Note: Descriptions are shown in the official language in which they were submitted.
PROCESS FOR THE IN-SITt~ PREPARATION OF LIPOSOMES AND
THEIR USE AS A 5USTAINED-RELEASE AEROSOL DELIVERY SYSI'EM
This invention relates to a process for the
preparation of liposomes and in particular to a simple,
rapid method of forming liposomes using a fluoro-
chlorocarbon propellent-based pressurised aerosol
device. The liposomes formed following delivery by the
10 pressurised aerosol device provide sustained-release of
medicament.
Liposomes are artificial spherules of
phospholipids composed of a series o concentric layers
alternated with aqueous compartments. Bangham et al
15 (J. Mol. Biol. 13 ~1) 238-59, 1965) fir~t described the
preparation of such multi-lamellar lipid vesicles and
since then a wide variety of methods have been reported
for the preparation of synthetic liposomes. Liposomes
were originally used as artificial models to study the
20 properties of biological membranes. However, the
practical potential of liposomes is that they are
capable of englobing or entrapping a wide range of
substances, e.g. drugs, to protect them from
degradation and/or to target them toward specific
25 organs. DrUgs and other molecules may be encapsulated
by two process~es. Hydrophilic species are then
entrapped within the aqueous phase whereas lipophilic
moieties associate with the concentric phospholipid
bilayers. Th~s, there as considerable interest in
30 developing commercially effective processes for the
production of liposomes.
Numerous methods have been proposed for the
production of liposomes. Liposomes have been prepared
using a method based on the evaporation of volatile
solvent from an ether/lipid/water dispersion. ~y
ultrasonication, a water in lipophilic solvent
dispersion was produced, from which the volatile
solvent was removed by either evaporation under reduced
pressure or by bubbling nitrogen through the mixture.
The small unilamellar vesicles produced by
10 ultrasonication possessed only a small aqueous
compartment ~25 nm diameter) and showed a low
e~ficiency in capturing biologically active mol~ecules.
A modif ied method was based on the removal of organic
solvent under reduced pressure to produce a lipid gel
15 which, on addition of excess aqueous phase, formed
vesicles of large volume capable of retaining
macromolecules with a high capture efficiency. Similar
large unilalaellar vesicles were also prepared by
utilising a calcium-induced structural change in the
20 lipid vesicles. However, this technique was limited to
a single pho~pholipid (phosphatidyl serine) and again
had a relatively low efficiency of encapsulation.
Injection of a solution of a lipid dissolved in
an organic phase such an ethanol or ether has been used
25 to produce unilamellar vesicles, but again low
encapsulation efficiencies were observed.
Centrifugation of a lipid/water/ether emulsion into an
aqueous phase has been suggested as a method for
preparing lipid vesicles, but the need for high speed
30 centrifugation is a disadvantage, Also a large amount
of the lipid/aqueous emulsion becsmes trapped at the
interface resulting in a low percentaye of material
entrapment. The entrapment of certain pharmaceuticals
~:25~
-- 3
in liquid vesicles has been achiev~d by freezing
or freeze-drying an aqueous phospholipid dispersion.
The complexity of the known processes for pre-
paring liposomes containing entrapped molecules, the sep-
aration of such liposomes from non-entrapped material
and the high cost bo-th in terms of time and money has
prevented effective commercial production of liposomes.
Furthermore serious stability problems are encountered
with the products of some processes and such products
must either be prepared immediately before use, which
is not always convenien-t, or must be stored under special
conditions, e.g. low temperature under ni-trogen.
Thus, -there ;s a demand Eor a simple efEective
method for the production oE liposomes and Eor a pack
1~ Eor carrylng out same.
Cn meet:ing this derrlancl, ttle pr~sent :Lnvent:Loll
provides a pack Eor use in preparing an aerosol which
comprises a single chamber containing a solution of sub-
stantially pure phospholipid and a therapeutically active
substance dissolved in a propellent material, the molar
ratio of phospholipid to the therapeu-tically active sub-
stance being greater than 1:1, the pack including an ar-
rangement for dispensing said solu-tion as a spray under
pressure developed by the propellent ma-terial.
This pack rnay be usecl in a process for the prep-
aration of liposomes which comprises spraying tnicro-f:ine
droplets of substan-tially pure phospholipid in a volatile
liquid carrier to impinge either upon or below an aqueous
surface thereby forming liposomes.
Thus, -the micro-fine droplets are generated
using a propellent-based pressurised aerosol delivery
system, the liquid carrier comprising the aerosol pro-
pellent which is conveniently a fluorochlorocarbon. This
system can be utilised to deliver drugs to the mucosal
i. ,~`
~568a!~
~ 3a -
surfaces within the lung and achieve sustained-release
from the liposomes which are produced in-s:itu.
The above process provides a simple rapid method
for producing liposomes. Discre-te micro-fine drople-ts,
generally having a diameter in the range 0.5 to 50 micron,
,
liquid carrier are sprayed onto or below an aqueous
surface. The liquid carrier evaporates and the contact
of the resulting solid phospholipid with the water
surface results in the spontaneous formation of
liposomes.
The process may be used to prepare entrapped
molecules, e.g. therapeutically active molecules within
the liposomes by simple admixture with the desired
compound with the phospholipid and lipid carrier. The
10 drug molecules must be dissolved in the formulation and
in the case of a propellent-based aerosol delivery
system, the drug may be dissolved either in the
propellent alone or in the presence oE a small
proportion of a co-solvent, e.g. alcoholsl particularly
15 ethanol. An alternative method of increasing the
solubility o~ hydrophilic drug molecules in
fluorocarbon propellents is to use an excipient which
forms an ion-pair with the drug molecule. Examples of
such excipients include dicetyl phosphate, benzalkonium
20 chloride, cetyl pyridinium chloride, etc.
The above-described process may be used to
entrap any drug molecule which may be solubilised in
the composition to entrap drug molecules within
liposomes. The entrapped drug molecules are gradually
25 Leleased from liposomes and accordingly these may be
used as a method of obtaining sustained release of drug
molecules. The rate of release of the drug molecule is
dependent UpOII the molecule itself, the amount of drug
entrapped and the particular formulation utilised.
The use of a propellent-based pressurised
aerosol delivery system allows preparation of the
liposomes spontaneously during use or immediately prior
to use thereby avoiding problems of poor stability on
.~
.j ,~ .
~5~
=5~
prolonged storage. Furthermore, the aerosol system is
capable of use in inhalation therapy to allow in situ
formation of liposomes entrapping therapeutically
active molecules on the moist surfaces of the lungs.
The phospholipids used in the invention may be
selected from a wide range including:
phosphatidylcholine (lecithin~ lPC)
phosphatidylglycerol (PG)
phosphatidylserine (PS)
phosphatidic acid (PA)
phosphatidylinositol lPI)
phosphatidylethanolamine (PE)
dipalmitoylphosphatidylglycerol ~DPPG) and
diacylphosphatidylcholine ~DAPC3.
one or more of the ~ollowing non-phospholipid
excipients may be included as Eonmulation aids to increase the stability
of the phospholipid bilayer or the controlthe surfac~ charge:
dicetylphosphate (DCP)
stearylamine lSA)
sphingomyelin (SM)
cholesterol (C)
distearyldimethyl ammonium chloride.
The phosp~ol~ids ~ust be substantially pure in
order to ensure uniform liposome formation. Preferably
25 the phospholipid is at least 80~ pure, more preferably
9o~ to 100% pure. A preferred phospholipid is purified
egg phosphatidylcholine (lecithin).
The volatile liquid carrier is preferably a
solvent for the phospholipid. Convenient carriers are
30 aerosol propellents, in particular fluorochlorocarbon
propellents, e.g. Propellent 11 ltrichloromono-
fluoromethane), Propellent 12 (dichlorodifluoromethane)
and Propellent 114 [dichlorotetrafluoroethane~.
~256~
-- 6
Suitable forn1ulatlolls for use with a pressurised aerosol
delivery system comprise 9~ to 99.9~ of one or
rnore fluorochlorocarbon propellents and O.l to 10% by
weight of one or more phospholipids plus formulation aids
if required.
The use oE phospholipids in fluorochlorocarbon-
based aerosol formulations is known and is disclosed in
British patent speciEication no. 2,00l,334 and German
Offenlegungsschrift no. 28 31 419. Aerosols from aqueous
phospholipid solutions are also known and are disclosed
in United States patent specification nos. 3,594,476 and
3,715,432. 11owever, these known Eormulations have not
been used to prepare liposomes and the phospholipid has
been present in the formulations as a surfactant in very
s111a]L a11~ounts and in impure ~orm, e.g. lS~ purity, to
aic1 t11e prepa~ati.on oE a clruy disp~rsi.or1, to stab:Llise
tl~ ac1u~c)us-, droplets agai11st qvaporal::ior1 or Eor therc
pc~u 1: :iC r~asolls .
In preEerrec1 e111bocli111er1ts oE tl1e lnvention, the
molar ratio of phospholipid to the tilerapeutically active
substance is at least 5:1. In a most preferred pack,
the molar ratio oE phospt1olipid to the therapeutically
active substance is in the range of lO:l to 20:1. The
pack may include a co-solvent Eor the therapeutically
active suhstance. Preferably, the solvent is anhydrous.
The invention will now be :illustrated wi-th ref-
erence to the following examples.-
~
~2568~
- 6 a -
In the accompanying drawings:
Fig. 1 is a representation of an aerosol sampling
device for use in providing humid conditions -to Eire an aero~
sol dose;
Fig. 2 is a miscible/immiscible phase diagram of
5~ w/w egg PC in Propellent 11, Propellent 12, Propellen-t
114;
Fig. 3 is a curve showing the effect of time on the
particle size of particles generated from aerosol containing
1~ w/w pure egg PC;
Fig. 4 is an electron micrograph of a receptor fluid
sample;
Fig. 5 is a curve showing the partition coefficient
against for a drug added to the liquid phase or aqueous phase
7 5 of a spray;
Fig. 6 ls a curve showing the entrapment oE sa:lbuta-
mol in DCP/PC liposormes against time;
Fig. 7 is a curve showing the partition coeEf:icient
for salbutarnol in liposorne (DCP/PC) against the molar rati.o
DCP/salbu-tarnol;
Fig. 8 is a representation of a multi-stage liquid
impinger for use to characterize an aerosol cloud according
to its par-ticle size distribution;
Fig. 9 is a curve showing the actual quantities of
PC delivered from difEerent pressure pachs containing egg
PC in blends;
Yig. 10 and 11 are cliagramms showing th~ MLI data
for a formulation emi-tted from a pressurized pack with diffe-
rent Pll/P12 ethanol blends;
Fig. 12 is a curve giving the percentage dug retention
against time for formulations containing differen-t ~ DCPC;
and
Fig. 13 is a curve showing the release of steroid
ester from diluted conven-tional and aerosolised liposome
preparations.
~S Ei~
~7~
Example 1
In vitro evidence to ~how the in-situ production o~
liposomes from a fluorozhlorocarbon propellent-based
aerosol device
.
Purified egg phosphatidyl choline (PC) was used
as the model phospholipid. Crude Pgg lecithin (BDH
Chemicals, England) containing 90~ egg PC was used as
the ~tarting material. The egg PC was purified and
10 recrystallised as described by Bangham et al, Methods
in Membrane Biology, editor E~D. Korn, 1~ page 68,
Plenum Press 1974, and was stored under aceton~ at
4C. The recrystallised egg PC was shown to be
chromatographically pure using a solvent ~ystem of
15 chloroform/methanol/water (14/6/1).
A pre-requisite for phospholipids to orientate
into a liposomal configuration is the presence of an
aqueous environment. An aerosol sampling device was
therefore designed to provide humid conditions into
20 which the aeeosol dose could be ~ired and is
illustrated in Figure 1 of the accompanying drawings.
The apparatus consisted of a 1 litre ~iltering flask 1,
containing a beaker partly filled with a known volume
of aqueous receptor fluicl 10. An intake tube 2
25 ~rotruded through its neck with one end 3 located just
above the receptor fluid surface and the other end 4
fitted with a medicinal aerosol oral adaptor 5 and
aerosol device 6. A means of sampling the receptor
flui~ was included.
The receptor fluid was glass distilled water
(pH 5.8) filtered through a 0.2 micron membrane
filter. To attain conditions within the flask of a
high relative humidity and 37C the flask was immersed
~:25iE;8~
up to the height of the side arm in a water bath 7
maintained at 37Co To ensure delivery of the ~ajority
of the aerosolised dose into the receptor fluid, air
flow through the apparatus was achievled via a tube ~
connec~ed to a Yacuum pump (Speedivac). A flow ra~e of
50 litre/min was monitored by a flow meter (Gap Ltd.).
A sampling syringe 9 was provided for obtaining samples
of liposome. ~o permit air flow, the aerosol adaptor
had an orifice 11 at the rear. The assembled apparatus
10 was positioned in a pre-equilibrated laminar air flow
cabinet to avoid contamination of the receptor fluid
with airborne particles.
Visual observations of a number of formulations
containing 5% w/w egg PC w~s made to allow the
15 construction of a miscible/immiscible phase diagram of
5~ w/w egg PC in Propellent llJPropellent 12/Propellent
114 blends at room temperature shown in Figure 2 of the
accompanying drawings. This triangular co-ordinate
graph was used to formulate two phase aerosols of a
20 specific vapour pressure containing up to 5% w/w egg PC
by selecting a propellent blend above the miscible/
immiscible interphase situated on the specific vapour
pressure contour.
Aerosols containing 1% w/w egg PC at vapour
25 ~ressures of 3.43 x 105 NJm2 and 4.79 x 105 N~m2 i50
and 70 psiaj (21C) were examined. 100 ml of receptor
fluid was dispensed into the flask and the apparatus
assembled. Sufficient time was allowed for
equilibration. The aerosol unit was shaken, primed and
30 placed in the oral adaptor. Air was drawn through the
apparatus and the valve actuated at 10 second intervals
for a previously determined number o times. Following
the final actuation, the air flow was shut off and
~2~;6~
9 ~
~amples of the receptor fluid were withdrawn and
analysed using photon correlation spectroscopy (~alvern
Model RR144, Malvern Instruments Ltd., Malvern, United
Kingdom)O ~he size of particles within the receptor
fluid was followed with time.
~ igure 3 of the ~ccompanying drawings shows the
effect of time on the particle size of particles
generated from aerosols containin~ 1% w~w pure egg PC
and possessing vapour pressures of 3.43 x 105 N/m~ and
10 4.79 x 105 N/m2 ~50 or 70 psia) at 21C, each point
representing the mean of three determinations with
~tandard error bars. ~he initial particle size was
dependent on vapour pressure; 882 nm for 3.43 x 105
N/m~ l50 psia) and 560 nm for 4.79 x 105 N/m2 ~70
15 psia). In each case the particle size decreased with
time equilibrating at approximately 90 to 100 minutes
to a size of 250 to 290 nm.
The 250 nm particles produced after loss of
propellent by evaporation were of similar particle size
~ and structural characteristics to the multi-lamellar
vesicles produced by a variety of methods of the prior
art.
Confirmation oE the_formation of liposomes using
25 electron microscoPY-
The formation of liposomes was confirmed byexamination of the receptor fluid samples after
negative staining with ammonia molybdate using
30 transmission electron microscopy. Figure 4 of the
accompanying drawings is an electron micrograph and
reveals clusters of aggregated multilamellar vesicles
ranging in si~e from 150 to 400 nm but collectively in
aggregates ~elow 1 micron in size.
'10=
Example 2
The partitionins sf a drug into li~osome~
Salbutamol - a hydrophilic co~pound
Using salbutamol hemisulphate and salbutamol
base as drug compounds, the partitioning of the drug
molecules into multi-lamellar vesicles (MLVs) produced
extemporaneously and produced spontaneously using an
10 aerosol delivery device was studied.
Extempoeaneous ~reparation of liposomes
Method3 for preparing of MLVs have been
extensively published ~e.g~ Juliano, R.L., Stamp, D.,
15 Biochem. Pharmacol. 27:21, 1978). An amount of pure PC
was weighed into a 50 ml flask and dissolved in a small
quantity of absolute ethanol. The organic solvent was
eotary evaporated at 40C (with the inclusion of small
volumes of acetone to encourage removal) to leave a
20 thin lipid film on the walls of the round bottom
flask. The so-called ~dry~ film was flushed with a jet
of nitrogen to ensure complete removal of the solvent.
The required amount ~10 ml) of aqueous phase was added
and the film allowed to hydrate by gently shaking at
25 '7C to form MLVs. The aqueous phase used was either
(a) 0.9~ w/v saline adjusted to pH 7.4 with O.lM sodium
hydroxide or (b~ physiologically iso-osmotic, phosphate
buffered saline ~P~S) at p~ 7.4. Salbutamol
hemisulphate being practically insoluble in ethanol was
30 added to the aqueous phase. Salbutamol base was
sufficiently soluble in ethanol to permit incorporation
into the lipid film prior to hydration. The final
concentration of lipid was 10 mg/ml and drug
~2S~8~l
'11=
1 mg/ml~ The liposome~drug ~uspensions were shaken at
37C for sufficient time to permit equilibration before
separation of liposo~es by centrifugation and assay for
drug content in the supernatent by ~PLC.
The enhancement_of the ~oportion of salbutamol
entr~pped wi~h the liposomes.
When salbutamol hemisulphate was added to the
aqueous phase during the extemporaneous manufacture of
10 MLVs, the drug partitioned into PC liposomes suspended
in iso-osmotic PBS, pH 7.4 to give an entrapment of
0.55 mg/mg percent with an apparent partition'
coefficient ~Kapp) of 5.83. The profiles for change of
partition coefficient with time with salbutamol base in
15 liposomes for drug added to the lipid phase or the
a~ueous phase are shown in Figure 5 of the accompanying
drawings and reveal that the degree of initial
liposomal association was greatest when the drug was
added to the lipid phase, although a rapid decline
20 occurred to equilibeium at approximately 3 hours. The
low entrapment efficiencies were of the order expected
for a hydrophillc species, fully ionised at pH 7.4 at 37C (the
pKa of the basic moiety of salbutamol is 10.3,
Newton D.W., Kluza, R.B., Drug Intell. Clin. Pharm.,
2:546, 1978). The insignificant effect of chol~sterol
on salbutamol partitioning (Figure 5~ suggested that
the salbutamol partitioned into the agueous channels
within the liposomes and was unassociated with the
lipid bilayer.
3D Hydrophobic species generally partition into
liposomes to a greater extent than hydrophilic
species. Formation of an ion-pair complex with a
lipophilic moiety represented by a method of conferring
~:256~30~
~12~
hydrophobicity to the salbutamol molecule. Dicetyl
pho~phate (DCP) incorporates into lecithin bilayers and
is routinely u~ed at concentrations below 10 ~ole ~ to
confer a negative charge to liposomes (Szoka, F.,
5 Papahad jop~ulos, D., Am. Rev. Biophys. Bioeng~ g:467,
1980). The effect of inclusion of DCP at 10, 20 and 30
mole ~ at 37C on the liposomal uptakes of ~albutamol
due to formation of a lipophilic ion-pair complex is
illustrated in Figures 6 and 7. Figure 6 represents a
10 plot of entrapment of salbutamol ~mg/mg %) in DCP/PC
liposomes at 37C against time for varying DCP
concentrations. Figure 7 represents a plot of the
partition coefPicient for salbutamol in liposome
~CP/PC) against molar ratio DCP/salbutamol. The
15 inclusion of 30% DCP caused a 175~ increase in
entrapment from 1.4 to 2.45 mg/mg %.
The in-situ preparation of liposomes usin~ a
Pressurised aerosol device.
_
The use of a more sophisticated multi-stage
liquid impinger (MLI) has been described (Bell, J.H.,
Brown, X., Glasby, J., J. Pharm. Pharmacol., 25~32P,
1973) to characterise an aerosol cloud according to its
particle size distribution. Figure 8 of the
25 accompanying drawings shows a multi-stage liquid
impinger which comprises a glass container 80 divided
into four sections (Stages 1 to 4) by glass sepaeation
plates 82, each section being in communication with
adjacent sections via conduits 84. The pressurised
30 aerosol container 86 is positioned at the throat 88 of
the apparatus. Glass collection plates 89 are positioned
on separation plate. A fixed volume /lC ml/ of the pre-
filtered /0.05 micrGn/ water was added to each stage to
686~l
- 13 -
insure that a moist scintered glass surface was presented
to the ~ir flowing through conduits 84. An outlet 90 is
provided in Stage 4 for communication, via a filter 92,
to a pump. In practice, air is drawn through the apparatus
by the pump so that 60 litres/minute enters the throat 88.
The MLI was calibrated in terms of effective
cut-off diameter by monitoring an aerosol cloud of methylene
blue particles produced from a 0.5~ ethanolic solution using
a spinning disc aerosol generator. The particles were
directed either into a calibrated 8-stage impactor (Andersen
Samplers, Inc., Georgia, U.S.A.) or into the MLI by an air-
stream generated by a vacuum situated downstream of the
sampling device. Data from MLI measurements made on pres-
surised aerosols at a range of vapour pressure and E'C
content are shown in the following Table 1.
5~8~l
~14=
Table 1
Depo~ition of aerosol emittec7 from pressurised
packs containing egg PC at 3.43 or 4.79 x 105 N/m2 ~50
or 70 psia) at 21~C in the multistage liquid impinger
apparatus.
~ach result ~expressed at a ~ retention of the
total aerosol) is a mean of three determinations.
Effective cut off diameter was assumed as 20
micron ~or the glass throat (Hallworth, G.W.,
Andrews, ~., J. Pharm. Pharmacol., 28:898, 1976) and
10 determined as 10.47 micron for Stage 1, 5.51 mi.cron for
Stage 2, 3.59 micron for Stage 3 and 1.25 micron ~or
Stage 4,
. _. __. _ .. _ . _.. _....... __.__ . ___ A_
V p 1 4.79 4 79 4-7~ 3-4
Egg PC % w/v 0.5 1 2 2 5
Adaptor 22.55 21.11 28.23 15.73 16.36
Throat 46.45 48.66 50.09 71.02 75.46
Stage 1 1.34 2.10 1.50 2.14 1.19
~tage 2 5 79 5.91 3.85 4.20 2.04
Stage 3 11.17 8.44 5.88 4.09 2.22
Stage 4 10.69 12.54 9.82 3.26 2.58
Filter 2.02 1.14 0.62 0.11 0.17
~ . . .
1) V.P. denotes the vapour pressure of the propellent
blend at 21C x 105 N/m2.
~s~
G15=
In terms of potential for liposome formulation,
the actual quantities of PC delivered within the
respirable range ( ~ 5 micron) from pressure pack~
oontaining eg~ PC in blends at 4.79 x 105 N/m2 at 21C
5 is reported in Figure 9 of the accompanying drawings.
An optimised formula for maximum delivery of PC In the
respirable range would contain 2~ w/w PC.
Two formulations, one containing DCP to form the
ion-pair with salbutamol (F2) and one containing no DCP
10 ~Pl) were prepared and eYaluated on the MLI.
Formulations Fl and F2 are tabulated below:
Content in gJ10 ml fill volume
E'l ~gl F2 ~)
15 Salbutamol 0.04 0.04
Egg phosph~tidylcholine 0.244 0.1957
Dicetyl phosphate _ 0.0597
~thanol 1.83 1.02
20 TrichloroflUoromethane (Pll)1.92 2.29
Dichlorodifluoromethane ~P12)8~20 9.16
Total 12.23 12.76
Density of liquid blend ~9/~1) 1.22 1.28
The MLI data for formulation F2 emitted from a
pressurised pack with Pll/P12/ethanol blends exhibiting
4.32 x 105 N/m~ (63 psig) at 25C are illustrated
diagramatically in Figure 10. Similar results were
30 obtained for formulation Fl when tested on the MLI.
Equilibrium partitioning data at 37C for
salbutamol in liposoJne formed on Stages 3 and 4 of the
MLI for deposited aerosol ~enerated from formulations Fl
~5 6
~16=
and F2 was evaluated. Negligible entrapment was
appar~nt for Fl, but for Y2 where the lipid component
containing 30 mole ~ DCP, partitioning of salbutamol
into the liposomes was observed. Entrapment
efficiencies were calculated as 2.67 + 0.69 mg~mg ~ for
Stage 3 and 2.65 + 0.78 mg/mg ~ for Stage 4. ~hese are
very similar to those described previously for the in
vitro liposome partitioning experiments using pre-formed
liposomes. It is concluded that the partitioning
10 characteristics observed with aerosol produced liposomes
are comparable with those observed using
extemporaneously prepared liposomes~
Example 3
Hydrocortisone octanoate - a lipophilic compound
Studies with liposomes (multi-lamellar vesicles,
MLVs) prepared by conventional methods illustrated that
20 hydrophobic drugs are incorporated into liposomes to a
higher degree than hydrophilic moieties (Juliano, R.L.,
Stamp. D., Biochem. Pharmacol. 27:21, 1976). For
example, the degree of liposomal incorporation of
steroidal esters can be increased by extending the
25 21~acyl chain length to yield partit.ion coefficients
greatly in favour of the liquid phase ~Shaw, I.H.,
Knight, V.G., Dingle, J.T., Biochem. J. 158:473, 1976;
Arrowsmith, M., Hadgraft, J., ~ellaway, I.W., Int. J.
Pharm., 14:191, 1983). ~ydrocortisone octanoate was
30 selected as an example of such a hydrophobic compound.
Radiolabelled compound (Amersham International, U.K.;
specif ic activity 83 Ci/mmol ) was used to permit
measurement of the drug efflux rate from liposomes
~ 25~
produced in-situ using a pressurised aerosol delivery
system.
Extemporaneous preparation of liposomes
~ixed films of e~g PC (25 mg) and the steroid
ester ~0c10 mg spiked with 0.83 uCi of the tritiatled
esterl were prepared in 50 ml round bottomed flasks
following evaporation under reduced pressure at 40C of
chloroform solutions on a rotary evaporator. To each
10 film, 7.5 ml of sterile 0.9% w/v saline was added and
the film h~drated at 40C to form an MLV stlspension
ln_sita preparation of li~osornes using a ~ressurised
aerosol devlce
L-alpha-phosphatid~lChOline, di[l-14C] palmitoyl
~14C-DPPC] of specific activity 112 mCi/mmol (Amersham
International, U.K.I was used in this Example.
Pressure packs (10 ml) containing 1~ w/w egg PC
(spiked with l.S9 ~Ci 14C-DPPC) and 1 mg of
20 hydrocortisone 21-octanoate (spiked with 4.15 ~Ci of the
tritiated steroid ester) i~ Pll/P12, 23/77 blend were
prepared. Following shaking and priming, the pressure
packs were secured in an inverted position in an oral
adaptor and depressed at 5 s intervals for 40
25 actuations. The emitted aerosol was directed into a
calibrated multistage li~uid impinger as described in
Example 2, each stage containing 10 ml of sterile 0.9~
w~v saline, at 60 litre/min via a glass throat. Aerosol
particles deposited on the glass throat~ actuator and
30 filter were removed with aliquots of ethanol and made to
25, 10 and 10 ml respectively. The liquid on each stage
was transferred to 10 ml Yolumetric flasks and made to
volume. Drug and lipid concentrations were determined
~18=
by liquid scintillation counting. Liposome suspensions
formed on Stages 3 and 4 were transferred to conioa
flasks for studies on drug entrapment and releaseO
All ~amples (1 ml) were incorporated int 10 ml
of cocktail T ~BD~, U.K. ) prior to oounti~ for 10
minutes in an LKB 1217 Rack Beta liquid scintillation
counter. Quench correction was carried out for 14C and
3~ using external standardisation resulting in counting
efficienCieS of 90~ for 14C and 30 to 35~ for 3~.
The partitioning of hydrocortisone 21-octanoate
between egg ~C and water was determined for the
conventional systems and Eor systems generated on Stages
3 and 4 of the MLI Pollowing e~uilibration (4~ h) at
370C by scintillation counting of duplicate aliquo~s of
15 the liposome suspension and of the supernatant obtained
by ultracentrifugation of a 3 ml sample at 195,~00 g for
1 hour.
The pattern of deposition E,~^oduced in an MLI
from a pressurized aerosol contain~ egg phospholipid
20-choline /pc/ and hydrocortisone 21-octanoate is displayed as
a histogram in figure 11 of the;accompanying drawings. No
significant difference occurred between the deposition
of egg PC and hydrocortisone 21-octanoate confirming
that the pack was composed of a homogeneous system. The
25 recoveries of egg PC and steroidal ester were ~ 9o%.
The respirable fraction of each component of the aerosol
cloud ti.e. that deposited on Stages 3 and 4 and filter)
was calculated as 20.91 ~ 2.82% for egg PC and
20.18 ~ 2.67~ for the steroid ester. This compares
30 favourably with the values obtained with traditional
suspension-type inhalation aerosols.
Table 2 reports the partitioning of
hydrocortisone 21-octanoate.
~251E~
1 9
TabIe 2
.. . ...
System Partition Co3efficient IK~
Aerosolised:
Stage 3 4 50
Stage 4 5.68
Conventional
5.50
From Table 2, it is apparent that the egg PC
liposome/water partition coefficient (K) for
conventional and aerosolised systems are
equivalent. This data demonstrates that the liposomes
20 formed in-situ have entrapped drug in a similar manner
to those peoduced extemporaneously. In addition, K was
independent of whether the liposome systems were
generated on Stage 3 or 4 of the impinger.
~56
-20~
Example 4
I~-vitro assessment of sustained-releiase from aqueous
liposome d spersions
s
a) Salbutamol - a hydrophilic compoulld
Because of the small quantities of phospholipid
reaching Stages 3 and 4 of the MLI, it was not possible
to study the efflux of salbutamol from liposomes
10 produce~ using a pressurised aerosol device. Instead,
eff}ux rates on liposomes produced extemporaneously were
studied using the method described in Example 1. The
liposome/drug sllspenslon at equilibrium ~approximately
15 ml~ was filtered :Ln a 400 ml ultrafiltration cell and
15 the liposome residue resuspended to the original volume
in fresh iso-osmotic PBS buffer, pH 7.4, maintained at
37C. Following stirring and transfer to a shaking
bath, efflux was monitored by separation of the aqueous
phase from the liposomes using ultrafiltration (PM10
20 membrane, Diaflo, Amicon, U.K.) and assay for free drug
by ~PLC (the lower limit of sensitivity of the assay was
1 ~g/ml salbutamol). Formulations containing 4 mg/
salbutamol egg PC and varying amounts of DCP were
examined and the results are recorded in Figure 12 which
25 .s a plot of percentage drug retention against time for
formulations containing 10, 20 and 30 mole ~ DCPC at
37C.
The efflux of salbutamol from liposomes was
dependent on DCP concentrations (Figure 12~. Release
3~ was more rapid from liposomes containing 20 mole % DCP
compared to 10 mole % DCP. Similar kinetics were
observed for efflux of salbutamol from liposomes
containing 20 and 30 mole ~ DCP. The estimated half
~2~
~21=
lives for data collected over 0 to 10 hours were:
DCP Content ~mole ~) ~al if~
10 259
20 ~4.1
30 24.6
Based on this data, a fluorocarbon based
pressurised aerosol s~stem containing salbutamol base
and DCP will exhibit significant sustained release
10 characteristics.
b~ HYdrocortisone 21-octa,noate - a 1~ ophilic com~ound
. . _ .
The use of radiolabel,l~d hydrocortisone
21-octanoate permitted the measurement of ~fflux of drug
15 from liposomes prsduced on Stages 3 and 4 of a
multi-stage liquid impinsel using a pressurised aerosol
device. The in-vitro efflux rate test method was as
described in Example 3 for salbutamol. The formulations
used were those used with hydrocortisone 21-octanoate in
20 Example 3. Figure 13 shows release of steroid ester
from diluted conventional or aerosQlised (Stage 4)
liposome preparations. In both systems, initial rapid
release of drug was apparentO Linear regression of
efflux profiles 2 hour post dilution resulted in half
25 ~ive~ of eEflux of 48.3 hours for the aerosolised system
and 50.2 hours for the conventional system.
Lengthening of the acyl 21-substituents has been
shown to have a great effect upon the partitioning
behaviour of csrtisone derivatives in DPPC
30 liposomes/water syst~ms and a partition coefficient
~lipid~aqueous, 37C) for the Cg ester oiE around
5 x 103 has been derived (Arrowsmith~ ~., Hadgraft, J.,
Kellaway, I.W., Int. J. Pharm. 14:191, 1983). Similar
~5E;i~
S22=
partitioning behaviour was shown in this work for
hydrocortisone 21-octanoate in e~g PC liposomes/water
systems. As similar steroid partitioning was apparent
in the conventional and aerosolised liposome
preparations, it is probable that liposomes produoed
from solution aerosols in the MLI are formed with
similar structural conformations as conventionally
prepared liposomes. Moreover, the similar steroid
partitioning in liposomes formed on Stages 3 ar.d 4 of
10 the impinger inferred that this occurred following
impaction on the aqueous surEace of various siæed
aerosol particles. It is generally accepted that
liposomes are ~ormed only when the phospholipicl and
aqueous medium are mixed at a temperature of higher than
15 the phase transition temperature of the resulting
hydrated form. ~ence, it is a valid assumption that
this will occur only with phospholipids of transition
temperatures less than that of the impaction medium.
Following dilution of equilibrated liposome
20 preparations, efflux of drug out of liposomes occurred
to re-establish the equilibrium partition coefficient.
After an initial phase of rapid release, the linearity
of the plots for both conventional and aerosolised
liposome preparations indicates that release proceeds by
2S first orde.r kinetics. These results show that sustained
release of drugs can be achieved from liposomes
generated from an aerosol system and, as shown by the
efflux half lives~ similar kinetics to conventional
prepared liposomes are produced. ~ence this mean~ of
30 producing liposomes in situ is useful in providing
sustained release o~ medicaments.
