Use of l-erdosteine compound

Abstract

The present invention relates to a use of L-erdosteine and, more specifically, to a use of L-erdosteine in the production of a pharmaceutical agent for at least one selected from among: mucolysis and expectoration in acute and chronic respiratory diseases; treating or ameliorating upper respiratory tract inflammation; treating or preventing acute and chronic bronchitis; treating or preventing cough; treating or preventing asthma; treating or preventing pneumonia; preventing pulmonary fibrosis; preventing bronchial and alveolar destruction; treating or preventing chronic obstructive pulmonary disease; enhancing resistance to bacterial infections of the upper respiratory tract; and ameliorating side effects caused by smoking. The present invention rapidly increases the blood concentration of Met-I, which is a metabolically active form of erdosteine, thereby exhibiting a rapid and excellent effect without toxicity, and thus has the effect of achieving the same effect at a lower dose than existing racemic compounds. In addition, since a lower dose can be used as compared to existing racemic compounds, the present invention also has the effect of reducing expected potential side effects.

Description

Uses of L-eldostane compounds

[0001] The present invention relates to the use of L-eldostane.

[0002] Erdosteine ​​is a compound with the IUPAC name 2-((2-oxo-2-((2-oxotetrahydrothiophen-3-yl)amino)ethyl)thio)acetic acid, and its representative commercial product is Erdos™. Erdosteine ​​is a component with mucolytic and expectorant effects, and is widely used as a treatment for acute and chronic respiratory diseases, including bronchitis. In particular, eldosteine ​​is known to have effects that other expectorants do not, such as promoting ciliary movement, antibacterial effects, anti-inflammatory effects, antioxidant effects, and synergistic effects when used in combination with antibiotics. More specifically, it inhibits the oxidation of α1-antitrypsin induced by smoking, preventing the destruction of bronchial alveoli, and increases physiological antibacterial substances in the body, such as immunoglobulin A and lysozyme, which are respiratory immune substances, to strengthen resistance to bacteria. It is also known to be effective for chronic obstructive pulmonary disease (COPD). When administered orally, eldosteine ​​is absorbed in the small intestine, metabolized in the liver, and excreted through the kidneys (absorption is not affected by food, and the maximum blood concentration is reached in about 1.4 hours, with a maximum blood concentration of about 2.5 ug / ml). In other words, eldosteine ​​is a type of prodrug that is activated by metabolism by liver enzymes after absorption. There are three known metabolites of eldostane: Met-Ⅰ, Met-Ⅱ, and Met-Ⅲ. Among them, the pharmacologically important active metabolite is the Met-Ⅰ (also called Metabolite-1, Metabolite-Ⅰ) compound as shown in the chemical formula Ⅰ below (including L-form, D-form, and their racemates centered on the missing carbon).

[0003] [Chemical Formula I]

[0004]

[0005] The Met-Ⅰ compound has two blocked sulfhydryl groups that are released after first-pass metabolism, which regulate mucus production and viscosity and increase mucus discharge. In particular, the free thiol group can exhibit an inhibitory effect on the action of free radicals generated by cigarette smoke, etc., and is recognized as the actual active substance of eldostane. Therefore, if the blood concentration of Met-I, the active substance of eldostane, can be increased rapidly and to a high concentration, it will serve as a highly desirable precursor drug. However, eldostane, which is currently commonly used, is a racemic compound consisting of a mixture of the D-form and the L-form, and is used as a drug without separating the enantiomers of the racemic compound.

[0006] [Prior Art Literature]

[0007] (Non-patent Document 1) Mario Cazzola et al., “Multifaceted Beneficial Effects of Erdosteine: More than a Mucolytic Agent”, Drugs (2020) 80:1799-.1809, https: / doi.org / 10.1007 / s40265-020-01412-x

[0008] The present invention provides an L-eldosteine ​​compound and its use, which exhibits excellent effects by rapidly and highly increasing the blood concentration of Met-I, an active metabolite, while being non-toxic and capable of producing the same effects at a lower dose than existing racemic compounds.

[0009] The present invention has been devised to solve the problems of the above-described prior art.

[0010] The present invention provides a use of an L-eldostane compound represented by the following chemical formula 1 in the manufacture of a medicament for at least one selected from the group consisting of dissolving mucus and expectorating phlegm in acute and chronic respiratory diseases, treating or improving upper respiratory tract inflammation, treating or preventing acute and chronic bronchitis, treating or preventing cough, treating or preventing asthma, treating or preventing pneumonia, preventing pulmonary fibrosis, preventing destruction of bronchial alveoli, treating or preventing chronic obstructive pulmonary disease, increasing resistance to upper respiratory tract infection bacteria, and improving side effects caused by smoking.

[0011] [Chemical Formula 1]

[0012]

[0013] In addition, the present invention provides a use of an L-eldosteine ​​compound as a prodrug of a compound represented by the following chemical formula 2.

[0014] [Chemical Formula 2]

[0015]

[0016] In addition, the present invention provides a pharmaceutical composition comprising an L-eldosteine ​​compound as an active ingredient and having at least one use selected from the group consisting of mucus dissolution and expectoration in acute and chronic respiratory diseases, treatment or improvement of upper respiratory tract inflammation, treatment or prevention of acute and chronic bronchitis, treatment or prevention of cough, treatment or prevention of asthma, treatment or prevention of pneumonia, prevention of pulmonary fibrosis, prevention of destruction of bronchial alveoli, treatment or prevention of chronic obstructive pulmonary disease, increased resistance to upper respiratory tract infection bacteria, and improvement of side effects caused by smoking.

[0017] In addition, a pharmaceutical composition characterized in that it comprises an L-eldosteine ​​compound in a daily dosage of 10 to 200 mg / kg is provided.

[0018] In addition, a pharmaceutical composition is provided, characterized by comprising 50 to 200 mg of an L-eldosteine ​​compound and a pharmaceutically acceptable excipient.

[0019] The present invention rapidly increases the blood concentration of Met-I, a metabolically active form of eldostane, resulting in rapid and superior efficacy. Furthermore, it is non-toxic and can achieve the same effect at lower doses than conventional racemic compounds. Furthermore, because lower doses are required compared to conventional racemic compounds, it can also reduce potential side effects.

[0020] Figure 1 is a photograph showing the results of a solubility test of the L-form, D-form, and racemate of eldostane.

[0021] Figure 2 is a graph showing the results of microsomal and S9 stability assay tests.

[0022] Figures 3 to 5 show the results of animal tests on pharmacokinetics after administration of L-Erdosteine ​​and Racemic-Erdosteine.

[0023] Figures 6 to 9 show the results of evaluating the bronchial relaxation effect when Racemic-Erdosteine ​​and L-Erdosteine ​​were applied to the bronchial tubes extracted from guinea pigs.

[0024] Figures 10 and 11 show the results of cytotoxicity and MUC5AC mRNA expression level evaluations, respectively, in the expectorant effect test of L-Erdosteine ​​and Racemic-Erdosteine.

[0025] Figures 12 and 13 show the histopathological examination section photographs and the analysis results thereof, respectively, in a test for the inhibitory effect of L-Erdosteine ​​and Racemic-Erdosteine ​​on lung inflammation.

[0026] Figure 14 shows the results of ROMO1 analysis in a test of the respiratory inflammation alleviation effect of L-Erdosteine ​​and Racemic-Erdosteine.

[0027] Hereinafter, the present invention will be described in detail.

[0028] In the present invention, the compound includes not only the compound itself, but also a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable hydrate or solvate thereof, and a pharmaceutically acceptable hydrate or solvate of a pharmaceutically acceptable salt thereof.

[0029] One aspect of the present invention is

[0030] It relates to the use of L-eldostane compounds.

[0031] The L-eldosteine ​​compound is a compound represented by the following chemical formula 1, and there is no example of it being used for the prevention or treatment of a disease.

[0032] [Chemical Formula 1]

[0033]

[0034] That is, although only D,L-eldostane (racemic-eldostane) has been used in the past for the prevention or treatment of diseases, there has been no example of the use of only L-eldostane compounds. Accordingly, the present invention provides a use of L-eldostane compounds for the prevention or treatment of diseases.

[0035] The use for preventing or treating the above diseases may include at least one selected from the group consisting of mucus dissolution and expectoration in acute and chronic respiratory diseases, treatment or improvement of upper respiratory tract inflammation, treatment or prevention of acute and chronic bronchitis, treatment or prevention of cough, treatment or prevention of asthma, treatment or prevention of pneumonia, prevention of pulmonary fibrosis, prevention of destruction of bronchial alveoli, treatment or prevention of chronic obstructive pulmonary disease, enhancement of resistance to upper respiratory tract infection bacteria, and improvement of side effects caused by smoking.

[0036] In addition, L-eldosteine ​​may be a kind of prodrug for an active metabolite that is activated by metabolism by liver enzymes after absorption. It may be a prodrug of a Met-Ⅰ compound represented by the following chemical formula 2, which is recognized as a practical active substance of eldosteine, such as having two blocked sulfhydryl groups that are released after first-pass metabolism in the liver, thereby regulating mucus production and viscosity and increasing mucus excretion, and in particular, a free thiol group that can exhibit an inhibitory effect on the action of free radicals generated by cigarette smoke, etc.

[0037] [Chemical Formula 2]

[0038]

[0039] Another aspect of the present invention is:

[0040] Contains L-eldostane compound as an active ingredient,

[0041] The present invention relates to a pharmaceutical composition for one or more uses selected from the group consisting of dissolving mucus and expectorating in acute and chronic respiratory diseases, treating or improving upper respiratory tract inflammation, treating or preventing acute and chronic bronchitis, treating or preventing cough, treating or preventing asthma, treating or preventing pneumonia, preventing pulmonary fibrosis, preventing destruction of bronchial alveoli, treating or preventing chronic obstructive pulmonary disease, increasing resistance to upper respiratory tract infection bacteria, and improving side effects caused by smoking.

[0042] The pharmaceutical composition may comprise an L-eldostane compound in a daily dosage of 10 to 200 mg / kg, preferably 15 to 150 mg / kg, more preferably 20 to 120 mg / kg, and even more preferably 25 to 100 mg / kg.

[0043] The pharmaceutical composition may comprise 50 to 200 mg of an L-eldosteine ​​compound and a pharmaceutically acceptable excipient. While conventional eldosteine ​​compounds have a single-administered dose of approximately 300 mg, the compound of the present invention may have a slightly reduced dose.

[0044] In addition, as can be understood through the examples and experimental examples described below, the L-eldostein compound has the characteristic of being converted into a metabolically active compound quickly and at a high concentration compared to the conventionally known D,L-eldostein compound (racemic-erdostein).

[0045] The L-eldosteine ​​compound of the present invention can be administered orally, parenterally, or locally as follows.

[0046] Oral administration (PO)

[0047] The compounds of the present invention can be administered orally, i.e., administered orally, where the term "oral" includes swallowing. By oral administration, the compounds of the present invention can enter the gastrointestinal tract, or be absorbed directly into the bloodstream from the mouth, such as by buccal or sublingual administration. Suitable compositions for oral administration may be in the form of solids, liquids, gels, or powders, and may have dosage forms such as tablets, lozenges, capsules, granules, and powders. Compositions for oral administration may optionally be enteric-coated, and exhibit delayed or sustained release through the enteric coating. That is, compositions for oral administration according to the present invention may be formulations having an immediate or modified release pattern. Liquid formulations may include solutions, syrups, and suspensions, and these liquid compositions may be contained within soft or hard capsules. These formulations may include a pharmaceutically acceptable carrier, such as water, ethanol, polyethylene glycol, cellulose, or oil. The formulations may also include one or more emulsifying and / or suspending agents. In tablet formulations, the amount of the active ingredient, the drug, may be present in an amount of from about 0.05% to about 95% by weight of the total weight of the tablet, more typically from about 2% to about 50% by weight of the formulation. The tablets may also contain a disintegrant in an amount of from about 0.5% to about 35% by weight, more typically from about 2% to about 25% by weight of the formulation. Examples of disintegrants that can be used include, but are not limited to, lactose, starch, sodium starch glycolate, crospovidone, croscarmellose sodium, maltodextrin, or mixtures thereof.Suitable lubricants included for manufacturing into tablets may be present in an amount of about 0.1 wt% to about 5 wt%, and talc, silicon dioxide, stearic acid, calcium, zinc or magnesium stearate, sodium stearyl fumarate, etc. may be used as lubricants, but the present invention is not limited to the types of these additives. Binders for manufacturing into tablets may include gelatin, polyethylene glycol, sugar, gum, starch, polyvinylpyrrolidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc., and suitable diluents for manufacturing into tablets may include mannitol, xylitol, lactose, dextrose, sucrose, sorbitol, starch, microcrystalline cellulose, etc., but the present invention is not limited to the types of these additives. Solubilizers that can be optionally included in the tablet can be used in an amount of about 0.1 wt% to about 3 wt% based on the total weight of the tablet, and for example, polysorbate, sodium lauryl sulfate, sodium dodecyl sulfate, propylene carbonate, diethylene glycol monoethyl ether, dimethyl isosorbide, polyoxyethylene glycolated natural or hydrogenated castor oil, HCOR™ (Nikkol), oleyl ester, Gelucire™, caprylic / caprylic acid mono / diglycerides, sorbitan fatty acid esters, Solutol HS™, etc. can be used in the pharmaceutical composition according to the present invention, but the present invention is not limited to specific types of such solubilizers.

[0048] Parenteral Administration

[0049] The compounds of the present invention can be administered directly into the bloodstream, muscle, or viscera. Suitable methods for parenteral administration include intravenous, intramuscular, subcutaneous intraarterial, intraperitoneal, intrathecal, and intracranial injections. Suitable devices for parenteral administration include injectors (including needle and needleless syringes) and infusion methods. Compositions for parenteral administration can be formulations having an immediate or modified release pattern, and the modified release pattern can be a delayed or sustained release pattern. Most parenteral formulations are liquid compositions, which are aqueous solutions containing the active ingredient according to the present invention, a salt, a buffer, an isotonic agent, etc. Parenteral formulations can also be prepared in a dry form (e.g., lyophilized) or as sterile non-aqueous solutions. These formulations can be used with a suitable vehicle, such as sterile water. Solubility-enhancing agents can also be used in the preparation of parenteral solutions.

[0050] Topical Administration

[0051] The compounds of the present invention can be administered topically, through the skin or transdermally. Formulations for topical administration include lotions, solutions, creams, gels, hydrogels, ointments, foams, implants, patches, and the like. Pharmaceutically acceptable carriers for topical administration formulations can include water, alcohol, mineral oil, glycerin, polyethylene glycol, and the like. Topical administration can also be carried out by electroporation, iontophoresis, phonophoresis, and the like. Compositions for topical administration can be formulations having an immediate or modified release pattern, and the modified release pattern can be a delayed or sustained release pattern.

[0052] Hereinafter, the present invention will be described in more detail with examples and experimental tests. However, it should be made clear that the following examples and experimental tests are intended only for the purpose of detailed explanation of the invention and are not intended to limit the scope of the rights herein.

[0053] Example

[0054] [Manufactured by (R)-2-methoxy-N-((R)-2-oxotetrahydrothiophen-3-yl)-2-phenylacetamide]

[0055]

[0056] Add 36 g of D,L-homocysteine ​​thiolactone hydrochloride and 3.4 L of chloroform and cool to 5°C. Add 33.2 g of triethylamine to the reactor while maintaining it at 5°C. Add 38 g of 1-hydroxybenzotriazole, 43.3 g of S-(+)-(2)-methoxyphenylacetic acid, and 54 g of 1-(3-dimethylaminopyrrolidone)-3-ethylcarbodiimide trichloride to the reactor while maintaining it at 5°C. Stir for 7 hours and 30 minutes while maintaining it at 0 to 5°C, then add distilled water to terminate the reaction. Wash with 5% sodium bicarbonate solution, 2 M hydrochloric acid solution, and saturated sodium chloride solution, dry over magnesium sulfate, and concentrate under reduced pressure. The crude product was purified by column chromatography (silica, ethyl acetate / hexane).

[0057] [Manufactured by (R)-3-aminodihydrothiophen-2(3H)-one]

[0058]

[0059] (R)-2-methoxy-N-((R)-2-oxotetrahydrothiophene-3-yl)-2-phenylacetamide 20 g, ethanol: 4 M HCl = 2:1 solution 1.2 L were added at room temperature. The temperature was increased, refluxed and stirred for 25 hours, and then cooled to room temperature. Distilled water and toluene were added to remove impurities from the organic layer. The aqueous layer was concentrated under reduced pressure. The crude product was recrystallized using isopropyl alcohol to obtain 7.24 g (55.6%) of (R)-3-aminodihydrothiophene-2(3H)-one.

[0060] [Manufacturing of L-Erdosteine]

[0061]

[0062] (R)-3-aminodihydrothiophene-2(3H)-one 9g, distilled water 21.6ml, and acetone 18ml were added at room temperature.

[0063] Cool to 0~10℃, add 4.92g of sodium bicarbonate, maintain the temperature and stir for 30 minutes. Add 9.29g of thiodiglycolic acid to the reactor while maintaining the temperature at 0~10℃. Stir for 3 hours at 0~10℃ and filter to obtain L-Erdosteine ​​(yield 82%).

[0064] While the L-Erdosteine ​​manufacturing method has been described in detail above, more specific details can be understood by referring to the contents of Korean Patent No. 2583743. The entire contents of Korean Patent No. 2583743 are hereby incorporated by reference herein.

[0065] Experimental example

[0066] In the experimental example results below, L-ERD represents L-eldosteine, DL-ERD represents D,L-eldosteine, and D-ERD represents D-eldosteine, respectively.

[0067] Solubility test

[0068] Dimethyl sulfoxide (DMSO) and methanol (MeOH) were used as solvents, and D-eldostane showed a solubility that was approximately 90% reduced compared to L,D-eldostane or L-eldostane. Figure 1 is a photograph showing the results of the solubility test.

[0069] Microsomal and S9 stability assay tests

[0070] To determine metabolic stability, microsomal and S9 stability assays were performed. The results are shown in Figure 2. For reference, the concentration was 100 μM and the reaction temperature was 45°C. The reaction temperature did not affect Met-Ⅰ production.

[0071] When the microsomal stability assay was performed, drug metabolism did not occur, but when metabolites were confirmed, L-eldosteine ​​increased slightly.

[0072] In the S9 fraction, which is a mixture of microsomes and cytosols, drug metabolism of eldosteine ​​proceeded, and it was confirmed that L-eldosteine ​​was metabolized faster than D,L-eldosteine. Met-Ⅰ, which is also produced through drug metabolism, was also analyzed to be high in L-eldosteine.

[0073] In vivo and in vitro (S9), the drug metabolism of L-eldostane proceeds faster than that of D,L-eldostane, and it was confirmed that the production of Met-Ⅰ with -SH (sulfhydryl group) released after first-pass metabolism was also higher in L-eldostane.

[0074] The S9 fraction stability test mimics the natural environment of liver tissue, closely replicating reactions occurring within actual tissue. Furthermore, the S9 fraction itself contains various enzymes involved in drug metabolism, primarily CYP enzymes expressed in hepatocytes, allowing the metabolic process to be identified through chemical reactions. Accordingly, the inventors of the present invention conducted a comparative evaluation of the production of metabolites M1 of L-Erdosteine ​​and Racemic-Erdosteine ​​using Rat S9 metabolic stability.

[0075] The analytical batch was suitable, and the drug metabolism using the S9 fraction was confirmed, and it was observed that the L-Erdosteine ​​treatment group increased the production of Erdosteine ​​metabolite 1 (Met-Ⅰ) at the same concentration compared to the Racemic-Erdosteine ​​treatment group.

[0076] The metabolic stability assay evaluates the metabolic reaction of the target substance, such as creating a reaction environment and promoting the reaction through cofactors (NADPH, UDPGA). However, the test substances L-Erdosteine ​​and Racemic-Erdosteine ​​were observed to produce metabolites even when the cofactor was not treated. This suggests that the chemical reaction caused by the metabolic enzymes present in the S9 fraction itself is naturally induced and affects drug metabolism, and it was observed that the production of metabolites increased depending on the concentration and time of the test substance from 0 to 6 hours of reaction.

[0077] As a result of the above, in the rat S9 fraction stability assay, the test substance L-Erdosteine ​​is judged to be a compound that increases the formation of Erdosteine ​​metabolite 1 (Met-Ⅰ), a metabolically active substance, compared to Racemic-Erdosteine.

[0078] Pharmacokinetic Test I

[0079] This study was conducted to evaluate the pharmacokinetic changes of the active metabolite M1 after a single oral administration of L-Erdosteine ​​and Racemic-Erdosteine ​​to Sprague-Dawley rats (Experimental animal: SD (Sprague-Dawley) rat, Test substance: L-erdosteine, DL-erdosteine, Dose (mg / kg / day): 50 mg / kg, Excipient: 5% DMSO + saline, Blood collection method: Jugular vein blood collection (heparin)). The results were as shown in Fig. 3 and Table 1.

[0080] Parameters50㎎ / ㎏50㎎ / ㎏L-ERDDL-ERDL-ERD-Met-IDL-ERD-Met-IAUC last (ng·hr / ml)109226064633.42002.9AUC inf (ng·hr / ml)11732653.247392048.8C max(ng / ml)383.11251.61531.3690.3T max (hr)0.50.522

[0081] As can be seen from the results, L-eldostane (L-ERD) was metabolized faster than D,L-eldostane (DL-ERD), which is similar to the results of the in vitro assay described above. In addition, because L-eldostane was metabolized faster, the quantitative value of the metabolite after the first pass through the liver also appears to be higher for L-eldostane. For reference, when proceeding with 100 mg / kg of saline as an excipient, D,L-eldostane did not dissolve, so a combination of 5% DMSO and saline was used.

[0082] As can be seen from the in vitro and in vivo pharmacodynamic results, the compound of the present invention has a relatively high blood AUC of Met-Ⅰ (Metabolite 1), an active metabolite of eldodostane, compared to racemic or D-form eldodostane, and it can be seen that the blood and tissue concentrations of the active metabolite can be maintained with only a small amount of intake.

[0083] Accordingly, it can be seen that it can exhibit relatively high activity in respiratory organ tissues such as the bronchial tubes and lungs, which are the target tissues, and also, when it has the same blood or tissue concentration characteristics, it can exhibit sufficient efficacy even with a smaller intake, so it can be expected that there will be a reduction in side effects (e.g., gastrointestinal disorders, which are the most clinically known side effects of eldosteine) due to a reduction in the dosage used.

[0084] Pharmacokinetic Test II

[0085] This study was conducted to evaluate the pharmacokinetic changes of the active metabolite M1 after a single oral administration of L-Erdosteine ​​and Racemic-Erdosteine ​​to Sprague-Dawley rats (Experimental animal: SD (Sprague-Dawley) rat, Test substance: L-erdosteine, DL-erdosteine, Dose (mg / kg / day): 25-100 mg / kg, Excipient: 5% DMSO + saline, Blood collection method: Jugular vein blood collection (heparin)). The test groups were composed of the following groups: group administered 5 mg / kg IV L-Erdosteine ​​(G1), group administered 25 mg / kg PO Racemic-Erdosteine ​​(G2), group administered 50 mg / kg PO Racemic-Erdosteine ​​(G3), group administered 100 mg / kg PO Racemic-Erdosteine ​​(G4), group administered 25 mg / kg PO L-Erdosteine ​​(G5), group administered 50 mg / kg PO L-Erdosteine ​​(G6), and group administered 100 mg / kg PO L-Erdosteine ​​(G7). Six animals were used for each group. The test items included observation of dead animals, general symptoms, body weight measurement, and PK parameter analysis, and the test results were as shown in Fig. 4 and Tables 2 to 5.

[0086]

[0087]

[0088]

[0089]

[0090] No deaths were observed during the observation period, and no significant changes in body weight were observed in any group (G1-G7) during the observation period. As a result of bioavailability (BA) analysis of Erdosteine, the bioavailability of Racemic-Erdosteine ​​in the 25 mg / kg PO, 50 mg / kg PO, and 100 mg / kg PO administration groups was calculated to be 78.90%, 77.61%, and 53.89%, respectively, and the bioavailability of L-Erdosteine ​​in the 25 mg / kg PO, 50 mg / kg PO, and 100 mg / kg PO administration groups was calculated to be 19.52%, 14.40%, and 15.42%, respectively. As a result of bioavailability analysis of Erdosteine ​​metabolite 1, the bioavailability of Racemic-Erdosteine ​​in the 25 mg / kg PO, 50 mg / kg PO, and 100 mg / kg PO administration groups was calculated to be 52.90%, 63.93%, and 39.60%, respectively, and the bioavailability of L-Erdosteine ​​in the 25 mg / kg PO, 50 mg / kg PO, and 100 mg / kg PO administration groups was calculated to be 73.08%, 66.60%, and 92.30%, respectively.

[0091] In summary, L-Erdosteine ​​was observed to have a bioavailability that was about 1 / 4 lower than that of its parent Erdosteine, Racemic-Erdosteine, but the bioavailability of its active metabolite, metabolite 1, was observed to be up to 52.70% higher for L-Erdosteine ​​than for Racemic-Erdosteine. Therefore, it is believed that L-Erdosteine ​​is more effective than Racemic-Erdosteine ​​at the same dose in the treatment or prevention of various diseases, such as acute and chronic respiratory diseases.

[0092] Pharmacokinetic Test III

[0093] To examine differences in pharmacokinetic responses between species, separate pharmacokinetic studies were conducted (Experimental animals: BALB / c mouse, SD (Sprague-Dawley) rat; Test substances: L-erdosteine, DL-erdosteine; Dose (mg / kg / day): BALB / c mouse 12.5 mg / kg, SD (Sprague-Dawley) rat 100 mg / kg; Excipient: 5% DMSO + saline; Blood collection method: Jugular vein blood collection (heparin)). The results were as shown in Fig. 5.

[0094] Although there were differences in the shape of specific curves and half-life between BALB / c mice and SD (Sprague-Dawley) rats, there was no difference in the tendency for the concentration of the active metabolite Met-Ⅰ to increase rapidly compared to the racemate L-erdosteine.

[0095] Evaluation of the bronchial relaxing effects of racemic-erdosteine ​​and L-erdosteine

[0096] This study was conducted to evaluate the bronchial relaxation effect when the test substances Racemic-Erdosteine ​​and L-Erdosteine ​​were administered to the bronchial tubes isolated from guinea pigs.

[0097] Histamine and carbachol were used as contractile agents to measure the bronchial relaxation effect, and the treatment concentrations of Racemic-Erdosteine ​​and L-Erdosteine ​​were treated to the final concentrations of 0, 0.5, 1.5, and 3.5 mg / mL for the relaxation effect. The treatment concentration of SNP (Sodium nitroprusside), a control substance for the relaxation effect, was treated at 100 μM. Specific pathogen-free (SPF) guinea pigs Hartley (sex: male, age (at acquisition): 5, number (at acquisition): 15, number (at organ extraction): 7) were used as experimental animals, and the test substance was administered directly to the organ bath containing the extracted bronchus at the highest concentration that was easy to prepare. The contractile substance histamine (3.2 × 10 -5 M) and carbachol (2×10 -7 M) was treated and when the contraction height reached a plateau, the test substance and control substance were treated to observe the relaxation response, and the relaxation rate by the test substance was calculated based on the base tension and the tension when only the contraction substance was treated. The measurement results were compared between groups through Student's t-test, a parametric multiple comparison procedure, using SPSS Statistics 12.0K for medical science. A comparison was performed between the vehicle control group and the test substance administration group, and a comparison of the test substances Racemic-Erdosteine ​​and L-Erdosteine ​​was performed, and a P value of 0.05 or higher was judged to be significant. EC 50 The Probit method was used to determine the concentration. The test results are shown in Figures 6 to 9.

[0098] In histamine-induced airway constriction, a significant increase in relaxation rate was observed when treated with L-Erdosteine ​​3.5 mg / mL, and a trend toward increase was observed when treated with Racemic-Erdosteine ​​3.5 mg / mL. In carbachol-induced airway constriction, a significant increase in airway relaxation rate was observed when treated with Racemic-Erdosteine ​​and L-Erdosteine ​​3.5 mg / mL.

[0099] In the relaxation rate measurement after endothelial cell removal to determine the effect on airway endothelial cells, a significant increase in airway relaxation rate induced by the constrictors histamine and carbachol was observed with 1.5 mg / mL and 3.5 mg / mL treatments of Racemic-Erdosteine ​​and L-Erdosteine.

[0100] EC of airway relaxation in response to histamine-induced constriction 50 Racemic-Erdosteine ​​was measured as 4.61 mg / mL, L-Erdosteine ​​as 4.34 mg / mL, and EC of airway relaxation in response to carbachol-induced contraction 50 Racemic-Erdosteine ​​was measured at 2.77 mg / mL and L-Erdosteine ​​was measured at 2.66 mg / mL. After removal of airway endothelial cells, the EC of airway relaxation in response to histamine-induced contraction 50 Racemic-Erdosteine ​​and L-Erdosteine ​​were measured as 1.76 mg / mL and 1.82 mg / mL, respectively, and the EC of airway relaxation in response to carbachol-induced contraction 50 Racemic-Erdosteine ​​was measured at 1.45 mg / mL and L-Erdosteine ​​was measured at 1.93 mg / mL.

[0101] As a result, it was determined that Racemic-Erdosteine ​​and L-Erdosteine ​​had equivalent airway relaxation effects on histamine and carbachol, regardless of the presence or absence of airway endothelial cells.

[0102] Expectorant effect test of Erdosteine ​​and Racemic-Erdosteine

[0103] The effect of test substances L-Erdosteine ​​and Racemic-Erdosteine ​​on MUC5AC production at different concentrations was evaluated using NCI-H292 cells (Tissue: Lung / mL, Media: RPMI1640, Supplier: Korea Cell Line Bank).

[0104] The test substances were prepared by dissolving L-Erdosteine ​​and Racemic-Erdosteine ​​in methanol at a concentration of 10.6 mg / mL each, and the inducer was prepared by dissolving PMA (Phorbol-12-myristate-13-acetate) in DMSO at a concentration of 1 mg / mL. The measurement results were prepared using SPSS statistics 12.0K for medical science. All data were confirmed to be normally distributed through the Kolmogorov-Smirnov test, and the induced control group (G2), untreated group (G1), and test substance-treated groups (G3-G8) were compared through parametric multiple comparison procedures. The analysis method was Student's t-test, and Welch's t-test was performed when the variances were not equal. All statistical analyses were considered statistically significant when P<0.05. MUC5AC expression levels were evaluated using qPCR. In addition, cytotoxicity (CCK-8 assay) was measured at different concentrations, and the concentration at which cell viability was 80% or higher in NCI-H292 cells was set as a non-cytotoxic concentration. The results were as shown in Fig. 10 (cytotoxicity) and Fig. 11 (mRNA expression level of MUC5AC).

[0105] PMA was observed to be non-cytotoxic at a concentration of 10 ng / mL, and L-Erdosteine ​​and Racemic-Erdosteine ​​were observed to be non-cytotoxic at all concentrations.

[0106] Meanwhile, a significant increase in MUC5AC mRNA expression was observed in the group treated with 10 ng / mL of PMA as a trigger substance (G2) compared to the untreated group (G1), and a statistically significant decrease in MUC5AC mRNA expression was observed in the groups treated with 1000 ng / mL, 500 ng / mL, and 250 ng / mL of L-Erdosteine ​​(G3-G5) compared to the group treated with 10 ng / mL of PMA as a trigger substance (G2), and a statistically significant decrease in MUC5AC mRNA expression was observed in the groups treated with 1000 ng / mL, 500 ng / mL, and 250 ng / mL of Racemic-Erdosteine ​​(G6-G8) compared to the group treated with 10 ng / mL of PMA as a trigger substance (G2).

[0107] From these results, it was confirmed that the production of MUC5AC was increased by PMA in NCI-H292 cells, and the expression level of MUC5AC was decreased when L-Erdosteine ​​and Racemic-Erdosteine ​​were treated at concentrations of 1000 ng / mL, 500 ng / mL, and 250 ng / mL, and thus it was judged to be effective for expectoration.

[0108] L-Erdosteine ​​and Racemic-Erdosteine: A Study on the Inhibitory Effects of Lung Inflammation

[0109] When L-Erdosteine ​​and Racemic-Erdosteine ​​were repeatedly administered orally for 21 days to a bleomycin-induced lung injury mouse model, the effect of suppressing lung inflammation was evaluated.

[0110] Each test substance of L-Erdosteine ​​and Racemic-Erdosteine ​​was weighed by dose, sequentially added with excipients (DMSO: saline) in a ratio of 5.0:95.0 (v / v), vortexed, and administered. The test animals used were mouse models (specific pathogen free (SPF) C57BL / 6N mice, C57BL / 6NCrljOri, gender: male, age: 7 weeks, weight range: 19.41 - 22.79 g). The administration method was oral administration (number of administrations: once / day, total of 21 times for 21 days, administration method: manually correct the dorsum of the animal and directly administer into the stomach using a 1 mL syringe equipped with a zonde, administration amount: 10 ml / kg / day). Lung injury was induced by exposing the upper airway through an incision along the midline of the neck, and spraying 1.25 mg / kg (50 μL) of bleomycin dissolved in saline into the lungs using a specially designed syringe through the upper airway, followed by suturing. Body weight was measured at the start of administration, twice daily thereafter, and on the day of autopsy.

[0111] The test groups were composed of normal group (G1), negative control group (G2), test substance L-Erdosteine ​​25 mg / kg / day administration group (G3), L-Erdosteine ​​50 mg / kg / day administration group (G4), L-Erdosteine ​​100 mg / kg / day administration group (G5), test substance Racemic-Erdosteine ​​25 mg / kg / day administration group (G6), Racemic-Erdosteine ​​50 mg / kg / day administration group (G7), and Racemic-Erdosteine ​​100 mg / kg / day administration group (G8). Eight animals were used in each group. The test items included observation of dead animals, general symptoms, and body weight, and histopathological examination (on the day of necropsy, the left lobe of the animal was removed, observed using a light microscope (Nikon, Japan), and photographed (Jena, Germany), and inflammatory cell numbers were analyzed). Statistical analysis was performed using SPSS statistics 12.0K for medical science. The comparison between the normal group (G1) and the negative control group (G2) in the parametric comparison procedures was performed between groups to determine the presence or absence of disease induction. The analysis method was Student's t-test, and Welch's t-test was performed when the variances were not equal. The comparison between the negative control group (G2) and the test substance administration groups (G3-G8) was performed using ONE-WAY ANOVA, and as a post-hoc test, Duncan's test was performed when the variances were equal, and Dunnett's test was performed when the variances were not equal. All statistical analyses were considered statistically significant if P<0.05. The test results are shown in Figures 12, 13, and Table 6.

[0112]

[0113] During the test period, no abnormal symptoms or deaths were observed, and no differences in body weight were observed between the test substance administration groups (G3-G8) compared to the negative control group (G2).

[0114] In the number of inflammatory cells, a statistically significant decrease was observed in the L-Erdosteine ​​50 and 100 mg / kg / day administration groups (G4 and G5) and the Racemic-Erdosteine ​​25 and 100 mg / kg / day administration groups (G6 and G8) compared to the negative control group (G2).

[0115] As a result, it was confirmed that repeated oral administration of L-Erdosteine ​​and Racemic-Erdosteine ​​for 21 days was effective in suppressing lung inflammatory responses in a bleomycin-induced lung injury mouse model. Furthermore, since pulmonary fibrosis is caused by long-term progression of inflammatory responses such as pneumonia, it can be expected that it will also have a long-term preventive effect on pulmonary fibrosis.

[0116] A study on the respiratory inflammation-relieving effects of L-Erdosteine ​​and Racemic-Erdosteine.

[0117] A comparative evaluation was conducted on the effect of alleviating respiratory tract inflammation by three repeated oral administrations of L-Erdosteine ​​and one other agent to an OVA (Albumin from chicken egg white)-induced asthma BALB / c mouse model.

[0118] The test substances used were L-Erdosteine ​​and Racemic-Erdosteine, the inducers used were OVA (Albumin from chicken egg white) and Alum (Aluminum hydroxide), and the positive control substance was Dexamethasone. Each test substance and positive control substance were weighed by volume, and the test substance and excipient (DMSO: saline) were sequentially added at a ratio of 5.0:95.0 (v / v), and the positive control substance was prepared by adding water for injection and vortexing. The test animals used were mouse models (specific pathogen free (SPF) C57BL / 6N mice, C57BL / 6NCrljOri, gender: male, age: 9 weeks, weight range: 21.38 - 26.18 g), and the administration method was oral administration (number of administrations: once / day, 3 times in total for 3 days, administration method: manually correct the dorsum of the animal and administer directly into the stomach using a 1 mL syringe equipped with a zonde, administration amount: 10 ml / kg / day). Body weight was measured at the start of administration, and thereafter twice / week and on the day of necropsy.

[0119] Asthma induction was performed according to the following procedure.

[0120] 1. Sensitization

[0121] 1.1. Cohort 1 animals were acclimatized for 4 days, and Cohort 2 animals for 11 days, and then sensitized by intraperitoneal administration of 50 μg / 0.1 mL / head of OVA twice on days -20 and -6. The normal group was administered the above excipient in the same manner.

[0122] 1.2. Preparation of sensitizing solution

[0123] 1.2.1. OVA (grade V chicken egg OVA (Sigma-Aldrich, St Louis, MO, USA)) was taken out of the refrigerator and left at room temperature for more than 30 minutes. Then, PBS was added to dissolve it to 10 mg / mL, and 1 mL of this solution was mixed with 9 mL of PBS to prepare a diluted OVA stock solution (1 mg / mL).

[0124] 1.2.2. 7.5 mL of OVA stock solution and 7.5 mL of alum were mixed in a 50 mL conical tube (0.5 mg / mL) and mixed well using a shaker for 30 minutes to complete the sensitizing solution.

[0125] 2. Cause

[0126] 2.1. The story was conducted on DAY 1, 2 and 3 (3 days in total).

[0127] 2.2. One hour after the test substance was administered, 10 mL (10 mg / mL) of 1% OVA was sprayed into the chamber to induce the challenge.

[0128] 2.3. The experiment was conducted for 60 minutes on days 1, 2, and 3, for a total of 3 days, and the test substance administration and experiment were conducted simultaneously for 3 days.

[0129] 2.4. At this time, 1 or 2 animals from each test group were placed in the same chamber, the lid was closed, and a silicone hose connected to the nebulizer was inserted into the cage hole to spray.

[0130] 2.5. The normal group was sprayed with the excipient in the same way.

[0131] 2.6. Preparation of the drug

[0132] 2.6.1. Take out OVA (grade III OVA (Sigma-Aldrich)) from the refrigerator, leave it at room temperature for more than 30 minutes, and weigh 600 to 800 mg into a 50 mL conical tube.

[0133] 2.6.2.OVA was dissolved in PBS to make 10 mg / mL, and then placed on ice to maintain refrigeration.

[0134] The test groups were composed of the normal group (G1), vehicle control group (G2), L-Erdosteine ​​12.5 mg / kg / day administration group (G3), 25 mg / kg / day administration group (G4), 50 mg / kg / day administration group (G5), Racemic-Erdosteine ​​12.5 mg / kg / day administration group (G6), 25 mg / kg / day administration group (G7), 50 mg / kg / day administration group (G8), and Dexamethasone 3 mg / kg / day administration group (G9, hereinafter referred to as positive control group), and 8 animals were used in each group. Statistical analysis was performed using SPSS statistics 12.0K for medical science. The comparison between the normal group (G1) and vehicle control group (G2) of the parametric comparison procedures was compared between groups to determine the presence or absence of asthma induction. One-way ANOVA was performed to compare the vehicle control group (G2) and the test substance administration groups (G3-G8), and Duncan's test was performed as a post-hoc test for equal variances and Dunnett's test for unequal variances. Student's t-test was performed to compare the normal group (G1) and the positive control group (G9), the vehicle control group (G2) and the L-Erdosteine ​​administration groups (G3-G5), and the Racemic-Erdosteine ​​administration groups (G6-G8), and Welch's t-test was performed for unequal variances. In addition, statistical exclusion of the western bolt measurement results was excluded using a box plot. All statistical analyses were considered statistically significant if P<0.05.

[0135] The test items were observation of dead animals and general symptoms (death, onset date and severity of symptoms were observed once a day during the administration and observation period and recorded for each individual, and the start date of administration was designated as DAY 1), body weight (measured upon acquisition and group separation, and measured twice a week thereafter and on the day of necropsy), and ROMO1 was analyzed using an ELISA Kit (LS Bio, Cat. LS-F35358) for BALF (bronchoalveolar lavage fluid). The test results were as shown in Fig. 14.

[0136] No abnormal symptoms or deaths were observed during the test period. No differences in body weight were observed between groups in any of the administration groups (G1-G9). A trend toward a decrease in ROMO1 in the lung lavage fluid was observed in the L-Erdosteine ​​12.5 mg / kg / day and 25 mg / kg / day administration groups (G3-G4) compared to the Racemic Erdosteine ​​12.5 mg / kg / day and 25 mg / kg / day administration groups (G6-G7).

[0137] As a result, a decrease in ROMO1 was observed by oral administration of L-Erdosteine ​​and Racemic-Erdosteine ​​three times in an OVA-induced asthma mouse model, suggesting that L-Erdostine is more effective than Racemic-Erdosteine ​​at the same dose in controlling and improving respiratory-related chronic inflammatory diseases such as chronic upper airway inflammation, asthma, and chronic obstructive pulmonary disease (COPD). For reference, the serum ROMO1 concentration in COPD patients is reported to be significantly higher than that in the healthy control group (Liang et al., "Increased Serum Romo1 Was Correlated with Lung Function, Inflammation, and Oxidative Stress in Chronic Obstructive Pulmonary Disease", Inflammation. 2019 Oct;42(5):1555-1560. doi: 10.1007 / s10753-019-01017-x.)

Claims

1. Use of an L-eldostane compound represented by the following chemical formula 1 in the manufacture of a medicament for one or more selected from the group consisting of dissolving mucus and expectorating in acute and chronic respiratory diseases, treating or improving upper respiratory tract inflammation, treating or preventing acute and chronic bronchitis, treating or preventing cough, treating or preventing asthma, treating or preventing pneumonia, preventing pulmonary fibrosis, preventing destruction of bronchial alveoli, treating or preventing chronic obstructive pulmonary disease, increasing resistance to upper respiratory tract infection bacteria, and improving side effects caused by smoking. [Chemical Formula 1] 2. Use of an L-eldosteine compound as a prodrug of a compound represented by the following chemical formula 2. [Chemical Formula 2] 3. Contains an L-eldosteine compound represented by the following chemical formula 1 as an active ingredient, A pharmaceutical composition for one or more uses selected from the group consisting of dissolving mucus and expectorating phlegm in acute and chronic respiratory diseases, treating or improving upper respiratory tract inflammation, treating or preventing acute and chronic bronchitis, treating or preventing cough, treating or preventing asthma, treating or preventing pneumonia, preventing pulmonary fibrosis, preventing destruction of bronchial alveoli, treating or preventing chronic obstructive pulmonary disease, increasing resistance to upper respiratory tract infection bacteria, and improving side effects caused by smoking. [Chemical Formula 1] 4. A pharmaceutical composition according to claim 3, characterized in that it comprises an L-eldosteine compound in a daily dosage of 10 to 200 mg / kg.

5. A pharmaceutical composition according to claim 3, characterized in that it comprises 50 to 200 mg of the L-eldosteine compound and a pharmaceutically acceptable excipient.

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