WO2012152707A1 - Microemulsions - Google Patents

Abstract

The present invention relates to preparations of substances in hydrophobic solvents in which they would not normally be soluble and to processes for obtaining these preparations. In particular, the invention relates to preparations of hydrophilic species in hydrophobic solvents such as oils and an amphiphile comprising a polyoxyethylene - 3 headgroup and a straight - chain hydrocarbon containing twelve carbons such as polyoxyethylene - 3 dodecyl ether with an HLB of 8.3. The use of these preparations in vaccines and other pharmaceutical compositions is also described.

Description

MIC OEMULSIONS

The present invention relates to preparations of substances in hydrophobic solvents in which they would not normally be soluble and to processes for obtaining these preparations. In particular, the invention relates to preparations of hydrophilic species in hydrophobic solvents such as oils.

The invention in particular applies to hydrophilic macromolecules that would not normally be soluble in oils or other hydrophobic solvents.

For many applications, e.g. in the pharmaceutical sciences, in food technology or the cosmetics industry, work with proteins and similar macromolecules presents problems because their hydrophilicity and high degree of polarity limit the extent to which they can interact with or incorporate into lipid phases. Many natural systems employ lipidic barriers (e.g. skin, cell membranes) to prevent access of hydrophilic molecules to internal compartments; the ability to disperse proteins in lipidic vehicles would open up a new route to introduction of these macromolecules into biological systems, whereby the lipid medium containing the protein can integrate with the hydrophobic constituents of barriers, instead of being excluded by them.

Another area where dissolution of proteins into oils may confer advantage is for the use of enzymes in organic phases. Enzymic syntheses are becoming increasingly important compared to chemical processes because of their much lower energy needs, greater substrate and product specificities, high yields, and the fact that many reactions are catalysed which are impossible by chemical means. Recent findings that enzymes can remain active in organic environments have opened up many additional possibilities. Thus, reactions involving lipophilic substrates and products may be catalysed effectively, and enzyme stability is often much greater than in aqueous environments, allowing them to be used in much more extreme conditions such as at high temperature. A very important aspect is that reactions involving hydrolytic enzymes such as lipases and peptidases can preferentially go in the reverse direction in low water environments, thus enabling the synthesis of a wide range of industrially important compounds. Another application is where a complex chain of reactions is involved in which the multiple catalytic units need to be maintained in close proximity to each other. Such might be the case in light-initiated redox reactions. An additional possibility is the controlled production of nanoparticulates in oil phase, using enzymes to induce mineralisation by action on organometallic substrates. The preparation of a stable dispersion of preformed nanoparticulates in oil phase may also be advantageous for the performance of certain surface- catalysed reactions. Dispersion of hydrophilic substances in oil phase rather than aqueous media confers other benefits in terms of increasing their stability with respect to temperature-mediated denaturation, hydrolysis, light sensitivity etc. Oils can be chosen which remain fluid over a wider temperature range than aqueous solutions, or that have a higher viscosity, resulting in greater protection against physical damage. In mixed-phase systems, sequestration of proteins in oil can limit mutually harmful interactions - e.g. oxidation - with water-soluble compounds.

Water- in-oil micro-emulsions are well known (for example WO95/13795). However, only certain organic phases, e.g. low molecular weight oils such as cyclohexane, octane or decane, can be used to make these compositions. Oil phases composed of longer chain, less volatile hydrocarbons such as squalane, squalene, mineral oil, or molecules where hydrocarbon chains are linked together (e.g. triglycerides) without the introduction of a polar component, are in general not readily amenable to forming water- in-oil microemulsions. In order to construct microemulsions using these oils, it is often necessary to combine a number of surfactants together with co- surfactants such as alcohols. However, for many pharmaceutical purposes, inclusion of volatile alcohols such as ethanol or pentanol is undesirable, since since there is a risk that the concentration of these components may change on storage (eg due to evaporation), or that they may induce destruction or denaturation of active agents incorporated therein, such as peptides or proteins.

In cases where such oils, containing higher molecular weight lipids, have been used to construct water-in-oil microemulsions, (e.g. for industrial purposes) these preparations do not contain significant quantities of proteins or other macromolecules encapsulated in the aqueous droplets. It is generally found that microemulsions composed of pharmaceutical acceptable agents, which can form using water alone, become unstable when protein is included in the aqueous phase. This is illustrated in the example 1 provided later. Encapsulation of proteins in oil phase is a very important method for achieving enhanced delivery of these proteins to specific organs in the body such as cells in the small intestine. In this respect, mineral oil has a greater degree of hydrophobicity than the shorter chain hydrocarbons, and microemulsions constructed of mineral oil as the single oil component are better at delivering macromolecules to antigen-presenting cells.

In cases where such micro-emulsions using mineral oil may be obtainable, the resulting composition is usually not capable of self-emulsifying, or the compound encapsulated within the discrete aqueous phase (i.e. the aqueous droplets) readily leaks out into the bulk aqueous phase when the formulation is dispersed in water. Both of these properties are highly disadvantageous if one is wanting to use the formulation as a carrier vehicle, for example in the field of vaccines administered parenterally or orally. Formulations in which discrete hydrophilic phases, dispersed as nano structures throughout an oil phase as a result of inclusion of amphiphiles, have been described before in the prior art (EP2044930, EP1597973 & WO2008/145183, all assigned to Nestec SA). However, there is no hydrophilic species entrapped within the discrete aqueous phase. Furthermore, in the methods described for manufacture of preparation of the Nestec, there is no way in which these could be adapted to include hydrophilic species within the oil phase without those same species being present in equal concentration in the aqueous phase external to the oil droplets. Thus, hydrophilic materials cannot be specifically entrapped within the oil phase. Indeed, the only 'active agents' incorporated into the oil in Nestec (WO2008/145183) are described as 'volatile aroma', all of which have a significant degree of lipophilicity, and whose retention within the formulations described is only partial, and temporary.

In the method of manufacture of the formulations described by Nestec, the oil comprising amphiphiles is added to a large volume of water, then emulsified very vigorously with the use of high-shear mechanical liquid processing equipment. Nestec also specify a requirement for use of amphiphiles with an HLB value of 10 or less.

The formulations described by Nestec have been created with different aims in mind. These include (i) modification of the ability of triglycerides to be digested by lipases, and (ii) improved absorption and controlled release of volatile agents.

Thus, for pharmaceutical purposes, particularly where targeted delivery to cells is desirable, new methods of preparing water-in-oil microemulsions are required which employ oils composed completely or substantially of hydrocarbon components, which employ pharmaceutically acceptable amphiphiles, which avoid the use of alcoholic co-surfactants, and are able to incorporate and retain significant levels of protein or other macromolecules in their aqueous phase. The methods of the present invention, are suited to creating compositions for use as carrier vehicles of pharmaceuticals and vaccine antigens, something which cannot be accomplished by Nestec, employing the methods described therein.

For the formation of oil-in-water emulsions, clearly defined rules have been established (initially by WC Griffin in: "Classification of Surface-Active Agents by 'HLB,'" J. Soc. ofCosm. Chemists 1 (1949): 31 1, and "Calculation of HLB Values of Non-Ionic Surfactants," J. Soc. of Cosm. Chemists 5 (1954): 259) setting out the relationship between hydrophilic-lipophilic balance (HLB) and the nature of the oil in order to form dispersions under any set of circumstances. Unfortunately, no such set of rules exists for the formation of water-in-oil microemulsions, and one skilled in the art has no way of predicting whether a particular amphiphile will work for any particular oil. This is especially the case with longer-chain or more hydrophobic oils, such as those described above.

Surprisingly, however, one amphiphile has now been discovered which is able to form hydrophilic species-containing micro-emulsions spontaneously in mineral oil, in spite of the fact that other amphiphiles of similar structure and HLB are unable to do so. This amphiphile is polyoxethylene 3 docecyl ether (POE3-C12).

Thus in the first aspect the present invention provides a composition comprising a hydrophilic species containing microemulsion comprising a high molecular weight hydrophobic phase, and an amphiphile comprising a polyoxyethylene 3 headgroup, and a straight-chain hydrocarbon containing twelve carbons as the sole emulsion-forming amphiphile. A microemulsion as used herein refers to a thermodynamically or kinetically stable liquid dispersion of an oil phase and a hydrophilic phase. The dispersed phase typically comprises small particles or droplets giving a transparent or translucent appearance, in contrast to regular emulsions which are turbid.

As used herein the term "hydrophobic phase" or "hydrophobic solvent" is defined as a non water-miscible oil composed completely or substantially of hydrocarbon components. A "high molecular weight hydrophobic phase" as used herein refers to a non- volatile hydrophobic phase, such as those having more than 10 carbon atoms per molecule. Thus this term does not include decane, octane, cyclohexane etc. Suitable hydrophobic phases include vegetable oils such as peanut oil, safflower oil, soya bean oil, cotton seed oil, corn oil, olive oil, almond oil, sesame oil, coconut oil, castor oil, chaulmoogra oil, persic oil, isopropyl myristate, mineral oil including light paraffin, squalene, squalane or mixtures thereof. Alternatively triglycerides including triglycerides of the fractionated plant fatty acids or mixtures thereof can be used. For examples mixtures of Caprylic, Capric, and Linoleic triglycerides such as Miglyol 818™ or mixtures of Propyleneglycol, dicarprylate and dicarprate such as Miglyol 840™ can be used. Other oils, which are normally solid at room temperature (eg tristearin, trilaurin or paraffin wax), can also be employed, if the step of addition of hydrophilic phase to oil is performed at a temperature above the melting point of the oil concerned.

In a second aspect of the present invention there is provided a process for the preparation of a composition, the process comprising mixing solutions of (a) polyoxethylene 3 docecyl ether dissolved in a hydrophobic solvent and (b) a hydrophilic species such as a protein dissolved in a hydrophilic phase to form a microemulsion. The amphiphile is dissolved in oil at a level of up to lOOmg/ml total solute in oil. The macromolecule, preferably an antigen and/or immunomodulator, is dissolved in water or other suitable aqueous phase such as DMSO, usually at a concentration of 10-20mg/ml. The aqueous phase is then added to the oil phase, preferably in the ratio of 1 : 10 vol/vol, giving a clear dispersion after brief mixing by vortexing. Thus, a small amount of liquid is added to the oil/amphiphile phase, which then forms a clear microemulsion simply by gentle mixing by hand. This is in contrast to the methods described by Nestec whereine the oil comprising amphiphiles is added to a large volume of water, then emulsified very vigorously with the use of high-shear mechanical liquid processing equipment.

The surprising nature of this discovery is highlighted by the fact that other amphiphiles of similar structure and virtually identical HLB are completely unable to performing the same function in terms of supporting the formation of micro-emulsions. Polyoxethylene 3 docecyl ether has an HLB of 8.3, and forms microemulsions with a wide range of hydrophilic phases, including transcutol, water and solutions of proteins in salt solution. In contrast, polyoxethylene 4 hexadecyl ether, with an HLB of 8.4 is completely unable to form water-in-oil microemulsions with any aqueous phase. Unexpectedly, however, microemulsions can be formed using mixtures of amphiphiles which have the same mean HLB as polyoxethylene 3 docecyl ether, providing that either the length of carbon chain, or the length of polyoxyethylene (POE) chain is conserved, similar to that of polyoxethylene 3 docecyl ether. Data supporting these observations are shown in the examples below.

Thus, a third aspect of this invention is a composition comprising a hydrophilic species containing microemulsion comprising a hydrophobic phase, and with a mixture of amphiphiles having a mean hydrophilic-lipophilic balance (HLB) between 8 and 8.5, and comprising either

(i) polyoxyethylene headgroups with between 2 and 6 subunits, and a straight- chain hydrocarbon containing twelve carbons, or

(ii) a polyoxyethylene 3 headgroup, and a straight-chain hydrocarbons containing between ten and fourteen carbons. No other amphiphilies are required to from the emulsion.

The amphiphiles can have polyoxyethylene headgroups with between 2 and 6 subunits, preferably 2, 3, 4, 5 or 6 POE subunits and a straight-chain hydrocarbon containing twelve carbons. Alternatively the amphiphiles can have a polyoxyethylene 3 headgroup, and a straight-chain hydrocarbon containing between ten and fourteen carbons, for example 10, 1 1, 12, 13 or 14 carbons. In a fourth aspect of the present invention there is provided a process for the preparation of a composition, the process comprising mixing solutions of

(a) a mixture of amphiphiles dissolved in a hydrophobic solvent and

(b) a hydrophilic species dissolved in a hydrophilic phase to form a microemulsion, wherein said mixture of amphiphiles has a mean hydrophilic- lipophilic balance (HLB) between 8 and 8.5, and comprising either

(i) polyoxyethylene headgroups with between 2 and 6 subunits, and a straight- chain hydrocarbon containing twelve carbons as the sole emulsion-forming amphiphiles, or

(ii) a polyoxyethylene 3 headgroup, and a straight-chain hydrocarbons containing between ten and fourteen carbons as the sole emulsion- forming amphiphiles.

The amphiphiles are dissolved in a hydrophobic solvent at a level of up to lOOmg/ml total solute in the hydrophobic solvent. The aqueous phase is then added to the hydrophobic phase, preferably in the ratio of 1 :10 vol/vol, giving a clear dispersion after brief mixing by vortexing.

Other molecules may be incorporated into the hydrophobic phase in order to improve the physical or chemical properties of the formulation (e.g. fluidity, viscosity, stability), or to confer enhanced biological performance to the formulation (e.g. improved immunogenicity). In one such embodiment, an adjuvant such as DDAB (Dimethyl Didodecyl Ammonium Bromide) is added. The invention described above is particularly suited to this, as it does not contain any negatively-charged amphiphiles which could interfere with the DDAB by interaction with the positive charge born by this molecule.

This water-in-oil microemulsion can be prepared using the methods described above by admixing the DDAB with the hydrophobic solvent.

The amphiphile and DDAB are dissolved in a hydrophobic solvent at a level of up to lOOmg/ml total solute in the hydrophobic solvent. The aqueous phase is then added to the hydrophobic phase, preferably in the ratio of 1 :10 vol/vol, giving a clear dispersion after brief mixing by vortexing.

Although microemulsions in mineral oil are not achievable using any low HLB non-ionic amphiphile on its own other than polyoxethylene 3 docecyl ether, it has now been found, that there is a certain ionic co-amphiphile which can be employed, which, in conjunction with other low HLB amphiphiles, will result in microemulsions being formed. This co-amphiphile is sodium docusate. Neither amphiphile is capable of forming a microemulsion on its own, but in combination, over a wide range of concentrations, microemulsions are readily prepared. Thus, a fifth aspect of the invention is an oil-in water microemulsion comprising (i) a hydrophobic phase comprising sodium docusate, and (ii) a solitary non-ionic amphiphile. As used herein a "solitary non-ionic amphiphile" is defined as an amphiphile which has a headgroup consisting of sorbitan, glycerol, or between 1 and 3 polyoxyethylene subunits i.e 1, 2, or 3 polyoxyethylene subunits, and a straight- chain hydrocarbon containing 12 to 16 carbons, i.e. 12, 13, 14, 15, or 16 carbons. Examples of suitable solitary non-ionic amphiphiles include polyoxethylene 2 hexadecyl ether (Brij 52), polyoxethylene 2 oleyl ether, polyoxethylene 3 hexadecyl ether, sorbitan lauryl ether (Span 20), sorbitan oleyl ether (glycomul), glycerol mono-oleate, lauroglycol.

In another aspect the invention provides an oil-in water microemulsion comprising (i) a hydrophobic phase comprising sodium docusate, and (ii) a mixture of non-ionic amphiphiles.

The mixture of non-ionic amphiphiles must have an average (mean) headgroup size and average (mean) chain length that corresponds to a solitary non-ionic amphiphile. Generally they can have a headgroup consisting of sorbitan, glycerol, or between 1 and 10 polyoxyethylene subunits, and a straight-chain hydrocarbon containing 8 to 20 carbons. For example the headgroup can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polyoxyethylene subunits. The straight-chain hydrocarbon can contain 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons.

In a further aspect of the invention is a method for forming a water in-oil microemulsion comprising mixing solutions of

(a) sodium docusate and a solitary non-ionic amphiphile or a mixture of non- ionic amphiphiles dissolved in a hydrophobic phase, and (b) a hydrophilic species dissolved in a hydrophilic phase to form a water-in-oil microemulsion.

The amphiphiles are dissolved in hydrophobic phase at a level of up to lOOmg/ml total solute in the hydrophobic phase. Experiments have shown that the combination of docusate and non-ionic amphiphile in a wide range of proportions gives micro-emulsions, whilst neither component does on its own. The proportion of amphiphile mixture made up by the non-ionic amphiphile component is preferably between 20 and 50% wt/vol, and more preferably between 30 to 40%. The optimal proportion in any one case depends specifically on the nature of the hydrophobic phase, the amphiphile and the hydrophilic solute, and may be readily determined by conduct of simple experiments as described below. The aqueous phase is then added to the hydrophobic phase phase, preferably in the ratio of 1 : 10 vol/vol, giving a clear dispersion after brief mixing by vortexing.

Additional agents may be added to the amphiphile mixture which may serve to broaden the range of ratios of non-ionic amphiphile and docusate over which a clear microemulsion can be formed. One such agent is phospholipid, either of synthetic or natural origin, for example egg or plant-derived lecithin such as soya lecithin and derivatives thereof. Other phospholipids may also be employed, having headgroups such as phosphocholine, phosphoglycerol, phosphophoethanol, phospho serine, or phosphate, and either one or two short, medium or long chain lipid tails. A preferred phospholipid is Soya Phosphatidylcholine (Soya-PC).

It is very much preferred that the preparations of the invention are optically clear and this can be monitored by measuring turbidity at visible wave lengths and, in some cases, by checking for sedimentation over a period of time. Typically the optical density at 620mm can be measured. A value of 0.2 or less, preferably 0.15 or less is considered to be clear.

In the present invention the term "hydrophilic species" relates to any species which is generally soluble in aqueous solvents but insoluble in hydrophobic solvents. This term excludes alkali metal borates such as potassium borates, antiwear agents such as zinc dithiophosphates, amine phosphates, phosphate esters, rust inhibitors, corrosion inhibitors, metal deactivators, bactericides and antifoam agents. The range of hydrophilic species of use in the present invention is diverse but hydrophilic macromolecules represent an example of a species that may be used.

A wide variety of macromolecules is suitable for use in the present invention. In general, the macromolecular compound will be hydrophilic or will at least have hydrophilic regions since there is usually little difficulty in solubilising a hydrophobic macromolecule in oily 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 or organelles. Examples of particular proteins which may be successfully solubilised by the method of the present invention include insulin, calcitonin, haemoglobin, cytochrome C, horseradish peroxidase, aprotinin, mushroom tyrosinase, erythropoietin, somatotropin, growth hormone, growth hormone releasing factor, galanin, urokinase, Factor ΓΧ, tissue plasminogen activator, superoxide dismutase, catalase, peroxidase, ferritin, interferon, Factor VIII and fragments thereof (all of the above proteins can be from any suitable source). Other macromolecules may be used are FITC-labelled dextran and RNA extract from Torulla yeast. The macromolecule can also be an antigen or immunogen, defined as any material capable of stimulating an immune response, and/or immuno-modulator, i.e. any material capable of enhancing (or in some cases down-regulating) the immune response generated towards an antigen or immunogen, especially for use in a vaccine composition.

The immunogen may be selected from, but not limited to, Diphtheria toxoid, tetanus toxoid, botulin toxoid, snake venom antigens, viral antigens e.g. Hepatitis virus A, B, C, D, or E antigens, whooping cough subunit, influenza A and/or B (either whole-killed, virus or protein subunits), H1N1 swine flu, H5N1 bird flu, polio virus, rotavirus, mumps, measles virus, chickenpox, meningitis, rubella, respiratory syncitial virus, HIV, EV71, dengue virus antigens, yellow fever antigens, human papilloma virus antigens, herpes virus HSV 1 or HSV2 antigens, ebola virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, West Nile virus, Japanese Encephalitis virus, hand-foot-and- mouth disease antigens, whole bacteria or extracts thereof e.g. BCG, other mycobacterial antigens, enteric disease pathogens and antigens thereof including for example cholera antigens, , enteric disease pathogens and antigens thereof including for example cholera antigens, Helicobacter pylori antigens, salmonella species, eschericia species, P aeruginosa, chlamydia species, neisseria species, yersinia species, fungi or fungal antigens, H. influenzae A or B (with or without carrier protein), protozoal antigens, e.g. malaria, leishmania, toxoplasma, trypanosoma, trematode antigens, e.g. schistosoma, cestode antigens e.g. from cysticerca, echinococcus, nematode antigens e.g. toxocara, hookworm and filarial, spirochete antigens e.g. borrelia species, surface membrane epitopes specific for cancer cells, and cell receptor targeting anti-inflammatory modulators, polymer conjugates of steroids, HLA antigens, pollens, dust mite antigens, bee stings or food allergens. Immunogens for use in down-regulating immune responses include glutamic acid dehydrogenase, insulin, or conjugates containing insulin subcomponents, for treatment of diabetes. In addition, the immunogen can be a collagen such as collagen type I or collagen type II. A formulation containing collagen type II is a promising candidate for oral down- regulation of immune responses in rheumatoid arthritis. The immunogens can be peptides, proteins, lipids, sugars, nucleic acids, or conjugates thereof.

In cases where an up-regulation of the immune response is desired, the immunogen may be combined with one or more other molecules (immunostimulants or adjuvants), co-entrapped within the same oil phase as the immunogen. Such adjuvants may include cholera toxin B fragment and analogues and derivatives thereof, E. coli heat labile toxin and analogues and derivatives thereof, BCG, CpG-containing oligonucleotide sequences, tetanus toxoid, diphtheria toxoid, bacterial lipid A (intact or detoxified), monophosphoryl lipid A. In one preferred embodiment the antigen is present with an immuno stimulant such as DDAB.

Usually a minimum concentration of 2.5mg macromolecule is incorporated into lml of the hydrophobic phase, so that the concentration of the macromolecule in the initial hydrophilic phase is at least lOmg/ml.

It may also be convenient to co-solubilise 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 may also be chemically conjugated with macromolecules and may thus be included in the compositions.

The process of the present invention allows encapsulation at a much lower amphiphile: protein ratio, as compared to the methods in the prior art, such as WO95/ 13795. This allows more of the macromolecule to be incorporated into the hydrophobic phase including oils such as triglycerides and mineral oil. In addition the retention of the macromolecules in the oil after dispersion in aqueous media is higher.

In addition to macromolecules, the process of the present invention is of use in solubilising smaller organic molecules. Examples of small organic molecules include glucose, carboxyfluorescein and many pharmaceutical agents, for example anti-cancer agents, but, of course, the process could equally be applied to other small organic molecules, for example vitamins or pharmaceutically or biologically active agents. In addition, compounds such as calcium chloride and sodium phosphate can also be solubilised using this process. Indeed, the present invention would be particularly advantageous for pharmaceutically and biologically active agents since the use of non aqueous solutions may enable the route by which the molecule enters the body to be varied, for example to increase bioavailability.

Another type of species that may 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 enables some of the properties of a colloidal metal such as colloidal gold, palladium, platinum or rhodium, to be retained even in hydrophobic solvents in which the particles would, under normal circumstances, aggregate. This could be particularly useful for catalysis of reactions carried out in organic solvents. It is very much preferred that the preparations of the invention are optically clear and this can be monitored by measuring turbidity at visible wavelengths and, in some cases, by checking for sedimentation over a period of time. Suitable methods such as measuring the optical density of the preparation at 620nm, are well known to the person skilled in the art. An optical density of 0.15 or less at 620nm indicates an optically clear solution.

After preparation of microemulsions by any of the methods described above, it is possible to reduce the quantity of the hydrophilic phase, ie. water enclosed in the aqueous droplets, for example by exposing the whole composition to vacuum, usually overnight at room temperature, or alternatively by passing the formulation through a molecular sieve capable of absorbing water molecules. This may be advantageous in cases where long shelf- life is required, and removal of free water can lead to reduction in degradation by hydrolysis. It may also be useful in reducing the chance of transfer of water from formulation to capsule shell, in cases where the oil is filled into a pharmaceutical capsule which takes up water.

Thus, a further aspect the invention provides a method of removal of the hydrophilic phase e.g. free water (either partially or completely) from the microemulsion compositions of the invention or obtained using any of the methods of the invention. This is preferably carried out by exposing the composition to vacuum, or by passing it through a molecular sieve. The composition prepared by the process of the invention or the hydrophobic preparation of the above aspects can be used to make a three-phase composition (specifically a water-in-oil-in water double emulsion). Thus in a further aspect the present invention provides a three phase composition comprising a dispersion of oil droplets within an aqueous solution, wherein said oil droplets comprise a composition or preparation of a water-in-oil microemulsion as described above.

The three-phase composition can be formed by contacting the water-in-oil microemulsion with an excess of a second aqueous solution. In a preferred embodiment the aqueous solution comprises gelatin or albumin. In the case of oils which are normally solid at room temperature (eg tristearin, trilaurin or paraffin wax) the step of addition of oil to hydrophilic phase is performed at a temperature above the melting point of the oil concerned.

In another aspect the invention provides a method for making a three phase composition comprising mixing a water-in-oil microemulsion of the invention in an aqueous solution. The microemulsion of the invention is added to a volume of aqueous solution, which is greater than that of the volume of microemulsion itself, in a suitable vessel. The contents of the vessel are rapidly mixed, e.g. by vortex-mixing, until a homogenous dispersion is obtained.

The water-in-oil-in-water double emulsions retain the solute inside the inner aqueous compartment with minimal leakage to the external aqueous compartment over varying periods of time. The oil used in this system is preferably mineral oil. If the outer aqueous phase is albumin, for example at a concentration of 50mg/ml, or gelatin up to a level of 20% w/w, then retention is further enhanced. Thus the aqueous solution preferably comprises gelatin or albumin. The higher the degree of retention the more suitable the formulation is as a vaccine delivery vehicle. The average size of the emulsion particles will depend on the exact nature of both the hydrophobic and the aqueous phases. However, it may be in the region of upto 2 μηι.

Dispersion of the water-in-oil microemulsion in the aqueous phase can be achieved by mixing, for example either by vigorous vortexing for a short time for example about 10 to 60 seconds, usually about 15 seconds, or by gentle mixing for several hours, for example using an orbital shaker. Dispersions containing the water-in-oil microemulsion preparations or compositions of the invention can also be used in the preparation of microcapsules. If the dispersion is formed from a gelatin-containing aqueous phase, the gelatin can be precipitated from the solution by coacervation by known methods and will form a film around the droplets of the hydrophile-containing hydrophobic phase. On removal of the aqueous solution, microcapsules will remain. This technology is known in the art, but has proved particularly useful in combination with the preparations of the present invention. One way in which the compositions of the present invention may be used is for the oral delivery to mammals, including man, of substances, which would not, under normal circumstances, be soluble in lipophilic solvents. This may be of use for the delivery of dietary supplements such as vitamins or for the delivery of biologically active substances, particularly proteins or glycoproteins, including insulin growth hormones and antigens.

In a further application, it is possible to encapsulate or microencapsulate, for example by the method described above, nutrients such as vitamins which can then be used, not only as human food supplements but also in agriculture and aquaculture, one example of the latter being in the production of a food stuff for the culture of larval shrimps.

In addition, the compositions find application in the preparation of pharmaceutical or other formulations for parenteral administration, as well as formulations for topical or ophthalmic use. For this application, it is often preferable to use an emulsion of the hydrophobic phase and an aqueous phase as described above. Many therapeutic and prophylactic treatments are intended for sustained or delayed release or involve a two component system, for example including a component for immediate release together with a component for delayed or sustained release. Because of their high stability, the preparations of the invention are particularly useful for the formulation of a macromolecule intended for sustained or delayed release.

The longer shelf life of the compositions of the present invention is a particular advantage in the pharmaceutical area.

The hydrophile-in-oil preparations of the invention may find application in the pharmaceutical or similar industries for flavour masking. This is a particular problem in the pharmaceutical industry since many drugs have unpleasant flavours and are thus unpopular with patients, especially children.

The compositions of the invention may be presented in unit dose forms containing a predetermined amount of each active ingredient per dose. Such doses can be provided in a single dose or as a number of discrete doses. The ultimate dose will of course depend on the condition being treated, the route of administration and the age, weight and condition of the patient and will be at the doctor's discretion.

The compositions of the invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). For suitable routes of administration, e.g. nasal, pulmonary, buccal and sublingual administration, the composition can be in the form of an aerosol. The aerosol can be formed of either oil droplets or oil-in- water droplets containing the hydrophobic preparation of the application.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; liquid emulsions.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

For applications to the eye or other external tissues, for example the mouth and skin, the formulations are preferably applied as a topical ointment or cream.

When formulated in an ointment, the active ingredient may be employed with either a paraffmic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

Pharmaceutical formulations adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the microemulsions comprising the active ingredient. Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

A further use is in the cosmetics industry where, again, hydrophobic preparations of hydrophilic compounds can very easily be incorporated into a cosmetic formulation. Examples of macromolecules that may be used in this way include those with moisturising or enzymatic action of some sort. The invention can also be used for the incorporation of proteins such as collagen into dermatological creams and lotions. Finally, the invention has numerous uses in the field of chemical and biological synthesis, for example, non-aqueous enzymatic synthesis.

The invention will now be further described with reference to the following examples. Example 1

Formation of a microemulsion in mineral oil with Polyoxyethylene 3 lauryl ether

1 lOOul of each of the amphiphiles in the table below was mixed with 400ul of mineral oil.

A. POE 3 myristyl ether

B. POE 3 lauryl ether

C. POE 4 cetyl ether

D. POE 4 myristyl ether

E. POE 3 decyl ether

F. POE 4 lauryl ether

G. POE 6 lauryl ether

H. POE 10 cetyl ether

I. POE 10 lauryl ether

3. 300ul of each oil solution was transferred to a fresh glass vial, and 30ul of distilled water added with rapid vortexing.

4. lOOul of each sample was transferred to the well of a clear plastic microplate, and the absorbance measured at 620nm. Mineral oil alone was used as a control.

5. The above steps were repeated using transcutol, or lysozyme (25mg/ml in phosphate-buffered saline) instead of distilled water. Table 1

The readings obtained are shown in the table above. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. POE 3 lauryl ether is the only amphiphile which will permit formation of microemulsions of water containing solutes such as proteins in mineral oil. It is significant that POE 4 cetyl ether, with an HLB identical to that of POE 3 lauryl ether, does not form microemulsions under these conditions. Therefore not all amphiphiles will form a microemulsion contain a protein. The prior art by Nestec specify a requirement for use of amphiphiles with an HLB value of 10 or less, while in the present invention it is made clear that the factor determining the formation of microemulsions or otherwise is not simply the HLB value alone. Indeed, it is shown in Table I above, that many amphiphiles with HLB values lower that 10 do not give the desired result. Example 2

Formation of a microemulsion in oil with a combination of non-ionic amphiphiles with POE 1 lauryl ether 1 400ul of each of the amphiphiles in the table below was mixed with 1600ul of mineral oil. A. POE 4 lauryl ether

B. POE 6 lauryl ether

C. POE 10 lauryl ether

800ul of POE 1 lauryl ether was mixed with 3200ul mineral oil.

2. Solutions of POE 1 lauryl ether were mixed in the proportions shown in the table below with each of the amphiphiles A, B and C in order to achieve solutions containing a percentage of each amphiphile equivalent to 100, 75, 62.5, 50, 37.5, 25 and 0% v/v.

3. 300ul of each oil solution was transferred to a fresh glass vial, and 30ul of lysozyme solution (25mg/ml) in phosphate buffered saline was added with rapid vortexing.

4. lOOul of each sample was transferred to the well of a clear plastic microplate, and the absorbance measured at 620nm.

For amphiphiles A and B (POE 4 and POE 6 lauryl ether respectively), admixtures of these with POE 1 lauryl ether at levels of 75% and 60% of the latter gave clear microemulsions. In the case of POE 10 lauryl ether, in contrast, no microemulsions were formed mixtures in any proportions.

Thus, a clear microemulsion is readily obtainable with a mixture of two non- ionic amphiphiles whose hydrophilic chain lengths span the range from 1 to 6 POE subunits.

Example 3 Formation of a microemulsion in oil with a combination of non-ionic amphiphiles with POE 1 lauryl ether

1. 400ul of each of the amphiphiles in the table below was mixed with 1600ul of mineral oil.

A. POE 3 decyl ether

B. POE 3 myristyl ether

2. Solutions of the two amphiphiles described above were mixed in equal proportions so that each amphiphile comprise 50% v/v of the total.

3. 300ul of the oil solution was transferred to a fresh glass vial, and 30ul of lysozyme solution (25mg/ml) in phosphate buffered saline was added with rapid vortexing.

4. lOOul of this sample was transferred to the well of a clear plastic microplate, and the absorbance measured at 620nm.

Optical density of mineral oil alone: 0.053

Optical density of sample from step 4 0.061 Thus, a clear microemulsion is readily obtainable with a mixture of two non- ionic amphiphiles whose lipid chain lengths span the range from 10 to 14 carbons.

Example 4

Formation of a microemulsion in oil with a combination of docusate and Polyoxyethylene 2 cetyl ether (Brij 52)

1 Into an 8ml glass screw-capped vial 500mg of sodium docusate, is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

2 Into an 8ml glass screw-capped vial 500mg of Brij 52 (polyoxyethylene

2 cetyl ether) is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

3 13 x 2ml glass screw-capped vials are labelled 1 to 13, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below. Table 2

4 The solutions were mixed well by vortexing and then 400 ul of each solution transferred into fresh 2ml vials.

5 To each sample 20ul of distilled water is added, and vortexed briefly.

6 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader.

7 The steps above were repeated using squalene and Miglyol 818 in place of mineral oil, and using lysozyme solution (25mg/ml in phosphate -buffered saline) in place of distilled water.

The readings obtained are shown in the table below. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor Brij 52 are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and Brij 52 are able to do so when Brij 52 comprises between 25 and 50% of the total amphiphile by weight. Similar observations apply for the other oils.

Example 5

Formation of a microemulsion in mineral oil with a combination of docusate and Glycomul

1 Into an 8ml glass screw-capped vial 500mg of sodium docusate, is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

2 Into an 8ml glass screw-capped vial 500ul of Glucomul is measured out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

3 13 x 2ml glass screw-capped vials are labelled 1 to 13, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below.

4 The solutions were mixed well by vortexing and then 400 ul of each solution transferred into fresh 2ml vials.

To each sample 20ul of distilled water is added, and vortexed briefly.

6 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader. 7 The steps above were repeated using squalene and Miglyol 818 in place of mineral oil, and using lysozyme solution (25mg/ml in phosphate -buffered saline) in place of distilled water. The readings obtained are shown in the table below. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor Glucomul are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and glycomul are able to do so when glycomul comprises between 40% and 50% of the total amphiphile by weight. Similar observations apply for the other oils.

Table 5

Example 6

Formation of a microemulsion in mineral oil with a combination of docusate and lauryl glycol 1 Into an 8ml glass screw-capped vial 500mg of sodium docusate, is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

2 Into an 8ml glass screw-capped vial 500ul of Lauroglycol FCC is measured out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

3 12 x 2ml glass screw-capped vials are labelled 1 to 12, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below.

4 The solutions were mixed well by vortexing and then 400 ul of each solution transferred into fresh 2ml vials.

5 To each sample 20ul of distilled water is added, and vortexed briefly. 6 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader. 7 The steps above were repeated using squalene and Miglyol 818 in place of mineral oil, and using lysozyme solution (25mg/ml in phosphate -buffered saline) in place of distilled water.

The readings obtained are shown in the table below. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor Lauroglycol FCC are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and lauryl glycol are able to do so when lauroglycol comprises between 40 and -70% of the total amphiphile by weight. Similar trends are seen with the other oils, although complete clarity is not achieved.

Table 3

Example 7 Formation of a microemulsion in mineral oil with a combination of docusate and glycerol mono-oleate

1 Into an 8ml glass screw-capped vial 500mg of sodium docusate, is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

2 Into an 8ml glass screw-capped vial 500ul of GMO (Glycerol monooleate) is measured out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

3 12 x 2ml glass screw-capped vials are labelled 1 to 12, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below.

Sample No: Docusate GMO % GMO

solution solution (wt/wt)

ul ul %

1 0 400 100

2 100 300 75

3 200 500 71.4

4 200 400 66.7

5 200 300 60

6 200 200 50

7 300 200 40

8 400 200 33.3

9 500 200 28.6

10 300 100 25

1 1 350 100 22.2

12 400 0 0 4 The solutions were mixed well by vortexing and then 400 ul of each solution transferred into fresh 2ml vials.

5 To each sample 20ul of distilled water is added, and vortexed briefly.

6 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader. 7 The steps above were repeated using squalene and Miglyol 818 in place of mineral oil, and using lysozyme solution (25mg/ml in phosphate -buffered saline) in place of distilled water.

The readings obtained are shown in the table below. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor GMO are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and GMO are able to do so over when the GMO comprises between 25 and 50% of total amphiphile by weight. Similar observations apply for the other oils, although the precise range may be different in different cases.

Table 4

Example 8

Formation of a microemulsion in mineral oil with a combination of docusate and Brij 52 containing the polymeric dye Poly R-476 1 Into an 8ml glass screw-capped vial 500mg of sodium docusate, is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml. 2 Into an 8ml glass screw-capped vial 500mg of Brij 52 (polyoxyethylene 2 cetyl ether) is weighed out, and 4.5ml of mineral oil added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml. 3 13 x 2ml glass screw-capped vials are labelled 1 to 13, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below. Tube No: Docusate Brij 52 % Brij 52

solution solution (wt/wt)

ul ul

1 0 400 100

2 200 400 66.7

3 200 300 60

4 200 200 50

5 300 200 40

6 400 200 33.3

7 500 200 28.6

8 300 100 25

9 350 100 22.2

10 400 100 20

1 1 450 100 18

12 500 100 16.7

13 400 0 0

4 The solutions were mixed well by vortexing and then 400 ul of each solution transferred into fresh 2ml vials.

5 To each sample 20ul of distilled water is added, and vortexed briefly.

6 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader.

The readings obtained are shown in table 5. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor Brij 52 are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and Brij 52 are able to do so when Brij 52 comprises between 25 and 50% of the total amphiphile by weight. Table 5

Example 9

Formation of a microemulsion in mineral oil with a combination of docusate and Brij 52 containing the polymeric dye Poly R-476 - leakage studies

1 Into an 8ml glass screw-capped vial 300mg of sodium docusate, is weighed out, and 2.7ml of cyclohexane added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

2 Into an 8ml glass screw-capped vial 300mg of Brij 52 (polyoxyethylene 2 cetyl ether) is weighed out, and 2.7ml of cyclohexane added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml. 3 5 x 2ml glass screw-capped vials are labelled 1 to 5, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below.

4 The solutions were mixed well by shaking and then frozen rapidly in the glycerol bath and then freezer, and then dried on the on the pump over night.

5 The following day 360ul of Mineral oil added to each sample, which are then placed on the roller mixer till reconstituted. To each sample 20ul of Poly -478 at lOOmg/ml solution added, and vortexed briefly.

6 In 2 ml vials 400ul of PBS and 20ul of the poly R oil formulation added. The samples were mixed vigorously till dispersed and then span down in the centrifuge at 3000rpm for 10 minutes. 7 200ul of the supernatant of each sample transferred onto to a separate well of a microplate, and the scattering and leakage level is evaluated by measuring optical density at 620nm and 492nm in a microplate reader. 8 Leakage level of Poly -478

Example 10

Formation of a microemulsion in mineral oil with a combination of docusate, Brij 52 and Soya PC containing the polymeric dye Poly R- 476 - leakage studies

Into an 8ml glass screw-capped vial 300mg of sodium docusate, is weighed out, and 2.7ml of cyclohexane added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

Into an 8ml glass screw-capped vial 300mg of Brij 52 (polyoxyethylene 2 cetyl ether) is weighed out, and 2.7ml of cyclohexane added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml.

Into an 8ml glass screw-capped vial 300mg of Soya phosphatidylcholine is weighed out, and 2.7ml of cyclohexane added. After screwing the cap on tightly the vial is shaken with warming until the contents are dissolved, to give a solution with a concentration close to lOOmg/ml. 4 5 x 2ml glass screw-capped vials are labelled 1 to 5, and into each tube solutions from steps 1 and 2 are dispensed in the volumes indicated in the table below.

5 The solutions were mixed well by shaking and then frozen rapidly in the glycerol bath and then freezer, and then dried on the on the pump over night.

6 The following day 450ul of Mineral oil added to each sample which are then placed on the roller mixer till reconstituted. To each sample 25ul of Poly -478 solution at lOOmg/ml added, and vortexed briefly.

7 In 2 ml vials 400ul of PBS and 20ul of the poly R oil formulation added. The samples were mixed vigorously till dispersed and then span down in the centrifuge at 3000rpm for 10 minutes.

8 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader.

% Brij 52 % Leakage

80 14.78%

60 4.1 1%

40 8.45%

20 17.01%

0 50.38% Example 11

Formation of a microemulsion in mineral oil with a combination of docusate and Brij 52 - aprotinin incorporation.

1 The samples prepared exactly as described in Example 6 (step 1-6) except 20ul of Aprotinin solution at lOOmg/ml was introduced instead Poly -478 solution.

2 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader.

The readings obtained are shown in the table below. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor Brij 52 are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and Brij 52 are able to do so when Brij 52 comprises between 25 and 50% of the total amphiphile by weight.

Example 12

Formation of a microemulsion in mineral oil with a combination of docusate, Brij 52 and Soya PC- aprotinin incorporation. 1 The samples prepared exactly as described in Example 7 (step 1-6) except 25ul of Aprotinin solution at lOOmg/ml was introduced instead Poly -478 solution.

2 200ul of each of the dispersions transferred to a separate well of a microplate, and the scattering is evaluated by measuring optical density at 620nm in a microplate reader.

The readings obtained are shown in the table below. An absorbance of less than 0.2 shows that a clear dispersion has been obtained, indicating that a microemulsion has been formed successfully. As can be seen, neither sodium docusate nor Brij 52 are able to assist in formation of microemulsions in mineral oil on their own, but a combination of docusate and Brij 52 are able to do so when Brij 52 comprises between 20 and 60% of the total amphiphile by weight.

Example 13

Formation of a microemulsion in mineral oil with a combination of docusate, Brij 52 and Soya PC- aprotinin incorporation and leakage studies

1 The samples prepared exactly as described in Example 7 (step 1-6) except 25ul of Aprotinin solution at lOOmg/ml was introduced instead Poly R- 478 solution. 2 In 2 ml vials 400ul of PBS and 20ul of the aprotinin oil formulation added. The samples were mixed vigorously till dispersed and then spun down in the centrifuge at 3000rpm for 10 minutes.

3 lOOul of the aqueous phase from each vial was transferred onto to a separate well of a microplate, and the concentration of protein measured using the Coomassie Brilliant Blue colorimetric assay at 570nm in a microplate reader. Standard aprotinin concentrations were employed for comparison, and calculations were performed to assess the quantity of protein which had leaked from the oil, as shown in the table below.

As can be seen, the combination of Soya PC, Brij 52 and sodium docusate, in all proportions tested, results in good retention of the protein in oil after dispersion.

Example 14 Formation of a microemulsion in mineral oil with a combination of docusate, Brij 52 and Soya PC- lysozyme incorporation and leakage studies

1 The samples prepared exactly as described in Example 7 (step 1-6) except 25ul of lysozyme solution at lOOmg/ml was introduced instead Poly R-

478 solution.

2 In 2 ml vials 400ul of PBS and 20ul of the lysozyme oil formulation added. The samples were mixed vigorously till dispersed and then spun down in the centrifuge at 3000rpm for 10 minutes. 3 lOOul of the aqueous phase from each vial was transferred onto to a separate well of a microplate, and the concentration of protein measured using the Coomassie Brilliant Blue colorimetric assay at 570nm in a microplate reader. Standard lysozyme concentrations were employed for comparison, and calculations were performed to assess the quantity of protein which had leaked from the oil, as shown in the table below.

As can be seen, the combination of Soya PC, Brij 52 and sodium docusate, in all proportions tested, results in good retention of the protein in oil after dispersion.

Example 15

Preparation of an oil phase containing Dimethyl Didodecyl Ammonium Bromide

To 222.2mg of dimethyl dioctadecyl ammonium bromide (DDA) 2.0ml of polyoxyethylene 3 lauryl ether was added, and mixed by vortexing to disperse. Once dispersed, 20.0ml of MINERAL OIL was added, and warmed to 70 C until a clear solution was obtained. To the warm solution 388.9ul of propylene glycol was added and mixed by vortexing. The solution was allowed to cool slowly to room temperature, whereupon the final mixture remained clear.

Example 16

Immune response to 'flu antigen generated by parenteral administration in oil To 1.2ml of oil phase, prepared as described in example 15, was added 60ul of PBS containing 'flu antigen at a concentration of lmg/ml. The mixture was vortexed rapidly for 10 seconds to give a clear opalescent dispersion (microemulsion). 0.1ml of the dispersion was injected sub-cutaneously into each of 5 albino mice in the dorsal region. Other groups of mice were prepared in the same way, in which the same, of a three-fold greater concentration of antigen was administered either alone, or in conjunction with alum. After 8 weeks, blood samples were taken from the tail vein, and the antibody levels against the 'flu antigen in the bloodstream were measured by ELISA. The relative levels of antibody generated, as expressed in terms of ELISA optical density at 405nm, are shown in Figure 1. As can be seen, a significant benefit was conferred by administration of the antigen in oil, as the antibody response generated by a dose of 5ug per animal is much greater than that obtained with three times that dose in alum.

Example 17

Immune response to malaria antigen generated by parenteral administration in oil

To 2ml of oil phase, prepared as described in example 15, was added lOOul of PBS containing a recombinant P. vivax MSP1 malaria fusion antigen at a concentration of 7mg/ml. The mixture was vortexed rapidly for 10 seconds to give a clear opalescent dispersion (microemulsion). The dispersion was injected sub-cutaneously in the inguinal regions of 3 macaque monkeys (4 x 0.15ml per animal). Each animal received a total of 200ug antigen on each day of administration. Other groups of monkeys were prepared in the same way, where the same quantity of antigen or oil was administered alone. Samples were administered three times, on day 0, day 42 and day 84. After 18 weeks, blood samples were taken from the tail vein, and the antibody levels against the malaria antigen in the bloodstream were measured by ELISA. The relative levels of antibody generated, expressed as titre logio, are shown in figure 2. As can be seen, a significant benefit was conferred by administration of the antigen in oil, as the antibody response generated is much greater than that obtained with antigen alone.

Claims

1. A composition comprising a hydrophilic species containing microemulsion, said microemulsion comprising a high molecular weight hydrophobic phase, and an amphiphile comprising a polyoxyethylene-3 headgroup, and a straight-chain hydrocarbon containing twelve carbons as the sole emulsion-forming amphiphile.

2. A composition of claim 1 wherein said hydrophilic species is selected from peptides, proteins, lipids, sugars, nucleic acids, steroids and/or conjugates of one or more of these agents in combination, and/or a conjugate with at least one medium- or long-chain hydrocarbon tail.

3. A process for the preparation of a composition of claim 1 or claim 2, the process comprising mixing solutions of (a) polyoxethylene 3 docecyl ether dissolved in a hydrophobic solvent and (b) a hydrophilic species dissolved in a hydrophilic phase to form a microemulsion.

4. A composition comprising a hydrophilic species containing microemulsion, said microemulsion comprising a high molecular weight hydrophobic phase, and with a mixture of amphiphiles having a net hydrophilic- lipophilic balance (HLB) between 8 and 8.5, and comprising either

(i) polyoxyethylene headgroups with between 1 and 6 subunits, and a straight- chain hydrocarbon containing twelve carbons, or

(ii) a polyoxyethylene 3 headgroup, and a straight-chain hydrocarbons containing between ten and fourteen carbons.

5. A composition of claim 4 wherein said hydrophilic species is selected from peptides, proteins, lipids, sugars, nucleic acids, steroids and/or conjugates of one or more of these agents in combination, and/or a conjugate with at least one medium- or long-chain hydrocarbon tail.

6. A process for the preparation of a composition of claim 4 or claim 5, the process comprising mixing solutions of

(a) a mixture of amphiphiles dissolved in a hydrophobic solvent and

(b) a hydrophilic species dissolved in a hydrophilic phase to form a microemulsion, wherein said mixture of amphiphiles has a net hydrophilic- lipophilic balance (HLB) between 8 and 8.5, and comprises either

(i) polyoxyethylene headgroups with between 2 and 6 subunits, and a straight- chain hydrocarbon containing twelve carbons as the sole emulsion-forming amphiphiles, or

(ii) a polyoxyethylene 3 headgroup, and a straight-chain hydrocarbons containing between ten and fourteen carbons as the sole emulsion-forming amphiphiles.

7. A composition as claimed in any one of claims 1, 2, 4, or 5 further comprising Dimethyl didodecyl ammonium bromide (DDAB).

8. A method of claim 3 or claim 6 for preparing a water-in-oil microemulsion of claim 7 wherein the DDAB is admixed with the hydrophobic solvent.

9. An oil-in water microemulsion comprising (i) a high molecular weight hydrophobic phase comprising sodium docusate, and (ii) a solitary non-ionic amphiphile.

10. An oil-in water microemulsion of claim 9 wherein the non-ionic amphiphile is selected from polyoxethylene 2 hexadecyl ether (Brij 52), polyoxethylene 2 oleyl ether, polyoxethylene 3 hexadecyl ether, sorbitan lauryl ether (Span 20), sorbitan oleyl ether (glycomul), glycerol mono-oleate, lauroglycol or mixtures thereof.

1 1. An oil-in water microemulsion comprising (i) a hydrophobic phase comprising sodium docusate, and (ii) a mixture of non-ionic amphiphiles.

12. An oil-in water microemulsion of any one of claims 9 to 11 where in the high molecular weight hydrophobic phase is selected from mineral oil, triglyceride, squalene or squalane or mixtures thereof.

13. A method for forming an oil-in water microemulsion of any one of claims 9 to 12 comprising mixing solutions of

(a) sodium docusate and a solitary non-ionic amphiphile or a mixture of non- ionic amphiphiles dissolved in a hydrophobic phase, and

(b) a hydrophilic species dissolved in a hydrophilic phase to form an oil-in-water microemulsion.

14. A process or method of any one of claims 3, 6, 8, or 13 further comprising removing the hydrophilic phase from the microemulsion composition.

15. A composition obtainable by the process or method of claim 14.

16. A composition of any one of claims 1, 2, 4, 5, 7, or 9 to 12 further comprising a phospholipid.

17. A composition of claim 15 wherein said phospholipid is Soya Phosphatidylcholine (Soy-PC).

18. A three phase composition comprising a dispersion of oil droplets within an aqueous solution, wherein said oil droplets comprise a composition of any one of claims 1, 2, 4, 5, 7, 9 to 12, 15 or 16.

19. A three phase composition of claim 16 wherein the aqueous solution comprises gelatin or albumin.

20. A method for making a three phase composition comprising mixing a water-in-oil microemulsion of any one of claims 1, 2, 4, 5, 7, 9 to 12, or 15 to 17 in an aqueous solution.

21. A pharmaceutical formulation comprising a composition of any one of claims 1, 2, 4, 5, 7, 9 to 12, or 15 to 19.

22. The pharmaceutical composition of claim 21 adapted for oral, intramuscular or subcutaneous administration..

23. A vaccine comprising a composition of any one of claims 1, 2, 4, 5, 7, 9 to 12, or 15 to 19.

24. The vaccine of claim 23 wherein said composition comprises a malaria antigen or an influenza antigen, or an enteric disease pathogen antigen.

25. A vaccine of claim 23 or claim 24 adapted for oral administration.

26. A capsule comprising a composition of any one of claims 1, 2, 4, 5, 7, 9 to 12, or 15 to 17.

27. A capsule of claim 25, wherein said capsule is enterically coated.

A vaccine comprising a capsule of claim 25 or claim 26.

29. The pharmaceutical composition of claim 21 comprising collagen or fragments, derivatives and analogues thereof for use in treating rheumatoid arthritis

30. A cosmetic formulation comprising a composition of any one of claims 1, 2, 4, 5, 7, 9 to 12, or 15 to 19.