(12) United States Patent
`Yasueda et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006274609Bl
`US 6,274,609 Bl
`Aug. 14,2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) AQUEOUS LIQUID PHARMACEUTICAL
`COMPOSITION CONTAINING AS MAIN
`COMPONENT BENZOPYRAN DERIVATIVE
`
`(75)
`
`Inventors: Shinichi Yasueda, Takarazuka; Tadashi
`Terai, Kobe; Takahiro Ogawa,
`Nishinomiya; Yoshinori Ii,
`Mishima-gun, all of (JP)
`
`(73) Assignees: Ono Pharmaceutical Co., Ltd.; Senju
`Pharmaceutical Co., Ltd., both of
`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.:
`
`09/463,211
`
`(22) PCT Filed:
`
`Jul. 15, 1998
`
`(86) PCT No.:
`
`PCT/JP98/03172
`
`§ 371 Date:
`
`Jan.21,2000
`
`§ 102(e) Date: Jan. 21, 2000
`
`(87) PCT Pub. No.: W099/04790
`
`PCT Pub. Date: Feb. 4, 1999
`
`(30)
`
`Foreign Application Priority Data
`
`Jul. 23, 1997
`
`(JP) ................................................... 9-196771
`
`Int. Cl? ..................................................... A61K 31/41
`(51)
`(52) U.S. Cl. .............................................................. 514/382
`(58) Field of Search ............................................... 514/382
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,976,760 * 3/1999 Sasatani eta!. ..................... 424/494
`
`FOREIGN PATENT DOCUMENTS
`
`0641569
`96/41628
`
`3/1995 (EP) .
`12/1996 (WO) .
`
`OTHER PUBLICATIONS
`
`Database WPI, Section Ch, Week 9621, Derwent Publica(cid:173)
`tions Ltd., London, GB; Class A96, AN 96-205438,
`XP002081469 & JP 08 073353 A (Ono Pharmaceutical)
`Mar. 19, 1996.
`
`* cited by examiner
`
`Primary Examiner-James H. Reamer
`(74) Attorney, Agent, or Firm-Wenderoth, Lind & Ponac
`L.L.P.
`
`(57)
`
`ABSTRACT
`
`In order to promote solubilization or suspension of 4-oxo-
`8-[ 4-( 4-phenylbutoxy)benzoylamino ]-2-(tetrazol-5-yl)-4H(cid:173)
`l-benzopyran or its hydrate (pranlukast) in water, at least
`one component selected from surfactants, water-soluble
`cellulose derivatives and water-soluble vinyl polymers is
`formulated together with pranlukast. Thus, it is possible to
`provide an aqueous liquid pharmaceutical composition con(cid:173)
`taining higher concentration of pranlukast and having good
`properties.
`
`37 Claims, 2 Drawing Sheets
`
`LUPIN EX1012, Page 1
`
`

`
`U.S. Patent
`
`Aug. 14,2001
`
`Sheet 1 of 2
`
`US 6,274,609 Bl
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`-
`
`•
`()
`QJ
`tl.l
`
`-Q
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`J
`
`.~
`+J
`
`~
`0
`·.-I
`tl.l
`~
`QJ
`01
`tl.l
`·.-I
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`I
`QJ p:;
`
`0
`.00001
`
`.0001
`
`.001
`
`.01
`
`.1
`
`1
`
`H PM C concentration (w/v%)
`
`Fig. 1
`
`LUPIN EX1012, Page 2
`
`

`
`U.S. Patent
`
`Aug. 14,2001
`
`Sheet 2 of 2
`
`US 6,274,609 Bl
`
`100
`
`80
`
`60
`
`s:=
`0
`-~
`.j..J
`I'd
`H
`-
`r-1
`·~ N
`~ ~
`-~ o:::t'
`0
`Q)
`•
`t,~ 40
`t> H
`0
`(1)
`s:=.O
`-~ s
`:::1
`tf.l
`0 c
`~-
`
`••
`
`20
`
`Q t - -
`
`Physiological
`saline
`solution
`
`Pranlukast
`
`Fig. 2
`
`LUPIN EX1012, Page 3
`
`

`
`US 6,274,609 Bl
`
`1
`AQUEOUS LIQUID PHARMACEUTICAL
`COMPOSITION CONTAINING AS MAIN
`COMPONENT BENZOPYRAN DERIVATIVE
`
`FIELD OF THE INVENTION
`
`The present invention relates to a pharmaceutical com(cid:173)
`position comprising as a main component 4-oxo-8-[ 4-( 4-
`pheny lbutoxy)benzoy lamina ]-2-( tetrazol-5 -yl)-4H -1-
`benzopyran or its hydrate. In particular, it relates to an
`aqueous liquid pharmaceutical composition containing the
`benzopyran derivative or its hydrate in higher concentration
`and having good properties.
`
`BACKGROUND OF THE INVENTION
`
`5
`
`2
`selected from surfactants and specific high-molecular
`weight substances. Thus, the present inventors have suc(cid:173)
`ceeded in preparing the desired aqueous liquid pharmaceu(cid:173)
`tical composition containing higher concentration of pran-
`lukast and having good properties, and have completed the
`present invention.
`That is, according to the present invention, there is
`provided an aqueous liquid pharmaceutical composition
`which comprises 4-oxo-8-[ 4-( 4-phenylbutoxy)
`10 benzoylamino ]-2-(tetrazol-5-yl)-4H-1-benzopyran or its
`hydrate and at least one component selected from
`surfactants, water-soluble cellulose derivatives and water(cid:173)
`soluble vinyl polymers.
`In particular, one aspect of the aqueous liquid pharma-
`15 ceutical composition of the present invention is an aqueous
`solution containing pranlukast and a surfactant. Preferably,
`the composition further contains a stabilizer.
`Another aspect of the aqueous liquid pharmaceutical
`composition of the present invention is an aqueous suspen-
`20 sian containing pranlukast and at least one component
`selected from the group consisting of water-soluble cellulose
`derivatives and water-soluble vinyl polymers. Optionally,
`the suspension may further contain a surfactant. Preferably,
`the suspension contains a relatively small amount of a
`25 surfactant together with the water-soluble high-molecular
`weight substance.
`These aqueous liquid pharmaceutical compositions have
`good re-dispersibility over a long time and are stable. In
`addition, they can contain higher concentration of pran-
`30 lukast. Then, they can be useful as eye drops, nasal drops,
`injectable preparations, internal medicine and the like and
`can be used as an eosinocyte infiltration inhibitor.
`
`4-0xo-8-[ 4-( 4-phenylbutoxy)benzoylamino ]-2-( tetrazol-
`5-yl)-4H-1-benzopyran is an antagonist toward leukotrienes
`(LTC4, LTD4, LTE4) and is known to be a medicine having
`excellent pharmacological activities on various allergic dis(cid:173)
`eases including asthma (JP-A 61-50977). Its 'h hydrate is
`called as "pranlukast" and is utilized as an anti-allergic
`medicine. Then, in the field of ophthalmology, it has been
`proposed to apply such a medicine to allergic eye diseases
`such as vernal keratoconjunctivitis, etc.
`In general, for preparing an aqueous liquid pharmaceuti(cid:173)
`cal composition such as water-soluble eye drops, it is
`considered that a pharmacologically active component
`should be present in concentration of about 0.01 to 0.1 %.
`However, 4-oxo-8-[ 4-( 4-phenylbutoxy)benzoylamino ]-2-
`(tetrazol-5-yl)-4H -1-benzopyran and its hydrate
`(hereinafter, they are referred to as "pranlukast" all together,
`unless otherwise stated) have very low water-solubility,
`which makes every difficult to prepare a useful aqueous
`liquid pharmaceutical composition thereof. Then, the use of
`polyvinylpyrrolidone and ~-cyclodextrin has been proposed 35
`to solubilize pranlukast (JP-A 8-73353).
`However, even if polyvinylpyrrolidone, which is a mate(cid:173)
`rial known to have strongest solubilization power, is used,
`pranlukast dissolves in water in concentration of, at highest,
`about 0.01 %.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`4-0xo-8-[ 4-( 4-phenylbutoxy)benzoylamino ]-2-(tetrazol-
`5-yl)-4H-1-benzopyran or its hydrate to be used in the
`present invention is not specifically limited but its 'h hydrate
`available from Ono Pharmaceutical Co., Ltd. as "pran-
`40 lukast" is suitable for the present invention.
`The amount of pranlukast to be formulated in the com(cid:173)
`position is not specifically limited but, in case of an aqueous
`solution, it is formulated in an amount of 0.2 w/v % or less,
`normally, 0.001 to 0.2 w/v %, preferably 0.005 to 0.1 w/v %
`45 based on the total weight of the composition and, in case of
`an aqueous suspension, 0.01 to 5.0 w/v %, preferably 0.1 to
`2.0 w/v % based on the total weight of the composition, in
`terms of its 'h hydrate.
`The surfactants to be used are at least one member
`selected from nonionic surfactants, cationic surfactants and
`anionic surfactants. As the nonionic surfactants, it is pre(cid:173)
`ferred to use those having HLB of 10 to 18. Examples of the
`nonionic surfactants include polyoxyethylene sorbitan fatty
`acid ester (e.g., Polysorbate 80, Polysorbate 60, Polysorbate
`55 40, etc.), polyoxyethylene hydrogenated castor oil (e.g.,
`polyoxyethylene hydrogenated castor oil 60, polyoxyethyl(cid:173)
`ene hydrogenated castor oil 50, polyoxyethylene castor oil
`40, etc.), polyoxyethylene alkylphenyl formaldehyde con(cid:173)
`densate (e.g., Tyloxapol, etc.), polyoxyethylene polyoxypro-
`60 pylene block copolymer (e.g., Poloxamer 188, Poloxamer
`403, etc.) and sucrose ester of fatty acid (e.g., Ryutosugar
`ester P-1570 and S-1570 manufactured by Mitsubishi
`Chemical Foods), among others.
`Examples of the cationic surfactants include quaternary
`65 ammonium salt (e.g., benzalkonium chloride, etc.), among
`others. Examples of the anionic surfactants include alkyl
`sulfate (e.g., sodium lauryl sulfate, etc.), among others.
`
`OBJECTS OF THE INVENTION
`
`The main object of the present invention is to promote
`solubilization or suspension of pranlukast in water, thereby
`providing an aqueous liquid pharmaceutical composition
`containing higher concentration of pranlukast and having
`good properties.
`This object as well as other objects and advantages of the
`present invention will become apparent to those skilled in 50
`the art from the following description with reference to the
`attached drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a graph illustrating the relation between hydrox(cid:173)
`ypropylmethyl cellulose (HPMC) concentration and time
`required for re-dispersion of pranlukast.
`FIG. 2 is a graph illustrating the results of the test for
`inhibitory activity on conjunctival eosinocyte infiltration
`hereinafter.
`
`SUMMARY OF THE INVENTION
`
`The present inventors have extensively studied to promote
`solubilization or suspension of pranlukast in water and,
`consequently, have found that the desired solubilization or
`suspension can be obtained by using at least one component
`
`LUPIN EX1012, Page 4
`
`

`
`US 6,274,609 Bl
`
`3
`The surfactant can be used alone or in combination of two
`or more thereof and, normally, in case of an aqueous
`solution, the surfactant(s) are formulated in an amount of 0.5
`to 8 w/v %, preferably 1 to 5 w/v %, more preferably 2 to
`4 w/v % based on the total weight of the composition. And, 5
`in this case, the proportion of the surfactant(s) to pranlukast
`is such that 5 to 100 parts, preferably 10 to 60 parts by
`weight of the surfactant(s) are formulated per 1 part of
`pranlukast ('h hydrate).
`In case that the composition is an aqueous suspension,
`optionally, the surfactant(s) are formulated in an amount of
`0.0001 to 0.2 w/v %, preferably 0.001 to 0.2 w/v %, more
`preferably 0.01 to 0.2 w/v% based on the total weight of the
`composition together with the water-soluble high-molecular
`weight substance as described hereinafter. And, in this case,
`the proportion of the surfactant(s) to pranlukast is such that
`0.0001 to 0.2 part, preferably 0.001 to 0.2 part, more
`preferably 0.01 to 0.2 part by weight of the surfactant(s) are
`formulated per 1 part of pranlukast ('h hydrate).
`In the present invention, when the composition is in the
`form of an aqueous solution, normally, pH of the composi(cid:173)
`tion is adjusted to 6 or higher, preferably 6 to 9, more
`preferably 6 to 8. Further, in addition to the surfactants, it is
`preferred to formulate at least one stabilizer selected from
`anti-oxidants and chelating agents in an amount of about 25
`0.001 to 0.1 w/v %, thereby improving stability of pran(cid:173)
`lukast.
`As such stabilizers, there are, for example, anti-oxidants
`such as butylated hydroxytoluene, butylated
`hydroxyanisole, etc. and chelating agents such as sodium
`edetate, etc. They can be used alone or in combination of two
`or more thereof.
`The water-soluble cellulose derivatives used in the
`present invention include at least one member selected from
`methylcellulose, carboxymethylcellulose, hydroxypropyl
`cellulose, hydroxyethyl cellulose, and hydroxypropylmethyl
`cellulose.
`The water-soluble vinyl polymers used in the present
`invention include at least one member selected from poly(cid:173)
`vinylpyrrolidone K25, polyvinylpyrrolidone K30, polyvi(cid:173)
`nyl pyrrolidone K90, polyvinyl alcohol and carboxyvinyl
`polymer.
`These water-soluble high-molecular weight substances
`can be used alone or in combination of two or more thereof.
`As described above, these water-soluble high-molecular
`weight substances can be used together with one or more
`surfactants, thereby further improving re-dispersibility of
`the aqueous suspension, in particular, after storage.
`In the present invention, the water-soluble cellulose 50
`derivatives and the water-soluble vinyl polymers function,
`in particular, as a suspending agent of the aqueous liquid
`pharmaceutical composition in the form of a suspension.
`Then, they are formulated in an amount of 0.00001 to 0.1
`w/v %, preferably 0.00005 to 0.05 w/v % based on the total 55
`weight of the composition. And, the proportion of the
`water-soluble high-molecular weight substance(s) topran(cid:173)
`lukast is such that 0.0001 to 0.1 part by weight, preferably
`0.0005 to 0.02 part by weight of the water-soluble high(cid:173)
`molecular weight substance(s) are formulated per 1 part by 60
`weight of pranlukast ('h hydrate).
`In a liquid pharmaceutical composition, such suspending
`agent is generally used in concentration ranging from 0.1 to
`4 w/v % based on the total weight of the composition. In
`view of this, it is very surprising that the suspending agent 65
`functions even in such a small amount as in the present
`invention.
`
`4
`When the aqueous liquid pharmaceutical composition is
`in the form of a suspension, pH of the composition may be
`within, for example, the range normally employed for eye
`drops (e.g., pH 4 to 9, preferably pH 5 to 8).
`If necessary, the aqueous liquid pharmaceutical compo-
`sition may further contain suitable additives, for example, an
`isotonic agent such as inorganic salt (e.g., sodium chloride,
`boric acid, potassium chloride, etc.) and polyhydric alcohol
`(e.g., glycerin, mannitol, sorbitol, etc.); a buffer solution
`10 such as borate buffer solution, phosphate buffer solution,
`acetate buffer solution, citrate buffer solution, Tris buffer
`solution, etc. and buffer agent such as amino acid (e.g.,
`glutamic acid, E-aminocapronic acid, etc.); a chelating agent
`such as sodium edetate, citric acid, etc.; a preservative such
`15 as quaternary ammonium salt (e.g., benzalkonium chloridie,
`benzethonium chloride, etc.), p-hydroxybenzoate (methyl
`p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl
`p-hydroxybenzoate, butyl p-hydroxybenzoate, etc.), sorbic
`acid, chlorobutanol, sodium edetate, boric acid, etc., and the
`20 like. Normally, an isotonic agent can be formulated in an
`amount of 0.5 to 6.5 w/v % based on the total weight of the
`composition. Likewise, 0.01 to 1.0 w/v % of a buffer and
`0.001 to 0.1 w/v % of a chelating agent can be formulated
`based on the total weight of the composition.
`The aqueous liquid pharmaceutical composition of the
`present invention can be prepared in the form of an aqueous
`solution or suspension such as eye drops, nasal drops,
`injectable preparations, internal medicine and the like
`according to a per se known method. For example, the
`30 composition can be prepared by adding the solubilizing
`agent and/or suspending agent, a buffer, an isotonic agent
`and a preservative to sterilized purified water and, if
`necessary, heating to dissolve them. The desired liquid
`pharmaceutical composition can be prepared by dissolving
`35 or suspending pranlukast in the resultant solution.
`The aqueous liquid pharmaceutical composition of the
`present invention has eosinocyte infiltration inhibitory activ(cid:173)
`ity and is useful as an eosinocyte infiltration inhibitor. Then,
`it can be used for prophylaxis and therapy of seasonal or
`40 year-round allergic conjunctivitis, vernal
`keratoconjunctivitis, atopic keratoconjunctivitis, giant pap(cid:173)
`illary conjunctivitis, contact blephroconjunctivits, keratitis,
`scleritis, uveitis, eye itch, allergic rhinitis, sneeze, nasal
`itching, nasal hypersensitivity, nasofrontal eczema, nasal
`45 obstruction and the like. In general, the composition is
`topically or systemically administered once to six times per
`day at a daily dosage of 20 to 100 ,ug/ml of pranlukast.
`The present invention will be further illustrated by the
`following experiments and preparation examples, but the
`present invention is not limited to these preparation
`examples. In the following experiments and preparation
`examples, "pranlukast" used is the 'h hydrate and all the
`"percents" are by weight unless otherwise stated.
`Experiment 1
`Study of solubilizing agents of pranlukast
`Method
`Pranlukast (manufactured by Ono Pharmaceutical Co.,
`Ltd.) was suspended in 0.1% borate buffer (pH 9 or 8) or
`0.1% phosphate buffer (pH 8 or 7) at concentration of 0.1
`w/v %. Likewise, pranlukast was suspended in each 0.5%
`solution of surfactants (polysorbate 80, polysorbate 60,
`polysorbate 40, polyoxyethylene hydrogenated castor oil60,
`Tyloxapol, benzalkonium chloride, and sodium lauryl
`sulfate), water-soluble vinyl polymers (polyvinyl pyrrolidi(cid:173)
`one K30, and polyvinyl alcohol), cyclodextrins
`(~-cyclodextrin, y-cyclodextrin, and 2HP-~-cyclodextrin)
`
`LUPIN EX1012, Page 5
`
`

`
`US 6,274,609 Bl
`
`5
`and caffeine in 0.1% phosphate buffer (pH 7) at concentra(cid:173)
`tion of 0.1 w/v %. Each suspension was filled in a glass
`ampoule and was shaken overnight (for about 15 hours) at
`25° C. After shaking, the suspension was filtered through a
`membrane filter of 0.45 ,urn pore size and pranlukast in the 5
`filtrate was determined by HPLC.
`Results
`Solubility of pranlukast is shown in Table 1.
`
`TABLE 1
`
`10
`
`6
`
`TABLE 2-continued
`
`Solubility of pranlukast
`(ug/ml)
`H
`
`Buffer
`
`0.1% acetate
`buffer (pH after
`shaking)
`
`5
`
`87.2
`(5.06)
`
`292.4
`(6.12)
`
`7
`
`8
`
`Buffers
`
`Surfactants
`
`Water(cid:173)
`soluble
`vinyl
`polymers
`Cyclodextrin
`(control)
`
`Additives
`
`0.1% borate buffer
`0.1% borate buffer
`0.1% phosphate buffer
`0.1% phosphate buffer
`polysorbate 80
`polysorbate 60
`polysorbate 40
`polyoxyethylene hydrogenated
`castor oil 60
`Tyloxapol
`benzalkonium chloride
`sodium lauryl sulfate
`polyvinyl pyrrolidone K30
`polyvinyl alcohol
`
`13-cyclodextrin
`y-cyclodextrin
`2HP-i)-cyclodextrin
`caffeine (control)
`
`Solubil-
`ity
`(ugiml)
`
`pH
`
`9
`8
`8
`7
`7
`7
`7
`7
`
`7
`7
`7
`7
`7
`
`7
`7
`7
`7
`
`129.3
`38.4
`72.5
`2.4
`719.6
`639.3
`628.6
`408.1
`
`551.0
`174.5
`223.3
`159.9
`99.2
`
`105.2
`94.9
`107.5
`63.0
`
`Experiment 3
`Relation between concentration of polysorbate 80 and
`15 polyoxyethylene hydrogenated castor oil 60, and solubility
`of pranlukast
`Method
`Polysorbate 80 or polyoxyethylene hydrogenated castor
`oil 60 (hereinafter abbreviated as HC0-60) was dissolved in
`20 0.1% phosphate buffer at concentration of 1.0%, 2.0%, 3.0%
`or 4.0%. Pranlukast (manufactured by Ono Pharmaceutical
`Co., Ltd.) was suspended in the resultant solution at con(cid:173)
`centration of 0.2%. Each suspension was filled in a glass
`ampoule and shaken overnight (for about 15 hours) at 25° C.
`25 After shaking, the suspension was filtered with a membrane
`filter of 0.22 ,urn pore size and pranlukast in the filtrate was
`determined by HPLC.
`Results
`Regarding each nonionic surfactant at each concentration,
`solubility of pranlukast is shown in Table 3.
`
`30
`
`40
`
`As seen from Table 1, solubility of pranlukast in the buffer
`was increased with the rise of pH. Further, solubility of
`pranlukast was increased by addition of the surfactants, 35
`water-soluble vinyl polymers, cyclodextrin and caffeine.
`Experiment 2
`Relation between solubilization of pranlukast and pH,
`when using polysorbate 80 as the solubilizing agent
`Method
`Polysorbate 80 was dissolved in 0.1% phosphate buffer
`and/or 0.1% acetate buffer at concentration of 0.5%. Pran(cid:173)
`lukast (manufactured by Ono Pharmaceutical Co., Ltd.) was
`suspended in the resultant solution at concentration of 0.2% 45
`and pH was adjusted to 5 to 8 with sodium hydroxide or
`hydrochloric acid. Each suspension was filled in a glass
`ampoule and shaken overnight (for about 15 hours) at 25° C.
`After shaking, the suspension was filtered with a membrane
`filter of 0.22 ,urn pore size and pranlukast in the filtrate was 50
`determined by HPLC.
`Results
`Solubility of pranlukast is shown in Table 2.
`As the rise in pH, solubility of pranlukast was increased. 55
`A difference in buffers was scarcely observed.
`
`TABLE 3
`
`Solubility of pranlukast
`(ug/ml)
`Concentration of additive (%)
`
`Additive
`
`pH
`
`1.0
`
`2.0
`
`3.0
`
`4.0
`
`Polysorbate
`80
`HC0-60
`
`7
`
`7
`
`1052.1
`
`1483.6
`
`1636.4
`
`1747.4
`
`718.9
`
`1119.9
`
`1370.7
`
`1525.3
`
`As seen from Table 3, solubility of pranlukast was
`increased as increase in the amount of polysorbate 80 and
`polyoxyethylene hydrogenated castor oil 60.
`Experiment 4
`Test for stability of aqueous solution of pranlukast
`Method
`According to the formulations in Table 4, solutions A to
`F were prepared. Each solution was filled in a 5 ml-glass
`ampoule and stored at 60° C. for 2 weeks. After 2 weeks,
`pranlukast in the solution was determined by HPLC and its
`residual rate was calculated.
`
`TABLE 4
`
`Formulation
`
`TABLE 2
`
`Solubility of pranlukast
`(ug/ml)
`H
`
`Buffer
`
`0.1% phosphate
`buffer (pH after
`shaking)
`
`5
`
`92.6
`(5.28)
`
`7
`
`719.6
`(6.98)
`
`8
`
`1147.4
`(7.86)
`
`252.9
`(6.00)
`
`Component
`
`A
`
`B
`
`60
`
`c
`
`D
`
`E
`
`F
`
`pranlukasut
`polysorbate
`80
`Tyloxapol
`HC0-60*
`boric acid
`BHf**
`
`65
`
`0.1 g
`
`0.1 g
`
`0.1 g
`
`0.1 g
`4.0 g
`
`0.1 g
`4.0 g
`
`0.1 g
`4.0 g
`
`4.0 g
`
`4.0 g
`
`4.0 g
`
`1.9 g
`
`0.01 g
`
`LUPIN EX1012, Page 6
`
`

`
`US 6,274,609 Bl
`
`7
`
`TABLE 4-continued
`
`Formulation
`
`Component
`
`A
`
`B
`
`c
`
`D
`
`E
`
`F
`
`sodium
`edetate
`sodium di(cid:173)
`hydrogen
`phosphate
`benzalkonium
`chloride
`0.1 N sodium
`hydroxide
`sterilized
`purified
`water
`pH
`
`0.1 g
`
`0.1 g
`
`0.1 g
`
`0.1 g
`
`0.1 g
`
`0.01 g
`
`0.005 g
`
`q.s.
`
`q.s.
`
`q.s.
`
`q.s.
`
`q.s.
`
`q.s.
`
`up to
`total
`100 ml
`7.0
`
`up to
`total
`100 ml
`7.0
`
`up to
`total
`100 ml
`7.0
`
`up to
`total
`100 ml
`7.0
`
`upt to
`total
`100 ml
`7.0
`
`up to
`total
`100 ml
`7.0
`
`*polyoxyethylene hydrogenated castor oil 60
`* *butylated hydroxytoluene
`
`Results
`The residual rate of pranlukast is shown in Table 5.
`
`TABLE 5
`
`Residual rate (%)
`
`8
`
`5
`
`Experiment 6
`Test for eye irritation
`Method
`According to the formulations of Table 6, aqueous solu-
`tions (formulations G and H) and suspension (formulation J)
`of pranlukast were prepared. Eye drops of each solution or
`suspension were applied to Japanese white male rabbits 8
`times per day (0.05 ml/once) and eye irritation was evalu-
`10 ated by the naked eye according to Draize method.
`
`15
`
`20
`
`25
`
`pranlukast
`polysorbate 80
`polyoxyethylene hydrogenated
`castor oil 60
`hydroxypropylmethyl
`cellulose
`butylated hydroxytoluene
`sodium dihydrogen phosphate
`sodium acetate
`sodium chloride
`methyl p-hydroxybenzoate
`propyl p-hydroxybenzoate
`0.1 N sodium hydroxide
`0.1 N hydrochloric acid
`serilized purified water
`
`TABLE 6
`
`G
`
`0.1 g
`4.0 g
`
`0.01 g
`0.1 g
`
`0.9 g
`
`H
`
`0.1 g
`
`4.0 g
`
`0.1 g
`
`0.9 g
`
`q.s.
`
`q.s.
`
`up to 100
`ml
`7.0
`
`up to 100
`ml
`7.0
`
`1.0 g
`
`0.005 g
`
`0.1 g
`0.9 g
`0.026 g
`0.014 g
`
`q.s.
`up to 100
`ml
`5.0
`
`A
`
`B
`
`c
`
`D
`
`E
`
`F
`
`pH
`
`100.0
`
`100.0
`
`100.0
`
`100.0
`
`100.0
`
`100.0
`
`99.6
`
`99.4
`
`98.9
`
`85.0
`
`97.5
`
`95.1
`
`30
`
`Immediately
`after
`preparation
`After two
`weeks
`
`45
`
`As seen from Table 5, when Tyloxapol and polyoxyeth- 35
`ylene hydrogenated castor oil 60 were used as the solubi(cid:173)
`lizing agents (formulations A, B and C), the residual rate of
`pranlukast was more than 98% and was stable. When
`polysorbate 80 was used as the solubilizing agent
`(formulation D), although stability of pranlukast was some- 40
`what lowered in comparison with the other surfactants,
`stability of more than 95% was obtained by adding the
`stabilizer, BHT or sodium edetate (formulations E and F).
`In each solution, no deposit of any insoluble material was
`observed.
`Experiment 5
`Relation between concentration of suspending agent and
`re-dispersibility
`Method
`Solutions of hydroxyporpylmethyl cellulose (hereinafter
`abbreviated as HPMC) ranging in concentration from
`0.0001 to 0.5% were prepared and pranlukast was added
`thereto at concentration of 0.5%. After standing at 25° C. for
`4 days, time required for re-dispersion was measured with a
`variable mix rotor VMR-5 (60 rpm, manufactured by Inch
`Co., Ltd.)
`Results
`FIG. 1 illustrates the relation between HPMC concentra- 60
`tion and time required for re-dispersion.
`As seen from FIG. 1, when concentration of HPMC
`(X-axis) was within the range of 0.00075 to 0.1 %, time
`required for re-dispersion of suspending particles of pran(cid:173)
`lukast (Y-axis) was relatively shorter. In addition, no aggre(cid:173)
`gation or caking of suspending particles of pranlukast was
`observed within this concentration range.
`
`50
`
`Results
`No eye irritation was observed in all the formulations.
`Experiment 7
`Conjunctival eosinocyte infiltration inhibitory activity
`Method
`1. Animals
`Hartley male guinea pigs (body weight: about 300 to 500
`g) purchased from Japan SLC were used. The animals were
`bred under conditions of a temperature of 23±2° C. and
`humidity of 55±15%. They were maintained on pellets
`(radiation sterilized Labo G standard manufactured by
`Nihon Nosan Kogyo) and sterilized water for animals ad
`libitum.
`2. Test medicine
`Pranlukast was suspended in a vehicle containing 0.1% of
`sodium dihydrogen phosphate, 0.9% of sodium chloride and
`0.1% polysorbate 80 (pH 7) at concentration of 1.0%. As a
`control, physiological saline solution was used.
`3. Procedure
`The guinea pigs were sensitized by administering a mix(cid:173)
`ture of 10 jAg of ovalbumin (hereinafter referred to as OA)
`and 30 mg of aluminum hydroxide gel intraperitoneally. 14
`Days after sensitization, 10 tAl of 2.5% OA antigen solution
`55 was instilled in both eyes of the guinea pigs to cause
`conjunctivitis (primary challenge). Likewise, 24 hours after
`the primary challenge, 2.5% OA antigen solution was
`instilled to cause conjunctivitis again (secondary challenge).
`After 6 hours, each guinea pig was slaughtered, the upper
`and lower lids were removed from the periosteum and the
`lids together with the eyeball were extracted. According to
`a conventional manner, the eyeball was fixed and embedded
`in paraffin to prepare a specimen for optical microscopic
`examination and cut into slices 3 jAm in thickness. The
`65 resultant pathological slice was stained by Luna stain, its
`part where most infiltration of conjunctival eosinocytes was
`observed was selected, and the number of eosinocytes in 5
`
`LUPIN EX1012, Page 7
`
`

`
`US 6,274,609 Bl
`
`9
`fields were counted which were not overlapped with one
`another under an optical microscope (magnifying power:
`400). The mean of the counts of the 5 fields was calculated
`(cells/0.04 mm2
`) to evaluate the medicine. The medicine
`was instilled in one eye at a dosage of 10 ,ul at 3, 2, 1 and 5
`0.5 hour prior to challenge of conjunctivitis, respectively.
`
`Results
`
`The results are shown in FIG. 2.
`FIG. 2 is a graph illustrating inhibitory effect of pran- 10
`lukast on conjunctival eosinocyte infiltration in guinea pig
`allergic conjunctivitis. The values in the graph represent the
`mean ± standard error. The symbol ** represents the sig(cid:173)
`nificant difference from physiological saline solution,
`p<O.Ol. As seen from FIG. 2, pranlukast showed significant 15
`inhibitory effect on conjunctival eosinocyte infiltration in the
`delayed type reaction.
`
`10
`
`Ingredient
`
`Pranlukast
`Sodium dihydrogen phosphate 2 hydrate
`Sodium edetate
`Benzalkonium chloride
`Tyloxapol
`Sodium chloride
`0.1 N sodium hydroxide
`Sterilized purified water
`pH
`
`Amount
`
`0.1 g
`0.2 g
`0.01 g
`0.01 g
`4.0 g
`0.9 g
`q.s.
`up to 100 ml
`7.0
`
`PREPARATION EXAMPLE 4
`
`Aqueous Solution
`
`PREPARATION EXAMPLE 1
`
`Aqueous Solution
`
`20
`
`According to a conventional method, an aqueous solution
`for eye drops and nasal drops having the following formu(cid:173)
`lation was prepared.
`
`According to a conventional method, an aqueous solution
`for eye drops and nasal drops having the following formu-
`lation was prepared.
`
`25
`
`Ingredient
`
`Pranlukast
`Sodium dihydrogen phosphate 2 hydrate
`Polysorbate 80
`Butylated hydroxytoluene
`Sodium edetate
`Benzalkonium chloride
`Sodium chloride
`0.1 N sodium hydroxide
`Sterilized purified water
`pH
`
`Amount
`
`0.1 g
`0.2 g
`4.0 g
`0.01 g
`0.01 g
`0.01 g
`0.9 g
`q.s.
`up to 100 ml
`7.0
`
`30
`
`35
`
`40
`
`Ingredient
`
`Pranlukast
`Sodium dihydrogen phosphate 2 hydrate
`Polysorbate 80
`Butylated hydroxytoluene
`Sodium edetate
`Benzalkonium chloride
`Boric acid
`Borax
`0.1 N sodium hydroxide
`Sterilized purified water
`pH
`
`Amount
`
`0.1 g
`0.2 g
`4.0 g
`0.01 g
`0.01 g
`0.01 g
`1.5 g
`0.3 g
`q.s.
`up to 100 ml
`7.0
`
`PREPARATION EXAMPLE 5
`
`PREPARATION EXAMPLE 2
`
`Aqueous Suspension
`
`Aqueous Solution
`
`According to a conventional method, an aqueous solution
`for eye drops and nasal drops having the following formu(cid:173)
`lation was prepared.
`
`According to a conventional method, an aqueous suspen(cid:173)
`sion for eye drops and nasal drops having the following
`45 formulation was prepared.
`
`Ingredient
`
`Amount
`
`50
`
`Pranlukast
`Sodium dihydrogen phosphate 2 hydrate
`Polyoxyethylene hydrogenated castor oil 60
`Sodium edetate
`Benzalkonium chloride
`Boric acid
`0.1 N sodium hydroxide
`Sterilized purified water
`pH
`
`0.1 g
`0.2 g
`2.0 g
`0.01 g
`0.01 g
`1.5 g
`q.s.
`up to 100 ml
`7.0
`
`55
`
`60
`
`PREPARATION EXAMPLE 3
`
`Aqueous Solution
`
`Ingredient
`
`Pranlukast
`Sodium acetate
`Hydroxypropylmethyl cellulose
`Sodium chloride
`Methyl p-hydroxybenzoate
`Propyl p-hydroxybenzoate
`0.1 N hydrochloric acid
`Sterilized purified water
`pH
`
`Amount
`
`1.0 g
`0.1 g
`0.005 g
`0.9 g
`0.026 g
`0.014 g
`q.s.
`up to 100 ml
`5.0
`
`PREPARATION EXAMPLE 6
`
`Aqueous Suspension
`
`According to a conventional method, an aqueous solution 65
`for eye drops and nasal drops having the following formu(cid:173)
`lation was prepared.
`
`According to a conventional method, an aqueous suspen(cid:173)
`sion for eye drops and nasal drops having the following
`formulation was prepared.
`
`LUPIN EX1012, Page 8
`
`

`
`US 6,274,609 Bl
`
`11
`
`12
`
`-continued
`
`Ingredient
`
`Pranlukast
`Sodium acetate
`HC0-60
`Sodium chloride
`Methyl p-hydroxybenzoate
`Propyl p-hydroxybenzoate
`0.1 N hydrochloric acid
`Sterilized purified water
`pH
`
`Amount
`
`1.0 g
`0.1 g
`0.1 g
`0.9 g
`0.026 g
`0.014 g
`q.s.
`up to 100 ml
`5.0
`
`PREPARATION EXAMPLE 7
`
`Aqueous Suspension
`According to a conventional method, an aqueous suspen(cid:173)
`sion for eye drops and nasal drops having the following
`formulation was prepared.
`
`Ingredient
`
`Pranlukast
`Sodium acetate
`Tyloxapol
`Sodium chloride
`Methyl p-hydroxybenzoate
`Propyl p-hydroxybenzoate
`Chlorobutanol
`0.1 N hydrochloric acid
`Sterilized purified water
`pH
`
`Amount
`
`1.0 g
`0.1 g
`0.1 g
`0.9 g
`0.026 g
`0.014 g
`0.3 g
`q.s.
`up to 100 ml
`5.0
`
`PREPARATION EXAMPLE 8
`
`Aqueous Suspension
`According to a conventional method, an aqueous suspen(cid:173)
`sion for eye drops and nasal drops having the following
`formulation was prepared.
`
`Ingredient
`
`Pranlukast
`Sodium acetate
`Hydroxypropylmethyl cellulose
`Sodium chloride
`Methyl p-hydroxybenzoate
`Propyl p-hydroxybenzoate
`0.1 N hydrochloric acid
`Sterilized purified water
`pH
`
`Amount
`
`0.5 g
`0.1 g
`0.0025 g
`0.9 g
`0.026 g
`0.014 g
`q.s.
`up to 100 ml
`5.0
`
`PREPARATION EXAMPLE 9
`
`Aqueous Suspension
`According to a conventional method, an aqueous suspen(cid:173)
`sion for eye drops and nasal drops having the following
`formulation was prepared.
`
`Ingredient
`
`Pranlukast
`Sodium acetate
`
`Amount
`
`0.1 g
`0.1 g
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Ingredient
`
`Hydroxypropylmethyl cellulose
`Sodium chloride
`Methyl p-hydroxybenzoate
`Propyl p-hydroxybenzoate
`0.1 N hydrochloric acid
`Sterilized purified water
`pH
`
`Amount
`
`0.0005 g
`0.9 g
`0.026 g
`0.014 g
`q.s.
`up to 100 ml
`5.0
`
`PREPARATION EXAMPLE 10
`
`Aqueous Suspension
`According to a conventional method, an aqueous suspen(cid:173)
`sion for eye drops and nasal drops having the following
`formulation was prepared.
`
`Ingredient
`
`Pranlukast
`Sodium acetate
`Methyl cellulose
`Sodium chloride
`Methyl p-hydroxybenzoate
`Propyl p-hydroxybehzoate
`0.1 N hydrochloric acid
`Sterilized purified water
`pH
`
`Amount
`
`1.0 g
`0.1 g
`0.005 g
`0.9 g
`0.026 g
`0.014 g
`q.s.
`up to 100 ml
`5.0
`
`PREPARATION EXAMPLE 11
`
`Aqueous Solution
`According to a conventional method, an aqueous solution
`for an injectable preparation having the following formula(cid:173)
`tion was prepared.
`
`Ingredient
`
`Pranlukast
`Polysorbate 80
`S