CA1256801A - Process for the in-situ preparation of liposomes and their use as a sustained-release aerosol delivery system - Google Patents

Abstract

ABSTRACT

PROCESS FOR THE IN-SITU PREPARATION OF LIPOSOMES AND
THEIR USE AS A SUSTAINED-RELEASE AEROSOL DELIVERY SYSTEM

A process for the preparation of liposomes which comprises spraying micro-fine droplets of a composition comprising substantially pure phospholipid in a volatile liquid carrier to impinge either upon or below an aqueous surface thereby forming liposomes at the said surface. The compositions may include drug molecules dissolved therein and upon formation of the liposomes drug molecules are entrapped. The entrapment of drug molecules in this manner provides a method of sustained release of drug molecules.

Description

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~

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. ,~`

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~ 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 .~
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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~

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.-~

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- 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.

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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.

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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 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.

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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.

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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.

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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.

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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~

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.

Claims (13)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A pack for 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 therapeutically active sub-stance being greater than 1:1, the pack including an ar-rangement for dispensing said solution as a spray under pressure developed by the propellent material.

2. A pack as claimed in claim 1, in which the molar ratio of phospholipid to the therapeutically active substance is at least 5:1.

3. A pack as claimed in claim 1, in which the molar ratio of phospholipid to the therapeutically active substance, is in the range of from 10:1 to 20:1.

4. A pack as claimed in claim 1 which addition-ally comprises a co-solvent for the therapeutically active substance.

5. A pack as claimed in claim 4, in which the solvent is anhydrous.

6. A pack according to claim 1, wherein the propellent material is a fluorochlorocarbon.

7. A pack according to claim 6, in which the fluorochlorocarbon is selected from trichloromonofluoro-methane, dichlorodifluorome-thane, dichlorotetrafluoro-ethane and mixtures thereof.

8. A pack according to claim 1, 2 or 3, in which the solution in the aerosol device comprises 90 to 99.9 propellent material having dissolved therein from 0.1 to 10% by weight phospholipid.

9. A pack according to claim 1, 2 or 3, in which the phospholipid is at least 80% pure.

10. A pack according to claim 1, 2 or 3, in which the phospholipid is 90 to 100% pure.

11. A pack according to claim 1, 2 or 3, in which the phospholipid is phosphatidyl choline.

12. A pack according to claim 1, wherein said spray comprises micro-fine droplets of a composition com-prising said substantially pure phospholipid in said pro-pellent material .

13. A pack according to claim 12, in which the micro-fine droplets have a particle size in the range 0.5 to 50 micron.