GROSS
`LINUUAL
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`translation of the following document, attached hereto:
`JP H02-124817 A (225 counterpart application)
`I declare that the foregoing is true and correct to the best of my knowledge.
`
`Executed on October 26, 2015
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`Alan F. Siegrist, CT
`CROSSLlNGUAL_. LLC
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`personally appeared AAIJ 9 - $(£«ll5‘i"—""' who proved to me on the basis of satisfactory
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`DAVID STURROCK
`"o.
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`Page 1 of 23
`
`SENJU EXHIBIT 2095
`
`LUPIN V. SENJU
`IPR20l5-01100
`
`

`
`JP H02-124817 A (1)
`
`
`
`
`
`(51) Int. Cl.5
`
` A61K
`31/195
`
`
`
`// C07C 229/40
`
`(54) Title of Invention:
`
`Claim of Priority
`
`(72) Inventor:
`
`(11) Unexamined Patent
`(19) Japan Patent Office (JP)
`Application Publication Number
`H2-124817
`(12) Kokai Patent Gazette (A)
`ID Symbol
`JPO File Nos.
`
` (43) Publication Date: May 14, 1990
`ABM
`7330-4C
`
`
`ABE
`7330-4C
`
`
`Request for examination: Not yet requested Number of claims: 6 (Total of 11 pages)
`Locally Administrable Therapeutic Composition for Inflammatory Disease
`(21) Application No.: H1-18549
`(22) Filing Date:
`January 27, 1989
`(32) January 27, 1988; (33) Japan (JP); (31) Application No. S63-16683
`Takahiro OGAWA
`8-23, Atagoyama, Nishinomiya-shi, Hyogo-ken
`Yoshikazu KURIBAYASHI
`1-2-10, Nakagō-cho, Nada-ku, Kobe-shi, Hyogo-ken
`Kazumichi USHIO
`1-6-13, Kamiōichi, Nishinomiya-shi, Hyogo-ken
`Akira OHTORI
`2-3454-11, Gakuen-Midorigaoka, Nara-shi, Nara-ken
`Senju Pharmaceutical Co., Ltd.
`3-6-1, Hirano-machi, Higashi-ku, Osaka-shi, Osaka
`A. H. Robins Company, Incorporated
`1407 Cummings Drive, Richmond, VA 23220, United States of America
`Suguru TAKEUCHI, Patent Attorney
`Specification
`(wherein R is hydrogen atom or a halogen atom), or a
`salt thereof or the hydrate of said acid or salt, wherein:
`1. Title of the Invention
`the aqueous solution contains a water-soluble polymer
` Locally Administrable Therapeutic Composition for
`and a sulfite salt and is stable at a pH of 6–9.
`Inflammatory Disease
`3) The
`locally
`administrable
`therapeutic
`composition for the treatment of inflammatory disease
`2. Claims
`according to claim 2 wherein the water-soluble polymer
`1) A locally administrable ophthalmic, otic or
`is
`polyvinylpyrrolidone,
`polyvinyl
`alcohol,
`nasal therapeutic composition for the treatment of
`carboxypropyl
`cellulose,
`hydroxyethyl
`cellulose,
`inflammatory disease which includes as its active
`hydroxypropyl cellulose or sodium polyacrylate.
`ingredient a benzoylphenylacetic acid of the formula:
`administrable
`therapeutic
`locally
`4) The
`composition for the treatment of inflammatory disease
`according to claim 2 wherein the concentration of the
`water-soluble polymer is in the range of about 0.1–
`10 W/W %.
`therapeutic
`administrable
`locally
`5) The
`composition for the treatment of inflammatory disease
`according to claim 2 wherein the sulfite salt is in the
`form of sodium, potassium, calcium or magnesium.
`6) The
`locally
`administrable
`therapeutic
`composition for the treatment of inflammatory disease
`according to claim 2 wherein the concentration of the
`sulfite salt is in the range of about 0.1–1 W/W %.
`3. Detailed Description of the Invention
`
`
`
`
`(wherein R is hydrogen atom or a halogen atom), or a
`salt thereof or the hydrate of said acid or salt.
`2) An aqueous locally administrable therapeutic
`composition for the treatment of inflammatory disease
`which includes as its active ingredient an aqueous
`solution of a benzoylphenylacetic acid of the formula:
`
`(72) Inventor:
`
`(72) Inventor:
`
`(72) Inventor:
`
`(71) Applicant:
`
`(71) Applicant:
`
`(74) Agent:
`
`
`
`– 99 –
`
`Page 2 of 23
`
`

`
`(Industrial Field of Utility)
`This invention relates to a locally administrable
`ophthalmic, otic or nasal therapeutic composition for
`inflammatory disease. More particularly, it relates to a
`locally administrable ophthalmic, otic or nasal
`therapeutic composition for
`inflammatory disease,
`which
`contains
`as
`its
`active
`ingredient
`a
`benzoylphenylacetic acid derivative, a salt thereof or
`the hydrate of said acid or salt.
`Another object of the present invention is to
`provide a
`stable
`locally administrable aqueous
`composition such as eye drops, otic composition and
`nasal composition containing the above compounds.
`
`(Prior Art)
`That certain benzoylphenylacetic acid derivatives,
`when orally administered, exhibit anti-inflammatory
`activity has been reported in detail in Journal of
`Medicinal Chemistry, Volume 27, pages 1370–88
`(1984),
`among
`others. Furthermore,
`Japanese
`Unexamined Patent Application Publication No. JP
`S62-126124 A describes pharmaceutical compositions
`for percutaneous administration which contain these
`compounds. However, none of the published literature
`– inclusive of the specification of the abovementioned
`patent application – contains any description indicating
`or suggesting that these medicinal substances are
`effective against inflammatory disease of the eye, nose
`or ear when they are administered topically.
`For the treatment, with topical application of drugs,
`of inflammatory ophthalmopathy such as uveitis and
`conjunctivitis which are most frequently observed in
`the ophthalmological field, steroidal drugs such as
`dexamethasone have so far been employed. Topical
`application of steroidal drugs to the eye has some
`apprehension of increasing intraocular pressure to cause
`glaucoma. And, there is a fear not only of causing
`corneal perforation when such steroidal drugs are
`applied to patients suffered from corneal herpes,
`corneal ulcer or the like, but also of induction of
`corneal herpes, keratomycosis, Pseudomonas infections
`and the like by the topical application of steroidal drugs.
`As there has been known such side effects as above,
`steroidal anti-inflammatory agents must be applied with
`particular care. In spite of such a situation, there is no
`known
`non-steroidal
`anti-inflammatory
`agent
`comparable with steroidal anti-inflammatory drugs in
`effectiveness
`for
`the
`treatment of
`inflammatory
`ophthalmopathy such as uveitis. Thus, in the present
`stage in this technical field, for the treatment of
`inflammatory ophthalmopathy, it is hardly possible not
`to use steroidal anti-inflammatory agents with particular
`care to avoid the side effects as abovementioned. Under
`such circumstances, it is natural that ophthalmological
`experts are awaiting the appearance of non-steroidal
`
`JP H02-124817 A (2)
`
`drugs which are effectively usable against uveitis or the
`like.
`
`(Problems the Invention is Intended to Solve)
`The present inventors investigated to find topically
`applicable drugs with lesser side effects and with
`superior effectiveness by which the topically applicable
`drugs that have been employed in the treatment of
`inflammatory ophthalmopathy,
`i.e. steroidal anti-
`inflammatory agents, can be replaced. As a result, the
`present
`inventors unexpectedly found
`that certain
`derivatives of benzoylphenylacetic acid are very
`effective
`in
`the
`treatment
`of
`inflammatory
`ophthalmopathy, especially of uveitis, by
`topical
`application, and that the effectiveness of such drugs is
`compatible with that of conventional steroidal anti-
`inflammatory drugs.
`Furthermore, since the inventors obtained the
`finding that there are some problems that the above-
`mentioned benzoylphenylacetic acid derivatives are
`unstable in an aqueous solution with the optimal pH
`range
`for
`a
`locally
`administrable
`therapeutic
`composition, they extensively investigated in search of
`the method for the preparation of a stable aqueous
`solution. As a result, we have succeeded in preparing a
`stable aqueous composition. Thus, the stable aqueous
`composition according to the invention is achieved
`based on the above finding.
`While a number of compounds falling under the
`category of non-steroidal anti-inflammatory agents are
`known, not all of them are effective in treating
`inflammatory eye diseases when topically administered
`to the eye. This is because there are several problems
`with them. First, when topically administered to the eye,
`a medicinal agent has to pass through the cornea so that
`it can reach the site of inflammation. Even when it has
`succeeded in arriving at the site of inflammation, it
`must remain there at a necessary concentration for a
`necessary period of time. If it fails to meet these
`requirements, it will be unable to produce the expected
`therapeutic effects. Furthermore, in case it is irritating
`to the eye, it is rather possible that the topical
`administration of the medicinal agent to the eye would
`cause exacerbation of symptoms. Therefore, great
`caution and much care are necessary in selecting a
`medicinal agent for topical administration to the eye.
`Furthermore, in case of administration in the form of
`eye drops, it goes without saying that it is desirable that
`the eye drop be stable for a long period of time in an
`aqueous solution without decomposition or forming
`foreign insoluble matter.
`Accordingly, it is an object of the invention to
`solve the above problems and provide a novel and
`useful agent for ophthalmic use or for otic or nasal
`therapeutic use.
`
`– 100 –
`
`Page 3 of 23
`
`

`
`Moreover, another object of the invention is to
`provide a sufficiently stable aqueous solution such as
`eye drops, otic solution and nasal solution which
`contains the above compounds when stored for a long
`period of time.
`
`(Means of Solving the Problem)
`Based on the above findings, the present invention
`solves the aforementioned problems, being a locally
`administrable ophthalmic, otic or nasal therapeutic
`composition for the treatment of inflammatory disease
`characterized in that it includes as its active ingredient a
`benzoylphenylacetic acid of the formula:
`
`
`(wherein R is hydrogen atom or a halogen atom), or a
`salt thereof or the hydrate of said acid or salt. In the
`formula, the halogen atom represented by R is, for
`example, fluorine, chlorine, bromine or iodine. The
`above compound to be used in accordance with the
`invention may be in a salt form. The salt includes alkali
`metal salts such as sodium salt and potassium salt,
`alkaline earth metal salts such as calcium salt and
`magnesium salt, among others, and any salt may
`suitably be used provided that it can attain the object of
`the invention. The compounds defined above may be
`obtained in the form of a hydrate depending on the
`conditions of synthesis, recrystallization and so forth,
`and such form may be used in practicing the invention
`without any inconvenience or trouble.
`Further, the above compounds may be unstable
`when stored in an aqueous solution for long periods of
`time, and there are some problems in the stability of an
`aqueous solution containing the compounds. Therefore
`the
`inventors extensively
`investigated stabilizing
`methods in order to enhance the stability. As a result,
`unexpectedly, they have succeeded in stabilizing the
`solution by incorporating a water-soluble polymer and
`sulfite and adjusting the pH to about 6–9.
`The compounds to be used as active ingredients in
`the topically administrable therapeutic compositions for
`inflammatory eye disease as well as nasal or otic
`disease in accordance with the invention (although such
`compositions are occasionally hereinafter referred to as
`“ophthalmic composition according to the present
`invention,” use of this abbreviation does not exclude
`the application of the composition in the nasal or otic
`fields) can be produced as described in the above-cited
`report in the Journal of Medicinal Chemistry, Volume
`27, pages 1370–88 (1984) or U.S. Pat. No. 1,136,375,
`for instance, or by a modification of the method
`described
`therein. The ophthalmic compositions
`according to the invention can be prepared in the form
`
`JP H02-124817 A (3)
`
`of eye drops, eye ointments and so on in the same
`manner as various known compositions for topical
`administration to the eye. Thus, a compound of the
`above formula or a mixture of two or more compounds
`of the above formula is preferably made up into an
`aqueous or non-aqueous solution or mixed with an
`ointment base suited for ophthalmic use. In this case, an
`aqueous base generally used in the production of
`ophthalmic preparations, for example sterile distilled
`water, is suitably used as the aqueous base and the
`solution state (pH) thereof is adjusted to a level suited
`for topical administration to the eye. It is desirable that
`an appropriate buffer should be added in adjusting the
`pH. The pH of the ophthalmic compositions according
`to the invention is selected with due consideration paid
`to the stability and topical eye irritation of the active
`ingredient, among others. According to the present
`invention, the stability of an aqueous composition
`containing the above compounds is remarkably so
`enhanced by incorporating a water-soluble polymer and
`sulfite, and adjusting the pH to 6–9, preferably about
`7.5–8.5. The eye irritation of the solution is not
`observed. A water-soluble
`polymer
`includes
`polyvinylpyrrolidone,
`carboxypropyl
`cellulose,
`hydroxyethyl
`cellulose, hydroxypropyl
`cellulose,
`polyvinyl alcohol, sodium polyacrylate and so on.
`Polyvinylpyrrolidone is preferred among them. The
`concentration of the water-soluble polymer is in the
`range of about 0.1 to 10 wt.%. The sulfite salt includes
`sodium sulfite, potassium sulfite, magnesium sulfite,
`calcium sulfite and so on. The concentration of sulfite is
`in the range of about 0.1 to 1.0 wt.%. The pH
`adjustment
`is generally conducted with sodium
`hydroxide or hydrochloric acid, for instance, and it is
`typical to form a buffer solution by combined use of,
`for example, sodium acetate, sodium borate or sodium
`phosphate and acetic acid, boric acid or phosphoric acid,
`respectively. The ophthalmic compositions according to
`the invention may further contain pharmaceutically
`active ingredients, such as an anti-inflammatory agent
`of another kind, an analgesic and an antimicrobial,
`unless they are unfit for the purpose of attaining the
`object of the invention. Examples of such anti-
`inflammatory agents are indomethacin and pranoprofen.
`Usable examples of the anti-microbial agents are the
`penicillins, cephalosporins, and synthetic antimicrobial
`agents of the quinolone-carboxylic acid series. Among
`these active ingredients for combined use with the
`active ingredient according to the invention, the anti-
`inflammatory agent is expected to be synergistic with
`said active ingredient in the ophthalmic compositions
`according to the invention. The analgesic is suited for
`the purpose of alleviating inflammation-associated pain,
`and the antimicrobial agent is suited for the purpose of
`preventing secondary infection. It is of course possible
`to incorporate active agents other than those mentioned
`
`– 101 –
`
`Page 4 of 23
`
`

`
`above in the ophthalmic compositions according to the
`invention unless the object of the invention cannot be
`attained due to the presence thereof.
`compositions
`In
`preparing
`the
`ophthalmic
`according to the invention as mentioned above, an
`isotonizing agent, a microbicidal agent or preservative,
`a chelating agent, a thickening agent and so forth may
`be added to the compositions in accordance with the
`general practice of ophthalmic preparation manufacture.
`The isotonizing agent includes, among others, sorbitol,
`glycerin, polyethylene glycol, propylene glycol, glucose
`and sodium chloride. The preservative includes para-
`oxybenzoic acid esters, benzyl alcohol, para-chloro-
`meta-xylenol, chlorocresol, phenethyl alcohol, sorbic
`acid and salts thereof, thimerosal, chlorobutanol, and
`the like. The chelating agent is, for example, sodium
`edetate, sodium citrate or sodium salt of condensed
`phosphoric
`acid.
`In preparing
`the ophthalmic
`compositions according to the invention in the form of
`eye ointments, the ointment base can be selected from
`among petrolatum, Macrogol, carboxymethylcellulose
`sodium, etc.
`The ophthalmic composition according to this
`invention is prepared by incorporating the active
`compound in a base or vehicle for topical application to
`the eye. To prepare a liquid preparation, the content of
`the active ingredient may range from about 0.001 to
`about 10% and is preferably in the range of about 0.01
`to about 5%. An ointment may be prepared by using the
`active compound in a concentration from about 0.001 to
`about 10%, preferably about 0.01 to about 5%. The
`ophthalmic composition of this invention may be
`administered
`in accordance with
`the
`following
`schedules. In the form of eye drops, one to several
`drops per dose are instilled with a frequency of once to
`4 times a day according to the clinical condition. In the
`form of eye ointment, about 0.1 g to 0.2 g per dose is
`applied to the corneal surface with a frequency of once
`to 4 times a day according to the clinical condition. Of
`course, the dosage may be adjusted according to
`symptoms.
`According to this invention, there can be obtained a
`stable aqueous composition such as otic composition or
`nasal composition. Other conventional methods can be
`used unless unsuitable for the object of this invention.
`Among others, an isotonizing agent, buffer solution and
`preservatives can be used. The concentrations of the
`compounds of the invention varies depending on
`symptoms and so on, and usually may be in the range of
`about 0.001 to about 10%, preferably about 0.01 to
`about 5%.
`The following experimental examples are given to
`demonstrate the efficacy of the ophthalmic composition
`of this invention and the stability of the aqueous
`compositions of the invention.
`Experimental Example 1
`
`JP H02-124817 A (4)
`
`Anti-inflammatory effect of the ophthalmic agent
`according
`to
`this
`invention
`on
`experimental
`ophthalmitis induced by bovine serum albumin in white
`rabbits
`[Animals]
`Seventeen male white rabbits weighing about 2 kg
`were used. They were fed 80 g of Labo RG-RO (Nosan
`Corporation) daily and had free access to tap water.
`[Test drug]
`Sodium 3-(4-bromobenzoyl)-2-aminophenylacetate
`monohydrate (hereafter referred to as Compound [I])
`was used as 0.5% and 0.1% ophthalmic solutions.
`These ophthalmic solutions had a pH value of 8.11 and
`and
`osmotic pressures of 310 mOsm/kg·H2O
`respectively. Bovine
`serum
`325 mOsm/kg·H2O,
`albumin (hereafter referred to as “BSA”) was dissolved
`in physiological saline to a concentration of 5% and
`sterilized by filtration. A 0.1 ml portion of the solution
`was injected into the central part of the vitreous body of
`both eyes using a 27G needle under anesthesia with
`0.4%
`oxybuprocaine
`hydrochloride
`to
`induce
`ophthalmitis (ophthalmitis I). After 28 days when the
`ophthalmitis I had nearly recovered, 2.5% BSA solution
`was administered in a dose of 25 mg/ml/kg into the
`auricular vein to cause ophthalmitis again (ophthalmitis
`II). The severity of ophthalmitis was rated according to
`the rating scale 1 ) of Yamauchi et al., based on the
`Draize Test in which an increased weight given to the
`internal segment of the eye. Observation was made with
`a frequency of once every one or two days during the
`peak period of inflammation and once every three or
`four days before and after the peak period for
`ophthalmitis I and 3, 6, 12 and 24 hours after
`intravenous injection of BSA for ophthalmitis II.
`[Results]
`Anti-inflammatory effect on ophthalmitis I
`Table 1 shows the sum of scores for respective
`parameters during a peak inflammatory period of 3 days
`after aseptic injection of 5% BSA into the central part
`of the vitreous body.
`Table 2 shows the amount of protein, white blood
`cell count and the concentration of prostaglandins in the
`anterior chamber aqueous humor.
`Anti-inflammatory effect on ophthalmitis II
`The administration of 2.5 ml/kg of 2.5% BSA
`solution into the auricular vein after 29 days when the
`inflammatory
`symptoms of ophthalmitis
`I had
`substantially subsided
`resulted
`in a
`relapse of
`inflammation after 3 hours in the physiological saline
`group, where both the external and internal segment of
`
`1) Hideyasu YAMAUCHI, Makoto INGU, Tadashi ISO and
`Anti-inflammatory
`effect
`of
`UDA:
`Kozo
`fluorometholone ophthalmic solution in experimental
`uveitis in rabbits, Folia Ophthalmologica Japonica, 24:
`969-79 (1973).
`
`– 102 –
`
`Page 5 of 23
`
`

`
`JP H02-124817 A (5)
`
`the eye after 12 hours showed inflammatory pictures
`similar to those observed at the peak of ophthalmitis I.
`These symptoms were still observed even after 24 hours.
`Table 3 shows the scores for respective parameters at 3,
`6, 12 and 24 hours after the intravenous injection of
`BSA. Table 4 shows the amount of protein, white blood
`cell count and the concentration of prostaglandins in the
`anterior chamber aqueous humor in ophthalmitis II.
`Administration of the test drug
`Visual observation was made on the day after
`injection of BSA into the vitreous body and with the
`animals arranged in the decreasing order of severity of
`ocular inflammation, grouping was carried out in such a
`manner that the intensity distribution would be uniform
`over the groups. Thus, a physiological saline group of 7
`animals, a 0.1% Compound [I] instillation group of 4
`animals and a 0.5% Compound [I] instillation group of
`5 animals were provided. After this grouping procedure,
`the test drugs and saline were respectively instilled into
`both eyes of the rabbits, 50 μl per dose, 4 times a day.
`For induction of ophthalmitis II, each drug was instilled
`into both eyes, 50 μl per dose, immediately after
`injection of BSA into the auricular vein and at 1-hour
`intervals thereafter, for a total of 14 times.
`Evaluation of results
`In ophthalmitis I, Compound [I] at concentrations
`of 0.1% and 0.5% caused a potent and dose-dependent
`inhibition for both the external and the internal segment
`of the eye. Furthermore, at both concentration levels,
`Compound [I] produced a substantially complete
`inhibition of prostaglandins in the aqueous humor in
`ophthalmitis I.
`In regard to the inhibitory effect on inflammatory
`symptoms, as evaluated by gross observation, which are
`induced by the intravenous injection of antigen, the
`Compound [I] according to this invention produced a
`substantially complete inhibition at both concentrations.
`As to white blood cell count, all drugs produced nearly
`the same degree of inhibition in both the internal and
`the external segments of the eye.
`For any of the drugs, no body weight suppression
`was observed even after 28 consecutive days of
`treatment. In the organs including the thymus, spleen,
`adrenal gland and so on, anatomically no abnormality
`was found.
`[Remainder of page intentionally left blank]
`
`– 103 –
`
`Page 6 of 23
`
`

`
`JP H02-124817 A (6)
`
`Conponnd[l]
`0.51(l0) "
`
`0.420.2 "(SLO)"
`
`1.5;t0.3 "($0.5)"
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`
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`
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`physiologicalsalineguupwaeobsetvedatthclcvelsof"=p<0_05,.2=p<0_0land'3=p<0_00l_
`Table 2
`
`1980 t 654
`
`5869 L‘: 2194
`55932306
`
`[Nous:o'rah1e2]
`Eachvaluc represents 1he:|:standanlcnor_ "1hatIheconcc|mationisk5sthanII1eassaylimit(0_4ng/ml); '1: significant
`difference fiumIhepI1ysiologia|lsalincgoupatp<0.05.
`
`Table3
`
`Conpound[I]
`0.51(l0) "
`
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`
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`
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`
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`0
`
`(100 )"
`
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`8.lt0.3 "(27.0)"
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`
`l3.l:l.l "($2.1)"
`2.8$0.8
`(-47.4)"
`
`l3.3t0.6 "($35.1)"
`2.Bt0.5 (-47.4)"
`
`2.81'0.| "($1.6)"
`2410.4 ''(((.2)"
`8.0tl.2 "(40.7)"
`
`3.3:0.3 "($4.8)"
`Z.9:0.3 "(32.6)"
`9.010.? "(33.3)"
`
`Z1.Et2.l "(35.7)"
`
`22,321.] "(-34.2)"
`
`Table4
`
`[Not$toTablu.-.3]
`EachvaluetepIrsa1tsfl|eistandatdumt.Themnnbasinpmu1d|cscslike( )"repIesa1tsfl1cnulnbu'ofcascs,andthcmlmbusin
`parentheses likc(
`)‘) mptesans the degree (%) of inhibilitn telative to the physiological saline group. Significant difiixcnccs fiom the
`physiologicalsalinegmupweleobsetvedatlhclcvclsof '=p<0.05and.2=p<0.0l.
`
`[Not$toTablc4]
`Eachvaltxcrqmscntsthcmcanistandardcnot:"mcansthattl|cconcemntionislessfl1anll1eassaylimi1(0.4ng/ml);"mcansasignifionm
`da1Iuencc&mnmepuysio1ogica1sa1incg;oupatme1cve1or"=p<o_o5ana”=p<o_o1_
`
`Page 7 of 23
`
`_104_
`
`

`
`JP H02-124817 A (7)
`
`was evaluated, and Experiment 3.b, in which the effect
`of indomethacin, the best known anti-inflammatory
`drug with strong cyclooxygenase inhibitory activity,
`was evaluated.
`Test drugs
`Solutions of the following formulations were used.
`a. Compound [I]
`0.001 0.0001%
`0.01
`0.1
`Compound [I]
`1.0
`1.0
`1.0
`1.0
`Boric acid
`q.s.
`q.s.
`q.s.
`q.s.
`Borax
`0.25
`0.25
`0.25
`0.25
`Sodium chloride
`0.02
`0.02
`0.02
`0.02
`Sodium edetate
`Benzalkonium chloride 0.005 0.005 0.005 0.005
`Tween 80
`0.3
`0.3
`0.3
`0.3
`(pH 8.0, osmotic pressure 310 mOsm/kg·H2O)
`b.
`Indomethacin
`Indomethacin
`0.5%
`Castor oil
`q.s.
`
`Animals
`A total of 28 male albino rabbits (4 rabbits ×
`7 groups) each with a body weight of about 2 kg were
`used. They had been confirmed, before the experiment,
`to have mydriatic response to 1% atropine for at least
`4 hours.
`Experimental Methods
`50 μl each of 1% atropine was instilled into both
`eyes of the animals one hour before the 1st paracentesis,
`in which 0.2 ml/eye of aqueous humor (primary
`aqueous humor) was collected. Topical application of
`50 μl each of the test drug solutions was conducted 30
`min before the paracentesis. Pupil diameter of each eye
`was measured with a slide caliper immediately before
`and 10 min after the paracentesis. The 2nd paracentesis
`was conducted 90 min after the 1st one, in which
`0.2 ml/eye of aqueous humor (secondary aqueous
`humor) was collected.
`
`
`
`
`Experimental Example 2
`The effect of the compounds according to this
`invention on carrageenin edema in rats
`Test drugs
`1. Sodium 3-(4-bromobenzoyl)-2-aminophenyl-acetate
`(hereinafter referred to as Compound [I])
`2. Sodium 3-(4-chlorobenzoyl)-2-aminophenyl-acetate
`(hereinafter referred to as Compound [II])
`3. Sodium
`3-benzoyl-2-aminophenyl-acetate
`(hereinafter referred to as Compound [III])
`Methods
`Using female Wistar rats weighing 100 g in groups
`of 5 animals or 10 eyes, 0.05 ml of 1% carrageenin
`(dissolved in physiological saline at 50°C) as an irritant
`was injected beneath the conjunctiva of both eyes to
`induce edema. Physiological saline, as a control, and
`test drugs were respectively instilled into both eyes 40
`and 20 minutes before and immediately after the
`injection of carrageenin, in the amount of 2.5 μl per
`dose. Four hours after the irritant treatment, each
`animal was sacrificed by cervical dislocation and in
`accordance with the method of Maistrello et al.2), the
`scalp was peeled off toward the eyelid and the
`edematous portion together with the skin was removed
`along the lid margin and weighed. The degrees of
`inhibition of carrageenin edema in the control group
`and drug treatment group are shown in Table 5. Each
`drug group showed a significant difference from the
`control group, indicating the effectiveness of the three
`compounds against acute ocular inflammation.
`Table 5
`Compound Concentration
`(%)
`1.0
`2.5
`1.0
`5.0
`–
`0.5
`1.0
`–
`
`Compound [I]
`
`Compound [III]
`
`
`Control
`Compound [III]
`
`
`Weight of
`edema*
`(mg)
`43.93±4.138
`36.32±3.308
`30.98±3.194
`32.80±2.409
`52.17±2.401
`37.52±2.423
`39.02±3.057
`59.47±3.057
`
`Degree of
`inhibition
`(%)
`16.9**
`31.3**
`41.4**
`37.9**
`–
`36.9**
`34.4**
`–
`
`Control
`[Notes to Table 5]
`*Each value represents the mean ± standard error for 10 groups.
`**Significant differences from the control group at p < 0.001.
`
`Experimental Example 3
`Effects on atropine-resistant miosis and on protein
`increase after paracentesis
`The experiment was divided into two parts, i.e.
`Experiment 3.a, in which the effect of Compound [I]
`
`2 ) Maistrello et al.: Quantitative Effect of Topically
`Applied Anti-inflammatory Agents on External Ocular
`Inflammation
`in Rats: Journal of Pharmaceutical
`Sciences, volume 62, pp. 1455–60 (1973).
`
`– 105 –
`
`Page 8 of 23
`
`

`
`JP H02-124817 A (3)
`
`Results
`
`As shown in Table 6, Compound [I] exhibited a
`dose-related
`inhibitory activity on miosis
`after
`paracentesis at
`the concentrations of 0.0001-0.1%,
`whereas little effect was observed with indomethacin at
`
`the concentrations as high as 0.5%. As shown in Table
`7, Compound [1] exhibited a strong and dose-related
`inhibitory activity on protein increase after paracentesis,
`in which the eflect of 0.01% of Compound [I] was
`equivalent to that of 0.5% indomethacin. It is well
`known that atropine-resistant miosis and protein
`increase in aqueous humor after paracentesis are caused
`by prostaglandin E2, which—is one of the most
`important chemical mediators of inflammation and is
`synthesized immediately after mechanical injury. The
`results,
`therefore.
`indicate that Compound [I] has
`stronger anti-inflammatory effect than indomethacin.
`[Remainder of page intentionally left blank]
`
`
` 1+
`
`T5tDmg
`
`Conoentntim(%)P|innryaqueoushuIIIn'
`
`500013131’?
`aqmoushnlmr
`
`Inhibi1ion(%)
`
`Table7
`
`0.89
`
`0.76
`0.39
`0.38
`
`0.44l+H-H-H’
`
`20.2liI.'!9
`
`6.13:t1.54 :3
`
`t-test
`
`tl : P<0.05
`
`t2 : P<0.0l
`
`t3 : P<0.001
`
`Page 9 of 23
`
`-106-
`
`

`
`
`
`
`
`JP H02-124817 A (9)
`
` Table 9
`4 weeks
`1 week
`2 weeks
`Formulation
`–
`–
`–
`B-1
`+
`–
`–
`B-2
`It was found that adding polyvinylpyrrolidone
`considerably prevented the appearance of red foreign
`insoluble matter, but on the fourth week, however,
`some foreign insoluble matter was observed.
`
`Experimental Example 6
`Next, as a result of searching for more stable
`formulations, the inventors obtained the finding that by
`further incorporating sodium sulfite in addition to
`polyvinylpyrrolidone,
`the stability was remarkably
`increased.
`Formulation
`Compound [I]
`Boric acid
`Borax
`EDTA-2Na
`Benzalkonium chloride
`Polysorbate 80
`Polyvinylpyrrolidone
`Sodium sulfite
`Sterile purified water
`
`
`B-3
`B-1
`0.1 g
`0.1 g
`1.25 g
`1.5 g
`q.s.
`q.s.
`0.02 g
`0.02 g
`0.007 g
`0.007 g
`0.15 g
`0.15 g
`2.0 g
`2.0 g
`0.2 g
`–
`To make 100 ml
`pH 8
`pH 8
`The percent remaining and appearance after 4
`weeks at 60° are as follows.
`Table 10
`Formulation Percent remaining (%) Appearance
`B-1
`93.4
`+
`B-3
`100.9
`–
`As shown in the results above, the change of
`appearance was observed in the formulation in which
`sodium sulfite was not incorporated, and the percent
`remaining increased by about 7%. By contrast, in the
`solution
`containing Compound
`[I]
`in which
`polyvinylpyrrolidone and sodium sulfite coexist, the
`change of appearance was not observed at all and the
`decomposition of Compound [I] was not observed
`either. It was found that the stability was remarkably
`enhanced. Thus, there can be successfully obtained a
`stable aqueous composition containing the compounds.
`
`
`
`Experimental Example 4
`Effects of the pH of Compound [I]
`Formulation
`0.1 g
`Compound [I]
`1.0 g
`Boric acid
`q.s.
`Borax
`0.25 g
`Sodium chloride
`0.02 g
`EDTA-2Na
`0.005 g
`Benzalkonium chloride
`0.3