by Copyright: law (Title 17’ £3.53
`
`.rial may be protect:
`
`international Jauma! of Pharmaceutics, 5t} (1989) L6
`Elsevler
`
`1:? 01665
`
`y..~
`
`Research Papers
`
`Hydrolysis of phoephatidylohollne
`in aqueous llposorne dispersions
`
`Mustal‘a Grit, Jan H. de Smidt, Anita Struijke and Dawn tin/5t. Crommeiin
`Department of Pharmaceutics, Faculty afPharmacy; University of Utrecht, Utrecht (The Nezhérimtdi)
`
`{Received 8 June 1988)
`(Acceptsd 8 July 1988)
`
`
`
`Summary
`
`Key words: Liposozne; Stability; Hydrolysis; Phosphatidyloholine
`
`
`
`
`The hydrolysis of phospltatil'
`“" oline was investigated as a fauction of pH, temperature, buffer concenzr' :ion and buffer species.
`
`The hydrolysis rate of phosphat: ylcholine inereased with increasing concentration of the buffer species;
`there was a linear
` roitttiot v
`concentration and at
`‘p between the buffer concentration and the observed rate constant. The pH profile at zero buffet
`
`on on the effect of the
`71?.” C thows a minimum hydrolysis rate at about pH 6.5. The data were analysed to obtain detailed inform:
`buffer species used on the stability: The relationship between the observed rate conStant and temperature could be deSCri‘oed
`adequately by the Arrhenius equation.
`
`
`(phosphofiipid component. Chemical decomposi—
`tion of phosphollpids (hydrolysis or oxidation)
`causes physical instability of the liposotne diaper“
`sions and might therefore interfere with the intro—
`duction of
`liposomes
`in therapy (locus and
`Kitztgawag 13374, Smolen and Shohet, 1974 and
`Kihat and Stricken 1985). Phospholipids can be
`hydrolysed to lyso~phospholipidsg these iyso-phos»
`pholiplds are also subject
`to further hydrolysis.
`2~lysophospholipids are the main initial hydrolysis
`products in aqueous dispersions (Fig. l) (Kemps
`and Crommoiin, 1988‘).
`
`CH2- 0 - R'
`l
`a" — o ., e - H
`
`l 0
`
`H2 » 0- wow - o — CHE one to” (crests
`
`introduction
`
`is
`it.
`From a pharmaceutical point of view,
`important
`to demonstrate that drugs or dosage
`forms are sufficiently stable, so that they can be
`stored for a reasonable period of time without
`changing into an inactive or toxic form
`Liposomes are under investigation as drug can
`rler systems for
`their potential
`to improve the
`therapeutic index of drugs to be used eg.
`in
`cancer chemotherapy or for tlte‘vtreatment of life-
`tlrroatening parasitic, viral or microbial infections.
`Liposomes are vesicular structures build up of
`lipid biiayere. For therapeutic purposes usually
`phosphatidylehoiine (PC)
`is used as
`the main
`
` .mdence: DEA. Ctormnelin, Department of Pharma-
`Oeutios, Faculty of Pharmacy, University of Utrecht, Croese—
`street 79, 3522 AD Utrecht, The Netherlands.
`
`Fig. ’1. Chemical structure of phosphatidylcholine (R"' and R”
`are the fatty acyl soh‘stitnents) and 2-iyso~phosphatidylcholine
`(R; is the fatty acyi substituont and R” is El
`
`‘373-5173/‘89/’$03.5(} © 1989 Eisovicr Science Fublishers RV. (Biomedical Division)
`
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`
`in this study, hydrolysis of PC as a function of
`pH,
`temperature and buffer concentration was
`investigated. The data were analysed to assess
`catalytic effects of
`the different hufi‘er species
`used.
`
`Materials and Methods
`
`Materials
`
`Soybean PC (Phospholipon ‘l00) was obtained
`from Natterrnann Gmhl-l, Cologne, EKG, and
`used as received. PC consisted of 90% PC, 50%
`
`lyso~compounds 5.096 free fatty acids and less
`than 0.1% water. The fatty acid composition or"
`PC, determined. by high performance liquid clue—
`matography (HPLC), was as follows: 3. .9’6 thyris—
`tic acid, 18.2% palnritic acid, 4.2% stearic acid,
`12.0% oleic acid, 61.
`9’6 linoleic acid. ()ther chem-
`icals were of analytical grade. All solutions were
`prepared with doubledistillerl water.
`
`Buffer solutions
`The following aqueous buffer solutions were
`used for the kinetic studies: pH 4vvvvv 5 acetate buffer,
`pH 5 to 6,5 citrate buffer and pH 7 to 9 Tris
`buffer, The pH was measured with a glass elec-
`trode and a pH meter (Type CG 817' T, Schott
`Gerate, 13.11.63. ionic strengths of the buffer solu—
`tions were 0.068, 0.125, 0.200 and 0.300 and ad—
`justed by manipulating the concentration of the
`buffer components.
`
`Preparation of the Ziposome dispersions
`E’C liposome dispersions were prepared by the
`“film” method (Sacha and Papahadjopoulos,
`l980). After formation of the phospholipid film in
`a round~hottom flash in a rotary evaporator at
`~ 50° C, the film was left under reduced pressure
`overnight. It was hydrated at ~ 50°C with the
`appropriate buffer solution and the pH of the
`dispersion was measured and adjusted,
`if necesm
`sary,‘ The initial PC concentration was 30 mh’i.
`Extrusion was carried out twice through a mom"
`hrane filter with. a pore size of 0.2 pm (Uni.~pore,
`Bio~Rad, Richmond, CA, USA.) 'l‘he vesicle di»
`ameter, determined by dynamic light scattering
`using the Maltrern PCS 2.4/2.3 software with a
`
`Malyern 4600 apparatus {Malvern Lid, Malvern,
`UK); equipped with a, 25 mW helium/ neon laser
`(NRC Corp, ’l‘okyo, Japan), was around 0.19 am,
`The dispersions were stored in the refrigerator
`overnight and extruded. again the next Clay. The
`pH of
`tlie dispersions was also measured and
`adjusted between the extrusions; the pH was fol-
`lowed during the studies on degradation kinetics
`for all samples; no changes were observed,
`
`Kinetic measurements
`
`The prepared liposome dispersions were filled
`into l nil ampoules under nitrogen atmosphere in
`an LAF cabinet and sealed. Ampoules were stored
`either in a constant temperature water bath or a
`constant
`temperature cabinet, which were equi—
`librated to the required temperature prior to use
`Samples were taken after appropriate time inter—
`vals and analysed by HPLC. Degradation kinetics
`was monitored at 40, 50, 60, 72 and 82°C.
`
`HPLC analysis of PC
`The HFLC analysis of FC was based on the
`method. described by Nasner and Kraus (1981); it
`was slightly modified. The HPLC system consisted
`of a solvent delivery system type 6000A, 23, W139
`710 B automatic sampling unit (both from Waters
`Associates, Milford, MA, USA.) and a variable
`wavelength detector
`(1Model SF 773, Kratos,
`Ramsey, NJ, USA.) The analytical {25 cm X 4.6
`mm) column was filled with Lichrosorh SI 60 (10
`um particles) packing material {Merck Darm~
`
`mm“:"""l""""l"""‘l
`O
`2
`4
`6 8101?
`minutes
`
`Fig. 2. HPLC chromatogram of phosphatidylchellne. For 0021'
`ditions see Materials and Methods. 1, solvent
`frontwifall)’
`acids; 2., phosphatidylcholine; '3, lysox-FC).
`
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`

`stndt, ERG.) PC separation was achieved with
`an eluent consisting of nwhexanemisopropnnoh
`water (2:451, 'v/v) at at flow rate of l inl/inin.
`Detection of PC was carried out at 206 1111‘}. A
`
`typical example of a chroznatogmm showing a
`partly hydrolysed dispersion is presented in Fi g. 2,
`Peak heights were used to quantify PC. Standard
`curves exhibited a linear response {r> 0.999) in
`the concentration range of il.l--—l mM, The total
`phosphorus content of
`the aniponles was den
`termined with the procedure of Fiske and Sub-
`haifow (1925).
`7
`
`Results
`
`A typical example of the obtained data. points
`during storage of PC liposoine dispersions is pre
`sented in Fig, 3, The disappearance of PC in
`buffered solutions
`followed pseudo first»order
`kinetics, This is indicated by the linearity of the
`seniiiogatithinic plots of PC concentration vs time
`(r in 0,993, From the slopes of these straight lines
`the pseudo first—order rate constants (km) were
`obtained.
`'
`
`The rate equation can he written as:
`
`_ fl : knitci
`at
`
`'
`
`m
`’
`
`’l‘he formatioii of fatty acids from the hydroly—
`sis of FC tends to change the pit during storage.
`
`log%remained
`
`.4.
`
`a; '
`"r""';""'1“"‘r““7““‘r““7“‘“‘rbr—1
`
`0
`190
`200
`300
`469
`50$
`time on
`the
`Fig, 3, Seinilogerithmic apparent
`firstuordet' plots for
`degradation of PC in pit 6,5 citrate buffer (ttmflifiéx). (e,
`820C;
`(:5, 2°C; n, 60°C; :1, 50°C). The lines were enlonv
`lated by linear regression analysis.
`
`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 he taken into account. (Connors, 1973), Suh~
`stantinl
`:ataiytic effects of the buffet" components
`in the stability studies were reported by investigm
`tors for other substances (Bennett, 1986; Carrey,
`1987). To evaluate the contribution of the buffer
`species, the km value can he exnressed as:
`
`kohs ' k0 + {Chilly-l '5" kOHi‘GHMi
`
`+ kbuffer ib‘lffer}
`
`(2)
`
`is the first~order rate constant for the
`where kc.
`degradation in water only, kH and I60“ are the
`seeondorder rate constants for proton» and hy-
`droxylvoataiysed degradation,
`respectively,
`and
`#1:},un is the sum of the second—order rate eon~
`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 investigated at con~
`stant pH and temperature (72° C), lint at different
`buffer concentrations. in this case, the term it buffer
`{buffer} in Eqn 2. is varied while the other terms
`are constant.
`
`For each pH value, plots of kc,“ against the
`buffer concentration yield a. straight line with an
`intercept equal to the rate constant neg“) at zero
`buffer concentration (llqn. 3) and 3 slope equal to
`the secondnoi'deif rate constant for catalysed de—
`gradation hy the buffer coinnonents. These kng
`values were used to obtain the pill profile of PC
`(Fig. 4).
`
`kdbs “m“ kn + kniH+i ’i" koniOH-‘i
`
`(3)
`
`For PC hydrolysis kinetics, plots of kg,“ against
`[H “l and [GH‘} yield straight lines with the slopes
`equal to kfi and ice“, respectively, and. the inter»
`cepts equal to kg. if ken {O‘Hfl << it}, {ill} then
`the catalytic effect of: the Oll‘ ions can he ne—
`glected. Conversely,
`if k” [ill] << k0“ {GH“]
`then the catalytic effect of the EV ions can be
`neglected.
`in buffer solutions, the buffer concentration is
`the sum of the concentrations of the buffer species.
`
`m..___—._.._AJ
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`il
`
`
`
`
`
`’1 0
`
`U7 .
`
`. WW"”]
`0.04
`(HIE
`0.08
`0.10
`0.02
`latter eencentratien (M)
`
`‘0
`
`Fig. 5. Effect of the buffer concentration of the degradation of
`PC in citrate buffer at 72°C. (Q. pH 5.0; 0.. pH 5.5; E), pH
`
`6.0} The lines were calculated by linear mgr
`"in analysis
`
`Each point represente the mean of at leagt twc ,, parate deter
`minaticns.
`
`-
`
`lkCiT‘" "' kw") and “(my _ kite)
`(k/‘w‘ ‘7‘ kin/to);
`in acetate, citrate and Tris buffers, respectively
`(Figure (3). The calculated rate constants for each
`buffer species are listed in Table l.
`The temperature dependence 0f the hydrolysis
`of PC was investigated in the pH range or" 4-9 and
`temperature range of {til-«82° C in the buffer sclw
`tions at. the ionic strength of 0068 (Fig-7). The
`
`010
`
`
`0.08 ‘
`
`
`
`stop-e
`
`Fig. 6. The relationship between the male fraction of the imifi
`in the buffer solution and the slope- cf the buffer cancentffi'
`ticn— km curves. (Lint acetate buffer; Q, lncittate buffer; $7
`in Tris buffer) The lines were calculated by linear regression
`analysis.
`
`
`
`~13
`
`kchsx10
`
`Fig. 4 Effect of pH. on the degradation of PC at 7'2“ C (buffer
`concentration :- 0). The lines were calculated by linear regress»
`Sion analysis.
`
`The concentration of each buffer component is
`equal to the mole fraction {f ) of the buffer coin
`pcnent,nmitiplied by the buffer concentration.
`Therefore, Eqn 2 can be transformed into the
`following equations for acetate (ll-lAc/Ac“, Eqn.
`4)., citrate (Ci3”"/’Ci2”, Eqn 5:) and Tris ("l‘risf/
`Tris, Eqn. 6) buffer systems (Beijnen. 198(2).
`
`ko‘cs 2 kits + {{km’ “‘ [CR/5.0)}: c +‘ kHAc}
`
`{butter}
`
`15mg '"‘
`
`31,54“ {(kcfi'“ “ kcr‘iifct’“ +-KCi2‘}
`
`{butter}
`
`‘
`
`knbs 3 Ma + {(k’l‘ris+
`
`k'l‘ris)f’l‘ris+ 'JF‘ ’C’E‘risi
`
`{buffer}
`
`(4)
`
`(5)
`
`(6)
`
`Plots of kabs against the buffer concentration
`yield straight lines. with slopes equal :0 (km—e
`
`ktlAc) fan" "l‘ kHAC? (kc: ‘” 1‘ C12") fCi3” + ken“ and
`(kmg fixings) fmswt- Aims in acetate, citrate and
`Tris buffers, respectively. Examples are shown in
`Fig. 5 fat hydrolysis of PC in citrate buffer at
`72.” C. These plots were drawn for 3 pH values in
`acetate buffer, 4 pH values in citrate buffer and. 5
`pH values in Tris buffer. Plots tf calculated slopes
`against the mole fraction of the. Ac“, Ci” and
`Tris?" ions yield straight lines with intercepts equal
`to km“, kc?" and km and slopes equal
`to
`
`000004
`
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`
`

`

`TABLE l
`
`TABLE 2
`
`Second-order rate constants for comb/sad degradation of PC at
`72 ° C
`
`
`ezzzrapies of
`Activation energies (Ea), frequency factors (A),
`activaiion (AS?) and probability factors (P) for degradation of
`PC as a function rig/'17" (3.1L 2 0, 068,1r
`
`
`
`4.0
`5.0 *
`5.0 H
`6.0
`as
`7.4
`8.0
`90
`
`2:74.0— «130.4
`9.4‘104
`4.1.5.
`1.2404
`99.0
`i,
`.10“
`—99.5
`7.7 . 103
`64.1
`12-10“
`“98.8
`l.2~ 104
`— 99.0
`9.0103
`«401.1
`
`,
`15 i- ‘
`55-10“5
`6.7-10'6
`0440’s
`4.5 310““
`6.9~l0"‘5
`{iii-10“”
`
`5.2.10"
`
`Standard deviations were typically ea 5%;
`** Citrate buffer.
`
`3‘ Acetate buffer.
`
`was analysed in more detail to find out whether
`probability factor (P) differed significantly from
`hydrolysis kinetics in a homogeneous; one—phase
`system. The entropy of activation, AS *, was
`calculated at 25 ° C from the frequency factor (A)
`obtained from Arrhenius equation by using lion. 8
`and the probability factor was calculated from the
`1 AS“ values by using Ego. 9 (Martin et ai, 1983):
`
`AN“ :R{lri A _ in kT/h)
`
`and
`
`P :2 eliS'V/R
`
`{8)
`
`(9)
`
`where k is the Boltzmann constant and h is
`
`Plane a constant. The obtained Arrhenius param-
`eters are listed in Table 2.
`
`Discussion
`
`Hydrolysis of PC in liposome vesicles first re"
`sults in iysovl’C and fatty acid formation, Further
`degradation, to smaller fragments occurs in a later
`stage (Kemps and Cromnieiin, 1988), The initial
`degradation products are likely to interact with
`the bilayer.
`it was not
`investigated how much
`lyso~PC and fatty acids can be take-01133 by the
`iiposornes before the vesicles disintegrate. Ap~
`parently, the degradation kinetics of PC were not
`affected by the presence of:
`lyso-cornpounds or
`fatty acids as straight pseudowfirsborder plots were
`
`
`
`
`
`k0
`kn
`k0”
`km"
`
`8.5‘1.0”4;l;1.7‘10‘4
`0.8.102 Jim-101"
`6.8402 iiiaoi-‘Z
`2.010"2 1». 1.4»10f
`
`
`
`Constants expressed in Mfijihfi‘, except for tire f st»order
`rate. constant k0 which is reported in h" 1,
`
`temperature on the decomposition is
`effect of
`expressed by the Arrhenius equation (7:):
`
`in kl,bs = in A — [Ba/RT
`
`{7)
`
`where A is the frequency factor, Ea is the activa—
`tion energy, R is the gas constant and T is the
`absolute temperature.
`Liposome dispersions are lyvo~piiase systems: a
`water phase and a biiayer phase can be discerned.
`The frequency factor for the iiposome dispersions
`
` p —2
`
`~3
`
`'4
`
`2.8
`
`-
`
`2.9
`
`I
`3.0
`
`I
`3.1
`
`3:2
`
`1/”? x 10'3
`
`Fig. ’7. Effect of the temperature on the hydrolysis of PC
`(it = 0068). (Q, pH 4.0; 0, pH 6.5; I, pll 8.0). The lines were
`calculated by linear regression analysis. Each point represents
`the mean, of at least two separate determinations.
`
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`
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`d
`
`the. pH
`ohtzlined it should be mentioned that
`tends to drop during storage unless proper buffers
`are used.
`
`During storage of the dispersions under aceel»
`created conditions a film was formed on the glass
`wall. This film could he retiispcrsed by vortexing.
`The PC concentration, before and after vortexinr‘15.,
`was similar. Therefore,
`the filni was mainly com—
`posed of non—PC. rnaterial Degradation kinetics of
`PC. were not alfeteal by this third phase forma»
`tion as no deviations from a straight line were
`observed
`
`General acidnbase catalysis was observed by the
`effect of acetate, citrate and 'l‘ris ions. This is
`indicated by the slopes of the plnl hydrolysis rate
`constant curves (slopes are equal to 0.4 for acid
`and base catalysis hydrolysis; Fig. 3) and the
`linear relationship between observed rate constant
`I and buffer concentration. From Table i 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 lable
`l. Similar protective effects of the acetic acid on
`the stability of cyclosidonrine have been described
`previously (Carrey, 198?). No attempt was made
`to disclose the mechanism of catalysis of protec»
`tion.
`
`The validity of Arrhenius law for the. hydrolysis
`of PC was investigated in the temperature range
`between 40 and 82° C. Begradation kinetics could
`be adequately described by Eqn. 7. Frokjaer et al.
`(1982) showed for distearoylphospbatitlylcholinc
`(DSPC) liposonies a break in the plot of in {Cabs vs
`l/"i‘ around the transition temperature (T0) of
`DSl’C: 55°C (Frolciaer et al,
`982). As men—
`tioned in the experimental section, soybean PC. is
`mainly composed of unsaturated fatty acids. The
`TC of this type ct" phospholipids is about ~15° C.
`(Szoka and Papalracljoponlos, 1980). This tempen
`attire range is above the transition temperature
`(1;) of soybean phosphatidylcholine. Therefore,
`no break. in the Arrhenius plot was observed (Fig.
`7).
`
`Further analysis of the frequency factor in the
`Arrhenius equation showed that probability lac“
`tors of the hydrolysis kinetics of PC in liposorne
`dispersions did not significantly differ from kinet~
`
`:ics inhomogeneous one--phaseaqueous solutions
`(Face: and Fearson 1961) Apparently the ester
`bonds (Eng. 1) are freely accessible for hydrolysis.
`From this study it can he decided that minimum
`degradation kineticsoccni at pH 6. 5 and thatlow
`buffer concentrations are favourable;[90% values
`at pl‘i 6.5 and at zero buffer concentration of 296
`clays at 6 ° C can be calculated. A lyso-PC content
`in PC vesicles of 10% (molar basis) increased the
`permeability dramatically (lnoue and liitagawa,
`197“,4 Kihat and Stricker l9t.1) Work is in pro
`gress to investigate the eftect ol' charge inducing
`agents and cholesterol on degradation kinetics and
`hilayer permeability.
`
`References
`
`3
`
`Reliant)...[Ha Chemica’ Smbih’w and fifiz‘omycin and Anthra-
`cycline Aniineopiastic Drugs, PhD. Thesis, University of
`Utrecht, Utrecht l986.
`Carrcy, C. F., Solution stability of ciclosidomine. J. Pharm.
`Sci, 76 (l987) 393397.
`Connors, RA, Reaction Mechanisms in Organic Anulyic'cai
`Chemistry, Wiley, New York, 1. 73, pp. 41301.
`Fiske C.H and Subharow Y. The colorimetric determination
`of phosphorus. J. Biol (View., 66 (1925) 375-400.
`Frost. A.A. and Pearson, R.G., Kinetics and Mechanism, Wi—
`ley. New York, 1961, pp. 100-«1'01.
`’
`Frekjaer. 8.. Hjorth, EL. and Warts. 0., Stability and storage
`of liposomes.
`In. Bundgaard,
`lei, Haggai Hansen, A. and
`Kofod,
`ll.
`(Eds). Opiimimtion of Drug Delivery,
`Monksgaard, Copenhagen, 1982, pp. 384—404.
`lnoue, K. and Kitagawa T Effect of exogenous lysolecithin
`on liposomal membranes' its relation to membrane fluidity.
`Biaclztm. Biopiiys. Acta, 363 (1974) 361»-37Z.
`Kemps, J'.lvi.A. and Crommelln l).l'. A. Chemische stabiliteit
`van fosfolipitlen in farniaceutische preparaten.l Hydrolysc
`van fosfolipiden in waterig milieu. Phase. Weekbi.,
`l23
`(1988) 353363.
`‘
`Kibat. PUG and Stricken ll, lageiungsstabilitat von Lip“-
`so'noimeisroren ans Soialeclilnricn thm Ind/13 (1985)
`ll84— H89.
`Martin A.N, Swarbiick,
`and Cammarata
`
`Pharmacy, Lea & Febiger Philadelphia, 1983 pp. 3..Il374‘
`Nasner, A. and Krausl..., Quantitative Bestin’nnung von
`Phophatidylcholin tnit Hilfe def HPLC.
`.Fetz‘e
`S6138?!
`Ans'trichmittel. 83 (1981) 70w73.
`Sine-ten, iii and Shohet, 8.3.3., Permeability changes inclu‘f-‘id
`by peroxidation in liposomes prepared from humftn
`erythrocyte lipids, Lipid Rein, l5 (l 9'74) 2‘73~280.
`Szoka. F. and Papahedjopoulcs, [1, Comparative propefi'lell
`and methods of preparation ot‘ lipid vesicles (liposomesl-
`Amati. Rev. Biophvr. Bioeng., 9 (1980) 461-508.
`
`000006
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