WO2024149826A1 - New pharmaceutical methionine formulation - Google Patents

Abstract

L-Methionine is a known essential and natural amino acid, which plays a critical role in cellular metabolic pathways and tissue functions in humans. Currently, there is not commercial or reported pharmaceutical form for use in human. To overcome difficulties in patients and particularly in pediatric use, the development of an oral and stable pharmaceutical presentation is needed. The inventors design an easy to prepare, stable pharmaceutical formulation of Methionine in the form of a powder ready to be orally used after mixing with a specific amount of aqueous solvent such as water. The methionine formulation was also optimized for palatability and acceptability like smell, taste and texture, as methionine is also well known for its strong unpleasant smelling and taste. Accordingly, the present invention relates to a pharmaceutical composition comprising methionine or a methionine-compound donor or any one of their pharmaceutically acceptable salts and uses thereof as a drug.

Description

NEW PHARMACEUTICAL METHIONINE FORMULATION

FIELD OF THE INVENTION:

The invention relates to the pharmaceutical field. In particular, the invention relates to pharmaceutical compositions comprising methionine or any one of its pharmaceutically acceptable salts.

BACKGROUND OF THE INVENTION:

L-Methionine is a known essential and natural amino acid, which plays a critical role in cellular metabolic pathways and tissue functions in humans.

Metabolic diseases such as those affecting methionine metabolisms could benefit from L-methionine treatment. Indeed, diseases caused by variants of MARS1 gene encoding for the methionine tRNA synthetase (MetRS; EC 6.1.1.10), has been described such as the Charcot- Marie-Tooth disease type 2, and the interstitial lung and liver disease (ILLD) which is associated with the MetRS mutation #615486 in OMIM web database. ILLD is characterized by systemic inflammation, a liver disease including elevated liver enzymes, steatosis, fibrosis and sometimes cirrhosis, and a specific type of primitive pulmonary alveolar proteinosis (PAP). ILLD and PAP caused by mutations in the MARS 1 gene has been described as being severe and usually manifesting in early childhood. It included dyspnea, cough, digital clubbing and severe respiratory distress often leading in death in childhood due to lung fibrosis, reaching 59% with half of them occurring before the age of two years (Hadchouel et al, Eur Respir J, 2022). Indeed, PAP accumulation of lipoproteins in the pulmonary alveoli occurs leading to lung disease and respiratory failure. In ILLD and PAP, MARS1 mutations result in MetRS enzymatic dysfunction leading to a lower binding affinity toward methionine and a reduced enzymatic MetRS activity so affecting protein traduction. A treatment with L- methionine or L-methionine donor compounds could help to increase plasmatic methionine concentration and then favor its cellular availability to ensure and/or restore cellular biochemical functions.

Currently, there is not commercial or reported pharmaceutical form for use in human. To overcome difficulties in patients and particularly in pediatric use, the development of an oral and stable pharmaceutical presentation is needed to ensure that the patients and medical staff have access to safe and accurate dosage forms of the active agent. The European Pediatric Formulations Initiatives (EuPFI) set out criteria for the choice of an oral form in children, including child acceptability, doses suitable for pediatric use, convenience of use with minimal impact on pediatric life-style, and efficacy and safety (excipients, stability, errors in administration).

Acceptability is defined by the EMA as the overall ability of the patient or caregiver to use a medicine. It includes the pharmaceutical characteristics of the medicine such as palatability, size and shape. Palatability is defined as the overall appreciation of a medicine towards its smell, taste, aftertaste and texture. Acceptability of drugs is of primary importance in children due to inherent characteristics of this population including swallowing difficulties and taste sensitivity. This is why a liquid form is particularly interesting in the pediatric population in view of their inability to swallow solid oral forms before the age of 6 years. In terms of palatability, methionine is a sulfur alpha-amino acid with a strong smell and a strong unpleasant taste. This is why a liquid formulation, enabling the addition of flavoring and sweetening agent is particularly interesting and mandatory for palatability acceptance. Indeed Methionine is also well known for its strong unpleasant smelling and taste (Laska, Chem. Senses. 2010). There is currently no validated pediatric pharmaceutical form of Met for the oral route.

The invention aims at provide an easily reproducible and stable powder and liquid pharmaceutical formulations of Methionine to be orally used.

SUMMARY OF THE INVENTION:

The invention relates to a pharmaceutical composition comprising comprising methionine or a methionine-compound donor or any one of their pharmaceutically acceptable salts.

In particular embodiment, the invention refers to a pharmaceutical composition comprising: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; ii) at least one flavoring agent; and iii) at least sweetening agent.

In particular, the invention relates to the pharmaceutical composition described above for use as a medicament.

In another aspect, the invention relates to a container comprising a pharmaceutical composition as defined above.

In another aspect, the invention relates to a method for preparing a liquid pharmaceutical composition of methionine as previously defined. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors design an easy to prepare, stable pharmaceutical formulation of Methionine in the form of a powder ready to be orally used after mixing with a specific amount of aqueous solvent such as water. The methionine formulation was also optimized for palatability and acceptability like smell, taste and texture, as Methionine is also well known for its strong unpleasant smelling and taste.

Pharmaceutical compositions of the invention and uses thereof

Accordingly, in a first aspect, the invention refers to a pharmaceutical composition comprising: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; ii) at least one flavoring agent; and iii) at least sweetening agent.

As used herein, the term “pharmaceutical composition” refers to a formulation of a pharmaceutical active which renders the biological activity of the active ingredient (methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts or enantiomers) therapeutically effective, but which does not include other ingredients which are obviously toxic to a subject to which the formulation are intended to be administered.

As used herein, the term “methionine”, has its general meaning in the art and refer to the compound 2-Amino-4-(methylthio)butanoic acid, which is of formula:

Methionine is an essential amino acid in human which plays a critical role in the metabolism, the angiogenesis, the growth of new blood vessels. According to the invention methionine includes all the isomers forms of methionine, including (S)-2-Amino-4- (methylthio)butanoic acid (also known as L-methionine) and (R)-2-Amino-4- (methylthio)butanoic acid (also known as D-methionine), and their racemates. Other particular forms of Methionine, and pharmaceutically acceptable salts thereof are also considered, including racemic and non-racemic compositions, comprising L-methionine and/or D-methionine.

As used herein, the term “methionine-donor compound”, has its general meaning in the art and refer to a compound that can donate a methyl group to another compound in a reaction catalyzed by methionine methyltransferase.

According to the invention the methionine-donnor compound is S-adenosyl-methionine (also known as “SAM”, “SAMe” or “AdoMet”).

As used herein, the term "pharmaceutically acceptable salts" of a compound thus means salts which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity of the parent compound. In a non-exhaustive manner, such salts may include: hydrates and solvates and pharmaceutically acceptable base addition salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion, an alkaline earth metal ion, or an aluminum ion ; is coordinated with a pharmaceutically acceptable organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

In particular, pharmaceutical acceptable salts of methionine or methionine-donor compound which are considered herein, and throughout the specifically, include those selected from R,S-(5^Z -Adenosyl)-L-methionine -toluenesulfonate salt (CAS Number : 52248-03-0), S-(5'-Adenosyl)-L-methionine Disulfate Salt; S-Adenosyl-L-Methionine iodide salt; S-(5^Z ; S- (5^Z -Adenosyl)-L-methionine-(S-methyl-13C)chloride; S-(5^Z -Adenosyl)-L-methionine chloride dihydrochloride; 2-hydroxy-4-(methylthio)butanoate (HMTBa), 2-hydroxy-4- (methylthio)butanoic acid, calcium salt (HMTBa-Ca, CAS Number : 4857-44-7).

Advantageously, the pharmaceutical composition according to the invention comprises a substantial amount of methionine or its pharmaceutically acceptable salts or methionine-donor compound.

In some embodiments, the pharmaceutical composition comprises L-methionine.

As used herein, the term “flavor” or “flavouring agent(s)” or “flavoring agent(s)” encompasses to the definition of flavor enhancer as laid down in point 14 of Annex 30 I of Regulation (EC) No 1333/2008 on food additives; which includes compounds/sub stances which enhance the existing taste and/or odor of a foodstuff. In particular, the flavor agent is selected in the group consisting of strawberry flavor, cherry flavor or blueberry flavor.

In preferred embodiment, the flavor agent is strawberry flavor.

In preferred embodiment, the flavor agent is not litchi flavor, cotton candy flavor and orange flavor.

Thus, in preferred embodiment, the invention refers to a pharmaceutical composition comprising: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; ii) strawberry flavor; and iii) at least sweetening agent.

As used herein, the term “sweetener(s)” or “sweetening agent(s)” refers to substances used to impart a sweet taste to foods or in table-top sweeteners. Such sub stances/ compounds are generally used as an additive in compositions to increase palatability. Sweeteners can be natural sweeteners or synthetic sweeteners. In a non-exhaustive manner, such sweeteners may include the common saccharide sweeteners, e.g., sucrose, fructose, glucose, and sweetener compositions comprising natural sugars, such as corn syrup (including high fructose corm syrup) or other syrups or sweetener concentrates derived from natural fruit and vegetable sources, semi-synthetic “sugar alcohoF sweeteners such as erythritol, isomalt, lactitol, mannitol, sorbitol, xylitol, maltodextrin, and the like, and artificial sweeteners such as aspartame, saccharin, acesulfame-K, advantame, cyclamate, sucralose, Neohesperidin dihydrochalchone, neotame, saccharin and alitame. Sweeteners also include cyclamic acid, mogroside, tagatose, maltose, galactose, mannose, sucrose, fructose, lactose, neotame and other aspartame derivatives, glucose, D-tryptophan, glycine, maltitol, lactitol, isomalt, hydrogenated glucose syrup (HGS), hydrogenated starch hydrolyzate (HSH), steviol glycosides such as stevioside, rebaudioside A and other sweet Stevia-based glycosides, carrelame and other guanidine-based sweeteners, etc. Alternative sweeteners include sucralose and fructose. Sucralose is a ‘high potency’ or ‘high intensity’ sweetener that is approximately 600 times as sweet as sucrose. Fructose (also known as “fruit sugar”) is a 6-carbon polyhydroxyketone monosaccharide sugar that is often found in plants and in honey. The monosaccharide is found in crystalline form, often referred to as D-fructose. Fructose can also be found as a component of other sweeteners such as high-fructose com syrup (HFCS), which is a mixture of glucose and fructose. Other types of natural sweeteners, include brazzein, curculin, erythritol, glycyrrhizic acid, mabilin/miraculin, mogrol glycosides, Luo Han Guo sweeteners, mogroside V, monatin, monellin, pentadin, and thaumatin. The term “sweeteners” or also includes combinations of sweeteners as disclosed herein.

In some embodiments, the sweetening agent is selected in the group consisting of sorbitol, saccharose, aspartame, xylitol and sucralose.

In some embodiments, the sweetening agent is sucralose.

In some embodiments, the pharmaceutical composition may further comprise at least one thickening agent.

As used herein, the term “Thickening agent” has its general meaning in the art and refers to substance added to composition to increase the viscosity and stability of a liquid composition without substantially changing its other properties.

Examples of thickening agents which are suitable for the pharmaceutical composition include xantham gum, sodium carboxymethyl cellulose (also known as sodium carmellose), methylcellulose, hydroxypropylmethyl cellulose, hydroxyethylmethyl cellulose, kaolin, hydroxypropyl cellulose, carboxymethylcellulose calcium, microcrystalline cellulose, arabic gum, guar gum, carrageenan, alginate, carbomer (Carbopol®) or their mixtures thereof.

In some embodiments, the thickening agents is xanthan gum and/or sodium carboxymethyl cellulose.

In some embodiments, the pharmaceutical composition may further comprise a preservative agent.

As used herein, the term “preservative agent” has its general meaning in the art and refers to substance added to composition to prevent or inhibit the growth of microbes.

Examples of preservative agents which are suitable for the pharmaceutical composition include sodium benzoate, benzoic acid, boric acid, sorbic acid and their salts thereof, benzyl alcohol, benzalkonium chloride, polidronium chloride (also known as Polyquad) parahydroxybenzoic acids and their alkyl esters, methyl and propyl parabens or their mixtures thereof.

In some embodiments, the pharmaceutical composition may further comprise at least one pH adjustment agent. As used herein, the term “pH adjustment agent” or “buffering agent” or « buffer » refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution. A pH adjustment agent helps maintain the pH of a given solution (i.e. the pharmaceutical composition) at or near to a pre-determined value, and the pH adjustment agents are generally chosen to complement the pre-determined value.

Examples of pH adjustment agent which are suitable for the pharmaceutical composition include ascorbic acid, acetic acid, tartaric acid, citric acid, trisodium citrate dehydrate, sodium citrate, potassium citrate, sodium phosphate such as monosodium phosphate and di sodium phosphate, tricalcium phosphate, calcium carbonate, sodium bicarbonate, calcium phosphate, carbonated calcium phosphate, acetic acid/sodium acetate buffer, magnesium hydroxide, hydrochloric acid, sodium hydroxide or their mixtures thereof.

In some embodiments, the pH adjustment agent(s) is selected from the group consisting of sodium citrate, citric acid, monosodium phosphate buffer, disodium phosphate buffer, acetic acid/sodium acetate buffer, sodium hydroxide, hydrochloric acid or their mixtures thereof.

In some embodiments, the pH adjustment agents are sodium citrate and/or citric acid.

Thus, in some embodiments, the pharmaceutical composition comprises or consists of: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; ii) at least one agent flavor; and iii) at least one sweetening agent; iv) at least one thickening agent; and/or at least one pH adjustment agent and/or a preservative agent.

In particular embodiment, the pharmaceutical composition comprises: L-methionine; sucralose; strawberry flavor; xantham gum; sodium carboxymethyl cellulose; sodium citrate; citric acid; and sodium benzoate.

In some embodiments, the pharmaceutical composition is substantially free, or even devoid of viable or revivable germs, potentially infectious, microbial known to those skilled in the art.

As used herein, the term “substantially free”, when used in relation to a given component of a composition (e.g. “a liquid pharmaceutical composition substantially free of germs"), refers to a composition to which essentially none of said component has been added. When a composition is “substantially free" of a given component, said composition suitably comprises no more than 0.001 wt % of said component, suitably no more than 0.0001 wt % of said component, suitably no more than 0.00001 wt %, more suitably no more than 0.000001 wt.

In some embodiments, the pharmaceutical composition is in powder form.

In some embodiments, the pharmaceutical composition further comprises an aqueous solvent.

Thus, in some embodiment, the invention refers to a liquid pharmaceutical composition comprising: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; and, ii) at least one flavor agent; and iii) at least one sweetening agent; and iv) an aqueous solvent.

In some embodiment, the invention refers to a liquid pharmaceutical composition comprising or consisting of: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; and, ii) at least one flavor agent; and iii) at least one sweetening agent; and iv) at least one thickening agent; and/or at least one pH adjustment agent and/or a preservative agent; and v) an aqueous solvent.

In some embodiments, the aqueous solvent is selected from water and aqueous mixtures of a polar organic solvent, for example an alcohol such as methanol, ethanol or the like, or apolar ether such as dioxane, tetrahydrofuran or similar.

According to some embodiments, the aqueous solvent is water (i.e. purified water).

In particular embodiment, the liquid pharmaceutical composition comprises methionine or L-methionine donor compound or its pharmaceutically acceptable salts at a concentration equal or superior to 150 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises methionine, or methionine-donor compound or their pharmaceutically acceptable salts at a concentration equal or superior to 100, 110, 120, 130, 140, 150, 160, 170, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 220, 230, 240 and 250 mg/mL.

In some embodiments, the liquid pharmaceutical composition comprises methionine, or methionine-donor compound or their pharmaceutically acceptable salts at a concentration of 200 mg/mL.

In particular embodiment, the methionine is L-methionine.

In some embodiments, the liquid pharmaceutical composition comprises sucralose at a concentration comprised between 0.5mg/mL and 2mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sucralose at a concentration of 1 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sucralose at a concentration of at least of 1 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sucralose at a concentration comprised between Img/mL and 2mg/mL.

In some embodiments, the liquid pharmaceutical composition comprises strawberry flavor at a concentration comprised between 10 mg/mL and 70 mg/mL, and more particularly between 10 mg/mL and 60 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises strawberry flavor at a concentration of 50 mg/mL.

In some embodiments, the liquid pharmaceutical composition comprises xanthan gum at a concentration comprised between 1 mg/mL and 3 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises xanthan gum at a concentration of 1, 2 or 3mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sodium carboxymethyl cellulose at a concentration comprised between 8mg/mL and 12mg/mL. In some embodiments, the liquid pharmaceutical composition sodium carboxymethyl cellulose in amount of 10 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises xanthan gum at a concentration comprised between 2mg/mL and 4mg/mL and sodium carboxymethyl cellulose at a concentration comprised between 8mg/mL and 12mg/mL. In some embodiments, the liquid pharmaceutical composition comprises xanthan gum at a concentration comprised between Img/mL and 3mg/mL and sodium carboxymethyl cellulose at a concentration comprised between 8mg/mL and 12mg/mL. In some embodiments, the liquid pharmaceutical composition comprises xanthan gum at a concentration of 3 mg/mL and sodium carboxymethyl cellulose in amount of 10 mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sodium benzoate at a concentration comprised between Img/mL and 4mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sodium benzoate at a concentration of 1, 2 or 3 mg/mL.

In some embodiments, the liquid pharmaceutical composition comprises sodium citrate at a concentration comprised between 5mg/mL and 25mg/mL and/or citric acid at a concentration comprised between 3mg/mL and 15mg/mL. In some embodiments, the liquid pharmaceutical composition comprises sodium citrate at a concentration of 17,3 mg/mL and/or citric acid at a concentration of 8,6 mg/mL.

In some embodiment, the liquid pharmaceutical composition comprises sodium citrate and/or citric acid at a concentration to obtain a PH of said ranging from 4.5 to 5 at 6°C, 23°C or 40°C. In some embodiment, the liquid pharmaceutical composition comprises sodium citrate and/or citric acid at a concentration to obtain a PH of 4.5 at 6°C, 23°C or 40°C.

In some embodiments, the liquid pharmaceutical composition comprises: i) L-methionine at a concentration of 200 mg/mL; ii) strawberry flavor at a concentration of 50 mg/mL; iii) sucralose at a concentration of 1 mg/mL; iv) xanthan gum at a concentration of 3 mg/mL and/or sodium carb oxy methyl cellulose in amount of 10 mg/mL v) sodium benzoate at a concentration of 3 mg/mL; vi) sodium citrate at a concentration of 17,3 mg/mL and/or citric acid cellulose at a concentration of 8,6 mg/mL; and vii) an aqueous solvent.

According to some embodiments, the pH of the liquid pharmaceutical composition ranges from 4 to 6 (including 4, 5, and 6) at 6°C, 23°C or 40°C. According to exemplary embodiments, the pH of the liquid pharmaceutical composition ranges from 4.5 to 5 at 6°C, 23°C or 40°C; for instance of about 4.87 at 6°C, 23°C or 40°C.

As used herein, the term “liquid composition” further includes liquid compositions stored and/or packaged in any recipient or compartment, sealed or not, which is suitable for pharmaceutical compositions, which may thus include any liquid composition stored in vials, bottles, intravenous (IV) bags, ampoules, cartridges and prefilled syringes. The liquid compositions which are particularly considered in the context of the present invention are those which are compatible with oral administration, and in particularly with pediatric or geriatric oral administration.

A solution compatible with pediatric oral administration have to comply with wide range of guideline, such as defined in European Pediatric Formulations Initiatives (EuPFI) guideline, EMA guideline or ASHP Guidelines (palatability, sterility, solubility, stability, viscosity,...). For example, acceptability of drugs is of primary importance in children due to inherent characteristics of this population including swallowing difficulties and taste sensitivity

As used herein, the term “palatability” refers to the organoleptic properties which include smell, taste, dose, volume and texture.

A treatment with L- methionine or L-methionine donor compounds could help to increase plasmatic methionine concentration and then favor its cellular availability to ensure and/or restore cellular biochemical functions.

Accordingly, in another aspect, the present invention refers to the pharmaceutical composition of the present invention for use as a medicament.

In particular embodiment, the present invention refers to the liquid pharmaceutical composition of the present invention for use as a medicament.

Thus, in particular embodiment, the present invention refers to a liquid pharmaceutical composition for use as a medicament, wherein the liquid pharmaceutical composition comprises or consists of: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; and, ii) at least one flavoring agent; and iii) at least one sweetening agent; and iv) at least one thickening agent; and/or at least one pH adjustment agent and/or a preservative agent; and v) an aqueous solvent.

In some embodiments, the methionine is L-methionine, the at least one flavoring agent is strawberry flavor, the at least one sweetening agent is sucralose, the at least one thickening agent are xanthan gum and/or sodium carboxymethyl cellulose, the at least one pH adjustment agent are sodium citrate and/or citric acid and the preservative agent is sodium benzoate. Accumulating preclinical evidence indicates that alterations in the methionine cycle play a pathogenetic role in liver disease. Recent studies shows that methionine supplementation can help reduce liver damage (Li et al, Target Ther. 2020).

Thus, in particular, the pharmaceutical composition of the invention is suitable for use for the treatment of liver disease, in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with metabolic disease. According to one embodiment, the liquid pharmaceutical composition is particularly suitable as a medicament for pediatric and geriatric populations. The pediatric population is defined herein as that group of the population between birth and 18 years of age. In some embodiments, the subject is a child that is less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 year(s) old. The geriatric population is defined herein as that group of the population aged 65 and over. In some embodiments, the subject is an elderly that is more than 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 year(s) old.

As used herein, the term “liver disease” refers to any disturbance of liver function that causes illness. The liver is responsible for many critical functions from protein production and blood clotting to cholesterol, glucose (sugar), and iron metabolism. When it becomes diseased or injured, the loss of those functions can cause significant damage to the body. Liver disease is also referred to as hepatic disease. According to the invention, liver disease includes nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fibrotic NASH, cirrhosis, such as, alcoholic liver cirrhosis and primary biliary cirrhosis (PBC), liver fibrosis, chronic hepatitis, i.e. chronic autoimmune hepatitis, chronic alcoholic hepatitis, viral hepatitis A, B, C, D, E and G, toxic metabolic liver damage, and fatty liver. The liver disease may result, for example, from infectious or autoimmune processes, from mechanical or chemical injury to the liver, or from cancer, all of which are included within the definition of “liver disease.” Chemical injury to the liver can be caused by a variety of toxins, such as alcohol, carbon tetrachloride, trichloroethylene, iron overdose, drug overdose, drug side-effects etc.

Furthermore, metabolic diseases affecting methionine metabolisms could benefit from methionine treatment which could help to increase plasmatic methionine concentration and then favor its cellular availability to ensure and/or restore cellular biochemical functions. Thus, in particular, the pharmaceutical composition of the invention is suitable for use for the treatment of methionine metabolism disorders, in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

As used herein, the term “methionine metabolism disorders” refers to disorder characterized by an abnormal deficit or use of methionine. According to the invention, methionine metabolism disorders includes pancreatitis, liver disease, growth failure, systemic inflammation, interstitial lung and liver disease (ILLD), pulmonary alveolar proteinosis (PAP), homocystinuria, atherosclerosis, methylenetetrahydrofolate reductase deficiency, homocystinuria-megaloblastic anemia, homocystinuria-megaloblastic anemia, sulfite oxidase deficiency and molybdenum cofactor deficiency.

In some embodiment, the methionine metabolism disorders is interstitial lung and liver disease (ILLD) related to MARS gene and/or protein mutations

In some embodiments, the ILLD is primary ILLD.

In some embodiment, the methionine metabolism disorders is pulmonary alveolar proteinosis (PAP) related to MARS gene and/or protein mutations.

As used herein, the term “interstitial lung and liver disease” (ILLD) refers to a rare lung and liver disorder characterized by an abnormal accumulation of surfactant-derived lipoprotein compounds within the alveoli of the lung leading to lung disease and respiratory failure, also known as “pulmonary alveolar proteinosis (PAP) ”, and liver failures. In the context of the invention, the ILDD or PAP is related to MARS gene and/or protein mutation.

As used herein, the term “MARS” refers to Methionyl-tRNA synthetase, is an enzyme cytoplasmic that in humans is encoded by the MARS gene. These enzymes play a critical role in protein biosynthesis by charging tRNAs with their cognate amino acids. The encoded protein is a component of the multi-tRNA synthetase complex and catalyzes the ligation of methionine to tRNA molecules. The naturally occurring human MARS has a nucleotide sequence as shown in Genbank Accession number NM 004990 and the naturally occurring human MARS protein has an amino acid sequence as shown in Genbank Accession number NP 004981. The naturally occurring murine MARS has a nucleotide sequence as shown in Genbank Accession numbers NM_001003913 and NM_001171582; and the naturally occurring murine MARS protein has an amino acid sequence as shown in Genbank Accession numbers NP OO 1003913 and NP_001165053. As used herein, the term “MARS gene and/or protein mutations” refers to any mutations in MARS gene and/or protein. As used herein, the term "mutation" has its general meaning in the art and refers to any detectable change in genetic material, e.g. DNA, RNA, cDNA, or in an amino acid sequence encoded by such a genetic material. This includes gene mutations, in which the structure (e.g. DNA sequence) of a gene is altered any gene as well as protein mutations, in which the amino- acid structure of the protein is altered. Generally, a mutation is identified in a subject by comparing the sequence of a nucleic acid or of a polypeptide expressed by said subject with the corresponding nucleic acid or polypeptide expressed in a control population. Typically, mutations are accessible in the Single Nucleotide Polymorphism Database (dbSNP), which is a free public archive for genetic variation within and across different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the National Human Genome Research Institute (NHGRI).

Typically, such mutation refers to at least one nucleotide and/or amino acid substitution(s), deletion(s) and/or insertion(s) in the MARS gene and/or protein sequence. In the context of the invention, the MARS gene and/or protein mutation refers to a double mutation Ala393Thr/Ser567Leu in MARS. Other MARS mutations were reported in the literature (Abuduxikuer PMID: 30271085, Sun PMID: 28148924, Van Meel PMID: 24103465, Rips PMID: 29655802, Alzaid PMID: 30723866) and are also located in the catalytic domain. Therefore, those mutations and all other mutations that may be identified in the future and that are located in the catalytic domain, mays benefit from this therapeutic intervention.

In some embodiments, the ILLD is primary ILLD.

Thus, the invention also refers to a method for treating ILLD related to MARS gene and/or protein mutations and/or PAP related to MARS gene and/or protein mutations in a subject in need thereof comprising administering a therapeutically effective amount of a liquid pharmaceutical composition comprising or consisting of: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; and, ii) at least one flavoring agent; and iii) at least one sweetening agent; and iv) at least one thickening agent; and/or at least one pH adjustment agent and/or a preservative agent; and v) an aqueous solvent.

In particular embodiment, the flavoring agent is strawberry flavor. In some embodiments, the subject is a child that is less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 year(s) old.

Cartilage tissue development can be promoted by the sulphur containing amino acid 1- methionine. Some study demonstrate the anti-arthritics effects of methionine (Wang et al, 3 Biotech. 2018).

Thus, in particular, the pharmaceutical composition of the invention is suitable for use for the treatment of arthritis, in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention.

As used herein, the term “arthritis” has its general meaning in the art and refers to disorders affecting joints. The main symptoms are joint pain and stiffness but include also redness, warmth and swelling. The most common types of arthritis are osteoarthritis and rheumatoid arthritis. According to the invention arthritis includes osteoarthritis, rheumatoid arthritis, gout, septic arthritis, ankylosing spondylitis juvenile idiopathic arthritis, still's disease, psoriatic arthritis.

As used herein, the term "treatment" or "treating" refer to prophylactic, palliative or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

As used herein, a “therapeutically effective amount” is intended for a minimal amount of active agent (i.e. methionine, methionine-donor compound and their pharmaceutically acceptable salts in pharmaceutical composition) which is necessary to impart therapeutic benefit to a patient. For example, a “therapeutically effective amount of the active agent” to a patient is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. methionine, methionine-donor compound and their pharmaceutically acceptable salts) into the subject, such as by ocular, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

In preferred embodiment, the pharmaceutical composition of the present invention is administered by an oral administration.

Containers of the invention

According to another aspect, the invention relates to a container comprising or containing the pharmaceutical composition of the invention as defined above.

Hence, accordingly, the invention further relates to a container comprising or containing the pharmaceutical composition, wherein the pharmaceutical composition comprises: i) methionine, or a methionine-compound donor or any one of their pharmaceutically acceptable salts; and, ii) at least one flavoring agent; and iii) at least one sweetening agent; and iv) at least one thickening agents.

In particular embodiment, the flavoring agent is strawberry flavor.

In particular embodiment, the pharmaceutical composition in the container further comprises: v) at least one pH adjustment agents; and vi) a preservative agent. In particular embodiment, the container comprises the pharmaceutical composition of the invention in powder form.

In particular embodiment, the container comprises the pharmaceutical composition of the invention in powder form allowing to reconstitute with an aqueous solvent the liquid pharmaceutical composition of the invention with desired concentrations as defined above (i.e L-methionine at a concentration of 200mg/mL).

In particular embodiment, the pharmaceutical composition in the container further comprises : vii) an aqueous solvent.

In other word, in particular embodiment, the container comprises the liquid pharmaceutical composition of the invention as defined above.

As used herein, the term “container” refers to any primary or secondary packaging material which is compatible with the storage of a liquid pharmaceutical composition. In particular, the container is compatible with an oral administration. In a non-exhaustive manner, such container may include single-dose containers, multi-dose containers, well-closed containers, airtight containers, light-resistant containers. Such containers may be formed, completely or in-part, in glass, plastics, rubbers, paper/card boards and metals. For example, glass containers may include or consist of Type-I glass, Type-II glass, Type-III glass or any other non parental usage glass. Plastic containers may include or consist of Urea formaldehyde (UF), Phenol formaldehyde, Melamine formaldehyde (MF), Epoxy resins (epoxides), Polyurethanes (PURs), Polyethylene, Polyvinylchloride, Polyethylene terepthalate (PET), Polyvinylidene chloride (PVdC), Polycarbonate Acrylonitrile butadiene styrene (ABS). Such containers may comprise or consist of vials, bottles, ampoules, cartridges and prefilled syringes.

Process of preparation of the pharmaceutical composition

According to another aspect, the invention relates to a method for preparing the pharmaceutical composition of the invention, as previously defined. Hence, the invention relates to a method for preparing the pharmaceutical composition of the invention comprising the steps of: a) providing methionine or methionine-donor compound or any one of their pharmaceutically acceptable salts in powder form b) bringing into contact said methionine or methionine-donor compound or any one of their pharmaceutically acceptable salts thereof in powder form, with strawberry flavor, at least one sweetening agent, and at least one thickening agents; thereby preparing the pharmaceutical composition.

In particular embodiment, at least one pH adjustment agents and/or a preservative agent are further bringing into contact to said methionine or methionine-donor compound or any one of their pharmaceutically acceptable salts thereof in step b).

In particular embodiment, the strawberry flavor, at least one sweetening agent, at least one thickening agents, and at least one pH adjustment agents and/or a preservative agent are in powder form.

In particular embodiment, an aqueous solvent are further bringing into contact to said methionine or methionine-donor compound or any one of their pharmaceutically acceptable salts thereof in step b) thereby preparing a liquid pharmaceutical composition

In particular, the method may further include a step c) of mixing the liquid pharmaceutical composition obtained at step b).

Hence, the invention relates to a method for preparing the liquid pharmaceutical composition of the invention, comprising the steps of: a) providing methionine or methionine-donor compound or any one of their pharmaceutically acceptable salts in powder form; and b) bringing into contact said methionine or methionine-donor compound or any one of their pharmaceutically acceptable salts thereof in powder form, with strawberry flavor, at least one sweetening agent, at least one thickening agents; at least one pH adjustment agents; a preservative agent; and an aqueous solvent thereby preparing the liquid pharmaceutical composition, and c) optionally mixing the composition obtained at step b), preferably in a manner suitable to obtain a solution.

Preferably, the above-mentioned methods further include introducing the obtained pharmaceutical composition at step b) or c) into a container; preferably a container suitable for oral administration.

The invention will be further illustrated by the following figures, tables and examples. However, these examples and Tables should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 21 Stability over time of methionine formulated powder at 4 ± 2°C (circle), 23 ± 2 °C (square), 40 ± 2°C (triangle) in glass pharmaceutical bottles and over time. (Remaining methionine concentration, calculated by the ratio of the concentration on the tested day to the concentration at day 0; values are mean ± SD; n = 3)

Figure 2: Stability of Methionine 200 mg/mL formulated suspension at 4 ±2°C (circle), 23 ± 2 °C (square), 40 ± 2°C (triangle) in glass pharmaceutical bottles and over time (Remaining methionine concentration, calculated by the ratio of the concentration on the tested day to the concentration at day 0; values are mean ± SD; n = 3).

Figure 3: pH value of the reconstituted Methionine 200 mg/mL formulated suspension stored at 4 ± 2°C (circle), 23 ± 2 °C (square), 40 ± 2°C (triangle) in glass pharmaceutical bottles and over time (for more visibility the points have been shifted for days 30 and days 45; standard deviation bars are not visible on the figure due to low data scatter; n =3)

EXAMPLE:

Material & Methods

Drugs and Chemicals

Pharmaceutical grade of L-Methionine pure powder was obtained from Fagron (Thiais, France), sodium benzoate and xanthan gum pharmaceutical powder were obtained from Cooper (Melun, France). Pharmaceutical sodium carmellose, sodium citrate and citric acid were supplied by Caelo (Hilden, Germany). Sucralose was provided by Inresa pharmaceutical (Bartenheim, France). Strawberry, orange, litchi, and candy floss flavors were purchased from Scrapcooking (Tours, France). Acetonitrile and ortho-phosphoric acid were of analytical grade, and purchased from VWR chemicals (Fontenay-sous-Bois, France). Distilled sterile water was purchased from Aguettant (Lyon, France).

Validation of stability-indicating liquid chromatography assay

Equipment and Analytical Conditions

Methionine assay was performed by a high-performance liquid chromatography (HPLC) system (Dionex Ultimate 3000, Thermo Scientific, Villebon-sur-Yvette, France) with HPG-3200SD quaternary pump and WPS-3000TSL autosampler, coupled to a MWD-3000 diode array detector (DAD). HPLC system data acquisition (e.g., peak time, area) was carried out using the Chromeleon® software (v6.80 SP2, Thermo Scientific). A Polaris® C18 column (250 x 4.6 mm; particle size, 5 pm; Agilent, USA) maintained at 30°C using a column heater was used. The mobile phase consisted of acetonitrile and water (0.5/99.5 v/v) adjusted to pH 2.1 with orthophosporic acid. The column temperature, the mobile phase, the flow rate, the injection volume and the detection for detection and quantification were set at 30 ± 2°C, 1.0 mL/min, 30 pL and 205 nm, respectively.

Validation of the HPLC Assay Method

The method was validated according to the international council for harmonisation (ICH) Q2R1 guidelines by assessing the linearity, accuracy, specificity and precision (6). Two stock solutions of Methionine were prepared daily over three days by diluting Methionine in dis-tilled water to a final concentration of 1 mg/mL, to independently prepare five calibration standard (between 160 and 240 pg/mL) and three quality controls (170; 190; and 230 pg/mL, recorded in triplicate). Each day, the slope, intercept and correlation coefficient (r2) were calculated to study linearity. Accuracy was determined using quality controls, and expressed as the percentage of recovery determined by the following equation: experimental concentration xlOO , with ± 5% as acceptance criteria.

Theoritical concentration

Precision was studied using quality controls recorded in triplicate on three independent days. The intra-days analysis was performed by calculating the relative standard deviation (RSD) on each quality control recorded in triplicate the same day (n=3). The inter-days analysis was performed by calculating the RSD on each quality control recorded over three days (n=9). Additionally, repeatability was estimated by recording 10 times a 200pg/mL Methionine solution and calculating the RSD.

The limit of detection (LOD) and limit of quantification (LOQ) for Methionine were evaluated based on response standard deviation and calibration curve slope based on the following equations: LOD = 3.3 x

s(slope) s(slope)

Matrix effect was assessed by comparing calibration curves and intercept of Methionine in water and Methionine in the presence of all excipients present in the formulation.

Intrinsic Stability of Methionine

The selectivity of the method was ensured so that no chromatographic peak of an excipient or degradation product coincided with that for Met. Chromatograms of Met in the presence of all formula excipients were visually inspected to detect changes in shape of the Met peak as well as by the analysis of the Met peak UV spectra recorded by DAD (200-400 nm).TFour stress conditions were applied on Methionine: acidic (1 M HC1), alkaline (1 M NaOH) and oxidative (0.03% H2O2) stress conditions for 72 h (H72) at 70°C, and photolytic stress for 32h (H32) with samples taken and analyzed at various time points. To perform the forced degradation study, a stock solution of Methionine with excipients as described in paragraph 2.3 was prepared in water (200 mg/mL of Met). This solution was then diluted in water to a final concentration of 400 pg/mL of Met, and finally diluted in equal parts either with aqueous IM HC1, IM NaOH and 0.03% H2O2 and ultrapure water. Photolytic stress studies were performed with a QSun-Xe-1 device (Q-Lab Corporation, Saarbriicken, Germany) equipped with a xenon lamp and a Day-light-Q filter (Q-Lab), which simulates natural sunlight (wavelength from 300 to 800 nm) operating in window mode and complying with ICH Q1B recommendations. The light beam obtained simulate CIE Standard Illuminant D65 (Q-Lab), and with irradiance set at a constant intensity constant (1.5 W/m²; 420 nm). The temperature was controlled and set at 23 ± 2°C.

Formulation powder

2.3. Preparation of Methionine Powder Formulation for Oral Suspension

A powder mixture ready to be reconstituted with water has been formulated to obtain a suspension of Met at 200mg/mL suitable for an oral administration in children.. It was composed of a mix of pharmaceutical grade Met, and pharmaceutical grade anhydrous excipients such as preservative (e.g. sodium benzoate), viscosifier and thickener (e.g. sodium carmellose, xanthan gum), pH buffer (sodium citrate and citric acid), sweetener (e.g. sucralose) and aroma (e.g. fruit flavor). To make the taste and odor of the preparation more attractive, several flavors were tested: orange, cotton candy, litchi and strawberry. Five adults rated each of the tested flavors on a subjective scale of 1 to 5, 5 being the highest score. Different amounts of sucralose (0.05 - 0.2% m/v) were tested by the similar taste scoring methodology.

Visual inspection of Met suspensions containing various amounts of xanthan gum (0.3- 0.5% w/v) was performed to assess particle distribution and agglomeration. The uniformity of Met content was determined after 10 seconds of manual bottle shaking; samples were taken (in triplicate) at three sampling levels: bottom, middle and top of the suspension. These samples were analyzed by HPLC assay for Met concentration (n = 3).

The final optimized Methionine powder oral formulation (200 mg/mL) was prepared by exactly weighed in-gredients (see Table 1). Powders were mixed and transferred into a 200 mL transparent type 3 glass bottle from SGD Pharma (Sucy en Brie, France). When needed, water should be added in two equal parts (e.g. twice 50 mL) and shaken vigorously for 1 min after the first half water addition. At the end, the mixture is shaken vigorously for one min until an homogeneous suspension is obtained.

Table 1 : Pharmaceutical Powder composition (for 100 mL of oral suspension)

Physicochemical stability experiments

Stability studies were performed under three storage conditions: 4 ± 2°C, 23 ± 2°C, and 40 ± 2°C. At day 0, three independent preparations were made and stored in each storage conditions. The Methionine concentration was calculated every day using a calibration curve made on the same day and validated by three quality controls prepared independently. The remaining Methionine concentration was calculated by the ratio of the concentration on the tested day to the concentration at day 0 (DO). Preparations were considered stable if organoleptic aspects were unchanged and Methionine concentration remained above 90% of the initial concentration.

Unreconstituted Methionine Formulation: Stability Experiments Over Time

A powder samples of each preparation were analyzed on days 0, 3, 7, 14, 21, 30, 45, 60, 75 and 90. After shaking, 200 mg of each sample were diluted into a 1,000 mL volumetric flask with distilled water and mixed for 10 minutes before being analyzed by HPLC.

Water Reconstituted Methionine Formulation: Stability Experiments Over Time

Following water addition and shaking (DO), aliquots of suspension from each preparation were obtained on days 0, 3, 7, 14, 21, 30, and 45. After shaking, a sample was collected and diluted to 1/1, 000th in water before being analyzed by HPLC. pH and Organoleptic Aspect

Potential hydrogen (pH) was measured with digital SevenEasy S20 pH meter (Mettler- Toledo, Viroflay, France) equipped with a calibrated InLab® Expert Pro-ISM pH sensor (Mettler-Toledo). A visual inspection was carried out by the examination of each sample under daylight and in front of a white background, under which aspect, color and odor of each sample were noted and after shaking for 30 s for Methionine suspension study.

Microbiological Stability Assessment

A microbiological stability study was conducted (Alpabio, Pargay-Meslay, France). To evaluate the efficacy of antimicrobial preservation of the reconstituted Methionine formulation in bottles, the test involved the artificial contamination of the sample formulation, using a graded inoculum of prescribed microorganisms according to the European Pharmacopeia v. 8.0 for non-sterile product. The inoculated product was kept at room temperature and away from light for 28 days. The number of microorganisms was monitored by sampling at defined time intervals by counts of the microorganisms in the sample taken. The product preservation properties were suitable if, under the test conditions, a significant reduction of microorganisms in the inoculated product occurred over the defined time intervals. Six collections type strains were included corresponding to three bacteria (i.e. Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 8739) and three fungi (i.e. Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Zygosaccharomyces rouxii IP 2021.92).

A neutralizing solution was used to ensure that any preservative effect of the formulation was neutralized at the moment of microbial enumeration, allowing the existent microorganisms to be recovered and counted in agar medium. For this purpose, a mixture of compounds was prepared using 30 g tween 80, 3 g soy lecithin, 1 g histidine, 1 g peptone for casein, 4.3g sodium chloride, 3.6 g potassium phosphate monobasic, 7.2 g phosphate disodium in 1,000 mL purified water. Tryptone agar medium and Sabouraud dextrose agar were used as culture media.

For each reference strain, one mL of a suspension containing between 1.102 and 1.103 colony -forming units (CFU)/mL was added to nine mL of the neutralizing agent, for the test in the absence of the product. In to two nine cm-diameter Petri dishes for each medium, one mL of the previous suspension was introduced separately. The same procedure was applied to the selected Methionine formulation. A count of the number of CFU per dish was performed after a maximum incubating time of five days at 30-38°C for soybean-casein digest agar, and a maximum incubating time of three days at 20-25°C for the Sabouraud dextrose agar. The method was considered validated when the number of CFUs counted in one mL of inoculated sample was at least 50% of that obtained in the control (neutralizing solution inoculated with each microorganism). At each time point, the log reduction in the number of viable microorganisms against the value obtained for the inoculum was calculated. This log reduction was then compared with the recommended values of the European Pharmacopeia.

In-use acceptability survey

After one year of Met suspension use, a questionnaire was given to the parents of 7 children (data not shown) To evaluate the appreciation of taste, the TASTY children facial expression scale (data not shown) was used by the parents [10],

Data analysis

Data analysis was performed using Excel (Microsoft, USA) and Prism (GraphPad Software, version 7.04, San Diego, CA, USA, 2017). Descriptive statistics for continuous variables were expressed as mean ± standard deviation (SD) unless otherwise specified.

Results

Validation of Stability-Indicating Liquid Chromatography Assay

Methionine calibration curve was linear between 160 pg/mL and 240 pg/mL (Data not shown) (y=0.294 (±0.01)x - 1.841 (±1.27); r2=0.994). The Methionine retention time was ~6.5 min. No matrix effect was demonstrated. As shown in Table 3, Methionine assay precision was < 1.55% and accuracy was no less than 98% for all Methionine concentration tested, and the repeatability was 0.6% (n=10). In addition, the accuracy profile was within the acceptability limits of 10% (Data not shown). The LOD and LOQ were 14 pg/mL and 42 pg/mL, respectively, which was acceptable for the concentration range measured. The forced degradation study showed a very high sensitivity of Methionine to oxidation, and to acidic conditions. However, no degradation products coeluted with Meth and no change in UV spectra was observed, showing the good specificity of the method and making it possible to qualify it as a stability indicator (Data not shown).

Met Formulation Optimisation

The quantities of some excipients were optimized to obtain a better acceptability in terms of odor, taste and uniformity of the content. Regarding odor and taste, a range of fruit flavors were tested from 1% to 6% (w/v). Of the fruit flavors tested, the 5% strawberry flavor was found to be satisfactory in masking the Met odor (data not shown). To improve taste, sucralose amount was tested from 0.05 to 0.2% (m/v). Below 0.1% of sucralose content, the preparation was not sufficiently sweet, while above 0.2% it was judged overly sweet (data not shown). The amount of xanthan gum was studied from 0.1% to 0.5%. The preparation was judged to be overly thick and improper to withdraw it with oral dispenser with an amount of xanthan gum higher than 0.3% (m/v). A visual inspection of the water re-constituted Met suspension containing xanthan gum at 0.3% showed a homogeneous particle distribution without powder agglomerate/cake over the 45 days of the study. After 10 seconds of gentle shaking three levels of sampling were performed and ana-lyzed for Met concentration. The mean relative error of Met expected concentration was 94.7 ± 5.2%, 93.6 ± 3.4%, and 93.3% ± 2.0%, for bottom, middle and top sample, respectively.

Regarding the other excipients, sodium benzoate was added at a regular concentration of 0,3% (m/v) for its antimicrobial properties at pH below 5, and citrate was added to the formulation for a target pH of 4.5 [11],

Stability of the bulk powders of Methionine in pharmaceutical bulk powders

A stability study was conducted for the unreconstituted “bulk” powder formulation of methionine stored at 4 ± 2°C, 23 ± 2°C and 40 ± 2°C after 3, 7, 14, 21, 30, 45, 54, and 90 days of powder storage in white glass pharmaceutical bottles (SGD Pharma; Sucy en Brie; France). The organoleptic aspects (i.e. color, odor) were assessed at each time and storage conditions. The physico-chemical stability of methionine in the bulk powder was immediately assessed after mixing and shaking with water and analyzed by a specific and validated stability indicating HPLC-UV method at each day of the experiments (in triplicate). Stability was defined as retention of at least 90% of the initial drug concentration. The organoleptic aspects (i.e. color, odor) were also assessed.

The unreconstituted formulation (bulk powders) of Methionine, stored between 2°C- 6°C, 21-25°C and 38-42°C retained its concentration around 100% of the baseline value at least 92 days after compounding (Table 2). No color or odor change occurred in any samples when the pharmaceutical powder bulk was stored between 2°C-6°C and 21-25°C.

Table 2. Methionine stability in the unreconstituted powder formulation

The unreconstituted Methionine powder formulation stored at 23 ± 2°C, 40 ± 2°C or 4 ± 2°C retained its concentration around 100 ± 10% of the baseline value at 92 days after compounding (Figure 1). No color or odor changes occurred in samples stored at 4 ± 2°C, remaining white and with a strawberry smell. In two out of three samples stored at 23 ± 2°C, a slightly change in color occurred at 92 days, the powders having taken a very light beige color. In samples stored at 40 ± 2°C, a change in color and odor occurred at day 14 in all three preparations, with a strong brown color and a heavy unpleasant smell.

Water reconstituted Methionine formulation : stability experiments

The stability of the water diluted methionine bulk powder formulation was conducted after day 0 (defined as the water mixing day of the bulk powders), 3, 7, 14, 21, 30 and 45 days after water solubilization and stored at : 4 ± 2°C, 23 ± 2°C and 40 ± 2°C in glass pharmaceutical bottles (SGD Pharma, Sucy en Brie, France) in triplicate at each temperature storage conditions. Stability was defined as retention of at least 90% of the initial drug concentration (Table 3). All experiments were performed in triplicate. pH was measured (Table 4) and the organoleptic aspects (i.e. color, odor) were assessed.

Table 3: Chemical stability of methionine in water reconstituted formulation (table 1) stored at 4-8°C, 21-25 °C, 38-42°C in glass pharmaceutical bottles and over time.

Table 4. pH value of the methionine water reconstituted bulk powders (see formula in

Table 1) stored at 2-6°C, 21-25 °C, 38-42°C in glass pharmaceutical bottles and over time.

The reconstituted Methionine formulation, stored between 4 ± 2°C, 23 ± 2°C and 40 ± 2°C retained its concentration around 100 ± 10% of the baseline value at 45 days after compounding (Figure 2). No pH change occurred in any samples stored between 4 ± 2°C, 23 ± 2°C and 40 ± 2°C for the reconstituted at 45 days after compounding (Figure 3). In samples stored between 4 ± 2°C or 23 ± 2°C, no color or odor change occurred, maintaining a white color and a strawberry smell. In samples stored between 40 ± 2°C, a change in color and odor occurred after 17 days, the samples having taken on a yellowish aspect.

Microbiological stability of the methionine formulation

The preservative effectiveness of the Met formulation against fungi and bacteria is presented in Table 6. The Met suspensions exhibited adequate preservative efficacy on days 14 and 28 against all 6 microorganisms, as shown by the measured log reduction for each of them, which meets the criteria of the European Pharmacopoeia (monograph 5.1.3). No growth was observed between day 14 and day 28. Over the sampling period, the microbial testing confirmed that all the Methionine formulation in bottles were within the acceptable criteria the European Pharmacopeia. For all replicates, the CFU count observed after incubation was less than 10 for total aerobic organisms and total yeasts and molds. The absence of Escherichia coli was also con-firmed at all time-points (Table 5).

Table 5: Microbiological stability results

Real-life survey : Acceptability of The Met suspension

The real-life survey included a panel of seven children treated with Met suspension for at least 1 year, and three patients had, at the time of the survey, a gastrostomy through which all nutritional intakes, including medications, were administered (data not shown). Prior to the use of Met suspension, five patients briefly experienced the use of oral Met treatment prepared in capsules requiring opening and dilution with water. Of these, all parents have preferred the handling of Met suspension over Met capsules and assigned a mean score of 4.7 ± 0.5 out of 5 regarding the ease/preference of using Met suspension. In the four patients without gastrostomy, the strawberry flavor was considered acceptable and parents assigned a mean score of 3.5 ± 1.7 out of 7 using the child's facial expression (data not shown). The odor of the Met suspension was rated at 3.1 ± 1.5 out of 5 (data not shown).

Discussion

Rare ILLD diseases characterized by double homozygous Ala393Thr/Ser576Leu mutations on MARS1 are particularly severe. Mortality reached 59% with about half of the deaths occurring before the age of two years despite repetitive whole-lung lavages and anti- inflammatory treatments (e.g. steroids) [3], Rescue of mutated MetRS activity has been demonstrated to be possible by increasing the plasmatic concentration of its physiological substrate [3,5], Promising results have been recently documented by oral Methionine supplementation in four children with PAP and multisystemic dysfunction related to MARS1 mutations with full remission of the disease in two patients and a clear on-going improvement in the two others [3], The posology of Methionine is adapted to the total body weight of the child, and according to the results of the Methionine plasma level. A gradual increase in doses is carried out at the start of treatment and throughout the child's development [3], Methionine supplementation as a liquid pharmaceutical form is particularly interesting for facilitating dose adjustments and infants’ acceptance.

Indeed, acceptability of drugs is crucial for children due to inherent characteristics of this population including swallowing difficulties and strong taste sensitivity. Acceptability as defined by drug agencies includes the pharmaceutical characteristics of the medicine such as palatability, size and shape. Palatability is defined as the overall appreciation of a medicine towards its smell, taste, aftertaste and texture. The attractiveness of pediatric medicinal products should be also carefully balanced between the risk of inadequate patient acceptability and accidental intake, and should be discussed with regards to all aspects of the medicine, the dosage form, the formulation, the strength as well as the primary and any secondary drug packaging. Unless otherwise justified, the palatability of a paediatric preparation should be satisfactory on its own merit, without mixing with food or drinks. However, paediatric preparations must not become too attractive to children (candy like) as this is known to increase the rate of accidental poisoning [7], In this regard, a powder for reconstitution exhibit a reduced risk of massive ingestion by children, and a secure childproof cap is also present on the bottles, even if the acute toxicity of oral Methionine is considered low [8],

In terms of palatability, Methionine is a sulfur amino acid with a strong and unpleasant smell and taste [6], Therefore, addition of sweetener and flavoring agents is of particular importance to improve both acceptability and palatability. In this regard, sucralose and several flavoring agents were tested: strawberry, orange, litchi, and candy floss. Only strawberry in combination with sucralose was judged acceptable and made it possible to partially mask the bad taste of Methionine (Table 6). The choice of suitable excipients in a paediatric medicinal product is also one of the key elements of its pharmaceutical development. Preserved preparations will generally be considered acceptable for children from birth provided that the preservatives and any other excipients can be considered safe for children in the target age group. Indeed, concerns are particularly raised against parabens, which are among the most used preservatives in medicines. In this regard, sodium benzoate was chosen as preservative in this formulation, the age of the treated population being over 56 days old. Critical product quality attributes to be considered for oral suspensions include physicochemical characteristics of the solution such as viscosity, potential for foaming, air entrapment, sedimentation, and sticking of the suspended active substance to the primary container and to the measuring device. Where sedimentation cannot be avoided, easy re-suspension with moderate shaking is required to reduce the risk of insufficient shaking and dosing errors due to inhomogeneous distribution of the active substance. The risks of under- and overdosing to the child because of inadequate shaking should be considered. In view of the relatively low water solubility of Methionine (~56 mg/mL) [9], an oral suspension was formulated. To ensure a rather good resuspension and homogeneity when taking the drug, as well as reducing the risk of false route through the lungs, xanthan gum and carmellose were added to the formulation. The homogeneity of the suspension has been checked by the determination of Met content after resuspension (10 sec of bottle manual shaking). The uniformity of the Met content was found to be acceptable, as no difference was found in the Met content de-pending on the sampling site within the bottles, and with no less than 93% of the ex-pected Met concentration. Furthermore, the suspension appears visually homogeneous without powder agglomeration after reconstitution and after gentle manual shaking before use. Under these conditions, ten seconds of agitation are recommended before the dose required for administration can be taken from the Met reconstituted suspension.

To develop such a pharmaceutical Methionine pediatric formulation a stability indicating analytical assay was validated for measuring Methionine. The method developed fulfills analytical and ICH requirements (e.g. linear, precise, and accurate), and was shown specific for Methionine stability assays as no interference peaks were detected as demonstrated by forced Methionine degradation studies with excipients. The results obtained from the stress degradation studies confirmed the sensitivity of Methionine for oxidation. The need for an antioxidant (e.g. vitamin C) was tested during the accelerated degradation test, without noticeable improvement (data not shown). The physico-chemical stability of the powder as well as of the suspension was satisfactory already without any specific antioxidant excipient.

We developed a 200 mg/mL Methionine bulk powder formulation for reconstitution. An oral Methionine suspension is obtained after water reconstitution by caregivers or family. The physico-chemical stability study showed a stability of 92 days at room temperature for the unreconstituted Methionine bulk powder formulation, and a stability of at least 45 days at refrigerated or ~22°C storage for the Methionine suspension. The microbiological criteria of the European Pharmacopoeia have been respected, which means that the Methionine preparation can be used without any risk of contamination if the storage conditions are followed. With this information, the pharmacy department can prepare the reconstituted formulation of Methionine and dispense it for inpatients on pediatric wards or for adults who cannot swallow a solid form. A measuring cup for water measurement as well as an instruction manual will also be provided to parents/caregivers to explain reconstitution and storage of the Methionine treatment.

Table 6: Result of the study of aromas on a panel of five adults using a subjective scale from 1 to 5; 5 being the best score.

A survey was given to the parents to evaluate acceptability of the Met formulation. It showed that all parents were satisfied with this Met suspension. Indeed, the survey indicated that the taste and smell of Met were improved. The taste and odor masking of Met could probably have been modified by the use of other strategies such as cyclodextrin or liposomal complexes. However, these strategies do not dispense with the addition of a flavoring which, in children, seems to be mandatory for a better oral acceptance, especially if the treatment is of long duration. Moreover, the choice was made to develop a formulation that could be more easily transposed to other pharmacy departments and without making the formulation too attractive (candy type), as the risks of overdose in Met are not well known

Conclusions

This study enabled the design of a Methionine formulation as a powder for oral suspension. This pharmaceutical form is more appropriate in terms of treatment administration and patient acceptability for the pediatric population. Indeed, it facilitates the gradual adaptation of dosages over time as well as the masking of the taste and smell of Methionine.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Laska, M. Olfactory Perception of 6 Amino Acids by Human Subjects. Chem. Senses

2010, 35, 279-287, doi: 10.1093/chemse/bjq017.

2. Hadchouel A, Drummond D, Pontoizeau C, Aoust L, Hurtado Nedelec MM, El Benna J, Gachelin E, Perisson C, Vigier C, Schiff M, Lacaille F, Molina TJ, Berteloot L, Renolleau S, Ottolenghi C, Treluyer JM, de Blic J, Delacourt C. Methionine supplementation for multi-organ dysfunction in MetRS-related pulmonary alveolar proteinosis. Eur Respir J. 2022 Apr 21;59(4):2101554. doi: 10.1183/13993003.01554-2021.

3. Li Z, Wang F, Liang B, Su Y, Sun S, Xia S, Shao J, Zhang Z, Hong M, Zhang F, Zheng S. Methionine metabolism in chronic liver diseases: an update on molecular mechanism and therapeutic implication. Signal Transduct Target Ther. 2020 Dec 4;5(l):280.

4. Wang S, Tian S, Li M, Li Z. Methionine attenuates the intensity of rheumatoid arthritis by downregulating NF-KB and iNOS expression in neonatal rats. 3 Biotech. 2018 Jul;8(7):303.

5. Comisso, M.; Hadchouel, A.; de Blic, J.; Mirande, M. Mutations in MARS Identified in a Specific Type of Pulmonary Alveolar Proteinosis Alter Methionyl-TRNA Synthetase Activity. FEBS J. 2018, 285, 2654-2661, doi: 10.1111/febs.14510.

6. Laska, M. Olfactory Perception of 6 Amino Acids by Human Subjects. Chem. Senses

2010, 35, 279-287, doi: 10.1093/chemse/bjq017.

7. EMA Pharmaceutical Development of Medicines for Paediatric Use Available online: https://www.ema.europa.eu/en/pharmaceutical-development-medicines- paediatric-use (accessed on 6 November 2022).

8. Garlick, P.J. Toxicity of Methionine in Humans. J. Nutr. 2006, 136, 1722S-1725S, doi: 10.1093/jn/136.6.1722S

9. PubChem Hazardous Substances Data Bank (HSDB) : 4317 Available online: https://pubchem.ncbi.nlm.nih.gov/source/hsdb/4317 (accessed on 6 November 2022)

10. Wagner, J. A.; Pabon, G.; Terrill, D.; Abdel-Rahman, S.M. Examining a New Scale for Evaluating Taste in Children (TASTY). J. Pediatr. Pharmacol. Ther. JPPT 2020, 25, 131-138, doi: 10.5863/1551-6776-25.2.131. 11. Sheskey, P.J.; Cook, W.G.; Cable, C.G. Handbook of Pharmaceutical Excipients;

Pharmaceutical Press, 2017; ISBN 978-0-85711-271-2.

Claims

CLAIMS:

1. A pharmaceutical composition comprising: i) methionine, or a methionine- compound donor or any one of their pharmaceutically acceptable salts; ii) at least one flavoring agent; and iii) at least sweetening agent.

2. The pharmaceutical composition of claim 1, comprising further at least one thickening agent; and/or at least one pH adjustment agent and/or a preservative agent.

3. The pharmaceutical composition of claim 1 or 2, comprising L-methionine.

4. The pharmaceutical composition of claim 1 or 2, wherein the methionine-donnor compound is S-adenosyl-methionine.

5. The pharmaceutical composition of anyone of claim 1 to 4, wherein the at least one flavoring agent is strawberry flavor.

6. The pharmaceutical composition of anyone of claim 1 to 5, wherein the sweetening agent is selected in the group consisting of sorbitol, saccharose, aspartame, xylitol and sucralose.

7. The pharmaceutical composition of anyone of claim 1 to 6, wherein the pharmaceutical composition comprises: L-methionine; sucralose; strawberry flavor; xantham gum; sodium carboxymethyl cellulose; sodium citrate; citric acid; and sodium benzoate.

8. The pharmaceutical composition of anyone of claim 1 to 7, wherein the pharmaceutical composition is in powder form.

9. The pharmaceutical composition of anyone of claim 1 to 8, wherein the pharmaceutical composition further comprises an aqueous solvent.

10. The liquid pharmaceutical composition of claim 9, wherein methionine or L- methionine donor compound or its pharmaceutically acceptable salts are at a concentration equal or superior to 150 mg/mL.

11. The liquid pharmaceutical composition of claim 9 or 10, wherein the pH of the liquid pharmaceutical composition ranges from 4 to 6 (including 4, 5, and 6) at 6°C, 23°C or 40°C.

12. The pharmaceutical composition of any one of claim 1 to 11 for use as a medicament.

13. A method for treating metabolic diseases affecting methionine metabolisms in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of the pharmaceutical composition of any one claims 1 to 11.

14. The method for treating metabolic diseases according to claim 13, wherein the metabolic disease affecting methionine metabolisms is liver disease, interstitial lung and liver disease (ILLD) related to MARS gene and/or protein mutations or pulmonary alveolar proteinosis (PAP) related to MARS gene and/or protein mutations.

15. A method for treating arthritis in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of the pharmaceutical composition of any one claims 1 to 11.