WO2016040650A1

WO2016040650A1 - Soluble guanylyl cyclase inhibition effects on lymphatic vasculature

Info

Publication number: WO2016040650A1
Application number: PCT/US2015/049450
Authority: WO
Inventors: Edward M. Schwarz; Christopher T. Ritchlin; Homaira RAHIMI
Original assignee: University of Rochester
Current assignee: University of Rochester
Priority date: 2014-09-10
Filing date: 2015-09-10
Publication date: 2016-03-17
Anticipated expiration: 2017-03-10

Abstract

The disclosure relates to methods and compositions for treating an inflammatory joint flare in a subject, a condition associated with a decreased lymphatic pulse, or an inflammatory joint condition. These methods including selecting a subject having an inflammatory joint flare, a decreased lymphatic pulse, or an inflammatory joint condition; and administering to the selected subject a soluble guanylyl cyclase ("sGC") inhibitor in an amount effective to treat the inflammatory joint flare, to increase the lymphatic pulse thereby treating the associated condition, or to treat the inflammatory joint condition.

Description

SOLUBLE GUANYLYL CYCLASE INHIBITION

EFFECTS ON LYMPHATIC VASCULATURE

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 62/048,660, filed September 10, 2014, which is hereby incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] Disclosed herein are methods and compositions for treating an inflammatory joint flare in a subject, a condition associated with a decreased lymphatic pulse, or an inflammatory joint condition. These methods and compositions involve the use of a soluble guanylyl cyclase ("sGC") inhibitor.

BACKGROUND OF THE DISCLOSURE [0003] Juvenile idiopathic arthritis ("JIA") is the most common rheumatic condition in childhood, with a prevalence of 16 to 150 per 100,000 in developed countries (Ravelli et al., "Juvenile Idiopathic Arthritis," Lancet. 369(9563): 767-78 (2007)). In the United States alone, a 2007 CDC study estimated a staggering one hundred thousand children seen for inflammatory polyarthropathies and synovitis (Sacks et al., "Prevalence of and Annual Ambulatory Health Care Visits for Pediatric Arthritis and Other Rheumatologic Conditions in the United States in 2001-2004," Arthritis Rheum. 57(8): 1439-45 (2007)). These numbers increase 10 fold in reference to adults with rheumatoid arthritis ("RA"). The most current data state an estimated 1.5 million people in the United States are afflicted with this life-long disease (Helmick et al., "Estimates of the Prevalence of Arthritis and Other Rheumatic Conditions in the United States: Part I," Arthritis Rheum. 58(1): 15-25 (2008)). The current approach to treat early and aggressively with an armamentarium consisting of anti-TNF medications, disease-modifying anti-rheumatic drugs, and non-steroidal anti-inflammatory medications has proven helpful to a great many patients. However, 40 percent of patients do not respond to anti-TNF therapy and those who do still develop episodes of flare throughout their lifetimes (Maini et al., "Therapeutic Efficacy of Multiple Intravenous Infusions of Anti-Tumor Necrosis Factor Alpha Monoclonal Antibody Combined with Low-Dose Weekly Methotrexate in Rheumatoid Arthritis," Arthritis Rheum. 41(9): 1552-63 (1998); Lipsky et al., "Infliximab and Methotrexate in the Treatment of Rheumatoid Arthritis: Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group," N. Engl. J. Med. 343(22): 1594-602 (2000); and Hyrich et al., "Predictors of Response to Anti-TNF- Alpha Therapy among Patients with Rheumatoid Arthritis: Results from the British Society for Rheumatology Biologies Register," Rheumatology (Oxford). 45(12): 1558-65 (2006)). Furthermore, current therapies continue to rely heavily on oral, injectable, and intravenous corticosteroids for immediate treatment when episodes of flare occur despite the many known systemic side effects of steroids, including avascular necrosis of bone, glaucoma, hypertension, and hyperglycemia, which may potentially cause more problems than the disease itself (Schacke et al., "Mechanisms Involved in the Side Effects of

Glucocorticoids," Pharmacol. Ther. 96(1): 23-43 (2002); Fisher et al., "Corticosteroid-Induced Avascular Necrosis: A Clinical Study of Seventy-Seven Patients," J. Bone Joint Surg. 53(5):859- 73 (1971)).

[0004] It would be desirable, therefore, to identify new therapies for the treatment of inflammatory joint conditions, particularly those associated with inflammatory joint flare or decreased lymphatic pulse. The present disclosure overcomes these and other deficiencies in the art.

SUMMARY OF THE DISCLOSURE

[0005] A first aspect of the disclosure relates to a method of treating an inflammatory joint flare in a subject that includes: selecting a subject having an inflammatory joint flare and administering to said subject a soluble guanylyl cyclase ("sGC") inhibitor in an amount effective to treat the inflammatory joint flare.

[0006] A second aspect of the disclosure relates to a method of treating a condition associated with a decreased lymphatic pulse that includes: selecting a subject having a decreased lymphatic pulse and administering to said subject an sGC inhibitor in an amount effective to increase the lymphatic pulse thereby treating said condition.

[0007] A third aspect of the disclosure relates to a method of treating an inflammatory joint condition that includes: selecting a subject having an inflammatory joint condition and administering to the subject an effective amount of an sGC inhibitor.

[0008] A fourth aspect of the disclosure relates to a pharmaceutical composition that includes a pharmaceutically acceptable carrier, an sGC inhibitor, and one or more additional therapeutic agents suitable for treatment of inflammatory joint flare, decreased lymphatic pulse, or an inflammatory joint condition. [0009] A fifth aspect of the disclosure relates to a therapeutic system that includes a first pharmaceutical composition comprising a pharmaceutically acceptable carrier and an sGC inhibitor; and a second pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more additional therapeutic agents suitable for treatment of inflammatory joint flare, decreased lymphatic pulse, or an inflammatory joint condition.

[0010] The current therapies for JIA and RA focus on inflammation in the joint itself, mediated by a dysfunctional immune system. The accompanying Examples demonstrate that lymphatics are a biomarker of arthritic flare, and provide evidence demonstrating that the nitric oxide/sGC receptor signaling pathway may be a key regulator of lymphatic function. In particular, the use of sGC inhibitors is shown to be successful in promoting lymphatic function (e.g., restoration of lymphatic pulse), which alone or in combination with other therapies should substantially improve the efficacy of arthritis treatments and treatments of other inflammatory conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A-1D show representative MRI and corresponding 3D volume renderings of the knee. Figure 1 A is a T2 fat-suppressed MRI image of an early rheumatoid arthritis ("RA") patient with asymmetric arthritic knee flare and increased lymphatic flow. Figure IB shows the 3D volume rendering of the inflammatory tissue (red; arrowhead) and large popliteal lymph node ("PLN") (blue, green, and yellow; arrows) corresponding to the 3D image of the early RA patient. Figure 1 C is a proton density weighted MRI image of a late RA patient with asymmetric knee flare and decreased lymphatic flow. Figure ID shows the 3D volume rendering of the inflammatory tissue (red; arrowhead) and collapsed PLN (blue, yellow; arrows) corresponding to the 3D image of the late RA patient.

[0012] Figures 2A-2J show representative histology and immunohistochemistry staining of the medial cross-section of the PLN. PLN tissue was harvested from either RA patients during total knee replacement surgery (Figures 2A-2E) or from human amputated legs that had evidence of knee osteoarthritis (Figures 2F-2J). H&E stained histology of the medial cross section of the PLN was photographed at 1.25X magnification (Figures 2A and 2F). Fluorescent immunohistochemistry with pseudocolor corresponding to antibody staining against LYVE-1

(Figures 2B and 2G), CD3 (Figures 2C and 2H), and CD 19 (Figures 2D and 21) are shown at 5X magnification. Figures IE and 1J show the merged fluorescent immunohistochemistry images. [0013] Figures 3A-3F show the evaluation of knee synovial volume and lymphatic pulse following B-cell depletion. Lymphatic drainage was evaluated in tumor necrosis factor- transgenic ("TNF-Tg") mice prior to (Figures 3A-3B) and following (Figures 3C-3D) treatment with anti-CD20. Figure 3E shows knee synovial volume measured in mice treated with placebo or a-CD20. Figure 3F shows the lymphatic pulse of mice treated with anti-CD20.

[0014] Figures 4A-4C show the co-localization of soluble guanylyl cyclase and alpha- smooth actin ("SMA") in PLN. Immunofluorescent microscopy was performed on fresh frozen histology sections of the PLN from TNF-Tg mice. Histology sections were stained for GCal (Figure 4A, red), a2 (Figure 4B, red), GC i (Figure 4C, red), and SMA (Figures 4A-4C, green). Co-localization is shown in Figures 4A-4C.

[0015] Figures 5A-5D illustrate the increase in Indocyanine Green ("ICG") uptake by lymphatic vessels with sGC inhibitor treatment. Figure 5D is a schematic image of a mouse hind limb and foot. Wild-type ("WT") mice (n=6) were injected with ICG in the foot pad and monitored for lymphatic flow. Control (Figure 5A) and sGC inhibitor (Figure 5B) treated mice were observed. After 30 minutes, an increased number of lymphatic vessels were observed

(Figure 5C, arrows) and some ICG appeared to extravasate into the interstitial space of the sGC treated leg (Figure 5C,*).

[0016] Figures 6A-B show the altered lymphatic rhythm and increased lymphatic pulse in mice treated with sGC inhibitor compared to control. Figure 6A is a representative graph of lymphatic pulse with control versus sGC inhibitor. Figure 6B shows the measured lymphatic pulse in control and sGC inhibitor treated mice.

[0017] Figures 7A-7F show that Gr-1+ cells are present in afferent lymphatic vessel to collapsed PLN and highly express iNOS. Intravital immunofluorescence microscopy was performed on draining lymphatic vessels afferent to PLNs by injecting fluorescein isothiocyanate (FITC)-conjugated anti-CDl lb, anti-Gr-1, anti-CD19, anti-CD3, or anti-CD45.2 antibody into the footpad of TNF-Tg or WT mice 2 hours prior to imaging (n=5/group). Immediately prior to imaging, Texas red-conjugated dextran beads were subcutaneously injected into the footpad to mark the draining lymphatic vessel, and an incision was made behind the knee to expose the PLNs and afferent lymphatics to the 5x objective lens of a fluorescent microscope. Figures 7A- 7B show immunofluorescence microscopic images of a representative lymphatic vessel afferent to the collapsed PLN, showing CD1 lb⁺ cells (Figure 7A) and Gr-1⁺ cells (Figure 7B). Figure 7C-7E are representative picomicrographs illustrating the lack of CD19⁺ cells (Figure 7C); lack of CD3⁺ cells (Figure 7D), and absence of hematopoietic CD45.2⁺ cells (Figure 7E) in lymphatic vessels afferent to a WT PLN. Histomorphometry of immunohistochemistry that stained for iNOS⁺ Gr-1⁺ cells in PLN was performed to quantify the number of Gr-1⁺, iNOS⁺ cells in WT, expanding, and collapsed PLN (Figure 7F), with the data presented as the mean ± S.D. (n=4; *p<0.05).

[0018] Figures 8A-8B are schematics of NO/sGC signaling in lymphatics during normal and inflamed conditions, as evidenced by the preceding results. Under normal conditions (Figure 8A), NO produced by eNOS in lymphatic endothelial cells ("LECs") in response to increased shear stress diffuses to the SMC layer of lymphatic collecting vessels and binds to sGC receptor, leading to dilation of the vessel. Dilation results in decreased shear stress turning off the eNOS signal to produce NO. Remaining NO diffuses away and the vessel contracts. During inflammation (Figure 8B), iNOS⁺ cells produce overwhelming amounts of NO, over-riding the normal process so that NO is continuously present, resulting in persistently dilated vessels that are unable to pulse. sGC inhibition prevents NO binding to its receptor and results in restoration of the pulse.

[0019] Figures 9A-9C show the contractility in lymphatics of TNF-Tg mice. Lymphatic collecting vessels were isolated and cannulated from TNF-Tg mice for ex vivo analysis in the absence (Figure 9B) and presence (Figure 9C) of Ca⁺ channel blocker BayK8644. Figure 9A shows TNF-Tg popliteal collecting lymphatic vessels tied to two glass pipettes for pressure control. Figures 9B-C show the raw tracing of TNF-Tg lymphatic contractions during a pressure step protocol. Input and output pressures (cmFtO, blue and red, respectively) are displayed on the top trace and are overlaid because they were changed simultaneously from 0.5 to 1, 2, 3, 5, 7, and 10 cmF^O. The bottom traces plot the inner diameter (μιη) measured continuously over time.

DETAILED DESCRIPTION OF THE DISCLOSURE [0020] As used herein, "patient" and "subject" are used synonymously, and are intended to refer to any animal that exhibits an inflammatory condition that is amenable to treatment in accordance with the methods disclosed herein. Preferably, the patient is a mammal. Exemplary mammalian patients include, without limitation, humans, non-human primates, dogs, cats, rodents (e.g., mouse, rat, guinea pig), horses, cattle and cows, sheep, and pigs.

[0021] The inflammatory condition to be treated using the methods disclosed herein include, but are in no way limited to, rheumatoid arthritis (RA), juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, Crohn's disease, autoimmune nephritis, primary biliary cirrhosis, psoriasis, acute pancreatitis, allograph rejection, allergic inflammation, inflammatory bowel disease, septic shock, osteoporosis, and cognitive deficits induced by neuronal inflammation. In one embodiment, the inflammatory condition is one that affects the joint, i.e., involves inflammation in or around the joint. Inflammatory conditions of the joint include rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, gout, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, adult Still's disease, viral arthritis, bacterial arthritis, and tuberculous arthritis.

[0022] As used herein, the treatment of an inflammatory joint condition includes (1) preventing the inflammatory joint condition, for example, preventing an inflammatory joint disease, condition or disorder in an individual that may be predisposed to the inflammatory joint disease, condition or disorder but does not yet experience or display the pathology or symptoms of such inflammatory joint disease, condition or disorder; (2) inhibiting the inflammatory joint condition, for example, inhibiting an inflammatory joint disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptoms of the inflammatory joint disease, condition or disorder (i.e., arresting further development of the pathology and/or symptoms or, alternatively, slowing progression of further development of the pathology or symptoms); and (3) ameliorating the inflammatory joint condition, for example, ameliorating an inflammatory joint disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptoms of inflammatory joint disease, condition or disorder (i.e., reversing the pathology and/or symptoms).

[0023] Thus, the treatment of the inflammatory joint condition may include preventing, inhibiting, or ameliorating one or more symptoms of the inflammatory joint condition (e.g., joint flare) as well as promoting other functions such as lymphatic pulse. Promoting lymphatic pulse connotes both reestablishment of lymphatic pulse in a region of the body where such pulse is/was lacking altogether, or enhancing the frequency of the lymphatic pulse in a region of the body where such pulse is/was diminished (i.e., relative to what would be considered normal across all individuals or normal for an individual patient prior to onset of the symptoms).

[0024] The therapeutic agents include an inhibitor or antagonist of soluble guanylyl cyclase ("sGC"). sGC is a heme-containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. sGC heterodimer is composed of an a-subunit and a shorter β-subunit which presents in the N-terminal the prosthetic heme group bound to histidine-105. Each subunit is composed of a heme -nitric oxide binding domain, a PAS-like domain, a coiled-coil bundle, and the C-terminal catalytic domain where turnover of GTP into cGMP occurs (Cary et al., Trends Biochem. Sci., 31 :231 (2006), which is hereby incorporated by reference in its entirety). The catalytic domain of sGC shows two sites at the interface of the a- and β-subunits that can accommodate small molecules: the GTP binding site, and an allosteric regulatory binding site (Allerston et al., PloS One 8:e57644 (2013); Zhang et al., Protein Sci. 6:903 (1997); Stasch et al., Nature 410:212 (2001), each of which is hereby incorporated by reference in its entirety). The two sites are different, and small molecules can bind to one or to both sites, thus showing a 1 : 1 or 1 :2 binding ratio.

[0025] Several classes of sGC inhibitors exist, including those that inhibit sGC activity act through oxidation of the heme moiety and those that bind directly to the catalytic domain of the enzyme.

[0026] Exemplary sGC inhibitors or antagonists that act via oxidation of the heme moiety include, without limitation, 3,7-bis(Dimethylamino)phenothiazin-5-ium (methylene blue); 6- anilino-5,8-quinolinedione (or LY-83583); [lH-l,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (or ODQ) (Moro et al., Proc. Natl. Acad. Sci. USA 93:1480 (1996) which is hereby incorporated by reference in its entirety); and 4H-8-Bromo-l,2,4-oxadiazolo(3,4-d)ben(b)(l,4)oxazin-l-one (or NS-2028) (Olesen et al., Br. J. Pharmacol. 123:299 (1998), which is hereby incorporated by reference in its entirety).

[0027] Exemplary sGC inhibitors or antagonists that bind directly to the catalytic domain of the enzyme include, without limitation, oxadiazolo[3,4-£]pyrazines and N2,N3- diphenylquinoxaline-2,3 -diamines as well as other compounds of the type described in Mota et al., Bioorg. Med. Chem. 23(17):5303-5310 (2015), which is hereby incorporated by reference in its entirety. Exemplary sGC inhibitors identified by Mota et al. include the following:

sipatrigine, N-(2,3-dichlorophenyl)quinazolin-4-amine; 4,5-bis((3- chlorophenyl)amino)phthalonitrile; N⁵,N⁶-bis(3-fluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine- 5,6-diamine; N⁵,N⁶-bis(2-nitrophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; l-(3-((3- ((4-acetylphenyl)amino)-6,7-dinitroquinoxalin-2-yl)amino)phenyl)ethan- 1 -one; 4,4'-((6- nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6-nitroquinoxaline-2,3- diyl)bis(azanediyl))diphenol; 6-nitro-N²,N³-diphenylquinoxaline-2,3-diamine; 4,4'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 2,3-bis((4- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide; and 2,3-bis((3- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide. The synthesis of these sGC inhibitors is fully described by Mota et al.

[0028] Other sGC inhibitors that bind directly to the enzyme can be identified using, e.g., a surface plasmon resonance-based assay with either full-length sGC or a smaller construct of the catalytic domain (sGCcat), as described in Mota et al., Bioorg. Med. Chem. Lett. 24: 1075 (2014), which is hereby incorporated by reference in its entirety. Screening of such agents that bind the catalytic domain for inhibition of sGC can then be carried out using the soluble guanylate cyclase assay to assess cGMP production, as described by Mota et al. {Bioorg. Med. Chem. Lett. 24:1075 (2014)) or Olesen et al. (Br. J. Pharmacol. 123:299 (1998)), each of which is hereby incorporated by reference in its entirety.

[0029] The use of additional agents in combination with sGC inhibitors or antagonists is also contemplated. Examples of these additional agents include, without limitation, VEGFR-3 agonists or gene therapy agents as described U.S. Patent No. 8,580,755 to Xing et al., which is hereby incorporated by reference in its entirety; VEGF inhibitors that inhibit VEGF directly or interfere with its signaling through VEGFR 1 or R2; traditional anti-inflammatory medication; steroid agents such as prednisone in various forms including orally or intra-articularly; an inhibitor of TNF-a; or a combination of any two or more thereof.

[0030] Exemplary VEGF inhibitors include, without limitation, antibodies directed to VEGF, VEGFR1 , VEGFR2, or the VEGF- VEGFR 1 or VEGF-VEGFR2 complex are described in U.S. Patent Publication No. 20020032313 to Ferrara et al., which is hereby incorporated by reference in its entirety; soluble VEGFR1 and R2 as set forth in U.S. Patent Publication No. 20030120038 to Kendall et al., which is hereby incorporated by reference in its entirety;

inhibitory VEGF variant proteins as described in U.S. Patent Publication Nos. 20060286636 to Shima et al. and 20050154187 to Shou et al., which are hereby incorporated by reference in their entirety; inhibitory fusion proteins directed to VEGF and VEGFR molecules as described in U.S. Patent Publication No. 20070253952 to Alvarez Vallina et al., which is hereby incorporated by reference in its entirety; aptamers against VEGF (see Ng et al., "Pegaptanib, A Targeted Anti- VEGF Aptamer for Ocular Vascular Disease," Nat Rev Drug Discovery 5(2): 123-32 (2006), which is hereby incorporated by reference in its entirety); and small molecule inhibitors of VEGF, VEGFR1, or VEGFR2 such as CP-547,632 (Pfizer Inc., NY, USA), AG13736 (Pfizer Inc.), ZD-6474 (AstraZeneca), AEE788 (Novartis), AZD-2171), VEGF Trap

(Regeneron/Aventis), Vatalanib (also known as PTK-787, ZK-222584: Novartis & Schering AG), IM862 (Cytran Inc. of Kirkland, Wash., USA); and angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif); and the compounds disclosed in

U.S. Pat. No. 6,534,524 to Kania et al., U.S. Pat. No. 6,235,764 to Larson et al., and U.S. Patent Publication No. 20070135489 to Huth et al., each of which is hereby incorporated by reference in its entirety. [0031] Exemplary anti-inflammatory medications include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticoids, disease modifying anti-rheumatic drugs, dihydrofolate reductase inhibitors (e.g., methotrexate), biologic response modifiers, and any combination thereof.

[0032] Exemplary NSAIDs include cyclooxygenase-2 (COX-2) inhibitors, non-limiting examples of which include nimesulide, 4-hydroxynimesulide, flosulide, meloxicam, celecoxib, and Rofecoxib (Vioxx), as well as non-selective NSAIDS such as diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac and tolmetin.

[0033] Exemplary analgesics include, without limitation, acetaminophen, oxycodone, tramadol, and propoxyphene hydrochloride.

[0034] Exemplary glucocorticoids include, without limitation, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, and prednisone.

[0035] Exemplary biological response modifiers include B-cell inhibitors, such as rituximab, or T cell activation inhibitors such as leflunomide, etanercept (Enbrel), or infliximab (Remicade).

[0036] Exemplary TNF-a inhibitors include anti-TNF-a antibody, a matrix

metalloproteinase inhibitor, a corticosteroid, a tetracycline TNF-a antagonist, a fluoroquinolone TNF-a antagonist, and a quinolone TNF-a antagonist.

[0037] Exemplary TNF-a antibodies include, without limitation, infliximab, etanercept,

CytoFAb, AGT-1, afelimomab, PassTNF, and CDP-870.

[0038] Exemplary corticosteroids include, without limitation, mometasone, fluticasone, ciclesonide, budesonide, beclomethasone, beconase, flunisolide, deflazacort, betamethasone, methyl-prednisolone, dexamethasone, prednisolone, hydrocortisone, Cortisol, triamcinolone, cortisone, corticosterone, dihydroxycortisone, beclomethasone dipropionate, and prednisone.

[0039] Exemplary tetracycline TNF-a antagonists include, without limitation, doxycycline, minocycline, oxytetracycline, tetracycline, lymecycline, and 4-hydroxy-4- dimethylaminotetracycline .

[0040] Exemplary fluoroquinolone TNF-a antagonists include, without limitation, norfloxacin, ofloxacin, ciprofloxacin, lomefloxacin, gatifloxacin, perfloxacin, and temafloxacin.

[0041] Exemplary quinolone TNF-a antagonists include, without limitation, vesnarinone and amrinone.

[0042] Other TNF-a antagonists include, without limitation, thalidomide, Onercept,

Pegsunercept, interferon-gamma, interleukin-1, pentoxyphylline, pimobeddan, lactoferrin, melatonin, nitrogen oxide, napthopyridine, a lazaroid, hydrazine sulfate, ketotifen, tenidap, a cyclosporin, peptide T, sulfasalazine, thorazine, glycyrrhizin, and L-carnitine.

[0043] The therapeutic agents described herein may be administered in any suitable manner or medically-accepted means for introducing a therapeutic directly or indirectly into a subject, including but not limited to orally, subcutaneously, intravenously, intramuscularly, parenterally, intrasynovially, intra-articularly, intraperitoneally, topically, transdermally, or by application to a mucosal surface. In one embodiment, the therapeutic agent is administered intrasynovially.

[0044] The therapeutic agents may also be delivered to the patient at multiple sites. The multiple administrations may be rendered simultaneously or over a period of several hours. In certain cases it may be beneficial to provide a continuous flow of the therapeutic composition. Additional therapy may be administered on a periodic basis, for example, daily, weekly or monthly.

[0045] The therapeutic agents may be formulated with uptake or absorption enhancers to increase their efficacy. Such enhancers include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS caprate and the like (see, e.g., Fix et al., "Strategies for Delivery of Peptides Utilizing Absorption-Enhancing Agents," J. Pharm. Sci. 85: 1282-1285 (1996); and Oliyai and Stella, "Prodrugs of Peptides and Proteins for Improved Formulation and Delivery," Ann. Rev. Pharmacol. Toxicol. 33:521-544 (1993), which are hereby incorporated by reference in their entirety).

[0046] The amount of therapeutic agent in a given dosage will vary according to the size of the individual to whom the therapy is being administered as well as the characteristics of the disorder being treated. For example, single dosages may contain between about 0.01 mg and about 1000 mg of the active agent. In exemplary treatments, it may be appropriate to administer up to about 50 mg/day, up to about 75 mg/day, up to about 100 mg/day, up to about 150 mg/day, up to about 200 mg/day, up to about 250 mg/day, or higher amounts such as 400 mg/day, 500 mg/day, 600 mg/day, 700 mg/day, 800 mg/day, 900 mg/day, and 1000 mg/day. These concentrations may be administered as a single dosage form or as multiple doses. Standard dose- response studies, first in animal models and then in clinical testing, will reveal optimal dosages for particular disease states and patient populations.

[0047] In one embodiment, suitable dosing and/or the effectiveness of therapy can be monitored in an individual subject by measuring lymphatic contraction rate (in pulses per minute) or lymph node volume (LNV) as surrogates for measuring the function of the lymph system. [0048] A further aspect relates to a pharmaceutical composition for practicing the therapeutic methods disclosed herein. The pharmaceutical composition includes a

pharmaceutically acceptable carrier along with an sGC inhibitor or antagonist and, optionally, one or more additional therapeutic agents.

[0049] The individual components of the pharmaceutical composition, i.e., the sGC inhibitor or antagonist and the additional therapeutic agents can be any of those described supra.

[0050] The pharmaceutical composition may also contain a carrier. Acceptable pharmaceutical carriers include solutions, suspensions, emulsions, excipients, powders, or stabilizers. The carrier should be suitable for the desired mode of delivery, discussed supra.

[0051] Pharmaceutical compositions suitable for injectable use (e.g., intravenous, intrasynovial, intra-arterial, intramuscular, etc.) may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Suitable carriers and/or excipients, include, but are not limited to sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier. 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 solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0052] Oral dosage formulations of the pharmaceutical composition can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Suitable carriers include 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, gum gragacanth, cornstarch, or gelatin; disintegrating agents such as cornstarch, potato starch, or alginic acid; a lubricant like stearic acid or magnesium stearate; and sweetening agents such as sucrose, lactose, or saccharine; and flavoring agents such as peppermint oil, oil of wintergreen, or artificial flavorings. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. [0053] An additional aspect relates to a therapeutic system for treating an antiinflammatory condition (or inflammatory joint flare or condition associated with a decreased lymphatic pulse). This therapeutic system includes a first pharmaceutical composition comprising a pharmaceutically acceptable carrier and an sGC inhibitor; and a second pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the (additional) therapeutic agents of the type described above.

[0054] Each of the first and second pharmaceutical compositions of the therapeutic system can take the forms of those described supra for the pharmaceutical composition that contains both active agents. Each composition of the therapeutic system is suitable for administering directly or indirectly into a subject, including but not limited to orally, subcutaneously, intravenously, intramuscularly, parenterally, intrasynovially, intra-articularly, intraperitoneally, topically, transdermally, or by application to a mucosal surface. The first and second pharmaceutical compositions may be introduced by the same route or by different routes, as needed.

EXAMPLES

[0055] The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope. Materials and Methods for Examples 1-5

[0056] Mice: The 3647 line of TNF-transgenic mice in a C57BL/6 background were originally obtained from Dr. George Kollias (Institute of Immunology, Alexander Fleming Biomedical Sciences Research Center, Vari, Greece). The TNF-Tg mice are maintained as heterozygotes, such that non-transgenic littermates are used as aged-matched wild type (WT) controls.

[0057] Contrast enhanced magnetic resonance imaging (CE-MRI) and data analysis:

CE-MRI images were followed by segmentation and quantification of knee synovial and lymph node volume.

[0058] Indocyanine Green Near-IR (ICG-NIR) Lymphatic Imaging: Lymphatic drainage was quantified by ICG-NIR. Indocyanine green was dissolved in distilled water and injected intradermally into the mouse footpad. ICG-NIR imaging was performed after ICG injection. Lymphatic pulsing frequency was quantified by analysis of NIR-ICG imaging of a region of interest. [0059] Intravital Inmmunofluorescent Microscope of lymphatics: Intravital

immunofluorescent microscopy was performed on draining lymphatic vessels afferent to PLN by injecting FITC conjugated anti-CDl lb, anti-Gr-1 , anti-CD19, anti-CD3, or anti-CD45.2 antibody into the footpad of TNF-Tg or wild type mice prior to imaging. Immediately prior to imaging, Texas red-conjugated dextran beads were subcutaneously injected into the footpad to mark the draining lymphatic vessel.

Example 1 - Asymmetric Knee Flare is Associated with PLI Collapse and the

Translocation of Bin Cells into the Paracortical Sinus Space of the Node

[0060] To validate lymphatic biomarkers in RA, the feasibility of detecting expanding and collapsed PLN using contrast enhanced magnetic resonance imaging ("CE-MRJ") and segmentations analysis was first demonstrated. CE-MRI was performed on RA patients with asymmetric knee flare (Figures 1A, 1C). A segmentation and threshold procedure was then used to determine synovial and lymph node volumes in the evaluated knee (Figures IB, ID).

[0061] CE-MRI imaging of an early RA patient knee experiencing arthritic flare with increased lymphatic flow confirmed the presence of an inflamed synovium, bone marrow edema in the femur and tibia, and a bright PLN (Figure 1A). 3D segmentation analysis of the same patient knee showed a region of uniformly distributed inflammatory tissue (red region

(arrowhead) of Figure IB) and an enlarged PLN (blue, green, and yellow regions (arrows) of Figure IB). In contrast, CE-MRI imaging and 3D volume rendering of a long-standing RA patient experiencing active flare with decreased lymphatic flow showed a patchy synovium (Figure 1C; red (arrowhead) in Figure ID) and a collapsed PLN (Figure 1C; blue and yellow (arrows) in Figure ID). These results are representative of the volumetric CE-MRI outcomes of knee synovium and PLN volumes observed in other patients.

[0062] To assess the cellular populations involved in RA, PLN was harvested from RA patients during total knee replacement surgery and processed for histology and

immunohistochemistry analysis. PLN was also processed from human amputated legs with evidence of knee osteoarthritis (OA). Histological analysis of LYVE⁺ (a lymph specific hyaluronic acid receptor), CD3⁺, and CD19⁺ cell populations showed large numbers of CD19⁺ B- cells in inflamed nodes (Bin cells) (Figures 2F-2J) in RA PLN as compared to OA PLN (Figures 2A-2E). LYVE⁺ expression on the surface of lymphatic vessels is associated with the accumulation of lymphatic fluid. Example 2 - B-Cell Depletion in a TNF-Tg Mouse Model Increases Lymph

Drainage from Inflamed Joints but does not Rescue Lymphatic Pulsing

[0063] The TNF-Tg model of chronic arthritis mimics RA in the delayed onset of disease, progression of destructive arthritis, and characteristics such as asymmetric arthritis. For these reasons, the TNF-Tg model is an ideal model for longitudinal studies investigating the progression of lymphatic dysfunction in the presence of chronic inflammation.

[0064] In mice, loss of lymphatic drainage is caused by loss of lymphatic pulse and clogging of efferent lymphatics by CD23⁺CD21^MCDld^M B cells ("Bin"). To directly test whether B-cell depleting therapy is able to ameliorate knee synovitis by restoring lymphatic drainage, the lymphatic flow in TNF-Tg mice treated with anti-CD20 antibody was evaluated.

[0065] Lymphatic drainage and pulsing were quantified using ICG-NIR lymphatic imaging. Treatment with anti-CD20 antibody allowed efficient drainage of ICG to the PLN (Figures 3A-3D). Synovial knee volume also decreased following treatment with anti-CD20 antibody, as compared to placebo (Figure 3E). These results confirmed that effective B-cell depletion therapy is achieved by increasing lymph drainage from inflamed joints.

[0066] Surprisingly, B-cell depletion treatment did not rescue lymphatic pulsing in TNF-

Tg mice (Figure 3F), indicating that the increase of ICG signal intensity observed in the lymphatic vessel following anti-CD20 antibody treatment is a result of the passive diffusion of ICG in the lymphatic vessel.

Example 3 - The Nitric Oxide Receptor sGC is Found in Lymphatic Vessels of

TNF-Tg Mice

[0067] Like blood vessels, lymphatic collecting vessels are comprised of an endothelial cell layer surrounded by a layer of smooth muscle cells ("SMCs"). NO molecules produced by endothelial cells diffuse to SMCs and bind to the nitric oxide receptor sGC. Following this binding, cGMP is activated in SMCs, acting on the contractile apparatus and leading to dilation of the vessel. Upon degradation of NO, the vessel contracts and the cycle repeats. While it is known that the sGC receptor, comprised of subunits alpha 1 and betal, is found in SMCs in blood vessels, its presence in SMCs surrounding the lymphatics has not been previously established. Immunofluorescence microscopy was used to confirm that sGC and alpha-smooth muscle actin subunits GCal (Figure 4A), GCa2 (Figure 4B), and GC i (Figure 4C) co-localize with SMA in the PLN of TNF-Tg mice. Example 4 - sGC Inhibition Alters Lymphatic Pulse in WT Mice

[0068] Pharmacological inhibition of the sGC receptor can be produced using the selective, irreversible inhibitor NS-2028. Eight month old wild type mice (n=6) were administered NS-2028 or vehicle control and measured for lymphatic pulse pressure from the footpad to the PLB using in vivo ICG-NI . An immediate visual difference was apparent between the limb given drug (Figure 5B) and the control limb (Figure 5A). The NS-2028 treated limb appeared to have increased ICG uptake in lymphatic vessels (Figure 5C) compared to control (Figure 5A). Measurement of the lymphatic pulse showed that the pulse activity was also clearly different from control, with an increased number of doublets and triplet spikes (Figure 6A). Quantification of the pulse showed a significant difference in lymphatic pulse pressure in the drug-tested leg, with increased lymphatic pulse (p<0.05) (Figure 6B). These results confirm that pharmacologic intervention via inhibition of sGC function enhances lymphatic pulse. Example 5 - Gr-1⁺ Cells are Present in Afferent Lymphatic Vessel to Collapsed

PLN and Highly Express iNOS

[0069] Prior to the onset of knee flare in TNF-Tg mice, the PLN expands as

inflammation causes an increase in lymphatic flow from the affected joint. At this point, the lymphatic vessels maintain their intrinsic lymphatic flow. However, at some point, the afferent lymphatic vessels lose their intrinsic pulse, the PLN collapses, and the knee flares (Figure 8B). The preceding Examples provide evidence that the collapse of the node is due to an

accumulation of a subtype of B cells in inflamed nodes (Bin cells), which translocate from the periphery of the node to the sinusoidal spaces. These accumulated B cells amass at the center of the node and block inflammatory cell and lymph egress from the joint. In addition, the lymph vessels lose their pulse via unknown mechanisms. Interestingly, passive lymphatic flow can be recovered following anti-CD20 B-cell depletion therapy that unclogs the PLN sinuses (Figures 3A-3D); however the recovery does not restore the lymphatic pulse in this chronic model of inflammatory arthritis (Figure 3F). This finding supports the belief that the intrinsic pulse has been lost due to a different mechanism.

[0070] NO plays a key role in the contraction and dilation cycle of the lymphatic vessels.

Figures 7A-7D show that macrophages that express inducible nitric oxide synthase (iNOS) are the primary cells moving through the lymphatic vessels efferent from inflamed joints in TNF-Tg mice. Figure 7E shows the absence of CD45.2⁺ cells in the PLN of WT mice. Figure 7F shows a significant increase in Grl⁺/iNOS⁺ cells in collapsed and expanded PLNs. These results are consistent with the fact that macrophage and granulocytes are the prominent cell types in the ankle pannus tissue of TNF-Tg mice, and that these cells travel through lymphatics. These results support the model for NO signalling in lymphocytes under normal conditions (Figure 8A) and during arthritic progression (Figure 8B) illustrated in Figure 8. During arthritic progression, activated iNOS expressing macrophages produce a high level of NO that squelches the normal NO signal from endothelial cells, leading to chronic dilation and loss of the lymphatic pulse.

Example 6 - NS-2028 does not Induce Adverse Systemic Effects in TNF-Tg Mice

[0071] To investigate possible cardiovascular side effects of NS-2028, wild type and

TNF-Tg mice (n=5) were treated systemically with 0.1 mg of NS-2028 administered by intraperitoneal injection daily for one week. No evidence of adverse systemic effects was observed at the dose given as measured by heart rate, blood pressure, and power Doppler evaluation of blood vasculature. In particular, no significant differences in heart rate, blood pressure, or power Doppler volume of blood vasculature were observed between wild-type and TNF-Tg mice treated with NS-2028.

Example 7 - Ex vivo Measurement of Lymphatic Properties [0072] Lymphatic collecting vessels were isolated and cannulated from TNF-Tg mice for ex vivo analysis in the absence (Figure 9B) and presence (Figure 9C) of Ca⁺ channel blocker BayK8644. Figure 9A shows TNF-Tg popliteal collecting lymphatic vessels tied to two glass pipettes for pressure control. Figures 9B-C show the raw tracing of TNF-Tg lymphatic contractions during a pressure step protocol. Input and output pressures (cmFLO, blue and red, respectively) are displayed on the top trace and are overlaid because they were changed simultaneously from 0.5 to 1, 2, 3, 5, 7, and 10 cmFLO. The bottom traces plot the inner diameter (μιη) measured continuously over time. These results demonstrate that the lymphatic vessel can be reliably used to measure lymphatic properties ex vivo. Prospective Example 8 - sGC Inhibition Will Improve Lymphatic Function

[0073] To expand on the demonstration of enhanced lymphatic pulse in response to administration of the sGC inhibitor NS-2028 (Example 4), NS-2028 and another sGC inhibitor (either ODQ or 4,4'-((6-nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol, see Mota et al., Bioorg. Med. Chem. 23(17):5303-5310 (2015), which is hereby incorporated by reference in its entirety) will be administered and other parameters of lymphatic function, including lymphatic clearance, will be measured. Wild type mice (n=5) will be treated for 6 weeks with drug versus placebo. The ex vivo flow chamber method (see Example 7) will be adopted to study the activity of lymphatics (e.g., lymphatic clearance and lymphatic pulse) in response to sGC inhibition in WT mice.

Prospective Example 9 - Effects of Inducible sGC-Knock Out

[0074] Following germline Flip recombination to convert sGCpitm2a(KOMP)Wtsi to sGC i^flox/flox, the recombinant mice will be crossed to SMMHC-CreER^T2 mice. The resulting strain will be used to demonstrate the effects of tamoxifen-inducible genetic sGC i loss of function in cells expressing SMMHC on the lymphatic pulse and lymphatic clearance using NIR- ICG imaging to measure lymphatic function. To confirm absence of NO activity, acetylcholine, a known stimulator of eNOS-derived NO production, will be used as a positive control. The ex vivo flow chamber method {see Example 7) will also be adopted to study the activity of lymphatics (e.g., lymphatic clearance and lymphatic pulse) before and after tamoxifen treatment.

Prospective Example 10 - Effect of sGC Inhibition on Lymphatic Pulse after PLN

Collapse in Chronic and Acute Murine Models of Arthritis

[0075] To target sGC inhibition as a treatment intervention and to determine the effect on disease status, sGC inhibitors will be administered for treatment of both serum-transfer and TNF- Tg murine models of acute and chronic arthritis, respectively. Treatment is expected to accelerate lymphatic drainage of inflamed joints by increasing the number of open efferent lymphatic vessels (Figure 5) and increasing the lymphatic pulse (Figure 6).

[0076] The serum-transfer model, in which the administration of sera from K/BxN mice to wild type mice results in antibody-mediated active joint inflammation similar to RA, is an acute model of arthritis. To evaluate the prophylactic and therapeutic effects of sGC inhibition on antibody mediated arthritis, wild type mice challenged with sera from K/BxN mice will be administered NS-2028 or 4,4'-((6-nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol {see Mota et al., Bioorg. Med. Chem. 23(17):5303-5310 (2015), which is hereby incorporated by reference in its entirety) prior to and 10 days following serum transfer. Mice will be imaged with CE-MRI and PD-US to assess synovial and lymph node inflmaation, NIR-ICG to measure lymphatic pulse and assess lymphatic function, and Micro-CT to quantify bone erosion prior to and following treatment. Histology of affected tissues (e.g., PLN, joint, and bone) will also be performed. [0077] As described above, the TNF-Tg mouse model of chronic arthritis mimics RA in the delayed onset of disease, progression of destructive arthritis, and characteristics such as asymmetric arthritis. To assess the prophylactic and therapeutic effects of sGC inhibition, TNF- Tg mice will be treated with NS-2028 or 4,4'-((6-nitroquinoxaline-2,3- diyl)bis(azanediyl))diphenol (see Mota et al., "A New Small Molecule Inhibitor of Soluble Guanylate Cyclase," Bioorg. Med. Chem. 23(17): 5303-5310 (2015), which is hereby incorporated by reference in its entirety) prior to and following PLN collapse. Drug effects on knee synovitis, lymphatic function, and bone erosion will be assessed by CE-MRI, NIR-ICG imaging, PD-US, micro-CT, and histology.

[0078] Arthritic flare can be induced in TNF-Tg mice using ultrasound and injection of microbubbles (see Bouta et al., "The Role of the Lymphatic System in Inflammatory-Erosive Arthritis," Semin. Cell Dev. Biol. 38:90-7 (2015), which is hereby incorporated by reference in its entirety), which leads to the loss of lymphatic pulse. In this method, the sGC inhibitor will be given both prophylactically and therapeutically to determine the effect on arthritic knee flare.

[0079] Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely 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

W I I VI IS CLAIMED:

1. A method of treating an inflammatory joint flare in a subject comprising:

selecting a subject having an inflammatory joint flare; and

administering to said subject a soluble guanylyl cyclase inhibitor in an amount effective to treat the inflammatory joint flare.

2. The method of claim 1, wherein the inflammatory joint flare is caused by rheumatoid arthritis.

3. The method of claim 1, wherein the inflammatory joint flare is caused by trauma to the joint.

4. The method of claim 1, wherein the inflammatory joint flare is caused by

osteoarthritis.

5. The method of claim 1, wherein the selected subject has a decreased or no lymphatic pulse.

6. The method of claim 1, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of 6-anilino-5,8-quinolinedione, lH-[l,2,4]Oxadiazolo[4,3-a]quinoxalin-l- one, 4H-8-Bromo-l,2,4-oxadiazolo(3,4-d)ben(b)(l,4)oxazin-l-one, and methylene blue.

7. The method of claim 1, wherein the soluble guanylyl cyclase inhibitor is an oxadiazolo[3,4-£]pyrazine or N2,N3-diphenylquinoxaline-2,3 -diamine that binds directly to the catalytic domain of the enzyme.

8. The method of claim 1, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of sipatrigine; N-(2,3-dichlorophenyl)quinazolin-4-amine; 4,5-bis((3- chlorophenyl)amino)phthalonitrile; N⁵,N⁶-bis(3-fluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine- 5,6-diamine; N⁵,N⁶-bis(2-nitrophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; l-(3-((3- ((4-acetylphenyl)amino)-6,7-dinitroquinoxalin-2-yl)amino)phenyl)ethan- 1 -one; 4,4'-((6- nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6-nitroquinoxaline-2,3- diyl)bis(azanediyl))diphenol; 6-nitro-N²,N³-diphenylquinoxaline-2,3-diamine; 4,4'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 2,3-bis((4- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide; and 2,3-bis((3- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide.

9. The method according to claim 1, wherein said administering is carried out intrasynovially or intra-articularly.

10. A method of treating a condition associated with a decreased lymphatic

pulse comprising:

selecting a subject having a decreased lymphatic pulse; and

administering to said subject a soluble guanylyl cyclase inhibitor in an amount effective to increase the lymphatic pulse thereby treating said condition.

11. The method of claim 10, wherein the subject has a lymphatic pulse below about 2.0 pulse per minute, about 1.8 pulse per minute, about 1.6 pulse per minute, about 1.5 pulse per minute, or about 1.4 pulse per minute.

12. The method of claim 10, wherein the subject has no lymphatic pulse.

13. The method of claim 10, wherein the condition is lymphedema.

14. The method of claim 10, wherein the condition is an inflammatory joint condition.

15. The method of claim 10, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of 6-anilino-5,8-quinolinedione, lH-[l,2,4]Oxadiazolo[4,3- a]quinoxalin-l-one, 4H-8-Bromo-l,2,4-oxadiazolo(3,4-d)ben(b)(l,4)oxazin-l-one, and methylene blue.

16. The method of claim 10, wherein the soluble guanylyl cyclase inhibitor is an oxadiazolo[3,4-£]pyrazine or N2,N3-diphenylquinoxaline-2,3 -diamine that binds directly to the catalytic domain of the enzyme.

17. The method of claim 10, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of sipatrigine; N-(2,3-dichlorophenyl)quinazolin-4-amine; 4,5-bis((3- chlorophenyl)amino)phthalonitrile; N⁵,N⁶-bis(3-fluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine- 5,6-diamine; N⁵,N⁶-bis(2-nitrophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; l-(3-((3- ((4-acetylphenyl)amino)-6,7-dinitroquinoxalin-2-yl)amino)phenyl)ethan- 1 -one; 4,4'-((6- nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6-nitroquinoxaline-2,3- 2 3

diyl)bis(azanediyl))diphenol; 6-nitro-N ,N -diphenylquinoxaline-2,3-diamine; 4,4'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 2,3-bis((4- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide; and 2,3-bis((3- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide.

18. The method of claim 10, wherein said administering is carried out at or near a blocked lymph node.

19. The method of claim 10, wherein said administering is carried out intrasynovially or intra-articularly.

20. The method of claim 10, wherein said administering is effective to increase lymphatic pulse to greater than about 2.1 pulse per minute, about 2.2 pulse per minute, about 2.3 pulse per minute, about 2.4 pulse per minute, about 2.5 pulse per minute, about 2.6 pulse per minute, about 2.7 pulse per minute, about 2.8 pulse per minute, about 2.9 pulse per minute, or about 3.0 pulse per minute.

21. A method of treating an inflammatory joint condition comprising:

selecting a subject having an inflammatory joint condition; and

administering to the subject an effective amount of a soluble guanylyl cyclase inhibitor.

22. The method of claim 21, wherein the inflammatory joint condition is rheumatoid arthritis, juvenile idiopathic arthritis, systemic lupus erythematosus, gout, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, adult Still's disease, or virally or bacterially-induced arthritis.

23. The method of claim 21, wherein said administering is carried out orally, subcutaneously, intravenously, intramuscularly, parenterally, intrasynovially, intraarticularly, intraperitoneally, topically, transdermally, or by application to a mucosal surface.

24. The method of claim 21, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of 6-anilino-5,8-quinolinedione, lH-[l,2,4]Oxadiazolo[4,3- a]quinoxalin-l-one, 4H-8-Bromo-l,2,4-oxadiazolo(3,4-d)ben(b)(l,4)oxazin-l-one, and methylene blue.

25. The method of claim 21, wherein the soluble guanylyl cyclase inhibitor is an oxadiazolo[3,4-£]pyrazine or N2,N3-diphenylquinoxaline-2,3 -diamine that binds directly to the catalytic domain of the enzyme.

26. The method of claim 21, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of sipatrigine; N-(2,3-dichlorophenyl)quinazolin-4-amine; 4,5-bis((3- chlorophenyl)amino)phthalonitrile; N⁵,N⁶-bis(3-fluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine- 5,6-diamine; N⁵,N⁶-bis(2-nitrophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; l-(3-((3- ((4-acetylphenyl)amino)-6,7-dinitroquinoxalin-2-yl)amino)phenyl)ethan- 1 -one; 4,4'-((6- nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6-nitroquinoxaline-2,3-

2 3

27. A pharmaceutical composition comprising:

a pharmaceutically acceptable carrier;

a soluble guanylyl cyclase inhibitor; and

one or more additional therapeutic agents suitable for treatment of inflammatory joint flare, decrease lymphatic pulse, or an inflammatory joint condition.

28. The pharmaceutical composition of claim 27, wherein the composition is suitable for administering orally, subcutaneously, intravenously, intramuscularly, parenterally, intrasynovially, intraarticularly, intraperitoneally, topically, transdermally, or by application to a mucosal surface.

29. The pharmaceutical composition of claim 27, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of 6-anilino-5,8-quinolinedione, 1H-

[l,2,4]Oxadiazolo[4,3-a]quinoxalin-l-one, 4H-8-Bromo-l,2,4-oxadiazolo(3,4- d)ben(b)(l,4)oxazin-l-one, and methylene blue.

30. The pharmaceutical composition of claim 27, wherein the soluble guanylyl cyclase inhibitor is an oxadiazolo[3,4-£]pyrazine or N2,N3-diphenylquinoxaline-2,3 -diamine that binds directly to the catalytic domain of the enzyme.

31. The pharmaceutical composition of claim 27, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of sipatrigine; N-(2,3- dichlorophenyl)quinazolin-4-amine; 4,5-bis((3-chlorophenyl)amino)phthalonitrile; N⁵,N⁶-bis(3- fluorophenyl)- [1 ,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; N⁵ _jlS^-bis^ -nitrophenyl)- [l ,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; l -(3-((3-((4-acetylphenyl)amino)-6,7- dinitroquinoxalin-2-yl)amino)phenyl)ethan-l -one; 4,4'-((6-nitroquinoxaline-2,3- diyl)bis(azanediyl))diphenol; 3,3'-((6-nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 6-nitro- N²,N³-diphenylquinoxaline-2,3-diamine; 4,4'-((6-(trifluoromethyl)quinoxaline-2,3- diyl)bis(azanediyl))diphenol; 3,3'-((6-(trifluoromethyl)quinoxaline-2,3- diyl)bis(azanediyl))diphenol; 2,3-bis((4-hydroxyphenyl)amino)-N-methylquinoxaline-6- carboxamide; and 2,3-bis((3-hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide.

32. The pharmaceutical composition of claim 27, wherein the soluble guanylyl cyclase inhibitor is present in an amount of between about 0.01 mg and about 1000 mg per unit dose.

33. The pharmaceutical composition of claim 27, wherein the one or more additional therapeutic agents comprise an anti- inflammatory agent, a VEGFR-3 agonist or gene therapy agent, a VEGF inhibitor, or an inhibitor of TNF-a.

34. The pharmaceutical composition of claim 33, wherein the one or more additional therapeutic agents is present in an amount of between about 0.01 mg and about 1000 mg per unit dose.

35. A therapeutic system comprising:

a first pharmaceutical composition comprising a pharmaceutically acceptable carrier and an soluble guanylyl cyclase inhibitor; and

a second pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more additional therapeutic agents suitable for treatment of inflammatory joint flare, decrease lymphatic pulse, or an inflammatory joint condition.

36. The therapeutic system of claim 35, wherein the first pharmaceutical composition and the second pharmaceutical composition are independently suitable for administering orally, subcutaneously, intravenously, intramuscularly, parenterally, intrasynovially, intraarticularly, intraperitoneally, topically, transdermally, or by application to a mucosal surface.

37. The therapeutic system of claim 35, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of 6-anilino-5,8-quinolinedione, 1H- [l,2,4]Oxadiazolo[4,3-a]quinoxalin-l-one, 4H-8-Bromo-l,2,4-oxadiazolo(3,4- d)ben(b)(l,4)oxazin-l-one, and methylene blue.

38. The therapeutic system of claim 35, wherein the soluble guanylyl cyclase inhibitor is an oxadiazolo[3,4-£]pyrazine or N2,N3-diphenylquinoxaline-2,3 -diamine that binds directly to the catalytic domain of the enzyme.

39. The therapeutic system of claim 35, wherein the soluble guanylyl cyclase inhibitor is selected from the group consisting of sipatrigine; N-(2,3-dichlorophenyl)quinazolin- 4-amine; 4,5-bis((3-chlorophenyl)amino)phthalonitrile; N⁵,N⁶-bis(3-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine; N⁵,N⁶-bis(2-nitrophenyl)-[l,2,5]oxadiazolo[3,4- b]pyrazine-5,6-diamine; l-(3-((3-((4-acetylphenyl)amino)-6,7-dinitroquinoxalin-2- yl)amino)phenyl)ethan-l-one; 4,4'-((6-nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'- ((6-nitroquinoxaline-2,3-diyl)bis(azanediyl))diphenol; 6-nitro-N²,N³-diphenylquinoxaline-2,3- diamine; 4,4'-((6-(trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 3,3'-((6- (trifluoromethyl)quinoxaline-2,3-diyl)bis(azanediyl))diphenol; 2,3-bis((4- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide; and 2,3-bis((3- hydroxyphenyl)amino)-N-methylquinoxaline-6-carboxamide.

40. The therapeutic system of claim 35, wherein the soluble guanylyl cyclase inhibitor is present in the first pharmaceutical composition is present in an amount of between about 0.01 mg and about 1000 mg per unit dose.

41. The therapeutic system of claim 35, wherein the one or more additional therapeutic agents in the second pharmaceutical composition comprises an anti-inflammatory agent, a VEGFR-3 agonist or gene therapy agent, a VEGF inhibitor, or an inhibitor of TNF-a.

42. The therapeutic system of claim 35, wherein the one or more additional therapeutic agents in the second pharmaceutical composition is present in an amount of between about 0.01 mg and about 1000 mg per unit dose.