Oral biofilm formation-inhibiting and / or dispersion-promoting agent

Abstract

To provide an oral cavity biofilm formation-inhibiting and / or dispersion-promoting agent which is stable and has a biofilm formation-inhibiting or dispersion-promoting action.SOLUTION: A composition for inhibiting the formation of oral biofilm and / or promoting the dispersion of oral biofilm, comprising one or more components selected from the group consisting of a basic amino acid salt of ascorbic acid 2-glucoside, an alkali metal salt of ascorbic acid 2-glucoside, and a basic amino acid salt of ascorbic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for inhibiting the formation and / or promoting the dispersion of oral biofilms. [Background technology]

[0002] Oral biofilms are communities of microorganisms that form on the surfaces of teeth and gums. Various types of bacteria exist in the oral cavity, forming a thin film called dental plaque. Without adequate brushing and oral hygiene, dental plaque accumulates in the grooves and crevices between the teeth and gums, known as periodontal pockets, forming a biofilm. Periodontal biofilms are formed when bacterial communities are bound together by sticky polymers, adhering to the tooth surface. This allows bacteria to multiply within the plaque and produce harmful metabolic substances that cause inflammation in the periodontal tissues. As periodontal disease progresses, biofilms develop further, potentially leading to the destruction of periodontal tissues and tooth loss. Periodontal biofilms require thorough brushing, regular dental checkups, and professional dental cleanings. Biofilm-controlling mouthwashes and specific medications prescribed by dentists are also available to restore oral health balance.

[0003] Ascorbic acids act as reducing agents, collagen synthesis promoters, and antioxidants, and among them, ascorbic acid, sodium ascorbate, and sodium or magnesium salts of ascorbic acid phosphate are known to be effective against periodontitis and the like (Patent Documents 1-2 and Non-Patent Documents 1-5). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-117697 [Patent Document 2] Japanese Patent Application Laid-Open No. 2018-95574 [Non-patent literature]

[0005] [Non-Patent Document 1] Japanese Journal of Periodontology 48(3):201-207,2006 [Non-patent document 2] Japanese Journal of Periodontology 46(2):137-142,2004 [Non-patent document 3] Japanese Journal of Dental Preservation 52(5):377-383,2009 [Non-patent document 4] J Periodontal 86:27-35,2015 [Non-patent document 5] Vitamins 94:553-556,2015 Summary of the Invention [Problem to be solved by the invention]

[0006] However, ascorbic acid and its derivatives are known to cause browning and have stability problems, and their effect on biofilms that cause periodontal disease is unknown. Therefore, an object of the present invention is to provide an agent for inhibiting the formation and / or promoting the dispersion of oral biofilms that is stable and has the effect of inhibiting the formation or promoting the dispersion of biofilms. [Means for solving the problem]

[0007] Therefore, the present inventors investigated the effects of various ascorbic acid derivatives on the formation and dispersion of oral biofilms and found that ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts are highly stable and have excellent oral biofilm formation-inhibiting effects and / or dispersion-promoting effects, and are useful as compositions for preventing and / or ameliorating periodontal disease.

[0008] That is, the present invention provides the following [1] to [9]. [1] A composition for inhibiting the formation and / or promoting the dispersion of oral biofilms, comprising one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts. [2] The composition according to [1], having a pH of 5.0 or more and 9.0 or less. [3] The composition according to [1] or [2], which is a composition for human use or a composition for mammals. [4] A composition for preventing and / or ameliorating periodontal disease, comprising one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts. [5] The composition according to [4], having a pH of 5.0 or more and 9.0 or less. [6] The composition according to [4] or [5], which is a composition for human use or a composition for mammals. [7] One or more ingredients selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts, used to inhibit the formation and / or promote the dispersion of oral biofilms, or to prevent and / or improve periodontal disease. [8] Use of one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts for the manufacture of an agent for inhibiting the formation and / or promoting the dispersion of oral biofilms, or an agent for preventing and / or improving periodontal disease. [9] A method for inhibiting the formation and / or promoting the dispersion of oral biofilms, or a method for preventing and / or ameliorating periodontal disease, characterized by administering one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts. [Effects of the Invention]

[0009] The composition of the present invention has excellent inhibitory effects on the formation and / or dispersion promotion of oral biofilms in humans or mammals, and is stable, and therefore is also useful as a composition for preventing and / or ameliorating periodontal disease. [Brief explanation of the drawings]

[0010] [Figure 1] The numbers of initially attached and aggregated viable bacteria are shown. In Figures 1 to 6, aminoASG represents ascorbic acid 2-glucoside arginine salt, and NaASG represents ascorbic acid 2-glucoside sodium salt. [Figure 2] The numbers of initially attached and aggregated dead bacteria are shown. [Figure 3] The total number of initially attached and aggregated bacteria is shown. [Figure 4] It exhibits biofilm formation inhibitory effect against Actinomyces oris. [Figure 5] It exhibits biofilm dispersal activity against Actinomyces oris. [Figure 6] It exhibits biofilm dispersal activity against Streptococcus gordonii. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present inventors have discovered that ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts are highly stable and have excellent oral biofilm formation inhibitory effects and / or dispersion-promoting effects, and are useful as compositions for the prevention and treatment of periodontal disease. Therefore, one aspect of the present invention is a composition for inhibiting the formation and / or promoting the dispersion of oral biofilms, which contains one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts. Another aspect of the present invention is a composition for preventing and / or ameliorating periodontal disease, which contains one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts.

[0012] Ascorbic acid in the present invention is a compound whose IUPAC name is (R)-3,4-dihydroxy-5-((S)-1,2-dihydroxyethyl)furan-2(5H)-one, and is also known as vitamin C or L-ascorbic acid. As mentioned above, ascorbic acid acts as a reducing agent, a collagen synthesis promoter, and an antioxidant. In particular, the excellent antioxidant effect of ascorbic acid inhibits melanin production in the skin, and it is known as an ingredient that prevents pigmentation such as age spots and freckles, and is widely used in pharmaceuticals, foods, and cosmetics.

[0013] Ascorbic acid 2-glucoside is a substance in which glucose is bound to the second carbon atom of ascorbic acid, and is considered a provitamin of ascorbic acid because it is metabolized in the body to become ascorbic acid. The basic amino acid salt of ascorbic acid 2-glucoside used in the present invention is a basic amino acid salt of ascorbic acid 2-glucoside. Here, the ascorbic acid 2-glucoside basic amino acid may be one or more selected from ascorbic acid 2-glucoside lysine salt, ascorbic acid 2-glucoside arginine salt, ascorbic acid 2-glucoside histidine salt, and ascorbic acid 2-glucoside tryptophan salt, of which ascorbic acid 2-glucoside lysine salt and ascorbic acid 2-glucoside arginine salt are more preferred. The molar ratio of ascorbic acid 2-glucoside to basic amino acid in the basic amino acid salt of ascorbic acid 2-glucoside is preferably 1:1.5 to 1.5:1, more preferably 1:1.2 to 1.2:1.

[0014] Examples of alkali metal salts of ascorbic acid 2-glucoside include sodium ascorbic acid 2-glucoside and potassium ascorbic acid 2-glucoside. The molar ratio of ascorbic acid 2-glucoside to the alkali metal salt in the alkali metal salt of ascorbic acid 2-glucoside is preferably 1:1.5 to 1.5:1, more preferably 1:1.2 to 1.2:1.

[0015] The basic amino acid ascorbate may be one or more selected from the group consisting of lysine ascorbate, arginine ascorbate, histidine ascorbate, and tryptophan ascorbate, of which lysine ascorbate and arginine ascorbate are more preferred. The molar ratio of ascorbic acid to basic amino acid in the basic amino acid salt of ascorbic acid is preferably 1:1.5 to 1.5:1, more preferably 1:1.2 to 1.2:1.

[0016] Ascorbic acid 2-glucoside basic amino acid salts can be produced by reacting ascorbic acid 2-glucoside with a basic amino acid. Specifically, ascorbic acid 2-glucoside and a basic amino acid are added to ion-exchanged water in which the dissolved oxygen concentration has been reduced by ultrasonication or the like, at 40 to 60°C, and the reaction is carried out by adjusting the amount of basic amino acid added and adjusting the pH to 5.22 to 5.55. After completion of the reaction, ascorbic acid 2-glucoside basic amino acid salts can be isolated as a powder by freeze-drying, spray-drying, or the like. Alkali metal ascorbic acid 2-glucoside can be produced by reacting ascorbic acid 2-glucoside with an alkali metal hydroxide, alkali metal carbonate, alkali metal bicarbonate, or the like. Specifically, ascorbic acid 2-glucoside and an alkali metal carbonate, or the like, are added to ion-exchanged water in which the dissolved oxygen concentration has been reduced by ultrasonication or the like at 20 to 60°C, and the reaction is carried out by adjusting the amount of alkali metal carbonate added by adjusting the pH to 4 to 7. After completion of the reaction, alkali metal ascorbic acid 2-glucoside can be isolated as a powder by freeze-drying, spray-drying, or the like. Ascorbic acid basic amino acid salts can be produced by reacting ascorbic acid with a basic amino acid. Specifically, ascorbic acid and a basic amino acid are added to ion-exchanged water in which the dissolved oxygen concentration has been reduced by ultrasonication or the like, at 20 to 60°C, and the reaction is carried out by adjusting the amount of basic amino acid added and adjusting the pH to 6 to 8. After completion of the reaction, the neutralized ascorbic acid basic amino acid salt can be isolated as a powder by freeze-drying, spray-drying, or the like.

[0017] The resulting powders of ascorbic acid 2-glucoside basic amino acid salt, ascorbic acid 2-glucoside alkali metal salt, and ascorbic acid basic amino acid salt are stable for extended periods and do not brown, making them useful as raw materials for incorporation into oral compositions and pharmaceutical compositions at high concentrations. In solutions, the upper concentration limit is inevitably limited due to the need to mix with other bases. Preserving raw materials in a liquid state requires the addition of preservatives, sterilization by filtering, filling into sterile containers, or storage at low temperatures. However, powdering allows for stable and compact storage at room temperature without the addition of other ingredients. Ascorbic acid 2-glucoside basic amino acid salts and the like are extremely stable and do not brown even in aqueous solution. This effect is completely unexpected, considering that browning occurs due to the Maillard reaction between ascorbic acid and ordinary amino acids.

[0018] As shown in the Examples below, a component selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts has an effect of inhibiting oral biofilm formation and / or promoting dispersion, and is useful as a composition for inhibiting oral biofilm formation and / or promoting dispersion, and as a composition for preventing and / or ameliorating periodontal disease. Here, the composition of the present invention can be intended for humans and mammals other than humans. As mentioned above, oral biofilms are aggregates of microorganisms that form on the surfaces of teeth and gums, and are the cause of the onset and progression of dental caries and periodontal disease. Therefore, inhibiting the formation and / or promoting the dispersion of oral biofilms is useful as an agent for preventing and / or ameliorating periodontal disease. Here, "inhibiting oral biofilm formation" refers to preventing or inhibiting the formation of a biofilm by microorganisms in the oral cavity, and "promoting the dispersion of oral biofilms" refers to dispersing or promoting the dispersion of a biofilm formed in the oral cavity. Furthermore, "preventing periodontal disease" refers to preventing or suppressing the onset of periodontal disease, and "ameliorating periodontal disease" refers to preventing the progression of symptoms of periodontal disease and improving the symptoms.

[0019] The oral biofilm formation inhibitor and / or dispersion promoter composition, or periodontal disease preventive and / or ameliorating composition of the present invention (hereinafter sometimes simply referred to as the composition of the present invention) may be in the form of a liquid, paste, etc., such as conventional oral compositions such as toothpaste, liquid toothpaste, lubricant toothpaste, and mouthwash, as well as in the form of lozenges, etc.

[0020] When the composition of the present invention is made into an oral composition or a troche, the content of the component (active ingredient of the present invention) selected from ascorbic acid 2-glucoside basic amino acid salt, ascorbic acid 2-glucoside alkali metal salt, and ascorbic acid basic amino acid salt is preferably 0.05% by mass or more and 10% by mass or less, more preferably 0.1% by mass or more and 8% by mass or less, and even more preferably 0.5% by mass or more and 5% by mass or less, of the total composition, from the viewpoint of inhibiting oral biofilm formation and promoting dispersion.

[0021] In addition to the active ingredient of the present invention, the composition of the present invention may contain other optional ingredients according to the dosage form, as needed, within the range that does not impair the effects of the present invention. For example, binders, thickeners, surfactants, sweeteners, preservatives, colorants, abrasives, fragrances, and other active ingredients other than the active ingredient of the present invention may be contained.

[0022] Examples of thickeners include sugar alcohols such as sorbitol, and polyhydric alcohols such as propylene glycol and glycerin. The content of the thickener in the total composition is preferably 5 to 50% by mass.

[0023] Examples of the binder include cellulose derivatives such as sodium carboxymethylcellulose, gums such as xanthan gum, carrageenan, etc. The content of the binder in the total composition is preferably 0 to 5% by mass, more preferably 0.1 to 5% by mass.

[0024] The surfactant may be an anionic surfactant, a cationic surfactant, a nonionic surfactant, or an amphoteric surfactant. Examples of anionic surfactants include alkyl sulfates such as sodium lauryl sulfate. Examples of nonionic surfactants include sugar alcohol fatty acid esters such as sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters; polyhydric alcohol fatty acid esters such as glycerin fatty acid esters, polyglycerin fatty acid esters, and polyoxyethylene glycerin fatty acid esters; polyoxyethylene fatty acid esters such as polyoxyethylene alkyl ethers and polyoxyethylene hydrogenated castor oil; and fatty acid alkanolamides. Examples of cationic surfactants include alkylammonium surfactants, and examples of amphoteric surfactants include betaine surfactants and imidazoline surfactants. The content of these surfactants in the total composition is preferably 0.1 to 10% by mass, more preferably 0.5 to 5% by mass.

[0025] Examples of sweeteners include saccharin sodium, etc., and examples of preservatives include parahydroxybenzoic acid esters, benzoic acid or salts thereof, etc. As colorants, dyes known for oral compositions can be used.

[0026] Other active ingredients (medicinal ingredients) may be known medicinal ingredients that are incorporated into oral compositions. Examples include fluorine-containing compounds such as sodium fluoride and sodium monofluorophosphate, anti-inflammatory agents such as tranexamic acid, epsilon aminocaproic acid, and allantoin, and enzymes such as dextranase. The active ingredients may be contained in effective amounts within a range that does not impair the effects of the present invention.

[0027] From the viewpoint of the stability of the active ingredient of the present invention, the pH of the composition is preferably 5.0 to 9.0, more preferably 5.5 to 8.5, and even more preferably 6.0 to 8.0 at 25° C. The pH of the composition may be adjusted using a known pH adjuster.

[0028] Another aspect of the present invention is one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts, which are used to inhibit the formation and / or promote the dispersion of oral biofilms or to prevent and / or ameliorate periodontal disease. Another aspect of the present invention is the use of one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts for the manufacture of an agent for inhibiting oral biofilm formation and / or promoting dispersion, or an agent for preventing and / or ameliorating periodontal disease. Furthermore, another aspect of the present invention is a method for inhibiting the formation and / or promoting the dispersion of oral biofilms, or a method for preventing and / or ameliorating periodontal disease, which comprises administering one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts. [Example]

[0029] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0030] [Production Example 1] Deionized water was degassed using ultrasonic waves at 0.5kW-1.0kW output. At a setting of 0.8kW, degassing was performed for 20-40 minutes. Degassing was confirmed by measuring with a dissolved oxygen meter until the dissolved oxygen concentration reached 0.5 ppm or less, yielding deionized water. 50ml of this water was heated to 45-60°C, preferably 50°C ± 0.5°C. 5.0g of ascorbic acid 2-glucoside was dissolved by stirring, and then 14.464mmol of arginine was added. The pH was preferably 5.22-5.55, more preferably 5.35. The molar concentration of arginine added was finely adjusted by adjusting the pH. This solution was freeze-dried to obtain a powder of ascorbic acid 2-glucoside arginine salt (7.55g, 90.03%). FT / IR spectra of the resulting powder of ascorbic acid 2-glucoside arginine salt were measured. The spectral results showed a peak at 1720cm. -1 (non-ionic COOH, antisymmetric stretching vibration) and characteristic band 1400 cm -1 was observed, confirming the introduction of arginine ionic bonds.

[0031] [Production Example 2] The ion-exchanged water used for dissolution was treated in the same manner as in Production Example 1. 100 ml of this ion-exchanged water was heated to 50°C ± 0.5°C, and 5.0 g of ascorbic acid 2-glucoside was added and dissolved by stirring. 17.97 mmol of lysine was then added. The pH was 5.1-5.6, but the molar concentration was finely adjusted to pH 5.3. This solution was freeze-dried to obtain ascorbic acid 2-glucoside lysine salt powder (7.62 g, 98.42%). [Production Example 3] 200 g of purified water was weighed into a 500 mL glass beaker. 34.2 g of ascorbic acid 2-glucoside powder was gradually added to the beaker while stirring with a magnetic stirrer and allowed to completely dissolve at room temperature. Sodium bicarbonate powder was gradually added to the beaker to adjust the pH of the solution to 5.0-8.0. Pure nitrogen gas was introduced into the solution from a nitrogen cylinder via a regulator at a flow rate of 150 mL / min from the beginning of the procedure. The nitrogen gas flow was stopped once the pH adjustment was complete. The aqueous solution was freeze-dried to obtain sodium ascorbic acid 2-glucoside powder. NMR analysis of the resulting powder confirmed its identity as sodium ascorbic acid 2-glucoside. [Production Example 4] 10 g of ascorbic acid was completely dissolved in 100 g of ion-exchanged water. While stirring, 10 g of L-arginine was gradually added until completely dissolved. The pH was confirmed to be between 6 and 8. The pH of the solution was 6.533 when prepared, and 6.489 after standing for 1 hour, showing little change and indicating a stable pH. The resulting solution was microfiltered through a 0.22-micrometer pore sterilizing filter and then freeze-dried (CDSH100A, Cell Diagnostics) to obtain arginine ascorbate powder (2.70 g).

[0032] Example 1 (Initial adhesion and aggregate formation inhibitory effect) The effect on the early stages of oral biofilm formation was examined over a short period of time up to one hour. (method) Actinomyces oris, a type of oral bacterium that serves as a scaffold for oral biofilm formation, was used as the initial adherent bacterium. The culture medium was collected by centrifugation and the concentration was adjusted with buffer. The bacteria were then inoculated into trypticase soy broth (TSBs) containing 0.25% sucrose on a glass-bottom dish coated with sterile human saliva. The culture was pre-incubated for 3 hours at 37°C in the presence of 5% carbon dioxide. After washing with sterile buffer, buffer or an ascorbic acid derivative was added. After incubation under the same conditions for 1 hour and washing with sterile buffer, the formed initial adherent and aggregated specimens were stained with LIVE / DEAD and observed under a confocal laser scanning microscope. (result) Figure 1 shows the number of initially attached and aggregated viable bacteria. With ascorbic acid 2-glucoside sodium salt, the number of viable bacteria was significantly reduced at 100 mg / mL. Furthermore, ascorbic acid 2-glucoside arginine salt at 100 mg / mL and ascorbic acid 2-glucoside sodium salt at 30 mg / mL tended to reduce the number of viable bacteria compared to the control (Figure 1). The number of initially attached and aggregated dead bacteria is shown in Figure 2. The number of dead bacteria increased with ascorbic acid 2-glucoside sodium salt and ascorbic acid 2-glucoside arginine salt compared to the control (Figure 2). The total number of live and dead bacteria is shown in Figure 3. A tendency for the number to decrease was observed with 100 mg / mL of ascorbic acid 2-glucoside sodium salt (Figure 3).

[0033] Example 2 (Biofilm formation inhibitory effect) The effect of inhibiting oral biofilm formation was examined over a long period of time, up to 16 hours. (method) Actinomyces oris, one of the oral bacteria that serves as a scaffold for oral biofilm formation, was used. The culture medium was collected by centrifugation and the concentration was adjusted with buffer. After that, TSBs were placed in a 96-well microplate coated with sterilized human saliva (1 hour, 4°C). Buffer or ascorbic acid derivatives were added and the plate was pre-incubated at 37°C for 16 hours in the presence of 5% carbon dioxide. The plate was washed with distilled water and dried, and then treated with 0.25% safranin solution for 15 minutes to stain the cell wall red. The plate was washed again with distilled water and dried. The dye was extracted with 70% ethanol, and the absorbance at 492 nm was measured using a microplate reader. (result) Ascorbic acid 2-glucoside arginine salt tended to inhibit biofilm formation against Actinomyces oris at concentrations of 30 to 60 mg / mL (Figure 4).

[0034] Example 3 (biofilm dispersal effect) We investigated whether it has the effect of dispersing formed biofilms. (method) According to standard procedures, biofilms were formed in a 96-well microplate using two types of oral bacteria, Actinomyces oris and Streptococcus gordonii, which serve as a scaffold for oral biofilm formation. After washing with sterile buffer, an ascorbic acid derivative sample was added. The plate was then cultured for 24 hours at 37°C in the presence of 5% carbon dioxide. The plate was then washed with distilled water and dried, and treated with 0.25% safranin solution for 15 minutes to stain the cell walls red. The plate was then washed with distilled water and dried, and the dye was extracted with 70% ethanol solution. The absorbance at 492 nm was measured using a microplate reader. (result) For Actinomyces oris, 1000 mg / mL of ascorbic acid 2-glucoside arginine salt and ascorbic acid 2-glucoside sodium salt dispersed the biofilm (Figure 5). For Streptococcus gordonii, 300, 600, and 1000 mg / mL of ascorbic acid 2-glucoside arginine salt dispersed the biofilm (Figure 6).

Claims

1. A composition for inhibiting the formation of and / or promoting the dispersion of oral biofilms, comprising one or more components selected from ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts.

2. 2. The composition according to claim 1, having a pH of 5.0 or more and 9.0 or less.

3. 10. The composition of claim 1, which is a human or mammalian composition.

4. A composition for preventing and / or ameliorating periodontal disease, comprising at least one component selected from the group consisting of ascorbic acid 2-glucoside basic amino acid salts, ascorbic acid 2-glucoside alkali metal salts, and ascorbic acid basic amino acid salts.

5. The composition according to claim 4, having a pH of 5.0 or more and 9.0 or less.

6. 5. The composition of claim 4, which is a human or mammalian composition.

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