A method for the preparation of liposomes, a pack for use therein and a composition for use in preparing a liposomal material

Method for preparing an aerosol spray containing liposomes, the package to be used therein and the use of the composition in the package

The present invention relates to a process for the preparation of aerosol sprays containing liposomes, to a package used in the preparation and to the use of the composition in a package. In particular, the invention relates to the preparation of preparations containing an active substance and a liposomal carrier using a pressurized aerosol system.

Certain therapeutically active compounds are administered to patients by inhalation, that is, they are administered to the respiratory tract through the nose or mouth as steam or gas, or as a mist together with a carrier material in the form of steam or gas. Similarly, in certain skin treatments, for example in the treatment of open wounds or sprains, in the treatment of dermatological conditions, or for cosmetic purposes, it may be desirable to administer the active compound by spraying it as a gas or vapor or mist. In any case, it is desirable that as much of the active agent as possible reach the desired site of action, and sometimes that the active compound remain intact for some time at the desired site of action. In other words, the active compound would be administered without being degraded upon transfer or in direct contact with the patient.

Thus, for example, when administering an anti-asthmatic bronchodilator compound known as bitolterol (described in GB Patent No. 1,298,771), it is desirable to administer the compound in a manner that achieves maximum bronchial penetration to achieve the most effective concentrations of active compound at the required sites of action.

The preparation of liposome preparations containing biologically active compounds has been described previously, and such preparations are described, for example, in GB Publication Nos. 1,575,343, 1,575,344 and 2,013,609A. However, in these known preparations, the liposomal material has previously been prepared and dried, typically by lyophilization (freeze-drying). The liposomal material is then reconstituted by mixing with water.

Aerosol preparations containing various cosmetic and therapeutic substances are also known, see for example GB Publication Nos. 780 885, 993 702,

302 671, 1,381,184, 1,516,195 and 2,001,334A. However, although these known aerosol formulations may contain lipid ingredients, these ingredients are invariably merely dispersing, suspending, emulsifying, or the like. Furthermore, such preparations are intended to produce either a dry spray so that no liposomal formation is possible, or in the case of cream formation as in publication No. 780 885, the preparations are in the form of an aqueous emulsion in which any lipid present is intended to act only as an emulsifier. '*. as an old or equivalent substance. In the same manner as described in GB Patent Publication Nos. 1,281,696 and^: 1,352,062, lecithin can be used in very small quantities

... in amounts in the photographic process as part of an aerosol developer preparation containing water, to separate the propellant from the developing solution.

In addition, U.S. Patent No. 3,594,476 discloses the preparation of lecithin aerosols for use in the lung. In the treatment of diseases, these aerosols may • contain other therapeutic agents such as antibiotics. In the process described, a suspension of DL-dipalmitoyl-Ct-lecithin is first prepared in water (or saline) with any drug dissolved in water or saline, then the suspension is nebulized, i.e. aerosolized, in an ultrasonic generator using a carrier . Thus, the method described requires the use of relatively expensive ultrasonic equipment to prepare the final mixture of lecithin and water, as well as the use of a mixture containing lecithin suspended in water. The ultrasound device may also be noisy, and may be unsuitable for use at all.

Further, U.S. Patent No. 4,371,451 discloses surface release preparations for lecithin-based cookware containing water, lecithin and dimethyl ether as a propellant. When the preparations in this publication are taken from aerosol containers, lecithin is obtained as a fast-drying and thin coating which does not foam, nor the liposomal material as such. Again, the preparations used for this purpose are prepared from a mixture of a suspension of solid lecithin in an aqueous carrier, i.e. lecithin is dispersed in a solution of dimethyl ether and water, or in a solution of dimethyl ether and aqueous ethanol. Similarly, cookware lubricants containing lecithin are described in GB Patent No. 1,463,964. However, in this publication, lecithin is mixed with a relatively large amount of oily substances to form a water-in-oil emulsion, and a sufficient amount to prevent liposome formation.

In our pending application No. 84-20850, published March 20, 1985, GB-2,145,107A, we have described a process for the preparation of liposomes which comprises contacting at least two separate components under pressure, said '35 ίο • '25 containing water and another the component containing a lipid material, and passing the mixture under pressure through a nozzle or the like to obtain an aerosol spray containing liposomes.

In the method of the previous application, liposomes are prepared in situ or without preparation using a pressurized aerosol system. To achieve this, the aqueous first component and the second component containing the dry lipid material are mixed by spraying to form a mist in which the liposomes may be either alone or as a liposomal carrier for one or more active agents, generally biologically active agents and / or agents useful in treatment or in the treatment of the human or animal body.

An advantage of the previous invention is that the active substance and water, and / or the propellant and water, can be practically kept separate until the desired dose is divided. It must be emphasized that by separating the substances which may interact with each other, leading to degradation, a sufficiently long service life can be achieved to enable the method to be used in many different practical contexts. At the same time, however, there may be certain connections where the resulting long lifespan is undesirable or necessary.

We have now surprisingly found that liposomes can be prepared without preparation by a much simpler method, for example for use in such contexts, by dissolving the lipid component in an organic solvent and combining the solution thus prepared with water to obtain a stock of combined material. The combination comprising a multi-dose system may be suitably mixed either before packaging and / or otherwise before use, for example by emulsifying the solution and water, and doses of the combination may be passed through a nozzle, e.g. a spray nozzle, to give separate doses as an aerosol spray. containing liposomes.

Accordingly, the present invention relates to a process for preparing liposomes, which comprises preparing a solution of a lipid component in an organic solvent, combining the solution thus prepared with another component containing water, forming a close mixture of the mixture, and passing a portion of the resulting mixture under pressure through a nozzle, e.g. to obtain an aerosol spray containing liposomes.

The method, packaging and use according to the invention are characterized by what is stated in claims 1, 9 and 14.

As in our previous application, in the present invention, liposomes are prepared without prior preparation using a pressurized aerosol system. However, in the present method, the aqueous component and the solution of the lipid component in the organic solvent are first combined to form a pool of combined material providing a multi-dose system (e.g., pre-packaged together) followed by a mixture, typically an aqueous emulsion that c an be passed through a nozzle under pressure. forming separate doses as a spray, If the liposomes may be on their own or as a liposomal carrier for one or more other substances. Thus, in a preferred embodiment of the present invention, a liposomal aerosol is formed comprising a liposomal carrier and a separate active agent that is generally biologically active and / or useful in treating or treating the human or animal body or plants. Such active agents are generally non-lipid agents, although in some cases the lipid may be a carrier for the active moiety, for example, associated therewith.

Alternatively, the invention may be used in the preparation and production of a liposomal aerosol as such, i.e. without a separate active compound or other substance. Such aerosols find a wide variety of uses, either in the preparation of liposomes per se for various applications, or in therapy. They are particularly useful in deliveries where newborns have difficulty breathing (respiratory distress syndrome), these can be helped to breathe more normally by administering a lipid to the bronchial area to compensate for the low levels of natural lung surfactants observed in these newborns.

However, a separate active ingredient is preferably present, which may be either in solution in the aqueous component or in solution in an organic solvent. On the other hand, with or without a separate active agent, a combination of a lipid solution and an aqueous component, which system generally contains more than a unit dose of active agent, is used to dispense unit doses as a spray.

In the method of the invention, the effect of incorporation into the mixture results in the formation of a liposomal material. Typically, the mixing step produces a mixture which, as such or during spraying, consists of an aqueous emulsion containing lipid micelles from which a liposomal material can be formed. In this regard, and because we do not want the present invention to be limited to any particular hypothesis, we believe that liposome formation occurs upon spraying upon decomposition of a previously formed aqueous emulsion containing lipid micelles as the organic solvent evaporates upon spraying and the stabilizing effect on the lipid phase . The single-walled micelles in the emulsion can open, and the lipid layers thus formed can adhere around the other intact micelles, forming a double-walled material, i.e., a liposome.

Further, when an active agent is present, the liposomal material contains the active agent in an aqueous medium either in an encapsulated form between lipid layers or layered between lipid molecules. By varying the conditions under which the mixture is formed and / or sprayed, the size of the liposomes and the relative proportions of free and bound active substance can vary over a wide range, the latter up to 100% binding. For example, by forming and / or spraying an aerosol mixture with varying degrees of mixing, for example simply mixing together the combined components, or under turbulent conditions which can be developed, for example by means of a mixing chamber and / or associated feed and / or spray nozzle, a spray can be prepared, with either a high proportion of free active substance - giving a rapid effect with a slow release active substance - or with a lower proportion of free active substance - giving a slower effect with a long-term and controlled release active substance. Thus, in the latter case, it is possible to prepare a preparation containing the active substance in which a large proportion of the material is obtained in a protected form at the desired site of action and / or to act in a delayed manner.

The method according to the invention is suitable for use in the preparation of preparations containing many types of biologically active substances and / or other substances which are useful in the treatment or treatment of the human or animal body or plants. Typically, the method can be used in the preparation of preparations containing therapeutically active compounds, including those used in veterinary medicine as well as biologically active reagents, cosmetically active substances, compounds having nutritional value, and other substances used in the treatment or treatment of the human or animal body. The preparations may also contain substances used in the treatment of plants, such as herbicides, pesticides, substances used as plant growth regulators and the like.

Suitable cosmetically active substances are products intended for the treatment of the skin and hair, for example moisturizers, artificial tanning agents (possibly in combination with colorants), flame retardants, antiperspirants, deodorants, lotions and refreshing, tinting, scaring, keratolytic and hair removal products scented water, extracts of animal and vegetable tissues, hair dyes, anti-dandruff agents, anti-sebum preparations, oxidizing agents, e.g., bleaching agents and reducing agents.

Therapeutically active substances include vitamins, steroids, e.g., corticosteroids, hormones, active peptides, enzymes, vaccines, anti-inflammatory agents, antibiotics and bactericides. However, the method of the invention is particularly suitable for the administration of therapeutically active compounds as inhalation therapy, such as bronchodilators and anti-asthmatic compounds, as well as antitumor agents. Such compounds, contained within or in a collection of liposomal carrier, are capable of penetrating the branches of the bronchi, thus advantageously modifying the efficacy of the therapy. Similarly, the method has particular advantages in the treatment of open wounds, e.g., intestinal ulcers, in which a liposomal carrier can also be used to simultaneously (promote) a modified effect, allowing treatment to be substantially limited to the ulcer without clear systemic disintegration.

<td></td><td>Of the above</td><td>in accordance with, and only</td><td>by way of illustration,</td>

<td> 5</td><td colspan="2">the following individual combinations may be mentioned:</td><td>seats and</td>

<td></td><td>Active compound</td><td>Type of compound</td><td>Method of administration</td>

<td> 10</td><td>Stanotsoli</td><td>Anabolic steroid</td><td>Local (e.g., open wound treatment)</td>

<td></td><td>Hydrocorticles</td><td>Anti-inflammatory steroid</td><td>Local</td>

<td> 15</td><td>betamethasone (and its esters, e.g. valerate)</td><td>Anti-inflammatory steroid</td><td>Local</td>

<td></td><td>bitolterol (and its salts, e.g. mesylate)</td><td>Katekolamiinibronkodilaattori</td><td>inhalation</td>

<td> 20</td><td>salbutamol</td><td>Katekolamiinibronkodilaattori</td><td>inhalation</td>

<td></td><td>theophylline</td><td>Ksanteenibronkodilaattori</td><td>inhalation</td>

<td></td><td>Natriumkromigly-</td><td>Non-steroidal</td><td>inhalation</td>

<td></td><td>cate</td><td>anti-asthmatic</td><td></td>

<td> ...25</td><td>N-acetylmuramyl</td><td>Macrophage activator</td><td>inhalation /</td>

<td> -</td><td>L-alanyl-D-isoglucose</td><td>substance (tumor left)</td><td>intranasal</td>

<td> ; · ;</td><td>tamamine (adjuvant</td><td rowspan="2">full dipeptide)</td><td rowspan="2">linen</td>

<td> . \</td><td>dipeptide)</td>

<td></td><td>propranolol</td><td>/ ^ - blocking agent and</td><td>Oral/-</td>

<td> 30</td><td></td><td>spermicide</td><td>intravaginal</td>

Of course, it will be apparent to those skilled in the art that numerous other active agents may be used. Still though topical, inhalation, intranasal and nasal_: .35 other routes of administration may be used whenever appropriate. For example, preparations prepared by the method of the invention and containing a suitable active ingredient may be administered directly to the eye or even intravenously.

In addition, mention may be made of biologically active substances which may also be therapeutically active, such as vaccines, or other biologically active substances which are useful as reagents, such as antigens and / or antibodies, e.g. anti-rabbit IgG. Reagents of this type may be useful in testing blood samples, typically for examining whether a disease is present or not.

The liposomal agent prepared according to the invention may be positively or negatively charged or neutral. The substance contains a lipid material in aqueous dispersion or suspension and may contain one or more other active substances. Furthermore, the lipid material may be in the form of a unilamellar and / or multilamellar lipid bilayer, which may contain any of said other active substances.

The liposomal material typically consists of any amphipathic lipid suitable for the manufacture of liposomes and generally has the formula:

XY wherein X is a polar hydrophilic group and Y is a non-polar hydrophobic group.

Preferably, however, the liposomal material contains a phospholipid, and more preferably it is a phospholipid containing phosphatidylcholine. For example, dipalmitoylphosphatidylcholine as such or in the form of lecithin; fosfadityylietanolamiinia; phosphatidylserine; fosfatidyylisfingomyeliiniä; fosfatidyylikardiolipiiniä; fosfatidyyliplasmalogeenia; phosphatidic acid; and / or phosphatidine cerebroside may be

<td></td><td>used as a lipid material. The liposomal material may also consist of a phospholipid, e.g. lecithin, alone or in combination with cholesterol and / or at least one compound which may provide a charge to the liposomal.</td>

<td> 5</td><td>material. Thus, for example, a charge can be obtained in a liposomal material using diacetyl phosphate or phosphatidic acid, which can give a negative charge to the lipid material, or stearylamine, which can give a positive charge to the lipid</td>

<td> 10</td><td>material. The use of the substances described above and their ratios, as well as the wide variety of materials available and the variations in their ratios, are known to those skilled in the art. In general,</td>

<td> 15</td><td>In ratios of lipid materials, the desired liposomal material is obtained, provided that the lipid component generally does not contain more than about 50% by weight of cholesterol and / or more than about 20% by weight of the charge-providing substance.</td>

<td> 20</td><td>However, by way of example only, in a preferred embodiment of the invention, the lipid component may contain phosphatidylcholine and cholesterol, typically in relative molar ratios of about 8: about 1-2, e.g., about 8.5: 1.</td>

<td> -25</td><td>In another preferred embodiment of the invention, the lipid component may contain phosphatidylcholine, cholesterol and disethyl phosphate, typically in relative molar ratios of about 8: about 1-2: about 1 to 0.05, e.g., about 8.5: 1: about 0.7 to 0.07.</td>

<td> 30</td><td>In a further embodiment of the invention, the lipid component may contain phosphatidylcholine, cholesterol and phosphatidic acid, typically in relative molar ratios of about 8: about 1-2: about 1 to 0.1, e.g., about 8.5: 1: about 0.1.</td>

In another preferred embodiment of the invention, the lipid component may contain phosphatidylcholine, cholesterol and stearylamine, typically in relative molar ratios of about 8: about 1-2: about 1 to 0.05, e.g., about 8.5: 1: about 0.06.

Alternatively, the lipid component may include, for example:

a mixture of phosphatidylcholine, e.g., lecithin, and phosphatidic acid, e.g., in a ratio of about 60: about 1;

a mixture of phosphatidylcholine, e.g., lecithin, and diacetylphosphate, e.g., in a ratio of about 15: about 1; or most preferably, lecithin alone.

The pH of the liposomal material produced in the invention is in the physiological range, so it is preferably between about 6.8 and 7.4, more preferably between about 7 and 7.4. A system comprising said solution and said aqueous component may also contain a buffering agent in an amount suitable to provide and maintain the desired pH, and a phosphate buffer typically containing sodium dihydrogen phosphate and disodium hydrogen phosphate is suitable for this purpose. However, in some cases, e.g. when the active substance consists of certain active compounds or reagents, such as a test tracer, the pH may be outside the physiological range. Thus, for example, in the case of hydrocortisone, the pH may be less than 5 for stability reasons.

The organic solvent used in the invention may be any solvent that dissolves the selected lipid component and that is otherwise suitable for the intended use. However, the solvent is preferably physiologically acceptable, and one in which both the lipid component and the other active substance are soluble.

Preferred solvents in this regard are relatively volatile organic solvents, for example alcohols such as ethanol and isopropyl alcohol, chloroform (subject to local regulations) and ether. The second component containing water may also contain small amounts of a compatible organic solvent such as ethanol, for example in an amount of up to 10% by weight.

In a preferred embodiment of the invention, the aerosol is prepared using one or more aerosol propellants. This propellant or propellants may be in a system containing said solution and said aqueous component and / or are fed separately. Preferably, however, a single system comprising one or two propellants, said solution and said aqueous component is used.

Typically, the propellant consists of a hydrocarbon or halogenated hydrocarbon or a mixture thereof. Examples of suitable propellants include propane, butane, compressed air, nitrogen, dimethyl ether, and propellant 11 (trichlorofluoromethane), although dichlorodifluoromethane and dichlorotetrafluoroethane or mixtures thereof are most preferred. The propellant can be prepared as a low pressure propellant that develops pressures between about 25 and 30 psi, such as a 20:80 mixture of dichlorodifluoromethane and dichlorotetrafluoroethane. Alternatively, the propellant may be a relatively high pressure propellant that always develops a pressure of about psi, such as a mixture of dichlorodifluoromethane and dichlorotetrafluoroethane in a weight ratio of 80:20.

In the method according to the invention, the selected preparation can be dispensed using various conventional spray valve systems. However, when the formulation contains the active agent to be dispensed in controlled discrete doses, the formulation is preferably allowed to discharge through the nozzle under pressure using a metering valve to provide the desired metered dose of that active agent with each actuation of the valve.

In the method according to the invention, the emulsion must also contain sufficient water to hydrate the lipids. Thus, although the weight ratio of water to lipid used may be as low as 2: 1, it is preferred to use a weight ratio of water to lipid of at least about 5: 1, more preferably at least about 10: 1, for example about 20: 1 or greater, e.g. 30: 1. In addition, sufficient organic solvent must be used to dissolve the lipid component to avoid the formation of a dispersion of solid lipid particles in water. Also, the amount of water in the mixture should generally be greater than the amount of propellant (if mixed therein), and preferably there is an excess of water of at least about 1.5: 1 with respect to any propellant mixed with it. Furthermore, any substances in the emulsion and / or their amounts must not be such as to interfere with the ability of any selected water / lipid mixture to form liposomes.

Thus, the following are preferred ranges of amounts for the various materials based on the weight of the entire system and the preferred weight ratios: Water - at least 45% and up to 97%, more preferably about 45% - 70%; Lipid - not more than 25%, more preferably about 2% to about 10%; Propellant - about 5% to about 50%, more preferably about 20% to 45%; Solvent - about 4% to 20%; Water / lipid - about 2: 1, more preferably about 5: 1 to 30: 1; Solvent / lipid - about 40: 1 or lower, e.g. 2: 1 or lower; Water / solvent - at least about 1: 1, more preferably at least about 4: 1; Water / propellant - about 1.1: 1 20: 1; Propellant / solvent - about 1.3: 1 to 12: 1.

The invention also comprises a kit for use in the manufacture of a liposomal aerosol, the kit comprising at least one chamber containing a solution of a lipid component in an organic solvent and an aqueous component, the first chamber and / or the second chamber containing at least one propellant, and the kit having a nozzle system. containing a lipid solution and an aqueous component, as a spray under pressure, developed with a propellant substance or substances.

Preferably, the kit contains the active substance as defined above in a solution of an organic solvent or in an aqueous component, e.g. water. Also preferred is that the outlet device is such that a measured amount of active substance can be taken out of the package, although a continuous flow valve can be used if desired. In general, the active agent, the lipid solution and the aqueous component, and the propellant are as described above in connection with the process of the invention.

In a package according to the invention, the first chamber may be an aerosol canister or bag, and any second chamber may be a second aerosol canister or bag. When two chambers are used, they are preferably arranged so that the first is inside the second (or vice versa), and the valve is located inside or above the outer canister or in the second chamber. However, even more preferably, the package according to the invention contains either a single canister with all the necessary ingredients and a propellant, or a canister with a flexible bag inside with the necessary ingredients, while the propellant is in the space between the bag and the canister. In the latter case, the ratio of water to propellant is generally close to the upper limit given above, about 20: 1, because much less propellant is usually required in a two-chamber package.

Alternatively, in a variation, the kit of the invention may comprise at least a first chamber containing a solution of the lipid component in an organic solvent, a system for supplying an aqueous component to the chamber, the first chamber, the second chamber and / or the aqueous component containing at least one propellant. through the nozzle as a spray under pressure, developed with propellant material or

- materials.

Such a modified kit can be used to make a kit that contains both the solution and the aqueous component at the time the kit is administered to a patient. In this way, water and lipid can be kept separate until the package is used, e.g., in pharmacy, the first chamber has a valve system that allows the aqueous component to enter the chamber during use. The patient (or physician) can then take the necessary unit doses from a pre-filled pack containing both the lipid solution and the aqueous component.

From the foregoing description, it will be appreciated that when using the present invention, the unit dose of active agent obtained with each use of the package described above will vary widely depending on a number of factors, for example:

1. Concentration of active substance in the packaging system;

2. The size of the metering valve used;

3. Discharge nozzle size; and

4. In the case of continuous flow valves, the duration of operation.

Furthermore, the size of the liposome can be controlled mainly (we believe) by the pressure developed by the propellant substance or substances and / or the viscosity of the mixture to be annealed. In general, the more vigorously the components are mixed, e.g., at the beginning by shaking, or during the discharge phase, the smaller the size of the liposomes. However, the size of the discharge or distribution nozzle of the valve used to dispense the mixed components also affects the size of the liposomes to be produced, and preferably the size of such a discharge nozzle ranges from about 350 to 450 microns.

On the other hand, once the parameters described above have been selected, the dosage level obtained can be readily ascertained, as can the amount of any active substance and the size of the liposomes obtained.

As will be appreciated by those skilled in the art of formulating active ingredients with carrier materials, the concentration of active ingredient in the liposomal carrier prepared in accordance with the present invention will have a clear effect on the daily dosage level selected for the entire formulation. Thus, such a daily dose level depends on the choice of preparation, which depends on the criteria presented, which are encountered in connection with any preparation.

Also disclosed is a composition for use in preparing multiple unit doses of liposomal material, the composition comprising an aqueous component and a solution of the lipid component in an organic solvent, the composition optionally comprising a propellant material or materials and / or an active agent as defined above.

In addition, a liposomal material or liposomal composition used in preparing a nebulizer by the method described herein, or for use in a package described herein is described.

The following examples illustrate the method, packaging and use of the invention.

In the examples given, the preparations contain ammonium molybdate as an electron-dense material, which was present only as a marker to facilitate the detection of liposomes and their diameter measurement using an electron microscope. It is, of course, clear to those skilled in the art of pharmaceuticals that ammonium molybdate, which is a rather toxic substance, must not be part of the actual preparation administered to the patient. The omission of ammonium molybdate has no detrimental effect on the concentration of the active substance, and thus in any of the preparations shown in the examples, the molybdate tracer can be omitted without disadvantage, and the results obtained can be obtained without it.

Also, while many different concentrations of active agent may be exemplified, and while these in turn provide many different concentrations of free and bound active agent, it will again be apparent to those skilled in the art that the dosage level and quality of the selected formulation for any package will depend on the desired dosage level. balance between unbound and bound active substance.

Thus, the active agent may be taken above or below (or even by) a known standardized dosage level to provide any variation in effect that can be increased by the selected concentration level.

i

Example 1

300 mg of egg lecithin and 4 mg of stanozolol were dissolved in 558 mg of ethyl alcohol (BP) and the solution thus obtained was transferred to a standardized 25 ml glass aerosol bottle (inhalation type). To this were added 9.0 ml of aqueous ammonium molybdate solution (0.5% by weight) and 3.0 g of dichlorodifluoromethane. The bottle was then closed with a standard 60 microliter measuring valve and a standard push-button with a diameter

450 micron nozzle, was installed in it. The flask was shaken to obtain an emulsion from an alcoholic and aqueous solution, and the emulsion thus obtained was sprayed as an aerosol. From the obtained aerosol spray, unilamellar and multilamellar liposomes with diameters ranging from 30 nm to 1000 nm were obtained. At each metering valve operation, 20 micrograms of stanozolol was released, of which 13 micrograms had been associated with liposomes.

Examples 2-6

The procedure of Example 1 was followed, but variations were performed as shown in the results in the following table.

Aqueous Valve Dosed Drug-Bound Drug Ethyl Alamycniuntnolyb Dose Number Amount Single Dose Amount Example Lecithin Stanozolol Cholate Date Solution Microliter Milligram Dose Microbial mg mg mg (0.5 wt%) in gin

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AC / F = Continuous flow valve with combined sectioning circuit as described in GB Patent Applications Nos. 84-20850 and 84-20851, now published GB 2 145 107A and GB '2 148 401A.

ND = not determined ίο

Examples 7-9

Three aerosol systems containing bitolterol mesylate were prepared as follows:

The lipid phase was prepared by combining a solution of lecithin (300 mg / ml) and bitolterol mesylate (1 mg / ml) in ethyl alcohol. Different amounts of lipid phase (0.5, 1.0 and 3.0 ml, respectively) were added to different amounts of 0.5% w / v ammonium molybdate solution in water (5.0, 5.0 and 3.0 ml, respectively) in separate aerosol bottles. Propellant 12, i.e. dichlorodifluoromethane (2.6 g), was added to each flask and the flasks were each sealed with a standard 15 mm, 100 microlitre metering valve to give three samples with lipid phase to aqueous phase ratios of 1:10, 1: 5 and 1: 1, respectively. and containing about 92%, about 85% and about 54% by weight of water, respectively, based on the total weight of the lipid and aqueous phases. The flasks were shaken vigorously to mix the phases.

In the first two samples (1:10 and 1: 5 lipid phase: aqueous phase ratios), a white emulsion was observed on a clear propellant phase. In the third sample, a clear solution was observed on top of the clear propellant layer.

Electron microscopic examination of condensed bitolterol mesylate aerosols was performed on samples by fusing the revealing liposomes present in all three nebulizers, albeit in a limited amount in a sample with a 1: 1 ratio of lipid phase to aqueous phase. Dilution of the sample significantly increased the number of liposomes produced, from which it can be concluded that the third sample contained insufficient aqueous phase to completely hydrate the lecithin in the lipid layer upon spraying.

As a result of the drug binding assays, 59, 60 and 10% by weight were obtained, which again indicates that the liposomes were not completely formed in the third sample. A very fine aerosol spray was obtained from all three samples using a pushbutton (manufactured by Coster) fitted with a radial feed connection to provide mechanical disintegration.