VOL. 215 NOS. 1-2
`
`14 MARCH 2001
`
`ISSN 0378-5173
`215 (1-2) 1-276 (2001)
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`Bass and Spangenberg
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`U.S. Patent No. 8,476,010
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`VJ The paper u'cd in thi' publication meets thc requirement~ of ANSI/NISO Z39.48-1992 (Permanence of Paper)
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`Printed 111 the Uni!cd Kin~dom
`
`Exh. 1009
`
`-
`
`

`
`I ntcrnatio n;ll Journal of Pharmm:cutic!> 215 CWO I) :!()7 2:!0
`
`international
`journal of
`pharmaceutics
`111111 .d..c1 tcr.Ctllll kx:;ttc ijphann
`
`Physical properties and stability of two emulsion
`fonnulations of propofol
`
`Jiho ng Ha n, Stanley S. Davis, C tive W ashin gton *
`
`.<:du111f 11/ l'llm·nlm·r•lftiml Srh•m·r•,\, Uuil'l'rsit,l' of Nu11inglw111. Uuir·r·rsity !'ark. Nollim.dunu. N(i7 :JRJ) Ul\
`Rccctvcd 0 I Ocwbcr 2000: r..:cdv..:d in rcvi~..:d form :!R Nowmhcr :!000: accepted 05 Dc..:cmhcr :wno
`
`\Vc have t:<lmparetl the phy'iical propcrtic:-. of two ~.:ommercial emulo.;ion formulation~ of the intrav..:nuus anaesthetic
`propofol. (l)ipri\'an • . 1\straZenc~.:a. and Propofol Intravenous l: mubion. Gen~ia Sieor Pharmac..:uticals) which appear
`10 tlillcr primarily in the additive conte111 and formulation pi I. Dipril'an • contains di-;otlium ctktatc and ha-; a pi I
`or 7 8.5. whit..: the Gen-.ia protlm:t contains S(}c.lium metabisulphite and is rormulatec.llll a pH or 4.5 (l.-1. Th..: alerage
`zeta potential of Oipriv;m• at pll 8 was - 50 mV while that of the Gen~m product at pH 4 5 was - -HlmV. Thi:-.
`information -;uggcsh that the physical stab11it) or Proporol lntntvcnOll'> l:.muhion :-.hould be lower than that of
`Diprivun •. Three random be~tt:hcs or both product:-. were ~ubjectcd to u range or \lability tesh. including shaking.
`thaw t:ycling. and the emulsion droplct 'i1..: c.lisuibution wa~ then a:-.:-.e:-.scc.l by dynamic
`thermal cycling. anc.l freeze
`light scaltering. light diffraction. ami electrical anc.l optical LOne sensing. !3oth emubi~lth initially showed narnm
`:.ubmicrometrc particle size distributions. An increased levd ~ll' droplets larger tiHtn S pm could be detected in
`Propofol l ntravenou~ bnulsion after a~ liule <IS 4 h shaking (300 -;trokc:. min at room ll.:mpcrature) and l'i:.iblc l'rce
`oil could be detected after 8 12 h shaking. In contrast. Oiprivan'' ~howell no increase in the large droplet ~.:ount after
`shak ing !'or time-. up to 16 h. A similar tlilTerence in the emul-.ions was found al'ler one freeze thaw cycle. with
`Propol'ol Intravenous Emulsion exhibiting extensive ~.:oalesccnce. while that or Diprivan'' was at the limits or
`detection. We ~.:ondude that these two pro~lucts have tliiTercnt physi<:al stability chantctcri~tks. and t hat this may in
`part be due lO the rec.luced zeta potential in Propol'ol Intravenous Emulsion compared to that or Diprivan'' . !'I 2001
`Published by Elsevier Science 13. V.
`
`Kr•yH·orck J>rnpofol: Dipriv;111 ' : Emul~ion: Stability
`
`• Curn.:,pundmg. author. I cl.: 1 ..J-1-11 "i-')S I 5():;\..J: J'ax: -t 4-J.
`115-951 'i I 0:!.
`L'-111wl mldrr·" : clivc.\\l"hin!,:tlmla nottingham.ac.uk !C'.
`wa,hingtun).
`
`I. Introduction
`
`Propofol (2.6 di-isopropylphcnol) is a widely
`ll!\~<.1 intr<I\CilOUs iliHIC~thctic (Glen and H unter,
`1984: Co~.:b,hou ct al.. 1990. 1991). Like many
`anaest hetics. propofol is hydrophobic. having a
`calculated partition cocl'fkicnt ( Log P, . .,1,.) of :un
`
`0~78-51 7:\ 01 "i- 'cc J'nmt mutter 1 20()1 Published by El~..:vicr Science B.V.
`I'll: SOJ7X - 51 7 )(()0)00Cl92 - X
`
`Exh. 1009
`
`

`
`208
`
`.1. !11111 1'1 a/.
`
`lnremmimml .Jmmmlof Plmrmacl!/1/ic.~ 1 15 (1001) 107 210
`
`and it presents lillie o pportunity for solubilization
`through salt formation. As a result it is formu(cid:173)
`lated as an oil-it1-water emulsion ( Diprivan ", As(cid:173)
`rraZeneca). in which the disperse phase is soya oil
`containing dissolved pro pofol. emulsified using
`lecithin. and having a mean droplet size of 150 -
`200 nm (VMD).
`Emulsions intended for intravenous use should
`have an extremely sma ll dro plet size .and be highly
`sta ble. since a ny large dro plets placed in the circu(cid:173)
`la tion nuay lodge in the pulmona ry capillaries a nd
`could potentially lead to an embolism (I lium ct
`al.. 1982). The exact size at which this phe(cid:173)
`nomeno n becomes importctnt is widely deba ted
`and pha rmacopoeia l limits on particulates in par-
`
`enterals a re vague. a lthough 5 pm is generally
`accepted as an upper limit. Oiprivan "', and intra(cid:173)
`venous feeding emulsions such as lntralipid. have
`p~u·ticle sizes much smaller
`than
`this. mean
`dro plet diameters being of the order of I 00-300
`nm. However. the systems a rc significa ntly poly(cid:173)
`disperse. and dro plet counting techniques can de(cid:173)
`tect a small nllln ber of pa nicles larger than I ~tm
`in the formulations. It is also evident that such
`emulsions must be formula ted with adequate
`ph ysical stability to prevent the droplet size distri(cid:173)
`bution evolving during the sto rage lifetime, and
`exceeding clinica lly sarc limits.
`Intravenous emulsions such HS Dipri van " arc
`stabilized with phospholi pids
`in
`the rorm of
`
`Naked Eye Inspectio n
`
`Microscopy
`
`Optical Couming
`
`Light Diffraction
`
`Electrical Zone Sensing
`
`Dynamic Light Scattering
`
`I~ JQ·K
`
`J0·7
`
`10·6
`
`IQ·S
`
`LQ-4
`
`JQ·3
`
`J0·2
`
`Fig. I. S..:nsitivity range~ or the panid..: charactcritalion techniques used in this study (exact values oepcnu on spccillc instrumcm)
`anti the sit.e distribution or" typical imravcnou~ emulsion.
`
`Size (m)
`
`Table I
`Properties OJ' Ull~trcsscd emulsion~
`
`l'mdm:l
`
`13atch Ill.~.
`
`Volume mean diamch.:r ( I)LS). nm
`pll
`Droplet count number > SJ•m ~~~ (Coulter Z2l
`
`Ditwivan"
`
`Propol'ol intl'avcnou~ cmubion
`
`Ull40 B
`
`K &179/A
`
`X91)151A
`
`99~~() 1
`
`991..:309
`
`99 1.:D I 2
`
`171 ± 2
`7.6
`120
`
`177 ± I
`7.5
`200
`- -
`
`172±2
`7.5
`160
`
`17tl± I
`5. 1
`J()()
`
`184 ± I
`5.5
`:100
`
`178± I
`5.6
`220
`
`Exh. 1009
`
`

`
`.1. Htm et a/.
`
`f11temational .loumalo( Pltamtllt'<•tttic-,· 215 (200 I ) 2117 :!10
`
`209
`
`Zeta potential (mV)
`
`Propof'Jl lnrr:.venous
`Ermtlston 1>H range
`
`Dtpnvan®pHrange
`
`0
`
`-10
`
`·20
`
`-30
`
`-40
`
`-50
`
`-60 ~----~------+------+------+------+------+------+----~
`4D
`2D
`SD
`7D
`3D
`SD
`6.0
`9.0
`10.0
`
`pH
`
`Fil!. 2. Zeta pmcntials or o.:mulsinn~ as a runclion or pi I. Closo.:u symbols: D iprivan • (thn:o.: bntch.:s). opcn symbol~. Propnl'nl
`ln~ra vcnous Emulsion (lhrco.: batchcs).
`
`z-avera ge
`diameter (nm)
`220
`
`Free Oil Free Oil
`
`Free Oil
`
`140 +-----r---~-----+----~----+-----r---~----~
`12
`10
`14
`16
`8
`6
`2
`4
`0
`Shaking time (hour)
`
`Fig. 3. :-avcragc dian1<:1o.:r or ..:mulsions by DLS as a !'unction or sh;1king tim.:. Closed symbok l)iprivan • (llm:o.: h;ncho.:~): 1.\p..:n
`symbols. l'mpo fol Intravenous Emul~ion (lhrO.:l' batc.:h..:s).
`
`Exh. 1009
`
`

`
`210
`
`.!. Han et af. / lllfemmirmal .!oumal IJ( Plwmwceutic.v 115 (1001) 10 7- 210
`
`puri fied egg lecithins. We and others have previ(cid:173)
`ously studied the stability or such emulsions in a
`number of pape1·s (Black and Popovich, 1981:
`
`Blllrnham et al.. 1983:
`1990,
`1991'
`1992:
`1992a.b. 1996) and
`
`Washington et al.. 1989.
`1990a,b.c.
`Washington.
`it
`i~ now well un-
`
`Counts/Ill, >5 J.lm
`
`2500
`
`Fig. 4. Count of larg.: droplets in emulsions by C'ouhcr Z2 a~ a function of ~hakin~ tim.:. Clo~cd symbpl~; DipriVilll " !IIH~:l· bill(,;hcs):
`open ~ym hol:;. J>ropofol Intravenous Emulsion (three batch.:~).
`
`Shaking time (hour)
`
`C oun t s/)..11,>5 )lm
`
`300 0
`
`Free Oin
`
`Fig. 5. Count of large droplets in emulsions by A ccusizcr 780 as a function o f shaking time. C losed symbols: Diprivan " (three
`batch.:s): open ~ymbols. l'ropofol Intravenous Emulsion (thr.:c batches).
`
`10
`Shaking tim e (hou r)
`
`12
`
`14
`
`16
`
`Exh. 1009
`
`

`
`J. lltm t•t a/.
`
`futt•rmaimwl Jn11ma/ o/' Plwrmun'llllc' 115 (21)()} J 207 220
`
`211
`
`Particle size (J.lm),
`<90%
`0.5
`
`0.3
`
`Free Oil
`
`0. 1 ~--~----~----~---+----+---~----~--~
`0
`2
`4
`6
`8
`10
`12
`1 4
`16
`Shaking time (h our)
`
`1-u!. 6. l)l)U of cmul~ll>ll~ by light c.Jill'ractiun by Coulter LSDO a~ a l'unclion ul 'hakmg time Cln...:d ,yml"K>I~: Diprh•an' (three
`ba~chc,): upcn ')mbok l'ropul'ol lntra\cnou' Emulsion (three batchc,).
`
`(a) 0 hours
`
`4hours
`
`8 hours
`
`(b) 0 hours
`
`4 hours
`
`8 hours
`
`Fig. 7 Pholtltnlcmgraph' or (a) Diprivan· anc.J (b) l'ropofl>l lntm\eii0\1'> Lmuhi\lll after II. 4 otnd ~ h .,hal.ing.
`
`tlcrstoo<.l that thc'c systems arc chargc-:>tabilized
`with a zeta potential of - 40 to - 50 mY at pll
`8. As a result. they show excellent stability in
`normal use. However, any !'actor which lowers the
`
`zeta potential may lead to instability and the
`resultant forma tion or large oil droplets. 1 mpor(cid:173)
`tant factor-; in thio; rega rd arc electro lytes and pi I
`(Washington. 1990b.c: Washingto n ct al.. 1993).
`
`Exh. 1009
`
`

`
`212
`
`J. /11111 t•/ ul.
`
`lntt•motional Joumul o/11/uii'J//IIt'l'lllil'.l' 2 I 5 (](}{)/} 207 220
`
`-u
`
`'-"
`
`Exh. 1009
`
`

`
`J. H1111 ''/ a/.
`
`lllt,•matiumd Jmmw/ td Phamlllt'('lltic.\ 115 (20(1/) 1/17 110
`
`The io niza tio n curve of lecithins reaches a point
`or zero charge (PZC) at approximately p i I 3 and
`conseqltenlly a lowering or p i I may reasonably
`be expected to lead to reduced physical st<tbility.
`Recently an alternative propo!'ol formulation
`to Dipriva n '' has appeared o n the U.S. market
`(Propofol Intravenous Emulsion. Gensia Sicor
`Pha rmaceuticals). This materia l is supe rficially
`similar to Diprivan '" in tha~ it consists or a soya
`o il based emulsion containing I 0 mg/ml propofol
`and having a mean droplet diameter or 150 200
`nm. Howeve r the formulation has a p ll in the
`range 4.5 6.4. necessary to ensu re the antimit:ro(cid:173)
`bial activity or its bisulphiuc additive. We would
`predict
`that.
`assuming
`that
`the
`let:ithin
`emulsi fiers are similar. this emulsion would show
`a different stability profile to that of' D ipri van "
`and therefore we have pe rformed a COlnprchen(cid:173)
`sive set of stability measurements to compare the
`two formulations. To assess the physical stability
`we used three widely accepted accelerated tests:
`
`shaking. freeze thaw cycling. and thermal cy(cid:173)
`cling. In order to measure properly the ef'f'ect
`that these processe:; had on the droplet size t.lis(cid:173)
`tribution it was necessary to usc a number or
`different particle size measurement
`techniques.
`The origi nal emulsions have na rrow size distribu(cid:173)
`tions which almost wholly lie below 1 pm. How(cid:173)
`ever after exposure to the accelen1ted stability
`test. the range or droplet sizes me~y potentially
`vary from hundreds or nanometrcs to millimc(cid:173)
`trcs. a span or lour orders or magnitude. Fig. I
`shows the measurement limits of the te<.:hniques
`used: it wi ll be readily apprcci<lled that a full
`asscssmcm requires :;cvera I d i rreren t measure(cid:173)
`ments. In particular it was lc lt that the Coulter
`Counter wus a n esscnti<tl technique since it pro(cid:173)
`vides droplet volume measurements. which arc
`traceable to primary standards. The remaining
`techniques arc all based on optical scatlcring and
`as a result may be sen siti ve to the optical prop(cid:173)
`erties or the emulsions.
`
`Particle s ize (nm)
`
`250
`
`200
`
`1 50+--------------+--------------+-------------~
`14
`2 1
`7
`0
`
`Dur ation (day)
`
`Fil!. 9. : ·avcraac diamctcr of cmubiotts by Dl.S a$ a !'unction ul' tlli.:rmal~.:~din!! tun..:. Chhed ..;ymbob: Dipri,an' (thrc..: batches):
`OJ~Il ~ymbol-;, -Prupul'ol ltHntvcnou~ Emul~ion (thrc..: bat~.:hc~l.
`
`Exh. 1009
`
`

`
`214
`
`J. I/ au t•t ol.
`
`lutematicmrtl Joumal u/ Pltw·mm·t·utic.~ 215 (2001) 207- 220
`
`Counts/J.LI, >5 ~m
`
`Fig. I 0 . Count of large droplet~ in .:mulsion ~ by Coulter Z2 il~ •• !'unction of thermal cycling time. Closed symbols: Diprivan • (three
`batdtcs): open ~ymbols. Prupol'ol Intravenous Emulsion (three batches).
`
`Duration (day)
`
`(a)
`
`(b)
`
`(c)
`
`(d)
`
`(e)
`
`(f)
`
`Fig. II. Phtllnmicrograph~ of l)iprivttn " Ca c) and l'rupol'ol lnt ravcntllt~ E:.mul~i<>n Cd
`
`I) after one freeze thaw cycle.
`
`Exh. 1009
`
`

`
`J. Jfau ('I a/.
`
`11111'/'1/alimw/ Joumal of' Pluii'J/Iacelrlics .215 (.200/) .207 1.20
`
`215
`
`2. Experimental section
`
`2.2. C/wroc t eri:m ion r I!CIIII iques
`
`2.1. Moll!riu/.1·
`
`Oiprivan '" (Batch nos. L8 140/ B. K8 179/A cmd
`X90 15/A) was supplied byAstraZeneca Mac(cid:173)
`clesfield (U K ). Propofo l Intravenous emu lsion
`(Batch nos. 99 E30 I. 99 E309. and 99 E3 12. Gensia
`Gensia Skor Pharmaceuticals. Irvine. California)
`was purc hased from material o n general sale in
`the U.S. T hree batches o f each fo rmula tio n were
`studied. Isotonic saline was p urchased fro m Beck(cid:173)
`man Coulter ( Luto n, UK). All ot he r chemicals
`used were purchased fro m Sigma Chemica l Com(cid:173)
`pany. Gillingham. UK. and were at leas t or A R
`grade. All materia ls were sto red in ac-cordance
`with the manufacturer's instructions.
`
`2.2. /. Zeta fU>fl!llliol
`Zeta po tentials were measured using a Malvern
`I nstrumcnts Zetasizer 4. which is based o n laser
`d o ppler velocimetry in an electric field. Values
`quoted are the average of fo ur measurements. The
`inst rument was va lida ted using the manufacturer's
`po lystyrene microsphere
`transfer
`standard
`(quoted -50 ±5 mV, measured
`- 46±2 mV)
`which is tracea ble to the
`1ST goe thite primary
`standard. The buffers used were: form ic acid/
`potassium hydroxide {pH 3.2): acetic acid/ potas(cid:173)
`sium hydrox ide (p H 4.3 and 5.3): potassium
`dihydrogcn phosphatc/diso dium hydrogen phos(cid:173)
`phate (pH 6.5 and 7.7) and boric acid/ borax (pH
`9.0). All buffers were made to estab)ished fonnu-
`
`511()
`
`~()()
`
`H ) ()
`
`:!!lll
`
`Jl)()
`
`I)
`
`I HI -111/B
`
`Kl! 17W,\
`
`X 'Jlll 5/ 1\
`
`')'l L~JO I
`
`991-:.30?
`
`'J'J J·:J 1:!
`
`Fig. 12. : -average \Jiamch:r or emulsions by DLS after one frcct.c 1haw cycle. Grcy bars: Diprivan ' (lhrcc batches): Fillc\.1 hars.
`J>ropofol l n1ravcnous Emu lsion (lhrcc balt:hcs).
`
`Exh. 1009
`
`

`
`(a)
`
`I
`10
`
`(b)
`
`10
`
`0.01
`
`0.1
`
`Volume (%)
`
`Diameter (J.UTI)
`
`().OJ
`
`Volume (%)
`
`0.1
`Oinmetcr (J.Lm)
`
`0.01
`
`0.1
`
`10
`
`Diameter (J.UTI)
`
`Fig. 1:1 . D rople t ~i/c di stribution~ !Coulter· LS23()} (a) u l"
`un~trc~~cd cmul~ion~: (h) after 6 h ~haking: and (c) al'tcr l lllC
`l'ri:c7c th;~w cycle. S(llid ~ymbok D iprivan " batch LR 140 13:
`empty ~ym bok Propofol l ntmvcnou~ Emul~ion balch 1)91:301 .
`
`lac (D a wson ct a l. , 1969) a nd we re diluted to a
`final t:atio n concentra tio n o f I n1M belo re usc.
`The p H quo ted is that o f the diluted buffer mea(cid:173)
`sured using a calibrated p H electro d e, and no t the
`p ublis hed va lue, which is no rmally incorrect due
`to the b uffer dilutio n facto r. We lhave previo usly
`used these buffers fo r the study o f trig lyceride
`emulsions and fo und tha t they d o no t give rise to
`d etecta ble e rro rs due lo specific ad sorptio n o f
`io ns.
`
`2.2.2. LiJ:hl d(/]i'auion
`Lig ht diffractio n was perfo rmed using a Co ul(cid:173)
`te r LS230 particle s ize ana lyser (Beckma n Coul(cid:173)
`te r, Luto n. U K) with the po la riza tio n inte nsity
`(' PI O S mo de') included in the analysis, a nd with
`an o ptical mod el using the refractive index o f soya
`o il. T he instrume nt was validated by the ma nu fa<.:(cid:173)
`turc r immedia tely prio r to the study. a nti several
`times during the trial. using polysty rene mic ro-
`
`216
`
`.1. /fan <'I a/.
`
`/nrermuioual .lmmw/of Plwmmcl!lllics 215 (200 /) 207 220
`
`Volume (%)
`
`·~! ~
`1~0 ~
`1-f-----< &~·1 I
`lil.._..._.~
`
`spheres (Co ulter Co rpo ra tio n , Mia mi, Flo rida.
`0.293 ~1111). Sa mple vo lumes o f 50- 200 ~d were
`diluted into the sample recirc ula to r until a P lDS
`intensity o f a pproximately 50'!1,, was achieved.
`Three sequential mea sureme nts (90 s each run)
`were mad e o n each ba tch.
`
`2.2.3. Optiml particle counting
`Particles larger than 1.5 ~1111 were counted with
`a PSS Nicomp Acc usizer 780 in the extinction
`m o de, in which the pa rticle s ize o f a dro plet is
`calculated fro m the lig ht blo ckage it produces in
`an o ptical sensor. The instrument was validated
`by
`the manufacturer immediately prio r to
`the
`study; calibra tio n was in the fo rm o r a calibra tio n
`rile whi<.:h is m a tched to the senso r. Pro pe r o pera(cid:173)
`ti o n o r
`the
`instrument wa s checked
`using
`polystyrene tnic rospheres (Duke Scientific. Pa lo
`Alto, Califo rnia) severa l times during the trial.
`The lower thresho ld was set to 1.7 pm: the mea(cid:173)
`surement time was 200 s w ith a now rate or
`60 ml/min. Fo r the unstressed emulsio ns a sample
`vo lume of 100 ~d was used but afte r the sta bility
`tests it was frequently nccessmy to predilute the
`samples to a void coincid ences be tween the numer(cid:173)
`o us large dro plets.
`
`2.2.4. Electrical =mw sensing
`D ro plet counting b y the e lectrica l zone m ethod
`was perfo rmed using a Coulter Z2 with a 50 ~·m
`a perture tube. This instrument p rovides a bsolute
`counts o f the number o f dro plets in a specified
`size range. ty pically fro m I to 10 ~un. The instru(cid:173)
`m ent was valid~tted by the lllcll1llfacturer immedi(cid:173)
`ately prio r
`to
`the study and
`the ca libra tio n
`c hec ked against po lystyrene microsphcrcs (Coul(cid:173)
`ter Corpo ratio n , Miami, Flo rida 9.932 pm ) sev(cid:173)
`eral times during the trial. The emulsio n was
`diluted to I :20 with isotonic saline, 40 ~tl of this
`mixture was added to 20 ml o f isoto nic saline in a
`co unting vial, a nd the dro plets in this mixture
`were counted w ith a 0.5 ml sample volume . This
`t:o rrespo nds to 50 nl o f th e origina l emulsio n
`being drawn uhro ugh the co unting o ririce. All
`samples were used immed ia te ly a fte r dilutio n a nd
`a ppropria te backgounds were subtracted .
`
`Exh. 1009
`
`

`
`J. llcm c•t a/.
`
`lntemlllimwl Joumctl 11{ Plwrman•IIIJI',\ 2 15 (2001) 207 :!20
`
`217
`
`2.2.5. Dy11cmJic li,::ht ~cauering (DLS)
`A Malvern 4700 DLS instrument was used to
`the
`:-aventge diameter
`(cllltllllants
`measure
`method) and droplet size distribution (analysis
`using CO T IN). 1 he in~trumenl oper<lles by
`measuring the diffusion coefficient or the droplets
`rrom the fluctuations in scattered light (Washing(cid:173)
`ton. 1992b} and is most sensitive to particles
`smaller than I ~1m. The instrument was validated
`against a poly~tyrcne microsphere standard (98
`nm. l ntert~u.:ial Dynamics Corporation. Portland.
`Oregon} every day prior to measurement. Samples
`or emtllsion were diluted into cylindrical counting
`cells until a count rate or 2 4 x 1 0~ counts per
`second was obtained. Datu were gathered ror 30 s
`counting periods with the correlator in linear se(cid:173)
`quenti<ll mode with automatic correlation time
`sclcdion. which normally resulted in a l'undamen(cid:173)
`tal sample time or 10 30 ps.
`
`2.2.6. p/1
`A Corning Model 7 pH meter was used with a
`combination glass SCE electrode to measure pH.
`The meter was checked and calibrated against
`standard pl-1 4 i CUll ami 7 ± I).() I butTers CScien(cid:173)
`tilic Laboratory Supplies. Nottingham). The elec(cid:173)
`trode was thoroughly rinsed. and its calibration
`checked. af'lcr every individual ~ample in order to
`avoid errors due to electrode oil contamination.
`
`2.1. 7. Ph rsiC'al appearmJe<'
`The pl;ysical appearance or the emulsions was
`recorded by microscopy (Olympus C H-2 micro(cid:173)
`scope). and by photography of' the scaled contain(cid:173)
`ers (Fuji DX7 digital camera) Hs necessary.
`
`2.3 .. s·whilily tests
`
`All testing was performed as :.oon as possible
`af'tcr the termination or the approprialC Slress test.
`All samples were tested in their original unopened
`containers on a single occasion and then di~­
`carded: no container was sampled more than
`once. l:::.m:h sample wm. evaluated by each instru(cid:173)
`ment in those cases where no f'ree oil was visible
`in the container. Samples showing a film or layer
`of oil visible to the naked eye without magnifica(cid:173)
`tion were considered to have broken and were not
`
`further measured. since we have found that the
`stati tical precision of such tests is poor. Further(cid:173)
`more. samples containing free oil normally cause
`subsequent exten~ive cleaning problems for the
`particle ~i.dng instru•nents.
`
`1.3.1. .%akin~
`Samples in the original unopened containers
`were ~ubjccted to shaking u ing a Burrell Model
`75 wrist-action shaker (Burrell Scientific. Pitts(cid:173)
`burgh} operating at 300 strokes/min at room tem(cid:173)
`perature (22 t 4°C). The shaking amplitude was
`set to its maximum value resulting in a bottle
`movement or approximately 8 cm. S<unples were
`shaken for 2. 4. 6, 8. I 0. 12 and 16 h and the
`position:.; or the vmiou~ batches was randomized
`on the shaker.
`
`2.3.2. Tflerlllal cycling
`Samples \Vcre stored in a Sanyo programm~tblc
`incubator (Model M I R 153. San yo Electric Co ..
`Japan) and subjected to a controlled thermal cycle
`or 8 h at 30°C followed by cooling to .t 8°C l'or
`16 h. Thi~ cycle was repeated on il daily basis and
`emulsion samples withdnmn at 7. 1-k and 21
`days. The heating cooling tunc or the incubator
`was approximately 30 min and its temperature
`precision was + 1°C. Thermal C)clcs were verified
`using a recording thermometer with K-type thcr(cid:173)
`mocou p le probe (I I anna I nst rum ems 11192804C
`with N/\MAS certified thennocouplc)
`
`2.3.3. Free:e tlta~t·
`Emulsions were lh.>Lcn in a convemional labo(cid:173)
`ratory freezer at - 20°C. They were <tgitatcd gen(cid:173)
`tly every )() min until completely solid and then
`stored rrozcn for a total or 8 h (i.e. inclutlinl!. the
`l'rcezing time) then th:nved at room tempcra7ure.
`
`3. Rcsull'i and di')cussicm
`
`The trial generated an exten-.ive array or data
`but ~pace prevent'> its complete presentation here.
`The complete data set is available electronically.
`Table I li'it'i the properties ol' the oriu.inal cmul(cid:173)
`-.ions. Samples from both manuf'acture;.s had sim(cid:173)
`ilar
`: -average
`diameter~ and
`sho"ed
`a
`comparahlc level of large droplets in all batches.
`
`Exh. 1009
`
`

`
`21R
`
`.1. //au 1!1 al.
`
`fllll!rlllllioual Joumalof Plmm wn•urics 115 (1001) 207 120
`
`However, Diprivan •· had a mean pH of 7.5 and
`Gensia Propofo l Emulsion had a mean pH of 5.3.
`Fig. 2 shows the zeta potentials ofrhe six batches
`or emulsions as a f'llnction of pH. T he trend is
`simila r to that previously reported fo r phospho(cid:173)
`lipid-stabilized pa renteral emulsions (Washington.
`1996) with a value of approximately -50 mY at
`pH 8 a nd a gntdual decline with decreasing pH.
`T his ~s due to changes in the io nization of the
`phospholipids; a sha rp surface pK., is not observed
`because the lecithin used is made up of a broad
`range o r compo nents with varying pK. Comparing
`the ze ta potential data to the emulsio n pH (Table
`I) indica tes that the mean zeta potential o r the
`emulsio n in the o rigina l container was - 50 mY in
`the case of Diprivan •· and - 40 mY in the case of
`Gensia Propofol Emulsion. This may not appear a
`pa rticula rly large difference and is fairly typical o r
`the level o r va riation observed in phospho lipid-sta(cid:173)
`bilized parenteral feeding emulsio ns, despite the
`difference in the fonTIUlation pH Of the emulsio ns.
`It sho uld be no ted tha t Diprivan'''' is formulated so
`that its 4elil po lenlinl doc,:s not va ry widely over its
`possible pH range, while the zeta po tential or
`Propo ro l lntravenOLIS Emulsio n can vary substan(cid:173)
`tially over its pH r<tnge (Fig. 2). As a result pH
`varia tions in Propofo l Intraveno us Emulsion will
`lead to much la rger changes in zeta po tential than
`co rrespo nding pH eha nges in Diprivan •·
`
`3. /. 1. Slwk i11g lest
`The results from the shaking test (DLS. Coulter
`Z2, Coulter LS230. PSS Accusizer) arc shown in
`Figs. 3 6. The :-average diameters of all the
`emulsions were simila r and did no t change signi fi(cid:173)
`cantly during the trial: all the variatio ns observed
`were within the limits of lo ng-term experimental
`erro r ( ± 5 nm ). All three batches of Propofo l
`Intra veno us Emulsion showed free oil a fter 8 12
`h shak ing. so DLS was not perfo rmed on these
`batches a fter these times. The a ppa rent insensitivity
`o r DLS to emulsion coalcscem;c is not unusual since
`this technique is only sensiti ve to dro plets smaller
`the\ I) approximately I pm. A small number o r laJ'gCI'
`droplets leads to a poorer qua lity of data filling but
`docs not generally in nuence the .:-average diameter
`despite the fact that such large d roplets contHin a
`significant mass of oil.
`
`The remaini111g instruments a re optimised fo r the
`detection of dro plets larger than approxima tely I
`~un, and they indica ted a rapid increase in the
`n umber of large droplets in Propofol Intravenous
`Emulsion with sha king. In contrast Diprivan "'
`showed no significant change over the dura(ion of
`t he study. Fig. 7 shows photomicrogra phs of all six
`batches of emu lsion after 8 h shaking. The a ppear(cid:173)
`ance of the Diprivan " samples was similar to that
`before shaking. while Propofo l Intravenous Emul(cid:173)
`sion showed a significan t number of large dro plets.
`After 16 h Propofol Intraveno us Emulsio n showed
`extensive free o il while Dipri van •· showed o nly a
`foam (Fig. 8). Foam formatio n is characteristic of
`emulsions in which no oil scpmation has occurred.
`since oil surface films invariably lead to rapid foam
`breakage (13ikcrman, 1973).
`
`3. 1.2. Thermal cycling
`None or the enwlsion batches showed a signifi(cid:173)
`cant change in either :-average droplet size o r large
`droplet count after 2 1 days thermal cycling. T ypical
`d ata (:-ctvcrage dia meter a nd Coulter Z2 large
`droplet count) are shown in Figs. 9 and I 0. respec(cid:173)
`'lively. The variatio ns in .:-average diameter arc
`typical of the lo ng-term precisio n o r the technique
`( ± 5 nm) and the va riations in the dro plet counts
`are consistent with the ra ndo m statistical variation
`in the droplet counting.
`
`3. I. 3. Free.:e- llw11'
`The behavio ur o r the emulsions under freeze(cid:173)
`thaw testing pa ralleled tha t o bserved during shak(cid:173)
`ing. with a single freeze thaw cycle causing a large
`increase in the droplet diameters of the Propofol
`Intraveno us Emulsion batches and only a very
`sma ll cha nge in the appeara nee or the Diprivan ''
`samples. Typical micrographs a rc shown in Fig. II .
`It is immediately obvious that the destabilizarion
`ca used by freeze- thawing was far more extensive
`than that ca used by shetking. since a large rraction
`or the Propo f'o l Intraveno us Emulsio n appeared to
`be made up of large droplets of broken emulsion.
`This is in contrast to the coalescence caused by
`shaking, and provides some insight into the mech(cid:173)
`anisms of destabiliza tion. In the case o r freeze(cid:173)
`thawing, the majo rity o r the oil droplets arc
`compressed together between the advancing ice
`
`Exh. 1009
`
`

`
`J. lfttll t'l ul.
`
`fllf('fllltlionul Journal t~/ 1'/tm·mun•tlfiC"'" :! 15 (:!(}(}I) :!07 220
`
`219
`
`crystals as the con