Classifications

• • 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
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
• • 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 present invention relates to a new use of fucoidin in the treatment of arthritis, in particular rheumatoid arthritis in mammals, including humans.
• the object of the present invention is to obtain a possibility of alleviating and/or treating the symptoms of arthritis in mammals including humans.
• leucocytes The recruitment of leucocytes from the vasculature and their extravasation into tissues is critical for a successful host response to tissue injury, but excessive accumulation of leucocytes may also cause pathologic tissue destruction.
• the infiltration of leucocytes during infection involves their adherence to endothelium, followed by extravasation into the tissues. This complex process is regulated, in part, by members of the selectin family of adhesion molecules, i.e. P-selectin (CD62P), L-selectin (CD62L), and E-selectin (CD62E) [8].
• P-selectin CD62P
• L-selectin CD62L
• E-selectin CD62E
• mice lacking each of the selectin types have been described, P-selectin-deficient mice have virtually no leucocyte rolling after extravasation of mesenteric venules, but regain part of the rolling potential later on [10]. In addition, the extravasation of neutrophils is delayed in thioglycollate- induced peritonitis and in skin wounds in these mice [10,11]. Fucoidin, a sulphated fucosylated polysaccharide from seaweed, binds to and blocks the function of L- and P-selectins [12,13].
• Fucoidin has been found to inhibit some pivotal in vivo processes, including leucocyte rolling and adhesion in ischemic and reperfusion injury [14], in lung inflammation [15], and in experimental meningitis [16,17]. Furthermore, fucoidin affects neutrophil extravasation into inflamed sites during the early stages of peritonitis [18], Treatment with dally subcutaneous injections of fucoidin had an ameliorating effect on collagen-induced arthritis in rats (H. Erlandsson-Harris, in manuscript). Since massive leucocyte infiltration is a prominent feature in the arthritic joint in both collagen- induced arthritis, considered an animal model for rheumatoid arthritis, and in S.
• the fucoidin-pretreated mice displayed somewhat higher bacterial growth in the kidneys (104 ⁇ 21xl0 7 CFU) as compared to animals treated with L-selectin mAb (86 ⁇ 15xl0 7 CFU) and non-treated, S. aureus inoculated controls (82 ⁇ 20xl0 7 CFU).
• the phagocytic activity of neutrophils and monocytes in peripheral blood of mice was determined by flow cytometry, The impact of fucoidin on phagocytosis of S. aureus by granulocytes and monocytes was analysed in vitro in peripheral blood monocytes and neutrophils originating from male NMRI mice. High concentrations of fucoidin (300 ⁇ g/ml) led to significantly decreased percentage of neutrophils being capable to phagocytose S. aureus as compared to the non-fucoidin exposed control neutrophils. In contrast, the phagocytic capacity of monocytes was unaffected by fucoidin (Table II).
• mice Analysis of phagocytosis in P-selectin deficient mice displayed a clearly decreased percentage of neutrophils (p ⁇ 0,05) and monocytes (n.s.) being capable of phagocytosing S. aureus as compared to the controls, Also, the phagocytic capacity of the granulocytes and monocytes was decreased in these mice (Table III).
• the selectin family of adhesion molecules is believed to mediate the earliest phase of leucocyte recruitment, rolling along the endothelium, which serves to tether the unstimulated leucocyte to the activated endothelial surface [26, 27].
• Neutrophils and monocytes/macrophages are responsible for eliminating bacteria during early stages of infection. Neutrophils are detected early in the synovial tissue and their numbers increase during, the destructive inflammatory process [2,28]. Nonetheless, the delay in recruitment of leucocytes into the joints contributed to a less severe course of arthritis.
• T-cells may also use P-selectin to migrate into inflamed joints, For example, emigration of Th 1 lymphocytes but not Th2 cells into inflamed tissues is preferentially mediated by P-selectin [31,32].
• T cells exert proinflammatory properties in S. aureus triggered arthritis [3].
• E-selectin mAbs block cytokine-induced leucocyte rolling in P-selectin deficient mice, but have little effect in wild-type controls, suggesting that P-and E-selectin are at least partially redundant and can replace each other as mediators of neutrophil rolling at inflammatory sites in vivo [9].
• Fucoidin-mediated blockade of leucocyte adhesion is dependent upon venular wall shear rate. Fucoidin is effective in inhibiting leucocyte adhesion when shear rates are relatively high [14,41]
• mice were obtained from B&K Universal AB (Stockholm, Sweden).
• P-selectin deficient mice C57BL/6J-Selp tmlBay
• controls C57BL/6J 000664
• mice were generated as described [44]. They were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in the animal facility at the Department of Rheumatology, University of G ⁇ teborg, under standard conditions of light and temperature and fed standard laboratory chow and water ad libitum. Male and female mice, 7-12 weeks old were used.
• the S. aureus strain LS-1 was originally isolated from a swollen joint of a spontaneously arthritic NZB/W mouse [45]. Bacteria were kept frozen at -20°C in phosphate buffered saline (PBS; 0.13 M NaCl, 10 mM sodium phosphate; pH 7.4) containing 5% bovine serum albumin and 10 % dimethylsulphoxide (C 2 H 6 0S), until used. The bacterial solution was thawed and washed in PBS before use. Viable Count was used to check the number of bacteria in each bacterial solution. Two hundred micro liters of bacterial solution were injected intravenously (i.v.) into one of the tail veins. Treatment with fucoidin
• Fucoidin is derived from the brown marine algae Fucus vesiculosus [46] and is a homopolymer of L-fucose and L-fucose-4-sulphate with a molecular mass between 100 and 150 kD, The sulphate content is 2.1 nmol S0 4 / ⁇ g fucoidin. Fucoidin (F-5631, Sigma
• Hybridoma cell line MEL- 14 obtained from the American Type Culture Collection (Rockville, MID), produces a rat Iowa antibody specific for mouse L-selectin.
• the cells were grown to a maximum density, immunoglobulins precipitated with 50% of saturated ammonium sulfate, dialyzed against phosphate-buffered saline (PBS; 0.13 M NaCl, 10 mM sodium phosphate (pH 7.4) and filter sterilized.
• the concentration of immunoglobulins was determined by a single radial immunodiffusion method [47].
• NMRI and P-selectin-deficient mice were injected intraperitoneally with 200 ⁇ g/mouse of mAb specific for L-selectin or an isotype-matched antibody starting one day prior to bacterial inoculation and then every day until the end of the experiment.
• mice P-selectin-deficient and fucoidin-treated mice were sacrificed 3 and 8 days after bacterial inoculation, respectively. One front and one hind paw were removed from the diseased animals. Histopathological processing included routine fixation, decalcification, paraffin embedding, and staining with haematoxylin and eosin. The slides were coded and evaluated by a blinded observer (M.V.), Specimens were evaluated with regard to synovial hypertrophy, cartilage and subchondral bone destruction, as well as occurrence of extra-articular manifestations [2]. By adding all scores from both limbs for each animal a histopathological score index was constructed.
• NMRI and P-selectin-deficient mice treated with L-selectin specific mAb or control antibody were sacrificed three days after bacterial inoculation .
• One front and one hind paw from each animal in all four groups were removed and demineralized by a procedure detailed in an earlier report [49].
• the demineralized specimens were mounted on cryostat chucks, frozen in isopentane prechilled by liquid nitrogen, and kept at -70°C until cryosectioned. Six ⁇ m-thick sections were cut frontally to permit simultaneous inspection on of most joints within the paw.
• kidneys were aseptically removed and homogenised in 10 ml of PBS. Appropriate dilutions were made and 0.1 ml of tissue suspension was plated onto blood agar plates containing 5 % horse blood. Bacteria from the talocrural and the radiocarpal joints were collected and transferred to agar plates, containing 7.5 % sodium chloride. After incubation for 48 hr at 37°C the bacterial colonies were counted and tested for catalase and coagulase activities.
• phagocytic cells The number and activity of phagocytic cells was determined with a commercial kit (Orpega Pharma, Heidelberg, Germany). Heparinized blood from naive NMRI mice and from P- selectin-deficient mice and controls was incubated with FITC labelled S. aureus strain LS-1 for 10 minutes at 37°C. Blood from NMRI mice was incubated with 0, 3, 30, and 300 ⁇ g/ml of fucoidin for 45 min at 20°C before incubation with bacteria. After bacterial incubation the samples were immediately placed on ice to stop phagocytosis. The samples were washed, membrane-bound FITC quenched and the cell membranes subsequently permeabilized.
• mice were sensitised by epicutaneous application of 50 ⁇ l of a mixture of absolute ethanol and acetone (11) containing 3 % 4-ethoxymethylene-2-phenyloxazolone (Sigma Chemical Co, St Louis, MO,) on the shaved abdomen and thorax skin. Seven days after sensitisation all mice were challenged by application of 15 ⁇ l 1 % oxazolone dissolved in olive oil on both sides of the right ear. One group of mice received 100 ⁇ l subcutaneously, corresponding to 1 mg of fucoidin one hour before sensitisation and then every day until challenge. A second group received fucoidin one hour before challenge, and another group received PBS alone.
• a mixture of absolute ethanol and acetone (11) containing 3 % 4-ethoxymethylene-2-phenyloxazolone (Sigma Chemical Co, St Louis, MO,) on the shaved abdomen and thorax skin. Seven days after sensitisation all mice were challenged by application of 15 ⁇ l 1 % oxa
• the thickness of the ear which is a measure of antigen-specific T cell responsiveness, was analysed before and 24 hours after challenge using an Oditest spring caliper (Kr ⁇ plin, Schluchtern, Germany) as previously described [51]. All challenges and measurements were performed under light anaesthesia. The intensity of the DTH reaction was expressed as (ear thickness 24 b - ear thickness 0 h ) x 10 "3 cm.
• Olive oil induces a strong granulocyte mediated but T cell independent inflammatory response in vivo [7,52]. Inflammation was induced by injection of 30 ⁇ l olive oil (Apoteksbolaget AB, G ⁇ teborg, Sweden) intradermally (id) in the hind footpad. NMRI mice received 1 mg of fucoidin subcutaneously in one experiment and in another 1 mg of fucoidin intravenously one hour before olive oil injection. The response to olive oil inflammation was also measured in P-selectin deficient mice. Footpad swelling was assessed 24 h after injection using an Oditest spring caliper. The intensity of the olive oil induced inflammation was expressed as (footpad thickness 24h - foot-pad thickness 0h ) x 10 "3 cm.
• test data obtained shows that the present invention will have an effect in the treatment of arthritis in humans as well.
• a further feature of the invention is to obtain a combination of fucoidin and an antibiotic agent, particularly at the treatment of septic arthritis, whereby the daily dose of antibiotic agent is dependent upon the recommended dose of the particular antibiotic agent selected, normally 50 to 2000 mg per 24 hrs, or 1 to 40 mg per kg body weight and 24 hrs.
• the compounds of the present invention can be administered in the form of oral, rectal, injection, or inhalatory preparations.
• Oral compositions normally exist as tablets, granules, capsules (soft or hard), or powders, either coated or uncoated products.
• coated products they may be merely enteric coated to provide for a more readily administered preparation, or as a sustained release coated composition, where the release of active compound will take place due to the dissolution of the coating, which dissolution is dependent on where in the gastro-intestinal tract one will have a release.
• the release can be controlled as to place and time. It may also be advantageous to coat the active compound if this is subject to degradation, such as by gastric acid, in order then to have the compound to pass the stomach.
• Tablets and capsules normally contain one dose of the active compound, i.e., the dose determined to fulfill the requirements of obtaining a therapeutically active level in serum or otherwise, either this is required once, twice or more times a day (24 hrs).
• Rectal compositions are normally prepared as suppositories, where the active compound is dissolved or dispersed in a waxy compound or fat having a melting temperature in the range of the body temperature, as to release the active compound when administered rectally.
• Preparations for injection are commonly made for subcutaneous, intramuscular, intravenous, or intra peritoneal administration.
• Injection solutions are normally provided with an adjuvant to facilitate absorption of the active compound.
• Preparations for inhalation are commonly present as powders which are administered either in pressurized containers with a dosing nozzle, or in an inhaler system where the powder is dosed in the system and then the patient is inhaling air through the apparatus to such degree that the powder becomes airborne and enters the respiratory tract, including the lungs.
• Inhalation preparation are normally used for inflammatory conditions in the respiratory tract including the lungs.
• compositions contain 0.5 to 99 % by weight of active compound, and the remainder is different inert, non-therapeutically active compounds which facilitate administration, preparation such as granulation, tableting, or storage. Such inert materials may, however, have a administratively positive effect.
• the active compound of the invention, fucoidin is administered in an amount of 1 to 100 mg per kilogram body weight depending on the condition of the patient, route of administration, age and body weight of the patient, and other considerations made by the physician.
• the most important aspect hereby is the serum concentration which may be 0.1 to 100 mM of active compound, in accordance with the present findings.
• Phagocytosing granulocytes (%) 18 + 3.3 17 ⁇ 2.5 11 ⁇ 1.8 8.5 ⁇ 1.5 b Fluorescence intensity 007 ⁇ 177 947 ⁇ 127 894 ⁇ 156 805 ⁇ 97
• Phagocytosing monocytes (%) 23 + 1.7 25 ⁇ 2.9 21 ⁇ 2.0 23 ⁇ 2.7 Fluorescence intensity 498 ⁇ 46 526 ⁇ 26 452 ⁇ 36 503 + 37
• T lymphocytes Clonal expansion of T lymphocytes causes arthritis and mortality in mice infected with toxic shock syndrome toxin-I-producing staphylococci. Eur. J, Immunol 1994. 24: 1161 - 1166.
• mice with the xid B-cell defect are less susceptible to developing Staphylococcus aureus induced arthritis. J. Immunol , 1995, 155: 2067-2076.
• Fucoidin a potent inhibitor of leucocyte rolling, prevents neutrophil influx into phorbol-ester- induced inflammatory sites in rabbit lungs. Am. J. Respir. Crit. Care. Med. 1996, 153:307-
• Fucoidin a polysaccharide inhibiting leucocyte rolling, attenuates inflammatory responses in experimental pneumococcal meningitis in rats, ⁇ euroscience Letters 1995, 191 : 1-4. 18. Preobrazhenskaya, M. L, Berman, A. E, Mikhailov, N. L, Ushakova, ⁇ . A.,
• Fucoidan inhibits leucocyte recruitment in a model peritoneal inflammation in rat and blocks interaction of P-selectin with its carbohydrate ligand, Biochem. Mol. Biol. Int. 1997. 43: 443-
• Fucoidin a potent inhibitor of L-selectin function, reduces contact hypersensitivity reaction in mice. Immunology Letters 1997. 59:47-51. 21. Tedder, T. E, Steeber, D, A. and Pizeucta, P.,
• Adhesion molecules mediating neutrophil migration to arthritis in vivo and across endothelium and connective tissue barriers in vitro. Inflamm. Res, 1998. 47: 123-132.
• the ⁇ 4 -integrin supports leucocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp, Med, 1996. 183: 1995-2006.
• the ⁇ 4 -integrin an alternative pathway for neutrophil recruitment? Immunol. Today 1999. 20: 545-550.
• P-and L-selectin mediate distinct but overlapping functions in endotoxin-induced leucocyte- endothelial interactions in the rat mesenteric microcirculation. J. Immunol. 1997. 159: 1977- 1986.
• P-selectin interacts with a ⁇ 2 -integrin to enhance phagocytosis. J. Immunol. 1994. 153, 3199- 3209.
• P-selectin ICAM-1 double mutant mice acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli. J. Clin. Invest. 1995. 95:1782- 1788.
• the accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model. Infect. Immun. 1993. 61 : 3879-3885.
• Mac-I a macrophage differentiation antigen identified by monoclonal antibody, Eur. J. Immunol. 1979.9: 301-306. 51. Carlsten, H., Nilsson, L. A. and Tarkowski, A.,

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