Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 0223~487 1998-04-21
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SOLUBILISATION METHODS
The present inven~ion relates to methods of solubilising
an agen~, eg biologically active materials, in an
amphiphile. In particular the invention relates to
methods of bringing biologically active substances used
for topical administration into association with
permeation aids.
It is a continuing objective of the pharmaceutical
industry to achieve high degrees of solubilisation of
biologically active materials in a variety of solvents.
There are several reasons for this need to achieve
solubilisation. For instance, achieving solubilisation in
particular solvents may improve bioavailibility. An
example of this would be the solubilisation of
biologically active materials in oils. Examples of
methods to achieve this can be found , for example, in
WO 95/13795, WO 96/17593 and WO 96/17594.
The methods disclosed in the above-noted patent
publications and applications include steps whereby the
biologically active materi~l is brought into association
with an amphiphile. There are also circumstances where it
would be desirable to achieve higher degrees of
solubilisation of biologically active molecules,
particularly water-soluble ones, in amphiphiles. Examples
include:
i) to improve dissolution characteristics in aqueous
media, eg. to aid in achieving rapid dissolution;
ii) to aid incorporation into low HLB systems such as oil
mlxtures; and
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iii) to bring about association between biologically
active substances and permeation aids, which are often
amphi~hiles, for topical use.
Thus, in a first aspect, the present invention provides
a method of solubilising an agent in an amphiphile which
method includes the steps of:
(i) bringing the agent and amphiphile into
association with each other in a common
solvent;
(ii) removing the common solventi and
(iii) heating the residue from step (ii);
In step (i), the agent and the amphiphile can suitably be
brought into association with each other by firstly
dissolving each one separately in the common solvent,
followed by mixing of the two resultant solutions.
The removal of the solven~ should be carried out at a
temperature such that the amphiphile/agent residue which
remains is in the solid state. The heating step should
then be sufficient to melt the solid amphiphile, and also
to convert the amphiphile/agent array from an ~'open~' form
to one which is more condensed.
The common solvent can be water, for example, and it can
be removed in step (ii) by, e.g. freeze drying,
centrifugal vacuum drylng or any other suitable method.
Suitably, in the above methods the amphiphile will be a
phospnolipla, for nstance lecithin, a glycolipid, a
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polyoxyethylene containing surfactant, a lipophilic
sulphate, betaine, a sarcosine containing surfactant,
Solulan 16, Solulan C24, polyoxye~hylene 40 stearate, one
of the Tween series of surfactants, one of the Span
', series of surfactants or a pegolated castor oil
derivative, e.g. Cremaphor EL35.
The "agent" is suitably a hydophilic species which is
generally soluble in aqueous solvents but insoluble in
1() hydropho~ic solvents. The range of hydrophilic species
of use in the present invention is diverse but
hydrophilic macromolecules represent an example of a
species which may be used.
l'i 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 assembiies of any of these including, in
some cases, whole cells or organelles. It may also be
2'l 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,
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horseraaish peroxidase, aprotinin, mushroom tyrosinase,
erythrcpoietin, somatotropin, growth hormone, growth
hormone releasing factcr, galanin, urokinase, Factor IX,
tissue ?lasminogen 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.
It seems that there is no upper limit of molecular weight
for the macromolecular compound since dextran having a
molecular weight of about 1,000,000 can easily be
solubilised by the proc-ess of the present invention.
In addi-ion to macromolecules, the process of the present
invention is of use in solubilising smaller organic
molecules. Examples c,f small organic molecules include
glucose, carboxyfluorescin and many pharmaceutical
agents, for example anti-cancer agents, but, of course,
the prccess 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
phospha~e 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 moiecule enters the body to be
varied, ~or example to increase bioavailability.
~nother Lype of species which may be included in the
hydropnobic compositions of the invention is an inorganic
materia: such as a small inorganic molecule or a
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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,
pallaaium, platinum or rhodium, tO be retained even in
5 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.
lt) The above-described method is particularly suitable for
achieving association between an agent which is for
topical administration and a permeation aid. An example
of the former is Zinc Acetate (ZnAc2).
l'i Particularly suitable amphiphiles are those which are
solid at room temperature, eg Solulan 16 and Solulan C24.
In other aspects the present invention provides:
i) a composition comprising an agent solubilised in an
amphiphile obtainable by any of the methods described
herein, particularly an agent for topical administration
solubilised in an aphiphile which is a permeation aid;
and
ii) the use of a composition of the invention in the
preparation of a medicament for topical administration,
particularly a composition for use in the treatment of
inflammation and/or ar~hritis wherein the active agent is
ZnAc2.
Preferred features of each aspect of the invention are as
for each other aspect mutat~ s mutandis.
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The inventlon wiii now be described with reference to the
followlng examples, which should not be construed as in
any way limiting the lnvention.
Example 1
(l) A solution of zinc acetate at a concentration of
lOOmg/ml was prepared by addition of lOOmg of ZnAc. to lml
of distilled water, and mixing at RT until dissolution
was achieved.
~2) A solution of Solulan 16 at a concentration of
lOOmg/ml was prepared by addition of 500mg of Solulan to
4.5ml of distilled wat-er and mixing at 60~C until
dissolution was achieved.
(3) Solutions from steps 1 & 2 were dispensed into 4ml
glass screw-capped vials as follows, and mixed well:
_
A B C
ZnAc. (Vol) 0.2ml 0.3ml 0.4ml
Solulan 16 (Vol) 1.8ml 1~7ml 1.6ml
ZnAc. (wt) 2Omg 3Omg 4Omg
Solulan 16 (wt) 180mg '70mg 160mg
25~Zn (wt:wt) 10 15 20
Ratio (wt:wt) 9:1 5.7:1 4:1
(4) The vials and contents were frozen in liquid
nitrogen and lyophilised overn~ght with a condenser
tempera~ure of -40~C, and a vacuum of O.lmBar.
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(5) The following day, the lyophilates were incubated at
+60~C on a heating block, to melt the solid cake of
Solulan S16.
5 (6) Because of the fact that the solutions solidified at
room temperature, dissolution or otherwise of ZnAc2 in
Solulan 16 was assessed visually by ~x~ml n; ng the
clarity or turbidity of the resulting liquid
formulations, rather than by recording optical densities.
1() Results of visual observations are recorded in the table
below:
Sample Ratio S16:Zn Optical Appearance
twt:wt) Clarity
A 9:1 +++ Clear Solution
B 5.7:1 - Cloudy Paste
C 4:1 - Cloudy Paste
Exam~le 2
2()
(1) Solutions of Solulan 16 and ZnAc2 were prepared as
above, and dispensed into 2ml glass screw-capped vials as
follows:
SUBSTITUTE SHEET (RULE 26)
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A B C D E F G
S16(~1) 0 20 40 80 120160 200
ZnAc2(~1) 2020 20 20 20 20 20
S16 (mg) 0 2 4 8 12 16 20
ZnAc2(mg) 2 2 2 2 2 2 2
Ratio 0:11:1 2:1 4:1 6:18:1 10:1
S16:Zn
(2) After lyophilisation and heating to 60~C, the
solubility of ZnAc2 in S16 was assessed visually as
described in Example 1. The results of observations are
given in the table below:
Sample A B C D E F G
Ratio (S16:Zn) 0 1 2 4 6 8 10
Observations * * * + + - -
after heating
*= Samples remained as white solids
+= Samples turned to a viscous glassy fluid
-= essentially clear free-flowing fluid
ExamDle 3
(1) A solution of Solulan C24 at a concentration of
100mg/ml was prepared by addition of 500mg of Solulan C24
to 4.5ml of distilled water and mixing at 60~C until
dissolution was achieved.
(2) 500mg CuAc2 was dissolved in 10ml of distilled water
SUBSTITUTE SHEET (RULE 26)
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to give a concentration of 50mg/ml.
(3) 150~1 of CuAc2 solution (7.5mg solid) and 925~1 of
Solulan C24 solution (92.5mg solid) were dispensed into
a 4ml glass screw-capped vial. The vial contents were
mixed well, frozen in liquid nitrogen and lyophilised
overnight.
(4) lOmg of CuAc7 was dispensed into a 2ml glass vial and
lOOmg of solulan C24 was added. The vials was capped
and heated to 60~C to melt the oil. The contents of the
tube were vortexed to disperse the CuAc2 in the oil, then
incubated at 60~C for eight hours. After incubation, the
contents of the vial consisted of a colourless oil
l'i solution on top of solid undissolved crystals of CuAc2.
(5) The following day, the contents of the lyophilised
tube was converted to a clear strong blue-coloured
solution by incubating in a heating block for 2 minutes
at 60~C.
(6) The vials from steps (4) and (5) were allowed to
stand at room temperature to allow any undissolved CuAc~
to sediment, before the oil solidified.
2'i
(7) 20~g of solid oil was taken off the surface of each
sample in step (6), and dissolved in 18o~l of distilled
water.
(8) The optical densities of the aqueous solutions
obtained in step (7) were measured at 65Onm, and
compared against reference solutions prepared by dilution
of CuAc1 solution from step 2 in distilled water. The
results are reported in the table below, where it is seen
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that a higher concentration of CuAc2 dissolved in the oil
can be measured by following the lyophilisation procedure
described above, than by simple mixing of the components.
Concentration in aqueous
solution(mg/ml)
Measured Theoretical
After 6.7 7.5
lyophilisation
After simple 0.8 lO.o
mlxlng
ExamDle 4
A virus suspension (Sabin strains, Types 1, 2, 3~
containing 5xlO~ particles/ml (spun to remove
contaminating protein) was diluted 50-fold by addition of
200~1 of the suspension to 9.9ml of distilled water,
yielding a concentration of 107 particles/ml. The
suspension was divided into four equal aliquots of 2.5ml,
and dispensed into 7ml screw-capped glass vials. 2.5ml of
distilled water was added to one aliquot of virus
particles and this group was labelled "W". 2.5ml of
Solulan C24 (lOOmg/ml) was added to another aliquot and
mixed gently. This group was labelled "S".
200~1 of each preparation was dispensed into 10 freeze-
drying vials, and the remainder in 100~1 aliquots into
other tubes as "pre-drying" controls. The controls were
stored overnight at +4~C. The freeze-drying vials were
placed in the centrifugal rotor of the freeze-dryer and
lyophilised overnight.
On the following day 100~1 of culture medium was added to
SUBSTITUTE SHEET (RULE 26)
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each vial in group ~W~ and mixed gently. The vials in
group "S" were sealed and heated to 60~C in a hot water
bath for 5 seconds to melt the Solulan C24, which
resulted in a claer solution. Upon cooling to room
temperature this material solidified. 90~1 of medium was
added to the vials of the "S" group to make the total
volume up to 100~1. 10~1 of sample was then transferred
from each of groups "S" and "W" to fresh lml vials and
lml of medium was added to each and mixed well.
11~
To fresh lml vials was added 4 x 20~1 of samples from
each of the pre-drying groups and lml of medium was added
to each. The contents of each vial were mixed well.
1'; The suspensions prepared as described herein were used to
perform 10-fold dilutions in Vero cell cultures,to
measure the viability of the polio virus present. The
results were expressed as the highest dilution at which
50~ cytopathic effects were observed.
2~)
Nature of Sample Highest Dilution at
which 50% CPE obser~ed
Non-dried control + water 10-~/10-6
2C, Non-dried control + Solulan C24 10-5/10-5
Freeze-dried control + water 10-2/10-2
Freeze-dried control + Solulan C24 10-6/10-a
