METHOD FOR IMPROVING EYE CONDITIONS

Abstract

Disclosed is a method for improving eye conditions comprising orally administering to humans, an ethanol-soluble component-containing degradation product obtained by degrading a comb with a protease and removing a solid.

Claims

1-6. (canceled)

7. A method for improving dry eye, comprising orally administering to a human in need thereof, an effective amount of a composition containing a degradation product obtained by degrading a comb with a protease and removing solids from the comb degraded with the protease, wherein; the degradation product contains an ethanol-soluble component in an amount of 70% by dry weight; and the degradation product contains a low-molecular weight hyaluronic acid having a molecular weight of 380 to 5000 in an amount of 95% by mass or more of total hyaluronic acid contained in the degradation product.

8. (canceled)

9. The method for improving dry eye according to claim 7, wherein the degradation product is a liquid product obtained by degrading a comb with a protease and removing a solid from the comb degraded with the protease, or a freeze-dried product of the liquid product.

10. (canceled)

11. The method for improving dry eye according to claim 9, wherein the liquid product or the freeze-dried product thereof contains a free amino acid.

12. The method for improving dry eye according to claim 9, wherein the degradation product is the liquid product.

13. The method for improving dry eye according to claim 9, wherein the degradation product is the freeze-dried product.

14. The method for improving dry eye according to claim 9, wherein the composition contains an excipient.

15. The method for improving dry eye according to claim 9, wherein the composition contains dextrin.

Description

EXAMPLES

[0047] The present invention is described more specifically with reference to Examples given below. The materials, the ratio thereof and the operations in the following Examples may be appropriately varied not overstepping the scope and the spirit of the present invention. Accordingly, the range of the present invention should not be interpreted limitatively by the specific examples shown below.

[0048] Component analysis of the compositions produced in this Example was carried out according to the following methods.

(1) Measurement of Water Content

[0049] One g of the composition was heated and dried at 105° C. for 3 hours, and the constant weight thereof was measured with a precision balance to quantify the water content thereof.

(2) Total Nitrogen Determination

[0050] The total nitrogen was quantitatively determined according to a semimicro-Kjeldahl method based on an AOAC method.

(3) Free Amino Acid Determination and Amino Acid Composition Analysis

[0051] The total free amino acid amount was quantified according to a ninhydrin method. For quantification, a calibration curve of leucine as a standard amino acid was formed and used. The composition of the free amino acid was analyzed using an amino acid automatic analyzer (manufactured by Hitachi Limited, L-8500 Model) equipped with a column for bioanalysis. In the analysis, 50 mg of the composition was dissolved in distilled water, dried into solid under reduced pressure using a rotary evaporator (60° C.), then eluted with 5 mL of 0.02 N hydrochloric acid, and filtered through filter paper and then through a germ-free filter, and 50 μL of the resultant filtrate was used as an analysis sample.

(4) Protein Determination

[0052] The total protein amount was determined according to a Lawry method. A bovine serum albumin was used for forming a standard calibration curve.

(5) N-acetyl-D-glucosamine Determination

[0053] The N-acetyl-D-glucosamine content was determined according to a Morgan-Elson method.

(6) Glucosaminoglycan Determination

[0054] The sample was analyzed through colorimetry according to a 2-nitrophenylhydrazine coupling method. For standard calibration curve formation, comb-derived sodium hyaluronate (manufactured by Wako Pure Chemical Corporation, HARC) and Streptococcus zooepidemicus-derived sodium hyaluronate (manufactured by Wako Pure Chemical Corporation, HASZ) were used.

(7) Measurement of Molecular Weight of Low-Molecular Hyaluronic Acid The molecular weight of hyaluronic acid was estimated through high-performance liquid chromatography (by Shimadzu Corporation) equipped with a differential refractometer (manufactured by Shimadzu Corporation, RID-10A Model). Columns of TSKgel G-2, 500PW.sub.XL (7.8 mm ID×30 cm) were used, and water was used as a mobile phase at a flow rate of 1 ml/min for analysis. As a molecular weight marker, four types of polyethylene glycol having a molecular weight of 400, 1000, 2000 or 6000 (manufactured by Aldrich Corp.) were used. The constituent weight ratio of each low-molecular hyaluronic acid was analyzed through high-performance liquid chromatography using samples of the pharmaceutical composition or dextrin alone, in which the peak area of dextrin was detracted from the peak area of the composition to determine the constituent weight ratio.

Production Example

[0055] One kg of freshly collected cock's combs were cut into small pieces of about 1 cm square, and thermally sterilized by steaming at 100° C. Food-derived enzymes mainly containing a protease were added to the small pieces and reacted at 45° C. for 1.5 hours, and then stirred and homogenized. Subsequently, rough solid fragments were removed by filtration to give a liquid degradation product (hereinafter referred to as “protease degradation product”). The protease degradation product had a pH of 6.5, a Brix value of 6.20 and a solid concentration of 5.91% by weight. The protease degradation product was freeze-dried and ground to be a freeze-dried powder of protease degradation product (composition 1). Dextrin in an amount of 3 equivalent times (as a ratio by mass) was added to the freeze-dried powder of protease degradation product to give a dextrin-added freeze-dried powder (composition 1′).

[Component Analysis of Composition]

[0056] The produced composition 1′ was analyzed for the constituent components thereof according to the above-mentioned method. The content of general components analyzed is shown in Table 1, the composition of free amino acids is shown in Table 2, and the analysis results of molecular weight of low-molecular hyaluronic acids are shown in Table 3. In Tables 1 to 3, “%” is “% by mass”.

TABLE-US-00001 TABLE 1 General Components % Water 2.2-2.6 Nitrogen 3.84 Total Protein 3.04 Free Amino Acid 4.08 N-acetylglucosamine 0 Dextrin (for food additive) 75.0

TABLE-US-00002 TABLE 2 Free Amino Acid Composition Amino Acid Content % ρ-serine 1.71 Taurine 3.30 Aspartic Acid* 2.94 Threonine* 1.30 Serine* 2.20 Glutamic Acid* 2.18 Glutamine 0.48 Sarcosine 1.81 Glycine* 2.26 Alanine* 3.52 Citrulline 0.92 α-Aminobutyric Acid 2.18 Cystine* 1.03 Methionine* 1.97 Cystine* 2.78 Leucine* 2.26 Isoleucine* 6.27 Tyrosine* 2.65 Phenylalanine* 3.30 β-aminoisobutyric Acid 5.45 Ornithine 1.05 Lysine* 1.17 1-Methylhystidine 0.78 Anserine 1.92 Arginine* 1.93 Identified Total Amino Acids 57.36 Unknown Amino Acids 42.64 *Protein composition ammo acid

TABLE-US-00003 TABLE 3 Estimated Molecular Weight, Constituent Unit Number and Constituent Weight Ratio of Low-Molecular HA Peak No. 1 2 3 4 5 Estimated Molecular Weight 5,000 1,520 1,140 760 380 Constituent Unit Number 13-14 4 3 2 1 Constituent Weight Ratio (%) 33 47 10 6 4

[0057] As shown in Table 2, among the free amino acids contained in the composition 1′, the content of isoleucine and β-aminoisobutyric acid was high, and then, alanine, phenylalanine, aspartic acid, cystine and tyrosine were contained much.

[0058] As shown in Table 3, the composition 1′ contained five types of low-molecular hyaluronic acids each having an estimated molecular weight of 5000, 1520, 1140, 760 and 380. When the molecular weight of one recurring unit of hyaluronic acid is about 400, the recurring unit number of each low-molecular hyaluronic acid is 13 to 14, 4, 3, 2 and 1 in that order from the largest molecular weight, and the mass ratio was 33%, 47%, 10%, 6% and 4%. Accordingly, it is known that the main components of the low-molecular hyaluronic acids are two components of a 4-molecular component having a molecular weight of about 1520, and a 13 to 14-molecular component having a molecular weight of about 5000. The content of the low-molecular hyaluronic acids having a molecular weight of 380 to 5000 in the composition 1′ was 13.4% by mass relative to the total amount of the composition 1′.

[Preparation of Composition-Containing Capsule Formulation]

[0059] A dextrin-added freeze-dried powder (composition 1′) prepared by adding dextrin to a freeze-dried, protease-degraded product powder in an amount of 3 times by equivalent (ratio by mass) was encapsulated in gelatin capsules to prepare a capsule formulation (hereinafter referred to as “protease-degraded product-containing capsules”). At this time, the amount of the freeze-dried, protease-degraded product powder that the capsules contained was 150 mg/capsule.

[Evaluation of Effect of Composition]

[0060] The effect of the freeze-dried powder of protease degradation product produced in Production Example was evaluated by panelists, healthy 12 men and women (6 men, 6 women) who were aware of daily dry eye and eyestrain. The age range of the panelists was 20 or more and less than 60. Specifically, before start of intake, each panelist was subjected to an eye lubricity test mentioned below. Subsequently, the panelists took protease degradation product-containing capsules, 4 capsules twice a day along with water or tepid water, and from the start of intake, after one week, after 2 weeks, after 3 weeks and after 4 weeks, the panelists were subjected to the same test. Here, the dosage of the capsule preparation corresponds to 1200 mg=600 mg as one intake a day×two times, of the freeze-dried powder of protease degradation product.

[0061] In the eye lubricity test, the panelists responded to specific items of DEQS (dry eye related quality of life score) by the Dry Eye Study Group, subjective symptom-related somesthetic VAS (visual analogue scale) questionnaire, and antiaging QOL common medical questionnaire by the Japanese Society of Anti-Aging Medicine, and were subjected to measurement of BUT (tear film break-up time) and subjected to an eyesight test and an intraocular pressure test. The test results before the start of intake and the test results after 4 weeks from the start of intake are shown in the following Tables 4 to 6, and a graph of the time-dependent response results of VAS Questionnaire is shown in the drawing.

[0062] Here, the score of the dry eye QOL questionnaire shown in Table 4 is QOL scores calculated based on the panelists' responses. QOL scores range 0 to 100, and a larger value means a higher severity level of dry eye symptoms, and indicates that the symptoms have a greater influence on the daily life and the mental aspect. The scores of the VAS questionnaire shown in Table 4 indicate a distance (mm) from the left end on a 100-mm line segment on which the panelists marked their own conditions in such a manner that the left end is the best condition (with no symptom) and the right end is the worst condition (one's worst-symptom ever). A larger score means a highest subjective symptom in every evaluation item.

[0063] In the physicochemical test shown in Table 5, test paper for Flores eye examination (by AYUMI Pharmaceutical Corporation) was used for detection of tear film; in the eyesight test, a space saving chart (ssc-370 Type D, by NIDEK Corporation) was used; and in the intraocular pressure test, a multifunction refractometer (MR-6000, by TOMEY Corporation) was used. In Table 5, a perforated card method was employed for decision of “superior eye” (dominant eye) and “non-superior eye” (non-dominant eye).

[0064] The scores of antiaging QOL common medical questionnaire shown in Table 6 are in five stages of the physical symptom of each item, “1: No, not at all”, “2: Yes, but little”, “3: Yes, a little”, “4: Yes, average”, and “5: Yes, highly”.

[0065] The values shown in Tables 4 to 6 and the drawing are average values of all the panelists. The significant level in each evaluation item is in a two-sided test for the variation after 4 weeks from the start of intake relative to before the start of intake, 5% (P value=0.05).

TABLE-US-00004 TABLE 4 Before/After Comparison before 4 Weeks Intake after Intake Subjective Symptom (average (average P value of Test Item value) value) Variation Dry Eye QOL QOL Score 52.40 23.60 0.002 Questionnaire VAS Ocular Dryness 67.70 34.70 0.001 questionnaire Ocular Roughness 46.50 25.10 0.021 Ocular Pain 51.90 27.70 0.017 Ocular Bleariness 68.60 32.00 0.002 Vision Sharpness 60.50 28.80 0.007 Depth of Sleep 65.30 31.90 0.013

TABLE-US-00005 TABLE 5 Before/After Comparison before 4 Weeks Intake after Intake (average (average P value of Physicochemical Test Item value) value) Variation Tear Film non-dominant eye 3.20 5.50 0.001 Breaking (sec) Time (BUT) average of both 3.63 5.54 0.023 eyes (sec) Vision Test corrected (average 1.38 1.58 0.044 of both eyes) Intraocular dominant eye 13.02 11.74 0.014 Pressure Test non-dominant eye 12.96 11.66 0.015 average of both eyes 12.99 11.70 0.004

TABLE-US-00006 TABLE 6 Before/After Comparison Antiaging QOL before 4 Weeks Common Medical Intake after Intake Questionnaire (physical (average (average P value of symptom test item) value) value) Variation Eye Tired 4.3 2.9 0.003 Eye Blurred 3.4 2.2 0.006 Eye Pain 3.1 1.8 0.006 Dizzy 2.3 1.3 0.016

[0066] As shown in Tables 4 to 6, when comparison was made between before the start of intake of protease degradation product-containing capsules and after 4 weeks from intake thereof, the dry eye subjective symptoms, the physical symptoms relating to eye condition (depth of sleep, test items shown in Table 6), the corrected eyesight and the intraocular pressure were significantly improved in 4 weeks after the intake. In addition, as shown in the drawing, the dry eye subjective symptoms were greatly relieved in one week after intake. From the above results, it is confirmed that, by oral intake of the degradation product produced by degrading combs with a protease, eye conditions can be improved.