U.S. patent number 5,595,756 [Application Number 08/172,140] was granted by the patent office on 1997-01-21 for liposomal compositions for enhanced retention of bioactive agents.
This patent grant is currently assigned to Inex Pharmaceuticals Corporation, University of British of Columbia. Invention is credited to Marcel B. Bally, Nancy L. Boman, Pieter R. Cullis, Lawrence D. Mayer.
United States Patent |
5,595,756 |
Bally , et al. |
January 21, 1997 |
Liposomal compositions for enhanced retention of bioactive
agents
Abstract
Liposomal compositions encapsulating bioactive agents and having
improved circulation longevity of the agents are disclosed. Such
liposomes combine a low pH of the solution in which a bioactive
agent is entrapped and a sugar-modified lipid or an amine-bearing
lipid, the combination of which enhances the retention of the
encapsulated bioactive agent and thereby promotes circulation
longevity. The present invention also discloses methods of making
and using such compositions.
Inventors: |
Bally; Marcel B. (Bowen Island,
CA), Boman; Nancy L. (Richmond, CA),
Cullis; Pieter R. (Vancouver, CA), Mayer; Lawrence
D. (North Vancouver, CA) |
Assignee: |
Inex Pharmaceuticals
Corporation (Vancouver, CA)
University of British of Columbia (Vancouver,
CA)
|
Family
ID: |
22626538 |
Appl.
No.: |
08/172,140 |
Filed: |
December 22, 1993 |
Current U.S.
Class: |
424/450; 264/4.1;
264/4.3 |
Current CPC
Class: |
A61K
9/1272 (20130101) |
Current International
Class: |
A61K
9/127 (20060101); A61K 009/127 () |
Field of
Search: |
;424/450 ;428/402.2
;264/4.1,4.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Allen, T. M., et al. (1987) "Large unilamellar liposomes with low
uptake into the reticuloendothelial system," FEBS Lett,
223(1):42-46. .
Allen, T. M., et al. (1989) "Liposomes with prolonged circulation
times: factors affecting uptake by reticuloendothelial and other
tissues," Biochim Biophys Acta, 981:27-35. .
Allen. T. M., et al. (1991) "Liposomes containing synthetic lipid
derivative of poly(ethlyene glycol) show prolonged circulation
half-lives in vivo," Biochim Biophys Acta, 1066:29-36. .
Allen, T. M., et al. (1991) "Uptake of liposomes by cultured mouse
bone marrow macrophages: influence of liposome composition and
size," Biochim Biophys Acta, 1061: 56-63. .
Allen, T. M., et al. (1992) "Stealth Liposomes: an improved
sutstained release system of 1-B-D-arabinofuranosylcytosine,"
Cancer Res, 52:2431-2439. .
Bally, M. B., et al. (1990) "Liposomes with entrapped doxorubicin
exhibit extended blood residence times," Biochim. Biophys. Acta,
1023:133-139. .
Boman, N. L., et al. (1993) "Optimization of the retention
properties of vincristine in liposomal systems," Biochim Biophys
Acta 1152:253-258. .
Boman, N. L., et al. 1994 "Liposomal vincristine which exhibits
increased drug retention and increased circulation longevity cures
mice bearing P388 tumors," Cancer Res., 54:2830-2833. .
Carter, S. K., et al. (1976) "Plant products in cancer
chemotherapy," Cancer Treat Rep. 60(8):1141-1155. .
Chakrabarti, A. C., et al. (1992) "Uptake of basic amino acids and
peptides into liposomes in response to transmembrane pH
gradients,"Biophys. J., 61:228-234. .
Chonn, A., et al. (1992) "Association of blood proteins with large
unilamellar liposomes in vivo: relation to circulation lifetimes,"
J. Biol Chem, 267(26):18759-18765. .
Derksen, J. T. P., et al. (1987) "Processing of different liposome
markers after in vitro uptake of immunoglobulin-coated liposomes by
rat liver macrophages," Biochim. Biophys. Acta, 931:33-40. .
Fichtner, I, et al. (1981) "Therapeutic evaluation of
liposome-encapsulated daunoblastin in murine tumor models,"
Neoplasma (Bratisl.), 28:141-149. .
Forssen, E. A., et al. (1981) "Use of anionic liposomes for the
reduction of chronic doxorubicin-induced cardiotoxicity," Proc.
Natl. Acad. Sci USA 78:1873-1877. .
Gabizon, A., et al. (1982) "Enhancement of adriamycin delivery to
liver metastatic cells with increased tumoricidal effect using
liposomes as drug carriers," Cancer Res. 43:4730-4735. .
Gabizon, A., et al. (1982) "Liposomes as in vivo carriers of
adriamycin: reduced cardiac uptake and preserved antitumor activity
in mice," Cancer Res. 42:4734-4739. .
Gabizon, A., et al. (1988) "Liposome formulations with prolonged
circulation time in blood and enhanced uptake by tumors," Proc.
Natl. Acad. Sci USA, 85:6949-6953. .
Gregoriadis, G., et al. (1975) "Treatment of tumour bearing mice
with liposome-entrapped actinomycin D prolongs their survival,"
Res. Commun. Chem. Pathol Pharmacol., 10(2):351-362. .
Herman, E. H., et al. (1983) "Prevention of chronic doxorubicin
cardiotoxicity in beagles by liposomal encapsulation," Cancer Res.,
43:5427-5432. .
Hunt, C. A., et al. (1979) "Retention of cytosine arabinoside in
mouse lung following intravenous administration in liposomes of
different size," Drug Metab. Dispos., 7:124-128. .
Jackson, D. V. Jr., et al. (1979) "Cytotoxic thresholds of
vincristine in a murine and a human leukemia cell line in vitro,"
Cancer Res., 39:4346-4349. .
Jackson, D. V., et al. (1986) "Moderate-dose vincristine infusion
in refractory breast cancer," Am. J. of Clin Onc., 9(5):376-378.
.
Jackson, D. V. Jr., et al. (1986) "Vincristine infusion in
refractory gynecologic malignancies," Gynecol. Onc., 25(2):212-216.
.
Klibanov, A. L., et al. (1990) "Amphipathic polyethyleneglycols
effectively prolong the circulation time of liposomes," FEBS Lett,
268(1):235-237. .
Klibanov, A. L., et al. (1991) "Activity of amphipathic
poly(ethylene glycol) 5000 to prolong the circulation time of
lipsomes depends on the liposome size and is unfavorable for
immunoliposome binding to target," Biochim Biophys Acta
1062:142-148. .
Kobayashi, T., et al. (1977) "Enhancement of anti-tumor activity of
1-.beta.-D-arabinofuranosylcytosine by encapsulation of liposomes,"
Int. J. Cancer, 20:581-587. .
Liu, D., et al. (1990) "pH-sensitive, plasma-stable liposomes with
relatively prolonged residence in circulation," Biochim Biophys
Acta 1022:348-354. .
Mayer, L. D., et al. (1989) "Influence of vesicle size, lipid
composition, and drug-to-lipid ratio on the biological activity of
liposomal doxorubicin in mice," Canc. Res., 49:5922-5930. .
Mayer, L. D., et al. (1990) "Strategies for optimizing liposomal
doxorubicin," J. of Liposome Res., 1(4):463-480. .
Mayer, L. D., et al. (1990) "Liposomal vincristine preparations
which exhibit decreased drug toxicity and increased activity
against murine L1210 and P388 tumors," Cancer Res. 50:575-579.
.
Mayer, L. D., et al. (1990) "Comparison of free and liposome
encapsulated doxorubicin tumor drug uptake and antitumor efficacy
in the SC115 murine mammary tumor," Cancer Lett., 53:183-190. .
Mayer, L. D., et al. (1990) "Characterization of liposomal systems
containing doxorubicin entapped in response to pH gradients,"
Biochim Biophys. Acta., 1025:143-151. .
Mayer, L. D., et al. (1993) "Identification of vesicle properties
that enhance the antitumour activity of liposomal vincristine
against murine L1210 leukemia," Cancer Chemother Pharmacol,
33:17-24. .
Mayhew, E., et al. (1985) "The use of liposomes as carriers of
therapeutic agents," Prog. Clin. Biol. Res. 172B:301-310. .
Mui, B. L. S., et al. (1993) "Osmotic properties of large
unilamerllar vesicles prepared by extrusion," Biophys. J.,
64:443-453. .
Olson, F., et al. (1979) "Preparation of liposomes of defined size
distribution by extrusion through polycarbonate membranes,"
Biochim. Biophys. Acta, 557:9-23. .
Owellen, R. J., et al. (1976) "Inhibition of tubulin-microtubule
polymerization by drugs of the vinca alkaloid class," Cancer Res.,
36:1499-1502. .
Owellen, R. J., et al. (1972) "The binding of vincristine,
vinblastine, and colchicine to tubulin," Biochem Biophys Res.
Commun. 47(4):685-691. .
Rahman, A., et al. (1982) "Doxorubicin-induced chronic
cardiotoxicity and its protection by liposomal administration,"
Cancer Res., 42:1817-1825. .
Senior, J., et al. (1991) "Influence of surface hydrophilicity of
liposomes on their interaction with plasma protein and clearance
from the circulation: studies with poly(ethylene glycol)-coated
vesicles," Biochim Biophys Acta, 1062:77-82. .
Sieber, S. M., et al. (1976) "Pharmacology of antitumor agents from
higher plants," Cancer Treat Rep., 60:1127-1139. .
Woo, S. Y., et al. (1983) "Liposomal methotrexate in the treatment
of murine L1210 leukemia," Cancer Drug Delivery, 1(1): 59-62. .
Chonn, A., et al. (1992) "Ganglioside G.sub.ml and hydrophilic
polymers increase liposome circulation times by inhibiting the
association of blood proteins," J. of Liposome Research,
2(3):397-410. .
Parr, M. J., et al. (1993) "The presence of G.sub.ml in liposomes
with entrapped doxorubicin does not prevent RES blockade,"
Biochimica et Biophysica Acta, 1168:249-252..
|
Primary Examiner: Kishore; Gollamudi S.
Attorney, Agent or Firm: Townsend and Townsend and Crew
Claims
We claim:
1. A composition comprising liposomes having encapsulated therein
both a bioactive agent and a buffered solution having a pH of 2 to
3, said liposomes containing a sugar-modified lipid which is a
ganglioside and cerebroside in an amount of about 10 mol
percent.
2. A composition according to claim 1 wherein said solution is pH
2.
3. A composition according to claim 1 wherein said sugar-modified
lipid is a ganglioside selected from the group consisting of
G.sub.M1,G.sub.M2 and G.sub.M3.
4. A composition according to claim 3 wherein said bioactive agent
is an anticancer agent.
5. A composition according to claim 3 wherein said solution is
buffered with citrate, maleate or glutamate.
6. A composition according to claim 3, further comprising a
pharmaceutically acceptable carrier or diluent.
7. A composition comprising liposomes produced by
(a) hydrating a lipid film in a buffered first solution having a pH
of 2 to 3 to yield liposomes, said lipid film containing a
sugar-modified lipid which is a ganglioside and cerebroside in an
amount of about 10 mol percent;
(b) contacting said liposomes with a second solution, said second
solution containing a bioactive agent to provide a liposomal
bioactive agent mixture;
(c) increasing the pH of said mixture in step (b) to a pH not above
the pK.sub.a of the bioactive agent to provide a liposomal mixture
having a pH differential; and
(d) incubating said mixture having a pH differential under
conditions and for a time sufficient to promote entrapping of said
bioactive agent by said liposomes, thereby producing liposomes that
encapsulate a bioactive agent and that contain a sugar-modified
lipid ganglioside and cerebroside in an amount of about 10 mol
percent.
8. A composition according to claim 7 in combination with a
pharmaceutically acceptable carrier or diluent.
9. A method for treating cancer in a warm-blooded animal,
comprising administering a therapeutically effective amount of a
composition according to claim 4 wherein said bioactive agent is an
anticancer agent.
10. A method for treating cancer in a warm-blooded animal,
comprising administering a therapeutically effective amount of a
composition according to claim 8 wherein said bioactive agent is an
anticancer agent.
Description
TECHNICAL FIELD
The present invention is generally directed toward liposomes having
improved retention of bioactive agents, and to methods for making
and using these compositions. This invention is more particularly
related to the enhanced retention of bioactive agents by liposomes
which include a sugar-modified lipid or an amine-bearing lipid and
which encapsulate a solution with a low pH.
BACKGROUND OF THE INVENTION
Despite enormous investments of financial and human resources, no
cure exists for a variety of diseases. For example, cancer remains
one of the major causes of death. A number of bioactive agents have
been found, to varying degrees, to be effective against tumor
cells. However, the clinical use of such antitumor agents has been
highly compromised because of treatment-limiting toxicities.
In order to decrease drug-induced toxic side effects, antitumor
agents have been encapsulated in liposomes. Liposomes are
completely closed lipid bilayer membranes containing an entrapped
aqueous volume. Liposomes may be unilamellar vesicles (possessing a
single membrane bilayer) or multilamellar vesicles (onion-like
structures characterized by multiple membrane bilayers, each
separated from the next by an aqueous layer). The bilayer is
composed of two lipid monolayers having a hydrophobic "tail" region
and a hydrophilic "head" region. The structure of the membrane
bilayer is such that the hydrophobic (non-polar) "tails" of the
lipid monolayers orient toward the center of the bilayer while the
hydrophilic (polar) "heads" orient toward the aqueous phase. The
current state of the art is such that liposomes may be reproducibly
prepared using a number of techniques.
Liposome encapsulation of various antitumor agents has been shown
to decrease drug-induced toxic side effects while maintaining or,
in some instances, increasing antitumor activity. Reduction of
toxicity results from the ability of liposomes to decrease drug
exposure, and subsequent damage, to susceptible tissues. The
mechanism of the antitumor activity of entrapped drugs is less well
understood, but may result from the capacity of liposomes to slowly
release encapsulated drug into the circulation or alternatively
passive targeting of liposomes and their contents to tumor sites. A
problem, however, with the encapsulation of antitumor agents is
that many of these drugs have been found to be rapidly released
from liposomes after encapsulation.
An example of an antitumor agent is vincristine, which is a member
of the Vinca alkaloid class and is derived from the periwinkle
plant. It is an important anticancer drug in that it displays
effectiveness against a wide variety of neoplasms including both
the Hodgkin's and non-Hodgkin's lymphomas, acute lymphoblastic
leukemia, embryonal rhabdomyosarcoma, neuroblastoma, breast
carcinoma, and Wilm's tumor. It is a cell-cycle specific drug which
arrests cell growth exclusively during metaphase by attaching to
the growing end of microtubules and terminating their assembly. For
this reason, it is advantageous to expose neoplastic cells to the
drug for prolonged periods of time. This effect has been
demonstrated in vitro by Jackson and Bender (Cancer Res. 39:4346,
1979), and has been confirmed using the murine L1210 leukemic cell
line (Mayer etal., Cancer Chemother. Pharmacol. 33:17-24, 1993).
The importance of this relationship in the treatment of human
malignancies is supported by clinical trials where patients
refractory to bolus vincristine therapy exhibited increased
response rates when the drug was administered as a 5-day
infusion.
Liposomal formulations of vincristine have been shown to exhibit
reduced toxicity and enhanced efficacy compared to free drug. The
antitumor activity of vincristine appeared to be dependent on the
circulation lifetime of the encapsulated drug. Circulation
longevity (of liposomally entrapped bioactive agent) in turn is
dependent, in part, on the rate of agent release from liposomes in
the blood. Therefore, enhancement of the retention of a bioactive
agent in liposomes is desirable as it will increase the circulation
lifetime of the encapsulated agent, thereby improving its
therapeutic activity.
Thus, there is a need in the art for liposomal bioactive agent
preparations with improved circulation longevity. The present
invention fulfills this need, and further provides other related
advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides a variety of
liposomal bioactive agent compositions, and methods of making and
using such compositions. In one aspect, the present invention
provides compositions comprising liposomes having encapsulated
therein both a bioactive agent and a buffered solution having a pH
of 2 to 3, the liposomes containing a sugar-modified lipid or an
amine-bearing lipid. In one embodiment, the composition
encapsulates a solution with a pH of 2. In another embodiment, the
lipid is selected from the group consisting of G.sub.M1,
stearylamine, sphingosine, and the amino lipids according to FIG.
6. In another embodiment, the bioactive agent is an anticancer
agent.
In another aspect, the present invention provides a method for the
production of liposomes encapsulating a bioactive agent, comprising
the steps of:
(a) hydrating a lipid film in a buffered first solution having a pH
of 2 to 3 to yield liposomes, the lipid film containing a
sugar-modified lipid or an amine-bearing lipid;
(b) contacting the liposomes with a second solution, said second
solution containing a bioactive agent;
(c) increasing the pH of the mixture from step (b) to a pH not
above the pK.sub.a of the bioactive agent; and
(d) incubating the mixture under conditions and for a time
sufficient to promote entrapping of the bioactive agent by the
liposome, thereby producing liposomes that encapsulate a bioactive
agent and that contain a sugar-modified lipid or an amine-bearing
lipid.
In one embodiment, the first solution has a pH of 2. In another
embodiment, the lipid is selected from the group consisting of
G.sub.M1, stearylamine, sphingosine, and the amino lipids according
to FIG. 6. In another embodiment, the bioactive agent is an
anticancer agent.
In another aspect, the liposomal bioactive agent compositions of
the present invention are combined with a pharmaceutically
acceptable carrier or diluent.
In yet another aspect of the present invention, methods are
provided for treating diseases in a warm-blooded animal. In one
embodiment, the method comprises administering a therapeutically
effective amount of an above-described composition for treating
cancer. In another embodiment, the method comprises treating cancer
by administering a therapeutically effective amount of a
composition produced by the above-described method.
These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-C graphically illustrate the influence of G.sub.M1
incorporation and entrapped vincristine on the plasma clearance
(panel A), liver uptake (panel B), and spleen uptake (panel C) of
100 nm DSPC/Chol liposomes. Liposomes were all administered i.v. at
a lipid dose of 20 mg/kg. Vincristine was encapsulated at a
drug-to-lipid ratio of 0.1:1 (wt:wt) using the transmembrane pH
gradient loading technique with liposomes prepared in 300 mM
citrate butler pH 4. G.sub.M1 was incorporated at a level of 10 mol
% in DSPC/Chol liposomes (45 mol % cholesterol). Liposomal lipid
was measured using the non-exchange, non-metabolizable lipid marker
.sup.14 C-cholesteryl hexadecyl ether. Lipid levels were determined
for drug free DSPC/Chol liposomes (O), DSPC/Chol liposomal
vincristine (.circle-solid.), and G.sub.M1 /DSPC/Chol liposomal
vincristine (.box-solid.). Error bars represent standard deviations
from at least four mice.
FIGS. 2A-B graphically illustrate the plasma clearance of
vincristine (panel A) and associated drug-to-lipid ratios (panel B)
as determined following i.v. administration in mice of liposomal
vincristine prepared using: DSPC/Chol pH 4 (.circle-solid.),
G.sub.M1 /DSPC/Chol pH 4 (.box-solid.), DSPC/Chol pH 2
(.tangle-solidup.) and G.sub.M1 /DSPC/Chol pH 2 (.diamond-solid.)
liposomes. Vincristine was encapsulated at a drug-to-lipid ratio of
0.1:1 (wt:wt) and was measured using .sup.3 H vincristine as a
tracer. Error bars represent standard deviations from at least four
mice.
FIGS. 3A-C graphically illustrate the drug clearance (panel A),
lipid clearance (panel B) and drug-to-lipid (panel C) ratios for
DSPC/Chol/Stearylamine (SA) when injected into BDF1 mice at a
vincristine dose of 2 mg/kg and a lipid dose of 20 mg/kg:
DSPC/Chol/SA prepared at pH 2 (O), and DSPC/Chol/SA prepared at pH
4 (.circle-solid.).
FIGS. 4A-C graphically illustrate the drug clearance (panel A),
lipid clearance (panel B), and drug-to-lipid (panel C) ratios for
DSPC/Chol/Aminolipid-1 (AL-1 ) when injected into BDF 1 mice at a
vincristine dose of 2 mg/kg and a lipid dose of 20 mg/kg:
DSPC/Chol/AL-1 prepared at pH 2 (O), and DSPC/Chol/AL-1 prepared at
pH 4 (.circle-solid.).
FIGS. 5A-C graphically illustrate the drug clearance (panel A),
lipid clearance (panel B), and drug-to-lipid (panel C) ratios for
DSPC/Chol/Sphingosine (sphingo) when injected into BDF1 mice at a
vincristine dose of 2 mg/kg and a lipid dose of 20 mg/kg:
DSPC/Chol/sphingo prepared at pH 2 (O), and DSPC/Chol/sphingo
prepared at pH 4 (.circle-solid.).
FIG. 6 depicts the chemical structures of the amino lipids
designated AL-1, AL-2, AL-3, AL-4, AL-5 and AL-6.
DESCRIPTION OF THE INVENTION
As noted above, the present invention provides liposomal bioactive
agent compositions, and methods of making and using such
compositions. An advantage of the compositions of the present
invention is that the retention of a bioactive agent in the
liposome is enhanced and, thereby, the circulation lifetime of the
bioactive agent is increased. In addition, the therapeutic activity
of such compositions is significantly improved.
The compositions of the present invention comprise liposomes which
encapsulate a bioactive agent in a solution having a low pH and
which contain a sugar-modified lipid or an amine-bearing lipid. The
disclosure of the present invention shows, unexpectedly, that
lowering the pH of the solution in which a bioactive agent is
entrapped within a liposome and including in the liposomal membrane
a sugar-modified lipid or an amine-bearing lipid synergistically
combine to significantly increase the bioactive agent concentration
in the plasma.
Compositions of the present invention may be formed in a variety of
ways, including by active or passive loading methodologies. For
example, bioactive agents may be encapsulated using a transmembrane
pH gradient loading technique. General methods for loading
liposomes with bioactive agents through the use of a transmembrane
potential across the bilayers of the liposomes are well known to
those in the art (e.g., U.S. Pat. Nos. 5,171,578 and 5,077,056).
However, as disclosed within the present invention, in the
embodiment where a transmembrane pH gradient is imposed across the
liposome membrane, the methodology is modified for lower internal
liposomal pH.
In brief, for example, lipids are first dissolved in an organic
solvent, such as ethanol, and gently heated (e.g., 60.degree. C.
for 30 minutes). The lipid components used in forming the liposomes
may be selected from a variety of vesicle-forming lipids, typically
including phospholipids and sterols (e.g., U.S. Pat. Nos. 5,059,421
and 5,100,662). Representative examples of lipids suitable for the
preparation of liposomes include cholesterol,
distearoylphosphatidylcholine, phosphatidylglycerol,
phosphatidylethanolamine, phosphatidylcholine, partially
hydrogenated phosphatidylcholine, dipalmitoylphosphatidyl glycerol,
dipalmityol-phosphatidylcholine, dioleylphosphatidylcholine, and
mixtures thereof. In addition, one or more lipids that contribute
to the retention of entrapped bioactive agents are included. Such
lipids include sugar-modified lipids and amine-bearing lipids.
Representative examples of sugar-modified lipids include
gangliosides, such as G.sub.M1, G.sub.M2 or G.sub.M3, and
derivatives thereof. Representative examples of amine-bearing
lipids include stearylamine, sphingosine, the amino lipids
according to FIG. 6, and derivatives thereof.
To the dissolved lipids, a pre-heated aqueous solution with a pH of
less than 4 is then added while vigorously mixing. For example, a
60.degree. C. solution containing 300 mM buffer is added, in a
ratio of 3 mL solution per 100 mg lipid, while vigorously
vortexing. Preferred buffers include citrate, maleate and
glutamate. Gitrate is particularly preferred. The pH of the
solution added to the lipids is less than 4, with a pH of about 2
to 3 preferred. A pH of about 2 is particularly preferred.
Following mixing, the resulting multilamellar vesicles ("MLVs") may
be heated (e.g., 60.degree. C. for an additional 30 min.) and
extruded through an extrusion device to convert the MLVs to
unilamellar liposome vesicles. The organic solvent used initially
to dissolve the lipids may be removed from the liposome preparation
by dialysis. The dialysis solution is a low pH solution identical
to that previously added to the dissolved lipids. The resulting
liposomes are substantially free of organic solvent and have an
interior pH of less than 4 (the exact pH dependent upon the pH of
the solution initially added to the dissolved lipids).
One or more bioactive agents may be entrappeal in the low pH
liposomes using transmembrane pH gradient loading. By raising the
pH of the solution external to the liposomes, a pH differential
will exist across the liposome bilayer. Thus, a transmembrane
potential is created across the liposome bilayer and a bioactive
agent is loaded into the liposomes by means of the transmembrane
potential. In brief, for example, low pH liposome vesicles and a
bioactive agent are mixed (e.g., to achieve an agent-to-lipid ratio
of about 0.1:1). The pH of the mixture (i.e., the pH external to
the liposomes) is generally raised to a pH approaching the pK.sub.a
of the agent (typically to neutrality or basic pH for many agents)
and then heated (e.g., 60.degree. C. for 10 min.) under conditions
and for a time sufficient to permit uptake of the bioactive agent
into the liposomes. For bioactive agents with amine groups capable
of being protonated, the pH is typically raised to about pH 7-9.
For example, for vincristine the pH is raised to pH 7.0-7.2 (e.g.,
using 0.5M Na.sub.2 HPO.sub.4). Alternatively, an agent may be
loaded passively into the low pH liposomes. Entrapment of a
bioactive agent may be determined using spectroscopic assays or
liquid scintillation counting (where radiolabeled), following
separation of liposomes from free (non-entrapped) bioactive agent
(e.g., by chromatography).
A wide variety of bioactive agents can be entrapped within the
liposomes according to the present invention. Such bioactive agents
include positively charged anticancer (antineoplastic) agents,
anti-malarial agents, calcium channel blockers, local anesthetics,
adrenergic antagonists, antiarrhythmics, cholinergic agents,
biogenic amines, antidepressants, antihistamines, antiprotozoan
agents, analgesics, and multiple drug resistance ("MDR")
inhibitors. Preferred anticancer agents include vincristine,
daunombicin, mitoxantrone, epimbicin, doxorubicin, vinblastine and
tamoxifen. Preferred anti-malarial agents include quinidine and
chloroquine. A preferred calcium channel blocker is verapamil.
Preferred local anesthetics include lidocaine, chlorpromazine,
prochlorperazine, trifluoperazine and dibucaine. Preferred
adrenergic antagonists include propranolol and timolol. A preferred
antiarrhythmic is quinidine. Preferred cholinergic agents include
pilocarpine, physostigmine and nicardapine. Preferred biogenic
amines include dopamine and serotonin. A preferred antidepressant
is imipramine. A preferred antihistamine is diphenhydramine. .A
preferred antiprotozoan agent is quinacrine. A preferred analgesic
is codeine. Preferred MDR inhibitors include prochlorperazine,
trifluoperazine, flupenthixol, tomoxifen and vindoline. It will be
evident to those of ordinary skill in the art that, although
certain agents are described as illustrative, numerous other agents
are also suitable within the liposome compositions of the present
invention.
Circulation longevity of liposomal bioactive agent compositions may
be assessed by plasma clearance studies. In brief, for example,
liposomes with or without loaded agent are injected into a tail
vein of a mouse. Typically, the liposomes will contain
radioactively labeled lipid (e.g., .sup.14 C-cholesteryl
hexadecylether), with or without radioactively labeled bioactive
agent. At various time points, blood is collected from
anaesthetized mice via cardiac puncture. The blood cells are
separated and the plasma assayed for liposomal lipid and/or
bioactive agent (e.g., using scintillation counting for
radiolabeled lipid or bioactive agent). The disclosure of the
present invention shows that incorporation into liposomes of a
sugar-modified lipid or an amine-bearing lipid in combination with
the use of an entrapped solution with a low pH results in an
unexpected improvement in bioactive agent circulation
lifetimes.
In a similar manner, biodistribution studies may be performed on
selected tissue/organs from injected mice by detecting liposomal
lipid and/or bioactive agent in digested, tissue homogenate
samples. The addition to liposomes of a sugar-modified lipid or
amine-bearing lipid (e.g., G.sub.M1 as shown in FIG. 1) reduces
significantly the liposomal lipid accumulation in liver and
spleen.
The effects of a liposomal bioactive agent composition of the
present invention may be examined in vitro or in vivo. For in vitro
antitumor studies, for example, a liposomal anticancer agent
composition can be contacted with a tumor cell line. In brief, such
a composition is incubated with cultured tumor cells and the
viability of the cells is measured to demonstrate the cytotoxicity
of the encapsulated anticancer agent. For in vivo studies, an
animal model system may be utilized. For example, for in vivo
antitumor studies, the P388 lymphocytic leukemia model can be used.
In brief, mice are injected i.p. with P388 cells. Liposomal
anticancer agent compositions or saline only are administered
(i.v.) 24 hours after tumor inoculation. .Animal weights and
mortality are monitored daily. As disclosed herein, the liposomal
anticancer agent compositions of the present invention (i.e.,
compositions which combine a reduced internal pH of the liposomes
and incorporation of a sugar-modified lipid or amine-bearing lipid)
exhibit significantly greater efficacy in the P388 tumor model. For
example, in the encapsulated vincristine embodiment which combines
a liposome internal pH of 2 with the presence of G.sub.M1 in the
liposome bilayer, administration of the composition at 2, 3 or 4
mg/kg to P388-inoculated mice consistently produced long term
survivors.
Liposome compositions of the present invention may be administered
to other warm-blooded animals, such as humans. Depending upon the
particular bioactive agent or combination of agents encapsulated,
such compositions may be used to treat diseases or induce
conditions in warm-blooded animals. Examples of uses of the
compositions of the present invention are for treating cancer, for
treating malaria, treating a disease for which a calcium channel
blocker is effective (e.g., supraventricular tachyarrhythmia),
inducing local anesthesia, treating a disease for which an
adrenergic antagonist is effective (e.g., hypertension and cardiac
arrhymias), treating arrhythmia, treating a disease for which a
cholinergic agent is effective (e.g., glaucoma), treating a disease
for which a biogenic amine is effective (e.g., treatment of shock),
treating depression, treating allergies, treating a disease
involving a protozoan (e.g., giardia), inducing analgesia, or
treating a disease for which a MDR inhibitor is effective (e.g.,
tumors resistant to chemotherapy). For administration to humans in
the treatment of afflictions, the prescribing physician will
ultimately determine the appropriate dose for a given human
subject, and this can be expected to vary according to the age,
weight, and response of the individual as well as the nature and
severity of the patient's symptoms. Due to the reduced toxicity of
liposome entrapped bioactive agents, dosages higher than those
normally used for the agents alone may be administered. It may be
desirable to initiate therapy with a dosage of about that typically
used for the agent alone and then increase the dosage as deemed
appropriate to the patient and the circumstances.
The sites and cells in the organism to which the agent is desired
to be delivered may determine the mode of administration. For
instance, delivery. to a specific external site may be most easily
accomplished by topical application. Such topical application may
be in the form of creams or ointments. Alternatively,
administration may be effected by absorption through epithelial or
mucocutaneous linings (e.g., nasal, oral, vaginal, rectal,
gastrointestinal, mucosa, etc.). The liposome compositions of the
present invention can be administered alone, but will generally be
administered in admixture with a pharmaceutical carrier selected
with regard to the intended route of administration and standard
pharmaceutical practice. They may be injected parenterally, for
example, intravenously, intramuscularly, or subcutaneously. For
parenteral administration, they are best used in the form of a
sterile aqueous solution which may contain other solutes, for
example, enough salts or glucose to make the solution isotonic.
For an oral mode of administration, liposome compositions of the
present invention can be used in the form of tablets, capsules,
lozenges, troches, powders, syrups, elixirs, aqueous solutions and
suspensions, and the like. In the case of tablets, carriers which
can be used include lactose, sodium citrate, and salts of
phosphoric acid. Various disintegrants such as starch, and
lubricating agents such as magnesium stearate, sodium lauryl
sulfate and talc, are commonly used in tablets. For oral
administration in capsule form, useful diluents are lactose and
high molecular weight polyethylene glycols. When aqueous
suspensions are required for oral use, certain sweetening and/or
flavoring agents can be added.
Alternatively, the liposome compositions of the present invention
may be used in vitro, e.g., as diagnostic agents. For example, an
agent possessing a reporter group may be entrappeal in liposomes to
which a targeting molecule is attached to the outermost layer.
Targeting molecules include antigens and antibodies, as well as a
number of other binding partners to molecules which may reside in a
sample to be tested in vitro. Dyes, such as rhodamine, merocyanine,
porphyrine and phthalocyanine, provide a convenient means to detect
the presence or absence of binding of a liposome to a sample.
Targeting molecules may be coupled to liposomes by a variety of
techniques (e.g., as described in U.S. Pat. No. 5,171,578). The
amount of the particular composition used will depend on the
sensitivity of the liposome-coupled targeting molecule to the
target components in the sample.
The following examples are offered by way of illustration and not
by way of limitation.
EXAMPLES
Example 1
PREPARATION OF LIPOSOMAL VINCRISTINE
"Oncovin" (vincristine sulfate) was obtained from the B.C. Cancer
Agency (Vancouver, British Columbia, Canada).
Distearoylphosphatidylcholine ("DSPC") was purchased from Avanti
Polar Lipids (Alabaster, AL), and was greater than 99% pure.
Monosialoganglioside G.sub.M1, cholesterol ("chol"), and all salts
were obtained from Sigma Chemical Company (St. Louis, Mo.).
Cholesteryl hexadecylether (.sup.14 C), a lipid marker that is not
exchanged or metabolized in vivo, was specially synthesized by
Amersham (Oakville, Ontario). Amino lipids AL-1 to AL-6, as
depicted in FIG. 6, were prepared as described in Example 4 below.
Female BDF1 mice (6-8 weeks old) were purchased from Charles .River
Laboratories.
DSPC/Chol (55:45; mol:mol), DSPC/Chol/G.sub.M1 (45:45:10;
mol:mol:mol), DSPC/Chol/Stearylamine (45:45:10), DSPC/Chol/AL-1
(45:45:10), or DSPC/Chol/Sphingosine (45:45:10) were prepared by
dissolving the lipid mixtures in 95% ethanol (1 mL/100 mg lipid).
The mixtures were then heated at 60.degree. C. for 30 min.
Subsequently, a preheated (60.degree. C.) solution of 300 mM citric
acid (pH 4 or pH 2) was added (3 mL buffer/100 mg total lipid)
while vigorously vortex mixing. The resulting multilamellar
vesicles ("MLVs") were heated at 60.degree. C. for an additional 30
min., followed by extrusion ten times through two polycarbonate
filters with 100 nm pores. The extrusion device, obtained from
Lipex Biomembranes (Vancouver, British Columbia, Canada), was also
maintained at 60.degree. C. Ethanol was removed from the liposome
preparation by dialyzing (Spectra/Por 2 dialysis tubing,
12,000-14,000 molecular weight cut-off against two changes (200 mL
dialysis buffer per 1 mL of sample) of 300 mM citric acid (pH 4 or
2) over a 24 h period. It has been determined that greater than
99.9% of the ethanol is removed using this procedure.
Vincristine was entrapped in the liposomes using a .DELTA.pH
loading procedure based upon that described by Mayer et al. (Cancer
Res. 50:575-579, 1990), but significantly modified by the use of a
low pH in the solution entrapped (e.g., pH 2 as described above).
Vesicles (25 mg/mL) were added to vincristine ("Oncovin" solution,
1 mg vincristine/mL) to achieve a drug-to-lipid ratio of 0.1:1. The
exterior pH of the liposome/vincristine mixture was raised to pH
7.0-7.2 with 0.5M Na.sub.2 HPO.sub.4 and immediately heated to
60.degree. C. for 10 min. Vincristine entrapment was determined by
column chromatography techniques (Mayer et al., Biochem.
27:2053-2060, 1988) using A.sub.297 (in ethanol/H.sub.2 O, 8/2) and
A.sub.815 spectroscopic assays for quantitation of vincristine and
lipid, respectively, or using liquid scintillation counting of
.sup.1 4 C-cholesteryl hexadecylether and .sup.3 H-vincristine.
Example 2
PLASMA CLEARANCE AND IN VIVO DRUG RELEASE
Plasma clearance studies were performed by injecting 20 mg lipid/kg
of drug loaded or empty liposomes (prepared according to Example 1
) via a lateral tail vein to female BDF1 mice (18-22 g). The
vincristine dose was therefore typically 2 mg/kg. Previous studies
have shown that this dose of vincristine, when entrapped in
liposomes, exhibits measurable levels of antitumor activity in L 12
10 and P388 ascites tumor models (Mayer et al., Cancer Res. 50:575,
1990). Four mice were used per time point. The mice were
anaesthetized at the indicated time points (i.p. mixture of
ketamine 160 mg/kg and xylazine 20 mg/kg). Blood was collected via
cardiac puncture and placed into EDTA coated microtainer tubes
(Becton Dickinson). The samples were then centrifuged (500 x g for
10 min) to pellet the blood cells and obtain plasma samples.
Liposomal lipid and/or vincristine were then assayed using
scintillation counting.
Biodistribution studies were performed on the same mice used for
plasma clearance studies. Following heart puncture, animals were
euthanized by cervical dislocation, and selected tissues were
removed from each animal and weighed. Saline was added to each
organ to achieve a 10% (w/v) homogenate using a Polytron
homogenizer (Brinkmann Instruments, Rexdale, Ont.). Tissue
homogenates (500 .mu.L) were digested with 500 .mu.L of "Solvable"
(DuPont Canada, Inc., Mississauga, Ont.) for 3 h at 50.degree. C.
Subsequently, the samples were cooled to room temperature before
decolorizing with 200 .mu.L of 30% hydrogen peroxide. Samples were
then counted using Picofluor (Packard) scintillation cocktail. The
statistical significance of both the plasma clearance and
biodistribution results were determined employing the student's
t-test.
The influence of G.sub.M1 incorporation and entrapped vincristine
on the circulation time of 100 nm DSPC/Chol liposomes is shown in
FIG. 1A. Two important conclusions can be derived from this data.
First, the circulation lifetime of liposomes with entrapped
vincristine is greater than control, drug-free, liposomes. This
effect cannot be achieved with free drug pre-treatment. Second,
incorporation of 10 mol % G.sub.M1 in 100 nm DSPC/Chol liposomal
vincristine results in a further increase in carrier circulation
lifetime. Plasma liposomal lipid levels are increased approximately
2.5-fold 24 h after i.v. administration when G.sub.M1 is
incorporated into DSPC/Chol liposomal vincristine. Liposomal lipid
accumulation in liver and spleen (FIG. 1B and C) is reduced
significantly when G.sub.M1 /DSPC/Chol liposomes are used to
encapsulate vincristine.
The influence of G.sub.M1 incorporation on circulating vincristine
levels over 24 h after i.v. administration is shown in FIG. 2..As
expected on the basis of data in FIG. 1, incorporation of G.sub.M1
into liposomes used to encapsulate vincristine resulted in
approximately a 3-fold increase in the level of drug achieved in
the plasma at 24 h (FIG. 2A). The circulating drug-to-lipid ratio
for these systems are shown in FIG. 2B, which shows that drug
release from liposomes in the plasma compartment is not influenced
by incorporation of G.sub.M1. For these liposomal vincristine
formulations, where drug was encapsulated using the pH gradient
loading procedure in liposomes prepared in 300 mM citrate buffer at
pH 4, greater than 90% of the encapsulated drug had been released
from circulating liposomes over the 24 h time course.
Retention of vincristine entrapped in 100 nm DSPC/Chol liposomes in
response to a transmembrane pH gradient can be improved by
decreasing the pH of the encapsulated citrated buffer from 4 to 2.
As shown in FIG. 2A, decreasing the interior pH from 4 to 2 results
in a 2.5 fold increase in the circulating vincristine levels
achieved at 24 h post i.v. injection. The change in intravesicular
pH did not influence the clearance of injected liposomes.
Therefore, the increased drug levels occur as a result of increased
drug retention. At every time point studied, higher drug-to-lipid
ratios were observed for vincristine encapsulated in liposomes
prepared at pH 2 (FIG. 2B).
Incorporation of G.sub.M1 in combination with the use of the pH 2
entrapped citrate buffer resulted in an unexpected improvement in
vincristine circulation lifetime. Plasma vincristine levels were
approximately 7.5-fold and 20-fold higher at 24 h than could be
achieved with comparable systems prepared in the absence of
G.sub.M1 using the pH 2 and pH 4 buffer, respectively. As shown in
FIG. 2B, G.sub.M1 /DSPC/Chol liposomes prepared at pH 2 exhibited
less than a 20% decrease in circulating drug-to-lipid ratio over 24
h.
The use of amine-containing lipids to promote retention of the
anti-cancer agent vincristine is illustrated in FIGS. 3-5. Selected
amine lipids were incorporated at the level of 5 mol % in liposomes
composed of DSPC/Chol. Large unilamellar liposomes were prepared as
described in Example 1 such that the liposomes entrapped 300 mM
citrate buffer at pH 4 (filled circles) or pH 2 (open circles) and
exhibited a mean particle size of less than 200 nm. Three examples
are provided including liposomes that contain stearylamine (FIG.
3), AL-1 (rac-1,2-dioleoyl-3-N,N-dimethylaminopropane, FIG. 4) and
sphingosine (FIG. 5). Drug retention characteristics were assessed
in vivo following i.v. administration of the liposomal vincristine
preparation in female BDF1 mice at a drug dose of 2 mg/kg (lipid
dose of 20 mg/kg). At the indicated time points (1, 4 and 24 h),
blood was collected from the mice (four mice per time point) via
cardiac puncture. The blood was immediately placed into EDTA coated
microtainers. Subsequently, plasma was prepared by centrifugation
of the collected blood. Liposomal lipid and vincristine in the
plasma were measured using radiolabeled markers (.sup.3
[H]-Vincristine and .sup.14 [C]-Cholesterylhexadecylether). The
resulting data demonstrates that significantly improved vincristine
retention is achieved when amine-containing lipids are incorporated
in liposomes with pH 2 citrate buffer. In contrast to the same
formulations prepared using pH 4 buffer, there is typically a 7-8
fold increase in the drug-to-lipid ratio observed in plasma
collected from animals 24 h after drug administration (Panel C,
FIGS. 3-5).
Example 3
ANTITUMOR ACTIVITY OF LIPOSOMAL VINCRISTINE
The antitumor effects of liposomal vincristine were monitored using
the P388 lymphocytic leukemia model. BDF1 mice (5 per group) were
injected i.p. with 1.times.10.sup.6 P388 cells. The indicated doses
of saline or liposomal vincristine were administered (i.v.) 24 h
after tumor inoculation. Animal weights and mortality were
monitored daily. Mean and median survival times as well as the
statistical significance of the results were determined using the
Mann-Whitney-Wilcoxon procedure.
Tumor efficacy studies were conducted to determine whether the
improved vincristine circulation lifetime achieved through the use
of G.sub.M1 and reduced internal pH improves the therapeutic
activity of the entrapped drug. Murine P388 antitumor activity of
the DSPC/Chol/G.sub.M1 pH 2 liposomal vincristine preparation was
compared to free drug and DSPC/Chol pH 4 liposomal vincristine
(Table I below).
The two liposomal preparations exhibited a 12-fold difference in
circulation half-life as estimated from the data in FIG. 2A. The
liposomal formulations were significantly more efficacious in the
P388 tumor model when compared to free drug. The liposomal
formulation which combined the use of G.sub.M1 and the reduced
internal pH exhibited remarkable activity in this model. This
formulation, when administered at 2, 3 and 4 mg/kg, consistently
produced long-term survival rates in excess of 50%. Similarly,
another liposomal formulation which combined the use of sphingosine
and reduced internal pH also consistently produced long-term
survival rates. No long-term survivors (>70 days) were obtained
with either free drug or the DSPC/Chol pH 4 liposomal vincristine
formulations. Drug induced weight loss data (% decrease in weight
on day 7) shown in Table I also suggest a decrease in drug toxicity
for the G.sub.M1 /DSPC/Chol pH 2 liposomes compared to DSPC/Chol pH
4 liposomes.
TABLE I
__________________________________________________________________________
P388 Antitumor Activity of Free and Liposomal Vincristine in BDFI
Mice Drug Lipid % wt Median Dose Dose change 60-day Survival Sample
(mg/kg) (mg/kg) on day 7 Survival (days) % ILS.sup.a L/F.sup.b
__________________________________________________________________________
Saline +9.8 0/10 10.0 control Free 1.0 10 +1.9 0/5 14.0 40
Vincristine 2.0 20 +0.5 0/5 15.0 50 3.0 30 -13.4 0/5 16.0 60 DSPC/
1.0 10 +2.8 0/5 22.0 120 1.57 Cholesterol 2.0 20 -2.1 0/10 27.0 170
1.80 pH 4.0 3.0 30 -12.0 0/10 31.0 210 1.94 Lipovinc 4.0 40 -24.9
0/10 32.0 220 -- DSPC/ 1.0 10 +3.3 1/5 20.0 100 1.43 Chol/G.sub.M1
2.0 20 +0.2 8/10 >70.0 ND.sup.c ND pH 2.0 3.0 30 -10.9 10/10
>70.0 ND ND Lipovinc 4.0 40 -14.4 10/10 >70.0 ND -- DSPC/ 2.0
20 +1.7 1/5 36.0 260 2.40 Chol/ 3.0 30 -7.0 3/5 >60.0 ND ND
Sphingosine 4.0 40 -18.5 3/5 >60.0 ND ND pH 2.0 Lipovinc
__________________________________________________________________________
.sup.a Percentage of ILS (increase in life span) values were
determined from median survival time comparing treated and saline
control groups. If greater than 50% of the animals survived for
greater than 70 days, median survival times and % ILS were not
calculated. .sup.b L/F (liposomal/free) values were calculated by
dividing the median survival time for the liposomal vincristine
group by the median survival time for the equivalent dosage of free
drug. .sup.c N.D. not determined.
Example 4
PREPARATION OF AMINO LIPIDS AL-1 TO AL-6
A. Synthesis of .+-.1,2-dioleoyl-3-N,N-dimethylamino-propane
(AL-1). This compound was prepared by the method of Leventis et al.
(Biochim. Biophys. Acta 1029:124-132, 1990). Three ml (35 mmol) of
oxalyl chloride was added to 1.0 g (3.5 mmol) oleic acid dissolved
in 10 ml benzene and stirred at room temperature for 1 h. After
removal of solvent and excess oxalyl chloride under vacuum, the
acid chloride was dissolved in 5 ml diethyl ether, and a further 5
ml of ether containing 0.20 g (1.7 mmol) of
3-N,N-dimethylamino-1,2-propanediol and 0.15 g pyridine was added.
The resulting mixture was stirred at room temperature for 30
minutes before quenching with 1 ml methanol and removing solvents
under vacuum. The crude product was dissolved in 50 ml hexane and
washed with 2.times.25 ml 0.1M potassium hydroxide in
methanol/water (1:1) followed by 25 ml 0.1M aqueous sodium
chloride. Drying over anhydrous sodium sulphate and removal of
hexane under vacuum gave a slightly yellow oil. Column
chromatography on silica gel (70-230 mesh), eluting with ethyl
acetate, gave 0.92 g (84%) of pure product (TLC, R.sub.f =0.5). The
structure of the product was confirmed by 200 MHz .sup.1 H-NMR. An
analogous procedure with decanoyl chloride was used to prepare
.+-.-1,2-didecanoyl-1-N,N-dimethylaminopropane (AL-6).
B. Synthesis of .+-.-1-oleoyl-2-hydroxy-3-N,N-dimethylaminopropane
(AL-2) and asymmetric .+-.-1,2-diacyl-3-N,N-dimethylaminopropanes
(AL3-ALS). Oleoyl chloride (3.5 mmol), prepared as above, was
dissolved in 5 ml THF and added to a five-fold excess of
3-N,N-dimethylamino-1,2-propanediol (2.0 g, 17 mmol) and 0.15 g
pyridine in 25 ml THF at 0.degree. C. Crude
1-monooleoyl-2-hydroxy-3-N,N-dimethylaminopropane (AL-2) was
isolated as above and purified by column chromatography on silica
gel using ethyl acetate/methanol (3:1) as eluant (R.sub.f 0.4).
Subsequent acylation with one equivalent of acetyl chloride,
butyryl chloride, or decanoyl chloride with reaction conditions,
extraction procedures, and purification as described above produced
AL-3, AL-4, and AL-5, respectively.
From the foregoing, it will be evident that, although specific
embodiments of the invention have been described herein for
purposes of illustration, various modifications may be made without
deviating from the spirit and scope of the invention.
* * * * *