URSODEOXYCHOLIC ACID-CONTAINING AGENT FOR TREATING OR PREVENTING PRESBYOPIA

Abstract

The present disclosure provides an agent for treating or preventing eye diseases such as presbyopia, comprising, as an active ingredient, ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof.

Claims

1-12. (canceled)

13. A method for treating or preventing presbyopia, comprising administering to a subject in need thereof an effective amount of ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein the ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof is administered ophthalmically.

15. The method according to claim 13, wherein the ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof is administered as an eye drop or an eye ointment.

16. The method according to claim 15, wherein the amount of ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof comprised in the eye drop or the eye ointment is 0.00001 to 10% (w/v).

17. The method according to claim 13, wherein the ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof is selected from ursodeoxycholic acid, tauroursodeoxycholic acid, glycoursodeoxycholic acid, ursodeoxycholic acid methyl ester, ursodeoxycholic acid ethyl ester, ursodeoxycholic acid n-propyl ester, ursodeoxycholic acid isopropyl ester, ursodeoxycholic acid n-butyl ester, ursodeoxycholic acid isobutyl ester, ursodeoxycholic acid sec-butyl ester, ursodeoxycholic acid tert-butyl ester, ursodeoxycholic acid n-pentyl ester, ursodeoxycholic acid n-hexyl ester, and a pharmaceutically acceptable salt thereof.

18. The method according to claim 13, wherein the ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof is selected from ursodeoxycholic acid, tauroursodeoxycholic acid, glycoursodeoxycholic acid, ursodeoxycholic acid methyl ester, ursodeoxycholic acid ethyl ester, ursodeoxycholic acid n-propyl ester, ursodeoxycholic acid isopropyl ester, and a pharmaceutically acceptable salt thereof.

19. The method according to claim 13, wherein the ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof is ursodeoxycholic acid or a sodium salt thereof.

20. The method according to claim 15, wherein the eye drop or the eye ointment comprises water, and an additive selected from ethyl pyruvate, sodium dihydrogenphosphate monohydrate, disodium hydrogenphosphate, hydroxypropyl methylcellulose, NaCl, and a mixture thereof.

21. A method for treating or preventing an eye disease accompanied by a decrease in lens elasticity, comprising administering to a subject in need thereof an effective amount of ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof.

22. A method for treating or preventing an eye disease accompanied by a decrease in accommodative function of the eye, comprising administering to a subject in need thereof an effective amount of ursodeoxycholic acid or an amide conjugate of ursodeoxycholic acid, or an ester thereof, or a pharmaceutically acceptable salt thereof.

Description

EXAMPLES

[0085] The results of pharmacological tests are shown below for a better understanding of the present invention and are not intended to limit the scope of the present invention.

[Pharmacological Test 1]

[0086] The effect of lipoic acid choline ester (EV06) on the lens elasticity was examined. The tests were conducted with reference to the methods described in InvestOphthalmol Vis Sci, 57, 2851-2863, 2016. EV06 is a compound represented by the following formula (2):

##STR00004##

(Preparation of Test Sample)

1) Preparation of Vehicle

[0087] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2PO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, and 0.5% (w/v) of NaCl was prepared.

2) Preparation of EV06 Sample

[0088] EV06 was sonicated with the addition of the vehicle to prepare a 5% (w/v) suspension. The resulting 5% (w/v) suspension was diluted with the vehicle to prepare a 1.5% (w/v) solution. Further, the resulting 1.5% (w/v) solution was diluted with the vehicle to prepare a 0.5% (w/v) solution. The total amount of each sample to be used in one day was prepared before use.

(Test Method)

[0089] 1) Each test sample (2.5 μL/eye) was instilled into the right eye of 8-month-old C57BL/6J mice with a Pipetman 3 times per day (around 9:00, 13:00 and 17:00) for 15 to 17 days.
2) After the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (Image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below.

[0090] The mean of the vehicle control group was based on 6 eyes and the mean of each EV06 sample group was based on 12 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each EV06 sample group−Mean charge in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0091] The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Lens elasticity improvement (μm) 0.5% EV06 sample 28.8 1.5% EV06 sample 47.3   5% EV06 sample 48.7

[0092] As shown in Table 1, all of the 0.5%, 1.5%, and 5% EV06 groups showed the increased lens diameter compared with the vehicle control group, which confirms that EV06 has an elasticity improving effect.

[Pharmacological Test 2]

[0093] The effect of sodium ursodeoxycholate on the lens elasticity was examined.

(Preparation of Test Sample)

1) Preparation of Vehicle

[0094] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

2) Preparation of Sodium Ursodeoxycholate Sample

[0095] Sodium ursodeoxycholate was sonicated with the addition of the vehicle to prepare a 1.5% (w/v) suspension. The resulting 1.5% (w/v) suspension was diluted with the vehicle to prepare a 0.5% (w/v) suspension. Further, the resulting 0.5% (w/v) suspension was diluted with the vehicle to prepare a 0.15% (w/v) suspension. The total amount of each sample to be used in one day was prepared before use.

3) Preparation of EV06 Sample

[0096] EV06 was sonicated with the addition of the vehicle to prepare a 1.5% (w/v) solution. The total amount of the sample to be used in one day was prepared before use.

(Test Method)

[0097] 1) Each test sample (2.5 μL/eye) was instilled into the right eye of 8-month-old C57BL/6J mice with a Pipetman 3 times per day (around 9:00, 13:00 and 17:00) for 12 to 15 days.
2) After the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS)).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (Image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. The mean of the vehicle control group was based on 5 eyes, the mean of each sodium ursodeoxycholate sample group was based on 10 eyes, and the mean of the EV06 sample group was based on 10 eyes.

Change in lens diameter Lens diameter in image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0098] The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Lens elasticity improvement (μm) 0.15% sodium 26.5 ursodeoxycholate sample  0.5% sodium 34.8 ursodeoxycholate sample  1.5% sodium 44.7 ursodeoxycholate sample 1.5% EV06 sample 38.2

[0099] As shown in Table 2, all, of the 0.15%, 0.5%, and 1.5% sodium ursodeoxycholate sample groups showed a potent lens elasticity improving effect. The lens elasticity improving effect of the 1.5% sample group was stronger than that of EV06 at the same concentration.

[Pharmacological Test 3]

[0100] The effect of ursodeoxycholic acid (free form) on the lens elasticity was examined.

(Preparation of Test Sample)

1) Preparation of Vehicle

[0101] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, and 0.5% (w/v) of NaCl was prepared.

2) Preparation of Ursodeoxycholic Acid Sample

[0102] Ursodeoxycholic acid was sonicated with the addition of the vehicle to prepare a 1.5% (w/v) suspension. The resulting 1.5% (w/v) suspension was diluted with the vehicle to prepare a 0.5% (w/v) suspension. Further, the resulting 0.5% (w/v) suspension was diluted with the vehicle to prepare a 0.15% (w/v) suspension. The total amount of each sample to be used in one day was prepared before use.

3) Preparation of EV06 Sample

[0103] EV06 was sonicated with the addition of the vehicle to prepare a 1.5% (w/v) solution. The total amount of the sample to be used in one day was prepared before use.

(Test Method)

[0104] 1) Each test sample (2.5 μL/eye) was instilled into the right eye of 8-month-old C57BL/6J mice with a Pipetman 3 times per day (around 9:00, 13:00 and 17:00) for 12 to 15 days.
2) After the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (Image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. The mean of the vehicle control group was based on 5 eyes, the mean of each ursodeoxycholic acid sample group was based on 10 eyes, and the mean of the EV06 sample group was based on 10 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0105] The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Lens elasticity improvement (μm) 0.15% ursodeozycholic 31.8 acid sample  0.5% ursodeoxycholic 39.4 acid sample  1.5% ursodeoxycholic 59.9 acid sample 1.5% EV06 sample 42.5

[0106] As shown in Table 3, all of the 0.15%, 0.5%, and 1.5% sodium ursodeoxycholate sample groups showed a potent lens elasticity improving effect. The lens elasticity improving effect of the 1.5% sample group was stronger than that of EV06 at the same concentration.

[Pharmacological Test 4]

[0107] The effect of once-daily instillation of ursodeoxycholic acid for 2 weeks on the lens elasticity was examined.

(Preparation of Test Sample)

1) Preparation of Vehicle

[0108] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

2) Preparation of Ursodeoxycholic Acid Sample

[0109] Ursodeoxycholic acid was sonicated with the addition of the vehicle to prepare a 3.0% (w/v) suspension. The resulting 3.0% (w/v) suspension was diluted with the vehicle to prepare a 1.0% (w/v) suspension. Further, the resulting 1.0% (w/v) suspension was diluted with the vehicle to prepare a 0.3% (w/v) suspension. The total amount of each sample to be used in one day was prepared before use.

3) Preparation of EV06 Sample

[0110] EV06 was sonicated with the addition of the vehicle to prepare a 1.5% (w/v) solution. The total amount of the sample to be used in one day was prepared before use.

(Test Method)

[0111] 1) Each test sample (2.5 μL/eye) was instilled into the right eye of 8-month-old C57BL/6J mice with a Pipetman once per day (QD; around 9:00), twice per day (BID; around 9:00 and 17:00), or 3 times per day (TID; around 9:00, 13:00 and 17:00) for 14 days.
2) After the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Reyence) was used to capture an image of the lens (image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. The mean of the vehicle control group was based on 5 eyes, the mean of each ursodeoxycholic acid sample group was based on 10 eyes, and the mean of each EV06 sample group was based on 10 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0112] The results are shown in Table 4.

TABLE-US-00004 TABLE 4 Lens elasticity improvement (μm) 0.3% ursodeoxycholic acid sample 2.8 (QD)   1% ursodeoxycholic acid sample 28.1 (QD)   3% ursodeoxycholic acid sample 30.4 (QD) 1.5% EV06 sample (QD) −3.6 1.5% EV06 sample (BID) 15.7 1.5% EV06 sample (TID) 29.5

[0113] As shown in Table 4, 1% ursodeoxycholic acid sample and 3% ursodeoxycholic acid sample caused a potent lens elasticity improvement when they were instilled once-daily while 1.51, EV06 sample instilled once-daily had no effect, which indicates that ursodeoxycholic acid has a more potent lens elasticity improvement effect compared with EV06.

[Pharmacological Test 5]

[0114] The effect of 1% ursodeoxycholic acid instilled once-daily for 1, 3, 7, 10, or 14 days on the lens elasticity was examined.

(Preparation of Test Sample)

[0115] 1) Preparation of Vehicle

[0116] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

[0117] 2) Preparation of 1% Ursodeoxycholic Acid Sample

[0118] Ursodeoxycholic acid was sonicated with the addition of the vehicle to prepare a 1.0% (w/v) suspension. The total amount of the sample to be used in one day was prepared before use.

(Test Method)

[0119] 1) The test sample (2.5 μL/eye) was instilled into both eyes of 8-month-old C57BL/6J mice with a Pipetman once per day (QD; around 13:30) for 1, 3, 7, 10, or 14 days.
2) Twenty-four hours after the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. Each mean of the untreated group and each ursodeoxycholic acid sample group was based on 9 or 10 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Untreated group  (Equation 2)

(Results)

[0120] The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Lens elasticity improvement (μm) 1% ursodeoxycholic acid sample 9.6 (1 day) 1% ursodeoxycholic acid sample 21.1  (3 days) 1% ursodecxycholac acid sample 27.4  (7 days) 1% ursodeoxycholic acid sample 38.1 (10 days) 1% ursodeoxycholic acid sample 34.2 (14 days)

[0121] As shown in Table 5, the 1% ursodeoxycholic acid sample instilled once-daily caused improvement in the lens elasticity according to the increase of the duration of instillation, and showed definitely an improvement in lens elasticity after instillation for 3 days. This suggests that ursodeoxycholic acid can early cause the effect.

[Pharmacological Test 6]

[0122] The effect of once-daily instillation of ursodeoxycholic acid methyl ester for 7 days on the lens elasticity was examined.

(Preparation of Test Sample)

[0123] 1) Preparation of Vehicle

[0124] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

[0125] 2) Preparation of Ursodeoxycholic Acid Methyl Ester Sample

[0126] Ursodeoxycholic acid methyl ester was sonicated with the addition of the vehicle to prepare a 0.3% (w/v) suspension, a 1.0% (w/v) suspension and a 3.0% (w/v) suspension. The total amount of each sample to be used in one day was prepared before use.

(Test Method)

[0127] 1) Each test sample (2.5 μL/eye) was instilled into both eyes of 7-month-old C57BL/6J mice with a Pipetman once per day (QD; around 13:30) for 7 days.
2) Twenty-four hours after the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (Image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. Each mean of the vehicle control group and each ursodeoxycholic acid methyl ester sample group was based on 9 or 10 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0128] The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Lens elasticity improvement (μm) 0.3% ursodeoxycholic acid 13.2 methyl ester sample   1% ursodeoxycholic acid 33.0 methyl ester sample   3% ursodeoxycholic acid 46.5 methyl ester sample

[0129] As shown in Table 6, 1% ursodeoxycholic acid methyl ester sample and 3% ursodeoxycholic acid methyl ester sample caused a potent lens elasticity improvement even when they were instilled once-daily. These results suggest that ursodeoxycholic acid methyl ester may also have a more potent lens elasticity improvement effect compared with 1.5% EV06.

[Pharmacological Test 7]

[0130] The effect of tauroursodeoxycholic acid instilled once-daily for 7 days on the lens elasticity was examined.

(Preparation of Test Sample)

1) Preparation of Vehicle

[0131] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

2) Preparation of Tauroursodeoxycholic Acid Sample

[0132] Tauroursodeoxycholic acid was dissolved with the addition of the vehicle to prepare a 1.0% (w/v) solution. The total amount of each sample to be used in one day was prepared before use.

(Test Method)

[0133] 1) Each test sample (2.5 μL/eye) was instilled into both eyes of 7-month-old C57BL/6J mice with a Pipetman once per day (QD; around 13:30) for 7 days.
2) Twenty-four hours after the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (Image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. Each mean of the vehicle control group and the tauroursodeoxycholic acid sample group was based on 10 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0134] The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Lens elasticity improvement (μm) 1% tauroursodeoxycholic 29.7 acid sample

[0135] The 1% tauroursodeoxycholic acid sample instilled once-daily caused a potent lens elasticity improvement as shown in Table 7 while the 1.5% EV06 sample instilled once-daily for 14 days had no effect as shown in Table 4, which suggests that tauroursodeoxycholic acid has a more potent lens elasticity improvement effect than EV06.

[Pharmacological Test 8]

[0136] The effect of glycoursodeoxycholic acid instilled once-daily for 7 days on the lens elasticity was examined.

(Preparation of Test Sample)

[0137] 1) Preparation of Vehicle

[0138] A vehicle comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (NaH.sub.2PO.sub.4H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

[0139] 2) Preparation of Glycoursodeoxycholic Acid Sample

[0140] Glycoursodeoxycholic acid was sonicated with the addition of the vehicle to prepare a 1.0% (w/v) suspension. The total amount of each sample to be used in one day was prepared before use.

(Test Method)

[0141] 1) Each test sample (2.5 μL/eye) was instilled into both eyes of 8-month-old C57BL/6J mice with a Pipetman once per day (QD; around 13:30) for 7 days.
2) Twenty-four hours after the final instillation, the mice were euthanized by carbon dioxide inhalation, and then the eyeballs were extracted and rinsed with Hank's balanced salt solution (HBSS).
3) The sclera near the optic nerve was cut with a razor, the lens was removed through the incision, and the removed lens was immersed in HBSS.
4) The lens was placed on a glass slide, and an all-in-one fluorescence microscope BZ-9000 (Keyence) was used to capture an image of the lens (image a).
5) Next, one cover glass (Corning.sup.(registered trade mark) 22×22 mm Square) was placed on the lens, and an image in which the thickness of the lens changed due to the weight was similarly captured (Image b).
6) A change in the lens diameter was calculated from Equation 1 wherein the lens diameter of Image a is subtracted from the lens diameter of Image b, as described below. Then, the lens elasticity improvement of each sample group compared with the vehicle control group was calculated from Equation 2 described below. Each mean of the vehicle control group and the glycoursodeoxycholic acid sample group was based on 9 to 10 eyes.

Change in lens diameter=Lens diameter in Image b of each test sample−Lens diameter in Image a of each test sample  (Equation 1)

Lens elasticity improvement of each sample group=Mean change in lens diameter of each Test sample group−Mean change in lens diameter of Vehicle control group  (Equation 2)

(Results)

[0142] The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Lens elasticity improvement (μm) 1% glycoursodeoxycholic 20.1 acid sample

[0143] The 1% glycoursodeoxycholic acid sample instilled once-daily caused a potent lens elasticity improvement as shown in Table 8 while the 1.5% EV06 sample instilled once-daily for 14 days had no effect as shown in Table 4, which suggests that glycoursodeoxycholic acid has a more potent lens elasticity improvement effect than EV06.

[Ocular Irritation Test]

(Preparation of Sample)

[0144] A vehicle (aqueous solution) comprising 0.1% (w/v) of ethyl pyruvate, 0.269% (w/v) of sodium dihydrogenphosphate monohydrate (Na.sub.2PO.sub.4.H.sub.2O), 0.433% (w/v) of disodium hydrogenphosphate (Na.sub.2HPO.sub.4), 0.2% (w/v) of hydroxypropyl methylcellulose, 0.5% (w/v) of NaCl was prepared.

(Test Method)

[0145] Group treated with an ophthalmic suspension of ursodeoxycholic acid 1% (w/v), 3% (w/v), and 10% (w/v) ursodeoxycholic acid ophthalmic suspensions were prepared in the same manner as in the above pharmacological tests. These ophthalmic suspensions and the vehicle were each instilled into the left eye of Japanese White rabbits at a dose of 50 μL/eye with pipette twice per day at a 6-hour interval for 2 weeks. One hour after the final instillation, ocular irritation of anterior segment of the eye was evaluated according to the McDonald-Shadduck method, and the lens was observed. The contralateral eye was untreated.

[0146] The ocular irritation of anterior segment of the eye was scored according to the following criteria:

+1: mild; +2: moderate; +3: severe.

(Test Result)

[0147] The test results are shown in Table 9. After the 2 week-repeated instillation, no abnormal findings were observed in eyes treated with the ophthalmic suspensions of ursodeoxycholic acid in the observation of ocular irritation of anterior segment of the eye and lens observation. Histopathological examination of the eyes showed no abnormal findings.

TABLE-US-00009 TABLE 9 1% 3% 10% Ursodeoxy- Ursodeoxy- Ursodeoxy- Ophthalmic cholic cholic cholic suspension Vehicle acid acid acid Number of animals 3 3 3 3 Ocular Conjunctival — — — — irritation hyperemia of Palpebral — — — — anterior conjunctival segment edema of Discharge — — — — the Corneal — — — — eye .sup.1) opacity Corneal — — — — epithelial disorder Lens — — — — Histopathological — — — — examination —: No noteworthy findings, .sup.1) Score of the instilled left eye and the number of the eye, 1 hour after the final instillation are described.

(Discussion)

[0148] It is shown that the ophthalmic suspensions of ursodeoxycholic acid is highly safe.

INDUSTRIAL APPLICABILITY

[0149] The agent of the present invention is useful for treating or preventing eye diseases such as presbyopia etc.