MXPA97004272A - Seized agents - Google Patents

Abstract

The invention provides the use of an agent to reduce the direct interaction between a hydrophobic phase and a hydrophilic phase, in which the hydrophobic phase is dispersed. Also provided are methods for preparing single phase hydrophobic preparations comprising a hydrophilic species, wherein said agent is added.

Description

KIDNAPPING AGENTS DESCRIPTION OF THE INVENTION The present invention relates to the use of certain compounds to aid in the retention of hydrophilic molecules, solubilized in a phase, in which they will not normally be soluble, in the hydrophobic phase when said hydrophobic phase is dispersed in a hydrophilic phase. In particular, the present invention relates to the use of such agents to assist in the retention of hydrophilic macromolecules in a hydrophobic phase, in which they will not normally be soluble. For many applications, v. gr., in pharmaceutical science, food technology or the cosmetics industry, work with similar proteins and macromolecules presents problems since their hydrophilic character and their high degree of polarity limit the degree to which they can interact with or incorporate into phases of lipid. Many natural systems employ lipid barriers (eg, skin, cell membranes) to prevent the entry of hydrophilic molecules into internal compartments; the ability to disperse proteins in lipid vehicles may open a new route for the introduction of these macromolecules into biological systems, so that the lipid medium containing the protein can be integrated with the hydrophobic constituents of barriers, instead of being excluded by they.

The dispersion of hydrophilic substances in the oil phase instead of aqueous media confers other benefits in terms of increases in its stability with respect to temperature-mediated denaturation, hydrolysis, sensitivity to light, etc. The oils can be chosen to remain fluid on a broader temperature scale than aqueous solutions, or to have a higher viscosity, resulting in greater protection against physical damage. In composite phase systems, the incorporation of hydrophobic substances in oil can mutually limit dangerous interactions, v. gr., oxidation, with other agents, both within the oil phase and in the aqueous phase. There are examples of formulations containing both macromolecules and oil and one such example is described in EP-A-0366277. The formulation described in this document is an emulsion having a hydrophobic as well as a hydrophilic phase, wherein the hydrophilic phase contains lipid-forming or kilomicron-forming lipids. However, the macromolecule dissolves in the hydrophilic phase, not in the hydrophobic phase. EP-A-0521994 also relates to a composition suitable for the oral delivery of macromolecules, which comprises a biologically active material in association with lecithin or a compound capable of acting as a precursor for lecithin in vivo. All of the compositions illustrated are formulations comprising a hydrophilic and a lipophilic phase. Again, in this prior art document, the macromolecule is dissolved in the hydrophilic phase rather than in the lipophilic phase. Although the formulations mentioned above contain both macromolecules and oils, it is important that in all cases, the macromolecule dissolves in the hydrophilic phase rather than in the lipophilic phase. Attempts to form true solutions of macromolecules in oils have met with limited success. Okahata et al. (J. Chem. Soc. Chem. Commun., 1988, 1392-1294) describe a process for solubilizing proteins in a hydrophobic solvent. However, in the protein arrangement surrounded by amphiphilic molecules, produced by said method, the authors established that the amphiphilic molecules reacted with the protein in the liquid medium through hydrogen bonding or via an electrostatic interaction to form a solid precipitate. United Kingdom patent application No. 9323588.5 describes a process by which a hydrophilic species can be solubilized in a hydrophobic solvent, in which it will not normally be soluble. The procedure is based on the surprising discovery that if a hydrophilic species is mixed with an amphiphile under certain conditions, the resulting composition will be readily soluble in lipophilic solvents such as oils. However, a potential problem with the product of said process is related to its use in the production of an emulsion, by dispersing the hydrophobic single-phase preparation in a hydrophilic phase, for example, water. In said dispersion, there will be a tendency, at least in some circumstances, for the solubilized hydrophilic species to "leak" into the hydrophilic phase, reversing the solubilization process. Thus, there is a need to reduce this effect and ensure that the hydrophilic species remains in the hydrophobic phase even when a hydrophobic phase is itself subsequently dispersed in a hydrophilic phase. Surprisingly, it has now been found that certain compounds can reduce the degree of direct interaction between a hydrophilic species solubilized in a hydrophobic phase, and a hydrophilic phase, in which the hydrophobic phase is subsequently dispersed, v. gr., as an emulsion. Thus, in a first aspect, the present invention provides the use of an agent to reduce the direct interaction between a hydrophilic species solubilized in a hydrophobic phase and a hydrophilic phase, in which the hydrophobic phase is dispersed. In the present invention, the term "agent" refers to any species, which is capable of reducing the direct interaction between a hydrophilic species solubilized in a hydrophobic phase, in which it could not be normally soluble, when the hydrophobic phase per se it is dispersed in a hydrophilic phase, for example, to form an emulsion, and the hydrophilic phase. In a second aspect, the present invention provides a single-phase hydrophobic preparation comprising a hydrophilic species solubilized in a hydrophobic solvent, in which it could not normally be soluble, and in addition an agent that reduces direct interaction between the hydrophobic species and a hydrophilic phase, in which the hydrophobic preparation is dispersed. It seems that although the hydrophilic species may be able to come into contact with individual molecules of the hydrophilic phase, it can not be contacted with the volume of the hydrophilic phase and, therefore, this results in a reduced "filtration" of the hydrophilic species towards the hydrophilic phase. In the present invention, the term "hydrophilic species" refers to any species, which is generally soluble in aqueous solvents but insoluble in hydrophobic solvents. Suitably, the agent can be: (i) an acidic lipid, for example, cholesterol hemisuccinate (Chems) or phosphatidic acid; or (ii) an emulsion stabilizer, which can not penetrate the hydrophobic phase, for example, a compound such as casein. In a third aspect, the invention also provides an agent for use in reducing the direct interaction between a hydrophilic species, solubilized in a hydrophobic solvent, in which it can not be normally soluble, and a hydrophilic phase, in which the phase hydrophobic is dispersed, v. gr., as an emulsion. Suitably, the agents described herein are used in a solubilization process as described in United Kingdom Patent Application No. 9323588.5. Thus, in a further aspect, the present invention provides a process for the preparation of a hydrophobic single-phase preparation comprising a hydrophilic species, in a hydrophobic solvent, the process comprising: (i) associating the hydrophilic species with a amphiphile in a liquid medium so that, in the liquid medium, there is no chemical interaction between the amphiphile and the hydrophilic species; (ii) remove the liquid medium to leave a disposition of amphiphilic molecules with their hydrophilic upper groups oriented towards the hydrophilic species; and, (iii) providing a hydrophobic solvent around the hydrophilic species / amphiphilic arrangement; wherein an agent, which reduces the direct interaction between the hydrophilic species and a hydrophilic phase, wherein the hydrophobic phase is dispersed, is added in one or more of the steps. Preferably, in this method the agent is added with the amphiphile in step (i), and is preferably an emulsion stabilizer, which can not penetrate the hydrophobic phase, v. g., a compound such as cholesterol hemisuccinate (Chems) or phosphatidic acid (PA). In the context of the present invention, the term "chemical interaction" refers to an interaction such as a covalent or ionic bond or a hydrogen bond. This does not pretend to include van der Waals forces or other interactions of that order of magnitude. In another aspect, the present invention provides a method for dispersing a hydrophobic single-phase preparation, comprising a hydrophilic species in a hydrophobic solvent, in a hydrophilic phase, which comprises the step of adding, to the hydrophilic phase, an agent which reduces the direct interaction between the hydrophilic species and the hydrophilic phase. In this method, the agent is preferably an emulsion stabilizer, which can not penetrate the hydrophobic phase, v. gr., a compound such as casein. A wide variety of macromolecules can be suitably solubilized according to the present invention. In general, the macromolecular compound will be hydrophilic or at least have hydrophilic regions, since usually there is very little difficulty in the solubilization of a hydrophobic macromolecule in oil solutions. Examples of suitable macromolecules include proteins and glycoproteins, oligo and polynucleic acids for example, DNA and RNA, polysaccharides and supramolecular assemblies of any of these including, in some cases, whole cells, organelles or viruses (complete or parts thereof). It may also be convenient to co-solubilize a small molecule, such as a vitamin, in association with a macromolecule, particularly a polysaccharide such as a cyclodextrin. Small molecules such as vitamin B12 can also be chemically conjugated with macromolecules and can thus be included in the compositions. In particular, when the macromolecule to be stabilized is a protein or polypeptide, the agent is preferably an acidic lipid. Examples of particular proteins, which can be successfully solubilized by the method of the present invention include insulin, calcitonin, hemoglobin, cytochrome C, horseradish peroxidase, fungus aprotinin tyrosinase, erythropoietin, somatotropin, growth hormone, growth hormone releasing factor, galanin, urokinase, Factor IX, tissue plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, Factor VIII, melanin, and fragments of the same (all the above proteins can be formed from any suitable source). Other macromolecules that can be used are dextran labeled with FITC and extract of yeast RNA Torulla. In addition to the macromolecules, the process of the present invention is used to solubilize smaller organic molecules. Examples of the small organic molecules include glucose, ascorbic acid, carboxyfluorescin, and many pharmaceutical agents, for example, anticancer agents, but, of course, the process can also be applied to other small organic molecules, for example, other vitamins or other pharmaceutically or biologically active agents. In addition, molecules such as calcium chloride and sodium phosphate can also be solubilized using the method of the invention. In fact, the present invention could be particularly advantageous for pharmaceutically and biologically active agents, since the use of non-aqueous solutions that can allow the route by which the molecule enters the body can be varied, for example, to increase bioavailability. Another type of species that can be included in the hydrophobic compositions of the invention, is an inorganic material such as a small inorganic molecule or a colloidal substance, for example, a colloidal metal. The process of the present invention allows some of the properties of a colloidal metal, such as gold, palladium, platinum, or colloidal rhodium, to be retained, even in hydrophobic solvents in which the particles could, under normal circumstances, be added. This can be particularly useful for catalysis of reactions carried out in organic solvents. There are numerous amphiphiles, which can be used in the present invention, and zwitterionic amphiphiles such as phospholipids are especially suitable. Phospholipids having a higher phosphatidyl choline group have been used with particular success and examples of such phospholipids include the same phosphatidyl choline (PC), lysophosphatidyl choline (lyso-PC), sphingomyelin, derivatives of any of these, by example, hexadecyl phosphocholine or amphiphilic polymers containing phosphoryl choline and halogenated amphiphiles, v. gr., fluorinated phospholipids. In the present invention, the terms phosphatidylcholine (PC) and lecithin are used interchangeably. Suitable natural lecithins can be derived from any suitable source, for example, egg and, in particular, soybeans. In most cases, it is preferred to select an amphiphile, which is chemically similar to the hydrophobic solvent chosen and this is discussed in detail below. The fact that the present inventors have found that zwitterionic amphiphiles such as phospholipids are particularly suitable for use in the process is a particular indication of the significant differences between the present invention and the method of Okahata et al. Significantly, the authors of that prior art document concluded that the anionic and zwitterionic lipids were completely unsuitable for use in their method, and it was established that they obtained a zero production of their complex using these lipids. The hydrophobic solvent of choice will depend on the purpose for which the composition is intended, on the type of species to be solubilized and on the amphiphile. Suitable solvents include non-polar oils such as mineral oil, squalane and squalene, long chain fatty acids with unsaturated fatty acids, such as oleic and linoleic acids., alcohols being preferred, particularly medium chain alcohols such as octanol and branched long chain alcohols such as phytol isoprenoids, v. g., nerol, and geraniol, terpineol, monoglycerides such as glycerol monooleate (GMO), other esters, for example, ethyl acetate, amyl acetate and bornyl acetate, diglycerides and triglycerides, particularly medium chain triglycerides and mixtures of the same, halogenated analogs of any of the foregoing including halogenated oils, v. g., long chain fluorocarbons or iodinated triglycerides, for example, lipidiol. Optimum results are usually obtained when the hydrophobic solvent and the amphiphile properly match. For example, with a solvent such as oleic acid, lyso-PC, it is a much more suitable choice of amphiphile than PC, while the opposite is true when the hydrophobic solvent is a triglyceride. Furthermore, in some cases, it has been found advantageous to add an amount of the amphiphile to the hydrophobic solvent, before it comes into contact with the hydrophilic species / amphiphilic arrangement. This ensures that the amphiphilic molecules are not separated from their positions around the hydrophilic species due to the high affinity of the amphiphile for the hydrophobic solvent. It is highly preferable that the preparations of the invention be optically transparent, and this can be inspected by measuring the turbidity at visible wavelengths and, in some cases, inspecting the sedimentation for a period. The orientation of the amphiphilic molecules towards an arrangement with their hydrophilic upper groups looking at the portions of a hydrophilic species can be achieved in various ways and particularly particularly suitable examples and methods are discussed in detail. In a first method, which has a starting point similar to the method described by Kirby et al., (Biotechnology, November 1984, 979-984, and Liposome Technology, Volume I, pages 19-27, Gregoriadis, Ed., CMC Press, Inc., Boca Raton, Florida, USA) a hydrophilic species is mixed with a dispersion of an amphiphile in a hydrophilic solvent, so that the molecules of the amphiphile form an assembly, in which the hydrophilic upper groups look out towards the hydrophilic phase, which contains the hydrophilic species. The hydrophilic solvent is then removed to leave a dry composition, in which the hydrophilic upper groups of the amphiphilic molecules are oriented towards the hydrophilic species. In the method described by Okahata et al., A solution of a protein was also mixed with a dispersion of an amphiphile in water. However, significantly, the authors of that document believed that it was necessary to obtain a precipitate, which could then be soluble in hydrophobic solvents. Since many of the preferred amphiphiles of the present invention do not form such a precipitate, Okahata and others concluded that they would not use it. In the process of the present invention, no precipitate is required and, in fact, it is generally believed that it is undesirable to allow the formation of a precipitate, since this results in a reduced production of the required product.

In this first method, it is preferred that the hydrophilic solvent is water, although other polar solvents may be used. The form taken by the amphiphilic assembly can be that of micelles, unilamellar vesicles, preferably small unilamellar vesicles, which are generally understood to have a diameter of about 25 nm, multilamellar vesicles or tubular structures, for example structures of snail cylinders, hexagonal phase, cubic or myelin phase. The form adopted will depend on the amphiphile, which is used and, for example, amphiphiles such as phosphatidyl choline (PC) tend to form small unilamellar vesicles, while lyso-phosphatidyl choline forms micelles. However, in all these structures, the hydrophobic ends of the amphiphilic molecules look inward toward the center of the structure, while the upper hydrophilic groups look outward toward the solvent where the hydrophilic species is dispersed. The weight ratio of amphiphile: hydrophilic species will generally be in the region of 1: 1 to 100: 1, preferably 2: 1 to 20: 1, and most preferably of 8: 1, approximately, for PC, and of 4 : 1 for smooth-PC. These relationships are only preferred relationships and, in particular, it should be noted that the upper limit is set by economic considerations, which means that it is preferable to use the minimum possible amount of amphiphile. The lower limit is a bit more critical, and it is likely that ratios of 2: 1 or less can be used only in cases where the hydrophilic species has a significant hydrophobic portion or is exceptionally large. Good performance is obtained when the solvent is removed quickly, and a convenient method for solvent removal is lyophilization, although other methods can be used. In some cases, it may be helpful to include salts in the hydrophilic solution, particularly if the hydrophilic species is a macromolecular compound such as a large protein. However, since the presence of larger amounts of inorganic salts tends to increase the formation of crystals and, therefore, to a hazy solution, it is preferred to use organic salts instead of inorganic salts, such as sodium chloride. Ammonium acetate is especially suitable for this purpose, since it has the additional advantage that it is easily removed through freeze drying. A second method for the preparation of a composition containing an arrangement of amphiphiles with their upper groups pointing towards the portions of the hydrophilic species, is to co-solubilize the hydrophilic species and the amphiphile in a common solvent, followed by the removal of the solvent. The product of the process of the invention is new, and therefore, in a further aspect of the invention, a single phase hydrophobic preparation comprising a hydrophilic species in a hydrophobic solvent obtainable through the process of the invention is provided. It is also desirable to include other constituents in the single phase hydrophobic preparation, in addition to the hydrophilic species. This is particularly appropriate when the hydrophilic species is a macromolecule and, in such case, the preparation may include for example, bile salts, vitamins or other small molecules, which bind to or otherwise be associated with the macromolecules. Although some macromolecule / amphiphile arrangements were described by Kirby et al., Supra, the described arrangements were all intermediain the formation of liposomes and, as discussed above, there has been no prior interest in non-liposomal or hydrophobic compositions comprising this type of entity. Therefore, the provisions of the present invention, in which the amphiphile is one that does not form small unilamellar vesicles and, therefore, is not expected to form liposomes, they're new. An advantage of the preparations of the present invention is that they are effectively anhydrous and, therefore, more stable to hydrolysis. In the case of proteins, they are also stable to freeze-thaw, and have greater stability at high temperatures, probably because water must be present in order for the protein to unfold and denature. This means that they can be expected to have a longer storage life than the aqueous preparations of the hydrophilic species. The solutions of the present invention are extremely versatile and have many applications. They can be used either alone, but are prebly combined with an aqueous phase to form an emulsion or a similar two-phase composition, which forms a further aspect of the invention. In this aspect of the invention, a two-phase composition comprising a hydrophilic phase and a hydrophobic phase is provided, the hydrophobic phase comprising a preparation of a hydrophilic species in a lipophilic solvent obtainable through the process, as described at the moment. Generally, in this type of composition, the hydrophobic phase will be dispersed in the hydrophilic phase. The two-phase compositions may be emulsions, which may be either transient or stable, depending on the purpose for which they are required. The average particle size of the emulsion will depend on the exact nature of both the hydrophobic phase and the aqueous phase. However, it can be in the region of 2 μm. The dispersion of the hydrophobic preparation in an aqueous phase can be achieved through mixing, for example, either through vigorous stirring for a short time, for example, from about 10 to 60 seconds, usually about 15 seconds, or through moderate mixing for several hours, for example using an orbital shaker.

In another aspect, the present invention provides a process for preparing a dispersion, e.g., an emulsion, of a hydrophobic phase, in which a hydrophilic species is solubilized, which comprises dispersing the hydrophobic phase into a hydrophilic phase, which has been added an agent that reduces the direct interaction between the hydrophilic species, when it is thus solubilized and the hydrophilic phase in which the hydrophobic phase is dispersed. Now, the invention will be described with reference to the following examples, which should not be construed as limiting the invention.

EXAMPLE 1 A borate regulator of 0.0015M was prepared, dissolving 60 mg of sodium tetraborate in 100 ml of distilled water, and adjusting the pH to 8.00. 5 mg of BAPNA was charged to a screw-capped B9 glass bottle and dissolved in 3 ml of methanol. 10 mg of trypsin was charged to a 15 ml plastic centrifuge and 10 ml of stirring borate buffer was added. The suspension was mixed in a roller mixer, then the material was centrifuged and the supernatant was decanted. The dilutions of aprotinin (50 μl / well) were dispensed along the rows of the microplate between 0 and 30 μg / ml concentration. The BAPNA solution, from the above, was diluted 20 times by adding 1 ml to 20 ml of pH buffer and 100 μl of a working BAPNA solution was then introduced into each well and mixed thoroughly. The plate was incubated with shaking at 37 ° C for 40 minutes and then read on a plate reader at 405 nm. After plotting the optical density due to the conversion of the substrate against the concentration of aprotinin in the cavity, an inflection was observed at the concentration at which aprotinin is just sufficient to neutralize the activity of the trypsin. The position of this inflection is moved according to the amount of additional aprotinin introduced into the cavities in the test sample, and this concentration can be inferred through comparison with normals. Thus, an indication of the proportion of aprotinin released from the oil and accessible to the aqueous phase can be obtained. Aprotinin was solubilized in Migiyol 818 by introducing a mixture of 100 μl of a soy phosphatidyl choline dispersion (100 mg / ml in distilled water), sound was applied as for the protocol of Example 4, and μl of a solution of aprotinin (20 mg / ml in distilled water), followed by the addition of 100 μl of Migiyol 818. The concentration of aprotinin was 5 mg / ml in oil. A control solution was prepared as before, in which aprotinin was omitted. Aqueous dispersions of these oils were prepared by stirring 10 μl of each oil with 1 ml of borate buffer for 10 seconds. The final concentration of aprotinin in these secondary dispersions was 0 and 50 μg / ml. The dispersions were diluted twice and added to the cavities of a microplate as described in the previous method, where dilutions of 0, 5, 10, 15 and 25 μg / ml were used. Normalized optical densities were reported in the Table presented below, and in the attached graph. The comparison with a normal control of 12.5 μg / ml indicates that at least 50% of aprotinin is released from the oil into the aqueous phase.

Nature of Aprotinin Oil Concentration +/- aprot 0 5 10 15 20 25 - / PC / M818 0.373 0.358 0.343 0.3 0.055 0 Aprot / PC / M818 0.269 -0.004 0.05 0.058 -0.03 0 12.5 μg / ml 0.337 0.299 0.135 -0.008 -0.017 0 aprotinin Aprot / PC / M818 0.332 0.264 0.201 -0.036 -0.055 dil. 2 times EXAMPLE 2 Aprotinin was solubilized in Migiyol 818, as described in the previous Example, except that the phospholipid dispersion contained 10% by weight of phosphatidic acid, in addition to the phosphatidyl choline. The dispersions were tested net, and compared with the control normal of 25 μg / ml and 12.5 μg / ml. Comparisons with these normals indicate that no more than 25% of the aprotinin was released into the aqueous phase.

Concentration of 20 15 10 aprotinin (μ / ml) pH regulator 0.134 0.26 0.273 0.283 0.277 25 μg / ml of 0.003 0.005 0.068 0.167 aprotinin 12.5 μg / ml of 0 -0.009 0.202 0.258 0.258 aprotinin PC: PA / M818 - 0.099 0.182 0.164 0.194 0.216 PC: PA / M818 - 50 0 0.059 0.182 0.219 0.245 μg / ml EXAMPLE 3 Aprotinin was solubilized in Migiyol 818 as described in the previous examples, except that the phospholipid dispersion contained 10% by weight of cholesterol hemisuccinate (Chems) in addition to phosphatidyl choline. The dispersions were tested neat, and compared with control normals of 25, 12.5 and 6.25 μg / ml. The comparison with these normals indicates that no more than 12.5% of the aprotinin is released into the aqueous phase. 60 min reading Apoprotinin concentration (μg / ml) Test samples 0 6 11 15 18 20 22 24 25 30 0 μg / ml 0.272 0.192 0 μg / ml 0.265 0.102 0.002 0.008 0.015 0.015 0.008 0.001 0 12.5 μg / ml 0.267 0.266 0.241 0.044 0.007 6.25 μg / ml 0.285 0.269 0.195 0.236 -0.003 PC: Chems / M818 / - 0.157 0.165 0.156 0.165 0.152 0.167 0.166 0.114 0.138 0 PC: Chems / M818 / 0.192 0.213 0.195 0.22 0.203 0.197 0.14 0.142 0.062 0 Aprot (50 μg / ml) EXAMPLE 4 An aqueous dispersion of soy phosphatidyl choline (PC soy) containing 100 mg / g of a suspension was prepared, completely flooded with nitrogen, and an amplitude of 8 micras was applied, peak to peak. Each aliquot was subjected to a total sound application time of 4 minutes, in 30 second pulses interspersed by cooling for 30 seconds in an ice mud bath. The resulting opalescent dispersion of small unilamellar vesicles (SUV) was then centrifuged for 15 minutes to remove the titanium particles. A colloidal gold solution was prepared as follows. 15 μl of 25 mM potassium carbonate, 15 μl of 1% tannic acid and 50 mg of trisodium citrate were developed to a total weight of 22.5 g with distilled water and 10 ml were transferred to a 25 ml corked glass flask ( A) and heated to 60 ° C in a water bath. 25 mg of gold chloride trihydrate was developed up to 250 mg with distilled water and 50 μl of the resulting solution was added to 40 ml of distilled water in a conical 50 ml conical glass flask (B) and heated to 60 ° C. C in the same b year of water. The contents of flask A were mixed with those of flask B, and heating was maintained for 75 minutes during which time a deep red colloidal gold solution was formed. After cooling to room temperature, a 10 ml portion was stabilized by mixing with 2 mg of bovine serum albumin. 1 ml of the stabilized gold solution was mixed with 0.6 ml of SUV, dried by freezing overnight and the resulting lyophthyl was dispersed by stirring with 300 mg of Migiyol 818. In one hour, a clear red dispersion was formed of colloidal gold in Migiyol. Three aliquots of 50 mg of this dispersion were added to small glass jars and then 500 mg of water, glucose solution regulated in its pH with phosphate (300 mM glucose containing 1 mM sodium phosphate, pH 7.4) and SUV were added to the jars separately. The mixtures were emulsified by stirring for 10 seconds and then observed. In one hour, the cream formation of the emulsions was started, and then it was left to rest overnight, this happened to a substantial degree. In all cases, a pink coloration of the lower aqueous phase was observed indicating the release of a proportion of the colloidal gold from the oil phase. However, the intensity of the color retention in the upper phase of oil emulsion was significantly higher, and that in the corresponding lower aqueous phase, in the preparation emulsified in the presence of SUV. Thus, the presence of the phospholipid SUV dispersion apparently served to reduce the loss of colloidal gold from the oil phase.

EXAMPLE 5 1ml of a 1% insulin solution (containing 2% acetic acid to aid dissolution) was mixed with 1μCi of 125 I-labeled insulin, followed by 3g of SUV-containing cholesterol hemisuccinate prepared as in Example 3 The mixture was freeze-dried and the resulting lyophilate was dispersed with 3 g of Migiyol 818, mixing for 4 hours on an orbital shaker to produce a clear dispersion of radiolabelled insulin in oil. Two aliquots of 200 mg of dispersion were each mixed with 800 mg of pH regulated saline with phosphate (PBS), and emulsified by stirring for 10 seconds and 30 seconds, respectively. Each of the resulting oil / water emulsions was diluted with an additional 9 ml of PBS and then centrifuged for 40 minutes at 80000 g to break it into its component fractions. The oil phase surface layer was carefully transferred to a bottle to count the gamma radioactivity due to insulin labeled with residual I125. The supernatant, containing any insulin labeled with I 125 liberated, was transferred to a separate container, leaving behind any pellets that could have formed. The pellet, representative portions of the supernatant and the fraction of residual oil, were all counted for radioactivity, making appropriate corrections for any contamination of the oil fraction with the supernatant. Of the two emulsions, the one that was stirred for 30 seconds showed 45.4% of the radiolabel retained within the oil phase and 3.0% was associated with the centrifuge pellet (assumed as liposomal by nature), while the corresponding figures for the second Scrambled emulsion were 43.3 and 1.3%, respectively. In contrast, in two separate experiments, where the SUV was used to prepare the oil dispersion were composed of pure soy PC instead of PC / soy Chems, the brand oil retentions were 31% and 28% , with an additional 1% in the pellet in each case. Thus, it appears that the inclusion of Chems in the amphiphilic systems used to prepare the protein in oil dispersions leads to an increased retention of the protein within the oil, when the latter is emulsified to form secondary dispersions of a / to.

EXAMPLE 6 Insulin labeled with 125 I was prepared and incorporated into oil phases, as described in Example 3, but using the compositions listed below.

Preparation No. Amphiphilic System Oil Phase 1 SUV of Soy PC Myglyol 818 2 PC soy / Phosphatidic acid Migiyol 818 (PA) SUV * PC / PA SUV were prepared as in Example 2. An aliquot of 100 mg of Preparation 1 and one of Preparation 2 were loaded into 10 ml centrifuge tubes. To each aliquot was added 1 ml of PBS. All were thoroughly stirred for 10 seconds and then diluted with 10 ml of PBS. The emulsions were then centrifuged and fractionated to their component phases as described in Example 2.

Preparation No. Dispersant% retention in each fraction Pella Oil Supernatant 1 PBS 27.6 1.5 70.9 2 PBS 26.1 20.9 53.0 The pellet obtained is believed to be composed of oil containing a high proportion of phospholipid. In the preparation containing phosphatidic acid, considerably less of the radiolabelled insulin was released into the aqueous supernatant than with the preparation containing just phosphatidyl choline only.

EXAMPLE 7 Insulin labeled with 125 I was prepared and incorporated into oil phases, as described in Example 2, but using the composition listed below.

Preparation No. Amphiphilic System Oil Phase SUV of PC soybean Oleic acid PC / PA SUV were prepared as in Example 2. A 100 mg aliquot of Preparation 1 was loaded into a 10 ml centrifuge tube, and 1 ml of a 0.5% casein solution was added and thoroughly stirred for 10 minutes. seconds. The contents of the tube were then diluted with 10 ml of 0.5% casein. The emulsion was then centrifuged and fractionated to its component phases, as described in Example 2.

Preparation No. Dispersant% retention in each fraction Oil Pella Supernatant 0.5% casein 63.3 4.7 32 As can be seen, a significant proportion of the radiolabeled insulin was retained within the oil phase.

EXAMPLE 8 0.8 ml of 25 mM calcium chloride was mixed with 0.8 ml of soy PC SUV prepared as in Example 1, dried by freezing overnight and the resulting lyophilate was mixed with 0 5 g of Migiyol 818 after To stand overnight, a completely transparent dispersion was formed. A portion of the dispersion was colored by mixing 150 mg together with approximately 0.17 mg Sudan 4 dye to form a clear, deep red solution. 2 ml of 1% sodium alginate was transferred to a glass test tube and briefly stirred, while, at the same time, the color oil dispersion was added from a Pasteur pipette. The resulting opaque emulsion was centrifuged at 500 g for 5 minutes and the pinkish, oil-rich upper phase was decanted from the underlying clear aqueous phase and examined under the light microscope. All the oil could now be seen to be present as numerous discrete, small drops, which showed no sign of coalescence. A proportion of the oil droplets appeared to be surrounded by an outer wall, which was presumed due to the formation of interfacial complex of the alginate by the calcium ions released from the oil droplets. After standing for 12 days, no signs of breakdown of the emulsion were observed, neither micro- nor macroscopically.

Claims (24)

1. - The use of an agent to reduce the direct interaction between a hydrophilic species solubilized in a hydrophobic phase, and a hydrophilic phase, in which the hydrophobic phase is dispersed, where the hydrophobic phase comprises an arrangement of amphiphilic molecules with their higher groups hydrophilic oriented towards the hydrophilic species dispersed in a hydrophobic solvent.

2 - The use according to claim 1, wherein the agent is an emulsion stabilizer, which can not penetrate the hydrophobic phase or an acid lipid.

3. The use according to claim 2, wherein the agent is cholesterol hemisuccinate (Chems), phosphatidic acid (PA) or casein.

4. A hydrophobic single-phase preparation comprising an arrangement of amphiphilic molecules with their hydrophilic upper groups oriented towards a hydrophilic species dispersed in a hydrophobic solvent, characterized in that the preparation comprises an agent that reduces the direct interaction between the hydrophilic species and a hydrophilic phase, in which the hydrophobic preparation is dispersed.

5. A process for the preparation of a hydrophobic single-phase preparation comprising a hydrophilic species in a hydrophobic solvent, the process comprising either: A. i) mixing the hydrophilic species with a solution or dispersion of the amphiphile in a solvent; ii) remove the solvent to leave a dry composition, in which the hydrophilic upper groups of the amphiphilic molecules are oriented towards the hydrophilic species; and iii) providing a hydrophobic solvent around the hydrophilic species / amphiphilic arrangement; or B. i) emulsifying a solution of the amphiphile in a hydrophobic solvent with a solution of the hydrophilic species in a hydrophilic solvent to give an emulsion; and ii) removing the hydrophobic solvent, characterized in that it is an agent that reduces the direct interaction between the hydrophilic species and a hydrophilic phase, in which the hydrophobic preparation is dispersed in one or all of the steps.

6. A method according to claim 5, wherein the agent is an acid lipid.

7. A process according to claim 6, wherein the agent is a cholesterol hemisuccinate (Chems) or phosphatidic acid (PA).

8. A method for dispersing a hydrophobic single-phase preparation comprising an arrangement of amphiphilic molecules with their hydrophilic upper groups oriented towards a hydrophilic species dispersed in a hydrophobic solvent, in a hydrophilic phase, characterized in that the process further comprises the step of adding an agent to the hydrophilic phase, which reduces the direct interaction between the hydrophilic species and the hydrophilic phase.

9. A process according to claim 8, wherein the agent is an emulsion stabilizer, which can not penetrate the hydrophobic phase.

10. A method according to claim 9, wherein the agent is casein.

11. The use according to any of the claims 1 to 3, a preparation according to claim 4 or a method according to any of claims 5 to 10, wherein the hydrophilic species is selected from the group consisting of proteins, glycoproteins, oligo- or polynucleic acids, polysaccharides and supramolecular assemblies of these.

12. The use, preparation or procedure according to claim 11, wherein the hydrophilic species is insulin, calcitonin, hemoglobin, cytochrome C, horseradish peroxidase, aprotonin, fungal tyrosinase, erythropoietin, somatrotopin, hormone growth, growth hormone release factor, galanin, urokinase, Factor IX, tissue plasminogen activator, peroxide dismutase, catalase, peroxidase, ferritin, interferon, Factor VIII, melanin, fragments of any of the foregoing, DNA, RNA, dextran labeled with FITC or vitamin B12.

13. The use, a preparation or the process according to any of claims 1 to 12, wherein the amphiphile is a phospholipid.

14. The use, preparation or process according to claim 13, wherein the phospholipid has an upper group of phosphatidyl choline.

15. The use, preparation or process according to claim 14, wherein the phospholipid is phosphatidyl choline (PA), lysophosphatidyl choline (lyso-PA), sphingomyelin, a derivative of one of the above, such as hexadecyl phosphocholine or an amphiphilic polymer containing phosphoryl choline.

16. A preparation or process according to any of claims 4 to 15, wherein the hydrophobic solvent comprises a long chain fatty acid, a medium chain alcohol, a branched long chain alcohol, a monoglyceride, diglyceride, medium chain triglyceride, or a long chain triglyceride.

17. A preparation or a process according to claim 16, wherein the phospholipid comprises PC and the hydrophobic solvent is a triglyceride, or wherein the phospholipid comprises lyso-PC and the hydrophobic solvent is oleic acid.

18. A method according to claim 5, wherein method A is used and in step (i), the hydrophilic species is mixed with a dispersion of the amphiphile in water.

19. A process according to claim 18, wherein the amphiphilic assembly comprises micelles, unilamellar vesicles, multilamellar vesicles, or a tubular structure, such as structures of snail cylinder type, hexagonal phase, cubic phase or myelin type

20. A process according to claim 18 or 19, wherein the water is removed by lyophilization.

21. A process according to claim 5, wherein method B is used and wherein the weight ratio of amphiphile to hydrophilic species is from about 1: 1 to 50: 1.

22. A process according to claim 21, wherein the emulsion is a water-in-oil emulsion.

23. A process according to claim 21 or 22, wherein the hydrophobic solvent is an organic ether of low melting point.

24. A hydrophobic single-phase preparation of a hydrophilic species in a hydrophobic solvent obtainable through a process according to any of claims 5, 7, or 11 to 23. 25.- A composition of two phases comprising a hydrophilic phase and a hydrophobic phase, wherein the hydrophobic phase comprises a preparation according to claim 4 or claim 24. 26.- A composition according to claim 25, wherein the hydrophobic phase is dispersed in a continuous hydrophilic phase. 27 - A composition according to claim 25 or 26, which is an emulsion. 28 - The use of a preparation according to claim 4, any of claims 1 to 17, a preparation according to claim 24 or a composition according to any of claims 25 to 28 in the oral delivery of a hydrophilic species. SUMMARY The invention provides the use of an agent to reduce the direct interaction between a hydrophobic phase and a hydrophilic phase, in which the hydrophobic phase is dispersed. Methods are also provided for preparing hydrophobic single-phase preparations comprising a hydrophilic species, wherein said agent is added.