WO1986001714A1

WO1986001714A1 - A liposomal sustained-release aerosol delivery system

Info

Publication number: WO1986001714A1
Application number: PCT/GB1985/000430
Authority: WO
Inventors: Brian Carman-Meakin; Ian Walter; Kellaway; Stephen James; Farr
Original assignee: Riker Laboratories, Inc.
Priority date: 1984-09-17
Filing date: 1985-09-17
Publication date: 1986-03-27
Also published as: CA1256801A; NZ213459A; ES547059A0; AU4866885A; ES8707859A1; JPS62500643A; EP0195809A1; ZA856969B; GB8423436D0

Abstract

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 which are entrapped upon formation of the liposomes. The entrapment of drug molecules in this manner provides a method of sustained release of drug molecules.

Description

A liposomal sustained-release aerosol delivery system

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 pressurised aerosol device provide sustained-release of medicament.

Liposomes are artificial spherules of phospholipids composed of a series of concentric layers alternated with aqueous compartments. Bangham et al (J. Mol. Biol. 13. (1) 238-59, 1965) first 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 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 organs. Drugs and other molecules may be encapsulated by two processes. Hydrophilic species are entrapped within the aqueous phase whereas lipophilic moieties associate with the concentric phospholipid bilayers. Thus, there is considerable interest in 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. By 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 ultrasonication possessed only a small aqueous compartment (25 nm diameter) and showed a low efficiency in capturing biologically active molecules. A modified method was based on the removal of organic solvent under reduced pressure to produce a lipid gel which, on addition of excess aqueous phase, formed vesicles of large volume capable of retaining macromolecules with a high capture efficiency. Similar large unilamellar vesicles were also prepared by utilising a calcium-induced structural change in the lipid vesicles. However, this technique was limited to a single phospholipid (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 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 centrifugation is a disadvantage. Also a large amount of the lipid/aqueous emulsion becomes trapped at the interface resulting in a low percentage of material entrapment. The entrapment of certain pharmaceuticals in liquid vesicles has been achieved by freezing or freeze-drying an aqueous phospholipid dispersion. The complexity of the known processes for preparing liposomes containing entrapped molecules, the separation of such liposomes from non-entrapped material and the high cost both 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 convenient, or must be stored under special conditions, e.g. low temperature under nitrogen.

British Patent Specification No. 2 145 107 A discloses a method for the preparation of liposomes in which at least two separate components are brought together under pressure, a first component comprising water and a second component comprising a lipid material. The components are then passed as a mixture under pressure through a nozzle or other arrangement to produce an aerosol spray containing liposomes. In addition, at least one of the first or second components preferably includes a separate active material, e.g. a drug molecule. The specification also discloses a pack for use in preparing a liposomal aerosol comprising at least a first and a second chamber, one chamber containing a first component comprising water and the other chamber containing a second component comprising a lipid material, and one or both of the chambers and/or a third chamber including a propellent material. The pack also includes an arrangement for dispensing as a spray a mixture of the first and second components fed from their respective chambers under pressure developed by the propellent material or materials.

The present invention provides an alternative method for the preparation of liposomes. According to the present invention there is provided a process for the preparation of liposomes which comprises spraying micro-fine droplets of substantially pure phospholipid in a volatile liquid carrier to impinge either upon or below an aqueous surface thereby forming liposomes.

In a preferred embodiment of the invention the micro-fine droplets are generated using a propellent-based pressurised aerosol delivery system, the liquid carrier comprising the aerosol propellent which is conveniently a fluorochlorocarbon. This system can be utilised to deliver drugs to the mucosal surfaces within the lung and achieve sustained-release from the liposomes which are produced in-situ.

The process of the invention provides a simple rapid method for producing liposomes. Discrete micro-fine droplets, generally having a diameter in the range 0.5 to 50 micron, of phospholipid in a volatile 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 of the desired compound with the phospholipid and lipid carrier. The drug molecules must be dissolved in the formulation and in the case of a propellent-based aerosol delivery = 5 =

system, the drug may be dissolved either in the propellent alone or in the presence of a small proportion of a co-solvent, e.g. alcohols, particularly ethanol. An alternative method of increasing the solubility of 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 chloride, cetyl pyridinium chloride, etc. The process of the invention 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 released 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 upon 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 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 process of the invention has significant advantages in its application to inhalation therapy compared to liposomes produced by contacting water with ϋpid material and drug prior to the production of an aerosol spray. The mean particle size of droplets issuing from the valve orifice of a pressurised aerosol pack is typically in the range 30 to 50 microns. Only particles in the range 2 to 7 microns are capable of reaching the lower regions of the lung and aerosols for use in inhalation therapy rely upon the rapid evaporation of the fluorochlorocarbon propellent from the droplets to produce a particle size reduction into the range 2 to 7 microns as the droplets are inhaled. The presence of water and any other liquid vehicles having high boiling points compared with the boiling points of fluorochlorocarbon propellents significantly reduces the ability of the droplets to reduce their particle size by evaporation of the liquid and therefore very few droplets are reduced in size sufficiently to fall within the respirable range. The present invention-relies predominantly upon the use of propellents in the formulations thereby producing droplets within the respirable range and liposomes are formed in-situ when the droplets contact an aqueous surface, e.g. the moist surface of the lungs.

In accordance with a further embodiment of the invention there is provided a pack for use in preparing an aerosol which comprises a single chamber containing a solution of substantially pure phospholipid and a therapeutically active substance dissolved in a propellent material, the molar ratio of phospholipid to the therapeutically active substance being greater than 1:1, the pack including an arrangement for dispensing said solution as a spray under pressure developed by the propellent material.

The molar ratio of phospholipid to the therapeutically active substance is generally at least 5:1 and normally within the range 10:1 to 20:1. Preferably, the solution is anhydrous.

The phospholipids used in the invention may be selected from a wide range including: phosphatidylcholine (lecithin) (PC) phosphatidylglycerol (PG) phosphatidylserine (PS) phosphatidic acid (PA) phosphatidylinositol (PI) phosphatidylethanola ine (PE) dipalmitoylphosphatidylglycerol (DPPG) and diacylphosphatidylcholine (DAPC) .

One or more of the following non-phospholipid excipients may be included as formulation aids to increase the stability of the phospholipid bilayer or to control the surface charge: dicetylphosphate (DCP) stearyla ine (SA) sphingomyelin (SM) cholesterol (C) distearyldimethyl ammonium chloride.

The phospholipids must be substantially pure in order to ensure uniform liposome formation. Preferably the phospholipid is at least 80% pure, more preferably 90% 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 aerosol propellents, in particular fluorochlorocarbon propellents, e.g. Propellent 11 (trichloro ono- fluoromethane) , Propellent 12 (dichlorodifluoromethane) and Propellent 114 (dichlorotetrafluoroethane) . Suitable formulations for use with a pressurised aerosol delivery system comprise 90 to 99.9% of one or more fluorochlorocarbon propellents and 0.1 to 10% by weight of one or more phospholipids plus formulation aids if required. The use of phospholipids in fluorochlorocarbon- based aerosol formulations is known and is disclosed in British Patent Specification No. 2 001 334 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. However, these known formulations have not been used to prepare liposomes and the phospholipid has been present in the formulations as a surfactant in very small amounts and in impure form, e.g. 15% purity, to aid the preparation of a drug dispersion, to stabilise the aqueous droplets against evaporation or for therapeutic reasons.

The invention will now be illustrated with reference to the following Examples.

Example 1 In vitro evidence to show the in-situ production of liposomes from a fluorochlorocarbon propellent-based aerosol device.

Purified egg phosphatidyl choline (PC) was used as the model phospholipid. Crude egg lecithin (BDH Chemicals, England) containing 90% egg PC was used as the starting material. The egg PC was purified and 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 acetone at 4°C. The recrystallised egg PC was shown to be chromatographically pure using a solvent system of 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 which the aerosol dose could be fired and is illustrated in Figure 1 of the accompanying drawings. The apparatus consisted of a 1 litre filtering flask 1, containing a beaker partly filled with a known volume of aqueous receptor fluid 10. An intake tube 2 protruded 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 fluid 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 37°C the flask was immersed up to the height of the side arm in a water bath 7 maintained at 37°C. To ensure delivery of the majority of the aerosolised dose into the receptor fluid, air flow through the apparatus was achieved via a tube 8 connected to a vacuum pump (Speedivac). A flow rate of 50 litre/min was monitored by a flow meter (Gap Ltd.). A sampling syringe 9 was provided for obtaining samples of liposome. To permit air flow, the aerosol adaptor had an orifice 11 at the rear. The assembled apparatus 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 was made to allow the construction of a miscible/immiscible phase diagram of 5% w/w egg PC in Propellent 11/Propellent 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 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 pressures of 3.43 x 10⁵ N/m² and 4.79 x 10⁵ N/m² (50 and 70 psia) (21°c) 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 placed in the oral adaptor. Air was drawn through the apparatus and the valve actuated at 10 second intervals for a previously determined number of times. Following the final actuation, the air flow was shut off and samples of the receptor fluid were withdrawn and analysed using photon correlation spectroscopy (Malvern Model RR144, Malvern Instruments Ltd., Malvern, united Kingdom) . The size of particles within the receptor fluid was followed with time.

Figure 3 of the accompanying drawings shows the effect of time on the particle size of particles generated from aerosols containing 1% w/w pure egg PC and possessing vapour pressures of 3.43 x 10⁵ N/m² and 4.79 x 105 N/m² (50 or 70 psia) at 21°C, each point representing the mean of three determinations with standard error bars. The initial particle size was dependent on vapour pressure; 882 nm for 3.43 x 10⁵ N/m² (50 psia) and 560 nm for 4.79 x 10⁵ N/m² (70 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 of the formation of liposomes using electron microscopy.

The formation of liposomes was confirmed by examination of the receptor fluid samples after negative staining with ammonia molybdate using transmission electron microscopy. Figure 4 of the accompanying drawings is an electron micrograph and reveals clusters of aggregated multilamellar vesicles ranging in size from 150 to 400 nm but collectively in aggregates below 1 micron in size.

Example 2

The partitioning of a drug into liposomes

Salbutamol - a hydrophilic compound

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 aerosol delivery device was studied.

Extemporaneous preparation of liposomes

Methods for preparing MLVs have been extensively published (e.g. Juliano, R.L., Stamp, D. , 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 rotary evaporated at 40°c (with the inclusion of small volumes of acetone to encourage removal) to leave a 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 37⁰c to form MLKs. The aqueous phase used was either (a) 0.9% w/v saline adjusted to pH 7.4 with 0.1M sodium hydroxide or (b) physiologically iso-osmotic, phosphate buffered saline (PBS) at pH 7.4. Salbutamol hemisulphate being practically insoluble in ethanol was 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 1 mg/ml. The liposome/drug suspensions were shaken at 37°C for sufficient time to permit equilibration before separation of liposomes by centrifugation and assay for drug content in the supernatent by HPLC.

The enhancement of the proportion of salbutamol entrapped within the liposomes.

When salbutamol hemisulphate was added to the aqueous phase during the extemporaneous manufacture of MLVs, the drug partitioned into PC liposomes suspended in iso-osmotic PBS, pH 7.4 at 37°C 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 liposomes for drug added to the lipid phase or the aqueous phase are shown in Figure 5 of the accompanying drawings and reveal that the degree of initial liposo al association was greatest when the drug was added to the lipid phase, although a rapid decline occurred to equilibrium at approximately 3 hours. The low entrapment efficiencies were of the order expected for a hydrophilic species, fully ionised at pH 7.4 (the pKa of the basic moiety of salbutamol is 10.3, Newton D.W., Kluza, R.B., Drug Intell. Clin. Phar . , 12:546, 1978). The insignificant effect of cholesterol on salbutamol partitioning (Figure 5) suggested that the salbutamol partitioned into the aqueous channels within the liposomes and was unassociated with-the lipid bilayer.

Hydrophobic species generally partition into liposomes to a greater extent than hydrophilic species. Formation of an ion-pair complex with a lipophilic moiety represented a method of conferring hydrophobicity to the salbutamol molecule. Dicetyl phosphate (DCP) incorporates into lecithin bilayers and is routinely used at concentrations below 10 mole % to confer a negative charge to liposomes (szoka, F., Papahadjopoulos, D. , Am. Rev. Biophys. Bioeng. 9:467, 1980). The effect of inclusion of DCP at 10, 20 and 30 mole % at 37°C on the liposomal uptakes of salbutamol due to formation of a lipophilic ion-pair complex is illustrated in Figures 6 and 7. Figure 6 represents a. plot of entrapment of salbutamol (mg/mg %) in DCP/PC liposomes at 37°C against time for varying DCP concentrations. Figure 7 represents a plot of the partition coefficient for salbutamol in liposome (DCP/PC) against molar ratio DCP/salbutamol. The inclusion of 30% DCP caused a 175% increase in entrapment from 1.4 to 2.45 mg/mg %. The in-situ preparation of liposomes using a pressurised aerosol device.

The use of a more sophisticated multi-stage liquid impinger (MLI) has been described (Bell, J.H., Brown, K., Glasby, J., J. Pharm. Pharmacol., 25:32P, 1973) to characterise an aerosol cloud according to its particle size distribution. Figure 8 of the accompanying drawings shows a multi-stage liquid impinger which comprises a glass container 80 divided into four sections (Stages 1 to 4) by glass separation plates 82, each section being in communication with adjacent sections via conduits 84. The pressurised aerosol container 86 is positioned at the throat 88 of the apparatus. Sintered glass collection plates 89 are positioned on each separation plate. A fixed volume (10 ml) of pre-filtered (0.05 micron) water was added to each stage to ensure that a moist sintered glass surface was presented to the air flowing through the 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 airstream generated by a vacuum situated downstream of the sampling device.

Data from MLI measurements made on pressurised aerosols at a range of vapour pressure and PC content are shown in the following Table 1. Table 1 Deposition of aerosol emitted 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. Each result (expressed as a % retention of the total aerosol) is a mean of three determinations.

Effective cut off diameter was assumed as 20 micron for the glass throat (Hallworth, G.W., Andrews, G., J. Pharm. Pharmacol., 28:898, 1976) and determined as 10.47 micron for Stage 1, 5.51 micron for Stage 2, 3.59 micron for Stage 3 and 1.25 micron for Stage 4.

V.P.¹ 4.79 4.79 4.79 3.43 3.43

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

Stage 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 21°C x 10⁵ N/m². =16=

In terms of potential for liposome formation, the actual quantities of PC delivered within the respirable range ( < 5 micron) from pressure packs containing egg PC in blends at 4.79 x 10⁵ N/m² at 21°C 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 ° (Fl) were prepared and evaluated on the MLI.

Formulations Fl and F2 are tabulated below:

Content in g/10 ml fill volume

Fl (g) F2 (g) 5 Salbutamol 0.04 0.04

Egg phosphatidylcholine 0.244 0.1957

Dicetyl phosphate - 0.0597

Ethanol 1.83 1.02 Trichlorofluoromethane (Pll) 1.92 2.29

Dichlorodifluoromethane (P12) 8.20 9.16

Total 12.23 12.76

Density of liquid blend (g/ml) 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/m² (63 psig) at 25°C are illustrated diagramatically in Figure 10. Similar results were obtained for formulation Fl when tested on the MLI.

Equilibrium partitioning data at 37©c for salbutamol in liposomes formed on Stages 3 and 4 of the MLI for deposited aerosol generated from formulations Fl ^{W0 86/β,7M} PCT/GB85. 0 .

=17=

and F2 was evaluated. Negligible entrapment was apparent for Fl, but for F2 where the lipid component contained 30 mole % DCP, partitioning of salbutamol into the liposomes was observed. Entrapment efficiencies

⁵ were calculated as 2.67 ^ 0.69 mg/mg % for Stage 3 and 2.65 ^ 0.78 mg/mg % for Stage 4. These are very similar to those described previously for the in vitro liposome partitioning experiments using pre-formed liposomes. It is concluded that the partitioning characteristics

10 observed with aerosol produced liposomes are comparable with those observed using extemporaneously prepared liposomes.

Example 3 15

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

²⁵21-acyl chain length to yield partition coefficients greatly in favour of the lipid phase (Shaw, I.H., Knight, V.G., Dingle, J.T., Biochem. J. 158:473, 1976; Arrowsmith, M. , Hadgraft, J., Kellaway, I.W., Int. J. Pharm., 14:191, 1983). Hydrocortisone octanoate was

30 selected as an example of such a hydrophobic compound. Radiolabelled compound (Amersham International, U.K.; specific activity 83 Ci/mmol) was used to permit measurement of the drug efflux rate from liposomes produced in-situ using a pressurised aerosol delivery system.

Extemporaneous preparation of liposomes Mixed films of egg PC (25 mg) and the steroid ester (O.10 mg spiked with 0.83 uCi of the tritiated ester) were prepared in 50 ml round bottomed flasks following evaporation under reduced pressure at 40°C of chloroform solutions on a rotary evaporator. To each film, 7.5 ml of sterile 0.9% w/v saline was added and the film hydrated at 40°C to form an MLV suspension.

In-situ preparation of liposomes using a pressurised aerosol device L-alpha-phosphatidylcholine, di[l-l⁴c] palmitoyl [14c-DPPC] of specific activity 112 mCi/mmol (Amersham International, U.K.) was used in this Example.

Pressure packs (10 ml) containing 1% w/w egg PC (spiked with 1.59 Ci 1 C-DPPC) and 1 mg of hydrocortisone 21-octanoate (spiked with 4.15 μCi of the tritiated steroid ester) in P11/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 actuations. The emitted aerosol was directed into a calibrated multistage liquid 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 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 volumetric flasks and made to volume. Drug and lipid concentrations were determined =19=

by liquid scintillation counting. Liposome suspensions formed on Stages 3 and 4 were transferred to conical flasks for studies on drug entrapment and release.

All samples (1 ml) were incorporated into 10 ml

⁵ of Cocktail T (BDH, U.K.) prior to counting for 10 minutes in an LKB 1217 Rack Beta liquid scintillation counter. Quench correction was carried out for ⁱ⁴c and 3H using external standardisation resulting in counting efficiencies of 90% for ⁱ⁴c and 30 to 35% for ³H.

10 The artitioning of hydrocortisone 21-octanoate between egg PC and water was determined for the conventional systems and for systems generated on stages 3 and 4 of the MLI following equilibration (48 h) at 37°C by scintillation counting of duplicate aliquots of

15 the liposome suspension and of the supernatant obtained by ultracentrifugation of a 3 ml sample at 195,000 g for 1 hour.

The pattern of deposition produced in a MLI from a pressurised aerosol containing egg phosphatidyl

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

25 composed of a homogeneous system. The recoveries of egg PC and steroidal ester were 90%. The respirable fraction of each component of the aerosol cloud (i.e. that deposited on Stages 3 and 4 and filter) was calculated as 20.91 ^ 2.82% for egg PC and

30 20.18 ^ 2.67% for the steroid ester. This compares favourably with the values obtained with traditional suspension-type inhalation aerosols.

Table 2 reports the partitioning of hydrocortisone 21-octanoate between egg PC liposomes and water at 37°C. Table 2

System Partition Coefficient (K) x 10-

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 formed in-situ have entrapped drug in a similar manner to those produced extemporaneously. In addition, K was independent of whether the liposome systems were generated on Stage 3 or 4 of the impinger.

Example 4

In-vitro assessment of sustained-release from aqueous liposome dispersions

a) Salbutamol - a hydrophilic compound

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 produced using a pressurised aerosol device. Instead, efflux rates on liposomes produced extemporaneously were studied using the method described in Example 1. The liposome/drug suspension at equilibrium (approximately 15 ml) was filtered in a 400 ml ultrafiltration cell and the liposome residue resuspended to the original volume in fresh iso-osmotic PBS buffer, pH 7.4, maintained at 37°c. Following stirring and transfer to a shaking bath, efflux was monitored by separation of the aqueous phase from the liposomes using ultrafiltration (PM10 membrane, Diaflo, Amicon, U.K.) and assay for free drug by HPLC (the lower limit of sensitivity of the assay was 1 ,ug/ml salbutamol). Formulations containing 4 mg/ml salbutamol egg PC and varying amounts of DCP were examined and the results are recorded in Figure 12 which is a plot of percentage drug retention against time for formulations containing 10, 20 and 30 mole % DCPC at 37°C.

The efflux of salbutamol from liposomes was dependent on DCP concentrations (Figure 12). Release 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 lives for data collected over 0 to 10 hours were:

DCP Content (mole %) Half life (hours) 10 259

20 24.1 30 24.6

Based on this data, a fluorocarbon based pressurised aerosol system containing salbutamol base and DCP will exhibit significant sustained release characteristics.

b) Hydrocortisone 21-octanoate - a lipophilic compound

The use of radiolabelled hydrocortisone 21-octanoate permitted the measurement of efflux of drug from liposomes produced on Stages 3 and 4 of a multi-stage liquid impinger 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 Example 3. Figure 13 shows release of steroid ester from diluted conventional or aerosolised (Stage 4) liposome preparations. In both systems, initial rapid release of drug was apparent. Linear regression of efflux profiles 2 hour post dilution resulted in half lives of efflux 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 cortisone derivatives in DPPC liposomes/water systems and a partition coefficient (lipid/aqueous, 37°C) for the CQ ester of around 5 x 10³ has been derived (Arrowsmith, M. , Hadgraft, J. , Kellaway, I.W., Int. J. Pharm. 14:191, 1983). Similar partitioning behaviour was shown in this work for hydrocortisone 21-octanoate in egg PC liposomes/water systems. As similar steroid partitioning was apparent in the conventional and aerosolised liposome preparations, it is probable that liposomes produced 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 and 4 of the impinger inferred that this occurred following impaction on the aqueous surface of various sized aerosol particles. It is generally accepted that liposomes are formed only when the phospholipid and aqueous medium are mixed at a temperature higher than the phase transition temperature of the resulting hydrated form. Hence, 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 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 first order 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. Hence this means of producing liposomes in situ is useful in providing sustained release of medicaments.

Claims

CLAIMS :

1. 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.

2. A process as claimed in Claim 1 in which the micro-fine droplets are generated by a pressurised aerosol device.

3. A process as claimed in Claim 2, in which the liquid carrier comprises the fluorochlorocarbon propellent.

4. A process as claimed in Claim 3, in which the fluorochlorocarbon propellent is selected from trichloromonofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethane and mixtures thereof.

5. A process as claimed in any one of Claims 2 to 4, in which the formulation in the aerosol device comprises 90 to 99.9% propellent having dissolved therein from 0.1 to 10% by weight phospholipid.

6. A process as claimed in any preceding claim, in which the phospholipid is at least 80% pure.

7. A process as claimed in Claim 5, in which the phospholipid is 90 to 100% pure.

8. A process as claimed in any preceding claim, in which the phospholipid is phosphatidyl choline.

9. A process as claimed in any preceding claim, in which the micro-fine droplets have a particle size in the range 0.5 to 50 micron.

10. A process as claimed in any preceding claim, in which the composition additionally comprises a therapeutically active substance which is entrapped within the liposome upon liposome formation.

11. A process as claimed in Claim 10, in which the therapeutically active substance is dissolved in the volatile liquid carrier optionally in the presence of a co-solvent.

12. A process as claimed in any preceding claim, in which the molar ratio of phospholipid to the therapeutically active substance is at least 5:1.

13. A pack for use in preparing an aerosol which comprises a single chamber containing a solution of substantially pure phospholipid and a therapeutically active substance dissolved in a propellent material, the molar ratio of phospholipid to the therapeutically active substance being greater than 1:1, the pack including an arrangement for dispensing said solution as a spray under pressure developed by the propellent material.

14. A pack as claimed in Claim 13, in which the molar ratio of phospholipid to the therapeutically active substance is at least 5:1.

15. A pack as claimed in Claim 13 or Claim 14, in which the molar ratio of phospholipid to the therapeutically active substance is in the range 10:1 to 20:1.

5

16. A pack as claimed in any one of Claims 13 to

15, which aditionally comprises a co-solvent for the therapeutically active substance.

10 17. A pack as claimed in any one of Claims 13 to

16, in which the solution is anhydrous.

15

20

25

30