US 2005.0049256A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0049256A1 LOrton et al. (43) Pub. Date: Mar. 3, 2005

(54) TREATMENT OF INFLAMMATORY Publication Classification AUTOIMMUNE DISEASES WITH ALPHA-ADRENERGIC ANTAGONSTS AND BETA-ADRENERGICAGONSTS (51) Int. Cl." ...... A61K 31/517; A61K 31/137 (76) Inventors: Dianne Lorton, Avondale, AZ (US); (52) U.S. Cl...... 514/252.17; 514/649; 514/651 Cheri Lubahn, Glendale, AZ (US) Correspondence Address: JENNINGS, STROUSS & SALMON, P.L.C. (57) ABSTRACT 201 E. WASHINGTON ST, 11TH FLOOR PHOENIX, AZ 85004 (US) The present invention discloses a novel compound and (21) Appl. No.: 10/928,437 method for the treatment of inflammatory autoimmune dis eases, for example, rheumatoid arthritis, using C.-adrenergic (22) Filed: Aug. 27, 2004 antagonists and B-adrenergic agonists in combination. Treat ment of animals, namely humans, with an O-adrenergic Related U.S. Application Data antagonist, preferably, phentolamine, and a B-adrenergic agonist, preferably terbutaline, in combination can Signifi (60) Provisional application No. 60/498.367, filed on Aug. cantly Suppress the joint destruction and inflammation due to 27, 2003. disease in these animals. Patent Application Publication Mar. 3, 2005 Sheet 1 of 5 US 2005/0049256A1

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TREATMENT OF INFLAMMATORY DMARDS. Use of one second-line drug followed by another AUTOIMMUNE DISEASES WITH once the drug being used is no longer effective or is not ALPHA-ADRENERGIC ANTAGONSTS AND tolerated well by the patient, is most widely practiced by BETA-ADRENERGICAGONSTS rheumatologists. Methotrexate and other immunosuppres Sive drugs, Such as cycloSporin and leflunomide were major CLAIM TO DOMESTIC PRIORITY advancements of the 1980s in treating rheumatoid arthritis. Methotrexate is currently the gold standard for treatment of 0001. This Application claims the benefit of priority of aggressive rheumatoid arthritis. However, its effectiveness U.S. Application Ser. No. 60/498.367, filed Aug. 27, 2003. wanes over time and can cause troublesome side effects, including liver damage, Sepsis, Severe anemia and bleeding. FIELD OF THE INVENTION 0007. The approved immunosuppressive drug, lefluno 0002 The present invention relates to an improved mide, was introduced for treating rheumatoid arthritis. This method for the treatment of inflammatory autoimmune dis drug relieves joint tenderneSS and Swelling, decreases joint ease, and more Specifically to a treatment for inflammatory pain and reduces indicators of global disease activity. While autoimmune disease, including rheumatoid arthritis, using leflunomide does not make patients more Susceptible to C.-adrenergic antagonists and B-adrenergic agonists in com infections, it can cause hair loSS, weight loss, hypertension, bination. diZZiness, and gastrointestinal Side effects. Advances in management of rheumatoid arthritis include the use of BACKGROUND OF THE INVENTION corticosteroids as anti-inflammatory and immunosuppres Sive agents. The main disadvantage of corticosteroids is that 0003 Rheumatoid arthritis (RA) is a chronic inflamma long-term use is limited due to adverse effects including tory autoimmune disease of the joints, producing pain and Weight gain, hyperglycemia, cataracts, Osteoporosis, and ultimate destruction of the joints. Rheumatoid arthritis is characterized by a chronic inflammatory response in the Stomach ulcers. joint that is directed by macrophages and T cells which 0008 Thus, treatment for patients with rheumatoid arthri invade affected joints. Production of proinflammatory cytok tis is imperfect. Accordingly, there is an urgent need for ines and other immune cell mediators by these immune cells treatments which have few if any side effects and that will results in the development of a proliferative invasive con be effective in Suppressing not only the inflammation but nective tissue derived from the Synovial membrane and its also prevent the bone and cartilage degeneration associated microvasculature that slowly erodes away the cartilage and with rheumatoid arthritis. bone tissues of joints. SUMMARY OF THE INVENTION 0004. While destruction of joint tissue is the most com mon feature of RA, the disease can exhibit significant 0009. The present invention discloses a method for the extra-articular manifestations, including cutaneous lesions, treatment of inflammatory autoimmune diseases by admin vasculitis, blood abnormalities, peripheral neuropathy, peri istering B-adrenergic agonists in combination with C.-adren carditis, arteritis of the Viscera, and pulmonary disease. This ergic antagonists. The present invention further discloses a indicates that RA is a Systemic disease that affects the compound useful in treating inflammatory autoimmune dis connective tissueS of major organs Systems. The Specific eases comprising B-adrenergic agonists in combination with etiology of RA remains elusive. Dysregulation of both C.-adrenergic antagonists. cell-mediated and humoral immunity are associated with 0010. In certain embodiments, the present invention con RA. The pathophysiology of RA intimately involves defects cerns a compound and method for treating rheumatoid in immune cell regulation. Although the exact mechanisms arthritis or other autoimmune diseases, Such as inflammatory have not been delineated, the functional activities of mac bowel disease, Krohn's disease, thyroiditis, fibromyalgia, rophages, B cells and T cells from both the joint and Systematic erythermatus, lupus, chronic fatigue Syndrome, lymphoid tissue are affected. and Type 1 diabetes, by the application of a therapeutically 0005. Unfortunately, current treatment strategies for RA effective dose of a f-adrenergic agonist, preferably a f-adr and other inflammatory autoimmune diseases are relatively energic agonist Such as terbutaline, coupled with a thera ineffective in preventing bone destruction. Conventional peutically effective dose of an O-adrenergic antagonist, anti-rheumatic drugs are classified into anti-inflammatory preferably C-, C., and C2-adrenergic receptor Subtypes Such drugs, Slow-acting drugs and corticosteroids. Only the slow as phentolamine, praZosin or yohimbine, to human Subjects acting drugs are thought to be capable of modifying the with the disease. Therapeutically effective doses of particu disease course of rheumatoid arthritis and are referred to as lar agonists or antagonists and the frequency of dosage disease modifying anti-rheumatic drugs (DMARD). Drugs administration are to be determined according to protocols from each of these classes are currently being used for understood by those skilled in the art. treatment of rheumatic diseases. Of these, the central focus 0011. Other B-adrenergic agonists useful in this novel has been and remains the DMARDS and nonsteroidal anti method of treatment include: metaproterenol, albuterol, iso inflammatory drugs (NSAID). NSAIDs effectively abolish etharine, pributerol, bitolterol, ritodrine, and Salmeterol. the Signs and Symptoms of joint inflammation and reduce Other C.-adrenergic antagonists useful in this novel method pain. They are not effective in preventing bone and cartilage of treatment include: yohimbine, regitine, prazosin, dox loss. The value of NSAIDs for treating rheumatic diseases is aZosin, tamsulosin, teraZosin, octopamine, phenoxyben limited by their side effects. Zamine, phentolamine, hydrochlorothiazide, 5-methyl ura 0006 Aggressive rheumatoid arthritis or early onset of pidil, chloroethylclonidine, bunazosin, alfuzosin, RS17053, joint destruction indicates the need for rapid treatment with BMY 7378, urapidil, L-765,314, nicergoline, ABT-866, US 2005/0049256 A1 Mar. 3, 2005

cyclazosin, A322312, A 119637, fiduxosin, JTH-601, imi 0018 FIG. 2 is a graph depicting the decreased dor loxan, 2 idopropoxyidazoxan, 2-methoxyidazoxan (RX Soplantar Swelling by day 28 in animals treated with vehicle, 821002), idazoxan, piperoxan, BRL 44408, beditin, atipam phentolamine and/or trebutaline. eZole, rawolscine, ARC 239, RS-79948, MK912, RS 79948, 0019 FIG. 3 illustrates the results of continuous treat UIC 14304 and ethoxyidazoxan. ment with vehicle, combination phentolamine/terbutaline, 0012. The f-adrenergic agonists and C.-adrenergic combination yohimbine/terbutaline or combination pra antagonists of the present invention may be administered to ZoSine/terbutiline drug therapies from initiated at disease a patient in a dosage form Selected from the group consisting onset, day 12 post-CFA challenge, and continued through of pills, tablets, capsules, caplets, Solutions, Suspensions, day 28, that reduced dorsoplantar footpad widths in arthritic Syrups, Suppositories, and aerosols. Additionally, the dosage rats when compared with vehicle treated controls. of the ?- (or f3-) and C.- (or C- or Cl-) adrenergic agonist 0020 FIGS. 4A-E shows the radiographic analysis of and antagonist, respectively, used may be in a Sustained ankle joints treated with vehicle and adrenergic drug com release form. The dosage form of the B-adrenergic agonist bination therapies described in FIG. 3. FIGS. 4A-E is an and the C.-adrenergic antagonistS may be administered by X-ray of normal non-arthritic rat limbs to be used for various routes including Sublingual, oral, intravenous, intra comparison. FIG. 4F illustrates the radiographic scores of muscularly, rectal, parenteral or Subcutaneous. the animals treated with yohimbine/terbutaline-, phentola 0013 The B-adrenergic agonists may be administered in mine/terbutaline- and prazosine/terbutaline compared with various Salt forms and may be Selected from the group the vehicle-treated AA rats. consisting of metaproterenol Sulfate; terbutaline Sulfate; 0021 FIGS. 5A-B shows an injury/sprouting response of albuterol Sulfate, isoetharine hydrochloride, isoetharine the NA nerves in the spleens from arthritic and non-arthritic meSylate; pributerol acetate; bitolterol meSylate; or ritodrine rats. FIG. 5A depicts the changes seen in the white pulp hydrochloride; and levalbuterol hydrochloride. The C-adr regions both hilar and distal. FIG. 5B depicts changes seen energic antagonists also may be administered in various Salt in the red pulp regions of the Spleens both hilar and distal. forms as well, and may be Selected from the group consist ing of phentolamine meSylate; regitine meSylate; prasoZin; teraZosin, doxazosin meSylate; and tamsulosin hydrochlo DETAILED DESCRIPTION ride. 0022. The present invention addresses one or more short 0.014. In one embodiment therapeutically effective doses comings or disadvantages in the available treatment regi for both the B-adrenergic agonist and C-adrenergic antago mens for rheumatoid arthritis or inflammatory autoimmune nist, respectively, range from about 1.0 to 10.0 mg, with a diseases through the use of a combination of B-adrenergic preferred range of about 2.0 to 5.0 mg, or even more agonists and C.-adrenergic antagonists. In preferred embodi preferably about 1.25 to 2.5 mg. In another embodiment the ments, the invention contemplates the use of B-adrenergic therapeutically effective dose of the compound is given three agonists and non-Specific Cl- or C- or C2-adrenergic antago times per day depending upon disease Severity and patient nists, and particularly terbutaline, phentolamine and pra responses to the drugs. In another embodiment of the present ZoSin, respectively, as agents to treat patients with rheuma invention method, therapeutically effective dosages of terb toid arthritis (RA) or inflammatory autoimmune diseases. utaline from about 1.0 to 10.0 mg coupled with therapeuti 0023 Cytokine contribution to RA pathology is based cally effective dosages of phentolamine from about 1.0 to largely on shifts in Th1 and Th2 cell cytokine patterns. The 10.0 mg are administered two or more times per day. nature and concentration of antigen exposure, the degree of antigen-induced activation of Subsets of CD4+ T helper (Th) 0.015 A further embodiment comprises administering a cells (known as Th1 and Th2 cells), and relative production therapeutically effective dose of B-adrengeric agonist fol of the different cytokines produced by Th1 and Th2 cells all lowed administering a therapeutically effective dose of an interact to determine whether an eventual immune reaction C.-adrenergic antagonist, or alternatively, administration of a is dominated by a cellular or humoral response. Th1 and Th2 therapeutically effective dose of B-adrenergic antagonist Subsets in mice and humans determine the cellular or followed by a therapeutically effective dose of B-adrenergic humoral dominance of a response by virtue of the pattern of agonist. Administration of the B-adrenergic agonist and cytokines that they produce. C.-adrenergic antagonist can occur within a 24-hour period, a 12-hour period, or preferably an 8-hour period, more 0024. Two distinct cytokine secretion patterns have been preferably an 4-hour period, or most preferably within a one defined among rodent CD4+ T cells. Type 1 helper (Th1) T hour period of each other. cells produce IL-2, gamma interferon (IFNY, and TNFB (lymphotoxin) and are important in the generation of BRIEF DESCRIPTION OF THE DRAWINGS delayed-type hypersensitivity (DTH) and cytotoxic responses. In contrast, Th2 cells Secrete IL-4, IL-5, IL-6 and 0016 FIGS. 1A=1D shows X-rays demonstrating joint IL-10, cytokines that are critical for regulating B cell destruction or lack thereof in arthritic animals treated daily growth, differentiation, antibody production, and immuno with i.p. injections Starting at 12 days post adjuvant injection globulin (Ig) isotype Switching. Th1 and Th2 cells are and continuing until day 28 with 1B vehicle, arthritic; 1C mature T cells derived from a precursor Tho cell that phentolamine, arthritic; 1D terbutaline, arthritic, and 1E primarily produces IL-2. phentolamine and terbutaline, arthritic. FIG. 1A is an X-ray 0025 Numerous studies suggest that the balance of of a normal non-arthritic rat limb used for comparison. cytokines produced by Th1/Th2 T cells play an important 0017 FIG. 1F shows the radiographic scores for the role in autoimmune disease development. Modulation of X-rays in FIGS. 1B-1E. Th1/Th2 cytokine profiles is accepted as having therapeutic US 2005/0049256 A1 Mar. 3, 2005 value, especially in diseaseS Such as RA, where a specific rophages markedly improved RA in clinical trials. Neutral causative agent has not been identified. Bypassing the caus izing antibodies against TNFC. given Systemically reduce ative agent and acting on the cytokine balance of Th1/Th2 clinical and histopathologic Signs of both collagen- and AA. cytokines might be a way to control autoimmunity and In TNFC. transgenic mice, this Same treatment combined chronic inflammation. with anti-IL-1 receptor therapy prevents the development of 0.026 Cytokines produced by one Th cell subset can arthritis. modulate the Synthesis of cytokines produced by the other 0032. Further, administration of cytokines that down Subsets. IFNY produced by antigen-stimulated Th1 cells can regulate macrophage function, IL-4 or IL-13, reduces joint inhibit the expansion of Th2 lymphocytes. This, in turn, has inflammation and destruction. In Streptococcal cell wall been shown to influence the Secretion of IgG2a. In contrast, induced arthritis, Systemic treatment with IL-4 reduced IL-4 and IL-10 produced by Th2 cells can inhibit growth of inflammation and joint destruction by inhibiting IL-1 and Th1 cells and production of Th1-type cytokines. Thus, DTH TNFC, and enhancing IL-1Ra. Conversely, neutralization of and antibody responses are regulated directly and indirectly IL-10 promoted macrophage-mediated joint pathology in by cytokines produced by both Th1 and Th2 cells. collagen-induced arthritis. In animals with fully expressed AA, treatment with bisphosphonate clodronate, a toxin taken 0027. In addition to antigen processing and presenting up into liposomal vesicles by macrophages and that Subse functions and their role in inflammation, macrophages are quently destroys them, Significantly reduced joint inflam also important regulators of cellular and humoral immune mation and destruction, giving this drug disease-modifying responsiveness. Macrophages regulate T cell proliferation in Status, with long-lasting effects after short-term therapy. a cell-cell contact manner and, through monokine Synthesis and release, play a critical role in driving Th1 and Th2 0033 Long-term loss of macrophages in lymphoid immune responses. organs correlated with the anti-arthritic effects. In fact, clodronate targeted macrophages in Secondary lymphoid 0028. Th1 cells are the predominate T cell subset in RA organs, rather then affected joints. ED3+ macrophages, joints, however, their function may be limited by other important for cell-cell interactions with T cells, were pref cytokines produced by macrophages in the Synovium. A erentially depleted in Spleen and LN. In contrast, clodronate dominant role for activated macrophages in RA disease treatment was only marginally effective when administered pathology is gaining acceptance. Altered macrophage func into affected joints. Additionally, repeated leukapheresis tion occurs in Adjuvant Arthritis (AA) and RA, and is effectively depleted circulating monocytes in RA patients contributory to defective T and B cell activity. Monocytes with consequent reduction in disease Symptoms. Thus, mac are a major Source of cytokines in the Synovial fluid in RA, rophages play a central role in Sustaining arthritis and particularly the proinflammatory mediators, IL-1 and TNFC. demonstrate the critical role for Systemic monocytes and In addition, IL-15, a cytokine with a Secondary Structure and macrophages and their Secreted cytokines in RA pathology. functional properties Similar to IL-2, is produced by mac rophages in the RAjoint. In the joint, IL-15 is chemotactic, 0034) The Central Nervous System (CNS) plays a role in promoting immune cells into the joint, and then Stimulating controlling levels of cytokines and the activation of different T cell proliferation. Elevated IL-12 concentrations in the RA T cell subsets is well documented. The Sympathetic Nervous Synovial fluid also are derived from macrophages. These System (SNS), through innervation of lymphoid tissues, cytokines should promote Th1 responses in the joint, includ provides a direct route for communication between the ing IFNY production by local Th1 cells. nervous and immune Systems. This innervation is regional and Specific, distributing along the vasculature and in the 0029 Macrophages that invade the joints in the chronic parenchyma adjacent to immune cells. Adrenergic receptors disease phase originate from the bone marrow and lymphoid are present on a variety of immune cells, including T and B tissues and are found in close proximity to T cell clusters that lymphocytes, and macrophages, and Stimulation of these resemble the lymphoid aggregates in joints. These macroph receptors can alter immune responses. age Subsets are activated based on expression of class II MHC antigens, high levels of macrophage mediators of 0035. Beta-adrenergic receptors are selectively tissue destruction, inflammatory cytokines in the joint, and expressed by Th1, but not by Th2, cell clones. In functional enhanced phagocytic and tumoricidal activity. Activated studies, Th1 and Th2 cells are differentially regulated by macrophages also are present in lymphoid tissues in RA B-AR stimulation. Th1 clones treated with terbutaline, a animal models. B-adrenergic receptor agonist, before activation with enriched populations of antigen-Specific B cells or anti-CD3 0030. In collagen- and AA, TNFC. mRNA is elevated in antibodies, inhibits both IL-2 and IFNY production by Th1 lymph nodes (LN) before the onset of arthritis, as well as cell clones, but does not affect IL-4 or IL-10 production by IFNY, a Th1 cytokine. Thus, TNFC. may prime disease Th2 cell clones. Additionally, terbutaline application relevant T cells present in LN. In TNFC. transgenic mice, reduced IL-2-dependent T cell proliferation consistent with elevated TNFC. levels produces a chronic erosive arthritis. the terbutaline-induced reduction in IL-2 production. Col Likewise, elevated circulating TNFC. levels occur with acute lectively, these findings Suggest differential regulation of Th flares of RA, Supporting an extra-articular Source of TNFC. cell Subset clones. Thus, in Vivo, 32-adrenergic receptors that is important in disease development. In contrast, IL-1 is distinguish Th1 and Th2 cells. Therefore, the differential more critically involved in local joint inflammation, Since its B-adrenergic receptor expression by Th1 and Th2 cells elevation in the Synovium parallels the onset of Synovitis, provides a mechanism for modulating clonal expansion of and intra-articular injection of IL-1B induces Synovitis. activated Th cells by sympathetic nerves. 0.031 Treatments aimed at either down-regulating mono 0036 Functionally, B-adrenergic receptor stimulation of nuclear phagocyte function or destruction of activated mac activated Th cell clones affects their ability to help B cells US 2005/0049256 A1 Mar. 3, 2005 produce Specific antibody isotypes. Terbutaline application histochemistry for catecholamines with morphametric to Th cells before activation inhibited IgG2a (but not IgG1) analysis and immunocytochemistry for thyrosine hydroxy production when cultured with B cell clones, blockable by lase. In AA rats, Sympathetic nerve density in the hilar addition of nadolol (B-adrenergic receptor antagonist) or by regions, where NA nerve fibers enter the Spleen, was exogenous administration of IFNY. Further, after B cells and increased by two-fold over that observed in vehicle treated Th cells are activated by exposure to a Th cell-dependent ratS. antigen in Vitro, f2-adrenergic receptor agonists enhance 0041. In contrast, there was a striking two-fold decline in antibody production by B cells, due to enhanced B cell the density of NA nerves in the Splenic regions distal to the proliferation and differentiation into antibody-Secreting hilus in arthritic rats compared with non-arthritic animals. In cells. both treatment groups, NA nerves distributed to the central 0037. The effect of B-adrenergic receptor stimulation on arterioles, white pulp regions, trabeculae and the capsule. B cell function results from a direct effect of AR agonists on However, NA nerve density in the white pulp was reduced, B cells. Thus, B-adrenergic receptor Stimulation enhances B but was increased in the red pulps in AA rats compared with cell function and that under conditions of enhanced Sympa non-AA rats. These findings indicate an injury/sprouting thetic tone, i.e. StreSS, prior to immune activation, both Th1 response with disease development whereby NA nerves die and B cell function will be diminished. In contrast, if back in distal regions and undergo a compensitory Sprouting Sympathetic outflow is enhanced, NE has a permissive effect response in the hilus. on humoral immunity. 0042. The redistribution of NA nerves from white pulp to 0038. The effect of chemical sympathectomy (SympX) red pulp Suggests these nerves signal activated immune cells with 6-OHDA on cytokine and antibody production after localized to the red pulp in the AA animals. Changes in keyhole limpet hemocyanin (KLH) immunization was adrenergic receptor expression, (decreased f-adrenergic examined in young adult mice with different Th dominance receptors on peripheral blood mononuclear cells and (e.g., BALB/cJ mice with a predominant Th2 cytokine increased C.-adrenergic receptors on monocytes) have been profile, and C57B1/6 mice with a predominant Th1 cytokine reported in RA patients. In the case of C.-adrenergic recep profile). SympX enhanced KLH-stimulated IL-2 and IFNY tor expression in monocytes from arthritic patients, periph production by Splenocytes in vitro, in both murine Strains. eral blood mononuclear cells of healthy donors did not Under the same conditions, IL-4 also is elevated in Spleno express functional O-adrenergic receptors, indicating that cyte cultures with cells from each Strain producing more of disease onset is associated with adrenergic receptor expres its dominant cytokine. Anti-KLH antibody titers for IgG1, Sion not normally expressed by these cells. IgG2a, and IgM were elevated in Serum from immunized, 0043 Stimulation of C.-adrenergic receptors on periph denervated C57B1/6, but not BALB/c mice. This is consis eral blood leukocytes of RA patients increased production of tent with a tonic inhibitory mechanism of immune regulation proinflammatory cytokines. Thus, monocyte C.-adrenergic by the SNS, and differential responsiveness by the two receptor expression is likely to contribute to the disease. Strains. Monocyte C.-adrenergic receptor expression can be induced 0039. The SNS plays a modulatory role to maintain by treatment with an fB-adrenergic receptor agonist, Sug immune System homeostasis, however, few studies have gesting that f2-adrenergic receptor Stimulation can regulate examined whether the SNS is playing a Significant role in monocyte C.-adrenergic receptor expression. Collectively, autoimmune diseases, Such as RA, where immune System this indicates a role for SNS modulation of immune function homeostasis is disrupted. Impaired Sympathetic nervous in RA. System function has been extensively described in RA using 0044) The effects of B-adrenergic agonists on immune cardiovascular tests during orthostatic StreSS, the Valsalva function results from binding of these ligands to B-adrener maneuver, deep breathing, pupil Size and perspiration. These gic receptorS expressed on lymphocytes and macrophageS/ changes are associated with increased urinary metabolites of monocytes to induce activation of GS proteins and elevated NE, Suggesting an increase in Sympathetic outflow. Defec levels of intracellular cAMP Cyclic AMP induces down tive function of SNS in patients with RA may contribute to Stream Signal modifications that dampen immune cell immune derangement and Severity of illness. In an AA rat responses. Elevation of intracellular cAMP activates protein model, 7 days after injection of adjuvant and preceding the kinase A (PKAO, which is followed by interfering with onset of inflammation, corticotrophin releasing hormones activation of Ras proteins). PKA interferes with Raf-1 (CRH) mRNA were increased in the PVN significantly binding to Ras, thus inhibiting downstream Signaling of above controls following immobilization StreSS. In contrast, immune responses. CRH mRNA levels in the PVN were significantly lower than in controls 14 days after adjuvant injection. This indicates a 0045. Additionally, B-adrenergic receptors can switch CRH induced increase in sympathetic outflow in early their coupling from GS proteins to Gi proteins, an effect that disease Stages, followed by a decrease in outflow in late AA is also mediated by PKA. PKA phosphorylation of the Stages. f2-adrenergic receptor leads to f-adrenergic receptor deSensitization, reduction cAMP production in response to 0040. The inventors have documented a a sprouting/ further stimulation, and causes the inhibition of the GS injury response in late disease Stages in lymphoid organs, as mediated adenylyl cyclase signal that Started the process. well as a decrease in NE concentrations in Spleens and LN Coupling of the f-adrenergic receptor to Gi proteins then of AA rats during late stages (FIGS. 5A and 5B). Noradr initiates a Second wave of Gi-mediated Signaling to activate energic (NA) innervation in Spleens of Lewis rats was mitogen activated protein (MAP) kinase pathways and pro examined 28 days following adjuvant treatment to induce mote functional responses initiated by Stimulators of arthritis or vehicle for the adjuvant using fluorescence immune responses. US 2005/0049256 A1 Mar. 3, 2005

0046) The effects of non-specific and specific C.-adrener model of rheumatoid arthritis. In certain embodiments, the gic antagonists on immune function have been Studied less. present invention concerns a method for treating rheumatoid Binding of an O-adrenergic agonist to C2-adrenergic recep arthritis or autoimmune diseases by the application of a tors expressed by immune cells results in activation of Gi therapeutically effective dose of a B-adrenergic agonist, and proteins. Activation of these G proteins results in activation preferably a B-adrenergic agonist Such as terbutaline, of MAP kinases in a Ras-dependent manner that is inde coupled with a therapeutically effective dose of an O.-, C.- or pendent of phospholipase activation or adenylyl cyclase. C2-adrenergic antagonist, and preferably phentolamine, pra Stimulation of the C2-adrenergic receptor induces phospho Zosin or yohimbine, to human Subjects with the disease. rylation of Shc by the Gi Bw subunit. Shc is one of three 0052 AS used herein, the term “treating a disease by the docking proteins, which function as platforms for assembly application of a therapeutically effective dose of a 3-adren of a Ras activation complex. Thus, C-adrenergic receptors ergic agonist' and "a therapeutically effective does of an O.-, can modulate immune responses by impacting MAP kinase C- or B-adrenergic antagonist' is used to Signify that the downstream signaling pathways of immune cells at the B-adrenergic agonist and C.-adrenergic antagonist is Supplied levels of Ras. to the patient in amounts, and for a period of time, that are 0047 Binding of an O-adrenergic agonist to C.-adrener effective to provide improvement in one or more of the gic receptors expressed by immune cells results in activation clinically measured parameters of the disease, particularly of pertussis toxin-insensitive Gq/11 proteins. Activation of disease parameters of cartilage and bone destruction. these G proteins leads to activation of an intracellular signal 0053 Several inflammatory autoimmune diseases are transduction pathway known to Stimulate phospholipase C proposed to be treatable by combined B-adrenergic agonists (PLC) and diacylglycerol (DAG) and inositol triphosphate and C.-adrenergic antagonists of the present invention, Such 3 (IP3) generation. This is followed by IP3 dependent as inflammatory bowel disease, Krohn's disease, fibromy increases in intracellular calcium and activation of protein algia, lupus, chronic fatigue Syndrome, and Type 1 diabetes. kinase C (PKC) by DAG. Activation of PKC results in The efficacy of the disclosed treatment is based on common activation of mitogen-activated protein (MAP) kinase acti mechanisms of immune dysfunction, inflammation targeting Vation. Activation of C.-adrenergic receptors thus can Specific organs in these diseases and the fact that a dying impact immune function by modulating MAP kinase path back of Sympathetic nervous System nerve fiberS Supplying ways known to be involved as intracellular Signal cascade pathways for induction of immune responses. Activation of immune organs have been documented in each of these the C-adrenergic receptor can also impact immune func disorders. tions by stimulation of nuclear factor of activated T cells 0054) To determine whether there has been an improve (NF-AT) transcription factors. ment in one or more of the clinically measured parameters of the disease, one would determine the value of Such a 0.048. At a functional level, drugs acting on adrenergic parameter in a given patient both before and during treat receptors expressed by lymphocytes and monocytes could ment. Various clinical Signs and Symptoms are known by impact rheumatoid arthritis by at least two different mecha those known to be skilled in the art as Suitable markers of nisms, by altering cytokine production or blocking mac disease Severity. Symptoms of inflammatory joint disease rophage functions. AS indicated above, shifting cytokine (compared with mechanical problems) should be assessed to profiles of Th cells towards Th2 cells significantly reduces gain evidence of active disease. These include degree of disease Severity. Similarly, drugs that inhibit macrophage joint pain, duration of morning Stiffness, Severity of fatigue, functions or neutralize TNFC. have a profound beneficial presence of actively inflamed joints on examination, and effect in reducing disease Severity. Stimulation of the Sym limitation of function. pathetic nervous System promotes a shift in Th cell cytokines profiles toward a Th2 response, thus inhibiting cell mediated 0055. However, the joint examination may not immunity. adequately reflect disease activity and Structural joint dam age. Periodic measurements of erythrocyte Sedimentation 0049. In addition, activation of macrophage B-adrenergic rate or C-reactive protein elevation, functional status (loss of receptors inhibits macrophage functions. However, interac motion, instability, malalignment, and/or deformity of tion of norepinephrine with macrophage/monocyte C.-adr affected joints) and radiographic examinations of involved energic receptors can Stimulate production of TNFC, a key joints should be performed. Functional Status assessment cytokine in promoting inflammation and bone destruction in can be performed with questionnaires known in the art Such RA. Presence of an O-adrenergic antagonist would block as the Arthritis Impact Measurement Scales or the Health this macrophage response. ASSessment Questionnaire. 0050. Further, NA innervation of secondary immune 0056. The present invention has the advantage of being a organs is lost as a part of the disease pathology. This results novel application and use of agents that are already in use in a loSS of a negative feedback System required for damp clinically in the treatment of various other disorders and ening immune and inflammatory responses during the acute ailments. The adrenergic agents of the present invention are and chronic disease Stages. This feedback System normally Safer and have fewer Side effects than drugs currently being is responsible for maintaining homeostasis of the immune used to treat rheumatoid arthritis and the other autoimmune System and may be critical for returning immune System diseases listed. Accordingly, Some f-adrenergic agonists functions back to normal levels in RA. considered to be of use in the present invention include 0051. Therefore, the present invention discloses -adren metaproterenol, albuterol, isoetharine, pirbuterol, bitoltrol, ergic agonists in combination with C.-adrenergic antagonists ritodrine, or Salbutamol, and preferably, terbutaline. that are useful in treating rheumatoid arthritis, as these drugs 0057. Some of the C.-adrenergic antagonists considered to are effective in treating adjuvant-induced arthritis, an animal be of use in the present invention include yohimbine, US 2005/0049256 A1 Mar. 3, 2005 regitine, prazosin, doxazosin, tamsulosin, teraZosin, octo occur in a patient with pheochromocytoma as a result of pamine, phenoxybenzamine, phentolamine, hydrochlorothi StreSS or manipulation during preoperative preparation and azide, 5-methyl urapidil, chloroethylclonidine, bunaZoSin, Surgical excision. Its use has been indicated for prevention alfuzosin, RS17053, BMY 7378, urapidil, L-765,314, nicer and treatment of dermal necrosis and Sloughing following goline, ABT-866, cyclazosin, A322312, A 119637, fidux IV administration or extravasation of norepinephrine or oSin, JTH-601, imiloxan, 2 idopropoxyidaZOXan, 2-meth dopamine as well. Phentolamine has been used to treat oxyidazoxan (RX 821002), idazoxan, piperoxan, BRL hypertensive crises secondary to MAO inhibitor/sympatho 44408, beditin, atipamezole, rawolscine, ARC 239, mimetic amine interactions and rebound hypertension on RS-79948, MK912, RS 79948, UIC 14304 and ethoxyida withdrawal of clonidine, propranolol or other antihyperten ZOX. Sives. It has also been used in combination with papaverine 0.058. The B-agonists and C-antagonists may be admin as an intracavemous injection for impotence. Alpha-adren istered to the patient in any pharmaceutically acceptable ergic antagonists are used for the treatment of hypertension, vehicle and by any route heretofore acceptable for these benign prostatic hyperplasia, refractory chronic heart failure agents. The preferred route of administration is orally, and management of raynaud's vasospasm. However, no although one may, if desired, choose to administer the adequate information exists on the use of C.-adrenergic agonists or antagonists intravenously, Sublingually, intra antagonists in arthritic patients. muscularly, Subcutaneously, or in a Sustained release form. 0063) To date, there has been no disclosure as to the 0059) As will be understood by those skilled in the art, combined use of a B-adrenergic agonist and a C-adrenergic the effective doses of the 13-agonist and C.-antagonist will antagonist (regardless of the receptor Subtype) in the treat depend upon the route of administration and the patient's ment of rheumatoid arthritis or inflammatory autoimmune Sensitivity to the particular f3- (or f3-) adrenergic agonist and diseases. The present invention discloses that a combination C.- (or C- or Cl-) adrenergic antagonist. Recommended of both these drugs is effective in the treatment of Such doses for both the B-adrenergic agonist and C.-adrenergic disorders, and particularly, in the treatment of rheumatoid antagonist, respectively, range from about 1.0 to 10.0 mg, arthritis. Other B-adrenergic agonists useful in this novel with a preferred range of about 2.0 to 5.0 mg, or even more method of treatment include: metaproterenol, albuterol, iso preferably about 1.25 to 2.5 mg given three times per day etharine, pributerol, bitolterol, ritodrine, and Salmeterol. depending upon disease Severity and patient responses to the Other C.-adrenergic antagonists useful in this novel method drugs. The dosages may be more effectively adjusted on an of treatment include: yohimbine, regitine, prazosin, dox individual basis as the disease Severity varies from patient to aZosin, tamsulosin, teraZosin, octopamine, phenoxyben patient. Zamine, phentolamine, hydrochlorothiazide, 5-methyl ura 0060. This invention comprises a novel method for the pidil, chloroethylclonidine, bunazosin, alfuzosin, RS17053, treatment of patients diagnosed as having a rheumatoid BMY 7378, urapidil, L-765,314, nicergoline, ABT-866, arthritis or inflammatory autoimmune disease with a com cyclazosin, A322312, A 119637, fiduxosin, JTH-601, imi bined use of a f-adrenergic agonist (preferably a C2-adren loxan, 2 idopropoxyidazoxan, 2-methoxyidazoxan (RX ergic agonist) and an O-adrenergic antagonist (preferably an 821002), idazoxan, piperoxan, BRL 44408, beditin, atipam C- or C2-adrenergic antagonist). This method comprises the eZole, rawolscine, ARC 239, RS-79948, MK912, RS 79948, administration of an effective dose of a B-adrenergic agonist UIC 14304 and ethoxyidazoxan. (preferably a f-adrenergic agonist) and an O.-adrenergic 0064. The f-adrenergic agonists and C.-adrenergic antagonist (preferably a C- or C2-adrenergic antagonist) to antagonists of the present invention may be administered to patients diagnosed as having rheumatoid arthritis or other a patient in a dosage form Selected from the group consisting inflammatory autoimmune disease. Even more particularly, of pills, tablets, capsules, caplets, Solutions, Suspensions, the method of the present invention comprises the admin Syrups, Suppositories, and aerosols. Additionally, the dosage istration of an effective dose of the B-adrenergic agonist of the ?- (or f32-) and C- (or C. - or C2-) adrenergic agonist terbutaline coupled with and effective dose of the O-adren and antagonist, respectively, used may be in a Sustained ergic antagonist, phentolamine (or more specifically C- or release form to cause the action of Such agonists and C2-adrenergic antagonists) to patients with rheumatoid antagonists to persist over a more prolonged period of time. arthritis or other inflammatory autoimmune disease. Such Sustained-release formulations are well known to those 0061 Terbutaline is known to be an agent of use as a skilled in the art. bronchodilator for treating or controlling acute or chronic bronchial asthma, exercise-induced bronchoSpasm, bronchi 0065. The B-adrenergic agonists may be administered in tis, emphysema, bronchiectasis or other obstructive pulmo various Salt forms. For example, the following are commer nary diseases, as well as a uterine relaxant in premature cially available: metaproterenol sulfate as “Alupent” (Boe labor. Treatment with B- or B-adrenergic agonists reduce hringer Ingelheim) or “Metaprel” (Dorsey); terbutaline sul disease Severity of demyelinating autoimmune diseases, fate as “brethaire' or Brethine” (Ciba-Geigy) or “Bricanyl” including multiple Sclerosis, myasthenia gravis, demyelinat (Merrell-Dow); albuterol sulfate as “Proventil” (Schering ing polyradiculoneuropathy, experimental allergic neuritis, Plough) or “Ventolin” (Glaxo); isoetharine hydrochloride as “Bronkosol” (Sterling) (Parke-Davis) or isoetharine mesy and post-infectious encephalomyelitis. For example, Salb late as “Bronkometer” (Sterling); pributerol acetate as utamol, a B-adrenergic agonist, reduces the Severity of “Max-air”; bitolterol mesylate as “Tomalate” (Sterling); or collagen-induced arthritis, an animal model of rheumatoid ritodrine hydrochloride as “Pre-Par” (Philips-Duphar) or arthritis. “Yutopar” (Astra); levalbuterol HCl as “Xopenex (Sepra 0.062 Phentolamine is known to be an agent of use in cor); Salmeterol as "Serevent” (GlaxoWellcome) or “Ser prevention or control of hypertensive episodes that may event Diskus” (GlaxoWellcome). US 2005/0049256 A1 Mar. 3, 2005

0.066 The C.-adrenergic antagonists also may be admin matory mediators important in disease progression. The istered in various Salt forms as well. For example, the present disclosure demonstrates that early in the disease following are commercially available: Phentolamine mesy process there is an increase in Sympathetic outflow that late (Bedford); Regitine mesylate (Ciba); prasozin as “Pra results in a large increase in release of norepinephrine from sozin” (Geneva, Goldline, Lederle, Major, Moor, Rugby, nerves targeting immune cells. Schein, Squibb, Zenith) or “Minipress” (Pfizer); terazosin as 0072 Stimulation of B-adrenergic receptors by this adr “Hytrin” (Abbott); doxazosin mesylate as “Cardura energic agonist is known to Stimulate expression of C.-adr (Roerig); and tamsulosin hydrochloride as “Flomax” (Boe energic receptor expression by monocytes/macrophages. hiringer Ingleheim). Activation of C.-adrenergic receptors by circulating norepi 0067. The dosage form of the f-adrenergic agonist and nephrine or epinephrine, or following B-agonist drug treat the C-adrenergic antagonists may be administered by Vari ments, promoteS production of proinflammatory mediators ous routes including Sublingual, oral, intravenous, rectal, by these macrophages, including tumor necrosis factor C. parenteral or Subcutaneous. Therapeutically effective doses The addition of the O.-adrenergic antagonist in this invention of particular agonists or antagonists and the frequency of is proposed to block the induction of proinflammatory dosage administration are to be determined according to mediators by activated macrophages. This is Supported by protocols understood by those skilled in the art. In one reports that C.-adrenergic receptors are expressed on mono embodiment of the present invention method, therapeuti cytes from arthritic patients but not expressed on monocytes cally effective dosages of terbutaline from about 1.0 to 10.0 from healthy donors, indicating that disease onset is asso mg coupled with therapeutically effective dosages of phen ciated with O-adrenergic receptor expression not normally tolamine from about 1.0 to 10.0 mg are administered two or expressed by these cells. more times per day. 0073 Stimulation of C.-adrenergic receptors on periph 0068 The treatment may also comprise administering a eral blood monocytes of rheumatoid arthritis patients therapeutically effective dose of B-adrengeric agonist fol increased production of proinflammatory cytokines. Further, lowed administering a therapeutically effective dose of an monocyte C.-adrenergic receptor expression can be induced C.-adrenergic antagonist, or alternatively, administration of a by treatment with a B-adrenergic agonist, Suggesting that therapeutically effective dose of C.-adrengeric antagonist B-adrenergic receptor Stimulation can regulate monocyte followed by a therapeutically effective dose of B-adrenergic C.-adrenergic receptor expression. agonist. Administration of the B-adrenergic agonist and 0074. In this manner, B-adrenergic agonists coupled C.-adrenergic antagonist can occur within a 24-hour period, with C.-adrenergic antagonists are believed to be likely to a 12-hour period, or preferably an 8-hour period, more provide a similar Suppressive effect on the excessive preferably an 4-hour period, or most preferably within a one immune responses found in other inflammatory autoimmune hour period of each other. diseases. By inhibiting cell mediated immunity through 0069. The exact mechanism by which the B- (or B-) shifting T helper lymphocyte production of cytokines that adrenergic agonist and C- (or C- or Y2-) adrenergic agonists promote humoral immunity, in the case of the B-adrenergic and antagonists, respectively, exert a Suppressive effect on agonist, and inhibiting production of proinflammatory disease Severity of rheumatoid arthritis or inflammatory mediators by macrophages, in the case of the C-adrenergic autoimmune diseases (e.g., inflammatory bowel disease, antagonists, these agonists and antagonists are believed to be Type 1 diabetes, lupus, and Krohn's disease) is unknown. likely to provide a similar Suppressive effect on the exces However, this present disclosure shows that there is an Sive cell mediated immune responses found in other inflam increase in Sympathetic outflow early in the disease that later matory autoimmune diseases. Thus, the f-adrenergic ago results in a loSS of Sympathetic nerve fibers that normally nists and C-adrenergic antagonists of the present treatment Signal immune cells in Secondary lymphoid tissue. These method may be useful in treating other inflammatory nerve fibers are necessary for maintaining immune System autoimmune diseases, Such as inflammatory bowel disease. homeostasis. 0075. The following example is included to demonstrate 0070 Beta-agonists replace the function of the lost sym preferred embodiments of the invention. It should be appre pathetic nerve fibers that occurs as the disease progresses. ciated by those of skill in the art that the techniques Stimulation of the Sympathetic nervous System is known to disclosed in the example which follow represent techniques promote or inhibit production of Specific cytokines by discovered by the inventor to function well in the practice of macrophages and/or T helper lymphocytes that shift the the invention, and thus can be considered to constitute immune response towards humoral immunity. When this preferred modes for its practice. However, those of skill in innervation to Secondary lymphoid immune organs is lost, the art should, in light of the present disclosure, appreciate the effect is to promote or inhibit production of Specific that many changes can be made in the Specific embodiments cytokine by macrophages and/or T helper lymphocytes that which are disclosed and still obtain a like or similar result shift the immune response towards cell mediated immunity, without departing from the Spirit and Scope of the invention. and in arthritic patients, increased disease Severity. The B-adrenergic agonist would be expected to shift cytokine EXAMPLE 1. production by immune cells toward a profile that favors 0076 Combined B-Adrenergic Agonist (Terbutaline) humoral immunity. and C-Adrenergic Antagonist Suppression of Adjuvant-In duced Arthritis in Rats 0071. The function of the C.-adrenergic antagonist is to block Stimulation of activated macrophage C.-adrenergic 0077. 1. Method receptors by circulating catecholamines, either norepineph 0078 Lewis rats with AA, a model for RA, were used to rine or epinephrine, that promote production of proinflam examine the combined action of the B-adrenergic receptor US 2005/0049256 A1 Mar. 3, 2005 agonist terbutaline and the non-Specific C-AR antagonist bone); (3) cartilage loss shown by narrowing of the joint phentolamine. AA was induced in adult male Lewis rats Spaces, and (4) heterotopic ossification defined as prolifera (200-225 gm) by a base of the tail intradermal injection of tion of new bone tissue (fine ossified line paralleling normal complete Freund's adjuvant (CFA). The CFA (0.03 g dried bone but not contiguous with calcified area of the bone and heat killed Mycobacterium butyricum (Difico, Detroit, itself). Mich.) emulsified in 10 ml sterile mineral oil) was prepared 0084. The radiographic scores for each category were by grinding the M. butyricum with a mortar and pestle until added for both hind limbs giving a maximum score of 40, the bacterial cell wall turned from a light beige to an and the individual Scores for each animal were then aver eggshell white powder. aged within the treatment groupS and expressed as a meanistandard error of the mean (SEM), and subjected to 007.9 The mineral oil was then slowly worked into the Kruskal-Wallis Statistical analysis (non-parametric statistic bacterial cell wall using the mortar and pestle. The Suspen equivalent to an ANOVA; p<0.05) followed by Dunn post Sion was treated with a Sonic dismembraner for 5 minutes to hoc testing. This experiment was replicated twice. Protocols ensure that the bacterial cell wall remained Suspended in the for the use and care of the animals in the Study were mineral oil for animal injections. While there was variability approved prior to beginning the experiments by the Sun in the Severity of disease development between the prepa Health Research Institute Animal Use and Care Committee rations of adjuvant, there was very little variability within and complied with NIH guidelines for the humane use and individual batches. All animals in each experiment were care of research animals. challenged with the same preparation of adjuvant and 100% of the animals developed arthritis. 0085 2. Results 0086 FIG. 2 illustrates that continuous treatment of 0080 Phentolamine (an O-adrenergic receptor antago adrenergic drug therapies from disease onset affected dor nist) and terbutaline (a f-adrenergic receptor agonist) were soplantar footpad widths in arthritic rats. All CFA-chal obtained from Sigma Chemical Company (St. Louis, Mo.). lenged rats developed AAbetween days 10-12 (FIG.2). Soft Both adrenergic drugs were dissolved in 0.01 mM ascorbic tissue SWelling was significantly decreased interbutaline- or acid in 0.9%. Sterile, endotoxin-free Saline. Adrenergic phentolamine-treated animals compared with the vehicle therapy with phentolamine and/or terbutaline or vehicle was treated AA rats by day 28 post-adjuvant injection (Terbuta Started on day 12 post-immunization, the time of disease line: Fozo–27.61, Day 28:ti-5.998, P-0.001) (Phentola onset, and continued until Sacrifice. The dose of phentola mine: Fo-30.50, ten-3.594, P-0.01) (FIG. 2). mine (500 ug/Kg/day) and/or terbutaline (600 ug/day) were Combination phentolamine and terbutaline treatment also administered by intraperitoneal (i.p.) injections twice a day Significantly reduced the Soft tissue SWelling in the hind in a total volume of 500 ul per injection. limbs by day 23, an effect which was maintained through day 28 compared with the vehicle-treated arthritic rats 0081. The inflammatory response in the arthritic rats was (PhenTerb: Fozo-30.04, Day 23: tries-3.363, P-0.05; assessed by routine methods previously described. Dor Day 25: te=3.557, P-0.01; Day 28: t—5.300, Soplantar width of the hind feet were measured using a P<0.001). These findings were replicated twice with similar Mitutoyo Corporation dial thickness gauge, beginning the results. day of CFA-immunization and continued approximately 0087 While inflammation was decreased in these treat every other day until sacrifice. The right and left footpads ment groups, we observed a greater effect for these drugs in from each animal were averaged together. The individual prevention of bone and cartilage destruction than on joint means for each animal were then averaged within each Swelling. FIGS. 1A-1E show that radiographic analysis of group and Subjected to a repeated measure two-way analysis the ankle joints revealed that vehicle-treated arthritic rats of variance (ANOVA; P-0.05) with Bonferroni post-hoc had visible Soft tissue SWelling, bone loSS, perioSteal bone testing. The animals were Sacrificed when Significant differ formation, narrowing of their joint Spaces, and a decreased ences between the dorsoplantar footpads of the groups bone density by day 28 (FIG. 1A-F). Radiographic scores became apparent in the late effector phase, 28 days post were significantly reduced with all adrenergic treatments adjuvant injection. compared with the vehicle controls (H=13.74, df=3: Phen tolamine (P<0.05), Terbutaline (P<0.05), or SH1293 0082 Prior to sacrifice the animals were anesthetized (P<0.01)) (FIG. 1F). Lower radiographic scores in phento with a 1.0 ml i.p.-injection of 8% chloral hydrate in sterile lamine or terbutaline treatment groups reflected a reduction Saline and radiographs were taken of their hind limbs to in the amount of Soft tissue SWelling, bone loSS, and joint assess disease Severity. Radiographs were taken using the Space narrowing. Terbutaline or phentolamine treatment following settings: 400 nN, 50 kVp, 0.4 second exposure resulted in an ~50% or 51% decrease in bone loss, respec time, at 40 centimeters and X-OMAT processor. X-rays were tively, compared to vehicle-treated AA rats. However, the evaluated using a grading Scale modified from Ackerman most dramatic effects on bone and cartilage loSS were and coworkers. observed in the combination phentolamine and terbutaline treatment, as they demonstrated an ~60% reduction in 0.083. In short, the radiographs were coded to obscure the radiographic Scores compared to vehicle-treated AA ani treatment groups, and then two independent observerS Sub mals. Although the differences in radiographic Scores com jectively rated each of the radiographs on the Scale: 0 pared between the adrenergic drug treatments were not (normal), 1 (slight), 2 (mild), 3 (moderate), and 4 (severe) Significant, this trend for the combination of phentolamine abnormalities in the tissue. The radiographs were Scored for and terbutaline to be more effective in preventing bone loSS each of the following characteristics: (1) Swelling as indi has been consistent in repeat experiments (FIG. 1F). These cated by the width of soft tissue shadows and alterations in dramatic bone sparing effects following the adrenergic drug the normal configuration of the Soft tissue planes; (2) treatments are illustrated in the radiographs (FIG. 1A-E). An osteoporosis as measured by bone density (recognized by X-ray of a normal non-arthritic joint (FIG. 1A) is included increases in radiolucency relative to uninvolved adjacent for comparison. US 2005/0049256 A1 Mar. 3, 2005

EXAMPLE 2 HBSS, centrifuged and re-suspended into complete RPMI 0088 Combined B-Adrenergic Agonist (Terbutaline) 1640 media supplemented with 5% fetal calf serum and 1% and O-Adrenergic Antagonist (Phentolamine) Treatment antibiotic/antimycotic. Promotes an Anti-inflammatory Cytokine Profile in the 0095 The prepared cells were counted using a hemocy Secondary Immune Organs and Peripheral Blood Mono tometer, then brought to the concentration of 2x106 cells/ nuclear Cells. ml. Two ml of the cell preparation were plated into 24 well plates (Falcon, Oxnard, Calif.), and in placed in an incubator 0089) 1. Method 7% CO., 37 C., for 24 h. After 24 h the culture Supernatants 0090. To determine if the adrenergic drug treatments were harvested and placed in the freezer at -70° C. until were able to modify cytokine patterns in the Secondary ELISA assay. lymphoid organs and Systemically in the blood, pro- and 0096 Optia kits for the detection of IL-10 and TNFC. anti-inflammatory cytokine profiles were obtained ex vivo were obtained from BD Pharmingen (Los Angeles, Calif.) from cells harvested from the draining (inguinal and popli and Sandwich ELISAS were used to measure the amount tial) lymph nodes, spleen and the PBMC from the animals cytokine released into the culture media. In brief: High described above. binding microtiter 96-well plates were pre-coated with 100 0.091 All tissue culture media and supplements were ul of capture antibody in coating buffer (0.1 M carbonate obtained from Gibco BRL (Rockville, Md.) unless otherwise buffer, pH 9.5) for IL-2, IL-4, IFNY and TNFC. and (0.2 M Stated. OPTIA Sandwich ELISA kits for IL-10 and TNFC. phosphate buffer, pH 6.5) for IL-10. The plates were sealed were purchased from Pharmingen (San Diego, Calif.). with plate film (Denville Scientific, South Plainfield, N.J.) and placed at 4 C. overnight. The plates were allowed to 0092. The spleen and lymph nodes were aseptically come to room temperature and washed with 0.1 M phos removed from the animal and placed into Hank's balanced phate buffered saline-0.5% Tween 20 (PBS-Tw20). salt solution (HBSS). The spleens were placed in a stoma cher bag and homogenized for 30 Seconds. Spleen cells were 0097. The non-specific binding was blocked using PBS triturated with a 10 ml pipette then washed with an addi Tw20 and 1% bovine serum albumin. Standard/sample was tional 10 ml of HBSS and passed through a nylon mesh then added to each well, the plates were Sealed and incu (Marsh Industries) to remove the extraneous connective bated overnight at 4 C. The plate was washed three times tissue. The collected cells were centrifuged and re-SuS with PBS-Tw20 and biotinylated secondary detection anti pended in 5 ml of a NHCl hypotonic buffer for 3 minto lyse body and Streptavidin/avidin enzyme conjugate (diluted in the red blood cells. The cells were washed 2x with 10 ml PBS-1% bovine serum albumin) were added to the wells and HBSS, centrifuged and re-suspended into complete RPMI the plates incubated for 1.5 h. The plates were then washed 1640 media supplemented with 5% fetal calf serum and 1% and developed with (TMB) reagent (Pharmingen, San antibiotic/antimycotic. Diego, Calif.) for 30 minutes. 0093. The draining (inguinal and poplitial) lymph nodes 0098. After color development the plates were stopped were placed into sterile etri dishes containing 5 ml HBSS. with addition of INSulfuric acid. Unknown sample cytokine The lymph nodes were teased apart using forcepS and levels were determined through comparison with a Standard triturated with a pipette. The homogenates then were passed curve present on each plate using an ELISA reader (Ceres through a nylon mesh, centrifuged and re-Suspended in 5 ml 900 HDI: BioTek Instruments Incorporated, Winooski, Vt.). HBSS. The cells then were centrifuged and re-suspended The concentrations were averaged for each treatment group at each time point, expressed as meantSEM in ng/ml, and into complete RPMI 1640 media Supplemented with 5% subjected to a one-way ANOVA (p<0.05) with Newman fetal calf serum and 1% antibiotic/antimycotic. Keuls post-hoc testing. 0094 PBMCs: Blood was collected using cardiac punc ture into a 7 ml lithium heparin vaccutainer tubes. The tubes 0099 Differences in cytokine production levels between were inverted 7 times and then spun down at 1000 rpm for treatment groups were determined by a one-way analysis of 15 minutes at 10 C. The buffy coat was removed using a 2 variance (ANOVA) for multiple groups. Means found to be ml pipette and placed into a Sterile 15 ml tube containing 10 significantly different were subjected to a Bonferroni Mul ml of a NHCl hypotonic buffer for 3 min to lyse the red tiple Comparison Test (t values are reported with the level of blood cells. The peripheral blood mononuclear cells Significance) post-hoc analysis to determine the Source of (PBMC) were centrifuged and re-suspended in 10 ml HBSS. the variance. The PBMC were washed three more times with 10 ml 01.00) 2. Results

Ratios of IL-10/TNFC. Following Adrenergic Drug Treatments

Vehicle Phentolamine Phen?Terb Terbutaline

PBL 0.2161 - 0.0315 O.8067 0.1762 1.2110 - 0.0446. O.4382 O.0471 DLN O.9642 - O.1980 1.096O. O.1961 0.8854 - 0.2122 1.5060 - 0.1642 SPL O4O90 O.O719 3.3350 0.6716* 5.0490 + 1.06906 2.1370 + 0.8285

Statistical analysis: One-way ANOVA with Bonferroni Multiple Comparison Test. N = 6, P < 0.001 compared with vehicle treated animals. US 2005/0049256 A1 Mar. 3, 2005

0101 These data demonstrate that following phentola 28: tries-3.312, P-0.05) (Prazosin (Terbutaline: Fis = mine treatment alone and in combination with terbutaline 88.42, Day 20: tra=3.403, P-0.05; Day 22: tra= treatment there is an increase in the ratio of anti- to pro 4.312, P-0.001; Day 24: t-5.747, P-0.001; Day 26: inflammatory cytokine production by macrophage cells in tra-5.297, P-0.001; Day 28: tri-6.353, the Secondary lymphoid organs and peripheral blood. These P<0.001). These findings were replicated twice with similar data demonstrate that adrenergic drug treatment reduces the results. amount of inflammatory mediators and provide a potential mechanism through which these drugs could be having their 0.108 b. Radiographic Scores from Arthritic Rats Started beneficial effects. on Adrenergic Drugs. On Day 12 0109 As shown in FIG. 4A-E, radiographic analysis of EXAMPLE 3 the ankle joints revealed that vehicle-treated arthritic rats had visible Soft tissue SWelling, bone loSS, perioSteal bone 0102 Combined B-Adrenergic Agonist (Terbutaline) formation, narrowing of their joint Spaces, and a decreased and the Non-Specific C.-Adrenergic Antagonist(Phentola bone density by day 28. Radiographic Scores were signifi mine), the Specific C1-Adrenergic Receptor Antagonist cantly reduced with these adrenergic treatments compared (prazosin) or the Specific O-Adrenergic Receptor Antago with the vehicle controls (H=17.78, df=3: combined phen nist (Yohimbine) Suppression of Adjuvant-Induced Arthritis tolamine and terbutaline (P<0.05), combined prazosin and in Rats 1. Method terbutaline (P<0.001). Lower radiographic scores in phen 0103 Lewis rats with AA were used to determine if the tolamine/terbutaline and praZosine/terbutaline treatment anti-inflammatory and bone sparing properties of the com groups reflected a reduction in the amount of Soft tissue bination treatment described above were being mediated by Swelling, bone loSS, perioSteal bone formation and joint blocking the C- and/or C-AR Subtypes. AA was induced in space narrowing (FIG. 4F). These dramatic bone sparing adult male Lewis rats as described above. Phentolamine (a effects following the adrenergic drug treatments are illus non-specific C-AR antagonist), prazosine (an C-AR antago trated in the radiographs in FIG. 4A-E. nist), yohimbine (an C-AR antagonist) and terbutaline (a f2-AR agonist) were obtained from Sigma Chemical Com EXAMPLE 4 pany (St. Louis, Mo.). All of the adrenergic drugs were dissolved in 0.01 mM ascorbic acid in 0.9% sterile, endot 0110. There is an Noradrenergic Nerve Injury/Sprouting oxin-free Saline. Adrenergic therapy with phentolamine and/ Response which Occurs in the Spleen of AA Rats with or terbutaline, praZoSine and/or terbutaline, yohimbine and/ Disease Development or terbutaline or vehicle was started on day 12 post 0111) 1. Methods immunization, the time of disease onset, and continued until Sacrifice. The dose of phentolamine (500 lig/day), prazosine 0112 Arthritis was induced in Lewis Rats using a single (2.5 mg/day), yohimbine (750 ug/day) and/or terbutaline base of the tail interdermal injection of complete Fruends (600 ug/day) were administered by i.p. injections twice a adjuvant as described above. day in a total volume of 500 ul per injection. 0113 a. Glyoxylic Acid Method of Histofluorescence for 0104. The inflammatory response in the arthritic rats was Catecholamines assessed and analyzed as described above. The animals were 0114. On day 28 post adjuvant injection the rats were Sacrificed when Significant differences between the dor given an anesthetic overdose of 8% chlorohydrate prior to Soplantar footpads of the groups became apparent in the late removal of their spleen tissue. Spleens from arthritic and effector phase, 28 days post-adjuvant injection. Radio non-arthritic rats were rapidly isolated and the blood vessels graphic analysis was also completed and analyzed as entering the Spleen Visualized. The Spleen and blood vessels described previously. This experiment was replicated twice. were dissected. NA nerves enter the Spleen in association Protocols for the use and care of the animals in the study with the blood vasculature (hilar regions). To obtain hilar were approved prior to beginning the experiments by the regions, parallel cross-sectional cuts were made on either Sun Health Research Institute Animal Use and Care Com Side of these blood vessels. Distal regions were taken mittee and complied with NIH guidelines for the humane between these blood vessels. Hilar and distal cross-sectional use and care of research animals. blocks were quickly frozen on dry ice and stored at -70° C. 01.05) 2. Results until further processing. 0106 a. Footpad Measurements from Arthritic Rats 0115 Fresh frozen sections from hilar and distal pieces of Started on Adrenergic Drugs on Day 12 spleen were cut at a thickness of 16 um on a cryostat at -20 C. Sections were melted onto gelatin coated Slides and 0107 Continuous treatment of combination adrenergic prepared for histofluorescence for localization of the cat drug therapies from disease onset affected dorsoplantar echolamines using a modification of the glyoxylic acid footpad widths in arthritic rats. All CFA-challenged rats condensation method (SPG). Three sections were mounted developed AAbetween days 10-12. As shown in FIG. 3, soft on each slide and immediately dipped into glyoxylic acid. tissue SWelling was Significantly decreased in yohimbine/ Dipped Sections were air dried under a stream of cold air terbutaline-, phentolamine/terbutaline- and praZosine/terb using a blow dryer for 15 minutes. Spleen Sections were utaline-treated animals compared with the vehicle-treated covered with several drops of mineral oil and the slide AA rats by day 28 post-adjuvant injection (Yohimbine/ placed on a copper plate in the oven at 95 C. for 2.5 terbutaline: Fis =17.21, Day 28:threat-3.353, P<0.05) minutes. Sections were examined and photographed on a (Phentolamine/Terbutaline: Fis =21.05, Day 24: the Zeiss fluorescence microScope equipped with epi-illumina Terb=3.330, P-0.05; Day 26: tie-2.975, P-0.05; Day tion accessories. US 2005/0049256 A1 Mar. 3, 2005

0116 b. Morphometric Analysis nerve fibers into the red pulp regions of the arthritic animals 0117 For quantification NA fiber density in the spleens compared to the non-arthritic animals (FIG. 5B). from AA and non-AA rats, one white pulp and one red pulp 0123 These data Support an injury response is taking region from three different Spleen Sections form the hilar and place in the white pulp region followed by a Subsequent distal region from 4 animals per groups was randomly Sprouting response of these NA fibers into the red pulp Selected. In the hilar Sections, the white pulp and red pulp regions. This nerve remodeling could have dramatic func region was Selected from an area 0.25 mm on either side of tional consequences, as the nerves are now potentially the apex of the Spleen Section. The distal Sections, the white Signaling immune cell populations which are at different pulp and red pulp areas chosen for assessment were taken activation States, thereby, could express different ratios of within 1 mm from either base point of the triangular spleen adrenergic receptor Subtypes. Sections. Red pulp areas used for assessment entirely filled 0.124 While the invention has been described with ref the photographic frame. White pulps in the hilar and distal erence to a particular embodiment, it will be understood by regions had fairly uniform diameters of their central arteri those skilled in the art that various changes may be made and oles. equivalents may be substituted for elements thereof without 0118 Central arteriole diameters ranged from 140 to 250 departing from the Scope of the invention. In addition, many lum in hilar sections and 70 to 130 um in distal sections in modifications may be made to adapt to a particular situation white pulp areas used to determine the volume density of NA or material to the teachings of the invention without depart fibers. The mean diameter for white pulp central arterioles in ing from the essential Scope thereof. Therefore, it is intended hilar Sections for non-arthritic and arthritic rats was that the invention not be limited to the particular embodi 182.02+6.43 um and 178.06+3.10 uM, respectively (means ment disclosed as best mode contemplated for carrying out expressed as MEAN standard error of the mean). In distal this invention, but that the invention will include all embodi Sections, the mean diameter of the central arterioles for the ments falling within the Scope of the appended claims. non-arthritic and arthritic rats was 95.59+5.18 um and 94.22+0.85 um, respectively. An attempt was made to select We claim: white pulps, to be used in assessing NA terminal Volume 1. A method for treating inflammatory autoimmune dis densities between treatment groups, So that the cut acroSS the ease in an animal comprising: central artheriole was in cross-section. administering a therapeutically effective dose of a com 0119) The volume density represents the volume of NA pound comprising an O-adrenergic antagonist, and a nerve terminals contained within the unit Volume of one B-adrenergic agonist, wherein inflammation in the ani photographic frame expressed as a percentage, using the mal is Suppressed. general formula for volume density: Vva,c=Pa/Pcx100, 2. The method in claim 1, wherein the inflammation is where Pe is the total volume of the tissue within the decreased. photographic frame (c) and Pa is the volume of NA nerve 3. The method in claim 1, wherein the C-adrenergic terminals (a) within the tissue area of the photographic antagonist is an O-adrenergic antagonist. frame. All fluorescent profiles in a Single white pulp or red 4. The method in claim 1, wherein the C-adrenergic pulp area from hilar and distal Sections were photographed antagonist is an C2-adrenergic antagonist. at the same magnification (50x; Selected Such that a single 5. The method in claim 1, wherein the C-adrenergic white pulp filled the frame) as a single 35 mm slide. antagonist is Selected from the group consisting of yohim 0120 Slides were projected onto a grid, 10x10 squares bine, regitine, praZoSin, doxazosin, tamsulosin, teraZosin, per inch using an AuS Jena Viewing Scope Set to magnify the Octopamine, phenoxybenzamine, phentolamine, hydrochlo Section 13x. The Size of the grid was based on the assump rothiazide, 5-methyl urapidil, chloroethylclonidine, tion that one NA varicosity would fill the area of a single bunazosin, alfuzosin, RS17053, BMY 7378, urapidil, L-765, Square of the grid. The lines of the interSections on the grid 314, nicergoline, ABT-866, cyclazosin, A322312, A 119637, in which the NA fluorescent nerve profiles projected were fiduXosin, JTH-601, imiloxan, 2 idopropoxyidaZOXan, counted. Points of intersection from all of the slides from 2-methoxyidazoxan, RX 821002, idazoxan, piperoxan, BRL each Section of Spleen were totaled. The percent volume 44408, beditin, atipamezole, rawolscine, ARC 239, density of the NA profiles per spleen was calculated based RS-79948, MK912, RS 79948, UIC 14304 and ethoxyida upon the actual magnification of the profiles when projected ZOX. onto the Screen, the Size of the grid, and the Volume density 6. The method in claim 1, wherein the B-adrenergic of each white or red pulp area taken from each Section. The agonist is a f-adrenergic agonist. differences in volume density of NA fibers among the two 7. The method in claim 1, wherein the B-adrenergic groups were analyzed by using a Student's t-Test (P<0.05). agonist is Selected from the group consisting of turbutaline, metaproternol, albutuol, isoetharine, pirbuterol, bitholtrol, 0121 2. Results ritodrine, and Salbutamol. 0122) The volume density of the NA nerves in the white 8. The method in claim 1, wherein the therapeutically pulp regions from the hilar spleen Sections was increased in effective dose of the compound comprises 1.0 to 10.0 mg of the arthritic rats compared to the non-arthritic rats (FIG. C.-adrenergic antagonist and 1.0 to 10.0 mg of B-adrenergic 5A). In contrast, in the distal regions of the Spleen, there was agonist. a reduction in the volume of NA nerves from the arthritic 9. The method in claim 1, wherein the therapeutically rats compared with the non-arthritic animals (FIG. 5A). effective dose of the compound comprises 2.0 to 5.0 mg of Additionally, in the red pulp regions from Spleen Sections C.-adrenergic antagonist and 2.0 to 5.0 mg of B-adrenergic from the hilar and distal regions there was a Sprouting of agonist. US 2005/0049256 A1 Mar. 3, 2005

10. The method in claim 1, wherein the therapeutically Octopamine, phenoxybenzamine, phentolamine, hydrochlo effective dose of the compound comprises 1.25 to 2.5 mg of rothiazide, 5-methyl urapidil, chloroethylclonidine, C.-adrenergic antagonist and 1.25 to 2.5 mg of B-adrenergic bunazosin, alfuzosin, RS17053, BMY 7378, urapidil, L-765, agonist. 314, nicergoline, ABT-866, cyclazosin, A322312, A 119637, 11. The method in claim 1, wherein the inflammatory fiduXosin, JTH-601, imiloxan, 2 idopropoxyidaZOXan, autoimmune disease is rheumatoid arthritis. 2-methoxyidazoxan, RX 821002, idazoxan, piperoxan, BRL 12. The method of claim 11, wherein joint destruction is 44408, beditin, atipamezole, rawolscine, ARC 239, Suppressed. RS-79948, MK912, RS 79948, UIC 14304 and ethoxyida 13. The method of claim 11, wherein joint destruction is ZOX. decreased. 34. The compound of claim 32, wherein the B-adrenergic 14. The method in claim 1, wherein the inflammatory agonist is Selected from the group consisting of turbutaline, autoimmune disease is inflammatory bowel disease. metaprotemol, albutuol, isoetharine, pirbuterol, bitholtrol, 15. The method in claim 1, wherein the inflammatory ritodrine, and Salbutamol. autoimmune disease is Krohn's disease. 35. The compound of claim 32, wherein the inflammatory 16. The method in claim 1, wherein the inflammatory autoimmune disease is rheumatoid arthritis. autoimmune disease is fibromyalgia. 36. The compound of claim 32, further comprising a 17. The method in claim 1, wherein the inflammatory pharmaceutically acceptable carrier. autoimmune disease is lupus. 37. The compound of claim 32, wherein the compound is 18. The method in claim 1, wherein the inflammatory capable of being administered in a form Selected from the autoimmune disease is chronic fatigue Syndrome. group consisting of pill, tablet, capsule, caplet, Solution, 19. The method in claim 1, wherein the inflammatory Suspension, Syrup, Suppository, and aerosol. autoimmune disease is Type 1 diabetes. 38. The compound of claim 32, wherein the compound is 20. The method in claim 1, wherein the animal is a capable of being administered in a route Selected from the mammal. group consisting of Sublingually, orally, intravenously, intra 21. The method in claim 17, wherein the mammal is a muscularly, rectally, parenterally, Subcutaneously, and Sub human. dermally. 22. The method of claim 1, wherein the compound is 39. The compound of claim 32, wherein the compound is administered in a form Selected from the group consisting of capable of being released over time. pill, tablet, capsule, caplet, Solution, Suspension, Syrup, 40. A method for treating inflammatory autoimmune Suppository, and aerosol. disease comprising: 23. The method of claim 1, wherein the compound is administered in a Sustained-release form. administering an O-adrenergic antagonist; and 24. The method of claim 1, wherein the route of admin administering a B-adrenergic agonist. istration is Selected from the group consisting of Sublin 41. The method of claim 40, wherein the B-adrenergic gually, orally, intravenously, intramuscularly, rectally, agonist is administered followed by the C-adrenergic parenterally, Subcutaneously, and Subdermally. antagonist 25. The method of claim 1, wherein the B-adrenergic 42. The method of claim 40, wherein the C-adrenergic agonist is administered in Salt form. antagonist is administered followed by the B-adrenergic 26. The method of claim 25, wherein the B-adrenergic agonist. agonist Salt form is Selected from the group consisting of 43. The method of claim 40, wherein the C-adrenergic metaproterenol Sulfate, turbutaline Sulfate, albuterol Sulfate, antagonist and B-adrenergic agonist are administered in the ioetharine hydrochloride, isoetharine meSylate, pributerol Same 24-hour time period. acetate, bitolerol meSylate, ritodrine hydrochloride, leval 44. The method of claim 40, wherein the y-adrenergic buterol hydrochloride, and salmeterol. antagonist and B-adrenergic agonist are administered in the 27. The method of claim 1, wherein the O.-adrenergic Same one-hour period. antagonist is administered in Salt form. 45. The method of claim 40, wherein the C-adrenergic 28. The method of claim 27, wherein the O.-adrenergic antagonist and B-adrenergic agonist are administered in the antagonist Salt form is Selected from the group consisting of Same one-hour time period. phentolamine meSylate, regitine meSylate, prasozin, tera 46. A method for treating rheumatoid arthritis comprising ZoSin, doxazosin meSylate, and tamsulosin hydrochloride. administering a compound comprising phentolamine and 29. The method of claim 1, wherein the therapeutically terbutaline. effective dose is administered two times per day. 47. The method of claim 46, wherein the compound 30. The method of claim 1, wherein the therapeutically comprises 1.0 to 10.0 mg of phentolamine. effective dose is administered three times per day. 48. The method of claim 46, wherein the compound 31. The method of claim 1, wherein the therapeutically comprises 1.0 to 10.0 mg of terbutaline. effective dose is administered more than three times per day. 49. The method of claim 46, wherein the compound is 32. A compound useful for treating inflammatory autoim administered more than one time per day. mune disease in humans comprising: 50. A compound useful in the treatment of rheumatoid an C.-adrenergic antagonist, and arthritis comprising: a f-adrenergic agonist. 0.01 mg to 100.0 mg of phentolamine; and 33. The compound of claim 32, wherein the O.-adrenergic 0.01 mg to 100.0 mg of terbutaline. antagonist is Selected from the group consisting of yohim bine, regitine, praZoSin, doxazosin, tamsulosin, teraZosin, k k k k k