WO2009111530A2 - Use of lipoxins to counteract the impact of anesthetic on inflammatory resolution - Google Patents

Use of lipoxins to counteract the impact of anesthetic on inflammatory resolution Download PDF

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WO2009111530A2
WO2009111530A2 PCT/US2009/035974 US2009035974W WO2009111530A2 WO 2009111530 A2 WO2009111530 A2 WO 2009111530A2 US 2009035974 W US2009035974 W US 2009035974W WO 2009111530 A2 WO2009111530 A2 WO 2009111530A2
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WO2009111530A3 (en
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Nan Chiang
Charles N. Serhan
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The Brigham And Women's Hospital, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/231—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having one or two double bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P23/00—Anaesthetics
    • A61P23/02—Local anaesthetics

Definitions

  • Anesthetics are generally administered when an individual undergoes surgery. Inflammation and would healing occur post surgery. It has not been well understood what impact the use of anesthetics have on the resolution at surgical sites. Often, the resolution at surgical sites can take significant periods of time until the patient is fully recovered. Therefore, a need exists for expediting resolution at surgical sites post surgery.
  • the present invention provides a surprising method for the increase of resolution (healing) in a subject's tissue subjected to an anesthetic.
  • the method comprises administering a therapeutically effective amount of lipoxin or a lipoxin analog to the subject prior to or during surgery, such that the subject's tissue subjected to the anesthetic resolve (heal) more quickly than without administration of a lipoxin or a lipoxin analog.
  • Lidocaine alters leukocyte infiltration during acute inflammation and delays resolution.
  • Mice were injected with zymosan A in the absence or presence of lidocaine (0.008% or
  • LXA 4 rescues lidocaine-delayed resolution.
  • Lidocaine alters pro- and anti-inflammatory proteins: proteomics and cellular proteins.
  • mice were injected with zymosan A in the absence or presence of lidocaine. Both lavage fluids (A) and cell pellets (C) were collected at indicated time points and proteins separated by two-dimensional gel electrophoresis. Changes in individual protein levels were measured by image analysis. Selected proteins that display significant differences between treatments are indicated by arrows, and identified by LC/MS/MS and peptide mapping (see Materials and Methods).
  • Lidocaine regulates selective pro- and anti-inflammatory cytokines/chemokines.
  • Volatile anesthetic isoflurane reduces leukocyte infiltration and promote resolution by shortening resolution interval.
  • mice were administered 1.4 MAC of isoflurane one hour prior to and after injection of zymosan A (lmg/ml, i.p.) (see timeline). The peritoneal lavages were collected at indicated time points.
  • mice were administered 1.4 Mac of isoflurane one hour prior to and after injection of zymosan A (lmg/ml, i.p.).
  • A The peritoneal lavage cells were collected at indicated time points and proteins separated by two-dimensional gel electrophoresis. Changes in individual protein levels were measured by image analysis. Selected proteins that display significant differences between treatments are denoted, and were identified by LC/MS/MS and peptide mapping.
  • B Peritoneal cell-free lavage fluids were collected. Cytokine and chemokine levels were determined and expressed as percent inhibition of zymosan A-induced cytokine/chemokine levels by isoflurane. *p ⁇ 0.05 when compared to mice treated with zymosan A alone. For raw values (pg/ml) of these selective cytokines, see Table 6.
  • Lidocaine alone without zymosan challenge did not alter peritoneal leukocyte numbers in this 4-24 h interval after administration.
  • lipoxin compounds can be utilized for expediting resolution at an anesthetized site post treatment. Ideally the lipoxin is administered prior to or during the procedure, however, post application can be undertaken as well. Background. Local and volatile anesthetics are widely used for surgery. It is not known whether anesthetics impinge on the orchestrated events in spontaneous resolution of acute inflammation. The present invention investigated whether a commonly used local anesthetic (lidocaine) and a widely used inhaled anesthetic (isoflurane) impact the active process of resolution of inflammation. Methods and Findings.
  • lidocaine delayed and blocked key events in resolution of inflammation.
  • Lidocaine inhibited both PMN apoptosis and macrophage uptake of apoptotic PMN, events that contributed to impaired PMN removal from exudates and thereby delayed the onset of resolution of acute inflammation and return to homeostasis.
  • Lidocaine did not alter the levels of specific lipid mediators, including pro-inflammatory leukotriene B 4 , prostaglandin E 2 and anti-inflammatory lipoxin A 4 , in the cell-free peritoneal lavages. Addition of a lipoxin A 4 stable analog, partially rescued lidocaine-delayed resolution of inflammation.
  • systematic proteomics was carried out using nanospray-liquid chromatography-tandem mass spectrometry. Lidocaine selectively up-regulated pro-inflammatory proteins including
  • lidocaine and isoflurane may underlie their opposite actions in resolution of inflammation, namely lidocaine delayed the onset of resolution (T max ), while isoflurane shortened resolution interval (Rz).
  • T max the onset of resolution
  • Rz isoflurane shortened resolution interval
  • Resolution agonists are agonists that not only block neutrophil (PMN) actions [5], but also stimulate non-phlogistic monocyte recruitment [6] and macrophage uptake of apoptotic PMN [7].
  • PMN neutrophil
  • resolution agonists have two main mechanisms of actions at the tissue level; they lower the numbers of infiltrating PMN to the inflamed sites and tissues; and they stimulate the active removal of debris and apoptotic PMN from the inflamed sites by non-phlogistic activation of macrophages [5]. Because it is important to study resolution of inflammation as a distinct process, we introduced resolution indices to a) quantitate the overall process; b) access the roles of specific mediators; and c) pinpoint mechanisms of pharmacological interventions in the resolution of inflammation.
  • duration the time point (T 50 ) when PMN numbers reduce to 50% of ⁇ max (R 50 ); and (iv) the resolution interval (R,): the time interval from the maximum PMN point ( ⁇ max ) to the 50% reduction point (R 50 ) [i.e. T 50 - T max ].
  • endogenous mediators such as resolvins and protectins accelerate resolution as evidenced by initiating the resolution of inflammation at earlier times (iT max and T 50 ) and/or shortening the resolution interval (iR,) [8,9].
  • the actions of these pro- resolution mediators sharply contrast those of agents and currently used therapeutics that are inhibitors and "resolution toxic".
  • this set of resolution indices can be utilized to evaluate the impact of endogenous mediators as well as potential new therapeutic agents in inflammatory resolution because they reflect the summation of tissue-level events that are multi-level cellular and molecular processes in resolution of inflammation.
  • lidocaine alters cellular infiltration in a self-limited spontaneously resolving murine peritonitis. For these analyses, we used our reported resolution map that was constructed using an unbiased systems approach that combined cell trafficking into inflammatory exudates and mass spectrometry-based proteomics and lipid mediator lipidomics of resolving exudates [8].
  • a microbial stimulus the yeast wall zymosan A
  • was administered intraperitoneally to initiate inflammation [15] was administered intraperitoneally to initiate inflammation [15], together with lidocaine given concomitantly.
  • lidocaine was introduced in order to determine if it significantly changed the signature of resolution map and indices in zymosan-initiated peritonitis.
  • Inflammatory exudates were collected at the indicated time intervals 4-72 h (Fig. IA).
  • Zymosan alone stimulated an acute increase in the total leukocyte numbers (i.e. PMN and mononuclear cells) present in the peritoneal exudates during the initial phase of inflammation (4 h after zymosan, 11.8 ⁇ 0.4 x 10 6 leukocytes), with a maximal infiltration at 12 h (30.0 ⁇ 2.5 x 10 6 leukocytes), followed by a decline or resolution as monitored to 72 h.
  • the time course of PMN infiltration followed a similar trend, peaking (17.5 ⁇ 2.5 x 10 6 PMN) at 12 h after zymosan challenge (Fig. IA).
  • lidocaine i.e. 0.08% (w/v) [16] administered with zymosan A significantly increased the number of total leukocytes by -49% within exudates at 4 h (p ⁇ 0.05).
  • the numbers of PMN continued to increase after 12 h and reached a maximum at 24 h.
  • the number of PMN in the exudate was significantly increased at this time point (-60% increase,
  • lidocaine directly delayed the spontaneous resolution of zymosan-initiated acute inflammation. Especially, lidocaine increased the dwell time of PMN present within the exudates, possibly blocking the clearance of PMN from the exudates in vivo (see below).
  • lidocaine is usually given before surgery [11,12].
  • mice were treated with lidocaine (0.08%) 15 min before initiation of acute inflammation by zymosan.
  • This prior exposure to lidocaine significantly potentiated zymosan-initiated leukocyte infiltration at 24 h by -40% (cf. zymosan alone, p ⁇ 0.0 ⁇ ).
  • lidocaine administration either just before or concomitant with zymosan, caused significant increases in the number of PMN present in exudates in the resolution phase of acute inflammation.
  • lipid mediators play a key role in resolution of inflammation [5] with some specifically switched on during the resolution phase to promote resolution [17].
  • key lipid mediators were monitored in murine exudates, including lipoxin (LX) A 4 , an antiinflammatory and pro-resolution mediator, and the pro-inflammatory LTB 4 and prostaglandin (PG) E 2 .
  • LX lipoxin
  • PG prostaglandin
  • the maximal levels present in cell-free lavages of the exudates of both LTB 4 and LXA 4 were obtained at 4 h. These subsequently subsided within 24 h (Fig. 2A). Lidocaine did not significantly alter the levels Of LXA 4 , LTB 4 or
  • PGE 2 present in these cell-free lavages of the peritoneal exudates.
  • these eicosanoids likely reflect the profile from resident peritoneal cells including macrophages as are less likely to report eicosanoids generated by the infiltrating leukocytes.
  • Lipoxins are potent agonists for resolution of inflamed tissues by regulating leukocyte infiltration, stimulating macrophage clearance of apoptotic PMN and also their exit via lymphatics [5,8,9]. Since LXA 4 can rescue inhibitor-imposed lesion with, for example, a selective COX-2 inhibitor [9], we questioned whether these resolution agonists impact leukocyte infiltration in lidocaine-treated mice. At 24 h, lidocaine (0.08%, -0.8 mg) administration increased, while ATLa (a stable analog for aspirin-triggered 15-epi-lipoxin A 4 ,
  • PMN apoptosis and their subsequent removal by macrophages are essential components of resolution at the tissue level [1,18]. Since lidocaine delayed PMN clearance in the resolution phase, we considered that lidocaine might have an impact on PMN apoptosis. To address this, peritoneal cells were collected at 24 h after zymosan challenge, well within the resolution phase, and labeled with FITC-annexin-V and PE-conjugated anti-
  • Gr-I Ab a specific cell surface marker for mouse PMN.
  • Peritoneal cells collected from mice receiving lidocaine (at both 0.08% and 0.008%) together with zymosan showed significantly decreased annexin- V + Gr- 1 + cells by 50% and 64%, respectively, indicating reduced PMN apoptosis (Fig. 3A).
  • lidocaine at 0.08% also reduced PMN apoptosis -40% (p ⁇ .01).
  • lidocaine impacts macrophage ingestion of PMNs.
  • a phagocytosis-based analysis in vivo Fig. 3B.
  • Exudate cells were collected at 24 h after zymosan challenge, and macrophages were labeled with the FITC-conjugated anti-F4/80 Ab. This was followed by permeabilization of these cells, allowing labeling of ingested PMN with PE-conjugated anti-Gr-1 Ab. Cells with positive staining of both F4/80 and Gr-I were then monitored by FACS analysis.
  • lidocaine has a direct impact on isolated macrophages.
  • pro-resolution mediators such as LXA 4 are potent stimulators of macrophage uptake of microbial particles, i.e., opsonized zymosan [9], in addition to stimulating the uptake of apoptotic PMN [7].
  • lidocaine at both doses 0.008% and 0.08%
  • lidocaine when added together with LXA 4 , significantly impaired LXA 4 -stimulated phagocytosis (Fig. 3C).
  • lidocaine can be considered "resolution toxic" because it impairs key components at the level of tissue resolution, namely PMN apoptosis and macrophage phagocytosis, and blocks the protective action of LXA 4 .
  • S100A8 and S100A9 are known to be abundant cytosolic proteins in human PMN that can be secreted and exhibit potent actions in inflammatory cell recruitment [20]. Also, SlOO proteins belong to a new group of damage-associated molecular pattern proteins and may function as "alarm/danger" signals to propagate inflammation [21]. To determine whether lidocaine impacts these proteins during inflammation-resolution, we carried out temporal- differential analysis of peritoneal exudate proteins collected from zymosan-challenged mice in the presence or absence of the anesthetic dose of lidocaine (0.08%).
  • S100A8/A9 complexes reflect, at least in part, the increases in PMN obtained in mice challenged with lidocaine and zymosan at 4 hours, compared to mice received zymosan alone.
  • lidocaine together with zymosan at 4 h gave significant up-regulation of several selective proteins compared to zymosan-challenged mice.
  • CRAMP cathelin-related anti -microbial peptide
  • the mouse homolog of antimicrobial protein LL-37 was increased approximately two-fold (Table 1).
  • CRAMP is also a documented chemotactic factor for PMN, monocytes, mast cells, and T cells [22].
  • exudate CRAMP/LL-37 may also contribute to increased PMN numbers obtained at 4 h in lidocaine-treated mice (Fig. IA).
  • lidocaine down-regulated galectin-1 -50% Fig. 4C and Table 1.
  • Galectin-1 inhibits PMN migration during PMN-endothelial interactions in vitro and in vivo [23].
  • Galectin-1 prolongs exposure of phosphatidylserine on the surface of leukocytes, suggesting a role in promoting PMN clearance [24]. Therefore, decreases in Galectin-1 levels from lidocaine-treated mice during resolution (i.e. 24 h) might also contribute to delayed PMN clearance, resulting in increased PMN dwell times in exudates (Fig. IA).
  • Lidocaine impacts chemical mediators in exudates
  • cytokines Production of both pro-inflammatory (e.g. IL-I ⁇ , IL-6, IL-12, TNF- ⁇ ) and antiinflammatory (e.g. IL-4, IL-10, and IL- 13) cytokines is essential in the control of inflammation [25].
  • pro-inflammatory e.g. IL-I ⁇ , IL-6, IL-12, TNF- ⁇
  • antiinflammatory e.g. IL-4, IL-10, and IL- 13
  • lidocaine gave similar results at 4 h, with significant, preferential reduction of anti-inflammatory cytokines, including IL-4, IL-10 and IL- 13 (Fig. 5B).
  • anesthetic dose of lidocaine decreased pro -inflammatory KC
  • lidocaine acts at several levels in acute inflammation, overall reducing what is coined the "cytokine/chemokine storm" observed in the early inflammatory response (4 h) (Fig. 5A).
  • lidocaine initially reduced the levels of most of the chemokines and cytokines induced by zymosan at 4 h, but increased their levels by 12 h (Table 2).
  • lidocaine-treated mice It is likely that the decreased levels of TGF- ⁇ contributed to impaired macrophage phagocytosis in lidocaine-treated mice (Fig. 3B), that can lead to delayed PMN clearance and their increased dwell time.
  • the impact of lidocaine was also evaluated in human whole blood ex vivo to access whether the murine system reflects human tissue events.
  • the anesthetic dose of lidocaine (0.08%) significantly diminished the levels of a panel of chemokines and cytokines in zymosan-stimulated human whole blood (Fig. 5D).
  • Isofiurane 1.4 MAC minimum alveolar concentration
  • Isoflurane significantly reduced zymosan-stimulated leukocyte infiltration at 12, 24 and 48 h (Fig. 6A).
  • T max isoflurane decreased maximal PMN numbers ( ⁇ ma ⁇ ) from 18.0 x 10 6 to 13.5 x 10 6 .
  • isoflurane dramatically reduced
  • isoflurane reduced cofilin-1 (Table 5), a major actin-depolymerization factor regulating actin dynamics and generation and maintenance of cell protrusions, key cellular events that are required for migration [26]. Therefore, during resolution of inflammation, isoflurane selectively regulates cellular proteins that are involved in cell migration and chemotaxis (i.e., CRAMP and cofilin-1). By comparison, isoflurane treatment increased SH3 domain-binding glutamic acid-rich like protein (SH3BGRL), which might have anti-oxidative and anti-inflammatory properties
  • mice selectively reduced zymosan-stimulated pro-inflammatory cytokine levels (IL- l ⁇ , IL-6, IL- 12, KC, JE [the mouse homolog of human MCP-I], MIP-Ia and Rantes) (Fig. 7B, Tables 6 and 7), but did not apparently affect the levels of cytokines IL-4, IL-IO and IL- 13 in the early inflammatory phase, 4 h after zymosan challenge (Fig. 7B).
  • zymosan-stimulated pro-inflammatory cytokine levels IL- l ⁇ , IL-6, IL- 12, KC, JE [the mouse homolog of human MCP-I], MIP-Ia and Rantes
  • lidocaine influences the immune system by reducing responses such as chemotaxis, microtubule assembly, phagocytosis, release of lysosomal enzymes and superoxide anion generation [31-33].
  • lidocaine can reduce inflammatory responses and protect tissues from local injury [34].
  • lidocaine worsens renal injury following ischemia-reperfusion by increasing necrosis and local inflammation [35].
  • lidocaine increases leukocyte numbers, which suggests an increase in PMN infiltration and/or increased viability of the leukocytes at the burn site [36]. Yet the clinical significance of these observations remains to be established.
  • lidocaine imposes a molecular lesion in resolution that delays the return to homeostasis. Specifically, lidocaine increases leukocyte accumulation in exudates, impairs the apoptosis of PMN and hampers ingestion of apoptotic PMN by macrophages in vivo. Summation of these multi-level actions in tissues significantly delays resolution of inflammation.
  • Aspirin for example, by way of initiating biosynthesis of endogenous lipid mediators (i.e., aspirin-triggered epimer of lipoxin A 4 [ATL]), promotes resolution [4,9]. Cyclin-dependent kinase and specific ERKl /2 inhibitors, in comparison, also promote resolution of inflammation by enhancing PMN apoptosis [37,38]. In contrast, COX or LOX inhibitors, by blocking the biosynthesis of key lipid mediators, dramatically impairs resolution [9,10]. In the peritoneal cell-free lavages, LXA 4 appeared in the early inflammatory phase, 4 h after zymosan challenge. PGE 2 , a signal that can activate the full
  • LXA 4 -biosynthetic capacity in vivo [17] was present in the peritoneum prior to peritonitis and elevated during the acute inflammatory response. Lidocaine did not alter either the magnitude or time course OfLXA 4 in a statistically significant fashion compared to the mice given zymosan alone. However, a trend towards reduction was observed at 4, 12 and 48 h. Of interest, when exogenous ATLa (a stable analog Of LXA 4 and ATL) was given together with lidocaine, it significantly reversed in part lidocaine' s delaying effects in the resolution of inflammation. Thus, pro-resolution mediators may have therapeutic potential in settings where sustained inflammation and impaired resolution are components of disease pathophysiology.
  • GABAA GABAA receptors
  • fMLP GABA reduced formyl peptide
  • SH3BGRL and other thioredoxin-like proteins might have anti-inflammatory properties, and contribute to the accelerated resolution in isoflurane-treated mice documented in the present report (Fig. 6).
  • LXA 4 stimulates IL-IO [9] as well as heme oxygenase- 1 [46,47] and, as indicated in the present report, was able to partially rescue the lidocaine-delayed resolution of inflammation (Fig.
  • Isoflurane and lidocaine gave opposite effects in the resolution of acute inflammation, as indicated by their differential impact in the resolution indices (Table 4). This reflects their distinct and selective impact on specific molecules involved in resolution of inflammation. For example, CRAMP protein levels were decreased in mice with isoflurane, contrasting with significant increases in CRAMP at 24 h as evoked by lidocaine, compared with mice given zymosan alone. Thus, it is likely that the reported chemotactic property of CRAMP [22] contributes to the observed opposing actions of isoflurane and lidocaine on peritoneal leukocyte infiltration. Also, isoflurane selectively reduced zymosan-stimulated pro- inflammatory cytokine levels (Fig.
  • the phagocytic index was defined in the cellular era and was used to determine the average number of bacteria ingested by phagocyte at single-cell level [48,49].
  • the resolution indices presented here expand the appreciation of the complexity of phagocytes at the tissue level and account for the summation of multi-level cellular and molecular events during resolution of inflammation.
  • the results of the recent study indicate that the local anesthetic lidocaine delays the onset of resolution.
  • lidocaine The impact of lidocaine is documented herein at multi-levels in resolution and reflects (i) increased exudate PMNs, (ii) impaired PMN apoptosis as well as their uptake by macrophages, (iii) modulating both pro- and anti-inflammatory proteins, including cytokines and chemokines. Dysregulation of resolution programs by lidocaine may have important unwanted consequences in both immune responses and host defense that were previously unappreciated.
  • lidocaine treatment male FVB mice (6-8 weeks; Charles River, Wilmington, MA) were administered lidocaine (0.08% or 0.008%) intraperitoneally together with 1 mg/ml zymosan A (i.p.) to evoke peritonitis [8] as in accordance with the Harvard Medical Area Standing Committee on Animals (protocol no. 02570).
  • ATLa a stable analog of aspirin-triggered
  • LXA 4 was prepared by total organic synthesis in the Organic Synthesis Core (P50- DEOl 6191).
  • mice were administered 1.4 MAC [50] of isoflurane for a 2 h period (from Ih before to 1 h after injection of zymosan, i.p.) (see timeline in Fig. 6).
  • mice were euthanized with an overdose of isoflurane, and peritoneal exudates were collected by lavaging with 5 ml sterile saline. Exudate cells and supernatants were obtained for analyses described below.
  • Venous blood (anticoagulated with 10 U/ml sodium heparin) was collected from healthy non- smoking volunteers who declared not to have taken any drugs for at least two weeks before the experiments. Informed consent was obtained from each volunteer. The protocol was approved by the Brigham and Women's Hospital Institutional Review Board (protocol no. 88-02642, approved 11/26/07). Heparinized whole blood was then incubated with either 0.008% or 0.08% of lidocaine in the presence of zymosan A (100 ⁇ g/ml) for 4 h, and plasma was collected by centrifugation at 2,000 rpm for 15 min.
  • zymosan A 100 ⁇ g/ml
  • the amounts of cytokines and chemokines levels were determined by multiplexed sandwich ELISA (SearchLight Proteome Array custom-designed by Pierce Boston Technology Center). Following a standard sandwich ELISA procedure, the entire plate is imaged to capture chemiluminescent signals generated at each spot within each well of the array.
  • the SearchLight CCD Imaging and Analysis System features image analysis software that calculates chemokine/cytokine concentrations (pg/ml) using pre-determined standard curves.
  • exudate cells were labeled with FITC-conjugated anti-annexin-V Ab (0.5 ⁇ g Ab/0.5xl0 6 cells, eBioscience) and PE-conjugated anti-mouse Gr-
  • Triton X-100 100 ⁇ l, 10 min. Permeabilized cells were then stained with PE- conjugated anti-mouse Ly-6G (0.5 ⁇ g/0.5xl0 6 cells). The F4/80 + Gr-l + cell population was determined by FACS.
  • murine peritoneal resident macrophages were collected and plated onto 24-well plates (lxl ⁇ 5 cells/well) and incubated with lidocaine (0.008% or 0.08%), LXA 4
  • FITC-zymosan (1 nM; Calbiochem) or both for 20 min.
  • FITC-zymosan 2.5 ⁇ l/well was then added to macrophages for 30 min. Supernatant was aspirated and extracellular fluorescence was quenched by adding trypan blue for 1 min. Cells were then washed and intracellular fluorescence was determined by a fluorescent plate reader.
  • the cell pellets were directly solubilized in the same lysis solution through sonication at 4 0 C.
  • the protein concentrations were measured in duplicate by a Bradford protein assay kit (Bio-Rad, Hercules, CA) in a 96-well plate format using bovine serum albumin as the standard.
  • Supernatant (25 ⁇ g) or cellular (50 ⁇ g) proteins from each animal were mixed with 125 ⁇ L of rehydration buffer containing 8M urea, 2% (w/v) CHAPS, 10 mM DTT, and 0.2% carrier ampholytes (pH 3-10), and then loaded onto nonlinear 7-cm, pH 3-10, IPG strips (Bio-Rad) through passive in-gel rehydration overnight. After iso-electric focusing for 10,000 V-h, the proteins in the IGP strips were reduced with dithiothreitol and alkylated with iodoacetamide.
  • the 2 nd dimension separation was then carried out using 10-14% SDS-PAGE (covering -MWlO to 200 kDa). Gels were stained with ProteomlQTM blue dye (Proteome Systems, Woburn, MA), and scanned with a GS-800 densitometer system (Bio-Rad). Image analysis was carried out with PDQuest software (version 8.0) (Bio-Rad). The differentially regulated protein spots were selected based on the normalized spot volumes.
  • LC-MS-MS proteomics The selected protein spots were excised and in-gel digested with sequencing grade trypsin (Promega, Madison, WI). Tryptic peptides were loaded onto a 2 ⁇ g capacity peptide trap (CapTrap; Michrom Bioresources, Auburn, CA) in 0.1% formic acid and 0.05% trifluoroacetic acid and separated by capillary liquid chromatography using a capillary column (75 ⁇ m x 5 cm x 3 ⁇ m; LC Packings, Amsterdam, The Netherlands) at 150 nl/min delivered by an Agilent 1 IOOLC pump (400 ⁇ l/min) and a flow splitter (Accurate, LC Packings).
  • CapTrap CapTrap; Michrom Bioresources, Auburn, CA
  • a mobile phase gradient was run using mobile phase A (2% acetonitrile/0.1 % formic acid), and B (80% acetonitrile/0.1% formic acid) from 0-10 min with 0-20% B followed by 10-90 min with 20-60% B.
  • Peptide mass and charge was determined on a
  • ThermoFinnigan Advantage ion-trap mass spectrometer (San Jose, CA) after electrospray ionization using end-coated spray Silicatip tip (ID 75 ⁇ m, tip ID 15 ⁇ m, New Objective) held at a spray voltage of 1.8 kV.
  • zoom scans and tandem mass spectra of parent peptide ions above a signal threshold of 2x10 4 were recorded with dynamic exclusion, using Xcalibur 1.3 data acquisition software (ThermoFinnigan).
  • Protein identification Proteins were identified by peptide mapping of tryptic peptide tandem mass spectra using TurboSequest (BioWorks 3.1 software, ThermoFinnigan against indexed Swiss-Prot protein database). Protein modifications that were taken into consideration included methionine oxidation and alkylation of cysteine with iodoacetamide. The search results were filtered by X C01T vs. charge with 1.5 for singly charged ions, 2.0 for doubly charged ions, and 2.5 for triply charged ions. A protein was considered identified when a minimum of two tryptic peptides were matched.
  • TGF-D levels was determined with ELISA using a monoclonal anti-TGF- ⁇ antibody (R&D Systems, Minneapolis, MN) recognizing the active forms of TGF- ⁇ (1, 2, and 3).
  • Eicosanoid ELISAs (LTB 4 , LXA 4 and PGE 2 ) were carried out following manufacturer's instructions (Neogen, Lexington, KY).
  • membrane After washing three times with TBST, membrane were incubated with HRP-linked anti-goat IgG (1 :5,000 dilution) for 1 h and the immunoreactive bands were developed by incubating with chemiluminescence substrates and visualized by exposure to an X-ray film.
  • PCR polymerase chain reactions
  • HotStar Master mix Qiagen
  • specific primers for mouse S100A9 sense: 5'- CCCTGACACCCTGAGCA AGAAG-3' and antisense 5'- TTTCCCAGAACAAAGGCCATTGAG-3 '.
  • Relative intensities of RT-PCR products were quantified and normalized by D-actin message levels using the public domain NIH image program (developed at the NIH, available on the Internet).
  • lipoxins and lipoxin analogs useful as therapeutic agents in treatment of the maladies described throughout this specification have the formulae encompassed by U.S. Patents 4,560,514, 5,441,951, 5,648,512, 5,650,435, 6,048,897 and 6,627,658, the contents of which are incorporated herein by reference in their entirety.
  • lipoxin analogs encompassed by the present invention include those having the following characteristics.
  • R 2 can be
  • the lipoxin analogs of this invention have the following structural formula I:
  • X is Ri, OR.i, or SRi; wherein Rj is (i) a hydrogen atom;
  • R a Q 2 R b wherein Q 2 is -O- or -S-; wherein R 3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R 4 is
  • R a Q a R b wherein Q a is O or S; wherein R a is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched;
  • the lipoxin analogs have the following structure II:
  • X is Ri, ORi, or SRi ; wherein Ri is (i) a hydrogen atom;
  • Q 2 is -O- or -S-; wherein R a is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R 4 is
  • R 5 is (a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
  • haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
  • the lipoxin analogs have the following structure III:
  • X is R 1 , ORi, or SRi ; wherein Ri is (i) a hydrogen atom;
  • Q 2 is -O- or -S-; wherein R 3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R 4 is
  • R 5 is (a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
  • lipoxin analogs have the following structural formula IV:
  • X is Ri, OR], or SRi ; wherein Rj is (i) a hydrogen atom;
  • R 5 is (a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
  • Q 3 is-O- or -S-; wherein R 3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched;
  • haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched; and wherein R 6 is (a) a hydrogen atom; (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; or
  • lipoxin analogs have the following structural formula V:
  • Ri is (i) a hydrogen atom
  • R 2 , R 3a , and R 3b are each independently: (a) a hydrogen atom; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
  • Q 2 is -O- or -S-; wherein R a is alkyl ene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y 2 is -OH, methyl, hydrogen, or — SH and wherein the other is
  • Z 1 , Z 11 , Z 111 , Z 1V and Z v are each independently selected from -NO 2 , -CN, -
  • lipoxin analogs have the structural formula VI:
  • R 3 is (a) a hydrogen atom
  • lipoxin analogs have the following structural formula VII:
  • the lipoxin analogs have the structural formula VIII:
  • the compounds have the structural formula X:
  • the compounds have the structural formula XI:
  • Q 2 is — O — or — S — ; wherein R a is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi is — OH, methyl, or — SH; wherein Y 2 is
  • Z 1 , Z 11 , Z 111 , Z lv and Z v are each independently selected from -NO 2 , -CN, -
  • Z 1 through Z v are as defined above; (d) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
  • the compounds of this invention have the following structural formulas:
  • R' is H or CH 3 ; and where the substituents at C* are in the R configuration.
  • the compounds of this invention have the following structural formulas:
  • lipoxins and lipoxin analogs useful to treat conditions described throughout the specification has the formula:
  • X is Ri, ORi, or SRi; wherein Ri is
  • R 3 Q 2 R b wherein Q 2 is -O- or -S-; wherein R a is alkyl ene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when R b is 0, then R b is a hydrogen atom; wherein R 4 is
  • Z 1 , Z 11 , Z 111 , Z 1V and Z v are each independently selected from -NO 2 , -CN, -
  • R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;
  • lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
  • X is Ri, ORi, or SRi; wherein Ri is
  • R 3 Q 2 R b wherein Q 2 is -O- or -S-; wherein R 3 is alkylene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when R b is 0, then R b is a hydrogen atom; wherein R 4 is
  • lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
  • X is Ri, ORi, or SRi; wherein Ri is
  • R a Q 2 R b wherein Q 2 is -O- or -S-; wherein R a is alkylene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein R b is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when R b is 0, then R b is a hydrogen atom; wherein R 4 is
  • Z 1 , Z n , Z m , Z, v and Z v are each independently selected from -NO 2 , -CN, -
  • R x is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group; wherein R 6 is (a) H;
  • lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
  • X is Ri, ORj, or SRi; wherein Ri is
  • lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
  • X is Ri, ORi, or SRi wherein Ri is
  • para-fluorophenyl and/or unsubstituted phenyl are preferred, e.g., 15- epi- 16-(para-fluoro)-phenoxy-LXA 4 , 16-(para-fluoro)-phenoxy-LXA 4 , 15-epi-l 6-phenoxy- LXA 4 or 16-phenoxy-LXA 4 .
  • the present invention is directed to pharmaceutical compositions including compounds having the formulae described throughout the specification and a pharmaceutically acceptable carrier.
  • a preferred compound is
  • Qi is a carbonyl
  • X is a hydroxyl or an -OR, wherein R is an alkyl group, i.e., methyl or ethyl groups, and R 4 is a hydrogen atom.
  • Yi is a hydroxyl and the carbon bearing the hydroxyl can have an R or S configuration.
  • the chiral carbon bearing the hydroxyl group e.g., Yi 1 is designated as a 15-epi-lipoxin as is known in the art.
  • the chirality of the carbons bearing the R 2 , R 3 , Q 3 and Q 4 groups can each independently be either R or S.
  • Q 3 and Q 4 have the chiralities shown in above-referenced structures.
  • R 4 is a hydrogen. In other preferred embodiments, R 6 is a hydrogen.
  • R 5 can be a substituted or unsubstituted, branched or unbranched alkyl group having between 1 and about 6 carbon atoms, preferably between 1 and 4 carbon atoms, most preferably between 1 and 3, and preferably one or two carbon atoms.
  • the carbon atoms can have substituents which include halogen atoms, hydroxyl groups, or ether groups. 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.
  • 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 diastereisomers as well as the racemic and optically resolved isomers.
  • the compounds useful in the present invention can be prepared by the following synthetic scheme: hydrogenation ⁇
  • acetylenic fragment can be prepared by the methods discussed in Nicolaou, K.C. et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100; Nicolaou, K.C. et al. (1989) J. Org. Chem. 54:5527; Webber,
  • the second fragment can be prepared by the methods of Raduchel, B. and Vorbruggen, H. (1985) Adv.
  • a “lipoxin analog” shall mean a compound which has an "active region” that functions like the active region of a "natural lipoxin", but which has a “metabolic transformation region” that differs from natural lipoxin.
  • Lipoxin analogs include compounds which are structurally similar to a natural lipoxin, compounds which share the same receptor recognition site, compounds which share the same or similar lipoxin metabolic transformation region as lipoxin, and compounds which are art-recognized as being analogs of lipoxin.
  • Lipoxin analogs include lipoxin analog metabolites.
  • the compounds disclosed herein may contain one or more centers of asymmetry. Where asymmetric carbon atoms are present, more than one stereoisomer is possible, and all possible isomeric forms are intended to be included within the structural representations shown.
  • Optically active (R) and (S) isomers may be resolved using conventional techniques known to the ordinarily skilled artisan. The present invention is intended to include the possible diastereomers as well as the racemic and optically resolved is
  • corresponding lipoxin and "natural lipoxin” refer to a naturally-occurring lipoxin or lipoxin metabolite.
  • an analog has activity for a lipoxin-specific receptor, the corresponding or natural lipoxin is the normal ligand for that receptor.
  • the corresponding lipoxin is LXA 4 .
  • an analog has activity as an antagonist to another compound (such as leukotriene C4 and/or leukotriene D4), which is antagonized by a naturally-occurring lipoxin, that natural lipoxin is the corresponding lipoxin.
  • Active region shall mean the region of a natural lipoxin or lipoxin analog, which is associated with in vivo cellular interactions.
  • the active region may bind the "recognition site" of a cellular lipoxin receptor or a macromolecule or complex of macromolecules, including an enzyme and its cofactor.
  • lipoxin A 4 analogs have an active region comprising C 5 — C 15 of natural lipoxin A 4 .
  • lipoxin B 4 analogs have an active region comprising C5-C14 of natural lipoxin B4.
  • a receptor may be isolated, on an intact or permeabilized cell, or in tissue, including an organ.
  • a receptor may be from or in a living subject, or it may be cloned.
  • a receptor may normally exist or it may be induced by a disease state, by an injury, or by artificial means.
  • a compound of this invention may bind reversibly, irreversibly, competitively, noncompetitively, or uncompetitively with respect to the natural substrate of a recognition site.
  • metabolic transformation region is intended to refer generally to that portion of a lipoxin, a lipoxin metabolite, or lipoxin analog including a lipoxin analog metabolite, upon which an enzyme or an enzyme and its cofactor attempts to perform one or more metabolic transformations which that enzyme or enzyme and cofactor normally transform on lipoxins.
  • the metabolic transformation region may or may not be susceptible to the transformation.
  • a nonlimiting example of a metabolic transformation region of a lipoxin is a portion Of LXA 4 that includes the C- 13, 14 double bond or the C-15 hydroxyl group, or both.
  • detectable label molecule is meant to include fluorescent, phosphorescent, and radiolabeled molecules used to trace, track, or identify the compound or receptor recognition site to which the detectable label molecule is bound.
  • the label molecule may be detected by any of the several methods known in the art.
  • labeled analog is further understood to encompass compounds which are labeled with radioactive isotopes, such as but not limited to tritium ( 3 H), deuterium ( 2 H), carbon ( 14 C), or otherwise labeled (e.g. fluorescently).
  • the compounds of this invention may be labeled or derivatized, for example, for kinetic binding experiments, for further elucidating metabolic pathways and enzymatic mechanisms, or for characterization by methods known in the art of analytical chemistry.
  • inhibitors metabolism means the blocking or reduction of activity of an enzyme which metabolizes a native molecule.
  • the blockage or reduction may occur by covalent bonding, by irreversible binding, by reversible binding which has a practical effect of irreversible binding, or by any other means which prevents the enzyme from operating in its usual manner on another lipoxin analog, including a lipoxin analog metabolite, a lipoxin, or a lipoxin metabolite.
  • resists metabolism is meant to include failing to undergo one or more of the metabolic degradative transformations by at least one of the enzymes which metabolize lipoxins.
  • Two nonlimiting examples Of LXA 4 analog that resists metabolism are 1) a structure which can not be oxidized to the 15-oxo form, and 2) a structure which may be oxidized to the 15-oxo form, but is not susceptible to enzymatic reduction to the 13,14- dihydro form.
  • the term "more slowly undergoes metabolism” means having slower reaction kinetics, or requiring more time for the completion of the series of metabolic transformations by one or more of the enzymes which metabolize lipoxin or lipoxin analogs.
  • a nonlimiting example of a LXA 4 analog which more slowly undergoes metabolism is a structure which has a higher transition state energy for C- 15 dehydrogenation than does LXA 4 because the analog is sterically hindered at the C- 16.
  • tissue is intended to include intact cells, blood, blood preparations such as plasma and serum, bones, joints, muscles, smooth muscles, and organs.
  • halogen is meant to include fluorine, chlorine, bromine and iodine, or fluoro, chloro, bromo, and iodo.
  • subject is intended to include living organisms susceptible to conditions or diseases caused or contributed bacteria and pathogens 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.
  • 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 therapeutic agent, in combination with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier 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.
  • 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'
  • 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.
  • pharmaceutically acceptable salts, esters, amides, and prodrugs 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.
  • 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.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like
  • non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like See, for example, Berge S. M., et al., "Pharmaceutical Salts," J. Pharm. ScL, 1977;66:1-19 which is incorporated herein by reference).
  • esters refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.
  • wetting agents 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.
  • antioxidants examples 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.
  • 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
  • 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.
  • 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. Pn 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.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • 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;
  • 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 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.
  • compositions 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.
  • 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.
  • 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 -butyl ene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solub
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • aqueous and nonaqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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.
  • 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 may be 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.
  • 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, chlorobutan
  • 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.
  • 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.
  • 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.
  • systemic administration means 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.
  • 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 maybe 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions of the invention include a "therapeutically effective amount” or a “prophylactically effective amount” of one or more of the therapeutic agent(s) 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 therapeutic agent 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.
  • 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 therapeutic agent 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 therapeutic agent of the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg.
  • 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.
  • the therapeutic agents of the present invention can be administered to the lung in the form of an aerosol of particles of respirable size (less than about 10 ⁇ m in diameter).
  • the aerosol formulation can be presented as a liquid or a dry powder.
  • particles can be prepared in respirable size and then incorporated into the suspension formulation containing a propellant.
  • 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.
  • 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 a therapeutic agent of the invention 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 VistalonTM (Exxon), RoyaleneTM (UniRoyal), bunaEP (Bayer).
  • diaphragms fashioned by extrusion, injection molding or compression molding from a thermoplastic elastomeric material such as FLEXOMERTM GERS 1085 NT polyolefin (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.
  • a condition susceptible of treatment by inhalation e.g., asthma, chronic obstructive pulmonary disease, etc. as described throughout the specification.
  • 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 therapeutic formulation contained within the packaging material.
  • This formulation contains an at least one therapeutic agent 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).
  • Suitable therapeutic agents include, for example, the lipoxin analogs described herein.
  • the invention features an article of manufacture that contains packaging material and at least one therapeutic agent 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.
  • the present invention provides a method for the increase of resolution in a subject's tissue subjected to an anesthetic, comprising the step of administering a therapeutically effective amount of lipoxin A 4 or a lipoxin analog, such that the subject's tissue subjected to the anesthetic resolve more quickly than without administration of a lipoxin A 4 or a lipoxin analog.
  • Gallos G Jones DR, Nasr SH, Emala CW, Lee HT (2004) Local anesthetics reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis.
  • Anesthesiology 101 902-911.

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Abstract

Use of lipoxin compounds for the resolution of tissues treated with a anesthetic is described. Lipoxins help facilitate the resolution phase of healing post treatment with an anesthetic. Pretreatment of the anesthetized site with a lipoxin helps to promote resolution of the tissue relative to untreated tissue.

Description

USE OF LIPOXINS TO COUNTERACT THE IMPACT OF ANESTHETIC ON
INFLAMMATORY RESOLUTION
CROSS-REFERENCE TO RELATED APPLICATION(S)
This application claims benefit under 35 U. S. C. § 119(e) to U.S. Serial No. 61/033,656, entitled "Use of Lipoxins to Counteract the Impact of Anesthetic on
Inflammatory Resolution" (attorney docket number 190141/US), filed March 4, 2008 by Charles N. Serhan, the contents of which are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This work was supported in part by NIH grants GM 38765 and P50-DE016191 and the German Research Council (DFG, Schw 1064/1-1). The U.S. Government therefore may have certain rights in the invention.
BACKGROUND
Anesthetics are generally administered when an individual undergoes surgery. Inflammation and would healing occur post surgery. It has not been well understood what impact the use of anesthetics have on the resolution at surgical sites. Often, the resolution at surgical sites can take significant periods of time until the patient is fully recovered. Therefore, a need exists for expediting resolution at surgical sites post surgery.
SUMMARY
The present invention provides a surprising method for the increase of resolution (healing) in a subject's tissue subjected to an anesthetic. The method comprises administering a therapeutically effective amount of lipoxin or a lipoxin analog to the subject prior to or during surgery, such that the subject's tissue subjected to the anesthetic resolve (heal) more quickly than without administration of a lipoxin or a lipoxin analog.
It has been surprisingly found that the administration of lipoxins prior to or during surgery, while under anesthesia, greatly reduces the time for the tissue to resolve. BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Figure 1. Lidocaine alters leukocyte infiltration during acute inflammation and delays resolution. (A) Mice were injected with zymosan A in the absence or presence of lidocaine (0.008% or
0.08%) and peritoneal lavages were collected at indicated time points. Total leukocytes were enumerated by light microscopy, and PMN and mononuclear cells determined by differential leukocyte counting. Results are expressed as the mean ± SEM from n=3-4. *p<0.05, **p<0.01, ***p<0.001 when compared to mice treated with zymosan A alone at the same time points. (B) Mice were injected with lidocaine (0.08%) 15 min prior to injection of zymosan A. Peritoneal lavages were collected at 24 h, and total leukocytes enumerated. Results are expressed as mean ± SEM from n=3. */><0.05, **/?<0.01 when compared to mice treated with zymosan A alone.
Figure 2. Lidocaine did not directly alter selective eicosanoid levels in cell-free exudates:
LXA4 rescues lidocaine-delayed resolution.
(A) Cell-free lavages from murine peritoneum were collected at indicated time points after zymosan challenge (1 mg/ml). LXA4, LTB4 and PGE2 amounts were determined by ELISA. Results are expressed as the mean ± SEM from duplicates of n=3, and were expressed as amounts (ng/ml). (B) Mice were injected with zymosan A together with lidocaine (0.08%),
ATLa (300 ng), or lidocaine plus ATLa. Peritoneal lavages were collected at 24 h, and total leukocytes enumerated. Results are expressed as mean ± SEM from n=3. *p=0.03 **p=0.01 when compared to mice treated with zymosan A alone. ***p=0.04 when compared to mice treated with zymosan A and lidocaine.
Figure 3. Lidocaine impairs PMN apoptosis and macrophage ingestion of PMN in vivo and zymosan in vitro.
(A) Apoptosis in vivo. Peritoneal cells were collected at 24 h or 48 h and labeled with FITC- annexin-V and PE-conjugated anti-Gr-1 Ab. The apoptotic PMN (annexin- V+Gr- 1+) are expressed as % of total PMN (Gr-I+). Results are the mean ± SEM from n=3-4. *p<0.0\ ,
**p<0.001. (B) Phagocytosis in vivo, {right) Representative dot plots of FACS analysis. In the non-permeabilized lavage cells, Gr-I+ represents PMN, and F4/80+ represents macrophages; and in the permeabilized cells, F4/80+Gr-l+ cell population represents macrophages with ingested PMN. {left) Results are expressed as the mean ± SEM from n=3- 4, and were expressed as percent of the F4/80+Gr-l+ cells. */><0.05. (C) Phagocytosis in vitro. Murine peritoneal resident macrophages were incubated with indicated compounds or vehicle alone for 20 min followed by addition of FITC-zymosan at a 10:1 ratio for 30 min. Cells were then quenched and fluorescence determined. Phagocytosis activity in the presence of 1 nM OfLXA4 was taken as 100%. Results are expressed as the mean ± SEM from n=3-4, and were expressed as % phagocytosis. */?<0.05, **p<0.01, compared to LXA4 alone.
Figure 4. Lidocaine alters pro- and anti-inflammatory proteins: proteomics and cellular proteins.
Mice were injected with zymosan A in the absence or presence of lidocaine. Both lavage fluids (A) and cell pellets (C) were collected at indicated time points and proteins separated by two-dimensional gel electrophoresis. Changes in individual protein levels were measured by image analysis. Selected proteins that display significant differences between treatments are indicated by arrows, and identified by LC/MS/MS and peptide mapping (see Materials and Methods).
(B) (Left) SlOO A9 protein levels. Supernatants from peritoneal lavages were subjected to Western blot analysis using an anti-S100A9 antibody. Relative intensities of immunoreactive bands were quantitated and normalized by albumin levels using an anti-albumin antibody. Data are expressed as mean ± SEM from n=3-4. *jc=0.02. (Right) SlOO A9 mRNA levels.
Peritoneal cells were collected and total RNA isolated for RT-PCR analysis using specific primers for mouse S100A9. Relative intensities of RT-PCR products were quantitated and normalized by b-actin message levels. Data are expressed as mean ± SEM from n=3-4.
*p<0.01.
Figure 5. Lidocaine regulates selective pro- and anti-inflammatory cytokines/chemokines.
(A, B) Mice were injected with zymosan alone or together with lidocaine (0.008% or 0.08%) for 4 h, and peritoneal cell-free lavage fluids collected. Cytokines and chemokines were expressed as (A) pg/ml or ng/ml in naϊve mice and mice treated with zymosan alone, (B) percent inhibition of zymosan A-induced cytokine/chemokine levels by lidocaine. The amounts of cytokines and chemokines levels were determined by multiplexed sandwich ELISA. (C) TGF-b (active form) levels were determined by ELISA. (D) Human heparinized whole blood was incubated with either 0.008% or 0.08% of lidocaine in the presence of zymosan A (100 μg/ml) for 4 h, and plasma was collected. The amounts of cytokines and chemokines levels were determined by multiplexed sandwich ELISA. Results are the mean from duplicate determinations of n=3-4. *p<0.05 when compared to mice treated with zymosan A alone (B, C) or human whole blood incubated with zymosan A alone (D).
Figure 6. Volatile anesthetic isoflurane reduces leukocyte infiltration and promote resolution by shortening resolution interval.
Mice were administered 1.4 MAC of isoflurane one hour prior to and after injection of zymosan A (lmg/ml, i.p.) (see timeline). The peritoneal lavages were collected at indicated time points. (A) Total leukocytes were enumerated by light microscopy, and PMN and mononuclear cells determined by differential leukocyte counting. Results are expressed as the mean ± SEM from n=3-4. *P<0.05 when compared to mice treated with zymosan A alone at the same intervals. (B) Resolution Indices were calculated with isoflurane as in Figure 8. Isoflurane treatment reduces the magnitude (Ψmax) of inflammation and accelerates resolution by shortening the resolution interval (R/.).
Figure 7. Isoflurane regulates cellular proteins - proteomic analysis.
Mice were administered 1.4 Mac of isoflurane one hour prior to and after injection of zymosan A (lmg/ml, i.p.). (A) The peritoneal lavage cells were collected at indicated time points and proteins separated by two-dimensional gel electrophoresis. Changes in individual protein levels were measured by image analysis. Selected proteins that display significant differences between treatments are denoted, and were identified by LC/MS/MS and peptide mapping. (B) Peritoneal cell-free lavage fluids were collected. Cytokine and chemokine levels were determined and expressed as percent inhibition of zymosan A-induced cytokine/chemokine levels by isoflurane. *p<0.05 when compared to mice treated with zymosan A alone. For raw values (pg/ml) of these selective cytokines, see Table 6.
Figure 8. Zymosan-initiated Peritonitis.
A resolution map is provided that defines the main quantitative indices. Figure 9. Lidocaine without Zymosan Challenge
Lidocaine alone without zymosan challenge did not alter peritoneal leukocyte numbers in this 4-24 h interval after administration.
Figure 10. Zymosan-initiated Peritonitis with Lidocaine Zymosan-initiated peritonitis causes the number of PMN to reach a maximum at 12 h.
DETAILED DESCRIPTION
The features and other details of the invention will now be more particularly described and pointed out in the claims. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. The principle features of this invention can be employed in various embodiments without departing from the scope of the invention.
Abbreviations used throughout the present application include the following and are included here for convenience. ATL, aspirin-triggered 15-epi-LX, 15 R-LX; ATLa, 15-epi- 16-(para-fluoro)-phenoxy-lipoxin A4
In the specification and in the claims, the terms "including" and "comprising" are open-ended terms and should be interpreted to mean "including, but not limited to. . . . " These terms encompass the more restrictive terms "consisting essentially of and "consisting of. It must be noted that as used herein and in the appended claims, the singular forms
"a", "an", and "the" include plural reference unless the context clearly dictates otherwise. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", "characterized by" and "having" can be used interchangeably. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes including describing and disclosing the chemicals, instruments, statistical analyses and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
It has been surprisingly discovered that lipoxin compounds, discussed infra, can be utilized for expediting resolution at an anesthetized site post treatment. Ideally the lipoxin is administered prior to or during the procedure, however, post application can be undertaken as well. Background. Local and volatile anesthetics are widely used for surgery. It is not known whether anesthetics impinge on the orchestrated events in spontaneous resolution of acute inflammation. The present invention investigated whether a commonly used local anesthetic (lidocaine) and a widely used inhaled anesthetic (isoflurane) impact the active process of resolution of inflammation. Methods and Findings. Using murine peritonitis induced by zymosan and a systems approach, we report that lidocaine delayed and blocked key events in resolution of inflammation. Lidocaine inhibited both PMN apoptosis and macrophage uptake of apoptotic PMN, events that contributed to impaired PMN removal from exudates and thereby delayed the onset of resolution of acute inflammation and return to homeostasis. Lidocaine did not alter the levels of specific lipid mediators, including pro-inflammatory leukotriene B4, prostaglandin E2 and anti-inflammatory lipoxin A4, in the cell-free peritoneal lavages. Addition of a lipoxin A4 stable analog, partially rescued lidocaine-delayed resolution of inflammation. To identify protein components underlying lidocaine' s actions in resolution, systematic proteomics was carried out using nanospray-liquid chromatography-tandem mass spectrometry. Lidocaine selectively up-regulated pro-inflammatory proteins including
S100A8/9 and CRAMP/LL-37, and down-regulated anti-inflammatory and some pro- resolution peptides and proteins including IL-4, IL-13, TGF-β and Galectin-1. In contrast, the volatile anesthetic isoflurane promoted resolution in this system, diminishing the amplitude of PMN infiltration and shortening the resolution interval (Rz) -50%. In addition, isoflurane down-regulated a panel of pro-inflammatory chemokines and cytokines, as well as proteins known to be active in cell migration and chemotaxis (i.e., CRAMP and cofilin-1). The distinct impact of lidocaine and isoflurane on selective molecules may underlie their opposite actions in resolution of inflammation, namely lidocaine delayed the onset of resolution (Tmax), while isoflurane shortened resolution interval (Rz). Conclusions. Taken together, both local and volatile anesthetics impact endogenous resolution program(s), altering specific resolution indices and selective cellular/molecular components in inflammation-resolution. Isoflurane enhances whereas lidocaine impairs timely resolution of acute inflammation.
Resolution of acute inflammation was widely held to be a passive event [I]. It is now clear that tissue resolution or its return from an inflammatory and/or disease state is an active process involving novel mediators [2,3]. Non-resolved inflammation can exacerbate tissue injury and may cause functional damage via abscess or scar formation [I]. An emerging body of evidence now indicates that anti-inflammation (i.e. inhibiting the cardinal signs of inflammation [I]) and pro-resolution, namely activating endogenous resolution programs [3] are distinct mechanisms in the control of inflammation [3, and for a recent consensus report, see ref 4]. Classic antiinflammatories are enzyme inhibitors and/or receptor antagonists such as inhibitors of cyclooxygenases (COX) and antagonists for leukotriene (LT) receptors.
Resolution agonists, in comparison, are also antiinflammatories, but act by different mechanisms than the classic ones [recently reviewed in ref 5].
Resolution agonists (such as lipoxins), for example, are agonists that not only block neutrophil (PMN) actions [5], but also stimulate non-phlogistic monocyte recruitment [6] and macrophage uptake of apoptotic PMN [7]. Hence resolution agonists have two main mechanisms of actions at the tissue level; they lower the numbers of infiltrating PMN to the inflamed sites and tissues; and they stimulate the active removal of debris and apoptotic PMN from the inflamed sites by non-phlogistic activation of macrophages [5]. Because it is important to study resolution of inflammation as a distinct process, we introduced resolution indices to a) quantitate the overall process; b) access the roles of specific mediators; and c) pinpoint mechanisms of pharmacological interventions in the resolution of inflammation.
To characterize resolution of inflammation in cellular and molecular terms, we established a resolution map and defined the main quantitative indices (Fig. 8) [8]. These indices chart and take into account (i) the magnitude of PMN tissue infiltration (maximal PMN, Ψmax); (ϋ) the time interval when numbers of PMN reach Ψmax within exudates (Tmax);
(iii) duration: the time point (T50) when PMN numbers reduce to 50% of Ψmax (R50); and (iv) the resolution interval (R,): the time interval from the maximum PMN point (Ψmax) to the 50% reduction point (R50) [i.e. T50 - Tmax]. Using this set of resolution indices, we demonstrated that endogenous mediators such as resolvins and protectins accelerate resolution as evidenced by initiating the resolution of inflammation at earlier times (iTmax and T50) and/or shortening the resolution interval (iR,) [8,9]. The actions of these pro- resolution mediators sharply contrast those of agents and currently used therapeutics that are inhibitors and "resolution toxic". These drugs/agents have an unwanted impact on resolution such as inhibitors of COX-2 [9,10] and lipoxygenases (LOX) [9]. Thus, this set of resolution indices can be utilized to evaluate the impact of endogenous mediators as well as potential new therapeutic agents in inflammatory resolution because they reflect the summation of tissue-level events that are multi-level cellular and molecular processes in resolution of inflammation.
Surgery itself initiates an inflammatory response [11], and local anesthetics, both topical and volatile, are widely used during surgery [12]. Some anesthetics (i.e., lidocaine and isoflurane) are reported to reduce inflammatory markers, including cytokines and chemokines [13,14]. Potential impact of these widely used anesthetics on resolution of inflammation has not been established. The actions of many widely used current drugs in the pharmacopeia on resolution of inflammation remain unknown because their resolution characteristics were not evaluated at the time of their classical development. Appropriate qualified models of resolution were simply not yet available [8,9]. Here, we report using an unbiased systems approach that the widely used local anesthetic, lidocaine, and a widely used volatile anesthetic, isoflurane, each impact in vivo the resolution of acute inflammation in opposite directions that were quantified using resolution indices. We also characterize their multi-level impact on key cellular and molecular components in resolution of inflammation.
Results
Local anesthetic lidocaine impairs resolution
We first determined whether lidocaine alters cellular infiltration in a self-limited spontaneously resolving murine peritonitis. For these analyses, we used our reported resolution map that was constructed using an unbiased systems approach that combined cell trafficking into inflammatory exudates and mass spectrometry-based proteomics and lipid mediator lipidomics of resolving exudates [8]. Here, a microbial stimulus, the yeast wall zymosan A, was administered intraperitoneally to initiate inflammation [15], together with lidocaine given concomitantly. Given the inflammation-resolution map as a background terrain, lidocaine was introduced in order to determine if it significantly changed the signature of resolution map and indices in zymosan-initiated peritonitis. Inflammatory exudates were collected at the indicated time intervals 4-72 h (Fig. IA). Zymosan alone, as expected, stimulated an acute increase in the total leukocyte numbers (i.e. PMN and mononuclear cells) present in the peritoneal exudates during the initial phase of inflammation (4 h after zymosan, 11.8 ± 0.4 x 106 leukocytes), with a maximal infiltration at 12 h (30.0 ± 2.5 x 106 leukocytes), followed by a decline or resolution as monitored to 72 h. The time course of PMN infiltration followed a similar trend, peaking (17.5 ± 2.5 x 106 PMN) at 12 h after zymosan challenge (Fig. IA). An anesthetic dose of lidocaine, i.e. 0.08% (w/v) [16] administered with zymosan A significantly increased the number of total leukocytes by -49% within exudates at 4 h (p<0.05). The increase in exudate PMN was -58% (p<0.05). In the mice treated with both lidocaine and zymosan, the numbers of PMN continued to increase after 12 h and reached a maximum at 24 h. As a result, in the presence of lidocaine, the number of PMN in the exudate was significantly increased at this time point (-60% increase,
/Kθ.01). In contrast, the patterns of mononuclear cell infiltrates did not appear to be significantly altered by lidocaine treatment in this time course (4-72 h). Even doses as low as 0.008% (w/v) lidocaine, when given together with zymosan, led to a significant increase in the accumulation of PMN at 24 h (-75% increase, p<0.001). Lidocaine alone without zymosan challenge did not alter peritoneal leukocyte numbers in this 4-24 h interval after administration (Fig. 9). These results suggested that lidocaine might hamper PMN clearance during the normal spontaneous resolution phase of acute inflammation.
As shown in Figures IA and 9, in the zymosan-initiated peritonitis, the number of PMN reached a maximum at 12 h. The time intervals between 12 h (Tmax) and 35 h (T50), exudate PMN decreased in number from 17.5 x 106 PMN (Ψmax; maximal PMN number) to
8.8 x 106 PMN (R50; essentially 50% reduction of PMN). This period of neutrophilic loss from the exudates is termed the resolution interval (R,) [8]. In mice treated with zymosan alone, R,- was -23 h {i.e., 12-35 h). When the resolution indices were calculated with lidocaine treatment together with zymosan, it was apparent that both the anesthetic (0.08%) and sub-anesthetic doses (0.008%) of lidocaine increased Ψmax and shifted the onset of R, from 12 h to a later time point (Tmax = 24 h) (see Fig. 9 and vide infra). These results demonstrate that lidocaine directly delayed the spontaneous resolution of zymosan-initiated acute inflammation. Especially, lidocaine increased the dwell time of PMN present within the exudates, possibly blocking the clearance of PMN from the exudates in vivo (see below).
Surgery can induce local inflammation via tissue injury, and lidocaine is usually given before surgery [11,12]. In order to mimic such a clinical scenario, mice were treated with lidocaine (0.08%) 15 min before initiation of acute inflammation by zymosan. This prior exposure to lidocaine significantly potentiated zymosan-initiated leukocyte infiltration at 24 h by -40% (cf. zymosan alone, p<0.0\). This is similar to the results obtained with mice that received lidocaine and zymosan together, which gave a -33% increase in the number of leukocytes present in the exudates when compared to zymosan alone (p<0.05, Fig. IB). Thus, lidocaine administration, either just before or concomitant with zymosan, caused significant increases in the number of PMN present in exudates in the resolution phase of acute inflammation.
Specialized lipid mediators play a key role in resolution of inflammation [5] with some specifically switched on during the resolution phase to promote resolution [17]. Here, key lipid mediators were monitored in murine exudates, including lipoxin (LX) A4, an antiinflammatory and pro-resolution mediator, and the pro-inflammatory LTB4 and prostaglandin (PG) E2. In this system, the maximal levels present in cell-free lavages of the exudates of both LTB4 and LXA4 were obtained at 4 h. These subsequently subsided within 24 h (Fig. 2A). Lidocaine did not significantly alter the levels Of LXA4, LTB4 or
PGE2 present in these cell-free lavages of the peritoneal exudates. Thus, these eicosanoids likely reflect the profile from resident peritoneal cells including macrophages as are less likely to report eicosanoids generated by the infiltrating leukocytes.
Lipoxins are potent agonists for resolution of inflamed tissues by regulating leukocyte infiltration, stimulating macrophage clearance of apoptotic PMN and also their exit via lymphatics [5,8,9]. Since LXA4 can rescue inhibitor-imposed lesion with, for example, a selective COX-2 inhibitor [9], we questioned whether these resolution agonists impact leukocyte infiltration in lidocaine-treated mice. At 24 h, lidocaine (0.08%, -0.8 mg) administration increased, while ATLa (a stable analog for aspirin-triggered 15-epi-lipoxin A4,
300 ng, i.p.) decreased exudates cell numbers, when compared with mice treated with zymosan alone. When ATLa was administered along with lidocaine and zymosan, it significantly reduced exudate leukocytes compared to mice received lidocaine and zymosan (p=0.04) (Fig. 2B). Thus, pro-resolution mediators, such as lipoxins, at much lower doses (by more than 3 log orders) partially rescued the defective resolution of inflammation caused by lidocaine. Lidocaine impairs PMN apoptosis and their removal by macrophages
PMN apoptosis and their subsequent removal by macrophages are essential components of resolution at the tissue level [1,18]. Since lidocaine delayed PMN clearance in the resolution phase, we considered that lidocaine might have an impact on PMN apoptosis. To address this, peritoneal cells were collected at 24 h after zymosan challenge, well within the resolution phase, and labeled with FITC-annexin-V and PE-conjugated anti-
Gr-I Ab, a specific cell surface marker for mouse PMN. Peritoneal cells collected from mice receiving lidocaine (at both 0.08% and 0.008%) together with zymosan showed significantly decreased annexin- V+Gr- 1+ cells by 50% and 64%, respectively, indicating reduced PMN apoptosis (Fig. 3A). At 48 h after zymosan challenge, lidocaine at 0.08% also reduced PMN apoptosis -40% (pθ.01).
It was next determined whether lidocaine impacts macrophage ingestion of PMNs. To this end, we carried out a phagocytosis-based analysis in vivo (Fig. 3B). Exudate cells were collected at 24 h after zymosan challenge, and macrophages were labeled with the FITC-conjugated anti-F4/80 Ab. This was followed by permeabilization of these cells, allowing labeling of ingested PMN with PE-conjugated anti-Gr-1 Ab. Cells with positive staining of both F4/80 and Gr-I were then monitored by FACS analysis. Of interest, cells collected from mice treated with lidocaine (0.08%) together with zymosan showed significantly reduced F4/80+Gr-l+ cells (18.0 ± 1.2%) when compared to those given only zymosan (28.4 ± 1.6%) (p<0.05, Fig. 3B). The low dose of lidocaine (0.008%) also gave decreased F4/80+Gr-l+ cells (24.8 ± 2.1%), albeit not significantly different from mice receiving zymosan alone. These results indicate that clinically used doses of lidocaine inhibit macrophage ingestion of apoptotic PMN in vivo, blocking their removal and resolution.
We also investigated whether lidocaine has a direct impact on isolated macrophages. To this end, we carried out in vitro phagocytosis of zymosan particles. This system represents recognition of microbes by the innate immune system [19]. Recently, we found that pro-resolution mediators such as LXA4 are potent stimulators of macrophage uptake of microbial particles, i.e., opsonized zymosan [9], in addition to stimulating the uptake of apoptotic PMN [7]. Of interest, lidocaine at both doses (0.008% and 0.08%), when added together with LXA4, significantly impaired LXA4-stimulated phagocytosis (Fig. 3C). Thus, lidocaine can be considered "resolution toxic" because it impairs key components at the level of tissue resolution, namely PMN apoptosis and macrophage phagocytosis, and blocks the protective action of LXA4.
Lidocaine regulates both anti- and pro-inflammatory proteins: proteomics
Using mass spectrometry-based resolution proteomics, we recently identified several components in inflammatory exudates, including haptoglobin, S100A9 and αl- macroglobulin, that may play active roles in promoting resolution of inflammation [8]. These proteins were identified by peptide mapping of in-gel digested proteins using capillary liquid chromatography-nanospray ion trap tandem mass spectrometry (nanospray-LC-MS-MS) and bioinformatics software (see Methods). Among these, S100A9 was present in exudates within 4 h of initiating inflammation and reached maximum levels at the onset of R, (12 h). These changes in S100A9 paralleled the time course of PMN infiltration (Fig. IA) [8]. Both S100A8 and S100A9 are known to be abundant cytosolic proteins in human PMN that can be secreted and exhibit potent actions in inflammatory cell recruitment [20]. Also, SlOO proteins belong to a new group of damage-associated molecular pattern proteins and may function as "alarm/danger" signals to propagate inflammation [21]. To determine whether lidocaine impacts these proteins during inflammation-resolution, we carried out temporal- differential analysis of peritoneal exudate proteins collected from zymosan-challenged mice in the presence or absence of the anesthetic dose of lidocaine (0.08%). Two time intervals were selected for analysis: one at 4 h within the early inflammatory phase, and the second at 24 h within the resolution phase, since lidocaine gave the most dramatic impact on PMN infiltration at these two time points (see Fig. IA). Four hours after zymosan challenge, both S100A8 and S100A9 were significantly increased in the presence of lidocaine (Fig. 4A and Table 1). This increase was verified from mice that received lidocaine together with zymosan by Western blot analysis that also demonstrated an increase in S100A9 proteins in exudates (Fig. 4B). Also S100A9 mRNA levels were higher in these mice compared to mice that received zymosan alone as determined with RT-PCR (Fig. 4B). Thus, it is likely that S100A8/A9 complexes reflect, at least in part, the increases in PMN obtained in mice challenged with lidocaine and zymosan at 4 hours, compared to mice received zymosan alone. We also determined, in parallel with exudate cells, changes in cell-associated proteins from these mice. As shown in Figure 4C, lidocaine together with zymosan at 4 h gave significant up-regulation of several selective proteins compared to zymosan-challenged mice. Among them, CRAMP (cathelin-related anti -microbial peptide), the mouse homolog of antimicrobial protein LL-37, was increased approximately two-fold (Table 1). CRAMP is also a documented chemotactic factor for PMN, monocytes, mast cells, and T cells [22]. Thus, exudate CRAMP/LL-37 may also contribute to increased PMN numbers obtained at 4 h in lidocaine-treated mice (Fig. IA). Moreover, we found that, within resolution at 24 h, lidocaine down-regulated galectin-1 -50% (Fig. 4C and Table 1). Galectin-1 inhibits PMN migration during PMN-endothelial interactions in vitro and in vivo [23]. In addition, Galectin-1 prolongs exposure of phosphatidylserine on the surface of leukocytes, suggesting a role in promoting PMN clearance [24]. Therefore, decreases in Galectin-1 levels from lidocaine-treated mice during resolution (i.e. 24 h) might also contribute to delayed PMN clearance, resulting in increased PMN dwell times in exudates (Fig. IA).
Lidocaine impacts chemical mediators in exudates
Production of both pro-inflammatory (e.g. IL-I β, IL-6, IL-12, TNF-α) and antiinflammatory (e.g. IL-4, IL-10, and IL- 13) cytokines is essential in the control of inflammation [25]. Here, we monitored a panel of chemokines and cytokines in exudates to assess whether lidocaine specifically regulates their levels. At 4 h after zymosan challenge, most cytokines and chemokines were dramatically up-regulated compared to naϊve mice (Fig. 5A). Of interest, both 0.08% and 0.008% lidocaine gave similar results at 4 h, with significant, preferential reduction of anti-inflammatory cytokines, including IL-4, IL-10 and IL- 13 (Fig. 5B). In addition, anesthetic dose of lidocaine decreased pro -inflammatory KC
(the murine homolog of human IL-8) without significant changes in other pro-inflammatory cytokines and chemokines in the exudates, suggesting that lidocaine acts at several levels in acute inflammation, overall reducing what is coined the "cytokine/chemokine storm" observed in the early inflammatory response (4 h) (Fig. 5A). In murine peritoneal exudates, lidocaine initially reduced the levels of most of the chemokines and cytokines induced by zymosan at 4 h, but increased their levels by 12 h (Table 2). Calculation of the ratios between pro- and anti-inflammatory cytokines (TNF-α / IL-10 or IL-6 / IL-10) indicated that lidocaine increased these ratios in the resolution phase (12 and 24 h after zymosan A injection) (Table 3). These changes in ratios (i.e. pro- / anti- inflammatory cytokines) likely contribute to the increased numbers of PMN present in the resolution phase (i.e. 12-24 h), compared to the mice not treated with lidocaine. We also found that lidocaine (0.08%) significantly decreased exudate levels of TGF-β at the late resolution phase, 48 h after zymosan challenge (Fig. 5C). It is likely that the decreased levels of TGF-β contributed to impaired macrophage phagocytosis in lidocaine-treated mice (Fig. 3B), that can lead to delayed PMN clearance and their increased dwell time. The impact of lidocaine was also evaluated in human whole blood ex vivo to access whether the murine system reflects human tissue events. The anesthetic dose of lidocaine (0.08%) significantly diminished the levels of a panel of chemokines and cytokines in zymosan-stimulated human whole blood (Fig. 5D).
Volatile anesthetic isoflurane promotes resolution
Isofiurane 1.4 MAC (minimum alveolar concentration) was administrated over a 2 h period (from 1 h before to 1 h after zymosan challenge) to mimic clinical use. Exudates were collected during inflammation-resolution to determine the potential impact of isoflurane in the resolution maps and indices. Isoflurane significantly reduced zymosan-stimulated leukocyte infiltration at 12, 24 and 48 h (Fig. 6A). When compared to mice that received zymosan alone, at the peak of inflammation, Tmax, isoflurane decreased maximal PMN numbers (Ψmaχ) from 18.0 x 106 to 13.5 x 106. In addition, isoflurane dramatically reduced
T50 from -34 h to -22 h, thus shortening R; by >50% from -22 h to -10 h (Fig. 6B). These results contrast with lidocaine' s impact on the resolution indices where lidocaine delayed the onset of resolution, i.e. Tmax (Table 4).
Isoflurane specifically regulates key exudate proteins
Since isoflurane gave the most dramatic reduction on zymosan-stimulated PMN infiltration at two time intervals (Fig. 6A), these intervals were selected for exudate proteomic analysis: First, the early resolution phase at 12 h, and second, the late resolution phase at 24 h after zymosan challenge (Fig. 7A). At 12 h after zymosan challenge, we found CRAMP, an anti -microbial protein and chemotactic factor was decreased -2-fold (Table 5) in mice that received 1.4 MAC isoflurane, contrasting with significant increase of CRAMP at 24 h mediated by lidocaine (Table 1). In addition, isoflurane reduced cofilin-1 (Table 5), a major actin-depolymerization factor regulating actin dynamics and generation and maintenance of cell protrusions, key cellular events that are required for migration [26]. Therefore, during resolution of inflammation, isoflurane selectively regulates cellular proteins that are involved in cell migration and chemotaxis (i.e., CRAMP and cofilin-1). By comparison, isoflurane treatment increased SH3 domain-binding glutamic acid-rich like protein (SH3BGRL), which might have anti-oxidative and anti-inflammatory properties
[27,28].
We monitored in the exudates a panel of chemokines and cytokines to examine whether isoflurane regulates their levels in vivo. Of interest, isoflurane-treated mice selectively reduced zymosan-stimulated pro-inflammatory cytokine levels (IL- lα, IL-6, IL- 12, KC, JE [the mouse homolog of human MCP-I], MIP-Ia and Rantes) (Fig. 7B, Tables 6 and 7), but did not apparently affect the levels of cytokines IL-4, IL-IO and IL- 13 in the early inflammatory phase, 4 h after zymosan challenge (Fig. 7B).
Discussion A systems approach to mapping the resolution of acute inflammation demonstrated that resolution is an active process [2,3] and a new terrain of cellular and molecular processes directed toward returning the tissue to homeostasis [8,29]. Using this differential-temporal and quantitative systems approach to analyze inflammation and its spontaneous resolution, we identified, for the first time, in the present report that widely used anesthetics impact the resolution of acute inflammation. Lidocaine, the first amino amide-type local anesthetic is well appreciated to affect depolarization in neurons by blocking the fast voltage gated sodium channels on cell membranes [30]. Earlier evidence from in vitro studies indicates that lidocaine influences the immune system by reducing responses such as chemotaxis, microtubule assembly, phagocytosis, release of lysosomal enzymes and superoxide anion generation [31-33]. In certain settings, lidocaine can reduce inflammatory responses and protect tissues from local injury [34]. On the other hand, lidocaine worsens renal injury following ischemia-reperfusion by increasing necrosis and local inflammation [35]. In burn wounds, lidocaine increases leukocyte numbers, which suggests an increase in PMN infiltration and/or increased viability of the leukocytes at the burn site [36]. Yet the clinical significance of these observations remains to be established. The results of the present studies demonstrate that lidocaine imposes a molecular lesion in resolution that delays the return to homeostasis. Specifically, lidocaine increases leukocyte accumulation in exudates, impairs the apoptosis of PMN and hampers ingestion of apoptotic PMN by macrophages in vivo. Summation of these multi-level actions in tissues significantly delays resolution of inflammation.
In this context, other currently and widely used therapeutic agents also affect resolution of inflammation. Aspirin, for example, by way of initiating biosynthesis of endogenous lipid mediators (i.e., aspirin-triggered epimer of lipoxin A4 [ATL]), promotes resolution [4,9]. Cyclin-dependent kinase and specific ERKl /2 inhibitors, in comparison, also promote resolution of inflammation by enhancing PMN apoptosis [37,38]. In contrast, COX or LOX inhibitors, by blocking the biosynthesis of key lipid mediators, dramatically impairs resolution [9,10]. In the peritoneal cell-free lavages, LXA4 appeared in the early inflammatory phase, 4 h after zymosan challenge. PGE2, a signal that can activate the full
LXA4-biosynthetic capacity in vivo [17], was present in the peritoneum prior to peritonitis and elevated during the acute inflammatory response. Lidocaine did not alter either the magnitude or time course OfLXA4 in a statistically significant fashion compared to the mice given zymosan alone. However, a trend towards reduction was observed at 4, 12 and 48 h. Of interest, when exogenous ATLa (a stable analog Of LXA4 and ATL) was given together with lidocaine, it significantly reversed in part lidocaine' s delaying effects in the resolution of inflammation. Thus, pro-resolution mediators may have therapeutic potential in settings where sustained inflammation and impaired resolution are components of disease pathophysiology. Increases in the ratios of IL-6/IL-10 are thought to signify pro- versus antiinflammatory response. This increase in ratio correlates with the severity of systemic inflammatory response syndrome and injury after trauma [39]. Since results from several studies indicate that the relationship between pro- and anti-inflammatory cytokines influences the severity of sepsis [40], and TNF D /IL- 10 ratios are used as an indicator for disease severity [41], we calculated the ratios of pro- to anti-inflammatory cytokines (Table 3) and found that lidocaine increased these values in the late phase (12 and 24 h after zymosan). Thus, changes in the balance between pro- and anti-inflammatory cytokines, rather than individual chemokines or specific cytokines, appear to contribute to the observed increases in PMN numbers obtained at 24 h with lidocaine. It is noteworthy that, during an inflammatory disease state, a complex network of interactions between different cytokines is likely to occur. The timing of cytokine release and the balance between pro- and anti-inflammatory cytokines is likely to contribute to the overall outcome and severity, as both pro- and anti-inflammatory mediators interact in highly specific ways. Along these lines, computational simulations were carried out to address these complex interactions in the setting of acute inflammation as well as simulate certain disease scenarios and the time course of cytokine levels in mice [42]. This approach may lead to in silico development of new therapeutics and real-time diagnostics. The volatile anesthetic isoflurane binds to gamma-aminobutyric acid type A
(GABAA) receptors, glutamate and glycine receptors, and inhibits conduction in activated potassium channels [30]. It is noteworthy that human peripheral mononuclear cells express several GABAA receptor subunits, and application of GABA reduced formyl peptide (fMLP)-stimulated increases in intracellular Ca2+ levels [43]. Thus, these GABA receptors may play a role in modulating immune responses. Along these lines, isoflurane is known to impact the inflammatory response, reducing inflammation in vivo [14], increasing leukocyte rolling velocities in mesenteric microcirculation [44], and decreasing activation of the L- selectin and D2-integrins CDl Ia and CDl Ib involved in these responses [45]. In the present report, we identified specific proteins in inflammatory exudates and cytoskeleton protein cofilin-1 that were reduced by volatile anesthetics and are known to be important in cell migration. In addition, isoflurane treatment increased SH3BGRL. The human homolog of SH3BGRL belongs to the thioredoxin-like protein superfamily [27]. Among them, thioredoxin-1 (TRX) is a small multifunctional protein with antioxidative and redox- regulating functions [27]. Serum TRX levels were elevated in patients with inflammatory bowel disease. Also, TRX significantly ameliorated DSS-induced colitis and colonic inflammation of IL-10 deficient mice [28]. Thus, SH3BGRL and other thioredoxin-like proteins might have anti-inflammatory properties, and contribute to the accelerated resolution in isoflurane-treated mice documented in the present report (Fig. 6). Of interest, LXA4 stimulates IL-IO [9] as well as heme oxygenase- 1 [46,47] and, as indicated in the present report, was able to partially rescue the lidocaine-delayed resolution of inflammation (Fig.
2B).
Isoflurane and lidocaine gave opposite effects in the resolution of acute inflammation, as indicated by their differential impact in the resolution indices (Table 4). This reflects their distinct and selective impact on specific molecules involved in resolution of inflammation. For example, CRAMP protein levels were decreased in mice with isoflurane, contrasting with significant increases in CRAMP at 24 h as evoked by lidocaine, compared with mice given zymosan alone. Thus, it is likely that the reported chemotactic property of CRAMP [22] contributes to the observed opposing actions of isoflurane and lidocaine on peritoneal leukocyte infiltration. Also, isoflurane selectively reduced zymosan-stimulated pro- inflammatory cytokine levels (Fig. 7B and Tables 6 and 7), which contrasts the events in mice treated with zymosan and anesthetic dose of lidocaine (0.08%), that significantly reduced anti-inflammatory IL- 13 (Fig. 5B). Hence, the changes in these exudates proteins might reflect the opposing actions of isoflurane and lidocaine in resolution of inflammation.
Historically, the phagocytic index was defined in the cellular era and was used to determine the average number of bacteria ingested by phagocyte at single-cell level [48,49]. By comparison, the resolution indices presented here expand the appreciation of the complexity of phagocytes at the tissue level and account for the summation of multi-level cellular and molecular events during resolution of inflammation. In conclusion, the results of the recent study indicate that the local anesthetic lidocaine delays the onset of resolution. The impact of lidocaine is documented herein at multi-levels in resolution and reflects (i) increased exudate PMNs, (ii) impaired PMN apoptosis as well as their uptake by macrophages, (iii) modulating both pro- and anti-inflammatory proteins, including cytokines and chemokines. Dysregulation of resolution programs by lidocaine may have important unwanted consequences in both immune responses and host defense that were previously unappreciated.
Clinical implications of the present observations might be far-reaching. Every serious surgical intervention unavoidably results in an inflammatory response. Severity of many postoperative surgical complications, particularly infection, are directly related to the degree and length of inflammatory response and resolution of such response during the postoperative period; therefore, hypothetically, accelerating resolution of postoperative inflammation should be helpful in the management of surgical patients during the postoperative period. Resolution of inflammation would become a resolution of many problems surgical patients face daily. The results of the present study offer new avenues, not only for continued studies in the cellular and molecular markers in resolution of inflammation, but also for future translational and clinical research. Also, combining pro-resolution molecules, such as LXA4 and ATL, together with lidocaine may be a useful strategy to rescue resolution of acute inflammation. In sharp contrast, the volatile anesthetic isoflurane accelerates resolution, shortening resolution interval. Together, these findings demonstrate, for the first time, the direct impact of anesthetics in the resolution of inflammatory challenge and the return of the local tissue to homeostasis. Materials and Methods
Murine acute inflammation.
For lidocaine treatment, male FVB mice (6-8 weeks; Charles River, Wilmington, MA) were administered lidocaine (0.08% or 0.008%) intraperitoneally together with 1 mg/ml zymosan A (i.p.) to evoke peritonitis [8] as in accordance with the Harvard Medical Area Standing Committee on Animals (protocol no. 02570). ATLa (a stable analog of aspirin-triggered
LXA4) was prepared by total organic synthesis in the Organic Synthesis Core (P50- DEOl 6191). For isoflurane treatment, mice were administered 1.4 MAC [50] of isoflurane for a 2 h period (from Ih before to 1 h after injection of zymosan, i.p.) (see timeline in Fig. 6). At indicated time points, mice were euthanized with an overdose of isoflurane, and peritoneal exudates were collected by lavaging with 5 ml sterile saline. Exudate cells and supernatants were obtained for analyses described below.
Human whole blood.
Venous blood (anticoagulated with 10 U/ml sodium heparin) was collected from healthy non- smoking volunteers who declared not to have taken any drugs for at least two weeks before the experiments. Informed consent was obtained from each volunteer. The protocol was approved by the Brigham and Women's Hospital Institutional Review Board (protocol no. 88-02642, approved 11/26/07). Heparinized whole blood was then incubated with either 0.008% or 0.08% of lidocaine in the presence of zymosan A (100 μg/ml) for 4 h, and plasma was collected by centrifugation at 2,000 rpm for 15 min. The amounts of cytokines and chemokines levels were determined by multiplexed sandwich ELISA (SearchLight Proteome Array custom-designed by Pierce Boston Technology Center). Following a standard sandwich ELISA procedure, the entire plate is imaged to capture chemiluminescent signals generated at each spot within each well of the array. The SearchLight CCD Imaging and Analysis System features image analysis software that calculates chemokine/cytokine concentrations (pg/ml) using pre-determined standard curves.
Differential leukocyte counts and FACS analysis.
Aliquots of exudate cells were prepared for determination of total and differential leukocyte counts. For determination of cellular composition (PMN vs. mononuclear cells), cells were blocked with anti-mouse CDl 6/32 blocking Ab (0.5 μg/O.5xlO6 cells) for 5 min and stained (20 min) with FITC-conjugated anti-mouse CD14 and PE-conjugated anti-mouse Ly-6G (0.5 μg/O.5xlO6 cells; clones rmC5-3 and RB6-8C5, respectively, from BD Pharmingen, San
Diego, CA). FACS analysis was then carried out.
Apoptosis and phagocytosis.
For determining PMN apoptosis in vivo, exudate cells were labeled with FITC-conjugated anti-annexin-V Ab (0.5 μg Ab/0.5xl06 cells, eBioscience) and PE-conjugated anti-mouse Gr-
1 (Ly-6G) Ab (0.5 μg Ab/0.5xl06 cells, eBioscience) for 20 min. The annexin- V+Gr- 1+ PMN population was determined by FACS.
For determining macrophage phagocytosis of apoptotic PMN in vivo, cells were blocked with anti-mouse CD 16/32 blocking Ab (0.5 μg/0.5xl06 cells) for 5 min, stained with FITC- conjugated anti-mouse F4/80 (0.5 μg/0.5xl06 cells) for 20 min, and then permeabilized with
0.1 % Triton X-100 (100 μl, 10 min). Permeabilized cells were then stained with PE- conjugated anti-mouse Ly-6G (0.5 μg/0.5xl06 cells). The F4/80+Gr-l+ cell population was determined by FACS.
For phagocytosis in vitro, murine peritoneal resident macrophages were collected and plated onto 24-well plates (lxlθ5cells/well) and incubated with lidocaine (0.008% or 0.08%), LXA4
(1 nM; Calbiochem) or both for 20 min. FITC-zymosan (2.5 μl/well) was then added to macrophages for 30 min. Supernatant was aspirated and extracellular fluorescence was quenched by adding trypan blue for 1 min. Cells were then washed and intracellular fluorescence was determined by a fluorescent plate reader.
Two-dimensional gel-based proteomics
Two-dimensional gel electrophoresis. Supernatants and cell pellets from peritoneal lavages were collected by centrifugation (15 min, 1,800 rpm) in the presence of protease inhibitors (Roche, Indianapolis, IN). Proteins in the supernatant were desalted by acetone precipitation, and the protein pellet was re-dissolved in a lysis solution containing 8 M urea,
4% w/v CHAPS, 40 mM Tris, and 65 mM dithiothreitol (DTT). The cell pellets were directly solubilized in the same lysis solution through sonication at 40C. The protein concentrations were measured in duplicate by a Bradford protein assay kit (Bio-Rad, Hercules, CA) in a 96-well plate format using bovine serum albumin as the standard. Supernatant (25 μg) or cellular (50 μg) proteins from each animal were mixed with 125 μL of rehydration buffer containing 8M urea, 2% (w/v) CHAPS, 10 mM DTT, and 0.2% carrier ampholytes (pH 3-10), and then loaded onto nonlinear 7-cm, pH 3-10, IPG strips (Bio-Rad) through passive in-gel rehydration overnight. After iso-electric focusing for 10,000 V-h, the proteins in the IGP strips were reduced with dithiothreitol and alkylated with iodoacetamide. The 2nd dimension separation was then carried out using 10-14% SDS-PAGE (covering -MWlO to 200 kDa). Gels were stained with ProteomlQ™ blue dye (Proteome Systems, Woburn, MA), and scanned with a GS-800 densitometer system (Bio-Rad). Image analysis was carried out with PDQuest software (version 8.0) (Bio-Rad). The differentially regulated protein spots were selected based on the normalized spot volumes.
LC-MS-MS proteomics. The selected protein spots were excised and in-gel digested with sequencing grade trypsin (Promega, Madison, WI). Tryptic peptides were loaded onto a 2 μg capacity peptide trap (CapTrap; Michrom Bioresources, Auburn, CA) in 0.1% formic acid and 0.05% trifluoroacetic acid and separated by capillary liquid chromatography using a capillary column (75 μm x 5 cm x 3 μm; LC Packings, Amsterdam, The Netherlands) at 150 nl/min delivered by an Agilent 1 IOOLC pump (400 μl/min) and a flow splitter (Accurate, LC Packings). A mobile phase gradient was run using mobile phase A (2% acetonitrile/0.1 % formic acid), and B (80% acetonitrile/0.1% formic acid) from 0-10 min with 0-20% B followed by 10-90 min with 20-60% B. Peptide mass and charge was determined on a
ThermoFinnigan Advantage ion-trap mass spectrometer (San Jose, CA) after electrospray ionization using end-coated spray Silicatip tip (ID 75 μm, tip ID 15 μm, New Objective) held at a spray voltage of 1.8 kV. After acquisition of the peptide parent ion mass, zoom scans and tandem mass spectra of parent peptide ions above a signal threshold of 2x104 were recorded with dynamic exclusion, using Xcalibur 1.3 data acquisition software (ThermoFinnigan).
Protein identification. Proteins were identified by peptide mapping of tryptic peptide tandem mass spectra using TurboSequest (BioWorks 3.1 software, ThermoFinnigan against indexed Swiss-Prot protein database). Protein modifications that were taken into consideration included methionine oxidation and alkylation of cysteine with iodoacetamide. The search results were filtered by XC01T vs. charge with 1.5 for singly charged ions, 2.0 for doubly charged ions, and 2.5 for triply charged ions. A protein was considered identified when a minimum of two tryptic peptides were matched.
ELISA -peptide and lipid mediators. Aliquots of supernatants were used to quantitate chemokines and cytokines using a
SearchLight Mouse Chemokine Array custom designed with Pierce Boston Technology Center. TGF-D levels was determined with ELISA using a monoclonal anti-TGF-β antibody (R&D Systems, Minneapolis, MN) recognizing the active forms of TGF-β (1, 2, and 3).
Eicosanoid ELISAs (LTB4, LXA4 and PGE2) were carried out following manufacturer's instructions (Neogen, Lexington, KY).
Western blot Supernatants from peritoneal lavages were collected and equal amounts of proteins were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) microporous membrane by electroblotting. Membranes were blocked in 5% non-fat milk in TBST (0.9% NaCl and 0.05% Tween-20 in 20 mM Tris/HCl, pH 7.4) and probed with a goat anti-mouse S100A9 polyclonal antibody (0.2 μg/ml, R&D Systems) for 1 hour. After washing three times with TBST, membrane were incubated with HRP-linked anti-goat IgG (1 :5,000 dilution) for 1 h and the immunoreactive bands were developed by incubating with chemiluminescence substrates and visualized by exposure to an X-ray film.
RT-PCR Murine peritoneal cells were collected, total RNA was isolated using TriZol reagent (GIBCO
BRL, Grand Island, NY) and reverse-transcribed followed by polymerase chain reactions (PCR) using HotStar Master mix (Qiagen) (95°C for 15 min, then 35 cycles of 95°C for 30 sec, 55 °C for 30 sec and 72°C for 60 sec) with specific primers for mouse S100A9 (sense: 5'- CCCTGACACCCTGAGCA AGAAG-3' and antisense 5'- TTTCCCAGAACAAAGGCCATTGAG-3 '). Relative intensities of RT-PCR products were quantified and normalized by D-actin message levels using the public domain NIH image program (developed at the NIH, available on the Internet).
Statistical approaches. All results were calculated and expressed as mean ± standard error of mean (mean ± SEM).
Group comparisons were carried out using one-way ANOVA or Student's t-test where appropriate, with P values <0.05 taken as statistically significant (sufficient to reject the null hypothesis).
LIPOXINS
In one aspect, lipoxins and lipoxin analogs useful as therapeutic agents in treatment of the maladies described throughout this specification have the formulae encompassed by U.S. Patents 4,560,514, 5,441,951, 5,648,512, 5,650,435, 6,048,897 and 6,627,658, the contents of which are incorporated herein by reference in their entirety. For example, lipoxin analogs encompassed by the present invention include those having the following characteristics.
The instant lipoxins comprising an "active region" and a "metabolic transformation region" as both terms are defined herein are generally of the following structure: wherein Ri can be
Figure imgf000025_0001
wherein Ri can be
Figure imgf000025_0002
Figure imgf000025_0003
Figure imgf000025_0004
Figure imgf000025_0005
Figure imgf000026_0001
and R2 can be
Figure imgf000026_0002
Figure imgf000026_0003
Figure imgf000026_0004
(forms
Figure imgf000026_0005
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000027_0003
Figure imgf000027_0004
Figure imgf000027_0005
(forms
Figure imgf000027_0006
In one embodiment, the lipoxin analogs of this invention have the following structural formula I:
Figure imgf000028_0001
wherein X is Ri, OR.i, or SRi; wherein Rj is (i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(iv) an aralkyl of 7 to 12 carbon atoms; (v) phenyl;
(vi) substituted phenyl
Figure imgf000028_0002
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein Qi is (C=O), SO2 or (CN); wherein Q3 is O, S or NH; wherein one of R2 and R3 is a hydrogen atom and the other is
(a) a hydrogen atom;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
(e) Ra Q2 Rb wherein Q2 is -O- or -S-; wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R4 is
(a) a hydrogen atom;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y2 is -OH, methyl, or -SH and wherein the other is
(a) a hydrogen atom
(b) CH3Zb where a+b=3, a=0 to 3, b=0 to 3; and each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; or
(d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Yi and Y2 taken together are
(a) =NH; or (b) =O; wherein R5 is
(a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
(b) -(CHz)n -R1 wherein n=0 to 4 and R1 is (i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) a phenyl; or
(iii) substituted phenyl
Figure imgf000030_0001
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(c) RaQaRb wherein Qa is O or S; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(d) -C(R111)(R1V)-R1 wherein R111 and RIV are each, independently: (i) a hydrogen atom; (ii) CH3Zb where a+b=3, a=0 to 3, b=0+3, and wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched; and wherein R6 is (a) a hydrogen atom;
(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched;
(c) a halogen.
In one embodiment of this invention, the lipoxin analogs have the following structure II:
Figure imgf000031_0001
wherein X is Ri, ORi, or SRi ; wherein Ri is (i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) a phenyl;
(vi) substituted phenyl
Figure imgf000031_0002
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule, such as but not limited to fluorescent labels; or (viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain or branched; wherein Q1 is (C=O), SO2 or (C=N); wherein Q3 is O, S or NH; wherein one of R2 and R3 is hydrogen and the other is (a) a hydrogen atom;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
wherein Q2 is -O- or -S-; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R4 is
(a) a hydrogen atom;
(b) alkyl of 1 to 6 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y2 is — OH, methyl, -H or -SH and wherein the other is
(a) a hydrogen atom;
(b) CHaZb where a+b=3, a=0 to 3, b=0 to 3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom; (c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Yi and Y2 taken together are
(a) =NH; or
(To) =O; wherein R5 is (a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
(b) — (CH2), - Ri wherein n=0 to 4 and R; is
(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) phenyl; or (iii) substituted phenyl
Figure imgf000033_0001
wherein Z1, Zn, Z1n, Z1V and Zv are each independently selected from -NO2, -CN, -C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
Figure imgf000033_0002
wherein Q3 is -O- or -S-; and wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(d) — C(Rin)(Rlv)—R, wherein R1n and R1V are each independently:
(i) a hydrogen atom; or
(ii) CH3Zb where a+b=3, a=0 to 3, b=0+3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom,
(e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
In one embodiment of this invention, the lipoxin analogs have the following structure III:
Figure imgf000034_0001
wherein X is R1, ORi, or SRi ; wherein Ri is (i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl;
(vi) substituted phenyl
Figure imgf000034_0002
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule; or
(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain or branched; wherein Qi is (C=O), SO2 or (C=N); wherein Q3 is O, S or NH; wherein one of R2 and R3 is hydrogen atom and the other is
(a) a hydrogen atom;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
wherein Q2 is -O- or -S-; wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R4 is
(a) a hydrogen atom; or
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y2 is hydroxyl, methyl, hydrogen or thiol and wherein the other is
(a) a hydrogen atom;
(b) CH3Zb where a+b=3, a 0 to 3, b=0 to 3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom ; (c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Yi and Y2 taken together are
(a) =NH; or
(b) =0; and wherein R5 is (a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
(b) — (CH2)n -R, wherein n=0 to 4 and R1 is (i) cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) phenyl;
(iii) substituted phenyl
Figure imgf000036_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
wherein Qa is -O- or -S-; wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; or Cd) -C(R111)(R1V)-R1 wherein R111 and R1V are each independently: (i) a hydrogen atom; or (ii) CH3Zb where a+b=3, a=0 to 3, b=0+3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom, (e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
In another embodiment of this invention, lipoxin analogs have the following structural formula IV:
Figure imgf000037_0001
wherein X is Ri, OR], or SRi ; wherein Rj is (i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl;
(vi) substituted phenyl
Figure imgf000037_0002
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule; or
(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain or branched; wherein Q1 is (C=O), SO2 or (CN); wherein Q3 is O, S or NH; wherein one of R2 and R3 is hydrogen and the other is
(a) a hydrogen atom; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or (e) RaQ2Rb wherein Q2 is -O- or-S-; wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R4 is
(a) a hydrogen atom; or
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y2 is — OH, methyl, or — SH and wherein the other is
(a) a hydrogen atom;
(b) CH3Zb where a+b=3, a=0 to 3, b=0 to 3, wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; or (d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Y] and Y2 taken together are
(a) =NH; or
(b) =O; wherein R5 is (a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
(b) — (CH2)n — R1 wherein n=0 to 4 and R; is
(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) phenyl; or (iii) substituted phenyl
Figure imgf000039_0001
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
wherein Q3 is-O- or -S-; wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(d) -C(R111)(R1V)-R1 wherein R111 and R1V are each independently: (i) a hydrogen atom; or (ii) CHaZb where a+b=3, a=0 to 3, b=0+3 and wherein each Z, independently, is a cyano, a nitro, or a halogen atom; or
(e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched; and wherein R6 is (a) a hydrogen atom; (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; or
(c) a halogen atom.
In another embodiment of this invention, lipoxin analogs have the following structural formula V:
Figure imgf000040_0001
wherein Ri is (i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl;
(vi) substituted phenyl
Figure imgf000040_0002
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule; or
(viii) an alkenyl of 2 to 8 carbon atoms, inclusive, straight chain or branched; wherein n = 1 to 10, inclusive; wherein R2, R3a, and R3b are each independently: (a) a hydrogen atom; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
wherein Q2 is -O- or -S-; wherein Ra is alkyl ene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y2 is -OH, methyl, hydrogen, or — SH and wherein the other is
(a) a hydrogen atom;
(b) CHaZb where a+b=3, a=0 to 3, b=0 to 3, and wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or Y1 and Y2 taken together are
(a) =NH; or (b) =0; wherein Y3 or Y4 is — OH, methyl, hydrogen, or — SH and wherein the other is
(a) a hydrogen atom;
(b) CHaZb wherein a+b=3, a=0 to 3, b=0 to 3, and wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or Y3 and Y4 taken together are (a) =NH; or
(Jo) =O; wherein Y5 or Y6 is — OH, methyl, hydrogen, or — SH and wherein the other is
(a) a hydrogen atom;
(b) CHaZb where a+b=3, a=0 to 3, b=0 to 3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom; (c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or Y5 and Y6 taken together are
(a) =NH; or
(b) =O; wherein R5 is
(a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
Cb) -(CHz)n -R1 wherein n = 0 to 4 and R1 is
(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive; (ii) phenyl; or
(iii) substituted phenyl
Figure imgf000042_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, -
C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
Figure imgf000042_0002
wherein Qa is — O — or — S — ; and wherein R3 is alkyl ene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is either alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched or substituted phenyl; Cd) -C(R111)(R1V)-R1 wherein R111 and Rlv are each independently: (i) a hydrogen atom; or (ii) CHaZb where a+b=3 , a=0 to 3 , b=0+3 , and wherein each Z, independently, is a cyano, a nitro, or a halogen atom; or (e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
In another embodiment of this invention, lipoxin analogs have the structural formula VI:
Figure imgf000043_0001
wherein R3 is (a) a hydrogen atom; or
(b) alkyl of 1 to 8 carbon atoms; wherein Rx is
(a) substituted phenyl
Figure imgf000043_0002
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(b) a substituted phenoxy
Figure imgf000044_0001
wherein Z1 through Zv are as defined above; or
Figure imgf000044_0002
wherein Z1 through Zv are as defined above.
In another preferred embodiment of this invention, lipoxin analogs have the following structural formula VII:
Figure imgf000044_0003
wherein Ra is
(a) a hydrogen atom; or
(b) an alkyl of 1 to 8 carbon atoms; wherein Rb and R0 are each independently:
(a) a hydrogen atom;
(b) a hydroxyl, or a thiol;
(c) a methyl or a halomethyl;
(d) a halogen;
(e) an alkoxy of 1 to 3 carbon atoms; wherein Rd and Re are each independently:
(a) a hydrogen atom;
(b) a hydroxyl, or thiol;
(c) a methyl or halomethyl;
(d) a halogen;
(e) an alkoxy of 1 to 3 carbon atoms; or
(f) an alkyls or haloalkyl of 2 to 4 carbon atoms, inclusive, which can be straight chain or branched.
In another preferred embodiment of this invention, the lipoxin analogs have the structural formula VIII:
Figure imgf000045_0001
wherein Ra is
(a) a hydrogen atom; or
(b) an alkyl of 1 to 8 carbon atoms; wherein Rb and R0 are each independently:
(a) a hydrogen atom;
(b) a hydroxyl or a thiol;
(c) a halomethyl;
(d) a halogen; (e) an alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched; or
(f) an alkoxy of 1 to 3 carbon atoms, inclusive; wherein Ra and Re are each independently:
(a) a hydrogen atom;
(b) a hydroxyl, or a thiol; (c) a methyl or a halomethyl;
(d) a halogen;
(e) an alkoxy of 1 to 3 carbon atoms, inclusive; or
(f) an alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which can be straight chain or branched. In another embodiment of this invention, the lipoxin analogs have the structural formula IX:
Figure imgf000046_0001
wherein R3 is
(a) a hydrogen atom; or
(b) an alkyl of 1 to 8 carbon atoms; wherein Rb and Rc are each independently:
(a) a hydrogen atom;
(b) a hydroxyl or thiol;
(c) a halomethyl;
(d) a halogen; (e) an alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;
(f) an alkoxy of 1 to 3 carbon atoms, inclusive; and wherein R5 is
(a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
(b) _(CH2)n, — R1 wherein n = 0 to 4 and R, is
(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) phenyl; or
(iii) substituted phenyl
Figure imgf000047_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - CC=O)-R1, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl; (C) RaQaRb wherein Qa is — O — or — S — ; wherein Ra is alkyl ene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is either alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched or substituted phenyl;
Figure imgf000047_0002
wherein R1n and R1V are each, independently: (i) a hydrogen atom; or (ii) CH3Zb where a+b=3, a=0 to 3, b=0+3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom; or (e) haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
In another preferred embodiment, the compounds have the structural formula X:
Figure imgf000048_0001
wherein R3 is
(a) a hydrogen atom; or
(b) alkyl of 1 to 8 carbon atoms, inclusive, straight chain or branched; and wherein Rb and Rc are each, independently:
(a) a hydrogen atom;
(b) a hydroxyl or a thiol;
(c) a halomethyl;
(d) a halogen;
(e) an alkyl of 1 to 3 carbon atoms, inclusive, straight chain or branched;
(f) an alkoxy of 1 to 3 carbon atoms, inclusive.
In another preferred embodiment, the compounds have the structural formula XI:
Figure imgf000049_0001
wherein Ra is
(i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched; or
(iii) a detectable label molecule; wherein n=l to 10, inclusive; wherein Y2, R3a, and R3b are each, independently:
(a) a hydrogen atom; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
wherein Q2 is — O — or — S — ; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi is — OH, methyl, or — SH; wherein Y2 is
(a) a hydrogen atom;
(b) CH3Zb where a+b=3, a=0 to 3, b=0 to 3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom; or
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; wherein Y3 and Y5 are each independently:
(a) a hydrogen atom;
(b) CHaZb wherein a+b=3, a=0 to 3, b=0 to 3 and wherein each Z, independently, is a cyano, a nitro, or a halogen atom; or (c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; wherein Y4 and Y6 are each, independently
(a) a hydrogen atom;
(b) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(c) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or (d) a hydroxyl or thiol; and wherein R5 is
(a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched; (b) — (CH2)n — R1 wherein n=0 to 3 and R, is (i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) phenyl; (iii) substituted phenyl
Figure imgf000050_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
Figure imgf000050_0002
wherein Qa is — O — or — S — ; wherein R3 is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is
(a) a substituted phenyl
Figure imgf000051_0001
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, -
C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl; (b) a substituted phenoxy
Figure imgf000051_0002
wherein Z1 through Zv are as defined above; or
Figure imgf000051_0003
wherein Z1 through Zv are as defined above; (d) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched.
In certain embodiments of this invention, the compounds of this invention have the following structural formulas:
Figure imgf000052_0001
Figure imgf000052_0002
Figure imgf000052_0003
Figure imgf000053_0001
Figure imgf000053_0002
Figure imgf000053_0003
10
Figure imgf000053_0004
Figure imgf000054_0001
Figure imgf000054_0002
Figure imgf000054_0003
Figure imgf000054_0004
where R' is H or CH3 ; and where the substituents at C* are in the R configuration.
In other preferred embodiments of this invention, the compounds of this invention have the following structural formulas:
Figure imgf000055_0001
Figure imgf000055_0002
Figure imgf000055_0003
10
Figure imgf000056_0001
Figure imgf000056_0002
Figure imgf000056_0003
10
Figure imgf000057_0001
Figure imgf000057_0002
Figure imgf000057_0003
Figure imgf000057_0004
where the substituents at the C* are in the R configuration.
It is to be understood that the carboxylic acids and esters of the invention can be converted, if necessary, into pharmaceutically acceptable salts. LIPOXINS HAVING PHENOXY OR THIOPHENOXY SUBSTITUENTS
In another aspect, lipoxins and lipoxin analogs useful to treat conditions described throughout the specification has the formula:
Figure imgf000058_0001
wherein X is Ri, ORi, or SRi; wherein Ri is
(i) a hydrogen atom;
(ϋ) an alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched; (iii) a cycloalkyl of 3 to 10 carbon atoms;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl;
(vi) substituted phenyl
Figure imgf000058_0002
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl; (vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein Qi is (C=O), SO2 or (CN), provided when Qi is CN, then X is absent; wherein Q3 and Q4 are each independently O, S or NH; wherein one of R2 and R3 is a hydrogen atom and the other is
(a) H;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or
(e) R3Q2Rb wherein Q2 is -O- or -S-; wherein Ra is alkyl ene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when Rb is 0, then Rb is a hydrogen atom; wherein R4 is
(a) H;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be a straight chain or branched;
wherein R5 is
Figure imgf000059_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, -
C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group; wherein Y] is -OH, methyl, -SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH3Zb where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen; wherein R6 is
(a) H;
(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; wherein T is O or S, and pharmaceutically acceptable salts thereof. In yet another aspect, lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
Figure imgf000060_0001
wherein X is Ri, ORi, or SRi; wherein Ri is
(i) a hydrogen atom;
(ϋ) an alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms;
(iv) an aralkyl of 7 to 12 carbon atoms;
(V) phenyl;
(Vi) substituted phenyl
Figure imgf000061_0001
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl; (vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein Qi is (C=O), SO2 or (CN), provided when Qi is CN, then X is absent; wherein one of R2 and R3 is a hydrogen atom and the other is (a) H;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or
(e) R3Q2Rb wherein Q2 is -O- or -S-; wherein R3 is alkylene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when Rb is 0, then Rb is a hydrogen atom; wherein R4 is
(a) H;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be a straight chain or branched; wherein R5 is
Figure imgf000062_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group; wherein Yi is -OH, methyl, -SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH3Zb where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen; wherein R6 is (a) H;
(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; wherein T is O or S, and pharmaceutically acceptable salts thereof. In still another aspect, lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
Figure imgf000062_0002
wherein X is Ri, ORi, or SRi; wherein Ri is
(i) a hydrogen atom; (ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl; (vi) substituted phenyl
Figure imgf000063_0001
wherein Z1, Zn, Z1n, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein Qi is (C=O), SO2 or (CN), provided when Qi is CN, then X is absent; wherein one of R2 and R3 is a hydrogen atom and the other is
(a) H;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or
(e) RaQ2Rb wherein Q2 is -O- or -S-; wherein Ra is alkylene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when Rb is 0, then Rb is a hydrogen atom; wherein R4 is
(a) H;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be a straight chain or branched; wherein R5 is
Figure imgf000064_0001
wherein Z1, Zn, Zm, Z,v and Zv are each independently selected from -NO2, -CN, -
C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group; wherein R6 is (a) H;
(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; wherein T is O or S, and pharmaceutically acceptable salts thereof. In yet another aspect, lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
Figure imgf000065_0001
wherein X is Ri, ORj, or SRi; wherein Ri is
(i) a hydrogen atom;
(ϋ) an alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms;
(iv) an aralkyl of 7 to 12 carbon atoms;
(V) phenyl;
(Vi) substituted phenyl
Figure imgf000065_0002
wherein Z1, Z11, Z111, Zlv and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl; (vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein Qi is (C=O), SO2 or (CN), provided when Qi is CN, then X is absent; wherein R4 is
(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be a straight chain or branched; wherein R5 is
Figure imgf000066_0001
wherein Z1, Z11, Z111, Z,v and Zv are each independently selected from -NO2, -CN, -C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group; wherein R6 is
(a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched; wherein T is O or S, and pharmaceutically acceptable salts thereof. In one aspect, lipoxins and lipoxin analogs useful as a therapeutic agent in the treatment of the maladies, disease states or conditions described throughout the specification has the formula:
Figure imgf000066_0002
wherein X is Ri, ORi, or SRi wherein Ri is
(i) a hydrogen atom;
(ϋ) an alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms;
(iv) an aralkyl of 7 to 12 carbon atoms;
(V) phenyl;
(Vi) substituted phenyl
Figure imgf000067_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl; (vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein R4 is
(a) H;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be a straight chain or branched; wherein R5 is
Figure imgf000067_0002
wherein Z1, Z11, Z111, ZIV and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group; and pharmaceutically acceptable salts thereof.
In preferred embodiments, X is ORi wherein Ri is a hydrogen atom, an alkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptable salt, Qi is C=O, R2 and R3, if present, are hydrogen atoms, R4 is a hydrogen atom or methyl, Q3 and Q4, if present, are both O, R6, if present, is a hydrogen atom, Yi, if present, is OH, T is O and R5 is a substituted phenyl, e.g.,
Figure imgf000068_0001
wherein Z1, Z11, Z111, ZIV and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl. In certain embodiments for R5, para-fluorophenyl and/or unsubstituted phenyl are preferred, e.g., 15- epi- 16-(para-fluoro)-phenoxy-LXA4, 16-(para-fluoro)-phenoxy-LXA4, 15-epi-l 6-phenoxy- LXA4 or 16-phenoxy-LXA4.
In still another aspect, the present invention is directed to pharmaceutical compositions including compounds having the formulae described throughout the specification and a pharmaceutically acceptable carrier. In one embodiment, a preferred compound is
Figure imgf000069_0001
In one embodiment, Qi is a carbonyl, X is a hydroxyl or an -OR, wherein R is an alkyl group, i.e., methyl or ethyl groups, and R4 is a hydrogen atom.
In other embodiments, Yi is a hydroxyl and the carbon bearing the hydroxyl can have an R or S configuration. In most preferred embodiments, the chiral carbon bearing the hydroxyl group, e.g., Yi1 is designated as a 15-epi-lipoxin as is known in the art.
In certain embodiments the chirality of the carbons bearing the R2, R3, Q3 and Q4 groups can each independently be either R or S. In preferred embodiments, Q3 and Q4 have the chiralities shown in above-referenced structures.
In preferred embodiments, R4 is a hydrogen. In other preferred embodiments, R6 is a hydrogen.
Additionally, R5 can be a substituted or unsubstituted, branched or unbranched alkyl group having between 1 and about 6 carbon atoms, preferably between 1 and 4 carbon atoms, most preferably between 1 and 3, and preferably one or two carbon atoms. The carbon atoms can have substituents which include halogen atoms, hydroxyl groups, or ether groups. 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 diastereisomers as well as the racemic and optically resolved isomers.
The compounds useful in the present invention can be prepared by the following synthetic scheme:
Figure imgf000070_0001
Figure imgf000070_0002
Figure imgf000070_0003
hydrogenation ►
(optional)
Figure imgf000070_0004
wherein X, Q1, Q3, Q4, R2, R3, R4, R5, R6, Yi and T are as defined above. Suitable methods known in the art to can be used to produce each fragment. For example, the acetylenic fragment can be prepared by the methods discussed in Nicolaou, K.C. et al. (1991) Angew. Chem. Int. Ed. Engl. 30:1100; Nicolaou, K.C. et al. (1989) J. Org. Chem. 54:5527; Webber,
S.E. et al. (1988) Adv. Exp. Med. Biol. 229:61; and U.S. Patent 5,441,951. The second fragment can be prepared by the methods of Raduchel, B. and Vorbruggen, H. (1985) Adv.
Prostaglandin Thromboxane Leukotriene Res. 14:263. As a consequence, the acetyl enic intermediates are also encompassed by the present invention as being useful for the treatments of the various maladies described herein.
A "lipoxin analog" shall mean a compound which has an "active region" that functions like the active region of a "natural lipoxin", but which has a "metabolic transformation region" that differs from natural lipoxin. Lipoxin analogs include compounds which are structurally similar to a natural lipoxin, compounds which share the same receptor recognition site, compounds which share the same or similar lipoxin metabolic transformation region as lipoxin, and compounds which are art-recognized as being analogs of lipoxin. Lipoxin analogs include lipoxin analog metabolites. The compounds disclosed herein may contain one or more centers of asymmetry. Where asymmetric carbon atoms are present, more than one stereoisomer is possible, and all possible isomeric forms are intended to be included within the structural representations shown. Optically active (R) and (S) isomers may be resolved using conventional techniques known to the ordinarily skilled artisan. The present invention is intended to include the possible diastereomers as well as the racemic and optically resolved isomers.
The terms "corresponding lipoxin" and "natural lipoxin" refer to a naturally-occurring lipoxin or lipoxin metabolite. Where an analog has activity for a lipoxin-specific receptor, the corresponding or natural lipoxin is the normal ligand for that receptor. For example, where an analog is a LXA4 specific receptor on differentiated HL-60 cells, the corresponding lipoxin is LXA4. Where an analog has activity as an antagonist to another compound (such as leukotriene C4 and/or leukotriene D4), which is antagonized by a naturally-occurring lipoxin, that natural lipoxin is the corresponding lipoxin.
"Active region" shall mean the region of a natural lipoxin or lipoxin analog, which is associated with in vivo cellular interactions. The active region may bind the "recognition site" of a cellular lipoxin receptor or a macromolecule or complex of macromolecules, including an enzyme and its cofactor. For example, lipoxin A4 analogs have an active region comprising C5 — C15 of natural lipoxin A4. Similarly, for example, lipoxin B4 analogs have an active region comprising C5-C14 of natural lipoxin B4. The term "recognition site" or receptor is art-recognized and is intended to refer generally to a functional macromolecule or complex of macromolecules with which certain groups of cellular messengers, such as hormones, leukotrienes, or lipoxins must first interact before the biochemical and physiological responses to those messengers are initiated. As used in this application, a receptor may be isolated, on an intact or permeabilized cell, or in tissue, including an organ. A receptor may be from or in a living subject, or it may be cloned. A receptor may normally exist or it may be induced by a disease state, by an injury, or by artificial means. A compound of this invention may bind reversibly, irreversibly, competitively, noncompetitively, or uncompetitively with respect to the natural substrate of a recognition site.
The term "metabolic transformation region" is intended to refer generally to that portion of a lipoxin, a lipoxin metabolite, or lipoxin analog including a lipoxin analog metabolite, upon which an enzyme or an enzyme and its cofactor attempts to perform one or more metabolic transformations which that enzyme or enzyme and cofactor normally transform on lipoxins. The metabolic transformation region may or may not be susceptible to the transformation. A nonlimiting example of a metabolic transformation region of a lipoxin is a portion Of LXA4 that includes the C- 13, 14 double bond or the C-15 hydroxyl group, or both. The term "detectable label molecule" is meant to include fluorescent, phosphorescent, and radiolabeled molecules used to trace, track, or identify the compound or receptor recognition site to which the detectable label molecule is bound. The label molecule may be detected by any of the several methods known in the art.
The term "labeled analog" is further understood to encompass compounds which are labeled with radioactive isotopes, such as but not limited to tritium (3H), deuterium (2H), carbon (14C), or otherwise labeled (e.g. fluorescently). The compounds of this invention may be labeled or derivatized, for example, for kinetic binding experiments, for further elucidating metabolic pathways and enzymatic mechanisms, or for characterization by methods known in the art of analytical chemistry. The term "inhibits metabolism" means the blocking or reduction of activity of an enzyme which metabolizes a native molecule. The blockage or reduction may occur by covalent bonding, by irreversible binding, by reversible binding which has a practical effect of irreversible binding, or by any other means which prevents the enzyme from operating in its usual manner on another lipoxin analog, including a lipoxin analog metabolite, a lipoxin, or a lipoxin metabolite.
The term "resists metabolism" is meant to include failing to undergo one or more of the metabolic degradative transformations by at least one of the enzymes which metabolize lipoxins. Two nonlimiting examples Of LXA4 analog that resists metabolism are 1) a structure which can not be oxidized to the 15-oxo form, and 2) a structure which may be oxidized to the 15-oxo form, but is not susceptible to enzymatic reduction to the 13,14- dihydro form.
The term "more slowly undergoes metabolism" means having slower reaction kinetics, or requiring more time for the completion of the series of metabolic transformations by one or more of the enzymes which metabolize lipoxin or lipoxin analogs. A nonlimiting example of a LXA4 analog which more slowly undergoes metabolism is a structure which has a higher transition state energy for C- 15 dehydrogenation than does LXA4 because the analog is sterically hindered at the C- 16. The term "tissue" is intended to include intact cells, blood, blood preparations such as plasma and serum, bones, joints, muscles, smooth muscles, and organs.
The term "halogen" is meant to include fluorine, chlorine, bromine and iodine, or fluoro, chloro, bromo, and iodo.
The term "subject" is intended to include living organisms susceptible to conditions or diseases caused or contributed bacteria and pathogens 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 therapeutic agent, 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. ScL, 1977;66:1-19 which is incorporated herein by reference).
The term "pharmaceutically acceptable esters" refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. The term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. 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. Pn 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 -butyl ene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, 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 may be 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 maybe 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 therapeutic agent(s) 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 therapeutic agent 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 therapeutic agent 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 therapeutic agent 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 therapeutic agents 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 therapeutic agents 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 a therapeutic agent of the invention 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 polyolefin (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 therapeutic formulation contained within the packaging material. This formulation contains an at least one therapeutic agent 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 therapeutic agents include, for example, the lipoxin analogs described herein.
More specifically, the invention features an article of manufacture that contains packaging material and at least one therapeutic agent 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.
The following paragraphs enumerated consecutively from one (1) through twenty (20) provide for various aspects of the present invention. In one embodiment, in a first paragraph
(1), the present invention provides a method for the increase of resolution in a subject's tissue subjected to an anesthetic, comprising the step of administering a therapeutically effective amount of lipoxin A4 or a lipoxin analog, such that the subject's tissue subjected to the anesthetic resolve more quickly than without administration of a lipoxin A4 or a lipoxin analog.
2. The method of paragraph 1, wherein the lipoxin A4 or lipoxin analog are administered prior to the administration of the anesthetic.
3. The method of paragraph 1 , wherein the lipoxin A4 or lipoxin analog are administered during the administration of the anesthetic. 4. The method of paragraph 1 , wherein the lipoxin A4 or lipoxin analog are administered after the administration of the anesthetic.
5. The method of paragraph 1 , wherein the lipoxin analog has the formula:
Figure imgf000084_0001
wherein R1, R2, X, OR1, SR1 ;Z,, Z11, Z111, Zn and Zv -ORx, Rx, Q1, Q3, R3, R3Q2Rb, Q2, Ra, Rb, R45Yi, Y2 ,CH3Zb, a, b, Z, R5, -(CH2)n -R1, n, R1, R3Q3Rb, Qa, — C(Rm)(Rlv) — R1, R]n, Riv, R3a, R-3b, Y3, Y4 Y5, Yβ, Rd, and Re (and any remaining substituents) are as defined above. 6. The method of paragraph 5, wherein the lipoxin analog is administered prior to the administration of the anesthetic.
7. The method of paragraph 5, wherein the lipoxin analog is administered during the administration of the anesthetic.
8. The method of paragraph 5, wherein the lipoxin analog is administered after the administration of the anesthetic.
9. The method of paragraph 1 , wherein the lipoxin analog is selected from the formulae I through XI as described above and pharmaceutically acceptable salts thereof.
10. The method of paragraph 9, wherein the lipoxin analog is administered prior to the administration of the anesthetic.
11. The method of paragraph 9, wherein the lipoxin analog is administered during the administration of the anesthetic.
12. The method of paragraph 9, wherein the lipoxin analog is administered after the administration of the anesthetic.
13. The method of paragraph 1, wherein the lipoxin analog has the formula:
Figure imgf000085_0001
14. The method of paragraph 13, wherein the lipoxin analog is administered prior to the administration of the anesthetic. 15. The method of paragraph 13, wherein the lipoxin analog is administered during the administration of the anesthetic.
16. The method of paragraph 13, wherein the lipoxin analog is administered after the administration of the anesthetic.
17. The method of any of paragraphs 1 through 4, further comprising a carrier with the therapeutically effective amount of lipoxin A4 or a lipoxin analog.
18. The method of any of paragraphs 5 through 8, further comprising a carrier with the therapeutically effective amount of the lipoxin analog.
19. The method of any of paragraphs 9 through 12, further comprising a carrier with the therapeutically effective amount of the lipoxin analog.
20. The method of any of paragraphs 13 through 16, further comprising a carrier with the therapeutically effective amount of the lipoxin analog.
References 1. Cotran RS, Kumar V, Collins T, editors (1999) Robbins Pathologic Basis of Disease. 6th ed. Philadelphia: W.B. Saunders Co. 1425 p.
2. Serhan CN, Clish CB, Brannon J, Colgan SP, Chiang N, et al. (2000) Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing. J Exp Med 192: 1197-1204.
3. Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, et al. (2002) Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter pro-inflammation signals. J Exp Med 196: 1025-1037.
4. Serhan CN, Brain SD, Buckley CD, Gilroy DW, Haslett C, et al. (2007) Resolution of inflammation: state of the art, definitions and terms. FASEB J 21 : 325-332.
5. Serhan CN, Chiang N (2007) Endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus. Br J Pharmacol: doi: 10.1038/sj.bjp.0707489. 6. Maddox JF, Serhan CN (1996) Lipoxin A4 and B4 are potent stimuli for human monocyte migration and adhesion: selective inactivation by dehydrogenation and reduction. J Exp Med 183: 137-146.
7. Godson C, Mitchell S, Harvey K, Petasis NA, Hogg N, et al. (2000) Cutting edge:
Lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. J Immunol 164: 1663-1667.
8. Bannenberg GL, Chiang N, Ariel A, Arita M, Tjonahen E, et al. (2005) Molecular circuits of resolution: Formation and actions of resolvins and protectins. J Immunol 174: 4345-4355.
9. Schwab JM, Chiang N, Arita M, Serhan CN (2007) Resolvin El and protectin Dl activate inflammation-resolution programmes. Nature 447: 869-874.
10. Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ, et al. (1999) Inducible cycloxygenase may have anti-inflammatory properties. Nat Med 5: 698-701.
11. Hawkins JM, Moore PA (2002) Local anesthesia: advances in agents and techniques.
Dent Clin North Am 46: 719-732, ix. 12. SaIo M (1992) Effects of anaesthesia and surgery on the immune response. Acta
Anaesthesiol Scand 36: 201-220. 13. Gallos G, Jones DR, Nasr SH, Emala CW, Lee HT (2004) Local anesthetics reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis. Anesthesiology 101 : 902-911. 14. Plachinta RV, Hayes JK, Cerilli LA, Rich GF (2003) Isoflurane pretreatment inhibits lipopolysaccharide-induced inflammation in rats. Anesthesiology 98: 89-95.
15. Winyard PG, Willoughby DA, editors (2003) Inflammation Protocols. Totowa, NJ:
Humana. 378 p.
16. Verleye M, Heulard I, Gillardin JM (2000) Phenazone potentiates the local anaesthetic effect of lidocaine in mice. Pharmacol Res 41 : 539-542.
17. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN (2001) Lipid mediator class switching during acute inflammation: signals in resolution. Nat Immunol 2: 612-619.
18. Savill J, Dransfield I, Gregory C, Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2: 965-975. 19. Underhill DM (2003) Macrophage recognition of zymosan particles. J Endotoxin Res 9:
176-180. 20. Roth J, Vogl T, Sorg C, Sunderkotter C (2003) Phagocyte-specific SlOO proteins: a novel group of proinflammatory molecules. Trends Immunol 24: 155-158. 21. Foell D, Wittkowski H, Vogl T, Roth J (2007) SlOO proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol 81 : 28- 37. 22. Kurosaka K, Chen Q, Yarovinsky F, Oppenheim JJ, Yang D (2005) Mouse cathelin- related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor- like 1 /mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant. J Immunol 174: 6257-6265.
23. Gil CD, Cooper D, Rosignoli G, Perretti M, Oliani SM (2006) Inflammation-induced modulation of cellular galectin-1 and -3 expression in a model of rat peritonitis.
Inflamm Res 55: 99-107.
24. Dias-Baruffi M, Zhu H, Cho M, Karmakar S, McEver RP, et al. (2003) Dimeric galectin-
1 induces surface exposure of phosphatidylserine and phagocytic recognition of leukocytes without inducing apoptosis. J Biol Chem 278: 41282-41293. 25. Nathan C (2002) Points of control in inflammation. Nature 420: 846-852.
26. DesMarais V, Ghosh M, Eddy R, Condeelis J (2005) Cofilin takes the lead. J Cell Sci
118: 19-26.
27. Mazzocco M, Maffei M, Egeo A, Vergano A, Arrigo P, et al. (2002) The identification of a novel human homologue of the SH3 binding glutamic acid-rich (SH3BGR) gene establishes a new family of highly conserved small proteins related to Thioredoxin
Superfamily. Gene 291 : 233-239.
28. Tamaki H, Nakamura H, Nishio A, Nakase H, Ueno S, et al. (2006) Human thioredoxin- 1 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production. Gastroenterology 131 : 1110-1121. 29. Serhan CN (2007) Resolution phases of inflammation: novel endogenous antiinflammatory and pro-resolving lipid mediators and pathways. Annu Rev Immunol 25: 101-137.
30. Golan DE, Tashjian AH, Jr., Armstrong EJ, Armstrong AW, editors (2007) Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy. 2nd ed. Baltimore: Lippincott Williams & Wilkins. 985 p. 31. Goldstein IM, Lind S, Hoffstein S, Weissmann G (1977) Influence of local anesthetics upon human polymorphonuclear leukocyte function in vitro. Reduction of lysosomal enzyme release and superoxide anion production. J Exp Med 146: 483-494.
32. Sasagawa S (1991) Inhibitory effects of local anesthetics on migration, extracellular release of lysosomal enzyme, and superoxide anion production in human polymorphonuclear leukocytes. Immunopharmacol Immunotoxicol 13: 607-622.
33. Cullen BF, Haschke RH (1974) Local anesthetic inhibition of phagocytosis and metabolism of human leukocytes. Anesthesiology 40: 142-146.
34. de Klaver MJM, Buckingham M-G, Rich GF (2003) Lidocaine attenuates cytokine- induced cell injury in endothelial and vascular smooth muscle cells. Anesth Analg 97: 465-470.
35. Lee HT, Krichevsky IE, Xu H, Ota-Setlik A, D'Agati VD, et al. (2004) Local anesthetics worsen renal function after ischemia-reperfusion injury in rats. Am J Physiol Renal Physiol 286: Fl 11 -F 119.
36. Yregard L, Cassuto J, Tarnow P, Nilsson U (2003) Influence of local anaesthetics on inflammatory activity postburn. Burns 29: 335-341.
37. Rossi AG, Sawatzky DA, Walker A, Ward C, Sheldrake TA, et al. (2006) Cyclin- dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis. Nat Med 12: 1056-1064.
38. Sawatzky DA, Willoughby DA, Colville-Nash PR, Rossi AG (2006) The involvement of the apoptosis-modulating proteins ERK 1/2, BcI -xL and Bax in the resolution of acute inflammation in vivo. Am J Pathol 168: 33-41.
39. Taniguchi T, Koido Y, Aiboshi J, Yamashita T, Suzaki S, et al. (1999) The ratio of interleukin-6 to interleukin-10 correlates with severity in patients with chest and abdominal trauma. Am J Emerg Med 17: 548-551. 40. Walley KR, Lukacs NW, Standiford TJ, Strieter RM, Kunkel SL (1996) Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 64: 4733-4738.
41. Gogos CA, Drosou E, Bassaris HP, Skoutelis A (2000) Pro- versus anti-inflammatory cytokine profile in patients with severe sepsis: a marker for prognosis and future therapeutic options. J Infect Dis 181 : 176- 180.
42. Chow CC, Clermont G, Kumar R, Lagoa C, Tawadrous Z, et al. (2005) The acute inflammatory response in diverse shock states. Shock 24: 74-84. 43. Alam S, Laughton DL, Walding A, Wolstenholme AJ. (2006) Human peripheral blood mononuclear cells express GABAA receptor subunits. MoI Immunol. 43: 1432-1442.
44. Hayes JK, Havaleshko DM, Plachinta RV, Rich GF (2004) Isoflurane pretreatment supports hemodynamics and leukocyte rolling velocities in rat mesentery during lipopolysaccharide-induced inflammation. Anesth Analg 98: 999-1006.
45. de Rossi LW, Horn NA, Buhre W, Gass F, Hutschenreuter G, et al. (2002) The effect of isoflurane on neutrophil selectin and b2-integrin activation in vitro. Anesth Analg 95: 583-587.
46. Nascimento-Silva V, Arruda MA, Barja-Fidalgo C, Villela CG, Fierro IM (2005) Novel lipid mediator aspirin-triggered lipoxin A4 induces heme oxygenase- 1 in endothelial cells. Am J Physiol Cell Physiol 289: C557-C563.
47. Biteman B, Hassan IR, Walker E, Leedom AJ, Dunn M, et al. (2007) Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing. FASEB J 21 : 2257-2266. 48. Brandt L (1967) Studies on the phagocytic activity of neutrophilic leukocytes. Scand J
Haematol Suppl 2: 1-126.
49. Tauber AI, Chernyak L (1991) Metchnikoff and the Origins of Immunology: From
Metaphor to Theory. New York: Oxford University Press. 247 p.
50. Azad SS, Bartkowski RR, Witkowski TA, Marr AT, Lessin JB, et al. (1993) A comparison of desflurane and isoflurane in prolonged surgery. J Clin Anesth 5: 122-
128.
One having ordinary skill in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein, including those in the background section, are expressly incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:
1. A method for the increase of resolution in a subject's tissue subjected to an anesthetic, comprising the step of administering a therapeutically effective amount of lipoxin A4 or a lipoxin analog, such that the subject's tissue subjected to the anesthetic resolve more quickly than without administration of a lipoxin A4 or a lipoxin analog.
2. The method of claim 1 , wherein the lipoxin A4 or lipoxin analog are administered prior to the administration of the anesthetic.
3. The method of claim 1 , wherein the lipoxin A4 or lipoxin analog are administered during the administration of the anesthetic.
4. The method of claim 1 , wherein the lipoxin A4 or lipoxin analog are administered after the administration of the anesthetic.
5. The method of claim 1, wherein the lipoxin analog has the formula:
Figure imgf000091_0001
wherein R1 if present can be
Figure imgf000091_0002
Figure imgf000092_0001
Figure imgf000092_0002
Figure imgf000092_0003
Figure imgf000092_0004
and R2 if present can be
Figure imgf000092_0005
Figure imgf000092_0006
Figure imgf000093_0001
(forms
Figure imgf000093_0002
Figure imgf000093_0003
Figure imgf000093_0004
Figure imgf000093_0005
Figure imgf000093_0006
Figure imgf000094_0001
(forms
Figure imgf000094_0002
wherein X is Ri, ORi, or SRi; wherein Ri is (i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbons atoms, inclusive, which can be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl;
(vi) substituted phenyl
Figure imgf000094_0003
wherein Z,, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - Q=O)-R1 , -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl; (vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive; wherein Q1 is (C=O), SO2 or (CN); wherein Q3 is O, S or NH; wherein one of R2 and R3 is a hydrogen atom and the other is
(a) a hydrogen atom;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
(e) RaQ2Rb wherein Q2 is -O- or -S-; wherein Ra is alkylene of O to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein Rb is alkyl of O to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein R4 is
(a) a hydrogen atom;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which can be straight chain or branched; wherein Yi or Y2 is -OH, methyl, or -SH and wherein the other is
(a) a hydrogen atom
(b) CHaZb where a+b=3, a=0 to 3, b=0 to 3; and each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched; or
(d) an alkoxy of 1 to 4 carbon atoms, inclusive; or Y] and Y2 taken together are
(a) =NH; or
(Jo) =O; wherein R5 is
(a) an alkyl of 1 to 9 carbon atoms which can be straight chain or branched;
(b) -(CH2)n -R, wherein n=0 to 4 and R1 is
(i) a cycloalkyl of 3 to 10 carbon atoms, inclusive;
(ii) a phenyl; or
(iii) substituted phenyl
Figure imgf000096_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(c) RaQaRb wherein Qa is O or S; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched;
Cd) -C(R111)(R1V)-R1 wherein R111 and R1V are each, independently:
(i) a hydrogen atom;
(ii) CHaZb where a+b=3, a=0 to 3, b=0+3, and wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(e) a haloalkyl of 1 to 8 carbon atoms, inclusive, and 1 to 6 halogen atoms, inclusive, straight chain or branched; and wherein R6 is
(a) a hydrogen atom;
(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched;
(c) a halogen; wherein R3a, and R3b are each independently:
(a) a hydrogen atom;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which can be straight chain or branched;
(c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which can be straight chain or branched; or
Figure imgf000097_0001
wherein Q2 is -O- or -S-; wherein Ra is alkylene of 0 to 6 carbons atoms, inclusive, which can be straight chain or branched; and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which can be straight chain or branched; wherein Y3 or Y4 is — OH, methyl, hydrogen, or — SH and wherein the other is
(a) a hydrogen atom;
(b) CHaZb wherein a+b=3, a=0 to 3, b=0 to 3, and wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or Y3 and Y4 taken together are
(a) =NH; or
(b) =O; wherein Y5 or Y6 is — OH, methyl, hydrogen, or — SH and wherein the other is
(a) a hydrogen atom;
(b) CH3Zb where a+b=3, a=0 to 3, b=0 to 3 wherein each Z, independently, is a cyano, a nitro, or a halogen atom;
(c) an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched;
(d) an alkoxy of 1 to 4 carbon atoms, inclusive, straight chain or branched; or Y5 and Y6 taken together are
(a) =NH; or (b) =O; wherein Ra is
(a) a hydrogen atom; or
(b) alkyl of 1 to 8 carbon atoms; wherein Rx is
(a) substituted phenyl
Figure imgf000098_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which can be a straight chain or branched, and hydroxyl;
(b) a substituted phenoxy
Figure imgf000098_0002
wherein Z1 through Zv are as defined above; or
Figure imgf000098_0003
wherein Z1 through Zv are as defined above. wherein Rb and Rc are each independently:
(a) a hydrogen atom;
(b) a hydroxyl, or a thiol;
(c) a methyl or a halomethyl;
(d) a halogen;
(e) an alkoxy of 1 to 3 carbon atoms; wherein Rd and Re are each independently:
(a) a hydrogen atom;
(b) a hydroxyl, or thiol;
(c) a methyl or halomethyl;
(d) a halogen;
(e) an alkoxy of 1 to 3 carbon atoms; or
(f) an alkyl or haloalkyl of 2 to 4 carbon atoms, inclusive, which can be straight chain or branched.
6. The method of claim 5, wherein the lipoxin analog is administered prior to the administration of the anesthetic.
7. The method of claim 5, wherein the lipoxin analog is administered during the administration of the anesthetic.
8. The method of claim 5, wherein the lipoxin analog is administered after the administration of the anesthetic.
9. The method of claim 1, wherein the lipoxin analog has the formula:
Figure imgf000099_0001
wherein X is Ri, ORi, or SRi; wherein Rj is
(i) a hydrogen atom;
(ii) an alkyl of 1 to 8 carbon atoms, inclusive, which may be straight chain or branched;
(iii) a cycloalkyl of 3 to 10 carbon atoms;
(iv) an aralkyl of 7 to 12 carbon atoms;
(v) phenyl;
(vi) substituted phenyl
Figure imgf000100_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - CC=O)-R1, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl;
(vii) a detectable label molecule; or
(viii) a straight or branched chain alkenyl of 2 to 8 carbon atoms, inclusive;
wherein Qi is (C=O), SO2 or (CN), provided when Qi is CN, then X is absent; wherein Q3 and Q4 are each independently O, S or NH; wherein one of R2 and R3 is a hydrogen atom and the other is
(a) H;
(b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;
(d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may be straight chain or branched; or
(e) R3Q2Rb wherein Q2 is -O- or -S-; wherein Ra is alkylene of 0 to 6 carbon atoms, inclusive, which may be straight chain or branched and wherein Rb is alkyl of 0 to 8 carbon atoms, inclusive, which may be straight chain or branched, provided when Rb is 0, then Rb is a hydrogen atom; wherein R4 is
(a) H;
(b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be a straight chain or branched;
wherein R^ is
Figure imgf000101_0001
wherein Z1, Z11, Z111, Z1V and Zv are each independently selected from -NO2, -CN, - C(=O)-Ri, -SO3H, a hydrogen atom, halogen, methyl, -ORx, wherein Rx is 1 to 8 carbon atoms, inclusive, which may be a straight chain or branched, and hydroxyl or a substituted or unsubstituted, branched or unbranched alkyl group;
wherein Yi is -OH, methyl, -SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH3Zb where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;
wherein R6 is (a) H;
(b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chain or branched;
wherein T is O or S, and pharmaceutically acceptable salts thereof.
10. The method of claim 9, wherein the lipoxin analog is administered prior to the administration of the anesthetic.
11. The method of claim 9, wherein the lipoxin analog is administered during the administration of the anesthetic.
12. The method of claim 9, wherein the lipoxin analog is administered after the administration of the anesthetic.
13. The method of claim 1 , wherein the lipoxin analog has the formula:
Figure imgf000102_0001
14. The method of claim 13, wherein the lipoxin analog is administered prior to the administration of the anesthetic.
15. The method of claim 13, wherein the lipoxin analog is administered during the administration of the anesthetic.
16. The method of claim 13, wherein the lipoxin analog is administered after the administration of the anesthetic.
17. The method of claim 1 , further comprising a carrier with the therapeutically effective amount of the lipoxin A4 or the lipoxin analog.
18. The method of claim 5, further comprising a carrier with the therapeutically effective amount of the lipoxin analog.
19. The method of claim 9, further comprising a carrier with the therapeutically effective amount of the lipoxin analog.
20. The method of claim 13, further comprising a carrier with the therapeutically effective amount of the lipoxin analog.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5441951A (en) * 1994-06-15 1995-08-15 Brigham & Women's Hospital Lipoxin compounds
US6329425B1 (en) * 1991-04-01 2001-12-11 Brigham And Women's Hospital Modulation of inflammation related to Columnar epithelia

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6329425B1 (en) * 1991-04-01 2001-12-11 Brigham And Women's Hospital Modulation of inflammation related to Columnar epithelia
US5441951A (en) * 1994-06-15 1995-08-15 Brigham & Women's Hospital Lipoxin compounds

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BLAITHIN MCMAHON ET AL.: 'Lipoxins: revelations on resolution.' TRENDS IN PHARMACOLOGICAL SCIENCES. vol. 22, no. 8, August 2001, pages 391 - 395 *
CAMILLA 1. SVENSSON ET AL.: 'Lipoxins and aspirin-triggered lipoxin inhibit inflammatory pain processing.' THE JOURNAL OF EXPERIMENTAL MEDICINE vol. 204, no. 2, 19 February 2007, pages 245 - 252 *
I.M. FIERRO ET AL.: 'Mechanisms in anti-inflammation and resolution: the role of lipoxins and aspirin-triggered lipoxins.' BRAZILIAN JOURNAL OF MEDICAL AND BIOLOGICAL RESEARCH. vol. 34, no. 5, 2001, pages 555 - 566 *
JAN M. SCHWAB ET AL.: 'Resolvin El and Protectin D1 activate inflammation-resolution programmes.' NATURE. vol. 447, 14 June 2007, pages 869 - 874 *
R. TYTHER ET AL.: 'The effect of the anaesthetic agent isoflurane on the rate of neutrophil apoptosis in vitro.' IRISH JOURNAL OF MEDICINAL SCIENCE. vol. 170, no. 1, January 2001, pages 41 - 44 *
SHENG-WEI JIN ET AL.: 'Posttreatment with aspirin-triggered lipoxin A4 analog attenuates lipopolysaccharide-induced acute lung injury in mice: the role of heme oxygenase-1.' ANESTHESIA & ANALGESIA. vol. 104, no. 2, February 2007, pages 369 - 377 *

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