WO2006054319A2

WO2006054319A2 - Use of chitooligomer formulations to modify abnormal activity of chitinase like proteins

Info

Publication number: WO2006054319A2
Application number: PCT/IS2005/000024
Authority: WO
Inventors: Olafur B. Einarsson; Jon M. Einarsson; Johannes Gislason; Finnbogi R. Thormodsson
Original assignee: Genis ehf
Current assignee: Genis ehf
Priority date: 2004-11-19
Filing date: 2005-11-21
Publication date: 2006-05-26
Anticipated expiration: 2007-05-19
Also published as: WO2006054319A3

Abstract

The present invention provides a use, a composition and a method for treatment of degenerative disorders or diseases in tissues or such as connective tissue, central nervous system, cardiovascular tissue or the immune system. The composition of the present invention comprises chito-oligomers for reduction of abnormally high expression of chitinase like proteins in patients suffering disorders or diseases associated with connective tissue, central nervous system, cardiovascular tissue or the immune system.

Description

USE OF CHITOOLIGOMER FORMULATIONS TO MODIFY ABNORMAL ACTIVITY OF CHITINASE LIKE PROTEINS

Background of the invention N-acetyl glucosamine and glucosamine

Glucosamine (GN or GIcN) is a modified glucose with NH₂ replacing the OH group on the carbon two in the sugar molecule. In animal cells, 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. In the next step, 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. These three amino sugars are important in glycosylation of proteins as well as building blocks for glycolipids, glycosaminoglycans (GAG), hyaluronan and 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

Hyaluronic acid (HA) 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. As the major constituent of the extracellular matrix, 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]. 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. In humans, there are at least seven types of hyaluronidase-like enzymes, several of which have been shown to possess tumor suppressor activity. 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

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 β-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.

Oligosaccharides of chitin and partially deacetylated chitin (CHOS) have been shown to possess biological activity in various experimental models and trials, both in animals and plants, demonstrating the importance of degree of deacetylation and polymerization [5], [6]. The biochemical mechanisms and processes behind these bioactive properties have not yet been revealed and in current literature, the discussions appear to be speculative.

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]. A demonstration of similar activity of eukaryotic HA synthases in vivo, would suggest novel functions for CHOS in vertebrates [2]. Yet it has been shown that 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]. Chitinase-like proteins (CLP) and their expression by mammalian genomes

Most chitinases are classified as belonging to the group of carbohydrate cleaving enzymes called Familylδ 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. During evolution, some of the family 18 chitinases genes have evolved into coding for new proteins serving new functions, both in plants and animals. These proteins are frequently classified as 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. Therefore we now refer to 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]. Also, 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]. This indicates that chitooligosaccharides (CHOS) with DP 4 or more are necessary to cause conformal changes in the protein resulting in a signal. However, 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.

Many authors have been able to demonstrate that the 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. These authors describe the crystal structure of the human chitotriosidase both free of ligand and bound with a chitooligosaccharide substrate and allosamidin inhibitor. The crystal structure of YKL-39, AMCase and Oviductin has not yet been published.

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. However, 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].

In embryonic development, YKL-40 seems to play an important role in endochondral bone formation and cartilage formation [27, 34]. In human osteophytic tissue, 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], Dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes, from human bone marrow show high expression of YKL-40 [34], Moreover, in primary culture of mouse costochondral chondrocytes, the master chondrogenic factor sox9 is markedly up regulated by YKL-40 [35]. Chondrocytes produce hyaluronan mainly by hyaluronan synthase-2 (HAS-2) and HAS-3 but not by HAS-I [36, 37], The possibility of chitin oligomer production in chondrogenesis has to be investigated.

Inflammation 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.

If inflammation of the affected site persists, released 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.

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.

However, in most tissues, 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.

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]. 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

10% fetal bovine serum FBS and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly high compared with the control culture group, indicating an increased osteoblast activity and bone formation [53]

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

Involvement of CLPs in human diseases

It appears that tissue specific expression of chitinase like proteins is associated with a wide range of diseases, especially diseases involving inflammation and degeneration or damaging of body tissues. The involvement of these increased expression levels of CLPs is increasingly being studied. Substantial amount of scientific data is already available in rheumatoid- and osteoarthritis and some other highly prevalent diseases in modern societies.

Rheumatoid- and osteoarthritis

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]. In osteoarthritis, only the YKL-39, not YKL- 40 seems to be up regulated in human chondrocytes which indicates a specific difference in expression of these closely related proteins [70, 73]. Injection of YKL-39 has shown to induce arthritis in mice [79] and this protein has also proved to be an autoantigen in rheumatoid arthritis along with YKL-40 [75, 78].

Asthma

ECF-L is expressed during inflammation as well as in allergy [80-84]. Recently, 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

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]. Recently, 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

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]. This was confirmed by [102] where YKL- 40 was only expressed in peritumoral macrophages but not in cancer cells in human small cell lung cancer. Interestingly, chitosan and especially chitooligosaccharides inhibited tumor-induced angiogenesis (blood vessel formation) in Ehriich ascites tumor in mice [108]. Also, [50, 51] was able to demonstrate involvement of YKL-40 in angiogenesis which indicates a role in endochondral bone formation.

Hepatic fibrosis Expression of YKL-40 is increased in hepatic fibrosis due to alcoholism [41, 45, 49]

Other diseases associated with elevated expression of CLPs

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 β-amyloid (AB) and interleukin-lβ (IL-lβ) is responsible for the pathology of Alzheimer's disease (AD). ϊfSiijsjLCj^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].

Increased expression of 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] . In Gaucher disease, enzyme- replacement therapy in humans resulted in a significant decrease in chitotriosidase activity [124] . Summary and conclusion

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). However, 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.

Summary of the invention

It is an object of the present invention to provide a use, a composition and a method for modulation of inflammation and/or treating prophylaxis and/or prevention of connective tissue diseases or degenerative disorders by modulating the regulation and or Expression of one or more C/CLP using chitooligomers formulations. 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.

Description of the invention

In a first aspect of the present invention a use of chitooligomers formulations for the manufacture of a medicament is provided 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.

In the present context 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. In reference to CLPs, 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.

In an embodiment of the present invention 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.

In the present context the term "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.

The term chito-oligomer formulations as used herein 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). As described herein and in the examples set forth, the composition is particularly useful for use as a medicament.

The term 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 Λ/-acetyl glucosamine and glucosamine, respectively.)

Preferably 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 degree of deacetylation DDA can also be expressed as the acetylation factor, F_A, where, e.g. DDA of 30% corresponds to F_A = 0.7.

The shorter chito-oligomers are postulated to be highly important for the activity of the composition of the invention. In a useful embodiment 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. In a certain embodiment 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. In certain embodiments 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. Such 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. In useful embodiments 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%. Depending on the manufacturing process, the composition may contain significant amounts of salt other than the salts of chito-oligomers, e.g. NaCI or KCI, but preferably the content of such extra salts is kept to a minimum. Depending on the process and conditions applied during the hydrolysis of the raw material polymer, some amount of 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, however, indicate that a certain amount of monomers, in particular NAG, present in compositions of the invention may have a positive synergistic effect.

The composition may further comprise a pharmaceutically acceptable excipient or diluent, a flavoring substance, a nutrient, or a colorant.

In an embodiment of the present invention, chitosan constitutes a portion of the medicament.

In an embodiment of the present invention, the mammal is a human and the C/CLP's of the present invention are also of human origin.

As mentioned, enzymatic hydrolysis of the chitin/chitosan is preferred to obtain the chito-oligomers, however the use of suitable mineral acids for depolymerization (e.g. hydrochloric acid or nitrous acid) is also encompassed by the current invention. Several 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 (enzyme/substrate ratio, temperature, pH, reaction time) 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).

In a second aspect of the present invention a method is provided 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. In the present context 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.

Relating to the use and the method of the present invention, 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. Further 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. . Further, said medical devices are represented by metallic or plastic joint implants including finger, knee, TMJ, stabilizing plates or other articulating joints. Furthermore, 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.

Further applications of the CO may be conducted via catheter for use for example in vertibralplasty or chronic delivery of insulin. Further application of catheter application may employ short term indwelling catheters for delivery of CO for short interval periods, typically used in conjunction with micropumps.

Yet further applications of the 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. Such forms include dry powder, granular, flake, fibrous and paste forms. However, the composition can also be contained in pills or capsules.

In an embodiment of the present invention a method is provided for administration of a biologically effective amount of a chitooligomer formulation to a human, that binds one or more C/CLP selected from the group consisting of: a) HC gp-39/YKL-40; b) YKL-39; c) ECF-L; d) Chitotriosidase; e) AMCase; and f) Oviductin, are of human origin.

Chitinase like proteins (C/CLP'S) 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 characterised as members of C/CLP's. 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.

In an embodiment of the present invention a method is provided 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

In an embodiment of the present invention a method is provided 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.

In an embodiment of the present invention a method is provided 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.

In an embodiment of the present invention a method is provided 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.

In relation to the use and the method of the present invention the term "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.

In an embodiment of the present invention a pharmaceutical composition is provided. The pharmaceutical composition comprises 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.

In an embodiment of the present invention a kit is provided 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.

In another embodiment of the present invention a method is provided 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.

In another embodiment of the present invention involvesadministration of a therapeutically acceptable level of chitooligomer formulations for the modulation of expression or activity of one or more C/CLP's

In an embodiment of the present invention 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. References

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Hepatology, 1999. 31(4): p. 741-746.

117. Beutler, E., et al., The Clinical Course of Treated and Untreated Gaucher- Disease - a Study of 45 Patients. Blood Cells Molecules and Diseases, 1995. 21(2): p. 86-108. 118. Giraldo, P., et al., Chitotriosidase genotype and plasma activity in patients with type 1 Gaucher's disease and their relatives (carriers and non- carriers). Haematologica, 2001. 86(9): p. 977-984.

119. Hollak, C. E. M., et al., Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. Journal of Clinical Investigation, 1994. 93(3): p. 1288-1292.

120. Wajner, A., et al., Biochemical characterization of chitotriosidase enzyme: comparison between normal individuals and patients with Gaucher and with Niemann-Pick diseases. Clinical Biochemistry, 2004. 37(10) : p. 893- 897. 121. Grosso, S., et al., Serum levels of chitotriosidase as a marker of disease activity and clinical stage in sarcoidosis. Scand J Clin Lab Invest., 2004. 64(1): p. 57-62. 122. Boot, R.G., et al., Marked elevation of the chemokine CCL18/PARC in Gaucher disease: a novel surrogate marker for assessing therapeutic intervention. Blood, 2004. 103(1) : p. 33-39.

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2005. 99(4): p. 396-402.

124. Cenarro, A,, et al., Plasma lipoprotein responses to enzyme-replacement in Gaucher's disease. The Lancet, 1999. 353(9153): p. 642-643.

Detailed description of the invention The present invention will now be discussed in further detail in the following figures and examples..

Figure 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.

Figure 2. The effect of CO (G010920) on human chondrocyte cell growth. Chondrocytes were grown in 16 wells for 7 days, then 500 μg/ml CHOS (G010920) were applied to 8 wells but only buffer (0 μg/ml CHOS) applied to the other 8 wells. The cells grown further for 7 days before analysis.

Figure 3. The effect of CO on fibroblast growth. Fibroblast density (no. of cells per field) as a function of time in days.|

Figure 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 μg/ml.

Figure 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 6. The Effects of Chitooligosaccarides in culture. The effects of different concentrations of CO on the pH level of the culture media after 1 hour (lower values) and 24 hours (higher values) (A), the optimal CO consentration (500 μg/ml) was used to measure the effects on cell growth (B) and migration of cells towards CO in 4 hours (C).

Figure 7. Time dependent effects of chitooligosaccharide treatment on the chitinase-like gene expression in Jurkat cells. The expression of the genes in untreated Jurkat cells (0) and in response to ChOS (G020418) (500 μg/ml) treatment for 1-6 hours. PCR amplified products of cDNA produced from mRNA isolated from Jurkat cell extract. Editium bromide stained 1% agarose gel, run for 30 min. /80 volts.

Figure 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 μg/ml CO (C), cells treated with 10-8 M PMA and 100 μg/ml CO (D). The cells clustered around calcofluor white particles of CO (E). Magnification, XlOO A-D and 630X E.

Figure 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).

Figure 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 μg/ml chitooligosaccaride (C). V=LadderV. EtdBr stained 1% agarose gel, 30 min./ 80 volt.

Figure 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 μg/ml chitooligosaccaride (C), determined by RT-PCR and normalized to GAPDH expression.

Figure 12. Semi-quantitative HCgp-39 gene expression determined with RT-PCR. By untreated (U) THP-I cells, in response to 10-8 M -PMA and 100 μg/ml chitooligosaccharide (C) treatment for 24 Hours, (*P<0.05, two-sided for C/PMA versus PMA treatment alone, and one-sided for C versus untreated. +/- SE, n=3, normalized to GAPDH expression.

Figure 13. The HCgp-39 secretion measured by EIA in the media of untreated (U) THP-I cells, in response to 10-8 M PMA and chitooligosaccaride (C) treatment for 4 days, (*P<0.05, two-sided for C/PMA versus PMA treatment alone). +/- SE, n= 9.

Figure 14. The effect of CO (G040823) on the HCgp-39 expression in monocytes (THP-I cells) as judged by ELISA.

Figure 15. Biogel P4 GPC analysis of production G010920. See Table 9 for MALDI-TOF analysis of numbered peaks.

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).

Figure 18. MALDI TOF MS of the crude CO mixture. Product G020418.

Figure 19. GPC (Biogel P4) analysis of CO after filtration (3000 Da cut-off). Product G020418

Figure 20. DP distributiton (%) of G040823 compared to G010920 as jugded by MALDI-TOF on crude CO product.

Figure 21. Homologue distribution (%) of G040823 compared to G010920 as jugded by MALDI-TOF on crude CO product.

Figure 22. Development of RA symptoms during a period where CHOS oral administration was stopped (day 0) and restarted (day 29). Based on data in Table 9.

Example 1. Biological evaluation of a Chitooligomer formulation General introduction

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. However, in our preclinical studies on rat and sheep models we have been able to demonstrate chondro- and osteogeneses in injured bone tissue treated with chitopolysaccharides, resulting in bone tissue regeneration through endochondral ossification.

In this document we present preliminary results demonstrating a growth promoting effect of CO (lot number G010920) on isolated human chondrocyte primary cultures.

Growth promoting effect of chitooligosaccharides on chondrocytes in culture

Introduction Chitooligosaccharides (CO) 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). 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.

Material and methods

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 μg/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.

Results

Number of cells per well (cell density) was increased in proportion to an increase in CO concentration, (50 - 500 μg/ml) with high degree of statistical significance as seen in Figure 1 and Table 1. At 1000 μg/ml there was a profound reduction in cell density (Figure 1). These were freshly isolated chondrocytes at first passing. It was apparent that when the experiment was repeated using more mature chondrocytes (third passing) no CHOS growth promoting effect was found. When the experiment was repeated with freshly isolated chondrocytes at first passing, a significant growth promoting effect (p<0.001) was again evident (Figure 2 and Table 2). This time the chondrocytes were grown in 16 wells for 7 days, then 500 μg/ml CHOS (G010920) were applied to 8 wells but only buffer (0 μg/ml CHOS) applied to the other 8 wells. The cells grown further for 7 days, then fixed, stained and the density estimated

Table 1. Chondrocyte density of culture on microplate after 5 weeks. Cell density is indicated as no. of cells per well. Mean and SD (n=8) are

Table 2. T-test of chondrocyte density of culture data shown in Figure 2.

t-test

Dependent Variable: cell density

Normality Test: Passed (P = 0,165) Equal Variance Test: Passed (P = 0,813)

Group Name N Missing Mean Std Dev SEM

CHOS 7 days after seeding 16 0 425,625 85 ,994 21,499

Control 16 1 283,667 71 ,006 18,334

Difference 141,958 t = 4,993 with 29 degrees of freedom. (P = <0,001)

95 percent confidence interval for difference of means: 83,806 to 200,111

The difference in the mean values of the two groups is greater than would be expected by chance; there is a statistically significant difference between the input groups (P = <0,001).

Power of performed test with alpha = 0,050: 0,999 Conclusions

These results show clearly that 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. However, there are indications that 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

Material and methods

In this experiment Genis CO, G040823 was used.

Plating fibroblast on gelatin coated culture disks, with or without CO.

PLATE PREPARATION:

Solution 1: Gelatin (0,1%) was solubilized in Hanks balanced salt solution (HBSS) by heating at 37 ⁰C for five minutes. Solution 2: Same as solution 1 with lOOOμg/ml chitooligosaccharides (CO) added. Both solutions were filter sterilized.

Culture plates (9 cm²; Nunc, Denmark) were coated by placing 0.5 ml of either solution in each plate, incubate for few sec, before pouring the excess solution out. The plates were stored with HBSS at 4 ⁰C.

FIBROBLAST PLATING AND COUNTING

Human fibroblasts from a patients were grown to confluence in a T25 culture flask, and then replated on the gelatin coated disks, at the concentration IxIO⁵ cells/ml, in RPMI 1640 medium supplemented with 10% fetal bovine serum and allowed to grow at 37⁰C in a moist, 5% CO₂ atmosphere. The number of cells was determined by counting the cells seen under the 1Ox objective of a light microscope. Eight plates were used for each experimental condition (n=8). RESULTS

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..

EXAMPLE 3 - CHITOOLIGOSACCHARIDES AS BLOCKERS FOR CHITINASE-A ACTIVITY; A MODEL FOR BIOACTIVITY AND BIOSTABILITY

Material and methods Chitooligosaccharides from Genis (lot No. G040823) were analysed for structure and sequence by MALDI-TOF mass spectrometry.

A purified chitinase A preparation from S. marcescens was a generous gift from Norway.

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 μM 4-MU-A3 in 50 mM phosphate buffer pH 7.4. Different concentrations of CO (0, 62.5, 125, 250, 500 and 1000 μg/ml) were made in the substrate buffer. For the assay, 25 μl of the Chit-A solution was mixed with 25 μl of the substrate, incubated at 37°C for 10 min. The reaction was stopped with 1.95 ml 0.2 M sodium bicarbonate buffer (Na₂CO₃). The formation of the product, 4-Methylumbelliferone (4-MU) was read for each reaction in a Perken-Elmer LS 5OB Fluorometer. The excitation wavelength was 380 nm (5 nm adjusting slit) and the emission wavelength was 460 nm (4 nm adjusting slit). Each reaction was read in triplicate. To estimate blocking, 50% inhibitory concentration (IC₅₀) was calculated for the COs, using non-linear fit equation, f=yθ+a*exp(-b*x), where x equals specific activity of the chitinase and f equals CO concentration (μg/ml). Affinity of COs was calculated as inverted IC₅₀. The formula used to determine affinity was (1/IC₅₀)*1000.

Results and discussion

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 IC₅₀ was calculated using non-linear fit equation, f=yθ+a*exp(-b*x), where x equals specific activity of the chitiπase and f equals CO concentration (μg/ml). The results are presented in Table 3. The IC₅₀ mean value is 79.3 μg/ml ± 0,45 μg/mi SD for the CO mixture tested (Table 3).The affinity of the CO to Chitinase A was determined using the formula (1/IC₅₀)*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 μg/ml, three product 4-MU formation

10 analysis of each sample, its mean and standard deviation (SD), calculated 4-MU in μM and nmoles and finally calculated mean and standard deviation (SD) of Chitinase A specific activity.

4-MU 4-MU Sp. Act. Sp. Act. O Cone. jl-MU 1 4-MU 2 4-MU 3 4-MU 1-3 4-MU 1-3

[μM] [nmol] Chit A Chit A

(μg/ml) 1 2 3 mean SD mean mean mean SD

1000 41.2 41.3 41.2 41.2 0.06 0.84 0.0418 95.3 0.13

. 500 50.3 49.6 50.3 50.1 0.40 1.05 0.0527 • 120.2 0.97

250 74.2 74.7 74.4 74.4 0.25 1.66 0.0828 188.7 0.64

125 105.6 106.3 106.5 106.1 0.47 2.44 0.1220 277.9 1.24

62.5 153.2 152.8 153.2 153.1 0.23 3.60 0.1799 410.0 0.62

0 273.6 274.6 276.1 274.8 1.26 6.60 0.3302 752.5 3.45

15 EXAMPLE 4 - CHITOOLIGOSACCHARIDES AFFECTS THE EXPRESSION OF CHITINASE-LIKE GENES IN JURKAT CELLS

Material and Methods

The jurkat Cell line (T-lymphoblastoid cells, ATCC) was maintained in RPMI-1640 medium supplemented with 5% FBS, 100 U penicillin /ml, 100 μg of

20 streptomycin/ml, incubated at 37 ⁰C and 5% CO₂. Cells were treated with 500 μg/ml of soluble ChOS (G020418) from Genis. The composition of ChOS comprises oligomers with mean molecular mass is 1.133 Da with an average length of n = 5,7 (degree of polymerization; DP). Migration of cells was evaluated in a Multiscreen chamber (Millipore). The upper and lower wells were separated

25 by polycarbonate membrane with 3 μm pores through which the cells migrate The tissue expression was observed in comercial cDNA libraries (Clontech). RNA isolation (Tri-reagent™) and cDNA construction (RevertAid™) was conducted according to manufactures instructions, RT-PCR was conducted with specific primer pairs to account for cross-hybridisation with closely related sequences,

30 splice variants and genomic DNA. Results

A general Chitinase-like gene expression was observed in the lung tissue, YKL-39 and HCgp-39 are expressed in the central nervous system as well (Figure 5). ChOS (500 μg/ml) did not affect the growth of Jurkat cells (Figure 6B)₇ but the results indicated significant (*p<0.05) migration by Jurkat cells towards ChOS (Figure 6C). Faint expression was observed in the Jurkat cell line for TSA1902- L/AMCase and chitotriosidase but substantial expression was observed for YKL-39 (Figure 7, O hours). This endogenous expression is altered in time dependant manner as a result of ChOS treatment (Figure 7); following 1-hour treatment, the expression is not detected for TSA1902/AMCase and following 4 hours for chitotriosidase. The expression diminished after 1 hour for YKL-39 but it is subsequently restored in 4 hours.

Discussion

Consistent with previous studies the 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. This preliminary data suggest that the chitinase-like gene expression can be down regulated in T-cells by 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

Materials and Methods

Cell line culture The THP-I Cell line (ATCC) was maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U pen./ml, 100 μg of strept./ml and incubated at 37⁰C with 5% CO₂. Soluble chitooligosaccharides (Genis ehf. Reykjavik, Iceland) at concentration of 100 μg/ml were added to some of the cells. The cells were then treated with 10^'8M Phorbol 12-myristate 13-acetate (PMA) for 4 days before harvesting. PMA was obtained from Sigma (P8139) dissolved in ethanol and stored at -20⁰C. A 50μl aliquot was taken out for cell counting and viability tests. They were mixed 1: 1 with 0.4% (w/v) trypan blue in PBS and after 2 min. at room temperature, the ratio of dead and living cells to evaluate counted in a haemocytometer. Viability (V) was evaluated by using the formula; V= (total number of cells - number of dead cells (stained))/(total number of cells + number of life cells (not stained)) xlOO.

Production of Chitooligosaccharides and cell attachment imaging Chitooligosaccharides were produced from clean high quality chitin made from shrimp cells. Briefly, chitin is partially deacetylated to render water-soluble chitosan. The chitosan polymers were then hydrolysed to oligomers with a fungal chitinase and ultra filtrated through a 1OkDa membrane. The oligomers were nanofiltrated (cut-off 500 Da) and spray-dried. Mean molecular mass of ChOS oligomers is 1.133 Da with an average length of n = 5,7 (degree of polymerisation; DP). To visualize the binding of cells to ChOS the powder was adjusted to insoluble chitosan particles. 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).

Extraction of nucleotides and Reverse Transcriptase Polymerase Chain Reaction Cells were introduced to ChOS at different times as indicated before RNA was isolated. Cells were washed with Hanks balanced salt solution (Gibco) before harvesting. Total RNA was prepared using standard Triagent assay (Molecular research center, Cincinnati, OH) as instructed by the manufacturer. RNA yield was determined by standard spectrophotometric assay (The mean ratio of

A260/A280 was 1.80). Total RNA (5 μg) from cells was reverse transcribed using revertAid™ First strand cDNA Synthesis kit (#1621 Fermentas), priming method used to generate them was by oligo(dT)₁s primers. For the 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/30μl 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 μl 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. No AB025009 (short), AB025008(Iong): forward, CCAGTCTGGTGGTGAATCCT reverse, CTGTG ACAGTTGGGG GATCT (short nt 79-249 and long nt 79-438), for Chitotriosidase (gene CHITl, Genbank Ace. No. NM003465) : forward, TGAATCCCAAGCTGAAGACC, and reverse, ACCTCGTATCCAG CATCCAC (nt 276- 590), for AMCase Genbank Ace. No AF290004, forward, CTTCATGCCTGACAACATCG, and reverse, TGCACCAGGACAGTGAAGAG (nt 222- 582), for Glyceraldehyde-3-phosphate dehydrogenase (gene GAPDH, Genbank Ace. No. BC013310) forward, CCACCCATGGCAAATTCCA and reverse TCTAGACGGCAGGTCAGGTCCACC (nt 272-851). Taq DNA polymerase (EP0072, Fermentas) was used for the PCR conducted in a conventional reaction. Briefly, denaturing at 95⁰C for 10 min, then 35-40 x ( denaturing at 95⁰C for 45 sec, annealing at 55-6O⁰C for 45 sec, extension at 72⁰C for 45 sec). The PCR mix contained conventional amount of ingredients; 1Ox buffer (IX), MgCL₂ 1-4 mM, dNTP 200 μM, forward and reverse primers 1,0 μM, 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. The products of the PCR procedure were visualized on a 1% agarose gel containing 1 μg/ml ethidium bromide in TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA). 2.4.4 Enzyme immunoassay and gene expression quantification

THP-I cell media was extracted from cells at times indicated and centrifuged. An enzyme immunoassay (ELISA) was conducted according to manufacture instructions (Quidel, METRA TM YKL-40 ELISA kit, cat. no. 8020). Briefly, 20 μl 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).

Results The phenotype changes of monocytes following activation

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 (100 μg/ml), 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). The effects are seen for cells treated with ChOS alone and with PMA as well (Figure ID). PMA treatment (ICT⁸ M) resulted in mixed sizes of cells indicating heterogeneous cell phenotypes (Figure 8B). Untreated cells range from 10 to 15 μm in diameter but larger cells in the PMA treated culture are 30 to 40 μm in diameter and attached to the vial base. The two to three fold enlargements in diameter of cells (Figure 8 B, white arrows) and surface attachment of cells indicates differentiated M0.

The expression of Chitotriosidase, HCqp-39, TSA1902 /AMCase and YKL-39 mRNA in tissues.

A strict tissue specific gene expression of CLPs was observed in normal human tissues (Figure 9). We analysed the expression of mRNA isolated from Aorta, whole brain, foetal brain, colon, lung, and testis for all the CLPs except Oviductin. All the CLP genes analysed were expressed in lung tissue. YKL-39 and HCgp-39 are expressed in the central nervous tissue as well. There are at least two known splice variants for TSA1902 gene transcripts; TSA1902-L (long) and TSA1902-S (short) and consistent with previously reported expression, both the expression of long and short isoforms of TSA1902 were observed in lungs but no signal was detected in any of the other tissues. Sequential alignment reveals that 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.

Table4. Human chitinase-like proteins (CLP), their tissue expression and alignment of the amino acid sequence responsible for the catalytic activity. The proteins are divided in two groups, based on amino acid differences (bold) in the catalytic site in compare to Chitotriosidase and their observed tissue specific expression. Foetal brain (FB), whole brain (WB). * The expression for Oviductin was not examined.

The expression changes of imRNA for Chitotriosidase, HCqp-39, TSA1902 /AMCase and YKL-39 in the THP-I cell line in response to chitooliqosaccarides and PMA treatment.

An endogenous gene expression was observed in the THP-I cell line for YKL-39, Chitotriosidase, AMCase and HCgp-39 (Figure 10). Total RNA was isolated after 24 hours treatment of ChOS and PMA before the RT-PCR analysis. PMA is known as an ample cell activator and the cells respond in different patterns of expression in response to ChOS and PMA. Interestingly, 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. These data prompted us to conclude that 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. We therefore tested the THP-I cells, which were negative for the duplication (results not shown).

Increased Chitotriosidase expression is believed to be linked to the differentiation of monocytes to M0. There was a consistent elevated Chitotriosidase expression as a result of PMA treatment, but both ChOS alone and synergetic ChOS / PMA treatment resulted in decreased Chitotriosidase expression (Fig 11 and 12). The decreased expression of AMCase as a result of PMA and ChOS treatment, stood out as the most dramatic response of all the CLPs.

Three separate batches of monocytes were cultured and some treated with PMA and/or ChOS for 24 hours and then total RNA was isolated for the RT-PCR analysis. The bands on the gels were quantified by the ImageQuant 5.1 software. The repeated RT-PCR analysis revealed significant (P<0.05) down regulation of HCgp-39 gene expression (Figure 12). The expression values are presented in arbitrary units indicating that the expression is down regulated by 53 % compared to the baseline level as a result of ChOS treatment in the monocytes. The opposite effects were seen as a result of combined PMA / ChOS treatment where the expression is significantly (P<0.05) upregulated. Altered HCqp-39 secretion by the THP-I cells in response to chitooliαosaccarides and PMA treatment. The CLPs have an endoplasmic reticulum signal peptide on the NH₂ termini indicating active secretion. Our results suggest that THP-I cells actively secrete HCgp-39 and that the secretion is affected by separate ChOS and PMA treatment by down regulation of gene expression. To further determine the effects of ChOS and PMA treatment on HCgp-39 secretion, nine (Figure 13) and four (Figure 147) individual batches of monocytes were cultured for 4 days and the media extracted from cells and analysed by ELISA. The ELISA analysis revealed significant down regulation (p<0.01) of HCgp-39 protein secretion for cells treated with ChOS (Figure 14 and Table 5). The opposite significant (P<0.05) enhancement of HCgp- 39 secretion was observed by the cells treated with both ChOS and PMA (Figure 13).

Table 5. Normality and t-test on data shown in Figure 14. t-test Wednesday, December 01, 2004, 14: 10: 14

Normality Test: Passed (P > 0.200)

Equal Variance Test: Passed (P = 0,572)

Group Name N Missing Mean Std Dev SEM Control 4 0 22.3501.113 0.557

CO 4 0 16.6881.828 0.914

Difference 5.662 t = 5.291 with 6 degrees of freedom. (P = 0.002) 95 percent confidence interval for difference of means: 3.044 to 8.281

The difference in the mean values of the two groups is greater than would be expected by chance; there is a statistically significant difference between the input groups (P = 0.002). Power of performed test with alpha = 0.050: 0.992

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). These results confirm that HCgp-39 is differentially expressed by monocytes and PMA activated monocytes in response to ChOS exposure indicating different expression response by monocytes and M0.

Discussion

The expression of 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. In this study we reveal the endogenous expression of CLPs in THP-I cells and demonstrate how the expression changes with activation of monocytes and ChOS treatment. 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 Mø.

Our first objective was to map the expression for the CLPs in an array of normal tissues. Consistent with previous studies the CLP genes are generally expressed in lungs. This extensive expression in normal lungs could indicate continuous expression rather than triggered immune response as a result of exposure to environmental factors. The observation could be an indication of more general role in various processes rather than unique response as a result of single condition. HCgp-39 and YKL-39 are the only CLPs which expression was detected in tissues other than lungs. HCgp-39 gene expression in whole brain underlines the need of further studies of its function in the central nervous system. Interestingly, HCgp-39 and YKL-39 have not been reported with chitinase activity like AMCase and Chitotriosidase indicating their functional distinction. Based on these two facts, we propose a categorisation of the human CLPs in two groups: those that have more diverse expression without chitinase activity and those with limited expression distribution but reported chitinase activity (see Table 4). Based on expression as well as functional indications the CLPs in humans might have similar functions as their CLP counterparts in other species. In Drosophila the

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. Thus, 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. In fact chitin has been reported to bind to mannose receptors leading to receptor mediated endocytosis of ChOS by Møs. 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.

Separate kinetics of mRNA expression was observed for different CLPs following activation of the THP-I cells. PMA or ChOS treatment alone decreased HCgp-39 expression, whereas PMA and ChOS together increased the HCgp-39 expression indicating monocyte/Mø differentiation. Enhanced HCgp-39 expression has been reported to indicate late stages of M0 differentiation. The different kinetics in CLP expression may illustrate the subsequent phenotypic changes taking place during maturation of in vitro cultured M0. Our data prompted us to conclude that ChOS could be acting as a co-activator with PMA in monocyte/Mø activation. These data does not enable us to predict the pattern of expression for each of the CLPs in different disease conditions, as the lifespan of M0 in tissues far exceeded the 4 days, the latest time tested in this in vitro experiment.

To the best of our knowledge, 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. The observed expression pattern of 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. In this way 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. However 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.

We can only be speculate regarding the expressional control of HCgp-39 in vivo. The precise mechanism causing the alteration of HCgp-39 and Chitotriosidase expression during different conditions is unknown. The composition of glycan structures of damaged or regenerated tissues could be acting in a similar manner as ChOS in vivo. These results could explain how endogenous ChOS affect the HCgp-39 expression in joint diseases. However, the exact physiological role for the CLPs in the various diseases is still unclear. 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. The fact that our ChOS material consist of soluble chitin oligomers, could mean that 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.

In summary, 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. We propose an involvement of 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.

Example 6 - Effect of CHOS on an RA patient

Introduction

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.

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. The inflammatory conditions and the pain reappeared gradually 3 to 8 weeks after discontinue of CHOS administration. In this example we describe the effect of CHOS on a rheumatoid arthritis patient

Material and methods

A human individual diagnosed with rheumatoid arthritis (RA) and receiving oral 10 administration of CHOS for an extensive period of time was monitored during cessation of the oral CHOS administration over a period of 29 days and for an additional period of 77 days after the oral CHOS administration was commenced. The subject was visited at specific time intervals, blood samples were drawn and the subject was asked to describe her condition verbally and rate the RA 15 sumptoms on a scale from 0-10.

Enzyme immunoassay

Blood samples were collected at different time during the period of the experiment and analyzed for 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.

Table 6. Results

The results show a marked relapse in the patients condition after cessation of the oral CHOS administration, but at day 29, the oral CHOS administration was commenced again and a dramatic improvement of the patient's symptoms were observed (Tab. 9 and Fig. 22)

Example 7 - Production of lot G010920

Methods 25 kg of Sodium hydroxide (does source make a difference?), was dissolved in 25 kg of water in a 80 L blender and heated to 70°C. 2.5 kg of Shrimp chitin (Genis ehf), was added and stirred (15 rpm) at 7O⁰C for 20 min. The slurry was then cooled with water, filtered through a cheese- cloth bag (200 x 40 cm) and washed for 10-15 minutes in running cold tap water (6°C). The chitin gel was transferred back into the blender, the pH was adjusted to 4 by addition of 30% HCl, and water was added to give a final volume of 80 L. 380 g (750 U/g) of Chitinase solution (Genis ehf, in house), was added and the gel was stirred for 16 hrs at 30⁰C. The enzyme was denatured by adjusting the pH to 7 and heating of the solution to 70⁰C for 10 minutes. After cooling, the oligomer solution was poured through a sieve of 280 μm mesh size. The solution was subjected to spray-drying, using a rotary atomizing spray-drying unit at an inlet air temperature of 190⁰C and an outlet air temperature of 8O⁰C. The atomizer rotor speed was 20,000 rpm. 2 kg of fine white CO powder was collected and kept at room temperature

Analysis of product.

Determination of water and ash content

A 4-5 g sample of spray-dried CO was analysed for water content by gravimetric analysis before and after incubating at 105⁰C for 3 hours. Ash content was determined by complete combustion at 800⁰C for 3 hours and calculated as percent weight of inorganic residue on a dry weight basis.

Determination of degree of deacetylation (PDA) by direct titration

CO (500 mg, moisture and ash corrected) was mixed with 125 ml 0.060 N HCI in a sealed Erlenmeyer flask and dissolved overnight at 22°C in a rotary shaker (150 rpm). Subsequently, 125 ml distilled water was added and the solution was shaken further for at least 15 min. 50.0 g of the solution were transferred to a beaker and titrated with 0.500 N NaOH solution, using a flow of 1.00 ml/min (HPLC pump). The pH was monitored between pH 1.8 to 9, and the DDA was calculated based on the volume of the NaOH consumed between the inflexion points of the titration curve, from pH 3.75 to pH 8.0, using the equation DDA = VoI (ml) NaOH * 16116 * 0.0500/100 mg chitosan. Each sample was titrated in triplicate.

DNS assay for determining the average degree of polymerisation (DP)

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. 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.

BioGel P4 Gel Permeation Chromatography analysis (GPC)

Two serial columns (Pharmacia), with Biogel P4, fine grade (BioRad, Munich, Germany), using 0.05 M ammonium acetate buffer adjusted with 0.23 M acetic acid to pH 4.2 as mobile phase. The flow rate was 27.7 ml/hr. Detection was done with Shimadzu RID 6A refractive index detector. Fractions were collected, appropriately combined, lyophilized prior to MALDI-TOF MS analysis.

MALDI-TOF Mass spectrometry analysis

Sample Preparation: Solutions of the G010920 CO [sample in H₂O (1 μL) were placed onto the target I and mixed with 1 μL of a 5 % solution of THAP or DHB in MeOH. After drying at room temperature, the sample was re-dissolved in 1 μL 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).

Results

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 A₂ (i.e.AA)and AD. Trimers (DP 3) contained A₂D as main product and A₃ 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

Production of lot G020418; a CO test lot. Material and Methods

Production

25 kg of Sodium hydroxide was dissolved in 25 kg of water in an 80 L blender and heated to 6O⁰C. 2.5 kg of Shrimp chitin from P. borealis (Genis ehf.)| was added and stirred (15 rpm) for 40 min. The slurry was then cooled with water and washed in a cheesecloth bag (200 x 40 cm) for 10-15 minutes. The chitin gel was transferred into a 200 L blender, the pH was adjusted to 4.0 by addition of 30% HCI, and water was added to give a volume of 100 L. 380 g (750 U/g) Chitinase enzyme solution was added and the gel was stirred for 22 hrs at 3O⁰C. The enzyme was denatured by adjusting the pH to 5.4 and heating of the solution to 8O⁰C for 10 min. After cooling, the CO solution was poured through a sieve of 280 μm mesh size. The solution was desalted using DSS LabStak M20 nanofiltration unit with 0.72 cm² 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 190⁰C and an outlet air temperature of 80⁰C. The fine white CO powder, 2.0 kg (80%) was collected and kept at room temperature.

Product analysis

The product was analysed by Genis quality standard procedures. See G020418 CoA for details.]

BioGel P4 Gel Permeation Chromatography analysis (GPC)

2.16 g of the CO powder was dissolved in 180 mL of 0.05 ammonium acetate buffer at pH 4.2. The resulting solution was filtered sequentially through a 0.8 μm, and a 0.2 μm cellulose acetate membrane (Schleicher & Schuell), and ultrafiltrated through a 3000 Da cut-off membrane (Amicon). The filtrate was lyophilised. The yield was 0.74 g (34%). The resulting powder (lots of 350 mg) were separated by gel permeation chromatography (GPC) on Biogel P4, fine grade (BioRad, MϋnichJ Germany). Column dimension: 5 x 200 cm; mobile phase 0.05 M ammonium acetate buffer, adjusted with 0.23 M acetic acid to pH 4.2; flow rate 60 mL/hour; Refractive index detector Shimadzu RID 6A. Fractions of 20 ml were collected, appropriately combined, concentrated to a small volume and finally lyophilized.

Mass Spectrometry

The lyophilised amac-]oligosaccharide derivatives were redissolved in 200-500 μL of methanol/water (v/v 50:50). An aliquot of the solution (0.5 μL) was mixed on the target) with 2 μL 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. For ionization, a nitrogen laser (337 nm, 3 ns pulse width, 3 Hz) was used. For optimization of the mass spectra, 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).

Results and Discussion

Product analysis

CO were prepared from water soluble| chitin (Lot. No. G020418, Genis ehf, Iceland, DDA 37% or F_A 0.63), see Genis CoA G020418 for further results.| Figure 18 shows the MALDI TOF MS of the CO mixture and Figure 19 shows the GPC chromatogram. Each fraction (Fl - FlO) was analyzed by MALDI TOF MS (MALDI TOF mass spectrum of GPC fraction F7, see below, others not shown). Table 7 shows the DP of each CO and homologues of each fraction as well as the mass distribution of fractions Fl - FlO.

Table 9, Composition and mass distribution of CO fractions Fl - FlO. GPC separation on Biogel P4. The yield is calculated from masses of the dialysed and dried fractions. Product G020418.

4. Production of lot G040323

Methods

2.5 kg of pure chitin flakes (source??) were deacetylated in 50 kg of 50% NaOH in water at 6O⁰C for 40 minutes, in an 80 L reactor (rpm 25). The slurry was then washed in running cold water (6°C) for 10-12 minutes in double bag cheesecloth, submerged a 350 Lj container of cold water. The deacetylated chitin was transferred to a 200 L blender and the pH adjusted to 3.8. Enzyme hydrolysis: 380 g (750 U/g) of Chitinase solution, was added and the reaction was allowed to continue for 22 h at 25°C (mixing speed of 50 rpm).

Ultrafiltration (UF) 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 (IMF) 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.

Table 9. The mass flow and yield of product G040823.

Results The spray-dried CO powder was analysed according to Genis quality lab standard procedure, see Genis CoA G040823 for further results]. Ash content (NaCI) is only 0.4 % [of the dry matter. The average DP is also lower than desired since all DP were retained during nanofiltration|.

Figure 20 shows the DP distribution and Figure 21 shows the homologue distribution, compared to the G010920 product. As seen in Figure 20, 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)_nDlA2 | found.

Claims

I. The 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.

2. The use according to claim 1, wherein the modulation involves a reduction of abnormally high activity and/or expression of the one or more C/CLP in a mammal.

3. The use according to claim 1, wherein the modulation involves an increase of abnormally low activity and/or expression of the one or more C/CLP in a mammal. ,

4. The use according to claims 1-3, wherein 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.

5. The use according to claims 1-4, wherein the modulation of the regulation of one or more C/CLP is for tissue repair or tissue regeneration.

6. The use according to claims 1-5, wherein the medicament is for an oral administration in a mammal.

7. The use according to claims 1-5, wherein the medicament is for a surgical administration to a tissue site in a mammal.

8. The use according to any of claims 1-7, wherein at least 50% of the chitooligomers have a chain length in the range of about 2-50, and wherein the degree of deacetylation of the chitooligomers is in the range of about 0-70%.

9. The use according to any of claims 1-8, wherein the degree of deacetylation of chitooligomers is in the range of about 30-50%.

10. The use according to any of claims 1-9, wherein at least about 60% of the chitooligomers have a chain length in the range of about 2-50.

II. The use according to any of claims 1-10, wherein at least about 75% of the chitooligomers have a chain length in the range of about 2-50.|

12. The use according to any of claims 1-11, wherein the effective amount of chitooligomers constitute in the range of 20-100% by weight of said chitooligomer formulation.

13. The use according to any of claims 1-12, wherein at least about 10 wt% of the chitooligomers have a chain length of 2 to 12.

14. The use according to any of claims 1-13, wherein about 15 to 75 wt% of the oligomers have a chain length of 2 to 12.

15. The use according to any of claims 1-14, wherein chitosan constitutes a portion of the medicament.

16. The use according to any of claims 1-15, wherein said chitooligomer formulation binds one or more C/CLP selected from the group consisting of: | a) HC gp-39/YKL-40; b) YKL-39; c) ECF-L; d) Chitotriosidase; e) AMCase; and f) Oviductin

17. The use according to any of claims 1-16, wherein the mammal is a human.

18. The use according to any of claims 1-17, wherein said C/CLP is of human origin.

19. The use according to any of claims 4-18, wherein the connective tissue diseases or degenerative disorders are selected from the group consisting of: a^') Paget's disease; b) osteoporosis; c) Gorham-Stout syndrome; d) arthritic diseases; e) osteoarthritis; f) rheumatoid arthritis; g) psoriatic arthritis; h) rheumatoid disease; and i) brittle bone disease j) osteoarthritis: k) atherosclerosis;

I) psoriasis; m) scleroderma; n) Gaucher disease; and o) beta-thalassemia.

20. 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 comprising the step of administering to the cell an effective amount of chitooligomer formulation.

21. The method of claim 20, wherein the cell is within a mammalian subject.

22. The method according to claims 20-21, wherein the animal subject has an abnormal activity and/or expression chitinase-like proteins.

23. The method according to claim 20, wherein the modulation involves a reduction of abnormally high activity and/or expression of the one or more C/CLP in a mammal.

24. The method according to claim 20, wherein the modulation involves an increase of abnormally low activity and/or expression of the one or more C/CLP in a mammal.

25. The method according to claims 20-24, wherein the modulation of the regulation of one or more C/CLP is for tissue repair or tissue regeneration.

26. The method according to claims 20-25, wherein the medicament is for an oral administration in a mammal.

27. The method according to claims 20-25, wherein the medicament is for a surgical administration to a tissue site in a mammal.

28. The method according to claims 20-27, comprising administering to a human a biologically effective amount of a chitooligomer formulation that binds one or more C/CLP selected from the group consisting of: a) HC gρ-39/YKL-40; b) YKL-39; c) ECF-L; d) Chitotriosidase; e) AMCase; and f) Oviductin

29. The method according to claims 20-28, wherein said C/CLP is of human origin.

30. The method according to claims 20-29, wherein 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

31. The method according to claims 20-29, wherein 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.

32. The method according to claims 20-29, wherein 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.

33. The method according to claims 20-29, wherein 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.

34. The method of claims 20-29, wherein said connective tissue disease or disorder is selected from the group consisting of: a) Paget's disease; b) osteoporosis; c) Gorham-Stout syndrome; d) arthritic diseases; e) osteoarthritis; f) rheumatoid arthritis; g) psoriatic arthritis; h) rheumatoid disease; and i) brittle bone disease j) osteoarthritis: k) atherosclerosis; I) psoriasis; m) scleroderma; n) Gaucher disease; and o) beta-thalassemia.

35, A pharmaceutical composition comprising a chitinase like protein modulating 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.

36. A kit 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, the kit provided sterile and at least comprising: an effective amount of a chitooligomer formulation, an applicator, and an instructional material for the use thereof.

37. The kit of claim 37 in combined use with other anti-inflammatory and pain relieving drugs selected from the following drug classes: a) analgesics; 2) nonsteroidal anti-inflammatory drugs (NSAIDs), including the COX-2 inhibitors;

3) glucocorticoids, which are more commonly know as steroids;

4) disease-modifying anti-rheumatic drugs (DMARDs), and;

5) new pipeline drugs including biological agents that target selective blocking of certain immune system functions.