KR19990022354A - Reverse fluorocarbon emulsion composition for drug delivery - Google Patents

Abstract

The present invention relates to polar liquid emulsions or microemulsions in perfluorine chemical reagents for use in the delivery of therapeutic or diagnostic agents. This composition is formed by blending a discontinuous aqueous phase, a continuous fluorocarbon phase and a non-fluorinated surfactant. In addition, polar liquid emulsions in fluorine chemical reagents can be used to form complex emulsions with an aqueous continuous phase. Such emulsions and microemulsions are suitable for administering medicaments including genetic material.

Description

Reverse fluorocarbon emulsion composition for drug delivery

Fluorocarbons, fluorine substituted hydrocarbons and perfluorocarbons, fluorocarbons in which all hydrogen atoms have been replaced with fluorine, have been widely used in the medical field as therapeutics and diagnostics. Such liquids are transparent, colorless, odorless, nonflammable, and nearly water insoluble. In addition, fluorocarbon liquids are denser than water and soft tissue, have low surface tension, and in most cases have low viscosity.

Fluorocarbons have desirable properties including biocompatibility, relatively low reactivity, and high oxygen entrainment. Brominated fluorocarbons have been found to exhibit radiopacity for any form of radiation. For example, US Pat. No. 3,975,512 to Long uses fluorocarbons, including brominated perfluorocarbons, as contrast enhancing media in radiomedical imaging. Commercially available fluorocarbon emulsion FLUOSOL® (Green Cross Corp., Osaka, Japan) has been used as an oxygen carrier during percutaneous lumen passage coronary angiogenesis. Fluorocarbon emulsions have also been used in diagnostic imaging applications, including nuclear magnetic resonance and ultrasound (US Pat. No. 5,114,703). Pure perfluorocarbons have also been used in the medical field. Imagent® GI, an FDA-approved diagnostic agent consisting of pure perfluorooctyl bromide (PFOB), is used to image the gastrointestinal pathway. Perfluorocarbons are also used in the ophthalmology field to treat large retinal tears (Aguilar et al., Retina, 15: 3-13) and are evaluated for use during liquid ventilation.

Therapeutic use of fluorocarbons as described above would have a greater advantage if used in combination with other agents or diagnostic agents. For example, poor blood circulation in the diseased part of the lung in general treatment of lung disease reduces the efficacy of drug delivery. However, pulmonary delivery of the biologic through the alveolar surface can be facilitated when done with liquid ventilation (see Wolfson et al., FASEB J., 4: A1105, 1990). Lung drug administration has been found to increase the biological response of some drugs when compared to intravenous administration (see Shaffer et al., Art. Cells, Blood Sub. Immob. Biotech., 22: 315, 1994).

Drug administration of the lung is also used for the treatment and / or diagnosis of diseases including respiratory distress syndrome (RDS), impaired pulmonary circulation, cystic fibrosis and lung cancer. The increased efficacy of drug delivery of the lungs through liquid ventilation can be attributed to the high electrodeposition coefficient of perfluorocarbons on the lung surface, an increase in alveolar surface area due to more effective lung swelling, and delivery of oxygen by perfluorocarbons.

The main problem associated with perfluorocarbon mediated drug delivery is that drugs are often insoluble on fluorocarbons. Conventional drug administration of the lungs includes preparation of crude dispersions of the drug and delivery by turbulence and spraying. Unfortunately, not all drugs can be delivered in this way.

Water inverse emulsions in perfluorocarbons have already been prepared using perfluorinated surfactants. The ability to stabilize these anti-emulsions with non-fluorinated biocompatible surfactants (ie, phospholipids) would provide an advantage.

Therefore, there is a need for a description of compositions and methods that can deliver fluorocarbon-coupled polar liquid soluble therapeutics and diagnostic agents in an efficient and reliable manner. The present invention focused on providing these needs with polar liquid emulsions, complex emulsions and microemulsions in fluorocarbons stabilized by biocompatible phospholipids or hydrogenated surfactants.

Summary of the Invention

The present invention provides a stable reverse (polar liquid in fluorocarbon) emulsion and a thermodynamically stable reverse microemulsion in a fluorocarbon continuous phase for delivery of a polar liquid soluble agent. Such emulsions overcome many of the disadvantages associated with heterogeneous crude drug dispersions in fluorocarbons. The present invention also provides a stable complex (polar liquid in fluorocarbons in polar liquids) emulsions.

Therefore, in a broad aspect the present invention

A dispersed liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent;

A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And

It consists of a fluorocarbon agent containing an effective amount of at least one non-fluorinated surfactant.

Another aspect of the invention relates to a thermodynamically stable formulation.

Another aspect of the invention

Providing a liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent;

Combining the liquid phase with a fluorocarbon phase consisting of an effective amount of one or more non-fluorinated surfactants and one or more lipophilic fluorocarbons to provide an emulsion formulation;

A method of making a therapeutic or diagnostic formulation comprising emulsifying the emulsion formulation to provide a therapeutic or diagnostic formulation.

Another aspect of the invention

A dispersed liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And a pharmaceutical emulsion containing an effective amount of one or more non-fluorinated surfactants;

A method of delivering a therapeutic or diagnostic agent to a patient, comprising administering the pharmaceutical emulsion to the patient.

In another embodiment, the reverse emulsions described above can be used to form water complex emulsions in fluorocarbons in water. Specifically, the reverse emulsion is dispersed in an aqueous solution containing at least one non-fluorinated surfactant. The non-fluorinated surfactant may be the same or different from that used to initially form the reverse emulsion.

The process for preparing the complex emulsion consists of the following steps:

a) providing a liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent;

b) combining the liquid phase with an fluorocarbon phase consisting of an effective amount of at least one non-fluorinated surfactant and at least one lipophilic fluorocarbon to provide an emulsion formulation;

c) emulsifying the emulsion formulation to provide a therapeutic or diagnostic reverse emulsion;

d) adding the therapeutic or diagnostic inverse emulsion to a second polar liquid which contains an effective amount of one or more non-fluorinated surfactants and which is the same as or different from the polar liquid; And

e) emulsifying the complex formulation to provide a complex emulsion.

In such complex emulsions, the external aqueous phase is continuous while the reverse emulsion is discontinuous. The combination emulsion may further contain inorganic salts, solvents, dispersants, buffers, carcinogenic agents, osmotic agents, nutritional agents, hydrophilic agents and lipophilic agents. These additives may be in the inner or outer aqueous phase, in the perfluorocarbon phase or at the interface. As used herein, a medicament is an agent that provides therapeutic or diagnostic utility when treating a patient.

Another aspect of the invention

A dispersed liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And an anti-emulsion having an effective emulsifying amount of at least one non-fluorinated surfactant;

It relates to a method for producing a pharmaceutical dispersion consisting of combining the reverse oil agent with a non-lipophilic fluorocarbon to form a dispersion.

Finally, in a broad aspect the present invention relates to a formulation containing a fluorine chemical reagent emulsion.

Such preparations

A dispersion liquid phase consisting of at least one polar liquid;

A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And

At least one non-fluorinated surfactant in an effective amount of emulsification.

In this preferred embodiment, the dispersed liquid phase consists of water, alcohols, alkyl sulfoxides, polyethylene glycols or mixtures thereof. In a particularly preferred embodiment, the alcohol is a short chain alcohol such as ethanol and the alkyl sulfoxide is dimethyl sulfoxide.

Preferably, the lipophilic fluorocarbons are halogenated fluorocarbons, halogenated perfluoroethers / polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbon ether diblocks or mixtures thereof. Advantageously, the halogenated perfluorocarbons are α, ω-dibromo-F-butane.

In addition, the fluorocarbon phase may further contain one or more additives capable of increasing the lipophilic phase of the fluorocarbon phase. Such additives are preferably non-surface active oils such as medium chain triglycerides, long chain triglycerides, silanes, silicone oils, hydrocarbons, freons, alkanes, squalene, fluorocarbon-hydrocarbon diblocks and lipophilic short chain fluorocarbons. Other surface active oils may be added to reduce the spontaneous curvature of the surfactant monolayer. Examples thereof include cholesterol, monoglycerides, diglycerides, long chain alcohols and sterols. Preferably, the fluorocarbons are brominated, chlorinated or iodide fluorocarbons.

According to another preferred embodiment, the therapeutic or diagnostic agent is a respiratory agent, antibiotic, anti-inflammatory drug, chemotherapeutic agent, anti-tumor drug, anesthetic, eye drop, cardiovascular agent, contrast agent, enzyme, nucleic acid, genetic protein or viral vector.

In a preferred embodiment, the non-fluorinated surfactant is alcohol, fatty acid salt, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, phosphatidic acid, phosphatidyl ethanolamine, N, N-dimethyl-phosphatidylethanolamine, phosphatidyl ethylene glycol, phosphatidyl Selected from the group consisting of methanol, phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol, phosphatidylthioethanol, dipitanoyl phosphatide, egg yolk phospholipid, cardiolipin, glyceroglycolipid, phosphatidylserine, phosphatidylglycerol and aminoethylphosphonolipide do. Preferably, the non-fluorinated surfactant contains at least one monounsaturated moiety. In a particularly preferred embodiment, the non-fluorinated surfactant is 1,2-dioleoylphosphatinic acid or 1,2-dioleoylphosphatidyl ethanolamine.

Advantageously, the non-fluorinated surfactant can have a low hydrophilic lipophilic balance. Such surfactants include SPANS®, BRIJs®, guerbet alcohol ethoxylates, dialkyl nonionic surfactants and dialkyl zwitterionic surfactants. . The emulsion may further contain surface active oils which can reduce spontaneous curvature of the surfactant film. Preferably, the surface active oil is monoglycerides, diglycerides, long chain alcohols or sterols.

Another embodiment of the invention is the administration of an emulsion to a patient. Those skilled in the art will understand that the emulsion of the present invention may be administered to a patient using a delivery device. Preferably, the delivery device is selected from the group consisting of intratracheal tubes, intrapulmonary catheter and nebulizer. It will also be appreciated that the present invention is particularly suitable for pulmonary delivery using partial liquid ventilation and spray injection methods.

In another embodiment, the present invention can be used to deliver a medicament. Preferably, the therapeutic or diagnostic agent incorporated is an antibiotic such as amoxicillin, nitrofuran, tetracycline, aminoglycosides, macrolides or clarithromycin. In selected embodiments, the infectious agent is Heliobacter pylori or Mycobacterium tuberculosis.

The present invention relates to a composition for delivery of therapeutic and diagnostic agents. More specifically, the present invention relates to polar liquid emulsions, complex emulsions and microemulsions in perfluorine chemical reagents.

1 is a photon of an inverse emulsion containing 1.0% w / v egg phosphatidylethanolamine, 90% v / v α, ω-dibromo-F-butane, 0.09% sodium chloride, 0.09% calcium chloride and 10% water It is particle size distribution obtained by correlation spectroscopy (PCS). The emulsion particle size is shown on the x-axis and the relative volume is shown on the y-axis.

2 shows the effect of the continuous phase refractive index (n _D ) on the reverse emulsion stability. Inverse emulsions containing α, ω-dibromo-F-butane (DBFB), trichlorotrifluoroethane (CFC-113), n-hexane, perfluorohexane (PFH) and mixtures thereof were analyzed. Volume fractions are shown on the x-axis and n _D is shown on the y-axis.

3 shows the effect of continuous phase molar volume (V _M ) on reverse emulsion stability. The continuous oil used for the reverse emulsion is shown on the x-axis and V _M is shown on the y-axis.

4 shows 1.0% w / v 1,2-dioleoylphosphatidylethanolamine, 0.21% w / v diolein, 90% v / v α, in the absence (Δ) and presence (0.0) of 0.051% gentamicin sulfate, Particle size distribution obtained by PCS of an inverse emulsion containing ω-dibromo-F-butane, 0.09% sodium chloride, 0.09% calcium chloride and 10% water is shown. Emulsion particle size is shown on the x-axis and relative volume is shown on the y-axis.

5 is a function of shear rate obtained from α, ω-dibromobutane inverse emulsion formulated in dispersed phases 5 (□), 10 (Δ), 15 (○), 20 (■) and 30 (●) vol%. The viscosity is shown. Dispersed phase 1,2-dioleoylphosphatidylethanolamine, sodium chloride and calcium chloride concentrations were set to 1.34 mM, 0.9% w / v and 0.9% w / v, respectively. Shear velocity is shown on the x-axis and emulsion viscosity is shown on the y-axis.

As noted above, the present invention provides a stable reverse (polar liquid in fluorocarbon) emulsion and a thermodynamically stable reverse microemulsion in a fluorocarbon continuous phase for delivery of polar liquid soluble drugs. The emulsions of the present invention overcome many of the disadvantages associated with heterogeneous crude drug dispersions in fluorocarbons. The present invention also provides a stable complex (polar liquid in fluorocarbons in polar liquids) emulsions, and methods of forming pharmaceutical nanoparticles.

In a preferred embodiment, the reverse emulsion or microemulsion system is a dispersed aqueous phase containing at least one polar liquid soluble therapeutic agent and / or a diagnostic agent, a continuous phase consisting of at least one fluorocarbon and at least one non-fluorinated interface Consisting of an active agent. In addition, fluorocarbons may contain one or more solutes that can increase the lipophilic on the fluorocarbons. As will be appreciated by those skilled in the art, a complex emulsion (liquid phase-fluorocarbon-liquid phase) can be prepared by combining the reverse emulsion formed with the continuous aqueous phase.

The main difference between microemulsions and conventional emulsions is thermodynamic stability. Given the calibration temperature, pressure and composition, the microemulsion will form spontaneously and will not thicken over time. Microemulsions are formed from components substantially the same as conventional emulsions, but the relative amounts of the disperse phases are generally less than conventional emulsions. Typically, the dispersed phase in the microemulsion will comprise less than 10% v / v, most preferably less than 5% v / v of the total emulsion volume, depending on its components.

The microstructure of the emulsion is preferably defined as a surfactant monolayer membrane at the water-oil interface. As will be appreciated by those skilled in the art, the term water is generally not limited to aqueous solutions when discussing emulsions. An important property of the surfactant film is its tendency to bend towards water or oil. The tendency of this surfactant film to be curved depends on, among other factors, the surfactant geometry (ie head group area, hydrocarbon tail chain length and volume), the penetration of the oil into the hydrocarbon tail of the surfactant and the degree of hydration of the hydrophilic head group. It can be explained quantitatively by spontaneous curvature (H ₀ ) which is an inherent property of the surfactant film. The signals and values of spontaneous curvature not only indicate that the formed emulsion exhibits a normal (oil in water) or reverse (water in oil) dispersion phase, but also indicates the extent to which it remains stable. Spontaneous curvature is considered positive if the membrane is likely to be curved towards the oil phase (o / w emulsion) and negative if the membrane is likely to be curved towards the aqueous phase (w / o emulsion).

In such compositions, emulsifiers or surfactants may be selected based on their geometry. In other words, surfactants having a small head group area and large tail volume (ie, inverted truncated or wedge shaped) are advantageous. Surface active oils may be added to the surfactant system to reduce spontaneous curvature of the surfactant monolayer. Examples thereof include monoglycerides and alcohols, in particular long chain alcohols, sterols and diglycerides. Certain inorganic salts may be added to reduce surfactant monolayer spontaneous curvature through the promotion of tight head group filling. Examples thereof include calcium, magnesium and aluminum salts.

Another embodiment of the invention relates to the formation of a substantially heterogeneous colloidal dispersion of a medicament in an non-lipophilic fluorocarbon, such as perfluorooctyl bromide. Other non-lipophilic fluorocarbons suitable for the present invention include perfluorooctyl chloride, F-octane and the like. As used herein, the term non-lipophilic refers to perfluorine chemical reagents having a relatively low lipophilicity. Preferred non-lipophilic fluorocarbons suitable for use in colloidal dispersions generally contain 6 or more carbon atoms. The colloidal dispersion preferably has particles having an average diameter of less than 3 μm, more preferably less than 1 μm. Particularly preferred embodiments consist of particles having an average diameter of less than 500 nm, in particular less than 100 nm. In selected embodiments, the reverse emulsion of the present invention is further combined with non-lipophilic liquid fluorocarbons. Because of the physical differences between inverse emulsions and non-lipophilic fluorocarbons, the agent undergoes a phase change to form an effective dispersion.

A. Discontinuous phase

In a preferred embodiment, the discontinuous (dispersed) phase contains at least one polar liquid for drug solubilization. While many polar liquids are suitable for the technology of the present invention, particularly preferred embodiments include water, short chain alcohols, dimethylsulfoxide, polyethylene glycol or mixtures thereof. In another preferred embodiment, the volume of the dispersed phase comprises about 0.05% to 70% of the total volume of the emulsion.

The disperse phase is also an inorganic salt, buffer, stabilizer, swelling and penetrating agent, nutritional agent, active ingredient, pharmaceutical active substance, genetic material, or other ingredients designed to enhance its various properties, including the stability, therapeutic efficacy and resistance of emulsions. It may contain. In a particularly preferred embodiment, the dispersed phase may consist of nucleic acid components such as RNA or DNA. The disperse phase may also include ions selected to stabilize the emulsion or encapsulated drug. For example, when the interfacial layer contains phosphatidylglycerol or phosphatidic acid, the emulsion stability can be increased by adding calcium or magnesium ions to the aqueous phase. In another example, any enzyme (eg, DNase) may retain more activity when certain ions are included for stabilization.

The disperse phase may contain additives (eg, long chain polar alcohols such as butanol) designed to inhibit Ostwald ripening (irreversible coarsening) in a reverse emulsion. To further improve the solubility of any drug in the emulsion of the present invention (e.g. Taxol®), ethanol, polyethylene glycol, water soluble Pluronics® or dimethylsulfoxy The lifting may be added in part or in whole to the dispersed phase. In order to further improve the stability to coalescence of the reverse emulsion, the emulsion viscosity can be increased by increasing the volume of the dispersed phase.

A composite water-oil-water emulsion comprising the reverse emulsion, dispersed in the form of globules in a continuous second polar liquid phase, may be expected. Such complex emulsions can be prepared by adding an inverse emulsion to a second polar liquid phase in which the at least one fluorinated or non-fluorinated surfactant is dispersed. The amount of surfactant used to form the complex emulsion will depend on the amount of polar liquid and inverse emulsion used. Generally, for polar liquid phases comprising 60% to 99.95% v / v inverse emulsions, the amount of surfactant used is from about 0.01% to about 10% w / v of the aqueous phase. Surfactants currently known in the art as good emulsifiers for oil emulsions in water can be used here. Examples thereof include phosphatidylcholine, egg yolk phospholipids and pluronics. The external polar liquid continuous phase may contain such additives as well as polar solvents including, for example, glycol, glycerol, dimethylformamide or dimethyl sulfoxide. These additives may be present in the second polar liquid phase or the oil phase, at the interface between the two phases or in both phases.

As described in more detail below, the emulsions of the present invention may deliver certain polar liquid soluble therapeutics and / or diagnostic agents. Preferred agents include antibiotics, antiviral agents, anti-inflammatory drugs, respiratory agents, genetic materials, anti-tumor drugs, anesthetics, contrast agents, eye drops and cardiovascular agents.

B. Continuous Phase

In a preferred embodiment, the reverse emulsion of the present invention contains about 40% to 99.95% v / v of a continuous oil phase consisting of one or more lipophilic fluorinated or perfluorinated organic compounds. The continuous fluorocarbon phase may consist of one or more fluorocarbons, perfluorocarbons or perfluorocarbon-hydrocarbon mixtures. Preference is given to highly lipophilic fluorocarbons which facilitate the dispersion of hydrocarbon surfactants in the fluorocarbon continuous phase. Generally, such lipophilic fluorocarbons contain halogen atoms (chlorine, bromine or iodine) or hydrocarbon components (eg C ₂ H ₅ ). In another preferred embodiment, the fluorocarbons contain up to eight carbon atoms. In a particularly preferred embodiment, the fluorocarbons contain 4 to 6 carbon atoms. The fluorocarbon molecules used in this emulsion may have a variety of structures, including straight or branched or cyclic structures, as described in Riess, J., Artificial Organs, 8 (1): 44-56, 1984. have.

There are many fluorocarbons that can be used in the present invention. Such fluorocarbons include halogenated perfluorocarbons (e.g., C _n F _{2n + 1} X, XC _n F _2n X where n is 2 to 8 and X is Cl, Br or I), Halogenated ethers or polyethers (e.g., XC _n F _2n OC _n F _2n X, XCF ₂ OCF ₂ CF ₂ OCF ₂ X, where n is 2 to 4 and X is Cl, Br or I), Fluorocarbon-hydrocarbon diblocks (eg, C _n F _{2n + 1} -C _m H _{2m + 1} , C _n F _{2n + 1} -CH = CH-C _m F _{2m + 1} ; n + m _&lt; 11, n = 3-8, m = 2-6) and fluorocarbon-hydrocarbon ether diblocks (eg, C _n F _{2n + 1} -OC _m H _{2m + 1} ; n + m <11, n = 3 -8, m = 2-6).

Other suitable fluorocarbons are brominated perfluorocarbons, for example 1-bromo-heptadecafluorooctane (C ₈ F ₁₇ Br), sometimes referred to as perfluorooctyl bromide or PFOB, currently US tradename Perflubronn known as perflubron®; α, ω-dibromo-F-butane; 1-bromopenta-decafluoroheptane (C ₇ F ₁₅ Br); 1-bromo-nonafluorobutane (C ₄ F ₉ Br); And 1-bromotridecafluorohexane (C ₆ F ₁₃ Br, sometimes known as perfluorohexyl bromide or PFHB). Other brominated fluorocarbons are described in US Pat. No. 3,975,512 to Long. Fluorocarbons having non-fluorine substituents such as perfluorooctyl chloride or perfluorooctyl hydride can be used in the present invention, as well as similar having other numbers of carbon atoms, for example 2 to 8 carbon atoms. It is anticipated that compounds may also be used.

Those skilled in the art will understand that esters, thioesters, amines, amides and other various modified fluorocarbon-hydrocarbon compounds are encompassed within the broad definition of fluorocarbon materials suitable for use in the present invention. It is also contemplated that forming a continuous phase from a mixture of fluorocarbons is within the scope of the present invention.

Useful fluorocarbons may be classified by other parameters. In one preferred embodiment, the fluorocarbons used in the continuous phase will have a critical solution temperature (CSTH) for hexanes of less than 10 ° C. In a particularly preferred embodiment, the selected fluorocarbons will have a CSTH of less than -20 ° C. In another preferred embodiment, the fluorocarbon will have a molar refractive index of less than about 50 cm 3, most preferably less than about 40 cm 3. In another preferred embodiment, the total chain length (n + m) of the fluorocarbons is less than 9, most preferably 6 or less. Indicating that fluorocarbons are particularly preferred may be obtained by measuring the refractive index n _D. In the emulsions of the invention, fluorocarbons having a refractive index in excess of 1.34 are particularly preferred.

The continuous oil phase may contain an amphiphilic oil to increase its hydrophilicity. Examples of suitable oils include hexane, triglycerides, freons (eg Freon-113) and squalene. The continuous phase may also contain additives (eg perfluoropolyethers such as Fomblins®) designed to stericly stabilize the reverse emulsion. Controlled or directed deposition of the emulsion dispersed phase content may be achieved by dilution with a less lipophilic oil phase. That is, high stability emulsions (stable for months) can be made to degrade within days or hours through the addition of less lipophilic compounds. This process can be performed prior to delivery or on site. In a preferred embodiment, less lipophilic compounds are added to the emulsion with extended storage stability just prior to administration. Such techniques can be advantageously used to adjust the delivery profile of the emulsion.

C. Emulsifier

A particular advantage of the emulsions described herein is the use of nonfluorinated surfactants for the formation of polar liquid emulsions or microemulsions in perfluorocarbons. All surfactants already used for the formation of water emulsions in perfluorocarbons were fluorinated. There is no literature published that water emulsions in fluorocarbons can be stabilized by hydrogenated surfactants. Significantly different from the reported agents, surfactants useful in the present invention include non-fluorinated lipid surfactants. In a preferred embodiment, these surfactants exhibit a geometry similar to an inverted truncated cone or wedge.

Surfactants are amphiphilic molecules containing both hydrophilic head groups and lipophilic tails. Surfactant Preferably, a monomolecular film is formed at the fluorocarbon / polar liquid (water) interface. The stability of the emulsion is controlled by the spontaneous curvature of the formed membrane. To form a water emulsion in stable fluorocarbons, the membrane must be curved towards water. In order to cause this phenomenon, the selected surfactants preferably have a small head group area and a large tail volume. Thus, uncharged (nonionic) surfactant head groups are preferred. Likewise, an increase in the degree of unsaturation in the surfactant bottoms predicts the possibility of reverse tanning. Thus, monofolled tails (eg oleoyl) are particularly preferred. Lysophospholipids containing a single fatty chain may also be used when complexed with divalent cations.

In a preferred embodiment, the reverse emulsion contains 0.01% to 10% w / v of a non-fluorinated surfactant or surfactant mixture. Because of their good biological properties, phospholipids are generally the most preferred class of hydrogenated surfactants. More particularly, phospholipids that are easy to select inverted hexagonal phases at low temperatures and concentrations are preferred. Thus, phosphatidylethanolamine, phosphatidic acid and the like are preferred. In a particularly preferred embodiment, the phospholipid has some molecular solubility in the continuous oil phase. Selected embodiments of the invention consist of phosphatidic acid or phosphatidylethanolamine containing one or more monounsaturated fatty acyl moieties. Most preferably, the phosphatidic acid or phosphatidylethanolamine is 1,2-dioleoylphosphatidic acid or 1,2-dioleoylphosphatidylethanolamine, respectively.

Other non-fluorinated surfactants suitable for use in the emulsions of the present invention include, but are not limited to, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, N, N-dimethyl-phosphatidylethanolamine, phosphatidyl ethylene glycol, phosphatidylmethanol , Phosphatidyl ethanol, phosphatidyl propanol, phosphatidyl butanol, phosphatidyl thioethanol, dipitanoyl phosphatide, cardiolipin, cholesterol, glyceroglycolipid, egg yolk phospholipid, fatty acid salt, phosphatidylserine, phosphatidylglycerol, aminoethyl phosphonolipide, Dipalmitoyl phosphatidylcholesterol, ether bound fats and dicetylphosphate.

Conventional detergents with low hydrophilic-lipophilic balance (about 2-10) may be used as surfactants. Such detergents include SPANS® (sorbitan tetraoleate, sorbitan tetrastearate, sorbitan tristearate, sorbitan tripalmitate, sorbitan trioleate and sorbitan distearate). And BRIJ® (for example, polyoxyethylene 2 stearyl ether). Guerbet alcohol ethoxylates including betaines and sulfobetaines, dialkyl nonionic surfactants and dialkyl zwitterionic surfactants may also be considered for use as emulsifiers. Also contemplated are other additives that promote steric stabilization of the anti-emulsion to aggregation. Preferred additives include block copolymers having low HLB.

A cosurfactant or surface active oil that reduces the spontaneous curvature of the emulsion formed will enhance its stability. Such additives include cholesterol, monoglycerides (eg monoolein), diglycerides (eg diolein) and alcohols (preferably long chains such as oleoyl alcohols). Because of the lack of any electrostatic repulsion properties in polar liquid droplets in fluorocarbons, addition of lipophilic or fluorofluoric steric stabilizers (eg polymers) is also envisaged. Such additives will help to reduce reverse tanning and coalescence. Optionally, small amounts of fluorinated or non-fluorinated dialkyl cationic surfactants can be incorporated into the interface membrane to improve cell targeting in gene therapy regimens.

D. Preparation of Emulsions

The preparation of the reverse emulsion involves the linking of a continuous fluorocarbon phase and a discontinuous polar liquid phase with a non-fluorinated surfactant. Preferably, the non-fluorinated surfactant is dispersed in fluorocarbons before mixing with the polar liquid. Emulsification requires a large amount of energy to convert an ideal immiscible system into a dispersed polar liquid phase consisting of small discrete droplets in a continuous fluorocarbon phase. Emulsification can be accomplished using techniques known in the art such as low energy mixers, sonicators or high energy mechanical homogenizers. Following formation, the reverse emulsion can be added to the polar continuous phase to provide a composite emulsion.

In ultrasonic emulsification, the probe is inserted into a mixture of fluorocarbons, emulsifiers, aqueous phases and therapeutic or diagnostic agents. Thereafter, an energy explosion is released from the tip of the probe.

In a mechanical emulsification process such as that performed by a Microfluidizer® device (Microfluidics, Newton, Mass.), The flow of mixed emulsion components is at high pressure (eg, 15,000 psi) and emulsion It is directed through the device under cavitation or high shear forces formed from the mechanical stresses applied to the fluid mixture forming the.

The emulsion formed is believed to consist of a polar solvent droplet of water surrounded by a surfactant film, dispersed on a continuous fluorocarbon. In selected embodiments, this structure of the polar liquid in the perfluorocarbon emulsion was confirmed by phase-controlled optical microscopy using an emulsion incorporating a water soluble dye. In addition, such emulsions can be readily diluted on the fluorocarbon phase, but not easily on the aqueous phase.

Inverse emulsions of the invention can be sterilized, for example, by autoclaving at 121 ° C. for 15 minutes or by filtration through a 0.22 μm filter.

Polar liquid emulsions in the fluorocarbons of the present invention can be administered in several ways, depending on the condition to be treated. For example, intranasal or pulmonary administration (ie, endotracheal tube, pulmonary catheter), partial liquid ventilation, misting or spraying are contemplated for the treatment of respiratory diseases and systemic administration (ie, intramuscular, subcutaneous, intraperitoneal, Oral) is contemplated for the treatment of systemic inflammation, infection (ie bacterial, viral, parasitic, fungal) and cardiovascular disease. Intraocular administration is contemplated for the treatment of intraocular diseases.

In addition, both complex and reverse tanning agents of the present invention may contain additives such as inorganic salts, solvents and dispersants, buffers, swelling and penetrating agents, nutritional agents, hydrophilic or lipophilic pharmacologically active substances. This entry agent may be present in either the polar liquid phase, the (outer) polar liquid phase, the oil phase, at the interface between the phases or in any phases.

Water complex emulsions in fluorocarbons in water of the invention, which can be administered intravenously, are antibiotics, Mycobacterium tuberculosis inhibitors, antimycobacterial agents, anticancer agents, mucolytic agents, antiviral and immunoactivators, pulmonary vascular substances or the above It may contain one more genetic material. Combination emulsions can also be administered using techniques selected from the group consisting of topical, subcutaneous, pulmonary, intramuscular, intraperitoneal, nasal, vaginal, rectal, ear, oral and ocular routes.

Preferred drugs that can be delivered using both combination and reverse tanning agents include anti-inflammatory drugs (eg sodium chromoline, Tilade®), chemotherapy drugs (eg cyclophospha) Meade, Lomustine® (CCNU), methotrexate, adriamycin, cisdiaminedichloroplatinum (cis-platin), antibiotics (penicillin, cephalosporin, macrolide, quinolone, tetracycline, chloramphenicol , Aminoglycosides), surfactants and bronchodilators.

Preferred bronchodilators include beta-2-agonists (ie terbutalin, metaproterenol sulfate, epinephrine hydrochloride, adrenaline, isoprenin, salbutamol, salmeterol, albuterol, formoterol); Choline inhibitors (e.g. ifpratropium bromide, oxytropium bromide), or glucocorticosteroids (ie beclomethasone, dipriopionate, triamcinolone acetonide, flunisolide, fluticasone, budesonide Are classified as). Antitumor drugs include adjuvants (eg, Ganite®, Zofran®); Antibiotic derivatives (eg doxorubicin hydrochloride, idamycin); Systemic antibiotics (eg, amikacin sulfate, gentamycin, streptomycin sulfate, cenysid, tobramycin); Antimetabolites (eg sodium methotrexate); And cytotoxic agents (e.g., cis-platin, platinum-AQ, taxol).

Cardiovascular agents suitable for the present invention include α / β adrenergic blockers (eg, Normodyne®, Trandate®); Angiotensin converting enzyme (ACE) inhibitors (eg, Vasotec®); Antiarrhythmic drugs (eg, Adenocard®, Bretylol); Beta blockers (e.g., Tenormin®); calcium channel blockers (e.g., Cardizem®); modulating agents (e.g., inocor lac Ate), bronchodilators (such as papaverine hydrochloride), and vasopressors (such as adrenaline chloride, intropine).

In a particularly preferred embodiment, the dispersed phase will contain the genetic material in the form of nucleic acid components such as DNA and RNA. Of course, the genetic material may be incorporated into both reverse tanning and combination tanning depending on the therapeutic or diagnostic scheme. Those skilled in the art will understand 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 can 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 locations include lung tissue, muscle tissue, lymphoid tissue, circulating cells, including T-cells and B-cells, and cells of the gastrointestinal pathway. It will also be appreciated that the above list is exemplary only and that the genetic materials described may be used to introduce genetic material anywhere in the organ.

Other drugs that can be used in the present invention include enzymes such as anesthetics (eg morphine sulfate), eye drops (eg polymyxin B sulfate, neomycin sulfate, gramicidine) and DNAse.

Selected embodiments of the invention can be used to deliver antibiotics to inhibit infection. In a particularly preferred embodiment, the reverse emulsion of the invention can be used to deliver antibiotics to the inner surface of the upper gastrointestinal route for the treatment of ulcers. There is increasing evidence that bacteria called Heliobacter pyrroli play a major role in the pathogenesis of several important gastroduodenal inflammations and tumor processes (Blaser, M., in Principles and Practice of Infectious Disease, Fourth Edition, GL Mandell et al. , eds., Churchill Livingstone, New York, pp. 1956-1964, 1995]. H, including amoxicillin, nitrofuran, tetracycline, aminoglycosides, imidazoles, macrolides and clarithromycin. Various antibiotics effective against pyrroli infections can be incorporated into the emulsions of the present invention. Other effective compositions include bismuth salts (PEPTO-BISMOL®), omeprazole, and hydrogen ion pump blockers. The resulting composition is administered orally to patients 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 above embodiments, the reverse fluorocarbon emulsion of the present invention may be added to perfluorooctyl bromide and other non-lipophilic fluorochemical reagents to form a dispersion of the incorporated drug. Preferably, the non-lipophilic fluorine chemical reagent will have a molar refractive index of greater than 40 cm 3 while the lipophilic continuous phase will have a molar refractive index of less than 40 cm 3. The reverse emulsion breaks because the continuous phase is no longer lipophilic enough to stabilize the emulsion. In the formulations formed, the discontinuous phase will preferably consist of solid ultrafine particles having an average diameter of about 3 μm or less, more preferably an average diameter of significantly less than 1 μm. In a particularly preferred embodiment, the formed fine particles have an average diameter of less than 500 nm and may have an average diameter on the order of 10 nm. The colloidal properties of the substantially heterogeneous dispersions of the present invention provide improved bioavailability due to their rapid dissolution at the target site.

The preparation of water emulsions in perfluorocarbons and water complex emulsions in fluorocarbons in water will be described in the following examples.

Example 1

Preparation of Water Station Emulsion in Fluorocarbon

10 ml of the following reverse tanning formulations were prepared:

1.0% w / v egg phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL)

90% v / v α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

0.09% calcium chloride (Sigma)

10% v / v distilled water for injection

100 watts of egg phosphatidylethanolamine (100 mg) using a Vibracell® sonicator (Sonics Materials, 30 mm od titanium probe) for about 1 minute (T = 5-10 ° C.) In α, ω-dibromo-F-butane (DBFB; 18 g). Thereafter, the electrolyte solution (1.0 mL, 10% v / v) was added dropwise during sonication. After the addition was complete, the counteremulsion was sonicated for at least 10 minutes in total. The electrolyte solution contained 0.9% w / v NaCl and 0.9% w / v CaCl ₂ .2H ₂ O. A milky emulsion of water in fluorocarbons was obtained. The particle size of the emulsion was analyzed by laser light scattering on a Nicomp 270 photon correlation spectrometer (Pacific Scientific). The analysis was done by cumulative method. Each emulsion sample was initially diluted with n-octane because the refractive indices of the continuous and disperse phases were about the same. The water inverse emulsion in the fluorocarbons formed had a median droplet size of about 450 ± 300 nm (FIG. 1). The inverse properties of the emulsions were confirmed by conductivity and stability after dilution with hydrocarbon oil (ie n-octane).

Example 2

Effect of Phospholipid Types on Inverse Emulsions

The emulsion formulation of Example 1 was modified by only changing the phospholipid type to test the ability of the various phospholipids to stabilize the reverse tanning. Emulsification procedures and conditions are as described in Example 1. The results are shown in Table I.

ingredient Amount (% w / v) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsification stability (day) Egg yolk phospholipid 1.0 530 10.0 3-7 Egg phosphatidylethanolamine 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 Dilinole oil phosphatidylcholine 1.0 210 100 14.0 2 1,2 Dilinoleylylphosphatidylethanolamine 1.0 200 110 10.0 2 Cardiolipin 1.0 150 65 12.0 5 1,2 dilinoleyl phosphatidylglycerol 1.0 180 85 7.5 2 1,2 dilinoleyl phosphatidylserine 1.0 460 330 6.0 2 1,2 dilinoleyl oilpatinic acid 1.0 130 60 〉 15.0 3 1,2 Dicaprylphosphatidylethanolamine 1.0 260 210 4.0 One 1,2 dilauroylphosphatidylethanolamine 1.0 435 350 6.0 4 1,2-dimyristoylphosphatidylethanolamine 1.0 2440 840 1.0 2 1,2-dioleoylphosphatidylethanolamine 1.0 450 100 〉 15.0 stability 1,2-dioleoylphosphatidylethanolamine 0.8 530 250 4.0 stability 1,2 dioleoylphosphatidylcholine 1.0 450 175 12.0 12 1,2 Dioleoylphosphatidic acid 1.0 430 180 10.5 stability 1,2 Dioleoylphosphatidic acid 0.8 700 300 5.0 stability 1,2 Diarachidonylphosphatidylethanolamine 0.8 240 85 〉 15.0 One 1-palmitoyl, 2-oleoyl phosphatidylethanolamine 1.0 500 110 8.0 stability 1-palmitoyl, 2-linoleoyl phosphatidylethanolamine 1.0 320 175 7.0 4 * S.D. - Standard Deviation

All phospholipids were obtained from Avanti Polar Lipids, except for egg yolk phospholipids obtained from Kabi Pharmacia, Stockholm, Stockholm. Particle size analysis was performed using the same procedures and conditions as in Example 1. The creaming time was measured spectrophotometrically by monitoring the transmittance of the emulsion filled cuvette. The creaming time is the time required for the emulsion filled cuvette to achieve a transmission from 0% to 100%. All samples were stored at 30 ° C. and monitored daily for emulsion stability (ie total phase separation).

It was found that formulations containing phospholipids with oleoyl fatty acid residues had improved properties compared to any other phospholipid mixture or phospholipid molecular species. The results also show that emulsions formulated with phospholipids having phosphatidylethanolamine or phosphatidic acid head groups showed improved stability. Both phosphatidic acid and phosphatidylethanolamine lipid systems have strong properties of forming an inverted, non-lamellar phase in which tight head group filling is desirable. On the other hand, phosphatidylcholine, phosphatidylglycerol and phosphatidylserine are more prone to head group filling and are more likely to take the lamellar phase.

Increasing the chain length and chain unsaturation lowers lamellar (L _α ) to the inverted hexagonal (H _II ) transition temperature. Thus, increasing chain length and chain unsaturation results in an increase in chain pressure that tends to reduce monolayer spontaneous curvature (H _o ). Increasing the degree of unsaturation, such as the conversion of 1,2-dioleoylphosphatidylethanolamine to 1,2-dilinoleoylphosphatidylethanolamine, imposes more severe filling pressures that can lead to the formation of new unwanted phases. easy. Therefore, the use of phospholipid surfactants with monounsaturated, for example, oleoyl, fatty acid residues and / or ethanolamine or phosphatidic acid head groups is preferred.

Example 3

Continuous effect on emulsion stability

5 ml of the following reverse tanning formulations were prepared:

0.5% w / v egg phospholipid (Kabi Pharmacia, Stockholm)

90% v / v oil or oil mixture (see list below)

0.09% Sodium Chloride (Sigma)

0.09% calcium chloride (Sigma)

10% v / v distilled water for injection

α, ω-dibromo-F-butane (DBFB), trichlorotrifluoroethane (CFC-113), perfluorooctyl bromide (PFOB), n-hexane, perfluorohexane (PFH) and mixtures thereof Containing reverse emulsions were prepared to examine the effect of the continuous phase on emulsion stability. The emulsification procedure and conditions described in Example 1 were followed. The emulsion was first visually checked to see if the oil was fully emulsified. The inverse properties of the emulsions were confirmed by stability after dilution with hydrocarbon oil, ie n-octane. The refractive index (n ₁₂ ) of the mixture was evaluated through the procedure described in Taslc et al; J. Chem. Eng. Data, 37: 310-313, 1992. Emulsion stability generally correlates with the refractive index n _D of the continuous phase (FIG. 2), and therefore oils or oil mixtures with n _D or n ₁₂ in excess of about 1.32 form stable inverse emulsions.

The preferred range of refractive indices will depend on the type of surfactant. The use of preferred surfactants, such as dioleoylphosphatidylethanolamine, can substantially reduce the acceptable refractive index values.

Example 4

Effect of Molar Volumes of Continuous Phase on Inverse Emulsion Stability

5 ml of the reverse tanning formulation described in Example 3 was prepared for each of the following oils: DBFB, CFC-113, PFOB, PFH, n-hexane, n-heptane, n-octane, n-decane, n-dodecane, n-heptadecane, chloroform (CHCl ₃ ), carbon tetrachloride (CCl ₄ ) and 1,6-dibromohexane. The emulsification procedure and conditions described in Example 1 were followed. The emulsion was first visually checked to see if complete emulsification occurred. The inverse properties of the emulsions were confirmed after dilution with hydrocarbon oil, ie n-octane. Emulsion stability is related to the molar volume (V _M ) of the continuous phase (FIG. 3), with oils having a V _M of less than about 190 formed a stable reverse emulsion. As mentioned above, the acceptable molar volume range for the continuous phase depends critically on the type of emulsifier. In general, high lipophilic fluorocarbons having a low molar volume are preferred.

Example 5

Inverse emulsion prepared with phospholipid / non-polar liquid formulation

The emulsion formulation of Example 1 was modified by changing the surfactant component to examine the effect of the nonpolar liquid additive on the properties of the phospholipid stabilized inverse emulsion. The emulsification procedure and conditions described in Example 1 were followed. Phospholipid concentrations were set at 1% w / v and additional 5, 10 or 25 mol% nonpolar lipids were incorporated. The results are shown in Tables IIa to IIg.

(Mono-olein) Primary surfactant Monoolein added amount (mol%) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (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 stability DOPE 10 435 220 7.5 stability DOPE 25 210 95 〉 15.0 stability DOPA 5 400 200 15.0 stability DOPA 10 500 125 14.5 stability DOPA 25 165 100 〉 15.0 stability ^* SD-standard deviation; DOPC = 1,2 dioleoylphosphatidylcholine; DOPE = 1,2 diole oil phosphatidylethanolamine; DOPA = 1,2 dioleoylphosphatidic acid

(Dioline) Primary surfactant Diolein added amount (mol%) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (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 stability DOPE 10 210 100 〉 15.0 stability DOPE 25 175 100 〉 15.0 stability DOPA 5 790 300 5.0 stability DOPA 10 860 425 7.0 stability DOPA 25 470 165 〉 15.0 stability

(Triolein) Primary surfactant Triolein added amount (mol%) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (days) DOPC 5 360 170 9.0 12 DOPC 10 250 130 9.0 4 DOPC 25 280 155 ? 12 DOPE 5 670 390 5.0 stability DOPE 10 470 210 10.0 stability DOPE 25 675 200 12.0 stability

(Medium chain triglycerides) Primary surfactant MCT oil added amount (mol%) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (days) DOPC 10 420 200 14.0 10 DOPC 25 470 200 N / D stability DOPE 5 670 285 〉 15.0 stability DOPE 10 963 650 10.0 stability DOPE 25 830 620 10.0 stability DOPA 10 700 350 〉 15.0 stability DOPA 25 690 480 〉 15.0 stability N / D = not detected

(cholesterol) Primary surfactant Cholesterol addition amount (% mol) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (days) DOPC 25 255 140 13.0 30 DOPE 25 170 100 〉 15.0 stability

(Squalene) Primary surfactant Squalene addition amount (mol%) Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (days) DOPC 10 200 100 13.0 15 DOPE 10 300 160 〉 15.0 stability

(Long chain alcohol) Primary surfactant Long chain alcohol Droplet median size (nm) SD ^* (nm) Creaming time (minutes) Emulsion stability (days) DOPE Decyl alcohol 220 110 〉 15.0 stability DOPE Oleoyl Alcohol 290 200 〉 15.0 stability

DOPC, DOPE and DOPA were obtained from Avanti Polar Lipids. Monoolein, diolein, decyl alcohol and oleoyl alcohol were obtained from Nu-Chek Prep (Elysian, MN). Cholesterol, triolein and squalene were obtained from Sigma. Medium chain triglycerides (MCT) were obtained from Karlshams (Janesville, Wis.). Particle size analysis was performed using the same procedures and conditions as described in Example 2.

Improved tanning properties were observed with DOPE or DOPA in combination with monoolein, diolein, cholesterol, squalene, decyl alcohol or oleyl alcohol. Improvements are noted by the reduction in the initial droplet size, which is a measure of the reduction in system coalescence. Similar or reduced tanning properties were observed with any combination of DOPC, triolein or MCTs. In general, emulsion properties improved with increasing nonpolar lipid content. The nonpolar component splits between phospholipid molecules to reduce monolayer spontaneous curvature (H _o ), thereby increasing the hydrocarbon chain volume and / or reducing the chain filling stress. The inability of triolein and MCTs to improve the stability of inverse tanning agents is due to the lack of amphiphilic properties necessary for it to split into surfactant monolayers. Triglycerides are simply dissolved in fluorocarbon oils.

Therefore, improved tanning properties are obtained when the phospholipid containing the reverse tanning agent is supplemented with amphiphilic nonpolar additives.

Example 6

Preparation of enzymes containing reverse tanning

Α, ω-Dibromo-F-butane (90% v / v), PULMOZYME® (Genentech, South San Francisco, CA) (10% v) according to the procedure outlined in Example 1 / v), 0.5% egg phosphatidylethanolamine (PE) or egg yolk phospholipids containing at least 15% w / w PE were prepared in water reverse emulsion in fluorocarbons containing enzyme. Pulmozyme contains 1.0 mg / mL Dornase alpha enzyme in saline. The water inverse emulsion in the resulting fluorocarbon containing enzyme is transparent with a median droplet size of about 300 nm. Encapsulated enzymes have been shown to maintain their activity (ie, entry into the nucleus, promotion of DNA destruction and final cell necrosis) by in vitro mononuclear cell macrophage culture analysis. A decrease in ex vivo sputum viscosity collected from cystic fibrosis patients after contact with an anti-emulsion containing puldomim was observed.

Of course, it will be appreciated that the incorporation of a polar liquid soluble therapeutic or diagnostic agent may be accomplished using an aqueous solution of drug instead of pulpyzim when the emulsion or microemulsion is formed. In doing so, the concentration of the agent in the emulsion or microemulsion can be adjusted simply by changing the drug concentration in the aqueous solution.

Example 7

Preparation of an Inverse Emulsion Containing Drugs in an Aqueous Phase

Using the emulsification procedure and conditions described in Example 1, 3 ml of the following reverse emulsion formulations containing the drug were prepared:

A: gentamicin sulfate inverse emulsion

0.051% w / v gentamycin 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 α, ω-Dibromo-F-butane (Exfluor)

0.09% Sodium Chloride (Sigma)

0.09% calcium chloride (Sigma)

10% v / v distilled water for injection

B: cis-platin reverse emulsion

0.025 w / v cis-platin (Sigma)

1.0% w / v 1,2-dioleoylphosphatidylethanolamine

0.21% w / v di-olein

90% v / v α, ω-dibromo-F-butane

0.09% sodium chloride

0.09% calcium chloride

10% v / v distilled water for injection

C: amikacin sulfate inverse emulsion

0.052% w / v amikacin sulfate (Sigma)

0.7% w / v egg PE (Avanti)

90% v / v α, ω-dibromo-F-butane

0.09% sodium chloride

0.09% calcium chloride

10% v / v distilled water for injection

D: terbutalin sulfate reverse emulsion

0.046% w / v Terbutalin Sulfate (Sigma)

1.0% w / v egg yolk phosphatide (Asahi, Tokyo, Japan)

90% v / v α, ω-dibromo-F-butane

0.09% sodium chloride

0.09% calcium chloride

10% v / v distilled water for injection

E: Tobramycin Sulfate Reversed Emulsion

0.03% w / v Tobramycin Sulfate (Sigma, St. Louis, Mo.)

1.0% w / v egg yolk phosphatide (Asahi, Tokyo, Japan)

90% v / v α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

10% v / v distilled water for injection

Particle size analysis was performed using the procedures and conditions described in Example 1. Creaming times were measured using the procedures and conditions described in Example 2. Particle size and creaming rate for amikacin sulfate and terbutalin sulfate will probably be improved when formulated with a DOPE / di-olein surfactant combination. A slight improvement in particle size distribution was observed in gentamycin sulfate containing emulsion compared to vehicle (FIG. 4). Table III shows the median droplet diameter and initial creaming time of the formulations.

Formulation Center droplet diameter (nm) Initial cream time (minutes) Gentamycin sulfate (0.051% w / v) 145 ± 70 〉 15.0 Tobramycin (0.03% w / v) 125 ± 80 〉 15.0 Cis-platin (0.025% w / v) 200 ± 100 〉 15.0 Amishcin sulfate (0.052% w / v) 600 ± 250 4.0 Terbutalin Sulfate (0.046% w / v) 460 ± 200 10.0

Example 8

Preparation of Complex Emulsion (Water / Fluorocarbon / Water)

Using the emulsification procedure and conditions described in Example 1, 5 ml of the following reverse tanning formulations were prepared:

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 α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

0.09% calcium chloride (Sigma, St. Louis, MO)

10% v / v distilled water for injection

60 mg egg yolk phospholipid (EYP) (Kabi Pharmacia, Stockholm, Sweden) was sonicated at 7 ° C. for about 2 minutes and dispersed in 2.4 g of distilled water for injection. Thereafter, a reverse emulsion (1.2 g) consisting of the components listed above was added dropwise to the EYP dispersion during sonication. After the addition was complete, the complex emulsion was further sonicated for 15 minutes. A milky emulsion with no free oil was obtained. The composite emulsion formed had an average particle size of 400 ± 200 nm (centrifugal sedimentation). The properties of the emulsion continuous phase were confirmed by conductivity and by dispersion into water.

Example 9

Preparation of Ethanol Containing Reverse Emulsion

Using the same emulsification procedure and conditions as described in Example 1, 5 ml of the following reverse tanning formulations were prepared:

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 α, ω-dibromo-F-butane (Exfluor, 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 distilled water for injection

A polar phase containing 25% v / v ethyl alcohol was added to the surfactant / fluorocarbon dispersion described in Example 1 to give an opal emulsion of milky light. The ethanol emulsion in the fluorocarbons formed had a median droplet size of 130 ± 35 nm.

Example 10

In Vitro Efficacy of Drugs Containing Inverse Emulsions

As described in Examples 1 and 7, 5 ml of the following drug containing reverse tanning and tanning vehicle were prepared:

Formulation A: Gentamicin Sulfate Reverse Emulsion Formulation

0.03% w / v gentamycin 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 α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

0.09% calcium chloride (Sigma, St. Louis, MO)

10% v / v distilled 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 α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

0.09% calcium chloride (Sigma, St. Louis, MO)

10% v / v distilled 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 α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

0.09% calcium chloride (Sigma, St. Louis, MO)

10% v / v distilled water for injection

Drug emulsion preparations containing antibiotics and various controls. It was tested for its antibacterial ability in Collie suspension cultures. To simulate bacterial infections in the lungs, Lee. Coli suspension cultures were maintained in well plates containing a monolayer of normal human bronchial / tracheal epithelial tissue cells. Drugs in the concentration range of 0.3 to 0.003 mg in 100 μL. Add to 1 mL medium containing collie / cell suspension. Fluorocarbon and tanning vehicle controls were added to a degree proportional to the extent present in the highest drug concentration sample. Plates were incubated overnight at 37 ° C. Each well was aspirated and diluted with two part LB medium. Diluted aliquot mixture (20 μL) was added to the LB plate. Incubate overnight at 37 ° C. for initial titration of the collie. Dilution was then carried out to determine the titer of each well. The results are shown in Table IV below.

Sample processing this. Collie titer (colony / mL) Do not process 6.3 E 7 Brine 7.0 E 7 α, ω-dibromo-F-butane 1.0 E 7 Reverse emulsion vehicle (Formulation C) 1.9 E 7 0.3 mg gentamycin sulfate in saline 0 0.03 mg gentamycin sulfate in saline 1.0 E 1 0.3 mg gentamycin sulfate in emulsion (Formulation A) 2.0 E 2 0.03 mg gentamycin sulfate in emulsion (Formulation A) 2.7 E 3 0.003 mg gentamycin sulfate in emulsion (Formulation A) 2.3 E 4 0.3 mg tobramycin sulfate in saline 0 0.03 mg tobramycin sulfate in saline 2.0 E 1 0.3 mg tobramycin sulfate in emulsion (Formulation B) 1.5 E 1 0.03 mg tobramycin sulfate in emulsion (Formulation B) 7.0 E 1 0.003 mg tobramycin sulfate in emulsion (Formulation B) 6.5 E 3

Negative controls, i.e. saline, α, ω-dibromo-F-butane, reverse tandem vehicle or untreated group, all showed no ability to inhibit bacterial growth. Drugs containing reverse tanning agents all exhibited equivalent antibacterial efficacy compared to their corresponding saline control. In addition, a dose dependent response of antibacterial ability was observed for the two drugs evaluated. This result illustrates that the efficacy of the drug is not inhibited by the surfactant monolayer or fluorocarbons.

Example 11

Preparation of Water Inverse Emulsion in Fluorocarbon by High Pressure Homogenization

15 ml of the following reverse tanning formulations were prepared:

1.0% w / v egg phosphatidylethanolamine

(Avanti Polar Lipids, Alabaster, AL)

90% v / v α, ω-dibromo-F-butane (Exfluor, Austin, TX)

0.09% sodium chloride (Sigma, St. Louis, MO)

0.09% calcium chloride (Sigma, St. Louis, MO)

10% v / v distilled water for injection

The surfactant, DBFB and saline solution were first dispersed by sonication or using a low shear method according to the procedures and conditions described in Example 1. Low shear methods have been developed for use in dispersion process-sensitive pharmacological preparations, such as DNA plasmids. In the low shear method, the surfactant and α, ω-dibromo-F-butane were dispersed in a low energy Tekmar type SD-1810 mixer at 10,000 revolutions / minute for about 1 minute. The dispersed phase was added dropwise during mixing. After the addition was complete, the reverse emulsion was mixed for a few more minutes. The sonicated or mixed emulsion was then further treated using an EmulsiFlex-CF homogenizer made by Avestin (Ottawa, Canada). The emulsion was homogenized using the following treatment conditions: 10 passes at 12K psi. A clear emulsion of water in a fluorine chemical reagent was obtained. Particle size analysis was performed by laser diffraction (Horiba LA-700, Kyoto, Japan) in the volume weighting mode. An equivalent portion of about 20-50 μL of each sample was diluted in 9-10 mL of n-dodecane. The refractive index ratio and fragment cells of distribution form 3, 1.1 were used. The reverse emulsion formed had a median droplet diameter of 200 ± 70 nm and 205 ± 70 nm.

Example 12

Stability of Inverse Emulsion Prepared by High Pressure Homogenization

Several stations in this study contain dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidine acid (DOPA) as the primary surfactant and 1,4-dibromofluorobutane (DBFB) as the oil. The tanning formulations were evaluated for their particle growth and hydrolysis stability. The effect of the addition of nonpolar additives such as cholesterol, monoolein, diolein and 1,3-diolein to DOPA and DOPE was evaluated. In addition, the stability of the gentamicin sulfate emulsion in DBFB was examined. 1,4-Dibromofluorobutane (DBFB) was obtained from Exfluor Corp. Cholesterol, gentamycin sulfate was 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 provided.

Using the emulsification procedure and conditions described in Example 11, 15 ml of the following emulsion formulations were prepared:

Component concentration

Brine solution ^* 10% v / v

DBFB 90% v / v

DOPE or DOPA 1% v / v

Non-polar additives, if included 10 mol% of primary surfactant

^The saline solution consists of 0.9% w / v NaCl and 0.9% w / v CaCl ₂ .2H ₂ O.

For drugs containing emulsions, gentamicin sulfate was dissolved in saline solution prior to sonication. No attempt was made to exclude oxygen or control temperatures during preparation or filling. Samples were stored and sealed in crimp cap vials at 5 ° C and 25 ° C. Reverse emulsion free fatty acid (FFA) concentrations were measured using spectrophotometry (Mahadevan, S., Dillard, C. J. and Tappel, A. L. Anal. Biochem .; 27 (1969) 387). Particle size analysis was performed using the same procedures and conditions as described in Example 1. The results are shown in Tables Va to Vc.

5 ℃ Emulsion ID Early 21d. 30d. 49d. 63d. 84d. 114d. 150d. 225d. DOPE 200 220 220 180 170 170 160 180 170 DOPA 305 350 240 4060 7610 8240 9140 n / d n / d DOPE / gentamycin 150 150 210 110 120 120 110 110 120 DOPE / Cholesterol 166 200 210 130 130 130 150 150 120 DOPA / monoolein 216 5310 3590 4920 5450 8430 8030 n / d n / d DOPA / Diolein 5968 5000 7220 6230 7200 8300 8780 n / d n / d DOPA / 1,3 diolein 304 3320 4890 6830 7360 8510 8870 n / d n / d n / d = not detected

25 ℃ Emulsion ID 14d. 21d. 30d. 49d. 63d. 77d. 84d. 100d. 114d. 150d. 225d. DOPE 200 190 230 190 180 250 8000 8740 13010 decomposition n / a DOPA 370 7550 6490 8210 8230 8250 10340 n / d 17400 n / d n / d DOPE / gentamycin 150 150 170 150 120 160 8020 8170 7950 8110 decomposition DOPE / Cholesterol 160 160 160 130 130 140 150 130 130 6000 n / d DOPA / monoolein 190 5430 6430 8210 8420 8500 8830 n / d 9800 n / d n / d DOPA / Diolein 3650 6600 7840 8130 8440 8670 9210 n / d 8720 n / d n / d DOPA / 1,3 diolein 420 6300 7440 8530 8600 8800 9840 n / d 9680 n / d n / d n / d = not detected

Free Fatty Acid Concentration (mEq / L) vs. Time Temperature DOPE DOPA DOPE / gentamycin DOPE / Cholesterol DOPA / monoolein DOPA / Diolein DOPA / 1,3 Olein Early 4.4 4.8 4.25 4.71 5.15 4.92 5.88 5 months 5 ℃ 5.26 7.94 6.59 6.28 6.55 7.99 10.01 5 months 25 ℃ 7.35 10.27 7.12 6.75 8.08 8.27 12.07

Similar initial median particle size (about 150-300 nm) was observed for all high pressure homogenized inverse emulsion formulations except DOPA / diolein. Inverse emulsions formulated using DOPE as the primary surfactant showed greater stability against coalescence and hydrolysis when compared to DOPA at both 5 ° C and 25 ° C. The greatest reverse emulsion stability at 25 ° C. was observed with the DOPE / cholesterol formulation. No significant particle growth occurred in the DOPE reverse tanning formulations stored at 5 ° C. for 225 days.

Particle growth of the reverse emulsion appeared to occur in two phases. The first phase of growth is characterized by the change in size of the median particle size that occurs for several days to one week. After the rapid growth phase, emulsion particle growth appears to deviate from the stable level for an extended period of time, followed by decomposition of the emulsion. Early rapid growth phases are indicative of union induced processes. However, the reason for the slowed emulsion growth at this point is unclear. The particle size measurement of such crude (> 1 μm) reverse emulsions can be inaccurate.

Example 13

Effect of Dispersed Phase Volume on Inverse Emulsion

The emulsion and emulsification procedures of Example 11 were modified by varying the dispersion and continuous phase volumes. Inverse emulsions with dispersed phases of 5, 10, 15, 20, 30, 40 and 50 vol% were prepared. The dispersed phase 1,2-dioleoyl phosphatidylethanolamine (DOPE) concentration in all tanning formulations was fixed at 1.34 mM. Samples were stored sealed in crimp cap vials at 25 ° C. Particle size analysis was performed using the same procedures and conditions as described in Example 1 above. Viscosity measurements were performed with a Brookfield model DV-II viscometer at 37 ° C. The results are shown in Table VI and FIG. 5.

Median particle diameter (nm) vs. time (days) Dispersion Phase v / v% Early 21d. 30d. 60d. 82d. 105d. 5 200 6300 11300 decomposition n / a n / a 10 200 190 180 160 180 170 15 180 190 150 120 140 120 20 170 150 140 130 120 130 30 150 120 150 130 150 150 40 170 150 n / d n / d n / d n / d 50 200 180 n / d n / d n / d n / d n / d = not detected

No viscosity measurements were taken for 40 and 50% v / v emulsions due to their high viscosity and insufficient sample volume. A noticeable drop in droplet stability was observed when reducing the dispersed phase concentration below 10%. As expected, the emulsion viscosity increased as the dispersed phase volume increased. Increasing the dispersed phase concentration increases the number of droplets per unit volume and therefore the viscosity, because the droplets are increasingly driven into the closed packed structure. Therefore, the emulsion rheological properties can be adjusted by varying the volume of the dispersed phase. It is also expected that the emulsion stability against coalescence (droplet growth) will be very simply suppressed by increasing the emulsion viscosity.

Example 14

Effect of Dispersion on Inverse Emulsion Stability

Only the dispersed phase composition was changed to modify the emulsion formulation and emulsification procedure of Example 11. Inverse emulsions were prepared containing deionized water, NaCl, 0.02M CaCl ₂ and 0.02M AlCl ₃ at various concentrations (0.02, 0.1, 0.2M). Particle size analysis was performed using the same procedures and conditions as described in Example 11. The results are shown in Table VII below.

Emulsion I.D. Center particle size (㎛) DI number 4.00 0.02M NaCl 0.52 0.1M NaCl 0.22 0.2M NaCl 0.14 0.02M CaCl ₂ 0.22 0.02M AlCl ₃ 0.25

Reduced emulsion particle diameter is observed as a function of NaCl concentration. The results also indicate that formulations containing either CaCl ₂ or AlCl ₃ formed an emulsion with a smaller particle size distribution compared to the NaCl concentration provided. Increasing the ion concentration of the dispersed phase lowers the lamellar (L _α ) to the inverted hexagonal (H _II ) transition temperature. This is done by reducing the hydration of the phosphate group, which then promotes an increase in head group interactions and a decrease in monolayer spontaneous curvature (H _o ). The effect of divalent and polyvalent ions on phase behavior is very complex and not well understood. However, its low binding constant has shown that it can have a large effect at low concentrations (see Seddon, JM, Biochem. Biophys. Acta, 1031 (1990) 1). Therefore, addition of small amounts of divalent or polyvalent salts may be advantageous.

Example 15

Relationship between Continuous Phase Chemical Properties and Inverse Emulsion Stability

3 ml of a reverse emulsion formulation was prepared using the same emulsification procedure and conditions as described in Example 1:

1% w / v 1,2-dioleoylphosphatidylethanolamine (DOPE)

(Avanti Polar Lipids, Alabaster, AL)

90% v / v oil or oil mixture (see Table VIII)

0.09% sodium chloride (Sigma Chemicals)

0.09% calcium chloride (Sigma Chemicals)

10% v / v water

Inverse emulsions were prepared with a wide range of oils to ascertain whether there is a correlation between continuous phase physicochemical properties and emulsion stability. The emulsification procedure and conditions described in Example 1 were followed. The emulsion was first visually checked to see if the oil was fully emulsified. The inverse properties of the emulsions were confirmed by stability after dilution with hydrocarbon oil, ie n-octane. Table II shows the 34 oils tested, their respective molar volume (V _m ), refractive index (n _D ²⁰ ), α-polarity (α), molar refractive index (R _m ), DOPE solubility, bromohexane critical The solution temperature (CST _BrHex ) and emulsion stability values are recorded. DOPE saturated oils were prepared by adding 50-600 mg of DOPE to 2 mL of oil and mixing gently for 1 week at room temperature. The solution was centrifuged at 4000 × g for 30 minutes, after which the DOPE saturated with oil was removed by syringe. DOPE content is described in Weers, JG, Ni, Y., Tarara, TE, Pelura, TJ, and Arlauskas, RA, The Effect of molecuclar Diffusion on Initial Particle Size Distributions in Phospholipid-Stabilized Fluorocarbon Emulsions; Colloids and Surfaces, 84 (1994) 81], by high performance liquid chromatography (HPLC) according to the method described. Samples were injected either as pure solutions or after dilution in 2-propanol: hexanes (1: 1 (v / v)). Quantification was made with reference to the external DOPE standard curve. The n _D ²⁰ value was measured using a manual refractometer when possible. α, R _m and n _D ²⁰ values <1.34 are described in Le, TD, and Weers, JG, QSPR and GCA Models for Predicting the Normal Boiling Points of Fluorocarbons; J. Phys Chem, 99 (1995) 6739. Le, TD, and Weers, JG, Group Contribution-Additivity and Quantum Mechanical models for Predicting the Molar Refractions, Indices of Refraction, and Boiling Points of Fluorochemicals; Calculated using the group influence activity model proposed by Le and Weers, described in J. Phys Chem, 99 (1995) 13909. CST _BrHex values were obtained from the technique of Le et al. Reported September 22, 1995. Emulsion stability is defined as the time required for the emulsion to fully decompose. Oil containing formulations that do not produce stable W / O dispersions upon sonication are defined as unstable.

Emulsion stability is strongly correlated with the continuous phase n _D ²⁰ (lipophilic) and its DOPE-solubility (Table VIII). There was no correlation with α, R _m , V _m or CST _BrHex . Stable reverse emulsion formation was caused by oils with n _D ²⁰ greater than about 1.34 and oils soluble in DOPE. Emulsion stability decreased dramatically over a small range of oil n _D ²⁰ as evidenced by comparing the stability of the DBFH (about 3 days) and DBFB (about 60 days) reverse emulsions. Continuous phase requirements (ie, n _D ²⁰ ) will also depend on the surfactant (s) and / or dispersed phase composition. The use of co-surfactants such as cholesterol and / or the addition of polyvalent ions such as AlCl ₃ can actually reduce the continuous phase n _D ²⁰ value required. The results are shown in Table VIII below.

Summary of Continuous Phase Oil Physicochemical Properties and Inverse Emulsion Stability compound Furtherance V _m (ml / mol) α-polarity (Å ³ ) R _m (cm 3) n _D ²⁰ DOPE solubility (w / w%) CST _BrHex (℃) Emulsion stability (days) Dibromohexane C ₆ H ₁₂ Br 153.83 10.1 43.09 1.507 n / d n / a ^* 〉 60 Carbon tetrachloride CCl ₄ 96.80 7.26 25.35 1.461 n / d n / a ^* 〉 60 chloroform CHCl ₃ 80.44 4.92 20.62 1.448 n / d n / a ^* 〉 60 n-hexadecane C ₁₆ H ₃₄ 292.95 16.5 75.54 1.434 1.10 n / a ^* 〉 60 CFC-113β26b C ₂ F ₃ ClBr ₂ 122.91 7.94 30.57 1.428 n / d n / d 40 n-dodecane C ₁₂ H ₂₆ 227.12 12.32 57.11 1.422 5.60 n / a ^* 〉 60 n-decane C ₁₀ H ₂₂ 194.92 10.23 47.9 1.411 11.20 n / a ^* 〉 60 n-octane C ₈ H ₁₈ 162.52 8.14 38.69 1.398 23.40 n / a ^* 〉 60 HCFC-132bβ2 C ₂ H ₂ F ₂ Br ₂ 100.02 5.08 23.84 1.385 22.30 n / d 60 CFC-316bc C ₄ F ₆ Cl ₄ 176.29 11.08 41.28 1.383 2.05 n / d 30 n-hexane C ₆ H ₁₄ 130.47 6.04 29.47 1.375 25.10 n / a ^* 〉 60 F6H12 C ₁₈ F ₁₃ H ₂₅ 391.33 20.03 88.07 1.367 n / d n / d 38 CFC-141b C ₂ F ₄ Cl ₂ 93.56 5.41 20.65 1.361 20.80 n / d 30 CFC-113 C ₂ F ₃ Cl ₃ 120.66 6.97 26.15 1.358 1.72 n / d 60 F6H10 C ₁₆ F ₁₃ H ₂₁ 357.95 17.94 78.85 1.356 n / d n / d 23 CFC-317mab C ₄ F ₇ Cl ₃ 165.23 9.53 35.78 1.353 BLQ n / d 40 DBFB C ₄ F ₈ Br ₂ 171.60 8.97 36.68 1.351 0.62 -25.7 60 DBFH C ₆ F ₁₂ Br ₂ 220.98 11.54 47.30 1345 BLQ -23.4 3 F6H8 C ₁₄ F ₁₃ H ₁₇ 326.00 15.84 69.64 1.344 n / d -31.1 3 F8H8 C ₁₆ F ₁₇ H ₁₇ 378.59 18.42 80.26 1.340 n / d -413 Instability F6H6 C ₁₂ F ₁₃ H ₁₃ 289.54 13.75 60.43 1.338 ¹ n / d -3.6 Instability FC-225 ca / cb C ₃ HF ₅ Cl ₂ 130.93 6.31 27.88 1.33 ¹ BLQ n / d Instability F8H6 C ₁₄ F ₁₇ H ₁₃ 338.62 16.32 71.05 1.33 ¹ n / d -14.4 Instability F8H4 C ₁₂ F ₁₇ H ₉ 304.67 14.23 61.83 1.32 ¹ n / d 14.4 Instability PFHE C ₈ F ₁₃ H ₅ 215.41 9.57 42.00 1.31 ¹ BLQ 24.8 Instability CFC-318-mbb C ₄ F ₈ Cl ₂ 161.30 7.98 30.28 1.31 ¹ n / d n / d Instability CFC-216ba C ₃ F ₆ Cl ₂ 138.98 6.70 25.96 1.30 ¹ n / d n / d Instability F4H3 C ₇ F ₉ H ₇ 190.63 8.04 35.98 1.30 ¹ n / d -41.5 Instability PFHB C ₆ F ₁₃ Br 211.76 9.51 39.59 1.299 n / d 33.2 Instability Purple Flubron C ₈ F ₁₇ Br 261.24 12.08 50.21 1.299 BLQ 68.1 Instability F4H2 C ₆ F ₉ H ₅ 173.73 7.00 31.38 1.29 ¹ n / d -1.9 Instability PFBB C ₄ F ₉ Br 164.76 6.93 28.97 1.28 ¹ BLQ -2.1 Instability PFH C ₆ F ₁₄ 200.11 7.47 31.88 1.251 ¹ BLQ 146.9 Instability ¹ n _D values are calculated by published techniques of Le and Weers. All other values are measured. N / a ^* = not available, oil is completely miscible with all bromoalkanes used. If measurable all values will be <-50 ° C. n / d = not detected; BLQ = below the quantification range

Example 16

Effect of Continuous Phase Refractive Index on Inverse Oil Life

Using the same emulsification procedure and conditions as described in Example 1, 5 ml of the following reverse tanning formulations were prepared:

1% w / v 1,2-dioleoylphosphatidylethanolamine (DOPE)

(Avanti Polar Lipids, Alabaster, AL)

90% v / v oil or oil mixture (see Table IX)

0.09% sodium chloride (Sigma Chemicals)

0.09% calcium chloride (Sigma Chemicals)

10% v / v water

Emulsion life and particle size distribution were determined as a function of the perflubronone / DBFB ratio. Each emulsion formulation was first visually checked to see if the polar phase remained emulsified for at least several hours. The inverse properties of the emulsions were confirmed by stability after dilution with hydrocarbon oil, ie n-octane.

Emulsions with a perflubronone / DBFB ratio of less than 0.55 were extremely unstable and immediately degraded. Samples were sealed and stored in crimp cap vials at 25 ° C. Particle size analysis was performed using the same procedures and conditions as described in Example 11. Emulsion life τ is defined as the time required for the emulsion to fully decompose. Oil containing formulations that do not produce stable W / O dispersions upon sonication are defined as unstable. Table IX shows n ₁₂ , median droplet diameter, and emulsion lifetimes in units of time for the tested perflubronone / DBFB ratio. The refractive index (n ₁₂ ) of the mixture is described in Taslc et al .; J. Chem. Eng. Data, 37: 310-313, 1992].

Stability of anti-emulsion formulated into ideal oil mixture _χ PFOB PFOB / DBFBn ₁₂ Median diameter (㎛) Emulsion life t (hours) 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 0.400 1.330 0.81 264 0.500 1.325 1.50 24 0.550 1.322 10.50 One 0.600 1.320 Instability Instability 0.700 1.314 Instability Instability 0.800 1.309 Instability Instability 0.850 1.307 Instability Instability One 1.299 Instability Instability

The τ value was found to correlate well with both the continuous phase n ₁₂ and the droplet diameter, reflecting the change in monolayer spontaneous curvature (H ₀ ) due to the penetration of the continuous phase molecule into the surfactant brush. The H ₀ value is reduced as a function of continuous lipophilic or n ₁₂ . At nearly tanning time about n ₁₂ = 1.320, a sharp increase in emulsion stability occurs with a small change in n ₁₂ . This effect has been observed for other surfactants [Kabalnov, A., and Weers, J., Macroemulsion Stability Within the Winsor III Region: Theory Versus Experiment; Langmuir, 12 (1996) 1931]. In addition, this result is in good agreement with the current emulsion stability theory of emulsion stability against changes in monolayer spontaneous curvature [Kabalnov, A., and Wennerstroem, H., Macroemulsion Stability: The Orientated Wedge Revisited; Langmuir, 12 (1996) 276]. Emulsion stability or longevity has also been found to correlate strongly with the emulsion average particle size and overall distribution. The emulsion having poor stability, τ ≦ 24 hours, had a large median diameter and had a wide particle distribution. As the DBFB / Perflubronn ratio increased, the distribution narrowed and the median diameter decreased.

Dilution of the stable counter-emulsion with a less lipophilic oil (ie perflubron) resulted in rapid coalescence and degradation of the emulsion. This fusion and degradation process reflects the reduced penetration of the continuous phase molecules into the surfactant brush. The ability to control the stability of the emulsion through the lipophilic of the continuous phase (n _D ) can be used to direct the deposition of the dispersed phase components.

Although the invention has been described with reference to particular preferred embodiments, the scope of the invention is defined by the following claims and should be inferred to include suitable equivalents.

Claims (39)

A dispersed liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And Fluorocarbon pharmaceutical formulations containing an effective amount of one or more non-fluorinated surfactants. The formulation of claim 1, wherein the formulation is a thermodynamically stable microemulsion. The formulation of claim 1 further comprising at least one disperse liquid phase additive selected from the group consisting of inorganic salts, buffers, stabilizers, swelling and infiltrating formulations and nutritional formulations. The method of claim 1, wherein the one or more lipophilic fluorocarbons are halogenated perfluorocarbons, halogenated perfluoroethers, halogenated polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbon ether diblocks and their Formulations selected from the group consisting of mixtures. The formulation of claim 4, wherein the lipophilic fluorocarbon is a halogenated perfluorocarbon and the halogenated perfluorocarbon is α, ω-dibromo-F-butane. The method of claim 1, wherein the one or more non-fluorinated surfactants are alcohols, fatty acids, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, phosphatidic acid, phosphatidyl ethanolamine, N, N-dimethyl-phosphatidylethanolamine, Phosphatidyl ethylene glycol, phosphatidylmethanol, phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol, phosphatidylthioethanol, diphytanoyl phosphatide, egg yolk phospholipid, cardiolipin, glyceroglipid, phosphatidylserine, phosphatidylglycerol and aminoethylphosphonolipid Formulation selected from the group consisting of. The formulation of claim 1, wherein said non-fluorinated surfactant has a low hydrophilic lipophilic balance. 8. The method of claim 7, wherein the non-fluorinated surfactants are both spans®, briss®, guerbet alcohol ethoxylates, dialkyl nonionic surfactants and dialkyls. An agent selected from the group consisting of ionic surfactants. The formulation of claim 1, wherein said non-fluorinated surfactant is a phospholipid consisting of monounsaturated fatty acid residues. 10. The formulation of claim 9, wherein said phospholipid is selected from the group consisting of dioleoyl phosphatidylethanolamine, dioleoylphosphatidic acid and combinations thereof. The method of claim 1, wherein the one or more polar liquid soluble therapeutics or diagnostic agents are respiratory agents, antibiotics, anti-inflammatory drugs, anti-tumor drugs, anesthetics, contrast agents, eye drops, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins And formulations thereof. The formulation of claim 1 further comprising an additive capable of reducing spontaneous curvature of the emulsion. 13. The formulation of claim 12 wherein said additive is selected from the group consisting of monoglycerides, diglycerides, alcohols, sterols, triglycerides, alkanes, Freons®, squalene and mixtures thereof. The method of claim 2, wherein the one or more lipophilic fluorocarbons are halogenated perfluorocarbons, halogenated perfluoroethers, halogenated polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbon ether diblocks and their Formulations selected from the group consisting of mixtures. The method of claim 2, wherein the one or more non-fluorinated surfactants are alcohols, fatty acids, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, phosphatidic acid, phosphatidyl ethanolamine, N, N-dimethyl-phosphatidylethanolamine Phosphatidyl ethylene glycol, phosphatidylmethanol, phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol, phosphatidylthioethanol, diphytanoyl phosphatide, egg yolk phospholipid, cardiolipin, glycerlgolipid, phosphatidylserine, phosphatidylglycerol and aminoethylphosphono A formulation selected from the group consisting of lipids. The method of claim 2, wherein the polar liquid soluble therapeutic or diagnostic agent is a respiratory agent, antibiotic, anti-inflammatory, anti-tumor drug, anesthetic, contrast agent, eye drop, cardiovascular agent, enzyme, nucleic acid, gene, viral vector, protein and combinations thereof. Formulation selected from the group consisting of. Providing a liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; Combining the liquid phase with a fluorocarbon phase consisting of an effective amount of one or more non-fluorinated surfactants and one or more lipophilic fluorocarbons to provide an emulsion formulation; A method of making a therapeutic or diagnostic formulation comprising emulsifying the emulsion formulation to provide a therapeutic or diagnostic formulation. The method of claim 17, wherein the therapeutic or diagnostic agent is thermodynamically stable. 18. The method of claim 17, wherein the polar liquid is selected from the group consisting of water, alcohols, polyethylene glycols, alkyl sulfoxides and mixtures thereof. 18. The method of claim 17, wherein the one or more lipophilic fluorocarbons are halogenated perfluorocarbons, halogenated perfluoroethers, halogenated polyethers, fluorocarbon-hydrocarbon diblocks, fluorocarbon-hydrocarbon ether diblocks and their And selected from the group consisting of mixtures. The method of claim 17, wherein the one or more polar liquid soluble therapeutics or diagnostic agents are respiratory agents, antibiotics, anti-inflammatory drugs, anti-tumor drugs, anesthetics, contrast agents, eye drops, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins And combinations thereof. 18. The method of claim 17, wherein the one or more non-fluorinated surfactants are alcohols, fatty acids, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, phosphatidic acid, phosphatidyl ethanolamine, N, N-dimethyl-phosphatidylethanolamine Phosphatidyl ethylene glycol, phosphatidylmethanol, phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol, phosphatidylthioethanol, diphytanoyl phosphatide, egg yolk phospholipid, cardiolipin, glycerlgolipid, phosphatidylserine, phosphatidylglycerol and aminoethylphosphono The method selected from the group consisting of lipids. 18. The method of claim 17, further comprising an additive capable of reducing spontaneous curvature of the emulsion. A therapeutic or diagnostic agent prepared according to the method of claim 17. A therapeutic or diagnostic agent prepared according to the method of claim 18. A therapeutic or diagnostic agent prepared according to the method of claim 22. A dispersed liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And a pharmaceutical emulsion containing an effective amount of one or more non-fluorinated surfactants; A method of delivering a therapeutic or diagnostic agent to a patient, comprising administering the pharmaceutical emulsion to the patient. The method of claim 27, wherein the pharmaceutical emulsion is a thermodynamically stable microemulsion. The method of claim 27, wherein the one or more non-fluorinated surfactants are alcohols, fatty acids, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, phosphatidic acid, phosphatidyl ethanolamine, N, N-dimethyl-phosphatidylethanolamine, Phosphatidyl ethylene glycol, phosphatidylmethanol, phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol, phosphatidylthioethanol, diphytanoyl phosphatide, egg yolk phospholipid, cardiolipin, glyceroglipid, phosphatidylserine, phosphatidylglycerol and aminoethylphosphonolipid The method is selected from the group consisting of. The method of claim 27, wherein the one or more polar liquid soluble therapeutics or diagnostic agents are respiratory agents, antibiotics, anti-inflammatory drugs, anti-tumor drugs, anesthetics, contrast agents, eye drops, cardiovascular agents, enzymes, nucleic acids, genes, viral vectors, proteins And combinations thereof. The method of claim 27, wherein the pharmaceutical emulsion is administered to the patient by a delivery device selected from the group consisting of an endotracheal tube, an intrapulmonary catheter, and an atomizer. The method of claim 27, wherein the pharmaceutical emulsion is administered to the patient by a route of administration selected from the group consisting of topical, subcutaneous, lung, intramuscular, intraperitoneal, nasal, vaginal, rectal, ear, oral and ocular routes. a) providing a liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; b) combining the liquid phase with an fluorocarbon phase consisting of an effective amount of at least one non-fluorinated surfactant and at least one lipophilic fluorocarbon to provide an emulsion formulation; c) emulsifying the emulsion formulation to provide a therapeutic or diagnostic reverse emulsion; d) adding said therapeutic or diagnostic inverse emulsion to a second polar liquid comprising an effective amount of at least one non-fluorinated surfactant and the same or different from the polar liquid to provide a complex formulation; And e) emulsifying the complex formulation to provide a complex emulsion. The method of claim 28, wherein the polar liquid soluble therapeutic or diagnostic agent is a respiratory agent, antibiotic, anti-inflammatory, anti-tumor drug, anesthetic, contrast agent, eye drop, cardiovascular agent, enzyme, nucleic acid, gene, viral vector, protein and combinations thereof. The method is selected from the group consisting of. A dispersed liquid phase consisting of at least one polar liquid and at least one polar liquid soluble therapeutic or diagnostic agent; A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And an anti-emulsion having an effective emulsifying amount of at least one non-fluorinated surfactant; A method for producing a pharmaceutical dispersion, comprising combining the inverse oil with an lipophilic fluorocarbon to form a dispersion. 36. The method of claim 35, wherein the polar liquid soluble therapeutic or diagnostic agent is a respiratory agent, antibiotic, anti-inflammatory, anti-tumor drug, anesthetic, contrast agent, eye drop, cardiovascular agent, enzyme, nucleic acid, gene, viral vector, protein and combinations thereof. The method is selected from the group consisting of. 36. The method of claim 35, wherein said non-lipophilic fluorocarbon is selected from the group consisting of brominated fluorocarbons, chlorinated fluorocarbons and F-alkanes. The method of claim 35, wherein the one or more non-fluorinated surfactants are alcohols, fatty acids, phosphatidylcholine, N-monomethyl-phosphatidylethanolamine, phosphatidic acid, phosphatidyl ethanolamine, N, N-dimethyl-phosphatidylethanolamine Phosphatidyl ethylene glycol, phosphatidylmethanol, phosphatidylethanol, phosphatidylpropanol, phosphatidylbutanol, phosphatidylthioethanol, diphytanoyl phosphatide, egg yolk phospholipid, cardiolipin, glycerlgolipid, phosphatidylserine, phosphatidylglycerol and aminoethylphosphono The method selected from the group consisting of lipids. A dispersion liquid phase consisting of at least one polar liquid; A continuous fluorocarbon phase consisting of at least one lipophilic fluorocarbon; And Fluorocarbon formulations containing an effective amount of one or more non-fluorinated surfactants.