(12) United States Patent
`US 6,624,193 B1
`(10) Patent N0.:
`Naka et al.
`(45) Date of Patent:
`Sep. 23, 2003
`
`US006624193B1
`
`(54) PREVENTIVE AND THERAPEUTIC AGENTS
`FOR EYE DISEASES
`
`(75)
`
`Inventors: Hiroaki Naka, Kobe (JP); Kazuhito
`Kawabata, Mishima-gun (JP); Hideki
`Tokushige, Kobe (JP)
`
`(73) Assignees: Ono Pharmaceutical C0., Ltd., Osaka
`(JP); Senju Pharmaceutical C0., Ltd.,
`Osaka (JP)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.:
`
`10/070,307
`
`(22) PCT Filed:
`
`Sep. 5, 2000
`
`(86) PCT No.:
`
`PCT/JP00/06014
`
`§ 371 (C)(1),
`(2), (4) Date: Apr. 2, 2002
`
`(87) PCT Pub. No.: W001/17527
`
`PCT Pub. Date: Mar. 15, 2001
`
`(30)
`
`Foreign Application Priority Data
`
`Sep. 6, 1999
`
`(JP)
`
`........................................... 11—251538
`
`(51)
`
`(52) US. Cl.
`
`Int. Cl.7 ........................ A01N 37/02; A01N 37/12;
`A61K 31/22; A61K 31/195; C07C 69/00
`....................... 514/546; 514/562; 514/604;
`514/618; 514/619; 560/138; 560/142
`(58) Field of Search ................................. 514/546, 562,
`514/604, 618, 619, 564; 560/138, 142
`
`(56)
`
`EP
`EP
`EP
`EP
`WO
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`
`347168
`0 519 354
`539223
`0 596 118
`90/04409
`
`12/1989
`12/1992
`4/1993
`5/1994
`5/1990
`
`OTHER PUBLICATIONS
`
`J. Cejkova et al., “Histochemical changes in the rabbit
`cornea and plasmin activity in the tear fluid during contact
`lens wear. Favourable influence of protease inhiitors (apro-
`tinin, PC5, elastatinal)” Histochemistry, vol. 97, No. 1,
`1992, pp. 69—76.
`A. Spierer et al., “The effect of 2—mercaptoacetyl—L—phe-
`nylalanyl—L—leucine, a specific inhibitor of Pseudomonas
`aeruginos a elastase, on experimental Pseudomonas keratitis
`in rabbit eyes,” Curr. Eye Res., vol. 3, No. 4, 1984, pp.
`645—650.
`
`Principles of Ambulatory Medicine, Fourth Edition. Barker
`et al, editors, pp. 943—952 and 1428—1431. Williams &
`Wilkins (1995).*
`Webster’s II New Riverside University Dictionary, p. 933,
`Houghton Mifflin Co. (1984).*
`Gregory D. Sloop, et al., “Acute Inflammation of the Eyelid
`and Cornea in Staphylococcus Keratitis in the Rabbit,”
`Invest. Ophthalmol. Vis. Sci., Feb. 1999, vol. 40, No. 2, pp.
`385—391.
`
`Jitka Cejkova, “Histochemical Study of Leukocyte Elastase
`Activity in Alkali—Burned Rabbit Cornea,” Opthalmic
`Research, 1997; 29, pp. 154—160.
`0. Schmut, et al., "PMN—Elastase—Bestimmung in der
`Tranenfliissigkeit bei Ulcus corneae,” Klin. Mbl. Augen-
`heilk. 188, 1986, pp. 593—595.
`Orhan Deger al., “Polymorphonuclear leukocyte elastase
`levels in patients With Behget’s disease,” Clinica Chimica
`Acta, 236, 1995, pp. 129—134.
`Nurettin Akyol, et al., “The importance of plasma polymor-
`phonuclear (PMN) elastase determination in patients With
`uveitis,” Acta Ophthalmol. Scandinavica,
`1997, pp.
`287—289.
`
`Kiyohiro Tsutsui, et al., “Increased Plasma Granulocyte
`Elastase Levels in Behget’s Disease, ” Journal of Rheuma-
`tology, 1998; 25:2, pp. 326—328.
`J .P. Barletta et al., “Inhibition of Pseudomonal Ulceration in
`Rabbit Corneas by a Synthetic Matrix Metalloproteinase
`Inhibitor,” Invest. Ophthalmol. Vis. Sci., 1996, vol. 37, No.
`1, pp. 20—28.
`
`* cited by examiner
`
`Primary Examiner—Richard L. Raymond
`Assistant Examiner—Zachary C. Tucker
`(74) Attorney, Agent, or Firm—Wenderoth, Lind & Ponack,
`L.L.P.
`
`(57)
`
`ABSTRACT
`
`The present invention provides a prophylactic and therapeu-
`tic medicament for ophthalmic diseases, especially oph-
`thalmic inflammatory diseases and corneal ulcer, comprising
`as an active ingredient a compound represented by the
`formula (I):
`
`(I)
`
`CONHCHZCOOH
`
`CH3
`|
`CH3—(|:—Coo
`CH3
`
`SOZNH
`
`or a pharmacologically acceptable salt or hydrate thereof.
`
`27 Claims, 9 Drawing Sheets
`
`Page 1 of 18
`
`LUPIN EX 1070
`
`LUPIN EX 1070
`
`Page 1 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 1 0f 9
`
`US 6,624,193 B1
`
`Fig. 1
`
`score
`
`Cornealopacity
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Days after endotoxin infusion
`
`Page 2 of 18
`
`Page 2 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 2 0f 9
`
`US 6,624,193 B1
`
`Fig. 2
`
`4.00
`
`3.50
`
`3.00
`
`2.50
`
`2.00
`
`score
`
`
`
`Cornealulcer
`
`1.50
`
`1.00
`
`0.50
`
`0.00
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Days after endotoxin infusion
`
`Page 3 of 18
`
`Page 3 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 3 0f 9
`
`US 6,624,193 B1
`
`+ Como!
`
`+1 1%OorrpomdA
`+0.1%belamefasone
`phosphaie
`
`
`
`Days after endotoxin infusion
`
`Fig. 3
`
`a:
`5
`8
`c
`.9
`iii
`N
`'C
`m
`'5O
`
`3>
`
`Page 4 of 18
`
`Page 4 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 4 0f 9
`
`US 6,624,193 B1
`
`Fig. 4
`
`I—tr—AL—IMOMI-h Total
`
`scoreofcornealinflammation
`
`Control.
`
`Compound A
`
`0.1 % Betamethasone
`phosphate
`
`Page 5 of 18
`
`Page 5 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 5 0f 9
`
`US 6,624,193 B1
`
`Fig. 5
`
`score
`
`
`
`Cornealopacity
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Days after alkaline exposure
`
`Page 6 of 18
`
`Page 6 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 6 0f 9
`
`US 6,624,193 B1
`
`Fig.6
`
`4.00
`
`3.50
`
`3.00
`
`2.50 -
`
`9
`
`OU 2
`
`(D
`
`2.00
`
`1.50
`
`1.00
`
`0.50
`
`0.00
`
`2 3
`
`(U
`
`Eo
`
`o
`
`Page 7 of 18
`
` 0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`Days after alkaline exposure
`
`Page 7 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 7 0f 9
`
`US 6,624,193 B1
`
`Fig. 7
`
`4.00
`
`3.50 '
`
`3.00
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Days after alkaline exposure
`
`0.50
`
`2.50
`
`2.00
`
`1.50
`
`1.00
`
`
`
`Vascularizationscore
`
`Page 8 of 18
`
`Page 8 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 8 0f 9
`
`US 6,624,193 B1
`
`Fig. 8
`
`5.00
`
`4.00
`
`3.00
`
`2.00
`
`1 .00
`
`0.00
`
`90
`§
`.8.3
`E
`as
`g
`0
`
`
`
`.
`.
`--o-- Physuologlcal
`saline group
`+Compound A group
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`Days after inoculation
`
`Page 9 of 18
`
`Page 9 of 18
`
`

`

`US. Patent
`
`Sep. 23, 2003
`
`Sheet 9 0f 9
`
`US 6,624,193 B1
`
`Fig. 9
`
`score
`
`Comealulcer
`
`Days after inoculation
`
`Page 10 of18
`
`Page 10 of 18
`
`

`

`US 6,624,193 B1
`
`1
`PREVENTIVE AND THERAPEUTIC AGENTS
`FOR EYE DISEASES
`
`This application is a 371 of PCT/JP00/06014 filed Sep.
`5, 2000.
`
`TECHNICAL FIELD
`
`The present invention relates to a prophylactic and thera-
`peutic medicament for ophthalmic diseases having a leuko-
`cyte (neutrophil)-derived elastase inhibitory activity.
`BACKGROUND OF THE INVENTION
`
`JP-B 5-81586 and JP-A5-194366 (corresponding to EP-A
`539223) disclose a compound represented by the formula
`(1)1
`
`(I)
`
`CONHCHZCOOH
`
`CH3
`|
`CH3—(|:—(:00
`CH3
`
`SOZNH
`
`(hereinafter referred to as a compound of Formula (I)) and
`a salt or hydrate thereof, which has a human neutrophil-
`derived elastase inhibitory activity and is effective for pre-
`venting and treating diseases such as pulmonary
`emphysema, atherosclerosis and rheumatoid arthritis.
`On the other hand, the ophthalmologic field also involves
`various diseases relating to leukocytes and their elastases.
`For example, ophthalmic infections, corneal traumas, cor-
`neal ulcers and uveitis may be mentioned in an ophthalmic
`infection, the cellular infiltration of leukocytes results in an
`intraocular abscess [Invest. Ophthalmol. Vis. Sci., 40,
`385—391 (1999)]. An alkaline trauma (erosion) which is one
`of corneal traumas allows leukocytes to, be infiltrated into
`corneal stromal cells at an early stage of the alkaline erosion,
`two to three weeks after which the elevation of leukocyte
`elastase activity is observed [Ophthalmic. Res., 29, 154—160
`(1997)]. Also in a case of corneal ulcers, a corneal wound or
`detachment results in the infiltration of leukocytes into a
`corneal stroma, which leads to the release or secretion of a
`protease such as an elastase or collagen [Klin. Monatsbl.
`Augenheilkd, 188, 593—595 (1986)]. An uveitis, especially
`Behcet’s disease, was reported to undergo an increase in a
`plasma leukocyte elastase [Clin. Chim. Acta 236:129—134
`(1995), Acta, Ophthalmol. Scand. 75:287—289 (1997),
`J .Reumatol. 25: 326—328 (1998)]. While leukocytes or their
`elastases were reported to be involved in the ophthalmic
`diseases mentioned above, no actual effect of the adminis-
`tration of an elastase inhibitor was reported.
`While in JP-A 5-221872 (corresponding to EP-A 519354)
`and JP-A 6-509232 (corresponding to EP-A 596118), a
`microbe-derived substance having human leukocyte elastase
`inhibitory activity is described generally to be useful as a
`prophylactic and therapeutic medicament against a corneal
`scar tissue formation or a fibroblast proliferation [eye solidi-
`fication (burn, mechanical or chemical damage,
`keratoconjunctivitis) and the like], it was not administered
`actually to verify its effect, and is different totally from a
`compound of Formula (I).
`OBJECTS OF THE INVENTION
`
`An objective of the present invention is to develop a
`prophylactic and therapeutic medicament for ophthalmic
`diseases containing as an active ingredient a compound of
`Formula (I).
`
`2
`This objective as well as other objectives and advantages
`of the present invention will be explained hereinafter with
`reference to the attached drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a graph showing the effect of an eye drop
`formulation of N-[o-(p-pivaloyloxybenzenesulfonyl-amino)
`benzoyl]glycine monosodium salt tetrahydrate (hereinafter
`referred to as Compound A) on an endotoxin-induced kerati-
`tis (effect on a corneal opacity). Each symbol represents a
`meanistandard deviation (n=4). A statistically significant
`difference from a control is analyzed with p<0.05 (Wilcoxon
`test, one-sided).
`FIG. 2 is a graph showing the effect of a CompoundAeye
`drop formulation on an endotoxin-induced keratitis (effect
`on a corneal ulcer). Each symbol
`represents a
`meanistandard deviation (n=4). A statistically significant
`difference from a control is analyzed with p<0.05 (Wilcoxon
`test, one-sided).
`FIG. 3 is a graph showing the effect of a CompoundAeye
`drop formulation on an endotoxin-induced keratitis (effect
`on a vascularization). Each symbol
`represents a
`meanistandard deviation (n=4).
`FIG. 4 shows the effect of a Compound A eye drop
`formulation 15 days after the challenge on an endotoxin-
`induced keratitis. Each column represents a mean=standard
`deviation (n=4). A statistically significant difference from a
`control is analyzed with p<0.05 (Wilcoxon test, one-sided).
`FIG. 5 is a graph showing the effect of a CompoundAeye
`drop formulation on an alkaline erosion keratitis (effect on
`a corneal opacity). Each symbol represents a mean=standard
`deviation (n=4).
`FIG. 6 is a graph showing the effect of a CompoundAeye
`drop formulation on an alkaline erosion keratitis (effect on
`a corneal ulcer). Each symbol represents a mean=standard
`deviation (n=4). A statistically significant difference from a
`control is analyzed with p<0.05 (Wilcoxon test, one-sided).
`FIG. 7 is a graph showing the effect of a CompoundAeye
`drop formulation on an alkaline erosion keratitis (effect on
`a vascularization). Each symbol represents a mean=standard
`deviation (n=4).
`FIG. 8 is a graph showing the effect of a CompoundAeye
`drop formulation on a pyocyanic corneal ulcer immediately
`after the inoculation of the microbe. Each symbol represents
`a mean=standard deviation (n=6). A statistically significant
`difference from a control is analyzed with p<0.05 (Wilcoxon
`test, one-sided).
`FIG. 9 is a graph showing the effects of the instillation of
`Compound A and lomefioxacin on a pyocyanic corneal ulcer
`one day after the inoculation of the microbe and later. Each
`symbol represents a meanistandard deviation (n=5—6). A
`statistically significant difference from a control is analyzed
`with * p<0.05 and ** p<0.01 (Steel test, one-sided).
`SUMMARY OF THE INVENTION
`
`The present inventors found out that a compound repre-
`sented by Formula (I) or a pharmacologically acceptable salt
`or hydrate thereof exhibits a marked prophylactic and thera-
`peutic effect against various ophthalmic diseases.
`Thus, the present invention provides a prophylactic and
`therapeutic medicament for ophthalmic diseases, especially
`ophthalmic inflammatory diseases and corneal ulcer, com-
`prising as an active ingredient a compound represented by
`Formula (I) or a pharmacologically acceptable salt or
`hydrate thereof.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Page 11 of18
`
`Page 11 of 18
`
`

`

`US 6,624,193 B1
`
`3
`The present invention also provides a method for pre-
`venting and treating an ophthalmic disease which comprises
`administering an active ingredient mentioned above to a
`mammal in need of a treatment for such ophthalmic disease.
`Furthermore,
`the present invention provides use of an
`active ingredient mentioned above in the manufacture of a
`prophylactic and therapeutic medicament for ophthalmic
`diseases.
`
`the present invention provides an eye drop
`Moreover,
`formulation in the form of an aqueous suspension of an
`active ingredient described above.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The prophylactic and therapeutic medicament according
`to the present invention is preferably in a dosage form for a
`local administration such as an eye drop formulation or an
`ophthalmic ointment, which is useful for preventing and
`treating various ophthalmic diseases such as ophthalmic
`infections (for example, corneal herpes, bacterial keratitis,
`bacterial conjunctivitis, mycotic keratitis, acanthamebic
`keratitis, infectious endophthalmitis, infectious corneal ulcer
`and the like), corneal trauma, cicatricial keratonjun ctival
`diseases (for example, alkaline erosive keratoconjunctivitis,
`Stevens-Johnson syndrome, ophthalmic pemphigoid and the
`like), corneal ulcer (for example, Mooren’s ulcer, corneal
`ulcer subsequent to chronic rheumatoid arthritis or collagen
`disease, Terrien’s margine degeneration, catarrhal corneal
`ulcer,
`infectious corneal ulcer and the like), vitamin A
`insufficiency-induced keratomalacia, necrotic keratitis, neu-
`roparalytic keratitis, diabetic keratophathy, keratoconjunc-
`tiva sicca, contact lens-induced keratoconjunctivitis, vernal
`conjunctivitis, allergic conjunctivitis, uveitis, Behcet’s
`syndrome,
`inflammation after cataract surgery and
`pseudopterygium, especially a keratoconjunctival inflam-
`matory disease (for example, corneal herpes, bacterial
`keratitis, bacterial conjunctivitis, mycotic keratitis, acan-
`thamebic keratitis, corneal
`trauma, alkaline erosive
`keratoconjunctivitis, corneal ulcer, vitamin A insufficiency-
`induced keratomalacia, necrotic keratitis, neuroparalytic
`keratitis, diabetic keratophathy, keratoconjunctiva sicca,
`contact
`lens-induced keratoconjunctivitis, vernal
`conjunctivitis, allergic conjunctivitis and the like).
`It
`is
`useful also for preventing and treating corneal ulcer
`(including various corneal ulcers described above and those
`induced otherwise), especially an infectious corneal ulcer.
`A compound of Formula (I) used as an active ingredient
`according to the present invention or a pharmacologically
`acceptable salt thereof is a known compound described in
`JP-B 5-81586, and can be produced, in accordance with the
`procedure described therein, by the amidation of
`p-pivaloyloxybenzenesulfonyl chloride followed by the con-
`version into a salt by a known method. The resultant
`compound may also be converted into a hydrate by a known
`method.
`
`A pharmacologically acceptable salt of a compound of
`Formula (I) may for example be an inorganic salt such as
`hydrochloride, hydrobromide, hydroiodide, sulfate, phos-
`phate and nitrate, an organic salt such as acetate, lactate,
`tartarate, benzoate, citrate, methanesulfonate,
`ethanesulfonate, benzenesulfonate,
`toluenesulfonate,
`isethionate, glucuronate and gluconate, an alkaline metal salt
`(sodium salt, potassium salt and the like), an alkaline earth
`metal salt (calcium salt, magnesium salt and the like), an
`ammonium salt, a pharmacologically acceptable amine salt
`(tetramethylammonium salt,
`triethylamine salt, methy-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`lamine salt, dimethylamine salt, cyclopentylamine salt, ben-
`zylamine salt, phenethylamine salt, piperidine salt, monoet-
`hanolamine salt, diethanolamine salt, tris(hydroxymethyl)
`aminomethane salt, lysine salt, arginine salt, N-methyl-D-
`glucamine salt and the like).
`One preferred especially as an active ingredient used in
`the present invention is a sodium salt tetrahydrate of a
`compound of Formula (I),
`i.e., N-[o-(p-
`pivaloyloxybenzenesulfonylamino)benzoyl]glycine mono-
`sodium salt tetrahydrate (described in Example 3 in JP-A
`5-194366 corresponding to EP-A 539223) represented by
`Formula (I-A):
`
`(I-A)
`
`CONHCHZCOO'Na+°4HZO
`
`CH3
`
`CH3—c—COO@SOZNH
`(EH,
`
`The prophylactic and therapeutic medicament for oph-
`thalmic diseases according to the present invention, on the
`basis of its leukocyte-derived elastase inhibitory activity, is
`useful in preventing and treating various ophthalmic dis-
`eases such as an ophthalmic infections (for example, corneal
`herpes, bacterial keratitis, bacterial conjunctivitis, mycotic
`keratitis, acanthamebic keratitis, infectious endophthalmitis,
`infectious corneal ulcer and the like), corneal trauma, cica-
`tricial keratoconjunctival diseases (for example, alkaline
`erosive keratoconjunctivitis, Stevens-Johnson syndrome,
`ophthalmic pemphigoid and the like), corneal ulcer (for
`example, Mooren’s ulcer, corneal ulcer subsequent
`to
`chronic rheumatoid arthritis or collagen disease, Terrien’s
`margine degeneration, catarrhal corneal ulcer,
`infectious
`corneal ulcer and the like), vitamin A insufficiency-induced
`keratomalacia, necrotic keratitis, neuroparalytic keratitis,
`diabetic keratophathy, keratoconjunctiva sicca, contact lens-
`induced keratoconjunctivitis, vernal conjunctivitis, allergic
`conjunctivitis, uveitis, Behcet’s syndrome,
`inflammation
`after cataract surgery and pseudopterygium, especially a
`keratoconjunctival inflammatory disease (for example, cor-
`neal herpes, bacterial keratitis, bacterial conjunctivitis,
`mycotic keratitis, acanthamebic keratitis, corneal trauma,
`alkaline erosive keratoconjunctivitis, corneal ulcer, vitamin
`A insufficiency-induced keratomalacia, necrotic keratitis,
`neuroparalytic keratitis, diabetic keratophathy, keratocon-
`junctiva sicca, contact
`lens-induced keratoconjunctivitis,
`vernal conjunctivitis, allergic conjunctivitis and the like). It
`is useful also for preventing and treating corneal ulcer
`(including various corneal ulcers described above and those
`induced otherwise), especially infectious corneal ulcer.
`The prophylactic and therapeutic medicament for oph-
`thalmic diseases according to the present invention can be
`mixed with a pharmacologically acceptable carrier, excipi-
`ent or diluent which is known per se and formulated by a
`method known per se into a pharmaceutical or a veterinary
`medicine in various oral or parenteral dosage forms such as
`tablets, capsules, granules, injection solutions, eye drops and
`ophthalmic ointments, and it is especially preferred to be
`used in a local dosage form, preferably an eye drop formu-
`lation or an ophthalmic ointment.
`The eye drop formulation may for example be aqueous
`formulations such as aqueous eye drops, aqueous suspension
`eye drops, viscous eye drops and solubilized eye drops as
`well as non-aqueous formulations such as non-aqueous eye
`drops and non-aqueous suspension eye drops, with an aque-
`
`Page 12 of 18
`
`Page 12 of 18
`
`

`

`US 6,624,193 B1
`
`5
`ous formulation being preferred. One preferred especially is
`an aqueous suspension eye drop formulation.
`The aqueous eye drop formulation may contain various
`additives incorporated ordinarily, such as buffering agents
`(e.g., phosphate buffers, borate buffers, citrate buffers, tart-
`arate buffers, acetate buffers, amino acids, sodium acetate,
`sodium citrate and the like), isotonicities (e.g., saccharides
`such as sorbitol, glucose and mannitol, polyhydric alcohols
`such as glycerin, concentrated glycerin, polyethylene glycol
`and propylene glycol, salts such as sodium chloride), pre-
`servatives or antiseptics (e.g., benzalkonium chloride, ben-
`zethonium chloride, p-oxybenzoates such as methyl
`p-oxybenzoate or ethyl p-oxybenzoate, benzyl alcohol,
`phenethyl alcohol, sorbic acid or its salt, thimerosal, chlo-
`robutanol and the like), solubilizing aids or stabilizing
`agents (e.g., cyclodextrins and their derivative, water-
`soluble polymers such as polyvinyl pyrrolidone, surfactants
`such as polysorbate 80 (Tween 80)), pH modifiers (e.g.,
`hydrochloric acid, acetic acid, phosphoric acid, sodium
`hydroxide, potassium hydroxide, ammonium hydroxide and
`the like), thickening agents (e.g., hydroxyethyl cellulose,
`hydroxypropyl cellulose, methyl cellulose, hydroxypropyl-
`methyl cellulose, carboxymethyl cellulose and their salts),
`chelating agents (e.g., sodium edetate, sodium citrate, con-
`densed sodium phosphate) and the like.
`The eye drop formulation in the form of an aqueous
`suspension may also contain suspending agents (e.g., poly-
`vinyl pyrrolidone, glycerin monostearate) and dispersing
`agents (e.g., surfactants such as tyloxapol and polysorbate
`80, ionic polymers such as sodium alginate) in addition to
`the additives listed above, whereby ensuring that the eye
`drop formulation is a further uniform microparticulate and
`satisfactorily dispersed aqueous suspension.
`When the eye drop formulation in the form of an aqueous
`suspension is produced, it is preferable to use a pH modifier
`to make the formulation acidic pH (pH4 to 5.5). Apreferred
`pH modifier is hydrochloric acid.
`The eye drop formulation in the form of an aqueous
`suspension preferably contains sodium citrate or sodium
`acetate as a buffering agent, concentrated glycerin and/or
`propylene glycol as an isotonicity and polyvinyl pyrrolidone
`as a suspending agent. A preferred dispersing agent is a
`surfactant and/or sodium alginate. Such surfactant is pref-
`erably tyloxapol or polysorbate 80.
`The ophthalmic ointment may employ an ointment base
`known per se, such as purified lanolin, petrolatum,
`plastibase, liquid paraffin, polyethylene glycol and the like.
`The prophylactic and therapeutic medicament of the
`present invention may be administered to a mammal which
`is or may be suffered from an ophthalmic disease (e.g.,
`human, rabbit, dog, cat, cattle, horse, monkey). While the
`administration route and the dose may vary depending on a
`symptom, age and body weight of a subject, the concentra-
`tion is about 0.001 to 5 (w/v) %, preferably about 0.01 to 3
`(w/v) % as a free form of a compound of Formula (I)
`contained in an aqueous eye drop formulation when given to
`an adult, and is given preferably 1 to 8 times a day with a
`single dose being one to several drops.
`When given as the ophthalmic ointment, the dose is about
`0.001 to 5 (w/v) %, preferably about 0.01 to 3 (w/v) % as a
`free form of a compound of Formula (I), and is given
`preferably 1 to 4 times a day as appropriate in view of the
`symptom.
`Unless the intended purpose of use is affected adversely,
`the prophylactic and therapeutic medicament of the present
`invention may contain or may be used together with other
`
`6
`for
`appropriate pharmacologically effective substances,
`example, steroidal anti-inflammatory agents
`(dexamethasone, prednisolone and the like), non-steroidal
`anti-inflammatory agents (diclofenac sodium, pranoprofen
`and the like), antiallergic agents (tranilast, ketotifen
`fumarate, sodium cromoglicate and the like), antihistamic
`agents (diphenhydramine hydrochloride and the like),
`glaucoma-treating agents (pilocarpine hydrochloride, phys-
`ostigmine salicylate, timolol, isopropylunoprostone and the
`like), antibiotics (gentamycin sulfate, fradiomycin sulfate,
`tobramycin, sulbenicillin, cefmenoxime, erythromycin,
`colistin, oxytetracycline, polymyxin B, chloramphenicol,
`micronomicin, dibekacin, sisomicin and the like), antibac-
`terial agents (sulfamethizole, sulfamethoxazole, ofloxacin,
`norfioxacin, lomefioxacin hydrochloride, enoxacin, ciprof-
`loxacin hydrochloride, cinoxacin, sparfioxacin, tosufioxacin
`tosylate, nalidixic acid, pipemidic acid trihydrate, pipemidic
`acid, fleroxacin,
`levofioxacin and the like), and antiviral
`agents (idoxuridine, acyclovir and the like), and antimycotic
`agents (pimaricin, fiuconazole, miconazole, amphotericin B,
`fiucytosine, itraconazole and the like).
`The prophylactic and therapeutic medicament of the
`present invention is used preferably together with at least
`one selected from the antibiotic, antibacterial, antiviral and
`antimycotic agents listed above in prophylaxis or therapy
`especially for an ophthalmic infection-induced inflammation
`or corneal ulcer.
`In such case, any of the antibiotic,
`antibacterial, antiviral and antimycotic agents can be com-
`bined with the prophylactic and therapeutic medicament of
`the present invention in a single formulation, or may be
`instilled separately. When being instilled separately,
`the
`prophylactic and therapeutic medicament of the present
`invention may be instilled simultaneously with any of the
`antibiotic, antibacterial, antiviral and antimycotic agents, or
`successively at a certain interval. When being instilled
`simultaneously, any of the prophylactic and therapeutic
`medicament of the present
`invention and the antibiotic,
`antibacterial, antiviral and antimycotic agents is first
`instilled and then preferably after a certain time period
`another agent is instilled whereby avoiding any escape of the
`agent given previously. Any of the antibiotic, antibacterial,
`antiviral and antimycotic agents listed above may also be
`given systemically by means of an oral or intravenous
`formulation.
`
`The present invention is further illustrated in detail by the
`following Experiments and Examples, which are not con-
`strued to limit the scope of the present invention.
`EXPERIMENT 1
`
`The effect of Compound A on an ophthalmic inflamma-
`tory disease was investigated as described below.
`The effect of Compound Awhen given as eye drops was
`investigated in a rabbit keratitis model using an endotoxin
`derived from Pseudomonas aeruginosa detected frequently
`in an ophthalmic infection as well as in a rabbit corneal
`alkaline erosion model.
`
`Materials and Methods
`
`(1) Animals
`Male Japanese albino rabbits each weighing about 2 to 2.5
`kg purchased from FUKUZAKI rabbit-raising association
`were used. Each animal was maintained at a temperature of
`2414° C. and a humidity of 55:15%.
`
`(2) Test Substances
`Compound Awas given as a 1.0% Compound Aeye drop
`formulation prepared by suspending Compound A in a
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`15
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`20
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`25
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`30
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`35
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`US 6,624,193 B1
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`7
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`formulation base (0.1% NaH2P04, 0.1% polysorbate 80 and
`0.9% NaCl, pH 5.0). As a positive control, a 0.1%
`betamethasone eye drop formulation (RinderonTM solution,
`Sionogi) was used. In a control group, the formulation base
`was given.
`
`(3) Methods
`1) Effect on Endotoxin-induced Keratitis
`16 Male Japanese albino rabbits each weighing 2 to 2.5 kg
`were used. The rabbits were divided into four groups each
`having 4 animals, which were anesthetized systemically by
`an intramuscular administration each of 1 ml/kg of an equal
`volume mixture of 5% ketamine hydrochloride and 2%
`xylazine hydrochloride. Each 10 pl of a 1% solution of
`Pseudomonas aeruginosa-derived endotoxin in physiologi-
`cal saline was infused into each corneal stroma of a rabbit.
`
`An anterior part of an eye was observed using a slit lamp
`every 5 days over a period from the day after the endotoxin
`infusion through the 30th day, and examined for the corneal
`opacity,
`the corneal ulcer and the vascularization, which
`were scored in accordance with the criteria shown in Table
`
`1. Each test substance was started to be instilled immediately
`after the endotoxin infusion, and then given 4 times a day in
`the volume of 20 pl every 2 hours.
`2) Effects on Alkaline Erosive Keratitis
`16 Male Japanese albino rabbits each weighing 2 to 2.5 kg
`were used. The rabbits were divided into four groups each
`having 4 animals, which were anesthetized systemically by
`an intramuscular administration each of 1 ml/kg of an equal
`volume mixture of 5% ketamine hydrochloride and 2%
`xylazine hydrochloride and also locally by an instillation of
`oxybuprocaine hydrochloride. A filter paper whose diameter
`was 10 mm and which had been immersed in 2N NaOH was
`
`brought into contact with the center of the right cornea of a
`rabbit for 1 minute to establish an alkaline erosion, and then
`the eye was rinsed immediately with 10 mL or more of
`physiological saline. The depth of the corneal ulcer and the
`vascularization were observed using a slit lamp every 5 days
`over a period from 5 days after the alkaline erosion through
`the 30th day, and scored in accordance with the criteria
`shown in Table 1. Each test substance was started to be
`
`instilled immediately after the alkaline erosion, and then
`given 4 times a day in the volume of 20 pl every 2 hours.
`
`Table 1
`
`Rabbit Keratitis Scoring Criteria
`remarks 1)
`Corneal opacity
`A) Degree
`0: No opacity
`1: Mild opacity but distinguishable anterior chamber
`2: Difficulty in distinguishing details of iris
`3: Almost no transparency in anterior chamber
`B) Corresponding size of corneal region
`1: 1/3 or less of entire
`2: 1/3 to 2/3 of entire
`3: 2/3 or more of entire
`* Corneal ulcer
`0: No corneal ulcer
`
`in depth from corneal surface
`1: Ulcer of less than 1/3
`toward inside of anterior chamber
`
`2: Ulcer of 1/3 or more and less than 2/3 in depth from
`corneal surface toward inside of anterior chamber
`
`3: Ulcer of 2/3 or more in depth from corneal surface
`toward inside of anterior chamber
`
`8
`
`4: Perforation in cornea
`* Vascularization’emarks 1)
`A) Length
`0: No vascularization into cornea
`
`1: Less than 1/3 from corneal limbus through center
`2: Less than 2/3 from corneal limbus through center
`3: 2/3 or more from corneal limbus through center
`B) Region
`0.5: Less than 1/3 of corneal circumference
`1:1/3 or more and less than 2/3 of corneal circumference
`
`2: 2/3 or more of corneal circumference Remarks 1) Each
`as score A x score B
`
`Results and Discussion
`
`1) Effects on Endotoxin-induced Keratitis
`FIGS. 1 to 3 show the change in the keratitis symptoms
`over a period from 5 to 30 days after the endotoxin infusion.
`In the control group, the severity of each symptom peaked
`on the 15th day, and then a gradual recovery was observed
`until the 30th day when almost all disappeared. In Com-
`pound A instillation group, inhibitory effects were observed
`on all of the evaluation items, i.e., the corneal opacity, the
`corneal ulcer and the vascularization, when compared with
`the control group. In the 0.1% betamethasone phosphate
`instillation group used as the positive control, the onset of
`the keratitis was inhibited almost completely over the obser-
`vation period. FIG. 4 shows the total score in each group on
`the 15th day when the severity of each symptom peaked, and
`revealed that the % inhibition in the CompoundAinstillation
`group when compared with the control group was 59.4%,
`with a statistically significant difference.
`Based on the results described above, the Compound A
`eye drop formulation was proven to be effective against
`various symptoms of the keratitis during an ophthalmic
`infection.
`
`While betamethasone phosphate used here as a positive
`control exhibited an extremely potent anti-inflammatory
`activity, its use is limited frequently in view of a side effect
`experienced as the exacerbation of an infection over a
`prolonged therapy with a steroid in a clinical case of the
`ophthalmic infections.
`Accordingly,
`the Compound A eye drop formulation
`expected to have a less risk of the exacerbation of an
`infection can serve as a hopeful agent against the ophthalmic
`infections.
`
`2) Effects on Alkaline Erosive Keratitis
`FIGS. 5 to 7 show the change in the keratitis symptoms
`over a period from 5 to 30 days after the corneal alkaline
`exposure. In the control group, the severity peaked on the 20
`to 25th day after the corneal alkaline exposure. In Com-
`pound A instillation group, a significant inhibitory effect on
`the corneal ulcer was observed on the 20th day, but no
`effects were noted on the vascularization or the corneal
`
`opacity. In the 0.1% betamethasone phosphate instillation
`group used as the positive control, a significant inhibitory
`effect was observed on the vascularization on the 15th day.
`EXPERIMENT 2
`
`Materials and Methods
`
`(1) Animals
`Male Japanese albino rabbits each weighing about 2 kg
`purchased from KITAYAMA LABE S CO., LTD. were used.
`Each animal was maintained at a temperature of 2313° C.
`and a humidity of 55:10%.
`
`(2) Test Substances
`Compound Awas given as a 1.0% Compound Aeye drop
`formulation prepared by suspending Compound A in a
`
`10
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`15
`
`20
`
`25
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`30
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`35
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`40
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`45
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`50
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`60
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`65
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`US 6,624,193 B1
`
`formulation base (0.1% sodium acetate, 0.1% polysorbate
`80 and 0.9% NaCl, pH 5.0). A 0.3% lomefloxacin (LFLX)
`hydrochloride was used as an.
`is antibacterial agent, and
`physiological saline *was used as a control.
`
`(3) Methods
`1) Excision of Nictitating Membrane
`After instilling 0.4% oxybuprocaine hydrochloride for a
`local anesthesia, a nictitating membrane was excised.
`2) Inoculation
`A causative microorganism used was a clinical isolate
`Pseudomonas aeruginosa strain No. ho-134. A rabbit