United States Patent
`(12)
`(10) Patent No.:
`US 7,112,565 B2
`Sawai et al.
`(45) Date of Patent:
`*Sep. 26, 2006
`
`
`US007112565B2
`
`(54) STABILIZED PHARMACEUTICAT.
`COMPOSITION IN LYOPHILIZED FORM
`
`(75)
`
`Inventors: Seiji Sawai, Takarazuka (JP); Akihiro
`Kasai, Ikoma (JP); Kazumi Ohtomo,
`Ibaraki (JP)
`
`(73) Assignee: Astellas Pharma Ine., Tokyo (JP)
`
`(*) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended. or adjusted under 35
`US.C. 154(b) by 307 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21) Appl. No.: 10/772,281
`
`(22) Filed:
`
`Feb. 6, 2004
`
`(65)
`
`Prior Publication Data
`US 2004/0157769 Al
`Aug. 12, 2004
`Related U.S. Application Data
`(62) Division of application No. 09/786,125, filed as appli-
`cation No. PCT/JP00/04381 on Jun. 29, 2000, now
`Pat. No. 6,774,104.
`
`(30)
`Jul. 1, 1999
`
`Foreign Application Priority Data
`(ID)
`ceecessceeeseveeeereeeeesreeeeen 11/187713
`
`G1)
`
`(52)
`
`(58)
`
`(56)
`
`JP
`
`Int. Cl.
`(2006.01)
`AGIK 38/00
`(2006.01)
`AGIK 38/12
`U.S. Ch. cece 514/9; 514/2; 514/11: 514/15;
`530/317; 530/323
`Field of Classification Search ......0......00..... None
`Sce application file for complete scarch history.
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`12/1994 Iwamoto et al.
`5,376,634 A
`10/1996 Obki et al.
`5,569,646 A
`8/1999 Floyd etal.
`5,942,510 A
`3/2001 Uedaet al.
`6,207,434 Bl
`6/2002 Kanasaki et al.
`6,399,567 Bl
`8/2004 Sawai et al. ......ee eee 514/9
`6,774,104 BL*
`FOREIGN PATENT DOCUMENTS
`3-193735
`8/1991
`
`
`
`JP
`JP
`JP
`
`JP
`wo
`wo
`WO
`
`WO
`
`3-240727
`6-51641
`9-301997
`
`10-507174
`WO 96/11210
`WO 97/39763
`WO 00/51564
`
`WO 00/51567
`
`10/1991
`7/1994
`11/1997
`
`T/A998
`4/1996
`10/1997
`9/2000
`
`2000
`
`® cited by
`
`examiner
`
`y
`Primary Examiner—B. Dell Chism
`(74) Attorney, Agent, or Firm—Oblon, Spivak, McClelland,
`Maier & Neustadt, P.C.
`
`(57)
`
`ABSTRACT
`
`Astabilized pharmaceutical composition in lyophilized form
`comprising: a cyclic polypeptide compoundrepresented by
`the general formula (1):
`
`2
`®
`
`NH
`
`(I
`®
`
`Or
`
`NH—R!
`
`Hy
`
`IIO
`
`wherein R’ is a hydrogen atom or an acyl group and R? and
`R? are, the sameordifferent, a hydrogen atom or a hydroxyl
`group, or a salt thereof and the stabilizer.
`
`23 Claims, No Drawings
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`1
`STABILIZED PHARMACEUTICAL
`COMPOSITION IN LYOPHILIZED FORM
`
`‘This application is a divisional of U.S. Ser. No. 09/786,
`125, filed Mar. 1, 2001, now U.S. Pat. No. 6,774,104, which
`is the national-stage under 35 U.S.C. §371 of PCT/JP00/
`04381, filed Jun. 29, 2000. This application also claims
`priority to JAPAN 11/187713, filed Jul. 1, 1999.
`TECHNICAL FIELD
`
`The present invention relates to a stabilized pharmaceu-
`tical composition in lyophilized form containing a cyclic
`polypeptide compound. More particularly,
`the present
`inventionrelates to a stabilized pharmaceutical composition
`in lyophilized form containing a cyclic polypeptide com-
`pound or its pharmaceutically acceptable salt and a stabi-
`lizer.
`The cyclic polypeptide compoundofthe present invention
`is represented by the general formula (1):
`
`R
`
`NH
`
`H3C
`
`HO Q
`
`N
`
`0)
`
`OH
`
`NH—R!
`
`‘Oo
`
`HO
`
`Oo
`
`JIN
`
`OIl
`
`0.
`
`CH;
`
`N
`
`‘OH
`
`30
`
`35
`
`40
`
`45
`
`wherein R' is a hydrogen atom or an acyl group and R? and
`R? are, the sameordifferent, a hydrogen atomor a hydroxyl
`group. The compoundhas an antimicrobial activity, particu-
`larly an antifungal activity and a B-1,3-glucan synthase
`i2
`inhibiting action, and is useful for preventing and treating 5
`various kinds ofinfectious diseases including Preumocystis
`carinii infection, e.g., carinii pneumonia.
`
`BACKGROUND ART
`
`Amongthe cyclic polypeptide compoundsrepresented by
`the above formula (1), a compound wherein R’* is a hydrogen
`atom and R* and R* are hydroxyl groups and a compound
`wherein R’, R? and R° are hydrogen atomsare obtained by
`a fermentation process disclosed by European Patent No.
`0462531 and processes disclosed by WO97/32975 and by
`W097/47738. A compound wherein R’ is an acyl group and
`its production process are disclosed by U.S. Pat. Nos.
`5,376,634 and 5,569,646 and WO96/11210 and WO99/
`40108.
`The cyclic polypeptide compounds(I) andtheir salts are
`generally unstable to light, humidity, acids, heat and thelike.
`
`60
`
`65
`
`2
`Therefore, desired is development of pharmaceutical prepa-
`rations in which the cyclic polypeptide compoundsandtheir
`salts are stabilized.
`
`DISCLOSURE OF INVENTION
`
`
`
`The present invention provides a stabilized pharmaceuti-
`cal composition in lyophilized form containing a cyclic
`polypeptide compound(1) or ils pharmaceutically accept-
`able salt and a stabilizer.
`
`The “acyl group”for R' in the formula(1) representing the
`cyclic polypeptide compound of the present invention is
`now explained. In the context of the present specification,
`“lower” means having one to six carbon atoms unless
`otherwise indicated.
`
`As examples of the acyl group, may be mentionedali-
`phatic acy] groups, aromatic acyl groups, aromatic-aliphatic
`acyl groups and heterocyclic acyl groups derived from
`aliphatic, aromatic, aromatic-aliphatic and heterocyclic car-
`boxylic acids.
`Examples ofthe aliphatic acyl groups include lower or
`higher alkanoyl groups such as formyl, acetyl, propanoyl,
`butanoyl, 2-methylpropanoyl, pentanoy], 2,2-dimethylpro-
`panoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl,
`undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, penta-
`decanoyl,
`hexadecanoyl,
`heptadecanoyl,
`octadecanoyl,
`nonadecanoyl, icosanoyl, etc.; cycloalkanoyl groups such as
`cyclopentanoyl and cyclohexanoyl;
`lower alkoxycarbonyl
`groups such as methoxycarbonyl, ethoxycarbonyl, t-butoxy-
`carbonyl,
`t-pentyloxycarbonyl, heptyloxycarbonyl,
`etc.;
`lower alkanesulfonyl groups
`such as methanesulfonyl,
`ethanesulfonyl, etc.; lower alkoxysulfonyl groups such as
`methoxysulfony!. ethoxysulfonyl, etc.; and the like.
`Examples of the aromatic acyl groups include aroyl
`groups such as benzoyl, toluoyl, naphthoyl and the like.
`Examples of the aromatic-aliphatic acyl groups include
`ar(lower)alkanoyl groups such as phenyl(C,—C,)alkanoyl
`(e.g., phenylacetyl, phenylpropanoyl, phenylbutanoyl, phe-
`nylisobulanoyl, phenylpentanoyl, phenylhexanoyl, etc.),
`naphthyl(C,—C,)alkanoyl
`(e.g., naphthylacetyl, naphthyl-
`propanoyl, naphthylbutanoyl, etc.) and the like; ar(lower)
`alkenoyl group such as phenyl(C,—C,)alkenoyl (e.g., phe-
`nylpropenoy],
`phenylbutenoyl,
`phenylmethacryloyl,
`phenylpentenoyl, phenylhexenoyl, etc.), naphthyl(C,—C,)
`alkenoyl (e.g., naphthylpropenoy!, naphthylbutenoyl, etc.
`and the like;
`ar (lower)alkoxycarbonyl groups such as phenyl(C,—C,)
`alkoxycarbonyl (e.g., benzyloxycarbonyl, etc.), fluorenyl
`(C,-C, alkoxycarbonyl (e.g., fluorenylmethoxycarbonyl,
`etc.) and the like;
`aryloxycarbonyl groups such as phenoxycarbonyl, naph-
`thoxycarbonyl, etc.;
`aryloxy(lower)alkanoy! groups such as phenoxyacety!, phe-
`noxypropionyl, etc.;
`arylcarbamoyl groups such as phenylcarbamoy], etc.;
`arylthiocarbamoyl groups such as phenylthiocarbamoyl,
`etc.;
`arylglyoxyloyl groups such as phenylglyoxyloyl, naphthylg-
`lyoxyloyl, etc.;
`arylsulfonyl groups which may be optionally substituted by
`a loweralkyl group such as phenylsulfonyl, p-tolylsulfo-
`nyl, etc.; and the like.
`Examples of the heterocyclic acyl groups include hetero-
`cyclic carbonyl groups such as thenoyl, furoyl, nicotinoyl,
`etc.;
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`5
`
`30
`
`35
`
`3
`heterocyclic(lower)alkanoyl groups such as heterocyclic
`acetyl, heterocyclic propanoyl, heterocyclic butanoyl,
`heterocyclic pentanoyl, heterocyclic hexanoyl, etc.;
`heterocyclic(lower)alkcnoyl groups such as heterocyclic
`propenoyl, heterocyclic butenoyl, heterocyclic pentenoyl,
`heterocyclic hexenoyl,etc.:
`heterocyclic glyoxyloy! and thelike.
`The acyl group for R' may have one or more suitable
`substituent(s). Among the above-mentioned examples for
`the acy! groups, an aroy! group which may have one or more
`suitable substituent(s) is particularly preferable.
`Examples of suitable substituents in the acyl group
`include a heterocyclic group substituted by an aryl group
`having a lower alkoxy group, a heterocyclic group substi-
`tuted by an aryl group having a lower alkoxy(lower)alkoxy
`group, a heterocyclic group substituted by an aryl group
`having a lower alkoxy(higher)alkoxy group, a heterocyclic
`group substituted by an aryl group having a cyclo(lower)
`alkyloxy group, a heterocyclic group substituted by an aryl
`group having a heterocyclic group, a heterocyclic group
`substituted by a cyclo(lower)alkyl group having a cyclo
`(ower)alkyl group, a heterocyclic group substituted by an
`aryl group having an aryl group substituted by a lower
`alkoxy(lower)alkoxy and a heterocyclic group substituted 5;
`by an aryl group having a heterocyclic group substituted by
`a cyclo(lower)alkyl group.
`Amongthese examples, preferred are an unsaturated 3- to
`8-membered heteromonocyclic group containing one to two
`oxygen atom(s) and one to three nitrogen atom(s) and
`substituted by phenyl having (C,-C,)alkoxy, an unsaturated
`condensed heterocyclic group containing one to two sulfur
`atom(s) and oneto three nitrogen atom(s) and substituted by
`phenyl having (C,-C,)alkoxy, an unsaturated 3- to 8-mem-
`bered heteromonocyclic group containing one to two sulfur
`atom(s) and oneto three nitrogen atom(s) and substituted by
`phenyl having (C,-C,)alkoxy(C,-C,alkoxy, an unsaturated
`3- to 8-membered heteromonocyclic group containing one to
`two sulfur atom(s) and one to three nitrogen alom(s) and
`substituted by phenyl having (C,—-C,)alkoxy(C,-C,,)
`alkoxy, a saturated 3- to 8-membered heteromonocyclic
`group containing one to four nitrogen atom(s) and substi-
`tuted by phenyl having (C,-C,)alkoxy(C,-C,,alkoxy, an
`unsaturated condensed heterocyclic group containing one to
`two sulfur atom(s) and one to three nitrogen atom(s) and
`substituted by phenyl having cyclo(C,-C,)alkyloxy, an
`unsaturated condensed heterocyclic group containing one to
`two sulfur atom(s) and one to three nitrogen alom(s) and
`substituted by phenyl, a saturated 3- to 8-membered hetero-
`monocyclic group containing one to two oxygen atom(s) and 5
`oneto three nitrogen atom(s), a saturated 3- to 8-membered
`heteromonocyclic group having one to four nitrogen atom(s)
`and substituted by cyclo(C,-C,)alkyl having cyclo(C,-C,)
`alkyl, an unsaturated 3- to 8-membered. heteromonocyclic
`group having one to two sulfur atom(s) and one to three
`nitrogen atom(s) and substituted by phenyl having phenyl
`substituted by (C,-C,alkoxy(C,-C,alkoxy, an unsaturated
`3- to 8-membered heteromonocyclic group containing one to
`two sulfur atom(s) and one to three nitrogen atom(s) and
`substituted by phenyl having a saturated 3- to 8-membered
`heteromonocyclic group which contains oneto four nitrogen
`atom(s) and is substituted by cyclo(C,-C,)alkyl, and an
`unsaturated. condensed heterocyclic group containing one to
`two sulfur atom(s) and one to three nitrogen atom(s) and
`substituted by phenyl having a saturated 3- to 8-membered
`heleromonocyclic group which contains oneto four nitrogen
`atom(s) and has cyclo(C,—C,)alkyl.
`
`40
`
`45
`
`4
`Among these, particularly preferred are an isoxazolyl
`group substituted by phenyl having pentyloxy, an imida-
`zothiadiazolyl group substituted by phenyl having penty-
`loxy, a thiadiazolyl group substituted by phenyl having
`methoxyhexyloxy, a thiadiazolyl group substituted by phe-
`nyl having methoxyoctyloxy, a thiadiazolyl group substi-
`tuted by phenyl having methoxyheptyloxy, an imidazothia-
`diavoly] group substituted by phenyl having cyclohexyloxy,
`an imidazothiadiazolyl group substituted by phenyl having
`dimethylmorpholino, a piperazinyl group substituted by
`phenyl having methoxyheptyloxy, a piperazinyl group sub-
`stituted by phenyl having methoxyoctyloxy, a piperaziny]l
`group substituted by cyclohexyl having cyclohexyl, a thia-
`diazoly! group substituted by phenyl having pheny! substi-
`tuted by mcthoxycthoxy, a thiadiazolyl group substituted by
`phenyl having pheny! substituted by methoxybutoxy,a thia-
`diavoly] group substituted by phenyl having phenyl substi-
`tuted by ethoxypropoxy, an imidazothiadiazolyl group sub-
`stituted by phenyl having piperazinyl
`substituted by
`cyclohexyl, an imidazothiadiazolyl group substituted by
`phenyl]having piperaziny! substituted by cyclohexyl, and the
`like.
`
`Accordingly, particularly suitable examples of the acyl
`group of R' maybe a benzoyl group having isoxazolyl
`substituted by phenyl having pentyloxy, a benzoyl group
`having imidazothiadiazolyl substituted by phenyl having
`pentyloxy, a benzoyl group having thiadiazolyl substituted
`by phenyl! having methoxyhexyloxy, a benzoyl group having
`thiadiazoly] substituted by phenyl having methoxyoctyloxy,
`a benzoyl group having thiadiazolyl substituted by phenyl
`having methoxyheptyloxy, a benzoyl group having imida-
`zothiadiazolyl substituted by phenyl having cyclohexyloxy,
`a benzoyl group having imidazothiadiazolyl substituted by
`pheny! having dimethylmorpholino, a benzoyl group having
`piperazinyl substituted by pheny] having methoxyheptyloxy,
`a benzoyl group having piperazinyl substituted by phenyl
`having methoxyoctyloxy, a benzoyl group having piperazi-
`nyl substituted by cyclohexyl having cyclohexyl, a benzoyl
`group having thiadiazolyl substituted by phenyl having
`phenyl]substituted by methoxyethoxy, a benzoyl group hav-
`ing thiadiazolyl substituted by phenyl having phenyl sub-
`stituted by methoxybutoxy, a benzoyl group having thiadia-
`zolyl substituted by phenyl having phenyl substituted by
`ethoxypropoxy, a benzoyl group having imidazothiadiazolyl
`substituted by phenyl having piperazinyl substituted by
`cyclohexyl, a benzoyl group having imidazothiadiazolyl
`substituted by phenyl having piperazinyl substituted by
`cyclohexyl, and the like.
`Particularly preferable examples of the acyl groups of R,
`are represented by the formulas:
`
`
`
`Oo
`
`0
`
`CDN—oO
`PDO
`// D4precisa
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`oO
`
`Oo
`
`5
`
`-continued
`
`o>
`—_
`a
`N
`s
`
`wa
`
`O(CH34yCH3
`
`N—N
`
`[ Dprecto
`
`8
`
`The cyclic polypeptide compounds(1) having the above-
`mentioned acyl groups maybe prepared from a compound
`having a hydrogen atom as R, and hydroxyl groups as R?
`and. R? or a compound having hydrogen atomsas R!, R? and
`R? according to the U.S. Pat. Nos. 5,376,634 and 5,569,646
`and WO96/11210 and WO99/40108.
`
`Suitable salts of the cyclic polypeptide compounds(1) are
`soluble in water and pharmaceutically acceptable salts
`including salts with bases and acid additionsalts. Such a salt
`
`
`
`6
`may beprepared by treating the cyclic polypeptide com-
`pound (1) with an appropriate base or acid according to the
`conventional method.
`
`As salts with bases, may be mentioned salts with inor-
`ganic bases such as alkali metal salts (e.g., sodiumsalts,
`potassium salts, etc.), alkaline earth metal salts (e.g., cal-
`cium salts, magnesium salts, etc.), ammonium salts and the
`like; salts with organic bases such as organic amine salts
`(e.g., triethylamine salts, diisopropylethylamine salts, pyri-
`dine salts, picoline salts, ethanolamine salts, triethanolamine
`salts, dicyclohexylamine salts, N,N'-dibenzylethylenedi-
`aminesalts, etc.); and the like.
`As acid addition salts, may be mentioned inorganic acid
`addition salts (c.g., hydrochlorides, hydrobromides, sulfates,
`phosphates, etc.); and organic carboxylic or sulfonic acid
`addition salts (e.g.,
`formales, acetates,
`trifluorvacetates,
`maleates, tartrates, fumarates, methnesulfonates, benzene-
`sulfonates,
`toluenesulfonates, etc.). Further, may also be
`mentioned salts with basic or acidic amino acids(e.g., salts
`with arginine, aspartic acid, glutamic acid, etc.).
`The cyclic polypeptide compounds (1) of the present
`invention also include possible conformers and a pair or
`moreof stereoisomers such as geometric isomers and optical
`isomers which may exist due to asymmetric carbon atoms.
`The preferable ones of the cyclic polypeptide compounds
`(1) are represented bythe following, formulas (II) to (V1):
`
`(to be continued on the next page)
`
`a)
`
`(>) () (>) ee
`
`N—O
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`N—N
`
`°
`
`N—N
`i
`\
`
`°
`
`-continued
`
`°gq
`
`Oo
`
`°
`
`
`
`.oa
`
`HN
`
`ay
`
`Z
`
`©as]
`
`°
`
`HO Q
`
`NI
`
`I
`
`N
`
`Q
`
`NH
`
`NaO—S—O HO
`
`OH
`
`NH
`
`0
`
`HO &
`
`NH
`
`N
`
`110.
`
`oO
`
`HN
`
`H3C
`
`oO.
`
`LN
`
`Oo
`
`NH
`
`O
`
`N
`
`NH
`
`OH
`
`o
`
`oO
`
`OH
`
`CH;
`
`OIL
`
`NaO—S—O
`
`HO
`
`ay
`
`(IV)
`
`0)
`
`O(CH)7OCH
`
`O(CHp)4CH3
`
`AMNEALEX. 1006
`
`fxa \
`
`Ne
`
`s
`
`OQ
`
`QO
`
`ogq
`
`o
`
`OIL
`
`CH;
`
`HO
`
`oO
`
`JIN
`
`NII
`
`O
`
`Zz
`
`‘4q
`
`°
`
`xCcQs Zzaooo
`°q
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`10
`
`-continued
`
`YeHN<<
`
`/
`
`(VI)
`
`—N
`
`Sprocct
`
`s
`
`The most preferable one is represented by the formula
`(II).
`The amountof the cyclic polypeptide compound(1) orits
`pharmaceutically acceptable salt contained in the composi-
`tion for a single unit dosage ofthe present invention is 0.1
`to 400 mg, more preferably 1
`to 200 mg, still more prefer-
`ably 10 to 100 mg,specifically 10, 15, 20, 25, 30, 35, 40, 45,
`50, 55, 60, 70. 75, 80, 85, 90, 95 and 100 mg.
`As the stabilizer, may be mentioned polysaccharides,
`disaccharides, sodium chloride and a combination thereof.
`Examples of the polysaccharide are dextran, starch, cel-
`lulose and hyaluronic acid; and examples ofthe disacharide
`are lactose, maltose and sucrose. The polysaccharide or
`disaccharide contained in the pharmaceutical composition of
`the present invention may be a-monohydrate, a-anhydride,
`B-anhydride or a combination thereof.
`The amountofthe stabilizer used in the pharmaceutical
`composition of the present
`invention should be at
`least
`sufficient for stabilizing the cyclic polypeptide compound(I)
`or its pharmaceutically acceptable salt in the composition. In
`order to stabilize the cyclic polypeptide compound(1), one
`part by weight of the stabilizer with respect to one part by
`weight of the cyclic polypeptide compound(I) or its phar-
`maceutically acceptable salt in the present composition is
`i2
`sufficient at least. he stabilizer mayalso serve as a carrier 5
`or an excipient. Thus the use amount ofstabilizer does not
`have a particular upper limit and may be determined in
`consideration of the weight or volume of the composition
`with respect to a unit dose of the compound and the like.
`However, such amount
`is preferably 0.4 to 50 parts by
`weight, more preferably 0.6 to 20 parts by weight, still more
`preferably 0.8 to 10 parts by weight with respect to one part
`by weight of the cyclic polypeptide compound (1) or its
`pharmaceutically acceptable salt, thoughit varies depending
`uponthe kind and the used amountof the cyclic polypeptide
`compound (I) or its pharmaceutically acceptable salt,
`its
`preparation form and/or the like. Specifically,
`it
`is more
`preferable that 1 to 20 parts, still more preferably 2 to 10
`parts by weight of the disaccharide are used with respect to
`one part by weight of the cyclic polypeptide compound (I)
`or its pharmaceutically acceptable salt. Specifically,
`it is
`morepreferable that 0.6 to 20 parts, still more preferably 0.8
`to 10 parts by weight of sodium chloride are used with
`
`respect to one part by weight of the cyclic polypeptide
`compound. (I) or its pharmaceutically acceptable salt.
`The pharmaceutical composition of the present invention
`may be produced according to methods known in the art
`with using additives if necessary. Here, Basic Lecture on
`Development ofPharmaceuticals XI 20 Production ofPhar-
`maceuticals (the second volume) (edited by Kyosuke Tsuda
`and Hisashi Nogami and published by Chizyo Shoten) is
`mentioned for reference. The lyophilized composition may
`be obtained by preparing an aqueous solution ofthe cyclic
`polypeptide compound(1) or its pharmaceutically accept-
`able salt and the stabilizer, optionally adding a pH adjustor
`(citric acid anhydrous, sodium hydroxide, ctc.) as required
`to attain pH 4.0-7.5, preferably pH 4.5-7.0, and then
`lyophilizing the resulting solution in vial according to a
`conventional method. Thus, the stabilized pharmaceutical
`composition in lyophilized form, when dissolved in purified
`water, preferably gives a solution of pH 4.0 to 7.5, more
`preferably pH 4.5 to 7.0.
`It
`is preferable that
`the thus
`prepared composition in lyophilized form is sealed and
`stored with shading. The lyophilized composition can be
`loaded in cach vial in the solution form before lyophilizing
`or in lyophilized powder form after lyophilizing.
`Since the cyclic polypeptide compoundis not satisfacto-
`rily stable to humidity, it is necessary that the lyophilized
`composition of the present
`invention contains 3.4% by
`weight or less of water, preferably 3.0%, more preferably
`2.0%.
`
`Usually the stabilized pharmaceutical compositionin lyo-
`philized form is dissolved in isotonic sodium chloride solu-
`tion as required and used as an injection solution. The
`pharmaccutical composition of the present invention may be
`used as an injection preparation which requires some com-
`pounding before use.
`
`BEST MODE FOR CARRYING OUT THE
`INVENTION
`
`The present invention is nowdescribed in further detail by
`way of examples and test examples, which should not be
`construed to limit
`the scope of the invention.
`In the
`examples, the compounds of formula (II) to (V1) are referred
`to as Compounds(II) to (VD, respectively.
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`11
`EXAMPLE 1
`
`12
`EXAMPLE9
`
`Compound(IT) 25 2
`Lactose 200 g
`anhydrous Citric acid in a suitable amount
`Sodium hydroxide in a suitable amount
`Lactose was dissolved in purified water (2000 ml) under
`heating below 50° C. After cooling below 20° C., the lactose
`solution was added with Compound (II) avoiding bubbling
`under gently stirrmg. After adding 2% aqueous citric acid
`solution (9.5 ml), the solution was added with 0.4% aqueous
`sodium hydroxide solution (about 24 ml) to adjust pH 5.5
`followed by diluting with purified water to make a given
`volume (2500 ml). The resulting solution was dispensed into
`1,000 vials of 10 mL volume, 2.5 ml per vial. The solution
`in the respective vials was lyophilized by using the lyo-
`philizer (RI.-603BS manufactured by Kyowa Shinku Co.,
`Ltd) by the conventional method to obtain lyophilized
`compositions each containing 25 mg of Compound(II).
`LXAMPLE 2
`
`Lyophilized compositions each containing 50 mg of Com-
`pound (11) were obtained in the same manneras in Example
`1 except that the amount of Compound(II) used was 50 g.
`EXAMPLE3
`
`Lyophilized compositions each containing 25 mg of Com-
`
`pound(II) are obtained in the same manner as in Example
`1 except that 150 2 of maltose is used instead of lactose.
`EXAMPLE 4
`
`Lyophilized compositions each containing 50 mg of Com-
`
`pound(II) are obtained in the same manner as in Example
`1 except that the amount of Compound (II) used is 50 g
`instead of 25 g and 250 g of sucrose is used instead of
`lactose.
`
`EXAMPLE 5
`
`Lyophilized compositions each containing 25 mg of Com-
`
`pound (II) are obtained in the same manner as in Example
`1 except that 25 g of sodium chloride is used instead of
`lactose.
`
`EXAMPLE 6
`
`Lyophilized compositions each containing 50 mg of Com-
`pound (V) are obtained in the same manneras in Example
`1 except that 50 g of Compound (V) is used instead of
`Compound(LD) and 50 g of sodiumchloride is used instead
`oflactose.
`
`EXAMPLE10
`
`Lyophilized compositions each containing 10 mg of Com-
`pound(V1) are obtained in the same manneras in Example
`1 except that 10 g of Compound (VI) is used instead of
`Compound (II) and 100 g of dextran is used instead of
`lactose.
`
`Test Example |
`
`Effect of Stabilizer in Stabilizing Lyophilized Compositions
`of Compound(1)
`10 mg of Compound(IJ) and, as a stabilizer, 100 mg of
`lactose or 9 mg of sodium chloride were dissolved com-
`pletely in 1 ml of water. The resulting solutions were
`lyophilized and maintained at 70° C. in glass vials. Nine
`days after, the resulting compositions were tested on their
`appearance,
`the residual amount of Compound (II), and
`others. As a control, used was a solution of Compound (ID
`without any stabilizers. The results are shown in Table 1.
`
`TABLE1
`
`Stabilizers Test Items
`
`6 hours
`
`After 9 days
`
`Control:
`nil
`
`Lactose
`(100 mg}
`
`40
`
`Sodium
`chloride
`(9 mg)
`
`Appearance
`pH*
`Residual amount (%)
`Water content (%}
`Appearance
`pH*
`Residual amount (%)
`Water content (%}
`Appearance
` pH*
`Residual amount (%)
`Water content (%}
`
`Slightly yellow mass
`White mass
`27
`TA
`8.0
`100.0
`—
`1.3
`White mass White mass
`64
`6.1
`100.0
`99.5
`1.0
`White mass White mass
`6.7
`6.3
`100.0
`759
`0.7
`—_
`
`*pH ofreconstituted solutions of compositions in 1 ml of water
`
`45
`
`Test Example 2
`
`Lyophilized compositions each containing 10 mg of Com-
`
`pound (II) are obtained in the same manner as in Example
`1 except that the amount of Compound(II) used is 10 g
`instead of 25 g and 100 g of dextran is used instead of
`lactose.
`
`LXAMPLE 7
`
`‘The similar tests were conducted in the same manner as
`
`in Test Example 1 except that 100 mg of maltose, 50 mg of
`sucrose or 50 mg of glucose was used as a stabilizer. The
`results are shown in Table 2.
`
`(to be continued on the next page)
`
`TABLE2
`
`Lyophilized compositions each containing 25 mg of Com-
`
`pound(II]) are obtained in the same manner as in Example
`1 except that 25 g of Compound (III) is used instead of
`Compound (II) and 200 g of maltose is used instead of
`lactose.
`
`EXAMPLE 8
`
`Lyophilized compositions cach containing 10 mg of Com-
`
`pound (IV) are obtained in the same manneras in Example
`1 except that 10 g of Compound(IV) is used instead of
`Compound(II) and the amount of lactose used is 100 g
`instead of 200 g.
`
`Stabilizers
`
`Test Items
`
`0 hours
`
`After 9 days
`
`Control: nil
`
`60
`
`Maltose
`(100 mg)
`
`Sucrose
`(50 mg)
`
`Appearance
`pH*
`Residual amount (%)
`Water content (%)
`Appearance
`pH*
`Residual amount (%)
`Water content (%)
`Appearance
`pH*
`Residual amount (%)
`Water content (%)
`
`White mass
`6.8
`100.0
`3.3
`While mass
`73
`100.0
`6.9
`White mass
`6.9
`100.0
`1.1
`
`White mass
`54
`<75.0
`—
`White mass
`6.7
`98.6
`—
`White melt
`7.0
`82.4
`—
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`TABLE, 2-continued
`
`Stabilizers
`Glucose
`(SU mg)
`
`Test. Items
`Appearance
`pH*
`Residual amount (%}
`Water content (%)
`
`0 hours
`White melt
`6.9
`100
`43
`
`After 9 days
`Brown melt
`3.6
`11
`
`—
`
`*pH ofreconstituted solutions of compositions in 1 ml of water
`
`the lyophilized
`1 and 2,
`As is obvious from Tables
`composition of Compound(II) and lactose, sodium chloride,
`maltose or sucrose wassignificantly stable as compared with
`the one not containing any stabilizers or containing other
`stabilizers.
`
`5
`
`16
`
`15
`
`Test Example 3
`
`14
`Asis obvious from Table 3, the lyophilized compositions
`of 10 mg of Compound(II) and various amount of lactose
`had no problem intheir stability.
`
`Test Example 4
`
`Stability of Lyophilized Compositions of 200 mg of Lactose
`and Compound(II) in Vial
`Tests were carried out in the same manner as in Test
`Example 1 except that 12.5 mg, 25 mg, 50 mg, 75 mg or 100
`mg of Compound(II) were used with 200 mg oflactose.
`Table 4 showsthe results of the tests on the residual amount
`of Compound(II) in the resulting compositions and thelike.
`Regarding all the compositions, their appearance is a while
`mass, the time for reconstitutional dissolution was 15 sec-
`onds, and the color and the clarity of reconstituted solutions
`of the compositions were colorless and transparent.
`
`TABLE 4
`Dependenceofthe Stability of Lyophilized Compositions of
`
`Compound (II) upon the Amount of Lactose Added
`Amount
`After 3
`Tesis were carried oul in the same manner as in Test
`of
`months
`Com-
`at 40° C,
`example 1 except that 20 mg, 50 mg, 100 mg or 200 mg of
`
`lactose were added asastabilizer. Table 3 shows the results pound and
`
`dap
`After 9 After 21
`a 75%
`of tests by observation of the appearance of compositions,
`added
`days at
`days at
`humid-
`(mg)
`70°C.
`60°C.
`ity
`Ohours
`Test
`the residual amount of Compound (II), the appearance of
`reconstituted solutions of compositions in 1 ml of water, and
`12.5
`6.15
`6.31
`6.08
`6.63
`pH*
`the like. Incidentally, it took 15 seconds to reconstitute the
`98.1
`O75
`99.6
`00.0
`Residual amount (%)
`3.95
`3.75
`2.71
`2.24
`Total
`impurities (%)
`compositions in 1 ml ofwater.
`1.3
`Water content (%)
`6.07
`6.11
`6.14
`6.37
`pH*
`99.3
`98.2
`101.2
`00.0
`Residual amount (%)
`4.03
`3.49
`2.68
`2.25
`‘otal
`impurities (%)
`After 3
`—_
`—_
`—_
`ll
`Water content (%)
`months at
`5.99
`6.00
`6.00
`6.26
`pH*
`40°C. anda
`After
`97.9
`97.3
`100.5
`00.0
`Residual amount (%
`75%
`9 days
`3.95
`3.68
`2.74
`2.25
`Total
`impurities (%)
`humidity
`at 70°C.
`0 hours
`—_
`—_
`—_
`1.2
`Water content (%)
`5.95
`5.96
`6.04
`6.13
`piil*
`Slightly White mass
`White
`98.1
`97.7
`99.0
`00.0
`Residual amount (%)
`yellowMass
`mass
`Total
`impurities (%)
`2.28
`4.14
`3.83
`2.76
`40
`Color* Colorless=White Colorless
`
`
`Water content (%)
`0.9
`—
`—
`—
`160
`pH*
`6.03
`5.92
`5.88
`5.85
`Clarity*
`Clear
`Not clear Clear
`Residual amount (%)
`00.0
`97.8
`96.7
`99.5
`pH*
`6.09
`3.03
`6.57
`Total
`impurities (%)
`2.46
`4.15
`3.92
`2.79
`
`Residual amount (%)—100.0 88.09 100.0
`
`Water content (%)
`1.3
`—
`—
`Total impurities (%)
`3.44
`12.3
`3.99
`Water content (%)
`1.2
`—
`50 Appearance
`White
`White
`mass
`mass
`Color*
`Colorless Colorless
`Clarity*
`Clear
`Clear
`pH*
`6.57
`5.56
`Residual amount (%)
`100.0
`96.7
`Total impurities (%)
`3.32
`737
`Water content (%)
`0.5
`—
`100 Appearance
`White
`White
`mass
`mass
`Color*
`Colorless Colorless
`Clarity*
`Clear
`Clear
`pH*
`6.58
`6.08
`Residual amount (%)
`100.0
`96.7
`Total impurities (%)}
`3.43
`7.08
`—_—
`Water content (%)
`0.3
`—
`White mass
`200 Appearance
`White
`White
`mass
`mass
`Colorless
`Color*
`Colorless Colorless
`Dependenceof the Stability of Lyophilized Compositions of
`Clear
`Clarity*
`Clear
`Clear
`Compound (II) upon the pH Value of the Solution of the
`5.36
`pH*
`6.78
`5.70
`
`Residual amount (%)=100.0 96.1 99.6 Composition before Lyophilizing
`
`
`‘lotal impurities (%}
`3.40
`730
`4.35
`10 mg of Compound (II) and, as a stabilizer, 100 mg of
`Water content (%)
`0.3
`—
`—
`lactose were dissolved completely in 1 ml of citrate-NaOH
`buffer having different pH value between pH 4.0 to 7.0. The
`resulling solutions having different pH values were lyo-
`philized and maintained at 70° C. in glass vials. Nine days
`
`‘TABLE 3
`
`—
`White mass
`Colorless
`Clear
`6.26
`99.8
`4.21
`—_—
`White mass
`Colorless
`Clear
`5.80
`99.6
`3.96
`
`Amount
`of
`lactose
`added
`Test Items
`(mg)
`20 Appearance
`
`
`
`*Color, clarity and pH ofreconstituted solutions of compositions in 1 ml
`of water
`
`25
`
`50
`
`75
`
`
`
`
`
`
`
`
`
`
`Items
`
`
`
`
`
`
`
`*pH of reconstituted solutions of compositions in 5 ml of purified water
`
`As is obvious from Table 4, all the lyophilized composi-
`tions were stable.
`
`Test Example 5
`
`Stability Test The pharmaceutical compositions obtained in
`Examples 1 and 2 were stored at room temperature. After 18
`months, the residual ratio of Compound (IT) was 98%in all
`the compositions.
`
`Test Example 6
`
`30
`
`35
`
`45
`
`60
`
`65
`
`AMNEALEX. 1006
`
`AMNEAL EX. 1006
`
`

`

`US 7,112,565 B2
`
`15
`after, the resulting compositions were tested on their pH and
`the residual amount of Compound (II). The results are
`shownin Table 5.
`
`TABLE5
`
`pH ofthe solution
`ofthe composition
`before lyophilizing
`5.0
`5.5
`6.0
`
`40
`
`45
`
`39
`0.2
`
`44
`0.2
`
`48
`03
`
`54
`03
`
`58
`03
`
`100
`
`6.5
`
`64
`04
`
`7.0
`
`6.8
`0.3
`
`40
`944
`
`45
`959
`
`49
`GIA
`
`5.8
`54
`98S 97.7
`
`6.4
`969
`
`6.8
`95.8
`
`0
`hours
`
`9
`days
`
`pH*
`Water
`content (%)
`Residual
`amount (%)
`pH*
`Residual
`amount (%)
`
`*pH ofreconstituted solutions of compositions in 5 ml!of purified water
`
`As is obvious from the table 5, the pharmaceutical com-
`position of the present invention is stable after lyophilizing
`the solution containing Compound(II) at pH 4.0 to 7.0 at
`least, preferably at pH 4.5 to 7.0.
`
`
`Test Example 7
`
`Dependence of the Stability of Lyophilized Compositions of
`Compound(II) upon the Water Content of the Composition
`10 mg of Compound (II) and, as a stabilizer, 50 mg of
`lactose were dissolved completely in 1 ml of water. ‘lhe
`resulting solutions were lyophilized and maintained at 70°
`C. in glass vials. Nine daysafter, the resulting compositions
`were tested on their pIJ, their water content and the residual
`amount of Compound(II). The results are shown in Table 6.
`
`TABLE 6
`
`Walter content al 0 hours (%
`
`0.9
`
`14
`
`2.6
`
`3.4
`
`5.1
`
`75
`2.5
`
`7A
`2.9
`
`7.1
`6.8
`3.6
`
`6.8
`43
`
`97.6
`
`98.1
`
`97.1
`
`92.7
`
`3.5
`5.4
`
`18.3
`
`0 hours
`After 9 days
`
`pH*
`pH*
`Water content
`(%)
`Residual
`amount (%)
`
`*pll of reconstituted sclutions of compositions in 1 ml of water
`
`Asis obvious from Table 6, the pharmaccutical compo-
`sition of the present invention is stable containing about
`3.5%, more particularly 3.4% by weight or less of water.
`According to the present invention, provided is a com-
`position in lyophilized form in whichthe cyclic polypeptide
`compound (1) or its pharmaceutically acceptable salt are
`stabilized by a stabilizer such as polysaccharide, disaccha-
`ride and sodium chloride.
`
`The mechanismofthe stabilization ofthe cyclic polypep-
`tide compound(I) or its pharmaceutically acceptable salt by
`the stabilizer such as polysaccharide, disaccharide and
`sodium chloride is still to be unknown,butit may be that the
`stabilizer adsorbs water in lyophilized cakes and that the
`stabilizer serves to disperse the compound or its pharma-
`ceutically acceptable salt uniformly in the composition.
`The cyclic po