EP3634448A1

EP3634448A1 - Phycocyanin composition for use in inhibiting bone resorption

Info

Publication number: EP3634448A1
Application number: EP18730315.1A
Authority: EP
Inventors: V ronique COXAM; Marie-Jeanne Davicco; Laurent LEOTOING; Fabien WAUQUIER
Original assignee: Institut National de la Recherche Agronomique INRA
Current assignee: Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Priority date: 2017-06-06
Filing date: 2018-06-05
Publication date: 2020-04-15
Also published as: US20210077574A1; WO2018224477A1; FR3066919B1; FR3066919A1

Abstract

The present invention relates to a phycocyanin composition for use in inhibiting bone resorption in humans or animals.

Description

TITLE OF THE INVENTION

PHYCOCYANINE COMPOSITION FOR USE IN INHIBITING

THE BONE RESORPTION FIELD OF THE INVENTION

The present invention relates to the field of inhibition of bone resorption in humans or animals.

STATE OF THE ART

The bone is not a static fabric. The bone undergoes constant remodeling through destruction and de novo synthesis of bone tissue in a complex process involving two main types of cells, osteoblasts that produce new bone tissue and osteoclasts that destroy bone.

The remodeling of the bone tissue coordinates the bone resorption and formation, thus ensuring the renewal of the skeleton, safeguarding its structure. When the system is in equilibrium, for example, before menopause, it ensures the integrity of the mechanical strength of the skeleton by preventing the excessive aging of its components. It is through the interaction of cellular play of osteoclasts and osteoblasts that this essential function is ensured. The remodeling process is always initialized by resorption, followed after a certain time by bone formation.

Osteoblasts, cells responsible for bone formation, differentiate from precursor cells and express and secrete structural proteins such as type 1 collagen, as well as enzymes (alkaline phosphatase) and many regulatory peptides. BMP (Bone Morphogenetic Proteins) (Stein G. et al, 1990, Curr Opin Cell Biol 2: 1018-1027, Harris S et al, 1994, J. Bone Miner Res Vol.9: 855-863). Osteoclasts are multinucleated cells that are responsible for bone loss, in a process commonly referred to as bone resorption.

In times of growth of humans or animals, the activity of bone formation predominates: it is the acquisition of bone capital. In adulthood, in humans or animals, the balanced action of osteoblasts and osteoclasts allows bone mass to be maintained over time and at the same time reshapes the bone tissue by resorption and de novo synthesis of the bone. 'bone.

However, with age, there is an imbalance in the process of bone remodeling, which results in bone loss, which is referred to as osteopenia.

Age-related osteopenia is a universal, non-pathological phenomenon in itself, but it is the field of osteoporosis, since the reduction of bone mass is the essential etiological factor in the genesis of this condition. However, it does not exclude the influence of other parameters such as, for example, skeletal architecture or propensity to fall.

Indeed, exacerbated, this osteopenia leads to fracture risk (mineral density below which the slightest shock is likely to cause a fracture) which determines the appearance of osteoporosis.

Osteoporosis (post-menopausal or senile) can therefore be conceived of as an infantile pathology whose prophylaxis could be based as well on (i) optimization of the bone capital (acquired during the growth of the individual), that ( ii) a slowing down of bone loss associated with aging.

There is a great heterogeneity in the value of the peak bone mass in individuals, as a result of variations in the bone growth process from an early age. Thus, the maximum bone mass reached before age 30, also known as peak bone mass, is highly variable from one individual to another. With age, individuals with low bone mass peak value are at a disadvantage.

Therefore early management of the individual should be favored, since the two critical phases for bone capital are:

- the period of growth, allowing the acquisition of the maximum bone mass (peak bone mass);

aging, conditioning the rate of loss of bone mass.

Among the pathological disorders related to an imbalance of bone metabolism, mention may in particular be made of osteoporosis, Paget's disease, bone loss or osteolysis observed near a prosthesis.

There are other factors that can increase bone loss leading to osteoporosis, such as cigarette smoking, alcohol abuse, lifestyle sedentary, low calcium intake, unbalanced diet or vitamin D deficiency.

These different causes of increased bone loss must be distinguished from causes related to diseases such as cancer or diabetes.

Among the disorders associated with abnormal bone resorption, the most common is osteoporosis, the most frequent manifestation of which is observed in women, after the onset of menopause.

Osteoporosis is a systemic skeletal disease characterized by a reduction in bone mass and deterioration of bone microarchitecture, associated with an increase in bone fragility and susceptibility to fracture.

According to the World Health Organization (WHO) clinical classification, the health status of an individual's bone is determined by the value of bone mineral density (BMD), as measured by bone densitometry, compared to a predetermined normal value, this predetermined normal value being that of the average peak bone mineral density of the 30-year-old population.

There are two types of osteoporosis, type I and type II, respectively.

Type I osteoporosis is six times more common in women than in men. Type I osteoporosis occurs mainly in a subgroup of postmenopausal women, aged 51 to 75 years, and is characterized by exaggerated bone loss predominant in the trabecular bone. Fractures of the vertebral bodies and the lower extremity of the forearm are the usual complications.

Type I osteoporosis is mainly related to the estrogen hormone deficiency of menopause, and to a certain extent also to andropause.

Type II osteoporosis affects a large population of men and women over 70 years of age and is associated with fractures of the femoral neck, upper extremity of the humerus and tibia, ie bone sites containing both cortical bone and trabecular bone. Circulating levels of parathyroid hormone (PTH) are often high.

Osteoporosis type II is twice as common in women.

Strategies to restore oestrogenic (and androgenic) impregnation by hormone replacement therapy were initially strongly encouraged, particularly to reduce the cohort of functional signs of menopause, and especially to reduce the vascular and bone risks caused by hormone deficiency. Nevertheless, for various reasons (contraindications, reluctance to prescribe hormones, increased risks of cancer, etc.), this type of prophylactic treatment is no longer applied as a first-line treatment. Health professionals are therefore currently relatively poor in terms of prophylactic tools. In particular, treatments related to bone remodeling insufficiency are sought among those who exert an inhibition of bone resorption. A variety of bone resorption inhibiting agents are known, and prescribed in particular in the treatment of osteoporosis, including age-related osteoporosis.

Among the bone resorption inhibitors, selective estrogen receptor modulators (or SERMs, for Selective Estrogenic Receptor Modulators), such as raloxifene, are known. However, this compound is devoid of effect at the fractures of the upper end of the femur (More study) and is contraindicated in patients with a history of thromboembolism. In addition, this treatment increases the frequency of hot flashes. Synthetic steroids, such as tibolone, which has estrogenic and progestative activity, are also known to have low androgenic properties, but may cause leucorrhea, vaginitis, mastodynia, and weight gain.

In fact, there are today many active compounds on the inhibition of bone resorption, among which we can mention the family of bisphosphonates, such as etidronate or alendronate (European Patent No. EP 210 728, patent application US Publication No. 2001/0046977), oxazolidinones thioamides (US Patent Application Publication No. 2002/0010341), or isoflavone compounds (US Patent Application Publication No. 2002/0035074).

Among the agents commonly used as inhibitors of bone resorption include alendronate, risedronate, ibandronate and zoledronic acid. Mention may also be made of denosumab, which is a monoclonal antibody targeting the RANK / RANKL system. The marketing authorizations as medicaments of these different compounds are restricted to a curative indication of osteoporosis. These bone resorption inhibitors are not currently prescribed for osteoporosis preventive activity. In addition, the use of these bone resorption inhibitors results in a variety of side effects such as (i) adverse gastrointestinal effects for alendronate, risedronate and ibandronate; (ii) oral lesions and osteonecrosis jaws, even fractures, for zoledronic acid, and (iii) cardiac damage to denosumab.

Although there is today a relatively large diversity of compounds to stimulate bone formation and / or inhibit bone resorption, health professionals express a constant need for new active compounds, in particular because of the limited success of current treatments. and important side effects with which they are associated.

In addition, given the chronic nature of certain states caused by an imbalance of bone metabolism, there is a need for new active compounds that can be used for a long period of time in humans or animals.

This is why health professionals, as well as the official regulatory authorities (report on osteoporosis in the European Community, 1998), recommend integrating complementary or even alternative validated therapies. In particular, a nutritional approach fully meets these criteria.

There is therefore a need expressed for new means for preventing or treating imbalances in bone metabolism, in particular new ways of preventing or treating bone resorption, in particular age-related bone resorption in humans or humans. 'animal. SUMMARY OF THE INVENTION

In some embodiments, said composition is a nutritional composition adapted for oral administration. Advantageously, said nutritional composition is intended to prevent bone loss, also called osteopenia, particularly in individuals in whom the occurrence of bone loss can be expected, for example because of age or bone loss likely to occur because of other factors, such as taking medication.

In certain other embodiments, said composition is a pharmaceutical composition for human or veterinary use.

In some embodiments, said nutritional composition or pharmaceutical composition is intended to prevent bone loss, also known as osteopenia. In certain embodiments, said nutritional composition or said pharmaceutical composition is intended to prevent the bone loss that occurs with aging or due to taking drugs, or to prevent bone loss occurring during certain diseases such as obesity, diabetes, thyroid pathologies and pathologies of the adrenal glands.

In some embodiments, said nutritional composition is adapted for administration, particularly oral administration, of a daily amount of from 0.01 to 10,000 mg of the phycocyanin compound.

In some embodiments, said nutritional composition is adapted for daily administration, particularly daily oral administration, ranging from 0.05 mg / kg to 1000 mg / kg, preferably from 1 mg / kg to 200 mg / kg. which includes from 10 mg / kg to 100 mg / kg.

It is recalled that on average, it is considered that a man or an adult woman weighs about 80 kg.

In some embodiments, said pharmaceutical composition is adapted for administration, particularly oral administration, of a daily amount of from 1 mg to 10,000 mg of the phycocyanin compound, which includes adaptation to daily administration ranging from from 4 mg to 8000 mg.

In some embodiments, said pharmaceutical composition is adapted for daily administration, particularly daily oral administration, ranging from 0.05 mg / kg to 1000 mg / kg, preferably from 1 mg / kg to 200 mg / kg. and preferably from 10 mg / kg to 100 mg / kg.

A phycocyanin composition according to the invention may be intended to prevent or treat any type of osteopenia.

In certain embodiments, said composition is intended to prevent or treat a pathology selected from type I or type II osteoporosis, secondary osteoporosis, Paget's disease, bone loss or osteolysis observed in the vicinity of a prosthesis. DESCRIPTION OF THE FIGURES

Figure 1 illustrates the results of measurement of bone mass in different groups of animals. Ordinate: bone mineral density (BMD) values, expressed in grams per cubic centimeter (g / cm ³ or g / cc). On the abscissa, the results for each of the groups of animals, from left to right of Figure 1: (i) SH-Control: control group having undergone pseudosurgery; (ii) OVX-Control: ovariectomy control group; (iii) OVX-Spirulina: ovariectomized group having received a diet supplemented with lysed spirulina; (iv) OVX-phycocyanin: ovariectomized group receiving a diet supplemented with purified phycocyanin. Figure 2 illustrates the results of measuring the proliferation of a mouse pre-osteoblast cell line (MC3T3-E1 line). On the ordinate: osteoblast proliferation index (corresponding to the cellular mitochondrial activity expressed in optical density variation per hour (Δ OD / h)). On the abscissa, the pre-osteoblast culture conditions, from left to right in FIG. 2: (i) C10%: culture medium supplemented with 10% (v / v) fetal calf serum; (ii) C2%: culture medium supplemented with 2% (v / v) fetal calf serum; (iii) Phi 00: culture medium supplemented with 100 μg / ml of purified phycocyanin, (iv) Ph250: culture medium supplemented with 250 μg / ml of purified phycocyanin, (v) Phi 000: culture medium supplemented with 1000 μg / ml purified phycocyanin, (vi) Ph2500: culture medium supplemented with 2500 μg / ml of purified phycocyanin.

Figure 3 illustrates the proliferation results of a mouse osteoclast line (line RAW264.7) exposed to different culture conditions. On the ordinate: Osteoclast proliferation index (corresponding to the cellular mitochondrial activity expressed in optical density variation per minute (Δ OD / min)). On the abscissa, the culture conditions of the osteoclasts, from the left to the right of FIG. 3: (i) C2%: culture medium supplemented with 2% (v / v) fetal calf serum; (ii) C10%: culture medium supplemented with 10% (v / v) fetal calf serum; (iii) Phy5: culture medium supplemented with 5 μg / ml of purified phycocyanin, (iv) PhylO: culture medium supplemented with 10 μg / ml of purified phycocyanin, (v) Phy25: culture medium supplemented with 25 μg / ml purified phycocyanin, (vi) PhylOO: culture medium supplemented with 100 μg / ml of purified phycocyanin, (vii) Phy250: culture medium supplemented with 250 μ / ηι1 of purified phycocyanin, (vi) PhylOOO: culture medium supplemented with 1000 μg / ml of purified phycocyanin.

Figure 4 illustrates the measurements of the enzymatic activity of tartrate-resistant acid phosphatase (TRAP) expressed by osteoclasts (cells of the Raw 264.7 line) in culture. On the ordinate: TRAP activity, expressed in optical density variation per hour and per milligram of proteins (Δ OD / h / mg proteins). On the abscissa, from the left to the right of Figure 5: the culture conditions of the following osteoclasts: (i) C: culture medium alone; (ii) RL: culture medium supplemented with 50 ng / ml RANK-L; (iii) RL5: RL medium supplemented with 5 μg / ml of purified phycocyanin; (iv) RL10: RL medium supplemented with 10 μg / ml of purified phycocyanin; (v) RL25: RL medium supplemented with 25 μg / ml of purified phycocyanin; (vi) RL50: RL medium supplemented with 50 μg / ml of purified phycocyanin; (vii) RL100: RL medium supplemented with 100 μg / ml of purified phycocyanin; (iii) Cl 00: medium alone supplemented with 100 μg / ml of purified phycocyanin.

Figure 5 illustrates the measurements of the enzymatic activity of tartrate-resistant acid phosphatase (TRAP) expressed by osteoclasts (cells of the Raw 264.7 line) in culture. On the ordinate: TRAP activity, expressed in optical density variation per hour and per milligram of proteins (Δ OD / h / mg proteins). On the abscissa, from the left to the right of FIG. 5: the culture conditions of the following osteoclasts: (i) C: culture medium alone; (ii) RL: culture medium supplemented with 50 ng / ml RANK-L; (iii) group of two bars. Right bar (gray):: RL5: RL medium supplemented with 5 μg / ml of purified phycocyanin; left bar (black): RL medium supplemented with the same dose of a phycocyanin 4 times purer (Sigma) (assay on the basis of a colorimetric analysis); (iv) group of two bars. Right bar (gray): RL10: RL medium supplemented with 10 μg / ml of purified phycocyanin; left bar (black): RL medium supplemented with the same dose of a phycocyanin 4 times purer (Sigma) (assay on the basis of a colorimetric analysis); (v) group of two bars. Right bar (gray): RL25: RL medium supplemented with 25 μg / ml of purified phycocyanin; left bar (black): RL medium supplemented with the same dose of a phycocyanin 4 times purer (Sigma) (assay on the basis of a colorimetric analysis) (vi) group of two bars. Right bar (gray): RL50: RL medium supplemented with 50 μg / ml of purified phycocyanin; left bar (black): middle RL supplemented with same dose of a phycocyanin 4 times purer (Sigma) (assay on the basis of a colorimetric analysis);

Figure 6 illustrates the expression of the TRAP coding gene in osteoclast cultures of RAW 264.7. On the ordinate: level of expression of the gene encoding TRAP, expressed in number of increases (fold increase). On the abscissa, from the left to the right of FIG. 7, the different culture conditions to which the cells are exposed: (i) Control: culture medium alone; (ii) RL: culture medium supplemented with 50 ng / ml RANK-L; (iii) RL + Phy5: RL medium supplemented with 5 μg / ml of purified phycocyanin; (iv) RL + PhylO: RL medium supplemented with 10 μg / ml of purified phycocyanin; (v) RL + Phy25: RL medium supplemented with 25 μg / ml of purified phycocyanin; (vi) RL + Phy50: RL medium supplemented with 50 μg / ml of purified phycocyanin; (vii) RL + PhylOO: RL medium supplemented with 100 μg / ml of purified phycocyanin.

Figure 7 illustrates the expression of the gene encoding Nox4 in RAW 264.7 osteoclast cultures. On the ordinate: level of expression of the gene coding Nox4, expressed in number of increases (fold increase). On the abscissa, from the left to the right of FIG. 8, the different culture conditions to which the cells are exposed: (i) Control: culture medium alone; (ii) RL: culture medium supplemented with 50 ng / ml RANK-L; (iii) RL + Phy5: RL medium supplemented with 5 μg / ml of purified phycocyanin; (iv) RL + PhylO: RL medium supplemented with 10 μg / ml of purified phycocyanin; (v) RL + Phy25: RL medium supplemented with 25 μg / ml of purified phycocyanin; (vi) RL + Phy50: RL medium supplemented with 50 μg / ml of purified phycocyanin; (vii) RL + PhylOO: RL medium supplemented with 100 μg / ml of purified phycocyanin.

Figure 8 illustrates the expression of the gene encoding NFATC1 in RAW 264.7 osteoclast cultures. On the ordinate: level of expression of the gene coding Nox4, expressed in number of increases (fold increase). On the abscissa, from the left to the right of FIG. 9, the different culture conditions to which the cells are exposed: (i) Control: culture medium alone; (ii) RL: culture medium supplemented with 50 ng / ml RANK-L; (iii) RL + Phy5: RL medium supplemented with 5 μg / ml of purified phycocyanin; (iv) RL + PhylO: RL medium supplemented with 10 μg / ml of purified phycocyanin; (v) RL + Phy25: RL medium supplemented with 25 μg / ml of purified phycocyanin; (vi) RL + Phy50: RL medium supplemented with 50 μg / ml of purified phycocyanin; (vii) RL + PhylOO: RL medium supplemented with 100 μg / ml of purified phycocyanin. DETAILED DESCRIPTION OF THE INVENTION

The inventors have shown that phycocyanin has an inhibitory effect on osteoclastic activity in in vivo and in vitro models of induced deregulation of bone metabolism.

Phycocyanin is a protein of the family of phycobiliproteins, which are present in some cyanobacteria. Phycocyanin consists of two protein subunits, the alpha and beta subunits, respectively. The phycocyanin has a plurality of bilefinic chromophoric moieties, wherein these chromophore groups are covalently bound to the phycocyanin via thioether linkages to cysteine residues. The phycocyanin has a bile group linked to the alpha subunit and two bile groups linked to the beta subunit. Phycocyanin is present in cyanobacteria, such as Spirulina platensis, as a complex mixture of trimers, hexamers and decamers (Romay et al, 2003, Current Protein and Peptide Science, Vol 4: 207-216). ).

Phycocyanin is therefore a protein found in extracts obtained from cyanobacteria of the genus Spirulina, and in particular from Spirulina platensis. Spirulina extracts have a complex composition. Such extracts typically comprise from 50% to 70% by weight of protein, from 4% to 7% by weight of lipids (83% of saponifiable fraction and 17% of unsaponifiable fraction), from 15% to 25% by weight of carbohydrates, in relation to the weight of the dry matter. Spirulina extracts also include water-soluble vitamins (eg, vitamins B1, B2, B6, B9, B12 and vitamin C), fat-soluble vitamins (eg beta-carotene, tocopherols), pigments, many minerals and trace elements (eg, calcium, phosphorus, magnesium, iron, zinc, copper, chromium, manganese, sodium, potassium). It is qualitatively based on the high protein content of spirulina extracts in food supplements, due to the presence of all the essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and valine). Spirulina extracts are known for a variety of health benefits, including immune system stimulation, antiviral, anticancer, antioxidant, detoxifying, and anti-hyperlipidemia properties. Spirulina extracts are marketed today as a dietary supplement, because of its many alleged beneficial effects. However, spirulina extracts can also induce adverse effects, such as the induction of impaired bone metabolism, leading to a reduction in bone mineral density, particularly in individuals with hormonal deficiency, and more specifically in individuals with estrogen deficiency (Ishimi et al., Biosci Biotechnol Biochem, Vol 70 (No. 2): 363-368).

It is therefore quite unexpected that the inventors have shown that phycocyanin, which is one of the protein components predominantly present in spirulina extracts, has, unlike extracts of spirulina, properties of inhibition of the reduction in bone metabolism observed in individuals with estrogen deficiency.

More specifically, the inventors have shown that a phycocyanin composition has properties of inhibition of osteoclastic activity in individuals suffering from an estrogen deficiency. It is thus shown in the examples that a phycocyanin composition makes it possible to inhibit the effect of reducing bone mineral density (demineralization process) in individuals with estrogen deficiency. In particular, it has been shown that individuals who have received a phycocyanin composition show an increase in the level of the OPG (osteoprotegerin) protein marker, compared with individuals who have not received this composition. It is also shown an improvement of the ratio OPG / RANKL, RANKL (for "Receptor Activator of Nuclear Factor Kappa B Ligand") being a protein synthesized by osteoblasts which acts on the differentiation and the activation of osteoclasts when it binds to RANK receiver. The increase in the level of expression of the OPG marker as well as the OPG / RANK-L ratio in individuals having received a phycocyanin composition is indicative of an inhibitory effect on the differentiation and activation of osteoclasts and a favorable orientation of bone metabolism (towards osteo-formation).

It is also shown that a phycocyanin composition induces an inhibition of the proliferation and differentiation of osteoclasts in vitro. In particular, a phycocyanin composition inhibits a protein marker of osteoclastic activity such as tartrate-resistant acid phosphatase (TRAP, for "Tartrate-Resistant Acid Phosphatase"). It is also shown that a phycocyanin composition inhibits the expression of genes that differentiate osteoclasts, such as genes trap (encoding the TRAP protein) and nfatcl (encoding the NFATC1 protein), as well as genes indicative of oxidative stress, such as NOX4 (encoding the NOX4 protein).

The inventors have also shown that a phycocyanin composition induces proliferation of osteoblasts.

In addition, as shown in the examples, the phycocyanin does not have a detectable effect mimicking the activity of an estrogen-like compound, also called "estrogen-like" effect, as evidenced by the low weight of uterine horns, as well as the lack of weight gain in treated rats. These results confirm the interest of phycocyanin for the prevention or treatment of bone resorption in humans or animals, especially in a situation of hormone deficiency. Due to the absence of an "estogen-like" effect caused by a phycocyanin composition within the meaning of the invention, such a composition can be administered indifferently to (i) the man or the woman and (ii) to any non-human animal, including any non-human mammal, male or female.

All of these experimental results show that the administration of a phycocyanin composition has a beneficial effect on bone metabolism in individuals with estrogen deficiency. These results show that a phycocyanin composition can prevent, at least in part, the bone loss that is caused by a reduction in oestrogenic impregnation.

Without wishing to be bound by any theory, the inventors believe that the activity of a phycocyanin composition on the bone metabolism of individuals with estrogen deficiency is mainly due to an activity of inhibition of bone resorption, although part of the activity of a phycocyanin composition may also be due to an activity of stimulating bone formation, since an induction of osteoblast proliferation is also shown.

In some embodiments, said phycocyanin composition is a nutritional composition. In preferred embodiments of a phycocyanin nutritional composition, such a composition is suitable for oral administration. By "phycocyanin" is meant according to the invention the protein referenced under the CAS No. 11016-15-2. Preferentially, it is a phycocyanin originating from a cyanobacterium of the species Spirulina platensis.

Phycocyanin means the entire protein, which does not include peptide fragments of this protein.

According to the invention, the term "phycocyanin composition" means a composition enriched in phycocyanin, that is to say a composition comprising at least 40% by weight of phycocyanin, relative to the total dry weight of the composition.

In the present description, the term "dry weight" means the dry weight of said composition. The weight of dry matter may be determined by those skilled in the art according to any known technique, including after removal of water by drying the composition in the oven until complete evaporation of the water which was initially contained in it. The drying step in the oven can be conventionally performed at a temperature ranging from 103 ° C to 110 ° C at atmospheric pressure.

According to the present disclosure, a phycocyanin composition comprising at least 40% by weight of phycocyanin, based on the dry weight of the composition, includes phycocyanin compositions comprising at least 41%, 42%, 43%, 44%, 45% , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 %, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% and 99% by weight of phycocyanin, relative to the total dry weight of the composition.

According to this definition, a phycocyanin composition comprises at most 60% by weight of compounds other than phycocyanin, based on the dry weight of the composition, which includes phycocyanin compositions comprising at most 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40% , 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23 %, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1% by weight of compounds other than phycocyanin, relative to the total dry weight of the composition.

In a phycocyanin composition according to the invention, the compounds other than phycocyanin may be compounds initially present in the starting material which has been used, for example in the starting material from spirulina, prior to the implementation of a process for extracting and enriching phycocyanin from the starting raw material.

A variety of compositions comprising at least 40% by weight of phycocyanin are commercially available. Mention may in particular be made of the following compositions: (i) C-Phycocyanin sold by Sigma-Aldrich under the reference No. P2172 and No. P6161, (ii) C-Phycocyanin (Spirulina sp) sold by the company Prozyme (Hayward, Canada) under the reference No. PB11, (iii) C-phycocyanin marketed by Soley Institute (Istanbul, Turkey) under the reference A620 / 280.

The phycocyanin can also be obtained by extraction from certain cyanobacteria such as the Cyanobacterium Arthrospira platensis, according to methods known to those skilled in the art. In order to obtain a phycocyanin composition from Arthrospira platensis, a person skilled in the art can in particular refer to the methods described by Moraes et al. (2011, Brazilian Journal of Chemical Engineering, Vol 28 (No. 1): 45-49), Kamble et al. (2013, J Applied Pharmaceutical Science, Vol 3 (No. 8): 149-153), Sivasankari et al. (International Journal of Current Microbiology and Applied Sciences, Vol 3 (No. 8): 904-909), Slimane et al. (2014, Bull Inst Natn Scientific Tech Sea of Salammbo, Vol 42: 13-15); Kumar et al. (2014, Plant Physiol J, Vol 19 (No. 2): 184-188), Manirafasha et al. (2017, Journal of Applied Phycology,: 1-10, doi: 10.1007 / sl0811-016-0989-y), Paswan et al. (2016, J Fluoresc, Vol 26 (2): 577-83, doi: 10.1007 / s 10895-015-1742-7); Chethana et al. (2015, J Food Sci Technol, Vol 52 (4): 2415-21, doi: 10.1007 / sl3197-014-1287-9), Seo et al. (2013, Int J Mol Sci Vol.14 (1): 1778-87, doi: 10.3390 / ijmsl4011778) or in PCT Application No. WO 2014/045177.

By inhibition of bone resorption is meant according to the invention, an inhibition of bone destruction activity by osteoclasts. To verify that the intake of phycocyanin, in humans or animals, inhibits bone resorption, a person skilled in the art can, for example, measure the urinary excretion of deoxypyridinoline, a decrease in the excretion of deoxypyridinoline being the reflection of an inhibition of the bone resorption (in: The biological markers of bone remodeling: pre-analytical variations and recommendations for their use., P. Garnero, F. Bianchi, Carlier PC, V. Genty, N. Jacob, S Kamel, C. Kindermans, E. Pluvier, M. Pressac & JC Souberbielle, Annals of Clinical Biology, 58 (6), 683-704 (2000)). In a preferred manner, to measure the inhibition bone resorption, one skilled in the art can measure the serum level of another protein marker, telopeptide C of type 1 collagen bypass (or "CTX" for "collagen type 1 cross-linked C-telopeptide") For the measurement of the serum level of CTX, one skilled in the art can refer to the article by Ganero et al. (2017. Mol Diagn Ther, doi: 10.1007 / s40291-017-0272-1). For the measurement of the serum level of CTX, a person skilled in the art may for example use commercially available measurement kits, such as the CTX kit (Osteoporosis test) marketed by LifeLabs®, the CTX test kit marketed by the Company. Roche, the ELISA test kit marketed under the reference No. LS-F20998 by LifeSpan BioSciences Inc., or the ELISA test kit marketed under the reference No. AC-02F1 by the company Immunodiagnosticsystem (IDS)

A nutritional composition or a therapeutic composition of phycocyanin as active compound is intended first of all for the prevention of bone loss due to an imbalance in the remodeling of bone tissue, in humans or animals, in particular in a mammal non-human, in particular a domestic mammal such as a dog or a cat, equines such as a horse, or in animals other than mammals such as avian species, which includes farmed avian species such as hens, geese, ducks, turkeys, pigeons etc.

The phycocyanin composition is particularly intended for individuals with symptoms of bone deficit (osteopenia), or likely to suffer from bone deficit, that is to say, an imbalance in the relationship between the formation of bone and resorption bone, which, if continued, induces a decrease in bone mass.

To determine whether a subject has a state of osteopenia (reduced bone mass), and therefore requires a supply of phycocyanin, the skilled person can refer in particular to the report of the World Health Organization (WHO) of 1994 Assessment of fracture risk and its application to screening for post-menopausal osteoporosis WHO Technical Series 843.

A nutritional or pharmaceutical composition for human or veterinary use according to the invention is useful for preventing the bone loss that occurs with aging.

In particular, a nutritional or pharmaceutical composition for human or veterinary use according to the invention is useful in the treatment of physiological situations such as osteoporosis type I or type II, secondary osteoporosis, Paget's disease, bone loss or osteolysis observed near a prosthesis.

Secondary osteoporosis includes bone resorption, which is caused by physiological conditions induced by the onset of diseases such as osteosarcoma, rheumatoid arthritis, rheumatoid spondylitis, malabsorption syndromes, hypogonadism, primary hyperparathyroidism, diabetes, obesity, chronic obstructive pulmonary disease, chronic liver disease, chronic renal failure, neurological disorders, AIDS, untreated hyperthyroidism and osteomalacia.

Thus, the treatment of secondary osteoporosis concerns the treatment of physiological situations of bone resorption which are not the cause of the disease to be treated but are a physiological imbalance concomitant with the disease to be treated. ,. In other words, the administration of a phycocyanin composition as defined in the present description to a patient afflicted with a specific pathology is not intended to exert a curative effect on the disease itself. The administration to a patient of a phycocyanin composition is therefore intended to inhibit bone resorption which is concomitant with the occurrence of said pathology.

A phycocyanin composition according to the invention may be in the form of a nutritional composition or in the form of a pharmaceutical composition, as described below.

Nutritional compositions of phycocyanin.

As already mentioned above, many disorders related to an imbalance of bone metabolism, such as osteoporosis, progressively evolve over a long period of time and require chronic treatments. Their prevention or treatment can therefore be achieved through a regular supply of phycocyanin, preferably in the form of a nutritional composition.

In particular, a regular nutritional intake of phycocyanin is useful for preventing bone loss that occurs during aging.

The invention relates to a nutritional composition for inhibiting bone resorption, characterized in that it comprises, as an active nutritional compound, phycocyanin. Nutritional composition is understood to mean a composition comprising, as active agent against bone resorption, a phycocyanin composition as defined in the present description and constituting a food composition or a dietary supplement which does not possess the characteristics of a drug.

A nutritional composition according to the invention may comprise from 1% by weight to 90% by weight of a phycocyanin composition as defined in the present description. The various uses of phycocyanin for the manufacture of a nutritional composition will be defined below in relation to the technical characteristics of said nutritional composition.

A nutritional composition according to the invention is preferably adapted for oral administration.

According to a first aspect, a nutritional composition according to the invention is a dietary food used for the maintenance of a good health of the man or the animal that ingests it.

Such a nutritional composition is also commonly referred to as a functional food, which is intended to be consumed, either as an integral part of the diet or as a dietary supplement, but whose phycocyanin content implies a physiological role going beyond the provision of the needs. basic nutrients.

A nutritional composition according to the invention includes food compositions enriched in phycocyanin.

In some aspects, said nutritional composition is intended to prevent bone loss that occurs with aging (osteopenia).

In other aspects, said nutritional composition is intended to prevent or treat disorders related to an imbalance in the ratio between the formation of bone and bone resorption.

According to still other aspects, said nutritional composition is intended to prevent physiological disorders chosen from osteopenia, type I or type II osteoporosis, secondary osteoporosis, Paget's disease and bone loss or osteolysis. observed near a prosthesis.

A nutritional composition, which comprises a phycocyanin composition as defined herein, can be in a wide variety of forms of food compositions and beverages, including juices (fruit or vegetable or seaweed), vegetable milks, oils, butters, margarines, fats vegetable, canned goods (eg tuna in oil), soups, milk-based preparations (yogurts, cottage cheese), ices, cheeses (eg cheeses preserved in oil), cooked products (such as bread , biscuits, pancakes and cakes), desserts, confectionery products, cereal bars, breakfast cereals, condiments, seasoning products (including spices and sauces).

In certain embodiments, a nutritional composition according to the invention may be in liquid form, for example in the form of an aqueous solution or in the form of an oily solution.

In other embodiments, a nutritional composition according to the invention may be in a solid form, for example in the form of a powder. In particular, the nutritional composition may be freeze-dried and be in the form of a powder. A nutritional composition according to the invention can also be in the form of a wide variety of products intended for animal feed, whether they are in wet form, in semi-wet form or in dry form, in particular in the form croquettes, granules.

In certain embodiments of a nutritional composition according to the invention, the phycocyanin composition as defined in the present description and which is included in said nutritional composition, is in the form of a product of extraction obtained from of a cyanobacterium belonging to the spirulina family, and in particular Spirulina platensis.

In other embodiments, a nutritional composition according to the invention is in the form of any product, in particular any beverage, for example a flavored beverage.

According to another aspect, in a nutritional composition according to the invention, the phycocyanin composition as defined in the present description can be produced by extraction or by synthesis. In particular, it is possible to use a recombinant phycocyanin produced by host cells transfected or transformed with a polynucleotide comprising an expression cassette encoding phycocyanin.

Preferably, a nutritional composition according to the invention comprises an amount of phycocyanin suitable for daily oral administration ranging from 0.05 mg to 10,000 mg. By way of illustration, in embodiments in which a nutritional composition is made using a phycocyanin composition having a phycocyanin content of 50 percent by weight based on the total weight of the phycocyanin composition, a nutritional composition is achieved. comprising 1000 mg of phycocyanin including 2000 mg of phycocyanin composition to the nutritional composition in preparation.

For consumption in humans, a nutritional composition according to the invention comprises a quantity of phycocyanin adapted to a daily intake of phycocyanin, provided by said composition, ranging from 1 mg to 10,000 mg, advantageously from 4 mg to 8000 mg , which includes 5 mg to 6,000 mg.

The results of the examples show an effectiveness of a phycocyanin composition as defined in the present description, for inhibiting bone resorption, when such a composition is administered in the mouse in a daily quantity of phycocyanin of 1 mg, that is to say because of a daily dose of 500 mg per kilogram of live weight.

Those skilled in the art can determine, using known methods, effective doses in other mammals, including humans (Hemon et al., Symposium "Science and Decision in Environmental Health", Sep 1996, Metz, France.ineris-00971997; Meyer C, eds., 2017, Dictionary of Animal Sciences. [On line] Montpellier, France, Cirad.

[05/21/2017]. <URL: http://dico-sciences-animales.cirad.fr/>).

According to these known methods, this means that a phycocyanin composition as defined in the present description is effective, for inhibiting bone resorption in humans, at a daily dose of phycocyanin of 55 mg per kilogram of live weight, ie daily dose of phycocyanin of (i) 4,400 mg for a human of 80 kgs and (ii) of 5,500 mg for a human of 100 kgs.

Thus, in certain embodiments, a phycocyanin composition as defined in the present description is suitable for a daily administration in humans of an amount of phycocyanin ranging from 4 mg to 10,000 mg, depending in particular on the weight of the considered individual.

Illustratively, a phycocyanin composition as defined herein is effective for inhibiting bone resorption in cats at a phycocyanin daily dose of 100 mg per kilogram of live weight. For example also, a phycocyanin composition as defined herein is effective, to inhibit bone resorption in dogs, at a daily dose of phycocyanin 70 mg per kilogram body weight.

As another illustration, a phycocyanin composition as defined in the present description is effective, for inhibiting bone resorption in the horse, at a daily dose of phycocyanin of 30 mg per kilogram of live weight.

In general, a phycocyanin composition as defined in the present description is preferably suitable for daily administration of phycocyanin ranging from 0.05 mg / kg to 1000 mg / kg, which ranges from 1 mg / kg to 200 mg / kg. mg / kg, for example from 10 mg / kg to 100 mg / kg.

In yet another aspect, the nutritional composition above may comprise other nutritional compounds, in combination with phycocyanin.

In some embodiments, particularly in embodiments wherein the phycocyanin composition is prepared by purifying phycocyanin from extracts, especially from cyanobacterial extracts, the phycocyanin composition may comprise, phycocyanin protein, also compounds that may consist of nutritional compounds, such as vitamins, lipids, carbohydrates, trace elements, mineral salts, or other phyto-constituents, which were initially contained in a crude extract departure.

In other embodiments, the nutritional composition comprises other nutritional compounds, which have been added to the starting phycocyanin composition.

Thus, the nutritional composition according to the invention may also comprise a source of calcium, for example in the form of a physiologically or physiologically acceptable inorganic compound, such as inorganic calcium salts (calcium chloride, calcium phosphate, sulfate Calcium oxide, calcium hydroxide, calcium hydroxide or calcium carbonate) or organic components containing calcium such as skim milk powder, calcium caseinate or organic calcium salts (calcium citrate, calcium maleate or their mixtures).

The amount of calcium contained in a nutritional composition according to the invention is suitable for a daily administration, provided by said composition, included between 100 mg and 1000 mg, preferably between 200 mg and 700 mg and most preferably between 300 mg and 600 mg of calcium.

A nutritional composition according to the invention may also comprise vitamins, such as vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, folic acid, thiamine, riboflavin, vitamin B6, vitamin B12, niacin, biotin or pantothenic acid.

A nutritional composition according to the invention may also comprise mineral elements and trace elements such as sodium, potassium, phosphorus, magnesium, copper, zinc, iron, selenium, chromium and molybdenum.

It may also include purified fibers such as agar, alginate, carob, carrageenan, gum arabic, guar gum, karaya gum, pectin or xanthan gum. these soils being in a hydrolyzed or unhydrolyzed form.

It may further include proteins, for example, nutritionally valuable proteins, such as protein hydrolysates, particularly milk protein hydrolysates, milk protein isolates containing micellar casein, meat protein hydrolysates. , vegetable proteins, etc.

It may also comprise energy source compounds, in particular one or more sources of carbohydrates chosen from maltodextrins, starch, lactose, glucose, sucrose, fructose, xylitol, fermentable sugars such as inulin and fructo-oligosaccharides, sorbitol and possibly fatty acids such as omega-3. In addition, a nutritional composition according to the invention may also comprise spices, aromatic herbs or other micro-nutriments such as other polyphenols, sterols, phenolic acids, stilbenes, carotenoids, iridoids, organo-sulfur and terpenes.

As already mentioned above, a phycocyanin composition for inhibiting bone resorption according to the invention may also be included in a pharmaceutical composition, as described below.

Human or veterinary pharmaceutical compositions according to the invention.

The subject of the invention is also a human or veterinary pharmaceutical composition for its use for inhibiting bone resorption, characterized in that it comprises, as active principle, a phycocyanin composition as defined in the present description.

In particular, the invention relates to the use of a phycocyanin composition for the manufacture of a pharmaceutical composition for human or veterinary use for the prevention or treatment of a pathology related to an imbalance of bone metabolism, and especially a pharmaceutical composition capable of inhibiting bone resorption. The uses of a phycocyanin composition for the manufacture of a pharmaceutical composition will be described in connection with the technical characteristics of said pharmaceutical composition hereinafter.

A pharmaceutical composition according to the invention comprises, as active principle, a phycocyanin composition as defined in the present description, in an amount adapted to inhibit bone resorption in individuals in need of such treatment.

In certain aspects, a human or veterinary pharmaceutical composition according to the invention is useful for preventing the bone loss that occurs during aging.

In some embodiments, a human or veterinary pharmaceutical composition according to the invention is useful in preventing bone loss that occurs during aging (osteopenia).

According to certain other aspects, a human or veterinary pharmaceutical composition according to the invention is useful for preventing or treating disorders or pathologies related to an imbalance in the ratio between the formation of bone and bone resorption.

In some embodiments, a pharmaceutical composition for human or veterinary use according to the invention is useful in the treatment of physiological disorders related to an imbalance of bone remodeling, such as osteopenia, osteoporosis type I or type 11, secondary osteoporosis, Paget's disease and bone loss or osteolysis observed near a prosthesis.

Secondary osteoporosis includes bone resorption, which is caused by physiological conditions induced by the onset of diseases such as osteosarcoma, rheumatoid arthritis, rheumatoid spondylitis, malabsorption syndromes, hypogonadism, primary hyperparathyroidism, diabetes, obesity, chronic obstructive pulmonary disease, chronic liver disease, insufficiency chronic renal failure, neurological disorders, AIDS, untreated hyperthyroidism and osteomalacia.

Thus, the treatment of secondary osteoporosis relates to the treatment of physiological bone resorption situations that are not the cause of a disease to be treated, but are concomitant with the disease to be treated. In other words, the administration of a phycocyanin composition as defined in the present description to a patient afflicted with rheumatoid arthritis is not intended to exert a curative effect on the disease itself. The administration to this patient of a phycocyanin composition exclusively aims to inhibit bone resorption which is concomitant with the occurrence of rheumatoid arthritis.

It may be a human or veterinary pharmaceutical composition, in particular for dogs or cats, equines such as horses, or in avian species, which includes farmed avian species such as hens. , geese, ducks, turkeys, pigeons etc.

The pharmaceutical composition according to the invention is in a form for oral, parenteral, intramuscular or intravenous administration.

In its form intended for human administration, a pharmaceutical composition according to the invention advantageously comprises an amount of phycocyanin suitable for daily administration of the active compound, provided by said composition, ranging from 0.01 mg to 10,000 mg.

For administration in humans, a pharmaceutical composition according to the invention comprises an amount of active compound adapted to a daily intake of phycocyanin, provided by said composition, ranging from 0.01 mg to 10 000 mg, preferably 1 mg at 8000 mg and most preferably from 10 mg to 6000 mg.

In general, a pharmaceutical composition as defined in the present specification is preferably suitable for daily administration ranging from 0.05 mg / kg to 1000 mg / kg, which ranges from 1 mg / kg to 200 mg / kg. for example from 10 mg / kg to 100 mg / kg.

A pharmaceutical composition according to the invention comprises a phycocyanin composition as defined in the present description in combination with at least one excipient selected from the group consisting of pharmaceutically acceptable excipients. Techniques for preparing pharmaceutical compositions according to the invention can easily be found by those skilled in the art, for example in the book Remmingston's Pharmaceutical Sciences, Mid. Publishing Co, Easton, PA, USA.

Physiologically acceptable adjuvants, vehicles and excipients are also described in "Handbook of Pharmaceutical Excipients, Second Edition, American Pharmaceutical Association, 1994".

In order to formulate a pharmaceutical composition according to the invention, those skilled in the art may advantageously refer to the latest edition of the European Pharmacopoeia or the United States Pharmacopoeia (USP).

Those skilled in the art may particularly advantageously refer to the fourth edition 2002 of the European Pharmacopoeia, or to the USP 25-NF 20 edition of the American Pharmacopoeia (U. S. Pharmacopeia).

A pharmaceutical composition as defined above is suitable for oral, parenteral, intramuscular or intravenous administration.

When the pharmaceutical composition according to the invention comprises at least one pharmaceutical or physiologically acceptable excipient, it is in particular a suitable excipient for administration of the composition orally or of a suitable excipient for administration of the parenteral composition.

The invention also relates to a method for preventing or treating a disorder related to an imbalance of bone metabolism, particularly a disorder associated with loss of bone mass, said method comprising a step in which patients are administered a therapeutically therapeutic amount. effective phycocyanin composition or a pharmaceutical composition containing phycocyanin.

A pharmaceutical composition comprising phycocyanin according to the invention is indifferently in a solid form or in a liquid form.

For oral administration, a solid pharmaceutical composition, in the form of tablets, capsules or capsules, will be preferred.

In the liquid form, a pharmaceutical composition in the form of an aqueous suspension or a lipid suspension, or in the form of a water-in-oil or oil-in-water emulsion, is preferred.

Solid pharmaceutical forms may comprise, as vehicles, adjuvants or excipients, at least one diluent, a flavor, a solubilising agent, an agent lubricant, a suspending agent, a binding agent, a disintegrating agent and an encapsulating agent, the identity and function of these various conventional excipients being fully documented in the European Pharmacopoeia or in the United States Pharmacopoeia (USP).

In some embodiments, a solid pharmaceutical composition includes those in the form of nanoparticles, including mesoporous silica nanoparticles, where appropriate functionalized; which allow controlled release, if appropriate delayed release, of the phycocyanin composition.

Such compounds are, for example, magnesium carbonate, magnesium stearate, talc, lactose, pectin, dextrin, starch, gelatin, cellulosic materials, cocoa butter, and the like.

The compositions in liquid form may also comprise water, optionally mixed with propylene glycol or polyethylene glycol, and optionally also coloring agents, flavorings, stabilizers and thickeners such as sugars in the liquid forms of the syrup type.

For the manufacture of a pharmaceutical composition according to the invention, the phycocyanin composition may be prepared in accordance with the teaching of the various patent documents mentioned above in the description.

The invention is further illustrated, without being limited, by the following examples.

EXAMPLES

A. MATERIALS AND METHODS

A. L. Animal study

A.1.1. Choice of the animal model

The experiment was conducted on ovariectomized mice at 8 weeks, a model adapted to the study of postmenopausal osteoporosis and having been used several times to test nutritional interventions or the potential of plant extracts. A.1.2. Legislative and technical aspects

Procedures related to animal testing have been approved by the Institute's Ethics Committee, respecting the recommendations of the European Union. The mice were housed in the Human Nutrition Unit pet shop, in a controlled environment: light cycle - darkness 12h-12h, room temperature between 20 and 22 ° C, relative humidity of 50-60%, accommodation conditions: one mouse per cage with water free access.

A standard diet (AIN 93) was provided by the Experimental Food Preparation Unit (UE0300 UPAE) of the INRA Center of Jouy-en-Josas. According to the experimental batches, the mice were supplemented by gavage with two different components:

a) In the first group, supplementation consists of 500 mg / kg live weight of lysed spirulina (10 mg / d / mouse), providing 1 mg of phycocyanin.

b) In the second case, the animals received 500 mg / kg live weight of phycocyanin purified from Spirulina (ie 10 mg / day / mouse). The dose of spirulina chosen was based on the fact that the food supplements available on the market bring up to 4 g of micro-algae per day for an average body weight of 60 kg. Regarding the extrapolation to the animal, with reference to the metabolic weight match (P ⁰ ^'75 ), a mouse will consume 12.5 mg / day of spirulina, ie 500 mg / kg body weight.

Forty-eight female C57BL / 6j mice, eight weeks old, were acquired from Janvier Laboratories (Saint-Berthevin, France). After 3 days of acclimation, the rodents were randomly divided into 4 groups of 12 animals:

- two groups received a standard regime called "control" (Ctrl),

the other two were exposed to the test gavages described above (freeze-dried spirulina or phycocyanin which was taken up in a volume of 250 μl of physiological saline, and then administered).

The power calculation determining the minimum number of animals required to demonstrate a significant effect was established by compiling data from the literature and previous studies (discriminant characterization of bone mineral density as the primary endpoint).

After 7 days of exposure to these diets, one of the two groups on the standard diet underwent pseudo-surgery (SH-Ctrl, placebo group). All other animals were ovariectomized and assigned to the following groups: OVX-Ctrl, OVX-Spirulina, OVX-Phycocyanin. Surgical procedures were performed under anesthesia (mixture of Imalgene 1000 (lg for 10mL) and Rompun 2% (0.5g of xylasin for 25mL)). The injected mixture (using a 26Gxl / 2 needle) is 1mL of imalgene, 0.5 mL of Rompun, supplemented to 10 mL with saline. These 10mL of solution are calculated for 1kg of mouse, ie 0.1g of ketamine and 0.01g of xylazine per kg of rodent. A drop of antibiotic is deposited on the wound after surgery to reduce the risk of infection, then the staples are laid. To limit pain upon awakening, a subcutaneous injection, containing a dose of buprenorphine (buprecar) at 0.1 mg / kg and metacam at 1 mg / kg, is performed after anesthesia. Upon waking, the animals had access to water containing paracetamol (2g / L) for 24 hours. Based on an average consumption of 3mL per day, this paracetamol solution allows the mouse to ingest 6mg of paracetamol over 24 hours, equivalent to 300mg / kg of body weight, the LD50 being fixed at 800mg / kg. body weight in intraperitoneal injection. During these 24 hours, the prostration, the movement of the animal and its food intake were controlled.

The different diets were initiated one week before the surgery. After 6 weeks of testing, the rodents were anesthetized. The injected mixture was the same as that used for ovariectomy (1mL of Imalgene, 0.5mL of Rompun, supplemented with 10mL with saline). The blood was then taken intracardiac in a Sarstedt tube and left at room temperature for 30 minutes. The tubes were then centrifuged for 5 min at 20 ° C and 10,000 x g. Serum was then collected and aliquoted into two different tubes (one for each serum label to be assayed) which were then stored in a freezer at -80 ° C until assayed. After blood collection, the animal was directly euthanized by cervical dislocation. The liver, spleen and uterus of the animals were collected and weighed to check for inflammation and to validate ovariectomy. The left and right femurs were also recovered. One out of every two femur was placed in a solution of formaldehyde and stored for one week at 4 ° C, then transferred to ethanol and finally stored again at 4 ° C until densitometer analysis. The other femur was kept directly in tubes in liquid nitrogen and then frozen at -80 ° C.

A.1.3. Analyzes performed

A.1.3.1. Animal weighing and consumption control

The weight of the animals was measured once a week. Consumption checks over 2 days were also carried out weekly. A.1.3.2. Echo MRI

The functioning of the Echo-MRI is based on the principle of nuclear magnetic resonance imaging which exploits the magnetic properties of atoms and allows to determine fat mass, lean mass, free water and total body water a small, unanesthetized live animal. Rodents enter a transparent plexiglass containment tube sized for body weight. The mouse goes back to the bottom of the tube. A slight stress is then applied using a piston to hold the animal in place at the bottom of the tube. The total time of contention does not exceed 2 minutes. Only one measurement is performed on each individual. Two evaluations of body composition were performed with EchoMRI-900: a first few days before ovariectomy, a second after 38 days of treatment.

A.1.3.3. Weight of the spleen and liver

At sacrifice, the spleen and liver are meticulously extracted from the murine and immediately weighed.

A.1.3.4. Biomarkers of bone metabolism

Bone resorption was evaluated using the RatLaps ™ EIA Immunodiagnostic Systems Elisa Kit, which quantifies telopeptide C of type I collagen (CTX 1), and through the Elisa Ret test. Mouse Trance / Rank L / TNFSF11 for Rank L.

For bone formation, the type I collagen propeptide (PINP) was measured using an IDS rat / mouse PINP EIA Elisa kit. This specific test makes it possible to determine the release of PINP during the synthesis of bone collagen. Osteoprotegerin was assayed using the kit provided by R & D Systems Quantikine Elisa Mouse OPG / TNFRSF 1 lb, an Elisa test. The procedures are provided by kit manufacturers.

A.1.3.5. Morphological analysis of bones

The morphological study of the femurs was performed using a micro-CT eXplore CT 120 scanner (GE Healthcare, Little Chalfont, United Kingdom) in the INSERM 990 Joint Research Unit. The acquisition consists of 360 shots on the chosen fabric. The left femurs are placed in a PBS buffer solution with 1 millisecond exposure per x-ray tube (100 kV and 50 mA). Images are reconstructed using a modified cone beam algorithm with an isotropic voxel 0.045 x 0.045 x 0.045 mm3. The obtained scans were analyzed with MicroViewH software version 2.3 (General Electric Healthcare Bio-Sciences, Pittsburgh, PA, USA). A calibration phantom of hydroxyapatite (SB3, Gamex RMI, WI, USA) serves as a reference for the conversion of gray levels to density values in hydroxyapatite. The trabecular bone of the distal femur was selected to determine bone mineral density and bone volume (BVF = bone volume / total volume) after definition of a cylindrical region (r = 0.7 mm). ) of interest at the center of the femur, starting at 0.1 mm from the growth plate and extending more than 0.32 mm in the proximal direction. Bone mineral density was estimated by the average of gray levels converted in the region of interest.

A.1.3.6. Statistical analyzes

The results are expressed as mean and standard error on the mean (SEM). The study was conducted on a total of 13 mice per group (5 groups in total), to allow a statistical analysis on at least 8 mice per group at the end of the study. Statistical analyzes are based on Fisher tests using XLSTAT software (ExcelStat Pro Software, Microsoft Office 2013).

A.2. In vitro study

A.2.1. Cell line and culture condition

MC3T3-E1 (immortalized mouse pre-osteoblast line) were cultured in sterile plates at the density of 3x10 ⁴ cells per cm ² . Cells were maintained in alphaMEM culture medium (GIBCO, Paisley, UK) supplemented with 1% penicillin / streptomycin (GIBCO, Paisley, UK) and 10% fetal calf serum (FBS, Lonza, Levallois- Perret, France). When the cells arrive at about 80% confluence, they were exposed to the different conditions: either the culture medium alone considered as the negative control (C-), or the medium containing 50 g / mL of ascorbic acid and 10 mM glycerophosphate alone (considered as the positive control) or, in the presence of phycocyanin at different concentrations (10, 25, 50, 100, 250

On the other hand, mouse RAW 264.7 (immortalized line) (ATCC, Washington, DC, USA) were used as pre-osteoclast and Rank-L-induced models. The cells were seeded in the wells at a density of 1 × 10 ⁴ cells / m ² and maintained in culture medium (α-minimal essential medium α-MEM; GIBCO, Paisley, UK) supplemented with 1% penicillin / streptomycin (GIBCO, Paisley, UK) and 10% fetal calf serum (FBS). When the cells arrived at about 80% confluence, they were exposed to different conditions: either the culture medium alone considered as the negative control (C-), or the culture medium containing either a culture medium alone considered as the negative control (C-), or culture medium containing 50 ng / mL of receptor activator of nuclear factor-kappa B ligand (RANK-L) (R & D Systems) alone (considered as the positive control), or in the presence of phycocyanin at different concentrations (5, 10, 25, 50, 100 μg / ml).

Both cell types were cultured at 37 ° C in a humid atmosphere at 5% CO 2 in air. The medium was changed every 2 days.

A. 2.2. Cell proliferation

The measurement of cell proliferation is based on a colorimetric assay. A yellow tetrazolium salt XTT is reduced by the succinate dehydrogenase activity of viable cells in the presence of an electron coupler reagent. The reaction produces an orange formazan salt.

RAW264.7 and MC3T3-E1 were inoculated into 96-well plates at a density of 3.5x10 ³ cells per well, then cultured for 2 hours with:

a) In the case of MC3T3-El, 10% serum alone, 2% serum alone or with phycocyanin at a concentration of 100, 250, 1000, 2500 μg / ml.

b) In the case of RAW 264.7, 10% serum alone, 2% serum alone or with phycocyanin at a concentration of 10, 25, 50, 100, 250,1000 μg / ml.

Cell viability was determined at 24 hours by a basée -based method using a cell proliferation kit (Sigma-Aldrich Cell Proliferation Kit II, St. Louis, MO, USA), following the manufacturer's recommendations. OD was determined at 450 nm.

A. 2.3. Measurement of alkaline phosphatase (ALP) activity

Alkaline phosphatase is an early marker of bone formation. The measurement of its activity is based on the ability of the enzyme to catalyze the hydrolysis of p-nitrophenylphosphate (p-NPP) to p-nitrophenol, a chromogen with an absorbance at 405 nm.

The enzymatic activity of ALP was measured on osteoblasts at 0, 2, 7 and 14 days according to the method previously published, adapted to our conditions experimental. The osteoblast culture is rinsed twice with PBS (Sigma-Aldrich, 38297 St. Quentin Fallavier, France), then placed at -20 ° C. Then the cells are lysed and homogenized in diethanolamine / magnesium chloride hexahydrate buffer (pH 9.8, Sigma-Aldrich). The cell lysate (ΙΟμΙ) is added to 200 μl of p-nitrophenyl phosphate solution (6 mg / ml) (Sigma-Aldrich, St. Louis, MO, USA). The absorbance is measured at 405 nm in kinetics (every 150s for 30 min), at 30 ° C., using an ELX808 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Finally protein measurement is performed using the BioRad protein assay (BioRad, Munich, Germany).

ALP is expressed in micromoles of p-nitrophenol per hour and per milligram of protein.

A. 2.4. Measurement of tartrate-resistant acid phosphatase activity (TRAP)

Resistant tartrate acid phosphatase is described as an enzymatic marker of osteoclasts that resorb bone tissue. This enzyme, also known as type 5 acid phosphatase, exists under iso forms 5a and 5β, the latter being exclusively recognized as the 5β active isoform of TRAP specific for osteoclasts. The inactive part of the 5β fraction is produced by macrophages.

TRAP activity is measured according to the standard method using a leukocyte acid phosphatase kit (Sigma Aldrich).

After 3 days of culture, the cells lysed with the NP 40 lysis buffer buffer are incubated at 37 ° C. in a p-nitrophenyl phosphate buffer, with 125 mM sodium acetate buffer (pH 5.2), and 100 mM p-nitrophenyl phosphate.

Absorbance is measured at 37 ° C to 405 nm in kinetics at 37 ° C using a plate reader

For each sample, the protein concentration is measured and Trap is expressed in OD / min / mg protein.

A. 2.5. TLD A and method of obtaining cDNAs

A.2.5.1. RNA extraction

Extraction, which aims to recover the necessary elements to analyze the expression of transcripts, takes place in several stages. The ribonucleic acids (RNAs) are purified following phenol-chloroform extraction (Trizol-Roche) and then assayed. A.2.5.2. Retro transcription of RNA

Strict aseptic conditions are required to avoid the application of false positives and contamination. To perform the retranscription, we used the high capacity cDNA reverse transcription kits (applied). For each sample, 20 μί of solution are prepared and deposited in the ice. The first step consists of preparing the 2X reverse transcription master mix, in ice, consisting of DNA polymerase (enzyme making the retro transcription of an RNA in cDNA), a buffer for optimizing the enzymatic activity, primers for fixing the polymerase on the RNA strand and of course nucleotides.

In a second phase, ΙΟμί of mix are added to ΙΟμί of a mixture of water and RNA extracted at the previous stage (the proportions within the mixture being adapted so that the same concentration of RNA is found in each of the conditions up to 500 ng), in the well of a 96-well plate.

After centrifugation of the plate, the sample is placed in thermocycler TC 512 TECHNE to initiate the reaction with a suitable program for the realization of TLDA.

10 minutes at 25 ° C, then 120 minutes at 37 ° C and finally 5 minutes at 85 ° C.

A.2.5.3. TLDA

The goal is to amplify the targeted sequence and quantify it. TLDA (or TaqMan® Array) technology makes it possible to rapidly determine the expression profile of a large number of genes or miRNA, in several samples, in a reduced reaction volume. Its high sensitivity and reproducibility make it a powerful tool for performing analyzes from very small quantities of starting material, and therefore suitable for micro genomics studies. TLDA® are micro-fluidic cards composed of 384 wells. They make it possible to simultaneously analyze from 1 to 8 samples out of 11 to 380 different primer systems per real-time PCR run (2h30).

In this study, we used TLDA cards specifically designed for the exploration of 48 genes involved in the bone resorption process in mice. For each sample, we prepared a solution of 100 μΐ held in ice with 50 μΐ TAqman® Fast Advanced Master Mix and 50 μΐ cDNA in water. Before being placed in the device (Applied Biosystems 7900 HT Real Time PCR System) and to perform the analysis using the SDS software, the cards are centrifuged twice for 1 minute at 1200 RPM.

A.2.5.4. Statistical analyzes

The results are expressed as means ± their standard errors (SEM).

All data were analyzed using XLSTAT software (Addinsoft, Paris, France).

Statistical analysis of the ANOVA type (parametric test) made it possible to highlight the differences between groups. If the result was considered significant (P <0.05), the multiple comparison Fisher test was then used to determine the specific differences between the means.

Example 1 In Vivo Effect of Phycocyanin on Bone Resorption

1.1. Validation of the study

The significant decrease in uterine horn weight in the OVX control group compared to SH-Ctrl, validates the quality of castration. In ovariectomized mice, this parameter was not significantly modified by the different experimental regimes.

1.2. Animal weighing and consumption control

Daily weight gain (GMQ) was assessed daily for the first 7 days of treatment, then weekly. From the third week, this parameter is significantly higher in OVX-Ctrl compared to SH-Ctrl.

The results obtained show that the different diets (or gavage) did not induce a significant variation of this criterion.

Consumption control, performed weekly during the experiment, shows no significant difference between the two groups submitted to the standard diet (SH-Ctrl and OVX-Ctrl). Castration does not affect food intake. It is the same in ovariectomized animals, receiving the standard diet and despite the force-feeding.

The results obtained did not show that the administration of phycocyanin caused a variation in the weight of the animals or in their food consumption. 1.3. Echo MRI

At the end of the experiment, fat mass was significantly higher in OVX-Ctrl compared to SH-Ctrl. On the other hand, consumption of the different diets did not have a statistically significant effect in ovariectomized animals.

Thus, the results did not show an effect of the administration of phycocyanin on the fat mass of the animals.

1.4. Weight of the spleen and liver

At sacrifice, in the OVX-Ctrl group this parameter is statistically higher than what is observed in the SH-Ctrl group. Inflammation following surgery could therefore be detected. On the other hand, it was not standardized by the experimental interventions since no significant difference is observed within the different OVX groups.

The different experimental conditions, with or without administration of phycocyanin, did not significantly alter the average weight of the livers.

1.5. Biomarkers of bone metabolism

For OPG, which is an indicative marker of bone resorption inhibition, significantly higher values are found in the SH-Ctrl group compared to P OVX-Ctrl, as well as in the OVX phycocyanin group. compared to the OVX-Ctrl group, compared to the OVX-Ctrl group.

As a result, the results show that phycocyanin at least partially normalizes the increase in the OPG marker that is induced by surgery.

Conversely, the RankL marker, which constitutes a marker indicative of bone resorption, is increased by castration. The administration of phycocyanin did not bring standardization of the level of this marker.

The OPG / RankL ratio, which is a bone remodeling index, is significantly higher in the SH-Ctrl group compared to the measured value of this ratio in the OVX-Ctrl group.

The OPG / RankL ratio is increased in ovariectomized rodents to which phycocyanin has been administered, which allows the metabolism to be favorably oriented for preservation of bone capital. With respect to the PINP marker, which is indicative of bone formation, no statistically significant difference is observed between the different experimental groups.

The same results are observed with respect to the marker CTX 1, which is a marker indicative of bone resorption.

1.6. Analysis of bone mass

Bone mass was measured in the different groups of animals. The results are shown in Figure 1.

The analysis of bone mineral density (BMD) confirms the decrease, conventionally observed, of this parameter in OVX-Ctrl mice compared to SH-Ctrl.

On the other hand, only the OVX group to which phycocyanin has been administered has a significantly higher bone mineral density than that of the OVX control lot.

These results show that the administration of phycocyanin blocks bone resorption in animals in which an ovariectomy hormone deficiency has been experimentally induced.

Example 2 In Vitro Confirmation of the Effects of Phycocyanin on Bone Resorption

The various experiments carried out in vitro were conducted with increasing doses of phycocyanin.

2.1. Cell proliferation

The proliferation of osteoblasts was measured by XTT after 24 hours of induction in the presence of different concentrations of phycocyanin.

The most important effect was observed in the cells exposed to the 100 μg / ml and 250 μg / ml phycocyanin doses, compared to the 2% control group. (Figure 2)

In contrast, the concentration of 2500 μg / ml was inhibitory, while the dose of 1000 μg / ml had no statistically significant effect. (Figure 2)

With regard to osteoclasts, proliferation was inhibited in a dose-dependent manner in cell cultures exposed to phycocyanin. (Figures 3 and 4) These results show that phycocyanin has an inhibitory effect on the proliferation of osteoclasts, which is in agreement with the in vivo results which show that phycocyanin has an inhibitory effect on bone resorption. 2.2. Measurement of Tartrate-Resistant Acid Phosphatase (TRAP) Activity

The activity of tartrate-resistant acid phosphatase (also known as Tartrate-Resistant-Acid Phosphatase or TRAP) was measured in osteoclast cultures. The results are shown in Figure 4.

The results of Figure 4 show that during osteoclast differentiation, the measurement of TRAP enzyme activity is significantly higher in the RankL control group than control. This phenomenon is inhibited in a dose-dependent manner by phycocyanin.

2.3. TLD A

To study the effect of phycocyanin on bone resorption, we measured the expression of different markers, respectively (i) the TRAP marker indicative of cell differentiation (Figure 6), (ii) the Nox4 marker indicative of oxidative stress ( Figure 7) and (iii) the NFATC1 marker which is a transcription factor (Figure 8).

TLDA analysis was performed on RA m extracted from cultures of the Raw 264.7 line, which is a model cell line of pre-osteoclasts. Cell cultures of the Raw 264.7 line were exposed to phycocyanin.

The results show :

a decrease in the level of specific markers of osteoclasts (such as TRAP (FIG. 6), the calcitonin receptor, metalloproteases),

oxidative stress, with a decrease in the expression of Nox 4 (see FIG. 7),

a decrease in the expression of the NFATC1 transcription factor, in the presence of a phycocyanin concentration of 10 μg / ml (see FIG. 8).

Summary of experimental results

The animal experiment carried out in ovariectomized mice, 8 weeks old, allowed to highlight a potentially beneficial action on the bone of a daily supplementation with phycocyanine, at the dose of 500 mg / kg body weight. In fact, the results of the measurement of femoral trabecular BMD indicate that this Nutritional intervention helps to prevent, in part, bone loss induced by reduced estrogen impregnation, as well as biological markers of bone remodeling such as OPG.

To validate these results in vivo, experiments were also conducted on immortalized murine lines of osteoblasts and osteoclasts exposed to various concentrations of phycocyanin. This compound essentially targets the differentiation and proliferation of osteoclasts with a dose dependent decrease in TRAP synthesis and a dose-dependent decrease in the expression of TRAP, MMP9, calcitonin receptor, and NOS 2 and NFATcl. To a lesser extent, there is pro-proliferative action on osteoblasts.

Claims

1. Phycocyanin composition for use in inhibiting bone resorption in humans or animals.

2. Phycocyanin composition for use according to claim

1, characterized in that said phycocyanin composition is included in a nutritional composition adapted for oral administration.

3. Phycocyanin composition for use according to claim

2, said composition being intended to prevent the bone loss that occurs with aging.

4. A phycocyanin composition for use according to claim 2, said composition being intended to prevent or treat pathologies selected from type I or type II osteoporosis, secondary osteoporosis, Paget's disease, and osteolysis. observed near a prosthesis.

5. phycocyanin composition for use according to one of claims 2 to 4, characterized in that said nutritional composition is in the form of food compositions, drinks, including juices (fruit or vegetable or seaweed) ), vegetable milks, oils, butters, margarines, vegetable fats, preserves (eg oil tuna), soups, milk-based preparations (yogurt, cottage cheese), ices, cheeses (eg cheeses preserved in oil), cooked products (such as bread, cookies, pancakes and cakes), desserts, confectionery products, cereal bars, breakfast cereals, condiments, food seasoning products (including spices and sauces).

6. phycocyanin composition for use according to one of claims 2 to 4, characterized in that said nutritional composition is in the form of a product for animal feed, in wet, semi-wet or dry form .

7. phycocyanin composition for use according to one of claims 1 to 6, characterized in that said phycocyanin composition is in the form of an extraction product obtained from spirulina.

8. phycocyanin composition for use according to one of claims 2 to 7, characterized in that said nutritional composition is adapted to daily administration ranging from 0.05 mg / kg to 1000 mg / kg, preferably from 1 mg / kg to 200 mg / kg, and preferably from 10 mg / kg to 100 mg / kg.

9. phycocyanin composition for use according to claim 1, characterized in that said phycocyanin composition is included in a human or veterinary pharmaceutical composition.

10. Phycocyanin composition for use according to claim 9, characterized in that said pharmaceutical composition is intended to prevent bone loss which occurs during aging.

11. phycocyanine composition for use according to claim 9, characterized in that said pharmaceutical composition is intended to prevent or treat a pathology related to an imbalance in the ratio between the formation of bone and bone resorption.

12. phycocyanin composition for use according to claim 9, characterized in that said pharmaceutical composition is intended to prevent or treat a pathology selected from type I or type II osteoporosis, secondary osteoporosis, Paget's disease, bone loss or osteolysis observed near a prosthesis.

13. A phycocyanin composition for use according to claim 9, characterized in that said pharmaceutical composition is in a form for oral, parenteral, intramuscular or intravenous administration.

14. A phycocyanin composition for use according to claim 9, characterized in that said pharmaceutical composition is suitable for a daily administration ranging from 0.05 mg / kg to 1000 mg / kg, advantageously from 1 mg / kg to 200 mg / kg. kg, and preferably from 10 mg / kg to 100 mg / kg.