(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1037
`
`

`

`
`
`in this study, hydrolysis of PC as: a function of
`pH temperature: and buffer concentration was.
`invest-timed lhe dam weae analysed to assess
`oatalylie elfeols of the oifferent buffer species
`used.
`
`Riaterials and Mefisods
`
`.Materials
`
`Soybean PC (Phospholipon 3.00) was obtained
`from Nanermann Gmblil, Cologne, EKG. and
`used as received. PC consisted of 90% FC, 50%
`
`lyso-eompounds 5.0% free now acids and less
`than. 0.1,0 \Vflbfll. The fatty acid composition of
`PC. determined by high performance liquid chro—
`matography (HPLC), was as follows: 3 .‘70 myris~
`tie acid, 13.2% palmitic acid, 4.2% steam: acid.
`12.0% oleic acid, {‘33. % linoleie acid. {Ether chom-
`ioals were of analytical, grade. All solutions were
`prepared with doublodistilled water.
`
`Buffer solutions
`The following aqueous holler solulloes were
`used for the kinetic studies: pH 4--~5 acetate buffer,
`pH 5 30 6,5 Citrate buffer and pH ‘7
`to 9 Tris
`buffer.
`’The pl‘l was measured with. a glass elec-
`trode and a, pHH meter (’l‘ype CG 8117 T, So3303:
`Ger’ate F .R.G.) Ionic $33mgflle ol’ Lhebull’el solu—
`tions wow 0 .068 0125, 0200 anti 0. 300 and ad-
`justed by manipulating the eoneeentrlion of the
`buffer eompooeols
`
`Preparation of the liposome dispersions
`PC liposome dispersions were prepared by the
`“film” method (Szoka and P'apahadjopoulos,
`1980). After formation of the phosplmléoid film in
`a round~bottom flask m a rotary evaporator at
`~ 50°C. the film was left under reduced pressure
`ovemight. It was hydraied at ~ 50%: with aha
`appreoriate buffer solution and the pH of the
`dispersion was measured 3.3363 afijusted,
`if nosesw
`sary. The lnillal PC eofieeolmlion was 30 mM.
`Extrusion was carried out twice 033031331 2. mem-~
`33323335: filter wltll. a pore slze of 07 33m (Uni.—pore
`BioRad Richmond CA USS.A.3 The vesicle (ii-v
`2.3116363, desermined by dynamic light scattering
`using the Malvem PCS 2.4/2.3 software with 3
`
`Malvem 4600 apparatus (Malvem Ltd. Malvern,
`UK.3 equipped with a. 25 mW helium/neon laser
`(NBC Corp, ’l‘okyo, Japan}, "W33 around 0.19 33m.
`The dispersions were stored in the refrigerator
`overnight and extruded again the next Clay. The
`pH of
`the dispersions was also omesured and
`adjusted between the extrusions; the pH was fol--
`lowed during the studies on (legradation lone-ties
`for all samples; no changes were observed.
`
`Kinetic n’zeasuremems
`
`The prepare-cl liposome dispersions were filled
`into 3 ml ampoules under nitrogen atmosphere in
`an LAF cabinet and sealed. Ampoules were stored
`either in a constant temperature waier oath or a
`constant
`temperature (3333333333, which were equi-
`librated to the required iemperature prior to use
`Samples were taken after appropriate time intern
`V2333 and analysed by HPLC. Degradation kinetics;
`was monitored at 40. 50, 60, 72 and 82°C.
`
`HPLC arméysis of PC
`The HPLC analysis of PC was based on the
`method. described by Nasner and limos (19813; it
`was slightly modified The HFLC system consisted
`of a solvent delivery system type 6000A. a, WES?”
`710 B automatic sampling unit (both from Waters
`Associates, Milford, MA. USA.) end a variable
`wavelength detector {Model SF 773,
`13213308.
`Ramsey, NJ, USA). The analytical {25 cm X 4.6
`mm} column was filled with Liclxrosorb SE 60 ('10
`p.333 particles) packing. maloial
`(Merck Dame
`
`F—"T—"T"""l"""T'""":""""'3
`O
`2
`4
`8
`8
`30 12
`minutes
`
`Fig.2HPLC chromatogram of phospardelwehue Fox coir:-
`
`ditiom see. 1'
`6313315 and Melhoos l. solvent fmr-HfBW
`
`2,, phosp hatédglcholinc; 3. lyso-PC)
`
`000002
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`
`
`
`

`

` i
`
`i
`
`no
`
`Therefore, it is necessary to keep the pH constant
`by using buffer solutions. The possible catalytic
`effect of the buffer on the degradation process has
`to be taken into account (Connors, 1973:). Sub~
`Stantial catalytic effects of the buffer components
`in the stability studies were reported hy investlgm
`tors for other substances (Beijnen, 1986; Cerrey,
`1987). To evaluate the contribution of the buffer
`species, the kuhs value can he expressed as:
`
`kohs ' kc + chlE'F-i “i" [Corrigyrl
`
`+ icbuffm. [bufferl
`
`('2)
`
`where k0 is the limb-order rate constant for the
`degradation in water only,
`JtH and kOH are the
`secondnorder rate constants for proton» and hy-
`droxyl-catztlysed degradation,
`respectively,
`and
`khurffir is the sum of the second—order rate con
`stants for the degradation catalysed by each of the
`buffer components.
`1
`The catalytic effects of the buffer components
`on the hydrolysis of PC were investigate at con~
`slant pH and temperature (72° C), but at different
`buffer concentrations. in this case the term it bum,
`{buffer} in Eqn. 2 is varied while the other terms
`are constant.
`
`For each pH 'ttlue, plots of km against the
`buffer concentration yield a. straight line with an
`intercept equal to the rate constant (kgbs) at zero
`buffer concentration (Eqri. 3} and 3 slope equal to
`the second—order rate constant for catalysed dew
`gradation by the buffer components. These .4:ng
`values were used to obtain the pl—l profile of PC
`(Fig. 4).
`
`ko‘os 3‘ kc kan+i 'i' koniQHHl
`
`{3)
`
`For PC hydrolysis kinetics, plots of kgbs against
`[H “} and [SET] yield straight lines with the slopes
`equal to kfi and {(0H, respectively, and the interw
`cepts equal to ['60. If ROE {0H1 <<.: kn {H'Fj then
`the catalytic effect. of: the Oll‘ ions can be ne—
`glected. Conversely,
`if kH-
`[H'*'} << kQH {0H1
`then the catalytic effect of the if ions can be
`neglected.
`In buffer solutions, the buffer concentration is
`the sum of the concentrations of the hiafi’er species.
`
`stadt, 11211.13). PC separation was achieved with
`an eluent consisting of n~hexanemisopropanoL
`water (2:4:l, v/v) at a flow rate of l .tnl/niin.
`Detection of PC was carried out at 206 run. A
`
`typical example of a chromatogram showing a
`partly hydrolysed dispersion is presented in Fig. 2.
`Peak heights were oscdf to quantify PC. Standard
`curves exhibited a linear response {r> 0.999) in
`the concentration range of G.l~-l. mM. The total
`phosphorus content of
`the aniponles was den
`terinincd with the procedure ol' Fiske and Sub—
`barow (1925).
`,
`
`Results
`
`A tynical example of the obtained data. points
`during storage of PC liposotne dispersions is pro
`seated.
`in Fig“ 33 The disappearance of PC in
`buffered solutions
`followed pseudo first»order
`kinetics, This is indicated by the linearity of the
`semilogartthniic plots of PC concentration vs time
`(r :2 0.9.9}. From the slopes of these straight lines
`the pseudo first—order rate constants (km) were
`obtained.
`'
`
`The rate equation can be written as:
`
`— 5%? = ignite}
`
`,
`
`{3)
`
`The formation of fatty acids from the hydroly-
`sis of PC tends to change the pH during storage.
`
`E 1
`(3
`
`’
`
`1 :7 ' """f""';""‘1“““r‘“‘7“‘“r"“1““’r"‘r—fil
`O
`100
`200
`360
`46%?
`ENE
`time (h)
`the
`fig, 3. Semllogarlthmic apparent
`firstuorder plots for
`degradation of PC in pH 6.5 citrate buffer (;t=0.068). (6,
`823C;
`4:}. PC; E. 60°C; :1, 59°C). The lines were calcnw
`hated by linear regression analysis.
`
`000003w
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`
` 1:!o
`
`E3
`
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`“\9
`{SP1
`2 .
`
`

`

`a M O
`
`1!)
`
`92
`
`PC!
`
`a....1....d.-.J
`lagk(h'1) .5:r17 atI?
`.2.a
`
`- a . o
`
`\Nfil
`a \&\
`
`//
`
`/n
`x
`
`Ramx10‘3
`
`. 3 . 2
`
`»r»T--»1»»»T»wmr--».»T-qu-wr-w-u-r-j
`3415678910
`pH
`
`Fig. 4, Effect of pH. on the degradation of PC at 7'2" C (buffer
`concentration = 0). Tim 113183 were calculated by linear regrcs»
`Sign analyééis.
`
`The. concentration of each buffer component is
`equal t0 the Izmir; fractlcm {f} of the. buffar can»
`pomnt multipliad lay the. buffer concentration.
`Therefore, Equ. 2 am he transfm‘msd into the
`following equations £01: acetate (MAC/Ac“, 5051,
`4)., citrate {C3i3"‘/Ci2", Eqr. 5:) and Tris ("l‘ris‘f/
`Tris, Eqn. 6) buffer systems (Bellman, i886).
`i
`r
`a
`\
`.
`'
`J
`kobs : ko’us + ile’ “‘ kliAu)./f:4w” “5' {CHAL L
`
`{buffer}
`
`(4)
`
`f'
`__ '7
`I
`Kama- __ ['UbS + l(kTris“
`
`-...
`
`'1
`f
`‘
`_
`i,
`h’l‘ris>f'l‘ris" + ":Tris ,4
`
`Plots 0? cabs against ths buffer concsxrxtration
`yield straight lines. with slopes equal to (kM—*
`kHAc) fl’kc" "l‘ kHAc.» Wei“ ”‘ 1'50"”) f€13“ + kCil“ and
`(16mg flaws) Jim“ + Aims in acetate, Citrate and
`Tris buffers. respectively. Examples are shown in
`Fig. 5 for hydrolysis at PC in citrate buffer at
`7'2." C. ’I‘hese glots were: drawn for 3 131-1 values in
`acatate buffer, 4 pH values in citrate buffer and. 5
`pH valuss in Tris bulky. Plots 0? calculated slams
`against the mole fraction of the. Ac“, Ci3” and
`Tri.s’*' ions yisld straight binds with ifltercepts equal
`to kg“,
`Icmz» and km and slopes equal
`to
`
`
`
`‘0
`
`WW
`9.06
`0.438
`0.04
`0.02
`Butter cancemratian (M)
`
`$2.1
`
`Fig. 5 Effect of" the buffer concentration of the degradation cf
`PC in citrate buffer at 72°C. ($5. pH 5.0; 0. pH 5.5; [3, pH
`
`Sign analysis:
`60). The lines were calculated by linear mgr
`Each paint represents the mean of at 153359. two gepamte deter
`minations.
`
`N
`
`la)
`kHAs)? like?" "A km“) and (kiwi _ 11‘"
`(km-
`in acetate, citrate and Tris buffers, respectivaly
`{Figure (3). The calculated rate constants for each
`buffer specks are listed in Table l.
`The tempcrature depefidenoe 0f the hydrolysis
`of ?C was investigated in the pH range or" 4.9 and
`temperature range of It’ll—82°C in the. buffer solu~
`tions at the ignic strength of 0068 (F2"’l Th6
`
`0,10
`
`0.08
`
`slope
`
`?
`
`Fig. 6, The \‘alationshjp between the male fraction of tha inns
`in the buffer solution and the slopes of the buffer concantféi'
`
`tiun~ kn,“ curves (Skin acetate buffer; Q, incittste buffer: $7
`in Tris buffer) 'llhfz llnes ware calculated by linear regressififi
`analysis.
`
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`

`'A‘AELE 3.
`
`TABLE 2
`
`
`
`NA 9 r-u
`Semnd»order rate constants far cataiys‘ed degradativn 03/" PC at
`M L
`
`
`
`
`Constants express-m in Ex “‘0th excepi for tha firstmrdcr
`rate ccnstant 12:0 which is repented in h ‘.
`
`effect. of temperature maths decomposition:
`0xprassed by the Arrhenius equatian (7:):
`
`is
`
`m k. m In A — fig/RT
`
`(7')
`
`where A is the freqncncy factor, Ea is the. 000%»
`tion energy, R is the gas constant and T is the
`absolute taxnperature.
`Liposome dispersions are tw0~phase systems: a
`water phase and a bilaye-a‘ phase can be discerned.
`The. frequency factor for the lipusame dispm‘sions
`
` ._.\
`
`2.3
`
`2.0
`
`3.4.
`
`3.2
`
`3.0
`m" x 10' 3
`
`of PC
`Fig. 7. Effect of the tempsrature on the hydro]
`
`as ware
`(0 ; 0.068). (Q, pH 4.0; 0, pH 6.5; m, pH 8.0). The
`calculated by linear régrcssien analysis. Each point represeats
`the mean. of at least two separate. determinations.
`
`000005
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`
`Aciivatian energies (Eu), frequemy factor/S {A}, emropies of
`
`activafion (AS?) and probablli’t
`facmrs (P) for degradation of
`PC as a function of pH ("Lt = 0,068).
`
`A
`E
`(0“)
`(k) ml")
`2.7 « 10
`29.7
`4.0
`9.4404
`48.5
`5.0 *
`5.0 * * 42.12210“
`6.0
`43.3
`1.1404
`0.5
`57.2
`7.7 . 103
`7.4
`42.3
`12-10"
`8.0
`41.9
`5.0 104
`9.0
`39.0
`9.02103
`
`' AS 8‘
`(J K“1 moi”)
`130.4
`41.5.
`99.0
`~ 99.5
`~ 04.1
`~98};
`— 99.0
`401.1
`
`P
`
`
`
`..
`6.9~10"‘6
`6.7-1'L_é
`5.240%
`
`Standard deviations were typicalty ca 5%; * Acetém buffer.
`*"“ Citrate: buffer.
`
`V518 analysed in mom detail to find out Whether
`pmbabflity factor (F) diffsmd significantly from
`hydrolysis kinetics in a homggmeaus, onewphase
`system. H13 entropy of activation, AS *, wag
`caicu‘taéed at 25 U C from the frequency facmr ( A)
`thainsd {mm Arrhenius equation by using Egg. 8
`and the pmbabflity factm was calculated from the.
`I 158* vaiues by using; Eqn, 9 (Martin at 01., 1983):
`
`AR“ :Rfln A ~10 ic'IVh)
`
`and
`
`- __ .A.S'*,./z<
`
`(8)
`
`(9)
`
`Where k is
`
`tbs Boltzmann constant amt h is
`
`Mariska constant. The (skimmed Arrhenius param-
`(D
`Ecters ar
`listen in Table 2.
`
`fiiswssinn
`
`Hydrolysis; 0? W3 in tipasnme vasictes first ra—
`suits in lyschC and fatty acid formation, Further
`degradation :0 smaiiet' fragments OCCUI‘S in a later
`stage: {Ramps and Crammafisl, 1988), The initial
`degradation products are.
`likely m interact with
`the biiaym.
`it was not
`investigawd how much
`lyso-PC and fatty acids can be taken. up by the
`liposonms before.
`tha chicles disintsgrats. Apv
`parently. the. dsgxmiation kinetics of PC wens not
`affected by the. presence 0!? lyso-wmpounds 01'
`fatty acids as straight pseudmfimmrdcr plots W51?
`
`
`
`
`
`
`

`

`
`
`
`(3
`
`the. pH
`it should he mentioned that
`obtained.
`tends to drop during storage unless proper buffers
`are used.
`
`During storage of the dispersions tinder accel»
`eratecl conditions a film was formed on the glass
`wall. This film could he reclispersed by vortexing.
`The. PC concentration, before and after vortexing,
`was similar Therefore,
`the film was mainly com"
`posed of non—PC. material. Degradation kinetics 0'
`PC. were not affected by this third phase former»
`tion as no deviations from a straight line were
`observed.
`
`General acid—base catalysis was observed by the
`effect of acetate, citrate and Tris ions. This is
`indicated by the slopes of the pH hydrolysis rate
`constant curves (slopes are equal to 0.4 for acid
`and base catalysis hydrolysis; Fig. 5) and the
`linear relationship between observed rate constant
`and butter concentration. From Table 1 an in-
`
`creasing catalytic effect can be read with increas-
`ing anionic charge of the citrate ions. Negative
`catalysis (a protective effect) was observed for
`acetic acid as indicated by the minus sign in 'l‘able
`l. Similar protective effects of the acetic acid on
`the stability of cyclosidomine have been described
`previously (Carrey. 1987‘). No attempt was made
`to disclose the mechanism of catalysis of protec-
`tion.
`
`The valltlity of Arrhenius law for the. hydrolysis
`of PC was investigated in the temperature range.
`between 40 anti 82° C. Degradation kinetics could
`be adequately described by Eon. 7. Frolcjaer et al.
`{1982) showed for (listestoyiphosphatitlylcholinc
`{DSPC} liposontcs a break in the plot of in km vs
`1/"? around the transition temperature (3:) ct
`DSPC: 55°C (FJFQli‘Kjaéll‘ et at... 1982). As men—
`tioned in the experimental section, soybean PC. is
`mainly composed of unsaturated fatty acids. The
`Tc of this type of phospholipids is about ~15“ C
`{Szoka and Papa‘nactioponios, 1.980). This temper~
`attire range is above the transition temperature
`(2:) of soybean phosphatidyloholine. ’l‘herefore,
`no break in the Arrhenius plot. \ 'as observed (Fig.
`7).
`
`Further analysis of the frequency factor in the
`Arrlienlu. equation showed that probability facn
`tors of the hydrolysis kinetics of PC in liposomc
`dispersions did not significantly differ from kinet~
`
`ics in homogeneous one~phase aqueous solutions
`(Frost and l’carson, 1961). Apparently the ester
`bonds (Fig. l) are freely accessible for hydrolysis.
`From this study it. can he decided that nn'niinom
`degradation kinetics occur at pH 6.5 and that low
`buffer concentrations are favourable; is,” values
`at pH 6.5 and at zero buffer concentration of 2%
`days at 6 ° C can be calculated. A lyso-PC content
`in PC vesicles of 10% (molar basis) increased the
`permeability dramatically (lnoue and Kitagawa,
`1974; Kibst and Stricker. 1955). Work is in pro-
`gress to investigate the effect of charge inducing
`agents and cholesterol on degradation kinetics and
`hilayer permeability.
`
`References
`
`‘
`
`Beijnen, Hi1, Chemical Stability and fi-fzftomycizt and Ant/tram
`eyeilne Ann‘neopiastlc Drugs, PhD. Thesis, University of
`Utrecht. Utrecht, l986.
`Carrey, CR, Solution stability of ciclosidomine. J. Pharm.
`Sci, 76 (1987) 393-697.
`Connors, EA, Reaction Mechanisms in Organic Analytical
`Chemistry, Wiley, Nth York, 1. 7'3, pg). 41401.
`Fiskc, CH. and Stibharow. Y, The colorimetric \ etermination
`of phosphorus, J. Biol. Chem.., 66 (1925) 375-400.
`Frost. AA. and Pearson. R.G., Kinetics and Mechanism, Wi—
`ley, New York, 1961, pp. 100-«1'01.
`’
`
`Frelg
`, 8.. Hjorth, EL. and Warts. 0., Stability and storag’l
`of liposomes.
`in. Bundgssrd, H... Banger Hansen. A. amt
`Kofod,
`ll.
`(Eds), Opiimz‘zation of Drug Delivery,
`Munksgnord. Copenhagen. 1982, pp. 384—404.
`lnoue. K. and Kitagawa. T, Efleot of exogenous lysoleoithin
`on llpcscmsl membranes: its relation to membrane fluidity.
`Btaciztm. Biophys. Acta. 363 (1974) 3617-372.
`Kcmps, .l'.lVi.A. and Crommelln, l')..l.A., Chemische stabiliteil
`
`van fostolipitlen in farm ..
`.
`, sche psepartiten. l. Hyrlrolyse
`van fosfollpiden in watcrlg milieu. Pizarro. Weefl:bl., ”3
`(1988) 35577363.
`Kibat. PG. and Stricken ll, lagerungsstabiiltlit Von Lip??-
`somtlispersioncn ans Sogaleclthinon. I‘hm‘m. Inch, 48 (1985)
`ll84—1189.
`J. and Cammarata, A, Physical
`Martin, AN, Swarbrick,
`Pharmacy, Lea & Febiger, Philadelphia, 1983. pp. 37l ~374-
`Nasner, A. anti Kraus,
`I... Quantitative Bestlmmung V011
`Phophaticlylcholin nnt Hilts tier HPU'I.
`Fat!!! Selle“
`Am'trichmitael. 33 @981) 70-73.
`Sine-ion, ME. and Sholiel, 3.3.5., Permeability changes motif-35
`
`by peroxidation in liposomes pren‘tretl
`From hum?!”
`
`erythrocyte lipids. Lipid Res, ’15 0974 2.7
`280.
`Szoka. F. and Papahadjopouics, 1)., Comparative properfie5
`and metltoals of preparation of llpicl vesicles (liposomesl-
`Amm, Rev. Biopr'tvs. Bioeng, 9 (1980) 461-508.
`
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