The invention provides a process for the preparation of a single phase hyudrophobic preparation comprising a hydrophilic species in a hydrophobic solvent wherein a compound which is: PA1 (a) a low molecular weight compound having at least some degree of polarity; and/or PA1 (b) a lipid-soluble organic acid; and/or PA1 (c) an amphiphile; and PA1 (d) glycerol or other polyhydric alcohols; is added during the process to aid solubilisation.

The present invention relates to the use of certain compounds as 
solubilisation aids for solubilising hydrophilic molecules in a 
hydrophobic phase in which they would not normally be soluble. In 
particular the present invention relates to the use of such solubilising 
aids for solubilising hydophilic macromolecules in a hydrophobic phase in 
which they would not normally be soluble. 
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 (eg 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. 
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--eg 
oxidation--with water-soluble compounds. 
There are examples of formulations containing both macromolecules and oil 
and one such example is disclosed in EP-A-0366277. The formulation 
disclosed in this document is an emulsion having both a hydrophobic and a 
hydrophilic phase, wherein the hydrophobic phase contains chylomicra or 
chylomicron-forming lipids. However, the macromolecule is dissolved 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 exemplified are formulations 
which comprise a hydrophilic and a lipophilic phase. Once 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 do contain both macromolecules 
and oils, it is significant that in all cases the macromolecule is 
dissolved in the hydrophilic 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, 1392-1394) disclose a 
process for solubilising proteins in a hydrophobic solvent. However, in 
the array of protein surrounded by amphiphile molecules produced by that 
method the authors stated that the amphiphile molecules reacted with the 
protein in the liquid medium by hydrogen bonding or via an electrostatic 
interaction to form a solid precipitate. 
UK patent application No. 9323588.5 discloses a process by which a 
hydrophilic species can be solubilised in a hydrophobic solvent in which 
it would not normally be soluble. The process relies on the surprising 
discovery that if a hydrophilic species is mixed with an amphiphile under 
certain conditions, the resultant composition will be readily soluble in 
lipophilic solvents such as oils. 
However, with some hydrophobic solvents, for example longer chain 
triglycerides, solubilisation is sometimes still difficult and there 
exists, therefore, a need for ways to increase the efficiency of 
solubilisation. 
Surprisingly it has now been found that certain compounds can aid the 
solubilisation of the hydophilic species and hence facilitate the 
formation of a single phase hydrophobic preparation. This is particularly 
useful when the hydrophobic solvent includes medium or longer chain 
triglycerides. 
Thus, in a first aspect the present invention provides a process for the 
preparation of a single phase hydrophobic preparation comprising a 
hydrophilic species, in a hydrophobic solvent, the process comprising: 
(i) associating the hydrophilic species with an amphiphile in a liquid 
medium such that, in the liquid medium, there is no chemical interaction 
between the amphiphile and the hydrophilic species; 
(ii) removing the liquid medium to leave an array of amphiphile molecules 
with their hydrophilic head groups orientated towards the hydrophilic 
species; and 
(iii) providing a hydrophobic solvent around the hydrophilic 
species/amphiphile array; 
wherein a compound which is; 
(a) a low molecular weight compound having at least some degree of 
polarity; and/or 
(b) a lipid-soluble organic acid; and/or 
(c) an amphiphile; and/or 
(d) glycerol or other polyhydric alcohols; 
is added at one or more of the above-noted stages (i)-(iii). 
In another aspect the present invention provides a process for the 
preparation of a single phase hydrophobic preparation comprising a 
hydrophilic species, in a hydrophobic solvent, the process comprising: 
(i) associating the hydrophilic species with a phosphoryl choline 
containing amphiphile in a liquid medium such that, in the liquid medium, 
there is no chemical interaction between the amphiphile and the 
hydrophilic species; 
(ii) removing the liquid medium to leave an array of amphiphile molecules 
with their hydrophilic head groups orientated towards the hydrophilic 
species; and 
(iii) providing a hydrophobic solvent around the hydrophilic 
species/amphiphile array; 
wherein a compound which is; 
(a) a low molecular weight compound having at least some degree of 
polarity; and/or 
(b) a lipid-soluble organic acid; and/or 
(c) a different amphiphile from that used above; and/or 
(d) glycerol or other polyhydric alcohols; 
is added at one or more of the above-noted stages (i)-(iii). 
Preferably, (a) described above for both aspects is a neutral lipid-soluble 
low molecular weight compound having at least some degree of polarity. 
The use of such compounds as described herein make easier the formation of 
a single phase species in which a hydophilic species is solubilised in a 
hydrophobic solvent in which it would not normally be soluble. This is 
particularly advantageous when the hydrophobic solvent is one or more 
longer chain triglycerides. However, even in situations where the 
hydrophobic solvent is not a longer chain triglyceride the use of such 
compounds will ease formation of a single phase preparation, and may, for 
instance, reduce the time required to produce such single phase 
preparations. 
Suitably, 
(a) can be a low molecular weight compound such as a carboxylic acid, an 
amino acid benzyl alcohol, ethanol, t-butanol, i-propanol, or glycerol 
mono-oleate; 
(b) can be a carboxylic acid, phenol, p-cresol, phenyl-boronic acid, benzyl 
boric acid, phenyl-sulphonic acid, phenyl-arsenic acid, benzoic acid, 
salicylic acid, acetic acid, sorbic acid, valearic acid, oleic acid and 
caproic acid; and 
(c) can be selected from cholesterol hemisuccinate (Chems) , 
.alpha.-tocopherol, .alpha.-tocopherol succinate (.alpha.TS) phosphatidic 
acid (PA),phosphatidyl-glycerol, phosphatidyl-inositol and lyso 
derivatives of any of the phosphatides. 
In the present invention the term "hydrophilic species" relates to any 
species which is generally soluble in aqueous solvents but insoluble in 
hydrophobic solvents. 
In a preferred embodiment the solubilisation aid is added at stage (i) 
and/or is provided with the hydrophobic solvent at stage (iii). 
The compounds are used at concentrations in the range of 0.1-75% of the 
total weight of preparation, preferably in the range 0.5-10%, and most 
preferably in the range 1-5%. 
In the context of the present invention, the term "chemical interaction" 
relates to an interaction such as a covalent or ionic bond or a hydrogen 
bond. It is not intended to include van der Waals forces or other 
interactions of that order of magnitude. 
Where the compound is added at stage (i) it is preferably selected from the 
group comprising amphiphiles or polyhydric alcohols. 
A wide variety of macromolecules can suitably be solubilised according to 
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, organelles or viruses (whole or parts 
thereof). 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. 
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 IX, tissue plasminogen 
activator, superoxide dismutase, catalase, peroxidase, ferritin, 
interferon, Factor VIII, melanin and fragments thereof (all of the above 
proteins can be from any suitable source). Other macromolecules which may 
be used are FITC-labelled dextran and RNA extract from Torulla yeast. 
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, ascorbic acid, carboxyfluorescin 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 other vitamins or pharmaceutically or biologically active agents. 
In addition molecules such as calcium chloride and sodium phosphate can 
also be solubilised using the process of the invention. 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 which 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. 
There are numerous amphiphiles which may be used in the present invention 
and zwitterionic amphiphiles such as phospholipids are among those which 
have been found to be especially suitable. Phospholipids having a 
phosphatidyl choline head group have been used with particular success and 
examples of such phospholipids include phosphatidyl choline (PC) itself, 
lyso-phosphatidyl choline (lyso-PC), sphingomyelin, derivatives of any of 
these, for example hexadecylphosphocholine or amphiphilic polymers 
containing phosphoryl choline and halogenated amphiphiles, e.g. 
fluoronated phospholipids. In the present application, the terms 
phosphatidyl choline (PC) and lecithin are used interchangeably. Suitable 
natural lecithins may be derived from any convenient source, for example 
egg and, in particular, soya. In most cases, it is preferable to select an 
amphiphile which is chemically similar to the chosen hydrophobic solvent 
and this is discussed in greater detail below. 
The fact that the present inventors have found zwitterionic amphiphiles 
such as phospholipids to be particularly suitable for use in the process 
is a further 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 anionic and zwitterionic lipids 
were completely unsuitable for use in their method and stated that they 
obtained zero yield 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 solubilised 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 being preferred, alcohols, 
particularly medium chain alcohols such as octanol and branched long chain 
alcohols such as phytol, isoprenoids, e.g. nerol and geraniol, terpineol, 
monoglycerides such as glycerol monooleate (GMO), other esters, e.g. ethyl 
acetate, amyl acetate and bornyl acetate, diglycerides and triglycerides, 
particularly medium chain triglycerides and mixtures thereof, halogenated 
analogues of any of the above including halogenated oils, e.g. long chain 
fluorocarbons or iodinated triglycerides, e.g. lipidiol. 
Optimum results are generally obtained when the hydrophobic solvent and the 
amphiphile are appropriately matched. For example, with a solvent such as 
oleic acid, lyso-PC is a more suitable choice of amphiphile than PC, 
whereas the converse is true when the hydrophobic solvent is a 
triglyceride. 
In addition, in some cases it has been found to be advantageous to add a 
quantity of the amphiphile to the hydrophobic solvent before it is brought 
into contact with the hydrophilic species/amphiphile array. This ensures 
that the amphiphile molecules are not stripped away from their positions 
around the hydrophilic species because of the high affinity of the 
amphiphile for the hydrophobic solvent. 
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. 
A hydrophile/amphiphile array in which the hydrophilic head groups of an 
amphiphile are orientated towards a hydrophilic species has been produced 
before but it has never been suggested that this type of composition may 
be soluble in lipophilic solvents. 
Kirby et al, in Bio/Technology, November 1984, 979-984 and in Liposome 
Technology, Volume I, pages 19-27, Gregoriadis, Ed., CRC Press, Inc., Boca 
Raton, Fla., USA describe a method for the preparation of liposomes in 
which a phospholipid is suspended in distilled water to form small 
unilamellar vesicles or multilamellar vesicles, mixed with the material to 
be entrapped and freeze dried. The mixture is then rehydrated to give 
liposomes. 
At the time of publication of this prior art there was extensive worldwide 
interest in the preparation of liposomes but the idea of producing a 
single phase hydrophobic preparation of a macromolecule seems either never 
to have been thought of or to have been dismissed as impossible or of 
little value. Certainly, there is no suggestion in any of the prior art 
that the intermediate arrays could be put to any other use than the 
preparation of liposomes. Even if a single phase hydrophobic preparation 
had been a desirable objective, the idea of adding a hydrophobic rather 
than a hydrophilic solvent would have been unlikely to have been taken 
seriously because there was a strong prejudice in the art against 
hydrophobic preparations of hydrophilic molecules. 
The orientation of amphiphile molecules into an array with their 
hydrophilic head groups facing the moieties of a hydrophilic species can 
be achieved in several ways and examples of particularly suitable methods 
are discussed in more detail below. 
In a first method, which has a similar starting point to the method 
described by Kirby et al, supra, a hydrophilic species is mixed with a 
dispersion of an amphiphile in a hydrophilic solvent, such that the 
amphiphile molecules form an assembly in which the hydrophilic head groups 
face outwards towards the hydrophilic phase which contains the hydrophilic 
species. The hydrophilic solvent is then removed to leave a dry 
composition in which the hydrophilic head groups of the amphiphile 
molecules are orientated 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 paper believed that it was necessary to obtain a 
precipitate which would then be soluble in hydrophobic solvents. Since 
many of the preferred amphiphiles of the present invention do not form 
such a precipitate, Okahata et al concluded that they would be of no use. 
In the process of the present invention, no precipitate is required and, 
indeed, it is generally thought to be undesirable to allow the formation 
of a precipitate since this results in a reduced yield 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 amphiphile assembly may be 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 cochleate cylinders, hexagonal phase, 
cubic phase or myelin type structures. The form adopted will depend upon 
the amphiphile which is used and, for example, amphiphiles such as 
phosphatidyl choline (PC) tend to form small unilamellar vesicles whereas 
lyso-phosphatidyl choline forms micelles. However, in all of these 
structures, the hydrophobic tails of the amphiphile molecules face inwards 
towards the centre of the structure while the hydrophilic head groups face 
outwards towards the solvent in which the hydrophilic species is 
dispersed. 
The weight ratio of amphiphile:hydrophilic species will generally be in the 
region of from 1:1 to 100:1, preferably from 2:1 to 20:1 and most 
preferably about 8:1 for PC and 4:1 for lyso-PC. 
These ratios are preferred ratios only and, in particular, it should be 
pointed out that the upper limit is set by economic considerations which 
mean that it is preferable to use the minimum possible amount of 
amphiphile. The lower limit is somewhat more critical and it is likely 
that ratios of 2:1 or below would only be used 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 the removal of the solvent is lyophilisation, 
although other methods can be used. 
In some cases, it may helpful to include salts in the hydrophilic solution, 
particularly if the hydrophilic species is a macromolecular compound such 
as a large protein. However, because the presence of larger amounts of 
inorganic salts tends to give rise to the formation of crystals and, 
hence, to a cloudy solution, it is preferred that organic salts are used 
rather than 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 by freeze drying. 
A second method for the preparation of a composition containing an array of 
amphiphiles with their head groups pointing towards the moieties of the 
hydrophilic species is to co-solubilise the hydrophilic species and the 
amphiphile in a common solvent followed by removal of the solvent. 
The product of the process of the invention is new since it makes possible 
the production of single phase hydrophobic preparations comprising a 
hydrophilic species which would not normally be soluble in a hydrophobic 
solvent. Therefore, in a further aspect of the invention there is provided 
a single phase hydrophobic preparation comprising a hydrophilic species in 
a hydrophobic solvent obtainable by the process of the invention. 
It may also be desirable to include other constituents in the single phase 
hydrophobic preparation in addition to the hydrophilic species. This is 
often particularly appropriate when the hydrophilic species is a 
macromolecule and, in that case, the preparation may include, for example, 
bile salts, vitamins or other small molecules which bind to or are 
otherwise associated with the macromolecules. 
Although some macromolecule/amphiphile arrays were disclosed by Kirby et 
al, supra, the arrays disclosed were all intermediates in the formation of 
liposomes and, as discussed above, there has been no previous interest in 
non-liposomal or hydrophobic compositions comprising this type of entity. 
Therefore, the arrays of the present invention in which the amphiphile is 
one which does not form small unilamellar vesicles and would therefore not 
be expected to form liposomes are new. 
One advantage of the preparations of the present invention is that they are 
essentially anhydrous and therefore stable to hydrolysis. They are also 
stable to freeze-thawing and have greater stability at high temperatures, 
probably because water must be present in order for the protein to unfold 
and become denatured. This means that they may be expected to have a much 
longer shelf life than aqueous preparations of the hydrophilic species. 
The solutions of the present invention are extremely versatile and have 
many applications. They may either be used alone or they may be combined 
with an aqueous phase to form an emulsion or similar two phase composition 
which forms yet a further aspect of the invention. 
In this aspect of the invention there is provided a two phase composition 
comprising a hydrophilic phase and a hydrophobic phase, the hydrophobic 
phase comprising a preparation of a hydrophilic species in a lipophilic 
solvent obtainable by a process as described herein. 
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 either be transient 
or stable, depending on the purpose for which they are required. 
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 2 .mu.m. 
Dispersion of the hydrophobic preparation in the aqueous phase can be 
achieved by mixing, for example either by vigourous 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. 
Emulsions containing the hydrophobic preparations of the invention can also 
be used in the preparation of microcapsules. If the emulsion 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 hydrophilic phase, microcapsules will remain. This 
technology is known in the art, but has proved particularly useful in 
combination with the preparations of the present invention. 
In other aspects the invention provides: 
(i) the use of; 
(a) a low molecular weight compound having at least some degree of 
polarity; and/or 
(b) a lipid-soluble organic acid; and/or 
(c) an amphiphile; and/or 
(d) glycerol or other polyhydric alcohols; 
in facilitating the solubilisation of a hydrophilic species in a 
hydrophobic solvent in which the hydrophilic species is not normally 
soluble; 
(ii) a compound which is: 
(a) a low molecular weight compound having at least some degree of 
polarity; and/or 
(b) a lipid-soluble organic acid; and/or 
(c) an amphiphile; and/or 
(d) glycerol or other polyhydric alcohols; 
for use in solubilising a hydrophilic molecule in a hydrophobic solvent in 
which it is not normally soluble; and 
(iii) the use of a compound which is; 
(a) a low molecular weight compound having at least some degree of 
polarity; and/or 
(b) a lipid-soluble organic acid; and/or 
(c) an amphiphile; and/or 
(d) glycerol or other polyhydric alcohols; 
in the preparation of an agent for facilitating the solubilisation of a 
hydrophilic species in a hydrophobic solvent in which the hydrophilic 
species is not normally soluble. 
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 and growth hormones. 
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 for use in topical or opthalmic applications. For this 
application, it is often preferable to use an emulsion of the oil solution 
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 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. 
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 which may be used in 
this way include those with antioxidant, 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.

EXAMPLE 1 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well receiving 50 .mu.l. In 
addition, all wells received soya phosphatidyl choline, dispersed in 
distilled water by probe sonication for ten minutes with cooling, at a 
concentration of 100 mg/ml, each well in a row of four receiving 100, 125, 
150 and 200 .mu.l respectively. The contents of the wells were mixed by 
gentle shaking, then frozen at -20.degree. C., then lyophilised overnight. 
The following day, various oils, with or without additives, were added to 
the wells in each row. The plate was shaken gently for several hours, and 
optical density measurements were taken at intervals with a plate-reader 
at 550 nm. A low absorbance value indicates a low level of scattering, and 
corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of addition of tertiary 
butanol to Miglyol 818 or sunflower oil in facilitating dispersion of 
aprotinin, using soya phosphatidyl choline as amphiphile, is demonstrated. 
The results, expressed in terms of optical density as a function of 
phosphatidyl choline concentration after removal of tertiary butanol by 
lyophilisation (at constant protein concentration) are given in the table 
and are shown in FIGS. 1 and 2. 
Initially, dispersions were performed by adding 100 .mu.l of the pure oil, 
or 200 .mu.l of a 50:50 vol:vol mixture of oil and t-butanol. After 
measurement of optical density, the samples were then frozen and the 
t-butanol was removed by lyophilisation. The OD of the resultant oils was 
measured again. Subsequent experiments have demonstrated that residual 
t-butanol in triglycerides after lyophilisation in no greater than 7% 
wt:wt. 
______________________________________ 
mg of PC per well 
OD @ 550 nm 10 12.5 15 20 
______________________________________ 
M818 alone 0.222 0.204 0.154 
0.089 
M818 + t-but 0.19 0.082 0.024 0.021 
Sunflower oil alone 0.157 0.197 0.222 0.215 
Sunflower oil + t-but 0.046 0.028 0.05 0.087 
______________________________________ 
EXAMPLE 2 
Aprotinin was dissolved in distilled water at a concentration of 10 mg/ml 
and dispensed into wells of a microplate, each well receiving 100 .mu.l. 
In addition, all wells received soya phosphatidyl choline, dispersed in 
distilled water by probe sonication for ten minutes with cooling, at a 
concentration of 100 mg/ml, each well in a row of eight receiving 125 
.mu.l. The contents of the wells were mixed by gentle shaking, then frozen 
at -20.degree. C., then lyophilised overnight. 
The following day, sunflower oil, with varying percentages of additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering, and corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of addition of glycerol 
mono-oleate, oleic acid and acetic acid to sunflower oil in facilitating 
dispersion of aprotinin, using soya phosphatidyl choline as amphiphile, is 
demonstrated. The results, expressed in terms of optical density as a 
function of concentration of additive (at constant protein and 
phosphatidyl choline concentration) are given in the table and FIG. 3. 
______________________________________ 
Additives 
% Additives wt:wt 
employed 0 0.15 0.3 0.55 1 1.33 2.5 5 
______________________________________ 
GMO 1.129 
0.477 
0.226 
OA 1.013 0.444 0.144 
Acetic Acid 1.088 0.588 0.586 0.304 0.229 0.113 0.069 0.068 
______________________________________ 
EXAMPLE 3 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well in a row of five 
receiving 12.5 .mu.l. In addition, soya phosphatidyl choline, dispersed in 
distilled water by probe sonication for ten minutes with cooling, was 
added to each well at a concentration of 100 mg/ml, wells in each row 
receiving 0, 25, 50, 75 and 100 .mu.l respectively. The contents of the 
wells were mixed by gentle shaking, then frozen at -20.degree. C., then 
lyophilised overnight. 
The following day, 100 .mu.l of sunflower oil, with or without additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of acetic 
acid, sorbic acid and oleic acid to sunflower oil (at a concentration of 
1% wt:vol) in facilitating dispersion of aprotinin, using soya 
phosphatidyl choline as amphiphile, is demonstrated. The results, 
expressed in terms of optical density as a function of phosphatidyl 
choline concentration (at constant protein concentration) are given in the 
table and FIG. 4. 
______________________________________ 
mg PC per +Oleic +Acetic 
+Sorbic 
well Oil alone acid acid acid 
______________________________________ 
2.5 0.124 0.052 0.021 0.025 
5 0.087 0.036 -0.002 -0.009 
7.5 0.1 0.073 0.04 0.005 
10 0.259 0.236 0.004 0.008 
______________________________________ 
EXAMPLE 4 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well in a row of five 
receiving 0, 12.5, 16.6, 25, and 50 .mu.l respectively. In addition, soya 
phosphatidyl choline, dispersed in distilled water by probe sonication for 
ten minutes with cooling, was added to each well at a concentration of 100 
mg/ml, wells in each row receiving 100 .mu.l. The contents of the wells 
were mixed by gentle shaking, then frozen at -20.degree. C., then 
lyophilised overnight. 
The following day, 100 .mu.l of sunflower oil, with or without additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of phenol, 
benzoic acid, caproic acid, valearic acid, acetic acid and sorbic acid to 
sunflower oil (at a concentration of 1% wt:vol) in facilitating dispersion 
of aprotinin, using soya phosphatidyl choline as amphiphile, is 
demonstrated. The results, expressed in terms of optical density as a 
function of protein oncentration (at constant phosphatidyl choline 
concentration) are given in the table and FIG. 5. 
______________________________________ 
Aprot 
(mg/well) 0 0.25 0.33 0.5 1 
______________________________________ 
Phenol 0.017 0.041 0.046 0.053 0.163 
Benzoic acid 0.024 0.019 0.023 0.035 0.145 
Caproic acid 0.018 0.031 0.029 0.041 0.151 
Valearic acid 0.02 0.016 0.019 0.039 0.132 
Acetic acid 0.031 0.023 0.016 0.04 0.105 
Sorbic acid 0.012 0.032 0.038 0.039 0.19 
Oil alone 0.155 0.1845 0.169 0.1915 0.322 
______________________________________ 
EXAMPLE 5 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well in a row of six 
receiving 0, 12.5, 16.6, 25, 33 and 50 .mu.l respectively. In addition, 
soya phosphatidyl choline, dispersed in distilled water by probe 
sonication for ten minutes with cooling, was added to each well at a 
concentration of 100 mg/ml, wells in each row receiving 100 .mu.l. The 
contents of the wells were mixed by gentle shaking, then frozen at 
-20.degree. C., then lyophilised overnight. 
The following day, 100 .mu.l of sunflower oil, with or without additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of valearic 
acid and triethylamine to sunflower oil (at a concentration of 1% wt:vol) 
in facilitating dispersion of aprotinin, using soya phosphatidyl choline 
as amphiphile, is demonstrated. The results, expressed in terms of optical 
density as a function of protein concentration (at constant phosphatidyl 
choline concentration) are given in the table and FIG. 6. 
______________________________________ 
Aprot (mg/well) 
0 0.33 0.5 0.66 1 
______________________________________ 
Oil alone 0.166 0.176 0.193 0.261 0.28 
Valeric acid 0.017 0.038 0.053 0.071 0.144 
Valeric acid + 0.021 0.023 0.045 0.062 0.134 
TEA 
TEA 0.254 0.152 0.206 0.24 0.381 
______________________________________ 
EXAMPLE 6 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well in a row of six 
receiving 0, 12.5, 16.6, 25, 33 and 50 .mu.l respectively. In addition, 
soya phosphatidyl choline, dispersed in distilled water by probe 
sonication for ten minutes with cooling, was added to each well at a 
concentration of 100 mg/ml, wells in each row receiving 100 .mu.l. The 
contents of the wells were mixed by gentle shaking, then frozen at 
-20.degree. C., then lyophilised overnight. 
The following day, 100 .mu.l of sunflower oil, with or without additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of benzyl 
boronic acid, benzoic acid and salicylic acid to sunflower oil (at a 
concentration of 1% wt:vol) in facilitating dispersion of aprotinin, using 
soya phosphatidyl choline as amphiphile, is demonstrated. The results, 
expressed in terms of optical density as a function of protein 
concentration (at constant phosphatidyl choline concentration) are given 
in the table and FIG. 7. 
______________________________________ 
Aprot 
(mg/well) 0 0.25 0.33 0.5 0.66 1 
______________________________________ 
Benzyl boric 
0.007 0.015 0.024 0.046 0.087 0.188 
acid 
Benzoic acid 0.002 0.008 0.014 0.045 0.08 0.169 
Salicylic 0.005 0.003 0.003 0.005 0.015 0.06 
acid 
Oil alone 0.04 0.137 0.172 0.236 0.275 0.285 
______________________________________ 
EXAMPLE 7 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well in a row of five 
receiving 0, 12.5, 25, 37.5 and 50 .mu.l respectively. In addition, soya 
phosphatidyl choline, dispersed in distilled water by probe sonication for 
ten minutes with cooling, was added to each well at a concentration of 100 
mg/ml, wells in each row receiving 100 .mu.l. The contents of the wells 
were mixed by gentle shaking, then frozen at -20.degree. C., then 
lyophilised overnight. 
The following day, 100 .mu.l of sunflower oil, with or without additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of benzoic 
acid, salicylic acid, p-cresol, benzoyl alcohol, nitrobenzene and acetic 
acid to sunflower oil (at a concentration of 1% wt:vol) in facilitating 
dispersion of aprotinin, using soya phosphatidyl choline as amphiphile, is 
demonstrated. The results, expressed in terms of optical density as a 
function of protein concentration (at constant phosphatidyl choline 
concentration) are given in the table and FIG. 8. 
______________________________________ 
Nature of Aprot (mg/well) 
facilitator 
0 0.25 0.5 0.75 1 
______________________________________ 
None 0.044 0.101 0.111 0.165 
0.244 
Benzoic Acid 0.006 0.01 0.025 0.052 0.112 
Salicylic Acid -0.004 0.005 0.007 0.028 0.095 
p Cresol -0.004 0.02 0.061 0.121 0.192 
Benzyl alcohol 0.017 0.03 0.051 0.165 0.229 
Nitro benzene 0.006 0.018 0.12 0.209 0.206 
Acetic Acid 0.037 0.036 0.0556 0.095 1.161 
______________________________________ 
EXAMPLE 8 
Wells of a microplate were filled with aprotinin and soya phosphatidyl 
choline as described in example 7, and lyophilised overnight. 
The following day, 100 .mu.l of jojoba oil, with or without additives, was 
added to the wells in each row. The plate was shaken gently for eighteen 
hours, and optical density measurements were taken with a plate-reader at 
550 nm. A low absorbance value indicates a low level of scattering of 
light, and corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of addition of salicylic 
acid to jojoba oil (at a concentration of 1% wt:vol) in facilitating 
dispersion of aprotinin, using soya phosphatidyl choline as amphiphile, is 
demonstrated. The results, expressed in terms of optical density as a 
function of protein concentration (at constant phosphatidyl choline 
concentration) are given in the table and FIG. 9. 
______________________________________ 
Aprot (mg/well) 
0 0.25 0.5 0.75 1 
______________________________________ 
Jojoba oil 0.249 0.539 0.798 0.744 
0.629 
Jojoba oil + sal 0.017 0.021 0.047 0.09 0.216 
______________________________________ 
EXAMPLE 9 
Wells of a microplate were filled with aprotinin and soya phosphatidyl 
choline as described in example 7, and lyophilised overnight. 
The following day, 100 .mu.l of squalane, with or without additives, was 
added to the wells in each row. The plate was shaken gently for eighteen 
hours, and optical density measurements were taken with a plate-reader at 
550 nm. A low absorbance value indicates a low level of scattering of 
light, and corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of addition of caproic 
acid, benzoic acid and phenol to squalane (at a concentration of 1% 
wt:vol) in facilitating dispersion of aprotinin, using soya phosphatidyl 
choline as amphiphile, is demonstrated. The results, expressed in terms of 
optical density as a function of protein concentration (at constant 
phosphatidyl choline concentration) are given in the table and FIG. 10. 
______________________________________ 
Nature of Aprot (mg/well) 
facilitator 
0 0.25 0.5 0.75 1 
______________________________________ 
None 0.735 0.495 1.004 1.014 
1.32 
caproic acid 0.059 0.024 0.022 0.03 0.11 
phenol 0.061 0.099 0.052 0.192 0.094 
benzoic acid 0.198 0.054 0.069 0.034 0.085 
ethanol 0.5 0.651 0.912 0.826 0.811 
______________________________________ 
EXAMPLE 10 
Wells of a microplate were filled with aprotinin and lyso-phosphatidyl 
choline as described in example 7, and lyophilised overnight. 
The following day, 100 .mu.l of phytol or octanol, with or without 
additives, was added to the wells in each row. The plate was shaken gently 
for eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of salicylic 
acid to phytol or octanol (at a concentration of 1% wt:vol) in 
facilitating dispersion of aprotinin, using lyso-phosphatidyl choline as 
amphiphile, is demonstrated. The results, expressed in terms of optical 
density as a function of protein concentration (at constant 
lyso-phosphatidyl choline concentration) are given in the table and FIG. 
11. 
______________________________________ 
Aprot (mg/well) 
0 0.25 0.5 0.75 1 
______________________________________ 
Phytol 0.023 0.147 0.533 0.636 
0.667 
Phytol + sal 0.011 0.009 0.005 0.003 0.179 
Octanol 0.013 0.044 0.021 0.302 0.741 
Octanol + sal 0.042 0.014 0.014 0.009 0.024 
______________________________________ 
EXAMPLE 11 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into wells of a microplate, each well in a row of six 
receiving 0, 12.5, 16.6, 26, 33 and 50 .mu.l respectively. Soya 
phosphatidyl choline was dispersed in distilled water by probe sonication 
for ten minutes with cooling, and sorbic acid was incorporated by mixing 
solid sorbic acid with one ml aliquots of the dispersed phospholipid to 
give concentrations of 1, 0.5, 0.25, 0.125 and 0.0625% in aqueous phase. 
100 .mu.l of each phospholipid suspension was added to a fresh row of six 
wells each. The contents of the wells were mixed by gentle shaking, frozen 
at -20.degree. C., then lyophilised overnight. 
The following day, 100 .mu.l of sunflower oil, with or without additives, 
was added to the wells in each row. The plate was shaken gently for 
eighteen hours, and optical density measurements were taken with a 
plate-reader at 550 nm. A low absorbance value indicates a low level of 
scattering of light, and corresponds to effective dispersion of protein in 
oil. 
Employing the method described above, the effect of addition of sorbic acid 
to the phosphatidyl choline dispersion at different concentrations in 
facilitating dispersion of aprotinin in sunflower oil is demonstrated. The 
results, expressed in terms of optical density as a function of protein 
and sorbic acid concentration (at constant phosphatidyl choline 
concentration) are given in the table and FIG. 12. 
______________________________________ 
Aprot.backslash.sorbic acid 
0 0.0625 0.125 0.25 0.5 1 
______________________________________ 
0.25 0.049 0.062 0.073 0.02 0.012 0.008 
0.5 0.147 0.113 0.14 0.085 0.042 0.004 
0.66 0.23 0.162 0.18 0.124 0.074 0.071 
1 0.366 0.271 0.251 0.198 0.127 0.143 
______________________________________ 
EXAMPLE 12 
Phospholipid dispersions were prepared as described in example 7 containing 
either 100 mg of soya phosphatidyl choline per ml of distilled water, or 
90 mg of phosphatidyl choline and 10 mg of phosphatidic acid per ml of 
distilled water. Wells of a microplate were filled with aprotinin and one 
or other of the phospholipid dispersions above as described in example 7, 
and lyophilised overnight. 
The following day, 100 .mu.l of Miglyol 818 or oleic acid was added to the 
wells in each row. The plate was shaken gently for eighteen hours, and 
optical density measurements were taken with a plate-reader at 550 nm. A 
low absorbance value indicates a low level of scattering of light, and 
corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of inclusion of 
phosphatidic acid in the phospholipid suspension in facilitating 
dispersion of aprotinin in Miglyol 818 or oleic acid is demonstrated. The 
results, expressed in terms of optical density as a function of protein 
concentration (at constant phospholipid concentration) are given in the 
table and accompanying FIGS. 13 and 14. 
______________________________________ 
Aprot (mg/well) 
0 0.25 0.5 0.75 1 
______________________________________ 
M818 
PC 0.014 0.014 0.036 0.073 0.238 
PC/PA 0.028 0.037 0.041 0.045 0.059 
Oleic acid 
PC 0.013 0.023 0.11 0.223 0.304 
PC/PA 0.04 0.04 0.045 0.048 0.087 
______________________________________ 
EXAMPLE 13 
Phospholipid dispersions were prepared as described in example 7 containing 
either 100 mg of soya phosphatidyl choline per ml of distilled water, or 
90 mg of phosphatidyl choline and 10 mg of phosphatidic acid per ml of 
distilled water. Wells of a microplate were filled with aprotinin and one 
or other of the phospholipid dispersions above as described in example 7, 
and lyophilised overnight. 
The following day, 100 .mu.l of cod liver oil was added to the wells in 
each row. The plate was shaken gently for eight hours, and optical density 
measurements were taken with a plate-reader at 550 nm. A low absorbance 
value indicates a low level of scattering of light, and corresponds to 
effective dispersion of protein in oil. 
Employing the method described above, the effect of inclusion of 
phosphatidic acid in the phospholipid suspension in facilitating 
dispersion of aprotinin in cod liver oil is demonstrated. The results, 
expressed in terms of optical density as a function of protein 
concentration (at constant phospholipid concentration) are given in the 
table and FIG. 15. 
______________________________________ 
Apoprotein Concentration 
Nature of oil +/- PA 
0 0.25 0.5 0.75 1 
______________________________________ 
PC/Cod liver 0.341 0.578 0.936 1.169 1.124 
PC:PA/Cod liver 0.119 0.339 0.198 0.174 0.756 
______________________________________ 
EXAMPLE 14 
Phospholipid dispersions were prepared as described in example 7 containing 
either 100 mg of soya phosphatidyl choline per ml of distilled water, or 
90 mg of phosphatidyl choline and 10 mg of either phosphatidic acid or 
cholesterol hemisuccinate per ml of distilled water. Wells of a microplate 
were filled with aprotinin and one or other of the phospholipid 
dispersions above as described in example 7, and lyophilised overnight. 
The following day, 100 .mu.l of squalane or sunflower oil was added to the 
wells in each row. The plate was shaken gently for eight hours, and 
optical density measurements were taken with a plate-reader at 550 nm. A 
low absorbance value indicates a low level of scattering of light, and 
corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of inclusion of 
phosphatidic acid or cholesterol hemisuccinate in the phospholipid 
suspension in facilitating dispersion of aprotinin in squalane or 
sunflower oil is demonstrated. The results, expressed in terms of optical 
density as a function of protein concentration (at constant phospholipid 
concentration) are given in the table and FIGS. 16 and 17. 
______________________________________ 
Apoprotein Concentration 
Nature of facilitator 
0 0.25 0.5 0.75 1 
______________________________________ 
Squalane 
PC alone 0.773 0.685 0.475 0.544 0.601 
PC + Chems 0.086 0.093 0.085 0.071 0.095 
PC + PA 0.074 0.033 0.032 0.032 0.051 
Sunflower oil 
PC alone 0.342 0.308 0.203 0.484 0.593 
PC + Chems 0.241 0.261 0.172 0.3 0.412 
PC + PA 0.168 0.336 0.079 0.06S 0.088 
______________________________________ 
EXAMPLE 15 
Phospholipid dispersions were prepared as described in example 7 containing 
either 100 mg of soya phosphatidyl choline per ml of distilled water, or 
90 mg of phosphatidyl choline and 10 mg of phosphatidic acid per ml of 
distilled water. Wells of a microplate were filled with aprotinin and one 
or other of the phospholipid dispersions above as described in example 7, 
and lyophilised overnight. 
The following day, 100 .mu.l of jojoba oil was added to the wells in each 
row. The plate was shaken gently for fifty three hours, and optical 
density measurements were taken with a plate-reader at 550 nm. A low 
absorbance value indicates a low level of scattering of light, and 
corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of inclusion of 
phosphatidic acid in the phospholipid suspension in facilitating 
dispersion of aprotinin in jojoba oil is demonstrated. The results, 
expressed in terms of optical density as a function of protein 
concentration (at constant phospholipid concentration) are given in the 
table and FIG. 18. 
______________________________________ 
Jojoba Oil 
Apoprotein Concentration 
Nature of facilitator 
0 0.25 0.5 0.75 1 
______________________________________ 
PC alone 0.439 0.435 0.623 0.546 
0.112 
PC + PA 0.154 0.16 0.08 0.073 0.087 
______________________________________ 
EXAMPLE 16 
Phospholipid dispersions were prepared as described in example 7 containing 
either 100 mg of soya phosphatidyl choline per ml of distilled water, or 
90 mg of phosphatidyl choline and 10 mg of .alpha.-tocopherol succinate 
per ml of distilled water. Wells of a microplate were filled with 25 .mu.l 
of aprotinin solution and one or other of the phospholipid dispersions 
above as described in example 7, and lyophilised overnight. 
The following day, 100 .mu.l of Miglyol 818 was added to the wells in each 
row. The plate was shaken gently for eighteen hours, and optical density 
measurements were taken with a plate-reader at 550 nm. A low absorbance 
value indicates a low level of scattering of light, and corresponds to 
effective dispersion of protein in oil. 
Employing the method described above, the effect of inclusion of 
.alpha.-tocopherol succinate in the phospholipid suspension in 
facilitating dispersion of aprotinin in Miglyol 818 is demonstrated. The 
results, expressed in terms of optical density as a function of protein 
concentration (at constant phospholipid concentration) are given in the 
table and FIG. 19. 
______________________________________ 
Aprot (mg/well) 0 0.5 
______________________________________ 
PC alone 0.051 0.085 
PC + tocopherol 0.036 0.037 
succinate 
______________________________________ 
EXAMPLE 17 
Aprotinin was dissolved in distilled water at a concentration of 20 mg/ml 
and dispensed into one set of five B2 glass vials (Group I) receiving 0, 
125, 250, 375 and 500 .mu.l respectively. In addition, 1 ml of soya 
phosphatidyl choline, dispersed in distilled water by probe sonication for 
ten minutes with cooling, was added to each vial at a concentration of 100 
mg/ml. A second set of vials (Group II) received 0, 62.5, 125, 187.5 and 
250 .mu.l of aprotinin solution as described above, together with 0.5 ml 
of soya phospholipid dispersion (100 mg/ml).The contents of the vials were 
mixed by gentle shaking, then frozen in liquid nitrogen, and lyophilised 
overnight. 
The following day, 1 ml of Miglyol 818 was added to each vial in Group I, 
and 0.5 ml of Miglyol 818 containing 10 mg salicylic acid per ml was added 
to all vials in Group II. All vials flushed with nitrogen, sealed and 
mixed at room temperature on a roller mixer until the oil dispersions in 
Group II (with salicylic acid as facilitator) were all essentially clear 
(three hours). 400 .mu.l of each of the oils from Group I were then 
transferred to fresh vials each containing 4 mg of dry solid salicylic 
acid, and the vials were sealed, flushed with nitrogen, and roller mixing 
continued. The tubes were incubated in this way for up to five days, and 
optical density measurements at 550 nm were taken on 100 .mu.l samples 
removed from the tubes at intervals, and dispensed into a plate-reader. A 
low absorbance value indicates a low level of scattering of light, and 
corresponds to effective dispersion of protein in oil. 
Employing the method described above, the effect of addition of salicylic 
acid (at a concentration of 1% wt:vol) either before or after mixing of 
oil with the protein/lipid complex in facilitating dispersion of 
aprotinin, using soya phosphatidyl choline as amphiphile, is demonstrated. 
The results, expressed in terms of optical density as a function of 
protein concentration (at constant phosphatidyl choline concentration) are 
given in the table and FIG. 20. 
______________________________________ 
Optical Density at 550 nm after 
incubation at room temperature for five days 
facilitator in oil 
facilitator 
Aprotinin no facilitator before addition added after oil 
______________________________________ 
0 0 0 0 
0.25 0.002 0 0 
0.5 0.443 0.004 0.017 
0.75 0.488 0.009 0.016 
1 0.866 0.002 0.128 
______________________________________ 
EXAMPLE 18 
An aqueous dispersion of soy phosphatidyl choline (soy PC) was prepared, 
containing 100 mg/g of suspension, flushed thoroughly with nitrogen, and 
sonicated at an amplitude of 8 microns peak to peak. Each aliquot was 
subjected to a total sonication time of 4 minutes, in pulses of 30 seconds 
interspersed by cooling for 30 seconds in an ice slurry bath. The 
resulting opalescent dispersion of small unilamellar vesicles (SUV) was 
then centrifuged for 15 minutes to remove particles of titanium. 
5 mg of lipase from Candida cylindericae was dissolved in distilled water 
to a concentration of 100 mg/g, and 50 microL aliquots (i.e. 0.5 mg lipase 
each) were added to small glass test tubes. To each tube was added 100 
.mu.l of SUV (ie 10 mg of PC) , and the contents mixed, shell-frozen in 
liquid nitrogen and freeze-dried overnight. 665 mg of linoleic acid was 
added to each lyophilate, mixed by vortexing and then left for 1 hour to 
disperse. To the resulting clear suspensions were added 335 mg of 
trilinolein followed by mixing. It was noted that addition of the 
triglyceride had no adverse effect on the clarity of the dispersions, 
whereas direct addition of trilinolein to such lyophilates would normally 
not allow such dispersion to take place. After incubating for 1 week at 
37.degree. C. there was no change in the clarity of the dispersions. Thus, 
solubilization of the protein in the presence of a long chain 
triglyceride, has been enabled by the presence of linoleic acid. 
EXAMPLE 19 
A solution of lipase from Mucor mehii, containing 8.9 mg protein/ml was 
distributed into aliquots of 0.1 ml (0.89 mg protein) in small glass 
vials. To each was added 200 mg of SUV containing 100 mg PC/g (i.e. 20 mg 
PC per vial) prepared as in example 1, and the mixtures were lyophilised 
overnight. 50% of the lyophilates were dispersed with 665 mg of oleic acid 
and the remainder with the same amount of linoleic acid. The dispersions 
were left for 3 hours by which time they were completely clear, and then 
335 mg trilinolein was added to the oleic acid dispersions and the same 
amount of triolein to the linoleic acid-based ones. Both types remained 
clear and were still so after 2 weeks of incubation at 37.degree. C. 
EXAMPLE 20 
Lyophilates prepared as above, each containing 1 mg of aprotinin (from 100 
microl of a 1% solution) and 20 or 30 mg soy PC (from 200 or 300 .mu.l of 
SUV respectively), were dispersed with sunflower oil containing 0, 10, 20 
and 30% oleic acid (w/w). All of those containing oleic acid became clear 
or slightly opalescent, while the oleic acid-free preparation remained as 
a turbid suspension. Similarly, a lyophilate mixed with the more saturated 
corn oil, containing 10% (w/w) oleic acid, formed a slightly opalescent 
dispersion while a control mixed with pure corn oil formed a turbid 
suspension. 
EXAMPLE 21 
Five columns of 4 rows of small test-tubes were set up. To all the tubes in 
each row, in the 1st, 2nd, 3rd and 4th rows, were added aliquots 
containing 0.36, 0.72, 1.08 and 1.44 mg aprotinin respectively (aprotinin 
was added as an aqueous solution containing 10 mg protein/ml). To every 
tube was then added 180 .mu.l of SUV containing 100 mg PC/ml (ie 18 mg PC 
added), prepared as in Example 1 The tube contents were mixed, 
shell-frozen and freeze-dried overnight. To all of the tubes in each of 
columns 1, 2, 3, 4 and 5, was then added 180 mg of sunflower oil 
containing 5, 3, 2, 1 and 0% oleic acid respectively. The tubes were mixed 
by vortexing and left to disperse overnight, after which the dispersions 
were transferred to a microtitre plate and absorbances read at 550 nm. The 
results are shown in Table 1. 
TABLE 1 
______________________________________ 
Effect of oleic acid on solubilisation of aprotinin in sunflower oil 
Aprotinin content 
Oleic acid content of sunf lower oil (%) 
(mg) 5 3 2 1 0 
______________________________________ 
0.36 0.029 0.018 0.023 0.022 
0.469 
0.72 0.052 0.054 0.048 0.06 0.128 
1.06 0.017 0.017 0.099 0.176 0.208 
1.44 0.182 0.182 0.162 0.104 0.286 
______________________________________ 
EXAMPLE 22 
Two rows of small test-tubes were set up. Into each tube of the first row 
was added 0.2 ml of 0.25% ascorbic acid solution (i.e. 0.5 mg ascorbic 
acid), and into the second, 0.2 ml of 0.125% solution (0.25 mg ascorbic 
acid). 60 .mu.l of soy PC SUV prepared as in Example 1, was added to each 
tube, and the contents shell-frozen and freeze-dried overnight. To the 
lyophilates in the 1st, 2nd, 3rd and 4th tube in each row was added 300 mg 
of sunflower oil solutions containing 1, 2, 3 and 4% of linoleic acid 
respectively. The tubes were vortexed briefly and then left to disperse. 
After 24 hours the dispersions were examined visually and the degrees of 
clarity listed on a score of 1 to 10. A score of 10 means completely clear 
while 1 means that apparently, no solubilization had taken place. The 
results are shown in Table 2. 
TABLE 2 
______________________________________ 
Effect of linoleic acid on solubilisation of 
ascorbic acid in sunflower oil 
Linoleic acid content 
Ascorbic acid of sunflower oil (%) 
content (mg) 1 2 3 4 
______________________________________ 
0.25 9+ 10 10 10 
0.5 7 8 10 10 
______________________________________ 
EXAMPLE 23 
A stock solution of 400 mM glycerol was prepared and diluted sequentially 
to give 200, 100, 50 and 25 mM solutions. Into each of 6 small test-tubes 
was added 200 .mu.l of a solution containing 18 mg of aprotinin/ml, and 
then across the row from left to right was added 75 .mu.l of distilled 
water, 25, 50, 100, and 200 mM glycerol respectively. To each tube was 
then added 300 .mu.l of soy PC SUV prepared as in Example 1, and the 
mixtures were shell-frozen, freeze-dried overnight and the lyophilates 
dispersed each with 300 mg of Miglyol 818. After vortexing and standing 
overnight, the dispersions were transferred to a microtitre plate and the 
absorbances measured at 550 nm. The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Effect of glycerol on solubilisation of Aprotinin in Miglyol 818 
Concentration of added glycerol (mM) 
0 25 50 100 200 400 
______________________________________ 
Absorption 0.152 0.073 0.052 0.037 0.05 0.361 
(550 nm) 
______________________________________ 
EXAMPLE 24 
i) 100 mg of ovalbumin was dissolved in 5 ml of distilled water. 
ii) 20 mg of proline, serine, glutamic acid and tyrosine were each 
dissolved in 1ml of distilled water. 
iii) Phospholipid was dispersed in distilled water at a concentration of 
250 mg/ml according to the method described in previous examples. 
iv) The solutions prepared in the steps above were dispersed into two ml 
glass vials as follows: 
______________________________________ 
Label 0 1 2 3 mg/vial 
______________________________________ 
PC (l) 90 90 90 90 22.5 
Ovalbumin (l) 100 100 100 100 2 
Amino acid (l) 0 12.5 25 50 0-1 
Amino acid (mg) 0 0.25 0.5 1.0 0-1 
______________________________________ 
v) The contents of all the tubes were mixed well, frozen in liquid nitrogen 
and hyophilised overnight. 
vi) The following day, 0.2 ml of Miglyol M840 was added to the contents of 
each vial and shaken at RT. 
vii) The following day, 501 samples were transferred to the wells of a 
microplate, and the optical densities measured at 600 nm wavelength. 
The measurements obtained are shown in the table below: 
______________________________________ 
0 1 2 3 
______________________________________ 
Glutamic acid 
0.31 0.197 0.194 
0.224 
Proline 0.27 0.196 0.163 0.15 
Serine 0.287 0.171 0.147 0.131 
Tyrosine 0.324 0.253 0.213 0.21 
______________________________________ 
These results are shown in FIG. 21. 
It can be seen that addition of the amino acids to the aqueous phase during 
incorporation of the protein into oil significantly reduced the turbidity 
of the final formulation, indicating an improvement in solubilisation due 
to the amino acids.