WO2018011419A2

WO2018011419A2 - Pharmaceutical use of alpha-l-guluronic acid

Info

Publication number: WO2018011419A2
Application number: PCT/EP2017/067920
Authority: WO
Inventors: Abbas Mirshafiey
Original assignee: HÄUSLER, Lars
Priority date: 2016-07-14
Filing date: 2017-07-14
Publication date: 2018-01-18
Also published as: DE102016113017A1; WO2018011419A3

Abstract

The invention relates to a uronic acid monomer for use in the treatment of immune system disturbances, inflammatory diseases, kidney diseases, allergies and neurodegenerative diseases, wherein the uronic acid monomer is α-L-guluronic acid or a derivative or pharmaceutically acceptable salt thereof, and said uronic acid monomer is administered at a dose of less than 50 mg/kg/day.

Description

Pharmaceutical use of alpha-L-guluronic acid

background

The invention relates to a uronic acid monomer for use in the treatment of disorders of the immune system, inflammatory diseases, kidney diseases, allergies and neurodegenerative diseases, wherein the uronic acid monomer is α-L-guluronic acid, a derivative or a pharmaceutically acceptable salt thereof and the uronic acid Monomer is administered in a dose of less than 50 mg / kg / d.

Rheumatoid arthritis is an inflammatory systemic disease that predominantly affects the joints. An older term for rheumatoid arthritis that is still commonly used is chronic polyarthritis. Over one million people in Germany suffer from rheumatoid arthritis. Around 2,000 new people fall ill each year.

The cause of rheumatoid arthritis is still not well known. It is believed that this is a so-called autoimmune disease, in which it comes to a false control of the body's defense system (the immune system) and this attacks cells of the body. The causes of the initial dysregulation of the immune system are still unknown. Scientists suspect that several factors are involved in the development of the disease. Infections with bacteria or viruses may play a role. Basically, the disease is not curable until today and accompanies the patient for the rest of his life.

For the treatment of rheumatoid arthritis currently preferred long-term drugs are used. The active ingredients used are combined due to the slow onset of their action with a faster-acting drug. These are so-called "cortisone-free anti-inflammatory drugs", so-called non-steroidal anti-inflammatory drugs, NSAIDs or non-steroidal antiinflammatory drugs, NSAID, which are among the aspirin-like drugs or cortisone.

The NSAIDs include, among others, the following active ingredients acetylsalicylic acid, diclofenac, indomethacin, piroxicam, rofecoxib, cefecoxib and meloxicam, which are marketed under different trade names.

NSAIDs have an anti-inflammatory and analgesic effect because they inhibit the function of all prostaglandins. A typical side effect of NSAIDs is that they cause the Gastric mucosa attack, which can lead to stomach and intestinal diseases, in the worst case to gastric and intestinal bleeding.

An influence on these prostaglandins is also attributed to other substances. Thus, EP 0506325 describes oligomers or polymers of L-guluronic acid and their epimer D-mannuronic acid in order to inhibit the production of cytokines such as interleukins IL-1, IL-6 and TNF.

IL-1 is known to inhibit the production of prostaglandins. The polysaccharides used were from the algae Laminaria digitata (copolymers "G-blocks" containing more than 90% L-guluronic acid), from the cells of Ascophyllum nodosum fruiting bodies (copolymers "M-blocks", more than 95% D -Mannannonic acid) or from cultures of Pseudomonas aeruginosa (polymannuronic acid).

The block copolymer alginic acid, which consists of varying proportions of mannuronic acid and guluronic acid, has long been used in the food and pharmaceutical industries because of its gelling properties. Its preparation and the preparation of the individual monomers is known in the art.

For the medical use of alginates, WO 01/661 19 describes a further application for the treatment of mucous membrane inflammations in the stomach.

Guan (Chem. Abstracts 1991, 1 14: 69045) describes the use of propylmannuronate sodium sulphate as an antithrombotic, hypolipidemic or as an agent suitable for the treatment of ischemic cardiovascular diseases.

US 5,952,308 describes a pharmaceutical composition which may include, inter alia, a mannuronic acid monomer as a complexing agent for a mineral to promote uptake of these minerals.

WO 01/56404 describes a process for the preparation of low molecular weight polymannuronates from marine algae, which is used against obesity and excessive cholesterol levels.

With respect to the monomers of alginic acid, DE 10247073 A describes the preparation and use of α-L-guluronic acid as an active ingredient in the treatment of diverse Diseases such as rheumatic diseases and kidney diseases. Nevertheless, these studies do not show optimized applications of αL-guluronic acid, eg with respect to the dose to be administered.

There is therefore a need for improved methods of administration and pharmaceutical use of α-L-guluronic acid.

The inventors of the present application solve this need by their surprising discovery that low-dose α-L-guluronic acid achieves comparable and / or improved effects as a higher dose in the treatment of immune system disorders, inflammatory diseases, kidney diseases, allergies and neurodegenerative diseases. It has been found that a dose of less than 50 mg / kg / d a-L-guluronic acid is sufficient to elicit this improved action of a-L-guluronic acid in patients.

Description of the pictures

Figure 1 shows the effect of α-L-guluronic acid (G2013) on the clinical score of experimental autoimmune encephalomyelitis (EAE). In the screening group, female C57BL / 6 mice (n = 6) were intraperitoneally administered (IP) from day 0 to day 20 after immunization with 40 mg / kg / d α-L-guluronic acid. The mice in treatment groups A and B were administered intraperitoneally with α-L-guluronic acid at 40 and 80 mg / kg / d from day 7 to day 20 post-immunization. The degree of the disease was assessed by a visual cumulative scoring system. Cumulative scores from day 10 to day 21 are reported as the mean + SEM. ^* P <0.05 is indicated at each data point by Mann-Whitney U test by comparing precautionary group and treatment groups against the control group.

Figure 2 shows representative light micrographs of histological samples of the central nervous system. (A) Hematoxylin-eosin staining (H & E staining) of brains shows that G2013 therapy can significantly suppress the progression of inflammation by limiting leukocyte infiltration. (B) Luxol fast blue staining (LFB staining) shows fewer demyelination sites in G2013 treated mice compared to the control group. N: normal; C: control group; P: prevention group; TA: treatment group A; TB: Treatment Group B. Figure 3 shows a table of comparative values of inflammatory criteria obtained from histological samples of the central nervous system from EAE mice.

Figure 4 shows the effect of α-L-guluronic acid (G2013) on the clinical score of collagen-induced arthritis (CIA) in therapeutic groups.

Figure 5 shows the effect of α-L-guluronic acid (G2013) on the expression of miR-155 in HEK-293hTLR4 cells.

Figure 6 shows the effect of α-L-guluronic acid (G2013) on the expression of SHIP-1 in HEK-293hTLR4 cells.

Figure 7 shows the effect of α-L-guluronic acid (G2013) on the expression of SOCS-1 in HEK-293hTLR4 cells.

Detailed description

In a first aspect, the present invention is directed to a uronic acid monomer for use in the treatment of immune system disorders, inflammatory diseases, kidney diseases, allergies and neurodegenerative diseases, wherein the uronic acid monomer is α-gluluronic acid, a derivative or a pharmaceutically acceptable salt thereof and the uronic acid monomer is administered at a dose of less than 50 mg / kg / d.

The term "uronic acid monomer", for the purposes of the present invention, is based on carboxylic acids which have formally been formed by oxidation of the primary hydroxyl group of monosaccharides (-CH ₂ -OH) to the carboxy group (-COOH) They belong to the sugar acids. Such a uronic acid monomer is α-L-guluronic acid. Α-Guluronic acid is a compound according to formula (I):

Formula (I)

-L-guluronic

(Gulua) The dose to be administered is less than 50 mg of uronic acid monomer (eg, αL-guluronic acid) per kilogram of body weight of a given patient per day (50 mg / kg / d). In preferred embodiments, the dose is less than 49 mg / kg / d, 48 mg / kg / d, 47 mg / kg / d, 46 mg / kg / d, 45 mg / kg / d, 44 mg / kg / d, 43 mg / kg / d, 42 mg / kg / d, 41 mg / kg / d, 40 mg / kg / d, 35 mg / kg / d, 30 mg / kg / d, 25 mg / kg / d, 20 mg / kg / d, 15 mg / kg / d or 10 mg / kg / d. In other preferred embodiments, the dose is 0.1-49.9 mg / kg / d, 1 -49.8 mg / kg / d, 3-48.5 mg / kg / d, 5-47 mg / kg / d , 8-45 mg / kg / d, 10-42.5 mg / kg / d or 15-39.9 mg / kg / d.

A patient according to the present invention may be a mammal, such as a rodent, a carnivore, a cloven-hoofed dog, an odd-toed ungulate, or a primate. In particularly preferred embodiments, the patient is a human.

Preferred pharmaceutically acceptable salts of α-L-guluronic acid are sodium α-L-guluronic acid, potassium α-L-guluronic acid, magnesium α-L-guluronic acid, calcium α-L-guluronic acid or a combination thereof.

The term "derivative" as used herein in reference to uronic acid monomer (eg, αL-guluronic acid) refers to a chemical substance or compound derived directly or by modification or partial substitution of α-L-guluronic acid substituents may be at most 4, at most 3, at most 2 or a substituent.

Inflammatory diseases as referred to in the application include autoimmune diseases, acute and chronic inflammations and osteoarthritis.

Autoimmune diseases as defined in the application include rheumatoid arthritis, juvenile arthritis, systemic lupus erythematosus, Sjogren's syndrome, progressive systemic scleroderma (PSS), polymyositis dermatomyositis, Behget's disease, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, and recurrent polychondritis.

Kidney diseases as referred to in the application include glomerulonephritis and glomerulosclerosis. Disorders of the immune system, in the sense of the application, include rejection reactions in transplants, allergic contact dermatitis and type I-IV hypersensitivity.

Neurodegenerative diseases, as defined in the application, include multiple sclerosis (MS) and Alzheimer's disease.

In preferred embodiments, the uronic acid monomer of the present invention is used in combination therapy for use with substances having hepatotoxic side effects. The term "hepatotoxic side effects" refers to the properties of chemicals that are toxic to hepatocytes (hepatic epithelial cells), which are known as hepatotoxic substances (hepatotoxic substances, hepatotoxins, liver toxins or liver toxins) and hepatotoxic substances that can damage liver hepatitis to necrosis or liver toxicity Cause liver dystrophy.

In further preferred embodiments, the uronic acid monomer is administered intraperitoneally, orally, buccally, rectally, intramuscularly, topically, subcutaneously, by inhalation, intraarticularly or intravenously.

In further further preferred embodiments, the uronic acid monomer is as ointment, cream, gel, paste, emulsion, tablet, Zäpfen, powder, powder, granules, lozenge, patch, patch, solution, foam, lotion, oil, shampoo, aerosol or spray in front.

The total active ingredient content of the abovementioned (drug) compositions according to the invention is preferably in the range from 0.05 to 90% by weight, preferably in the range from 0.1 to 50% by weight, particularly preferably 0.5 to 20% by weight. %, in each case based on the total weight of the (drug) agent. The proportion of active substance is determined in the manner known to the person skilled in the art, depending on the respectively selected dosage form, of the selected active ingredient (s), and the dose suitable for the respective therapeutic purpose. The therapeutically suitable dosages of the individual active ingredients are known to the person skilled in the art.

Said (drug) agents may contain known in the art additional ingredients such as excipients (carriers, skin protectants, disinfectants, surfactants, etc.). Suitable adjuvants are, for example, the following: particulate carriers (eg talc, zinc oxide, starch, starch derivatives, diatomaceous earth); gel-forming substances (eg gelatin, tragacanth, cellulose derivatives, alginates, polyacrylic acid); Humectants (eg urea, glycerol, propylene glycol), pressure-sensitive adhesive polymers (eg polyacrylates, as well as adhesive resins); Ointment bases (eg vaseline, fats, cellulose derivatives, polyacrylic acid, polyethylene glycols); Emulsifiers (eg wool wax, sorbitan esters, monoglycerides); Preservatives (eg benzalkonium chloride), antioxidants (eg butylhydroxyanisole), thickeners (eg hydroxypropylmethylcellulose), pH corrigents; Binders (for example polyvinylpyrrolidone, starch, hydroxypropylmethylcellulose, polyethylene glycols), fillers (eg microcrystalline cellulose, sorbitol), dyes, flavorings, sweeteners (eg sorbitol, aspartame); Solvents (eg water, ethanol, ethanol-water mixtures); Solubilizers (eg glycerol, propylene glycol); Skin penetration enhancers (eg, propylene glycol); Plasticizers (eg sorbitol, glycerol, phthalic acid esters); Wetting agents (eg, sodium lauryl sulfate, polysorbates); synthetic and natural oils (eg medium chain triglycerides); Propellants for aerosol or foam sprays (eg norflurane, cryoflurane, dichlorofluoromethane, trichlorofluoromethane, propane, butane, isobutane, nitrogen).

The use of uronic acid monomers according to the invention can be carried out together with other, preferably chemically pure, drugs and / or herbal medicines, or the pharmaceutical preparation of the invention may contain such other, preferably chemically pure, drugs or drugs.

Examples:

Example 1 Induction of Multiple Sclerosis (MS) and Therapeutic Treatment

Experimental autoimmune encephalomyelitis (EAE) is the most widely used experimental model for the study of multiple sclerosis (MS). This was induced in C57BL / 6 mice by immunization with a Hooke kit. To this end, thirty female C57BL / 6 mice (10 weeks old), weighing 18-20 g, were purchased from the Experimental Animal Center of the Pasteur Institute of Iran. The mice were kept with access to food (pellets) and water according to the Institute's guidelines. The mice were randomly divided into five groups (6 mice each) as follows: I: normal group (PBS treated); II: control group (MOG ₃₅ -55-induced EAE and PBS treated); III: Prevention group (MOG ₃ 5-55-induced EAE and 40 mg / kg / d G2013 treated from day 0-20); IV: Treatment group A (MOG ₃₅ -55-induced EAE and 40 mg / kg / d G2013 treated by day 7-20) and V: treatment group B (MOG ₃₅ -55-induced EAE and 80 mg / kg / d G2013 treated by Day 7-20). The EAE was tested by the Hooke kit (Hooke Laboratories, Inc., USA) to study the induced pathogenesis and treatment of MS in the animal model. The kit consists of two components; Antigen (MOG35-55) in an emulsion with complete Freund's adjuvant (CFA) in two pre-filled syringes and a vial of lyophilized pertussis toxin (PTX). The freeze-dried PTX was dissolved in PBS on day 0. The mice were each injected with an emulsion of 0.1 ml subcutaneously on the upper and lower back. About 2 hours after injection of the emulsion, the first dose of PTX (0.1 ml / mouse) was injected intraperitoneally (IP). This was repeated 24 hours later as a second dose of PTX (0.1 ml / mouse). The αL-guluronic acid (G2013) (manufactured in the Pathobiology Department of the TUMS) was administered to the precaution group from day 0 to day 20 after immunization in the specific dose of 40 mg / kg / d IP. The mice of treatment groups A and B were given 40 and 80 mg / kg / d G2013 from day 7 post-immunization until day 20 IP. The mice of the normal and control groups received PBS (0.1 ml of each mouse, daily). IP injection into the mice of all groups was done on six consecutive days per week. Mice were monitored daily and scored for clinical scores. Clinical score of EAE mice was defined as follows: 0: no clinical signs; 1: flabby tail; 2: flaccid tail and weakness of the hind legs; 3: flaccid tail and complete hindleg paralysis (most common) or flaccid tail with paralysis of anterior and posterior leg; 4: flaccid tail, complete hind leg and partially frontal paralysis; 5: Mouse rolls spontaneously in the cage or was found dead because of paralysis.

For histopathological analysis, on day 21 after immunization, all mice were anesthetized and blood samples taken from their hearts. For histopathological assessment, the mice were killed after their anesthesia and brains and cerebellum removed to fix them then in neutral 10% formalin, as well as paraffin embedded, cut (8 μηη thickness) and stained with hematoxylin-eosin (H & E) to assess the inflammatory criteria in meningeal and parenchymal tissue and to stain with Luxol-fast-blue (LFB) to differentiate demyelination. All stained samples were analyzed blindly on a slide by an expert pathologist.

For nitrite evaluation, N0 ₂ in the serum samples was examined by the Griess method based on the assessment of the N0 ₂ final product. The Griess method uses a colorimetric reaction for the measurement of N0 ₂ (nitrite) in an aqueous solution. Gries's reagent was mixed by dissolving 1 g of sulfanilamide in 100 ml of 5% phosphoric acid prepared with 0.1 g of naphthylethylenediamine HCl (NED) in 100 ml of distilled water. The serum sample (50 μΙ) was mixed with 50 μΙ Griess reagent for 10 min at room temperature. The absorbance was measured on an ELISA reader instrument at 540 nm. The concentration of nitrite was determined by a standard curve of 0.1 M sodium nitrite in distilled water.

The results in conjunction with clinical findings indicate that the clinical course and the degree of the disease differ between the prevention group, the treatment groups and the control group. The mean scores of the disease were higher in the control mice than in the preventive and treatment groups (Figure 1). Also, the EAE incidence was lower and the onset of EAE was delayed in the screening group and treatment groups compared to the control mice. These effects resulted in significant clinical improvement and delayed disease progression during the 21 days of observation, indicating that α-L-guluronate (G2013) inhibits the progression of EAE.

Representative images of H & E and LFB stained tissue sections from all groups show that EAE mice receiving G2013 have significantly reduced inflammatory criteria and demyelination compared to control mice (Figure 2). The results in Figure 3 illustrate that the degree of inflammation observed in histopathological studies of the central nervous system (CNS) correlates with the clinical grade of EAE.

NO production was significantly reduced in the precautionary group (10.61 ± 1, 80 μΜ / dL, p> 0.05) and treatment group B (10.71 ± 1.47μΜ / άΙ_, p> 0.05) compared to the control mice (13.00 ± 1.00 μΜ / dL), but did not significantly reduce NO production in treatment group A (1 1.16 ± 2.15μΜ / dl, p> 0.05). In the normal group, NO production was 5.85 ± 0.53μΜ. Therefore, G2013 causes a decrease in NO concentration in serum and this was consistent with the clinical findings. In summary, these data indicate that a-L-guluronic acid (G2013) therapy can reduce the progression of the disease in an experimental model of MS.

Example 2: Therapeutic effect of α-L-guluronic acid (G2013) in an experimental model of rheumatoid arthritis

Collagen-induced arthritis (CIA) as an experimental model of rheumatoid arthritis was induced in DBA / 1J mice and the therapeutic efficacy of aL Guluronic acid (G2013) was tested in these animals in Radboudumc, Experimental Rheumatology Department of Radboudumc, The Netherlands. Male DBA / 1J mice were obtained from Janvier-Elevage (Le Genest St. Isle, France). All mice were maintained in filter-top cages under specific pathogen-free conditions. Standard food and water were provided to them ad libitum. The mice used were between 10 and 14 weeks old. All animal procedures were approved by the Institutional Ethics Committee in Radboudumc, Nijmegen. Freund's adjuvant (FCA) and Mycobacterium tuberculosis (strain H37Ra) were obtained from Difco, Detroit, MI. Type II bovine collagen (CM) was prepared by the inventors as described by Joosten (Joosten LAB, et al, 1996). To induce CIA, bovine type II collagen (CM) was diluted in 0.05 M acetic acid at a concentration of 2 mg / ml and emulsified in equal volumes with Freund's Complete Adjuvant (FCA) (2 mg / ml M. tuberculosis). , The DBA / 1J mice were immunized intradermally at the base of the tail with 100 μg CM. On day 21, the mice received an intraperitoneal (ip) booster injection of 100 μg CM dissolved in phosphate buffered saline (PBS). The onset of arthritis usually occurred a few days before or after the booster injection. Mice were examined visually three times per week visually for arthritis manifestations in the peripheral joints, and disease activity ratings were assigned as previously described (van den Berg WB, et al, 1994). The clinical grade of arthritis (arthritis score) was graded on a scale of 0-2 for each paw, consistent with changes in redness and swelling in the toes or in other parts of the paws. G2013 treatment was started before the onset of clinical symptoms of the disease (day 21) or during established arthritis (macroscopic arthritis scores of 0.25 to 1, 25 on a scale of 0-8) and continued for 14 days. Treatment with G2013 was administered at a dose of 25 or 50 mg / kg / d by intraperitoneal (ip) injection. Anti-TNF (Enbrel) was used as a positive control of the treatment and injected IP 3 times a week. For measurement of cytokines, Luminex multi-analyte technology was used in combination with the Miliplex Cytokine / Chemokine Magnetic Bead Panel kit (Millipore) according to the manufacturer's protocol to measure the levels of IL-1, IL-6, IL-10 and KC to measure cytokines in treated and untreated mouse sera. For histological analysis, isolated joints were fixed in 10% formalin, 5% decalcified formic acid for 4 days and then dehydrated and embedded in paraffin. Standard 7 mm frontal sections were mounted on SuperFrost microscope slides (Menzel-Gläser, Braunschweig, Germany). Hematoxylin and eosin (H & E) staining and safranine O staining were performed to study joint pathology. The pathological grade in the joints was on a scale of 0-3 (0 = no pathology, 3 = maximum Cellularity) as previously described (Lubberts, Arthritis Rheum 2004) for the following five parameters: arthritis, proteoglycan (PG) depletion from the cartilage matrix, chondrocyte death, cartilage surface erosion and bone erosion. Histopathological changes were evaluated on five semiserial sections with a distance of 140 mm. The evaluation was done blind in by two independent observers.

The results in the low dose (25 mg / kg, G2013) IP injection therapeutic group demonstrate a therapeutic efficacy of αL-guluronic acid (G2013) by mitigating the progression of the disease in the experimental model of rheumatoid arthritis (RA) compared to the control group and the anti-TNF treated animals in the treatment of RA (Figure 3).

Example 3: Immunosuppressive effects of α-L-guluronic acid (G2013) on the expression of microRNA 155 and its cytosolic targets

In inflammatory and autoimmune diseases, gene expression of microRNA 155 and its cytosolic targets that mediate signal transduction of miR-155 (eg, SHIP-1 and SCOS-1) is useful for determining the degree of disease progression and therapeutic effects of drugs. HEK293 hTLR4 cells, a human embryonic kidney line, were transfected into RPMI 1640 medium (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA), 1X penicillin-streptomycin antibiotic and 2 mM L-glutamine at 37 ° C cultured under a humidified atmosphere at 95% air and 5% C0 ₂ . Counting and determining viability were done using trypan blue. Cells were seeded in 6-well plates at a density of 1x10 ⁶ cells per well. After culturing in normal growth medium for 24 hours, the cells were treated with α-L-guluronic acid (G2013) for 24 hours as follows: low dose (5 μg / ml), high dose (15 μg / ml), and pure medium (control). , Total RNA was extracted from the control and treated samples using the Hybrid R Mini Kit (GeneAll, South Korea) according to the manufacturer's instructions. The purity of the isolated RNA was checked ND-1000 (Isogen Life Science) per NanoDrop® to ensure spectrophotometric ratios of A ₂ A ₂ 6o nm 8o nm ~ 2 and A ₂ 6o nm / A ₂₃ _nm o ^. 2 After isolation of the RNA from the cells, cDNA was prepared using the RT-for-RCR kit (Gene All, South Korea). The amplification of SHIP1 (forward: 5'-GCC TAC ACC AAG CAG AAA GC-3 '; reverse: 5'-GGA CCG TTC TTG GAG ACA AA-3') and SOCS1 (forward: 5'-AGA GCT TCG ACT GCC TCT TC-3 '; reverse: 5'-GAT GCG CTG GCG GCA CAG CT-3') mRNA was amplified by quantitative PCR (QPCR) (Applied Biosystem) with SYBR Premix Ex Tq (Takara, Japan). The conditions for the PCR were as follows: 95 ° C for 3 minutes (1 cycle) and 94 ° C for 20 s, 60 ° C for 30 s and 72 ° C for 40 s (40 cycles). The relative mRNA abundance was determined by normalizing to ß-actin mRNA using 2 ^A -AACT method determines (Ct refers to the threshold value). The miRNA cDNA was generated using MystiCq® MicroRNA cDNA Synthesis Mix (Sigma-Aldrich) according to the manufacturer's instructions. The qPCR was performed using a specific miR-155 primer, 5'-UUA AUG CUA AUC GUG AUA GGG GU-3 '(Sigma-Aldrich), and SYBR Premix EX Tq (Takara, Japan). The conditions for the qPCR were as follows: 95 ° C for 30 s, 94 ° C for 5 s, 60 ° C for 30 s, and 70 ° C for 15 s (40 cycles). The One-Step Real Time (Applied Biosystem) thermocycler was used for the qPCR and the data was analyzed using GraphPad Prisma 5 software based on the one-way Anova variance method and Student's t-test. Relative miRNA frequency was determined by normalization against U6 snRNA using the 2 ^Λ - ΔΔΟΤ method.

The results of aL-guluronic acid treated cells show a significant decrease in expression of microRNA 155 compared to the control group (Figure 4) and a significant increase in gene expression of SHIP-1 (Figure 5) and SOCS-1 (Figure 6) compared to control group. This shows the immunosuppressive properties of o L-guluronic acid (G2013).

Example 4: Effect of α-L-guluronic acid (G2013) on the differentiation, maturation and function of human dendritic cells

Conventional DC-based immunosuppressants have notable side effects to increase the risk of infectious diseases and cancer. The aim of these experiments is to demonstrate the safe use of α-L-guluronic acid, so that there are no effects on the differentiation, maturation and function of dendritic cells. The in vitro differentiation of human monocytes into dendritic cells (DCs) was performed as described by Sreevalsan with minor modifications [Sreevalsan T, 2009]. Six human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Mediatech Cellgro) density gradient centrifugation. Monocytes were purified from peripheral blood mononuclear cells using anti-CD14 microbeads (Miltenyi Biotec). The monocytes (> 95% purity) were incubated at 37 ° C in 24-well plates (700,000 cells per well) in 3 ml serum-free AIM V medium (Invitrogen) containing 100 ng / ml human granulocyte-macrophage colony stimulating factor (GM-CSF) (PeproTechs) and human interleukin-4 (IL-4, 20 ng / ml, R & D Systems). The cells were cultured with two different dosages of G2013. On the one hand a 6 μg well low dosage solution and on the other hand a 12 μg well high dosage solution. 6 wells were selected as control (not treated with G2013). A total of 0.5 ml of fresh medium with GM-CSF and IL-4 was added to the cell cultures on the third day. To induce DC maturation, on the fifth day, the cell cultures were contacted with lipopolysaccharide (LPS, 1 μg ml, Catalog No. L2654, Sigma-Aldrich) for 24 hours. Two doses of aL-guluronic acid (G2013) solution were added to the wells 4 hours prior to LPS addition. The harvested cells were washed twice in PBS and resuspended in 100 μM PBS supplemented with 0.5% bovine serum albumin (BSA) and 0.1% sodium azide for staining. The cells were incubated for 10 minutes at room temperature with an Fc receptor blocking solution (BioLegend, San Diego, USA). The cells were then incubated at 4 ° C for 30 min with the following mouse monoclonal anti-human antibodies (MAb): FITC-conjugated mAbs against the cell surface molecules CD83, CD14 and PE-conjugated mAbs against CD1a, CD86 and PECY5- conjugated mAbs to MHCII (eBiosciences, USA). In all experiments, isotype controls were used that were appropriate mAbs of the same Ig class or subclass. After staining, the cells were washed twice in PBS supplemented with 0.5% BSA and 0.1% sodium azide and resuspended in PBS with 0.99% paraformaldehyde. Flow cytometry was performed on a Cytomics FC-500 cytometer (Beckman Coulter) and all subsequent analyzes were analyzed by the FlowJo software (Tree Star). DC cytokine production was detected in each of the supernatants of DC cultures (IL-12p70 and IL-10). Supernatants were collected and frozen at -70 ° C until use. Cytokine concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (BenderMed System, Austria) according to the manufacturer's instructions.

The expression of CD14 and CD1a (as a differentiation marker) was studied in monocytes and immature dendritic cells with two different doses (6 and 12 μg) of α-L-guluronic acid (G2013). The results show that there are no significant differences between the control group and the groups treated with G2013 solution.

In order to determine if the effect of two different doses (6 and 12 μg / well) of a G2013 solution has effects on the expression of CD83, CD86 and MHC II (as maturation markers), DCs were cultured in the presence of these two different doses. The expression of MHC-II and the co-stimulatory molecules was assessed by means of Flow cytometry analyzed. The data show that the expressions of the co-stimulatory molecules and MHC-II were not significant differences between the control group and the groups treated with G2013.

The supernatants of cultured DCs were collected and cytokine concentrations were measured by the ELISA method. The results show that there was no significant difference in cytokine concentrations (IL-12p70 and IL-10) between the control group and the groups treated with G2013 (eg 6 g / well (P = 0.068) and 12 μg A well, respectively ( P = 1, 4) in mature dendritic cells.The results show that aL-guluronic acid (G2013), as a safe drug, has no detrimental effect on the differentiation, maturation, and function of dendritic cells and can be used as a novel immunosuppressant. that has no or fewer side effects (eg increasing the risk of infectious diseases and cancer) than comparable products on the market.

Claims

Claims:

1 . Uronic acid monomer for use in the treatment of disorders of the

Immune system, inflammatory diseases, renal diseases, allergies and neurodegenerative diseases, wherein the uronic acid monomer is α-L-guluronic acid, a derivative or a pharmaceutically acceptable salt thereof, and the uronic acid monomer is administered at a dose of less than 50 mg / kg / d.

2. The uronic acid monomer for use according to claim 1, wherein the

Inflammatory diseases include autoimmune diseases, acute and chronic inflammation and osteoarthritis.

3. The uronic acid monomer for use according to claim 2, wherein the

Autoimmune Diseases Rheumatoid Arthritis, Juvenile Arthritis, Systemic Lupus Erythematosus, Sjogren's Syndrome, Progressive Systemic Scleroderma (PSS), Polymyositis Dermatomyositis, Behget's Disease, Ankylosing Spondylitis,

Include psoriatic arthritis, Reiter's syndrome, and recurrent polychondritis.

4. The uronic acid monomer for use according to claim 1, wherein the

Renal diseases include glomerulonephritis and glomerulosclerosis.

5. The uronic acid monomer for use according to claim 1, wherein the disorders of the immune system rejection reactions in transplants, allergic

Contact dermatitis and hypersensitivity include Type I-IV.

6. The uronic acid monomer for use according to claim 1, wherein the

neurodegenerative diseases include multiple sclerosis (MS) and Alzheimer's disease.

7. The uronic acid monomer for use according to claim 1 for combination therapy with substances having hepatotoxic side effects.

8. The uronic acid monomer for use according to any one of claims 1-7, wherein the uronic acid monomer is selected from the group consisting of sodium αL-guluronic acid, potassium αL-guluronic acid, magnesium αL-guluronic acid, calcium αL-guluronic acid and a combination thereof is selected.

The uronic acid monomer for use according to any one of claims 1-8, wherein the uronic acid monomer is administered intraperitoneally, orally, buccally, rectally, intramuscularly, topically, subcutaneously, inhalatively, intraarticularly or intravenously.

The uronic acid monomer for use according to any one of claims 1 to 9, wherein the uronic acid monomer is an ointment, cream, gel, paste, emulsion, tablet, suppository, powder, powder, granule, lozenge, patch, patch, solution, Foam, lotion, oil, shampoo, aerosol or spray is present.