U.S. patent application number 10/046326 was filed with the patent office on 2002-10-10 for liposome-entrapped topoisomerase inhibitors.
This patent application is currently assigned to Alza Corporation. Invention is credited to Colbern, Gail T., Slater, James L., Working, Peter K..
Application Number | 20020146450 10/046326 |
Document ID | / |
Family ID | 22301756 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146450 |
Kind Code |
A1 |
Slater, James L. ; et
al. |
October 10, 2002 |
Liposome-entrapped topoisomerase inhibitors
Abstract
A composition for administration of a therapeutically effective
dose of a topoisomerase inhibitor I or topoisomerase I/II inhibitor
is described. The composition includes liposomes having an outer
surface and an inner surface defining an aqueous liposome
compartment, and being composed of a vesicle-forming lipid and of a
vesicle-forming lipid derivatized with a hydrophilic polymer to
form a coating of hydrophilic polymer chains on both the inner and
outer surfaces of the liposomes. Entrapped in the liposomes is the
topoisomerase inhibitor at a concentration of at least about 0.10
.mu.mole drug per .mu.mole lipid.
Inventors: |
Slater, James L.; (Palo
Alto, CA) ; Colbern, Gail T.; (Pacifica, CA) ;
Working, Peter K.; (Burlingame, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
Alza Corporation
|
Family ID: |
22301756 |
Appl. No.: |
10/046326 |
Filed: |
October 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10046326 |
Oct 19, 2001 |
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09419189 |
Oct 15, 1999 |
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6355268 |
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60104671 |
Oct 16, 1998 |
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Current U.S.
Class: |
424/450 ;
514/253.02; 514/283 |
Current CPC
Class: |
B82Y 5/00 20130101; A61K
31/00 20130101; A61K 31/4745 20130101; A61K 9/1271 20130101; A61K
31/496 20130101; A61K 31/473 20130101; A61P 43/00 20180101; A61P
35/00 20180101; A61K 9/1278 20130101 |
Class at
Publication: |
424/450 ;
514/253.02; 514/283 |
International
Class: |
A61K 031/496; A61K
031/4745; A61K 009/127 |
Claims
It is claimed:
1. A composition for treating a tumor in a subject, comprising
liposomes composed of a vesicle-forming lipid and between about
1-20 mole percent of a vesicle-forming lipid derivatized with a
hydrophilic polymer, said liposomes being formed under conditions
that distribute the polymer on both sides of the liposomes' bilayer
membranes; and entrapped in the liposomes, a topoisomerase
inhibitor at a concentration of at least about 0.10 .mu.mole drug
per .mu.mole lipid, said liposomes having an inside/outside ion
gradient sufficient to retain the topoisomerase inhibitor within
the liposomes at the specified concentration.
2. The composition of claim 1, where the topoisomerase inhibitor is
a topoisomerase I inhibitor selected from the group consisting of
camptothecin and camptothecin derivatives.
3. The composition of claim 2, wherein the camptothecin derivative
is selected from the group consisting of 9-aminocamptothecin,
7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin,
10,11-methlyenedioxycamptothecin,
9-amino-10,11-methylenedioxycamptotheci- n and
9-chloro-10,11-methylenedioxycamptothecin.
4. The composition of claim 2, wherein the camptothecin derivative
is selected from the group consisting of irinotecan, topotecan,
(7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camptothecin,
7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-camptothecin
and 7-(2-N-isopropylamino)ethyl)-(20S)-camptothecin.
5. The composition of claim 1, wherein the topoisomerase inhibitor
is a topoisomerase I/II inhibitor selected from the group
consisting of
6-[[2-(dimethylamino)-ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-o-
ne dihydrochloride, azotoxin and
3-methoxy-11H-pyrido[3',4'-4,5]pyrrolo[3,-
2-c]quinoline-1,4-dione.
6. The composition of claim 1, wherein the hydrophilic polymer is
polyethyleneglycol having a molecular weight between 500-5,000
daltons.
7. The composition of claim 1, wherein the liposomes further
include a vesicle-forming lipid having a phase transition
temperature above 37.degree. C.
8. The composition of claim 7, wherein the vesicle-forming lipid is
selected from the group consisting of hydrogenated soy
phosphatidylcholine, distearoylphosphatidylcholine and
sphingomyelin.
9. The composition of claim 7, wherein the liposomes are composed
of 20-94 mole percent hydrogenated soy phosphatidylcholine and 1-20
mole percent distearoylphosphatidylethanolamine derivatized with
polyethyleneglycol and 5-60 mole percent cholesterol.
10. The composition of claim 7, wherein the liposomes are composed
of 30-65 mole percent hydrogenated soy phosphatidylcholine, 5-20
mole percent distearoylphosphatidylethanolamine derivatized with
polyethyleneglycol and 30-50 mole percent cholesterol.
11. The composition of claim 7, wherein the liposomes are composed
of 20-94 mole percent distearoylphosphatidycholine and 1-20 mole
percent distearoylphosphatidylethanolamine derivatized with
polyethyleneglycol.
12. The composition of claim 1, wherein the liposomes include a
polyanionic polymer within the liposomes, said polymer capable of
forming a complex with said topoisomerase inhibitor.
13. The composition of claim 12, wherein said polyanionic polymer
is selected from dextran sulfate, chondroitin sulfate A,
polyvinylsulfuric acid, and polyphosphoric acid.
14. A composition for administration of a topoisomerase inhibitor,
comprising liposomes composed of vesicle-forming lipids and having
an inside/outside ion gradient effective to retain the drug within
the liposomes; and entrapped in the liposomes, the topoisomerase
inhibitor at a concentration of at least about 0.20 .mu.mole drug
per pmole lipid.
15. The composition of claim 14, wherein the toposiomerase
inhibitor is a topoisomerase I inhibitor selected from
MPE-camptothecin, topotecan and (7-(2-N-isopropylamino) ethyl)
-(20S)-camptothecin.
16. The composition of claim 14, wherein the toposiomerase
inhibitor is a topoisomerase I/II inhibitor selected from the group
consisting of
6-[[2-(dimethylamino)-ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-o-
ne dihydrochloride and
3-methoxy-11H-pyrido[3',4'-4,5]pyrrolo[3,2-c]quinol-
ine-1,4-dione.
17. The composition of claim 14, wherein the liposomes further
include a polyanionic polymer within the liposomes, said polymer
capable of forming a complex with said topoisomerase inhibitor.
18. A method of treating a tumor in a subject, comprising preparing
liposomes composed of vesicle-forming lipids including between 1-20
mole percent of a vesicle-forming lipid derivatized with a
hydrophilic polymer chain, said liposomes being formed under
conditions that distribute the polymer on both sides of the
liposomes' bilayer membrane, said liposomes containing a
topoisomerase inhibitor entrapped in the liposomes at a
concentration of at least about 0.10 mole per .mu.mole lipid, the
liposomes having an inside/outside ion gradient sufficient to
retain the topoisomerase inhibitor within the liposome at the
specified concentration; and administering the liposomes to the
subject.
19. The method of claim 18, wherein preparing further comprises
entrapping the inhibitor in the liposomes by remote loading.
20. The method of claim 19, wherein entrapping further comprises
entrapping the topoisomerase inhibitor in the liposomes using an
ammonium sulfate gradient.
21. The method of claim 19, wherein entrapping further comprises
entrapping the topoisomerase inhibitor in the liposomes using a
polyanionic polymer trapping agent.
22. The method of claim 21, wherein said polyanionic polymer is
selected from dextran sulfate, chondroitin sulfate A,
polyvinylsulfuric acid, and polyphosphoric acid.
23. The method of claim 18, wherein preparing further includes
preparing the liposomes with a vesicle-forming lipid having a phase
transition temperature above 37 .degree. C.
24. The method of claim 23, wherein the vesicle-forming lipid is
selected from the group consisting of hydrogenated soy
phosphatidylcholine, distearoylphosphatidylcholine and
sphingomyelin.
25. The method of claim 24, wherein the liposomes are composed of
20-94 mole percent distearoylphosphatidycholine and 1-20 mole
percent distearoylphosphatidylethanolamine derivatized with
polyethyleneglycol.
26. The method of claim 24, wherein the liposomes are composed of
30-65 mole percent hydrogenated soy phosphatidylcholine, 5-20 mole
percent distearoylphosphatidylethanolamine derivatized with
polyethyleneglycol and 30-50 mole percent cholesterol.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/104,671, filed Sep. 16, 1998, incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a liposome composition
having an entrapped topoisomerase inhibitor.
BACKGROUND OF THE INVENTION
[0003] Next to heart disease, cancer is the major cause of death in
the United States, causing over 500,000 fatalities annually
(Katzung, B., "Basic and Clinical Pharmacology", 7.sup.th Edition,
Appleton & Lange, Stamford Conn., 1998, p. 882). With present
methods of treatment, one-third of patients are cured with local
measures, such as surgery or radiation therapy, which are quite
effective when the tumor has not metastasized by the time of
treatment. Earlier diagnosis might lead to increased cure of
patients undergoing such local treatments. However, in many cases,
early micrometastasis is a characteristic feature of the neoplasm,
indicating that a systemic approach such as chemotherapy may be
required, often along with a local treatment method, for effective
cancer management.
[0004] Cancer chemotherapy can be curative in certain disseminated
neoplasms that have undergone either gross or microscopic spread by
the time of diagnosis. These include testicular cancer, diffuse
large cell lymphoma, Hodgkin's disease and choriocarcinoma as well
as childhood tumors such as acute lymphoblastic leukemia. For other
forms of disseminated cancer, chemotherapy provides a palliative
rather than curative therapy. Effective palliative therapy results
in temporary clearing of the symptoms and signs of cancer and
prolongation of useful life. Advances in cancer chemotherapy have
recently provided evidence that chemical control of neoplasia is
possible for a number of cancers.
[0005] One category of drugs used for cancer therapy is
topoisomerase inhibitors. These compounds inhibit the action of
topoisomerase enzymes which play a role in the replication, repair,
genetic recombination and transcription of DNA. An example of a
topoisomerase inhibitor is camptothecin, a natural compound that
interferes with the activity of topoisomerase I, an enzyme involved
in DNA replication and RNA transcription. Camptothecin and the
camptothecin analogues topotecan and irinotecan are approved for
clinical use.
[0006] Camptothecin and its analogues are effective in cancer
chemotherapy by interfering with the breakage/reunion actions of
topoisomerase I. The compounds stabilize and form a reversible
enzyme-camptothecin-DNA ternary complex which prevents the reunion
step of the breakage/union cycle of the topoisomerase reaction.
[0007] One problem with camptothecin is its water insolubility,
which hinders the delivery of the drug. Numerous analogues of
camptothecin have been prepared to improve the compound's water
solubility. Another problem with camptothecin and its analogues is
that the compounds are susceptible in aqueous environments to
hydrolysis at the .alpha.-hydroxy lactone ring. The lactone ring
opens to the carboxylate form of the drug, a form that exhibits
little activity against topoisomerase I.
[0008] Various approaches to improving the stability of
camptothecin and its analogues have been described. One approach
has been to entrap the compounds in liposomes.
[0009] Burke (U.S. Pat. No. 5,552,156) describes a liposome
composition intended to overcome the instability of camptothecin
and its analogues by entrapping the compounds in liposomes having a
lipid bilayer membrane which allows the compound to penetrate, or
intercalate, into the lipid bilayer. With the compound intercalated
into the bilayer membrane, it is removed from the aqueous
environment in the core of the liposome and thereby protected from
hydrolysis.
[0010] One problem with this approach is that the liposomes are
quickly removed from the bloodstream by the reticuloendothelial
system (RES), preventing delivery, and preferably accumulation, at
the tumor site.
[0011] Subramanian and Muller (Oncology Research, 7(9):461-469
(1995)) describe a liposome formulation of topotecan and report
that in liposome-entrapped form, topotecan is stabilized from
inactivation by hydrolysis of the lactone ring. However, the
biological activity of the liposome-entrapped drug in vitro has
only 60% of the activity of the free drug.
[0012] Lundberg (Anti-Cancer Drug Design, 13:453 (1998)) describes
two lipophilic, oleic acid ester derivatives of camptothecin
analogues which are entrapped in liposomes and intercalated into
the bilayer for stabilization of the lactone ring. Daoud
(Anti-Cancer Drugs, 6:83-93 (1995)) describes a liposome
composition including camptothecin, where the drug is also
intercalated into the lipid bilayer. The liposomes in both of these
references are prepared conventionally, where the drug is passively
entrapped in the liposomes to sequester the drug in the lipid
bilayer membrane for stabilization. Using this method of
preparation it is difficult to achieve a sufficient drug load in
the liposomes for clinical efficacy.
[0013] Accordingly, there is still a need in the art for a liposome
formulation which (i) includes a topoisomerase inhibitor, such as
camptothecin and its analogues; (ii) remains in the bloodstream for
a prolonged period of time; (iii) retains antitumor activity; and
(iv) includes a sufficient drug load for clinical relevance.
SUMMARY OF THE INVENTION
[0014] Accordingly, it is an object of the invention to provide a
topoisomerase inhibitor composition for improved cancer
therapy.
[0015] It is another object of the invention to provide a liposome
composition for administration of a topoisomerase inhibitor for
antitumor therapy.
[0016] In one aspect, the invention includes a composition for
treating a tumor in a subject, comprising liposomes composed of a
vesicle-forming lipid and between about 1-20 mole percent of a
vesicle-forming lipid derivatized with a hydrophilic polymer. The
liposomes are formed under conditions that distribute the polymer
on both sides of the liposomes' bilayer membranes. Entrapped in the
liposomes is a topoisomerase I inhibitor or a topoisomerase I/II
inhibitor at a concentration of at least about 0.10 .mu.mole drug
per .mu.mole lipid. The liposomes have an inside/outside ion
gradient sufficient to retain the topoisomerase I inhibitor or
topoisomerase I/II inhibitor within the liposomes at the specified
concentration.
[0017] In one embodiment, the topoisomerase inhibitor is a
topoisomerase I inhibitor selected from the group consisting of
camptothecin and camptothecin derivatives. For example, the
camptothecin derivative can be 9-aminocamptothecin,
7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin,
10,11-methlyenedioxycamptothecin,
9-amino-10,11-methylenedioxycamptothecin or
9-chloro-10,11-methylenedioxy- camptothecin. In other embodiments,
the camptothecin derivative is irinotecan, topotecan,
(7-(4-methylpiperazinomethylene)-10,11-ethylenedio-
xy-20(S)-camptothecin,
7-(4-methylpiperazinomethylene)-10,11-methylenediox-
y-20(S)-camptothecin or
7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin.
[0018] In another embodiment, the topoisomerase inhibitor is a
topoisomerase I/II inhibitor, such as
6-[[2-(dimethylamino)-ethyl]amino]--
3-hydroxy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride, azotoxin
or
3-methoxy-11H-pyrido[3',4'-4,5]pyrrolo[3,2-c]quinoline-1,4-dione.
[0019] The hydrophilic polymer included in the liposome composition
can be polyvinylpyrrolidone, polyvinylmethylether,
polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacry- lamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,
hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol
and polyaspartamide.
[0020] In a preferred embodiment, the hydrophilic polymer is
polyethyleneglycol having a molecular weight between 500-5,000
daltons.
[0021] In still another embodiment, the liposomes further include a
vesicle-forming lipid having a phase transition temperature above
37.degree. C.
[0022] In yet another embodiment, the vesicle-forming lipid is
hydrogenated soy phosphatidylcholine, distearoyl
phosphatidylcholine or sphingomyelin. One preferred liposome
composition is composed of 20-94 mole percent hydrogenated soy
phosphatidylcholine, 1-20 mole percent distearoyl
phosphatidylcholine derivatized with polyethyleneglycol and 5-60
mole percent cholesterol.
[0023] Another preferred composition is 30-65 mole percent
hydrogenated soy phosphatidylcholine, 5-20 mole percent distearoyl
phosphatidylcholine derivatized with polyethyleneglycol and 30-50
mole percent cholesterol.
[0024] In another aspect, the invention includes a composition for
administration of a topoisomerase I inhibitor or a topoisomerase
I/II inhibitor, comprising liposomes composed of vesicle-forming
lipids and having an inside/outside ion gradient effective to
retain the drug within the liposomes. Entrapped in the liposomes is
the topoisomerase I inhibitor or the topoisomerase I/II inhibitor
at a concentration of at least about 0.20 .mu.mole drug per
.mu.mole lipid.
[0025] In another aspect, the invention includes a method of
treating a tumor in a subject, comprising preparing liposomes
composed of vesicle-forming lipids including between 1-20 mole
percent of a vesicle-forming lipid derivatized with a hydrophilic
polymer chain, the liposomes being formed under conditions that
distribute the polymer on both sides of the liposomes' bilayer
membrane. The liposomes contain a topoisomerase I inhibitor or a
topoisomerase I/II inhibitor entrapped in the liposomes at a
concentration of at least about 0.10 mole per .mu.mole lipid, the
liposomes having an inside/outside ion gradient sufficient to
retain the topoisomerase I inhibitor or topoisomerase I/II
inhibitor within the liposome at the specified concentration. The
liposomes are then administered to the subject.
[0026] In one embodiment of this aspect, the method further
includes entrapping the topoisomerase I inhibitor or topoisomerase
I/II inhibitor in the liposomes by remote loading, for example, via
an ammonium sulfate gradient.
[0027] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1A is a plot of the blood circulation lifetime of
liposome-entrapped MPE-camptothecin (solid circles), taken as the
percent of injected dose as a function of time, compared to the
free form of the drug (solid squares);
[0029] FIG. 1B shows the blood concentration of MPE-camptothecin,
as a function of time, in hours, after administration of
liposome-entrapped MPE-camptothecin (solid circles) and of free
(non-liposomal) MPE-camptothecin (solid squares) to rats;
[0030] FIG. 2A is a plot showing the body weight of mice, in grams,
as a function of days after tumor inoculation with an HT29 colon
tumor. The animals were treated on days 10, 16 and 23 after tumor
inoculation with liposome-entrapped MPE-camptothecin at dosages of
24 mg/kg (closed circles), 15 mg/kg (closed triangles) and 6 mg/kg
(closed squares) and with free MPE-camptothecin at doses of 24
mg/kg (open circles), 15 mg/kg (open triangles) and 6 mg/kg (open
squares);
[0031] FIG. 2B is a plot showing tumor volume, in mm.sup.3, as a
function of days after inoculation with an HT29 colon tumor. The
animals were treated on days 10, 16 and 23 after tumor inoculation
with liposome-entrapped MPE-camptothecin at dosages of 24 mg/kg
(closed circles), 15 mg/kg (closed triangles) and 6 mg/kg (closed
squares) and with free drug at doses of 24 mg/kg (open circles), 15
mg/kg (open triangles) and 6 mg/kg (open squares);
[0032] FIG. 3A is a plot showing the body weight of mice, in grams,
as a function of days after inoculation with an HT29 colon
tumor.
[0033] The animals were treated on days 9, 16 and 23 after tumor
inoculation with liposome-entrapped MPE-camptothecin at dosages of
5 mg/kg (open triangles), 3 mg/kg (open inverted triangles), 1
mg/kg (open diamonds), 0.5 mg/kg (open circles) and 0.1 mg/kg (open
squares) and with free MPE-camptothecin at a dose of 20 mg/kg
(closed squares);
[0034] FIG. 3B is a plot showing tumor volume, in mm.sup.3, as a
function of days after inoculation with an HT29 colon tumor. The
animals were treated on days 9, 16 and 23 after tumor inoculation
with liposome-entrapped MPE-camptothecin at dosages of 5 mg/kg
(open triangles), 3 mg/kg (open inverted triangles), 1 mg/kg (open
diamonds), 0.5 mg/kg (open circles) and 0.1 mg/kg (open squares)
and with free MPE-camptothecin at a dose of 20 mg/kg (closed
squares);
[0035] FIGS. 4A-4B are plots showing the plasma concentration of
topotecan as a function of time, in hours, after administration of
liposome-entrapped topotecan (solid triangles) and of free
(non-liposomal) topotecan (solid squares) to rats at dosages of 2
mg/kg (FIG. 4A) and 5 mg/kg (FIG. 4B);
[0036] FIG. 5A is a plot showing the body weight of mice, in grams,
as a function of days after inoculation with an HT29 colon tumor.
The animals were treated on days 9, 16 and 23 after tumor
inoculation with liposome-entrapped topotecan at dosages of 2 mg/kg
(diamonds), 5 mg/kg (circles), 8 mg/kg (open squares);
liposome-entrapped MPE-camptothecin at 4 mg/kg (triangles); free
topotecan at a dose of 25 mg/kg (inverted triangles) and saline
(closed squares);
[0037] FIG. 5B is a plot showing tumor volume, in mm.sup.3, as a
function of days after inoculation with an HT29 colon tumor. The
animals were treated on days 9, 16 and 23 after tumor inoculation
with liposome-entrapped topotecan at dosages of 2 mg/kg (diamonds),
5 mg/kg (circles), 8 mg/kg (open squares); liposome-entrapped
MPE-camptothecin at 4 mg/kg (triangles); free topotecan at a dose
of 25 mg/kg (inverted triangles) and saline (closed squares);
[0038] FIG. 6 is a plot of plasma concentration of CKD602 as a
function of time, in hours, after administration of
liposome-entrapped CKD602 (solid circles) and of free
(non-liposomal) topotecan (solid squares) to rats at a dosage of 1
mg/kg;
[0039] FIG. 7A is a plot showing the body weight of mice, in grams,
as a function of days after inoculation with an HT29 colon tumor.
The animals were treated on days 9, 16 and 23 after tumor
inoculation with liposome-entrapped CKD602 at dosages of 4 mg/kg
(diamonds), 2 mg/kg (circles), 1 mg/kg (open squares);
liposome-entrapped MPE-camptothecin at 4 mg/kg (triangles); free
CKD602 at a dose of 20 mg/kg (inverted triangles) and saline
(closed squares); and
[0040] FIG. 7B is a plot showing tumor volume, in mm.sup.3, as a
function of days after inoculation with an HT29 colon tumor. The
animals were treated on days 9, 16 and 23 after tumor inoculation
with liposome-entrapped CKD602 at dosages of 4 mg/kg (diamonds), 2
mg/kg (circles), 1 mg/kg (open squares); liposome-entrapped
MPE-camptothecin at 4 mg/kg (triangles); free CKD602 at a dose of
20 mg/kg (inverted triangles) and saline (closed squares).
DETAILED DESCRIPTION OF THE INVENTION
[0041] I. Definitions
[0042] Unless otherwise indicated, the terms below have the
following meaning:
[0043] "Effective amount" or "effective dose" refers to the amount
necessary or sufficient to inhibit undesirable cell growth, e.g.,
prevent undesirable cell growth or reduce existing cell growth,
such as tumor cell growth. The effective amount can vary depending
on factors known to those of skill in the art, such as the type of
cell growth, the mode and regimen of administration, the size of
the subject, the severity of the cell growth, etc. One of skill in
the art would be able to consider such factors and make the
determination regarding the effective amount.
[0044] "Therapeutically effective antitumor therapy" refers to a
therapy which is effective to maintain or decrease the size, e.g.,
volume, of a primary tumor or metastatic tumor. "Topoisomerase I
inhibitor" refers to any compound that inhibits or reduces the
action of topoisomerase I enzyme.
[0045] "Topoisomerase I/II inhibitor" refers to any compound that
inhibits or reduces the action of both topoisomerase I enzyme and
topoisomerase II enzyme.
[0046] "Topoisomerase inhibitor" refers to a topoisomerase I
inhibitor or a topoisomerase I/II inhibitor.
[0047] "MPE-camptothecin" refers to
7-(4-methyl-piperazino-methylene)-10,1-
1-ethylenedioxy-20(S)-camptothecin.
[0048] "Topotecan" refers to
9-dimethyl-aminomethyl-10-hydroxycamptothecin- .
[0049] "CKD-602" refers to
7-(2-(N-isopropylamino)ethyl)-(20S)-camptotheci- n.
[0050] II. Liposome Composition
[0051] The present invention is directed to a liposome composition
for administration of a topoisomerase I inhibitor or a
topoisomerase I/II inhibitor. In studies performed in support of
the invention, three topoisomerase inhibitors were entrapped in
liposomes and characterized in vivo: topotecan,
7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20-
(S)-camptothecin (referred to herein as "MPE-camptothecin") and
7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (referred to
herein as "CKD-602"). The drugs were entrapped in liposomes by
remote loading to achieve a high drug load stably retained in the
liposomes, as will be described. In vivo studies with the
formulations demonstrated that the liposome composition achieves a
surprising and unexpected degree of improvement in therapeutic
activity when compared to therapy with the topoisomerase inhibitor
in free form. More specifically, and as will be described below,
the dose of the liposome-entrapped topoisomerase I inhibitor
MPE-camptothecin required to achieve therapeutic antitumor therapy
is about 20 times lower than the dose required when the drug is
administered in free form.
[0052] In this section, the liposome composition will be described,
including methods for preparing the liposomes.
[0053] A. Liposome Components
[0054] Liposomes suitable for use in the composition of the present
invention include those composed primarily of vesicle-forming
lipids. Vesicle-forming lipids can form spontaneously into bilayer
vesicles in water, as exemplified by the phospholipids. The
liposomes can also include other lipids incorporated into the lipid
bilayers, with the hydrophobic moiety in contact with the interior,
hydrophobic region of the bilayer membrane, and the head group
moiety oriented toward the exterior, polar surface of the bilayer
membrane.
[0055] The vesicle-forming lipids are preferably ones having two
hydrocarbon chains, typically acyl chains, and a head group, either
polar or nonpolar. There are a variety of synthetic vesicle-forming
lipids and naturally-occurring vesicle-forming lipids, including
the phospholipids, such as phosphatidylcholine,
phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,
and sphingomyelin, where the two hydrocarbon chains are typically
between about 14-22 carbon atoms in length, and have varying
degrees of unsaturation. The above-described lipids and
phospholipids whose acyl chains have varying degrees of saturation
can be obtained commercially or prepared according to published
methods. Other suitable lipids include glycolipids and sterols such
as cholesterol.
[0056] Cationic lipids are also suitable for use in the liposomes
of the invention, where the cationic lipid can be included as a
minor component of the lipid composition or as a major or sole
component. Such cationic lipids typically have a lipophilic moiety,
such as a sterol, an acyl or diacyl chain, and where the lipid has
an overall net positive charge. Preferably, the head group of the
lipid carries the positive charge. Exemplary cationic lipids
include 1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-[l-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydrox-
yethylammonium bromide (DMRIE);
N-[1-(2,3,-dioleyloxy)propyl]-N,N-dimethyl- -N-hydroxy
ethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)
propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3
[N-(N',N'-dimethylaminoethane) carbamoly] cholesterol (DC-Chol);
and dimethyldioctadecylammonium (DDAB).
[0057] The cationic vesicle-forming lipid may also be a neutral
lipid, such as dioleoylphosphatidyl ethanolamine (DOPE) or an
amphipathic lipid, such as a phospholipid, derivatized with a
cationic lipid, such as polylysine or other polyamine lipids. For
example, the neutral lipid (DOPE) can be derivatized with
polylysine to form a cationic lipid.
[0058] In another embodiment, the vesicle-forming lipid is selected
to achieve a specified degree of fluidity or rigidity, to control
the stability of the liposome in serum and to control the rate of
release of the entrapped agent in the liposome.
[0059] Liposomes having a more rigid lipid bilayer, or a liquid
crystalline bilayer, are achieved by incorporation of a relatively
rigid lipid, e.g., a lipid having a relatively high phase
transition temperature, e.g., above room temperature, more
preferably above body temperature and up to 80.degree. C. Rigid,
i.e., saturated, lipids contribute to greater membrane rigidity in
the lipid bilayer. Other lipid components, such as cholesterol, are
also known to contribute to membrane rigidity in lipid bilayer
structures.
[0060] On the other hand, lipid fluidity is achieved by
incorporation of a relatively fluid lipid, typically one having a
lipid phase with a relatively low liquid to liquid-crystalline
phase transition temperature, e.g., at or below room temperature,
more preferably, at or below body temperature.
[0061] Vesicle-forming lipids having a main phase transition
temperatures from approximately 2.degree. C.-80.degree. C. are
suitable for use as the primary liposome component of the present
composition. In a preferred embodiment of the invention, a
vesicle-forming lipid having a main phase transition temperature
above about 37.degree. C. is used as the primary lipid component of
the liposomes. In another preferred embodiment, a lipid having a
phase transition temperature between about 37-70.degree. C. is
used. By way of example, the lipid distearoyl phosphatidylcholine
(DSPC) has a main phase transition temperature of 55.1.degree. C.
and the lipid hydrogenated soy phosphatidylcholine (HSPC) has a
phase transition temperature of 58.degree. C. Phase transition
temperatures of many lipids are tabulated in a variety of sources,
such as Avanti Polar Lipids catalogue and Lipid Thermotropic Phase
Transition Database (LIPIDAT, NIST Standard Reference Database
34).
[0062] The liposomes also include a vesicle-forming lipid
derivatized with a hydrophilic polymer. As has been described, for
example in U.S. Pat. No. 5,013,556 and in WO 98/07409, which are
hereby incorporated by reference, such a hydrophilic polymer
provides a surface coating of hydrophilic polymer chains on both
the inner and outer surfaces of the liposome lipid bilayer
membranes. The outermost surface coating of hydrophilic polymer
chains is effective to provide a liposome with a long blood
circulation lifetime in vivo. The inner coating of hydrophilic
polymer chains extends into the aqueous compartments in the
liposomes, i.e., between the lipid bilayers and into the central
core compartment, and is in contact with the entrapped compound
after the compound is loaded via remote loading. As will be
illustrated below, the liposome formulation having a surface
coating of hydrophilic polymer chains distributed on the inner and
outer liposome surfaces provides for a topoisomerase I inhibitor or
topoisomerase I/II inhibitor composition where the compound is
retained in the liposomes for improved therapeutic activity.
[0063] Vesicle-forming lipids suitable for derivatization with a
hydrophilic polymer include any of those lipids listed above, and,
in particular phospholipids, such as distearoyl
phosphatidylethanolamine (DSPE).
[0064] Hydrophilic polymers suitable for derivatization with a
vesicle-forming lipid include polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,
hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,
and polyaspartamide. The polymers may be employed as homopolymers
or as block or random copolymers.
[0065] A preferred hydrophilic polymer chain is polyethyleneglycol
(PEG), preferably as a PEG chain having a molecular weight between
500-10,000 daltons, more preferably between 500-5,000 daltons, most
preferably between 1,000-2,000 daltons. Methoxy or ethoxy-capped
analogues of PEG are also preferred hydrophilic polymers,
commercially available in a variety of polymer sizes, e.g.,
120-20,000 daltons.
[0066] Preparation of vesicle-forming lipids derivatized with
hydrophilic polymers has been described, for example in U.S. Pat.
No. 5,395,619. Preparation of liposomes including such derivatized
lipids has also been described, where typically, between 1-20 mole
percent of such a derivatized lipid is included in the liposome
formulation. It will be appreciated that the hydrophilic polymer
may be stably coupled to the lipid, or coupled through an unstable
linkage which allows the coated liposomes to shed the coating of
polymer chains as they circulate in the bloodstream or in response
to a stimulus.
[0067] B. Topoisomerase Inhibitor
[0068] The liposomes of the invention include a topoisomerase
inhibitor entrapped in the liposome. Entrapped is intended to
include encapsulation of an agent in the aqueous core and aqueous
spaces of liposomes. It will be appreciated that for compounds
having some hydrophobicity, entrapment in the lipid bilayer(s) of
the liposomes may also occur.
[0069] Topoisomerases catalyze the introduction and relaxation of
superhelicity in DNA. Several types of enzymes with varying
specifities are known to be important in the replication of DNA, as
well as in the repair, genetic recombination and transcription of
DNA. The simplest topoisomerases, designated topoisomerase I, relax
superhelical DNA, a process that is energetically spontaneous. The
gyrases, which are known as topoisomerase II, catalyze the
energy-requiring and ATP-dependent introduction of negative
superhelical twists into DNA. In DNA replication, topoisomerases I
and II have the function of relaxing the positive superhelicity
that is introduced ahead of the replicating forks by the action of
helicases. In addition, gyrases introduce negative twists into
segments of DNA that allow single-strand regions to appear.
[0070] Topoisomerase inhibitors, then, are compounds that inhibit
topoiosmerase activity. Compounds known as topoisomerase I
inhibitors have activity against topoisomerase I, and the
topoiosmerase II inhibitors have activity against topoisomerase II.
Some compounds have activity against both topoisomerase I and
topoisomerase II and are known as topoisomerase I/II
inhibitors.
[0071] Preferred topoisomerase I inhibitors for use in the present
invention are camptothecin and analogs of camptothecin.
Camptothecin is a pentacyclic alkaloid initially isolated from the
wood and bark of Camptotheca acuminata, a tree indigenous to China
(Wall, M. E. et al., J. Am. Chem. Soc., 94:388 (1966)).
Camptothecin exerts its pharmacological effects by irreversibly
inhibiting topoisomerase I. Methods for the synthesis of
camptothecin and camptothecin analogs or derivatives are known, and
are summarized and set forth in U.S. Pat. No. 5,244,903, which is
herein incorporated by reference in its entirety.
[0072] Analogues of camptothecin include SN-38
((+)-(4S)-4,11-diethyl-4,9--
dihydroxy-1H-pyrano[3',4':6,7]-indolizino[1,2-b]quinoline-3,14(4H,12H)-dio-
ne); 9-aminocamptothecin; topotecan (hycamtin;
9-dimethyl-aminomethyl-10-h- ydroxycamptothecin); irinotecan
(CPT-11; 7-ethyl-10-[4-(1-piperidino)-1-pi-
peridino]-carbonyloxy-camptothecin), which is hydrolyzed in vivo to
SN-38); 7-ethylcamptothecin and its derivatives (Sawada, S. et al.,
Chem. Pharm. Bull., 41(2):310-313 (1993));
7-chloromethyl-10,11-methylene-dioxy- -camptothecin; and others
(SN-22, Kunimoto, T. et al., J. Pharmacobiodyn., 10(3):148-151
(1987); N-formylamino-12,13,dihydro-1,11-dihydroxy-13-(beta-
-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione
(NB-506, Kanzawa, F et al., Cancer Res., 55(13):2806-2813 (1995);
DX-8951f and lurtotecan (GG-211 or
7-(4-methylpiperazino-methylene)-10,11-
-ethylenedioxy-20(S)-camptothecin) (Rothenberg, M. L., Ann. Oncol.,
8(9):837-855 (1997)) and
7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (CKD602, Chong Kun
Dang Corporation, Seoul Korea).
[0073] Topoisomerase inhibitors having activity against both
topoisomerase I and topoisomerase II include
6-[[2-(dimethylamino)-ethyl]amino]-3-hydro-
xy-7H-indeno[2,1-c]quinolin-7-one dihydrochloride, (TAS-103,
Utsugi, T., et al., Jpn. J. Cancer Res., 88(10):992-1002 (1997))
and
3-methoxy-11H-pyrido[3',4'-4,5]pyrrolo[3,2-c]quinoline-1,4-dione
(AzalQD, Riou, J. F., et al., Mol. Pharmacol., 40(5):699-706
(1991)).
[0074] In one embodiment of the invention, the topoisomerase I
inhibitor administered is the pharmacologically active enantiomer
of a camptothecin analogue having a chiral center. The enantiomer
can be resolved from the racemic mixture using techniques known to
those of skill in the art.
[0075] C. Method of Preparing the Liposome Composition
[0076] The liposomes may be prepared by a variety of techniques,
such as those detailed in Szoka, F., Jr., et al., Ann. Rev.
Biophys. Bioeng. 9:467 (1980), and specific examples of liposomes
prepared in support of the present invention will be described
below. Typically, the liposomes are multilamellar vesicles (MLVs),
which can be formed by simple lipid-film hydration techniques. In
this procedure, a mixture of liposome-forming lipids and including
a vesicle-forming lipid derivatized with a hydrophilic polymer are
dissolved in a suitable organic solvent which is evaporated in a
vessel to form a dried thin film. The film is then covered by an
aqueous medium to form MLVs, typically with sizes between about 0.1
to 10 microns. Exemplary methods of preparing derivatized lipids
and of forming polymer-coated liposomes have been described in
co-owned U.S. Pat. Nos. 5,013,556, 5,631,018 and 5,395,619, which
are incorporated herein by reference.
[0077] The therapeutic agent of choice can be incorporated into
liposomes by standard methods, including (i) passive entrapment of
a water-soluble compound by hydrating a lipid film with an aqueous
solution of the agent, (ii) passive entrapment of a lipophilic
compound by hydrating a lipid film containing the agent, and (iii)
loading an ionizable drug against an inside/outside liposome ion
gradient, termed remote loading. Other methods, such as reverse
evaporation phase liposome preparation, are also suitable.
[0078] In the present invention, a preferred method of preparing
the liposomes is by remote loading. In the studies performed in
support of the invention, three exemplary topoisomerase I
inhibitors were loaded into pre-formed liposomes by remote loading
against an ion concentration gradient, as has been described in the
art (U.S. Pat. No. 5,192,549) and as described in Example 1. In a
remote loading procedure, a drug is accumulated in the liposomes'
central compartment at concentration levels much greater than can
be achieved with other loading methods. In a preferred embodiment
of the invention, the topoisomerase I inhibitor or topoisomerase
I/II inhibitor is loaded into the liposomes to a concentration of
at least about 0.10 .mu.mole drug per .mu.mole lipid, more
preferably of at least about 0.15 .mu.mole drug per .mu.mole lipid,
most preferably of at least about 0.20 .mu.mole drug per .mu.mole
lipid. The liposomes prepared in support of the invention contained
MPE-camptothecin, topotecan or CKD602. As set forth in Example 1,
these compounds were loaded into the liposomes by remote loading,
discussed below, to a drug concentration level of greater than 0.20
.mu.mole drug per .mu.mole lipid (see the table in Example 1).
[0079] Liposomes having an ion gradient across the liposome bilayer
for use in remote loading can be prepared by a variety of
techniques. A typical procedure is as described above, where a
mixture of liposome-forming lipids is dissolved in a suitable
organic solvent and evaporated in a vessel to form a thin film. The
film is then covered with an aqueous medium containing the solute
species that will form the aqueous phase in the liposome interior
spaces.
[0080] After liposome formation, the vesicles may be sized to
achieve a size distribution of liposomes within a selected range,
according to known methods. The liposomes are preferably uniformly
sized to a selected size range between 0.04 to 0.25 .mu.m.
[0081] Small unilamellar vesicles (SUVs), typically in the 0.04 to
0.08 .mu.m range, can be prepared by extensive sonication or
homogenization of the liposomes. Homogeneously sized liposomes
having sizes in a selected range between about 0.08 to 0.4 microns
can be produced, e.g., by extrusion through polycarbonate membranes
or other defined pore size membranes having selected uniform pore
sizes ranging from 0.03 to 0.5 microns, typically, 0.05, 0.08, 0.1,
or 0.2 microns. The pore size of the membrane corresponds roughly
to the largest size of liposomes produced by extrusion through that
membrane, particularly where the preparation is extruded two or
more times through the same membrane. The sizing is preferably
carried out in the original lipid-hydrating buffer, so that the
liposome interior spaces retain this medium throughout the initial
liposome processing steps.
[0082] After sizing, the external medium of the liposomes is
treated to produce an ion gradient across the liposome membrane,
which is typically a lower inside/higher outside concentration
gradient. This may be done in a variety of ways, e.g., by (i)
diluting the external medium, (ii) dialysis against the desired
final medium, (iii) molecular-sieve chromatography, e.g., using
Sephadex G-50, against the desired medium, or (iv) high-speed
centrifugation and resuspension of pelleted liposomes in the
desired final medium. The external medium which is selected will
depend on the mechanism of gradient formation and the external pH
desired, as will now be considered.
[0083] In the simplest approach for generating an ion gradient, the
hydrated, sized liposomes have a selected internal-medium pH. The
suspension of the liposomes is titrated until a desired final pH is
reached, or treated as above to exchange the external phase buffer
with one having the desired external pH. For example, the original
medium may have a pH of 5.5, in a selected buffer, e.g., glutamate
or phosphate buffer, and the final external medium may have a pH of
8.5 in the same or different buffer. The internal and external
media are preferably selected to contain about the same osmolarity,
e.g., by suitable adjustment of the concentration of buffer, salt,
or low molecular weight solute, such as sucrose.
[0084] In another general approach, the gradient is produced by
including in the liposomes, a selected ionophore. To illustrate,
liposomes prepared to contain valinomycin in the liposome bilayer
are prepared in a potassium buffer, sized, then exchanged with a
sodium buffer, creating a potassium inside/sodium outside gradient.
Movement of potassium ions in an inside-to-outside direction in
turn generates a lower inside/higher outside pH gradient,
presumably due to movement of protons into the liposomes in
response to the net electronegative charge across the liposome
membranes (Deamer, et al., 1972).
[0085] In another more preferred approach, the proton gradient used
for drug loading is produced by creating an ammonium ion gradient
across the liposome membrane, as described, for example, in U.S.
Pat. No. 5,192,549. Here the liposomes are prepared in an aqueous
buffer containing an ammonium salt, typically 0.1 to 0.3 M ammonium
salt, such as ammonium sulfate, at a suitable pH, e.g., 5.5 to 7.5.
The gradient can also be produced by using sulfated polymers, such
as dextran ammonium sulfate or heparin sulfate. After liposome
formation and sizing, the external medium is exchanged for one
lacking ammonium ions, e.g., the same buffer but one in which
ammonium sulfate is replaced by NaCl or a sugar that gives the same
osmolarity inside and outside of the liposomes.
[0086] After liposome formation, the ammonium ions inside the
liposomes are in equilibrium with ammonia and protons. Ammonia is
able to penetrate the liposome bilayer and escape from the liposome
interior. Escape of ammonia continuously shifts the equilibrium
within the liposome toward the right, to production of protons.
[0087] The topoisomerase inhibitor is loaded into the liposomes by
adding the drug to a suspension of the ion gradient liposomes, and
the suspension is treated under conditions effective to allow
passage of the compound from the external medium into the
liposomes. Incubation conditions suitable for drug loading are
those which (i) allow diffusion of the derivatized compound, with
such in an uncharged form, into the liposomes, and (ii) preferably
lead to high drug loading concentration, e.g., 5-500 mM drug
encapsulated, more preferably between 20-200 mM, most preferably
between 50-300 mM.
[0088] The loading is preferably carried out at a temperature above
the phase transition temperature of the liposome lipids. Thus, for
liposomes formed predominantly of saturated phospholipids, the
loading temperature may be as high as 60 C. or more. The loading
period is typically between 15-120 minutes, depending on
permeability of the drug to the liposome bilayer membrane,
temperature, and the relative concentrations of liposome lipid and
drug.
[0089] With proper selection of liposome concentration, external
concentration of added compound, and the ion gradient, essentially
all of the compound may be loaded into the liposomes. For example,
with a pH gradient of 3 units (or the potential of such a gradient
employing an ammonium ion gradient), the final internal:external
concentration of drug will be about 1000:1. Knowing the calculated
internal liposome volume, and the maximum concentration of loaded
drug, one can then select an amount of drug in the external medium
which leads to substantially complete loading into the
liposomes.
[0090] Alternatively, if drug loading is not effective to
substantially deplete the external medium of free drug, the
liposome suspension may be treated, following drug loading, to
remove non-encapsulated drug. Free drug can be removed, for
example, by molecular sieve chromatography, dialysis, or
centrifugation.
[0091] In another embodiment of the invention, the topoisomerase
inhibitor is loaded into preformed liposomes which include in the
liposome interior a trapping agent effective to complex with the
topoisomerase inhibitor and enhance retention of the compound. In a
preferred embodiment, the trapping agent is a polyanionic polymer,
e.g., a molecule consisting of repetitive units of preferably
similar chemical structure and having ionizable groups, that is,
chemical functional groups capable of electrolytic dissociation
resulting in the formation of ionic charge, and preferably an
anionic charge. Polymers having a molecular weight over a broad
range are suitable, from 400-2,000,000 Daltons.
[0092] The polyanionic polymer is entrapped in the liposomes during
lipid vesicle formation. Upon loading of a drug into the pre-formed
liposomes, the polymer serves to trap or retain the drug within the
liposomes. In the studies described herein, dextran sulfate was
used as an exemplary polyanionic polymer. Dextran sulfate is a
polymer of anhydroglucose with approximately 2.3 sulfate groups per
glucosoyl residue. It is composed of approximately 95%
alpha-D-(1-6) linkages and the remaining (1-3) linkages account for
the branching of dextran. The polymer is readily available in
molecular weights ranging from 5,000 to 500,000 Daltons. However,
other polymer are suitable including sulfated, sulfonated,
carboxylated or phosphated hydrophilic polymers. For example,
sulfated proteoglycans, such as sulfated heparin, sulfated
polysaccharids, such as sulfated cellulose or cellulose
derivatives, carrageenin, mucin, sulfated polypeptides, such as
polylysine with sulfated amine groups, glycopeptides with
sulfonate-derivatized saccharide or peptide subunits, and
hyaluronic acid. Chondroitin sulfates A, B and C, keratin sulfates,
dermatan sulfates are also contemplated. The polymer can also be a
neutral polymer modified to include an anionic functional group.
For example, amylose, pectin, amylopectin, celluloses, and dextran
can be modified to include an anionic subunit. Polymers bearing a
sulfo group such as polyvinylsulfate, polyvinylsulfonate
polystyrenesulfonate and sulfated rosin gum are also suitable.
[0093] Preparation of liposomes which include such a trapping agent
is described with respect to Example 4. In this example, the
polyanionic polymer dextran sulfate is entrapped in the liposomes
by adding the liposome lipids, which are first dissolved in
ethanol, to a solution of dextran sulfate ammonium salt and mixed
to form liposomes having dextran sulfate ammonium salt entrapped
within the liposomes. The external media was exchanged to establish
an ammonium ion gradient across the liposomes for remote loading of
drug.
[0094] III. In vivo Administration of the Composition
[0095] Liposomes were prepared in support of the invention as
described in Example 1. The topoisomerase I inhibitors
(7-(4-methylpiperazino)-methyle-
ne)-10,11-ethylenedioxy-20(S)-camptothecin), referred to herein as
"MPE-camptothecin"; topotecan; and
7-(2-(N-isopropylamino)ethyl)-(20S)-ca- mptothecin, referred to
herein as "CKD-602", were loaded into liposomes under an ammonium
sulfate ion concentration gradient. The liposomes were composed of
hydrogenated soy phosphatidylcholine, cholesterol and polyethylene
glycol derivatized to distearoyl phosphatidylethanolamine
(PEG-DSPE) in a molar ratio 55.4:39:5.6.
[0096] The table in Example 1 summarizes the drug to lipid ratios
for the liposome formulations prepared. The calculated liposomal
drug concentrations for the three compounds, based on an extruded
liposome captured volume of 0.9 .mu.l/.mu.mole lipid, are 284 mM
for MPE-camptothecin, 264 mM for topotecan and 298 for CKD-602.
Based on an extruded liposome captured volume of 1.5 .mu.l/.mu.mole
lipid, the calculated liposomal drug concentrations are 189 mM for
MPE-camptothecin, 174 mM for topotecan and 198 for CKD-602. The in
vivo studies performed with each drug will now be described.
[0097] 1. In vivo Administration of MPE-Camptothecin
[0098] The long-circulating, PEG-coated liposomes containing
MPE-camptothecin were administered to rats to determine the blood
circulation lifetime of the drugs in liposome-entrapped form.
[0099] The pharmacokinetic profile of the liposome-entrapped drug
and of the free drug are shown in FIG. 1A as the percent of
injected dose as a function of time. As can be seen, the blood
circulation time of the topoisomerase I inhibitor in
liposome-entrapped form (solid circles) is significantly longer
than the free form of the drug (solid squares). For
MPE-camptothecin, the blood circulation half-life of the
liposome-entrapped drug was 14 hours, compared to about 50 minutes
for the free drug. The blood clearance of the liposome-entrapped
drug in rats was approximately 35-fold lower and the area under the
curve was approximately 1250-fold higher than that of the free
drug. Analytical results indicate that essentially all the drug
remains entrapped in the liposomes in the bloodstream.
[0100] FIG. 1B shows the concentration of MPE-camptothecin in whole
blood after administration of the liposome formulation (solid
circles) and of the free drug to rats. The longer circulation
lifetime results in a higher concentration of the drug in the
blood.
[0101] The anti-tumor efficacy of the MPE-camptothecin liposome
formulation was determined in xenograft tumor models, where
homozygous nude mice were inoculated with human tumor cells of
colon, HT29 origin. Surprisingly, these toxicity and antitumor
efficacy studies showed that liposomal MPE-camptothecin was
significantly more toxic than the free form of the drug at
equivalent doses. These studies and the results will now be
described.
[0102] Liposomes were prepared as set forth in Example 1 to include
entrapped MPE-camptothecin. Nude mice with HT-29 colon xenografts
were treated with liposome-entrapped MPE-camptothecin at dosages of
24 mg/kg, 15 mg/kg and 6 mg/kg or with free MPE-camptothecin at the
same dosages. Treatment began 10 days after tumor inoculation and
doses were administered at days 10, 16 and 23. The tumor volume in
each animal was assessed during and following treatment as
described in Example 2.
[0103] The body weight of each test animal and the tumor volume of
each animal are shown, respectively in FIGS. 2A and 2B, where
animals were treated with liposomal entrapped MPE-camptothecin at
dosages of 24 mg/kg (closed circles), 15 mg/kg (closed triangles)
and 6 mg/kg (closed squares) and with free MPE-camptothecin at
doses of 24 mg/kg (open circles), 15 mg/kg (open triangles) and 6
mg/kg (open squares).
[0104] With respect to the animals treated with the
liposome-entrapped MPE-camptothecin, all of the animals dosed with
15 mg/kg and 24 mg/kg died after two doses due to drug-related
toxicity, with most deaths on day five after the first dose. All of
the animals treated with 6 mg/kg liposome-entrapped
MPE-camptothecin survived until administration of the third dose on
day 23, after which five of the ten animals died within a few days.
The toxicity of the liposome-entrapped MPE-camptothecin is
reflected in the greater body weight losses, as seen in FIG.
2A.
[0105] In contrast, all of the animals treated with the free form
of the drug survived the study, with the exception of one animal in
the 24 mg/kg dosing group that died a few days after the third dose
on day 23.
1 TABLE 1 Number of Number Surviving Animals of after after after
Dose Test dose 1 dose 2 dose 3 Treatment mg/kg Animals (day 9) (day
16) (day 23) Saline na 20 20 20 20 free MPE-camptothecin 24 10 10
10 9 free MPE-camptothecin 15 10 10 10 10 free MPE-camptothecin 6
10 10 10 10 liposome-entrapped 24 10 1 0 0 liposome-entrapped 15 10
5 0 0 liposome-entrapped 6 10 10 10 5
[0106] With respect to antitumor activity of the formulations, the
liposome-entrapped MPE-camptothecin was more effective than the
free form of the drug in inhibiting tumor growth, despite its
greater toxicity. This can be seen in FIG. 2B, where the 6 mg/kg
dose of liposome-entrapped MPE-camptothecin was significantly more
effective in inhibiting tumor growth (log growth rate of -0.026)
than even the highest dose level of free MPE-camptothecin 5 (24
mg/kg, log growth rate 0.0048).
[0107] The complete and partial remission of the tumors in the test
animals was monitored and is presented in Table 2. Complete
remission of a tumor is defined as the elimination of tumor mass
until the end of the experiment. A partial remission is defined as
a tumor volume of less than 50% of the peak tumor volume for an
individual animal.
2TABLE 2 Dose Complete Partial Treatment mg/kg Remission.sup.1
Remission.sup.2 Saline 0/20 0/20 free MPE-camptothecin 24 3/10 1/10
free MPE-camptothecin 15 2/10 0/10 free MPE-camptothecin 6 0/10
0/10 liposome-entrapped 24 --.sup.3 --.sup.3 liposome-entrapped 15
--.sup.3 --.sup.3 liposome-entrapped 6 10/10 na.sup.4
.sup.1complete remission defined as elimination of tumor mass until
experiment termination. .sup.2partial remission defined as a tumor
volume of less than 50% of the peak tumor volume for an individual
animal. .sup.3all 10 animals in test groups died after the second
dose on day 16. .sup.4na = not applicable
[0108] As can be seen in Table 2, the liposome-entrapped
MPE-camptothecin at a dose of 6 mg/kg was effective to cause a
complete remission of tumors in all 10 test animals. This effect
was observed within five days after the second treatment on day 16.
As noted above, five of the test animals in the 6 mg/kg
liposome-entrapped test group died shortly after the third
dose.
[0109] In the surviving five animals, the tumors did not recur by
the end of the study, approximately 30 days after the final
treatment on day 23. Data is unavailable for the animals treated
with 15 mg/kg and 24 mg/kg liposome-entrapped MPE-camptothecin,
since all of the animals in these test groups died due to
drug-related toxicity, as noted above.
[0110] Administration of MPE-camptothecin in free form at a dose of
24 mg/kg resulted in 3 animals with complete tumor remission and 1
animal with partial tumor remission, as seen in Table 2.
[0111] Comparison of the results observed for the drug administered
in free form and in liposome-entrapped form indicate that the drug
is more potent when administered in liposome-entrapped form. In
fact, the liposome-entrapped drug is at least four times more
potent than the free form of the drug, as can be seen by comparing
the results obtained for a 6 mg/kg of liposome-entrapped
MPE-camptothecin dosage to a 24 mg/kg free MPE-camptothecin dosage
(FIG. 2B, Table 2). It is clear from these results that the dose of
liposome-entrapped MPE-camptothecin required for therapeutically
effective anti-tumor therapy is four times lower than the dose
required when the drug is administered in free form.
[0112] Example 2 describes the details of a second study to
determine the maximum tolerated dose and the lowest effective dose
of the liposome-entrapped MPE-camptothecin. In this study,
liposomes were prepared as described in Example 1 and the liposome
formulation was administered to test animals at drug dosages of 0.1
mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg and 5 mg/kg. The free drug was
administered at 20 mg/kg as a comparison.
[0113] Table 3 summarizes the number of test animals in each group,
specifying the number of animals surviving at each dosing phase of
the study. As seen in the table, all of the control, saline treated
animals and all of the animals treated with free MPE-camptothecin
survived for the duration of the study. Of the ten animals treated
with 5 mg/kg liposome-entrapped MPE-camptothecin, four of the
animals died of drug-related toxicity and one additional animal
died of apparently nonspecific causes after the third dose. One of
the ten animals in the test group receiving 3 mg/kg
liposome-entrapped MPE-camptothecin died after the second dose, but
the death was not considered due to drug treatment because of the
absence of any correlating signs of toxicity. All other animals
treated with liposome-entrapped MPE-camptothecin survived the
entire study duration.
3 TABLE 3 Number of Number Surviving Animals of after after after
Dose Test dose 1 dose 2 dose 3 Treatment mg/kg Animals (day 9) (day
16) (day 23) Saline 20 20 20 20 free MPE-camptothecin 20 10 10 10
10 liposome-entrapped 5 10 10 10 5 liposome-entrapped 3 10 10 9 9
liposome-entrapped 1 10 10 10 10 liposome-entrapped 0.5 10 10 10 10
liposome-entrapped 0.1 10 10 10 10
[0114] The results of the study are shown in FIGS. 3A-3B, where
FIG. 3A shows the body weight of mice, in grams, as a function of
days after inoculation with the HT-29 colon tumor. The animals were
treated on days 9, 16 and 23 after tumor inoculation with liposomal
entrapped topoisomerase I inhibitor at dosages of 5 mg/kg (open
triangles), 3 mg/kg (open inverted triangles), 1 mg/kg (open
diamonds), 0.5 mg/kg (open circles) and 0.1 mg/kg (open squares)
and with free drug at a dose of 20 mg/kg (closed squares). As can
be seen in FIG. 3A, body weight changes were dose-related and,
these changes were correlated with other observations of
toxicity.
[0115] FIG. 3B is a similar plot showing tumor volume, in mm.sup.3,
as a function of days after tumor inoculation, where the dosages
are represented by the same symbols as in FIG. 3A. FIG. 3B shows
that both the 5 mg/kg and 3 mg/kg dose levels of liposome-entrapped
MPE-camptothecin were more therapeutically effective in inhibiting
tumor growth than the 20 mg/kg dose of the free drug.
[0116] Treatment with 20 mg/kg of free MPE-camptothecin (log growth
rate of 0.011) was approximately equivalent in antitumor activity
to the 1 mg/kg dosage level of the drug in liposome-entrapped form
(log growth rate of 0.017).
[0117] Table 4 summarizes the complete and partial tumor remission
in the test animals.
4TABLE 4 Complete Partial Dose Remis- Remis- Treatment mg/kg
sion.sup.1 sion.sup.2 Saline 0/20 0/20 free MPE-camptothecin 20
0/10 1/10 liposome-entrapped MPE-camptothecin 5 10/10 na.sup.3
liposome-entrapped MPE-camptothecin 3 7/10 1/10 liposome-entrapped
MPE-camptothecin 1 0/10 0/10 liposome-entrapped MPE-camptothecin
0.5 0/10 1/10 liposome-entrapped MPE-camptothecin 0.1 0/10 0/10
.sup.1Complete remission defined as elimination of tumor mass until
experiment termination. .sup.2Partial remission defined as a tumor
volume of less than 50% of the peak tumor volume for an individual
animal. .sup.3na = not applicable
[0118] There were no complete tumor remissions in the animals
treated with 20 mg/kg of free MPE-camptothecin. In contrast, all
ten of the animals treated with liposome-entrapped MPE-camptothecin
at the 5 mg/kg dosage level had complete remissions.
[0119] At the 3 mg/kg dosage, seven of the animals had complete
remission of their tumor.
[0120] The results from the study of Example 3 shows that antitumor
activity of the liposome-entrapped topoisomerase inhibitor
MPE-camptothecin is significantly better when compared to the free
form of the drug, indicating that the liposome-entrapped form was
about 20-fold more potent since the antitumor activity of the free
drug at a dose of 20 mg/kg was most comparable to the activity of a
1 mg/kg dose of the liposome-entrapped form of the drug. That the 3
mg/kg and 5 mg/kg liposome-entrapped MPE-camptothecin dosages were
significantly more effective in antitumor therapy than the 20 mg/kg
dose of the drug in free form indicates that the therapeutic index
of the drug entrapped in liposomes is approximately four-fold to
five-fold higher than the drug in free form.
[0121] 2. In vivo Adminstration of topotecan
[0122] In another study performed in support of the invention,
topotecan was entrapped in liposomes composed of DSPC and mPEG-DSPE
in a 95:5 molar ratio, as described in Example 4. Early studies,
not reported here, indicated that topotecan was not readily
retained in the liposomes. The lipid bilayer was selected to use a
single component phospholipid having an acyl chain length close to
DSPE in the mPEG-DSPE component. Such a bilayer has minimal packing
defects which arise from imperfections in nearest neighbor
interactions in a solid phase bilayer, which have reduced lateral
and rotational mobility relative to fluid bilayers. In addition, a
dextran-sulfate loading battery was used in order to achieve
precipitation of the topotecan in the liposome interior. Other
polymers, in particular polyanionic polymers, are suitable for this
purpose, such as chondroitin sulfate A, polyvinylsulfuric acid, and
polyphosphoric acid.
[0123] The pre-formed liposomes containing dextran ammounim sulfate
in the central compartment were loaded with topotecan as described
in Example 4. After loading, unentrapped drug was removed by
diafiltration and the liposomes were characterized. The liposomes
were loaded to a drug:lipid ratio of 0.238 and the liposomes had an
average particle diameter of 87 nm.
[0124] The liposomes containing topotecan were administered
intraveneously to rats to determine the blood circulation lifetime.
FIGS. 4A-4B show the plasma concentration of topotecan as a
function of time after administration to rats. FIG. 4A compares the
concentration of liposome-entrapped topotecan administered at 2
mg/kg (solid triangles) to the concentration of free topotecan
administered at the same dosage (solid squares). FIG. 4B compares
the two forms of the drug at a dosage of 5 mg/kg. The calculated
pharmacokinetic parameters are given in Table 5.
5 TABLE 5 Dosage = 2 mg/kg Dosage = 5 mg/kg Free Liposome- Free
Liposome- Parameter Topotecan Entrapped Topotecan Entrapped lasma
Cmax (.mu.g/mL) 2.89 54.5 8.23 119.3 AUC (.mu.g/mL h) 0.57 523 1.57
1140 T 1/2 (h) 0.20 7.2 0.30 9.8 CL (mL/h) 887 0.96 820 1.10 Vol.
Dist. (mL) 173 9.2 278 17.5 elimination rate 3.45 0.096 2.33 0.071
constant (1/h)
[0125] The data in Table 5 shows that the liposome-entrapped drug
has a significantly longer circulation time than the free form of
the drug.
[0126] The efficacy of the liposomes was determined in another
study. As described in Example 4, the liposomes were administered
to mice bearing a subcutaneous xenograft tumor. Tumor-bearing mice
were randomized into six treatment groups of 12 mice for treatment
with one of the following: saline, liposome-entrapped
MPE-camptothecin 4 mg/kg; free topotecan 25 mg/kg; liposome
entrapped topotecan at drug dosages of 2 mg/kg, 5 mg/kg or 8 mg/kg.
All treatments were administered as intravenous bolus injections
given weekly for 3 treatments, specifically on days 9, 16 and
23.
[0127] The tumor size in each animal was measured twice weekly
during the study to evaluate therapeutic efficacy. Body weight of
each animal was monitored twice weekly to assess toxicity of the
formulations. The results are shown in Tables 6 and 7 and in FIGS.
5A-5B.
6TABLE 6 Complete Partial Non- Dose Remis- Remis- Respon- Treatment
mg/kg sion.sup.1 sion.sup.2 sive.sup.3 Saline 0 0 12
liposome-entrapped MPE- 4 8 4 0 camptothecin free topotecan 25 0 1
11 liposome-entrapped topotecan 2 1 2 9 liposome-entrapped
topotecan 5 2 8 2 liposome-entrapped topotecan 8 7 3 2
.sup.1Complete remission defined as elimination of tumor mass until
experiment termination. .sup.2Partial remission defined as a tumor
volume of less than 50% of the peak tumor volume for an individual
animal. .sup.3Non-responsive defined as a tumor volume equal to or
greater than initial tumor volume.
[0128] As can be seen from FIGS. 5A and Table 6, left untreated the
tumors grew at a rate of 17.8 mm.sup.3 per day for the duration of
the study. The animals treated with liposome-entrapped
MPE-camptothecin (positive control animals) experienced a tumor
growth rate -1.2 mm.sup.3 per day for the duration of the study.
Animals treated with nonencapsulated topotecan, which was
administered at 25 mg/kg somewhat below the maximum tolerated
dosage (MTD) of 40 mg/kg, had tumor growth of 14.1 mm.sup.3 per
day. Animals treated with liposome-entrapped topotecan had tumor
growth of 0.9 mm.sup.3 per day for a dosage of 2 mg/kg, -1.9
mm.sup.3 per day for a dosage of 5 mg/kg and -0.8 mm.sup.3 per day
for a dosage of 8 mg/kg. The negative growth rate indicates
regression of tumor size below the starting tumor volume.
[0129] The size of treated tumors as a function of the size of
control tumors (%T/C) was examined for all treatment groups and is
summarized in Table 6. The National Cancer Institute defines
significant anti-tumor activity as a %T/C less than 42.
7TABLE 7 Dose % T/C.sup.1 % T/C % T/C Treatment mg/kg Day 29 Day 33
Day 36 liposome-entrapped MPE- 4 1.8 0.6 1.9 camptothecin free
topotecan 25 82.8 79.0 85.9 liposome-entrapped topotecan 2 19.5
12.9 16.3 liposome-entrapped topotecan 5 10.5 5.6 5.6
liposome-entrapped topotecan 8 2.0 2.2 2.2 .sup.1% T/C defined as
the average tumor volume at day indicated over the average tumor
volume of the control, saline treated animals.
[0130] 3. In vivo Adminstration of CKD-602
[0131] Example 5 describes another study conducted in support of
the invention using the topoisomerase inhibitor CKD-602. The drug
was remotely loaded into liposomes against an ammonium-sulfate
gradient with dextran as a trapping agent. The liposome lipid
composition was identical to that used for the study using
topotecan--HSPC and mPEG-DSPE in a 95/5 mole ratio.
[0132] FIG. 6 is a plot showing the plasma concentration of CKD-602
as a function of time after administration to rats at a dosage of 1
mg/kg. The liposome-entrapped form of the drug (solid circles) had
a calculated half-life of 9.8 hours and an AUC of 274 .mu.g/mL/hr.
The free form of the drug had a calculated half-life of 0.2 hours
and an AUC of 0.37 .mu.g/mL/hr.
[0133] Therapeutic efficacy of the CKD-602 formulation was
evaulated using mice hearing a HT-20 colon cancer xenograft.
Seventy-two mice were inoculated with HT-29 tumor cells and nine
days later were randomized into six treatment groups. The animals
in each group were treated with one of the following formulations:
saline, liposome-entrapped MPE-camptothecin 4 mg/kg; free CKD-602
20 mg/kg; liposome entrapped CKD-602 at drug dosages of 1 mg/kg, 2
mg/kg or 4 mg/kg. All treatments were administered as intravenous
bolus injections given weekly for 3 treatments, specifically on
days 11, 18 and 25.
[0134] The tumor size in each animal was measured twice weekly
during the study to evaluate therapeutic efficacy. Body weight of
each animal was monitored twice weekly to assess toxicity of the
formulations. The results are shown in Tables 8 and 9 and in FIGS.
7A-7B.
8TABLE 8 Complete Partial Non- Dose Remis- Remis- Respons-
Treatment mg/kg sion.sup.1 sion.sup.2 ive.sup.3 Saline 0/10 0/10
10/10 liposome-entrapped MPE- 4 6/10 0/10 4/10 camptothecin free
CKD602 20 0/6 0/6 6/6 liposome-entrapped CKD602 1 2/10 7/10 1/10
liposome-entrapped CKD602 2 6/10 2/10 2/10 liposome-entrapped
CKD602 4 4/4 0/4 0/4 .sup.1Complete remission defined as
elimination of tumor mass until experiment termination.
.sup.2Partial remission defined as a tumor volume of less than 50%
of the peak tumor volume for an individual animal.
.sup.3Non-responsive defined as a tumor volume equal to or greater
than initial tumor volume.
[0135] As can be seen in Table 8 and in FIG. 7B, the animals
treated with saline experienced continuous tumor growth, at a rate
of 15.45 mm.sup.3 per day for the duration of the study. The
animals treated with the liposome-entrapped MPE-camptothecin
(positive control animals)had a tumor growth rate of -0.63 mm.sup.3
per day for the duration of the study. Animals treated with free,
unentrapped CKD602 had tumor growth of 15.21 mm.sup.3 per day.
Animals treated with liposomal CKD602 had tumor growth of -2.21
mm.sup.3 per day for animals treated with a dose of 1 mg/kg, -0.96
mm.sup.3 per day for a dose of 2 mg/kg and -2.37 mm.sup.3 per day
for a dose of 4 mg/kg. The negative growth rate indicates
regression of tumor size below the starting tumor volume.
[0136] The size of treated tumors as a function of the size of
control tumors (%T/C) was examined for all treatment groups and is
summarized in Table 9. The National Cancer Institute defines
significant anti-tumor activity as a %T/C less than 42.
9TABLE 9 Dose % T/C.sup.1 % T/C % T/C Treatment mg/kg Day 29 Day 33
Day 36 liposome-entrapped MPE- 4 2.9 2.3 1.6 camptothecin free
CKD602 20 129.1 120.1 99.9 liposome-entrapped CKD602 1 11.4 7.7 4.4
liposome-entrapped CKD602 2 4.8 2.8 1.6 liposome-entrapped CKD602 4
1.0 1.3 0.9 .sup.1% T/C defined as the average tumor volume at day
indicated over the average tumor volume of the control, saline
treated animals.
IV. EXAMPLES
[0137] The following examples illustrate methods of preparing,
characterizing, and using the composition of the present invention.
The examples are in no way intended to limit the scope of the
invention.
Materials
[0138] The topoisomerase inhibitor
(7-(4-methyl-piperazino-methylene)-10,1-
1-ethylenedioxy-20(S)-camptothecin trifluoroacetate (GI147211)
(MPE-camptothecin), was provided by Glaxo Research Institute,
Research Triangle Park, N.C. CKD602
(7-(2-(N-isopropylamino)ethyl)-(20S)-camptothe- cin) was provided
by Chong Kun Dang Corporation, Seoul Korea. Topotecan
(Hycamtin.RTM.) was purchased commercially.
[0139] Materials for preparation of the liposomes and all other
reagents were from commercially available sources.
Methods
[0140] Animal Studies: Homozygous nude mice were obtained from
Taconic Farms (Germantown, N.Y.) and allowed to acclimate for 7
days prior to initiation of the experiment. Animals were housed in
appropriate isolated caging with ad lib sterile rodent food and
acidified water and a 12:12 light:dark cycle. Animals were
randomized into treatment groups prior to tumor inoculation based
on body weight. Randomization was confirmed based on tumor size
immediately prior to initiation of treatment.
[0141] Tumors: Tumors were inoculated by trochar placement of
fragments from rapidly growing tumors on donor animals. The human
colon cancer cell line, HT-29, was used to initiate subcutaneous
xenograft tumors. Cultured cells were trypsinized, washed, counted
and resuspended at 50 million cells per mL normal growth media.
Tumors were inoculated by injection of 0.1 mL (5 million cells) at
the back of the neck. Tumors were allowed to grow to an average
size of 100 mm.sup.3 prior to initiation of treatment.
[0142] Monitoring: All animals were observed daily for general
well-being throughout the experiments. Animals were weighed prior
to tumor inoculation and weekly thereafter. Tumors were measured
twice weekly throughout the experiment, beginning 5-10 days after
tumor inoculation. Any animal observed to have 15% or greater
weight loss from the initial starting weight and any animal
observed to have greater than 4,000 mm.sup.3 tumor volume were
excluded from the study.
Example 1
Preparation of Liposomes with Entrapped Topoisomerase Inhibitor
[0143] Liposomes were prepared and loaded with a selected
topoisomerase inhibitor as follows.
[0144] A. Liposome Preparation
[0145] The lipids hydrogenated soy phosphaticylcholine (HSPC),
cholesterol (Chol) and mPEG-DSPE (at a ratio of 56.4:38.3:5.3
mol/mol) were dissolved in ethanol at 65.degree. C. in a 250 mL
round bottom. The lipids were agitated continuously for at least 30
minutes at 65.degree. C. The total lipid concentration in ethanol
solution was 3.7 g total lipid per 10 mL ethanol.
[0146] The dissolved lipid solution was transferred to another 250
mL round bottom flask containing 100 mL of 250 mM ammonium sulfate
solution equilibrated to 65.degree. C. The ethanol:lipid:ammonium
sulfate hydration mixture was mixed continuously for at least one
hour while maintaining the temperature using a 65.degree. C. water
bath to form oligolamellar ethanol hydration liposomes.
[0147] The oligolamellar liposomes were size reduced using a Lipex
thermobarrel extruder to pass the hydration mixture through
polycarbonate membranes with known pore size dimensions. The
mixture was passed 5 times through a 0.20 .mu.m pore diameter
membrane, followed by 10 passes through a 0.10 .mu.m pore diameter
membrane. The extruded liposomes contained ammonium sulfate within
the interior aqueous compartment(s) of the liposomes, as well as in
the exterior aqueous bulk phase medium in which they are suspended.
The sized liposomes were stored in the refrigerator until
diafiltration preceding the remote loading procedure.
[0148] 100 mg of a selected topoisomerase inhibitor,
MPE-camptothecin, CKD-602 or topotecan, was dissolved in 40 mL 10%
sucrose solution to yield a concentration of 2.5 mg/mL. After
dissolution, the solution was passed through a 0.20 .mu.m filter to
remove insoluble particulates.
[0149] B. Remote Loading of Liposomes
[0150] Ammonium sulfate and ethanol were removed from the external
bulk aqueous phase immediately prior to remote loading by hollow
fiber tangential flow diafiltration with a 100 KDa nominal
molecular weight cutoff cartridge. Constant feed volume was
maintained, and at least seven exchange volumes were used resulting
in liposomes suspended in an exterior aqueous phase comprised of
10% sucrose.
[0151] After diafiltration, the liposomes were mixed with a
selected drug solution at a ratio (drug solution:liposomes) of 1:4
(vol/vol) and rapidly warmed to 65.degree. C. using a
pre-equilibrated jacketed vessel containing water. The temperature
of the mixture was maintained at 65.degree. C. for 40 to 60
minutes, after which the mixture was rapidly cooled in an ice-water
bath. After remote loading, a sample of the liposomes was taken to
check for the presence of crystals, to determine percent
encapsulation and to measure the mean particle diameter.
[0152] Unencapsulated drug was removed from the bulk phase medium
by hollow fiber tangential flow diafiltration using a 100 kDa
nominal molecular weight cutoff cartridge. At least eight exchange
volumes were used, resulting in liposomally encapsulated drug
suspended in an external aqueous phase comprised of 10% sucrose 10
millimolar Histidine pH 6.5.
[0153] The final liposome preparation was sterile filtered using a
0.22 .mu.m cellulose acetate syringe filter and stored refrigerated
and protected from light until use.
[0154] C. Characterization of Liposomes
[0155] Percent encapsulation was determined using size exclusion
chromatography to compare the percent drug in the void volume
(liposomal encapsulated) to the total drug (void volume plus
included volume). Drug concentration in the column fractions was
determined by absorbance. Mean particle diameter was determined
using quasielectric laser light scattering (QELS). The total lipid
concentration was assayed at the post-sterile filtration stage in
order to determine the drug to lipid ratio. Liposomes loaded with
MPE-camptothecin, topotecan and
7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin (CKD-602) were
prepared and characterized. The results are shown in the table
below.
10 liposome-entrapped liposome-entrapped liposome-entrapped
Parameter MPE-camptothecin Topotecan CKD-602 lot no. 221AZ43A
221AZ43B 221AZ53 Total Lipid 17.81 .mu.mol/mL 15.97 .mu.mol/mL
14.079 .mu.mol/mL concentration Drug concentration 2.69 mg/mL 1.72
mg/mL 1.77 mg/mL (4.55 .mu.mol/mL) (3.76 .mu.mol/mL) (3.77
.mu.mol/mL) drug:lipid ratio 0.26 0.24 0.27 (mol/mol) (1:3.92)
(1:4.25) (1:3.73) Mean Particle 99 nm 95.4 nm 96.7 nm diameter
Percent Encapsulation 96.4% 99.9% 95.3%
Example 2
In vivo Efficacy of Liposome-entrapped MPE-Camptothecin
[0156] Liposomes containing entrapped MPE-camptothecin were
prepared as described in Example 1. The liposome entrapped drug and
the free drug were diluted in 5% dextrose in water as required to
achieve the desired concentrations.
[0157] Nude mice were inoculated with the human colon cancer cell
line HT-29 as described above in the methods section. Seventy mice
were randomized to one of seven treatment groups as follows: free
drug at 24 mg/kg, 15 mg/kg or 6 mg/kg; liposome entrapped drug at
24 mg/kg, 15 mg/kg or 6 mg/kg; saline. Treatment was initiated when
average tumor volume was approximately 75 mm.sup.3 on day 10
post-tumor inoculation. All treatments were administered as
intravenous bolus injections given weekly for 3 treatments,
specifically on days 10, 16 and 23.
[0158] Tumor size during and following each experiment was used as
the primary evaluation of therapeutic efficacy. Body weight was
evaluated to assess toxicity. All tumor bearing animals were
observed following cessation of treatment, until euthanized based
on criteria above. Experiments were concluded when a majority of
control tumors achieved the maximal allowed volume (4,000
mm.sup.3).
[0159] Tumor size in each animal was measured repeatedly at various
time points, thus these measurements were regarded as correlated
information. Since the tumor sizes over time after treatment were
of interest, repeated measurement analyses was done for each data
set. By examining the data, a log transformation seemed reasonable.
Let Y denote the original tumor measurement, let Z=log(Y+1). After
transforming data, repeated measurement analyses was done for the
transformed data Z. The SAS procedure PROC MIXED was used. The log
growth rate for each treatment group was calculated and used to
compare the different treatment groups. Statistical significance
was declared at the 0.05 level, but due to multiple comparisons,
adjustment to the type I error were done and a P-value of
<0.0033 indicated a statistically significant difference in any
designated comparison.
[0160] The results are summarized in Tables 1 and 2 and in FIGS.
2A-2B.
Example 3
Dose Finding Study for Liposome-entrapped MPE-Camptothecin
[0161] Liposomes containing entrapped MPE-camptothecin were
prepared as described in Example 1. The liposome entrapped drug and
the free drug were diluted in 5% dextrose in water as required to
achieve the desired concentrations.
[0162] Nude mice were inoculated with the human colon cancer cell
line HT-29 as described above in the methods section. Seventy mice
were randomized to one of seven treatment groups as follows:
liposome entrapped drug at 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg,
5 mg/kg or 20 mg/kg; and saline. Treatment was initiated when
average tumor volume was approximately 75 mm.sup.3 on day 9
post-tumor inoculation. All treatments were administered as
intravenous bolus injections given weekly for 3 treatments,
specifically on days 9, 16 and 23.
[0163] The tumor size was evaluated and analyzed as described in
Example 2, and the results are shown in Tables 3 and 4 and in FIGS.
3A-3B.
Example 4
In vivo Efficacy of Liposome-Entrapped Topotecan
[0164] A. Liposome Preparation
[0165] Liposomes containing topotecan were prepared as follows.
[0166] The lipids distearoylphospatidylcholine (DSPC) and
(N-(carbonyl-methoxypolyethylene glycol
2000)-1,2-distearoyl-sn-glycero-3- -phosphoethanolamine)
(mPEG-DSPE) were combined at a molar ratio of 95:5 and dissolved in
ethanol at 70.degree. C. using continuous agitation. The lipid
concentration in the ethanol solution was 8.9 grams per 10 mL
ethanol.
[0167] Dextran sulfate-ammonium salt was prepared by ion exchange
chromatography using dextran sulfate sodium salt as the starting
material. A 100 mg/mL solution of dextran sulfate ammonium salt was
prepared by dissolving dextran sulfate sodium salt in water and
adjusting the solution pH to 5 using ammonium hydroxide.
[0168] 100 mL of dextran sulfate solution was heated to 70.degree.
C. and combined with the ethanol solution of lipid while mixing to
form oligolamellar liposomes. The temperature of the oligolamellar
ethanol hydration liposome dispersion was maintained at 70.degree.
C. for one hour with continuous mixing.
[0169] The post hydration mixture was heated to 70 degrees and size
reduced using a Lipex thermobarrel extruder through a series of
polycarbonate membranes to arrive at a particle size near 100 nm
mean particle diameter. Typically, the sequence involved 5 passes
through an 0.2 .mu.m pore diameter membrane, followed by 10 passes
through an 0.1 .mu.m pore diameter membrane.
[0170] Unentrapped dextran sulfate polymer and remaining ethanol
were removed from the external bulk aqueous phase immediately prior
to the active drug loading step with eight volume exchanges using
350 mM sodium chloride solution, followed by eight volume exchanges
using a 10% sucrose solution. The diafiltration cartridge employed
had a specified nominal molecular weight cutoff of 100,000
Daltons.
[0171] A solution of topotecan was prepared at a concentration of
2.5 mg/mL in 10% sucrose. The drug solution and diafiltered
liposomes were combined at a volume ratio of 4:1, and the
temperature of the resulting mixture was raised to 70.degree. C.
and maintained with constant stirring for one hour. Active drug
loading was terminated by rapidly cooling the post-loading mixture
using an ice water bath.
[0172] Unentrapped drug was removed by diafiltration employing a
cartridge having nominal molecular weight cutoff of 100,000
Daltons. Typically, 8-10 volume exchanges were employed using 10%
sucrose 10 mM Histidine pH 6.5 as the exchange buffer.
[0173] Drug concentration was adjusted to the final value by
assaying for potency with a uv-vis absorbance measurement and
diluting accordingly.
[0174] The final process step involved sterile grade filtration
employing a 0.22 .mu.m filter prior to filling vials.
[0175] B. Liposome Characterization
[0176] Percent encapsulation was determined using size exclusion
chromatography to determine the percent drug in the void volume
("liposomal drug") to the total amount recovered in both the
included and void volume fractions. Drug concentration was
monitored using uv-vis absorbance spectrophotometry. Mean particle
diameter was determined using quasielastic laser light scattering.
Total lipid was determined using phosphorous assay.
[0177] The results are summarized in the table below.
11 Liposome-entrapped Parameter Topotecan total lipid 17.2 mg/mL
total drug 2.1 mg/mL drug:lipid ratio (mol:mol) 0.238 mean particle
diameter 87.3 nm percent encapsulation 98.8
[0178] C. In vivo Pharmacokinetics and Efficacy
[0179] Seventy two mice were inoculated with HT-29 cancer cells as
described above in the methods section. Nine days after tumor
inoculation, the animals were treated weekly with one of the
following intravenous treatments: saline; liposome-entrapped
MPE-camptothecin 4 mg/kg; free topotecan 25 mg/kg; liposome
entrapped topotecan at drug dosages of 2 mg/kg, 5 mg/kg or 8 mg/kg.
All treatments were administered as intravenous bolus injections
given weekly for 3 treatments, specifically on days 9, 16 and
23.
[0180] The tumor size was evaluated and analyzed as described in
Example 2, and the results are shown in Tables 6 and 7 and in FIGS.
5A-5B.
Example 5
In vivo Efficacy of Liposome-Entrapped CKD-602
[0181] A. Liposome Preparation and Characterization
[0182] Liposomes containing CKD-602 were prepared as described in
Example 4, except using a drug solution of CKD-602. The liposomes
were characterized as described in Example 4 and the results are
summarized below.
12 Liposome entrapped Parameter CKD-602 total lipid 12.5 mg/mL
total drug 2.07 mg/mL drug:lipid ratio (mol:mol) 0.315 mean
particle diameter 92.8 nm percent encapsulation 94.7
[0183] B. In vivo Pharmacokinetics and Efficacy
[0184] Seventy two mice were inoculated with HT-29 cancer cells as
described above in the methods section. Eleven days after tumor
inoculation, the animals were treated weekly with one of the
following intravenous treatments: saline, liposome-entrapped
MPE-camptothecin 4 mg/kg; free CKD602 20 mg/kg; liposome entrapped
CKD602 at drug dosages of 1 mg/kg, 2 mg/kg or 4 mg/kg.
[0185] All treatments were administered as intravenous bolus
injections given weekly for 3 treatments, specifically on days 11,
18 and 25.
[0186] The tumor size in each animal was measured twice weekly
during the study to evaluate therapeutic efficacy. Body weight of
each animal was monitored twice weekly to assess toxicity of the
formulations. The results are shown in Tables 8 and 9 and in FIGS.
7A-7B.
[0187] Although the invention has been described with respect to
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications can be made without
departing from the invention.
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