EP4106869A1

EP4106869A1 - Collagen hydrolysate as active substance against periodontitis or gingivitis

Info

Publication number: EP4106869A1
Application number: EP20839280.3A
Authority: EP
Inventors: Stephan Hausmanns; Hans-Ulrich Frech; Steffen Oesser; Tonja Lipp
Original assignee: Gelita AG
Current assignee: Gelita AG
Priority date: 2020-02-18
Filing date: 2020-12-17
Publication date: 2022-12-28
Also published as: DE102020104279A1; KR20220143012A; WO2021164925A1; US20220387545A1; CA3168164A1; AU2020430016A1; MX2022009473A; CN115279457A; BR112022014640A2; JP2023515440A

Abstract

The invention relates to a collagen hydrolysate for use as an active substance in the treatment and/or prophylaxis of periodontitis or gingivitis.

Description

Collagen hydrolyzate as an active ingredient against periodontitis or gingivitis

The present invention relates to collagen hydrolyzate for use as an active ingredient in the treatment and / or prevention of periodontitis or gingivitis.

Periodontitis is an inflammation of the so-called tooth supporting apparatus, i.e. the entire tissue that holds the tooth in the jawbone is affected by this disease. Colloquially, but not medically correct, this disease is often referred to as periodontal disease.

In contrast, gingivitis is only an inflammation of the gums (gingiva) or the gum margin. Often, however, gingivitis is a preliminary stage of periodontitis and can develop into such, especially if it is not treated. Therefore, in the context of the present description, whenever a treatment or prevention of periodontitis is mentioned, it always includes treatment or prevention of gingivitis, unless the context indicates otherwise.

In industrialized countries, periodontitis and gingivitis are among the most common diseases. In Germany around 23 million people are affected.

Periodontitis can be acute, but very often it takes a chronic course. Since it is usually painless in the early stages, it is often recognized too late, often in patients between the ages of 40 and 50. Typical symptoms are redness, swelling, bleeding gums and receding gums. The latter leads to an increased sensitivity to pain in the exposed tooth necks. At a more advanced stage, periodontitis can lead to loosening of the teeth and ultimately to tooth loss. In addition to the negative and sometimes serious effects on the teeth holding apparatus or teeth, periodontitis can also have systemic effects. On the one hand, the inflamed tooth-supporting apparatus facilitates the penetration of pathogens into the bloodstream, and on the other hand, the inflammation-promoting messenger substances are poured out from the affected tissue. As a result, chronic periodontitis leads to a sometimes significantly increased risk of cardiovascular diseases, respiratory diseases, heart attacks and strokes.

A causal treatment of periodontitis is not yet known. The course of the disease can, however, be contained, in particular by regular mechanical removal of supragingival and subgingival dental plaque, with surgical opening of the gums being necessary in the advanced stage. In some cases it is also advisable to administer antibiotics. Overall, this treatment of periodontal disease is lengthy and complex.

Against this background, the invention is based on the object of proposing an active substance for the treatment and / or prevention of periodontitis or gingivitis.

To solve this problem, according to the present invention, the use of collagen hydrolyzate is proposed as a corresponding active ingredient. The effectiveness of collagen hydrolyzate in the treatment of periodontal disease was clearly demonstrated on the basis of a double-blind, placebo-controlled clinical study, the results of which are presented in detail below.

For collagen hydrolyzate, physiological effects have been known for a long time, especially in connection with osteoporosis or joint complaints. Positive effects of collagen hydrolyzate on the health of the skin have already been described, for example in the international patent application WO 2012/065782 A2. Nevertheless, the high effectiveness of collagen hydrolyzate in the treatment of periodontal disease is surprising. Collagen hydrolyzate is a degradation product of animal raw materials, which are also used as food, and is completely harmless to health and has no known harmful side effects. It does not require any legal approval as a medicinal product, but can, in particular, be placed on the market and used in the form of a dietary supplement. Within the scope of the present invention, the use of collagen hydrolyzate as a food supplement, as an over-the-counter drug (OTC) or as a prescription drug (in particular in combination with other active ingredients) is encompassed. Independent of this classification, the collagen hydrolyzate is a therapeutic agent.

The collagen hydrolyzate is conveniently administered orally. It is known that the peptides of the collagen hydrolyzate are at least to a certain extent absorbed in the intestine, even with relatively high molecular weights of up to 10,000 Da.

The specific form of administration of the collagen hydrolyzate can be that of a powder, a solution, a tablet or a capsule.

Further preferred forms of administration are chewing gum, in particular based on compressed tablets, lozenges or similar products that remain in the mouth for a longer period of time, as well as drinkable mouthwashes. In addition to the systemic effect, this type of administration also enables an intensive local action of the collagen hydrolyzate on the gums.

The daily dose of the administered collagen hydrolyzate, in particular in the case of oral administration, is favorably from approx. 1 to approx. 20 g, preferably from approx. 2 to approx. 15 g, more preferably from approx. 3 to approx. 10 g. An effectiveness could be shown in the clinical study with a daily dose of 5 g collagen hydrolyzate. A preferred embodiment of the invention relates to the treatment of periodontitis or gingivitis, ie the administration of the collagen hydrolyzate to a patient with a corresponding disease, in particular with chronic periodontitis. In this case, the administration, particularly in the case of an advanced disease, is advantageously carried out in combination with another therapy for periodontal disease. This additional therapy includes, in particular, the mechanical removal of supragingival and subgingival plaque (biofilm and tartar). Even if the administration of collagen hydrolyzate according to the invention does not replace the removal of the plaque, it can positively influence the course of the disease and significantly reduce the symptoms and complaints of periodontitis. Under certain circumstances, a severe course of the disease, which ultimately also requires surgical measures, can be prevented by the administration of collagen hydrolyzate.

Another embodiment of the invention relates to the prevention of periodontitis or gingivitis. In this context, it is particularly advantageous to administer collagen hydrolyzate to a patient who has one or more risk factors for these diseases. The risk factors for periodontitis include, in particular, a weakened immune system, tobacco smoke, hormonal changes (e.g. menopause) and certain genetic diseases (e.g. Down syndrome). Existing gingivitis can generally be viewed as a risk factor for periodontitis, so that treatment of gingivitis by administering collagen hydrolyzate is also a prevention of periodontitis.

The collagen hydrolyzate as the active ingredient according to the invention typically has an average molecular weight of 500 to 15,000 Da, preferably 1,000 to 8,000 Da, more preferably 1,500 to 5,000 Da, most preferably 1,800 to 2,200 Da. This information always means the weight average molecular weight, which can be determined in particular by gel permeation chromatography.

The collagen hydrolyzate is preferably produced by enzymatic hydrolysis of a collagen-containing starting material. Be for this hydrolysis in particular endopeptidases or exopeptidases of microbial or plant origin are used. By suitable selection of the peptidases and the hydrolysis conditions, collagen hydrolysates can be produced in the particular molecular weight range desired.

The collagen-containing starting material is usually selected from the skin or bones of vertebrates, preferably from mammals or birds, and in particular from the skin of cattle or pigs (beef split or pig skin) or of sheep. Alternatively, the collagen-containing starting material can be selected from the skin, bones and / or scales of fish, in particular cold or warm water fish.

The collagen hydrolyzate can either be produced in a one-step process from these starting materials or via the intermediate gelatin, in which case both type A and type B gelatin can be used.

The collagen hydrolyzate is preferably produced by the successive action of at least two endoproteases with a different specificity, in particular of at least two different metalloproteases and / or serine proteases, ie of proteases which have the amino acid sequence of the collagen molecules before or after certain ami nosacids split. The metalloproteases and / or serine proteases are advantageously enzymes from the microorganisms Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Aspergillus oryzae and Aspergillus melleus.

By selecting suitable endoproteases, not only can a specific molecular weight distribution of the collagen hydrolyzate be obtained, but the type of amino acids at the termini of the peptides contained in the hydrolyzate is also influenced. In this regard, it is preferred, for example, if at least 50% of the N-terminal amino acids of the collagen hydrolyzate are hydrophobic amino acids, in particular alanine, leucine and isoleucine. As an alternative to enzymatic hydrolysis, the collagen hydrolyzate can be produced in the context of the invention by recombinant gene expression. By using natural collagen sequences, especially from cattle or pigs, and expressing them in genetically modified cells (e.g. yeast, bacteria or plant cells, especially tobacco), products can be produced which are essentially identical to the hydrolysis products of the corresponding collagen-containing raw materials. It is possible, please include to keep a narrower or precisely predetermined molecular weight distribution.

The present invention also relates to a therapeutic method for the treatment and / or prevention of periodontal disease or gingivitis, comprising the administration of collagen hydrolyzate to a patient. Advantages and preferred embodiments of this method have already been described in connection with the collagen hydrolyzate according to the invention.

The effectiveness of collagen hydrolyzate in the treatment of periodontitis is explained in more detail on the basis of the clinical study described below and on the basis of cell tests on gingival fibroblasts.

1. Clinical study

1.1 Study desian

The double-blind, placebo-controlled clinical study was carried out at the Department of Periodontology at the University Hospital Würzburg with 43 patients who were receiving regular treatment there for existing periodontitis (removal of subgingival plaque two to four times a year). All study participants had at least three teeth with a gingival index of 1 or 2 (see below) and a probing depth of the gingival pockets of at least 3 mm. The participants were between 35 and 70 years old and had a BMI between 24 and 30. The participants were randomly divided into a treatment group with 23 patients and a placebo group with 20 patients, whereby neither the participants nor the treating staff were informed about the classification of the individual participants.

The participants in the treatment group received a daily dose of 5 g collagen hydrolyzate for the entire study period of 90 ± 14 days. A collagen hydrolyzate with an average molecular weight of approx.

2,000 Da is used, which is sold by the applicant GELITA AG under the name VERISOL B. The production of VERISOL B essentially corresponds to the production process described in WO 2012/065782 A2.

Instead of the collagen hydrolyzate, the participants in the placebo group received a corresponding daily dose of a placebo, which was indistinguishable from the collagen hydrolyzate in terms of packaging, texture and taste.

At the beginning of the study (VI on day 0), all study participants were examined in detail and a number of parameters and indices were determined that characterize the status of periodontitis. The patients then received professional teeth cleaning with mechanical removal of supragingival and subgingival plaque.

The same parameters and indices were determined again in a second examination (V2 after 60 ± 14 days) and in a third examination at the end of the study period (V3 after 90 ± 14 days). Not all studies could be carried out on all of the original 43 study participants; the number of participants in the two groups examined is given in the following analysis. A comparison of the changes in the determined parameters and indices between the treatment group and the placebo group allows conclusions to be drawn about the effectiveness of the collagen hydrolyzate in the treatment of periodontitis.

1.2 probing blood

During bleeding on probing (BoP), a periodontal probe is inserted into the gingival pockets. Any bleeding that occurs here is a sure sign of acute inflammation.

The probing bleeding in examinations VI, V2 and V3 was carried out with a PCP-11 periodontal probe, the result being that the percentage of all gingival pockets in which bleeding occurred was recorded as BoP [%] for each patient. The results are shown in Table 1 below.

Table 1: Probing bleeding (BoP) These results show that the percentage of bleeding gingival pockets decreased in all patients during the study period, but significantly more so in the participants in the treatment group than in the participants in the placebo group.

1.3 Plaque Control Record

To determine the plaque control record (PCR) according to O'Leary et al. In 1972 the plaque is stained and its extent is visually recorded along the entire gumline (i.e. on all sides of the tooth). The parameter results from the number of plaque-positive areas in relation to the total number of areas assessed in percent.

The results for the PCR [%] in examinations VI, V2 and V3 are shown in Table 2 below.

Table 2: Plaque Control Record (PCR) The results show that the PCR decreased in all patients during the study period, but significantly more so in the participants in the treatment group than in the participants in the placebo group.

1.4 Gingival index

The gingival index (Gl) according to Löe 1967 (modified from Lobene et al. 1986) results from the visual assessment of the acute inflammation of the gums on the buccal side according to the following scheme:

Grade 0: no inflammation

Grade 1 color change of a gum area, but not the entire gumline or the gingival papilla Grade 2: color change that includes the entire gumline or the gingival papilla

Grade 3: as in Grade 2, but pronounced reddening

For each patient, the gingival index Gl is the mean value of the indexes for all teeth. The test results for the gingival index are shown in Table 3 below.

Table 3: Gingival index (Gl)

The results show that the gingival index of all patients decreased during the study period, i.e. the acute degree of gingival inflammation decreased. However, this decrease is significantly greater in the participants in the treatment group than in the participants in the placebo group.

1.5 PISA score

The PISA score indicates the extent of the periodontal inflamed surface area in mm ² . The PISA score can be calculated from the parameters described above; the results are shown in Table 4 below.

Table 4: PISA score The results show a significant reduction in the PISA score for all patients during the study period, but the change in this parameter is significantly more pronounced in the participants in the treatment group than in the participants in the placebo group.

1.6 Summary

Using the parameters probing bleeding, plaque control record, gingival index and PISA score, the clinical study shows that the administration of 5 g of collagen hydrolyzate a day achieved a significantly greater improvement in periodontitis than in the placebo group. This result was achieved over a relatively short period of three months, with both groups receiving the same conventional treatment (supragingival and subgingival removal of plaque).

The results clearly demonstrate the effectiveness of collagen hydrolyzate as an active ingredient in the treatment and / or prevention of periodontitis or gingivitis.

2. Cell experiments on gingival fibroblasts

The gums are made up of gingival fibroblasts (gum cells) and the extracellular matrix formed by these cells, which contains various types of collagen and proteoglycans. In the case of periodontitis or gingivitis, there is typically a disruption of the balance between the biosynthesis and the breakdown of these matrix proteins. Stimulation of the synthesis of matrix proteins by the fibroblasts can thus counteract periodontitis or gingivitis.

In the cell experiments described below, the stimulating effect of collagen hydrolyzates with various mean molecular weights on the synthesis of the matrix proteins type I collagen, biglycan and decorin in human gingival fibroblasts was investigated in vitro. This was done by determining the expression of the corresponding mRNA using Real-time PCR and a semi-quantitative evaluation (based on a control without collagen hydrolyzate).

The human gingival fibroblasts were isolated from surgically removed gums by enzymatic digestion and cultivated in HAMs F12 medium, which was supplemented with 10% fetal calf serum, 20 U / ml penicillin-streptomycin and 50 pg / ml partricin. After a confluence of 80% had been reached, the culture medium was exchanged for fresh medium which had been supplemented with 0.5 mg / ml collagen hydrolyzate.

The cell tests were carried out with three different collagen hydrolyzates: (1) a bovine collagen hydrolyzate with an average molecular weight of approx. 2,000 Da, which was also used in the above clinical study, (2) a porcine collagen hydrolyzate with an average molecular weight of approx. 3,000 Da and (3) a bovine collagen hydrolyzate with an average molecular weight of approx. 6,000 Da.

After the fibroblasts had been incubated for 24 h in the presence of the respective collagen hydrolyzate (or without collagen hydrolyzate in the case of the control), the cells were harvested and lysed, and then the entire RNA was extracted and precipitated.

The results of the semiquantitative evaluation of the amounts of mRNA for type I collagen, biglycan and decorin are shown in the figures.

They show in detail:

Figure 1: Stimulation of the synthesis of matrix proteins by a bovine

Collagen hydrolyzate with approx. 2,000 Da;

FIG. 2: Stimulation of the synthesis of matrix proteins by a porcine collagen hydrolyzate with approx. 3,000 Da; and Figure 3: Stimulation of the synthesis of matrix proteins by a bovine

Collagen hydrolyzate with approx. 6,000 Da.

All three examined collagen hydrolyzates lead accordingly to a clear stimulation of the synthesis of the extracellular matrix proteins type I collagen, biglycan and decorin by human gingival fibroblasts in vitro. This effect is most pronounced in the case of the collagen hydrolyzate with an average molecular weight of approx. 2,000 Da, and this particularly with regard to the stimulation of the synthesis of type I collagen and decorin.

This result confirms the effectiveness of collagen hydrolyzate in the treatment and / or prevention of periodontal disease or gingivitis. At least one of the mechanisms of action is the strengthening of the extracellular matrix of the gums through an increased synthesis of matrix proteins under the action of collagen hydrolyzate.

Claims

Patent claims

1. Collagen hydrolyzate for use as an active ingredient in the treatment and / or prevention of periodontal disease or gingivitis.

2. Collagen hydrolyzate for use according to claim 1, wherein the collagen hydrolyzate is a food supplement or a drug.

3. Collagen hydrolyzate for use according to one of the preceding claims, wherein the collagen hydrolyzate is to be administered orally.

4. collagen hydrolyzate for use according to claim 3, wherein the collagen hydrolyzate is to be administered in the form of a powder, a solution, a tablet or a capsule.

5. collagen hydrolyzate for use according to any one of the preceding claims, wherein the collagen hydrolyzate is to be administered in a daily dose of approx. 1 to approx. 20 g, preferably in a daily dose of approx. 2 to approx. 15 g, more preferably in a daily dose of approx. 3 to approx. 10 g.

6. Collagen hydrolyzate for use according to one of the preceding claims, wherein the collagen hydrolyzate is to be administered to a patient with periodontitis or gingivitis, in particular with chronic periodontitis.

7. collagen hydrolyzate for use according to claim 6, wherein the collagen hydrolyzate is to be administered in combination with another therapy of parodon titis or gingivitis, in particular a mechanical removal of supra- and subgingival plaque.

8. collagen hydrolyzate for use according to any one of claims 1 to 5, wherein the collagen hydrolyzate is to be administered to a patient in whom one or more risk factors for periodontitis or gingivitis are present.

9. Collagen hydrolyzate for use according to one of the preceding claims, wherein the collagen hydrolyzate has an average molecular weight of 500 to 15,000 Da, preferably 1,000 to 8,000 Da, more preferably 1,500 to 5,000 Da, most preferably 1,800 to 2,200 Da.

10. Collagen hydrolyzate for use according to one of the preceding claims, wherein the collagen hydrolyzate is produced by enzymatic hydrolysis of a collagen-containing starting material.

11. Collagen hydrolyzate for use according to claim 10, wherein the col-containing starting material is selected from skin or bones of vertebrates, preferably from mammals, birds or fish, in particular from the skin of cattle or pigs.

12. Collagen hydrolyzate for use according to claim 10 or 11, wherein the collagen hydrolyzate is produced by the successive action of at least two endoproteases with a different specificity, in particular of at least two different metalloproteases and / or serine proteases.

13. Collagen hydrolyzate for use according to claim 12, wherein the metalloproteases and / or serine proteases are selected from enzymes from the microorganisms Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Aspergillus oryzae and Aspergillus melleus.

14. Collagen hydrolyzate for use according to claim 12 or 13, wherein at least 50% of the N-terminal amino acids of the collagen hydrolyzate are hydrophobic amino acids, in particular alanine, leucine and isoleucine.

15. Collagen hydrolyzate for use according to one of claims 1 to 9, wherein the collagen hydrolyzate is produced by recombinant gene expression.

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