WO2000056298A2 - Traitement de la maladie de la lyme au moyen de formulations de glycosaminoglycans polysulfates - Google Patents

Traitement de la maladie de la lyme au moyen de formulations de glycosaminoglycans polysulfates Download PDF

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WO2000056298A2
WO2000056298A2 PCT/US2000/006382 US0006382W WO0056298A2 WO 2000056298 A2 WO2000056298 A2 WO 2000056298A2 US 0006382 W US0006382 W US 0006382W WO 0056298 A2 WO0056298 A2 WO 0056298A2
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composition
psgag
gag
burgdorferi
antibiotic
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PCT/US2000/006382
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WO2000056298A3 (fr
WO2000056298A9 (fr
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Richard P. Lawrence
Ralf A. Lange
Mary Jane Helenek
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Luitpold Pharmaceuticals, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to the treatment of Lyme disease with polysulfated glycosaminoglycan compositions, particularly for the treatment of connective tissue disorders, including arthritis and inflammatory joint conditions associated with Lyme disease.
  • the present invention relates to a polysulfated glycosaminoglycan (PSGAG) composition useful for the treatment of Lyme disease associated arthritis and for prophylactic treatment against Lyme disease associated arthritis and joint disorders in patients.
  • PSGAG polysulfated glycosaminoglycan
  • Lyme disease was first recognized in the United States in 1975, after an outbreak of arthritis near Lyme, Connecticut. Since then reports of Lyme disease have increased dramatically, from 491 cases in 1982 to 12,801 in 1997, and has quickly become the most common vector-transmitted illness in the USA (National Center for Infectious Diseases, Division of Vector-Borne Infectious Diseases: Public Information Guide, ww.cdc.gov/ncidooVdiseases/lyme/lyme.htm, March 3, 1999 and Simon et al, Immunol Today (1991) 12(1):11-16). In endemic areas, its occurrence is extremely common in children (Prose et al,
  • Lyme disease is spread by the bite of ticks of the genus Ixodes that are infected with Borerrelia burgdorferi.
  • the disease is characterized as a multisystemic disorder, in which the invasive spirochete has a predilection for collagenous tissue which results in one of the major clinical manifestation of the disease; arthritis (Grab et al, FEMS Microbiol Lett (1996) 144(l):39-45).
  • Lyme disease is usually treated by antibiotics, however, symptoms may continue to recur, including in about 10% of patients who develop chronic (unremittant for 6 months or more) knee involvement (National Center for Infectious Diseases, Division of Vector-Borne Infectious Diseases: Public Information Guide, www.cdc.gov/ncidod/diseases/fyme/lyme.htm, March 3, 1999 and Merck Manual (Berkow et al, eds) 1992, 16 th edition, ⁇ 1, pages 154-157, Merck Research Laboratories, Merck & Co., Inc., Rahway, N.J.).
  • pathogenic microbes are known to cause infections in joints or bones. These forms of reactive arthritis may involve adherence to mammalian host tissues through sulfated carbohydrates, a pathway which has been suggested to be an important trait in microbial pathogenesis.
  • pathogens include mycobacteria (Menozzi et al, JExp Med (1996) 184(3):993-101)
  • Candida (Dupont et al, J Infect Dis (1985) 152(3):577-591), gonococci (Chen et al, JExp Med (1995) 182(2):511-517), staphylococci (Gallenga et al, Opthalmologica (1998) 212(3):184-187) and Haemophilus influenzae (Noel et al, Infect Immun (1994) 62(9):4028-4033).
  • DGI disseminated gonococcal infections
  • T cells and macrophages have a direct, effector role in the pathogenesis of Lyme arthritis (Du Chateau et al, Infect Immun (1996) 64(7):2540-2547 and Sigal LH., Ann Rev Immunol (1997) 15:63-92).
  • severe destructive arthritis was induced in recipients of macrophages from R. burgdorferi vaccinated animals (Du Chateau et al, Infect Immun (1996) 64(7):2540-2547).
  • cytokines and lymphokines e.g., IL-12 inhibitors, CD40 ligand inhibitors, IL-1 inhibitors, Nf-kappa B inhibitors
  • IL-12 inhibitors e.g., CD40 ligand inhibitors, IL-1 inhibitors, Nf-kappa B inhibitors
  • IL-12 inhibitors e.g., CD40 ligand inhibitors, IL-1 inhibitors, Nf-kappa B inhibitors
  • antibiotics such as oral doxycyclin, oral amoxicillin and probenecid.
  • aspirin or other non-steroidal anti-inflammatory drugs e.g., idomethacin
  • NSATD nonsteroidal anti-inflammatory drugs
  • salicylates e.g., aspirin
  • gold compounds in addition to NSAIDs
  • Penicillimine also can also be given at this stage if gold treatments are unsatisfactory.
  • Moderate to severe arthritis requires, as a first treatment, the use of compounds such as hydroxychloroquine and sulfasalazine.
  • Polysulfated glycosaminoglycan is a semi-synthetic glycosaminoglycan (GAG) prepared by extracting GAGs from cartilage, for example, bovine tracheal cartilage, and then polysulfating the GAGs.
  • GAGs are polysaccharides composed of repeating disaccharide units.
  • the GAG present in PSGAG is principally composed of chondroitin sulfate, containing about 3 to 4 sulfate esters per disaccharide unit.
  • PSGAG has several biochemical effects on injured or degenerative joints and has been classified as a chondroprotective compound, because PSGAG has been found to be incorporated in the cartilage matrix upon inj ection.
  • PSGAG has also been shown to inhibit various catabolic enzymes which degrade glycosaminoglycans competitively, including elastase, stromelysin, metalloproteases, cathepsin Bi, hyaluronidases and collagenases.
  • PSGAG has also been demonstrated to indue synthesis of collagen, proteoglycans and hyaluronic acid by chondrocytes and synoviocytes. Further, anti-inflammatory activity has been demonstrated for these molecules.
  • GAGs in the treatment of non-Lyme disease related connective tissue disorders also has been disclosed in the medical and patent literature, including methods of treating damage corneal, uterine or cartilage tissues and/or treating rheumatism (see Klundas, U.S. Patent No. 5,036,056 and Akoi et al, U.S. Patent No. 5,470,578).
  • these disclosure are limited to repair and/or replacement of GAG loss in the extracellular matrix, and the administration of GAGs would not have been expected to inhibit, for example, the contributory effects of elastases and chondroitinases on connective tissue degeneration as these GAGs themselves would be substrate for such enzymes.
  • the literature also describes both the preparation and use of sulfated chondroitin sulfate, including the use of such polysulfated chondroitin for therapy in humans and animals as an antiarthritic treatment (see Wolf U.S. Patent No. 4,534,066).
  • PSGAG sulfated chondroitin sulfate
  • PSGAG previously was marketed in Europe for the treatment of degenerative joint disorders, and reportedly was most effective as long as there is no extensive full thickness loss of articular cartilage.
  • the intramuscular treatment regimen in humans was 50 mg twice weekly for up to 15 injections. That European formulation of PSGAG was taken off the market in 1994 because two deaths occurred in the treated patient group.
  • PSGAGs may be safely administered to humans with appropriate care. For example, it is now known that PSGAG should not be administered intra-articularly in the presence of active joint inflammation.
  • the ADEQUAN® Canine formulation is recommended for intramuscular injection for the control of signs associated with non-infectious degenerative and/or traumatic arthritis of canine synovial joints.
  • the ADEQUAN® i.m. formulation is recommended for the intramuscular treatment of non-infectious degenerative and/or traumatic joint dysfunction and associated lameness of the carpal and hock joints in horses.
  • the ADEQUAN® CANINE formulation is a formulation of PSGAG for intramuscular administration to dogs. It is sold in a solution of 100 mg/mL in a 5 mL preserved solution.
  • ADEQUAN® Canine contains 100 mg of PSGAG, 0.9% v/v benzyl alcohol as a preservative, and water for injection q.s. to 1 mL.
  • Sodium hydroxide and/or hydrochloric acid are added when necessary to adjust pH.
  • a second product, ADEQUAN® i.m. formulation is a formulation of PSGAG for intramuscular use in horses, which is sold in a solution of 500 mg/5 mL.
  • Each 5 milliliters of the ADEQUAN® i.m. formulation contains 500 mg of PSGAG and water for injection q.s.
  • Sodium hydroxide and/or hydrochloric acid may be added when necessary to adjust the product's pH.
  • PSGAG is chemically similar to the glycosaminoglycans in articular cartilage matrix.
  • PSGAG is a potent proteolytic enzyme inhibitor and diminishes or reverses the pathologic processes of traumatic or degenerative joint disease which result in a net loss of cartilage matrix components.
  • PSGAG improves joint function by reducing synovial fluid protein levels and increasing synovial fluid hyaluronic acid concentration, for example, in traumatized equine carpal and hock joints.
  • PSGAG is characterized as a
  • PSGAG disease modifying osteoarthritis drug.
  • PSGAG inhibits certain catabolic enzymes that have an increased activity in inflamed joints, and enhances the activity of some anabolic enzymes.
  • PSGAG has been shown to significantly inhibit serine proteinases, which have been demonstrated to play a role in the Interleukin-1 mediated degradation of cartilage proteoglycans and collagen.
  • PSGAG is reported to be an inhibitor of Prostaglandin E2 (PGE2) synthesis.
  • PGE2 has been shown to increase the loss of proteoglycan from cartilage.
  • PSGAG also has been reported to inhibit some catabolic enzymes such as elastase, stromelysin, metalloproteases, cathepsin Bl, and hyaluronidases, which degrade collagen, proteoglycans, and hyaluronic acid in degenerative joint disease.
  • catabolic enzymes such as elastase, stromelysin, metalloproteases, cathepsin Bl, and hyaluronidases, which degrade collagen, proteoglycans, and hyaluronic acid in degenerative joint disease.
  • the anabolic effects of PSGAG include the compound's ability to stimulate the synthesis of protein, collagen, proteoglycans, and hyaluronic acid in various cells and tissues in vitro.
  • Cultured human and rabbit chondrocytes have shown increased synthesis of proteoglycan and hyaluronic acid in the presence of PSGAG.
  • PSGAGs have shown a specific potentiating effect on hyaluronic acid synthesis by synovial membrane cells in vitro.
  • Lyme disease progresses, successful treatment with antibiotics becomes less reliable because of the persistent presence of the spirochete, coupled with an activated immune system. Therefore, what is needed is a more optimal therapy for the various stages of Lyme disease which can: 1) exploit the mechanism of pathogen binding to the infected host's tissue components; 2) repair and replace host ground substance; or 3) modulate immune activation and/or inflammatory responses.
  • the present invention relates to a prophylactic or therapeutic treatment for Lyme disease-associated arthritis (LDAA) in a human or mammalian subject, and includes the step of administering an effective amount of a glycosaminoglycan (GAG) composition to a subject in need thereof, particularly a polysulfated GAG (PSGAG).
  • GAG glycosaminoglycan
  • PSGAG polysulfated GAG
  • the composition is administered subsequent to the onset of erythema chronicum migrans (ECM).
  • compositions of the present invention may be administered systemically or at the site of swelling, such as by injection at such a site.
  • treatment of LDAA involves coadministering an effective amount of a composition comprising a PSGAG and therapeutic amounts of one or more other compounds that also are effective against R. burgdorferi.
  • Particularly contemplated other compounds include antibiotic such as tetracycline, doxycycline, penicillin, amoxicillin, probenecid and ceftriaxone or a combination of antibiotics such as amoxicillin and probenecid.
  • Other contemplated compounds include antibodies, antibody conjugates, chemokine-inhibitors and cytokine inhibitors.
  • Contemplated PSGAG compounds have a molecular weight range between about 3,000 to 15,000 daltons. A preferred range is about 4,000 to 10,000 daltons and a more preferred range is about 5,000 to 8,000 daltons. Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon considering the following detailed description of the invention which describes presently preferred embodiments thereof.
  • Fig. 1. presents a HP-GPC chromatogram for PSGAG.
  • the present invention provides significant improvements in the therapeutic treatment of Lyme disease associated arthritis and associated inflammatory symptoms, particularly involving connective tissue and joint disorders through the use of product formulations which include semisynthetic polysulfated glycosaminoglycans (PSGAGs).
  • PSGAGs semisynthetic polysulfated glycosaminoglycans
  • Such compositions have been discovered to possess enhanced biochemical activities for such purposes in comparison to naturally occurring glycosaminoglycans (GAGs).
  • the present invention provides for a prophylactic treatment for Lyme disease associated arthritis using an injectable form of a PSGAG.
  • PSGAG is a biological polymer with a molecular weight range of about 3,000 to 16,000 Daltons.
  • the invention relates to the use of this select fraction of PSGAG with a distinct molecular weight distribution and poly-ionic properties.
  • Polysulfated glycosaminoglycan (PSGAG) is a poly-anionic mucopolysaccharide that enhances the inhibition of catabolic enzymes while stimulating anabolic pathways for regeneration of damaged cartilage.
  • Compositions of PSGAG are useful in the prevention and treatment of inflammatory symptoms associated with degenerative joint diseases. Humans and animals infected with Lyme disease caused by the spirochete, R.
  • PSGAG inhibits the attachment of spirochetes, such as R. burgdorferi, to joint (connective) tissues. Since PSGAG enhances the inhibition of neutral protease and inhibits the attachment of R. burgdorferi, to joint (connective) tissues, PSGAG alone or in combination with a suitable antibiotic may be used to treat Lyme disease associated arthritis.
  • Treatment comprises systemic administration or direct treatment of specific sites with an effective amount of PSGAG.
  • treatment comprises co-administration of PSGAG and other compounds that are therapeutically effective against R. Burgdorferi.
  • Preferred compounds that are -Ineffective against R. Burgdorferi include antibiotics, antibodies and chemokine and cytokine inhibitors.
  • ECM is a red circular patch that appears usually three days to one month after the bite of an infected tick at the site of the bite. The patch then expands, and sometimes many patches appear that vary in shape. Common sites for such patches are the thigh, groin, trunk and the armpits.
  • Co-administered means that two or more different compositions may be administered to a patient simultaneously or sequentially as long as the compositions are administered in a time frame in which the patient derives therapeutic benefit from such two or more compositions.
  • Connective tissue means any area of the musculoskeletal system where there is a relatively large portion of extracellular matrix in relation to cells. For example, joints are considered connective tissues.
  • LDAA Low Density Associated Arthritis
  • LDAA Low Density Associated Arthritis
  • these symptoms occur within weeks to months of onset of erythema chronicum mi grans (ECM).
  • protection or “therapeutically effective” generally mean that the PSGAG administered is effective to inhibit significantly the attachment of infective microorganisms to their target tissue or cells in an infected host; or that the administered PSGAG is effective to significantly repair and replace host ground substance in the presence of Lyme disease infection; or to significantly reduce the infected host's immunopathological response to the infection.
  • PSGAG means the semi-synthetic biological polymer composed of repeating disaccharides units of D-glucuronic acid sulfate and N-acetyl- galactosamine 4, 6 polysulfate. 2. Pharmacology of PSGAG:
  • -PSGAG was distributed to all tissues and body fluids investigated -after intra-articular injection, maximum drug concentrations were found a) after 3 hours in the adrenals, thyroids, peritoneal fluid, lungs, eyes, aorta, articular cartilage, synovial fluid and spinal cord b) after 6 hours in kidneys, stomach, testes, adipose tissue, skeletal muscle and brain c) after 24 hours in liver, spleen, bone marrow, salivary glands, thymus, pancreas, skin and heart, -the maximum concentration of drug after intra-articular injection of 1 mg/kg in rabbits (7) in the various organs was given at (all values in dpm/mg or mL): a) kidneys 4782 b) adrenals 439, liver 416, spleen 380, bone marrow 250, thyroids 108, and salivary glands 100 c) thymus 64, pancreas 57, peritoneal fluid 54,
  • PSGAG is bound to serum proteins in human blood (6,8,9).
  • the drug binds to both albumin and chi- and beta-globulins and the extent of the binding is stated to be 30-40% (8).
  • the drug exists in both the bound and free form in the blood stream (8).
  • the synovial membrane is not a significant barrier to distribution of PSGAG from the blood stream to the synovial fluid (4,6,8,9,10). Distribution from the synovial fluid to the cartilage takes place by diffusion (11). In the articular cartilage, the drug is deposited into the cartilage matrix (11) and appears to be bound to macromolecules; perhaps to proteoglycans (11) or other non-collagenous proteins (1).
  • Metabolism of PSGAG in humans and rabbits includes desulfation and depolymerization (4, 6, 7, 9); metabolites include sulfate, oligosaccharide and monosaccharides. In rabbits, metabolism is reported to take place in the liver, spleen, and bone marrow (7). Metabolism may also occur in the kidneys (13). No metabolism of the drug has been observed in the knee joint of rats (16). It is, however, known that glycosaminoglycans are metabolized by chondrocytes. It has been postulated that PSGAG can be degraded in granulocytes (13) and that extracellular degradation may take place (14).
  • PSGAG administered intramuscularly and not protein bound or bound to other tissues is excreted (7). This excretion is primarily via the kidneys, with a very small proportion in the feces. In rabbits, 55.3% of intramuscularly administered PSGAG was excreted in the urine and 1.7 % in the feces within 2 days (4). In humans, 35-40% of a 50 mg intramuscular dose of PSGAG was excreted via the kidneys within 12 hours of administration (6).
  • PSGAG has several biochemical effects on injured or degenerative joints. The sum of these effects is called chondroprotection: the inhibition of degradation and effects favorable to repair of articular cartilage (1). Drugs having these effects are classified as chondroprotective.
  • PSGAG binds to macromolecules in the extracellular matrix of cartilage; the identity of the binding site is thought to be either a proteoglycan (16) or other noncollagenous protein (1).
  • This activity in damaged cartilage with a depleted proteoglycan content has been described as a repair process (17,18,19). It is possible that this binding effect may improve the biochemical properties of damaged cartilage matrix, such as water binding capacity, or may protect damaged matrix components from further enzymatic degradation.
  • PSGAG ability of PSGAG to inhibit a broad range of catabolic enzymes has been reported in both in vitro and in vivo test systems. This is significant because enzymatic degradation of collagen, proteoglycans, and hyaluronic acid is a critical element of the pathogenesis of degenerative joint disease (1). Many of the most important of these catabolic enzymes are inhibited by fluid or tissue concentrations of PSGAG as low as about 0.1 to 2 ug/mL in some cases (1,4). The mechanism of enzyme inhibiting varies with the type of enzyme. For many of the glycosidases, which degrade glycosaminoglycans and hyaluronic acid, the inhibition is competitive; the drug acts as a competitive substrate for the enzyme (13).
  • Stromelysin is a neutral protease which degrades proteoglycans at low concentrations.
  • PSGAG was the only drug tested which significantly inhibited stromelysin at concentrations readily achievable in equine joint tissues (24).
  • PSGAG at concentrations of 0.05-0.5 ug/mL inhibited the activity of the enzyme (41).
  • PSGAG collagen degrading enzymes
  • These enzymes include cathepsin Bi (14,25,26) and elastase (see above).
  • PSGAG also inhibits enzymes which degrade hyaluronic acid; the glycoanohydrolases or "hyaluronidases” (13,27,28,29), betaglucuronidase (13,27,28,29,30), and beta-N-acetylglucosaminodases (13,18,28,30).
  • hyaluronidases 13,27,28,29
  • betaglucuronidase 13,27,28,29,30
  • beta-N-acetylglucosaminodases 13,18,28,30.
  • PSGAG inhibits enzymes which destroy hyaluronic acid, proteoglycans and collagen.
  • Degenerative joint disease is characterized by a net loss of these critical components (1).
  • Anabolic Effects in Damaged or Diseased Joint Tissue PSGAG has been reported to stimulate the endogenous synthesis of collagen, proteoglycans and hyaluronic acid by chondrocytes or synoviocytes. The stimulation of proteoglycan and hyaluronate in increased amounts and with higher molecular weight by PSGAG in cultured human chondrocytes has been reported (32). These studies were confirmed in lapine chondrocytes by another investigator (1).
  • PSGAG has also been shown to be anti-inflammatory in at least two important pathways.
  • PSGAG was shown to inhibit the biosynthesis of the eicosanoid prostaglandin E2 (39).
  • This potent inflammatory mediator plays an important role in pain and inflammation in joint disease (1).
  • a dose dependent decrease in the release of toxic oxygen radicals from human monrophils by PSGAG has been reported (40).
  • Toxic oxygen radicals are implicated in the early breakdown of hyaluronic acid in the synovial fluid of inflamed joints (1).
  • Lyme disease may be treated by various therapeutic methods that include the administration of PSGAG. It is contemplated that PSGAG may be coadministered or combined in dosage forms with other anti-Lyme agents including antibiotics, anti-mflammatories, antibodies against R. Burgdorferi epitopes and antibodies against cytokines and chemokines. In a preferred embodiment, PSGAG may be co-administered with the following antibiotics: tetracycline, deoxycycline, penicillin, amoxicillin, ceftriaxone and probenecid. For example, antibodies against R. burgdorferi may be useful in the treatment of this disease, particularly in combination with the administration of PSGAG. Useful antibodies may be generated by standard methods.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies, as well as chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library.
  • Various procedures known in the art may be used for the production of such antibodies and fragments.
  • antibodies may be generated which comprise antibody conjugates (U.S. Patent No. 5,612,016).
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al, 1985, in Monoclonal Antibodies and Cancer Therapy. Alan R. Liss, Inc., pp. 77-96). All of these techniques are incorporated herein by reference. Techniques described in U.S. Patent No. 4,946,778, incorporated herein by reference, for the production of single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
  • antibodies to cytokines or cytokines can be co-administered with PSGAG.
  • Cytokines or chemokines can be obtained from any source, most preferably from humans.
  • the following chemokines are envisaged: RANTES (U.S. Patent No. 5,451, 660), IP-10 and mig (U.S. Patent No. 5,871, 723), MCP-1 (U.S. Patent No. 5,707,815), gro-alpha and IL-8 (U.S. Patent No. 5,831,032).
  • the cytokine IL-12 is preferred.
  • antibodies to IL-12 in soluble form, would typically be administered in a single dosage of between 10 mg and 20 mg/kg of body weight.
  • 500 mg to 1000 mg can be given P.O.
  • 10 mg to 20 mg/kg of body weight can be administered as a single or as a weekly intravenous injection.
  • the age, weight and condition of the individual must be considered in determining a final dose.
  • 500 mg to 1000 mg can be microencapsulated as described for slow release over a four to eight week period.
  • dosages are best optimized by the practicing physician and methods for determining dosages are described for example, in Remington's Pharmaceutical Sciences (24).
  • Suitable carriers for oral administration of antibodies to cytokines or lymphokines include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants.
  • Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans.
  • Suitable liquid carriers may be water, pharmaceutically accepted oils, or a mixture of both. The liquid can also contain other suitable pharmaceutical additions such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents. Examples of suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a pH-regulated gel.
  • the antibodies may be contained in enteric coated capsules that release antibodies into the intestine to avoid gastric breakdown. In a preferred embodiment antibodies are directed to OspA, OspB (U.S.
  • Anti-OspA and anti-OspB polypeptide antibodies of this invention may be generated by infection of a mammalian host with B. burgdorferi, or by immunization of a mammalian host with an OspA or OspB polypeptide.
  • Such antibodies may be polyclonal or monoclonal, it is preferred that they are monoclonal.
  • Methods to produce polyclonal and monoclonal antibodies are well known to those of skill in the art. For a review of such methods, see Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988), and D. E. Yelton, et al, Ann. Rev. of Biochem., 50, pp. 657-80 (1981).
  • OspA and OspB antibodies are combined as pharmaceutical compositions with carriers and adjuvants in their dosage forms.
  • the pharmaceutical compositions of OspA and OspB antibodies may be in a variety of conventional depot forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions. The preferred form depends upon the intended mode of administration and prophylactic application.
  • Such dosage forms may include pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • carriers and adjuvants include, for example, RIBI, ISCOM, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
  • Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
  • Mincing Frozen cartilage is minced. The source of the cartilage is bovine trachea.
  • Degreasing The minced cartilage is heated in hot water (80 °C) for twenty minutes. The cartilage's fat is separated from the hot tissue via centrifugation. 2400 kg of frozen cartilage is reduced to 1800 kg after this process. It has lost 25%) of its weight.
  • Drying The moist granules of degreased cartilage are dehydrated using hot air (80°C) for 22 hours. The 1700 kg is further reduced to 500 kg. This represents a 60% loss in weight.
  • Formaldehyde Treatment 500 kg of intermediate 1 A - 54.65 kg (50.6 liters) is weighted out, added and the mixture then agitated for twenty minutes. The treated cartilage is transferred to PVC drums and stored for 4-9 weeks at room temperature.
  • In-Process Specifications The only in-process test is for microbial limits. The limit should be less than 10 micro-organisms per gram with the absence of any harmful species.
  • NEUTRALIZATION The extract is transferred to another vessel and the pH is adjusted to 7.2 with hydrochloric acid. The extract is then clarified by filtration.
  • a volume of about 5600 liters is evaporated under a vacuum to a volume of 400-480 liters.
  • SOLVENT PRECIPITATION Sodium chloride is added to the reduced extract. The pH is adjusted to 7.5 with hydrochloric acid. Four volumes of methanol is added to one volume of extract. The product is allowed to precipitate and settle overnight. The next day the supernate liquid is drawn from the top of the vessel and the precipitated product is suspended in the remaining liquid.
  • CENTRIFUGATION The suspension is centrifuged at 400 rpm. The precipitate is collected on polypropylene filter cloth.
  • DRYING The precipitate is dried for three hours at 80 °C at atmospheric pressure, then under vacuum for 64 hours.
  • GRINDING 25 kg portions are ground in ball mills for 38 hours. The balls are removed and the powder transferred to drums lined with double polyethylene bags.
  • ACID SOLVENT PRECIPITATION The hot reaction mixture is added to 260 liters of methanol and mixed for 5 minutes. A precipitate settles and the supernatant is withdrawn. Sodium chloride is added and the precipitate is dissolved in hot water to a volume of 160 liters. 320 liters of methanol is added and the PSGAG precipitates again. The supernatant is again withdrawn.
  • Glycosaminoglycan precipitated via the addition of methanol. After settling overnight the supernatant is withdrawn and the precipitate is suspended in the remaining solution. The suspension is centrifuged under a continuous stream of nitrogen. The precipitate is removed and the process repeated three times.
  • FILTRATION The dried product is dissolved in a sufficient amount of water. The pH is adjusted to 7.0 and after standing overnight the solution is filtered.
  • PRECIPITATION FROM NEUTRAL SOLUTION Four volumes of methanol is added to one volume of filtrate. The pH is maintained at 7. The precipitate is allowed to settle for at least 3 hours. The supernatant is removed and the suspension centrifuged under a continuous stream of nitrogen. The filter cake is rinsed with methanol.
  • the filter is dried in the fluid bed drier using hot air.
  • the dried precipitate is collected in PVC drums lined with double polyethylene bags.
  • Depolymerization Sufficient amounts of Intermediate 3 and sodium chloride are dissolved in water. EDTA and sodium hydroxide are added. The pH is adjusted to 6.05 and the solution is heated to 90 °C. To the hot solution, a hydrogen peroxide solution is stirred in and the temperature maintained for 115 minutes. The solution is then cooled to 25 °C.
  • Precipitation/Drying The solution is transferred to another vessel. Sodium chloride is added and the Polysulfated Glycosaminoglycan is again precipitated using methanol. The methanol is recovered and the precipitate collected via filtration. The precipitate is dried as described (IV) 4 above.
  • Ion exchange treatment The solution is filtered through a glass column containing a cation exchange resin. The void volume is discarded and a total of 440 liters of filtrate collected.
  • Acid precipitation The solution is split and transferred to two separate vessels. Sodium chloride is added to each vessel and the Polysulfated Glycosaminoglycan is again precipitated using methanol. The methanol is recovered and the precipitate collected via filtration. The steps “Filtration”, “Ion Exchange Treatment” and “Acid Precipitation” are repeated two more times.
  • Neutral precipitation Sodium chloride is added and the pH is adjusted to 7.5 with sodium hydroxide. A sufficient amount of methanol is added to precipitate the Polysulfated Glycosaminoglycan. The precipitate is recovered via filtration.
  • Second filtration The filter cake is dissolved in situ within the closed filter with distilled water. The resulting solution is filtered through a 0.45 micron filter which is integrity tested both before and after use via the bubble point method.
  • First membrane filtration Sodium chloride and EDT A are added to the solution. The pH is adjusted to 7.5 with hydrochloric acid. The solution is filtered through an in-line combination of a plate filter and a 0.2 micron cartridge membrane filter. The membrane filter should be integrity tested prior to and after use.
  • Polysulfated Glycosaminoglycan is recovered via filtration and washed with methanol. The filtrate is then redissolved in distilled water and more sodium chloride added.
  • Second membrane filtration The solution is filtered with a steam- sterilized cartridge membrane filter equipment into a sterile buffer vessel, then combined with 50 liter distilled water washings of the filter housing. The pH is adjusted to 7.5.
  • Drying The solution is fed to a spray drier, with an air entrance temperature at 140 and an exit temperature of 85 °C.
  • the dried product is collected in a drum lined with double polyethylene bags, then transferred to the quarantine area for sampling and examination by Q.C.
  • the yield of the final product is about 64 to 72 kg of white powder.
  • Example 2 Characterization of the PSGAG Compounds
  • the unique poly-anionic properties of PSGAG is determined by electrophoresis.
  • a 70 mm cellulose acetate (CA) membrane, saturated with 0.2 N hydrochloric acid is suspended with contact between the cathode and anode of an electrophoresis chamber.
  • a test solution of PSGAG containing about 1 microgram of PSGAG is applied to the cathode end of the strip.
  • a current of about 65 mA is applied for 65 minutes.
  • the CA membrane is removed from the tank, then stained with a 0.1% toluidine blue solution for 5 minutes.
  • the CA membrane is rinsed with a sufficient quantity of water to remove the excess toluidine blue.
  • PSGAG appears as only one violet-blue spot at a distance of 3 cm from its starting point.
  • the cathode is the negative terminal of the electrophoresis chamber.
  • Anionic compounds such as PSGAG, under an applied current will migrate towards the chamber's anode (i.e., its positive terminal).
  • the poly-anionic property of PSGAG was compared to hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, heparin sulfate and keratan sulfate using electrophoresis. The results are as follows:
  • the molecular weight distribution of PSGAG is determined by High Pressure Gel Permeation Chromatography (HP-GPC).
  • HP-GPC High Pressure Gel Permeation Chromatography
  • the HP-GPC system uses a high efficiency gel filtration column packed with packed with a bonded diol-coated silica gel with a particle size of 7 microns. The column's molecular weight range is between 100 to 50,000 Daltons.
  • a mobile phase consisting of a dilute solution of sodium sulfate (0.05 M) is pumped at a constant flow rate, pressure and temperature.
  • Samples of the PSGAG are diluted to 5 mg per mL with the mobile phase and a portion of the resulting solution (10 ⁇ L) is injected into the HP-GPC system.
  • a refractive index detector measures the substance's peak response which is proportional to the substance's concentration.
  • a computerized GPC software program interprets the data and calculates the relative: weight average molecular weight (M w ), number average molecular weight (M n ) and polydispersity index (M w /M n ) of the sample. These values are based on commercially available molecular weight standards ranging from 1,000 to 47,300 Daltons which are used to calibrate the system immediately prior to sample analysis.
  • PSGAG is a semi-synthetic biological polymer composed of repeating disaccharide units of D-glucuronic acid sulfate and N-acetyl-D-galactosamine 4,6 polysulfate. It exhibits a uniform molecular weight distribution curve characterized as follows:
  • the preferred dosage form for human use is as a sterile, isotonic solution prepared by dissolving a suitable quantity of PSGAG in water for injection, where the final concentration of PSGAG is between about 50 mg/mL and about 125 mg/mL of PSGAG, preferably between about 75 mg/mL and about 100 mg/mL.
  • a sufficient amount of sodium chloride is added to adjust the solution's osmolality to an isotonic range of about 270 to about 330 mOmol/kg.
  • suitable antimicrobial preservatives such as benzyl alcohol, methyl paraben and/or propyl paraben may be added at effective concentrations during formulation.
  • the bulk solution is sterilized via filtration though a 0.2 micron membrane filter, then aseptically filled into suitable sterile, pyrogen free ampules or vials.
  • PSGAG The content of PSGAG in the injection is determined by its hexuronic acid content.
  • PSGAG is composed of repeating disaccharide units of D- glucuronic acid sulfate and N-acetyl-D-galactosamine 4,6 polysulfate. Upon acid digestion, the D-glucuronic acid sulfate contained in PSGAG's repeating disaccharide units is hydrolyzed to hexuronic acid. Based on its hexuronic acid content, the injection contains about 90.0 to about 110.0% of its labeled amount of PSGAG.
  • the protein content and endotoxin content in PSGAG has been reduced from 0.1 % w/w and 2.5 EU /mg respectively to 0.05% and 0.5 EU/mg.
  • Example 4 Treatment of Human Lyme Disease Patients
  • the preferred method of treating inflammatory symptoms associated with the chronic, destructive arthritis caused by the spirochete, R. burgdorferi is a suitable injectable formulation of PSGAG administered intra-articularly, intramuscularly, subcutaneously, or by local infiltration at a dose of about 0.5 mg to about 5 mg , preferably about 1 mg to about 4 mg, or more preferably about 2 mg to about 3 mg PSGAG per kilogram body weight twice a week over a seven to eight week period.
  • the PSGAG dosage in mg per kilogram body weight administered is sufficient to provide a minimum therapeutic level of about 0.1 ⁇ g/mL of PSGAG throughout its seven to eight week treatment regime.
  • PSGAG Since upon administration, PSGAG is distributed to all tissues and body fluids, this minimum therapeutic level of about 0.1 ⁇ g/mL throughout its seven to eight week treatment regime will aid in the inhibition of the attachment ofR. burgdorferi, to joint and other connective tissues. Therefore, PSGAG alone or in combination with a suitable antibiotic may be used to inhibit the attachment of R. burgdorferi, to joint and other connective tissues.
  • Greiling H Biochemical investigations of the mode of action of Arteparon. IX Europ Cong Rheumatol Eular Basil 1980 pp 11-18 14. Kruze D, Fehr K, Boni A: Effects of antirheumatic drugs on cathepsin Bl from bovine spleen. Z Rheumatol 35:95-102 1976 15. Enislidis AC: Tissue distribution of tritium labeled glycosaminoglycan polysulfate following intraarticular injection. Med Welt 23: 733-735 1972 16.
  • Puhl W The effect of intraarticular injections of Arteparon in osteoarthritis. IX Europ Cong Rheumatol Eular Basil 1980 pp 91-97
  • Trnavsky K Effects of Arteparon on the collagenolytic activity of cathepsin Bl. IX Europ Cong Rheumatol Eular Basil 1980 pp 27-29
  • Verbruggen G, Veys E M The effect of sulfated glycosaminoglycan on the proteoglycan metabolism of synovial lining cells. Acta Rheumatol Belgica 1 :75-92 1977
  • Verbruggen G Veys E M: Proteoglycan metabolism of connective tissue cells. An in vitro technique and its relevance to in vivo conditions. In Degenerative Joints; Test Tubes, Tissues, Models, and Man Amsterdam Excerpta Medica 1982 pp 113-126 33.
  • Adam M Krabcova M, Musilova J, Pescova V, Brettschneider I,
  • Glade M Polysulfated glycosaminoglycan accelerates net synthesis of collagen and proteoglycans by arthritic equine cartilage tissues and chondrocytes. Am J Vet Res 51:5779-785 1990 36. Nishikawa H, Mon I, Umemoto J,: Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovia. Arch Biochem Biophys 240:146-148 1985

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Abstract

L'invention porte sur des compositions et sur des procédés de traitement de la maladie de la Lyme associée à des symptômes articulaires et arthritiques, ces procédés de traitement étant effectués au moyen de glycosaminoglycans polysulfatés.
PCT/US2000/006382 1999-03-19 2000-03-13 Traitement de la maladie de la lyme au moyen de formulations de glycosaminoglycans polysulfates WO2000056298A2 (fr)

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Cited By (1)

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EP0418827A1 (fr) * 1989-09-19 1991-03-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Vaccin contre la maladie de Lyme
US5036056A (en) * 1987-07-08 1991-07-30 Martin Kludas Methods for treating damaged corneal, uterine, or cartilage tissue
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US5036056A (en) * 1987-07-08 1991-07-30 Martin Kludas Methods for treating damaged corneal, uterine, or cartilage tissue
EP0418827A1 (fr) * 1989-09-19 1991-03-27 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Vaccin contre la maladie de Lyme
EP0441003A2 (fr) * 1989-12-20 1991-08-14 Lawrence Goldman Medicaments ophtalmiques
WO1998024477A1 (fr) * 1996-12-06 1998-06-11 Amgen Inc. Therapie combinee utilisant un inhibiteur de l'il-1 pour traiter les maladies induites par l'il-1
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002032407A2 (fr) * 2000-10-19 2002-04-25 Hans-Knöll-Institut für Naturstoff-Forschung e.V. Utilisation de derives d'acide hyaluronique pour inhiber les arthrites inflammatoires
WO2002032407A3 (fr) * 2000-10-19 2003-11-20 Knoell Hans Forschung Ev Utilisation de derives d'acide hyaluronique pour inhiber les arthrites inflammatoires

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