WO2003090758A1 - Method for treating inflammatory disorders - Google Patents

Method for treating inflammatory disorders Download PDF

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Publication number
WO2003090758A1
WO2003090758A1 PCT/EP2003/004342 EP0304342W WO03090758A1 WO 2003090758 A1 WO2003090758 A1 WO 2003090758A1 EP 0304342 W EP0304342 W EP 0304342W WO 03090758 A1 WO03090758 A1 WO 03090758A1
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Prior art keywords
deoxy
fluoro
inhibitor
administered
glycosylation
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PCT/EP2003/004342
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French (fr)
Inventor
Vishwas Sadashiv Ganu
Shou-Ih Hu
Earl F. Kimble
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Novartis Ag
Novartis Pharma Gmbh
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Priority to AU2003227681A priority Critical patent/AU2003227681A1/en
Publication of WO2003090758A1 publication Critical patent/WO2003090758A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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]

Definitions

  • the invention relates to methods for the treatment of individuals suffering from inflammatory diseases by the administration to such a subject of an effective amount of an inhibitor of N-glycosylation of proteins, where the inhibitor is not glucosamine, D- mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6- phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose.
  • the invention in one aspect, is directed to methods for inhibiting the production of inflammatory cytokines, for inhibiting the production of non-cytokine inflammatory mediator molecules, for decreasing the amount of matrix metalloprotease activity, for treating a subject suffering from inflammation in one or more bodily tissues, or for treating a subject suffering from an inflammatory disease in a subject in need of such treatment, comprising administering to said subject an effective amount of an inhibitor of N- glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D- galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl- D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose, to achieve the aforesaid effects.
  • the invention is directed to methods for identifying compounds that will inhibit the production of inflammatory mediator molecules, inflammatory cytokines, or that will inhibit MMP-13, aggrecanase or other matrix metalloprotease activity or production in the tissues of a mammal, comprising determining that a compound not known to be an inhibitor of an enzyme involved in the N-glycosylation of proteins is an inhibitor of at least one enzyme involved in the N-glycosylation of proteins, to yield a previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins; and testing the previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins in an assay system to determine if the previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins inhibits a member selected from the group consisting of (a) the production by cells or tissues of a mammal of inflammatory mediator molecules (b) the production of inflammatory cytokines, (c) the production of MMP-13, (d) aggrecanases, or (e) matrix metalloprot
  • the present inventors have surprisingly and unexpectedly discovered that subjects suffering from inflammatory diseases can be treated by the administration to such a subject of an effective amount of an inhibitor of N-glycosylation of proteins, where the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2- deoxy-D-galactose or mannose.
  • 2-deoxy-2-fluoro-d-hexoses are analogs of hexose sugars.
  • Radioactive 2-deoxy-2-fluoro-d- glucose (FDG) is an agent currently administered to humans for use in positron emission tomography (PET).
  • unlabeled 2-deoxy-2-fluoro-d-hexoses including FDG and 2-deoxy-2-fluoro-d-mannose (FDM)
  • FDG 2-deoxy-2-fluoro-d-mannose
  • FDM 2-deoxy-2-fluoro-d-mannose
  • Other compounds with N-glycosylation-inhibiting activity would be expected to have similar antiinflammatory activity.
  • Non-limiting examples of inflammatory cytokines produced by stimulated chondrocytes are IL-6, IL-8, and IL-18.
  • Nonlimiting examples of glycosylated enzymes involved in inflammatory processes are matrix metalloproteases such as MMP-9 (92 kd gelatinase) and MMP-1 (collagenase-1), inducible nitric oxide synthase, and PC-1 (PC- 1: plasma cell antigen 1 or nucleotide pyrophosphatase/phosphodiesterase, aka NPPl).
  • MMP-9 92 kd gelatinase
  • MMP-1 collagenase-1
  • PC-1 PC-1
  • the production of any other glycosylated enzymes involved in inflammatory processes, where the enzymes contain an Asn-X-Thr or Asn-X-Ser motif in the primary sequence can also be modulated by the methods and compositions of the present invention.
  • Non-limiting examples of non-cytokine inflammatory mediator molecules are transforming growth factor-beta, insulin-like growth factor, and insulin-like growth factor-binding proteins.
  • symptoms of osteoarthritis includes joint pain and aching, limited range of motion and instability, radiographic evidence of the erosion of the articular cartilage, joint space narrowing, sclerosis of the subchondral bone, and osteophytes (spurs).
  • N-glycosylation-inhibiting compounds of the invention can be administered to a subject suffering from inflammation orally, topically, through an inhalation device, or as an injectable.
  • the compounds can be administered alone in aqueous solution or in combination with, e.g., a hyaluronic acid polymer to patients with rheumatoid or osteoarthritis or other inflammatory disorders (e.g. psoriasis, dermatitis, respiratory diseases or cardiovascular or ophthalmic conditions etc.).
  • the compounds of the invention can be administered alone or in combination with other anti-inflammatory drugs to treat conditions where MMP, NO, and cytokines are either known or expected to play a role in the pathogenesis of the disease (e.g. psoriasis, dermatitis, respiratory diseases or cardiovascular or inflammatory or neovascular ophthalmic conditions).
  • MMP, NO, and cytokines are either known or expected to play a role in the pathogenesis of the disease (e.g. psoriasis, dermatitis, respiratory diseases or cardiovascular or inflammatory or neovascular ophthalmic conditions).
  • Analogs of glucose, mannose, and ribose that are substituted at either or both of positions 2 and 4 of the sugar chains with other halogens are also within the scope of the invention, as are therapeutic methods that utilize such analogs.
  • the invention relates to the administration of a pharmaceutical composition
  • a pharmaceutical composition comprising inhibitors of N-glycosylation of proteins, preferably 2-deoxy-2-fluoro-d- hexoses and analogs thereof, in conjunction with one or more pharmaceutically acceptable carriers, for any of the therapeutic effects discussed above.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions encompassed by the invention may be administered by any number of routes including, but not limited to, oral, intraarticular, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0. 1 %-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 ED 50 .
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions, or locally administered doses, may be administered every 3 to 4 days, every week, or once every two weeks, or less frequently depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.001 g up to a total dose of about 1.5 g, depending upon the route of administration.
  • dosages can be given in multiples of 25 mg, i.e., 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 1000 mg, 1500 mg, etc. or in multiples of 10 mg. Total dosage can range between any of the aforementioned dosages.
  • the present invention is also directed to methods for identifying antiinflammatory compounds by first identifying previously unknown inhibitors of N-glycosylation of proteins, and then assaying the identified inhibitors for antiinflammatory activity. Some assays to determine whether compounds are inhibitors of N-glycosylation of proteins are detailed below, and such assays also include determination of N-glycosylation of the glucose transporter in cells.
  • assays are well-known in the art, e.g., assays for detecting inhibition of UDP-N-acetylglucosamine:dolichol-phosphate N- acetylglucosamine-1 -phosphate transferase, the first enzyme involved in the dolichol pathway of protein N-glycosylation and assays for detecting inhibition of a mannosyl transferase, an enzyme that catalyzes the transfer of GDP-d-mannose to dolichol-phosphate (Dol-P) or to Dol-P-(GlcNAc) 2 .
  • In vivo assays to determine whether a compound is possesses antiinflammatory activity or cartilage protecting activity include using the STR/ort mouse model of spontaneous osteoarthritis (Altman, F.P., Ann. Rheum. Dis. 1981 ;40:303-6), using a guinea pig spontaneous osteoarthritis model (Meacock, S.C. et al., J. Exp. P ⁇ t/wZ. (Oxford) 1990;71:279-93), and using surgical models in various mammals, such as rabbits (Bohr, H. Acta Orthop. Scand. 1976;47:558-65), in addition to the paw edema model set out herein.
  • Confluent primary human chondrocytes were isolated from cartilages of osteoarthritic patients undergoing joint replacement surgery.
  • Cells were cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 ⁇ g/ml BSA/5.5 mM d-glucose.
  • the cells are pre-treated for 1 h at 37°C with 2-deoxy-2-fluoro-d-glucose (FDG, mol. wt. 182.12) and 2- deoxy-2-fluoro-d-mannose (FDM, mol. wt. 182.12) followed by stimulation with 2 ng/ml IL-l ⁇ plus 2 ng/ml OSM.
  • FDG 2-deoxy-2-fluoro-d-glucose
  • FDM 2- deoxy-2-fluoro-d-mannose
  • MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed by an ELISA assay using a commercially available kit.
  • 2 ng/ml IL-l ⁇ plus 2 ng/ml oncostatin M (OSM) is added to all groups except unstimulated cells.
  • FDG or FDM Treatment with FDG or FDM in doses ranging from 50 ⁇ M to 1 mM, the compounds inhibit cytokine induced production of nitric oxide and prostaglandin E 2 (PGE2) in a dose- dependent fashion.
  • PGE2 prostaglandin E 2
  • FDG and FDM also inhibit matrix metalloprotease activity in a dose- dependent fashion.
  • bovine chondrocytes The inhibitory effect of FDG and FDM on chondrocyte activation is also observed with bovine chondrocytes in a similar dose-dependent manner.
  • the bovine chondrocytes are assayed as follows: confluent primary bovine chondrocytes obtained from bovine articular cartilage are cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 ⁇ g/ml BSA/5.5 mM d-glucose. The cells are pre-treated for 1 h at 37°C with 2-deoxy-2- fluoro-d-glucose (FDG, mol. wt. 182.12) followed by stimulation with 2 ng/ml IL-l ⁇ plus 2 ng/ml OSM.
  • FDG 2-deoxy-2- fluoro-d-glucose
  • MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed by an ELISA assay using a commercially available kit.
  • FDG and FDM are known to interfere with the enzymes in the biosynthesis of N-linked (asparagine) glycoproteins.
  • N-linked (asparagine) glycoproteins To determine whether general inhibition of protein glycosylation will inhibit the production of inflammatory mediator molecules and/or inhibit the activity of proteases known to be involved in inflammation, we tested the efficacy of tunicamycin, a known specific inhibitor of the 1 st step in the biosynthesis of N-linked protein glycosylation. Tunicamycin, at concentrations above about 1 nM and in a dose dependent fashion, inhibits cytokine-induced MMP activty and NO and PGE-2 production by bovine chondrocytes.
  • the assay is performed as follows: Confluent primary bovine chondrocytes from bovine articular cartilage are cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 ⁇ g/ml BSA/5.5 mM d-glucose. The cells are pre- treated for 1 h at 37°C with tunicamycin (Sigma Chem. Co., St. Louis, MO) followed by stimulation with 2 ng/ml IL-l ⁇ plus 2 ng/ml OSM. After 48 h, the media is harvested and analyzed for MMP activity, nitric oxide, and PGE-2.
  • MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed by an ELISA assay using a commercially available kit.
  • 2 ng/ml IL-l ⁇ plus 2 ng/ml OSM is added to all groups except unstimulated cells. It is believed that this is the first demonstration that tunicamycin can suppress cytokine-induced production of inflammatory mediators by chondrocytes of any type.
  • Example 4 Since tunicamycin is a known inhibitor of UDP-N-acetylglucosamine:dolichyl-phosphate N-acetyl glucosamine- 1 -phosphate transferase (GPT), the results above led the inventors to investigate whether inhibition of other enzymes in the biosynthetic pathway for N- glycosylated proteins could lead to identification of novel inhibitors to control production of inflammatory and cartilage degrading mediators by chondrocytes.
  • GPT UDP-N-acetylglucosamine:dolichyl-phosphate N-acetyl glucosamine- 1 -phosphate transferase
  • the enzyme upstream of GPT is ccl-4 mannosyltransferase, which catalyzes the transfer of GDP-d-mannose to (GlcN) 2 -PP-Dol. It is found that inhibition of this enzyme also leads to inhibition of cytokine induced mediator production by chondrocytes.
  • 2-deoxy-d-glucose (2DG) interferes with the glycosylation of N-linked glycoproteins. The hypothesis that 2- deoxy-d-glucose inhibits cytokine induced chondrocyte activation by interfering with the formation of N-glycosylation of proteins or intermediates necessary to produce cytokine stimulated production of mediators is tested.
  • d-mannose also tested is the ability of d-mannose to reverse the effect of 2DG. It is found that 2DG dose dependently inhibited MMP activity, nitric oxide, and PGE-2 activity, d-mannose but not d-glucose reverse the inhibitory effects of 2DG on the production of inflammatory mediators (MMP, nitric oxide, and PGE-2) by chondrocytes. Neither d-mannose or d-glucose has any effect on the production of the mediators themselves.
  • the assay is performed as follows: Confluent primary bovine chondrocytes obtained from bovine articular cartilage are cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 ⁇ g/ml BSA/5.5 mM d-glucose. The cells are pre-treated for 1 h at 37°C with 2DG, 2DG plus 1 M d-mannose (Mnos), 2DG plus 1 mM d-glucose (Glc), 1 mM d- mannose, and 1 mM d-glucose followed by stimulation of all groups, except unstimulated cells, with 2 ng/ml IL-l ⁇ plus 2 ng/ml OSM.
  • MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed using a commercially available ELISA kit.
  • the sugar derivatives FDG and FDM showed an ability to inhibit PGE-2 production by cytokine-stimulated chondrocytes. Since PGE-2 is an inflammatory molecule, it was tested whether FDG could inhibit inflammation in vivo. In the following in vivo model in rats, FDG, 2DG, and d-glucosamine (GlcN) are tested for efficacy in inhibiting edema formation in rat paws. It is determined that FDG is more potent than glucosamine and 2DG in inhibiting edema formation in rats.
  • GlcN, DG or FDG at 100 mg/kg are dosed intraperitoneally into SD male rats (5/group).
  • Fifteen minutes after compound administration 0.1 ml of 1% carrageenan solution in saline is injected into the subplantar region of the left hind paw to induce inflammation (an equal volume of saline, 0.1 ml, is injected into the subplantar region of the right hind paw).
  • the rats are euthanatized three hours after carrageenan or saline injection and both hind paws cut off at the hairline and weighed on an electric balance.
  • the amount of edema in the inflamed paw is determined by subtracting the weight of the non-inflamed paw (right) from the weight of the inflamed paw (left).
  • the percent inhibition caused by a compound is determined for each rat as percent paw weight gained as compared to the vehicle-treated group average.
  • Inflammation is inhibited in DG- and FDG-treated rats to a significantly greater extent than in GlcN-treated rats. Vehicle-treated rats show an average increase in paw weight of 1.35 ⁇ 0.8 grams. The above results confirm the ability of FDG to inhibit inflammation in vivo.
  • IC-21 cells mouse peritoneal macrophages
  • 2DG, FDG, and FDM are stimulated with LPS in the presence or absence of 2DG, FDG, and FDM, and TNF ⁇ produced 24 h later is quantitated.
  • FDG, FDM, and 2-amino-2-deoxy-d-glucose are tested in an in vivo model of TNF ⁇ production in mice to determine whether they can inhibit in vivo TNF ⁇ production.
  • FDG, FDM or GlcN at 30 mg/kg are dosed intraperitoneally into C57BL/6J mice (5/group).
  • TNF ⁇ production is stimulated with an intraperitoneal injection of LPS/D-galactosamine.
  • the mice are bled and plasma obtained.
  • the plasma is used to determine TNF ⁇ levels using ELISA kits. NaCl- treated mice have between 500 and 1700 pg ml of plasma TNF ⁇ in two separate studies.
  • FDG and FDM are functionally active agents, in vivo, for the inhibition of inflammation and cartilage degradation by virtue of their ability to inhibit MMP activity, the production of nitric oxide, prostaglandin E2, and the pro-inflammatory cytokine TNF ⁇ .
  • Aggrecanase is an enzyme that degrades proteoglycan, a substrate residing in articular cartilage. There are two forms, aggrecanase- 1 and aggrecanase-2. These enzymes are implicated in the degradation of articular cartilage in osteoarthritis. It is determined that N- glycosylation inhibitors inhibit the accumulation of mRNA for aggrecanase- 1 and -2.
  • Primary bovine chondrocytes are treated with cytokines (IL-l ⁇ plus oncostatin M) alone or in combination with 1 ⁇ M tunicamycin (Tn), 10 mM d-glucosamine (GlcN), or 1 mM 2- deoxy-d-glucose (2-DG).
  • chondrocytes are isolated from the cartilage of osteoarthritic patients undergoing joint replacement surgery.
  • the cells are treated with IL-l ⁇ plus oncostatin M in the presence and absence of FDG and FDM (at concentrations of 50 ⁇ M, 100 ⁇ M, 300 ⁇ M, and 1000 ⁇ M, when present).
  • FDG and FDM at concentrations of 50 ⁇ M, 100 ⁇ M, 300 ⁇ M, and 1000 ⁇ M, when present.
  • the production of MMP-13 and IL-6 by the chondrocytes is quantitated by ELISA. It is determined that FDG inhibits the production of IL-6 and MMP-13 at a concentration of 50 ⁇ M and above, and that FDM inhibits the production of IL-6 at a concentration of 300 ⁇ M and above and inhibits production of MMP-13 at a concentration of 100 ⁇ M and above.

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Abstract

Disclosed herein are methods for treating inflammatory disorders, compositions for treating inflammatory disorders, and methods for identifying compounds that will treat inflammatory disorders.

Description

METHOD FOR TREATING INFLAMMATORY DISORDERS
The invention relates to methods for the treatment of individuals suffering from inflammatory diseases by the administration to such a subject of an effective amount of an inhibitor of N-glycosylation of proteins, where the inhibitor is not glucosamine, D- mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6- phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose.
Summary of the Invention
The invention, in one aspect, is directed to methods for inhibiting the production of inflammatory cytokines, for inhibiting the production of non-cytokine inflammatory mediator molecules, for decreasing the amount of matrix metalloprotease activity, for treating a subject suffering from inflammation in one or more bodily tissues, or for treating a subject suffering from an inflammatory disease in a subject in need of such treatment, comprising administering to said subject an effective amount of an inhibitor of N- glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D- galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl- D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose, to achieve the aforesaid effects.
In another aspect, the invention is directed to methods for identifying compounds that will inhibit the production of inflammatory mediator molecules, inflammatory cytokines, or that will inhibit MMP-13, aggrecanase or other matrix metalloprotease activity or production in the tissues of a mammal, comprising determining that a compound not known to be an inhibitor of an enzyme involved in the N-glycosylation of proteins is an inhibitor of at least one enzyme involved in the N-glycosylation of proteins, to yield a previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins; and testing the previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins in an assay system to determine if the previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins inhibits a member selected from the group consisting of (a) the production by cells or tissues of a mammal of inflammatory mediator molecules (b) the production of inflammatory cytokines, (c) the production of MMP-13, (d) aggrecanases, or (e) matrix metalloprotease activity or production.
Detailed Description of the Invention
The present inventors have surprisingly and unexpectedly discovered that subjects suffering from inflammatory diseases can be treated by the administration to such a subject of an effective amount of an inhibitor of N-glycosylation of proteins, where the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2- deoxy-D-galactose or mannose.
2-deoxy-2-fluoro-d-hexoses are analogs of hexose sugars. Radioactive 2-deoxy-2-fluoro-d- glucose (FDG) is an agent currently administered to humans for use in positron emission tomography (PET). It has now been unexpectedly discovered that, in particular, unlabeled 2-deoxy-2-fluoro-d-hexoses, including FDG and 2-deoxy-2-fluoro-d-mannose (FDM), can be used for inhibiting the production of inflammatory cytokines in a subject, for inhibiting the production of non-cytokine inflammatory mediator molecules in a subject, for decreasing the amount of matrix metalloprotease activity in a subject, for treating a subject suffering from inflammation in one or more bodily tissues, or for treating a subject suffering from an inflammatory disease, due to their ability to inhibit the N-glycosylation of proteins by cells. Other compounds with N-glycosylation-inhibiting activity would be expected to have similar antiinflammatory activity.
Non-limiting examples of inflammatory cytokines produced by stimulated chondrocytes are IL-6, IL-8, and IL-18. Nonlimiting examples of glycosylated enzymes involved in inflammatory processes are matrix metalloproteases such as MMP-9 (92 kd gelatinase) and MMP-1 (collagenase-1), inducible nitric oxide synthase, and PC-1 (PC- 1: plasma cell antigen 1 or nucleotide pyrophosphatase/phosphodiesterase, aka NPPl). The production of any other glycosylated enzymes involved in inflammatory processes, where the enzymes contain an Asn-X-Thr or Asn-X-Ser motif in the primary sequence, can also be modulated by the methods and compositions of the present invention.
Non-limiting examples of non-cytokine inflammatory mediator molecules are transforming growth factor-beta, insulin-like growth factor, and insulin-like growth factor-binding proteins.
As used herein, the term "symptoms of osteoarthritis" includes joint pain and aching, limited range of motion and instability, radiographic evidence of the erosion of the articular cartilage, joint space narrowing, sclerosis of the subchondral bone, and osteophytes (spurs).
The N-glycosylation-inhibiting compounds of the invention, either alone or in a pharmaceutically acceptable preparation can be administered to a subject suffering from inflammation orally, topically, through an inhalation device, or as an injectable. In injectable form, the compounds can be administered alone in aqueous solution or in combination with, e.g., a hyaluronic acid polymer to patients with rheumatoid or osteoarthritis or other inflammatory disorders (e.g. psoriasis, dermatitis, respiratory diseases or cardiovascular or ophthalmic conditions etc.). The compounds of the invention can be administered alone or in combination with other anti-inflammatory drugs to treat conditions where MMP, NO, and cytokines are either known or expected to play a role in the pathogenesis of the disease (e.g. psoriasis, dermatitis, respiratory diseases or cardiovascular or inflammatory or neovascular ophthalmic conditions). Analogs of glucose, mannose, and ribose that are substituted at either or both of positions 2 and 4 of the sugar chains with other halogens are also within the scope of the invention, as are therapeutic methods that utilize such analogs.
Thus, the invention relates to the administration of a pharmaceutical composition comprising inhibitors of N-glycosylation of proteins, preferably 2-deoxy-2-fluoro-d- hexoses and analogs thereof, in conjunction with one or more pharmaceutically acceptable carriers, for any of the therapeutic effects discussed above. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
The pharmaceutical compositions encompassed by the invention may be administered by any number of routes including, but not limited to, oral, intraarticular, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0. 1 %-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions, or locally administered doses, may be administered every 3 to 4 days, every week, or once every two weeks, or less frequently depending on half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from 0.001 g up to a total dose of about 1.5 g, depending upon the route of administration. When taken orally, dosages can be given in multiples of 25 mg, i.e., 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 1000 mg, 1500 mg, etc. or in multiples of 10 mg. Total dosage can range between any of the aforementioned dosages.
The present invention is also directed to methods for identifying antiinflammatory compounds by first identifying previously unknown inhibitors of N-glycosylation of proteins, and then assaying the identified inhibitors for antiinflammatory activity. Some assays to determine whether compounds are inhibitors of N-glycosylation of proteins are detailed below, and such assays also include determination of N-glycosylation of the glucose transporter in cells. Other such assays are well-known in the art, e.g., assays for detecting inhibition of UDP-N-acetylglucosamine:dolichol-phosphate N- acetylglucosamine-1 -phosphate transferase, the first enzyme involved in the dolichol pathway of protein N-glycosylation and assays for detecting inhibition of a mannosyl transferase, an enzyme that catalyzes the transfer of GDP-d-mannose to dolichol-phosphate (Dol-P) or to Dol-P-(GlcNAc)2. In vivo assays to determine whether a compound is possesses antiinflammatory activity or cartilage protecting activity include using the STR/ort mouse model of spontaneous osteoarthritis (Altman, F.P., Ann. Rheum. Dis. 1981 ;40:303-6), using a guinea pig spontaneous osteoarthritis model (Meacock, S.C. et al., J. Exp. Pαt/wZ. (Oxford) 1990;71:279-93), and using surgical models in various mammals, such as rabbits (Bohr, H. Acta Orthop. Scand. 1976;47:558-65), in addition to the paw edema model set out herein.
EXAMPLES
Example 1
Confluent primary human chondrocytes were isolated from cartilages of osteoarthritic patients undergoing joint replacement surgery. Cells were cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 μg/ml BSA/5.5 mM d-glucose. The cells are pre-treated for 1 h at 37°C with 2-deoxy-2-fluoro-d-glucose (FDG, mol. wt. 182.12) and 2- deoxy-2-fluoro-d-mannose (FDM, mol. wt. 182.12) followed by stimulation with 2 ng/ml IL-lβ plus 2 ng/ml OSM. After 48 h, the media is harvested and analyzed for MMP activity, nitric oxide, and PGE-2. MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed by an ELISA assay using a commercially available kit. 2 ng/ml IL-lβ plus 2 ng/ml oncostatin M (OSM) is added to all groups except unstimulated cells.
Treatment with FDG or FDM in doses ranging from 50 μM to 1 mM, the compounds inhibit cytokine induced production of nitric oxide and prostaglandin E2 (PGE2) in a dose- dependent fashion. FDG and FDM also inhibit matrix metalloprotease activity in a dose- dependent fashion.
Example 2
The inhibitory effect of FDG and FDM on chondrocyte activation is also observed with bovine chondrocytes in a similar dose-dependent manner. The bovine chondrocytes are assayed as follows: confluent primary bovine chondrocytes obtained from bovine articular cartilage are cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 μg/ml BSA/5.5 mM d-glucose. The cells are pre-treated for 1 h at 37°C with 2-deoxy-2- fluoro-d-glucose (FDG, mol. wt. 182.12) followed by stimulation with 2 ng/ml IL-lβ plus 2 ng/ml OSM. After 48 h, the media is harvested and analyzed for MMP activity, nitric oxide, and PGE-2. MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed by an ELISA assay using a commercially available kit.
Example 3
FDG and FDM are known to interfere with the enzymes in the biosynthesis of N-linked (asparagine) glycoproteins. To determine whether general inhibition of protein glycosylation will inhibit the production of inflammatory mediator molecules and/or inhibit the activity of proteases known to be involved in inflammation, we tested the efficacy of tunicamycin, a known specific inhibitor of the 1st step in the biosynthesis of N-linked protein glycosylation. Tunicamycin, at concentrations above about 1 nM and in a dose dependent fashion, inhibits cytokine-induced MMP activty and NO and PGE-2 production by bovine chondrocytes. The assay is performed as follows: Confluent primary bovine chondrocytes from bovine articular cartilage are cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 μg/ml BSA/5.5 mM d-glucose. The cells are pre- treated for 1 h at 37°C with tunicamycin (Sigma Chem. Co., St. Louis, MO) followed by stimulation with 2 ng/ml IL-lβ plus 2 ng/ml OSM. After 48 h, the media is harvested and analyzed for MMP activity, nitric oxide, and PGE-2. MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed by an ELISA assay using a commercially available kit. 2 ng/ml IL-lβ plus 2 ng/ml OSM is added to all groups except unstimulated cells. It is believed that this is the first demonstration that tunicamycin can suppress cytokine-induced production of inflammatory mediators by chondrocytes of any type.
Example 4 Since tunicamycin is a known inhibitor of UDP-N-acetylglucosamine:dolichyl-phosphate N-acetyl glucosamine- 1 -phosphate transferase (GPT), the results above led the inventors to investigate whether inhibition of other enzymes in the biosynthetic pathway for N- glycosylated proteins could lead to identification of novel inhibitors to control production of inflammatory and cartilage degrading mediators by chondrocytes.
The enzyme upstream of GPT is ccl-4 mannosyltransferase, which catalyzes the transfer of GDP-d-mannose to (GlcN)2-PP-Dol. It is found that inhibition of this enzyme also leads to inhibition of cytokine induced mediator production by chondrocytes. 2-deoxy-d-glucose (2DG) interferes with the glycosylation of N-linked glycoproteins. The hypothesis that 2- deoxy-d-glucose inhibits cytokine induced chondrocyte activation by interfering with the formation of N-glycosylation of proteins or intermediates necessary to produce cytokine stimulated production of mediators is tested. Also tested is the ability of d-mannose to reverse the effect of 2DG. It is found that 2DG dose dependently inhibited MMP activity, nitric oxide, and PGE-2 activity, d-mannose but not d-glucose reverse the inhibitory effects of 2DG on the production of inflammatory mediators (MMP, nitric oxide, and PGE-2) by chondrocytes. Neither d-mannose or d-glucose has any effect on the production of the mediators themselves. These observations demonstrate that inhibition of a αl-4 mannosyltransferase or a mannosyltransferase whose activity is modulated by exogenously added d-mannose leads to inhibition of mediators produced by cytokine stimulated chondrocytes. It is believed that the inventors are the first to demonstrate that inhibitors of a mannosyltransferase inhibits the production of inflammatory mediators by chondrocytes or other cell types.
The assay is performed as follows: Confluent primary bovine chondrocytes obtained from bovine articular cartilage are cultured in 48 well plates in media containing DMEM/10 mM HEPES/50 μg/ml BSA/5.5 mM d-glucose. The cells are pre-treated for 1 h at 37°C with 2DG, 2DG plus 1 M d-mannose (Mnos), 2DG plus 1 mM d-glucose (Glc), 1 mM d- mannose, and 1 mM d-glucose followed by stimulation of all groups, except unstimulated cells, with 2 ng/ml IL-lβ plus 2 ng/ml OSM. After 48 h, the media is harvested and analyzed for MMP activity, nitric oxide, and PGE-2. MMP activity is assayed after trypsin activation, nitric oxide is measured by Greiss reaction, and PGE-2 is assayed using a commercially available ELISA kit.
Example 5
As noted above, the sugar derivatives FDG and FDM showed an ability to inhibit PGE-2 production by cytokine-stimulated chondrocytes. Since PGE-2 is an inflammatory molecule, it was tested whether FDG could inhibit inflammation in vivo. In the following in vivo model in rats, FDG, 2DG, and d-glucosamine (GlcN) are tested for efficacy in inhibiting edema formation in rat paws. It is determined that FDG is more potent than glucosamine and 2DG in inhibiting edema formation in rats.
GlcN, DG or FDG at 100 mg/kg are dosed intraperitoneally into SD male rats (5/group). Fifteen minutes after compound administration, 0.1 ml of 1% carrageenan solution in saline is injected into the subplantar region of the left hind paw to induce inflammation (an equal volume of saline, 0.1 ml, is injected into the subplantar region of the right hind paw). The rats are euthanatized three hours after carrageenan or saline injection and both hind paws cut off at the hairline and weighed on an electric balance. The amount of edema in the inflamed paw is determined by subtracting the weight of the non-inflamed paw (right) from the weight of the inflamed paw (left). The percent inhibition caused by a compound is determined for each rat as percent paw weight gained as compared to the vehicle-treated group average. Inflammation is inhibited in DG- and FDG-treated rats to a significantly greater extent than in GlcN-treated rats. Vehicle-treated rats show an average increase in paw weight of 1.35 ± 0.8 grams. The above results confirm the ability of FDG to inhibit inflammation in vivo.
Example 6
To determine whether FDG and FDM have the potential to inhibit the production of TNFα in vitro, IC-21 cells (mouse peritoneal macrophages) are stimulated with LPS in the presence or absence of 2DG, FDG, and FDM, and TNFα produced 24 h later is quantitated. 2DG, FDG, and FDM inhibit TNFα production by -50-60 % (n=3) at 100 μM.
FDG, FDM, and 2-amino-2-deoxy-d-glucose (glucosamine, GlcN) are tested in an in vivo model of TNFα production in mice to determine whether they can inhibit in vivo TNFα production. FDG, FDM or GlcN at 30 mg/kg are dosed intraperitoneally into C57BL/6J mice (5/group). One hour later, TNFα production is stimulated with an intraperitoneal injection of LPS/D-galactosamine. One hour after stimulation, the mice are bled and plasma obtained. The plasma is used to determine TNFα levels using ELISA kits. NaCl- treated mice have between 500 and 1700 pg ml of plasma TNFα in two separate studies. Normal mice (no LPS/D-galactosamine stimulation) have 18 pg/ml of plasma TNFα. FDG and FDM at 30 mg/kg inhibit TNFα production by between about 50 and 65%, respectively. This is significantly greater than the inhibition by 30 mg/kg of GlcN. Thus, FDG and FDM are functionally active agents, in vivo, for the inhibition of inflammation and cartilage degradation by virtue of their ability to inhibit MMP activity, the production of nitric oxide, prostaglandin E2, and the pro-inflammatory cytokine TNFα.
Example 7
Aggrecanase is an enzyme that degrades proteoglycan, a substrate residing in articular cartilage. There are two forms, aggrecanase- 1 and aggrecanase-2. These enzymes are implicated in the degradation of articular cartilage in osteoarthritis. It is determined that N- glycosylation inhibitors inhibit the accumulation of mRNA for aggrecanase- 1 and -2. Primary bovine chondrocytes are treated with cytokines (IL-lβ plus oncostatin M) alone or in combination with 1 μM tunicamycin (Tn), 10 mM d-glucosamine (GlcN), or 1 mM 2- deoxy-d-glucose (2-DG). After 24 h, mRNA is isolated and RT-PCR is performed using appropriate primers based on cDNA sequences of these two enzymes. Tunicamycin and 2- deoxy-d-glucose are potent inhibitors of the accumulation of aggrecanase mRNA. These findings suggest that N-glycosylation inhibitors will also inhibit cartilage degradation by inhibiting aggrecanase. Example 8
Primary human chondrocytes are isolated from the cartilage of osteoarthritic patients undergoing joint replacement surgery. The cells are treated with IL-lβ plus oncostatin M in the presence and absence of FDG and FDM (at concentrations of 50 μM, 100 μM, 300 μM, and 1000 μM, when present). The production of MMP-13 and IL-6 by the chondrocytes is quantitated by ELISA. It is determined that FDG inhibits the production of IL-6 and MMP-13 at a concentration of 50 μM and above, and that FDM inhibits the production of IL-6 at a concentration of 300 μM and above and inhibits production of MMP-13 at a concentration of 100 μM and above.

Claims

What is claimed is:
1. A method for inhibiting the production of inflammatory cytokines in a subject in need of such treatment, comprising administering to said subject an effective amount of an inhibitor of N-glycosylation of proteins, wherein the inhibitor is not glucosamine, D- mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6- phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose, to inhibit the production of inflammatory cytokines in said subject.
2. A method for inhibiting the production of non-cytokine inflammatory mediator molecules in a subject in need of such treatment, comprising administering to said subject an effective amount of an inhibitor of N-glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy- D-galactose or mannose, to inhibit the production of non-cytokine inflammatory mediators in said subject.
3. A method for decreasing the amount of matrix metalloprotease activity in a subject in need of such treatment, comprising administering to said subject an effective amount of an inhibitor of N-glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy- D-galactose or mannose, to decrease the amount of matrix metalloprotease activity in said subject.
4. A method for treating a subject suffering from inflammation in one or more bodily tissues, comprising administering to said subject an effective amount of an inhibitor of N-glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N- acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose, to decrease inflammation in said one or more bodily tissues.
5. A method for treating a subject suffering from an inflammatory disease, wherein said subject exhibits one or more symptoms of said inflammatory disease, comprising administering to said subject an effective amount of an inhibitor of N-glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose, to ameliorate one or more of said symptoms of said inflammatory disease.
6. The method of claim 1, wherein said inhibitor of N-glycosylation of proteins is a non-radioactive 2-deoxy-2-fluoro-d-hexose sugar.
7. The method of claim 2, wherein said inhibitor of N-glycosylation of proteins is a non-radioactive 2-deoxy-2-fluoro-d-hexose sugar.
8. The method of claim 3, wherein said inhibitor of N-glycosylation of proteins is a non-radioactive 2-deoxy-2-fluoro-d-hexose sugar.
9. The method of claim 4, wherein said inhibitor of N-glycosylation of proteins is a non-radioactive 2-deoxy-2-fluoro-d-hexose sugar.
10. The method of claim 5, wherein said inhibitor of N-glycosylation of proteins is a non-radioactive 2-deoxy-2-fluoro-d-hexose sugar.
11. The method of claim 1, wherein said effective amount of a non-radioactive-2- deoxy-2-fluoro-d-hexose compound is administered in multiple doses and is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d-mannose.
12. The method of claim 2, wherein said effective amount of a non-radioactive-2- deoxy-2-fluoro-d-hexose compound is administered in multiple doses and is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d-mannose.
13. The method of claim 3, wherein said effective amount of a non-radioactive-2- deoxy-2-fluoro-d-hexose compound is administered in multiple doses and is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d-mannose.
14. The method of claim 4, wherein said effective amount of a non-radioactive-2- deoxy-2-fluoro-d-hexose compound is administered in multiple doses and is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d-mannose.
15. The method of claim 5, wherein said effective amount of a non-radioactive-2- deoxy-2-fluoro-d-hexose compound is administered in multiple doses and is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d-mannose.
16. The method of claim 11, wherein said multiple doses are administered once a day on multiple successive days.
17. The method of claim 12, wherein said multiple doses are administered once a day on multiple successive days.
18. The method of claim 13, wherein said multiple doses are administered once a day on multiple successive days.
19. The method of claim 14, wherein said multiple doses are administered once a day on multiple successive days.
20. The method of claim 15, wherein said multiple doses are administered once a day on multiple successive days.
21. The method of claim 11, wherein said multiple doses are administered from two to four times a day on multiple successive days.
22. The method of claim 12, wherein said multiple doses are administered from two to four times a day on multiple successive days.
23. The method of claim 13, wherein said multiple doses are administered from two to four times a day on multiple successive days.
24. The method of claim 14, wherein said multiple doses are administered from two to four times a day on multiple successive days.
25. The method of claim 15, wherein said multiple doses are administered from two to four times a day on multiple successive days.
26. The method of claim 1, wherein said inhibitor of N-glycosylation of proteins is administered in combination with one or more members selected from the group consisting of steroidal antiinflammatory drugs and non-steroidal antiinflammatory drugs.
27. The method of claim 2, wherein said inhibitor of N-glycosylation of proteins is administered in combination with one or more members selected from the group consisting of steroidal antiinflammatory drugs and non-steroidal antiinflammatory drugs.
28. The method of claim 3, wherein said inhibitor of N-glycosylation of proteins is administered in combination with one or more members selected from the group consisting of steroidal antiinflammatory drugs and non-steroidal antiinflammatory drugs.
29. The method of claim 4, wherein said inhibitor of N-glycosylation of proteins is administered in combination with one or more members selected from the group consisting of steroidal antiinflammatory drugs and non-steroidal antiinflammatory drugs.
30. The method of claim 5, wherein said inhibitor of N-glycosylation of proteins is administered in combination with one or more members selected from the group consisting of steroidal antiinflammatory drugs and non-steroidal antiinflammatory drugs.
31. The method of claim 1, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered orally.
32. The method of claim 2, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered orally.
33. The method of claim 3, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered orally.
34. The method of claim 4, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered orally.
35. The method of claim 5, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered orally.
36. The method of claim 1, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered intraarticularly.
37. The method of claim 2, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered intraarticularly.
38. The method of claim 3, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered intraarticularly.
3 . The method of claim 4, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered intraarticularly.
40. The method of claim 5, wherein said inhibitor of N-glycosylation of proteins is selected from the group consisting of 2-deoxy-2-fluoro-d-glucose and 2-deoxy-2-fluoro-d- mannose, said effective amount of a non-radioactive-2-deoxy-2-fluoro-d-hexose compound is administered in multiple doses, and said effective amount is administered intraarticularly.
41. A method of identifying compounds that will inhibit the production of inflammatory mediator molecules, inflammatory cytokines, or that will inhibit MMP-13, aggrecanase or matrix metalloprotease activity in the tissues of a mammal, comprising: determining that a compound not known to be an inhibitor of an enzyme involved in the N- glycosylation of proteins is an inhibitor of at least one enzyme involved in the N- glycosylation of proteins, to yield a previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins; and testing the previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins in an assay system to determine if the previously unknown inhibitor of an enzyme involved in the N-glycosylation of proteins inhibits a member selected from the group consisting of the production by cells or tissues of a mammal of inflammatory mediator molecules the production of inflammatory cytokines, the production of glycosylated enzymes involved in inflammatory processes, or matrix metalloprotease activity.
42. The method of claim 41, wherein the enzyme involved in the N-glycosylation of proteins is a mannosyl transferase.
43. A method for treating osteoarthritis in a subject in need of such treatment, comprising administering to said subject an amount of an inhibitor of N-glycosylation of proteins, wherein the inhibitor is not glucosamine, D-mannosamine, D-galactosamine, their biocompatible acid addition salts, glucosamine-6-phosphate, N-acetyl-D-glucosamine, the sugars 2-deoxy-D-glucose, 2-deoxy-D-galactose or mannose, effective to diminish at least one symptom of osteoarthritis in said subject.
PCT/EP2003/004342 2002-04-26 2003-04-25 Method for treating inflammatory disorders WO2003090758A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2012016935A1 (en) 2010-08-02 2012-02-09 Centrum Für Angewandte Nanotechnologie (Can) Gmbh Seven carbon (c-7) sugars derivatives and their use
WO2012077828A1 (en) * 2010-12-08 2012-06-14 学校法人日本医科大学 Therapeutic agent for inflammatory diseases, which can inhibit function of inflammatory cytokine
JP6043997B2 (en) * 2010-12-08 2016-12-14 田中　信之 Inflammatory disease therapeutic agent that suppresses the function of inflammatory cytokines

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