.rial may be protect:
`
`by Copyright: law (Titte 1?’ Ujié
`
`1;.~.'emarz'0nal Journal 0fPr'1ai‘mace'm’ics,
`)‘:‘.':sr;='v'ier
`
`it} (1989) L6
`
`ll? 01665
`
`..;
`
`Research Papers
`
`E-lyclroiysis of ;3hospha.tidy’loholine
`in aqueous il.p0SOt‘t’1t3 dispersions
`
`Mustafa Grit, Jan H. de Smidt, Anita Struijke and Daall EA. Crommelin
`Départmem‘ of Plzc:z'nzac.4=uz’ics, Faculty afPr'1armary, Un.z'.ner.sz‘zy of Uirwizt, U.fr.r2cr’1z ("T323 1\’ezhe::'iar::is)
`
`{Received 8 June 1988)
`(Accepted 8 July 1.988)
`
`Key words: Liposome; Stability; Hydrolysis; Phosphatidylcholiiie
`
`Summary
`
`
`The hydrolysis of phc-s-phatid_' "" Olifie was investigated as a fimctiozt of pH, temperature, buffer concentr tion and bttfler species.
`The hydrolysis rate
`phosphat:
`,1 ‘nc-line increased with increasing stoncentrati-:>n of the buffer species;
`there was a linear
`
`relation.
`‘p ‘oetween the buffer concentration amt the observed rate constzim. The pH p.\'o.t’ile at 'z.ero buffs, cotmcemmtion and at
`
`ion on (E11: effect of the:
`72° C shows 2; minimum hydrolysis rate at about pH 6.5.
`date: were analysed to obtain detailed lI1f()1‘tEl-'
`buffer spcci-as used on the stability: The t'elst,ionship between the Ol’)S€1‘V6d
`rate constzmt and temperature could be riescribed
`adequately by the Arrhenius equation.
`
`
`
`(phosphc-)lipid component. Chemical decomposi-
`tion of phospholipids (hydrolysis or oxidation)
`causes physical inst; bility of the liposome cEis'per~
`sions and might therefore interfere with the intro-
`c‘=.ucti.on of
`liposomes
`in t‘z1ei'ajpy
`(lnoue and
`Kitztgaws, 1974, Smol-en and Shohet, 1974 and
`Kihat and Stmioker, 1985). Phospliolipids can be
`hydrolysed to lyso~pliospholipids; these lyso-ph<;ss--
`pholipids are also subject
`to fu:“:her hydrolysis.
`2~lysophosp-hcilipids are the main initial hydrolysis
`pifoductis in aqueous dispersions (Fig. 1) (Kemps
`and Crommelin, 1988).
`
`CH2—O-R’
`!
`R" — 0 -v C - H
`
`i C
`
`H2 ~ 0 - F’O(O!-1’) - C) — CH2 CH2 N+ (-.’)H3)3
`
`Chemical structure of pliosphatidytcholine (R"' and R”
`Fig.
`are the fatty acyl sul7'st.ituents) and 2-lyso~phosphatidylcholine
`(IV is the fatty acyl substltuent and R” is H;
`
`fixttrovciuetion
`
`is
`it
`F1‘o1n 8. pharitiaceutical. point. of View,
`important
`to demonstrate that drugs or dosage
`forms are su.€fi.<:iently
`stable, so that; they can be
`stored for a reasonable period of time without
`cthanging into an 7ma.<:t.ive or toxic form.
`Liposomes are under investigation as drug can
`trier systems for
`their potential
`to improve the
`therapeutic index of drugs to be used e.g.
`in
`cancer chemotherapy or for the‘tre22tment of life-
`thma.teni'ng 'pai'a.sitic, Viral or microbial infections.
`Liposomes are vesicular structures builczt up of
`lipid bilayers. For tlieiajpetttic purposes usually
`plmsphatidylcholine (PC)
`is used as
`the main
`
`1 C
`
`orie .);.derz::e: D.J.A. Crommelizi, Department of Pharma-
`‘cseutics, Faculty
`Phmmaey, Uttiyersity of Utrecht, Ct'oose—
`straat '79, 3522 AD Utrecht, The Netlierlaitds.
`.2
`
`378--5173’/'89/‘$03.50 © 1989 Elsevicr Salome Fublisliers l$.V. (Biomedical Division)
`
`000001
`
`WAKBM 1037
`
`
`
`
`
`
`
`A—_,-—-.....4....,.....1~,-~.4._..__——--A--------A«-------»—-------
`
`000001
`
`

`
`
`
`/.
`
`in this study, hydrolysis of PC as a function of
`pH,
`temperature and buffer C011CtiI1t3T£i.ti()IE was
`investigates}. The data were analysed to assess
`eatziiytie effects of
`the different buffer" species
`used.
`
`Materiais and Metiieds
`
`Mater*itais
`
`Soybean. PC (Phesphoiipori 1.00) was obtained
`from Nattemiarm Gmhi-1, Cologne, F.R.G, and
`used as received. PC consisted of .90% PC, 5.0%
`
`1yso~oompoimds, 5.0% free fatty acids and less
`than 0.1% water. The fatty acid eempositioii of
`PC, determined. by high performance liquid ehro~
`rhatogitaphy (HPLC), was as foimws:
`myris—
`tie acid, 18.2% pahiiitie acid, 4.2% stearie acid,
`12.0% oieie acid, 61. % iirieieie acid. Gther ehem-
`ieais were of analytical grade. Ail sohitieiis were
`prepared with deuh1e~disti11eci water.
`
`Buffer saiutions
`The following aqueous buffer solutions were
`used for the kinetic studies: pH 4--V5 acetate buffer,
`pH 5 to 6,5 citrate ‘buffer and pH '7 to 9 Tris
`buffer. The pH was measured with a giass elec-
`trode and a pH meter (Type CC: 817' T, Sehott
`Gerate, F.R.G.). Ionic strengths of the buffer solu-
`tions were 0.068, 0.125, 0.200 and 0.300 and ad-
`justed by manipulating the i3(3}.'1C6'ntI’£‘.§,iOn of the
`buffer eempoheiits.
`
`Preparation of the lipasome di.s';;er5ions
`INC) liposeme dispersions were prepared by the
`“filth” methed (Szoka and Papahadjopouios,
`1980). After formation of the phesphoiipid fihn in
`a roimd~hottorn flask 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 heces~
`sary. The iriitiai PC eorieeiitrat;:ien was 30 mitt.
`Extrusion was carried out twice through a mem-
`brane filter with. a pore size of 0.2 grin (Uiii.~pore_,
`Bie~.R.ad, Riehinond, CA, ‘U.S.A.). The vesicle di»
`ameter, determined by dyrianiie light scattering
`usixrig the Maivem PCS 2.4/2,3 software with a
`
`Maiverra 4600 apparatus {Maivern Ltd, Maivern,
`U.K.), equipped with a, 25 mW helium/iieon laser
`(NRC Corp, Toiryo, Japan), was around 0.19 am.
`The dispers.ieiis were stored in the refrigerator
`overnight arid extruded. again the
`day, The
`pH of
`the dispersiens was also measured and
`adjusted between the t3Xti‘E1S'iGl1S; the pH was foi-
`iowed duririg the stitches on ciegra.dat.ion kinetics
`for all samples; no changes were observed.
`
`!'t'7z'm2z‘z'e:- mezasuremems
`
`The prepared iiposerne dispersions were filled
`into 1 mi ampouies under nitrogen atmosphere in
`an LAF cabinet and seated. Ampoules were stored
`eithei' in a constant tempei'atui'e water hath or a
`constant
`temperature eahiriet, which were equi~
`iibrated to the required. temperature prior to use
`Samples were taken after appropriate time intern
`vets and ariaiysed by HPLC. Degradation kinetics
`was monitored. at 40, 50, 60, 72 and 82°C.
`
`HPLC arzc1(vsi,s of PC
`based on the
`The HFLC ariaiysis of FC
`method. described by Nasner and Kraus (1981); it
`was slightly modified. The HPLC system consisted
`of a soivem deiivery system type 6000A, a. W‘tSP
`710 B automatic samphhg tiiiit (both from Waters
`Associates, Milford, MA, U.S.A.) and a variable
`wavelength detector {Model SF 773, Kratos,
`Ramsey, NJ, U.S.A.). The ariaiytieai (25 cm X 4.6
`rrmi) eohiirm was fitted with Liehroserh SI 60 (10
`_um particles) pztctkizig iriateiiiai
`(Merck, Darm~
`
`I"""T"""T"""F"""1""""1""""3
`O
`2
`4
`6
`8
`10
`12
`minutes
`
`Fig. 2. HPLC chrorriatogram cf phosphatidyieheliiie. Fer corr
`ditioiis see i‘vEateri:3.1s and Methods. 1, soivem trorit+ fatt)’
`acids; 2., phosphatidylchoiine; 3, 1}/so--PC).
`
`000002
`
`
`
`000002
`
`

`
`l'*'.R..G.). PC separation was achieved with
`stadt,
`an eiuent consisting of n~hexane~isopropati.ol-
`water (_2:4:l, V/V) at £1 flow rate of 1 tn}/inin.
`Detection of PC was oa.i‘ri.ed out at 206 nm. A
`
`typical example of a ehroznatogram showing a
`partly hydrolysed dispersion is presented in Fig. 2.
`Peak heights were used to quantify PC. Stanciard‘
`curves exhibited a linear response (r> 0.999) in
`the concentration range of
`l’l1.lVl. The total
`phosphorus content of
`the ampoules was de»
`termined with the procedure of Fiske and Sub-
`barow (1925).
`;
`
`ltestiltts
`
`A typical example of the obtained data. points
`during storage of PC liposoine dispersions is pr.e~
`seated.
`in Fig,
`33 The disappearaiice of PC in
`buffered solutions
`foiloweti pseudo firsnorder
`lciiieticts, This is indicated by the linearity of the
`seniilogarithniie plots of PC concentration vs time
`(r
`0.9.9}. From the slopes of these straight. lines
`the pseudo f.irst—or(ler rate constants (km) were
`obtained.
`*
`
`The rate equation can be written as:
`
`—
`
`at
`
`= kobslljcl
`
`
`
`at
`’
`
`The l‘orniati.oii of fatty acids f1‘OII1 the hydroly~
`sis of PC tends to change the pit during storrage.
`
`tog%remained
`
`0
`
`1G0
`
`409
`
`50$
`
`300
`200
`time (ts)
`the
`Fig, 3, Se1niloga1'ithniio apparent
`lirst~oi'dei' plots for
`(Q,
`degradatiozi of PC in pH 6,5 citrate ‘buffer (ltwfltiééi).
`82°‘C;
`<3», 2°C; E. 60°C; :1, 50°C). The lines were eaten»
`iated by linear regression analysis.
`
`1.x...................
`
`
`
`Th.eret‘ore, it is necessary to keep the pH eonstant
`by using buffer soltit.io_tis. The possible catalytic
`effect of the buffer on the degradation process has
`to be taken into 8tC?(30llill. (Connors,
`il973), Su‘o~
`stanti.al. catalytic effects of the buffer components
`in the stability studies were reported by investiga~
`tors for other substances {Bei_inen, 1986; Carrey,
`1987). To evaluate the cotitrihutioii of the l>ui€fer
`species, the km value can he exggaressed as:
`
`kohs
`
`k0 + 'i*"HiH+l '5" kotilgflml
`
`+ klvuffer lbufferi
`
`where k0 is the first~order rate constant. for the
`degradation in water only, kH and ice“ are the
`seeonchorder rate constants for proton» and hy-
`droxyl~oatalysed clegrad.ation,
`respectively,
`and
`k,,um,, is the sum of the secotid.—oa‘der rate oon~
`stants for the €i6gI'£l{lt1E.i0I1 catalysed by teach of the
`buffer eornponents.
`1
`The catalytic effects of the hiiffer coinpo;rieht.s
`on the hydrolysis of PC were it1'vest.igated at con~
`stant pH and temperature (72° C), but at different
`buffer concentrations. In this case, the term /6 -,,._,m,_,
`{buffer} in Eqn. 2. is varied while the other terms
`are constant.
`
`For each pl-i value, plots of kfibs a.ga.inst. the
`buffer concentration yield a. straight line with an
`intercept equal to the rate constant (;’1c;,,_,) at zero
`buffet‘ concentration (liqn. 3) and
`slope equal to
`the seoondnorder rate constant for catalysed de-
`gradation hy the buffer coinnozients. These kg“
`values were used. to obtain the pill profile of PC
`(Fig. 4).
`
`kcias ‘"“ "C0 +
`
`'i" k0}i‘l0H":l
`
`
`
`l3). 2
`
`I‘
`Fol‘ PC hydrolysis ldnetlcts, plots of km against
`[H “} and [0H‘} yield straight lines with the slopes
`equal to kfi and 52:05, respectively, and the inter»
`cepts equal to leg. ll’ ken lOH"} ~<< kg {EV} then
`the catalytic effect. of the Oil‘ ions can he ne-
`glected. Conversely,
`if kt” [Hf] << istgfl
`{{)3S:l"']
`than the catalytic effect of the EV ions can be
`neg;lec:t.ed.
`In buffer solutions, the buffer eonee:ntrat.ion is
`the simi of the concentrations of the biaffer species.
`
`k
`000003
`
`000003
`
`

`
`ES
`
`4
`
`-2.0
`
`3.
`
`'\e
`
`/1’
`/4
`
`‘T oz 4-§
`
`5.
`
`X 3
`
`
`
`~13
`
`kcbsX16)
`
`10
`
`U7 .
`
`.
`
`T“"‘T““““.“““‘i“““"!""""‘]
`ELSE
`0.03
`0.10
`0.04
`0.02
`utter eencentratitm (M)
`
`‘Q
`
`-
`
`Fig. 5. Effect of the buffer concentration cf the degradation of
`PC
`citrate buffer at 72°C. (Q, pH 5.0; 0. pH 5.5; E), pH
`
`6.0)‘ The lines were Calculated by lineal’ regx‘
`"in analysis’
`Each point represents the mean of at least
`., pafate deter
`min-ations.
`
`
`
`ikcif“ "' k(3i7"'> find (kTris+ ‘ 'i\’5';'g:-.l
`(‘I6/Kc" ink!-1A0);
`in acetate, citrate and Tris buffeifs, respectively
`(Figure 6). The calculated rate constants for each
`buffer speczties are listed in ‘Table 1.
`The temperatture dependence of the hwlrolysis
`of PC was investigated in the pH range of 4--«9 anal
`temperatme range of 40--—82,° C in the buffer solu~
`tions at the ionic strength of 0.068 (Fig.7). The
`
`0.10
`
`
`
`0.08 ‘
`
`slope»
`
`Fig. 6. The relationsliip between the mute fraction of the ions
`in the buffer solution and the siope
`the buffer eem:.erttra—
`tion~ kc-[,3 curves. (L‘J,Vin acetate buffer; Q, ti1.citt'at,e litxfferl 3:
`in Tris buffer) Thu": lines were ::a‘tc1ilated by linear regressifin
`analysis.
`
`Effect of pH. on the degradation of PC at 7'2“ C (buffer
`Fig.
`coiicetitration = 0). The lines were calculated '-by linear l‘v'Sg1'CS~
`sion analysis.
`
`The concentration of each buffer component is
`equal to the mole fraction (1 f ) of the buffer com~
`pertentim'uit,:iplied by the buffer concentration
`Therefore,
`2 can be ta'ansfa:>i'med into the
`fe‘ticwi.tig equations for acetate (it-1A.c,/‘ Ac“, Eqn,
`4), citrate (Ci3""/Ciz”,
`5) and Tris ("i‘ris+/
`Tris, Eqn. 6) buffer systems (Beijtien, 1986).
`
`kc‘-ns 2 /(SIDS + {{k‘A::’ “‘ knAc)f.
`
`”‘HAe-,i
`
`{buffer}
`
`(4)
`
`k-(tbs
`
`$135+ {(k€Z1i3"""kCi"‘)fCi3" +~kci2‘}
`
`[buffer]
`
`‘
`
`knlts 3 k<';t,~.e. + {(k'r.«is+
`
`k’i‘ris>‘f'=.*ras*' "F "5't‘:is
`
`{buffer}
`
`Plots of icabs against the buffer COI1(i€I.’.\lt‘EitlO1'.‘t
`yield st.raig,h.tt ‘times with slopes equal to UCAC--“
`
`’i‘:1~i;’&c) 215- "F i‘5it.2\~.~,.~ (kc: *" "'5cz*‘~‘) fC13” + kct’-“ 33353
`(ic~ms+ —,it'.T,iS) f-mS++ zkms in acetate, citrate and
`Tris buffers, respectively. Examples are shown in
`Fig. 5 for hyd.t'olysis of PC in citrate buffer at
`’/'27
`These plots were clrawn for 3 pH values in
`acetate buffet‘, 4 pH values in citrate buffer and 5
`pH values in Tris buffer. Plots
`cal«;ru.late<l slopes
`against the mole f;racti<m of the Ac“, Ci” and
`’Tri.s"" ions yield straight lines with intercepts equal
`to kg”, km».
`am": kms and slopes equal
`to
`
`000004
`
`000004
`
`

`
`TABLE 1;
`
`S'‘e<:ond-order rate constants far cata¢’ySea' degr.-1a'at1'orr of PC at
`72 ° C
`
`TABLE 2
`
`ezzzropies of
`(Ea), frequency factors (A),
`Activ./ltian erzergies
`activation (./33*.) am’! probability fa::tm'.v (P) for degradatfart of
`PC as a function of pH (ta = C’,068}
`
`
`
`1.5 - 1-3
`5.5-1{)"‘5
`6.7-nré
`(».4=10"5
`4.5 »10"“
`6.9-10"‘
`5.7!-in-6
`5.2-1:34
`
`fatty acids as straight pseucio»i’i1'st~erdervpiots were
`
`4.0
`5.0 *
`5.0 **
`6.0
`as
`7.4
`8.0
`9.0
`
`
`
`130.4
`2.7«1o—
`-31.5
`9.440‘
`99.0
`1.2404
`—99.5
`1.140“
`64.1
`73.7 V 103
`12-10“ was
`1.9. 104
`—99.o
`9.0403
`101.1
`
`St;-indard deviations were typically
`"“* Citrate buffer.
`
`5%;
`
`"‘ Acetate buffer.
`
`was anaiysecl in more detail to find out whether
`probabiiity factor (P) djiffexreci’ s:ig:eifieant1y from
`hydrolysis kinetics in a homogeneous, one-phase
`system. The entropy of actwation, AS *, was
`eaieiiiated at 25 ° C from the frequency factor (A)
`obtained from A1:rlieni.us equation by using Eqn. 8
`and the probabiiity facter was calculated from the
`A AS“" values by using F.qo.. 9 (jiviztrtizi et 221., 1983):
`
`[.'xt'* :R(1n A -111 icl”/I4.)
`
`and
`
`-E’i$€?L’:LAS'\"’:/12
`
`where k is the ‘Boltzmann constant anti
`
`I:
`
`is
`
`Pianeics constant. The csbtaineci Arrhenius param~
`eters are iisted in Table 2.
`
`Biscussiczi
`
`Hydrolysis of PC in liposome vesicles first re-
`sults in 1yso~PC and fatty acid fox‘mat:ion, Further
`degratlmion to smaller fragments occurs in a later
`stage (Kenips and Cromnielin, 1988), The initiai
`degradation products" are likely to interact with
`the ‘biiayer. It was not
`izivestigateetl how much
`iyso~PC and fatty acids can be taken up by the
`liposomes before the Vesicles disintegrate. A53»
`parentiy, the degradat.io11 kinetics of PC were not
`affected by the presence 01": iyso-compounds or
`
`to
`kn
`W,
`kw
`MAG
`/’.;Ci—2
`kci»-2
`](Td5+
`k’E‘ris
`
`as-iio*““_—i»1K7«1e‘4
`:::».x.1c-2 $9.1-102
`e.2's,1c-2
`-_+_—.e.4«1-'7-fl
`2.o=10“f ;+;1.4»1ejf
`-6.0-1.oj-+4.4-i
`
`'
`
`'5
`
`1.1‘ u
`
`4'2"
`
`Coxistants expressed in ix/i""‘h"1‘, except for the f st»order
`rate constant Ito which
`reported in 11" ‘.
`
`temperature on the deeomposiititm is
`effect of
`expressed by the Arrhenius equation (7):
`
`In km = in A — Ea/RT
`
`where A is the frequency factor, Ea is the a.c:.iva—
`tion energy, R is the gas constant and T is the
`absolute temperature.
`Liposonie dispersions are two-«pliase systems: a
`water phase and a bilayer phase can be discerned.
`The frequency factor for the iiposome dispemions
`
` (_.. -2
`
`~:a
`
`EU}S}Q
`
`.56
`U?
`
`0~
`
`*4
`
`2.8
`
`-
`
`2.9
`
`I
`3.0
`
`I
`3.1
`
`3:2
`
`1/1" x W3
`
`Fig.‘ '7. Effect of the temperature on the hysir()]ysi.<s of PC
`(gt = 0068). (8, pH 4.0; 0, pH 6,5; , pH 8.0). The lines were
`caicuiateti by linear regression analysis. Each point represents
`the meam, of at least two separate deterrninations.
`
`000005
`
`000005
`
`

`
`the pH
`that
`obtained. it should he inentionecl
`tends to drop during storage unless proper buffers
`are used,
`
`During storage of the dispersions under accel-
`erateci conditions a filrni was formed on the glass
`wall. This film could he reciispersed by vortexi.:r1.g.
`The PC concenti'a.ti.on, before and after vortexing,
`was similar. Therefore,
`the film was mainly com-
`posed of no:n~l’C niaterial. Degradation kinetics of
`PC were not at‘t'e-steal by this third phase former»
`tion as no deviations from a straight line were
`observed.
`
`General acidnbase catalysis was observed by the
`effect of acetate, citrate and Tris ions‘ This is
`imlicated by the slopes of they plri hydroljysis rate
`constant curves (slopes are equal to 0.4 for acid
`and ‘ease catalysis hy'drolysi.s;
`5) and the
`linear relationship between observed rate constant
`and buffer cottcentration. From Table 1 an in-
`
`creasing catalytic effect can he read with increas-
`ing anionic charge of the citrate ions. Negative
`catalysis {a protective effect) was Ol1“iSt1t“V6Ci
`tot‘
`acetic acid as i..odicat.ed by the minus sign in Table
`l. Similar protective effects of the acetic acid on
`the .~:tal>:ility of cyciosidonune have been described
`previously (Carrey, 198?). No attempt
`made
`to disclose the niechanisin of cataiysis of protec»
`tion.
`
`for the hydrolysis
`The validity of Arrhenius
`of PC was investigated in the temperature range
`‘tzetween 40 and 82° C £>egrad.ation kinetics could
`be adequately described by Eqn. 7. Fnzshiaer et al.
`(1982) showed for distearoyiphosphatidylcholine
`(DSPC) iiposonies a break in the plot of in kabs vs
`1/T around the transition teniperatnre (T9) of
`DSl’C.: 55°C (Fgtokjaer et al..,
`i982). As men-
`tioned in the experiinental section, soylzean PC. is
`niainty composed of unsaturated fatty acids. The
`I’: of this type of phosphoiipids is about -15“ C.
`{Szoka and Pajpaliadjopoiilos, 10980).
`'l“liis tempen
`ature range is above the transition tetnperat.ure
`-(IQ) of soybean phosphatidyldnoline. Therefore,
`no hireak in the Art°henins plot was ol>sez'ved (Fig,
`7).
`
`Further analysis of the frequency factor in the
`Arrhenius equation ‘showed that probability fats»
`tors of the hydrolysis kinetics of PC in liposonie
`dispersions did not significantly differ front l<inet~
`
`ics in homogeneous one-phase aqueous solutions
`(Frost and l’e-arson, 1961). Apparently the ester
`bonds (Fig. 1) are freely accessible for hys:h'olys:is.
`From this study it can he decided that rniniinutn
`degradation kinetics occur at pH 6.5 and that low
`‘buffer concentrations are favourable;
`values
`at pH 6.5 and at zero bn.ffer concentration of 296
`days at 6 ° C can be calculated. A lyso-PC content
`in PC vesicles of 10% (molar basis) increased the
`taerrneahility diarnaticaily (lnoue and Kitagawa,
`1t97'4; Kibat and Stricker,
`'.t,985j), Work is in pro-
`gress to investigate the effect of charge inducing
`agents and cholesterol on deg;t’adation t<:ii:ietics and
`biiayer pernieahility.
`
`References
`
`‘
`
`.l.H., Chernieal Stability: and Mz?z‘amycirz and Anthra-»
`Beijnen,
`cyciine Aniiizeopiastic Drugs, PhD. Thesis, Universzity of
`Utrecht, Utrecht, i986.
`C;-mey, C.F., Solution stability of ciclosidomine. J. Pharm.
`Sczl, 76 (1987) 393-~-397.
`in (.'.1'rg'anic /irzazlyiical
`Connors,
`l<L.A,, Rea<:titm J‘z{ecka:'si.vr.-is
`Chemistry, Wiley, New York, 1. 73, pp. 41»-101.
`Fiske, CH. and Subbarow, Y., The colorimetric determination
`of phosphorus, J. Biol. Chem.., 66 (1925) 375-~-400.
`Frost, AA. and Pearson, R.G., Kinetics and Mecr'umism, Wi-
`ley, New Y'ozk_. 1961, pp. 100--‘101.
`0
`Frekjaer, S.. Hjorth, EL. and Warts. 0., Stability and stora§>;".
`of liposomest In Bundgsard, H.,
`Hanseng A. and
`Kofod,
`ii.
`(Eds), Opiirnization of Drug Delivery,
`Mtinksgaard, Copenhagen, 1982, pp“ 384—40=‘i.
`Inoue, K. and Kitagawa, T., Effect of exogenous lysoiecithin
`on lip-osomal membranes: its reh"-.t.ion to membrane fluidity.
`Biaclztm. Biophys. Acta, 363 (1974) 361»-372.
`Kemps, .l.M.A. and Cronunelln, l')..l'.A., Chemische stabiliteit
`van fosfolipiden in farniaceutische preparaten. I. Hydrolysc
`van fosfolipiden, in watevig milieu. Phrnrrz. Wee.I’«:.5i.,
`‘:23
`(1988) 355»363.
`‘
`Kibat. PG. and Strieker, H, Lagerungsstabilitat Von Lipo-
`sorndispersionen
`Sojaleeitliinen. I’i1arm.
`Im2’., 48 (1985)
`’ll84—'l189.
`
`3’. and Cammarata, A, Phy "£7511
`Martin, A.‘N., Swa1'b1'ick,
`Pharmacy, Lea & Febiger, Philadelphia, 1983, pp. 371»-3'74.
`‘Nasner, A. and Kraus, L, Quantitative Bestimrnung V011
`Pliophaticiylcholin niit Hilfe der HEEL‘; Fette Seiiflfl
`An,s'tr'ic}2mitte!. 83 (1981) 70w73.
`Sine-ten,
`and Shohet, S,l'£.Jt9 Pei'meahiiity changes indu~'.‘-r:s'3
`by p6l‘€)XidatiO11
`in liposomes prepared from humafi
`erythrocyte iipids, _I...'?,r2i(i Res., 15 (l 9'74) 2‘73~28G,
`Szoka. F. and Pap-ahedjopoulcs, 1)., Comparative prope1’i'i55
`and methods of preparation of lipid vesieies (liposomeS)-
`Anrzza, Rev. Biop.hys. Bz'oeng., 9 (1980) 46'.’--508.
`
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