WO2002049617A2

WO2002049617A2 - Self forming, thermodynamically stable liposomes and their applications

Info

Publication number: WO2002049617A2
Application number: PCT/US2001/050118
Authority: WO
Inventors: Dan D. LASIC; Brian Keller Keller
Original assignee: Biozone Laboratories Inc
Current assignee: Biozone Laboratories Inc
Priority date: 2000-12-20
Filing date: 2001-12-19
Publication date: 2002-06-27
Anticipated expiration: 2003-06-20
Also published as: US20050249797A1; JP2002212106A; US20070042032A1; DE60138566D1; EP1343475A2; JP4497765B2; US7150883B2; ATE429901T1; EP1343475B1; US20110064791A1; AU2002232788A1; WO2002049617A3; US6610322B1; US6958160B1; US7718190B2; JP2010001318A

Abstract

A liposome suspension forms spontaneously upon adding a lipid composition to an aqueous solution. The lipid composition comprises a single lipid or a mixture of lipids that have appropriate packing parameters, that includes polyethyleneglycol, and that has a melting temperature which allows it to be in liquid form when mixed with the aqueous solution. Such liposome suspensions are useful for a variety of purposes, including the delivery of therapeutic agents.

Description

S P E C I F I C A T I O N

TITLE OF INVENTION

SELF FORMING, THERMODYNAMICALLY STABLE LIPOSOMES AND THEIR APPLICATIONS

FIELD OF THE INVENTION

The present invention relates to liposomes. More particularly, the present invention relates to liposomes which form spontaneously upon mixing of lipids and an

aqueous solution, and applications thereof.

BACKGROUND OF THE INVENTION

Liposomes are self-closed colloidal particles in which membranes composed of

one or more lipid bilayer(s) encapsulate a fraction of the aqueous solution in which they are suspended. The surfaces of bilayers are hydrophilic while the interior of bilayers,

which contain hydrocarbon chains, are hydrophobic. Because of the different

microenvironments in their structure, liposomes can encapsulate hydrophilic molecules,

bind molecules on the bilayer surfaces or dissolve hydrophobic molecules into the middle of the bilayer. Their ability to incorporate many types of molecules has resulted in

applications for drug delivery, diagnostics, cosmetics, cosmeceuticals and nutraceuticals.

Liposomes are manufactured by several different methods. Typically, the process

begins when a lipid or combination of lipids are dissolved in an organic solvent. Upon removal of organic solvents and hydration, large multilamellar vesicles (MLVs) are formed. For some applications, small unilamellar vesicles (SUVs) may be desired.

SUVs can be produced from MLVs by several techniques including sonication, extrusion

through membranes with well-defined pores, French press extrusion and

ho ogenization.

Problems associated with liposomes include colloidal instability, difficulty in

scale-up sterilization, and variability between batches in manufacturing. Liposome

preparation and manufacturing typically involves removal of organic solvents followed

by extrusion or homogenization. These processes may expose liposomal components to

extreme conditions such as elevated pressures, elevated temperatures and high shear conditions which can degrade lipids and other molecules incorporated into the liposomes.

Liposome preparations are often characterized by very heterogeneous

distributions of sizes and number of bilayers. Conditions optimized on a small scale normally do not scale up well and preparation of large-scale batches is cumbersome and

labor intensive.

Another issue associated with liposomes for medical uses is sterilization. Among

heat sterilization, ethanol oxide exposure, gamma irradiation and sterile filtration, only the last technique is suitable for liposomes and then only for liposomes smaller than

about 100 nanometers (nm). Filtration of liposomes poses many difficulties.

Another problem for liposome applications is colloidal stability. Liposomes in suspension can aggregate and fuse upon storage, heating and addition of various

additives. Because of these stability problems, liposomes are often lyophilized. Lyophilization is costly and time consuming. Upon reconstitution, size distributions

often increase and encapsulated materials may leak out from the liposomes.

It is therefore desirable to develop new methods and materials which address these problems with current liposome formulations.

BRIEF DESCRIPTION OF THE INVENTION

A liposome suspension forms spontaneously upon adding a lipid composition to an aqueous solution. The lipid composition comprises a single lipid or a mixture of

lipids that have appropriate packing parameters, that includes polyethyleneglycol, and

that has a melting temperature which allows it to be in liquid form when mixed with the

aqueous solution. Such liposome suspensions are useful for a variety of purposes,

including the delivery of therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute a part of

this specification, illustrate one or more embodiments of the present invention and, together with the detailed description, serve to explain the principles and

implementations of the invention.

In the drawings:

FIG. 1 is a diagram depicting the cross-section of a liposome made of lipid molecules. FIG. 2 is a space-filling diagram of a lipid molecule having a polar head group

and nonpolar hydrocarbon chains.

FIG. 3 is a diagram depicting a cross-section of a micelle made of lipid

molecules.

FIG. 4 is a diagram depicting a cross-section of a structure made of lipid

molecules with large tails relative to the head groups.

FIG. 5 is a diagram showing the molecular structure of PEG-12 Glyceryl Dioleate

or Hetoxamate GDO-12.

DETAILED DESCRIPTION

Embodiments of the present invention are described herein in the context of a self-forming, thermodynamically stable liposomes and their applications. Those of ordinary skill in the art will realize that the following detailed description of the present invention is illustrative only and is not intended to be in any way limiting. Other embodiments of the present invention will readily suggest themselves to such skilled persons having the benefit of this disclosure. Reference will now be made in detail to implementations of the present invention as illustrated in the accompanying drawings. The same reference indicators will be used throughout the drawings and the following detailed description to refer to the same or like parts.

In the interest of clarity, not all of the routine features of the implementations described herein are shown and described. It will, of course, be appreciated that in the

development of any such actual implementation, numerous implementation-specific decisions must be made in order to achieve the developer's specific goals, such as

compliance with application- and business-related constraints, and that these specific goals will vary from one implementation to another and from one developer to another.

Moreover, it will be appreciated that such a development effort might be complex and

time-consuming, but would nevertheless be a routine undertaking of engineering for

those of ordinary skill in the art having the benefit of this disclosure.

Those of ordinary skill in the art will realize that the following description of the present invention is illustrative only and not in any way limiting. Other embodiments of

the invention will readily suggest themselves to such skilled persons having the benefit

of this disclosure.

Most, if not all, known liposome suspensions are not thermodynamically stable.

Instead, the liposomes in known suspensions are kinetically trapped into higher energy

states by the energy used in their formation. Energy may be provided as heat, sonication,

extrusion, or homogenization. Since every high-energy state tries to lower its free

energy, known liposome formulations experience problems with aggregation, fusion, sedimentation and leakage of liposome associated material. A thermodynamically stable

liposome formulation which could avoid some of these problems is therefore desirable.

The present invention teaches liposome suspensions which are thermodynamically stable at the temperature of formation. The formulation of such

suspensions is achieved by employing a composition of lipids having several fundamental properties. First, the lipid composition must have packing parameters which

allow the formation of liposomes. Second, as part of the head group, the lipid should include polyethyleneglycol (PEG) or any polymer of similar properties which sterically

stabilizes the liposomes in suspension. Third, the lipid must have a melting temperature which allows it to be in liquid form when mixed with an aqueous solution.

By employing lipid compositions having the desired fundamental properties, little

or no energy need be added when mixing the lipid and an aqueous solution to form liposomes. When mixed with water, the lipid molecules disperse and self assemble as

the system settles into its natural low free energy state. Depending on the lipids used, the

lowest free energy state may include small unilamellar vesicle (SUN) liposomes,

multilamellar vesicle (MLV) liposomes, or a combination of SUVs and MLVs.

Lipid compositions suitable for use in the invention may include compositions

comprising only a single type of lipid molecule as well as compositions made up of more

than one lipid. As will be appreciated by those skilled in the art, both types of

compositions may be quantified according to cited fundamental properties.

One required fundamental property is the ability to form liposomes by virtue of

having the proper packing parameters. Packing parameters are relative measures of a

given lipid composition, and depend on factors such as size relationships between lipid

head groups and lipid hydrocarbon chains, charge, and the presence of stabilizers such as cholesterol. (Israelachvili, DD Lasic, Liposomes: From Physics to Applications, Elsevier, pp 51, 1993).)

To form a lipid bilayer, lipid head groups and hydrocarbon chains must organize

themselves so that the radius of curvature results in a liposome (see FIG. 1). If the hydrocarbon chains are too small relative to the head group, the radius of curvature will be too large and micelles will be produced (see FIG. 3). If the hydrocarbon chains are

too large relative to the head groups, the radius of curvature will be of the opposite sign

and liposomes cannot form (see FIG. 4).

FIG. 1 is a diagram depicting the cross-section of a liposome made of lipid

molecules. Liposome 10 comprises a lipid bilayer, made of lipid molecules (e.g., 12, 14,

16, 18), enclosing an aqueous space 20.

FIG. 2 is a space-filling diagram of a lipid molecule having a polar head group

and nonpolar hydrocarbon chains. Lipid molecule 22 is comprised of a hydrophilic

group 24 and a hydrophobic tail 26. Hydrophobic tail 26 may comprise two hydrocarbon

chains 32, 34. While its chemical bonds allow the lipid molecule to be flexible, the head

group generally fills an area of diameter 28 while the tail fills an area of diameter 30.

Because lipid molecules must be organized in a bilayer to form a liposome, the ratio of

the head group diameter to the tail diameter can be neither too large nor too small if

liposome formation is to occur.

FIG. 3 is a diagram depicting a cross-section of a micelle made of lipid

molecules. Micelle 36 is composed of lipid molecules (e.g., 38, 40). Because the tail groups of the lipid molecules have small diameters relative to the head groups, the lipid molecules organize with a small radius of curvature, and a bilayer cannot form.

FIG. 4 is a diagram depicting a cross-section of a structure made of lipid molecules with large tails relative to the head groups. In FIG. 4, it can be seen that structure 42 forms when lipids (e.g., 44) having large tails relative to the head groups are mixed in aqueous solution. Again, the size ratio between head groups and tails makes

bilayer formation impossible.

While FIGS. 1, 3 and 4 have illustrated the basic principle of packing parameters using a single type of lipid molecule, it will be appreciated that the same principle applies

to mixtures of lipids. For example, a lipid which has hydrocarbon chains too small to

form liposomes as a single species can be mixed with cholesterol to result in a

composition which has the proper packing parameters. As another example, a lipid

which by itself has the proper packing parameters may form liposomes incorporating limited amounts of other lipids which, by themselves, do not have proper packing

parameters. Both single lipids and mixtures of lipids have packing parameters that may

be calculated by known methods, (ref) In general, liposome compositions which allow

liposome formation have packing parameter measurements of P_a between about .84 and

.88 and P_v between about .88 and .93.

P_a is the packing parameter with respect to surface and Pv is packing parameter

with respect to volume (DD Lasic, Liposomes: From Physics to Applications, Elsevier,

pp 51, 1993). The parameters are derived from the equations HC_a/T_a= P_a and HC_V/T_V≡P_V where HC_a is the hydrocarbon chain area, T_a is the total area of the molecule, HC_V is the

volume of the hydrocarbon chains and T_v is the volume of the whole molecule.

Packing parameters can be calculated for mixtures of lipids, since ideal mixing of lipids results in arithmetic average of their individual characteristics. For instance

HC_a/T_a≡P_a of a binary mixture, in the case of ideal mixing can be expressed as: <P_a>=X₁Pι+X₂P₂, Xι+ X₂=l More generally in the case of i lipids composing a given mixture can be

represented by:

<P_a>=Σ₁X₁P, andΣ₁X₁=l where X_! is the mole fraction of the lipid in the mixture and Pi is the packing parameter

with respect to surface of that lipid.

FIG. 5 is a diagram showing the molecular structure of PEG-12 Glyceryl Dioleate

or Hetoxamate GDO-12 which includes a glycerol backbone 46, a PEG chain 48, and a

tail group 50 having two hydrocarbon chains. Together, the backbone 46 and the PEG

chain 48 comprise the head group of the molecule. PEG-12 GDO is an preferred lipid

molecule for use in the present invention, in part because its head group has the proper

size in relation to its tail group.

Two general factors influence the size of the head group in a lipid molecule. One

is the actual physical size of the head group. For example, employing a longer PEG

chain would make the head group larger. The other is the charge associated with the

head group. For example, if the PEG chain was conjugated to the backbone by a

phosphodiester bond the phosphate would impart a charge to the head group, effectively

increasing its size. In the present invention, non-phospolipids are preferred so that the

general means of varying head group size is by varying the length of the PEG chain.

However, those skilled in the art will recognize that the effective size of the head group

could be varied in other ways, for example by using a different backbone other than glycerol or adding a linker such as phosphate between the glycerol backbone and the PEG chain. The size of the tail of a lipid is mostly influenced by the length of hydrocarbon

chains and degree of saturation in the lipid chain. Single chain lipids will generally not form liposomes, though they may be incorporated into liposomes composed of lipids with two chains. Similarly, lipids with one long chain and one short chain may have

relatively small tail sizes. Those skilled in the art will recognize that such lipids may be

used to form liposomes in the present invention, especially if sterols such as cholesterol

are used to stabilize the bilayer.

A second fundamental property of lipid compositions suitable for use in this

invention is that the head group of at least one lipid must include a PEG chain. PEG

stabilizes the liposomes by creating a steric barrier at the outer surface of the liposomes.

Preferably, the PEG chain have a molecular weight between about 300 Daltons and 5000 Daltons, although those skilled in the art will recognize that differing concentrations of

PEG on the liposome surface as well as differing chain lengths may be used to stabilize

the liposomes.

A third fundamental lipid property is that the lipid composition must have a melting temperature which allows the composition to be in liquid form when mixed with

an aqueous solution. Generally, this means that the lipid composition should have a

phase transition temperature of between about 0°C and 100°C. As with packing

parameters, melting temperatures may be determined for mixtures of lipids.

Related to melting point of a lipid is bending elastic modulus. Generally

preferred in the present invention are lipids with a bending elastic modulus that allows the lipid to be sufficiently flexible to form liposomes in an aqueous solution without the

need for large energy inputs. If the bending elastic modulus is too great, the lipid will be

too rigid to spontaneously form liposomes in an aqueous solution. Preferably, the bending elastic modulus is between about 0 kt and 15 kt. More preferably, the bending

elastic modulus is between about 1 kt and 10 kt. The bending elastic modulus is largely

determined by the backbone. Glycerol provides an ideal backbone for the present

invention.

Table 1 shows a number of lipids which have been tested as single lipids for

suitability for the present invention. Lipids were tested at 2 weight percent in aqueous

solution. Note that GDL means glycerol dilaurate, GDO means glycerol dioleate, GDM

means glycerol dimyristate, GDP means glycerol dipalmitate, and GDS means glycerol

distearate. For each lipid, the number after "PEG" indicates the numbers of C^O subunits in the PEG chain. The unsaturated dioleate lipids have similar packing^'

parameters to the saturated dimyristate lipids.

The table shows that lipids which possess the required properties will

spontaneously form liposomes when mixed in an aqueous solution. For example, PEG-

12 GDM spontaneously forms liposomes at all temperatures tested since it is a liquid at

those temperatures and includes PEG in addition to having packing parameters within the

required ranges. Similarly, PEG-12 GDO, which shares nearly identical properties to PEG-12 GDM, spontaneously forms liposomes at all temperatures tested.

PEG-12 GDS shows one example of the requirement that the lipid be liquid at the

temperature of liposome formation. While this lipid has the required packing parameters

as well as including PEG, it does not spontaneously form liposomes until the temperature of liposome formation is high enough for the lipid to be in liquid form.

The GDM series of lipids illustrates the importance of proper packing parameters. While these lipids all include PEG and are in liquid form at 60 degrees, only PEG-12 GDM has the proper packing parameters to allow spontaneous liposome formation. The GDS lipid series at 60 degrees illustrates the same point. Those skilled in the art can practice the present invention by using knowledge of the required properties to predict and create lipid compositions which will spontaneously

form liposomes. Foe example, certain PEG lipids which form micelles can form

liposomes in mixtures with sterols, such as cholesterol, because of cholesterol' s effect on

packing parameters and melting point. For instance, the mixture of PEG-45 GDS and

cholesterol forms liposomes. Similarly, the size of the head group may be changed to affect packing parameters, for example by varying the size of the PEG chain or by varying the concentration of PEG-containing lipids in the lipid composition.

It will be appreciated that, while liposomes form spontaneously at the temperature

of formation, cooling of liposomes after formation results in liposomes in trapped kinetic states. To minimize distortions in liposome structure upon such cooling, sterols such as

cholesterol may be mixed with the lipid before liposome formation. It has been observed

that cholesterol may be dissolved in PEG-12 GDO at up to about 10 percent by weight.

Utility

The present invention is useful in a variety of situations, and provides advantages

over the prior art in several different ways. Problems with liposome preparation,

reproducibility, colloidal stability, sterilization, and storage may be reduced by

employing the invention.

Since the liposomes of the present invention are self-forming, liposome preparation entails merely mixing the lipid with an aqueous solution. In general,

liposome formation is scale dependant. It is simple, in the case of the present invention, to scale up from test batches to large batches. Because the liposomes of the present invention exist in the lowest energy state

that the lipid can exist in while in aqueous solution, reproducibility of liposome

formation is no problem. A defined lipid, lipid mixture, or lipid/compound mixture will

form similar liposomes every time when mixed with the same aqueous solution. It

should be noted, however, that above critical concentrations (around 20% weight to volume for most lipids) non-liposomal structures will begin to form in aqueous solution.

Aggregation and fusion may occur with liposomes in thermodynamically trapped

states. Because the liposomes of the present invention are in the lowest energy state, they

do not aggregate and fuse.

Because the liposomes of the present invention are small, they can be sterile

filtered. Also, the lipids may be heat sterilized prior to liposome formation.

As with any liposome dispersion, these liposomes can be lyophilized in the

presence of appropriate cryoprotectants. Even in liquid form, the liposomes are

colloidally stable because they are a thermodynamically stable system. Also, because of

their self-forming nature, the liposomes need not be stored at all. Instead, the lipid may be stored and constituted into liposomes as needed.

Applications of the present liposomes include the delivery of therapeutics and

other compounds, use in cosmetics, and use in drug delivery screening. Since the liposomes include an aqueous space, a hydrophobic region within the bilayer, and sites for covalent attachment (e.g., on the PEG chain or the backbone), many

types of compounds may be encapsulated by the liposomes. Such compounds include

compounds ranging from hydrophilic to hydrophobic, including many insoluble

compounds. These liposomes may substitute for currently available Cremophor® and

Solutol®.

For delivery of intravenous drugs, the drug may be provided in a sealed container

co-dissolved or co-mixed along with the lipid. The container may also contain inert gas

to reduce decomposition of the drug and lipid. Prior to administration an aqueous solution is added to the container, thereby forming liposomes with encapsulated drug.

Drugs suitable for such a mode of administration include: proteins, peptides, nucleic

acids, antineoplastic agents, anti-inflammatories, anti-infectives, gastrointestinal agents,

biological and immunologic agents, dermatologic agens, ophthalmic and otic agents,

diagnostic aids, nutrients and nutritional agents, hematological agents, endocrine and metabolic agents, cardiovasculars, renal and genitourinary agents, respiratory agents,

central nervous system agents.

In one aspect, the invention includes a method of preparing liposomes. The method comprises providing an aqueous solution; providing a lipid solution, where the

solution has a P_a between about .84 and .88, a P_v between about .88 and .93, and where at

least one lipid in the solution includes a polyethyleneglycol (PEG) chain; and combining the lipid solution and the aqueous solution. The PEG chain preferably has a molecular weight between about 300 Daltons and 5000 Daltons. Kinetic energy, such as shaking or vortexing, may be provided to the lipid solution and the aqueous solution. The lipid solution may comprise a single lipid. The lipid may comprise dioleolglycerol-PEG-12,

either alone or as one of the lipids in a mixture. The method may further comprise

providing an active compound; and combining the active compound with the lipid

solution and the aqueous solution.

The active compound may be selected from the group comprising proteins,

peptides, nucleic acids, antineoplastic agents, anti-inflammatories, anti-infectives,

gastrointestinal agents, biological and immunologic agents, dermatologic agents,

ophthalmic and otic agents, diagnostic aids, nutrients and nutrtitional agents, hematological agents, endocrine and metabolic agents, cardiovasculars, renal and

genitourinary agents, respiratory agents, central nervous system agents.

In another aspect, the invention includes a liposome suspension. The suspension

comprises one or more lipids, where the lipids as an aggregate have a P_a between about .84 and .88, a P_v between about .88 and .93 and a melting temperature of between about

0 to 100 degrees centigrade; and where at least one lipid includes a polyethyleneglycol

(PEG) chain. The PEG chain preferably has a molecular weight between about 300

Daltons and 5000 Daltons. The suspension may comprise a single lipid. The lipid may comprise dioleolglycerol-PEG-12. The suspension may further comprise an active

compound, which may be select from the group described above.

In another aspect, the invention includes a composition for combining with an aqueous solution to form a liposome suspension. The composition comprises one or

more lipids, where the lipids as an aggregate have a P_a between about .84 and .88, a P_v between about .88 and .93 and a melting temperature of between about 0 to 100 degrees centigrade; and where at least one lipid includes a polyethyleneglycol (PEG) chain. The

PEG chain preferably has a molecular weight between about 300 Daltons and 5000

Daltons. The composition may comprise a single lipid. The composition may comprise

dioleolglycerol-PEG-12. The composition may further comprise an active compound selected from the group above. The composition may be provided in a sealed container,

where the container also contains an inert gas to prevent oxidative degradation.

In another aspect, the invention includes a method of intravenously administering a therapeutic compound. The method comprises providing a composition including one

or more lipids, where the lipids as an aggregate have a P_a between about .84 and .88, a P_v

between about .88 and .93 and a melting temperature of between about 0 to 100 degrees

centrigrade; and where at least one lipid includes a polyethyleneglycol (PEG) chain; providing an active compound; providing an aqueous solution; combining the

composition, compound and solution to form a liposome suspension; and administering

the liposome suspension intravenously. The method may further comprise providing

kinetic energy to the liposome suspension. The method may also include providing the

composition in a sealed container containing an inert gas. The PEG chain preferably has

a molecular weight between about 300 Daltons and 5000 Daltons. The composition may comprise a single lipid. The lipid may comprise dioleolglycerol-PEG-12. The active

compound may be selected from the group above.

In another aspect, the invention includes a method of solubilizing an active compound. The method comprises providing a composition including one or more

lipids, where the lipids as an aggregate have a P_a between about .84 and .88, a P_v between about .88 and .93 and a melting temperature of between about 0 to 100 degrees centigrade; and where at least one lipid includes a polyethyleneglycol (PEG) chain;

providing the active compound; providing an aqueous solution; and combining the active compound, the lipid and the aqueous solution to form a liposome suspension. The

method may further comprise providing kinetic energy to the liposome suspension. The

method may include providing the composition in a sealed container containing an inert

gas. The PEG chain preferably has a molecular weight between about 300 Daltons and

5000 Daltons. The composition may comprise a single lipid. The lipid may comprise

dioleolglycerol-PEG-12. The active compound may be selected from the group above.

In another aspect, the invention includes a method of orally administering a

therapeutic compound. The method comprises providing a composition including one or

more lipids, where the lipids as an aggregate have a P_a between about .84 and .88, a P_v

centigrade; and where at least one lipid includes a polyethyleneglycol (PEG) chain;

providing an active compound; providing an aqueous solution; combining the

the liposome suspension orally in the form selected from the group comprising a two

piece hard gelatin capsule, a soft gelatin capsule, or drops.

The compositions may be administered topically, inter-orally, vaginally or

rectally.

While embodiments and applications of this invention have been shown and

described, it would be apparent to those skilled in the art having the benefit of this

disclosure that many more modifications than mentioned above are possible without departing from the inventive concepts herein. The invention, therefore, is not to be

restricted except in the spirit of the appended claims.

EXAMPLES

Example 1 : Spontaneous liposomes

Single lipids were tested for their ability to spontaneously form liposomes when mixed with an aqueous solution. PEG-12 GDO was obtained from Global 7 (New

Jersey). All other lipids were synthesized for the experiment. For each experiment,

water and lipid were separately equilibrated at the appropriate temperature. Two weight

percent lipid was added to the aqueous solution, and the solution was analyzed for the

presence of liposomes by optical light microscopy, cryo TEM, freeze fracture TEM, or

hot stage microscopy. The results of the experiments are shown in Table 1 above.

Example 2: Formation of multilamellar

liposomes with cholesterol

Cholesterol, 10 Wt. %, was added to PEG-12 Glyceryl Dioleate and heated to

about 60° -70°C, for 15-20 minutes. Water, at room temperature, was added to the

heated lipid solution. The mixture was left overnight. Examination of the preparation under a optical microscopic with polarized light at 100 power and 600 power showed

mulitlamellar liposomes in the size range of 20nm to 40nm. No crystals of cholesterol were observed.

Example 3: Spontaneous liposomes for intravenously administering therapeutic compounds

Ingredient Conc.(%wt)

Methotrexate 5

PEG-12 GDO 10

DI Water 85

Methotrexate (Sigma) was dissolved in PEG-12 Glyceryl Dioleate and gently

mixed for about 5 minutes. The resultant mixture was a clear solution. Deionized water

was slowly added to the solution and gently mixed. The resultant mixture was a yellow, opaque solution. The preparation was examined under a light microscope with polarized

light and showed a suspension of liposomes.

Example 4: Spontaneous liposomes for intravenously administered therapeutic

compound and as a drug solubilization vehicle.

Ingredient Cone. (wt%)

Cyclosporine A 0.77

PEG-12 GDO 4.30

DI Water 95.23

Cyclosporin A (Sigma 49H4066) was mixed with PEG-12 Glyceryl Dioleate by vortexing and sonication for 10 minutes. Water was added and gently mixed. Examination under optical microscope at 600 power showed mulitlamelar liposomes and

crystals of cyclosporin A. Example 5: Spontaneous liposomes with active compounds for dermatology.

Ingredient Conc.(wt)

PEG-12 GDO 18 g

Betamethasone diproprionate 50 mg

Cholesterol 100 mg

Uniphen-23® 1.5 mg

Water 80.35 g

Weighed amounts of PEG-12 Glyceryl Dioleate, Betamethasone diproprionate

and cholesterol were combined and heated to 50°C while mixing. Uniphen-23® and water were combined an heated to 50°C. When mixtures reached temperature they were

commingled while stirring gently. Mixture was cooled to room temperature while

stirring. Examination by optical microscope at lOOx and 600x showed a suspension of

multilamellar liposomes.

Example 6: Spontaneous liposomes with active compounds for topical anesthesia.

Ingredient Cone.

Tetracaine 2 g

PEG-12 GDO 20 g

Uniphen-23® 1.5 g

Water 76.5g

Tetracaine, PEG-12 Glyceryl Dioleate, and Uniphen-23® were mixed together and heated to 40°C while stirring. Water was heated to 40 degrees C and added to the tetracaine solution while stirring gently. Mixture was cooled to room temperature. Examination by electron microscope showed LUV's and MLVs.

Example 7: Spontaneous liposomes for intravenous and topical formulations

Tretinoin (all-trans retinoic acid), 6mg, was dissolved in 500ul of PEG-12

Glyceryl Dioleate. Dissolution was complete. Distilled water, 4.5ml, was added to the

mixture and gently mixed. This yielded a concentration of 1 mg/ml. Examination by optical microscope showed multilamellar liposomes in the size range of lOOnm to

200nm. This solution can easily be incorporated into a cream, gel or lotion dosage form.

While embodiments and applications of this invention have been shown and

disclosure that many more modifications than mentioned above are possible without

departing from the inventive concepts herein. The invention, therefore, is not to be

restricted except in the spirit of the appended claims.

Claims

CLAIMS What is claimed is:

1. A method of preparing liposomes, the method comprising:

providing an aqueous solution; providing a lipid solution, where the solution has a P_a between about .84 and .88,

a P_v between about .88 and .93; and where at least one lipid in the solution includes a

polyethyleneglycol (PEG) chain, and combining the lipid solution and the aqueous solution.

2. The method of claim 1, where the PEG chain has a molecular weight between

about 300 Daltons and 5000 Daltons.

3. The method of claim 1, further comprising:

providing kinetic energy to the lipid solution and the aqueous solution.

4. The method of claim 1, where the lipid solution comprises a single lipid.

5. The method of claim 1, where the lipid comprises dioleolglycerol-PEG-12.

6. The method of claim 1, further comprising: providing an active compound; and

combining the active compound with the lipid solution and the aqueous solution.

7. The method of claim 6, where the active compound is selected from the group comprising proteins, peptides, nucleic acids, antineoplastic agents, anti-inflammatories,

anti-infectives, gastrointestinal agents, biological and immunologic agents, dermatologic agents, ophthalmic and otic agents, diagnostic aids, nutrients and nutritional agents,

hematological agents, endocrine and metabolic agents, cardiovasculars, renal and genitourinary agents, respiratory agents, central nervous system agents.

8. A liposome suspension, the suspension comprising:

one or more lipids, where the lipids as an aggregate have a P_a between about .84

and .88, a P_v between about .88 and .93 and a melting temperature of between about 0 to 100 degrees centigrade; and where at least one lipid includes a polyethyleneglycol (PEG)

chain.

9. The suspension of claim 8, where the PEG chain has a molecular weight between

about 300 Daltons and 5000 Daltons.

10. The suspension of claim 8, where the suspension comprises a single lipid.

11. The suspension of claim 8, where the lipid comprises dioleolglycerol-PEG-12.

12. The suspension of claim 8, further comprising:

an active compound.

13. The suspension of claim 12, where the active compound is selected from the group comprising proteins, peptides, nucleic acids, antineoplastic agents, anti- inflammatories, anti-infectives, gastrointestinal agents, biological and immunologic

agents, dermatologic agents, ophthalmic and otic agents, diagnostic aids, nutrients and nutritional agents, hematological agents, endocrine and metabolic agents, cardiovasculars, renal and genitourinary agents, respiratory agents, central nervous

system agents.

14. A composition for combining with an aqueous solution to form a liposome

suspension, the composition comprising:

and .88, a P_v between about .88 and .93 and a melting temperature of between about 0 to

100 degrees centigrade; and where at least one lipid includes a polyethyleneglycol (PEG) chain.

15. The composition of claim 14, where the PEG chain has a molecular weight

between about 300 Daltons and 5000 Daltons.

16. The composition of claim 14, where the composition comprises a single lipid.

17. The composition of claim 14, where the lipid comprises dioleolglycerol-PEG-12.

18. The composition of claim 14, further comprising:

an active compound.

19. The suspension of claim 18, where the active compound is selected from the group comprising proteins, peptides, nucleic acids, antineoplastic agents, anti- inflammatories, anti-infectives, gastrointestinal agents, biological and immunologic

agents, dermatologic agents, ophthalmic and otic agents, diagnostic aids, nutrients and

nutritional agents, hematological agents, endocrine and metabolic agents, cardiovasculars, renal and genitourinary agents, respiratory agents, central nervous

system agents.

20. The composition of claim 14, where the composition is in a sealed container, said

container also containing an inert gas.

21. A method of intravenously administering a therapeutic compound, the method comprising:

providing a composition including one or more lipids, where the lipids as an

aggregate have a P_a between about .84 and .88, a P_v between about .88 and .93 and a

melting temperature of between about 0 to 100 degrees centigrade; and where at least one lipid includes a polyethyleneglycol (PEG) chain;

providing an active compound;

providing an aqueous solution;

combining the composition, compound and solution to form a liposome

suspension; and administering the liposome suspension intravenously.

22. The method of claim 21, further comprising:

providing kinetic energy to the liposome suspension.

23. The method of claim 21, where said providing a composition includes providing the composition in a sealed container containing an inert gas.

24. The method of claim 21, where the PEG chain has a molecular weight between about 3000 Daltons and 5000 Daltons.

25. The method of claim 21, where the composition comprises a single lipid.

26. The method of claim 21, where the lipid comprises dioleolglycerol-PEG-12.

27. The method of claim 21, where the active compound is selected from the group comprising proteins, peptides, nucleic acids, antineoplastic agents, anti-inflammatories,

anti-infectives, gastrointestinal agents, biological and immunologic agents, dermatologic

agents, ophthalmic and otic agents, diagnostic aids, nutrients and nutritional agents,

hematological agents, endocrine and metabolic agents, cardiovasculars, renal and

genitourinary agents, respiratory agents, central nervous system agents.

28. A method of solubilizing an active compound, the method comprising:

providing a composition including one or more lipids, where the lipids as an

aggregate have a P_a between about .84 and .88, a P_v between about .88 and .93 and a melting temperature of between about 0 to 100 degrees centigrade; and where at least

one lipid includes a polyethyleneglycol (PEG) chain;

providing the active compound;

providing an aqueous solution; and

combining the active compound, the lipid and the aqueous solution to form a liposome suspension.

29. The method of claim 28, further comprising:

providing kinetic energy to the liposome suspension.

30. The method of claim 28, where said providing a composition includes providing

the composition in a sealed container containing an inert gas.

31. The method of claim 28, where the PEG chain has a molecular weight between

about 300 Daltons and 5000 Daltons.

32. The method of claim 28, where the composition comprises a single lipid.

33. The method of claim 28, where the lipid comprises dioleolglycerol-PEG-12.

34. The method of claim 28, where the active compound is selected from the group

comprising proteins, peptides, nucleic acids, antineoplastic agents, anti-inflammatories,

genitourinary agents, respiratory agents, central nervous system agents.

35. A method of orally administering a therapeutic compound, the method

comprising:

providing a composition including one or more lipids, where the lipids as an

melting temperature of between about 0 to 100 degrees centigrade; and where at least

one lipid includes a polyethyleneglycol (PEG) chain;

providing an active compound;

providing an aqueous solution; combining the composition, compound and solution to form a liposome suspension; and

administering the liposome suspension orally in the form selected from the group

comprising a two piece hard gelatin capsule, a soft gelatin capsule, or drops.

36. A method of orally or sublingually administering a therapeutic compound, the

method comprising:

providing a composition including one or more lipids, where the lipids as an

one lipid includes a polyethyleneglycol (PEG) chain;

providing an active compound;

providing an aqueous solution;

combining the composition, compound and solution to form a liposome

suspension; and administering the liposome suspension orally by spraying it into

the mouth.

37. A method of administering a therapeutic compound, the method comprising:

providing a composition including one or more lipids, where the lipids as an

one lipid includes a polyethyleneglycol (PEG) chain;

providing an active compound;

providing an aqueous solution;

combining the composition, compound and solution to form a liposome

suspension; incorporating the composition into a form selected from the group comprising a cream, lotion, and gel; and administering the liposome suspension.