WO2006113236A2 - Novel chemical inhibitors of neutrophil activation through the sac-dependent pathway - Google Patents

Abstract

The present invention relates to a method of treating an inflammatory disorder in a subject. The method involves administering to a subject an effective amount of a compound that modulates soluble adenylyl cyclase, thereby treating the inflammatory disorder in the subject. The present invention also relates to a method of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by neutrophils. The method involves contacting adherent neutrophils with an effective amount of a compound that modulates soluble adenylyl cyclase.

Description

NOVEL CHEMICAL INHIBITORS OF NEUTROPHIL ACTIVATION THROUGH THE sAC-DEPENDENT PATHWAY

[0001] This application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/671,408, filed April 14, 2005, which is hereby incorporated by reference in its entirety.

[0002] The subject matter of this application was made with support from the

National Institutes of Health (Grant Nos. AI46382, GM62328, HD42060, HD38722,

HL46849, and HL64774). The U.S. Government may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to methods of treating an inflammatory disorder in a subject by administering to a subject an effective amount of a compound that modulates soluble adenylyl cyclase. The present invention also relates to methods of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by neutrophils by contacting adherent neutrophils with an effective amount of a compound that modulates soluble adenylyl cyclase.

BACKGROUND OF THE INVENTION

[0004] Neutrophils, the most abundant leukocytes in blood, provide a critical element of host defense. Defects in neutrophil numbers, migration across endothelium or bactericidal mechanisms lead to life-threatening infections. At the same time, neutrophil activation is a major contributor to inflammatory tissue damage (Weiss, "Tissue Destruction by Neutrophils," NEnglJMed 320:365-376 (1989)). Thus, it is a goal of anti-inflammatory therapy to target neutrophils, but it is a challenge to do so without impairing host defense.

[0005] Two families of neutrophil products play key roles in host defense and tissue damage. Activated neutrophils degranulate to release antibiotic proteins, including proteases that degrade connective tissue. Activated neutrophils also secrete reactive oxygen intermediates (ROI), products of phox, a multi-component enzyme whose catalytic flavocytochrome is a transmembrane protein of the granules. ROI contribute directly to bacterial killing (Fang, "Antimicrobial Reactive Oxygen and Nitrogen Species: Concepts and Controversies," Nat Rev Microbiol 2:820-832 (2004)) but also promote tissue damage by: triggering K⁺ influx into the phagolysosome, desorbing proteases from their proteoglycan bed (Fang, "Antimicrobial Reactive Oxygen and Nitrogen Species: Concepts and Controversies," Nat Rev Microbiol 2:820-832 (2004)); activating matrix metalloproteinases (Peppin et al., "Activation of the Endogenous Metalloproteinase, Gelatinase, by Triggered Human Neutrophils," Proc Natl Acad Sci USA 83:4322-4326 (1986)); inactivating anti-proteases (Weiss, "Tissue Destruction by Neutrophils," N Engl J Med 320:365-376 (1989)); and activating NF-κB and API, transcription factors for numerous genes with proinflammatory products, including chemokines that attract more neutrophils. Thus, inhibition of neutrophil ROI production would be expected to reduce inflammatory proteolysis through multiple mechanisms.

[0006] Maximal neutrophil activation can be induced artificially with phorbol myristate acetate (PMA) or physiologically by inflammatory peptides and proteins, such as tumor necrosis factor (TNF) (Nathan, "Neutrophil Activation on Biological Surfaces. Massive Secretion of Hydrogen Peroxide in Response to Products of Macrophages and Lymphocytes," J Clin Invest 80:1550-1560 (1987)). The soluble, physiologic agonists are only effective if the neutrophils are adherent to extracellular matrix proteins via integrins (Nathan et al., "Cytokine-Induced Respiratory Burst of Human Neutrophils: Dependence on Extracellular Matrix Proteins and CD11/CD18 Integrins," J Cell Biol 109: 1341 - 1349 (1989)). Adherence (Kruskal et al., "Spreading of Human Neutrophils is Immediately Preceded by a Large Increase in Cytoplasmic Free Calcium," Proc Natl Acad Sci USA 83:2919-2923 (1986)) and TNF (Richter et al., "Tumor Necrosis Factor-Induced Degranulation in Adherent Human Neutrophils is Dependent on CDl lb/CD18-Integrin-Triggered Oscillations of Cytosolic Free Ca2+," Proc Natl Acad Sci USA Sl ':9472-9476 (1990)) combine to trigger oscillatory elevations of intracellular Ca²⁺, which in neutrophils responding to N-formylpeptide take the form of spatially restricted, moving waves (Kindzelskii et al., "Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study," J Immunol 170:64-72 (2003)). However, it remains unclear how TNF activates neutrophils.

[0007] The present invention is directed to overcoming these deficiencies in the art.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a method of treating an inflammatory disorder in a subject. The method involves administering to a subject an effective amount of a compound that modulates soluble adenylyl cyclase, where the compound has the following formula:

where:

R₁ is H, OH, alkyloxy, or halogen;

R₂ and R₅ are H or halogen;

R₃ is H or OH;

R₄ is H, alkyloxy, or halogen;

R₆ is H or alkyl; and

R₇ is H or CH₂R₈, where R₈ is H, alkyl, or substituted or unsubstituted phenyl, with the proviso that at least one OfR₁, R₂, R₃, and R₄ is a halogen, thereby treating the inflammatory disorder in the subject.

[0009] The present invention also relates to a method of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by neutrophils. The method involves contacting adherent neutrophils with an effective amount of a compound that modulates soluble adenylyl cyclase, where the compound has the following formula: - A -

where:

R₁ is H, OH, alkyloxy, or halogen;

R₂ and R₅ are H or halogen;

R₃ is H or OH;

R₄ is H, alkyloxy, or halogen;

R₆ is H or alkyl; and

R₇ is H or CH₂R₈, where R₈ is H₅ alkyi, or substituted or unsubstituted phenyl, with the proviso that at least one OfR₁, R₂, R₃, and R₄ is a halogen.

[0010] The present invention identifies, through chemical screening, a pyrazolone that reversibly blocks activation of phagocyte oxidase ("phox") in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spares the activation of phox by phorbol ester or bacteria, bacterial killing,

TNF-induced granule exocytosis and phox assembly, and endothelial transmigration.

The pyrazole's mechanism of action can be traced to inhibition of TNF-induced intracellular Ca²⁺ elevations and a non-transmembrane ("soluble") adenylyl cyclase

(sAC) is identified in neutrophils as a Ca²⁺-sensing source of cAMP. A sAC inhibitor mimicks the pyrazolone' s effect on phox. Both compounds block TNF-induced activation of Rapl A, a phox-associated GTPase regulated by cAMP. Thus, TNF turns on phox through Ca²⁺-triggered, sAC-dependent activation of Rapl A. This pathway offers opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures IA-E identify compounds that inhibit the neutrophil respiratory burst in response to TNF. Figure IA depicts chemical structures and concentration-response curves. Compounds were added at 37°C for 30 min before stimulation with TNF (squares) or PMA (triangles). H₂O₂ release at 90 min is displayed as % H₂O₂ release seen with TNF or PMA in the presence of the vehicle, DMSO. Figure IB illustrates the reversibility of the inhibition of TNF triggered H₂O₂ release by compound 2 (2-[3-chloro-phenyl]-5-phenyl-2,4-dihydro-pyrazol-3-one). Neutrophils incubated with DMSO (D), compound 2 or compound 5 were left unmanipulated (black bars) or washed (open bars) with buffer before both sets were plated and stimulated with TNF. Other neutrophils from which the compounds had been washed off were re-exposed to the compounds (hatched bars). Figure 1C shows the influence of compound 2 after the onset of respiratory burst. At times indicated by arrows, compound 2 was added to neutrophils already undergoing a TNF-triggered respiratory burst. The subsequent release of additional H₂O₂ is indicated by lines. Figure ID illustrates the inhibition by compound 2 of the respiratory burst triggered by fMLF but not by bacteria. Neutrophils were preincubated with DMSO (D), compound 2 or compound 5 and stimulated with fMLF (100 nM), L. monocytogenes or S. enterica., H₂O₂ release is depicted at the time it reached plateau. Results for Figures IA-C are means ± SEM for triplicates. Figure IE shows the impact on degramilation. Neutrophils were stimulated and their supernates assayed for lactoferrin (LTF) and myeloperoxidase (MPO) as a percent of that released in the absence of any compound. Each dot is the mean of duplicates in one experiment. Horizontal bars are group means. Unless indicated otherwise, TNF and PMA are each used at 100 ng/ml in all figures.

[0012] Figure 2 shows the impact of compound 2 on bacterial killing by neutrophils. Survival of bacteria was measured in culture only (open bars) or in culture with neutrophils for 30 min (Salmonella) or 60 min (Listeria) (black symbols). Survival of bacteria in the presence of DMSO (D) or compound 2 is shown in colony forming units (CFU) x 10^"3/ml (for Listeria) or CFU x lO^/ml (for Salmonella). Results are means ± SEM for duplicates in a representative experiment of two performed.

[0013] Figure 3 illustrates the impact of inhibitors on neutrophil spreading.

Neutrophils were plated on FBS-coated glass coverslips, incubated or not with each compound at 37°C for 30 min before stimulation with TNF, PMA, or an equal volume of buffer (no stimulus, NS). After 30 min, the cells were fixed and photographed with phase-contrast microscopy (10Ox).

[0014] Figure 4 illustrates the interaction of neutrophils with TNF-activated human umbilical vein endothelial cell (HUVEC) monolayers. Neutrophils were incubated with or without each compound at room temperature for 30 min, layered on collagen-supported HUVECs that had not been stimulated (gray bars) or had been activated for 12-18 h with TNF (50 pg/ml) (black bars), and allowed to transmigrate at 37°C for 30 min. Unbound cells were washed off with PBS and the cultures fixed and stained. Neutrophils on top of and beneath the HUVEC were counted microscopically. The total number of neutrophils per field was scored as adherent (left panel). The proportion of the total that was beneath the HUVEC was scored as having undergone transendothelial migration (TEM) (right panel). Means ± SEM for 6 replicates are shown in one of three similar experiments, each with a different donor of neutrophils and HUVEC. Pretreatment of neutrophils with compound 2 blocked neither the adhesion of the cells to nor their migration across monolayers of TNF- activated HUVECs. Moreover, pretreatment of HUVECs with compound 2 did not influence the integrity of the monolayer nor the cells' ability to respond to TNF by supporting adhesion and transmigration of untreated neutrophils. Similarly, a 2-day incubation with compound 2 did not affect the morphology or adherence of primary mouse peritoneal exudate macrophages, nor the ability of TNF to synergize with IFNγ in inducing them to release nitric oxide. The experiments with degranulation of neutrophils and activation of HUVEC and macrophages demonstrated that compound 2 inhibited only a selective aspect(s) of TNF signal transduction. [0015] Figures 5A-D show the impact of inhibitors on biochemical events involved in TNF-induced activation of adherent neutrophils. Figure 5A shows the effect on overall protein tyrosine phosphorylaton. Adherent neutrophils were incubated for 30 min with DMSO (D), compound 2 or compound 5 and, then, not stimulated (NS) or treated with TNF for 30 min. Total cell lysates (TCL) were separated by SDS-PAGE and western-blotted (WB) with anti-phosphotyrosine (PY) antibody. Figure 5B shows the Src activity. Recombinant Src (40 Units, UBI, Calgary, Canada) was incubated for 30 min with DMSO (D), compound 2, compound 5, or a known Src kinase inhibitor, pp2 (10 μM, Calbiochem, La Jolla, CA) (Hanke et al., "Discovery of a Novel, Potent, and Src family-Selective Tyrosine Kinase Inhibitor. Study of Lck- and FynT-Dependent T cell Activation," J Biol Chem 271 :695-701 (1996), which is hereby incorporated by reference in its entirety) and assayed for its ability over 10 min at 30°C to incorporate ³²P from ATy³²P into a synthetic substrate peptide from p34^cdo2 purchased from UBI. Background was measured by omitting Src (sub only). Figure 5C shows the Syk activity. Left: Syk was immunoprecipϊtated (IP) from adherent neutrophils treated as in Figure 5A and western blots (WB) carried out with anti-phosphotyrosine antibody (PY) (upper panel) or anti-Syk antibody (lower panel). Right: Activity of recombinant Syk (60 ng, UBI) was measured as for Src. Compound 2 did inhibit by about 50% the TNF- triggered tyrosine phosphorylation of Syk within neutrophils. Thus, compound 2 appears to affect an event upstream of Syk, rather than Syk itself. Figure 5D illustrates TNF-induced phosphorylation of endogenous Pyk2. Total cell lysates (TCL) from adherent neutrophils treated as indicated were western blotted (WB) with antibody specific for Pyk2 phosphorylated on tyrosine 402 (PY402) (upper panel) and with antibody for total Pyk2 (lower panel). In Figures 5A and 5C-D, migration of markers is indicated on the left by their M₁- in kDa.

[0016] Figures 6A-B show the impact of inhibitors on components of the phox complex. Figure 6A illustrates the translocation of p47^phox to membranes. Neutrophils were incubated with DMSO (D) or compound 2 at 37°C for 30 min and stimulated with TNF (T), PMA (P), or buffer (not stimulated, NS). After 40 min, cells were lysed and membrane fractions collected by ultracentrifugation, separated by SDS-PAGE and western-blotted with anti-p47^phox antibody (Leto et al., "Characterization of Neutrophil NADPH Oxidase Factors p47-Phox and p67-Phox From Recombinant Baculoviruses," J Biol Chem 266:19812-19818 (1991), which is hereby incorporated by reference in its entirety). Figure 6B shows the activation of Rap IA. Lysates of neutrophils, that had been pretreated for 30 min with DMSO (D), compound 2, KH7 (25 μM), or P site inhibitor (25 μM) and then stimulated for 30 min with TNF (T) or buffer alone (NS), were incubated with agarose beads coupled to recombinant RalGDS-Rap binding domain (RBD) to affinity purify GTP- bound Rap IA. Beads were boiled in SDS sample buffer and the supernatant subjected to SDS-PAGE and western blot with anti-Rapl A antibody (upper row). Western blot of total cell lysates (lower row) served as a control for equal input into the RBD affinity purification.

[0017] Figures 7A-F show sAC as a critical element of TNF signaling in neutrophils. Figure 7 A is a Western blot. Neutrophils were lysed and subjected to SDS-P AGE/Western blot with anti-sAC mAb R21. Lane 1 : lysate of cells transfected with sperm isoform of sAC. Lanes 2, 3: neutrophil lysates treated with (lane 2) or without (lane 3) protease inhibitor DFP. Figure 7B shows images from immunofluorescence microscopy. Neutrophils were stained with or without biotinylated anti-sAC mAb R41 followed by streptavidin-Alexafluor 594. Figure 7C shows the effects of sAC inhibitor, KH7, and tmAC inhibitor, "ddAdo", on the respiratory burst. Compounds were added to adherent neutrophils at 37°C for 30 min before stimulation with TNF (squares) or PMA (triangles) and H₂θ₂ release measured as in Figure IA. Figure 7D shows sAC activity in neutrophils (5 x 10⁶/well) preincubated 30 minutes with the phosphodiesterase inhibitor, isobutylmethylxanthine (DBMX) (100 μM), and either DMSO (D)₅ compound 2, or KH7, then stimulated with ionomycin (1 μM). cAMP accumulation was measured at 0 min (open bars) and 2 min (black bars) after stimulation. Figure 7E illustrates the impact on TNF triggered Ca²⁺ elevation in Fluo-3/AM-loaded neutrophils. Left panels are images of adherent neutrophils pretreated with DMSO (D) or compound 2 for 30-60 minutes and then treated with TNF or no stimulus (NS). Average relative fluorescence intensity (RFI) for all cells in similar microscopic fields is plotted as a function of time on the right. Arrow marks time of addition of TNF. Some cells were pretreated with KH7 (25 μM), as indicated. Figure 7F shows a model that incorporates new findings for TNF signaling in neutrophils.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention relates to a method of treating an inflammatory disorder in a subject. The method involves administering to a subject an effective amount of a compound that modulates soluble adenylyl cyclase, where the compound has the following formula:

where:

R₁ is H, OH₅ alkyloxy, or halogen; R.₂ and R₅ are H or halogen; R₃ is H or OH;

R₄ is H, alkyloxy, or halogen; ' R₆ is H or alkyl; and

R₇ is H or CH₂R₈, where R₈ is H, alkyl, or substituted or unsubstituted phenyl, with the proviso that at least one OfR₁, R₂, R₃, and R₄ is a halogen, thereby treating the inflammatory disorder in the subject.

[0019] In mammalian cells, two classes of adenylyl cyclase generate cAMP.

Transmembrane adenylyl cyclases (tmACs) are tethered to the plasma membrane and regulated by heterotrimeric G proteins in response to hormonal stimuli (for review, see Hanoune et al., "Regulation and Role of Adenylyl Cyclase Isoforms," Annu. Rev. Pharmacol. Toxicol. 41:145-174 (2001), which is hereby incorporated by reference in its entirety). A second source of cAMP, the more recently described "soluble" adenylyl cyclase (sAC), resides in discrete compartments throughout the cell (Zippin et al., "Compartmentalization of Bicarbonate-Sensitive Adenylyl Cyclase in Distinct Signaling Microdomains," FASEB J. 17:82-84 (2003), which is hereby incorporated by reference in its entirety) and is regulated by the intracellular signaling molecules, bicarbonate (Chen et al., "Soluble Adenylyl Cyclase as an Evolutionarily Conserved Bicarbonate Sensor," Science 289:625-628 (2000), which is hereby incorporated by reference in its entirety) and calcium (Jaiswal et al., "Calcium Regulation of the Soluble Adenylyl Cyclase Expressed in Mammalian Spermatozoa," Proc. Natl. Acad. ScL USA 100:10676-10681 (2003); Litvin et al., "Kinetic Properties of 'Soluble' Adenylyl Cyclase. Synergism Between Calcium and Bicarbonate," J Biol. Chem. 278:15922-15926 (2003), which are hereby incorporated by reference in their entirety).

[0020] cAMP elicits its cellular effects by activation of three known classes of effector proteins: exchange proteins activated by cAMP (EPAC), cyclic nucleotide gated ion channels, and protein kinase A (PKA). A subset of these targets resides at the plasma membrane, where they exist in macromolecular signaling complexes that also include a G protein coupled receptor, its transducing G protein, and the source of cAMP, a tmAC isoform (Davare et al., "A Beta2 Adrenergic Receptor Signaling Complex Assembled With the Ca²⁺ Channel Cavl.2," Science 293:98-101 (2001), which is hereby incorporated by reference in its entirety). The cAMP generated by tmACs appears to act locally (Rich et al., "Cyclic Nucleotide-Gated Channels Colocalize With Adenylyl Cyclase in Regions of Restricted cAMP Diffusion," J Gen. Physiol. 116:147-161 (2000); Rich et al., "A Uniform Extracellular Stimulus Triggers Distinct cAMP Signals in Different Compartments of a Simple Cell," Proc. Natl. Acad. Sd. USA 98:13049-13054 (2001); Zaccolo et al., "Discrete Microdomains With High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes," Science 295:1711-1715 (2002), which are hereby incorporated by reference in their entirety), most likely restricted by phosphodiesterase "firewalls" (Zaccolo et al., "Discrete Microdomains With High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes," Science 295:1711-1715 (2002); Mongillo et al., "Fluorescence resonance Energy Transfer-Based Analysis of cAMP Dynamics in Live Neonatal Rat Cardiac Myocytes Reveals Distinct Functions of Compartmentalized Phosphodiesterases," Cir Res 95(l):65-75 (2004), which are hereby incorporated by reference in their entirety), which define the limits of these cAMP signaling microdomains. However, targets of cAMP do not solely reside at the plasma membrane. EPAC is localized to the nuclear membrane and mitochondria (Qiao et al., "Cell Cycle-Dependent Subcellular Localization of Exchange Factor Directly Activated by cAMP," J. Biol. Chem. 277:26581-26586 (2002), which is hereby incorporated by reference in its entirety), and PKA is tethered throughout the cell by a class of proteins called AKAP (A-kinase-anchoring proteins; Michel et al., "AKAP Mediated Signal Transduction," Annu. Rev. Pharmacol. Toxicol. 42:235-257 (2002), which is hereby incorporated by reference in its entirety). The observation that cAMP does not diffuse far from tmACs (Bacskai et al., "Spatially Resolved Dynamics of Camp and Protein Kinase A Subunits in Aplysia Sensory Neurons," Science 260:222- 226 (1993); Zaccolo et al., "Discrete Microdomains With High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes," Science 295:1711-1715 (2002), which are hereby incorporated by reference in their entirety) reveals that there must be another source of cAMP modulating the activity of these distally localized targets.

[0021] Soluble adenylyl cyclase (sAC; Buck et al., "Cytosolic Adenylyl

Cyclase Defines a Unique Signaling Molecule in Mammals," Proc. Natl. Acad. Sd. USA 96:79-84 (1999); U.S. Patent No. 6,544,768 to Buck et al.; International Publication No. WO 01/85753, which are hereby incorporated by reference in their entirety) is widely expressed in mammalian cells (Sinclair et al., "Specific Expression of Soluble Adenylyl Cyclase in Male Germ Cells," MoI. Reprod. Dev. 56:6—11 (2000), which is hereby incorporated by reference in its entirety). Unlike tmACs, sAC is G protein insensitive (Buck et al., "Cytosolic Adenylyl Cyclase Defines a Unique Signaling Molecule in Mammals," Proc. Natl. Acad. ScL USA 96:79-84 (1999), which is hereby incorporated by reference in its entirety), and among mammalian cyclases, it is uniquely responsive to intracellular levels of bicarbonate (Chen et al., "Soluble Adenylyl Cyclase as an Evolutionarily Conserved Bicarbonate Sensor," Science 289:625-628 (2000), which is hereby incorporated by reference in its entirety). The ubiquitous presence of carbonic anhydrases ensures that the intracellular bicarbonate concentration (and sAC activity) will reflect changes in pH (Pastor-Soler et al., "Bicarbonate-Regulated Adenylyl Cyclase (sAC) is a Sensor That Regulates pH-Dependent V- ATPase Recycling," J. Biol. Chem. 278:49523-49529 (2003), which is hereby incorporated by reference in its entirety) and/or CO₂. Because CO₂ is the end product of energy-producing metabolic processes, sAC is poised to function as a cell's intrinsic sensor of metabolic activity (Zippin et al., "CO(2)/HCO(3)(-)-Responsive Soluble Adenylyl Cyclase as a Putative Metabolic Sensor," Trends Endocrinol. Metab. 12:366-370 (2001), which is hereby incorporated by reference in its entirety). sAC possesses no transmembrane spanning domains (Buck et al., "Cytosolic Adenylyl Cyclase Defines a Unique Signaling Molecule in Mammals," Proc. Natl. Acad. Sci. USA 96:79-84 (1999), which is hereby incorporated by reference in its entirety) and is distributed to subcellular compartments containing cAMP targets (Zippin et al., "Compartmentalization of Bicarbonate-Sensitive Adenylyl Cyclase in Distinct Signaling Microdomains," FASEB J. 17:82-84 (2003), which is hereby incorporated by reference in its entirety) that are distant from the plasma membrane. sAC was also found localized inside the mammalian cell nucleus (Zippin et al., "Compartmentalization of Bicarbonate- Sensitive Adenylyl Cyclase in Distinct Signaling Microdomains," FASEB J. 17:82-84 (2003), which is hereby incorporated by reference in its entirety). [0022] cAMP has been well known as a ubiquitous second messenger molecule affecting many different cellular functions, although the source of cAMP in certain cellular processes and its connection to those processes have remained undefined.

[0023] Soluble adenylyl cyclase (sAC) mentioned herein refers to any of the alternatively spliced isoforms generated from either of the two human genes. The s ACl locus resides at Iq24 in the human genome. Two specific transcripts from this locus, referred to as sAQ and sACg, are highly expressed in male germ cells (U.S. Patent No. 6,544,768 to Buck et al., which is hereby incorporated by reference in its entirety). In addition, there is a large number of unique alternatively spliced isoforms expressed in somatic tissues. There is a second sAC locus, at 6p21, which is also alternatively spliced to generate a number of putative sAC2 isoforms (see GenBank Accession Numbers NT_007592, NT_007592, NT_007180, NT_007204, NT_007234, NT_007239, NT_007255, NT_007358, NT_007402, NT_007412, NT_007432, NT__007454 NT_019430, NT023358, NT_023406, NT_023407, NT_023409, NT_023412, NT_023424, NT_023426, NT_023429, NT_023430, NT_023484, NT__023513, NT_026301, NT_026302, NT_027038, NT_027048, NTJ327049, NT_027050, NT_027051, NT_027052, NT_027059, NT_028206, NT_028223, NT__029311, NT_029314, NT_029316, NT_029319, NT_031804, NT_033943, and NT_034881, which are hereby incorporated by reference in their entirety). The homology between sACl and sAC2 is above 40% within the catalytic domains, while it diminishes to ~30% in the sequences beyond the catalytic domains (which are of unknown function). Expression of sAC2 transcripts has been demonstrated in a number of human tissues, and at least a subset of the sAC-selective compounds used in the method of the present invention may recognize these presumptive sAC2 isoforms. The sAC-selective compounds used in the methods of the present application encompass compounds that modulate the various sACl and/or sAC2 alternatively spliced isoforms.

[0024] In one embodiment of the present invention, the compound that modulates soluble adenylyl cyclase has the following formula:

KH7

[0025] Other suitable compounds for use in the above method of the present invention include, but are not limited to:

KH7.05

KH7.06

KH7.07

KH7.11

KH7.12

KH7.13

and

KH7.15

[0026] In all embodiments of this method of the present invention, the above compounds are administered to a subject under conditions effective to treat the inflammatory disorder.

[0027] The compounds used according to the methods of the present invention can be administered alone or as a pharmaceutical composition, which includes the compound(s) and a pharmaceutically-acceptable carrier. The compounds of the present invention are typically provided as a pharmaceutical composition. The pharmaceutical composition can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions, or emulsions. Typically, the composition will contain from about 0.01 to 99 percent, preferably from about 5 to 95 percent of active compound(s), together with the carrier. [0028] The compounds of the present invention, when combined with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, whether in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes, for example, that of the nose, throat, and bronchial tubes including, for example, by inhalation. [0029] For most therapeutic purposes, the compounds can be administered orally as a solid or as a solution or suspension in liquid form, via injection as a solution or suspension in liquid form, or via inhalation of a nebulized solution or suspension. The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the compounds of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these compounds are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.

[0030] For injectable dosages, solutions or suspensions of these materials can be prepared in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose, and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0031] For use as aerosols, the compound in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer. [0032] For transdermal routes, the compound is present in a carrier which forms a composition in the form of a cream, lotion, solution, and/or emulsion. The composition can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

[0033] AU mammals are suitable subjects for use in methods of the present invention, including, but not limited to, humans.

[0034] Suitable disorders to be treated or prevented in all aspects of the present invention present herein above or below are disorders in which a major pathogenic role is assigned to inflammation, including, without limitation, ischemia- reperfusion injury (occlusive and embolic stroke and myocardial infarction, type I diabetes mellitus, asthma, chronic obstructive pulmonary disease, gout, pre-term labor, sarcoidosis, ulcerative colitis, rheumatoid arthritis, osteoarthritis, xenograft rejection, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, pemphigus, chronic obstructive pulmonary disease, systemic lupus erythematosus, atopic dermatitis, vasculitides (Wegener's Syndrome, Goodpasture's Syndrome, giant cell arteritis, polyarteritis nodosa), multiple sclerosis, Alzheimer's Disease, and Crohn's Disease (regional enteritis).

[0035] The method of the present invention is also useful for treating diseases of infectious origin in which inflammation may contribute as much to pathology as does microbial toxicity, including, without limitation, sepsis syndrome, poststreptococcal glomerulonephritis, hepatitis C, Neisseria! or Pneumococcal meningitis, Helicobacter pylori gastritis, influenza virus pneumonia, tuberculosis, leprosy (tuberculoid form), filariasis, cystic fibrosis, bacterial dysentery, and Chagas Disease {^Trypanosoma cruzi).

[0036] Additional conditions or disorders encompassed by the methods of the present invention are diseases of diverse origin in which post-inflammatory fibrosis is a major cause of pathology. These diseases include, without limitation: schistosomiasis, idiopathic pulmonary fibrosis, hepatic cirrhosis (post-viral or alcoholic), radiation-induced pulmonary fibrosis, chronic allograft rejection, and bleomycin-induced pulmonary fibrosis.

[0037] Another aspect of the present invention is a method of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by the neutrophils. This method involves contacting adherent neutrophils with an effective amount of a compound that modulates soluble adenylyl cyclase. Examples of such compounds, as well as the formulations and modes of administration of such compounds, are described above.

[0038] This aspect of the present invention may be carried out by contacting adherent neutrophils in vitro using methods known in the art, including, but not limited to, adding the compounds described herein above to adherent neutrophils in a suitable cell culture system. This aspect of the present invention may also be carried out by contacting neutrophils in vivo as described above.

[0039] In this method of the present invention, where contacting with a compound of the present invention inhibits respiratory burst in adherent neutrophils, the respiratory burst can be triggered by an protein effector agent, such as a chemokine, a cytokine, a complement component, a secreted or shed bacterial product, or a bacterial cell wall component.

[0040] Suitable chemokines for this aspect of the present invention include, without limitation, macrophage inflammatory protein-1 (MIP-I) and interleukin-8

(IL-8).

[0041] Suitable cytokines for this aspect of the present invention include, without limitation, tumor necrosis factor (TNF), lymphotoxin, granulocyte-specific colony stimulating factor (G-CSF), and granulocyte/macrophage-specific colony stimulating factor (GM-CSF).

[0042] Suitable complement components for this aspect of the present invention include, without limitation, the chemoattractant complement component

C5a.

[0043] Suitable secreted or shed bacterial products of this aspect of the present invention include, without limitation, amino-terminally formylated peptides, such as

N-formyl-methionyl-leucyl-phenylalanine (fMLF) .

EXAMPLES

[0044] The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope. Example 1 - Cells

[0045] All cells were used under Institutional Review Board-approved protocols. Neutrophils were isolated from heparinized (10 U/ml) blood of healthy, consenting adult donors to >95% purity using Polymorphprep™ (Axis-Shield PoC AS, Norway) according to the manufacturer's instructions. Contaminating erythrocytes were lysed by hypotonic shock for 45 seconds with 0.2% saline. Neutrophils were resuspended in Krebs Ringer phosphate with glucose (KRPG) formulated as described in De Ia Harpe et al., "A Semi-Automated Micro- Assay for H₂O₂ Release by Human Blood Monocytes and Mouse Peritoneal Macrophages," J. Immunol. Methods 78:323-36 (1985), which is hereby incorporated by reference in its entirety. HUVEC were isolated from umbilical cords and cultured as described in Muller et al., "A Human Endothelial Cell-Restricted, Externally Disposed Plasmalemmal Protein Enriched in Intercellular Junctions," J Exp Med 170:399-414 (1989), which is hereby incorporated by reference in its entirety. Peritoneal macrophages were isolated from C57BL/6 mice four days after injection of 2 ml of thioglycollate broth (4%) and cultured as described in De Ia Harpe et al., "A Semi- Automated Micro- Assay for H₂O₂ Release by Human Blood Monocytes and Mouse Peritoneal Macrophages," J. Immunol. Methods 78:323-36 (1985), which is hereby incorporated by reference in its entirety.

Example 2 — Methods For Measuring H₂O₂ Release and Degranulation

[0046] The neutrophil respiratory burst was measured as described in De Ia

Harpe et al., "A Semi- Automated Micro-Assay for H₂O₂ Release by Human Blood Monocytes and Mouse Peritoneal Macrophages," J. Immunol. Methods 78:323-36 (1985), which is hereby incorporated by reference in its entirety. Briefly, 96-well flat- bottomed plates (Primaria, Falcon, Bedford, MA) were coated with 50 μl/well of fetal bovine serum (FBS) in 5% CO₂ in air at 37°C for at least 1 h and washed three times with 0.9% saline. 1.5 x 10⁴ neutrophils were added to triplicate wells containing 100 μl of reaction mixture (2.4 nniol scopoletin, 0.5 μg horseradish peroxidase (HRP), and 1 niM NaN₃) and stimulated with either buffer control, TNF (Preprotech, Rocky Hill, NJ)₅ or phorbol myristate acetate (PMA), each at 100 ng/ml. The reduction of scopoletin by H₂O₂ was recorded every 15 min on a plate-reading fluorometer until H₂O₂ release reached plateau. The amount of H₂O₂ released was calculated as described in De Ia Harpe et al., "A Semi-Automated Micro- Assay for H₂O₂ Release by Human Blood Monocytes and Mouse Peritoneal Macrophages," J Immunol. Methods 78:323-36 (1985), which is hereby incorporated by reference in its entirety. The supernatant from the H₂O₂ release assay with 1.5 x 10^ neutrophils was used to measure degranulation using lactoferrin (LTF) or myeloperoxidase (MPO) ELISA kit (Oxis International, Inc., Portland, OR). Spontaneous cell death from the same supernatant was measured using Cytotoxicity Detection Kit (Roche Molecular Biochemicals, Indianapolis, IN), an assay for lactate dehydrogenase (LDH). The total cell content of LF, MPO, and LDH was determined after lysing cells with 1% Triton XlOO.

Example 3 — Automated Assay Screening

[0047] A collection of 15,000 compounds with drug-like characteristics from

ChemDiv Labs (San Diego, CA) was screened robotically in the High Throughput Screening Facility at Rockefeller University to identify specific inhibitors of TNF- rriggered H₂O₂ release in human neutrophils. KH7 was identified in the same library during an independent screen for sAC. H₂O₂ release was measured as above with the following modifications. Black, instead of clear, 96-well tissue culture plates (Falcon, catalog number 353945) were used to reduce background fluorescence as measured in a Perkin-Elmer Fusion microplate reader (Boston, MA). A Titertek Multidrop 96/384 dispenser (SSI Robotics, Tustin, CA) was used for bulk reagent dispensing, Bi-Tek EIx 405 Select system for plate washing, and a PerkinElmer MiniTrakV liquid handling dispenser for the delivery of compound aliquots to each well. Percent inhibition by each compound was calculated using the formula: ([TO- T90] x 100)/(Tc0-Tc90), where TO and T90 are fluorescence readings at each well at 0 and 90 min, respectively, and TcO and Tc90 are the mean fluorescence readings in the compound-free control wells at 0 and 90 min, respectively. Compounds that showed >90% inhibition of the TNF-triggered respiratory burst were retested with both TNF and PMA as stimuli to eliminate non-specific, toxic chemicals and protein kinase C inhibitors. Example 4 - Test of Reversibility of Inhibition

[0048] Neutrophils were incubated with each compound at 37°C for 30 min in

FBS coated tubes (Falcon, catalog number 352063), washed twice with cold KRPG (5 min each at 4°C), and plated in 96 well plates, before immediate stimulation with TNF or an equivalent volume of KRPG buffer as a control. On a separate plate, compounds were added back to the neutrophils from which compounds had been washed off, and cells were stimulated to evaluate the neutrophils' capacity to respond. Neutrophils incubated with each compound without washing were also stimulated for comparison.

Example 5 - Bacteria-Triggered Respiratory Burst

[0049] Neutrophils were incubated with DMSO or each compound for 30 min at 37°C and then exposed to 10% autologous serum-opsonized Salmonella enterica var. Typhimurium (ATCC 14028s) ox Listeria monocytogenes (ATCC 104035) at a multiplicity of infection of 0.5 bacteria per neutrophil. H₂O₂ release was measured as described in Example 2.

Example 6 - Bacterial Killing

[0050] Neutrophils were preincubated with or without compound 2 ((2-[3- chloro-phenyl]-5-phenyl-2,4-dihydro-pyrazol-3-one) for 30 min at 37°C and then exposed to 10% autologous serum-opsonized Salmonella enterica var. Typhimurium (ATCC 14028s) ox Listeria monocytogenes (ATCC 15323) at a multiplicity of infection of 0.5 bacteria per neutrophil. Neutrophils were lysed at the indicated times with 1% sodium deoxycholate for assays with Salmonella or 1% Triton XlOO for assays with Listeria. Bacteria recovered from each condition were grown in LB agar plate at 37° C overnight and the colonies were counted.

Example 7 - Morphology

[0051] Acid-washed glass coverslips were placed in a 12 well tissue culture plate, coated with FBS in 5% CO₂ at 37°C for at least 1 h and washed three times with

0.9% saline. Neutrophils (2 x 10 ) were added to each well in 1 ml of reaction mixture and incubated or not with each compound at 37°C for 30 min before stimulation with TNF (100 ng/ml), PMA (100 ng/ml), or an equal volume of KRPG. Cells were fixed with 2% paraformaldehyde and 3.7% formadehyde buffer, and photographed through a phase-contrast microscope. Results are typical of those based on inspecting living cells at 15 min intervals over 2 hours.

Example 8 - Transendothelial Migration Assay

[0052] As described in Muller et al., "A Human Endothelial Cell-Restricted,

Externally Disposed Plasmalemmal Protein Enriched in Intercellular Junctions," J Exp Med 170:399-414 (1989), which is hereby incorporated by reference in its entirety, HUVEC were isolated and cultured in Ml 99 medium (Gibco, Carlsbad, CA) supplemented with 20% normal human serum, penicillin, and streptomycin. Experiments used cells at passage two that were cultured on hydrated type I collagen gels in 96-well plates. Where shown, HUVEC were stimulated with TNF (50 pg/ml) in 5% CO₂ at 37°C for the final 12-18 hours. Neutrophils were isolated from the peripheral blood of healthy adult volunteers by density gradient sedimentation in a discontinous gradient of Ficoll (Amersham Pharmacia, Uppsala, Sweden) and Histopaque (Sigma-Aldrich, St. Louis, MO), washed in Hanks' balanced salt solution with 0.1% human serum albumin, resuspended to 0.5 x 10⁶ cells/mL, and added to HUVEC monolayers. Test compounds were incubated with the neutrophils at room temperature for 30 min before TEM and allowed to remain during the duration of the assay. After neutrophils were allowed to transmigrate for 30 minutes at 37°C, nonadherent neutrophils were washed off with PBS and the remaining adherent and transmigrated cells were fixed with the endothelial monolayer by incubation overnight in 2.5% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in 0.1 sodium cacodylate buffer. HUVECs and neutrophils were differentially stained with Wright- Giemsa stain, and adherent and transmigrated cells were counted in multiple fields from the six replicates of each condition tested. Total adhesion was calculated as the total number of cells, both adherent and transmigrated, per high-powered field. The percentage of adherent cells that transmigrated below the endothelial layer was called % TEM. Example 9 - Immunoprecipitation and Western Blot

[0053] Tissue culture plates (Primaria, Falcon) were coated with 3ml of FBS in 5% CO₂ at 37°C for at least 1 h and washed twice with 0.9% saline. Neutrophils

₆ (15 x 10 ) were added to each plate containing 4 ml of reaction mixture, incubated with each compound at 37°C for 30 min, and stimulated with either buffer control or TNF (100 ng/ml). When cells were fully spread (20-40 min), they were treated with 5 mM diisopropylfluorophosphate to inhibit serine proteases and lysed with either 125 μl of SDS lysis buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% SDS, 1 mM PMSF, 1 mM Na pyrophosphate, 1 mM NaF₃I mM vanadate, 5 μg/ml each of aprotinin, leupeptin, chymostatin, pepstatin A) for Western blot or with 200 μl of non- denaturing modified RIPA buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton-XlOO, 1 mM PMSF, 1 mM Na pyrophosphate, 1 mM NaF, 1 mM vanadate, 5 μg/ml each of aprotinin, leupeptin, chymostatin, pepstatin A) for immunoprecipitation. The cell lysate was passed through 26-gauge needle six times to shear DNA and centrifuged at 20,000 x g for 15 min to remove cell debris and DNA. The protein concentration was determined using a Bio-Rad (Hercules, CA) kit. Then, cell lysates were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH). The membranes were incubated with 5% milk in TBST (100 mM Tris-HCl, pH 7.5, 9% NaCl, 0.1% Tween-20) for 1 h at 37°C and then overnight at 4⁰C with anti-phosphotyrosine antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Syk Ab (Transduction Laboratories, Bedford, MA), anti-phospho-specifϊc Pyk2 Abs (Biosource, Camarillo, CA), or anti-Pyk2 Ab (Upstate Biotech, Lake Placid, NY). sAC protein was immunoblotted with monoclonal antibody R21 and immunostained with monoclonal antibodies R21, R41, and R52. Membranes were washed with TBST and incubated with secondary antibody conjugated with HRP in 5% milk in TBST for 1 h at 37°C. After further washing with TBST, bound antibody was detected by enhanced chemiluminescence (ECL, Pierce, Rockford, IL).

Example 10 - in vitro Kinase Assay for Src and Syk

[0054] Kinase assays were performed according to the instructions provided by UBI, from which recombinant Src and Syk were purchased. Briefly, after incubation of recombinant Src (40 U, UBI) with each compound at room temperature for 30 min, Src kinase reaction buffer (100 mM Tris-HCl, pH 7.2, 125 mM MgCl₂, 25 mM MnCl₂, 2 mM EGTA, 0.25 mM NaVO₄, 2 mM DTT), Src kinase substrate peptide (375 μM, UBI), and [γ-³²P]ATP were added to the reaction mixture. After incubation at 30°C for 10 min, the reaction was stopped by addition of 40% TCA. After transferring each sample to a P81 paper square (UBI), and un-incorporated radioactivity was washed off with 0.75% phosphoric acid and acetone, the assay square was read in scintillation counter. For Syk kinase assay, reaction buffer contained 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1 mM NaVO₄, 0.15 M 2-mercaptoethanol and substrate was poly (Glu₄-Tyr)(4:l) (CSI Bsiointerantional, Mumbai, India). Each condition was done in duplicate.

Example 11 - Translocation of p47^phox

[0055] Neutrophil lysates were prepared as in Example 6 with 2.5 %

TritonX-100 lysis buffer (final concentraion of 1% TritonX-100 after dilution in plate with residual KRPG). The cell lysate was passed through 26 gauge needle six times to shear DNA and centrifuged at 20,000 x g for 15 min to remove cell debris and DNA. The supernatant was centrifuged again at 100,000 x g for 1 hour to pellet membrane fraction. The pellet was washed with PBS and resuspended in 2% TritonX-100 lysis buffer. Membrane fraction from each condition was separated by SDS-PAGE and Western blotted with anti-ρ47^phox antibody.

Example 12 - cAMP Accumulation Assay

[0056] Neutrophils (5 x 10⁶/well) were plated in FBS-coated 24 well tissue culture plate (Corning Incorporated, Corning, NY) in 500 μl of KRPG. The cells were pretreated with 3-isobutyl-l-methylxanthine (IBMX, 100 μM) and compound 2 (5 μM) or KH7 (25 μM) for 30 minutes. Ionomycin (1 μM, Sigma) was added to each well to stimulate calcium release, and cAMP was allowed to accumulate for 0 or 2 minutes at 37°C. The assay was stopped, cells were lysed by adding 500 μl of 0.2 N HCl, and cAMP concentration was determined using the Correlate-EIA Direct cAMP Kit (Assay Designs, Inc., Ann Arbor, MI). Example 13 - Rapl Activation Assay

[0057] Lysates of neutrophils that had been pretreated for 30 min with DMSO

(D), compound 2, KH7 (25 μM) or ddAdo (25 μM; Calbiochem) and then stimulated for 30 min with TNF (T) or buffer alone(NS) were incubated with agarose beads coupled to recombinant RalGDS-Rap binding domain (RBD) to affinity purify GTP- bound Rapl . Beads were boiled in SDS sample buffer and the supernatant was subjected to SDS-PAGE and Western blot with ami Rapl antibody (Santa Cruz Biotechnology, Inc.). Western blot of total cell lysates served as a control for equal input into the RBD affinity purification.

Example 14 — Immunofluorescence Staining of sAC in Human Neutrophils

[0058] Neutrophils were fixed and permeabilized with 3.3% paraformaldehyde, 0.05% glutaraldehyde, and 0.25 mg/ml saponin in PBS for 5 min at room temperature. The reactions were stopped with the same volume of 20 mM glycine buffer. The cells were washed three times with PBS. After permeabilization, cells were blocked in 2% BSA for at least 1 hour. Cells were subsequently stained with biotin-conjugated anti-sAC (R21, R41, or R52) monoclonal antibody (1:250) overnight in 2% BSA, 0.01% Triton X-100; washed 5 times in 2% BSA; stained for 45 min with streptavidin Alexa Fluor 594 (Molecular Probes, Eugene, OR) in 2% BSA, 0.01% Triton X-100; and mounted with gelvatol/diaminobicyclooctane (Sigma-Aldrich). Fluorescent images were recorded by a digital camera (Hamamatsu, Bridgewater, NJ) connected to an inverted epifluorescent microscope (Nikon, Melville, NY).

Example 15 - Determination of Neutrophil Intracellular Free Ca²⁺ ([Ca²⁺]O

[0059] The Ca²⁺-sensitive fluorescent probe Fluo-3/AM (Molecular Probes) was used for determination of changes in [Ca ];. Neutrophils were incubated in the dark for 40 min at room temperature with Fluo-3/AM (10 μM) in Ml 99 (Gibco) containing 0.025% pluronic acid, 2.5 mM probenecid, 0.5% human serum albumin (HAS), and 20 mM HEPES. Cells were washed twice and incubated with Ml 99 containing 2.5 mM probenecid, 0.5% HSA, and 20 mM HEPES in the dark for 20 min at room temperature to allow hydrolysis of the dye ester. IxIO⁵ PMN with DMSO₅ compound 2 or KH7 were plated in 35-mm glass-bottomed coverslip dishes (Mat Tek Corporation, Ashland, MA) precoated with FBS and allowed to settle for 10 min. Changes in neutrophil [Ca²⁺]i in response to TNF (100 ng/ml) and/or ionomycin (1 μM) were measured using a Zeiss Axiovert 200M widefield microscope (Thornwood, NY). Digitized images were captured every 5 s before and after TNF stimulation through a charge-coupled device camera controlled by MetaMorph software (Universal Imaging, Downingtown, PA). Quantitative analysis of images was performed with MetaMorph software. Briefly, after background subtraction, the total averaged intensity per field was measured per time point. Relative fluorescence intensities per field for each time point were plotted.

Example 16 - Screening of Chemical Library For Chemical Inhibitors of TNF- Triggered HjO₂ Release

[0060] About 15,000 compounds were screened for those that blocked H₂O₂ release by TNF-treated, adherent neutrophils but spared H₂O₂ release when the same cells were triggered by PMA. The PMA counterscreen was used to exclude compounds that were toxic, inhibited phox or protein kinase C, or interfered with the assay. Four compounds were both selectively inhibitory and consistently effective with neutrophils from different donors. Compound 2 (2-[3-chloro-phenyl]-5-phenyl- 2,4-dihydro-pyrazol-3-one) was selected for analysis. Compound 5 shares its core structure with two of the other three inhibitory compounds, but had no effect on H₂O₂ release and was used as a control. Concentration-response curves of compound 2 using neutrophils from three donors yielded a mean 50% inhibitory concentration (IC₅₀) ± SEM of 1.6 ± 0.4 μM for H₂O₂ release (Figure IA). A new screen of 1100 congeners of compound 2 also identified compound 2', with IC₅₀24 ± 6 nM (Figure IA). At 5 μM, compound 2 yielded > 95% inhibition of TNF-triggered H₂O₂ release with cells from all donors tested. This concentration was used for all further experiments.

[0061] When neutrophils were treated with compound 2 and then washed, they regained responsiveness to TNF (Figure IB). The cells remained susceptible to inhibition when compound 2 was added back (Figure IB). Thus, inhibition by compound 2 was fully reversible. When compound 2 was added to neutrophils that were already releasing H₂O₂ in response to TNF, the respiratory burst slowed quickly (Figure 1C). Thus, the process inhibited by compound 2 was needed not just to launch the TNF-triggered respiratory burst but also to sustain it.

Example 17 - Interaction With Bacteria or Bacterial Product

[0062] Next, the effects of compound 2 on neutrophils' responses to bacteria and to the major neutrophil stimulant released by E. coli, formylated methionyl- leucyl-phenylalanine (fMLF), was tested. H₂O₂ release triggered by fMLF was blocked by compound 2. However, compound 2 did not inhibit H₂O₂ release triggered by Salmonella enterica var. Typhimurium and only slightly reduced the respiratory burst elicited by Listeria monocytogenes (Figure ID). Nor did compound 2 interfere with killing of these pathogens by neutrophils (Figure 2).

Example 18 - Degranulation of Neutrophils

[0063] Degranulation delivers the phox flavocytochrome to phagosomal and plasma membranes (Bjerrum et al., "Dual Granule Localization of me Dormant NADPH Oxidase and Cytochrome b559 in Human Neutrophils," Eur J Haematol 43:61-11 (1989), which is hereby incorporated by reference in its entirety), where the cytosolic components of phox are recruited. Therefore, compound 2 might block activation of phox by blocking degranulation. However, compound 2 did not block TNF-induced exocytosis of the specific granule marker lactoferrin and only slightly inhibited release of the azurophil granule marker myeloperoxidase (Figure IE).

Example 19 - Influence on Neutrophil Spreading

[0064] The large-scale respiratory burst triggered by TNF or fMLF requires that neutrophils spread on extracellular matrix (Nathan, "Neutrophil Activation on Biological Surfaces. Massive Secretion of Hydrogen Peroxide in Response to Products of Macrophages and Lymphocytes," J Clin Invest 80:1550-1560 (1987); Nathan et al., "Cytokine-Induced Respiratory Burst of Human Neutrophils: Dependence on Extracellular Matrix Proteins and CDl 1/CDl 8 Integrins," J Cell Biol 109:1341-1349 (1989), which are hereby incorporated by reference in their entirety). Pretreatment with compound 2 allowed neutrophils to spread normally on serum- coated glass in response to PMA. With TNF as a stimulus in compound 2-pretreated cells, spreading was initiated, suggesting that compound 2 did not interfere with binding of TNF and transmission of signals to the cytoskeleton. However, compound 2 caused TNF-triggered spreading to halt prematurely, suggesting inhibition of a later stage in TNF signaling (Figure 3).

Example 20 - Transmigration of Neutrophils

[0065] Interference with spreading of neutrophils in response to TNF raised the question whether compound 2 might block migration across endothelium, the process by which neutrophils enter tissues. However, pretreatment of neutrophils with compound 2 blocked neither the adhesion of the cells to, nor their migration across monolayers of TNF-activated human umbilical vein endothelial cells (HUVECs) (Figure 4). Moreover, pretreatment of HUVECs with compound 2 did not influence the integrity of the monolayer nor the cells' ability to respond to TNF by supporting adhesion and transmigration of untreated neutrophils. Similarly, a 2-day incubation with compound 2 did not affect the morphology or adherence of primary mouse peritoneal exudate macrophages, nor the ability of TNF to synergize with IFNγ in inducing them to release nitric oxide. The experiments with degranulation of neutrophils and activation of HUVEC and macrophages demonstrated that compound 2 inhibited only a selective aspect(s) of TNF signal transduction.

Example 21 — Effects on Protein Tyrosine Phosphorylation

[0066] Neutrophil responses to TNF require protein tyrosine phosphorylation involving Syk (Yan et al., "Signaling by Adhesion in Human Neutrophils: Activation of the p72syk Tyrosine Kinase and Formation of Protein Complexes Containing p72syk and Src Family Kinases in Neutrophils Spreading Over Fibrinogen," J Immunol 158:1902-1910 (1997); Mocsai et al., "Syk is Required for Integrin Signaling in Neutrophils," Immunity 16:547-558 (2002), which are hereby incorporated by reference in their entirety), Src family kinases, Hck and Fgr (Lowell et al., "Deficiency of Src Family Kinases p59/61hck and p58c-fgr Results in Defective Adhesion-Dependent Neutrophil Functions," J Cell Biol 133:895-910 (1996), which is hereby incorporated by reference in its entirety), and Pyk2 (Han et al., "Critical Role of the Carboxyl Terminus of Proline-Rich Tyrosine Kinase (Pyk2) in the Activation of Human Neutrophils by Tumor Necrosis Factor: Separation of Signals for the Respiratory Burst and Degranulation," J Exp Med 197:63-75 (2003); Fuortes et al., "Role of the Tyrosine Kinase pyk2 in the Integrin-Dependent Activation of Human Neutrophils by TNF," J Clin Invest 104:327-335 (1999), which are hereby incorporated by reference in their entirety). However, treatment of adherent neutrophils with compound 2 had little impact on overall protein tyrosine phosphorylation induced by TNF (Figure 5A). Nor did 2 inhibit recombinant Src (Figure 5B), Syk (Figure 5C) or TNF -induced (auto)phosphorylation of Pyk2 on tyrosine 402 (Figure 5D), which is critical for Pyk2's kinase activity (Sieg et al., "Pyk2 and Src-Family Protein-Tyrosine Kinases Compensate for the Loss of FAK in Fibronectin-Stimulated Signaling Events But Pyk2 Does Not Fully Function to Enhance FAK- Cell Migration," EMBO J 17:5933-5947 (1998), which is hereby incorporated by reference in its entirety).

Example 22 - Translocation of p47^phox

[0067] Cytosolic components of phox— p40^phox, p47^phox, and p67^phox— form a

Mr -240,000 complex (Park et al., "The Cytosolic Components of the Respiratory Burst Oxidase Exist as a M(r) Approximately 240,000 Complex That Acquires a Membrane-Binding Site During Activation of the Oxidase in a Cell-Free System," J Biol Chem 267:17327-17332 (1992); Wientjes et al., "Interactions Between Cytosolic Components of the NADPH Oxidase: p40phox Interacts With Both p67phox and p47phox," Biochem J317 (Pt 3):919-924 (1996), which are hereby incorporated by reference in their entirety) in resting neutrophils, with p47^phox serving as an essential adaptor. The complex translocates to the membrane upon activation. Compound 2 had no effect on TNF-induced translocation of p47^phox to the membrane (Figure 6A). Since compound 2 inhibited neither the assembly of phox (Figure 6A) nor its catalytic activity when triggered by PMA (Figure IA), compound 2 might interfere with a later stage in phox activation, namely, TNF-dependent activation of one of the GTPases bound to phox, Rac2 (Knaus et al., "Regulation of Phagocyte Oxygen Radical Production by the GTP-Binding Protein Rac 2," Science 254:1512-1515 (1991), which is hereby incorporated by reference in its entirety) or Rap IA (Quinn et al., "Association of a Ras-Related Protein With Cytochrome b of Human Neutrophils," Nature 342: 198-200 (1989), which is hereby incorporated by reference in its entirety). Rac2-deficient neutrophils have severe migration defects (Ambruso et al., "Human Neutrophil Immunodeficiency Syndrome is Associated With an Inhibitory Rac2 Mutation," Proc Natl Acad Sd USA 97:4654-4659 (2000), which is hereby incorporated by reference in its entirety), while compound 2-treated neutrophils migrated normally (Figure 4). Thus, Rac2 was unlikely to be the locus of regulation by compound 2, and Rap IA was investigated.

Example 23 - Rapl Activation Assay

[0068] Rapl A is bound stoichiometrically to the phox flavocytochrome in a

GTP- and phosphorylation-dependent manner (Bokoch et al., "Inhibition of Rapl A Binding to Cytochrome b558 of NADPH Oxidase by Phosphorylation of Rap IA," Science 254:1794-1796 (1991), which is hereby incorporated by reference in its entirety). Treatment of neutrophils with fMLF or GM-CSF activated RaplA, that is, converted it to the GTP-bound state (M'Rabet et al., "Activation of the Small GTPase Rapl in Human Neutrophils," Blood 92:2133-2140 (1998), which is hereby incorporated by reference in its entirety). It was found that TNF treatment of adherent neutrophils also activated RaplA, and compound 2 blocked TNF-induced RaplA activation (Figure 6B).

[0069] RaplA can be activated by a guanine nucleotide exchange protein,

Exchange protein directly activated by cAMP (Epac) (de Rooij et al., "Epac is a Rapl Guanine-Nucleotide-Exchange Factor Directly Activated by Cyclic AMP," Nature 396:474-477 (1998); Kawasaki et al., "A Family of cAMP-Binding Proteins That Directly Activate Rapl," Science 282:2275-2279 (1998), which are hereby incorporated by reference in their entirety). The cAMP that activates Epac can arise from the long-studied G protein-regulated, transmembrane adenylyl cyclases (tmACs) or from a recently discovered non-transmembrane adenylyl cyclase, sAC, which is regulated by intracellular Ca²⁺ and/or bicarbonate (Chen et al., "Soluble Adenylyl Cyclase as an Evolutionarily Conserved Bicarbonate Sensor," Science 289:625-628 (2000); Jaiswal et al., "Calcium Regulation of the Soluble Adenylyl Cyclase Expressed in Mammalian Spermatozoa," Proc Natl Acad Sd USA 100:10676-10681 (2003); Litvin et al., "Kinetic Properties of 'Soluble' Adenylyl Cyclase. Synergism Between Calcium and Bicarbonate," J. Biol. Chem, 278:15922-15926 (2003), which are hereby incorporated by reference in their entirety). tmACs were considered unlikely candidates for neutrophil activation, since their pharmacologic stimulation inhibits most neutrophil functions, including TNF-induced activation of phox (Nathan et al., "Tumor Necrosis Factor and CDl 1/CDl 8 (beta 2) Integrins Act Synergistically to Lower cAMP in Human Neutrophils," J Cell Biol 111:2171-2181 (1990), which is hereby incorporated by reference in its entirety). Thus, it was examined whether neutrophils contain sAC.

Example 24 - Anti-sAC Western Blots with PMN Lysates and Impact of

Inhibitors of Adenylyl Cyclases on TNF Triggered RB and Ca Release

[0070] Imrnunoblot revealed sAC in highly purified neutrophil preparations

(Figure 7A). Neutrophils themselves were the source of the immunoreactivity because they were uniformly and specifically stained by three monoclonal antibodies, each directed against a different epitope of sAC (Figure 7B). The granular pattern of staining raised the possibility that sAC may reside near phox, but definitive analysis of sAC's subcellular localization awaits immuno-electron microscopy. Next, pharmacological reagents were selected to distinguish between possible sources of cAMP in a mammalian cell. A chemical screen identified a specific inhibitor of recombinant human sAC, 2-(lH-berizoimidazole-2-ylsulfanyl)-propionic acid (5- bromo-2-hydroxy-benzylidene)-hydrazide (KΗ7), that spares tmACs. Reciprocally, a concentration range was determined in which 2'5' dideoxyadenosine (ddAdo) (Onda et al., "Type-Specific Regulation of Adenylyl Cyclase. Selective Pharmacological Stimulation and Inhibition of Adenylyl Cyclase Isoforms," J Biol Chem 276:47785- 47793 (2001), which is hereby incorporated by reference in its entirety) acts as a selective inhibitor of tmACs, sparing sAC. Like compound 2, KH7 inhibited TNF- triggered activation of Rap IA (Figure 6B) and suppressed TNF-triggered H₂O₂ release with an IC_5O ± SEM of 4.7 ± 0.3 μM, while sparing PMA-triggered H₂O₂ release (Figure 7C). In contrast, the selective inhibitor of tmACs, ddAdo, had no effect on activation of Rapl (Figure 6B) nor on the TNF-induced respiratory burst (Figure 7C). These findings suggest that TNF-triggered activation of Rapl is mediated by sAC, not by tmACs. [0071] Unlike KH7, compound 2 did not inhibit recombinant sAC. Thus, compound 2 might block a step upstream of sAC, such as TNF-induced elevation of intracellular Ca²⁺. It was first demonstrated that neutrophil sAC could respond to elevations of intracellular Ca²⁺. Ionomycin, a Ca²⁺ ionophore, induced production of cAMP in neutrophils, and the ionomycin-induced cAMP production was blocked by the sAC inhibitor, KH7 (Figure 7D), indicating that sAC was responsible for the rise in cAMP. Compound 2 had no effect on the ionomycin-induced production of cAMP, confirming that compound 2 does not inhibit endogenous sAC. To test whether . compound 2 blocked TNF induced Ca²⁺ elevation, adherent neutrophils were loaded with a Ca²⁺-sensitive fluorochrome and their fluorescence was studied via videomicroscopy. TNF triggered an almost instantaneous elevation of intracellular Ca²⁺ that persisted throughout the observation period (~2 min) (Figure 7E). The elevation appeared nearly constant when expressed as average fluorescence intensity per microscopic field in fields containing hundreds of cells. However, inspection of individual cells revealed asynchronous, irregular oscillations, as noted by others (Schumann et al., "Recombinant Human Tumor Necrosis Factor Alpha Induces Calcium Oscillation and Calcium- Activated Chloride Current in Human Neutrophils. The Role of Calcium/Cahnodulin-Dependent Protein Kinase," J Biol Chem 268:2134- 2140 (1993), which is hereby incorporated by reference in its entirety). Pre-treatment of the cells with compound 2 substantially suppressed the TNF-induced increase in intracellular Ca²⁺ (Figure 7E), although compound 2 had no effect on Ca²⁺ elevation induced artificially by ionomycin. hi contrast, pre-treatment with KH7 did not suppress the TNF-induced increase in intracellular Ca²⁺ (Figure 7E). It was proposed that compound 2 be named "neucalcin-1" for neutrophil calcium inhibitor, and compound 2' be named "neucalcin-2".

[0072] These findings outline a pathway for TNF signal transduction that is distinct from the known routes leading to transcriptional regulation and apoptosis. Figure 7F outlines a working model: exposure of adherent neutrophils to TNF leads by an unknown mechanism to elevation of intracellular Ca²⁺ (Richter et al., "Tumor Necrosis Factor-Induced Degranulation in Adherent Human Neutrophils is Dependent on CDl lb/CD18-Integrin-Triggered Oscillations of Cytosolic Free Ca²⁺," Proc Natl Acad Sd USA %7 -.9472-9416 (1990); Schumann et al., "Recombinant Human Tumor Necrosis Factor Alpha Induces Calcium Oscillation and Calcium- Activated Chloride Current in Human Neutrophils. The Role of Calcium/Calmodulin-Dependent Protein Kinase," J Biol Chem 268:2134-2140 (1993), which are hereby incorporated by reference in their entirety), which stimulates sAC (Litvin et al., "Kinetic Properties of "Soluble" Adenylyl Cyclase. Synergism Between Calcium and Bicarbonate," J Biol Chem 278:15922-15926 (2003); Jaiswal et al, "Calcium Regulation of the Soluble Adenylyl Cyclase Expressed in Mammalian Spermatozoa," Proc Natl Acad Sd USA 100:10676-10681 (2003), which are hereby incorporated by reference in their entirety) to generate cAMP. The elevated cAMP causes activation of Rap IA, perhaps via Epac. Activated Rapl A turns on phox, whose components have been pre- assembled in response to TNF, fMLF, and other soluble, physiologic agonists. This proposed pathway is independent of the previously described TNF signaling cascade involving phosphatidylinositol 3-kinase and tyrosine kinases, which mediates degranulation and the degranulation-dependent assembly of phox (Mocsai et al., "Syk is Required for Integrin Signaling in Neutrophils," Immunity 16:547-558 (2002); Fuortes et al., "Role of the Tyrosine Kinase pyk2 in the Integrin-Dependent Activation of Human Neutrophils by TNF," J Clin Invest 104:327-335 (1999), which are hereby incorporated by reference in their entirety). Thus, the sAC-dependent and sAC-independent pathways converge on phox. The two chemically distinct inhibitors used in this study, neucalcin and KH7, appear to act at two different points in the sAC-dependent pathway (Figure 7F).

[0073] It may seem paradoxical that in previous studies, direct measurement of cAMP levels in TNF-treated neutrophils did not reveal an elevation; on the contrary, TNF prolonged a decline in cAMP that was induced when neutrophils adhered to biological surfaces (Nathan et al., "Tumor Necrosis Factor and CDl 1/CD18 (beta 2) Integrins Act Synergistically to Lower cAMP in Human Neutrophils," J Cell Biol 111:2171-2181 (1990), which is hereby incorporated by reference in its entirety). Those observations can be reconciled with the new observations disclosed herein by proposing that the elevations of cAMP required to sustain the activation of phox by TNF are spatially limited and quantitatively insufficient to nullify the fall in cAMP proceeding at the same time in a larger cellular compartment.

[0074] Opposing effects of cAMP have been reported on protein kinase B, depending on whether the cAMP effector is protein kinase A or Epac (Mei et al., "Differential Signaling of Cyclic AMP: Opposing Effects of Exchange Protein Directly Activated by Cyclic AMP and cAMP-Dependent Protein Kinase on Protein Kinase B Activation," J Biol Chem 277:11497-11504 (2002), which is hereby incorporated by reference in its entirety). A similar situation may pertain to Rap IA. Pharmacologically induced, global, sustained elevations in cAMP that suppress neutrophil functions are likely to activate protein kinase A, which can phosphorylate Rapl A, blocking its association with phox (Bokoch et al., "Inhibition of Rapl A Binding to Cytochrome b558 of NADPH Oxidase by Phosphorylation of Rapl A," Science 254: 1794-1796 (1991), which is hereby incorporated by reference in its entirety). In contrast, since TNF-induced Ca²⁺ elevations are oscillatory (Richter et al., "Tumor Necrosis Factor-Induced Degranulation in Adherent Human Neutrophils is Dependent on CDl lb/CD18-Integrin-Triggered Oscillations of Cytosolic Free Ca²⁺," Proc Natl Acad Sd USA 87:9472-9476 (1990); Schumann et al., "Recombinant Human Tumor Necrosis Factor Alpha Induces Calcium Oscillation and Calcium- Activated Chloride Current in Human Neutrophils. The Role of Calcium/Calmodulin-Dependent Protein Kinase," J Biol Chem 268:2134-2140 (1993), which are hereby incorporated by reference in their entirety) and probably localized (Kindzelskii et al., "Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study," J Immunol 170:64-72 (2003), which is hereby incorporated by reference in its entirety), sAC-mediated production of cAMP and the ensuing activation of Rapl A may also be oscillatory and localized, consistent with observations that Rapl A must undergo cycles of association with and dissociation from phox to sustain phox activity (Kindzelskii et al., "Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study," J Immunol 170:64- 72 (2003); MaIy et al., "Activated or Dominant Inhibitory Mutants of Rapl A Decrease the Oxidative Burst of Epstein-Barr Virus-Transformed Human B Lymphocytes," J Biol Chem 269:18743-18746 (1994), which are hereby incorporated by reference in their entirety). The kinetic studies in Figure 1 C are consistent with the concept that sustained H₂O₂ release in response to TNF requires repetitive Rapl A activation. [0075] In sum, the following has been identified: (1) a new enzyme in neutrophils, sAC; (2) a new pathway for TNF signal transduction, centered on sAC; (3) the ability of endogenous sAC to sense Ca²⁺ elevations; (4) a functional role for the Ca²⁺ elevations in adherent, TNF-stirnulated neutrophils; (5) a likely role for Rap IA in intact neutrophils as a regulator of assembled phox; (6) evidence that cAMP can not only inhibit but also activate neutrophils; and (7) chemical agents, neucalcins, that may serve as tools to discover which Ca²⁺ channels are activated by TNF and how. These findings help generate a list of targets whose inhibition might allow selective intervention in neutrophil functions. Finally, this work shows that it is feasible to block the activation of phox by inflammatory factors without interfering with neutrophil migration or phox-dependent killing of ingested bacteria. Blocking non-bacterial activation of phox might lead to preservation of oxidation-sensitive anti- proteases, reducing proteolysis at sites of inflammation.

[0076] Although the invention has been described in detail, for the purpose of illustration, it is understood that such detail is for that purpose and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method of treating an inflammatory disorder in a subject comprising: administering to a subject an effective amount of a compound that modulates soluble adenylyl cyclase, said compound having the following formula:

wherein:

R₁ is H, OH, alkyloxy, or halogen;

R.₂ and R₅ are H or halogen;

R₃ is H or OH;

R₄ is H, alkyloxy, or halogen;

R₆ is H or alkyl; and

R₇ is H or CH₂R₈, wherein R₈ is H, alkyl, or substituted or unsubstituted phenyl, with the proviso that at least one of Ri, R₂, R₃, and R₄ is a halogen,

thereby treating the inflammatory disorder in the subject.

2. The method according to claim 1 , wherein the compound has the following formula:

3. The method according to claim 1 , wherein the compound has the following formula:

4. The method according to claim 1, wherein the compound has the following formula:

5. The method according to claim 1 , wherein the compound has the following formula:

6. The method according to claim 1, wherein the compound has the following formula:

7. The method according to claim 1, wherein the compound has the following formula:

8. The method according to claim 1, wherein the compound has the following formula:

9. The method according to claim 1 , wherein the compound has the following formula:

10. The method according to claim 1, wherein the compound is administered as part of a composition further comprising a pharmaceutically- acceptable carrier.

11. The method according to claim 1 , wherein said administrating is carried out orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intravesical instillation, by intracavitary, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membrane.

12. The method according to claim 1, wherein the inflammatory disorder is selected from the group consisting of ischemia-reperfusion injury, occlusive and embolic stroke, myocardial infarction, type I diabetes mellitus, asthma, chronic obstructive pulmonary disease, gout, pre-term labor, sarcoidosis, ulcerative colitis, rheumatoid arthritis, osteoarthritis, xenograft rejection, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, pemphigus, chronic obstructive pulmonary disease, systemic lupus erythematosus, atopic dermatitis, vasculitides, Wegener's Syndrome, Goodpasture's Syndrome, giant cell arteritis, polyarteritis nodosa, multiple sclerosis, Alzheimer's Disease, Crohn's Disease regional enteritis, sepsis syndrome, poststreptococcal glomerulonephritis, hepatitis C, Neisseria! or Pneumococcal meningitis, Helicobacter pylori gastritis, influenza virus pneumonia, tuberculosis, tuberculoid leprosy, filariasis, cystic fibrosis, bacterial dysentery, Chagas Disease (Trypanosoma cruzi), schistosomiasis, idiopathic pulmonary fibrosis, hepatic cirrhosis, radiation-induced pulmonary fibrosis, chronic allograft rejection, and bleomycin-induced pulmonary fibrosis.

13. The method according to claim 1, wherein the subject is a mammal.

14. The method according to claim 13, wherein the mammal is a human.

15. A method of inhibiting respiratory burst in adherent neutrophils without inhibiting neutrophil degranulation in or bacterial killing by neutrophils, said method comprising: contacting adherent neutrophils with an effective amount of a compound that modulates soluble adenylyl cyclase, said compound having the following formula:

wherein:

R₁ is H, OH, alkyloxy, or halogen;

R₂ and R₅ are H or halogen;

R₃ is H or OH;

R₄ is H, alkyloxy, or halogen; R₆ is H or alkyl; and

R₇ is H or CH₂R₈, wherein R₈ is H, alkyl, or substituted or unsubstituted phenyl, with the proviso that at least one OfR₁, R₂, R₃, and R₄ is a halogen.

16. The method according to claim 15, wherein the compound has the following formula:

17. The method according to claim 15, wherein the compound has the following formula:

18. The method according to claim 15, wherein the compound has the following formula:

19. The method according to claim 15, wherein the compound has the following formula:

20. The method according to claim 15, wherein the compound has the following formula:

21. The method according to claim 15, wherein the compound has the following formula:

22. The method according to claim 15, wherein the compound has the following formula:

23. The method according to claim 15, wherein the compound has the following formula:

24. The method according to claim 15, wherein said contacting neutrophils is carried out in vitro.

25. The method according to claim 15, wherein said contacting neutrophils is carried out in vivo.

26. The method according to claim 15, wherein said contacting with a compound inhibits respiratory burst in adherent neutrophils triggered by an agent selected from the group consisting of a chemokine, a cytokine, a complement component, a secreted or shed bacterial product, and a bacterial cell wall component.

27. The method according to claim 26, wherein said agent is a chemokine selected from the group consisting of macrophage inflammatory protein- 1 (MIP- 1 ) and interleukin-8 (IL-8).

28. The method according to claim 26, wherein said agent is a cytokine selected from the group consisting of tumor necrosis factor (TNF), lymphotoxin, granulocyte-specific colony stimulating factor (G-CSF), and granulocyte/macrophage-specific colony stimulating factor (GM-CSF).

29. The method according to claim 26, wherein said agent is a complement component that is a chemoattractant complement component C5a.

30. The method according to claim 26, wherein said agent is a secreted or shed bacterial product that is an amino-terminally formylated peptide.