Note: Descriptions are shown in the official language in which they were submitted.
CA 02361946 2001-11-13
DELAYED DRUG RELEASE USING THERMOSENSITIVE LIPOSOME
TPChnical FiPlri
S This invention is directed toward methods of delivering a therapeutic agent
to a target
tissue in a mammal using long-circulating thermosensitive liposomes combined
with a mild
hyperthermic treatment.
Background of the Invention
The successful treatment of breast tumors, head and neck tumors, prostate
tumors and
other deep seated tumors (malignant or benign) within the human body is a
difficult task. The
main objective of the treatment is to reduce in size or completely remove the
tumor mass by one
or more modalities available at the treatment facility. The most common
modalities are surgery,
radiation therapy and chemotherapy. Surgical treatment of breast cancer often
involves
substantial disfigurement, and surgery for other deep seated cancers often
creates complications
for surrounding vital organs and healthy tissue. Radiation therapy of deep
seated tumors also puts
surrounding healthy tissues at risk.
It has long been established that incorporation of membrane rigidification
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 the
liposome. Conventional
approaches to liposome formulation dictate inclusion of substantial amounts
(e.g. 30-45 mol %)
cholesterol or equivalent membrane rigidification agents (such as other
sterols) into liposomes.
More recently, means for providing targeted release of liposome contents via
the use of
"thermosensitive" drug carriers have been developed (for example, see Yatvin
et al., Science
202:1290 (1978); 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
CA 02361946 2001-11-13
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, the efficacy of liposomes targeted to diseased sites by hyperthermia
depends on the
stability of the liposome in the blood stream (when liposomes are administered
to the circulatory
system), and on the amount of active agent released by the liposome at the
target site. For
example, liposomes described by Yatvin et al, Science 202:1290 ( 1978)
released only a portion of
the drug carried by the liposome at hyperthermic temperatures.
While the use of thermosensitive liposomes in hyperthermia therapy is
promising, the
tissue targeting effect depends upon how stably the liposomes administered
circulate through the
circulatory system at the normal body temperature and how much the liposomes
release the drug
at the site of tumor at the temperature of hyperthermia. The thermosensitive
liposomes reported
so far have problems with respect to the stability and the release by heating,
and may not be
expected to show their full effect.
For example, the liposomes described in Science, 202, 1290 (1978) release only
a small
amount of the drug at the temperature of hyperthermia, and the liposomes
described in J. Urol.,
135, 162 (1986) release a certain amount of the drug already at a temperature
too low for
effective treatment conditions. Thus the liposomes prepared by the
conventional methods have
problems to be solved with respect to release by heating and stability.
Other liposomes, particularly liposomes containing a substantial amount of
cholesterol,
require an elevated temperature (generally above 45° C) to release the
drug, which presents
disadvantages in that collateral tissue damage may occur. 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 sufficiently
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
2
CA 02361946 2001-11-13
Cevc, G. (1) & (2) Biochimica et Biophysica Acta (1990)1029:91-97(1) & (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. As a result, currently
available liposomes
either suffer from low stability and low drug retention rates, or are not able
to release their
contents at the 'mildly' hyperthermic temperatures needed to be useful in
human therapy.
Summary of Invention
The present inventors provide a method which allows improved delivery of an
active
agent to a tissue of interest in a mammal. Provided is a method of delivering
an agent to a site of
interest in a subject by administering thermosensitive liposomes containing an
active agent to a
subject, allowing an extended time period for liposome localization to the
site of interest, and
subsequently administering a hyperthermic treatment at the site of interest to
cause the release of
the liposome contents. The period between the administration of liposomes and
the
administration of a hyperthermic treatment is preferably at least about 4
hours, but may be at least
about 8, 12, 24 or 48 hours.
The 'delaying' of the hyperthermia treatment which allows improved tissue
localization is
enabled by the discovery of means for preparing thermosensitive liposomes
having phase
transition temperatures at 'mildly' hyperthermic temperatures, extended
circulation longevity and
improved drug retention. The inventors provide liposomes prepared. in the
absence of cholesterol
that can be made to behave comparably to cholesterol-containing liposomes
through the
incorporation of a hydrophilic polymer conjugated lipid. These results are
contrary to the
previous wisdom concerning liposomes prepared without cholesterol. The methods
set forth
below are based on the finding that liposomes having phase transition
temperatures useful for
thermosensitive applications display drug retention and circulation
longevities that are
comparable or better than cholesterol containing liposomes in mouse models of
disease if the
temperatures of the mice are maintained below the phase transition temperature
of the liposomes.
Results of this observation are set forth in Example 3.
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
3
CA 02361946 2001-11-13
temperatures mildly hyperthermic to a subject's body temperature to a
cholesterol-containing
liposome.
Further, it has been discovered that the hydrophilic polymer stabilization
effects due to
use of PEG-modified lipid incorporation are, surprisingly, not substantially
dependent on the
concentration of the polymer (concentrations as low as 0.5 mol% PEG-2000 can
cause a greater
than 15-fold increase in Area-Under-the Curve (AUC) when compared to the same
liposome
prepared without the PEG-lipid) or polymer molecular weight (PEG 350 at
concentrations of 5
mol% can cause a greater than 25 fold improvement in AUC when compared to the
same
liposome prepared without the PEG lipid and this increase in AUC is comparable
to the greater
than 38-fold improvement observed when using 5 mol% PEG 2000). The resulting
liposomes
exhibit much enhanced longevity of the liposomes while in blood circulation.
These results are
contrary to the previous wisdom concerning the incorporation of PEG-lipids
into a liposome.
The methods disclosed herein allow the controlled release of an active agent
from a
liposome upon administration of hyperthermia. This controlled release has
heretofore not been
possible with liposomes having long circulation times and/or sufficient drug
retention. The
treatment method allows both higher levels of drug to be administered, due to
reduced drug
toxicity in liposomes, and greater drug efficacy, due to selective liposome
localization in a target
tissue, preferably in the intercellular fluid of a tumor. The methods will
allow comparable or
better toxicity and tissue localization than methods using liposomes
containing substantially the
same lipids and in the same ratio but containing at least 20mo1% cholesterol,
which do not have
the thermosensitivity characteristics of the present liposomes.
The invention therefore discloses a method of administering an agent to a
mammalian
subject, comprising:
(a) administering to the bloodstream of a subject thermosensitive liposomes
comprising a
therapeutic agent, said liposomes having a phase transition temperature
greater than that of the
body of the subject to be treated, and less than 45°C;
(b) administering a hyperthermic treatment to a localized site on the body of
the subject a
mild hyperthermic treatment at a time point at least 4 hours after
administration of the liposomes
of step (a); and
(c) said hyperthermic treatment delivered in an amount and for a time
sufficient to cause
the release of enapsulated therapeutic agent from the administered liposomes.
The invention provides improved treatment methods by allowing the administered
liposomes to circulate in the bloodstream of the subject until a desired
biodistribution of the
4
CA 02361946 2001-11-13
liposomes is achieved. An agent of interest such as a therapeutic or
diagnostic agent can thereby
be exposed to a target surface or target tissue in the subject mammal,
Also provided is a method of preparing an agent for localization in a target
tissue by
extravasation of liposomes containing the agent into the target tissue, when
the agent is
administered by intravenous injection, comprising:
entrapping the agent in liposomes which are characterized by:
(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 mol % of one or more vesicle-forming lipids;
wherein the liposome displays a drug:lipid ratio in the bloodstream at a time
point at least
4 hours after administration to 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 containing
substantially the same lipids
and in the same proportions but with at least 20 mol % cholesterol.
In another aspect, the invention discloses a method of localizing a
therapeutic agent in a
target tissue in a subject by extravasation of liposomes containing the
therapeutic agent into the
target tissue comprising:
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids;
(d) having a therapeutic agent encapsulated therein; and
injecting the composition intravenously in the subject in an amount effective
to localize a
therapeutically effective quantity of the therapeutic agent in the target
tissue. This method may
achieve localization of the liposomes in the target tissue, 4 hours after
intravenous administration,
that is comparable or better than that of liposomes containing substantially
the same lipids and in
the same proportions but with at least 20 mol % cholesterol.
In a further aspect, the invention provides a method of administering a
therapeutic agent
to a target tissue in a subject, comprising:
providing a composition of liposomes 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;
CA 02361946 2001-11-13
(b) from about 2 to about 15 mol % hydrophilic polymer-conjugated lipid; and
(c) up to about 38 mol % of one or more vesicle-forming lipids; and
(d) having a therapeutic agent encapsulated therein
injecting the composition intravenously to the subject in an amount effective
to localize a
therapeutically effective quantity of the agent in the target tissue; and
administering hyperthermia to the target tissue at least 4 hours after
injection the
composition intravenously to the subject in a dose sufficient to cause release
of therapeutic agent
contained in liposomes.
Another aspect of the invention relates to a method of administering a
therapeutic agent to
a target tissue in a subject, comprising:
selecting a subject suffering from a neoplastic disease;
providing a composition of liposomes 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 mol % of one or more vesicle-forming lipids; and
(d) having a therapeutic agent encapsulated therein
injecting the composition intravenously to the subject in an amount effective
to localize a
therapeutically effective quantity of the agent at a neoplastic lesion; and
administering hyperthermia to a neoplastic lesion at least 4 hours after
injection the
composition intravenously to the subject in a dose sufficient to cause release
of therapeutic agent
contained in liposomes.
In any of the methods of the invention, the liposome administered to the
subject will most
preferably contain substantially no cholesterol, or will contain less than 1
mol % cholesterol, or
more preferably essentially no cholesterol. As discussed herein, the absence
of cholesterol will
generally provide optimal phase transition at temperatures which are 'mildly
thermosensitive'
resulting in optimal release of encapsulated agent from liposomes.
Furthermore, the liposome for use in the methods of the invention preferably
comprise at
least 60, 70, 80 or 90 mol % of a phospholipid comprising two saturated fatty
acids. Most
preferably, the liposomes comprise : (a) at least 60, 70, 80 or 90 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 mol % of one or more vesicle-
forming lipids. A
CA 02361946 2001-11-13
particularly useful phospholipid is dipalmitoylphosphatidylcholine (DPPC). The
hydrophilic
polymer-conjugated lipid is preferably a PEG lipid.
As further described herein, the agent encapsulated in the liposome may be any
suitable
agent, including preferably a therapeutic agent (drug) or a diagnostic agent.
According to the methods of the invention, the period of time between liposome
administration and administration of the hyperthermic treatment will generally
be sufficient such
that increased accumulation of therapeutic agent in a target tissue (for
example a tumor) is
achieved when compared with free therapeutic agent (e.g. delivered in a non-
liposomal
formulation). The hyperthermic treatment is preferably administered at least
4, 8, 12, 24 or 48
hours after administration of the liposomes of step (a). The hyperthermic
treatment is preferably
delivered locally at a tumor site, at a site of inflammation or at a site of
infection. The
hyperthermic treatment preferably comprises administering to a tissue of
interest a temperature
greater than that of the body of a subject to be treated but less than
45°C, and more preferably a
temperature is between 39° C and 42° C.
According to preferred methods of the invention, the liposomes for use in
accordance
with the methods of the invention display: (i) a circulation longevity at a
fixed time point after
administration to a mammal is which is comparable to or better than the
circulation longevity in a
mammal at said fixed time point of a liposome containing substantially the
same lipids and in the
same proportions but with at least 20 mol % cholesterol; and/or (ii) when
encapsulating a drug, a
drug:lipid ratio in the bloodstream at a fixed time point after administration
to 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 containing substantially the same lipids and in the same proportions
but with at least 20
mol % cholesterol. The fixed time point is preferably at least 4, 8 ,12, 18,
24, 36 or 48 hours after
liposome administration.
As discussed, while any suitable liposome composition in accordance with the
methods of
the invention can be used, the inventors provide particularly preferred
liposome compositions.
The invention thus encompasses a composition 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 mol % of one or more vesicle-forming lipids, providing that
the
liposome contains substantially no cholesterol;
CA 02361946 2001-11-13
wherein the liposome displays a comparable or greater circulation longevity,
or when
encapsulating a drug displays a comparable or greater circulation longevity
and/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.
Preferably, in accordance with the invention, the fixed time point is a time
point at least about 4,
8, 12, 18, 24, 36 or 48 hours after administration of the liposomes to the
subject. It will be
appreciated that said time point may also be a time point greater than about
4, 8, 12, 18, 24, 36 or
48 hours after administration of the liposomes to the subject.
The liposome will preferably contain substantially no cholesterol, or may
optionally contain less than 1 mol % cholesterol or may contain essentially 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 42° C.
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
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.
It will be appreciated that any suitable method for determining the
circulation longevity
and/or drug retention of a liposome can be used. In this specification, the
term "retention" with
8
CA 02361946 2001-11-13
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. A
drug: lipid ratio or retention time which is deemed 'comparable' will depend
on the
circumstances, but is preferably at least 60 %, 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 and/or free of PEG. The 'time point' is generally a
number as
measured in hours, minutes, etc.
Liposomes for use in the present methods may be prepared using known and
conventional
techniques. Selection or design of liposomes having the desired circulation
longevity and drug
retention characteristics can be obtained according to methods described
herein. 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 as well as the techniques
presented in Examples
1 to 5. 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.
CA 02361946 2001-11-13
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.
1 S 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 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 5, 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:
CA 02361946 2001-11-13
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
PEG2000-
DSPE): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polyethylene glycol
2000]; DSPE-
PEG 750 (or 750PEG-DSPE or PEG750-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]; 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.
As used herein, the term "hyperthermia" refers to the elevation of the
temperature of a
subject's body, or a part of a subject's body, compared to the normal
temperature of the subject.
Such elevation may be the result of a natural process (such as inflammation)
or artificially
induced for therapeutic or diagnostic purposes.
In mammals, a normal body temperature is ordinarily maintained due to the
thermoregulatory center in the anterior hypothalamus, which acts to balance
heat production by
body tissues with heat loss. "Hyperthermia" refers to the elevation of body
temperature above the
hypothalamic set point due to insufficient heat dissipation. In contrast to
hyperthermia, "fever"
refers to an elevation of body temperature due to a change in the
thermoregulatory center. The
overall mean oral temperature for a healthy human aged 18-40 years is 36.8.+-
Ø4°C. (98.2.+-
Ø7°F.). See, e.g., Harrison's Principles of Internal Medicine (Fauci
et al., Eds.) 14th Edition,
McGraw-Hill, New York, p. 84 (1998).
As used herein, "hyperthermic administration" of an active agent refers to its
administration in conjunction with the use of clinical hyperthermia in the
subject at a preselected
target site, to deliver a larger amount of active agent to the target site
compared to that which
would result from the administration of the active agent in the absence of
hyperthermia.
As used herein, "solid tumors" are those growing in an anatomical site other
than the
bloodstream (in contrast to blood-borne tumors such as leukemias). Solid
tumors require the
formation of small blood vessels and capillaries to nourish the growing tumor
tissue.
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
11
CA 02361946 2001-11-13
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
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 liposomes are described in copending international
patent application
10 PCT/CA01/00655, which is incorporated herein by reference.
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, sulfate, amino or
sulfhydryl 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
lipid
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, polylactie
acid, polyglycolic acid, polyacrylic acid and copolymers thereof, as well as
those disclosed in
12
CA 02361946 2001-11-13
United States Patents 5,013,556 and 5,395,619, the disclosures of which are
incorporated herein
by reference. 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. Regarding liposomes containing polymer grafted
lipids in the vesicle
membrane, 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, the
disclosures of all of the
references being incorporated herein by reference in their entireties. 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 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 between 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
13
CA 02361946 2001-11-13
chains of which being independently selected from the group consisting of
caproyl (6:0), octanoyl
(8:0), capryl (10:0), lauroyl (12:0), myristoyl (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 (C16)
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), the disclosures of which are incorporated
herein by
reference.
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 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.
The liposomes may comprise amphipathic lipids in addition to those described
above, but
preferably no substantial amount of cholesterol. ' Such lipids include
sphingomyelins, glycolipids,
14
CA 02361946 2001-11-13
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 sources or 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), the disclosures of
which are
incorporated herein by reference.
As shown in Example 1, 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.
Advantageous methods of designing and preparing thermosensitive and/or
substantially
cholesterol free liposomes having improved circulation longevity and/or drug
retention
characteristics are described in the copending Application No. [] titled
"Improved
cholesterol-free liposomes" filed on the same day as the present application
and the disclosure of
which is incorporated herein by reference.
In summary, this method comprises:
(i) comparing a drug retention property 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
comparable to or
improved over that of the substantially equivalent cholesterol-containing
liposome of step (b).
CA 02361946 2001-11-13
In preferred aspects, said method comprises the steps of:
(a) preparing a liposome having a phase transition temperature greater than
that of
the body of a subject to be treated, and less than 45°C;
(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;
(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 a separate non-human mammal;
(e) determining drug:lipid ratios in the blood of the mammals at least one
fixed time
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) having 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.
The liposome of the steps (a) in the method of designing liposomes will
typically
comprise at least 60, 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, wherein at least
one of said acyl chains
has 16 carbon atoms. Most preferably, the liposome comprises DPPC, most
preferably at least 30,
50, 60, 80 or 95 mol% DPPC. Said liposome may further comprises one or more
phospholipids
selected from the group consisting of: I, PA and PE. As in the liposomes of
the invention, the
hydrophilic polymer-conjugated lipid is preferably a PEG-lipid, preferably
having a molecular
weight from about 100 to about 5000 daltons, or from about 1000 to about 5000
daltons. The
PEG lipid will be present in the liposome of step (a) from about 2 to about 10
mol % PEG-lipid.
Moreover, preferably the liposome of step (a) will contain substantially no
cholesterol and/or 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, or
more preferably between 39° C and 41 ° C.
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 at least one polymer-conjugated lipid, are prepared. A physiologically
acceptable buffer is
16
CA 02361946 2001-11-13
used for 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
180 nm 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. 80-
140 nm liposomes 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 SOnm 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.
Active agents may be loaded into liposomes using passive and active loading
methods
described herein.
Passive methods of encapsulating active agents
Therapeutic agents may be encapsulated using passive methods of encapsulation.
Passive
methods of encapsulating active agent in liposomes involve encapsulating the
agent during the
synthesis of the liposomes. In this method, the active agent 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.
17
CA 02361946 2001-11-13
(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.
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 agent 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 an 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 active agent.
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 therapeutic agent or other agent to be encapsulated.
Once the agent
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 active
agent 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.
18
CA 02361946 2001-11-13
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 active agent to
cross the liposome membrane and conversion of the moiety to a charged form
causes the active
agent 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 active agents 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
19
CA 02361946 2001-11-13
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 US patent no.
5,939,096).
In other aspects, charge attraction methods may be utilized to actively load
therapeutic
agents. Charge attraction 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.
CA 02361946 2001-11-13
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.
The liposomes will have 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 a mildly hyperthermic conditions, which are
generally understood
to be between that of the body of a subject to be treated and about
45°C, or more preferably
between that of the body of a subject to be treated and about 43°C, or
more preferably between
that of the body of a subject to be treated and about 42°C.
The terms "drug", "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, active
agent includes 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-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
21
CA 02361946 2001-11-13
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.
The anti-neoplastic agent which may be used is any compound, including the
ones listed
herein, which can be stably entrapped in liposomes at a suitable loading
factor and administered
at a therapeutically effective dose (indicated below in parentheses after each
compound). These
include amphipathic anti-tumor compounds such as the plant alkaloids
vincristine (1.4 mg/m2),
vinblastine (4-18 mg/m2) and etoposide (35-100 mg/m2), and the anthracycline
antibiotics
including doxorubicin (60-75 mg/m2), epirubicin (60-120 mg/m2) and
daunorubicin (25-45
mg/m2). The water-soluble anti-metabolites such as methotrexate (3 mg/m2),
cytosine
arabinoside (100 mg/m2), and fluorouracil (10-15 mg/kg), the antibiotics such
as bleomycin (10-
units/m2), mitomycin (20 mg/m2), plicamycin (25-30 µg/2) and dactinomycin
(15
µg/m2), and the alkylating agents including cyclophosphamide (3-25 mg/kg),
thiotepa (0.3-0.4
mg/Kg) and BCNU (150-200 mg/m2) are also useful in this context. Preferred
examples, the
15 plant alkaloids exemplified by vincristine and the anthracycline
antibiotics including doxorubicin,
daunorubicin and epirubicin are preferably actively loaded into liposomes, to
achieve drug/lipid
ratios which are several times greater than can be achieved with passive
loading techniques. Also
the liposomes may contain encapsulated tumor-therapeutic peptides and protein
drugs, such as IL-
2, and/or TNF, and/or immunomodulators, such as GM-CSF, which are present
alone or in
20 combination with anti-neoplastic agents, such as an anthracycline
antibiotic drug.
Other examples of agents that can be used according to the invention are shown
in Table
1.
22
CA 02361946 2001-11-13
Table 1
CLASS TYPE OF AGENT 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, Wilms'
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,
Methylmelamines 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 Malignant pancreatic
insulinoma,
(streptozotocin) malignant carcinoid
Triazines Dacarbazine (DTIC;Malignant melanoma,
Hodgkin's
dimethyltriazenoimidazole-disease, soft-tissue
sarcomas
Antimetabolites Folic Methotrexate Acute lymphocytic
Acid Analogs leukemia,
(amethopterin) choriocarcinoma, mycosis
fungoides, breast,
head and neck,
lung, osteogenic sarcoma
Pyrimidine Analogs Fluouracil (5-fluorouracil;Breast, colon, stomach,
pancreas,
5-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
23
CA 02361946 2001-11-13
lymphomas, breast,
testis
Vincristine Acute lymphocytic
leukemia,
neuroblastoma, Wilms'
tumor,
rhabdomyosarcoma,
Hodgkin's
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,
Wilins' 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,
C) colon, 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
24
CA 02361946 2001-11-13
Medroxyprogesterone
acetate Megestrol acetate
Estrogens Diethylstilbestrol Breast, prostate
Ethinyl estradiol (other
preparations available)
Antiestrogen Tamoxifen Breast
Androgens Testosterone propionate Breast
Fluoxymesterone (other
preparations available)
Antiandrogen Flutamide Prostate
Gonadotropin- Leuprolide Prostate
releasing hormone
analog
For medical applications, formulations of the liposomes of the present
invention for
parenteral administration are preferably in a sterile aqueous solution
optimally comprised of
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.
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 of a subject
(intravenous or "IV") or subcutaneous administration of liposomes. Where
liposomes according
to the present invention are used in conjunction with hyperthermia, the
liposomes may be
administered by any suitable means that results in delivery of the liposomes
to the treatment site.
For example, liposomes may be administered intravenously and thereby brought
to the site of a
tumor by the normal blood flow; heating of this site results in the liposomal
membranes being
heated to the phase transition temperature so that the liposomal contents are
preferentially
released at the site of the tumor.
In accordance with the present invention, the anti-tumor or anti-neoplastic
agent of choice
is entrapped within a liposome according to the present invention; the
liposomes are formulated to
be of a size known to penetrate the endothelial and basement membrane
barriers. The resulting
liposomal formulation can be administered parenterally to a subject in need of
such treatment,
preferably by intravenous administration. Tumors characterized by an acute
increase in
permeability of the vasculature in the region of tumor growth are particularly
suited for treatment
CA 02361946 2001-11-13
by the present methods. Administration of liposomes is followed by heating of
the treatment site
to a temperature that results in release of the liposomal contents.
Where site-specific treatment of inflammation is desired, effective liposome
delivery of
an active agent requires that the liposome have a long blood halflife, and be
capable of
penetrating the continuous endothelial cell layer and underlying basement
membrane surrounding
blood vessels adjacent to the site of inflammation. Liposomes of smaller sizes
have been found to
be more effective at extravasation through the endothelial cell barrier and
into associated
inflamed regions. See, e.g., U.S. Pat. No. 5,356,633 to Woodle et al. In
accordance with the
present invention, the anti-inflammatory agent of choice is entrapped within a
liposome according
to the present invention; the liposomes are formulated to be of a size known
to penetrate the
endothelial and basement membrane barriers. The resulting liposomal
formulation can be
administered parenterally to a subject in need of such treatment, preferably
by intravenous
administration. Inflamed regions characterized by an acute increase in
permeability of the
vasculature in the region of inflammation, and by a localized increase in
temperature, are
particularly suited for treatment by the present methods.
It will further be appreciated that the liposomes of the present invention may
be utilized to
deliver of anti-infective agents to sites of infection, via the bloodstream.
The use of liposomes
containing a vesicle-forming lipid derivatized with a hydrophilic polymer, and
having sizes
ranging between 0.07 and 0.2 microns, to deliver therapeutic agents to sites
of infection is
described in published PCT patent application WO 93/19738. In accordance with
the present
invention, the anti-infective agent of choice is entrapped within a liposome
having a membrane
according to the present invention, and the resulting liposomal formulation is
administered
parenterally to a subject, preferably by intravenous administration. If
desired, localized
hyperthermia may be induced at the site of infection to cause the preferential
release of liposomal
contents at that site.
Upon administration, the liposomes are allowed time to reach the site of
disease.
Preferably, where liposome are administered to the bloodstream time is allowed
for liposomes to
extravasate into target tissues, e.g. tumors, inflamed tissues, infected
tissues. The thermosensitive
liposomes of the invention allow extended stability such that extended
circulation times and drug
retention are obtained. The invention thus comprises allowing an extended
period of time before
administering hyperthermic treatment to a tissue, which has heretofore not
been possible.
The invention thus comprises administering a mild hyperthermic treatment to a
subject at
least about 4, 6, 8, 12, or 18 hours, or more preferably at least about 24,
36, or 48 hours following
26
CA 02361946 2001-11-13
the administration of the liposomes to the subject. The hyperthermic treatment
is preferably
administered locally, such as to a disease site, so as to provide a localized
release of active agent
from liposomes.
Hyperthermia can be administered by any suitable method. Several methods and
apparati
are known and available. Preferably a minimally invasive RF, microwave, or
ultrasound based
hyperthermia delivery system is used to administer the hyperthermic treatment.
The
hyperthermic treatment is thus preferably minimally invasive and targeted,
capable for example
of targeting large tumor masses or large-volume infected or arthritic tissue
or other diseased
tissue deep within the body. The hyperthermia delivery system thus produces
heat which
activates thermosensitive liposomes and releases drugs in targeted tissue in
accordance with the
invention. Adaptive phased array systems are available, further described in
U.S. Patent No.
5,510,888, Fenn, et al, Int J Hyperthermia 15 (1): 45-61 (1999); and Gavrilov
et al, Int J
Hyperthermia 15(6): 495-507 ( 1999), the disclosures of which are incorporated
herein by
reference, and are available from Celsion Corp (Maryland). Other hyperthermia
delivery devices
include a surface temperature controlled microwave ring radiator embedded in
an contoured
epoxy plaque base, which allowed circulation of water inside the plaque to
cool the tissue surface
and provide deeper heating field penetration for tumor treatment, described in
Huang et al
(Cancer Res. 54: 5135-5143 (1994) the disclosure of which is incorporated
herein by reference).
Although minimally invasive systems which allow specific localization of heat
at tissues deep
within the body are preferred, other less sophisticated or surface based
heating devices can also
be used. Such systems are particularly useful for animal studies. For example,
a Positive
Temperature Coefficient Thermistor (PTC) heater, (Tokyo Denki Kagaku, Tokyo)
can be used on
the tumor surface (Katsumi et al, J. Pharm. Sciences 80(6): 522-525 (1991),
incorporated herein
by reference). Alternatively, for testing, an animal can be treated in a water
bath such as used to
heat implanted leg or flank tumors. In another example, a gas can be used as a
hyperthermic
potentiator by incorporating it in a liposome for use with ultrasound imaging
devices. Devices
specially designed for administering ultrasonic hyperthermia are described
U.S. Pat. Nos.
4,620,546, 4,658,828, and 4,586,512, the disclosures of which are incorporated
herein by
reference.
Hyperthermia delivery apparati can thus be used to heat a target site where
release of
liposome contents is to be stimulated. For the treatment of solid tumors, for
example, an
apparatus is used to administer hyperthermia to the solid tumor, thereby
causing the release of
drug at or near the tumor site. Similarly, for the treatment of inflammatory
and infectious disease,
27
CA 02361946 2001-11-13
hyperthermia will be focused on one or more sites of inflammation or
infection. Hyperthermia
dosage and intensity required to release liposome contents can be readily
determined by the
skilled person. Commercially available apparati permit for example the
targeting of tumors in the
liver, breast, rectum, cervix, pancreas, lung, and other areas deep within the
torso. Hyperthermia
can also be applied to the target site prior to administration of the
therapeutic agent, followed by a
second application of heat after at least 4 hours post administration.
Assessment of hyperthermia treatment
The treatment methods of the invention can readily be compared to conventional
methods. Commonly used studies examine tumor drug uptake and tumor growth
delay. For each
of tumor drug uptake and tumor growth delay, two ratios can be calculated:
1 ) (HT + Lip)/(Lip), comparing the endpoint reached when treated with
thermosensitive
liposomes combined with hyperthermia treatment (HT + Lip) to liposomes without
hyperthermia
(Lip); and
2) (HT + Lip)/(HT + drug), comparing the endpoint reached when treated with
hyperthermia and thermosensitive liposomes to that of hyperthermia and free
drug.
For drug uptake studies, the endpoint used to calculate the therapeutic ratios
are the tumor
drug levels achieved at the end of a study. The endpoint used to calculate the
therapeutic ratios
for the tumor growth delay studies can be either the number of days to reach a
predetermined
tumor volume or the tumor volumes reached at the end of the study. For tumor
growth delay
studies, liposomes can be administered to a mouse via tail vein, and flank or
leg tumors are heated
in a water bath. Tumor volumes can be followed over time until a certain tumor
size is attained or
time point is reached (Maekawa et al, Cancer Treatment Reports 71:1053-1059
(1987); and
Nishimura et al, Radiation Res. 122:161-167 (1990), the disclosures of which
are incorporated
herein by reference).
Tumor models are well known in the art, and any suitable tumor type of test
animal can
be used. Preferably mice are used. Commonly used tumors include C-26 colon
carcinoma, J-
6456 lymphoma, B16F10 melanoma, Meth A fibrosarcoma, RIF-1, Walker
carcinosarcoma 256
in liver and rous sarcoma virus induced glioma.
Assessment of circulation longevity
28
CA 02361946 2001-11-13
The liposomes according to the invention result in enhanced longevity
(circulation time).
Preferably, a liposome or lipid carrier of this invention will be made such
that the lipid dose
remaining in the bloodstream of an animal at one or more selected time points
at least 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 and retention of drug in the bloodstream as
shown in Figure 1.
This observation is contrary to conventional wisdom concerning the ability of
thermosensitive
liposomes to display extended circulation longevity and drug retention (see
the review article:
Dewirst et al. (1999) Int. J. Hyperthermia 15(S): 345-370). This increased
stability was due to the
maintenance of the body temperature of the mice below the Tm of the liposomes.
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, which is comparable to or better than the
circulation longevity
in a mammal of a 'reference' liposome containing substantially the same lipids
and in the same
proportions but with at least 20 mol % cholesterol. These cholesterol-
containing 'reference'
liposome may contain a hydrophilic polymer-conjugated 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.
For liposome circulation longevity, the data obtained from a model animal
system can be
reasonably extrapolated to humans and veterinary animals of interest.
Liposomes 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.
Assessment of drug retention: drug: lipid ratio
29
CA 02361946 2001-11-13
The ability of the liposomes to retain an amphipathic anti-tumor drug while
circulating in
the bloodstream over the extended periods after administration are also an
important factor for
liposomes which are to reach and enter a site such as a distant solid tumor.
An extended period
can be for example at least about 4, 6, 12, 18, 24, 36 or 48 hour period, that
is the time allowed in
the present method between liposome administration and stimulated release of a
therapeutic
agent. For example, a therapeutic agent can be encapsulated into DPPC-PEG
liposomes of the
invention, and the drug:lipid ratio can be determined at one or more specified
time points after
administration of the liposomes to a mammal, preferably a mouse. Blood is
removed from the
mammal at fixed time intervals such as 1, 2, 3, 4, 8, 12, 18, 24 or 48 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 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.
A liposome in accordance with the invention preferably displays a drug:lipid
ratio at a
fixed time point after administration to 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 containing
substantially the
same lipids and in the same proportions but with at least 20 mol %
cholesterol.
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 [3H]-
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 C02
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. [3H]- and [14C]-CHE labels are
available from
NEN/Dupont.
It will be appreciated however that any 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
CA 02361946 2001-11-13
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. As used in the context of determining a drug:lipid
ratio, the term drug
refers generally to the active agent encapsulated in the liposome
administered.
Assessment of extravasatioh into Tumors
As mentioned, the high stability/high drug retention liposomes of the
invention allow
methods of treatment which maximize benefit from liposome extravasation into a
site of disease,
e.g. tumor, infectious disease or inflammation. Liposomes can extravasate
through the
endothelial cell barrier and underlying basement membrane separating a
capillary from for
example the tumor cells supplied by the capillary. This feature is optimized
in liposomes with
sizes between about 50nm and 200nm in diameter, although liposomes with
smaller size are also
expected to extravasate. However, in the examples provided herein, liposomes
have sizes of
about 80 nm to 140nm are expected to allow a su~ciently large drug-carrying
capacity.
Previous studies with cholesterol-containing non-thermosensitive liposomes
have
indicated that delivery to the tumor offers advantages in drug targeting (see
for example U.S.
Patent No. 5,510,888). A study which can be used to demonstrate the ability of
preferred
liposomes to reach a target site involves inoculating mice subcutaneously with
the J-6456
lymphoma, which forms a solid tumor mass of about 1 cm3 after one to two
weeks. The animals
are then injected either with free drug or drug loaded into the liposomes of
the invention,
preferably substantially cholesterol-free PEG-liposomes. The tissue
distribution (heart, muscle,
and tumor) of the drug can then assayed at 4, 8, 12, 18, 24, 36 and/or 48
hours after drug
administration, as shown in Example 4 or according to conventional techniques
known in the art.
Drug levels accumulated in a target tissue such as a tumor can thereby be
compared to drug levels
in other tissues, and can serve to demonstrate that the liposomes of the
invention result in drug
accumulating at higher levels in the tumor site as compared to heart, muscle,
liver, etc. than free
drug or drug encapsulated in liposomes containing substantially the same
lipids and in the same
proportions as the thermosensitive liposomes of the invention but comprising
at least 20mo1%
cholesterol.
Preferably the liposomes of the invention show increased drug accumulation
into the
target tissue (e.g. tumor, site of inflammation or site of infection).
Preferably, the target tissue
contains at least 2, 4, 8, 10, 20, 50 or 100 times more drug compared with
healthy muscle and at
31
CA 02361946 2001-11-13
least 2, 4, 6, 8, 10, 20 or 100 times the amount in heart following
administration of the liposome
of the invention (for example, at 12, 18, 24 or 48 hours post administration).
To demonstrate that increase in drug accumulation are due to the entry of
intact liposomes
into the extravascular region of a tumor, tumor tissue can be separated into
cellular and
supernatant (intercellular fluid) fractions, and the presence of liposome-
associated and free drug
in both fractions can be determined. To assay liposome-associated drug, the
supernatant is passed
through a gel filtration column to remove free drug (Gabizon, A et al, (1989)
J. Natl. Cancer Inst.
81, 1484-1488), and the drug remaining in the supernatant is assayed. Further
demonstration of
liposome extravasation into tumor cells can be obtained by direct microscopic
observation of
liposome distribution in normal liver tissue and in solid tumors, at a time
point, e.g. 12, 18, 24, 48
hours, after IV injection of liposomes of the invention.
Assessment of tumor Localization and treatment e~cacy
The liposomes of the invention provide means for achieving improved
localization of an
anti-tumor, anti-inflammatory or anti-infective agents specifically in a
target tissue or region by
virtue of the extended lifetime of the liposomes in the bloodstream and a
liposome size which
allows both extravasation into tumors, a relatively high drug carrying
capacity and minimal
leakage of the entrapped drug during the time required for the liposomes to
distribute to and enter
the tumor (the first 24-48 hours following injection). In a preferred
embodiment, the liposomes
thus provide an effective method for localizing a compound selectively to a
solid tumor, by
entrapping the compound in such liposomes and injecting the liposomes IV into
a subject. In this
case, for an IV injected liposome (and its entrapped anti-tumor drug) to reach
the tumor site it
must leave the bloodstream and enter the tumor. In one embodiment, the method
is used for tumor
treatment by localizing an anti-tumor drug selectively in the tumor. The anti-
tumor drug which
may be used is any compound, including the ones listed herein, which can be
stably entrapped in
liposomes at a suitable loading factor and administered at a therapeutically
effective dose
(indicated below in parentheses after each compound).
Studies to compare treatment efficacy of delayed release methods using the
liposomes of
the invention to conventional methods can be carried out in animal models as
described above.
Alternatively, studies may also measure endpoints such as tumor growth or
survival. For
example, in one comparison animals can be treated with free drug or drug
entrapped in liposomes
of the invention. In another comparison, animals can be treated with drug
entrapped in liposomes
32
CA 02361946 2001-11-13
of the invention with the delivery of hyperthermia immediately after liposome
administration (e.g.
5, 10, 30 minutes post administration), or at extended times as in the
"delayed-release" methods
of the invention (e.g. at least 4, 8, 12, 18, 24, 36 or 48 hours post
administration). The study may
assess for example percent survivors over a period of a certain number of days
(for example a
100-day period) or tumor growth delay following tumor implantation.
Since reduced toxicity has been observed in model animal systems and in a
clinical
setting in tumor treatment by doxorubicin entrapped in conventional liposomes
(as reported, for
example, in U.S. Pat. No. 4,797,285), studies can be carried out to determined
the degree of
toxicity protection provided in the treatment method of the present invention.
In one example,
animals are injected intravenously with increasing doses of a drug in free
form or entrapped in
conventional or thermosensitive liposomes of the invention, and the maximum
tolerated dose
(MTD) for the various drug formulations is determined. Preferably, entrapment
in liposomes of
the invention provides an MTD for the drug which is comparable or better than
that seen in
liposomes containing substantially the same lipids and in the same proportions
but containing at
least 20mo1% cholesterol. Preferably, entrapment in liposomes of the invention
provides an MTD
for the drug which is at least 2, 4, 10, 20 or 100 times better than with free
drug.
It will be appreciated that the ability to localize a compound selectively in
a tumor, by
liposome extravasation, can also be exploited for improved targeting of an
imaging agent to a
tumor, such as for tumor diagnosis. Here the imaging agent, typically a
radioisotope in chelated
form, or a paramagnetic molecule, is entrapped in liposomes, which are then
administered IV to
the subject being examined. After a selected period, typically 12 to 24 or 24
to 48 hours, the
subject is then monitored, for example by gamma scintillation radiography in
the case of the
radioisotope, or by nuclear magnetic resonance (NMR) in the case of the
paramagnetic agent, to
detect regions of local uptake of the imaging agent.
Also, as noted above, it is anticipated that long circulating thermosensitive
liposomes
would be useful for delivery of anti-infective drugs to regions of infections.
Sites of infection, like
tumors, often exhibit compromised leaky endothelial barriers--as evidenced by
the fact that
edema (fluid uptake from the bloodstream) is quite often found at these sites.
It is expected that
liposomes containing antibiotics (such as aminoglycosides, cephalosporins, and
beta lactams)
would improve drug localization at sites of infection, thereby improving the
therapeutic index of
such agents--particularly ones which exhibit dose-related toxicities, such as
the aminoglycosides.
33
CA 02361946 2001-11-13
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 of 3H-
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 moles 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 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.
34
CA 02361946 2001-11-13
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-PEG2000 (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-PEGZ000
(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 1 h 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-PEG2000 (90:4 mol%) liposomes via the tail vein. Mice
were
subsequently placed 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-
PEG2000 (90:4 mol%) liposomes via the tail vein. Mice were terminated after l
Omin and 1h. Blood
was collected by cardiac puncture into EDTA-coated microtainers.
CA 02361946 2001-11-13
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 H2S04. 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, ~,em: 550
nm).
Doxorubicin-containing DPPC:DSPE-PEG2000 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-PEG2000 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
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
36
CA 02361946 2001-11-13
(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 the
hyperthermia 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 after hyperthermia,
blood samples
are quantitated for levels of doxorubicin and lipid as in Example 3.
Example 5
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 3rd and 20th 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 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.
37
