WO2008009798A1 - Composition based on d-glucosamine, on lactoferrin and on chondroitin sulphate for preventing and/or treating degenerative joint diseases - Google Patents

Abstract

The invention relates to a composition intended to be administered orally for preventing and/or treating degenerative joint diseases. Said composition comprises, as active components, an effective amount of a mixture of D-glucosamine, of lactoferrin and of chondroitin sulphate.

Description

COMPOSITION BASED ON D-GLUCOSAMINE, LACTOFERRIN AND CHONDROITINE SULFATE FOR THE PREVENTION AND / OR TREATMENT OF JOINT DEGENERATIVE DISEASES

The invention relates to the treatment and prevention of degenerative joint diseases, and relates more particularly to compositions intended to be administered orally, such as a medicament, a food supplement and / or a medicinal product, with a view to preserve the health of the articular cartilages especially with regard to chronic and / or acute inflammations. Articular cartilage is a connective tissue consisting of four main constituents: water (about 70-80% by weight), proteoglycans (about 10-15%), collagen (of the order of 10-15%) and cells (about 1%). Among the cells present, chondrocytes are responsible for the constant and perpetual construction and remodeling of cartilage.

Resistant and elastic, articular cartilage has the role of reducing friction between moving joints. Also, by accumulating synovial fluid and rejecting it wisely, it fulfills the function of hydraulic damper with respect to shocks affecting the joints. Degenerative joint diseases such as rheumatoid arthritis, arthritis psoriasis and osteoarthritis (osteoarthritis) are the cause of profound disorders in the structure and integrity of articular cartilage. In particular, there is an imbalance in arthritic individuals between the synthesis and degradation of articular cartilage proteoglycans, in part caused by the depolymerization of glycosaminoglycan precursors.

Degenerative joint disease is most often caused by chronic joint inflammation that results in chronic and irreversible disruption of the joint. In addition to pain, symptoms also result in abnormal joint stiffness (especially on awakening) and / or joint swelling and / or deformity of the joints.

Also, it is known that acute (non-chronic) inflammation can induce the destruction of articular cartilage by evolving into chronic inflammation, but these are relatively rare cases.

At present, the cause (s) of the chronic inflammations responsible for these degenerative joint diseases are unclear. The factors determining the remission of acute inflammations of the joints, or those responsible for the establishment of an osteoarthritis state, are not known either.

Even today, the treatments provided in this type of disease consist almost exclusively in the palliative prescription of drug preparations which combine steroidal anti-inflammatory drugs (in particular, corticosteroids) or non-steroids, and analgesic compounds, to reduce inflammation and pain of patients. Together with these palliative treatments, which only aim to relieve pain and / or curb the disease, surgical techniques of reconstruction and replacement (with prostheses) of the affected joints have been developed. In addition to these inefficient palliative treatments and these heavy surgical operations, have recently appeared compositions of the drug type, food supplements or nutraceuticals, developed to regenerate and / or structurally strengthen the articular cartilages with respect to osteoarthritis. JP 2005 06806 discloses an oral pharmaceutical composition characterized by the combination of lactoferrin with one or more molecule (s) or extract (s) for the treatment of arthritis.

US 6,924,273 proposes a method for stimulating or enhancing the production of articular proteoglycans which is based on the oral administration of a composition prepared from hyaluronic acid, and optionally supplemented with a quantity of glucosamine sulfate and / or chondroitin sulfate and / or vitamins (A, C, D and / or E) and / or biotin. Hyaluronic acid and glucosamine are described as precursors for the synthesis of cartilaginous proteoglycans.

For the same purpose, WO 98/44929 describes a composition comprising hydrolyzed collagen fractions (of molecular weight between 1 and 300 kDa, and preferably between 10 and 80 kDa) in combination with glucosamine.

EP 0 704 216 discloses a medicament composition in the form of an orally administrable gel, comprising as an active substance with respect to the synthesis of cartilaginous proteoglycans, an effective amount of an organic salt. or inorganic chondroitin sulfate.

The aim of the invention is to propose a novel composition of the medicinal product type, food supplement or foodstuff, intended for the prevention and treatment of degenerative joint diseases and has improved efficacy.

It is an object of the invention to provide a composition having a generally noticeable effect on preventing and alleviating the deterioration of articular cartilage that generally develops in patients with degenerative joint disease. In particular, the invention aims to provide a composition capable of blocking / slowing the establishment and development of arthritis, especially when it is induced by collagen type IL

Also, the object of the invention is to propose a composition having protective properties with respect to chondrocytes, and whose biological activity results in an improvement in the survival of these cells when they are subjected to an inflammatory environment and / or mechanical stress.

To this end, the invention relates to a composition intended to be administered orally for preventing and / or treating degenerative joint diseases, comprising, as active components, an effective amount of D-glucosamine, lactoferrin and chondroitin sulfate.

The invention results first of all from the demonstration made by the inventors that the particular combination of these three active agents makes it possible to significantly reduce the risk of contracting a degenerative joint disease. For this purpose, the inventors have in particular carried out tests to assess the sensitivity with respect to the contraction of arthritis induced by type II collagen of mice subjected to a diet supplemented or not with an active ingredient composition according to the invention. The invention also results from the observation according to which, in degenerative joint diseases, a chronic inflammatory state and / or a mechanical stress does not induce (not) a simple severe deterioration of the components of the cartilaginous matrix of the joints (collagen and proteoglycans). It promotes the gradual establishment of disruption of the behavior of chondrocytes, almost exclusive cells of the cartilaginous and primordial tissues in the process of repair and regeneration of cartilage. This disruption of chondrocyte behavior results in an imbalance between the synthesis and the degradation of the components of the cartilaginous matrix. However, the inventors have succeeded in demonstrating an exceptional and unexpected synergistic protective effect of D-glucosamine and lactoferrin with respect to the apoptotic evolution of chondrocytes subjected to an inflammatory environment, in particular chronic. Moreover, the inventors have also found that this synergistic effect of D-glucosamine and lactoferrin on the survival of chondrocytes is significantly amplified by chondroitin sulfate.

In addition, the inventors have shown, by means of an arthritic index test, that this exceptional synergistic effect of D-glucosamine associated with lactoferrin and chondroitin sulfate is found on a murine model. Likewise, the inventors have demonstrated this exceptional synergistic effect of D-glucosamine associated with lactoferrin and chondroitin sulfate on the plasma concentration of TNF-α in mice treated with IL-type collagen. Thus, in addition to the conventional treatments which are aimed at the reduction of joint inflammations and / or the restoration of the cartilaginous matrix, in which patients are prescribed anti-inflammatories and / or compounds which are preferred for their precursor effect and / or stimulator of proteoglycan metabolism and / or articular collagen, a composition according to the invention makes it possible in particular to advantageously improve the survival and integrity of chondrocytes with respect to inflammatory events characteristic of arthropathies.

An oral composition according to the invention, which is therefore characterized in that it comprises an effective amount of D-glucosamine, lactoferrin and chondroitin sulfate, may consist of a drug or a food supplement. A composition in accordance with the invention may be produced in galenic forms and various formulations, in particular:

in the form of a mixture of solid and divided particles, for example an effervescent or non-effervescent powder, the setting of which requires that it be mixed with water,

- in the form of an effervescent tablet or not, to swallow with a little water, to suck or chew,

in the form of a solution, for example, a suspension, a syrup, a liquid composition embedded in a hard or soft capsule.

An oral administration composition according to the invention may also consist of a food product, that is to say an ordinary, solid and / or liquid food composition, which incorporates an effective amount of a mixture of D- glucosamine, lactoferrin and chondroitin sulfate. Whatever the form in which the composition of the invention is presented (medicament, dietary supplement, medicament), besides said mixture of D-glucosamine, lactoferrin and chondroitin sulfate, it further comprises at least one additive and / or at least one pharmaceutically acceptable and / or food acceptable excipient, in particular to obtain an orally administrable galenic form. These additives and / or excipients are, for example, binders, lubricants, sweeteners, diluents, excipients, effervescent agents, coating agents, etc. With regard to the active compounds, to prepare a composition according to as D-glucosamine, it is advantageous to use at least one glucosamine salt chosen from: glucosamine hydrochloride (in particular in the form of D-glucosamine HCl), glucosamine sulphate (especially in the form of D-sulphate), glucosamine 2KC1).

Preferably, only glucosamine hydrochloride is used.

Advantageously and according to the invention, a D-Glucosamine with a molecular weight of the order of 215 Da, derived from the chitin extracted from the crustacean shell, is used.

With regard to lactoferrin, it is possible to use lactoferrin compositions originating from animal milk, prepared using conventional purification and extraction techniques (such as ultrafiltration, ion exchange column chromatography, chromatography). on steric exclusion columns ...). These compositions more or less rich in lactoferrin generally correspond to particular protein fractions, purified from milk of bovine origin. Advantageously and according to the invention, a lactoferrin whose molecular weight is of the order of 80 kDa is used. The lactoferrin powder is prepared from cow's milk. In terms of dry matter weight, it advantageously has a lactoferrin content at least of the order of 40%. Preferably, this content is at least 50%. Typically, it is a composition of pure lactoferrin at least 85%.

As chondroitin sulfate, it is advantageous to use a chondroitin sulfate extracted from the selacian cartilage. Advantageously and according to the invention, this chondroitin sulfate has a molecular weight of the order of 30-50 kDa.

Advantageously, in a composition according to the invention, the chondroitin sulfate is present in the form of a powder which, in terms of weight of dry matter, has an effective content of chondroitin sulfate at least of the order of 90%.

The inventors have found that, in addition to improving the synergistic effect of D-glucosamine and lactoferrin on the survival of chondrocytes subjected to a chronic inflammatory environment, chondroitin sulfate also reinforces the proteoglycan matrix of cartilage. articular.

Advantageously, a composition according to the invention has a weight proportion lactoferrin / D-glucosamine (effective weight of active compounds) of between 1:40 and 20: 1. By way of example, this proportion is advantageously of the order of 1: 10 or of the order of 1: 1.

Advantageously, in a composition according to the invention, D-glucosamine and lactoferrin are present in the form of two mixed powders. The D-glucosamine powder is prepared from the chitin extracted from the crustacean shell. In terms of dry matter weight, it advantageously has an effective content of D-glucosamine of at least about 99%.

Advantageously, in a composition according to the invention, the weight ratio chondroitin sulfate / D-glucosamine (in terms of effective weight of the active compounds) is between 1: 5 and 100: 1, more particularly between 1: 3 and 20: 1 It is preferably of the order of 1: 2. According to a particularly preferred embodiment, a composition according to the invention comprises, as active components, an effective amount of a mixture of lactoferrin, D-glucosamine and chondroitin sulfate, with a lactoferrin / D weight ratio. glucosamine / chondroitin sulfate of the order of 1: 10: 5.

According to another preferred embodiment, a composition according to the invention comprises, as active components, an effective amount of lactoferrin, D-glucosamine and chondroitin sulfate, with a lactoferrin / D-glucosamine / chondroitin weight ratio. sulfate of the order of 1: 17.5: 5.

According to another preferred embodiment, a composition according to the invention comprises, as active components, an effective amount of lactoferrin, D-glucosamine and chondroitin sulfate, with a lactoferrin / D-glucosamine / chondroitin weight ratio. sulfate of the order of 1: 37.5: 10.

According to another preferred embodiment, a composition according to the invention comprises, as active components, an effective amount of a mixture of lactoferrin, D-glucosamine and chondroitin sulfate with a lactoferrin / D-weight proportion. glucosamine / chondroitin sulfate of the order of 1.2: 1: 1.

According to these embodiments of the invention, with these particular proportions of active agents according to the invention, the effective amounts of D-glucosamine in the daily intake as well as the amounts of total daily intake (dosages) for a given 70 kg of man are presented in Table 1 below.

Table 1

Accordingly, a composition according to the invention comprises an effective amount of D-glucosamine, lactoferrin and chondroitin sulfate determined for a daily dose corresponding to an effective amount of D-glucosamine (dosage) of between 125 mg and 1850 mg, in particular of the order of 550 mg, for an adult of the order of 70 kg.

Also, a composition according to the invention comprises an effective amount of D-glucosamine, lactoferrin and chondroitin sulfate determined for a total daily intake (dosage) of between 320 mg and 2390 mg, in particular of the order of 1000 mg, for an adult of the order of 70 kg.

The invention also relates to a composition for oral administration for preventing and / or treating degenerative joint diseases, and the use of a mixture of glucosamine, lactoferrin and chondroitin sulfate for the preparation of a A medicament for preventing and / or treating degenerative joint diseases, particularly osteoarthritis, characterized in combination by all or some of the features mentioned above or hereafter.

Other objects, features and advantages of the invention will appear on reading the detailed description which follows and which presents some of the tests carried out by the inventors in the context of the present invention. invention. The results obtained from these tests alone make it possible to conclude from the overall protective effect exercised by the compositions according to the invention with respect to the development of degenerative joint diseases, in general, and with regard, on the one hand, apoptotic evolution of chondrocytes subjected to a chronic inflammatory environment and secondly with respect to a murine arthritic model subjected to a chronic inflammatory treatment, in particular. The experimental results are presented in the form of appended graphical representations, among which:

FIG. 1 is a rod representation relating to tests showing the sensitivity of chondrocytes subjected to an inflammatory environment, and validating the in vitro study model used to test the potential effects of various active agents on the survival of chondrocytes cultured in an inflammatory environment,

FIG. 2 is a representation in the form of curves illustrating the dose-dependent effect of the active agents tested on the survival of chondrocytes subjected to an inflammatory environment,

FIGS. 3 a and 3b illustrate, in the form of rods, the results of tests demonstrating the synergistic effect of D-glucosamine and lactoferrin on the survival of chondrocytes subjected to an inflammatory environment,

FIG. 4 is a rod representation summarizing the comparative in vitro measurements of the effects of a reduction of inflammation specific to the use of ibuprofen, with respect to the effects of particular compositions in accordance with the invention, on the survival of chondrocytes subjected to an inflammatory environment,

FIG. 5 is a rod representation relating to the result of arthritic index (AI) tests carried out on mice,

FIG. 6 is a rod representation relating to the result of a quantification test of TNF-α (Tumor Necrosis factor-α) measured in mouse plasma, FIG. 7 is a rod representation relating to the result of a quantification test of INF-γ (interferon-γ) produced by mice.

FIG. 8 is a rod representation relating to the photometric result (OD) of an ELISA test for the quantification of total IgG of mice in an acellular extract derived from mouse articulation.

1 / - Experience of apoptosis of chondrocytes subjected to an inflammatory environment.

During joint inflammation, inflammatory cells and immune pool produce a large number of cytokines such as TNFa (tumor necrosis factor α), IL-I I ¹ (interleukin-1) and INFγ (γ interferon ). These cytokines act concomitantly and are responsible for the development of inflammation, which actively contributes to the destruction of the articular matrix, notably by inducing the death of chondrocytes. a) Reconstitution of culture medium reproducing the effects of inflammation.

The inventors have reconstituted, in vitro, a chondrocyte culture affected by an inflammatory reaction. The culture medium comprises DMEM supplemented with 10% of calf serum, penicillin and streptomycin (100 μg / ml).

According to a first operating mode, the inflammatory state is mimicked by adding to the previously exposed standard medium, a quantity of

CYTOMIX, a cytokine cocktail comprising, among others, TNFα, IL-1 and INFγ. To do this, per milliliter of standard medium were added 60 ng Cytomix ^®.

According to a second operating mode, the inflammatory state is mimicked by the presence, in the standard medium, of macrophages having previously been activated by bacterial exotoxins. This complementary approach of the previous one allows to reproduce in vitro, the interconnection between the chondrocytes of the matrix articular and inflammatory pool cells, and thereby reproduce conditions close to those encountered in vivo.

The chondrocytes are cultured in these different culture media for 48 hours before appreciating the proportion of dead cells by apoptosis:

a standard medium (st.),

a standard medium containing macrophages in the inactivated state

(st / Mac ^" ),

a standard medium supplemented by CYTOMIX (st.lCytom.),

a standard medium with activated macrophages (st./Mac).

The degenerative chondrocyte process was evaluated by measuring apoptotic death using an APOPTAG kit. This kit makes it possible to visualize the DNA breaks and to quantify, by a fluorescence microscope reading, the percentage of cells in apoptosis.

The results obtained are summarized in Table 2 below, and are also presented in the form of rods in FIG.

Table 2

Under standard conditions (chondrocyte culture in a medium without inflammatory stress), apoptosis of chondrocytes occurs for about 10% of cells after 48 hours. This rate is of the order of 22%, when the chondrocytes are co-cultured with macrophages (not activated). Under the effect of an inflammation simulated by the presence of CYTOMIX ^® in the culture medium, or by the presence of activated macrophages, the chondrocytes die by apoptosis after 72 hours. About 80% of chondrocytes die in less than 48 hours. On the one hand, these results confirm that an inflammatory reaction has the effect of inducing apoptosis of chondrocytes.

On the other hand, they demonstrate that the complementation of a culture medium by CYTOMDC makes it possible to reproduce, in vitro, the in vivo effects of an inflammatory reaction on the chondrocytes in a relatively similar manner. This pro-inflammatory culture medium can be advantageously used as a study model to test the potential effects of different agents and substances on the survival of chondrocytes cultured in an inflammatory environment. b) Study of the protective effect of D-glucosamine and / or lactoferrin and / or chondroitin sulfate

Chondrocytes are cultured in a medium

(R) standard supplemented with CYTOMIX, possibly in the presence of the different substances to be tested: D-glucosamine, lactoferrin and chondroitin sulfate.

After a 48-hour culture period, the proportion of

(K) dead cells by apoptosis is evaluated using an APOPTAG kit.

Initially, D-glucosamine, lactoferrin and chondroitin sulfate were individually studied for their effects on chondrocyte survival, depending on their concentration in the culture medium. The studies were carried out in particular with a

D-Glucosamine derived from chitin extracted from the shell of crustaceans. This D-glucosamine, with a molecular weight of the order of 215 Da, is marketed in particular in powder form which, in terms of dry matter weight, has an effective content of D-glucosamine of at least about 99% . With regard to lactoferrin, its effect has been tested with two types of lactoferrin compositions:

1) a substantially pure lactoferrin (Lf) composition in the form of a powder; this lactoferrin composition contains at least 97% (by weight of dry matter) of proteins originating from cow's milk, and of which at least 90% corresponds to a native lactoferrin with a molecular weight of the order of 80 kDa;

2) a co-isolate (co-Is), also in powder form, which contains at least 90% (by weight of dry matter) proteins from cow's milk, and 45-60% of which corresponds to a native lactoferrin of molecular weight of the order of 80 kDa (the remaining protein part is essentially represented by the other milk proteins such as lactoperoxidase, β-lactoglobulin and lactalbumin).

As regards chondroitin sulfate, the tests were carried out in particular with a chondroitin sulfate of molecular weight of the order of 30-50 kDa, extracted from selacian cartilage. This chondroitin sulfate is commercially available in the form of a powder which, in terms of dry matter weight, has an effective chondroitin sulfate content of at least about 90%. The previously mentioned compositions of D-glucosamine, lactoferrin and chondroitin sulfate are especially marketed by the company BIO SERAE, France.

Table 3 below presents the results obtained, expressed as a percentage of dead cells by apoptosis, after 48 hours of culture in an inflammatory environment, as a function of the concentration of the factors tested (expressed in micrograms of the factor composition) per milliliter. of culture medium.

Table 3

The corresponding results are also shown in FIG. 2 in the form of dose-effect curves.

In view of this first series of results, it is found that D-glucosamine (D-gluco) has a protective effect of chondrocytes which is a function of its dose. This effect is measurable at the concentration of 0.001 μg / ml, to reach a maximum effect at around 10 μg / ml. Under the same conditions, lactoferrin, used in the form of a highly concentrated composition (Lf), also has a protective effect of chondrocytes. In comparison with D-glucosamine, its effect is nevertheless more limited.

Tested with a composition of less concentration, in this case the co-isolate (co-Is.), The protective effect of lactoferrin is not significant under the experimental conditions, when this substance is tested separately from other active or potentially active agents.

The same observation is made with chondroitin sulfate, when tested separately from other active or potentially active agents. In a second step, the protective effects of the following binary mixtures were tested:

- D-glucosamine + lactoferrin,

ainsi que l'effet protecteur du mélange ternaire :- D-glucosamine + chondroitin sulphate, - D-glucosamine + lactoferrin (in the form of a composition Lf

as well as the protective effect of the ternary mixture:

- D-glucosamine + lactoferrin + chondroitin sulfate. Tables 4 and 5 below show the results obtained.

Les représentations graphiques en bâtonnets des figures 3a et 3b, élaborées à partir de données numériques sélectionnées du tableau 3, illustrent l'effet synergique entre la D-glucosamine et la lactoferrine.

The bar graphs of Figures 3a and 3b, developed from selected numerical data in Table 3, illustrate the synergistic effect between D-glucosamine and lactoferrin.

Table 5

c) Comparative study of the effects of a reduction of the inflammation generated by ibuprofen and the effects of particular compositions in accordance with the invention on the survival of chondrocytes subjected to an inflammatory environment Ibuprofen is a non-inflammatory steroidal properties also analgesic, antipyretic.

Ibuprofen is widely prescribed in the treatment of arthropathies, despite its potential adverse effects: gastritis, stomatitis, abdominal pain, ulceration of the digestive tract, jaundice, headache, drowsiness ... It is also accompanied by many contraindications, for example in case of a history of gastric or duodenal ulcer, in case of gastric or renal insufficiency, in case of pregnancy or breastfeeding ...

The inventors have compared the effects of a reduction of inflammation caused by ibuprofen with the chondroprotective effects of different compositions according to the invention, on the survival of chondrocytes cultured in an inflammatory environment.

For this purpose, the chondrocytes were co-cultured with macrophages previously activated by bacterial exotoxins, in different culture media:

1) a standard medium containing ibuprofen at a molar concentration of 1 μM,

2) standard media comprising D-glucosamine (0.01 μg / ml) and / or lactoferrin (0.001 μg / ml) and / or chondroitin sulfate (1 μg / ml).

The proportion of dead cells was assessed after 48 hours of culture.

The results obtained are summarized in Table 6 below, and are also presented, in graphical form, in FIG.

Table 6

The results obtained show that D-glucosamine, lactoferrin and chondroitin sulfate, when tested alone (at the indicated concentration) do not show an effectiveness far from being comparable to that of a reduction of the generated inflammation. by ibuprofen. However when these substances are used in combination, according to the invention, the effects are at least comparable.

2 / - Experience of induction of arthritis by collagen type II - Identification of active agents with protective effects. The inventors conducted a set of tests in the mouse to quantify the protective effects of various oral compositions containing lactoferrin and / or D-glucosamine and / or chondroitin sulfate with respect to induced arthritis. by collagen (collagen type II).

For this purpose, the inventors have used male DBA (H-2 ^q ) mice, 6-8 weeks old, marketed by IFFA-CREDO (Les Oncins, France).

Each mouse was subjected to a specific diet, including lactoferrin and / or D-glucosamine and / or chondroitin sulfate, for 15 days before it was inoculated with effective doses of collagen. type II for the purpose of inducing an arthritic condition.

For the purpose of comparison, the same experiments were also carried out by replacing the previous active agents with known antiinflammatories conventionally used to treat degenerative joint diseases, in particular ibuprofen, aspirin and indomethacin. The induction of arthritis was performed with a type II collagen solution emulsified with an equal volume of CFA (GlBCO-BRL, Germany), injected at the base of the tail of each animal. A "boost" is carried out 3 weeks later, using a type II collagen solution emulsified in incomplete Freund's adjuvant. The mice were followed 85 days later and probed for clinical symptoms of arthritis. a) Determination of IgG2a anti-collagen type II (anti-CII IgG2a) at the joints and serum of arthritic mice.

Assays were performed with the ELISA method. For this purpose, 96-well microtiter plates were coated with type II collagen, and left overnight for PBS. The wells were then loaded with 100 μl of sera, extracted from the joints and diluted in PBS. Different dilutions of the sera were applied.

After further intensive washing, goat anti-mouse IgG antibodies conjugated to alkaline phosphatase (PharMingen) were added to the wells.

The wells were again washed extensively, before revealing the antigenic antibody complexes (anti-mouse IgG), using a conventional developing substrate (ELISA kit, SIGMA). The concentration of antibody-antigen complexes was assessed by an optical density measurement at 410 nm.

Table 7 below shows the OD corresponding to the IgG2a anti-collagen type II assays carried out on arthritic mice, by a type II collagen induction induced after 15 days of an ordinary diet supplemented with an the following substances:

- ibuprofen,

a chondroitin sulfate / D-glucosamine mixture, 50:50 (by dry weight), a first composition (Comp.T) particular according to the invention, comprising a lactoferrin / D-glucosamine / chondroitin sulfate mixture, 1: 37 , 5: 10,

a second particular composition (Comp.2) according to the invention, comprising a lactoferrin / D-glucosamine / chondroitin sulfate mixture, 1: 17.5: 5.

FIG. 8 shows the rods representative of the photometric determination (OD) of type II anti-collagen IgG2a made in mice rendered arthritic by a type II collagen induction after an ordinary food diet lasting 15 days and supplemented with 5 mg daily of any of the following substances:

- control solution (C), - Chondroitin sulfate (Cs),

- D-glucosamine (D Gluco),

Lactoferrin (Cl),

- Particular composition according to the invention comprising a mixture of Lactoferrin and D-glucosamine, 1.2: 1 (D Gluco + Cl),

- Particular composition according to the invention comprising a mixture of lactoferrin, D-glucosamine and chondroitin sulfate 1.2: 1: 1 (LGC),

50 μM ibuprofen (Ib), 50 μM indomethacin (In),

Aspirin 50 μM (As). b) Determination of TNFα induced after type II collagen treatment.

In parallel with the IgG2a anti-CII assay, the inventors have also been interested in TNFα (tumor necrosis factor α), an inflammatory cytokine used here as a marker of the severity of arthritic symptoms caused in mice.

The TNFα assay results obtained are also shown in Table 7 for the compositions lactoferrin, D-glucosamine, chondroitin sulfate 1: 37.5: 10 and 1: 17.5: 5 below and in FIG. lactoferrin, D-glucosamine, chondroitin sulfate 1,2: 1: 1 composition.

Table 7

Figure 6 shows the rods representative of the TNF-α assay. performed on mice rendered arthritic by a type II collagen induction after an ordinary food diet lasting 15 days and supplemented with 5 mg per day of one of the following substances:

- control solution (C),

- chondroitin sulfate (Cs),

D-Glucosamine (D-Gluco), Lactoferrin (Cl),

- Particular composition according to the invention comprising a mixture of Lactoferrin and D-glucosamine, 1.2: 2, (D Gluco + Cl),

- Particular composition according to the invention comprising a mixture of lactoferrin, D-glucosamine and chondroitin sulfate 1.2: 1: 1 (LGC),

Ibuprofen 50 μM (Ibu),

Indomethacin 50 μM (Ind),

Aspirin 50 μM (Asp). These few results demonstrate very significantly a protective effect exerted by the compositions of the invention with respect to the establishment and development of arthropathies. Indeed, on the one hand, the production of TNF-α by lymphoid spleen cells in response to collagen II is substantially reduced, indicating that a composition according to the invention (Comp 1 and Comp 2 and LGC) exerts a control over the mechanisms of inflammation characteristic of an arthritic condition. On the other hand, there is also a decrease in the level of type II anti-collagen IgG2a, in mice fed with a composition according to the invention, compared to mice following an ordinary diet. d) Evaluation of the arthritic index after type II collagen treatments

The inventors have also conducted a series of tests aimed at measuring the frequency of occurrence and the severity of the arthritic symptoms (pronounced edema and swelling of the joints) caused in the mouse by IL-type collagen treatment. The results are given on a subjective scale. graduated from 1 to 3.

FIG. 5 clearly describes a synergistic inhibitory effect characteristic of the composition according to the invention lactoferrin / D-glucosamine / chondroitin sulfate 1.2: 1: 1 (LGC) on the inflammation induced by collagen type II in comparison with the following treatments:

- control solution (C),

- chondroitin sulfate (Cs),

- D-glucosamine (D Gluco), - lactoferrin (Cl),

D-glucosamine + lactoferrin, 1: 1.2 (D Gluco + Cl),

- lactoferrin + D-glucosamine + chondroitin sulfate in lactoferrin / D-glucosamine / chondroitin sulfate proportions, 1.2: 1: 1 (LGC)

The inventors have also found, by means of statistical analyzes (not shown here), that a diet supplemented with a composition in accordance with the invention makes it possible to reduce significantly the risk for mice tested to develop arthropathy. 20 to 55% of the mice fed a diet supplemented with a composition according to the invention develop the symptoms of arthritis following an injection of type II collagen: mice that developed arthropathy had inflammatory symptoms and moderate clinical criteria . For mice with a regular diet, this proportion varies between 84% and 100% with severe symptoms (swelling of the joint, edema). e) Assay of INF-γ in mice

FIG. 7 shows the rods representative of the INF-γ assay carried out in arthritic (right) or non-arthritic (left) mice by means of treatment with type II collagen after an ordinary dietary diet. a duration of 15 days supplemented with 5 mg per day of one of the following substances with 5 mg of composition per day:

- control solution (C), - chondroitin sulfate (Cs),

D-glucosamine (D-Gluco),

Lactoferrin (Cl),

- Particular composition according to the invention comprising a mixture of Lactoferrin and D-glucosamine 1,2: 1 (D Gluco + Cl), - Particular composition according to the invention comprising a mixture of lactoferrin, D-glucosamine, chondroitin 1.2: 1: 1 sulfate (LGC),

Ibuprofen (Ibu) 50 μM,

Indomethacin (Ind) 50 μM,

Aspirin (Asp) 50 μM. In mice for which inflammation was not induced with type II collagen, it was clearly observed that there was a significant decrease in the level of INF-γ basal comparable to the decrease observed with the controls Ibuprofen, Indomethacin and Aspirin. . On the other hand, in the mice for which the inflammation was induced with type II collagen, a marked decrease in the level of INF-γ was observed in the mice treated with the lactoferrin, D-glucosamine and chondroitin sulfate 1 composition. , 2: 1: 1, this The decrease in the production of INF-gamma is greater than that obtained with the control compositions.

Claims

II - Composition intended to be administered orally for preventing and / or treating degenerative joint diseases, comprising, as active components, an effective amount of D-glucosamine, lactoferrin and chondroitin sulfate. 2 / - The composition of claim 1, characterized in that said D-glucosamine consists of at least one glucosamine salt selected from glucosamine hydrochloride and glucosamine sulfate. 3 / - Composition according to one of claims 1 or 2 characterized in that said D-glucosamine is exclusively glucosamine hydrochloride. Al - Composition according to one of claims 1 to 3, characterized in that said D-glucosamine has a molecular weight of the order of 215 Da. 5 / - Composition according to one of claims 1 to 4, characterized in that said lactoferrin corresponds to a particular protein fraction purified from milk of bovine origin. 6 / - Composition according to claim 5, characterized in that said lactoferrin has a molecular weight of the order of 80 kDa. 11 - Composition according to one of claims 1 to 6, characterized in that said chondroitin sulfate is extracted from the cartilage of selachian. 8 / - Composition according to one of claims 1 to 7, characterized in that said chondroitin sulfate has a molecular weight of the order of 30-50 kDa. 91 - Composition according to one of claims 1 to 8, characterized in that it has a lactoferrin / D-glucosamine weight ratio of between 1:40 and 20: 1. 10 / - Composition according to one of claims 1 to 9, characterized in that it has a lactoferrin / D-glucosamine weight proportion of the order of 1:10. 11 / - Composition according to one of claims 1 to 9, characterized in that it has a weight ratio lactoferrin / D-glucosamine of the order of 1: 1. 12 / - Composition according to one of claims 1 to 11, characterized in that it has a weight ratio chondroitin sulfate / D-glucosamine of between 1: 5 and 100: 1. 13 / - Composition according to one of claims I h 12, characterized in that it has a weight ratio chondroitin sulfate / D-glucosamine of the order of 1: 2. 14 / - Composition according to one of claims 1 to 13, characterized in that it comprises, as active components, an effective amount of lactoferrin, D-glucosamine and chondroitin sulfate, with a weight proportion lactoferrin / D -glucosamine / chondroitin sulfate of the order of 1: 10: 5. 15 / - Composition according to one of claims 1 to 13, characterized in that it comprises, as active components, an effective amount of lactoferrin, D-glucosamine and chondroitin sulfate, with a weight proportion lactoferrin / D -glucosamine / chondroitin sulfate of the order of 1.2: 1: 1. 16 / - Composition according to claims 1 to 15, characterized in that it comprises an effective amount of lactoferrin, D-glucosamine and chondroitin sulfate determined for a daily dose corresponding to an effective amount of D-glucosamine of between 125 mg and 1850 mg, in particular of the order of 550 mg, for an adult of the order of 70 kg. 17 / - Composition according to claim 16, characterized in that it comprises an effective amount of D-glucosamine, lactoferrin and chondroitin sulfate determined for a total daily intake of between 320 mg and 2390 mg, in particular of the order 1000 mg, for an adult of the order of 70 kg. 18 / - Composition according to one of claims 1 to 17, characterized in that it further comprises at least one additive and / or at least one excipient acceptable from a pharmaceutical and / or food. 19 / - Composition according to one of claims 1 to 18, characterized in that it is a preparation selected from: a drug, a food supplement, a food. 20 / - Use of a composition according to one of claims 1 to 19 for the preparation of a medicament for preventing and / or treating degenerative joint diseases. 21 / - Use of a composition according to one of claims 1 to 20 for the preparation of a medicament for preventing and / or treating osteoarthritis.