Note: Descriptions are shown in the official language in which they were submitted.
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A method for preuaration of vesicles loaded with biological
structures biopo lvmers and/or oliaomers
The invention is relqted to a method for preparation of
vesicles loaded with biological structures, biopolymers
and/or - oligomers, a formulation comprising vesicles loaded
with biological structures, biopolymers and/or -oligomers
obtainable according to the method of the invention, a
medicament comprising the formulation of the invention as
well as a method of treating diseases administering the
medicament of the invention.
Several attempts have been tried to use lipid vesicles formed
by natural or synthetic phospholipids as vehicles for the
administration of effective substances.
Grey, A. and Morgan, J. report that liposomes were first
described nearly a quarter of a century ago and have been
useful models for studying the physical chemistry of lipid
bilayers and the biology of the cell membrane. It was also
realised that they might be used as vehicles for the delivexy
of drugs but clinical application have been slow to emerge.
Proposed clinical uses have included vaccine adjuvancy, gene
transfer and diagnostic imaging but the major effort has been
WO 95/04523 PCT/EP94/02242
2 -
in the development of liposomes as targetable drug carriers
in the treatment of malignancy. Although based on good in
vitro data and animal studies, the strategies have been
mostly impractical due to the predominant but unwanted
uptake by the reticuloendothelial system and the limited
extent of extravasation. The same features have nonetheless
been turned to advantage in the case of amphotericin B which
has recently become the first liposomally formulated agent
to be licensed for parenteral use. Liposomal doxorubicin is
currently also being evaluated in clinical trials. The early
evidence suggests that while liposomal encapsulation may not
greatly enhance their efficacy the toxicity of these agents
is greatly attenuated (A. Gray, J. Morgan, "Liposomes in
Haematology" in Blood Reviews, 1991, 5, 258 - 271).
Liposomes have been used in biological systems such as plasma
extravascular space like reticuloendothelial system to more
access celluar uptake of liposomes. Liposomes were loaded
with amphotericin which is an effective but toxic antifungal.
Antitumor agents like adriamycine have also be incorporated
into liposomes. Vaccines and adjuvants as well as biological
response modifiers like lymphocines and so on were studyed
in encapsulated form. Liposomes are discussed in field of
a gene transfert as vehicles.
N. Sakuragawa et al. report in Thrombosis Research 38, 681 -
685, 1985, 1988 Clinical Hematology 29 (5) 655 - 661, that
liposomes containing factor VIII have been prepared for oral
administration to patients which are suffering from von
Willebrand's disease. The encapsulation was carried out by
dissolving the protein factor VIII concentrates in an
aprotinin containing solution and transferred into lecithin
coated flasks. After drying the flasks by rotation for 30
min under negative pressure liposomes were formed which
entrapped factor VIII concentrates. The liposome solution
was centrifuged yielding 40 s of factor VIII entrapped in
liposomes.
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Another method for entrapment of drugs in liposomes is
based on dehydration - rehydration. This is described by C.
Kirby and G. Gregoriadis in Bio/Technology, November 1984,
pages 979 - 984. In this preparation the entrapments can be
increased by using additional lipid. Disclosed is the use
of cholesterol as being of positive influence of the drug
entrapment. Since cholesterol is involved in the pathobio-
chemistry of some disorders, administration of cholesterol
containing vesicles is not harmless at all.
Obj ect of the present invention is to provide a method for
encapsulating biological structures, biopolymers and/or
oligomers particularly those being pharmaceutically active
into lipid membrane vesicles giving higher encapsulation of
the respective substance. A further object is the
preparation of a formulation particularly a medicament
having a higher efficiency.
Surprisingly, one object of the invention is solved by a
method for preparation of vesicles loaded with biological
structures, biopolymers and/or -oligomers comprising the
step of co-drying a fraction of amphiphatic material and a
fraction of biological structures, biopolymers and/or
-oligomers wherein said fraction of amphiphatic material is
present in an organic solvent which is miscible with water
and said fraction of biological structures, biopolymers
and/or -oligomers is present in an aqueous medium.
According to one aspect of the invention there is a method
for preparation of vesicles loaded with at least one of
biological cell-structures, biopolymers and -oligomers, by
co-drying a fraction of amphiphatic material and a fraction
of the at least one biological cell-structures, biopolymers
and -oligomers wherein said fraction of amphiphatic
material is present in an organic solvent which is miscible
with water and said fraction of the at least one biological
cell-structures, biopolymers and -oligomers is present in
an aqueous medium, the method consisting of the following
steps:
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a) solubilizing the arnphiphatic material in said
organic solvent being miscible with water
(fraction A) ;
b) solubilizing the at least one of biological cell-
structures, biopolymers and -oligomers in said
aqueous medium being physiologically compatible
having a salt content equivalent to up to 5% by
weight of sodium chloride solution (fraction B);
c) mixing the fractions A and B together;
d) lyophilizing the fraction obtained thereby; and
e) taking up the product of step d) in an aqueous
medium to give a dispersion of vesicles loaded
with said biological cell-structures, biopolymers
or -oligomers.
Brief Description of the Drawings
Figure 1 describes the results of the in vivo induction of
DTH by skin test to membrane vaccines (STM) compared to in
vitro simulation (SI) by STM;
Figure 2 represents the factor IX calibration curve
clotting assay; and
Figure 3 is the schematic diagram of the method according
to one embodiment of the invention.
Liposomes can be classified according to various
parameters. For example, when size and number of lamellae
(structural parameters) are used, three major types of
liposomes have been described: Multilamellar vesicles
(MLV), small unilamellar vesicles (SW) and large
unilamellar vesicles (LUV). MLV are the species which form
spontaneously on hydration of dried phospholipids above
their gel to liquid crystalline phase transition
temperature (Tm). Their size is heterogenous
CA 02168656 2004-03-16
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and their structure resembles an onion skin of alternating,
concentric aqueous and lipid layers.
SUV are formed from MLV by sonication and are single
layered. They are the smallest species with a high surface-
to-volume ratio and hence have the lowest capture volume of
aqueous space to weight of lipid.
A third type of liposome LUV has a large aqueous
compartment and a single (unilamellar) or only a few
(oligolamellar) lipid layers.
Further details are disclosed in D. Lichtenberg and Y.
Barenholz, Liposomes: Preparation, Characterization, and
Preservation, in Methods of Biochemical Analysis, Vol. 33,
pp. 337 - 462 (1988).
As used herein the term "loading" means any kind of
interaction of the biopolymeric substances to be loaded,
for example, an interaction such as encapsulation, adhesion
(to the inner or outer wall of the vesicle) or embedding in
the wall with or without extrusion of the biopolymeric
substances.
As used herein, the term "liposome" is intended to include
all spheres or vesicles of any amphiphatic compounds which
may spontaneously or non-spontaneously vesiculate, for
example phospholipids where at least one acyl group
replaced by a complex phosphoric acid ester. The most of
triacylglycerol is suitable and most common phospholipids
for the present invention are the lecithines (also referred
to as phosphatidylcholines (PC)), which are mixtures of the
diglycerides of stearic, palmitic, and oleic acids linked
to the choline ester of phosphoric acid. The lecithines are
found in all animals and plants such as eggs, soybeans, and
animal tissues (brain, heart, and the like) and can also be
produced synthetically. The source of the phospholipid or
WO 95/04523 b 3 6 tjPCT/EP94/02242
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its method of synthesis are not critical, any naturally
occurring or synthetic phosphatide can be used.
Examples of specific phosphatides are L-a-(distearoyl)
lecithin, L-a-(diapalmitoyl) lecithin, L-a-phosphatide acid,
L-a-(dilauroyl)-phosphatidic acid, L-a(dimyristoyl)
phosphatidic acid, L-a(dioleoyl)phosphatidic acid, DL-a(di-
palmitoyl) phosphatidic acid, L-a(distearoyl) phosphatidic
acid, and the various types of L-a-phosphatidylcholines
prepared from brain, liver, egg yolk, heart, soybean and the
like, or synthetically, and salts thereof. Other suitable
modifications include the controlled peroxidation of the
fatty acyl residue cross-linkers in the phosphatidylcholines
(PC) and the zwitterionic amphiphates which form micelles
by themselves or when mixed with the PCs such as alkyl
analogues of PC.
The phospholipids can vary in purity and can also be hydro-
genated either fully or partially. Hydrogenation reduces the
level of unwanted peroxidation, and modifies and controls
the gel to liquid/crystalline phase transition temperature
(Tn,) which effects packing and leakage.
The liposomes can be "tailored" to the requirements of any
specific reservoir including various biological fluids,
maintains their stability without aggregation or chromato-
graphic separation, and remains well dispersed and suspended
in the injected fluid. The fluidity in situ changes due to
the composition, temperature, salinity, bivalent ions and
presence of proteins. The liposome can be used with or
without any other solvent or surfactant.
Another important consideration in the selection of phospho-
lipid is the acyl chain composition thereof. Currently, it
is preferred that it has an acyl chain composition which is
characteristic, at least with respect to transition
temperature (Tm) of the acyl chain components in egg or
WO 95/04523 PCT/EP94/02242
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soybean PC, i. e., one chain saturated and one unsaturated
or both being unsaturated. However, the possibility of using
two saturated chains is not excluded.
The liposomes may contain other lipid components, as long
as these do not induce instability and/or aggregation and/or
chromatographic separation. This can be determined by routine
experimentation.
A variety of methods for producing the modified liposomes
which are unilamellar or multilamellar are known and avail-
able:
1. A thin film of the phospholipid is hydrated with an
aqueous medium followed by mechanical shaking and/or
ultrasonic irradition and/or extrusion through a suit-
able filter;
2. dissolution of the phospholipid in a suitable organic
solvent, mixing with an aqueous medium followed by
removal of the solvent;
3. use of gas above its critical point U. e., freons and
other gases such as COZ or mixtures of CO2 and other
gaseous hydrocarbons) or
4. Preparing lipid detergent mixed micelles then lowering
the concentration of the detergents to a level below
its critical concentration at which liposomes are
formed (Lichtenberg, Barenholz, 1988).
In general, they produce liposomes with heterogeneous sizes
from about 0.02 to 10 m or greater. Since liposomes which
are relatively small and well defined in size are preferred
for use in the present invention, a second processing step
defined as "liposome down sizing" is for reducing the size
and size heterogeneity of liposome suspensions.
WO 95/04523 PCT/EP94/02242
~
- 716 u r ~r3
The liposome suspension may be sized to achieve a selective
size distribution of vesicles in a size :range less than about
m and preferably to be < 0.4 m. Liposomes in this range
can readily be sterilized by filtration through a suitable
filter. Smaller vesicles also show less a tendency to aggre-
gate on storage, thus reducing potentially serious blockage
or plugging problems when the liposome is injected intraveno-
usly. Finally, liposomes which have been sized down to the
submicron range show more uniform distribution. -
Several techniques are available for reducing the sizes and
size heterogeneity of liposomes, in a manner suitable for
the present invention. Ultrasonic irradiation of a liposome
suspension either by standard bath or probe sonication
produces a progressive size reduction down to small uni-
lamellar vesicles (St,TVs) between 0.02 and 0.08 m in size.
Homogenization is another method which relies on shearing
energy to fragment large liposomes into smaller ones. In a
typical homogenization procedure the liposome suspension is
recirculated through a standard emulsion homogenizer until
selected liposome sizes, typically between about 0.1 and 0.5
m are observed. In both methods, the particle size distri-
bution can be monitored by conventional laser-beam particle
size determination.
Extrusion of liposomes through a small-pore polycarbonate
filter or equivalent membrane is also an effective method
for reducing liposome sizes down to a relatively well-defined
size distribution whose average is in the range between about
0.02 and 5 m, depending on the pore size of the membrane.
Typically, the suspension is cycled through one or two
stacked membranes several times until the desired liposome
size distribution is achieved. The liposome may be extruded
through successively smaller pore membranes, to achieve a
gradual reduction in lipsome size.
~: I vC~~)Jfj
- 8 -
Centrifugation and molecular sieve chromatography are other
methods which are available for pro(ducing a liposome
suspension with particle sizes below a selected threshold less
than 1 m. These two respective methods involve preferential
removal of large liposomes, rather than conversion of large
particles to smaller ones. Liposome yields are correspondingly
reduced.
The size-processed liposome suspension may be readily
sterilized by passage through a sterilizing membrane having
a particle discrimination size of about 0.4 m, such as a
conventional 0.45 m depth membrane filter. The liposomes are
stable in lyophilized form and can be reconstitued shortly
before use by taking up in water.
In a preferred embodiment the method of the invention
comprises the steps:
- a) solubilizing amphiphatic material in a polar-protic sol-
vent being miscible with water (fraction A),
alternatively, dried lipids or lipid mixture can be used
in any form (powder, granular, etc.) directly,
- b) solubilizing biopolymers and/or oligomers in an aqueous
medium being physiologically compatible, optionally
having a salt content equivalent to up to 5% by weight,
preferably 1.5 % by weight of sodium chloride solution
(fraction B)
- c) mixing together the fractions A and B
- d) drying the fraction obtained in step c) with a method
retaining the functional properties of said biological
structures, biopolymers and/or oligomers.
In general, the lipids mentioned above are suitable to be used
for forming lipid membrane vesicles. In particular
saturated, unsaturated phospholipids and combinations thereof
are advantageously used according to the method of the in-
vention. Dimyristoyl phosphatidyl choline (DMPC) and/or
AAAENDED SHCfT
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- 9 -
dimyristoyl phosphatidyl glycerol (DMPG) are used more pre-
ferabLy for forming of the lipid vesicles. Preferably, the
molar ratio of the lipids DMPC : DMPG is be.tween 120 and
20 : 1.
According to the method of the invention the organic solvent
being miscible with water is a polar-protic solvent having
solubilizing properties such as for example aliphatic alcohols
with lower number of carbon atoms so long as they mix with
aqueous systems and do not affect adversely the effectivity
of the biological structures, biopolymers and/or -oligomers
to be encapsulated. Suitable alcohols are e. g. methanol,
ethanol, propanol and/or preferably t-butanol.
Biological structures to be encapsulated according to the
invention are any structures of higher order built up by
various components and/or substructures. Examples for these
structures are whole cells, such as natural or transformed
B-cells, cell organelles, such as ribosoms or
mitochondriae.Virions or particles such as hepatitis B surface
antigen (HBsAg) particles. Biopolymers and/or -oligomers to
be encapsulated according to the inventior.i are any substances
having effects in human or animal systems. Preferred are
substances as proteins such as enzymes, proenzymes, cofactors,
such as those of the blood clotting system, antigens, anti-
bodies, factors of the immune system such as complement
factors, peptides such as hormones, nucleotides and/or nucleic
acids such as genomic DNA for use in gene therapy, RNA such
as mRNA=, rRNA, tRNA, antisense RNA and the like.
It is unterstood by the skilled person that the amount of
organic polar-protic solvent miscible with water is strongly
dependent on its interference with the substance to be en-
capsulated to the liposomes. For example, for HBsAg 50% is
tolerable while factor IX (which is a clotting factor) is to
be encapsulated as an amount of approximately 30% of tert.-
butanol is tolerable. This may strongly vary with the nature
AAENDEC' S'icE i
4 ~ iJUU
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of the substance to be encapsulated. For example, if factor
IX which is a clotting factor is to be encapsulated an amount
of about 30% of tertiary butanol is tolerable, whereas, factor
VIII is much more sensitive to the impact of tert.-butanol.
In this case an amount of less than 10% of tert.-butanol is
preferred. The percentage of t-butanol in these examples is
based on percent by volume calculated for final concentration.
According to the method of the invention it is preferred to
keep the biopolymers and/or -oligomers in a medium having an
ionic strength corresponding up to a about 5% sodium chloride
concentration with or without cryoprotectant which is a
pharmaceutically acceptable agent such as lactose, sucrose
or trehalose, preferably the medium for solubilizing, dis-
solving or dispersing the biological structures, biopolymers
and/or -oligomers is an aqueous solutiori of about 0.9% by
weight sodium chloride and/or an isoosmotic cryoprotectant.
According to the invention any method for drying is suitable
so long as the effectivity of the biological structures,
biopolymers and /or -oligomers are not affected a versely
by the selected drying method. The function of the iDiologicai
structures, biopolymers and/or oligomers; to be loaded are
mostly retained when mild drying conditions are selected.
For example removing of the solvents of the solution of the
biological structures, biopolymers and/or -oligomers and the
lipids is favourably achieved by drying under reduced
pressure at slightly elevated temperatures at maximum. The
resistance of the active substances to be loaded depend
strongly on the stability of the respective biopolymer and/or
-oligomer. For example, nucleic acids are more stable versus
impact of heat on their structure and function than proteins.
The latter are more sensitive to heat-denaturation. A very
preferred method for co-drying of the f:ractions according
to the invention is the method of lyophilisation (freeze
4 V'.. !
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WO 95/04523 2168656 PCT/EP94/02242
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drying). This method is a mild drying procedure for almost
all of the active biological structures, biopolymers and/or
oligomers which become liposomal loaded according to the
invention.
According to the method of the invention the product obtained
as described above in dry form is taken up in an aqueous
medium. Thereby, liposomes formed become loaded with the
respective biological structures, biopolymers and/or oligo-
mers. The system typically forms a dispersion.
According to the method of the invention a novel formulation
is provided comprising lipid membrane vesicles loaded with
biological structures, biopolymers and/or -oligomers. The
formulation of the invention preferably is in a solid state,
which is available a~ter the co-drying of fraction A and B,
eventually having other pharmaceutically acceptable vehicles
and/or adjuvants as well as other pharmaceutically active
agents.
Another preferred embodiment of the formulation of the
invention comprises a solution of the fraction in an aqueous
medium obtainable according to the method of the invention.
Preferably, the aqueous medium for taking up the dry fraction
of the formulation contains a balanced salt content in order
to adjust the conditions of the formulation in such a manner
that the aqueous solution thus obtained can readily be used
as a medicament. Typically, the formulation tends to form
a dispersion after being taken up into water.
Thus, the medicament of the invention is basically the
formulation obtainable by the method of the invention but
being adapted to a way of administration which is suitable
for the treatment or prophylaxis of the respective disease.
For example, the medicament of the invention can be
administered by topical, oral, intravenous, pulmonary, intra-
WO 95/04523 . ~ ' PCT/EP94/02242
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peritoneal, intranasal, rectal, intraocular, buccal,
subcutaneous and intramuscular ways of application.
A method of treatment and/or prophylaxis of diseases by
administering an effective amount of the medicament according
to the invention is provided. It is understood by the skilled
person that the dosage is depending on the concentration of
the effective substances as well as their efficiency.
According to the method of treatment and/or prophylaxis of
the invention preferably a dosage of up to 2, 000 mg vesicles
(e. g. phospholipid liposomes) /kg body weight is administered
to the patient. The accurate dosage can vary dramatically.
The variation, however, depends on e. g. the type and
efficacy of the substance encapsulated in the liposomes, the
efficiency of the encapsulation reaction itself (being high
with the method of the invention), the kind of administration
and the like. The respective parameters can be easily
optimized by the person skilled in the art and can be
regarded as being routine experiments.
The invention is further explained by the following non-
limiting examples.
Example 1
Preparation of samples of anti-HBV liposomal vaccine
The following samples of vaccine, designated samples 1, 2,
3 and 4 were prepared using the method of the invention.
Sample 1: A mixture of DMPC : DMPG in a molar ratio of 9 : 1
respectively was prepared in tert.-butanol. An aqueous HBsAg
solution such as 0.9%- NaCl in 1: 1 (v/v) was added. The
final HBsAg: phospholipids (w/w) ratio was 0.0015. The
solution was frozen and dried by lyophilization. A dry powder
was obtained which was reconstituted before use with double
distilled sterile pyrogen-free water. Multilamellar liposomes
WO 95/04523 2 1 b 8, 6 5 6 PCT/EP94/02242
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were formed; loading efficiency of HBsAg was 97%-. "Empty
liposomes" were prepared similarly by mixing 1 vol of aqueous
solution of 0.9% NaCl with 1 vol of lipid solution in
tertiary butanol.
The extent of HBsAg exposure on the liposome surface of
sample 1 and liposome size was determined. It was found that
the size of these lipsomes was 4.5 m and the exposure of
the antigen on the liposome surface was tested. It was found
that the titer of antibodies which was developed was high
and sufficient to protect against infection by HBV (see Table
1) . The titer was similar to that obtained in mice that were
vaccinated with the same antigen using aluminum hydroxide
based vaccine except for the high dose of injected antigen
(2.5 g) in which the liposomal vaccine was inferior: in-
jection of this dose to mice in the control group stimulated
the highest titer of antibodies.
Sample 2: Liposomes loaded with HBsAg and "empty liposomes"
were prepared as described for sample 1. A group of seven
Balb/c mice, six weeks old, were vaccinated by 0.09 g HBsAg
loaded in liposomes which were diluted with "empty liposomes"
and 0.9k NaCl. The final injection volume was 0.5 ml/mice,
which included also 1 mg/kg mice of the immunomodulator MTP-
PE in POPC/DOPS (7 : 3 mole ratio) liposomes. After 35 days
the level of anti-HBs in the mice was measured. The titer
of antibodies was twice the titer which developed after
injecting the same dose of antigen without MTP-PE (sample
1).
Sample 3: Liposomes loaded with HBsAg and identical "empty
liposomes" were prepared as described for sample 1 with one
difference in that the aqueous solution used for lipid
hydration also contained 5%- lactose. The liposomes were
frozen and dried. A powder was obtained which was re-
constituted before use with sterile pyrogen-free bidistilled
water. The liposomes were characterized for their size,
WO 95/04523 PCT/EP94/02242
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~ ~ "D
percentage of antigen loading and the extent of antigen
exposure on the liposome surface. The immunization efficacy
of the preparation was tested in Balb/c mice, six weeks old.
The mice were divided into three groups, five mice in each
group, and the animals were vaccinated using three doses of
antigen: 0.09 g, 0.27 g, O.81 g, respectively. Anti-HBs
was measured after 35 days (see Table 1). A high titer of
antibodies was observed which should be sufficient to protect
against HBV infection.
Injecting this preparation in low doses of antigen (0.09 g)
to mice resulted in development of the highest titer of
antibodies, compared with the titer which was obtained with
all other preparations including the mice group which was
vaccinated with the commonly used aluminum hydroxide-based
vaccine having identical HBsAg.
Sample 4: Liopsomes loaded with HBsAg were prepared as
described for sample 3. Three groups of five Balb/c mice,
six weeks old, were vaccinated with four doses of HBsAg at
a level of 0.09 g, 0.27 g, 0.81 g, respectively. The total
injection volume was 0.5 ml/mice. The liposomes were diluted
with PBS only and not with "empty liposomes" and therefore
the amount of lipid varied and increased with increasing
protein level. After 35 days the mice were bled and their
serum antibody titer was determined. The results show a high
titer of antibodies which should be sufficient to protect
against infection by HBV.
0
Table 1
Summary of anti-HBs titer (mIU/ml) usina the linosomal vaccine samales
described in Sxamwle 1
Sample No. m HBsAg injected
0.09 0.27 0.81 2.5
1 52.4 f 18.6 426.7 t 206.3 4,953.2 1,211.5 6,692.0 t 854.5
2 106.1 t 16.5 - - -
3 193.3 t 69.1 1,664.6 t 392.8 2,701.4 203.6 - -~
cn
N
4 55.0 t 17.3 895.9 t 384.6 1,527.7 166.6 - ~-'
Cr.
O-N
Control Alum- 40.0 t 13.6 396.6 t 73.1 6,749.3 2,342.5 17,465.3 t 2,967.0
Lr~
based vaccine
=
A
NN
WO 95/04523 PCT/EP94/02242
16 -
Example 2
Stability of liposomal HBsAa vaccine after storage at various
temperatures
As described above hepatitis vaccines known in the art used
aluminum hydroxide as adjuvant and stabilizer. The dis-
advantage of the aluminum hydroxide-based vaccines is that
they cannot be frozen nor can they be stored beyond 8 C.
These vaccines thus have to be stored between 2 - 8 C to
maintain their efficacy.
There are three parameters to demonstrate stability of a
vaccine under different conditions:
1. Efficiency (measure immunogenicity).
2. Chemical stability (measssure hydrolysis of lipids;
measure protein to lipid ratio).
3. Physical stability (measure size of particle).
The stability of the vaccine was tested after storage at
three temperature (a) -20 C, (b) 2 - 6 C and (c) room
temperature.
The results obtained were as follows:
(a) The vaccine stored at -20 C was effective after 1 month
or more and was chemically and physically stable after
1.5 years and more.
(b) The vaccine stored at 2 - 6 C was effective after 1
month and more and was chemically and physically stable
after 1.5 years and more.
(c) The vaccine stored at room temperature was chemically
and physically stable after 1.5 years or more.
WO 95/04523 PCT/EP94/02242
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These results demonstrate that the vaccine of the invention
in form of liposoms is stable over a wide temperature range.
Since the current hepatitis vaccines lose their immuno-
genicity during freezing it is unexpected that the liposom-
vaccine of the invention retians its activity both during
the freezing step of the freeze drying process and also
during storage of the vaccine below 0 C.
Thus, the advantage of HBV vaccine of the invention is
evident. It does not need to be stored in a refrigerator and
is not sensitive to freezing. the distribution of such a
vaccine is grealty simplified especially in third world
countries where the need for a vaccine against hepatitis B
is greatest; additionally a vaccine which may be frozen aids
distribution in countries such as Russia and China were the
ambient temperature is often below freezing.
Applicants have thus produced a novel liposomal based HBsAg
vaccine which is stable both below zero degrees and at room
temperature, i. e. the vaccine may be stored under suboptimal
conditions.
Example 3
Preoaration and characterization of factor-IX-loaded lipo
somes
Two different methods of liposome preparation will be com-
pared for stability and Factor IX encapsulation.
(a) Dehydrated Reydrated Vesicles (DRV's)
(b) Lipid and drug co-solubilization in an organic solvent.
(a) Dehydrated Rehydrated Vesicles (DRV's)
WO 95/04523 PCT/EP94/02242
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-is -
Preparation of multilamellar vesicles loaded with Factor-IX
by the DRV method require the following steps: preparation
of small unilamellar vesicles (SUV's) in bidistilled water,
mixing them with a solution of factor IX previously dialyzed
against amino acids and flash-frozen the mixture. After
lyophilization, multilamellar vesicles loaded with Factor-IX
were obtained by rehydrating the preparation with bidistilled
water, then stepwise saline is added, until the final lipo-
somes concentration was reached. At this point the multi-
lamellar vesicles can be sized by extrusion to obtain oligo-
lamellar or small unilamellar vesicles.
Rehydration of lyophilized material with minimal volume
results in an increase of the overall concentration of the
factor. After liposomes are formed the solution can be
further diluted without affecting the loading efficiency,
and this is reflected in the concentration of the material
that is actually loaded. Since liposomes are osmotically
active, losses of material on exposure to hypotonic media
during all manipulations subsequent to hydrating were
minimzed by dialyzing the Factor before mixing with the SUV's
to obtain a lower osmolarity in the liposome interior during
the rehydration step.
(b) Lipid and druct co-solubilization in an organic solvent
In this preparation lipid solubilized in tert.-butanol is
mixed with an aqueous solution of the factor to obtain an
homogeneous solution. The solution is frozen and the solvent
removed by lyophilization. Mulitlamellar vesicles loaded with
Factor-IX are obtained by hydration of the dry mixture,
firstly in small volume of bidistilled water, then stepwise
with saline, until the final liposome concentration is
reached. At this point the multlamellar vesicles can be sized
by extrusion to obtain oligolamellar or small unilamellar
vesicles.
CA 02168656 2004-03-16
WO 95104523 PCT/EP94102242
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Determination of factor IX activity
Factor IX activity was measured by a clotting assay. In this
assay the percent of factor IX activity-can be determined
by the degree of correction obtained when a dilution of the
tested sample is added t-o the factor IX Deficient Plasma
(purchased from Baxter ~Dia;gndgtics Inc. ). The measuring
instrument is called ACCAuttgmated Coagulation Laboratory
from Instrumentation Labortory (Italy).
A calibration curve was first constructed for the clotting
assay of factor IX, using appropriate dilutions of a stock
solution of ca. 50 U/ml. Figure 2 shows a good fit to a
linear regression (R2 = 0.989).
Liposomes containing fac-tor IX were pelleted by centri-
fugation in an Eppendorffucentrifuge at 12,000 g for 10 min
and the factor IX activitiy was determined in the super-
natants and pellet. The pellet was solubilized- prior analysis
with Triton X-100. A concentration dependency on factor IX
activity with Tritor?X100 was found. 1t TritonX100 (final
concentration) caused a 50% loss of activity, while no loss
was observed at 0.2o.In general, the total activity of the
factor was recuperated, namely, the activity of the super-
natants and pellet was always similar or even higher than
the inital activity of the preparation. The loading
efficiency was higher than 80%.
ExamQl e 4
Melanoma treatment of human natients by ligosomal vaccine
containing Allogenic Human Melanoma vaccine prepared using
tertiary butanol.
Vaccine preparation: A mixture of DMPC : DMPG in a molar
ratio of 9: 1 respectively was dissolved in tert.-butanol
WO 95/04523 PCT/EP94/02242
1~ - 20 -
in a 1 : 6.7 w/v ratio. The mixture was heated and stirred
until the lipids were dissolved. After sterile filtration,
sterile water was added to the organic mixture until a 1:
1 (v/v) ratio between the tert.-butanol and the water was
reached. An aqueous solution of the melanomic membrane
mixture was added to a 1 : 750 protein : phospholipids (w/w)
ratio. This final mixture was divided in single doses of ig
phospholipids and each one was frozen and dried by lyophili-
zation. A dry powder was obtained and stored at -70 C. Prior
to application liposomes were formed by rehydration in double
distilled, sterile and pyrogen-free aqueous solution con-
taining 0.9t NaCl to obtain a liposome dispersion of 10%
phospholipid concentration. After reconstitution, this
liposomes had an average size of 1 m and an average phospho-
lipid : protein ratio of 765 : 1.
Treatment:(see enclosed)
A three arm randomized study for the treatment of metastatic melanoma by asi
alone and either systemic
or regional interleukin-2
Clinical and immunologial
Evaluation: eligible:
PTS with metastatic disease;
ECOP PS 1 - 2: no previous
Immunother.; positive to 3/7
antigens (merieux). =
Cimetidine Vaccine in liposomes
800 mg X 2 / daily 200 g protein/sile, (se),
4 weeks at 2 sites, 10 weekly
immunizations
R
A Melanoma vaccine only-given on day 1. ,N
N A cimetidine 800 mg X 2 / daily.
0~
D
ON
O B Melanoma vaccine only given on day 1, followed by Li-
M IV IL-2, at one million units/msq. on consecutive days 2, 3, 4. C3a
I cimetidine 800 mg X 2 / daily.
z A C Melanoma vaccine given on day 1, followed by subcutaneous
T IL-2 at vaccine site, concomitantly, IL-2 dose: 50.000 units/
I site at two sites, on days 1, 2 & 3. Cimetidine 800 mg X 2 0 daily.
N
PAT PROT TREATMENT DISEASE RESPONSE OUTCOME
#1 A CIM+4X V CC Lung PD(2m)Lung/Br/Liver Dead (4m)
#2 A CIM+2X VACC SC PD (2m) Brain Dead (6m)
#3 A CIM+5X VACC LN/LIVER PD(2m)LN/Liver Alive (6m)
#4 A CIM+5X VACC LN/Liver PD(2m)LN/Liver Alive (6m)
}
#1 B CIM+10X VACC+IL2 S Liver PD(3.5m)/Liver/Bone Dead (8m)
#2 B CIM+10X VACC+IL2 S LN/Bone PD(4m) LN/Bone Alive (16m)
#3 B CIM+10X VACC-IL2 S LN PD(4m) LN/Lung Alive (13m)
N
#4 B ICIM+6X VACC+IL2 S LN PD? (sepsis) Dead (2.5m)
#1 C CIM+10X VACC+IL2 R LN/Lung CR(8m) LN/Lung Alive (13m) NED
#2 C CIM+10X VACC+IL2 R LN CR(9m) LN Alive (13m) NED
#3 C CIM+10X VACC+IL2 R LN/Liver PD(3m) LN/Liver Dead (6m)
#4 C CIM+10X VACC+IL2 R LN/Lung PR(4m) LN(PR)/Lung(CR) Alive (12m) Surg
NED
#5 C CIM+10X VACC+IL2 R SC/Liver/Bone PR(5m) SC(PR)/Liver(CR) Dead(9m) PD
Brain
#6 C CIM+10X VACC+IL2 R SC/Lung MixR(5m) Alive (llm) IPL NED
SC (PD) /Lung (CR)
L::t7 C CIM+7X VACC+IL2 R SC/LN/Liver PD(3m) SC/Liver Dead (5m)
C CIM+5X VACC+IL2 R SC/Lung/Liver PD(1.5m) SC/Liver/Lung Alive (2m)
WO 95/04523 ~ 168656 PCT/EP94/02242
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CR = complete response
PR = partial response
SC = stable condition
m = month
(1) Allogeneic human melanoma vaccine was prepared from
membranes of six melanoma cell lines which express
both class I and II MHC antigens and MAAs (by R24
and P97) MoAbs) ;
(2) Membranes were loaded in liposomes consisting of
DMPC : DMPG in a 9 : 1 molar ratio, were tested for
sterility, pyrogenicity and tumorigenicity in nude
mice;
(3) 16 PTS were treated by vaccine: 4 - vaccine only
(A); 4 - vaccine + systemic IL2 (B); and 8 - by
vaccine + low-dose, regional (C) IL2;
(4) Clinical responses (regression of metastases) were
observed in 5 of 8 PTS in arm C of the protocol;
(5) The above clinical responses correlated with de novo
induction of cutaneous DTHI to membrane vaccine pre-
paration (STM) and in vitro MLTC (proliferative)
responses to STM;
(6) Augmented cytolytic responses against melanoma cell
lines were observed in the majority of vaccine-
treated PTS, but these were were not MHC-restricted,
nor did they show any correlation with clinical re-
sponses;
(7) Selective anti-melanoma cytolytic responses
following IVS were observed when 18 h - instead of 4
CA 02168656 2004-03-16
WO 95/04523 PCT/EP94/02242
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h assay was used, suggesting CD4, T-cell response,
also corroborated by surface markers study;
(8) In parallel patients were vaccinated with the same
antigens given as alumm based vaccine without any
response.
Example 5: Candidemia treatment in mice by liposomal
vaccine containing Candida ribosomes
Vaccine preparation:
DMPC : DMPG at a 9 1 molar ratio were dissolved in
tert.-butanol in a 1 10 (w : v) ratio and the lipid
mixture was pre-warmed to dissolve the lipids completely.
An aqueous ribosomal mixture containing 1.5 mg ribo-
somes/ml (determined by Orcinolly was added to the lipids
at a 1: 100 w/w final ratio. In some cases Lipid-A was
added at this stage as an adjuvant in a 1 : 1,000 lipid-A
to phospholipids molar ratio. This suspension was frozen
and lyophilized in aliquouts of 0.5 g phospholipids and
the dry powder was stored at =20 C. Prior application
liposomes were formed by adding two aliquots of 0.5 ml
volume of double distilled, sterile and pyrogen free
aqueous solution containing 0.9% NaCl.
Treatment:
Four groups of five Balb/c mice, six weeks old, were
vaccinated with a one single dose of 100 g ribosomes. Two
weeks later a booster injection was given and twenty
eight days after the first immunization the mice were
challenged by intravenous infection with 104 Candida
albicans cells.
Group 1: buffer (TMB) and IFA (incomplete Freund
adj uvant ) .
~ l b8 6 50"
WO 95/04523 PCT/EP94/02242
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Group 2: ribosomal mixture and IFA
Group 3: liposomes containing ribosomes
Group 4: liposomes containing ribosomes and lipid-A.
This experiment was repeated twice and the results are
summarized in the following table.
Group 1 Group 2 Group 3 Group 4
Mortality 6/9 2/10 0/10 0/10
Percentage 67% 20% 0% 0%
Example 6: Preparation of liposomes containing anti-
haemophilic factor IX
Liposomes Preparation:
Purified egg yolk phosphatidylcholine was dissolved in
tert.-butanol at various ratios and the mixture was
slightly warmed until the phospholipid was dissolved.
Double distilled sterile, pyrogen free water was added
until the desired ratio between the organic solvent and
the water was reached. An aqueous so:Lution of salt free
Factor IX (OCTANYNE adjusted pH 7.4 was added to the
suspension under continuous mixing and subsequently lyo-
philized. The ratio of the total protein to phospholipid
was 1..400 (w/w). The dry mixture was stored at 4 C.
Liposomes of 1 m average size were pr.epared by hydrating
the powder with aliquots of sterile, pyrogen-free double
distilled water and mixing well between the additions.
The last addition consisted of saline to raise the salt
concentration to isosmotic conditions.
