U.S. patent application number 14/030563 was filed with the patent office on 2014-05-01 for targeted liposomes.
This patent application is currently assigned to Georgetown University. The applicant listed for this patent is Georgetown University. Invention is credited to Esther H. CHANG, SangSoo Kim, Antonina Rait.
Application Number | 20140120157 14/030563 |
Document ID | / |
Family ID | 50547457 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120157 |
Kind Code |
A1 |
CHANG; Esther H. ; et
al. |
May 1, 2014 |
TARGETED LIPOSOMES
Abstract
The present invention is in the field of drug delivery, and
specifically, cationic liposome-based drug delivery. In
embodiments, this invention provides methods of making
ligand-targeted (e.g., antibody- or antibody fragment-targeted)
liposomes useful for the delivery of liposomes to tumors, including
brain tumors. In embodiments, the liposomes deliver temozolomide
across the blood-brain barrier for treatment of primary or
metastatic brain tumors. Additional cancers that can be treated
with the liposomes include neuroendocrine tumors, melanoma,
prostate, head and neck, ovarian, lung, liver, kidney, breast,
urogenital, gastric, colorectal, cervical, vaginal, angiosarcoma,
liposarcoma, rhabdomyosarcoma, choriocarcinoma, pancreatic,
retinoblastoma and other types of cancer. In another embodiment the
liposomes deliver melphalan for the treatment of multiple myeloma,
other tumors of the blood or other solid tumors. In still other
embodiments the liposomes can deliver other drugs such as
pemetrexed or irinotecan for treatment of cancer or drugs including
atropine for treatment of organophosphate poisoning.
Inventors: |
CHANG; Esther H.; (Potomac,
MD) ; Kim; SangSoo; (Gaithersburg, MD) ; Rait;
Antonina; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Georgetown University |
Washington |
DC |
US |
|
|
Assignee: |
Georgetown University
Washington
DC
|
Family ID: |
50547457 |
Appl. No.: |
14/030563 |
Filed: |
September 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61767453 |
Feb 21, 2013 |
|
|
|
61702796 |
Sep 19, 2012 |
|
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Current U.S.
Class: |
424/450 ;
424/178.1 |
Current CPC
Class: |
A61K 9/1272 20130101;
A61K 9/1271 20130101; A61K 9/0019 20130101; A61K 45/06
20130101 |
Class at
Publication: |
424/450 ;
424/178.1 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of preparing a targeted active agent cationic liposome
complex, comprising: (a) preparing a lipid solution comprising one
or more cationic lipids in ethanol; (b) preparing a solution of an
active agent selected from temozolomide, melphalan and atropine;
(c) mixing the lipid solution with the solution of active agent;
(d) injecting the mixture of lipid and active anent into an aqueous
solution, thereby forming an active agent cationic liposome; (e)
mixing the active agent cationic liposome with a ligand to form the
targeted active agent cationic liposome, wherein the ligand is
directly complexed with, but not chemically conjugated to, the
cationic liposome.
2. The method of claim 1, wherein the ligand is an antibody, an
antibody fragment or a protein.
3. The method of claim 2, wherein the ligand is a single chain Fv
antibody fragment.
4. (canceled)
5. (canceled)
6. The method of claim 1, wherein the lipid solution comprises
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
7. The method of claim 1, wherein the solution of active agent is
prepared at a concentration of about 1 mM to about 200 mM.
8. (canceled)
9. The method of claim 1, wherein the molar ratio of lipid:active
agent is about 0.1:1 to about 5:1.
10. (canceled)
11. (canceled)
12. The method of claim 1, wherein the weight ratio of ligand:lipid
is about 0.01:1 to about 0.5:10.
13-24. (canceled)
25. A method of treating cancer in a patient, comprising
administering to the patient a targeted active agent cationic
liposome complex, wherein the targeted active agent cationic
liposome complex comprises: (a) a cationic liposome comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) an active
agent selected from temozolomide and melphalan; and (c) a ligand
directly complexed with, but not chemically conjugated to, the
cationic liposome.
26. The method of claim 25, wherein the ligand is an antibody, an
antibody fragment or a protein.
27. The method of claim 26, wherein the ligand is a single chain Fv
antibody fragment.
28. (canceled)
29. The method of claim 25, wherein the active agent is
administered to the patient at a dose of about 10 mg/m.sup.2 to
about 500 mg/m.sup.2.
30. (canceled)
31. The method of claim 25, wherein the molar ratio of lipid:active
agent in the cationic liposomes is about 0.1:1 to about 5:1.
32. (canceled)
33. (canceled)
34. The method of claim 25, wherein the weight ratio of
ligand:lipid in the cationic liposome is about 0.01:1 to about
0.5:10.
35. (canceled)
36. (canceled)
37. The method of claim 25, wherein the administration is
intravenous (IV), intratumoral (IT), intralesional (IL), sublingual
(SL), aerosal, percutaneous, oral, endoscopic, topical,
intramuscular (IM), intradermal (ID), intraocular (IO),
intraperitoneal (IP), transdermal (TD), intranasal (IN),
intracereberal (IC), intraorgan (e.g. intrahepatic), slow release
implant, or subcutaneous administration, or via administration
using an osmotic or mechanical pump
38. The method of claim 25, wherein the cancer is head and neck
cancer, breast cancer, prostate cancer, pancreatic cancer, brain
cancer, neuroendocrine cancer, cervical cancer, lung cancer, liver
cancer, kidney cancer, liposarcoma, angiosarcoma, rhabdomyosarcoma,
choriocarcinoma, melanoma, retinoblastoma, ovarian cancer, vaginal
cancer, urogenital cancer, gastric cancer, colorectal cancer,
multiple myeloma or a cancer of the blood.
39. The method of claim 38, wherein the brain cancer is a glioma,
astrocytoma or a glioblastoma.
40. The method of claim 25, further comprising administering an
additional different therapy to the patient in combination with the
targeted active agent cationic liposome complex.
41. The method of claim 40, wherein the additional different
therapy comprises administration of a chemotherapeutic agent, a
small molecule, radiation therapy or a nucleic acid-based
therapy.
42. The method of claim 41, wherein the nucleic acid-based therapy
comprises administration of a cationic liposome complex comprising
an antisense oligonucleotide, an siRNA, an miRNA, a plasmid DNA or
an shRNA
43-54. (canceled)
55. A method of treating cancer in a patient, comprising
administering to the patient a targeted active agent cationic
liposome complex prepared by the method of claim 1.
56. The method of claim 55, wherein the ligand is an antibody, an
antibody fragment or a protein.
57. The method of claim 56, wherein the ligand is a single chain Fv
antibody fragment.
58. (canceled)
59. The method of claim 55, wherein active agent is administered to
the patient at a dose of about 10 mg/m.sup.2 to about 500
mg/m.sup.2.
60. (canceled)
61. The method of claim 55, wherein the molar ratio of lipid:active
agent is about 0.1:1 to about 5:1.
62. (canceled)
63. (canceled)
64. The method of claim 55, wherein the weight ratio of
ligand:lipid is about 0.01:1 to about 0.5:10.
65. (canceled)
66. (canceled)
67. The method of claim 55, wherein the administration is
intravenous (IV), intratumoral (IT), intralesional (IL), aerosal,
percutaneous, oral, endoscopic, topical, intramuscular (IM),
intradermal (ID), sublingual (SL), intraocular (IO),
intraperitoneal (IP), transdermal (TD), intranasal (IN),
intracereberal (IC), intraorgan (e.g. intrahepatic), slow release
implant, or subcutaneous administration, or via administration
using an osmotic or mechanical pump
68. The method of claim 55, wherein the cancer is head and neck
cancer, breast cancer, prostate cancer, pancreatic cancer, brain
cancer, neuroendocrine cancer, cervical cancer, lung cancer, liver
cancer, kidney cancer, liposarcoma, angiosarcoma, rhabdomyosarcoma,
choriocarcinoma, melanoma, retinoblastoma, ovarian cancer, vaginal
cancer, urogenital cancer, gastric cancer, colorectal cancer,
multiple myeloma or a cancer of the blood.
69. (canceled)
70. The method of claim 55, further comprising administering an
additional different therapy to the patient in combination with the
targeted active agent cationic liposome complex.
71. The method of claim 70, wherein the additional different
therapy comprises administration of a chemotherapeutic agent, a
small molecule, radiation therapy or a nucleic acid-based
therapy.
72. The method of claim 71, wherein the nucleic acid-based therapy
comprises administration of a cationic liposome complex comprising
an antisense oligonucleotide, an siRNA, an miRNA, a plasmid DNA or
an shRNA
73-153. (canceled)
154. A method of treating organophosphate poisoning in a patient,
comprising administering to the patient a targeted atropine
cationic liposome complex, wherein the targeted atropine cationic
liposome complex comprises: (a) a cationic liposome comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) atropine;
and (c) a ligand directly complexed with, but not chemically
conjugated to, the cationic liposome.
155. The method of claim 154, wherein the ligand is an antibody, an
antibody fragment or a protein.
156. The method of claim 155, wherein the ligand is a single chain
Fv antibody fragment.
157. (canceled)
158. The method of claim 154, wherein the atropine is administered
to the patient at a dose of about 1 mg to about 10 mg.
159. (canceled)
160. The method of claim 154, wherein the molar ratio of
lipid:atropine in the cationic liposome: atropine is about 0.1:1 to
about 5:1.
161. (canceled)
162. (canceled)
163. The method of claim 154, wherein the weight ratio of
ligand:lipid in the cationic liposome is about 0.01:1 to about
0.5:10.
164. (canceled)
165. (canceled)
166. The method of claim 154, wherein the administration is
intravenous (IV), intratumoral (IT), intralesional (IL), sublingual
(SL), aerosal, percutaneous, oral, endoscopic, topical,
intramuscular (IM), intradermal (ID), intraocular (IO),
intraperitoneal (IP), transdermal (TD), intranasal (IN),
intracereberal (IC), intraorgan (e.g. intrahepatic), slow release
implant, or subcutaneous administration, or via administration
using an osmotic or mechanical pump.
167. The method of claim 154, wherein the liposome crosses the
blood-brain barrier.
168-240. (canceled)
241. The method of claim 25, wherein the liposome crosses the
blood-brain barrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Patent Application Nos. 61/702,796, filed Sep. 19,
2012, and 61/767,453, filed Feb. 21, 2013, the disclosures of each
of which are incorporated by reference herein in their
entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is in the field of drug delivery, and
specifically, cationic liposome-based drug delivery. In
embodiments, this invention provides methods of making
ligand-targeted (e.g., antibody- or antibody fragment-targeted)
liposomes useful for the delivery of liposomes to tumors, including
brain tumors. In embodiments, the liposomes deliver temozolomide
across the blood-brain barrier for treatment of primary or
metastatic brain tumors. Additional cancers that can be treated
with the liposomes include, but are not limited to, neuroendocrine
tumors, melanoma, prostate, head and neck, ovarian, lung, liver,
breast, kidney, urogenital, gastric, colorectal, vaginal, cervical,
liposarcoma, angiosarcoma, rhabdomyosarcoma, choriocarcinoma,
pancreatic, retinoblastoma, multiple myeloma and other types of
cancer. In another embodiment the liposomes deliver melphalan for
the treatment of multiple myeloma, other tumors of the blood or
other solid tumors. In still other embodiments the liposomes can
deliver other drugs such as atropine, pemetrexed or irinotecan
across the blood-brain barrier.
BACKGROUND OF THE INVENTION
[0004] Primary brain tumors, and particularly gliomas, are one of
the most difficult cancers to treat. In addition to primary tumors,
metastatic brain cancer from a variety of primary sources
[predominately lung (60%), breast (20%) and melanoma (10%)], is
diagnosed in over 150,000 patients a year (Newton H and Malkin M
(2010) Neurologic Complications of Systemic Cancer and
Antineoplastic Therapy. Informa Healthcare). Thus, there is a
critical need for improved therapies for brain cancers, which is
confirmed by the fact that the NCI has made brain cancers one of
its top 5 funding priorities. The lack of improvement in the
prognosis of patients with brain cancer over the last few years,
despite recent advances in drug discovery and development of
targeted therapies, is due in large part to the inability of the
therapeutics to cross the blood-brain barrier (BBB) (Blakeley, J.
(2008): Drug delivery to brain tumors. Current Neurology &
Neuroscience Reports, 8:235-241).
[0005] The current standard of therapy for glioblastoma multiforme
(GBM) is surgical resection, followed by radiotherapy and
chemotherapy with Temozolomide (TMZ). TMZ, a second-generation
alkylating (methylating) agent causes cytotoxic DNA lesions, is
also approved for treatment of anaplastic astrocytoma (AA) and is
in clinical trials for treatment of brain metastases from other
non-CNS solid tumors. The mechanism of action and pharmacological
properties have been recently reviewed (Tentori L and Graziani G
(2009) Recent Approaches to Improve the Antitumor Efficacy of
Temozolomide. Current Medicinal Chemistry 16: pp 245-257; and
Mrugala M M, Adair J and Kiem H P (2010) Temozolomide: Expanding
Its Role in Brain Cancer. Drugs of Today 46: pp 833-846). TMZ is
relatively well tolerated (Jiang G, Wei Z P, Pei D S, Xin Y, Liu Y
Q and Zheng J N (2011) A Novel Approach to Overcome Temozolomide
Resistance in Glioma and Melanoma: Inactivation of MGMT by Gene
Therapy. Biochemical and Biophysical Research Communications 406:
pp 311-314), however myelosuppression, neutropenia and
thrombocytopenia are among its side effects and therapeutic dosages
are limited by these. Extended TMZ dosing regimens were also found
to provoke lymphocytopenia and opportunistic infections (Tentori L
and Graziani G (2009) Recent Approaches to Improve the Antitumor
Efficacy of Temozolomide. Current Medicinal Chemistry 16: pp
245-257). The extensive tissue distribution that results from the
non-tumor specific uptake of the orally administered TMZ is a major
cause of these side effects. Thus, tumor-targeting delivery of TMZ
could help reduce these adverse events.
[0006] TMZ has shown survival benefit in a subset of GBM patients,
however this median increase is only 2.5 months compared to
radiation alone (Chamberlain M C (2010) Temozolomide: Therapeutic
Limitations in the Treatment of Adult High-Grade Gliomas. Expert
Review of Neurotherapeutics 10: pp 1537-1544). Recent studies have
also indicated that 60-75% of GBM patients and 50% of AA patients
do not benefit from TMZ (Chamberlain M C (2010) Temozolomide:
Therapeutic Limitations in the Treatment of Adult High-Grade
Gliomas. Expert Review of Neurotherapeutics 10: pp 1537-1544). The
failure of chemotherapy can be attributed to a number of factors
including, short half-life in circulation, efflux of drugs from the
tumor by p-glycoprotein, resistance of the tumors to the drug and
failure to cross the blood-brain barrier. The primary mechanism of
resistance to TMZ is overexpression of
O.sup.6-methylguanine-DNA-methyl transferase (MGMT), which repairs
the TMZ-induced DNA lesion by removing the O.sup.6-guanine adducts
(Mrugala M M, Adair J and Kiem H P (2010) Temozolomide: Expanding
Its Role in Brain Cancer. Drugs of Today 46: pp 833-846). Thus, a
means to down modulate MGMT activity, for example via the tumor
specific delivery of the p53 tumor suppressor gene, would enhance
the therapeutic effect of TMZ.
[0007] There is, therefore, an urgent need to develop new therapies
for treatment of brain and other cancers. The present invention
fulfills these needs by providing a cationic-liposome-based drug
delivery system for delivery of temozolomide.
BRIEF SUMMARY OF THE INVENTION
[0008] In embodiments, methods of preparing a targeted temozolomide
cationic liposome complex are provided. Such methods suitably
comprise preparing a lipid solution comprising one or more cationic
lipids in ethanol, preparing a solution of temozolomide, mixing the
lipid solution with the solution of temozolomide, injecting the
mixture of lipid and temozolomide into an aqueous solution, thereby
forming a temozolomide cationic liposome, and mixing the
temozolomide cationic liposome with a ligand to form the targeted
temozolomide cationic liposome, wherein the ligand is directly
complexed with, but not chemically conjugated to, the cationic
liposome.
[0009] Suitably, the ligand is an antibody (targeted for example,
but not limited to, to the Transferrin receptor, Folate receptor,
HER-2 receptor, the vesicular glutamate transporter type 1 [VGluT1]
receptor), an antibody fragment or a protein such as Transferrin or
Folate, including a single chain Fv antibody fragment, such as an
anti-transferrin receptor single chain Fv (TfRscFv).
[0010] Suitably the solution of temozolomide is prepared in
dimethyl sulfoxide (DMSO). Suitably the solution of temozolomide is
prepared at a concentration of about 1 mM to about 200 mM, or about
50 mM to about 200 mM.
[0011] In embodiments the lipid solution comprises
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phospsphothanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol.
[0012] Suitably the molar ratio of lipid:Temozolomide,
lipid:melphalan lipid:atropine or lipid:irinotecan is about 0.1:1
to about 5:1, more suitably about 0.5:1 to about 2:1 or about 1:1.
Suitably the concentration of the liposome is about 1 mM to about 2
mM, or about 2 mM to about 10 mM
[0013] In embodiments, the weight ratio of ligand:lipid is in the
range of about 1:10 to about 1:50, suitably about 1:20 to about
1:40 (w:w). Suitably, the weight ratio of ligand:lipid is about
0.01:1 to about 0.05:1, more suitably about 0.03:1 to about 0.04:1.
In embodiments the weight ratio of TfRscFv:lipid is about
0.033:1.
[0014] Suitably, a method of preparing a targeted temozolomide
cationic liposome complex is provided. In embodiments, the methods
comprise preparing a lipid solution comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol in ethanol, preparing a solution of
Temozolomide in DMSO, water or other appropriate solvent such as a
buffer solution such as a Phosphate buffer or a HEPES buffer or a
TRIS buffer, mixing the lipid solution with the solution of
temozolomide, injecting the mixture of lipid and temozolomide into
an aqueous solution, thereby forming a temozolomide cationic
liposome, mixing the temozolomide cationic liposome with an
anti-transferrin receptor single chain Fv (TfRscFv) to form the
targeted temozolomide cationic liposome complex, wherein the
TfRscFv is directly complexed with, but not chemically conjugated
to, the cationic liposome.
[0015] In another embodiment, a method of preparing a targeted
melphalan cationic liposome complex is provided. In embodiments,
the methods comprise preparing a lipid solution comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol in ethanol, preparing a solution of melphalan
in DMSO, water or other appropriate solvent such as a buffer
solution such as a Phosphate buffer or a HEPES buffer or a TRIS
buffer, mixing the lipid solution with the solution of melphalan,
injecting the mixture of lipid and melphalan into an aqueous
solution, thereby forming a melphalan cationic liposome, mixing the
melphalan cationic liposome with a ligand, including for example,
an anti-transferrin receptor single chain Fv (TfRscFv), to form the
targeted melphalan cationic liposome complex, wherein the ligand
(e.g., TfRscFv) is directly complexed with, but not chemically
conjugated to, the cationic liposome.
[0016] In another embodiment, a method of preparing a targeted
atropine cationic liposome complex is provided. In embodiments, the
methods comprise preparing a lipid solution comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol in ethanol, preparing a solution of atropine in
ethanol, DMSO, water or other appropriate solvent such as a buffer
solution such as a Phosphate buffer or a HEPES buffer or a TRIS
buffer, mixing the lipid solution with the solution of atropine,
injecting the mixture of lipid and atropine into an aqueous
solution, thereby forming a atropin cationic liposome, mixing the
atropine cationic liposome with a ligand, including for example, an
anti-transferrin receptor single chain Fv (TtRscFv), to form the
targeted atropine cationic liposome complex, wherein the ligand
(e.g., TfRscFv) is directly complexed with, but not chemically
conjugated to, the cationic liposome.
[0017] In another embodiment, a method of preparing a targeted
irinitecan cationic liposome complex is provided. In embodiments,
the methods comprise preparing a lipid solution comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol in ethanol, preparing a solution of irinotecan
in ethanol, DMSO, water or other appropriate solvent such as a
buffer solution such as a Phosphate buffer or a HEPES buffer or a
TRIS buffer, mixing the lipid solution with the solution of
irinotecan, injecting the mixture of lipid and irinotecan into an
aqueous solution, thereby forming an irinotecan cationic liposome,
mixing the irinotecan cationic liposome with a ligand, including
for example, an anti-transferrin receptor single chain Fv
(TfRscFv), to form the targeted atropine cationic liposome complex,
wherein the ligand (e.g., TfRscFv) is directly complexed with, but
not chemically conjugated to, the cationic liposome.
[0018] In further embodiments, methods of treating cancer in a
patient, comprising administering to the patient a targeted
temozolomide cationic liposome complex are provided. Suitably the
targeted temozolomide cationic liposome complex comprises a
cationic liposome comprising 1,2-dioleoyl-3-trimethylammonium
propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE) or a mixture of dimethyldioctadecylammonium bromide (DDAB)
and DOPE, with or without cholesterol, temozolomide and a ligand
complexed with, but not chemically conjugated to, the cationic
liposome.
[0019] In further embodiments, methods of treating cancer in a
patient, comprising administering to the patient a targeted
melphalan cationic liposome complex are provided. Suitably, the
targeted melphalan cationic liposome complex comprises a cationic
liposome comprising 1,2-dioleoyl-3-trimethylammonium propane
(DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or
a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE,
with or without cholesterol, melphalan and a ligand complexed with,
but not chemically conjugated to, the cationic liposome.
[0020] In further embodiments, methods of treating cancer in a
patient, comprising administering to the patient a targeted
atropine cationic liposome complex are provided. Suitably, the
targeted atropine cationic liposome complex comprises a cationic
liposome comprising 1,2-dioleoyl-3-trimethylammonium propane
(DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or
a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE,
with or without cholesterol, atropine and a ligand complexed with,
but not chemically conjugated to, the cationic liposome.
[0021] In further embodiments, methods of treating cancer in a
patient, comprising administering to the patient a targeted
irinotecan cationic liposome complex are provided. Suitably, the
targeted atropine cationic liposome complex comprises a cationic
liposome comprising 1,2-dioleoyl-3-trimethylammonium propane
(DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or
a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE,
with or without cholesterol, irinotecan and a ligand complexed
with, but not chemically conjugated to, the cationic liposome.
[0022] In embodiments, the administration is intravenous (IV),
intratumoral (IT), intralesional (IL), aerosal, percutaneous, oral,
endoscopic, topical, intramuscular (IM), intradermal (ID),
intraucular (IO), iitraperiwineal (IP), sublingual (SL),
transdermal (TD), intranasal (IN), intracereberal (IC), intraorgan
(e.g. intrahepatic), slow release implant, or subcutaneous
administration, or via administration using an osmotic or
mechanical pump
[0023] Suitably the cancer being treated is a brain cancer, for
example a glioma, glioblastoma or an astrocytoma.
[0024] In other embodiments, the cancer being treated is, but is
not limited to, a primary or metastatic brain tumor, neuroendocrine
tumors, melanoma, prostate, head and neck, ovarian, lung, kidney,
liver, breast, vaqginal, urogenital, gastric, colorectal, cervical,
liposarcoma, angiosarcoma, rhabdomyosarcoma, choriocarcinoma,
pancreatic, retinoblastoma, multiple myeloma and other types of
cancer.
[0025] In another embodiment the liposomes deliver melphalan for
the treatment of multiple myeloma.
[0026] In still other embodiments the liposomes can deliver other
drugs such as atropine, pemetrexed or irinotecan and/or derivatives
thereof.
[0027] In embodiments, the methods further comprise administering
an additional therapy to the patient in combination with the
targeted temozolomide, melphalan, atropine, premetrexed or
irinotecan cationic liposome complex. Suitably the additional
therapy comprises administration of a chemotherapeutic agent, a
small molecule, radiation therapy or a nucleic acid-based therapy.
In embodiments, the nucleic acid-based therapy comprises
administration of a cationic liposome complex comprising a plasmid
DNA expressing wild-type p53, or an oligonucleotide such as an
siRNA, an miRNA or an shRNA. In embodiments the additional therapy
comprises administration of any molecule that down-regulates,
modifies or otherwise negates the effect of MGMT in the cancer
cell. In additional embodiments the additional therapy comprises
administration of any molecule that interferes with the production
of acetylcholine, or binding of acetylcholine to its receptor.
[0028] In embodiments, methods of treating brain cancer in a
patient, comprising administering to the patient a targeted
temozolomide cationic liposome complex are provided. Suitably the
targeted temozolomide cationic liposome complex comprises a
cationic liposome comprising 1,2-dioleoyl-3-trimethylammonium
propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE or a mixture of dimethyldioctadecylammonium bromide (DDAB)
and DOPE, with or without cholesterol, temozolomide and an
anti-transferrin receptor single chain Fv (TfRscFv) complexed with,
but not chemically conjugated to, the cationic liposome.
[0029] Methods of treating cancer in a patient, are also provided,
suitably comprising administering to the patient a targeted
temozolomide cationic liposome complex or targeted inirotecan
cationic liposome complex prepared by the methods described
herein.
[0030] In still other embodiments, methods of treating multiple
myeloma in a patient, comprising administering to the patient a
targeted cationic liposome complex, are provided. Suitably the
targeted melphalan cationic liposome complex comprises a cationic
liposome comprising 1,2-dioleoyl-3-trimethylammonium propane
(DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE or
a mixture of dimethyldioctadecylammonium bromide (DDAB) and DOPE,
with or without cholesterol,), melphalan and an anti-transferrin
receptor single chain Fv (TfRscFv) directly complexed with, but not
chemically conjugated to, the cationic liposome.
[0031] In still other embodiments, methods of treating any cancer
in a patient, comprising administering to the patient a targeted
cationic liposome complex are provided. Suitably the targeted
melphalan cationic liposome complex comprises a cationic liposome
comprising 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol, melphalan and an anti-transferrin receptor
single chain Fv (TfRscFv) directly complexed with, but not
chemically conjugated to, the cationic liposome.
[0032] Methods of treating cancer in a patient, are also provided,
suitably comprising administering to the patient a targeted
melphalan cationic liposome complex prepared by the methods
described herein.
[0033] Methods are also provided of treating a brain cancer in a
patient, comprising administering to the patient a cationic
liposome complex comprising a cationic liposome comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol, a plasmid DNA expressing wild-type p53 and an
anti-transferrin receptor single chain Fv (TfRscFv) directly
complexed with, but not chemically conjugated to, the cationic
liposome; and temozolomide.
[0034] In still other embodiments, methods of treating any cancer
in a patient, comprising administering to the patient a targeted
cationic liposome complex are provided. Suitably the targeted
irinotecan cationic liposome complex comprises a cationic liposome
comprising 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or a mixture
of dimethyldioctadecylammonium bromide (DDAB) and DOPE, with or
without cholesterol, irinotecan and an anti-transferrin receptor
single chain Fv (TfRscFv) directly complexed with, but not
chemically conjugated to, the cationic liposome.
[0035] Methods of treating cancer in a patient, are also provided,
suitably comprising administering to the patient a targeted
irinotecan cationic liposome complex prepared by the methods
described herein.
[0036] Further embodiments, features, and advantages of the
embodiments, as well as the structure and operation of the various
embodiments, are described in detail below with reference to
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0037] FIG. 1 shows U251 cell survival versus concentration of
Temozolomide (TMZ) for free TMZ and three cationic liposomes
comprising TMZ, as described herein.
[0038] FIG. 2 shows U87 cell survival versus concentration of TMZ
for free TMZ and cationic liposomes comprising TMZ, as described
herein.
[0039] FIG. 3A shows T98G cell survival versus concentration of TMZ
for free TMZ and a targeted cationic liposome comprising TMZ, as
described herein.
[0040] FIG. 3B shows KMS-11 cell survival versus concentration of
TMZ for free TMZ and a targeted cationic liposome comprising TMZ,
as described herein.
[0041] FIG. 4A shows fluorescence intensity of the brain tumors
after systemic delivery of a targeted cationic
liposome-fluorescently labeled oligonucleotide using the
Maestro.TM. in vivo fluorescence imaging system.
[0042] FIG. 4B shows magnetic resonance imaging (MRI) images of
U87MG-luc2 glioblastoma tumor xenografts in response to treatment
with free TMZ and a TfRscFv targeted cationic liposome-TMZ complex
(scL-TMZ).
[0043] FIG. 5 shows the size of brain tumors measured by MRI after
treatment with free TMZ or scL-TMZ.
[0044] FIG. 6 shows bioluminescence imaging of tumors in mice
treated with free TMZ and scL-TMZ.
[0045] FIG. 7 shows additional bioluminescence imaging of tumors in
mice treated with free TMZ and scL-TMZ.
[0046] FIG. 8 shows the quantification of bioluminescence signal
intensities of tumors in mice treated with free TMZ and
scL-TMZ.
[0047] FIG. 9 shows the quantification of bioluminescence signal
intensities of tumors in mice treated with free TMZ and
scL-TMZ.
[0048] FIG. 10 shows body weight measurements of mice during and
after treatment with free TMZ and scL-TMZ.
[0049] FIG. 11 shows a Kaplan-Meier plot demonstrating long term
survival for animals treated with free TMZ and scL-TMZ.
[0050] FIG. 12 shows a Kaplan-Meier plot demonstrating long term
survival for animals treated with free TMZ and scL-TMZ at different
doses.
[0051] FIG. 13 shows weights of brain tumors demonstrating the
effect of free TMZ and scL-TMZ.
[0052] FIG. 14 shows results of flow cytometric analysis for the
level of apoptosis as determined by cleaved caspase-3 antibody
staining of single cells isolated from brain tumors.
[0053] FIG. 15 shows tumor size for free TMZ and scL-TMZ treated
mice.
[0054] FIG. 16 shows body weight of mice treated with free TMZ and
scL-TMZ.
[0055] FIG. 17 shows TUNEL staining of CD133+ CSCs and CD133-
non-CSCs isolated from subcutaneous T98G xenograft tumors after
treatment.
[0056] FIG. 18 shows levels of apoptosis assessed by cleaved
caspase-3 antibody staining of SSEA-1+ CSCs from subcutaneous T98G
brain tumors after treatment.
[0057] FIG. 19 shows T98G cell survival versus concentration of TMZ
for free TMZ, scL-TMZ, as described herein and the combination
scL-TMZ along with targeted cationic liposomes expressing the p53
gene.
[0058] FIG. 20 shows tumor-targeted delivery of systemically
administered scL-6FAM-ODN to U251 xenograft brain tumors.
[0059] FIG. 21 shows flow cytometric analysis of scL-delivered
6FAM-ODN uptake in CD133+ and CD133- non-CSC cells isolated from
U251 xenograft tumors after systemic administration.
[0060] FIG. 22 shows tumor specific targeting of CSCs in IC GBM by
scL-Delivered ODN after systemic administration.
[0061] FIG. 23 shows XTT assay after the addition of the TMZ to the
cells and IC.sub.50 values.
[0062] FIG. 24 shows bioluminescence imaging of U87 IC tumors using
Xenogen showing the synergistic effect of the combination of
SGT-53+TMZ.
[0063] FIG. 25 shows the synergistic effect of SGT-53 plus TMZ on
intracranial (IC) U87 glioblastoma muliforme (GBM).
[0064] FIG. 26 shows the synergistic Effect of the combination of
systemically administered scL-p53 plus TMZ.
[0065] FIG. 27 shows Kaplan-Meier plots demonstrating SGT-53
sensitization to TMZ treatment significantly enhances survival in
an intracranial U87 model of GBM.
[0066] FIG. 28 shows Kaplan-Meier plots demonstrating SGT-53
sensitization to TMZ treatment significantly enhances survival in
an intracranial TMZ Resistant Model of GBM (T98G Cells).
[0067] FIG. 29 shows percent of tumor cells in apoptosis
post-treatment as indicated.
[0068] FIG. 30 shows down modulation of MGMT expression in T98G
TMZ-resistant brain tumor cells and SQ xenograft tumors by systemic
complex p53 gene therapy.
[0069] FIG. 31 shows down modulation of MGMT expression in T98G
TMZ-resistant intracranial brain tumors by systemic
TfRscFv-cationic liposome-p53 plasmid DNA (scL-p53) complex gene
therapy.
[0070] FIG. 32 shows a proposed dosing schedule for scL-p53.
[0071] FIG. 33 shows KMS-11 cell survival versus concentration of
melphalan (MEL) for free MEL and a targeted cationic liposome
comprising MEL, as described herein.
[0072] FIG. 34 shows comparison of unencapsulated MEL, with Lip-MEL
without the targeting moiety and with the full TfRscFv-cationic
liposome-melphalan complex (scL-MEL), as well as with liposome
only.
[0073] FIG. 35 shows comparison of unencapsulated MEL, with fresh
and lyophilized scL-MEL complex.
[0074] FIG. 36 shows the effect of targeted scL-p53 therapy on
KMS-11 cells.
[0075] FIG. 37 shows the effect of free (unencapsulated) MEL alone,
scL-MEL alone, or the combination of free (unencapsulated) or scL
encapsulated MEL plus scL-p53 on KMS-11 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0076] It should be appreciated that the particular implementations
shown and described herein are examples and are not intended to
otherwise limit the scope of the application in any way.
[0077] The published patents, patent applications, websites,
company names, and scientific literature referred to herein are
hereby incorporated by reference in their entireties to the same
extent as if each was specifically and individually indicated to be
incorporated by reference. Any conflict between any reference cited
herein and the specific teachings of this specification shall be
resolved in favor of the latter. Likewise, any conflict between an
art-understood definition of a word or phrase and a definition of
the word or phrase as specifically taught in this specification
shall be resolved in favor of the latter.
[0078] As used in this specification, the singular forms "a," "an"
and "the" specifically also encompass the plural forms of the terms
to which they refer, unless the content clearly dictates otherwise.
The term "about" is used herein to mean approximately, in the
region of, roughly, or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
20%.
[0079] Technical and scientific terms used herein have the meaning
commonly understood by one of skill in the art to which the present
application pertains, unless otherwise defined. Reference is made
herein to various methodologies and materials known to those of
ordinary skill in the art.
[0080] In embodiments, methods of preparing targeted temozolomide
cationic liposome complexes are provided. Suitably, the methods
comprise preparing a lipid solution comprising one or more cationic
lipids in ethanol. A solution oftemozolomide is prepared. The lipid
solution is mixed with the solution of temozolomide. The mixture of
cationic lipid and temozolomide is injected into an aqueous
solution, thereby forming a temozolomide cationic liposome. The
temozolomide cationic liposome is then mixed with a ligand to form
the targeted temozolomide cationic liposome complex. Suitably, the
ligand is directly complexed with, but not chemically conjugated
to, the cationic liposome.
[0081] In embodiments, methods of preparing targeted melphalan
cationic liposome complexes are provided. Suitably, the methods
comprise preparing a lipid solution comprising one or more cationic
lipids in ethanol. A solution of melphalan is prepared, suitably in
absolute ethanol containing enough hydrochloric acid to facilitate
dissolving the melphalan. The lipid solution is mixed with the
solution of melphalan. The mixture of cationic lipid and melphalan
is injected into an aqueous solution, thereby forming a melphalan
cationic liposome. The melphalan cationic liposome is then mixed
with a ligand to form the targeted melphalan cationic liposome
complex. Suitably, the ligand is directly complexed with, but not
chemically conjugated to, the cationic liposome.
[0082] In embodiments, methods of preparing targeted atropine,
irinotecan or premetrexed cationic liposome complexes are provided.
Suitably, the methods comprise preparing a lipid solution
comprising one or more cationic lipids in ethanol. A solution of
atropine, irinotecan or premetrexed is prepared in an appropriate
solvent such as absolute ethanol, DMSO, water or a buffer solution
such as a Phosphate buffer or a HEPES buffer or a TRIS buffer. The
lipid solution is mixed with the solution of atropine, irinotecan
or premetrexed. The mixture of cationic lipid and atropine,
irinotecan or premetrexed is injected into an aqueous solution,
thereby forming a cationic atropine, irinotecan or premetrexed
liposome. The atropine, irinotecan or premetrexed cationic liposome
is then mixed with a ligand to form the targeted atropine,
irinotecan or premetrexed cationic liposome complex. Suitably, the
ligand is directly complexed with, but not chemically conjugated
to, the cationic liposome.
[0083] The terms "complex," "nanocomplex," "liposome complex" and
"cationic liposome complex" and "cationic immunoliposome complex"
are used interchangeably throughout to refer to the cationic
liposomes of the present invention.
[0084] Described throughout are various active agents that can be
encapsulated in the liposome complexes described. It is to be
understood that derivates of such active agents (e.g.,
hydrochloride salts as well as other derivates) are also
encompassed by the disclosure.
[0085] As described herein, temozolomide is a second-generation
alkylating (methylating) agent causes cytotoxic DNA lesions, and is
also approved for treatment of anaplastic astrocytoma (AA). The
structure of temozolomide shown below, has an empirical formula:
C.sub.6H.sub.6N.sub.6O.sub.2.
##STR00001##
In embodiments, a salt of temozolomide, e.g., an HCl salt, can also
be used in the methods described herein.
[0086] Suitably, the solution of temozolomide (TMZ) is prepared in
dimethyl sulfoxide (DMSO) or other appropriate solvent. The
solution of TMZ can be prepared at any desired concentration. In
embodiments, the concentration of TMZ in the solution is about 0.1
mM to about 500 mM, more suitably about 1 mM to about 200 mM, about
50 mM to about 200 mM, about 50 mM to about 100 mM, or about 50 mM,
about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM,
about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150
mM, about 160 mM, about 170 mM, about 180 mM, or about 200 mM.
[0087] Melphalan is an antineoplastic agent belonging to the class
of nitrogen mustard alkylating agents. An alkylating agent adds an
alkyl group (C.sub.nH.sub.2n+1) to DNA. It attaches the alkyl group
to the guanine base of DNA, at the number 7 nitrogen atom of the
imidazole ring. The structure of melphalan shown below, has an
empirical formula: C.sub.13H.sub.18C.sub.12N.sub.2O.sub.2.
##STR00002##
[0088] Suitably, the solution of melphalan is prepared in absolute
ethanol containing enough hydrochloric acid to facilitate
dissolving the melphalan or other appropriate solvent. The solution
of melphalan can be prepared at any desired concentration. In
embodiments, the concentration of melphalan in the solution is
about 0.1 mM to about 500 mM, more suitably about 1 mM to about 200
mM, about 50 mM to about 200 mM, about 50 mM to about 100 mM, or
about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM,
about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140
mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, or
about 200 mM.
[0089] Irinotecan is the hydrochloride salt of a semisynthetic
derivative of camptothecin, a cytotoxic, quinoline-based alkaloid
extracted from the Asian tree Camptotheca acuminata. Irinotecan, a
prodrug, is converted to a biologically active metabolite
7-ethyl-10-hydroxy-camptothecin (SN-38) by a
carboxylesterase-converting enzyme. One thousand-fold more potent
than its parent compound irinotecan, SN-38 inhibits topoisomerase I
activity by stabilizing the cleavable complex between topoisomerase
I and DNA, resulting in DNA breaks that inhibit DNA replication and
trigger apoptotic cell death. Because ongoing DNA synthesis is
necessary for irinotecan to exert its cytotoxic effects, it is
classified as an S-phase-specific agent. Empirical Formula (Hill
Notation) C.sub.33H.sub.38N.sub.4O.sub.6
##STR00003##
##STR00004##
[0090] The terms "ironotecan" and "irinotecan hydrochloride" are
used interchangeably throughout.
[0091] Suitably, the solution of irinotecan is prepared in DMSO or
other appropriate solvent. The solution of irinotecan can be
prepared at any desired concentration. In embodiments, the
concentration of irinotecan in the solution is about 0.1 mM to
about 500 mM, more suitably about 1 mM to about 200 mM, about 50 mM
to about 200 mM, about 50 mM to about 100 mM, or about 50 mM, about
60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about
110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM,
about 160 mM, about 170 mM, about 180 mM, or about 200 mM.
[0092] Suitably, the solution of atropine or premetrexed is
prepared in an appropriate solvent such as absolute ethanol, DMSO,
water or a buffer solution such as a Phosphate buffer or a HEPES
buffer or a TRIS buffer. The solution of atropine, or premetrexed
can be prepared at any desired concentration. In embodiments, the
concentration of atropine, or premetrexed in the solution is about
0.1 mM to about 500 mM, more suitably about 1 mM to about 200 mM,
about 50 mM to about 200 mM, about 50 mM to about 100 mM, or about
50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about
100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM,
about 150 mM, about 160 mM, about 170 mM, about 180 mM, or about
200 mM.
[0093] A wide variety of lipids are useful in the methods described
herein. Published PCT application WO 99/25320 describes the
preparation of several cationic liposomes. Examples of suitable
lipids include phosphatidylcholine (PC), phosphatidylserine (PS),
as well as mixtures of dioleoyltrimethylammonium propane (DOTAP)
and dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol
(chol); a mixture of dimethyldioctadecylammonium bromide (DDAB) and
DOPE with or without cholesterol. The ratio of the lipids can be
varied to optimize the efficiency of loading of the TMZ, melphalan,
atropine, pemetrexed or irinotecan and uptake in the specific
target cell type. The liposome can comprise a mixture of one or
more cationic lipids and one or more neutral or helper lipids. A
desirable ratio of cationic lipid(s) to neutral or helper lipid(s)
is about 1:(0.5-3), preferably 1:(1-2) (molar ratio). Exemplary
lipids for use in preparing the cationic liposomes described herein
are well known in the art and include, for example,
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
[0094] Examples of ratios of various lipids useful in the practice
of methods described herein include, but are not limited, to:
TABLE-US-00001 LipA DOTAP/DOPE 1:1 molar ratio LipB DDAB/DOPE 1:1
molar ratio LipC DDAB/DOPE 1:2 molar ratio LipD DOTAP/Chol 1:1
molar ratio LipE DDAB/Chol 1:1 molar ratio LipG DOTAP/DOPE/Chol
2:1:1 molar ratio LipH DDAB/DOPE/Chol 2:1:1 molar ratio (DOTAP =
1,2-dioleoyl-3-trimethylammonium propane, DDAB =
dimethyldioctadecylammonium bromide; DOPE =
dioleoylphosphatidylethanolamine; chol = cholesterol).
[0095] As described herein, the lipids are suitably prepared in
ethanol (e.g., absolute ethanol) prior to preparing the complexes
described herein.
[0096] Following solubilization in ethanol of the lipid components
of the complexes, the appropriate amount of TMZ, melphalan
atropine, irinotecan or premetrexed, dissolved in DMSO, ethanol,
hydrochloric acid or other suitable solvent such as a buffer
solution such as a Phosphate buffer or a HEPES buffer or a TRIS
buffe, is added to the lipid mixture. Suitably, the lipid mixture
is maintained at a temperature of about 50-60.degree. C., prior to
and during the addition of the TMZ, melphalan, atropine, irinotecan
or premetrexed.
[0097] To prepare the liposomes, the solution of lipids and TMZ,
melphalan, atropine, irinotecan or premetrexed is injected into an
aqueous solution to form the liposomes. As used herein "injected"
means to force or drive the solution of lipids and TMZ, melphalan,
atropine, irinotecan or premetrexed into an aqueous solution.
Suitably, the aqueous solution is water, though additional buffers
and salts can be present in the aqueous solution. In embodiments,
the aqueous solution is endotoxin free LAL reagent water (suitably
having an endotoxin content of <0.005 EU/ml) (BioWhittaker).
Suitably the injection is carried out utilizing a syringe or
similar device to produce the liposomes. In embodiments, the
aqueous solution is stirred rapidly during addition of the
lipid/TMZ, melphalan, atropine, irinotecan or premetrexed solution
so as to facilitate liposome formation.
[0098] It has been unexpectedly found that no extrusion or
sonication is required to form the liposomes having the desired
size and Zeta Potential characteristics, according to the methods
described herein. In embodiments, evaporation, sonication, milling
and/or extrusion of the liposomes is specifically excluded from the
disclosed methods. In further embodiments, the methods of preparing
targeted cationic liposomes described throughout suitably consist
of or consist essentially of the recited elements. In such
embodiments, addition of steps such as evaporation, sonication
and/or extrusion, are considered a material alteration to such
methods and thus are specifically excluded from such methods that
consist essentially of the recited elements.
[0099] Preparation of liposomes by mixing the lipids (in
Chloroform) together, evaporating to dryness and reconstituting
with water containing the drug in solution (a common procedure for
liposome encapsulation of drugs), did not produce a homogeneous
population. Measurement by light scattering gave poor results, with
the quality report indicating that the cumulant fit error was high,
thus the data quality was too poor for cumulant analysis, and the
sample too polydisperse for cumulant analysis. The Z-Average (d-nm)
for this preparation was 743.9 nm (number average).
[0100] The temozolomide, melphalan, atropine, irinotecan or
premetrexed cationic liposomes formed according to the injection
methods described herein are then mixed with a ligand to form the
targeted temozolomide, melphalan, atropine, irinotecan or
premetrexed cationic liposomes. As described throughout, the ligand
is directly complexed with, but not chemically conjugated to, the
cationic liposome. In other embodiments, the ligand can be
chemically conjugated to the cationic liposome.
[0101] As used herein the term "ligand" refers to any suitable
targeting moiety that can be either chemically conjugated to, or
directly associated/complexed with, but not chemically conjugated
to, the cationic liposomes. In embodiments where the ligand is
directly associated/complexed with, but not chemically conjugated
to the cationic liposomes, no linker, spacer or other bridging
molecule is used to complex the ligands to the liposomes. Exemplary
ligands for use in the practice of the present invention include,
but are not limited to, proteins (e.g., transferrin or folate),
peptides (e.g., L-37pA), antibodies, antibody fragments (including
Fab' fragments and single chain Fv fragments (scFv)) and sugars
(e.g., galactose), as well as other targeting molecules.
[0102] In exemplary embodiments, a whole antibody or an antibody
fragment can be used as the ligand to make the complexes of this
invention. In a suitable embodiment, an antibody fragment is used,
including Fab fragments and single chain Fv fragments (scFv) of an
antibody. One suitable antibody is an anti-Transferrin receptor
(anti-TfR) monoclonal antibody, and a suitable antibody fragment is
an scFv based on an anti-TfR monoclonal antibody (TfRscFv). An scFv
contains the complete antibody binding site for the epitope of the
TfR recognized by this MAb as a single polypeptide chain of
approximate molecular weight 26,000. An scFv is formed by
connecting the component VH and VL variable domains from the heavy
and light chains, respectively, with an appropriately designed
peptide, which bridges the C-terminus of the first variable region
and N-terminus of the second, ordered as either VH-peptide-VL or
VL-peptide-VH. Additional ligands, such as those described
throughout, can also be used in the practice of the present
invention.
[0103] In one embodiment, a cysteine moiety is added to the
C-terminus of the scFv. Although not wishing to be bound by theory,
it is believed that the cysteine, which provides a free sulfhydryl
group, may enhance the formation of the complex between the
antibody and the liposome in both the chemically conjugated and
non-chemically conjugated embodiments. With or without the
cysteine, the protein can be expressed in E. coli inclusion bodies
and then refolded to produce the antibody fragment in active
form.
[0104] Suitable ligands, for example, proteins/peptides, antibody
or antibody fragments, are those which will bind to the surface of
the target cell, and preferably to a receptor that is
differentially expressed on the target cell. The ligands are mixed
with the cationic liposome at room temperature and at a ligand
(e.g., protein, antibody or antibody fragment):lipid ratio
(weight:weight) in the range of about 1:10 (0.1:1) to about 1:50,
suitably about 1:20 to about 1:40 (w:w). Suitably, the weight ratio
of ligand:lipid is about 0.1:10 to about 0.5:10, about 0.3:10 to
about 0.4:10, or about 0.33:10, including any ratio within these
ranges. The ligand (e.g., the protein/peptide, antibody or antibody
fragment) and the liposome are allowed to incubate at room
temperature for a short period of time, typically for about 10-15
minutes.
[0105] The size of the liposome complex is typically within the
range of about 5-1000 nm as measured by dynamic light scattering
using a Malvern ZETASIZER.RTM. 3000 or a Malvrn ZETASIZER.RTM.
NANO-ZS. See U.S. Published Patent Application No. 2003/0044407 and
U.S. patent application Ser. No. 11/520,796, the disclosures of
which are incorporated by reference herein in their entireties. The
size of the liposomes is demonstrated by a single peak,
representing a homogenous size population. More suitably, the size
of the liposome complex prior to the addition of the ligand is in
the range of about 5 nm to about 500 nm, about 5 nm to about 300
nm, about 5 nm to about 200 nm, about 5 nm to about 100 nm, about
10 nm to about 70 nm, or about 20 nm to about 60 nm. The size of
the liposome complex following addition of the ligand is suitably
in the range of about 5 nm to about 800 nm, about 10 nm to about
500 nm, about 20 nm to about 400 nm, about 20 nm to about 200 nm,
or about 30 nm to about 200 nm.
[0106] Suitably the liposomes described herein have a positive Zeta
Potential. Suitably the Zeta Potential of the liposomes prior to
the addition of the ligand are about 1 mV to about 200 mV, about 1
mV to about 100 mV, about 10 mV to about 100 mV, about 20 mV to
about 60 mV, or about 30 mV to about 50 mV. Suitable the Zeta
Potential of the liposomes following the addition of the ligand are
about 1 mV to about 200 mV, about 1 mV to about 100 mV, about 10 mV
to about 80 mV, about 10 mV to about 60 mV, or about 25 mV to about
50 mV.
[0107] In embodiments, liposomes used to form the complex as
described herein are sterically stabilized liposomes. Sterically
stabilized liposomes are liposomes into which a hydrophilic
polymer, such as PEG, poly(2-ethylacrylic acid), or
poly(n-isopropylacrylamide (PNIPAM) has been integrated. Such
modified liposomes can be particularly useful, as they typically
are not cleared from the bloodstream by the reticuloendothelial
system as quickly as are comparable liposomes that have not been so
modified. To make a sterically stabilized liposome complex of the
present invention, a cationic liposome comprising temozolomide,
melphalan, atropine, irinotecan or premetrexed is prepared as
above. To this liposome is added a solution of a PEG polymer in a
physiologically acceptable buffer at a ratio of about 0.1:100 (nmol
of PEG:nmol of liposome), suitably, about 0.5:50, for example,
about 1:40 (nmol of PEG:nmol of liposome). The resultant solution
is incubated at room temperature for a time sufficient to allow the
polymer to integrate into the liposome complex. The ligand (e.g.,
protein/peptide, antibody or antibody fragment) then is mixed with
the stabilized liposome complex at room temperature and at a ligand
(e.g., protein):lipid ratio in the range of about 1:5 to about 1:40
(w:w).
[0108] As described herein, the ligand (e.g., protein/peptide,
antibody or antibody fragment) is suitably directly associated
(complexed) with the liposome via an interaction (e.g.,
electrostatic, van der Walls, or other non-chemically conjugated
interaction) between the ligand and the liposome. In general, a
linker or spacer molecule (e.g., a polymer or other molecule) is
not used to attach the ligands and the liposome when non-chemically
conjugated.
[0109] As described herein, in additional embodiments, the ligand
(e.g., protein/peptide, antibody or antibody fragment) is
chemically conjugated to the cationic liposomes, for example, via a
chemical interaction between the cationic liposome which contains a
maleimidyl group or other sulfhydryl-reacting group, and a sulfur
atom on the ligand (e.g., protein/peptide, antibody or antibody
fragment). Such methods of direct chemical conjugation are
disclosed in U.S. patent application Ser. No. 09/914,046, filed
Oct. 1, 2001, the disclosure of which is incorporated by reference
herein in its entirety.
[0110] Suitable ratios of lipid:temozolomide for use in the methods
and liposomes are described throughout. In exemplary embodiments,
the molar ratio of lipid:temozolomide is about 0.1:1 to about
1:100, about 0.05:1 to about 1:50, about 1:1 to about 1:20, about
2:1 to about 10:0.1, about 0.5:1 to about 2:1, or about 1:1.
[0111] Suitable ratios of lipid:melphalan for use in the methods
and liposomes are described throughout. In exemplary embodiments,
the molar ratio of lipid:melphalan is about 0.1:1 to about 1:100,
about 0.5:1 to about 1:50, about 1:1 to about 1:20, about 2:1 to
about 10:0.1, about 0.5:1 to about 2:1, or about 1:1.
[0112] Suitable ratios of lipid:atropine, irinotecan or premetrexed
for use in the methods and liposomes are described throughout. In
exemplary embodiments, the molar ratio of lipid:atropine,
irinotecan or premetrexed is about 0.1:1 to about 1:100, about
0.5:1 to about 1:50, about 1:1 to about 1:20, about 2:1 to about
10:0.1, about 0.5:1 to about 2:1, or about 1:1.
[0113] Encapsulation efficiency for the TMZ liposomes is suitably
in the range of about 20% to about 100%, about 20% to about 80%,
about 20% to about 60%, or about 30% to about 55%, encapsulated
TMZ. This is a surprising and unexpected result of the ethanol
injection method for encapsulating TMZ in the liposomes.
[0114] Encapsulation efficiency for the melphalan liposomes is
suitably in the range of about 20% to about 100%, about 20% to
about 80%, about 20% to about 60%, or about 20% to about 40%,
encapsulated melphalan. This is a surprising an unexpected result
of the ethanol injection method for encapsulating melphalan in the
liposomes.
[0115] Encapsulation efficiency for the atropine, irinotecan or
premetrexed liposomes is suitably in the range of about 20% to
about 100%, about 20% to about 80%, about 20% to about 60%, or
about 20% to about 40%, encapsulated atropine, irinotecan or
premetrexed. This is a surprising an unexpected result of the
ethanol injection method for encapsulating atropine, irinotecan or
premetrxed in the liposomes.
[0116] In additional embodiments, the liposomes can also comprise
endosomal disrupting peptides, such as the K[K(H)KKK].sub.5-K(H)KKC
(HoKC) (HK) (SEQ ID NO: 1) peptide manufactured by Sigma-Genosys
(The Woodlands, Tex.), associated with the liposomes. The endosomal
disrupting peptide HoKC may help the release of TMZ, melphalan,
atropine, pemetrexed or irinotecan from the endosomes into the
cytoplasm of the cells. In such embodiments, the liposomes suitably
also comprise MPB-DOPE at 5 molar percent of total lipid. Since the
HoKC peptide (K[K(H)KKK].sub.5-K(H)KKC) carries a terminal
cysteine, MPB-DOPE is included to allow conjugation of the peptide
to the liposome. The Lip-HoKC liposomes were prepared using the
coupling reaction between the cationic liposomes carrying the
maleimide group (Lip-MPB) and the peptide. An aliquot of 0.1 mmol
of the peptide with a free thiol group on cysteine was added to 2
mmol of Lip-MPB in 10 mM HEPES, pH 7.4, solution and rotated at
room temperature (20-30 r.p.m.) for 2 h.
[0117] The liposomal complexes prepared in accordance with the
present invention can be formulated as a pharmacologically
acceptable formulation for in vivo administration. The complexes
can be combined with a pharmacologically compatible vehicle or
carrier. The compositions can be formulated, for example, for
intravenous administration to a mammal, for example a human patient
to be benefited by administration of the TMZ, melphalan, atropine,
irinotecan or premetrexed in the complex. The complexes have an
inherent size so that they are distributed throughout the body
following i.v. administration. Alternatively, the complexes can be
delivered via other routes of administration, such as intratumoral
(IT), intralesional (IL), sublingual (SL), aerosal, percutaneous,
oral, endoscopic, topical, intramuscular (IM), intradermal (ID),
intraocular (IO), intraperitoneal (IP), transdermal (TD),
intranasal (IN), intracereberal (IC), intraorgan (e.g.
intrahepatic), slow release implant, or subcutaneous
administration, or via administration using an osmotic or
mechanical pump. Preparation of formulations for delivery via such
methods, and delivery using such methods, are well known in the
art.
[0118] The complexes can be optimized for target cell type through
the choice and ratio of lipids, the ratio of ligand (e.g.,
protein/peptide, antibody or antibody fragment) to liposome, the
ratio of ligand and liposome to TMZ, melphalan, atropine,
irinotecan or premetrexed and the choice of ligand.
[0119] The complexes made in accordance with the methods of this
invention can be provided in the form of kits for use in the
delivery of TMZ, melphalan, atropine, irinotecan or premetrexed.
Suitable kits can comprise, in separate, suitable containers, the
targeted TMZ, melphalan, atropine, irinotecan or premetrexed
cationic liposome complexes (suitably dried, lyophilized powders)
and water or a suitable buffer. The components can be mixed under
sterile conditions in the appropriate order and administered to a
patient within a reasonable period of time, generally from about 30
minutes to about 24 hours, after preparation. Liposomes are
suitably prepared in sterile water-for-injection, along with
appropriate buffers, osmolarity control agents, etc. The complete
complex is suitably formulated as a dried powder (lyophilized)
(see, e.g., U.S. Published Patent Application No. 2005/0002998, the
disclosure of which is incorporated by reference herein in its
entirety).
[0120] The cationic liposome complexes of the present invention
suitably comprise an anti-transferrin receptor single chain
antibody molecule (TfRscFv) on their surface. It has been
determined that this targeting molecule enhances delivery across
the blood-brain barrier and targeted delivery to brain cancer
cells. The targeted liposomes can also be used to treat other
cancers in the body and to deliver other drugs.
[0121] Also provided are cationic liposome complexes prepared
according to the methods described throughout. For example,
ligand-targeted (e.g., protein/peptide, antibody- or antibody
fragment-targeted) cationic liposome complexes comprising a
cationic liposome, a ligand (e.g., protein/peptide, antibody or
antibody fragment), and TMZ, melphalan, atropine, pemetrexed or
irinotecan, wherein the ligand is directly complexed/associated
with, but not chemically conjugated to the cationic liposome, are
provided.
[0122] The TMZ, melphalan, atropine, irinotecan or premetrexed can
be encapsulated within the cationic liposome (i.e., in the
hydrophilic, aqueous interior of the liposomes), contained within a
hydrocarbon chain region of the cationic liposome, associated with
an inner or outer monolayer of the cationic liposome (e.g., the
head-group region), or any combination thereof. Suitably, the
cationic liposomes of the present invention are unilamellar
liposomes (i.e. a single bilayer), though multilamellar liposomes
which comprise several concentric bilayers can also be used. Single
bilayer cationic liposomes of the present invention comprise an
interior aqueous volume in which TMZ, melphalan, atropine,
irinotecan or premetrexed can be encapsulated. They also comprise a
single bilayer which has a hydrocarbon chain region (i.e., the
lipid chain region of the lipids) in which TMZ, melphalan,
atropine, irinotecan or premetrexed can be contained. In addition,
TMZ, melphalan, atropine, irinotecan or premetrexed can be
complexed or associated with either, or both, the inner monolayer
and/or the outer monolayer of the liposome membrane (i.e., the
head-group region of the lipids). In further embodiments, TMZ,
melphalan, atropine, irinotecan or premetrexed can be
encapsulated/associated/complexed in any or all of these regions of
the cationic liposome complexes of the present invention.
[0123] In further embodiments, pharmaceutical compositions
comprising the ligand-targeted cationic liposome complexes
described throughout are provided. In suitable embodiments, the
pharmaceutical compositions further comprise one or more excipients
selected from the group consisting of one or more antibacterials
(e.g., amphotericin B, chlortetracycline, gentamicin, neomycin),
one or more preservatives (e.g., benzethonium chloride, EDTA,
formaldehyde, 2-phenoxyethanol), one or more buffers (e.g.,
phosphate buffers, sodium borate, sodium chloride), one or more
surfactants (polysorbate 20, 80), one or more protein stabilizers
(e.g., albumin, lactose, potassium glutamate), sugars e.g. sucrose
or dextrose, and adjuvants (e.g., aluminum hydroxide, aluminum
phosphate). Additional excipients are well known in the art and can
be readily used in the practice of the present invention.
[0124] Also provided are pharmaceutical compositions comprising a
first ligand-targeted cationic liposome complex comprising a
cationic liposome, a ligand (e.g., protein/peptide, antibody or
antibody fragment), and TMZ, melphalan, atropine, irinotecan or
premetrexed wherein the ligand is directly complexed/associated
with, but not chemically conjugated to the cationic liposome. In
further embodiments, the ligand can be chemically conjugated to the
cationic liposome. The pharmaceutical compositions also suitably
comprise a second different ligand-targeted cationic liposome
complex comprising a cationic liposome, a ligand (e.g.,
protein/peptide, antibody or antibody fragment), and one or more
nucleic acid molecules (including plasmid DNA, siRNA, miRNA, shRNA
or antisense nucleic acids), wherein the ligand is directly
complexed/associated with, but not chemically conjugated to the
cationic liposome. (See U.S. Pat. No. 7,780,822 and US Published
Patent Application No. 2007/0065449, the disclosures of which are
incorporated by reference herein in their entireties). In further
embodiments, the compositions can also comprise a ligand-targeted
cationic liposome complex comprising a ligand (e.g.,
protein/peptide, antibody or antibody fragment), and one or more
small molecules (see U.S. Published Patent Application No.
2007/0231378, the disclosure of which is incorporated by reference
herein in its entirety) or one or more imaging agents (see U.S.
Published Patent Application No. 2007/0134154, the disclosure of
which is incorporated by reference herein in its entirety), wherein
the ligand is directly complexed/associated with, but not
chemically conjugated to the cationic liposome. In further
embodiments, the ligand can be chemically conjugated to the
cationic liposome in such compositions.
[0125] Also provided are pharmaceutical compositions comprising a
first ligand-targeted cationic liposome complex comprising a
cationic liposome, a ligand (e.g., protein/peptide, antibody or
antibody fragment), and TMZ, melphalan, atropine, irinotecan or
premetrexed wherein the ligand is directly complexed/associated
with, but not chemically conjugated to the cationic liposome. In
further embodiments, the ligand can be chemically conjugated to the
cationic liposome. The pharmaceutical compositions also suitably
comprise a second ligand-targeted cationic liposome complex
comprising a cationic liposome, a ligand (e.g., protein/peptide,
antibody or antibody fragment), and one or more nucleic acid
molecules (including plasmid DNA, siRNA, miRNA, shRNA or antisense
nucleic acids), one or more small molecules, or one or more imaging
agents (including superparamagnetic iron oxide, or gadolinium)
wherein the nucleic acid molecules, small molecules, or imaging
agents down-regulates, modifies or otherwise negates the effect of
MGMT in the cancer cell.
[0126] In further embodiments, methods of treating cancer in a
patient are provided. Suitably, such methods comprise administering
to a patient one or more of the targeted cationic liposome
complexes as described herein. Suitably the complexes are prepared
according to the methods described throughout.
[0127] In embodiments, the methods of treatment comprise
administering to a patient a targeted Temozolomide, irinotecan or
melphalan cationic liposome complex, wherein the cationic liposome
complex comprises a cationic liposome comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or comprising
dimethyldloadecylammonlum bromide (DDAB) and DOPE, with or without
cholesterol, Temozolomide, irinotecan or melphalan, and a ligand
complexed with, but not chemically conjugated to, the cationic
liposome.
[0128] As described throughout, the ligand is suitably an antibody,
an antibody fragment or a protein, including a single chain Fv
antibody fragment. In exemplary embodiments, the single chain Fv
antibody fragment is an anti-transferrin receptor single chain Fv
(TtRscFv).
[0129] Also provided of methods of treating organophosphate
poisoning (i.e., nerve gas poisoning) in a patient, comprising
administering to the patient a targeted atropine cationic liposome
complex, wherein the targeted atropine cationic liposome complex
comprises cationic liposome comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), atropine and
a ligand directly complexed with, but not chemically conjugated to,
the cationic liposome. Exemplary ligands are described herein.
[0130] Suitably the temozolomide is administered to the patients
utilizing the methods described herein at a dose of about 1
mg/m.sup.2 to about 1000 mg/m.sup.2, more suitably at a dose of
about 10 mg/m.sup.2 to about 500 mg/m.sup.2, or about 50 mg/m.sup.2
to about 400 mg/m.sup.2, about 80 mg/m.sup.2 to about 300
mg/m.sup.2, about 50 mg/m.sup.2 to about 250 mg/m.sup.2, about 50
mg/m.sup.2 to about 250 mg/m.sup.2, or about 50 mg/m.sup.2, about
60 mg/m.sup.2, about 70 mg/m.sup.2, about 80 mg/m.sup.2, about 90
mg/m.sup.2, about 100 mg/m.sup.2, about 110 mg/m.sup.2, about 120
mg/m.sup.2, about 130 mg/m.sup.2, about 140 mg/m.sup.2, about 150
mg/m.sup.2, about 160 mg/m.sup.2, about 170 mg/m.sup.2, about 180
mg/m.sup.2, about 190 mg/m.sup.2, about 200 mg/m.sup.2, about 210
mg/m.sup.2, about 220 mg/m.sup.2, about 230 mg/m.sup.2, about 240
mg/m.sup.2, about 250 mg/m.sup.2, about 260 mg/m.sup.2, about 270
mg/m.sup.2, about 280 mg/m.sup.2, about 290 mg/m.sup.2, or about
300 mg/m.sup.2.
[0131] Suitably the melphalan is administered to the patients
utilizing the methods described herein at a dose of about 1
mg/m.sup.2 to about 500 mg/m.sup.2, more suitably at a dose of
about 1 mg/m.sup.2 to about 100 mg/m.sup.2, or about 1 mg/m.sup.2
to about 50 mg/m.sup.2, about 1 mg/m.sup.2 to about 30 mg/m.sup.2,
about 5 mg/m.sup.2 to about 20 mg/m.sup.2, or about 6 mg/m.sup.2 to
about 16 mg/m.sup.2, or about 1 mg/m.sup.2, about 2 mg/m.sup.2,
about 3 mg/m.sup.2, about 4 mg/m.sup.2, about 5 mg/m.sup.2, about 6
mg/m.sup.2, about 7 mg/m.sup.2, about 8 mg/m.sup.2, about 9
mg/m.sup.2, about 10 mg/m.sup.2, about 11 mg/m.sup.2, about 12
mg/m.sup.2, about 13 mg/m.sup.2, about 14 mg/m.sup.2, about 15
mg/m.sup.2, about 16 mg/m.sup.2, about 17 mg/m.sup.2, about 18
mg/m.sup.2, about 19 mg/m.sup.2, about 20 mg/m.sup.2, about 21
mg/m.sup.2, about 22 mg/m.sup.2, about 23 mg/m.sup.2, about 24
mg/m.sup.2, or about 25 mg/m.sup.2.
[0132] Suitably the, irinotecan or premetrexed is administered to
the patients utilizing the methods described herein at a dose of
about 1 mg/m.sup.2 to about 1000 mg/m.sup.2, more suitably at a
dose of about 10 mg/m.sup.2 to about 500 mg/m.sup.2, or about 50
mg/m.sup.2 to about 400 mg/m.sup.2, about 80 mg/m.sup.2 to about
300 mg/m.sup.2, about 100 mg/m.sup.2 to about 250 mg/m.sup.2, or
about 50 mg/m.sup.2, about 60 mg/m.sup.2, about 70 mg/m.sup.2,
about 80 mg/m.sup.2, about 90 mg/m.sup.2, about 100 mg/m.sup.2,
about 110 mg/m.sup.2, about 120 mg/m.sup.2, about 130 mg/m.sup.2,
about 140 mg/m.sup.2, about 150 mg/m.sup.2, about 160 mg/m.sup.2,
about 170 mg/m.sup.2, about 180 mg/m.sup.2, about 190 mg/m.sup.2,
about 200 mg/m.sup.2, about 210 mg/m.sup.2, about 220 mg/m.sup.2,
about 230 mg/m.sup.2, about 240 mg/m.sup.2, about 250 mg/m.sup.2,
about 260 mg/m.sup.2, about 270 mg/m.sup.2, about 280 mg/m.sup.2,
about 290 mg/m.sup.2, or about 300 mg/m.sup.2.
[0133] In embodiments, the atropine is administered to the patients
(suitably intramuscularly) utilizing the methods described herein
at a dose of about 0.01 mg to about 100 mg, more suitably at a dose
of about 0.1 mg to about 50 mg, or about 1 mg to about 40 mg, about
1 mg to about 30 mg, about 1 mg to about 20 mg, about 1 mg to about
10 mg, about 2 mg to about 6 mg, or about 1 mg, about 2 mg, about 3
mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg,
about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg,
about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg,
about 19 mg or about 20 mg.
[0134] As described herein, in embodiments, the molar ratio of
lipid in the cationic liposome:temozolomide, melphalan, atropine,
irinotecan or premetrexed for use in the methods described herein
is about 0.1:1 to about 5:1. More suitably, the molar ratio of
lipid in the cationic liposome:temozolomide, melphalan, atropine,
irinotecan or premetrexed is about 0.1:1 to about 1:100, about
0.5:1 to about 1:50, about 1:1 to about 1:20, about 2:1 to about
10:0.1, about 0.5:1 to about 2:1, or about 1:1.
[0135] The weight ratio of ligand:lipid in the cationic liposome
for use in the methods described herein is suitably about 0.01:1 to
about 0.5:10. Suitably, the weight ratio of ligand:lipid in the
cationic liposome is about 0.1:10 to about 0.5:10, about 0.3:10 to
about 0.4:10, or about 0.33:10, including any ratio within these
ranges.
[0136] Suitable methods of administration include, but are not
limited to, intravenous (IV), intratumoral (IT), intralesional
(IL), aerosal, percutaneous, oral, endoscopic, topical,
intramuscular (IM), sublingual (SL), intradermal (ID), intraocular
(IO), intraperitoneal (IP), transdermal (TD), intranasal (IN),
intracereberal (IC), intraorgan (e.g. intrahepatic), slow release
implant, or subcutaneous administration, or via administration
using an osmotic or mechanical pump. They can be administered as a
bolus or as an infusion. In additional embodiments, the ligand can
be chemically conjugated to the cationic liposome using the various
methods described herein or otherwise known in the art.
[0137] Exemplary cancers that can be treated using the methods
described herein include, but are not limited to, cancers of the
head and neck, breast, prostate, pancreatic, brain, including
glioblastoma and astrocytoma, neuroendocrine, cervical, lung,
liver, kidney, liposarcoma, rhabdomyosarcoma, choriocarcinoma,
angiosarcoma, melanoma, retinoblastoma, ovarian, vaginal,
urogenital, gastric, colorectal cancers, multiple myeloma and
cancers of the blood.
[0138] As described herein, it has been surprisingly found that the
targeted cationic liposomes prepared by the disclosed methods are
able to cross the blood-brain barrier. Generally, this barrier is a
significant hindrance to treatments designed to treat cancers and
other diseases or conditions of the brain or other treatments
designed to deliver drugs to the brain. Thus, in embodiments, the
methods described in herein are useful in the successful treatment
of primary and metastatic brain cancers, including gliomas
glioblastomas and astrocytomas, and in general to deliver drugs
across the blood-brain barrier
[0139] In another embodiment the methods described herein can also
be used as a treatment for organophosphate poisoning.
[0140] As described herein, it has been surprisingly found that the
targeted cationic liposomes prepared by the disclosed methods are
able to efficiently deliver enough TMZ to target tumor cells that
are resistant to standard unencapsulated TMZ to overcome their
inherent resistance (which may be due to activated MGMT) resulting
in these tumor cells now responding to TMZ.
[0141] As described herein, it has been surprisingly found that the
targeted cationic liposomes prepared by the disclosed methods are
able to induced cell death (apoptosis) in tumor cells that are
resistant to the killing effects of TMZ administered without the
targeted cationic liposomes.
[0142] As described herein, it has been surprisingly found that the
targeted cationic liposomes prepared by the disclosed methods are
able to induced apoptosis in cancer stem cells (CSC) as well as
differentiated cancer cells (non-CSC) in tumors irrespective of
their response to TMZ when it is administered without the targeted
cationic liposomes.
[0143] As described herein, it has been surprisingly found that the
level of apoptosis induced by the targeted cationic liposomes
prepared by the disclosed methods is at least equal to if not
proportionally greater in cancer stem cells (CSC) than in
differentiated cancer cells (non-CSC) in tumors.
[0144] As described herein, it has been surprisingly found that
treatment of tumors in mammals with the targeted cationic liposomes
prepared by the disclosed methods not only induce tumor growth
inhibition, but also result in tumor regression and that this
response can be maintained even after the treatment has ended.
[0145] As described herein, it has been surprisingly found that
treatment of tumor cells with the targeted cationic liposomes
prepared by the disclosed methods not only induce tumor cell growth
inhibition, but also result in tumor regression and that this
response can be maintained even after the treatment has ended.
[0146] In suitable embodiments, the methods further comprise
administering an additional different therapy to the patient in
combination with the targeted temozolomide cationic liposome
complex. Exemplary therapies that can be utilized include,
administration of chemotherapeutic agent, small molecule, radiation
therapy or a nucleic acid-based therapy. Exemplary chemotherapeutic
agents include, but are not limited to, docetaxel, mitoxantrone,
doxorubicin and gemcitabine. Exemplary small molecules include, but
are not limited to, imatinib mesylate (GLEEVEC.TM.), Erlotinib
hydrochloride (TARCEVA.TM.), Sunitinib Malate (SU11248, SUTENT.TM.)
and Gefitinib (IRESSA.TM.). Exemplary nucleic acid-acid based
therapies (including tumor suppressor genes, antisense
oligonucleotides, siRNA, miRNA, or shRNA) include those disclosed
in U.S. Published Patent Application No. 2007/0065499 and U.S. Pat.
No. 7,780,882, the disclosures of each of which are incorporated by
reference herein in their entireties. In suitable embodiments, the
nucleic acid-based therapy comprises administration of a cationic
liposome complex comprising plasmid DNA encoding the wtp53 gene and
targeted via TfRscFv (scL-p53), as described in U.S. Pat. No.
7,780,882.
[0147] Also provided are methods treating a brain cancer of a
patient, comprising administering to the patient a cationic
liposome complex as described in U.S. Pat. No. 7,780,882. Suitably,
the complex comprises a cationic liposome comprising
1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a plasmid DNA
expressing wild-type p53 and an anti-transferrin receptor single
chain Fv (TfRscFv) directly complexed with, but not chemically
conjugated to, the cationic liposome. The methods further comprise
administering Temozolomide or scL-TMZ, suitably before, at the same
time or after administration of the cationic liposome complex.
[0148] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein may be made without
departing from the scope of the invention or any embodiment
thereof. Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention.
Example 1
Preparation of Cationic Liposomes Comprising Temozolomide
[0149] Materials:
[0150] DOTAP (1,2-dioleoyl-3-trimethylammonium propane, chloride
salt) [0151] Obtained from Avanti Polar Lipids, Inc. Cat. #890890E,
MW 698.55 [0152] Concentration: 25 mg/mL ethanol solution [0153]
Dilute lipid to 20 mg/ml with absolute ethanol before use
[0154] DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) [0155]
Obtained from Avanti Polar Lipids, Inc. Cat. #850725E, MW 744.04
[0156] Concentration: 25 mg/mL ethanol solution. [0157] Dilute
lipid to 20 mg/ml with absolute ethanol before use
[0158] Temozolomide (TMZ, M.W. 194.15), powder [0159] Obtained from
Sigma, Cat. #T2577-100 mg [0160] Dissolve TMZ in pure DMSO to
desired concentration. For example, 19.415 mg/ml=100 mM of TMZ; 28
mg/ml=144.218 mM of TMZ
[0161] Ultra-pure, endotoxin free LAL Reagent Water (e.g.
BioWhittaker, Cat. #W50-500, endotoxin <0.005 EU/ml)
[0162] Injector: Hamilton Gastight Syringe, 1 ml (Hamilton #81230)
with a 22 gauge needle, part #81365)
Procedure:
[0163] 1) Fresh TMZ solution is prepared by dissolving TMZ in DMSO
to the desired concentration by vortexing at high speed for 5-10
mins (must be clear). The solution is held at room temperature
until used to mix with lipids.
[0164] 2) Place lipid solutions at 37.degree. C. for 10-15 min. The
lipid solutions are then placed in a 65.degree. C. water bath with
occasional shaking for 5 min.
[0165] 3) To prepare the Lip-TMZ: Place a brown glass bottle with
stir bar on a hot plate set to 50.degree. C. to 60.degree. C. While
stirring at high speed without splashing, add the lipids and TMZ to
the bottle in the following order. It should be noted that other
component ratios and concentrations as described herein can be
prepared using the same protocol as shown below.
[0166] For 0.5:1 (Lip:TMZ) molar ratio (2 mM TMZ in formulation)
[0167] DOTAP 87.5 .mu.l (of 20 mg/ml)=2.5 .mu.mol or 1.75 mg [0168]
DOPE 93.75 .mu.l (of 20 mg/ml)=2.5 .mu.mol or 1.875 mg [0169] Add
TMZ soln., 100 .mu.l (of 19.41 mg/ml)=10 .mu.mol,
[0170] Continue stir for 3 min. after all 3 are added
[0171] For 1:1 (Lip:TMZ) molar ratio (2 mM TMZ in formulation)
[0172] DOTAP 175 .mu.l (of 20 mg/ml)=5 .mu.mol or 3.5 mg [0173]
DOPE 187.5 .mu.l (of 20 mg/ml)=5 .mu.mol or 3.75 mg [0174] Add TMZ
soln., 100 .mu.l (of 19.41 mg/ml)=10 .mu.mol,
[0175] Continuously stir for 3 min. after all 3 added
[0176] For 1:1 (Lip:TMZ) molar ratio (8 mM TMZ in formulation)
[0177] DOTAP 560 .mu.l (of 25 mg/ml)=20 .mu.mol or 14 mg [0178]
DOPE 600 .mu.l (of 25 mg/ml)=20 .mu.mol or 15 mg [0179] Add TMZ
soln., 277.36 .mu.l (of 28 mg/ml)=40 .mu.mol,
[0180] Continuously stir for 3 min. after all 3 added
[0181] For 2:1 (Lip:TMZ) molar ratio (2 mM TMZ in formulation)
[0182] DOTAP 350 .mu.l (of 20 mg/ml)=10 .mu.mol or 7 mg [0183] DOPE
375 .mu.l (of 20 mg/ml)=10 .mu.mol or 7.5 mg [0184] Add TMZ soln.,
100 .mu.l (of 19.41 mg/ml)=10 .mu.mol,
[0185] Continuously stir for 3 min. after all 3 added
[0186] 4) Warm 4 mL LAL water to 65.degree. C. in water bath in
brown glass bottle with stir bar. Immediately prior to addition of
the Lipid-TMZ solution, move the bottle to a hot plate
(50.degree.-60.degree. C.). Stir water at high speed with no
splashing for a few seconds to remove bubbles from the stir
bar.
[0187] 5) Keep the water on the hot plate. Continue stirring the
water at high speed (without splashing) during lipid addition.
After mixing lipids and TMZ as above, immediately and as rapidly as
possible, using the Hamilton syringe for injection, inject the
mixture into the hot water on the hot plate (50.degree.-60.degree.
C.) directly into the center of the vortex. Continue stirring on
high speed (without splashing) for 1 min after the addition of the
lipid mixture while loosely covered.
[0188] 6) Move the glass bottle to a room temperature stir plate
and continue to stir slowly until the loosely covered solution
cools down to 20-25.degree. C. (room temperature).
[0189] 7) Adjust the volume to 5 ml with room temperature LAL
water.
[0190] 8) Filter the solution using a 0.22 .mu.m pore Milex GV
filter if desired.
[0191] 9) Measure particle size and zeta potential if desired.
[0192] Results of these preparation methods demonstrate
approximately at least 30-55% loading, of TMZ and liposomes having
a particle size of about 20-60 nm and a Zeta Potential of about 30
to 50 mV.
Example 2
Preparation of scL-TMZ without Chemical Conjugation (by Simple
Mixing)
[0193] Using the TMZ-comprising cationic liposomes prepared
according to the procedure described in Example 1, the ligand
targeted TMZ cationic liposome complex as described herein is
prepared by simple mixing of the components and without chemical
conjugation. The preparation of the complexes was in accordance
with the following general procedure:
[0194] To the liposome-water (or buffer) the appropriate amount of
targeting moiety is added to give the desired ratio and mixed by
gentle inversion 5-10 seconds. The targeting moiety can be a ligand
including but not limited to transferrin or folate, or other
proteins. It can also be an antibody or an antibody fragment that
targets a cell surface receptor including, but not limited to the
transferrin or HER-2 receptor (e.g., TfRscFv). This mixture is kept
at room temperature for 10-15 minutes (again inverted gently for
5-10 seconds after approximately 5 minutes). To yield the desired
final volume the targeting moiety-Lip-TMZ admixture is mixed with
any volume (including none) of water (suitably deionized water) or
a buffer of any pH including, but not limited to, Tris buffers,
HEPES buffers or Phosphate Buffered Saline, required to give a
desired volume and inverted gently for 5-10 seconds to mix. This
mixture is kept at room temperature for 10-15 minutes (again
inverted gently for 5-10 seconds after approximately 5
minutes).
[0195] Typically, for use in an in vitro assay, it is desirable
that the amount of TMZ in the final complex is in the range of
about 1 .mu.M to 300 .mu.M per well; for in vivo use, it is
desirable to provide about 1 mg/kg to about 50 mg/kg of TMZ per
injection. For use in vivo dextrose or sucrose is added last to a
final concentration of about 1-50% (V:V) dextrose or sucrose,
suitably 5% dextrose or 10% sucrose, and mixed by gentle inversion
for 5-10 seconds. This mixture is kept at room temperature for
10-15 minutes (again inverted gently for 5-10 seconds after
approximately 5 minutes).
[0196] A specific example at a suitable ratio of 1:30 (antibody
fragment:liposome, w:w) and 1:1 Liposome:TMZ (molar ratio) is as
follows: For a final volume of approximately 700 uL, at a TMZ
concentration of 25 mg/kg/injection, mix 319 .mu.L of Lip:TMZ (8 mM
stock) with 305 .mu.L of antibody fragment (at an anti-transferrin
receptor single chain antibody fragment [TfRscFv] concentration of
0.2 mg/mL). Add 6 .mu.L of water or buffer and, as the last step,
70 .mu.L of 50% Dextrose or no water or buffer and 140 uL of 50%
sucrose.
[0197] A second specific example at a preferred ratio of 1:30
(antibody fragment:liposome, w:w) and 1:1 Liposome:TMZ (molar
ratio) is as follows: For a final volume of approximately 1.8 mL,
at a TMZ concentration of 25 mg/kg/injection, mix 1276 .mu.L of
Lip:TMZ (2 mM stock) with 305 .mu.L of antibody fragment (at an
anti-transferrin receptor single chain antibody fragment [TfRscFv]
concentration of 0.2 mg/mL). 39 .mu.L of water or buffer is added
and 180 .mu.L of 50% Dextrose is added as the last step.
[0198] Another specific example at a preferred ratio of 1:30
(antibody fragment:liposome, w:w) and 1:1 Liposome:TMZ (molar
ratio) is as follows: For a final volume of approximately 400
.mu.L, at a TMZ concentration of 5 mg/kg/injection, mix 280 .mu.L
of Lip:TMZ (2 mM stock) with 64 .mu.L of antibody fragment (at an
anti-transferrin receptor single chain antibody fragment [TfRscFv]
concentration of 0.2 mg/mL). 16.5 .mu.L of water or buffer is added
and 40 .mu.L of 50% Dextrose is added as the last step.
[0199] The size (number average) of the final complex prepared by
the methods is between about 10 to 800 nm, suitably about 20 to 400
nm, most suitably about 25 to 200 nm with a zeta potential of
between about 1 and 100 mV, more suitably 10 to 60 mV and most
suitably 25 to 50 mV as determined by dynamic light scattering
using a Malvern Zetasizer ZS. This size is small enough to
efficiently pass through the tumor capillary bed, or cross the
blood brain barrier, and reach the tumor cells.
Example 3
Determination of the Percent Encapsulation of TMZ in the scL-TMZ
Complex
[0200] To determine the percent of the TMZ encapsulated in the
scL-TMZ complex, we prepared scL-TMZ as described in Example 1 with
2 mM stock Lip:TMZ. Various amounts of complex were prepared
ranging from 126 to 560 ul scL-TMZ. The scL-TMZ complex was
subsequently diluted to a Lip:TMZ concentration of 0.5 mM. The
initial concentration of TMZ was checked by measuring the
absorbance of the scL-TMZ complex at 320 nm using a Beckman
spectrophotometer. A standard curve of TMZ concentrations spanning
0.001 to 0.1 mM TMZ was also generated by measuring absorbance at
320 nM using DMSO as the blank. Free TMZ was separated from
complexed scL-TMZ by filtration through a Vivaspin.RTM. 500, 5 kDa
MWCO (GE Healthcare, UK). 200 ul of the diluted scL-TMZ complex was
loaded onto the filter and centrifuged at 14,000 g for 15 min at
room temperature. The flow through was collected and the volume
(175 ul), and optical density at 320 nm were determined. Endotoxin
free LAL water (175 ul) was added to the filter which was mixed by
inversion 10 times and again centrifuged as above. The flow through
was again collected, volume and OD measured as above. The bound
complex on the filter was recovered by addition of 100 ul of
Endotoxin free LAL water to the filter. After inversion 10 times
the filter was placed upside down in the collection tube and
centrifuged at 1,000 g for 2 minutes and the OD measured. The TMZ
concentrations of the loaded scL-TMZ complex, each of the flow
through samples and the retained sample were determined from the
standard curve and the amount of TMZ in each calculated, correcting
for volume of each sample and for dilution. In all cases, all of
the unencapsulated TMZ was recovered in the initial flow through
with nothing detected in the wash sample. Multiple experiments were
performed and the average percent encapsulation was found to be
38.7.+-.3.7% (mean.+-.S.E.)
[0201] The identical procedure was employed to test the
encapsulation when 8 mM stock
[0202] Lip:TMZ was prepared. In this instance the volume of scL-TMZ
prepared as described in Example 1 was 350 ul. Multiple experiments
were performed. The percent encapsulation of TMZ, 39.2.+-.3.7%
(mean.+-.S.E.), was virtually identical to that obtained with 2 mM
Lip:TMZ. Moreover, the size of the scL-TMZ was determined using the
Malvern Zetasizer ZS after recovery from the Vivaspin.RTM. filter
and compared to the initial size obtained immediately
post-preparation prior to loading on the column. There was no
change evident, with the initial and final sizes of the scL-TMZ
complex being 75.99 nm and 76.93 nm, respectively.
Example 4
In Vitro Efficacy of Targeted Cationic Liposomes Comprising
Temozolomide
[0203] Human glioblastoma multiforme (GBM) cell lines U87MG and
T98G were obtained from ATCC (Manassas, Va.). U87 is derived from a
grade IV glioblastoma, and carries wtp53 (Van Meir E G, Kikuchi T,
Tada M, Li H, Diserens A C, Wojcik B E, Huang H J S, Friedmann T,
Detribolet N and Cavenee W K (1994) Analysis of the P53 Gene and
Its Expression in Human Glioblastoma Cells. Cancer Research 54: pp
649-652). A version of U87MG that stably expresses the luciferase
gene has been obtained from Caliper Life Sciences for use in in
vive studies where tumor growth and response will be monitored by
the IVIS.RTM. Imaging System, Xenogen. The human GBM cell line U251
was obtained from the Division of Cancer Treatment and Diagnosis
Tumor Repository, National Cancer Institute-Frederick (Frederick,
Md.). Cells were maintained at 37.degree. C. in a 5% CO.sub.2
atmosphere in modified IMEM (Gibco, Grand Island, N.Y.; U87 and
U87MG-luc2 cells), MEM (Mediatech Manassas, Va.; T98G cells), or
RPMI 1640 medium (Gibco; U251 cells) supplemented with 10%
heat-inactivated fetal bovine serum (Omega Scientific, Tarzana,
Calif.), 2 mmol/L L-glutamine (Mediatech, Manassas, Va.), and 50
.mu.g/mL each of penicillin, streptomycin, and neomycin (PSN).
Cells were grown to 70-80% confluence before the next passage or
further experiments through trypsinization using TrypLE Express
(Gibco). Sodium
3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benze-
ne sulfonate (XTT) was purchased from Polysciences (Warrington,
Pa.).
[0204] The human multiple myeloma cell line KMS-11 was maintained
at 37.degree. C. in a 5% CO.sub.2 atmosphere in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum 2 mmol/L
L-glutamine and 50 .mu.g/mL each of penicillin, streptomycin, and
neomycin (PSN). Cells were grown to 70-80% confluence before the
next passage or further experiments. These cells grow in suspension
and not as monolayers.
[0205] U87MG is categorized as being sensitive to TMZ (Patil R,
Portilla-Arias J, Ding H, Inoue S, Konda B, Hu J W, Wawrowsky K A,
Shin P K, Black K L, Holler E and Ljubimova J Y (2010) Temozolomide
Delivery to Tumor Cells by a Multifunctional Nano Vehicle Based on
Poly(Beta-L-Malic Acid). Pharmaceutical Research 27: pp 2317-2329).
This is a well established orthotopic mouse model of GBM (Liu Y,
Lang F, Xie X, Prabhu S, Xu J, Sampath D, Aldape K, Fuller G and
Puduvalli V K (2011) Efficacy of Adenovirally Expressed Soluble
TRAIL in Human Glioma Organotypic Slice Culture and Glioma
Xenografts. Cell Death & Disease 2). U87MG cells reproducibly
develop tumors within 10 days when 5.times.10 cells are
intracranially injected in athymic nude mice. The mice succumb to
tumor burden within 30-40 days. T98G is also isolated from a human
glioblastoma. However, this cell line is known to be resistant to
TMZ (Patil R, Portilla-Arias J, Ding H, Inoue S, Konda B, Hu J W,
Wawrowsky K A, Shin P K, Black K L, Holler E and Ljubimova J Y
(2010) Temozolomide Delivery to Tumor Cells by a Multifunctional
Nano Vehicle Based on Poly(Beta-L-Malic Acid). Pharmaceutical
Research 27: pp 2317-2329) and carries a mutant form of the p53
gene (Van Meir E G, Kikuchi T, Tada M, Li H, Diserens A C, Wojcik B
E, Huang H J S, Friedmann T, Detribolet N and Cavenee W K (1994)
Analysis of the P53 Gene and Its Expression in Human Glioblastoma
Cells. Cancer Research 54: pp 649-652). T98G xenograft tumors are
induced via subcutaneous inoculation of 5-10.times.10.sup.6 cells
in Matrigel.TM. (Torres S, Lorente M, Rodriguez-Formes F,
Hernandez-Tiedra S, Salazar M, Garcia-Taboada E, Barcia J, Guzman M
and Velasco G (2011) A Combined Preclinical Therapy of Cannabinoids
and Temozolomide Against Glioma. Molecular Cancer Therapeutics 10:
pp 90-103). Both cell lines have elevated TfR expression (Sang H,
Kelley P Y, Hatton J D and Shew J Y (1989) Proto-Oncogene
Abnormalities and Their Relationship to Tumorigenicity in Some
Human Glioblastomas. Journal of Neurosurgery 71: pp 83-90).
[0206] Studies were carried out to compare the efficacy of standard
free (unencapsulated) TMZ; and unliganded TMZ-containing liposomes
(Lip-TMZ) The Lip-TMZ was prepared as described above in Example 1
using a liposome concentration of 2 mM. The zeta potentials of the
Lip-TMZ molecules ranged from 35.6-40.1 mV. The TMZ concentration
used was varied from 1 to about 250 uM. The ratios of Liposome to
TMZ was 0.5:1, 1:1 or 2:1 (molar ratio).
[0207] Human brain tumor derived U251 cells were plated in
triplicate at 2.times.10.sup.3 per well in a 96-well plate.
Following overnight incubation, the medium was replaced with
serum-free medium, overlaid with 100 .mu.L of indicated
concentrations of either Lip-TMZ, or free TMZ, incubated for 5 h,
and then supplemented with fetal bovine serum. After incubation for
an additional 91 h, cell viability was determined by the XT assay
as described previously (Rait A, Pirollo K F, Rait V, et al.
Inhibitory effects of the combination of HER-2 antisense
oligonucleotide and chemotherapeutic agents used for the treatment
of human breast cancer. Cancer Gene Ther 2001; 8:728-39.). Formazan
absorbance, which correlates to cell viability, was measured at 450
nm using a microplate reader (Bio-Rad, Hercules, Calif.). The
IC.sub.50 value, the drug concentration resulting in 50% cell kill,
was interpolated from the graph of the log of drug concentration
versus the fraction of surviving cells.
[0208] FIG. 1 demonstrates that in human brain tumor (GBM) cell
line U251, compared with the effects of free TMZ, in vitro
treatment with the liposome encapsulated TMZ as described herein
resulted in a significant reduction in IC.sub.50 values in human
GBM cells. 50% of the cells were killed at TMZ concentrations of
only 46.3 .mu.M, 28.8 .mu.M and 16 .mu.M, when these concentrations
of TMZ were encapsulated in the targeted cationic-liposome-TMZ
complex at Lip/TMZ molar ratios of 0.5:1, 1:1 and 2:1,
respectively. In contrast these concentrations of TMZ had virtually
no cell killing effect when not part of the scL-TMZ complex. The
higher the ratio of Lip to TMZ, the greater the increase in cell
killing effect, yielding a lower IC.sub.50 value. It is well known
by those familiar with the art that free, unencapsulated TMZ is the
form of the drug most commonly used to treat tumors in
patients.
[0209] Similar results are shown in FIG. 2 with human GBM tumor
cell line U87 comparing free TMZ and the Lip-TMZ at molar ratios of
Liposome to TMZ of 1:1 and 2:1. Once again the unencapsulated TMZ
has virtually no cell killing effect on these brain tumor cells. In
contrast when encapsulated in the Liposome at molar ratios of 1:1
or 2:1 (Lip:TMZ) using the method of this invention, TMZ
concentrations of only 11.4 and 21.5 .mu.M, respectively resulted
in significant tumor cell death.
Example 5
Increased Effect of scL-TMZ on Tumor Cells Compared to Free
(unencapsulated) TMZ
[0210] The scL-TMZ complex was prepared as described above in
Examples 1-2 using an anti-transferrin receptor single-chain
antibody fragment (TfRscFv) as the targeting moiety, a Lip:TMZ
molar ratio of 1:1(size=46.2 nm; zeta potential=42.5 mV) (liposome
concentration=2 mM) and an TfRscFv to Liposome ratio if 1:30 (w:w).
The size of the scL-TMZ complex was about 27.5 nm. The in vitro
cell killing ability of the scL-TMZ was compared to free,
unencapsulated TMZ in TMZ resistant human brain tumor cell line
T98G using the XTT assay.
[0211] FIG. 3A shows that tumor targeting scL-TMZ complex, has
significantly improved anti-cancer efficacy compared to free TMZ.
The free TMZ has an IC.sub.50 value >1000 .mu.M. In contrast,
when prepared according to the methods described herein and
delivered to the tumor cell by means of the tumor-targeting
complex, at least 20 fold less TMZ effectively kills the cancer
cells. This is especially significant as this GBM cell line is well
known in the art to be resistant to the killing effects of TMZ.
This reversal of resistance is due to the efficient delivery and
uptake of the TMZ payload into the tumor cell by means of the
binding of the targeting ligand (protein, antibody or antibody
fragment) to its receptor on the cell and the triggering of uptake
through active transport mechanisms like receptor mediated
endocytosis. This process "floods" the cells with drug overcoming
the mechanisms the tumor cell has in place to repair the DNA damage
caused by TMZ (such as upregulation of MGMT), and/or the mechanisms
to pump the TMZ out of the cells. Thus the cell dies.
[0212] There are a number of ramifications as a result of the
tumor-targeting delivery of TMZ via the targeted cationic liposomes
described herein.
[0213] 1) Increased efficacy means less drug needs to be delivered
to the patient to see improved anti-tumor effect.
[0214] 2) Tumor-specific delivery (tumor specificity) will decrease
the deleterious side-effects currently associated with TMZ as the
drug will not be taken up by non-target cells.
[0215] 3) The efficient delivery of TMZ to the tumor cells
overcomes the resistance to TMZ inherent in a significant
population of brain tumors (GBM and astrocytomas) and other cancer
types including, but not limited to, prostate cancer, multiple
myeloma, lung cancer, liver cancer, ovarian cancer, pancreatic
cancer, head and neck cancer, kidney cancer, stomach cancer, and
melanoma. This reversal of resistance broadens the scope of use for
TMZ as an anti-cancer treatment.
[0216] FIG. 3B demonstrates that the method of this invention is
not limited to sensitization of brain tumor cells to TMZ. The
killing effects of scL-TMZ were compared to that of free
(unencapsulated TMZ) in multiple myeloma cell line KMS-11. The
scL-TMZ complex was prepared with different increasing doses of TMZ
(0 to 100 uM TMZ) as described above using an anti-transferrin
receptor single-chain antibody fragment (TfRscFv) as the targeting
moiety, a Lip:TMZ molar ratio of 1:1 (liposome concentration=8 mM)
and an TfRscFv to Liposome ratio if 1:30 (w:w). The size of the
scL-TMZ complex was about 143 nm. The in vitro cell killing ability
of the scL-TMZ was compared to free, unencapsulated TMZ in multiple
mycloma cell line KMS-11. Transfection was performed and the
viability of the KMS-11 cells 48 hours post-transfection was
assessed. TMZ has not previously been used to treat multiple
myeloma, thus, it was very surprising that delivery of TMZ to these
cells by encapsulation in the scL complex resulted in significant
cell death, even at a very low concentration (25 uM) of TMZ.
[0217] The identical methodology and procedures described above
were used to prepare Lip-TMZ and scL-TMZ and the resultant scL-TMZ
nanocomplex was also used to transfect prostate (DU145), lung
(A549), ovarian (Hey-A8), pancreatic (PANC-1) and hepatocellular
carcinoma (HEP-G2) cells. In all cases there was a significant
increase in tumor cell response to the TMZ when it was encapsulated
in the scL nanocomplex when compared to free (unencapsulated) TMZ,
the current standard method of delivery.
Example 6
TfRscFV Liposomes Crossing the Blood Brain Barrier
[0218] Studies were performed to determine if the TfRscFv targeted
liposome (scL) complex (scL) can cross the blood-brain barrier
(BBB) and target tumors after i.v injection. Brain tumors were
induced in nude mice by intracranial inoculation of
5.times.10.sup.5 U87 cells. Three weeks later, the mice were I.V.
injected with uncomplexed free Cy5-ODN, scL-Cy5-ODN, or unliganded
Lip-Cy5-ODN (without the targeting moiety; unL-Cy5-ODN) (25
.mu.g/mouse) (2 mice/group). Twenty-four hours post-injection, mice
were euthanized and tumor-bearing brains imaged using the
Maestro.TM. in vivo fluorescence imaging system. Fluorescence
intensity of the brain tumors were compared using the Maestro.TM.
software. I.V. injection of scL-Cy5-ODN resulted in a strong
fluorescence signal specifically in the brain tumor (FIG. 4A). In
contrast, only low levels of fluorescence were observed in the
tumors after injection of either free Cy5-ODN or unL-Cy5-ODN. This
result demonstrates the ability ofscL to cross the BBB and
efficiently deliver a payload to brain tumors.
Example 7
Efficacy of scL-TMZ in Animal Models of Brain Cancer Compared to
Free, Unencapsulated TMZ
[0219] Intra cranial GBM tumors were induced in 5-6 week old female
athymic nude mice by stereotaxic inoculation of U87MG-luc2 cells
that stably carry the luciferase gene. Seven to ten days
post-inoculation, tumors were evaluated by bioluminescence using
Xenogen IVIS in vivo imaging system (Caliper Life Sciences) and
mice were evenly divided into treatment groups. Treatment was
initiated on the day of randomization (day 0). Animals were
injected intravenously (i.v.) via the tail vein with 5 mg/kg (per
injection per mouse) of TMZ alone or TfRscFv-targeted TMZ cationic
liposome complex (scL-TMZ). The scL-TMZ complex was prepared as
described above in Examples 1-2 using an anti-transferrin receptor
single-chain antibody fragment (TfRscFv) as the targeting moiety, a
Lip:TMZ molar ratio of 1:1 (liposome concentration=2 mM) and an
TfRscFv to Liposome ratio of 1:30 (w:w). Mice were injected twice
per week with each reagent for 5 weeks. Control animals (Vehicle)
received Liposome only (no TMZ, no TfRscFv). The sizes of the
scL-TMZ complexes iv injected into the mice during this study were
found to average about 100.5+14.7 nm (number average)
(Mean+S.E.).
[0220] Assessment of In Vivo Efficacy:
[0221] The in vivo response to treatment was evaluated based upon
the changes in tumor growth, body weight change, and overall
survival. Tumor growth was monitored by bioluminescence imaging
(BLI) using Xenogen IVIS in vive imaging system before, during, and
after the treatments at the indicated date. U87MG-luc2 cells were
genetically engineered to express luciferase gene which results in
the emission of bioluminescence signal when treated with the
substrate luciferin. The bioluminescence intensity of the brain
tumors, a measure of tumor size/growth, was measured and compared
between treatment groups. Half-way through treatment (after mice
received 3 weeks of treatment with each reagent), all animals were
scanned with magnetic resonance imaging (MRI) to evaluate the brain
tumor. The animal imaging was done with a 7T with a Bruker Biopsin
(Billerica, Mass.), using a respiratory gated (BioPac Physiological
Data Monitor) T1-Weighted, 2 dimensional Turbo Multi-slice
Multi-echo imaging sequences. Tumor volume was calculated from the
MRI scan and compared between treatment groups. Body weight change
was monitored weekly. Overall survival was recorded and plotted by
Kaplan-Meier method.
[0222] FIG. 4B shows the comparison of in vivo anti-tumor efficacy
of the scL-TMZ and free, unencapsulated TMZ on intracranial
U87MG-luc2 glioblastoma tumor xenografts. The brain tumors were
imaged using MRI before treatment began and again after the mice
had received 3 weeks of treatment (6 injections). The outlined
areas indicate the glioblastoma tumors. Over this 3 week period the
tumors in the control mice grew significantly larger as expected.
As this cell line is known to be responsive to TMZ, some tumor
growth inhibition was expected and was evident in the mice that
received free TMZ. However, in the animals that received the
scL-TMZ, not only was tumor growth inhibition evident, but tumor
regression had also occurred, even over this short period of
treatment, indicating the increased effectiveness of the scL-TMZ as
a therapeutic agent. This was an unexpected result.
[0223] A comparison of the tumor sizes in the animals of all three
groups is shown graphically in FIG. 5 and show the consistent
dramatic response and small tumor size of the mice treated with the
scL-TMZ.
[0224] FIG. 6 shows the Bioluminescence (BLI) imaging via Xenogen
of a representative animal from each group followed from
pre-treatment through the treatment period and post-treatment. The
intensity of the Bioluminescence signal, which correlates to tumor
size, is shown in a color map: Red color=a stronger signal, Violet
color=a weaker signal. Free TMZ, the current method of
administration for brain tumors, was able to control the growth of
the tumor for .about.2.5 weeks. However, once treatment ended after
5 weeks, significant tumor growth occurred. In fact, recurrence was
even evident at day 31 of treatment. In contrast, in the animal
that was treated with scL-TMZ, not only was the tumor growth
inhibition maintained throughout treatment and post treatment, but
an unexpected result of tumor regression (see Day 51) was observed
that lasted at least 2 weeks after the end of treatment. FIG. 7
shows an additional comparison of the results. A graphic
representation of the BLI signal intensities from the mice in FIG.
6 is shown in FIG. 8 (bar at bottom of graph indicates the duration
of treatment). A similar plot of the signal intensities over time
for all of the mice in each group is given in FIG. 9. Here again
the unexpected result of lack of tumor recurrence after the end of
treatment in the scL-TMZ treated group is evident. This is
unexpected since tumor recurrence is a common problem in cancers of
all types including brain cancers.
[0225] The lack of toxicity of the scL-TMZ treatment is shown by
body weight measurements during treatment and post-treatment (FIG.
10). The steep decrease in weight of the animals in the Vehicle
(Day 21) and Free TMZ (Day 51) groups is caused by the advanced
disease state. Compared to these other groups, the animals treated
with scL-TMZ evidenced no decrease in weight over the course of the
experiment and even gained weight at the end. This demonstrates the
lack of toxicity of this approach.
[0226] The long term survival of the animals in this experiment is
shown in a Kaplan-Meier plot (FIG. 11). All of the mice in the
Vehicle group had died by day 30. Although the free TMZ extended
the lifespan of these mice compared to the control group, there was
a significant increase in long term survival after treatment with
scL-TMZ compared the Free TMZ group.
[0227] In a second experiment, different doses/number of injections
per week of scL-TMZ, prepared as described herein, were compared.
Groups of mice bearing U87MG-luc2 glioblastoma intracranial
xenograft tumors were iv tail vein injected for 5 weeks with
scL-TMZ at: 2.5 mg/kg one injection per week; 5 mg/kg one injection
per week; or 5 mg/kg two injections per week and survival
determined. The Kaplan-Meier plot in FIG. 12 shows a dose dependent
response and that even a single injection at a dose of 5 mg/kg can
extend the life span of mice bearing intracranial brain tumors.
Furthermore injections at a lower dose of TMZ can also be effective
if the number of injections/week is increased.
[0228] Results Summary: Compared to the treatment with free TMZ,
intravenous treatment with scL-encapsulated TMZ resulted in a
robust inhibition against tumor growth monitored by either MRI or
BLI, and prolonged the survival in an intracranial U87MG-luc2 GBM
tumor xenograft model. However, no significantly increased toxicity
assessed by body weight change was observed in scL-TMZ treated
animals compared to those of free TMZ treated animals. Also, an
unexpected result was the maintenance of the tumor response,
including tumor regression, for at least 2 weeks after treatment
had ended.
Example 8
In Vivo Induction of Apoptosis by scL-TMZ in Cancer Stem Cells and
Differentiated Cancer Cells
[0229] Intracranial U87MG-luc2 tumor was induced in 5-6 week old
female athymic nude mice as described above. Three weeks after
inoculation, tumor bearing mice were randomly divided into groups
and treatment was started. Animals were injected i.v. via the tail
vein with 5 mg/kg (per injection per mouse) of TMZ alone or TMZ
encapsulated in tumor targeting liposome complex. The scL-TMZ
complex was prepared as described above using an anti-transferrin
receptor single-chain antibody fragment (TfRscFv) as the targeting
moiety, a Lip:TMZ molar ratio of 1:1 (liposome concentration=2 mM)
and an TfRscFv to Liposome ratio if 1:30 (w:w). Control animals
(Vehicle) received Liposome only (no TMZ, no TfRscFv). Prepared as
described above, the sizes of the scL-TMZ liposomes iv injected
into the mice during this study were found to average 85.5+4.96 nm
(number average) (Mean+S.E.).
[0230] The mice were treated two times per week. After receiving 3
injections, all animals were euthanized and brains were harvested.
Brain tumors were carefully dissected from normal brain tissue and
weighed. The in vivo anti-tumor efficacy was evaluated by assessing
the level of apoptosis. After weighing the tumors, single-cell
suspensions were obtained from the tumors by collagenase digestion
in Hank's balanced solution containing 1 mg/mL collagenase (Roche)
and 2 mmol/L DNase I (Sigma) 1 h at 37.degree. C. The fractionated
cells were passed through a 70-.mu.m cell strainer (Fisher
Scientific, Pittsburgh, Pa.) and washed with PBS. To determine the
level of apoptosis, single cells were stained with antibodies for
cleaved caspase-3 (Cell Signaling Technology, Danvers, Mass.), and
human CD133 (Miltenyi Biotec). The labeled cells were analyzed by
flow-activated cell sorting (FACS) on BD FACS Aria flow cytometer
(BD Biosciences, San Jose, Calif.).
[0231] Assessment of in vivo efficacy: The anti-tumor efficacy was
assessed by evaluating the induction of apoptosis in the
intracranial U87MG-luc2 brain tumors. The weights of the brain
tumors after 3 iv injections of Vehicle (liposome only, no TMZ, no
TfRscFv), Free unencapsulated TMZ or scL-TMZ are shown in FIG. 13.
Unexpectedly, even after only three injections a difference in the
tumor size between the free TMZ and scL-TMZ is evident.
[0232] Cancer stem cells (CSC) are often responsible for tumor
recurrence and resistance to chemotherapy. Because of the
unexpected finding described above wherein the tumor growth
inhibition and even regression were observed after the end of
treatment with scL-TMZ (but NOT with free TMZ), the level of
apoptosis was assessed in cancer stem cells, as well as in
differentiated cancer cells (non-CSC) in these tumors. FIG. 14
shows the results of flow cytometric analysis for the level of
apoptosis as determined by cleaved caspase-3 antibody staining of
single cells isolated from the brain tumors. CD133, a marker of GBM
cancer stem cells (CSCs) was used to distinguish CSCs from
differentiated cancer cells. The CSC population (CD133+) clearly
show a significant increase in the % of cells undergoing apoptosis
compared to free TMZ. Thus the scL-TMZ can target and efficiently
transfect CSCs resulting in significant tumor cell death.
[0233] Results Summary: In an intracranial U87MG-luc2 GBM tumor
xenograft model, intravenous treatment with scL-TMZ resulted in a
significant inhibition of tumor growth demonstrated by tumor weight
compared to those treated with free TMZ. In addition, intravenous
treatment with scL-TMZ resulted in a significantly increased
induction of apoptosis not only in CD133- differentiated cancer
cells but also in CD133+ CSCs.
Example 9
In Vivo Efficacy of scL-TMZ in TMZ Resistant Brain Cancer Cell Line
T98G Subcutaneous Xenograft Tumors
[0234] T98G GBM tumor cells are known to be resistant to treatment
with TMZ. For the TMZ-resistant GBM tumor model, subcutaneous T98G
xenografts were used. T98G xenograft tumors were induced in female
athymic nude mice by the subcutaneous injection of T98G cells or
tumor particles suspended in Matrigel collagen basement membrane
(BD Biosciences, San Jose, Calif.) on the lower back above the
tail, two sites per mouse. When the subcutaneous T98G tumors
reached .about.100 to 300 mm.sup.3, the mice were randomly divided
into groups and i.v. injected with 25 or 66 mg/kg (per injection
per mouse) of free TMZ or 25 mg/kg (per injection per mouse) TMZ
encapsulated in tumor targeting complex (1:1 molar ratio). The
scL-TMZ complex was prepared as described above in Example 1 using
an anti-transferrin receptor single-chain antibody fragment
(TfRscFv) as the targeting moiety, a Lip:TMZ molar ratio of 1:1
(liposome concentration=8 mM) and an TfRscFv to Liposome ratio if
1:30 (w:w). Control animals (Vehicle) received Liposome only (no
TMZ, no TfRscFv). Prepared by the methods described above, the
sizes of the scL-TMZ complexes iv injected into the mice during
this study were found to average 130.8+13.5 nm (number average)
(Mean+S.E.). The mice were treated once per day for 5 consecutive
days. They were euthanized 48 hours after the last injection and
the tumors harvested. Treatment was started on day 0.
[0235] Assessment of in vive efficacy: The in vivo response of T98G
subcutaneous tumors to treatment with either free TMZ or scL-TMZ
complex was evaluated based upon the changes in tumor growth, body
weight change, and induction of apoptosis. The size of each tumor
was measured and tumor volume (L.times.W.times.H) in mm.sup.3 was
plotted versus time. Body weight change was also monitored during
injection. The in vivo efficacy was further evaluated by assessing
the level of apoptosis by TUNEL assay or cleaved caspase-3 staining
with flow cytometry. Forty eight hours after the last injection the
mice were euthanized and tumors harvested. Single-cell isolation
was performed as described above. To determine the level of
apoptosis, single cells were stained either for TUNEL assay or with
antibodies for cleaved caspase-3, human CD133 and SSEA-1. SSEA-1 is
known to be a marker for CSCs in GBM tumors. The labeled cells were
analyzed by FACS.
[0236] The tumor size (volume in mm.sup.3) of the tumors over this
short period of time is shown in FIG. 15. Here again there is a
significant difference in growth of these TMZ resistant tumors
between those animals that received the free TMZ and the scL-TMZ
complex, both administered at 25 mg/kg, in which there is
significant tumor growth inhibition. As T98G tumors are known to be
resistant to TMZ it is novel and unexpected that TMZ could control
tumor growth. As discussed above for use in vitro, this reversal of
resistance is due to the efficient delivery and uptake of the TMZ
payload into the tumor cell by means of the binding of the
targeting ligand (protein, antibody or antibody fragment) to its
receptor on the cell and the triggering of uptake via through
active transport mechanisms like receptor mediated endocytosis.
This process "floods" the cells with drug overcoming the mechanisms
the tumor cell has in place to repair the DNA damage caused by TMZ
(such as upregulation of MGMT), and/or the mechanisms to pump the
TMZ out of the cells. Thus the tumor cell and consequently the
tumor dies. Based upon this in vivo data, the same mechanism works
in vivo and thus demonstrates the potential use of the scL-TMZ
complex as an anticancer agent for brain and other tumors,
including those currently resistant to TMZ. The lack of toxicity of
the iv administered scL-TMZ is indicated by the minimal change in
body weight over the short term of this experiment (FIG. 16).
[0237] The level of apoptosis in the tumors from animals that had
been iv injected with 66 mg/kg (per injection per mouse) of free
TMZ or 25 mg/kg (per injection per mouse) TMZ encapsulated in tumor
targeting complex was assessed 8 hours post-injection by TUNEL
staining of CD133+ CSCs and CD133- non-CSCs isolated from
subcutaneous T98G xenograft tumors. As shown in FIG. 17, even
though the animals were treated with more than double the dose of
free TMZ compared to the amount of TMZ encapsulated in scL, the
level of apoptosis induced by scL-TMZ was more than 5 fold higher
in the non-CSCs and 8 fold higher in the CSCs.
[0238] Similar results were obtained when the level of apoptosis
was assessed by cleaved caspase-3 antibody staining of SSEA-1+ CSCs
from the same subcutaneous T98G brain tumors (FIG. 18). SSEA-1,
another marker of CSCs was used to distinguish CSCs from
differentiated cancer cells. Here also the scL-TMZ induced a 5 fold
(SSEA-1-) and 9.5 fold (SSEA-1+) higher level of apoptosis than
free TMZ even though more than twice the amount of free TMZ was
administered.
[0239] Results Summary: Compared to the treatment with free TMZ,
intravenous treatment with scL-TMZ (at the same or even lower dose
of TMZ) resulted in a significantly enhanced growth inhibition
against TMZ-resistant T98G tumor xenografts. However, no
significantly increased toxicity based upon body weight change was
observed in scL-TMZ treated animals compared to those of free TMZ
treated animals. In addition, intravenous treatment with scL-TMZ
resulted in a significantly increased level of apoptosis not only
in CD133- and SSEA-1- differentiated cancer cells, but also in
CD133+ or SSEA-1+ CSCs.
[0240] These results demonstrate that delivery of TMZ by scL can
induce massive apoptosis and overcome the inherent resistance of
tumors cells (including, but not limited to brain, multiple
myeloma, lung cancer, prostate cancer, liver cancer, ovarian
cancer, pancreatic cancer, head and neck cancer, kidney cancer,
melanoma, stomach cancer) to this drug.
Example 10
Combination Therapy for TMZ-Resistant Tumors
[0241] Although the first-line chemotherapeutic agent Temozolomide
(TMZ) has shown benefit in patients with brain tumors, it also has
significant therapeutic dose limiting toxicities (Villano J L,
Seery T E and Bressler L R (2009) Temozolomide in Malignant
Gliomas: Current Use and Future Targets. Cancer Chemotherapy and
Pharmacology 64: pp 647-655), including myelosuppression. Thus,
ways to tumor-target TMZ so that it is specifically and efficiently
delivered to, and taken up by, tumor cells in the brain thereby
reducing non-specific toxicities, would be of significant benefit
to those patients who are currently candidates for use of this
drug.
[0242] However, even if efficiently delivered to the tumor cells,
one significant drawback to the widespread use of TMZ for
glioblastomas and other brain cancers is that a significant percent
of tumors are resistant to TMZ. This is primarily due to
overexpression of O.sup.6-methylguanine-DNA-methyl transferase
(MGMT), which repairs the TMZ-induced DNA lesions by removing the
O.sup.6-guanine adducts (Mrugala M M, Adair J and Kiem H P (2010)
Temozolomide: Expanding Its Role in Brain Cancer. Drugs of Today
46: pp 833-846), thus negating the therapeutic action of TMZ.
Therefore, it is imperative to develop ways to overcome this
resistance.
Tumor-Targeting scL-p53 Nanocomplex for Gene Therapy
[0243] As described in U.S. Pat. No. 7,780,822, the disclosure of
which is incorporated by reference herein in its entirety, a
delivery system carrying a plasmid DNA encoding the wtp53 gene and
targeted via TfRscFv (scL-p53) has been successfully developed.
scL-p53 has also been developed for use in combination with
chemotherapy/radiation to increase the tumor response to these
standard therapeutic modalities.
[0244] Although TMZ is a first-line chemotherapeutic for the
treatment of brain tumors, only a subset of GBM patients respond to
this drug. Based on the work of Stupp et al., (Stupp R, Hegi M E,
Mason W P, van den Bent M J, Taphoorn M J B, Janzer R C, Ludwin S
K, Allgeier A, Fisher B, Belanger K, Hau P, Brandes A A, Gijtenbeek
J, Marosi C, Vecht C J, Mokhtari K, Wesseling P, VIIIa S,
Eisenhauer E, Gorlia T, Weller M, Lacombe D, Caimcross J G and
Mirimanoff R (2009) Effects of Radiotherapy With Concomitant and
Adjuvant Temozolomide Versus Radiotherapy Alone on Survival in
Glioblastoma in a Randomised Phase III Study: 5-Year Analysis of
the EORTC-NCIC Trial. Lancet Oncology 10: pp 459-466) as well as
that of Hegi et al, (Hegi M E, Diserens A, Gorlia T, Hamou M, de
Tribolet N, Weller M, Kros J M, Hainfellner J A, Mason W, Mariani
L, Bromberg J E C, Hau P, MirimanoffR O, Cairncross J G, Janzer R C
and Stupp R (2005) MGMT Gene Silencing and Benefit From
Temozolomide in Glioblastoma. New England Journal of Medicine 352:
pp 997-1003) two distinct groups of patients were indentified
regarding response to TMZ treatment: those with a downregulated
MGMT promoter with better prognosis and those with an active MGMT
promoter with worse prognosis.
[0245] Thus, development of a means to down-regulate MGMT would
increase the number of patients that respond to TMZ. There have
been a number of reports indicating that increasing wtp53
expression could down-regulate expression of DNA repair genes such
as MGMT (Bocangel D, Sengupta S, Mitra S, Bhakat K.K (2009)
P53-Mediated Down-Regulation of the Human DNA Repair Gene
O6-Methylguanine-DNA Methyltransferase (MGMT) Via Interaction With
Spl Transcription Factor. Anticancer Research; Harris L C, Remack I
S, Houghton P J and Brent T P (1996) Wild-Type P53 Suppresses
Transcription of the Human O6-Methylguanine-DNA Methyltransferase
Gene. Cancer Research 56: pp 2029-2032; Srivenugopal K S, Shou J,
Mullapudi S R S, Lang F F, Rao J S and Ali-Osman F (2001) Enforced
Expression of Wild-Type P53 Curtails the Transcription of the
06-Methylguanine-DNA Methyltransferase Gene in Human Tumor Cells
and Enhances Their Sensitivity to Alkylating Agents. Clinical
Cancer Research 7: pp 1398-1409). The use of the scL-p53
nanocomplex, shown to efficiently target primary and metastatic
tumors and to cross the BBB, should be an effective means to
overcome the MGMT induced resistance to TMZ observed in a
significant percentage of GBM and other tumors, thus broadening the
application of this drug for use in, and improving the prognosis
of, patients with primary and metastatic brain tumors. Moreover,
since TMZ is also being evaluated for use in other non-brain
refractory or advanced malignancies including pancreatic,
neuroendocrine and areodigestive tract cancers (Tentori L and
Graziani G (2009) Recent Approaches to Improve the Antitumor
Efficacy of Temozolomide. Current Medicinal Chemistry 16: pp
245-257.), treatment with scL-p53 will enhance the potential of TMZ
to be an effective therapeutic agent for a variety of cancers.
scL-TMZ and scL-p53 Combination Therapy
[0246] Described herein is the use of the combination of scL-TMZ
and scL-p53. The development of scL-TMZ for use as a monotherapy
will be of benefit to patients that currently are candidates for
TMZ treatment. However, the combinatorial approach will have an
even greater therapeutic potential. The decreased tumor resistance
due to scL-p53, along with the improved properties that result from
tumor-targeted delivery of scL-TMZ, would result in converting
currently TMZ unresponsive brain tumors (and possibly other
cancers) to responsive. Therefore, this approach has the potential
to be developed into a new, less toxic, more effective therapeutic
regimen for the treatment of GBM and other cancers.
[0247] Experimental Approach
[0248] The experiments are designed to demonstrate development of a
new, more effective treatment regimen for GBM with use of scL-TMZ,
both alone and when used in combination with scL-p53.
Human Brain Tumor Cell Lines and in Vivo Models
[0249] Human brain cancer cell lines U87MG and T98G were described
above. A version of U87MG that stably expresses the luciferase gene
has been obtained from Caliper Life Sciences for use in in vivo
studies where tumor growth and response will be monitored by the
IVIS.RTM. Imaging System Xenogen.
Imaging Protocol
[0250] The MR imaging for brain tumors will be performed on a 7T
Bruker Biopsin (Billerica, Mass.) horizontal spectrometer/imager
with a 20 cm bore equipped with 100 gauss/cm microimaging gradients
and run by Paravision 4.0 software. The imaging protocol is a
T1-weighted Turbo rapid acquisition with rapid enhancement
three-dimensional imaging sequence as previously described
(Haggerty T, Credle J, Rodriguez O, Wills J, Oaks A W, Masliah E
and Sidhu A (2011) Hyperphosphorylated Tau in an
Alpha-Synuclein-Overexpressing Transgenic Model of Parkinson's
Disease. European Journal of Neuroscience 33: pp 1598-1610).
Demonstration of In Vivo Efficacy of scL-TMZ Alone and in
Combination with scL-p53
[0251] In these studies, the U87-Luc orthotopic intracranial (TMZ
sensitive) and T98G (TMZ resistant) subcutaneous tumor models will
be used to examine the effect of scL-TMZ alone and in combination
with scL-p53 on tumor growth and/or regression. It should be noted
that although U87MG has wt p53, an increased in vive response is
observed when U87 intracranial tumors are treated with the
combination of scL-p53 and free TMZ. Groups of mice (6
mice/group/tumor model) will receive i.v (tail vein) injections of
Free TMZ alone, scL-TMZ alone, scL-p53 alone, scL-p53 plus free TMZ
or scL-TMZ. Untreated mice will serve as controls. The p53 dose
will be 30 ug/mouse/injection, and TMZ will be used at 5 mg/kg.
Both treatments will be administered twice weekly for 5 weeks.
Tumor growth inhibition/regression will be assessed by size for
T98G and with the Xenogen for the intracranial tumors where tumor
volume will be determined by MRI. Xenogen/MRI imaging will be done
pre-treatment, once/week during treatment and immediately after
treatment has ended. Half-way through treatment, tumors and various
normal organs and tissues will be taken from 3 mice in each group
and coded. Half of each tissue will be used for the analysis of
cancer stem cell targeting and the remainder examined by histology
for markers of apoptosis (Tunnel, Caspase -3) and for proliferation
marker Ki67. One day after treatment has ended, 3 mice will be
humanely euthanized and necropsied by a commercial CRO
(BioReliance, Rockville Md.) to look for differences in myelotoxic
effects and lymphopenia associated with TMZ.
In Vitro Results
[0252] To test the hypothesis that treatment with scL-p53 could
down modulated MGMT activity and sensitize TMZ resistant brain
tumors to this drug, a preliminary XTT cell survival assay was
performed. TMZ resistant T98G cells were plated at 2.times.10.sup.3
per well in a 96-well plate and transfected with scL-p53 in
combination with either Free TMZ or scL-TMZ. The cells were also
transfected with just free TMZ or just scL-TMZ. The XTT assay was
performed 90 h later and the IC.sub.50 values (the concentration
yielding 50% growth inhibition) determined. Transfection with
scL-p53 in combination with either free or scL complexed TMZ
resulted in an increased level of response compared to single agent
TMZ in this known TMZ resistant cell line (Patil R, Portilla-Arias
J, Ding H, Inoue S, Konda B, Hu J W, Wawrowsky K A, Shin P K, Black
K L, Holler E and Ljubimova J Y (2010) Temozolomide Delivery to
Tumor Cells by a Multifunctional Nano Vehicle Based on
Poly(Beta-L-Malic Acid). Pharmaceutical Research 27: pp 2317-2329)
(FIG. 19). Moreover, compared to free TMZ alone, transfection with
the scL-TMZ nanocomplex resulted in a significant level of
chemosensitization to the drug.
Example 11
In Vivo Targeting of Cancer Stem Cells by Systemically Administered
scL-Complex in a Mouse Brain Tumor Model
[0253] Human brain tumor xenografts were induced in nude mice by
subcutaneous inoculation of U251 cells. Three weeks later, the mice
were I.V. injected with 6FAM-ODN (100 .mu.g/mouse) administered as
either uncomplexed free 6FAM-ODN, scL-6FAM-ODN (scL-ODN), or the
unliganded Lip-6FAM-ODN (the liposome without the targeting moiety)
(LIP-ODN). 24 hours post-injection, the tumors were imaged using
the Maestro.TM. in vivo fluorescence imaging system to determine
the level of fluorescence in the tumors. After Maestro.TM. imaging,
single cells were isolated from the tumors by enzyme digestion, and
the amount of 6FAM-ODN uptake in CD133+ (Cancer Stem Cell (CSC))
and CD133- (non-CSC) cells analyzed and quantitated by FACS.
Systemic administration of both free 6FAM-ODN and unliganded
Lip-ODN resulted in very little, if any, fluorescence in the
tumors. In contrast, a strong fluorescence signal was evident in
the tumor from the mouse that received the scL-ODN nanocomplex
(FIG. 20).
[0254] More importantly, this significant difference was also
reflected in the transfection efficiency of CSCs (FIG. 21). Whereas
less than 10% of the CSC and Non-CSC cells were transfected with
the free or unliganded 6FAM-ODN, >60% of both CSC and non-CSC
cell populations demonstrated the presence of the payload after
I.V. injection. Gray histograms represent untreated controls. These
findings confirm that with systemic administration, the scL
delivery system described herein can target and efficiently deliver
payloads to CSCs in vivo.
Example 12
Tumor Specific Targeting of CSCs in IC GBM by scL-Delivered ODN
After Systemic Administration
[0255] FIG. 22 shows the comparison of in vivo delivery efficiency
of payload delivered by tumor-targeting complex, non-targeting
complex, and payload itself without the delivery system in an
animal model of intracranial U87MG-luc2 glioblastoma multiforme
(GBM) brain tumors. Fluorescently labeled (Cy5) oligonucleotide
(Cy5-ODN) was encapsulated in tumor-targeted complexes
(scL-Cy5-ODN) (prepared as described in U.S. Pat. No. 7,780,882) or
complexes without tumor-targeting ligand (Lip-Cy5-ODN). Twenty five
micrograms of Cy5-ODN (free or encapsulated with or without the
targeting moiety) were injected intravenously to each animal
bearing a U87MG-luc2 intracranial tumor. At 60 hr after injection,
the animals were euthanized and tumors were harvested to assess the
efficiency of delivery to cancer stem cells (CSCs) in these
intracranial tumors by flow cytometry using markers for cancer stem
cells, CD133 and SSEA, both of which are known by those familiar
with the art to be markers of CSC in general (CD133+) and CSC in
brain tumors (SSEA-1+). Single cells were isolated from brain
tumors and subjected to FACS analysis after staining with CSC
marker antibodies (CD133+ or SSEA-1+). The shift in the curve in
each histogram in FIG. 22 represents Cy5-ODN uptake in cancer stem
cells. Only the curves representing the CSCs isolated from the mice
receiving the scL-Cy5-ODN demonstrated a significant shift,
indicating that neither the free Cy5-ODN, nor the Lip-Cy5-ODN
(without the targeting moiety) efficiently transfected CSCs in the
brain tumor.
Example 13
In Vitro Sensitization of Brain Tumor cells to Temozolomide (TMZ)
by scL-p53
[0256] To assess the ability of scL delivered wtp53 to sensitize
brain tumor cells to first-line chemotherapeutic agent TMZ, human
brain tumor derived U87 and U251 cells were treated with TMZ alone,
or the combination of TMZ plus scL-p53 (prepared as described in
U.S. Pat. No. 7,780,882). As a control, cells were also treated
with the combination of TMZ and the scL delivery system carrying
the same vector used to construct the pSCMVp53 plasmid, but without
the p53 insert (scL-vec). The cells were plated in a 96-well plate
and treated 24 hours later with scL-p53 or scL-vec. 6 hours
post-transfection, the TMZ was added in increasing concentrations.
The XTT assay was performed 144 h after the addition of the TMZ to
the wells and the IC.sub.50 values (the concentration yielding 50%
growth inhibition) determined. As these two cell lines are known to
be sensitive to TMZ, it was not unexpected that there was some
response to TMZ alone. However, as shown in FIG. 23, there is a
significant increase in sensitization to TMZ when the cells are
transfected with wtp53 delivered by the scL delivery system when
compared to TMZ alone for both cell lines. Minimal to no
sensitization (U87 and U251, respectively) was observed with the
complex carrying the empty vector, demonstrating that the effect is
due to the p53 and not the delivery system.
[0257] However, as only a subset of brain tumor patients respond to
TMZ it was more critical to assess the ability of scL-p53 to
sensitize TMZ resistant tumors to this chemotherapeutic agent.
Thus, to assess the ability of scL delivered wtp53 to sensitize TMZ
resistant brain tumor cells to this first-line chemotherapeutic
agent, human brain tumor derived LN-18 and T98G cells were treated
with TMZ alone, or the combination of TMZ plus scL-p53 (FIG. 23).
As a control, cells were also treated with the combination of TMZ
and the scL delivery system carrying the same vector used to
construct the pSCMVp53 plasmid, but without the p53 insert
(scL-vec). The cells were plated in a 96-well plate and treated 24
hours later with scL-p53 or scL-vec. 24 hours post-transfection,
the TMZ was added in increasing concentrations. The XTT assay was
performed 72 h after the addition of the TMZ to the wells and the
IC.sub.50 values (the concentration yielding 50% growth inhibition)
determined. As shown in FIG. 23 with LN-18 cells, after
transfection with scL-p53 these TMZ resistant cells are now
responding to even very low doses of TMZ. More than 50% of the
cells are killed at a dose of TMZ as low as .about.50 uM compared
to TMZ alone, in which no significant cell death is observed until
a dose of .about.1000 uM. With the T98G cells (FIG. 23), although
not as responsive as to TMZ as LN-18 after treatment with scL-p53,
these highly resistant cells are also sensitized to the killing
effects of this drug. The cells treated with scL-p53 prior to
exposure to TMZ have an IC.sub.50 of 600 uM while those receiving
TMZ only do not reach IC.sub.50 until the TMZ dose is .about.2000
uM. As above, there is minimal or no effect on the response of the
cells to TMZ after transfection with the control scL-vec indicating
that the response in these resistant cell lines is due to the
presence of wtp53.
Example 14
In Vivo Sensitization of Brain Tumor Cells to Temozolomide (TMZ) by
Systemically Administered scL-p53
[0258] Tumor Regression in an Intracranial (IC) Mouse Model of
Brain Cancer Induced by Systemic Treatment with the combination of
scL-n53 plus TMZ
[0259] An experiment was performed to examine tumor growth
inhibition induced by the sensitization of IC brain tumors to TMZ
by systemic administration of scL-p53 prepared as described in U.S.
Pat. No. 7,780,882. U87MG-Luc xenograft brain tumors were induced
in nude mice by intracranially inoculating U87MG-luc cells. This
cell line, obtained from Caliper Life Sciences, has been modified
to stably express the Luciferase gene. 10 days post-inoculation,
tumor-bearing animals were i.v. tail vein injected with TMZ alone
(5.0 mg/kg/injection), scL-p53 alone (30 ug DNA/mouse/injection) or
TMZ in combination with scL-p53. As a control, one group received
PBS (vehicle). All i.v. injections were administered 2.times./week
to a total of 10 injections. To assess tumor response,
bioluminescence imaging (BLI) was performed using IVIS.RTM. Imaging
System's Xenogen. FIG. 24 is a comparison of in vivo anti-tumor
efficacy of the various groups. Bioluminescence signals which
correlate to tumor size are shown in a color map. Red color (at the
top of the scale bar): the stronger signal, Violet color (at the
bottom of the scale bar): the weaker signal. The bioluminescence
intensity of the brain tumors, a measure of tumor size/growth, was
compared between groups using Xenogen Living Imagem software and is
plotted over time in FIG. 25. The horizontal bar indicates the
duration of treatment (Last treatment=Day 24).
[0260] While TMZ alone and scL-p53 alone had some minimal effect on
IC tumor growth during treatment, the tumors in both groups rapidly
increased in size after the end of treatment. In contrast, the
tumors in the group of mice that received the combination of
scL-p53 and TMZ displayed not only tumor growth inhibition, but
tumor regression during treatment. More significantly, this
regression continued for more than 20 days after treatment had
ended.
[0261] To confirm the bioluminescence findings, the mice were also
imaged by MRI, without contrast agent, before and after the mice
received 3 weeks of treatment. The tumor regression observed in the
combination treatment group by bioluminescence was also observed
here. In FIG. 26 the outlines indicate the glioblastoma tumors. It
is evident that instead of increasing in size post treatment as is
evident in the single agent treatment groups, any residual tumor is
barely detectable in the these animals that received both scL-p53
and TMZ. Therefore, this experiment demonstrates that the presence
of scL-delivered wtp53 can sensitize GBM tumors to TMZ leading to
significant tumor response (regression) not just tumor growth
inhibition.
Significantly Increased Survival of Mice Bearing Intracranial GBM
Tumors after Treatment with the Combination of scL-p53 and TMZ
[0262] As the above experiments demonstrated significant tumor
responses, including regression post-treatment, the effect of this
combination treatment on survival was next assessed. U87MG-Luc
xenograft brain tumors were induced in nude mice as described
above. 10 days post-inoculation, tumor-bearing animals were i.v.
tail vein injected with TMZ alone (25.0 mg/kg/injection), scL-p53
alone (30 ug DNA/mouse/injection) (prepared as described in U.S.
Pat. No. 7,780,882) or TMZ in combination with scL-p53. As a
control, one group received PBS (vehicle). All i.v. injections were
administered 2.times./week to a total of 10 injections. The animals
were monitored 2-3 times/week and euthanized when moribund. The
results, analyzed by Kaplan-Meier method, are shown in FIG. 27 and
Table 1 below. The gray bar in FIG. 27 indicates the duration of
treatment. Although TMZ alone was able to prolong survival for a
period of time in this TMZ responsive cell line, all of the mice
succumbed to their tumor by .about.day 155 with a Median Survival
of 112 days. However, the survival time was extended considerably
by the addition of scL-p53 to the treatment regimen. In these
animals 60% of the mice were still surviving at day 210. Therefore,
the % survival prolongation for mice receiving this combination
regimen was >740 times that of the untreated mice, 500 times
that of scL-p53 alone and almost twice that of TMZ alone. Thus,
this adding scL-p53 to treatment with TMZ results in a significant
increase in long term survival.
TABLE-US-00002 TABLE 1 Median Survival Log Survival Prolongation
Rank Treatment n (Days) (%)* P-value Untreated 4 25 -- -- scL-p53 5
35 40 0.0117 TMZ 5 112 348 0.0088 TMZ + scL-p53 5 >210 >740
0.0058 *Determine as a ratio of the median survival of untreated
GBM xenografts
Significantly Increased Survival of Mice Bearing TMZ Resistant
Intracranial GBM Tumors After treatment with the Combination of
scL-p53 and TMZ
[0263] The in vitro studies described above indicated that
transfection of TMZ resistant brain tumor cells could be sensitized
to TMZ by transfection of scL-p53. Thus an experiment was performed
to assess survival of mice bearing intracranial tumors derived from
TMZ resistant human GBM cell line T98G. Athymic nude mice were
intracranially inoculated with T98G human glioblastoma cell line.
Ten days after the inoculation, animals were imaged by MRI and
evenly divided into 3 groups. Treatment was started immediately
after imaging. The animals were iv treated with 100 mg/m.sup.2 of
TMZ once a day for 14 consecutive days, iv administered scL-p53 (30
ug DNA/injection) (prepared as described in U.S. Pat. No.
7,780,882) twice weekly for 2 weeks, or the combination of both
treatments. Survival was monitored. The results, analyzed by
Kaplan-Meier method, are shown in FIG. 28. The number of mice
surviving/group at day 14 is indicated for each group. Over this 2
week time of treatment a significant number of animals succumbed to
their disease in the two groups that received TMZ or scL-p53 as a
single agent. In contrast, 4 of 5 mice that had received the
combination therapy were still surviving. Thus, this small efficacy
experiment confirms the in vitro data and indicates that treatment
with scL-p53 can sensitize previously resistant GBM tumors to
TMZ.
Example 15
Enhanced Apoptosis in Intracranial Brain Tumors by the Combination
of scL-p53 and TMZ
[0264] Tumor suppressor p53 is known to play a role in the
apoptotic pathway. To begin to evaluate the mechanism responsible
for the increase in tumor cell response and increase in animal
survival observed with the combination of scL-p53 and TMZ, the
level of apoptosis induced in intracranial U87MG-luc2 brain tumors
after various treatments was determined using Annexin V-FITC and
Flow Cytometry. U87MG-luc2 brain tumors were induced as described
above. 10 days post inoculation of the cells, the mice were treated
with either TMZ alone (5 mg/kg per injection per mouse, 2
injections per week), scL-p53 alone (30 ug DNA per injection per
mouse, 2 injections per week) (prepared as described in U.S. Pat.
No. 7,780,882) or the combination of scL-p53 and free TMZ. Each
animal received a total of 3 injections after which the animals
were euthanized, single cell population isolated from the tumors
and subjected to the Annexin V assay.
[0265] As shown in FIG. 29, there is a significant increase in the
percent of the tumors cells in apoptosis after treatment with the
combination of scL-p53 and TMZ compared to either treatment alone.
Thus, these results indicate that uptake of systemically
administered scL-p53 by the IC tumors results in an enhanced
apoptotic response to chemotherapeutic agent TMZ.
Example 16
Treatment of TMZ Resistant GBM Tumors with scL-p53 Downmodulates
MGMT Expression In Vitro and In Vivo
[0266] The primary mechanism of resistance to TMZ is over
expression of O.sup.6-methylguanine-DNA-methyl transferase (MGMT),
which repairs the TMZ-induced DNA lesion by removing the
O.sup.6-guanine adducts. Thus, a means to down modulate MGMT
activity would enhance the therapeutic effect of TMZ. A number of
reports have indicated that increasing wtp53 expression could
down-regulate expression of DNA repair genes such as MGMT and
increase the sensitivity of tumor cells to alkylating agents such
as TMZ. The in vitro and in vivo data described in the Examples
above indicated that treatment with scL-p53 could reverse
resistance to TMZ in brain tumor cells. One possible mechanism for
this sensitization is p53 dependent down modulation of MGMT. Uptake
of scL-delivered wtp53 was examined to determine if it had an
effect on the level of MGMT expression in TMZ-resistant T98G human
glioblastoma cells in vitro and in in vivo subcutaneous xenograft
tumors. T98G cells were transfected with scL-p53 (prepared as
described in U.S. Pat. No. 7,780,882). 16 and 24 hours
post-transfection, the cells were harvested, protein isolated and
40 ug micrograms total protein was electrophoretically fractionated
using a Nu-PAGE Precast 4-12% gradient gel, transferred to
nitrocellulose membrane, and probed for expression of MGMT and
GAPDH by Western blot analysis. The signal was detected by ECL
reagent (FIG. 30).
[0267] For the in vivo experiment shown in FIG. 30, scL-p53 (30 ug
DNA/injection/mouse) was i.v. injected three times over a 24 hr
period. At 16 and 24 hours after the last scL-p53 treatment, the
mice were euthanized, tumors harvested, and protein extracted. Four
mice were harvested at each time point. One group as a control did
not receive SGT-53. 40 ug micrograms total protein was
electrophoretically fractionated using a Nu-PAGE Precast 4-12%
gradient gel, transferred to nitrocellulose membrane, and probed
for expression of MGMT and GAPDH by Western blot analysis. The
signal was detected by ECL reagent.
[0268] In vitro, there was complete down modulation of MGMT
expression by 16 hours, which lasted as long as 24 hours
post-transfection. Similarly, in the in vivo study, a significant
decrease in the expression of MGMT was evident at 16 hours, with
virtual elimination of the protein in two of the animals. By 24
hours after the last injection, virtually complete down modulation
of MGMT was evident in all of the mice treated with SGT-53.
Consistent expression of GAPDH protein demonstrated equal protein
loading. The lack of MGMT to repair the DNA damage induced by TMZ
in these tumors, along with the exogenous SGT delivered wtp53's
positive effect on the apoptotic pathway, likely plays a role in
overcoming the resistance of T98G to the killing effects of
TMZ.
[0269] More significantly, similar results were observed in an
intracranial tumor model with T98G (FIG. 31). In this experiment,
scL-p53 (at 30 ug DNA/mouse) was i.v. injected only once. At
various time points between 16 and 72 hours after the scL-p53
injection, mice were euthanized, tumors harvested, and protein
extracted. One group as a control (UT) did not receive SGT-53. 40
ug micrograms total protein was electrophoretically fractionated
using a Nu-PAGE Precast 4-12% gradient gel, transferred to
nitrocellulose membrane, and probed for expression of p53, MGMT and
GAPDH by Western blot analysis. The signal was detected by ECL
reagent.
[0270] At 16 and 24 hours after the single scL-p53 i.v. injection,
an increase in the level of p53 protein is evident as compared to
the UT animal indicating the presence of the exogenous p53. By 43
and 72 hours this signal had decreased back to a level similar to
that of the UT control. More, importantly, as observed with the
subcutaneous tumors, in these Intracranial tumors a significant
decrease in expression of MGMT was observed at both 43 and 72 hours
after treatment with scL-p53. This timing for the observed decrease
in MGMT signal is consistent with the mechanism of action of p53 in
sensitizing cells to TMZ by interfering with DNA repair
mechanisms.
Example 17
Combination Treatment of scL-p53 and TMZ in Patients with
Glioblastoma or Gliosarcoma
[0271] Standard administration of temozolomide requires a daily
dose of Temozolomide for 21 days. To optimize effectiveness of the
potential chemosensitization of scL-p53, in a preferred embodiment,
scL-p53 treatment will begin 1 day before temozolomide treatment.
Pre-clinical studies have shown that the wtp53 tumor suppressor
gene delivered by the scL-p53 complex functions to sensitize tumors
to the chemotherapeutic agent, making them more responsive to the
drug. Thus, it is critical that p53 is being expressed when
temozolomide is administered in order to have the benefit of the
scL-p53. Using the proposed schedule shown in FIG. 32., scL-p53 is
being expressed at the start of temozolomide treatment.
[0272] In an alternate embodiment, two scL-p53 treatments will be
administered before the start of temozolomide treatment. Here,
scL-p53 will be administered on the same scheduled indicated in the
table in FIG. 33, beginning on Day 1. However, the first TMZ
treatment will not be until Day 6. Temozolomide will be
administered orally at 100-250 mg/m.sup.2 every day (including
weekends) for 21 days (from day 6 to day 26).
Example 18
Preparation of Targeted Cationic Liposomes Comprising Melphalan
(MEL)
Materials:
[0273] DOTAP (1,2-dioleoyl-3-trimethylammonium propane, chloride
salt) [0274] Obtained from Avanti Polar Lipids, Inc. Cat. #890890E,
MW 698.55 [0275] Concentration: 25 mg/mL ethanol solution [0276]
Dilute lipid to 20 mg/ml with absolute ethanol before use
[0277] DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) [0278]
Obtained from Avanti Polar Lipids, Inc. Cat. #850725E, MW 744.04
[0279] Concentration: 25 mg/mL ethanol solution. [0280] Dilute
lipid to 20 mg/ml with absolute ethanol before use
[0281] Melphalan Hydrochloride (MeI), powder (M Wt=341.7) [0282]
Obtained from Sigma, M2011-100 mg, [0283] Dissolve in absolute
ethanol to a concentration of 50 mg/ml, with 10-15 ul of 6N HCl to
aid in dissolution
[0284] Ultra-pure, endotoxin free LAL Reagent Water (e.g.
BioWhittaker, Cat. #W50-500, endotoxin <0.005 EU/ml)
[0285] Injector: Hamilton Gastight Syringe, 1 ml (Hamilton #81230)
with a 22 gauge needle, part #81365)
Procedure:
[0286] 1. Fresh MeI solution is prepared each time by dissolving
MeI in absolute ethanol to a concentration of 50 mg/ml (146.3 mM)
with the addition of 6N HCl (.about.10-15 uL) by vortexing at high
speed until dissolved (must be clear). Hold at room temperature
until used to mix with lipids (Step 3 below).
[0287] 2. Place lipid solutions at 37.degree. C. for 10-15 min,
following which place the lipid solutions in a 65.degree. C. water
bath with occasional shaking for 5 min.
[0288] 3. To prepare the Lip-MeI: Place a brown glass bottle with
stir bar on a hot plate set to 50.degree. C. to 60.degree. C. While
stirring at high speed without splashing, add the lipids and MeI to
the bottle in the following order (important):
[0289] For 1:1 (Lip:Mel) molar ratio
[0290] DOTAP 175 .mu.l (of 20 mg/ml)=5 .mu.mol or 3.5 mg
[0291] DOPE 187.5 .mu.l (of 20 mg/ml)=5 .mu.mol or 3.75 mg
[0292] Add MeI soln., 68.3 .mu.l (of 50 mg/ml)=10 .mu.mol,
[0293] Continuously stir for 3 min. after all 3 have been added
[0294] 4. In the meantime, warm 4,569 uL LAL water to 65.degree. C.
in water bath in brown glass bottle with stir bar. Immediately
prior to addition of the Lipid-MeI solution, move the bottle to a
hot plate (50.degree.-60.degree. C.). Stir water at high speed with
no splashing for a few sec to remove bubbles from the stir bar.
[0295] 5. Keep the water on the hot plate. Continue stirring the
water at high speed (without splashing) during lipid addition.
After mixing lipids and MeI as above (Step 2), immediately and as
rapidly as possible, using the Hamilton syringe for injection,
inject the mixture into the hot water on the hot plate
(50.degree.-60.degree. C.) directly into the center of the vortex.
Continue stirring on high speed (without splashing) for 1 min after
the addition of the lipid mixture while loosely covered.
[0296] 6. Move the glass bottle to a RT stir plate, and, continue
to stir slowly until the loosely covered solution cools down to
20-25.degree. C. (room temperature)
[0297] 7. Adjust the volume to 5 ml with room temperature LAL
water.
[0298] 8. Filter the solution using a 0.22 .mu.m pore Milex GV
filter.
[0299] 9. Measure particle size and zeta potential if desired.
[0300] Results of these preparation methods demonstrate liposomes
having a particle size of about 20-60 nm and a Zeta Potential of
about 10 to 50 mV.
Example 19
Preparation of Targeted Cationic Liposome Containing Melphalan
(scL/MEL) Without Chemical Conjugation (by Simple Mixing)
[0301] Using the MEL-comprising cationic liposomes prepared
according to the procedure described above, the ligand targeted MEL
cationic liposome complex as described herein is prepared by simple
mixing of the components and without chemical conjugation. The
preparation of the complexes was in accordance with the following
general procedure:
[0302] To the liposome-water (or buffer) the appropriate amount of
targeting moiety is added to give the desired ratio and mixed by
gentle inversion 5-10 seconds. The targeting moiety can be a ligand
including but not limited to transferrin or folate, or other
proteins. It can also be an antibody or an antibody fragment that
targets a cell surface receptor including, but not limited to the
transferrin or HER-2 receptor (e.g., TfRscFv). This mixture is kept
at room temperature for 10-15 minutes (again inverted gently for
5-10 seconds after approximately 5 minutes). To yield the desired
final volume the targeting moiety-Lip-MEL admixture is mixed with
any volume (including none) of water (suitably deionized water) or
a buffer of any pH including but not limited to, Tris buffers,
HEPES buffers or Phosphate Buffered Saline, required to give a
desired volume and inverted gently for 5-10 seconds to mix. This
mixture is kept at room temperature for 10-15 minutes (again
inverted gently for 5-10 seconds after approximately 5
minutes).
[0303] Typically, for use in an in vitro assay, it is desirable
that the amount of MEL in the final complex is in the range of
about 1 .mu.M to 30 .mu.M per well; for in vivo use, it is
desirable to provide about 1 mg/kg to about 50 mg/kg of MEL per
injection. For use in vive dextrose or sucrose is added last to a
final concentration of about 1-50% (V:V) dextrose or sucrose,
suitably 5% dextrose or 10% sucrose, and mixed by gentle inversion
for 5-10 seconds. This mixture is kept at room temperature for
10-15 minutes (again inverted gently for 5-10 seconds after
approximately 5 minutes).
[0304] A specific example for in vitro transfection at a suitable
ratio of 1:30 (antibody fragment:liposome, w:w) and 1:1
Liposome:MEL (molar ratio) is as follows: For a final volume of
approximately 600 uL, mix 250 L of Lip:TMZ (2 mM stock) with 56.7
.mu.L of antibody fragment (at an anti-transferrin receptor single
chain antibody fragment [TtRscFv] concentration of 0.21 mg/mL). Add
293.3 .mu.L of water or buffer.
[0305] The size (number average) of the final complex prepared by
the method is between about 10 to 800 nm, suitably about 15 to 400
nm, most suitably about 20 to 200 nm with a zeta potential of
between about 1 and 100 mV, more suitably 5 to 60 mV and most
suitably 10 to 50 mV as determined by dynamic light scattering
using a Malvern Zetasizer ZS. This size is small enough to
efficiently pass through the tumor capillary bed, or cross the
blood brain barrier, and reach the tumor cells.
[0306] The complex prepared as described above containing dextrose
or sucrose (1-50%, volume to volume) can also be lyophilized to
dryness and stored at room temperature, 2-8.degree. C., or -20 to
-80.degree. C. The samples are reconstituted with water prior to
use. The size (number average) of the final lyophilized complex
after reconstitution is between about 10 to 800 nm, suitably about
15 to 400 nm, more suitably about 20 to 200 nm and most suitably 50
to 150 nm with a zeta potential of between about 1 and 100 mV, more
suitably 5 to 60 mV and most suitably 10 to 50 mV as determined by
dynamic light scattering using a Malvern Zetasizer ZS. These
complexes retain at least 80% of the original biological
activity.
Example 20
Increased Effect of Lip/MEL on Tumor Cells Compared to Free
(unencapsulated) MEL
[0307] The scL-MEL nanocomplex was prepared as described above
using an anti-transferrin receptor single-chain antibody fragment
(TfRscFv) as the targeting moiety, a Lip:MEL molar ratio of 1:1 and
0.75:1 (sizes=30 and 25 nm, respectively) (liposome concentration=2
mM) and an TfRscFv to Liposome ratio if 1:30 (w:w). The size of the
scL-MEL nanocomplex was 45 and 57 nm, respectively. The in vitro
cell killing ability of the scL-MEL was compared to free,
unencapsulated MEL in KMS-11 cells.
[0308] For these in vitro cell survival studies, the human multiple
myeloma cell line KMS-11 was used. These are non-adherent cells and
grow in suspension. 4.times.10.sup.5 cells in 2.6 ml of serum free
media were incubated in a sterile 50 ml centrifuge tube with 400 ul
of the scL-MEL (at either ratio) or unencapsulated MEL for one
hour. Following this incubation, the medium was supplemented with
fetal bovine serum to a final concentration of 10% (0.3 ml/tube).
After incubation for an additional 47 h, cell viability was
determined by Trypan Blue counting of the cells. To accomplish
this, one part trypan blue solution is mixed with 1 part cell
suspension (1:1 dilution) in a 2 mL eppendorf tube (e.g. 200 .mu.L
trypan blue is mixed with 200 .mu.L cell suspension). Using a
hemacytometer and a microscope the number of viable cells
(unstained) and dead cells (stained) are counted. The average
number of cells per 0.1 mm.sup.3 is calculated and the number of
cells per mL determined. The percent of viable cells is calculated
as follows:
Viable Cells %-(Number of viable cells/Number of Cells)*100
[0309] The results are plotted and the IC.sub.10 and IC.sub.30
values, the drug concentration resulting in 50% and 30% cell kill,
respectively, was interpolated from the graph of the drug
concentration versus the percent of viable cells.
[0310] FIG. 33 shows that tumor targeting scL-MEL nano complex, in
which the MEL is encapsulated in the liposome, has significantly
improved anti-cancer efficacy compared to unencapsulated MEL. The
unencapsulated MEL has an IC.sub.50 value of 17.6 uM. In contrast,
when encapsulated in the liposome via the method of this invention
at a 1:1 molar ratio of Liposome to MEL, and delivered to the tumor
cell by means of the tumor-targeting nanocomplex of this invention
at approximately 2 fold less MEL will effectively kill the cancer
cells (IC.sub.50 of 9.6 uM). Although not as dramatic, there was
also a 30% decrease in the IC.sub.50 values between the
unencapsulated MEL and scL-MEL when the Lip-MEL was prepared at a
Liposome:MeI ratio of 0.75:1 (molar ratio). Transfection with the
Liposome alone did not result in any significant cell kill
(IC.sub.50>33 .mu.M) indicating that the sensitization observed
with the scL-MEL is not a result of non-specific cell kill by the
liposome. Thus a variety of different molar ratios of liposome to
Melphalan when used in the methods of this invention will result in
a compound which when complexed to the targeting moiety by simple
mixing and without chemical conjugation using the methods of this
invention will result in a complex that has unexpected enhanced
efficacy against multiple myeloma cells.
[0311] Similar results are shown in FIG. 34 comparing
unencapsulated MEL, with Lip-MEL as described herein without the
targeting moiety and with the full scL-MEL nanocomplex, as well as
with liposome only. The Lip-MEL and scL-MEL were prepared as
described in Examples 1 and 2 at molar ratios of Liposome to MEL of
1:1)(liposome concentration=2 mM). An anti-transferrin receptor
single-chain antibody fragment (TfRscFv) was used as the targeting
moiety, with a TtRscFv to Liposome ratio of 1:30 (w:w). Once again
the liposome only has virtually no cell killing effect on these
multiple myeloma cells. In contrast, even without the targeting
moiety, when encapsulated in the Liposome at molar ratios of 1:1
(Lip:MEL) using the methods described herein, there was a
significant decrease in the IC.sub.50 value compared to free
(unencapsulated) MEL. This level of sensitization was even greater
(almost 2 fold) when the full scL-MeI complex was used. This level
of sensitization of multiple myeloma cells after encapsulation in
liposomes via the method of this invention is unexpected.
Example 21
Maintenance of Biological Activity of scL-MEL After Lyophilization
and Reconstitution
[0312] The scL-MEL complex containing sucrose (final
concentration=10%) was prepared as described above using an
anti-transferrin receptor single-chain antibody fragment (TfRscFv)
as the targeting moiety, a Lip:MEL molar ratio of 1:1 (size=21 nm)
(liposome concentration=2 mM) and an TfRscFv to Liposome ratio if
1:30 (w:w). This complex was subsequently lyophilized and the
samples stored in a desiccator at 2-8.degree. C. After one week of
storage the lyophilized scL-MeI complex was reconstituted with
endotoxin free water and used to transfect KMS-11 cells as
described above. The size of the reconstituted scL/MEL was 56 nm
(number average), within the preferred size range of the complex
prepared by the method of this invention when freshly prepared.
Thus, lyophilization did not significantly alter the size of the
complex. The cell killing ability of the lyophilized/reconstituted
scL-MEL was compared with freshly prepared scL-MEL and free
(unencapsulated) MEL. The results are shown in FIG. 35. Both the
IC.sub.50 and IC.sub.20 values for the freshly prepared and
lyophilized scL-MEL nanocomplex are virtually identical. Thus, this
lyophilized complex is also able to maintain at least 80% of its
biological activity.
Example 22
Combination Therapy with scL-MEL and Tumor Suppressor Gene p53
[0313] Restoration or activation of the tumor suppressor p53
pathway has been shown to induce apoptosis (programmed cell death),
in multiple myeloma cells (Ludwig H, Beksac M, Blade J, Boccadoro
M, Cavenagh J, Cavo M, et al. Current MM treatment strategies with
novel agents: a European perspective. Oncologist 2010; 15(1):6-25;
Hurt E M, Thomas S B, Peng B, Farrar W L. Reversal of p53
epigenetic silencing in multiple myeloma permits apoptosis by a p53
activator. Cancer Biology & Therapy 2006; 5:1154-60).
Furthermore, studies investigating the molecular causes of multiple
myeloma disease have shown that myeloma cells often have healthy
(i.e., unmutated) p53 genes but very little p53 protein.
Restoration of p53 levels slows the growth of multiple myeloma
cells and causes their death. Thus p53 gene therapy is a logical
treatment strategy for multiple myeloma.
Tumor-Targeting scL-p53 Nanocomplex for Gene Therapy
[0314] As described in U.S. Pat. No. 7,780,822, the disclosure of
which is incorporated by reference herein in its entirety, a
delivery system carrying a plasmid DNA encoding the wtp53 gene and
targeted via TfRscFv (scL-p53) has been successfully developed.
Systemic administration of scL-p53 results in high levels of wtp53
expression in numerous different tumor types. scL-p53 has also been
developed for use in combination with chemotherapy/radiation to
increase the tumor response to these standard therapeutic
modalities by inducing apoptosis.
[0315] The use of the scL-p53 nanocomplex, shown to efficiently
target and efficiently deliver wtp53 to both primary and metastatic
tumors, should be an effective means to increase the levels of p53
protein in multiple myeloma cells.
scL-MEL and scL-p53 Combination Therapy
[0316] Described herein is the use of the combination of scL-MEL
and scL-p53. The development of scL-MEL for use as a monotherapy
will be of benefit to patients in that we have shown the
unexpectedly high increase in multiple myeloma cell death after
treatment with scL-MEL as compared the unencapsulated MEL, the form
that is currently used for treatment. However, as increasing
expression of p53 in multiple myeloma cells also has therapeutic
potential, the combinatorial approach will have an even greater
therapeutic potential.
Experimental Approach
[0317] The experiments are designed to demonstrate development of a
new, more effective treatment regimen for multiple myeloma with use
of scL-MEL, when used in combination with scL-p53.
In Vitro Results
[0318] To test the hypothesis that treatment with scL-p53 could
result in multiple myeloma cell death a preliminary cell viability
assay was performed as described above. Human multiple myeloma cell
line KMS-11, described above, was transfected with scL-p53 alone.
scL-p53 was prepared by simple mixing of the components in a
defined order as described in U.S. Pat. No. 7,780,822. The complex
was prepared with increasing doses of a plasmid encoding the normal
human wtp53 gene. Plasmid DNA doses ranged from 0 to 1.3 ug DNA.
The KMS-11 cells were transfected using the identical procedure
described above. The percent of viable cells was determined 24 h
post-transfection and the IC.sub.50 and IC.sub.20 values
determined. Transfection with scL-p53 resulted in a dose-dependent
level of cell death indicating that increasing the expression of
wtp53 in multiple myeloma cells by itself can result in significant
level of cell death (FIG. 36). This is unexpected since the reports
in the art regarding modulation of p53 expression in multiple
myeloma have all employed either additional activating agents or
indirectly, not directly, affected p53 by blocking expression of
other proteins.
[0319] The combination of scL-MEL and scL-p53, both of which were
shown to increase the level of cell death on their own were
transfected simultaneously to assess the response of KMS-11 cells
to this combination therapy. KMS-11 cells were transfected using
the procedure described above. scL/MEL was prepared as described
above using an anti-transferrin receptor single-chain antibody
fragment (TfRscFv) as the targeting moiety, a Lip:MEL molar ratio
of 1:1 (liposome concentration=2 mM) and an TfRscFv to Liposome
ratio if 1:30 (w:w). Cells (4.times.10.sup.5 per tube) were
transfected with different scL/MEL complexes containing increasing
doses of MEL. The scL-p53 was prepared as previously described
(U.S. Pat. No. 7,780,822). The DNA dose used for all transfections
was 0.2 ug/4.times.10.sup.s cells. This dose was based upon the
data from FIG. 36 wherein the IC.sub.20 was found to be a dose of
approximately 0.2 ug. The IC.sub.20 was used to allow detection of
an additive or synergistic effect of the two treatments. The KMS-11
cells were transfect with Free (unencapsulated) MEL alone, scL-MEL
alone, or the combination of Free (unencapsulated) or scL
encapsulated MEL plus scL-p53 (FIG. 37).
[0320] When compared to free MEL alone, transfection with the
scL-MeI complex resulted in a significant level of
chemosensitization to the drug. Moreover, when used in combination
with either free or scL complexed MEL the addition of scL-p53 was
able to significantly improve the response of the KMS-11 cells to
this chemotherapeutic agent, with the combination of scL-MEL and
scL-p53 being the most effective. Compared to the IC.sub.50 of
unencapsulated MEL, the standard form used as a therapeutic
(IC.sub.50=10.9) the IC.sub.50 of the scL-MEL plus scL-p53 was
4.57, a greater that 2 fold increase in cell death.
[0321] Although these studies have been performed in vitro, it is
fully expected that similar results (increased multiple myeloma
cell death) will also occur when scL-MeI and scL-p53 are
administered systemically in combination to human patients. The
dose of scL-p53 is expected to be between 2.4 and 3.6 mg/infusion
with twice weekly infusions for 5 weeks. The scL-MEL will be
administered as a single intravenous Infusion of a dose of between
6 and 16 mg/m.sup.2 at two week intervals for four doses.
[0322] The unexpected level of enhancement of the combination
observed here indicates the potential of this combination approach
as a new therapeutic modality for the treatment of multiple myeloma
in human patients.
Example 23
Preparation of Cationic Liposomes Comprising Atropine
Materials:
[0323] DOTAP (1,2-dioleoyl-3-trimethylammonium propane, chloride
salt) [0324] Obtained from Avanti Polar Lipids, Inc. Cat. #890890E,
MW 698.55 [0325] Concentration: 25 mg/mL ethanol solution [0326]
Dilute lipid to 20 mg/ml with absolute ethanol before use
[0327] DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) [0328]
Obtained from Avanti Polar Lipids, Inc. Cat. #850725E, MW 744.04
[0329] Concentration: 25 mg/mL ethanol solution. [0330] Dilute
lipid to 20 mg/ml with absolute ethanol before use
[0331] Atropine, powder (M Wt=289.37) [0332] Obtained from Sigma
[0333] Dissolve in absolute ethanol to a concentration of 100
mM.
[0334] Ultra-pure, endotoxin free LAL Reagent Water (e.g.
BioWhittaker, Cat. #W50-500, endotoxin <0.005 EU/ml)
[0335] Injector: Hamilton Gastight Syringe, 1 ml (Hamilton #81230)
with a 22 gauge needle, part #81365)
Procedure:
[0336] 1. Fresh Atropine solution is prepared each time by
dissolving Atropine in absolute ethanol to a concentration of 100
mM by vortexing at high speed until dissolved (must be clear). Hold
at room temperature until used to mix with lipids (Step 3
below).
[0337] 2. Place lipid solutions at 37.degree. C. for 10-15 min,
following which place the lipid solutions in a 65.degree. C. water
bath with occasional shaking for 5 min.
[0338] 3. To prepare the Lip-Atropine: Place a brown glass bottle
with stir bar on a hot plate set to 50.degree. C. to 60.degree. C.
While stirring at high speed without splashing, add the lipids and
Atropine to the bottle in the following order (important):
[0339] For 1:1 (Lip:Atropine) molar ratio
[0340] DOTAP 175 .mu.l (of 20 mg/ml)=5 .mu.mol or 3.5 mg
[0341] DOPE 187.5 .mu.l (of 20 mg/ml)=5 .mu.mol or 3.75 mg
[0342] Add Atropine soln., 100 .mu.l (of 100 mM)=10 mmol,
[0343] Continuously stir for 3 min. after all 3 have been added
[0344] 4. In the meantime, warm 4,569 uL LAL water to 65.degree. C.
in water bath in brown glass bottle with stir bar. Immediately
prior to addition of the Lipid-Atropine solution, move the bottle
to a hot plate (50.degree.-60.degree. C.). Stir water at high speed
with no splashing for a few sec to remove bubbles from the stir
bar.
[0345] 5. Keep the water on the hot plate. Continue stirring the
water at high speed (without splashing) during lipid addition.
After mixing lipids and Atropine as above (Step 2), immediately and
as rapidly as possible, using the Hamilton syringe for injection,
inject the mixture into the hot water on the hot plate
(50.degree.-60.degree. C.) directly into the center of the vortex.
Continue stirring on high speed (without splashing) for 1 min after
the addition of the lipid mixture while loosely covered.
[0346] 6. Move the glass bottle to a RT stir plate, and, continue
to stir slowly until the loosely covered solution cools down to
20-25.degree. C. (room temperature)
[0347] 7. Adjust the volume to 5 ml with room temperature LAL
water.
[0348] 8. Filter the solution using a 0.22 .mu.m pore Milex GV
filter.
[0349] 9. Measure particle size and zeta potential if desired.
Results of these preparation methods demonstrate liposomes having a
particle size of about 20-100 nm and a Zeta Potential of about 10
to 50 mV.
Example 24
Preparation of Targeted Cationic Liposomes Containing Atropine
Without Chemical Conjugation (By Simple Mixing)
[0350] Using the Atropine-comprising cationic liposomes prepared
according to the procedure described above in Example 23, the
ligand targeted Atropine cationic liposome complex as described
herein is prepared by simple mixing of the components and without
chemical conjugation. The preparation of the complexes was in
accordance with the following general procedure.
[0351] To the liposome-water (or buffer) the appropriate amount of
targeting moiety is added to give the desired ratio and mixed by
gentle inversion 5-10 seconds. The targeting moiety can be a ligand
including but not limited to transferrin or folate, or other
proteins. It can also be an antibody or an antibody fragment that
targets a cell surface receptor including, but not limited to the
transferrin or HER-2 receptor (e.g., TfRscFv). This mixture is kept
at room temperature for 10-15 minutes (again inverted gently for
5-10 seconds after approximately 5 minutes). To yield the desired
final volume the targeting moiety-Lip-Atropine admixture is mixed
with any volume (including none) of water (suitably deionized
water) or a buffer of any pH including, but not limited to, Tris
buffers, HEPES buffers or Phosphate Buffered Saline, required to
give a desired volume and inverted gently for 5-10 seconds to mix.
This mixture is kept at room temperature for 10-15 minutes (again
inverted gently for 5-10 seconds after approximately 5
minutes).
[0352] For use in vivo dextrose or sucrose is added last to a final
concentration of about 1-50% (V:V) dextrose or sucrose, suitably 5%
dextrose or 10% sucrose, and mixed by gentle inversion for 5-10
seconds. This mixture is kept at room temperature for 10-15 minutes
(again inverted gently for 5-10 seconds after approximately 5
minutes).
[0353] The size (number average) of the final complex prepared by
the method is between about 10 to 800 nm, suitably about 15 to 400
nm, most suitably about 20 to 200 nm with a zeta potential of
between about 1 and 100 mV, more suitably 5 to 60 mV and most
suitably 10 to 50 mV as determined by dynamic light scattering
using a Malvern Zetasizer ZS. This size is small enough to
efficiently pass through the tumor capillary bed, or cross the
blood brain barrier.
Example 25
Preparation of Cationic Liposomes Comprising Irinotecan
Hydrochloride
[0354] Materials
[0355] DOTAP (1,2-dioleoyl-3-trimethylammonium propane, chloride
salt) [0356] Obtained from Avanti Polar Lipids, Inc. Cat. #890890E,
MW 698.55 [0357] Concentration: 25 mg/mL ethanol solution [0358]
Dilute lipid to 20 mg/ml with absolute ethanol before use
[0359] DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) [0360]
Obtained from Avanti Polar Lipids, Inc. Cat. #850725E, MW 744.04
[0361] Concentration: 25 mg/mL ethanol solution. [0362] Dilute
lipid to 20 mg/ml with absolute ethanol before use
[0363] Irinotecaa HCl (IH) analytical sample (slightly yellow
powder) [0364] Obtained from ScinoPharm Taiwan, Ltd. [0365]
Dissolve in DMSO to a concentration of 50 mg/ml
[0366] Ultra-pure, endotoxin free LAL Reagent Water (we use
BioWhittaker, [0367] Cat. #W50-500; endotoxin <0.005 EU/mL)
[0368] Injector Hamilton Gastight Syringe, 1 ml (Hamilton #81230)
with a 22 gauge needle, part #81365)
[0369] Procedure
[0370] 1. Warm lipids and IH solutions to 37.degree. C. for 10-15
min.
[0371] 2. Using a stir bar and hot plate, mix together for 5 min in
a foil covered brown glass bottle without splashing the following
in this order (important): [0372] DOTAP 175 .mu.l (of 20 mg/ml)=5
.mu.mol [0373] DOPE 187.5 .mu.l (of 20 mg/ml)=5 mmol [0374] IH
135.5 .mu.l (of 50 mg/ml)=10 .mu.mol
[0375] Before mixing, warm up the hot plate to about 50.degree. C.
Keep plate warm (about 50.degree. C.) during the 5 min mixing.
[0376] 3. In the meantime, warm .about.4.35 ml water to 65.degree.
C. in foil covered brown glass bottle with stir bar.
[0377] 4. After mixing for 5 min as above (Step 2), warm the
mixture of DOTAP, DOPE, and IH to 50-60.degree. C. in water bath
established at 65.degree. C. (should take less than 5 min).
[0378] 5. Place the water form Step 3 on a warm (about 50.degree.
C.) plate. Stir water for a few see to remove bubbles from the stir
bar.
[0379] 6. Using Hamilton syringe quickly inject the warm mixture of
lipids and IH into the water, stirring on high speed (without
splashing) for 2 min loosely covered.
[0380] 7. Turn off the heat and continue to stir until the loosely
covered solution cools down to 20-25.degree. C. (room
temperature)
[0381] 8. Adjust the volume to 5 ml with room temperature LAL
water.
[0382] 9. Filter the solution using a 0.22 .mu.m pore Milex GV
filter.
[0383] 10. Measure particle size and zeta potential.
[0384] Results of these preparation methods demonstrate liposomes
having a particle size of about 20-100 nm and a Zeta Potential of
about 10 to 50 mV.
Example 26
Preparation of Targeted Cationic Liposomes Containing Irinotecan
Hydrochloride (IH) Without Chemical Conjugation (By Simple
Mixing)
[0385] Using the Irinotecan-comprising cationic liposomes prepared
according to the procedure described above in Example 25, the
ligand targeted IH cationic liposome complex as described herein is
prepared by simple mixing of the components and without chemical
conjugation. The preparation of the complexes was in accordance
with the following general procedure.
[0386] To the liposome-water (or buffer) the appropriate amount of
targeting moiety is added to give the desired ratio and mixed by
gentle inversion 5-10 seconds. The targeting moiety can be a ligand
including but not limited to transferrin or folate, or other
proteins. It can also be an antibody or an antibody fragment that
targets a cell surface receptor including, but not limited to the
transferrin or HER-2 receptor (e.g., TfRscFv). This mixture is kept
at room temperature for 10-15 minutes (again inverted gently for
5-10 seconds after approximately 5 minutes). To yield the desired
final volume the targeting moiety-Lip-Irinotecan admixture is mixed
with any volume (including none) of water (suitably deionized
water) or a buffer of any pH including, but not limited to, Tris
buffers, HEPES buffers or Phosphate Buffered Saline, required to
give a desired volume and inverted gently for 5-10 seconds to mix.
This mixture is kept at room temperature for 10-15 minutes (again
inverted gently for 5-10 seconds after approximately 5
minutes).
[0387] For use in vivo dextrose or sucrose is added last to a final
concentration of about 1-50% (V:V) dextrose or sucrose, suitably 5%
dextrose or 10% sucrose, and mixed by gentle inversion for 5-10
seconds. This mixture is kept at room temperature for 10-15 minutes
(again inverted gently for 5-10 seconds after approximately 5
minutes).
[0388] The size (number average) of the final complex prepared by
the method is between about 10 to 800 nm, suitably about 15 to 400
nm, most suitably about 20 to 200 nm with a zeta potential of
between about 1 and 100 mV, more suitably 5 to 60 mV and most
suitably 10 to 50 mV as determined by dynamic light scattering
using a Malvern Zetasizer ZS. This size is small enough to
efficiently pass through the tumor capillary bed, or cross the
blood brain barrier.
Example 27
Enhanced Sensitization of Tumor Cells to Irinotecan Hydrochloride
(IH) when Delivered by the scL Nanocomplex
[0389] Although IH has been approved by the FDA for use in
treatment of human colorectal cancers, as well as gastric and
non-small cell lung cancers, the uptake of the drug by normal,
non-tumor cells results in toxic side effects ranging from severe
diarrhea (which can require hospitalization) to immunosuppression.
A means to enhance efficacy while reducing these side effects would
have significant clinical impact. We have previously shown that
encapsulation of antisense oligonucleotides, siRNA and even small
molecules by our tumor-targeting scL nanocomplex results in
increased sensitization of various types of tumor cells to these
therapeutic agents when compared to the free, unencapsulated agent.
A more specific tumor-targeted delivery of IH will reduce the side
effects that can occur from non-specific delivery to normal cells,
and will also enable a reduction in the amount of drug required to
result in effective killing of cancer cells. We performed
experiments which unexpectedly demonstrated that this approach
could be applied to IH.
[0390] A. In Vitro Studies
[0391] To determine whether the effect of the chemotherapeutic drug
IH to tumor cells is enhanced when delivered by this
tumor-targeting nanocapsule we compared the IC.sub.50 values (the
concentration yielding 50% growth inhibition) of free IH to scL-IH
in a number of human tumor cell lines. For these studies,
2-2.5.times.10.sup.3 cells/well of human colon cancer cell line
HT-29, human pancreatic cancer cell line PANC-1 or human
hepatocellular carcinoma cell line Hep G2 cells the appropriate
growth medium were plated in a 96-well plate. After 24 hours, the
media was replaced with serum-free medium, overlaid with 100 .mu.L
of increasing concentrations of either scL-IH nanocomplex, prepared
as described above in Example 26, unliganded nanocomplex (L-IH),
free IH or LipA only in serum-free medium. The cells were,
incubated for 5 hours and then supplemented with FBS. After
incubation for an additional 19 hours at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2, the wells were washed
with IMEM without phenol red and a cell-viability XTT-based assay
was performed. Formazan absorbance, which correlates to cell
viability, was measured at 450 nm using a microplate reader. The
IC.sub.50 was interpolated from the graph of the log of drug
concentration versus the fraction of surviving cells.
[0392] Colon cancer is one of the clinical indications for use of
IH. At the low dose levels used, the free IH shows no significant
effect on HT-29 human colon cancer cells (IC.sub.50 value of >10
uM). In contrast, delivery of IH via the scL nanocomplex resulted
in an significant increase in the level of cell kill with an
IC.sub.50 value of 1.5 uM. This is an unpectedly high increase in
sensitivity of at least 10 fold over unencapsulated (free) IH.
While the liposome-IH minus the targeting moiety displays a small
level of improvement over free TH, it is significantly less
effective than that of the scL-IH complex containing the TfRscFv
targeting molecule. This is due to the efficient uptake of the
complex into the cells as a result of the binding of the TfRscFv to
the Tf receptor on the cells triggering receptor mediated
endocytosis of the complex and trafficking of the scL encapsulated
IH efficiently into the cells. Moreover, as with Free IH the
liposome alone had no effect on cell kill indicating that the
toxicity observed with scL-IH is not due to non-specific
cytotoxicity, but is in fact due to the enhanced uptake of IH into
the cells.
[0393] Similar results were observed when this approach was tested
in two other human tumor cell lines, Hep G2 and PANC-1. IN both
cases there is no effect of Free IH on the cells at the low doses
tested. In neither case is an IC.sub.50 value achieved when the
cells are treated with Free IH. However, significant cell kill is
also unpectedly observed when the same doses of IH are administered
as part of the scL nanocomplex. Here also, as was observed in the
HT-29 cells, in addition to no effect of the liposomes alone, a
slight effect was observed with unliganded Liposome-IH, albeit
significantly less than that with the targeted scL nanocomplex.
[0394] In order to demonstrate that the cell kill observed was
tumor cell specific, we also performed an in vitro experiment in
which non-cancerous lung fibroblast cells (IMR-90) were also
treated with the scL-IH and the controls described above. The
results show no sensitization by the scL-IH in these normal cells.
These results demonstrate the tumor cell specificity of the scL
nanocomplex. Therefore, we can successfully incorporate
chemotherapeutic agent IH into the scL nanocomplex leading to
enhanced and tumor cell specific cells killing by doses at which
free IH is ineffective.
[0395] B. In Vivo Studies
[0396] The studies described above demonstrated that inclusion of
IH within the scL nanocomplex could significantly enhance its tumor
cell killing effect in various human tumor cell types compared to
free IH in vitro. However, in order to assess the translational
potential of this nanotechnology, it is necessary to demonstrate
that scL-IH has similar effects in vivo.
[0397] In our initial in vivo experiment, we assessed the
anti-tumor efficacy of various molar ratios of liposome to IH.
Tumors of human colon tumor cell line HT-29 were induced in female
athymic nude mice (5-10 animals/group) by the subcutaneous
injection of 3.12.times.10 cells/site/mouse suspended in
Matrigel.RTM. collagen basement solution. When the tumors reached
.about.100 mm.sup.3 treatment was initiated. Groups of mice were
intravenously injected with the scL-IH complex, prepared as
described above in Example 26, at molar ratios of Liposome to IH of
0.5:1, 1:1 and 2:1, with an IH concentration of 10 mg/kg. Groups of
animals were also treated with Free IH, scL without IH, the
nanocomplex without the targeting moiety (Lip-IH), as well as a
group of untreated mice. The mice were IV injected twice weekly to
a total of 15 injections. The results demonstrated that free IH has
minimal effect on tumor growth compared to untreated tumors. The
same minimal level of tumor response is evident when the scL-IH
nanocomplex was prepared at liposome to IH molar ratios of 0.5:1
and 2:1. In contrast, there was almost complete inhibition of tumor
growth when the mice were treated with the scL-IH nanocomplex
prepared at a molar ratio of 1:1. Furthermore, the unliganded L-IH
nanocomplex had virtually no anti-tumor effect, demonstrating the
importance of the tumor-targeting moiety. Thus, in the remainder of
the studies the optimal ratio of 1:1 (Liposome:IH) was used in
preparation of the scL-IH nanocomplex.
[0398] To be utilized as a clinical agent, the scL-IH nanocomplex
must be available in a stable form with a reasonable shelf-life.
Thus, development of a lyophilized form of the scL-IH is an
important step leading to the commercialization of our scL
nanocomplex therapeutic agent. We have previously shown that we
could produce a lyophilized form of the scL nanocomplex carrying
plasmid DNA, siRNA and even contrast agent Magnevist. Thus we also
compared the anti-tumor efficacy of the freshly prepared and a
lyophilized form of scL-IH in three tumor models, mice bearing
either HT-29 colon, PANC-1 pancreatic, or Hep G2 hepatocellular
carcinoma xenograft tumors.
[0399] In the HT-29 animal experiment, the tumors were induced by
injection of 5.times.10.sup.5 cells in Matrigel.RTM. and the
animals treated as described above with scL-IH at an IH
concentration of 10 mg/kg and prepared at a Liposome:IH ratio of
1:1. Here also the mice received a total of 15 intravenous
injections. The results are similar to that described above with
significant tumor growth inhibition by the scL-IH, with only
minimal effect from free IH and the nanocomplex minus the targeting
moiety. More significantly, the lyophilized/reconstituted form of
scL-IH yielded an identical anti-tumor response as the freshly
prepared scL-IH.
[0400] Similar results were observed in mice bearing PANC-1
xenograft tumors. In this experiment the subcutaneous tumors were
induced by inoculation of 200 ul of tissue obtained from serially
passaged PANC-1 xenograft tumors in Matrigel.RTM.. Here each animal
received a total of eleven intravenous injections of the various
solutions, also at an IH concentration of 10 mg/kg and using the
Liposome:IH molar ratio of 1:1.
[0401] As with the other two tumor models, the use of a lyophilized
form of the scL-IH nanocomplex in the Hep G2 xenograft tumor model
resulted in an identical pattern of anti-tumor efficacy as was
observed when the mice were treated with the freshly prepared
scL-IH nanocomplex. In this experiment, the tumors were induced by
the subcutaneous injection of 5.times.10.sup.6 Hep G2 cells in
Matrigel.RTM.. As above, the animals were intravenously injected
with the solutions at an IH concentration of 10 mg/kg and using the
Liposome:IH molar ratio of 1:1. The mice received a total of twelve
injections. Even over the short term of this study, there is
significantly greater anti-tumor efficacy of the scL delivered IH
in this rapidly growing tumor model when compared to the Free IH,
in addition to the identical pattern between freshly prepared and
lyophilized/reconstituted scL-IH.
[0402] Therefore, the above experiments show, through the
significant anti-tumor effect observed in three separate mouse
models of human cancer, the unexpected high increase in efficacy of
the scL-IH compared to the currently used free IH. Moreover, the
instant inventors have successfully produced a usable Lyophilized
formulation of scL-IH that retains it biological activity after
lyophilization/reconstitution, enhancing the translational
potential of this therapeutic complex.
[0403] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein can be made without
departing from the scope of any of the embodiments.
[0404] It is to be understood that while certain embodiments have
been illustrated and described herein, the claims are not to be
limited to the specific forms or arrangement of parts described and
shown. In the specification, there have been disclosed illustrative
embodiments and, although specific terms are employed, they are
used in a generic and descriptive sense only and not for purposes
of limitation. Modifications and variations of the embodiments are
possible in light of the above teachings. It is therefore to be
understood that the embodiments may be practiced otherwise than as
specifically described.
[0405] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
* * * * *