Title of Invention	STABLE LIPOSOMAL NEBULIZING INHALATION FORMULATIONS FOR CONTROLLED PULMONARY DELIVERY SYSTEMS
Abstract	The present invention discloses a stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in the treatment of asthma to give controlled drug delivery for pulmonary systems. This invention is more particularly relates to liposomal carriers in the < form of nebulizing inhalation formulations that can carry the drug into deep lung and maintain it at the site of action for a prolonged therapeutic effect.

Title of Invention

STABLE LIPOSOMAL NEBULIZING INHALATION FORMULATIONS FOR CONTROLLED PULMONARY DELIVERY SYSTEMS

Abstract

The present invention discloses a stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in the treatment of asthma to give controlled drug delivery for pulmonary systems. This invention is more particularly relates to liposomal carriers in the < form of nebulizing inhalation formulations that can carry the drug into deep lung and maintain it at the site of action for a prolonged therapeutic effect.

Full Text	FORM 2 THE PATENTS ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION [See section 10; rule 13] "STABLE LIPOSOMAL NEBULIZING INHALATION FORMULATIONS FOR CONTROLLED PULMONARY DELIVERY SYSTEMS" (a)BAJAJAMRITA (b) 201, Namita, Gulmohar Road, No 4, Juhu Scheme, Mumbai- 400 049. India (c) Indian National (a) KHALEANUBHA (b) 17-Nandadeep, Pandurang Wadi, 5th Road, Goregaon (East), Mumbai-400 063. India (c) Indian National The following specification particularly describes the nature of this invention and the manner in which it has to be performed. Technical Field of the Invention: The present invention relates to stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in the treatment of asthma to give controlled drug delivery to pulmonary systems. This invention is more particularly relates to liposomal nebulizing inhalation formulations carriers that can carry the drug into deep lung and maintain it at the site of action to give a prolonged therapeutic effect. Background and Prior art: Inhalation aerosols have been employed to treat localized disease states within bronchi. Since pulmonary route of administration can directly deliver therapeutic agents to the diseased regions whilst reducing their distribution to other organs, it provides an excellent example of targeted drug therapy. A more favourable therapeutic index can be obtained for the treatment of pulmonary diseases when drugs are administered by inhalation rather than by the oral route. Bronchodilators, anti-inflammatory agents, mucolytics, antiviral agents are all routinely given as aerosolised formulations. The possibility of systemic absorption of proteins and peptides via the airways has currently stimulated interest in pulmonary delivery systems. A significant disadvantage of many existing inhaled drugs is the relatively short duration of resultant clinical effects and most medications in aerosol form require at least 3-4 times daily administration. Although the onset of action is very rapid, the duration is often short lived as the drug can be removed from the lungs through various clearance mechanisms. A reduction in the frequency of dosing would be convenient, particularly for chronic treatments such as those for asthma. Most bronchodilators, p2-agonists have the potential to induce significant cardiovascular side effects such as hypotension and tachycardia due to stimulation of the p2-adrenoreceptors in the systemic circulation and cross reactivity with cardiac pi- adrenoreceptors. Controlled release of such drugs in the pulmonary region would be particularly beneficial since they could be delivered to and retained at the targeted receptors for a prolonged period of time and thus minimize the biodistribution through systemic circulation. 2 Drugs for inhalation therapy are administered in aerosol form of fine particles dispersed in a gas. An obvious prerequisite for therapeutic efficacy is the ability of the aerosolized drug to reach the peripheral airways. Typically only 10% of inhaled dose reaches the lower airways due to complex functional anatomy of the respiratory tract that prevents the entry of particulate matter. The extent of aerosol deposition in the pulmonary tract is difficult to predict because particles can come in vastly different shapes & sizes. To account for shape and size, aerosol particles are described by their aerodynamic diameter. In recent years several strategies to improve drug delivery to respiratory system have been developed e.g. aerosolised microspheres, nanoparticles and large porous particles. Particulate carriers such as liposomes have many attractive features for pulmonary drug delivery. Liposomes are phospholipid vesicles that are biologically inert in nature, devoid of any antigenic, pyrogenic or allergic reactions and their components can be utilized as those of biological membranes. Liposomes have an aqueous volume, which is entirely enclosed by a membrane composed of lipid molecule. The drug molecules can either be encapsulated in aqueous phase or intercalated into the lipid bilayers. Liposomal encapsulation of compounds can localize and maintain the drug levels in the lungs for an extended period of time. Pharmacologically, there is also evidence that liposomal drugs can produce selective and prolonged effects with decreased systemic toxicity. Physicochemical properties of the entrapped species, the lipid composition of the vesicles and the site of pulmonary deposition are the factors essential in designing a liposomal formulation. Lyophilization is an excellent method to extend the shelf-life of liposomes. Lyophilization and reconstitution of drug-containing liposomes has been achieved without significant effect upon liposome size, release properties, ion gradients or trapping ability. In a study, more than 90% of entrapped drug is retained upon rehydration. Nebulization is probably the simplest and most effective way to deliver liposomes to the respiratory tract; nebulizers employ compressed gas or ultrasound to generate aerosols from aqueous solutions or suspensions of drugs. 3 US 5340587 discloses a system includes a liposome composition comprises beta-adrenoreceptor antagonist in a liposome entrapped form and a device for aerolizinga a metered quantity of the composition. US4895719 discloses a method and apparatus for administering dehydrated liposomes by inhalation. EPO158441 discloses aerosol formulations in aerosol droplet form from water/lipid/ethanol. Jet nebulizer assembly for home administration of drugs in aerosols disclosed in US patent application 2003 0157032. Small particle liposome aerosols for delivery of anti-cancer drugs disclosed in US patent application 20020102296. An inhaler device for use with a medicinal fluid canister is disclosed in US patent application 2004000307. US patent application 20030157032 describes pharmaceutical aerosol formulations comprising therapeutic agent in the form of coated particles in presence of surfactant in a suspension form for the administration by pulmonary route. US 4895719 disclose a system and method for administering a drug, at a selected dose, via the respiratory tract. Spray-dried liposome particles containing the selected dose of the entrapped drug are released into the air in aerosolized form, either by entrainment in an air or propellant stream, or by release from a pressurized can containing a suspension of the liposomes in a fluorchlorocarbon solvent. Liposomes are recognized as a DDS that can improve the therapeutic efficacy and safety of a wide range of compounds. Three decades of research into liposomal drug delivery has led to vastly improved technology in terms of drug capture, vesicle stability and the design of the formulations for special tasks. In parallel, remarkable advances have been made in 4 understanding and controlling behavior in vivo. For future successful development and commercialization of liposomal products key issues of stability, reproducibility, high encapsulation efficiency, particle size control, sterility assurance and scale-up are being successfully addressed. Further, the utility of liposome formulation technology at the floor of industry is to be explored by simplifying, standardizing and optimizing method of preparation and enhancing the storage stability of the product. The objective of the invention is to develop liposomal pulmonary drug delivery systems. Physicochemical properties of the entrapped species, the lipid composition of the vesicles and the site of pulmonary deposition are the factors essential in designing a liposomal formulation that will not only optimize pharmacokinetic and pharmacological profiles but also satisfy pharmaceutical requirements. The present invention overcomes or minimizes many of the problems which have limited systemic drug delivery by inhalation. Liposomal encapsulation of compounds can localize and maintain the drug for longer period of time. Nebulization is the simplest and most effective way to deliver the liposomes to the respiratory tract. The present invention is to develop a controlled release inhalation formulation of salbutamol with liposomal carrier system, establish its safety & study its efficacy in comparison with conventional aerosol inhalation formulations. Objective of the Invention: One objective of the invention is to develop a controlled release inhalation formulation of salbutamol sulphate with liposomal carrier system for a prolonged therapeutic effect. Other objective of the invention is to lyophilize the salbutamol liposomes to improve their long term stability. Other objective of the invention is to establish its safety of the salbutamol liposomes via 5 pulmonary route. Another objective of the invention is to establish its efficacy in comparison to the conventional marketed nebulizing solution formulations. Further objective of the invention is to study the deposition pattern of the drug in the pulmonary region. SUMMARY: The present invention discloses to a stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in nebulizing device for controlled drug delivery for pulmonary systems, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.01% to 0.02%; a-tocoferol-0.005% to 0.01%; and phosphate buffer pH (6.4). This invention is more particularly relates to nebulizing inhalation formulations containing liposomal carriers that can carry the drug into deep lung and maintain it at the site of action for a prolonged therapeutic effect. Three batches of four different formulations Nl, N2, N3 and N4 were prepared and subjected to stability studies. Formulations Nl and N2 are the final optimized formulations contained 0.005% and 0.010% of the anti-oxidant, butylated hydroxyl anisole respectively. Formulations N3 and N4 contained the anti-oxidant, a-tocoferol 0.01% and 0.02% respectively. As liposomal preparations are prone to oxidation, quantity of anti-oxidant plays an important role in the stability of the product. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate, in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; butylated hydroxyl anisole 0.005% in phosphate buffer pH (6.4). 6 A stable liposomal nebulizing inhalation formulation of salbutamol sulphate , in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.010% in phosphate buffer pH (6.4). A stable liposomal nebulizing inhalation formulation of salbutamol sulphate, in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; a-tocoferol 0.01% in phosphate buffer pH (6.4). A stable liposomal nebulizing inhalation formulation of salbutamol sulphate ,in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; a-tocoferol 0.02% in phosphate buffer pH (6.4). The phospholipid is selected from phospholipon 90H, the hydrogenated form of phosphatidylcholine, and phospholipon 90G, the phosphatidyline choline enriched lecithin but, more preferably phospholipid is phospholin 90G. The solvent used to dissolve the lipid is selected from chloroform and methanol, but preferably chloroform. Thin film hydration method is selected for the preparation of salbutamol liposomes. The type of lipid, its concentration, drug to lipid ratio, concentration of other additives such as cholesterol, anti-oxidants, pH of the hydrating buffer, salt or base form of the drug were found to affect the drug entrapment as well as drug release from the liposomes to more or less extent. Rotation speed of rotovapour flask, quantity of glass beads, solvent system, shaking and sonication period, ratio of the batch size and the capacity of the rotary flask etc. are the process parameters that need to be optimized for liposomal preparation. The preparation of liposomes is optimized with respect to the solvent system, glass bead quantity and visicle sizing technique in which, the optimized ratio of glass beads and solvent quantity is 2:3 as it gives liposomes in the size of 10-20 (xm Drug to lipid ratio of 1:10 has showed the maximum drug entrapment in the liposomes. The salbutamol liposomes are found to be bilayered, circular structures under electron microscopy and the size of the liposomes observed are 1 to 5 urn The percentage drug entrapment in the aqueous layer of liposomes is 50 to 52%. The rate of diffusion of the salbutamol liposomes are extended for a period of 9 to 10 hrs. The net respirable fraction is found to be 20 to 24%. 7 The nebulizing formulation of salbutamol liposomes is prepared with isotonicity adjustments by calculating sodium chloride equivalents for all the ingredients. The pH of the phosphate buffer is 6.3 to 6.4 suitable to prepare isotonic nebulizing dispersions. The structure of the liposomes is bilamellar and trilamellar under optical microscopy. The percent drug entrapment in liposomes is 48 to 52%. The in vitro studies showed, 65-70% drug release by the end of 6hrs, 85-90% by the end of 8hrs and the entire drug is released by the end of 9-10hrs. The nebulizing formulation comprises anti oxidants such as butylated hydroxy anisole and a-tocoferol. The salbutamol liposomal nebulizing dispersions are stable at 5°C, 25°, 60% RH for one year. The shelf life for the product is 12months when stored between 5-8°C. Guinea pig is used as animal model for toxicity studies of the liposomal inhalations. Formulations did not show any chronic toxicity, as there were no significant histopathological changes in the lungs, after administration of nebulized liposomal dispersions for prolonged period of 3 weeks, two times a day. Acute toxicity studies at a dose level, 7 times more than that of therapeutic dose showed dose dependent reversible degeneration in the histopathology. At higher dose levels, which are 14 times more than therapeutic dose, irreversible damage to lungs is observed. The anti-histamic effect of pharmacodynamic studies such as branchial hyper reactivity and whole body plethysmography is in vivo. Nose-only method is the technique used for in-vivo evaluation of performance of aerosol in animals. Salbutamol liposomes are lyophilized to improve their long term stability. The percent protection of liposomal nebulizing solution of salbutamol sulphate to histamine induced asthmatic attack is 7 times more than marketed formulations when challenged immediately as well as 4hrs. Detailed Description: The present invention relates to a stable pharmaceutical aerosol formulation which can be administered by the pulmonary route. The use of aerosols for the administration of 8 medicaments by peripheral aerial pathways has been known for several decades. Targeted drug delivery for pulmonary diseases can be achieved by administration via inhalation route. Higher therapeutic index is achieved as aerosol inhalations deliver therapeutic agents to the diseased regions of lungs whilst reducing their distribution to other organs. The deposition of aerosol can be improved with the help of particulate carriers that can carry the drug deep into the lungs and maintain it at the site of action for a longer period; liposomes are one of such drug delivery carriers. Their suitability lies in their natural origin, similar to those in lung secretions. Inhalation aerosols are generally administered by nebulizers, pressurized Meter Dose Inhalers (MDI) or Dry Powder Inhalers (DPI). Experimental The experimental is carried out in the following steps 1) Preparation of salbutamol liposomes 2) Characterization of salbutamol liposomes 3) Preparation of nebulising dispersions 4) Stability studies of the liposomal nebulising dispersions 5) lyophilization of salbutamol liposomes 6) Toxicity studies of liposomal inhalations 7) Pharmacodynamic studies on liposomal nebulizing solutions Example 1 Preparation of Salbutamol Liposomes: A variety of lipids is used in the preparation of liposomes. 'Thin Film Hydration method' is a method of choice for preparation of liposomes. Phospholipon® 90 H, the hydrogenated form of phosphatidylcholine gave liposomal vesicles with large size of 20-30 um. Phospholipon® 90 G; the Phosphatidylcholine enriched lecithin gave maximum liposomes in the range 10-20 um. 9 In order to prepare salbutamoi -loaded liposomes, salbutamoi base as well as salbutamoi sulphate are used. In case of salbutamoi base liposomes, the dispersions are found to discolour gradually by the end of three weeks. Comparative study of liposomal dispersions prepared with various lipids-food grade lecithin, phospholipon 90H and phospholipon 90 G is given in table 1. A series of formulations of liposomal dispersions (Fl to F7 with salbutamoi base and Gl G7 with salbutamoi sulphate) is prepared with various concentrations of the base as well as salbutamoi sulphate ranging from 0.15 to 1.5% as described under table 2. The liposomal dispersions are studied for physical appearance, texture, settling rate, redispersibility and optical microscopy. TABLE - 1 Comparative Study of Liposomal Dispersions prepared with Various Lipids - Food Grade Lecithin, Phospholipon 90H® and Phospholipon 90G® Settling Rate + Slow ++Moderate +++ Fast Redispersibility + Poor ++ Good +++ Excellent Formul ation Colour Texture Settlin gRate Redispersi bility VesicleSizeRange Changes after 3 weeks FoodGradeLecithin Yellowis h Not smooth & presence of agglomerates +++ + 40-50um Turned light brown and formed hard cake. Phospholipon90H® W hite Floccu lated appearance +++ +++ 20-30pm Separated into three layers to give cracked appearance Phosph olipon White Smooth dispersion + +++ 10-20μm WeU dispersed, 10 90G® whitecoloureddispersion. TABLE 2 Liposomal Formulations with salbutamol base and salbutamol sulphate. Formulation Percentage Fl F2 F3 F4 F5 F6 F7 Phospholipon 90G® 0.1 0.25 0.5 0.75 10. 1.25 1.50 Salbutamol base 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Formulation Gl G2 G3 G4 G5 G6 G7 Phospholipon 90G® 0.1 0.25 0.5 0.75 10. 1.25 1.50 Salbutamol sulphate 0.1 0.1 0.1 0.1 0.1 0.1 0.1 11 Liposomes prepared with salbutamol sulphate are showed satisfactory physical properties. Various concentrations of base and sulphate salt of salbutamol formulations (Fl to F7 and Gl to G7) are incorporated as indicated in table 3 and 4. Physical characteristics are studied and optical microscopy is also performed. TABLE - 3 Physico-Chemical Characterization of Liposomal Formulations Prepared With Salbutamol Base. Formul ation Fl F2 F3 F4 F5 F6 F7 Colour Off-white Off-white Off-white Yellowish White Yellowish White Yellowish Yellowis h Texture Smooth Smoot h Smoot h Thicksuspension Agglomer ates Agglomer ates Agglom erates Settling Rate + + ++ +++ +++ ++++ ++++ Redispe rsibility +++ +++ +++ ++ ++ + + Microsc opy Multi lamellar Multilamellar Multilamellar Clusters Clusters Clusters Clusters Vesicle size 10-20um 10-20um 10-20pm 20-30μm 20-30μm 20-30μm 20-30pm After 3 weeks Yellow YeUo w Brown ish Brownish Brownish Brown Brown Settling Rate + Slow ++ Moderate +++ Fast Redispersibility + Poor ++ Good +++ Excellent 12. TABLE 4 Physico-Chemical Characterization of Liposomal Formulations prepared with Salbutamol sulphate. Formula tion Gl G2 G3 G4 G5 G6 G7 Colour White White White White White White White Texture Smooth Smooth Smooth Thick suspension Agglo merates Agglom erates Agglo merates Settling Rate + + + ++ +++ ++++ ++++ Redispers ibility +++ +++ +++ ++ ++ + + Microscopy Multila mellar Multila mellar Multila mellar Multilamell ar Cluste rs Clusters Clusters Vesicle size 10-20pm 10-20pm 10-20pm 20-30pm 20-30pm 20-30pm 20-30pm After 3 weeks White White White White White White White Settling Rate + Slow ++ Moderate +++ Fast Redispersibility + Poor ++ Good +++ Excellent Process is optimized with respect to the solvent system (liposomal formulations with different solvent blends such as HI, H2, H3), glass bead quantity (formulations with varying proportions of glass beads such as II, 12, 13 and 14) and vesicle sizing technique(optimization of viscicle sizing with different shaking times of formulations Jl, J2, J3,and J4). The details are given in table 5. As a solvent system for lipid film formation, chloroform alone is found to give desired film uniformity as compared to its combination with methanol. 13 TABLE - 5 Process Optimization for Preparation of Liposomal Dispersions of Salbutamol sulphate TABLE - 5 A Solvents Liposomal Formulations with Different Solvent Blends HI H2 H3 Chloroform 1 2 1 Methanol 1 1 0 Film Uniformity + ++ +++ TABLE - 5 B Formulations with varying Proportions of Glass Beads Formulation 11 12 13 14 Glass Beads '■ solvent 1 = 3 2:3 1 = 2 0:1 Uniformity of Film + +++ ++ - Separation of Vesicle Aggregates + +++ ++ - Vesicle Size (v) 30 16 24 42 TABLE - 5 C Optimization of Vesicle Sizing Technique Formulation Jl J2 J3 J4 Shaking Time 0 2hrs 4hrs 6hrs Number of Particles of size l-5p.m 5 - 10n m 8 16 10 18 14 1 Dispersion broke Uniformity of Film + Moderate ++ Good +++ Excellent - Poor 14 Glass beads: solvent quantity ratio when studied, 2: 3 ratio is found to be the optimum as it gave liposomes size in the range of 10-20um and vesicular clusters are separated. A standardized method of mechanical shaking and sonication is proved to be a suitable technique for vesicle sizing. Four hours shaking with intermittent sonication for 1 min, after every 30min of shaking gives desired vesicle size, which is l-3μm whereas excessive shaking resulted into breaking of the dispersion. The formula is optimized for the basic components like lipid concentration and drug to lipid ratio. Formulations Kl, K2, K3, K4 and K5 with varying concentrations of phospholipon 90G with salbutamol sulphate is prepared and observed the physical properties of the formulations and size of the liposomes. The physical property of the salbutamol sulphate liposomes is observed under electron microscopy. Electron micrographs of the salbutamol sulphate liposomes confirmed the circular bilamellar structure of the viscicle size 1-3 [i as shown in fig 1 and 2. Formulations of LI, L2 and L3 are prepared with varying proportions of drug to lipid ratio and observed the physico -chemical studies. Formulation with 1 % phospholipon 90G gives the optimum results in terms of the physical properties and the liposome vesicle size is given in table 6. TABLE - 6 Formula Optimization for the Liposomes of Salbutamol Sulphate. TABLE 6 A Varying Concentrations of Phospholipon 90G Formulation Percentage Kl K2 K3 K4 K5 Phospholipon 90G 0.1 0.5 1.0 1.25 1.5 Salbutamol sulphate 0.1 0.1 0.1 0.1 0.1 TABLE - 6 B Physical Observations of Liposomal Formulations ofSalbutamol sulphate prepared with Phospholipon 90G. 15 Formulation Kl K2 K3 K4 K5 Colour White White White White White Texture Thin suspension Thin suspension Smooth Susp. Thick suspen. Gel like consistency Settling Rate +++ ++ + + - Redisper-sibility ++ +++ +++ ++ Not apphcable Microscopy Multilamellar Multilamellar Multilamellar Clusters Clusters oflargevesicles Vesicle Size l-5μm l"5μm 2-3μm 10-20μm 10-30μm Settling Rate + Slow ++ Moderate +++Very Fast Redispersibility + Poor ++ Good +++ Excellent TABLE - 6 continued Formula Optimization of the Liposomal Preparations of Salbutamol Sulphate. TABLE -6 C Formulations with Varying Proportionsof Drug to Lipid Formulation LI L2 L3 Phospholipon 90G 1 part 1 part 1 part Salbutamol sulphate 5 parts 10 parts 20 parts Physico-chemical Study of Formulations Prepared with Varying Proportions of Drug to Lipid Formulation LI L2 L3 Appearance White colour suspension White colour suspension White colour suspension 16 Redispersibility +++ +++ ++ Settling Rate + + ++ % Drug entrapment 30.62 42.55 36.21 Settling Rate + Slow ++ Moderate +++ Very Fast Redispersibility + Poor ++ Good +++ Excellent % Drug entrapment values were obtained by taking average of six observations. Drug: lipid ratio 1:10 has shown the maximum drug entrapment in the liposomes (table 6). The formulation is also found to have satisfactory physical properties. Optimization of other components of the liposomal formulation such as cholesterol concentration, butylated hydroxyl anisole (BHA) concentration and pH of the hydrating buffer is carried out with the help of 3 Factorial Design. The concentrations of the three components are varied at three different levels of low, medium and high. The concentration of BHA has slight effect on the percent drug entrapment where as pH of hydrating buffer has significant effect and cholesterol concentration has no significant effect on the percent drug entrapment. The factorial design model is further reduced to 23 by considering the three variables at two levels, low and high. They are studied in terms of the percent drug entrapment. It is observed from these studies that cholesterol concentration in the formulation has significant effect on the percent drug released from the liposomes. The pH of the hydrating buffer also has some effect on the percent drug released. However, BHA concentration has absolutely no significant effect on this study parameter. The antioxidant BHA is replaced by another antioxidant a-tocoferol. From the previous studies, cholesterol concentration is optimized to 0.25% and hence the study design is reduced to 2 factorial levels. Concentration of a-tocoferol and pH of the hydrating buffer are varied at two levels, high and low and the formulations are studied in terms of the percent drug entrapment and percent drug released. Both a-tocoferol concentration and pH of the hydrating buffer have slight effect on the percent drug entrapped. Release of 17 the drug from liposomes is mainly affected by the pH of the hydrating buffer. The pH of the buffer 6.4 has shown maximum drug entrapment and the release is extended for 10 hours. The effect of process variables viz. rotation speed and ratio of batch volume to rotary flask capacity is studied for the formulations prepared by optimized formula. The maximum drug entrapment is found at a lower rotation speed of 30 rpm where as with the maximum rotation speed, the drug entrapment is found to minimum. Optimum ratio of batch volume: rotary flask volume is found to be 1: 6.5. Maximum drug entrapment is obtained at this ratio. Morphology of the liposomes, its drug entrapment, the percent drug released from liposomes is determined by the components of the formulation as well as the processing parameters. The final formulations of salbutamol liposomes prepared for stability studies and optimized process variables are shown in table 7. TABLE- 7 Ingredients I II III IV Phospholipon@90G 1.0% 1.0% 1.0% 1.0% Cholesterol 0.25% 0.25% 0.25% 0.25% Salbutamol sulphate 0.1% 0.1% 0.1% 0.1% Chloroform 100ml 100ml 100ml 100ml Butylated Hydroxy Anisol 0.01% 0.02% - - a-tocoferol - - 0.005% 0.01% Phosphate buffer pH 6.4 q.s 100ml 100ml 100ml 100ml Optimized Process: 18 Method: Thin Film Hydration Glass beads: 40g Rotation Speed: 30 rpm Vesicle Sizing: Four hours mechanical shaking with intermittent cycles of 1 min sonication after every 30min. shaking. As formulations with BHA and a-tocoferol did not show much difference in the drug entrapment and release profile with respect to their concentration used as well as no added advantages over the other, salbutamol liposomal formulations with both the antioxidants having two different concentrations are further subjected to accelerated stability studies. Example 2 Characterization of Salbutamol Liposomes: The liposomes are characterized for their physical attributes such as shape, size and size distribution, lamellarity, percent drug entrapped and drug release profile. To determine the percent drug entrapment in the salbutamol liposomes, a technique of centrifugation is adopted for separating unentrapped drug, followed by colorimetric estimation of the drug content. Percent drug entrapment for salbutamol liposomes are found in the range of 50- 52%. The DSC thermogram is carried out for various liposomes such as empty liposomes along with cholesterol, without cholesterol, salbutamol liposomes along with cholesterol and again without cholesterol. From the DSC thermograms of liposomal dispersions of salbutamol confirmed that cholesterol gets into the lipid bilayers and gives rigidity to the lipoidal walls and salbutamol sulphate is situated in the aqueous compartment of the liposomes. Transmission Electron Microscopy studies are confirmed the bilamellar structure of the vesicles. The vesicle size of the liposomes is in the range of 1-2 um. The Dynamic Light Scattering studies have confirmed the size of the liposomes, which is in the range of 1-3μm. 19 The rate of diffusion is studied through a diffusion bag showed release extended over the period of 9-10 hours. The unentrapped portion of the drug, which is about 50%, available within first 2hours. Subsequent release of the drug from the entrapped portion is about 70% in 6 hours and 85-90% by the end of 8 hours. In-vitro pulmonary deposition studies are carried out by Twin Impinge apparatus. It is found that, 72-77 % of drug deposited in the device and 2-4 % in the stage I. The Net Respirable Fraction, which is indicated by stage II, is found to be 20-24%. Example 3 Preparation of Nebulizing solution: Nebulizing solutions are usually formulated in water, although other cosolvents such as glycerine, propylene glycol and ethanol may be used. Nebulizing solutions needs to be iso-osmotic and hence an osmotic agent has to be added in the formulation. For preparing nebulizing solution of the salbutamol liposomes, isotonicity adjustments are done by calculating sodium chloride equivalents for all the ingredients. However, the additional quantity of sodium chloride required for isotonicity adjustment is taken care by the sodium chloride from the phosphate buffer pH 6.4 and hence, no extra sodium chloride is needed in the formulation. Formulations are prepared and evaluated for physico-chemical characteristics. White colour, fine, uniform dispersion with pH in the range of 6.3-6.5 is prepared. Optical microscopy has showen bi-tri lamellar structure. Laser diffraction studies by Malvern Mastersizer has showed that 90% of the particles are in the range 1-3 urn. Electron Microscopy has confirmed the bilamellar structure of the liposomes. Percent drug entrapment in liposomes is found in the range of 48-52%. Total drug content is 99-102%. In-vitro diffusion studies have showed the release of about 40% of the dose in first 2 hours, which is the unentrapped portion immediately available for the action. By the end of 6 hours, 65-70% of salbutamol sulphate is released and 85-90% of salbutamol sulphate 20 is released by the end of 8 hours and by the end of 9-10 hours, the entire drug is released. Example 4 Stability studies of the Liposomal Nebulizing solution: Three batches of four different formulations Nl, N2, N3 and N4 are prepared and subjected to stability studies. Formulations Nl and N2 are the final and optimized formulations contained 0.005% and 0.010% of the anti-oxidant, butylated hydroxyl anisole (BHA) respectively. Formulations N3 and N4 contained another anti- oxidant, a- tocoferol 0.01% and 0.02% respectively. As liposomal preparations are prone to oxidation, the quantity of anti-oxidants plays an important role in the stability of the product. Hence, stability study is carried out with two different anti-oxidants at two levels of concentrations. The four formulations i.e. formulations containing two different antioxidants with two different concentrations are subjected to accelerated stability studies at 5°C, 25°C at 60% RH and 40°C at 75% RH. The samples are withdrawn at the end of 1, 2 and 3 months and evaluated for physical appearance, redispersibility, drug entrapment, drug content, particle size and release profile. The stability data of 3 months is summarized under tables 8-9. The stability study is further continued up to at 5°C and 25°C at 60% RH. Samples are with drawn at the end of 6 and 12 months and analyzed the results, which are given in tables 10-13. 21 TABLE- 8: 3 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing BHA 0.005% in Formulation (Nl) and BHA 0.01% in Formulation (N2), at different conditions of temperature s.No Test Formulation Nl (BHA 0.005%) Formulation N2 ( BHA 0.0 1%) Initial 5°C 25°C, 60% RH 40°C,75%RH Initial 5°C 25°C,60%RH 40°C,75%RH 1 Appea ranee White Dispersion Off-white dispersi on Yello wish white disp. White Dispersion Off-white dispers ion Yello wish Disper sion 2 Redisp ersibility +++ +++ +++ +++ +++ +++ +++ +++ 3 %Drug Conte nt 99.51 101.14 100.23 100.89 101.16 99.63 100.44 99.72 4 DrugEntrapment 52.58 % 50.14 % 49.28% 24.36 % 50.87 % 51.68 % 52.78 % 24.75 % 5 Particl e Size 2.62u 2.94u 3.04u 15.96n 2.52u 3.25u 3.09u 16.63u 6 Releas eProfile T30 13.6 % 16.24 % 15.42% 29.30 % 14.42 % 16.73 % 15.20 34.12 T3 49.72 % 48.37 % 50.12% 83.14 % 45.14 % 51.15 % 46.02 % 88.90 T6 74.22 % 79.93 % 81.45% _ 69.89 % 78.40 % 70.01 % T8 94.61 % 93.12 % 95.17% _ 90.36 % 92.85 % 91.91% T10 103.2 8% 106.21 % 104.26% — 105.55 % 106.77 % 103.23 % — +++ Excellent Each observation is an average of three batches subjected to stability studies. 22 TABLE- 9 : 3 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing a-Tocoferol 0.01%» in Formulation (N3). and 0.02% in Formulation (N4), at different conditions of temperature. s.No Test Formulation N3 (a -Tocoferol 0.01%) Formulation N4 (a -Tocoferol 0.02%) Initial 5°C 25°C,60%RH 40°C,75%RH Initial 5°C 25°C,60%RH 40°C,75%RH 1 Appear an ce White Dispersion Yellowishwhitedispersion. White Dispersion Yellowishwhitedispersion 2 Redisper sibility +++ +++ +++ +++ +++ +++ +++ +++ 3 % Drug content 100.26 101.12 99.64 101.06 100.32 99.74 100.05 99.45 4 DrugEntrapment 52.11 ■% 50.85% 51.76 % 24.16 % 54.18 % 51.0 8% 50.13 % 25.14 % 5 Particle Size 2.57u 2.97n 3.09u 12.34u 3.12u 3.04 3.14u 17.13u 6 Release ProfileT30 19.04 % 18.07 17.55 29.87 16.43 % 17.5 2 16.25 30.45 T3 48.55 % 50.77 47.12 83.40 49.77 % 46.2 9 45.62 78.84 T6 72.81 % 77.15 74.86 107.03 71.90 % 75.2 4 74.41 108.45 T8 96.20 % 94.13 95.10 — 94.75 % 93.65 94.26 _ T10 — 16.14 16.70 — 112.40 % 105.22 108.12 — +++ Excellent Each observation is an average of three batches subjected to stability studies 23 TABLE-10: 6 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing BHA 0.005% in Formulation (Nl) and BHA 0.01% in Formulation (N2), at different conditions of temperature S. No. Test Formulation Nl (BHA 0.005%) Formulation N2 (BHA 0.01%) Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH 1 Appearance White Dispersion White Dispersion 2 Redispersibility +++ +++ +++ +++ +++ +++ 3 %Drug Content 101.21 100.69 100.42 101.01 99.58 99.84 4 Drug Entrapment 52.58% 49.65% 47.97% 50.87% 50.06% 46.55% 5 Particle Size 2.62fi 2.92^ 1.23n 2.52n 2.84u 2.03u 6 Release Profile T30 13.6% 15.32% 14.25% 14.42% 13.33% 16.04% T3 49.72% 46.31% 48.93% 45.14% 46.79% 49.96% T6 74.22% 72.19% 73.11% 69.89% 71.09% 74.27% T8 94.61% 91.20% 92.46% 90.36% 92.44% 95.68% T10 103.28 % 101.67% 103.88% 105.55% 100.57 % 105.12% +++ Excellent Each observation is an average of three batches subjected to stability studies TABLE- 11: 6 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing a-Tocoferol 0.01% in Formulation (N3) and a -Tocoferol 0.02% in Formulation (N4), at different conditions of temperature s.No Test Formulation N3 (a -Tocoferol 0.01%) Formulation N4 (a -Tocoferol 0.02%) Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH 1 Appearanc e White Dispersion White Dispersion 2 Redispersi bilty +++ +++ +++ +++ +++ +++ 3 % Drug content 100.23 99.70 101.22 100.41 99.66 101.14 4 DrugEntrapment 52.11% 48.21% 46.10% 54.18% 49.90% 47.15% 5 Particle Size 2.62^1 1.96u 2A2\i 2.52^1 2.61u 3.31u 24 6 Release ProfileT30 13.6% 15.21% 17.80% 14.42% 16.02% 15.94% T3 49.72% 47.26% 46.03% 45.14% 49.17% 51.44% T6 74.22% 70.24% 74.12% 69.89% 72.25% 74.23% T8 94.61% 91.62% 93.68% 90.36% 92.42% 94.55% T10 103.28% 101.35% 104.37% 105.55% 104.19% 107.86% +++ Excellent Each observation is an average of three batches subjected to stability studies TABLE-12: 12 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing BHA 0.005% in Formulation (Nl) and BHA 0.01% in Formulation (N2), at different conditions of temperature s.No. Test Formulation Nl (BHA 0.005%) Formulation N2 (BHA 0.01%) Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH 1 Appear an ce White Dispersion White Dispersion 2 Redispers ibilty +++ +++ +++ +++ +++ +++ 3 % Drug content 101.07 100.80 100.41 100.61 101.22 99.10 4 DrugEntrapment 52.58% 50.87% 42.52% 51.81% 48.61% 41.95% 5 Particle Size 2.62ja. 1.51u 3.21u 2.52u 2.14u 3.15u 6 Release ProfileT30 13.6% 15.32% 17.52% 14.42% % 13.96% 18.40% T3 49.72% 53.21% 54.10% 45.14% 43.58% 47.69% T6 74.22% 76.22% 74.64% 69.89% 73.28% 77.55% T8 94.61% 98.11% 96.17% 90.36% 94.79% 92.06% T10 103.28% 106.16% 105.37% 105.55% 101.84% 106.88% 25 TABLE- 13 : 12 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing in a -Tocoferol 0.01% Formulation (N3) and a -Tocoferol 0.02% Formulation (N4), at different conditions of temperature s.No. Test Formulation N3 (a-Tocoferol 0.01%) Formulation N4 (a-Tocoferol 0.02%) Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH 1 Appear an ce White Dispersion White Dispersion 2 Redispers ibilty +++ +++ +++ +++ +++ +++ 3 % Drug content 100.66 101.81 99.57 100.17 101.02 100.71 4 DrugEntrapment 52.11% 48.32% 42.12% 54.18% 47.41% 41.89% 5 Particle Size 2.62u 2.34 1 10.12μ 2.52μ 2.55μ 24.33μ 6 Release ProfileT30 13.6% 16.02% 19.56% 14.42% 15.75% 18.28% T3 49.72% 52.66% 56.27% 45.14% 48.32% 49.62% T6 74.22% 72.32% 79.31% 69.89% 73.20% 77.74% T8 94.61% 91.42% 98.54% 90.36% 92.11% 97.43% T10 103.28% 101.65% 108.36% 105.55% 103.13% 107.05% +++ Excellent Each observation is an average of three batches subjected to stability studies The liposomal nebulizing dispersions of salbutamol sulphate are found to be stable at 5°C and 25°C, 60% RH. The liposomal nebulizing dispersions of salbutamol sulphate are found to be more stable at 5°C for 12 months. Leakage of the entrapped drug is observed at 40°C at 75% RH and found to increase with the increasing exposure, a-tocoferol is found to be more acceptable anti-oxidant, in comparison with BHA. 26 Example 5 Lyophilization: Lyophilization of liposomal dispersion of salbutamol results in a dry, waxy mass. Almost 12-15% of the entrapped drug is lost during the process of lyophilization. DSC thermograms of lyophilized liposomes showed the formation of a new entity when compared with the individual DSC scans. Scanning Electron Microscopy has confirmed the bilamellar structure of the liposomes even after the process of lyophilization. The lyophilized liposomes of salbutamol sulphate can not be formulated as MDI preparations due to its poor dispensability in the propellant HFA 134a. Liposomal nebulizing solution for reconstitution can be formulated with 10-15 %, escape of the drug from the liposomes. The lamellar structure of the liposomes in the reconstituted product is unaltered but, showed slight increase in the vesicle size from 1-5 umto 5-10 urn. Example 6 Toxicity studies of liposomal inhalations Guinea pig is used as animal model for toxicity studies of the liposomal inhalations. Ultrasonic nebulizer with nose applicator is found to be convenient for administration of the test nebulizing substance to the animals. The formulations did not show any chronic toxicity, as there are no significant histopathological changes in the lungs, after administration of nebulized liposomal dispersions for prolonged period of 3 weeks, two times a day. Acute toxicity studies at a dose level 7 times more than that of therapeutic dose showed dose dependent reversible degeneration in the histopathology. At a higher dose level, which is 14 times more than the therapeutic dose, irreversible damage to lungs is observed. This indicates high margin of safety. The placebo studies have confirmed mild to moderate congestion and interstitial pneumonia at the same dose levels indicating the safety of the excipients even at higher 27 concentrations. Example 6 Pharmacodynamic studies on Liposomal Nebulizing Solutions: Two methods are applied in order to study the efficacy of the liposomal inhalation of salbutamol. a. Bronchial Hyper reactivity by Whole Body exposure method and Nose- Only Exposure method. b. Whole Body Plethysmography. Bronchial Hyper reactivity: i) Whole Body Exposure method: The marketed sample has showed percent protection to bronchial hyper-reactivity in the range of 160-180%, 20min after administration; where as the percent protection by the liposomal nebulizing solution of salbutamol sulphate is in the range of 275-290%. When histamine challenge is given after 8 hours, the percent protection to bronchial hyper-reactivity of the marketed preparation is reduced to 85-95%; where as the liposomal nebulizing solution of salbutamol sulphate showed the percent protection between 165-180%. ii) Nose Only Exposure Method: The protection to bronchial hyper-reactivity is found to improve by Nose-Only Exposure Method. The marketed preparation has showed 225-235%) protection, immediately 30 min after the administration of the drug; where as that for liposomal nebulizing solution, the protection to bronchial hyper-activity is in the range of 380-395%), showing better protection. The anti-asthmatic effect after 8 hours is found to reduce around 130%) for the 28 marketed preparation, where as for the liposomal preparation it is found to be around 225%. The percent protection is calculated as= Pre convulsion time- Pre convulsion time (Treated) (Untreated) Percent protection—- X 100 Pre convulsion Time (Untreated) It is clearly revealed from the studies that after the treatment with the antiasthmatic formulations in the form of liposomal nebulizing dispersions, there is a decrease in the pulmonary airway resistance when challenged by histamine, 30min as well as 8 hours after the administration. This is indicated the potential of proposed controlled release pattern obtained from the salbutamol liposomal nebulizing dispersions. b. Whole Body Plethysmography: The average percent protection after 30 minutes of the histamine challenge is found to be 86.49% and 73.83%, for the marketed conventional nebulizing preparation and the liposomal nebulizing solution respectively. The average percent protection after 4 hours is found to be 7.16% and 51.28% for the marketed preparation and the liposomal nebulizing solution respectively. The study performed using Whole Body Plethysmometer showed that the conventional marketed inhalation and liposomal nebulizing dispersion has good antiasthmatic effect immediately after the inhalation. The liposomal nebulizing solution of salbutamol sulphate has showed better percent protection to the histamine induced asthmatic attack in the guinea pigs, immediately after 30min as well as after 4 hours. 29 When compared with the marketed preparation, the liposomal preparation has showed the percent protection almost 7 times more than the marketed preparation, when challenged immediately and after 4 hours. Thus, the prolonged therapeutic effect of the liposomal preparation is confirmed. Description of drawing: Fig 1 and Fig 2 illustrates the physical appearance of the salbutamol sulphate liposomes under electron microscopy. Electron micrographs of the salbutamol sulphate liposomes confirmed the circular bilammellar structure of the viscicles and the viscicles size is in the range of 1-3 um. "While the present invention is described above in connection with preferred or illustrative embodiments, these embodiments are not intended to be exhaustive or limiting of the invention. Rather, the invention is intended to cover all alternatives, modifications and equivalents included within its spirit and scope, as defined by the appended claims". 30 We claim 1. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in nebulizing device for controlled drug delivery for pulmonary systems, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.005% to 0.01%; a-tocoferol- 0.01% to 0.02%; in phosphate buffer pH (6.4). 2. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1 %; chloroform-100ml; butylated hydroxyl anisole 0.005% in phosphate buffer pH (6.4). 3. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.010% in phosphate buffer pH (6.4). 4. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1 %; chloroform-100ml; a-tocoferol 0.01% in phosphate buffer pH (6.4). 5. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; a-tocoferol 0.02% in phosphate buffer pH (6.4). 6. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 5 wherein the said phospholipid is selected from phospholipon 90H, the hydrogenated form of phosphatidylcholine, and phospholipon 90G, the phosphatidyline choline enriched lecithin. 7. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 6 wherein the said phospholipid is phospholin 90G. 31 8. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 7 wherein the solvent used to dissolve the lipid is selected from chloroform and methanol but preferably chloroform. 9. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 8 wherein the preparation of liposomes is optimized with respect to the solvent system, glass bead quantity and vesicle sizing technique. 10. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 9, wherein the said optimized ratio of glass beads and solvent quantity is 2:3 as it gives liposomes in the size range of 10-20um 11. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 10 wherein the drug to lipid ratio of 1:10 is showed the maximum drug entrapment in the liposomes. 12. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 11 wherein the size of the said liposome is 1 to 5 urn under electron microscopy. 13. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 12 wherein the rate of diffusion of the said salbutamol liposomes are extended for a period of 9 to 10 hrs. 14. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 13 wherein the net respirable fraction is found to be 20 to 24%. 15. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 14, wherein the said bebulizing formulation of salbutamol sulphate is prepared with isotonicity adjustments by calculating sodium chloride equivalents for all the ingredients. 16. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 15, wherein the pH of the phosphate buffer is 6.3 to 6.4 suitable to prepare isotonic nebulizing dispersions. 17. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 16 wherein the structure of the said liposomes is bilamellar and trilamellar under optical microscopy. 32 18. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 17 wherein the percent drug entrapment in liposomes is 48 to 52%. 19. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 18, wherein the said salbutamol liposomes are found to be bilayered, circular structures under electron microscopy. 20. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 19, wherein the percentage drug entrapment in the aqueous layer of liposomes is 50 to 52%. 21. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 20, wherein, in vitro studies showed, 65-70% drug release by the end of 6hrs, 85-90% by the end of 8 hrs., and the entire drug is released by theend of 9-10hrs. 22. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 21, wherein the said nebulizing formulation comprises anti oxidants such as butylated hydroxy anisole and a-tocoferol. 23. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 22, wherein the said salbutamol liposomal nebulizing dispersions are stable at 5°C, 25°, 60% RH for one year. 24. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 23, wherein the shelf life for the product is 12months when stored between 5-8°C. 25. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 24, wherein Guinea pig is used as animal model for toxicity studies of the above said liposomal inhalations. 26. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 24, wherein the said formulations did not show any chronic toxicity, as there are no significant histopathological changes in the lungs, after administration of nebulized liposomal dispersions for prolonged period of 3 weeks, two times a day. 33 27. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 24, wherein acute toxicity studies at a dose level 7 times more than that of therapeutic dose showed dose dependent reversible degeneration in the histopathology. 28. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 25, wherein irreversible damage to lungs observed at higher dose levels, which is 14 times more than therapeutic dose. 29. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 26 wherein, pharmacodynamic studies such as bronchial hyper reactivity and whole body plethysmography to study the antiasthmatic effect is in vivo. 30. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 27, wherein, the nose-only method is the technique used for in-vivo evaluation of performance of aerosol in animals. 31. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 28, wherein, the said salbutamol liposomes are lyophilized to improve their long term stability. 32. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 29 wherein, the percent protection of liposomal nebulizing solution of salbutamol sulphate to histamine induced asthmatic attack is 7 times more than marketed formulations when challenged by histamine immediately as well as 4 hrs after the administration. 33. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate for controlled drug delivery to pulmonary systems as substantially described herein with reference to the foregoing examples lto 7. 34 Abstract: The present invention discloses a stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in the treatment of asthma to give controlled drug delivery for pulmonary systems. This invention is more particularly relates to liposomal carriers in the

Full Text

FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
[See section 10; rule 13]
"STABLE LIPOSOMAL NEBULIZING INHALATION FORMULATIONS FOR CONTROLLED PULMONARY DELIVERY SYSTEMS"
(a)BAJAJAMRITA
(b) 201, Namita, Gulmohar Road, No 4, Juhu Scheme, Mumbai- 400 049. India
(c) Indian National

(a) KHALEANUBHA
(b) 17-Nandadeep, Pandurang Wadi, 5th Road, Goregaon (East), Mumbai-400 063. India
(c) Indian National
The following specification particularly describes the nature of this invention and the manner in which it has to be performed.

Technical Field of the Invention:
The present invention relates to stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in the treatment of asthma to give controlled drug delivery to pulmonary systems. This invention is more particularly relates to liposomal nebulizing inhalation formulations carriers that can carry the drug into deep lung and maintain it at the site of action to give a prolonged therapeutic effect.
Background and Prior art:
Inhalation aerosols have been employed to treat localized disease states within bronchi. Since pulmonary route of administration can directly deliver therapeutic agents to the diseased regions whilst reducing their distribution to other organs, it provides an excellent example of targeted drug therapy. A more favourable therapeutic index can be obtained for the treatment of pulmonary diseases when drugs are administered by inhalation rather than by the oral route. Bronchodilators, anti-inflammatory agents, mucolytics, antiviral agents are all routinely given as aerosolised formulations. The possibility of systemic absorption of proteins and peptides via the airways has currently stimulated interest in pulmonary delivery systems.
A significant disadvantage of many existing inhaled drugs is the relatively short duration of resultant clinical effects and most medications in aerosol form require at least 3-4 times daily administration. Although the onset of action is very rapid, the duration is often short lived as the drug can be removed from the lungs through various clearance mechanisms. A reduction in the frequency of dosing would be convenient, particularly for chronic treatments such as those for asthma.
Most bronchodilators, p2-agonists have the potential to induce significant cardiovascular side effects such as hypotension and tachycardia due to stimulation of the p2-adrenoreceptors in the systemic circulation and cross reactivity with cardiac pi- adrenoreceptors. Controlled release of such drugs in the pulmonary region would be particularly beneficial since they could be delivered to and retained at the targeted receptors for a prolonged period of time and thus minimize the biodistribution through systemic circulation.
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Drugs for inhalation therapy are administered in aerosol form of fine particles dispersed in a gas. An obvious prerequisite for therapeutic efficacy is the ability of the aerosolized drug to reach the peripheral airways. Typically only 10% of inhaled dose reaches the lower airways due to complex functional anatomy of the respiratory tract that prevents the entry of particulate matter. The extent of aerosol deposition in the pulmonary tract is difficult to predict because particles can come in vastly different shapes & sizes. To account for shape and size, aerosol particles are described by their aerodynamic diameter.
In recent years several strategies to improve drug delivery to respiratory system have been developed e.g. aerosolised microspheres, nanoparticles and large porous particles. Particulate carriers such as liposomes have many attractive features for pulmonary drug delivery. Liposomes are phospholipid vesicles that are biologically inert in nature, devoid of any antigenic, pyrogenic or allergic reactions and their components can be utilized as those of biological membranes. Liposomes have an aqueous volume, which is entirely enclosed by a membrane composed of lipid molecule. The drug molecules can either be encapsulated in aqueous phase or intercalated into the lipid bilayers.
Liposomal encapsulation of compounds can localize and maintain the drug levels in the lungs for an extended period of time. Pharmacologically, there is also evidence that liposomal drugs can produce selective and prolonged effects with decreased systemic toxicity. Physicochemical properties of the entrapped species, the lipid composition of the vesicles and the site of pulmonary deposition are the factors essential in designing a liposomal formulation.
Lyophilization is an excellent method to extend the shelf-life of liposomes. Lyophilization and reconstitution of drug-containing liposomes has been achieved without significant effect upon liposome size, release properties, ion gradients or trapping ability. In a study, more than 90% of entrapped drug is retained upon rehydration.
Nebulization is probably the simplest and most effective way to deliver liposomes to the respiratory tract; nebulizers employ compressed gas or ultrasound to generate aerosols from aqueous solutions or suspensions of drugs.
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US 5340587 discloses a system includes a liposome composition comprises beta-adrenoreceptor antagonist in a liposome entrapped form and a device for aerolizinga a metered quantity of the composition.
US4895719 discloses a method and apparatus for administering dehydrated liposomes by inhalation. EPO158441 discloses aerosol formulations in aerosol droplet form from water/lipid/ethanol.
Jet nebulizer assembly for home administration of drugs in aerosols disclosed in US patent application 2003 0157032.
Small particle liposome aerosols for delivery of anti-cancer drugs disclosed in US patent application 20020102296.
An inhaler device for use with a medicinal fluid canister is disclosed in US patent application 2004000307.
US patent application 20030157032 describes pharmaceutical aerosol formulations comprising therapeutic agent in the form of coated particles in presence of surfactant in a suspension form for the administration by pulmonary route.
US 4895719 disclose a system and method for administering a drug, at a selected dose, via the respiratory tract. Spray-dried liposome particles containing the selected dose of the entrapped drug are released into the air in aerosolized form, either by entrainment in an air or propellant stream, or by release from a pressurized can containing a suspension of the liposomes in a fluorchlorocarbon solvent.
Liposomes are recognized as a DDS that can improve the therapeutic efficacy and safety of a wide range of compounds. Three decades of research into liposomal drug delivery has led to vastly improved technology in terms of drug capture, vesicle stability and the design of the formulations for special tasks. In parallel, remarkable advances have been made in
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understanding and controlling behavior in vivo.
For future successful development and commercialization of liposomal products key issues of stability, reproducibility, high encapsulation efficiency, particle size control, sterility assurance and scale-up are being successfully addressed. Further, the utility of liposome formulation technology at the floor of industry is to be explored by simplifying, standardizing and optimizing method of preparation and enhancing the storage stability of the product.
The objective of the invention is to develop liposomal pulmonary drug delivery systems. Physicochemical properties of the entrapped species, the lipid composition of the vesicles and the site of pulmonary deposition are the factors essential in designing a liposomal formulation that will not only optimize pharmacokinetic and pharmacological profiles but also satisfy pharmaceutical requirements.
The present invention overcomes or minimizes many of the problems which have limited systemic drug delivery by inhalation. Liposomal encapsulation of compounds can localize and maintain the drug for longer period of time. Nebulization is the simplest and most effective way to deliver the liposomes to the respiratory tract.
The present invention is to develop a controlled release inhalation formulation of salbutamol with liposomal carrier system, establish its safety & study its efficacy in comparison with conventional aerosol inhalation formulations.
Objective of the Invention:
One objective of the invention is to develop a controlled release inhalation formulation of salbutamol sulphate with liposomal carrier system for a prolonged therapeutic effect.
Other objective of the invention is to lyophilize the salbutamol liposomes to improve their long term stability.
Other objective of the invention is to establish its safety of the salbutamol liposomes via
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pulmonary route.
Another objective of the invention is to establish its efficacy in comparison to the conventional marketed nebulizing solution formulations.
Further objective of the invention is to study the deposition pattern of the drug in the pulmonary region.
SUMMARY:
The present invention discloses to a stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in nebulizing device for controlled drug delivery for pulmonary systems, wherein the said formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.01% to 0.02%; a-tocoferol-0.005% to 0.01%; and phosphate buffer pH (6.4). This invention is more particularly relates to nebulizing inhalation formulations containing liposomal carriers that can carry the drug into deep lung and maintain it at the site of action for a prolonged therapeutic effect.
Three batches of four different formulations Nl, N2, N3 and N4 were prepared and subjected to stability studies. Formulations Nl and N2 are the final optimized formulations contained 0.005% and 0.010% of the anti-oxidant, butylated hydroxyl anisole respectively.
Formulations N3 and N4 contained the anti-oxidant, a-tocoferol 0.01% and 0.02% respectively. As liposomal preparations are prone to oxidation, quantity of anti-oxidant plays an important role in the stability of the product.
A stable liposomal nebulizing inhalation formulation of salbutamol sulphate, in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; butylated hydroxyl anisole 0.005% in phosphate buffer pH (6.4).
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A stable liposomal nebulizing inhalation formulation of salbutamol sulphate , in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.010% in phosphate buffer pH (6.4).
A stable liposomal nebulizing inhalation formulation of salbutamol sulphate, in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; a-tocoferol 0.01% in phosphate buffer pH (6.4).
A stable liposomal nebulizing inhalation formulation of salbutamol sulphate ,in which the formulation comprises, Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; a-tocoferol 0.02% in phosphate buffer pH (6.4).
The phospholipid is selected from phospholipon 90H, the hydrogenated form of phosphatidylcholine, and phospholipon 90G, the phosphatidyline choline enriched lecithin but, more preferably phospholipid is phospholin 90G.
The solvent used to dissolve the lipid is selected from chloroform and methanol, but preferably chloroform.
Thin film hydration method is selected for the preparation of salbutamol liposomes. The type of lipid, its concentration, drug to lipid ratio, concentration of other additives such as cholesterol, anti-oxidants, pH of the hydrating buffer, salt or base form of the drug were found to affect the drug entrapment as well as drug release from the liposomes to more or less extent. Rotation speed of rotovapour flask, quantity of glass beads, solvent system, shaking and sonication period, ratio of the batch size and the capacity of the rotary flask etc. are the process parameters that need to be optimized for liposomal preparation.
The preparation of liposomes is optimized with respect to the solvent system, glass bead quantity and visicle sizing technique in which, the optimized ratio of glass beads and solvent quantity is 2:3 as it gives liposomes in the size of 10-20 (xm
Drug to lipid ratio of 1:10 has showed the maximum drug entrapment in the liposomes. The salbutamol liposomes are found to be bilayered, circular structures under electron microscopy and the size of the liposomes observed are 1 to 5 urn The percentage drug entrapment in the aqueous layer of liposomes is 50 to 52%. The rate of diffusion of the salbutamol liposomes are extended for a period of 9 to 10 hrs. The net respirable fraction is found to be 20 to 24%.
7

The nebulizing formulation of salbutamol liposomes is prepared with isotonicity adjustments by calculating sodium chloride equivalents for all the ingredients.
The pH of the phosphate buffer is 6.3 to 6.4 suitable to prepare isotonic nebulizing dispersions. The structure of the liposomes is bilamellar and trilamellar under optical microscopy. The percent drug entrapment in liposomes is 48 to 52%.
The in vitro studies showed, 65-70% drug release by the end of 6hrs, 85-90% by the end of 8hrs and the entire drug is released by the end of 9-10hrs.
The nebulizing formulation comprises anti oxidants such as butylated hydroxy anisole and a-tocoferol.
The salbutamol liposomal nebulizing dispersions are stable at 5°C, 25°, 60% RH for one year. The shelf life for the product is 12months when stored between 5-8°C. Guinea pig is used as animal model for toxicity studies of the liposomal inhalations. Formulations did not show any chronic toxicity, as there were no significant histopathological changes in the lungs, after administration of nebulized liposomal dispersions for prolonged period of 3 weeks, two times a day.
Acute toxicity studies at a dose level, 7 times more than that of therapeutic dose showed dose dependent reversible degeneration in the histopathology.
At higher dose levels, which are 14 times more than therapeutic dose, irreversible damage to lungs is observed.
The anti-histamic effect of pharmacodynamic studies such as branchial hyper reactivity and whole body plethysmography is in vivo.
Nose-only method is the technique used for in-vivo evaluation of performance of aerosol in animals.
Salbutamol liposomes are lyophilized to improve their long term stability. The percent protection of liposomal nebulizing solution of salbutamol sulphate to histamine induced asthmatic attack is 7 times more than marketed formulations when challenged immediately as well as 4hrs.
Detailed Description:
The present invention relates to a stable pharmaceutical aerosol formulation which can be administered by the pulmonary route. The use of aerosols for the administration of
8

medicaments by peripheral aerial pathways has been known for several decades. Targeted drug delivery for pulmonary diseases can be achieved by administration via inhalation route. Higher therapeutic index is achieved as aerosol inhalations deliver therapeutic agents to the diseased regions of lungs whilst reducing their distribution to other organs.
The deposition of aerosol can be improved with the help of particulate carriers that can carry the drug deep into the lungs and maintain it at the site of action for a longer period; liposomes are one of such drug delivery carriers. Their suitability lies in their natural origin, similar to those in lung secretions.
Inhalation aerosols are generally administered by nebulizers, pressurized Meter Dose Inhalers (MDI) or Dry Powder Inhalers (DPI).
Experimental
The experimental is carried out in the following steps
1) Preparation of salbutamol liposomes
2) Characterization of salbutamol liposomes
3) Preparation of nebulising dispersions
4) Stability studies of the liposomal nebulising dispersions
5) lyophilization of salbutamol liposomes
6) Toxicity studies of liposomal inhalations
7) Pharmacodynamic studies on liposomal nebulizing solutions
Example 1
Preparation of Salbutamol Liposomes:
A variety of lipids is used in the preparation of liposomes. 'Thin Film Hydration method' is a
method of choice for preparation of liposomes. Phospholipon® 90 H, the hydrogenated form of
phosphatidylcholine gave liposomal vesicles with large size of 20-30 um. Phospholipon® 90
G; the Phosphatidylcholine enriched lecithin gave maximum liposomes in the range 10-20 um.
9

In order to prepare salbutamoi -loaded liposomes, salbutamoi base as well as salbutamoi sulphate are used. In case of salbutamoi base liposomes, the dispersions are found to discolour gradually by the end of three weeks. Comparative study of liposomal dispersions prepared with various lipids-food grade lecithin, phospholipon 90H and phospholipon 90 G is given in table 1. A series of formulations of liposomal dispersions (Fl to F7 with salbutamoi base and Gl G7 with salbutamoi sulphate) is prepared with various concentrations of the base as well as salbutamoi sulphate ranging from 0.15 to 1.5% as described under table 2. The liposomal dispersions are studied for physical appearance, texture, settling rate, redispersibility and optical microscopy.
TABLE - 1
Comparative Study of Liposomal Dispersions prepared with Various Lipids - Food Grade
Lecithin, Phospholipon 90H® and Phospholipon 90G®
Settling Rate + Slow ++Moderate +++ Fast
Redispersibility + Poor ++ Good +++ Excellent

Formul ation Colour Texture Settlin gRate Redispersi bility VesicleSizeRange Changes after 3 weeks
FoodGradeLecithin Yellowis h Not smooth & presence of agglomerates +++ + 40-50um Turned light brown and formed hard cake.
Phospholipon90H® W hite Floccu lated appearance +++ +++ 20-30pm Separated into three layers to give cracked appearance
Phosph olipon White Smooth dispersion + +++ 10-20μm WeU dispersed,
10

90G® whitecoloureddispersion.
TABLE 2
Liposomal Formulations with salbutamol base and salbutamol sulphate.

Formulation Percentage
Fl F2 F3 F4 F5 F6 F7
Phospholipon 90G® 0.1 0.25 0.5 0.75 10. 1.25 1.50
Salbutamol base 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Formulation Gl G2 G3 G4 G5 G6 G7
Phospholipon 90G® 0.1 0.25 0.5 0.75 10. 1.25 1.50
Salbutamol sulphate 0.1 0.1 0.1 0.1 0.1 0.1 0.1
11

Liposomes prepared with salbutamol sulphate are showed satisfactory physical
properties. Various concentrations of base and sulphate salt of salbutamol formulations
(Fl to F7 and Gl to G7) are incorporated as indicated in table 3 and 4. Physical
characteristics are studied and optical microscopy is also performed.
TABLE - 3
Physico-Chemical Characterization of Liposomal Formulations
Prepared With Salbutamol Base.

Formul ation Fl F2 F3 F4 F5 F6 F7
Colour Off-white Off-white Off-white Yellowish White Yellowish White Yellowish Yellowis h
Texture Smooth Smoot h Smoot h Thicksuspension Agglomer ates Agglomer ates Agglom erates
Settling Rate + + ++ +++ +++ ++++ ++++
Redispe rsibility +++ +++ +++ ++ ++ + +
Microsc opy Multi lamellar Multilamellar Multilamellar Clusters Clusters Clusters Clusters
Vesicle size 10-20um 10-20um 10-20pm 20-30μm 20-30μm 20-30μm 20-30pm
After 3 weeks Yellow YeUo w Brown ish Brownish Brownish Brown Brown
Settling Rate + Slow ++ Moderate +++ Fast
Redispersibility + Poor ++ Good +++ Excellent
12.

TABLE 4
Physico-Chemical Characterization of Liposomal Formulations
prepared with Salbutamol sulphate.

Formula tion Gl G2 G3 G4 G5 G6 G7
Colour White White White White White White White
Texture Smooth Smooth Smooth Thick suspension Agglo merates Agglom erates Agglo merates
Settling Rate + + + ++ +++ ++++ ++++
Redispers ibility +++ +++ +++ ++ ++ + +
Microscopy Multila mellar Multila mellar Multila mellar Multilamell ar Cluste rs Clusters Clusters
Vesicle size 10-20pm 10-20pm 10-20pm 20-30pm 20-30pm 20-30pm 20-30pm
After 3 weeks White White White White White White White
Settling Rate + Slow ++ Moderate +++ Fast
Redispersibility + Poor ++ Good +++ Excellent
Process is optimized with respect to the solvent system (liposomal formulations with different solvent blends such as HI, H2, H3), glass bead quantity (formulations with varying proportions of glass beads such as II, 12, 13 and 14) and vesicle sizing technique(optimization of viscicle sizing with different shaking times of formulations Jl, J2, J3,and J4). The details are given in table 5. As a solvent system for lipid film formation, chloroform alone is found to give desired film uniformity as compared to its combination with methanol.
13

TABLE - 5
Process Optimization for Preparation of Liposomal Dispersions of Salbutamol sulphate

TABLE - 5 A Solvents Liposomal Formulations with Different Solvent Blends
HI H2 H3
Chloroform 1 2 1
Methanol 1 1 0
Film Uniformity + ++ +++
TABLE - 5 B Formulations with varying Proportions of Glass Beads
Formulation 11 12 13 14
Glass Beads '■ solvent 1 = 3 2:3 1 = 2 0:1
Uniformity of Film + +++ ++ -
Separation of Vesicle Aggregates + +++ ++ -
Vesicle Size (v) 30 16 24 42
TABLE - 5 C Optimization of Vesicle Sizing Technique
Formulation Jl J2 J3 J4
Shaking Time 0 2hrs 4hrs 6hrs
Number of Particles of size l-5p.m 5 - 10n m 8 16 10 18 14 1 Dispersion broke
Uniformity of Film + Moderate ++ Good
+++ Excellent - Poor
14

Glass beads: solvent quantity ratio when studied, 2: 3 ratio is found to be the optimum as it gave liposomes size in the range of 10-20um and vesicular clusters are separated.
A standardized method of mechanical shaking and sonication is proved to be a suitable technique for vesicle sizing. Four hours shaking with intermittent sonication for 1 min, after every 30min of shaking gives desired vesicle size, which is l-3μm whereas excessive shaking resulted into breaking of the dispersion.
The formula is optimized for the basic components like lipid concentration and drug to lipid ratio. Formulations Kl, K2, K3, K4 and K5 with varying concentrations of phospholipon 90G with salbutamol sulphate is prepared and observed the physical properties of the formulations and size of the liposomes. The physical property of the salbutamol sulphate liposomes is observed under electron microscopy. Electron micrographs of the salbutamol sulphate liposomes confirmed the circular bilamellar structure of the viscicle size 1-3 [i as shown in fig 1 and 2. Formulations of LI, L2 and L3 are prepared with varying proportions of drug to lipid ratio and observed the physico -chemical studies. Formulation with 1 % phospholipon 90G gives the optimum results in terms of the physical properties and the liposome vesicle size is given in table 6. TABLE - 6 Formula Optimization for the Liposomes of Salbutamol Sulphate.

TABLE 6 A Varying Concentrations of Phospholipon 90G
Formulation Percentage
Kl K2 K3 K4 K5
Phospholipon 90G 0.1 0.5 1.0 1.25 1.5
Salbutamol sulphate 0.1 0.1 0.1 0.1 0.1
TABLE - 6 B Physical Observations of Liposomal Formulations ofSalbutamol sulphate prepared with Phospholipon 90G.
15

Formulation Kl K2 K3 K4 K5
Colour White White White White White
Texture Thin suspension Thin suspension Smooth Susp. Thick suspen. Gel like consistency
Settling Rate +++ ++ + + -
Redisper-sibility ++ +++ +++ ++ Not apphcable
Microscopy Multilamellar Multilamellar Multilamellar Clusters Clusters oflargevesicles
Vesicle Size l-5μm l"5μm 2-3μm 10-20μm 10-30μm
Settling Rate + Slow ++ Moderate +++Very Fast
Redispersibility + Poor ++ Good +++ Excellent
TABLE - 6 continued
Formula Optimization of the Liposomal Preparations of Salbutamol Sulphate.

TABLE -6 C Formulations with Varying Proportionsof Drug to Lipid
Formulation LI L2 L3
Phospholipon 90G 1 part 1 part 1 part
Salbutamol sulphate 5 parts 10 parts 20 parts
Physico-chemical Study of Formulations Prepared with Varying Proportions of Drug to Lipid
Formulation LI L2 L3
Appearance White colour suspension White colour suspension White colour suspension
16

Redispersibility +++ +++ ++
Settling Rate + + ++
% Drug entrapment 30.62 42.55 36.21
Settling Rate + Slow ++ Moderate +++ Very Fast
Redispersibility + Poor ++ Good +++ Excellent
% Drug entrapment values were obtained by taking average of six observations.
Drug: lipid ratio 1:10 has shown the maximum drug entrapment in the liposomes (table 6). The formulation is also found to have satisfactory physical properties.
Optimization of other components of the liposomal formulation such as cholesterol concentration, butylated hydroxyl anisole (BHA) concentration and pH of the hydrating buffer is carried out with the help of 3 Factorial Design. The concentrations of the three components are varied at three different levels of low, medium and high.
The concentration of BHA has slight effect on the percent drug entrapment where as pH of hydrating buffer has significant effect and cholesterol concentration has no significant effect on the percent drug entrapment.
The factorial design model is further reduced to 23 by considering the three variables at two levels, low and high. They are studied in terms of the percent drug entrapment. It is observed from these studies that cholesterol concentration in the formulation has significant effect on the percent drug released from the liposomes. The pH of the hydrating buffer also has some effect on the percent drug released. However, BHA concentration has absolutely no significant effect on this study parameter.
The antioxidant BHA is replaced by another antioxidant a-tocoferol. From the previous studies, cholesterol concentration is optimized to 0.25% and hence the study design is reduced to 2 factorial levels. Concentration of a-tocoferol and pH of the hydrating buffer are varied at two levels, high and low and the formulations are studied in terms of the percent drug entrapment and percent drug released. Both a-tocoferol concentration and pH of the hydrating buffer have slight effect on the percent drug entrapped. Release of
17

the drug from liposomes is mainly affected by the pH of the hydrating buffer. The pH of the buffer 6.4 has shown maximum drug entrapment and the release is extended for 10 hours.
The effect of process variables viz. rotation speed and ratio of batch volume to rotary flask capacity is studied for the formulations prepared by optimized formula. The maximum drug entrapment is found at a lower rotation speed of 30 rpm where as with the maximum rotation speed, the drug entrapment is found to minimum.
Optimum ratio of batch volume: rotary flask volume is found to be 1: 6.5. Maximum drug entrapment is obtained at this ratio.
Morphology of the liposomes, its drug entrapment, the percent drug released from liposomes is determined by the components of the formulation as well as the processing parameters.
The final formulations of salbutamol liposomes prepared for stability studies and optimized process variables are shown in table 7.
TABLE- 7

Ingredients I II III IV
Phospholipon@90G 1.0% 1.0% 1.0% 1.0%
Cholesterol 0.25% 0.25% 0.25% 0.25%
Salbutamol sulphate 0.1% 0.1% 0.1% 0.1%
Chloroform 100ml 100ml 100ml 100ml
Butylated Hydroxy Anisol 0.01% 0.02% - -
a-tocoferol - - 0.005% 0.01%
Phosphate buffer pH 6.4 q.s 100ml 100ml 100ml 100ml
Optimized Process:
18

Method: Thin Film Hydration
Glass beads: 40g
Rotation Speed: 30 rpm
Vesicle Sizing: Four hours mechanical shaking with intermittent cycles of 1 min
sonication after every 30min. shaking.
As formulations with BHA and a-tocoferol did not show much difference in the drug entrapment and release profile with respect to their concentration used as well as no added advantages over the other, salbutamol liposomal formulations with both the antioxidants having two different concentrations are further subjected to accelerated stability studies.
Example 2
Characterization of Salbutamol Liposomes:
The liposomes are characterized for their physical attributes such as shape, size and size
distribution, lamellarity, percent drug entrapped and drug release profile. To determine
the percent drug entrapment in the salbutamol liposomes, a technique of centrifugation is
adopted for separating unentrapped drug, followed by colorimetric estimation of the drug
content. Percent drug entrapment for salbutamol liposomes are found in the range of 50-
52%.
The DSC thermogram is carried out for various liposomes such as empty liposomes along
with cholesterol, without cholesterol, salbutamol liposomes along with cholesterol and
again without cholesterol. From the DSC thermograms of liposomal dispersions of
salbutamol confirmed that cholesterol gets into the lipid bilayers and gives rigidity to the
lipoidal walls and salbutamol sulphate is situated in the aqueous compartment of the
liposomes.
Transmission Electron Microscopy studies are confirmed the bilamellar structure of the vesicles. The vesicle size of the liposomes is in the range of 1-2 um. The Dynamic Light Scattering studies have confirmed the size of the liposomes, which is in the range of 1-3μm.
19

The rate of diffusion is studied through a diffusion bag showed release extended over the period of 9-10 hours. The unentrapped portion of the drug, which is about 50%, available within first 2hours. Subsequent release of the drug from the entrapped portion is about 70% in 6 hours and 85-90% by the end of 8 hours.
In-vitro pulmonary deposition studies are carried out by Twin Impinge apparatus. It is found that, 72-77 % of drug deposited in the device and 2-4 % in the stage I. The Net Respirable Fraction, which is indicated by stage II, is found to be 20-24%.
Example 3
Preparation of Nebulizing solution:
Nebulizing solutions are usually formulated in water, although other cosolvents such as
glycerine, propylene glycol and ethanol may be used. Nebulizing solutions needs to be
iso-osmotic and hence an osmotic agent has to be added in the formulation. For preparing
nebulizing solution of the salbutamol liposomes, isotonicity adjustments are done by
calculating sodium chloride equivalents for all the ingredients. However, the additional
quantity of sodium chloride required for isotonicity adjustment is taken care by the
sodium chloride from the phosphate buffer pH 6.4 and hence, no extra sodium chloride is
needed in the formulation.
Formulations are prepared and evaluated for physico-chemical characteristics. White colour, fine, uniform dispersion with pH in the range of 6.3-6.5 is prepared. Optical microscopy has showen bi-tri lamellar structure. Laser diffraction studies by Malvern Mastersizer has showed that 90% of the particles are in the range 1-3 urn. Electron Microscopy has confirmed the bilamellar structure of the liposomes. Percent drug entrapment in liposomes is found in the range of 48-52%. Total drug content is 99-102%.
In-vitro diffusion studies have showed the release of about 40% of the dose in first 2 hours, which is the unentrapped portion immediately available for the action. By the end of 6 hours, 65-70% of salbutamol sulphate is released and 85-90% of salbutamol sulphate
20

is released by the end of 8 hours and by the end of 9-10 hours, the entire drug is released.
Example 4
Stability studies of the Liposomal Nebulizing solution:
Three batches of four different formulations Nl, N2, N3 and N4 are prepared and
subjected to stability studies. Formulations Nl and N2 are the final and optimized
formulations contained 0.005% and 0.010% of the anti-oxidant, butylated hydroxyl
anisole (BHA) respectively. Formulations N3 and N4 contained another anti- oxidant, a-
tocoferol 0.01% and 0.02% respectively. As liposomal preparations are prone to
oxidation, the quantity of anti-oxidants plays an important role in the stability of the
product.
Hence, stability study is carried out with two different anti-oxidants at two levels of concentrations.
The four formulations i.e. formulations containing two different antioxidants with two different concentrations are subjected to accelerated stability studies at 5°C, 25°C at 60% RH and 40°C at 75% RH. The samples are withdrawn at the end of 1, 2 and 3 months and evaluated for physical appearance, redispersibility, drug entrapment, drug content, particle size and release profile. The stability data of 3 months is summarized under tables 8-9. The stability study is further continued up to at 5°C and 25°C at 60% RH. Samples are with drawn at the end of 6 and 12 months and analyzed the results, which are given in tables 10-13.
21

TABLE- 8: 3 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing BHA 0.005% in Formulation (Nl) and BHA 0.01% in Formulation (N2), at different conditions of temperature

s.No Test Formulation Nl (BHA 0.005%) Formulation N2 ( BHA 0.0 1%)
Initial 5°C 25°C, 60% RH 40°C,75%RH Initial 5°C 25°C,60%RH 40°C,75%RH
1 Appea ranee White Dispersion Off-white dispersi on Yello wish white disp. White Dispersion Off-white dispers ion Yello wish Disper sion
2 Redisp ersibility +++ +++ +++ +++ +++ +++ +++ +++
3 %Drug Conte nt 99.51 101.14 100.23 100.89 101.16 99.63 100.44 99.72
4 DrugEntrapment 52.58 % 50.14 % 49.28% 24.36 % 50.87 % 51.68 % 52.78 % 24.75 %
5 Particl e Size 2.62u 2.94u 3.04u 15.96n 2.52u 3.25u 3.09u 16.63u
6 Releas eProfile T30 13.6 % 16.24 % 15.42% 29.30 % 14.42 % 16.73 % 15.20 34.12
T3 49.72 % 48.37 % 50.12% 83.14 % 45.14 % 51.15 % 46.02 % 88.90
T6 74.22 % 79.93 % 81.45% _ 69.89 % 78.40 % 70.01 %
T8 94.61 % 93.12 % 95.17% _ 90.36 % 92.85 % 91.91%
T10 103.2 8% 106.21 % 104.26% — 105.55 % 106.77 % 103.23 % —
+++ Excellent
Each observation is an average of three batches subjected to stability studies.
22

TABLE- 9 : 3 Months Stability Profile of the Liposomal Nebulizing Solution
Formulations containing a-Tocoferol
0.01%» in Formulation (N3). and 0.02% in Formulation (N4), at different conditions of
temperature.

s.No Test Formulation N3 (a -Tocoferol 0.01%) Formulation N4 (a -Tocoferol 0.02%)
Initial 5°C 25°C,60%RH 40°C,75%RH Initial 5°C 25°C,60%RH 40°C,75%RH
1 Appear an ce White Dispersion Yellowishwhitedispersion. White Dispersion Yellowishwhitedispersion
2 Redisper sibility +++ +++ +++ +++ +++ +++ +++ +++
3 % Drug content 100.26 101.12 99.64 101.06 100.32 99.74 100.05 99.45
4 DrugEntrapment 52.11 ■% 50.85% 51.76 % 24.16 % 54.18 % 51.0 8% 50.13 % 25.14 %
5 Particle Size 2.57u 2.97n 3.09u 12.34u 3.12u 3.04 3.14u 17.13u
6 Release ProfileT30 19.04 % 18.07 17.55 29.87 16.43 % 17.5 2 16.25 30.45
T3 48.55 % 50.77 47.12 83.40 49.77 % 46.2 9 45.62 78.84
T6 72.81 % 77.15 74.86 107.03 71.90 % 75.2 4 74.41 108.45
T8 96.20 % 94.13 95.10 — 94.75 % 93.65 94.26 _
T10 — 16.14 16.70 — 112.40 % 105.22 108.12 —
+++ Excellent
Each observation is an average of three batches subjected to stability studies
23

TABLE-10: 6 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing BHA 0.005% in Formulation (Nl) and BHA 0.01% in Formulation (N2), at different conditions of temperature

S. No. Test Formulation Nl (BHA 0.005%) Formulation N2 (BHA 0.01%)
Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH
1 Appearance White Dispersion White Dispersion
2 Redispersibility +++ +++ +++ +++ +++ +++
3 %Drug Content 101.21 100.69 100.42 101.01 99.58 99.84
4 Drug Entrapment 52.58% 49.65% 47.97% 50.87% 50.06% 46.55%
5 Particle Size 2.62fi 2.92^ 1.23n 2.52n 2.84u 2.03u
6 Release Profile T30 13.6% 15.32% 14.25% 14.42% 13.33% 16.04%
T3 49.72% 46.31% 48.93% 45.14% 46.79% 49.96%
T6 74.22% 72.19% 73.11% 69.89% 71.09% 74.27%
T8 94.61% 91.20% 92.46% 90.36% 92.44% 95.68%
T10 103.28 % 101.67% 103.88% 105.55% 100.57 % 105.12%
+++ Excellent
Each observation is an average of three batches subjected to stability studies
TABLE- 11: 6 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing a-Tocoferol 0.01% in Formulation (N3) and a -Tocoferol 0.02% in Formulation (N4), at different conditions of temperature

s.No Test Formulation N3 (a -Tocoferol 0.01%) Formulation N4 (a -Tocoferol 0.02%)
Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH
1 Appearanc e White Dispersion White Dispersion
2 Redispersi bilty +++ +++ +++ +++ +++ +++
3 % Drug content 100.23 99.70 101.22 100.41 99.66 101.14
4 DrugEntrapment 52.11% 48.21% 46.10% 54.18% 49.90% 47.15%
5 Particle Size 2.62^1 1.96u 2A2\i 2.52^1 2.61u 3.31u
24

6 Release ProfileT30 13.6% 15.21% 17.80% 14.42% 16.02% 15.94%
T3 49.72% 47.26% 46.03% 45.14% 49.17% 51.44%
T6 74.22% 70.24% 74.12% 69.89% 72.25% 74.23%
T8 94.61% 91.62% 93.68% 90.36% 92.42% 94.55%
T10 103.28% 101.35% 104.37% 105.55% 104.19% 107.86%
+++ Excellent
Each observation is an average of three batches subjected to stability studies
TABLE-12: 12 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing BHA 0.005% in Formulation (Nl) and BHA 0.01% in Formulation (N2), at different conditions of temperature

s.No. Test Formulation Nl (BHA 0.005%) Formulation N2 (BHA 0.01%)
Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH
1 Appear an ce White Dispersion White Dispersion
2 Redispers ibilty +++ +++ +++ +++ +++ +++
3 % Drug content 101.07 100.80 100.41 100.61 101.22 99.10
4 DrugEntrapment 52.58% 50.87% 42.52% 51.81% 48.61% 41.95%
5 Particle Size 2.62ja. 1.51u 3.21u 2.52u 2.14u 3.15u
6 Release ProfileT30 13.6% 15.32% 17.52% 14.42% % 13.96% 18.40%
T3 49.72% 53.21% 54.10% 45.14% 43.58% 47.69%
T6 74.22% 76.22% 74.64% 69.89% 73.28% 77.55%
T8 94.61% 98.11% 96.17% 90.36% 94.79% 92.06%
T10 103.28% 106.16% 105.37% 105.55% 101.84% 106.88%
25

TABLE- 13 : 12 Months Stability Profile of the Liposomal Nebulizing Solution Formulations containing in a -Tocoferol 0.01% Formulation (N3) and a -Tocoferol 0.02% Formulation (N4), at different conditions of temperature

s.No. Test Formulation N3 (a-Tocoferol 0.01%) Formulation N4 (a-Tocoferol 0.02%)
Initial 5°C 25°C, 60% RH Initial 5°C 25°C, 60% RH
1 Appear an ce White Dispersion White Dispersion
2 Redispers ibilty +++ +++ +++ +++ +++ +++
3 % Drug content 100.66 101.81 99.57 100.17 101.02 100.71
4 DrugEntrapment 52.11% 48.32% 42.12% 54.18% 47.41% 41.89%
5 Particle Size 2.62u 2.34 1 10.12μ 2.52μ 2.55μ 24.33μ
6 Release ProfileT30 13.6% 16.02% 19.56% 14.42% 15.75% 18.28%
T3 49.72% 52.66% 56.27% 45.14% 48.32% 49.62%
T6 74.22% 72.32% 79.31% 69.89% 73.20% 77.74%
T8 94.61% 91.42% 98.54% 90.36% 92.11% 97.43%
T10 103.28% 101.65% 108.36% 105.55% 103.13% 107.05%
+++ Excellent
Each observation is an average of three batches subjected to stability studies
The liposomal nebulizing dispersions of salbutamol sulphate are found to be stable at 5°C and 25°C, 60% RH. The liposomal nebulizing dispersions of salbutamol sulphate are found to be more stable at 5°C for 12 months. Leakage of the entrapped drug is observed at 40°C at 75% RH and found to increase with the increasing exposure, a-tocoferol is found to be more acceptable anti-oxidant, in comparison with BHA.
26

Example 5
Lyophilization:
Lyophilization of liposomal dispersion of salbutamol results in a dry, waxy mass. Almost 12-15% of the entrapped drug is lost during the process of lyophilization. DSC thermograms of lyophilized liposomes showed the formation of a new entity when compared with the individual DSC scans.
Scanning Electron Microscopy has confirmed the bilamellar structure of the liposomes even after the process of lyophilization. The lyophilized liposomes of salbutamol sulphate can not be formulated as MDI preparations due to its poor dispensability in the propellant HFA 134a.
Liposomal nebulizing solution for reconstitution can be formulated with 10-15 %, escape of the drug from the liposomes. The lamellar structure of the liposomes in the reconstituted product is unaltered but, showed slight increase in the vesicle size from 1-5 umto 5-10 urn.
Example 6
Toxicity studies of liposomal inhalations
Guinea pig is used as animal model for toxicity studies of the liposomal inhalations.
Ultrasonic nebulizer with nose applicator is found to be convenient for administration of
the test nebulizing substance to the animals.
The formulations did not show any chronic toxicity, as there are no significant histopathological changes in the lungs, after administration of nebulized liposomal dispersions for prolonged period of 3 weeks, two times a day.
Acute toxicity studies at a dose level 7 times more than that of therapeutic dose showed dose dependent reversible degeneration in the histopathology. At a higher dose level, which is 14 times more than the therapeutic dose, irreversible damage to lungs is observed. This indicates high margin of safety.
The placebo studies have confirmed mild to moderate congestion and interstitial pneumonia at the same dose levels indicating the safety of the excipients even at higher
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concentrations.
Example 6
Pharmacodynamic studies on Liposomal Nebulizing Solutions:
Two methods are applied in order to study the efficacy of the liposomal inhalation of
salbutamol.
a. Bronchial Hyper reactivity by Whole Body exposure method and Nose- Only
Exposure method.
b. Whole Body Plethysmography.
Bronchial Hyper reactivity:
i) Whole Body Exposure method:
The marketed sample has showed percent protection to bronchial hyper-reactivity in the range of 160-180%, 20min after administration; where as the percent protection by the liposomal nebulizing solution of salbutamol sulphate is in the range of 275-290%.
When histamine challenge is given after 8 hours, the percent protection to bronchial hyper-reactivity of the marketed preparation is reduced to 85-95%; where as the liposomal nebulizing solution of salbutamol sulphate showed the percent protection between 165-180%.
ii) Nose Only Exposure Method:
The protection to bronchial hyper-reactivity is found to improve by Nose-Only Exposure Method. The marketed preparation has showed 225-235%) protection, immediately 30 min after the administration of the drug; where as that for liposomal nebulizing solution, the protection to bronchial hyper-activity is in the range of 380-395%), showing better protection.
The anti-asthmatic effect after 8 hours is found to reduce around 130%) for the
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marketed preparation, where as for the liposomal preparation it is found to be around 225%.
The percent protection is calculated as=
Pre convulsion time- Pre convulsion time
(Treated) (Untreated)
Percent protection—- X 100
Pre convulsion Time (Untreated)
It is clearly revealed from the studies that after the treatment with the antiasthmatic formulations in the form of liposomal nebulizing dispersions, there is a decrease in the pulmonary airway resistance when challenged by histamine, 30min as well as 8 hours after the administration.
This is indicated the potential of proposed controlled release pattern obtained from the salbutamol liposomal nebulizing dispersions.
b. Whole Body Plethysmography:
The average percent protection after 30 minutes of the histamine challenge is
found to be 86.49% and 73.83%, for the marketed conventional nebulizing
preparation and the liposomal nebulizing solution respectively. The average
percent protection after 4 hours is found to be 7.16% and 51.28% for the marketed
preparation and the liposomal nebulizing solution respectively.
The study performed using Whole Body Plethysmometer showed that the
conventional marketed inhalation and liposomal nebulizing dispersion has good
antiasthmatic effect immediately after the inhalation.
The liposomal nebulizing solution of salbutamol sulphate has showed better
percent protection to the histamine induced asthmatic attack in the guinea pigs,
immediately after 30min as well as after 4 hours.
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When compared with the marketed preparation, the liposomal preparation has showed the percent protection almost 7 times more than the marketed preparation, when challenged immediately and after 4 hours. Thus, the prolonged therapeutic effect of the liposomal preparation is confirmed.
Description of drawing:
Fig 1 and Fig 2 illustrates the physical appearance of the salbutamol sulphate liposomes under electron microscopy. Electron micrographs of the salbutamol sulphate liposomes confirmed the circular bilammellar structure of the viscicles and the viscicles size is in the range of 1-3 um.
"While the present invention is described above in connection with preferred or illustrative embodiments, these embodiments are not intended to be exhaustive or limiting of the invention. Rather, the invention is intended to cover all alternatives, modifications and equivalents included within its spirit and scope, as defined by the appended claims".
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We claim
1. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate for
use in nebulizing device for controlled drug delivery for pulmonary systems,
wherein the said formulation comprises,
Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.005% to 0.01%; a-tocoferol- 0.01% to 0.02%; in phosphate buffer pH (6.4).
2. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as
claimed in claim 1, wherein the said formulation comprises,
Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1 %; chloroform-100ml; butylated hydroxyl anisole 0.005% in phosphate buffer pH (6.4).
3. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as
claimed in claim 1, wherein the said formulation comprises,
Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform-100ml; butylated hydroxyl anisole 0.010% in phosphate buffer pH (6.4).
4. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as
claimed in claim 1, wherein the said formulation comprises,
Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1 %; chloroform-100ml; a-tocoferol 0.01% in phosphate buffer pH (6.4).
5. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as
claimed in claim 1, wherein the said formulation comprises,
Phospolipon 90G-1%; cholesterol- 0.25%; salbutamol sulphate 0.1%; chloroform- 100ml; a-tocoferol 0.02% in phosphate buffer pH (6.4).
6. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 5 wherein the said phospholipid is selected from phospholipon 90H, the hydrogenated form of phosphatidylcholine, and phospholipon 90G, the phosphatidyline choline enriched lecithin.
7. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 6 wherein the said phospholipid is phospholin 90G.
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8. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 7 wherein the solvent used to dissolve the lipid is selected from chloroform and methanol but preferably chloroform.
9. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 8 wherein the preparation of liposomes is optimized with respect to the solvent system, glass bead quantity and vesicle sizing technique.
10. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 9, wherein the said optimized ratio of glass beads and solvent quantity is 2:3 as it gives liposomes in the size range of 10-20um
11. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 10 wherein the drug to lipid ratio of 1:10 is showed the maximum drug entrapment in the liposomes.
12. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 11 wherein the size of the said liposome is 1 to 5 urn under electron microscopy.
13. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 12 wherein the rate of diffusion of the said salbutamol liposomes are extended for a period of 9 to 10 hrs.
14. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 13 wherein the net respirable fraction is found to be 20 to 24%.
15. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 14, wherein the said bebulizing formulation of salbutamol sulphate is prepared with isotonicity adjustments by calculating sodium chloride equivalents for all the ingredients.
16. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 15, wherein the pH of the phosphate buffer is 6.3 to 6.4 suitable to prepare isotonic nebulizing dispersions.
17. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 16 wherein the structure of the said liposomes is bilamellar and trilamellar under optical microscopy.
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18. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 17 wherein the percent drug entrapment in liposomes is 48 to 52%.
19. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 18, wherein the said salbutamol liposomes are found to be bilayered, circular structures under electron microscopy.
20. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claims 1 to 19, wherein the percentage drug entrapment in the aqueous layer of liposomes is 50 to 52%.
21. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 20, wherein, in vitro studies showed, 65-70% drug release by the end of 6hrs, 85-90% by the end of 8 hrs., and the entire drug is released by theend of 9-10hrs.
22. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 21, wherein the said nebulizing formulation comprises anti oxidants such as butylated hydroxy anisole and a-tocoferol.
23. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 22, wherein the said salbutamol liposomal nebulizing dispersions are stable at 5°C, 25°, 60% RH for one year.
24. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 23, wherein the shelf life for the product is 12months when stored between 5-8°C.
25. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 24, wherein Guinea pig is used as animal model for toxicity studies of the above said liposomal inhalations.
26. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 24, wherein the said formulations did not show any chronic toxicity, as there are no significant histopathological changes in the lungs, after administration of nebulized liposomal dispersions for prolonged period of 3 weeks, two times a day.
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27. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 24, wherein acute toxicity studies at a dose level 7 times more than that of therapeutic dose showed dose dependent reversible degeneration in the histopathology.
28. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 25, wherein irreversible damage to lungs observed at higher dose levels, which is 14 times more than therapeutic dose.
29. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 26 wherein, pharmacodynamic studies such as bronchial hyper reactivity and whole body plethysmography to study the antiasthmatic effect is in vivo.
30. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 27, wherein, the nose-only method is the technique used for in-vivo evaluation of performance of aerosol in animals.
31. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 28, wherein, the said salbutamol liposomes are lyophilized to improve their long term stability.
32. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate as claimed in claim 1 to 29 wherein, the percent protection of liposomal nebulizing solution of salbutamol sulphate to histamine induced asthmatic attack is 7 times more than marketed formulations when challenged by histamine immediately as well as 4 hrs after the administration.
33. A stable liposomal nebulizing inhalation formulation of salbutamol sulphate for controlled drug delivery to pulmonary systems as substantially described herein with reference to the foregoing examples lto 7.

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Abstract:
The present invention discloses a stable liposomal nebulizing inhalation formulation of salbutamol sulphate for use in the treatment of asthma to give controlled drug delivery for pulmonary systems. This invention is more particularly relates to liposomal carriers in the

STABLE LIPOSOMAL NEBULIZING INHALATION FORMULATIONS FOR CONTROLLED PULMONARY DELIVERY SYSTEMS

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