Note: Descriptions are shown in the official language in which they were submitted.
W096/09037 2 1 9 9 0 1 8 PCT~S94/10812
METHOD OF PRODUCING A LYOPHILIZED LIPOSOME PRODUCT
The present invention is generally directed to a
method of producing lyophilized liposomes and particularly to a
method in which an organic solvent, typically used for dissolving
the lipid and other components of the process, is eliminated.
This enables the liposomes to be lyophilized in a more efficient
and less costly manner.
Methods of forming liposome vesicles for the
association of a bioactive agent are well known. As used herein
the term "association" shall mean bioactive agent which is
encapsulated within the liposome and bioactive agent which, while
not encapsulated, remains with the liposome and is not readily
separated therefrom.
Some methods of forming liposomes employ an organic
solvent to dissolve a lipid alone or the lipid and a bioactive
agent such as a drug. For example, in Bally et al., U.S. Patent
No. 5,077,056, lipids are dissolved in an organic solvent and
combined with an aqueous medium to form liposomes. Then a
bioactive agent such as a drug is loaded into the preformed
liposomes using a transmembrane concentration gradient. On the
other hand, in Lenk et al., U.S. Patent No. 5,082,664, a lipid
and a bioactive agent are dissolved together in an organic
solvent, and combined with an aqueous medium to form liposomes
associated with the bioactive agent. In particular, the lipid
and the bioactive agent (e.g. lipophilic drugs such as the
prostagl~n~;n~) are co-dissolved in an aqueous-miscible organic
solvent such as ethanol, then added slowly to an aqueous
solution, which may additionally contain a drying protectant
and/or a buffer, as discussed in the Lenk et al. patent. Both of
these patents are hereby incorporated by reference into the
present disclosure.
Another method for forming liposomes employs ethanol
injection and is discussed in Batzri et al., Biochem. Bio~hys.
2 1 990 1 8
W096/09037 PCTnUS94/10812
Acta. 298:1015 (1973). The ethanol injection method has been
used to form liposomes having associated therewith a lipophilic
or hydrophilic bioactive agent. When forming liposomes
containing a lipophilic bioactive agent (e.g. prostaglandin), an
optional preservative and the bioactive agent are added to the
ethanol containing lipid. The resulting mixture is then slowly
added to an aqueous medium. This process forms liposomes
entrapping the aqueous medium. Ethanol injection processes, as
well as other liposome formation processes, using a desalted
charged lipid are disclosed in Popescu et al., U.S. Patent No.
5,154,930, incorporated by reference into the present
specification. A method of controlling size distribution of
resultant liposomes in an ethanol infusion process is discussed
in Aitcheson et al., U.S. Patent No. 4,994,213.
For the formation of liposomes having a hydrophilic
bioactive agent associated therewith (e.g. aminoglycosides, such
as gentamicin), the bioactive agent is added to the aqueous
phase. The lipid and ethanol are combined to form a solution
which is added to the aqueous phase and the resulting mixture is
processed to form liposomes. The aqueous phase may be a solution
of one or more drying protectants with or without a preservative.
The liposome preparations prepared by such methods
typically contain liposomes having a wide variety of particle
sizes. It is often desirable to reduce the size of the larger
liposomes to obtain a single-modal size distribution encompassing
a desired mean particle size. The term "single-modal size
distribution" as used herein shall mean that most of the
liposomes have a particle size within a continuous range of
particle sizes encompassing the mean particle size. The term
"mean particle size" shall mean the sum of the diameters of each
liposome of the population divided by the total number of
1 lposomes .
Size reduction to obtain a single-modal size
distribution can be achieved by a number of methods such as by
extrusion through a filter, as described in Pieter Cullis et al.,
U.S. Patent No. 5,008,050, incorporated herein by reference.
wo g6togo37 2 1 9 9 0 1 8 PCT/USg4/10812
A method of sizing liposomes by filtration through a
200 nm UniporeTM polycarbonate filter is discussed in Szoka,
Proc. Natl. Acad. Sci. U.S.A. 75:4194-8 (1978). A size-
processing method based on liposome extrusion through a series of
uniform straight-pore type polycarbonate membranes is described
in Hunt et al., U.S. Patent No. 4,529,561.
U.S. Patent No. 4,737,323, describes a method for
sizing liposomes by extrusion through an asymmetric ceramic
filter. Such filters are designed for operation at relatively
high pressure, and can be backflushed to prevent clogging. U.S.
Patent No. 4,927,637, describes a method of sizing liposomes by
passing them through a polymer filter having a web-like
"tortuous-path" construction.
An alternative type of filter medium is described in
Furneaux et al., U.S. Patent No. 4,687,551. This patent
discloses a filter sheet comprising an anodic al-]mlnl-m oxide film
having branched pores extending from one surface of the film to
the other. The film is unique in that it includes a system of
larger pores extending in from one face and a system of smaller
pores extending in from the other face. The system of larger
pores interconnects with the system of smaller pores such that
the inner ends of one or more smaller pores are joined to the
inner end of a larger pore and there are substantially no larger
pores that terminate within the film.
The application of an aluminum oxide porous film to
the size reduction of liposomes is disclosed in Royden M. Coe et
al., U.S. Serial No. 771,267 filed on October 4, 1991.
Homogenization is another method for size reducing
liposomes. In a simple homogenization method, a suspension of
liposomes is repeatedly pumped under high pressure through a
small orifice or reaction chamber until a desired size
distribution is achieved.
In such liposome-forming methods, the resulting
liposomes may be dehydrated or lyophilized by any method known in
the art, so that the size and contents are maintained during the
drying procedure and through rehydration. It has been found that
wo 96/0go37 2 1 9 9 0 1 8 PCTrUS94/10812
one group of drying protectants, the saccharides, when included
in the liposome formulations, are especially useful at
maintaining the liposome particle size after rehydration.
For the purpose of rehydration of the dehydrated or
lyophilized product, an aqueous solution such as distilled water
with or without buffer may be added. The pH gradient may be
established by adding a relatively acidic aqueous solution to the
formulation. Reconstitution may proceed at a temperature of
about 20~ to 70~C and the solutions diluted as needed and
10 ~m; n ' stered.
These methods of lyophilization, however, are not as
efficient as desired because of the presence of residual organic
solvent in the liposome product prior to lyophilization. The
solvent may make it more difficult and time consuming to
lyophilize the product due to the need for a lower primary drying
temperature. Elimination of the organic solvent may be
beneficial, for example, because the product may have a higher
glass transition temperature and primary drying could take place
at higher temperatures.
SUMMARY OF THE lNv~NlION
The present invention is directed to a process
employing no added organic solvent for the production of a
lyophilized liposome product. The process comprises combining at
least one lipid with an aqueous solution containing a drying
protectant in the absence of added organic solvent and
lyophilizing the resulting mixture to form the liposome product.
The liposome product may contain a bioactive agent. The
temperature needed to lyophilize the final product may not be as
low as previously required in systems using an organic solvent.
As a result, the time and cost of the lyophilization procedure
may be significantly reduced over processes which employ an
organic solvent.
In accordance with the present invention, there is provided
a method of forming a lyophilized liposome product comprising:
W096/09037 i 2 1 q 9 0 1 8 PCT~S94/10812
(a) without adding organic solvent, combining at least one
lipid with an aqueous solution containing a drying
protectant to form a mixture;
(b) agitating the mixture to form a population of
liposomes; and
(c) lyophilizing the processed mixture containing said
population of liposomes to form the lyophilized
liposome product.
In a particular embodiment of the present invention, a
bioactive agent is associated with the liposomes. The invention
is also directed to the lyophilized liposome product produced by
the above method, and to compositions of liposomes produced by
reconstituting the lyophilized product. The invention further
provides a lyophilized liposome product comprising a lipid-
encapsulated bioactive agent having a reduced level of organicsolvent, and preferably substantially absent organic solvent.
DETAILED DESCRIPTION OF THE lNv~NlION
The present invention is premised, inter alia, on the
discovery that a lyophilized liposome product can be made without
adding organic solvent. Processing of liposomes in accordance
with the present invention eliminates the time and cost of adding
the solvent, as well as removing the solvent during
lyophilization.
The liposomes of the present invention are prepared
by adding a lipid to an aqueous solution containing a drying
protectant. A particular type of lipid material for use in this
invention is one which is amphipathic in character. Hydrophilic
character can be imparted to the molecule through the presence of
phosphato, carboxylic, sulphato, amino, sulfhydryl, nitro, and
other like groups. Hydrophobicity can be conferred by the
inclusion of groups that include, but are not limited to, long
chain saturated and unsaturated aliphatic hydrocarbon groups and
such groups substituted by one or more aromatic, cycloaliphatic
or heterocyclic group. The preferred amphipathic compounds are
phosphoglycerides, representative examples of which include
phosphatidylcholine, phosphatidylethanolamine, lysophosphatidyl-
21 990t 8
W096l09037 PCT~S94/10812
choline, lysophosphatidylethanolamine, phosphatidylserine, phos-
phatidylinositol, phosphatidic acid, dimyristoylphosphatidyl-
glycerol and diphosphatidylglycerol alone or in combination with
other lipids. Synthetic saturated compounds such as dimyristoyl-
phosphatidylcholine, dipalmitoylphosphatidylcholine, ordistearoylphosphatidylcholine or unsaturated species such as
dioleoylphosphatidylcholine or dilinoleoylphosphatidylcholine
might also be usable. Other compounds lacking phosphorus, such
as members of the sphingolipid and glycosphingolipid families,
are also within the group designated as lipid.
A variety of cholesterols and other sterols and their
water soluble derivatives have also been used to form liposomes;
see specifically Janoff et al., U.S. Patent No. 4,721,612 and
references referred to therein, all of which are incorporated
herein by reference. Various tocopherols and their water soluble
derivatives have also been used to form liposomes, as disclosed
in Janoff et al. U.S. Patent No. 4,861,580, incorporated herein
by reference. Preferred of this group are cholesterol
hemisuccinate and tocopherol hemisuccinate.
The drying protectants which are employed for
lyophilization in accordance with the present invention are
selected from saccharides such as sucrose, dextrose, maltose,
mannose, galactose, raffinose, trehalose, lactose, as well as
polyhydric alcohols such as mannitol, and mixtures thereof.
Other drying protectants which can be employed in the present
process include albumin, dextrans, or polyvinyl alcohol. Maltose
is particularly preferred.
The concentration of the drying protectants is
generally in the range of from about 1 to 20% by weight,
preferably about 5 to 10% by weight, based on the weight of the
aqueous phase. The polyhydric alcohol, when present and used in
addition to the saccharides, is preferably provided at a
concentration of up to 2% by weight, more preferably about 1~ by
weight, based on the weight of the aqueous phase. The preferred
polyhydric alcohol is mannitol.
The bioactive agents which may be encapsulated within
the lipid bilayer include nucleic acids, polynucleotides,
W096/09037 2 1 9 9 0 1 8 PCTnUS94110812
antibacterial compounds, antiviral compounds, tumoricidal
compounds, proteins, toxins, enzymes, hormones, neurotrans-
mitters, glycoproteins, immunoglobulins, immunomodulators, dyes,
radio labels, radio-opaque compounds, fluorescent compounds,
polysaccharides, cell receptor binding molecules, anti-
inflammatories, antiglaucomic agents, mydriatic compounds, local
anesthetics, and the like. Spçcific examples of such active
agents and their incorporation into liposomes can be found in
Lenk et al., U.S. Patent No. 4,522,803; Fountain et al., U.S.
Patent No. 4,588,578i Janoff et al., U.S. Patent No. 4,861,580
and 4,897,394i and Lenk et al., U.S. Patent No. 5,082,664; each
of which is incorporated herein by reference.
The bioactive agents which find particularly effective
application to the present invention are lipophilic bioactive
agents, particularly arachidonic acid metabolites including their
structural analogs and synthetic enzyme inhibitors. One class of
such arachidonic acid metabolites is the group of bioactive
agents known as prostagl~n~'n~ including, but not limited to
prostaglandin El.
Hydrophilic bioactive agents, such as the
aminoglycoside antibiotics and their structural analogs, are
examples of hydrophilic bioactive agents. These include
gentamicin, streptomycin, dihydrostreptomycin, tobramycin,
neomycin B, paromycin, ribostamycin, lividomycin, kanamycin,
viomycin, sisomicin, netilmicin and amikacin, as well as
analogues and derivatives thereof. Gentamicin is the preferred
aminoglycoside antibiotic.
The process of forming liposomes, in accordance with
the present invention is essentially the same for lipophilic and
hydrophilic bioactive agents. For bioactive agent associated
liposomes, an optional preservative such as disodium EDTA and the
bioactive agent (e.g. prostaglandin E1 or gentamicin) are added
to an aqueous medium, preferably a solution of a drying
- protectant, most preferably a maltose solution at a preferred
concentration of about 5 to 10~ by weight based on the total
weight of the aqueous phase. The liposomes are prepared at a
2 1 990 1 8
W096/09037 PCTnUS94/10812
temperature above the phase transition temperature of the lipid
membrane.
The resulting bulk liposomes, whether or not the
bioactive agent is associated therewith, may if desirable,
undergo size reduction. Size reduction may be accomplished by
utilizing any one of the methods described hereinbefore to obtain
a single-modal size distribution of liposomes encompassing a
desired mean particle size.
The size reduction of the liposomes is preferably
conducted by extruding the liposomes through filters having
straight through or tortuous paths, according to the procedure
disclosed in U.S. Serial No. 07/771,267 filed October 4, 1991,
using an AnoporeTM filter or by homogenization such as by the use
of a Microfluidizer to form a single-modal size distribution,
preferably having a mean particle size in the range of no more
than 200 nm, most preferably 150 to 190 nm.
The bulk liposomes produced by the process of the
present invention may be separated from unassociated bioactive
agent, if necessary, as well as from free lipid, salts and water
by the common technique of ultrafiltration such as disclosed in
Munir Cheryan, Ultrafiltration Handbook, pp. 205-213 and 377,
Technomic Publishing Company (1986).
Diafiltration is one such ultrafiltration system in
which permeable solutes are removed by the addition of fresh
solvent or other solution to the feed liquid. The rem~-n-ng
liquid (the retentate) containing non-permeated substances
including the desired liposome product is recovered. A preferred
method of diafiltration is disclosed in Lenk, et al., PCT
Published Application No. W089/00846, the disclosure of which is
incorporated herein by reference.
Diafiltration systems typically employ a filter device
having one or more primary pathways formed by a porous filter
composition. The filter device has a rated pore size such that
generally materials having a size equal to or less than the rated
pore size will be able to pass through the filter device via
narrower secondary pathways. Generally, the larger components
WO 96/09037 2 1 9 9 0 1 ~3 PCrlUS94/10812
will remain in the primary pathways and pass through the filter
device as part of the liquid retentate. When liposomes are
prepared using a diafiltration system, the liposomes pass out of
the filter device through the primary pathways while the
5 permeable solutes pass through the narrower secondary pathways.
The dehydration or lyophilization of the liposomes of
the present invention may be performed by any methods known in
the art for dehydrating or lyophilizing liposomes. For
dehydration, for example, the liposomes may be dried according to
the procedures of Janoff et al., U.S. Patent No. 4,880,635,
incorporated herein by reference.
The liposomes of the invention are preferably
lyophilized by first pre-cooling the liposomes in a vessel at a
temperature of from about 0 to 8~C and then freezing the pre-
15 cooled liposomes to a temperature of from about -50 to -38~C,
preferably about -40~C. Thereafter, the pressure of the vessel
is reduced while raising the shelf temperature to a temperature
of from about -18 to -22~C, preferably about -20~C until the
product and shelf temperature equilibrate. Once the primary
2 0 drying stage is completed, secondary drying is commenced by
raising the shelf temperature to about 36 to 40~C, preferably
about 38~C, and maint~;nlng that temperature until the water
content is reduced to below about 2~ by weight, preferably to
below about l~ by weight. The lyophilized liposome formulation
25 prepared in this manner in the absence of an organic solvent may
be stable for at least one year when stored at temperatures of up
to 25~C.
When the lyophilized liposomes are to be used,
rehydration can be accomplished by adding an aqueous solution,
30 e.g., distilled water, water for injection (WFI), or buffer or
aqueous solution of appropriate pH, as described above, to the
liposomes, and gently agitating them to rehydrate and suspend
them. The rehydration may be performed at about room
temperature, that is 25~C. If the bioactive agent was
35 incorporated into the liposomes prior to dehydration, and no
further composition changes are desired, the rehydrated liposomes
W096/09037 2 1 9 9 0 1 8 PCTnUS94/10812
can be used directly in the therapy following known procedures
for administering liposome associated drugs.
During preparation of the liposomes as described
above, organic solvents are not used to suspend the lipids and/or
the active agent, such as prostaglandin or gentamicin. It being
understood, however, that minor amounts of residual solvent may
be present in components used to make the liposomes including
the lipids and perhaps the bioactive agent. Accordingly, the
final liposome product contains no residual organic solvent other
than very small amounts which may be present in the raw materials
used to make the liposomes.
The resulting liposome product may be freeze dried at
higher temperatures than liposomes containing an organic solvent
such as ethanol.
For example, a bioactive agent such as
prostaglandin can be added to an aqueous solution containing a
drying protectant, such as maltose and mixed in a reactor
equipped with an impeller. The lipid, such as egg
phosphatidylcholine, can then be added to the reaction vessel
without mixing. After the addition, mixing can be commenced
again to produce a liquid medium containing a heterogeneous (non-
uniform) size distribution of liposomes associated with the
bioactive agent. In preferred embodiments, much of the bioactive
agent is encapsulated as part of the aqueous phase within the
liposomes.
The resulting bulk liposome medium can be extruded
through a filter, such as a branched-pore al-~m;nl~m oxide filter,
and then sterilized by filtration to form a single-modal size
distribution of liposomes.
The resulting liposomes can then be lyophilized, for
example, as follows. The liposomes can be placed in a freeze
dryer that is preferably pre-cooled to about 5~C and then frozen
by lowering the shelf temperature to preferably about -42~C.
Once the liposomes reached -40~C, primary drying can be initiated
by lowering the pressure of the vessel, preferably to about 0.150
mm Hg and raising the shelf temperature, preferably to about 20
W096/09037 2 1 9 9 0 1 8 PCTnUS94/10812
- 11
~C, which is preferably maintained until the product and shelf
temperature equilibrate. Upon completion of the primary
drying cycle, secondary drying can be commenced by raising the
shelf temperature, preferably to about 38~C and preferably
maintaining that temperature for about 7-8 hours. According to
the following example, this process can result in 99.6% liposomes
having a size between 50 nm and 450 nm, and the lyophilized
liposome product having a water content of 0.9%.
The liposomes resulting from the processes of the
present invention can be used therapeutically in m~mm~l S,
including man, in the treatment of infections or conditions which
require the sustained delivery of the drug in its bioactive form.
Such conditions include, but are not limited to, disease states
such as those that can be treated with prostagl~n~;n~ or
aminoglycosides.
The process of the present invention is capable of
producing a single-modal size distribution of liposomes under
less severe and time consuming conditions than are possible when
the liposomes are prepared using an organic solvent.
EXAMPLE 1
20.0 ~g of prostaglandin El (PGEl) was added to 800 mL
of aqueous solution containing 880 mg/mL of maltose and mixed for
10 minutes in a 3 liter ApplikonTM reactor equipped with 3 baffles
and a Lightn;nTM R-lO0 impeller.
8.8 mg of egg phosphatidylcholine were added to the
reaction vessel without mixing. After the addition, mixing was
commenced again with the impeller rotating at the rate of l,995
rpm for 30 minutes to produce a liquid medium containing a
heterogeneous (non-uniform) size distribution of liposomes
associated with the PGEl. In particular, much of the PGEl is
encapsulated as part of the aqueous phase within the liposomes.
The resulting bulk liposome medium was then extruded
through a lO0 nm backed AnoporeTM branched-pore aluminum oxide
filter (manufactured by Whatman Corp. of Banbury Oxon, United
WO 96/09037 2 1 9 9 0 l 8 rcr/usg4/lo8l2
Kingdom) and then sterilized by filtration using a 220 nm
MillipakTM 100 filter to form a single-modal size distribution of
liposomes.
The resulting liposomes were then lyophilized in the
5 following manner:
(1) 5 mL of liposomes in a 20 mL vial were placed in an
FTSTM freeze dryer and pre-cooled to 5~C;
(2) The pre-cooled product was then frozen by lowering the
shelf temperature to -42~C;
(3) Once the liposomes reached -40~C, primary drying was
initiated by lowering the pressure of the vessel to
0.150 mm Hg and raising the shelf temperature to 20~C
which was maintained until the product and shelf
temperature equilibrated; and
(4) Upon completion of the primary drying cycle, secondary
drying was commenced by raising the shelf temperature
to 38~C and maintaining that temperature for 7-8
hours. An analysis of the resulting lyophilized
product is shown in Table 1.
TABLE
Water Content (96) 0.9
pH 4.2
Osmolality (mosmol/kg) 302
Particle Size Mean (nm) 158 nm
% c 50 nm 0.2
50 nm c g6 c 450 nm 99.6
450 nm 0.2
Total PGEl (llg/vial) 94
Free PGEl g~ 2
Total Phospholipid (mg/vial) 44
