AU2013203609B2 - Method for screening drug efficacious in treating dry eye and/or keratoconjunctival disorders and pharmaceutical composition obtained thereby - Google Patents

Abstract

Provided are a method for screening a drug which is efficacious in treating dry eye with a dry eye severity 5 level 3 or higher that is defined in 2007 report of the International Dry Eye Workshop and/or keratoconjunctival disorders, and a pharmaceutical composition comprising said drug. Also provided is a method for treating dry eye with a dry eye severity level 3 or higher that is defined in 2007 10 report of the International Dry Eye Workshop and/or keratoconjunctival disorders by using the aforesaid drug.

Description

1 DESCRIPTION (Title of the Invention) Method for screening drug efficacious in treating dry eye and/or keratoconjunctival disorders and pharmaceutical 5 composition obtained thereby This application is a divisional application of Australian Application No. 2010323566 the specification of which as originally filed are incorporated herein in their entirety 10 by reference. [Technical Field] [0001] The present invention provides a method for screening an agent being useful for the treatment of dry 15 eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to the report of the International Dry Eye WorkShop (DEWS Report) (2007) and a pharmaceutical composition comprising the agent. The present invention further provides a method for the 20 treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007) using the agent. [Background Art] 2 00021 DEWS Report (2007) is a review of current knowledge on dry eye by 70 international experts of dry eye and subcommittees of DEWS provides details of dry eye including definition and classification, epidemiology, 5 diagnosis and monitoring, and management and therapy. The report is published in "The Ocular Surface (APRIL 2007, VOL 5, NO.2 pp. 65-204)" (non-patent literature 1, herein incorporated by reference) and can be viewed at a website (www.tearfilm.org). 10 [ 00031 According to DEWS Report (2007), dry eye is defined as follows (see "The Definition and Classification of Dry Eye Disease" (p75-92)): "Dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film 15 instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface." [ 00041 Since dry eye is a multifactorial disease as defined above, treatments of dry eye vary depending on its 20 conditions and symptoms. DEWS Report (2007) classifies dry eye severity level into 4 levels (see "Management and Therapy of Dry Eye Disease" (p163-178) and Table 2 (p173)) and provides treatment recommendations for each severity level (see "Management and Therapy of Dry Eye Disease" and 25 Table 4) (p174)).

3 00051 According to DEWS Report (2007), there are several agents recommended for dry eye severity levels 1 and 2. However, for dry eye severity level 3 or more, particularly for lesion of ocular surface such as cornea 5 and conjunctiva, there is no recommendable agent other than serum. Further, serum should be derived from blood of a patient to be treated (that is, autologous serum) and should be diluted appropriately when administered to eyes. [ 00061 At present, substantially, there is no effective 10 agent for dry eye of dry eye severity level 3 or more according to DEWS Report (2007). Therefore, it has been required to establish an animal model representing dry eye according to the classification of DEWS Report (2007), in particular dry eye of dry eye severity level 3 or more 15 according to the report, specifically lesion of ocular surface such as cornea and conjunctiva. Also, by using the animal model for drug screening, it has been required to provide a pharmaceutical composition being useful for dry eye of dry eye severity level 3 or more according to DEWS 20 Report (2007), specifically lesion of ocular surface such as cornea and conjunctiva. [Prior art references] [ 00071 25 [ Non-patent literature] The Ocular Surface (APRIL 2007, VOL I'H-\AYL\Intenvoven\NRPottbl\DCC\AYL\6552277_.doc-7/28/2014 4 5, NO.2 pp. 65-204) [Summary of Invention] [0008] In one aspect, the present invention provides a 5 method for screening an agent being useful for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007). In another aspect, the present invention provides a pharmaceutical composition for the treatment of dry eye and/or corneal and 10 conjunctival lesion as defined above. [0009] In one or more embodiments, the present invention may provide: e A method for screening an agent being useful for the treatment of dry eye and/or corneal and conjunctival lesion of 15 dry eye severity level 3 or more according to the report of the International Dry Eye WorkShop (DEWS Report) (2007), which comprises preparing a model rabbit of corneal and conjunctival lesion having corneal and conjunctival epithelial abrasion by 20 instilling n-heptanol solution to an eye of a rabbit; and administering a test agent to the eye of the model rabbit and determining an effect of restoring corneal tissue of the test agent. a A pharmaceutical composition for the treatment of dry eye 25 and/or corneal and conjunctival lesion of dry eye severity l'H:\AYL\Intenvoven\NRPortbl\DCC\AYL\6552277_l.do-7/28/2014 5 level 3 or more according to DEWS Report (2007), which comprises an agent obtained by the method of (1). e The pharmaceutical composition of (2), wherein the agent is human albumin. 5 e A pharmaceutical composition for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007), which comprises human albumin as an active agent. 0 The pharmaceutical composition of (4) , wherein human 10 albumin is human serum albumin. a A method for the treatment of dry eye and/or corneal and conjunctival lesion, which comprises administering an effective amount of an agent obtained by the method of (1) to a patient having dry eye and/or corneal and conjunctival lesion of dry 15 eye severity level 3 or more according to DEWS Report (2007). 0 A method for the treatment of dry eye and/or corneal and conjunctival lesion, which comprises administering an effective amount of human albumin to a patient having dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or 20 more according to DEWS Report (2007). * Use of an agent obtained by the method of (1) for the manufacture of a pharmaceutical composition for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007). 25 e Use of human albumin for the manufacture of a 1'H.AYL\Intenvovcn\NRPortbl\DCC\AYL\6552277l1.doc-7/28/2014 6 pharmaceutical composition for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007). 5 [Effect of the invention] [0010] According to the instant application, it has become possible to provide a method for screening an agent being useful for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more 10 according to DEWS Report (2007) . The agent determined as having an effect of restoring corneal tissue according to the screening method of the present invention is useful for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report 15 (2007). Further, according to the instant application, it has become possible to provide a method for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007) by using the agent.

7 [Description of Embodiments] [0011] According to DEWS Report (2007), dry eye severity level is classified as follows (see "Management and Therapy 5 of Dry Eye Disease" (p163-178) and Table 2 (p173)). Table 1] Dry eye severity grading scheme Discomfort, severity Mild and/or episodic Moderate episodic or Severe frequent or Severe and/or & frequency occurs under environ chronic, stress or no constant without disabling and constant stress stress strces None or episodic mild Annoying arid/or activity Anoying, chronic iind/ Constat aind/or Visual symptoms fatigue limiting episodic or constant limiting possibly disabling activity Conjunctival injection None to mild None to mild +/ +/++ Conjrutival sLaining None to Mild Variable Moderate to marked Marked Corneal stairing None to mild Variable Marked central Severe punctate (severity/ ocation) erosions Filamentary keratitis, Filamentary keratitis, Cornnal/tnar signs None to mild Mild debris, I meniscus mucus clumping, mucus clumping, T tear debris I teardebris, ulceraton LI d/meibom ian glands MGD variably present MGD variably present Frequent Tn sis, keratinization, TFBUT (sec) Variable <10 < 5 Immediate Schirmer score Variable i10 5 2 (nim/5 nin) *Mst have signs AND symptoms TBUT: fluorescein tear break-up time. MGD: meibomian glad disease The "dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report 10 (2007) " as used in herein refers to dry eye corresponding to dry eye severity level 3 or more according to the above classification, and/or corneal and conjunctival lesion showing any of signs and symptoms of such dry eye. The term "about" as used herein, when used with any 15 numerical number, refers to ±7- 30% and ±7- 20%, and preferably +/- 10%.

8 00121 The model rabbit of corneal and conjunctival lesion used in the screening method of the present invention is prepared by instilling n-heptanol solution to an eye of a rabbit. Generally, the model rabbit may be 5 prepared by anesthetizing a rabbit by intramuscular injection of an anesthetic agent and then by ocular administration of a topical anesthetic agent, instilling n heptanol solution to one of the eyes of the rabbit such that the n-heptanol solution spreads over cornea and bulbar 10 conjunctiva, and forcing the rabbit to blink. [ 00131 Species of rabbit used in the present invention may be, but not limited to, New Zealand white, Japanese White, and Dutch, and New Zealand white is preferably used. The anesthetic agent administered by intramuscular 15 injection may include ketamine, xylazine, and medetomidine, and preferably, ketamine and xylazine are used in combination. The topical anesthetic agent for ocular administration may include oxybuprocaine, lidocaine, and bupivacaine, and oxybuprocaine is preferably used. 20 N-heptanol solution instilled into an eye is a solution consisting of n-heptanol only, or a mixture of n heptanol and ethanol. The mixture of n-heptanol and ethanol is preferred because it has improved hydrophilicity and can easily spread over ocular surface and efficiently 25 induce lesion. Ratio of n-heptanol and ethanol is about 9: 9 1 to about 7: 3, preferably about 8: 2. The applying volume of n-heptanol solution is about 0.01 to about 0.1 ml, preferably about 0.03 to about 0.05 ml in total and it is preferably instilled in several times (2-4 times) such that 5 the n-heptanol solution efficiently spreads over cornea and bulbar conjunctiva. After instillation of n-heptanol solution to an eye, the rabbit is forced to blink (1-5 times, preferably 2-4 times), and forced to close the eye for several minutes 10 (about 0.5 to about 5 min, preferably about 1 to about 3 min). As a result, corneal and conjunctival epithelial abrasion is occurred. According to the present invention, a stable model of corneal and conjunctival lesion can be prepared. 15 [ 00141 By determining an effect of restoring corneal tissue of a test agent by using the model rabbit of corneal and conjunctival lesion of the present invention, it is possible to screen an agent being useful for the treatment of dry eye and/or corneal and conjunctival lesion of dry 20 eye severity level 3 or more according to DEWS Report (2007) . [00151 In the screening method of the present invention, frequency of ocular administration of a test agent is generally, but not limited to, 1-6 times per day. To 25 determine potency of a test agent regarding the effect of 10 restoring corneal tissue, frequency of ocular administration per day is preferred to be lower and preferably twice per day in general. Duration of ocular administration of a test agent may vary depending on the 5 factors such as timing of the test and species of the rabbit. Generally, the effect of a test agent can be determined in 10 days from the start of its ocular administration. [00161 Generally, the effect of restoring corneal tissue 10 of a test agent may be determined as follows. Conditions of cornea and conjunctiva are observed by staining an eye with a dye before and after instillation of n-heptanol solution; before first-time administration of the test agent on each day; after second-time administration of the 15 test agent on each of days 4, 5, and 6; and one day after the final administration of the test agent. The dye for staining may include fluorescein, rose bengal, and lissamine green and fluorescein is preferably used. Degree of corneal lesion is scored by observing and photographing 20 conditions of corneal staining. A high staining score represents a high degree of corneal lesion and a low staining score represents a low degree of corneal lesion. [00171 For scoring, any conventional method may be used. For example, cornea is divided into 5 areas (central, upper, 25 temporal, nasal and lower areas) and staining intensity of 11 each area is scored out of 3 and the total score (out of 15) is used for determination. Staining intensity is scored 0 for no staining; 1 for partial staining; 2 for staining of about two-third of the area; and 3 for overall 5 staining. [00181 In the present invention, the effect of restoring corneal tissue of a test agent is determined to be significant when significant difference between the staining score showing degree of corneal lesion of the eye 10 administered with the test agent and that of the eye administered with saline is determined at each observation of cornea and conjunctiva and the significance level is less than 5%. Significant difference may be determined by any method appropriately, and for example, by Paired 15 Student's t-test. [ 0019] For agents each recommended for dry eye severity level 1, 2 or 3 in DEWS Report (2007), the effect of restoring corneal tissue was confirmed by using the model rabbit of corneal and conjunctival lesion of the present 20 invention. Agents recommended in DEWS Report (2007) for dry eye severity levels 1 and 2, such as a formulation containing viscous component (Hyalein@ mini ophthalmic solution 0.3%: Santen Pharmaceutical (Japan): approved in Japan) or a formulation containing electrolyte 25 (Cationorm@: Novagali Pharma (France): approved in EU) for 12 dry eye severity level 1, and a formulation containing an anti-inflammatory agent (Restasis@: Allergan ( us ) approved in US) for dry eye severity level 2, did not show a significant effect of restoring corneal tissue. On the 5 other hand, serum, which is recommended for dry eye severity level 3 (human serum pools: Kohjin Bio (Japan)) showed a significant effect of restoring corneal tissue. Accordingly, the method of determining the effect of restoring corneal tissue by using the model rabbit of 10 corneal and conjunctival lesion of the present invention is understood to be useful for screening an agent being useful for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007). 15 [ 00201 Further, according to the screening method of the present invention, it has been revealed that human serum albumin has a significant effect of restoring corneal tissue in the model rabbit of corneal and conjunctival lesion of the present invention, similar to the agent 20 recommended in DEWS Report (2007) for dry eye severity level 3 (serum). Therefore, humen albumin is understood to be useful for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007). 25 [ 00211 In the present invention, human albumin is 13 preferably human serum albumin. Any human serum albumin may be used in the present invention, on condition that it has purity for general medical purpose. Specifically, it is preferred to contain albumin 80% or more when analyzed by 5 electrophoresis. In addition, it is preferred to be heat treated for inactivation of virus or others. Particularly, human serum albumin commercially available as a medicament is preferred. Recombinant human serum albumins produced by microorganisms are also preferably used for the present 10 invention. Methods for preparing recombinant human serum albumins using gene-recombination technology are well known in the art. For example, a vector containing a gene encoding human serum albumin is prepared and introduced into host cells such that the cells are transformed. Then, 15 transformed cells producing human serum albumin are selected and cultured, and from the cells or the culture media thereof, human serum albumin is isolated and purified. The host cell may include microorganisms commonly used in the art for protein production, such as yeasts and E. Coli. 20 Examples of yeasts may include those belonging to the genus Kluyveromyces, Saccharomyces and Pichia, and in partucular, Kluyveromyces laactis, Saccharomyces cerevisiae and Pichia pastoris are preferably used. It is still preferable to use Kluyveromyces laactis CBS2360 strain, Saccharomyces 25 cerevisiae AH22 strain (a, his 4, leu 2, can 1) and Pichia 14 pastoris GTS115 strain (his 4) as the host yeast. [ 00221 As used herein, the term "treatment" or "treating" refers to any means of control of the conditions, including prevention, cure and relief of the conditions and 5 arrestation or relief of development of the condition. [ 00231 The pharmaceutical composition of the present invention may be in any dosage form for topical administration to eyes. It is particularly preferred to be ophthalmic solutions or eye drops. 10 [ 00241 In case of the composition of the present invention is formulated as an ophthalmic solution comprising human albumin as an active agent, the composition may contain human albumin in an amount of about 1 to 1000 mg/ml (0.1-100w/v%), more preferably about 10 to 15 1000 mg/ml(1-100w/v%), and especially, about 10 to 250 mg/ml (1-25w/v%) . The composition may further contain a pharmaceutically acceptable diluent. [ 00251 As used herein, the "pharmaceutically acceptable diluent" may be any diluent which is used for ophthalmic 20 composition known to persons skilled in the art, for example, water, physiological saline, artificial tear solution, and the like. The pharmaceutical composition of the present invention may further comprise various components that are generally used in ophthalmic 25 compositions, such as stabilizers, sterilizers, buffering 15 agents, isotonic agents, chelating agents, pH adjusters, surfactants, and the like. When the composition is formulated as an ophthalmic solution or eye drops, the pH of the composition is preferably adjusted from 5 to 8. 5 [ 00261 The pharmaceutical composition of the present invention may be administered in an amount of about 1 to about 100 pl/eye, preferably about 10 to about 50 pl/eye, and more preferably about 30 to 50 pl/eye for one administration. 10 [ 00271 In a different aspect, the present invention provides use of human albumin for the manufacture of a pharmaceutical composition for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007). 15 [ 00281 In a further different aspect, the present invention provides a method for the treatment of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007), which comprises administering to a subject in need threrof an 20 effective amount of human albumin. [ 00291 In these methods of the present invention, the "effective amount" of human albumin is an amount required for the desirable treatment, and may be selected an optimum according to the patient's symptoms, age, sex, body weight, 25 diet, other drugs used in combination and various factors 16 which are recognized by persons skilled in the medical field. This effective amount may also vary depending on the kind or activity of humna alubmin, in addition to the above factors. In these methods of the present invention, 5 the pharmaceutical composition may be administered in an amount of about 1 to about 100 pl/eye, preferably about 10 to about 50 pl/eye and more preferably about 30 to 50 pl/eye, about 1 to about 20 times per day and more preferably, 1 to 10 times per day, but it is not intended 10 to limit the scope of the invention. [ 00301 The pharmaceutical composition used in the present invention may be co-administered with other pharmaceutically active compound unless it does not interfere with the effect of the present invention. The 15 term "co-administer" may include to administer the other pharmaceutically active compound before, simultaneously with and after administering the pharmaceutical composition of the present invention. When the other pharmaceutically active compound is administered simultaneously with the 20 pharmaceutical composition of the present invention, the composition of the present invention and the other compound may be formulated together in single dosage form or respectively in separate dosage forms. For example, artificial tear solution, polysaccharide sulfate such as 25 hyaluronic acid and chondroitin sulfate, cyclosporine, 17 glutathione, flavin-adenine nucleotide sodium, corticosteroid, tetracycline, vitamin A (retinol) and its derivatives such as vitamin A esters including retinol palmitate and retinol acetate, and cell growth factor such 5 as hepatocyte growth factor(HGF), epidermal growth factor(EGF), fibroblast growth factor(FGF), insulin-like growth factor(IGF), nerve growth factor(NGF), platelet derived growth factor(PDGF), transforming growth factor(TGF-a and TGF-B) and keratinocyte growth factor(KGF) 10 may be co-administered with the pharmaceutical composition of the present invention. [ 00311 The present invention will be further explained in view of test examples shown below. The test examples should not be used for limiting the scope of the instant 15 application in any means. [Examples] [00321 Test Example 1 (Preparation of corneal and conjunctival lesion model) 20 Rabbits (std:NZW) was anesthetized by intramuscular injection of ketamine (10 mg/kg) and xylazine (4 mg/kg) and then by ocular administration of 0.4% oxybuprocaine (2 drops per eye). The rabbits were held in a recumbent position and instilled 0.04 ml (0.01 ml X 4 times) of n 25 heptanol solution (n-heptanol: ethanol = 8: 2), prepared 18 just before use, into one of eyes such that the n-heptanol solution spreads over cornea and bulbar conjunctiva. Then, the rabbits were forced to blink 3 times and to close the eyes for 2 min after the third blinking. The both eyes 5 were given this process. Thus, models of corneal and conjunctival lesion were prepared. 00331 (Administration of test agents) Starting from the day of preparation of corneal and 10 conjunctival lesion, serum or 0.3% sodium hyaluronate formulation was administered repeatedly 2 times per day for 10 days. Saline was administered in a same manner to the other eyes than those administered with the test agent. They were all administered in a volume of 50 tl/eye/time. 15 For use as serum, human serum pools (Kohjin Bio (Japan)) was sterilized by filtration (MILLEX GV, PVDF, 0.22 tm, MILLIPORE) and diluted 5 times with saline according to the method known to prepare serum ophthalmic solution used in autologous serum therapy for dry eye. 20 The 0.3% sodium hyaluronate formulation was Hyalein@ mini ophthalmic solution 0.3% (Santen Pharmaceutical (Japan)). 00341 (Test groups) 25 [ Table 2] 19 number of eyes Group Test agent nme fee (number of cases) 1 Serum 6 eyes (6 animals) 2 0.3% sodium hyaluronate 6 eyes (6 animals) The rabbits were allocated to each group by stratified randomization on body weight. 00351 (Determination of corneal lesion) 5 Conditions of cornea and conjunctiva were observed before treatment of n-heptanol solution (one day before the first administration of the test agent); just after treatment of n-heptanol solution; before first-time administration of the test agent on each day; after second 10 time administration of the test agent on each of days 4, 5, and 6; and one day after the final administration of the test agent. Two 1tl of 1% fluorescein was instilled into the eyes. After 2 mins, staining of cornea and conjunctiva was 15 photographed and observed by a digital microscope (VHX-900, KEYENCE CORPORATION (Japan)) with a cobalt blue filter (FUJI FILTER BPB45) in the light emitting section and a yellow filter (FUJI FILTER SC52) in front of the lense, and then degree of corneal lesion was scored. For scoring, 20 each cornea was divided into 5 areas (central, upper, temporal, nasal and lower areas). Staining intensity with fluorescein of each area was scored out of 3 and the total score (out of 15) was used for determination. Staining 20 intensity was scored 0 for no staining; 1 for partial staining; 2 for staining of about two-third of the area; and 3 for overall staining. The 1% fluorescein was prepared by dissolving 5 fluorescein sodium salt with saline. 00361 (Result) Scores of fluorescein staining of cornea after the treatment of n-heptanol solution were shown in Table 3. In 10 all animals, scores of fluorescein staining of cornea before the treatment of n-heptanol solution were 0. As shown in Table 3, in the eyes administered with serum, a significant effect of restoring corneal epithelium (p<0.05) was observed on day 4 of the treatment of lesion. 15 On the other hand, no effect of restoring corneal epithelium was observed with the administration of 0.3% sodium hyaluronate. 00371 Table 3] 21 CD m m C, co LO ts O o oio 0 o 10 c A 0 o L Q O CO c 1 0 .00 - 0 C O o 0 1 o Co C1- 01 0l 01 0 1 C 0 0 C 0100 10 xo N 4-o OO 0 cN C- Co N- 0 o cN - + 04 10 Co o L ) o o C Co 00 LZL C O C0 N o 210 N- 0 1 - - o0 e0 czc C> t t-- e N- C_ N o Co 0 I1 0 - 0> c0 L C A - - 0 06 - ,-' 0 0 0 o N- 10 N- o cc0 O 71 Qo S N- o0 0 o 0 0 Co 0 -~ 0 0 o Co 0 01 0 C 6o => C CO o -0 C N C o .. o:) 4-1 1 C 4 Co K 0o C 00 ~ C 1= N- C C - N- - 0 IC 4- c - - 6 : C/Co UC -~ ',CO 10 ~ 0 0 C~~ 0 0 0 0 0 0~~~ 0 0 0 0 o ,z.C/I 0 0 0 0. 3 0 ~ ~ ~ ~ c 0 0 ' z 0 22 [00381 Test Example 2 Preparation of corneal and conjunctival lesion model, administration of test agents, and determination of corneal lesion were performed as described in Test Example 1. 5 The test agent was 0.05% cyclosporine formulation or cationic emulsion. As 0.05% cyclosporine formulation, Restasis@ (Allergan, Inc (US) ) was used. As cationic emulsion, Cationorm@ (Novagali Pharma SA (France)) was used. 10 [ 00391 (Test groups) Table 4] number of eyes Group Test agent nme fee (number of cases) 1 0.05% cyclosporine 6 eyes (6 animals) 2 cationic emulsion 6 eyes (6 animals) The rabbits were allocated to each group by stratified randomization on body weight. 15 [ 00401 (Result) Scores of fluorescein staining of cornea after the treatment of n-heptanol solution were shown in Table 5. In all animals, scores of fluorescein staining of cornea 20 before the treatment of n-heptanol solution were 0. As shown in Table 5, administration of any of 0.05% cyclosporine and cationic emulsion did not show an effect of restoring corneal epithelium.

23 [00411 According to the results of Test Examples 1 and 2, it was confirmed that the agent recommended in DEWS Report (2007) for dry eye severity level 3 (serum), but not the agents recommended for dry eye severity levels 1 and 2, 5 shows a significant effect of restoring corneal tissue in the determination system using the model rabbit of the present invention. Accordingly, the model rabbit of corneal and conjunctival lesion of the present invention is understood to be useful as a model animal representing dry 10 eye according to the classification of DEWS Report (2007), particularly dry eye of dry eye severity level 3 or more according to the report, specifically lesion of ocular surface such as cornea and conjunctiva. [ 00421 15 [ Table 5] 24 tN c t cm t- - t- o Q c5 C Q0 co - cq Cm C) -Y 0' cM CM cm cM cM - c0 - ) C CM 0 C2 - CM -i LQ cM cM - - CMi CM 0 o1 -aC 0 C C I-- 60 c- 20 4 - -t OO CN CD 02 CO CM C O -U 4-N CC CD CO CFl LQ T L0 o C CO L - L0 -* Q0 L CC o LO LO cL CL N M ) C 7C -l~ [-- a w r- CI C CC cl OD C G N- LO F- N LC C LO LO cc C I CM L C +) C T c~ o c.c-2 C CD C C O 4O 4- - LoD = N- r- to L) C LO Q C) 0 C 1 c0 - CM C C' 6 CGu -. O* r M,,C 2 CO O e* c ~ CDCO CDl CD CDC CZ 10 CMM LC LO 0 CD C C) cM CM 0 ) ' C 6 -t C cM C) O V)) t C,, ) C) CD CIO0 uO CO C C 0 C O) 2 2cL 66 6 610 C6 CD- C0 C> C) 0 CM N- O C) C 0 L 6 L 6 L6 6 UL2 ' CD0 0 C - -- 0 La C. La C. LO C Q O ;-C CD CM CD O~ ~~~~ 7G GW Q M C) r) C) C) C) r/ o 0 CD O - C) 000 10 )oI c)to-H 1 C o C ) ;o;, ; CD O CO C C ) CC (C c10 C 2 2 2+ o 0c E CD-i C0 0 yC 0 rD 20 0 C O o 000 C L 0 CM c O .

C)

0 CO )0 0 C O w C C 0 0 C (J Cc >. 0- 0 0 0 cC 25 [00431 Test Example 3 Preparation of corneal and conjunctival lesion model, administration of test agents, and determination of corneal lesion were performed as described in Test Example 1. 5 The test agent was 10% nHSA. For use as 10% nHSA, Kenketsu albumin-Wf (25% human serum albumin formulation, Mitsubishi Tanabe Pharma) was diluted with sodium chloride solution such that osmotic pressure was almost equal to saline. 10 [ 00441 (Test groups) Table 6] number of eyes Group Test agent nme fee (number of cases) 1 10% nHSA 6 eyes (6 animals) 00451 (Result) 15 Scores of fluorescein staining of cornea after the treatment of n-heptanol solution were shown in Table 7. In all cases, scores of fluorescein staining of cornea before the treatment of n-heptanol solution were 0. As shown in Table 7, scores of fluorescein staining of 20 cornea were significantly lowered with the administration of 10% nHSA compared to saline on days 4, 4.5, 5, 5.5, 6 and 10 of the treatment of lesion, indicating the effect of restoring corneal epithelium of 10% nHSA (Days 4, 5, 5.5, 6, 26 and 10: p<0.05; Day 4.5: p<0.01). 00461 According to the above result, it is understood that human serum albumin has an effect of restoring corneal 5 tissue equal to or more than that of serum which is recommended in DEWS Report (2007) for dry eye severity level 3 (Test Example 1). 00471 Table 7] 27 CD2 m Z>T 4-10 c o co ol co co c 0 co

C.

Ct Co* CO o c G 10 C O +1 Ct CoD ccz LO 0l CO Q+ 0 C . O .0 .- t C, t C C-. NoGo o- o10 0 "0 (0 Go 0 C *C czC/ 4- dR 4 C o o o - o - O N 10 o 0 LQ oL Q CD ~ C = Q~ IF CCD 0C C CD Q 0 Ct * o -~-- - z: -~ - Ct z Q Co Co 0 28 00481 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as 5 an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 10 [ 00491 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of 15 a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (25)

1. A method for the treatment of dry eye and/or corneal and conjunctival lesion, which comprises administering an effective 5 amount of human albumin to a patient having dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007).

2. A method according to claim 1, wherein the albumin is 10 recombinant human albumin.

3. A method according to claim 1 or 2 wherein the human albumin is human serum albumin. 15

4. A method according to any one of claims 1 to 3 wherein the human albumin is administered in an amount of from 1 to 100 ptl/eye.

5. A method according to any one of claims 1 to 4 wherein the 20 human albumin is administered in an amount of from 10 to 50 ptl/eye. 'H:\AYL\Intenoven\NRPortbl'DCC\AYL\65522771.doc-7/28/2014 30

6. A method according to any one of claims 1 to 5 wherein the human albumin is administered in an amount of from 30 to 50 ptl/eye. 5

7. A method according to any one of claims 1 to 6 wherein the human albumin is administered at a concentration of from 1 to 25 w/v%.

8. A method according to any one of claims 1 to 7 wherein the 10 human albumin is administered at a concentration of 10 w/v%.

9. A method according to any one of claims 1 to 8 wherein the human albumin is administered from 1 to 20 times per day. 15

10. A method according to any one of claims 1 to 9 wherein the human albumin is administered from 1 to 10 times per day.

11. Use of human albumin for the manufacture of a pharmaceutical composition for the treatment of dry eye and/or 20 corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007).

12. Use according to claim 11, wherein the albumin is 1'H:\AYL\Tntenvoven\NRPortbl\DCC\AYL\6552277_I.doc-7/28/2014 31 recombinant human albumin.

13. Use according to claims 11 or 12 wherein the human albumin is human serum albumin. 5

14. Use according to any one of claims 11 to 13 wherein the human albumin is administered in an amount of from 1 to 100 ptl/eye. 10

15. Use according to any one of claims 11 to 14 wherein the human albumin is in a concentration of from 1 to 25 w/v%.

16. Use according to any one of claims 11 to 15 wherein the human albumin is in a concentration of 10 w/v%. 15

17. Use according to any one of claims 11 to 16 wherein the human albumin is administered from 1 to 20 times per day.

18. A pharmaceutical composition when used for the treatment 20 of dry eye and/or corneal and conjunctival lesion of dry eye severity level 3 or more according to DEWS Report (2007), comprising human albumin as an active agent. I'H:\AYL\Intenvoven\NRPortbl\DCC\AYL\6552277_I.doc-7/28/2014 32

19. A pharmaceutical composition according to claim 18, wherein the albumin is recombinant human albumin.

20. The pharmaceutical composition of Claims 18 or 19, wherein 5 the human albumin is human serum albumin.

21. A pharmaceutical composition according to any one of claims 18 to 20 wherein the human albumin is in a concentration of from 1 to 25 w/v%. 10

22. A pharmaceutical composition according to any one of claims 18 to 21 wherein the human albumin is in a concentration of 10 w/v%. 15

23. A method according to claim 1 substantially as hereinbefore described with reference to any one of the Examples and/or Figures.

24. Use according to claim 11 substantially as hereinbefore 20 described with reference to any one of the Examples and/or Figures.

25. A pharmaceutical composition when used according to claim I'H:\AYL\Ienvoven\NRPortbl\DCC\AYL\6552277_1.doc-7/28/2014 33 18 substantially as hereinbefore described with reference to any one of the Examples and/or Figures.