WO2004084911A2 - Modulation de reponses immunitaires par administration de roxithromicine ou de son derive - Google Patents

Modulation de reponses immunitaires par administration de roxithromicine ou de son derive Download PDF

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WO2004084911A2
WO2004084911A2 PCT/JP2004/004049 JP2004004049W WO2004084911A2 WO 2004084911 A2 WO2004084911 A2 WO 2004084911A2 JP 2004004049 W JP2004004049 W JP 2004004049W WO 2004084911 A2 WO2004084911 A2 WO 2004084911A2
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cells
macrolide antibiotic
disease
rxm
production
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PCT/JP2004/004049
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WO2004084911A3 (fr
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Chikao Morimoto
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Y's Therapeutics Co., Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to modulation of immune responses by administration of compositions comprising macrolide antibiotics and use of such compositions in methods for treating or preventing diseases or disorders involving transendothelial migration of T cells and activated T cells, proinflammatory cytokine production from T cells, and IL-6 production from macrophages, particularly arthritic and/or rheumatic disorders such as rheumatoid arthritis.
  • erythromycin has been reported to be effective in the treatment of chronic lower respiratory tract diseases, including diffuse panbronchiolitis and bronchial asthma (2, 3).
  • EM may have anti- inflammatory properties in addition to its antimicrobial effects.
  • These immunomodulatory effects are the result of leukocyte activation, such as stimulation of phagocytosis (4, 5), natural killer activity (5, 6), production of superoxide anion (5), and neutrophil chemotaxis (2, 7-9).
  • Roxithromycin a new macrolide antibiotic, has a 14-member macrocycline ring structure which resembles that of erythromycin (10). RXM is characterized by rapid and complete absorption after oral administration, resulting in high serum levels (11).
  • RXM modifies the function of neutrophils (12, 13) and keratinocytes (14). RXM also affects lymphocyte functions. In particular, RXM has been shown to affect proliferation induced by mitogens and purified protein derivative (PPD) (15). Furthermore, proliferation and cytokine secretion induced by mitogens have been modified by RXM (10, 16).
  • T cell receptor T cell receptor
  • CD3/TcR CD3/TcR
  • costimulatory signals are indispensable for full activation of T cells, which is characterized by T cell proliferation and cytokine production. Therefore, the triggering of costimulatory signals plays an important role in the generation of hypersensitive immune reaction.
  • the costimulatory signals can be provided by a number of accessory molecules, such as CD28/CTLA-4 (18-20).
  • CD26 a novel costimulatory molecule, CD26, that is preferentially expressed on CD4 + memory T cells (18, 19, 21-23), and is predicted to be involved in the functions of effector T cells which migrate to the focus of inflammation in immuno-mediated diseases and disorders (24) (25).
  • the present inventor examined the effect of RXM on T cell proliferation and cytokine production through different costimulatory signaling pathways. Specifically, the effects of RXM on T cell migration and proinflammatory cytokine production by T cell and macrophages were investigated. The therapeutic effect of RXM on inflammation in mice with collagen-induced arthritis (CIA) and patient's with rheumatoid arthritis (RA) was also studied. The results herein demonstrate that RXM specifically inhibits proinflammatory cytokine production by T cells and macrophages, inhibits T cell migration, and inhibits the development and/or symptoms of arthritis in mice and humans.
  • CIA collagen-induced arthritis
  • RA rheumatoid arthritis
  • RXM was shown to inhibit the development of collagen-induced arthritis, serum IL-6 levels, the migration of leukocytes into affected joints or synovial membrane and the destruction of bones and cartilage in a mouse model of CIA. Furthermore, RXM was shown to reduce clinical symptoms of RA as well as indices of RA activity in patients. Accordingly, roxithromycin therapy may serve as an effective treatment for arthritic and/or rheumatic disorders, such as rheumatoid arthritis.
  • one aspect of the invention is to provide a method for treating or preventing a disease or disorder in which transendothelial migration of T cells and activated T cells, pro- inflammatory cytokine production from T cells, or IL-6 production from macrophages is implicated, the method comprising the step of administering a therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a patient or mammal in need thereof.
  • the disease or disorder to be treated or prevented using the composition and methods of the invention is an arthritic or rheumatic disorder, including rheumatoid arthritis, osteoarthritis, and infectious, psoriatic and/or viral arthritis; Crohn's disease; graft-versus-host disease after allo-bone marrow transplantation; heart failure; graft rejection; atrial myxoma; multiple myeloma; Castleman's disease; glomerulonephritis including mesangial proliferative glomerulonephritis; osteoporosis; EBV-positive lymphoma; systemic lupus erythmatosis; collagenosis; ulcerative colitis; autoimmune hemolytic anemia; hepatitis including active chronic hepatitis; gout; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; ankly
  • the one or more macrolide antibiotics comprise a therapeutically effective amount of a 14-membered macrolide antibiotic administered in conjunction with a therapeutically effective amount of one or more non-macrolide antibiotics.
  • the one or more macrolide antibiotics are administered in combination or conjunction with a therapeutically effective amount of one or more non- macrolidic antibiotics, and/or a therapeutically effective amount of one or more anti- inflammatory compounds or immunomodulatory agents.
  • the anti-inflammatory compound or immunomodulatory drug comprises interferon; interferon derivatives comprising betaseron, .beta.-interferon; prostane derivatives comprising iloprost, cicaprost; glucocorticoids comprising cortisol, prednisolone, methylprednisolone, dexamethasone; immunsuppressives comprising cyclosporine A, FK-506, methoxsalene, thalidomide, sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitors comprising zileutone, MK-886, W ⁇ -50295, SC-45662,
  • the present invention provides a method of treating or preventing arthritic or rheumatic disorders, the method comprising the step of administering a therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a patient or mammal in need thereof.
  • the one or more macrolide antibiotics may be administered directly to an afflicted region or via systemic administration as well as orally.
  • the one or more macrolide antibiotics may be administered in combination or conjunction with a therapeutically effective amount of one or more non-macrolidic antibiotics and/or a therapeutically effective amount of one or more anti-inflammatory compounds and/or a therapeutically effective amount of one or more immunomodulatory agents, such as those described above.
  • the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
  • the macrolide antibiotic comprises a 14-member macrocycline ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
  • the present invention provides a method of inhibiting the development or progression of an arthritic or rheumatic disorder comprising the step of administering a therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a patient or mammal in need thereof.
  • the one or more macrolide antibiotics may be administered directly to an afflicted region or via systemic administration as well as orally.
  • the one or more macrolide antibiotics may be administered in combination or conjunction with a therapeutically effective amount of one or more non-macrolide antibiotics and/or a therapeutically effective amount of one or more anti- inflammatory compounds and/or a therapeutically effective amount of one or more immunomodulatory agents, such as those described above.
  • the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
  • the macrolide antibiotic comprises a 14-member macrocycline ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
  • a method for inhibiting bone and cartilage destruction comprising the step of administering a therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a patient or mammal in need thereof.
  • the one or more macrolide antibiotics may be administered directly to an afflicted region or via systemic administration as well as orally.
  • the one or more macrolide antibiotics may be administered in combination or conjunction with a therapeutically effective amount of one or more non-macrolide antibiotics and/or a therapeutically effective amount of one or more anti-inflammatory compounds and/or a therapeutically effective amount of one or more immunomodulatory agents, such as those described above.
  • the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
  • the macrolide antibiotic comprises a 14-member macrocycline ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
  • a method for inhibiting leukocyte migration into an arthritic joint or synovial membrane comprising the step of administering therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a patient or mammal in need thereof.
  • the one or more macrolide antibiotics m y be administered directly to an afflicted region or via systemic administration as well as orally.
  • the one or more macrolide antibiotics may be administered in combination or conjunction with a therapeutically effective amount of one or more non-macrolide antibiotics and/or a therapeutically effective amount of one or more anti- inflammatory compounds and/or a therapeutically effective amount of one or more immunomodulatory agents, such as those described above.
  • the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
  • the macrolide antibiotic comprises a 14-member macrocycline ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
  • a method for inhibiting transendothelial migration of T cells and activated T cells and/or production of proinflammatory cytokines, including IL-6 or TNF-alpha and/or NF- ⁇ B, from T cells comprising the step of contacting one or more T cells or activated T cells with one or more macrolide antibiotics.
  • the one or more macrolide antibiotics comprise a 14- membered macrolide antibiotic.
  • the 14-membered macrolide antibiotic is roxithromycin or a derivative thereof.
  • a method for inhibiting IL-6 production from macrophages comprising the step of contacting one or more macrophages with one or more macrolide antibiotics.
  • a method for inhibiting the activation of NF- ⁇ B in a cell comprising the step of contacting one or more T cells or macrophage cells with one more macrolide antibiotics.
  • the one or more macrolide antibiotics comprise a 14-membered macrolide antibiotic.
  • the 14-membered macrolide antibiotic is roxithromycin or a derivative thereof.
  • a pharmaceutical composition comprising one or more macrolide antibiotics and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition is used to treat or prevent a disease or disorder in which transendothelial migration of T cells and activated T cells, pro-inflammatory cytokine production including, inter alia, the production of TNF-alpha, and/or NF- B from T cells, or IL-6 from macrophages, is implicated.
  • the therapeutically effective amount of macrolide antibiotic administered is that amount sufficient to reduce or inhibit the production of pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 from macrophages.
  • the reduction or inhibition of the production of pro-inflammatory cytokine production including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 from macrophages occurs without inhibition of IL-2, IFN-gamma, IL-4 and IL-5 by the T cells.
  • the therapeutically effective amount of macrolide antibiotic administered to a mammal in need thereof is that amount sufficient to reduce or inhibit the production of pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 from macrophages, provided that said amount does not first, simultaneously or subsequently cause inhibition of IL-2, IFN-gamma, IL-4 and IL-5 by the T cells.
  • the reduction or inhibition of the production of pro-inflammatory cytokines including, inter alia, the production of TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 from macrophages is on the order of about 10-20% reduction or inhibition.
  • the reduction or inhibition is on the order of 30-40% reduction or inhibition. In another embodiment, the reduction or inhibition is on the order of 50-60% reduction or inhibition. In yet another embodiment, reduction or inhibition is on the order of 75%-100% reduction or inhibition of the production of pro-inflammatory cytokine production including, inter alia, TNF- alpha and/or NF- ⁇ B from T cells, or IL-6 production from macrophages. It is intended herein that by recitation of such specified ranges, the ranges recited also include all those specific integer amounts between the recited range. For example, in the range about 75% and 100%, it is intended to also encompass 76 to 99%, 77%-98%, etc, without actually reciting each specific range.
  • the disease or disorder treated or prevented using the therapeutically effective amount of the pharmaceutical composition of the invention is an arthritic or rheumatic disorder, including rheumatoid arthritis, osteoarthritis, and infectious, psoriatic and/or viral arthritis; Crohn's disease; graft-versus-host disease after allo-bone marrow transplantation; heart failure; graft rejection; atrial myxoma; multiple myeloma; Castleman's disease; glomerulonephritis including mesangial proliferative glomerulonephritis; osteoporosis EBV-positive lymphoma; systemic lupus erythmatosis; collagenosis; ulcerative colitis autoimmune hemolytic anemia; hepatitis including active chronic hepatitis; gout artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma encephalomyelitis;
  • the present invention provides a method of relieving or ameliorating pain or symptoms associated with any one or more of the above-identified diseases or disorders in a mammal suffering such, the method comprising the step of administering to the mammal in need thereof a therapeutically effective pain or symptom-reducing amount of a pharmaceutical composition comprising one or more macrolide antibiotics, either alone or in combination with one or more other non-macrolide antibiotics, and/or one or more anti- inflammatory compounds or immunomodulatory agents; and a pharmaceutically acceptable carrier or excipient.
  • the one or more macrolide antibiotics of the present invention are administered directly to an affected region or lesion or by way of systemic administration.
  • the pharmaceutical compositions are administered orally, systemically, via an implant, intravenously, topically, or intrathecally.
  • the subject or mammal is a human. In other embodiments of the methods of the present invention, the subject or mammal is a veterinary mammal.
  • the phrase "therapeutically effective amount,” means that amount of the pharmaceutical composition that provides a therapeutic benefit in the treatment, prevention, or management of pain associated with the particular disease or disorder.
  • macrocyclic lactone ring refers to a ring having a lactone moiety and more than seven carbon atoms.
  • arthritis literally means joint inflammation (from the Greek, “arth” meaning joint and “itis” meaning inflammation) and encompasses a wide range of different inflammatory conditions, ranging from relatively mild forms of tendinitis (as in "tennis elbow") and bursitis to crippling systemic forms, such as rheumatoid arthritis.
  • rheumatism and “rheumatic disorders” refer to general disease conditions characterized by painful, often multiple, local inflammations, usually of the joints and muscles but also extending sometimes to the deeper organs, such as the heart.
  • the present invention is directed to a method for treating or preventing a disease or disorder in which transendothelial migration of T cells and activated T cells, proinflammatory cytokine production from T cells, or IL-6 production from macrophages is implicated, comprising the step of administering to a subject in need thereof a composition comprising a therapeutically effective amount of one or more macrolide antibiotics or a derivative thereof.
  • the diseases or disorders contemplated for treatment or prevention with the methods of the present invention include, inter alia, arthritic and rheumatic disorders.
  • the diseases or disorders include, but are not limited to, an arthritic or rheumatic disorder, including rheumatoid arthritis, osteoarthritis, and infectious, psoriatic and/or viral arthritis; Crohn's disease; graft-versus-host disease after allo-bone marrow transplantation; heart failure; graft rejection; atrial myxoma; multiple myeloma; Castleman's disease; glomerulonephritis including mesangial proliferative glomerulonephritis; osteoporosis; EBV-positive lymphoma; systemic lupus erythmatosis; collagenosis; ulcerative colitis; autoimmune hemolytic anemia; hepatitis including active chronic hepatitis; gout; arlherosclerosis; psoriasis; al
  • the present invention is directed to a method of treating or preventing an arthritic or rheumatic disorder, the method comprising the step of administering a therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a subject in need thereof.
  • the subject suffers from arthritis, particularly rheumatoid arthritis.
  • the present invention is directed to a method of inhibiting the development or progression of an arthritic or rheumatic disorder comprising the step of administering a therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a subject in need thereof.
  • the subject suffers from a rheumatic disorder such as rheumatoid arthritis.
  • inhibiting the development or progression of an arthritic or rheumatic disorder encompass full or partial reduction, amelioration and/or inhibition of one or more clinical symptoms of the disorder, but not limited to, pannus formation, synovial membrane proliferation, leukocyte migration and inflammation of the joints (resulting in joint swelling and tenderness), and bone and cartilage destruction, to name a few.
  • Degree of inflammation can be indexed by measuring serum levels of C-reactive protein (CRP).
  • CRP C-reactive protein
  • the present invention is directed to a method for inhibiting bone and cartilage destruction comprising the step of administering to a subject in need thereof a therapeutically effective amount of a composition comprising one or more macrolide antibiotics.
  • the subject suffers from arthritis, particularly rheumatoid arthritis.
  • the present invention provides a method for inhibiting leukocyte migration into an arthritic joint or synovial membrane comprising the step of administering therapeutically effective amount of a composition comprising one or more macrolide antibiotics to a patient or mammal in need thereof.
  • the subject suffers from arthritis, particularly rheumatoid arthritis.
  • a method for inhibiting transendothelial migration of T cells and activated T cells or production of proinflammatory cytokines including IL-6 or TNF-alpha from T cells comprising contacting one or more T cells and activated T cells with one or more macrolide antibiotics.
  • a method is provided for inhibiting IL-6 production from macrophage comprising contacting one or more macrophages with one or more macrolide antibiotics.
  • a method is provided for inhibiting the activation of NF- ⁇ B in a cell comprising contacting one or more macrophage cells with one more macrolide antibiotics.
  • a pharmaceutical composition comprising one or more macrolide antibiotics, wherein the pharmaceutical composition is used to treat or prevent a disease or disorder in which transendothelial migration of T cells and activated T cells, pro-inflammatory cytokine production from T cells, or IL-6 production from macrophages is implicated.
  • the one or more macrolide antibiotics comprise a 14-membered ring structure macrolide antibiotic.
  • the 14-membered macrolide antibiotic is roxithromycin or a functional derivative thereof.
  • the invention also provides for the use of one or more macrolide antibiotics comprising a
  • a composition comprising a therapeutically effective amount of one or more macrolide antibiotics necessary to treat or prevent a disease or disorder in which transendothelial migration of T cells and activated T cells, pro-inflammatory cytokine production including, inter alia, TNF-alpha, and/or NF- ⁇ B from T cells, or IL-6 production from macrophages is implicated.
  • the amount of macrolide antibiotic administered to a mammal in need thereof is that amount sufficient to reduce or inhibit the production of pro- inflammatory cytokine including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 from macrophages.
  • the reduction or inhibition of the production of pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF-KB from T cells, or IL-6 from macrophages occurs without first, simultaneously or subsequently causing inhibition of IL-2, IFN-gamma, IL-4 and IL-5 by the T cells.
  • the therapeutically effective amount of macrolide antibiotic administered to a mammal in need thereof is that amount sufficient to reduce or inhibit the production of pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 production from macrophages provided that said amount does not first, simultaneously or subsequently cause inhibition of IL-2, IFN-gamma, IL-4 and IL-5 by the T cells.
  • pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 production from macrophages provided that said amount does not first, simultaneously or subsequently cause inhibition of IL-2, IFN-gamma, IL-4 and IL-5 by the T cells.
  • the therapeutically effective amount of macrolide antibiotic administered to a mammal in need thereof is that amount sufficient to reduce or inhibit the production of pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF- ⁇ B from T cells, or IL-6 from macrophages, but that said amount may either first, simultaneously or subsequently cause inhibition of IL-2, IFN-gamma, IL-4 and IL-5 by the T cells.
  • Macrolide antibiotics include, for example, but are not limited to, those described by Bryskier et al, e.g., a lipophilic molecule with a characteristic central lactone ring bearing 12 to 17 atoms, fewer than 5 and preferably no double bonds, and preferably no nitrogen atoms.
  • erythromicin and erythromicin derivatives 14-membered ring structure macrolidic antibiotics.
  • a group of somewhat atypical macrolide antibiotics are lankacidin derivatives, 17 membered-ring macrocyclic antibiotics which do not have sugars fixed to the aglycone ring.
  • Another group of somewhat atypical macrolide antibiotics are azalide compounds which contain an endocyclic nitrogen, namely azalide, within the aglycone ring.
  • the one or more macrolide antibiotics contemplated for use in the methods of the present invention preferably comprise a 14-membered ring structure macrolide antibiotic or derivatives thereof that may be used separately or as mixtures of two or more macrolide and/or non- macrolide antibiotics.
  • the antibiotics of the present invention may be combined with one or more pharmaceutically acceptable compounds such as carriers and/or excipients.
  • a particularly preferred macrolide antibiotic for use in the compositions and methods of the present invention is roxithromycin or a functional derivative thereof.
  • the phrase "functional derivative" refers to compounds that retain the one or more biologically significant activities of roxithromycin associated with treating or preventing arthritic and rheumatic disorders.
  • examples of such biologically significant activities include, but are not limited to, the ability to (a) inhibit proinflammatory cytokine production by T cells and macrophages, (b) inhibit transendothelial migration of T cells and activated T cells, (c) inhibit the development or progression of an arthritic or rheumatic disorder, (d) inhibit serum IL-6 levels, (e) inhibit the migration of leukocytes into arthritic joints or synovial membrane, (f) inhibit the destruction of bones and cartilage in arthritic or rheumatic subjects, (g) inhibit pannus formation, and (h) reduce or ameliorate clinical symptoms of an arthritic or rheumatic disorder or indices of arthritic or rheumatic activity.
  • the determination of such activities can be conducted by methods well known to those skilled in the art and include those described in the examples herein.
  • macrolide antibiotics that may be used in combination with the preferred roxithromycin macrolidic antibiotic include, inter alia, but are not limited to, the following synthetic, semi-synthetic or naturally occurring microlidic antibiotic compounds: methymycin, neomethymycin, YC-17, litorin, erythromycin A to F, oleandomycin, roxithromycin, dirithromycin, flurithromycin, clarithromycin, davercin, azithromycin, josamycin, kitasamycin, spiramycin, midecamycin, rokitamycin, miokamycin, lankacidin, and the derivatives of these compounds.
  • erythromycin and compounds derived from erythromycin belong to the general class of antibiotics known as "macrolides.”
  • examples of preferred erythromycin and erythromycin-like compounds include: erythromycin, clarithromycin, azithromycin, and troleandomycin.
  • Additional antibiotics other than the macrolidic antibiotics described above, which are suitable for use in the methods of the present invention include, for example, but are not limited to, any molecule that tends to prevent, inhibit or destroy life and as such, and as used herein, includes anti-bacterial agents, anti-fungal agents, anti-viral agents, and anti-parasitic agents. These agents may be isolated from an organism that produces the agent or procured from a commercial source (e.g., pharmaceutical company, such as Eli Lilly, Indianapolis, Ind.; Sigma, St. Louis, Mo.).
  • Anti-bacterial antibiotic agents include, but are not limited to, penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides, and fluoroquinolones (see Table below).
  • antibiotic agents include, but are not limited to, Penicillin G (CAS Registry No.: 61-33-6); Methicillin (CAS Registry No.: 61-32-5); Nafcillin (CAS Registry No.: 147-52-4); Oxacillin (CAS Registry No.: 66-79-5); Cloxacillin (CAS Registry No.: 61-72-3); Dicloxacillin (CAS Registry No.: 3116-76-5); Ampicillin (CAS Registry No.: 69-53-4); Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin (CAS Registry No.: 34787-01-4); Carbenicillin (CAS Registry No.: 4697-36-3); Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin (CAS Registry No.
  • Anti-fungal agents include, but are not limited to, terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clolrimazole, benzoic acid, salicylic acid, and selenium sulfide.
  • Anti-viral agents include, but are not limited to, amantadine hydrochloride, rimantadin, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idojoiridine, ribavirin, sorivudine, trifluridine, valacyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine.
  • Anti-parasitic agents include, but are not limited to, pirelhrins/piperonyl butoxide, permethrin, iodoquinol, metronidazole, diethylcarbamazine citrate, piperazine, pyrantel pamoate, mebendazole, thiabendazole, praziquantel, albendazole, proguanil, quinidine gluconate injection, quinine sulfate, chloroquine phosphate, mefloquine hydrochloride, primaquine phosphate, atovaquone, co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidine isethionate.
  • immunomodulatory drugs or agents it is meant, e.g., agents which act on the immune system, directly or indirectly, e.g., by stimulating or suppressing a cellular activity of a cell in the immune system, e.g., T-cells, B-cells, macrophages, or other antigen presenting cells (APC), or by acting upon components outside the immune system which, in turn, stimulate, suppress, or modulate the immune system, e.g., hormones, receptor agonists or antagonists, and neurotransmitters; immunomodulators can be, e.g., immunosuppressants or immunostimulants.
  • anti-inflammatory drugs it is meant, e.g., agents which treat inflammatory responses, i.e., a tissue reaction to injury, e.g., agents which treat the
  • Anti-inflammatory or immunomodulatory drugs or agents suitable for use in this invention include, but are not limited to, interferon derivatives, e.g., betaseron, .beta. -interferon; prostane derivatives, e.g., compounds disclosed in PCT/DE93/0013, e.g., iloprost, cicaprost; glucocorticoid, e.g., cortisol, prednisolone, methylprednisolone, dexamethasone; immunsuppressives, e.g., cyclosporine A, FK-506, methoxsalene, thalidomide, sulfasalazine, azathioprine, methotrexate; lipoxygenase inhibitors, e.g., zileutone, MK-886, WY-50295, SC- 45662, SC-41661A, BI-L-357; leukotriene antagonist
  • Figure 1 depicts the effect of RXM on the proliferative response of peripheral T cells.
  • the cpm value in the case of T cells without any stimuli was near the background level (data not shown).
  • Mean cpm values ⁇ SD from triplicate samples were plotted from 4 different donors. RXM did not significantly inhibit the proliferative response of T cells induced by above stimuli.
  • Figure 2 depicts the effect of RXM on the production of IL-2 (A) and IFN- ⁇ (B) by the peripheral T cells.
  • the T cells were stimulated for 24 hours, and the culture supernatants were assayed for the concentration of IL-2 and IFN- ⁇ by ELISA. Mean values ⁇ SD from triplicate samples were plotted. The data are representative of 4 independently performed experiments. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
  • FIG. 3 depicts the effect of RXM on the production of IL-4 (A) and IL-5 (B) by the peripheral T cells.
  • the T cells were stimulated for 24 hours, and the culture supernatants were assayed for the concentration of IL-4 and IL-5 by ELISA. Mean values ⁇ SD from triplicate samples were plotted. The data are representative of 4 independently performed experiments. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
  • Figure 4 depicts the effect of RXM on the production of IL-6 (A) and TNF- ⁇ (B) by the peripheral T cells.
  • the T cells were stimulated for 24 hours, and the culture supernatants were assayed for the concentration of IL-6 and TNF- ⁇ by ELISA. Mean values ⁇ SD from triplicate samples were plotted. The data are representative of 3 independently performed experiments. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown). The p values calculated by two-tailed Student's t test were indicated in each figure (between 0 and 1.4 ⁇ M, 0 and 14 ⁇ M, 0 and 28 ⁇ M of RXM, respectively).
  • Figure 5 depicts the effect of RXM on the production of IL-6 and TNF- ⁇ by macrophages stimulated with LPS. Macrophages were stimulated by LPS (1 g/ml) for 8 hours, and the culture supernatants were assayed fur the concentration of IL-6 and TNF- ⁇ , by ELISA. Mean values ⁇ SD from triplicate samples were plotted. The data are representative of 3 independently performed experiments. The p values calculated by two-tailed Student's t test were indicated in each figure (between 0 and 1.4 ⁇ M, 0 and 14 ⁇ M, 0 and 28 ⁇ M of RXM, respectively).
  • FIG. 6 depicts the inhibitory effect of RXM on transendothelial migration (chemokinesis) of PHA-activated T cells.
  • ECV304 cells were grown on Transwell cell culture inserts for 48 hours. After washing of the monolayers with the assay medium (0.6% BSA RPMI1640), PHA-activated T cells were layered onto them with or without 3 different concentrations of RXM. T cells that spontaneously migrated to the lower chambers without any exogenous chemokines were counted in each experiment by flow cytometry. Bars show the mean values ⁇ SD of three different experiments.
  • the p values calculated by two-tailed Student's t test were indicated in each figure (p ⁇ 0.01 between 0 and 14 ⁇ M, p ⁇ 0.001 between 0 and 28 ⁇ M of RXM, respectively). The data are representative of 5 different donors.
  • Figure 7 depicts the effect of RXM therapy on the development of CIA.
  • Figure 8 depicts the effect of RXM treatment on the serum IL-6, INF- ⁇ and type II collagen antibody levels in CIA mice.
  • the sera of CIA mice were collected on the day of first and second day and day 7, 14, and 21 after second immunization of collagen.
  • Serum levels of cytokines and type II collagen antibody were assayed by ELISA.
  • Mean value ⁇ SD from 8 mice were plotted.
  • IL-4 and TNF- ⁇ levels were always below the background levels (data not shown).
  • the p values calculated by two-tailed Student's t test were indicated in the figure (between 0, 400 and SOO ⁇ g/day).
  • Figure 9 depicts the results of HE staining of ankle joint of CIA mice. Ankle joints of CIA mice on day 21 after second immunization of collagen were collected. The ankle joints of CIA mice with 0, 200, and 800 ⁇ g of RXM treatment were stained by eosin and hematoxilyn. Pharmacology
  • the pharmaceutical compositions of the present invention can be used in both veterinary medicine and human therapy.
  • the magnitude of a prophylactic or therapeutic dose of the pharmaceutical composition of the invention in the acute or chronic management of pain associated with diseases or disorders wherein transendothelial migration of T cells and activated T cells, proinflammatory cytokine production from T cells, or IL-6 production from macrophage is implicated will vary with the severity of the condition to be treated and the route of administration.
  • the dose, and perhaps the dose frequency will also vary according to the age, body weight, and response of the individual patient.
  • the total daily dose range of the active ingredient of this invention is generally between about 1 and 1000 mg per 70 kg of body weight per day, or about 10 and 800 mg per 70 kg of body weight per day, preferably between about 50 and 500 mg per 70 kg of body weight per day, and more preferably between about 300 and 150 mg per 70 kg of body weight per day.
  • the ranges cited also include all those integer amounts between the recited range. For example, in the range about 1 and 500, it is intended to encompass 2 to 499, 3-498, etc, without actually reciting each specific instance.
  • the actual preferred amounts of the active ingredient will vary with each case, according to the species of mammal, the nature and severity of the particular affliction being treated, and the method of administration. It is also understood that doses within those ranges, but not explicitly stated, such as 30 mg, 50 mg, 75 mg, 150 mg, etc. are encompassed by the stated ranges, as are amounts slightly outside the stated range limits.
  • each daily dose is a unit dose, i.e., tablet, cachet or capsule, which contains between about 1 mg to 1000 mg of the active ingredient, or pharmaceutical composition, about 10 mg to 800 mg of the active ingredient, or pharmaceutical composition, preferably about 50 mg to 500 mg, and more preferably about 100 mg to 300 mg of the active ingredient (i.e., excluding excipients and carriers).
  • the daily dose may include two or more unit doses, i.e., tablets, cachets or capsules, to be administered each day.
  • compositions of the present invention are periodically administered to an individual patient as necessary to improve symptoms of the particular disease or disorder being treated.
  • the length of time during which the compositions are administered and the total dosage will necessarily vary with each case, according to the nature and severity of the particular affliction being treated and the physical condition of the subject or patient receiving such treatment.
  • Suitable routes include, for example, oral, rectal, parenteral (e.g., in saline solution), intravenous, topical, Iransdermal, subcutaneous, intramuscular, by inhalation, and like forms of administration may be employed.
  • Suitable dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, patches, suppositories, and the like, although oral dosage forms are preferred.
  • compositions used in the methods of the present invention include the active ingredients described above, and may also contain pharmaceutically acceptable carriers, excipients and the like, and optionally, other therapeutic ingredients.
  • the composition is dissolved in a vegetable oil, such as olive oil or peanut oil, and, optionally, encapsulated in a gelatin capsule.
  • a preferred method of administering pharmaceutical compositions of the present invention is orally, in the form of a gelatin capsule.
  • pharmaceutically acceptable salt refers to a salt prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, sulfuric, and phosphoric.
  • Appropriate organic acids may be selected, for example, from aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic, stearic, sulfanilic, algenic, and galacturonic.
  • organic acids may be selected, for example, from aliphatic, aromatic, carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic
  • inorganic bases for potential salt formation with the sulfate or phosphate compounds of the invention, include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.
  • Appropriate organic bases may be selected, for example, from N,N-dibenzylethylenediamine, chloroprocaine, chorine, diethanolamine, ethylenediamine, meglumaine (N-melhylglucamine), and procaine.
  • compositions for use in the methods of the present invention include compositions such as suspensions, solutions and elixirs; aerosols; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like, in the case of oral solid preparations (such as powders, capsules, and tablets), with ⁇ y
  • oral solid preparations being preferred over the oral liquid preparations.
  • the most preferred oral solid preparations are capsules.
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques.
  • compositions for use in the methods of the present invention may also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, the disclosures of each of which are hereby incorporated by reference in their entirety.
  • compositions for use in the methods of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, or aerosol sprays, each containing a predetermined amount of the active ingredient, as a powder or granules, as creams, pastes, gels, or ointments, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association the carrier with the active ingredient which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing in a suitable machine the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression or molding, optionally, with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form, such as powder or granules, optionally mixed with a binder (e.g., carboxymethylcellulose, gum arabic, gelatin), filler (e.g., lactose), adjuvant, flavoring agent, coloring agent, lubricant, inert diluent, coating material (e.g., wax or plasticizer), and a surface active or dispersing agent.
  • Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • Figure 1 depicts the effect of RXM on the proliferative response of peripheral T cells stimulated with CD3, CD3 plus CD28, CD3 plus PMA, and CD3 plus CD26.
  • Figure 2 depicts the effect of RXM on the production of IL-2 and IFN- ⁇ by peripheral T cells stimulated with CD3 plus CD28, CD3 plus PMA, and CD3 plus CD26.
  • Figure 3 depicts the effect of RXM on the production of IL-4 and IL-5 by peripheral T cells stimulated with CD3 plus CD28, CD3 plus PMA, and CD3 plus CD26.
  • F ⁇ giB ⁇ re 4 depicts the effect of RXM on the production of IL-6 and TNF- ⁇ by peripheral T cells stimulated with CD3 plus CD28, CD3 plus PMA, and CD3 plus CD26.
  • Figiir® 5 depicts the effect of RXM on the production of IL-6 and TNF- ⁇ by macrophages stimulated with LPS.
  • Figfflir® 6 depicts the inhibitory effect of RXM on transendothelial migration (chemokinesis) of PHA-activated T cells.
  • Figure 7 depicts the effect of RXM therapy on the development of CIA.
  • Figure 8 depicts the effect of RXM treatment on the serum IL-6, INF- ⁇ and type II collagen antibody levels in CIA mice.
  • Figure 9 depicts the HE staining of ankle joint of CIA mice.
  • Human PBMC were isolated from healthy volunteer donors by Ficoll-Hypaque (Pharmacia Biotech Inc., Piscataway, NJ) density gradient centrifugation (21). Unfractionated mononuclear cells were separated into an E rosette-positive (E + ) population and were used as resting T cells. Monocytes were depleted by adherence to plastic plates for 24 hours at 37°C followed by incubation with 5 mM L-leucine methyl ester HCL (Sigma Chemical Co., St. Louis, MO) for 1 hour. The monoclonal antibody (mAb) OKT3 was obtained via American Tissue Culture Collection (ATCC, Rockville, MD).
  • ATCC American Tissue Culture Collection
  • Anti-CD26 (1F7) and anti-CD28 mAb, 4B10 were developed in the laboratory of the present inventor as previously described (19, 21).
  • Roxithromycin (RXM) was generously supplied by Eizai Ltd., Tokyo, Japan and was dissolved in DMSO and further diluted in the culture media consisting of RPMl-1640 and 10% fetal calf serum (FCS).
  • PBS phosphate-buffer saline
  • Cytoldne production assays Antibody-coated plates and purified T cells were prepared in the same manner as described above, with the exception of OKT3 concentration being 0.5 g/ml. Cytokine production by T cells was assayed in triplicates in 96 well, flat-bottomed plates as described above for the T cell proliferation assay. After 24 hours of incubation, culture supernatants were subjected to ELISA (IL-2 and IL-4: Biosource International, Camarillo, CA; IFN- ⁇ and IL-5: R&D systems, Minneapolis, MN) to measure the levels of IL-2, IFN- ⁇ , IL-4 and IL-5 as well as TNF- ⁇ and IL-6.
  • IL-2 and IL-4 Biosource International, Camarillo, CA
  • IFN- ⁇ and IL-5 R&D systems, Minneapolis, MN
  • macrophages were enriched from E cells by adherence to plastic plates. Macrophages (1 x 10 6 /ml) were suspended in 10% FCS-RPMI1640 and stimulated with 1 ⁇ g/ml of LPS (Sigma). After 8-hour culture, culture supernatants were harvested and were subjected to ELISA (TNF- ⁇ and IL-6: R&D systems, Minneapolis, MN). The serum levels of IL-6, TNF- ⁇ , IFN- ⁇ , and IL-4 were also detected using the ELISA kit described above.
  • the trypan blue dye exclusion test was used to assess cell viability. In all experiments, the viability was found to be over 95% at each point measured (data not shown).
  • Transendothelial migration activity was assessed using a kind of boyden-chamber assay as described previously with some modifications (26).
  • the human endothelial cell line ECV304 obtained from ATCC was pre-cultured to make monolayer sheet on Transwell cell culture inserts with 3.0 ⁇ m pore size (Corning Coslar, Cambridge, MA), for 48 hours.
  • RXM was first dissolved in DMSO and further diluted in the assay medium consisting of RPMI1640 and 0.6% bovine serum albumin, then added to culture plates in a final volume of 600 ⁇ l (the lower chamber) just before migration assay.
  • PHA-activated T cells (1 x 10 fi cells/well) were added to each insert in a volume of 200 ⁇ l simultaneously with the same concentration of RXM as in the corresponding culture wells (the upper chamber).
  • the spontaneous migration (chemokinesis) assay was performed at 37°C for 8 hours with or without RXM, then harvested and counted by flow cytometry (FACS Calibur, Nippon Becton-Dickinson, Tokyo, Japan) for 1 minute.
  • the mAbs used for this experiment were anti-CD3 (OKT3), anti-CDlla (BD PharMingen, NJ, USA), anti-CD25 (BD PharMingen), anti-CD26 (1F-7), anti-CD29 (4B4), anti-CD44 (ED PharMingen), and anti-CD47 (BD PharMingen).
  • Bovine type II collagen (Collagen Research Center, Tokyo Japan) was dissolved at 4mg/ml in 0.05M acetic acid and then emulsified with an equal volume of Complete Freund's Adjubant (DIFCO).
  • DIFCO Complete Freund's Adjubant
  • 100 ⁇ l of the immunogen were injected intradermally into 8-week-old mice at the tail base. After 3 weeks, the mice received the same dose of immunogen s.c. The arthritis developed within 10 days of second immunization. These mice were kept under specific pathogen-free conditions in a clean room at the Animal Research Center, Institute of Medical Science, University of Tokyo.
  • RXM was dissolved in 5% Alabic Gum in 0.9% NaCl and different doses of RXM
  • Serum of CIA mice was collected on the day of first end second immunization and day 7, 14, and 21 after second immunization.
  • Cytokine of IL-6, TNF- ⁇ , IL-4, and IFN- ⁇ was assayed by ELISA.
  • Type II collagen antibody levels are assayed by ELISA (Chondrex, Washington , USA). The antibody levels were compared by 490nm O.D.
  • mice were killed by CO 2 asphyxiation and hind paws taken from CIA mice 3 weeks after the second immunization were fixed in 10% phosphate-buffered formalin (pH 7.4), decalcified in 10% EDTA and embedded in paraffin. Sections (4 ⁇ m) were stained with hematoxylin and eosin. For immunohistochemical analysis, synovial tissues from the ankle joints were embedded in Tissue-Tec ornithine calbamyl transferase compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at -80°C.
  • Tissue-Tec ornithine calbamyl transferase compound Miles, Elkhart, IN
  • the sections were then incubated with 10% normal goat serum for lh at room temperature and treated with rabbit anti-human IFN- ⁇ Ab, rabbit anti mouse TNF- ⁇ Ab, or normal rabbit serum overnight at 4°C. They were subsequently incubated with biotinylated goat anti-rabbit IgG, treated with 0.3% hydrogen peroxide in methanol, and incubated with HRP-labeled streptavidin. Bound Abs were visualized with 0.5mg/ml 3,3'-diaminobenzidine tetrahydrochloride in PBS (pH 7.4) and 0.02% hydrogen peroxide, then they were stained with hamatoxylin.
  • CRP also known as C-Reactive Protein
  • CRP is a test which measures the concentration in blood serum of a special type of protein produced in the liver that is present during episodes of acute inflammation or infection.
  • doctors can utilize the CRP test to assess the effectiveness of a specific arthritis treatment and monitor periods of disease flareup.
  • serum CRP was measured by the laser nephelometry (LN) method and less than 0.5 ⁇ g/ml is considered to be normal. 13.
  • Example 1 Effect off RXM on T cell proliferation through different eosliimiilatoiy pathways
  • roxithromycin RXM
  • three different costimulatory pathways were used. The first one includes the phorbol ester PMA; the second involves stimulation through one of the representative T cell costimulatory molecules, CD28; and the third is mediated by CD26, which is preferentially expressed on CD4 + memory T cells. All 3 stimuli were combined with submitogenic doses of anti-CD3 mAb.
  • T cells were stimulated with immobilized anti-CD3 mAb alone, anti-CD3 mAb and anti-CD26 mAb, anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb with PMA in the presence of various concentrations of RXM.
  • the stimulus through CD3 alone resulted in the induction of low levels of T cell proliferation.
  • Marked T cell proliferation was observed when stimulation through CD3 was combined with an additional second signal, such as anti-CD26 mAb, anti-CD28 mAb, or PMA.
  • RXM did not virtually inhibit T cell proliferation at any tested concentration (1.4 ⁇ M to 28 ⁇ M) from different donors. It should be noted that at higher concentration (28 ⁇ M), only slight inhibition of T cell proliferation was observed at certain donor.
  • Example 2 Effect of RXM on Thl-type cytokine production throughdifferent costimulatory pathways
  • cytokine production assays peripheral T cells did not secrete any detectable cytokines when stimulated with CD3 alone, therefore the results with CD3 alone were omitted in the figures.
  • Fig. 2A RXM, even at 28 ⁇ M, did not inhibit IL-2 production under all costimulatory conditions in three different donors.
  • Example 4 Effect of RXM on proinflammatory cytokine production through different costimulatory pathways
  • Example 5 Effect of RXM on proinflammatory cytokine production by macrophages Since macrophages play a role in the host defense against infections and in the local modulation of immune and inflammatory responses (29), the effect of RXM on the production of the proinflammatory cytokines IL-6 and TNF- ⁇ by macrophages was also examined.
  • macrophages isolated from PBMC were stimulated by LPS (1 g/m ⁇ ) and the culture supernatants were assayed for IL-6 and TNF- ⁇ production.
  • the preliminary time course experiment showed that 8 hours after stimulation was optimal for LPS-stimulated proinflammatory cytokine production by macrophages. Therefore, a different time point was chosen from the case with T cells, which was 24 hours after stimulation.
  • RXM inhibited both IL-6 and TNF- ⁇ production by macrophages at a dose dependent manner.
  • T cell migration was significantly inhibited in a range from 14 ⁇ M to 28 ⁇ M in a dose-dependent manner when RXM was present during the endothelial migration assay (p ⁇ 0.05).
  • RXM at dose of 1.4 ⁇ M did not inhibit T cell migration at 5 different donors but from 14 ⁇ M to 28 ⁇ M of RMX, T cell migration was always inhibited.
  • pretreated ECV304 with various concentrations of RXM for 48 hours, and then washed and exposed to PHA-stimulated T cells RXM did not affect the migration of T cells through ECV304 even at the highest concentration tested (28 ⁇ M) (data not shown).
  • RXM 100 ⁇ g, 200 ⁇ g, 400 ⁇ g, and 800 ⁇ g treated mice and control mice, clear statistical differences of suppressing disease scores were observed (p ⁇ 0.05 and p ⁇ 0.01). It should be noted that in RXM 400 ⁇ g/day and 800 ⁇ g/day groups, disease scores were markedly inhibited, but the differences in the disease scores between both groups did not reach statistical differences after 14 days treatment.
  • Example 8 Effect of RXM treatment on the serum IL-6, TNF- ⁇ , IL-4 and IFN- ⁇ levels and the type II collagen antibody levels
  • type II collagen antibody levels (Fig.SC)
  • a type II collagen antibody was detected after day 7 on the second immunization of collagen, but RXM treatment did not affect the serum titer of this antibody. Therefore, RXM treatment, or especially, 400 ⁇ g and 800 ⁇ g RXM treatment significantly inhibited the production of IL-6 in the serum of CIA mice.
  • Example 9 ISM inhibits leukocyte migration and bone destruction in affected joints off CIA mice Based on the findings that RXM inhibits the development of CIA as well as serum levels of IL-6, immunohistochemical analyses were performed on the inflamed joints and synovial tissue from those mice.
  • Example 10 Effect of RXM treatment on patients with rheumatoid arthritis
  • RXM inhibited T cell migration, it was postulated that it may also be useful in the treatment of RA. Likewise, the prevention of the development of CIA in mice by RXM and inhibition of in vivo serum IL-6 strongly support the suggestion that RXM may be effective for the treatment of RA.
  • RA rheumatoid arthritis
  • This patient was treated with 6 mg of predonine, 1000 mg Azurufizin EN per day and 8 mg MTX per week. Since her serum levels of C-reactive protein (CRP) were continuously high (1.8 to 2.2 ⁇ g/ml) and RA activity was increased, she received 150 mg Roxithromycin per day in addition to the above medications. One month later, her CRP level was decreased from 1.8 to 1.5 ⁇ g/ml and some clinical improvement of RA activity was observed.
  • CRP C-reactive protein
  • This patient was treated with 5 mg predonine, 1000 mg Azurufizin EN, 200 mg Rimatil per day and 6 mg MTX per week.
  • Proximal interphalgeal (PIP) joint swelling and tenderness was observed in his right third and fourth fingers as well as his left third finger. Accordingly, he received 150 mg Roxithromycin per day in addition to the above medications.
  • PIP Proximal interphalgeal
  • This patient was treated with 5 mg predonine, 1000 mg Azurufizin EN per day and 7.5 mg MTX per week. Since left wrist joint swelling and tenderness was observed and her serum
  • CRP levels were increased to 0.6 ⁇ g/ml, she received 150 mg Roxithromycin per day in addition to the above medications. One month later, her CRP level was down to 0.4 ⁇ g/ml and left wrist joint swelling and tenderness were decreased.
  • This patient was treated with 8 mg predonine, 1000 mg Azurufizin EN and 200 mg Rimatil per day. Since she presented with multiple joint swelling and tenderness with a serum
  • the present inventor demonstrated that RXM clearly inhibited production of the proinflammatory cytokines, TNF- ⁇ and IL-6 by T cells and macrophages, with virtually no effect on the production of the Thl type cytokines IL-2 and IFN- ⁇ and Th2 type cytokines IL-4 and IL-5 by T cells stimulated by costimulatory stimuli such as CD28 and CD26.
  • RXM inhibited T cell migration.
  • RXM treatment of CIA mice showed that RXM inhibited the development of CIA, serum IL-6 levels and the migration of leukocytes into affected joint or synovial membrane and the destruction of bones and cartilages.
  • CD28 and CD26 Their costimulations for CD28 and CD26 are used for anti-CD28 from Genzyme and anti-CD26 from Biodesign but the instant CD28 and CD26 mAbs arc 4B10 mAb and 1F7 mAb that have been developed by the inventor. Moreover, others investigators have reported that other macrolides such as midecamycin, clarithromycin and josamycin inhibited both Thl type and Th2 type cytokine such as IL-2, IL-4 and IL-5 productions by T cells stimulated by ConA (31). Regarding proinflammatory cytokine production such as TNF- ⁇ , EM and RXM are reported to inhibit TNF- ⁇ production by macrophages by LPS-stimulation (32, 33).
  • other macrolides such as midecamycin, clarithromycin and josamycin inhibited both Thl type and Th2 type cytokine such as IL-2, IL-4 and IL-5 productions by T cells stimulated by ConA (31).
  • RXM inhibited TNF- ⁇ production by LPS-stimulated macrophages (32, 33)
  • the present inventor are the first report that RXM specifically inhibited proinflammatory cytokine production such as IL-6 and TNF- ⁇ by T cells stimulated by several costimulatory stimuli such as CD28 and CD26 but did not inhibit IL-2, TNF- ⁇ , IL-4 and IL-5 by T cells.
  • RXM treatment inhibited the development of arthritis in CIA mice as well as in vivo serum level of IL-6 in CIA mice in a dose dependent manner.
  • bronchial asthma is a T cell-mediated inflammatory disorder and selective recruitment of CD4 T cells into sites of inflammation may contribute to the development of different pathological conditions (36, 37).
  • Current studies suggest that TNF- ⁇ which is produced in considerable quantities in asthmatic airways, may potentially be involved in the development of bronchial hyper-responsiveness by directly altering the contractile properties of the airway smooth muscle (ASM) (38, 39).
  • IL-6 has regulation of IgE synthesis. Increased levels of IL-6 have been detected in the blood and bronchoalveolar lavage after bronchial challenge of patients with asthma and bronchial biopsies of these patients reveal an increased expression of IL-6 (40).
  • TNF- ⁇ and IL-6 production by T cells and macrophages may also have important therapeutic implications for bronchial asthma. It is reported that EM suppresses NF- KB activation in T cells (41). Since NF- ⁇ B is involved in gene expression for a variety of mediators including IL-2, IL-6, IL-8, TNF- ⁇ and GM-CSF (42), therefore it is conceivable that RXM also appears to suppress NF- ⁇ B activation. Further studies are needed to define the mechanism of the specific inhibition of IL-6 and TNF- ⁇ but not IL-2 by RXM.
  • CIA joint inflammation and cartilage and bone destruction depend on the level of TNF- ⁇ and IL-6 in the affected joints (42, 55).
  • RXM inhibited the development of CIA through inhibiting the production of proinflammatory cytokines such as IL-6 and TNF- ⁇ as well as leukocyte migration.
  • the effectiveness of RXM in inhibiting the development of CIA is supported by the fact that RXM inhibited in vitro proinflammatory cytokines production by T cells and macrophages as well as T cell migration.
  • TNF- ⁇ has been validated as a good target for treatment and to date, two biological agents that target TNF- ⁇ have been licensed for clinical use (42). These are inflixmab, anti-TNF- ⁇ mAb (45) and etanercept, an engineered p75 TNF receptor dimmer linked to the Fc portion of human IgGl (46).
  • therapies inhibiting TNF- ⁇ in patients with active RA result in rapid and sustained improvement in symptoms and signs of disease, improvement in the quality of life and protection of joints from structural damage (42, 47).
  • anti-TNF- ⁇ treatment has been reported in effective in Crohn's disease (48).
  • IL- 6 regulates the production of acute-phase proteins by hepatocytoes and activates osteoclasts to absorb bone (49, 50).
  • a humanized anti-IL-6R mAb has been used to treat patients with severe RA and clinical improvements have been reported (51).
  • T cells and macrophages at the inflammatory sites of such diseases and proinflammatory cytokines as TNF- ⁇ and IL-6 play a key role in triggering and maintaining pathophysiology of the above disease
  • P CM may be effective in treating these disorders. Effectiveness of RXM in inhibiting the development of CIA mice and suppression of serum IL-6, inhibiting leukocyte migration and bone and cartilage destruction strongly support the above conclusion.
  • TNF- ⁇ may play a role in the pathophysiology of disease
  • RXM may also be useful for treatment of such conditions.
  • roxithromycin specifically inhibits pro- inflammatory cytokine productions by T cells and macrophages; inhibits T cell migration; inhibits the development of collagen-induced arthritis; inhibits serum IL-6 levels; inhibits the migration of leukocytes into affected joint or synovial membrane and the destruction of bones and cartilages in a mouse model of CIA; and ameliorates clinical symptoms in patients with RA.
  • macrolide antibiotics such as roxithromycin and derivatives thereof, have multiple research and clinical utilities.
  • the macrolide antibiotic compositions of the present invention may be used to treat or prevent diseases or disorders associated with transendothelial migration of T cells and activated T cells, pro-inflammatory cytokine production from T cells, or IL-6 production from macrophages.
  • compositions of the present invention find utility in treating or preventing arthritic or rheumatic disorders including, but not limited to, rheumatoid arthritis; osteoarthritis, and infectious, psoriatic and/or viral arthritis; Crohn's disease; graft-versus-host disease after allo-bone marrow transplantation; heart failure; graft rejection; atrial myxoma; multiple myeloma; Castleman's disease; glomerulonephritis including mesangial proliferative glomerulonephritis; osteoporosis; EBV-positive lymphoma; systemic lupus erythmatosis; collagenosis; ulcerative colitis; autoimmune hemolytic anemia; hepatitis including active chronic hepatitis; gout; arlherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; ankly
  • the macrolide antibiotic of the present invention may be administered alone or in combination with other therapeutic agents and administered orally, systemically, via an implant, intravenously, topically, or intrathecally.
  • B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2, Proc Natl Acad Sci U S A 88, 6575-9., 1991.
  • Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6, Proc Natl Acad Sci U S A
  • Negrusz-Kawecka M., [The role of TNF-alpha in the etiopathogenesis of heart failure],

Abstract

L'invention concerne en général la modulation de réponses immunitaires par administration de compositions comprenant des antibiotiques de macrolide tels que la roxithromycine, et l'utilisation de ces compositions dans des méthodes permettant de traiter ou de prévenir des maladies ou des troubles impliquant une migration transendothéliale de lymphocytes T et de lymphocytes T activés, la production de cytokine pro-inflammatoire à partir de lymphocytes T et la production de IL-6 à partir de macrophages. L'invention concerne également l'utilisation de ces compositions antibiotiques de macrolide pour traiter ou prévenir les troubles arthritiques et rhumatoïdes, tels que l'arthrite rhumatoïde, et pour inhiber le développement ou la progression d'un ou de plusieurs symptôme(s), tels qu'un trouble impliquant un oedème articulaire, une prolifération synoviale et une douleur à la pression, la formation d'un pannus synovial et la destruction osseuse et cartilagineuse.
PCT/JP2004/004049 2003-03-24 2004-03-24 Modulation de reponses immunitaires par administration de roxithromicine ou de son derive WO2004084911A2 (fr)

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CN102095697A (zh) * 2010-12-07 2011-06-15 桂林理工大学 一种测定罗红霉素的方法
WO2017174593A1 (fr) 2016-04-07 2017-10-12 Universite Claude Bernard Lyon 1 Nouvelles compositions antivirales pour le traitement de la grippe

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