CA2222063A1 - Reverse fluorocarbon emulsion compositions for drug delivery - Google Patents

Abstract

A polar liquid-in-perfluorochemical emulsion or microemulsion for use in delivery of therapeutic or diagnostic agents. These compositions are formed by combining a discontinuous aqueous phase, a continuous fluorocarbon phase and a nonfluorinated surfactant. Further, the polar liquid-in-fluorochemical emulsions may be used to form multiple emulsions having an aqueous continuous phase. Such emulsions and microemulsions are suitable for the administration of pharmaceutical agents including genetic material.

Description

WO 96/40057 PCT~US96/09064 REVERSE FLUOROCARBON EMULSION COMI~S~ I IONs FOR DRUG DELIVERY
Field o~ the Invention The present invention relates to compositions for delivery of therapeutic and diagnostic agents. More speci~ically, the invention relates to polar liquid-in-per~luorochemical emulsions, multiple emulsions, and microemulsions.
Backqround o~ the Invention Fluorocarbons, fluorine substituted hydrocarbons, and per~luorocarbons, ~1uorocarbons in which all of the hydrogen atoms have been replaced with fluorine, have found wide applications in the medical field as therapeutic and diagnostic agents. These liquids are clear, colorless, odorless, nonflammable and essentially insoluble in water. In addition, fluorocarbon liquids are denser than water and soft tissue, have low surface tension and, for the most part, low viscosity.
Fluorocarbons possess desirable characteristics including biocompatibility, relatively low reactivity and high oxygen carrying capacity. Brominated fluorocarbons have been shown to exhibit radiopacity for certain types of radiation. For example, U.S. Patent No. 3,975,512 to Long uses fluorocarbons, including brominated per~luorocarbons, as a contrast enhancement medium in radiological imaging. The commercial fluorocarbon emulsion FLUOSOL~ (Green Cross Corp., Osaka, Japan) has been used as an oxygen carrier during percutaneous translllm; n~l coronary angioplasty. Fluorocarbon emulsions have also been used in diagnostic imaging applications including nuclear magnetic resonance and ultrasound (U.S.
Patent No. 5,114,703). Neat per~luorocarbons have also found medical applications. Imagent~ GI, an FDA-approved diagnostic agent comprising neat per~luorooctyl bromide (PFOB), is used for imaging the gastrointestinal tract. Per~luorocarbons also have ophth~lm;c applications for treating giant retinal tears WO 96/40057 PCTAUS9G/O~OG~
(Aguilar et al., Retina , lS:3-13) and are being evaluated for use during liguid ventilation applications.
While impressive, the aforementioned therapeutic fluorocarbon applications would be of greater benefit if used in conjunction with other drugs or diagnostic agents. For example, in the current treatment of lung disease, poor vascular circulation of diseased portions of the lung re~ces the effectivenes6 of drug delivery. It has been shown, however, that pulmonary delivery of biological agents through the alveolar surface can be facilitated when accomplished in conjunction with liquid ventilation (Wolfson et al., FASEB J., 4:A1105, 1990). It has also been shown that pulmonary drug ~m; n; stration can increase the biological response of some drugs when compared to intravenous administration. (Shaffer et al., Art. Cells, Blood Sub. & Immob. Biotech., 22:315, 1994).
plllmon~ry drug ~m; n; stration also has applications in the treatment and/or diagnosis of disorders including respiratory distress syndrome (R~S), impaired pulmonary circulation, cystic fibrosis and lung cancer. The increased efficacy of plllmon~y drug delivery via liquid ventilation may be due to high spre~;ng coe~ficients of per~luorocarbons on the plllmon~y surface, an increase in alveolar surface area due to more effective lung inflation, and the delivery of oxygen by the perfluorocarbon.
A major problem associated with per~luorocarbon-mediated drug delivery is that the drugs are often insoluble in the fluorocarbon phase. Current methods of plllmon~y drug administration involve the preparation of crude dispersions of the drug and delivery by turbulent flow and nebulization.
Unfortunately, not all drugs can be delivered in this m~nner.
Reverse water-in-perfluorocarbon emulsions have been prepared previously using perfluorinated surfactant. The ability to stabilize these reverse emulsions with nonfluorinatedbiocompatible surfactants (i.e. phospholipids) would provide an advantage.

Accordingly, there is a need in the art for compositions and methods capable of delivering fluorocarbon-associated polar liquid-soluble therapeutic and diagnostic agents in an effective, reliable m~nne~. The pre~ent invention addresses this need by providing polar liquid-in-fluorocarbon emulsions, multiple emulsions, and microemulsions stabilized by biocompatible phospholipids or hydrogenated surfactants.

Summarv of the Invention The present invention provides stable reverse (polar liquid-in-fluorocarbon) emulsions and thermodynamically stable reverse microemulsions in a fluorocarbon continuous phase for the delivery of polar liquid-soluble pharmaceutical agents.
These emulsions overcome many of the difficulties associated with heterogeneous crude drug dispersions in fluorocarbons.
Further, the present invention also provides stable multiple (polar liquid-in fluorocarbon-in-polar liquid) emulsions.
Accordingly, in a broad aspect the invention comprises a fluorocarbon pharmaceutical formulation comprising:
a disperse liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent;
a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant.
Another aspect of this part of the invention is directed to thermodynamically stable formulations.
Yet another aspect of the invention consists of method for preparing a therapeutic or diagnostic formulation comprising:
providing a li~uid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent;
combining said liquid phase with an effective emulsifying amount of at least one nonfluorinated surfactant and a WO96/40057 PCT~S~G/O~C61 fluorocarbon phase comprising at least one lipophilic fluorocarbon to provide an emulsion formulation; and emulsifying said emulsion formulation to produce a therapeutic or diagnostic formulation.
5Still another aspect is directed toward the delivery a - therapeutic or diagnostic agent to a patient comprising:
providing apharmaceutical emulsion comprising a disperse liquid phase, said liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or lOdiagnostic agent; a continuous fluor.ocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant;
and ~m;n~stering said pharmaceutical emulsion to a patient.
15In another embodiment, the reverse emulsions described may be used to form a multiple water-in-fluorocarbon-in-water emulsion. Specifically, the reverse emulsions are dispersed in an aqueous solution containing at least one nonfluorinated surfactant. The nonfluorinated surfactant may be the same or 20different as the one used to initially form the reverse emulsion.
The process for the preparation of a multiple emulsion comprises the ~ollowing steps:
a) providing a liquid phase comprising at least one 25polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent;
b) combining said liquid phase with an effective emulsifying amount of at least one nonfluorinated surfactant and a fluorocarbon phase comprising at least one lipophilic 30fluorocarbon to provide an emulsion formulation;
c) emulsifying said emulsion formulation to produce a therapeutic or diagnostic reverse emulsion;
d) adding said therapeutic or diagnostic reverse emulsion to a second polar liquid comprising an effective 35emulsi~ying amount of at least one non~luorinated surfactant Wo96/40057 PCT~S96/09064 to provide a multiple formulation wherein said second polar liquid is the same or different than said polar liquid; and e) emulsifying said multiple formulation to produce a multiple emulsion.
In such a multiple emulsion, the external aqueous phase is continuous while the reverse emulsion is discontinuous.
The multiple emulsions may further comprise one or more additives such as mineral salts, solvents, dispersants, buffer agents, oncotic agents, osmotic agents, nutritive agents, hydrophilic pharmaceutical agents, and lipophilic pharmaceutical agents. These additives may be in with the internal or external aqueous phase, the perfluorocarbon phase, or at the interfaces. As used herein, a pharmaceutical agent is an agent that provides therapeutic or diagnostic value when treating a patient.
Yet still another aspect of the present invention is directed toward methods of preparing pharmaceutical dispersions comprising:
providing a reverse emulsion having a disperse liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent; a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant; and combining said reverse emulsion with a non-lipophilic fluorocarbon to form a dispersion.
Finally, in a broad aspect the invention is directed to $ormulations containing fluorochemical emulsions.
Such formulations comprise:
a disperse liquid phase comprising at least one polar liquid;
a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one non$1uorinated surfactant.

Wo 96/40057 PCTIUS96/09064 In the preferred embodiments described above, the disperse liquid phase comprises water, alcohols, alkyl sulfoxides, polyethylene glycols, or mixtures thereof. In particularly preferred embodiments, the alcohols are short chain alcohols such as ethanol and the alkyl sulfoxide is dimethylsulfoxide .
Preferably, the lipophilic fluorocarbon is a halogenated f luorocarbon, halogenated perf luoroether/polyether, fluorocarbon-hydrocarbon diblock, fluorocarbon-hydrocarbon ether diblock or mixture thereof. Advantageously, the halogenated perfluorocarbon is ~,cl~-dibromo-F-butane.
In addition, the fluorocarbon phase may further comprise one or more additives capable of increasing the lipophilicity o~ the ~luorocarbon phase. These additives are pre~erably non-surface active oils, such as medium-chain triglycerides, long-chain triglycerides, silanes, silicone oils, hydrocarbons, Freons, ~ 1 k~nes, squalene, f luorocarbon-hydrocarbon diblocks and lipophilic short-chain fluorocarbons.
Other surface-active oils may be added to decrease the spontaneous curvature of the surfactant monolayer. These include cholesterol, monoglycerides, diglycerides, long-chain alcohols and sterols . Prei~erably, the f luorocarbon is a brominated, chlorinated or iodinated f luorocarbon .
According to other preferred ernbodiments, the therapeutic or diagnostic agent is respiratory agent, antibiotic, anti-inf lammatory, chemotherapeutic agent, antineoplastic agent, anesthetic, ophth~lm;c agent, cardiovascular agent, imaging agent, enzyme, nucleic acid, gene protein or viral vector.
In preferred embodiments the nonfluorinated surfactant is selected from the group consisti.ng of alcohols, salts of fatty acids, phosphatidylcholines, N-monomethyl-phosphatidylethanol~m~ nes, phosphatidic acids, phosphatidyl ethanolamines, N, N-dimethyl-pho,sphatidyl -ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, egg yolk phospholipids, cardiolipins, glycerglycolipids, phosphatidylserines, phosphatidylglycerols and aminoethylphosphonolipids. Preferably, the nonfluorinated surfactant contains at least one mono-unsaturated moiety. In particularly preferred embodiments the nonfluorinated surfactant is 1,2 dioleoylphosphatic acid or 1,2 dioleoyphosphatidyl ethanolamine.
Advantageously, the nonfluorinated surfactant may a low hydrophilic lipophilic balance. Such surfactants include SPANS~, BRIJs~, Guerbet alcohol ethoxylates, dialkyl nonionic surfactants and dialkylzwitterionic surfactant~. The emulsion may further comprise a surface active oil capable of decreasing the spontaneous curvature of the surfactant film.
Preferably, the surface active oil is a monoglyceride, diglyceride, long-chain alcohol or sterol.
Another embodiment of the invention is administering the emulsion to the patient. Those skilled in the art will appreciate that the emulsion of the present invention may be ~m; n; stered to the patient using a delivery device.
Preferably the delivery device is selected from the group consisting of an endotracheal tube, an intrapulmonary catheter, and a nebulizer. It will further be appreciated that the present invention is particularly suited for pulmonary delivery using partial liquid ventilation and aerosolization.
In yet other embodiments the present invention may be used to deliver pharmaceutical formulations. Preferably, the incorporated therapeutic or diagnostic agent is an antibiotic such as an amoxicillin, a nitrofuran, a tetracycline, an aminoglycoside, a macrolide or clarithromycin. In selected embodiments, the infective agent is Heliobacter pylori or Mycobacterium tuberculosis.

srief DescriPtion of the Fiqures Figure 1 is the particle size distribution obtained by photon correlation spectroscopy (PCS) of a reverse emulsion cont~;n;ng 1.0~ w/v egg phosphatidylethano1Am;ne, 90~ v/v ~,~-dibromo-F-butane, 0.09~ sodium chloride, 0.09~ calcium chloride and 10~ water. The emulsion particle size is shown on the x-axis and the relative volume is shown on the y-axis.
Figure 2 shows the effect of the Continuous Phase Refractive Index (nD) on reverse emul~ion stability. Reverse emulsions containing ~,~-dibromo-F-butane (DBFB), ~richlorotrifLuorQethane (CFC-113),n-h~,perfluorohexane (PFH) and mixtures of each were analyzed. The volume fraction is shown on the x-axis and nD is shown on the y-axis.
Figure 3 shows the effect of Continuous Phase Molar Volume (VM) on reverse emulsion stability. The oil o~ the continuous phase used in reverse emulsion formation is shown on the x-axis and VM is shown on the y-axis.
Figure 4 shows the particle size distribution obtained by PCS of a reverse emulsion cont~;n;ng 1.0~ w/v 1,2-dioleoylphosphatidyl-ethanolamine, 0~21~ w/v diolein, 90~ v/v ~,~-dibromo-F-butane, 0.09~ sodium chloride, 0.09~ calcium chloride and 10~ water in the absence (~) and presence (-) of 0.051~ gentamicin sulfate. The emulsion particle size is shown on the x-axis and the relative volume is shown on the y-axis.
Figure 5 shows the viscosity as a function of shear rate obtained from ~-~-dibromobutane reverse emulsions ~ormulated with 5 (0), 10 (~), 15 (0), 20 (-) and 30 (-) percent dispersed phase by volume. The dispersed phase 1,2 dioleoylphosphatidylethanolamine, sodium chloride andcalcium chloride concentrations were fixed at 1.34 mM, 0.9~ w/v and 0.9~ w/v respectively. The shear rate is shown on the x-axis and the emulsion viscosity is shown on the y-axis.

Detailed DescriPtion of the Invention As described above, the presentinvention provides stable reverse (polar liquid-in-fluorocarbon) emulsions and thermodynamically stable reverse microemulsions in ~luorocarbon continuous phase for the delivery of polar WO 96/40057 PCT~US96/09064 liquid-soluble drugs. The emulsions of the present invention overcome many of the difficulties associated with heterogeneous crude drug di5persions in fluorocarbons. The present invention also provides stable multiple (polar li~uid-in fluorocarbon-in-polar liquid) emulsions, and methods for forming pharmaceutical nanoparticulates.
In preferred embodiments the reverse emul~ion or microemulsion systems comprise a disper8e aqueous phase containing one or more polar liquid-soluble therapeutic and/or diagnostic agents, a continuous phase comprising at least one fluorocarbon and at least one nonfluorinated surfactant. In addition, the fluorocarbon may contain one or more solutes capable of increasing the lipophilicity of the fluorocarbon phase. As will be appreciated by those skilled in the art, a multiple emulsion (liquid phase-fluorocarbon-liquid phase) may be produced by combining the formed reverse emulsion with a continuous aqueous phase.
A major difference between a microemulsion and a "conventional" emulsion is thermodynamic stability. Given the correct temperature, pressure and composition, microemulsions will form spontaneously and will not coarsen over time.
Microemulsions are formed from substantially the same components as '~conventional" emulsions, yet the relative amount o~ disperse phase is generally smaller than in conventional emulsions. Typically, in microemulsions, the disperse phase will comprise less than 10~ v/v, and most preferably, depending on its components, less than 5~ v/v of the total emulsion volume.
An emulsion's microstructure is preferably defined as a surfactant monolayer film at a water-oil interface. As will be appreciated by those skilled in the art, the term "water" is not limited to aqueous solutions when discussing emulsions generally. An important property of a surfactant film is its tendency to curve toward either the water or the oil. This tendency of the surfactant film to curve can be ~uantitatively described by the spontaneous curvature (Ho)~

~, _ g _ W O 96/40057 PCT/US~61'~3064 an intrinsic property of the surfactant film which depends on the surfactant geometry (i.e., headgroup area, hydrocarbon tail chain length and volume), the degree of penetration of the oil into the surfactant's hydroc~hon tails, and the degree of hydration of the hydrophilic head groups among other factors. The sign and value of the spontaneous curvature not only dictates whether the resulting emulsion will exhibit a normal (oil-in-water) or reverse (water-in-oil) disperse phase system, but also the degree to which it will remain stable. The spontaneous curvature is considered positive if the film tends to curve toward the oil phase (o/w emulsion), and negative if the film tends to curve toward the aqueous phase (w/o emulsion).
In such compositions, the emulsifiers or surfactants may be chosen based on their geometry, i.e. surfactants are favored which have a small headgroup area and a large tail volume (i.e. an inverted truncated cone or wedge). Surface-active oils may be added to the surfactant system to decrease the spontaneous curvature of the surfactant monolayer. These include, for example, monoglycerides and alcohols, especially long chain alcohols, sterols and diglycerides. Specific mineral salts may also be added to reduce the surfactant monolayer spontaneous curvature through the promotion of tight headgroup packing. These include, for example, calcium, magnesium, and al~lm;nllm salts.
Another embodiment of the invention is the formation of a substantially homogeneous colloidal dispersion of a pharmaceutical agent in a non-lipophilic fluorocarbon such as perfluorooctyl bromide. Other non-lipophilic fluorocarbons that are compatible with the present invention include perfluorooctyl chloride, F-octane, and the like. As used herein, the term"non-lipophilic" refers toperfluorochemicals having a relatively low measured lipophilicity. Preferred non-lipophilic fluorocarbons suitable for use in the colloidal dispersions generally contain at least six carbon atoms. The W O 96/40057 PCTAJS96/09~
colloidal disper~ion preferably has particles with an average diameter of less than 3 ~m and more preferably of less than 1 ~m. Particularly preferred embodiments comprises particulates having an average diameter less than 500 nm and especially less than 100 nm. In seleCted embodiments, the reverse emulsions of the invention are further combined with a non-lipophilic liquid fluorocarbon. Because of physical differences between the reverse emulsions and the non-lipophilic fluorocarbon, the pharmaceutical agent undergoes a phase change to form an efficacious dispersion.

A. The Discontinuous Phase In a preferred embodiment, the discontinuous (disperse) phase comprises at least one polar liquid for drug solubilization. While numerous polar liquids are compatible with the teachings of the present invention, particularly preferred embodiments incorporate water, short-chain alcohols, dimethylsulfoxide, polyethylene glycols, or mixtures thereof.
In another preferred embodiment, the volume of the disperse phase comprises between about 0.05~ and 70~ of the total volume of the emulsion.
The disperse phase may also contain additives such as mineral salts, buffers, stabilizers, oncotic and osmotic agents, nutritive agents, active principals, pharmaceutically active substances, genetic material, or other ingredients designed to enhance various characteristics of the emulsions including their stability, therapeutic efficacy andtolerance.
In particularly preferred embodiments the disperse phase may comprise a nucleic acid moiety such as RNA or DNA. The disperse phase may also incorporate selected ions to stabilize the emulsion or the encapsulated drug. For example, if the interfacial layer contains phosphatidylglycerol or phosphatidic acid, emulsion stability may be increased by the addition of calcium or magnesium ions into the aqueous phase.
In other instances, certain enzymes (e.g. DNase), may retain more activity when specific ions are included for stability.

W O 96/40057 PCT~US96/09064 The disperse phase may also contain additives (e.g.
longer chain polar alcohols such as butanol) designed to suppress Ostwald ripening (irreversible coarsening) in the reverse emulsions. To further improve the solubility of certain drugs (e.g. Taxol~) in the emulsions of the invention, ethanol, polyethylene glycols, water-soluble Pluronics~, or dimethylsulfoxide may be added in part or in whole to the disperse phase. To further improve the stability of reverse emulsions against coalescence, the emulsion viscosity may be increased by increasing the dispersed phase volume.
Multiple water-oil-water emulsions c~~ ising the reverse emulsions described above, dispersed in the form of globules in a continuous second polar liquid phase, are also contemplated. Such a multiple emulsion may be prepared by addition of a reverse emulsion to an second polar liquid phase in which is dispersed at least one fluorinated or non-fluorinated surfactant described hereinabove. The amount of surfactant employed in the formation of multiple emulsions will depend on the quantity of polar liquid and reverse emulsion used. In general, for a polar liquid phase constituting 60~ to 99.95~ v/v of a reverse emulsion, the amount of surfactant used is between about 0.01~ and about 10~
w/v of the aqueous phase. Surfactants that are presently known in the art as good emulsifiers for oil-in-water emulsions can be employed here. These include, for example, phosphatidylcholines, egg yolk phospholipids, and Pluronics.
The external polar liquid continuous phase can also contain polar solvents including, for example, glycol, glycerol, dimethylformamide, or dimethylsulfoxide, as well as the additives described above. These additives may be present in the second polar liquid phase, the oily phase, at the interface between the phases, or in both of the phases.
As will be discussed in more detail below, the emulsions o~ the present invention are capable of delivering any desired polar liquid-soluble therapeutic and/or diagnostic agent(s).
Preferred pharmaceutical agents include antibiotics, -12- v W O 96/40057 PCT~US96/05~6 antivirals, anti-inflammatories, respiratory agents, genetic material, antineoplastics, anesthetics, imaging agents, ophth~l m; c agents and cardiovascular agents.
B. The Continuous PhasQ
In a preferred embodiment, the reverse emulsions of the invention contain between about 40~ and 99.95~ v/v of a continuous oily phase, comprising at least one lipophilic fluorinated or perfluorinated organic compound. The continuous fluorocarbon phase may comprise one or more fluorocarbons, perfluoroc~hnn~ or perfluoroc~hon-hydroc~hnn mixtures. Highly lipophilic fluorocarbons which facilitate dispersion of the hydrocarbon surfactants in the fluorocarbon continuous phase are preferred. In general, such lipophilic fluorocarbons contain a halogen atom (chlorine, bromine, or lS iodine) or a hydrocarbon moiety (e.g., C2Hs). In another preferred embodiment, the fluorocarbon contains up to eight carbon atoms. In a particularly preferred embodiment, the fluorocarbon contains between four and six carbon atoms.
Fluorocarbon molecules used in these emulsions may have various structures, including straight or branched chain or cyclic structures, as described in Riess, J., Artificial Organs, 8(1):44-56, 1984.
There are a number of fluorocarbons that are contemplated for use in the present invention. These fluorocarbons include halogenated perfluorocarbons (e.g., CnF2n~lX, XCnF2nX, wherein n=2-8, X is Cl, Br or I), halogenated ethers or polyethers (e.g., XCnF2nOCnF2nX, XCF20CF2CF20CF2X, wherein n=2-4, and X is Cl, Br or I), fluorocarbon-hydrocarbon diblocks (e.g., CnF2n~1-CmH2~ CnF2nll~CH=CH~CmF2m~li n+m~11, n+3-8, m=2-6) and fluorocarbon-hydrocarbon ether diblocks (e-g- CnF2n+l~~~CmH2mtl;
n+m~11, n=3-8, m=2-6).
Other suitable fluorocarbons may be selected from brominated perfluorocarbons such as l-bromo-heptadecaflurooctane (C~F17Br), sometimes designated perfluorooctyl bromide or "PFOB", now known by the U.S.
adopted name "perflubron~"); ~,~-dibromo-F-butane; 1-bromopenta-decafluoroheptane (C7Fl5Br); 1-bromo-nonafluorobutane (C~FgBr); and 1-bromotridecafluoroh~x~ne (C6F13Br, sometimes known as perfluorohexyl bromide or "PFHB").
Other brominated fluorocarbons are disclosed in U.S. Patent No. 3,975,512 to Long. It is also contemplated that fluorocarbons having nonfluorine substituents, such as perfluorooctyl chloride, orperfluorooctyl hydride may be used in the present invention as well as similar compounds having different numbers of carbon atoms, e.g., 2-8 carbon atoms.
Those skilled in the art will appreciate that esters, thioesters, ~m; nes, amides, and other variously modified fluorocarbon-hydrocarbon compounds are also ~ncomr~sed within the broad de$inition of "fluorocarbon" materials suitable for use in the present invention. Furthermore, forming the continuous phase from mixtures of fluorocarbons is also contemplated as being within the scope of the present invention.
Useful fluorocarbons may also be classified by other parameters. In one preferred embodiment, the fluorocarbon used in the continuous phase will have a critical solution temperature versus hexane (CSTH) of less than 10~C. In a particularly preferred embodiment, t:he selected fluorocarbon will have a CSTH of less than -20~C. In another preferred embodiment, the fluorocarbon will have a molar refractivity less than about 50 cm3 and, most preferably, less than about 40 cm3. In yet another preferred embodiment, the total chain length of the fluorocarbon (nlm) is less than nine, most preferably six or less. Indication of which fluorocarbons are particularly preferred may also be obtained by measuring the refractive index nD. In the emulsions of the present invention fluorocarbons having a re~ractive index greater than 1.34 are particularly preferred.
The continuous oily pha~e may also contain "no~mrh~philic" oils to increase its lipophilicity. Suitable oils include, for example, h~x~ne, triglycerides, Freons (e.g. Freon-113) and squalene. The continuous phase may also contain additives (e.g., perfluoropolyethers such as Fomblins~) designed to sterically stabilize the reverse emulsions. Controlled or directed deposition of the emulsion dispersed phase contents may be achieved by dilution with a less lipophilic oil phase. That is, a highly stable emulsion (stable for months) may be made to break in a matter of days or hours through the addition of a less lipophilic compound.
The process may be performed prior to delivery or in-situ. In a preferred embodiment the less lipophilic compound is added to an emulsion having prolonged storage stability just prior to administration. Such techniques may be advantageously used to control the delivery profile of the emulsion.

C. The Emulsifying Agent A particular advantage of the emulsions disclosed herein is the use of nonfluorinated surfactants for the formation of a polar liquid-in-perfluorocarbon emulsion or microemulsion.
All of the surfactants previously used for the formation of water-in-perfluorocarbon emulsions have been fluorinated.
There is no suggestion in the literature that water-in-fluorocarbon emulsions can be stabilized by hydrogenated surfactants. In sharp contrast to reported formulations, surfactants useful in the present invention include nonfluorinatedlipidicsurfactants. In preferred embodiments, these surfactants exhibit a geometry resembling an inverted truncated cone or wedge.
Surfactants are amphiphilic molecules that contain both a hydrophilic ~head group" and a lipophilic "tail". The surfactant preferably forms a monomolecular film at the fluorocarbon/polar liquid (water) interface. The stability of the emulsion is controlled by the spontaneous curvature of the resulting film. For stable water-in-fluorocarbon emulsions to form, the film must bend toward the water. For this to occur, surfactants chosen preferably have small head group areas and large tail volumes. Thus, uncharged (nonionic) surfactant headgroups are preferred. Similarly, W O 96/40057 PCT~US~6/0~064 increasing the degree of unsaturation in the suriactant tail area favors reverse emulsion formation. Accordingly, monosaturated tails (e.g. oleoyl) are particularly preferred.
Lysophospholipids cont~;n;ng a single lipid chain may also be used when complexed with a divalent cation.
In preferred embodiments, the reverse emulsion contains between o.O1~ and 10~ w/v of a nonfluorinated surfactant or mixture of surfactants. Because of their excellent biocharacteristics, phospholipids are generally the most preferred class of hydrogenated surfactants. More particularly, phospholipids that tend to adopt the reverse hexagonal phase at low temperatures and concentrations are favored. Accordingly, phosphatidylethanol~m; n~ and phosphatidic acids and the like are preferred. In particularly preferred embodiments, the phospholipid ha6 some molecular solubility in the continuous oily phase. Selected embodiments of the invention comprise, phosphatidic acids or phosphatidylethanolamines cont~;n;~g at least one mono-unsaturated fatty acyl moiety. Most preferably, the phosphatidic acid or phosphatidylethanolamine is 1,2-dioleoylphosphatidicor 1,2-dioleoylphosphatidylethanolamine respectively.
Other nonfluorinated surfactants suitable for use in the emulsions of the present invention include, but are not limited to, phosphatidylcholines, N-monomethyl-phosphatidylethanol~m;ne~, N,N-dimethyl- phosphatidyl-ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, cardiolipins, cholesterol, glyceroglycolipids, egg yolk phospholipids, salts of fatty acids, phosphatidylserines, phosphatidylglycerols, aminoethylphosphonolipid, dipalmitoyl phosphatidylcholesterol, ether-linked lipids and dicetylphosphate.

W O 96/40057 PCT~US96/09064 Conventional detergents with low hydrophilic-lipophilic balance (ca. 2-10) may also be used as urfactants. Such detergents include SPANS~ (sorbitan tetraoleate, sorbitan tetrastearate, sorbitan tristearate, sorbitan tripalmitate, sorbitan trioleate, and sorbitan distearate) and the BRIJ~
family (e.g. polyoxyethylene 2 stearyl ether). Guerbet alcohol ethoxylates, dialkyl nonionic surfactants and - dialkylzwitterionic surfactants including bet~;n~s and sulfobetaine~ are also contemplated for use as emulsifying agents. In addition, other additives which promote steric stabilization of reverse emulsions versus flocculation are anticipated. Preferred additives include block copolymers with low HBB.
Cosurfactants or surface active oils that decrease the spontaneous curvature of the resulting emulsion will ~nh~n~e its stability. Such additives include cholesterol, monoglycerides (e.g. monoolein), diglycerides (e.g. diolein), and alcohols (preferably long chain, e.g. oleoyl alcohol).
Because polar liquid-in-fluorocarbon droplets lack any electrostatic repulsive properties, the addition of lipophilic or fluorophilic steric stabilizers (e.g. polymers) is also contemplated. Such additives will help reduce emulsion flocculation and coalescence. Optionally, small amounts of fluorinated or nonfluorinated dialkyl cationic surfactant may be incorporated into the interfacial film to improve cell targeting in gene therapy applications.

D. Preparation o~ Emulsions The preparation of reverse emulsions involves the continuation of the nonfluorinated surfactant with the continuous fluorocarbon phase and discontinuous polar liquid phase. Preferably, the nonfluorinated surfactant is dispersed in the fluorocarbon prior to mixing with the polar liquid.
Emulsification requires large amounts of energy to convert a two-phase immiscible system into a disperse polar liquid phase comprising small discontinuous droplets in a continuous WO 96/40057 PCT~US9G/~9064 fluorocarbon phase. Emulsification may be achieved using techniques known in the art such as a low energy mixer, sonifier or high energy mechanical homogenizer. Following ~ormation the reverse emulsion may be added to a polar continuous phase to provide a multiple emulsion.
In sonication emulsification, a probe is inserted into a mixture comprising ~luorocarbon, emulsi~ier, aqueous phase and therapeutic or diagnostic agent. Bursts o~ energy are then released from the tip of the probe.
In a mechanical emulsi~ication process such as that per~ormed by a Microfluidizer~ apparatus (Microfluidics, Newton, MA), streams o~ the mixed emulsion components are directed through the apparatus at high velocity and under high pressure (e.g. 15,000 psi), and the high shear ~orces or cavitation resulting from the mechanical stress applied to the fluid mixture produces the emulsion.
It is thought the resulting emulsions consist o~ polar solvent droplets of water surrounded by a ~ilm of the surfactant, dispersed in the continuous fluorocarbon phase.
In selected embodiments this structure of polar liquid-in-per~luorocarbon emulsions has beencon~irmedbyphase-contrast optical microscopy using an emulsion incorporating a water soluble dye. Moreover, such emulsions can be easily diluted in a ~luorocarbon phase, but do not readily dilute in an aqueous phase.
The reverse emulsions o~ the present invention may be sterilized, ~or example, by autoclaving at 121~C for 15 minutes or by filtration through a 0.22 ~m ~ilter.
The polarliquid-in-fluorocarbon emulsions o~ the present invention may be ~m;n;stered in various ways, depending on the disorder to be treated. For example, intranasal or intraplllm~n~ry administration (i.e. endotracheal tube, pulmonary catheter), partial liquid ventilation, aerosolization, or nebulization is contemplated for treatment or respiratory disorders; systemic administration (i.e.
intramuscular, subcutaneous, intraperitoneal, oral) is W O 96/40057 PCTrUS96/09064 contemplated for treatment of sy6temic inflammation, infections (i.e. bacterial, viral, parasitic, fungal) and cardiovascular disease. Intraocular A~m; n; Btration is contemplated for treatment of intraocular disorders.
Additionally, both the multiple emulsions and reverse emulsions of the present invention may contain additives such as mineral salts, solvents and dispersants, buffer agents, ~ oncotic and osmotic agents, nutritive agents, hydrophilic or lipophilic pharmacologically active substanCes. The additives may be present in either the polar liquid phase, the (external) polar liquid phase, the oily phase, at the interface between the phases, or in any of the phases.
The multiple water-in-fluorocarbon-in-water emulsion of the present invention, which may be A~m; n; stered intravenously, may further comprise antibiotic, tuberculostatic, antimycobacterial, anticancer, mucolytic, antiviral, and ;mmllnoactive agents, pl-lmon~ry vasoactive substances, or genetic material as previously described.
Further multiple emulsion may be ~m; n; stered using a technique selected from the group consisting of topical, subcutaneous, plllmonAry, intramuscular, intraperitoneal, nasal, vaginal, rectal, aural, oral and ocular routes.
Preferred drugs that may be delivered using both multiple emulsions and reverse emulsions include anti-inflammatory agents (e.g., sodium Cromolyn, Tilade~), chemotherapeutics (e.g., cyclophosphamide, Lomustine~ (CCNU), Methotrexate, Adriamycin, Cisdiaminedichloroplatinum (cis-platin), antibiotics (Penicillins, Cephalosporins, Macrolides, Quinolones,Tetracyclines,Chloramphenicol,Aminoglycosides), surfactants and bronchodilators.
Preferred bronchodilators are classified as beta-2-agonists (i.e., Terbutaline, Metaproterenol sulfate, Epinephrine hydrochloride, Adrenaline, Isoprenaline, Salbutamol, Salmeterol, Albuterol, Formoterol);
anticholinergics (e.g., Ipratropium bromide, Oxitropium bromide), or glucocorticosteroids (i.e., Beclomethasone CA 02222063 l997-ll-24 WO96/40057 PCT~S96/09064 diprioprionate, Triamcinolone acetonide, Flunisolide, Fluticasone, Budesonide). Antineoplastics include adjuncts (eg., Ganite~, Zofran~); antibiotic derivatives (e.g., Doxorubicin hydrochloride, Idamycin~; systemic antibiotics (e.g., Amikacin sulfate, Gentamicin, Streptomycin sulfate, Cefonicid, Tobramycin); antimetabolites (e.g., sodium Methotrexate); and cytotoxic agents (e.g., Cis-Platin, Platinol-AQ, Taxol).
Cardiovascular agents compatible with the invention include ~/~ adrenergic blockers (e.g., Normodyne~, Trandate~); Angiotensin Converting Enzyme (ACE) inhibitors (e.g., Vasotec~); Antiarrhythmics (e.g., Adenocard~, Bretylol); Beta Blockers (e.g., Tenormin~); calcium ch~nnel blockers (e.g., Cardizem~); inotropic agents (e.g., Inocor Lactate); vasodilators (e.g., Papaverine hydrochloride); and vasopressors (e.g., Adrenalin chloride, Intropin).
In particularly preferred embodiments the disperse phase will comprise genetic material in the form of nucleic acid moieties such as DNA and RNA. Of course the genetic material may be incorporated in both reverse emulsions and multiple emulsions dep~n~;ng on the therapeutic or diagnostic strategy.
Those skilled in the art will appreciate that the present invention is particularly useful for the introduction and expression of selected genes or gene fragments when performing gene therapy. In particular, the emulsions of the present invention may be used to introduce genetic material in the form of cDNA, plasmids, expression vectors including viral vectors, mRNA, tRNA and anti-sense constructs to selected target sites. Exemplary target sites include pulmonary tissue, muscle tissue, lymphatic tissue, circulating cells including T-cells and B-cells and the cells of the gastrointestinal tract. It will further be appreciated that the foregoing lists are exemplary only and that the disclosed emulsions may be used to introduce genetic material anywhere in the organism.

WO 96/40057 PCT~US96/09064 Other drugs contemplated for use in the present invention include anesthetics (e.g., morphine sulfate), oph~h~lm;c agents (e.g., Polymyxin B sulfate, Neomycin sulfate, Gramicidin) and enzymes such as DNAse.
Selected embodiments of the present invention may be used to deliver antibiotics to combat infection. In particularly preferred embodiments, the reverse emulsions of the invention ~ may be used to deliver antibiotics to the lining of the upper gastrointestinal tract for treatment of ulcers. Increasing evidence indicates that a bacterium called Heliobacter pylori plays a central role in the etiology of several important gastroduodenal inflammatory and neoplastic processes (Blaser, M., in Principles and Practice of Infectious Disease, Fourth Edition, G.L. Mandell et al., eds., Churchill Livingstone, New York, pp. 1956-1964, 1995). Various antibiotics effective against H. pylori infections may be incorporated into the emulsions of the invention, including amoxicillin, nitrofurans, tetracyclines, aminoglycosides, imidazoles, macrolides and clarithromycin. Other effective compositions include bismuth salts (PEPTO-BISMOL~) and omeprazole, a hydrogen ion pump blocker. The resulting composition is administered orally to a patient in need of ulcer treatment.
In a preferred embodiment, three or four of these antibiotics are administered simultaneously for 10 to 14 days.
In addition to the previously described embodiments the reverse fluorocarbon emulsions of the invention may be added to perfluorooctyl bromide and other non-lipophilic fluorochemicals to create dispersions of an incorporated drug.
Preferably, the non-lipophilic fluorochemicals will have molar refractivity values greater than 40 cm3 while the lipophilic continuous phase has a molar refractivity value less than 40 cm3. The reverse emulsion breaks because the continuous phase is no longer lipophilic enough to stabilize the emulsion. In the resulting ~ormulation the discontinuous phase will pre~erably comprise solid microparticulates having average diameters on the order of 3 ~m or less, and more pre~erably WO 96/40057 PCT~US~6/O~OC
having average diameters signi~icantly less than 1 ~m. In particularly preferred embodiments the formed particulates have an average diameter less than 500 nm and may have average diameters on the order of ten of nanometers. The colloidal nature of the substantially homogeneous dispersions of the present invention provide ~nhAnced bioavailability due to their rapid dissolution at the target site.
Preparation o~ water-in-perfluorocarbon and multiple water-in-fluorocarbon-in-water emulsions are described in the following examples.

Example 1 PreParation of reverse water-in-~luorocarbon emulsion Ten milliliters of the ~ollowing reverse emulsion formulation was prepared:

1.0~ w/v egg phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL) go~ v/v ~,~-dibromo-F-butane (Ex Fluor, Austin, TX) 0.09~ sodium chloride (Sigma, St. Louis, MO) 0.09~ calcium chloride (Sigma) 10~ v/v water for injection.
Egg phosphatidylethanolamine (100 mg) was dispersed in ~,~-dibromo-F-butane (DBFB; 18 g) with a Vibracell~ sonicator (Sonics Materials, 30 mm o.d. titanium probe) at a power o~
100 watts for approximately l minute (T=5-10~C). An electrolyte solution (1.0 mL, 10~ v/v) was then added dropwise during sonication. A~ter the addition was complete, the reverse emulsion was sonicated for a total of not less than 10 minutes. The electrolyte solution contained 0.9~ w/v NaCl and o.9~ w/v CaCl2-2H2O. A milky water-in-~luorocarbon emulsion was obtained. Particle size of the emulsions was analyzed via laser light scattering on a Nicomp 270 photon correlation spectrometer (Pacific Scientific). Analysis was W O 96/400~7 PCTrUS96/09064 by the method of cumulants. Each emulsion sample was first diluted with n-octane since the refractive indices of the continuous and dispersed phases are nearly equal. The resulting reverse water-in-fluorocarbon emulsion had a mean droplet size of about 450~300 nm (Figure 1). The reverse character of the emulsion was established by con~lctivity and by stability after dilution with a hydrocarbon oil (i.e., n-octane).

Example 2 Effect of Nature of Phospholipid on Reverse Emulsions The emulsion formulation of Example 1 was modified by changing only the nature of the phospholipid in order to ~x~m; ne the ability of various phospholipids to stabilize reverse emulsions. The emulsification procedure and conditions were those described in Example 1. The results are shown in Table I.

WO 96/40057 PCT~US9G~

TABLE I
1"yl, ~ It Amount Droplet S.D.~ Creaming Emuision Ske TimeStability Mean (% wh) (nm) (nm) (min.) (Days) Egg Yolk Phua~ ' 1.0 530 10.0 3-7 Egg rhu.~ 'yluL~,anold" ,e 0.75 450 100 6.0 3-7 80% EPC/20% EPE 1.0 200 95 ~15.0 3-7 50% EPC/50% EPE 1.0 230 120 8.5 3-7 20% EPC/80% EPE 1.0 230 110 8.5 3-7 1,2 '", ' Jl,~JIlu~tlll ~' lyl~J -" ,e 1.0 210 100 14.0 2 101,2 ''~t JI~Jl.O ~' '- 'yl~,U. ' ~ ~e 1.0 200 110 10.0 2 Cardiolipin 1.0 150 6512.0 5 1,2', ' 11,ullu .~.l ~- l~l~ly_e,ul 1.0 180 857.5 2 1,2'' lùle~JI~JIIG~JI ~ lyls_.i"e 1.0 460 330 6.0 2 1,2' J4~1lùspl, ' Add 1.0 130 60~15.0 3 15 1,2 ~ JhUa~JII 'yl_Uhal,uld", ,~ 1.0 260 210 4.0 1,2 dilaL,,uJl~Jl,ost,l lyl~U,a"ul.l" ,e 1.0 435350 6.0 4 1,2 ' J.i,tuJI~.IIuapL lyl~L,d" ' ,~ 1.0 2440 840 1.0 2 1,2 ~ JI~Jho;."l ~- 'yluU~dl -'; ,e 1.0 450 100 ~15.0 Stable1,2 d- ' ~ bo5~1, 'yl-uhall~ ~e 0.8 530 250 4.0 Stable 1,2 ~ ' Jl~,hua~ll 'ylul ,~ 1.0 450 175 12.0 12 1,2 ~ 4~ho~pl, ' " Add 1.0 430 180 10.5 Stable 1,2'~ ~rl,ul,~ ,' ' ' Add 0.8 700 300 5.0 Stable 1,2 dialaullid~ IJl~hos~l, lyluUla" ' ,~ 0.8 240 85 ~15.0 1-palmitoyl, 2-oleoyl l,l,u.~l, ' lyLU,a~ ' ,e 1.0 500 110 8.0 Stable 251-palmitoyl, 2-r ,ol~u,l ~hu:r~l ~- 'ylulha,~ 1.0 320 175 7.0 4 ~S.D. - standard deviabion All phospholipids were obtained ~rom Avanti Polar Lipids, 30except ~or egg yolk phospholipid which was obt~in~ ~rom Kabi Pharmacia (Stockholm, Sweden). Particle size analysis was per~ormed using the identical procedure and conditions described in Example 1. Creaming times were determined spectrophotometrically by monitoring the percent transmittance 35of an emulsion-filled cuvette. The creaming time is the time required for the emulsion-~illed cuvette to go from 0~ to 100~
transmittance. All samples were stored at 30~C and monitored daily ~or emulsion stability (i.e., total phase separation).
It can be seen that the ~ormulations cont~;n;ng 40phospholipids with oleoyl ~atty acid moieties had improved properties when compared to any other phospholipid mixture or phospholipid molecular species. Additionally, the results W O 96/40057 PCT~US96/03C6 show that emulsions formulated with phospholipids having phosphatidylethanolamine or phosphatidic acid head groups exhibited enhanced stability. Both phosphatidic acid and phosphatidylethanolamine lipid systems have a strong t~n~ncy to form inverted, non-lamellar phases, where a tight headgroup packing i8 favorable. Phosphatidylcholines~
phosphatidylglycerols and phosphatidylserines, on the other hand, have a somewhat more expanded head group packing and tend to adopt lamellar phases.
Increasing both the ~h~;nlength and chain unsaturation lowers the lamellar (L~) to inverted hexagonal (H~) transition temperature. Thus, by increasing the ch~;nlength and chain unsaturation, an increased chain pressure results, which tends to decrease the monolayer spontaneous curvature (Ho)~
Increasing the unsaturation, e.g., 1,2-dioleoylphos-phatidylethanolamine to 1,2-dilinoleoylphosphatidyl-ethanolamine, further tends to impose more severe packing constraints that can lead to the formation of new undesirable phases. Therefore, the use of a phospholipid surfactant with mono-unsaturated, e.g., oleoyl, fatty acid moieties and/or ethanolamine or phosphatidic acid headgroups are preferred.

WO96/40057 PCT~S96/09064 Examplo 3 Effect of the Continuous Phase on Emulsion Stability Five mL of the following reverse emulsion ~ormulations were prepared:
0.5~ w/v egg yolk phospholipid (Kabi Pharmacia, Stockholm) 90~ v/v oil or oil mixture (see list below) 0.09~ v/v sodium chloride (Sigma) 0.09~ v/v calcium chloride (Sigma) 10~ v/v water for injection Reverse emulsions cont~;n;ng ~,~-dibromo-F-butane (DBFB), trichlorotrifluoroethane (CFC-113), perfluorooctyl bromide ~PFOB), n-hexane, per~luorohexane (PFH) and mixtures of each were prepared to ~x~m;ne the effect of the continuous phase on emulsion stability. The emulsification procedure and conditions described in Example 1 were followed. The emulsions were first checked visually to determine whether the oils were completely emulsified. The reverse character of the emulsion was established by stability after dilution with hydrocarbon oil, i.e., n-octane. Refractive indices of mixtures (nl2) were estimated via the procedure described by Taslc et al. (J. Chem. Eng. Data, 37:310-313, 1992). Emulsion stability generally correlated with the re~ractive index (nD) (Figure 2) of the continuous pha~e, whereby oils or oil mixtures with nD or n1z greater than approximately 1.32 formed stable reverse emulsions.
The preferred range of refractive indices will depend upon the nature of the surfactant. The use of preferred surfactants such as dioleoylphosphatidylethanolamine may, in fact, decrease the acceptable refractive index values.

W O 96/40057 PCT~US96/09064 Example 4 Effect of Molar Volume of the Continuous Phase on Reverse Emulsion StabilitY

Five mL of the reverse emulsion formulation described in Example 3 was prepared for each of the following oils: DBFB, CFC-113, PFOB, PFH, n-heY~ne~ n-heptane, n-octane, n-decane, n-dodecane, n-heptadecane, chloroform (CHCl3), c~hon tetrachloride (CCl~) and 1,6-dibromoh~Y~n~. The emulsification procedure and conditions described in Example 1 were followed. The emulsions were first checked visually to determine whether complete emulsification had occurred.
The reverse character of the emulsion was verified by dilution with hydrocarbon oil, i.e., n-octane. Emulsion stability was related to the molar volume, (VM), of the continuous phase (Figure 3), whereas oils with a VM of less than about 190 formed stable reverse emulsions. As mentioned above, the acceptable molar volume range for the continuous phase is critically dependent on the nature of the emulsifying agent.
In general, highly lipophilic fluorocarbons with low molar volumes are preferred.

Example 5 Reverse Emulsions Pre~ared with a PhosPholi~id/Nonpolar Li~id Combination The emulsion formulation of Example 1 was modified by changing the surfactant ingredients to ~m;ne the effect non-polar lipid additives have on properties of phospholipid-stabilized reverse emulsions. The emulsi~ication procedure and conditions described in Example 1 were followed. The phospholipid concentration was fixed at 1~ w/v and an .

W O 96/40057 PCTrUS~G~
additional 5, 10 or 2S mole~ non-polar lipid was incorporated.
The results are shown in Tables IIa to IIg.

Table lla (McnD~
5Primary Surfactant Amount Droplet Credl~l .9 Emulsion M~n~ ', Size SD~ rlme Stability Added Mean (Mole %) (nm) (nm) (min.) (Days) DOPC 5 380 170 5.0 30 DOPC 10 330 150 6.0 30 DOPC 25 250 115 >15.0 30 DOPE 5 600 195 9.0 Stable 1 0 DOPE 10 435 220 7.5 Stable DOPE 25 210 95 >15.0 Stable DOPA 5 400 200 15.0 Stable DOPA 10 500 125 14.5 Stable DOPA 25 165 100 >15.0 Stable Table llb (Diolein~
Primary SurfactantAmount Droplet Cr~dn lg Emulsion Diolein Size SD~ Time Stability Added Mean (Mole %) (nm) (nm) (min.) (Days) DOPC 5 225 120 11.0 12 DOPC 10 205 110 12.0 12 DOPC 25 230 130 15.0 12 DOPE 5 325 120 15.0 Stable DOPE 10 210 100 ~15.0 Stable DOPE 25 175 100 ~15.0 Stable 2 5 DOPA 5 790 300 5.0 Stable DOPA 10 860 425 7.0 Stable DOPA 25 470 165 >15.0 Stable 3 0 Table llc (Triolein) Primary SurfactantAmount Droplet Cr~dlll .9Emulsion Triolein Size SD~ rlme Stability Added Mean (Mole %) (nm) (nm) (min.) (Days) DOPC 5 360 170 9.0 12 DOPC 10 250 130 9.0 4 DOPC 25 280 155 ? 12 3 5 DOPE 5 670 390 5.0 Stable DOPE 10 470 210 10.0 Stable DOPE 25 675 200 12.0 Stable Table lld (Medium Chain T.iyly. erida) Primary SurfactantAmount Droplet Cr~d,. ,9 Emulsion MCT Oil Ske SD~Time Stability Added Mean (Mole %) (nm) (nm) (min.) (Days) DOPC 10 420 20014.0 10 DOPC 25 470 200N/D Stable DOPE 5 670 285~15.0 Stable DOPE 10 963 65010.0 Stable DOPE 25 830 62010.0 Stable DOPA 10 700 350>15.0 Stable DOPA 25 690 480~15.0 Stable Table lle (Cl--le ~.I) Primary SurfactantAmount Droplet Crt:dll ,9Emulsion Ct~- ul Ske SD~Time Stability Added Mean (Mole %) (nm) (nm) (min.) (Days) DOPC 25 255 14013.0 30 DOPE 25 170 100~15.0 Stable Table llf (~
Primary Sur~actantAmount Droplet Cledlll' 19Emulsion Sq~ ~ ,e Ske SD~Time Stability Added Mean (Mole %) (nm) (nm) (min.) (Days) 2 5 DOPC 10 200 10013.0 15 DOPE 10 300 160~15.0 Stable 3 0 Table llg (Long Chain Alcoho~e) Primary Sur~actant Long Droplet Cr~dlll ,9Emulsion Chain Ske SD~Time Stability Alcohol Mean (nm) (nm) (min.) (Days) DOPE Decyl Alcohol 220 110~15.0 Stable DOPE Oleoyl Alcohol 290 200~15.0 Stable ~S.D. - S~dllddll.] deY;d~iun~ N/D = not de~:", ,ed DOPC = 1 2 - JI~Jhos~ulldlidylul ,e DOPE = 1 2 ' '~ JI~ hospl Idlidyl~llldll~ldlll' ,e; DOPA = 1 2 d ~ hosuh Acid W O 96/40057 PCT~US9GlO~CCq DOPC, DOPE and DOPA were obtained ~rom Avanti Polar Lipids. Monoolein, diolein, decyl alcohol and oleoyl alcohol were obtained ~rom Nu-Chek Prep (Elysian, MN). Cholesterol, triolein and squalene were ~rom Sigma. Medium chain triglycerides (MCT) were ~rom Karlsh~mn~ (Janesville, WI).
Particle size analysis was per~ormed using the identical procedure and conditions described in Example 2.
Improved emulsion properties were observed with DOPE or DOPA in combination with monoolein, diolein, cholesterol, squalene, decyl alcohol or oleoyl alcohol. The improvements are noted by decreases in the initial droplet size, which is a measure o~ decreased coalescence in the system. Similar or A;m;n;shed emulsion properties were observed with any combination o~ DOPC, triolein or MCTs. In general, the emulsion characteristics improved with increasing nonpolar lipid content. ~onpolar components decrease the monolayer spontaneous curvature (Ho) by partitioning between phospholipid molecules, thereby increasing the hydrocarbon chain volume and/or relieving chain packing stress. The ine~ectiveness of triolein and MCTs to improve the stability o~ reverse emulsions is because they lack the amphiphilic nature required to partition into the sur~actant monolayer.
Triglycerides are simply dissolved in the ~luorocarbon oil.
There~ore, improved emulsion characteristics are obt~;neA
when a phospholipid cont~;n;ng reverse emulsion ~ormulation is supplemented with an amphiphilic non-polar additive.

Examplo 6 Pre~aration of enzyme containin~ reverse emulsion Reverse water-in-~luorocarbon emulsions cont~;n;n~ an enzyme were prepared with ~,~-di~romo-F-butane (90~ v/v), PULMOZYME~ (Genentech, South San Francisco, CA) (10~ v/v), 0.5~ o~ either egg phosphatidylethanolamine (PE) or egg yolk phospholipid containing at least 15~ w/w PE ~ollowing the procedure outlined in Example 1. Pulmozyme contains 1.0 mg/mL
dornase alfa enzyme in saline. The resulting reverse water-in-fluorocarbon emulsion cont~;n;ng enzyme was transparent with a mean droplet size of about 300 nm. The encapsulated enzyme was shown by an in vitro monocyte macrophage cell culture assay to retain its activity (i.e., entry into the nucleus, promotion of DNA breakdown and eventual cell death).
Decreases in ex vivo sputum viscosity collected from cystic fibrosis patients was observed after contact with reverse emulsions cont~;n;ng Pulmozyme.
Of course, it will be understood that the incorporation of polar liquid-soluble therapeutic or diagnostic agents can be accomplished by using an aqueous solution of the drug in place of the Pulmozyme when the emulsions or microemulsions are formed. In doing so, the concentration of the agent in the emulsion or microemulsion can be controlled simply be varying the concentration of drug in the aqueous solution.

Example 7 Pre~aration of reverse emulsions containinq a drua in the aaueous phase Three mL of the following reverse emulsion formulations cont~;n;ng drugs were prepared using the emulsification procedure and conditions described in Example 1.

A: Gentamicin sulfate reverse emulsion 0.051~ w/v Gentamicin sulfate (Sigma) 1.0~ w/v 1,2 Dioleoylphosphatidylethanolamine (DOPE; Avanti) 0.21~ w/v Di-olein (Nu-Chek Prep, Elysian, MN) 90~ v/v ~,~-Dibromo-F-butane (Exfluor) 0.09~ sodium chloride (Sigma) 0=09~ c-alril_m chloride (Sigma) 10~ v/v water for injection B: Cis-platin reverse emulsion 0.025~ w/v Cis-platin (Sigma) CA 02222063 l997-ll-24 WO96/400~7 PCT~S96/09064 1.0~ w/v 1,2 Dioleoylphosphatidylethanolamine 0.21% w/v Di-olein 90~ v/v ~,~-Dibromo-F-butane 0.09~ sodium chloride 0.09~ calcium chloride 10~ v/v water for injection C: Amikacin sul~ate reverse emulsion 0.052~ w/v Amikacin sul~ate (Sigma) 0.7~ w/v Egg PE (Avanti) 90~ v/v ~,~-Dibromo-F-butane 0.09~ sodium chloride 0.09~ calcium chloride 10~ v/v water ~or injection D: Terbutaline sulfate reverse emulsion 0.046~ w/v Terbutaline sul~ate (Sigma) 1.0~ w/v Egg yolk phosphatide (Asahi, Tokyo, Japan) 90~ v/v ~,~-Dibromo-F-butane 0.09~ sodium chloride 0.09~ calcium chloride 10~ v/v water for injection E: Tobramvcin sul~ate reverse emulsion 0.03~ w/v Tobramycin sulfate (Sigma, St. Louis, MO) 1.0~ w/v Egg Yolk phosphatide (Asahi, Tokyo, Japan) 90~ v/v ~,~-Dibromo-F-butane (Ex Fluor, Austin, TX) 0.09~ Sodium chloride (Sigma, St. Louis, MO) 10~ v/v Water ~or injection Particle size analysis was per~ormed using the procedure and conditions described in Example 1. Creaming times were measured using the procedure and conditions described in Example 2. The particle size and cre~m; ng rates ~or amikacin sul~ate and terbutaline sul~ate will most likely improve when ~ormulated with DOPE/di-olein sur~actant combination. A
slight improvement in particle size distribution was observed in gentamicin sul~ate containing emulsions compared to the vehicle (Figure 4). Table III shows the mean droplet diameters and initial creaming times o~ the ~ormulations.

CA 02222063 l997-ll-24 W 096/40057 PCT~US96~q Table III
Formulation Mean Droplet Initial CreA~ng Diameter Time (nm) (min.) Gentamicin Sulfate (0.051~w/v) 145 i 70 ~15.0 Tob. ~cin (0.03~ w/v) 125 i E~0 ~15.0 5Cis-platin (o. 025~ w/v) 200 i 100 ~15.0 Amikicin Sulfate (0. 052~ w/v) 600 i 250 4.0 Terbut~ n~ Sulfate (0.046~ w/v) 460 + 200 10.0 Example 8 Preparation of multiple emulsion (water / ~luorocarbon / water) Five mL of the following reverse emulsion ~ormulation was prepared using the identical emulsi~ication procedure and conditions described in Example 1:

1.0~ w/v 1,2 Dioleoylphosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL).
0.21 % w/v Di-olein (Nu-Chek Prep, Elysian, MN).
90~ v/v a,w-Dibromo-F-butane (Ex Fluor, Austin, TX).
0.09~ Sodium chloride (Sigma, St. Louis, MO).
0.09~ Calcium chloride (Sigma, St. Louis, MO).
10~ v/v Water ~or injection.

60 mg egg yolk phospholipid (EYP) (Kabi Pharmacia, Stockholm, Sweden) was dispersed in 2.4g. water for injection by sonication ~or approximately 2 minutes at 7"C. The reverse emulsion (1.2 g.) comprising the components listed above was then added dropwise to the EYP dispersion during sonication.
After the addition was complete, the multiple emulsion was the sonicated ~or an additional 15 minutes. A milky emulsion was obtained with no visible ~ree oil. The resulting multiple emulsion had a median particle size o~ 400 + 200 nm (centri~ugal sedimentation). The character o~ the emulsion continuous phase was established by conductivity and by being dispersible with water.
Example 9 Pre~aration of ethanol containinq reverse emulsion Five milliliters of the following reverse emulsion formulation was prepared using the identical emulsification procedure and conditions described in Example 1:
1.0~ w/v 1,2 Dioleoylphosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL).
0.21 ~ w/v Di-olein (Nu-Chek Prep, Elysian, MN).
90~ v/v ~,~-Dibromo-F-butane (Ex Fluor, Austin, TX).
0.09~ Sodium chloride (Sigma, St. Louis, MO).
0.09~ Calcium chloride (Sigma, St. Louis, MO).
2.5~ v/v Ethyl Alcohol (Spectrum, New Brunswick, NJ).
7.5~ v/v Water for in~ection.

The polar phase cont~;n;ng 25~ v/v ethyl alcohol was added to the surfactant/fluorocarbon dispersion as described in Example I. An opalescent reverse emulsion was obtained.
The resulting ethanol-in-fluorocarbon emulsion had a mean droplet size of 130 + 35 nm.
Example 10 In vitro efficacy of drua containinq reverse emulsions Five mL of the following drug cont~;n;ng reverse emulsions and emulsion vehicle were prepared as described in examples 1 and 7:

Formulation A: Gentamicin sulfate reverse emulsion formulation.

WO 96/40057 PCT~US9G/0~3 0.03~ w/v Gentamicin sulfate (Sigma, St. Louis, MO) 1.0~ w/v 1,2 Dioleoylphosphatidylethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL).
0.21 ~ w/v Di-olein (Nu-Chek Prep, Elysian, MN).
90~ v/v ~,~-Dibromo-F-butane (Ex Fluor, Austin, TX).
0.09~ Sodium chloride (Sigma, St. Louis, MO).
0.09~ Calcium chloride (Sigma, St. Louis, MO).
10~ v/v Water for injection.

Formulation B: TobramYcin sulfate reverse emulsion formulation.

0.03~ w/v Tobramycin sulfate (Sigma, St. Louis, MO) 1.0~ w/v Egg yolk phosphatide (Asahi, Tokyo, Japan).
90~ v/v ~,~-Dibromo-F-butane (Ex Fluor, Austin, TX).
0.09~ Sodium chloride (Sigma, St. Louis, MO).
0.09~ Calcium chloride (Sigma, St. Louis, MO).
10~ v/v Water for injection.

Formulation C: Reverse emulsion vehicle formulation.

1.0~ w/v 1,2 Dioleoylphosphatidylethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL).
0.21 ~ w/v Di-olein (Nu-Chek Prep, Elysian, MN).
90~ v/v ~,~-Dibromo-F-butane (Ex Fluor, Austin, TX).
0.09~ Sodium chloride (Sigma, St. Louis, MO).
0.09~ Calcium chloride (Sigma, St. Louis, MO).
10~ v/v Water for injection.
The drug emulsion formulations containing antibiotics and various controls were tested in an E.coli suspension culture for their antibacterial ability. To mimic the bacterial infection in the lung, the E.coli suspension culture was maintained in a well plate containing a monolayer CA 02222063 l997-ll-24 WO96/40057 PCT~S96/09064 of normal human bronchial/tracheal epithelial cells. Drug concentrations ranging from 0.3 to 0.003 mg in lOO~L were added to a 1 mL culture media cont~; n; ng the E.coli/cell suspension. Fluorocarbon and emulsion vehicle controls were added at levels that were proportional to levels present in the highest drug concentration samples. The plates were then incubated overnight at 37~C. Each well was aspirated and diluted with two parts LB media. The diluted culture mixture (20~L) was added to an LB plate and incubated overnight at 37~C for an initial titration of E.coli. Subsequent dilutions were made to determine the titer in each well. The results are shown in Table IV below.

T~3~E IV
Sample Treatment E.coli Titer (colonies/m~) No Treatment 6.3 E 7 Saline 7.0 E 7 20~,~-Dibromo-F-butane 1.0 E 7 Reverse Emulsion Vehicle (Formulation C)1.9 E 7 0.3mg Gentamicin Sul~ate in Saline o 0.03mg Gentamicin Sul~ate in Saline 1.0 E 1 0.3mg Gentamicin Sul~ate in Emulsion 2.0 E 2 25(Formulation A) 0.03mg G~nt~m; cin Sul~ate in Emulsion 2.7 E 3 (Formulation A) 0.003mg G~nt~m;cin Sul~ate in Emulsion 2.3 E 4 (Formulation A) 300.3mg Tobramycin Sul~ate in Saline o 0.03mg Tobramycin Sul~ate in Saline 2.0 E 1 0.3mg Tobramycin Sul~ate in Emulsion 1.5 E 1 (Formulation B) 0.03mg Tobramycin Sul~ate in Emulsion 7.0 E
35(Formulation B) 0.003mg Tobramycin Sul~ate in Emulsion 6.5 E 3 (Formulation B) The negative controls, i.e., saline, ~,~-Dibromo-F-butane, reverse emulsion vehicle or no treatment, all showed no ability to inhibit bacterial growth. The drug cont~; n; ng reverse emulsion formulations all ~mo~Rtrated equivalent antibacterial efficacy compared with their corresponding W O 96/40057 PCT~US96/09064 saline controls. In addition, a dose dependent response of antibacterial ability was observed for the two drugs evaluated. These results illustrate that the effectiveness of the drug was not inhibited by sUrfaCtant monolayer or fluorocarbon.

Example 11 Pre~aration of reverse water-in-fluorocarbon emulsions bv hiqh pressure homoqenization Fifteen milliliters of the following reverse emulsion formulation was prepared:

1.0% w/v egg phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL).
90% v/v ~,w-dibromo-F-butane (Ex Fluor, Austin, TX).
0.09~ w/v sodium chloride (Sigma, St. Louis, MO).
0.09~ w/v calcium chloride (Sigma, St. Louis, MO).
10~ v/v water for injection.

The surfactants, DBFB and saline solution were first dispersed by sonication following the procedure and conditions described in Example 1 or using an alternative low shear method. The low shear method was developed for use in dispersing process-sensitive pharmacological agents, such as DNA plasmid. In the low shear method the surfactants and the ~,w-dibromo-F-butane were dispersed with a low energy Te~mar Type SD-1810 mixer (Cincinnati, OH) at 10,000 revolutions/minute for approximately 1 minute. The dispersed phase was then added dropwise during mixing. After the addition was complete, the reverse emulsion was mixed for an additional minute. The sonicated or mixed emulsion was then further processed using an EmulsiFlex-CF homogenizer made by Avestin (Ottawa, Canada). The emulsions were homogenized using the following processing conditions: 10 passes at 12K

WO 96/40057 PCTrUS96/09064 p5i. Transparent water-in-l~luorochemical emulsions were obtained. Particle size analysis was done by laser diffraction (Horiba LA-700, Kyoto, Japan) in the volume weighted mode. Approximately a 20 to 50 ~L aliquot of each S sample was diluted in 9 to 10 mL of n-dodecane. The distribution shape "3", refractive index ratio of 1.1 and the fraction cell was used. The resulting reverse emulsions had mean droplet diameters of 200 + 70 nm and 205 i 70 nm respectively.
Bxample 12 Stabilit~ of Reverse Emulsions Prepared by Hiqh Pressure Homoqenization In this study several reverse emulsion formulations containing dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidic acid (DOPA) as the primary surfactant and 1,4 dibromofluorobutane (DBFB) as the oil for were evaluated for their particle growth and hydrolytic stability. The addition o~ non-polar additives such as cholesterol, monoolein, diolein and 1,3-diolein to DOPA and DOPE were evaluated for their effect. In addition, the stability o~ a gentamicin sulfate-in-DBFB emulsion was also ~X~m; ned~ The 1,4 dibromofluorobutane (DBFB), was obt~;ne~ from Exfluor Corp. Cholesterol, gentamicin sulfate were obtained from Sigma Chemicals. Dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidic acid (DOPA) were obtained from Avanti Polar Lipids. Monoolein, diolein and 1,3-diolein were obtained from NuChek Prep. All materials were used as received.
Fifteen milliliters ofthe followingemulsion formulation was prepared using the emulsification procedure and conditions described in Example 11.
Com~onent Concentration W O 96/40057 PCT~US96/09064 Saline Solution* 10~ v/v DBFB 90~ v/v DOPE or DOPA 1~ v/~
Non-Polar Additive 10 mole~ of Primary Surfactant (if included) * The saline solution consists of 0.9~ w/v NaCl, and 0.9~ w/v CaCl2--2HzO) .

In the case of the drug cont~;n;ng emulsion the gentamicin sulfate was dissolved in the saline solution prior to sonication. No attempts to exclude oxygen or control temperature was made during manufacturing or filling. Samples were stored and sealed in crimp cap vials at 5 ~ 25~C.
Reverse emulsion free fatty acid (FFA) concentration was determined using a Spectrophotometric method (Mahadevan, S., Dillard, C.J. and Tappel, A.L., Anal. Biochem.; 27 (1969) 387). Particle size analysis was performed using the identical procedure and conditions described in Example A. The results are shown in Tables Va through Vc.

W O 96/40057 PCT~US9G/03064 ~ABLE Va: 5~C

EmulsionlD Initial21d.30d. 49d. 63d. 84d. 114d. ISOd. 22Sd.

DOPA 305 3502404060761082409140 nld n/d DOPEl(i~ 150 150210 110 120120 110 110 120 DOPEI~ ' a' 166 200210 130 130130 IS0 IS0 120 1 0 DOPA/ ' ~ 216531035904920 54S084308030 n/d n/d DOPAtdioldn S968 50007220623072008300 8780 n/d nld DOPA11,3 diolein 304 33204890683073608510 8870 nld nld nld = not determined 1 5 TABLE Vb: 25-C

EmulsionlD 14d.21d.30d.49d. 63d. Md.84d.100d.114d. 150d. 22Sd.

DOPE 200190 230 190180 2S08000874013010Brol~e n/a 2 0 DOPA 370755064908210 823082S010340n/d 17400 nld nld DOPEIG- ~- 150 150170 150 1201608020817079S08110Brol~e DOPEI~ ' o' 160160 160 130130 140 150 130 130 6W0 nld DOPA/ ~ ~ 1905430 64308210 842085008830n/d 9800 n/d n/d DOPA/dioldn 3650 6600784081308440 86709210 n/d8720 n/d n/d 2 5DOPA11,3 dloldn 420630074408530 860088009840 n/d 9680 n/d n/d n/d = no~ determined TABLE Vc 3 0 Free Fatty Acid r - (mEq/L)vs. Time & Tem,oer ture DOPE DOPA DOPE/Gentamicin DOPE/Cholesterol DOPA/monolein DOPA/diolein DOPA/1.3dioleit Initial 4.4 4.8 4.25 4.71 5.15 4.92 5.88 5Mo. 5-C 5.26 7.94 6.59 6.28 6.55 7.99 10.01 3 55Mo. 25-C 7.35 10.27 7.12 6.75 8.08 8.27 12.07 W O 96/40057 PCT~US9~ .61 S;m;l~~ initial m~;~n particle diameters (ca. 150-300 nm) were observed for all high pressure ho,.,oy~ized reverse emulsion formulations except for DOPA/diolein. Reverse emulsions formulated with DOPE as the primary surfactant exhibited greater stability to coarsening and hydrolysis when comr~ed with DOPA at both 5~C and 25~C. The greatest reverse emulsion stability at 25~C was observed with the DOPE/cholesterol ~ormulation. No signi~icant particle growth has occurred in the DOPE reverse emulsion formulations stored at 5~C for 225 days.
The particle growth o~ reverse emulsions appears to occur in two phases. The first phase of growth is characterized by an order o~ magnitude change in m~ n particle diameter that occurs over a period of a few days to a week. After the rapid growth phase, the emulsion particle growth appears plateau off ~or an extended period and is followed by the breakage of the emulsion. The initial rapid growth phase is indicative of a coalescence driven process.
However, it is unclear at this point why the emulsion growth slows down. One possible explanation could be that the particle size measurements of these coarse (~l~m) reverse emulsions are inaccurate.

Example 13 Ef~ect of Dis~ersed Phase Volume on Reverse Emulsions The emulsion formulation and the emulsification procedure o~ Example 11 was modi~ied by changing the dispersed and continuous phase volumes. Reverse emulsions with dispersed phase volume percentages o~ 5, 10, 15, 20, 30, 40, and 50 were prepared. The dispersed phase 1,2, dioleoyl phosphatidylethanolamine (DOPE) concentration in all emulsion preparations was ~ixed at 1.34mM. Samples were sealed and stored in crimp cap vials at 25OC. Particle size analysis Wo96/40057 PCT~S~6/u~6 was performed using the identical procedure and conditions described in Example A (above). Viscosity measurements were performed with a Brookfield model DV-II viscometer at 37~C.
The results are shown in Table VI and Figure 5.

TAB~E VI
Median Particle Diameter (nm) vs. Time (days) 25-CDispe~sed Pbase v/v9~ al21 d.30 d. 60 d. 82 d. 105 d.

200 630011300bn~cnla n/a 180 1~,0 150 120 140 120 170 150 nld nld nld n/d 200 180 n/d n/d nld n/d n/d = not determined Viscosity measurements were not performed on the 40 and lS 50% v/v emulsions due to their high viscosity and insufficient sample volume. A dramatic decrease in droplet stability was observed when decreasing the dispersed phase concentration below 10%. As anticipated the emulsion viscosity increased with increasing dispersed phase volume.
By increasing the dispersed phase concentration the number of droplets per unit volume increases and thus the viscosity increases since the droplets are ~orced more and more into a close packed configuration. Therefore, by varying the dispersed phase volume the emulsion rheological characteristics can be controlled. Additionally, one might anticipate emulsion stability to coalescence (droplet growth) to be suppressed significantly simply by increasing the emulsion viscosity.

CA 02222063 l997-ll-24 WO96/40057 PCT~S96/09064 Example 14 Effect of Dispersed Phase on Reverse Emulsion StabilitY

The emulsion formulation and the emulsification procedure of Example 11 was modified by changing only the dispersed phase composition. Reverse emulsions cont~;n;ng with deionized water, various NaCl conc~ntrations (0.02, 0;1, 0.2M), 0.02M CaCl2 and 0.02M AlC13 were prepared. Particle size analysis was performed using the identical procedure and conditions described in Example 11. The results are shown in Table VII

TAB~E VII

Emulsion I.D. ~; ~n Particle Diameter (~m) DI Water 4.00 0.02M NaCl 0.52 O.lM NaCl 0.22 0.2M NaCl 0.14 0.02M CaCl2 0.22 0.02M AlCl3 0.25 Decreased emulsion particle diameters were observed as a function o~ NaCl concentration. Additionally, the results show that formula,tions cont~;n;ng either CaCl2 or AlCl3 produced emulsions with smaller particle size distributions when compared with a given NaCl concentration. Increasing the ion concentration o~ the dispersed phase lowers the lamellar (La) to inverted hexagonal (HII) transition temperature. It does so by reducing the hydration o~ the phosphate groups, which in turn promotes increased headgoup interactions and a decrease in monolayer spontaneous curvature (Ho)~ The ef~ect of di- and polyvalent ions on the phase behavior is quite complicated and not well understood.

WO 96/40057 PCTAUS~GI'~0~4 However, due to their low binding constants it has been shown that they can have a great e~ect at low concentration~
(Seddon, J.M., Biochem. Biophys. Acta, 1031 (1990) 1).
There~ore, the addition o~ small amounts o~ di- or polyvalent salts may be advantageous.

Example 15 Relationshl~ between continuous phase chemical ~roPerties and reverse emulsion stability:

Three mL o~ the ~ollowing reverse emulsion ~ormulation was prepared using the identical emulsi~ication procedure and conditions described in Example 1:

l~ w/v 1,2 dioleoyl phosphatidyleth~nn~m;ne (DOPE) (Avanti Polar Lipids, ~l~h~ter~ AL).
90~ v/v oil or oil mixture (see Table VIII).
0.09~ w/v sodium chloride (Sigma Chemicals) 0.09~ w/v calcium chloride (Sigma Chemicals).
10~ v/v water Reverse emulsions were prepared with a wide range o~
oils to determine i~ a correlation between the continuous phase physicochemical properties and emulsion stability could be made. The emulsi~ication procedure and conditions described in Example l were ~ollowed. The emulsions were ~irst checked visually to determine whether the oils were completely emulsi~ied. The reverse character of the emulsion was esta~lished by stability a~ter dilution with hydrocarbon oil, i.e., n-octane.
Table II lists the 34 oils ~m; ned and their respective molar volume (Vm), index o~ re~raction (nDZ~), ~-polarizability (~), molar re~ractivity (Rm)~ DOPE

W O 96/40057 PCT~US~6~5 solubility, bro~he~ne critical solution temperature (CSTBrH,X) and emulsion stability values. Oils saturated with DOPE were prepared by ~;n~ 50 to 600mg of DOPE into 2 mL oil and gently mixed at room temperature for 1 week.
The solutions were centrifuged at 4000 x g for 30 minutes, after which the DOPE saturated oils were lel.l~v~d by syringe. The DOPE content was determined by high performance liquid chromatography (HPLC) following a previously described method (Weers, J.G., Ni, Y., Tarara, T.E., Pelura, T.J., and Arlauskas, R.A., ~The Effect of molecular Diffusion on Initial Particle Size Distributions in Phospholipid-Stabilized Fluorocarbon Emulaions";
Colloids and Surfaces, 84 (1994) 81.). Samples were injected as neat solutions or after dilution in 2-propanol:h~ne {1:1 (v/v)}. Quantitation was made by reference to external DOPE st~n~rd curves. The nD20 values were measured using a hand refractometer when possible. The ~, Rm and nD'~ values c 1.34 were calculated using the group contribution-activity models proposed by Le and Weers (Le, T.D., and Weers, J.G., ~QSPR and GCA
Models for Predicting the Normal Boiling Points of Fluorocarbons"; J. Phys Chem, 99 (1995) 6739. Le, T.D., and Weers, J.G., "Croup Contribution-Additivity and Quantum Mechanical models for Predicting the Molar Refractions, Indices of Refraction, and Boiling Points of Fluorochemicals~'; J. Phys Chem, 99 (1995) 13909.). The CSTBr~eX values were taken from Le et al. technical report dated 22 September 1995. Emulsion stability was defined as the time necessary for the emulsion to completely break. Formulations cont~;n;ng oils which did not produce a stable W/O dispersion upon sonication were noted as ~unstable".
Emulsion stability strongly correlated with the continuous phase nD20 (lipophilicity) and it's DOPE-- 35 solubility (Table VIII). No correlation could be made CA 02222063 l997-ll-24 W O 96/40057 PCTAJS96~S~61 with ~, Rm ,Vm, or CST~3rHex Stable reverse emulsion ~ormation occurred with oils having nD2D greater than approximately 1.34 and with oils in which DOPE was soluble. The emulsion stability decreased sharply over a small range o~ oil nD20 as evi~nc~ when comparing the stability o~ the DBFH (ca. 3 days) and DBFB (ca. 60 days) reverse emulsions. The continuous phase requirements (i.e., nD20) will also depend upon the surfactant(s) and/or the disperse phase composition. The use o~ co-sur~actants such as cholesterol and/or the addition o~ polyvalent ions such as AlCl3 may, in ~act, decrease the required continuous phase nD20 values. The results are presented in TABLE VIII immediately below.

~ ~3 e, A A A A ~ A A A 8 ~ A ~ ~ 8 ~ ~ 8 _I - ~ 2 2 2 2 2 2 2 2 ~ 2 2 ~ 2 2 ~ 2 E
8. ~ ~ ~ ~ ~ ~ o ~ ~ ~ o '~ Cq H O
E

~ ~ o ~ O ~_ .

~~ -- '~ ~ ''' ~; ~ ~ J o O o O ~ s ~ ~ ~ ~ ~ 8 ~ 8 ~ ~ m -- ; ~ 5 8 ~ ~ 8 ~ ~ u y .7 D 81 "

W O 96/40057 PCT~US96/'0506 ~ =0 ~ g 'f9 ~ '~ ~0 0 0 o o ~ ~ -- ~

2 ~ 'o -, 3~ 2 ~
' _ o ~ ,., ", ~ _ _ o o ~

:~: o o ~ 8 ~ c~ c~ ~. ~ ~ ~ ~ ' .

. ~ ~ ~ ~ ~ o~ ~ g ~ ~ 8 c ~ ' s E ~ o ~ ~ ~ ~~ ~ ~ ~ 8 u E

W O 96/40057 PCT~US96/09064 Example 16 Effect of Continuous Phase Index of Refraction on Reverse Emulsion Lifetime Five milliters of the following reverse emul~ion formulation was prepared using the identical emulsification procedure and conditions described in Example 1:

1~ w/v 1,2 dioleoyl phosphatidylethanoamine (DOPE) (Avanti Polar Lipids, Alabaster, AL).
90~ v/v oil or oil mixture (see Table IX).
0.09~ w/v sodium chloride (Sigma Chemicals).
0.09~ w/v calcium chloride (Sigma Chemicals).
10~ v/v water Emulsion lifetime and particle size distribution were determined as a function of Perflubron/DBFB ratio. Each emulsion formulation was first checked visually to determine .whether the polar phase r~ml~ne~ e.m..ul~ified for at lGa~t hour. The reverse character of the emulsion was established by stability after dilution with hydrocarbon oil, i.e., n-octane.
Emulsions with Perflubron/DBFB ratios less then 0.55 were extremely unstable and broke immediately. Samples were sealed and stored in crimp cap vials at 25~C. Particle size analysis was performed using the identical procedure and conditions described in Example 11. The emulsion lifetime, T, iS defined as the time for the emulsion to completely break. Formulations containing oils which did not produce a stable W/O dispersion upon sonication were noted as "unstablen. Table IX shows the nl2, median droplet diameter and lifetime of the emulsion in hours for the Perflubron/DBFB
ratios ~mi ned~ Refractive indices of mixtures (n12) was CA 02222063 l997-ll-24 WO96/40057 PCT~S96/09064 estimated via the procedure described by Taslc et al. (J.
Chem. Eng. Data, 37:310-313, 1992).

Table IX
Stability of reverse emulsions ~ormulated with binary oil mixtures XPFOB PFOB/DBFB l~edi n EmulsionLifetime ~ Di meter(~m)t (houls) 0.000 1.351 0.41 3816 0.100 1.346 0.55 3816 0.200 1.340 0.63 2040 0.300 1.335 0.71 528 150.400 1.330 0.81 264 0.500 1.325 1.50 24 0.550 1.322 10.50 O.oOO 1.320 uost~blc uns~ble 0.700 1.314 unsslble unstllble 200.800 1.309 unstable unst~ble 0.850 1.307 unsblble uns~ble 1.299 uust~ble ulls~ble The value o~ T was ~ound to correlate well with both the continuous phase nl2 and the droplet diameter, which is a re~lection o~ changes to the monolayer spontaneous curvature (Ho) due to the degree o~ penetration o~ the continuous phase molecules into the sur~actant "brush". The value of Ho decreases as a ~unction of the continuous phase lipophilicity or nlz. In the vicinity o~ the emulsion breakage point, ca. nl2 = 1.320, a sharp increase in the emulsion stability occurs over a small change of nl2. This effect has been observed for other sur~actants (~h~lnov~ A., and Weers, J., "Macroemulsion Stability Within the Winsor III
Region: Theory Versus Experiment:"; Langmuir, 12 (1996) 1931). Moreover, these results qualitatively agree well with CA 02222063 l997-ll-24 Wo96/40057 PCT~S96/09064 current emulsion stability theories relating emulsion stability to changes in the monolayer spontaneous curvature (Kabalnov, A., and Wennerstrom, H., " Macroemulsion Stability: The Orientated Wedge Revisited"; Langmuir, 12 (1996) 276). The emulsion stability or lifetime was also found to correlate well with the emulsion m~ n particle size and overall distribution. Emulsions with poor stability, T 5 24h, had broad particle distributions with large median diameters. As the DBFB/Perflubron ratio increased the distributions narrowed and the median diameter decreased.
The dilution of a stable reverse emulsion with a less lipophilic oil (i.e., perflubron) results in the rapid coarsening and the breakage of the emulsion. This coarsening and breakage process is a reflection of the decreased penetration of the continuous phase molecules into the surfactant "brush". The ability to control the stability of an emulsion through the lipophilicity of the continuous phase (nD) could be used to direct the deposition of the dispersed phase components.
Although the invention has been described with reference to particular preferred em~odiments, the scope of the invention is defined by the following claims and should be construed to include reasonable equivalents.

Claims (39)

WE CLAIM:

1. A fluorocarbon pharmaceutical formulation comprising:
a disperse liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent;
a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant.

2. The formulation of claim 1 wherein said formulation is a thermodynamically stable microemulsion.

3. The formulation of Claim 1 further comprising one or more disperse liquid phase additives selected from the group consisting of mineral salts, buffers, stabilizers, oncotic and osmotic agents and nutritive agents.

4. The formulation of Claim 1, wherein said at least one lipophilic fluorocarbon is selected from the group consisting of halogenated perfluorocarbons, halogenated perfluoroethers, halogenated polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbon ether diblocks and mixtures thereof.

5. The formulation of Claim 4, wherein said lipophilic fluorocarbon is a halogenated perfluorocarbon and halogenated perfluorocarbon is .alpha.,.omega.-dibromo-F-butane.

6. The formulation of Claim 1, wherein said at least one nonfluorinated surfactant is selected from the group consisting of alcohols, salts of fatty acids, phosphatidylcholines, N-monomethyl-phosphatidylethanolamines, phosphatidic acids, phosphatidyl ethanolamines, N,N-dimethyl-phosphatidyl-ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, egg yolk phospholipids, cardiolipins, glycerglycolipids, phosphatidylserines, phosphatidylglycerols and aminoethylphosphonolipids.

7. The formulation of Claim 1, wherein said nonfluorinated surfactant has a low hydrophilic lipophilic balance.

8. The formulation of Claim 7, wherein said nonfluorinated surfactant is selected from the group consisting of SPANS~, BRIJs~, guerbet alcohol ethoxylates, dialkyl nonionic surfactants and dialkylzwitterionic surfactants.

9. The formulation of Claim 1, wherein said nonfluorinated surfactant is a phospholipid comprising a monounsaturated fatty acid moiety.

10. The formulation of Claim 9, wherein said phospholipid is selected from the group consisting of dioleoyl phosphatidylethanoamine, dioleoylphosphatidic acid and combinations thereof.

11. The formulation of Claim 1 wherein said at least one polar liquid soluble therapeutic or diagnostic agent is selected from the group consisting of respiratory agents, antibiotics, anti-inflammatories, antineoplastics, anesthetics, imaging agents, ophthalmic agents, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins and combinations thereof.

12. The formulation of Claim 1, further comprising an additive capable of decreasing the spontaneous curvature of the emulsion.

13. The formulation of Claim 12, wherein said addative is selected from the group consisting of monoglycerides, diglycerides, alcohols, sterols, triglycerides, alkanes, Freons~, squalene, and mixtures thereof.

14. The formulation of Claim 2, wherein said at least one lipophilic fluorocarbon is selected from the group consisting of halogenated perfluorocarbons, halogenated perfluoroethers, halogenated polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbonether diblocks and mixtures thereof.

15. The formulation of Claim 2, wherein said at least one nonfluorinated surfactant is selected from the group consisting of alcohols, salts of fatty acids, phosphatidylcholines, N-monomethyl-phosphatidylethanolamines, phosphatidic acid, phosphatidyl ethanolamine, N,N-dimethyl-phosphatidyl-ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, egg yolk phospholipids, cardiolipins, glycerglycolipids, phosphatidylserines, phosphatidylglycerols and aminoethylphosphonolipids.

16. The formulation of Claim 2, wherein said polar liquid soluble therapeutic or diagnostic agent is selected from the group consisting of respiratory agents, antibiotics, anti-inflammatories, antineoplastics, anesthetics, imaging agents, ophthalmic agents, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins and combinations thereof.

17. A method for generating a therapeutic or diagnostic formulation comprising:
providing a liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent;
combining said liquid phase with an effective emulsifying amount of at least one nonfluorinated surfactant and a fluorocarbon phase comprising at least one lipophilic fluorocarbon to provide an emulsion formulation; and emulsifying said emulsion formulation to produce a therapeutic or diagnostic formulation.

18. The method of claim 17 wherein said therapeutic or diagnostic formulation is thermodynamically stable.

19. The method of claim 17 wherein said polar liquid is selected from the group consisting of water, alcohols, polyethylene glycols, alkyl sulfoxides, and mixtures thereof.

20. The method of claim 17 wherein said at least one lipophilic fluorocarbon is selected from the group consisting of halogenated perfluorocarbons, halogenated perfluoroethers, halogenated polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbon ether diblocks and mixtures thereof.

21. The method of claim 17 wherein said at least one polar liquid soluble therapeutic or diagnostic agent is selected from the group consisting of respiratory agents, antibiotics, anti-inflammatories, antineoplastics, anesthetics, imaging agents, ophthalmic agents, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins and combinations thereof.

22. The method of claim 17 wherein said at least one nonfluorinated surfactant is selected from the group consisting of alcohols, salts of fatty acids, phosphatidylcholines, N-monomethyl-phosphatidylethanolamines, phosphatidic acids, phosphatidyl ethanolamines, N,N-dimethyl-phosphatidyl-ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, egg yolk phospholipids, cardiolipins, glycerglycolipids, phosphatidylserines, phosphatidylglycerols and aminoethylphosphonolipids.

23. The method of claim 17 further comprising an additive capable of decreasing the spontaneous curvature of the emulsion.

24. A therapeutic or diagnostic formulation prepared according to the method of claim 17.

25. A therapeutic or diagnostic formulation prepared according to the method of claim 18.

26. A therapeutic or diagnostic formulation prepared according to the method of claim 22.

27. A method for delivering a therapeutic or diagnostic agent to a patient comprising:
providing a pharmaceutical emulsion comprising a disperse liquid phase, said liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent; a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant; and administering said pharmaceutical emulsion to a patient.

28. The method of claim 27 wherein said pharmaceutical emulsion is a thermodynamically stable microemulsion.

29. The method of claim 27 wherein said at least one nonfluorinated surfactant is selected from the group consisting of alcohols, salts of fatty acids, phosphatidylcholines, N-monomthyl-phosphatidylethanolamines, phosphatidic acids, phosphatidyl ethanolamines, N,N-dimethyl-phosphatidyl-ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, egg yolk phospholipids, cardiolipins, glycerglycolipids, phosphatidylserines, phosphatidylglycerols and aminoethylphosphonolipids.

30. The method of claim 27 wherein said at least one polar liquid soluble therapeutic or diagnostic agent is selected from the group consisting of respiratory agents, antibiotics, anti-inflammatories, antineoplastics, anesthetics, imaging agents, ophthalmic agents, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins and combinations thereof.

31. The method of Claim 27, wherein said pharmaceutical emulsion is administered to the patient using a delivery device, said delivery device selected from the group consisting of an endotracheal tube, an intrapulmonary catheter, and a nebulizer.

32. The method of Claim 27, wherein said pharmaceutical emulsion is administered to a patient using a route of subcutaneous selected from the group consisting of topical, subcutaneous, pulmonary, intramuscular, intraperitoneal, nasal, vaginal, rectal, aural, oral and ocular routes.

33. A process for the preparation of a multiple emulsion comprising the following steps:
a) providing a liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent;
b) combining said liquid phase with an effective emulsifying amount of at least one nonfluorinated surfactant and a fluorocarbon phase comprising at least one lipophilic fluorocarbon to provide an emulsion formulation;
c) emulsifying said emulsion formulation to produce a therapeutic or diagnostic reverse emulsion;
d) adding said therapeutic or diagnostic reverse emulsion to a second polar liquid comprising an effective emulsifying amount of at least one nonfluorinated surfactant to provide a multiple formulation wherein said second polar liquid is the same or different than said polar liquid; and e) emulsifying said multiple formulation to produce a multiple emulsion.

34. The method of Claim 28, wherein said polar liquid soluble therapeutic or diagnostic agent is selected from the group consisting of respiratory agents, antibiotics, anti-inflammatories, antineoplastics, anesthetics, imaging agents, ophthalmic agents, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins and combinations thereof.

35. A method of preparing a pharmaceutical dispersion comprising: providing a disperse liquid phase comprising at least one polar liquid and at least one polar liquid-soluble therapeutic or diagnostic agent; a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant; and combining said reverse emulsion with a non-lipophilic fluorocarbon to form a dispersion.

36. The method of Claim 35, wherein said polar liquid soluble therapeutic or diagnostic agent is selected from the group consisting of respiratory agents, antibiotics, anti-inflammatories, antineoplastics, anesthetics, imaging agents, ophthalmic agents, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins and combinations thereof.

37. The method of Claim 35, wherein said non-lipophilic fluorocarbon is selected from the group consisting of brominated fluorocarbons, chlorinated fluorocarbons, and F-alkanes.

38. The method of claim 35 wherein said at least one nonfluorinated surfactant is selected from the group consisting of alcohols, salts of fatty acids, phosphatidylcholines, N-monomethyl-phosphatidylethanolamines, phosphatidic acids, phosphatidyl ethanolamines, N,N-dimethyl-phosphatidyl-ethanolamines, phosphatidyl ethylene glycols, phosphatidylmethanols, phosphatidylethanols, phosphatidylpropanols, phosphatidylbutanols, phosphatidylthioethanols, diphytanoyl phosphatides, egg yolk phospholipids, cardiolipins, glycerglycolipids, phosphatidylserines, phosphatidylglycerols and aminoethylphosphonolipids.

39. A fluorocarbon formulation comprising:

a disperse liquid phase comprising at least one polar liquid;
a continuous fluorocarbon phase comprising at least one lipophilic fluorocarbon; and an effective emulsifying amount of at least one nonfluorinated surfactant.