WO2004033472A1 - Procede de production d'acide uronique monomere, en particulier d'un acide d-mannuronique et d'un acide d-guluronique, - Google Patents

Procede de production d'acide uronique monomere, en particulier d'un acide d-mannuronique et d'un acide d-guluronique, Download PDF

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WO2004033472A1
WO2004033472A1 PCT/DE2003/003349 DE0303349W WO2004033472A1 WO 2004033472 A1 WO2004033472 A1 WO 2004033472A1 DE 0303349 W DE0303349 W DE 0303349W WO 2004033472 A1 WO2004033472 A1 WO 2004033472A1
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acid
alkyl
pharmaceutical composition
substituted
uronic
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PCT/DE2003/003349
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WO2004033472A8 (fr
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Bernd Helmut Adam Rehm
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Westfälische Wilhelms-Universität Münster
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Priority claimed from DE10253514A external-priority patent/DE10253514A1/de
Priority claimed from DE10261693A external-priority patent/DE10261693A1/de
Application filed by Westfälische Wilhelms-Universität Münster filed Critical Westfälische Wilhelms-Universität Münster
Priority to AU2003277820A priority Critical patent/AU2003277820A1/en
Publication of WO2004033472A1 publication Critical patent/WO2004033472A1/fr
Publication of WO2004033472A8 publication Critical patent/WO2004033472A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/033—Uronic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the invention relates to a process for the preparation of monomeric uronic acids, in particular mannuronic acid and guluronic acid, preferably from microbially produced polymannuronate or polyguluronate, and to the polyuronic acid and derivatives thereof prepared in this way.
  • monomeric uronic acids in particular mannuronic acid and guluronic acid, preferably from microbially produced polymannuronate or polyguluronate
  • polyuronic acid and derivatives thereof prepared in this way.
  • the use of these substances for the production of a pharmaceutical composition which has an anti-inflammatory action and can be used for the treatment of disorders of the immune system, in particular of joint inflammation, and for the production of a cosmetic composition which contain these compounds is also described.
  • Rheumatoid arthritis is an inflammatory systemic disease that mainly affects the joints.
  • An older term for rheumatoid arthritis that is still used frequently is chronic polyarthritis. Over a million people in Germany suffer from rheumatoid arthritis. Every year around 2,000 new people contract it.
  • rheumatoid arthritis is still not exactly known. It is believed that it is a so-called autoimmune disease, in which the body's immune system (the immune system) malfunctions and attacks cells in its own body. The causes of the initial immune system malfunction are still unknown. Science suspects that several factors are involved in the development of the disease. possibly infections with bacteria or viruses play a role. Basically, the disease is not curable until today and accompanies the patient for the rest of his life.
  • NSAID non-steroidal anti-inflammatory drugs
  • NSAID non-steroidal anti-inflammatory drugs
  • the NSAIDs include the following active ingredients acetylsalicylic acid, diclofenac, indomethacin, piroxicam, rofecoxib, cefecoxib and meloxicam, which are sold under various trade names.
  • NSAIDs have anti-inflammatory and analgesic effects because they inhibit the formation of prostaglandins or affect their effects.
  • a typical side effect of NSAIDs is that they attack the gastric mucosa, which can lead to stomach and intestinal diseases, and in the worst case, to gastric and intestinal bleeding.
  • oligomers or polymers of L-guluronic acid and their epi he D-mannuronic acid are described 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 made 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” containing more than 95% D-mannuronic acid included) or isolated from cultures of Pseudomonas aeruginosa (polymannuronic acid).
  • the block copolymer alginic acid which consists of changing proportions of mannuronic acid and guluronic acid, has long been used in the food and pharmaceutical industries due to its gelling properties.
  • WO 01/56404 describes a process for the production of low molecular weight polymannuronates from marine algae, which is used to combat obesity and excessive cholesterol levels.
  • Microorganisms are usually preserved and are of high molecular weight.
  • Such high molecular weight block polymers have a high viscosity and are poorly soluble in water. As a result, the use of these high molecular weight polymers, for example in high concentrations in foods, is very restricted.
  • 10-900 kDa include acidic / alkaline hydrolysis [Haug,
  • the monomer produced by the process is preferably D-mannuronic acid or L-guluronic acid or derivatives thereof.
  • the polyuronic acid can have protective groups in the 2- and / or 3-position of the uronic acid monomer.
  • the protective groups protect the hydroxyl groups present on the uronic acid from undesirable side reactions.
  • the protective groups selected from the group consisting of Ci-C ⁇ Q carboxylate, especially formate, acetate, pyruvate, lactate, acyl, -C-C 2 o-alpha-aminocarboxylate, C ⁇ -C 2 oN -Acyl- compounds such as N-formyl, N-acetyl, N-propionyl, sulfonate and phosphate.
  • the acid function of the uronic acid monomer in the polymer can also be protected and in the form of an ester of a C 1 to C 2 carboxylic acid or amino acid, an alkali metal or optionally substituted quaternary ammonium salt.
  • the polyuronic acids used in the process according to the invention are preferably obtained from a plant or microbial source. Alginates and, in particular, cell supernatants from bacterial cultures are among the possible sources. Preferred are the polyuronic acid-producing bacterial strains whose epimerase gene and preferably also the 0-transacetylase gene are inactive or have been inactivated.
  • the epimerase-negative Pseudomonas strain of the species Pseudomonas putida which produces the polyuronic acid is preferred.
  • a Pseudomonas strain is used which is both epimerase negative and O-transacetylase negative.
  • the epimerase-negative Pseudomonas strain can be cultivated in a fermenter, and the fermentation broth can be subjected to a treatment to remove the insoluble constituents.
  • the treatment for separating the insoluble constituents comprises filtering, in particular ultrafiltration, centrifuging and combinations of the aforementioned methods.
  • the filtrate is subjected to a precipitation treatment, preferably an alcoholic precipitation treatment.
  • a precipitation treatment preferably an alcoholic precipitation treatment.
  • the polyuronic acid obtained in this way can be redissolved and treated with unspecific proteases and deoxyribonucleases to remove protein and deoxyribonucleic acid impurities. Low molecular weight contaminants can then be removed by dialysis and / or ultrafiltration. For further treatment, the polyuronic acid can then be dried by lyophilization.
  • the polyuronic acid is then hydrolyzed under acidic conditions, the hydrolysis preferably being carried out at 100-140 ° C. in an acidic solution, preferably dilute HCl, at a pH of 1-5.
  • an acidic solution preferably dilute HCl, at a pH of 1-5.
  • these protective groups can be subjected to acidic hydrolysis of the polymer under alkaline conditions, preferably weakly alkaline Conditions in the range between at a pH of 9 to 13 are split off, and then the solution obtained can be purified, for example dialyzed, in order to facilitate the work-up and purification of the polymer.
  • the protective groups can also remain on the polymers or be modified if this results in properties, in particular improved pharmacological properties, which can be advantageous when the polymers or monomers, in particular pharmaceutical use, are used later.
  • the invention therefore also encompasses derivatives of the uronic acid monomers shown in formula (I) below, which can be substituted in positions 1 to 4 and on the acid group (R5), and their use as a pharmaceutically active ingredient in a pharmaceutical or in a cosmetic composition.
  • R 1 and R are hydroxyl and R 2 and R 3 are selected from the group consisting of hydroxyl, C 1 -C 2 o Carboxylate, especially formate, acetate, pyruvate, lactate, acyl, -C-C 2 o-alpha-aminocarboxylate and -C-C 2 oN-acyl compounds such as N-formyl, N-acetyl, N-propionyl, N-lactate, and R 5 is hydroxyl, -O-alkali metal and -0- (quaternary ammonium), which can also be substituted by one or more C_-C 2 o ⁇ alkyl, the C ⁇ -C 3 o Alkyl radical can be straight-chain, cyclic or branched and can be substituted by one or more heteroatoms, preferably halogen, which can be used as
  • R 1 to R 4 are preferably selected from the group consisting of the hydroxy, carboxylate of an organic acid with up to 20, preferably 10 carbon atoms, such as acetate, butyrate, pyruvate, alpha-amino acids, N-acyl compounds such as N-acetylglucosamine.
  • the uronic acid monomers are particularly preferably only substituted in the 2 and / or 3 position of the sugar ring.
  • the uronic acid monomer or a derivative thereof which is obtainable by the process according to the invention, as a pharmaceutically active ingredient for producing a pharmaceutical composition or as an ingredient in a cosmetic composition
  • the uronic acid monomer preferably mannuronic acid, guluronic acid or a pharmacologically active derivative thereof.
  • Pharmacologically active derivatives which can be prepared by derivatizing the polymers or monomers of uronic acid are particularly preferred.
  • the modification of the uronic acid monomers can be achieved by enzymatic modification or by chemical derivatization methods, such as those used in inorganic or organic chemistry.
  • derivatives of uronic acids are therefore also detected which are straight-chain on the acid group by salt formation, ester or amide formation with a C 1 to C 2 -alkyl alcohol or amine , branched, cyclic and / or unsaturated, or the like are converted into derivatives.
  • the use of Protecting groups for the hydroxyl groups as stated above for R 1 to R 4 are preferred.
  • the uronic acid onomer is usually in the pyranose form as a hemiacetal between the Cl and C5.
  • the uronic acid monomer is preferably particularly suitable at least in the 2- and / or 3-position for the attachment of a protective group, the solubility behavior of the uronic acid monomer being able to be influenced by the choice of the protective group.
  • protective groups e.g. the enzymatic modifications which are catalyzed by transferases or aldolases, preferably by O-transacetylase (AlglJF) and acetyl-CoA.
  • the solution of the uronic acid polymer obtained after the deprotection has been eliminated is neutralized after dialysis and the hydrolysis of the polymer is split off under acidic conditions, preferably weakly acidic conditions in the range between at a pH of 1 to 5, and the monomer-pure uronic acid Monomer isolated from the neutral solution.
  • monomer-pure means a purity of more than 99%, preferably 100%, of a monomer.
  • polymers which are composed of different monomers. "However, then such polymers are preferred, more than 50% of a Monomers included.
  • various alginates can also be used, which are then used to prepare the monomers.
  • alginates from brown algae with a guluronic acid content of 50% and more can be used.
  • these monomer mixtures must be subjected to a further purification step after the hydrolysis, for example in the form of a separation by means of column chromatography processes using chiral column materials.
  • the uronic acid monomer thus obtained or a derivative thereof has beneficial properties in the treatment of various diseases, particularly inflammatory diseases.
  • NSAIDs inhibit the enzymes from the group of cyclooxygenases, of which the enzymes COX-1, COX-2, COX-3, PCOX-la and PCOX-lb are known to date.
  • the cyclooxygenases catalyze the formation of prostaglandins from cleavage products of the membrane phospholipids.
  • Use as a pharmaceutical includes the use of the uronic acid monomers, in particular D-mannuronic acid and L-guluronic acid, mixtures thereof or their pharmacologically active derivatives as a pharmaceutically active substance in the treatment of disorders of the immune system including the effect as an immunosuppressant, rheumatic diseases, kidney diseases, inflammation of the Genitourinary system, multiple sclerosis, allergies, Alzheimer's and cardiovascular diseases.
  • the rheumatic diseases include in particular rheumatic arthritis, juvenile arthritis, systemic and lupus erythe atodes.
  • kidney diseases include glomerulonephritis, glomerulosclerosis and renal vein thrombosis.
  • the immune system disorders include rejection reactions in transplants and autoimmune diseases.
  • the uronic acid monomers can be used for combination therapy with substances that have hepatotoxic side effects.
  • Epimerization of D-mannuronic acid for example, by liver epimerases provides glucuronic acid.
  • the glycosides of glucuronic acid, the glucuronides can metabolize many of the body's own animals (e.g. steroid hormones and Bilirubin) as well as foreign substances, especially the phenolic compounds (e.g. pharmaceuticals), are excreted in the urine after coupling to glucuronic acid (so-called glucuronidation or formation of so-called glucuronic acid conjugates). Because of these detoxifying properties, the uronic acid monomers, in particular guluronic acid after epimerization, can also be used for cancer prevention.
  • PPAR Activate
  • the NSAIDs can change the transcription rate of the
  • Ratio of BAX: BCL-X L in the cell increased.
  • the good tolerance of the uronic acid monomers or the derivatives thereof at higher doses and long-term applications not only enables special use for cancer prevention but also generally the preventive use of uronic acid monomers in healthy individuals in order to prevent diseases and thus increase the life expectancy of the individual.
  • This preventive application is particularly suitable for groups of people who have an increased risk of illness, be it due to extraordinary stress on the body in adolescence or a genetic predisposition to certain diseases, such as cancer, in particular breast cancer or Parkinson's.
  • the inventors assume that the main targets for the binding of D-mannuronic acid are the mannose receptor (MR) and the endol ⁇ O receptor from the mannose receptor family. Possible accessory receptors for this molecule are presumably the Toll-like receptor 2 (TLR2), the Toll-like receptor 4 (TLR4), the CDllb / CD18 (Mac-1 or CR3) and P-selectin.
  • MR mannose receptor
  • TLR2 Toll-like receptor 2
  • TLR4 Toll-like receptor 4
  • CDllb / CD18 Mac-1 or CR3
  • P-selectin P-selectin.
  • the mannose receptor family is a subset of the C-type lectin superfamily (WI Weis, et al.; 1998) and comprises four members: the mannose receptor (MR), the M-type phospholipase A2 receptor (PLA2R), the DEC-205 / gp200-MR6 and the Endol ⁇ O, where MR and Endol ⁇ O are multifunctional receptors. So far it has only been shown that the MR and Endol80 bind monosaccharides (L.East and CM. Isacke; 2002).
  • the C-type lectin superfamily is a large group of transmembrane receptors and soluble proteins.
  • Mannose receptor family members are unique within this superfamily because they alone contain multiple C-type lectin-like domains (CTLD's) within a single polypeptide backbone.
  • CTLD C-type lectin-like domains
  • all receptors in this family are characterized by an N-terminal cysteine-rich domain, followed by a single fibronectin type II (FNII) domain.
  • FNII fibronectin type II
  • the receptors all end in short cytoplasmic domains that contain motives for interacting with the cellular endocytosis machinery (L. East and CM. Isacke; 2002).
  • the invention is further illustrated by the following examples.
  • a 0.5% polymannuronate solution was prepared and adjusted to a final concentration of 0.1 M NaOH. This solution was stirred at 20 ° C for 3 hours to remove the acetyl groups. A pH of 10 was then adjusted with HCl and dialysis was carried out against 100 times the volume of distilled water. The polymannuronate thus deacetylated was now completely hydrolyzed. For this purpose, the pH was adjusted to 2.3 with HCl and the incubation was carried out at 135 ° C. for 4 h. After the subsequent neutralization with hydrochloric acid, the sample was lyophilized and the residue obtained was made available for further experiments. The residue was analyzed by TLC and anion exchange chromatography.
  • indomethacin The active ingredient indomethacin (hereinafter referred to only as indomethacin) is used as a reference substance, which has long been known as NSAID and is available on the market as Amuno ® and Indomet-ratiopharm ® .
  • the first treatment of the test groups took place 15 days after the induction.
  • the first experimental group was not treated.
  • the second test group was administered 40 mg / kg D-mannuronic acid intraperitoneally.
  • the third test group was administered indomethacin in a concentration of 2 mg / kg in a suspension of 0.5% methyl cellulose and 0.025% Tween 80.
  • Treatment with D-mannuronic acid or indomethacin was repeated daily until the 25th day as the last day of the trial. During this time, volume measurements of the paws of both the treated right paw and the untreated contralateral paw (negative control) were carried out every two days. In addition, some animals were killed for histological examination of the contralateral paw. 1 shows the effect of indomethacin (adjuvant + indo) and D-mannuronic acid (adjuvant + M2000) compared to the untreated control (adjuvant). After only 10 days of treatment, the paw volume of the animals treated with D-mannuronic acid decreased by 57% compared to the untreated paw. The anti-inflammatory effect of the treatment with D-mannuronic acid is therefore comparable to the effect of the treatment with indomethacin, in which the paw volume (paw oedema) was reduced by 60% (P ⁇ 0.01).
  • both the paws of animals from the second and third experimental groups and animals in which the arthritis had not been induced were examined histologically.
  • the rear extremities were separated under the knee joint, skinned and fixed in 1% formaldehyde solution.
  • the limbs were decalcified, cut and stained with hematoxylin for purple staining of the cell nucleus and with eosin for pink staining of the collagen and the cytoskeleton of the cells.
  • the joint connections of the tarsal and metatarsophalangeal joints were examined microscopically.
  • the synovium, cartilage and soft tissue in each joint were examined for synovial hyperplasia, inflammation, edema, and bone and cartilage degradation.
  • the untreated test group 1 shows clear morphological features of arthritis in numerous joints 25 days after the induction of arthritis (FIG. 2b). Significant cellular infiltration of the joints was already evident on day 14 without any signs of bone or cartilage degradation (results not shown).
  • the paws of Lewis rats from the 2nd experimental group showed both a reduced inflammatory cell infiltration and a reduced number of osteoclasts in the bone beneath the cartilage. The damage caused by the edema and the bone loss in the paws are largely reduced.
  • test groups 2nd treated with D-guluronic acid
  • 3rd treated with indomethacin
  • Test group 1 untreated
  • MMP-2 matrix metalloproteinase type 2
  • a Fibrosarcoma cell line (WEHI 164) with an initial density of 2 * 10 4 cells / chamber in RPMI-1640 base medium (5% CO 2 , 37 ° C, moisture saturated atmosphere).
  • the basic medium will be under other 5% fetal calf serum, 100 U / ml penicillin and 100 ⁇ g / ml streptoycin added.
  • D-mannuronic acid and the medicinal products dexamethasone, piroxicam and diclofenac are added in different doses (10 to 200 ⁇ g / ml) to overnight cultures of the cell line described above. Untreated cell cultures are used for the control batches (all test batches in triplicate). The treated (test group) and untreated (control group) cells are cultivated overnight under the conditions described above. The groups are then subjected to colorimetric studies and zymoanalysis.
  • the cells from the overnight cultures described above are washed three times with ice-cold phosphate-buffered saline (PBS) and then fixed in 5% formaldehyde solution.
  • the fixed cells are washed three times with ice-cold phosphate-buffered saline (PBS) and then stained with 1% crystal violet solution.
  • the stained cells are washed again, then lysed and solubilized in 33% acetic acid solution.
  • the color intensity is then read out at 580 nm.
  • a modified Heussen and Dowdle method is used to detect gelatinase (collagenase type IV or MMP-2) and MMP-9 (gelatin zymography).
  • the samples from the individual test batches are analyzed in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page) under non-reducing conditions for 3 hours at a constant 80 volts, the gel containing 2 mg / ml of gelatin.
  • SDS-Page sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • To remove the SDS from the gel the gel washed three times in 2.5% TritonXIOO solution after electrophoresis. The electrophoresis gel is then incubated at 37 ° C.
  • Tris-HCl gelatinase activation buffer pH 7.4 which contains 10 mM CaCl 2 and then stained with 0.5% Coomassie blue solution. After intensive decolorization, the proteolyzed areas in the gel appear as clear bands against a blue background.
  • a UVI Pro-Gel documentation system based on gray scale comparisons is used for the quantitative evaluation of both the surface and the intensity of the lysed areas as a measure of the enzyme activity.
  • the resulting values for the gray levels of treated cell cultures are compared with those of untreated cell cultures and expressed as "relative expression" of the gelatinolytic activity.
  • kidney functions of the test animals after 12 injections of D-mannuronic acid (30 mg / kg / 48 h) into the abdominal cavity are measured using measurements of the serum creatinine (S. Creat) content, urea nitrogen (BUN), and protein secretion by the urine (U.Pro), the urea content (U.Urea) and the plasma concentration of triglycerides (S.Trig) and cholesterol (S.Chol).
  • the stained tissue sections from the kidney tissue are examined with a light microscope.
  • the kidney tissue is first fixed in 10% formalin solution and then embedded in paraffin. Sections of the tissue are made with an ultramicrotome, which are stained with hematoxylin-eosin and Schiff's reagent. Glomerular lesions are then rated on a 0-3 scale (0, negative; 1, easy; 2, medium; 3, severe) using the following
  • the changes in body temperature are continuously monitored during the twelve times injection of D-mannuronic acid solution in a concentration of 30 mg / kg / 48h with the help of thermocouples.
  • the results of the temperature measurements are compared to those of untreated animals.
  • the Student's T-test is used for the statistical analysis of the individual test results (P ⁇ 0.05).
  • the results of the proliferation experiments with a fibrosarcoma cell line are shown in FIG. 4.
  • the comparison of the cell number in the cell cultures that have been treated with D-mannuronic acid with those that have been treated with steroidal and non-steroidal drugs (dexamethasone, piroxicam and diclofenac) shows the good tolerability of D-mannuronic acid at high doses and is suitable therefore, in comparison to other anti-inflammatory drugs, also for long-term applications.
  • 5 shows the effect of D-mannuronic acid, dexamethasone, piroxicam and diclofenac-treated cell cultures on the gelantinolytic activity of MMP-2.
  • the inhibitory effect of D-mannuronic acid on the activity of MMP-2 shows a possible mechanism of action of D-mannuronic acid in combating inflammatory reactions.
  • This example illustrates the therapeutic effect of D-mannuronic acid in kidney diseases using artificially induced nephrotic syndromes in rats.
  • the nephrotic syndromes are induced by an intravenous injection of adriamycin (7.5 mg / kg Adriablastina; Farmitalia, Milan, Italy) through the tail vein.
  • Treatment of groups T1 and T2 is started six days after induction.
  • the experimental animals in group T1 are given D-mannuronic acid in a dose of 30 mg / kg body weight, while the experimental animals in group T2 are administered 0.3 mg / kg piroxicam.
  • T1 and T2 are given 5 injections of the composition described above every day for 14 days. They also get 9 Injections of the same type given every 48 hours, so that the treatment is stopped after 28 days. For further investigations, the animals are sacrificed 43 days after the start of treatment.
  • the determination of the side effects on kidney function are determined on the basis of the serum and urine determinants (serum creatinine content, the urea nitrogen content, the protein secretion by the urine, the urea content and the plasma concentration of triglycerides and cholesterol), as was already the case in exemplary embodiment 2 has been described.
  • serum and urine determinants serum creatinine content, the urea nitrogen content, the protein secretion by the urine, the urea content and the plasma concentration of triglycerides and cholesterol
  • FIGS. 7 and 8 The results of the investigations are shown in FIGS. 7 and 8. It can be seen that the treatment of nephrotic syndrome in rats with D-mannuronic acid shows effects comparable to those of piroxicam already on the market. Protein secretion by urine (U.Prot) is significantly lower in the animals treated with D-mannuronic acid (T1) than in the untreated patient group (P) and the group treated with piroxicam (T2) (Fig. 7) , The histological examinations of the kidney tissue using the parameters mentioned above show a significant reduction in the glomerular changes in the test group treated with D-mannuronic acid in comparison with the untreated patient group (P) (FIG. 8).
  • Example of the use of the anti-inflammatory agent D-mannuronic acid for the treatment of T-cell-mediated autoimmune diseases Example of the influence on multiple sclerosis can be estimated. This is demonstrated by the treatment of autoimmune encephalon ayelitis (EAE) in rats, which is induced by the myelin-based protein (MBP) and serves as an animal model for multiple sclerosis (MS).
  • EAE autoimmune encephalon ayelitis
  • MBP myelin-based protein
  • MS multiple sclerosis
  • CFA Complete Freund's Adjuvant
  • the rats are weighed daily and examined for neurological peculiarities.
  • the data are presented as an "average clinical picture" (mean clinical score).
  • a total of 18 injections (ip) of the solutions described are administered to both groups. The injections are started one day before the immunization (day -1) and continued until the 16th day after the immunization (day +16). On the 21st day after the immunization, 4 rats from each group are removed from the lymph nodes and placed in Hank's solution. Then they are pressed through a mesh sieve to obtain a lymph node cell suspension.
  • the reaction of the cell suspension to MBP and the Mitogen ConA is tested in growth experiments.
  • the cells of the lymph nodes are washed and inoculated in microtiter plate cultures with a concentration of 2 * 10 5 cells / chamber. 5 or 10 ⁇ g / ml MBP or 2 ⁇ g / ml Con A are added to the cells.
  • the test batches are carried out 4 times each and 200 ⁇ l of stimulation medium are added to each test batch, the 1 mM L-glutamine, 5 * 10 ⁇ 5 M 2-mercaptoethanol (ME) and 1% fresh gene-identical serum, the with and has been prepared without antibodies. After 72 h of cultivation at 37 ° C.
  • the cultures 5-bromo-2'-deoxyuridine (BrdU) are fed. 2 hours after the addition, the cell growth in the individual test batches is quantified using a standard cell growth ELISA kit (Boehringer Mannheim, Germany). The resulting color development is proportional to the concentration of BrdU in the DNA-synthesizing cells of the individual cell cultures. The absorption is determined using an optical densitometer.
  • the first neurological peculiarities occur 10 days after the immunization of EAE (FIG. 9) and reach their maximum 13 days after the immunization. From the 12th day after the immunization, the mean body weight also decreased more in the EAE control group than in the EAE group (FIG. 10).
  • the average clinical picture of the EAE group was significantly lower than that of the EAE control group.
  • the lowering of the IL-6 level in the blood serum of rats is another example that demonstrates the anti-inflammatory effect of D-mannuronic acid.
  • the rat nephrotic syndrome is the result of an injection of adriamycin.
  • D-mannuronic acid and piroxicam were administered to the experimental animals (patient groups were treated with D-mannuronic acid (Tl) and piroxicam (T2)) 6 days after the onset of the disease in the same amounts as described in use example 3.
  • Tl D-mannuronic acid
  • T2 piroxicam
  • the treatment is also ended here after 28 days.
  • the animals were killed 43 days after the start of the treatment and the serum was examined for the amount of IL-6 present in the serum using the IL-6 ELISA kit. The results show (FIG.
  • the cells were treated with 80 ⁇ g / ml D-mannuronic acid, dexamethasone and piroxicam for 24 hours.
  • a further control group was examined that remained untreated. After the 24 hours had elapsed, they were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100.
  • the APO-BRDU TM apoptosis kit was carried out with these cells.
  • the cell nuclei have been stained with fluorescein and propidium iodide.
  • the cell number and the cell phase in which they are located were examined using a flow cytometer (FACSCalibar) from Becton Dickinson (USA). The results are expressed in percent of apoptotic cells.
  • the results of the flow cytometry can be seen in FIG.
  • the individual graphs represent the following control approaches on the left-hand side from top left to bottom left: (1) negative control of the non-apoptotic cells supplied with the test kit become; (2) positive control of the apoptotic cells supplied with the test kit; (3) Fibrosarcoma cells not treated with dexamethasone, piroxicam or D-mannuronic acid (WHI 164).
  • the individual graphs on the right-hand side show the following experimental approaches from top right to bottom right: (4) cells treated with dexamethasone; (5) cells treated with piroxicam; and (6) cells treated with D-mannuronic acid.
  • the apoptosis rate of the test batches shows an apoptosis rate of 17.42% in comparison with the control batches with treatment with D-mannuronic acid, with treatment with dexamethasone of 20.92% and with treatment with piroxicam of 28.15%.
  • the results show a reduced apoptosis rate when treated with D-mannuronic acid, which even goes slightly beyond that of cells treated with dexamethasone and piroxicam.

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  • Physical Education & Sports Medicine (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé de production d'acides uroniques monomères, en particulier un acide mannuronique et un acide guluronique, de préférence à partir de polymannuronate ou de polyguluronate produits de manière microbienne et leurs dérivés pharmacologiquement actifs. L'invention concerne également l'utilisation desdites substances dans la production d'une composition pharmacteutique, qui agit de manière antiphlogistique et qui peut être utilisée dans le traitement de l'arthrite.
PCT/DE2003/003349 2002-10-09 2003-10-08 Procede de production d'acide uronique monomere, en particulier d'un acide d-mannuronique et d'un acide d-guluronique, WO2004033472A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003277820A AU2003277820A1 (en) 2002-10-09 2003-10-08 Method for producing monomer uronic acids, especially d- mannuronic acid and d-guluronic acid and derivatives thereof, and the use of the same as medicaments

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
DE10247073A DE10247073B4 (de) 2002-10-09 2002-10-09 Verfahren zur Herstellung von monomeren Uronsäuren, insbesondere D-Mannuronsäure und D-Guluronsäure und deren Verwendung als Arzneimittel
DE10247073.1 2002-10-09
DE10253514A DE10253514A1 (de) 2002-11-16 2002-11-16 Verfahren zur Herstellung von monomeren Uronsäuren, insbesondere D-Mannuronsäure und D-Guloronsäure und deren Verwendung als Arzneimittel
DE10253514.0 2002-11-16
DE10261693A DE10261693A1 (de) 2002-10-09 2002-12-30 Verfahren zur Herstellung von monomeren Uronsäuren, insbesondere D-Mannuronsäure und D-Guluronsäure, Derivaten davon und deren Verwendung als Arzneimittel
DE10261693.0 2002-12-30

Publications (2)

Publication Number Publication Date
WO2004033472A1 true WO2004033472A1 (fr) 2004-04-22
WO2004033472A8 WO2004033472A8 (fr) 2004-08-26

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PCT/DE2003/003349 WO2004033472A1 (fr) 2002-10-09 2003-10-08 Procede de production d'acide uronique monomere, en particulier d'un acide d-mannuronique et d'un acide d-guluronique,

Country Status (2)

Country Link
AU (1) AU2003277820A1 (fr)
WO (1) WO2004033472A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110151773A (zh) * 2019-05-13 2019-08-23 深圳大学 寡聚古罗糖醛酸在预防和治疗Tau蛋白病药物中的应用

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Publication number Priority date Publication date Assignee Title
WO1995024497A2 (fr) * 1994-03-11 1995-09-14 Fidia Advanced Biopolymers S.R.L. Preparation enzymatique de polysaccharides
WO2001056404A1 (fr) * 2000-02-03 2001-08-09 Kbp Co., Ltd. Polymannuronate de faible poids moleculaire

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WO1995024497A2 (fr) * 1994-03-11 1995-09-14 Fidia Advanced Biopolymers S.R.L. Preparation enzymatique de polysaccharides
WO2001056404A1 (fr) * 2000-02-03 2001-08-09 Kbp Co., Ltd. Polymannuronate de faible poids moleculaire

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CHANDA N P ET AL: "Alginic acids in Lessonia trabeculata: characterization by formic acid hydrolysis and FT-IR spectroscopy", CARBOHYDRATE POLYMERS, APPLIED SCIENCE PUBLISHERS, LTD. BARKING, GB, vol. 46, no. 1, September 2001 (2001-09-01), pages 81 - 87, XP004247274, ISSN: 0144-8617 *
F. SMITH ET AL.: "Synthesis of uronic acids. Part II. 2:3:4-Trimethyl derivatives of mannuronic, glucuronic and galacturonic acids", JOURNAL OF THE CHEMICAL SOCIETY, 1944, pages 131 - 134, XP008028256 *
HEYRAUD A ET AL: "An enzymatic method for preparation of homopolymannuronate blocks and strictly alternating sequences of mannuronic and guluronic units", CARBOHYDRATE RESEARCH, ELSEVIER SCIENTIFIC PUBLISHING COMPANY. AMSTERDAM, NL, vol. 308, no. 3-4, April 1998 (1998-04-01), pages 417 - 422, XP004181405, ISSN: 0008-6215 *
J. SZEJTLI: "The stepwise total hydrolysis of alginic acid", ACTA CHIMICA ACADERNIAE SCIENTIARUM HUNGARICAE, vol. 47, 1966, pages 301 - 310, XP008028265 *
OTTERLEI M ET AL: "INDUCTION OF CYTOKINE PRODUCTION FROM HUMAN MONOCYTES STIMULATED WITH ALGINATE", JOURNAL OF IMMUNOTHERAPY, RAVEN PRESS, NEW YORK, NY, US, vol. 10, no. 4, August 1991 (1991-08-01), pages 286 - 291, XP000882645, ISSN: 1053-8550 *
R.G. AULT ET AL.: "Preparation of d-mannuronic acid and its derivatives", JOURNAL OF THE CHEMICAL SOCIETY, 1935, pages 517 - 518, XP008028255 *
S.N. DANILOV, L.I. RASTORGUEVA: "Hydrolysis of alginic acid and derivatives of D-mannuronic acid", ZHURNAL OBSHCHEI KHIMII, vol. 25, 1955, pages 1549 - 1557, XP008028267 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110151773A (zh) * 2019-05-13 2019-08-23 深圳大学 寡聚古罗糖醛酸在预防和治疗Tau蛋白病药物中的应用

Also Published As

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WO2004033472A8 (fr) 2004-08-26
AU2003277820A1 (en) 2004-05-04

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