COMPOSITION FOR PREVENTING OR TREATING ASTHMA, RHINITIS OR CONJUNCTIVITIS, COMPRISING N-ACYL AMINO ACID AS ACTIVE INGREDIENT

Abstract

The present disclosure relates to a composition for preventing, alleviating or treating asthma, rhinitis and/or conjunctivitis using an N-acylamino acid, which has almost no side effect on the human body. The composition of the present disclosure has an excellent effect of alleviating the symptoms of asthma by exhibiting alleviation effects on both inflammatory response in the airway and airway fibrosis, which are major clinical symptoms of asthma, and thus can be effectively used in drugs or foods for alleviating asthma. In addition, it can be usefully used in drugs or foods for alleviating rhinitis and conjunctivitis since it significantly alleviates the clinical symptoms of rhinitis and conjunctivitis.

Claims

1.-8. (canceled)

9. A method for preventing, alleviating or treating asthma, rhinitis or conjunctivitis, comprising administering a composition comprising an effective amount of an N-acylamino acid or a pharmaceutically acceptable salt thereof to a subject.

10. The method according to claim 9, wherein the N-acylamino acid is N-acylalanine or N-acyltryptophan.

11. The method according to claim 10, wherein the N-acylalanine is N-acetylalanine or N-oleoylalanine.

12. The method according to claim 10, wherein the N-acyltryptophan is N-acetyltryptophan or N-oleoyltryptophan.

13. The method according to claim 9, wherein the composition is a pharmaceutical composition, a food composition or a cosmetic composition.

14. The method according to claim 9, wherein the composition is administered orally, intranasally or intraocularly.

15. The method according to claim 9, wherein the acyl is formyl (HCO—), acetyl (CH.sub.3CO—), propionyl (C.sub.2H.sub.5CO—), butyryl (C.sub.3H.sub.7CO—), valeryl (C.sub.4H.sub.9CO—), pentanoyl (CH.sub.3(CH.sub.2).sub.3CO—), palmitoyl (C.sub.15H.sub.31CO—), stearoyl (C.sub.17H.sub.33CO—), oleoyl (C.sub.17H.sub.31CO—), oxalyl (—CO—CO—), malonyl (—COCH.sub.2CO—), succinyl (—CO(CH.sub.2).sub.2CO—), benzoyl (C.sub.6H.sub.5CO—), toluoyl (CH.sub.3—C.sub.6H.sub.4—CO—), salicyloyl (HO—C.sub.6H.sub.4—CO—), cinnamoyl (C.sub.6H.sub.5CH═CHCO—), naphthoyl (C.sub.10H.sub.7CO—), phthaloyl (CO—C.sub.6H.sub.4—CO—), furoyl (C.sub.5H.sub.3O.sub.2—), undecanoyl (CH.sub.3(CH.sub.2).sub.9CO—) or docosenoyl.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 shows H&E staining images of lung tissue showing the inhibition of inflammation by oral administration of N-acyl-L-alanine, N-oleoyl-L-alanine, N-acyl-L-tryptophan and N-oleoyl-L-tryptophan (OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

[0067] FIG. 2 shows Masson's trichrome staining images of lung tissue showing the inhibition of bronchial fibrosis by oral administration of N-acyl-L-alanine, N-oleoyl-L-alanine, N-acyl-L-tryptophan and N-oleoyl-L-tryptophan (OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

[0068] FIG. 3 shows the inhibition of Th2 cytokine (IL4) secretion by cells in the airway by N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan (*p<0.05 vs ovalbumin group; OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

[0069] FIG. 4 shows the inhibition of Th2 cytokine (IL5) secretion by cells in the airway by N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan (*p<0.05 vs ovalbumin group; OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

[0070] FIG. 5 shows the inhibition of Th2 cytokine (IL13) secretion by cells in the airway by N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan (*p<0.05 vs ovalbumin group; OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

[0071] FIG. 6 shows the inhibition of rhinitic response by N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan (FIG. 6A: nasal rubbing, FIG. 6B: sneezing; *p<0.05 vs ovalbumin group; OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

[0072] FIG. 7 shows the inhibition of allergic conjunctivitis by N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan (FIG. 7A: images of eye, FIG. 7B: clinical grade; *p<0.05 vs ovalbumin group; OVA=ovalbumin, NAA=N-acetyl-L-alanine, NOA=N-oleoyl-L-alanine, NAT=N-acetyl-L-tryptophan, NOT=N-oleoyl-L-tryptophan).

BEST MODE

[0073] Hereinafter, the present disclosure is described in further detail through examples. These examples are provided only to illustrate the present disclosure more specifically, and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.

EXAMPLES

Preparation Example 1. Preparation of Experimental Animals

[0074] Specific pathogen-free BALB/c mice (6-week-old, male) were purchased from Doul Biotech (Osan, Korea). Experiment was started when the mice were 7- to 10-week old. The mice were housed in a laminar flow cabinet and were given water and feed ad libitum.

Preparation Example 2. Preparation of Drugs

[0075] As the drugs used in the present disclosure, N-acetyl-L-alanine and N-acetyl-L-tryptophan were purchased from Sigma (MO, USA), and N-oleoyl-L-alanine was purchased from Cayman (MI, USA). N-Oleoyltryptophan was synthesized as follows. First, for preparation of methyl oleoyl L-tryptophanate, a mixture of oleoic acid (2.36 mmol), L-tryptophan methyl ester hydrochloride (2.60 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (2.60 mmol), hydroxybenzotriazole (HOBt) (2.60 mmol) and triethylamine (11.8 mmol) dissolved in dichloromethane was stirred at room temperature for 12 hours. The reaction mixture was concentrated, diluted with saturated NaHCO.sub.3 solution, and then extracted 3 times with ethyl acetate. The extracted organic solvent layer was combined and washed with brine. After washing with 1 N HCl and then with brine, the organic solvent layer was dried on anhydrous MgSO.sub.4, concentrated, and then purified by medium-pressure liquid chromatography (MPLC) using n-hexane and ethyl acetate. Yield was 73% and the data for the prepared methyl oleoyl L-tryptophanate are as follows. .sup.1H NMR (500 MHz, chloroform-d) δ 8.18 (s, 1H), 7.56 (dd, J=7.9, 1.0 Hz, 1H), 7.39 (dt, J=8.1, 0.9 Hz, 1H), 7.29 (s, 1H), 7.22 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.14 (ddd, J=8.0, 7.0, 1.0 Hz, 1H), 7.00 (d, J=2.4 Hz, 1H), 5.99 (d, J=7.9 Hz, 1H), 5.37 (qd, J=4.1, 2.1 Hz, 2H), 5.00 (dt, J=8.0, 5.3 Hz, 1H), 3.72 (s, 3H), 3.39-3.28 (m, 2H), 2.20-2.13 (m, 2H), 2.08-1.99 (m, 5H), 1.60 (t, J=7.4 Hz, 2H), 1.34-1.28 (m, 24H), 0.92-0.89 (m, 3H). LC-MS (ESI), calcd for C.sub.30H.sub.46N.sub.2O.sub.3 482.4, found m/z 483.4 (M+H.sup.+). For preparation of N-oleoyltryptophan from the methyl oleoyl L-tryptophanate, a solution of the methyl oleoyl L-tryptophanate (0.37 mmol) dissolved in tetrahydrofuran was stirred at room temperature for 12 hours after adding NaOH (1.48 mmol) solution. The reaction mixture was extracted with dichloromethane after adding water. The aqueous layer was adjusted to pH 1 by adding 1 N HCl, and then extracted 3 times with ethyl acetate. The extracted organic solvent layer was dried on anhydrous MgSO.sub.4, and then concentrated. Yield was 86%, and the data for the prepared N-oleoyltryptophan are as follows. .sup.1H NMR (500 MHz, chloroform-d) δ 8.22 (s, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.40 (dt, J=8.1, 0.9 Hz, 1H), 7.23 (ddd, J=8.1, 6.9, 1.2 Hz, 1H), 7.15 (ddd, J=8.0, 7.1, 1.0 Hz, 1H), 7.08 (d, J=2.4 Hz, 1H), 5.37 (qd, J=5.3, 4.6, 2.3 Hz, 2H), 4.95 (dt, J=7.3, 5.6 Hz, 1H), 3.45-3.33 (m, 2H), 2.17-2.10 (m, 2H), 1.59-1.51 (m, 2H), 1.38-1.26 (m, 26H), 0.91 (d, J=6.8 Hz, 3H). C.sub.29H.sub.44N.sub.2O.sub.2 468.3, found m/z 469.3 (M+Hi).

Preparation Example 3. Induction of Asthma in Mice

[0076] The mice were sensitized by intraperitoneally injecting 0.02 mg of ovalbumin (OVA) and alum (1 mg) (Sigma-Aldrich) as an immunologic adjuvant twice on days 0 and 14. For 4 days from day 28, the mice were placed in a plastic nebulizer container of a regular size and were made to inhale OVA in the form of an aerosol by spraying 5% OVA for 30 minutes (airway challenge).

Preparation Example 4. Induction of Rhinitis in Mice

[0077] The mice were sensitized by intraperitoneally injecting 0.02 mg of ovalbumin (OVA) and alum (1 mg) (Sigma-Aldrich) as an immunologic adjuvant twice on days 0 and 14. For 7 days from day 21, the mice were topically stimulated by injecting 10 μL of a solution of 100 μg of OVA dissolved in 20 μL of phosphate-buffered saline into both nasal cavities, once a day. After the final injection of OVA (day 27), symptom score was recorded by summing the numbers of nasal rubbing and sneezing. The symptom score was recorded for 15 minutes, and the result was compared between the groups. The result is shown in FIG. 6.

Preparation Example 5. Induction of Conjunctivitis in Mice

[0078] The mice were sensitized by intraperitoneally injecting 0.02 mg of ovalbumin (OVA) and alum (1.5 mg) (Sigma-Aldrich) as an immunologic adjuvant twice on days 0 and 7. For 12 days from day 14, conjunctivitis was induced by injecting 10 μL of a solution of 100 μg of OVA dissolved in 20 μL of phosphate-buffered saline into each eye, once a day.

Preparation Example 6. Measurement of in Inflammatory Cells in Bronchus

[0079] After anesthetizing the mouse and inserting a tube into the bronchus, the bronchus was washed with a buffer and cells were obtained therefrom. The cells were centrifuged, suspended at 1×10.sup.4 cells/100 μL, and fixed by centrifuging with a cytospin. Then, the number of eosinophils, monocytes, neutrophils and macrophages were measured by staining with Diff-Quik.

Preparation Example 7. Staining of Lung Tissue

[0080] For histological assay, tissues taken out from the lung were fixed with formalin and embedded in paraffin. The embedded tissue was sliced to 4-μm thickness and subjected to 1) hematoxylin and eosin (H&E) staining for investigation of the degree of inflammation and 2) Masson's trichrome staining for investigation of the degree of pulmonary fibrosis.

Preparation Example 8. Measurement of Clinical Grading of Conjunctival Edema and Flare

[0081] 24 hours after the final injection of OVA (day 25), the clinical grading of conjunctival edema and flare (redness) was assessed using a portable slit lamp (Carl Zeiss, Oberkochen, Germany). The assessment was performed in a blinded fashion 2 hours after OVA injection. Each clinical parameter was scored on a scale of 0 to 4+(0 absent, 1+minimal, 2+mild, 3+moderate, 4+severe) as described in Merayo-Lloves, J., Calonge, M, and Foster, C. S. 1995. Experimental model of allergic conjunctivitis to ragweed in guinea pig. Curr. Eye Res. 14: 487-494. A negative control group was given no treatment.

Example 1. Asthma-Inhibiting Effect of N-Acetyl-L-Alanine, N-Oleoyl-L-Alanine, N-Acetyl-L-Tryptophan and N-Oleoyl-L-Tryptophan

Example 1-1. Inhibition of Inflow of Inflammatory Cell into Airway of Asthma-Induced Mice by Administration of N-Acetyl-L-Alanine, N-Oleoyl-L-Alanine, N-Acetyl-L-Tryptophan and N-Oleoyl-L-Tryptophan

[0082] Neutrophils, macrophages, monocytes and eosinophils were observed as inflammatory cells in the airway. Each of N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan was dissolved in 10 mL of phosphate-buffered saline to 3.5 μmol/kg and was orally administered for 7 days from day 25, once a day. As seen from Table 1, the orally administration of N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan significantly inhibited the inflow of eosinophils into the airway.

TABLE-US-00001 TABLE 1 Bronchoalveolar lavage fluid parameters WBC (×10.sup.2 WBC Differential Counting (%) WBC Differential Counting (k/uL) Group Values cells/μL) MAC NEU MONO EOS MAC NEU MONO EOS Normal Mean 0.05 90.0 8.5 1.6 0.0 0.04 0.00 0.00 0.00 S.D. 0.03 6.5 6.7 2.0 0.0 0.03 0.00 0.00 0.00 Min 0.02 78.5 1.9 0.0 0.0 0.02 0.00 0.00 0.00 Max 0.12 98.1 21.5 5.3 0.0 0.11 0.01 0.01 0.00 N 8 8 8 8 8 8 8 8 8 OVA Mean 0.61 68.9 3.6 1.8 25.8 0.42 0.02 0.01 0.15 S.D. 0.25 4.7 1.7 0.9 4.1 0.20 0.01 0.01 0.06 Min 0.40 59.9 0.9 0.0 22.1 0.28 0.01 0.00 0.09 Max 1.14 74.2 6.1 2.6 32.0 0.83 0.03 0.03 0.27 N 8 8 8 8 8 8 8 8 8 OVA + NAA Mean 0.39 81.8 4.9 3.3 10.1 0.32 0.02 0.01 0.04 S.D. 0.15 7.0 1.9 1.6 6.2 0.14 0.01 0.00 0.03 Min 0.22 70.7 1.4 1.4 2.8 0.17 0.01 0.01 0.01 Max 0.69 91.6 6.5 6.5 21.3 0.63 0.03 0.02 0.11 N 8 8 8 8 8 8 8 8 8 OVA + NOA Mean 0.38 87.3 5.5 3.8 3.4 0.34 0.02 0.01 0.01 S.D. 0.13 7.3 2.6 2.8 2.4 0.13 0.01 0.00 0.00 Min 0.19 70.9 2.2 1.8 1.8 0.13 0.01 0.01 0.01 Max 0.56 93.3 10.5 10.5 8.1 0.52 0.03 0.02 0.02 N 8 8 8 8 8 8 8 8 8 OVA + NAT Mean 0.47 71.8 4.1 3.9 20.1 0.33 0.02 0.02 0.10 S.D. 0.18 7.1 2.0 2.4 8.3 0.14 0.01 0.01 0.05 Min 0.16 62.1 2.0 0.0 12.3 0.11 0.01 0.00 0.03 Max 0.76 83.1 6.6 8.0 35.9 0.58 0.05 0.04 0.18 N 8 8 8 8 8 8 8 8 8 OVA + NOT Mean 0.43 74.3 5.1 3.6 17.0 0.32 0.02 0.01 0.07 S.D. 0.07 10.7 3.2 2.0 9.5 0.08 0.01 0.01 0.04 Min 0.27 61.8 1.9 1.9 5.3 0.18 0.01 0.01 0.02 Max 0.52 86.2 11.4 7.7 32.3 0.45 0.05 0.03 0.14 N 8 8 8 8 8 8 8 8 8 OVA = ovalbumin, NAA = N-acetyl-L-alanine, NOA = N-oleoyl-L-alanine, NAT = N-acetyl-L-tryptophan, NOT = N-oleoyl-L-tryptophan, MAC = macrophages, NEU = neutrophils, MONO = monocytes, EOS = eosinophils, N = number of mice, S.D. = standard deviation, Min = minimum value, Max = maximum value.

Example 1-2. Investigation of Degree of Inflammation by H&E Staining of Lung Tissue

[0083] Lung tissue was stained with H&E after oral administration of the drug for a week. While a normal group (normal) showed almost no inflammatory cells around the airway and an OVA-challenged group (OVA) showed many inflammatory cells gathered around the airway as well as narrowing of the bronchus and blood vessel formation around the bronchus (FIG. 1), the groups to which N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan or N-oleoyl-L-tryptophan was orally administered (OVA+NAA, OVA+NOA, OVA+NAT, OVA+NOT) showed remarkably inhibited inflammation. In particular, N-oleoyl-L-alanine showed the highest inhibition of inflammation (FIG. 1).

Example 1-3. Measurement of Cytokines in Bronchial Wash

[0084] The concentration of several cytokines (TNF-α, IL-4, IL-5, IL-13) in bronchial wash was measured using an ELISA kit (R&D Systems, Minneapolis, Minn., USA).

Example 2. Bronchial Obstruction-Inhibiting Effect of N-Acetyl-L-Alanine, N-Oleoyl-L-Alanine, N-Acetyl-L-Tryptophan and N-Oleoyl-L-Tryptophan

[0085] Lung tissue was stained with Masson's trichrome after oral administration of the drug for a week. Whereas a normal group (normal) showed almost no fibrosis around the airway and an OVA-challenged group (OVA) showed a high degree of fibrosis around the airway (FIG. 2), the groups to which N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan or N-oleoyl-L-tryptophan was orally administered (OVA+NAA, OVA+NOA, OVA+NAT, OVA+NOT) showed inhibited fibrosis. In particular, N-oleoyl-L-alanine showed the highest inhibition (FIG. 2).

Example 3. Inhibition of Th2 Cytokine (IL4, IL5 and IL13) Secretion in Airway by N-Acetyl-L-Alanine, N-Oleoyl-L-Alanine, N-Acetyl-L-Tryptophan and N-Oleoyl-L-Tryptophan

[0086] Th2 cytokines, IL-4, IL-5 and IL-13, are known to be involved in airway inflammation, bronchial hypersensitivity and IgE antibody production mediated by eosinophils. 3.5 μmol/kg N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan or N-oleoyl-L-tryptophan dissolved in 10 mL of phosphate-buffered saline was orally administered once a day, for 7 days from day 25 after the induction of asthma, and the cytokines were measured. The oral administration of N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan significantly inhibited IL-4, IL-5 and IL-13 (FIG. 3, FIG. 4 and FIG. 5).

Example 4. Rhinitic Response-Inhibiting Effect of N-Acetyl-L-Alanine, N-Oleoyl-L-Alanine, N-Acetyl-L-Tryptophan and N-Oleoyl-L-Tryptophan

[0087] Ovalbumin (OVA) of Preparation Example 3 was used as an antigen to induce allergic rhinitis. After OVA challenging from day 21 to day 26, 10 μL of each of N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan dissolved in phosphate-buffered saline to 10 μM was intranasally administered 12 hours later. After the final injection of OVA (day 27), symptom score was recorded by summing the numbers of nasal rubbing and sneezing. The symptom score was recorded for 15 minutes, and the result was compared between the groups. The result is shown in FIG. 6. Whereas a normal group showed almost no nasal rubbing or sneezing and an OVA-challenged group showed remarkably increased nasal rubbing or sneezing (FIG. 6A and FIG. 6B), the groups to which N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan or N-oleoyl-L-tryptophan was intranasally administered showed remarkably decreased numbers of nasal rubbing and sneezing (FIG. 6A and FIG. 6B).

Example 5. Conjunctivitis-Inhibiting Effect of N-Acetyl-L-Alanine, N-Oleoyl-L-Alanine, N-Acetyl-L-Tryptophan and N-Oleoyl-L-Tryptophan

[0088] Ovalbumin of Preparation Example 4 was used as an antigen to induce conjunctivitis. After challenging 100 μg of ovalbumin into the eye from day 15 to 26, 10 μL of each of N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan and N-oleoyl-L-tryptophan dissolved in phosphate-buffered saline to 100 μM was intraocularly administered 30 minutes and 12 hours later. On day 26, the clinical grading of conjunctival edema and flare (redness) was assessed using a portable slit lamp (Carl Zeiss, Oberkochen, Germany). The clinical grade of each test group was compared, and the result is shown in FIG. 7. Whereas a normal group showed almost no symptom of conjunctivitis and an OVA-challenged group showed an increased clinical grade of conjunctivitis (FIGS. 7A and 7B), the groups to which N-acetyl-L-alanine, N-oleoyl-L-alanine, N-acetyl-L-tryptophan or N-oleoyl-L-tryptophan M was intraocularly administered showed remarkably increased clinical grades (FIGS. 7A and 7B).

[0089] While the specific exemplary embodiments of the present disclosure have been described in detail above, it is obvious to those having ordinary knowledge in the art that such detailed descriptions are merely specific examples and the scope of the present disclosure is not limited by them. It is to be appreciated that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.