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Claims  |
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It is claimed:
1. A liposome composition effective to extend, to at least 24 hours, the
period of effective activity of a therapeutic compound which can be
administered intravenously in a therapeutically effective amount and which
is cleared in free form from the bloodstream with a halflife of less than
about 4 hours, comprising
liposomes (i) composed of vesicle-forming lipids and between 1-20 mole
percent of a vesicle-forming lipid derivatized with a polymer selected
from the group consisting of polyethyleneglycol, polyacetic acid and
polyglycolic acid, and (ii) having a selected mean particle diameter in
the size range between about 0.1 to 0.4 microns, and
the compound in liposome-entrapped form,
for intravenous administration at a dose of the composition which contains
an amount of the compound in liposome-entrapped form which is at least
three times such therapeutically effective amount.
2. The composition of claim 1, wherein the hydrophilic polymer is
polyethyleneglycol having a molecular weight between about 1,000-5,000
daltons.
3. The composition of claim 2, wherein the polymer is derivatized to a
phospholipid.
4. The composition of claim 1, wherein the polymer is selected from the
group consisting of polyacetic acid and polyglycolic acid.
5. A liposome composition effective to extend, to at least 48 hours, the
period of therapeutic activity of a polypeptide which can be administered
intravenously in a therapeutically effective amount, which is cleared in
free form from the bloodstream with a halflife of less than about 4 hours,
and whose therapeutically active blood concentration is in the
picogram-nanogram/ml concentration range, comprising
liposomes (i) composed of vesicle-forming lipids and between 1-20 mole
percent of a vesicle-forming lipid derivitized with a polymer selected
from the group consisting of polyethyleneglycol, polyacetic acid and
polyglycolic acid, and (ii) having a selected mean particle diameter in
the size range between about 0.1 to 0.4 microns, and
the polypeptide in liposome-entrapped form,
for intravenous administration at a dose of the composition which contains
an amount of the polypeptide liposome-entrapped form which is at least
three times such therapeutically effective amount.
6. The composition of claim 5, wherein the hydrophilic polymer is
polyethyleneglycol having a molecular weight between about 1,000-5,000
daltons.
7. The composition of claim 5, wherein the polypeptide is a peptide hormone
which is therapeutically active at a plasma concentration in the
picogram/ml range, and the liposome composition is effective to release
the hormone in a therapeutically effective dose for a period of at least
five days after intravenous administration of the composition.
8. The composition of claim 7, wherein the peptide hormone is vasopressin.
9. The composition of claim 5, wherein the compound is a protein selected
from the group consisting of superoxide dismutase, glucocerebrosidase,
asparaginase, adenosine deaminase, interferons (alpha, beta, and gamma),
interleukin (1,2,3,4,5,6,7), tissue necroses factor (TNF-alpha, beta),
colony stimulating factors (-CSF (macrophage), G-CSF (granulocyte), GM-CSF
(granulocyte, macrophage), TPA, prourokinase, and urokinase, HIV-1
vaccine, hepatitis B vaccine, malaria vaccine, and melanoma vaccine,
erythropoietin (EPO), factor VIII, bone growth factor, fibroblast growth
factor, nerve growth factor, platelet-derived growth factor, tumor growth
factors (alpha, beta), somatomedin C (IGF-1), and a ribosome inhibitor
protein.
10. The composition of claim 9, wherein the protein is macrophage colony
stimulating factor.
11. A method of extending, to at least 24 hours, the period of effective
activity of a therapeutic compound which can be administered intravenously
in a therapeutically effective amount, and which has a halflife in the
bloodstream in free form of less than about 4 hours, comprising
providing a liposome composition containing liposomes (i) composed of
vesicle-forming lipids and between 1-20 mole percent of a vesicle-forming
lipid derivitized with ah polymer selected from the group consisting of
polyethyleneglycol, polyacetic acid and polyglycolic acid, and (ii) having
a selected mean particle diameter in the size range between about 0.1 to
0.4 microns, and the compound at least about 70% in liposome-entrapped
form, and
administering the liposome composition intravenously to a subject at a dose
which contains an amount of the compound which is at least three times
such therapeutically effective amount.
12. The method of claim 11, wherein the hydrophilic polymer is
polyethyleneglycol having a molecular weight between about 1,000-5,000
daltons.
13. The method of claim 11, wherein the polymer is selected from the group
consisting of polylactic acid and polyglycolic acid.
14. The method of claim 11, wherein the compound is a peptide hormone which
is therapeutically active at a plasma concentration in the
picogram-to-nanogram/ml range, and said administering is effective to
release the hormone in a therapeutically effective dose for a period of at
least five days.
15. The method of claim 14, wherein the peptide hormone is vasopressin.
16. The method of claim 11, wherein the compound is a protein selected from
the group consisting of superoxide dismutase, glucocerebrosidase,
asparaginase, adenosine deaminase, interferons (alpha, beta, and gamma),
interleukin (1,2,3,4,5,6,7), tissue necroses factor (TNF - alpha, beta),
colony stimulating factors (M-CSF (macrophage), G-CSF (granulocyte),
GM-CSF (granulocyte, macrophage), TPA, prourokinase, and urokinase, HIV-1
vaccine, erythropoietin (EPO), factor VIII, bone growth factor, fibroblast
growth factor, nerve growth factor, platelet-derived growth factor, tumor
growth factors (alpha, beta), somatomedin C (IGF-1), and a ribosome
inhibitor protein.
17. The method of claim 16, wherein the protein is macrophage colony
stimulating factor.
18. A liposome composition effective to extend, to at least one week, the
period of effective activity of a therapeutic compound which can be
administered in a therapeutically effective amount, comprising
liposomes (i) composed of vesicle-forming lipids and between 1-20 mole
percent of a vesicle-forming lipid derivitized with a polymer selected
from the group consisting of polyethyleneglycol, polyacetic acid and
polyglycolic acid, and (ii) having a selected mean particle diameter in
the size range between about 0.1 to 0.4 microns, and
the compound in liposome-entrapped form,
for subcutaneous administration at a dose of the composition which contains
an amount of the compound in liposome-entrapped form which is at least ten
times such therapeutically effective intravenously administered amount.
19. The composition of claim 18, wherein the compound is a polypeptide
selected from the group consisting of superoxide dismutase,
glucocerebrosidase, asparaginase, adenosine deaminase, interferons (alpha,
beta, and gamma), interleukin (1,2,3,4,5,6,7), tissue necrosis factor (TNF
- alpha, beta), colony stimulating factors (M-CSF (macrophage), G-CSF
(granulocyte), GM-CSF (granulocyte, macrophage), TPA, prourokinase, and
urokinase, HIV-1 vaccine, hepatitis B vaccine, malaria vaccine, and
melanoma vaccine, erythropoietin (EPO), factor VIII, bone growth factor,
fibroblast growth factor, nerve growth factor, platelet-derived growth
factor, tumor growth factors (alpha, beta), somatomedin C (IGF-1), and a
ribosome inhibitor protein.
20. The composition of claim 19, wherein the polypeptide is vasopressin.
21. A method of extending, to at least one week, the period of effective
activity of a therapeutic compound which can be administered in a
therapeutically effective amount, comprising
providing a liposome composition containing liposomes (i) composed of
vesicle-forming lipids and between 1-20 mole percent of a vesicle-forming
lipid derivitized with a polymer selected from the group consisting of
polyethyleneglycol, polyacetic acid and polyglycolic acid, and (ii) having
a selected mean particle diameter in the size range between about 0.1 to
0.4 microns, and the compound at least about 70% in liposome-entrapped
form, and
administering the composition subcutaneously to a subject at a dose which
contains an amount of the compound in liposome-entrapped form which is at
least ten times such therapeutically effective intravenously administered
amount.
22. The method of claim 21, wherein the compound is a peptide hormone
selected from the group consisting of superoxide dismutase,
glucocerebrosidase, asparaginase, adenosine deaminase, interferons (alpha,
beta, and gamma), interleukin (1,2,3,4,5,6,7), tissue necroses factor
(TNF-alpha, beta), colony stimulating factors (M-CSF (macrophage), G-CSF
(granulocyte), GM-CSF (granulocyte, macrophage), TPA, prourokinase, and
urokinase, HIV-1 vaccine, hepatitis B vaccine, malaria vaccine, and
melanoma vaccine, erythropoietin (EPO), factor VIII, bone growth factor,
fibroblast growth factor, nerve growth factor, platelet-derived growth
factor, tumor growth factors (alpha, beta), somatomedin C (IGF-1), and a
ribosome inhibitor protein.
23. The method of claim 22, wherein the polypeptide is vasopressin.
24. A liposome composition composed of vesicle-forming lipids and a
vesicle-forming lipid derivatized with a hydrophilic polymer selected from
the group consisting of polylactic acid and polyglycolic acid.
25. A lipid composition composed of a vesicle-forming lipid having a polar
head group, and a polylactic acid moiety derivatized to said head group.
26. A lipid composition composed of a vesicle-forming lipid having a polar
head group, and a polyglycolic acid moiety derivatized to said head group. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The present invention relates to a liposome composition and method for
administering a therapeutic compound into the bloodstream over an extended
period.
REFERENCES
Allen, T. M., (1981) Biochem. Biophys. Acta 640. 385397.
Allen, T. M., and Everst, J. (1983) J. Pharmacol. Exp. Therap. 226.
539-544.
Ashwell, G., and Morell, A. G. (1974) Adv. Enzymology 41, 99-128.
Banga, A. K., et al., Int J Pharm, 48:15 (1988).
Czop, J. K. (1978) Proc. Natl. Acad. Sci. U.S.A. 74:3831.
Durocher, J. P., et al. (1975) Blood 45:11.
Ellens, H., et al. (1981) Biochim. Biophys. Acta 674:10-18.
Gabizon, A. Huberty, J. Straubinger, R. and Papahadjopoulos, D. (1988-1989)
J. Liposome Resh. 1, 123-135.
Gregoriadis, G., and Ryman, B. E. (1974) Eur. J. Biochem. 24, 485-491.
Gregoriadis, G., and Neerunjun, D. (1974) Eur. J. Biochem. 47, 179-185.
Gregoriadis, G., and Senior, J. (1980) FEBS Lett. 119, 43-46.
Greenberg, J. P., et al (1979) Blood 53:916.
Hakomori, S. (1981) Ann. Rev. Biochem 50, 733-764.
Hwang, K. J., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77:4030.
Jonah, M. M., et al. (1975) Biochem Biophys. Acta 401, 348.
Juliano, R. L., and Stamp, D. (1975) Biochem. Biophys Res. Commun. 63.
651-658.
Karlsson, K. A. (1982) In: Biological Membranes, Vol. 4, D. Chapman (ed.)
Academic Press, N.Y., pp. 1-74.
Kimelberg, H. K., et al. (1976) Cancer Res. 36,2949-2957.
Lee, K. C., et al., J. Immunology 125:86 (1980).
Lee, V. H. L., Pharm Int, 7:208 (1986).
Lee, V. H. L., Biopharm Manuf, 1:24 (1988).
Lopez-Berestein, G., et al. (1984) Cancer Res. 44, 375-378.
Okada, N. (1982) Nature 299:261.
Poste, G., et al., in "Liposome Technology" Volume 3, page 1 (Gregoriadis,
G., et al, eds.), CRC Press, Boca Raton (1984);
Poznansky, M. J., and Juliano, R. L. (1984) Pharmacol. Rev. 36. 277-336.
Richardson, V. J., et al. (1979) Br. J. Cancer 40, 3543.
Scherphof, T., et al. (1978) Biochim. Biophys. Acta 542, 296-307.
Senior, J., and Gregoriadis, G. (1982) FEBS Lett. 145, 109-114.
Senior, J., et al. (1985) Biochim. Biophys. Acta 839, 1-8.
Szoka, F., Jr., et al. (1978) Proc. Natl. Acad. Sci. U.S.A. 75:4194.
Szoka, F., Jr., et al. (1980) Ann. Rev. Biophys. Bioeng. 9:467.
Woodruff, J. J., et al. (1969) J. Exp. Med. 129:551.
BACKGROUND OF THE INVENTION
With recent advances in biotechnology, the development of medicinal
peptides or proteins has become an integral part of the pharmaceutical
industry (Lee, 1986, 1988). Several therapeutic proteins have been
successfully produced through recombinant DNA technology, such as human
growth hormone, human insulin, .alpha.-interferon, interleukin-2, TPA, and
a variety of peptide vaccines, all of which are now commercially available
(Banga). As oral administration generally does not result in therapeutic
responses, the parenteral route is preferred However, when administered
parenterally, most peptides and proteins have an extremely short half-life
in the bloodstream, typically less than 2 hours, and thus require large
doses and multiple daily injections or infusions. Often, the therapeutic
regimens employed require close medical supervision and are difficult for
most patients to accept.
Liposomes have been proposed as a carrier for intravenously (IV)
administered compounds. However, the use of liposomes for slow release of
liposome-entrapped material into the bloodstream has been severely
restricted by the rapid clearance of liposomes from the bloodstream by
cells of the reticuloendothelial system (RES). Typically, the RES will
remove 80-95% of IV injected liposomes within one hour, and effectively
remove circulating liposomes from the bloodstream within of 4-6 hours.
A variety of factors which influence the rate of RES uptake of liposomes
have been reported (e.g., Gregoriadis, 1974; Jonah; Gregoriadis, 1972;
Juliano; Allen, 1983; Kimelberg, 1976; Richardson; Lopez-Berestein; Allen,
1981; Scherphof; Gregoriadis, 1980; Hwang; Patel, 1983; Senior, 1985;
Allen, 1983; Ellens; Senior, 1982; Hwang; Ashwell; Hakomori; Karlsson;
Schauer; Durocher; Greenberg; Woodruff; Czop; and Okada). Briefly,
liposome size, charge, degree of lipid saturation, and surface moieties
have all been implicated in liposome clearance by the RES. However, no
single factor identified to date has been effective to provide long blood
halflife, and more particularly, a relatively high percentage of liposomes
in the bloodstream than 1 day or more after IV administration.
One factor which does favor longer liposome lifetime in the bloodstream is
small liposome size, typically in the size range of small unilamellar
vesicles (SUVs): 0.03-0.07 microns. However, the intravesicular volume of
SUVs is quite limited, to the extent that loading SUVs with a peptides or
proteins in a therapeutically effective dose range is not practical for
parenteral administration.
SUMMARY OF THE INVENTION
It is therefore one general object of the invention to provide a liposome
composition and method for administering a therapeutic compound for an
extended period in the bloodstream.
The invention includes, in one aspect, a liposome composition effective to
extend to at least 24 hours, the period of effective activity of an
therapeutic compound which can be administered intravenously in a
therapeutically effective amount, and which has a blood halflife, in free
form, of less than about 4 hours. The composition includes liposomes (i)
composed of vesicle-forming lipids and between 1-20 mole percent of a
vesicle-forming lipid derivatized with a biocompatible hydrophilic
polymer, and (ii) having a selected mean particle diameter in the size
range between about 0.1 to 0.4 microns, and the compound in
liposome-entrapped form. The composition is intended for intravenous
administration at a dose which contains an amount of the
liposome-entrapped compound which is at least three times the
therapeutically effective dose for the compound in free form.
In one preferred embodiment, the hydrophilic polymer is polyethyleneglycol
having a molecular weight between about 1,000-5,000 daltons, and the
polymer is derivatized with the polar head group of a phospholipid, such a
phosphatidylethanolamine (PE). Alternatively, the polymer may be other
suitable biocompatible hydrophilic polymers, such as polylactic acid and
polyglycolic acid.
Also in one preferred embodiment, the composition is effective to extend to
at least 48 hours, the period of therapeutic activity of an intravenously
injected polypeptide which can be administered intravenously in a
therapeutically effective amount. The polypeptide may be a peptide or
protein, such as superoxide dismutase, glucocerebrosidase, asparaginase,
adenosine deaminase, interferons (alpha, beta, and gamma), interleukin
(1,2,3,4,5,6,7), tissue necrosis factor (TNF - alpha, beta), colony
stimulating factors (M-CSF (macrophage), G-CSF (granulocyte), GM-CSF
(granulocyte, macrophage), TPA, prourokinase, and urokinase, HIV-1
vaccine, hepatitis B vaccine, malaria vaccine, and melanoma vaccine,
erythropoietin (EPO), factor VIII, bone growth factor, fibroblast growth
factor, insulin-like growth factor, nerve growth factor, platelet-derived
growth factor, tumor growth factors (alpha, beta), somatomedin C (IGF-1),
and a ribosome inhibitor protein, which is therapeutically active when
administered intravenously. Where the polypeptide is active in the
picogram/ml range, such as is vasopressin, the composition is effective to
deliver a therapeutically effective amount of the peptide into the
bloodstream for a period of between 5-10 days.
Also forming part of the invention is a method for extending to at least 24
hours, the period of effective activity of an therapeutic compound which
can be administered intravenously in a therapeutically effective amount,
and which has a halflife in the blood, in free form, of less than about 4
hours. In this method, a liposome composition of the type described above
is administered intravenously to a subject at a dose which contains an
amount of the compound which is at least three times such therapeutically
effective amount
Also disclosed is a liposome composition effective to extend to at least
one week, the period of effective activity of an therapeutic compound
which can be administered intravenously in a therapeutically effective
amount. The composition includes liposomes (i) composed of vesicle-forming
lipids and between 1-20 mole percent of a vesicle-forming lipid
derivatized with a biocompatible hydrophilic polymer, and (ii) having a
selected mean particle diameter in the size range between about 0.07-0.15
microns, and the compound in liposome-entrapped form. The composition is
intended for subcutaneous administration at a dose which contains an
amount of the liposome-entrapped compound which is at least ten times such
therapeutically effective intravenously administered amount.
The liposome composition is used in a method for extending the period of
release of a therapeutic compound, preferably a polypeptide, in a
therapeutically active amount, for a period of at least 2 weeks.
In another aspect, the invention includes a liposome composition composed
of vesicle-forming lipids and a vesicle-forming lipid derivatized with
polylactic acid or polyglycolic acid, and a lipid composition composed of
a vesicle-forming lipid having a polar head group, and a polylactic acid
or polyglycolic acid moiety derivatized to the lipid's head group.
These and other objects and features of the present invention will become
more fully apparent when the following detailed description of the
invention is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a general reaction scheme for derivatizing a
vesicle-forming lipid amine with a polyalkyl-ether;
FIG. 2 is a reaction scheme for preparing phosphati-dylethanolamine (PE)
derivatized with polyethyleneglycol via a cyanuric chloride linking agent;
FIG. 3 illustrates a reaction scheme for preparing phosphatidylethanolamine
(PE) derivatized with polyethyleneglycol by means of a diimidazole
activating reagent;
FIG. 4 illustrates a reaction scheme for preparing phosphatidylethanolamine
(PE) derivatized with polyethyleneglycol by means of a trifluoromethane
sulfonate reagent;
FIGS. 5A, 5B and 5C illustrate a vesicle-forming lipid derivatized with
polyethylene glycol through a peptide, ester and disulfide linkage
respectively.
FIG. 6 illustrates a reaction scheme for preparing phosphatidylethanolamine
(PE) derivatized with polylactic acid;
FIG. 7 is a plot of liposome retention time in the blood, expressed in
terms of percent injected dose as a function of hours after IV injection,
for PEG-PE liposomes containing different amounts of phosphatidylglycerol;
FIG. 8 is a plot similar to that of FIG. 7, showing retention times in the
blood of liposomes composed of predominantly unsaturated phospholipid
components;
FIG. 9 is a plot similar to that of FIG. 7, showing retention times in the
blood of PEG liposomes (solid triangles) and conventional liposomes (solid
circles);
FIG. 10 is a plot of blood lifetimes of PEG-liposomes sized by extrusion
through 0.1 micron (solid squares), 0.2 micron (solid circles), and 0.4
micron (solid triangles) polycarbonate membranes;
FIG. 11 is a plot of blood retention times in liposomes containing a
vesicle-forming lipid derivatized with polylactic acid (solid squares) and
polyglycolic acid (open triangles);
FIG. 12 shows urine flow rates in rats, as a percentage of predosage rate,
after surgery and IV administration of saline (control, open circles) and
of aqueous solutions of vasopressin at total doses of 0.2 g (closed
squares), 0.8 .mu.g (closed triangles), and 2 .mu.g (closed circles);
FIG. 13 shows urine flow rates in rats, as a percentage of predosage rate,
after surgery and IV administration of saline (control, open circles) and
of PEG-liposomes containing entrapped vasopressin at total doses of 2
.mu.g (closed squares), 8 .mu.g (closed tiangles), and 24 .mu.g (closed
circles);
FIG. 14 shows urine flow rates in rats, as a percentage of predosage rate,
after surgery and IV administration of saline (control, open circles) and
of PEG-liposomes containing entrapped vasopressin at a total dose of 8
.mu.g and mole percent of cholesterol in the liposomes of 33% (closed
circles), 16% (closed triangles), and 0% (closed squares);
FIG. 15 shows the blood clearance kinetics of free macrophage-colony
stimulating factor (M-CSF) (solid triangles), PEG-liposomes containing 30
mole percent cholesterol (solid triangles), and M-CSF associated with the
PEG-liposomes (solid circles);
FIG. 16 shows the blood clearance kinetics of free M-CSF (solid triangles),
cholesterol-free PEG-liposomes (solid triangles), and M-CSF associated
with the PEG-liposomes (solid circles);
FIG. 17 is a plot of percent release of M-CSF into the blood from PEG
liposomes containing 30 (solid circles) and 0 (solid triangles) mole
percent cholesterol;
FIG. 18A shows urine flow rates in rats, as a percentage of predosage rate,
after surgery and subcutaneous administration of saline (control, open
circles) and free vasopressin, in an amount 2 .mu.g (solid triangles), 25
.mu.g (solid circles), and 50 .mu.g (solid squares), and 100 .mu.g (solid
diamonds);
FIG. 18B shows urine flow rates in rats, as a percentage of predosage rate,
after surgery and subcutaneous administration of saline (control, open
circles) and vasopressin entrapped in PEG-liposomes, in an amount 25 .mu.g
(solid triangles), 100 .mu.g (solid, circles), and 400 .mu.g (solid
diamonds); and
FIGS. 19A and 19B show the change in PEG-liposome size, as a function of
homogenization time, for liposome particles in an homogenized suspension.
DETAILED DESCRIPTION OF THE INVENTION
I. Preparation of Derivatized Lipids
FIG. 1 shows a general reaction scheme for preparing a vesicle-forming
lipid derivatized with a biocompatible, hydrophilic polymer, as
exemplified by polyethylene glycol (PEG), polylactic acid, and
polyglycolic acid, all of which are readily water soluble, can be coupled
to vesicle-forming lipids, and are tolerated in vivo without toxic
effects. The hydrophilic polymer which is employed, e.g., PEG, is
preferably capped by a methoxy, ethoxy or other unreactive group at one
end, or is one in which one end is more reactive than the other, such as
polylactic acid.
The polymer is activated at one end by reaction with a suitable activating
agent, such as cyanuric acid, diimadozle, anhydride reagent, or the like,
as described below. The activated compound is then reacted with a
vesicle-forming lipid, such as phosphatidylethanol (PE), to produce the
derivatized lipid.
Alternatively, the polar group in the vesicle-forming lipid may be
activated for reaction with the polymer, or the two groups may be joined
in a concerted coupling reaction, according to known coupling methods. PEG
capped at one end with a methoxy or ethoxy group can be obtained
commercially in a variety of polymer sizes, e.g., 500-20,000 dalton
molecular weights.
The vesicle-forming lipid is preferably one having two hydrocarbon chains,
typically acyl chains, and a polar head group. Included in this class are
the phospholipids, such as phosphatidylcholine (PC), PE, phosphatidic acid
(PA), phosphatidylinositol (PI), and sphingolipids such as sphingomyelin
(SM), where the two hydrocarbon chains are typically between about 14-22
carbon atoms in length, and have varying degrees of unsaturation. Also
included in this class are the glycolipids, such as cerebrosides and
gangliosides.
Another vesicle-forming lipid which may be employed is cholesterol and
related sterols In general, cholesterol may be less tightly anchored to a
lipid bilayer membrane, particularly when derivatized with a high
molecular weight polyalkylether, and therefore be less effective in
promoting liposome evasion of the RES in the bloodstream.
More generally, and as defined herein, "vesicle-forming lipi" is inene to
include any amphipathic lipid having hydrophobic and polar head group
moieties, and which (a) by itself can form spontaneously into bilayer
vesicles in water, as exemplified by phospholipids, or (b) is stably
incorporated into lipid bilayers in combination with phospholipids, with
its hydrophobic moiety in contact with the interior, hydrophobic region of
the bilayer membrane, and its polar head group moiety oreinted toward the
exterior, polar surface of the membrane. An example of a latter type of
vesicle-forming lipid is cholesterol and cholesterol derivatives, such as
cholesterol sulfate and cholesterol hemisuccinate.
According to one important feature of the invention, the vesicle-forming
lipid may be a relatively fluid lipid, meaning that the lipid phase has a
relatively low liquid-to-liquid crystal phase-transition temperature,
e.g., at or below room temperature, or relatively rigid lipid, meaning
that the lipid has a relatively high melting temperature, e.g., up to
50.degree. C. As a rule, the more rigid, i.e., saturated lipids,
contribute to greater membrane rigidity in a lipid bilayer structure and
also contribute to greater bilayer stability in serum. Other lipid
components, such as cholesterol, are also known to contribute to membrane
rigidity and stability in lipid bilayer structures. Phospholipids whose
acyl chains have a variety of degrees of saturation can be obtained
commercially, or prepared according to published methods.
FIG. 2 shows a reaction scheme for producing a PE-PEG lipid in which the
PEG is derivatized to PE through a cyanuric chloride group. Details of the
reaction are provided in Example 1. Briefly, methoxy-capped PEG is
activated with cyanuric chloride in the presence in sodium carbonate under
conditions which produced the activated PEG compound in the figure. This
material is purified to remove unreacted cyanuric acid. The activated PEG
compound is reacted with PE in the presence of triethyl amine to produce
the desired PE-PEG compound, also shown in the figure. The yield is about
8-10% with respect to initial quantities of PEG.
The method just described may be applied to a variety of lipid amines,
including PE, cholesteryl amine, and glycolipids with sugar-amine groups.
A second method of coupling a polyalkylether, such as capped PEG to a lipid
amine is illustrated in FIG. 3. Here the capped PEG is activated with a
carbonyl diimidazole coupling reagent, to form the activated imidazole
compound shown in FIG. 3. Reaction with a lipid amine, such as PE leads to
PEG coupling to the lipid through an amide linkage, as illustrated in the
PEG-PE compound shown in the figure Details of the reaction are given in
Example 2.
A third reaction method for coupling a capped polyalkylether to a lipid
amine is shown in FIG. 4. Here PEG is first protected at its OH end by a
trimethylsilane group. The end-protection reaction is shown in the figure,
and involves the reaction of trimethylsilylchloride with PEG in the
presence of triethylamine. The protected PEG is then reacted with the
anhydride of trifluoromethyl sulfonate (FIG. 4) to form the PEG compound
activated with trifluoromethyl sulfonate. Reaction of the activated
compound with a lipid amine, such as PE, in the presence of triethylamine,
gives the desired derivatized lipid product, such as the PEG-PE compound,
in which the lipid amine group is coupled to the polyether through the
terminal methylene carbon in the polyether polymer. The trimethylsilyl
protective group can be released by acid treatment, as indicated at H+ in
the figure, or by reaction with a quaternary amine fluoride salt, such as
the fluoride salt of tetrabutylamine.
It will be appreciated that a variety of known coupling reactions, in
addition to those just described, are suitable for preparing
vesicle-forming lipids derivatized with hydrophilic polymers such as PEG,
polylactic acid, or polyglycolic acid. For example, the sulfonate
anhydride coupling reagent illustrated in FIG. 4 can be used to join an
activated polyalkylether to the hydroxyl group of an amphipathic lipid,
such as the 5'-OH of cholesterol. Other reactive lipid groups, such as an
acid or ester lipid group may also be used for coupling, according to
known coupling methods. For example, the acid group of phosphatidic acid
can be activated to form an active lipid anhydride, by reaction with a
suitable anhydride, such as acetic anhydride, and the reactive lipid can
then be joined to a protected polyalkylamine by reaction in the presence
of an isothiocyanate reagent.
In another embodiment, the derivatized lipid components are prepared to
include a labile lipid-polymer linkage, such as a peptide, ester, or
disulfide linkage, which can be cleaved under selective physiological
conditions, such as in the presence of peptidase or esterase enzymes or
reducing agents such as glutathione present in the bloodstream. FIG. 5
shows exemplary lipids which are linked through (A) peptide, (B), ester,
and (C), disulfide containing linkages. The peptide-linked compound can be
prepared, for example, by first coupling a polyalkylether with the
N-terminal amine of the tripeptide shown, e.g., via the reaction shown in
FIG. 3. The peptide carboxyl group can then be coupled to a lipid amine
group through a carbodiimide coupling reagent conventionally. The ester
linked compound can be prepared, for example, by coupling a lipid acid,
such as phosphatidic acid, to the terminal alcohol group of a
polyalkylether, using alcohol via an anhydride coupling agent.
Alternatively, an short linkage fragment containing an internal ester bond
and suitable end groups, such as primary amine groups can be used to
couple the polyalkylether to the amphipathic lipid through amide or
carbamate linkages. Similarly, the linkage fragment may contain an
internal disulfide linkage, for use in forming the compound shown at C in
FIG. 5.
FIG. 6 illustrates a method for derivatizing polylactic acid with PE. The
polylactic acid is reacted, in the presence of PE, with
dicyclohexylcarboimide (DCCI), as detailed in Example 2. Similarly, a
vesicle-forming lipid derivatized with polyglycolic acid may be formed by
reaction of polyglycolic acid or glycolic acid with PE in the presence of
a suitable coupling agent, such as DCCI, also as detailed in Example 2.
The vesicle-forming lipids derivatized with either polylactic acid or
polyglycolic acid form part of the invention herein. Also forming part of
the invention are liposomes containing these derivatized lipids, in a 1-20
mole percent.
II. Preparation of Liposome Composition
A. Lipid Components
The lipid components used in forming the liposomes of the invention may be
selected from a variety of vesicle-forming lipids, typically including
phospholipids and sterols. As will be seen, one requirement of the
liposomes of the present invention is long blood circulation lifetime. It
is therefore useful to establish a standardized measure of blood lifetime
which can be used for evaluating the effect of lipid components on blood
halflife.
One method used for evaluating liposome circulation time in vivo measures
the distribution of IV injected liposomes in the bloodstream and the
primary organs of the RES at selected times after injection. In the
standardized model which is used herein, RES uptake is measured by the
ratio of total liposomes in the bloodstream to total liposomes in the
liver and spleen, the principal organs of the RES. In practice, age and
sex matched mice are injected intravenously (IV) through the tail vein
with a radiolabeled liposome composition, and each time point is
determined by measuring total blood and combined liver and spleen
radiolabel counts, as detailed in Example 6.
Since the liver and spleen account for nearly 100% of the initial uptake of
liposomes by the RES, the blood/RES ratio just described provides a good
approximation of the extent of uptake from the blood to the RES in vivo.
For example, a ratio of about 1 or greater indicates a predominance of
injected liposomes remaining in the bloodstream, and a ratio below about
1, a predominance of liposomes in the RES. For most of the lipid
compositions of interest, blood/RES ratios were calculated at 1, 2, 3, 4,
and 24 hours.
The liposomes of the present invention include 1-20 mole percent of the
vesicle-forming lipid derivatized with a hydrophilic polymer, described in
Section I. According to one aspect of the invention, it has been
discovered that blood circulation halflives in these liposomes are largely
independent of the degree of saturation of the phospholipid components
making up the liposomes. That is, the phospholipid components may be
composed of predominantly of fluidic, relatively unsaturated, acyl chains,
or of more saturated, rigidifying acyl chain components. This feature of
the invention is seen in Example 7, which examines blood/RES ratios in
liposomes formed with PEG-PE, cholesterol, and PC having varying degrees
of saturation (Table 4). As seen from the data in Table 5 in the example,
high blood/RES ratios were achieved with in substantially all of the
liposome formulations, independent of the extent of lipid unsaturation in
the bulk PC phospholipid, and no systematic trend, as a function of degree
of lipid saturation, was observed.
Accordingly, the vesicle-forming lipids may be selected to achieve a
selected degree of fluidity or rigidity, to control the stability of the
liposomes in serum and the rate of release of entrapped drug from the
liposomes in the bloodstream and/or tumor. The vesicle-forming lipids may
also be selected, in lipid saturation characteristics, to achieve desired
liposome preparation properties. It is generally the case, for example,
that more fluidic lipids are easier to formulate and down size by
extrusion or homogenization than more rigid lipid components. In general,
more fluidic lipids (low transition temperature) are preferred because of
high compound-release rates in the bloodstream.
Similarly, it has been found that the percentage of cholesterol in the
liposomes may be varied over a wide range without significant effect on
observed blood/RES ratios The studies presented in Example 8A, with
reference to Table 6 therein, show virtually no change in blood/RES ratios
in the range of cholesterol between 0-30 mole percent.
Cholesterol, or related cholesterol derivatives may be important, however,
in regulating the rate of release of liposome entrapped therapeutic
compounds into the bloodstream. The studies reported in Examples 15 and
16, for example, indicate that the rate of release of encapsulated
polypeptide (peptide or protein) from liposomes in vitro (in the presence
of human serum) or in vivo (in the bloodstream) is strongly dependent on
cholesterol concentration PEG-liposome formulations containing high
cholesterol (e.g., 30 mole percent or greater) release very little peptide
or protein into serum in vitro, whereas decreasing amounts of cholesterol
produce increasing loss of encapsulated polypeptide Similarly, and as
described below, increased cholesterol in intravenously administered
PEG-liposomes produced reduced release of encapsulated compound into the
bloodstream (Example 16) and reduced physiological effect (Example 15).
Thus, in accordance with one feature of the invention, the rate of release
of compound from long-circulating liposomes can be controlled by the
percent cholesterol included in the liposomes.
It has also been found, in studies conducted in support of the invention,
that blood/RES ratios are also relatively unaffected by the presence of
charged lipid components, such as phosphatidylglycerol (PG). This can be
seen from FIG. 7, which plots percent loss of encapsulated marker for
PEG-PE liposomes containing either 4.7 mole percent PG (triangles) or 14
mole percent PG (circles). Virtually no difference in liposome retention
in the bloodstream over a 24 hour period was observed
In one embodiment, the liposomes are formulated to contain diglyceride at a
mole ratio of up to 25 mole percent or more total liposome lipids Such
liposomes are characterized by rapid liposome breakdown in the
bloodstream, with release of encapsulated material, and the rate of
breakdown can be selectively controlled by the percent of diglyceride
included in the liposomes. The ability of the such liposomes to avoid
uptake by the RES, and at the same time, to break down in the bloodstream
over a period of 2-12 hours or more in the bloodstream provides a
composition for achieving delayed release of an intravenously administered
drug over a several hour period, and which also avoids drug accumulation
predominantly in the RES tissues
The vesicle-forming lipid derivatized with a hydrophilic polymer is present
in an amount preferably between about 1-20 mole percent, on the basis of
moles of derivatized lipid as a percentage of total moles of
vesicle-forming lipids. It will be appreciated that a lower mole ratio,
such as 0.1 mole percent, may be appropriate for a lipid derivatized with
a large molecular weight polymer, such as one having a molecular weight
greater than 100 kilodaltons. As noted in Section I, the hydrophilic
polymer in the derivatized lipid preferably has a molecular weight between
about 200-20,000 daltons, and more preferably between about 1,000-5,000
daltons. Example 8B, which examines the effect of very short ethoxy ether
moieties on blood/RES ratios indicates that polyether moieties of greater
than about 5 carbon ether are required to achieve significant enhancement
of blood/RES ratios.
B. Preparing the Liposome Composition
The liposomes may be prepared by a variety of techniques, such as those
detailed in Szoka et al, 1980. One method for preparing drug-containing
liposomes is the reverse phase evaporation method described by Szoka et al
and in U.S. Pat. No. 4,235,871. The reverse phase evaporation vesicles
(REVs) have typical average sizes between about 2-4 microns and are
predominantly oligolamellar, that is, contain one or a few lipid bilayer
shells. The method is detailed in Example 5A. This method is generally
preferred for preparing liposomes with encapsulated proteins high
encapsulation efficiencies (up to 50%) are possible, and thus protein loss
or problems of recovery and purification of non-encapsulated protein are
reduced.
Multilamellar vesicles (MLVs) can be formed by simple lipid-film hydration
techniques. In this procedure, a mixture of liposome-forming lipids of the
type detailed above dissolved in a suitable solvent is evaporated in a
vessel to form a thin film, which is then covered by an aqueous medium, as
detailed in Example 5B. The lipid film hydrates to form MLVs, typically
with sizes between about 0.1 to 10 microns.
In accordance with one important aspect of the invention, the liposomes for
intravenous injection are prepared to have substantially homogeneous sizes
in a selected size range between about 0.1 and 0.4, and preferably 0.1 to
0.2 micron size ranges. Liposomes in this size range have sufficiently
high encapsulation volumes for carrying therapeutically effective amounts
of the compound to be administered. At lower liposome sizes, the ratio of
liposome-encapsulated compound to free compound may be too low to achieve
a requisite initial dose level of liposome-encapsulated compound in the
bloodstream or may not remain in circulation due to extravasation. At the
same time, 0.1-0.4 micron liposomes are small enough to give long b | | |
