WO2002069984A2

WO2002069984A2 - Use of hyaluronic acid uronides for the treatment of inflammatory processes

Info

Publication number: WO2002069984A2
Application number: PCT/DE2002/000688
Authority: WO
Inventors: Peter-Jürgen Müller; Albert Härtl; Waltraud Hertel; Jörg-Hermann Ozegowski
Original assignee: Hans-Knöll-Institut für Naturstoff-Forschung e.V.; Friedrich-Schiller-Universität Jena
Priority date: 2001-03-02
Filing date: 2002-02-21
Publication date: 2002-09-12
Also published as: EP1408990A2; DE10111165A1; WO2002069984A3

Abstract

The invention relates to the intracorporeal use of highly active, low molecular weight hyaluronic acid uronides of biotechnological origin for the treatment of inflammatory processes and fostering health in humans and animals.

Description

Use of hyaluronic acid uronides for the treatment of inflammatory processes

The invention relates to the use of systemically and intracorporeally topical hyaluronic acid uronides for the treatment of inflammatory processes and for promoting health in humans and animals. The invention can be used with utility in the pharmaceutical industry as well as the food and feed industries.

It is known that hyaluronic acid in humans and animals, preferably the monogastric animal, has a multitude of effects which promote health, well-being and appearance. The high-molecular hyaluronic acids mainly cause mechanical effects in the body. In particular, because of their viscoelastic properties in the synovial fluid, they develop an effect that lubricates the joints (US 4,801,619, US 5,972,909, US 5,079,236). In the subcutaneous tissue, the high-molecular hyaluronic acid causes the turgor, which is responsible for the firmness and thus for the youthful appearance. Degradation processes, in which low-molecular oligohyaluronic acids are formed, allow the equilibrium concentration of hyaluronic acid in the organs to reach a low level, which is detrimental to health. This deficit can be compensated for by an increased incorporeal supply of high-molecular but also of low-molecular hyaluronic acid.

According to JP 5.111.367, a food can be obtained from cockscombs by subjecting the tissue to various cleaning and digestion procedures, which also leads to a partial breakdown of the hyaluronic acid. It is pointed out that hyaluronic acid in humans and animals develops a multitude of beneficial effects, which promote health, in particular in the case of traumatic tissue symptoms in the joint areas, and also the condition and appearance.

A number of property rights describes the topical application of hyaluronic acid and its salts in arthritis through intra-articular injection. US 4,808,576 protects systemically acting injection preparations which can be injected or applied topically dermally at locations which are not identical to the location of the traumatic tissue changes. The sites of action in the healing of traumatized tissue differ from the application sites, e.g. the treatment of arthritis of the joints in accordance with US 4,808,576 does not require intra-articular application.

Hyaluronic acid is also proposed as a tolerance-improving component in peritoneal perfusion solutions (JP 1.151.462).

DE 19853066 protects low-molecular fragments of hyaluronic acid (oligohyaluronic acids) for the production of vaccines, produced by digestion with hyaluronidase from high-molecular hyaluronic acid, and DE 198339113 also uses low-molecular hyaluronic acid produced by digestion with hyaluronidase in the production of dendritic cells. In addition to the injectable preparations, oral preparations are also proposed. The patents state that the polymeric compounds pass through the gastrointestinal tract and are absorbed largely undamaged in the small intestine through the intestinal wall and via the blood or lymph in the Body are transported. The increased supply of mucopolysaccharides leads to a health-promoting systemic effect, especially in organs in which there is a deficiency in hyaluronic acid.

Oral administration of hyaluronic acid is e.g. described in JP 5.111.367. The hyaluronic acid is obtained from cockscombs by subjecting the tissue to various cleaning and digestion procedures. JP 9.262.057 describe a special food that i.a. Contains hyaluronic acid and is recommended for halitosis.

According to JP 10.165.138, a food that promotes health and beauty contains special mucopolysaccharides and nucleic acids. The mucopolysaccharides consist of a complex of hyaluronic acid, chondroitin sulfate and collagen as well as nucleic acids.

WO 97/25051 describes preparations with hyaluronic acid to be taken orally to prevent or prevent diseases and therapeutic preparations consisting of hyaluronan, a hyaluronic acid which additionally contains protein of animal origin crosslinked with formalin, in particular bird protein. JP 11.124.401 protects the production of oligohyaluronic acids with molar masses less than 10,000 D with a hyaluronic acid-degrading enzyme and uses ultrafilter modules with a cut-off not less than 10,000 D when removing the enzyme. JP 09.098.739 also describes the production of low molecular weight hyaluronic acid. The oligohyaluronic acids or low molecular weight hyaluronic acids are used as a food additive or for pharmaceutical and cosmetic applications, e.g. B. proposed with effect against aging of the skin. RU 2.137.402 describes a special additive to dietary food, consisting of dried algae biomass with the addition of hyaluronic acid and possibly antioxidants, vitamin C and starch.

JP 10.056.983 uses hyaluronic acid or cockerel extracts to improve the texture of gel-like foods. JP 4.197.145 uses hyaluronic acid in an amount of 0.05 to 0.8% for the preparation of starchy foods such as pastries, cooked rice or cakes. This way, an improved structure is obtained after the baking process and the food stays fresh longer.

In the case of hyaluronic acid obtained from animals, there is the serious disadvantage that the supply of hyaluronic acid, which is inherently beneficial for health, can at the same time lead to the transfer of infectious or allergenic material such as viruses, prions or nucleic acids. The animal proteins that are present as contaminants whenever the hyaluronic acid is obtained from animal material can lead to an antigenic reaction. Furthermore, there is a risk that when collecting the perishable animal organs such. B. cockscombs can lead to microbial infections in the material in which microbial toxins are transmitted to the consumer. This danger is not eliminated even in animal material that has been treated with formalin to prevent microbial infections. On the contrary; animal binding proteins that bind to glycosaminoglycans can thereby additionally be chemically crosslinked. They are then immobilized on this material as impurities which are difficult to dissolve and cannot be removed by any cleaning procedures, such as, for example, commercially bundled hyaluronan. It is also disadvantageous that the effect of the hyaluronic acid obtained in this way on the traumatic tissue is only insufficiently characterized.

The tissue absorption of the preparations of higher molecular weight hyaluronic acid usually used with molecular weights between about 100,000 D and about 2,500,000 D is relatively low due to the high molecular weights; the greater part is excreted via the digestive tract. Another general disadvantage of high-molecular hyaluronic acid is that it can only be used in injectables due to the high viscosity of its aqueous solutions up to a concentration of about 20 mg / ml.

It can be assumed that the smaller fragments of hyaluronic acid are probably better absorbed than high-molecular hyaluronic acid and thus reach the places with deficiency symptoms or with traumatic tissue changes via the bloodstream or the lymphatic system. In the cases where low molecular weight hyaluronic acid (oligohyaluronic acids) with molar masses less than 100,000 D are used, hydrolytic cleavage of the hyaluronic acid with hyaluronidase was always carried out in order to break down the high molecular weight hyaluronic acid hydrolytically into small fragments with the addition of water. The hyaluronidase is currently generally obtained from animal tissue, in particular from cattle testicles. The use of hyaluronidase of animal origin such as e.g. from cattle testicles due to the risk of transmission of infectious material from the animal to the user.

As an alternative, the enzyme hyaluronate lyase was used in WO 0.038.647 in order to produce low-molecular hyaluronic acid fragments. Due to the specific cleavage mechanism of this enzyme, a double bond is formed on the glucuronyl residue in the Δ-4,5-position. The cleavage of hyaluronic acid with hyaluronate lyase accordingly leads to chemical cleavage products which differ from the hydrolysis products of hydrolysis with hyaluronidase. The double bonds after cleavage with hyaluronate lyase change the physiological properties of the cleavage products. For example, it is responsible for the special radical-binding properties of uronide. Because of this effect, WO 0.038.647 describes the epidermal topical application of formulations with hyaluronic acid uronides to protect, maintain or reconstruct the normal function and structure of human and animal skin and / or to prevent environmental, including UV-related Skin or mucous membrane damage described by UV radiation.

It is the aim of the invention to effectively promote the healing of traumatic tissue or inflammation, for example rheumatoid arthritis, or to prevent the occurrence of such phenomena with their known negative effects on the state of health and the disadvantages of known procedures in the care of humans and animals to deal with hyaluronic acid or oligohyaluronic acids as the active ingredient.

The object on which the objective is based is achieved according to the invention by the use of hyaluronic acid uronides as active ingredients for the treatment of inflammatory processes in human and animal tissues, the Uronids are administered intracorporeally and act systemically. According to the invention, uronides of hyaluronic acid with molecular weights less than 80,000 D, preferably with molecular weights between 5,000 and 30,000 D, are used. The uronides contain less than 0.5%, less than 0.05% proteins for applications in injectables.

A characteristic feature of the hyaluronic acid uronides is their in vitro proven, previously unknown effect of being more angiogenic and anti-inflammatory than corresponding amounts of high-molecular hyaluronic acid or the low-molecular hyaluronic acid obtained with hyaluronidase. An advantage of the supply of angiogenic hyaluronic acid uronides in people and animals with undersupplied hyaluronic acid is that they improve the growth of blood vessels in traumatized tissues or promote health or accelerate healing. The intracorporeal administration of the uronides means that the rate of degradation of the body's own high-molecular hyaluronic acid, which is a pool for the body's own low-molecular hyaluronic acid, which has beneficial physiological effects, is reduced. The supply of external hyaluronic acid uronides has a positive influence on the pool of high molecular hyaluronic acid because the natural outflow to the low molecular hyaluronic acids is reduced.

Another advantage of using the uronides is that they do not produce any disadvantageous viscosity in aqueous solution for the use according to the invention.

The use of the uronides according to the invention is new; So far, hyaluronic acid uronides have not been proposed for oral applications or as injectables, infusion solutions or rinsing solutions. The angiogenic and anti-inflammatory properties of uronides have not been described. The epidermal topical applications as free radical scavengers or the use as protection against UV rays are expressly not part of the present invention.

Possibilities according to the invention for intracorporeal application of the uronides are the use in the form of an injection preparation, a rinsing solution or an infusion solution, with which the uronids reach the body directly without going through the digestive tract. Flushing solutions are solutions that flush body cavities and wound cavities to achieve a dual effect, removing harmful substances, and aiding healing. When used in the form of an injection, isotonic sterile solutions are suggested which contain between 1 mg / ml and 200 mg / ml hyaluronic acid uronides.

When used as perfusion, infusion or rinsing solutions, these contain between 0.1 g / l and 100 g / l uronides as well as auxiliary substances and possibly other active substances or medicines. Physiological saline is preferably used as the solvent. Another way according to the invention for indirect administration is to add the uronides to food or feed. For this purpose, concentrated uronides are present in non-perishable or storage-stable, unmixable and defined form in the foodstuffs or feedstuffs in various stages of the food or feed preparation. They can also be used directly in food supplements. The uronides are proposed as food supplements in solid form, for example as powders, pellets, capsules or tablets or liquid formulations such as beverage additives, ready-made drinks or pasty, gel-like or semi-solid form. Without thereby restricting the invention, up to 30 g uronide per day without adverse health effects per 80 kg body weight are recommended for human applications. In further versions, β-sitosterol, α-tocopherol, vitamins and / or lecithin are added. For monogastric animals, higher doses can be used depending on their weight. The possibility of realizing such a high daily dose represents a clear advantage of the invention compared to the oral intake of high-molecular or high-viscosity hyaluronic acid known per se.

The uronides used in accordance with the invention are in any case produced in a manner known per se by the action of an endogenously cleaving hyaluronate lyase from microorganisms, for example with an endo-hyaluronate lyase from a Streptococcus. The purified enzyme is placed in a solution of high molecular hyaluronic acid with molecular weights greater than 500,000 D. The concentration of hyaluronic acid in the solutions is between 1 g / l to 20 g / l. The starting solutions can be prepared by dissolving hyaluronic acid in water or buffer solutions. A more economical way of manufacturing is to start from hyaluronic acid solutions that are produced in the manufacturing process of hyaluronic acid. The advantage of the last production variant is that a drying step can be avoided. The cleavage reaction is interrupted by increasing the temperature above 50 ° C. by denaturing the hyaluronate lyase.

The reaction and the end of the reaction are monitored photometrically via the formation of a double bond in the Δ-4,5-position on the glucuronyl radical with an absorption maximum at 232 nm. The molecular weights are determined using a laser light scattering method.

Following the cleavage, it is possible to hydrolyze the protein impurities present in solution, including the denatured hyaluronate lyase, by adding proteases, in particular microbial or vegetable origin. Neutral proteases of plant or microbial origin are preferably used as proteases. The optionally added proteases or other high-molecular impurities such as lipoproteins still present in the hyaluronic acid can be removed by ultrafiltration with an ultrafilter module whose cut-off can be a maximum of 0.7 to 0.3 times lower than the average molar mass of the uronides. The uronide-free retentate is discarded and the run is concentrated with an ultrafilter. For concentration and partial purification, the solution or the retentate, which contains the uronides and possibly cleavage products of the protease treatment, is concentrated by ultrafiltration via ultrafilter modules. Surprisingly, it was found that the ultrafilter must preferably have a cut-off for retentate formation which is 15 to 35 times lower than the average molar mass of the uronides present after the enzymatic degradation. Enrichment with an ultrafilter of 1 kD gives a concentrated uronide solution with an average molecular weight between 20,000 and 30,000 D. Experience has shown that the use of ultrafilters with a larger cut off, for example of 5 kD instead of 1 kD, leads to considerable losses of the uronides with molecular weights of 20,000 to 30,000 D in the concentrate. Uronides with molecular weights greater than 80,000, on the other hand, can be cleaned with an ultrafilter with a cut off of 5 kD. In the production of a uronide with molecular weights between 20,000 and 30,000 D suitable for oral applications, the step of protein removal by ultrafiltration can be omitted if sufficiently clean hyaluronic acid preparations are used.

The uronide-containing concentrates are brought into a solid form by one of the measures such as precipitation in alcohols such as ethanol, freeze drying or spray drying.

After freeze-drying, a white solid product is obtained, which easily dissolves in water. In one embodiment, the concentrate is precipitated in ethanol or in isobutanol. The solid product is collected and dried.

Example 1

The angiogenic effect of the hyaluronic acid uronides is demonstrated with a test for vascularization on the chorionallantois membrane (CAM) of the chicken embryo ex ovo. Uronides with molecular weights of 30 kD, 100 kD and 235 kD are placed on a CAM irritated with 0.25% sodium dodecyl sulfate (SDS) and the angiogenic reactions are assessed subjectively after 24 hours. Fertilized chicken eggs are incubated for 3 days under standard conditions. On the third day of incubation, the eggs are disinfected with 70% ethanol. After dipping in ethanol, the eggs are air dried for a few minutes. During this time the embryo positions itself in the normal position. The hatching eggs are opened at the bottom and the contents are carefully transferred into sterile petri dishes (20 mm high, 200 mm diameter). 2.5 ml cell culture medium (89.5% Eagles minimum essential medium, 10% fetal calf serum, 500 lU / ml penicillin and 50 μg / ml streptomycin) are added to each petri dish. The shellless chicken embryos are incubated further in the incubator at 36.5 ° C and over 96% humidity and 0.7% CO ₂ concentration.

Test sequence:

The CAM is damaged from the 8th day of breeding with 0.25% SDS (20μl) in two different places. About an hour later, test solution (20μl) is superficially applied to one site of damage, the second remains untreated as a control. After 24 hours, the CAM reaction and the formation of new vessels are assessed verbally on the color monitor image. Test documentation:

The angiogenesis of the injured CAM is documented with the ^' Stemi 2000-C, Schott cold light source ^' KL 1500 ", color camera 2 1-CCD, type: MC-1009S, color monitor and JVC video cassette recorder SR-S388E. The 30 kD, 100 kD and 235 kD hyaluronic acid uronides significantly accelerate the angiogenic reactions in the first 24 hours after CAM irritation of the ex ovo-incubated chicken embryos after SDS treatment.

Embodiment 2

2.1 Production of uronides with an average molecular weight of 25 kD

50 g of hyaluronic acid are dissolved in 4.9 L of 0.01 M acetate buffer pH 7.0 at 4 ° C. overnight with stirring. The solution is then heated to 30 ° C. in a thermostat while stirring. For cleavage, 100 ml of a hyaluronate lyase solution containing 1000 U of hyaluronate lyase / ml of 0.01 M acetate buffer pH 7.0 are added to this solution. Hyaluronate lyase activity is determined according to the method described above. The 100 ml hyaluronate lyase solution is mixed in as quickly as possible, and the viscosity of the hyaluronic acid solution drops rapidly after only 3 minutes. It is incubated with stirring for 1 h at 30 ° C. Samples are continuously taken during the cleavage and their absorbance measured at 232 nm in 1 cm cuvettes. After incubation for one hour, the hyaluronate lyase activity is inactivated by heating to 75 ° C. for 5 minutes. After cooling the solution to 40 ° C., the uronides are concentrated in an ultrafiltration through an ultrafilter with a cut off of 1 kD (0.07 m ² ) to 1/10 of the starting volume. The oligosaccharides and disaccharides are separated off at the same time. The concentrate is lyophilized and the molecular weight is determined therefrom.

2.2 Production of uronides with an average molecular weight of 80 kD

50 g of hyaluronic acid are dissolved in 4.9 L of 0.01 M acetate buffer pH 7.0 at 4 ° C. overnight with stirring. The solution is then heated to 30 ° C. in a thermostat while stirring. For cleavage, 100 ml of a hyaluronate lyase solution containing 100 U of hyaluronate lyase / ml of 0.01 M acetate buffer pH 7.0 are added to this solution. Hyaluronate lyase activity is determined according to the method described above. The 100 ml hyaluronate lyase solution is mixed in as quickly as possible, the viscosity of the hyaluronic acid solution dropping rapidly after 10 minutes. It is incubated with stirring for 1 h at 30 ° C. Samples are continuously taken during the cleavage and their absorbance measured at 232 nm in 1 cm cuvettes. After incubation for one hour, the hyaluronate lyase activity is inactivated by heating to 75 ° C. for 5 minutes. After cooling the solution to 40 ° C., the uronides in the minisette are concentrated to 1/5 of the starting volume by ultrafiltration through a 5 kD ultrafilter (0.07 m ² ). The oligosaccharides and disaccharides are separated off at the same time. The concentrate is lyophilized and the molecular weight is determined therefrom. 2.3 Determination of the molecular weight

Molecular weight is determined using HPLC chromatography on PSSG gel using a HEMA Bio 2000 7.5 x 300 mm column.

2.4 Determination of hyaluronate lyase

Solutions:

0.1 M acetate buffer pH 6.0:

8.1 g of sodium acetate are dissolved in 100 ml of distilled water and the pH is adjusted to pH 6.0 with 1 M HCl and to 11 with distilled water. Replenished water.

Hyaluronic acid solution:

20 mg hyaluronic acid are dissolved in 100 ml 0.1 M acetate buffer pH 6.0

CTA solution:

2.5 g of cetyltrimethylammonium bromide are dissolved in 100 ml of 2% (2 g / 100 ml) sodium hydroxide solution with gentle heating.

Enzyme assay:

50 μl of 0.1 M acetate buffer pH 6.0 are placed in a microtiter plate. Then a geometric dilution series of 50 μl sample solution is added and 50 μl hyaluronic acid solution added. This approach is incubated at 37 ° C for 30 min. Then 200μl CTA solution are added. After 10 minutes, the plate is measured in the ELISA reader at 600 nm. The reciprocal dilution at which 50% of the hyaluronic acid is broken down indicates the activity of the hyaluronate lyase.

Definition of the unit:

One hyaluronate lyase unit corresponds to the activity, the 0.05 mg hyaluronic acid in 30

Minutes splits.

Embodiment 3

The hyaluronic acid is cleaved in accordance with embodiment 2.1. The ultrafiltration step is modified. 50 Anson units of the protease bromelain (Merck Eurolab GmbH) are added. The mixture is stirred at room temperature for 2 hours. The solution is then ultrafiltered through an ultrafilter with a cut off of 20 kD. The run is then concentrated to a retentate volume of 3 l with an ultrafilter with a cut off of 1 kD. The concentrate is then freeze-dried and taken in orally in the form of solutions and orally as a solution or addition to food and feed. Embodiment 4

10 g of uronides with an average molecular weight of 20 to 30 kD are mixed with 90 g of dry milk powder, 0.2 g of lecithin, 0.02 g of tocopherol, 1 g of β-sitosterol and 2 g of sucrose by grinding together and then taken orally as a dietary supplement.

Embodiment 5

90 g of cereal starch are mixed with 10 g of hyaluronic acid uronide in the form of their sodium salt and 0.1 g of a vitamin mixture and taken orally as a dietary supplement.

Embodiment 6

To prepare an injection solution containing about 50 mg / ml, 50 g of hyaluronic acid uronide with an average molecular weight of 25,000 D in the form of its sodium salt are dissolved in 1 liter of pyrogen-free physiological saline. The solution is sterile filtered, filled into ampoules as a solution for injection and injected into humans or animals.

Embodiment 7

To prepare a solution for applications in peritoneal perfusion, 1 g of glucose, 2 g of glycerol and 1 g of hyaluronic acid uronide in the form of their sodium salt are dissolved in one liter of water for injection and isotonic with saline and sterile filtered.

Embodiment 8

100 g hyaluronic acid uronide in the form of its sodium salt are dissolved with 100 ml of physiological saline and mixed with 0.02% potassium sorbate, 5 g glucose and 0.1% sorbic acid. The acidity is adjusted to a pH of 5.2 with dilute citric acid. Up to 10 ml of the solution are recommended daily for oral intake by an adult.

Claims

claims

1. Use of hyaluronic acid uronides as active ingredients for the treatment of inflammatory processes in human and animal tissues.

2. Use according to claim 1, characterized in that the hyaluronic acid uronides have molecular weights between 1,000 D and 80,000 D and a protein content of less than 0.5%.

3. Use according to claim 1, characterized in that the hyaluronic acid uronides are supplied intracorporeally.

4. Use according to claim 1, characterized in that the hyaluronic acid uronides are substances which are produced from hyaluronic acid or hyaluronate of biotechnical origin by the action of microbial hyaluronate lyases.

5. Use according to claim 1, characterized in that the hyaluronic acid uronides are used in intravenous, subcutaneous or intramuscular isotonic injection solutions which contain the uronides in concentrations between 1 mg / ml and 200 mg / ml and which are less than 0.05 % Protein, based on the uronides used.

6. Use according to claim 1, characterized in that the hyaluronic acid uronides are used in the form of isotonic perfusion, rinsing or infusion solution which contain the uronides in concentrations between 0.1 mg / ml and 100 mg / ml

7. Use according to claim 1, characterized in that the hyaluronic acid uronides are added to foods or animal feed.

8. Use according to claim 1, characterized in that the hyaluronic acid uronides are used in food supplements and these optionally contain tocopherols and / or β-sitosterol.

9. Use according to claim 1, characterized in that the hyaluronic acid uronides are used in dissolved form.

10. Use according to claim 1, characterized in that the hyaluronic acid uronides are used in solid form as powder, pellets, capsules or tablets or in pasty to gel-like semi-solid form.