WO2023105086A1

WO2023105086A1 - Use of glycosylated sugar alcohols

Info

Publication number: WO2023105086A1
Application number: PCT/EP2022/085394
Authority: WO
Inventors: Rama Krishna GUDIMINCHI
Original assignee: Gudiminchi Rama Krishna
Priority date: 2021-12-10
Filing date: 2022-12-12
Publication date: 2023-06-15

Abstract

The present invention provides sugar based small molecules that are capable of modulating protein-protein interactions. The sugar based small molecules may be used in the treatment of diseases which may preferably be associated with protein-protein interactions, specifically with plasmin-inhibitor interactions

Description

USE OF GLYCOSYLATED SUGAR ALCOHOLS

Description

Field of the Invention

[0001] The present invention relates to the field of (poly)glycosylated sugar alcohols capable of suppressing the plasmin inhibition by its inhibitor. The said (poly)glycosylated sugar alcohols are useful in the treatment of various disorders and diseases mediated by plasmin inhibition. The use of said (poly)glycosylated sugar alcohol as active ingredient in a pharmaceutical formulation for the prevention and/or treatment of disease.

Background Art

[0002] Protein-protein interactions (PPIs) are at the center of virtually all life processes. On the cellular level, PPIs mediate cellular signal transduction, regulate transcription and translation, and fulfil transport functions within and outside of cells. Therefore, modulation of PPIs in diseases with the intention to disturb a given PPI and inhibit the aberrant signaling pathway could result in potential new forms of therapy. Thus, pharmacological targeting of PPIs is of high importance. Researchers have predominantly focused on small molecules because of their ease of administration and good bioavailability. Serine protease-serine protease inhibitor (SERPIN) interaction is one such PPI. Plasmin is a major fibrinolytic serine protease enzyme generated by converting its zymogen plasminogen by fibrinolytic activators in the presence or absence of fibrin. Plasmin also regulates other physiological and pathological events like immunomodulation and inflammation in addition to fibrinolysis. Plasmin activity in the blood is regulated by either plasmin generation or by plasmin inhibition. Plasmin inhibition is caused by SERPINs. Extrinsic inhibition of plasmin may lead to increased pathology caused by reduced proteolytic digestion of pathogens, attenuated inflammation and defense mechanism, and inhibited fibrinolysis. Disruption of the protein-protein interaction between plasmin and its inhibitor could be a most promising approach for preventing and/or treating diseases which are associated with plasmin activity. [0003] Spectroscopic in vitro activity assays developed based on different chromophore, fluorophore or chemiluminescence substrates to estimate the activities of a protease enzyme, preferably plasmin, trypsin, thrombin etc., in a given sample are available that can be used to screen for inhibitors of the desired enzyme (Miszta et al., Int. J. Mol. Sci. (2021), 22:22758). Quantitative measurement of plasmin activity and kinetics of its inhibition is possible through simple in vitro assays that have been recently developed. The assessment of plasmin activity allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. [0004] Despite advances in the underlying understanding of the biology of proteinprotein interactions and its contribution to disease, there is an ongoing need for a new, effective way of disrupting the protein-protein interactions which may be the cause of various diseases. Thus, a molecule that is able to disrupt the proteinprotein interaction could be promising in providing a prophylactic and therapeutic effect in the treatment of diseases and their diagnosis.

[0005] Sugar alcohols (also called glycitols, polyols, polyhydric alcohols, polyalcohols, or alditols) are organic compounds, typically derived from sugars, containing one hydroxyl group attached to each carbon atom, e.g., sorbitol, xylitol, glycerol, arabitol, inositol, maltitol, and lactitol. The number of carbon atoms may vary between 2 and 24. Majorly sugar alcohols find applications in the food industry as a replacement for conventional sugars due to their relatively lower sweetness and lower energy content.

[0006] Attachment of glucose or other sugar to a molecule (glycosylation) with a glycosidic bond is a common practice by nature. As a result, glycosylated products often display unique physical, chemical and biological properties when compared to its aglycone part. However, the unique features of a glycosylated molecule are greatly determined by the site- and anomeric configuration (a/p) of the glycosidic bond. Glycosylated sugar alcohol 2-O-a-D-glucopyranosyl-sn-glycerol is a natural osmolyte found in bacteria and plants. Whereas l-O-palmitoyl-2-O-oleoyl-3-O-( a - D-glucopyranosyl)-glycerol and l-O-myristoyl-2-O-oleoyl-3-O-( a -D- glucopyranosyl)-glycerol isolated from algae have notable biological effects, including significant fibrinolysis, or inhibition of tumors (Wu et al. Mar. Drugs (2009), 7:85-94). The regio- and stereo-specific glycosylation of a sugar alcohol is of particular interest to add structural and functional diversity.

[0007] US8288353B2 relates to the chemical production of (poly)glucopyranosyl- xylitol (xylitylglucoside). (Poly)glucopyranosyl-xylitol is chemically produced and sold under the tradename Aquaxyl® as a mixture of xylitylglucoside (-50% max), anhydroxylitol, and xylitol. The xylitylglucoside is not fully characterized yet. However, since the product is produced via a chemical method it is assumed that the xylitylglucoside product more likely is an anomeric mixture of 1/5-O-D- glucopyranosyl-xylitol (1/5GX).

[0008] Aquaxyl® is used in the cosmetic industry due to its capability to reinforce the synthesis of essential lipids and proteins involved in the organization of the stratum corneum. Moreover, the synthesis of dermal macromolecules, such as hyaluronic acid and chondroitin sulfate that can “trap" water, also increased in fibroblast culture (Leite e Silva et al. (2009) J. Cosmet. Dermatol. 8:32-39).

[0009] Since the commercially available (poly)gl ucopyra nosyl-xy lito I product is not pure due to the incapacity of chemical processes to easily produce the single isomer, attempts were already made to produce a single isomer of well-defined glycosylated sugar alcohols by employing enzymatic methods. Enzymatic production of (poly)gl ucopyra nosyl-xylitol is more attractive over the chemical approach due to the simple reaction steps (no protection and deprotection steps), high regio- and stereo-selectivity, and low production cost. Several enzymes derived naturally can perform such transfer reactions.

[0010] JP2004222507A relates to the production of different oligosaccharide sugar alcohols having one molecule of glucose which is p-glycoside-bonded to the sugar alcohol at 2^nd position resulting in a 2/4- -D-glucopyranosyl-xylitol. The method comprises the use of cellobiose phosphorylase. W09007002A provides a method for producing a-glycoside bonded sugar alcohols from glucose and sugar alcohols in the presence of alpha-glucosidase. 2/4-O-a-D-glucopyranosyl-xylitol (2/4aGx) is produced by sucrose phosphorylase (Kitao and Sekine, Biosci. Biotech. Biochem. (1992) 56:2011-2014). Site selective synthesis of a- or p-anomers of (poly)glucopyranosyl-xylitol is already known from the literature. However, low-cost production with higher yields and no by-products is still a challenge for commercial production using enzymes.

Summary of invention

[0011] It is the object of the present invention to provide a glycosylated sugar alcohol molecule capable of disrupting the protein-protein interaction of plasmin and its inhibitor without affecting the plasmin’s activity.

[0012] The object is solved by the subject-matter of the present invention.

[0013] The present invention relates to novel types of sugar based small molecules that are useful in the treatment of various disorders and diseases which may be caused by plasmin inhibition. The invention therefore relates to the use of glycosylated sugar alcohol molecule in the prevention and/or treatment of diseases.

[0014] Specifically, the present invention relates to (poly)glucopyranosyl sugar alcohol molecule for use in the prevention and/or treatment of a disease which is associated with inhibited plasmin activity.

[0015] The inhibited plasmin activity may be caused by a plasmin-inhibitor interaction.

[0016] According to one embodiment of the invention, the (poly)glucopyranosyl sugar alcohol molecule disrupts said plasmin-inhibitor interaction and subsequent formation of plasmin-inhibitor complex, and restore the plasmin activity.

[0017] The (poly)glucopyranosyl sugar alcohol molecule according to the invention is sugar alcohol molecule comprising at least one sugar alcohol moiety and at least one glucopyranose (glucose) moiety which may be linked through a glycosidic linkage. The sugar alcohol moiety may be selected from the group consisting of sorbitol, xylitol, glycerol, arabitol, inositol, maltitol, and lactitol. In one embodiment the sugar alcohol moiety is xylitol.

[0018] The sugar alcohol moiety and the glucopyranose moiety maybe linked through an a- or a p-glycosidic linkage, preferably a p-glycosidic linkage.

[0019] The glycosidic linkage between glucopyranose moiety and sugar alcohol moiety may be a (1— ”1) or (1— ”2) or (1— ”3) or (1— ”4) or (1— ”5) or (1— ”6) linkage, preferably a (1— ”2) or (1— ”4).

[0020] According to one embodiment of the invention the (poly)glucopyranosyl sugar alcohol molecule is a (poly)glucopyranosyl-xylitol. The (poly)glucopyranosyl sugar alcohol molecule may be 1-O-a-D-glucopyranosyl-xylitol, 1-O-p-D- glucopyra nosy I -xylitol, 2-O-a-D-glucopyra nosy I -xylitol, 2-O-p-D-glucopyranosyl- xylitol, 3-O-a-D-glucopyranosyl-xylitol, 3-O-p-D-glucopyranosyl-xylitol, 4-0-a-D- glucopyra nosy I -xylitol, 4-O-p-D-glucopyranosyl-xylitol, 5-O-a-D-glucopyranosyl- xylitol, or 5-O-p-D-glucopyranosyl-xylitol.

[0021] The plasmin inhibitor may form a reversible or irreversible complex with plasmin. The plasmin inhibitor may be a serine protease inhibitor selected from the group consisting of a₂ antiplasmin, aprotinin, miropin, crmA, plasminostrepsin and soybean trypsin inhibitor.

[0022] According to one embodiment of the invention the (poly)glucopyranosyl sugar alcohol molecule as described herein disrupts the plasmin-inhibitor interaction and subsequent formation of plasmin-inhibitor complex and thereby restore the activity of plasmin.

[0023] The disease associated with plasmin-inhibitor interaction is selected from the group consisting of fibrinolysis, immunomodulation, autoimmune disease, cancer, infectious disease, neurodegenerative disease, gum disease and skin disorders.

[0024] One embodiment of the invention relates to a pharmaceutical composition comprising a (poly)glucopyranosyl sugar alcohol molecule as the active ingredient. [0025] The pharmaceutical composition comprising as described herein comprises a 1-O-a-D-glucopyranosyl-xylitol, 1-O-p-D-glucopyranosyl-xylitol, 2-0-a-D- glucopyra nosy I -xylitol, 2-O-p-D-glucopyra nosy I -xylitol, 3-O-a-D-glucopyranosyl- xylitol, 3-O-p-D-glucopyranosyl-xylitol, 4-O-a-D-glucopyranosyl-xylitol, 4-O-p-D- glucopyranosyl-xylitol, 5-O-a-D-glucopyranosyl-xylitol, or 5-O-p-D-glucopyranosyl- xylitol, or a derivative thereof, as active ingredient.

[0026] According to one embodiment of the invention, the pharmaceutical composition comprises conventional excipients and/or carriers.

[0027] The pharmaceutical composition may be formulated in the form of creams, oils, lotions, gels, sprays, pastes, rinses, drops, chewing gums, lozenges, band aids, tablets, capsules, and syrups.

[0028] According to the present invention there is provided a method for regio- and stereo-selective synthesis of well-defined and isomerically pure glycosylated sugar alcohol molecules.

[0029] According to the present invention there is provided an in vitro screening method for determining if a test agent disrupts formation of plasmin-inhibitor complex.

[0030] According to the present invention there is identified a sugar based small molecule which is capable of modulating plasmin-inhibitor interactions with the help of the provided in vitro screening method.

[0031] According to the present invention the use of the identified glycosylated sugar alcohol molecule in disease prevention.

[0032] According to the present invention the use of the identified glycosylated sugar alcohol molecule in diagnostic tests.

[0033] According to the present invention the use of the identified glycosylated sugar alcohol molecule as active ingredient in formulations. Brief description of drawings

[0034] Fig. 1 depicts chemical and biochemical routes for the synthesis of different (poly) gluco pyra nosy I -xylitol products.

[0035] Fig. 2 Time course of amidolysis of S-2251 in the presence of plasmin, plasmin + aprotinin, and plasmin + aprotinin + 2/4pGX (2-O-p-D-glucopyranosyl- xylitol or 4-O-p-D-glucopyranosyl-xylitol).

[0036] Fig. 3 shows a relative comparison of the spectroscopic properties of different assay mixtures for determining the suppression of the inhibitory activity of an inhibitor (aprotinin) of plasmin by different (poly)gl uco pyra nosyl-xy litol molecules.

[0037] Fig. 4 shows the concentration dependent suppression of the inhibitory activity of an inhibitor of plasmin preferably by a (po ly)gl ucopy ra nosy l-xylitol (2/4pGX). Suppression of the inhibitory activity of (A) aprotinin (B) soybean trypsin inhibitor (STI) on plasmin activity.

[0038] Fig. 5 is a schematic representation of the hypothetical mechanism of the disruption of the interactions between plasmin and its inhibitor by a (poly) gluco pyra nosy I -xylitol (2/4 GX).

Detailed description of the Invention

[0039] The present invention provides (poly)glycosylated sugar alcohol molecules that are capable of disrupting protein-protein interaction such as a serine protease-inhibitor complexes. The (poly)glycosylated sugar alcohol molecules are thus capable to suppress the serine protease inhibition by a corresponding inhibitor and consequently release functionally active protease. Due to their capability to disrupt protein-protein interactions, the (poly)glycosylated sugar alcohol molecules may be used in the treatment and/or prevention of various disorders and diseases which are caused by an inhibited serine-protease, such as plasmin. Diseases which may be caused by inhibited serine-proteases are for example, infectious, inflammatory, or immunomodulatory diseases, and fibrinolysis. The glycosylated sugar alcohol molecule may thus be used as an active ingredient in pharmaceutical and/or cosmetic preparations.

[0040] “Inhibitor" as used herein refers to a molecule that binds to a protein, e.g., to an enzyme and hinders its activity.

[0041] The term “serine protease inhibitor complex" as used herein refers to an inhibitor which is interconnected (reversible or irreversible) to a serine protease. Due to the complex formed between the enzyme and the inhibitor, the activity of the enzyme is inhibited. According to the invention, the (poly)glycosylated sugar alcohol molecules are capable to disrupt a complex formed by plasmin, a serine protease, and a corresponding inhibitor to restore the activity of plasmin.

[0042] The term “serine protease inhibitor interaction" as used herein refers to an inhibitor which interacts with a serine protease. Due to the interactions between the enzyme and the inhibitor, the activity of the enzyme is impaired. According to the invention, the (poly)glycosylated sugar alcohol molecules are capable to interfere with the interaction of plasmin, a serine protease, and a corresponding inhibitor in order to restore the impaired activity of plasmin.

[0043] The terms “(poly)glycosylated sugar alcohol" and “(poly)glucopyranosyl sugar alcohol" as used herein are interchangeable.

[0044] According to present invention the (poly)glycosylated sugar alcohol molecule consists of one or more glucosyl residue(s) and one or more sugar alcohol residue(s).

[0045] The glycosyl moiety refers to one glucosyl residue or to a chain of glucosyl residues.

[0046] The sugar alcohol moiety is preferably selected from the group consisting of sorbitol, xylitol, glycerol, arabitol, inositol, maltitol, and lactitol, more preferably a xylitol. Sugar alcohol moiety may comprise one or more of said sugar alcohols.

[0047] The glycosyl moiety and the sugar alcohol moiety may be linked through a site-selective glycosidic linkage in a specific anomeric configuration (a or p). The glycosidic linkage between glycosyl moiety and sugar alcohol moiety may be a (a 1 ►

[0048] According to the invention, the sugar based small molecule may be (poly)glucopyranosyl-xylitol, specifically 1-O-a-D-glucopyranosyl-xylitol, or 1-O-p- D-glucopyranosyl-xylitol, or 2-O-a-D-glucopyranosyl-xylitol, or 2-O-p-D- glucopyranosyl-xylitol, or 3-O-a-D-glucopyranosyl-xylitol, or 3-O-p-D- glucopyranosyl-xylitol, or 4-O-a-D-glucopyranosyl-xylitol, or 4-O-p-D- glucopyranosyl-xylitol, or 5-O-a-D-glucopyranosyl-xylitol, or 5-O-p-D- glucopyranosyl-xylitol. The sugar based small molecule may be 2-O-a-D- glucopyranosyl-xylitol, or 2-O-p-D-glucopyranosyl-xylitol, or 4-0-a-D- glucopyranosyl-xylitol, or 4-O-p-D-glucopyranosyl-xylitol. According to one embodiment of the invention, the sugar based small molecule is 2-O- -D- glucopyranosyl-xylitol or 4-O-p-D-glucopyranosyl-xylitol.

[0049] A further embodiment of the invention relates to a derivative of the sugar based small molecule. The derivative may be an acyl derivative or a fatty acid or lipid derivative.

[0050] Xylitol, a five-carbon achiral sugar alcohol, is one of the most common sugar alcohols occurring naturally in fruits and vegetables, and is widely accepted as safe for human consumption. In addition to its relatively lower energy value and equal sweetness, its metabolism in human body is independent of insulin, making xylitol as excellent artificial sweetener for diabetics. Moreover, xylitol inhibits infections of the middle ear and teeth. However, the actual mechanism is not clearly known.

[0051] Glycosylation of xylitol theoretically yields six different isomers of (poly)gl ucopy ra nosy l-xy litol molecules depending on the position (regioselectivity) 01, or 02 or 03 on xylitol and a- or p- configuration (stereo-selectivity) of the glycosidic linkage between glucose and xylitol namely, 1/5-0-a-D- glucopyranosyl-xylitol or 1/5-O-p-D-glucopyranosyl-xylitol or 2/4-O-a-D- glucopyranosyl-xylitol or 2/4-O-p-D-glucopyranosyl-xylitol or 3-0-a-D- glucopyranosyl-xylitol or 3-O-p-D-glucopyranosyl-xylitol.

[0052] Suitable synthetic routes to generate (poly)glucopyranosyl-xylitol are well known in the art, e.g., see US8288353B2.

[0053] Introduction of a glycosidic bond between xylitol and glucose with high regio- and stereo-selectivity demands excellent control over the glucosyltransfer reaction by the catalyst. Enzymes have been proven to be the best in dealing with such reactions with high precision over chemical catalysts (see JP2004222507A). [0054] Different isomers of (poly)gl ucopyra nosyl-xylitol molecule may be synthesized in a suitable reaction medium by allowing xylitol as a glycosyl acceptor to react with an appropriate glycosyl donor under suitable pH and temperature conditions in the presence of a chemical or a biochemical catalyst that can introduce the said glycosidic linkage between acceptor and donor selectively. The glycosyl donor is preferably a cheap and renewable raw material selected from the group consisting of, but not limited to, starch, dextrin, maltodextrin, maltose, cellulose, cellobiose, sucrose, glucose-l-phosphate, etc. The biochemical catalyst is a synthetic or natural molecule composed of one or more short or long chains of amino acids which acts as a catalyst to bring about a specific biochemical reaction. Suitable biochemical catalysts preferably selected from, but not limited to, transglycosylating enzymes such as glucosidases, 1,4-a-glucanotransferases, cyclodextrin glucanotransferases, transglucosyl amylases, sucrose phosphorylases, cellobiose phosphorylases, cellodextrin phosphorylases, laminaribiose phosphorylases, maltose phosphorylases, p-l-2-glucanphosphorylases, sophorooligoglucan phosphorylases, glucansucrases etc. The biochemical catalysts may be used in pure form, crude extract form, immobilized form or whole-cells. The biochemical catalysts may be produced in the lab or procured from the market. Thus, produced isomers of (poly)gl ucopyra nosyl-xylito I molecules are preferably isolated in a pure form from the reaction mixtures after completion of the reaction. [0055] Following isomers of (poly)glucopyranosyl-xylitol, namely 2/4-O-a-D- glucopyranosyl-xylitol, 2/4-O-p-D-glucopyranosyl-xylitol and 3-0-a-D- glucopyranosyl-xylitol, have been synthesized enzymatically and isolated in pure form.

[0056] Well-known serine proteases are plasmin, trypsin, chymotrypsin, TMPRSS2, kallikrein, urokinase, elastase, collagenase, thrombin, cathepsin, subtilisin, etc. According to the present invention, the serine protease plasmin is of specific interest.

[0057] The serine protease enzyme plasmin may be complexed with a suitable inhibitor. The inhibitor may be a synthetic or natural molecule composed of one or more short or long chains of amino acids that can regulate the biochemical reaction of the said serine protease by inhibition of its activity. Said inhibitor is a serine protease inhibitor (SERPIN). The serine protease inhibitor may be selected from the group consisting of pepstatin, lima bean trypsin inhibitor, aprotinin, ovomucoid, soyabean trypsin inhibitor, miropin, plasminostrepsin, a₂-antiplasmin, tranexamic acid, and caproic acid. According to the present invention, a₂-antiplasmin, aprotinin, miropin, plasminostrepsin or soyabean trypsin inhibitor as potent inhibitor of the serine protease enzyme plasmin are of specific interest.

[0058] Plasmin activity may be inhibited by a complex formed between plasmin and a corresponding inhibitor. The impaired plasmin activity may be the cause for various diseases. These diseases may be treated by administering a compound which is capable to disrupt the plasmin-inhibitor complex and to restore plasmin activity. Surprisingly, it was found by the inventor, that (poly)glycosylated sugar alcohol molecules are capable to disrupt plasmin-inhibitor interactions and to restore plasmin activity.

[0059] Thus, (poly)glycosylated sugar alcohol compounds may be used in the prevention and/or treatment of diseases which are caused by impaired plasmin activity. According to the invention, 1-O-a-D-glucopyranosyl-xylitol, 1-O- -D- glucopyra nosy I -xylitol, 2-O-a-D-glucopyranosyl-xylitol, 2-O-p-D-glucopyranosyl- xylitol, 3-O-a-D-glucopyranosyl-xylitol, 3-O-p-D-glucopyranosyl-xylitol, 4-0-a-D- glucopyra nosy I -xylitol, 4-O-p-D-glucopyranosyl-xylitol, 5-O-a-D-glucopyranosyl- xylitol, and 5-O-p-D-glucopyranosyl-xylitol, are for use in the prevention and/or treatment of diseases caused by impaired plasmin activity. One embodiment of the invention relates to 2-O-a-D-glucopyranosyl-xylitol, 2-O-p-D-glucopyranosyl- xylitol, 4-O-a-D-glucopyranosyl-xylitol, or 4-O-p-D-glucopyranosyl-xylitol for use in the prevention and/or treatment of diseases caused by impaired plasmin activity. [0060] The (poly)glycosylated sugar alcohol compound may be used as active pharmaceutical ingredient. The active ingredient may be selected from the group consisting of 1-O-a-D-glucopyranosyl-xylitol, 1-O-p-D-glucopyranosyl-xylitol, 2-O- a-D-glucopyranosyl-xylitol, 2-O-p-D-glucopyra nosy I -xylitol, 3-O-a-D- glucopyranosyl-xylitol, 3-O-p-D-glucopyranosyl-xylitol, 4-O-a-D-glucopyranosyl- xylitol, 4-O-p-D-glucopyranosyl-xylitol, 5-O-a-D-glucopyranosyl-xylitol, and 5-O-p- D-glucopyranosyl-xylitol, preferably a 2-O-a-D-glucopyranosyl-xylitol, a 2-O-p-D- glucopyranosyl-xylitol, 4-O-a-D-glucopyranosyl-xylitol, or a 4-O-p-D- glucopyranosyl-xylitol, of specific interest.

[0061] The modulation of a serine protease-inhibitor interaction is intended to regulate, adjust or adapt said serine protease-inhibitor interaction for a beneficial effect. The said modulation is preferably a suppression or disruption of the serine protease-inhibitor interaction. Serine protease-inhibitor interactions between, e.g., but not limited to, plasmin-a₂ antiplasmin, plasmin-aprotinin, plasmin-miropin, plasmin-soyabean trypsin inhibitor, trypsin-soyabean trypsin inhibitor, trypsinovomucoid, TMPRSS2-aprotinin, etc.

[0062] According to one embodiment of the present invention the (poly)glycosylated sugar alcohol molecule that can disrupt serine protease-inhibitor interaction is 2-O-p-D-glucopyranosyl-xylitol or a 4-O-p-D-glucopyranosyl-xylitol, and wherein the serine protease is plasmin. [0063] Among the different compounds tested, namely 2-0-a-D-glucopyranosyl- xylitol, 2-0-p-D-glucopyranosyl-xylitol, 3-O-a-D-glucopyranosyl-xylitol, 4-O-a-D- glucopyranosyl-xylitol, 4-O-p-D-glucopyranosyl-xylitol, the (poly)glycosylated sugar alcohols that are connected with p 1— >2 or p 1— >4 glycosidic linkage showed a significant interference with serine protease-inhibitor complex resulting in disruption of serine protease-inhibitor interactions releasing active serine protease. [0064] According to one embodiment of the present invention the (poly)glycosylated sugar alcohol molecule specifically disrupts the plasmin- aprotinin, and plasmin-soyabean trypsin inhibitor complexes whereupon the plasmin activity is restored.

[0065] The results suggest that the (poly)glycosylated sugar alcohol molecule successfully disrupts the plasmin-inhibitor complexes.

[0066] The (poly)glycosylated sugar alcohol molecules are serine protease specific and not inhibitor specific.

[0067] Specifically, according to the present invention the (poly)glycosylated sugar alcohol molecule disrupts the serine protease complex and releases the active serine protease. This suggest that the (poly)glycosylated sugar alcohol molecule interferes with binding sites on the serine protease and not with the catalytic site. [0068] The sugar based small molecules according to the invention may be used in the treatment of diseases wherein modulation of serine protease-inhibitor interaction is crucial. According to one embodiment of the invention the serine protease is plasmin which may be associated with an inhibitor thereby forming a serine protease inhibitor complex. Diseases which are associated with a serine protease-inhibitor-interaction may be selected from the group consisting of, but not limited to, fibrinolysis, immunomodulation, autoimmune disease, cancer, infectious disease, neurodegenerative disease, gum disease and skin disorders, and the like.

[0069] Inhibition of plasmin is often named as cause for diseases related to fibrinolysis, infections, immunomodulation, neurodegenerative. Inhibition of plasmin may lead to increased pathology caused by reduced proteolytic digestion of pathogens, attenuated inflammation and defense mechanism, inhibited fibrinolysis. Inhibited fibrinolysis leads to the blood loss. [0070] Fibrin deposition due to the inhibition of plasmin may lead to neurovascular damages which may be the cause for neurovascular diseases such as Alzheimer’s disease.

[0071] For example, Tannerella forsythia is a bacterial pathogen which may cause gum disease. The bacterial pathogen secretes miropin, a serine protease inhibitor in order to protect cell envelope proteins against degradation by proteolytic enzymes. This inhibition process helps the pathogen to invade and survive in the host.

[0072] CrmA, a further serine protease inhibitor, is secreted by cowpox virus. CrmA inhibits the plasmin activity and increases the infectivity by suppressing the host’s inflammatory response.

[0073] Plasminostrepsin, another serine protease inhibitor is produced by Streptomyces antifibrino/yticus. Plasminostrepsin is also a potent plasmin inhibitor. [0074] Ticks are obligate hematophagous arthropods and act as vectors for a great variety of pathogens, including viruses, bacteria, protozoa, and helminths. Ticks become infected with these microorganisms by feeding on infective hosts (human or animal) and, after a cycle of biological development, later inject the microorganisms into the new host during their subsequent blood meal. Ticks secrete serine protease inhibitors that inhibit the plasmin activity to attenuate the inflammation on biting site which is beneficial for the feeding ticks.

[0075] The (poly)glycosylated sugar alcohol molecule may be used in prevention and treatment of disease or conditions which are associated with plasmin-inhibitor interactions. The administered (poly)glycosylated sugar alcohol molecule may be able to disrupt the plasmin-inhibitor complex and thereby restore plasmin activity. [0076] The (poly)glycosylated sugar alcohol molecule may be comprised in a pharmaceutical composition. The pharmaceutical composition may be used in the prevention and/or treatment of a disease which is associated with a plasmininhibitor complex and impaired plasmin activity.

[0077] The pharmaceutical composition may be a pharmaceutical formulation. The pharmaceutical formulation may be selected from the group consisting of, but not limited to, creams, oils, lotions, gels, sprays, pastes, rinses, drops, chewing gums, lozenges, band aids, tablets, capsules, syrups, and the like.

Examples [0078] The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.

Example 1 - Synthesis of various (poly)glucopyranosyl-xylitol products

[0079] The reaction mixture, containing 1 M of xylitol and 1 M of glucosyl donor e.g., cyclodextrin or sucrose or glucose-l-phosphate and 30 U/mL of glucosyltransferase enzyme e.g., cyclodextrin glucanotransferase or sucrose phosphorylase or cellobiose phosphorylase in water, was incubated at 30 or 50° C under constant mixing for 48 to 72 h. After completion of the reaction, the reaction mixture was heated to 95° C and incubated for 10 min to stop the reaction. The reaction mixture was treated with amyloglucosidase and yeast as per the need for several hours to eliminate the unwanted byproducts from the reaction medium. The reaction mixture majorly containing (poly)gl ucopyra nosy l-xy litol and small amounts of unconverted xylitol is diluted 5-folds with water, centrifuged at 5.000 rpm for 30 min, heat treated at 95° C for 10 min, filtered through 0.25 p membrane and concentrated to 10-folds under vacuum on a rotary evaporator. The concentrate was passed through carbomcelite (1:1) column to isolate the respective (poly)gl ucopyra nosy l-xy litol in highly pure form. The fractions that contained majority of the product were pooled, concentrated under vacuum on a rotary evaporator and freeze-dried. NMR analysis of the pure product was performed for the structural identification of the product.

Example 2 - T ansglucosylation products analysis and characterization

[0080] Synthesis of the (poly)glucopyranosyl-xylitol products and consumption of xylitol were analyzed using H PLC coupled to an Rl detector. Separation was performed with a YMC Polyamine II column 250 mm x 4.6 mm operated at 25° C. An isocratic elution was performed at a constant flow rate of 1 ml/min using water that contained varied amounts of acetonitrile. The substrate consumption and product formation were confirmed by analyzing the changes in peak areas in H PLC chromatograms. Authentic standards of xylitol and donor substrates were used for peak identification and quantification. The retention times of product peaks were first identified based on changes in peak areas of the samples from time courses of the synthesis reaction. The structural properties of the products later confirmed by ^TH and ¹³C nuclear magnetic resonance (NMR) analysis of the purified products.

Example 3 -Testing of (poly)glucopyranosyl-xylitol for modulation of plasmininhibitor interactions

[0081] A mixture of 100 pL of albumin buffer solution, 23 pL of

(poly)gl ucopy ra nosy l-xy litol stock solution or different dilutions of the stock solution or 23 pL of glucose stock solution or 23 pL of xylitol stock solution or 23 pL of ultra-pure water, 1 pL of plasmin solution and 1 pL aprotinin solution were added per well in a 96-well plate. The plate was shaken for 10 sec and incubated for 10 min at 37° C. 25 pL of chromogenic substrate S-2251™ (Chromogenix) was added. The hydrolysis rate of S2251 was measured following the optical density at 405 nm in time at 37° C. Activity is expressed as OD/min. Each sample was measured in triplicate.

[0082] Albumin buffer solution = 140 mM NaCI, 60 mM H EPES, 0.02% NaN₃, 5 mg/mL BSA (pH 7.35).

[0083] Plasmin stock solution (1.58 mg/ml; 169.1 nkat/mg). Two-fold diluted solution were freshly generated from the stock solution in water before performing the assay.

[0084] S-2251™ solution: 3.5 mg dissolved in 5 ml of ultra-pure water.

[0085] Aprotinin (6,300 Kallikrein Inactivator Units/mg): 5 pM Aprotinin stock is generated in 0.1% BSA solution.

[0086] Soybean Trypsin Inhibitor (STI): 0.7 pM soybean trypsin inhibitor stock is generated in 0.1% BSA solution.

[0087] (Poly)glucopyranosyl-xylitol stock solution: Pure lyophilized (poly)glucopyranosyl-xylitol products were used to generate 200 mM stock solution in water and different concentrations were generated by diluting with water.

[0088] Similarly, the stock solutions of glucose and xylitol were also produced.

Claims

1. A (poly)glucopyranosyl sugar alcohol molecule or derivative thereof for use in the prevention and/or treatment of a disease caused by impaired plasmin activity.

2. The (poly)glucopyranosyl sugar alcohol molecule for use according to claim 1, wherein the impaired plasmin activity is caused by a plasmin-inhibitor interaction.

3. The (poly)glucopyranosyl sugar alcohol molecule for use according to claim 1 or 2, wherein said (poly)glucopyranosyl sugar alcohol molecule interferes with said plasmin-inhibitor interaction and restore the plasmin activity.

4. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 3, wherein said derivative is an acyl or fatty acid or lipid derivative.

5. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 4, wherein said (poly)glucopyranosyl sugar alcohol molecule consists of at least one sugar alcohol moiety and at least one glucose moiety may be linked through a glycosidic linkage.

6. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 5, wherein said sugar alcohol moiety is selected from the group consisting of sorbitol, xylitol, glycerol, arabitol, inositol, maltitol, and lactitol, preferably a xylitol.

7. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 6, wherein said sugar alcohol moiety and glucose moiety maybe linked through an a- or a p-glycosidic linkage, preferably a p-glycosidic linkage.

8. The (poly)glucopyranosyl sugar alcohol molecule for use according to claim 7, wherein said glycosidic linkage between glucose moiety and sugar alcohol moiety is (1— ”1) or (1— ”2) or (1— ”3) or (1— ”4) or (1— ”5) or (1^6), preferably a (1^2) or (1^4).

9. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 7, wherein said (poly)glucopyranosyl sugar alcohol molecule is a (poly)glucopyra nosy I -xylitol.

10. The (poly)glucopyranosyl sugar alcohol molecule for use according claim 9, wherein said (poly)glucopyranosyl sugar alcohol molecule is 1-0-a-D- gl ucopyra nosy I -xylitol, 1-0-0- D-gl ucopyra nosy I -xylitol, 2-0-a-D- glucopyra nosy I -xylitol, 2-0-0-D-gl ucopyra nosy I -xylitol, 3-0-a-D- gl ucopyra nosy I -xylitol, 3-0- - D-gl ucopyra nosy I -xylitol, 4-0-a-D- gl ucopyra nosy I -xylitol, 4-0-0- D-gl ucopyra nosy I -xylitol, 5-0-a-D- gl ucopyra nosy I -xylitol, 5-0-0- D-gl ucopyra nosy I -xylitol. The (poly)glucopyranosyl sugar alcohol molecule for use according claim 9, wherein said (poly)glucopyranosyl sugar alcohol molecule is 2-0-0-D- glucopyranosyl-xylitol, or 4-0-0-D-glucopyranosyl-xylitol. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 11, wherein said plasmin inhibitor forms a reversible or irreversible complex with plasmin. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 12, wherein the said plasmin inhibitor is a serine protease inhibitor selected from the group consisting of a₂ antiplasmin, aprotinin, miropin, crmA, plasminostrepsin and soybean trypsin inhibitor. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 13, wherein the (poly)glucopyranosyl sugar alcohol molecule is capable to interfere with the plasmin-inhibitor interaction thereby restoring the activity of plasmin. The (poly)glucopyranosyl sugar alcohol molecule for use according to any one of claims 1 to 14, wherein the disease caused by impaired plasmin activity is selected from the group consisting of fibrinolysis, immunomodulation, autoimmune disease, cancer, infectious disease, neurodegenerative disease, gum disease and skin disorders. A pharmaceutical composition comprising a (poly)glucopyranosyl sugar alcohol molecule as the active ingredient. The pharmaceutical composition according to claim 16, wherein said (poly)glucopyranosyl sugar alcohol molecule is 1-O-a-D-glucopyranosyl- xylitol, 1-0-0-D-glucopyranosyl-xylitol, 2-O-a-D-glucopyranosyl-xylitol, 2-0-0- D-gl ucopyra nosy I -xylitol, 3-O-a- D-gl ucopyra nosy l-xylitol, 3-0-0-D- gl ucopyra nosy I -xylitol, 4-O-a- D-gl ucopyra nosy I -xylitol, 4-0-0-D- glucopyranosyl-xylitol, 5-O-a- D-gl ucopyra nosy I -xylitol, 5-0-0-D- gl ucopyra nosy I -xylitol. 17 The pharmaceutical composition comprising according to claim 16, wherein the pharmaceutical composition is selected from the group consisting of creams, oils, lotions, gels, sprays, pastes, rinses, drops, chewing gums, lozenges, band aids, tablets, capsules, and syrups. A cosmetic composition comprising a (poly)glucopyranosyl sugar alcohol molecule as the active ingredient.