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WO2006054319A2 - Utilisation de formulations a base de chito-oligomeres pour modifier l'activite anormale des proteines de type chitinase - Google Patents

Utilisation de formulations a base de chito-oligomeres pour modifier l'activite anormale des proteines de type chitinase Download PDF

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WO2006054319A2
WO2006054319A2 PCT/IS2005/000024 IS2005000024W WO2006054319A2 WO 2006054319 A2 WO2006054319 A2 WO 2006054319A2 IS 2005000024 W IS2005000024 W IS 2005000024W WO 2006054319 A2 WO2006054319 A2 WO 2006054319A2
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ykl
clp
disease
mammal
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WO2006054319A3 (fr
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Olafur B. Einarsson
Jon M. Einarsson
Johannes Gislason
Finnbogi R. Thormodsson
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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
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • Glucosamine is a modified glucose with NH 2 replacing the OH group on the carbon two in the sugar molecule.
  • glucosamine is only found in two forms; as glucosamine-6-phosphate (GN-6-P) and N-acetyl glucosamine (NAG or GIcNAc).
  • the amino sugar GN-6-P is synthesized from glutamine and fructose-6-phosph ' ate (F-6-P). This reaction is catalyzed by glucosamine synthase as the rate limiting step in amino sugar biosynthesis.
  • GN- 6-P is the precursor to all hexosamines and hexosamine derivatives.
  • GN-6-P is acetylated by acetyl coenzyme A to N-acetyl glucosamine (NAG or GIcNAc).
  • NAG can subsequently be converted into N-acetyl galactosamine or N- acetyl mannosamine.
  • GAG glycosaminoglycans
  • hyaluronan hyaluronan
  • proteoglycans Hence, in eucariotic biochemistry, these compounds constitute a dynamic metabolic pool of aminosugars playing vital roles in a variety of crucial metabolic pathways.
  • Hyaluronic acid is a polymer composed of repeating dimeric units of glucuronic acid and N acetyl glucosamine.
  • the polymer is mostly of extremely high molecular weight (up to several million daltons) and forms the core of complex proteoglycan aggregates found in extracellular matrix and together with lupricin, it comprices the major constituent of synovial fluid. Thus, it helps lubricating and preserving the joint by providing a viscous consistency to the synovial fluid.
  • HA contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions, especially those including its primary receptor in vivo, CD44. Upregulation of CD44 itself is widely accepted as a marker of lymphocyte cell activation.
  • HA is synthesized by a class of integral membrane proteins called hyaluronan synthases (HAS), but in vertebrates, three types HAS have been identified; HASl, HAS2, and HAS3. These enzymes lengthen hyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide. The synthesis occurs at the inner face of the plasma membrane by membrane bound HA synthases (HAS), and is subsequently extruded to the extracellular space. However, HA can also re-enter the cell, and can even translocate to the nucleus [1, 2].
  • HAS membrane bound HA synthases
  • HA is produced in large quantities during wound repair [3] and NAG has been shown to stimulate the synthesis of HA by mesothelial cells and fibroblasts in a dose-dependent manner [4].
  • Hyaluronan is degraded by a family of enzymes called hyaluronidases.
  • hyaluronidases a family of enzymes called hyaluronidases.
  • the CD44 receptor is known to participate in cell adhesion interactions which are required for proliferation of tumor cells and it is suggested that HA may be involved in oncogenesis through its interactions with the CD4 receptor.
  • Chitin is one of the main components in the cell walls of fungi, exoskeletons of insects and other arthropods, and in specific tissues and organs of some other animals. It is a polysaccharide, made out of units of N-acetyl glucosamine (N- acetyl-D-glucos-2-amine). These are linked together in ⁇ -1,4 fashion, similar to the glucose units that make up cellulose.
  • Chitosan is a derivative of chitin, produced through N-deacetylation of the chitin polymer and the degree of deacetylation will heavily influence the physicochemical and biological properties of the polymer. Only partially deacetylated chitin will have significantly different solubility properties compared to more extensively deacetylated chitosan and same can be said about the biochemical properties of the polysaccharide.
  • chitin synthases and hyaluronic synthases In an evolutionary perspective, chitin is an older biopolymer than HA, but HA can be regarded as progeny of the chitin polymer. Thus, genes expressing chitin synthesizing enzymes are more ancient than genes expressing HA synthesizing enzymes (HAS). It is now evident that the two enyme systems are closely related ant that the HA synthases have evolved from the chitin synthases gene family [2]. In vertebrates, the three HA synthases (HASl, HAS2 and HAS3) are encoded by three distinct genes which have been identified by complementing HA-deficient cell lines [7-11]. HASl, HAS2 and HAS3 have been shown to possess distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis [11].
  • a mouse HA synthase (HASl) is capable to synthesizing HA in vitro, when it is supplied with UDP-GIcA and UDP-NAG [12]. However, when HASl is incubated with UDP-NAG alone, it synthesizes chitin-oligosaccharides (ChOS) [12].
  • ChOS chitin-oligosaccharides
  • chitin oligosaccharides are produced in vivo during the development of vertebrates (Xenopus, zebrafish and mouse), where the chitin synthase-like DG42/HAS subfamily synthesizes both CHOS and HA during cell differentiation. These natural CHOS have been shown to be vital for a normal anterior/posterior axis formation in the late gastrula, prior to neurolation [2, 12-17] reviewed in [18].
  • chitinases are classified as belonging to the group of carbohydrate cleaving enzymes called Familyl ⁇ Glycosidases. These family 18 chitinases are expressed in most organisms from bacteria to mammals and are endo-chitinases, possessing a strong chitin cleaving activity and require adjacent N-Acetyl
  • Glucosamine moieties as a substrate recognition site before they can hydrolyze the polymer down to smaller sub units.
  • CLPs chitinase like proteins
  • CLPs In humans, six CLPs have been described. These are YKL-40 (HC gp-39), YKL-39, ECF-L (YmI), Chitotriosidase, Acitic Mammalian Chitinase (AMCase; two subforms TSA1902-L and -S) and Oviductin. Al! exept AMCase (TSA1902-L) and Chitotriosidase are inactive (silent) as chitinases. The CLPs have conserved their chitin catalytic domain (their ability to bind chitin) but many have lost their catalytic activity (the ability to cut chitin) by one amino acid substitution in the catalytic domain.
  • this protein domain As the chitin binding domain where at least 5 monosaccharide units are needed in order to bind properly (full affinity binding).
  • the crystal structure of human YKL-40 (HC gp-39) has been worked out by [19] and the goat YKL-40 by [20].
  • the structure of the human YKL-40 along with CHOS (A9) lying in its binding domain indicates that unlike the chitinases, binding of the oligosaccharide ligand to the YKL-40 induces a large conformational change in the protein-Iigand complex, which may well reflect the signaling nature of this protein [19].
  • chitin fragments of four or more GIcNAc residues occupy the central part of the groove of YKL-40 and therefore the specificity of the binding depends on the length of the chitin fragments [21].
  • data from [19] revealed about 50 times stronger binding of DP 6 (A6) than DP 4 (A4) to pure YKL-40 demonstrating DP 4 oligomers to be a rather poor ligand.
  • CLPs play a role as cell signaling proteins (growth promoters or cytokines) in many tissues, but although the crystallographic three-dimensional structure of human [19, 21] and goat YKL- 40 [20] has been described, the site and mode of binding to cell surface receptors is not yet known.
  • the structure of ECF-L (YmI) has been work out by [22] and the crystal structure of chitotriosidase has been described by [21].
  • the chitotriosidase is known to be active against chitin, and is inhibited by allosamidin, a known inhibitor of the family 18 chitinases.
  • YKL-40 or human chondrocyte glycoprotein 39 (HC gp-39, gp38k, CHI3L1) is the most studied member of mammalian family 18 chitinase like protein [23, 24]. It has been characterized as a heparin and chitin-binding lectin [24, 25] without chitinase activity [23, 25] and is secreted by chondrocytes, human synovial cells, osteoblasts, osteocytes, macrophages and neutrophils [26, 27].
  • YKL-40 was first isolated from cow whey [28] but later it was identified in human osteoblasts (bone forming cells) [29] and in human chondrocytes (cartilage forming cells) [23] where it was shown to increase expression in patients with rheumatoid arthritis [23].
  • Analysis in serum by RIA revealed increased expression of YKL-40 in patients with various forms of inflammatory and degenerative joint disease [30] and the protein has been postulated as a new marker for joint disorders [31].
  • Transcriptional regulation of the YKL-40 gene (CHI3L1) has revealed that the gene is a marker gene for late stages of human macrophage differentiation [32].
  • CHOS have shown to significantly inhibit nitric oxide (NO) production in inflammatory activated macrophages [33].
  • Pure CHOS (A6) and CHOS (A5) also inhibited NO production but with less potency.
  • pure CHOS of A4, A3, A2 and the monomer, A and D had little effect on NO production by the activated cells [33], These results coincide with the conformational changes of YKL-40 mentioned above [19].
  • YKL-40 seems to play an important role in endochondral bone formation and cartilage formation [27, 34].
  • the expression of YKL-40 mRNA was intense in flattened, end- stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation [27],
  • DECs Dedifferentiated chondrocytes
  • mesenchymal stem cells capable of differentiating into chondrocytes, from human bone marrow show high expression of YKL-40 [34]
  • the master chondrogenic factor sox9 is markedly up regulated by YKL-40 [35].
  • Inflammation is the first response of the immune system to infection and has two main components - exudative and cellular.
  • the exudative component involves the movement of fluid from blood vessels (usually containing many important proteins such as fibrin and immunoglobulins (antibodies).
  • the cellular component involves the movement of white blood cells from blood vessels into the inflamed tissue,
  • the white blood cells or leucocytes take on an important role in inflammation; they extravasate from the capillaries into tissue, and act as phagocytes, picking up bacteria and cellular debris. They may also aid by walling off an infection and preventing its spread.
  • cytokines IL-I and TNF will activate endothelial cells to upregulate receptors VCAM-I, ICAM-I, E-selectin, and L-selectin for various immune cells. Receptor upregulation increases extravasation of neutrophils, monocytes, activated T-helper and T-cytotoxic, and memory T and B cells to the infected site.
  • Cytokines are small protein molecules that regulate communication among immune system cells and between immune cells and those of other tissue types and are actively secreted by immune cells as well as other cell types in response to external stimuli.
  • the same cytokine can have different effects on a cell, depending on the state of the cell. For instance, there are several known cytokines that have both stimulating and suppressing action on lymphocyte cells and immune response. To make things even more complicated, cytokines often regulate the expression of other cytokines (either upwards or downwards), often triggering cascades of other cytokines. The cytokines in these cascades can interact with each other and the cells that produced them in complicated fashions to form cytokine networks. Cytokines often act together in ways that are synergistic or antagonistic.
  • CLPs During inflammation and in many inflammatory diseases, an elevated expression of CLPs has been shown to occur along with expression of the proinflammatory cytokines, but the role of these proteins in the inflammatory processes is not yet discovered. As suggested by the complexity of the inflammatory cascade and the complex involvement of the inflammatory cytokines, it can be expected that the involvement of CLPs could be equally complex and the same protein might contribute to inflammation in one particular situation and tissue environment, but assert a tissue regenerative effect in an other situation/tissue.
  • Tissue repair Repair mechanism of degenerated or damaged tissue usually occurs according to a stepwise process initiated by a) inflammation involving defense mechanisms of the immune system and stimulation of fibroblasts and endothelial cells to activate the b) proliferative phase, characterized by angiogenesis and fibroplasia which then turns into c) maturation and remodeling phase which can take considerable time and involves remodeling of the extracellular matrix.
  • this repair mechanisms are not capable of reestablish the functionality of the normal tissue but a scar will be formed which is fundamentally different from the surrounding specialized tissue, be it a skin tissue, internal organ like liver tissue, cardiovascular tissue, neural tissue, bone tissue or cartilage tissue.
  • YKL-40 plays a role in tissue remodeling, [19, 38-41] especially in connective tissue remodeling or degradation of extracellular matrix. [19, 24].
  • YKL-40 is a growth factor for connective tissue cells where it increases the growth of fibroblastic cell lines in a dose-dependent way [42, 43]. In this manner the protein is possibly involved in scar tissue formation during the healing of a tissue. It is up regulated in cirrhotic liver diseases such as hepatitis C virus (HCV) [44] and is suspected to trigger fibrosis and is known as a fibrosis serum marker [41, 45-49]. It has proved to be a potent migration factor for endothelial cells [50] and vascular smooth muscle cells [51] such as in endochondral'bone formation.
  • HCV hepatitis C virus
  • YKL-40 is undetectable in the chondrocytes of normal articular cartilage [27], but YKL-40 expression in guinea pig chondrocytes (GPC), rabbit chondrocytes (RC), and rabbit synoviocytes (RS) was higher in dividing cells than in confluent cells, suggesting a participation of YKL-40 in cell cycle events [43]. Chondrocyte culture experiments have shown that YKL-40 production increases to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury [52].
  • osteoblast cell line In human osteophytic tissue the chitinase-like protein YKL-40 is expressed intensively in end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation [27]. Proliferating osteoblasts express low to moderate YKL-40 levels and mature osteocytes are negative [27]. The authors suggest not only cartilage degeneration but increased osteogenesis in osteoarthritis [27]. In osteoblast cell line, supplemented with
  • alkaline phosphatase (ALP) activity was significantly high compared with the control culture group, indicating an increased osteoblast activity and bone formation [53]
  • Sox9 In chondrogenesis, the master chondrogenic transcriptor factor Sox9 is expressed in all prechondrogenic and chondrocytic cells during embryonic development and has been shown to play a crucial role in cartilage formation. However, during inflammation, interleucin-1 (IL-I) and tumor necrosis factor-D (TNF-D) strongly inhibit Sox9 [54]. suggesting suppression of chartilage regeneration during inflammation and inhibition of endochondral ossification pathway in bone formation
  • YKL-40 Involvement of YKL-40 in various diseases has been reported. Strong up regulation of the protein has been widely reported in rheumatoid arthritis (RA) [23, 55-68] and in osteoarthritis (OA) [40, 63, 69-73], reviewed in [74]. In rheumatoid arthritis the YKL-40 protein is known as an autoantigen [55, 56, 58- 60, 62, 67, 75, 76]. Successful attempts have been made to increase immunotolerance to YKL-40 by inoculation of the protein [62, 77]. Increased expression (up regulation) of YKL-39 is both linked to osteoarthritis and rheumatoid arthritis [70, 73, 75, 78].
  • ECF-L is expressed during inflammation as well as in allergy [80-84].
  • AMCase was shown to be involved in asthma [85, 86].
  • the authors found that if the AMCase protein was blocked with an antibody or with an inhibitor that prevents it from digesting chitin, the asthma-like inflammation was decreased [85]. It was also found that the protein was present in the airways of human asthmatics but not in control lungs [85], The authors hope that blocking AMCase in human asthmatics will have a similar beneficial effect on the human disease [85].
  • Patents has been filed on this matter where allosamidins, chitinase blockers made of NAG reduce the expression and enzymic activity of AMCase in mouse lungs [87, 88].
  • Atherosclerosis is characterized by infiltration of monocytes into the subendothelial space of the vessel wall and subsequent lipid accumulation of activated macrophages.
  • a strong up-regulation of both YKL-40 and chitotriosidase by activated macrophages has been demonstrated in atherosclerosis during the lipid accumulation phase [71, 89-91].
  • Osteoporosis Osteoclasts are essentially involved in bone resorbsion (breakdown of the bone in bone remodelling), but in osteoporosis decreased activity of osteoblasts (bone forming cells) and increased activity of osteoclasts result in bone demineralization. Hence, in treating osteoporosis, focus has been on restoring the balance between osteoblasts (bone formation) and osteoclasts (bone resorption), mainly by inhibiting osteoclastic bone resorbsion. In an osteoporosis rat model, oral administration of low molecular weight chitosan (LMWC) resulted in restoration of bone mineral density [92].
  • LMWC low molecular weight chitosan
  • ECF-L has been identified as a novel osteoclast stimulating factor shown by a dose-dependent osteoclast formation response to ECF-L in mouse bone marrow cultures [93]. Hence, evidences suggest that ECF-L is likely to play a central role in bone remodeling and osteoporosis. Cancer
  • YKL-40 Increased expression of YKL-40 has been widely reported in serum in various human cancer patients [38, 39, 47, 70, 94-100]. These are breast cancer,[38, 99] lung cancer,[39, 101, 102] ovarian cancer [96, 98, 103], colorectal cancer [94], human glioma, [47], glioblastoma, [104-106] chondrosarcoma [107] and renal cell carcinoma [97]. It is not known which cells are responsible for the YKL- 40 production in these cancers, but studies in glioblastoma indicate that activated macrophages are involved [104, 105].
  • Hepatic fibrosis Expression of YKL-40 is increased in hepatic fibrosis due to alcoholism [41, 45, 49]
  • the seronegative spondyloarthropathies are a group of multisystem inflammatory disorders that share a variety of clinical, radiologic, and genetic features. They include ankylosing spondylitis, psoriatic arthritis, Reiter's disease, and arthritis associated with inflammatory bowel disease.
  • the common features include: 1) a common association with the genetically determined histocompatibility antigen HLA-B27, 2) Inflammation at the bony insertion of ligaments and tendons (the enthesis). This process is characterized radiographically by shallow erosions followed by bony proliferation and eventually ankylosis of the joint, 3) Spinal predilection resulting in sacroiliitis, spondylitis and in advanced cases complete spinal fusion known as bamboo spine. In ankylosing spondylitis involvement of the hips, shoulders and knee is quite common [109]. When looking at the molecular signalling pathways of these disorders, they remind strongly of chitinase like protein related disorders [HO].
  • a chronic inflammatory response is evident in astrocytoma cells and/or microglia in both Alzheimer and multiple sclerosis, both representing severe degeneration of the central nervous system involving inflamatory cytokines (Interleukins, TNF-a etc.) secreted by microglia cells.
  • a chronic inflammatory response associated with ⁇ -amyloid (AB) and interleukin-l ⁇ (IL-l ⁇ ) is responsible for the pathology of Alzheimer's disease (AD).
  • ⁇ fSiijsjLCj ⁇ e ⁇ water soluble chitosan (WSC) in human astrocytoma cells [111]
  • YKL-40 was found to be up regulated in schizophrenia [112], as well as in purulent meningitis [113]. In the meningitis, the levels of the YKL-40 was 10-fold higher in cerebrospinal fluid than the corresponding levels in serum, indicating that YKL-40 may be produced by activated macrophages within the central nervous system [113].
  • chitotriosidase has been linked to beta-thalassemia, [114, 115] as well as to Gaucher disease [116-120] and sarcoidosis [121] .
  • the Gaucher disease can be effectively treated by costly intravenous infusions with recombinant glucocerebrosidase, the deficient enzyme. The result is a decrease in chitotriosidase blood level [122] .
  • Increased expression of YKL-40 is also found in patients with pulmonary sarcoidosis [123] .
  • enzyme- replacement therapy in humans resulted in a significant decrease in chitotriosidase activity [124] .
  • the current evidence demonstrates a substantial involvement of CLPs in a range of various degenerative diseases involving inflammation, tissue repair and tissue remodeling.
  • Several authors have suggested methods to inhibit these proteins in one way or another, mainly by using antibodies or chitin specific inhibitors (patent applications US 2005/0214297 Al and WO 03/009808 A2).
  • the current evidence also suggests a role of some of the CLPs in embryonic development such as endochondral bone formation and possibly in other tissue regeneration processes.
  • the HA synthases (HAS 1, HAS 2, and HAS 3), which are genetically derived from chitin synthases, have also been shown to play a vital role in anterior/posterior axis development in the early embryo and there are strong indications that the HAS 1 enzyme specifically synthesizes chitin oligosaccharides during this phase of embryonic development, which in turn is the first indication of a role of chitin chemical structure in human development and health. This might suggest a yet hidden link between the role of chitin and CLPs in human development and health. It is therefore of great interest to evaluate the possible role of chitin chemical structures in human methabolism and cell signaling mechanisms, looking into their possible role in degenerative diseases, their therapeutic value and their general role in human health.
  • the present invention shows that chitooligomers formulations prevent Fibroblasts from invading a tissue site and allow for regeneration of tissue instead of repair.
  • the chitooligomers formulations of the present invention are shown to bind to the citin-binding site of the C/CLP's, which indicates that the formulations can block the activity of the proteins.
  • the formulation not only modulates the activity of the proteins, but also regulates their gene expression.
  • a pronounced effect of the formulations is further shown in patients suffering from disorders of the connective tissue, indicating that the C/CLP's play a role in the etiology of the diseases.
  • a use of chitooligomers formulations for the manufacture of a medicament for modulating the activity and/or expression of one or more C/CLP in a mammal.
  • the modulation involves may involve a reduction of abnormally high activity or and/or expression of the one or more C/CLP as well as an increase of abnormally low activity and/or expression of the one or more C/CLP in a mammal.
  • the term "activity” refers to a biochemical process interaction of a protein or CO leading to a biological or biochemical response or action within the target cell or organ.
  • this term comprises both gene expression of the protein and the activity of the protein within the cell or organ including the functional translation, formation and activity of the protein and its ability to interact with other biomolecules within or in the environment of the cell or organ.
  • the use includes the modulation of the regulation of one or more C/CLP is for modulation of inflammation and/or treating prophylaxis and/or prevention of connective tissue diseases or degenerative disorders.
  • the modulation of the regulation of one or more C/CLP according to the use or the method of the present invention may further be useful for tissue repair.
  • tissue repair refers to healing of a tissue accurs either by regeneration of the damaged tissue cells, preserving the structure and functionality of the original tissue, or by repair of the tissue damage through activation of a three phase wound healing cascade. This involves inflammation, proliferation with activation of fibroblasts, and maturation. The wound healing cascade will leave a scar in the repaired tissue interupting the continuum of the tissue architecture.
  • This invention deals with a modulation of the healing of a tissue suppressing the fibroblast activation and stimulating the proliferation and activation of tissue specific progenetor cells capable of regenerating the injured tissue.
  • chito-oligomer formulations refer to oligomers and polymers of one or both of /V-acetyl glucosamine (NAG) and glucosamine, i.e. oligomer chains with a minimum chain length of 2 (dimers).
  • NAG /V-acetyl glucosamine
  • glucosamine i.e. oligomer chains with a minimum chain length of 2 (dimers).
  • the composition is particularly useful for use as a medicament.
  • homologue defines equal length chito-oligomer chains with the same ratio of monomers, which may have different sequence, i.e., the homologue A3D2 may comprise e.g., the chito-oligomer sequences A-A-A-D-D and A-A-D-A-D.
  • a and D refer to ⁇ /-acetyl glucosamine and glucosamine, respectively.
  • the degree of deacetylation of the chito-oligomers is in the range of about 0-70%, such as 10-60%, 20-70%,10-50%,20-60%, 20-50%30-60%, 30- 50% and more preferably in the range of about 30-50%, and even more preferably about 35-50%, such as about 40-50%, including about 40% or about 50%.
  • the shorter chito-oligomers are postulated to be highly important for the activity of the composition of the invention.
  • at least about 10 wt% of the chito-oligomers of the composition have a chain length of 2 to 12, more preferably at least 15 wt%, including at least 25 wt%, and even more preferably at least 50 wt% of the chito-oligomers have a chain length of 2 to 12.
  • about 15 to 75 wt% of the chito-oligomers have a chain length of 2 to 12, such as about 50 wt% of the chito-oligomers, preferably about 15 to 75 wt% of the chito-oligomers have a chain length of 2 to 9.
  • at least 50 wt% of the chito-oligomers have a chain length in the range of 2 to 15, such as at least 60 wt%, including at least 70%, or at least about 80%.
  • the chito-oligomer composition may conveniently be provided in an essentially dry form comprising a powder, flakes or fibrous material which can be capsulated or dissolved or suspended in an aqueous solution for intake.
  • a composition may consist of substantially only the aforementioned chito-oligomers, i.e. in the range of about 80 - 100 wt% of the chito-oligomers.
  • the composition comprises in the range of 20-100% by weight of said chito- oligomers, including about 25 - 95 wt%, such as about 50 - 90 wt%.
  • the composition may contain significant amounts of salt other than the salts of chito-oligomers, e.g.
  • compositions of the invention typically present in the compositions of the invention, such as in the amount of 0- 60 wt% of the total saccharide amount, such as less than about 50 wt%, but preferably the monomers are less than about 40 wt% of the total saccharide amount, and such as less than about 25 wt%, including less than about 20 wt%.
  • monomers of glucosamine and NAG are typically present in the compositions of the invention, such as in the amount of 0- 60 wt% of the total saccharide amount, such as less than about 50 wt%, but preferably the monomers are less than about 40 wt% of the total saccharide amount, and such as less than about 25 wt%, including less than about 20 wt%.
  • Our test results indicate that a certain amount of monomers, in particular NAG, present in compositions of the invention may have a positive synergistic effect.
  • composition may further comprise a pharmaceutically acceptable excipient or diluent, a flavoring substance, a nutrient, or a colorant.
  • chitosan constitutes a portion of the medicament.
  • the mammal is a human and the C/CLP's of the present invention are also of human origin.
  • chitinase active enzymes are available and may be employed in this regard, e.g. chitinase (EC no. 3.2.1.14) available from Sigma- Aldrich, also, lysozyme (EC no. 3.2.1.17) is found to have chitinase activity (see, e.g., U.S. Patent No. 5,262,310).
  • the enzyme incubation conditions may be varied, depending on the specific activity and optimum reaction conditions of the employed enzyme. As demonstrated in Example 2 (see Sample 1 and 2; production), conditions may be optimized to obtain a desired ratio of small to medium-sized chito-oligomers. Longer chito-oligomers and polymers (DP 30 and higher) may optionally be separated from the desired short and medium length chito-oligomers, either by preparative chromatography, or by precipitation at a high pH (about pH 9 or higher).
  • a method for modulation of inflammation and/or treating prophylaxis and/or prevention of connective tissue diseases or degenerative disorders by modulating the regulation of one or more C/CLP in a cell.
  • the method comprises the step of administering to the cell an effective amount of chitooligomer formulations.
  • the cells are within a mammalian subject and wherein the animal subject has an abnormal activity and/or expression chitinase-like proteins.
  • the modulation involves may involve a reduction of abnormally high activity or and/or expression of the one or more C/CLP as well as an increase of abnormally low activity and/or expression of the one or more C/CLP in a mammal.
  • the CO formulations enjoy several properties that allow for a variety of application methods.
  • the preferred method of administration is via oral ingestion.
  • Alternative methods of administration include subdermal administration including subcutaneous and intramuscular injections.
  • COs may be compounded to be inserted subdermally as a slow release therapeutic.
  • the CO formulations can also be administered via a medical implant product, defined as a Class I, II or III regulated medical device by the US Food and Drug Administration where the medical implant typically is installed during a surgical procedure either for repair from trauma, disease, resection or revision.
  • Said medical devices may be permanently placed or for temporary use.
  • said medical implants may comprise technology that decompose over a desired time frame.
  • the CO may be used in conjunction with other medical implants either as a coating or as a component.
  • said medical devices are represented by metallic or plastic joint implants including finger, knee, TMJ, stabilizing plates or other articulating joints.
  • the COs may be applied as a coating to similar medical devices. Said example would be as a surface coating of an implantable medical device such as an intramedullary rod or other stabilizing devices such as reconstruction plates or screws.
  • CO may be conducted via catheter for use for example in vertibralplasty or chronic delivery of insulin.
  • catheter application may employ short term indwelling catheters for delivery of CO for short interval periods, typically used in conjunction with micropumps.
  • CO may occur via transdermal transport patches or microneedles, typically between 100 and 1,000 micrometers long. This method of application can be enhanced with the used of localized ultrasonic enhancement.
  • the pharmaceutical composition shall preferably be in a form suitable for oral administration, such as a dry form which can be readily dissolved, e.g. in a glass of water.
  • a dry form which can be readily dissolved, e.g. in a glass of water.
  • Such forms include dry powder, granular, flake, fibrous and paste forms.
  • the composition can also be contained in pills or capsules.
  • Chitinase like proteins share a high sequence homology and a structural relationship with family 18 glycosyl hydrolases (F18 family).
  • HC gp- 39/YKL-40 (CHI3L1: chitinase 3-like 1 (cartilage glycoprotein-39), GP39, YKL40, HC-gp39, HCGP-3P), YKL-39 (CHI3L2: chitinase 3-like 2, YKL39, YKL-39), ECF- L/T1902 (CHIA: eosinophil chemotactic cytokine, ECF-L, AMCase, TSA1902), Chitinase 1 (CHITl : chitinase 1 (chitotriosidase), CHIT) and Oviductin (OVGPl : oviductal glycoprotein 1, 12OkDa (mucin 9, oviductin), MUC9) have all been
  • C/CLP's lack catalytic activity because of single point mutation in their catalytic domain but they maintain their oligosaccharide binding ability, which usually involves 5-8 chito-oligosaccharide. C/CLP's bind chito-oligomers.
  • a method for the treatment of prophylaxis and/or prevention of connective tissue diseases or degenerative disorders comprises inhibiting synovial hyperplasia associated with an arthritic disease in a mammal
  • a method for the treatment of prophylaxis and/or prevention of connective tissue diseases or degenerative disorders comprises inhibiting cartilage damage associated with an arthritic disease in a mammal.
  • a method for the treatment of prophylaxis and/or prevention of connective tissue diseases or degenerative disorders comprises inhibiting infiltration of inflammatory cells associated with an arthritic disease in a mammal.
  • a method for the treatment of prophylaxis and/or prevention of connective tissue diseases or degenerative disorders comprises inhibiting bone deterioration associated with an arthritic disease in a mammal.
  • connective tissue diseases or degenerative disorders refers to conditions selected from the group consisting of, but not limited to: a) Paget's disease; b) osteoporosis; c) Gorham-Stout syndrome; d) arthritic diseases; e) osteoarthritis; 0 rheumatoid arthritis; g) psoriatic arthritis; h) rheumatoid disease; and i) brittle bone disease.
  • a pharmaceutical composition comprising an effective amount of chitooligomers for modulation of abnormal activity and/or expression of chitinase like proteins in patients for modulating inflammation and/or treating prophylaxis and/or prevention of connective tissue diseases or degenerative disorders.
  • kits for modulation of inflammation and/or treating prophylaxis and/or prevention of connective tissue diseases or degenerative disorders in a mammal by modulating the regulation of one or more C/CLP, said kit at least comprising: an effective amount of an chitinase-like molecule inhibitor, an applicator, and an instructional material for the use thereof.
  • a method for treating inflammatory and degenerative disorders or diseases in tissues or systems selected from the group containing connective tissue, central nervous system, cardiovascular tissue or the immune system in a mammal wherein said disease is associated with an increased level of chitinase, said method comprising administering an effective amount of a chitooligomer formulation to said mammal, thereby treating said inflammatory disease in said mammal.
  • the use, the method and the kits of the present invention add to the cure or releave of symptoms of a disease or disorder in a combination with other anti-inflammatory and pain relieving drugs selected from the following drug classes: a) analgesics; b) nonsteroidal anti-inflammatory drugs (NSAIDs), including the COX-2 inhibitors; c) glucocorticoids, which are more commonly know as steroids; d) disease-modifying anti-rheumatic drugs (DMARDs), and; e) new pipeline drugs including biological agents that target selective blocking of certain immune system functions.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • glucocorticoids which are more commonly know as steroids
  • DMARDs disease-modifying anti-rheumatic drugs
  • new pipeline drugs including biological agents that target selective blocking of certain immune system functions.
  • Renkema, G. H., et al., Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 giycosyl hydrolases secreted by human macrophages. European Journal of
  • HC-gp39 The chitinase 3-like protein human cartilage glycoprotein 39 (HC-gp39) stimulates proliferation of human connective-tissue cells and activates both extracellular signal-regulated kinase- and protein kinase B-mediated signalling pathways.
  • Peltomaa R., et al., Increased level of YKL-40 in sera from patients with early rheumatoid arthritis: a new marker for disease activity.
  • chitinase 3-like protein 1 is up regulated in osteoarthritic chondrocytes.
  • YmI is a neutrophil granule protein that crystallizes in p47phox-deficient mice.
  • ECF-L Eosinophil Chemotactic Factor-L
  • FIG. 1 The effect of chito-oligomers (G010920-1K) on human chondrocyte cell growth.
  • the cells are grown on a microplate (96 well format) for 5 weeks. See Materials and Method and Table 1 for details.
  • FIG. 1 The effect of CO (G010920) on human chondrocyte cell growth. Chondrocytes were grown in 16 wells for 7 days, then 500 ⁇ g/ml CHOS (G010920) were applied to 8 wells but only buffer (0 ⁇ g/ml CHOS) applied to the other 8 wells. The cells grown further for 7 days before analysis.
  • FIG. 3 The effect of CO on fibroblast growth. Fibroblast density (no. of cells per field) as a function of time in days.
  • FIG. 4 The blocking effect of CO lot G040823 on chitinase A.
  • the graph indicates specific activity of the enzyme as a function of CO concentration. Based on data in Table 3.
  • the IC50 was calculated as 79 ⁇ g/ml.
  • FIG. 5 The expression pattern of chitinase-like genes in normal human tissues. Bands represent PCR amplificated cDNA maid from mRNA isolated from Aorta, whole brain, fetal brain, colon, Lung, and testis. Editium bromide stained 1% agarose, run for 30 min./ 80 v. Neg. control; water, genomic DNA (gDNA).
  • Figure 7 Time dependent effects of chitooligosaccharide treatment on the chitinase-like gene expression in Jurkat cells.
  • FIG. 8 Effects of CO on THP-I cells in culture incubated for 48 h. Untreated cells (A), Cells treated with 10-8 M PMA with enlarged cells out of focus attached at the bottom of the well (B), with 100 ⁇ g/ml CO (C), cells treated with 10-8 M PMA and 100 ⁇ g/ml CO (D). The cells clustered around calcofluor white particles of CO (E). Magnification, XlOO A-D and 630X E.
  • FIG. 9 The expression pattern of HCgp-39, YKL-39, TSA1902 S (inactive AMCase) and TSA1902 L (actice AMCase) and Chitotriosidase in normal human tissues. Bands represent PCR amplified cDNA maid from mRNA isolated from
  • Aorta whole brain, foetal brafn, colon, Lung, and testis. Etbr stained 1% agarose, run for 30 min./ 80 v. Neg. control; water, genomic DNA (gDNA).
  • FIG. 10 RT-PCR results showing the expression of HCgp-39, YKL-39, AMCase, and Chitotriosidase compared to GAPDH in untreated (U) THP-I cells and in response to 10-8 M PMA and 100 ⁇ g/ml chitooligosaccaride (C).
  • V LadderV.
  • FIG. 11 Relative expression of HCgp-39, YKL-39, AMCase and Chitotriosidase in untreated (U) THP-I cells and in response tolO-8 M PMA and 100 ⁇ g/ml chitooligosaccaride (C), determined by RT-PCR and normalized to GAPDH expression.
  • FIG. 12 Semi-quantitative HCgp-39 gene expression determined with RT-PCR.
  • U THP-I cells
  • C chitooligosaccharide
  • FIG. 14 The effect of CO (G040823) on the HCgp-39 expression in monocytes (THP-I cells) as judged by ELISA.
  • Figure 16 Homologue distribution of sample G010920-1K as judged by MALDI- TOF (crude sample). Al and Dl are not shown.
  • Figure 17. DP distribution of product G010920-1K as judged by MALDI-TOF (based on the data in Figure 15).
  • FIG. 20 DP distributiton (%) of G040823 compared to G010920 as jugded by MALDI-TOF on crude CO product.
  • Glucosamine a monosugar frequently occurring in the chitooligosccharides (CO) produced by Genis, is generally used as an oral treatment of osteoarthritis (OA).
  • This aminosugar has been claimed to induce regeneration of damaged cartilage tissue in OA, but no significant evidence has yet been presented to prove this theory.
  • Chitooligosaccharides are oligosaccharides made from chitin through hydrolysis. Chitin forms a large family of oligosaccharides composed from of two different monosaccharides, N-acetyl glucosamine (GIcNAc) and glucosamine (GIcN).
  • GIcNAc N-acetyl glucosamine
  • GcN glucosamine
  • the bioactivity of CO on chondrocytes, among other cells has been studied and indicates growth promotional effect.
  • the material used in these studies is a crude mixture of CO thus no information exist on which fractions of the CO do carry the bioactivity.
  • Genis ehf is developing a method of preparing water soluble oligosaccharides from shrimp chitin using chitinolytic enzymes. Fractionation of different oligosaccharides and in depth analysis of their structure will enable a thorough characterization of their bioactive properties.
  • the chitooligosaccharides (G010920) were manufactured by Genis ehf. Human chondrocytes (Cell-Lining GmbH, Germany) where shipped frozen, thawed and cultured in 96 well microplates for, supplemented with different concentrations of CO (0, 10, 50, 100, 500 and 1000 ⁇ g/ml). After two weeks and five weeks of incubation, the cells were fixed in -20° C methanol and HE stained. Finally, cells were photographed in each well through a microscope and the photographs used to count the cells and evaluate there appearance.
  • chondrocytes were grown in 16 wells for 7 days, then 500 ⁇ g/ml CHOS (G010920) were applied to 8 wells but only buffer (0 ⁇ g/ml CHOS) applied to the other 8 wells. The cells grown further for 7 days, then fixed, stained and the density estimated
  • CHOS have a growth promoting effect on chondrocytes in culture.
  • the observed change in appearance of the more rapidly growing cells could be due to the direct effect of CO on the cell phenotype.
  • the CHOS induce cell growth in a dose-dependent manner.
  • CHOS only induce early stage chondrocytes, since ceil cultures at a later stage do not respond to CHOS.
  • Chondrocyte culture experiments have shown that YKL-40 production increases to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. We suspect that CO could possibly work through YKL-40 and the Sox9 signaling pathway in injured cartilage. Attempts are now being made to develop this cell model further.
  • Example 2 The effect of chitooligosaccharides on fibroblast growth
  • Solution 1 Gelatin (0,1%) was solubilized in Hanks balanced salt solution (HBSS) by heating at 37 0 C for five minutes.
  • Solution 2 Same as solution 1 with lOOO ⁇ g/ml chitooligosaccharides (CO) added. Both solutions were filter sterilized.
  • HBSS Hanks balanced salt solution
  • CO chitooligosaccharides
  • Figure 3 shows the average number of cells counted in each wells for three consecutive days. There was a significant reduction of growth in the presence of CO over the period examined, indicating the capacity of CHOS to suppress fibroblast growth and proliferation..
  • a standard substrate for chitinase 4-methylumbelliferyl-beta-D-N,N'- triacetylchitotrioside (4-MU-A3), a chitin tetramer (A4) analogue, was purchased from Sigma, USA.,.
  • Chitinase A 0.5 nM (500 pM) in 0.1 mg/ml BSA, 50 mM phosphate buffer pH 7.4 was used (Chit-A solution.).
  • the substrate solution was 40 ⁇ M 4-MU-A3 in 50 mM phosphate buffer pH 7.4.
  • Different concentrations of CO (0, 62.5, 125, 250, 500 and 1000 ⁇ g/ml) were made in the substrate buffer.
  • 25 ⁇ l of the Chit-A solution was mixed with 25 ⁇ l of the substrate, incubated at 37°C for 10 min. The reaction was stopped with 1.95 ml 0.2 M sodium bicarbonate buffer (Na 2 CO 3 ).
  • the pH for the blocking experiments was adjusted to pH 7.4 in order to free the amine groups of COs from protons. This pH also better resembles the physiological pH of the blood, better reflecting the behaviour of the COs in the human body. Earlier pilot experiments performed at pH 5.5, the optimal activity at for chitinase, indicated low blocking activities of the COs due to protonation of amine groups of COs.
  • Table 3 shows the blocking effect of CO lot G040823 on chitinase A.
  • Figure 4 shows CO blocking of the chitinase activity along with the non linear fit.
  • the IC 50 mean value is 79.3 ⁇ g/ml ⁇ 0,45 ⁇ g/mi SD for the CO mixture tested (Table 3).
  • the affinity of the CO to Chitinase A was determined using the formula (1/IC 50 )*1000, resulting in a mean value of 12.6 ⁇ 0.071 SD.
  • No cleavage products were detected in the CO mixture after the chitinase treatment using MALDI-TOF mass spectrometry, indicating good biological stability of the homologues in the CO mixture.
  • Table 3 The blocking effect of CO lot G040823 on chitinase A.
  • the Table shows different chitooligomer concentration in ⁇ g/ml, three product 4-MU formation
  • the jurkat Cell line (T-lymphoblastoid cells, ATCC) was maintained in RPMI-1640 medium supplemented with 5% FBS, 100 U penicillin /ml, 100 ⁇ g of
  • ChOS soluble ChOS
  • Migration of cells was evaluated in a Multiscreen chamber (Millipore). The upper and lower wells were separated
  • RNA isolation Tri-reagentTM
  • cDNA construction RevertAidTM
  • chitinase-like genes are generally expressed in Lungs. This extensive expression in normal lungs indicates constant expression rather than triggering immune response as a result of environmental factors, HCgp-39 and YKL-39 expression in fetal brain could indicate their role in development like their Drosophila's counterparts, but HCgp-39 is expressed in whole brain as well.
  • ChOS treatment In that way ChOS could be a new solution to reduce the effects of AMCase in Th2 inflammation of asthma.
  • Example 5 Activation dependent Human cartilage glycoprotein-39 expression of monocytes in response to chito oligosaccaridesj
  • THP-I Cell line culture The THP-I Cell line (ATCC) was maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U pen./ml, 100 ⁇ g of strept./ml and incubated at 37 0 C with 5% CO 2 . Soluble chitooligosaccharides (Genis ehf. Reykjavik, Iceland) at concentration of 100 ⁇ g/ml were added to some of the cells. The cells were then treated with 10 '8 M Phorbol 12-myristate 13-acetate (PMA) for 4 days before harvesting. PMA was obtained from Sigma (P8139) dissolved in ethanol and stored at -20 0 C. A 50 ⁇ l aliquot was taken out for cell counting and viability tests.
  • PMA Phorbol 12-myristate 13-acetate
  • V Viability
  • the ChOS was dissolved in water, adjusted to pH 7.4, centrifuged (3000 x g) the precipitate collected and freeze- dried to render chitosan particles.
  • Calcofluor staining was performed as follows: Cells were spun down and washed 3X in 0.01 M phosphate-buffered saline (PBS). After harvesting they were resuspended in 0.2% serum in PBS solution in 1: 1 ratio. The ceils were then smeared on superfrost (Menzel) microscope slides (76x26mm) and allowed to dry followed by fixing in 4% Para formaldehyde for 15 min. Then the micro slides were incubated in calcofluor white solution (SC15-100, Dalynn Biologicals) for 30 min. Finally, after 3X washing in PBS, samples were immersed with antifade reagent (P-7481, Molecular probes) and examined with a phase contrast laser scanning light microscope (Axiskop 2 mot plus, Zeiss).
  • RNA yield was determined by standard spectrophotometric assay (The mean ratio of
  • RNA was reverse transcribed using revertAidTM First strand cDNA Synthesis kit (#1621 Fermentas), priming method used to generate them was by oligo(dT) 1 s primers.
  • tissue cDNA complementary DNA was synthesized with conventional method, using the HybriZAP-2.1 cDNA synthesis kit (Stratagene, La JoIIa, CA). These libraries were maid from mRNA originated from human tissues; whole brain (Clontech, 6516-1), fetal brain (Clontech, 6525-1), colon (Origen cat. nr.
  • HM-1015 Lung (Clontech, 6524-1), testis (Clontech , 6535-1), Aorta (Clontech, 6572) and the priming method used to generate them were Oligo dT.
  • polymerase chain reaction (PCR) was performed using 100 ng/30 ⁇ l reaction of cellular cDNA and oligonucleotide primers were designed with Primer3 program (http://frodo.wi.mit.edu/cgi- bin/primer3/primer3_www.cgi) specific for the conserved sequences.
  • the primer sequences were compared to cDNA database by the Blastn program at the National Institute of Health (NCBI) homepage (http://www.ncbi.nlm.nih.gov). Sequence alignment was conducted by using the Clustal W program at http://workbench.sdsc.edu. For PCR from tissue libraries a standard amount of 5 ⁇ l cDNA was used.
  • the primer sequences for the cDNA sequences were: For YKL- 39 (gene CHI3L2, Genbank Ace. No.
  • BC011460 forward, CATCTATTCATTCGCCAGCA and reverse, AG CCTTTCCTTG GTG GATTT (nucleotides (nt) 216-553), for HCgp-39 (gene CHI3L1, Genbank Ace. No. BC039132): forward, TGTGAAGGCGTCTCAAACAG , reverse, TCTGGGTGTTGGAGGCTATC (nt 25-343), for TSA1902-S and TSA1902-L, gene TSA1902, Genbank Ace.
  • the PCR mix contained conventional amount of ingredients; 1Ox buffer (IX), MgCL 2 1-4 mM, dNTP 200 ⁇ M, forward and reverse primers 1,0 ⁇ M, Taq 1,25 units/ reaction. All primers were confirmed to yield the expected products under these conditions. PCR was conducted with specific primer pairs to account for cross-hybridisation with closely related sequences, splice variants and genomic DNA. Genomic DNA was isolated by Puregen TM kit (D-5000, Gentra systems) from the THP-I cells.
  • THP-I cell media was extracted from cells at times indicated and centrifuged.
  • An enzyme immunoassay was conducted according to manufacture instructions (Quidel, METRA TM YKL-40 ELISA kit, cat. no. 8020). Briefly, 20 ⁇ l of cell media was used for each reaction and the exact amount of protein calculated by comparison to HCgp-39 standards. Densitometry analysis of scanned gels was used to quantify the PCR products. Optical density values of all bands were compared by GAPDH and normalized for the value obtained for each sample on the gel. Each value was calculated as a percentage of mean peak analysed with Quantity one 4.1.0 software. The relative expression is shown as averages with corresponding standard errors. Statistical significance was found by student paired t-test, for the results, p values of 0.05> p >0.01 in were considered significant (*)(two-sided or one-sided tests indicated for each analysis).
  • the cells appeared normal based on microscopic examination.
  • the human hematopoietic cell line THP-I grows as non-adherent in suspension but can be induced to differentiate into cells with M0 like properties like adherence and enlargement.
  • the treatment with ChOS did not affect the cell growth or viability in culture (data not shown).
  • ChOS did however have dramatic effects on the morphology of the cell culture. These effects are seen as clustering of the cells (Figure 8C).
  • the clustering can be explained by attachment of cells to the chitooligosaccharides. The attachment was viewed when the insoluble fraction (chitosan) of the ChOS was used stained with calcofluor white ( Figure 8E).
  • TSA1902-L (AB025008) has a 99% identical nucleotide sequence to acidic mammalian chitinase (AMCase) (AF290004) in humans and the mouse AMCase (AF290003) has 81% identical nucleotide sequence to human TSA1902-L/AMCase.
  • the TSA1902-L/AMCase expression however, is inconsistent with the previously undetected expression in the lungs of normal mice. For HCgp-39, the strongest signal was detected in foetal brain and no expression by the CLP genes was detected in colon, testis or aorta. Interestingly, the observed tissue expression distribution of the CLPs goes in hand with two or more amino acid changes in the catalytic site of the proteins (Table 4.). We propose a categorisation of the CLPs in to two groups based on these differences.
  • CLP Human chitinase-like proteins
  • PMA is known as an ample cell activator and the cells respond in different patterns of expression in response to ChOS and PMA.
  • PMA or ChOS treatment alone resulted in decreased HCgp-39 expression but PMA and ChOS synergistically increased the HCgp-39 expression indicating monocyte/ M0 differentiation.
  • the different kinetics in CLP expression may illustrate the subsequent phenotypic changes taking place during maturation of in vitro cultured M0.
  • ChOS could be acting as a co-activator with PMA in monocyte / M0 activation.
  • a frequently inherited 24 bp duplication in exon 10 of the CHITl gene results in Chitotriosidase deficient individuals.
  • the duplication results in cryptic 3 'splice site and cultured M0 from recessive individuals contain very little mRNA and almost no Chitotriosidase protein.
  • the ELISA results confirm the effect on HCgp-39 expression seen by ChOS and PMA treatment detected in the RT-PCR analysis.
  • the assay was quantified by measuring the standard of known HCgp-39 concentration, presented in nanomoles per millilitre.
  • the HCgp-39 average activity was 21.1 nmol/ml in the media from untreated cells and 23.4 nmol/ml in the media from cells treated with ChOS and PMA ( Figure 13).
  • CLPs has been linked to several pathological conditions were a common feature is the infiltration of monocytes into the affected tissue sites and subsequent differentiation into activated M0.
  • the expression of HCgp-39 by M0 has been directly linked to conditions like arteriosclerosis and glioblastoma multiforme.
  • ChOS treatment of monocytes is followed by diminished secretion of the HCgp-39 protein.
  • the opposite response was observed by simultaneous treatment with ChOS and PMA.
  • the dramatic changes for HCgp-39 expression as a result of ChOS and PMA treatment indicate active expression control when monocytes differentiate to M ⁇ .
  • IDGF 's are highly expressed in the fat body, like HCgp-39, which was suggested as marker of fibrosis in alcoholic cirrhosis. Also, HCgp-39 and YKL-39 expression in foetal brain could indicate their role in development like the Drosophila counterparts.
  • AMCase has been reported as an important mediator of asthmatic Th2 inflammation in mice. The mouse AMCase has 81% identity to the human homologue and whether their function is exactly similar in both species can only be speculated. In similar way HCgp-39 has been reported with chemotactic activity in humans like the AMCase homologue in mice which has been shown to be an cell stimulating factor expressed in the lung and stomach.
  • the proposed categorization could be helpful in predicting the similarities of CLPs in different species and to get an overview of their functions as well as distribution.
  • Phenotype changes by cells were observed as a result of PMA treatment indicating M0 differentiation.
  • the ChOS exposure resulted in dramatic change of the culture as a result of binding of cells to ChOS and subsequent clustering of cells.
  • the basis for the binding of cells is not clear but M0 posses receptors for mannose/GlcNAG oligosaccharides.
  • chitin has been reported to bind to mannose receptors leading to receptor mediated endocytosis of ChOS by M ⁇ s.
  • the results show that the monocytes have binding affinity to ChOS as well as PMA activated cells indicating that the function is not dependant on the M0 properties.
  • HCgp-39 expression has not been reported in primary monocytes. This expression for HCgp-39 in untreated THP-I cells is consistent with previous reported studies, and could be explained as a result of their malignant origin. Interestingly, increased HCgp-39 expression has been described in several cancer types, such as cells originating from glioma tissue. Since HCgp-39 has been reported as a chemotactic factor, it could act as a secondary factor in cancer affecting metastasis of cancer cells. Similarly, HCgp-39 could be an important factor in the formation of neointima during arteriosclerosis since it has been reported as chemotactic factor for smooth muscle cells.
  • HCgp-39 and YKL-39 in normal tissues indicates other functions in addition to response to parasites and conditions in malignant tissue.
  • the autoimmune response to HCgp-39 proteins in patients suffering from osteoarthritis might be a result of an ancient defence reaction.
  • HCgp- 39 might aid the recognition of chitin containing parasites.
  • Our results show that ChOS exposure results in down regulation of the CHITl gene in monocytes.
  • PMA and ChOS exposure together results in the up regulation of the CHITl gene indicating that differentiation to M0 is prerequisite for monocytes to respond to chitin motifs by CLP up regulation. Based on these results, the protective role of CLPs against chitin containing pathogens cannot be ruled out.
  • the CLPs have binding sites for chitin motifs at the carboxyl termini and a chitinase reaction site at their amino termini, but due to alterations in the amino acid sequence in the reaction site they have reduced or nonexistent chitinase activity. It has been proposed that Chitotriosidase and HCgp-39 recognize hyaluronan as a substrate and interfere with its synthesis and local concentration. Furthermore, it has been suggested that even though HCgp-39 has lost its chitinase activity, the reaction site still retains its carbohydrate-binding ability where the binding is followed by conformational chance of the protein.
  • ChOS material consist of soluble chitin oligomers
  • they can simulate endogenous extracellular signal control of tissue regeneration through CLPs.
  • the influence on expression is perhaps more prominent explanation for the overall effects of ChOS on the activity of CLPs than the previously proposed function of ChOS as chitinase inhibitors.
  • Our results show that the ChOS affect the expression of each CLP differently, which might reflect their refined expressional control.
  • the findings could demonstrate how endogenous ChOS functions in the control of the CLP expression in vivo.
  • the results support the ChOS involvement in the pathogenesis of the diseases by altering the expression of the CLPs.
  • activated M0 have been shown to express CLPs in various conditions and our study revealed phenotypic changes in culture and expression differences of HCgp-39, Chitotriosidase, AMCase and YKL-39 during the activation of monocytes.
  • the considerable expression in normal lungs indicates consistent expression rather than triggering response to external factors.
  • the selective CLP expression in foetal brain couid imply a role during the development of the central nervous system but a more basic role of CLPs as response factors to chitin containing organism cannot be ruled out.
  • ChOS on CLP expression similar to endogenous factors in the modulation of extracellular matrix during tissue remodelling and tissue regeneration. This represents a new way of looking at short endogenous chitin structures as controlling elements of CLPs expression.
  • YKL-40 Involvement of YKL-40 in various diseases has been reported. Strong up regulation of the protein has been widely reported in rheumatoid arthritis (RA) and in osteoarthritis (OA), reviewed in. In rheumatoid arthritis the YKL-40 protein is known as an autoantigen. Therefore, down regulation of YKL-40 in RA patients should reduce attacking of the immune system on this antigen and thus result in a relief of RA symptoms. Successful attempts have been made to increase immunotolerance to YKL-40 by inoculation of the protein.
  • RA rheumatoid arthritis
  • OA osteoarthritis
  • Genis ehf has found indications of anti-inflammatory effects of CHOS in human rheumatoid arthritis patients.
  • the relief of pain occurs in 20 to 30 days after 5 administration of 2 grams of CHOS a day.
  • this example we describe the effect of CHOS on a rheumatoid arthritis patient
  • RA rheumatoid arthritis
  • HC gp-39 (YKL-40) concentration by enzyme immunoassay (ELISA) (Quidel, METRA TM YKL-40 ELISA kit, cat. no. 8020) 20 according to the manufacturer protocol.
  • ELISA enzyme immunoassay
  • a 4-5 g sample of spray-dried CO was analysed for water content by gravimetric analysis before and after incubating at 105 0 C for 3 hours. Ash content was determined by complete combustion at 800 0 C for 3 hours and calculated as percent weight of inorganic residue on a dry weight basis.
  • the average degree of polymerization (DP value) of the 0.50 % oligomer solution was measured by a sugar reducing end assay using 3,5-dinitrosalicylic acid (DNS) as a reagent and glucose as a standard. This method is originally described by Miller. A volume of 1.00 ml of chitosan oligomer solution (5.00 mg/ml, moisture and ash corrected in 0.5% acetic acid), was mixed with 2.00 ml of DNS reagent, boiled for 8 min, cooled and centrifuged at 2000 x G for 3 min.
  • DNS 3,5-dinitrosalicylic acid
  • the optical density of the supernatant was measured in a spectrophotometer at 540 nm and the average DP-value was calculated using the absorbance of 1.00 mg/ml (5.55 mM) glucose as a standard. Water (1.00 ml in 2.00 ml DNS solution) served as a blank at 540 nm. The average molecular weight used for DP calculation was 200 Da. Each sample was assayed in duplicate.
  • Sample Preparation Solutions of the G010920 CO [sample in H 2 O (1 ⁇ L) were placed onto the target I and mixed with 1 ⁇ L of a 5 % solution of THAP or DHB in MeOH. After drying at room temperature, the sample was re-dissolved in 1 ⁇ L of MeOH to yield a thin layer of very fine crystals when dried at room temperature.
  • Mass spectra were recorded with a Bruker Reflex II Instrument (Bruker Daltonik, Bremen, Germany).
  • the spray-dried CO sample was analysed for ash and water content.
  • the ash content (99.8% NaCI as judged by titration)] was 13.8 (w/w) and water 8.3% (w/w).
  • the degree of deacetylation (DDA) was 43.6% ⁇ 0.5% (SD).
  • Biogel P4 GPC followed by MALDI-TOF analysis (Table 6) showed the monomer (DP 1) being mainly /V-acetyl glucosamine (GIcNAc or A) with minor appearance of glucosamine (GIcN or D).
  • Dimers (DP 2) were a mixture Of A 2 (i.e.AA)and AD.
  • Trimers (DP 3) contained A 2 D as main product and A 3 as a minor product.
  • the sequence of the main trimer product was determined to be DAA. Longer COs (DP 4 to DP 20) were found in smaller quantity, as judged by the Biogel P4 analysis ( Figure 15 and Table 6). Existence of longer oligomers was confirmed by both Biogel P4 and MALDI-TOF MS analysis as indicated in Figure 15, 16 and 17 and Table 6. Table 7. MALDI-TOF MS of Biogel P4 GPC peaks shown in Figure 1. Each numbered peak was collected and analysed by MALDI-TOF MS. The table shows fraction number and homologues of each fraction. Product G010920
  • the enzyme was denatured by adjusting the pH to 5.4 and heating of the solution to 8O 0 C for 10 min. After cooling, the CO solution was poured through a sieve of 280 ⁇ m mesh size. The solution was desalted using DSS LabStak M20 nanofiltration unit with 0.72 cm 2 of 500 Da cut-off membranes at pH 4.8. The solution was then subjected to spray drying, using a rotary atomizing spray-drying unit at an inlet air temperature of 190 0 C and an outlet air temperature of 80 0 C. The fine white CO powder, 2.0 kg (80%) was collected and kept at room temperature.
  • the lyophilised amac-]oligosaccharide derivatives were redissolved in 200-500 ⁇ L of methanol/water (v/v 50:50). An aliquot of the solution (0.5 ⁇ L) was mixed on the target) with 2 ⁇ L of a solution of DHB as a matrix (15 mg x mL '1 ) in 30% aqueous ethanol, and the drop was dried under gentle stream of air. Crystallization of the matrix occurred usually spontaneously. In some cases, crystallization was observed only after diluting the original sample solution ca. 5- fold with methanol/water (v/v 50:50).
  • MALDI TOF mass spectra were recorded on a Bruker Reflex II (Bruker Daltonik, Bremen, Germany) in the positive ion mode.
  • a nitrogen laser (337 nm, 3 ns pulse width, 3 Hz) was used.
  • the laser was aimed either at the cental area of the sample or at the outmost edge of the crystal rim. All spectra were measured in the reflector mode using external calibration (Angiotensin II).
  • Ultrafiltration was done on a Helicon SS50, (PTGC, 10 kDa MWCO) spiral- wound ultrafiltration membrane (Millipore, USA) using tangential flow filtration in a Millipore PUF-200-FG pilot module.
  • the hydrolysate was passed through the membranes, retaining the enzyme and molecules of >10 kDa. The retentate was discarded and the filtrate kept.
  • Nanofiltration was used in order to desalt the filtrate, using a semi automated pilot unit Type R (GEA Filtration, Germany).
  • Thin-film membranes type DK4040F, (150-300 Da MWCO), supplied by Osmonics, Germany, were applied.
  • Sodium CNa + ) and chlorate (Cl " ) ions were filtrated and discarded.
  • the pH of the liquid was 3.4 until at the end of NF, pH was adjusted to pH 8 (NaOH).
  • the resulting salt free CO retentate (conductivity 0.45mS/cm), 80 kg was spray dried as described, resulting in 2.08 kg of dry CO referred to as G040823.
  • Table 8 shows the mass flow and yield of product.
  • Figure 20 shows the DP distribution and Figure 21 shows the homologue distribution, compared to the G010920 product.
  • the DP5-10 percent of the G040823 is higher than of G010920.
  • a uniform distribution of DPs was found. Mainly two homologues of each DP were detected with the exception of triads, for which one main homologue of (D2Al) n DlA2

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Abstract

La présente invention concerne une utilisation, une composition et un procédé pour le traitement des troubles ou maladies dégénératifs des tissus ou du tissu conjonctif, du système nerveux central, des tissus cardiovasculaires ou du système immunitaire. La composition de cette invention comprend des chito-oligomères visant à réduire l'expression anormalement élevée de protéines de type chitinase chez des patients souffrant de troubles ou de maladies associés au tissu conjonctif, au système nerveux central, aux tissus cardiovasculaires ou au système immunitaire.
PCT/IS2005/000024 2004-11-19 2005-11-21 Utilisation de formulations a base de chito-oligomeres pour modifier l'activite anormale des proteines de type chitinase Ceased WO2006054319A2 (fr)

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