Note: Descriptions are shown in the official language in which they were submitted.
CA 02361914 2001-11-13
IMPROVED CHOLESTEROL-FREE LIPOSOMES
Technical Field
This invention is directed toward improved thermosensitive liposomes having
phase
transition temperatures at mildly hyperthermic conditions which have improved
drug retention and
circulation longevity, and their uses in the treatment of disease.
Background of the Invention
Liposomes and other lipid-based carrier systems have been extensively
developed and
analyzed for their ability to improve the therapeutic index of drugs by
altering the pharmacokinetic
and tissue distribution properties of drugs. This approach is aimed at
reducing exposure of healthy
tissues to therapeutic agents while increasing drug delivery to a diseased
site.
I 5 Some drugs, and in particular, many anti-neoplastic drugs, are knownto
have a short
half life in the bloodstream such that their parenteral use is not optimized.
The use of lipid-based
carriers such as liposomes for site-specific delivery of such drugs via the
bloodstream presents
possible means to improve the use of such drugs. However, the use of liposomes
for site-specific
delivery is limited by the rate of clearance of liposomes from the blood, for
example by cells of the
Mononuclear phagocytic system (MPS). Furthermore, drugs encapsulated into
liposomes are often
not retained in the liposome after intravenous administration. In order for
therapeutic effectiveness of
liposome encapsulated drugs to be realized, such drugs must be effectively
retained within a
liposome after intravenous administration and the liposomes must have a
sufficient circulation
lifetime to permit the desired drug delivery.
It has long been established that incorporation of membranerigidification
agents such as
cholesterol into a liposomal membrane enhances circulation lifetime of the
liposome as well as
retention of drugs within the liposome. Inclusion of cholesterol in liposomal
membranes has been
shown to reduce release of drug after intravenous administration (for example,
see: United States
Patents 4,756,910; 5,077,056; 5,225,212; and 5,843,473; Kirby, C., et al.
(1980) Biochem. J.
186:591-598; and, Ogihara-Umeda, I. and Kojima, S. (1989) Eur. J. Nucl. Med
15:617-7).
Generally, cholesterol increases bilayer thickness and fluidity while
decreasing membrane
permeability, protein interactions, and lipoprotein destabilization of
theliposome. Conventional
approaches to liposome formulation dictate inclusion of substantial amounts
(e.g. 30-45mo1 %)
cholesterol or equivalent membrane rigidification agents (such as other
sterols) into liposomes.
CA 02361914 2001-11-13
More recently, means for providing targeted release of liposome contents via
the use of
"thermosensitive" drug carriers have been developed (for example, see United
States Patent
6,200,598; and, Gaber, M., et al. (1996) Int. J. Radiation Oncology Biol.
Phys. 36:1177-1187).
Thermosensitive liposomes are designed to have a phase transition temperature
slightly above body
temperature so that the liposomes remain in a gel state while in circulation
but exceed the phase
transition temperature upon application of heat to a patient's body or
specific tissues. When heated,
the liposome releases an encapsulated drug because the liposome bilayer
becomes much more
permeable above the transition temperature. However, since cholesterol has the
effect of broadening
the phase transition temperature (inclusion of about 30 mol % or more
cholesterol will usually
eliminate phase transition entirely) thermosensitive liposomes are made
without cholesterol. Further,
to have a phase transition temperature su~ciently close to normal human body
temperature (e.g. 40-
45°C), the lipid composition of the liposome is carefully tailored. A
preferred lipid for use in
thermal-sensitive liposomes is DPPC, which has an acyl chain length of 16
carbon atoms.
Incorporation of any substantial amount of lipids having longer acyl chain
lengths will raise the phase
transition temperature of the liposome beyond the point of usefulness in
thermosensitive applications.
While circulation lifetime of a thermosensitive liposome may be enhanced by
inclusion of PEG-
conjugated lipids into the liposome just as in more conventional liposomes
(see: United States Patent
5,843,473; Unezaki, S., et al. (1994) Pharm. Res. 11:1180-5; Maruyama, K., et
al. (1993) Biochimica
et Biophysica Acta 1149:209-206; Blume, G. and Cevc, G. ~l> & c2~ Biochimica
et Biophysica Acta
(1990)1029:91-97~'~ & (1993) 1146:157-168~2~), thermosensitive liposomes
exhibit poor drug
retention in vivo. It is apparent that liposomes with surface conjugated PEG
moieties still require
cholesterol to exhibit optimal circulation behavior and that these liposomes
would exhibit inferior
characteristics for therapeutic applications in vivo.
Summary of Invention
In the present invention, the inventors have provided liposomes which,
prepared in the
absence of cholesterol, can be made to behave comparably to cholesterol-
containing liposomes
through the incorporation of a hydrophilic polymer conjugated lipid. The
present invention provides
a method of preparing or selecting liposomes using a testing format based on
the comparison of a
cholesterol-free liposome having a phase transition temperatures mildly
hyperthermic to a subject's
body temperature to a cholesterol-containing liposome. By providing preferred
liposome
compositions and guidance on how to select liposomes, the inventors allow for
increased liposome
stability and drug retention properties.
3 5 The methods set forth below are based on the finding that liposomes having
phase transition
temperatures useful for thermosensitive applications display enhanced drug
retention properties and
2
CA 02361914 2001-11-13
circulation longevities if the temperature of recipient is maintained below
the phase transition
temperature of the liposomes. Results of this observation are set forth in
Example 3.
Further, the inventors provide liposomes in which the hydrophilic polymer
stabilization
effects due to use of PEG-modified lipid incorporation are not substantially
dependent on the
concentration of the polymer or polymer molecular weight. The inventors
provide that
concentrations as low as 0.5 mol% PEG-2000 can cause a significant increase in
Area-Under-the
Curve (AUC) when compared to the same liposome prepared without the PEG-lipid
and that PEG
350 at concentrations of S mol% can cause a significant improvement in AUC
when compared to the
same liposome prepared without the PEG lipid, the increase in AUC being
comparable to the
improvement observed when using 5 mol% PEG 2000. The resulting liposomes
provide much
enhanced longevity of the liposomes while in blood circulation.
'This invention provides a liposome comprising a drug, the liposome identified
or prepared
by a process comprising:
(i) comparing drug retention or blood circulation longevity of
(a) a liposome having a drug encapsulated therein, said liposome 1) having a
phase transition temperature greater than that of the body of a subject to be
treated but
less than 45°C and/or 2) being substantially free of cholesterol, and
(b) a substantially equivalent (e.g. containing substantially the same lipids
and in
the same proportions) cholesterol-containing liposome having a drug
encapsulated
therein; and
(ii) identifying a liposome of step (a) demonstrating drug retention or blood
circulation
longevity comparable to or improved over that of the substantially equivalent
cholesterol-
containing liposome of step (b). The method optimally comprises the additional
step of preparing
liposomes so identified for use.
Preferably the liposome of the invention is both substantially cholesterol-
free and has a phase
transition temperature greater than that of the body of a subject to be
treated but less than 45°C.
Additionally, this invention provides a method of designing or selecting a
liposome for
design, the liposome comprising an encapsulated drug, comprising the steps o~
(a) providing a liposome having a drug encapsulated therein, said liposome 1 )
having a
phase transition temperature greater than that of the body of a subject to be
treated but less than 45°C
and/or 2) being substantially free of cholesterol;
(b) providing a liposome having a drug encapsulated therein, said liposome
containing substantially the same lipids and in the same proportions as the
liposome in (a) with at
least 20 mol % cholesterol;
(c) comparing drug retention or blood circulation longevity of the liposomes
of steps
(a) and (b);
CA 02361914 2001-11-13
(d) identifying a liposome of step (a), which liposome demonstrates comparable
or
improved drug retention or blood circulation longevity compared to a liposome
containing
substantially the same lipids and in the same proportions with at least 20 mol
% cholesterol.
Preferably, drug retention time of a liposome compositions is compared, which
preferably
involves determining the drug:lipid ratio of the liposome after administration
of said liposome to the
bloodstream a non-human mammal. However, in other embodiments of the methods
of the invention,
circulation longevity of liposome compositions are compared instead of or in
addition to comparing
drug retention. Thus, optionally, in the methods described herein, the
invention may encompass
determining the proportion of liposome present in the bloodstream of an animal
at least one fixed
time point subsequent to administration instead of or in addition to
determining the drug:lipid ratio.
In preferred embodiments, the invention provides a method for designing or
selecting a
liposome for an improved liposome composition, comprising the steps of
(a) providing a liposome having a drug encapsulated therein, said liposome 1)
having a
phase transition temperature greater than that of the body of a subject to be
treated but less than 45°C
and/or 2) being substantially free of cholesterol;
(b) providing a liposome having a drug encapsulated therein, said liposome
containing
substantially the same lipids and in the same proportions as the liposome in
(a) with at least 20 mol
cholesterol;
(d) administering the liposomes of (a) and (b) to the bloodstream of a non-
human
mammal;
(e) for the liposomes of each of (a) and (b), determining drug:lipid ratios in
the blood of
the mammals at least one fixed time point subsequent to administration; and
(f) comparing the drug:lipid ratios in the blood of the mammals so determined;
and
(g) identifying a liposome of step (a) demonstrating a drug:lipid ratio at a
fixed time
point in a mammal is which is comparable to or better than the drug:lipid
ratio in a mammal at said
fixed time point of a liposome of step (b) containing substantially the same
lipids and in the same
proportions as the liposome in step (a) with at least 20 mol % cholesterol.
In further aspects, the invention provides a method for designing or selecting
aliposome for
an improved liposome composition, said method comprising the steps o~
(a) providing a liposome 1) containing substantially no cholesterol and/or 2)
having a
phase transition temperature greater than that of the body of a subject to be
treated and less than
45°C;
(b) providing a liposome containing substantially the same lipids and in the
same
proportions as the liposome in (a) with at least 20 mol % cholesterol;
(c) encapsulating the drug into the liposomes of (a) and (b);
4
CA 02361914 2001-11-13
(d) administering the liposomes of (a) and (b) after encapsulation of the drug
to the
bloodstream of a non-human mammal;
(e) determining drug:lipid ratios in the blood of the mammals at least one
fixai time
point subsequent to administration; and
(f) comparing the drug:lipid ratios in the blood of the mammals so determined;
and
(g) identifying a liposome of step (a) demonstrating a drug:lipid ratio at a
fixed time
point in a mammal is which is comparable to or better than the drug:lipid
ratio in a mammal at said
fixed time point of a liposome of step (b) containing substantially the same
lipids and in the same
proportions as the liposome in step (a) with at least 20 mol % cholesterol.
In yet further aspects, the invention provides a method for designing or
selecting aliposome
for an improved liposome composition, said method comprising the steps o~
a) providing a liposome having a drug encapsulated therein, said liposome 1 )
having a phase
transition temperature greater than that of the body of a subject to be
treated but less than 45°C and/or
2) being substantially free of cholesterol;
b) assessing the drug:lipid ratios of the liposome of step (a) after
administration of said
liposome to the bloodstream of a non-human mammal;
c) comparing the drug:lipid ratio of step (b) so assessed to the drug:lipid
ratio of a liposome
containing substantially the same lipids and in the same proportions as the
liposome in (a) with at
least 20 mol % cholesterol;
wherein the liposome of step (a) demonstrates a drug:lipid ratio at a fixed
time point in a
mammal which is comparable to or better than the drug:lipid ratio in a mammal
at said fixed time
point of a liposome of step (c).
It will be appreciated that the drug:lipid ratio obtained for the liposome of
step (c) can be
obtained by administering the liposome of step (c) to the bloodstream of a non-
human mammal, or
by consulting literature providing the drug:lipid ratio for said liposome
composition at particular time
points and under particular conditions.
As mentioned, in the preferred methods of the invention preferably, the
drug:lipid ratio of the
liposome after administration of said liposome to the bloodstream a non-human
mammal is
determined. However, in other embodiments of the methods of the invention,
circulation longevity of
liposome compositions are compared instead of or in addition to comparing
drug:lipid ratio. Thus, it
will be appreciated that in the methods of the invention, 'drug:lipid ratio'
may be substituted for
'drug retention property' if desired.
The methods according to the invention of designing or selecting aliposome can
thus further
comprise additional steps to improve the liposome complex, or can involve
repeating any or all of the
aforementioned steps. It will also be appreciated that in the steps relating
to 'providing' a liposome,
the term providing may be substituted with the term 'preparing'. Liposomes
having desired lipid
CA 02361914 2001-11-13
composition and proportion can be prepared according to known methods, several
examples of which
are provided herein, or can be obtained from commercial suppliers.
Phase transition temperature are preferably between about the temperature the
body of a
subject to be treated and 45° C, between about 38° C and
45° C, between 38° C and 43° C, and yet
more preferably between 39°C and 41°C. Most preferably, the
subject to be treated is a human.
Optionally, the subject to be treated is a non-human mammal. In general, an
optimal phase transition
temperature is preferably that at which mild hyperthermic conditions can cause
release of liposome
contents without causing damage or other effects adverse to the intended
treatment, to the vasculature
of a patient.
It will be appreciated that any suitable method for deterrnning the
circulation longevity
and/or drug retention of a liposome can be used. In this specification, the
term "retention" with
respect to a drug or other agent encapsulated in a liposome refers to
retention of the drug in a
liposome while the liposome is present in the bloodstream of a mammal. This
term does not refer to
a measure of drug that may be loaded or incorporated into a liposome or the
ability of a liposome to
retain the drug in ex vivo conditions. Most preferably, the methods of the
invention for assessing
drug retention comprise determining the drug:lipid ratio at least one time
point upon administration to
a non-human mammal. Circulation longevity is preferably expressed in terms of
portion (percent) or
lipid dose remaining in the blood of a mammal at a given time point. As used
herein, a drug:lipid
ratio or retention time which is deemed 'comparable' will depend on the
circumstances, but is
preferably at least 5%, 10%, 20%, 40%, 50%, 70%, 80%, 90%, or more preferably
95% of the drug
retention time or drug:lipid ratio of a reference (e.g. cholesterol-
containing) liposome. As further
discussed herein, cholesterol-containing reference liposome will contain the
same lipids and in the
same proportions as substantially cholesterol-free liposomes of the invention,
but will contain at least
20 mol % cholesterol. These cholesterol-containing reference liposomes may
contain a hydrophilic
polymer-conjugated lipid such as PEG, or may be free of hydrophilic polymer-
conjugated lipid
andlor free of PEG. The 'time point' is generally a number as measured in
hours, minutes, etc.
A particularly suitable non-human mammal for use in the aforementioned method
for
comparing drug:lipid ratios is the mouse. The liposome compositions to be
compared, that is the
liposomes of steps (a) and (b) in the aforementioned methods, will each
typically be administered to a
separate (that is, individual) non-human mammal for determination of
drug:lipid ratios. However, it
can also be envisioned to compare drug lipid ratios for both compositions of
steps (a) and (b) in the
same non-human mammal if means (e.g. detectable labels) are used to
distinguish each of the
liposomes and drugs from one another. The drug encapsulated in the liposomes
is preferably the
same drug for both liposomes to compared, but may also be different drugs so
long as the drugs have
similar retention properties in a liposome.
CA 02361914 2001-11-13
Preferably, the amount of cholesterol in the liposome at (b) will be about 30
to about 50 mol
%. Preferably, the drug:lipid ratios will be determined in step (e) at a
series of intervals subsequent to
administration with the comparison at (f) being of the ratios determined over
the series of intervals.
'This invention also provides methods for determining whether retention of a
particular drug
is enhanced by elimination of cholesterol from a liposome. This method allows
conditions to be
standardized during thermosensitive liposome design such that improvement can
be evaluated more
accurately.
This invention further provides a method for determining whether retention of
a drug in a
liposome may be improved, as well as a method for designing or selecting an
improved liposome
composition. Said methods comprising the steps of:
(a) preparing a liposome 1) having a phase transition ~mperature greater than
that of
the body of a subject to be treated but less than 45°C and/or 2) being
substantially free of cholesterol;
(b) preparing a liposome containing substantially the same lipids and in the
same
proportions as the liposome in (a) with at least 20 mol % cholesterol;
I 5 (c) encapsulating the drug into the liposomes of (a) and (b);
(d) administering the liposomes of (a) and (b) after encapsulation of the drug
to the
bloodstream of separate non-human mammals;
(e) determining drug:lipid ratios in the blood of the mammals at least one
fixed time
point subsequent to administration; and
(f) comparing the ratios so determined for each mammal, wherein an increase in
drug:lipid ratio in a mammal in which liposomes of (a) were administered as
compared to drug:lipid
ratio in a mammal in which liposomes of (b) were administered, is indicative
of improvement in drug
retention.
In principle, any suitable liposome composition may be used, as long as the
liposome has
the required phase transition temperatures. However, due to the well known
effects of cholesterol
on phase transition, liposomes of the invention will generally contain little
or no cholesterol.
Preferably, liposome of the invention, including liposomes for use in step (a)
of the preceding
methods, will comprise at least 60, 70, 80, 85, 90 or 95 mol % of a
phospholipid having two
saturated fatty acids, wherein at least one of the acyl chains has 16 carbon
atoms. A preferred
phospholipid with acyl chains of 16 carbon atoms is
dipalmitoylphosphatidylcholine (DPPC).
More preferably, liposomes for use in step (a) in the method above will have
at least about 80, at
least about 85, and even more preferably, at least 90 mol % of such a
phospholipid. Preferably,
DPPC is the predominant phospholipid. The remainder of the liposome may
comprise one or
more amphipathic lipids suitable for use in liposomes, but substantially no
cholesterol.
Preferably, such other lipids will include a hydrophilic polymer-conjugated
lipid. Preferably, the
amount of such polymer-conjugated lipids present in the liposome will be from
about 1 to about
7
CA 02361914 2001-11-13
15 mol %. Liposomes of the invention and liposomes for use in step (a)
comprise a hydrophilic
polymer-conjugated lipid. Preferably, the hydrophilic polymer-conjugated lipid
is a PEG-lipid,
preferably having a molecular weight from about 100 to about 5000 daltons, or
from about 1000
to 5000 daltons. Preferably the liposome comprises 2 to about 15 mol %, or 5
to about 10 mol
hydrophilic polymer-conjugated lipid.
Liposomes for use in the above method may be prepared using known and
conventional
techniques. Determination of phase transition temperatures, encapsulation of
drug into liposomes
(liposome loading), administration of liposomes, and determining drug:lipid
ratios from blood may
be carried out according to known and conventional techniques.
The above-described method may be used to select a liposome formulation to
achieve
optimal drug retention. Accordingly, this invention also provides a
combination of a liposome and a
drug wherein the liposome is a liposome as described above with respect to
step (a) and the drug is an
anti-neoplastic agent which exhibits greater retention in such a liposome,
when the above-described
method is performed. By "combination", it is meant that the drug is
encapsulated in the liposome or
is segregated but associated with the liposome (such as in a commercial
package or kit comprising
the liposome and the drug). Preferably, the liposome is one having the
preferred characteristics of
liposomes of step (a) as described above.
This invention also provides improved drug retention in liposomes for specific
drugs which
previously exhibited poor retention in conventional cholesterol-containing
liposomes. Also provided
are novel cholesterol-free liposome formulations that are particularly suited
for use in this invention.
This invention also provides novel liposomes which are particularly suitable
for use in this
invention. The invention provides in preferred aspects a liposome comprising:
(a) at least 60 mol % of a phospholipid comprising two saturated fatty acids,
the acyl
chain of each being the same or different, at least one of said acyl chains
having 16 carbon atoms;
(b) from about 2 to about 15 mol % hydrophilic polymer-conjugated lipid; and
(c) up to about 38 mot % of one or more vesicle-forming lipids, providing that
the
liposome contains substantially no cholesterol; wherein the liposome displays
a comparable or
greater circulation longevity, or when encapsulating a drug displays a
comparable or greater
circulation longevity or drug:lipid ratio, at a fixed time point upon
administration to a mammal than a
liposome containing substantially the same lipids and in the same proportions,
but with at least 20
mol % cholesterol.
In other embodiments, the liposome will comprise at least 70, 80, 85, 90 or 95
mol % of a
phospholipid comprising two saturated fatty acids, the acyl chain of each
being the same or different,
at least one of said acyl chains having 16 carbon atoms, preferably wherein
the phospholipid is
DPPC.
CA 02361914 2001-11-13
Preferably, the liposome will contain substantially no cholesterol.
Preferably, the liposome
will have a phase transition temperature preferably between about the
temperature the body of a
subject to be treated and 45° C, between about 38° C and
45° C, between 38° C and 43° C, and yet
more preferably between 39° C and 41° C.
This invention also provides the novel liposomes of this invention in
combination with a
drug and the use of such liposomes as a carrier for a drug encapsulated in the
liposome. Such drugs
include most preferably anti-neoplastic, anti-inflammatory or anti-infective
agents.
Brief Description of the Drawings
Figure 1: A graph showing lipid dose remaining in the blood of mice after
intravenous injection
of radiolabelled (a) a 90: 4 molar ratio of DPPC: DSPE-PEG2000 liposomes (80-
100 mmoles
total lipid) with and without thermal control (squares and triangles
respectively), (b) a 55:45:4
mol ratio of DSPC: cholesterol: DSPE-PEG2000 liposomes (triangles) into female
Balb/c mice as
a function of time.
Figure 2: A graph showing doxorubicin: lipid remaining in the blood of mice
after intravenous
injection of radiolabelled (a) a 90: 4 molar ratio of DPPC: DSPE-PEG2000
liposomes (80-100
mmoles total lipid) with and without thermal control (squares and triangles
respectively) into
female Balb/c mice as a function of time.
Detailed Description of the Invention
As mentioned above, the inventors have provided cholesterol-free liposomes,
more
particularly cholesterol-free liposomes suitable for thermosensitive
applications in animals which can
be made to be at least as stable in circulation and having drug retention
characteristics comparable or
better than their cholesterol-containing counterparts. In addition to
providing such liposome
compositions, the inventors have provided a method of preparing and selecting
such cholesterol-free
liposomes having advantageous properties.
The inventors have provided a means for the design wherein a liposome during
development
is tested by comparison with a similar liposome composition containing
cholesterol. As an additional
advantage, the inventors provide a means for assessing drug retention and/or
in vivo serum stability
based on the known properties of cholesterol containing liposomes, use of such
a liposome as a
reference liposome allows testing conditions to be carefully assessed.
As mentioned, the inventors provide liposomes in which the hydrophilic polymer
stabilization effects due to use of PEG-modified lipid incorporation are not
substantially dependent
CA 02361914 2001-11-13
on the concentration of the polymer or polymer molecular weight. The inventors
provide that
concentrations as low as 0.5 mol% PEG-2000 can cause a significant (preferably
greater than S, 10,
or 15-fold increase in Area-Under-the Curve (AUC) when compared to the same
liposome prepared
without the PEG-lipid. Provided also is that for example PEG 350 at
concentrations of 5 mol% can
cause a significant (preferably greater than 10, 15 or 25 fold) improvement in
AUC when compared
to the same liposome prepared without the PEG lipid, and this increase in AUC
is comparable to a
significant (preferably greater than 10, 15, 25 or 38-fold) improvement
obtained when using 5 mol%
PEG 2000). The resulting liposomes provide much enhanced longevity of the
liposomes while in
blood circulation.
Throughout this specification, the following abbreviations have the indicated
meaning.
PEG: polyethylene glycol; PEG preceded or followed by a number: the number is
the molecular
weight of PEG in Daltons; PEG-lipid: polyethylene glycol-lipid conjugate; PE-
PEG: polyethylene
glycol-derivatized phosphatidylethanolamine; PA: phosphatidic acid; PE:
phosphatidylethanolamine; PC: phosphatidylcholine; PI: phosphatidylinositol;
DSPC: 1,2-
distearoyl-sn-glycero-3-phosphocholine; DSPE-PEG 2000 (or 2000 PEG-DSPE or
PEGZ~-DSPE):
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol 2000];
DSPE-PEG 750 (or
750PEG-DSPE or PEG~SO-DSPE): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[polyethylene glycol 750]; DPPE-PEG2000: 1,2-dipalmaitoyl-sn-glycero-3-
phosphoethanolamine-
N-[polyethylene glycol 2000); DAPC: 1,2-arachidoyhrn-glycero-3-phosphocholine;
DBPC: 1,2-
dibehenoyl-sn-glycero-3-phosphocholine; CH or Chol: cholesterol; DPPC: 1,2-
dipalmaitoyl~sn-
glycero-3-phosphocholine; HEPES: N-[2-hydroxylethyl]-piperazine-N-[2-
ethanesulfonic acid].
As used in this specification and the appended claims, the singular forms "a,"
"an" and "the"
include plural references unless the context clearly dictates otherwise.
The term "cholesterol-free" as used herein with reference to a liposome means
that a
liposome is prepared in the absence of cholesterol, or that the liposome
contains substantially no
cholesterol, or that the liposome contains essentially no cholesterol. The
term "substantially no
cholesterol" allows for the presence of an amount of cholesterol that is
insufficient to significantly
alter the phase transition characteristics of the liposome (typically less
than 20 mol % cholesterol).
20 mol % or more of cholesterol broadens the range of temperatures at which
phase transition occurs,
with phase transition disappearing at higher cholesterol levels. Preferably, a
liposome having
substantially no cholesterol will have about 15 or less and more preferably
about 10 or less mol
cholesterol. The term "essentially no cholesterol" means about 5 or less mol
%, preferably about 2 or
less mol % and even more preferably about 1 or less mol % cholesterol. Most
preferably, no
cholesterol will be present or added when preparing "cholesterol-free"
liposomes. Cholesterol free
and liposomes having substantially no cholesterol are described incopending
international patent
application PCT/CA01/00655, which is incorporated herein by reference.
CA 02361914 2001-11-13
The term "liposome" as used herein means vesicles comprised of one or more
concentrically
ordered lipid bilayers encapsulating an aqueous phase. Formation of such
vesicles requires the
presence of "vesicle-forming lipids" which are amphipathic lipids capable of
either forming or being
incorporated into a bilayer structure. The latter term includes lipids that
are capable of forming a
bilayer by themselves or when in combination with another lipid or lipids. An
amphipathic lipid is
incorporated into a lipid bilayer by having its hydrophobic moiety in contact
with the interior,
hydrophobic region of the membrane bilayer and its polar head moiety oriented
toward an outer,
polar surface of the membrane. Hydrophilicity arises from the presence of
functional groups such as
hydroxyl, phosphate, carboxyl, sulphate, amino or sulflrydryl groups.
Hydrophobicity results from
the presence of a long chain of aliphatic hydrocarbon groups.
The term "hydrophilic polymer-lipid conjugate" refers to a vesicle-forming
lipil covalently
joined at its polar head moiety to a hydrophilic polymer, and is typically
made from a lipid that has a
reactive functional group at the polar head moiety in order to attach the
polymer. Suitable reactive
functional groups are for example, amino, hydroxyl, carboxyl or formyl. The
lipid may be any lipid
described in the art for use in such conjugates other than cholesterol.
Preferably, the lipid is a
phospholipid such as PC, PE, PA or PI, having two acyl chains comprising
between about 6 to about
24 carbon atoms in length with varying degrees of unsaturation. Most
preferably, the lipid in the
conjugate is a PE, preferably of the distearoyl form. The polymer is a
biocompatible polymer
characterized by a solubility in water that permits polymer chains to
effectively extend away from a
liposome surface with sufficient flexibility that produces uniform surface
coverage of a liposome.
Preferably, the polymer is a polyalkylether, including polymethylene glycol,
polyhydroxy propylene
glycol, polypropylene glycol, polylactic acid, polyglycolic acid, polyacrylic
acid and copolymers
thereof, as well as those disclosed in United States Patents 5,013,556 and
5,395,619. Conventional
liposomes suffer from a relatively short half life in the blood circulation
due to their rapid uptake by
macrophages of the liver and spleen (organs of the reticuloendothelial system
or RES), and therefore
do not accumulate in leaky tumor tissue. Liposome preparations have been
devised which avoid rapid
RES uptake and which have increased circulation times. See, e.g., Allen, UCLA
Symposium on
Molecular and Cellular Biology, 89:405 (1989); Allen et al., Biochim. Biophys.
Acta 1066:29
(1991); Klibanov et al., FEBS Letters 268:235 (1990); Needham et al., Biochim.
Biophys. Acta
1108:40 (1992); Papahadjopoulos et al., Proc. Natl. Acad. Sci. USA 88:11460
(1991); Wu et al.,
Cancer Research 53:3765 (1993); Klibanov and Huang, J. Liposome Research 2:321
(1992); Lasic
and Martin, Stealth Liposomes, In: Pharmacology and Toxicology, CRC Press,
Boca Raton, Fla.
(1995). See also U.S. Pat. No. 5,225,212 to Martin et al.; U.S. Pat. No.
5,395,619 to Zalipsky et al.
regarding liposomes containing polymer grafted lipids in the vesicle membrane.
The presence of
polymers on the exterior liposome surface decreases the uptake of liposomes by
the organs of the
RES. A preferred polymer is polyethylene glycol (PEG). Preferably the polymer
has a molecular
CA 02361914 2001-11-13
weight between about 1000 and 5000 daltons. The conjugate may be prepared to
include a releasable
lipid-polymer linkage such as a peptide, ester, or disulfide linkage. The
conjugate may also include a
targeting ligand. Mixtures of conjugates may be incorporated into liposomes
for use in this
invention. The term "PEG-conjugated lipid" as used herein refers to the above-
defined hydrophilic
polymer-lipid conjugate in which the polymer is PEG.
The term "phase transition temperature" is the temperature or range of
temperatures at which
a liposome changes from a gel state to a liquid crystalline state. A
convenient method for measuring
phase transition temperature is to monitor energy absorption while heating a
preparation of liposomes
and noting the temperature or range in temperatures at which there is an
energy absorbance.
The predominant vesicle-forming lipid in liposomes of this invention are
responsible for
achieving phase transition temperatures of betweefi the body temperature of a
subject to be treated
(e.g. human or non-human mammal) and 45°C. Preferably, the lipid is a
phospholipid such as PC,
PE, PA or PI. The preferred phospholipid is PC. When selecting lipids,
precautions should be taken
since phase separation may occur if acyl chain lengths of these lipids differ
by four or more
methylene groups. Preferably the lipid will have two saturated fatty acids,
the acyl chains of which
being independently selected from the group consisting ofcaproyl (6:0),
octanoyl (8:0), capryl
(10:0), lauroyl (12:0), mirystoyl (14:0) and palmitoyl (16:0).
As mentioned, liposomes used according to the invention comprise a lipid
possessing a gel-
to-liquid crystalline phase transition temperature in the hyperthermic range,
and preferred are
phospholipids whose acyl groups are saturated. A particularly preferred
phospholipid is
dipalmitoylphosphatidylcholine (DPPC). DPPC is a common saturated chain (C 16)
phospholipid
with a bilayer transition of 41.5° C. (Blume, Biochemistry 22:5436
(1983); Albon and Sturtevant,
Proc. Natl. Acad. Sci. USA 75:2258 (1978)). Thermosensitive liposomes
containing DPPC and other
lipids that have a similar or higher transition temperature, and that can be
mixed with DPPC (such as
1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG)
(Tc=41.5° C.) and 1,2-Distearoyl-
sn-Glycero-3-Phosphocholine (DSPC) (Tc=55.1° C.)) have been studied.
Kastumi Iga et al, Intl. J.
Pharmaceutics, 57:241 (1989); Bassett et al, J. Urology, 135:612 (1985); Gaber
et al, Pharmacol. Res.
12:1407 ( 1995).
As demonstrated in the Examples, a preferred example of a liposome formulation
of the
invention was prepared having a 90: 4 molar ratio of DPPC: DSPE-PEG2000.
Generally, preferred
liposomes of the invention comprise at least 60 mole % of a phospholipid.
Preferably, DPPC is the
predominant lipid. Most preferably the liposomes comprise at least 30, 40, 50,
60, 70, 80, 85, 90 or
95 mole/% DPPC. It will be appreciated, however, that any other suitable lipid
composition may be
used according to the invention and that the liposomes of the invention need
not be limited to
liposomes comprising DPPC. Moreover, it is often practice to prepare liposomes
comprising several
12
CA 02361914 2001-11-13
different lipids (e.g. to achieve optimal stability and drug retention
characteristics, or as a surface
active agent). Thus, liposomes of the invention may comprise lipids which by
themselves would not
have the desired transition temperatures so long as~the lipid (for example a
hydrophilic polymer lipid
conjugate) does not destabilize the membrane at processing temperatures where
the bilayer is in the
liquid phase, nor at physiological temperatures where the bilayer is in the
gel phase. For example,
other phase compatible components such as DSPE, DSPE-PEG or DSPC can
optionally be included
a liposome. Preferably, however, DSPC is not the predominant lipid (e.g. the
main lipid component
of liposome bilayer material) and more preferably DSPC is present at less than
40 mole/%, less than
20, 10 or 5 mole/%, or the liposome is essentially free of DSPC.
Preferably, the liposomes which are to be designed according to the methods of
this
invention, and the liposome compositions of the invention may comprise
amphipathic lipids in
addition to those described above, but no substantial amount of cholesterol.
Such lipids include
sphingomyelins, glycolipids, ceramides and phospholipids. Such lipids may
include lipids having
therapeutic agents, targeting agents, ligands, antibodies or other such
components which are used in
liposomes, either covalently or non-covalently bound to lipid components.
Methods of preparation
The liposomes that are the subject of the methods of the invention can be
obtained from
commercial sourcesor can be prepared according to known methods, as described
herein or otherwise
known.
Liposomes of the present invention or for use in the present invention may be
generated by a
variety of techniques including lipid film/hydration, reverse phase
evaporation, detergent dialysis,
freeze/thaw, homogenation, solvent dilution and extrusion procedures. Various
known techniques
are provided for example in U.S. Pat. No. 4,235,871; Published PCT
applications WO 96/14057;
New RRC, Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-
104; Lasic D D,
Liposomes from physics to applications, Elsevier Science Publishers,
Amsterdam, 1993; Liposomes,
Marcel Dekker, Inc., New York (1983).
As shown in Example l, liposome comprising having a 90: 4 molar ratio of DPPC:
DSPE-
PEG2000 were prepared. The liposomes were administrated to mice as detailed,
and circulation
longevity was assessed as shown in Example 3.
It will be appreciated that any suitable method for producing the liposomes of
the invention
can be used. A non-limiting example is provided for illustration as follows.
Liposomes having a desired molar ratio of lipids, comprising [at least one
phospholipid] and
3 S at least one polymer-conjugated lipid, are prepared. A physiologically
acceptable buffer is used for
13
CA 02361914 2001-11-13
formation of the liposome, for example citrate having an acid pH of typically
about pH 2 to about pH
6, about pH 3 to pH 5, and most preferably at about pH 4.
Once the liposomes are prepared with the entrapped acidic buffer, the
liposomes can be sized
to a desired size range. Liposomes of this invention or for use in this
invention are typically greater
than SOnm in diameter, more preferably between about SOnm and about lpm in
diameter. However,
preferred liposomes of this invention will be less than about 200 nm,
preferably less than about 160
nm, and more preferably less than about 140 nm in diameter. 100-140 nm
liposomes (cholesterol-
free liposomes tend to be slightly larger than cholesterol containing ones)
are employed in the
Examples below. Liposomes are typically sized by extrusion through a filter
(e.g. a polycarbonate
filter) having pores or passages of the desired diameter. A liposome
suspension may also be sonicated
either by bath or probe down to small vesicles of less than about 0.05 microns
in size.
Homogenization may also be used to fragment large liposomes into smaller ones.
In both methods
the particle size distribution can be monitored by conventional laser-beam
particle size discrimination
or the like.
Therapeutic agents may be loaded into liposomes using passive and active
loading
methods described herein.
Passive methods of encapsulating therapeutic agents
Therapeutic agents may be encapsulated using passive methods of encapsulation.
Passive
methods of encapsulating therapeutic agents in liposomes involve encapsulating
the agent during
the synthesis of the liposomes. In this method, the drug may be membrane
associated or
encapsulated within an entrapped aqueous space. This includes a passive
entrapment method
described by Bangham et al., (J. Mol. Biol. 12, (1965), 238) where the aqueous
phase containing
the agent of interest is put into contact with a film of dried vesicle-forming
lipids deposited on the
walls of a reaction vessel. Upon agitation by mechanical means, swelling of
the lipids will occur
and multilamellar vesicles (MLV) will form. Using extrusion, the MLV's can be
converted to
large unilamellar vesicles (LUV) or small unilamellar vesicles (SUV) following
sonication.
Another method of passive loading that may be used includes that described by
Deamer et al
(Biochim. Biophys. Acta 443, (1976), 629). This method involves dissolving
vesicle-forming
lipids in ether and, instead of first evaporating the ether to form a thin
film on a surface, this film
being thereafter put into contact with an aqueous phase to be encapsulated,
the ether solution is
directly injected into said aqueous phase and the ether is evaporated
afterwards, whereby
liposomes with encapsulated agents are obtained. A further method that may be
employed is the
Reverse Phase Evaporation (REV) method described by Szoka & Papahadjopoulos
(P.N.A.S.
(1978) 75: 4194) in which a solution of lipids in a water insoluble organic
solvent is emulsified in
an aqueous carrier phase and the organic solvent is subsequently removed under
reduced pressure.
14
CA 02361914 2001-11-13
Other methods of passive entrapment that may be used subjecting liposomes to
successive
dehydration and rehydration treatment, or freezing and thawing; dehydration
was carried out by
evaporation or freeze-drying. This technique is disclosed by Kirby et al
(Biotechnology,
November 1984, 979-984). Also, Shew et al (Biochim. Et Biophys. Acta 816
(1985), 1-8)
describe a method wherein liposomes prepared by sonication are mixed in
aqueous solution with
the solute to be encapsulated, and the mixture is dried under nitrogen in a
rotating flask. Upon
rehydration, large liposomes are produced in which a significant fraction of
the solute has been
encapsulated.
Active methods of encapsulating therapeutic agents
Therapeutic agents in accordance with this invention may be encapsulated using
active
methods of encapsulation. Active loading involves the use of transmembrane
gradients across
the liposome membrane to induce uptake of a therapeutic agent after the
liposome has been
formed. This can involve a gradient of one or more ions including Na+, K+, H+,
and/or a
protonated nitrogen moiety. Active loading techniques that may be used in
accordance with this
invention include pH gradient loading, charge attraction, and drug shuttling
by an agent that can
bind to the drug.
Liposomes may be loaded according to the pH gradient loading technique.
According to
this technique, liposomes are formed which encapsulate an aqueous phase of a
selected pH.
Hydrated liposomes are placed in an aqueous environment of a different pH
selected to remove or
minimize a charge on the drug or other agent to be encapsulated. Once the drug
moves inside the
liposome, the pH of the interior results in a charged drug state, which
prevents the drug from
permeating the lipid bilayer, thereby entrapping the drug in the liposome.
To create a pH gradient, the original external medium is replaced by a new
external
medium having a different concentration of protons. The replacement of the
external medium can
be accomplished by various techniques, such as, by passing the lipid vesicle
preparation through a
gel filtration column, e.g., a Sephadex column, which has been equilibrated
with the new medium
(as set forth in the examples below), or by centrifugation, dialysis, or
related techniques. The
internal medium may be either acidic or basic with respect to the external
medium.
After establishment of a pH gradient, a pH gradient loadable agent is added to
the mixture
and encapsulation of the agent in the liposome occurs as described above.
PH gradient loading may be carried out according to methods described in US
patent nos.
5,616,341; 5,736,155 and 5,785,987 the disclosures of which are incorporated
herein by
reference.
Therapeutic agents that may be loaded using pH gradient loading comprise one
or more
ionizable moieties such that the neutral form of the ionizable moiety allows
the drug to cross the
CA 02361914 2001-11-13
liposome membrane and conversion of the moiety to a charged form causes the
drug to remain
encapsulated within the liposome. Ionizable moieties may comprise, but are not
limited to
comprising, amine, carboxylic acid and hydroxyl groups. PH gradient loadable
agents that load in
response to an acidic interior may comprise ionizable moieties that are
charged in response to an
acidic environment whereas drugs that load in response to a basic interior
comprise moieties that
are charged in response to a basic environment. In the case of a basic
interior, ionizable moieties
including but not limited to carboxylic acid or hydroxyl groups may be
utilized. In the case of an
acidic interior, ionizable moieties including but not limited to primary,
secondary and tertiary
amine groups may be used.
Preferably, the pH gradient loadable agent is a drug and most preferably an
anti-
neoplastic agent. Examples of some of the antineoplastic agents which can be
loaded into
liposomes by this method and therefore may be used in this invention include
but are not limited
to anthracyclines such as doxorubicin, daunorubicin, mitoxanthrone,
idarubicin, epirubicin and
aclarubicin; antineoplastic antibiotics such as mitomycin and bleomycin; vinca
alkaloids such as
vinblastine, vincristine and vinorelbine; alkylating agents such as
cyclophosphamide and
mechlorethamine hydrochloride; campthothecins such as topotecan, irinotecan,
lurtotecan, 9-
aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin; purine and
pyrimidine
derivatives such as 5-fluorouracil; cytarabines such as cytosine arabinoside.
This invention is not
to be limited to those drugs currently available, but extends to others not
yet developed or
commercially available, and which can be loaded using the transmembrane pH
gradients.
Various methods may be employed to establish and maintain a pH gradient across
a
liposome all of which are incorporated herein by reference. This may involve
the use of
ionophores that can insert into the liposome membrane and transport ions
across membranes in
exchange for protons (see for example US patent no. 5,837,282). Buffers
encapsulated in the
interior of the liposome that are able to shuttle protons across the liposomal
membrane and thus
set up a pH gradient (see for example US patent no 5,837,282) may also be
utilized. These
buffers comprise an ionizable moiety that is neutral when deprotonated and
charged when
protonated. The neutral deprotonated form of the buffer (which is in
equilibrium with the
protonated form) is able to cross the liposome membrane and thus leave a
proton behind in the
interior of the liposome and thereby cause a decrease in the pH of the
interior. Examples of such
buffers include methylammonium chloride, methylammonium sulfate,
ethylenediammonium
sulfate (see US patent no. 5,785,987) and ammonium sulfate. Internal loading
buffers that are
able to establish a basic internal pH, can also be utilized. In this case, the
neutral form of the
buffer is protonated such that protons are shuttled out of the liposome
interior to establish a basic
interior. An example of such a buffer is calcium acetate (see LJS patent no.
5,939,096).
16
CA 02361914 2001-11-13
In other aspects, charge attraction methods may be utilized to actively load
therapeutic
agents. Charge amaction mechanisms for drug loading involves creating a
transmembrane potential
across the membrane by creating a concentration gradient for one or more
charged species. Thus, for
a drug that is negatively charged when ionized, a transmembrane potential is
created across the
membrane that has an inside potential which is positive relative to the
outside potential. For a drug
that is positively charged, the opposite transmembrane potential would be
used.
Following a separation step as may be necessary to remove free drug from the
medium
containing the liposome, the liposome suspension is brought to a desired
concentration in a
pharmaceutically acceptable carrier for administration to the patient or host
cells. Many
pharmaceutically acceptable carriers may be employed in the compositions and
methods of the
present invention. A variety of aqueous carriers may be used, e.g., water,
buffered water, 0.4% saline,
0.3% glycine, and the like, and may include glycoproteins for enhanced
stability, such as albumin,
lipoprotein, globulin, etc. Generally, normal buffered saline (135-150 mM
NaCI) will be employed as
the pharmaceutically acceptable carrier, but other suitable carriers will
suffice. These compositions
may be sterilized by conventional liposomal sterilization techniques, such as
filtration. The
compositions may contain pharmaceutically acceptable auxiliary substances as
required to
approximate physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting
agents and the like, for example, sodium acetate, sodium lactate, sodium
chloride, potassium
chloride, calcium chloride, etc. These compositions may be sterilized
techniques referred to above or
produced under sterile conditions. The resulting aqueous solutions may be
packaged for use or
filtered under aseptic conditions and lyophilized, the lyophilized preparation
being combined with a
sterile aqueous solution prior to administration.
The concentration of liposomes in the carrier may vary. Generally, the
concentration will be
about 20-200 mg/ml, usually about 50-150 mg/ml, and most usually about 75-125
mg/ml, e.g., about
100 mg/ml. Persons of skill may vary these concentrations to optimize
treatment with different
liposome components or for particular patients. For example, the concentration
may be increased to
lower the fluid load associated with treatment.
Thus, in the present method, one step comprises providing a liposome of the
invention
having a phase transition temperature greater than that of the body of a
subject to be treated. The
liposome is generally stable at body temperature but is capable of releasing
an encapsulated drug at
mildly hyperthermic conditions, which are generally understood to be between
about 37°C and 45°C,
or more preferably between about 37°C and 43°C.
Another step comprises providing a 'reference' liposome containing
substantially the same
lipids and in the same proportions as the liposome of the invention, but
containing at least 20 mol
cholesterol. The cholesterol-containing liposome may contain a hydrophilic
polymer-conjugated
17
CA 02361914 2001-11-13
lipid such as PEG, or may be free of hydrophilic polymer-conjugated lipid
and/or free of PEG. The
'time point' is generally a number as measured in hours, minutes, etc.
The liposome compositions to be compared will generally have encapsulated
therein a
therapeutic agent. The therapeutic agent contained in each of the liposome
compositions to be
compared will preferably be the same agent; however, it will be appreciated
that agents that are not
identical but have structural similarities or having similar or identical
liposome loading, stability or
retention properties can also be used. The liposomes may be provided, when
obtained from
commercial sources for example, with a drug already encapsulated therein.
Typically, however, the
method will involve preparing the liposomes to be compared, and encapsulating
a drug into each of
the liposomes compositions to be compared.
In an alternative, albeit less preferable embodiment, an agent which is not
itself a therapeutic
molecule may be used in the methods of the invention. Generally, to be useful
in the present method,
the marker will have physical properties allowing it to be indicative of drug
behavior in the liposome.
That is, the step of encapsulating a drug into one or both of liposomes (e.g.
the thermosensitive
and/or substantially cholesterol-free liposomes of the invention, and the
cholesterol-containing
reference liposomes) can be substituted with the step of encapsulating a
marker molecule, and the
steps of determining and comparing drug:lipid ratios can be substituted with
determining and
comparing marker: lipid ratios. Alternatively, said comparing step may
comprise comparing a
marker:lipid ratio to a drug:lipid ratio where the drug and marker have
similar or substantially
identical physical properties or retention properties in a liposome.
The terms "drug" and "therapeutic agent" as used herein generally refer to
moieties used in
therapy and for which liposome-based delivery is desirable. Active agents
(including drugs,
therapeutic agents or other agents) suitable for use in the present invention
include therapeutic agents
and pharmacologically active agents, nutritional molecules, cosmetic agents,
diagnostic agents and
contrast agents for imaging. Included are small molecule therapeutics as well
as nucleic acids,
polynucleotides, polypeptides or any other suitable agents. As used herein,
activeagents include
pharmacologically acceptable salts of active agents. Suitable therapeutic
agents include, for example,
antineoplastics, antitumor agents, antibiotics, antifungals, anti-inflammatory
agents,
immunosuppressive agents, anti-infective agents, antivirals, anthelminthic,
and antiparasitic
compounds. The term "anti-neoplastic agent" as used herein refers to chemical
moieties having an
effect on the growth, proliferation, invasiveness or survival of neoplastic
cells or tumours. In treating
tumors or neoplastic growths, suitable compounds may include alkylating
agents, antimetabolities,
anthracycline antibiotics (such as doxorubicin, daunorubicin, carinomycin, N-
acetyladriamycin,
rubidazone, 5-imidodaunomycin, N30 acetyldaunomycin, and epirubicin) and plant
alkaloids (such
as vincristine, vinblastine, vinorelbine, etoposide, ellipticine and
camptothecin). Other suitable agents
include paclitaxel (Taxol); docetaxol (taxotere); mitotane, cisplatin, and
phenesterine. Anti-
18
CA 02361914 2001-11-13
inflammatory therapeutic agents suitable for use in the present invention
include steroids and non-
steroidal anti-inflammatory compounds, such as prednisone, methyl-
prednisolone, paramethazone,
11-fludrocortisol, triamciniolone, betamethasone and dexamethasone, ibuprofen,
piroxicam,
beclomethasone; methotrexate, azaribine, etretinate, anthralin, psoralins;
salicylates such as aspirin;
and immunosuppresant agents such as cyclosporine. Antiinflammatory
corticosteroids and the
antiinflammatory and immunosuppressive agent cyclosporine are both highly
lipophilic and are
suited for use in the present invention. Other examples of agents that can be
used according to the
invention are shown in Table 1.
19
CA 02361914 2001-11-13
Table 1
CLASS TYPE OFAGENT NONPROPRIETARY DISEASE (Neoplastic)
NAMES
(OTHER NAMES)
Alkylating Agents NitrogenMechlorethamine Hodgkin's disease,
Mustards (HN2) non-Hodgkin's
lymphomas
Cyclophosphamide Acute and chronic
lymphocytic
Ifosfamide leukemias, Hodgkin's
disease, non-
Hodgkin's lymphomas,
multiple
myeloma, neuroblastoma,
brest,
ovary, lung, Wihns'
tumor, cervix,
testis, soft-tissue
sarcomas
Melphalan (L-sarcolysin)Multimple myeloma,
breast, ovary
Chlorambucil Chronic lymphocytic
leukemia,
primary macroglobulinemia,
Hodgkin's disease,
non-Hodgkin's
lymphomas
Ethylenimenes and HexamethylmelamineOvary Thiotepa Bladder,
breast,
Methyhnelamines ovary
Alkyl Sulfonates Busulfan Chronic granulocytic
leukemia
Nitrosoureas Carmustine (BCNU) Hodgkin's disease,
non-Hodgkin's
lymphomas, primary
brain tumors,
multiple myeloma,
malignant
melanoma
Lomustine (CCNU) Hodgkin's disease,
non-Hodgkin's
lymphomas, primary
brain tumors,
small-cell lung
Semustine (methyl-CCNU)Primary brain tumors,
stomach,
colon
Streptozocin (streptozotocin)Malignant pancreatic
insulinoma,
malignant carcinoid
Triazines Dacarbazine (DTIC;Malignant melanoma,
Hodgkin's
dimethyltriazenoimidazole-disease, soft-tissue
sarcomas
Antimetabolites Folic Methotrexate (amethopterin)Acute lymphocytic
Acid Analogs leukemia,
choriocarcinoma, mycosis
fungoides, breast,
head and neck,
lung, osteogenic sarcoma
Pyrimidine Analogs Fluouracil (5-fluorouracil;Breast, colon, stomach,
5- pancreas,
FU) ovary, head and neck,
urinary
bladder
Floxuridine (fluorode-premalignant skin
lesions (topical)
oxyuridine; FudR)
Cytarabine (cytosineAcute granulocytic
and acute
arabinoside) lymphocytic leukemias
Purine Analogs and Mercaptopurine(6- Acute lymphocytic,
acute
Related Inhibitors mercaptopurine; granulocytic and chronic
6-MP)
granulocytic leukemias
Thioguanine(6-thioguanine;Acute granulocytic,
acute
TG) lymphocytic and chronic
granulocytic leukemias
Pentostatin(2- Hairy cell leukemia,
mycosis
deoxycoformycin) fungoides, chronic
lymphocytic
leukemia
Natural Products Vinca Vinblastine (VLB) Hodgkin's disease,
Alkaloids non-Hodgkin's
lymphomas, breast,
testis
Vincristine Acute lymphocytic
leukemia,
neuroblastoma, Wilms'
tumor,
rhabdomyosarcoma,
Hodgkin's
CA 02361914 2001-11-13
disease, non-Hodgkin's lymphomas,
small-cell lung
Epipodophyllotoxins Etoposide Testis, small-cell
lung and other
Tertiposide lung, breast, Hodgkin's
disease, non-
Hodgkin's lymphomas,
acute
granulocytic leukemia,
Kaposi's
sarcoma
Antibiotics Dactinomycin (actinomycinChoriocarcinoma, Wilms'
tumor,
D) rhabdomyosarcoma,
testis, Kaposi's
sarcoma
Daunorubicin (daunomycin;Acute granulocytic
and acute;
rubidomycin) lymphocytic leukemias
Doxorubicin Soft-tissue, osteogenic
and other
sarcomas; Hodgkin's
disease, non-
Hodgkin's lymphomas,
acute
leukemias, breast,
genitourinary,
thyroid, lung, stomach,
neuroblastoma
Bleomycin Testis, head and neck,
skin,
esophagus, lung and
genitourinary
tract; Hodgkin's disease,
non-
Hodgkin's lymphomas
Plicamycin (mithramycin)Testis, malignant
hypercalcemia
Mitomycin (mitomycinStomach, cervix, colon,
C) breast,
pancreas, bladder,
head and neck
Enzymes L-Asparaginase Acute lymphocytic
leukemia
Biological Response Interferon alfa Hairy cell leukemia,
Kaposi's
Modifiers sarcoma, melanoma,
carcinoid,
renal cell, ovary,
bladder, non-
Hodgkin's lymphomas,
mycosis
fungoides, multiple
myeloma,
chronic granulocytic
leukemia
Miscellaneous Platinum Cisplatin (cis-DDP)Testis, ovary, bladder,
Coordination , head and
Agents Complexes Carboplatin neck, lung, thyroid,
cervix,
endometrium, neuroblastoma,
osteogenic sarcoma
Anthracenedione Mitoxantrone Acute granulocytic
leukemia, breast
Substituted Urea Hydroxyurea Chronic granulocytic
leukemia,
polycythemia vera,
essental
thrombocytosis, malignant
melanoma
Methyl Hydrazine Procarbazine(N- Hodgkin's disease
Derivative methylhydrazine,
MIH)
Adrenocortical Mitotane (o,p'-DDD)Adrenal cortex
Suppressant AminoglutethimideBreast
Hormones and AdrenocorticosteroidsPrednisone (severalAcute and chronic
other lymphocytic
Antagonists equivalent preparationsleukemias, non-Hodgkin's
available) lymphomas, Hodgkin's
disease,
breast
Progestins HydroxyprogesteroneEndometrium, breast
caproate
Medroxyprogesterone
acetate Megestrol
acetate
Estrogens DiethylstilbestrolBreast, prostate
Ethinyl estradiol
(other
preparations available)
Antiestrogen Tamoxifen Breast
Androgens Testosterone propionateBreast
Fluoxymesterone
(other
preparations available)
21
CA 02361914 2001-11-13
Antiandrogen Flutamide Prostate
Gonadotropin-releasing Leuprolide Prostate
hormone analog
The methods of the invention comprise providing and comparing a first liposome
having a
phase transition temperature at temperatures mildly hyperthermic to the body
of a subject to be
treated, and a second liposome comprising substantially the same lipids and in
the same proportions
as the first liposome, but comprising cholesterol (e.g. preferably at least 20
mole/%).
Liposomes of this invention may be formulated for parenteral administration in
a suitable
carrier such as a sterile aqueous solution. The carrier may comprise
excipients known to be tolerated
by warm-blooded animals. When performing the assay method of this invention, a
mammal such as
a mouse will be injected with a liposome formulation and blood is removed from
the mouse at fixed
time intervals such as 1, 2, 3, 4, 8, 12, 18 or 24 hours post-administration.
A convenient means for
obtaining blood at a fixed time interval is by cardiac puncture. Following
removal of whole blood,
the plasma is isolated and subjected to suitable techniques known in the art
for measuring the amount
of lipid and drug present. For example, the lipid component may be
radioactively labeled and the
plasma subjected to liquid scintillation counting. The amount of drug can be
determined for example
by a spectraphotometric assay.
It will be appreciated that the drug:lipid ratios of the liposomes can be
determined according
to any suitable method. One convenient method for determining the lipid
component is liquid
scintillation counting. For example, liposomes are labeled with ~H]-CHE as a
non-exchangeable,
non-metabolizeable lipid marker. The liposome are injected to a mouse via the
lateral tail vein with a
lipid dose of 50 mg/kg and an injection volume of 200 pL into ~ 22 g female CD-
1 mice. At various
times, three mice from each group are terminated by COz asphyxiation. Blood is
collected by cardiac
puncture, and placed into EDTA-coated or heparin-coated microtainer collection
tubes (Becton-
Dickinson). After centrifuging the blood samples at 4°C for 15 minutes
at 1000 x g, plasma is
isolated. Aliquots of the plasma obtained are counted directly in 5.0 mL
scintillation fluid. ~H]- and
['4C]-CHE labels are available from NEN/Dupont. It will be appreciated however
that any other
suitable method of determining the drug retention time of a liposome can be
used. In this
specification, the term "retention" with respect to a drug or other agent
encapsulated in a liposome
refers to retention of the drug in a liposome while the liposome is present in
the bloodstream of a
mammal. This term does not refer to a measure of drug that may be loaded or
incorporated into a
liposome or the ability of a liposome to retain the drug inex vivo conditions.
The liposomes according to the invention result in enhanced longevity
(circulation time)
while the liposome or lipid carrier of this invention is present in the
bloodstream of a warm blooded
22
CA 02361914 2001-11-13
animal. Preferably, a liposome or lipid carrier of this invention will be made
such that the amount
that would remain in the bloodstream of an animal at 4, 6, 12, 18, 24, 36 or
48 hours after intravenous
administration is at least about 10%, 20%, 40%, 50%, 60%, 70%, 80% or 90% of
the amount
administered. Example 3 demonstrates the circulation longevity of exemplary
liposomes of the
invention at specified time points after administration. These DPPC-DSPE-
PEG2000-liposomes
demonstrated substantial stability in the bloodstream as shown in Figure 1. In
another aspect,
liposomes in accordance with the invention may display a circulation
longevity, preferably the
proportion of injected liposome remaining in the bloodstream at a fixed time
point after
administration to a mammal, is which is comparable to or better than the
circulation longevity in a
mammal of a liposome containing substantially the same lipids and in the same
proportions but with
at least 20 mol % cholesterol. For liposome circulation longevity, the data
obtained from a model
animal system can be reasonably extrapolated to humans and veterinary animals
of interest.
Liposome uptake by liver and spleen has been found to occur at similar rates
in several mammalian
species, including mouse, rat, monkey, and human (Gregoriadis, G., and
Neerunjun, D. (1974) Eur. J.
Biochem. 47, 179-185; Jonah, M. M., et al. (1975) Biochem. Biophys. Acta 401,
336-348;
Kimelberg, H. K., et al. (1976) Cancer Res. 36,2949-2957; Juliano, R. L., and
Stamp, D. (1975)
Biochem. Biophys. Res. Commun. 63. 651-658; Richardson, V.J., et al. (1979)
Br. J. Cancer 40,
3543; Lopez-Berestein, G., et al. (1984) Cancer Res. 44, 375-378). This result
likely reflects the fact
that the biochemical factors which appear to be most important in liposome
uptake by the RES--
including opsinization by serum lipoproteins, size-dependent uptake effects,
and cell shielding by
surface moieties--are common features of all mammalian species which have been
examined.
Optionally, any or all of the steps of the method of designing or preparing
liposomes of the
invention can be repeated. For example, the steps of providing liposomes and
comparing retention
properties of cholesterol-free or thermosensitive liposomes of the invention
and cholesterol-
containing liposomes can be repeated. With these iterations, adjustments to
the respective liposome
compositions can be made. In this way, liposomes can be optimized and the
resulting compositions
can be assessed for drug retention and/or circulation longevity properties.
It will be appreciated that a panel of liposomes can be prepared, liposomes of
said panel
having a phase transition temperatures greater than that of the body of a
subject to be treated,
preferably above 37°C but less than 45°C, having differences in
lipids and in lipid proportions, so
long as at least one of the liposomes of said panel is compared against a
liposome comprising
substantially the same lipids and in the same proportions, but comprising
cholesterol (e.g. preferably
at least 20 mole %). A panel of liposomes can comprise for example at least 2,
3, 4, 5, 8, 10, 20, 50,
100 different liposome compositions.
A liposome having the aforementioned phase transition properties, preferably a
liposome that
is substantially cholesterol-free can thus be identified, which liposome has
drug retention
23
CA 02361914 2001-11-13
characteristics comparable or better than a similar cholesterol-containing
liposome. In the context of
identifying said liposome, the terms 'identifying' and 'selecting' can be used
interchangeably. This
liposome with a drug incorporated therein can be used as such as a medicament
for treatment of a
subject, or can be the subject of further optimization or design steps to
modify the composition as
desired. In particular, steps of adjusting the liposome composition can be
carried out, and the steps of
the method of the present invention can be repeated to compare drug:lipid
ratios with a comparable
cholesterol-containing liposome. In other aspects, the improved liposome
obtained using the method
of the invention can also be used with different drugs. For example, it will
be appreciated that
different drugs having similar retention properties to the drug or marker used
in the drug:lipid ratio
comparison of the present method can be incorporated the liposome in place of
the drug used in the
drug:lipid comparison. Furthermore, if desired, tests can be carried out to
compare drug:lipid ratios
of the two drugs in the thermosensitive liposomes of the invention.
Administration and therapeutic use
The liposomes of the invention allows the,use of thermosensitive liposomes in
combination
with hyperthermia at the desired target site, permitting improved targeting at
'mildly hyperthermic'
temperatures which otherwise do not cause damage to the patient. Applications
of such liposomes
have been reported for example in Magin and Weinstein In: Liposome Technology,
Vol. 3,
(Gregoriadis, G., ed.) p. 137, CRC Press, Boca Raton, Fla. (1993); Gaber et
al., Intl. J. Radiation
Oncology, Biol. Physics, 36(5):1177 (1996).
Liposome of the present invention may be administered to warm-blooded animals
to be
treated, including humans. Liposomes of the present invention may be
administered using methods
that are known to those skilled in the art, including but not limited to
delivery into the bloodstream
(e.g. administered intravenously) of a subject or subcutaneous administration
of liposomes. These
liposomes may be used to treat a variety of diseases in warm-blooded animals,
the application of
which depending on the particular bioactive agent incorporated in the
liposome. Examples of
medical uses of the compositions of the present invention include but are not
limited to treating
cancer, treating cancer, inflammation, treating bacterial, fungal or parasitic
infections. For treatment
of human ailments, a qualified physician will determine how the compositions
of the present
invention should be utilized with respect to dose, schedule and route of
administration using
established protocols. Such applications may also utilize dose escalation
should bioactive agents
encapsulated in liposomes of the present invention, exhibit reduced toxicity
to healthy tissues of the
subject.
3 5 For medical applications, formulations of the liposomes of the present
invention for
parenteral administration are preferably in a sterile aqueous solution
optimally comprised of
24
CA 02361914 2001-11-13
excipients known to be tolerated by warm-blooded animals. For oral or topical
applications, the
liposome and lipid carrier compositions of the present invention may be
incorporated in vehicles
commonly used for the respective applications such as but not limited to
creams, salves, ointments
and slow release patches for topical medical applications and tablets,
capsules, powders, suspensions,
solutions and elixirs for oral applications.
The liposomes and lipid carrier compositions of the present invention may also
be used for
diagnostic purposes where the controlled release of liposome contents can
provide improved delivery
of distribution of a diagnostic agent. Liposomes of the present invention can
be administered as a
parenteral agent to warm-blooded animals to detect the presence of specific
disease sites or markers
of disease. Such compositions may contain imaging agents including, but not
limited to,
radionuclides, magnetic resonance contrast agents and heavy atom contrast
agents.
EXAMPLES
Example 1
Preparation of liposomes
Solutions of DPPC, DSPE-PEG2000 and cholesterol in chloroform were combined to
give a
90: 4 molar ratio of DPPC: DSPE-PEG2000 (80-100 pmoles total lipid), and a
55:45:4 mol ratio of
DSPC: cholesterol: DSPE-PEG2000 with 50,000 dpm/mg lipid of3H-cholesteryl
hexadecyl ether
(3CHE) as a radiolabelled marker. The resulting mixture was dried under a
stream of nitrogen gas
and placed in a vacuum pump overnight. The samples were then hydrated with 300
mM citrate pH
4.0 and subsequently passed through an extrusion apparatus (Lipex
Biomembranes, Vancouver, BC)
10 times with 1 X 80 nm and 1 X 100 nm polycarbonate filters at 55 °C.
Average liposome size was
determined by quasi-elastic light scattering using a NICOMP 370 submicron
particle sizer at a
wavelength of 632.8 nm.
Example 2
Encapsulation of drug
For each of the radiolabelled solutions of Example 1, a 90: 4 molar ratio of
DPPC: DSPE-
PEG2000 (80-100 pmoles total lipid), and a 50:45:5 mol ratio of DSPC:
cholesterol: DSPE-
PEG2000, the solution was run down a Sephadex G50 column equilibrated with HBS
(20 mM
HEPES, 150 mM NaCI, pH 7.45) in order to create a transmembrane pH gradient by
exchange of the
exterior buffer. Resulting pH gradient liposomes were combined with
doxorubicin to give a final
CA 02361914 2001-11-13
concentration of 5 mM lipid and 1 mM doxorubicin (0.2:1 drug:lipid ratio) in a
final volume of 1 mL
adjusted with HBS. The resulting mixture was incubated at 37°C prior to
assaying the amount of
encapsulated doxorubicin. At various time points, samples were fractionated on
a 1 mL mini-
Sephadex G-50 spin column to remove unencapsulated doxorubicin. The voided
fraction was
assayed for liposomal lipid by scintillation counting. To measure levels of
doxorubicin, a defined
volume of the eluant was adjusted to 100 pL followed by addition of 900 pL, of
1% Triton X-100 to
dissolve the liposomal membrane. The sample was heated until cloudy in
appearance and the
Abs480 was measured after equilibration at room temperature. Concentrations of
doxorubicin were
calculated by preparing a standard curve.
Example 3
Administration of thermosensitive liposomes and assessment of circulation
longevity and drug:
lipid ratio
DPPC: DSPE-PEG2000 (90:4 mol %) liposomes were prepared and loaded with
doxorubicin
as outlined in the methods of Example 1 and 2 respectively.
Non-thermally controlled mice were treated with cholesterol-free liposomes as
follows:
Adult female Rag-2 mice were injected with DPPC: DSPE-PEGz~o (90:4 mol%)
liposomes via the
tail vein. Mice were killed and blood was collected by cardiac puncture into
EDTA-coated
microtainers at 10 min, 1h, 2h, and 4h after treatment.
Non-thermally controlled mice were treated with cholesterol-containing
liposomes as
follows:
Adult female Rag-2 mice were injected with DSPC: Cholesterol:DSPE-PEGz~
(55:45:4 mol%) via
the tail vein. Mice were killed and blood was collected by cardiac puncture
into EDTA-coated
microtainers 1h and 4h after treatment.
Thermally controlled mice were treated with cholesterol-free liposomes as
follows:
Late time points (2 and 4 hours post injection):
Twelve mice were anaesthetized for at least 1h with ketamine/xylazine (160/10
mg/kg). Mice were
placed in groups of four mice per cage in a temperature controlled cage
incubator that was pre-heated
at 37°C. After being fully anaesthetized, mice were removed from the
cage incubator and injected
with DPPC:DSPE-PEGZOOO (90:4 mol%) liposomes via the tail vein. Mice were
subsequently placed
26
CA 02361914 2001-11-13
back in the cage incubator with the heat turned off. After 2h, the mice had
fully recovered from the
anesthesia and their body temperature was therefore not controlled thereafter.
Mice were terminated
at 2h and 4h post injection and blood was collected by cardiac puncture into
EDTA-coated
microtainers.
Early time points (10 minutes and 1 hour post injection):
The remaining 12 mice were anaesthetized as described above and placed
individually in a custom-
made mouse-incubator that was preheated at 37°C. Mice were injected
with DPPC: DSPE-PEC~ooo
(90:4 mol%) liposomes via the tail vein. Mice were terminated after lOmin and
1h. Blood was
collected by cardiac puncture into EDTA-coated microtainers.
Lipid and plasma doxorubicin concentrations were determined as follows:
Plasma was separated by centrifugation at 750g for lOmin and the lipid
concentration in plasma was
determined by liquid scintillation counting. Doxorubicin was extracted and
quantified as follows:
A defined volume of plasma was adjusted to 200 mL with distilled water and the
following
reagents were added: 600 mL of distilled water, 100 mL of a 10% sodium dodecyl
sulfate
solution, and 100 mL of 10 mM HZS04. To the resulting mixture, 2mL of
isopropanol/chloroform
(1:1 vol/vol) was added and mixed vigorously. Samples were frozen at -
20°C overnight or at
80°C for 1 hour to promote protein aggregation, brought to room
temperature, mixed again and
centrifuged at 3000 rpm for 10 minutes. The bottom organic layer was removed
and assayed by
fluorescence spectroscopy (~,eX: 470 nm, 7<,em: 550 nm).
Doxorubicin-containing DPPC:DSPE-PEGZOOO liposomes exhibited extended
circulation
longevity (Figure 1) and enhanced drug retention (Figure 2) in temperature
controlled mice similar to
the cholesterol-containing formulation and in contrast to doxorubicin-
containing DPPC:DSPE-
PEGzooo liposomes administered to mice without thermal control. At 4h after
injection, the body
temperature of thermally controlled mice was likely increased to temperature
at which liposomes
start releasing the drug (39°C) since the anesthetic wore off starting
at 1.5h - 2h after injection. The
body temperature in mice can increase to values up to 40.5°C as a
stress-response in non anesthetized
mice, which may explain why lipid and drug levels were decreased 4h after
administration (due to
lack of thermal control).
Results depicted in Figures 1 and 2 are contrary to observations set forth in
the state of the art
(see review article: Kong et al. (1999) Int. J. Hyperthermia 15(5): 345-370).
Most likely this is due to
the absence of thermal control in previous studies described in the art.
Example 4
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CA 02361914 2001-11-13
Delayed release of cholesterol-free, thermosensitive liposomes
DPPC: DSPE-PEG2000 (95: 5 mol %) liposomes are prepared and loaded with
doxorubicin
as outlined in the materials and methods of Example 1 and 2 respectively.
The resulting doxorubicin loaded liposomes are administered to a
mouse(however, the body
temperatures of the mice cannot be controlled according to the methods of
Example 3 due to time
points in the study beyond 1 hr) in a final volume of 200 pL immediately after
preparation (within 1-
2 hrs). Subsequent to administration, local hyperthermia (42°C) at the
tumor site using a
radiofrequency oscillator or a water bath (with specially designed holders
that allow the tumor to be
placed in a water bath) is started at 4, 6, 12,18,24,36 and 48 hours after
administration and continued
for a set period of time (typically not exceeding 2 hrs). Just prior to and
after thehyperthermia
treatment, blood is collected and tumors excised. Lipid levels are measured by
liquid scintillation
counting. To determine drug levels, tumors are frozen at-70 °C and
extracted with chloroform and
silver nitrate to determine doxorubicin concentrations (Cummings et al. (1986)
Br. J. Cancer 53: 835-
838). Samples may also be extracted with only chloroform for comparison to
determine the amount
of doxorubicin bound to DNA or RNA (thereby giving a measure of released
drug). Concentrations
of doxorubicin in tumour samples are quantified using high performance liquid
chromatography.
In order to determine drug and lipid levels, in the blood afterhyperthermia,
blood samples are
quantitated for levels of doxorubicin and lipid as in Example 3.
Example S
Assessing survival time upon administration of liposomal doxorubicin
DPPC: DSPE-PEG2000 (95: 5 mol %) liposomes are prepared and loaded with
doxorubicin as outlined in the materials and methods of Example 4 (except
temperature control is
not possible at time points greater than 1 hour according to the methods of
Example 3).
P388/wt cells are maintained by passage in vivo (in the peritoneum) of BDF-1
female
mice. Cells are only used for experiment between the 3~d and 20'h passage.
Cells are harvested 7
days post inoculation, diluted in Hepes Buffered Saline (HBS) to 2 x 106
cells/mL, and 0.5 mL is
injected intraperitoneally into BDF-1 mice. Two days after tumor cell
inoculation, BDF1 female
mice are administered by intravenous administration one of the following: HBS;
doxorubicin (1
mg/kg); DPPC: DSPE-PEG2000 (95: 5 mol %) liposomes (1 mg/kg) loaded with
doxorubicin.
Percent survival is calculated based on 4 mice per group.
Although the foregoing invention has been described in some detail by way of
illustration and
example for purposes of clarity of understanding, it will be readily apparent
to those of skill in the art
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CA 02361914 2001-11-13
in light of the teachings of this invention that changes and modification may
be made thereto without
departing from the spirit or scope of the appended claims. All patents, patent
applications and
publications referred to herein are incorporated herein by reference.
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