WO2003061668A1

WO2003061668A1 - Use of pulmonary surfactant for the prevention of infectious diseases

Info

Publication number: WO2003061668A1
Application number: PCT/GB2003/000235
Authority: WO
Inventors: Derek Woodcock; Jim Thompson; John Staniforth; Joy Conway
Original assignee: Britannia Pharmaceuticals Limited
Priority date: 2002-01-17
Filing date: 2003-01-17
Publication date: 2003-07-31
Also published as: GB0201028D0

Abstract

Infections caused by inhaling airborne pathogens and toxic particles, such as Bacillus anthracis, are prevented by administering to the respiratory system a powdered surface active phospholipid (SAPL) composition, preferably comprising a mixture of phosphatidyl choline (PC) and phosphatidyl glycerol (PG).

Description

USE OF PULMONARY SURFACTANT FOR THE PREVENTION OF INFECTIOUS DISEASES.

Background of the Invention

This invention relates to pharmaceutical products based on surface active phospholipids (SAPLs) for use in the prevention of infectious diseases, in particular diseases caused by inhalation of infectious or toxic airborne particles.

SAPLs are used clinically for the treatment of respiratory distress syndrome (RDS) in neonates. In this role, it has been assumed that the SAPL functions by reducing the high surface tension forces at the air- water interface within the alveoli, thereby reducing the pressure needed to expand the lungs, see Bangham et al., Colloids & Surfaces, 10 (1984), 337 to 341.

EP-0 528 034-A (Tokyo Tanabe) describes the use of pulmonary surface active material as an ingredient of an anti-asthmatic, which is in the form of a liquid or suspension for injection or spraying into the patient's air way.

WO 99/27920 (Britannia) describes the use of phospholipids to treat disorders of the middle ear, and WO 99/51244 (Britannia) describes the use of phospholipids to prevent surgical adhesions. WO 00/30654 (Britannia) describes a combination product for treating asthma, comprising a powdered surface active phospholipid composition and an anti-asthma drug.

There are a number of surface-active lipid compositions available commercially, but most of these are solvent extracts from bovine or porcine lungs. As a result, they are expensive and run the risk of transmitting pathogens or pirogues of animal origin to the patient. All of these compositions are currently administered 'wet', i.e. the surfactant is dispersed in saline and given as either a bolus or as a droplets from a nebulizer.

Summary of the Invention

The present invention is based on the appreciation that administration of a surface- active phospholipid (SAPL) to the respiratory system, in a manner and in a sufficient amount, typically by oral or nasal inhalation, forms and maintains at least a partial SAPL coating on surfaces of the respiratory tract or nasopharynx, to provide a alveolar surface of the lung can prevent inhaled infectious particles from binding to pulmonary membranes and/or prevents toxins released from infectious particles from reaching the membranes.

One example of an inhalable airborne infectious particle is the anthrax bacterium, where the disease resulting from inhalation is much more deadly than infection caused by skin contact. The bacterium Bacillus anthracis secretes a toxin made up of three proteins: protective antigen (PA), oedema factor (OF) and lethal factor (LF). PA binds to cell-surface receptors on the host cell's membranes. After being cleaved by a protease, PA binds to OF and LA and mediates their transportation into the cytosol where they exert their pathogenic effects.

Brief Description of the Drawings

Fig. A shows the apparatus used for the barrier assay; Fig. 1 shows the effect of mucolytics on transport of beads through thin model sputum;

Fig. 2 shows the effect of mucolytics on transport of beads through thick model sputum;

Fig. 3 shows the effect of surfactants on the transport of beads through thin model sputum;

Fig. 4 shows the effect of surfactants on the transport of beads through thin model sputum;

Fig. 5 shows the effect of surfactants on the transport of beads through thin model sputum; Fig. 6 shows the effect of surfactants on the transport of beads through thin model sputum;

Fig. 7 shows the effect of surfactants on the transport of beads through thick model sputum;

Fig. 8 shows the effect of surfactants on the transport of beads through thin model sputum.

Detailed Description of the Invention

This invention postulates that the barrier layer formed on, for example, the pulmonary membranes by administration of an SAPL by inhalation prevents the PA secreted by Bacillus anthracis from binding to cell surface receptors and so inhibits the transport of OF and LA into the cytosol.

This invention postulates that a barrier layer is formed on the respiratory tract and lung surfaces by inhalation of SAPL. This presents toxic and infectious particles contacting the surfaces and becoming active.

The invention may also be used to prevent infection from person to person as well as inhalation from environmental sources for example infection from: Viruses e.g., varicella, Influenza, Rubeola, Rubella, Variola, Bacteria e.g. Whooping coug, Meningitis, Diptheria, Pneumonia, Tuberculosis. Environmental sources, psittacosis, Legionnaires disease, Acute allergic alveolitis, aspergillosis, Histoplasmosis, Coccidicirnycosis.

To deliver the SAPL to the lungs, this invention advantageously employs a dry, preferably synthetic, SAPL composition, and delivers it dry. The preferred dry SAPL composition is prepared from phosphatidyl choline (PC) and phosphatidyl glycerol (PG). Particularly favourable SAPL compositions are prepared from synthetic dipalmitoyl phosphatidyl choline (DPPC) co-precipitated with PG in the weight ratio of 6:4 to 8:2, especially about 7:3. The composition can advantageously be administered to the respiratory system as a dry powder since it spreads extremely rapidly on water, much faster than exogenous extracts from the lung and surfactant which is dispersed in aqueous media.

The phospholipids used in accordance with the invention have diacyl substituents on the phosphatidyl groups. As in their natural counterparts, the diacyl groups may comprise identical or different, saturated or unsaturated acyl radicals, generally C14- 22, especially C 16-20, acyl radicals. Thus the phospholipids may comprise, by way of acyl radicals, the saturated radicals palmitoyl C16:0 and stearoyl C18:0 and or the unsaturated radicals oleoyls C18:l and C18:2 . The phospholipids used in the compositions in accordance with the invention more particularly comprise two identical saturated acyl radicals, especially dipalmitoyl and distearoyl, or a mixture of phospholipids in which such radicals predominate, in particular mixtures in which dipalmitoyl is the major diacyl component. Thus PC and PG may be used may be used with the same diacylphosphatidyl profile as in PC and PG extracted from human or animal sources, but if synthetic sources are used the dipalmitoyl component may predominate, as in the DPPC mentioned above.

As also mentioned above, the SAPL compositions are preferably protein free, but in some circumstances the presence of naturally occurring proteins associated with PC and PG in vivo may be tolerated. For example the presence of apoproteins A, B, C and D may be tolerated in SAPL compositions for human use.

DPPC can be prepared synthetically by acylation of glycerylphosphorylcholine using the method of Baer & Bachrea - Can. J. of Biochem. Physiol 1959, 37, page 953 and is available commercially from Sigma (London) Ltd. The PG may be prepared from egg phosphatidyl-choline by the methods of Comfurions et al, Biochem. Biophys Acta 1977,488, pages 36 to 42; and Dawson, Biochem J. 1967,102, pages 205 to 210.

When co-precipitated with DPPC from a common solvent such as chloroform, PG forms with DPPC a fine powder which spreads rapidly over the surfaces of the airways and lungs. The most preferred composition of the invention contains DPPC and a phosphatidyl glycerol derived from egg phosphatidyl choline, which results in a mixture of C 16, C 18 (saturated and unsaturated) and C20 (unsaturated) acyl groups.

While not wishing to be limited to the following theory it is believed that an effective barrier on e.g. pulmonary membranes is formed by administering exogenous SAPL in a form which displays two properties. Firstly, it spreads rapidly over the surface of the incumbent fluid for widespread distribution throughout the lung. Secondly, it then absorbs to the epithelial surface to repair/fortify the semi- permeable barrier of natural surfactant.

Although the honeycomb structure of aveoli makes it difficult to conduct absorption studies, it has been demonstrated in studies using radio-labelled DPPC that this SAPL will absorb to bronchial epithelium and that PG potentates this absorption by a factor of 2-3. A mixture of 7:3 DPPC:PG provides essentially optimal absorption levels. Once having attained the condition described above, one of the factors which will reduce the life of the lining or coating of SAPL will be the presence of enzymes such as phospholipase capable of digesting DPPC and/or PG. Such enzymes only attack the laevo-rotatory form which constitutes a naturally occurring form. Accordingly, it is preferred that the SAPLs used in the present invention preferably contain the dextro-rotatory form or at least comprise the racemic mixture which is obtained by synthetic routes.

The SAPL compositions preferably used in accordance with the present invention are finely-divided, solid powders and are described in detail in our co-pending PCT applications WO 99/27920 and WO 00/30654, the whole contents of which are incorporated by reference. However in summary, our above applications indicate that an important feature of the SAPL compositions that are usable in the present invention is that they are in the form of a powder, that is, it is in solid form. The "dry" surfactant has a high surface activity.

Preferably, the SAPL composition has two components. Suitably the first component of the SAPL comprises one or more compounds selected from the group consisting of diacyl phosphatidyl cholines. Examples of suitable diacyl phosphatidyl cholines (DAPCs), are dioleyl phosphatidyl choline (DOPC); distearyl phosphatidyl choline (DSPC) and dipalmitoyl phosphatidyl choline (DPPC). Most preferably, the first component is DPPC.

The second component may comprise one or more compounds selected from the group consisting of phosphatidyl glycerols (PG); phosphatidyl ethanolamines (PE); phosphatidyl serines (PS); phosphatidyl inositols (PI) and chlorestyl palmitate (CP).

Phosphatidyl glycerol (PG) is a preferred second component. PG is also a preferred second component because of its ability to form with the first component, especially PC and particularly DPPC, a very finely-divided, dry powder dispersion in air.

The composition advantageously comprises a diacyl phosphatidyl choline and a phosphatidyl glycerol. The phosphatidyl glycerol is advantageously a diacyl phosphatidyl glycerol. The acyl groups of the phosphatidyl glycerol, which may be the same or different, are advantageously each fatty acid acyl groups which may have from 14 to 22 carbon atoms. In practice, the phosphatidyl glycerol component may be a mixture of phosphatidyl glycerols containing different acyl groups. The phosphatidyl glycerol is expediently obtained by synthesis from purified lecithin, and the composition of the acyl substituents is then dependent on the source of the lecithin used as the raw material. It is preferred for at least a proportion of the fatty acid acyl groups of the phosphatidyl glycerol to be unsaturated fatty acid residues, for example, mono-or di-unsaturated C18 or C20 fatty acid residues.

Preferred acyl substituents in the phosphatidyl glycerol component are palmitoyl, oleoyl, linoleoyl, linolenoyl and arachidonoyl. The medicament preferably comprises dipalmitoyl phosphatidyl choline and phosphatidyl glycerol, with the phosphatidyl moiety of the phosphatidyl glycerol advantageously being obtainable from the phosphatidyl moiety of egg lecithin.

The compositions are administered in a dry, finely-divided state, preferably using a delivery device such as described in our above co-pending applications, or by directly introducing the aerosolised powder, e.g. by an endotracheal tube (which may be coated to improve transport of SAPL), into the lungs. The dosage and/or period of administration should be long enough to maintain an effective barrier layer over the surface of the lung prior to and/or during and/or after an expected exposure to airborne pathogens or toxic particles.

The particle size of the SAPL should be sufficiently small to reach the lungs when introduced into the subject's airways, e.g. in the form of an aerosolised powder. Generally, the particle size should be less than 10 micron, preferably less than 5 micron e.g. 2-4 micron. Aerosolised powder of this later size range can be introduced via an endotracheal tube having a 2-3 mm diameter. By this technique a fine particle dose of about 25~50mg can be successfully introduced into the lungs.

"Finely divided" as used herein means that the material has a particle size distribution which is such that at least a major proportion by weight of the particles are small enough to enter into a patient's airways and, preferably, deep into the lungs when inhaled. In practice, the first and second components preferably each have a particle size distribution which is such that not less than 90%, by weight, of the particles of those components in combination, and more preferably of each of the first and second components, have a particle size of not greater than lOμm, and especially of not greater than 5μm. Advantageously, the median particle size of the combined first and second components, and more preferably of each of the first and second components is not more than lOμm, and preferably not more than 5μm. The median particle size may be less than 3μm, for example, about 1.2μm. It may be desirable in some circumstances for the particles to have a median particle size of at least 0.5μm. The size of the particles may be calculated by laser diffraction, or by any other method by which the aerodynamic diameter of particles can be determined. "Median particle size" as used herein means mass median aerodynamic diameter (MMAD). The MMAD may be determined using any suitable method, for example, using a Multi-Stage Liquid Impinger in accordance with the method described in European Pharmacopoeia (supplement 1999) 2.9.18 (Aerodynamic assessment of fine particles). Alternatively, the size distribution of the particles may be characterised by their volume mean diameter (VMD). Advantageously, the VMD is not more than lOμm, for example not more than 5μm, and preferably less than 3μm. Finely divided dry powders of this kind (which may be described as fumed powders) can be adsorbed onto the surfaces of lung tissue and are believed, in use, to become bound to the epithelium.

To obtain a mixture in which the particle size is suitable for use in the device of the invention, the phospholipid components may be dissolved in a suitable solvent, for example ethanol, the solution filtered and vacuum-dried, and the solid product size- reduced to obtain particles of the desired size. During size-reduction, care should be taken to protect the mixture from moisture, oxygen, direct heat, electrostatic charge and microbial contamination.

Whilst a barrier layer is preferably formed prior to exposure to infectious particles, given that it may be difficult to predict that exposure will take place, it may well be possible to begin administration only during exposure or after exposure has taken place. However the formation of the barrier will still be effective if it can even partially inhibit contact of particles with the membranes or reduce the binding of proteins that mediate the transport of toxins.

Other theories of the mechanism of action of SAPLs in the lungs also point to the efficacy of SAPLs in preventing infectious particles from binding with lung surfaces. For example, it has been proposed that the efficacy of SAPL in treating asthma is that the administration of SAPL in excess of natural levels has a "flushing" effect whereby the spreading SAPL sweeps or washes allergens away from the pulmonary surfaces. Another theory proposes that particles of SAPL "float" over the alveolar surfaces, rising up through the airways entrapping and carrying allergens with them, to be eventually swallowed and inactivated by gastric fluid.

These mechanisms, in addition to the barrier theory favoured by the present inventors, all point to the efficacy of SAPLs in preventing infectious or toxic particles from binding to pulmonary surfaces.

The effect of SAPL to prevent infectious or toxic particles contacting the surfaces of the lung airways, by reinforcing the barrier properties of mucus, is illustrated by the Examples below.

Example 1

Assay of Barrier properties of Sputum and added Surfactants

Method

The barrier assay was used to measure the ability of an agent to increase the transport of fluorescent beads (200 nm in diameter) through a layer of sputum. The structure of the chamber is shown in Figure A. The apparatus consists of a chamber with two compartments (1,2) separated by an 8 μm polycarbonate filter (3). The upper compartment (1) is provided with wells (4) containing sputum, fluorescent beads (5) and the agent under investigation. The lower compartment (2) is provided with wells (6) containing phosphate buffered saline. Following the incubation period the chamber is dismantled and the fluorescence of the solution contained in the lower wells (4) due to the presence of transported beads (7) is measured.

Results

A model sputum was used as a barrier to the transport of fluorescent beads in the assay. This model sputum contained DNA, mucin and actin. In each experiment a phosphate buffered saline (PBS) control was performed. This involved the addition of PBS to the upper well in the presence of fluorescent beads to indicate the maximum possible transport of the beads achievable. This represents 100% transport. Two types of model sputum have been used: thick and thin preparations. The composition of these models is detailed below. The assay has been validated using both thick and thin model sputum in the presence of a mucolytic agent, DNase.

Experiment 1

Thin model sputum (3.33 mg/ml DNA, 8.3 mg/ml mucin, 0.33 mg/ml actin) was treated with DNase and fluorescent beads added (10% v/v). 15 μl of the DNase preparation was added to upper wells of the 48-well chamber in quadruplicate. The chamber was centrifuged briefly at 1000 rpm to remove air bubbles and incubated at 37°C, with shaking, in the dark for 1 hour. Following incubation the fluorescence of the solution in the lower wells was measured.

Figure 1 shows that thin model sputum acts as a 50% barrier to the transport of fluorescent beads when compared with the PBS control. The DNase mucolytic (2.9 μg/ml DNase) increased transport from 50% to approximately 80%. 50 μg/ml DNase completely restored the transport of beads to the level seen in the PBS control. (* P < 0.05 compared with model sputum only)

Experiment 2

Thick model sputum (5 mg/ml DNA, 30 mg/ml mucin, 0.33 mg/ml actin) was treated with DNase and fluorescent beads added (10% v/v). 15 μl of the preparation was added to upper wells of the 48-well chamber in quadruplicate. The chamber was centrifuged briefly at 1000 rpm to remove air bubbles and incubated at 37°C, with shaking, in the dark for 1 hour. Following incubation the fluorescence of the solution in the lower wells was measured.

Figure 2 shows that thick model sputum allows the transport of only 35% of beads compared with the PBS control. Addition of 2.9 μg/ml DNase increases transport to 63%o, and 50 μg/ml DNase almost fully restores transport with 95% compared to the PBS control. (* P < 0.05 compared with model sputum only) This indicates that the two types of model sputum provide different barriers to transport and both are appropriate models for testing barrier properties of added agents. Surfactants A and B were tested in the barrier assay using the same method. The effect of mixing the Surfactants with the thin model sputum and sprinkling the Surfactants on top of sputum was investigated, as models for administration of powdered SAPL to the airways by inhalation. Incubation periods of 1 hour and 17 hours were tested to ensure that any time-dependent effects were observed.

Surfactant A:

A 7:3 by wt mixture of DPPC and PG obtained by coprecipitation from a common solvent; mean particle size range of about 10-16 microns with 90% of the particles within about 20-50 microns.

Surfactant B:

A 7:3 by wt mixture of DPPC and PG obtained by coprecipitation from a common solvent; mean particle size of 2 microns with 95% below 5 microns.

Experiment 3

Thin model sputum (3.33 mg/ml DNA, 8.3 mg/ml mucin, 0.33 mg/ml actin) was mixed with fluorescent beads (10% v/v). This mixture was then mixed directly with Surfactant, or added directly to wells and Surfactant sprinkled on top. In each case 15 μl of the preparation was added to upper wells of the 48- well chamber in quadruplicate. Chamber centrifuged briefly at 1000 rpm to remove air bubbles and incubated at 37°C, with shaking, in the dark for 1 hour. Following incubation the fluorescence of the solution in the lower wells was measured.

Figure 3 shows that after an incubation period of 1 hour, the Surfactants that had been mixed with the model sputum and beads caused a decrease in transport. Surfactant A decreased transport from 30% to 9%, and Surfactant B decreased transport to 12%. The addition of Surfactant powder to the surface of model sputum surprisingly showed little effect, with Surfactant A causing a decrease in transport from 30% to 27%, and Surfactant B resulting in a slight increase in transport from 30% to 36%. The model was later adjusted to investigate this.

Experiment 4 Thin model sputum (3.33 mg/ml DNA, 8.3 mg/ml mucin, 0.33 mg/ml actin) was mixed with fluorescent beads (10% v/v). This mixture was then mixed directly with Surfactant, or added directly to wells and Surfactant sprinkled on top. In each case 15 μl of the preparation was added to upper wells of the 48- well chamber in quadruplicate. Chamber centrifuged briefly at 1000 rpm to remove air bubbles and incubated at 37°C, with shaking, in the dark for 17 hours. Following incubation the fluorescence of the solution in the lower wells was measured.

The results obtained following an incubation period of 17 hours are shown in Figure 4. Thin model sputum was less effective as a barrier with a longer incubation time. Over a 1-hour incubation period thin model sputum reduced the transport of beads to 30% of the PBS control (Figure 3), whereas over a 17-hour incubation period thin model sputum only reduced transport to 65% of the PBS control (Figure 4). Shorter incubation periods were therefore used for subsequent assays. In Figure 4 mixing the Surfactants with model sputum caused a decrease in transport; Surfactant A reduced transport from 65% to 29% and Surfactant B reduced it from 65% to 43%. The addition of Surfactant to the surface of model sputum again showed no apparent effect; Surfactant A increased transport from 65% to 66% and Surfactant B caused no change in transport.

The results obtained in Figures 3 and 4 indicated that the effects of sprinkling surfactant on the surface of model sputum required further investigation. For the next experiment a 4-well chamber was used in which each well has a much larger surface area. This allowed a larger quantity of surfactant to be sprinkled on the surface of the sputum. However, as shown in Figure 5, the use of larger wells diminished the barrier function of the thin model sputum. Model sputum only reduced transport of beads to 95% of that of the PBS control. Addition of 20 mg of Surfactant A and Surfactant B resulted in 95% and 94% transport respectively.

Experiment 5

Thin model sputum (3.33 mg/ml DNA, 8.3 mg/ml mucin, 0.33 mg/ml actin) was mixed with fluorescent beads (10% v/v) and 500 μl added per upper well. 20 mg Surfactant was sprinkled on top. The 4-well chamber was incubated at 37°C, with shaking, in the dark for 3.5 hours. Following incubation the fluorescence of the solution in the lower wells was measured.

In an attempt to continue to sprinkle larger quantities of surfactant on the surface of the model sputum the 4-well chambers were re-used. Also the beads were laid on top of the sputum layer in the well prior to sprinkling the surfactant on top. This more closely mimics the in vivo effect of inhaled dust particles and inhaled Surfactant powder. The incubation time was shortened to increase the barrier function of sputum in these large volume chambers.

Experiment 6

500 μl of thin model sputum (3.33 mg/ml DNA, 8.3 mg/ml mucin, 0.33 mg/ml actin) was added to wells. 50μl beads were added on top of sputum, and 20 mg Surfactant sprinkled on top of the beads. The 4-well chamber was incubated at

37°C, with shaking, in the dark for 40 mins. Following incubation the fluorescence of the solution in the lower wells was measured.

The addition of beads to the well after model sputum was shown to increase the barrier function of the sputum (Figure 6). Model sputum decreased transport to 54% of the PBS control. This remained unaltered by the addition of 20 mg Surfactant A, however, addition of 20 mg Surfactant B reduced transport to 26% of the PBS control.

This experiment was repeated using a thicker preparation of model sputum to increase the barrier function (Figure 7). Model sputum was added to wells and incubated with 20 mg of pumactant for a range of time periods prior to the addition of beads. This was performed in an attempt to alter the model sputum before the addition of beads and make the assay more sensitive. There was no PBS control in this experiment due to a shortage of wells in the chamber so the thick sputum model alone is given as the 100% value in Figure 7. Surfactant A was shown to reduce the transport of beads through the thick model sputum despite pre-incubation of the model sputum and Surfactant A. 20, 40 and 60 minute pre-incubation times resulted in a reduction in transport from 100% to 54%, 51% and 69% respectively, in contrast to the surprising lack of effect reported above. Experiment 7

500 μl of thick model sputum (5 mg/ml DNA, 30 mg/ml mucin, 0.33 mg/ml actin) was added to wells. 20 mg Surfactant was sprinkled on top of the beads and incubated at 37°C for a range of time periods. 50 μl of beads were added following the incubation and the 4-well chamber incubated at 37°C, with shaking, in the dark for 10 mins. Following incubation the fluorescence of the solution in the lower wells was measured.

In the next experiment the beads were mixed with surfactant and added on top of the model sputum layer in a 48-well chamber.

Experiment 8

15 μl of thin model sputum (3.3 mg/ml DNA, 8.3 mg/ml mucin, 0.33 mg/ml actin) was added to wells and the chamber centrifuged briefly at 1000 rpm to remove air bubbles. 10 μl of beads, into which had been dissolved 1 mg Surfactant, was added on top of the sputum layer. The 48-well chamber was incubated at 37°C, with shaking, in the dark for 1 hour. Following incubation the fluorescence of the solution in the lower wells was measured.

Figure 8 shows that with the beads added on top of the thin model sputum layer the transport of beads was reduced to 64% of the PBS control. Addition of Surfactant A mixed with beads reduced transport from 64% to 3%, and Surfactant B reduced it to 17%.

Overall the test results show support the concept of the invention that inhaled SAPL powder improves the barrier function of sputum to prevent inhaled particles passing through the sputum to contact the underlying lung surfaces.

Example 2

The study was used to evaluate the effect of an exogenous surfactant agent on the early and late asthmatic responses (EAR and LAR). In a randomized, single-blind, cross-over study seven, mild, allergic asthmatic subjects (18-60 yr) with an EAR and LAR to a specific inhaled antigen were enrolled. The active treatment involved 2 doses of 400mgs of surfactant (respirable dose lOOmg as a dry powder SAPL inhalation, consisting of dipalmitoyl phosphatidyl choline and unsaturated phosphatidyl glycerol 7:3 by wt) given at 8 and 0.5 hrs prior to BPT allergen challenge. The placebo treatment involved exactly the same procedure but empty vials were used instead. The two treatment days were separated by 3 weeks.

The surfactant treatment abolished the EAR (early asthmatic response) in all 7 subjects and the surfactant was well tolerated with no adverse events.

The study supports the postulate that administration of an exogenous surfactant agent as a powder provides a physical barrier in the lung to prevent the interaction of an inhaled particles e.g. allergens, with the epithelial lining of the airways. The data also indicates that the SAPL powder can be administered at high doses, without any irritability issues for the patient.

Claims

CLAIMS:

1. Use of surface active phospholipids to prepare a surface active phospholipid (SAPL) composition for administering to the respiratory or nasal system of a mammal by oral or nasal inhalation to reduce the risk of infections in mammals.

2. Use according to claim 1 in which the SAPL is administered as a dry finely divided powder.

3. Use according to claim 1 or 2 in which the SAPL composition comprises phosphatidyl choline (PC) and phosphatidyl glycerol (PG).

4. Use according to claim 3, in which the PC is dipalmitoylphosphatidyl choline (DPPC) or a mixture of phosphatidyl cholines in which DPPC is the predominant component.

5. Use according to claim 3 or 4 in which the SAPL comprises a blend of PC or DPPC and PG in which the weight ratio of PC or DPPC to PG is from 6:4 to 8:2.

6. Use according to claim 5 in which the ratio is about 7:3.

7. Use according to any one of the preceding claims in which the SAPLs are prepared synthetically.

8. Use according to any one of the preceding claims in which the PC, DPPC and/or PG comprise the dextro-rotatory form.

9. Use according to claim 1 in which the SAPL composition is a dry, finely- divided solid which is a blend of a first component consisting of one or more phosphatidyl cholmes and a second component selected from one or more phosphatidyl glycerols, phosphatidyl ethanolamines, phosphatidyl serines, phosphatidyl inositols, and chlorestyl palmitate.

10. A method of reducing the risk of infections in mammals which comprises administering by oral or nasal inhalation to the respiratory or nasal system of a mammal, before, during or after exposure to inhalable infectious or toxic particles, a surface active phospholipid (SAPL) composition in a sufficient amount and for a sufficient period to form and maintain an at least partial coating of the SAPL on the surfaces of the respiratory tract or nasopharynx.

11. A method according to claim 10 in which the SAPL is administered as a dry finely divided powder.

12. A method according to claim 10 or 11 in which the SAPL composition comprises phosphatidyl choline (PC) and phosphatidyl glycerol (PG).

13. A method according to claim 12, in which the PC is dipalmitoylphosphatidyl choline (DPPC) or a mixture of phosphatidyl cholines in which DPPC is the predominant component.

14. A method according to claim 12 or 13 in which the SAPL comprises a blend of PC or DPPC and PG in which the weight ratio of PC or DPPC to PG is from 6:4 to 8:2.

15. A method according to claim 14 in which the ratio is about 7: 3.

16. A method according to any one of claims 10 to 15 in which the SAPLs are prepared synthetically.

17. A method according to any one of claims 10 to 16 in which the PC, DPPC and/or PG comprise the dextro-rotatory form.

18. A method according to claim 10 in which the SAPL composition is a dry, finely-divided solid which is a blend of a first component consisting of one or more phosphatidyl cholines and a second component selected from one or more phosphatidyl glycerols, phosphatidyl ethanolamines, phosphatidyl serines, phosphatidyl inositols, and chlorestyl palmitate.