Lipid based composition containing diacylglycerol, phospholipid, polar liquid and biologically active material

A biologically active composition containing (a) a diacyl glycerol, (b) a phospholipid and, optionally, (c) a polar liquid in such proportions that they together form an L2-phase or a cubic liquid crystalline phase, in which a biologically active material is dissolved or dispersed. A method for preparing the composition by mixing (a), (b) and, optionally, (c) for forming an L2-phase or a cubic liquid crystalline phase, the biologically active material being added before, during or after the formation of said phase. Use of the L2-phase or the cubic liquid crystalline phase for encapsulating a biologically active material for obtaining a preparation is provided, which yields a controlled release of the biologically active material.

This application is a 3717 PCT/SEQS /00593 filed May 24, 1995. 
TECHNICAL FIELD 
The present invention relates to the field of polar lipids and, more 
specifically, to a new biologically active composition, which is based on 
polar lipids and has been found to yield significant improvements as 
compared with previously known lipid-based compositions used as carriers 
for biologically active materials. More concretely, the invention relates 
to a new composition whose L2-phase and cubic liquid crystalline phase 
each confer advantages in the controlled release of biologically active 
material encapsulated therein, as compared with previously known, similar 
L2-phases and cubic phases, respectively, and are mutually positioned in a 
phase diagram so as to also confer valuable advantages for some specific 
applications in which special demands are placed on the release of 
biologically active materials. 
BACKGROUND OF THE INVENTION 
Polar lipids are amphiphilic molecules, i.e. they are both "hydrophilic" 
and "hydrophobic". When placed in an aqueous solution, they must thus 
unite in some way or another to form different kinds of aggregates. The 
most well-known of the aggregates formed in water is probably the spheric 
micelle, which typically contains 50-100 lipid molecules arranged in such 
a manner that their hydrocarbon tails (the hydrophobic part) form the 
interior of the micelle concerned and the polar main groups (the 
hydrophilic part) act as a shield against the surrounding water. 
The micelle, however, is only- one of many different types of aggregate 
formed. It is also possible to find both bar-shaped micelles and 
reversed-type micelles (L2), which are also called microemulsions in which 
water forms the internal phase. 
A number of liquid crystalline structures are also normally found in 
systems of polar lipid and water. These comprise hexagonal phases of the 
normal type (HI) and of the reversed type (HII), as well as the lamellar 
phase (L.alpha.). The lamellar structure provides, at an excess of water, 
liposomes, which are spheroidal shells of lipid bilayers. These have been 
studied to a very large extent and have been used in the release of 
pharmaceutical preparations, for example in chemotherapy of cancer. The 
first liposome product on the market contains amphoteracin and is intended 
for treatment of infections. 
Many cubic crystalline phases are said to be both water- and 
oil-continuous, i.e. bicontinuous. These phases consist of lipid bilayers 
whose centre forms a minimum surface, separating two water channel 
systems. There are four main positions in the phase diagram, in which 
cubic phases can be found. Cubic phases of normal topology (oil-in-water) 
can be found either adjacent the micellar solution (in many cases between 
the normal micellar solution (L1) and the hexagonal HI-phases), or between 
the HI-phase and the lamellar L.alpha.-phase. In the first-mentioned case, 
the structures are considered to be anisotropic micellar aggregates. In 
the last-mentioned case, the structures seem to be permanently 
bicontinuous. Cubic phases may also be found between the L.alpha.- and the 
HII-phase. In this case, the structures are also bicontinuous. Cubic 
liquid crystalline phases occurring in the fourth possible position in the 
phase diagram, i.e. between the HII-phase and the reverse micellar 
solution (L2), have been studied by J. M. Seddon in Biochemistry, Vol. 29, 
No. 34, 1990, pp 7997-8002, and by V. Luzzati et al, in Biochemistry 1992, 
31, pp 279-285, but these articles do not concern the properties which 
these phases have been found to possess in the release of biologically 
active materials. 
Furthermore, certain compositions are per se known, whose L2-phase, 
hexagonal phase and/or cubic phase (formed when mixing water and 
amphiphilic lipids) have been used for the release of biologically active 
materials. As examples of publications illustrating this, mentioned can be 
made of U.S. Pat. No. 4,388,307, U.S. Pat. No. 5,143,934, U.S. Pat. No. 
5,151,272, U.S. Pat. No. 5,196,201, U.S. Pat. No. 5,262,164, EP,B1, 
314,689, WO 92/20377 and the article by T. Norling et al. in J. Clin. 
Periodontol 1992, 19, pp 687-692. This does not correspond to the 
composition of the L2-phase and the cubic phase for the composition 
according to the present invention, the mutual positioning of the two 
phases or their advantageous properties in the controlled release of 
biologically active materials. 
SUMMARY OF THE INVENTION 
According to the present invention, a new composition is provided, which is 
so made up and has such properties that there are a number of advantages 
as compared with the previously known compositions within this field. 
Thus, it has surprisingly been found that the biocompatibility of a 
composition according to the invention is better than that of the 
previously known compositions of this type based on monoglycerides 
(monoacyl glycerol). Besides it has been unexpectedly found that the 
release profile of the biologically active material from the composition 
according to the present invention is more advantageous than that of the 
previously known glyceride-based compositions used for this purpose. In 
addition to this, the L2-phase and the cubic phase have such a mutual 
positioning in a phase diagram that the L2-phase may be used for specific 
applications of a biologically active material in situ in mammals, 
especially humans, by the L2-phase swelling when contacting body fluid or 
added polar liquid, such that a phase transition to the corresponding 
cubic liquid crystalline phase occurs. In practice, this means that a 
casting in situ can be accomplished in the specific position in which the 
application of the biologically active material is desired. 
A first object of the invention thus is to provide a lipid composition, 
which yields a controlled release of a biologically active material 
encapsulated therein. 
A further object of the invention is to provide a lipid composition, Which 
has improved biocompatibility compared with previously known 
glyceride-based systems. 
One more object of the invention is to provide a composition, which yields 
an enhanced release profile for a biologically active material as compared 
with previously known monoacyl-glycerol-based systems. 
One more object is to provide a lipid composition, in which the carrier of 
the biologically active material is biodegradable. 
A still further object of the invention is to provide a lipid composition, 
for which the phase transition from the L2-phase to the cubic liquid 
crystalline phase can be used to "cast" the desired biologically active 
material in situ where the intended effect should occur. 
Other objects and advantages of the invention will be apparent to those 
skilled in the art from the following, detailed description of the 
invention. 
The objects of the invention are achieved by a glycerol-ester-based 
composition comprising 
(a) at least one diacyl glycerol, wherein the acyl groups, which are the 
same or different, are each derived from an unsaturated fatty acid having 
16-22 carbon atoms, or from a saturated fatty acid having 8-10 carbon 
atoms, 
(b) at least one phospholipid selected from glycerophophatides and 
sphingophosphatides, wherein the acyl groups, which are the same or 
different, are each derived from a fatty acid having 14-22 carbon atoms, 
and 
optionally, (c) at least one polar liquid selected from the group 
consisting of water, glycerol, ethylene glycol and propylene glycol, 
the proportions between the components (a), (b) and optionally (c) being 
such that they together form an L2-phase or a cubic liquid crystalline 
phase, wherein (c) is an optional component in the case of L2-phase and a 
requisite component in the case of cubic liquid crystalline phase, and the 
biologically active material being dissolved or dispersed in said L2-phase 
or said cubic liquid crystalline phase. 
In general, the unsaturated fatty acid for the diacyl glycerol (a) should 
be liquid at room temperature and it should preferably contain 16-20 
carbon atoms. A particularly advantageous fatty acid in this context is 
one having 18 carbon atoms. Of these fatty acids having 18 carbon atoms, 
oleic acid and linolic acid are of special interest, and oleic acid is the 
one most preferred. Diacyl glycerol (a) from saturated fatty acids in 
liquid state implies, however, that the chain length of the fatty acid is 
shorter, for example 8 or 10 carbon atoms. So-called medium chain length 
triglycerides may be used as starting material for the preparation. 
In many cases it is, however, not necessary to use or even, if anything, 
preferred from the economical point of view not to use the diacyl glycerol 
in pure or synthesised form, but to use a natural product containing 
essentially the same, for example in which the desired diacyl glycerol is 
obtained by esterification/reesterification between glycerol and vegetable 
or animal oils or fatty acids therefrom. Preferred examples of such oils 
are canola, maize, cottonseed, olive, rape-seed, soybean, safflower, 
sunflower, linseed and tall oil. 
The used phospholipid is, as mentioned above, generally based on a fatty 
acid having 14-22 carbon atoms. A preferred carbon atom content of said 
fatty acid is, however, 16-20, while 16 or 18 carbon atoms are especially 
preferred. Also in this case, a phospholipid derived from a natural 
product in the form of vegetable or animal raw materials may, however, 
also be concerned. Examples of preferred such raw materials are soybean, 
rape-seed and egg. 
However, the phospholipid may of course also be an entirely synthetic 
product. 
As preferred examples of glycerophosphatides, mention can be made of 
lecithines and cephalines which are based on choline and, respectively, 
ethanolamine or serine. Especially preferred is phosphatidyl choline. An 
especially preferred sphingophosphatide is sphingomyelin which is also 
based on choline. Specific examples of phospholipids are 
dioleylphosphatidyl choline and dioleylphosphatidyl ethanolamine. 
In respect of the unsaturated fatty acid for the diacyl glycerol (a), the 
term "unsaturated" includes both monounsaturated and polyunsaturated, i.e. 
said acid can contain one or more unsaturated valences. 
The fatty acid for the phospholid (b) can be saturated, monounsaturated 
and/or polyunsaturated. A saturated fatty acid of special interest is one 
having 16 carbon atoms, while a monounsaturated fatty acid of special 
interest is one having 18 carbon atoms. 
The polar liquid used in the inventive composition is preferably water, but 
this water may also be wholly or partially replaced with another polar 
liquid, such as glycerol, ethylene glycol and/or propylene glycol. The 
release rate of the biologically active material may thus be, for example, 
adjusted by varying the proportions of the used polar liquids. By water is 
meant not only pure water, but of course also, for instance, an isotonic 
aqueous solution or a body fluid, especially in the case of casting in 
situ in or on the tissue of humans or animals. 
The exact composition of the L2-phase and/or the cubic liquid crystalline 
phase is, of course, taken from a phase diagram, and the desired release 
rate of the biologically active material which is to be encapsulated is 
readily determined by a person skilled in the art by simple routine 
testing. The enclosed FIGURE illustrates, as will be described in more 
detail below, the phase diagram for a specific system within the scope of 
the present invention, which means that the exact composition of the 
L2-phase and/or the cubic phase can be taken from this diagram for exactly 
this specific system. Since the inventive idea has now been presented, the 
expert will probably have no difficulties in preparing the corresponding 
phase diagram for other specific systems within the scope of the invention 
and determining the exact position of the desired L2-phase and/or cubic 
phase. 
However, it may be generally said that the weight ratio of diacyl 
glycerol:phosphatidyl choline is in the range from 95:5 to 50:50, 
preferably from 90:10 to 70:30. 
The weight content of the polar liquid (c), based on the total weight of 
(a) plus (b) plus (c), is generally in the range of 0-20%, especially 
0-15%, such as 0-10% for the L2-phase and 5-15% for the cubic phase. Thus, 
in the case of L2-phase, the content of the polar liquid (c) may even be 
0, i.e. even the diacyl glycerol and the phospholipid mixed together may 
give the desired L2-phase. In many cases, it is however the matter of a 
certain content of polar liquid in the L2-phase, and in the cubic liquid 
crystalline phase, said liquid is of course always present. Small amounts 
of water may also be bound to the phospholipid and, thus, be added via the 
phospholipid. 
As indicated above, an especially preferred embodiment of the inventive 
composition is represented by the case in which the cubic liquid 
crystalline phase is obtained by swelling of the corresponding L2-phase 
with the polar liquid. Of course, this means in such a case that the 
L2-composition is selected such that the adding of the stated liquid, e.g. 
body fluid, yields a phase transition from the L2-phase to the cubic 
liquid crystalline phase. Other phase transitions are, however, also 
within the scope of the invention, if desired. In some cases, a reversed 
hexagonal chase can be formed in addition to the cubic phase. 
By the above-mentioned phase transition, the cubic liquid crystalline phase 
can be cast in situ where the desired controlled release of the 
biologically active material is wanted. The enormous advantages this may 
confer in some specific cases for application of special medical 
preparations for achieving, for instance, a local effect, will be easily 
understood by the expert. 
The invention is not restricted to one or a few specific biologically 
active materials, but is generally applicable to the different types of 
biologically active materials which have been present earlier in similar 
contexts. However, it will be easily realised that new fields of 
application for certain types of medical preparations will open up by 
means of the present invention, especially when using the phase transition 
L2 to cubic phase. By "biologically active material" or the like is, in 
the present case, like before, meant a compound or composition which, when 
present in an effective amount, reacts with and/or affects living cells 
and organisms. This includes also a biological material which requires a 
controlled release for vaccination purposes. Preventing the release of 
medicals having a bad taste is a further application of the invention. 
An interesting group of compounds for encapsulation according to the 
present invention is, however, the group of pharmaceutical compounds, such 
as antibiotics, antimycotics, proteins, peptides, steroids, local 
anesthetics, chemotherapeutants and antiviral substances. 
Whether the biologically active material is dissolved or dispersed depends, 
of course, on its solubility in the phase mentioned, i.e. whether it is 
water-soluble, lipid-soluble etc, but to this part of the invention, the 
prior art technique applies, as well as to where in the chase concerned 
the resolution/dispersion takes place in reality. 
The content of the biologically active material is of course dependent on a 
number of different factors which are well-known to the expert, especially 
the desired degree of activity. The content must therefore be determined 
by the expert in each individual case. However, it may be generally said 
that the biologically active material is present in an amount of 0.01-30%, 
especially 0.1-5% for soluble substances, and 5-30% for a dispersion, the 
percentages being expressed as percent by weight based on the weight of 
the total composition. 
The biologically active composition is, as indicated above, of special 
interest as pharmaceutically acceptable composition. Of course, this also 
means that it is prepared in a conventional manner for the desired 
administration. 
Examples of specific applications in which the biologically active 
composition according to the present invention may be used are 
when administering antibiotics (e.g. tetracycline, metronidazole), for 
example when treating parodontosis; 
for enhancing the absorption of proteins/peptides (e.g. cyclosporins); 
when treating infections in and adjacent mucous membranes; 
in parenteral administration of drugs, especially via the L2-phase (for 
example vaccine), and 
in depot preparation for e.g. steroids or antibiotics. 
However, these applications are in no way restrictive to the invention, 
since this is primarily directed to new carriers for active substances, 
and not to the substances as such. 
According to another aspect of the invention, a method is provided for 
preparing the above described composition, the method comprising the steps 
of mixing the diacyl glycerol (a), the phospholipid (b) and, optionally, 
the polar liquid (c) in such amounts that an L2-phase, alternatively a 
cubic liquid crystalline phase, is formed, the mixing procedure for 
providing said L2-phase or said cubic liquid crystalline phase occurring 
according to per se known principles, which need not be described here. 
The addition of the biologically active material is carried out before, 
during or after the formation of said L2-phase or said cubic liquid 
crystalline phase. Regarding the addition of the active material to said 
phase "before" the formation thereof, it should also be said for the sake 
of clarity that this means of course that the material can be added from 
the beginning to one of the components (a), (b) or (c), before they are in 
turn mixed for obtaining the desired L2-phase or cubic phase. 
Besides, according to that described above, the cubic liquid crystalline 
phase is possibly formed in the position in which the controlled release 
of the biologically active material is desired, viz. by the presence or 
addition of the polar liquid (c), for example in the form of body fluid. 
Moreover, the preferred embodiments which have been described above in 
connection with the inventive composition apply to the method according to 
the invention. 
Finally, the invention also relates to the use of an L2-phase or a cubic 
liquid crystalline phase according to the above definitions for 
encapsulating a biologically active material for obtaining a preparation, 
which yields a controlled release of the biologically active material. 
Such a preparation can also imply that the lipid-based composition is 
dispersed in an aqueous solution by prior art technique. 
Also in respect of the use, the preferred embodiments are those described 
in more detail above. 
It may also be added that in the present case, the term "biocompatibility" 
has the generally recognised meaning ability of a material to give a 
biologically acceptable, or good, response in a host organism in a 
specific application. More concretely, the lipid mixture must not cause 
undesirable tissue reactions in the form of infection, inflammation or 
other rejection phenomena. Examples of undesirable reactions are swelling, 
pain or the formation of connective tissue capsules. It is also important 
that the lipid mixture affects the inherent healing power as little as 
possible, i.e. such that the natural healing process is not disturbed or 
affected to any decisive degree.

EXAMPLES 
The present invention will finally be further illustrated by means of the 
following working examples regarding the preparation of some specific 
compositions according to the invention and their properties in the 
release of biologically active material. 
EXAMPLE 1 
Phospholipid in the form of a purified soybean phosphatidyl choline (about 
95%) and diacyl glycerol derived from sunflower oil and containing 80% 
oleic acid, based on the total fatty acid content, were weighed in the 
ratio of 15:85 in an injection bottle (10 ml), which was filled with inert 
atmosphere and sealed. The sample was allowed to assume equilibrium at 
room temperature and was centrifuged back and forth, thereby providing a 
homogeneous mixture of the components. The L2-phase was formed in the form 
of a homogeneous, mobile oil. 
EXAMPLE 2 
1 g of the L2-phase prepared according to Example 1 above was transferred 
to a test tube, which was centrifuged such that all the material was 
obtained on the bottom. 9 g of an aqueous solution of Pluronic F68 (1.1 
weight/weight BASF) were added to the test tube. The sample was created 
with an ultrasonic rod (Branson sonifier 250, microtip) for 1+2 min at a 
power of 52 W+56 W. A stable, milky emulsion was formed in this treatment 
of the two phases in the sample. 
EXAMPLE 3 
A fine-grain powder of metronidazol benzoate (7% by weight) was mixed with 
the L2-phase prepared from the above-mentioned phosphatidyl choline and 
diacyl glycerol in the ratio of 3:7. The dispersion was of relatively low 
viscosity, but when adding water to the dispersion, the sample solidified. 
The particle dispersion of metronidazol benzoate in the L2-phase was 
studied under microscope, magnified 200 times. This study showed that the 
particles were well dispersed without any formation of aggregates. 
EXAMPLE 4 
The biocompatibility and the biodegradability of liquid crystalline phases, 
which may function as depot preparations for drugs, were studied in a 
so-called abdomen plug model on rat. The model is developed for testing 
implant material, for example plastics, but has also appeared to function 
satisfactorily for the evaluating of depot preparations. This model means 
that the material to be studied is inserted between the straight abdominal 
muscle and the peritoneum in male rats (Sprague-Dawley, 350-400 g). 
The samples studied were obtained from soybean phosphatidyl choline and 
sunflower oil glycerides. The lipids were first swelled with a 
physiological salt solution to liquid crystalline phases, which were 
implanted. These liquid crystalline phases can be in equivalence with an 
excess of water and are suitable for testing in this model. 
The animals were put to death after 10 and 30 days, and the size of the 
connective tissue capsule and the amount of residual sample were noted. 
The thickness of the capsule may be taken as a measure of the 
biocompatibility of the material. The results after 10 days are summarised 
in the table below. The measure of the capsule thickness is relative and 
concerns the number of squares in the microscope. 
After 30 days, all lipid implants had decreased in size or disappeared 
completely, which proves that the lipids are biodegradable. 
______________________________________ 
Lipid Capsule thickness 
______________________________________ 
monoacyl glycerol 2.0 
phospholipid/diacyl glycerol 1:1 
0.4* 
______________________________________ 
*the capsule was partly fragmented 
EXAMPLE 5 
Biodegradable systems for the controlled release of bioactive systems 
should have a degradation profile which conforms with the release, since 
otherwise an empty carrier, which serves no purpose, may remain in the 
tissue. This is especially the case with water-soluble substances, which 
are released by diffusion. Therefore, a study was made of the effect of 
water on particles of a water-soluble colourant (methylene blue) which was 
dispersed in two different lipid carrier systems. 
Particles of methylene blue were thus mixed with two different L2-phases, 
viz. a first one from the system monoacyl glycerol (sunflower oil)/water 
and a second one from the system phospholipid/diacyl glycerol from Example 
3. Both L2-phases formed a highly viscous cubic liquid crystalline phase 
at an excess of water. In the first case, the cubic phase was both water- 
and oil-continuous, whereas the cubic phase formed in the latter case was 
oil-continuous only. 
The samples were studied under microscope, magnified 200 times, after 
addition of water, and the following phenomena were noted: 
a distinct release of methylene blue from the dispersion to water occurred 
from the monoacyl-glycerol-based system. The dispersion of methylene blue 
in the L2-phase of diacyl glycerol/phospholipid demonstrated no such 
release, when an excess of water was present. 
The release of drugs from the system phospholipid/diacyl glycerol can, 
consequently, be assumed to be controlled more by the degradation shown in 
vivo in Example 4 than by diffusion, whereas the release of drugs from a 
liquid crystalline phase based on monoglyceride will be more difficult to 
control, since this is more dependent on the properties of the 
pharmaceutical preparation, such as molecular size and solubility. 
EXAMPLE 6 
In this example a dispersion of a local anesthetic was prepared by 
dispersing a fine powder of lidocaine hydrochloride in a liquid 33:67 
mixture of phosphatidyl choline from soy and diacyl glycerol from tall oil 
to an L2-phase containing 40% by weight of lidocaine-HCl. The dispersion 
was relatively mobile and was sprayable. The preparation solidified when 
sprayed into water and a sustained dissolution of the lidocaine-HCl 
crystals was obtained as compared with the case when said crystals were 
directly mixed with water. 
EXAMPLE 7 
Cytotoxity tests were performed in the following way. Two liquid 
crystalline phases having cubic structures but different compositions were 
tested in a way that is utilized in the biomaterial field (ISO 
109935:1992(E)) to test whether plastic materials release anything that is 
toxic to cells. Both these cubic phases can be in equilibrium with water 
without being dispersed and can thus be tested as solid bodies. 
Samples from the cubic phases having an area of 3,14 cm.sup.2 were 
contacted with water (4 ml) at 37.degree. C. for more than 24 hours. The 
aqueous phase was then separated from the cubic phase and admixed with 
growth media to which a cell culture was added. The evaluation was then 
performed with reference to growth inhibition of fibroblasts. The results 
were as follows: 
______________________________________ 
Composition of cubic phase 
Growth inhibition 
______________________________________ 
Monoolein* from tall oil 
65% Yes 
Water 35% 
Diolein* frorn tall oil 
60% No 
Phosphatidicyl choline from soy 
30% 
Water 10% 
______________________________________ 
*Purified by molecular distillation