Patent Application
20230131906
The invention relates to muscle preservation. In particular, the invention relates to bacteria specific to the gut microbiota and to compositions containing them for preventing and/or treating pathological muscle loss or a disease characterized by pathological muscle loss.
Pathological muscle loss is well known to those skilled in the art and refers to muscular atrophy, a decrease in muscle volume (Gordon et al. Therapeutic approaches for muscle wasting disorders. Pharmacol Ther. 2007 March; 113(3):461-87). This muscle loss generally concerns the striated skeletal muscles, which are the muscles under voluntary control. Several pathologies are characterized by muscle loss, in particular amyotrophy, pre-sarcopenia, sarcopenia, rhabdomyolysis, cachexia.
Amyotrophy is a decrease in the volume of skeletal and cardiac striated muscles, that is to say, the number of fibers composing these muscles (long cells) decreases.
Sarcopenia is a syndrome characterized by age-related muscle loss that can lead to deterioration in muscle strength and physical performance.
Rhabdomyolysis is a disease in which cells of the skeletal muscles rapidly degrade and release their contents into the bloodstream. Some of these breakdown products of damaged muscle cells are harmful to the kidneys and can lead to kidney failure in particular. The disease is characterized by several symptoms, such as muscle aches, vomiting and confusion, and can be life threatening in some cases. Some animals may be affected, in particular horses.
Cachexia is a profound weakening of the body characterized by weight loss, fatigue and muscular atrophy in particular, linked to very significant malnutrition.
Muscular atrophy can be caused in particular by the synthesis of norleucine in the body. Other markers of muscle wasting are known as albumin, pre-albumin, C-reactive protein and myoglobin.
Norleucine is a non-proteinogenic amino acid that, when present, takes the place of another amino acid, methionine, during protein synthesis. This replacement of methionine by norleucine is toxic for the cells, and it in particular prevents the renewal of muscle cells, decreases the growth of muscle fibers and reduces muscle development (Bogosian et al. Biosynthesis and Incorporation Into Protein of Norleucine by Escherichia Coli, J Biol Chem 1989 Jan. 5; 264(1):531-9. Norleucine particularly affects organs where protein replacement is important, such as muscle. Thus, the muscles experience low-dose norleucine toxicity. As an example, Jeffery Escobar et al. 2010 demonstrated that the administration of leucine to piglets has a positive effect on protein synthesis in skeletal muscle and that norleucine does not have a positive effect on protein synthesis in muscle. They also show that the chemical structure of leucine gives it these properties.
Norleucine can be synthesized by bacteria in the side product pathway of the branched chain amino acid synthesis pathway from pyruvate and alpha-ketobutyrate.
Currently, the toxicity of norleucine is studied very little and no treatment for pathological muscle loss or a disease characterized by pathological muscle loss is based on a reduction in norleucine toxicity, despite the involvement of this amino acid in ubiquitous metabolic pathways.
Albumin is a soluble protein that is measured to check the amount of protein in the blood. A low albumin concentration is a marker of malnutrition, which is accompanied by significant muscle loss.
C-reactive protein is a protein that shows the patient's inflammatory state, which is observed during muscle loss due to cachexia in particular. A high concentration of C-reactive protein is a marker of cachexia, which is accompanied by significant muscle loss.
Myoglobin is a protein capable of transporting oxygen to the muscles, therefore essential for the work and development of the muscles. A high concentration of plasma myoglobin is a marker of muscle damage or rhabdomyolysis in polytrauma, infectious diseases, muscular dystrophy, myopathy.
Furthermore, existing treatments for pathological muscle loss or diseases characterized by pathological muscle loss are not effective. Indeed, these essentially consist of physical exercises during repeated sessions several times a week, an optimal protein intake to stimulate protein synthesis, the installation of an infusion containing a saline solution, alkalinization of the urine, dialysis, surgery of the injured region, which does not provide a satisfactory result. In addition, the treatments are often heavy, like for the treatment of rhabdomyolysis, which consists of abundant intravenous infusions, and which may require dialysis or hemofiltration in more serious cases.
One article focused on the study of the gut microbiota in patients with primary sarcopenia, and in particular the effects of physical activity on the microbiota. The effects of administering probiotics such as FOS and inulin to improve symptoms of muscle weakness and sarcopenia have also been discussed, although there is no evidence of a distinct gut microbiota in patients with sarcopenia (Andrea Ticinesi et al. Aging gut microbiota at the cross-road between nutrition, physical frailty, and sarcopenia: Is there a gut-muscle axis?. 2017). However, the study does not show any particular interest for certain strains, nor any use of bacteria according to the invention.
Thus, there is a significant need for an effective treatment of pathological muscle loss and diseases characterized by pathological muscle loss that is capable of acting on the various markers at the origin of the disease, and in particular on the synthesis of norleucine, that is easy to administer and that has no side effects.
This is the objective of the present invention, which, to respond to this need, focuses on the use of particular bacteria of the human gut microbiota, namely bacteria of the family Christensenellaceae.
Bacteria of the family Christensenellaceae, including the genus Christensenella, have already been studied and described. This is the case in particular for Christensenella minuta, Christensenella massiliensis and Christensenella timonensis. Christensenella minuta in particular was described for the first time in 2012. In 2014, a study showed that it was the most heritable taxon in a cohort of British twins and that its presence is associated with a low body mass index. This correlation between Christensenella minuta and low body mass index was then observed in a dozen studies published since 2014 in geographically diverse populations. Finally, patent application WO/2018/162738 describes the use of bacteria from the Christensenellaceae family in the treatment of obesity and excess weight
Surprisingly, according to the invention, the bacteria of the Christensenellaceae family, when they are administered to humans or animals, are capable of acting on the markers of pathological muscle loss to prevent or treat this disease or diseases characterized by muscle loss, such as amyotrophy, sarcopenia, pre-sarcopenia, rhabdomyolysis, cachexia.
Therefore, the subject of the invention is a bacterium of the family Christensenellaceae, for its use in the prevention and/or treatment of muscular atrophy in humans or animals.
Advantageously, such a bacterium, when it is administered to a human being or an animal exhibiting pathological muscle loss or a disease characterized by muscle loss, is capable of acting on the molecules produced in excess during this pathological state such as in particular norleucine, and also possibly against albumin and/or pre-albumin and/or protein C and/or myoglobin, allowing an increase in protein synthesis and production of muscle cells to regain lost muscle mass.
In addition, it is a bacterium naturally present in the gut microbiota and whose administration does not cause side effects.
Preferably, the bacteria for use according to the invention are administered within compositions. Thus, the invention also relates to compositions comprising at least one bacterium of the family Christensenellaceae for its use in the prevention and/or treatment of pathological muscle loss and/or a disease characterized by pathological muscle loss in humans or animals.
Other features and advantages will become apparent from the detailed description of the invention that follows.
Within the meaning of the invention, “pathological loss of muscle mass” or “muscle loss” or “pathological muscle loss” means an abnormal, pathological decrease in muscle mass due to a decrease in protein synthesis, in particular of skeletal muscle mass.
Within the meaning of the invention, “disease characterized by pathological muscle loss” or “disease characterized by muscle loss” means a disease whereof one of the symptoms is a loss of muscle mass. These may in particular include amyotrophy, sarcopenia, pre-sarcopenia, rhabdomyolysis or cachexia.
Within the meaning of the invention, “marker” of a disease means a molecule or a substance whose assay makes it possible to follow the evolution of said disease.
The invention relates to the use of at least one bacterium of the Christensenellaceae family in the prevention and/or treatment of pathological muscle loss and/or a disease characterized by pathological muscle loss in humans or animals.
The invention therefore relates to a bacterium of the Christensenellaceae family for its use in the prevention and/or treatment in humans or animals of pathological muscle loss or of a disease characterized by muscle loss chosen in particular from amyotrophy, sarcopenia, pre-sarcopenia, rhabdomyolysis and cachexia, in particular in humans or animals exhibiting pathological muscle loss or a disease characterized by pathological muscle loss with a synthesis of at least norleucine and/or of at least one marker chosen from: albumin and/or pre-albumin and/or C-reactive protein and/or myoglobin.
According to the invention, the bacteria of the Christensenellaceae family, when they are administered to a human or an animal exhibiting a muscle loss or a disease characterized by muscle loss, are capable of acting on the molecules produced in excess during this pathological state, in particular on the production of norleucine and/or of at least one marker chosen from: albumin and/or pre-albumin and/or C-reactive protein and/or myoglobin.
In the case of pathological muscle loss, in particular in diseases characterized by muscle loss that exhibit synthesis of norleucine, and/or synthesis of at least one marker chosen from: albumin and/or pre-albumin and/or C-reactive protein and/or myoglobin, their decrease is a sign of increased protein synthesis and increased muscle cell production.
When pathological muscle loss or disease characterized by pathological muscle loss presents with hyperproduction of norleucine, its decrease is a sign of reduced pathways for the synthesis of branched chain amino acid byproducts, that is to say, this production is less stimulated. From then on, the production of excessive norleucine responsible for its toxicity is slowed down and the system gradually returns to normal. The physiological mechanism of muscle synthesis normalizes and muscle development increases.
The useful bacteria according to the invention are administered to humans or animals in an amount effective for an action on at least one of these markers of pathological muscle loss, i.e., to reduce the production of at least one of these markers in the body.
According to a suitable embodiment, the bacterium or bacteria are administered at a dose of 109 to 1012 colony-forming units (CFU) per day, regardless of the weight of the person or animal. It will preferably be a single dose, i.e., administered once daily, or a dose before each meal (three times a day).
The useful bacterium or bacteria according to the invention are bacteria of the Christensenellaceae family, preferably of the genus Christensenella. It may be, in particular, Christensenella massiliensis, Christensenella timonensis and/or Christensenella minuta. According to a particularly suitable variant, it is Christensenella minuta.
These bacteria can be isolated from human stools for example according to the protocols published by Morotomi et al. 2012 (Morotomi, M., Nagai, F. & Watanabe, Y. Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposal of Christensenellaceae fam. nov. INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY 62, 144-149 (2012)) and NDongo et al. 2016 (Ndongo, S., Dubourg, G., Khelaifia, S., Fournier, P. E. & Raoult, D. Christensenella timonensis, a new bacterial species isolated from the human gut. New Microbes and New Infections 13, 32-33 (2016)). These documents also describe the culture methods of the bacteria that are useful according to the invention.
The useful bacteria according to the invention are preferably administered to the human or animal in a composition.
Thus, the invention also relates to compositions comprising at least one bacterium of the family Christensenellaceae for its use in the prevention and/or treatment of pathological muscle loss and/or a disease characterized by pathological muscle loss in humans or animals, in particular in persons or animals exhibiting norleucine synthesis and/or at least one marker chosen from: albumin and/or pre-albumin and/or C-reactive protein and/or myoglobin.
The bacteria are present in an effective amount in the composition, allowing an effect on the loss of muscle mass of the treated persons or animals.
Preferably, the useful composition according to the invention comprises 106 to 1012 colony-forming units (CFU) of bacteria of the family Christensenellaceae per daily dose of composition to be administered. Preferably, this corresponds to a daily dose of bacteria to be administered, regardless of the weight of the person or the animal. Preferably, this daily dose is administered once per day.
The useful composition according to the invention may be in liquid form. It may in particular comprise bacteria of the Christensenellaceae family and a culture medium for said bacteria that makes it possible to preserve them, such as, for example, Columbia anaerobic medium enriched with sheep blood, or an equivalent medium not containing an animal byproduct.
When the compositions are in liquid form, they are preferably frozen, maintained at −20° C. in a sealed bag.
According to one variant, the useful composition according to the invention may be in solid form. In this case, the bacteria may be present in freeze-dried form, and the compositions may also comprise excipients such as, for example, microcrystalline cellulose, lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum, calcium lactate, magnesium sulphate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin, galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, sorbitol and/or glycerol.
The useful compositions according to the invention may in particular be in the form of powder, microencapsulated powder, gelcap, capsule, tablet, lozenge, granules, emulsion, suspension or suppository. According to a particularly suitable embodiment, they may be in a gastro-resistant form, such as a coated tablet containing microencapsulated bacteria.
When the compositions are in solid form, they are preferably packaged in capsules or in a coating hermetically sealed against light and oxygen, maintained at an ambient temperature of between 15° C. and 40° C. and a humidity level between 3% and 70%.
The bacteria can be used alive, or inactivated, for example by heat, exposure to an appropriate pH, gamma radiation or high pressure.
They can all be alive or all inactivated.
Preferably, at least one part is made up of living bacteria are alive, in particular at least 50% (by number), even more preferably at least 90% (by number).
Thus, according to a suitable embodiment, the bacteria present in the useful composition according to the invention are at least 50% living bacteria (by number), preferably at least 90% living bacteria (by number), and even more preferably all living.
The useful bacteria according to the invention, and in particular the compositions that include them, can be administered orally, topically, through the lungs (inhalation) or rectally.
The useful compositions according to the invention, in addition to the useful bacteria according to the invention, can comprise other compounds, such as:
The invention is now illustrated by examples of useful bacteria according to the invention, methods of culturing these bacteria, examples of compositions containing them and test results demonstrating the effectiveness of the bacteria of the Christensenellaceae family on pathological muscle loss and diseases associated with pathological muscle loss.
The Christensenella minuta bacteria can be cultured according to the operating protocol described below.
1/ Dissolve a dehydrated RCM (“Reinforced Clostridial Medium”) medium in distilled water
2/ Add 0.5 mL/L of resazurin-Na solution (0.1% w/v)
3/ Bring to a boil and cool to room temperature while injecting a gaseous mixture of 80% N2 and 20% CO2
4/ Spread the medium under the same gaseous atmosphere in anoxic Hungate-type tubes or in serum vials, then autoclave
5/ Before use, add 1.0 g of sodium carbonate per liter from a sterile anoxic stock solution prepared with a gaseous mixture of 80% N2 and 20% CO2
6/ Check the pH of the medium after autoclaving and adjust the pH between 7.3 and 7.5, using a sterile anoxic stock solution of sodium bicarbonate (5% w/v) prepared in a gaseous atmosphere at 80% N2 and at 20% CO2.
The Christensenella massiliensis bacteria can be cultured according to the operating protocol described below.
1/ Prepare a carboxymethylcellulose (N2/CO2) medium by following the instructions below provided by DSMZ (Deutsche Sammlung von Mikroorganismen und Zell-kulturen), presented in Table 1.
2/ Dissolve the different constituents listed in the table above, except cysteine, carbohydrates and carbonate.
3/ Boil the medium for 1 min, then let it cool to room temperature under a gaseous atmosphere containing 80% N2 and 20% CO2.
4/ Add 0.5 g/L of L-cysteine-HCl×H2O and pour it under the same gaseous atmosphere into Hungate-type tubes (for strains requiring meat particles, introduce these first into the tube; use 1 part meat particles to 4 or 5 parts liquid).
6/ After autoclaving, add glucose, cellobiose, maltose and starch from sterile anoxic stock solutions prepared with 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under gaseous mixtures at 80% N2 and 20% CO2.
7/ Adjust the pH of the medium to 7, if necessary.
The composition of the meat filtrate is shown in Table 2.
The meat filtrate is prepared as follows.
a/ Use lean beef or horse meat.
b/ Remove fat and connective tissue before chopping.
c/ Mix the meat, water and NaOH, then boil for 15 minutes with stirring.
d/ Allow to cool to room temperature, remove fat from the surface and filter, retaining meat particles and filtrate.
e/ Add water to the filtrate to a final volume of 1000.0 mL.
The bacteria must be grown under anaerobic conditions at 37° C.
The Christensenella timonensis bacteria can be cultivated according to the same procedure as that described in Example 2 for Christensenella massiliensis.
An example of a useful composition according to the invention in liquid form is a composition comprising Christensenella minuta 109 CFU/mL in the RCM anaerobic culture medium described above, modified to contain no product of animal origin and enriched with 5% glycerol.
The composition of Example 4 was obtained from an RCB (“research cell bank”) prepared with Christensenella minuta 1010 CFU/mL and stored frozen at −20° C. in a bag sealed to oxygen.
The frozen composition must be warmed to room temperature until a liquid form is found before use.
An example of a useful composition according to the invention in freeze-dried form can be obtained by lyophilization of the composition of Example 4 in the frozen state.
The objective of this study is to demonstrate the effect of bacteria of the Christensenellaceae family on norleucine in vitro. The study was carried out on norleucine, responsible for the toxicity causing muscle wasting.
Norleucine is a non-proteinogenic amino acid that replaces methionine during protein synthesis. This replacement is toxic: it has been shown to lead to a decrease in the stability of cytochrome P450 (Cirino, P. C., Tang, Y., Takahashi, K., Tirrell, D. A. & Arnold, F. H. Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity. Biotechnol. Bioeng. (2003). doi:10.1002/bit.10718) and a lack of stimulation of muscle synthesis in pigs (Escobar, J. et al. Leucine and alpha-ketoisocaproic acid, but not norleucine, stimulate skeletal muscle protein synthesis in neonatal pigs. J. Nutr. 140, 1418-24 (2010)).
This non-proteinogenic amino acid can be synthesized by different bacterial pathways, including the branched chain amino acid synthesis pathway from pyruvate and alpha-ketobutyrate, where norleucine is a by-product.
The procedure of the study is described below.
Relative peak area=(metabolite peak area)/(internal standard peak area×amount of sample).
The relative amount of norleucine and the relative abundance of Christensenella spp were analyzed and correlated, obtaining a linear regression of R=−0.45 (n=18).
The results are shown in Table 3.
Christensenella spp (×10−2)
There is a negative correlation between bacteria of the Christensenellaceae family and norleucine, which demonstrates a protective effect of the bacteria of the Christensenellaceae family against the toxicity of norleucine in the enzymatic reactions catalyzed by cytochrome P450 and muscle synthesis inhibition.
Thus, the bacteria of the Christensenellaceae family are capable of acting by reducing the production of markers of pathological muscle loss or of a disease characterized by pathological muscle loss, in particular of norleucine. They can therefore be used to prevent and/or treat pathological muscle loss or diseases characterized by muscle loss.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR1906843 | Jun 2019 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/067892 | 6/25/2020 | WO |
