WO2008063772A2

WO2008063772A2 - Resolvin d series and protectin d1 mitigate acute kidney injury

Info

Publication number: WO2008063772A2
Application number: PCT/US2007/080960
Authority: WO
Inventors: Jeremy S. Duffield; Song Hong; Charles N. Serhan; Joseph V. Boneventre
Original assignee: The Brigham And Women's Hospital Inc.
Priority date: 2006-10-13
Filing date: 2007-10-10
Publication date: 2008-05-29
Also published as: WO2008063772A3; WO2008063772A9

Abstract

The use of DHA (docosahexaenoic acid) analogs termed 'resolvins' to treat and, more importantly, to prevent kidney injury is disclosed.

Description

RESOLVIN D SERIES AND PROTECTIN Dl MITIGATE ACUTE KIDNEY INJURY

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims priority to U.S. Provisional Application No. 60/829,407, filed October 13, 2006, the content of which is incorporated in the entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work leading to this invention was supported in part by Senior Fellowship from the National Kidney Research Fund grants DK-73299, DK- 39773, DK-38452, GM-38765, DK-074448, and P50 DE-016191 from the National Institutes of Health. The U.S. Government therefore may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to previously unknown uses of therapeutic agents derived from novel signaling and biochemical pathways that use docosahexaenoic acid (DHA), a polyunsaturated fatty acid (PUFA) as a precursors to the production of bioactive novel endogenous products that control physiologic events in inflammation and resolution in vascular endothelial reactions and neural systems (brain). More specifically, the present invention relates to di- and trihydroxy potent bioactive products termed "Resolvins" and "Protectins" which are derived from polyunsaturated fatty acids. In addition, therapeutic stable analogs of resolvins of the D series (docosahexaenoic acid) and protectins that could enhance their biologic properties are described that can be used to expedite resolution by inhibiting the pro-inflammatory amplification of leukocyte entry.

BACKGROUND OF THE INVENTION

Injury to the kidney, even of a relatively modest degree, is a major health burden accounting for many hospital admissions and high morbidity and mortality (2-4). Although there have been advances in the management of acute kidney injury over the past 50 years, treatment is largely supportive. In many cases, there is complete failure or only partial failure of resolution with important implications for mortality and in some cases progression to end stage renal disease (5). Over the last decade it has been appreciated that acute kidney injury is an inflammatory disease, with inflammation contributing to small vessel congestion and the perpetuation of the functional deficiencies (6-10). Even with repair and regeneration following injury, emerging evidence indicates that post- inflammatory scarring of the kidney may be a major factor in chronic renal disease (11-13). It has also been postulated that ischemic inflammatory kidney injury is a central component of many progressive diseases of the kidney (14-16). In murine models of ischemic and toxic acute kidney injury, the innate immune response plays a key role in disease progression as demonstrated by the protective effect of antibodies blocking integrin and integrin receptor interactions necessary for diapedesis of leukocytes as well as the protective effect of depletion of complement factors such as C5a (6, 8). Similar to the innate inflammatory response in other settings, neutrophils predominate in the first 24h of injury, being replaced subsequently by monocytes/macrophages and T cells (6, 17-19).

To date, several compounds have been shown in animal models to ameliorate acute kidney injury, but in clinical trials these reagents have had limited efficacy (20-23). One of the features of injury to the kidney may be redundancy in pro-inflammatory pathways. Therefore blockade of one pathway and/or target alone may be ineffective. The focus of therapy has to date been primarily directed at pro-inflammatory factors and there has been little attention to the understanding of the endogenous factors normally involved in the resolution of inflammation in an attempt to facilitate this process therapeutically.

A need therefore exists for an improved understanding of the function of these materials in physiology as well as the isolation of bioactive agents that can serve to eliminate or diminish various disease states or conditions, such as those associated with kidney injury/inflammation. BRIEF SUMMARY OF THE INVENTION

The therapeutic agents of the invention useful to treat inflammation of the kidney, such as kidney injury include, for example:

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wherein a bond depicted as represents either a cis or trans double bond: wherein P₁, P₂ and P₃, if present, each individually are protecting groups, hydrogen atoms or combinations thereof; wherein Ri, R₂ and R₃, if present, each individually are substituted or unsubstituted, branched or unbranched alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted, branched or unbranched alkylaryl groups, halogen atoms, hydrogen atoms or combinations thereof; wherein Z is -C(O)OR^d, -C(O)NR^CR^C, -C(O)H, -C(NH)NR^CR^C, -C(S)H, -C(S)OR^d, -C(S)NR^CR^C, -CN; each R^a, if present, is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl; each R^b, if present, is a suitable group independently selected from the group consisting of =0, -OR^d, (C1-C3) haloalkyloxy, -OCF₃, =S, -SR^d, =NR^d, =N0R^d, -NR^CR^C, halogen, -CF₃, -CN, -NC, -OCN, -SCN, -NO, -NO₂, =N₂, -N₃, -S(O)R^d, -S(O)₂R^d, -S(O)₂OR^d, -S(O)NR^CR^C, -S(O)₂NR^CR^C, -OS(O)R^d, -OS(O)₂R^d, -OS(O)₂OR^d, -OS(O)₂NR⁰R⁰, -C(O)R^d, -C(0)0R^d, -C(O)NR^CR^C, -C(NH)NR^CR^C, -C(NR^a)NR°R°, -C(NOH)R^a, -C(NOH)NR⁰R⁰, -OC(O)R^d, -0C(0)0R^d, -OC(O)NR⁰R⁰, -OC(NH)NR^CR°, -0C(NR^a)NR^cR^c, -[NHC(0)]_«R^d, -[NR^aC(O)]«R^d, -[NHC(0)]«0R^d, -[NR^aC(0)]«0R^d, -[NHC(O)J_nNR⁰R⁰, -[NR^aC(0)]«NR°R^c, -[NHC(NH)J_nNR⁰R⁰ and -[NR^aC(NR^a)]_nNR°R°; each R°, if present, is independently a protecting group or R^a, or, alternatively, each R^c is taken together with the nitrogen atom to which it is bonded to form a 5 to 8 -membered cycloheteroalkyl or heteroaryl which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R^a or suitable R^b groups; each n, independently, if present, is an integer from O to 3; each R^d, independently, if present, is a protecting group or R^a; in particular, Z is a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile; wherein X, if present, is a substituted or unsubstituted methylene, an oxygen atom, a substituted or unsubstituted nitrogen atom, or a sulfur atom; wherein Q, if present, represents one or more substituents and each Q individually, if present, is a halogen atom or a branched or unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl, alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group; wherein U, if present, is a branched or unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxycarbonyloxy, and aryloxycarbonyloxy group; and pharmaceutically acceptable salts thereof.

In certain embodiments, Z is a pharmaceutically acceptable salt of a carboxylic acid, and in particular is an ammonium salt or forms a prodrug.

In certain embodiments, P₁, P₂, and P₃, if present, each individually are hydrogen atoms and Z is a carboxylic acid or ester. In other embodiments, X is an oxygen atom, one or more P's are hydrogen atoms, and Z is a carboxylic acid or ester. In still other embodiments, Q is one or more halogen atoms, one or more P's are hydrogen atoms, and Z is a carboxylic acid or ester.

In certain embodiments, R₁, R₂ and R₃, if present, are each individually lower alkyl groups, such as methyl, ethyl, and propyl and can be halogenated, such as trifluoromethyl. In one aspect, at least one of Ri, R₂ and R₃, if present, is not a hydrogen atom. Generally, Z is a carboxylic acid and one or more P's are hydrogen atoms.

In certain embodiments, when OP₃ is disposed terminally within the resolvin analog, the protecting group can be removed to afford a hydroxyl. Alternatively, in certain embodiments, the designation of OP₃ serves to denote that the terminal carbon is substituted with one or more halogens, i.e., the terminal C-22 carbon, is a trifluoromethyl group, or arylated with an aryl group that can be substituted or unsubstituted as described herein. Such manipulation at the terminal carbon serves to protect the resolvin analog from omega P450 metabolism that can lead to biochemical inactivation.

In certain embodiments, P₁, P₂, and P₃, if present, each individually are hydrogen atoms and Z is a carboxylic ester. In other embodiments, Pi, P2, and P₃, if present, each individually are hydrogen atoms and Z is not carboxylic acid.

In one aspect, the compounds described herein are isolated and/or purified, in particular, compounds in which P₁, P₂, and P3, if present, each individually are hydrogen atoms and Z is a carboxylic acid, are isolated and or purified.

In certain aspects of the invention, particular compounds are not included; these include the even numbered compounds identified above by Roman numbers, i.e., II, IV, VT, VIII, X, etc. through LVIII.

In one aspect, the resolvins described herein that contain epoxide, cyclopropane, azine, or thioazine rings within the structure also serve as enzyme inhibitors that increase endogenous resolvin levels in vivo and block "pro" inflammatory substances, their formation and action in vivo, such as leukotrienes and/or LTB₄.

Another embodiment of the present invention is directed to pharmaceutical compositions of the novel compounds described throughout the specification useful to treat kidney injury and related conditions.

The present invention also provides methods to treat various disease states and conditions, including for example, kidney injury and related conditions.

The present invention also provides packaged pharmaceuticals that contain the novel mono, di- and tri-hydroxy DHA derivatives described throughout the specification for use in treatment with various kidney disease states and conditions.

While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Endogenous DHA, Resolvin D series, and Protectin Dl in murine renal tissues and plasma 24h after ischemic injury. A, Concentration of DHA in kidney (LH panel) and plasma (RH panel) from sham operated mice, mice 24h following I/R kidney injury (vehicle), and mice 24h following I/R injury with an infusion of DHA. B, The molecular structures of DHA-derived 17S-HDHA, PDl, RvDl and RvD2, and RvD3. C, DHA-derived products in kidney from sham-operated mice, mice 24h post kidney I/R injury, and mice 24h post I/R injury with an infusion of DHA. D, DHA-derived compounds in plasma from sham-operated mice, mice 24h post kidney I/R injury, and mice 24h post I/R injury with an infusion of DHA. Identification and quantification were conducted via LC-UV-MS-MS-based informatics. Mice received 5 μg DHA/g body weight or an equivalent volume of its vehicle. Details are shown in the Methods. Sham-operated mice served as control. Data represent the mean from two independent experiments. E, MS/MS spectrum at m/z 375 showing the production of RvD2 in mouse plasma 24h following ischemia/reperfusion. F, MS/MS spectrum at m/z 359 showing endogenous generation of PDl in mouse kidney 24h after ischemia.

FIGURE 2. Kidneys are partially protected from ischemic injury with Protectinl, Resolvin D series, but not the precursor DHA. A, Mice with renal I/R injury were treated with vehicle (white), with DHA (125 μg/mouse), PDl (3.5 μg/mouse) or the intermediate 17S-HDHA (17.5 μg/mouse), and plasma creatinine was measured 24h following injury. Note that DHA does not protect kidneys from renal injury. B, Mice were treated with PDl (3.5 μg [shaded] or 35 μg/mouse (25)), RvDs (3.5 μg [shaded] or 35 μg/mouse (25)) and plasma creatinine measured 24h later. Note a dose-dependent increase in protection with both compounds. C, Mice were treated with PDl (3.5 μg [shaded] or 35 μg/mouse (25)), RvDs (3.5 μg [shaded] or 35 μg/mouse (25)), and plasma creatinine measured 48h later. Note that at high concentrations both compounds result in more rapid declines in plasma creatinine from 24 to 48 hr than vehicle, but at lower doses RvDs-treated mice show similar plasma creatinine levels at 24h and 48h after injury, albeit in both cases lower than vehicle-treated animals (*P < 0.05, **P < 0.01 compared to vehicle-treated controls).

FIGURE 3. Histological demonstration of protection from ischemic injury by PDl or RvDs. Forty-eight hours following ischemic injury, kidneys were sectioned and stained by the PAS method. At higher magnification (X200) of the outer medullary region, vehicle-treated kidneys show widespread proximal tubular necrosis, with complete loss of tubular cells in some areas, lumina filled with debris and marked flattening of tubular cells in distal tubules and thick ascending limbs. In addition, many apoptotic cells are seen in the distal tubules, and interstitial cell infiltrate is visible. At lower power (X40), the whole outer medullar area shows confluent necrosis as distinguished by pale pink (arrows), and more proximally many tubules show proteinaceous debris (intense pink). By comparison kidneys from mice treated with both PDl and RvDs show much milder disease. Many fewer proximal tubules show necrosis (X200), many tubules have an intact brush border and those with necrosis show small amounts of intratubular debris alongside intact surviving tubule cells. Distal tubules and ascending limbs are normal in appearance without tubule cell flattening and with fewer apoptotic cells. At low power (X40) there is little evidence of necrotic debris or proteinaceous debris.

FIGURE 4. RvDs and PDl reduce myeloperoxidase activity in whole kidney 24h and 48h after ischemic injury. A, Percentage reduction of myeloperoxidase (MPO) activity in kidneys, 24h following I/R, treated with 17S- HDHA (17.5 mg/mouse), PDl or parent compound DHA, compared with vehicle-treated mice undergoing I/R (white bar). B, Percentage reduction in MPO activity in kidneys 48h following I/R treated with PDl (3.5 μg/mouse [shaded] or 35 μg/mouse (52)) or RvDs (3.5 μg/mouse [shaded] or 35 μg/mouse (52)). All values represent mean ± SEM from 3-4 different mice. When MPO levels for RvDs, PDl or DHA treatments were compared with vehicle-treated disease control there was a significant reduction (** P < 0.01).

FIGURE 5. Both neutrophils and monocytes are reduced by PDl and RvDs in the kidney following ischemic injury. Kidney sections 48h following I/R injury were immunolabeled with anti-CDl Ib (myeloid leukocytes), anti- CD68 (macrophages) or anti-GRl antibodies (neutrophils). The entire outer medullary region of sagittal sections was photographed at X200 in serial images and the percentage area occupied by red or green color was measured by computerized morphometry. Compared with vehicle-treated kidneys, both Protectin Dl- and Resolvin D-treated kidneys showed many fewer myeloid leukocytes at 48h following injury, as detected by CDl Ib (upper panels and upper right graph). To determine whether this was due to a reduction in recruitment of neutrophils or monocytes, sections were labeled for monocytes/macrophages (green) detecting CD68 and neutrophils (red) detecting GR-I (lower two immunocytochemistry panels). At 48h both types of leukocyte were present in increased numbers in the vehicle-treated kidneys, and anti-CD68 antibody-labeled interstitial cells predominated slightly over anti-GR-1 -labeled cells. When sections were assessed by morphometry, the area of neutrophils, stained with anti-GR-1 antibodies, was markedly reduced in kidneys treated with both compounds (middle graph) as was the area of monocytes stained with anti- CD68 antibodies (lower graph). Thus, both neutrophil and monocyte recruitment is reduced by both RvDs and Protectin Dl . ** P < 0.01, * P < 0.05 compared to vehicle-treated controls.

FIGURE 6. D series Resolvins and synthesized Resolvin Dl, administered after the onset of ischemic injury partially protect kidneys from ischemic injury. A, Mice with renal I/R injury were treated with vehicle, PDl (10.0 μg), RvDs (10.0 μg) or RvDl (lO.Oμg) ten minutes after both clamps removed from kidneys and plasma creatinine measured 24h later. B, Mice with renal FR injury were treated with vehicle, PDl (10.0 μg), RvDs (10.0 μg) or RvDl (lO.Oμg) ten minutes after both clamps removed from kidneys, and plasma creatinine measured 48h later. Note at this dose RvDs-treated mice experienced lower peak creatinine at 24h compared with vehicle treated mice and synthesized RvDl-treated mice had more marked protection at both 24h and 48h. PDl treated mice at this dose showed no evidence of significant protection at 24h or 48h (*P < 0.05 compared to vehicle-treated controls).

FIGURE 7. D series Resolvins and Protectin Dl do not protect tubular epithelial cells from toxic injury, but can limit macrophage activation. A, Confluent wells of cultured LLC-PKl (pig proximal tubule cells) treated with H2O2 (500 μM) for 6h released similar amounts of LDH into supernatants regardless of prior administration of docosanoids or arachidonic acid (AA) at concentrations ranging from 0.5 nM to 500 nM. Baseline proportionate release of LDH was 0.073 ± 0.019. B, Cultured bone marrow-derived macrophages were activated with LPS, 100 ng/ml, for lOmin followed by administration of docosanoids in doses ranging from 0.05 nM to 50 nM, for 24h. Each (DHA, RvDl, and PDl) gave significant reduction in TNFa release into the medium when applied at a concentration of 50 nM (*P < 0.001). However, only RvDl and PDl showed a dose-dependent reduction in TNFa release, suggesting that only at very high concentrations is DHA able to stop macrophage activation. Control macrophage cultures generated a concentration of 1762pg/ml in response to LPS FIGURE 8. D series Resolvins and Protectin Dl limit interstitial fibrosis that persists following recovery from ischemia-reperfusion kidney injury. A, 15d following I/R injury, with and without treatment, kidneys were assessed for interstitial fibrosis in the outer medulla in 3 μm sections stained with Gomori's trichrome. Note turquoise-stained material in the interstitium corresponding to fibrosis. B, The entire outer medulla of each kidney was imaged sequentially and the area of kidney sections occupied by turquoise-stained material was assessed objectively and blinded analyses were performed using computerized morphometry. * P < 0.05 compared with vehicle.

DETAILED DESCRIPTION

The present invention is drawn to methods for treating or preventing a kidney injury in a subject by administration of a combination of a polyunsaturated fatty acid(s) (PUF A(s)) and aspirin, i.e., polyunsaturated fatty acids including C22:6 (DHA). In one embodiment, the omega fatty acid, e.g., C22:6, and an analgesic, such as aspirin, are administered at two different times.

Resolvins are natural counter regulatory lipid mediators in host defense mechanisms that protect host tissues from effector cell mediated injury and over amplification of acute inflammation to dampen the inflammatory response, i.e., counterregulative. Some known chronic inflammatory diseases may represent the loss of and/or genetically program low resolvin edogenous responders and/or levels. The resolvin analogs described throughout the specification can be used to replace, enhance and/or treat the loss of these substances therapeutically and thereby pharmacologically resolve inflammation by inhibiting leukocyte recruitment and amplification, namely inhibition of the amplification of inflammation.

The present invention is also drawn to methods for treating disease states or conditions that are associated with inflammation (hence "resolving"), the recruitment of neutrophils, leukocytes and/or cytokines are included within the general scope of kidney injury.

Administration of RvDs or PDl to mice prior to the ischemia resulted in a reduction in functional and morphological kidney injury. Initiation of RvDs and RvDl administration 10 min after reperfusion also resulted in protection of the kidney as measured by serum creatinine 24 and 48 hr later. Interstitial fibrosis after I/R was reduced in mice treated with RvDs. Both RvDs and PDl reduced the number of infiltrating leukocytes and blocked toll-like receptor-mediated activation of macrophages. Thus, the renal production of Resolvins and Protectins, a previously unrecognized endogenous anti-inflammatory response, may play an important role in protection against and resolution of acute kidney injury. These data have therapeutic implications for potentiation of recovery from acute kidney injury.

Recently it has been recognized that endogenous anti-inflammatory lipid mediators can be generated which are short-lived autacoids derived from precursor omega-3 fatty acids (24, 25). Production of these mediators can be enhanced by aspirin. These novel families of compounds were termed Resolvins and Protectins (26) since they derive from docosahexaenoic acid (DHA) and act to resolve inflammation (24). During the resolution of inflammation in mice treated with aspirin (ASA), endothelial-neutrophil (PMN) interactions were required for the generation of some of these compounds by human cells (24). PMNs generate both bioactive D series Resolvins and Protectins from DHA and precursors present in exudates (24, 27).

The present invention provides evidence of endogenous production of anti-inflammatory D series resolvin (RvDs) and protectin Dl in the kidney following ischemic acute kidney injury. The precursor DHA is present in kidneys before and after ischemic acute kidney injury. Increased amounts of resolvins and protectins were generated in the post-ischemic kidney, and administration of these compounds protected kidneys from ischemic injury, reducing leukocyte influx and the post-ischemic increase in serum creatinine as well as reducing post-ischemic kidney fibrosis. It is of particular interest that initiation of adminstration of resolvin Dl and RvDs after reperfusion also provide functional protection of the kidney as measured by serum creatinine 24 and 48 hr later. Based on these results, it is believed that endogenous anti-inflammatory compounds directed at resolution of the inflammatory response play an important role in the natural course of acute kidney injury (28)(28). Dysregulation or inadequacy of this response may be responsible for delay in recovery or inability to recover from acute kidney injury as seen in many cases in humans. These newly identified DHA-derived lipid mediators serve as a new paradigm for the design of effective therapeutics to treat patients with acute kidney injury and hasten their recovery improving morbidity and mortality in these patients.

Terms and abbreviations used throughout the specification include:

DHA: docosahexaenoic acid;

17S-HDHA: 17S^r-hydroxy-4Z, IZ, 1OZ, 13Z, \5E, 19Z-docosahexaenoic acid;

LC-UV-MS-MS: liquid chromatography-UV diode array detector-tandem mass spectrometry;

LO: lipoxygenase;

LX: lipoxins;

PDl : Protectin Dl, 10i?,175-dihydroxy-docosa-4Z,7Z,l l_J£',13£,15Z,19Z- hexaenoic acid, also termed neuroprotectin Dl (NPDl) when produced in neural tissues (see ref. (I));

PLP : paraformaldehyde-L-lysine-periodate;

RvDs: Resolvin D series; RvDl: 75,8,175-trihydroxy-docosa-4Z,9£,l l£,13Z,15£,19Z-hexaenoic acid;

RvD2 : 75, 16 , 175-trihydroxy-docosa-4Z, SE, 1 OZ, 1 IE, 14E, 19Z-hexaenoic acid;

RvD3: 45,1 l,17S,-trihydroxy-docosa-5,7£',9£',13Z,15£',19Z-hexaenoic acid;

RvD4: 45,5, 175-trihydroxy-docosa-6_J£',8£', 1 OZ, 13Z, 15E, 19Z-hexaenoic acid;

RvD5 : 75, 175-dihydroxy-docosa-4Z,8.£, 1 OZ, 13Z, 15,E, 19Z-hexaenoic acid;

RvD6: 45,175-dihydroxy-docosa 5£,7Z,10Z,13Z,15£,19Z-hexaenoic acid.

"AlkyJ" by itself or as part of another substituent refers to a saturated or unsaturated branched, straight-chain or cyclic monovalent hydrocarbon radical having the stated number of carbon atoms (i.e., C1-C6 means one to six carbon atoms) that is derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne. Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl, prop-1-yn-l-yl , prop-2-yn-l-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-l-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl , but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-1-en-l-yl, cyclobut-l-en-3-yl, cyclobuta-l,3-dien-l-yl, but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl, etc.; and the like. Where specific levels of saturation are intended, the nomenclature "alkanyl," "alkenyl" and/or "alkynyl" is used, as defined below. In preferred embodiments, the alkyl groups are (C1-C6) alkyl.

"Alkanyi" by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic alkyl derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-l-yl (isobutyl), 2-methyl-propan-2-yl (ϊ-butyl), cyclobutan-1-yl, etc.; and the like. In preferred embodiments, the alkanyl groups are (C1-C6) alkanyl.

"Alkenyl" by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-l-yl , prop-l-en-2-yl, prop-2-en-l-yl, prop-2-en-2-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl ; butenyls such as but-1-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-1-en-l-yl, cyclobut-l-en-3-yl, cyclobuta-l,3-dien-l-yl, etc.; and the like. In preferred embodiments, the alkenyl group is (C2-C6) alkenyl.

"Alkynyl" by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-l-yl , prop-2-yn-l-yl, etc.; butynyls such as but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl , etc.; and the like. In preferred embodiments, the alkynyl group is (C2-C6) alkynyl.

"Alkyldiyl" by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic divalent hydrocarbon group having the stated number of carbon atoms {i.e., C1-C6 means from one to six carbon atoms) derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene or alkyne, or by the removal of two hydrogen atoms from a single carbon atom of a parent alkane, alkene or alkyne. The two monovalent radical centers or each valency of the divalent radical center can form bonds with the same or different atoms. Typical alkyldiyl groups include, but are not limited to, methandiyl; ethyldiyls such as ethan-l,l-diyl, ethan-l,2-diyl, ethen-l.l-diyl, ethen-l,2-diyl; propyldiyls such as propan-l,l-diyl, propan-l,2-diyl, propan-2,2-diyl, propan-l,3-diyl, cyclopropan-l,l-diyl, cyclopropan-l,2-diyl, prop-l-en-l,l-diyl, prop-l-en-l,2-diyl, prop-2-en-l,2-diyl, prop-l-en-l,3-diyl, cycloprop- 1 -en- 1 ,2-diyl, cycloprop-2-en- 1 ,2-diyl, cycloprop-2-en- 1 , 1 -diyl, prop-l-yn-l,3-diyl, etc.; butyldiyls such as, butan- 1,1 -diyl, butan-l,2-diyl, butan-l,3-diyl, butan- 1,4-diyl, butan-2,2-diyl, 2-methyl-propan- 1,1 -diyl, 2-methyl-propan- 1 ,2-diyl, cyclobutan- 1 , 1 -diyl; cyclobutan- 1 ,2-diyl, cyclobutan- 1,3 -diyl, but- 1 -en- 1,1 -diyl, but-l-en-l,2-diyl, but- 1 -en- 1,3 -diyl, but- 1 -en- 1 ,4-diyl, 2-methyl-prop- 1 -en- 1 , 1 -diyl, 2-methanylidene-propan- 1 , 1 -diyl, buta-l,3-dien-l,l-diyl, buta-l,3-dien-l,2-diyl, buta-l,3-dien-l,3-diyl, buta- 1 ,3-dien- 1 ,4-diyl, cyclobut-1 -en-1 ,2-diyl, cyclobut- 1 -en- 1 ,3 -diyl, cyclobut-2-en- 1 ,2-diyl, cyclobuta- 1 ,3-dien- 1 ,2-diyl, cyclobuta- 1 ,3-dien- 1 ,3-diyl, but- 1-yn- 1,3 -diyl, but- 1-yn- 1,4-diyl, buta-l,3-diyn-l,4-diyl, etc.; and the like. Where specific levels of saturation are intended, the nomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Where it is specifically intended that the two valencies are on the same carbon atom, the nomenclature "alkylidene" is used. In preferred embodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also preferred are saturated acyclic alkanyldiyl groups in which the radical centers are at the terminal carbons, e.g., methandiyl (methano); ethan-l,2-diyl (ethano); propan- 1,3 -diyl (propano); butan- 1,4-diyl (butano); and the like (also referred to as alkylenos, defined infra).

"Alkyleno" by itself or as part of another substituent refers to a straight-chain saturated or unsaturated alkyldiyl group having two terminal monovalent radical centers derived by the removal of one hydrogen atom from each of the two terminal carbon atoms of straight-chain parent alkane, alkene or alkyne. The locant of a double bond or triple bond, if present, in a particular alkyleno is indicated in square brackets. Typical alkyleno groups include, but are not limited to, methano; ethylenos such as ethano, etheno, ethyno; propylenos such as propano, prop[l]eno, propa[l,2]dieno. prop[l]yno, etc.; butylenos such as butano, but[l]eno, but[2]eno, buta[l,3]dieno, but[l]yno, but[2]yno, buta[l,3]diyno, etc.; and the like. Where specific levels of saturation are intended, the nomenclature alkano, alkeno and/or alkyno is used. In preferred embodiments, the alkyleno group is (C1-C6) or (C1-C3) alkyleno. Also preferred are straight-chain saturated alkano groups, e.g., methano, ethano, propano, butano, and the like.

"Heteroalkyl," Heteroalkanyl" Heteroalkenyl," Heteroalkynyl," Heteroalkyldiyl" and "Heteroalkyleno" by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkyleno groups, respectively, in which one or more of the carbon atoms are each independently replaced with the same or different heteratoms or heteroatomic groups. Typical heteroatoms and/or heteroatomic groups which can replace the carbon atoms include, but are not limited to. -O-, -S-, -S-O-, -NR'-, -PH-, -S(O)-, -S(O)₂-, -S(Oj NR'-, -S(O)₂NR'-, and the like, including combinations thereof, where each R' is independently hydrogen or (C1-C6) alkyl.

"Cycloalkyl" and "Heterocycloalkyl" by themselves or as part of another substituent refer to cyclic versions of "alkyl" and "heteroalkyl" groups, respectively. For heteroalkyl groups, a heteroarom can occupy the position that is attached to the remainder of the molecule Typical cycloalkyl groups include, but art. not limited to, cyclopropyl: cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyis such as cyclopentanyl and cyclopentenyl; cyclonexyjs such as cyclohexanyl and cyclohexenyl; and the like. Typical heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl (e.g., tetiahydroturan-2- yl, cettah/drofuran-3-yl. etc.), piperidinyl (e.g., piperidin-1-yl, piperidin-2-yl, etc J, morpholinyl (e.g., morpholin-3-yl. morpholin-4-yl, etc.), piperazinyl (e.g., piperazm-i-yl, piperazin-2-yl, etc.), and the like.

"Acyclic Heteroatomic Bridge" refers to a divalent bridge in which the backbone atoms are exclusively heteroatoms and/or heteroatomic groups. Typical acyclic heteroatomic bridges include, but are not limited to, -0-, -S-, -S- O-, -NR^"-, -PH-, -S(O)-, -S(O)₂-. -S(O) NR'-, -S(O)₂NR'-, and the like, including combinations thereof, where each R' is independently hydrogen or (Cl -C6) alkyl. "Parent Aromatic Ring System" refers to an unsaturated cyclic or polycyclic ring system having a conjugated π electron system. Specifically included within the definition of "parent aromatic ring system" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, tetrahydronaphthalene, etc. Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and the like, as well as the various hydro isomers thereof.

"Ary_l" by itself or as part of another substiruent refers to a monovalent aromatic hydrocarbon group having the stated number of carbon atoms (i.e., C5- C15 means from 5 to 15 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, αs-indacene, 5-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like, as well as the various hydro isomers thereof. In preferred embodiments, the aryl group is (C5-C15) aryl, with (C5-C10) being even more preferred. Particularly preferred aryls are cyclopentadienyl, phenyl and naphthyl.

"Arylaryl" by itself or as part of another substiruent refers to a monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a ring system in which two or more identical or non-identical parent aromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. Where the number of carbon atoms in an arylaryl group are specified, the numbers refer to the carbon atoms comprising each parent aromatic ring. For example, (C5-C15) arylaryl is an arylaryl group in which each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parent aromatic ring system of an arylaryl group is independently a (C5-C15) aromatic, more preferably a (C5-C10) aromatic. Also preferred are arylaryl groups in which all of the parent aromatic ring systems are identical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

"Biaryi" by itself or as part of another substituent refers to an arylaryl group having two identical parent aromatic systems joined directly together by a single bond. Typical biaryi groups include, but are not limited to, biphenyl, binaphthyl, bianthracyl, and the like. Preferably, the aromatic ring systems are (C5-C15) aromatic rings, more preferably (C5-C10) aromatic rings. A particularly preferred biaryi group is biphenyl.

"Arylalkyl" by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-l-yl, 2-phenylethen-l-yl, naphthylmethyl, 2-naphthylethan-l-yl, 2-naphthylethen-l-yl, naphthobenzyl, 2-naphthophenylethan-l-yl and the like. Where specific alkyl moieties are intended, the nomenclature arylalkanyl, arylakenyl and/or arylalkynyl is used. In preferred embodiments, the arylalkyl group is (C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C6) and the aryl moiety is (C5-C15). In particularly preferred embodiments the arylalkyl group is (C6-C13), e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) and the aryl moiety is (C5-C10). "Parent Heteroaromatic Ring System" refers to a parent aromatic ring system in which one or more carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Typical heteroatoms or heteroatomic groups to replace the carbon atoms include, but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc. Specifically included within the definition of "parent heteroaromatic ring systems" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Also included in the definition of "parent heteroaromatic ring system" are those recognized rings that include common substituents, such as, for example, benzopyrone and 1-methyl- 1,2,3,4-tetrazole. Typical parent heteroaromatic ring systems include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.

"Heteroaryi" by itself or as part of another substituent refers to a monovalent heteroaromatic group having the stated number of ring atoms (e.g., "5-14 membered" means from 5 to 14 ring atoms) derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryi groups include, but are not limited to, groups derived from acridine, benzimidazole, benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like, as well as the various hydro isomers thereof. In preferred embodiments, the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred.

"Heteroaryl-Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a ring system in which two or more identical or non-identical parent heteroaromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent heteroaromatic ring systems involved. Typical heteroaryl-heteroaryl groups include, but are not limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified, the numbers refer to the number of atoms comprising each parent heteroaromatic ring systems. For example, 5-15 membered heteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which each parent heteroaromatic ring system comprises from 5 to 15 atoms, e.g., bipyridyl, tripuridyl, etc. Preferably, each parent heteroaromatic ring system is independently a 5-15 membered heteroaromatic, more preferably a 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroaryl groups in which all of the parent heteroaromatic ring systems are identical.

"Biheteroaryi" by itself or as part of another substituent refers to a heteroaryl-heteroaryl group having two identical parent heteroaromatic ring systems joined directly together by a single bond. Typical biheteroaryi groups include, but are not limited to, bipyridyl, bipurinyl, biquinolinyl, and the like. Preferably, the heteroaromatic ring systems are 5-15 membered heteroaromatic rings, more preferably 5-10 membered heteroaromatic rings. "Heteroarylalkyl" by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp³ carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/or heteroarylalkynyl is used. In preferred embodiments, the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and the heteroaryl moiety is a 5-15-membered heteroaryl. In particularly preferred embodiments, the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3) alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

"Halogen" or "Halo" by themselves or as part of another substituent, unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

"Haloalkyi" by itself or as part of another substituent refers to an alkyl group in which one or more of the hydrogen atoms is replaced with a halogen. Thus, the term "haloalkyi" is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls. For example, the expression "(C1-C2) haloalkyi" includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1 ,2-difluoroethyl, 1 , 1 , 1 -trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that are commonly used in the art to create additional well-recognized substituent groups. As examples, "alkyloxy" or "alkoxy" refers to a group of the formula -OR", "alkylamine" refers to a group of the formula -NHR" and "dialkylamine" refers to a group of the formula — NR"R", where each R" is independently an alkyl. As another example, "haloalkoxy" or "haloalkyloxy" refers to a group of the formula -OR'", where R'" is a haloalkyi.

"Protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group may be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3^rd Ed., 1999, John Wiley & Sons, NY and Harrison et ah, Compendium of Synthetic Organic Methods, VoIs. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC") and the like. Representative hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is either acylated (esterifled) or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPS groups), glycol ethers, such as ethylene glycol and propylene glycol derivatives and allyl ethers.

Throughout the following descriptions, it should be understood that where particular double bonding is depicted, it is intended to include both cis and trans configurations. Exemplary formulae are provided with specific configurations, but for completeness, the double bonds can be varied. Not every structural isomer is shown in efforts to maintain brevity of the specification. However, this should not be considered limiting in nature. Additionally, where synthetic schemes are provided, it should be understood that all cis/trans configurational isomers are also contemplated and are within the scope and purvue of the synthesis. Again, particular double bonding is depicted in exemplary manner.

The present invention provides compounds and pharmaceutical compositions useful for the treatment of kidney inflammatory conditions, having the formula:

Pi and P₂ each individually are protecting groups, hydrogen atoms or combinations thereof.

Ri and R₂ each individually are substituted or unsubstituted, branched or unbranched alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted, branched or unbranched alkylaryl groups, halogen atoms, hydrogen atoms or combinations thereof.

Z is -C(O)OR^d, -C(O)NR^CR^C, -C(O)H, -C(NH)NR^CR^C, -C(S)H, -C(S)OR^d, -C(S)NR⁰R⁰, -CN; each R^a, if present, is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl; each R^b, if present, is a suitable group independently selected from the group consisting of =0, -OR^d, (C1-C3) haloalkyloxy, -OCF₃, =S, -SR^d, =NR^d, =N0R^d, -NR^CR^C, halogen, -CF₃, -CN, -NC, -OCN, -SCN, -NO, -NO₂, =N₂, -N₃, -S(O)R^d, -S(O)₂R^d, -S(O)₂OR^d, -S(O)NR⁰R⁰, -S(O)₂NR⁰R⁰, -OS(O)R^d, -OS(O)₂R^d, -OS(O)₂OR^d, -OS(O)₂NR⁰R⁰, -C(O)R^d, -C(0)0R^d, -C(O)NR⁰R⁰, -C(NH)NR⁰R⁰, -C(NR^a)NR°R^c, -C(NOH)R³, -C(NOH)NR⁰R⁰, -OC(O)R^d, -0C(0)0R^d, -OC(O)NR⁰R⁰, -OC(NH)NR⁰R⁰, -0C(NR^a)NR°R^c, -[NHC(O)]«R^d, -[NR^aC(0)]_MR^d, -[NHC(0)]_«0R^d, -[NR^aC(0)]_π0R^d, -[NHC(O)]_πNR°R^c, -[NR²C(O)]JSIR⁰R⁰, -[NHC(NH)J_nNR⁰R⁰ and -[NR^aC(NR^a)]«NR⁰R°; each R°, if present, is independently a protecting group or R^a, or, alternatively, each R^c is taken together with the nitrogen atom to which it is bonded to form a 5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R^a or suitable R^b groups; each n, independently, if present, is an integer from O to 3; each R^d, independently, if present, is a protecting group or R^a; and pharmaceutically acceptable salts thereof.

In certain embodiments, Pj and P₂ are hydrogen atoms, Ri and R₂ each individually are methyl groups or hydrogen atoms or combinations thereof, and Z is carboxylic acid or a carboxylic ester.

In particular, Z is a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile.

In certain embodiments Z is carboxylic acid, a carboxylic ester, or pharmaceutically acceptable carboxylic acid salt.

P₁, P₂ and P₃ each individually are protecting groups, hydrogen atoms or combinations thereof and R₁, R₂ and Z are as defined above.

In certain embodiments, Pi, P₂ and P₃ each are hydrogen atoms, Ri and R₂ each individually are methyl groups or hydrogen atoms or combinations thereof and Z is a carboxylic acid or a carboxylic ester.

In certain aspects the designation of OP₃ serves to denote that the terminal carbon is substituted with one or more halogens (I, Cl, F, Br, mono, di or tri substitution) to form, for example, a trifluoromethyl group, or is an aryl group or phenoxy group that can be substituted or unsubstituted as described herein.

In certain embodiments Z is carboxylic acid, a carboxylic ester, or pharmaceutically acceptable carboxylic acid salt. The present invention provides compounds and pharmaceutical compositions useful for the treatment of kidney inflammatory conditions, having the formula:

Pi, P₂, P3, Ri and Z are as defined above.

In an embodiment, Pi, P₂ and P₃ each are hydrogen atoms, Ri is a methyl group or a hydrogen atom and Z is a carboxylic acid or a carboxylic ester.

Pi. ?_2> ?3, R-i and Z are as defined above.

In a particular embodiment, Pi, P₂ and P3 each are hydrogen atoms, Ri is a methyl group or a hydrogen atom and Z is a carboxylic acid or a carboxylic ester. In particular, Z is a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile.

P₁, P₂, P₃ and Z are as defined above.

R₁, Ra and R3, each individually are substituted or unsubstituted, branched or unbranched alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted, branched or unbranched alkylaryl groups, halogen atoms, hydrogen atoms or combinations thereof.

In a particular embodiment, P₁, P₂ and P₃ each are hydrogen atoms, R₁, R₂ and R₃ are each hydrogen atoms and Z is a carboxylic acid or a carboxylic ester.

The present invention also provides compounds and pharmaceutical compositions useful for the treatment of kidney inflammatory conditions, having the formula:

P₁, P₂, R₁, R₂ and Z are as defined above.

In certain embodiments, Pi and P₂ are hydrogen atoms, Ri and R₂ each individually are methyl groups or hydrogen atoms or combinations thereof and Z is carboxylic acid or a carboxylic ester.

The present invention further provides compounds and pharmaceutical compositions useful for the treatment of kidney inflammatory conditions, having the formula:

Pi, P₂, P₃, R₁, R₂ and Z are as defined above.

In a particular embodiment, Pi, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester.

In certain aspects the designation Of OP₃ serves to denote that the terminal carbon is substituted with one or more halogens (I, Cl, F, Br, mono, di or tri substitution) to form, for example, a trifluoromethyl group, or is an aryl group or phenoxy group that can be substituted or unsubstituted as described herein. In particular, Z is a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile.

Pi₅ ?₂, P3_> R] and Z are as defined above.

Q represents one or more substituents and each Q, independently, is a hydrogen atom, a halogen atom or a branched or unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl, aJkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group.

In one particular aspect, P₁, P₂ and P₃ each are hydrogen atoms. Ri is a methyl group or a hydrogen atom, each Q is a hydrogen atom and Z is a carboxylic acid or a carboxylic estei.

In particular, Z is a carboxylic acid, ester, amide, thiocarbamate. carbamate, thioester, thiocarboxamide or a nitrile. In certain embodiments Z is carboxylic acid, a carboxylic ester, or pharmaceutically acceptable carboxylic acid salt.

Pi, P₂, P3, Ri and Z are as defined above.

In one embodiment, P₁, P₂ and P₃ each are hydrogen atoms, Ri is a methyl group or a hydrogen atom, and Z is a carboxylic acid or a carboxylic ester.

Pi, P₂, P₃, Ri, Q and Z are as defined above.

In one particular embodiment, Pi, P₂ and P₃ each are hydrogen atoms, R is a methyl group or a hydrogen atom, each Q is a hydrogen atom and Z is a carboxylic acid or a carboxylic ester.

Pi, P₂, Ri, R₂ and Z are as defined above.

U is a branched or unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxycarbonyloxy, and aryloxycarbonyloxy group.

In one aspect, Pi, and P₂ each are hydrogen atoms, Ri and R₂ each individually are methyl groups or hydrogen atoms or combinations thereof, U is a trifluoromethyl group and Z is a carboxylic acid or a carboxylic ester.

Pi, P₂ and Z are as defined above.

In certain embodiments, Pi and P₂ are hydrogen atoms and Z is carboxylic acid or a carboxylic ester. In certain embodiments, when Pi and P₂ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

The analogs are designated as 7, 17-diHDHAs. In certain aspects, the chiral carbon atom at the 7 position (C-7) has an R configuration. In another aspect, the C-7 carbon atom preferably has an S configuration. In still another aspect, the C-7 carbon atom is as an R/S racemate. Additionally, the chiral carbon atom at the 17 position (C- 17) can have an R configuration. Alternatively, the C- 17 carbon can have an S configuration. In still yet another aspect, the C-17 carbon can preferably exist as an R/S racemate. Exemplary analogs include, for example, IS, Il R/S- diHDHA, 75^r,17R/5-dihydroxy-docosa- 4Z,8E, 1 OZ, 13Z, 1 SE, 19Z-hexaenoic acid.

Pi, P₂, P₃ and Z are as defined above.

In certain embodiments, P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when Pi, P₂ and P₃ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

Pi and Z are as defined above.

X is a substituted or unsubstituted methylene, an oxygen atom, a substituted or unsubstituted nitrogen atom, or a sulfur atom. In one embodiment, Pi is a hydrogen atom, X is an oxygen atom and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when Pi is a hydrogen atom and Z is a carboxylic acid, the compound is either isolated and/or purified.

Pi, P2, P3 and Z are as defined above.

The analogs are designated as 7, 8, 17-trihydroxy-DHAs. In certain embodiments, the chiral carbon atom at the 7 position (C-7) has an R configuration. In other embodiments, the C-7 carbon atom preferably has an S configuration. In still other embodiments, the C-7 carbon atom is as an R/S racemate. In certain aspects, the chiral carbon atom at the 8 position (C-8) has an R configuration. In another aspect, the C-8 carbon atom has an S configuration. In still another aspect, the C-8 carbon atom preferably is as an R/S racemate. Additionally, the chiral carbon atom at the 17 position (C- 17) can have an R configuration. Alternatively, the C-17 carbon can preferably have an S configuration. In still yet another aspect, the C- 17 carbon can exist as an R/S racemate. In a particular embodiment, Pi, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when Pi, P₂ and P₃ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

Pi, P₂, P₃ and Z are as defined above.

In a particular embodiment, Pi, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when Pi, P₂ and P₃ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

The analogs are designated as 7, 16, 17-trihydroxy-DHAs. In certain embodiments, the chiral carbon atom at the 7 position (C-7) has an R configuration. In other embodiments, the C-7 carbon atom preferably has an S configuration. In still other embodiments, the C-7 carbon atom is as an R/S racemate. In certain aspects, the chiral carbon atom at the 16 position (C- 16) has an R configuration. In another aspect, the C- 16 carbon atom has an S configuration. In still another aspect, the C- 16 carbon atom preferably is as an R/S racemate. Additionally, the chiral carbon atom at the 17 position (C-17) can have an R configuration. Alternatively, the C- 17 carbon can preferably have an S configuration. In still yet another aspect, the C- 17 carbon can exist as an R/S racemate.

Pi, X and Z are as defined above.

In one embodiment, Pi is a hydrogen atom, X is an oxygen atom and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when Pi is a hydrogen atom and Z is a carboxylic acid, the compound is either isolated and/or purified.

P₁, P₂, P3 and Z are as defined above.

In a particular embodiment, P₁, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when Pi, P₂ and P₃ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

The analogs are designated as 4, 11, 17-trihydroxy-DHAs. In certain embodiments, the chiral carbon atom at the 4 position (C-4) has an R configuration. In other embodiments, the C-4 carbon atom preferably has an S configuration. In still other embodiments, the C-4 carbon atom is as an R/S racemate. In certain aspects, the chiral carbon atom at the 11 position (C-11) has an R configuration. In another aspect, the C-I l carbon atom has an S configuration. In still another aspect, the C-11 carbon atom preferably is as an R/S racemate. Additionally, the chiral carbon atom at the 17 position (C- 17) can have an R configuration. Alternatively, the C-17 carbon can preferably have an S configuration. In still yet another aspect, the C-17 carbon can exist as an R/S racemate.

P₁, P₂ and Z are as defined above.

In certain embodiments, Pi and P₂ are hydrogen atoms and Z is carboxylic acid or a carboxylic ester. In certain embodiments, when P₁, and P₂ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

Pi, ?2, P3 and Z are as defined above.

In a particular embodiment, Pi, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester. In certain embodiments, when P₁, P₂ and P₃ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

P₁, P₂, P₃, Q and Z are as defined above.

In one particular aspect, Pi, P₂ and P₃ each are hydrogen atoms, each Q is a hydrogen atom and Z is a carboxylic acid or a carboxylic ester.

Pi, P₂, P₃ and Z are as defined above.

In one embodiment, Pi, P₂ and P₃ each are hydrogen atoms and Z is a carboxylic acid or a carboxylic ester.

The present invention still further provides compounds and pharmaceutical compositions useful for the treatment of kidney inflammatory conditions, having the formula:

Pu P_∑; P3) Q and Z are as defined above.

In one particular embodiment, P₁, P₂ and P3 each are hydrogen atoms, each Q is a hydrogen atom and Z is a carboxylic acid or a carboxylic ester.

Pi, P₂, U and Z are as defined above.

In one aspect, Pi, and P2 each are hydrogen atoms, U is a trifluoromethyl group and Z is a carboxylic acid or a carboxylic ester.

The present invention, further pertains to dihydroxy-docosahexaenoic acid analogs useful to treat kidney inflammatory conditions (diHDHA) having the formula

The analogs are designated as 10, 17-diHDHAs. Pi and P₂ are as defined above and can be the same or different. Z is as defined above and in particular can be a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile. The broken double bond line indicates that either the E or Z isomer is within the scope of the analog(s). In certain aspects, the chiral carbon atom at the 10 position (C-IO) has an R configuration. In another aspect, the C-10 carbon atom has an S configuration. In still another aspect, the C-10 carbon atom preferably is as an R/S racemate. Additionally, the chiral carbon atom at the 17 position (C- 17) can have an R configuration. Alternatively, the C- 17 carbon can preferably have an S configuration. In still yet another aspect, the C- 17 carbon can exist as an R/S racemate. In one example, the present invention includes 10,17S-docosatriene, 10,175- dihydroxy-docosa- 4Z,7Z,1 l£,13,15£,19Z-hexaenoic acid analogs such as 1 Oi^-OCH₃, 175-HDHA, 1 OR/S, methoxy- 1 IS hydroxy-docosa-4Z,7Z, 1 IE, 13 , 15.E, 19Z-hexaenoic acid derivatives.

In certain embodiments, when Pi and Pi are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

In still yet another embodiment, the present invention pertains to diHDHA analogs useful to treat kidney inflammatory conditions having the formula

The analogs are designated as 4, 17-diHDHAs. P₁, P₂ and Z are as defined above. Pi and P₂ can be the same or different. In particular, Z can be a carboxylic acid, ester, amide, thiocarbamate, carbamate, thioester, thiocarboxamide or a nitrile. In certain aspects, the chiral carbon atom at the 4 position (C -4) has an R configuration. In another aspect, the C-4 carbon atom preferably has an S configuration. In still another aspect, the C-4 carbon atom is as an R/S racemate. Additionally, the chiral carbon atom at the 17 position (C- 17) can have an R configuration. Alternatively, the C- 17 carbon can have an S configuration. In still yet another aspect, the C- 17 carbon can preferably exist as an R/S racemate.

In certain embodiments, when Pi and P₂ are hydrogen atoms and Z is a carboxylic acid, the compound is either isolated and/or purified.

For example, the present invention includes 4S, Il RJS- diHDHA, 4S, 17R/S'-dihydroxy-docosa-5.E,7Z, 1 OZ, 13Z, 15E, 19Z-hexaenoic acid analogs .

It should be understood that "Z" can be altered from one particular moiety to another by a skilled artisan. In order to accomplish this in some particular instances, one or more groups may require protection. This is also within the skill of an ordinary artisan. For example, a carboxylic ester (Z) can be converted to an amide by treatment with an amine. Such interconversion are known in the art.

In one aspect of the invention, the compound(s) of the invention are substantially purified and isolated by techniques known in the art. The purity of the purified compounds is generally at least about 90%, preferably at least about 95%, and most preferably at least about 99% by weight (100% by weight) based on analytical measurements such has GC, MS, or ¹HNMR, etc. This applies to all isolated and/or purified compounds throughout the specification.

In certain embodiments, the subject compounds are purified, e.g., substantially separated from other compounds or isomers that are present in a cellular environment where resolvins are produced or that are present in crude products of synthetic chemical manufacturing processes. In certain embodiments, a purified compound is contaminated with less than 25%, less than 15%, less than 10%, less than 5%, less than 2%, or even less than 1% of cellular components (proteins, nucleic acids, carbohydrates, etc.), chemical byproducts, reagents, and starting materials, and the like. In certain embodiments, a purified compound is contaminated with less than 25%, less than 15%, less than 10%, less than 5%, less than 2%, or even less than 1% of other resolvins and/or other isomers of the compound. The addition of pharmaceutical excipients, other active agents, or other pharmaceutically acceptable additives is not understood to decrease the purity of a compound as this term is used herein.

The compounds described throughout the specification can be administered alone or in combination with a pharmaceutically acceptable carrier.

In the DHA analogs, it should be understood that reference to "hydroxyl" stereochemistry is exemplary, and that the term is meant to include protected hydroxyl groups as well as the free hydroxyl group. In certain embodiments, the C- 17 position has an R configuration. In other embodiment, the C- 17 position has an S configuration. In other aspects, certain embodiments of the invention have an R configuration at the C-18 position.

In certain aspects of the present invention, ASA pathways generate R>S and therefore, 4S₅ 7S,8R/S, 1 IR, 16S, 17R. With respect to species generated from the 15-LO pathway the chirality of C- 17 is S , C- 16 is R, preferably R.

The hydroxyl(s) in the DHA analogs can be protected by various protecting groups (P), such as those known in the art. An artisan skilled in the art can readily determine which protecting group(s) may be useful for the protection of the hydroxyl group(s). Standard methods are known in the art and are more fully described in literature. For example, suitable protecting groups can be selected by the skilled artisan and are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups include methyl and ethyl ethers, TMS or TIPPS groups, acetate (esters) or propionate groups and glycol ethers, such as ethylene glycol and propylene glycol derivatives.

For example, one or more hydroxyl groups can be treated with a mild base, such as triethylamine in the presence of an acid chloride or silyl chloride to facilitate a reaction between the hydroxyl ion and the halide. Alternatively, an alkyl halide can be reacted with the hydroxyl ion (generated by a base such as lithium diisopropyl amide) to facilitate ether formation.

The compounds can be prepared by methods provided herein and in US Patent Applications 09/785,866, filed February 16, 2001, entitled "Aspirin Triggered Lipid Mediators" by Charles N. Serhan and Clary B. Clish, 10/639,714, filed August 12, 2003, entitled "Resolvins: Biotemplates for Novel Therapeutic Interventions" by Charles N. Serhan and PCT Applications WO 01/60778, filed February 16, 2001, entitled "Aspirin Triggered Lipid mediators" by Charles N. Serhan and Clary B. Clish and WO 04/014835, filed August 12, 2003, entitled "Resolvins: Biotemplates for Novel Therapeutic Interventions" by Charles N. Serhan and US Patent No. 6,949, 664, by Nicos Petasis entitled "Trihydroxy polyunsaturated eicosanoid" as well as several of the references noted herein, the contents of which are incorporated herein by reference in their entirety.

It should also be understood that for the DHA analogs, not all hydroxyl groups need be protected. One, two or all three hydroxyl groups can be protected. This can be accomplished by the stoichiometric choice of reagents used to protect the hydroxyl groups. Methods known in the art can be used to separate the di- or tri-protected hydroxy compounds, e.g., HPLC, LC, flash chromatography, gel permeation chromatography, crystallization, distillation, etc. It should be understood that there are one or more chiral centers in each of the above-identified compounds. It should understood that the present invention encompasses all stereochemical forms, e.g., enantiomers, diastereomers and racemates of each compound. 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 resolvin analogs depicted throughout the specification contain acetylenic and/or ethylenically unsaturated sites. Where carbon carbon double bonds exist, the configurational chemistry can be either cis (E) or trans (Z) and the depictions throughout the specification are not meant to be limiting. The depictions are, in general, presented based upon the configurational chemistry of related DHA compounds, and although not to be limited by theory, are believed to possess similar configuration chemistry.

Throughout the specification carbon carbon bonds in particular have been "distorted" for ease to show how the bonds may ultimately be positioned relative one to another. For example, it should be understood that acetylenic portions of the resolvins actually do include a geometry of approximately 180 degress, however, for aid in understanding of the synthesis and relationship between the final product(s) and starting materials, such angles have been obfuscated to aid in comprehension.

Known techniques in the art can be used to convert the carboxylic acid/ester functionality of the resolvin analog into carboxamides, thioesters, nitrile, carbamates, thiocarbamates, etc. and are incorporated herein. The appropriate moieties, such as amides, can be further substituted as is known in the art.

In general, the resolvin analogs of the invention are bioactive as alcohols. Enzymatic action or reactive oxygen species attack at the site of inflammation or degradative metabolism. Such interactions with the hydroxyl(s) of the resolvin molecule can eventually reduce physiological activity as depicted below:

R "protecting group"

Bioactive "inactive metabolite" serves to increase bioactivity

The use of "R" groups with secondary bioactive alcohols, in particular, serves to increase the bioavailability and bioactivity of the resolvin analog by inhibiting or diminishing the potential for oxidation of the alcohol to a ketone producing an inactive metabolite. The R "protecting groups" include, for example, linear and branched, substituted and unsubstituted alkyl groups, aryl groups, alkylaryl groups, phenoxy groups, and halogens.

Generally the use of "R protection chemistry" is not necessary with vicinal diols within the resolvin analog. Typically vicinal diols are not as easily oxidized and therefore, generally do not require such protection by substitution of the hydrogen atom adjacent to the oxygen atom of the hydroxyl group. Although it is generally considered that such protection is not necessary, it is possible to prepare such compounds where each of the vicinal diol hydroxyl groups, independently, could be "protected" by the substitution of the hydrogen atom adjacent to the oxygen atom of the hydroxyl group with an "R protecting group" as described above. 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 "subject" is intended to include living organisms susceptible to conditions or diseases caused or contributed bacteria, pathogens, disease states or conditions as generally disclosed, but not limited to, throughout this specification. 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, i.e., at least one DHA analog, in combination with a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

In certain embodiments, 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, esters, amides, and prodrugs" as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use of the compounds of the invention. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. (See, for example, Berge S. M., et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19 which is incorporated herein by reference).

The term "pharmaceutically acceptable esters" refers to the relatively nontoxic, 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.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for intravenous, oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in- oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

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.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitol, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Such solutions are useful for the treatment of conjunctivitis.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Intravenous injection administration is preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

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.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of ordinary skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

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.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

The pharmaceutical compositions of the invention include a "therapeutically effective amount" or a "prophylactically effective amount" of one or more of the DHA analogs of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., a diminishment or prevention of effects associated with various disease states or conditions. A therapeutically effective amount of the DHA analog may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the DHA analog and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a DHA analog of the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

Delivery of the DHA analogs of the present invention to the lung by way of inhalation is an important method of treating a variety of respiratory conditions (airway inflammation) noted throughout the specification, including such common local conditions as bronchial asthma and chronic obstructive pulmonary disease. The DHA analogs can be administered to the lung in the form of an aerosol of particles of respirable size (less than about 10 μm in diameter). The aerosol formulation can be presented as a liquid or a dry powder. In order to assure proper particle size in a liquid aerosol, as a suspension, particles can be prepared in respirable size and then incorporated into the suspension formulation containing a propellant. Alternatively, formulations can be prepared in solution form in order to avoid the concern for proper particle size in the formulation. Solution formulations should be dispensed in a manner that produces particles or droplets of respirable size.

Once prepared an aerosol formulation is filled into an aerosol canister equipped with a metered dose valve. The formulation is dispensed via an actuator adapted to direct the dose from the valve to the subject.

Formulations of the invention can be prepared by combining (i) at least one DHA analog in an amount sufficient to provide a plurality of therapeutically effective doses; (ii) the water addition in an amount effective to stabilize each of the formulations; (iii) the propellant in an amount sufficient to propel a plurality of doses from an aerosol canister; and (iv) any further optional components e.g. ethanol as a cosolvent; and dispersing the components. The components can be dispersed using a conventional mixer or homogenizer, by shaking, or by ultrasonic energy. Bulk formulation can be transferred to smaller individual aerosol vials by using valve to valve transfer methods, pressure filling or by using conventional cold-fill methods. It is not required that a stabilizer used in a suspension aerosol formulation be soluble in the propellant. Those that are not sufficiently soluble can be coated onto the drug particles in an appropriate amount and the coated particles can then be incorporated in a formulation as described above.

Aerosol canisters equipped with conventional valves, preferably metered dose valves, can be used to deliver the formulations of the invention. Conventional neoprene and buna valve rubbers used in metered dose valves for delivering conventional CFC formulations can be used with formulations containing HFC-134a or HFC-227. Other suitable materials include nitrile rubber such as DB-218 (American Gasket and Rubber, Schiller Park, 111.) or an EPDM rubber such as Vistalon (Exxon), Royalene^™ (UniRoyal), bunaEP (Bayer). Also suitable are diaphragms fashioned by extrusion, injection molding or compression molding from a thermoplastic elastomeric material such as FLEXOMER^™ GERS 1085 NT polyolefm (Union Carbide).

Formulations of the invention can be contained in conventional aerosol canisters, coated or uncoated, anodized or unanodized, e.g., those of aluminum, glass, stainless steel, polyethylene terephthalate.

The formulation(s) of the invention can be delivered to the respiratory tract and/or lung by oral inhalation in order to effect bronchodilation or in order to treat a condition susceptible of treatment by inhalation, e.g., asthma, chronic obstructive pulmonary disease, etc. as described throughout the specification.

The formulations of the invention can also be delivered by nasal inhalation as known in the art in order to treat or prevent the respiratory conditions mentioned throughout the specification.

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.

The invention features an article of manufacture that contains packaging material and a DHA analog formulation contained within the packaging material. This formulation contains an at least one DHA analog and the packaging material contains a label or package insert indicating that the formulation can be administered to the subject to treat one or more conditions as described herein, in an amount, at a frequency, and for a duration effective to treat or prevent such condition(s). Such conditions are mentioned throughout the specification and are incorporated herein by reference. Suitable DHA analogs are described herein. More specifically, the invention features an article of manufacture that contains packaging material and at least one DHA analog contained within the packaging material. The packaging material contains a label or package insert indicating that the formulation can be administered to the subject to asthma in an amount, at a frequency, and for a duration effective treat or prevent symptoms associated with such disease states or conditions discussed throughout this specification.

Materials and Methods

In vivo experimental protocols

Kidney ischemia/reperfusion model. Male 23-28 g BALB/C mice (Charles River) fed on standard chow were anesthetized with pentobarbital (65 mg/g) IP, shaved on both flanks, and prepared by cleaning skin with Betidine. The animals were placed prone on temperature-controlled heating pads linked to a rectal probe (Harvard Apparatus Co.). Core mouse temperature was stabilized between 36.7⁰C and 37.3°C. Mice were given reagents by tail vein injection. When compounds were administered intravenously the interval between injection and clamping of kidney vessels was 10 min. Longitudinal incisions were made over both kidneys in the mid scapular line, the muscle wall divided by blunt dissection. The left kidney was exposed. A microaneurysm clamp (Roboz) was placed across the renal pedicle to occlude artery and vein. Dusking of the hue of the kidney was confirmed after which the clamped kidney was returned to the retroperitoneum, and the skin held closed. The right kidney was then exposed and the renal pedicle clamped within one min of the placement of the left kidney clamp.

For both kidneys the clamps were allowed to remain for precisely 30 min. Clamps were then removed and re-perfusion confirmed visually. Kidneys were again returned to the retroperitoneum, and the skin wound closed with clips. Prior to closure of the right flank wound, a pocket was created by blunt dissection between the fascial planes separating dermis and muscle, pointing cranially toward the high thoracic spine. Primed, warmed, pre-prepared osmotic mini- pumps were placed in this space with the exit point facing cranially, and the skin wound closed. Mice were transferred to a warmed cage until they had fully recovered from anesthesia and given 1 ml of PBS (37°C) intraperitoneally (IP). All mice were given another 1 ml PBS (37°C) IP 24h later following tail bleeds. One hundred microliters of blood were collected by tail tip incisions and capillary tube withdrawal.

Experimental protocols with docosahexaenoic acid, resolvins and protectins. Mice (n=5 per group [weight matched]) received 200-300 μl of docosahexaenoic acid (DHA) (125 μg/mouse), Protectin Dl (3.5 or 35 μg/mouse), 175-Hydroxy DHA (17S-HDHA) (17.5 μg/mouse), or vehicle (0.9% NaCl saline containing 0.01% delipidated bovine serum albumin (BSA)). Half of each solution was injected into the lateral tail vein 10 min prior to bilateral clamping of renal pedicles. The other half was infused subcutaneously via ALZET osmotic-pump (Durect Co., Cupertino, CA) at a flow rate of 8 μl/h (model 2001D) for 24h or 1 μl/h (model 1003D) for 72h. Each treatment group was matched with a vehicle-treated control group. In a second series of experiments, cohorts of mice (n=7 per group) received Protectin Dl (3.5 μg or 35 μg/mouse), RvD (3.5 or 35 μg/mouse), or vehicle (0.9% NaCl saline containing 0.01% BSA). Half of each solution was injected into the lateral tail vein 10 min prior to bilateral clamping of renal pedicles. Another half was infused subcutaneously via ALZET osmotic-pump (model 2001D) at 8 μl/h until mice were euthanized at 24h. In a third series of experiments, mice (n=5 per group) were treated identically to the second series with the exception that the reagents were administered by a 1003D minipump at a flow rate of 1 μl/h continuously over 48h. Tail bleeds were performed at 24h, and mice euthanized at 48h. hi a fourth series of experiments animals were treated as in the 2^nd and 3^rd except that the infusion via osmotic-pump lasted for 72h and the mice were allowed to recover from ischemia/reperfusion injury for 15 days at which time they were euthanized.

A fifth series of experiments was conducted to evaluate if treatment post- reperfusion would have any beneficial effects. Mice (n=4 per group [weight matched]) received 200 μl of RvDs (10 μg/mouse), RvDl (10 μg/mouse), Protectin Dl (10 μg/mouse), or vehicle. In these studies, half of each solution was injected intraperitoneally 10 min. after release of the bilateral clamps on the renal pedicles. The other half was infused subcutaneously via ALZET osmotic- pump (1003D) as above. Sham-operated mice also received compounds administered identically. All studies were carried out in accordance with a protocol approved by the Harvard Center for Animal Resources and Comparative Medicine.

Lipidomic analysis of Tissue Resolvin D series and Protectin Dl

Blood was extracted with 2 volumes of methanol (~ 4°C). Renal tissues were homogenized and extracted in methanol on ice. The tissue and blood extracts were cleaned with Cl 8 solid phase extraction cartridge (300 mg, Alltech, Deerfield, IL) (25), (29). The analyses were conducted using a LC-UV-MS/MS (liquid chromatography-UV-ion trap tandem mass spectrometer) (LCQ, ThermoFinnigan, San Jose, CA) equipped with a LUNA Cl 8 column (100 mm x 2 mm x 5 μm) (24, 25, 30) The column was eluted at 0.2 ml/min. The mobile phase gradient was as follows: from 0 to 8 min, phase A (methanol:water:acetic acid=65:35:0.01 by volume); from 8.01 min to 30 min, linearly changing from A to methanol; and from 30.01 to 35 min, methanol. The photodiode-array ultraviolet detector (UV) scanned from 200 to 400 run. The electrospray voltage for the mass spectrometer LCQ was 4.3 kV. The ion trap analyzer typically scanned from m/z 200 to 800 in MS mode and from m/z 95 to 380 in MS/MS mode.

Characterization of RvDs, PDl and related products

RvDs and 17S-hydroxy-DHA (HDHA) were generated using DHA (Cayman Chemical, Ann Arbor, MI) and 15-lipoxygenase (25, 31, 32). The enzymatically derived preparations were isolated via Cl 8 solid phase extraction (SPE) followed by HPLC on a Cl 8 column (150 mm x 2 mm x 5μm) (Phenomenex, Torrance, CA), which was eluted with 70% methanol. RvD and 17S-HDHA were isolated using HPLC, collected, and characterized (25). Protectin Dl was prepared by total organic synthesis and was quantified by both physiochemical and biological properties (33). The solutions were taken to dryness with N₂ gas and suspended in ethanol as stock solutions. Immediately prior to I/R experiments, each stock solution was diluted with saline (0.9% NaCl) or saline containing delipidated endotoxin-free BSA (0.01% BSA) (34). The composition of the RvD series compounds (RvDs) was 1:2:1 (RvDl :RvD2:RvD3), reflecting their relationship in vivo. These results were confirmed with RvDl prepared by total organic synthesis, matched with enzymatic and biologically generated RvDl that will be reported elsewhere (Sun et al personal communication). The synthetic RvDl and PDl were provided by Professor Nicos P. Petasis at Department of Chemistry in the University of Southern California at Los Angeles. The total organic synthesis of PDl was reported (35).

Assessment of kidney injury

Preparation of kidneys for analysis. Cohorts of mice were euthanized at 24h, 48h, or 15d. Tissues were either flushed with ice-cold PBS to remove erythrocytes and circulating leukocytes or perfusion-fixed in situ with paraformaldehyde-L-lysine-periodate (PLP) solution using techniques previously described (11). Unfixed kidneys were snap-frozen in liquid N₂ and stored at -80⁰C. Fixed tissues were transferred from PLP to 18% sucrose solution in PBS after 2h. 18h later they were frozen in OCT (optimal cutting temperature) compound and stored at -80⁰C. Tissues for staining were fixed in 10% neutral buffered formalin for 12h, transferred to 70% ethanol and embedded in paraffin wax. Imm unofluorescence. Five-micron cryotome-cut paraformaldehyde-L- lysine-periodate (PLP)-fixed sagittal sections were pre-blocked with Fc-block (Pharmingen), then immunolabeled with anti-GR-1 phycoerythrin (PE) fluorescent antibodies, or anti-CD 1 Ib PE fluorescent antibodies (EBioscience) and anti-CD68-FITC antibodies (Serotec), all at 1:200 dilution in 10% rabbit serum for 2h at room temperature. Sections were washed in PBSx3, then mounted with Vectashield including DAPI (200 ng/ml). Sections were viewed by fluorescence microscopy (X200) and serial images captured using identical settings, covering the entire section. All images were assessed quantitatively for percentage area of kidney positive for a particular stain using Methods previously described (36). Briefly, digital images were assessed using Fovea Pro software. A range of hues, saturations and intensities were selected to selectively include the positively stained cells only. These settings were applied to each image, giving a percentage area of the image positive for the stain. For each kidney the average for the each whole sagittal section was obtained by recording the area for each captured image.

Staining of tissue sections. Three-micron paraffin sagittal sections were stained with PAS or the Masson's trichrome method. PAS-stained sections from each kidney were assessed blindly for histological severity of disease using the following established scale (6): 1+, Normal; 2+, Mitosis and necrosis of individual cells; 3+, Necrosis of all cells in adjacent proximal tubules, with survival of surrounding tubules; 4+, Necrosis confined to the distal third of the proximal tubules with a band of necrosis extending across the inner cortex; 5+, Necrosis affecting all three segments of the proximal tubules. Each kidney was ascribed a disease severity value. The quantity of blue-stained collagen deposition was assessed in kidneys 15 d after ischemic injury using quantitative morphometry measurements as described above. The quantity of collagen was determined as the % area of the total kidney area.

Quantitative Myeloperoxidase assay. Snap-frozen kidney samples were homogenized at 4 C in potassium phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide, sonicated, freeze-thawed X3, then sonicated again. The suspension was centrifuged at 12,000 g for 15 min, and 20 μl of supernatant was added to 900 μl of potassium phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine dihydrochloride (Sigma) and 0.0006% hydrogen peroxide (Sigma). The rate of change in absorbance at 460 ran was monitored at 23°C at 90-second intervals (37).

Creatinine measurements. Plasma was obtained by either tail bleeds as described or cardiac puncture into pre-heparinized tubes. Plasma was separated by centrifugation (800 X g for 10 min.). Thirty-five-microliter aliquots of plasma in duplicate were used for measurement with a Beckmann II Creatinine analyzer (38).

Mouse activity score. Mice were assessed 24h following ischemia/reperfusion for well-being using a standardized assay ranging from 1 to 3 (1+, lazy, slow movement; 2+, intermediate level of activity; 3+, active movement or searching) (39) . This time reflects the peak of the plasma creatinine.

In Vitro Assays

Tubule cell injury. LLC-PK₁ , swine kidney epithelial cells with proximal tubule characteristics, were obtained from ATCC and passaged in DMEM (Gibco) with glutamine and 10% FCS. For the experiments cells were grown to confluence in 24-well plates (Corning) in 1.0 ml medium containing 1% FCS. Experiments were carried out in triplicate. Cells were treated with 500 μM H₂O₂ and simultaneously one of the active reagents, DHA, Protectin Dl, RvDs or the essential fatty acid, arachidonic acid (AA), in one of the following molar concentrations: 0.5 μM, 50 μM, 500 μM. In previous experiments we had determined that 500 μM H₂O₂ resulted in approximately 50% LDH release 6h later (40). Thus, after 6h of incubation, 100 μl of supernatant was collected and cells lysed in the remaining medium with 0.1% TritonX-100. The proportion of total cellular lactate dehydrogenase (LDH) released into the supernatant was used as a measure of injury and LDH was quantitated using a standard colorimetric LDH-specific assay (40).

Macrophage TNFa production. Primary bone marrow-derived macrophages were cultured as previously described (41). Day 7 mature macrophages were plated into 24-well plates (2.5 x 10⁵/well). Each well had 500 μl of DMEM/F12 containing 10% FCS. Each experiment was performed in triplicate. One hundred ng/ml of LPS (E. coli 0127:B8 Sigma) was added to each well together with simultaneous addition of one of the active reagents, DHA, Protectin Dl, or RvD in one of the following molar concentrations: 0.05 nM, 0.5 nM, 50 nM, or 500 nM in PBS 0.01% delipidated BSA or an equal volume of vehicle alone. Macrophages were cultured for 24h, supernatants harvested and assayed for TNFα generation using a mouse TNFα sandwich ELISA (R&D Systems). The concentration of TNFα was normalized for the total protein in each well of macrophages which was determined by dissolving cells in 100 μl of Complete lysis buffer (Roche) containing 0.1% TritonX-100. Two microliters were diluted 50-fold; the sample was mixed with 9 parts of protein assay solution (Biorad) and protein quantitated colorimetrically at 595 nm and compared with an albumin control.

Statistics

Values are expressed as mean ± sem. Differences among groups were assessed by ANOVA and between groups by Students' t-test. Significant differences between groups are denoted: * P < 0.05, ** P < 0.01.

Ischemia/reperfusion acute kidney injury results in biosynthesis and release of D series resolvins and protectins.

In order to explore the role of endogenously generated omega-3 DHA, and lipids derived from exogenously introduced omega-3 DHA, on recovery after injury to the kidney, DHA (62.5 μg/mouse) or vehicle was administered as a bolus to mice prior to ischemia and then infused by osmotic-pump (dose 62.5 μg /mouse) over the succeeding 24h. The impact of renal ischemia/reperfusion in the presence and absence of concurrent administration of DHA on the biosynthesis and release of D series resolvins and protectins was studied using LC-UV- MS/MS-based informatics (Fig. 1). In contrast to sham-treated mice, ischemia followed by 24h reperfusion triggered in kidney tissue the endogenous biosynthesis and/or release of the precursor DHA into the plasma (Fig. IA). The DHA-derived lipid mediator, Protectin Dl , and its biosynthetic intermediate, 17S-HDHA, and to a lesser extent RvDl and RvD3, were also generated by the postischemic kidney, but not by sham-operated kidney (Fig. 1 , C and F). In plasma, endogenous DHA was markedly elevated by ischemia/reperfusion (Fig. IA) and there was an increase in the intermediate 17S-HDHA. In contrast to kidney tissue, however, there was no increase in plasma levels of Protectin Dl or RvDl in the absence of added DHA, whereas both RvD2 and RvD4 and to a lesser extent RvD3 levels were increased in plasma (Fig. 1, D and E). Both RvD5 and RvD6 were also identified at low levels in plasma after ischemia/reperfusion in the vehicle group (Fig. IZ)). Increased levels of 17S- HDHA were found in post-ischemic kidneys in both the vehicle and DHA-treated groups, providing that ischemia/reperfusion induced activity of the enzyme 15- lipoxygenase (Fig. 1, A and Q, which is instrumental in transforming DHA to 17S-HDHA in vivo in mouse tissues (25, 34).

Exogenous administration of DHA to mice exposed to kidney ischemia/reperfusion not only increased the amounts of unesterified DHA in both plasma and renal tissue (Fig. IA), but increased the tissue levels of RvD6 and RvD2. In addition the level of the precursor 17S-HDHA was increased, as was the level of RvDl and to a lesser extent RvD3. Protectin Dl was notably not increased by DHA infusion (Fig. IQ. Exogenous administration of DHA following ischemic kidney injury led to de-no vo generation of Protectin Dl and RvDl in plasma. RvD2 concentration was increased in plasma as were the levels of other resolvins including RvD3, RvD5 and RvD6 (Fig. IQ. Of interest, administration of DHA did not directly change RvD4 levels in plasma or in kidney tissue within this interval. The levels of 17S-HDHA were also increased in plasma with administration of DHA (Fig. ID). The identification of the DHA-derived bioactive mediators described above was carried out using a liquid chromatography-tandem mass spectrometry (LC-MS-MS) informatics approach and generating ion chromatograms from LC- MS-MS (Fig. 1, E and F). The MS/MS spectrum at m/z 375 acquired at a LC retention time of 4.8 min on a representative plasma sample from mice 24h following ischemia/reperfusion is consistent with the structure of RvD2 with diagnostic ions at m/z 109 (129-2H₂O-2H), 123 (142-H₂O-H), 131 (129+2H), 203 (247-CO₂-H), 209 (263-3H₂O), 217 (233+2H-H₂O), 227 (263-2H₂O), 233, 241 (276-2H₂O+H), 247 (246+H), 261 (263-2H), 277 (276+H), 287 (306-H₂O- H), 293 (M-H-2H₂O-CO₂-2H), 313 (M-H-H₂O-CO₂), 331 (M-H-CO₂), 339 (M- H-2H₂O), 357 (M-H-H₂O-CO₂), and 375 (M-H) (Fig. IE) (25). The complete stereochemistry of neuroprotectin Dl/protectin Dl was recently established (42). PDl was identified in mouse kidney 24h following ischemia based on the MS/MS spectrum at m/z 359, which possesses diagnostic ions at m/z 359 (M-H), 341 (M-H-H₂O), 323 (M-H-2H₂O), 315 (M-H-CO₂), 297 (M-H-H₂O-CO₂), 289, and 277 (M-H-2H₂O-CO₂-2H). Ions consistent with the carbon 10 and carbon 17 alcohol-containing positions were observed at m/z 153, 163 (182-H-H₂O), 181 (182-H), 205, 217 (216-CO₂-H), 243 (261-H₂O), 245 (261-H₂O+2H), and 261 (Fig. IF) (25). Since RvDs and PDl display activity of anti-inflammatory autacoids in inflammatory settings (33), it was next considered whether RvDs and PDl function as anti-inflammatory agents in ischemia/reperfusion-induced kidney injury.

Administration of RvDs and PDl protects kidneys from ischemic acute kidney injury

Since local inflammation is a prominent component in the pathophysiology of acute kidney injury (9) and DHA is the precursor of RvDIs and PDl, it was evaluated whether DHA was an effective therapy for ischemic kidney injury. Mice were given DHA by IP injection immediately prior to induction of bilateral ischemia/reperfusion injury. At reperfusion, DHA therapy was continued over 25h by subcutaneous osmotic-pump infusion. At 24h, levels of plasma creatinine (a quantitative marker of renal failure) were measured (Fig. 2A). Neither creatinine levels nor kidney histology (examined at 24h, data not shown) were different in the DHA-treated mice compared with the vehicle- treated mice. Furthermore, when histological injury was determined by a semiquantitative injury severity score determined by viewing PAS-stained kidney sections, there was no significant difference between DHA-treated and vehicle- treated sections (4.2 ± 0.3 vs. 4.0 ± 0.2).

The D series resolvins display potent anti-inflammatory activity (31, 34, 43). These compounds were administered to mice in amounts related to their endogenous formation (1:2: 1, RvDl:RvD2:RvD3; vide supra) 10 min prior to ischemia of both kidneys and during the subsequent 48h interval via a subcutaneous mini pump. Half of the total dose of Resolvin D (RvDs) was given by infusion pump (Alzet). At both a total dose of 3.5 μg (140 ng/g body weight) or 35 μg (1.4 μg/g body weight), RvDs limited functional renal injury at 24h as reflected by a lower plasma creatinine when compared with vehicle-treated animals (Fig. 2B). RvDs had no effect on plasma creatinine levels in sham- operated mice (data not shown).

Protectin Dl administered to mice as above also protected kidneys. Both RvDs and Protectin Dl not only attenuated peak creatinine levels at 24h, but also had additional actions on the renal resolution process between 24h and 48h. Plasma creatinine levels returned to near normal at 48h in PDl -treated mice and in mice treated with 35 μg of RvDs, whereas in vehicle-treated mice plasma creatinine levels remained markedly elevated (Fig. 1C). A lower total dose of 3.5 μg (140 ng/g) had less efficacy compared with the higher dose of 35 μg (1.4 μg/g) at 24h. Furthermore, PAS-stained sections of postischemic kidney showed enhanced tubule cell survival, decreased renal inflammation and decreased capillary occlusion in mice treated with either 35 μg PDl or RvDs (Fig. 3, Table I). From a behavioral perspective, mice with acute kidney injury secondary to ischemia exhibited reduced activity during the first 24 h of reperfusion. Mice treated with either RvDs or Protectin Dl exhibited increased activity (Table II) compared with vehicle-treated mice, providing further evidence for the protective actions of these mediators.

TABLE I. Histological injury score for kidneys of mice treated with vehicle, Protectin Dl, or Resolvin Ds

vehicle PDl RvDs

score 4.25 ± 0.17* 3.25 ± 0.29 3.5 ± 2.4

The dose of Protectin Dl was 35 μg and RvDs was 35 μg. Scores were determined at 48h post-injury (see Materials and Methods for details). Sham operated mice had histological injury score of 1.0 irrespective of treatment. *P=0. 017 (ANOVA)

TABLE II. Mouse activity score following ischemia in mice treated with Protectin Dl or Resolvin Ds

vehicle PDl RvDs

score 2.1 ± 0.2* 2.8 ± 0.3 2.7 ± 0.4

The dose of Protectin Dl was 35 μg and RvDs was 35 μg. Activity scores were determined at 24h. Sham operated mice had activity scores of 3.0 irrespective of treatment. * P = 0.03 (ANOVA).

Resolvins and protectin Dl reduce leukocyte accumulation

Since leukocytes are implicated in the pathophysiology of ischemic kidney disease (36, 37) two independent methods were used to quantify the actions of Protectin Dl and RvDs on leukocyte involvement in kidney injury. Tissue myeloperoxidase (MPO) activity in whole kidney was assessed and leukocytes were stained with specific antibodies for immunocytochemistry. Kidney MPO activity was decreased in mice treated with DHA, 17S-HDHA, PDl, and RvDs compared with vehicle-treated mice at both 24 and 48h (Fig. 4, A and B). Both exogenous PDl and RvDs resulted in a greater than 50% reduction in MPO activity 48h into the recovery from I/R. High-dose RvDs reduced MPO activity by 80% compared with vehicle (Fig. AB). Further, PDl reduced MPO activity by 67% 24h after ischemia. It is important to note that, while high-dose DHA reduced MPO activity by 41% (Fig. AA), it did not prevent the rise in creatinine seen with ischemia/reperfusion as discussed above (Fig. 2).

The degree of monocyte and neutrophil infiltration was assessed quantitatively by morphometry using antibodies to CDl Ib, which is expressed by both PMNs and monocytes, antibodies to GR-I, which is expressed by PMNs, and antibodies to CD68, which is expressed by monocytes (Fig. 5). Both Protectin Dl and RvDs treatments resulted in quantitatively less area of CDlIb, GR-I, and CD68 immunostaining objectively assessed by morphometry (36). Thus, tissue PMNs and monocytes were reduced by both PDl and RvDs (Fig. 5).

Administration of RvDs or RvDl after onset of ischemic injury is protective

The unique compounds described herein demonstrated efficacy in protecting kidneys from severe injury and promoting resolution. In the above experiments, however, the injured kidneys were exposed to the compounds prior and subsequent to onset of injury. To determine whether Resolvins and Protectins retained efficacy when reaching the kidney after the onset of injury, further studies were designed. In these, resolvins and protectins were administered after ischemic injury. Furthermore, the compounds were given IP rather than intravenously (followed by subcutaneous infusion) to slow the rate at which they might gain access to the kidneys. Kidneys treated with RvDs or synthesized RvDl exhibited marked protection from development of acute renal failure (Fig. 6). PDl given after ischemic injury did not significantly alter the course of acute renal failure compared with vehicle-treated mice.

RvDs and PDl limit leukocyte activation but do not directly protect proximal tubule cells from oxidative agents

To study the impact of these novel renoprotective DHA-derived mediators in more detail, their actions were addressed in vitro. The generation of oxygen free radicals and hydrogen peroxide are implicated in the pathogenesis of both ischemic and toxic acute kidney injury (44, 45). To model tubular cell injury observed in vivo, RvDs and PDl were tested to determine whether they protected cultured proximal tubular epithelial cells from H₂O₂ injury using an established assay for cell injury (40). The effects of arachidonic acid (AA) and DHA were examined in parallel. None of the anti-inflammatory RvDs and PDl or AA or DHA, at concentrations as high as 500 nM, had any effect on the H₂O₂-induced release of LDH from cultured tubular cells (Fig. 7A). In each case, despite the presence of relatively high concentrations of RvDs and PDl, approximately 25% of total LDH was released from cells by H₂O₂ exposure. By contrast, in cells not treated with H₂O₂, 7.3 ± 1.9% of total LDH was released in control conditions. LDH release from cells not treated with H₂O₂ was not affected by these compounds (not shown). These results suggest that the actions of these mediators in the kidney were not the direct result of tubule cell protection from oxidative influences.

In addition to H₂O₂, the monocyte/macrophage pro-inflammatory cytokine TNFα has also been implicated in the pathogenesis of ischemic acute kidney injury (46, 47). Cultured macrophages were prepared that were derived from bone marrow and tested whether DHA, D series Resolvins, or Protectin Dl, administered simultaneously with a pro-inflammatory stimulus LPS, limited TNFα generation in response to cell activation (Fig. 75). At low concentrations, DHA itself had no suppressive effect on TNFα release in macrophages by LPS. At higher concentrations DHA reduced TNFα release by -22.0 ± 1.6% (Fig. 75). By contrast, RvDl afforded a dose-dependent reduction in LPS-induced TNFα release, and had notable actions on TNFα release at concentrations 100 X lower than those required with its precursor, DHA. Protectin Dl also lowered LPS- induced TNFα release, although this was less marked than RvDl , suggesting that both active compounds have anti-inflammatory activities directed at monocytes/macrophages .

RvDs and PDl limit post-ischemic interstitial kidney fibrosis

Even though there is recovery of the kidney functionally following renal pedicle clamping and reperfusion, as measured by serum creatinine, histology reveals incomplete resolution with progressive interstitial fibrosis over weeks (11). This fibrosis may result from persistence of inflammatory leukocytes, in particular interstitial macrophages (48). Interstitial fibrosis and persistent leukocyte infiltration (chronic inflammation) are the harbingers of scarring and chronic renal failure. As many as forty percent of patients with acute kidney injury are left with worsening of their baseline kidney status and chronic kidney disease (4, 49). RvDs and PDl were assessed to determine whether they could limit the deposition of interstitial collagens that contribute to fibrosis. Kidney sections stained for collagen by Gomori's trichrome method were assessed by quantitative computerized morphometry to assess the area of collagen deposition 15d after ischemia/reperfusion. RvDl treatment for 72h after ischemia resulted in 44 + 17% less deposition of collagen whereas Protectin Dl was less effective (a non-significant reduction of 21 + 12% in reducing scarring at 15d (Fig. 8)).

It has been demonstrated for the first time that two families of newly identified anti-inflammatory mediators, D series Resolvins and Protectin Dl, are generated in the kidney in response to ischemia/reperfusion. These mediators are generated by stereospecific enzymatic pathways both in the presence and absence of exogenous administration of precursor DHA. When infused peripherally in mice, they markedly attenuate ischemic kidney injury as well as reduce fibrosis. One of the actions of these compounds in the kidney is to limit the influx of leukocytes and another is to limit activation of leukocytes. Therefore the pathways described counter-regulate the pro-inflammatory, cellular and molecular signaling that results from ischemia/reperfusion. This represents a new paradigm in the pathophysiology of acute kidney injury, which often leads to acute renal failure. Hence, these anti-inflammatory and counterregulatory influences may be critical to the resolution of damage and limitation of chronic fibrosis resulting from this damage.

More Protectin Dl than RvD is produced with ischemia/reperfusion, especially in the absence of exogenously administered DHA. RvD and Protectin Dl displayed profound protective effects within 24h of I/R, when PMN are important that persist for 48h (monocyte dominated). D series resolvins were apparently more effective than Protectin Dl in reducing scarring 15d after ischemia and are much more effective at reducing injury when initially administered after reperfusion. Experiments with epithelial cells in vitro suggest that protection in vivo is not the result of a direct effect on the tubular epithelial cells of the kidney but more likely related to the ability of these mediators to down-regulate components of the inflammatory response involving infiltrating cells.

The present invention provides that endogenously generated RvDs and PDl protect from ischemic injury in the kidney. PDl, also known as NPDl when generated in neuronal tissues (1, 42), is protective in the brain (34). There is currently a need for effective therapies for diseases in humans resulting from ischemia to the kidney as well as brain, both of which are characterized by uncontrolled local inflammation, which is believed to contribute to acute and chronic functional impairment (3, 34). RvDs and Protectin Dl both limit infiltration of leukocytes and also limit activation of leukocytes in both postischemic organs. It is possible that the compounds of the invention may have additional cellular sites of actions in the kidney, i.e., on the endothelium and vascular tone, as well as interstitial fibroblasts since they are also antifibrotic.

In an experimental model of stroke there was active generation of both Protectin Dl and Resolvin Dl, the former peaking 10 h after injury, and the latter peaking 24h after injury (34). Infusion of PDl into ventricles of the brain following stroke markedly attenuated stroke area as measured 48h after ischemia (34). RvDs and PDl in sterile peritonitis are generated during the resolution phase, and administration of both RvDs and PDl not only limited neutrophil influx but also limited both chemokines and pro-inflammatory cytokines in the inflammatory exudates (27).

It is notable that the endogenous level of the biosynthetic precursor of RvDs and PDl , DHA, was increased in response to injury. This likely reflects an activation of cellular lipases (i.e., PLA₂) enzymes, which can cleave DHA from phospholipids. Since increased levels of DHA in tissue and plasma alone does not account for the increased generation of D series resolvins and PDl found with ischemia/reperfusion, renal ischemic injury may upregulate the local formation of RvDs and PDl (25, 34). Endogenously generated anti-inflammatory mediators with or without exogenous DHA administration may be present in insufficient quantities in normal mice during acute kidney injury to protect the organ from innate immune-mediated injury.

The present invention demonstrates that compounds of the invention such as RvDs and synthesized RvDl are effective in attenuating established renal injury. These findings indicate that these pro-resolution-of-inflammation compounds may not simply block the activation of the inflammatory response pathways but that they are bioactive during the acute injury phase and can actively counteract established inflammation and injury. The fact that equimolar administration of RvDl appears more potent in protecting kidneys from injury than the mixture of RvDs in the post injury administration of compounds suggests that RvDl might be more potent than RvD2 and RvD3. Further studies will be required to determine whether RvDl is more potent at blocking neutrophil activation or chemotaxis. The present invention provides that PDl in the kidney is not efficacious in promoting resolution once the injury is established. This may reflect the reduced efficacy of PDl after established disease onset, and point to a distinct mechanism of action from the Resolvin D series, or possibly reflects inadequate bioavailability. In the studies in which PDl is effective, doses of both 3.5μg and 35μg were effective whereas in the post-injury studies a single dose of 10 μg was chosen due to limitation of availability of the compound. It is possible that after injury onset higher doses of PDl are required to achieve therapeutic levels in the injured kidneys. Further studies will be required.

Earlier studies focused on the actions of compounds of the invention such as RvDs and PDl on PMN activity and chemotaxis (25, 28, 34, 42). The present invention also implicate these compounds in limiting macrophage activity showing, for the first time, that LPS-induced activation of macrophages is reduced by PDl and to a lesser extent by RvDs even when administered at the same time as LPS-induced activation, without the necessity for pre-treatment. Since LPS acts through toll-like receptors (TLR) 4 and to a lesser extent TLR 2 it is likely that these mediators impinge on TLR-activation pathways (50, 51). Further studies are required to determine whether blockage of macrophage activation has effects on many pathways or is limited to TLR-mediated pathways.

In summary, the present invention demonstrates activation of DHA- biosynthetic pathways in the kidney resulting in the local appearance of antiinflammatory mediators following ischemic injury. Administration of compounds of the invention, such as D series resolvins and PDl, sharply reduced renal injury in a model of acute kidney injury. Thus a previously unrecognized endogenous anti-inflammatory response to injury may play an essential role in resolution of acute kidney injury. Moreoveran antifibrotic action of these mediators was uncovered and found that they also act on macrophages. Dysregulation of these response(s) may underlie a delay in recovery or inability to recover in many cases of acute renal failure in humans. Administration of mimetics of these endogenously produced biotemplates retaining antiinflammatory and antifibrotic activities may be of therapeutic importance in treating acute kidney injury.

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Although the present invention has been described with reference to preferred embodiments, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention. All publications and references cited herein, including those in the background section, are expressly incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method to treat or prevent a kidney inflammatory condition, comprising administering to a subject a compound selected from:

XVIII

-115-

XXXIX

XXXXII

XXXXIII

XXXXIV

XXXXV

XXXXVI

XXXXVIII

XXXXIX

wherein a bond depicted as represents either a cis or trans double bond; wherein Pi, P₂ and P₃, if present, each individually are protecting groups, hydrogen atoms or combinations thereof; wherein R₁, R₂ and R₃, if present, each individually are substituted or unsubstituted, branched or unbranched alkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted, branched or unbranched alkylaryl groups, halogen atoms, hydrogen atoms or combinations thereof; wherein Z is -C(O)OR^d, -C(O)NR^CR^C, -C(O)H, -C(NH)NR^CR^C, -C(S)H, -C(S)OR^d, -C(S)NR^CR^C, -CN; each R^a, if present, is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, moφholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl; each R^b, if present, is a suitable group independently selected from the group consisting of =0, -OR^d, (C1-C3) haloalkyloxy, -OCF₃, =S, -SR^d, =NR^d, =N0R^d, -NR^CR^C, halogen, -CF₃, -CN, -NC, -OCN, -SCN, -NO, -NO₂, =N₂, -N₃, -S(O)R^d, -S(O)₂R^d, -S(O)₂OR^d, -S(O)NR^CR^C, -S(O)₂NR^CR^C, -OS(O)R^d, -OS(O)₂R^d, -OS(O)₂OR", -OS(O)₂NR⁰R⁰, -C(0)R^d, -C(0)0R^d, -C(O)NR⁰R⁰, -C(NH)NR°R^C, -C(NR^a)NR°R°, -C(NOH)R³, -C(NOH)NR⁰R⁰, -0C(0)R^d, -0C(0)0R^d, -OC(O)NR⁰R⁰, -OC(NH)NR⁰R⁰, -OC(NR^a)NR^cR⁰, -[NHC(0)]«R^d, -[NR^aC(0)]_nR^d, -[NHC(0)]_n0R^d, -[NR^aC(0)]_n0R^d, -[NHC(O)]^NR⁰R⁰, -[NR^aC(0)]_nNR^cR°, -[NHC(NH)J_nNR⁰R⁰ and -[NR^aC(NR^a)]_«NR^cR^c; each R°, if present, is independently a protecting group or R^a, or, alternatively, each R° is taken together with the nitrogen atom to which it is bonded to form a 5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally include one or more of the same or different additional heteroatoms and which may optionally be substituted with one or more of the same or different R^a or suitable R^b groups; each n, independently, if present, is an integer from O to 3; each R^d, independently, if present, is a protecting group or R^a; wherein X, if present, is a substituted or unsubstituted methylene, an oxygen atom, a substituted or unsubstituted nitrogen atom, or a sulfur atom; wherein Q, if present, represents one or more substituents and each Q individually, if present, is a halogen atom or a branched or unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, amino, hydroxy, cyano, carboxyl, alkoxycarbonyloxy, aryloxycarbonyloxy or aminocarbonyl group; wherein U, if present, is a branched or unbranched, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkoxy, aryloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, alkoxycarbonyloxy, and aryloxycarbonyloxy group; wherein the double bond configurations of the compounds can be either cis or trans; and pharmaceutically acceptable salts thereof.

2. The method of claim 1, wherein Z is a carboxylic acid, ester, a pharmaceutically acceptable carboxylic acid salt, or prodrug thereof.

3. The method of claim 1 , further comprising a pharmaceutically acceptable carrier.

4. The method of any of claims 1 through 3, wherein the compound is purified.