Inhibition of TNF-(&agr;)-initiated neutrophil response

The impact of lipoxin A4 (LXA4) and aspirin-triggered-lipoxins (ATL) was investigated in tumor necrosis factor (TNF&agr;)-initiated neutrophil (PMN) responses in vitro and in vivo using metabolically stable LX analogs. At concentrations as low as 1-10 nM, the LXA4 and ATL analogs each inhibited TNF&agr;-stimulated superoxide anion generation and IL-1&bgr; release by human PMN.

BACKGROUND OF THE INVENTION

Lipid and protein mediators of inflammation such as cytokines and chemokines have a profound impact on the formation and actions of each other (Serhan, C. N., J. Z. Haeggstr m, and C. C. Leslie. 1996. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J. 10:1147-1158). In particular, the cytokines TNF and IL-1 play major roles in inflammation, septic shock and tissue injury. PMN perform a range of well-appreciated specialized functions, including chemotaxis, generation of reactive oxygen species and biosynthesis of potent lipid mediators (Weiss, S. J. 1989. Tissue destruction by neutrophils. N. Engl. J. Med. 320:365-376). In this regard, TNF stimulates PMN to transcribe and release cytokines such as IL-1 , enhances leukotriene biosynthesis, and up-regulates adhesion molecules (Marucha, P. T., R. A. Zeff, and D. L. Kreutzer. 1991. Cytokine-induced IL-1 gene expression in the human polymorphonuclear leukocyte: transcriptional and post-transcriptional regulation by tumor necrosis factor and IL-1. J. Immunol. 147:2603-2608). Since PMN represent approximately 70% of the peripheral blood leukocytes and are in many instances the initial cell type recruited to interstitial sites, they are now considered a significant source of proinflammatory cytokines including TNF and IL-1 . These as well as other PMN-derived cytokines and chemokines can, in turn, affect the course of inflammatory and immune responses (Lloyd, A. R., and J. J. Oppenheim. 1992. Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunology Today 13:169-172). In certain clinical settings, including respiratory distress syndrome, myocardial reperfusion injury, gout and rheumatoid arthritis, PMN contribute to ongoing damage of host tissues (Weiss, S. J. 1989. Tissue destruction by neutrophils. N. Engl. J Med. 320:365-376; Hachicha, M., P. H. Naccache, and S. R. McColl. 1995. Inflammatory microcrystals differentially regulate the secretion of macrophage inflammatory protein-1 and interleukin-8 by human neutrophils: A possible mechanism of neutrophil recruitment to sites of inflammation in synovitis. J. Exp. Med. 182:2019-2025; Hansen, P. R. 1995. Role of neutrophils in myocardial ischemia and reperfusion. Circulation 91:1872-1885). Thus, it is of interest to understand the complex relationships between lipid mediators and TNF -evoked PMN responses in order to gain insight for new approaches in controlling these events.

SUMMARY OF THE INVENTION

The present invention pertains to methods for modulating a disease or condition associated with TNF initiated polymorphoneutrophil (PMN) inflammation. The methods include administration to a subject, an effective anti-inflammatory amount of a lipoxin analog having the formula described infra, such that the TNF initated PMN inflammation is modulated.

The present invention also pertains to methods for treating TNF initiated polymorphoneutrophil (PMN) inflammation in a subject. The methods include administration of an effective anti-inflammatory amount of a lipoxin analog described infra, such that TNF initiated polymorphoneutrophil (PMN) inflammation is treated.

The present invention further pertains to methods for modulating a disease or condition associated with TNF initiated cytokine activity in a subject. The methods include, administration of an effective anti-TNF amount of a lipoxin analog described infra, such that a disease or condition associated with TNF initiated cytokine activity, is modulated.

The present invention further relates to methods for treating TNF initiated cytokine activity in a subject. The methods include administration of an effective anti-TNF amount of a lipoxin analog described infra, such that TNF initiated cytokine activity, e.g., inflammation, is treated.

The present invention also relates to methods for modulating a disease or condition associated with TNF initiated IL-1 activity in a subject. The methods include administration of an effective anti-inflammatory amount of a lipoxin analog described infra, such that a disease or condition associated with TNF initiated IL-1 , is modulated.

The present invention further pertains to methods for treating TNF initiated IL-1 activity in a subject. The methods include administration of an effective anti-TNF amount of a lipoxin analog described infra, such that TNF initiated IL-1 activity, e.g., inflammation, is treated.

In preferred embodiments, the methods of the invention are performed in vitro or in vivo.

In another aspect, the present invention is directed to a packaged pharmaceutical composition for treating a the activity or conditions listed above in a subject. The packaged pharmaceutical composition includes a container holding a therapeutically effective amount of at least one lipoxin compound having one of the formulae described infra and instructions for using the lipoxin compound for treating the activity or condition in the subject.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.

The impact of lipoxin A 4 (LXA 4 ) and aspirin-triggered-lipoxins (ATL) was investigated in tumor necrosis factor (TNF )-initiated neutrophil (PMN) responses in vitro and in vivo using metabolically stable LX analogs. At concentrations as low as 1-10 nM, the LXA 4 and ATL analogs each inhibited TNF -stimulated superoxide anion generation and IL-1 release by human PMN. These LXA 4 -ATL actions were time- and concentration-dependent and proved selective for TNF , since these responses were not altered with either GM-CSF or zymosan-stimulated cells. TNF -induced IL-1 gene expression was also regulated by both anti-LXA 4 -receptor antibodies and LXA 4 -ATL analogs. In murine air pouches, 15 R/S-methyl-LXA 4 dramatically inhibited TNF -stimulated leukocyte trafficking, as well as the appearance of both macrophage inflammatory peptide-2 and IL-1 , while concomitantly stimulating IL-4 in pouch exudates. Together, these results indicate that both LXA 4 and ATL regulate TNF directed neutrophil actions in vitro and in vivo and stimulate IL-4 in exudates, which plays a pivotal role in immune responses.

Transcellular biosynthesis of lipoxin A 4 during adhesion of platelets and neutrophils in experimental immune complex glomerulonephritis. Kidney Int. 47:1295-1302). LXA 4 is also present in nasal lavage fluids of aspirin-sensitive asthmatics and is generated by leukocytes from patients with asthma and rheumatoid arthritis (Chavis, C., I. Vachier, P. Chanez, J. Bousquet, and P. Godard. 1996. 5(S),15(S)-Dihydroxyeicosatetraenoic acid and lipoxin generation in human polymorphonuclear cells: dual specificity of 5-lipoxygenase towards endogenous and exogenous precursors. J. Exp. Med. 183:1633-1643; Thomas, E., J. L. Leroux, F. Blotman, and C. Chavis. 1995. Conversion of endogenous arachidonic acid to 5,15-diHETE and lipoxins by polymorphonuclear cells from patients with rheumatoid arthritis. Inflamm. Res. 44:121-124). Like most autacoids and lipid mediators, LX are rapidly biosynthesized, act within a local microenvironment, and are rapidly enzymatically inactived. To advance the understanding of LX and ATL roles in vivo, metabolically stable LX analogs were designed that resist rapid inactivation and mimic the in vitro actions of naturally occurring LX and ATL (Serhan, C. N., J. F. Maddox, N. A. Petasis, I. Akritopoulou-Zanze, A. Papayianni, H. R. Brady, S.P. Colgan, and J. L. Madara. 1995. Design of lipoxin A 4 stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 34:14609-14615). It has been unexpectedly discovered that these compounds are potent inhibitors of TNF -driven PMN-associated inflammatory events in vitro as well as in vivo. Moreover, LXA 4 -ATL inhibit MIP-2 and IL-1 yet stimulate the local appearance of IL-4 within exudates.

The present invention pertains to methods for modulating a disease or condition associated with TNF initiated polymorphoneutrophil (PMN) inflammation. The methods include administration to a subject, an effective anti-inflammatory amount of a lipoxin analog having the formula described infra, such that the TNF initated PMN inflammation is modulated.

The present invention also pertains to methods for treating TNF initiated polymorphoneutrophil (PMN) inflammation in a subject. The methods include administration of an effective anti-inflammatory amount of a lipoxin analog described infra, such that TNF initiated polymorphoneutrophil (PMN) inflammation is treated.

The present invention further pertains to methods for modulating a disease or condition associated with TNF initiated cytokine activity in a subject. The methods include, administration of an effective anti-TNF amount of a lipoxin analog described infra, such that a disease or condition associated with TNF initiated cytokine activity, is modulated.

The present invention further relates to methods for treating TNF initiated cytokine activity in a subject. The methods include administration of an effective anti-inflammatory amount of a lipoxin analog described infra, such that TNF initiated cytokine activity, e.g., inflammation, is treated.

The present invention also relates to methods for modulating a disease or condition associated with TNF initiated IL-1 activity in a subject. The methods include administration of an effective anti-TNF amount of a lipoxin analog described infra, such that a disease or condition associated with TNF initiated IL-1 , is modulated.

The present invention further pertains to methods for treating TNF initiated IL-1 activity in a subject. The methods include administration of an effective anti-TNF amount of a lipoxin analog described infra, such that TNF initiated IL-1 activity, e.g., inflammation, is treated.

In preferred embodiments, the methods of the invention are performed in vitro or in vivo.

In another aspect, the present invention is directed to a packaged pharmaceutical composition for treating a the activity or conditions listed above in a subject. The packaged pharmaceutical composition includes a container holding a therapeutically effective amount of at least one lipoxin compound having one of the formulae described infra and instructions for using the lipoxin compound for treating the activity or condition in the subject.

In one embodiment, compounds useful in the invention have the formula (I)

wherein X is R 1 , OR 1 , or SR 1 ;

wherein R 1 is

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms;

(iv) an aralkyl of 7 to 12 carbon atoms;

wherein Z i , Z i , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein r x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;

wherein Q 1 is (C O), SO 2 or (CN), provided when Q 1 is CN, then X is absent;

wherein Q 3 and Q 4 are each independently O, S or NH;

wherein one of R 2 and R 3 is a hydrogen atom and the other is

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

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or

(e) R a Q 2 R b wherein Q 2 is O or S ; wherein R a is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when R b is 0, then R b is a hydrogen atom;

wherein R 4 is

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

wherein R 5 is

wherein Z i , Z ii , Z iii , Z iv , and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR, wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;

wherein Y 1 , is OH, methyl, SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH a Z b where a b 3, a 0 to 3, b 0 to 3 and Z is cyano, nitro or a halogen;

wherein R 6 is

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

wherein T is O or S, and pharmaceutically acceptable salts thereof.

In another embodiment, compounds useful in the invention have the formula (II)

wherein X is R 1 , OR 1 , or SR 1 ;

wherein R 1 is

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms;

(iv) an aralkyl of 7 to 12 carbon atoms;

wherein Z i , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;

wherein Q 1 is (C O), SO 2 or (CN), provided when Q 1 is CN, then X is absent;

wherein one of R 2 and R 3 is a hydrogen atom and the other is

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

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or

(e) R a Q 2 R b wherein Q 2 is O or S ; wherein R a is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when R b is 0, then R b is a hydrogen atom;

wherein R 4 is

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

wherein R 5 is

wherein Z i , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R a , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;

wherein Y 1 is OH, methyl, SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH a Z b where a b 3, a 0 to 3, b 0 to 3 and Z is cyano, nitro or a halogen;

wherein R 6 is

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

wherein T is O or S, and pharmaceutically acceptable salts thereof.

The invention is also directed to useful lipoxin compounds having the formula (III)

wherein X is R 1 , OR 1 , or SR 1 ;

wherein R 1 is

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms;

(iv) an aralkyl of 7 to 12 carbon atoms;

wherein Z 1 , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;

wherein Q 1 is (C O), SO 2 or (CN), provided when Q 1 is CN, then X is absent;

wherein one of R 2 and R 3 is a hydrogen atom and the other is

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

(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or

(e) R a Q 2 R b wherein Q 2 is O or S ; wherein R b is alkylene of 0 to 6 carbons atoms, inclusive, which may be straight chain or branched and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when R b is 0, then R b is a hydrogen atom;

wherein R 4 is

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

wherein R b is

wherein Z 1 , Z ii , Z iii , Z iv , and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;

wherein R 6 is

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

wherein T is O or S, and pharmaceutically acceptable salts thereof.

The invention is further directed to useful lipoxin compounds having the formula (IV)

wherein X is R 1 , OR 1 , or SR 1 ;

wherein R 1 is

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms;

(iv) an aralkyl of 7 to 12 carbon atoms;

wherein Z i , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;

wherein Q 1 is (C O), SO 2 or (CN), provided when Q 1 is CN, then X is absent;

wherein R 4 is

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

wherein R 5 is

wherein Z i , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;

wherein R 6 is

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

wherein T is O or S, and pharmaceutically acceptable salts thereof.

The invention is further directed to useful lipoxin compounds having the formula (V)

wherein X is R 1 , OR 1 , or SR 1 ;

wherein R 1 is

(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain or branched;

(iii) a cycloalkyl of 3 to 10 carbon atoms;

(iv) an aralkyl of 7 to 12 carbon atoms;

wherein Z 1 , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, Or x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;

(vii) a detectable label molecule; or

(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;

wherein R 4 is

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

wherein R 5 is

wherein Z i , Z ii , Z iii , Z iv and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;

wherein R 6 is

(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; and

In preferred embodiments, X is OR 1 wherein R 1 is a hydrogen atom, an alkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptable salt, Q 1 is C O, R 2 and R 3 , if present, are hydrogen atoms, R 4 is a hydrogen atom or methyl, Q 3 and Q 4 , if present, are both O, R 6 , if present, is a hydrogen atom, Y 1 , if present, is OH, T is O and R 5 is a substituted phenyl, e.g.,

wherein Z i , Z ii , Z iii , Z iv , and Z v are each independently selected from NO 2 , CN, C( O) R 1 , SO 3 H, a hydrogen atom, halogen, methyl, OR x , wherein R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl. In certain embodiments for R 5 , para-fluorophenyl and unsubstituted phenyl are excluded, e.g., 15-epi-16-(para-fluoro)-phenoxy-LXA 4 , 15-epi-16-phenyoxy-LXA 4 , 16-(para-fluoro)-phenoxyl-LXA 4 , and/or 16-phenoxy-LXA 4 . The compounds encompassed by U.S. Pat. No. 5,441,951 are excluded from certain aspects of the present invention.

In preferred embodiments, Y 1 , is a hydroxyl and the carbon bearing the hydroxyl can have an R or S configuration. In most preferred embodiments, the chiral carbon bearing the hydroxyl group, e.g., Y 1 , is designated as a 15-epi-lipoxin as is known in the art.

In certain embodiments the chirality of the carbons bearing the R 2 , R 3 , Q 3 and Q 4 groups can each independently be either R or S. In preferred embodiments, Q 3 and Q 4 have the chiralities shown in structures II, III, IV or V.

In preferred embodiments, R 4 is a hydrogen. In other preferred embodiments, R 6 is a hydrogen.

Additionally, R 5 can be a substituted or unsubstituted, branched or unbranched alkyl group having between 1 and about 6 carbon atoms, preferably between 1 and 4 carbon atoms, most preferably between 1 and 3, and preferably one or two carbon atoms. The carbon atoms can have substituents which include halogen atoms, hydroxyl groups, or ether groups.

The compounds useful in the present invention can be prepared by the following synthetic scheme:

In still another aspect, the present invention is directed to pharmaceutical compositions including compounds having the above-described formulae and a pharmaceutically acceptable carrier. In one embodiment, a preferred compound is

In a preferred embodiment, the pharmaceutical carrier is not a ketone, e.g., acetone.

In one embodiment, the antiinflammatories of the invention can be incorporated into a shampoo or a body cleansing product, e.g., a soap, for cleansing of the scalp and/or body. The use of these compounds in a shampoo or soap product can be used to treat psoriasis, seborrheic dermatitis, pustular dermatosis and dandruff. Thus the compounds are useful for modulating TNF PMN or cytokine inflammation associated with such conditions.

A lipoxin analog shall mean a compound which has an active region that functions like the active region of a natural lipoxin , but which has a metabolic transformation region that differs from natural lipoxin. Lipoxin analogs include compounds which are structurally similar to a natural lipoxin, compounds which share the same receptor recognition site, compounds which share the same or similar lipoxin metabolic transformation region as lipoxin, and compounds which are art-recognized as being analogs of lipoxin. Lipoxin analogs include lipoxin analog metabolites. The compounds disclosed herein may contain one or more centers of asymmetry. Where asymmetric carbon atoms are present, more than one stereoisomer is possible, and all possible isomeric forms are intended to be included within the structural representations shown. Optically active (R) and (S) isomers may be resolved using conventional techniques known to the ordinarily skilled artisan. The present invention is intended to include the possible diastereiomers as well as the racemic and optically resolved isomers.

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 LXA 4 specific receptor on differentiated HL-60 cells, the corresponding lipoxin is LXA 4 . Where an analog has activity as an antagonist to another compound (such as a leukotriene), which is antagonized by a naturally-occurring lipoxin, that natural lipoxin is the corresponding lipoxin.

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 A 4 analogs have an active region comprising C 5 -C 15 of natural lipoxin A 4 . Preferred lipoxin B 4 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 or permeabilized 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 LXA 4 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 ( 3 H), deuterium ( 2 H), carbon ( 14 C), 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 chemistry.

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 LXA 4 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 LXA 4 analog which more slowly undergoes metabolism is a structure which has a higher transition state energy for C-15 dehydrogenation than does LXA 4 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. In certain aspects, the compounds of the invention do not include halogenated compounds, e.g., fluorinated compounds.

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

When the compounds of the present invention are administered as pharmaceuticals, to humans and mammals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

In certain embodiment, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term pharmaceutically acceptable salts in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

The term pharmaceutically acceptable esters refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. In a preferred embodiment, the ester is not a methyl ester (See, for example, Berge et al., supra.).

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

The phrases systemic administration, administered systematically, peripheral administration and administered peripherally as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 0.1 to about 40 mg per kg per day. For example, between about 0.01 microgram and 20 micrograms, between about 20 micrograms and 100 micrograms and between about 10 micrograms and 200 micrograms of the compounds of the invention are administered per 20 grams of subject weight.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.

Materials and Methods

Human and mouse recombinant TNF and human recombinant GM-CSF were obtained from Boehringer Mannheim (Indianapolis, Ind.). Dulbecco's PBS (Mg and Ca -free), RPMI-1640 and FCS were purchased from Bio whittaker Inc. (Walkersville, Md.). Ficoll-Hypaque was from Organon Teknika Corp. (Durham, N.C.) and Hank's balanced salt solution was purchased from Gibco BRL (Grand Island, N.Y.). Serum bovine albumin, dextran, antibiotics, L-glutamine, cytochrome C, superoxide dismutase and zymosan were obtained from Sigma (St. Louis, Mo.). The assessment of human IL-1 in supernatants was performed by using an immunometric assay with acetylcholine esterase (Cayman Chemical, Ann Arbor, Mich.). Murine IL-1 was assessed using an ELISA from Endogen (Woburn, Mass.). ELISAs for IL-4 and IL-10 were from Amersham (Arlington Heights, Ill.); MIP-2 and IL-13 ELISAs were from R&D Systems (Minneapolis, Minn.). LXA 4 and ATL metabolically stable analogs were prepared and characterized, including nuclear magnetic resonance spectroscopy, as in (14). Concentrations of each LX analog were determined using an extinction coefficient of 50,000 M 1 .cm 1 just prior to each experiment. Where indicated, statistical analyses were performed using Student's non-paired t-test (two-tailed), and significance (*) was considered to be attained when P was <0.05.

Preparation of Human PMN Suspensions and Superoxide-anion Generation

Venous blood from healthy donors was collected using sterile conditions using acid citrate dextrose (ACD) as an anticoagulant, and PMN were isolated as in (15). PMN were suspended in cold (4 C.) Hank's medium (supplemented with 1.6 mM Ca , 0.1% FCS, 2 mM L-glutamine, 1% penicillin, and 2% streptomycin, pH 7.4). Cell preparations were >98% PMN as determined by Giemsa-Wright staining; cell viability was >98% as determined by trypan blue exclusion and light microscopy. To examine superoxide production, PMN (1.0 10 6 /ml) were placed at 37 C. (3 min) and then exposed to either vehicle (0.1% ethanol) or synthetic LXA 4 , 15R/S-methyl LXA 4 or 16-phenoxy-LXA 4 for 5 min at 37 C. Before adding TNF (50 ng/ml), PMN were incubated with cytochrome C (0.7 mg/ml) for 10 min at 37 C. Superoxide dismutase-dependent reduction of cytochrome C was terminated by rapidly placing tubes in an ice-water bath. The extent of cytochrome C reduction in each supernatant was determined at 550 nm in reference to control values obtained when superoxide dismutase was added before a stimulus or vehicle control. Cytochrome C reduction was quantitated using the extinction coefficient of 21.1/mmol/L.

RNA Isolation and Northern Blot Analysis

Total RNA extraction and Northern blot analyses were performed as in (7). pSM320 vector containing cDNA for IL-1 was purchased from ATCC.

Murine Air Pouches

Six to eight week old male BALB/c mice were obtained from Taconic Farms (Germantown, N.Y.). Air pouches were raised on the dorsum by s.c. injection of 3 ml of sterile air on day 0 and day 3. All experiments were conducted on day 6 (16). Individual air pouches (one per mouse) were injected with either vehicle alone (0.1% ethanol), TNF , 15 R/S-methyl-LXA 4 or TNF plus 15 R/S-methyl-LXA 4 , and each was suspended in 1 ml endotoxin-free PBS immediately before injection into pouch cavities. At given intervals, the mice were sacrificed and individual air pouches were lavaged three times with sterile PBS (1 ml). The exudates were centrifuged at 2000 RPM (5 min) and the supernatants were removed. Cell pellets were suspended in PBS (200 l) for enumeration and assessed for viability. Fifty l of each cell suspension was mixed with 150 l 30% BSA and then centrifuged onto microscope slides at 500 RPM for 5 min using a cytospin centrifuge, air dried, and stained with Giemsa-Wright.

Inhibition of TNF -stimulated Superoxide Generation

Suppression of TNF -stimulated IL-1 Release

PMN express and release interleukin-1 , which is a potent proinflammatory cytokine (Dinarello, C. A. 1996. Biologic basis for interleukin-l in disease. Blood 87:2095-2147). Therefore, the actions of native LXA 4 and its analogs on TNF -induced IL-1 release were investigated. Incubation of PMN with physiologically relevant concentrations of TNF , GM-CSF or phagocytic particles (zymosan) resulted in a concentration dependent increase in the levels of IL-1 present in supernatants. Approximate EC 50 for each agonist were: TNF , 10 ng/ml; GM-CSF, 10 U/ml; and zymosan, 100 g/ml. Native LXA 4 specifically inhibited TNF -induced IL-1 release (FIG. 2 A), while similar amounts of IL-1 were released in the presence or absence of LXA 4 when PMN were exposed to either GM-CSF or zymosan.

PMN were exposed to increasing concentrations of 15 R/S-methyl-LXA 4 , 16-phenoxy-LXA 4 or native LXA 4 in the presence of TNF (10 ng/ml) or vehicle alone. At a concentration of 100 nM, 15 R/S-methyl-LXA 4 inhibited 60% of IL-1 release, and 16-phenoxy-LXA 4 at equimolar levels gave approximately 40% inhibition (values comparable to those obtained with native LXA 4 ). Time course and concentration dependence were carried out with 15 R/S-methyl LXA 4 (FIG. 2 B). At 10 nM, 15 R/S-methyl-LXA 4 gave clear statistically significant inhibition, which was evident within 6 h and more prominent after 24 h (FIG. 2 B). Inhibition of IL-1 by these LX analogs was, at least in part, the result of a down-regulation in gene expression, since the IL-1 messenger RNA levels in cells treated with TNF (10 ng/ml) plus 15 R/S-methyl-LXA 4 (100 nM) were decreased by approximately 60% when compared to cells treated with TNF alone (FIG. 3 ). Therefore, since IL-1 and TNF are two cytokines that are considered important in inflammation, the inhibition of IL-1 observed ( FIGS. 1 & 2 ) suggested that 15 R/S-methyl-LXA 4 might exert a potent in vivo anti-cytokine action (vide infra).

Involvement of LXA 4 Receptor

To investigate whether the LXA 4 receptor (LXA 4 -R) was involved in the regulation of TNF -stimulated IL-1 release, the rabbit polyclonal antibodies against a portion of the third extracellular domain (ASWGGTPEERLK) of LXA 4 -R prepared earlier were used (Fiore, S., and C. N. Serhan. 1995. Lipoxin A 4 receptor activation is distinct from that of the formyl peptide receptor in myeloid cells: inhibition of CD11/18 expression by lipoxin A 4 -lipoxin A 4 receptor interaction. Biochemistry 34:16678-16686). PMN were incubated with 50 g/ml of either preimmune protein-A purified IgG or IgG directed against LXA 4 -R for 1 h at 4 C. prior to exposure to TNF (10 ng/ml) and 15 R/S-methyl-LXA 4 (100 nM). Anti-LXA 4 -R antibodies prevented IL-1 release by TNF , suggesting that the third extracellular loop plays a crucial role in LXA 4 receptor activation (FIG. 4 ). 15 R/S-methyl-LXA 4 inhibited about 50% of IL-1 release. When added together, anti-LXA 4 -R antibodies and 15R/S-methyl-LXA 4 in the presence of TNF did not further inhibit IL-1 appearance, and neither anti-LXA 4 -R antibodies nor 15 R/S-LXA 4 alone stimulated significant amounts of IL-1 to appear within supernatants. The results of these experiments are two fold: first, they indicated that the inhibitory action of 15 R/S-methyl-LXA 4 is transduced via the LXA 4 receptor and second, that the anti-LXA 4 -R antibodies alone activate the LXA 4 receptor and lead to inhibition of IL-1 release.

Inhibition of TNF -directed Leukocyte Trafficking In Vivo

Since TNF evokes leukocyte infiltration in a chemokine-dependent fashion in the murine six day air pouch, the impact of 15 R/S-methyl-LXA 4 in this model was evaluated to determine whether LXA 4 or ATL also intersects the cytokine-chemokine axis in vivo (Tessier, P. A., P. H. Naccache, I. Clark-Lewis, R. P. Gladue, K. S. Neote, and S. R. McColl. 1997. Chemokine networks in vivo: involvement of C-X-C and C-C chemokines in neutrophil extravasation in vivo in response to TNF- . J. Immunol. 159:3595-3602; Sin, Y. M., A. D. Sedgwick, E. P. Chea, and D. A. Willoughby. 1986. Mast cells in newly formed lining tissue during acute inflammation: a six day air pouch model in the mouse. Ann. Rheum. Dis. 45:873-877). 15 R/S-methyl-LXA 4 is the most subtle modification to native LXA 4 and ATL structure with addition of a methyl at carbon 15. Murine TNF (10 ng/ml) caused a transient infiltration of leukocytes to the air pouch in a time-dependent fashion with maximal accumulation at 4 h. 15 R/S-methyl-LXA 4 at 25 moles inhibited the TNF -stimulated recruitment of leukocytes to the air pouch by 62% (FIG. 5 ). Inhibition was evident at 1 h, and maximal between 2 h and 4 h. At these intervals, a more than 60% reduction in leukocyte infiltration was noted that remained significantly reduced at 8 h ( FIG. 5 , insert). Injection of pouches with either vehicle or the analog alone did not cause a significant leukocyte infiltration. Also, inflammatory exudates were collected 4 h after injection with vehicle alone, TNF , 15 R/S-methyl-LXA 4 alone, or TNF plus 15 R/S-methyl-LXA 4 , and cell types were enumerated. In the six-day pouches given TNF , PMN constituted the major cell type present within the exudates at 4 h and ranged from 80 to 85% of total cell number. Administration of both 15 R/S-methyl-LXA 4 and TNF into the six day air pouch cavity inhibited migration of PMN and eosinophils/basophils as well as mononuclear cells (Table I). Of interest, administration of 15 R/S-methyl-LXA 4 alone evoked a small but statistically significant increase in mononuclear cell influx (Table I), a result which is consistent with earlier in vitro observations in which specific stimulation of monocyte and inhibition of PMN chemotaxis have been observed (Maddox, J. F., M. Hachicha, T. Takano, N. A. Petasis, V. V. Fokin, and C. N. Serhan. 1997. Lipoxin A 4 stable analogs are potent mimetics that stimulate human monocytes and THP-1 cells via a G-protein linked lipoxin A 4 receptor. J. Biol. Chem. 272:6972-6978).

Since MIP-2 is the major chemokine involved in recruiting PMN to the air pouch following injection of TNF , the action of 15 R/S-methyl-LXA 4 in this TNF -induced chemokine/cytokine axis was determined. MIP-2 and IL-1 are important pro-inflammatory cytokines, and IL-4, IL-10 and IL-13 possess immunomodulatory properties (Isomaki, P., and J. Punnonen. 1997. Pro- and anti-inflammatory cytokines in rheumatoid arthritis. Ann. Med. 29:499-507; Volpert, O. V., T. Fong, A. E. Koch, J. D. Peterson, C. Waltenbaugh, R. I. Tepper, and N. P. Bouck. 1998. Inhibition of angiogenesis by interleukin 4. J. Exp. Med. 188:1039-1046). Exudates from selected time intervals were collected and cell-free supernatants assessed for the presence of these murine cytokines. TNF induced maximal detectable amounts of MIP-2 and IL-1 within 90 minutes (not shown). 15 R/S-methyl-LXA 4 (25 nmoles) inhibited TNF -stimulated MIP-2 and IL-1 release by 48% and 30% respectively (FIG. 6 ). 15 R/S-methyl-LXA 4 alone in the air pouch did not stimulate MIP-2 or IL-1 release. In sharp contrast, 15 R/S-methyl-LXA 4 stimulated the appearance of IL-4 within the exudates. This stimulation of IL-4 was observed both in the absence as well as presence of TNF . Neither IL-10 nor IL-13 was detected within the pouch exudates. These results demonstrate that administration of 15 R/S-methyl-LXA 4 modified the cytokine-chemokine axis in TNF -initiated acute inflammation, and of interest this re-orientation of the cytokine-chemokine axis paralleled the reduction in leukocyte infiltration.

Several different strategies have been explored in an attempt to attenuate nondesirable action of TNF in inflammatory diseases and ischemia/reperfusion injury including treatment of patients suffering from rheumatoid arthritis with specific Fc portion of monoclonal antibodies directed against TNF -receptor (26). Different steroidal and nonsteroidal drugs to alleviate the pain and the severity of inflammatory responses are extensively used (Marriott, J. B., M. Westby, and A. G. Dalgleish. 1997. Therapeutic potential of TNF- inhibitors old and new. DDT2:273-282). However, certain clinical settings such as reperfusion injury are still not well controlled, and new therapeutic agents are needed. The present results indicate that LXA 4 and ATL, as evidenced by the actions of their metabolically stable analogs (16-phenoxy-LXA 4 and 15 R/S-methyl-LXA 4 ), are potent cytokine-regulating lipid mediators that can also impact the course of inflammation initiated by TNF and IL- . These two cytokines are considered to be key components in orchestrating the rapid inflammatory-like events in ischemia/reperfusion (within minutes to hours), and are major cytokines in rheumatoid arthritis and many other chronic diseases. Of interest, in an exudate and skin wound model, 15 R/S-methyl-LXA 4 not only inhibited the TNF -elicited appearence of IL-1 and MIP-2, but also concomitantly stimulated IL-4 (FIGS. 5 - 6 ). This represents the first observation that lipoxins induce upregulation of a potential anti-inflammatory cytokine such as IL-4. Hence, it is of particular interest that IL-4 inhibits PMN influx in acute antibody-mediated inflammation and inhibits H 2 O 2 production by IFN -treated human monocytes (Saleem, S., Z. Dai, S. N. Coelho, B. T. Konieczny, K. J. M. Assmann, F. K. Baddoura, and F. G. Lakkjs. 1998. IL-4 is an endogenous inhibitor of neutrophil influx and subsequent pathology in acute antibody-mediated inflammation. J. Immunol. 160:979-984; Lehn, M., W. Y. Weiser, S. Engelhorn, S. Gillis, and H. G. Remold. 1989. IL-4 inhibits H 2 O 2 production and antileishmanial capacity of human cultured monocytes mediated by IFN- . J. Immunol. 143:3020-3024). IL-4 is also an active antitumor agent and most recently was shown to be a potent inhibitor of angiogenesis (Volpert, O. V., T. Fong, A. E. Koch, J. D. Peterson, C. Waltenbaugh, R. I. Tepper, and N. P. Bouck. 1998. Inhibition of angiogenesis by interleukin 4. J. Exp. Med. 188:1039-1046). It is thus likely that the increase in IL-4 levels stimulated by metabolically stable LX analogs may mediate in part some of the in vivo impact of LXA 4 and aspirin-triggered 15-epi-LXA 4 , a finding that opens a new understanding of the relationship between antiinflammatory cytokines and lipid mediators.

In conclusion, LXA 4 and aspirin-triggered-LXA 4 appear to be involved in controlling both acute as well as chronic inflammatory responses. The results presented here support the notion that aspirin may exert in part its beneficial action via the biosynthesis of endogenous aspirin-triggered-LXA 4 that can in turn act directly on PMN and/or the appearance of IL-4. Thus, LX-ATL can protect host tissues via multi-level regulation of proinflammatory signals.

REFERENCES

4. Lloyd, A. R., and J. J. Oppenheim. 1992. Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response. Immunology Today 13:169-172.

21. Fiore, S., and C. N. Serhan. 1995. Lipoxin A 4 receptor activation is distinct from that of the formyl peptide receptor in myeloid cells: inhibition of CD11/18 expression by lipoxin A 4 -lipoxin A 4 receptor interaction. Biochemistry 34:16678-16686.

One having ordinary skill in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein, including those in the background section, are expressly incorporated herein by reference in their entirety.