WO2009023196A1 - Modulation of immune responses by administration of roxithromycin or its derivative - Google Patents

Modulation of immune responses by administration of roxithromycin or its derivative Download PDF

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Publication number
WO2009023196A1
WO2009023196A1 PCT/US2008/009642 US2008009642W WO2009023196A1 WO 2009023196 A1 WO2009023196 A1 WO 2009023196A1 US 2008009642 W US2008009642 W US 2008009642W WO 2009023196 A1 WO2009023196 A1 WO 2009023196A1
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Prior art keywords
hexopyranosyloxy
methyl
olide
trideoxy
xylo
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PCT/US2008/009642
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French (fr)
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Chikao Morimoto
Peter H. Cheung
Kazuo Tsuzuki
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Y's Therapeutics Co, Ltd.
The University Of Tokyo
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Priority to JP2010520002A priority Critical patent/JP2010535776A/en
Priority to EP08795247A priority patent/EP2188281A1/en
Priority to CA2733706A priority patent/CA2733706A1/en
Publication of WO2009023196A1 publication Critical patent/WO2009023196A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/08—Antibacterial agents for leprosy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins

Definitions

  • the present invention relates generally to compounds useful for modulation of immune responses, compositions comprising the compounds, and methods of use of such compositions for treating or preventing diseases or disorders associated with modulation of immune responses.
  • EM erythromycin
  • Roxithromycin a new macrolide antibiotic
  • RXM 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).
  • New macrolide antibiotics are described in WO2004/084911 and (70-71).
  • 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). Thus, there is increasing evidence that macrolides have a variety of biologic activities, apart from their antibacterial actions (17). [0006] In the initial stage of immune response, a certain antigen is engaged by a T cell receptor (TcR), followed by various cytokine release. However, this process alone is not sufficient to induce all events that accompany T cell activation.
  • TcR T cell receptor
  • costimulatory signals that occur through additional T cell surface molecules, which are independent of the CD3/TcR (18). These 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 (19-21).
  • CD26 a novel costimulatory molecule, that is preferentially expressed on CD4 + memory T cells (19, 20, 22-24), 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 (25) (26).
  • RXM was also 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, therapy by roxithromycin and its derivatives 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 but not limited to an 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; ence
  • the present invention relates to a compound of the following formula (I):
  • Rl is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-IO alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
  • R2 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-10 alkyloxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl
  • R3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when Rl is hydrogen or methyl, and that R3 is not cladinose when Rl is methyl and R2 is methyl; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
  • R 1 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
  • R 2 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-IO alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible
  • R 3 is selected from the group consisting of cladinose, Cl-IO alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-IO alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S.
  • Rl is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkyloxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
  • R2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkyloxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-5 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 al
  • R3 is selected from the group consisting of cladinose, C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when Rl is hydrogen or methyl, and that R3 is not cladinose when Rl is methyl and R2 is methyl.
  • R 1 is selected from the group consisting of hydrogen, C2-5 alkenyl, C2-C5 alkynyl, Cl- 5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6- 10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
  • R 2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R 1 and R 2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 alkyn
  • R 3 is selected from the group consisting of cladinose, C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R 3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
  • R 1 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, Cl-5 alkylcarbonyl, Cl-5 alkoxycarbonyl, Cl-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
  • R 2 is selected from the group consisting of hydrogen, Cl-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, Cl-5 alkylcarbonyl, Cl-5 alkoxycarbonyl, Cl-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R 1 and R 2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-5 alkoxy, carboxyl, Cl-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8
  • R 3 is selected from the group consisting of Cl-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-5 alkoxy, carboxyl, Cl-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
  • the present invention relates to a compound of the following formula (II):
  • Xl is selected from the group consisting of:
  • X2 is selected from the group consisting of:
  • said compound is not roxithromycin (5-(3,4,6-Trideoxy-3- dimethylamino- ⁇ -D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O-methyl- ⁇ -L- ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2-rnethoxyethoxy)methoxyimino-
  • the present invention relates to a compound of the following formula (III):
  • R , ⁇ is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-IO alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-IO alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
  • R 2 is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, and
  • R 3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R 2 is not hydrogen when R 1 is hydrogen or methyl, and that R 3 is not cladinose when R 1 is methyl and R 2 is methyl; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
  • the present invention relates to a compound selected from the group consisting of:
  • the present invention relates to a compound selected from the group consisting of:
  • the present invention relates to a compound selected from the group consisting of:
  • the present invention is the compound of 5-(3,4,6-Trideoxy-3- benzenesulfonamido- ⁇ -D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O-methyl- ⁇ -L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2-methoxyethoxy)methoxyirnino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide.
  • the present is the compound of 5-(3,4,6-Trideoxy-3- dimethylamino- ⁇ -D-xylo-hexopyranosyloxy)-3-benzoyloxy-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6, ⁇ , 10, 12-hexamethylpentadecan- 13-olide.
  • the compound of formula (IV) may be a compound of formula (IV):
  • the compound of formula (H) in another embodiment, alternatively, may be a compound of formula (V):
  • the present invention also provides a pharmaceutical composition for the treatment of a bacterial infection in a mammal, comprising a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition for the treatment of a disorder in which proinflammatory cytokine production is implicated in a mammal, comprising a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier.
  • a method of treating a bacterial infection in a mammal comprising administering to said mammal a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier, is provided.
  • a method of treating a disorder in which proinflammatory cytokine production is implicated in a mammal comprising administering to said mammal a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier, is provided.
  • the present invention relates to use of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof for treating a bacterial infection in a mammal.
  • the present invention relates to use of the above compound or a pharmaceutically acceptable salt, prodrug for treating a disorder in which proinflammatory cytokine production is implicated in a mammal.
  • the disease or disorder to be treated or prevented using the compound, composition and methods of the invention is an infectious disorder such as bacterial, fungal or viral infection, or an arthritic or rheumatic disorder, 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; artherosclerosis; psoriasis; atopic dermatiti
  • the one or more compounds of the invention 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 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 in conjunction with a therapeutically effective amount of one or more non- macrolide antibiotics, and/or a therapeutically effective amount of one or more antiinflammatory 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, WY-50295, SC-45662, SC-41661A, BI-L-357; leukotriene antagonists; peptide derivatives comprising ACTH and analogs thereof; soluble TNF-receptors; TNF- antibodies; soluble receptors of interle
  • 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 antibiotics comprise a 14-member macrocylcine 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 antiinflammatory 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 antibiotics comprise a 14-member macrocylcine 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 antibiotics comprise a 14-member macrocylcine 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 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 antiinflammatory 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 antibiotics comprise a 14-member macrocylcine 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-member macrolide antibiotic.
  • the 14-member 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- member macrolide antibiotic.
  • the 14-member 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.
  • 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 proinflammatory 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. In another embodiment, 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.
  • 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;
  • 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 present invention provides a method of treating a disease or disorder associated with modulation of immune response comprising administering a macrolide antibiotic that has an antibiotic activity less than roxithromycin and an increased inhibition of TNFalpha and IL-6 production from T cells and macrophages in comparison to roxithromycin.
  • the macrolide antibiotic can have one or more of the following properties: an IC50 of less than about 15 ⁇ M for TNFalpha; less than about 12 ⁇ M for IL-6; or a minimum inhibiting concentration of at least about 100 ⁇ M for Staphylococcus aureus FDA 209P.
  • the present invention provides a use of a compound of formulae (I)-(V) or a pharmaceutically acceptable salt or prodrug thereof for treating a bacterial infection or a disorder in which proinflammatory cytokine production is implicated in a mammal.
  • the present invention includes methods of making a compound of formulae (I)-(V) using the methods described herein.
  • 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.
  • 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.
  • Figure 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.
  • Figure 5 depicts the effect of RXM on the production of IL-6 and TNF- ⁇ by macrophages stimulated with LPS.
  • Figure 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 8A-8C 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.
  • Figure 10 depicts the effect of RXM and Its derivatives on cytokine production of IL-2, IL- 17A and INF- ⁇ .
  • Figure 11 depicts the effect of RXM and Its derivatives on cytokine production of IL-6 and TNF- ⁇ .
  • Figure 12 depicts the effect of RXM and Its derivatives on cytokine production of IL-4, IL-5 and IL-10.
  • 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 tendonitis (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.
  • Cl-10 alkyl means a branched or straight carbon chain having 1 to 10 carbons, and includes for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neo-pentyl, isopentyl, 1,2- dimethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 1-ethylbutyl, 2- ethylbutyl, isoheptyl, octyl, or isooctyl, etc.
  • it is the one having 1 to 5 carbons and for example, is methyl or ethyl.
  • C2-10 alkenyl includes straight-chain or branched-chain mono- or poly-unsaturated aliphatic hydrocarbon radicals having 2 to 10, for example 2-5 carbons containing at least one carbon-carbon double bond.
  • alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E 5 Z-, Z 5 E- and Z,Z-hexadienyl and the like.
  • C2-10 alkynyl includes straight-chain or branched-chain mono- or poly-unsaturated aliphatic hydrocarbon radicals having 2 to 10, for example 2-5 carbons containing at least one carbon-carbon triple bond.
  • alkynyl radicals include, but are not limited to, ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like.
  • Cl-10 alkylcarbonyl means a carbonyl group having a straight or branched carbon chain having 1 to 10 carbons, and includes for example, hormyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, or octanoyl, etc.; for example, is the one having 1 to 5 carbons; and for example, is acetyl or propionyl.
  • Cl-10 alkoxycarbonyl means alkoxycarbonyl having 1 to 10 carbons, and includes methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, isopentyloxycarbonyl, neopentyloxycarbonyl, hexyloxycarbonyl, heptyloxycarbonyl, or octyloxycarbonyl, etc.; for example, is methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl; and for example, is methoxycarbonyl.
  • Cl-10 alkylsulfonyl means an alkylsulfonyl group having 1 to 10 carbons, and includes particularly, methylsulfonyl, ethylsulfonyl, butylsulfonyl, hexylsulfonyl, or octylsulfonyl, etc., and for example, is methylsulfonyl.
  • C6-C10 arylsulfonyl means an arylsulfonyl group having 6 to 10 carbons, such as phenylsulfonyl (SO 2 Ph), 1 -naphthylsulfonyl, or 2-naphthylsulfonyl.
  • C3-8 cycloalkyl means a cycloalkyl group having 3 to 8 carbons, and includes particularly, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl; and is for example cyclopropyl.
  • aryl means particularly a carbocyclic aryl group such as phenyl or naphthyl, or heteroaryl such as pyridyl or furyl, and for example, is phenyl.
  • arylcarbonyl means benzoyl, 4-methoxybenzoyl, or 3- trifluoromethylbenzoyl, etc., and for example, is benzoyl.
  • aryloxycarbonyl means phenoxycarbonyl, naphthyloxycarbonyl, 4- methylphenoxycarbonyl, 3-chlorophenoxycarbonyl, or 4-methoxyphenoxycarbonyl, etc.; and for example, is phenoxycarbonyl.
  • aralkoxycarbonyl means benzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, or 3-trifluoromethylbenzyloxycarbonyl, etc.; and for example, is benzy loxy carbony 1.
  • Compound (I) and compound (II) according to the present invention may form acid addition salts. Further, it may form salts with bases, depending on the species of the substituent.
  • These salts are not restricted insofar as they are pharmaceutically acceptable, and include particularly, mineral salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate or sulfate, etc.; organic sulfonates such as methanesulfonate, 2- hydroxyethanesulfonate or p-toluenesulfonate, etc.; and organic carbonates such as acetate, trifluoroacetate, propionate, oxalate, citrate, malonate, succinate, glutarate, adipate, tartrate, maleate, malate, or mandelate, etc.
  • salts with bases salts with inorganic bases such as sodium salts, potassium salts, magnesium salts, calcium salts or alminium salts, and salts with organic bases such as methylamine salts, ethylamine salts, lysine salts or ornithine salts, etc. are included.
  • phrases "pharmaceutically acceptable salt(s)" includes salts of acidic or basic groups which may be present in the compounds of the present invention.
  • the compounds of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • the acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, ptoluenesulfonate and pamoate salts.
  • the compounds of the present invention that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in
  • Those compounds of the present invention that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations.
  • Examples of such salts include the alkali metal or alkaline earth metal salts and, particularly, the calcium, magnesium, sodium and potassium salts of the compounds of the present invention.
  • Certain compounds of the present invention may have asymmetric centers and therefore exist in different enantiomeric and diastereomic forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of the present invention, and mixtures thereof, and to all pharmaceutical compositions and methods of treatment that may employ or contain them.
  • the present invention includes the compounds of the present invention, and the pharmaceutically acceptable salts thereof, wherein one or more hydrogen, carbon or other atoms are replaced by isotopes thereof.
  • Such compounds may be useful as research and diagnostic tools in metabolism pharmacokinetic studies and in binding assays.
  • the compounds of this invention including the compounds of formula (I) or (II) include pharmaceutically acceptable derivatives or prodrugs thereof.
  • a "pharmaceutically acceptable derivative” or “pharmaceutically acceptable prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention that, upon administration to a recipient, is capable of providing directly or indirectly a compound of this invention or a metabolite or residue thereof.
  • Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient, enhance delivery of the parent compound to a given biological compartment, increase solubility to allow administration by injection, after metabolism or alter rate of excretion.
  • Compounds of this invention described herein such as the compounds of formula (I) and (II) can be converted into prodrugs through, for example, free amino, amido, hydroxy or carboxylic groups.
  • prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of a compound of formula I.
  • amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-am inobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.
  • prodrugs can be derivatized as amides or alkyl esters.
  • the amide and ester moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.
  • Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates and phosphoryloxymethyloxycarbonyls, as outlined in D. Fleisher et al., Advanced Drug Delivery Reviews, 19:115 (1996).
  • Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs and sulfate esters of hydroxy groups.
  • the compounds of this invention also include pharmaceutically acceptable salts of the compounds described herein, such as the compounds of formulae (I) and (II).
  • pharmaceutically acceptable salt as used herein, unless otherwise indicated, includes salts of acidic or basic groups that may be present in the compounds of the present invention.
  • 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; artherosclerosis; psoriasis
  • 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.
  • the phrases “treating or preventing” and “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 for inhibiting IL-6 production from macrophage comprising contacting one or more macrophages with one or more macrolide antibiotics.
  • a method 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 14-membered macrolide antibiotic or derivatives thereof, as previously defined above, in the manufacture of a medicament to treat or prevent an arthritic or rheumatic disorder, 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; artherosclerosis; ps
  • 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-KB 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- ⁇ B 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. Several amino and/or neutral sugars are preferably fixed to the lactone ring.
  • One group of macrolide antibiotics are 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 can be a functional derivative of roxithromycin.
  • 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.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1); Imipenem (CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.: 78110-38-0); Cepha
  • Ciprofloxacin (CAS Registry No.: 85721-33-1); Ofloxacin (CAS Registry No. 82419-36-1); Enoxacin (CAS Registry No.: 74011-58-8); Lomefloxacin (CAS Registry No. 98079-51-7); Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No. 564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CAS Registry No. 60-54-8); Amikacin (CAS Registry No.: 37517-28-5); Gentamicin (CAS Registry No.:
  • Kanamycin (CAS Registry No.: 8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CAS Registry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1); Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CAS Registry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8); Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethyl succinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate (CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS Registry No.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643- 22-1); Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.: 61036- 64-4); Chloramphenicol (CAS Registry No.
  • Anti-fungal agents include, but are not limited to, terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid, and selenium sulfide.
  • Anti-viral agents include, but are not limited to, amantadine hydrochloride, rimantadin, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine, valacyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine.
  • Anti-parasitic agents include, but are not limited to, pirethrins/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.,
  • 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-41661 A, BI-L-357; leukotrien
  • peptide derivatives e.g., ACTH and analogs; soluble TNF-receptors; TNF-antibodies; soluble receptors of interleukines, other cytokines, T-cell-proteins; antibodies against receptors of interleukines, other cytokines, and T-cell-proteins.
  • 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.
  • 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.
  • unit dose is meant to describe a single dose, although a unit dose may be divided, if desired.
  • any suitable route of administration may be employed for providing the patient with an effective dosage of the composition according to the methods of the present invention, oral administration is preferred.
  • Suitable routes include, for example, oral, rectal, parenteral (e.g., in saline solution), intravenous, topical, transdermal, 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.
  • 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, choline, diethanolamine, ethylenediamine, meglumaine (N-methylglucamine), 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 the 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.
  • 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.
  • FIG. 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).
  • FIG 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 RPMII640), PHA-activated T ceils 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 800 ⁇ 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.
  • Figure 10 depicts the effect of RXM and its derivatives on the production of IL-2, IL-17 A and IFN- y 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. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
  • FIG 11 depicts the effect of RXM and its derivatives on the production of IL-6 and TNF- a 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- a by ELISA.
  • the cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
  • FIG 12 depicts the effect of RXM and its derivatives on the production of IL-4, IL-5 and IL-IO 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, IL-5 and IL-10 by ELISA.
  • the cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
  • 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 or by Sigma (R4393) and was dissolved in DMSO or methanol (WAKO, #137- 01823) and further diluted in the culture media consisting of RPM 1-1640 and 10% fetal calf serum (FCS).
  • Derivatives of RXM was synthesized by Nard Institute Co. Ltd., Japan and was dissolved in DMSO or methanol and further diluted in the culture media consisting of RPMl- 1640 and 10% fetal calf serum (FCS).
  • PBS phosphate-buffer saline
  • the cell suspension supplemented with 5 ng/ml of PMA was applied into OKT3-coated wells.
  • Cells were incubated at 37°C in 5% CO 2 humidified atmosphere for 3 days.
  • Cells were pulsed with 1 ⁇ Ci/well of [ 3 H]-thymidine (ICN Radiochemicals, Irvine, CA) 8 hours prior to harvest onto a glass-fiber filter (Wallac, Turku, Finland), and the incorporated radioactivity was quantitated by a liquid scintillation counter (Wallac).
  • 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.
  • IL-2 and IL-4 Biosource International, Camarillo, CA; IFN- ⁇ and IL-5: R&D systems, Minneapolis, MN; IL-17A: eBIOSCIENCE,#88-7176-88; IL-IO: BD, #555157
  • IL-2 and IL-4 Biosource International, Camarillo, CA
  • IFN- ⁇ and IL-5 R&D systems, Minneapolis, MN
  • IL-17A eBIOSCIENCE,#88-7176-88
  • IL-IO BD, #555157
  • TNF- ⁇ and IL-6 production by macrophages macrophages were enriched from E cells by adherence to plastic plates.
  • Macrophages (1 x 10 6 /ml) were suspended in 10% FCS-RPMI 1640 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 ELlSA kit described above.
  • Transendothelial migration activity was assessed using a kind of boyden-chamber assay as described previously with some modifications (39).
  • 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 Costar, Cambridge, MA), for 48 hours.
  • RXM was first dissolved in DMSO and further diluted in the assay medium consisting of RPMI 1640 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 6 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.
  • PHA-activated T cells were incubated with or without the indicated doses of RXM at 37 0 C for 8 hours in 5% CO 2 /95% air and then washed. All of the cells were incubated with mAb against various cell surface molecules (1 ⁇ g/ml) on ice for 30 minutes. The cells were further incubated with fluorescein isothiocyanate labeled goat anti-mouse IgG (Sigma) after being washed 3 times with PBS containing 2% newborn calf serum and 0.02% sodium azide (Sigma).
  • the mAbs used for this experiment were anti-CD3 (OKT3), anti-CD 1 Ia (BD PharMingen, NJ, USA), anti-CD25 (BD PharMingen), anti-CD26 (1F-7), anti-CD29 (4B4), anti-CD44 (ED PharMingen), and anti-CD47 (BD PharMingen).
  • mice Male DBA/1 J mice were purchased from Japan Charles River Breeding Laboratories (Tokyo, Japan). Bovine type Il 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). For the primary immunization, 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.
  • DIFCO Complete Freund's Adjubant
  • the legs were scored as follows: 0, normal; 1, erythema and mild swelling confined to the ankle joint or toes; 2, erythema and mild extending from the ankle to the midfoot; 3, erythema and severe swelling extending from ankle to the metatarsal joints; 4, ankylosing deformation with joint swelling (67).
  • the disease score for each mouse was calculated as the sum of the scores for the two hind legs.
  • RXM was dissolved in 5% Alabic Gum in 0.9% NaCl and different doses of RXM (lOO ⁇ g, 200 ⁇ g, 400 ⁇ g, and 800 ⁇ g) in 5% Alabic Gum in 0.9% NaCl were orally given to 5 different groups comprising 8 mice. 5% Alabic Gum in 0.9% NaCl alone were also orally given to control mice group. The RXM or 5% Alabic Gum in 0.9% NaCl was orally given to mice every day up to day 14 after second immunization of type II collagen.
  • 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 0 C.
  • Tissue-Tec ornithine calbamyl transferase compound Miles, Elkhart, IN
  • the sections were then incubated with 10% normal goat serum for Ih at room temperature and treated with rabbit anti-human IFN - ⁇ Ab, rabbit anti mouse TNF- ⁇ Ab, or normal rabbit serum overnight at 4 0 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.
  • Example 1 Effect of RXM on T cell proliferation through different costimulatory 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.
  • AU 3 stimuli were combined with submitogenic doses of anti-CD3 mAb.
  • Purified 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
  • Th-2 type CD4 + T cells may play a role in allergic disorders such as asthma (27, 28)
  • the effect of RXM on the Th2-type cytokine production of IL-4 and IL-5 was examined. As shown in Fig. 3A and B, RXM did not inhibit IL-4 and IL- 5 production under each costimulatory conditions at any tested doses (1.4 ⁇ M to 28 ⁇ M). Thus, the results herein demonstrated that RXM did not inhibit the Th-2 type cytokine production in the instant experimental systems.
  • Example 4 Effect of RXM on proinflammatory cytokine production through different costimulatory pathways
  • 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.
  • Fig.8A serum IFN- ⁇ levels
  • Fig.8B serum IL-6 levels were increased in control CIA mice on day 7, and then decreased and after day 14 reached to the undetectable level.
  • serum IL-6 levels were inhibited on day7 in a dose dependent manner, and especially in 400 ⁇ g and 800 ⁇ g RXM treated CIA mice, serum IL-6 levels were significantly inhibited on day7 (p ⁇ 0.05).
  • Serum IL-4 and TNF- ⁇ levels could not be detected due to the low sensitivity of detection kits in these CIA mice groups.
  • type II collagen antibody levels could not be detected due to the low sensitivity of detection kits in these CIA mice groups.
  • 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 RXM inhibits leukocyte migration and bone destruction in affected joints of CIA mice
  • Roxithromycin 1 (2.0 g, 2.39 mmol) was dissolved 4:1 MeOH:H 2 O (35 mL) and sodium acetate (0.98 g, 11.95 mmol) was added. The mixture was stirred under argon while being heated to 51°C(degrees Celsius). After about 3 min, solid iodine (0.595 g, 2.34 mmol) was added to the mixture. The solution turned intense orange color. In approximately 10 min, IM NaOH (700 ⁇ L) was added to the reaction mixture. After another 20 min, another portion of IM NaOH (700 ⁇ L) was added, and then in 30 min an additional portion of IM NaOH (350 ⁇ L) was added.
  • the mixture was stirred under argon while being heated to 51°C(degrees Celsius). After about 3 min, solid iodine (0.112 g, 0.442 mmol) was added to the mixture. The solution turned intense orange color. In approximately 10 min, 150 ⁇ L of IM NaOH was added to the reaction mixture. After another 20 min, another portion of 150 ⁇ L of IM NaOH was added, and then in 30 min a 75 ⁇ L portion of IM NaOH was added. The mixture was stirred at 51°C(degrees Celsius) for 5 h, at which point it became almost colorless. The reaction mixture was then poured into 500 mL of 5% NH 4 OH solution.
  • a sample for biological testing was further purified by recrystallization.
  • Free base (20 mg) was dissolved in of acetone (ca. 0.5 mL) and 10% NH 4 OH solution (15 mL) was added. The solution was allowed to stand for 48 h at 0 °C(degrees Celsius), resulting in formation of colorless crystals, which were filtered and dried in vacuo, yielding about 3 mg of colorless solid.
  • a sample for biological testing was purified as follows: Crude 4 (252 mg, 0.311 mmol) and Et 3 N (0.43 mL, 3.1 mmol) in CH 2 Cl 2 at 0 0 C was treated with trifluoroacetic anhydride (0.14 mL, 0.96 mmol), and the mixture was allowed to warm to ambient temperature. After 2h, the mixture was washed with saturated aqueous NaHCO 3 , dried (Na 2 SO 4 ), filtered, and concentrated. The residue was dissolved in anhydrous MeOH and allowed to stand at ambient temperature for 18 h.
  • the product was shown to be free of the N-Me analog by 1 H - ⁇ MR, which showed absence of the N-Me singlet at 2.22 ppm that is observed for the authentic N-Me analog. Spiking the product with the N-Me analog, and taking the 1 H-NMR spectrum of the mixture demonstrated that this singlet was clearly resolved from other signals.
  • PBMC peripheral blood mononuclear cells
  • 96-welled plates (MaxiSorp, #460984 from Nalge NUNC International, Rochester, NY) were sterilized with 70% ethanol and then coated with 100 ⁇ l of PBS containing an anti-huCD3 mAb, 1 ⁇ g/ml (OKT3 with #16-0037 from eBioscience, San Diego CA) and 5 ⁇ g/ml of an anti-huCD28 mAb, (#MAB342 from R&D System, Minneapolis, CA). The plates were covered and store at 4° over night. At day 1, the plates were washed 3x with 200 ⁇ l of PBS each time.
  • PBMC Allcells, #PB003F, Berkeley, CA
  • PBMC Allcells, #PB003F, Berkeley, CA
  • Cell density and viability were determined using a hemocytometer and trypan blue dye.
  • the vial content were transferred to a 50 ml conical tube (#21008-178, VWR International) with 5 ml of pre-warmed media (RPMI- 1640, Media Tech with 10% FBS and benzonase nuclease #80108-808, EMD bioscience at 228 ⁇ l per liter of media) added.
  • the media volume was adjusted to 20 ml accordingly.
  • the cells were centrifuged at 20Og for 15 minutes (Sorvall Legend R/T # 75004377 at 900 rpm).
  • This assay is based on the liquid dilution method.
  • the test compounds were dissolved in dimethylsulfoxide (DMSO) to reach a final concentration of 10 mM, and several diluted aliquots were prepared from the 10 mM solution and stored at -20°C(degrees Celsius).
  • the strain of Staphylococcus aureus FDA 209P was incubated in 117 micrococcus medium (Ml 17).
  • the inhibition of growth was determined by transillumination using microplate reader (settings: OD492) which allowed the minimum inhibiting concentrations (MIC) to be evaluated expressed in microM format. The results were summarized in Table 1.
  • test compounds were dissolved in DMSO to reach a final concentration of lmg/mL and stored in a refrigerator.
  • the bacterial strains of Enterococcus and Staphylococcus, Streptococcus, Haemophilus and Moraxella were incubated in Mueller Hinton Broth and Brain Heart Infusion, respectively, and the seeding culture was adjusted to 10 6 CFU/ml in two-fold concentration of the growth medium.
  • the DMSO solution of test compound was diluted in two-fold series manner with distilled water, and transferred into assigned well of microplate (lOOmicroL/well).
  • each well was seeded with lOOmicroL of the culture and after incubating for twenty-four hours at 37°C(degrees Celsius), the inhibition of growth was determined by transillumination using microplate reader (Measurement Filter 540 nm) which allowed the MIC to be evaluated expressed in micrograms/mL. In addition the growth inhibition was confirmed by visual inspection. The results were shown in Table 2. Table 2.
  • Example 13 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
  • roxithromycin 150mg per day
  • 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 C-reactive protein (CRP)
  • 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 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 CRP level of 2.7 ⁇ g/ml, she received 150 mg Roxithromycin per day in addition to the above medications. One month later, her CRP level was down to 1.9 ⁇ g/ml and multiple joints swelling and tenderness were decreased.
  • the anti-inflammatory effect of the compounds of the present invention were tested by monitoring the inhibitory effect of the compound in the release of pro-inflammatory cytokines, TNF ⁇ and IL-6 in anti-CD3/antiCD28 mAbs stimulated peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • 96-welled plates (MaxiSorp, #460984 from Nalge NUNC Internatinal, Rochester, NY) were sterilized with 70% ethanol and then coated with 100 ⁇ l of PBS containing an anti-huCD3 mAb, 1 ⁇ g/ml (OKT3 with #16-0037 from eBioscience, San Diego CA) and 5 ⁇ g/ml of an anti-huCD28 mAb, (#MAB342 from R&D System, Minneapolis, CA). The plates were covered and store at 4° over night. At day 1, the plates were washed 3x with 200 ⁇ l of PBS each time.
  • PBMC Allcells, #****, Berkeley, CA
  • PBMC PBMC (Allcells, #****, Berkeley, CA) were thawed quickly in a 37°C water bath.
  • Cell density and viability were determined using a hemocytometer and trypan blue dye.
  • the vial content were transferred to a 50 ml conical tube (#21008-178, VWR International) with 5 ml of pre-warmed media (RPMI-1640, Media Tech with 10% FBS and benzonase nuclease #80108-808, EMD bioscience at 228 ⁇ l per liter of media) added.
  • the media volume was adjusted to 20 ml accordingly.
  • the cells were centrifuged at 20Og for 15 minutes (Sorvall Legend R/T # 75004377 at 900 rpm).
  • 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 ThI 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 ThI type and Th2 type cytokine such as IL-2, IL-4 and IL-5 productions by T cells stimulated by ConA (44). Regarding proinflammatory cytokine production such as TNF- ⁇ , EM and RXM are reported to inhibit TNF- ⁇ production by macrophages by LPS-stimulation (45, 46).
  • other macrolides such as midecamycin, clarithromycin and josamycin inhibited both ThI type and Th2 type cytokine such as IL-2, IL-4 and IL-5 productions by T cells stimulated by ConA (44).
  • RXM inhibited TNF- ⁇ production by LPS-stimulated macrophages (45, 46)
  • 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 1L-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 (49, 50).
  • 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) (51, 52).
  • 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 (53).
  • 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- ⁇ B activation in T cells (54). Since NF- ⁇ B is involved in gene expression for a variety of mediators including IL-2, IL-6, IL-8, TNF- ⁇ and GM-CSF (55), therefore it is conceivable that RXM also appears to suppress NF- KB activation. Further studies are needed to define the mechanism of the specific inhibition of IL-6 and TNF- ⁇ but not IL-2 by RXM.
  • 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 (55). These are inflixmab, anti-TNF- ⁇ mAb (58) and etanercept, an engineered p75 TNF receptor dimmer linked to the Fc portion of human IgGl (59).
  • RXM 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.
  • derivatives of RXM were synthesized and were investigated to decrease antibiotics activities, to increase inhibition of TNF- ⁇ and IL-6 production, and to decrease cytotoxicity.
  • the compounds of 5-1 and 8-B can be used to inhibit the production of TNF- ⁇ and IL-6 without having an increased cytotoxicity or antibiotics activity.
  • compound 5-1 is superior to RXM, because it inhibits the production of TNF- ⁇ and IL-6 production better than RXM and its antibiotics activity is 100 times less than RXM.
  • roxithromycin and/or its derivatives 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; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; anklyo
  • Macrolide antibiotics of the present invention find further utility in relieving or ameliorating the pain or symptoms associated with any one or more of the above-identified diseases or disorders.
  • 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.
  • new potent drugs for anti rheumatoid arthritis show to reduce antibiotic activities, production of macrolide antibiotics and to increasing inhibition of TNF- ⁇ and IL-6 without cytotoxicity.
  • B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2, Proc Natl Acad Sci USA 88, 6575-9., 1991.

Abstract

Provided are compounds of formula (III) useful for modulation of immune responses, compositions comprising the compounds, and methods of use of such compositions for treating diseases or disorders involving an immune response. In certain embodiments, the compounds are useful for the treatment of diseases or disorders associated with transendothelial migration of T cells and activated T cells, and proinflammatory cytokine production from T cells and macrophages. Diseases or disorders that can be treated include arthritic and rheumatic disorders, such as rheumatoid arthritis.

Description

MODULATION OF IMMUNE RESPONSES BY ADMINISTRATION OF ROXITHROMYCIN OR ITS DERIVATIVE
Related Application
[0001] This application claims priority benefit of U.S. Provisional Application No. 60/964,296, filed August 10, 2007, which is hereby incorporated by reference in its entirety.
I. TECHNICAL FIELD
[0002] The present invention relates generally to compounds useful for modulation of immune responses, compositions comprising the compounds, and methods of use of such compositions for treating or preventing diseases or disorders associated with modulation of immune responses.
IL BACKGROUND OF THE INVENTION
[0003] There is increasing evidence that macrolides have a variety of biologic activities, apart from their antibacterial actions (1). Recently, low-dose and long-term erythromycin (EM) has been reported to be effective in the treatment of chronic lower respiratory tract diseases, including diffuse panbronchiolitis and bronchial asthma (2, 3). However, the mechanism of this drug remains unclear. 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).
[0004] Roxithromycin (RXM), 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). New macrolide antibiotics are described in WO2004/084911 and (70-71).
[0005] In vitro investigation revealed that 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). Thus, there is increasing evidence that macrolides have a variety of biologic activities, apart from their antibacterial actions (17). [0006] In the initial stage of immune response, a certain antigen is engaged by a T cell receptor (TcR), followed by various cytokine release. However, this process alone is not sufficient to induce all events that accompany T cell activation. Accumulating evidence suggests the presence of so-called costimulatory signals that occur through additional T cell surface molecules, which are independent of the CD3/TcR (18). These 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 (19-21). The present inventor have established the identity of a novel costimulatory molecule, CD26, that is preferentially expressed on CD4+ memory T cells (19, 20, 22-24), 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 (25) (26).
[0007] In view of the above, there is continuing need in the art to understand and ultimately modulate the immune response. Accordingly, the present inventor has satisfied this long felt need with the following invention.
III. SUMMARY OF THE INVENTION
[0008] The effect of RXM and its derivates on T cell proliferation and cytokine production through different costimulatory signaling pathways was studied. Specifically, the effects of RXM and its derivatives 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 patients with rheumatoid arthritis (RA) was also studied. The results herein demonstrate that RXM and/or its derivatives specifically inhibit proinflammatory cytokine production by T cells and macrophages, inhibit T cell migration, and inhibit the development and/or symptoms of arthritis in mice and humans. RXM was also 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, therapy by roxithromycin and its derivatives may serve as an effective treatment for arthritic and/or rheumatic disorders, such as rheumatoid arthritis. [0009] Accordingly, 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.
[0010] In one embodiment, the disease or disorder to be treated or prevented using the composition and methods of the invention is an arthritic or rheumatic disorder, including but not limited to an 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; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; Sjogren's syndrome; unstable angina; myocardial infarction; treatment after coronary stent placement; or IL-6 related diseases.
[0011] In one embodiment, the present invention relates to a compound of the following formula (I):
Figure imgf000004_0001
wherein
Rl is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-IO alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-10 alkyloxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when Rl is hydrogen or methyl, and that R3 is not cladinose when Rl is methyl and R2 is methyl; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
[0012] In another embodiment, in the above formula (I),
R1 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-IO alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, Cl-IO alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-IO alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S.
[0013] In yet another embodiment, in the above formula (I),
Rl is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkyloxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkyloxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-5 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when Rl is hydrogen or methyl, and that R3 is not cladinose when Rl is methyl and R2 is methyl.
[0014] In other embodiment, in the above formula (I),
R1 is selected from the group consisting of hydrogen, C2-5 alkenyl, C2-C5 alkynyl, Cl- 5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6- 10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R1 and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
[0015] In one embodiment, in the above formula (I),
R1 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, Cl-5 alkylcarbonyl, Cl-5 alkoxycarbonyl, Cl-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, Cl-5 alkylcarbonyl, Cl-5 alkoxycarbonyl, Cl-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R1 and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-5 alkoxy, carboxyl, Cl-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of Cl-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-5 alkoxy, carboxyl, Cl-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
[0016] In one embodiment, the present invention relates to a compound of the following formula (II):
Figure imgf000008_0001
wherein Xl is selected from the group consisting of:
Figure imgf000008_0002
X2 is selected from the group consisting of:
Figure imgf000009_0001
with the proviso that said compound is not roxithromycin (5-(3,4,6-Trideoxy-3- dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L- ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2-rnethoxyethoxy)methoxyimino-
2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide); or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
[0017] In one embodiment, the present invention relates to a compound of the following formula (III):
Figure imgf000009_0002
wherein
R ,ι is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-IO alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-IO alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when R1 is hydrogen or methyl, and that R3 is not cladinose when R1 is methyl and R2 is methyl; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
[0018] For example, the present invention relates to a compound selected from the group consisting of:
5-(3,4,6-Trideoxy-3-N-ethylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-N-(2-methylpropyl)methylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-N-benzylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-N-(4-methoxybenzyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan- 13-olide; 5-(3,4,6-Trideoxy-3-N-(4-chlorobenzyl)methylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-N-(3-pyridylmethyl)rnethylarnino-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6sl l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-ethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-(2-methylpropylamino)-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 1 1,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-benzylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-[3,4,6-Trideoxy-3-(4-methoxybenzylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-[3,4,6-Trideoxy-3-(4-chlorobenzylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6-dideoxy-3- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan- 13-olide;
5-[3,4,6-Trideoxy-3-(3-pyridylmethylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-acetamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3- O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-benzenesulfonarnido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 1 1,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-acetoxy-6,l l,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan- 13- olide; 5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6, 1 1,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13- olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-phenylacetoxy-6,l l,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13- olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3-pyridylacetoxy)- 6, 11 , 12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10,12- hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3- methoxyphenyacetoxy)-6, 11,12-trihydroxy-9-(2-methoxyethoxy)rnethoxyimino- 2,4,6,8, 10, 12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(4- methoxyphenylacetoxy)-6, 11 , 12-trihydroxy-9-(2-methoxyethoxy)methoxyimino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; and
5-(3,4,6-Trideoxy-3-propylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
[0019] In another embodiment, the present invention relates to a compound selected from the group consisting of:
5-(3,4,6-Trideoxy-3-N-ethylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-O-methy l-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-N-(2-methylpropyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methy l-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-N-(4-chlorobenzyl)methylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; 5-(3,4,6-Trideoxy-3-N-(3-pyridylmethyl)rnethylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-ethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-(2-methylpropylamino)-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-[3,4,6-Trideoxy-3-(3-pyridylmethylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-acetamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-acetoxy-6,l l,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13- olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3-pyridylacetoxy)- 6, 11 , 12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10,12- hexamethylpentadecan-13-olide; and
5-(3,4,6-Trideoxy-3-propylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 1 1,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan-l 3-olide; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
[0020] In yet another embodiment, the present invention relates to a compound selected from the group consisting of:
5-(3,4,6-Trideoxy-3-benzenesulfonamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan-l 3-olide; and 5-(3,4,6-Trideoxy-3-dimethylarnino-β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6, 11,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13- olide; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
[0021] In other embodiment, the present invention is the compound of 5-(3,4,6-Trideoxy-3- benzenesulfonamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O-methyl- α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2-methoxyethoxy)methoxyirnino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide.
[0022] In yet another embodiment, the present is the compound of 5-(3,4,6-Trideoxy-3- dimethylamino-β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,δ, 10, 12-hexamethylpentadecan- 13-olide.
[0023] In the embodiments herein, wherein the compound of formula (I) is described, in another embodiment, alternatively, the compound may be a compound of formula (IV):
Figure imgf000014_0001
(IV)
[0024] In the embodiments herein, wherein the compound of formula (H) is described, in another embodiment, alternatively, the compound may be a compound of formula (V):
Figure imgf000015_0001
[0025] The present invention also provides a pharmaceutical composition for the treatment of a bacterial infection in a mammal, comprising a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier.
[0026] Further, the present invention provides a pharmaceutical composition for the treatment of a disorder in which proinflammatory cytokine production is implicated in a mammal, comprising a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier.
[0027] Moreover, according to the present invention, a method of treating a bacterial infection in a mammal, comprising administering to said mammal a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier, is provided.
[0028] Also, according to the present invention, a method of treating a disorder in which proinflammatory cytokine production is implicated in a mammal, comprising administering to said mammal a therapeutically effective amount of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable carrier, is provided. [0029] Further, the present invention relates to use of the above compound or a pharmaceutically acceptable salt, prodrug, or solvate thereof for treating a bacterial infection in a mammal.
[0030] Also, the present invention relates to use of the above compound or a pharmaceutically acceptable salt, prodrug for treating a disorder in which proinflammatory cytokine production is implicated in a mammal.
[0031] In one embodiment, the disease or disorder to be treated or prevented using the compound, composition and methods of the invention is an infectious disorder such as bacterial, fungal or viral infection, or an arthritic or rheumatic disorder, 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; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; Sjogren's syndrome; unstable angina; myocardial infarction, or for treatment after coronary stent placement; or IL-6 related diseases.
[0032] In another embodiment, the one or more compounds of the invention 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.
[0033] In another embodiment, 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.
[0034] In another embodiment, the one or more macrolide antibiotics are administered in combination or in conjunction with a therapeutically effective amount of one or more non- macrolide antibiotics, and/or a therapeutically effective amount of one or more antiinflammatory compounds or immunomodulatory agents.
[0035] In certain embodiments of the method of the present invention, 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, WY-50295, SC-45662, SC-41661A, BI-L-357; leukotriene antagonists; peptide derivatives comprising ACTH and analogs thereof; soluble TNF-receptors; TNF- antibodies; soluble receptors of interleukines, other cytokines, T-cell-proteins; antibodies against receptors of interleukines, other cytokines, T-cell-proteins; and calcipotriols and analogues thereof taken either alone or in combination.
[0036] In another aspect, 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. Likewise, 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.
[0037] In a particular embodiment, the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
[0038] In a preferred embodiment, the macrolide antibiotics comprise a 14-member macrocylcine ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
[0039] In yet another aspect, 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. As above, the one or more macrolide antibiotics may be administered directly to an afflicted region or via systemic administration as well as orally. Likewise, 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 antiinflammatory compounds and/or a therapeutically effective amount of one or more immunomodulatory agents, such as those described above.
[0040] In a particular embodiment, the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
[0041] In a preferred embodiment, the macrolide antibiotics comprise a 14-member macrocylcine ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
[0042] In a further aspect of the invention, a method for inhibiting bone and cartilage destruction is provided, 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. As above, the one or more macrolide antibiotics may be administered directly to an afflicted region or via systemic administration as well as orally. Likewise, 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.
[0043] In a particular embodiment, the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
[0044] In a preferred embodiment, the macrolide antibiotics comprise a 14-member macrocylcine ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
[0045] In yet another aspect of the invention, a method for inhibiting leukocyte migration into an arthritic joint or synovial membrane is provided, the method 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. As above, the one or more macrolide antibiotics may be administered directly to an afflicted region or via systemic administration as well as orally. Likewise, 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 antiinflammatory compounds and/or a therapeutically effective amount of one or more immunomodulatory agents, such as those described above.
[0046] In a particular embodiment, the patient or mammal is afflicted with arthritis, more particularly rheumatoid arthritis.
[0047] In a preferred embodiment, the macrolide antibiotics comprise a 14-member macrocylcine ring structure macrolide antibiotic, such as roxithromycin or a derivative thereof.
[0048] In yet another aspect of the invention, 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-κB, from T cells is provided, the method comprising the step of contacting one or more T cells or activated T cells with one or more macrolide antibiotics. In a particularembodiment of the invention, the one or more macrolide antibiotics comprise a 14-member macrolide antibiotic. In a further embodiment, the 14-member macrolide antibiotic is roxithromycin or a derivative thereof.
[0049] In yet a further aspect of the invention, a method for inhibiting IL-6 production from macrophages is provided, comprising the step of contacting one or more macrophages with one or more macrolide antibiotics. In yet another aspect of the invention, a method for inhibiting the activation of NF-κB in a cell is provided, comprising the step of contacting one or more T cells or macrophage cells with one more macrolide antibiotics.. In a particularembodiment of the invention, the one or more macrolide antibiotics comprise a 14- member macrolide antibiotic. In a further embodiment, the 14-member macrolide antibiotic is roxithromycin or a derivative thereof.
[0050] In yet another aspect of the invention, a pharmaceutical composition is provided, the 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-κB from T cells, or IL-6 from macrophages, is implicated.
[0051] 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-κB from T cells, or IL-6 from macrophages. In each of the methods of the present invention, the reduction or inhibition of the production of pro-inflammatory cytokine production including, inter alia, TNF-alpha and/or NF-κB 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.
[0052] Accordingly, in one aspect of the invention, 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-κB 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.
[0053] In one embodiment, the reduction or inhibition of the production of proinflammatory cytokines including, inter alia, the production of TNF-alpha and/or NF-κB from T cells, or IL-6 from macrophages, is on the order of about 10-20% reduction or inhibition. In another embodiment, 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-κB 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.
[0054] In another aspect of the invention, 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; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; and Sjogren's syndrome.
[0055] In yet another aspect, 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.
[0056] In one embodiment, the present invention provides a method of treating a disease or disorder associated with modulation of immune response comprising administering a macrolide antibiotic that has an antibiotic activity less than roxithromycin and an increased inhibition of TNFalpha and IL-6 production from T cells and macrophages in comparison to roxithromycin. In certain embodiments, the macrolide antibiotic can have one or more of the following properties: an IC50 of less than about 15 μM for TNFalpha; less than about 12 μM for IL-6; or a minimum inhibiting concentration of at least about 100 μM for Staphylococcus aureus FDA 209P.
[0057] In yet another embodiment, the present invention provides a use of a compound of formulae (I)-(V) or a pharmaceutically acceptable salt or prodrug thereof for treating a bacterial infection or a disorder in which proinflammatory cytokine production is implicated in a mammal. [0058] In yet another aspect of the invention, the present invention includes methods of making a compound of formulae (I)-(V) using the methods described herein.
[0059] In one embodiment of the invention, 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. In another embodiment of the invention, the pharmaceutical compositions are administered orally, systemically, via an implant, intravenously, topically, or intrathecally.
[0060] In certain embodiments of the methods of the present invention, 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.
[0061] It is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention.
IV. BRIEF DESCRIPTION OF THE FIGURES
[0062] 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.
[0063] Figure 2 depicts the effect of RXM on the production of IL-2 and IFN-γ by peripheral T cells stimulated with CD3 plus CD28, CD3 plus PMA, and CD3 plus CD26.
[0064] 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.
[0065] Figure 4 depicts the effect of RXM on the production of IL-6 and TNF-α by peripheral T cells stimulated with CD3 plus CD28, CD3 plus PMA, and CD3 plus CD26.
[0066] Figure 5 depicts the effect of RXM on the production of IL-6 and TNF-α by macrophages stimulated with LPS.
[0067] Figure 6 depicts the inhibitory effect of RXM on transendothelial migration (chemokinesis) of PHA-activated T cells.
[0068] Figure 7 depicts the effect of RXM therapy on the development of CIA. [0069] Figure 8A-8C depicts the effect of RXM treatment on the serum IL-6, INF-γ and type II collagen antibody levels in CIA mice.
[0070] Figure 9 depicts the HE staining of ankle joint of CIA mice.
[0071] Figure 10 depicts the effect of RXM and Its derivatives on cytokine production of IL-2, IL- 17A and INF-γ.
[0072] Figure 11 depicts the effect of RXM and Its derivatives on cytokine production of IL-6 and TNF-α.
[0073] Figure 12 depicts the effect of RXM and Its derivatives on cytokine production of IL-4, IL-5 and IL-10.
V. DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0074] The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and not intended to be limiting.
[0076] As used herein, 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.
[0077] The term "macrolide antibiotic", as used herein, refers to an antibiotic containing a macrocyclic lactone ring. The phrase "macrocyclic lactone ring", as used herein, refers to a ring having a lactone moiety and more than seven carbon atoms. [0078] The term "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 tendonitis (as in "tennis elbow") and bursitis to crippling systemic forms, such as rheumatoid arthritis. There are pain syndromes like fibromyalgia and arthritis-related disorders, such as systemic lupus erythematosus, that involve every part of the body. There are forms of the disease, such as gout, that almost nobody connects with arthritis, and there are other conditions - like osteoarthritis - that many people think is the only form of the disease. The common denominator for all these conditions is joint and musculoskeletal pain, which is why they are grouped together as "arthritis." Often the pain is a result of inflammation of the joint lining. In many forms of arthritis, the synovium becomes inflamed and thickened, producing extra fluid which contains inflammatory cells. The inflamed synovium and fluid can damage the cartilage and underlying bone.
[0079] The terms "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.
1. Compounds
[0080] The term "Cl-10 alkyl", as used herein, unless otherwise indicated, means a branched or straight carbon chain having 1 to 10 carbons, and includes for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neo-pentyl, isopentyl, 1,2- dimethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 1-ethylbutyl, 2- ethylbutyl, isoheptyl, octyl, or isooctyl, etc. For example, it is the one having 1 to 5 carbons and for example, is methyl or ethyl.
[0081] The term "C2-10 alkenyl", as used herein, unless otherwise indicated, includes straight-chain or branched-chain mono- or poly-unsaturated aliphatic hydrocarbon radicals having 2 to 10, for example 2-5 carbons containing at least one carbon-carbon double bond. Examples of alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E5Z-, Z5E- and Z,Z-hexadienyl and the like.
[0082] The term "C2-10 alkynyl", as used herein, unless otherwise indicated, includes straight-chain or branched-chain mono- or poly-unsaturated aliphatic hydrocarbon radicals having 2 to 10, for example 2-5 carbons containing at least one carbon-carbon triple bond. Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like.
[0083] The term "Cl-10 alkylcarbonyl" means a carbonyl group having a straight or branched carbon chain having 1 to 10 carbons, and includes for example, hormyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, or octanoyl, etc.; for example, is the one having 1 to 5 carbons; and for example, is acetyl or propionyl.
[0084] The term "Cl-10 alkoxycarbonyl" means alkoxycarbonyl having 1 to 10 carbons, and includes methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, isopentyloxycarbonyl, neopentyloxycarbonyl, hexyloxycarbonyl, heptyloxycarbonyl, or octyloxycarbonyl, etc.; for example, is methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl; and for example, is methoxycarbonyl.
[0085] The term "Cl-10 alkylsulfonyl" means an alkylsulfonyl group having 1 to 10 carbons, and includes particularly, methylsulfonyl, ethylsulfonyl, butylsulfonyl, hexylsulfonyl, or octylsulfonyl, etc., and for example, is methylsulfonyl.
[0086] The term "C6-C10 arylsulfonyl" means an arylsulfonyl group having 6 to 10 carbons, such as phenylsulfonyl (SO2Ph), 1 -naphthylsulfonyl, or 2-naphthylsulfonyl.
[0087] The term "C3-8 cycloalkyl" means a cycloalkyl group having 3 to 8 carbons, and includes particularly, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl; and is for example cyclopropyl.
[0088] The term "aryl" means particularly a carbocyclic aryl group such as phenyl or naphthyl, or heteroaryl such as pyridyl or furyl, and for example, is phenyl.
[0089] The term "arylcarbonyl" means benzoyl, 4-methoxybenzoyl, or 3- trifluoromethylbenzoyl, etc., and for example, is benzoyl.
[0090] The term "aryloxycarbonyl" means phenoxycarbonyl, naphthyloxycarbonyl, 4- methylphenoxycarbonyl, 3-chlorophenoxycarbonyl, or 4-methoxyphenoxycarbonyl, etc.; and for example, is phenoxycarbonyl. [0091] The term "aralkoxycarbonyl" means benzyloxycarbonyl, 4- methoxybenzyloxycarbonyl, or 3-trifluoromethylbenzyloxycarbonyl, etc.; and for example, is benzy loxy carbony 1.
[0092] Compound (I) and compound (II) according to the present invention may form acid addition salts. Further, it may form salts with bases, depending on the species of the substituent. These salts are not restricted insofar as they are pharmaceutically acceptable, and include particularly, mineral salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate or sulfate, etc.; organic sulfonates such as methanesulfonate, 2- hydroxyethanesulfonate or p-toluenesulfonate, etc.; and organic carbonates such as acetate, trifluoroacetate, propionate, oxalate, citrate, malonate, succinate, glutarate, adipate, tartrate, maleate, malate, or mandelate, etc. As salts with bases, salts with inorganic bases such as sodium salts, potassium salts, magnesium salts, calcium salts or alminium salts, and salts with organic bases such as methylamine salts, ethylamine salts, lysine salts or ornithine salts, etc. are included.
[0093] The phrase "pharmaceutically acceptable salt(s)", as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. The compounds of the present invention that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, ptoluenesulfonate and pamoate salts. The compounds of the present invention that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
[0094] Those compounds of the present invention that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts and, particularly, the calcium, magnesium, sodium and potassium salts of the compounds of the present invention. [0095] Certain compounds of the present invention may have asymmetric centers and therefore exist in different enantiomeric and diastereomic forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of the present invention, and mixtures thereof, and to all pharmaceutical compositions and methods of treatment that may employ or contain them.
[0096] The present invention includes the compounds of the present invention, and the pharmaceutically acceptable salts thereof, wherein one or more hydrogen, carbon or other atoms are replaced by isotopes thereof. Such compounds may be useful as research and diagnostic tools in metabolism pharmacokinetic studies and in binding assays.
[0097] The compounds of this invention, including the compounds of formula (I) or (II) include pharmaceutically acceptable derivatives or prodrugs thereof. A "pharmaceutically acceptable derivative" or "pharmaceutically acceptable prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention that, upon administration to a recipient, is capable of providing directly or indirectly a compound of this invention or a metabolite or residue thereof. Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient, enhance delivery of the parent compound to a given biological compartment, increase solubility to allow administration by injection, after metabolism or alter rate of excretion.
[0098] Compounds of this invention described herein such as the compounds of formula (I) and (II) can be converted into prodrugs through, for example, free amino, amido, hydroxy or carboxylic groups. Examples of such prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of a compound of formula I. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvalin, beta-alanine, gamma-am inobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.
[0099] Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. The amide and ester moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates and phosphoryloxymethyloxycarbonyls, as outlined in D. Fleisher et al., Advanced Drug Delivery Reviews, 19:115 (1996). Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in R. P. Robinson et al., J. Medicinal Chemistry, 39:10 (1996).
[0100] The compounds of this invention also include pharmaceutically acceptable salts of the compounds described herein, such as the compounds of formulae (I) and (II). The term "pharmaceutically acceptable salt", as used herein, unless otherwise indicated, includes salts of acidic or basic groups that may be present in the compounds of the present invention.
[0101] Compounds of the invention may exist in tautomeric form. All tautomers of the compounds described herein, such as the compounds of formulae (I) and (II), are included.
2. Compositions and Methods
[0102] In one aspect, 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.
[0103] The diseases or disorders contemplated for treatment or prevention with the methods of the present invention include, inter alia, arthritic and rheumatic disorders. Examples of 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; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; Sjogren's syndrome; unstable angina; myocardial infarction; treatment after coronary stent placement; or IL-6 related diseases.
[0104] In another aspect, 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. In a particular embodiment, the subject suffers from arthritis, particularly rheumatoid arthritis.
[0105] In yet another aspect, 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. In a particular embodiment, the subject suffers from a rheumatic disorder such as rheumatoid arthritis.
[0106] In the context of the present invention, the phrases "treating or preventing" and "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). Thus, "treating or preventing an arthritic or rheumatic disorder" may further encompass a reduction in measured serum CRP levels or other indices of arthritic or rheumatic activity.
[0107] Accordingly, in a further aspect, 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. In a particular embodiment, the subject suffers from arthritis, particularly rheumatoid arthritis.
[0108] Likewise, in yet a further aspect, 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. In a particular embodiment, the subject suffers from arthritis, particularly rheumatoid arthritis.
[0109] In yet another aspect of the invention, a method is provided 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.
[0110] In yet another aspect of the invention, a method is provided for inhibiting IL-6 production from macrophage comprising contacting one or more macrophages with one or more macrolide antibiotics. In yet another aspect of the invention, a method is provided for inhibiting the activation of NF-κB in a cell comprising contacting one or more macrophage cells with one more macrolide antibiotics.
[0111] In yet another aspect of the invention, a pharmaceutical composition is provided 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.
[0112] In each of the specific aspects recited above, the one or more macrolide antibiotics comprise a 14-membered ring structure macrolide antibiotic.
[0113] In one embodiment, the 14-membered macrolide antibiotic is roxithromycin or a functional derivative thereof.
[0114] The invention also provides for the use of one or more macrolide antibiotics comprising a 14-membered macrolide antibiotic or derivatives thereof, as previously defined above, in the manufacture of a medicament to treat or prevent an arthritic or rheumatic disorder, 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; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; Sjogren's syndrome; unstable angina; myocardial infarction; treatment after coronary stent placement; or IL-6 related diseases..
[0115] In each aspect of the invention, a composition is provided 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-KB 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-κB from T cells, or IL-6 from macrophages. In each of the methods of the present invention, the reduction or inhibition of the production of pro-inflammatory cytokines including, inter alia, TNF-alpha and/or NF-κB 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.
[0116] Accordingly, in one aspect, 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-κB 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.
[0117] In another aspect, however, 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-κB 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.
[0118] With respect to macrolide antibiotics in general, macrolide antibiotics have been described by Bryskier, et al (27) and Omura (28).
[0119] Macrolide antibiotics, as used herein, 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. Several amino and/or neutral sugars are preferably fixed to the lactone ring. One group of macrolide antibiotics, are 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.
[0120] 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.
[0121] A particularly preferred macrolide antibiotic for use in the compositions and methods of the present invention can be a functional derivative of roxithromycin. Herein, 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. In the context of the present invention, 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.
[0122] Other examples of 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. Thus, 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.
[0123] 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.).
[0124] 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). Examples of 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.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1); Imipenem (CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.: 78110-38-0); Cephalothin (CAS Registry No.: 153-61-7); Cefazolin (CAS Registry No.: 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5); Cefamandole formate sodium (CAS Registry No.: 42540-40-9); Cefoxitin (CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.: 55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefmetazole (CAS Registry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7); Cefprozil (CAS Registry No.: 92665-29-7); Loracarbef (CAS Registry No.: 121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone (CAS Registry No.: 62893-19-0); Cefotaxime (CAS Registry
No. 63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftriaxone (CAS Registry No. 73384-59-5); Ceftazidime (CAS Registry No.: 72558-82-8); Cefepime (CAS Registry No. 88040-23-7); Cefixime (CAS Registry No.: 79350-37-1); Cefpodoxime (CAS Registry No. 80210-62-4); Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No. 79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin (CAS Registry No. 70458-96-7); Ciprofloxacin (CAS Registry No.: 85721-33-1); Ofloxacin (CAS Registry No. 82419-36-1); Enoxacin (CAS Registry No.: 74011-58-8); Lomefloxacin (CAS Registry No. 98079-51-7); Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No. 564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CAS Registry No. 60-54-8); Amikacin (CAS Registry No.: 37517-28-5); Gentamicin (CAS Registry No.:
1403-66-3); Kanamycin (CAS Registry No.: 8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CAS Registry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1); Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CAS Registry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8); Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethyl succinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate (CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS Registry No.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643- 22-1); Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.: 61036- 64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin (CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.: 738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin (CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1); Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS Registry No.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin (CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations of Piperacillin and Tazobactam; and their various salts, acids, bases, and other derivatives. [0125] Anti-fungal agents include, but are not limited to, terbinafine hydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole, miconazole nitrate, flucytosine, fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid, and selenium sulfide.
[0126] Anti-viral agents include, but are not limited to, amantadine hydrochloride, rimantadin, acyclovir, famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine, valacyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine.
[0127] Anti-parasitic agents include, but are not limited to, pirethrins/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.
[0128] In another aspect, in the method of the present invention, one may, for example, supplement the composition by administration of a therapeutically effective amount of one or more an anti-inflammatory or immunomodulatory drugs or agents. By "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. By "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 immune, vascular, or lymphatic systems.
[0129] 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-41661 A, BI-L-357; leukotriene antagonists, e.g., compounds disclosed in DE 40091 171 German patent application P 42 42 390.2; WO 9201675; SC-41930; SC-50605; SC-51 146; LY 255283 (29) ; LY 223982 (30); U-75302 and analogs, e.g., described by J. Morris et al. (31) and C. E. Burgos et al. (32); B. M. Taylor et al. (33); compounds disclosed in U.S. Pat. No. 5,019,573; ONO-LB-457 and analogs, e.g., described by K. Kishikawa et al.(34); M. Konno et al. (35); WF-1 1605 and analogs, e.g., disclosed in U.S. Pat. No. 4,963,583; compounds disclosed in WO 9118601 , WO 91 18879; WO 91 18880, WO 91 18883, antiinflammatory substances, e.g., NPC 16570, NPC 17923 described by L. Noronha-Blab. et al.(36); NPC 15669 and analogs described by R. M. Burch et al.(37); S. Pou et al.(38); peptide derivatives, e.g., ACTH and analogs; soluble TNF-receptors; TNF- antibodies; soluble receptors of interleukines, other cytokines, T-ce 11 -proteins; antibodies against receptors of interleukines, other cytokines, and T-cell-proteins. peptide derivatives, e.g., ACTH and analogs; soluble TNF-receptors; TNF-antibodies; soluble receptors of interleukines, other cytokines, T-cell-proteins; antibodies against receptors of interleukines, other cytokines, and T-cell-proteins.
[0130] The invention as described herein can be illustrated further by the references to the figures that are provided.
Pharmacology
[0131] 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. In general, 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. [0132] It is intended herein that by recitation of such specified ranges, 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.
[0133] 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. Generally, then, 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). If desired, the daily dose may include two or more unit doses, i.e., tablets, cachets or capsules, to be administered each day.
[0134] In general, the 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.
[0135] It is further recommended that children, patients aged over 65 years, and those with impaired renal or hepatic function initially receive low doses, and that they then be titrated based on individual response(s) or blood level(s). It may be necessary to use dosages outside these ranges in some cases, as will be apparent to those of ordinary skill in the art. Further, it is noted that the clinician or treating physician will know, with no more than routine experimentation, how and when to interrupt, adjust, or terminate therapy in conjunction with individual patient response.
[0136] The term "unit dose" is meant to describe a single dose, although a unit dose may be divided, if desired. Although any suitable route of administration may be employed for providing the patient with an effective dosage of the composition according to the methods of the present invention, oral administration is preferred. Suitable routes include, for example, oral, rectal, parenteral (e.g., in saline solution), intravenous, topical, transdermal, 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.
[0137] The pharmaceutical 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. In one embodiment, for example, the composition is dissolved in a vegetable oil, such as olive oil or peanut oil, and, optionally, encapsulated in a gelatin capsule. For human therapy, a preferred method of administering pharmaceutical compositions of the present invention is orally, in the form of a gelatin capsule.
[0138] The term "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. Examples of such 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, choline, diethanolamine, ethylenediamine, meglumaine (N-methylglucamine), and procaine.
[0139] The 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 the oral solid preparations being preferred over the oral liquid preparations. The most preferred oral solid preparations are capsules. [0140] Because of their ease of administration, 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.
[0141] In addition to the common dosage forms set out above, the 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.
[0142] Pharmaceutical 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. In general, 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.
[0143] For example, 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. Those skilled in the art will know, or will be able to ascertain with no more than routine experimentation, appropriate pharmacological carriers for said pharmaceutical compositions.
B. Detailed Description of the Drawings [0144] 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.
[0145] Figure 2 depicts the effect of RXM on the production of IL-2 (A) and IFN-γ (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-γ 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).
[0146] Figure 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).
[0147] Figure 4 depicts the effect of RXM on the production of IL-6 (A) and TNF-α (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-α 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 μM, 0 and 14 μM, 0 and 28 μM of RXM, respectively).
[0148] Figure 5 depicts the effect of RXM on the production of IL-6 and TNF-α by macrophages stimulated with LPS. Macrophages were stimulated by LPS (1 μg/ml) for 8 hours, and the culture supernatants were assayed fur the concentration of IL-6 and TNF-α, 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 μM, 0 and 14 μM, 0 and 28 μM of RXM, respectively).
[0149] Figure 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 RPMII640), PHA-activated T ceils 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 μM, p<0.001 between 0 and 28 μM of RXM, respectively). The data are representative of 5 different donors.
[0150] Figure 7 depicts the effect of RXM therapy on the development of CIA. Disease scores of CIA mice measured from the dayO to day21 after the second immunization. Sum of scores were plotted from 4 different doses 100, 200, 400, and 800μg of RXM treatment groups (each: n=8) and control group (n=8). Mean values±SD from 8 mice were plotted. The p values calculated by two-tailed Student's t test were indicated in the figure (between 0, 100, 200, 400 and 800 μg/day).
[0151] Figure 8 depicts the effect of RXM treatment on the serum IL-6, INF-γ 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. Serum levels of INF-γ (A), IL-6(B), and type II collagen antibody levels(C) were plotted from 4 different doses of 100, 200, 400, and 800 μg of RXM treatment groups (each: n=8) and control group (n=8). Mean value±SD from 8 mice were plotted. IL-4 and TNF-α 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 800μg/day).
[0152] 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 μg of RXM treatment were stained by eosin and hematoxilyn. [0153] Figure 10 depicts the effect of RXM and its derivatives on the production of IL-2, IL-17 A and IFN- y 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 -γ by ELISA. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
[0154] Figure 11 depicts the effect of RXM and its derivatives on the production of IL-6 and TNF- a 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- a by ELISA. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
[0155] Figure 12 depicts the effect of RXM and its derivatives on the production of IL-4, IL-5 and IL-IO 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, IL-5 and IL-10 by ELISA. The cytokine levels in the case of T cells stimulated with CD3 alone were always the background level (data not shown).
III. EXEMPLARY EMBODIMENTS
[0156] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.
A. Materials and Methods
1. Cells and reagents
[0157] 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). 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 or by Sigma (R4393) and was dissolved in DMSO or methanol (WAKO, #137- 01823) and further diluted in the culture media consisting of RPM 1-1640 and 10% fetal calf serum (FCS). Derivatives of RXM was synthesized by Nard Institute Co. Ltd., Japan and was dissolved in DMSO or methanol and further diluted in the culture media consisting of RPMl- 1640 and 10% fetal calf serum (FCS).
2. T cell proliferation assays
[0158] One hundred micro liters of phosphate-buffer saline (PBS) containing 0.05 μg/ml of OKT3 with or without 5 μg/ml of 1F7, or 5 μg/ml of 4B10 mAb, and incubated overnight at 4°C, as described previously (22). Prior to use, these plates were washed once with 200 μl of PBS. Highly purified T cells were resuspended at 1 x 105 in 200 μl of RPMI medium containing 10% FCS, along with 4 different concentrations of RXM (0, 1.4, 14 and 28 μM). To assess PMA stimulation, the cell suspension supplemented with 5 ng/ml of PMA was applied into OKT3-coated wells. Cells were incubated at 37°C in 5% CO2 humidified atmosphere for 3 days. Cells were pulsed with 1 μCi/well of [3H]-thymidine (ICN Radiochemicals, Irvine, CA) 8 hours prior to harvest onto a glass-fiber filter (Wallac, Turku, Finland), and the incorporated radioactivity was quantitated by a liquid scintillation counter (Wallac).
3. Cytokine production assays
[0159] 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 μg/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-γ and IL-5: R&D systems, Minneapolis, MN; IL-17A: eBIOSCIENCE,#88-7176-88; IL-IO: BD, #555157) to measure the levels of IL-2, IFN-γ, IL-17A, IL-4, IL-5 and IL-IO as well as TNF-α and IL-6. For TNF-α and IL-6 production by macrophages, macrophages were enriched from E cells by adherence to plastic plates. Macrophages (1 x 106/ml) were suspended in 10% FCS-RPMI 1640 and stimulated with 1 μg/ml of LPS (Sigma). After 8- hour culture, culture supernatants were harvested and were subjected to ELISA (TNF-α and IL-6: R&D systems, Minneapolis, MN). The serum levels of IL-6, TNF-α, IFN-γ, and IL-4 were also detected using the ELlSA kit described above.
4. Assessment of cell viability
[0160] 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).
5. Transendothelial migration assay
[0161] Transendothelial migration activity was assessed using a kind of boyden-chamber assay as described previously with some modifications (39). The human endothelial cell line ECV304 obtained from ATCC was pre-cultured to make monolayer sheet on Transwell cell culture inserts with 3.0 μm pore size (Corning Costar, Cambridge, MA), for 48 hours. RXM was first dissolved in DMSO and further diluted in the assay medium consisting of RPMI 1640 and 0.6% bovine serum albumin, then added to culture plates in a final volume of 600 μl (the lower chamber) just before migration assay. PHA-activated T cells (1 x 106 cells/well) were added to each insert in a volume of 200 μl 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.
6. Immunofluorescence analysis
[0162] PHA-activated T cells were incubated with or without the indicated doses of RXM at 370C for 8 hours in 5% CO2/95% air and then washed. All of the cells were incubated with mAb against various cell surface molecules (1 μg/ml) on ice for 30 minutes. The cells were further incubated with fluorescein isothiocyanate labeled goat anti-mouse IgG (Sigma) after being washed 3 times with PBS containing 2% newborn calf serum and 0.02% sodium azide (Sigma). The mAbs used for this experiment were anti-CD3 (OKT3), anti-CD 1 Ia (BD PharMingen, NJ, USA), anti-CD25 (BD PharMingen), anti-CD26 (1F-7), anti-CD29 (4B4), anti-CD44 (ED PharMingen), and anti-CD47 (BD PharMingen).
7. Induction of CIA [0163] Male DBA/1 J mice were purchased from Japan Charles River Breeding Laboratories (Tokyo, Japan). Bovine type Il 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). For the primary immunization, 100 μl 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.
8. Assesment of CIA disease severity
[0164] After physical examination, the legs were scored as follows: 0, normal; 1, erythema and mild swelling confined to the ankle joint or toes; 2, erythema and mild extending from the ankle to the midfoot; 3, erythema and severe swelling extending from ankle to the metatarsal joints; 4, ankylosing deformation with joint swelling (67). The disease score for each mouse was calculated as the sum of the scores for the two hind legs.
9. Oral treatment of Roxithromycin (RXM) for CIA mice
[0165] RXM was dissolved in 5% Alabic Gum in 0.9% NaCl and different doses of RXM (lOOμg, 200μg, 400μg, and 800μg) in 5% Alabic Gum in 0.9% NaCl were orally given to 5 different groups comprising 8 mice. 5% Alabic Gum in 0.9% NaCl alone were also orally given to control mice group. The RXM or 5% Alabic Gum in 0.9% NaCl was orally given to mice every day up to day 14 after second immunization of type II collagen.
10. ELISA assay of cytokines and type II collagen levels in CIA mice
[0166] 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-α, IL-4, and IFN-γ 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.
11. Histology and immunohisotchemistry
[0167] Mice were killed by CO2 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 μm) 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 -800C. Serial cryostat sections (8 μm) were air-dried, fixed with cold 4% phosphate-buffered paraformaldehyde (pH7.4), and washed with 10 mM Tris-HCl (pH7.5) containing 150 mM NaCl and 0.1% saponin. Saponin permeabilizes the membrane of cells and intracellular organelles, thereby allowing the detection of intracellular cytokines. This technique gives positive nuclear staining in conventional immunohistchemical analysis, thus showing that cytokines can be stained mostly around the nucleus (68). The sections were then incubated with 10% normal goat serum for Ih at room temperature and treated with rabbit anti-human IFN -γ Ab, rabbit anti mouse TNF-α Ab, or normal rabbit serum overnight at 40C. 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.
12. Serum CRP Assay
[0168] CRP, also known as C-Reactive Protein, 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. In cases of inflammatory rheumatic disorders, such as rheumatoid arthritis, doctors can utilize the CRP test to assess the effectiveness of a specific arthritis treatment and monitor periods of disease flareup. Herein, serum CRP was measured by the laser nephelometry (LN) method and less than 0.5 μg/ml is considered to be normal.
13. Statistical analysis
[0169] Statistical analysis was performed by two-tailed student's t test for all the assays (3H-thymidine incorporation assay, ELISA, and transendothelial migration assay). The comparison was made between drug free control and the each dose of reagents. Statistical differences of the ankle width and the paw width of the CIA mice were assessed by Student's t test and the disease score by the Mann- Whitney U test. However, statistical analysis was not performed for RXM derivatives. B. Results
Example 1: Effect of RXM on T cell proliferation through different costimulatory pathways
[0170] To determine the effect of roxithromycin (RXM) on T cell proliferation, 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. AU 3 stimuli were combined with submitogenic doses of anti-CD3 mAb.
[0171] Purified 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. As shown in Fig. 1, 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. Under these conditions, RXM did not virtually inhibit T cell proliferation at any tested concentration (1.4 μM to 28 μM) from different donors. It should be noted that at higher concentration (28 μM), 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
[0172] Next, to examine the effect of RXM on cytokine production following stimulation with the agents described above, the levels of IL-2 and IFN-γ in the culture supernatants were measured. In 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. As shown in Fig. 2A, RXM, even at 28 μM, did not inhibit IL-2 production under all costimulatory conditions in three different donors. In addition to IL-2, RXM did not show any apparent effect on the production of IFN-γ at any tested concentrations (1.4 μM to 28 μM) (Fig.2B). These findings therefore indicated that RXM did not inhibit the Th-I type cytokine production in the instant experimental conditions. Example 3: Effect of RXM on Th2-type cytokine production through different costimulatory pathways
[0173] Since Th-2 type CD4+ T cells may play a role in allergic disorders such as asthma (27, 28), the effect of RXM on the Th2-type cytokine production of IL-4 and IL-5 was examined. As shown in Fig. 3A and B, RXM did not inhibit IL-4 and IL- 5 production under each costimulatory conditions at any tested doses (1.4 μM to 28 μM). Thus, the results herein demonstrated that RXM did not inhibit the Th-2 type cytokine production in the instant experimental systems.
Example 4: Effect of RXM on proinflammatory cytokine production through different costimulatory pathways
[0174] Next, the effect of RXM on the production of such proinflammatory cytokines as IL-6 and TNF-α was examined. As shown in Fig. 4, production of the proinflammatory cytokines IL-6 (A) mid TNF-α (B) was significantly inhibited by RXM in a dose-dependent manner under each costimulatory condition. Therefore RXM inhibited proinflammatory cytokine productions by T cells stimulated by the instant costimulatory conditions.
Example 5: Effect of RXM on proinflammatory cytokine production by macrophages
[0175] Since macrophages play a role in the host defense against infections and in the local modulation of immune and inflammatory responses (42), the effect of RXM on the production of the proinflammatory cytokines IL-6 and TNF-α by macrophages was also examined. For this purpose, macrophages isolated from PBMC were stimulated by LPS (1 μg/ml) and the culture supernatants were assayed for IL-6 and TNF-α 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. As shown in Fig. 5, RXM inhibited both IL-6 and TNF-α production by macrophages at a dose dependent manner. Example 6: Effect of RXM on transendothelial migration of activated T cells
[0176] To gain insights into the effect of RXM on T cell extravasation in vivo, its effect on transendothelial migration of PHA-stimulated T cells was examined. Since resting T cells hardly migrate through the endothelial cell monolayer, activated T lymphocytes were used for the migration assay, with T cell activation being accomplished with PHA-treatment (10 μg/ml). These pre-activated T cells spontaneously migrate from the upper chamber to the lower chamber through the endothelial monolayer, which is therefore regarded as chemokinesis. As shown in Fig. 6, T cell migration was significantly inhibited in a range from 14 μM to 28 μM in a dose-dependent manner when RXM was present during the endothelial migration assay (p<0.05). As compared to the proinflammatory cytokine productions by T cells and macrophages, RXM at dose of 1.4 μM did not inhibit T cell migration at 5 different donors but from 14 μM to 28 μM of RMX, T cell migration was always inhibited. When 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 μM) (data not shown).
Example 7: Effect of RXM therapy on the development of CIA
[0177] Since RXM inhibited proinflammatory cytokine production such as IL-6 and TNF-α by peripheral T cells and macrophages in vitro as well as the migration of T cells, the effects of RXM on the pathophysiology of CIA were investigated. The mice were immunized twice with type II collagen to induce arthritis. Oral treatment of RXM was started after second immunization of type II collagen, and daily treatment of RXM was continued up to day 14. During the course of the disease, the disease score described in the material & methods were assessed on the identified day. As shown in Fig 7, disease scores were suppressed in a dose dependent manner after 7 days treatment. In RXM 100 μg, 200 μg, 400 μg, and 800 μg 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 μg/day and 800 μg/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.
[0178] These results suggest that RXM treatment clearly inhibited the development of CIA. Example 8: Effect of RXM treatment on the serum IL-6, TNF-α, IL-4 and IFN-γ levels and the type II collagen antibody levels
[0179] Since RXM inhibited the production of IL-6 and TNF-α by T cells and macrophages in vitro and IL-6 and TNF-α appear to be involved in the development of CIA (68), the serum levels of IL-6, TNF-q, IL-4, and IFN-γ in CIA mice on dayO, day 7, day 14 and day 21 were examined after RXM treatment.
[0180] Regarding serum IFN-γ levels (Fig.8A), it appears that treatment of RXM did not affect the serum level of IFN-γ. As shown in Fig.8B, serum IL-6 levels were increased in control CIA mice on day 7, and then decreased and after day 14 reached to the undetectable level. In contrast, in RXM treated CIA mice, serum IL-6 levels were inhibited on day7 in a dose dependent manner, and especially in 400 μg and 800 μg RXM treated CIA mice, serum IL-6 levels were significantly inhibited on day7 (p<0.05).
[0181] Serum IL-4 and TNF-α levels could not be detected due to the low sensitivity of detection kits in these CIA mice groups. Regarding type II collagen antibody levels
(Fig.8C), 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.
[0182] Therefore, RXM treatment, or especially, 400 μg and 800 μg RXM treatment significantly inhibited the production of IL-6 in the serum of CIA mice.
Example 9: RXM inhibits leukocyte migration and bone destruction in affected joints of CIA mice
[0183] 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.
[0184] As shown Fig.9, in CIA mice without treatment of RXM, a large numbers of cells of leukocytes origin, synovial membrane proliferation and pannus formation as well as the destruction of bone and cartilage observed in control ClA mice whereas in CIA mice treated with 200 μg and 800 μg RXM per day, the infiltration of leukocytes as well as cartilage and bone destruction and pannus formation was strongly suppressed in a dose dependent manner. Especially in 800 μg RXM treated CIA mice, no cartilage and bone destruction, pannus formation and synovial membrane proliferation was observed.
[0185] These results indicated that RXM inhibited the leukocyte migration as well as cartilage and bone destruction.
Example 10: Synthetic Protocols
General.
[0186] Crude products were purified by preparative reverse-phase HPLC using a Phenomenex Luna 250x10 mm C-18 5μm column with Shimadzu LC-8A series HPLC pumps and PDA detector, and Shimadzu fraction collector. One of the two following buffer systems was used for purifications based on peak resolutions observed in trial injections: (1) TFA buffer system: A: water/0.05% TFA; B: acetonitrile. (2) ammonium acetate buffer system: A: 10 mmol ammonium acetate solution in water; buffer B: acetonitrile.
[0187] Analytical LC-MS analysis was performed on one of two systems:
(I) A system comprised of Shimadzu LC-10ADvp series HPLC pumps, a dual wavelength UV detector, a Gilson 215 autosampler, and a PE/Sciex API 150EX mass spectrometer. A Phenomenex Luna 4.6 x 50 mm, 5μm C18 column was used, with HPLC mobile phases consisting of (A) 0.05% trifluoroacetic acid in HPLC grade water and (B) 0.05% trifluoroacetic acid in HPLC grade acetonitrile. A gradient from 10% to 100% B over 4 min was performed with 5 μL injections for each sample.
(2) An HPl 100 HPLC system equipped with a SEDEX 75 ELS detector and PE Sciex API 150EX mass spectrometer. A Phenomenex Prodigy 3.2 x 100 mm, 5μm Cl 8 column was used, with HPLC mobile phases consisting of (A) 0.1% acetic acid in HPLC grade water and (B) 0.1% acetic acid in HPLC grade acetonitrile (B). A gradient from 10% to 100% B over 5 min was performed with 5 μL injections for each sample.
[0188] 1H and 13C spectra were recorded using a Varian Mercury-300 spectrometer, or a Bruker Avance 400 MHz spectrometer. The samples were dissolved in either d4-Methanol with 0.05% v/v TMS, or (J6-DMSO with 0.05% v/v TMS. About 5-10 mg of sample per 0.6 mL of solvent was used for 1H-NMR, and about 30 - 60 mg of sample per 0.6 mL of solvent for 13C-NMR. All spectra were recorded at room temperature unless otherwise noted. Characterization of NMR spectra as "Consistent with assigned structure" generally refers to satisfactory spot checks of spectra for expected changes relative to the structure and spectra of representative close analogs.
Derivatives of Desmethyl-Roxithromycin.
[0189] Derivatives of Desmethyl-Roxithromycin were prepared according to Scheme 1.
Scheme 1
Figure imgf000052_0001
Figure imgf000052_0003
Figure imgf000052_0002
5-(3,4,6-Trideoxy-3-methylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)-methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (2) [0190] The procedure was based on that reported for demethylation of Erythromycin reported by Freiberg (Freiberg, L. A. US Patent 3 725 385, 1970.). Roxithromycin 1 (2.0 g, 2.39 mmol) was dissolved 4:1 MeOH:H2O (35 mL) and sodium acetate (0.98 g, 11.95 mmol) was added. The mixture was stirred under argon while being heated to 51°C(degrees Celsius). After about 3 min, solid iodine (0.595 g, 2.34 mmol) was added to the mixture. The solution turned intense orange color. In approximately 10 min, IM NaOH (700 μL) was added to the reaction mixture. After another 20 min, another portion of IM NaOH (700 μL) was added, and then in 30 min an additional portion of IM NaOH (350 μL) was added. The mixture was stirred at 51°C(degrees Celsius) for 5 h, at which point it became almost colorless. The reaction mixture was then poured into of 3% NH4OH solution (100 mL) and kept overnight at 5°C (degrees Celsius). The suspension was extracted with CH2C12 (3 x 70 mL), then the organic fractions were combined, washed with 3% NH4OH (50 mL) and brine (50 mL), dried over sodium sulfate, and evaporated in vacuo to produce a white foamy solid. This solid (1.49 g, 1.78 mmol) was dissolved in (4: 1) MeOH:H2O (20 mL) and sodium acetate (0.181 g, 2.21 mmol) was added. The mixture was stirred under argon while being heated to 51°C(degrees Celsius). After about 3 min, solid iodine (0.112 g, 0.442 mmol) was added to the mixture. The solution turned intense orange color. In approximately 10 min, 150 μL of IM NaOH was added to the reaction mixture. After another 20 min, another portion of 150 μL of IM NaOH was added, and then in 30 min a 75 μL portion of IM NaOH was added. The mixture was stirred at 51°C(degrees Celsius) for 5 h, at which point it became almost colorless. The reaction mixture was then poured into 500 mL of 5% NH4OH solution. The suspension was extracted with CH2Cl2 (4 x 25 mL), and the organic fractions were combined, washed with 5% NH4OH solution (25 mL), then with brine (25 mL), dried over sodium sulfate and evaporated in vacuo to produce a white solid. Yield: 1.29 g (68%). LC/MS >98% (ELSD); calcd. for C40H74N2Oi5 822.5, found 823.7 (M+H)+. 13C NMR (CD3OD) was also recorded: δ ppm 175.95, 172.09, 102.25, 97.08, 96.74, 83.38, 80.12, 78.17, 76.95, 74.77, 74.52, 72.100, 71.88, 70.45, 68.01, 67.71, 65.52, 59.97, 58.29, 48.97, 45.25, 39.63, 37.94, 36.99, 35.12, 32.14, 27.11, 26.19, 21.42, 20.92, 20.54, 18.32, 16.28, 15.55, 14.17. The most notable change was a decrease in intensity of ca. 39 ppm signal (assigned to N(CH3)2 in 1). To unambiguously demonstrate the absence of RXM (1), a spiking experiment was performed, whereby the spectrum of isolated 2 in DMSO-dό was compared with the spectrum of isolated 2 intentionally mixed ("spiked") with 1. The spectrum of isolated 2 showed an absence of key peaks attributable to 1.
5-(3,4,6-Trideoxy-3-N-(2-methylpropyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-0-methyI-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9- (2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (3-B) [0191] Compound 2 (0.123 g, 0.15 mmol) was dissolved in 5 mL of MeOH and stirred at 45°C(degrees Celsius) in a 2-necked flask under flow of argon. To the solution, isobutyraldehyde (0.041 mL, 0.45 mmol, 3.0 eq) and acetic acid (0.015 mL, 0.26 mmol) were added. The mixture was stirred for 4 h (imine formation), and then NaBH(OAc)3 (0.191 g, 0.9 mmol) was added in one portion. Stirring was continued for 72 h, at which point LC/MS of the reaction mixture (ELS detection) showed about 80% conversion to the product. Addition of more of aldehyde, acetic acid and reducing agent did not improve the conversion to product. Solvent was removed in vacuo, then crude product was dissolved in CH2Cl2 (20 mL) and extracted with saturated sodium bicarbonate (2 X 10 mL) and brine (1 X 10 mL). The CH2Cl2 fraction was dried over sodium sulfate and concentrated. The resulting crude material was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.030 g (24%) of the title compound as a colorless solid. LC/MS >98 % (ELSD); calcd. for C44H82N2Oi5 878.6, found 879.5 (M+H)+. 1H- and 13C-NMR spectra were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-N-ethylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)-methoxyimino-2,4,6,8,10,12-hexamethylpentadccan-13-olide (3-A). [0192] Using a procedure adapted from that described above for 3-B, the title compound was prepared from 2 and acetaldehyde on 0.15 mmol scale. Modifications included use of 1 equiv. OfCH3CHO, addition OfMgSO4 (20 mg) to assist imine formation, and use of 3 equiv.. OfNaBH(OAc)3. Reaction was performed at O°C(degrees Celsius) for 2 h, with slow warming to room temperature. Total reaction time was 18 h. Workup as described above for 3-B and reverse-phase HPLC purification (using TFA buffer) afforded 42 mg (30%) of the product as a colorless solid. The product was dissolved in 5 mL CH2Cl2 and washed two times with 5 mL of sodium bicarbonate solution; then recovered as free base by drying in vacuo. LC/MS 96 % (ELSD); calcd. for C42H78N2Oi5 850.5, found 851.3 (M+H)+. IH- and 13C-NMR spectra were consistent with the assigned structure. Intermediate 2 was identified as the principal impurity.
[0193] A sample for biological testing was further purified by recrystallization. Free base (20 mg) was dissolved in of acetone (ca. 0.5 mL) and 10% NH4OH solution (15 mL) was added. The solution was allowed to stand for 48 h at 0 °C(degrees Celsius), resulting in formation of colorless crystals, which were filtered and dried in vacuo, yielding about 3 mg of colorless solid. LC/MS 99 % (ELSD); calcd. for C42H78N2Oi5 850.5, found 851.3 (M+H)+.
5-(3,4,6-Trideoxy-3-N-benzylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (3-C) [0194] The title compound was prepared from 2 and benzaldehyde on 0.486 mmol scale using the procedure described above for 3-B, except that 4 equiv. of NaBH4 were used for reduction and reaction time was 13 h. Purification of the crude material by reverse-phase HPLC (TFA buffer) yielded 0.124 g of the title compound, >99% pure (ELSD). After sodium bicarbonate wash, 61.4 mg, (12%) of the title compound was recovered as the free base. LC/MS >99 % (ELSD); calcd. for C47H80N2O15912.6, found 913.3 (M+H)+. 1H- and 13C- NMR spectra were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-N-(4-methoxybenzyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9- (2-methoxyethoxy)methoxyimino-2,4,6,8,10jl2-hexamethylpentadecan-13-olide (3-D) [0195] Using a procedure modified from the one described above for 3-B, the title compound was prepared from 2 and/7-anisaldehyde on 0.122 mmol scale. Modifications included use of trimethyl orthoformate (TMOF) as solvent (2 mL), use OfMgSO4 (50 mg) as dehydrating agent, and use of 6 equiv. OfNaBH(OAc)3. The reaction was carried out for 42 h (18 h for imine formation, 24 h for reduction) at room temperature. Purification of the crude material by reverse-phase HPLC (TFA buffer) and subsequent bicarbonate wash yielded 37 mg (32%) of the title compound. LC/MS >99 % (ELSD); calcd. for C48H82N2Oi6942.6, found 943.3 (M+H)+. 1H- and 13C-NMR spectra were consistent with the assigned structure. 5-(3,4,6-Trideoxy-3-N-(4-chIorobenzyl)methylamino-β-D-xyIo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyloxy)-6,H,12-trihydroxy-9- (2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (3-E) [0196] Using a procedure modified from that described above for 3-B, the title compound was prepared from 2 and p-chlorobenzaldehyde on 0.15 mmol scale. Modifications included use of 4A molecular sieves for dehydration and 7.5 equiv. of NaBH(OAc)3 for reduction. Reaction time was 27 h. Purification of the crude material by reverse-phase HPLC (TFA buffer) yielded 0.60 g of the product. After sodium bicarbonate wash, 43.0 mg (31%) of the title compound was recovered as a colorless solid. LC/MS >99 % (ELSD); calcd. for C47H79ClN2Oi5946.5, found 947.7 (M+H)+. 1H- and 13C-NMR spectra were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-N-(3-pyridylmethyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyIoxy)-6,ll,12-trihydroxy-9- (2-nicthoxycthoxy)methoxyimino-2,4,6,8,10,12-hcxamethylpentadecan-13-olide (3-F) [0197] Using a procedure modified from that described above for 3-B, the title compound was prepared from 2 and 3-pyridylcarboxaldehyde on 0.15 mmol scale. Modifications included use TMOF (2 mL) as solvent, use OfMgSO4 (100 mg) as dehydrating agent, and use of 8 equiv. OfNaBH(OAc)3. Reaction was carried out for 20 h (3 h for imine formation, 17 h for reduction) at room temperature. Purification of the crude material by reverse-phase HPLC (TFA buffer) and sodium bicarbonate wash yielded 63.6 mg (46%) of the title compound as a colorless solid. LC/MS >99 % (ELSD); calcd. for C46H792N3Oi5913.6, found 914.3 (M+H) +. 1H- and 13C-NMR spectra were consistent with the assigned structure.
Derivatives of bis-desmethyl-Roxithromycin.
[0198] Derivatives of bis-desmethyl-Roxithromycin were prepared according to Schemes 2
- 5. Scheme 2
h , NaQMe
Figure imgf000057_0002
Figure imgf000057_0001
4
+ 2
(ca.10% by '3C NMR)
Scheme 3
Crude 4
Figure imgf000058_0001
5-1 (R = SO2Ph)
Scheme 4
Ac2O, Et3N Crude 4 *—!—* 5-H (R = Ac)
Scheme 5
1. RCHO
Crude 4 5-A-G
2. NaBH4
Purification by preparative HPLC or via the N-trif luoroacetamide by the sequence (a) (CF3CO)2O, Et3N; (b) MeOH; (c) Preparative HPLC; (d) K2CO3, MeOH
Figure imgf000058_0002
5-(3,4,6-Trideoxy-3-amino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O- methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2-inethoxyethoxy)- methoxyimino-2,4,6,8,10,12-hexaniethylpentadecan-13-olide (4). [0199] Sodium metal (276 mg, 12.0 mmol) was added to anhydrous MeOH (70 mL) in a 250 mL round-bottomed flask. The flask was chilled in an ice bath (0 0C) and stirred under N2 until all of the sodium metal was consumed (ca. 15 min). Crude amine 2 (1.93 g, 2.35 mmol) was dissolved in ca. 10 mL of anhydrous MeOH and added via syringe. I2 (solid) (2.98 g, 1 1.7 mmol) was added in one portion. The dark orange solution was stirred at 0 0C for 5.5 h. A quench solution was prepared by dissolving Na2S2O3 (5.0 g) and cone. NH4OH (4 mL) in 220 mL H2O. The quench solution was added to the reaction mixture at 0 °C until the mixture became colorless (ca. 75 mL). The colorless solution was concentrated by rotary evaporation to approximately 50% of the original volume. The resulting white suspension was extracted with CHCl3 (4 x 25 mL). The CHCl3 layers were washed with a 1 : 1 mixture of brine : 4% aq. NH4OH (1 x 25 mL), dried over anhydrous Na2SO4, filtered, concentrated, and dried under high vacuum for 18 h to provide 1.58 g of a colorless foam. LC/MS calcd. for C39H72N2Oi5808.49, found (M+H)+ 809.65. LC/MS showed the presence of m/e 823.85 (corresponding to (M+H)+ for 2) co-eluting with the desired product. The 13C NMR (CD3OD) was recorded. Compared to the spectrum for 2, the most notable changes were reductions in the signals at 61.07 ppm and 33.16 ppm and a new signal at 59.37 ppm. Based on changes in the relative peak intensities, the amount of unreacted 2 was estimated to be ca. 10-20%. This material was used directly for preparation of derivatives 5.
[0200] A sample for biological testing was purified as follows: Crude 4 (252 mg, 0.311 mmol) and Et3N (0.43 mL, 3.1 mmol) in CH2Cl2 at 0 0C was treated with trifluoroacetic anhydride (0.14 mL, 0.96 mmol), and the mixture was allowed to warm to ambient temperature. After 2h, the mixture was washed with saturated aqueous NaHCO3, dried (Na2SO4), filtered, and concentrated. The residue was dissolved in anhydrous MeOH and allowed to stand at ambient temperature for 18 h. The resulting mixture was purified by silica gel chromatography (5% MeOH /1% NEt3 in CH2Cl2 as eluent) to yield 168 mg of trifuoroacetamide as a colorless solid. LC/MS 99.5% (ELSD); calcd. for C4iH7iF3N2Oi6 904.48, found (M+H)+ 905.77; the N-Me side product was observed by LC/MS as a trace contaminant. The trifuoroacetamide (ca. 100 mg) was dissolved in a mixture of MeOH (7.1 mL) and H2O (0.47 mL). Solid K2CO3 (133 mg, ca. 5.2 equiv.) was added and the mixture was stirred at ambient temperature for 18 h. The MeOH was removed by rotary evaporation, and the residue was partitioned in CHCl3, H2O (5 mL), and brine (5 mL). The aqueous layer was extracted with CHCl3 (3 x 10 mL), and the combined CHCI3 layers were washed with brine (1 x 10 mL), dried over Na2SO4, filtered, and concentrated. The product was transferred to a scintillation vial with MeOH, the MeOH was evaporated, and H2O (ca. 1 mL) was added. The solution was frozen and lyophilized to provide 0.109 g (overall 42% yield) of the title compound as a colorless powder. LC/MS 99% (ELSD); calcd. for C39H72N2Oi5 808.49, found 809.83 (M+H)+. 1H- and 13C-NMR were consistent with the assigned structure. 1H-NMR spiking experiments were attempted to assess the absence of the -NMe derivative, but the methyl peaks were poorly resolved.
5-(3,4,6-Trideoxy-3-benzenesulfonamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)-methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5.1)
[0201] Crude 4 (166 mg, 0.205 mmol) in 2.1 mL CH2Cl2 (2.1 mL) was treated with Et3N (0.070 mL, 0.51 mmol) and the mixture was chilled to 0 0C. Benzenesulfonyl chloride (0.027 mL, 0.22 mmol) was added dropwise and the mixture was allowed to warm to ambient temperature with magnetic stirring under N2. The reaction was monitored by LC/MS. After 2.5 h, no change in the reaction mixture was observed by LC/MS. The mixture was diluted with CH2Cl2 (10 mL ) and washed with saturated NaHCO3 (1 x 10 mL), dried over Na2SO4, and concentrated to provide crude title compound. LC/MS analysis indicated baseline separation of desired product from the N-Me benzenesulfonamide contaminant. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.027 g (14%) of the title compound as a colorless solid. LC/MS 99% (ELSD; absence of the N-Me contaminant was confirmed); calcd. for C45H76N2Oi7S 948.49, found 949.75 (M+H)+. 1H- and 13C-NMR were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-acetamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-
3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,lljl2-trihydroxy-9-(2-methoxyethoxy)- methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-H)
[0202] Using a procedure analogous to that described above for 5-1, crude 4 (180 mg, 0.222 mmol) and Et3N (0.080 mL, 0.56 mmol) in CH2Cl2 (2.2 mL) treated with acetic anhydride
(0.022 mL, 0.23 mmol) provided the crude title compound. LC/MS indicated a resolved peak corresponding to N-Me-contaminant (M+H)+ 865.95. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.030 g (16%) of the title compound as a colorless solid. LC/MS 97% (ELSD; absence of the N-Me contaminant was confirmed); calcd. for C4IH74N2Oi6850.50, found (M+H)+ 851.65. 1H- and 13C-NMR were consistent with the assigned structure.
5-(3,4,6-Tiϊdeoxy-3-benzylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)-methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-D) [0203] Crude 4 (174 mg, 0.216 mmol) was dissolved in anhydrous MeOH (2.2 mL) and benzaldehyde (0.033 mL, 0.32 mmol) was added. The mixture was stirred under N2 at ambient temperature for 18 h. Then NaBH4 (29 mg, 0.77 mmol) was dissolved in MeOH (ca. 1.0 mL) and added to the reaction mixture. After stirring at ambient temperature for 2 h under N2, LC/MS analysis (ELS detection) indicated the presence of the title compound as the major product. The reaction was quenched with 2% aq. NH4OH (ca. 0.2 mL). The solution was concentrated and the residue was partitioned in 2% NH4OH (ca. 10 mL) and CHCb. The aqueous layer was extracted with CHC13 (3 x 10 mL). Brine was added as necessary to inhibit emulsions during the extractions. The combined organic layers were washed with brine (I x ca. 30 mL), dried over Na2SO4, filtered, and concentrated to provide the crude product as a colorless foam. Crude yield: 222.7 mg. The product was purified by preparative reverse- phase HPLC(ammonium acetate buffer) to yield 0.044 g (23%) of the title compound as a colorless solid. LC/MS 99% (ELSD); calcd. for C46H78N2O15898.54, found 899.88 (M+H)+. 1H- and 13C-NMR were consistent with the assigned structure. The product was shown to be free of the N-Me analog by 1H -ΝMR, which showed absence of the N-Me singlet at 2.22 ppm that is observed for the authentic N-Me analog. Spiking the product with the N-Me analog, and taking the 1H-NMR spectrum of the mixture demonstrated that this singlet was clearly resolved from other signals.
5-(3,4,6-Trideoxy-3-propylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)-methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-B) [0204] The reductive alkylation was performed as described above for 5-D, except that the propionaldehyde was added as a 2.0 M solution in MeOH. Thus, treatment of crude 4 (193 mg, 0.24 mmol) with propionaldehyde (2.0 M in MeOH, 0.155 mL, 0.3 mmol), and NaBH4 (32 mg, 0.83 mmol) afforded 217 mg of crude title compund. LC/MS calcd. for C42H78N2O)5 850.54, found (M+H)+ 851.65. The crude product was purified via the trifiuoroacetamide according to the following procedure. Crude title compound (217 mg, 0.255 mmol) was dissolved in CH2Cl2 (2.6 mL) and Et3N (0.36 mL, 2.55 mmol). The mixture was chilled to 0 0C and trifluoroacetic anhydride (0.1 15 mL, 0.81 mmol) was added dropwise. The mixture was allowed to warm to ambient temperature. After 2 h, LC/MS analysis indicated complete consumption of starting material and formation of higher retention time products. The mixture was diluted with 5 mL CHCl3 and washed with saturated NaHCO3 (1 x 10 mL), dried over Na2SO4, filtered, and concentrated. Anhydrous MeOH (5 mL) was added to the crude products and the mixture was stirred at ambient temperature for 18 h. The MeOH was removed by rotary evaporation to provide 208 mg of the crude trifiuoroacetamide as a light yellow foam. LC/MS analysis indicated baseline separation of the desired trifiuoroacetamide from -NMe side-products. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield ca. 0.030 g of trifluoroaceamide as a colorless solid. LC/MS 100% (ELSD); calcd. for C44H77F3N2O16946.52, found 947.81 (M+H)+. The trifiuoroacetamide (ca. 30 mg) was treated with K2CO3(23 mg) in a mixture of MeOH (1.2 mL) and H2O (0.072 mL) as described above under purification of 4 to provide 0.020 g (overall 10% yield) of the title compound as a colorless solid. LC/MS 97% (ELSD); calcd. for C42H78N2Oi5 850.54, found (M+H)+ 851.65. 1H- and 13C-NMR were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-ethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)-methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-A)
[0205] The reductive alkylation was carried out in an analogous manner to the procedure described above for 5-B. Thus, treatment of crude 4 (159 mg, 0.197 mmol) with acetaldehyde (2.0 M in MeOH, 0.123 mL, 0.39 mmol), and NaBH4 (26.1 mg, 0.69 mmol) afforded 156 mg of the crude amine. LC/MS calcd. for C4]H76N2O15 836.52, found 837.85 (M+H)+. The crude product was purified via the trifiuoroacetamide as described above for purification of n- propyl analog 5-B. Treatment of the crude product (156 mg, 0.187 mmol) in CH2Cl2 (2 mL) with Et3N (0.26 mL, 1.9 mmol), and trifluoroacetic anhydride (0.082 mL, 0.56 mmol) provided 160 mg of crude trifluoroacetamide after MeOH treatment. Analytical LC/MS indicated baseline separation of the desired trifluoroacetamide from -NMe side-products. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield ca. 100 mg of the trifluoroacetamide as a colorless solid. LC/MS 100% (ELSD); calcd. for C43H75F3N2O16932.51, found 933.61 (M+H)+. The trifuoroacetamide (ca. 100 mg) was hydrolyzed with K2CO3 (80 mg) in a mixture of MeOH (4.2 mL) and H2O (0.25 mL) as described above for purification of 4 to afford 10 mg (overall 6% yield) of the title compound as a white solid. LC/MS 96% (ELSD) ; calcd. for C4)H76N2Oi5 836.52, found 837.75 (M+H)+. 1H- and 13C-NMR were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-(2-methylpropylamino)-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-C) [0206] The procedures described for 5-D were used. Crude 4 (176 mg, 0.218 mmol), isobutyraldehyde (0.040 mL, 0.44 mmol), and NaBH4 (29 mg, 0.77 mmol) provided 184 mg of crude amine. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.033 g (17%) of title compound as a colorless solid. LC/MS 99% (ELSD) calculated for C43H80N2O15864.56, found (M+H)+ 865.65. 1H- and 13C-NMR were consistent with the assigned structure. 1H -NMR spiking experiments confirmed the absence of the -NMe side-product.
5-[3,4,6-Trideoxy-3-(4-methoxybenzylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-E)
[0207] The procedures described for 5-D were used. Crude 4 (137 mg, 0.169 mmol),/?- anisaldehyde (0.031 mL, 0.25 mmo), and NaBH4 (23 mg, 0.61 mmol) provided 202 mg crude amine. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.030 g (19%) of title compound as a colorless solid. LC/MS 99% (ELSD) calculated for C47H80N2O16 928.55, found (M+H)+ 929.85. 1H- and 13C-NMR were consistent with the assigned structure. 1H-NMR spiking experiments confirmed the absence of the - NMe side-product. 5-[3,4,6-Trideoxy-3-(4-chlorobenzylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6-dideoxy- 3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-F) [0208] The procedures described for 5-D were used. Crude 4 (152 mg, 0.188 mmol), 4- chlorobenzaldehyde (40 mg, 0.28 mmol), and NaBH4 (25 mg, 0.66 mmol) provided 166 mg crude product. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.030 g (17%) of title compound as a colorless solid. LC/MS 99% (ELSD) calculated for C46H77ClN2O15 932.50, found (M+H)+ 933.64. 1H- and 13C-NMR were consistent with the assigned structure. IH-NMR spiking experiments confirmed the absence of the -NMe side-product.
5-[3,4,6-Trideoxy-3-(3-pyridylmethylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,ll»12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (5-G)
[0209] The procedures described for 5-D were used. Crude 4 (161 mg, 0.199 mmol), nicotinaldehyde (0.028 mL, 0.30 mmol), and NaBH4 (27 mg, 0.71 mmol) provided 161 mg crude product. The product was purified by preparative reverse-phase HPLC (ammonium acetate buffer) to yield 0.027 g (15%) of title compound as a colorless solid. LC/MS 99% (ELSD) calculated for C45H77N3O15 899.54, found (M+H)+ 900.82. 1H- and 13C-NMR were consistent with the assigned structure. 1H-NMR spiking experiments confirmed the absence of the -NMe side-product.
Derivatives of des-cladinose Roxithromycin.
[0210] These analogs were prepared according to Scheme 6.
Scheme 6
Figure imgf000065_0001
Figure imgf000065_0002
Figure imgf000065_0003
Figure imgf000065_0004
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3,6,ll,12- tetrahydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan- 13-olide (6).
[0211] The procedure is based on that reported for removal of cladinose from Clarithromycin (LeMahieu, R. A.; Carson, M.; Kierstead, R. W. J. Med. Chem., 17, 1974, pp. 953 - 956.). Roxithromycin 1 (1.12 g, 1.34 mmol) was dissolved in 1% HCl - MeOH (50 mL). The solution was stirred under argon at room temperature for 12 h. The reaction was judged complete by LC/MS (ELSD), and 50 mL of saturated sodium bicarbonate solution was added to the reaction mixture. Volatile components were removed by rotary evaporation, and the cloudy aqueous solution was extracted with CH2Cl2 (3 x 50 mL). Organic fractions were combined and extracted with 3 N HCl (3 x 40 mL), then the aqueous layer was treated with 3N NaOH solution to pH = 8 - 9. The resulting basic solution was then extracted with CH2Cl2 (4 x 40 mL), and the organic fractions were combined, dried over sodium sulfate, and evaporated to afford 0.90 g (98%) of the title compound as a colorless solid. LC/MS >99 % (ELSD); calcd. for C33H62N2O12678.4, found 679.5 (M+H)+. 13C NMR (CD3OD) showed relevant changes from starting material, including the disappearance of a strong signal at 35.12 ppm assigned to cladinose position 2", and the disappearance of a signal at 96.73 ppm assigned to cladinose position 1". The 1H-NMR spectrum was also recorded.
5-(3,4,6-Trideoxy-2-0-acetyl-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3,6,ll,12- tetrahydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan- 13-olide (7)
[0212] Compound 6 (0.872 g, 1.285 mmol) was dissolved in 12 mL Of CH2Cl2, and to this solution was added solid sodium carbonate (0.136 g, 1.285 mmol). The reaction mixture was flushed with argon and acetic anhydride (0.182 mL, 1.928 mmol) was added via syringe. The reaction mixture was stirred at room temperature for 5.5 h, at which time LC/MS indicated complete conversion to the product. CH2Cl2 (50 mL) was added to the mixture, and the solution was washed with saturated sodium carbonate and brine. The solvent was removed in vacuo, giving 0.852 g (92%) of the title compound as a white foamy solid. LC/MS >95 % (ELSD); calcd. for C35H64N2On 720.4, found 721.3 (M+H)+. 13C NMR (CD3OD) δ ppm (new signals from acetyl are highlighted in bold): 175.62, 172.36, 171.18, 99.51, 97.31, 84.48, 77.77, 77.12, 74.79, 74.43, 72.01, 71.53, 70.98, 68.75, 68.16, 63.43, 58.32, 44.35, 39.76, 37.02, 34.44, 30.48, 27.20, 25.79, 21.83, 20.74, 20.43, 18.24, 16.52, 15.17, 14.64, 10.20, 8.27.
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyIoxy)-3-acetoxy-6,ll,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13- olide (8-A)
[0213] Compound 7 (0.100 g, 0.139 mmol) was dissolved in pyridine (1.8 mL) and Ac2O
(0.375 mL, 3.98 mmol) was added. The solution was heated in a closed vial at 71°C(degrees Celsius) for 18 h, at which time the reaction was judged complete by LC/MS. The solvent was removed in vacuo and the residue was dissolved in ethyl acetate (30 mL) and washed with saturated sodium bicarbonate (2 X 15 mL) and brine (1 X 15 mL). The organic phase was concentrated to dryness, and the residue was dissolved in MeOH (10 mL) and re fluxed for 6 h. Solvent was removed in vacuo and the residue was purified by reverse-phase prep- HPLC (TFA buffer). Following conversion to the free base with bicarbonate as described for 3-B, 73.9 mg (73%) of the title compound was obtained as white fluffy solid. LC/MS >99% (ELSD); calcd. for C35H64N2O)3 720.4, found 721.5 (M+H)+. 13C NMR (CD3OD) δ ppm: 173.84, 171.86, 171.18, 101.52, 97.15, 82.81, 78.92, 77.37, 74.62, 74.12, 71.85, 70.73, 69.18, 68.54, 67.99, 65.75, 58.21, 42.81, 41.22, 36.55, 36.27, 29.96, 26.94, 25.42, 21.51, 20.27, 19.92, 18.08, 16.34, 14.59, 14.43, 9.10, 8.24. The 1H-NMR spectrum was consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3-pyiϊdylacetoxy)- 6,1142-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12- hexamethylpentadecan-13-olide (8-D)
[0214] Compound 7 (0.120 g, 0.168 mmol) and N,N-dimethylaminopyridine (DMAP, 0.021 g, 0.168 mmol) were dissolved in CH2Cl2 (1 mL) and chilled to 0 °C(degrees Celsius). Meanwhile, 3-pyridylacetic acid hydrochloride (0.092 g, 0.555 mmol) was dissolved in 2 mL CH2Cl2 and placed in 2-necked, round-bottomed flask. This solution was cooled to -15 0C (degrees Celsius) and flushed with argon. To this mixture, triethylamine (0.078 mL, 0.555 mmol) and pivaloyl chloride (0.069 mL, 0.555 mmol) were added. The mixture was stirred for 20 min, and then the chilled solution of 9 and DMAP was added via syringe. The mixture was stirred for about 5 h, at which point the completion of the reaction was confirmed by LC/MS. To the reaction mixture was added 20 mL Of CH2Cl2, and the organic phase was washed with small amounts of sodium bicarbonate and brine solutions (twice each). The solvent was removed in vacuo to a yellow oil. The compound was purified by flash chromatography (silica gel, 1.5 cm x 6" column) using 7% MeOH/ CH2Cl2 as eluent. The product was detected in fractions 6 - 9 (8 mL each). Following combination of the fractions and evaporation of solvent, 0.140 g (99%) of the 2'-acetyl derivative of the title compound was obtained as white crystals. LC/MS > 98% (ELSD); calcd. for C42H69N3O]4 839.5, found 840.53 (M+H)+. 13C NMR (CD3OD) δ ppm (signals assigned to the pyridylacetyl moiety are highlighted in bold): 182.37, 174.28, 172.58, 171.48, 150.34, 148.39, 139.14, 131.59, 124.70, 103.19, 101.70, 97.78, 83.86, 80.66, 78.06, 75.25, 74.92, 72.49, 71.34, 71.26, 69.51, 68.63, 65.54, 58.87, 43.61, 40.27, 38.59, 37.43, 34.30, 31.37, 28.26, 27.59, 26.18, 22.13, 20.85, 20.18, 18.76, 16.99, 15.30, 15.09, 10.68, 9.14.
[0215] The above intermediate (0.125 g, 0.149 mmol) was dissolved in 20 mL of methanol and refluxed for 5 h. LC/MS indicated complete removal of 2 '-acetyl group and reaction was worked up by removing all volatile components in vacuo to afford as yellowish oil. The sample was purified by reverse-phase HPLC (TFA buffer). Following conversion to the free base with bicarbonate as described for 3-B, 70 mg (45%) of the title compound was obtained as a white crystalline fluffy solid. LC/MS >99% (ELSD); calcd. for C40H67N3O13797.5, found 798.5 (M+H) +. 13C NMR (CD3OD) δ ppm: 173.53, 171.87, 170.09, 147.27, 145.31, 144.31, 142.65, 126.32, 101.10, 97.16, 95.67, 83.45, 80.53, 77.53, 74.60, 74.20, 71.85, 70.73, 69.18, 68.49, 68.01, 65.62, 58.19, 42.89, 37.19, 36.74, 29.88, 26.91, 25.45, 21.47, 19.91, 18.07, 16.34, 14.76, 14.26, 9.99, 8.39. Proton NMR shows a signal from pyridine around 8 - 9 ppm as expected.
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-phenylacetoxy- 6,ll,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12- hexamethylpentadecan-13-olide (8-C), 5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo- hexopyranosyloxy)-3-(3-methoxyphenyacetoxy)-6,ll,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide (8-E), 5- (3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyIoxy)-3-(4- methoxyphenylacetoxy)-6,ll,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-
2,4,6,8,10,12-hexamethylpentadecan-13-olide (8-F)
[0216] These compounds were prepared using the procedure described above for 8-D, affording crude products in 85 - 95% yield. Crude products were purified by reverse-phase HPLC (TFA buffer), typically affording 15 - 30% of pure products.
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-phenylacetoxy-
6,ll,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12- hexamethylpentadecan-13-olide (8-C)
[0217] Yield 27.9 mg, 15%. LC/MS 95% (ELSD); calcd. for C41H68N2O13796.5, found
797.5 (M+H)+. 1H- and 13C-NMR spectra were consistent with the assigned structure. 5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3- methoxyphenyacetoxy)-6,ll»12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-
2,4,6,8,10,12-hexamethylpentadecan-13-olide (8-E)
[0218] Yield 37.9 mg, 28%. LC/MS 99% (ELSD); calcd. for C42H70N2Oi4826.5, found 827.3 (M+H)+. 1H- and 13C-NMR spectra were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(4- methoxyphenylacetoxy)-6,ll>12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-
2,4,6,8,10,12-hexamethylpentadecan-13-olide (8-F)
[0219] Yield 25.9 mg, 15%. LC/MS 100% (ELSD); calcd. for C42H70N2O14826.5, found
827.3 (M+H)+. 1H- and 13C-NMR spectra were consistent with the assigned structure.
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6,ll,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13- olide (8-B)
[0220] Benzoic acid proved to be unreactive with the coupling agent pivaloyl chloride, so an alternative procedure was used. Compound 7 (0.100 g, 0.139 mmol), benzoic acid (0.085 g, 0.695 mmol), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (0.125 g, 0.653 mmol), DMAP (0.017 g, 0.139 mmol) and N,N-diisopropylethlamine (0.149 mL, 0.834 mmol) were dissolved in 3 mL OfCH2Cl2 and left stirring in a capped vial at room temperature for 6 days. The reaction was monitored by LCMS, and after six days about 60% conversion was achieved. (In previous trial reactions, no further conversion was observed upon addition of more reagents and/or extension of reaction time up to 8 days). The reaction mixture was washed with sodium bicarbonate, and the residue after solvent evaporation was purified by reverse-phase HPLC (TFA buffer). The recovered acetyl intermediate was dissolved in fresh anhydrous methanol (2 mL) and refluxed for 5 h. Evaporation of methanol and thorough drying of the sample yielded 29.6 mg (28%) of the title compound as a colorless solid. LC/MS >99% (ELSD); calcd. for C40H66N2O13 782.5, found 783.7 (M+H)+. IH- and 13C-NMR spectra were consistent with the assigned structure. Example 11: Test of inhibitory effect for pro-inflammatory cytokine
[0221] The anti-inflammatory effect of the compounds of the present invention were tested by monitoring the inhibitory effect of the compound in the release of pro-inflammatory cytokines, TNFα and IL-6 in anti-CD3/antiCD28 mAbs stimulated peripheral blood mononuclear cells (PBMC) as follows:
Determination of inhibitory effect of RXM analogues in the release of TNFα and IL6 from anti-CD3 and anti-CD28 stimulated human PBMC
[0222] At day zero, 96-welled plates (MaxiSorp, #460984 from Nalge NUNC International, Rochester, NY) were sterilized with 70% ethanol and then coated with 100 μl of PBS containing an anti-huCD3 mAb, 1 μg/ml (OKT3 with #16-0037 from eBioscience, San Diego CA) and 5μg/ml of an anti-huCD28 mAb, (#MAB342 from R&D System, Minneapolis, CA). The plates were covered and store at 4° over night. At day 1, the plates were washed 3x with 200 μl of PBS each time. PBMC (Allcells, #PB003F, Berkeley, CA) were thawed quickly in a 37°C water bath. Cell density and viability were determined using a hemocytometer and trypan blue dye. The vial content were transferred to a 50 ml conical tube (#21008-178, VWR International) with 5 ml of pre-warmed media (RPMI- 1640, Media Tech with 10% FBS and benzonase nuclease #80108-808, EMD bioscience at 228 μl per liter of media) added. The media volume was adjusted to 20 ml accordingly. The cells were centrifuged at 20Og for 15 minutes (Sorvall Legend R/T # 75004377 at 900 rpm). After centrifugation, the supernatant was removed with 2-3 ml of media left behind. The cell pellet was resuspended gently. The media volume was re-adjusted to 40 ml with media containing no benzonase nuclease accordingly and cells were centrifuged at 20Og for 15 minutes again. The supernatant was removed with approximately 5 ml of media left behind. The cell density was determined and adjusted to 105 cells per 270 μl. A total volume of 270 μl cells and 30 μl of 10x tested compound in 10% DMSO was added into each well of a 96-welled plate. Plate contents were rocked for mixing, covered and incubated for another 24 hr in a 37°C CO2 incubator for another 24 hours. At day two, 96-welled plates were centrifuged at 1500 rpm for 15 minutes and 220 μl of supernatant from each was carefully removed without disturbing the cell pellet. A 100 μl of that was transferred to ELlSA plates provided by R&D system (#QTA00B) for TNF-α ELISA and another 100 μl was transferred to ELISA plates provided by Biosource (#KHC0061C, Camarillo, CA) for IL6 ELISA. The results were summarized in Table 1.
Table 1.
Figure imgf000071_0001
Example 12: Evaluation of antibacterial activities
Determination of antibacterial activity against Staphylococcus aureus FDA 209P
[0223] This assay is based on the liquid dilution method. The test compounds were dissolved in dimethylsulfoxide (DMSO) to reach a final concentration of 10 mM, and several diluted aliquots were prepared from the 10 mM solution and stored at -20°C(degrees Celsius). The strain of Staphylococcus aureus FDA 209P was incubated in 117 micrococcus medium (Ml 17). The test compound dilution (5microL) was transferred into assigned polypropylene tube and the seeding culture (500microL) of OD550= 0.0001 was added to the tube. After incubation over night at 37°C(degrees Celsius) with agitation, 200microL of culture was transferred into assigned wells of microplate. The inhibition of growth was determined by transillumination using microplate reader (settings: OD492) which allowed the minimum inhibiting concentrations (MIC) to be evaluated expressed in microM format. The results were summarized in Table 1.
Determination of antibacterial spectrum
[0224] This measurement is based on the liquid dilution method. The test compounds were dissolved in DMSO to reach a final concentration of lmg/mL and stored in a refrigerator. The bacterial strains of Enterococcus and Staphylococcus, Streptococcus, Haemophilus and Moraxella were incubated in Mueller Hinton Broth and Brain Heart Infusion, respectively, and the seeding culture was adjusted to 106 CFU/ml in two-fold concentration of the growth medium. The DMSO solution of test compound was diluted in two-fold series manner with distilled water, and transferred into assigned well of microplate (lOOmicroL/well). Then, each well was seeded with lOOmicroL of the culture and after incubating for twenty-four hours at 37°C(degrees Celsius), the inhibition of growth was determined by transillumination using microplate reader (Measurement Filter 540 nm) which allowed the MIC to be evaluated expressed in micrograms/mL. In addition the growth inhibition was confirmed by visual inspection. The results were shown in Table 2. Table 2.
MIC (microg/mL)
K*
Figure imgf000073_0001
Example 13: Effect of RXM treatment on patients with rheumatoid arthritis
[0225] Since 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.
[0226] Accordingly, four patients with rheumatoid arthritis (RA), aged 32 to 62, were treated with low dose of roxithromycin (150mg per day) every day. Mild clinical responses were obtained in all four patients after 1 month of treatment.
Case 1 - 32 year old woman (A.K.)
[0227] 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 μg/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 μg/ml and some clinical improvement of RA activity was observed.
Case 2 - 62 year old man (O.Y.)
[0228] 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. One month later, all PIP joint swelling of the noted joints as well as tenderness were decreased.
Case 3 - 43 year old woman (T.Y.)
[0229] 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 μg/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 μg/ml and left wrist joint swelling and tenderness were decreased. Case 4 - 53 year old woman (T.H.)
[0230] 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 CRP level of 2.7 μg/ml, she received 150 mg Roxithromycin per day in addition to the above medications. One month later, her CRP level was down to 1.9 μg/ml and multiple joints swelling and tenderness were decreased.
Example 14 : Evaluation of cytotoxicity
[0231] The anti-inflammatory effect of the compounds of the present invention were tested by monitoring the inhibitory effect of the compound in the release of pro-inflammatory cytokines, TNFα and IL-6 in anti-CD3/antiCD28 mAbs stimulated peripheral blood mononuclear cells (PBMC).
[0232] At day zero, 96-welled plates (MaxiSorp, #460984 from Nalge NUNC Internatinal, Rochester, NY) were sterilized with 70% ethanol and then coated with 100 μl of PBS containing an anti-huCD3 mAb, 1 μg/ml (OKT3 with #16-0037 from eBioscience, San Diego CA) and 5μg/ml of an anti-huCD28 mAb, (#MAB342 from R&D System, Minneapolis, CA). The plates were covered and store at 4° over night. At day 1, the plates were washed 3x with 200 μl of PBS each time. PBMC (Allcells, #****, Berkeley, CA) were thawed quickly in a 37°C water bath. Cell density and viability were determined using a hemocytometer and trypan blue dye. The vial content were transferred to a 50 ml conical tube (#21008-178, VWR International) with 5 ml of pre-warmed media (RPMI-1640, Media Tech with 10% FBS and benzonase nuclease #80108-808, EMD bioscience at 228 μl per liter of media) added. The media volume was adjusted to 20 ml accordingly. The cells were centrifuged at 20Og for 15 minutes (Sorvall Legend R/T # 75004377 at 900 rpm). After centrifugation, the supernatant was removed with 2-3 ml of media left behind. The cell pellet was resuspended gently. The media volume was re-adjusted to 40 ml with media containing no benzonase nuclease accordingly and cells were centrifuged at 20Og for 15 minutes again. The supernatant was removed with approximately 5 ml of media left behind. The cell density was determined and adjusted to 105 cells per 270 μl. A total volume of 270 μl cells and 30 μl of 10x tested compound in 10% DMSO was added into each well of a 96-welled plate. Plate contents were rocked for mixing, covered and incubated for another 24 hr in a 370C CO2 incubator for another 24 hours. At day two, 96-welled plates were centrifuged at 1500 rpm for 15 minutes and 220 μl of supernatant from each was carefully removed without disturbing the cell pellet. A 100 μl of that was transferred to ELISA plates provided by R&D system (#QTA00B) for TNF-α ELISA and another 100 μl was transferred to ELISA plates provided by Biosource (#KHC0061C, Camarillo, CA) for IL6 ELISA. The results shows Table 3.
[0233] Example 15 Effect of RXM and Its derivatives on cytokine production
[0234] Due to solubility of 5-1 and 8-B, dissolution method was modified. RXM and its derivatives were dissolved in Methanol with sonication. The other methods were same as described in Materials and Methods. These results, as shown in Figs. 10, 11 and 12 showed that cytokine production of 5-1 is almost same as RXM.
Table 3
Figure imgf000076_0001
C. Discussion
[0235] Herein, the present inventor demonstrated that RXM clearly inhibited production of the proinflammatory cytokines, TNF-α and IL-6 by T cells and macrophages, with virtually no effect on the production of the ThI type cytokines IL-2 and IFN -γ and Th2 type cytokines IL-4 and IL-5 by T cells stimulated by costimulatory stimuli such as CD28 and CD26. In addition RXM inhibited T cell migration. More importantly, 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.
[0236] The previous studies showed that RXM could not inhibit ConA-induced T cell proliferation but could inhibit both ConA-induced IL-2 and IL-4 production by T cells (16). Moreover, same groups reported that RXM inhibited Th2 type cytokine IL-4 and IL-5 but not ThI type cytokine IL-2 and IFN-γ by T cells stimulated with the same costimulatory stimuli used by the present inventor (43). At present, the precise reasons for the difference between the inventor results and those of Konno et al (16) and Asano et al (43) are not clear. The discrepancy may be due to the difference in the method for stimulation. 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 ThI type and Th2 type cytokine such as IL-2, IL-4 and IL-5 productions by T cells stimulated by ConA (44). Regarding proinflammatory cytokine production such as TNF-α, EM and RXM are reported to inhibit TNF-α production by macrophages by LPS-stimulation (45, 46). More recently, Guchelaar et al reported that EM inhibited TNF-α and IL-6 production induced by heat-killed streptococcus pneumonia in human whole blood ex-vivo (47). It should be noted that their results are not clear whether EM inhibited TNF-α and IL-6 production by T cells or macrophages or both or whether EM inhibited only TNF-α and IL-6 production but did not inhibit other cytokines such as IL-2, IFN-γ, IL-4 and IL-5.
[0237] Although it is reported that RXM inhibited TNF-α production by LPS-stimulated macrophages (45, 46), the present inventor are the first report that RXM specifically inhibited proinflammatory cytokine production such as IL-6 and TNF-α by T cells stimulated by several costimulatory stimuli such as CD28 and CD26 but did not inhibit 1L-2, TNF-γ, IL-4 and IL-5 by T cells. Moreover, 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.
[0238] Systemic administration of macrolide antibiotics (EM and RXM) have been shown to be effective in the treatment of lower and upper airway inflammatory diseases such as bronchial asthma and diffuse pan-bronchiolitis (2, 46-48). Although these inflammatory diseases have been reported to be successfully treated with low-dose administration of macrolide antibiotics, which cannot be expected to act as antibacterial agents, the precise mechanisms of effectiveness by this therapy are not well understood. The present findings of the inventor that RXM inhibited specifically the production of proinflammatory cytokines such as TNF-α and IL-6 by T cells and macrophages could explain at least the partial mechanisms of anti-inflammatory effects of RXM and effectiveness of RXM on such disorders.
[0239] Moreover, recently it has been reported that the 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 (49, 50). Current studies suggest that TNF-α 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) (51, 52). 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 (53). Therefore, inhibition of TNF-α and IL-6 production by T cells and macrophages as well as inhibition of T cell migration by RXM may also have important therapeutic implications for bronchial asthma. It is reported that EM suppresses NF-κB activation in T cells (54). Since NF-κB is involved in gene expression for a variety of mediators including IL-2, IL-6, IL-8, TNF-α and GM-CSF (55), therefore it is conceivable that RXM also appears to suppress NF- KB activation. Further studies are needed to define the mechanism of the specific inhibition of IL-6 and TNF-α but not IL-2 by RXM. [0240] In CIA, as well as in RA, joint inflammation and cartilage and bone destruction depend on the level of TNF-α and IL-6 in the affected joints (55, 68). By treatment of CIA mice with RXM, the present inventor showed that RXM inhibited the development of CIA through inhibiting the production of proinflammatory cytokines such as IL-6 and TNF-α 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.
[0241] At the site of inflammation in the affected RA synovium, infiltration of leukocytes, especially T cells from blood vessels is the initial step necessary for the development of the RA lesion (56). The mechanism of inflammatory cell infiltration into the tissues consists of the following multistep process: rolling, triggering, adhesion, and migration (57). Accordingly, inhibition of these processes could be the objective RA treatment. Since RXM was shown to inhibit T cell migration, it was postulated that RXM 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. The case study data presented herein represent the first experimental evidence confirming this hypothesis.
[0242] Advances in the understanding of the pathogenesis of RA, based on studies of human tissues and criterional models of disease, have led to the identification of a number of molecular targets for immunetherapeutic intervention. Of these, TNF-α has been validated as a good target for treatment and to date, two biological agents that target TNF-α have been licensed for clinical use (55). These are inflixmab, anti-TNF-α mAb (58) and etanercept, an engineered p75 TNF receptor dimmer linked to the Fc portion of human IgGl (59). Therapies inhibiting TNF-α 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 (55, 60). Moreover, anti-TNF-α treatment has been reported in effective in Crohn's disease (61). IL-6 regulates the production of acute- phase proteins by hepatocytoes and activates osteoclasts to absorb bone (62, 63). In a preclinical study, a humanized anti-IL-6R mAb has been used to treat patients with severe RA and clinical improvements have been reported (64). [0243] Since T cells and macrophages at the inflammatory sites of such diseases and proinflammatory cytokines as TNF-α and IL-6 play a key role in triggering and maintaining pathophysiology of the above disease, RXM 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. Moreover, in addition to RA and Crohn's disease, in several other disorders such as other arthritic or rheumatic disorders (56), graft-versus-host disease (GVHD) after allo-BMT (65), heart failures (66), and Castleman's disease (69), TNF-α may play a role in the pathophysiology of disease, RXM may also be useful for treatment of such conditions.
[0244] Based on above considerations, derivatives of RXM were synthesized and were investigated to decrease antibiotics activities, to increase inhibition of TNF-α and IL-6 production, and to decrease cytotoxicity. In one embodiment, the compounds of 5-1 and 8-B can be used to inhibit the production of TNF-α and IL-6 without having an increased cytotoxicity or antibiotics activity.
[0245] For example, compound 5-1 is superior to RXM, because it inhibits the production of TNF-α and IL-6 production better than RXM and its antibiotics activity is 100 times less than RXM.
D. Industrial Applicability
[0246] The results herein clearly demonstrate that roxithromycin and/or its derivatives 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. Thus, macrolide antibiotics, such as roxithromycin and derivatives thereof, have multiple research and clinical utilities.
[0247] For example, 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. More particularly, the 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; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; Sjogren's syndrome; unstable angina; myocardial infarction; treatment after coronary stent placement; or IL-6 related diseases.
[0248] Macrolide antibiotics of the present invention find further utility in relieving or ameliorating the pain or symptoms associated with any one or more of the above-identified diseases or disorders.
[0249] 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.
[0250] Moreover, new potent drugs for anti rheumatoid arthritis show to reduce antibiotic activities, production of macrolide antibiotics and to increasing inhibition of TNF-α and IL-6 without cytotoxicity.
£. Conclusion
[0251] The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration it is believed that one skilled in the pharmaceutical art can, given the preceding description, utilize the present invention to its fullest extent, using no more than routine experimentation. Therefore any examples are to be construed as merely illustrative and not a limitation on the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.
VI. REFERENCES
[0252] All publications, including, but not limited to, patents and patent applications cited in this specification, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
1. Nelson. S.. Summer. W. R, Terry, P. B.. Warr, G. A. and Jakab, G. J., Erythromycin- induced suppression of pulmonary antibacterial defenses. A potential mechanism of superinfection in the lung, Am Rev Respir Dis 136, 1207- 12., 1987.
2. Kadota,. J., Sakito, O., Kohno, S., Sawa, H., Mukae, H., Oda, H., Kawakami, K., Fukushima, K., Hiratani, K. and Hara, K., A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis, Am Rev Respir Dis 147, 153-9., 1993.
3. Miyatake, H., Suzuki, K., Taki, F., Takagi, K. and Satake, T., Effect of erythromycin on bronchial hyperresponsiveness in patients with bronchial asthma, Arzneimittelforschung 41, 552-6., 1991.
4. Yokota, T., Suzuki, E. and Arai, K., Cefpodoxime proxetil, its in vitro antibacterial activity, affinity to bacterial penicillin-binding proteins, and synergy of bactericidal activity with serum complement and mouse-cultured macrophages. Drugs Exp CHn Res 14. 495-500. 1988.
5. Fraschini, F., Scaglione, F., Ferrara, F., Marelli, O., Braga, P. C. and Teodori, F., Evaluation of the immunostimulating activity of erythromycin in man, Chemotherapy 32, 286-90, 1986.
6. Mikasa, K., Sawaki, M., Konishi, M., Egawa, S., Yoneda, T., Yagyu, Y., Fujimura, M., Hamada, K., Kunimatsu, M. and Narita, N., The effect of erythromycin treatment on natural killer (NK) cell activity in patients with chronic lower respiratory tract infections, Kansenshogaku Zasshi 63, 811-5., 1989. 7. Ras, GJ. , Anderson, R., Eftychis, H.A., Koch, U., Theron, A., Van Wyk, H.A. and Olivier, L. R., Chemoprophylaxis with erythromycin stearate or amoxycillin in patients with chronic bronchitis— effects on cellular and humoral immune functions, SAfr Med J 66, 955-8., 1984.
8. Aho, P. and Mannisto, P.T., Effects of two erythromycins, doxycycline and phenoxymethylpenicillin on human leucocyte chemotaxis in vitro, J Antimicrob Chemother 21 Suppl D, 29-32., 1988.
9. Anderson, R., Erythromycin and roxithromycin potentiate human neutrophil locomotion in vitro by inhibition of leukoattractant-activated superoxide generation and autooxidation, J/«/ec/ Z)/5 159,966-73., 1989.
10. Yoshimura, T., Kurita, C, Yamazaki, F., Shindo, J., Morishima, L5 Machida, K., Sumita, T., Horiba, M. and Nagai, H., Effects of roxithromycin on proliferation of peripheral blood mononuclear cells and production of lipopolysaccharide-induced cytokines, Biol Pharm Bull 18, 876-81., 1995.
11. Young, R A., Gonzalez, J. P. and Sorkin, E. M., Roxithromycin. A review of its antibacterial activity, pharmacokinetic properties and clinical efficacy, Drugs 37, 8-41., 1989.
12. Noma, T., Hayashi, M., Yoshizawa, L, Aoki, K., Shikishima, Y. and Kawano, Y., A comparative investigation of the restorative effects of roxihromycin on neutrophil activities, Int J Immunopharmacol 20, 615-24., 1998.
13. Mitsuyama, T., Furuno, T., Hidaka, K. and Hara, N., Modulatory effect of roxithromycin on human neutrophil function, Res Exp Med 196, 301-7, 1996.
14. Wakita, H., Tokura, Y., Furukawa, F. and Takigawa, M., The macrolide antibiotic, roxithromycin suppresses IFN-gamma-mediated immunological functions of cultured normal human keratinocytes, Biol Pharm Bull 19, 224-7., 1996. 15. Konno, S., Adachi, M., Asano, K., Okamoto, K. and Takahash, T., Inhibition of human T- lymphocyte activation by macrolide antibiotic, roxithromycin, Life Sci 51, L231-6, 1992.
16. Konno, S., Asao, K., Kurokawa, M., Ikeda, Kl, Okamoto, K. And Adachi, M., Antiasthmatic activity of a macrolide antibiotic, roxithromycin: analysis of possible mechanisms in vitro and in vivo, Int Arch Allergy Immunol 105, 308-16., 1994.
17. Nelson. S.. Summer. W. R, Terry, P. B.. Warr, G. A. and Jakab, G. J., Erythromycin- induced suppression of pulmonary antibacterial defenses. A potential mechanism of superinfection in the \ung, Am Rev Respir Dis 136, 1207-12.,1987.
18. Robey, E. and Allison, J. P., T-cell activation: integration of signals from the antigen receptor and costimulatory molecules, Immunol Today 16,306-10., 1995.
19. Green, J. M., Noel, P.J., Sperling, A. I., Walunas, T. L., Gray, G. S., Bluestone, J. A. and Thompson, C. B., Absence of B7-dependent responses in CD28-deficient mice, Immunity 1, 501-8., 1994.
20. Gimmi, C. D., Freeman. G. J., Gribben, J. G., Sugita, K., Freedman, A. S., Morimoto, C. and Nadler, L. M., B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2, Proc Natl Acad Sci USA 88, 6575-9., 1991.
21. Linsley, P. S. and Ledbetter, J. A., The role of the CD28 receptor during T cell responses to antigen, Annu Rev Immunol 11, 191-212, 1993.
22. Morimoto, C, Torimoto, Y., Levinson, G., Rudd, C. E., Schrieber, M., Dang, N. H., Letvin, N. L. and Schlossman, S. F., 1F7, a novel cell surface molecule, involved in helper function of CD4 cells, J Immunol 143, 3430-9., 1989.
23. Dang, N. H., Torimoto, Y., Deusch, K., Schlossman, S. F. and Morimoto, C, Comitogenic effect of solid-phase immobilized anti-lF7 on human CD4 T cell activation via CD3 and CD2 pathways, J Immunol 144, 4092-100., 1990. 24. Tanaka, T., Kameoka, J., Yaron, A., Schlossman, S. F. and Morimoto, C, The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzymatic activity. Proc Natl Acad Sci USA 90, 4586-90., 1993.
25. Hafler, D. A., Fox, D. A., Manning, M. E., Schlossman, S. F., Reinherz, E. L. and Weiner, H. L., In vivo activated T lymphocytes in the peripheral blood and cerebrospinal fluid of patients with multiple sclerosis, N EnglJ MedZll, 1405-11., 1985.
26. Nakao, H., Eguchi, K., Kawakami, A., Migita, K., Otsubo, T., Ueki, Y., Shimomura, C, Tezuka, H., Matsunaga, M., Maeda, K. and et al., Increment of TaI positive cells in peripheral blood from patients with rheumatoid arthritis, J Rheumatol 16, 904- 10., 1989.
27. Bryskier, A., Agouridas, C, and Chantot, J. F., Structure and Activity in The New Macrolides, Azalides, And Streptogramins: Pharmacology And Clinical Applications, 3,3-11 (Neu, H. C, Young, L. S., and Zinner, S. H., eds., 1993.
28. Omura, S., Macrolide Antibiotics—Chemistry, Biology and Practice 1984.
29. D. K. Herron et al., FASEB J. 2: Abstr. 4729, 1988.
30. D. M. Gapinski et al. J. Med. Chem. 33: 2798-2813, 1990.
31. J. Morris et al. Tetrahedron Lett. 29: 143-146, 1988.
32. C. E. Burgos et al., Tetrahedron Lett. 30: 5081-5084, 1989.
33. B. M. Taylor et al., Prostaglandins 42: 211-224, 1991.
34. K. Kishikawa et al., Adv. Prostagl. Thombox. Leukotriene Res. 21: 407-410, 1990.
35. M. Konno et al., Adv. Prostagl. Thrombox. Leukotriene Res. 21: 411-414, 1990.
36. L. Noronha-Blab. et al., Gastroenterology 102 (Suppl.): A 672, 1992. 37. R. M. Burch et al., Proc. Nat. Acad. ScL USA 88: 355-359, 1991.
38. S. Pou et al., Biochem. Pharmacol. 45: 2123-2127, 1993.
39. Iwata, S., Yamaguchi, N., Munakata, Y., Ikushima, H., Lee, J. F., Hosono, O., Schlossman, S. F. and Morimoto, C, CD26/dipcptidyl peptidase IV differentially regulates the chemotaxis of T cells and monocytes toward RANTES: possible mechanism for the switch from innate to acquired immune response, lnt Immunol 11, 417- 26., 1999.
40. Kapsenberg, M. L., Wierenga, E. A., Bos, J. D. and Jansen, H. M., Functional subsets of allergen-reactive human CD4+ T cells, Immunol Today 12, 392-5., 1991.
41. Romagnani, S., Lymphokine production by human T cells in disease states. Annu Rev Immunol 12, 227-57, 1994.
42. Johnston, R B., Jr., Current concepts: immunology. Monocytes and macrophages, N Engl J Med 31S, 747-52.,1988.
43. Asano, K., Kamakazu, K., Hisamitsu, T. and Suzak, H., Modulation of Th2 type cytokine production from human peripheral blood leukocytes by a macrolide antibiotic, roxithromycin, in vitro, lnt Immunopharmacol λ, 1913-21., 2001.
44. Morikawa, K., Zhang, J., Νonaka, M. and Morikawa, S., Modulatory effect of macrolide antibiotics on the ThI- and Th2-type cytokine production, lnt J Antimicrob Agents 19, 53-9., 2002.
45. Suzaki, H., Asano, K., Ohki, S., Kanai, K., Mizutani, T. and Hisamitsu, T., Suppressive activity of a macrolide antibiotic, roxithromycin, on pro- inflammatory cytokine production in vitro and in vivo, Mediators lnflamm 8, 199-204,1999.
46. lino, Y., Toriyama, M., Kudo, K., Νatori, Y. and Yuo. A., Erythromycin inhibition of lipopolysaccharide-stimulated tumor necrosis factor alpha production by human monocytes in vitro, Ann Otol Rhinol Laryngol Suppl 157, 16-20., 1992. 47. Guchelaar, H. J., Schultz, M. J., van der Poll, T. and Koopmans, R. P., Pharmacokinetic- pharmacodynamic modeling of the inhibitory effect of erythromycin on tumour necrosis factor-alpha and interleukin-6 production, Fundam Clin Pharmacol 15, 419-24., 2001.
48. Itkin, I. H. and Menzel, M. L., The use of macrolide antibiotic substances in the treatment ofasthma, JΛ//ergy 45, 146-62., 1970.
49. Robinson, D. S., Hamid, Q., Ying. S., Tsicopoulos, A., Barkans, J., Bentley, A. M., Corrigan, C, Durham. S. R. and Kay, A. B., Predominant TH2-like bronchoalveolar T- lymphocyte population in atopic asthma, N Engl J MY/ 326, 298-304., 1992.
50. Ying, S., Humbert, M., Barkans, J., Corrigan, C. J., Pfister, R., Menz, G., Larche, M., Robinson, D. S., Durham, S. R and Kay, A. B., Expression of IL-4 and IL-5 mRNA and protein product by CD4+ and CD8+ T cells, eosinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics, J Immunol 158, 3539- 44., 1997.
51. Baeuerle, P. A. and Henkel, T., Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 12, 141-79, 1994.
52. Amrani, Y., Chen, H. and Panettieri, R A., Jr., Activation of tumor necrosis factor receptor 1 in airway smooth muscle: a potential pathway that modulates bronchial hyper-responsiveness in asthma? Respir Res 1, 49-53, 2002.
53. Hirano, T., Interleukin 6 and its receptor: ten years later, Int Rev Immunol 16, 249-84, 1998.
54. Aoki, Y. and Kao, P. N., Erythromycin inhibits transcriptional activation of NF-kappaB, but not NFAT, through calcineurin-independent signaling in T cells, Antimicrob Agents Chemother 43, 2678-84., 1999.
55. Harris, E. D., Jr., Rheumatoid arthritis. Pathophysiology and implications for therapy, N Engl J Med 322, 1277-89., 1990. 56. Mackay, C. R. and Imhof, B.A., Cell adhesion in the immune system, Immunol Today 14, 99-102., 1993.
57. Taylor, P. C, Williams, R. O. and Maini, R. N., Immunotherapy for rheumatoid arthritis, Curr Opin Immunol 13, 611-6., 2001.
58. Knight, D. M., Trinh, H., Le, J., Siegel, S., Shealy, D., McDonough, M., Seal Ion, B., Moore, M. A., Vilcek, J., Daddona, P. and et al., Construction and initial characterization of a mouse-human chimeric anti-TNF antibody, MoI Immunol 30, 1443-53., 1993.
59. Weinblatt, M. E., Kremer, I. M., Bankhurst, A. D., Bulpitt, K. J., Fleischmann, R. M., Fox, R. I., Jackson, C. G., Lange, M. and Burge, D. J., A trial of etanercept, a recombinant tumor necrosis factor receptoπFc fusion protein in patients with rheumatoid arthritis receiving methotrexate, N EnglJ Med 340, 253-9., 1999.
60. Lipsky, P. E., van der Heijde, D. M., St Clair, E. W., Furst, D. E., Breedveld, F. C, Kalden, J. R., Smolen. J. S., Weisman, M., Emery, P., Feldmann, M., Harriman. G. R. and Maini, R. Ν., Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group, NEnglJMed343, 1594-602., 2000.
61. Kam, L. Y. and Targan, S. R., TNF-alpha antagonists for the treatment of Crohn's disease, Expert Opin Pharmacother 1, 615-22., 2000.
62. Castell, J. V., Gomez-Lechon, M. J., David, M., Hirano, T., Kishimoto, T. and Heinrich, P. C, Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hep atocytes. FEBS Lett 232, 347-50., 1988.
63. Tamura, T., Udagawa, N., Takahashi, N., Miyaura, C, Tanaka, S., Yamada, Y., Koishihara, Y., Ohsugi, Y., Kumaki, K., Taga, T. and et al., Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6, Proc Natl Acad Sci USA 90,1 1924-8.,1993. 64. Yoshizaki, K., Nishimoto. N., Mihara, M. and Kishimoto, T., Therapy of rheumatoid arthritis by blocking IL-6 signal transduction with a humanized anti-IL-6 receptor antibody, Springer Semin Immunopathol 20, 247-59, 1998.
65. Jacobsohn, D. A., Novel therapeutics for the treatment of graft-versus-host disease, Expert Opin Investig Drugs 11, 1271-80., 2002.
66. Negrusz-Kawecka, M., [The role of TNF-alpha in the etiopathogenesis of heart failure], Pol Merkuriusz Lek 12, 69-72., 2002.
67. Rosloniec, E.F., M. Cremer, A. Kang and L. K. Myers. Collagen-induced arthritis. In current Protocols in Immunology. J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds. Wiley, New York, pl5.5.1, 1996.
68. Marinova-Mutafchieva, L. R. O. Williams, L. J. Mason, C. Mauri, M. Feldmann, and R. N. Maini. Dynamics of proinflammatory cytokine expression in the joints of mice with collagen-induced arthritis (CIA). Clin. Exp. Immunol. 107:507,1997.
69. Katsume A, Saito H, Yamada Y, Yorozu K, Ueda O, Akamatsu K, Nishimoto N, Kishimoto T, Yoshizaki K and Ohisugi Y. Anti-interleukin 6(IL-6) receptor antibody suppress castleman's disease like symptoms emerged in IL-6 transgenic mice. Cytokine 21 304-311, 2002.
70. Urasaki Y, Nori M, Iwata S, Sasaki T, Hosono O, Kawasaki H, Tanaka H, Dang NH, Ikeda E, and Morimoto C. Roxithromycin specifically inhibits development of collagen induced arthritis and production of proinflammatory cytokines by human T cells and macrophages. J. Rheumatol. 32(9): 1765-74, 2005.
71. Urasaki Y, Nori M, Iwata S, Sasaki T, Hosono O, Kawasaki H, Tanaka H, Dang NH, and Morimoto C. Roxithromycin specifically inhibits development of collagen-induced arthritis and proinflammatory cytokines by human T cells and macrophages. Arthritis and Rheumatism, 50(N9): 370, 2004.

Claims

VII. CLAIMSWhat is claimed is:
1. A compound of the following formula (III):
Figure imgf000091_0001
wherein
R1 is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-IO alkylcarbonyl, Cl-IO alkoxycarbonyl, Cl-IO alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-IO alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when R1 is hydrogen or methyl, and that R3 is not cladinose when R1 is methyl and R2 is methyl; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
2. A compound of the following formula (I):
Figure imgf000092_0001
wherein
R1 is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-IO alkylcarbonyl, Cl-IO alkoxycarbonyl, Cl-IO alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, C2- ClO alkynyl, Cl-10 alkylcarbonyl, Cl-IO alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkyny! and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5- tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, C 1-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S; with the proviso that R2 is not hydrogen when R1 is hydrogen or methyl, and that R3 is not cladinose when R1 is methyl and R2 is methyl; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
3. A compound according to claim 2, wherein
R1 is selected from the group consisting of hydrogen, Cl-IO alkyl, C2-10 alkenyl, Cl-IO alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C6-C10 arylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, Cl-10 alkyl, C2-10 alkenyl, Cl-10 alkylcarbonyl, Cl-10 alkoxycarbonyl, Cl-10 alkylsulfonyl, C3-8 cycloalkyl, C6-10 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl, alkenyl, alkynyl and aryl moieties in the Rl and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-8 alkoxy, carboxyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, and C6-10 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, Cl-10 alkylcarbonyl, and C6-10 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with hydroxyl, amino, fluorine, chlorine, bromine, Cl-8 alkoxy, carboxyl, Cl-8 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said Cl-10 alkylcarbonyl and C6-10 arylcarbonyl are, where possible, optionally replaced by O, N or S.
4. A compound according to claim 2, wherein
R1 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R1 and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
5. A compound according to claim 4, wherein
R1 is selected from the group consisting of hydrogen, C2-5 alkenyl, C2-C5 alkynyl, Cl- 5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6- 10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R1 and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of cladinose, C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
6. A compound according to claim 4, wherein
R1 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-C10 arylsulfonyl, C6-10 aryloxycarbonyl, and carbamoyl;
R2 is selected from the group consisting of hydrogen, C 1-5 alkyl, C2-5 alkenyl, C2-C5 alkynyl, C 1-5 alkylcarbonyl, C 1-5 alkoxycarbonyl, C 1-5 alkylsulfonyl, C3-8 cycloalkyl, C6-8 aryl, C6-10 arylcarbonyl, C6-10 aryloxycarbonyl, and carbamoyl; wherein the alkyl alkenyl, alkynyl and aryl moieties in the R1 and R2 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, and C6-8 aryl are, where possible, optionally replaced by O, N or S;
R3 is selected from the group consisting of C 1-5 alkylcarbonyl, and C6-8 arylcarbonyl, wherein alkyl and aryl moieties in the R3 may be optionally substituted with aryl, hydroxyl, amino, fluorine, chlorine, bromine, C 1-5 alkoxy, carboxyl, C 1-5 alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, or 5-tetrazolyl, wherein one to three carbons of said C 1-5 alkylcarbonyl and C6-8 arylcarbonyl are, where possible, optionally replaced by O, N or S.
7. A compound of the following formula (II):
Figure imgf000095_0001
wherein X1 is selected from the group consisting of:
Figure imgf000096_0001
Figure imgf000096_0002
Figure imgf000096_0003
X ^2 is selected from the group consisting of:
Figure imgf000096_0004
with the proviso that said compound is not roxithromycin; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
8. A compound selected from the group consisting of:
5-(3,4,6-Trideoxy-3-N-ethylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-N-(2-methylpropyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-N-benzylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-N-(4-methoxybenzyl)rnethylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-N-(4-chlorobenzyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-N-(3-pyridylmethyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-ethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-(2-methylpropylamino)-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide;
5-(3,4,6-Trideoxy-3-benzylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan- 13-olide;
5-[3,4,6-Trideoxy-3-(4-methoxybenzylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-[3,4,6-Trideoxy-3-(4-chlorobenzylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexaιnethylpentadecan- 13-olide;
5-[3,4,6-Trideoxy-3-(3-pyridylmethylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopy ^nOSyIoXy)-0, 1 1,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-acetamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-benzenesulfonamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-acetoxy-6, 11,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13 - olide;
5-(3,4,6-Trideoxy-3-dimethylarnino-β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6,l 1,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13- olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-phenylacetoxy- 6,11,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10,12- hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3-pyridylacetoxy)- 6,l l,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12- hexamethylpentadecan-13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3- methoxyphenyacetoxy)-6, 11,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(4- methoxyphenylacetoxy)-6, 11,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; and
5-(3,4,6-Trideoxy-3-propylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
9. A compound selected from the group consisting of:
5-(3,4,6-Trideoxy-3-N-ethylmethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; 5-(3,4,6-Trideoxy-3-N-(2-methylpropyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-N-(4-chlorobenzyl)methylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6- dideoxy-3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxy imino-2,4,6,8, 10, 12-hexamethy lpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-N-(3-pyridylmethyl)methylamino-β-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-ethylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-(2-rnethylpropylamino)-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy- 3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-[3,4,6-Trideoxy-3-(3-pyridylmethylamino)-β-D-xylo-hexopyranosyloxy]-3-(2,6- dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-acetamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl- 3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-acetoxy-6,l l,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-13- olide;
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-(3-pyridylacetoxy)- 6,11, 12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10,12- hexamethy lpentadecan- 13-olide; and
5-(3,4,6-Trideoxy-3-propylamino-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C- methyl-3-0-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10,12-hexamethylpentadecan- 13-olide; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
10. The compound of claim 2, wherein formula (I) is selected from the group consisting of: 5-(3,4,6-Trideoxy-3-benzenesulfonamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3- C-methyl-3-O-methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyim ino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; and
5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6,l 1,12- trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13- olide; or a pharmaceutically acceptable salt, prodrug, or solvate thereof.
11. The compound of claim 2, wherein the compound is 5-(3,4,6-Trideoxy-3- benzenesulfonamido-β-D-xylo-hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O- methyl-α-L-ribo-hexopyranosyloxy)-6, 11,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide.
12. The compound of claim 2, wherein the compound is 5-(3,4,6-Trideoxy-3-dimethylamino- β-D-xylo-hexopyranosyloxy)-3-benzoyloxy-6,l l,12-trihydroxy-9-(2- methoxyethoxy)methoxyimino-2,4,6,8,10,12-hexamethylpentadecan-l 3-olide.
13. A pharmaceutical composition comprising a compound of claim 2 or a pharmaceutically acceptable salt, prodrug, or solvate thereof, and a pharmaceutically acceptable excipient.
14. The pharmaceutical composition of claim 13, wherein said composition comprises an effective amount for the treatment of reducing or inhibiting transendothelial migration of T cells and activated T cells; proinflammatory cytokine production by T cells; or IL6 production by macrophages.
15. The pharmaceutical composition of claim 13, wherein said compound is selected from the group consisting of a. 5-(3,4,6-Trideoxy-3-benzenesulfonamido-β-D-xylo- hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo- hexopyranosyloxy)-6, 1 1, 12-trihydroxy-9-(2-methoxyethoxy)methoxy imino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; and b. 5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3- benzoyloxy-6,l l,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino-2,4,6,8,10,12- hexamethylpentadecan-13-olide; or a pharmaceutically acceptable salt, prodrug, or solvate of (a) and (b).
16. The pharmaceutical composition of claim 14, wherein said reduction or inhibition is within a range selected from the group consisting of a. a range of about 10% to 20%; b. a range of about 30% to 40%; c. a range of about 50% to 60%; and d. a range of about 75% to 100%.
17. The pharmaceutical composition of claim 14, wherein said effective amount does not first, simultaneously or subsequently cause inhibition of the production by T cells of one or more cytokines selected from the group consisting of IL2, IFN gamma, 1L4 and IL5.
18. The pharmaceutical composition of claim 13, wherein said composition is suitable for administration directly to an affected region or lesion; systemically; orally; via an implant; intravenously; topically; or intrathecally.
19. The pharmaceutical composition of claim 13 suitable for administration to a human.
20. The pharmaceutical composition of claim 13 suitable for administration to a non-human.
21. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition comprises an effective amount of the compound for the treatment of a bacterial infection in a mammal.
22. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is for the treatment of a disorder in which proinflammatory cytokine production is implicated in a mammal.
23. The pharmaceutical composition of claim 13 further comprises a second macrolide antibiotic.
24. The pharmaceutical composition of claim 13, wherein the composition further comprises one or more non-macrolide antibiotics, anti-inflammatory compounds, or immunomodulatory agents.
25. A method of treatment of a disease or disorder associated with modulation of immune response comprising administering to a patient or mammal in need thereof a therapeutically effective amount of a composition comprising a compound of claim 2 and a pharmaceutically acceptable excipient.
26. The method of claim 25, wherein the disease or disorder implicates transendothelial migration of T cells and activated T cells; proinflammatory cytokine production by T cells; or IL6 production by macrophages.
27. The method of claim 26, wherein said disease or disorder is an arthritic or rheumatic disorder, 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; artherosclerosis; psoriasis; atopic dermatitis; pulmonary diseases associated with granuloma; encephalomyelitis; anklyosing spondylitis; bursitis and tendonitis; carpal tunnel syndrome; chronic back injury; diffuse idiopathic skeletal hyperostosis (DISH); fibromyalgia; lyme disease; Paget's disease; polymyalgia rheumatica; polymyositis and dermatomyositis; Raynaud's phenomenon; Reiter's syndrome; repetitive stress injury; scleroderma; Behcet's syndrome; Sjogren's syndrome; unstable angina; myocardial infarction; treatment after coronary stent placement; or IL-6 related diseases.
28. The method of claim 25, wherein said compound is selected from the group consisting of a. 5-(3,4,6-Trideoxy-3-benzenesulfonamido-β-D-xylo- hexopyranosyloxy)-3-(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo- hexopyranosyloxy)-6, 1 1,12-trihydroxy-9-(2-methoxyethoxy)methoxyimino- 2,4,6,8, 10, 12-hexamethylpentadecan- 13-olide; and b. 5-(3,4,6-Trideoxy-3-dimethylamino-β-D-xylo-hexopyranosyloxy)-3- benzoy loxy-6, 11,12-trihydroxy-9-(2-methoxyethoxy)methoxy imino-2,4,6,8, 10,12- hexamethylpentadecan-13-olide, or a pharmaceutically acceptable salt, prodrug, or solvate of (a) or (b).
29. The method of claim 25, wherein the disease or disorder is arthritic or a rheumatic disorder.
30. The method of claim 29, wherein said disorder is rheumatoid arthritis.
31. The method of claim 25, wherein said composition is administered directly to an affected region or lesion; systemically; orally; via an implant; intravenously; topically; or intrathecally.
32. The method of claim 25, wherein said composition is administered in combination or conjunction with one or more non-macrolide antibiotics, anti-inflammatory compounds, or immunomodulatory agents.
33. The method of claim 32, wherein 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, WY-50295, SC- 45662, SC-41661A, BI-L-357; leukotriene antagonists; peptide derivatives comprising ACTH and analogs thereof; soluble TNF-receptors; TNF-antibodies; soluble receptors of interleukines, other cytokines, T-cell-proteins; antibodies against receptors of interleukines, other cytokines, T-cell-proteins; and calcipotriols and analogues thereof taken either alone or in combination.
34. The method of claim 25, wherein said composition is administered in combination or conjunction with one or more macrolide antibiotics.
35. The method of claim 25, wherein said mammal is a human.
36. The method of claim 25, wherein said mammal is a non-human.
37. The method of claim 25, wherein the disease or disorder is pain or symptom associated with arthritic or rheumatic disorders.
38. The method of claim 25, wherein the method comprises inhibiting the transendothelial migration of T cells and activated T cells or the production of IL6 or TNFalpha or NFkappaB from T cells.
39. The method of claim 25, wherein the method comprises inhibiting IL6 production from macrophages.
40. The method of claim 25, wherein the method comprises inhibiting the activation of NFkappaB in a T cell or macrophage.
41. The method of claim 25, wherein the disease or disorder is a bacterial infection in a mammal.
42. The method of claim 25, wherein the disorder is a disorder in which proinflammatory cytokine production is implicated in a mammal.
43. A method of treating a disease or disorder associated with modulation of immune response comprising administering a macrolide antibiotic that has an antibiotic activity less than roxithromycin and an increased inhibition of TNFalpha and IL-6 production from T cells and macrophages in comparison to roxithromycin.
44. The method of claim 43, wherein said macrolide antibiotic has an IC50 of less than about 15 μM for TNFalpha and less than about 12 μM for IL-6 and a minimum inhibiting concentration of at least about 100 μM for Staphylococcus aureus FDA 209P.
45. Use of a compound according to claim 2 or a pharmaceutically acceptable salt or prodrug thereof for treating a bacterial infection in a mammal.
46. Use of a compound according to claim 2 or a pharmaceutically acceptable salt or prodrug thereof for treating a disorder in which proinflammatory cytokine production is implicated in a mammal.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107531737A (en) * 2015-03-25 2018-01-02 哈佛大学的校长及成员们 The synthesis of desosamine
US11084876B2 (en) 2013-02-08 2021-08-10 Amgen Research (Munich) Gmbh Anti-leukocyte adhesion for the mitigation of potential adverse events caused by CD3-specific binding domains
WO2021183758A1 (en) * 2020-03-12 2021-09-16 Zoetis Services Llc Immunomodulating urea azalides
WO2023038852A1 (en) * 2021-09-07 2023-03-16 Zoetis Services Llc Immunomodulating azalides
RU2811591C1 (en) * 2020-03-12 2024-01-15 ЗОЕТИС СЕРВИСИЗ ЭлЭлСи Immunomodulatory azalides based on urea

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3098205A1 (en) * 2018-04-17 2019-10-24 Shanghai Tonglian Pharmaceutical Co., Ltd. Medicament for preventing and/or treating pain and/or fever, composite product and use thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0216169A2 (en) * 1985-08-28 1987-04-01 Beecham Group Plc Erythromycin derivatives
EP0284203A2 (en) * 1987-02-24 1988-09-28 Beecham Group Plc Erythromycin derivatives, process for their preparation and their pharmaceutical use
EP0487411A1 (en) * 1990-11-21 1992-05-27 Roussel Uclaf Erythromycin derivatives, their preparation, intermediates obtained and their application as medicaments
EP0619320A1 (en) * 1991-12-27 1994-10-12 Taisho Pharmaceutical Co. Ltd 5-o-desosaminylerythronolide derivative
WO2005085266A2 (en) * 2004-02-27 2005-09-15 Rib-X Pharmaceuticals, Inc. Macrocyclic compounds and methods of making and using the same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0216169A2 (en) * 1985-08-28 1987-04-01 Beecham Group Plc Erythromycin derivatives
EP0284203A2 (en) * 1987-02-24 1988-09-28 Beecham Group Plc Erythromycin derivatives, process for their preparation and their pharmaceutical use
EP0487411A1 (en) * 1990-11-21 1992-05-27 Roussel Uclaf Erythromycin derivatives, their preparation, intermediates obtained and their application as medicaments
US5444051A (en) * 1990-11-21 1995-08-22 Roussel Uclaf Erythromycin compounds
EP0619320A1 (en) * 1991-12-27 1994-10-12 Taisho Pharmaceutical Co. Ltd 5-o-desosaminylerythronolide derivative
WO2005085266A2 (en) * 2004-02-27 2005-09-15 Rib-X Pharmaceuticals, Inc. Macrocyclic compounds and methods of making and using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KONNO S-I ET AL: "Influences of roxithromycin on cell-mediated immune responses", LIFE SCIENCES, PERGAMON PRESS, OXFORD, GB, vol. 51, no. 10, 1 January 1992 (1992-01-01), pages PL107 - PL112, XP023741424, ISSN: 0024-3205, [retrieved on 19920101] *
YOUNG R A ET AL: "ROXITHROMYCIN. ÖA REVIEW OF ITS ANTIBACTERIAL ACTIVITY, PHARMACOKINETIC PROPERTIES AND CLINICAL EFFICACY", DRUGS, ADIS INTERNATIONAL LTD, vol. 37, 1 January 1989 (1989-01-01), pages 8 - 41, XP000569145, ISSN: 0012-6667 *

Cited By (10)

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US11084876B2 (en) 2013-02-08 2021-08-10 Amgen Research (Munich) Gmbh Anti-leukocyte adhesion for the mitigation of potential adverse events caused by CD3-specific binding domains
CN107531737A (en) * 2015-03-25 2018-01-02 哈佛大学的校长及成员们 The synthesis of desosamine
JP2018510878A (en) * 2015-03-25 2018-04-19 プレジデント アンド フェローズ オブ ハーバード カレッジ Synthesis of desosamine
EP3277701A4 (en) * 2015-03-25 2019-03-06 President and Fellows of Harvard College Synthesis of desosamines
US11008358B2 (en) 2015-03-25 2021-05-18 President And Fellows Of Harvard College Synthesis of desosamines
WO2021183758A1 (en) * 2020-03-12 2021-09-16 Zoetis Services Llc Immunomodulating urea azalides
US11771677B2 (en) 2020-03-12 2023-10-03 Zoetis Services Llc Immunomodulating urea azalides
RU2811591C1 (en) * 2020-03-12 2024-01-15 ЗОЕТИС СЕРВИСИЗ ЭлЭлСи Immunomodulatory azalides based on urea
RU2811591C9 (en) * 2020-03-12 2024-03-26 ЗОЕТИС СЕРВИСИЗ ЭлЭлСи Immunomodulatory azalides based on urea
WO2023038852A1 (en) * 2021-09-07 2023-03-16 Zoetis Services Llc Immunomodulating azalides

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