WO1992003123A1 - Liposome alternative bilayer formulations - Google Patents

Liposome alternative bilayer formulations Download PDF

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Publication number
WO1992003123A1
WO1992003123A1 PCT/US1991/005978 US9105978W WO9203123A1 WO 1992003123 A1 WO1992003123 A1 WO 1992003123A1 US 9105978 W US9105978 W US 9105978W WO 9203123 A1 WO9203123 A1 WO 9203123A1
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component
vesicles
liposomes
lipid
cholesterol
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PCT/US1991/005978
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French (fr)
Inventor
Paul S. Uster
Luke S. S. Guo
Yolanda P. Quinn
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Liposome Technology, Inc.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • A61K9/1271Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Abstract

Lipid bilayer vesicles are formed by combining a micelle-forming anionic or zwitterionic surfactant with either (i) a single-aliphatic chain lipid, or (ii) cholesterol or a cholesterol analogue or derivative, at a weight ratio of between about 1:2 and 2:1. The vesicles can be prepared without the use of organic solvents, and generally employ relatively inexpensive components.

Description

LIPOSOME ALTERNATIVE BILAYER FORMULATIONS

Field of the Invention

The present invention relates to lipid bilayer struct: res formed by a binary mixture of a micelle- forming surfactant and a single-aliphatic chain lipid.

References

Anderson, R. L., et al., Invest. Dermatol. 58:369 (1972).

Brockerhoff, H., et al., Biochim. Biophys. Acta 691:227 (1982).

Fendler, J. H., "Membrane Mimetic Chemistry", Wiley Interscience, New York, NY (1982).

Gebicki, J. M., et al., Nature 243:232 (1973).

Gebicki, J. M., et al., Chem. Phys. Lipids 16:142 (1976).

Hargreaves, W. R., et al., Biochemistry 17:3759 (1978).

Johnston, D. S., et. al., Biochim. Biophys. Acta 602, p. 57, (1980).

Lemp, M. A., et al., Int. Ophthalmol Clin. 13:185 (1973). Lawrence, D. J., et al., Ann. NY Acad. Sci. 106:646 (1963).

Martin, F. J., et al., U. S. Patent No. 4,752,425, issued June 21, 1988.

O'Brien, D. F., et. al., J. Polym. Sci. Polym. Lett. Ed. 19, p. 95 (1981).

Regen, S. L., et. al., J. Am. Chem. Soc. 102, p. 6638 (1980).

Sjogren, H., et al., Surv. Ophthalmol. 16:145

(1971).

Szoka, F., Jr. et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980).

Taylor, J. L., U. S. Patent No. 4,394,372, issued July 19, 1983.

ϋster, P. S., et al., U. S. Patent No. 4,828,837, issued May 9, 1989.

Background of the Invention

A variety of lipid bilayer vesicles, or liposomes, have been described. Typically, bilayer vesicles are composed of phospholipids or mixtures of phospholipids and secondary lipophilic components, such as cholesterol (e.g., Szoka).

Liposomes composed of phospholipids are commonly prepared by hydrating a film of vesicle-forming lipids. Typically, a phospholipid or phospholipid mixture

dissolved in an organic solvent is dried to a thin film, and the film is hydrated by addition of water. The multilamellar vesicles whicw form on hydration have a heterogenous size distribution, and typically show encapsulation efficiency of between about 5-15% (the percent of soluble material present in the hydrating medium which becomes encapsulated in the liposomes) .

Another standard method for producing liposomes is by solvent injection. Here a solution of vesicle-forming lipids, such as phospholipids or phospholipids plus cholesterol, in an organic solvent, are injected into an aqueous medium. The lipids in the organic solvent form liposomes upon mixing with water, and the solvent is removed from the aqueous phase, e.g., by reduced

pressure.

Other methods of preparing liposomes using organic solvents to dissolve the vesicle-forming phospholipids have also been proposed, e.g., by a reverse-phase

evaporation method (Szoka) . One limitation of these methods is the requirement for an organic solvent for the lipids. Such solvents may be incompatible with enzymes or other biomolecules or drugs which are to be incorporated into the liposomes. Further, organic solvents add to the expense and inconvenience of liposome

production, and may be difficult to remove as

contaminants from the final liposome preparation.

Another drawback of conventional liposome

preparation methods is the relatively high cost of purified phospholipid components. Further, phospholipids may undergo peroxidative changes on storage, leading to loss of encapsulated material and/or to toxic lipid products.

Methods of forming liposomes with surfactant components, rather than phospholipids, have also been

proposed. U.S. Patent No. 4,217,344 describes liposomes prepared from polyoxyethylene acyl ethers, sorbitan alkyl esters, and polyoxyethylene sorbitan fatty acid esters.

PCT patent application PCT/US88/00722 describes multilamellar lipid vesicles formed from polyoxyethylene cetyl ether or cetyl amine surfactants, and between 20-50 percent sterol. Also, related PCT patent application PCT/US88/00721 discloses a method of preparing

surfactant/sterol liposomes under conditions which lead to high aqueous volume encapsulation. A lipophilic phase used in the method is composed of a polyoxyethylene acyl ether or a polyglycerol acyl ether surfactant and a sterol and a charge producing amphiphile. With the lipophilic phase maintained above the phase transition temperature of the melting point of the surfactant, the lipophilic phase is combined with an excess of an aqueous phase under high stress, i.e., high shear conditions.

Another related PCT patent application

PCT/US88/00723 describes a preparation of paucilamellar lipid vesicles formed by mixing one of a number of specified surfactants with an aqueous medium under high shear conditions. The surfactants disclosed are

apparently capable of forming lipid bilayer structures in the absence of lipid compounds, although the vesicles may also be formulated to contain sterol and charged

amphiphile compounds.

The present invention describes novel vesicle compositions based on stable, safe, and inexpensive starting materials which, when combined under the appropriate conditions, spontaneously form multilamellar vesicles without the use of organic solvents, specialized

apparatus, or high-shear-mixing conditions.

Summary of the Invention

It is one object of the invention to provide novel lipid bilayer vesicles. These vesicles are composed of (a) an anionic or zwitterionic surfactant which, when dispersed alone in water at a temperature above the surfactant phase transition temperature, is in a micellar phase, and (b) a second lipid selected from the

following: (i) a single-aliphatic chain lipid which, when dispersed alone in water at a temperature above the lipid transition temperature, is in a lipid emulsion phase, and which is an acid, ester, or alcohol; or (ii) a sterol, in particular cholesterol or a cholesterol analog or derivative. In these vesicles the weight ratio between components (a) and (b) is between about 1:2 to 2:1.

Preferred embodiments of the invention include the above vesicles where component (a) is a quaternary phosphate surfactant, such as MONAQUAT P-TL, or a

zxritterionic surfactant, such as cocoamidopropyl betaine or lauroamidopropyl betaine.

Another preferred embodiment includes the above vesicles where component (a) is an anionic surfactant, such as N-methyl cocyl taurate or N-methyl oleyl taurate.

Component (b) of the vesicles of the present

invention can be selected from the group consisting of the following fatty alcohols, or their ester or acid derivatives: lauryl, myristyl, palmityl, palmitoleyl, stearyl, oleyl, linoleyl, arachidatyl, and arachidonyl.

In one embodiment of the present invention component (a) is N-methyl cocyl taurate and component (b) is oleic acid.

In another aspect of the present invention, the aliphatic-chain of component (b) is greater than about 12 carbons in length: the aliphatic chain can be either saturated or unsaturated. For example, component (b) can be retinol or retinoic acid.

In one embodiment, the vesicles of the present invention comprise N-methyl cocyl taurate, oleic acid, and minoxidil at a weight percent of approximately

3.7:2.5:2, respectively. In another specific embodiment the vesicles comprise N-methyl cocyl taurate, oleic acid, and hydrocortisone at a weight ratio of approximately 1.4:0.9:1, respectively.

Further, component (b) can be cholesterol,

cholestanol, cholesterol sulfate and another cholesterol analog or derivative. The invention further includes a method of preparing lipid-bilayer vesicles by combining components (a) and (b) in an aqueous medium in a weight ratio of between 1:2 to 2:1. Usually component (a) is suspended in the aqueous medium to form a micelle solution; component (b) is then added as either an aqueous suspension or a solid. The two components can, however, be mixed in a

substantially dry form before the addition of aqueous medium. The liposomes of the present invention

spontaneously form upon combining (a) and (b) in the aqueous medium, generally requiring mixing only with a magnetic stirrer. Alternatively, the components (a) and (b) can be combined in an organic solvent, the solvent removed to form a dried mixture of the two components, and liposomes formed by hydrating the dried mixture.

A variety of substances, (i.e., active agents), can be incorporated in either the aqueous or lipid phase of the liposomes of the present invention. Two preferred agents are minoxidil and hydrocortisone.

Brief Description of the Drawings

Figure 1 shows the result of Sephadex size exclusion chromatography of inulin-loaded tauranol: oleic acid liposomes.

Figure 2 shows the result of Sephadex size exclusion chromatography of sucrose-loaded tauranol: oleic acid liposomes.

Figure 3 shows the data for 3H-Inulin leakage out of tauranol: oleic acid MLVs during storage at 4°C.

Figure 4 shows the data for the pH stability of tauranol: oleic acid MLVs during storage at 50°C.

Figure 5 shows the data comparing transdermal uptake of tauranol: oleic acid: Minoxidil liposomes versus

ROGAINE. Figure 6 shows the data comparing transdermal uptake of tauranol: oleic acid: Minoxidil liposomes versus

Minoxidil/lauryl sulfosuccinate compositions.

Figure 7 shows the structure of a typical phosphate quaternary compound, "MONAQUAT P-TL".

Figure 8 shows the results of Sephadex size

exclusion chromatography of 3H-Inulin-loaded MLVs composed of a zwitterionic surfactant and a non-ionic sterol.

Figure 9 shows the results of Sephadex size

exclusion chromatography of 14C-Sucrose-loaded MLVs composed of a zwitterionic surfactant and a non-ionic sterol.

Figure 10 shows the results of Sephadex size

exclusion chromatography of 3H-Inulin-loaded MLVs composed of an anionic surfactant and a non-ionic sterol.

Figure 11 shows the results of Sephadex size

exclusion chromatography of 14C-Sucrose-loaded MLVs

composed of an anionic surfactant and a non-ionic sterol.

Figure 12 illustrates the stability of liposomes generated by the ABF system over a 70 day period when stored at 4°C or 50°C.

Detailed Description of the Invention

I. Preparing Liposome Compositions

The present invention describes new and useful liposome systems for entrapping or otherwise

incorporating active ingredients of cosmetic, diagnostic and therapeutic purpose. This invention provides all the useful attributes of liposomes without the disadvantages of glycerophospholipid hydrolysis, peroxidation, and cost.

The typical bilayer forming system has been

considered to be a double chain amphipathic compound which by itself can form a closed lamellar bilayer phase. Examples include the glycerophospholipids, and synthetic, double chain compounds which are described in the

"membrane-mimetic" systems of Fendler. The instant application describes an alternative bilayer formulation (ABF). The instant ABF system is a combination of two or more components which do not typically, by themselves, form closed multi-lamellar vesicles (MLVs) but which, under defined conditions, spontaneously self-assemble into vesicles. The ABF system of the present invention need not be polymerized by chemical or photochemical means to self-assemble into a bilayer (Regen et. al., Johnston et. al., and O'Brien et. al.). Nor does the ABF system require use of mechanically generated high-shear conditions.

A. Lipid Components

An ABF system is s mixture of two or more

appropriate amphipathic components. The first component ((a)) is a surfactant which forms micelles when added to aqueous solution above the surfactant's phase transition temperature and is composed of one or three or more, but not two, aliphatic chains. These aliphatic chains may be saturated, unsaturated, or substituted in other ways, such as by ethoxylation: typically, the aliphatic chain contains greater than about 12 carbons. To component (a) a sufficient quantity of a second component (b) is added where (b) is (i) a single-aliphatic chain lipid, or (ii) a sterol, in particular cholesterol or a cholesterol analog or derivative.

In general, candidates for component (a) may be screened by evaluating their ability to form a clear solution of micelles when dispersed alone in water at a temperature above the surfactant's phase transition temperature. Suitable candidates for component (a) include the following: lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl- sarcosine; linoleyl-, myristyl-, or cetyl- betaine; lauroamidopropyl-, cocoamidopropyl-,

linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl = MACKAM LMB-LS, available from Mclntyre Chemical Co.); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or sodium methyl oleyl-taurate; and, the MONAQUAT series (Mona Industries, Inc., Paterson, NJ).

Single-aliphatic chain lipid candidates for

component (b) have the characteristics that when they are dispersed alone in water, at a temperature above the 1 pid transition temperature, they are in a lipid

emulsion phase. Suitable candidates for component (b) include the ester, alcohol, and acid forms of the

following fatty acids: laurate, myristate, palmitate, palmitoleic acid, stearate, oleic acid, linoleic acid, arachidate, arachidonic acid, and other single-aliphatic chain acids. Further candidates include the ester, alcohol, and acid forms of the retinols, in particular, retinol and retinoic acid. Generally for component (b) the aliphatic chain contains greater than about 12 carbons and can be either saturated or unsaturated.

Sterol lipid candidates for component (b) have the characteristics that they do not readily disperse under the same conditions as component (a) and they form a flocculate or precipitate when added alone to aqueous solution. Suitable candidates for component (b) include cholesterol, cholestanol, cholesterol sulfate and other cholesterol analogs and derivatives.

Double chain glycerophospholipids qualify as neither component (a) nor (b), although they can be incorporated into liposomes constructed by the instant alternative bilayer formulation. B. Liposome Preparation

The alternative bilayer formulation of the present invention provides a straight-forward method of preparing durable liposomes. In general, component (a) is

dispersed in water followed by the addition of component (b). Component (b) can be added directly or as an aqueous suspension. When component (b) is a powder, e.g., cholesterol, it can be added as a solid. Further, components (a) and (b) may be combined before the

addition of aqueous medium. The temperature of the aqueous solution can be raised to facilitate

solubilization of the components.

Alternatively, the components can be mixed together, in appropriate proportions, in an organic solvent

suitable to dissolve both components. The organic solvent is then evaporated and the residue can be

rehydrated by the addition of an aqueous medium.

The combination of components (a) and (b), prepared by one of the above described methods, is then mixed using a magnetic stirrer and the MLVs spontaneously generate. The liposomes can be sized by a variety of methods, including filtration (see below). The weight ratio of component (a) to component (b) is typically between about 1:2 to 2:1.

Example 1A describe the formation of liposomes using one anionic surfactant and a single-aliphatic chain lipid, resulting in liposomes having a net negative charge. Although these liposomes show some aggregation before sizing, after sizing they remain dispersed.

Examples 1B and 1C describe the formation of liposomes using one zwitterionic surfactant and a non-ionic sterol resulting in liposomes having a net neutral charge. Two important features of these preparations were that there was no apparent aggregation of the liposomes and the liposomes were relatively homogeneous in size. Similarly, liposomes formed by using an anionic surfactant and cholesterol, resulting in liposomes having a net negative charge, also show size homogeneity and no apparent aggregation (Example ID).

Other surfactants useful as component (a) are the phosphate quaternary compounds (PQCs: such as MONAQUAT P-TL and MONAQUAT P-TS). PQCs are synthetic surfactants containing a phosphate moiety in the molecule. These surfactants have been used as surface active and forming agents and are commercially available from Mona

Industries, Inc. These surfactants are chemically stable and amphoteric in nature exhibiting exceptionally low oral toxicity and low ocular irritation. In water or aqueous solutions, both MONAQUAT P-TL and MONAQUAT P-TS dissolve completely and form micelles. Figure 7 shows the structure of MONAQUAT P-TL, an exemplary MONAQUAT.

When a PQC surfactant is mixed with amphiphiles that possess a relative large hydrophobic volume (such as cholesterol, cholestanol, alpha-tocopherol, retinol, ergocalciferol, fatty acids, fatty alcohols, diglyceride, corticosteroids and some nonsteroidal anti-inflammatory agents, flurbiprotein, ibuprofen, and indomethathin) closed vesicles form spontaneously during hydration.

Experiments performed in support of the present invention demonstrated formation of lipid vesicles using MONAQUAT

P-TL and cholesterol as evidenced by the following. When MONAQUAT P-TL and cholesterol are combined in the range of 3:1 to 1:3 mole percent by the method of the present invention, lipid vesicles were formed. Formation of the lipid vesicles was confirmed by the presence of spherical and tubular lamellar structures under a phase-contrast microscope. Also, when MONAQUAT P-TL and cholesterol are mixed in a 2:1 mole percent ratio at a final lipid concentration of 37.5 μmoles/ml, the resulting MLVs have an entrapment efficiency of about 4-5% for 14C-sucrose.

Further, the MONAQUATs combine well with single-chain lipids (e.g. fatty alcohols, acids, or esters) to generate alternative bilayer liposomes.

An important feature of the present invention is that the safety of the components used in the formulation of the liposomes are well established. For example, when the liposomes are used for topical application the safety of the components used to form the liposomes can be verified in the CTFA Cosmetic Ingredient Dictionary.

Entrapment efficiency, another factor to be

considered in selecting the lipid composition, refers to the total amount of an agent which can be loaded into liposomes, expressed as a ratio of the agent per mole per liposome lipid. Example 2A describes the trapping of water soluble agents in liposomes composed of a anionic surfactant and a single-aliphatic chain lipid. Figures 1 and 2 show the results of column chromatography resolving the liposome encapsulated aqueous phase marker from the free aqueous phase marker. These results indicate an approximately 11.7% encapsulation efficiency for inulin (Figure 1) and an approximately 14.2% encapsulation efficiency for sucrose (Figure 2).

Example 2B describes the trapping of water soluble agents in liposomes composed of a zwitterionic surfactant and a non-ionic sterol. Figures 8 and 9 show the results of column chromatography resolving the liposome

encapsulated aqueous phase marker from the free aqueous phase marker. These results indicate an approximately

23% encapsulation efficiency for inulin (Figure 8) and an approximately 19.8% encapsulation efficiency for sucrose (Figure 9). Example 2C describes similar entrapment studies of inulin and sucrose in liposomes composed of an anionic surfactant and a non-ionic sterol. Figures 10 and 11 show the results of column chromatography resolving the liposome encapsulated aqueous phase marker from the free aqueous phase marker. These results indicate, for the anionic formulation, an approximately 25.5% encapsulation efficiency for inulin (Figure 10) and an approximately 24.5% encapsulation efficiency for sucrose (Figure 11).

The above results show, for both the zwitterionic and anionic formulations, the ability of the ABF system to effectively encapsulate water soluble agents.

Accordingly, other water soluble agents having

therapeutic value can also be incorporated into the ABF liposomes by incorporating them into the hydrating solution. Representative water soluble agents include minoxidil, acyclovir, gentamycin, pentamidine, insulin, epidermal growth factor, and lipocortin.

The present invention allows markedly improved active ingredient loading into liposomes of drugs which are poorly soluble in aqueous solution. For example, using the ABF liposomes of the present invention, normal minoxidil aqueous solubility of about 0.3% can be boosted to at least 2% by making a Taurenol WS HP/Oleic

Acid/Minoxidil (3.7/2.5/2, w/w/w) liposome formulation (Example 4). It is possible to achieve 5% drug loading by weight by increasing the dry weight of each excipient proportionately.

The liposomes of the present invention have enabled formulation of drugs, such as minoxidil, at pH 6;

previous liposome vehicles have had to be formulated at pH 5 to achieve the desired drug loading (e.g., U. S.

Patent No. 4,828,837). Further, the tauranol/oleic acid/minoxidil liposomes (Example 4) have excellent pH stability during long term storage (Figure 4): there were no large pH shifts, indicative of ester-hydrolysis, which have been observed with other liposome formulation of minoxidil.

The usefulness of the invention is further supported by Figure 5 which compares the in vitro percutaneous absorption across hairless mouse skin of an ABF/minoxidil composition to that of the Upjohn NDA formulation: the ABF vehicle gives remarkably improved drug flux across mouse skin. Further, the ABF liposome vehicle gives improved drug flux relative to other liposome minoxidil formulations (Figure 6).

ABF liposomes also have diagnostic applications. A variety of diagnostic agents can be encapsulated by this system, including, nucleic acids, immunoglobins, enzymes, reporter molecules, and enzyme substrates.

An important consideration for liposomes loaded with any agent is liposome stability. Figure 3 (Example 3A) illustrates the stability of liposomes generated by the ABF system. Over a 35 day period at 4°C there was only a 17% loss of loaded inulin from the ABF liposomes. Figure 12 (Example 3B) further illustrates the stability of liposomes generated by the ABF system. Over a 70 day period at 4°C there was only a 26% loss of loaded inulin from the ABF liposomes; at 50°C over the same period there was only a 37% loss. These data show that ABF liposomes have good stability even at elevated

temperatures. Typically to obtain an acceptable level of stability at elevated temperatures, rigid-lipids, having high phase transition temperatures, have been used. The ABF liposomes provide an alternative for to such rigid-lipid liposomes.

The above data show that ABF liposomes have

excellent stability. Conventional phospholipid vesicles are unstable at room temperature due to oxidation and hydrolysis of the lipids. Their use, therefore, is limited to products which require storage at an ambient temperature.

An alternative to aqueous phase incorporation into ABF liposomes is the incorporation of agents into the lipid phase of the MLVs. Agents suitable for

incorporation into the lipid phase include amphiphiles which possess relatively large hydrophobic volume, such as cholesterol, cholestanol, alpha-tocopherol, retinol, ergocalciferol, fatty acids, fatty alcohols, and

corticosteroids; further, some non-steroidal antiinflammatory agents, such as flurbiprotein, ibuprofen, and indomethacin, also function in this capacity.

For example, up to 1.1% solubilized hydrocortisone liposome preparations have been demonstrated for ABF liposomes. The hydrocortisone (or other suitable lipid soluble drug) is added after the component (a) solution has clarified, and before the addition of component (b). Hydrocortisone has been incorporated into Tauranol: Oleic Acid liposomes at a weight ratio of Tauranol WS-HP/Oleic Acid USP/Hydrocortisone ratio of 1.4/0.9/1 (w/w/w).

These hydrocortisone containing liposomes were prepared as in Example 1A: the hydrocortisone was added after the Tauranol solution clarified, and before the addition of the oleic acid. At the above weight ratio the liposome vehicle is saturated with drug. The amount of drug loading may be scaled up by a proportionate increase of all lipid phase excipients.

C. Liposome Sizing

The liposome suspension may be sized to achieve a selective size distribution of vesicles. The sizing serves to eliminate larger liposomes and to produce a defined size range having optimal pharmacokinetic

properties. Several techniques are available for reducing the sizes and size heterogeneity of liposomes. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to less than about 0.05 microns in size. In a typical homogenization procedure, MLVs are recirculated through a standard emulsion homogenizer until selected liposome sizes are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particles size discrimination.

Extrusion of liposomes through a small-pore polycarbonate membrane is an effective method for reducing liposome sizes down to a relatively well-defined size distribution depending on the pore size of the membrane. Typically, the suspension is cycled through the membrane several times until the desired liposome size

distribution is achieved. One such filter is the 0.45 μm Acrodisc filter, as used in Example 1. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.

Centrifugation and molecular sieve chromatography are other methods which are available for producing a liposome suspension with particle sizes below a selected threshold of 1 micron or less. These two methods both involve preferential removal of larger liposomes, rather than conversion of large particles to smaller ones:

liposome yields are correspondingly reduced.

D. Removing Non-encapsulated Agent

Free agent, i.e., drug present in the bulk aqueous phase of the medium, is preferably removed to increase the ratio of liposome-entrapped to free agent. Several methods are available for removing free agent from a liposome suspension. Sized liposome suspensions can be pelleted by high-speed centrifugation, leaving free agent and very small liposomes in the supernatant. Another method involves concentrating the suspension by ultrafiltration, then resuspending the concentrated liposomes in an agent-free replacement medium. Alternatively, gel filtration can be used to separate larger liposome particles from solute (free agent) molecules. Also, some agents can be removed using ion-exchange or affinity chromatography to bind the agent in free form, but not in liposome-bound form.

II. Formulations and Therapeutic Uses

The instant invention defines novel vesicle compositions based on stable, safe, and inexpensive raw

materials. These novel lipid vesicles can be used for a variety of formulations and therapeutic applications.

Therapeutic compounds can be incorporated into the ABF liposomes in either the aqueous or lipid phase. A. Ophthalmic Uses

In one general application, the liposomal

formulation is used to produce a sustained drug release at the ocular tissue site. Ophthalmic drugs suitable for delivery in liposomal-entrapped form for sustained

release include: antiviral agents, such as fluorouracil, iodouridine, trifluorouridine, vidarabine, azidothymidine, robavirin, phosphonoformate, phosphonoacetate, and acyclovir; anti-allergic agents such as cromolyn, cemetidine, naphazoline, lodoxamide, and

phenylephinephrine; anti-inflammatory agents such as prednisolone, dexamethasone, and supraphen; and antiglaucoma agents which act by lowering intraocular

pressure, such as carbacol, N-demethylcarbacol,

pilocarpine, anti-glaucoma agents which act as

cholinesterase inhibitors, such as isoflurophate, exothioiodate, and demecarium bromide, and anti-glaucoma agents which act as beta-blockers, such as timolol, depaxolol, meti-pranalol, levobunalol, and celiprolol.

According to one advantage of the liposome

suspension, the amount of drug which is delivered in drop form may be substantially higher than that achievable in free solution form; accordingly, undesired side effects related to high free drug concentrations are reduced.

The liposomes suspensions are also useful in

ophthalmic for treating dry eye. This condition, which is characterized by poor moisture retention on the eye, has a number of distinct etiologies, including poor water-secretion by the lacrimal gland (Sjogren), poor mucin secretion by goblet cells, (Lemp), vitamin A deficiency (Lawrence), and alteration of film-forming lipids as a result of chronic blepharitis. These filmforming lipids are primarily long-chain alcohols and acids and cholesterol esters, which are required for forming a stable preocular tear film (Anderson); such compounds are easily incorporated into the liposomes of the present invention.

Another important consideration in the choice of lipid components is to minimize the extent of oxidative lipid damage which can occur on storage, particularly where, as is usual, the formulation will be stored over a several month period at room temperature. It is also important to minimize lipid hydrolysis which occurs on storage, and again particularly at room temperature or above. Oxidative damage and hydrolysis of the lipids in conventional phospholipid vesicle formulations is a common problem.

The surfactants used as component (a) of the ABF liposomes are very stable and are not susceptible to hydrolytic and oxidative damage as are phospholipids. In particular, ABF liposomes have outstanding stability even when stored at 50°C for 70 days (Example 3B, Figure 12).

Still another consideration in the choice of lipid components is achieving good optical clarity in a

liposome suspension. Both the size and the stability of the liposomes, in terms of aggregative effects, affect the optical clarity of a formulation. The ABF formulations of the present invention have good size

distribution and maintain good dispersion after sizing (Examples 1B, 1C, and 1D) resulting in good liposome dispersion. Specifically, with the MACKAM LMB-LS

(lauroamidoproply betaine):cholesterol formulation, no aggregation of the liposomes was observed after the formulation had been stored for one week (Example 1C).

B. Topical Uses

ABF liposome compositions, when applied topically, can provide controlled release of a variety of topical medications, such as anti-bacterial or anti-fungal agents, and steroids, and can also serve as a source of moisturizing lipids.

(i) Pastes and Foams

Paste or foam formulations of the liposomes provide advantages of (1) relatively good stability on storage, (2) high drug capacity and (3) a high ratio of liposome-entrapped to free drug, particularly for water-soluble, liposome-permeable drugs. Liposome pastes or foams are suitable for application to burned or broken skin, ocular tissue, and in body cavities, where the high viscosity of the material helps maintain the material at the site of application. The concentrate for paste or foam formulations is preferably formed by ultrafiltration with

continued recycling of the liposome suspension material. Liposome paste formulations produced by the

invention are well suited for use as topical ointments and creams, without additional processing.

Liposome foams can be prepared using conventional two-chamber propellant devices, such as are used for cosmetic foams, such as shaving cream. Here a heavy liposome suspension contained in one chamber is mixed with propellant gas contained in a second chamber, and the gassified mixture or foam is expelled under the propellant release pressure through a discharge nozzle. U.S. Patent #3,326,416 describes a two-chamber propellant foam device which could be readily adapted for use in liposome foam generation. (ii) Gels

under conditions Of low ionic strength, charged

liposomes in an aqueous suspension repel each other and, as a result, the liposomes become ordered in the solution giving a viscous consistency to the liposome suspension, i.e. a gel-like consistency, such is the case for ABF liposomes of the present invention which are formulated with anionic surfactants. An increase in the viscosity of charged-liposome suspensions can be obtained by ultrafiltration.

Under conditions of increased ionic strength, as when a gel-like liposome suspension is applied to the skin, charge-shielding by the ions of the liposome surface charges results in a reduction of the viscosity of the liposome suspension. This effect is useful for topical application of liposome suspensions since it allows a gel to more easily disperse on the skin.

The liposome formulations of the present invention wherein one component is a zwitterionic surfactant

(component (a) ) and the other (component (b) ) is

uncharged at neutral pH (e.g., cholesterol or a single- aliphatic chain ester or alcohol), have a net neutral charge at neutral pH. Under conditions of low ionic strength the pH of the liposome suspension can be changed away from the zwitterion's neutral state toward either the more acidic or more basic pK, of the zwitterion; such a change results in acquired charged on the liposome surface. As discussed above the charged liposomes become more ordered in the suspension, resulting in an increased viscosity of the suspension.

Such a liposome suspension has the advantage that it can be prepared and more easily manipulated, e.g.

filtered, while the suspension is in a more fluidic form. The pH of the suspension can then be altered to result in a more gel-like consistency. Also, as noted above, a subsequent increase in the ionic strength will result in charge-shielding and an accompanying reduction in the suspension's viscosity.

C. Aerosolized Liposomes

Aerosolized liposomes, or liposome sprays are a convenient vehicle for applying the liposomes to the nasal or oral mucosa. In one simple embodiment, the liposomes are formulated as a dilute aqueous suspension, and sprayed from a conventional pump or squeeze spray bottle. Alternatively, paste formulations provide an ideal storage form for liposomes where the entrapped drug is water-soluble and liposome-permeable, e.g., where drug equilibration of the drug between encapsulated and bulk aqueous compartments occurs. The present invention provides a simple means for forming such a paste

directly, or with little additional water removal. For drug administration, the paste is diluted just prior to treatment, preferably to between about 10-30 volume percent, and the diluted suspension is atomized in a form suitable for inhalation, before significant drug equilibration can occur.

In more elaborate embodiments, the ABF liposomes are formulated for use with fluorocarbon propellant solvents in a pressurized canister system: several suitable propellant solvents are disclosed in co-owned PCT

Application, WO 86/06959, for "Liposome Inhalation System and Method", published December 4, 1986, which is incorporated by reference herein. Briefly, the liposomes may be suspended in the propellant in powdered or aqueous paste form, or combined in paste or powdered form with the propellant during propellant release from the

pressurized canister.

Exemplary drugs for delivery to the nasal mucosa in liposomal form include anti-allergens, anti-histamines, such as benadryl, diphenhydramine HCl, clemastine

fumarate, promethazine HCl, and triprolidine HCl;

vasconstrictors, such a metaraminolbitartrate,

epinephrine, norepinephrine, phenylephrine HCl, and ephedrine; and peptide hormones, such as insulin, calcitonin, growth hormone, epidermal growth factor, atrial natriuretic peptide, vasopressin, and oxytocin.

Exemplary drugs for delivery to the oral mucosa include anesthetics, such as benzocaine, lidocaine HCl, dyclonine HCl; and antiviral or antibacterial agents, such as amantadine HCl, fluorouracil, iodouridine, gentamicin, erythromycin, cephalosporin, and

tetracycline. D. Solid Matrix Formulations

The high-retention liposomes of the invention can be embedded or encapsulated within several types of solid matrix supports, either to protect the liposomes from rapid clearance or breakdown and/or to provide slow release the liposomes from the matrix into the region of tissue mucosa. One type of matrix is a suppository designed either to be melted or dissolved in a body cavity, to release the embedded liposomes. Conventional materials and preparation methods for suppositories would be suitable, to the extent the liposomes are not exposed to transient temperatures above about 60°C.

Biocompatible polymers, such as collagen,

polylysine, polylactic acid, polymethylacrylate,

polyurethanes, polyglycolic acid, hydroxypropcellulose, agar and agarose, are also suitable bulk carriers for liposomes of the invention. Methods for preparing these polymers in cross-linked and/or gel form are well known, and the methods can be readily adapted to incorporate liposomes, again with the proviso that transient temperatures above about 60°C are avoided. Many of the

polymers, such as agar, collagen, and polyurethanes can be formulated in permeable cross-linked structures which allow liposome movement through and out of the matrices at a selected rate. Matrices of this type are suitable for drug delivery in body cavities, where the matrix can be held in place over an extended period, or for ocular use, where an implant can take the form of a clear lens or the like. Other polymer compositions, like polylactate, can be formulated as a biodegradable solid which release the entrapped slowly over an extended polymer degradation period. Such matrices are suitable for liposome release in the mouth or stomach. Some of the polymer compositions, such as polylysine, can be

polymerized in a liposome suspension to form a polymer shell about individual liposomes, to form a coating which, for example, would protect the liposomes from rapid breakdown in the stomach. E. Parenteral Drug Administration

Liposomes having sizes less than about 0.4 microns and predominance of liposome-entrapped drug are ideally suited for parenteral administration of the drug, either by intravenous or intramuscular route. The present invention provides a simple, efficient method for

preparing liposome suspensions of this type.

While preferred embodiments, uses, and methods of practicing the present invention have been described in detail, it will be appreciated that various other uses, formulations, and methods of practice as indicated herein are within the contemplation of the present invention. MATERIALS

The following sources were used for materials described in the Examples: Cholesterol, Croda, Inc. (New York, NY); All MACKAMS, Mclntyre Chemical Co. (Chicago, IL); all MONAQUATS, Mona Industries (Paterson, NJ);

Taurenol WS, Finetex, Inc. (Elmwood Park, NJ); all radiolabeled compounds, Amersham, Inc. (Arlington

Heights, IL); Oleic Acid, J. T. Baker Inc. (Philipsburg, NJ); and Minoxidil, The Upjohn Co. (Kalamazoo, MI). EXAMPLE 1

Production of Multilammelar Vesicles A. Taurenol WS: Oleic Acid MLVs

This example describes the spontaneous generation of liposomes using an anionic surfactant and a single-aliphatic chain lipid by mixing the components in an aqueous buffer. In general, 1.4 to 1.6 part Tauranol was added to 1 part oleic acid by weight with a total of 6.2% lipid solids to generate the liposomes.

To produce a 10 ml batch of liposomes 0.37 gm of Taurenol WS, powder (N-methylcocoyltaurate) was added to 10 ml hydrating solution (.01% 100mg/ml DTPA, 0.02%

NaAzide in distilled water). The mixture was then stirred until clear. To this clear solution 0.25 gm Oleic Acid (liquid) were added. This mixture was then stirred for 1 hour using magnetic stirring. The resulting multilayer vesicles (MLVs) were sized through 0.45 μm Acrodiscs (Gelmar Sciences, Inc, Ann Arbor, MI) 3 times and

filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography column.

Macroscopically, the formulation had the appearance of skimmed milk. It was very white, non-foamy, and did not have the pearlescent appearance of egg

phosphatidylcholine MLVs. Microscopically, the MLVs were small, thin-walled, and had a slight degree of

aggregation before sizing.

B. MACKAM 35HP (cocoamidopropyl betaine) Cholesterol MLVs

This example describes the spontaneous generation of liposomes using a zwitterionic mono-alkyl betaine

surfactant and a non-ionic sterol by mixing the

components in an aqueous buffer. In general, 1 part of 35HP was added to 1 part cholesterol by weight with a total of 10% solids to generate the liposomes.

To produce a 10 ml batch of liposomes 1.43 gms

MACKAM 35HP solution (35% solids) was added to 8.07 mis hydrating solution (0.01% 100 mg/ml DTPA, 0.02% NaAzide in distilled water). To this solution 0.50 gm of cholesterol was added. The solution was then mixed with

magnetic stirring for 1 hour. The resulting multilayer vesicles (MLVs) were sized through 0.45 μm Acrodiscs

(Gelman Sciences, Inc., Ann Arbor, MI) 3 times and

filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography column.

Macroscopically, the liposome formulation was opaque white and had a consistency slightly thicker than water. Microscopically, the MLVs were very uniform in size, (predominantly in the range of 5-10 μm in diameter), very thick-walled (as seen through a polarizer), and the MLVs had no apparent aggregation or crystals.

C. MACKAM LMB-LS (lauroamidopropyl Betaine) Cholesterol MLVs.

This example describes the spontaneous generation of liposomes using a zwitterionic mono-alkyl betaine

surfactant and a non-ionic sterol by mixing the solid components in an aqueous buffer. In general, 5 parts of LMB-LS were added to 3 parts cholesterol by weight with a total of 10% solids to generate the liposomes.

To produce a 10 ml batch of liposomes 1.95 gm MACKAM LMB-LS solution (32% solids) was combined with 8.05 ml hydrating solution (0.01% 100mg/ml DTPA, 0.02% NaAzide in distilled water). To this solution 0.375 gm cholesterol were added. This solution was then mixed with magnetic stirring for 30 minutes. The resulting MLVs were sized through 0.45 μm Acrodiscs 3 times and filtered through a 10 ml volume Sephadex™ G-150-120 gel column.

Macroscopically, the liposome formulation was thick in consistency and very white in color. No precipitation was observed over a one week time span.

Microscopically, the MLVs were round and well-distributed in the 1-10 urn size range; there was no apparent aggregation.

D. Taurenol WS: Cholesterol Liposomes

This example describes the spontaneous generation of liposomes using an anionic mono-alkyl surfactant and a non-ionic sterol by mixing the solid components in an aqueous buffer. This formulation results in liposomes having a net negative charge. In general, 4 parts of Taurenol were added to 5 parts cholesterol by weight with a total of 5% solids to generate the liposomes. To produce a 10 ml batch of liposomes, 222 mg

Taurenol WS (N-methyl cocoyl taurate; Finetex, Inc., Elmwood Park, NJ) were added to 10 ml hydrating solution (.01% 100 mg/ml DTPA, .02% NaAzide, in distilled H2O). This solution was stirred until clear and then 278 mg of cholesterol were added. The solution was mixed with magnetic stirring on low heat for 20 minutes. After heating stirring was continued without heat for 2 hours. The resulting multilayer vesicles (MLVs) were sized through .45 μm Acrodiscs 3 times and filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion

chromatography column.

Macroscopically, the above formulation was very white, somewhat "creamy". Microscopically, these MLVs were round and well-distributed in size, ranging from 1- 30 um in diameter, with no apparent aggregation.

In the above procedure heat and stirring were required for completely dissolving the cholesterol crystals.

EXAMPLE 2

Trapping Studies of Multilammelar Vesicles A. Taurenol WS: Oleic Acid Multilammelar Vesicles. This example describes the formation of Tauranol: oleic acid liposomes with the accompanying entrapment of water-soluble compounds.

1. Inulin

Liposomes were prepared as in Example 1A, with the exception that the hydrating solution contained 1 mg/ml inulin. The inulin was radioactively labelled (3H) to serve as an aqueous marker and 14C-cholesterol served as a lipid marker. The mixture was sized as in Example 1A and filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography column (Figure 1). The resulting inulin-loaded MLVs showed a 11.7% encapsulation of the total inulin present. The MLVs had a captured volume of 1.77 gm water/gm solids.

2. Sucrose

Liposomes were prepared as in Example 1A, with the exception that the hydrating solution contained 1 mg/ml sucrose. The sucrose was radioactively labelled (14C) to serve as an aqueous marker and 3H-cholesterol served as a lipid marker. The mixture was sized as above and

filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography column (Figure 2). The

resulting sucrose-loaded MLVs showed a 14.2%

encapsulation of the total sucrose present. The MLVs had a captured volume of 2.15 gm water/gm solids.

B. MACKAM LMB-LS (lauroamidoproplv betaine) Cholesterol MLVs.

This example describes the formation of lauroamidopropyl betaine : cholesterol liposomes with the

accompanying entrapment of water-soluble compounds.

1. Inulin

Liposomes were prepared as in Example 1C, with the exception that the hydrating solution contained 1 mg/ml inulin. The inulin was radioactively labelled (3H) to serve as an aqueous marker and 14C-cholesterol served as a lipid marker. The mixture was. sized as in Example 1C and filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography column (Figure 8). The

resulting inulin-loaded MLVs showed a 23% encapsulation of the total inulin present. The MLVs had a captured volume of 2.07 gm water/gm solids.

2. Sucrose

Liposomes were prepared as in Example 1C, with the exception that the hydrating solution contained 1 mg/ml sucrose. The sucrose was radioactively labelled (14C) to serve as an aqueous marker and 3H-cholesterol served as a lipid marker. The mixture was sized as above and

filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography COIUΠL (Figure 9). The

resulting sucrose-loaded MLVs showed a 19.8%

encapsulation of the total sucrose present. The MLVs had a captured volume of 1.80 gm water/gm solids.

C. Taurenol WS: Cholesterol Liposomes with Entrapment of Sucrose or Inulin.

This example describes the formation of Taurenol WS: Cholesterol liposomes with the accompanying entrapment of water-soluble compounds.

1. Inulin

Liposomes were prepared as in Example ID, with the exception that the hydrating solution contained 1 mg/ml inulin. The inulin was radioactively labelled (3H) to serve as an aqueous marker and 14C-cholesterol served as a lipid marker. The mixture was sized as in Example ID and filtered through a 10 ml volume G-150-120 SEPHADEX size exclusion chromatography column (Figure 10). The

resulting inulin-loaded MLVs showed a 25.5% encapsulation of the total inulin present. The MLVs had a captured volume of 4.85 gm water/gm solids.

2. Sucrose

Liposomes were prepared as in Example 1D, with the exception that the hydrating solution contained 1 mg/ml sucrose. The sucrose was radioactively labelled (14C) to serve as an aqueous marker and 3H-cholesterol served as a lipid marker. The mixture was sized as above and

filtered through a 10 ml volum' G-150-120 SEPHADEX size exclusion chromatography column (Figure 11). The resulting sucrose-loaded MLVs showed a 24.5%

encapsulation of the total sucrose present. The MLVs had a captured volume of 4.66 gm water/gm solids. EXAMPLE 3

Flux studies of Inulin-loaded Liposomes

A. Taurenol WS:Oleic Acid Liposomes

This example describes studies of the stability of the Taurenol WS: Oleic Acid liposomes at 4°C over a 35 day period. The flux studies were conducted using 1 mg/ml inulin-loaded MLVs prepared as in Example 2A.

Radioactively labelled inulin (3H) was used as an aqueous marker and 14C-labelled cholesterol as a lipid marker. Liposomes were isolated by column chromatography as in Example 2A. The liposomes were held at 4°C for the time course of the study. The percent loss of the aqueous marker from the liposomes (flux) is calculated from the change in the 3H/14C ratio. The results of the flux study are presented in Table 1.

Table 1

Sample Type 0 day 7 day 14 day 21 day 28 day 35 day 4°C regime 0% nd 2% 10% 9% 17%

Figure 3 shows a plot of the data, log10(%3H retained) vs. days. This data appears to fit a linear regression where the slope equals -2.24533X10-3, the Y intercept is 2.008, and the correlation coefficient (R-Val) is 0.91574 (values calculated from the data in Table 1 using the SIGMAPLOT program).

B. Taurenol WS: Cholesterol Liposomes.

This example describes studies of the stability of the Taurenol WS: Cholesterol liposomes at 4°C and 50°C over a 70 day period. The flux studies were conducted using 1 mg/ml inulin-loaded MLVs prepared as in Example 2C.

Radioactively labelled inulin (3H) was used as an aqueous marker and 14C-labelled cholesterol as a lipid marker. Liposomes were isolated by column chromatography as in Example 2C. The liposomes were held at either 4°C or 50°C for the time course of the study. The percent loss of the aqueous marker from the liposomes (flux) is calculated from the change in the 3H/14C ratio. The results of the flux study are presented in Table 2.

Table 2

Sample type 0 day 7 day 14 day 21 day 28 day 70 day 4° C regime 0% 17% 17% 19% 15% 26%

50° C regime 0% 37% 37% 33% - 37%

Figure 12 shows a plot of the data, log10(%3H

retained) vs. days; the data for both regimes are biphasic.

EXAMPLE 4

Production of Tauranol: Oleic Acid Liposomes containing Minoxidil

This example describes the use of tauranol: oleic acid liposomes to solubilize minoxidil.

Liposomes were produced essentially as described in Example 1A. Thirty seven grams of tauranol was added to 5.33 mis of glass distilled water containing 0.01% DTPA. This solution was stirred until it cleared: 10 minutes at approximately 50°C. To titrated the pH, 0.31 g of 1.0 N HCl was added to the solution. The minoxidil (0.2 g) was then slowly added to the solution with gentle

stirring. The temperature of the solution was raised to 63°C and the solution remained somewhat cloudy. The temperature of the solution was then raised to

approximately 73°C an the solution stirred for 5 minutes, resulting in a clear solution. This solution was then incubated at approximately 76°C for 5 minutes more. To this solution 0.26 g of oleic acid was added with gentle stirring and the solution removed from heat. When this solution cooled to room temperature it was a gel.

The total weight of the gel was increased to 10 g by the addition of glass distilled water containing 0.01% DTPA. The pH of the resulting suspension was determined to be 6.09 using an Orion pH Meter. Macroscopically, the suspension was milky and slightly viscous.

Microscopically, the MLVs were small, heterogeneously sized, and there was no apparent aggregation.

Liposomes from three batches of Tauranol: Oleic Acid: Minoxidil liposomes, produced as in Example 4, were stored at 50°C for 28 days. Samples were removed at days 0, 7, 14, 21, and 28 for evaluation of their pH. Figure 4 presents the data which illustrate the excellent storage stability of the instant formulation.

EXAMPLE 5

Transdermal Penetration of Tauranol: Oleic Acid:

Minoxidil Liposomes

This example describes the transdermal uptake of the Tauranol: Oleic Acid: Minoxidil liposomes of the present invention compared to other forms of Minoxidil.

A. Experimental Protocol

The transdermal cell used for measuring skin

penetration has upper and lower chambers which are separated by a skin patch. The lower chamber is designed to permit continuous flow-through of saline, which collects drug penetrating from the outer side of the skin (exposed to the upper chamber) through the skin and into the saline in the lower chamber. An infusion pump is used to move through the chamber at a controlled rate (about 4 ml/hour).

Female hairless mice, strain HRS/hr, were obtained from Simonsen (Gilroy, CA). The animals were 7-8 weeks old, and weighed 20-30 gm when used. After sacrifice, three 2 cm diameter skin patches were removed from each animal. The patches were individually mounted in the cell, and held sealed against the lower chamber by an O- ring which is pressed against the patch by clamping.

Typically 14C-labelled Minoxidil was used in all of the formulations.

Prior to adding the drug solution to the skin, a phosphate-buffered saline solution was pumped through the system, at a flow rate of about 5 ml/hr for one hour. Fractions were collected continuously from the outlet side of the lower chamber, and dispensed into vials in a fraction collector. Collection time per fraction was one hour. Fractions were collected for up to 24 hours after the drug solution was applied to the skin membrane.

After the test period, the skin patch is washed several times, and removed. The hourly fractions, wash fractions obtained at the end of the experiment, and the skin patch itself were counted for radioactivity by

conventional scintillation counting methods.

B. Control Skir Penetration Test

The control vehicle was ROGAINE obtained from Upjohn Co. The ROGAINE formulation contains 2% minoxidil in an ethanol/propylene glycol/water solvent vehicle, and was labeled with tritiated minoxidil before testing. One hundred fifty μl samples were applied to skin patches and the uptake of minoxidil across the skin monitored as described. Typical results for a 24-hour test period are shown in Figure 5, where the control drug data is

indicated by the open squares in the figure. As seen, the rate of uptake of the drug in the control formulation is substantially linear over the test period, and reaches a cumulative maximum, at the end of the test period, of about 30 μg/cm2, corresponding to about 0.5-1.0% of the total drug applied to the skin.

C. Results

(1) Minoxidil/Tauranol/Oleic acid liposomes compared to ROGAINE.

The 1.7% Minoxidil/Tauranol/Oleic acid liposomes, prepared as in Example 4, and ROGAINE (Upjohn Co.) were tested for transdermal uptake, using the experimental methods described above. Three runs were made for each formulation at each time point. The results, expressed in terms of cumulative μg drug uptake (X 10-3)/cm2 of skin patch, are shown in Figure 5, where the data for the ROGAINE composition is shown in open squares and the data for the 1.7% Minoxidil/Tauranol/Oleic acid liposomes is shown as open triangles. The solid lines flanking the data lines are the 95% prediction intervals calculated based on the triplicate data points.

It is clear from Figure 5 that the liposome

formulation of the present invention gives greater drug transdermal penetration than the control drug

formulation: the final cumulative dose corresponds to about 1,800 μg.

(2) Minoxidil/Tauranol/Oleic acid liposomes compared to Minoxidil/Laureth Sulfosuccinate.

The Minoxidil/Tauranol/Oleic acid liposomes,

prepared as in Example 4, and 2% Minoxidil/Laureth

Sulfosuccinate compositions, prepared as described in U. S. Patent No. 4,828,837, were tested for transdermal uptake, using the experimental methods describe above.

It is clear from Figure 6 that the liposome

formulation of the present invention gives greater drug transdermal penetration than the 2% Minoxidil/Laureth Sulfosuccinate composition: the final cumulative doses corresponds, respectively, to about 1,800 μg and about 100 μg.

While the invention has been described with

reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

Claims

IT IS CLAIMED:
1. Lipid bilayer vesicles comprising:
(a) an anionic or zwitterionic surfactant which, when dispersed alone in water at a temperature above the surfactant's phase transition temperature, is in a micellar phase, and
(b) a second lipid selected from the group
consisting of (i) a single-aliphatic chain lipid which, when dispersed alone in water at a temperature above the lipid transition temperature, is in a lipid emulsion phase, and which is an acid, ester, or alcohol, and (ii) cholesterol or a cholesterol analog or derivative;
where the weight ratio between components (a) and (b) is between about 1:2 to 2:1.
2. The vesicles of claim 1, wherein component (a) is a zwitterionic surfactant.
3. The vesicles of claim 2, wherein the
zwitterionic surfactant is selected from the group consisting of quaternary phosphate surfactants.
4. The vesicles of claim 3, wherein component (a) is a quaternary phosphate surfactant selected from the group consisting of MONAQUAT P7TL and MONAQUAT P-TS.
5. The vesicles of claim 2, wherein the
zwitterionic surfactant is selected from the group consisting of cocoamidopropyl betaine and
lauroamidopropyl betaine.
6. The vesicles of claim 1, wherein component (a) is an anionic surfactant.
7. The vesicles of claim 6, wherein the anionic surfactant is selected from the group consisting of N- methyl cocyl taurate, and N-methyl oleyl taurate.
8. The vesicles of claim 1, wherein the component (b) is selected from the group consisting of the
following fatty alcohols: lauryl, myristyl, palmityl, palmitoleyl, stearyl, oleyl, linoleyl, arachidatyl, and arachidonyl.
9. The vesicles of claim 1, wherein the component (b) is selected from the group consisting of the
following fatty acids: lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, arachidatic, and arachidonic.
10. The vesicles of claim 1, wherein component (a) is N-methyl cocyl taurate, and component (b) is oleic acid.
11. The vesicles of claim 1, wherein the aliphatic-chain of component (b) is greater than about 12 carbons in length.
12. The vesicles of claim 11, wherein the
aliphatic-chain of component (b) is saturated.
13. The vesicles of claim 11, wherein the
aliphatic-chain of component (b) is unsaturated.
14. The vesicles of claim 13, wherein component (b) is selected from the group consisting of retinol and retinodc acid.
15. The vesicles of claim 1, which further comprise N-methyl cocyl taurate, oleic acid, and minoxidil at a weight percent of approximately 3.7:2.5:2, respectively.
16. The vesicles of claim 1, which further comprise N-methyl cocyl taurate, oleic acid, and hydrocortisone at a weight ratio of approximately 1.4:0.9:1, respectively.
17. The vesicles of claim 1, wherein component (b) is cholesterol.
18. A method of preparing lipid-bilayer vesicles, comprising:
combining in an aqueous medium in a weight ratio of between 1:2 to 2:1
(a) an anionic or zwitterionic surfactant which, when dispersed alone in water at a
temperature above the surfactant phase transition temperature, is in a micellar phase, and
(b) a second lipid selected from the group consisting of (i) a single-aliphatic chain lipid which, when dispersed alone in water at a
temperature above the lipid transition temperature, is in a lipid emulsion phase, and which is an acid, ester, or alcohol, and (ii) cholesterol or a cholesterol analog or derivative; and
mixing components (a) and (b) in the aqueous medium until lipid bilayer vesicles are formed.
19. The method of claim 18, wherein said combining includes adding an aqueous micellar suspension of
component (a) to component (b).
20. The method of claim 18, wherein the aqueous suspension of component (a) is added to component (b) in substantially dry form.
21. The method of claim 18, wherein component (a) is added to component (b) before the addition of the aqueous medium.
22. The method of claim 18, wherein the components (a) and (b) are separately used to prepare aqueous suspensions, and the aqueous suspension of component (a) is added to an aqueous suspension of component (b).
23. The method of claim 18, wherein said combining includes mixing components (a) and (b) in an organic solvent, removing the solvent to form a dried mixture of the two components, and hydrating the dried mixture by the addition of water.
24. The method of claim 18, wherein the two
components are mixed in substantially dry form before the addition of aqueous medium.
25. The method of claim 18, where said combining includes the addition of an active agent to the aqueous medium containing component (a) before the addition of component (b).
26. The method of claim 18, wherein the lipid bilayer vesicles carry an active agent.
27. The method of claim 26, where the active agent is minoxidil.
28. The method of claim 26, where the active agentis hydrocortisone.
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