USE OF A MURAMYL PEPTIDE IN THE TREATMENT OF JUVENILE GROWTH DELAYS

Abstract

The present invention relates to the use of at least one muramyl peptide, such as muramyl dipeptide or one of its analogs, in the treatment of skeletal growth retardation in humans or juvenile animals. Figure for abstract: None

Description

Title of the invention: USE OF A MURAMYL PEPTIDE IN TREATMENT DELAYS IN JUVENILE GROWTH 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 juvenile growth retardation, particularly in humans or malnourished animals. Prior art

[0002] Linear and weight-bearing growth is an inherent capacity of all juvenile multicellular organisms. In mammals, postnatal growth is controlled by the somatotropic axis, where growth hormone (GH) signals the liver and peripheral tissues to produce insulin-like growth factor 1 (IGF-1) in order to promote systemic growth, that is, the coordinated growth of organs, tissues, and the skeleton. Thus, IGF-1 is the primary 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 fuels to growing tissues. Consequently, insulin also influences the development and growth of animals preferentially through support for weight gain.

[0003] Juvenile 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 growth 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 respectively 36% and 56% of children are affected (United Nations Children's Fund, World Health Organization, The World Bank. UNICEFWHO-World Bank). Joint Child Malnutrition Estimates). Once established, growth retardation and its systemic effects generally persist into adulthood and endure.

[0004] Muramyl peptides are constituents of peptidoglycans in bacterial cell walls that are released into the body during the degradation 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 (Nod-like receptor) family 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 discovered 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). It is also known that muramyl dipeptides activate macrophages and other immune system cells to kill cancer cells (Z. Jakopin, Current Medicinal Chemistry, 2013, 20(16), 2068-2079).Although numerous therapeutic applications of muramyl dipeptide or its derivatives have already been proposed, its use for the treatment of juvenile growth retardation in humans or chronically malnourished animals has not yet been described.

[0005] The present invention falls precisely within the restoration of conditions allowing to restore better skeletal growth, or even to restore normal skeletal growth (also called linear or longitudinal), particularly in a context of acute undernutrition or chronic undernutrition.

[0006] It was in this context that it was surprisingly discovered that muramyl peptides, and in particular muramyl dipeptide (MDP) and its chemical analogues, could be advantageously used in the treatment of juvenile growth retardation in humans or animals, especially when such growth retardation is due to chronic undernutrition. It has thus been demonstrated that oral administration of MDP, or one of its analogues such as mifamurtide, or murabutide, have a positive effect on skeletal growth (linear or longitudinal) in a juvenile mouse model subjected to a nutritionally deficient diet mimicking chronic undernutrition (examples 1 and 2).

[0007] 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 that have been or are subjected to chronic undernutrition.

[0008] Another objective of the invention is to provide such compositions, which can also be used in a therapeutic context.

[0009] Another objective of the invention is to provide a method of therapeutic treatment. Objects of the invention

[0010] The present invention has as its first object 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.

[0011] According to this first object, the composition may further contain at least one adjuvant to promote growth, in particular juvenile growth, said adjuvant being in particular chosen from bacterial strains having an intestinal tropism, in particular strains of commensal species or of species present in the intestinal microbiota of the target species, or acceptable as a probiotic.

[0012] 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.

[0013] A second object of the invention is a method of treating skeletal growth delays (or linear growth or longitudinal growth) in humans or juvenile animals, comprising administering to a subject in need, a composition according to the invention comprising at least one muramyl peptide, optionally in association with at least one adjuvant to promote growth, in particular skeletal growth. Definitions

[0014] According to the invention, an analogue or chemical analog of muramyl dipeptide is a chemical species that differs from muramyl dipeptide only by the replacement of one atom or group of atoms by another, and which exhibits physicochemical and biological properties similar to those of muramyl dipeptide.

[0015] The definition of juvenile skeletal growth retardation according to the World Health Organization (WHO) is that the "height for age" value is less than two standard deviations from the median of the growth norms of a healthy child.

[0016] A juvenile subject is a subject that has not reached sexual maturity.

[0017] 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 size of the snout or mouth to the base of the tail in mammalian animals or by body length in vertebrates and invertebrates.

[0018] According to the present invention, "juvenile growth" does not include weight gain, whether related 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 analogues for animals possessing an exoskeleton but lacking 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").

[0019] 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, individuals are in a state of "stunting" or "short stature," that is, a state of impaired or even irreversible growth retardation, unlike cases of acute undernutrition where juvenile growth retardation, as defined by this invention, can be reversible. Detailed description

[0020] 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.

[0021] According to a particular and preferred embodiment of the invention, said human or animal is undernourished, the latter being either acute or chronic.

[0022] 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-yD-Glu, CAS No. 53678-77-6) and analogues of muramyl dipeptide.

[0023] MDP is commercially available in lyophilized form (powder), notably from InvivoGen Europe (Toulouse, France). It is of bacterial origin and may be in purified or semi-purified form, such as, for example, MDP bound to fragments of bacterial cell wall.

[0024] Muramyl dipeptide analogues are synthetic peptides, among which we can mention in particular mifamurtide (or muramyl tripeptide phosphatidyl ethanolamine, also called 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.

[0025] According to a preferred embodiment of the invention, the muramyl dipeptide analogue is chosen from murabutide and mifamurtide.

[0026] According to a preferred embodiment of the invention, said at least one muramyl peptide is muramyl dipeptide.

[0027] Said at least one muramyl peptide generally represents about 10 to 100 pg per unit dose of composition, more particularly about 20 to 80 pg, and preferably about 25 to 65 pg, per unit dose of composition.

[0028] 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.

[0029] According to a particular and preferred embodiment of the invention, the composition further comprises at least one adjuvant to promote growth, in particular juvenile growth.

[0030] According to this particular embodiment, said adjuvant can be chosen from bacterial strains having an intestinal tropism, bacterial constituents, postbiotics, food supplements, nutrients, and mixtures thereof.

[0031] By bacteria having an “intestinal tropism”, we mean a bacterium having the ability to pass the gastric barrier and which is able to persist in the intestines.

[0032] According to an advantageous feature of the invention, said bacterium can promote the production of IGF-1 in humans or animals that are treated with the composition according to the invention.

[0033] The bacterial strains having an intestinal tropism usable in the composition according to the invention are in particular strains of commensal species or of species present in the intestinal microbiota of the target species or acceptable as a probiotic.

[0034] 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.

[0035] 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.

[0036] The bacterial strains are preferably chosen from bacteria belonging to the following families: Lactobacillaceae, Streptoccaceae, Enterococcaceae, Leuconostocaceae, and Bifidobacteriaceae.

[0037] Among such strains, we can more particularly mention strains of the genus Lactobacillus, in particular of one of the following species, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus.

[0038] More specifically, these are bacteria belonging to the species Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus. According to one modality, the bacterial strain is chosen from among the species Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus casei.

[0039] According to one embodiment, the bacterial strain is chosen from L. plantarum WJL, L. plantarum IGFL1 deposited on July 19, 2017 at the CNCM (National Collection of Microorganism Cultures - Pasteur Institute) under number 1-5217, L. plantarum IGFL2 deposited on July 19, 2017 at the CNCM under number 1-5218, L. plantarum G821 deposited on May 11, 2015 at the CNCM under number 1-4979, 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.

[0040] Among such strains, preference is given to L. plantarum WJL, L. plantarum IGFL1 deposited at the National Collection of Microorganism Cultures (Institut Pasteur) under registration number CNCM 1-5217 on July 19, 2017, L. plantarum G821 deposited at the National Collection of Microorganism Cultures (Institut Pasteur) under registration number CNCM 1-4979 on May 11, 2015, L. plantarum IGFL2 deposited at the National Collection of Microorganism Cultures (Institut Pasteur) under registration number CNCM 1-5218 on July 19, 2017, and L. plantarum NIZO2877.

[0041] The WJL strain (Eun-Kyoung Kim et al., Genome Announcements, November / December 2013, vol. 1, no. 6 e00937-13, GenBank AUTE00000000, Lactobacillus plantarum WJL, whole genome shotgun sequencing project) was initially isolated and can be isolated from Drosophila (JH Ryu et al., Science 2008, 19: 777-782).

[0042] 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.

[0043] Strain G821 was obtained by experimental evolution (i.e. by selection of natural variants having accumulated mutations induced naturally during DNA replication processes) of strain NIZ02877.

[0044] 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. Other examples of suitable strains are as follows: L. casei ATCC 393, L. casei L919 (Koryszewska-Baginska A. et al., September 26, 2013, Genome Announc), 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).

[0046] 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.

[0047] Within the composition according to the present invention, the bacterial strain(s) may be present in a quantity of about 105 to 1012, in particular about 10 to 10, preferably about 10 to 10 colony-forming bacterial cells (CFU, from the English expression "Colony forming Unit"), per gram of composition.

[0048] 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 usable 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.

[0049] According to a particular and preferred embodiment of the invention, the composition comprises purified bacterial cell walls of L. plantarum WJL and / or fragments of purified bacterial cell walls of L. plantarum WJL.

[0050] Within the composition according to the present invention, the bacterial constituent(s) may be present in an amount of about 0.02 pg to 200 mg, in particular about 0.2 pg to 2 mg, preferably about 20 pg, per unit dose of composition.

[0051] When the composition according to the invention contains, as an adjuvant, 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.

[0052] 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. According to 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.

[0053] The present invention also relates to a method for treating skeletal growth delays in a human or juvenile animal subject, in particular in an undernourished subject, said method comprising the administration to a subject in need of it, of a composition comprising at least one muramyl peptide and as defined according to the first object of the invention.

[0054] As previously indicated, said method may be a therapeutic treatment or a probiotic (or nutritional supplement) treatment when the composition also contains at least one bacterium with intestinal tropism as an adjuvant.

[0055] Thus, the present invention also relates to a probiotic or postbiotic treatment method to promote skeletal growth in an animal or human subject, in particular an undernourished subject, said method comprising the administration to said subject of a composition comprising at least one muramyl peptide and in the case of probiotic treatment at least one bacterium with intestinal tropism as an adjuvant.

[0056] The method according to the invention comprises administering a sufficient quantity of a composition conforming to the first object of the invention and such that 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 involve 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 depending on the subject's age. The frequency of administration will range from one dose (single or divided) daily to one dose monthly. Typically, the frequency of administration will range from one dose (single or divided) daily to one dose weekly, or even every 2, 3, 4, 5, or 6 days. Each dose (single or divided) will represent, in particular, several grams to several tens of grams of the composition.

[0057] Other features and advantages of the invention will become apparent from the detailed description of the following examples, as well as from the attached Figures 1 to 12, which illustrate 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 with a placebo or mifamurtide (Example 2 and Figures 7 to 12), and on which:

[0058] [Fig. 1]: represents the size (in cm) of the animals treated either with placebo, or with MDP, or with murabutide as a function of the number of days after birth.

[0059] [Fig.2]: represents the relative size (in %) of the animals treated either with the placebo, either by MDP or by murabutide depending on the number of days after birth.

[0060] [Fig.3]: represents the growth rate (in cm / day) of animals treated either by the placebo, either by MDP or by murabutide.

[0061] [Fig.4]: represents the femur length (in mm) of animals treated with either placebo, either by the MDP, or by the murabutide.

[0062] [Fig.5]: represents the size of the tibia (in mm) of the animals treated either with placebo, either by the MDP, or by the murabutide.

[0063] [Fig.6]: represents the circulating quantity of the growth factor IGF-1 (in ng / ml) animals treated either with placebo, MDP, or murabutide.

[0064] [Fig.7]: represents the size (in cm) of the animals treated either with the placebo or with the mifamurtide depending on the number of days after birth.

[0065] [Fig.8]: represents the relative size (in %) of the animals treated with either placebo, either by mifamurtide depending on the number of days after birth.

[0066] [Fig.9]: represents the growth rate (in cm / day) of animals treated either by the placebo, or mifamurtide.

[0067] [Fig. 10]: represents the size of the femur (in mm) of the animals treated either with placebo or with mifamurtide.

[0068] [Fig. 11]: represents the size of the tibia (in mm) of the animals treated either with placebo or with mifamurtide.

[0069] [Fig. 12]: represents the circulating quantity of the growth factor IGF-1 (in ng / ml) of animals treated either with placebo or with mifamurtide. EXAMPLES

[0070] The raw materials used in the examples are as follows:

[0071] - Muramyl dipeptide sold under the name MDP by the company InvivoGen Europe, 5, rue Jean Rodier, F-31400, Toulouse, France;

[0072] - Murabutide, sold under the name Murabutide by the company InvivoGen Europe, 5, rue Jean Rodier, F-31400 Toulouse, France.

[0073] - Mifamurtide, sold under the name Mifamurtide by the company CliniSciences, 74 Rue des Suisses, 92000 Nanterre, France.

[0074] - Dimethyl sulfoxide (DMSO), sold under the name Dimethyl sulfoxide by Merck, Darmstadt, Germany.

[0075] - Maltodextrin, sold under the name Maltodextrin by the company Merck, Darmstadt, Germany

[0076] EXAMPLE 1: Demonstration of the effects of administering different muramyl peptides in a chronically malnourished mouse model

[0077] In this example, the effects of several compositions according to the present invention comprising isomolar amounts of MDP or murabutide (MDP Weight Mean Molar Mass (Mw) = 492.5 g / mol, Murabutide Mw = 548.6 g / mol) were tested in a chronically malnourished mouse model, namely:

[0078] - a composition A comprising 25 pg of MDP in 30 pL of placebo composition,

[0079] - a composition B comprising 28 pg of murabutide in 30 pL of composition placebo.

[0080] These compositions were compared to a placebo composition containing only a cryoprotectant (Maltodextrin) resuspended in 30 pL of sterile saline buffer. 1.1 Preparation of compositions

[0081] 15 pg of cryoprotectant were resuspended in 30 pL of sterile saline buffer to generate a dose of placebo composition.

[0082] 25 pg of MDP were resuspended in 30 pL of placebo composition to obtain composition A has a final MDP concentration of 25 pg / 30 pL.

[0083] 28 pg of Murabutide were resuspended in 30 pL of placebo composition for the composition B at a final murabutide concentration of 28 pg / 30 pL (isomolar with 25 pg / 30 pL of MDP). 1.2 Animal Preparation

[0084] Conventional C57Bl / 6j laboratory mice were bred at the Gnotobiology Laboratory for more than 10 generations. The mice were kept in IVC cages (Tecniplast, Italy), exposed to 12:12 hour light-dark cycles, supplied with tap water, and fed ad libitum with the sterile diet VI124-300 (Ssniff Spezialdiâten GmbH) (irradiated ~25 kGy, Bioster, Czech Republic). The mice were mated, and after birth, the litter size was reduced to 6 pups per mother. On day 21, the male mice were weaned onto the experimental low-protein, low-fat 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 a low-protein, low-fat diet were treated five times a week with placebo, composition A containing MDP, or composition B containing Murabutide. At the end of the experiment, food and bedding were removed at 8:00 a.m., and the mice were sacrificed after a five-hour fast by isoflurane inhalation and cervical dislocation. All animals were sacrificed within one hour, between 1:00 and 2:00 p.m. 1.3 Administration of compositions

[0085] The daily treatment dose was 30 pL of placebo, or for MDP, 25 pg in 30 pL of placebo, and for murabutide, 28 pg in 30 pL 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

[0086] 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). The femur and tibia bones were dissected at the time of sacrifice, fixed in 4% paraformaldehyde (PB S) overnight at 4°C, washed with PB S, and stored in 70% ethanol. Bone length was measured using a digital caliper (Festa).

[0087] At the end of the experiment, the blood was collected and left to coagulate at room temperature for 2 hours. The sera were separated by centrifugation (2,000 x g for 5 minutes, 4 °C) and stored at -80 °C until use. The levels IGF-1 levels were measured using the Mouse / Rat IGF-1 Quantikine ELIS A kit (R&D Systems) according to the manufacturer's instructions.

[0088] The data were analyzed using the following statistical test: One way ANOVA with Tukey's multiple comparisons test. 1.5 Results

[0089] The results obtained are presented in the attached Figures 1 to 6.

[0090] Figure 1 shows the change in size (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 2 shows the change in relative growth (% change) as a function of the number of days after birth for each of the tested compositions, using the same symbols as in Figure 1. Figure 3 shows the change in the growth rate (in cm / day) for each of the tested compositions. Figure 4 shows the femur length (in mm) for each of the tested compositions. Figure 5 shows the tibia length (in mm) for each of the tested compositions. Figure 6 shows the IGF-1 level in the blood (in ng / ml) for each of the tested compositions.

[0091] 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. In particular, 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 did not reach statistical significance.Finally, circulating IGF-1 levels 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.

[0092] EXAMPLE 2: Demonstration of the effects of mifamurtide administration in a chronically malnourished mouse model

[0093] 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 pg / day in 30 pL of placebo.

[0094] This composition was compared to a placebo composition containing only maltodextrin (cryoprotectant) resuspended in 28.75 pL of saline buffer and 1.25 pL of DMSO. 1.1 Preparation of compositions

[0095] 15 pg of cryoprotectant were dissolved in 1.25 pL of DMSO and 28.75 pL of sterile saline buffer to generate a dose of placebo composition.

[0096] 10 mg of Mifamurtide were resuspended in 200 pL of DMSO in order to obtain a 50 pg / pL solution of Mifamurtide. 1.25 pL of this solution were resuspended in 28.75 pL of placebo composition to obtain a composition having a final Mifamurtide concentration of 62.5 pg / 30 pL (isomolar with 25 pg / 30 pL of MDP). 1.2 Animal Preparation

[0097] Conventional C57Bl / 6j laboratory mice were bred at the Gnotobiology Laboratory for more than 10 generations. The mice were kept in IVC cages (Tecniplast, Italy), exposed to 12:12 hour light-dark cycles, supplied with tap water, and fed ad libitum with the sterile diet VI124-300 (Ssniff Spezialdiâten GmbH) (irradiated ~25 kGy, Bioster, Czech Republic). The mice were mated, and after birth, the litter size was reduced to 6 pups per mother. On day 21, the male mice were weaned onto the experimental low-protein, low-fat 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 a low-protein, low-fat diet were treated five times a 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 8:00 a.m., and the mice were sacrificed after a five-hour fast by isoflurane inhalation and cervical dislocation. All animals were sacrificed within one hour between 1:00 p.m. and 2:00 p.m. 1.3 Administration of compositions

[0098] Each of the compositions was administered by pipette onto the mouse's tongue. The mouse was held until the composition had been swallowed completely. 1.4 Description of tests performed

[0099] 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 length gain, where body length on day 21 (weaning) was set at 100%. The rate of length growth was calculated as dividing the absolute length gain from day 21 to day 56 by the number of days (i.e., 35). The femur and tibia bones were dissected at the time of sacrifice, fixed in 4% paraformaldehyde-PB S overnight at 4°C, washed with PB S, and preserved in 70% ethanol. Bone length was measured using a digital caliper (Festa).

[0100] 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 (2,000 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 ELIS A kit (R&D Systems) according to the manufacturer's instructions.

[0101] The data were analyzed using the following statistical test: unpaired t-test. 1.5 Results

[0102] The results are presented in the attached Figures 7 to 12. Figure 7 shows the change in height (in cm) as a function of the number of days after birth for each of the compositions tested: composition with mifamurtide: curve with empty triangles, placebo composition: curve with crosses. Figure 8 shows the change in relative growth (% change) as a function of the number of days after birth for each of the compositions tested, using the same symbols as in Figure 7. Figure 9 shows the change in the growth rate (in cm / day) for each of the compositions tested. Figure 10 shows the femur length (in mm) for each of the compositions tested. Figure 11 shows the tibia length (in mm) for each of the compositions tested. Figure 12 shows the IGF-1 level in the blood (in ng / ml) for each of the compositions tested.

[0103] 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-containing composition compared to those treated with the placebo composition.

Claims

Demands

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

2. Composition for use according to claim 1, characterized in that said at least one muramyl peptide is selected from muramyl dipeptide and analogues of muramyl dipeptide, 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. Composition for use according to any one of claims 1 to 2, characterized in that said at least one muramyl peptide is muramyl dipeptide.

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

5. Composition for use according to any one of claims 1 to 4, characterized in that said composition further comprises at least one growth-promoting adjuvant, said adjuvant being selected from strains having intestinal tropism, bacterial constituents, food supplements, nutrients, and mixtures thereof.

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

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

8. Composition for use according to any one of claims 5 to 7, characterized in that said strains are selected from L. plantarum WJL, L. plantarum IGFL1, L. plantarum IGFL2, L. plantarum G821, 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 mixtures thereof.

9. Composition for use according to claim 5, characterized in that the bacterial constituents are selected from inactivated bacteria, bacterial lysates, purified bacterial cell walls and purified bacterial cell wall fragments from bacteria having intestinal tropism, peptidoglycans and teichoic acids.

Images