Compositions of liposomes and ß2-receptor active substances
DESCRIPTION
Field of the invention
The present invention relates to a novel pharmaceutical composition in dry powder form and is particularly concerned with liposomal formulations of ß2-receptor active substances for inhalation.
The object of the invention is to provide a pharmaceutical composition consisting of a dry powder comprising a ß2-receptor active substance encapsulated into liposomes. By encapsulating a ß2-receptor active substance into liposomes, it is possible to prolong the retention of this group of substances in the lung and hence to increase the duration and efficacy of the anti-inflammatory, broncho-dilating and anti-allergic activities.
One of the major problems in the development of a pharmaceutical liposomal formulation is the long-time stability. Aqueous liposome dispersions have a limited physical stability since the liposomes can aggregate resulting in a change in the size distribution. Furthermore, if the encapsulated drug is hydrophilic it may be lost into the external aqueous phase. In addition, there is a potential risk for chemical degradation of the lipid components and the pharmacologically active substance in an aqueous milieu. The problem concerning stability can to large extent be solved if a dry solid composition is developed.
The following ß2-receptor active substances are examples of substances which can be used in accordance with the present invention: terbutaline, salbutamol, mabuterol, fenoterol.
orciprenaline, formoterol, isoprenaline, isoetharine, clenbuterol, hexoprenaline, procaterol, 1-(4-hydroxyphenyl-2-[1,1-dimethyl-3-{2-methoxy-phenyl)propylamino]-ethanol, 1-(3,5-dihydroxy-phenyl)-2-[1,1-dimethyl-3-(2-methoxyphenyl)-propylamino]-ethanol, 1-(3,4-dihydroxyphenyl)-2-[1,1¬-dimethyl-3-(2-methoxyphenyl)propylamino]ethanol, (4-hydroxy-α-[[[6-(4-phenylbutoxy)-hexyl]-amino]-methyl]-1,3-benzyl-dimethanol, pharmacologically acceptable salts thereof and compounds of similar pharmacological properties. Preferred pharmacologically acceptable salts of B2-receptor substances are salts with physiologically acceptable acids. Suitable acids which may be used are, for example, hydrochloric, hydrobromic, sulfuric, fumaric, citric, tartaric, maleic or succinic acid. The B2-receptor active substance which is particularly preferred is terbutaline sulphate.
Background art
Inhalation of ß2-receptor active substances is used for the treatment of allergic and inflammatory conditions in the respiratory tract, like asthma and airway hyperresponsiveness. However, the treatment suffers from the disadvantage that it has a limited duration of action. For example, the bronchodilating effect of inhaled terbutaline sulphate administered during the evening is lost during the late night which might result in a new asthmatic attack during the sleeping period.
Liposomes are widely described in the litterature and their general structure is well known; they are structures composed of concentric rings of lipid bilayers. Dehydrated liposomes are described in International Application WO86/01103 (Liposome Co.). Liposomes have been used as carriers for different kinds of pharmacologically active drugs in order to improve the therapeutic efficacy. Drug-loaded liposomal formulations are however generally intended for subcutanous, intravenous or oral administration. Drug encapsulated into liposomes intended specifically for inhalation are for instance described in European Patent Applications 158441 (Phares), 84898 (Fison) and 0170642 (Draco) and in International Application WO86/01714 (Riker).
Disclosure of the invention
The lipid materials used in the present invention may be any of those conventionally used in liposomal formulations. Usually the main liposome-forming component is a phosholipid, including synthetic lecithins and natural lecithins,
e.g. those derived from egg and soyabean. The phase-transition temperature (Tc) of the phospholipid can have a marked influence on the retention of the liposome encapsulated substance in the target organ. It is therefore favourable to use well-defined synthetic phospholipids. Dimyristoyl phosphatidylcholine, DMPC (Tc - 23 ºC), dipalmitoyl phosphatidylcholine, DPPC (Tc - 41 ºC) and distearoyl phosphatidylcholine, DSPC (Tc - 55 °C), either alone or in combination are preferred to the natural lecithins. It is known that DPPC is the main phospholipid in the natural lung-surfactant. By the use of pure synthetic phospholipids the risk of undesired immunological reactions is minimized.
In addition to the main liposome-forming component other lipids may be used to optimize the properties of the formulation. Examples of such additives are cholesterol and components which provide positive or negative charge.
Cholesterol, or carbohydrate derivatives thereof in a proportion up to 50 % w/w of the total lipids may be incorporated to modify the membrane structure rendering it more fluid or more rigid and thereby influence the release properties of the entrapped pharmacologically active material. Cholesterol also has a positive effect on the stability of the liposomes during lyophilization.
Components which provides a negative or positive charge may be incorporated in a proportion up to 30 % w/w of the total lipids. They will provide an electrostatic stabilization of the liposome dispersion and may also optimize the uptake of the liposomes in the target cells. Examples of negatively charged lipophilic substances are phosphatic acid, dicetyl phosphoric acid, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol and phosphatidyl ethanolamine. Examples of positively charged lipophilic substances are stearylamine, stearylamine acetate and cetylpyridinium chloride.
The initial stages of the preparation of liposomes according to the present invention may conveniently follow any procedure which results in the encapsulation of a hydrophilic substance into liposomes.
Examples of such methods are the
A reverse phase evaporation (US Patent 4,235,871)
B dehydration-rehydration method (Kirby, C and Gregoriadis, G; Bio/Technology, Nov 1984, 979-984)
C film method (Bangham et al J Mol Biol 1965, 13, 238-252)
D freeze-drying method (UK Patent 1,573,343)
Method A
The lipid materials are dissolved in an organic solvent and the ß2-receptor active substance is dissolved in an aqueous phase. The two solutions are mixed to produce an emulsion of the water-in-oil type. The organic solvent is removed and the resulting gel is suspended in an aqueous solution to give a liposome dispersion.
Method B
The lipid material is dissolved in a solvent e.g. chloroform or t-butanol, and are evaporated to a thin lipid film (chloroform) or freeze-dried (t-butanol). Distilled water is added and the temperature is raised. The final temperature will be above the phase-transition temperature of the lipid material. The resulting liposome dispersion is mixed with an aqueous solution of a ß2-receptor active substance. It is often appropriate to use 0.1 to 10 parts by weight of ß2-receptor active substance per part of lipid material.
The mixture is freeze-dried, and the dry material is dispersed in a minimal amount of distilled water. The temperature is raised above the phase-transition temperature of the lipid material. After equilibration, the dispersion is diluted with additional aqueous solution. The resulting liposomes will be in a range of sizes (50 nm - 10 μm ) .
Method C
The lipid materials are dissolved in a solvent, e.g. chloroform or ethanol and the solvent is evaporated. Liposomes are formed by adding an aqueous solution of a ß2-receptor active substance and raising the temperature above the phase-transition temperature of the lipid material. It is often appropriate to use 0.1 to 10 parts by weight of ß2-receptor active substance per part of lipid material. The concentration of ß2-receptor active substance during liposome formation should be 1 - 100 mg/ml. The resulting liposomes will be in a range of sizes (50 nm - 10 μm) .
Method D
The lipids and the ß2-receptor active substance are dissolved in a solvent, e.g. a mixture of t-butanol and water, and freeze-dried. It is often appropriate to use 0.1 to 10 parts by weight of ß2-receptor active substance per part of lipid material. The resulting freeze-dried powder is dispersed in a minimal amount of distilled water and the temperature is raised. The final temperature will be above the phase-transition temperature of the lipid material. After equilibration, the dispersion is diluted with additional aqueous solution. The resulting liposomes will be in a range of sizes (50 nm to 10 μm) .
Regardless the method used for formation of the liposomes there will be significant amounts of drug not encapsulated
into the liposomes but remaining in the continuous aqueous phase. It may be desirable to remove the drug (or a fraction of it) from the continuous phase and this is conveniently done either by dialysing the liposomal formulation against a drug-free aqueous phase, by centrifugation of the liposome dispersion of by chromatography using an ion-exchange resin capable of selectivly binding the non-entrapped ß2 -receptor active substance.
Preparation of dry liposomal powder containing ß2-receptor active substance
Since aqueous dispersions of liposomes have a limited stability, it may be favourable to remove the solvent from preparations intended for long-time storage. The dehydration can be performed in a number of different ways, e.g. spray-drying and lyophilization. Lyophilization is particulary preferred. In that case the liposome dispersions described in the present invention are mixed with a cryoprotective agent such as a carbohydrate, e.g. lactose or threhalose at the concentration of 0 to 95 % by weight of the final composition and are rapidly frozen in liquid nitrogen. We find the rapid freezing step important to preserve the liposomal structure. After lyophilization a dry powder suitable for long-time storage is obtained. The liposome dispersion can be reconstituted after addition of an aqueous solution to the said powder.
Determination of the percentage ß2-receptor active substance associated with liposomes
The equilibrated liposome dispersion containing the ß2-receptor active substance is, if necessary, diluted with an appropriate aqueous solution (distilled water, saline etc) and centrifuged at 25000 g to 100000 g for 15 ain to 1
hour. Aliquots of the supernatant and the liposomal pellet (suspended in distilled water) are dissolved in t-butanol and assayed in a Varian DMS 100 spectrophotometer at the following wavelengths; terbutaline sulphate 280 nm, salbutamol 276 nm, mabuterol 240 nm, 1-(4-hydroxyphenyl)--2-[1,1-dimethyl-3-(2-methoxyphenyl) propylamino]-ethanol, 270 nm and procaterol 257 nm. The percentage of ß2-receptor active substance encapsulated in the liposomes (the encapsulation efficacy) is calculated as follows:
Working examples
The present invention is exemplified but in no way limited by the following examples.
Compositions
Example 1
DPPC (10 mg), DPPA (Dipalmitoyl phosphatic acid) (1 mg) and cholesterol (10 mg) are mixed in a glass tube. All components are dissolved in chloroform. The solvent is evaporated by the use of N2 , which results in a thin film of the lipid components on the inner surface of the glass tube. 1 ml of an aqueous solution of terbutaline sulphate (10 mg/ml) is added to the lipids. Liposomes are formed by shaking the glass tube at 60 ºC for 30 minutes. The encapsulation of terbutaline sulphate into the liposomes was 10 % according to the method described above.
Example 2
One gram of Epikuron 200H (Lucas Meyer, Hamburg) and one gram of terbutaline sulphate are dispersed, under gentle heating, in t-butanol (30 ml) and distilled water is added until the components are completely dissolved. The solution is frozen and lyophilized. 40 mg of the dry lyophilized powder is dispersed in 200 μl distilled water and the dispersion is heated (60 ºC) for 30 minutes. Thereafter 2.8 ml distilled water is added. The percentage of terbutaline sulphate encapsulated into the liposomes was 51 % according to the method described above.
Example 3
In a 50 ml round bottom flask 60 mg of DPPC and 60 mg of cholesterol are dissolved in 10 g chloroform. 60 mg of terbutaline sulphate is dissolved in 1 g of distilled water. The terbutaline sulphate solution is added to the flask and the two solutions are emulsified with an Ultra-Turrax. The resulting emulsion is evaporated on a Buchi rotary evaporator until a gel is formed. To the gel 3 g of distilled water is added and the sample is mixed until a liposome dispersion forms. The encapsulation of terbutaline sulphate into the liposomes was 38 % according to the method described above.
Examples 4 - 14
The desired quantities of the appropriate lipids (see below) are mixed in a glass tube. All components are dissolved in a small quantity of chloroform and evaporated to dryness to leave a thin lipid film on the inner surface of the glass tube. Distilled water (4 ml) is added to the lipid film and liposomes are formed by sonificating the sample at elevated temperature. Terbutaline sulphate is
dissolved in 2 ml distilled water. The liposome dispersion and the drug solution are mixed, frozen and lyophilized. The dry product is dispersed in 100 μl distilled water per 10 mg phospholipid. Liposomes are formed by heating (60 °C) the sample for 30 minutes. The encapsulation of drug into the liposomes is determined according to the method described above.
The following liposome compositions were prepared using the above general procedure:
Encapsulation efficacy (%)
4) DPPC 10 mg 43
Terbutaline sulphate 10 mg
5) DPPC 10 mg
Cholesterol 10 mg 40
Terbutaline sulphate 10 mg
6) DPPC 10 mg
Stearylamine 1 mg
Cholesterol 10 mg 43
Terbutaline sulphate 10 mg
7) DPPC 10 mg
Phosphatidyl serine 1 mg
Cholesterol 10 mg 36
Terbutaline sulphate 10 mg
8) DPPC 9 mg
DPPA 1 mg
Cholesterol 10 mg 42
Terbutaline sulphate 10 mg
9) DPPC 9 mg
DPPA 1 mg
Cholesterol 1 mg 31
Terbutaline sulphate 10 mg
10) DPPC 9 mg
DPPA 1 mg 28
Terbutaline sulphate 10 mg
11) DMPC 9 mg
DPPA 1 mg 47
Cholesterol 10 mg
Terbutaline sulphate 10 mg
12) DSPC 9 mg
DPPA 1 mg
Cholesterol 10 mg 64
Terbutaline sulphate 10 mg
13) Egg-lecithin 40 mg
DPPA 5 mg 37
Cholesterol 50 mg
Terbutaline sulphate 50 mg
14) Egg-lecithin 9 mg
DPPA 1 mg 40
Cholesterol 10 mg
Terbutaline sulphate 10 mg
Examples 15 - 18
Liposomes with various ß2-receptor active substances are prepared according to the method described in examples 4 -14 and the fraction of the drug encapsulated into the liposomes is determined according to the method described above.
Encapsulation efficacy (%)
15) DPPC 40 mg
DPPA 4 mg 38
Cholesterol 40 mg
Mabuterol 40 mg
16) DPPC 40 mg
DPPA 4 mg 45
Cholesterol 40 mg
Salbutamol 40 mg
17) DPPC 40 mg
DPPA 4 mg
Cholesterol 40 mg 20
1-(4-hydroxyphenyl)-2- [1,1-dimethyl-3-(2-methoxyphenyl) propylamino]- ethanol 40 mg
18) DPPC 40 mg
DPPA 4 mg
Cholesterol 40 mg 35
Procaterol 40 mg
Examples 19 - 22
Liposome dispersions are prepared according to the method described in examples 4 - 14. The amount of lipid material was kept constant while the amount of ß2-receptor active substance (in this case terbutaline sulphate) was varied.
The following liposome compositions were prepared:
Encapsulation Absolute amount efficacy (%) of terbutaline sulphate encapsulated into liposomes (mg)
19) DPPC 9 mg
DPPA 1 mg 57 2.9
Cholesterol 10 mg
Terbutaline sulphate 5 mg
20) DPPC 9 mg
DPPA 1 mg 39 3.9
Cholesterol 10 mg
Terbutaline sulphate 10 mg
21) DPPC 9 mg
DPPA 1 mg 27 5.4
Cholesterol 10 mg
Terbutaline sulphate 20 mg
22) DPPC 9 mg
DPPA 1 mg 19 5.7
Cholesterol 10 mg
Terbutaline sulphate 30 mg
Dry powder
Example 23
Liposomes are prepared according to examples 4 - 14. The liposome dispersion (100 μl) is diluted to 1.5 ml with an aqueous solution of lactose (100 mg/ml). The dispersion is flash-frozen by dripping it into liquid nitrogen and is then lyophilized. The dry powder is dispersed in distilled water and the encapsulation of terbutaline sulphate into the liposomes is calculated according to the method described above.
Encapsulation efficacy (%)
DPPC 10 mg
Cholesterol 10 mg 20
Terbutaline sulphate 10 mg
Example 24
Liposomes are prepared according to examples 4 - 14. The liposome dispersion (100 μl ) is diluted to 5 ml with an 0.9 % NaCl solution and centrifuged at 25000 g for 15 min. The pellet is suspended in 1.5 ml of an aqueous solution of lactose (100 mg/ml) The dispersion is flash-frozen by dripping it into liquid nitrogen and is then lyophilized. The dry powder is dispersed in distilled water and the encapsulation of terbutaline sulphate into the liposomes is calculated according to the method described above.
Encapsulation efficacy (%)
DPPC 10 mg
Cholesterol 10 mg 26
Terbutaline sulphate 10 mg
Example 25
11 g Epikuron 200H and 11 g terbutaline sulphate were dissolved in a mixture of 154 g t-butanol and 66 g distilled water under gentle heating. The solution was flash-frozen by dripping it into liquid nitrogen and was then lyophilized. 2.5 g of the resulting powder was dispersed in 197.5 g of an aqueous solution of lactose (3.8 weight %). Liposomes were formed by heating (maximum temperature 60°C) the sample for approximately 30 minutes during stirring. The liposome dispersion was spray-dried with a Buchi 190 Mini Spray-Dryer using an inlet temperature of 159°C. When an aqueous solution was added to the spray-dried powder, a liposome dispersion was reformed.
Release of ß2-receptor active substance from liposomes
Substantially all the non-encapsulated drug is removed from the continuous aqueous phase by centrifugation at 25000 g for 15 minutes and redispersion of the pellet in saline (0.9 % NaCl solution). The liposome dispersion ( 4 ml ) is placed in a dialysis bag (Spectrapor Membran Tubing). The rate of release of ß2-receptor active substance from the
liposomes is determined by measuring the amount of drug in the liposomes after dialysis at 37 °C against 100 ml of saline. After various times the dialysis is stopped and the amount of ß2-receptor active substance in the dialysis bag is measured according to the method described above.
The results of this study are shown in Table 1.
Table 1 DIALYSIS OF FREE AND LIPOSOME ENCAPSULATED B2-RECEPTOR ACTIVE SUBSTANCES.
Test pre% in dia lysis bag at various times paration 1-1. 5 h 3-4 h 6-7 h 16-22 h
TERB 52 17 n.d. 4
TERB-LIP n.d. 82 79 72
MAB-LIP 94 74 62 43
TERB: Free terbutaline sulphate
TERB-LIP: Liposome encapsulated (DPPC, DPPA, cholesterol, 10:1:10 w/w) terbutaline sulphate
MAB-LIP: Liposome encapsulated (DPPC, DPPA, cholesterol, 10:1:10 w/w) mabuterol
n.d.: not determined
Already after 3 hours an equilibrium between the aqueous phase inside the dialysis bag and the external aqueous phase is established when terbutaline sulphate is dialysed. On the other hand, liposome-encapsulated mabuterol and terbutaline sulphate do not reach any equilibrium even after 20 hours of dialysis. These results show that it is possible to obtain a local retention of the active substance by encapsulation into liposomes.
Biological Tests
A Preparation of formulations for administration
DPPC (40 mg), DPPA (4 mg ) and cholesterol (40 mg) are mixed in a glass tube. The components are dissolved in chloroform. The solvent is evaporated by the use of N resulting in a thin film of the lipid components on the inner surface of the glass tube. Distilled water (4 ml) is added to the lipids. Formation of the liposomes is performed by sonication at a temperature above the phase transition temperature.
40 mg of terbutaline sulphate (or an other ß2-receptor active substance) dissolved in 2 ml distilled water is added to the liposomal dispersion and the mixture is frozen and freeze-dried.
The freeze-dried powder is hydrated in 400 μl distilled water at 60 °C for 30 minutes and diluted to appropriate concentration with saline. Approximately 40 % of the drug was encapsulated into the liposomes. This formulation was used for determination of anti-edema activity of Sephadex treated rats.
The liposome dispersion was centrifuged at 25000 g for
15 minutes in order to obtain a formulation where almost 100 % of the drug is encapsulated into the liposomes. This formulation was used for determination of protective effect against a histamine-elicited bronchospasm in guinea-pigs.
B Anti-inflammatory effect
Intratracheal instillation of Sephadex beads into rats leads to bronchial and also to alveolar inflammation (Källström, L. et al. Agents and Actions 1985 vol 17, 3/4, 355). This provokes interstitial lung edema, which in
creases the lung weight, and the inflammation can be graded as the increase of the lung weight compared to a saline-instilled control group. The lung edema formation can be counteracted by pretreatment with ß2-receptor active substances, preferably by local adminstration as intratracheal instillation or by inhalation. Ideally an anti-inflammatory action should be obtained only at the site of drug application in the lung, but not in the rest of the body.
The differentiation between drug actions in the treated lung region and outside this area can be tested in the following way. Spraguo Dawley rats (240 g) were slightly anaesthetized with ether and the ß2-receptor active preparation (in liposomes suspended in saline) in a volume of 0.5 ml/kg was instilled into just the left lung lobe. Two hours later a suspension of Sephadex (5 mg/kg in a volume of 1 ml/kg) was instilled in the trachea well above the bifurcation so that the suspension reached both the left and right lung lobes. 2 hours after Sephadex instillation the test preparation in a volume of 0.5 ml/kg was instilled into the left lung lobe. 16 to 20 hours later the rats were killed and the left and right lung lobes were dissected out and weighed separately. Control groups got saline instead of the test preparations and saline instead of Sephadex suspension to determine the weight of non-drug treated Sephadex edema and the normal lung weight.
As stated above an ideal ß2-receptor active substances should have a high pharmacological activity at the site of application in lung, but a low activity outside this area. Therefore, in the selected model an optimal preparation should have a high anti-edema activity in the locally pretreated left lung lobe and less activity in the right lung half.
The results of a comparative study is given in Table 2. The pharmacological profile of a liposomal formulation of terbutaline sulphate is compared that of free terbutaline sulphate.
Table 2 EFFECT OF FREE AND LIPOSOME ENCAPSULATED TERBUTALINE SULPHATE ON SEPHADEX INDUCED LUNG EDEMA IN RAT (N-6)
Preparation and dose % Inhibition of lung edema
in treated mg/kg left lobe in right lobe
TERB 0.1 20 23
1 43 41*
10 95** 81**
TERB-LIP 0.01 36 5
0.1 79* 47
1 98** 65**
TERB: Free terbutaline sulphate
TERB-LIP: Liposome encapsulated (DPPC, DPPA, cholesterol, 10:1:10 w/w) terbutaline sulphate
** P < 0.05, 0.01, respectively, in comparison with control group
The liposomal formulation of terbutaline sulphate had a more selective activity for the application site in the lung than free terbutaline sulphate. The two test formulations more or less completely blocked the edema of the left lung lobe but the liposomal formulation was surprisingly coupled to only a moderate protective activity in the other lung lobe wheras free terbutaline sulphate completely blocked the edema of the right lung as well.
In addition to the observed separation between the anti-edema activity in the left and right lung lobe, terbutaline sulphate encapsulated into liposomes also shows a surprisingly high absolute potency for the action of the left lung lobe (100 times more potent than free terbutaline sulphate).
Additional tests in this model have shown that procaterol, mabuterol and salbutamol encapsulated into liposomes show the same anti-edema profile as terbutaline sulphate encapsulated into liposomes, i.e. a 100-fold potentiation of the anti-edema activity at the site of application compared with free terbutaline sulphate.
C Bronchospasmolytic effect
Inhalation of aerosolized histamine to concious guinea pigs produces a dyspnea. The concentration of histamine to be aerosolized can be selected to produce a defined dyspnotic breathing within 2 min of exposure to histamine. Animals pretreated with inhaled bronchospasmolytic drug can be protected from the dyspnotic breathing (animals which withstand the dyspnea for more than 2 min). By administering ß2-receptor active substances at different time intervals before the histamine provocation and by measuring the protective effect it is possible to determine the duration of the activity of the substance.
Guinea pigs were exposed for 15 min to aerosolized terbutaline sulphate or to aerosolized liposome encapsulated terbutaline sulphate generated from a MA2 nebulizer with a terbutaline sulphate concentration of 1 x 10-3 M of the two formulations. The animals were exposed to the bronchospasmolytic agent 1, 2, 5 and 10 hours before the histamine challenge. The results of this study are given in Table 3.
Table 3 EFFECT OF FREE AND LIPOSOME ENCAPSULATED TERBUTALINE SULPHATE ON HISTAMINE INDUCED DYSPNEA IN GUINEA PIG.
Test preProtective effect (sec) at various times paration 1h 2h 5h 10h
TERB 306 195 154 108 TERB-LIP 185 200 374* 150
TERB: Free terbutaline sulphate
TERB-LIP: Liposome encapsulated (DPPC, DPPA, cholesterol, 10:1:10 w/w) terbutaline sulphate
* = P < 0.05 in comparison with free terbutaline sulphate
Free terbutaline sulphate shows rapid onset of the a protective effect against histamine. A corresponding concentration of liposome-encapsulated terbutaline sulphate appears to have a delay in developing the same protective effect as the free terbutaline sulphate. When administered 2 hours before challenge the two used formulations of terbutaline sulphate have the same effect. However, there is only a limited protective effect of terbutaline sulphate when adminstered 5 hours before histamine provocation whereas liposome-encapsulated terbutaline sulphate surprisingly shows a maximal protection when the formulation is adminstered at this time. It can be concluded from this study that encapsulation of terbutaline sulphate into liposomes gives a prolonged duration of the bronchospasmolytic activity compared with equal amount of the free drug.