WO2012017406A1

WO2012017406A1 - Exogenous pulmonary surfactant preparation comprising a phospholipid and an adjuvans

Info

Publication number: WO2012017406A1
Application number: PCT/IB2011/053471
Authority: WO
Inventors: Rinti Banerjee; Geetanjali Chandrashekhar Chimote
Original assignee: Indian Institute of Technology Bombay
Current assignee: Indian Institute of Technology Bombay
Priority date: 2010-08-05
Filing date: 2011-08-04
Publication date: 2012-02-09
Anticipated expiration: 2013-02-05
Also published as: TW201208690A

Abstract

The present invention describes exogenous surfactant preparations and surface- active drug delivery systems that can be used for the treatment of lung surfactant dysfunction in respiratory distress syndrome (RDS) and respiratory tract infections such as tuberculosis. The surface-active drug delivery systems relate to a drug loaded exogenous pulmonary surfactant vesicle. The vesicle can be a liposome. The vesicles achieve low surface tensions on film compression in the presence of mycobacterial lipids and address the surfactant dysfunction associated with RDS and respiratory tract infections such as tuberculosis. The invention provides for pulmonary surfactant replacement and can be administered as an aerosol or intratracheal instillation for RDS and respiratory tract infections such as tuberculosis, and can include drugs.

Description

EXOGENOUS PULMONARY SURFACTANT PREPARATION COMPRISING A PHOSPHOLIPID AND AN

ADJUVANS

FIELD OF INVENTION

The present invention relates to exogenous surfactant preparations and surface- active drug delivery systems that can be used to mitigate the effects of lung/pulmonary surfactant dysfunction or deficiency in respiratory distress syndrome (RDS) and respiratory tract infections such as pulmonary tuberculosis. The systems provide for delivery of drugs as aerosols or intratracheal instillations. BACKGROUND OF THE INVENTION

Tuberculosis affects about 8-9 million people every year. Tuberculosis predominantly affects the human lungs causing what is known as pulmonary tuberculosis. Pulmonary tuberculosis is a mycobacterial infection caused by acid-fast bacilli Mycobacterium tuberculosis. Primarily, pulmonary tuberculosis affects the lungs where it is associated with areas of focal or widespread atelectasis, and granuloma formation. As the disease progresses, pulmonary tuberculosis ultimately culminates into partial or complete lung collapse. In pulmonary tuberculosis, the acid-fast bacilli Mycobacterium tuberculosis, shed their cell wall lipids at the pulmonary air-aqueous interface. This results in development of lung surfactant dysfunction. Mycobacterial lipids like mycolic acid and cord factor present at the pulmonary air-aqueous interface biophysically inhibit surfactant lipids and result in attainment of higher surface tension (Colloids Surf. B Biointerfaces, 2005, 45, 215-223). Pulmonary tuberculosis is associated with decreased lung compliance, areas of atelectasis and lung collapse (Tubercle, 1951 , 32, 108-1 10). Pulmonary surfactant deficiency or dysfunction results in increased alveolar minimum surface tension on expiration, decreased lung compliance and areas of alveolar atelectasis (Curr. Sci., 2002, 82, 420-428). Advanced stages of pulmonary tuberculosis are associated with respiratory distress/failure that requires medical intervention in the form of ventilator support and administration of effective surfactants. If left unattended, the disease condition progresses to fatal outcome. Respiratory failure and distress arising from widespread involvement of lung in pulmonary tuberculosis/multidrug resistant tuberculosis constitutes an unmet medical need and require therapeutic intervention. Currently, there is no effective and practical therapy for associated pulmonary surfactant dysfunction in tuberculosis which relieves symptoms of breathlessness, decreased work capacity, and correction of surfactant dysfunction.

Another problem associated with pulmonary tuberculosis is the tuberculosis chemotherapy itself. Conventional antitubercular therapy in the form of daily oral tablets is associated with the inability of antitubercular drugs to reach target tissues, short residence time in target organs and an undesirable biodistribution, giving rise to adverse effects. Pulmonary tuberculosis, remains inaccessible or poorly accessible to orally administered antitubercular drugs. The year long anti-tubercular chemotherapy is associated with dose related side effects, which leads to poor patient compliance and emergence of multidrug resistant tuberculosis. The current tuberculosis regimen consists of daily intake of isoniazid, rifampicin, ethambutol and pyrizinamide given as once daily oral tablets over a period of 9 months. The current anti-tubercular drug therapy is associated with drug dosage related side effects like nausea, vomiting, Gl disturbances, flu like syndrome, deranged liver function, nephrotoxicity and ototoxicity. These side effects lead to incomplete treatment which further complicates the problem and leads to the development of multi-drug resistant tuberculosis. Apart from the plethora of problems associated with pulmonary tuberculosis, the current antitubercular therapy fails to bring about early sputum conversion from a bacillary carrier stage to a bacilli free stage. This shortcoming results in rapid disease transmission, especially in overcrowded population pockets. Hence, there is a need to develop drug delivery systems which will decrease the drug dosage and dosage related adverse events.

Respiratory distress syndrome (RDS) refers to a set of pulmonary disorders associated with deficiency or dysfunction of the pulmonary surfactant and manifests clinically in the form of decreased lung compliance. Lung compliance is the ability of the lungs to stretch during a change in volume relative to an applied change in pressure. Compliance is calculated using the following equation, where AV is the change in volume, and ΔΡ is the change in pleural pressure (C = ΔΡ/Δ\ ). RDS results in difficulty in breathing.

Chest radiographs of patients affected with respiratory distress syndrome show loss of lung volume and focal or widespread alveolar collapse (atelectasis). Respiratory distress syndrome affecting the new born (usually preterm new born) is known as neonatal respiratory distress syndrome (NRDS) while respiratory distress affecting the adults is known as acute respiratory distress syndrome. Pulmonary surfactant deficiency or dysfunction is treated by intratracheal administration of commercially available exogenous surfactant replacements (Bose et al., 1990). Exogenous surfactant replacements consist of synthetic or animal derived phospholipid mixtures containing surfactant-associated proteins. They are designed to mimic the endogenous pulmonary surfactant. Pulmonary surfactant is a lipoprotein complex synthesized by type II alveolar cells in vivo and is responsible for reducing pulmonary air-aqueous surface tension to near zero values during expiration, essential for preventing alveolar collapse (Biochimica et Biophysica Acta - Molecular Basis of Disease, 1998, 1408, 79-89).

For an effective surfactant function, it is critical that the surfactants quickly adsorb to the air-water interface, maintain near zero surface tension on end expiration and stabilize the terminal airways. Animal derived surfactants, such as Curosurf™, Survanta™, Neosurf™, fulfil the aforementioned criteria but are expensive, carry the risk of disease transmission and allergic reactions. Synthetic surfactants are expensive but do not manifest the aforementioned problems associated with animal derived surfactants. Synthetic surfactants however do not exhibit the quality of quick adsorption and spreading. Spreading is the ability of the surfactant to uniformly line the entire lung surface even though it is administered at one site. Synthetic lung surfactants currently available in the market, such as Exosurf Neonatal™, ALEC™ (ALEC: Artificial lung expanding compound), do not have this ability. Hence, there is a need for synthetic surfactants which fulfil the aforementioned criteria and are free from the drawbacks associated with animal derived surfactants.

Moreover, in respiratory infections like tuberculosis there is pre-existing lung surfactant dysfunction. Hence, the physical presence of the aerosolised drugs in the lungs should not result in elevation of pulmonary minimum surface tension, which will ultimately worsen the pre-existent lung surfactant dysfunction.

Development of inhalable therapy as an aerosol in diseased patient (a subject or a patient who is a confirmed case of tuberculosis) and as intratracheal instillation in acute and severely ill patients (subjects or patients who require admission in intensive care units and exhibit respiratory distress requiring medical intervention in the form of ventilatory life support) requiring hospitalisation according to this invention ameliorates this problem by direct delivery of the drugs to the site of action and reduction in drug dosage. Since pulmonary tuberculosis is also associated with lung surfactant dysfunction, the inhalable therapy not only needs to be compatible with the surfactant but also mimic the surfactant so as to allow opening of the atelectatic alveoli. Currently, there is no effective and practical therapy for associated pulmonary surfactant dysfunction in tuberculosis which relieves symptoms of breathlessness, decreased work capacity, and correction of surfactant dysfunction. The present invention addresses this need.

Exogenous surfactant preparations and surface-active drug delivery systems of the present invention have the ability to relieve symptoms of breathlessness, decreased work capacity due to correction of surfactant dysfunction in pulmonary tuberculosis at the pulmonary air-aqueous interface.

In this invention exogenous surfactant preparations and surface-active drug delivery systems are to be administered as an aerosol or an intra-tracheal instillation depending upon the clinical need.

SUMMARY OF THE INVENTION

The invention relates to exogenous surfactant preparations and surface-active drug delivery systems which can be used in the treatment of lung surfactant dysfunction in respiratory distress syndrome (RDS) and respiratory tract infections such as pulmonary tuberculosis.

The exogenous surfactant preparations comprise varying combinations of phospholipids and adjuvants. The phospholipids are saturated phospholipid dipalmitoylphosphatidylcholine (DPPC) either alone or in combination with other phospholipids like dipalmitoylphoshatidylethanolamine (DPPE). Turmeric oil or curcumin is used as an adjuvant.

The exogenous surfactant preparations optionally contain an anti-infective agent particularly an anti-tubercular agent and such preparations function as surface-active drug delivery systems. Accordingly, the invention also relates to surface-active drug delivery systems which contain anti-infective agents such as anti-tubercular drugs. The surface-active drug delivery systems are exogenous surfactant based drug delivery systems for pulmonary delivery of anti-infectives such as anti-tubercular agents for treatment of respiratory tract infections like pulmonary tuberculosis. The surface-active drug delivery systems can perform dual action of an exogenous surfactant replacement effective in lung surfactant dysfunction associated with pulmonary tuberculosis and other infectious diseases such as pulmonary infections which comprise of pneumonias, broncho-pneumonias arising from different pathological organisms; and as an inhalable drug carrier for frontline antitubercular drugs or other anti-infective drugs.

An embodiment of the invention relates to surface-active drug delivery systems that relate to antitubercular drug loaded exogenous pulmonary surfactant vesicles. The vesicles refer to liposomes in micron size and nano size range (nanocarriers). The liposomes are made of phospholipid mixtures with adjuvants in varying proportions. These vesicles can be drug loaded and can carry one or more drugs together and are delivered by simple nebulization process. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the effect of formulation 1 B and formulation 2A with and without mycolic acid on minimum surface tension values.

Figure 2 shows the sustained release profiles of formulation 1 B.

Figure 3a shows the in vitro deposition in lower impingement chamber with five minute nebulisation cycle of formulation 1 B.

Figure 3b shows the in vitro deposition patterns in the lower impingement chamber with five minute nebulisation of formulation 2A.

Figure 4a shows the particle size distribution of formulation 1 B.

Figure 4b shows the particle size distribution of formulation 2B.

Figure 5 shows the % capillary opening simulation terminal airway opening using formulation 1 B and formulation 2A.

Figure 6 shows the adsorption potential of formulation 1 B and formulation 2A.

Figure 7 shows the effect of formulation 3A and formulation 4A and other commercially available surfactants on minimum surface tension in presence of mycolic acid.

Figure 8 shows the adsorption profile of a formulation 3A and commercially available surfactants.

Figure 9 shows the % of capillary opening obtaining using a formulation 3A and formulation 4A and other commercially available surfactants.

Figure 10a shows the particle size distribution of formulation 3B.

Figure 10b shows the particle size distribution of formulation 3A.

Figure 1 1 shows the in vitro hemolysis profile of formulation 1 B and formulation 2A. Figure 12 shows the viability percentage of L929 cells after 24 hours incubation with formulation 3A and formulation 1 B. DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it has to be understood that this invention is not limited to particular embodiments. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which the invention belongs.

Definitions

Phospholipids: Phospholipids are a class of lipids which contain a diglyceride, a phosphate group, and a simple organic molecule such as choline.

Adjuvant: An adjuvant is a pharmacological agent that modifies or enhances the effect of other agents used in a formulation.

Formulations: Pharmaceutical preparations consisting of one or more phospholipids (containing two or more phospholipids in different w/w ratios) mixed with adjuvants of herbal oils like turmeric oil and anti-infective drugs like anti-tubercular drugs, physically existing as liposomes.

Exogenous surfactant preparations: Aforementioned formulations which exhibit surface activity at pulmonary air-aqueous interface similar to that exhibited by natural pulmonary surfactant.

Surface active drug delivery system: Drug delivery system which exhibit surface activity mimicking the pulmonary surfactant (pulmonary surfactant refers to the phospholipid mixture which lines the pulmonary air-aqueous interface) is referred to as a surface active drug delivery system. In the context of the present invention, the surface active drug delivery systems are exogenous surfactant preparations containing one or more antitubercular drugs and said drug delivery system acts as a carrier for the drugs.

Lung surfactant deficiency: Conditions where the natural pulmonary surfactant is absent or present in lower quantities than normal.

Lung surfactant dysfunction: Conditions where the natural pulmonary surfactant though present are suboptimal in function. Vesicles/Liposomes: The terms "vesicle" and "liposomes" can be used interchangeably and they refer to self assembled lipid bilayers which entrap a hydrophilic core.

Anti-infective drugs: These drugs include those anti-infective drugs that are conventionally used to treat infectious disorders like pulmonary infections and pneumonias.

Anti-tubercular drugs: These drugs include those anti-tubercular drugs that are conventionally used to treat tuberculosis. A few examples of conventionally used anti-tubercular drugs include isoniazid, rifmpicin, pyrazinamide or ethambutol.

The invention relates to exogenous surfactant preparations and surface-active drug delivery systems which can be used in the treatment of lung surfactant dysfunction in respiratory distress syndrome (RDS) and respiratory tract infections such as pulmonary tuberculosis. As has been indicated herein above that the surface active drug delivery systems are exogenous surfactant preparations containing one or more antitubercular drugs. Accordingly, the surface active drug delivery systems function both as an exogenous surfactant preparation and as a carrier for drugs. Such formulations can be used for reducing surfactant dysfunction and for simultaneously causing sustained release and homogenous distribution of drugs in the lungs when used in the treatment of respiratory tract infections. The invention also acts as an exogenous surfactant preparation per se which can be used in the treatment of conditions of lung surfactant dysfunction like pulmonary tuberculosis.

Accordingly, in one embodiment, the invention relates to a method for treating lung surfactant dysfunction or lung surfactant deficiency in a subject comprising administering to the subject the formulation of the present invention which may be an exogenous surfactant preparation without anti-tubercular drugs or the exogenous surfactant preparation with anti-tubercular drugs (the surface-active drug delivery systems). As has been indicated herein above, the lung surfactant dysfunction may be caused due to respiratory tract infections such as pulmonary tuberculosis or respiratory distress syndrome (RDS); and the lung surfactant deficiency may be caused due to respiratory distress syndrome (RDS).

The exogenous surfactant preparations comprise varying combinations of phospholipids and adjuvants. The phospholipids are saturated phospholipids such as dipalmitoylphosphatidylcholine (DPPC) which is used either alone or in combination with other phospholipids like dipalmitoylphoshatidylethanolamine (DPPE). Turmeric oil or curcumin is used as an adjuvant. Adjuvant refers to a pharmacological agent that modifies the effect of other agents. In this invention, the adjuvant, turmeric oil or curcumin, enhances the adsorption and spreading qualities of the exogenous surfactant preparations.

The invention relates to surface-active drug delivery systems which contain anti- infective agents such as anti-tubercular drugs. The surface-active drug delivery systems are exogenous surfactant based drug delivery systems for pulmonary delivery of anti-infectives such as anti-tubercular drugs for the treatment of respiratory tract infections like pulmonary tuberculosis.

An embodiment of the invention relates to surface-active drug delivery systems that relate to antitubercular drug loaded exogenous pulmonary surfactant vesicles, which serve dual function of being a biocompatible inhalable drug delivery system for pulmonary tuberculosis and an exogenous surfactant replacement for treatment of lung surfactant dysfunction associated with tuberculosis. The vesicles refer to liposomes in micron size and nano size range (nanocarriers). The liposomes are made of phospholipid mixtures with adjuvants in varying proportions. These vesicles can be drug loaded and can carry one or more drugs together and are delivered by simple nebulization process.

The surface-active drug delivery systems are non-invasive, direct lung delivery systems, which cause significant reduction in the drug dosage administered. The surface-active drug delivery systems can perform dual action of an exogenous surfactant replacement effective in lung surfactant dysfunction associated with pulmonary tuberculosis and other infectious diseases such as pulmonary infections which comprise of pneumonias, broncho-pneumonias arising from different pathological organisms; and an inhalable drug carrier for frontline antitubercular drugs or other anti-infective drugs. For effective delivery of drugs to the alveoli and normal breathing, it is essential to maintain terminal airway patency. Lung surfactant has a crucial role in maintaining airway patency.

The invention also relates to exogenous surfactant preparations without antitubercular drugs and are used for the treatment of lung surfactant dysfunction in respiratory tract infections and respiratory distress syndrome (RDS). These exogenous surfactant preparations can be adapted for an adjunct therapy in pulmonary tuberculosis and as a surfactant in critical care cases of exacerbations in pulmonary tuberculosis. These exogenous surfactant preparations relate to exogenous pulmonary surfactant vesicles. These vesicles refer to liposomes in micron and nano size. These exogenous surfactant preparations can be administered by intra-tracheal route, orally inhaled or intranasal routes.

The formulations of the present invention include both "exogenous surfactant preparations" and "surface active drug delivery systems" and comprise vesicles which are made up of mixture of at least DPPC, and turmeric oil or curcumin. The formulations may also include DPPE, isoniazid, ethambutol, rifampicin, and/or lactose in varying combinations and ratios as explained below. The formulations exhibit action of relieving lung surfactant dysfunction and respiratory distress associated with lung infections including advanced stages of pulmonary tuberculosis (which will reduce the need of ventilatory support and respiratory distress in pulmonary tuberculosis) and RDS. The formulations can be used to deliver drugs directly to the lungs, thereby reducing the drug dosage associated toxicity and improving the reach of drugs to the affected area in the lungs. Formulations of this invention can be used to deliver anti-tubercular drugs. Formulations of this invention can also be used to deliver drugs for treatment of other lung infections such as the lung infections arising from pulmonary infectious disease causing pathogens such as Staphylococcus aureus, Streptococcus, Klebsiella, Pneumococcus, Pseudomonas aeruginosa; fungal infections such as aspergillosis and other pulmonary parasitic infections. The therapy has the potential to cause early sputum conversion, which can directly decrease the community disease burden. Moreover, the surface-active drug delivery systems of the present invention have the potential to cause significant dose reduction over the conventional oral antitubercular drug therapy. This is an advantage as it not only decreases the dose related side effects but also makes the therapy more acceptable to the patients. This can increase patient compliance and decrease the problems associated with non-compliance which include development of tuberculosis relapse (in the same patient i.e. the subject being treated for tuberculosis) and multi-drug resistant strain of Mycobacterium tuberculosis. The formulations can also be administered as intratracheal suspension in cases of advanced tuberculosis/RDS when a patient is on ventilatory support. It can be supplied to the end user as a lyophilized pellet that can be reconstituted prior to its use. In an embodiment of the invention "exogenous surfactant preparations" comprises disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC) and turmeric oil. In the formulation, DPPC is the major component (up to but not limited to about 94-95 % by weight). DPPC is obtained from a synthetic source. Turmeric oil constitutes up to but not limited to about 5-6 % by weight and can be replaced by curcumin at the same percentage by weight.

In another embodiment of the invention, the formulation comprises of a second phospholipid, dipalmitoylphosphatidylethanolamine (DPPE) that is used in combination with DPPC. In this embodiment, the formulation comprises DPPC: DPPE in a ratio of 9:1 by weight and about 5-6 % by weight of turmeric oil.

In another embodiment of the invention, the formulation comprises DPPC: DPPE in a ratio of 9:1 by weight and about 5-6 % by weight of curcumin. This formulation can be used with or without anti-infective drugs such as the anti-tubercular drugs.

Anti-infective drugs such as the anti-tubercular drugs can be added to the aforementioned formulations, to act as surface-active drug delivery systems to improve the reach of drugs to areas of alveolar collapse. The surface active drug delivery systems comprise varying combinations of phospholipids, adjuvants and anti-tubercular drugs. Other embodiments of the invention include surface active drug delivery systems comprising either DPPC and one or more anti-tubercular drugs or DPPC, DPPE and one or more anti-tubercular drugs along with adjuvants. Non limiting examples of anti-tubercular drugs that can be included in the formulation include hydrophilic drugs such as isoniazid, ethambutol and ampicillin; and hydrophobic drugs such as aminoglycosides, fluoroquinolones and rifampicin.

In an aspect of the invention, hydrophilic anti-tubercular drugs such as isoniazid and ethambutol can be added.

In another aspect of the invention, hydrophobic anti-tubercular drugs such as rifampicin can be added.

Hydrophobic drugs refer to drugs which do not tend to dissolve in, mix with, or be wetted by water or do so in limited quantities. Hydrophilic drugs refer to drugs which tend to or dissolve in water.

Other anti-tubercular drugs which can be used are pyrizinamide, moxifloxacine, and/or rifabutin.

A formulation of the invention is prepared by thin film hydration method as described below. The thin lipid film is hydrated using physiological saline containing about but not limited to 2 mM calcium adjusted to pH of 7.4. Turmeric oil is added to the aqueous phase. If curcumin is used instead of turmeric oil, it is added along with phospholipid during thin film formation. The formulation when carrying anti-tubercular drugs is optimized to carry antitubercular drugs like isoniazid, rifampicin, and ethambutol (isoniazid, ethambutol in the aqueous phase and rifampicin with DPPC while formation of thin film).

The formulations when carrying anti-tubercular drugs such as isoniazid, rifampicin and ethambutol act as surface active drug delivery systems and exhibit entrapment efficiency of isoniazid upto but not limited to 25 %, upto but not limited to 15 % for rifampicin and upto but not limited to 55 % for ethambutol. These formulations had a hydrodynamic diameter of 1 -5 μιη before extrusion through polycarbonate membrane and acquired a hydrodynamic diameter of 400-800 nm after extrusion through polycarbonate filters (500 nm) (Figure 4b).

In an aspect, the surface active drug delivery system exhibits sustained delivery of drugs.

On nebulization for a period of five minutes the formulations when carrying anti- tubercular drugs exhibit in vitro deposition in the range of but not limited to 12 - 43 % for the three different anti-tubercular drugs (isoniazid, rifampicin and ethambutol) (Glaxo Type single stage impactor, Copley Instruments, Nottingham Ltd) (Figure 3a, 3b). These formulations were able to overcome mycobacterial lipid induced surfactant inhibition (Figure 1 ).

The formulations with or without the anti-tubercular drugs can be stored at 4^QC for a period of 30 days without loss of surfactant activity or aggregation of particles. The formulations can be stabilized as a lyophilized pellet using for example lactose as cryoprotectant. The stability of the formulations can be extended up to 6 months.

As described below, formulations with or without the anti-tubercular drugs exhibited in vitro antimycobacterial activity in the BACTEC460 method used for testing of mycobacterial sensitivity.

As described below, formulations with or without the anti-tubercular drugs exhibit biocompatibility when tested for cytocompatibility using L929cells (ASTM standard), haemocompatibility and absence of complement activation.

The formulations of the present invention in the absence of antitubercular drugs have the capability to overcome mycobacterial lipid, mycolic acid and cord factor induced surfactant inhibition. The formulations reached a minimum surface tension of 1 -7 mN/m in presence of mycobacterial lipids. This is lower than the values of minimum surface tension upto 18 mN/m reached by commercially available surfactants in presence of mycolic acid as shown in Figure 7. As explained above, near zero minimum surface tension is essential for maintaining alveolar stability. Thus formulations of this invention improve the resistance of surfactant composition to mycobacterial cell wall lipid (mycolic acid) induced inhibition.

The formulations (surface active drug delivery system) of the present invention in the presence of antitubercular drugs cause statistically significant improvement in surfactant adsorption (< 30 mN/m) to the interface within one second. This is an improvement over that of DPPC which reaches surface tension of > 30 mN/m (Figure 6) over a period of one second to 30 minutes and is at par with that exhibited by animal derived surfactants.

The formulations (exogenous surfactant preparations) of the present invention in the absence of antitubercular drugs are able to overcome the poor adsorption exhibited by commercially available protein free surfactants and perform as well as animal derived surfactants in the absence of surfactant associated proteins (Figure 8).

The formulations of the present invention in the presence and absence of antitubercular drugs have statistically significant ability to maintain terminal airway patency of 100 % as compared to DPPC alone and other synthetic exogenous surfactants (Figure 5 and Figure 9 respectively). This is better than other protein free surfactants and comparable to animal derived surfactants containing surfactant associated proteins.

The formulations of the present invention contain liposomes in either micron size or nanosize. The formulations include aqueous liposomal dispersion of micron size 1 -4 μιη as well as nanosized vesicles of 400-600 nm in diameter. The nanosized vesicles may be formed by downsizing the micron sized liposomes by extrusion (Figure 10a). Methods of preparing nanosized vesicles like freeze thawing, reverse phase evaporation; high-pressure homogenization may also be used. Formulations of this invention can be aerosolized by using a jet nebulizer to form aerosols of mass median aerodynamic diameter of 1 -10 μιη.

Process of preparation of surface active drug delivery systems.

For the formulation with the anti-tubercular drugs, the drugs were added to the lipids at a ratio of 1 :1 w/w. The ratio can also be changed to drug: lipid ratio of 0.5:1 and lower, and higher to 1 :2, 1 :3, 1 :4 and 1 :5 w/w. As a representative example of surface active drug delivery system, the drugs added included isoniazid, rifampicin, and ethambutol which were added at ratio of 1 :2:3 w/w respectively to the formulation such that the total drug: lipid is maintained at 1 :1 w/w or lower. Rifampicin was added to chloroform-methanol solution containing the phospholipids (either DPPC: DPPE in 9:1 w/w ratio or DPPC only). The phospholipids when used in combination the ratio can also be changed to 8:2 and lower or 10:1 and higher. The solvents were evaporated under vacuum at 40^QC for an hour to form a thin film. This film was further hydrated for a limited period of 1 -2 hours at 40^QC to 45^QC with continuous rotation using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium. The antitubercular drugs, isoniazid and ethambutol, and the adjuvant, turmeric oil were added to the hydrating medium. After formation of vesicles the unencapsulated anti-tubercular drugs were removed by centrifugation at 25,000 g to 35,000 g for fifteen minutes at 4^QC. The unencapsulated drugs refer to the drugs which are not retained by the vesicles. The pellet thus obtained was reconstituted using physiological saline containing 2 mM calcium chloride as the aqueous hydrating medium adjusted to pH of 7.4. The vesicle suspension thus formed has a hydrodynamic particle size in the range of 1 -5 μιη.

In an alternate procedure, the vesicle suspension can be extruded through polycarbonate membranes having a nanometer pore size, for example, 500 nm pore size or homogenised to form the nanovesicles.

Alternatively, other methods of preparing nanovesicles like freeze thawing, high pressure homogenization, microfluidization and reverse phase evaporation may also be used.

For aerosol formation, the formulations of the present invention can be nebulized using a jet nebulizer at the air flow rate of upto but not limited to 10 L/min.

Process of preparation of exogenous surfactant preparations.

Weighed amounts of DPPC or a mixture of DPPC and DPPE in the ratio of 9:1 w/w were dissolved in a chloroform-methanol (ratio) 2:1 solution. The phospholipid ratio can also be changed to 8:2 and lower or 10:1 and higher. Other solvents like methanol may be used instead of the chloroform-methanol solution.

The solvent was evaporated under vacuum at 40°C for ten minutes to one hour and a thin film of the lipid was formed. This film was further hydrated for a period of 1 -2 hours at 40°C to 45°C with continuous rotation using physiological saline containing about but not limited to 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium. Turmeric oil was added to the aqueous hydrating phase (5 to 6 % by weight of the formulation). The vesicle suspension thus formed has a hydrodynamic diameter of 1 -4 μιη.

In another embodiment, the vesicle suspension can be extruded through polycarbonate membranes of having a nanometer (nm) pore size, for example, 500 nm pore size to form nano size liposomes.

For purposes of the description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in are approximations that may vary depending upon the desired properties to be obtained by the present invention. Also, it should be understood that any numerical range recited herein is intended to include all subranges subsumed therein. For example, a range of "1 to 10" is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

The invention is further understood by reference to the following Examples, which are intended to be purely exemplary of the invention. The present invention is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only. Any methods that are functionally equivalent to those described in the Examples are within the scope of the invention. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications fall within the scope of the appended claims. Example 1

Preparation of surface active vesicles carrying anti-tubercular drugs.

For preparation of the vesicles which carry anti-tubercular drugs, DPPC (20 mg) was used with rifampicin (6.6 mg). The aforesaid ingredients were dissolved in 20 ml of chloroform: methanol mixture (2:1 v/v ratio) in a round bottom flask. The solvents were evaporated under vacuum at 40^QC for an hour to form a thin film. This film was further hydrated for a limited period of 1 hour at 45^QC with continuous rotation using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium. The antitubercular drugs isoniazid (3.3 mg) and ethambutol (9.9 mg) were added to the hydrating medium. After formation of vesicles the unencapsulated anti-tubercular drugs were removed by centrifugation at 25,000 g for fifteen minutes at 4^QC. The pellet thus obtained was reconstituted using physiological saline containing 2 mM calcium chloride as the aqueous hydrating medium adjusted to pH of 7.4. The vesicle suspension thus formed has a hydrodynamic particle size in the range of 1 -5 μιη. This process led to the formation of micron sized drug loaded surface-active exogenous pulmonary surfactant vesicles. This is referred to as formulation 1 A.

Another formulation was prepared using DPPC (20 mg) and turmeric oil (1 mg) and anti-tubercular drugs. DPPC (20 mg) and rifampicin (6.6 mg) were dissolved in 20 ml of chloroform: methanol mixture (2:1 v/v ratio) in a round bottom flask. The solvents were evaporated under vacuum at 40²C for an hour to form a thin film. This film is further hydrated for a limited period of 1 hour at 45²C with continuous rotation using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium. The antitubercular drugs, isoniazid (3.3 mg) and ethambutol (9.9 mg) and turmeric oil were added to the hydrating medium. The vesicles formed by the above described process attained particle size with hydrodynamic diameter of 2-3 μιη. This formulation herein after will be referred to as Formulation 2A.

Formulation 1 A and the formulation 2A were extruded through polycarbonate membranes of 500 nm pore size or homogenized to form the surface-active exogenous pulmonary surfactant vesicles in nanosize range which are referred to as formulation 1 B and formulation 2B respectively.

The drugs were added to the lipids at 1 :1 w/w ratio in preparation process for formulation 1 A, formulation 1 B, formulation 2A and formulation 2B. This drugilipid ratio can also be changed to 0.5:1 and lower. The drugs added were isoniazid, rifampicin, and ethambutol which were added at a ratio of 1 :2:3 w/w respectively to the formulation such that the total drug: lipid is maintained at 1 :1 w/w or lower. Example 2

Effect of surface active vesicles containing antitubercular drugs (formulation 1 B and formulation 2A) on mycolic acid induced fluidisation.

The mycobacterial lipid, mycolic acid was mixed with formulation 1 B and formulation 2A. Mycolic acid (27 g) was mixed with either formulation 1 B or formulation 2A and was then pre-dissolved in chloroform: methanol mixture (2:1 v/v) at concentration of 1 mg/ml at 1 :1 w/w ratio. Mycolic acid (27 g) was mixed with commercially available surfactant preparations [Cosurf, Survanta, ALEC (Artificial Lung Expanding Compound) and Exosurf Neonatal] in 1 :1 w/w ratio. In formulation 1 B, the surfactant preparation refers to DPPC at 27 g in 9:1 w/w ratio with 27 g of the isoniazid: rifampicin: ethambutol mixture at 1 :2:3 ratios by weight. In formulation 2A, the surfactant preparation refers to DPPC at 25.6 g + turmeric oil at 1 .35 g with 27 g of the isoniazid: rifampicin: ethambutol mixture at 1 :2:3 ratios by weight (Figure 1 ). The mixture was then deposited on the air-aqueous interface of a Wilhelmy balance maintained at subphase temperature of 37^QC (for closer simulation of body temperature), it forms monolayers. The subphase consisted of physiological saline containing about but not limited to 2 mM calcium chloride adjusted to pH of 7.4. Sub phase pH and temperature conditions are adopted to simulate physiological conditions in vivo. The monolayers were allowed 30 minutes for solvent evaporation and subsequently compressed to 85 % of the initial surface area. Minimum surface tension was recorded at this point (Figure 1 ). The most important property of pulmonary surfactant is to achieve low values of surface tension on compression at the air-aqueous interface. Near zero surface tension values were observed on compression of films of the natural surfactant (J. Appl. Physiol., 1977, 43, 198-203). In the isotherms, minimum surface tension was achieved at maximal compression as seen in figure 1 . It is a reflection of the minimum surface tension values achieved by the surfactant on end expiration. Near zero minimum surface tension is essential for maintaining alveolar stability. As seen in figure 1 , only formulation 2A was able to reach the desired minimum surface tension of < 1 imN/m and formulation 1 B was able to decrease the minimum surface tension to 13 imN/m, which was significant improvement over all the other examined commercial surfactants which reached higher minimum surface tensions in presence of mycolic acid.

The commercially available surfactants used were: Curosurf: Curosurf is an animal derived surfactant of porcine origin. Suspension is a sterile, non-pyrogenic pulmonary surfactant intended for intratracheal use only. It is an extract of natural porcine lung surfactant consisting of 99 % polar lipids (mainly phospholipids) and 1 % hydrophobic low molecular weight proteins (surfactant associated proteins SP-B and SP-C).

Survanta: Bovine lung derived surfactant. It is an intratracheal suspension for surfactant replacement therapy used by health care professionals for prevention and treatment of respiratory distress syndrome (RDS) in premature infants. It is extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin added to it. ALEC: ALEC (Artificial Lung Expanding Compound) is a synthetic animal derived surfactant, which is a mixture of DPPC and Phosphatidyl glycerol (PG) in 7:3 ratio w/w.

Exosurf Neonatal: is a synthetic surfactant used in earlier times (now obsolete) consisting of colfosceril, cetyl alcohol, and tyloxapol combination.

The formulations of the invention carrying anti-tubercular drugs do not interfere with the model lung surfactant and in fact improves its surface active function in presence of mycobacterial cell wall lipids, MA (mycolic acid) and DPPC.

Example 3

Encapsulation efficiency of antitubercular drugs in formulation 1 B and formulation 2A.

After formation of vesicles of formulation 1 B and formulation 2A, the unencapsulated anti-tubercular drugs were removed by centrifugation at 25,000g for fifteen minutes at 4^QC. The pellet thus obtained was reconstituted using physiological saline containing 2 mM calcium chloride as the aqueous hydrating medium adjusted to pH of 7.4. The drug content was estimated for formulation 1 B and formulation 2A by analysing the free drug available in the supernatant obtained by centrifuging formulation 1 B and formulation 2A respectively. The supernatants were analyzed for free drug content using HPLC. The amount of anti-tubercular drugs in the supernatant (w) were then subtracted from the total amount of anti-tubercular drugs added during the liposome formation process (W). (W-w) will give the amount of anti- tubercular drugs entrapped in the pellet. The absence of anti-tubercular drug precipitation and sedimentation was ensured during the whole process. Aqueous solubilities of the three anti-tubercular drugs, isoniazid, ethambutol and rifampicin were also taken into account to eliminate errors occurring due to drug precipitation and sedimentation.

The encapsulation efficiencies with the different formulations were as follows, rifampicin encapsulation efficiency was 13.68±2.45 % in formulation 2A. 23.88 ± 1 .45 % encapsulation efficiency was observed with isoniazid and 51 .76 ± 1 .58 % with ethambutol with formulation 2A in its nanosize vesicle form. Encapsulation efficiency for isoniazid, rifampicin and ethambutol in formulation 1 B nanovesicles was 28.04 ±1 .65, 16.60 ± 1 .84, 59.47 ± 2.45 % respectively. Example 4

Drug release profile of the anti-tubercular drugs from the Formulation 1 B.

Figure 2 compares the in vitro release profiles of anti-tubercular drugs from the Formulation 1 B and the unformulated antitubercular drugs at 37^QC and at pH 7.4 (physiological pH). The unencapsulated drugs and drug release from formulation 1 B was evaluated by dialysis. Statistically significant and marked increase in the cumulative release of the antitubercular drugs from formulation 1 B was observed as compared to the drug release profile exhibited by unformulated antitubercular drugs at 37^QC. Example 5

In vitro deposition of surface active vesicles containing antitubercular drugs in simulated glass lung model.

The nebulizer cup was filled with formulation 1 B and subjected to nebulization. 80 μg of encapsulated isoniazid was deposited in the lower impingement chamber, which corresponds to 1 1 .5 ± 0.61 % of the total encapsulated isoniazid. 20.46 ± 4.14 % of encapsulated ethambutol corresponding to 912.6 μg was deposited in the lower impingement chamber. 371 .5 μg of rifampicin corresponding to 44.07 ± 0.27 % of the total encapsulated rifampicin was deposited in the lower impingement chamber, with five minute nebulization cycle for formulation 1 B. Higher amounts of antitubercular drugs can be deposited by increasing the nebulization period. The values in percentages are expressed as means ± standard deviation of three sets of experiments. As against the formulated drugs, free/unencapsulated anti-tubercular drugs failed to deposit in the twin impinger model. Formulation 2A was subjected to nebulization and the pulmonary drug deposition potential was estimated using the twin stage glass impinger model. This is shown in Figure 3a. Higher amounts of antitubercular drugs can be deposited by increasing the nebulization time, unencapsulated free drugs are not deposited in the chamber even with extended nebulization period.

Figure 3b represents the percentage of drugs deposited in the lower impingement chamber with five-minute nebulization cycle. In general for formulation 1 B, formulation 2A and formulation 2B upto but not limited to 42.17 % of rifampicin deposition is observed in five minute of nebulization. Upto but not limited to 21 .45 % deposition was observed in the lower impingement chamber for ethambutol. Total amount of isoniazid deposited in the lower impingement chamber was upto but not limited to 16.20 %. As against the encapsulated drugs no deposition was observed upon nebulization of the free/unformulated drugs for the same period.

All the drugs were deposited in amounts greater than the minimum inhibitory concentration, which is essentially required for bacterial killing. Therapeutic plasma levels were 3.4-1 1 .7 pg/ml for rifampicin, 2-4 pg/ml for ethambutol while isoniazid reaches a plasma concentration of 2.7-5.6 g/ml when given at oral daily dose of 600 mg, 900 mg, and 300 mg respectively (Int. J. Pharm., 2004, 276, 41 ). Thus the aerosol comprises liposomes of either formulation 1 B or formulation 2A within the respirable range and the formulations had the capability to reach the lower impingement chamber upon aerosolization suggestive of the ability of the particles to reach the alveoli.

Example 6

Particle sizing of formulations with antitubercular drugs

The formulation 1 A, formulation 1 B, formulation 2A and formulation 2B have a particle size with hydrodynamic diameter of 2-4 μιη before extrusion through polycarbonate membranes or before downsizing them by any other alternative procedures like high pressure homogenization or extrusion or sonication. The formulations formed were extruded through polycarbonate membranes of 500 nm diameters to form the nanovesicles in nanometer size range. Hydrodynamic diameter and size distribution of the formulations was determined using photon correlation spectroscopy (PCS) by Zeta Plus (Brookhaven Instrument Corporation, USA). This system utilizes a laser beam to investigate colloidal suspension. The laser beam hits the colloidal suspension and results in scattering of light. These intensity fluctuations were picked up by a detector and used for further analysis to obtain the particle size, which corresponds to the geometric diameter of the particle. As seen in Figure 4a and Figure 4b, formulation 1 A and formulation 2A attain a particle size of 1 -5 μιη before extrusion and 400-500 nm after extrusion, where they are referred to as formulation 1 B and formulation 2B.

Example 7

Airway opening and patency achieved by surface active formulations with antitubercular drugs (formulation 1 B and formulation 2A.)

The liposomal concentration of formulation 1 B and formulation 2A was 1 mg/ml of physiological saline containing 2 mM calcium chloride maintained at pH of 7.4. As seen in Figure 5, all the formulations exhibited 100 % capillary opening suggestive of the capability of the formulations to maintain terminal airway opening.

Example 8

Adsorption of surface active vesicles with antitubercular drugs (formulation 1 B and formulation 2A)

Figure 6 illustrates the comparison of adsorption surface tensions of formulation 1 B and formulation 2A with that of DPPC only formulation. The surface tensions achieved at 0, 0.01 , 0.5 and 30 minutes have been compared. The surface tension achieved at 0.01 minute is of particular relevance and the formulations achieving lower values of surface tension at 0.01 minute are considered superior. Formulation 1 B and formulation 2A exhibited instantaneous reduction in surface tension as against slow and significantly lower reduction in surface tension exhibited by DPPC only formulation, emphasizing the efficacy of the formulations to quickly spread across the lung surface delivering antitubercular drugs, resulting in uniform pulmonary drug distribution. Example 9

Preparation of formulation 3A, formulation 3B, formulation 4A and formulation 4B. The surface active vesicles were also developed in the absence of drugs as pulmonary surfactant replacements. Weighed amount of DPPC (20 mg) was dissolved in a chloroform-methanol 2:1 solution (10 ml). The solvent was evaporated under vacuum and a thin film of the lipid was formed.

This film was further hydrated for a limited period of 1 hour at 45²C with continuous rotation using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium.

Turmeric oil was added to the aqueous hydrating medium at 5 % by weight of the formulation (0.5 mg).

This process led to the formation of micron sized surface-active exogenous pulmonary surfactant vesicles. This is referred to as formulation 3A.

Another formulation was also prepared. Weighed amounts of DPPC (9 mg) and DPPE (1 mg) in 9:1 ratio by weight were dissolved in a chloroform-methanol 2:1 solution (10ml). The solvent was evaporated under vacuum and a thin film of the lipid was formed. This film was further hydrated for a limited period of 1 hour at 45^gC with continuous rotation using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium. Turmeric oil was added to the aqueous hydrating medium at 5 % by weight of the formulation (0.5 mg). This process led to the formation of micron sized surface-active exogenous pulmonary surfactant vesicles. This is referred to as formulation 4A.

Formulation 3A and formulation 4A were extruded through polycarbonate membranes of 500 nm pore size or homogenized to form the surface-active exogenous pulmonary surfactant vesicles in nanosize range which are referred to as formulation 3B and formulation 4B respectively.

Example 10

Ability of formulation 3A and formulation 4A (in the absence of drugs) to nullify the inhibitory effect of mycobacterial lipid-mycolic acid.

The surfactant formulations tested below refer to the formulation 3A and formulation 4A.

The mycobacterial lipid, mycolic acid was mixed with the respective formulations (mycolic acid and formulations were pre-dissolved in choloroform: methanol mixture at concentration of 1 mg/ml) at 1 :1 w/w ratio. Mycolic acid (27 g) was mixed with equivalent weights of commercially available surfactants or formulation 3A or formulation 4A. The commercially available surfactants used were:

Curosurf: Curosurf is an animal derived surfactant of porcine origin. Suspension is a sterile, non-pyrogenic pulmonary surfactant intended for intratracheal use only. It is an extract of natural porcine lung surfactant consisting of 99 % polar lipids (mainly phospholipids) and 1 % hydrophobic low molecular weight proteins (surfactant associated proteins SP-B and SP-C).

Survanta: Bovine lung derived surfactant. It is an intratracheal suspension for surfactant replacement therapy used by health care professionals for prevention and treatment of respiratory distress syndrome (RDS) in premature infants. It is extracted from minced cow lung with additional DPPC, palmitic acid and tripalmitin added to it. ALEC: (Artificial Lung Expanding Compound) is a synthetic animal derived surfactant, which is a mixture of DPPC and Phosphatidyl glycerol (PG) in 7:3 ratio w/w.

Exosurf Neonatal: is a synthetic surfactant used in earlier times (now obsolete) consisting of Colfosceril, cetyl alcohol, and tyloxapol combination

The mixtures prepared above were then deposited on the air-aqueous interface of a Wilhelmy balance maintained at subphase temperature of 37 °C. The subphase consisted of physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4. Sub phase pH and temperature conditions were adopted to simulate physiological conditions in vivo. The monolayers were allowed 30 minutes for solvent evaporation and subsequently compressed to 85 % of the initial surface area. Minimum surface tension was recorded at this point. The most important property of pulmonary surfactant is to achieve low values of surface tension on compression at the air-aqueous interface. Near zero surface tension values were observed on compression of films of the natural surfactant (J. Appl. Physiol., 1977, 43, 198-203). For a surface active drug delivery system and an efficacious surfactant system, it is essential that the formulations reach minimum surface tension of < 10 imN/m independently as well as in presence of inhibitor substances physically present like mycolic acid at the air-aqueous interface which are seen in infectious conditions like tuberculosis. In figure 7, the black bars denote the minimum surface tensions achieved by the formulations alone in the absence of any inhibitory agents. The grey bars denote the minimum surface tensions achieved by the formulations in the presence of mycolic acid which acts as an inhibitor for surfactant function and is present in tuberculosis. Survanta™ and Curosurf™ are commercially available animal derived surfactants and ALEC™ and Exosurf Neonatal™ are synthetic surfactants. Formulation 3A and formulation 4A attain minimum surface tension of 0 - 3 imN/m, which is comparable to animal derived surfactants like Survanta™ and Curosurf™ and superior to synthetic surfactants like ALEC™ and Exosurf Neonatal™. The formulation 3A and formulation 4A retain their ability to attain significantly lower minimum surface tension in presence of mycolic acid (as seen by low values of minimum surface tension in the presence of mycolic acid), which is not exhibited by animal derived surfactants as well as synthetic surfactants (as seen by the high values of minimum surface tensions achieved by Survanta™, Curosurf™, ALEC™ and Exosurf Neonatal™ in the presence of mycolic acid).

Example 1 1

Adsorption profile of formulation 3A (in the absence of drugs).

The ability of formulation 3A (surface active vesicles without any drugs) to adsorb to air-aqueous interface from bulk was tested at 37^QC using a modified Langmuir- Blodgett instrument (figure 8). It consists of a 2 cm diameter Teflon trough to which 3ml of formulation 3A (1 mg/ml) was added. Surface tension was monitored continuously from force exerted on sandblasted Wilhelmy gold plate dipped into interface (American Journal of Physiology, 1972, 223, 715). Adsorption of formulation 3A was measured at 37^QC for 30 minutes. Instantaneous adsorption of the exogenous surfactants ensures quick and widespread spreading of the surfactant on the pulmonary surface. As observed in figure 8, formulation 3A exhibited instantaneous reduction in surface tension as against slow and significantly lower reduction in surface tension exhibited by other synthetic surfactants, emphasizing the efficacy of formulation 3A.

Example 12

Airway opening with formulation 3A and formulation 4A (in the absence of drugs). The ability of the surface active vesicles without any drugs to maintain airway patency was evaluated by using capillary surfactometer. The commercial surfactants Survanta™ and Curosurf™ are available as pellets to be resuspended in the sterile physiological saline. The concentration of commercial surfactants and formulation 3A and formulation 4A for the experiment was 1 mg/ml of physiological saline containing about but not limited to 2 mM calcium chloride maintained at pH of 7.4. As seen in figure 9, the formulation 3A and formulation 4A exhibited 100 % capillary opening suggestive of the capacity of the fomulations to maintain terminal airway opening. 100 % capillary opening is a desired quality to maintain terminal airway patency, especially in cases of respiratory distress.

Example 13

Particle sizing of formulation 3A, formulation 3B, formulation 4A and formulation 4B (in the absence of drugs): Hydrodynamic diameter.

Hydrodynamic diameter and size distribution of the formulation 3A and formulation 3B (surface active vesicles without any drugs) was determined using photon correlation spectroscopy (PCS) by Zeta Plus (Brookhaven Instrument Corporation, USA). This system utilizes a laser beam to investigate colloidal suspension. The laser beam hits the colloidal suspension and results in scattering of light. These intensity fluctuations are picked up by a detector and used for further analysis to obtain the particle size, which corresponds to the geometric diameter of the particle. The formulation 4A and formulation 4B also exhibit particle size profile similar to that of formulation 3A and formulation 3B. The data shown in Figure 10a is for formulation 3B and the data shown in Figure 10b is for formulation 3A. Example 14

Stability of formulation 3B and formulation 1 B (in the absence or presence of drugs respectively).

The stability studies were done to evaluate the efficacy of formulation 3B and formulation 1 B as surfactants and as pulmonary drug delivery systems. For stability studies the samples were stored in a screw capped bottles in a refrigerator at 4^QC away from direct light for a period of one month. Another way the aforesaid formulations were stored was in form of a lyophilized pellet, using lactose as cryoprotectant. Since lactose is included in the list of accepted inhalable ingredient and its safety is known it was used for lyophilization purposes. Lyophilization was done at -40^QC. The formulations were prepared as mentioned in Example 1 and Example 9 and lactose in graded amounts from 10 mg upto but not limited to 100 mg was added in the hydrating medium. As disclosed in tables 1 and 2 the formulations retain their stability profile as a suspension for a period of one month and it can be extended upto 6 months. Table I: Stability profile of the formulation 3B (Stored at 4°C as a suspension)

Table 2: Stability of the formulation 1 B (Stored at 4 °C as a suspension)

Example 15

Mycobacterial sensitivity of formulations in the absence or presence of drugs (formulation 3A, formulation 1 B and formulation 2A).

Mycobacteria were sensitive to formulation 3A, formulation 1 B and formulation 2A. Formulation 3A, formulation 1 B and formulation 2A were tested at concentrations comparable to the minimum inhibitory concentrations of the free drugs. Minimum inhibitory concentrations of isoniazid, rifampicin, and ethambutol are 0.1 μg/ml, 2 μg/ml, and 2.5 μg/ml respectively. Table 3: Mycobacterial susceptibility towards the formulations 3A, formulation 1 B and formulation 2A.

(Drugs entrapped were isoniazid, rifampicin and ethambutol, liposomal suspension added to BACTEC 460 set up for mycobacterial susceptibility,)

Example 16

Biocompatibility of formulation 1 B and formulation 2A. Haemocompatibility

The haemocompatibility assay was performed as per the protocol provided in Journal of Ethnopharmacology, 2001 , 74,239-243.

Lungs are highly vascular organs rich in red cells or erythrocytes. Red cells if lysed or affected due to inhalation of toxic fumes, medications or diseases result in the development of adult respiratory distress syndrome, an almost fatal condition. Red blood cells were used to model cell membranes to evaluate effect of the antitubercular drugs loaded liposomes on erythrocytes, which are abundantly present in the alveoli. If the drug loaded liposome formulation is detrimental to the erythrocytes, it will lyse the erythrocytes, releasing hemoglobin. Presence of red cell components like hemoglobin, fractured red cell membranes cause surfactant inhibition at the air-aqueous interface. Hemolysis of erythrocytes is an indication of the toxicity of the formulation.

Hemolysis percentage in all the formulations of the present invention was near zero. Representative results of formulation 1 B and formulation 2A are shown (Figure 1 1 ). This implies that the erythrocyte membrane integrity was not compromised by the formulations (formulation 1 B and formulation 2A) at the concentrations tested (i.e. 0.1 -1 mg/ml). Red blood cells remain intact in normotonic solutions like normal saline and in contact with biocompatible materials. This indicates that formulation 1 B and formulation 2A did not contain any material, which caused rupture of the RBC membrane, qualifying them as biocompatible biomaterials and suggestive of their safety.

Cytocompatibility.

The cytocompatibility assay was performed as per the protocol provided in J Immunol Methods 1997; 208:151-158.

Viability of fibroblast cells in presence of all the concentrations of formulation 3A and formulation 1 B tested is more than 95 % (Figure 12). Morphology of fibroblast cells is unaltered and monolayer confluence was present. At the evaluated concentrations the formulation 3A and formulation 1 B do not show any cytotoxic effect.

Complement activation study.

The complement activation assay was performed as per the protocol provided in Journal of Biomedical Materials Research Part A, 2009, 89A, 281 -292.

Level of C5a in plasma was measured by ELISA following incubation of formulation 3A, formulation 1 B and formulation 2A with plasma for evaluation of complement activation due to either antitubercular drugs or DPPC. No significant change in C5a level occurs due to antitubercular drug loaded DPPC liposomes at 1 mg/ml DPPC concentration. C5a was measured as C5a desArg which is a cleaved form of C5a. Increased level of C5a occurs in plasma due to activation of complement systems is expected on exposure to toxins and with biomaterials having poor biocompatibility. Cobra venom factor served as a positive control for the study and exhibited a rise in the C5a desArg value to 3,000 ng/ml as against the 1 -7 ng/ml of C5a desArg value observed in plasma from normal healthy adults. All the formulations of the present invention exhibited absence of complement activation and activity.

Claims

We claim:

1 . An exogenous surfactant preparation comprising at least one phospholipid, an adjuvant and optionally one or more anti-tubercular drugs.

2. The exogenous surfactant preparation as claimed in claim 1 , wherein the preparation comprises at least one phospholipid and an adjuvant selected from turmeric oil or curcumin.

3. The exogenous surfactant preparation as claimed in claim 1 or 2, wherein the phospholipid is dipalmitoylphosphatidylcholine (DPPC).

4. The exogenous surfactant preparation as claimed in claim 1 or 2, wherein the phospholipid is a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE).

5. The exogenous surfactant preparation as claimed in any one of the preceding claims 1 to 4, wherein said preparation comprises phospholipids up to 95 % by weight of the preparation.

6. The exogenous surfactant preparation as claimed in claim 2, wherein the adjuvant is turmeric oil.

7. The exogenous surfactant preparation as claimed in claim 6, wherein the turmeric oil is present in an amount of about 5 % to about 6 % by weight of the preparation.

8. The exogenous surfactant preparation as claimed in claim 2, wherein the adjuvant is curcumin.

9. The exogenous surfactant preparation as claimed in claim 8, wherein curcumin is present in an amount of about 5 % to about 6 % by weight of the preparation.

10. The exogenous surfactant preparation as claimed in any one of the preceding claims 2 to 9, wherein said preparation comprises one or more anti-tubercular drugs selected from rifampicin, isoniazid or ethambutol or a combination thereof.

1 1 . The exogenous surfactant preparation as claimed in claim 10, wherein the ratio of the antitubercular drugs to phospholipid ranges between 0.5: 1 and 1 : 5 w/w.

12. The exogenous surfactant preparation as claimed in claim 10 or 1 1 , wherein said preparation functions as a surface-active drug delivery system for the treatment of pulmonary tuberculosis.

13. The exogenous surfactant preparation as claimed in any one of the preceding claims 10 to 12 is a surface-active drug delivery system which exhibits sustained delivery of anti-tubercular drugs.

14. The exogenous surfactant preparation as claimed in claim 13, wherein the surface active drug delivery system delivers the anti-tubercular drugs over a period of 24 hours.

15. A method for treating lung surfactant dysfunction or lung surfactant deficiency in a subject comprising administering to the subject the exogenous surfactant preparation as claimed in any one of the claims 1 to 1 1 .

16. The method according to claim 15, wherein the lung surfactant dysfunction results from pulmonary tuberculosis or respiratory distress syndrome (RDS).

17. The method according to claim 15, wherein lung surfactant deficiency results from respiratory distress syndrome (RDS).

18. The method according to claim 15, wherein the exogenous surfactant preparation is administered intratracheal^ or as an aerosol.

19. A process for preparing an exogenous surfactant preparation comprising a phospholipid, dipalmitoylphosphatidylcholine (DPPC) or a mixture of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) and an adjuvant selected from turmeric oil or curcumin, which process comprises the steps of:

a. dissolving a solution of DPPC, or DPPC and DPPE in 9:1 ratio by weight in a chloroform-methanol 2:1 solution;

b. evaporating the solvent under vacuum to form thin film of the phospholipids;

c. hydrating the thin film of phospholipids for 1 to 2 hours at 45^gC with continuous rotation using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 to obtain hydrating medium; and d. adding the adjuvant, turmeric oil or curcumin to the aqueous hydrating medium to obtain the exogenous surfactant preparation.

20. A process for preparing a surface-active drug delivery system comprising a phospholipid, dipalmitoylphosphatidylcholine(DPPC) or a mixture of phospholipids, DPPC and dipalmitoylphosphatidylethanolamine (DPPE); an adjuvant selected from turmeric oil or curcumin; and one or more anti- tubercular agents; said process comprising the steps of:

a) dissolving DPPC; or DPPC and DPPE in 9:1 ratio in a chloroform- methanol 2:1 solution with a hydrophobic antitubercular drug;

b) evaporating the solvent under vacuum at 40^gC for ten minutes to form a thin film of the phospholipid;

c) hydrating the thin film for 1 to 2 hours at 45^gC using physiological saline containing 2 mM calcium chloride adjusted to pH of 7.4 as the aqueous hydrating medium; and

d) adding one or more hydrophilic antitubercular drugs and turmeric oil or curcumin to the aqueous hydrating medium to obtain the surface-active drug delivery system.

21 . The process according to claim 20, wherein the hydrophobic antitubercular drug is rifampicin.

22. The process according to claim 20, wherein the hydrophilic antitubercular drugs are isoniazid and ethambutol.

23. Exogenous surfactant preparation as claimed in any one of the claims 1 to 1 1 for use in the treatment of lung surfactant dysfunction or lung surfactant deficiency.

24. Exogenous surfactant preparation for use according to claim 23, wherein the lung surfactant dysfunction results from pulmonary tuberculosis or respiratory distress syndrome (RDS).

25. Exogenous surfactant preparation for use according to claim 23, wherein lung surfactant deficiency results from respiratory distress syndrome.

26. Exogenous surfactant preparation for use according to claim 23, wherein the preparation is administered intratracheal^ or as an aerosol.

27. Use of the exogenous surfactant preparation as claimed in any one of the claims 1 to 1 1 for the manufacture of a medicament for the treatment of lung surfactant dysfunction or lung surfactant deficiency.