΜ, 120 h) or phorbol myristate acetate (PMA) (20 nM, 48 h). Before performing binding assays, cells were washed twice in phosphate buffered saline (PBS), Ca2'1 - and Mg2+- free, enumerated and suspended at 20 x 106 cells/ml in Tris buffer (10 mM), pH 7.4 (Fiore, S. et al. (1992) J. Biol. Chem. 267:16168). Nitro blue tetrazolium reduction was performed to monitor induction of polymorphonuclear phenotype as in (Imaizumi, M. and Breitman, T.R. (1986) Blood 67:1273), and cell adherence was determined for induction of macrophage-like phenotype (Collins, S.J. (1987) Blood 70:1273). Human umbilical vein endothelial cells (HUVEC) maintained in culture third passage were obtained from Dr. M. Gimbrone (Brigham and Women's Hospital Department of Pathology).

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PMN, platelet and RBC isolation from peripheral blood. Human PMN were obtained by the modified Boyum method (Boyum, A. (1968) Scan. J. Clin. Lab. Invest. 21:77) from fresh heparinized blood after venipuncture of healthy normal Volunteers. Suspensions in PBS were monitored for cell number and viability by their ability to exclude trypan blue both exceeding 98%. Red blood cells were obtained from 10 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 as previously described (Serhan, C.N. and Sheppard, K.-A. (1990) J. Clin. Invest. 85:772).

Ligand binding. 3H-LXA4 and 3H-LTB4 binding was performed 10 essentially as in Fiore, S. et al. (1992) J. Biol. Chem. 267:16168. After suspending cells in Tris buffer 10 mM (pH 7.4, Ca2+ 2.5 mM, Mg2+ 1.2 mM), aliquots (0.5 ml) were incubated (20 min at 4°C) with 3H- ligands alone (0.3 nM) or in the presence of increasing concentrations of homoligands or other compounds (3-300 nM). The incubations were rapidly centrifuged (60 sec, 12,000 g) on silicon oil (d=1.028), and cell-associated radioactivity was determined by liquid scintillation counting (Wallac 1409, Pharmacia, Piscataway, NJ). Binding experiments with HUVEC cells were performed in 12 well plates with 3.5x105 cells/well. After 10 min, wells were washed twice 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 using the Ligand program (Elsevier-Biosoft, Cambridge, UK).

PLD activity. Human PMN and FIL-60 cells (50 x 106 cells/ml) prepared as above were incubated with 3H-myristic acid or 3H-palmitic acid (8 pCi/50 x 106 cells) for 40-60 min at 37°C in PBS. Cell uptake ranged between 60-80% of added label, 7,1 ± 4.2% (n=10; mean ± S.D.) and 32.6 ± 10.3% (n=6; mean ± S.D.) into total phospholipid classes of PMN and HL-60 cells, respectively. Incubations were carried out at 37°C (10 x 106 cells/ml PBS). Agonists were added in 50 pL PBS or with 1:10 (v/v) EtOH:PBS for phospbatidylethanol (PEt) formation as in Billah, M.M. et al.

(1989) J. Biol. Chem. 264:17069. At indicated times, incubations were stopped by adding 3.5 ml of ice cold CHCl3/MeOH (2/5, v/v) containing l-14C-arachidonic acid (5,000 cpm) used here as internal standard to quantitate extraction recoveries. Samples were extracted using a modified Bligh and Dyer extraction as in (Serhan, C.N. and Sheppard, K.-A. (1990) J. Clin. Invest. 85:772). Organic phases, concentrated in 50 pL of CHCl3/MeOH (8/2, v/v), were spotted onto linear K6D TLC plates developed with the organic phase of ethyl acetate/isooctane/acetic acid/water (110/50/20/100, vfaMv) for 50 min (Billah, M.M. et al. (1989) J. Biol. Chem. 264:17069). In this system, phosphatidic acid (PA) gave an Rf = 0.1 ± 0.04 and PEt gave an Rf = 0.56 ± 0.04; n = 38 ± S.D. that were clearly separated from other phospholipids (that remained at the origin)

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-75or neutral lipids (Rf= 0.75-0.90). Lipids were visualized with iodine vapor and identified by co-elution with authentic standards that were also spotted and chromatographed in each TLC plate. Regions corresponding to PA, PEt, and internal standards were scraped and quantified by liquid scintillation counting. In addition to 3H-myristate or 3H-palmitate labeling, both [l-i4C]arachidonate (0.25 pCi/30 x 106 PMN) and 32ΡγΑΤΡ (20 pCi/50 x 106 PMN) labeled cells were used to monitor LXA4. stimulated formation of PA and PEt generation. In these experiments, PA was resolved using ethyl acetate/isooctane/acetic acid (45/15/10, v/v/v) as solvent system (Bocckino, S.B. etal. (1989) Anal. Biochem. 180:24) and gave an Rf = 0.46 ± 0.03 (n =; 15). All values reported for PEt formation in Tables 4 and 5 were calculated by subtracting the dpm obtained in the presence of agonist(s) alone minus those measured in the presence of agonist(s) and 0.5% EtOH.

Impact of IAP and staurosporine on LXA^-induced PLD activity. PLD activity assays, performed as described (vide supra), were preceded by cell exposure to either IAP or staurosporine. IAP treatment of PMN was performed as previously described (Nigam, S. et al. (1990) J. Cell Physiol. 143:512), and HL-60 cells were incubated in the presence or absence of IAP (300 ng/ml) for 2 h at 37°C (Kanaho, Y. et al. (1992) J. Biol. Chem. 267:23554). Aliquots (107 cells/0.5 ml) were added to 0.4 ml buffer. Next, incubated cells were exposed to either 100 μΐ of vehicle (PBS, EtOH

0.04%) or LXA4 (10-7 M and 10~9 M), in the presence or absence of 0.5% EtOH.

Staurosporine (100 nM) was added to cell suspensions for 5 min at 37°C before addition ofLXA4.

RESULTS

After preparation of synthetic [11,12-3H]LXA4, its specific binding to promyelocytic cells (HL-60) was characterized and direct comparisons with specific binding of [14,15-3H]LTB4 were performed. When routine phenotypic markers were monitored, untreated HL-60 cells displayed a low level of specific binding for both 3H30 LXA4 and 3H-LTB4 ligands. Differentiation induced by exposure to either DMSO (1.12%) or retinoic acid (1 μΜ) for 5 days was accompanied with a three to fivefold increase in specific binding for both radioligands. PMA-treated cells (20 nM, 48 h) displaying characteristics of macrophagic-like phenotype, i.e. NBT negative cells that adhere toplastic (see Imaizumi, M. and Breitman, T.R. (1986) Blood67:1273; Collins,

S.J. (1987) Blood 70:1233), also led to the appearance of specific binding with both 3HLXA4 and 3H-LTB4. Equilibrium binding with 3H-LXA4 at 4°C was reached at 10 min that remained virtually unchanged for the next 20 min.

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To assess whether induction of specific binding for both 3H- ligands required 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 labeled eicosanoids, suggesting inhibition of de novo protein biosynthesis also blocked appearance of specific binding sites. The impact of protease and glycosidase treatments was assessed with both differentiated HL-60 cells and human PMN for 3H-LXA4 specific binding. Protease treatment reduced specific binding and provided additional evidence in support of a protein component of LXA4 specific binding sites.

Results from isotherm binding assays (4°C, 10 min) with differentiated HL-60 ceils and [11,12-3H]LXA4 (0.1-30 nM) showed that [11,12-3H]LXA4 specific binding sites gave a Kd = 0.6 ± 0.3 nM. A nonlinear portion of the Scatchard plot was observed for concentrations observed with LXA4 specific binding with human PMN (Fiore, S. et al. (1992) J. Biol. Chem. 267T6168). Results obtained here with LTB4 specific binding with HL-60 cells, Kd = 0.12 nM, are essentially in agreement with values recently reported by Harada (Xie, M. et al. (1991) J. Clin. Invest. 88:45), namely Kd = 0.23 nM.

To further characterize the interactions of 3H-LXA4 with its specific binding sites, competition binding experiments were performed with differentiated HL60 cells. LXA4, LXB4, LTB4, LTC4 and the leukotriene receptor antagonists SKF 104353 (LTD4 antagonist; Harada, Y. (1990) Hiroshima J. Med. Sci. 39:89) and ONO4057 (LTB4 antagonist; Gleason, J.G, et al. (1987) J. Med. Chem. 30:959) were assessed as potential competing ligands. Neither LXB4, LTB4, nor trihydroxyheptanoic acid (methyl ester) (300 nM) were able to displace 3H-LXA4 specific binding with differentiated HL-60 cells while LTC4 caused ~30% decrease of specific binding when added in 3 iog molar excess. The finding that LXA4 (300 nM) was unable to compete with 3H-LTB4 (0.3 nM) binding to differentiated HL-60 cells suggests that LXA4 and LTB4 interact with separate classes of specific binding sites. Leukotriene receptor antagonists SKF 104353 and ONO-4057 did not displace 3H-LXA4 binding with differentiated HL-60 cells, but SKF 104353 and LTD4 were f ffective in competing the specific 3H-LXA4 binding with HUVEC. HUVEC displayed a Kd of 11.0 ± 2.6 nM and a Bmax of 2.5 x 10-10 M for 3FI-LXA4, and virtually identical values were calculated for LTD4 competition. In the case of 3H-LTB4, HUVEC did not specifically bind LTB4, but non-specific cell association was evident with this IH-Iigand (n-3; not shown). Specific association of 3H-LXA4 was not evident among several other cell types surveyed. Here, neither washed platelets, RBCs, the β-cell (Raji), nor T-cell (Jurkat) cultured cell lines displayed specific binding for 3H-LXA4. Taken together, these results indicate that LXA4 interacts with unique binding sites in differentiated HL-60 cells that is not

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PCT/US97/16342 sensitive to either leukotris e receptor antagonist (SKF 104353 or ONO-4057). In HUVEC, 3H-LXA4 specific binding was sensitive to both LTD4 and SKF 104353 but not ONO-4057, suggesting that3H-LXA4 specific binding in this cell type may reflect its interaction with putative LTD4 receptors.

LXA4 rapidly stimulates phosphatidic acid formation in human neutrophils (Nigam, S. et al. (1990) J. Cell Physiol. 143:512). To determine if 3H-LXA4 binding confers PLD activation, PEt and PA were monitored in both PMN and HL-60 cells. Results indicated that LXA4 stimulates PLD activity in these cell types with similar temporal responses. PMN exposed to LXA4 (10'10 M) rapidly generated the ethanol trapping product PEt within 60 s that declined to baseline levels by 5 min. In the absence of added EtOH, PEt was not formed at statistically significant levels. A biphasic concentration dependence was obtained for PEt formation in both PMN and differentiated HL-60 cells. An apparent maximal response was noted with the concentration range of ~10'9-l 010 M LXA4, and a second peak of activity was observed at IO'7 M LXA4. Below ϊ O'8 M, both the chemotactic peptide FMLP and LXA4 gave results of similar magnitude with FMLP appearing to be slightly more potent with PMN from some donors. To evaluate the potential contribution of other biosynthetic pathways, LXA4-induced PA formation was also examined in both 32PyATP and [1 14C]-arachidonate-labeled PMN. 32P-labeled PA was evident in statistically significant levels only after 30 min of exposure to LXA4 (1 O'7 M). Similar results were obtained with 14C-labeled PA formation derived from ,4C-arachidonate-labeled precursors.

These findings indicated that LXA4 can also stimulate other routes of PA formation in PMN but only after 30 min of exposure.

Only differentiated HL-60 cells (expressing specific binding sites for 3H25 LXA4) incubated with LXA4 rapidly generated PEt that was evident within 30 sec. Undifferentiated HL-60 cells incubated with LXA4 (10-9 M) did not rapidly generate PEt. The concentration dependence with these cells also gave a biphasic response with LXA4 and gave an apparent maximum at 10'9 M. To investigate possible signal transduction events involved in LXA4-mediated PLD activation, PMN and HL-60 cells were next exposed to either IAP or staurosporine. Results indicate that the LXA4mediated PLD activity evoked within the lower concentration range (10'9-10',θ M) was sensitive to IAP treatment in both cell types and, similarly, tlie PLD activity stimulated at higher concentrations of LXA4 (107 M) was inhibited by staurosporine. Thus, in both cellular systems at concentrations below 108 M, LXA4 rapidly interacts with specific binding sites that trigger PLD activity and hence confers a functional response, while within submicromolar concentrations of LXA4, it may stimulate additional processes that can also lead to activation of PLD.'

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Example 4: Lipoxin Bioactivity Assays

Several of the preferred LX analogs (shown structurally as compounds 1 through 8 above) were prepared by total synthesis as described in Example 1. Following preparation and isolation of these compounds via HPLC, compounds were first assessed to determine whether they retain biological activity using the neutrophil adhesion assay and epithelial cell transmigration assays (as described in Nash, S et al., (1987) J. Clin. Invest. 80:1104-1113; Nash, S et al., (1991) J. Clin. Invest. 87: 1474-1477; Parkos, C.A. etal., (1991) J. Clin. Invest. 88:1605-1612; Parkos, C.A. etal. (1992) J. Cell. Biol. 117:757-764; Madara J.L. et al., (1992) J. Tiss. Cult. Meth. 14:209-216).

Compounds 1 through 8 (IO-? - 10’^θΜ) were found to inhibit neutrophil adhesion to endothelial cells and their transmigration on epithelial cells. The acetylenic precursors ( compound 1,3, 5 and 7) were found to be physically more stable than their tetraene counterparts. Compound 7, which did not have an alcohol group in the Cl5 position or other modifications in the series, showed no biological activity in the assays.

It would therefore appear that a substituent in the Cl 5 position of LX is necessary for the biological activity of at least LXA4 analogs. 15-methyl-LX A4 (compound 2) also proved to inhibit polymorphonuclear (PMN) adhesion triggered by leukotriene B4 (LTB4) to human endothelial cells with an IC5Q of ~ 1 nM. LX analogs 1 through 8 were 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 for inhibition induced by LXA4 or other analogs. The results in these neutrophil-containing bioassays indicate that LXA4 analogs with modifications in C15-C20 positions retain their biological action and can inhibit PMN transmigration and adhesion events.

The bio-half-life of compounds 1-8 was assessed using phorbol estertreated human promyelocytic leukemia (HL-60) cells as described in Example 2. These cells converted more than 95% of LX A4 within five minutes of its addition to the cell incubation. LXA4 in this system was rapidly transformed to I5-OXO-LXA4. However, in the same assay, 15-methyl-LXA4 (compound 2) and cyclohexyl-LXA4( compound 4) were quantitatively recovered in the incubation medium at times up to two hours.

These results illustrate that modification in the carbon 20 through the carbon 15 positions prevents the further metabolism of LXA4 by leukocytes.

In addition, the stability of the acetylenic methyl ester LXA4 (compound

1) was recovered essentially intact after 60 minutes of incubation in whole blood (37°C) ex vivo, as assessed after extraction and reverse phase HPLC. When taken together, these results indicate that LX analogs retain biological action and are resistant to further metabolism in vitro.

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Example 5: Effect of 15-epi-Lipoxins' on Cell Proliferation

MATERIALS and METHODS

Synthetic (5S,67?,15R)-trihydroxy-7,9,13-/ra/?5-ll-cw-eicosatetraenoate:

carboxymethyl ester (1 S-epi-LXA^methyl ester) was prepared by total organic synthesis and was a gift of Prof. N. A. Petasis (Department of Chemistry, University of Southern California). 15-epi-LXA4 free acid was obtained by saponification of 15-epiLXA4-methyl ester in tetrahydrofUran with LiOH (0.1M) at 4°C for 24 h. Synthetic eicosanoid reference samples were from Cascade Biochem Limited (Reading,

Berkshire, England). Inhibitors of 5-LO (Rev 5901 isomer) and cytochrome P450 (17octadecaynoic acid, 17-ODYA) activities were from Biomol (Plymouth Meeting, PA). Radiolabeled ([^¾) dCTP and ((¾]) arachidonic acid and methyl-thymidine were from Dupont NEN (Boston, MA). Ionophore (A23187), ASA, 3,(4,5-dimethylthiazoyl15 2-yl) 2,5 (diphenyl-tetrazolium bromide) (MTT) and guanidinium isothyocyanate were purchased from Sigma Chemical Co (St. Louis, MO). Recombinant human interleukin 1 β (IL-1 β) was obtained from R&D systems (Minneapolis, MN), Dulbecco's phosphatebuffered saline containing both CaCl2 (0.6 mM) and MgCb (1.0 mM) (pH 7.4) (DPBS2+), fetal bovine serum (FBS), penicillin and streptomycin were from Bio

Whittaker (Walkersville, MD). Hank's balanced salt solution (HBSS) and F-12K nutrient mixture were from Gibco Laboratories (Grand Island, NY). High-pressure liquid chromatography (HPLC) grade solvents and cesium chloride were purchased from JT Baker (Phillipsburg, NJ), methyl formate was from Eastman Kodak Co (Rochester, NY) and Sep-Pak C j g cartridges were from Waters Associates (Milford, MA). Diazomethane was prepared from N-methyl-N'-nitro-N-nitroguanidine purchased from Aldrich Chemical Company (Milwaukee, WI). N,0-bis (trimethylsilyl) trifluoroacetamide (BSTFA) was from Pierce (Rockford, IL). First strand cDNA synthesis kit and other molecular biology reagents were from Promega (Madison. WI). Oligonucleotide primers were purchased to Integrated DNA Technologies (Coralville, IA).

Cell isolation and culture:

Human type II epithelial A549 cells from human lung carcinoma and normal human skin fibroblast (breast) were obtained from the American Type Culture

Collection (Rockville, MD). The A549 cell line originated from a human alveolar cell carcinoma and was a useful cell line because it was easily assessable and could be maintained in culture without contaminating tissue macrophages. (Lieber, M., et al.

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-80PCT/US97/16342 (1976) A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Ini. J. Cancer Fibroblasts were utilized within their limited window of viability and their passage numbers were recorded (results are reported for cells from 3-5 passages). Epithelial A549 cells were seeded into T-75 cn/ tissue culture flasks and maintained in F-12K medium supplemented with 10% heatinactivated FBS, penicillin (50 U/mL) and streptomycin (50 (g/mL). Human PMN from healthy donors who had not taken ASA or other medications for at least two weeks were obtained by Ficoll-Hypaque gradient centrifugation and dextran sedimentation, (Boyum A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 7789.), and suspended in DPBS+ at pH 7.4. Viability of A549 cells and PMN was determined by their ability to exclude trypan blue and were 95 ± 2 and 97 ± 1%, respectively. These values were not significantly altered during the reported incubations.

Incubation Conditions:

In incubations involving permeabilized A549 cells (prepared by a rapid freezethaw cycle), IL-Ιβ-treated (1 ng/ml, 24 h) A549 cells (1.5x10^ cells/ml) were pretreated for 20 min with either vehicle (0.1% EtOH), ASA (500 (M; used throughout), 5 (M 17ODYA, an inhibitor of cytochrome P450, (Muerhoff A.S., et al. (1989) Prostaglandin and fatty acid co and (co-l)-oxidation in rabbit lung: acetylenic fatty acid mechanismbased inactivators as specific inhibitors. J. Biol. Chem. 244: 749-756.), or 5 mM Rev 5901 isomer, a 5-LO inhibitor, subjected to two cycles of rapid freezing in a dry Ice25 acetone bath and thawing to room temperature (full cycle <20 min), and incubated with arachidonic acid (20 (M) for 20 min at 37°C in 4 ml of DPBS2+. In experiments involving radiolabeled arachidonic acid, incubations were initiated with the addition of [3fi]-arachidonic acid (0.25 (Ci/ml) plus unlabeled arachidonic acid (20 (M) for 20 min at 37 °C.

For time-course experiments involving the generation of 15-HETE from endogenous sources (see FIG. 2B), intact A549 cells were exposed to IL-1 p (1 ng/ml) for up to 48 h and then treated with vehicle (containing 0.1% EtOH) or ASA for 20 min, followed by addition of ionophore A23jg7 (5 μΜ) in 4 mi of HBSS for 30 min at 37 °C.

In coincubation experiments, confluent A549 cells were exposed to IL-1 β (I ng/ml, 24 h), washed in HBSS and treated with vehicle alone or ASA for 20 min and arachidonic acid (20 (M) for 60 seconds at 37°C. Coincubations were performed by

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-81 PCT/US97/16342 adding PMN to A549 cell monolayers followed by costimulation with ionophore A23187 (5 μΜ) in 4 ml of HBSS for 30 min at 37°C,

Analysis of eicosanoids:

Incubations were stopped with 2 volumes cold MeOH containing prostaglandin B2 (200 ng) and products were extracted using Sep-Pak Cjg cartridges. Materials which eluted in the methyl formate fractions were concentrated under a stream of N2 and scanned for ultraviolet-absorbing material (in methanol) with a model 8452 spectrophotometer (Hewlett Packard Co, Palo Alto, CA) prior to injection into a reversed phase (RP)-HPLC system. This system consisted of a dual pump gradient (LKB, Bromma, Sweden), a diode array detector (Hewlett-Packard 1040M series II) and a HPLC^D ChemStation software. The collected UV data were recalled at 300 nm to monitor conjugated tetraenes, at 270 nm for trienes and 234 for monoHETEs. All UV spectra were acquired using step = 4 nm, Bw = 10 nm, and range = 220-360 nm with a sampling interval of 0.96 s.

The monohydroxy eicosanoids (i. e. 5-, 12- and 15-HETE) from A549 cells were analyzed using a Ultrasphere-ODS column (5 pm, 4.6 mm x 25 cm) (Beckman Instruments, Fullerton, CA) was eluted with MeOH/^O/acetic acid (65:35:0.01; v/v/v) as phase one ((q -20 min), and a linear gradient with MeOH/acetic acid (99.9:0.1, v/v) as phase two (20-45 min) at a flow rate of 1.0 ml/min. The R- and S-enantiomers of 15HETE were resolved and identified using a chiral HPLC system similar to that reported by Hawkins et al. (1988). (Hawkins et al. (1988) Resolution of enantiomers of hydroxyeicosatetraenoate derivatives by chiral phase high-pressure liquid chromatography. Anal. Biochem. 173: 456-462.) Briefly, after RP-FIPLC material eluting beneath 15-HETE peak was extracted with chloroform and converted into methyl ester by ethereal diazomethane treatment, chiral analysis was performed with a Bakerbond DNBPG (covalent) chiral column (5 pm, 4.6 mm x 25 cm) (JT Baker, Phillipsburg, NJ) eluted with n-hexane/2-propanol (100:0.4; v/v) at a flow rate of 0.8 ml/min. When indicated, generation of 15-HETE from endogenous sources was monitored by radioimmunoassay (RIA). The antibody was raised against 155-HETE with 0.1% crossreactivity at 50% B/Bq 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 C]g (3.9 x 300 nm) column eluted with an isocratic mobile phase MeOH/^O/acetic acid (60:40:0.01;

v/v/v) with a flow rate of 0.6 ml/min or an Altex Ultrasphere ODS column (5 pm, 10

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-82PCT/US97/16342 mm x 25 cm) eluted with MeOH/H2O/acetic acid (65:35:0.01; v/v/v) at a flow of 3 ml/min. Peptidoleukotrienes (LTC4 and LTD4) eluted in the MeOH fractions from SepPak cartridge extractions were resolved with a Beckman Ultrasphere-ODS column eluted with MeOH/H2O/acetic acid (65:35:0.01; vNIv), pH 5.7, at 1 ml/min. Incubations of PMN with 15Λ-ΗΕΤΕ were stopped with MeOH and methyl formate fractions of the

Sep-Pak C j § extracted products were injected into an Altex Ultrasphere ODS column (5 pm, 10 mm x 25 cm) eluted with MeOH/H2O/acetic acid (65:35:0.01; v/v/v) using a flow of 3 ml/min. The material beneath peaks absorbing at 300 nm were individually collected after RP-HPLC and analyzed by gas chromatography-mass spectrometry (GC10 MS) employing a Hewlett-Packard 5890 GC series II equipped with a 5971A massselective quadrapole detector as in Claria, J. and Serhan, C.N. (1995) Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interaction. Proc. Nail Acad. Sci. USA 92:9475-9479, herein expressly incorporated by reference.

Reverse Transcription (RT) and PCR:

Total RNA was obtained from A549 cells by the guanidinium isothiocyanatecesium chloride method and cDNA was produced by RT. Oligonucleotide primers were constructed from published sequences of PGHS-1 and PGHS-2 ((5'-TGC CCA GCT

CCT GGC CCG CCG CTT-3' (sense), 5'-GTG CAT CAA CAC AGG CGC CTC TTC3' (antisense)), and (5'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3' (sense) and 5-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 CCA

GCG GTA ACA AGG GAA CCT GAC CTC-3’ (antisense)), 12-LO, (Funk, C.D. and FitzGerald, G.A. (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 CTG ATG TTT 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 OCAS' (sense) and 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (antisense). PGHS-1, PGHS-2 and GAPDH samples were amplified for 25 cycles of denaturation at 94 °C for 1 min, annealing at 58°C for 2 min and extension at 72 °C for 3 min. 5-, 12- and 15-LO were amplified at 94°C (1 min), 55°C (2 min) and 72°C (2.5 min) for 35 cycles. PCR products were analyzed by electrophoresis in 2% agarose gel and their identity was monitored by restriction enzyme analysis. For detection of specific PCR-amplified

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-83PCT/US97/16342 targets, 0.5 (Ci of [-^pjdCTP (3000 Ci/mmol) was added to the PCR mixture and the products were quantified by phosphorimager using Image-Quant programming (Molecular Dynamics, San Lorenzo, CA).

Cell Proliferation:

A microcuiture 3,(4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyl-tetrazolium bromide) (MTT) assay, (Marshall, NJ., et al. (1995) A critical assessment of the use of microcuiture 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 cell proliferation. A549 cells and fibroblasts from exponential-phase maintenance cultures were counted and dispensed within replicate 96-well culture plates in 100 μΐ volumes of medium (-2000 cells/well). Following 24 hours at 37 °C, the culture medium was removed, and fresh medium (100 μΐ) containing either the compounds (5-1000 nM) or vehicle (medium plus 0.15% ethanol) was added to 4 replicates for each condition studied and the culture plates were then incubated for up to 96 hours at 37 °C in a 5% CC>2 atmosphere. At the end of this period, 25 μΐ of freshly prepared MTT in HBSS (5 mg/ml) was added to the wells and the plates were incubated for 4 hours at 37 °C. Dye solution was aspirated, wells were washed once with HBSS and dye taken up by the cells was extracted in 100 μΐ of isopropyl alcohol: 1 NHC1 (96:4, v/v) and quantitated at 570 nm using a microplate reader (Molecular Devices, Menlo Park, CA). In some experiments, cells were grown in 12-well culture plates, treated as above and enumerated using a Neubauer hemocytometer. Viability was assessed routinely using trypan blue exclusion assay. For the A549 cells and fibroblasts, a linear relation was established for the MTT values and cell number within the range of the experiments shown (r ~ 0,995, P < 0.005). A549 cells grown for 72 hours in the presence of the compounds (5-1000 nM) or vehicle (0.15% EtOH) were lysed with 0.25 NNaOH and the cellular protein content was determined by applying the Bio-Rad (Richmond, CA) microassay method using bovine serum albumin as standard. The mean cellular protein content in resting A549 cells was 46,6 ± 1.5 pg/cell.

Thymidine Incorporation and DNA synthesis:

A549 cells (—2 x 10^ cells/ml) were seeded in 96-well plates, allowed to settle for 24 hours and grown for an additional 72 hours in the presence of compounds (51000 nM) or vehicle (medium plus 0.15% ethanol). Twenty-four hours before the assay, (Ci/ml of methyl-pHjthymidine (specific activity 6.7 Ci/mmol) were added to each

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-84PCT/US97/16342 well. (See Cybulsky et al. (1992) Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells. Am, J. Physiol. 262 (Renal Fluid Electrolyte Physiol.3Y): F639-F646.) After pulse-labeling, each well was washed four times with cold DPBS2+, and the cells were lysed with NaOH (0.25 N) and radioactivity measured.

The Student's /-test was used for statistical analysis and differences were considered significant at a P value (0.05).

RESULTS

Eicosanoids are formed by initial oxygenation of arachidonic acid by PGHS or LO enzymatic pathways. (Samuelsson B., et al. (1987) Leukotrienes and Lipoxins: structures, biosynthesis, and biological effects. Science 237: 1171-1176.) To assess which of the eicosanoid-generating enzymes are present and/or regulated by cytokines in A459 cells, mRNA levels ofPGHS-1 and -2 and 5-, 12- and 15-LO from A549 cells grown in the presence or absence of IL-1 p were monitored by RT-PCR followed by phosphorimager analysis. As illustrated in FIG. 1 A, mRNA levels for PGHS-2 were significantly increased (~ twofold) after stimulation of A549 cells with IL-1 p. In contrast, mRNA levels for PGHS-1 and 5-LO were not significantly altered after exposure of the cells to cytokine (FIG. ΙΑ). A549 cells failed to show either 15- or 12LO expression before or after cytokine induction (FIG. 1 A). The absence of 15-LO mRNA in A549 cells, was further confirmed by performing the RT-PCR in parallel with human lung tissue and peripheral blood monocyte RNA, which are known positive and negative sources, (cf. Funk C.D. and FitzGerald G.A. (1991) Eicosanoid forming enzyme 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 airway epithelial cells, IL-lp-stimulated A549 ceils (1.5x1cells/ml) were permeabilized and incubated with arachidonic acid, and the products formed were extracted and analyzed by RP-HPLC. The chromatographic profile revealed the presence of a major product with strong UV absorbance at 234 nm which coeluted with synthetic 15-HETE. Aiso, when pHJ-arachidonic acid was added to IL-lp-treated A549 cells, radiolabeled material was recovered beneath the peak coeluting with 15-HETE (FIG. IB). In these experiments, the formation of either 5- or 12-HETE was not consistently observed. ASA treatment of A549 cells led to a marked increase in the formation of 15-HETE, while incubation of permeabilized A549 cells with 17-ODYA, a reported inhibitor of P450 eicosanoid metabolism, (Muerhoff, A..S. et al. (1989) Prostaglandin and fatty acid ω and

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85(co-l)-oxidation in rabbit lung: acetylenic fatty acid mechanism-based inactivators as specific inhibitors, J. Biol. Chem. 244:749-756), resulted in ~50% reduction in 15-HETE (FIG. 2A). 17-ODYA is a potent inhibitor of P450 eicosanoid metabolism and does not selectively inhibit either cyclooxygenase or LO activity . (See Muerhoff et al. and supplier's supporting materials.) The 5-LO inhibitor (Rev-5901 isomer) did not alter the amount of 15-HETE produced by A549 cells in a statistically significant fashion. Heatdenatured A549 cells reduced the quantities of 15-HETE by ~90%, suggesting an enzymatic component in its formation. Production of 15-HETE from endogenous sources of arachidonate was also obtained from intact A549 cells (25.0 ± 10.0 ng/10? cells) treated with IL-1 β (1 ng/ml) for 24 hours. Taken together these results indicate that 15-HETE is the main monohydroxy product generated by A549 cells and suggest that acetylated PGHS-2 and cytochrome P450 each contributes to its biosynthesis.

To investigate the time-course for generation of 15-HETE from endogenous sources of arachidonate, intact A549 cells were exposed to IL-1 β (1 ng/ml) for up to 48 h and the amount of immunoreactive 15-HETE present in the cell supernatant was monitored by means of a specific RIA. In resting conditions, A549 cells produced significant levels of immunoreactive 15-HETE (19.1 ± 10.5 ng/lO? cells, n = 3, d = 2). These values were unchanged by addition of ionophore A23137 (5 (M) in the absence of IL-1 β (FIG. 2B). Also, in the absence of ionophore stimulation, addition of IL-1 β to

A549 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 β plus A23J87 stimulation of A549 cells led to a marked increase in the production of 15-HETE (FIG. 2B). The maximal levels were found at 24 h hf exposure to the cytokine with the levels of 15-HETE declining thereafter.

Because the stereochemistry of the alcohol in 15-HETE produced by A549 cells was of interest, the relative amounts of individual R and S enantiomers of 15-HETE generated by IL-1 β-treated A549 cells were examined using a chiral phase HPLC analysis. (See Methods.) PGHS-2 as well as cytochrome P450 are enzymes other than 15-LO that can each convert arachidonic acid to 15-HETE. ASA-acetylated PGHS-2derived 15-HETE carries its carbon (C)-15 alcohol group mainly in the R configuration. (Holtzman, M.L, et al., (1992) Identification of a pharmacologically distinct prostaglandin H synthase in cultured epithelial cells, J. Biol. Chem. 267:21438-21445). Here, 15-HETE produced by IL-1 β-primed A549 cells was converted to its methyl ester and subjected to SP-HPLC chiral analysis. The 15-HETE from activated A549 cells was 65% in the R and 35% in the 5 configuration (FIG. 3). Pretreatment of A549 cells with ASA (20 min, 37 °C) resulted in a 3-fold increase in the amounts of 15Λ-ΗΕΤΕ whereas formation of 15S-HETE remained unaltered (FIG. 3). In the presence of ASA, 15RWO 98/11049

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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 ρ-primed A549 cells in presence of ASA was in the R configuration.

Transcellular eicosanoid biosynthesis is an important means of amplifying lipid mediators as well as generating new mediators. (Marcus, A. J. (1995) Aspirin as prophylaxis against colorectal cancer, N. Engl. J. Med. 333:656-658.) Costimulation of human endothelial cells and PMN after ASA treatment results in the formation of a new class of bioactive eicosanoids. (Claria, J. and Serhan, C.N. (1995) Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions, Proc. Natl. Acad. Sci. USA 92:9475-9479.) These novel eicosanoids were identified as 15-epi-LXs and their biosynthesis involve leukocyte transformation of ASA-triggered endothelial-derived 157?-HETE. In view of these results, it is possible that formation of new eicosanoids by transcellular biosynthesis also occurs during epithelial cell-PMN interactions. To test this hypothesis, confluent A549 cells were exposed to IL-1 β (1 ng/ml, 24 h), treated with ASA and costimulated with PMN. FIG. 4A shows a representative HPLC profile of material obtained from stimulated cells exposed to ASA, which revealed the presence of four major products with strong UV absorbance when plotted at 300 nm. On-line spectral analysis of these products showed that they each displayed a triplet of absorbing bands characteristic of conjugated tetraene-containing chromophores indicative of the LX basic structure (maxima at 301 nm and shoulders at 288 and 316 ±2 nm) (FIGs. 4B and 4C).

LXA4 and 15-epi-LXA4 were identified in the chromatographic profiles on the basis of coelution with synthetic standards and the presence of the characteristic chromophore. In these coincubations, 15-epi-LXA4 accounted for ~88% of the total amount of LXA4 detected which is in agreement with the observation that the majority of 15-HETE generated by IL-1 β-primed A549 cells exposed to ASA is predominantly in the R configuration. (Cf. FIGs. 3 and 4A, 4B and 4C). In this RP-HPLC system, 15-epi1 l-trans-EXA^ and LXB4 coeluted, as did 1 l-/raw-LXA4 and 15-epi-LXB4 (not shown). These LX isomers were not further resolved by HPLC and are denoted in the profile beneath peaks labeled as peaks A and B, respectively (FIG. 4A). The compounds beneath peaks A and B did resolve as OTMS, methyl ester derivatives in GC-MS (vide infra). The products were present in ~8:2 ratio in favor of their 157? epimers (n=3). Compound C (FIG. 4A) did not coelute with any of the previously identified LXs and was also present in the RP-HPLC profile from activated PMN incubated with 157?HETE (data not shown, n = 5). Material eluting beneath Compound C matched the physical properties of Compound III that was recently isolated from endothelial cellPMN interactions. (Cf Claria, J. and Serhan, C.N. (1995) Aspirin triggers previously

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-87undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions, Proc. Natl. Acad. Sci. USA 92:9475-9479.) Although the complete stereochemistry of compound C remains to be determined, the UV spectral data and chromatographic mobilities suggest that it may be the 15-epimer form ofl-cis-M-trans-UAN^.

(Nicolaou, K.C., et al. (1989) Identification of a novel 7-cis-l 1-translipoxin A4, generated by human neutrophils: total synthesis, spasmogenic activities and comparison with other geometric isomers of LXs A4 and B4, Biochim, Biophys. Acta 1003:44-53.) Thus, the LXs generated during epithelial (A549 cell)-PMN costimulation after ASA treatment were predominantly 15-epi-LX (FIG. 4A).

In addition to the ability to produce LXs, coincubations of activated PMN with A549 cells also generate significant amounts (~ 8 times more than tetraene-containing LXs) of peptidoleukotrienes (pLTs; LTC4, and LTD4) in the absence of ASA (FIGs. 5A and 5B). The amounts of both LXs and pLTs produced in these coincubations were dependent upon individual cell ratios (FIG. 5A and 5B, insets). Exposure of airway epithelial A549 cells to ASA (20 min) before addition of PMN led to an increase in the formation of LXs and a decrease in pLTs (FIGs. 5A and 5B). Neither PMN nor A549 cells incubated separately, in the absence or presence of ASA, generate detectable levels of LXs or pLTs (FIGs. 5 A and 5B and data not shown). Taken together, these results indicate that, during A549 cell-PMN interactions, both LXs and pLTs originate from transcellular routes.

LXs are vasodilators and potent regulators of leukocyte responses, such as inhibition of chemotaxis, adhesion to endothelial cells and transmigration across epithelium. (See Serhan, C.N. (1994) Lipoxin biosynthesis and its impact in inflammatory and vascular events, biochim. Biophys. Acta 1212.T-25.) In contrast, pLTs possess both vasoconstrictor and proinflammatory actions as well as stimulate the growth of several cell types including fibroblasts, smooth-muscle and glomerular epithelial cells, (Baud, L., et al. (1985) Leukotriene C4 binds to human glomerular epithelial 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 block LTC4-stimulated hematopoiesis. (Serhan, C.N. (1994) Lipoxin biosynthesis and its impact in inflammatory and vascular events. Biochim. Biophys. Acta 1212.T -25.)

Because ASA enhances 15-epi-LX formation and inhibits pLT biosynthesis (FIGs, 5A and 5B), these eicosanoids may play counterregulatory actions on cell proliferation and contribute to ASA's protective mechanisms in human cancer. To this end, the effect of these LO products on epithelial cell proliferation (FIGs. 6A and 6B) was tested and their actions was compared to that of dexamethasone, a well-established inhibitor, employing a soluble microculture tetrazolium (MTT) assay. (Alley, M.C., et al. (1988) Feasibility

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-88PCT/US97/16342 of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res, 48:589-601.) These experiments were performed with synthetic 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 in FIGs. 6A and 6B, LXA4 and LXB4 inhibited A549 cell proliferation in a time- (A) and dose-dependent (B) fashion. LXA4 and LXB4 as well as dexamethasone [1 μΜ] inhibited A549 cell proliferation after 72 and 96 hours of treatment (FIG. 6A). After 72 hours, LXA4 shared the anti-proliferative properties (FIG. 6A) observed for dexamethasone with these cells. (Cf. Croxtall, J.D., and Flower R.J. (1992) Lipocortin 1 mediates dexamethasone-induced growth arrest of the A549 lung adenocarcinoma cell line. Proc. Natl. Sci. USA 89:3571-3 575.) The half maximum inhibition (IC5Q) for LXA4 was ~8011M compared to that of dexamethasone, which was ~7 nM. LXB4 at concentrations of 0.5 and 1 μΜ was (3 times more active than either LXA4 or dexamethasone (FIG. 6B). Of interest, both LXA4 and LXB4 showed essentially equal potency for blocking A549 cell growth when each was added repeatedly (i.e., 24-hour intervals) to the cells for 3 consecutive days (data not shown, n = 3, d = 4), suggesting that LX may be inactivated by these epithelial cells. Results from additional experiments employing direct cell enumeration (FIG. 7B) and measurement of total cellular protein content (data not shown, n = 3, d==4) paralleled those obtained with MTT assay, thus confirming the anti-proliferative actions of LXs in A549 cells. Furthermore, a blockage of DNA synthesis, as determined by ^H-thymidine incorporation, occurred when A549 cells were exposed for 72 hours to concentrations of 50 nM or higher of LXB4 or dexamethasone (FIG. 7A). After incubation of A549 cells with the compounds, the viability 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 of concentrations used in these experiments.

The 15-hydroxy epimeric forms of LXA4 and LXB4 (15-epi-LXA4 and 15-epiLXB4, 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 the following Table 1.

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-89Table 1: LX and 15-epi-LX actions on cell proliferation

<td> Compound [10'7</td><td> % inhibition</td><td> P value*</td>

<td> Vehicle</td><td> 1.6 + 10.3</td><td> NS</td>

<td> Dexamethasone</td><td> 42.1+5.4</td><td> <0.005</td>

Cells (2000 A549 cells/well) were grown in 96-well plates and exposed to vehicle (0.15% vol/vol in EtOH/F-12K media) or equimolar concentrations (107 M) of LXA4,

15-epi-LXA4, LXB4,15-epi-LXB4, or dexamethasone for 72 hours at 37 °C, Values represent + SEM from 3 to 7 experiments performed in quadruplicate and are expressed as percent inhibition of cell growth. *P values denot statical differences as compared to cells alone. & P<0.001 for 15-epi-LXB4·

At equimolar levels (100 nM), 15-epi-LXA4 inhibited A549 cell growth to a similar 10 extent as LXA4. On the other hand, 15-epi-LXB4 isolated from conversion of 157?HETE by activated PMN and added back to the A549 cells gave a more potent antiproliferative 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 and less prominent ions at 482 (M+-100) pt was not possible to obtain its molecular ion because of its low abundance]. Thus, the predominant material beneath the peak labeled B in FIG. 4B was consistent with that of 15-epi-LXB4, which gave a shorter C value and separated from LXB4 as OTMS, methyl ester derivative in GC-MS analysis. The mass spectra of 15-epi-LXB4 and LXB4 were essentially identical (not shown) but their C values were distinct. Material eluting beneath the peak denoted as C (isolated from activated PMN incubated with 15P-HETE) also showed a mild inhibitory action on epithelial cell growth (18 + 2% inhibition proliferation, n = 3, d = 4), In sharp contrast, 15-epi-/ram-LXA4, 11-/tww-LXB4, 8,9-acetylenic-LXB4, peptidoleukotrienes (LTC4 and LTD4) and the LX precursors (155- and 15Ρ-ΗΕΊΈ) each tested at 10’^ -10^ M were not able to significantly inhibit A549 cell proliferation (data not shown, n = 3-5, d = 4). These results indicate that LX and 15-epi-LX gave a stereoselective action in blocking cell proliferation in A549 cells. LXA4 and LXB4 was tested with human skin fibroblasts to determine if they were antiproliferative for this ceil type. At 100 nM, both LXA4 and LXB4 inhibited proliferation of fibroblasts. LXB4 gave 38.0 ( 7.5%

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-90PCT/VS97/16342 inhibition and LXA4 10.7 ( 1.8% compared to dexamethasone (29.7 (0.4%) as positive control (n=3).

The presence of an active cytochrome P450 enzyme system in human airway

A549 cells, (Vogel, et al. (1994) Transforming growth factor-βΐ inhibits TCDD-induced cytochrome P450IA1 expressions in human lung cancer A549 cells. Arch. Toxicol. 68: 303-307.), together with the results that inhibition of P450 as well as heat denaturing blocks 15-HETE generation in these cells (FIG. 2 A) suggests that this enzyme system in epithelial cells also contributes to 15-HETE biosynthesis and the generation of 15-epilipoxin by transcellular routes (FIG. 8). Taken together, these observations (FIGs, 1-4) establish the existence of two separate enzymatic pathways (i.e. ASA-acetylated PGHS2 and cytochrome P450), which can initiate the formation of 15-epi-lipoxin during airway epithelial cell-PMN interactions (FIG. 8). Also, it should be noted that, in view of ASA's ability to induce P450 enzymes, Pankow, D. et al, (1994) Aeetylsalicylic acid inducer of cytochrome P-450 2E1? Arch. Toxicol. 68: 261-265, it is possible that these two independent routes may act in concert to generate 15-epi-lipoxin.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

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PCT/US97/16342

-93 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4

AGATCATCTC TGCCTGAGTA TCTT (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATGGGTCTCT ACCGCATCCG CGTGTCCACT (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TG'OLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 50

CACCCAGCGG TAACAAGGGA ACCTGACCTC (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

WO 98/11049

-94PCT/US97/16342 (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

AGTTCCTCAA TGGTGCCAAC 20 (2) INFORMATION FOR SEQ ID NO: 8;

(i, SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

ACAGTGTTGG GGTTGGAGAG 20 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION; SEQ ID NO:9:

GAAGACCTGA TGTTTGGCTA CC 22 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

WO 98/11049

-95PCT/US97/16342 (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES (xi,) SEQUENCE DESCRIPTION: SEQ ID NO:10:

AGGGTTCTCA TCTCCCGG 18 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: NO ' (Xi) SEQUENCE DESCRIPTION; SEQ ID NO:11:

CCACCCATGG CAAATTCCAT GGCA 24 (2) INFORMATION FOR SEQ ID NO :12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH; 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

TCTAGACGGC AGGTCAGGTC CACC

WO 98/11049

-96PCT/US97/16342

Contents167

77 members in 11 offices

This recordMember, by publication datePriority claim1 more office is in the list below

Priority claims8

Priority claims
Document	Office	Kind	Date
71261096	United States of America	A	1996-09-13
71261096	United States of America	A	1996-09-13
9716342	United States of America	W	1997-09-15
9716342	United States of America	W	1997-09-15
08712610	–	–	–
PCTUS9716342	–	–	–
US19960712610	–	–	–
WO1997US16342	–	–	–

Members77

Family members, oldest first
Document	Office	Kind
CA2164951A1	Canada	A1	1994-12-22
CA2448302A1	Canada	A1	1994-12-22
WO9429262A1	World Intellectual Property Organization (WIPO)	A1	1994-12-22
AU7110994A	Australia	A	1995-01-03
US5441951A	United States of America	A	1995-08-15
EP0703897A1	European Patent Office (EPO)	A1	1996-04-03
JPH08512023A	Japan	A	1996-12-17
US5648512A	United States of America	A	1997-07-15
CA2265708A1	Canada	A1	1998-03-19
WO9811049A1	World Intellectual Property Organization (WIPO)	A1	1998-03-19
AU4271097AThis record	Australia	A	1998-04-02
AU692453B2	Australia	B2	1998-06-11
EP0927150A1	European Patent Office (EPO)	A1	1999-07-07
US6048897A	United States of America	A	2000-04-11
AU723321B2	Australia	B2	2000-08-24
JP2001500866A	Japan	A	2001-01-23
US2001031882A1	United States of America	A1	2001-10-18
US6316648B1	United States of America	B1	2001-11-13
US2002010351A1	United States of America	A1	2002-01-24
EP0927150B1	European Patent Office (EPO)	B1	2002-04-10
AT215924T	Austria	T	2002-04-15
ATE215924T1	Austria	T1	2002-04-15
EP1201639A2	European Patent Office (EPO)	A2	2002-05-02
DE69711894D1	Germany	D1	2002-05-16
US2002082435A1	United States of America	A1	2002-06-27
US2002091279A1	United States of America	A1	2002-07-11
DK0927150T3	Denmark	T3	2002-07-22
US2002111505A1	United States of America	A1	2002-08-15
PT927150E	Portugal	E	2002-08-30
DE69711894T2	Germany	T2	2002-11-14
ES2175460T3	Spain	T3	2002-11-16
EP1201639A3	European Patent Office (EPO)	A3	2003-01-02
US2003032827A1	United States of America	A1	2003-02-13
US6569075B2	United States of America	B2	2003-05-27
CA2265708C	Canada	C	2003-06-10
US2003134901A1	United States of America	A1	2003-07-17
US6620919B2	United States of America	B2	2003-09-16
US6635776B2	United States of America	B2	2003-10-21
US6653493B2	United States of America	B2	2003-11-25
CA2164951C	Canada	C	2004-02-10
US6750360B2	United States of America	B2	2004-06-15
JP2005089472A	Japan	A	2005-04-07
JP2005097314A	Japan	A	2005-04-14
US6887901B1	United States of America	B1	2005-05-03
US2005148668A1	United States of America	A1	2005-07-07
US2006069158A1	United States of America	A1	2006-03-30
EP1657233A1	European Patent Office (EPO)	A1	2006-05-17
US7288569B2	United States of America	B2	2007-10-30
US7294728B2	United States of America	B2	2007-11-13
JP4060366B2	Japan	B2	2008-03-12
EP1201639B1	European Patent Office (EPO)	B1	2008-08-13
AT404522T	Austria	T	2008-08-15
ATE404522T1	Austria	T1	2008-08-15
EP0703897B1	European Patent Office (EPO)	B1	2008-08-20
AT405539T	Austria	T	2008-09-15
ATE405539T1	Austria	T1	2008-09-15
DE69738918D1	Germany	D1	2008-09-25
DE69435125D1	Germany	D1	2008-10-02
PT1201639E	Portugal	E	2008-10-03
DK1201639T3	Denmark	T3	2008-11-24
PT703897E	Portugal	E	2008-11-25
DK0703897T3	Denmark	T3	2008-12-01
ES2312499T3	Spain	T3	2009-03-01
ES2313715T3	Spain	T3	2009-03-01
JP2009073810A	Japan	A	2009-04-09
EP2058293A1	European Patent Office (EPO)	A1	2009-05-13
EP2107050A1	European Patent Office (EPO)	A1	2009-10-07
US2009258945A1	United States of America	A1	2009-10-15
US2009270498A1	United States of America	A1	2009-10-29
US7741369B2	United States of America	B2	2010-06-22
US7825271B2	United States of America	B2	2010-11-02
JP4625310B2	Japan	B2	2011-02-02
JP4625311B2	Japan	B2	2011-02-02
JP2011148815A	Japan	A	2011-08-04
JP4819847B2	Japan	B2	2011-11-24
CA2448302C	Canada	C	2012-10-30
US2012277311A1	United States of America	A1	2012-11-01