SE459471B - LIPOSOMAL AEROSOL COMPOSITION AND PROCEDURES AND PACKAGING FOR PREPARATION THEREOF - Google Patents

Description

. VI '459 471 liga places of action.

It has previously been proposed to prepare liposome compositions containing biologically active compounds and such compositions are disclosed, for example, in British Patent Nos. 1,575,343, 1,575,344 and 2,013,609 A. However, in these known compositions the liposomal material is one which - prepared and dried, generally by lyophilization (freeze-drying).

The liposomal material is then regenerated by mixing with water. Aerosol compositions and therapeutic materials British Patent No. 780 containing a variety of cosmetics are also known - compare, for example, those 885, 993 702, 1,302,671, 1,381,184, 1 S16 195 and 2,001,334 A. Although such known aerosol compositions may contain lipids constituents, these constituents are constantly present only as dispersants, suspending agents, emulsifying agents or the like.

In addition, these compositions are intended to give either a dry spray so that no liposomal formation is possible or, as in the case of powder substrates according to patent no. 780 885, as an emulsified premix of any lipid and water.

In addition, as stated in U.S.P. No. 3,594,476, proposed the preparation of lecithin aerosols useful for the treatment of lung diseases, which may optionally contain other therapeutic agents such as antibiotics.

In the proposed method of preparation, a suspension of DL dipalmitoyl-α-lecithin is first prepared in water (or saline), with any drug dissolved in the water or saline, which suspension is then nebulized, i.e. formed into an aerosol, in an ultrasonic generator equipped with a carrier air stream. The proposed method is thus based on a relatively expensive ultrasound output of the final mixture as well as the use of an initial mixture comprising lecithin suspended in water. The ultrasonic equipment may also be noisy and would be unsuitable equipment for the production of the necessity of lecithin and water, - 459 471 to use in general.

In addition, as stated in U.S.P. No. 4,371,451 proposed the preparation of lecithin-based pan-releasing compositions comprising water, lecithin and dimethyl ether as propellants. Although in this context the solution is distributed from an aerosol container, the stated method is based on the use of an initial mixture of lecithin dispersed in water. What is also sought is a quick-drying and thin coating of lecithin that does not foam rather than a liposomal material - as such.

We have now surprisingly found that using a pneumatic aerosol system, liposomal materials can be formed without preparation from separate aqueous and lipid components and that active materials of various kinds can be administered with improved effect by distributing them as a sprayed mixture with a liposomal carrier formed in situ when the spray mixture is distributed. Such liposomal materials formed in situ when a spray mixture is distributed are also useful per se, for example in the treatment of lung diseases, as is known in the art.

Accordingly, the present invention provides a method of making liposomes, which method comprises pressurizing together at least two separate components, a first component comprising water and a second component comprising a lipid material, and passing the mixture under pressure through a nozzle or a other device and thereby give an aerosol spray containing liposomes.

The method of the invention provides for the preparation of liposomes in situ or extemporaneously using a pneumatic aerosol system. According to the invention, an aqueous first component and a second component comprising dry lipid material are mixed to produce a spray, in which the liposomes may be present as such or as a liposomal carrier for one or more other materials. According to a preferred embodiment of the invention, a liposomal aerosol is prepared comprising liposomal carrier and a separate active material which is generally one which is biologically active and / or useful for the treatment or care of the human or animal body.

Such an active material is generally a non-lipid material, although in some cases a lipid may be used to carry an active group, for example, attached thereto.

Alternatively, the invention may be used to prepare and provide a liposomal aerosol as such, i.e. without a separate active compound or other material. Such aerosols could be applied in several ways, either to produce liposomes per se for a variety of application areas or in therapy. They would be especially useful in childbirth as newborns with respiratory distress syndrome could obtain respiratory assistance more normally by administering a lipid material to their bronchial area to supplement the low levels of naturally occurring lung surfactants found in these children. However, a separate active material is provided which may initially be present in either the first or more preferably the second component. Such an active material may be present as a suspension, e.g. a dry powder, in the first or second component but preferably in solution in one of the components. By using a solution of the active material, it is possible to avoid any sedimentation which would otherwise be the case with a suspension and any subsequent variations in the dose level depending on sedimentation.

In the method according to the invention, the effect of forming and / or spraying the mixture leads to the production of a liposomal material. Thus, we believe that as the mixture is formed, an emulsion is formed comprising lipid micelles in suspension in water, which then yield liposomes upon spraying. In addition, the active material, when present in the liposomal material, comprises active material carried by an aqueous medium either trapped in encapsulated form between the lipid layers or sandwiched between the lipid molecules. By varying the conditions under which the two components are mixed and / or sprayed, the size of the liposomes and the relative proportions of free and incorporated active material can be varied within wide limits in the latter case up to about 100% incorporation. Thus, for example, by shaping and / or spraying the aerosol mixture under varying degrees of mixing activity, for example provided by turbulent conditions generated in, for example, a mixing chamber and / or feed device connected thereto and / or in a spray nozzle, a spray can be formed either as one with a high proportion of free active material which gives a rapid effect with a reserve of slowly releasing active material - or one with a lower proportion of free active material, which gives a slower effect with a more prolonged and controlled release of active material. Thus, in the latter case, a composition may be prepared comprising active material in which a large proportion of the material is capable of being carried in a "protected" form to the intended site of action and / or otherwise acting in a sustained release manner.

The method of the invention can be applied to the preparation, in a form suitable for immediate administration, of compounds comprising a variety of biologically active materials and / or materials useful in the treatment or care of the human or animal body. In general, the methods can be used for the preparation of compositions comprising therapeutically active compounds, including those used in veterinary treatments, as well as biologically active reagents, cosmetically active materials, nutritional compounds and other materials used to treat or care for humans. - or the animal's body. 459 471 Suitable cosmetically active materials include products intended for skin and hair care, such as wetting agents, artificial tanning agents (possibly together with dyes), water-soluble sunscreens, perspiration agents, deodorants, refreshing agents, toning agents, anti- astringents and opioids, keratolytic and depilatory products, perfumed water, animal and plant extracts, water-soluble hair dyes, anti-dandruff agents, anti-seborrheic agents, oxidizing agents, reducing agents. for example bleaches and Therapeutically active compounds which may be mentioned include vitamins, steroids, e.g. corticosteroids, peptides, enzymes, vaccines, antibiotics and bactericides. hormones, active inflammatory agents, anti- However, the method of the invention is particularly useful for the administration of therapeutically active compounds used in inhalation therapy such as bronchodilators and anti-asthmatic compounds as well as anti-tumor agents. Such compounds including 1 or with the liposomal carrier can penetrate the bronchial tree, thus enabling the effectiveness of the therapy to be successfully modified. Likewise, in the treatment of skin lesions or other dermatological treatments, or in cosmetic treatment, the liposomal carrier can be used to give (or promote) a modified effect, for example a delayed release effect and thus enable a certain level of treatment to be maintained during a longer period of time.

In accordance with the above and by way of example, the following specific compounds and modes of administration may be mentioned: Active compound Stanozolol Hydrocortisone (and its esters) Betamethasone (and its esters eg its valerate) Bitolterol (and its esters eg its mesylate) Salbutamol Theophylline Sodium chromium glycate N-acetyl-muramyl-L-alanyl-D-isoglutamine (adjuvant dipeptide) Propranolol 459 471 7 Administration class Association route Anabolic steroid Topical Anti-inflammatory Topical steroid Anti-inflammatory Topical steroid Catecholamine Inhalation bronchodilator Xanthene Inhalation bronchodilator Non-steroidal Inhalation anti-asthmatic Macrophage active Inhalation / ringing agent intranasal (anti-tumor dipeptide) B-blocking agent Intravaginal and contraceptive A person skilled in the art should of course understand that a variety of other active materials can be used as well as other administra- method. For example, the compositions prepared according to the method of the invention containing appropriate active materials may be administered directly, for example to the eye or even intravenously.

In addition, mention may be made of biologically active materials which may also be therapeutically active such as vaccines or other biologically active materials useful as reactants such as antigens and / or antibodies, e.g. anti-rabbit IgG. Reagents of this kind can be used as markers in, for example, test blood samples, especially to detect the presence or absence of disease.

The liposomal material prepared according to the invention 459 471 may be one which is positively or negatively charged or one which is neutral. The material comprises particulate lipid material present in aqueous dispersion or suspension and may comprise one or more other active materials. In addition, the lipid material may be in the form of unilamellar and / or multilamellar lipid bilayers which associated therewith may carry any of the above-mentioned active materials.

The liposamal material generally comprises a phospholipid and is preferably a phospholipid comprising a phosphatidylcholine, e.g. dipalmitoyl phosphatidylcholine as such or in the form of lecithin; a phosphatidylethanolamine; or a phosphatidylserine.

The liposomal material may be formed from a phospholipid alone or together with cholesterol and / or at least one compound which may give charge to the liposomal material. Thus, for example, charge can be provided on the liposomal material by using dicetyl phosphate or phosphatidic acid, which can give a negatively charged lipid material, or stearylamine, which can give a positively charged lipid material.

The use of the above materials and the proportions thereof, as well as the wide variety of materials and the variations in proportions that can be used, are well known to one skilled in the art. However, for example, in a preferred embodiment of the invention, a second component may comprise a phospholipid consisting of a phosphatidylcholine and cholesterol, generally in relative molar proportions of about 8 about 1 to about 2, e.g. According to another preferred embodiment of the present invention, the second component may comprise a phospholipid consisting of a phosphatidylcholine, cholesterol and dicetyl phosphate, generally in relative molar proportions of about 8: from about 1 to about 2: from about 1 to about 0.05, e.g. about 8.5: l -. from about 0.7 to about 0.07.

According to a further preferred embodiment of the invention, the second component may comprise a phospholipid consisting of a phosphatidylcholine, cholesterol and phosphatidic acid, generally in relative molar proportions of about 8: from about 1 to about 2: from about 1 to about 0.1, e.g. about 8.5: 1: about 0.1.

According to yet another preferred embodiment of the invention, the second component may comprise a phospholipid consisting of a phosphatidylcholine, cholesterol and stearylamine, generally in relative molar proportions of about 8: from about 1 to about 2: from about 1 to about 0.05, .ex. about 8.5: 1: to about 0.06.

Alternatively, the second component may comprise, for example: a mixture of a phosphatidylcholine, e.g. lecithin and phosphatidic acid, e.g. in a ratio of about 60: about 1; or a mixture of a phosphatidylcholine, e.g. lecithin, and di-. cetyl phosphate, e.g. in a ratio of about 15: about 1.

The aqueous liposomal material prepared according to the invention should preferably have a pH in the physiological range and thus preferably in the range from about 6.8 to about 7.4, most preferably from about 7 to about 7.4. It may also contain a buffering agent or buffer system in an amount suitable for determining and maintaining the desired pH and very suitable for this purpose is a phosphate buffer, which may generally comprise sodium dihydrogen phosphate and disodium hydrogen phosphate. In addition, such a buffer may preferably be included in the aqueous first component.

However, in some cases, e.g. when the active maua fi aletine- * 459 471 takes a reagent as a test marker, the pH is outside the physiological range.

Depending on the constituents of the second component, it may be necessary or preferred to include a physiologically acceptable liquid vehicle, preferably one in which both the lipid material and any other active material are soluble. Preferred liquid vehicles are relatively volatile organic solvents, for example ethanol, chloroform (subject to local restrictions), ether and isopropyl alcohol.

In addition, the first component may also comprise a physiologically acceptable liquid vehicle in addition to water. Such a vehicle may be a volatile organic solvent, preferably a water-miscible organic solvent, of which ethanol is preferred.

According to a preferred embodiment of the invention, the first and second components are mixed under pressure using one or more aerosol propellants, which may be a component of one or both components and / or provided separately.

However, the propellant is preferably a component of the second component and the pressure is applied to the first component via a propellant which is kept separate therefrom.

In general, one or more propellants may comprise a hydrocarbon or halogenated hydrocarbon or a mixture of such hydrocarbons, examples of such propellants are propane, butane, dimethyl ether or propellant II (trichlorofluoromethane) although dichlorodifluoromethane or dichlorotetrafluoroethane or mixtures thereof are preferred. . The propellant may be a propellant prepared as a low pressure propellant and develops pressures up to, for example, about 1.75 to about 2.1 kP / cm 2 such as a 20:80 w / w mixture of dichlorodifluoromethane and dichlorotetrafluoroethane or the propellant may be a relatively high pressure propellant which develops a pressure of approximately 4.9 kP / cm 2 such as an 80:20 w / w mixture of dichlorodifluoromethane and dichlorotetrafluoroethane. Of course, it should be clear that relative dose ratios such as between the components can be controlled and varied inter alia depending on the fuel pressure used for each of the components.

According to the method of the invention, it is preferred that the first and second components each be provided isolated from the second, that each of the components be delivered in controlled separate doses to a mixing chamber under its own propellant pressure and that the mixture thus formed flow out of the mixing chamber through an orifice under the pressure of at least one of said propellants. Preferably, the first and second components should also be mixed in a manner that provides a measured dose of any of said active materials. Most preferably, the second component should be delivered as a metered dose to a metered dose of the first component and preferably brought into contact with at least the main mass of the first component at a variety of locations within said metered dose. Thus, in particular, the method according to the invention preferably comprises in sequence the following steps, namely: the first component is delivered to a mixing chamber in a measured amount; a measured amount of the second component is delivered to the mixing chamber in a manner that provides turbulent mixing of the two components, preferably by introducing the second component into the mixing chamber at a plurality of locations; and the mixture flows out of the mixing chamber.

Conveniently, the measured amount of the first component is controlled by delivering the first component to the mixing chamber in an amount that fills the chamber, and by selecting chamber volume, the desired measured amount of the first component is obtained.

The invention also comprises a package intended for use in the manufacture of a liposomal aerosol, which package comprises at least a first and a second chamber, a chamber containing a first component comprising water, which may constitute a buffered aqueous material and the second the chamber contains a second component comprising a lipid material, one of the components optionally comprising, preferably in suspension or solution therein, said active material, e.g. For use in the treatment or care of the human or animal body And one or both of the chambers and / or a third chamber comprising a fuel material, the package comprising a device for distribution, such as a spray, a mixture of the first and second components fed into from the respective chamber under pressure generated by the fuel material or materials.

Preferably, the dispensing device is such that the mixing ratio of the components is controlled (for example to a measured level or to within a narrowly measured range ratio), whereby the desired measured dose of any of said active materials can be dispersed. Once again, it is preferred that the active material be in suspension or solution in the second component comprising a lipid material, and most preferably the active compound should be in solution, to avoid sedimentation. In general, the active material, the first and second components and the propellant are as described above for the method according to the invention.

Preferably, the package according to the invention comprises an outflow mouth and a mixing chamber and a valve device for allowing said first and second components to be delivered from their respective chambers under pressure generated by one or more propellants in the mixing chamber and for controlling the components ratio (for example to a measured level or within a narrowly measured ratio range) whereby the desired measured mixing dose, i.e. active material plus liposome carrier or liposome per se, can be dispersed through the nozzle orifice.

Preferably, said valve device comprises a device (for example of two valves) for sequentially allowing the first component to be delivered to the mixing chamber to fill the chamber, delivering a measured dose of the second component to the mixing chamber in a manner which provides turbulent mixing of the two components and outflow of the mixture through said nozzle orifice. Preferably, the valve devices also comprise a device which allows the second component to be delivered to the mixing chamber at a plurality of locations within the chamber.

If desired, additional mixing devices may also be provided either upstream or downstream of the mixing chamber to create additional intimate and / or turbulent mixing conditions to promote emulsion formation prior to effluent through the effluent orifice. Thus, for example, the outflow orifice device may itself comprise a subordinate mixing chamber in which the mixture is fed in such a way that further turbulent conditions are formed, e.g. by being tangentially fed into the chamber via one or a plurality of inlet openings before the outflow. In the package according to the invention, the first chamber may be a first aerosol can or bag, and the second chamber may be a second aerosol can or bag, and the cans or the other first and second closed chambers may be arranged: valve device and mixing chamber arranged between or above the cans or the other chambers; Side by side with the respective valve device and mixing chamber arranged between or over the cans or the other chambers; or The first in the second (or vice versa) with valve devices and mixing chambers arranged within or above the outer can or second chamber. Aerosol dispensers using this type of arrangement are described, for example, in U.S. Pat. Patent Nos. 3,325,056 and 3,326,416.

In the package according to the invention an outer magazine is provided, e.g. a (cylindrical) container, within which the closed chambers can be arranged and through which the valve devices can be operated. In such a package it is preferred that the second component comprises a propellant and is arranged in an inner sucked chamber with rigid walls, that the first component is arranged in an outer closed chamber with flexible walls, preferably surrounding the inner chamber, that the magazine is a magazine with rigid walls and that the area between the outer closed chamber and the surrounding magazine contains a fuel. An especially preferred two-component packaging device suitable for providing these embodiments is that described in European Patent Nos. 84-10-45891.1 and British Patent No. 2148401 both based on Swiss Patent No. 664943.

However, if desired, the inner chamber device may be the same as the outer chamber device, i.e. with the inner chamber formed as a chamber with flexible walls surrounded by a chamber with rigid walls containing propellant. As will be apparent from the foregoing, in the practice of the present invention, the dosage level of the active material dispensed through each operation of the package described above may vary widely depending on a variety of factors, in particular: 1. The active concentration materials of the first and / or second component; and 2. The mixing ratio between the first and second components.

The mixing ratio of the components depends on the size of the measured amounts of the first and second components provided for mixing, which in turn depends on the particular mixing and / or valve devices used. In the particular case of the particularly preferred device mentioned above, the mixing chamber preferably has a volume of from about 25 or about 50 to about 100 e.g. 75 to 100 microliters and the valve that controls the measurement of the second component preferably distributes to the mixing chamber from about 25 or about 50 to about 100 microliters per activation.

We assume that the liposome size is mainly controlled by the pressure generated by the propellant or propellants and / or by the viscosity of the components. This generally means that the more strongly the components are mixed, the smaller the size of the liposomes. However, the size of the discharge or distribution orifice in the valve used to distribute the mixed components can also affect the size of the liposomes produced, and such an effluent orifice preferably has a size of from about 350 to about 450 microns.

On the other hand, once the above parameters have been determined, the resulting dose level can be readily determined as well as the degree of incorporation of any active material and the size of the liposomes produced.

As any person familiar with the preparation of active materials -familiar with pharmaceutical preparations with carrier materials will recognize, according to the present invention, the level of incorporation of active material within the liposomal carrier may have a pronounced effect on the initial (date) dose level selected for the whole. the preparation. Consequently, this initial dose level becomes a preparation choice depending on the calculated criteria to be met by the particular preparation.

The invention further comprises a liposomal material or a liposomal composition prepared as a spray according to a method described herein or by using a package as described herein.

The following examples illustrate the method and aerosol packaging of the invention.

In the specific examples, a majority of the formulations comprise ammonium ylobdate as an electron-tight material, which was incorporated only as a marker to facilitate the tracking of liposomes and the measurement of their diameter using an electron microscope. It should, of course, be understood by all, that ammonium molybdate, which is moderately toxic, should not form part of a current working preparation for administration to a patient. As can be seen from a comparison between, for example, Example 19 and the example below, the exclusion of ammonium molybdate has no detrimental effect on the level of incorporation of active ingredient and in fact the level of incorporation is somewhat higher in Example 20. Consequently, in any of the formulation examples the molybdate marker is excluded without delay and the given results can be obtained in its absence.

Although a variety of levels of active ingredient are exemplified and although this in turn provides a variation of levels of free and incorporated active ingredient per effect, it should again be understood by one of ordinary skill in the art that the dose level and type of formulation chosen for which preferably special work packaging depends on the particular desired dose range to be achieved, as well as any necessary or desired balance between - 459 471 l7 not incorporated and incorporated active material.

For example, assuming that an average person on average applies a cream formulation to the skin in an amount of 2.2 mg / cm 2 and assuming that a simple aerosol spray or "shot" covers 25 cm 2 of skin, the following comparison can be made between exemplified and standard compositions: Formulation Application level ( micrograms / cm 2) - Stanozolol cream (0.1%) 2.3 Stanozolol aerosol from Example 21 4.0 Hydrocortisone cream (1.0%) 23 Hydrocortisone aerosol. according to Example 25 4.8 Betamethasone cream (0.1%) 2.5 Betamethasone aerosol according to Example 27 0.64 Thus, the active material can be distributed at a total dose level above or below (or even at) the known standard. - the level to give any desired variations in effect, which variation in effect is improved by the selected degree of incorporation.

Likewise, in the preparations intended for inhalation, the total dose level can be varied depending on the desired effect. As illustrated, for example, in Example 31, salbutamol can be effectively distributed per impact as 125 micrograms of slow release material and 125 micrograms of free material compared to 100 micrograms per impact from a standard dispenser. In addition, as illustrated by Examples 29 and 30, the active material may be administered at much higher or much lower dose levels per exposure than by standard - i.e. 933 micrograms versus 200 micrograms (standard) for bitolterol in Example 29 and 200 micrograms versus 2 mg (standard) for sodium cromoglycate in Example 30 - although in both cases the dose level can be adjusted to the standard level 459 471 18 by changing the total amount of active material which b is honored by the other resp. first component.

Example 1 240 mg of egg lecithin and 28 mg of cholesterol were dissolved in 4 ml of ethanol and the solution was added to 25 g of a 20 -. 80 w / w mixture of dichlorodifluoromethane and dichlorotetrafluoroethane propellants. The mixture was then sealed in a first container equipped with a valve. 25 ml of a 3.3 mM aqueous sodium phosphate solution, pH 7.2, was introduced into an aluminum tube which was then sealed in a separate container equipped with a valve and containing 25 g of the above propellant. The containers were connected via a mixing chamber and each actuation on the valve mechanism released part of the lipid solution and part of the phosphate solution to the mixing chamber and then to the atmosphere outside via a narrow mouth. The resulting aerosol contained multilamellar and unilamellar liposomes with a mean diameter of from 40 nm to 1000 nm.

Example 2 The procedure of Example 1 was repeated except that the first container equipped with a valve contained a mixture of 240 mg of egg lecithin, 28 mg of cholesterol and 4 mg of phosphatic acid dissolved in 4 ml of ethanol, together with 25 g of a 40: 60 w / w weight - mixture of dichlorodifluoromethane and dichlorotetrafluoroethane propellants.

The resulting device could be used to make an aerosol containing negatively charged multilamellar and unilamellar liposomes with an average diameter of from 40 nm to 1000 nm. 19 459 471 Exemgel 3 The procedure of Example 1 was repeated except that the first container equipped with a valve contained a mixture of 240 mg of egg lecithin, 28 mg of cholesterol and 1.6 mg of stearylamine dissolved in 4 ml of ethanol, together with 25 g of a 20: 80 w / w mixture of dichlorodifluoromethane and dichlorotetraethane propellants.

The resulting device could be used to produce enerosol, containing positively charged multilamellar and unilamellar liposomes with an average diameter of from 40 nm to 1000 nm.

Example 4 The procedure of Example 1 was repeated except that the first container equipped with a valve contained a mixture of 240 mg of egg lecithin and 4 mg of phosphatidic acid dissolved in 4 ml of ethanol, together with 25 g of a 20:80 w / w mixture of di - chlorodifluoromethane and dichlorotetrafluoroethane propellants.

The resulting device could be used to make an aerosol containing negatively charged multilamellar and unilamellar liposomes with an average diameter of from 40 nm to 1000 nm. Example 5 The procedure of Example 1 was repeated except that the first container equipped with a valve contained a mixture of 24 mg of dipalmitoylphosphatidylcholine, 2.8 mg of cholesterol, 2 mg of dicetyl phosphate and 2 mg of bitolterol mesylate dissolved in 4 ml of ethanol, together with 25 mg. g of a 40:60 w / w mixture of dichlorodifluoromethane and dichlorotetrafluoroethane propellants.

The resulting device could be used to produce a 459 471 ′ ′ aerosol containing negatively charged multilamellar and unilamellar liposomes with an average diameter of from 40 nm to 1000 nm. In addition, more than 50% of the bitolterol mesylate was incorporated into the liposomes.

Example 6 The procedure of Example 1 was repeated except that the first container equipped with a valve contained a mixture of 240 mg of egg lecithin, 28 mg of cholesterol, 2 mg of dicetyl phosphate and mg of bitolterol mesylate dissolved in 4 ml of ethanol, g of a 40: together with 60 wt. / weight mixture of dichlorodifluoromethane and dichlorotetrafluoroethane propellants.

The resulting device could be used to provide an aerosol containing multilamellar and unilamellar liposomes with a diameter of from 40 nm to 1000 nm. In addition, more than 50% of the bitolterol mesylate was incorporated into the liposomes.

Example 7 50 mg of egg lecithin was dissolved in ethyl alcohol (96% BP) and the volume was supplemented to 1.0 ml with ethyl alcohol to give a lipid component. The resulting solution was transferred to a glass vessel and 7.0 g of dichlorodifluoromethane was added. The glass vessel was then sealed with a 65 microliter measuring valve connected to a 75 microliter mixing chamber arranged to receive lipid components which flow out through the measuring valve and in themselves form part of a second valve system.

The mounting device thus formed was placed in a second flexible vessel (a tubular bag) containing 40 ml of ammonium molybdate solution (0.5% w / v in deionized water) to provide a water component, which component is in communication with the mixing chamber with the second valve in its closed position. The second vessel was then pressed into a magazine containing 2.0 g of dichlorodifluoromethane propellant in a manner whereby the second valve system (connected to the bag and the mixing chamber) could, here activated itself, also activate the first valve for outflow of lipid components into the mixing chamber. thereby providing an outflow of mixed components from the entire aerosol magazine structure, e.g. as described in the aforementioned British Patent No. 2148401, based on Swiss Patent No. 664,943. A standard spray head with a 450 μm orifice was fitted to the second valve system.

The resulting aerosol magazine construct could be used to prepare an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 400 nm.

Example gel 8 The procedure of Example 7 was repeated except that the glass vessel was equipped with a 50 microliter measuring valve connected to a 100 microliter mixing chamber. The resulting aerosol magazine construct could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 1000 nm.

Example 9 The procedure of Example 7 was repeated except that the glass vessel was equipped with a 100 microliter measuring valve connected to a 100 microliter mixing chamber. The resulting aerosol magazine construct could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 50 nm to 1000 nm. Example 10 60.0 mg of egg lecithin and 7.0 mg of cholesterol were dissolved in 1.0 ml of ethyl alcohol (96% BP). The resulting solution was transferred to a glass vessel and 6.0 g of a 20: §0 w / w mixture of dichlorodifluoromethane and dichlorotetrafluoroethane was added thereto.

The glass vessel was then sealed with a 50 microliter measuring valve connected to a 100 microliter mixing chamber as in Example 7.

The structure thus formed was placed in a second, flexible vessel containing 40 ml of 3.3 mM aqueous sodium phosphate solution at pH 7.2. As in Example 7, the second vessel was then pressed into an aerosol magazine containing 2.0 g of dichlorodifluoromethane propellant in a manner whereby the second valve system allowed the spray to flow out of the entire aerosol magazine assembly. A standard 450 μm orifice spray head was then fitted to the second valve system¿ The resulting aerosol magazine design could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 1000 nm.

Examples 11 to 15 The procedure of Example 10 was repeated using in a glass vessel a solution of the following ingredients dissolved in 1.0 ml of ethyl alcohol (96% BP), namely: Example No. 11. 60.0 mg egg lecithin, 1.0 mg phosphatidic acid and 7.0 mg cholesterol. l2. 60.0 mg egg lecithin, 0.4 mg stearylamine and 7.0 mg cholesterol. - 459 471 23 13. 60.6 mg egg lecithin and 1.0 mg phosphatidic acid. 14. 0.5 mg bitolterol mesylate, 6.0 mg dipalmitoylphosphatidylcholine, 0.5 mg dicetyl phosphate and 0.7 mg cholesterol. . 5.0 mg bitolterol mesylate, 60.0 mg egg lecithin, 0.5 mg dicetyl phosphate and 7.0 mg cholesterol.

The resulting aerosol magazine construct for each of Examples 11 to 15 could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 1000 nm.

Example gel 1600 mg egg lecithin and 6.0 mg stanozolol were dissolved in ethyl alcohol (96% BP) and the volume was supplemented to 2.0 ml with ethyl alcohol. The resulting solution was transferred to a glass vessel and 9.0 g of dichlorodifluoromethane was added. The glass vessel was then sealed with a 50 microliter measuring valve connected to a 100 microliter mixing chamber as in Example 7.

The structure thus formed was placed in a second flexible vessel containing 40 ml of ammonium molybdate solution (0.5% w / w in deionized water). As in Example 7, the second vessel was then pressed into an aerosol canister containing 2.0 g of dichlorodifluoromethane propellant in such a way that the second valve system allowed spray to flow out of the entire aerosol canister assembly. A standard spray head with a 450 μm orifice was then fitted to the second valve system.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 110 nm.

Of the 50.0 micrograms of stanozolol released by each actuation of the second valve system, an amount of 21.6 micrograms was shown to be associated with the liposomes. 459 4 ~ 71 24 Example 17 The procedure of Example 16 was repeated except that the solution filled in the glass vessel contained 450 mg of egg lecithin and 60 mg of stanozolol dissolved in ethyl alcohol (96% BP) with the volume supplemented to 3.0 ml with ethyl alcohol, 4.5 g dichlorodifluoromethane was added to the vessel and the spray head had a mouth of 360 μm.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 44 nm to 440 nm.

Of the 500.0 micrograms of stanozolol released by each actuation of the second valve system, an amount of 35.4 micrograms was shown to be associated with the liposomes.

Example 18 The procedure of Example 17 was repeated except that the measuring valve was a 100 microliter measuring valve and the spray head had a 450 U orifice.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 25 nm to 190 nm.

Of the 1000.0 micrograms of stanozolol released by each actuation of the second valve system, 170.5 micrograms were found to be associated with the liposomes.

Example 19 The procedure of Example 16 was repeated except that the solution filled in the glass vessel contained 900 mg of egg lecithin and 60 mg of stanozolol dissolved in ethyl alcohol (96% BP) with the volume supplemented to 3.0 ml with ethyl alcohol and 4.5 g of dichlorodifluoromethane additive. to the vessel. ~ 459 471 The resulting aerosol container design could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 350 nm.

Of the 500.0 micrograms of stanozolol released by each effect on the other valve system, an amount of 81.3 micrograms was shown to be associated with the liposomes.

Example 20 The procedure of Example 19 was repeated except that the second vessel contained only 40 ml of deionized water.

Of the 500 micrograms of stanozolol released by each action on the other valve system, 85.0 micrograms were shown to be associated with the liposomes.

Example 21 The procedure of Example 20 was repeated except that the solution in the glass vessel contained 12 mg of stanozolol.

The resulting aerosol container design could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 40 nm to 350 nm.

Of the 100 micrograms of stanozolol released by each actuation of the second valve system, 76.8 micrograms were found to be associated with the liposomes.

Example 22 The procedure of Example 21 was repeated except that the second vessel contained 40 ml of a solution of ethyl alcohol (1% v / v) in deionized water.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 30 nm to 500 nm. Of the 100 micrograms of stanozolol released by each actuation of the second valve system, an amount of 77.3 micrograms was shown to be associated with the liposomes.

Example gel 23 The procedure of Example 21 was repeated except that the second vessel contained 40 ml of a solution of ethyl alcohol (5% v / v) in deionized water.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 30 nm to 500 nm.

Of the 100 micrograms of stanozolol released by each actuation on the other valve system, 76.3 micrograms were shown to be associated with the liposomes.

Example Gel 24 The procedure of Example 21 was repeated except that the second vessel contained 40 ml of a solution of ethyl alcohol (10% v / v) in deionized water.

The resulting aerosol container design could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 30 nm to 500 nm.

Of the 100 micrograms of stanozolol released by each effect on the system, 66.6 micrograms were shown to be associated with the liposomes.

Example gel The procedure of Example 17 was repeated except that the solution in the glass vessel contained 900 mg of egg lecithin and 14.4 mg of hydrocortisone BP dissolved in ethyl alcohol (96% BP) with volume supplemented to 3.0 ml with ethyl alcohol. 27 459 471 The resulting aerosol container design could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 44 nm to 220 nm.

Of the 120 micrograms of hydrocortisone BP released by each actuation on the other valve system, 54.0 micrograms were shown to be associated with the liposomes.

Example gel 26 The procedure of Example 16 was repeated except that the solution in the glass vessel contained 841 mg of egg lecithin, 54.8 mg of dicetyl phosphate and 14.4 mg of hydrocortisone BP dissolved in ethyl alcohol (96% BP) with the volume supplemented to 3.0 ml with ethyl alcohol and 4.5 g of dichlorodifluoromethane added to the vessel.

Of the 120 micrograms of hydrocortisone BP released by each effect on the other valve system, 54.6 micrograms was shown to be associated with the liposomes.

Example gel 27 The procedure of Example 26 was repeated except that the solution in the glass vessel contained 841 mg of egg lecithin and 4.8 mg of betamethasone-valued BP / USP / NF dissolved in ethyl alcohol (96% BP) by volume supplemented to 3.0 ml with ethyl alcohol.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 20 nm to 300 nm.

In addition, 16 micrograms of betamethasone valerate was released through each actuation of the second valve system. 459 471 28 Example 28 The procedure of Example 27 was repeated except that betamethasone was excluded from the solution in the glass vessel.

The resulting aerosol container design could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 30 nm to 1000 nm.

Example 29 The procedure of Example 26 was repeated except that the solution in the glass vessel contained 841 mg of egg lecithin and 112 mg of bitol tertrol mesylate dissolved in ethyl alcohol (96% BP) with the volume supplemented to 3.0 ml with ethyl alcohol.- The aerosol container construction obtained could be used. to give an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 25 nm to 250 nm.

Of the 933 micrograms of bitolterol mesylate released by each action on the second valve system, an amount of 545 micrograms was shown to be associated with the liposomes.

Example 30 The procedure of Example 28 was repeated except that the second vessel contained 40 ml of a solution of sodium cromoglycate USP XX, BP '80 (2 mg / ml) and ammonium molybdate (5 mg / ml) in 0.1 M sodium phosphate buffer at pH 7. 4.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 35 nm to 700 nm.

Of the 200 micrograms of sodium cromoglycate released by each effect on the system, 22 micrograms were shown to be associated with the liposomes.

- I 459 471 29 Example 31 The procedure of Example 18 was repeated except that 60 mg of stanozolol was replaced with 15 mg of salbutamol base BP.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes with diameters of from 30 nm to 300 nm.

Of the 250 micrograms of salbutamol released by each effect on the other valve system, 125 micrograms were shown to be associated with the liposomes.

Example 32 The procedure of Example 28 was repeated except that the second vessel contained 10 ml of a solution of N-acetylmuramyl-L-alanyl-D-isoglutamine / NALAG / (100 micrograms / ml) and ammonium molybdate (5 mg / ml) in deionized water.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes. In addition, 10 micrograms of NALAG was released through each actuation on the other valve system.

Example 33 841 mg of egg lecithin was dissolved in ethyl alcohol (96% BP) and the volume was supplemented to 3.0 ml with ethyl alcohol. The resulting solution was transferred to a glass vessel and 4.5 g of dichlorodifluoromethane was added. The glass vessel was then sealed with a 50 microliter measuring valve connected to a 100 microliter mixing chamber, as in Example 7.

The construct thus formed was placed in a second flexible vessel containing 35 ml of a dilution (50 microliters / ml) of anti-rabbit IgG (1/64 titer against 1/640 normal rabbit serum as determined by agar block precipitation titration) and ammonium. -71 molybdate (5 mg / ml) in deionized water. The second vessel was re-pressed as in Example 7 into a container containing 2.0 g of dichlorodifluoromethane propellant in such a way that the second valve system could allow the spray to flow out of the entire aerosol container structure. A standard spray head with a 450 μm orifice was then fitted to the second valve system.

The resulting aerosol container construction could be used to provide an aerosol spray containing unilamellar and multilamellar liposomes. 5 microliters of anti-rabbit IgG (1/64 titer) - was released by each action on the other valve system.

It is to be understood that the invention is not limited to the details of the specific examples set forth above. Thus, for example, a variety of other lipid materials can be used to form the desired liposomal material and a variety of active materials can be carried therewith. Several other aerosol devices can also be used, especially one of the other specific devices mentioned above. In addition, the required pressure can be generated by means of devices other than the use of one or more aerosol propellants.

In general, it is apparent from the foregoing description that the invention encompasses in total any method of in situ preparation of liposomes using a pneumatic aerosol system whether or not the liposomes include or have a separate active ingredient and a package for use.

Claims (20)

- '459 471 31 PATENT REQUIREMENTS A method of preparing liposomes from a lipid material and water, characterized in that at least two separate components are brought together under pressure, a first component comprising water and a second component comprising a lipid material and that the mixture is passed under pressure through a nozzle or another device thereby delivering an aerosol spray containing liposomes. 2. A method according to claim 1, characterized in that at least one of said first or second components comprises an active material which is biologically active and / or useful in the treatment or care of the human or animal body as a therapeutically * or cosmetically active material. . 3. A method according to claim 2, characterized in that the active material is in solution in the first or second component. 4. A method according to claim 2 or claim 3, characterized in that the active material is a bronchodilator, an anti-asthmatic compound, an anti-tumor agent, an anti-inflammatory agent, a contraceptive agent or an anabolic steroid. . 5. A method according to claim 2 or claim 3, characterized in that the active material is an antigen, antibody or other biologically active reagent. 6. A method according to any one of the preceding claims, characterized in that the lipid material in the second component is a phospholipid material selected to provide a positively charged, a negatively charged or a neutral liposome. 459 471 32 7. A method according to any one of the preceding claims, characterized in that the second component comprises a physiologically acceptable liquid vehicle and / or that the first component comprises a physiologically acceptable liquid vehicle in addition to water. 8. A method according to any one of the preceding claims, characterized in that the first and the second component are mixed under pressure generated by at least one aerosol propellant which may be a constituent of one or both components. and / or provided separately. 9. A method according to claim 8, characterized in that a propellant is a component of the second component and that pressure is applied to the first component via a propellant held separately therefrom. 10. l0. A method according to any one of the preceding claims, characterized in that said first and second components are each provided isolated from each other, that each of the components is provided in controlled discrete doses to a mixing chamber under their own fuel pressure and that it so The mixture formed flows out of the mixing chamber through an orifice under pressure from at least one of said propellants. 11. ll. A method according to any one of the preceding claims, characterized in that the second component is provided in a metered dose to a metered dose of the first component. A method according to claim 10 or claim 11, R characterized therefrom, further - in succession the first component is delivered to the mixing chamber to fill the chamber, that a measured dose of the second component is delivered to the mixing chamber in a manner which gives turbulent mixing of the two components and that the mixture flows out of the mixing chamber. - - 459 471 33 A package for use in the manufacture of a liposomal aerosol, said package comprising at least a first and a second chamber, one or both chambers and / or a third chamber comprising a propellant material and characterized in that the first chamber contains a first component comprising water and the second chamber contains a second component comprising a lipid material and that the package comprises a device for distributing, as a spray, a mixture of the first and the second component fed from their respective chambers under pressure - developed of the fuel material or materials. Packaging according to claim 13, characterized in that at least one of said first or second components comprises an active material which is biologically active and / or useful in the treatment or care of the human or animal body and that the distribution device is one according to which mixing ratio of the components can be controlled whereby the desired measured dose of the active material can be distributed. A package according to claim 13 or claim 14, characterized in that the package also comprises an outflow mouth and a mixing chamber and valve device to enable said first and second components to be delivered from their respective chambers under pressure developed by one or more fuel into the mixing chamber and check the condition of the components whereby the desired measured mixing dose can be distributed through the effluent orifice. A package according to claim 15, characterized in that said valve device comprises a device for sequentially enabling the first component to be delivered to the mixing chamber to fill the chamber, a metered dose of the second component being delivered to the mixing chamber. the chamber in a manner which gives turbulent mixing of the two components and that the mixture can flow out through said outflow mouth. A package according to claim 15 or claim 16, characterized in that a device is supplied which comprises the valve device comprising the second component to the mixing chamber at a plurality of locations within the chamber. Packaging according to any one of claims 15 to 17, characterized in that the first and second chambers are closed chambers arranged one in the other, that the valve device and the mixing chamber are arranged in and / or over the outer chamber. Packaging according to claim 18, characterized in that the second component comprises a propellant and is arranged in an inner closed chamber with rigid walls, that the first component is arranged in an outer closed chamber with flexible walls, that the package comprises a outer magazine with rigid walls within which the chambers are arranged and through which the valve device can be operated and that the space between the outer closed chamber and the surrounding magazine comprises a propellant. Liposomes or a liposomal composition prepared as a spray according to a method according to any one of claims 1 to 12 or by the use of a package according to any one of claims 13 to 19.