WO1987001586A1

WO1987001586A1 - Aerosol

Info

Publication number: WO1987001586A1
Application number: PCT/GB1986/000549
Authority: WO
Inventors: James Arthur Hayward
Original assignee: Biocompatibles Limited
Priority date: 1985-09-17
Filing date: 1986-09-17
Publication date: 1987-03-26
Also published as: EP0240511A1; GB8522964D0; JPS63501014A

Abstract

An aerosol comprising a carrier fluid and suspended therein liposomes comprising a phospholipid, a therapeutic or diagnostic agent for inhalation being entrapped in the liposomes.

Description

AEROSOL

This invention relates to an aerosol comprising liposomes.

Aerosols have achieved wide application in clinical respiratory science (cf. Clarke and Pavia, 1984). Therapeutic aerosols have been used for centuries 5 in the treatment of asthma and are still used increasingly in the treatment of chronic obstructive airway diseases. Ventilation-imaging by inhalation of radioaerosols is commonly used in clinical diagnoses.

Aerosol deposition is affected by the aerodynamic properties of the inhaled solutes and by the physiology and anatomy of the airway. Particles more

10 than 5 microns in diameter tend to impact against the wall^'s of the larger conducting airways whereas particles smaller than 5 microns in diameter may penetrate to the terminal airways as far as the alveoli. The physiological factors which determine the nature of aerosol deposition include lung volumes, flow rates and regional variations in ventilation determined by airflow

15 obstruction and gravity (Chamberlain et al., 1983). Generally speaking, the fate of aerosolised drugs is either clearance from conducting airways by mucociliary action (in which case the drug is either swallowed or removed at the anterior nares) or by absorptive processes.

^" _- Drug absorption in the lung may be by diffusion-limited processes or by

20- phagocytosis and subsequent entry into the lymphatic system. In the case of the former, several barriers to diffusion may be involved. Entry into the vascular system requires passage across: a) the surfactant layer (discussed below) and the aqueous hypophase with which it is associated; b) the alveolar epithelium via a transcellular path (wherein transepi helial transport occurs via iπgestion on the

25 alveolar side of the epithelium and ejection on the perivascular side) or via a paracellular path (wherein transepithelial diffusion occurs passively through intercellular junctions) (Jones, 1984); and, c) the capillary endothelium. Despite the presence of these multiple barriers, the removal of pharmacological and diagnostic solutes from the lung is frequently rapid enough to limit their

30 efficacy. ^' Phospholipids have been identified in the secretions of every division of the respiratory system (Lopez-Vidriero, 1984). The film facing the alveolar space is rich in highly surface active phospholipids. Pulmonary surfactant is composed of 90 % lipid, most (75 %} of which is phosphatidylcholine (PC). This phospholipid class is present in sufficient quantity to form a monomolecular film over the entire alveolar surface. The surfactant properties of PC lower the surface tension at the air-alveolar interface; the hydrocarbon chains form a hydrophobic surface which acts as a water repellant and helps to keep the alveolar surface dry. Clinical evidence leaves no doubt that surfactants are vital to the well-being of the lung (Hill, 1983). Surfactant deficiency is associated with respiratory distress syndrome (RDS), a life-threatening disease occurring in infants and adults. Clinical trials have documented the efficacy of surfactant replacement (Robertson, 1983) wherein the active ingredient is a saturated PC. Synthetic bilayers of PC, prepared by dispersing small amounts of the dried lipid in aqueous solutions, have been extensively studied as models of cellular membranes (Chapman, 1982) . That phospholipid dispersions could behave as osmometers, that is, form sealed containers, was first demonstrated in 1965

(Bangham et al., 1965). The capacity to form sealed containers has since focused much attention on the potential of liposomes as pharmacological capsules (Gregoriadis, 1984) . Liposomes have been utilised in a variety of parenteral and topical delivery routes, but have not been utilised to prolong solute delivery to the lung. The present invention concerns liposome aerosols especially those containing entrapped drugs and/or imaging agents with the explicit purpose of localising and prolonging delivery to the lung by imposing an additional, natural barrier to the diffusion of free solutes. A previous study (Geiger et al., 1975) has examined the fate of nebulised liposomes of PC as a therapeutic replacement for surfactant deficiency in RDS. Nebulisation and inhalation of PC led to its deposition within the lung. Alveolar deposition is not restricted by the aerodynamic properties of liposomes as their diameters are essentially always submicronic. Nebulised PC was detectable in rat alveoli up to 12 hours post inhalation (Geiger et al. , 1975). As the alveolar level of inhaled lipid decreased, systemic levels of the lipid were found to increase. These results provide firm evidence that nebulised phospholipids are -capable of reaching.the terminal airways whereupon they slowly enter the circulation.

However it is not easy to nebulise liposomes_. without rupturing all the liposomes and thereby releasing their entrapped payload. It has now surprisingly been found that liposomes including phospholipids are suitable for aerosolisation and may be nebulised without undue rupturing of the liposomes. Accordingly the present invention provides an aerosol comprising a carrier fluid and suspended therein liposomes comprising a phospholipid, a therapeutic or diagnostic agent for inhalation being entrapped in the liposome. Preferably the phospholipid is a phosphatidyl choline derivative, e.g. distearoyl, dipal itoyl or dimyristoylphosphatidyl choline.

The carrier fluid is typically an aqueous medium in the form of droplets containing a number of liposomes, the droplets being suspended in a propellant gas suitable for inhalation, preferably compressed air. These microcapsules can be employed to prolong both the retention of an agent within the lung and its systemic availability. The phospholipid itself may have therapeutic value as a replacement for, or supplement to, the natural surfactant present in the lung.

The invention also provides a liposomal formulation for administration by inhalation as an aerosol, the liposomes comprising a phospholipid and a therapeutic or diagnostic agent having activity in or being capable of absorption from the respiratory tract.

The invention further provides a process for producing aerosolised liposomal formulations which comprises dispersing liposomes, comprising a phospholipid, a therapeutic or diagnostic agent for inhalation being entrapped in the liposomes, in a carrier fluid.

Known aerosolisation or nebulisation techniques may be exploited using aqueous suspensions of liposomes and a suitable carrier gas such as compressed air. In certain circumstances it may be advantageous to conduct the aerosolisation such that a proportion of the liposomes are ruptured thus providing a two phase release of the therapeutic agent such that a rapid initial phase where the agent from ruptured liposomes is followed by a slower release from intact liposomes.

Herein we propose aerosolised liposomes as vehicles for delivery of therapeutic and diagnostic agents in respiratory medicine. This invention is contingent upon the demonstration of liposomal integrity and latency during the nebulisation process. In support of our invention, we provide below evidence for solute entrapment within nebulised, liposomal microcapsules.

Aerosolised liposomes according to the invention may be used for diagnostic and therapeutic applications. The nature of the entrapped solute and the lipid composition of the liposomes being varied according to the intended use. In therapeutic embodiments an advantage of aerosolised liposomes is the therapeutic value of the carrier itself: phospholipid surfactants have an obligatory role in respiratory function. Among the diagnostic applications for aerosolised liposomes are imaging, functional assessment and provocation or challenge testing. The inclusion of gamma-emitting radionuclides (such as TC ) or radiopacifiers within their enclosed volume is useful for ventilation imaging. Site-directed liposomes may be prepared by attachment of a tissue-specific molecule, such as a monoclonal antibody, to the external face of the liposomes; this technique can be used, for instance, in targetting neoplastic foci. Rates of mucociliary clearance may be determined with non-targeted liposomes which, because of their small size, have access to small airways. Provocation tests, in which the patient is challenged with a suspected allergen, may be enhanced by the use of liposomal carriers. The antigenicity of many allergens is increased by incorporation in liposomes, thereby permitting lower challenge doses of the antigen.

Aerosolised liposomes may be used therapeutically in those cases where either the drug itself exhibits toxic effects after clearance from the lung or the efficacy of the drug is diminished by a too-rapid clearance. One example of this is in the case of corticosteroids used in treatment of asthma and bronchitis, the systemic effects of such drugs include a number of undesirable side-effects such as suppression of the hypothalamic-pituitary-adrenal axis and the development of Cushingoid features, but these and similar side-effects may be limited by the prolonged confinement of an aerosolised drug within the lung as is afforded by entrapment of the steroid within liposomes. Similarly therapeutic aerosolrsed liposomes may be used to deliver prophylactic agents (such as sodium cromoglycate) , mucolytics (such as ri-acetylcysteine) , antibiotics, prostaglandins, vaccines, local and general anaesthetics, bronchodilators, bronchoconstrictors and methyl xanthines. The gradual vascular drainage of entrapped drugs from the lung may also serve as a mechanism for controlled systemic delivery.

Rates of therapeutic or diagnostic agent delivery may be controlled by the lipid composition. A new development in this field is the inclusion of diacetylenic, butadienic, vinylic, acryloylic and methacryloylic moieties into the fatty acyl chains of synthetic lipids forming the liposome. Therapeutic and diagnostic agent delivery from such liposomes may be regulated by controlling the extent of polymerisation of the polymerisable lipids.

The invention will now be illustrated with reference to the following Examples in which the sodium salt of carboxyfluorescein is used as a model for the therapeutic or diagnostic agent to facilitate study of the properties of the liposomes.

EXAMPLE 1

Distearoylphosphatidylcholine (DSPC) and cholesterol (1:1, moleimole) were dried from chloroform solution onto the walls of a glass tube under a steady stream of Ng. The dried film was dispersed in 8 mis (10 H final lipid concentra tion) of Tris-buffered saline (TBS; 0.06 M Tris, 0.09 M NaCl, pH 7.5) containing 250 mM of the sodium salt of carboxyfluorescein (CF) at 60°C with vigorous vortexing. The dispersion was sonicated at room temperature for 5 minutes with a probe sonicator. Extravesicular CF was removed by five-times centrifugation (5 minutes/room temperature/15, 000 X g) and washing in TBS. The final pellet was redispersed in 10 ml TBS to yield 8 mM total lipid. EXAMPLE 2

Large unila ellar vesicles of dimyristoylphosphatidyl choline (DMPC) wer prepared by the solvent-injection method of Deamer (1984). The aqueous medium wa CF in TBS, and extravesicular (i.e. non-entrapped) solute was removed as describe above.

EXAMPLE 3

Nebulisation of the suspensions of liposomes of Examples 1 and 2 was performed with a turret nebuliser

_1 (Medic Aid, Ltd.) operated at 61 min (compressed air), although other methods of nebulisation may be employed.

The configuration of this nebuliser is such that greater than 99.9% of the aerosol is returned to the reservoir via a system of baffles. Thus, the integrity of aerosolised liposomes could be assessed by sampling the nebuliser reservoir as a function of time. The integrity of nebulised liposomes was assessed by electron microscopy of negatively stained samples. The extent of entrapment of CF was determined by relief of self-quenching according to the method of Senior and Gregoriadis (1984). Vesicle stability is reflected by the X Latency which is defined as:

Intravesicular CF

% Latency X 100 %

Total CF. CF latencies were determined as a function of nebulisation time and are given in Table I

TABLE I

Table I. %CF latency as a function of nebulisation time. The date presented in Table I indicate that there is essentially no effect of nebulisation on liposome stability over a period of 5 minutes. Neither did nebulisation affect the appearance of the liposomes as revealed by electron microscopy.

However, the initial latencies of the two preparations are considerably different, and reflect the dependence of liposome permeability on lipid composition. Our observations of the dependence of initial latency upon lipid composition is similar to that observed by others (Senior and Gregoriadis, 1984). Permeability decreases with increasing chain length, extent of saturation and cholesterol concentration. Thus, the time-dependent release of an entrapped solute may be controlled by lipid composition.

Literature Cited

Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252.

Chamberlain, M.J., Morgan, W.K.C. and Vinitski, S. (1983) Clin. Sci.64, 69-78.

Chapman, D. (1982) in (Brown, G.H., ed.) Advances in Liquid Crystals, Vol. 5, Academic Press, New York.

Clarke, S.W. and Pavia, D., eds., Aerosols and the Lung, Butterworths, London, 1984.

Deamer, D.W. (1984) in (Gregoriadis, G., ed.) Liposome Technology, CRC Press, Florida. Gieger, K., Gallagher, M.L. and Hedley-White, J. (1975) J. Applied Physiol. 39, 759-766.

Gregoriadis, G. , ed., Liposome Technology, CRC Press, Florida, 1984.

Hi 11s, B.A. (1983) in (Cos mi , E.V. and Scarpel 1 i, E.M., eds.) Pulmonary Surfactant System, Elsevier, Amsterdam. Jones, J.G. (1984) in (Clarke, S.W. and Pav a, D., eds.) Aerosol s and the Lung, Butterworths, London.

Lopez-Vidriero, M.T. (1984) in (Clarke, S.W. and Pavia, D., eds.) Aerosols and the Lung, Butterworths, London, 1984.

Robertson, B. (1983) in (Cosmi, E.V. and Scarpelli, E.M., eds.) Pulmonary Surfactant System, Elsevier, Amsterdam.

Senior, J. and Gregoriadis, G. (1984) in (Gregoriadis, G., ed.)' Liposome Technology, CRC Press, Florida.

Claims

1. An aerosol comprising a carrier fluid and suspended therein liposomes comprising a phospholipid, a therapeutic or diagnostic agent for inhalation being entrapped in the liposomes.

2. An aerosol according to claim 1 wherein the carrier fluid is an aqueous medium in the form of droplets suspended in a propellant gas suitable for inhalation.

3. An aerosol according to claim 2 wherein the aqueous medium is a buffered saline solution.

4. An aerosol according to any preceding claims wherein the phospholipid is a phosphatidyl choline derivative.

5. An aerosol according to claim 4 -wherein the phosphatidyl choline derivative is distearoyl, dipalmitoyl or dimyristoylphosphatidyl choline.

6. An aerosol according to any one of claims 1 to 5 comprising a diagnostic agent.

7. An aerosol according to claim 6 wherein the diagnostic agent is an imaging agent or a suspected allergen.

8. An aerosol according to any one of claims 1 to 5 comprising a therapeutic agent.

9. An aerosol according to claim 8 wherein the therapeutic agent is a corticosteroid, a prophylactic agent, ^'a mucolytic agent, an antibiotic, bronchodilator, bronchoconstrictor, prostaglandin or vaccine or a local or general anaesthetic, or a methylxanthine.

10. A liposome formulation for administration by inhalation as an aerosol, the liposomes comprising a phospholipid and a therapeutic or diagnostic agent having activity in or capable of being absorbed from the respiratory tract.

11. A liposome formulation according to claim 10 comprising liposomes dispersed in an aqueous medium.

12. A liposome formulation according to claim 11 wherein the aqueous medium is a buffered saline solution.

13. A liposome formulation according to any one of claims 10 to 12 wherein the phospholipid is ^'a phosphatidyl choline derivative.

14. A liposome formulation according to claim 13 wherein the phosphatidyl choline derivative is distearoyl, dipalmitoyl or dimyristoylphosphatidyl choline.

15. A liposome formulation according to any one of claims 10 to 14 comprising a diagnostic agent.

16. A liposome formulation according to claim 15 wherein the diagnostic agent is an imaging agent or a suspected allergen.

17. A liposome formulation according to any one of claims 10 to 14 comprising a therapeutic agent.

18. A liposome formulation according to claim 17 wherein the therapeutic agent is a corticosteroid, a prophylactic agent, a mucolytic agent, an antibiotic, bronchodilator, bronchoconstrictor, prostaglandin or vaccine or a local or general anaesthetic, or a methylxanthine.

19. A method for producing an aerosol according to any one of claims 1 to 9 comprising nebulising a liposome formulation according to any one of claims 10 to 18.

20. A method for treating a human or non-human mammal by therapy or diagnosis comprising administering an effective non-toxic amount of an aerosol formulation according to any of claims 1 to 9.