USE OF A MURAMYL EPETID TO TREAT SKELETAL GROWTH DELAY

Description

Technical field

[0001] The present invention relates to the use of at least one muramyl peptide, such as muramyl dipeptide or one of its analogues, in the treatment of skeletal growth retardation in a juvenile subject, particularly in humans or undernourished animals. State of the art

[0002] Linear and weight growth is an inherent capacity of all juvenile multicellular organisms. In mammals, postnatal growth is controlled by the somatotropic axis where growth hormone (GH, from the English " Growth Hormone " orders the liver and peripheral tissues to produce insulin-like growth factor 1 (IGF-1, from the English " Insulin-like Growth Factor-1"), in order to promote systemic growth, that is, the coordinated growth of its organs, tissues, and skeleton. Thus, IGF-1 is the main factor controlling skeletal growth, also called linear or longitudinal growth. After IGF-1, insulin is the body's main anabolic hormone, regulating the metabolism of carbohydrates, lipids, and proteins. While the somatotropic axis (GH / IGF-1) is of paramount importance during growth, coordinated regulation of metabolism by insulin is necessary to provide substrates and fuel to growing tissues. Consequently, insulin also influences the development and growth of animals preferentially by supporting weight gain.

[0003] Childhood growth retardation is characterized by a reduced growth rate during postnatal development. It manifests as a reduction in weight (weight gain) and a decrease in height (skeletal growth, also called linear or longitudinal growth) at a given age compared to the healthy population of the same age. It is a primary manifestation linked to acute malnutrition or chronic undernutrition and / or recurrent enteric infections, such as diarrhea and helminthiasis, in early childhood and even before birth, due to maternal malnutrition during fetal development. In 2012, it was estimated that 162 million children under the age of 5, or 25%, suffered from growth retardation.More than 90% of children suffering from stunting worldwide live in Africa and Asia, where 36% and 56% of children are affected, respectively (United Nations Children's Fund, World Health Organization, The World Bank, UNICEF-World Bank Joint Child Malnutrition Estimates). Once established, stunting and its systemic effects generally persist into adulthood and are long-lasting.

[0004] Muramyl peptides are components of peptidoglycans in bacterial cell walls that are released into the body during the degradation, growth, or division of bacteria constituting the gut microbiota. They are considered natural regulators of immunity. According to numerous studies, muramyl peptides are the minimal biologically active fragments that initiate the immune response after interacting with intracellular receptors of the NLR family (from the English expression " NOD-like Receptors " of innate immunity (NOD1, NOD2, NALP3, etc.). Among these, muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-isoglutamine) is a synthetic immunoreactive molecule consisting of N-acetylmuramic acid attached to a short amino acid chain of -Ala-D-isoGln. It was first identified in bacterial cell wall peptidoglycan as an active component of Freund's complete adjuvant (FCA). In 1974, MDP was found to be the minimal structure required for the efficacy of FCA, one of the most potent and widely used adjuvants in experimental animal models. MDP and its derivatives have been shown to exhibit significant immunomodulatory properties, via one of the PRRs (" Pattern Recognition Receptors "), the NOD2 receptor (from the English expression " Nucleotide-binding Oligomerization Domain 2(Girardin S. et al., J. Biol. Chem., 2003, 278(11), p. 8869-72; F. Coulombe et al, 2012 PLoS ONE, 7(5): Article ID e36734). Muramyl dipeptides are also known to activate macrophages and other immune system cells to kill cancer cells (Z. Jakopin, Current Medicinal Chemistry, 2013, 20(16), 2068-2079).

[0005] Furthermore, US patent application 2015 / 141352 describes the use of muramyl dipeptide (MDP) or a pharmaceutical composition containing it to prevent bone loss, promote bone regeneration, and support bone formation. Among the bone disorders that can be treated by systemic administration of MDP is rickets, a growth and bone formation disorder observed in infants and young children suffering from acute malnutrition, primarily due to vitamin D and calcium deficiency. Rickets is characterized by stunted weight gain and insufficient calcification of bones and cartilage.

[0006] Patent application FR3020949 describes a composition comprising an intestinal-tropic strain of Lactobacillus for use in promoting juvenile growth in cases of malnutrition.

[0007] Although many therapeutic applications of muramyl dipeptide or its derivatives have already been proposed, its use for the treatment of skeletal growth delays in humans or chronically undernourished juvenile animals has not yet been described.

[0008] The present invention falls precisely within the restoration of conditions enabling the restoration of better skeletal growth, or even the restoration of normal skeletal growth (also called linear or longitudinal), particularly in a context of chronic undernutrition, and especially in a situation of protein deficiency.

[0009] It is in this context that it was surprisingly discovered that muramyl peptides, and in particular muramyl dipeptide (MDP) and its chemical analogs, could be advantageously used, notably orally or via inhalation, in the treatment of skeletal growth retardation in humans or juvenile animals, especially when this growth retardation is due to chronic undernutrition, particularly protein deficiency, i.e., a daily protein intake lower than the recommended daily allowance (RDA). As a guide, the RDA for protein in children between 0 and 36 months is approximately 9 to 12 g / day, 15 to 30 g / day for children aged 4 to 10 years, and 30 to 50 g / day for boys aged 11 to 18 years and 30 to 40 g / day for girls aged 11 to 18 years.It has thus been demonstrated that oral administration of MDP, or one of its analogues such as mifamurtide or murabutide, has a positive effect on skeletal growth (linear or longitudinal) in a juvenile mouse model subjected to a protein-deficient nutritional diet and mimicking chronic undernutrition (examples 1 and 2).

[0010] The present invention thus aims to propose new compositions enabling the improvement of skeletal growth (linear or longitudinal) in juvenile subjects, human or animal, or even the restoration of normal growth, particularly in humans or animals who have been or are subjected to chronic undernutrition, and in particular to a protein deficiency.

[0011] Another objective of the invention is to provide such compositions, which can also be used in a therapeutic context. Objects of the invention

[0012] The present invention is defined in the claims. The first object of the present invention is a composition comprising at least one muramyl peptide, for use in the treatment of skeletal growth delays (or linear growth or longitudinal growth) in a human or juvenile animal.

[0013] According to this first objective, the composition may also contain at least one adjuvant to promote growth, particularly juvenile growth, said adjuvant being in particular chosen from bacterial strains having an intestinal tropism, in particular strains of commensal species or species present in the intestinal microbiota of the target species, or acceptable as a probiotic.

[0014] Thus, the indication of the composition according to the present invention may be therapeutic or health, as a drug or as a pharmaceutical composition, or nutritional, in particular as a food supplement, or postbiotic, or even probiotic when said composition contains at least one bacterial strain with intestinal tropism.

[0015] A second object of the invention is the non-therapeutic use of a composition according to the invention comprising at least one muramyl peptide and at least one bacterium belonging to the families Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae, And Bifidobacteriaceae as an adjuvant, said bacterium being live or inactivated, in a non-therapeutic probiotic treatment method to promote skeletal growth in an animal or human subject. Definitions

[0016] A muramyl dipeptide analogue or chemical analog is a chemical species that differs from muramyl dipeptide only by the replacement of one atom or group of atoms by another, and that exhibits physicochemical and biological properties similar to those of muramyl dipeptide.

[0017] The World Health Organization (WHO) definition of juvenile skeletal growth retardation is that the "height for age" value is less than two standard deviations from the median growth norms of a healthy child.

[0018] A juvenile subject is a subject that has not reached sexual maturity.

[0019] In this description, the terms "skeletal growth," "longitudinal growth," and "linear growth" are synonymous with "juvenile growth." This growth can be basically measured by height in a human, or by the length from the snout or mouth to the base of the tail in mammals, or by body length in vertebrates and invertebrates.

[0020] "Juvenile growth" does not include weight gain, whether attributed to an increase in muscle mass and / or an increase in fat mass. Conversely, juvenile growth within the meaning of the present invention can be assessed by an increase in bone length or body length and / or an increase in the production of IGF-1 and / or its functional analogs in animals with an exoskeleton but also without a bony skeleton such as invertebrates, and such as peptides belonging to the family of insulin-like factors (" insulin-like peptides " And " insulin-like growth factors ".

[0021] Undernutrition (or malnutrition) is a state of significant food deficiency characterized by insufficient food intake to meet an individual's daily nutritional needs and energy expenditure, resulting in nutritional and metabolic deficiencies. Undernutrition can be chronic or acute. In the case of chronic undernutrition, and particularly in the case of protein deficiency, individuals are in a state described as " stunting or of short stature ", that is to say, in a state of altered or even irreversible growth retardation, unlike cases of acute undernutrition (such as rickets) where growth retardation, particularly weight retardation, can be reversible. Detailed description

[0022] The first object of the invention is a composition comprising at least one muramyl peptide, for use in the treatment of skeletal growth retardation in a human or juvenile animal.

[0023] According to a particular and preferred embodiment of the invention, the human or animal is chronically undernourished. Most preferably, this state of chronic undernutrition is a protein deficiency.

[0024] According to a preferred embodiment of the invention, said at least one muramyl peptide is selected from muramyl dipeptide (or MDP or MurNAc-L-Ala-γ-D-Glu, CAS No. 53678-77-6) and analogues of muramyl dipeptide.

[0025] MDP is commercially available in lyophilized form (powder), notably from the company InvivoGen Europe (Toulouse, France). It is of bacterial origin and can be found in purified or semi-purified forms, such as MDP bound to fragments of bacterial cell wall.

[0026] Muramyl dipeptide analogues are synthetic peptides, among which we can mention mifamurtide (or muramyl tripeptide phosphatidyl ethanolamine, also known as MTP-PE) such as, for example, the product sold under the trade name Mepact by the company Takeda, murabutide (or muramyl dipeptide butyl ester) (InvivoGen Europe, Toulouse, France), 6-O-stearoyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine (or L18-MDP), muropeptide MurNAc-Ala-D-isoGln-Ly (M-TriLYS, InvivoGen Europe, Toulouse, France) and their mixtures.

[0027] According to a preferred embodiment of the invention, the muramyl dipeptide analogue is chosen from murabutide and mifamurtide.

[0028] According to a preferred embodiment of the invention, said at least one muramyl peptide is muramyl dipeptide.

[0029] Said at least one muramyl peptide generally represents from 10 µg to about 100 mg per unit dose of composition, more particularly from about 50 µg to about 50 mg, preferably from about 100 µg to about 10 mg, and even more preferably from 0.5 to 1 mg per unit dose of composition.

[0030] The number of doses to be administered and the frequency of administration of these doses are of course to be adjusted according to the size, weight of the human or animal subject to be treated, as well as the severity of the growth retardation and the bioavailability of the muramyl peptide(s) administered.

[0031] According to the present invention, a unit dose is understood to mean the quantity of composition according to the invention that can be administered, per dose, to a subject to be treated, regardless of the total mass of this unit dose.

[0032] According to a particular and preferred embodiment of the invention, the composition further comprises at least one adjuvant to promote growth, particularly in a juvenile subject.

[0033] According to this particular embodiment, said adjuvant may be chosen from bacterial strains having intestinal tropism, bacterial constituents, postbiotics and mixtures thereof.

[0034] By bacteria having an “intestinal tropism”, we mean a bacterium that has the ability to pass the gastric barrier and is able to persist in the intestines.

[0035] According to an advantageous feature of the invention, said bacterium can promote the production of IGF-1 in humans or animals treated with the composition according to the invention.

[0036] The bacterial strains with intestinal tropism that can be used in the composition according to the invention include strains of commensal species or species present in the intestinal microbiota of the target species or acceptable as a probiotic.

[0037] Defined in 2001 by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO), probiotics are live microorganisms which, when ingested in sufficient quantities, exert positive effects on health, beyond the usual nutritional effects.

[0038] According to the invention, postbiotics are inactivated compounds based on inactivated microbial / bacterial cells or isolated inactivated cellular components (such as short-chain fatty acids, peptides, proteins, bacterial cell wall components or enzymes), with or without metabolites, which also offer health benefits.

[0039] The bacterial strains are chosen from bacteria belonging to the following families: Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae, And Bifidobacteriaceae.

[0040] Among such strains, we can particularly mention strains of the genus Lactobacillus, in particular one of the following species, Lactobacillus delbrueckii, Lactobacillus plantarum (or according to the new name) Lactiplantibacillus plantarum), Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei, And Lactobacillus rhamnosus .

[0041] More specifically, these are bacteria belonging to the species Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei, And Lactobacillus rhamnosus Depending on the method, the bacterial strain is chosen from among the species Lactobacillus plantarum, Lactobacillus fermentum, And Lactobacillus casei.

[0042] According to one embodiment, the bacterial strain is chosen from L. plantarum WJL deposited at the CNCM (National Collection of Microorganism Cultures - Pasteur Institute) on February 15, 2017 under number I-5169, L. plantarum IGFL1 filed on July 19, 2017 with the CNCM under number I-5217, L. plantarum IGFL2 filed on July 19, 2017 with the CNCM under number I-5218, L. plantarum G821 filed on May 11, 2015 with the CNCM under number I-4979, L. plantarum NIZO2877, L. casei ATCC 393, L. casei L919, L. paracasei ATCC25302, L. paracasei Shirota, L. fermentum ATCC9338, L. rhamnosus L900, L. rhamnosus L908, L. rhamnosus GG, and their mixtures.

[0043] Among such strains, we prefer L. plantarum WJL filed with the CNCM on February 15, 2017 under number I-5169, L. plantarum IGFL1 filed with the CNCM under registration number I-5217 on July 19, 2017. L. plantarum G821 filed with the CNCM under registration number I-4979 on May 11, 2015, L. plantarumIGFL2 filed with the CNCM under registration number I-5218 on July 19, 2017, and L. plantarum NIZO2877.

[0044] The IGFL1 strain was obtained by experimental evolution (i.e., by selection of natural variants having accumulated mutations induced naturally during DNA replication processes) of the WJL strain.

[0045] The G821 strain was obtained by experimental evolution (i.e., by selection of natural variants having accumulated mutations induced naturally during DNA replication processes) of the NIZ02877 strain.

[0046] The IGFL2 strain was obtained by experimental evolution (i.e., by selection of natural variants having accumulated mutations induced naturally during DNA replication processes) of the NIZ02877 strain.

[0047] Other examples of suitable strains include: L. casei ATCC 393, L. casei L919 (Koryszewska-Baginska A. et al., September 26, 2013, Genome Announcement), L. paracasei ATCC25302, L. paracasei Shirota (Yuki N et al., Int J Food Microbiol. April 1, 1999; 48(1):51-7), L. fermentum ATCC9338, L. rhamnosus L900 (Aleksandrzak-Piekarczyk T. et al., Genome Announc, August 15, 2013), L. rhamnosus L908 (Koryszewska-Baginska A. et al., February 20, 2014, Genome Announc), L. rhamnosus GG (Kankainen M. et al., Proc Natl Acad Sci USA, October 6, 2009).

[0048] The composition according to the invention may comprise the bacterial strain(s) in live form. It may be a bacterial suspension, which can be frozen and thawed before use, or a lyophilized powder, which can be used as is or after rehydration in a suitable liquid vehicle. It may then comprise a conventional lyophilization excipient.

[0049] Within the composition according to the present invention, the bacterial strain(s) may be present in an amount of approximately 10⁵ to 10¹², in particular approximately 10⁶ to 10¹², preferably approximately 10⁸ to 10¹² colony-forming bacterial cells (CFU, from the English expression " Colony forming unit "), per gram of composition.

[0050] The composition according to the invention may also include the bacterial strain(s) in inactivated form. The inactivation of the bacterial strain(s) may be achieved by any suitable technique well known to those skilled in the art, such as, for example, bacteriolytic or bacteriostatic chemical treatment or physical treatments by pressure, radiation, heat, or ultrasound.

[0051] When the composition according to the invention contains bacterial constituents as an adjuvant, these may, for example, be selected from inactivated bacteria, bacterial lysates, purified bacterial cell walls and fragments of purified bacterial cell walls from bacteria with intestinal tropism, peptidoglycans, and teichoic acids. Intestinal-tropic bacteria suitable for preparing purified bacterial cell walls or fragments of purified bacterial cell walls may, in particular, be selected from bacterial strains with intestinal tropism as described above.

[0052] According to a particular and preferred embodiment of the invention, the composition comprises purified bacterial cell walls of L. plantarum WJL and / or purified bacterial cell wall fragments of L. plantarum WJL.

[0053] Within the composition according to the present invention, the bacterial constituent(s) may be present in an amount of about 0.02 µg to 200 mg, in particular about 0.2 µg to 2 mg, preferably about 20 µg, per unit dose of composition.

[0054] The composition according to the invention may further contain, as an additional ingredient, one or more food supplements and / or nutrients; these may in particular be chosen from carbohydrates, lipids, peptides other than muramyl peptides, proteins, fatty acids, amino acids such as L-glutamine, vitamins, minerals, and prebiotics such as, for example, acacia gum fibers, etc.

[0055] The composition according to the present invention may be administered orally or via inhalation and may be presented, for example, as a powder, capsule or tablet, an oral solution, a nasal spray, or an airborne spray. In a particular embodiment of the invention, said form of administration is gastro-resistant. This embodiment is particularly suitable when the composition contains at least one bacterial strain as an adjuvant in addition to said at least one muramyl peptide. Indeed, this presentation allows it to pass through the stomach undegraded and then release said bacteria in the intestine.

[0056] The present invention also relates to the use of a composition comprising at least one muramyl peptide and at least one bacterium belonging to the families Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae, And Bifidobacteriaceaeas an adjuvant, said bacterium being live or inactivated, in a non-therapeutic probiotic treatment method to promote skeletal growth in an animal or human subject.

[0057] According to the use in accordance with the invention, the method comprises administering a sufficient quantity of a composition conforming to the first object of the invention and as defined above. The quantity and frequency of administration will depend, in particular, on the severity of the skeletal growth retardation, the age, and the condition of the patient or animal. The method will comprise administering, in one or more doses, which may be staggered over the subject's growth period (until puberty or sexual maturity), doses of the composition according to the invention. The doses may, in particular, be divided to facilitate administration, especially according to the subject's age. The frequency of administration is, in particular, between one dose (single or divided) daily and one dose monthly.Typically, the frequency of administration will range from one dose (single or divided) every day to one dose every week, or even every 2, 3, 4, 5, or 6 days. Each dose (single or divided) represents several grams to several tens of grams of the composition.

[0058] Other features and advantages of the invention will become apparent from the detailed description of the following examples, as well as from the figures 1 to 14 attached figures that represent the different effects of a treatment on the juvenile growth of young mice treated daily for 5 weeks after weaning with a placebo, MDP, or murabutide (Example 1 and figures 1 to 6 ) or by a placebo or mifamurtide (Example 2 and figures 7 to 12 ), or by a placebo, MDP alone, or a combination of MDP and bacteria Lactiplantibacillus plantarum WJL strain or Lactiplantibacillus plantarum WJL strain alone (example 3 and Figures 13 and 14 ) and on which: [ Fig.1 ] : represents the size (in cm) of animals treated either with placebo, MDP, or murabutide as a function of the number of days after birth. [ Fig. 2 ] : represents the relative size (in %) of animals treated with either placebo, MDP, or murabutide as a function of the number of days after birth. [ Fig.3 ] : represents the growth rate (in cm / day) of animals treated either with placebo, MDP, or murabutide. [ Fig. 4 ] : represents the size of the femur (in mm) of the animals treated either with placebo, MDP, or murabutide. [ Fig. 5 ] : represents the size of the tibia (in mm) of animals treated either with placebo, MDP, or murabutide. [ Fig. 6 ]: represents the circulating amount of IGF-1 growth factor (in ng / ml) in animals treated with either placebo, MDP, or murabutide. [ Fig. 7 ] : represents the size (in cm) of the animals treated either with placebo or with mifamurtide according to the number of days after birth. [ Fig. 8 ] : represents the relative size (in %) of animals treated either with placebo or with mifamurtide as a function of the number of days after birth. [ Fig. 9 ] : represents the growth rate (in cm / day) of animals treated either with placebo or with mifamurtide. [ Fig. 10 ] : represents the size of the femur (in mm) of animals treated either with placebo or with mifamurtide. [ Fig. 11 ] : represents the size of the tibia (in mm) of animals treated either with placebo or with mifamurtide.[ Fig. 12 ] : represents the circulating amount of IGF-1 growth factor (in ng / ml) in animals treated with either placebo or mifamurtide. [ Fig. 13 ] : represents the growth gain (in %) in animals treated either with MDP alone or with bacteria Lactiplantibacillus plantarum WJL strain alone, or by the association of MDP + bacteria Lactiplantibacillus plantarum WJL strain, compared to animals treated with placebo. [ Fig. 14 ] : represents the percentage improvement in circulating IGF-1 levels (in %) in animals treated either with MDP alone or with bacteria Lactiplantibacillus plantarum WJL strain alone, or by the association of MDP + bacteria Lactiplantibacillus plantarum WJL strain, compared to animals treated with placebo. EXAMPLES

[0059] The raw materials used in the examples are as follows: Muramyl dipeptide, sold under the name MDP by InvivoGen Europe, 5 rue Jean Rodier, F-31400 Toulouse, France; Murabutide, sold under the name Murabutide by InvivoGen Europe, 5 rue Jean Rodier, F-31400 Toulouse, France; Mifamurtide, sold under the name Mifamurtide by CliniSciences, 74 Rue des Suisses, 92000 Nanterre, France; Dimethyl sulfoxide (DMSO), sold under the name Dimethyl sulfoxide by Merck, Darmstadt, Germany; Maltodextrin, sold under the name Maltodextrin by Merck, Darmstadt, Germany. Bacteria Lactiplantibacillus plantarum strain WJL (filed with the CNCM on February 15, 2017 under number 1-5169). EXAMPLE 1: Demonstration of the effects of administering different muramyl peptides in a chronically malnourished mouse model

[0060] In this example, the effects of several compositions according to the present invention comprising isomolar amounts of MDP or murabutide (MDP M) were tested. assemolar mean weight (Mw) = 492.5 g / mol, Murabutide Mw = 548.6 g / mol) in a chronically undernourished mouse model, namely: composition A comprising 25 µg of MDP in 30 µL of placebo composition, composition B comprising 28 µg of murabutide in 30 µL of placebo composition.

[0061] These compositions were compared to a placebo composition containing only a cryoprotectant (Maltodextrin) resuspended in 30 µL of sterile saline buffer. 1.1 Preparation of compositions

[0062] 15 µg of cryoprotectant were resuspended in 30 µL of sterile saline buffer to generate a dose of placebo composition.

[0063] 25 µg of MDP were resuspended in 30 µL of placebo composition to obtain composition A at a final MDP concentration of 25 µg / 30 µL.

[0064] 28 µg of Murabutide were resuspended in 30 µL of placebo composition for composition B at a final Murabutide concentration of 28 µg / 30 µL (isomolar to 25 µg / 30 µL of MDP). 1.2 Animal Preparation

[0065] Conventional C57Bl / 6j laboratory mice were bred at the Gnotobiology Laboratory for over 10 generations. The mice were kept in IVC cages (Tecniplast, Italy), exposed to 12:12 hour light-dark cycles, fed tap water, and fed ad libitumMice were fed the sterile diet V1124-300 (Ssniff Spezialdiäten GmbH) (irradiated ~25 kGy, Bioster, Czech Republic). They were mated, and after birth, litter size was reduced to 6 pups per mother. On day 21, male mice were weaned onto the low-protein, low-fat experimental diet (3.5 kcal / gram, protein: 4.1% by weight, carbohydrates: 78% by weight, fat: 2% by weight) and monitored regularly until 56 days postpartum (adulthood in mice). Mice weaned onto the low-protein, low-fat diet were treated 5 times per week with placebo, composition A containing MDP, or composition B containing Murabutide. At the end of the experiment, food and bedding were removed at 8 a.m. and the mice were sacrificed after 5 hours of fasting by isoflurane inhalation and cervical dislocation.All the animals were sacrificed within the hour between 1 p.m. and 2 p.m. 1.3 Administration of compositions

[0066] The daily treatment dose was 30 µL of placebo, or for MDP, 25 µg in 30 µL of placebo, and for murabutide, 28 µg in 30 µL of placebo. Each composition was administered by pipette onto the mouse's tongue. The mouse was held until the entire composition had been swallowed. 1.4 Description of tests performed

[0067] For body length measurement, mice were briefly anesthetized with isoflurane (Piramal Healthcare, UK). The anesthetized mouse was held by the tail, and the nose-to-anus length was measured using a ruler. Relative length was calculated as a percentage gain in length, where the body length on day 21 (weaning) was set at 100%. The rate of length growth was calculated by dividing the absolute length gain from day 21 to day 56 by the number of days (i.e., 35). Femoral and tibial bones were dissected at the time of sacrifice, fixed in 4% paraformaldehyde-PBS overnight at 4°C, washed with PBS, and stored in 70% ethanol. Bone length was measured using a digital caliper (Festa).

[0068] At the end of the experiment, the blood was collected and allowed to coagulate at room temperature for 2 hours. The sera were separated by centrifugation (2000 x g for 5 minutes, 4 °C) and stored at -80 °C until use. IGF-1 levels were measured using the Mouse / Rat IGF-1 Quantikine ELISA kit (R&D systems) according to the manufacturer's instructions.

[0069] The data were analyzed using the following statistical test: One way ANOVA with Tukey's multiple comparisons test. 1.5 Results

[0070] The results obtained are presented on the Figures 1 to 6 attached.

[0071] There figure 1 represents the evolution of height (in cm) as a function of the number of days after birth for each of the tested compositions: composition A with MDP: curve with empty squares, composition B with murabutide: empty circles and curve with crosses: placebo composition. figure 2represents the evolution of relative growth (% change) as a function of the number of days after birth for each of the compositions tested and using the same symbols as for the figure 1 . There figure 3 represents the evolution of the growth rate (in cm / day) for each of the tested compositions. figure 4 represents the femur length (in mm) for each of the tested compositions. figure 5 represents the size of the tibia (in mm) for each of the tested compositions. figure 6 represents the level of IGF-1 in the blood (in ng / ml) for each of the compositions tested.

[0072] These results indicate significantly increased relative growth, total length, and growth rate in mice receiving composition A containing MDP or composition B containing Murabutide compared to mice receiving the placebo composition. Specifically, the femur of animals treated with composition A containing MDP was significantly longer than that of animals treated with the placebo composition, with a positive trend following Murabutide treatment that did not reach statistical significance. Tibia length also showed a positive trend of increased growth following MDP and Murabutide treatments compared to placebo treatments, but this trend did not reach statistical significance.Finally, circulating levels of IGF1 are significantly increased in animals treated with murabutide, and a positive but non-significant trend is observed in animals treated with MDP compared to animals treated with placebo. EXAMPLE 2: Demonstration of the effects of mifamurtide administration in a chronically malnourished mouse model

[0073] In this example, the effects of a composition according to the present invention comprising isomolar amounts of a muramyl peptide mifamurtide (Mw = 1237.50 g / mol) were tested in a chronically undernourished mouse model, namely: Mifamurtide 62.5 µg / day in 30 µL of placebo.

[0074] This composition was compared to a placebo composition containing only maltodextrin (cryoprotectant) resuspended in 28.75 µL of saline buffer and 1.25 µL of DMSO. 2.1 Preparation of compositions

[0075] 15 µg of cryoprotectant were dissolved in 1.25 µL of DMSO and 28.75 µL of sterile saline buffer to generate a dose of placebo composition.

[0076] 10 mg of Mifamurtide were resuspended in 200 µL of DMSO to obtain a solution of 50 µg / µL Mifamurtide. 1.25 µL of this solution were resuspended in 28.75 µL of placebo composition to obtain a composition having a final Mifamurtide concentration of 62.5 µg / 30 µL (isomolar with 25 µg / 30 µL of MDP). 2.2 Animal Preparation

[0077] Conventional C57BI / 6j laboratory mice were bred at the Gnotobiology Laboratory for over 10 generations. The mice were kept in IVC cages (Tecniplast, Italy), exposed to 12:12 hour light-dark cycles, fed tap water, and fed ad libitummice were bred using the sterile diet V1124-300 (Ssniff Spezialdiäten GmbH) (irradiated ~25 kGy, Bioster, Czech Republic). After birth, the litter size was reduced to 6 pups per mother. On day 21, male mice were weaned onto the low-protein, low-fat experimental diet (3.5 kcal / gram, protein: 4.1 wt%, carbohydrates: 78 wt%, fat: 2 wt%) and monitored regularly until 56 days postpartum. Mice weaned onto the low-protein, low-fat diet were treated 5 times per week with either the placebo composition or the composition containing mifamurtide as prepared in section 1.1 above. At the end of the experiment, food and bedding were removed at 8 a.m. and the mice were sacrificed after 5 hours of fasting by isoflurane inhalation and cervical dislocation.All the animals were sacrificed within the hour between 1 p.m. and 2 p.m. 2.3 Administration of compositions

[0078] Each composition was administered via pipette onto the mouse's tongue. The mouse was held until the entire composition had been swallowed. 2.4 Description of tests performed

[0079] For body length measurement, mice were briefly anesthetized with isoflurane (Piramal Healthcare, UK). The anesthetized mouse was held by the tail, and the nose-to-anus length was measured using a ruler. Relative length was calculated as a percentage gain in length, where the body length on day 21 (weaning) was set at 100%. The rate of length growth was calculated by dividing the absolute length gain from day 21 to day 56 by the number of days (i.e., 35). Femoral and tibial bones were dissected at the time of sacrifice, fixed in 4% paraformaldehyde-PBS overnight at 4°C, washed with PBS, and stored in 70% ethanol. Bone length was measured using a digital caliper (Festa).

[0080] At the end of the experiment, the blood was collected and allowed to coagulate at room temperature for 2 hours. The sera were separated by centrifugation (2000 x g for 5 minutes, 4 °C) and stored at -80 °C until use. IGF-1 levels were measured using the Mouse / Rat IGF-1 Quantikine ELISA kit (R&D systems) according to the manufacturer's instructions.

[0081] The data were analyzed using the following statistical test: unpaired t-test. 2.5 Results

[0082] The results are presented on the Figures 7 to 12 attached. The figure 7 represents the evolution of height (in cm) as a function of the number of days after birth for each of the tested compositions: composition with mifamurtide: curve with empty triangles, placebo composition: curve with crosses. figure 8represents the evolution of relative growth (% change) as a function of the number of days after birth for each of the compositions tested and using the same symbols as for the figure 7 . There figure 9 represents the evolution of the growth rate (in cm / day) for each of the tested compositions. Figure 10 represents the femur length (in mm) for each of the tested compositions. figure 11 represents the size of the tibia (in mm) for each of the tested compositions. figure 12 represents the level of IGF-1 in the blood (in ng / ml) for each of the compositions tested.

[0083] These results indicate significantly increased relative growth, total length, and growth rate in mice receiving the mifamurtide-containing composition compared to mice receiving the placebo composition. Femur and tibia lengths, as well as circulating IGF-1 levels, were significantly increased in animals treated with the mifamurtide composition compared to those treated with the placebo composition. EXAMPLE 3 : Demonstration of the effects of combined administration of MDP and an intestinal-tropic bacterium in a chronically undernourished mouse model

[0084] In this example, we tested the effects of different compositions: a composition P (placebo) containing only a cryoprotectant (15 µg of maltodextrin) resuspended in 30 µL of sterile saline buffer, a composition A according to the present invention comprising MDP alone at a dose of 25 µg / day in 30 µL of placebo, a composition AB according to the present invention comprising MDP at a dose of 25 µg / day and live bacteria Lactiplantibacillus plantarum WJL strain at a dose of 2 x 10⁸ CFU / day in 30 µL of placebo, a comparative composition B not forming part of the invention comprising only bacteria Lactiplantibacillus plantarum WJL strain at a dose of 2.10 8< CFU / day in 30 µL of placebo. 3.1 Preparation of compositions

[0085] Compositions P and A were prepared as shown above in example 1.

[0086] Composition AB was prepared like composition A but also adding 2 x 10⁸ CFU of the bacteria Lactiplantibacillus plantarum WJL strain in the 30 µL of placebo in addition to the 25 µg of MDP.

[0087] Composition B was prepared by adding only 2 x 10⁸ CFU of the bacteria Lactiplantibacillus plantarum WJL strain in 30 µL of placebo. 3.2. Animal Preparation

[0088] The tests were carried out on male mice with each of the compositions P, A, AB and B above, according to the protocol indicated in example 1, point 1.1 above (period of 56 days). 3.3. Administration of compositions

[0089] The daily treatment dose was 30 µL of composition P (placebo) or 25 µg of MDP alone in 30 µL of placebo (composition A), or 25 µg of MDP and 2 x 10⁸ CFU of bacteria Lactiplantibacillus plantarum WJL strain in 30 µL of placebo (composition AB) or 2 x 10⁸ < CFU of bacteria Lactiplantibacillus plantarum WJL strain in 30 µL of placebo (composition B).

[0090] Each composition was administered via pipette onto the mouse's tongue. The mouse was held until the entire composition had been swallowed. 3.4. Description of tests performed

[0091] The length size of the mice was measured weekly for each of the groups of mice treated with compositions P, A, AB and B according to the method indicated above in point 1.4 of example 1.

[0092] The gain in length growth at day 56 (in %) of animals treated with compositions A, AB and B was calculated relative to the length growth measured at day 56 on mice treated with the placebo composition according to the protocol indicated above in point 1.4 of example 1.

[0093] The circulating IGF-1 level was determined for each of the lots, according to the protocol indicated above in example 1, point 1.4. The percentage improvement in the circulating IGF-1 level for each of the compositions tested (A, AB and B) was then calculated relative to the circulating IGF-1 level measured on the mice in the lot treated with the placebo composition (100%).

[0094] The numerical data from the measurements were analyzed using appropriate statistical methods (one-way ANOVA with Tukey's multiple comparisons test) to test the effects of each of the compositions A, AB, and B, and any potential cumulative effects between treatment with MDP alone and with the bacteria. Lactiplantibacillus plantarum WJL strain alone or the MDP + bacteria combination Lactiplantibacillus plantarum WJL strain. 3.5. Results

[0095] The results obtained are reported on the Figures 13 and 14 attached.

[0096] There figure 13 represents the % improvement in linear growth for each of the compositions tested compared to the placebo.

[0097] There figure 14 represents the % improvement in circulating IGF-1 levels for each of the compositions tested compared to placebo.

[0098] These results confirm the positive impact of MDP on the relative linear growth rate compared to the placebo condition. They also show that administration of the AB composition, including the combination of MDP and bacteria, Lactiplantibacillus plantarum strain WJL significantly increases the effect of treatment on the relative linear growth rate compared to each of the components administered alone ( Figure 13 This result is corroborated by the study of circulating IGF-1 levels in mice that received the AB composition, i.e., the MPD + bacteria combination Lactiplantibacillus plantarum WJL strains for which a significantly more pronounced effect is observed than when each component is administered alone.

[0099] Therefore, the presence of intestinal-tropic bacteria, such as bacteria Lactiplantibacillus plantarum The WJL strain significantly improves the action of muramyl peptides on skeletal growth.

Claims

1. A composition comprising at least one muramyl peptide, for use in the treatment of skeletal growth retardation in a juvenile human or animal.

2. The composition for use according to claim 1, characterised in that said at least one muramyl peptide is selected from muramyl dipeptide and muramyl dipeptide analogues, said analogues being selected from mifamurtide, murabutide, 6-O-stearoyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, muropeptide MurNAc-Ala-D-isoGln-L, and mixtures thereof.

3. The composition for use according to claim 1 or 2, characterised in that said at least one muramyl peptide is muramyl dipeptide.

4. The composition for use according to any of claims 1 to 3, characterised in that said at least one muramyl peptide represents from 10 µg to 100 mg per unit dose of composition.

5. The composition for use according to any of claims 1 to 4, characterised in that said composition additionally comprises at least one adjuvant for promoting growth, said adjuvant being selected from bacteria belonging to the Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae and Bifidobacteriaceae families, bacterial constituents, postbiotics and mixtures thereof.

6. The composition for use according to claim 5, characterised in that said adjuvant is selected from bacteria belonging to the Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae and Bifidobacteriaceae families.

7. The composition for use according to claim 5 or 6, characterised in that said strains are selected from the Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus casei species.

8. The composition for use according to any of claims 5 to 7, characterised in that said strains are selected from L. plantarum WJL, L. plantarum IGFL1, L. plantarum IGFL2, L. plantarum G821, L. plantarum NIZ02877, L. casei ATCC 393, L. casei L919, L. paracasei ATCC25302, L. paracasei Shirota, L. fermentum ATCC9338, L. rhamnosus L900, L. rhamnosus L908, L. rhamnosus GG, and mixtures thereof.

9. The composition for use according to claim 5, characterised in that the bacterial constituents are selected from inactivated bacteria, bacterial lysates, purified bacterial walls and purified bacterial wall fragments of bacteria with intestinal tropism, peptidoglycans and teichoic acids.

10. The composition for use according to any of the preceding claims, characterised in that it contains, as an additional ingredient, one or more food supplements and / or nutrients selected from carbohydrates, lipids, peptides other than muramyl peptides, proteins, fatty acids, amino acids, vitamins, minerals and prebiotics.

11. The composition for use according to any of the preceding claims, characterised in that the subject is a chronically undernourished human or animal subject due to protein deficiency.

12. Non therapeutic use of a composition comprising at least one muramyl peptide and at least one bacteria belonging to the Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae and Bifidobacteriaceae families as an adjuvant, said bacteria being alive or inactivated, for promoting skeletal growth in an animal or human subject.