Transthyretin tetramer stabilizer and agent for preventing or suppressing the progression of transthyretin amyloidosis

Abstract

Provided are a transthyretin tetramer stabilizer and an agent for preventing or suppressing the progress of transthyretin amyloidosis.　The present invention relates to a transthyretin tetramer stabilizer containing glabridin, glabrene, and glabrol and an agent for preventing or suppressing the progress of transthyretin amyloidosis, the agent containing glabridin, glabrene, and glabrol.

Description

[Technical Field]

[0001] First, the present invention relates to a transthyretin tetramer stabilizer for stabilizing a transthyretin tetramer.

[0002] Secondly, the present invention relates to a prophylactic or inhibitory agent for preventing or inhibiting the progression of transthyretin amyloidosis, a condition in which amyloid fibrils are deposited as a result of abnormal aggregation of transthyretin. [Background technology]

[0003] Transthyretin is a β-sheet-rich homotetrameric protein, with each subunit consisting of 127 amino acid residues. Transthyretin is known to bind to and transport thyroxine (T4) in blood and cerebrospinal fluid. The transthyretin tetramer has two thyroxine-binding sites (T4-binding sites) at the junction between dimers (Non-Patent Document 1).

[0004] Although transthyretin tetramers are normally stable under physiological conditions, factors such as genetic abnormalities and aging can cause instability and dissociation into monomers, resulting in misfolding and aggregation of amyloidogenic intermediates, which then undergo amyloidogenesis and lead to the deposition of amyloid fibrils, resulting in the clinical symptoms of amyloidosis. Examples of transthyretin amyloidosis include familial amyloid polyneuropathy (FAP), in which genetically mutated transthyretin (e.g., V30M mutant transthyretin) amyloidizes and deposits in various organs throughout the body, causing organ damage; and senile systemic amyloidosis (SAA), in which wild-type transthyretin amyloidizes with age and deposits in the heart, tendons, and other organs, causing disease.

[0005] Stabilization of the transthyretin tetramer and inhibition of amyloid formation can be achieved by using small molecule compounds that bind to the T4 binding site of transthyretin, and although various pharmaceuticals have been developed, they have side effects and the high cost of each dose places a heavy burden on patients. Furthermore, transthyretin amyloidosis is not only difficult to cure once it develops, but also has a high incidence rate with age, so the development of preventive measures is gaining attention.

[0006] Non-patent literature 1 and non-patent literature 2 describe that two molecules of glabridin, a polyphenol of licorice glabra, bind to and stabilize the transthyretin tetramer, thereby inhibiting the formation of amyloid fibrils.

[0007] Licorice is a plant of the genus Glycyrrhiza in the family Fabaceae, which is widely distributed in China, Europe, Russia, Afghanistan, Iran, Pakistan, etc., and its roots have a long history of use as food and herbal medicine. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Yokoyama, T. et al., Biol. Pharm. Bull. 41, 979-984 (2018) [Non-patent document 2] Yokoyama, T. et al., J. Med. Chem. 57, 1090-1096 (2014) Summary of the Invention [Problem to be solved by the invention]

[0009] Since stabilizing the transthyretin tetramer is effective in preventing or inhibiting the progression of transthyretin amyloidosis, if a stabilized formulation of the transthyretin tetramer could be developed and taken before the onset of the disease, it would be possible to prevent transthyretin amyloidosis. Therefore, an object of the present invention is to provide a transthyretin tetramer stabilizer and an agent for preventing or inhibiting the progression of transthyretin amyloidosis. [Means for solving the problem]

[0010] As mentioned above, Non-Patent Documents 1 and 2 describe that glabridin alone contributes to the stabilization of the transthyretin tetramer, but the stabilizing effect of a mixture containing other polyphenol compounds in addition to glabridin on the transthyretin tetramer has not been investigated. As a result of extensive research, the present inventors have found that a polyphenol mixture containing glabridin, glabrene, and glabrol is particularly effective as an active ingredient in a transthyretin tetramer stabilizer and an agent for preventing or suppressing the progression of transthyretin amyloidosis, and have completed the following invention.

[0011] (1) A transthyretin tetramer stabilizer containing glabridin, glabrene, and glabrol. (2) The agent according to (1), further containing 4'-O-methylglabridin. (3) In HPLC analysis under the following conditions a), the properties b), c) and d) are: a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B), column: ODS column, flow rate: 1.0 mL / min, temperature: 40 °C, detector: UV detector, detection wavelength: 282 nm, b) the ratio of the glabrene peak intensity to the glabridin peak intensity is 38% or more and 41% or less; c) the ratio of the glabridin peak intensity to the glabriol peak intensity is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity to glabridin peak intensity is 15% or more and 20% or less; The agent according to (2), which exhibits any one or more of the following. (4) The agent according to any one of (1) to (3), wherein the content of glycyrrhizinic acid is 0.005% by weight or less. (5) A transthyretin amyloidosis preventive or progression inhibitor containing glabridin, glabrene, and glabrol. (6) The agent according to (5), wherein the transthyretin amyloidosis is senile systemic amyloidosis or familial amyloid polyneuropathy. (7) The agent according to (5) or (6), further containing 4'-O-methylglabridin. (8) In HPLC analysis under the following conditions a), the properties b), c) and d) are: a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B), column: ODS column, flow rate: 1.0 mL / min, temperature: 40 °C, detector: UV detector, detection wavelength: 282 nm, b) the ratio of the glabrene peak intensity to the glabridin peak intensity is 38% or more and 41% or less; c) the ratio of the glabridin peak intensity to the glabriol peak intensity is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity to glabridin peak intensity is 15% or more and 20% or less; The agent according to (7), which exhibits any one or more of the following. (9) The agent according to any one of (1) to (8), which is in the form of a tablet, capsule, granule, or powder. (10) The agent according to any one of (1) to (9), wherein the content of glabridin per intake or dosage unit is 4 mg to 1200 mg.

[0012] (12) Use of a polyphenol mixture containing glabridin, glabrene, and glabrol for the manufacture of a composition for stabilizing transthyretin tetramer. The composition is preferably in the form of a tablet, capsule, granule, or powder, and preferably contains 4 mg to 1200 mg of glabridin per intake or dosage unit. (13) Use of a polyphenol mixture containing glabridin, glabrene, and glabrol for the manufacture of a medicine for stabilizing transthyretin tetramer. The medicine is preferably in the form of an oral preparation such as a tablet, capsule, granule, powder, etc. The medicine preferably contains 4 mg to 1200 mg of glabridin per intake or dosage unit. (14) contacting a polyphenol mixture containing glabridin, glabrene, and glabrol with transthyretin tetramer present in vitro; and inhibiting the monomerization of the transthyretin tetramer and / or the formation of amyloid fibrils from the transthyretin tetramer; A method for stabilizing a transthyretin tetramer in vitro, comprising: (15) Administering a polyphenol mixture containing glabridin, glabrene, and glabrol to a subject in need of stabilization of transthyretin tetramer; and inhibiting monomerization of the transthyretin tetramer and / or amyloid fibril formation from the transthyretin tetramer in the subject; A method for stabilizing a transthyretin tetramer in a subject, comprising: Preferably, the polyphenol mixture is administered orally to the subject in the form of an oral preparation such as tablets, capsules, granules, or powder, so that the total amount of polyphenols in the polyphenol mixture is 0.01 to 100 mg / kg body weight, preferably 0.1 to 30 mg / kg body weight per day. (16) A polyphenol mixture containing glabridin, glabrene, and glabrol for stabilizing transthyretin tetramers in vitro or in vivo. (17) The use according to (12), the use according to (13), the method according to (14), the method according to (15), or the polyphenol mixture according to (16), wherein the polyphenol mixture further contains 4'-O-methylglabridin. Here, the polyphenol mixture further containing 4'-O-methylglabridin has the following characteristics b), c) and d) in HPLC analysis under the following condition a): a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B), column: ODS column, flow rate: 1.0 mL / min, temperature: 40 °C, detector: UV detector, detection wavelength: 282 nm, b) the ratio of the glabrene peak intensity to the glabridin peak intensity is 38% or more and 41% or less; c) the ratio of the glabridin peak intensity to the glabriol peak intensity is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity to glabridin peak intensity is 15% or more and 20% or less; It is more preferable that the compound exhibits one or more of the following: (18) The use according to (12), the use according to (13), the method according to (14), the method according to (15), or the polyphenol mixture according to (16), wherein the content of glycyrrhizinic acid in the polyphenol mixture is 0.005% by weight or less.

[0013] (19) Use of a polyphenol mixture containing glabridin, glabrene, and glabrol for producing a composition for preventing or inhibiting the progression of transthyretin amyloidosis. The composition is preferably in the form of a tablet, capsule, granule, or powder, and preferably contains 4 mg to 1200 mg of glabridin per intake or dosage unit. (20) Use of a polyphenol mixture containing glabridin, glabrene, and glabrol for the manufacture of a medicine for preventing or inhibiting the progression of transthyretin amyloidosis. The medicine is preferably in the form of an oral preparation such as a tablet, capsule, granule, powder, etc. The medicine preferably contains 4 mg to 1200 mg of glabridin per intake or dosage unit. (21) Administering a polyphenol mixture containing glabridin, glabrene, and glabrol to a subject in need of prevention or suppression of progression of transthyretin amyloidosis; and inhibiting monomerization of transthyretin tetramers and / or amyloid fibril formation from transthyretin tetramers in the subject; A method for preventing or inhibiting the progression of transthyretin amyloidosis in a subject, comprising: Preferably, the polyphenol mixture is administered orally to the subject in the form of an oral preparation such as tablets, capsules, granules, or powder, so that the total amount of polyphenols in the polyphenol mixture is 0.01 to 100 mg / kg body weight, preferably 0.1 to 30 mg / kg body weight per day. (22) A polyphenol mixture containing glabridin, glabrene, and glabrol for preventing or inhibiting the progression of transthyretin amyloidosis. (23) The use according to (19), the use according to (20), the method according to (21), or the polyphenol mixture according to (22), wherein the transthyretin amyloidosis is senile systemic amyloidosis or familial amyloid polyneuropathy. (24) The use according to (19), the use according to (20), the method according to (21), or the polyphenol mixture according to (22), wherein the polyphenol mixture further contains 4'-O-methylglabridin. Here, the polyphenol mixture further containing 4'-O-methylglabridin has the following characteristics b), c) and d) in HPLC analysis under the following condition a): a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B), column: ODS column, flow rate: 1.0 mL / min, temperature: 40 °C, detector: UV detector, detection wavelength: 282 nm, b) the ratio of the glabrene peak intensity to the glabridin peak intensity is 38% or more and 41% or less; c) the ratio of the glabridin peak intensity to the glabriol peak intensity is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity to glabridin peak intensity is 15% or more and 20% or less; It is more preferable that the compound exhibits one or more of the following: (25) The use according to (19), the use according to (20), the method according to (21), or the polyphenol mixture according to (22), wherein the content of glycyrrhizinic acid in the polyphenol mixture is 0.005% by weight or less.

[0014] This specification includes the disclosure of Japanese Patent Application No. 2020-169842, from which this application claims priority. [Effects of the Invention]

[0015] The transthyretin tetramer stabilizer according to the present invention can stably maintain the transthyretin tetramer and inhibit monomerization and amyloid fibril formation.

[0016] The agent for preventing or inhibiting the progression of transthyretin amyloidosis according to the present invention can prevent or inhibit transthyretin amyloidosis such as familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SAA). [Brief explanation of the drawings]

[0017] [Figure 1]The left side of Figure 1 shows the ratio of transthyretin monomer to total transthyretin (monomer / total) in serum incubated with the polyphenol mixture-containing medium-chain fatty acid triglyceride solution and glabridin at 0, 10, and 50 μM glabridin concentrations, respectively, in Example 3. The right side of Figure 1 shows the ratio of transthyretin tetramer to total transthyretin (tetramer / total) in serum incubated with the polyphenol mixture-containing medium-chain fatty acid triglyceride solution and glabridin at 0, 10, and 50 μM glabridin concentrations, respectively, in Example 3. Open circles indicate the ratio in serum to which glabridin was added. Closed circles indicate the ratio in serum to which a polyphenol mixture-containing medium-chain fatty acid triglyceride solution was added. [Figure 2] Figure 2 shows the ratio of transthyretin monomer to tetramer (monomer / tetramer) in plasma obtained over time after humans ingested a polyphenol mixture-containing medium-chain fatty acid triglyceride solution (600 mg / day) for four weeks in Example 4. A lower ratio indicates higher stability of the transthyretin tetramer. DETAILED DESCRIPTION OF THE INVENTION

[0018] <Polyphenols> The transthyretin tetramer stabilizer and agent for preventing or inhibiting the progression of transthyretin amyloidosis according to the present invention contain glabridin, glabrene, and glabrol as active ingredients, and more preferably further contain 4'-O-methylglabridin. In this specification, the combination of polyphenols used as active ingredients may be referred to as a "polyphenol mixture." The present inventors have found that the polyphenol mixture has a significantly higher transthyretin tetramer-stabilizing effect than glabridin, which has long been known to have a transthyretin tetramer-stabilizing effect, when used alone.

[0019] Each polyphenol contained in the polyphenol mixture is a compound group classified as prenylflavonoid, and has a structure in which one or more C5 isoprene units are bonded to diphenylpropane.

[0020] The polyphenol mixture may further comprise glycycoumarin, glycyrol, glycyrin, liquiritigenin, glycolicone, 3'-hydroxy-4'-O-methylglabridin, glyurallin B, licocoumarone, gancaonin I, dehydroglyasperin D, echinatin, isolicoflavonol, dehydroglyasperin C, glyasperin B B), glycyrrhisoflavanone, lupiwighteone, glyasperin D, semilicoisoflavone B, and other polyphenols.

[0021] Further examples of the other polyphenols include genistein, daidzein, quercetin, rutin, catechin, epigallocatechin gallate, hesperidin, nobiletin, tyrosol, hydroxytyrosol, oleuropein, naringenin, caffeic acid, apple polyphenols, tea polyphenols, and gallic acid. The other polyphenols are preferably genistein, daidzein, quercetin, rutin, catechin, epigallocatechin gallate, hesperidin, nobiletin, naringenin, caffeic acid, apple polyphenols, and tea polyphenols. These other polyphenols may be used alone or in combination of two or more.

[0022] Each polyphenol in the polyphenol mixture may exist in the form of a salt, ester, glycoside, or the like. Examples of polyphenol salts include salts with acids acceptable for the final use, such as pharmaceuticals, foods, beverages, and feeds, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid; or salts with bases acceptable for the final use, such as alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts with suitable organic ligands, e.g., quaternary ammonium salts. Examples of polyphenol esters include fatty acid esters, specifically esters with long-chain fatty acids such as oleic acid, palmitic acid, stearic acid, linoleic acid, and linolenic acid, and short- or medium-chain fatty acids such as acetic acid and butyric acid. Examples of polyphenol glycosides include glycosides in which monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, and the like are bound as sugar components.

[0023] The polyphenol mixture may be contained in the transthyretin tetramer stabilizer and transthyretin amyloidosis preventive or progression inhibitor according to the present invention as a licorice hydrophobic extract or a purified product thereof.

[0024] Each polyphenol in the polyphenol mixture may be chemically synthesized. Each polyphenol in the polyphenol mixture may also be extracted from a biological sample such as a plant, microorganism, or animal, and purified as necessary. Each polyphenol in the polyphenol mixture may also be produced by fermentation using a microorganism capable of producing the polyphenol. The microorganism capable of producing the polyphenol may be a genetically modified microorganism or a wild-type microorganism.

[0025] In a preferred embodiment, the polyphenol mixture has the following characteristics b), c) and d) in HPLC analysis under the following condition a): a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B), column: ODS column, flow rate: 1.0 mL / min, temperature: 40 °C, detector: UV detector, detection wavelength: 282 nm, b) the ratio of the glabrene peak intensity (peak area) to the glabridin peak intensity (peak area) is 38% or more and 41% or less; c) The ratio of the glabrol peak intensity (peak area) to the glabridin peak intensity (peak area) is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity (peak area) to glabridin peak intensity (peak area) is 15% or more and 20% or less; It is preferable that the mixture exhibits one or more of the above, more preferably two or more of the above, and particularly preferably all of the above.

[0026] The ratio of the glabrene peak intensity (peak area) to the glabridin peak intensity (peak area) is not particularly limited as long as it is within the above-mentioned range, but the lower limit of the peak intensity is preferably 38% or 39%, and the upper limit is preferably 41% or 40%.

[0027] The ratio of the glabrol peak intensity (peak area) to the glabridin peak intensity (peak area) is not particularly limited as long as it is within the above-mentioned range, but the lower limit of the peak intensity is preferably 44% or 45%, and the upper limit is preferably 47% or 46%.

[0028] The ratio of the 4'-O-methylglabridin intensity (peak area) to the glabridin peak intensity (peak area) is not particularly limited as long as it is within the range described above, but the lower limit of the peak intensity is preferably 15%, 16%, or 17%, and the upper limit is preferably 20%, 19%, or 18%, for example.

[0029] In the above a), the gradient of mobile phase A and mobile phase B is preferably such that the ratio of mobile phase A to the total amount of mobile phase A and mobile phase B is constant at 50% (v / v) for 20 minutes from the start of the analysis, increases at a constant rate from 20 minutes to 75 minutes to reach 80% (v / v), remains constant at 100% (v / v) from 75 minutes to 80 minutes, and remains constant at 50% (v / v) from 80 minutes to 100 minutes. In the above a), the ODS column may be, for example, YMC J'sphere ODS-H80 (YMC Corporation). The size of the ODS column can be, for example, an inner diameter of 4.6 mm and a length of 250 mm.

[0030] Methods for measuring polyphenol content include, for example, colorimetric methods such as the ferric tartrate method, Prussian blue method, Folin-Ciocalteu method, and Folin-Denis method, as well as component-by-component measurement by HPLC. Any of these methods may be used, but the Folin-Denis or Folin-Ciocalteu method is often used to measure the total amount of polyphenols. In the case of the Folin-Denis or Folin-Ciocalteu method, a calibration curve can be prepared using a standard substance, and the content can be calculated in terms of the standard substance. For example, glabridin can be used as the standard substance. Specifically, the content of polyphenol components contained in the transthyretin tetramer stabilizer and transthyretin amyloidosis preventive or progression inhibitor of the present invention can be calculated in terms of glabridin, for example, by the method described in the Examples below.

[0031] <Licorice Hydrophobic Extract> As described above, the polyphenol mixture can be used in the form of a licorice extract. In this embodiment, the licorice used as the raw material for the extract is not particularly limited as long as it is a plant belonging to the genus Glycyrrhiza. Specific examples of licorice include Glycyrrhiza uralensis (G. uralensis), Glycyrrhiza inflata, Glycyrrhiza glabra, G. eurycarpa, and G. aspera. Preferred are G. uralensis, G. inflata, and G. glabra, with G. glabra being more preferred.

[0032] The plant part used to obtain the licorice extract is not particularly limited, and any of the whole licorice plant, leaves, stems, roots (rhizomes), flowers, seeds, etc. may be used.

[0033] Since glabridin, glabrene, and glabrol are hydrophobic, a hydrophobic licorice extract can be used as the licorice extract.

[0034] The licorice hydrophobic extract containing the polyphenol mixture may be an extract obtained by extracting hydrophobic components from licorice. Examples of the licorice hydrophobic extract include an extract containing a licorice extracting solvent, a concentrate or dried product of the extract obtained by removing part or all of the extracting solvent from the extract, or a processed product of the extract, concentrate, or dried product. Examples of the processed product include a dilution of the extract, concentrate, or dried product, and a processed product obtained by concentrating or purifying (including crude purification) the polyphenols from the extract, concentrate, or dried product to increase the concentration of the polyphenols. The method for obtaining the licorice hydrophobic extract from licorice is not particularly limited. For example, the hydrophobic components can be obtained by extraction with an organic solvent from licorice root, its powder, or cultured licorice cells. Alternatively, the hydrophilic components of licorice root can be extracted and removed using water or an alkaline aqueous solution, and then the hydrophobic components of the licorice root can be extracted with an organic solvent from the licorice residue or the dried residue. Alternatively, the hydrophobic extract obtained by the above method can be further extracted with a different organic solvent.

[0035] The organic solvent used as the extraction solvent is preferably one approved for use in the production and processing of pharmaceuticals, foods, food additives, etc. Examples include organic solvents such as alcohols (e.g., ethanol), esters (e.g., ethyl acetate), ketones (e.g., acetone), and hydrocarbons (e.g., hexane), as well as oils and fats (e.g., medium-chain fatty acid triglycerides). The organic solvent is preferably an alcohol, ketone, or oil and fat, and specifically, ethanol, acetone, medium-chain fatty acid triglycerides, etc. The organic solvent may be used alone or in a mixture of two or more. Water-containing solvents of these organic solvents may also be used. However, in order to keep the content of glycyrrhizinic acid (glycyrrhizin), which will be described later, low, it is preferable that the extraction solvent have a low water content.

[0036] The licorice hydrophobic extract may be used as it is after extraction using an organic solvent, or may be crudely or purified by further purification steps such as column treatment, deodorization treatment, decolorization treatment, etc.

[0037] The content of polyphenols in the licorice hydrophobic extract is not particularly limited, but is preferably 50% by weight or more, more preferably 60% by weight or more, and more preferably 70% by weight or more.

[0038] <Transthyretin tetramer stabilizer> The transthyretin tetramer stabilizer according to the present invention has the effect of stabilizing the transthyretin tetramer and inhibiting the monomerization of transthyretin. By stabilizing the transthyretin tetramer, the amyloid formation of transthyretin can be inhibited. The present inventors have found that the polyphenol mixture has a significantly higher transthyretin tetramer-stabilizing effect than glabridin alone, which has previously been known to have the effect of stabilizing the transthyretin tetramer.

[0039] The origin of the transthyretin to be stabilized is not particularly limited, but it is usually a mammal, preferably a human. The amino acid sequence of transthyretin may be wild-type or may contain a mutation.

[0040] In one embodiment, the transthyretin tetramer stabilizer of the present invention is administered to a subject as a pharmaceutical, food or drink, or the like, or is ingested by the subject, and can be used to stabilize transthyretin tetramers in the subject's body, for example, in the blood or cerebrospinal fluid.

[0041] Another embodiment of the present invention is Administering a polyphenol mixture comprising glabridin, glabrene, and glabrol to a subject in need of transthyretin tetramer stabilization; and inhibiting monomerization of the transthyretin tetramer and / or amyloid fibril formation from the transthyretin tetramer in the subject; a method for stabilizing a transthyretin tetramer in said subject, comprising: Regarding.

[0042] The transthyretin tetramer stabilizer and the method for stabilizing a transthyretin tetramer according to the present invention are intended for humans or non-human animals requiring stabilization of the transthyretin tetramer, preferably humans. Non-human animals include, for example, farmed animals, pet animals, and sports animals. Farmed animals include, but are not limited to, livestock such as horses, cows, pigs, sheep, goats, camels, and llamas; laboratory animals such as mice, rats, guinea pigs, and rabbits; and poultry such as chickens, ducks, turkeys, and ostriches. Pet animals include, but are not limited to, dogs and cats. Sports animals include, but are not limited to, racehorses. It is particularly preferred that the non-human animal is a mammal.

[0043] The frequency and dosage of administration of the transthyretin tetramer stabilizer according to the embodiment to a subject may be adjusted as appropriate depending on the age, sex, condition, etc. of the subject. The daily dosage of the transthyretin tetramer stabilizer may be adjusted as appropriate, and may be, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 30 mg / kg body weight, per day in terms of the total amount of polyphenols. The number of administrations per day may also be adjusted as appropriate, and may be, for example, one or more times, two or more times, or five or fewer times. The above-mentioned dosage and dosage frequency examples are particularly preferred when the subject is an adult. The route of administration may be oral or parenteral, with oral administration being preferred.

[0044] In another embodiment, the transthyretin tetramer stabilizer of the present invention can be used to stabilize a transthyretin tetramer in an ex vivo sample containing the transthyretin tetramer, such as a sample derived from a body fluid containing the transthyretin tetramer, including blood and cerebrospinal fluid, or a solution containing the transthyretin tetramer.

[0045] Yet another embodiment of the present invention comprises: contacting a polyphenol mixture containing glabridin, glabrene, and glabrol with transthyretin tetramer present in vitro; and inhibiting the monomerization of the transthyretin tetramer and / or the formation of amyloid fibrils from the transthyretin tetramer; A method for stabilizing transthyretin tetramers in vitro, comprising: Regarding.

[0046] The transthyretin tetramer stabilizer may be any composition containing the polyphenol mixture, or may be a composition further containing one or more other components (for example, a composition or pharmaceutical for stabilizing the transthyretin tetramer). Preferred forms of the transthyretin tetramer stabilizer are described below.

[0047] <Agents for preventing or slowing the progression of transthyretin amyloidosis> When administered to a subject in need of prevention or inhibition of the progression of transthyretin amyloidosis, the agent for preventing or inhibiting the progression of transthyretin amyloidosis according to the present invention stabilizes the transthyretin tetramer and inhibits transthyretin monomerization, thereby preventing or inhibiting the progression of transthyretin amyloidosis. The present inventors have found that the polyphenol mixture has a significantly higher transthyretin tetramer-stabilizing effect than glabridin alone, which has traditionally been known to have the effect of stabilizing the transthyretin tetramer, and therefore has a significantly higher activity in preventing or inhibiting the progression of transthyretin amyloidosis.

[0048] Another embodiment of the present invention is Administering a polyphenol mixture containing glabridin, glabrene, and glabrol to a subject in need of prevention or inhibition of progression of transthyretin amyloidosis; and inhibiting monomerization of transthyretin tetramers and / or amyloid fibril formation from transthyretin tetramers in the subject; a method for preventing or inhibiting the progression of transthyretin amyloidosis in a subject, comprising: Regarding.

[0049] In one embodiment, the subject of the agent for preventing or inhibiting the progression of transthyretin amyloidosis according to the present invention and the method for preventing or inhibiting the progression of transthyretin amyloidosis according to the present invention is a human or non-human animal in need of prevention or inhibition of the progression of transthyretin amyloidosis, preferably a human. Specific examples of the subject are as described above for the transthyretin tetramer stabilizer.

[0050] The administration frequency and dosage of the transthyretin amyloidosis preventive or progression inhibitor according to the above-described embodiment to a subject may be adjusted as appropriate depending on the subject's age, sex, condition, etc. The daily dosage of the transthyretin amyloidosis preventive or progression inhibitor may be adjusted as appropriate, and may be, for example, 0.01 to 100 mg / kg body weight, preferably 0.1 to 30 mg / kg body weight, per day in terms of the total amount of polyphenols. The number of administrations per day may also be adjusted as appropriate, and may be, for example, one or more and five or fewer times. The above-described dosage and administration frequency examples are particularly preferred when the subject is an adult. The route of administration may be oral or parenteral, with oral administration being preferred.

[0051] The agent for preventing or suppressing the progression of transthyretin amyloidosis may contain the polyphenol mixture, and may also be a composition containing one or more other components (for example, a composition or pharmaceutical for preventing or suppressing the progression of transthyretin amyloidosis). Preferred forms of the agent for preventing or suppressing the progression of transthyretin amyloidosis will be described later.

[0052] Examples of transthyretin amyloidosis that can be prevented or progress inhibited include senile systemic amyloidosis and familial amyloid polyneuropathy.

[0053] <Preferred embodiments of the transthyretin tetramer stabilizer and the agent for preventing or suppressing the progression of transthyretin amyloidosis> In the following description, the transthyretin tetramer stabilizer and the agent for preventing or suppressing the progression of transthyretin amyloidosis are collectively referred to as the "agent of the present invention."

[0054] The agent of the present invention may contain the polyphenol mixture. It may consist solely of the polyphenol mixture, or it may be a composition containing the polyphenol mixture and one or more other ingredients. The one or more other ingredients may be, for example, one or more ingredients acceptable for use in foods (regular foods, foods for specified health uses, foods with functional claims, dietary supplements, etc.), pharmaceuticals (human pharmaceuticals or non-human animal pharmaceuticals), quasi-drugs, cosmetics, or feed (livestock feed or pet food). Examples of the one or more other ingredients acceptable for use in foods, pharmaceuticals, quasi-drugs, cosmetics, or feed include the ingredients other than the polyphenol mixture that may be contained in the agent of the present invention, as described below.

[0055] The total content of glabridin, glabrene, and glabrol, or glabridin, glabrene, glabrol, and 4'-O-methylglabridin in the agent of the present invention is not particularly limited, but the lower limit is 0.1 wt% or more, preferably 1 wt% or more, more preferably 4 wt% or more, 5 wt% or more, 9 wt% or more, or 10 wt% or more. The upper limit of the total content in the agent of the present invention is not particularly limited, and a higher content is preferable, but from the viewpoint of containing necessary amounts of other active ingredients, it is preferably 99 wt% or less, more preferably 90 wt% or less.

[0056] The content of glabridin in the agent of the present invention is not particularly limited, but the lower limit is preferably 0.1% by weight or more, 1% by weight or more, or 4% by weight or more. The upper limit is also not particularly limited, but is preferably 90% by weight or less, 85% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less, from the viewpoint of mixing with other polyphenols and components.

[0057] When the agent of the present invention is ingested by or administered to a subject, the agent of the present invention preferably contains 0.40 to 4000 mg, more preferably 4 mg to 1200 mg, of glabridin per intake or administration unit. When the agent of the present invention is a food product, a single intake or administration unit refers to the amount ingested in one sitting. For example, when the agent of the present invention is packaged in a bottle or can in a single-serving form, or when the agent is packaged individually, a single intake or administration unit refers to one package unit. When the agent of the present invention is a pharmaceutical product, a single intake or administration unit refers to a recommended single dose.

[0058] In a preferred embodiment of the agent of the present invention, the content of glycyrrhizinic acid (also called glycyrrhizin) is preferably equal to or less than the content of polyphenols in the composition, and even more preferably equal to or less than the content of glabridin, by weight. Depending on the extraction conditions, licorice hydrophobic extract may contain components other than polyphenols, such as the hydrophilic component glycyrrhizin. In agents of the present invention containing licorice hydrophobic extract as a polyphenol mixture, the content of glycyrrhizin is preferably low from the viewpoint of safety when ingested or administered over a long period of time. Preferably, the agent of the present invention is substantially free of glycyrrhizin or has a low content of glycyrrhizin, for example, 0.005% by weight or less, preferably 0.001% by weight or less.

[0059] The agent of the present invention may further contain a medium-chain fatty acid triglyceride. From the viewpoint of ease of handling, the polyphenol mixture or the licorice hydrophobic extract containing the polyphenol mixture is preferably dissolved in a medium-chain fatty acid triglyceride before use. The medium-chain fatty acid triglyceride used in this case is not particularly limited as long as it is composed of fatty acids having 6 to 12 carbon atoms. However, triglycerides composed of saturated fatty acids having 8 or 10 carbon atoms are preferred, and triglycerides composed mainly of saturated fatty acids having 8 carbon atoms are more preferred. The proportion of fatty acids constituting the medium-chain fatty acid triglyceride is not particularly limited. However, the proportion of fatty acids having 8 to 10 carbon atoms is preferably 50% by weight or more, more preferably 70% by weight or more. Furthermore, medium-chain fatty acid triglycerides having a specific gravity of 0.94 to 0.96 at 20°C and a viscosity of 23 to 28 cP at 20°C are particularly preferred. These medium-chain fatty acid triglycerides may be naturally derived or may be prepared by transesterification or the like.

[0060] The medium-chain fatty acid triglyceride may be a glycerin fatty acid ester containing a medium-chain fatty acid triglyceride, preferably a glycerin fatty acid ester containing 50% by weight or more of a medium-chain fatty acid triglyceride, and more preferably a glycerin fatty acid ester containing 70% by weight or more of a medium-chain fatty acid triglyceride.

[0061] Furthermore, the agent of the present invention may further contain a partial glyceride in addition to the medium-chain fatty acid triglyceride, or a partial glyceride of a medium-chain fatty acid may be used instead of the medium-chain fatty acid triglyceride. The partial glyceride is a glycerin fatty acid ester containing a partial glyceride, preferably a glycerin fatty acid ester containing 50% by weight or more of a partial glyceride, and more preferably a glycerin fatty acid ester containing 70% by weight or more of a partial glyceride. The partial glyceride here refers to a diglyceride (1,2-diacylglycerol, 1,3-diacylglycerol) or a monoglyceride (1-monoacylglycerol, 2-monoacylglycerol), either of which may be used, or a mixture of two or more of them may be used. From the viewpoint of processability, a diglyceride is preferred. The partial glyceride may be naturally derived or may be prepared by transesterification or the like. The fatty acid residue constituting the partial glyceride is exemplified by those having 4 to 24 carbon atoms, with medium-chain fatty acid residues having 8 to 10 carbon atoms being particularly preferred, and saturated fatty acids, unsaturated fatty acids, etc. can be selected depending on the application. For example, unsaturated fatty acids are preferred when fluidity is required, and saturated fatty acids are preferred when plasticity is required. Branched fatty acids such as isostearic acid can also be used.

[0062] In addition to the polyphenol mixture, the agent of the present invention may contain other ingredients used in formulation, such as excipients, disintegrants, lubricants, binders, antioxidants, colorants, anti-aggregating agents, absorption enhancers, solubilizers for active ingredients, stabilizers, oils and fats, and viscosity adjusters.

[0063] The excipient is not particularly limited, but examples thereof include sucrose, lactose, glucose, corn starch, mannitol, crystalline cellulose, calcium phosphate, calcium sulfate, magnesium sulfate, and the like.

[0064] The disintegrant is not particularly limited, and examples thereof include starch, agar, calcium citrate, calcium carbonate, sodium hydrogen carbonate, dextrin, crystalline cellulose, carboxymethyl cellulose, tragacanth, and the like.

[0065] The lubricant is not particularly limited, but examples thereof include talc, magnesium stearate, polyethylene glycol, silica, and hydrogenated vegetable oil.

[0066] The binder is not particularly limited, but examples thereof include ethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, tragacanth, shellac, gelatin, gum arabic, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, and sorbitol.

[0067] The antioxidant is not particularly limited, but examples thereof include ascorbic acid, tocopherol, sodium hydrogen sulfite, sodium thiosulfate, sodium pyrosulfite, and citric acid.

[0068] The coloring agent is not particularly limited, but examples thereof include those permitted to be added to pharmaceuticals and foods.

[0069] The anti-aggregating agent is not particularly limited, but examples thereof include stearic acid, talc, light anhydrous silicic acid, and hydrous silicic acid dioxide.

[0070] The absorption enhancer is not particularly limited, but examples thereof include surfactants such as higher alcohols, higher fatty acids, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyglycerin fatty acid esters.

[0071] The solubilizing agent for the active ingredient is not particularly limited, but examples thereof include organic acids such as fumaric acid, succinic acid, and malic acid.

[0072] The stabilizer is not particularly limited, but examples thereof include benzoic acid, sodium benzoate, ethyl parahydroxybenzoate, and propylene glycol.

[0073] The oil and fat component is not particularly limited, and examples thereof include vegetable oils such as corn oil, rapeseed oil, high eruption rapeseed oil, soybean oil, olive oil, safflower oil, cottonseed oil, sunflower oil, rice bran oil, perilla oil, perilla oil, linseed oil, evening primrose oil, cacao butter, peanut oil, palm oil, and palm kernel oil; animal oils such as fish oil, beef tallow, lard, milk fat, and egg yolk oil; oils and fats obtained by fractionating, hydrogenating, interesterifying, or the like using these as raw materials; and mixed oils thereof.

[0074] The viscosity modifier is not particularly limited, but examples thereof include beeswax, Japan wax, lanolin, microcrystalline wax, and liquid paraffin.

[0075] The agent of the present invention may be in the form of a food (ordinary food, food for specified health uses, food with functional claims, dietary supplement, etc.), a pharmaceutical (a pharmaceutical for human use or a pharmaceutical for non-human animals), a quasi-drug, a cosmetic, or a feed (livestock feed or pet food), and is preferably in the form of a food or a pharmaceutical.

[0076] When the agent according to the present invention is in the form of a food, pharmaceutical, quasi-drug, feed, or feedstuff, it may be in the form of an orally ingested preparation. Examples of orally ingested preparations include tablets, capsules (hard capsules, microcapsules, soft capsules), granules, powders, chewable preparations, syrups, and liquids that can be orally ingested. When a capsule is prepared, the capsule base material is not particularly limited, and may include gelatin derived from cow bone, cowhide, pig skin, fish skin, or the like, as well as other base materials such as carrageenan, which can be used as a food additive, seaweed-derived products such as alginic acid, plant seed-derived products such as locust bean gum and guar gum, microbial-derived products such as pullulan and curdlan, and manufacturing agents containing cellulose.

[0077] The agent according to the present invention may be in the form of a common food, including, but not limited to, beverages such as milk drinks, soft drinks, energy drinks, and beauty drinks, confectioneries such as chewing gum, chocolate, candy, jelly, cakes, biscuits, and crackers, frozen desserts such as ice cream and frozen desserts, noodles such as udon, Chinese noodles, spaghetti, and instant noodles, fish paste products such as kamaboko, chikuwa, and half pieces, seasonings such as dressing, mayonnaise, and sauce, bread, ham, rice porridge, cooked rice, soup, various retort foods, and various frozen foods.

[0078] When the agent of the present invention is in the form of a food other than a pharmaceutical that can be taken for health maintenance, such as a food for specified health uses, a food with functional claims, or a dietary supplement, the agent of the present invention may be packaged, and the package may be labeled with a function related to stabilizing transthyretin tetramers or preventing or inhibiting the progression of transthyretin amyloidosis. The package is not particularly limited, and examples thereof include a box, a container, packaging film, and wrapping paper. Furthermore, the function to be labeled on the package may be expressed differently as long as it is a similar function.

[0079] Furthermore, the agent according to the present invention may be in the form of a parenteral preparation. For example, it may be in a form to be applied directly to the skin. In this case, the dosage form is not particularly limited, and examples include those in the form of creams, pastes, jellies, gels, emulsions, or liquids obtained by dissolving or mixing and dispersing the above-mentioned components in a suitable base (ointments, liniments, lotions, sprays, etc.), those in which the above-mentioned composition is dissolved or mixed and dispersed in a base and then spread on a support (poultices, etc.), and those in which the above-mentioned composition is dissolved or mixed and dispersed in an adhesive and then spread on a support (plasters, tapes, etc.).

[0080] When the agent according to the present invention is in the form of a quasi-drug, the quasi-drug refers to a quasi-drug defined in the "Act on Securing Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc.", and examples thereof include oral preparations (liquid preparations such as extracts, elixirs, syrups, tinctures, and lemonades, and solid preparations such as capsules, granules, pills, powders, and tablets). [Example]

[0081] Example 1 <Sample preparation> 63.9 g of an ethanol solution containing 135 mg of glabrene, 921 mg of glabridin, 184 mg of glabrol, and 160 mg of 4'-O-methylglabridin was mixed with 18.8 g of medium-chain triglyceride (Actar M2; Riken Vitamin Co., Ltd., fatty acid composition C8:C10 = 99:1), and the ethanol was removed by vacuum concentration. The resulting 28.7 g of solution was filtered by suction to remove the insoluble matter, which was then washed with hexane. The resulting oil was added to the filtrate. 4.5 g of medium-chain triglyceride was added to 26.2 g of the recovered filtrate to obtain 30.7 g of a polyphenol mixture-containing medium-chain triglyceride solution (containing 1.4 g of the polyphenol mixture).

[0082] The polyphenol content in the medium-chain triglyceride solution was measured by the Folin-Denis polyphenol analysis method using glabridin (a commercially available reagent) as a standard substance. The glycyrrhizin content was confirmed to be 0.005% by weight or less using the following analytical method.

[0083] (HPLC conditions for glycyrrhizinic acid analysis) Column: YMC J'sphere ODS-H80, inner diameter 4.6 mm x length 250 mm (YMC Co., Ltd.) Column temperature: 40℃ Mobile phase A: Acetonitrile Mobile phase B: 20mM phosphoric acid aqueous solution Gradient: The ratio of mobile phase A to the total amount of mobile phase A and mobile phase B is kept constant at 36% for 10 minutes from the start of analysis, then increased at a constant rate to 45% after 10 minutes and 50 minutes, then kept constant at 100% from 50 minutes to 55 minutes, and then kept constant at 36% from 55 minutes to 75 minutes. Flow rate: 1mL / min Wavelength: UV254nm Sample injection volume: 20 μL

[0084] <Analysis of peak intensity of each polyphenol> 1 g of the polyphenol mixture-containing medium-chain fatty acid triglyceride solution was dissolved in HPLC-grade methanol, and the total volume was adjusted to 100 mL. (HPLC conditions for polyphenol analysis) Column: YMC J'sphere ODS-H80, inner diameter 4.6 mm x length 250 mm (YMC Co., Ltd.) Column temperature: 40℃ Mobile phase A: acetonitrile:methanol (1:1 = v / v) Mobile phase B: 20mM phosphoric acid aqueous solution Gradient: The ratio of mobile phase A to the total volume of mobile phase A and mobile phase B is kept constant at 50% (v / v) for 20 minutes from the start of analysis, then increased at a constant rate to 80% (v / v) after 20 minutes and 75 minutes, then kept constant at 100% (v / v) from 75 minutes to 80 minutes, and then kept constant at 50% (v / v) from 80 minutes to 100 minutes. Flow rate: 1.0mL / min Wavelength: UV282nm Sample injection volume: 20 μL

[0085] <Analysis results> In HPLC analysis under the above conditions using the polyphenol mixture-containing medium-chain fatty acid triglyceride solution as a sample, the peak intensity (peak area) of glabridin was 100%, and the peak intensity of glabrene, glabrol, and 4'-O-methylglabridin were 38%, 44%, and 19%, respectively. The contents of each component per gram of the polyphenol mixture-containing medium-chain triglyceride solution were 4.4 mg of glabrene, 30.0 mg of glabridin, 6.0 mg of glabrol, and 5.2 mg of 4'-O-methylglabridin. The contents of each component were determined using calibration curves prepared from the HPLC analysis results of commercially available standards of known concentrations of each component.

[0086] <Polyphenol content analysis> The polyphenol content was measured by the Folin-Denis method using glabridin (a commercially available reagent) as a standard substance, and the total polyphenol content in 1 g of the polyphenol mixture-containing medium-chain fatty acid triglyceride solution was found to be 239.1 mg.

[0087] Example 2 <Sample preparation> 64.0 g of each of the polyphenol mixture-containing ethanol solutions 1 to 4, which were prepared so that the peak intensities (peak areas) of glabrene, glabrol, and 4'-O-methylglabridin relative to glabridin in HPLC analysis under the above conditions were as shown in the table below, was mixed with 19.0 g of medium-chain triglyceride, and the ethanol was removed by vacuum concentration. 4.5 g of medium-chain triglyceride was then added to the vacuum concentrate to obtain a polyphenol mixture-containing medium-chain triglyceride solution.

[0088] [Table 1]

[0089] Example 3 <Transthyretin tetramer stabilization test 1> Blood samples were collected from three middle-aged and elderly individuals to obtain serum samples. The polyphenol mixture-containing medium-chain fatty acid triglyceride solution and glabridin obtained in Example 1 were added to each serum sample (each at a concentration of 0, 10, or 50 μM as glabridin), incubated at 25°C for 30 minutes, and then denatured with urea. Using these samples, transthyretin (tetramer, dimer, and monomer) in the blood was quantified by electrophoresis and Western blotting. The stabilizing effect was assessed by the ratio of monomer to total transthyretin (monomer / total) and the ratio of tetramer to total transthyretin (tetramer / total). The left side of Figure 1 shows the results of the analysis of monomer / total. The right side of Figure 1 shows the results of the analysis of tetramer / total.

[0090] As shown in Figure 1, when glabridin was added alone, the ratio of monomer to total transthyretin did not change, and the ratio of tetramer to total transthyretin did not increase, up to an added concentration of 50 μM, and no transthyretin stabilizing effect was observed. On the other hand, when a medium-chain fatty acid triglyceride solution containing a polyphenol mixture was added, the ratio of monomer to total transthyretin decreased and the ratio of tetramer to total transthyretin increased (transthyretin stabilizing effect) at the maximum concentration.

[0091] Example 4 <Transthyretin tetramer stabilization test 2> Three healthy subjects ingested the polyphenol mixture-containing medium-chain fatty acid triglyceride solution obtained in Example 1 for four weeks (600 mg / day intake), and blood samples were collected over time to obtain plasma. Each plasma sample was incubated at 25°C for 30 minutes and then denatured with urea. Using these samples, transthyretin (tetramer and monomer) in the blood was quantified by electrophoresis and Western blotting, and the stabilizing effect was examined based on the ratio of monomer to tetramer (monomer / tetramer). The results are shown in Figure 2. As shown in Figure 2, the ratio of monomers to tetramers in the medium-chain fatty acid triglyceride solution containing the polyphenol mixture decreased after two weeks, and specifically, the effect of decreasing the proportion of monomers and increasing the proportion of tetramers (transthyretin stabilization) was observed. From the above, it can be seen that the polyphenol mixture can stabilize the transthyretin tetramer, reduce the proportion of the monomer contained in the body, and increase the tetramer. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A transthyretin tetramer stabilizer comprising glabridin, glabrene, glabrol and 4'-O-methylglabridin.

2. In HPLC analysis under the following condition a), the properties b), c) and d) were confirmed: a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B); column: ODS column; flow rate: 1.0 mL / min; temperature: 40°C; detector: UV detector; detection wavelength: 282 nm; b) the ratio of the glabrene peak intensity to the glabridin peak intensity is 38% or more and 41% or less; c) the ratio of the glabridin peak intensity to the glaubrol peak intensity is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity to glabridin peak intensity is 15% or more and 20% or less; The agent according to claim 1, which exhibits any one or more of the following.

3. 3. The agent according to claim 1, wherein the content of glycyrrhizinic acid is 0.005% by weight or less.

4. An agent for preventing or suppressing the progression of transthyretin amyloidosis, comprising glabridin, glabrene, glabrol and 4'-O-methylglabridin.

5. The agent according to claim 4, wherein the transthyretin amyloidosis is senile systemic amyloidosis or familial amyloid polyneuropathy.

6. In HPLC analysis under the following condition a), the properties b), c) and d) were confirmed: a) Mobile phase: gradient of acetonitrile:methanol = 1:1 (mobile phase A) and 20 mM phosphoric acid (mobile phase B); column: ODS column; flow rate: 1.0 mL / min; temperature: 40°C; detector: UV detector; detection wavelength: 282 nm; b) the ratio of the glabrene peak intensity to the glabridin peak intensity is 38% or more and 41% or less; c) the ratio of the glabridin peak intensity to the glaubrol peak intensity is 44% or more and 47% or less; d) the ratio of 4'-O-methylglabridin intensity to glabridin peak intensity is 15% or more and 20% or less; The agent according to claim 4, which exhibits any one or more of the following.

7. The agent according to any one of claims 1 to 6, which is in the form of a tablet, capsule, granule or powder.

8. The agent according to any one of claims 1 to 7, wherein the content of glabridin per intake or administration unit is 4 mg to 1200 mg.

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