Title of Invention

METHOD FOR IMPROVING STABILITY AND SHELF-LIFE OF LIPOSOME COMPLEXES

Abstract A method for preparing a stable cell-targeting complex comprising a ligand and a cationic liposome encapsulating a therapeutic or diagnostic agent comprises (a) combining the complex with a solution comprising a stabilizing amount of sucrose and (b) lyophilizing the resultant solution to obtain a lyophilized preparation; wherein, upon reconstitution, the preparation retains at least about 80% of its pre-lyophilization activity.
Full Text METHOD FOR IMPROVING STABILITY AND
SHELF-LIFE OF LIPOSOME COMPLEXES
[001] This application claims priority from U.S.
provisional application number 60/475,500, filed June 4,
2003 incorporated herein by reference in its entirety.
Field of the Invention
[002] This invention relates to a method of preparing a
stable complex comprising a ligand and a cationic liposome
encapsulating a therapeutic or diagnostic agent.
Background of the Invention
[003] All references cited herein are incorporated by
reference in their entirety.
[004] Cationic liposomes are composed of positively
charged, lipid bilayers and can be complexed to negatively
charged, naked DNA by simple mixing of lipids and DNA such
that the resulting complex has a net positive charge. The
complex can be bound to and taken up by cells in culture
with moderately good transfection efficiency. Cationic
liposomes have been proven to be safe and efficient for in
vivo gene delivery.
[005] Liposomes carr be used to target tumor cells by
modifying the liposomes so that they selectively deliver
their payload to tumor cells. Surface molecules can be used
to target liposomes to tumor cells because the type and/or
number of molecules that decorate the exterior of tumor
cells differ from those on normal cells. For example, if a
liposome has a folate or transferrin (Tf) molecule on its
surface, it will home to cancer cells that have levels of
the folate or transferrin receptor which are higher than
those on normal cells.
[006] In addition to the use of ligands that are
recognized by receptors on tumor cells, specific antibodies
also can be attached to the liposome surface, enabling them
to be directed to specific tumor surface antigens
(including, but not limited to, receptors). These
"immunoliposomes" can deliver therapeutic drugs to a
specific cell population. It has been found, for example,
that anti-HER-2 monoclonal antibody (Mab) Fab fragments
conjugated to liposomes could bind specifically to a breast
cancer cell line, S-BR-3, that over-expresses HER-2 (Park,
J.W., et al. PNAS 92:1327-1331 (1995)). The immunoliposomes
were found to be internalized efficiently, and the anchoring
of anti-HER-2 Fab fragments enhanced their inhibitory
effects. The combination of cationic liposome-gene transfer
and immunoliposome techniques appears to be a promising
system for targeted gene therapy.
[007] A ligand-targeted liposomal delivery system for
DNA gene therapy possessing selective tumor targeting and
aigh transfection efficiency has been described in the art.
S.u, L., et al. Human Gene Therapy 8:467-475 (1997); Xu, L. ,
et al., Human Gene Therapy 10:2941-2952 (1999); and Xu, L. ,
et al., Tumor Targeting 4:92-104 (1999). This system has
peen improved through use of an anti-transferrin receptor
single chain (TfRscFv) antibody fragment as the targeting
Ligand in the complex (Xu, L., et al. Molecule Medicine
7:723-734 (2001); Xu, L., et al. Molecular Cancer
Therapeutics 1:337-346 (2002)). The TfRscFv is formed by
connecting the component VH and VL variable domains from the
Light and heavy chains,; respectively, with an appropriately
designed linker peptide. The linker bridges the C-terminus
of the first variable region and N-terminus of the second,
ordered as either VH-linker-VL or VL-linker-VH. The binding
site of an scFv can replicate both the affinity and
specificity of its parent antibody bonding site.
[008] Conventional treatments for cancer involve
chemotherapy and/or radiation treatments. Incorporating
into these conventional cancer therapies a new component
which results in sensitization of tumors to the chemotherapy
or radiation therapy would have great clinical relevance,
lowering the effective doses of both types of anti-cancer
modalities and correspondingly lessening the severe side
effects often associated with these treatments.
[009] Initial studies with liposome complexes as
described above have shown that the complexes are efficient
in delivering diagnostic or therapeutic agents to the target
cells of interest. It is impractical to administer the •
complexes to a patient immediately upon their preparation.
It would be desirable to provide targeted liposome complexes
that upon lyophilization and storage at 2-8°C, -2 0°C or -
80°C remain stable for at least six months and can be
reconstituted without a significant loss of activity.
[010] Previous reports have indicated that a two-
component complex (lipid and.DNA but without targeting
ligand or proteins) could be lyophilized in the presence of
mono- or di-saccharides and still maintain their biological
activity and a particle size appropriate for gene therapy
(Li, B., et al., Journal of Pharmaceutical Sciences 89:355-
364 (2000) and Molina, M.D.C. et al. Journal of
Pharmaceutical Sciences 90:1445-1455 (2001); Allison, S.D.,
et al. Biochemical et Biophysical Acta 1468:127-138 (2000)).
In addition, Tf linked through PEG to a PEI-DNA poiyplex
retained some biological activity after freezing and thawing
(Kursa, M. et al., Bioconjugate Chemistry 14:222-231
(2003)). It is important to note that this complex was not
lyophilized, no indication of possible length of storage or
condition given, and sugar (glucose) , if included, was added
after thawing. This polymer complex requires the Tf to be
linked to the polymer through a PEG molecule.
Summary of the Invention
[0011] A method for preparing a stable complex comprising
a ligand and a cationic liposome encapsulating a therapeutic
or diagnostic agent or reporter gene comprises:
combining a complex comprising a ligand and a cationic
liposome encapsulating a diagnostic or therapeutic agent
or reporter gene with a solution comprising a stabilizing
amount of sucrose; and
lyophilizing the resultant solution of complex and
sucrose to obtain a lyophilized preparation;
wherein, upon reconstitution, the preparation retains
at least about 80% of its pre-lyophilization activity.
[012] In a preferred embodiment, the preparation retains
at least about 85% of its pre-lyophilization activity, and
more preferably, at least about 90% of its prelyophilization
activity.
Brief Description of the Figures
[013] Figure 1 shows the size (nm) of the freshly
prepared and lyophilized complexes (Tf-LipA-Luc and TfRscFv-
LipA-Luc) containing 5% dextrose or 5% sucrose.
[014] Figure 2A and 2B shows the in vitro transfection
efficiency, given as relative light units (RLU) per ug
protein, of freshly prepared and lyophilized complexes (Tf-
LipA-Luc and TfRscFv-LipA-Luc) containing 5% dextrose or 5%
sucrose in DU145 human prostate cells.
[015] Figure 3 shows the comparison of the in vitro
transfection efficiency of freshly prepared and lyophilized
TfRscFv-LipA-Luc complex (RLU/ug protein) containing 5% or
10% sucrose in DU145 human prostate cancer cells.
[016] Figure 4A and 4B, respectively, show DU145
prostate and PANCI pancreatic xenograft tumor targeting by
lyophilized ligand-liposome plasmid DNA complexes.
[017] Figure 5 demonstrates in a human prostate cancer
(DU145) xenograft mouse model that the tumor targeting
ability of laboratory prepared TfRscFv-LipA-p53 complex with
10% sucrose is maintained after lyophilization and storage
at 2°-8°C for up to six months.
[018] Figure 6 shown the batch to batch tumor targeting
and transfection efficiency consistency of lyophilized
TfRscFv-LipA-p53 complexes with 10% sucrose in a DU145
xenograft mouse model.
[019] Figure 7 shows the tumor targeting and
transfection efficiency of five different commercially
prepared and lyophilized batches of TfRscFv-LipA-p53 with
10% sucrose in a DU145 xenograft mouse model.
[020] Figure 8 shows the iestimated percent of
uncomplexed TfRscFv in various fresh and lyophilized
TfRscFv-LipA-p53 complexes by non-denaturing gel
electrophoresis.
[021] Figure 9 shows the in vitro comparison of cell
growth inhibition between lyophilized and freshly made
TfRscFv-LipA-AS-HER-2 complexes in MDA-MB-435 breast cancer
cells.
[022] Figure 10 shows the in vitro comparison of PANC-1
chemosensitization by freshly prepared or lyophilized
TfRscFv-LipA-AS HER-2 complexes.
[023] Figure 11 shows the in vitro down modulation of
HER-2 expression in MDA-MB-435 human breast cancer cells by
TfRscFv-LipA-AS HER-2 with 10% sucrose after lyophilization
and storage at 2°-8°C for up to six months.
Detailed Description of the Invention
[024] In accordance with the present invention, the
stability of lyophilized complexes of a ligand and a
liposome encapsulating a diagnostic or therapeutic agent can
be increased by combining the complexes prior to
lyophilization with an aqueous solution of a stabilizing
amount of sucrose. The sucrose solution can be simply
sucrose in water or a buffer can be included, such as PBS,
HEPES, TRIS or TRIS/EDTA. Typically the sucrose solution is
combined with the complex to a final concentration of about
1% to about 80% sucrose, typically 1% to about 50% sucrose,
such as 1% to about 10%, 20%, 30% or 40% sucrose, preferably
about 5% to 10% sucrose, and most preferably about 10%
sucrose. The lyophilized preparation is stable within a
range of from about 2-8°C to about -80°C for a period of at
least 6 months without losing significant activity.
Preferably the preparation is stable for a period of at
least about 6-12 months. -Upon reconstitution, the complexes
retain at least about 80% of their pre-lyophilization
activity, preferably at least about 85% of their pre-
lyophilization activity and most preferably at least about
90 - 95% of their pre-lyophilization activity.
[025] Previous reports have indicated that a mixture of
lipid and DNA could be lyophilized in the presence of mono
or disaccharides and maintain biological activity (Li, B.,
et al., Journal of Pharmaceutical Sciences 89:355-364 (2000)
and Molina, M.D.C. et al. Journal of Pharmaceutical Sciences
90:1445-1455 (2001); Allison, S.D., et al. Biochemical et
Biophysical Acta 1468:127-138. (2000)). It is unexpected,
however, that a three component complex consisting of 1) a
protein (e.g transferrin), including even a protein which is
an antibody or antibody fragment (e.g., anti-transferrin
receptor single chain antibody fragment, TfRscFv); 2)a
liposome and 3) a therapeutic nucleic acid molecule (e.g. a
plasmid DNA, an antisense oligonucleotides molecule or even
an siRNA molecule) also could be lyophilized and retain both
its size and biological activity after reconstitution.
[026] The liposome complexes typically are administered
intravenously. For intravenous injection, a 50% dextrose
solution conventionally has been added to the ligand-
liposome complexes to a final concentration of 5%. It now
surprisingly has been found that by combining freshly
>
prepared (i.e., a complex that is no more than about 1 to
about 24 hours old) ligand-liposome complexes with a
solution of sucrose, rather than dextrose, the activity and
shelf life of the three component complexes (including those
with an antigen targeting entity) following lyophilization
and reconstitution can be significantly increased.
[027] The three component complexes can simply be mixed
with a sucrose solution prior to lyophilization. Typically
the solution comprises about 50 to about 100% sucrose by
weight, preferably about 50% by weight sucrose.
Lyophilization can be in accordance with any conventional
procedure that reduces the moisture content of the complex
to less than about 1.3%. One preferred procedure comprises
lyophilizing the complex-containing solution at -50°C to -
60°C, 20-50 millitorr, preferably 25 millitorr, for 12 to 60
hours, preferably 20-48 hours, then storing the lyophilized
preparation between about 2-8°C and about -80°C. In another
preferred procedure, vials containing the solution-of
complex are loaded into a commercial type lyophilizer at
ambient temperature, then the temperature is ramped to -45°C
+. 3°C over 1 hour and held at that temperature for three
hours. The condenser then is chilled at -80°C or colder and
the vacuum is set to 50 micron Hg. The shelf temperature is
then ramped to -35° ± 3°C over 1 hour and once there held at
this temperature for about 36-72 hours, preferably about 48
hours. The shelf temperature then is ramped to 20° ± 3°C
over 4 hours and held at this temperature for about 6 to
about 48 hours, preferably about 12 hours. At the end of
this process the chamber pressure is restored to atmospheric
with nitrogen (passed through an appropriate sterilizing
microbial retentive filter) and the vials stoppered.
[028] The lyophilized complexes can be reconstituted by
the addition of sterile, endotoxin-free water equal to the
volume of solution prior to lyophilization. The dried
complexes dissolve rapidly with gentle rocking. No
appreciable changes in size of the complex or zeta potential
occurs due to the lyophilization or storage.
[029] ' Suitable complexes which can be mixed with a
sucrose solution, lyophilized and reconstituted are cell-
targeting ligand/liposome/therapeutic, reporter or
diagnostic molecule complexes that are capable of cell-
targeted, systemic delivery of a variety of types of $
therapeutic or diagnostic molecules for use in treating or
diagnosing diseases. The target cell preferably is a cancer
cell, but can be a non-cancer cell as well. Preferred
cancer target cells include prostate, pancreatic, breast,
head and neck, ovarian, liver and brain cancers and
melanoma. It is well known to one of ordinary skill in the
art that most types of cancer cells, including, but not
limited to, those listed above, overexpress the receptor for
transferring and folate and that these receptors also
rapidly recycle in cancer cells (Li, H., and Qian,.Z.M.,
Medicinal research Reviews 2(3):225-250 (2000); Qian, Z.M.,
et al., Pharmacological Reviews 54(4):561-587 (2002);
gosselin, M.A., and Lee, P.J., Biotechnology annual Reviews
8:103-131 (2002)) .
[030] Desirably, the therapeutic molecule is a gene,
polynucleotide, such as plasmid DNA, . DNA fragment,
oligonucleotide, oligodeoxynucleotide, antisense
oligonucleotide, chimeric RNA/DNA oligonucleotide, RNA,
siRNA, ribozyme, viral particle, growth factor, cytokine,
immunomodulating agent, or other protein, including proteins
which when expressed present an antigen which stimulates or
suppresses the immune system. Preferred therapeutic agents
are nucleic acid molecules, preferably DNA or siRNA
molecules. A preferred DNA molecule is one which encodes a
gene such as a wild type p53 molecule, an Rb94 molecule, an
Apoptin molecule, an E6F6 molecule or an antisense molecule.
A preferred HER-2 antisense oligonucleotide is against the
HER-2 gene and has the sequence 5' -TCC ATG GTG CTC ACT-3' .
A preferred siRNA molecule is one which acts against HER-2
mRNA. Other preferred therapeutic molecules can be
determined by one of ordinary skill in the art without undue
experimentation.
[031] As noted above, the target cell alternatively can
be a non-cancer cell. Preferred non-cancer target cells
include dendritic cells, endothelial cells of the blood
vessels, lung cells, breast cells, bone marrow cells and
liver cells. Undesirable, but benign, cells can be
targeted, such as benign prostatic hyperplasia cells, over-
active thyroid cells, lipoma cells, and cells relating to
autoimmune diseases, such as B cells that produce antibodies
involved in arthritis, lupus, myasthenia gravis, squamous
metaplasia, dysplasia and the like.
[032] Alternatively, the agent can be a diagnostic agent
capable of detection in vivo following administration.
Exemplary diagnostic agents include electron dense material,
magnetic resonance imaging agents and radiopharmaceuticals.
Radionuclides useful for imaging include radioisotopes of
copper, gallium, indium, rhenium, and technetium, including
isotopes 64Cu, 67Cu, 111In, 99mTc, 67Ga or 68Ga. Imaging agents
disclosed by Low et a.l. in U.S. Patent 5,688,488,
incorporated herein by reference, are useful in the
liposomal complexes described herein.
[033] The ligand can be any ligand the receptor for
which is differentially expressed on the target cell.
Examples include transferrin, folate, other vitamins, EGF,
insulin, Heregulin, RGD peptides or other polypeptides
reactive to integrin receptors, antibodies or their
fragments. A preferred antibody fragment is a single chain
Fv fragment of an antibody.
[034] The antibody or antibody fragment is one which
will bind to a receptor on the surface of the target cell,
and preferably to a receptor that is differentially
expressed on the target cell. One preferred antibody is an
anti-TfR monoclonal antibody and a preferred antibody
fragment is an scFv based on an anti-TfR monoclonal
antibody. Another preferred antibody is an anti-HER-2
monoclonal antibody, and another preferred antibody fragment
is an scFv based on an anti-HER-2 monoclonal antibody.
[035] The ligand is mixed with the liposome at room
temperature and at a ligand:liposome ratio in the range of
about 1:0.001 to 1:500 (ug:nmole), preferably about 1:10 to
about 1:50 (ug:nmole). The therapeutic agent is mixed with
the cationic liposome at room temperature and at an
agent:lipid ratio in the range of about 1:0.1 to about 1:50
(ug:nmole) , preferably about 1:10 to about 1:24 (ug:nmole).
In complexes, for example, in which the ligand is
transferrin and the therapeutic agent is plasmid DNA, useful
ratios of therapeutic agent to liposome to ligand typically
are within the range of about 1 ug: 0.1-50 nmoles: 0.1 -100
µg, preferably 1 ug: 5-24 nmoles: 6-36 ug, most preferably
about 1 ug: 10 nmoles: 12.5 ug. If the ligand is TfRscFv,
useful ratios of ligand to liposome typically are within the
range of about 1:5 to 1:40 (µg:µg), preferably 1:30 (µg:µg) ,
and the ratio of plasmid DNA to liposome typically-is within
the range of about 1:6 to 1:20 (µg:µg), preferably 1:14
(µg:µg) . If the therapeutic agent is an oligonucleotide
(ODN) rather than plasmid DNA, typical ratios of ligand,
liposome and the ODN are 0.1 nmole to 3 6 nmole
(ODN: liposome) and 0.1 µg to 100 µg (ligand: liposome) ,
preferably 0.5 nmoles to 20 nmoles (ODN:liposome) and 0.5 µg
to 50 µg (ligand:liposome), most preferably 1 nmole to 15
nmole (ODN:liposome) and 1 ug to 30 µg (ligand:liposome) .
If the therapeutic agent is an siRNA, useful ratios of the
components, can be 0.1 ug to 30 nmole (siRNA:liposome) and
0.1 ug to 100 µg (TfRscFv:liposome), preferably 1 µg to 7
nmole (siRNA: lipsosome) and 1 µg to 30 µg
(TfRscFv:liposome).
[036] A wide variety of cationic liposomes are useful in
the preparation of the complexes. Published PCT application
WO99/25320, incorporated herein by reference, describes the
preparation of several cationic liposomes. Examples of
desirable liposomes include those that comprise a mixture of
dioleoyltrimethylammonium phosphate (DOTAP) and
dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol
(chol), or a mixture of dimethyldioctadecylammonium bromide
(DDAB) and DOPE and/or cholesterol. The ratio of the lipids
can be varied to optimize the efficiency of uptake of the
therapeutic molecule for 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 l:(l-2) (molar
ratio).
[037] In one embodiment, the liposome used to form the
complex is a sterically stabilized liposome. Sterically
stabilized liposomes are liposomes into which a hydrophilic
polymer, such as PEG, poly(2-ethylacrylic acid) or poly(n-
isopropylacrylamide) (PNIPAM) have been integrated. Such
modified liposomes can be particularly useful when complexed
with therapeutic or diagnostic agents, as they typically are
not cleared from the blood stream by the reticuloendothelial
system as quickly as are comparable liposomes that have not
been so modified. In a second embodiment, the liposome used
to form the complex is also bound to a peptide composed of
histidine and lysine (either branched or linear) where the
peptide is at least about 10 amino acids in length,
typically between about 10 and 1000 amino acids in length,
and is composed of 5-100% histidine and 0-95% non-histidine
amino acids; preferably at least 10% of the non-histidine
amino acids are lysine. Most preferably the peptide is
about thirty-one amino acids, approximately 20% of which are
histidine and approximately 80% of which are non-histidine.
Of these, at least 75% are lysine and at least one is a
terminal cysteine. A preferred peptide has the structure
5'-K[K(H)-K-K-K]5-K(H)-K-K-C-3' and can be covalently
conjugated to the liposome through the terminal cysteine and
a maleimide group in the liposome. In such complexes, the
ratios of the components typically can be as follows: ligand
to HK-iiposome (ug:' ug) of 1:5 to 1:40, preferably, 1:30 and
DNA to HK-liposome (ug:nmole) of 1:1 to 1:20, preferably
1:14.
[038] The complexes can be prepared by mixing the
ligand-liposome and the therapeutic or diagnostic agent
together, slowly inverting the resultant solution a number
of time or stirring the solution at a speed where a vortex
just forms in the solution for a period ranging from about
10 seconds to about 10 minutes, preferably 15 seconds to
about 2 minutes.
[039] The complexes can be administered in combination
with another therapeutic agent, such as either a radiation
or chemotherapeutic agent. The therapeutic agent, or a
combination of therapeutic agents, can be administered
before or subsequent to the administration of the complex,
for example within about 12 hours to about 7 days.
Chemotherapeutic agents include, but are not limited to, for
example, doxorubicin, 5-fluorouracil (5FU), cisplatin
(CDDP) , docetaxel, gemcitabine, paclitaxel, vinblastine,
etoposide (VP-16), cantptothecia, actinomycin-D, mitoxantrone
and mitomycin C. Radiation therapies include gamma
radiation, X-rays, UV irradiation, microwaves, electronic
emissions and the like.
[040] The invention is further illustrated by the
following examples which are provided for illustrative
purposes and are not intended to be limiting.
Examples
Example 1
Preparation of Fresh and Lvophilized
Complexes with Carbohydrate and In Vitro
Assessment of Activity and Size
[041] Initial experiments were performed to test both
the size and the in vitro transfectioh efficiency of the
ligand-liposome nucleic acid complexes made with
carbohydrate before and after lyophilization. Two separate
ligands, Tf and TfRscFv, were tested. The complexes were
made using the methodology described in U.S. Patent
Application 09/601,444 and published U.S. Patent
Applications 09/914,046 and 10/113,927 [See also, Xu, L., et
al. Human Gene Therapy 10:2941-2952 (1999); Xu, L., et al.,
Human Gene Therapy 13:469-481 (2002); and Xu, L., et al.,
Molecular Cancer Therapeutics 1:337-346 (2002)]. In each
complex, the liposome was a 1:1 ratio of DOTAP:DOPE,
identified herein as Liposome A (LipA). The DNA used was a
plasmid carrying a gene encoding the firefly luciferase
gene. In all cases the carbohydrate solution was added as
the last step in preparation of the complex.
[042] A series of 8 complexes was made. Four contained
Tf as the ligand (at a ratio of DNA:LipA:Tf of 1 ug:10
nmoles:12.5 ug) ; four contained TfRscPv as the ligand (at a
ratio of DNA:LipA:TfrscFv of 1 µg:14 nmoles:0.34 ug) . The
solutions containing the ligand-liposome and the DNA were
mixed together, slowly inverted 10 times, and the resultant
solution was held at room temperature for 15 minutes prior
to the addition of an aqueous solution of dextrose or
sucrose in water to a final concentration of 5%. Each
resultant admixture was inverted 10 times and then held at
room temperature for 15 minutes prior to lyophilization or
transfection.
[043] The solutions to be lyophilized were lyophilized
using a Virtis Benchtop 3L lyophilizer at 25 millitorr for
24 hours, at -55°C and then stored overnight at -80°C prior
to reconstitution. After reconstitution with a volume of
water equal to the volume of solution prior to
lyophilization, the container holding each solution was
slowly inverted 10 times and held at room temperature for 60
minutes. After this time the reconstituted complex could'be
kept at 2°-8°C for up to 24 hours. The size of the
complexes before and after lyophilization were measured by
dynamic laser light scattering using a Malvern Zetasizer
3000H.
[044] The results of the sizing (number average) are
shown in Figure 1. When Tf was the ligand there was an
approximate 10 fold increase in size after lyophilization in
the presence of 5% dextrose. While the size of the fresh
complex with 5% sucrose was slightly larger than that with
5% dextrose, there was essentially no change pre/post
lyophilization in the presence of sucrose. A similar
pattern was observed when TfRscFv was used as the ligand.
Here also use of 5% sucrose gave much better post-
lyophilization results.
[045] The fresh and lyophilized complexes also were
assessed for their transfection efficiency in a human
prostate tumor cell line DU145. The transfection
efficiencies of the lyophilized complexes (with Tf and
TfRscFv) upon reconstitution and corresponding freshly made
solution of the same complex were compared. The results of
the transfection efficiency pre- and post- lyophilization is'
shown in Figures 2A and 2B. When the ligand was Tf, the
efficiency dropped after lyophilization to about 60% of that
of the freshly prepared complex for the preparation
containing 5% dextrose, whereas the lyophilized preparation
containing'5% sucrose retained about 80% of its initial
activity. The pattern was similar with the TfRscFv ligand.
The lyophilized complex containing 5% dextrose dropped to
about 50% of the fresh activity whereas about 90% of the
activity was retained when 5% sucrose was used (Fig 2B) .
Thus, sucrose is a more efficient stabilizer than dextrose.
[046] The sugar/complex ratio was further optimized to
improve the stability and maintain particle size. Complexes
with 5% and 10% sucrose were compared. The amount.of
plasmid DNA also was increased to 20µg, the amount
customarily with used for a singe injection in the in vivo
studies discussed below. After lyophilization as described
above, the transfection efficiency of the complex containing
10% sucrose was -95% of that seen with the fresh complex
prepared the conventional way with 5% dextrose solution.
Figure 3 shows a comparison of transfection efficiency
between complexes prepared with 5% and 10% sucrose. The in
vitro transfection efficiency was best with the lyophilized
complex containing 10% sucrose. This was found to be true
independent of whether the protein or the antibody fragment
was used as targeting ligand. The size of the complexes
containing 10% sucrose also were assessed before and after
lyophilization using the conditions given above. There was
no significant difference between the sizes of the complexes
made with 10% sucrose, either before and after
lyophilization, as compared to the conventional freshly
prepared complex made with 5% dextrose. Here also this was
found to be the case independent of the targeting ligand.
[047] Thus, the presence of 10% sucrose in a
reconstituted liposome complex preparation resulted in
higher maintenance of biological activity and size than that
obtained with comparable reconstituted preparations
containing either 5% dextrose or 5% sucrose.
Example 2
In Vivo Human Prostate Tumor Targeting by Lyophilized
Complex After Storage at 2-8°C for One Week
[048] In vivo tumor targeting of a liposome complex with
10% sucrose (freshly made or lyophilized and stored for 1
week at 2-8°C was tested using enhanced green fluorescence
protein (E6FP) as the reporter gene in the complex.. The
complex was TfRscFv-Liposome A-pEGFP' where liposomeA is
D0TAP:D0PE (1:1). The ratio of the three components was 0.3
ug:14 nmoles:l ug (TfRscFv:Liposome: DNA). The complex was
prepared and lyophilized as described above in Example 1.
Post-lyophilization, the complex was stored refrigerated at
2-8°C for one week. The samples were reconstituted by the
addition of endotoxin'free water to a volume equal to that
prior to lyophilization as described in Example 1.'
[049] Mice bearing DU145 xenograft tumors of at least 50
mm3 were i.v. injected 3 times over 24 hours with various
complexes (freshly made preparation with 5% dextrose;
freshly made preparation with 10% sucrose and a
reconstituted lyophilized preparation with 10% sucrose which
prior to reconstitution had been held refrigerated for 1
week at 2-8°C all with TfRscFv as the targeting ligand).
After 48 hours, the animals were sacrificed, tumor and liver
excised, protein isolated and Western analysis performed
using anti-EGFP Ab (COVANCE). As shown in Figure 4A, the
lyophilized and reconstituted complex with 10% sucrose had a
comparable level of gene expression compared to either of
the freshly prepared complexes. More significantly, while a
high level of exogenous gene expression was evident in the
tumors, almost no EGFP was expressed in the livers of the
mice, demonstrating the tumor specific nature of the
complex, and that this tumor targeted specificity was
maintained after lyophilization, storage at 2°-8°C for at
least one week and reconstitution.
Example 3
In Vivo Human Pancreatic Tumor Targeting bv
Lyophilized complex After Storage at -80°C for One Month
[050] The stability of the lyophilized complex also was
tested after one month of storage at -80°C by targeting to
pancreatic cancer xenograft tumors. The'complex (the same
complex and ratio as described above in Example 2) was
prepared with 10% sucrose ad lyophilized as described in
Example 1. Post-lyophilization the samples were stored at -
80°C for one month, theii reconstituted with endotoxin free
water as described in example 1.
[051] As shown in Figure 4B, the tumors from mice i.v.
injected 3 times over a 24 hour period (as in Example 2
above) showed an even higher level of EGFP gene expression
than found after injection with the freshly prepared
complex. Again, very little or no expression was seen in
the liver.
Example 4
Long-Term Stability of the Lyophilized Complex
Stored as 2-8°C as Assessed by Size and
In vitro Transfection Efficiency
[052] To increase the potential of our TfRscFv-liposome-
DNA complex as a viable clinical therapeutic, a means of
increasing its stability was developed, thus maintaining its
tumor-targetability, and shelf-life. Some of our studies
have indicated that the lyophilized complex'with 10% sucrose
as the excipient could be stored successfully at either -
20°C, -80°C or 2-8°C. For convenience for use in the clinic
the preferred method of storage is 2-8°C. To determine the
length of time the lyophilized complex can be stored at 2-
8°C without loss of biological activity, the in vitro
transfection efficiency of complex lyophilized and stored at
2-8°C for 1, 4 and 6 months was evaluated. The complex was
TfRscFv-Liposome A-p53 where liposome A,is DOTAP:DOPE (1:1).
The ratio of the three components was 0.3 µg: 14 nmoles: 1
µg (TfRscFv: Liposome A: DNA) which is equivalent to 0.34
µg:10 µg:l µg. 10% sucrose was used as the excipient. The
DNA in the complex was a plasmid vector containing -1.7 Kb
cDNA sequence coding for human wild-type p53. The complex
was prepared, lyophilized and reconstituted at the
appropriate time after storage at 2-8°C as described in
Example 1. Size (number parameter) and Zeta Potential were
determined using a Malvern 3000H Zetasizer. Functional
activity was assessed using a luciferase co-transfection
assay. Human prostate cancer DU145 cells were co-
transfected with BP100 plasmid DNA and with the complexes.
BP100 plasmid carries the luciferase gene under the control
of a wtp53 inducible promoter. Thus, the level of
functional p53 in the transfected cells is reflected by the
level of luciferase activity. 24 hours after transfection,
the cells were lysed and luciferase activity assayed, using
the Promega Luciferase Reagent according to manufacturing
protocol. As shown in Table 1, the luciferase activity,
size and zeta potential of the complexes are consistent
between the freshly prepared complex and complexes
lyophilized and stored at 2-8°C for up to six months.
Table 1
Comparison of Lvophilized Complex
With Freshly Made Complex
In Vivo Tumor Specific Targeting of the Svstemically
Administered Lvophilized Complex After Storage at 2-8°C .
[053] The fresh and lyophilized complexes, prepared,
stored at 2-8°C for 1, 4, or 6 months, and then
reconstituted for the studies described in Example 4 were
also tested in vivo for their ability to reach and transfect
human prostate xenograft tumors after systemic (i.v.)
administration. Athymic nude mice bearing subcutaneous
human prostate tumor cell line DU145 xenograft tumors of at
least 100 mm3 were i.v. injected three times over 24 hours
with complex (fresh or lyophilized and reconstituted) in an
amount equivalent to 40 ug of DNA per injection in a final
volume of' 0.8 mL. At 48 hours after the last injection the
animals are humanely euthanized, the organs removed, protein
isolated and expression determined by Western Analysis as
described by Xu, L. et al., Tumor Targeting 4:92-104'(1999).
Other methods commonly known in the art alternatively could
have been used. 80ug of total protein lysate was
loaded/lane of a 12% SDS-polyacrylamide gel. After the gel
was run, protein was transferred to nitrocellulose membrane
and probed with an anti-p53 mouse monoclonal antibody
(Oncogene Research Products).
[054] The results of the in vivo tumor targeting in mice
are shown in Fig.5. The levels of p53 expression were
similar between those in the tumor from animals receiving
the freshly prepared complex and those from animals
receiving each of the lyophilized complexes even six months
after lyophilization. p53 expression levels in all tumors
were significantly higher than those in the liver,
demonstrating that the tumor specificity after i.v.
administration is maintained even after 6 months storage at
2-8°C.
Batch to Batch Consistency and
Stability After Lyophilization
[055] it is important to establish that multiple batches
of complex, prepared and lyophilized at different times on
the same day and prepared and lyophilized on different days
have similar sizes and levels of transfection efficiency.
The complex TfRscFv-Liposome A-p53, where liposome A is
DOTAP:DOPE (1:1) was prepared, lyophilized, stored and
reconstituted as described in Example 1. The ratio of the
components and the p53 DNA were as described In Example 4.
[056] Functional expression of multiple TfRscFv-LipA-p53
complexes either freshly prepared or lyophilized was
evaluated in prostate cancer DU145 cells. In vitro activity
was assessed using the BP100 plasmid and the luciferase
assay as described above in Example 4. On 5 different days
at least two independent samples were prepared and
lyophilized. After being stored at 2-8°C for 2 weeks, the
samples were reconstituted as in Example 1 and tested in
vitro and in vivo. In vitro, the luciferase activity
(RLU/ug: Relative Light units per ug of protein in cell) was
assayed, using the Promega Luciferase Reagent as described
in the manufacture's protocol, and the zeta potential and
the particle size (number parameter)of each batch were also
measured on a Malvern 3000H Zetasizer. As shown in Table 2,
by number parameter, size of the majority of the
preparations falls into the 400-700 nm range and the zeta
potentials are all in the positive, range. Thus, different
complexes made on different days have consistent behavior.
[057] The samples also have been evaluated in vivo in
DU145 xenograft bearing athymic nude mice as described in
Example 5. To demonstrate that the ligand-liposome-DNA
complex employing TfRscFv as the targeting entity maintains
tumor specificity, Western analysis was employed (Figure 6).
these independent batches of TfRscFv-LipA-p53 complex were
tested in the DU145 human prostate subcutaneous xenograft
mouse model. Nude mice carrying DU145 tumors of ~50-100 mm3
were intravenously injected three times over a 24 hour
period with the complexes (40ug DNA/injection). Twenty-four
hours after the last injection the animals were sacrificed
and the tumor and liver excised. Protein was isolated. 80
µg of total protein lysate were loaded/lane of a 12% SDS-
polyacrylartd.de gel. After the gel was run, protein was
transferred to nitrocellulose membrane and probed with an
anti-p53 mouse monoclonal antibody (Oncogene Research
Products) . The membrane was subsequently probed for GAPDH
levels to demonstrate equal loading. High p53 expression
levels are evident in the tumor from the mice receiving the
complexes containing each of the various batches (Fig.6).
Similar level of p53 expression were observed in the tumors
either with the freshly prepared or lyophilized TfRscFv
complex. In contrast, very low p53 expression is observed
in the mice not treated with the complex. The tumor
specificity is demonstrated by the very low levels of
expression in the liver. An all instances, there is a 5-10
fold difference in expression between the tumor and liver.
Compared to the level of p53 in the untreated tumor, all
preparations gave strong p53 signal in the tumors of treated
mice but not in the corresponding livers in the same mice.
Therefore, these studies indicate that lyophilization of the
complete complex is feasible and may be able to overcome the
problem of stability and shelf-life.
Example 7
Lyophilization by a Commercial Manufacturer:
In Vitro and In Vivo Testing
[058] For a lyophilized complex to be useful in treating
human patients it is necessary to show that the process of
complex preparation in the presence of 10% sucrose could be
transferred and successfully performed on a large scale by a
commercial manufacturing entity. The complexes were
prepared by stirring, under contract and a confidentiality
agreement, by Cardinal Health, Albuquerque, NM. The DNA
solution was added to the TfRscFv:liposome solution while
stirring at a speed where a vortex was just forming in the
solution for 3 0 seconds to 1 minute. This solution was held
at room temperature for 10-20 minutes, after which an
aqueous solution of 50% sucrose was added with stirring as
above for 30 seconds to 1 minute to a final concentration of
10% and held at room temperature for 10 - 20 minutes. The
commercially prepared batches ranged in size from 50-1000
ml. The lyophilization protocol using a Hull lyophilizer at
this commercial facility was as follows:
• 10 mL vials, each containing 5mL of complex with 10%
sucrose, were loaded at ambient temperature.
• The shelf temperature was ramped to -45aC over 1 hour.
Once the shelf temperature reached -45 ± 30C, the
product was held for 3 hours.
• At this point, the condenser was chilled to -55aC or
colder and the vacuum was set to 50 micron Hg.
• The shelf temperature was than ramped to -35SC over 1
hour.
• Once the shelf temperature reached -352C, the product
was held for 48 hours.
• The shelf temperature was ramped to 202C over 4 hours
and the product was held for 12 hours.
At the end of the cycle, the chamber pressure was
restored to atmospheric with nitrogen, NF filtered
through an appropriate sterilizing microbial retentive
filter.
• The product was stoppered, labeled and stored at 2-8aC.
[059] Five different batches of the TfRscFv-LipA-p53
complex were prepared by the commercial entity. An example
of the in vitro luciferase activity, size and zeta potential
of representative commercially prepared batches are shown in
Table 3. The size zeta potential and level of luciferase
activity of the commercially prepared and lyophilized
complexes was comparable to that of the complex freshly
prepared in the laboratory.

[060] To compare the five batches, mice bearing DU145
xenografts were treated as described in Example 5 (at 40 µg
DNA/injection in 0.8mL). Each mouse received three i.v.
injections over 24 hours. Forty-eight hours after the last
injection the animals were sacrificed and organs harvested.
All five batches show high levels of p53 expression by
Western Analysis that were comparable to that of the freshly
prepared complex and significantly higher than that observed
in either untreated tumor or liver (Fig.7). Therefore, this
technology can be successfully transferred to commercial
manufacturers.
Example 8
Consistency of Percent of Uncompleted TfRscFv
Levels in the Lyophilized Complex
[061] To further assess the stability of the lyophilized
complex, the amount of uncomplexed ligand was determined
after storage at 2-8°C for up to six months. To evaluate
the amount of uncomplexed TfRscFv present in the TfRscFv-
LipA-p53 complex, 4%-20% gradient non-denaturing and non-
reducing polyacrylamide gel electrophoresis followed by
Western analysis was employed using methods commonly known
to one skilled in the art (Fig.8). A polyclonal rabbit
antibody against the TfRscFv protein was used as the first
antibody (produced by Animal Pharm, Healdsburg,,CA) and a
HRP-labeled mouse anti-rabbit monoclonal antibody (Sigma) as
the second antibody. Freshly made or lyophilized complexes
containing 134 ng of TfRscFv in each were prepared and
lyophilized as described in Example 1. The lyophilized
samples were stored at 2-8°C for 1, 4, or 6 months, after
which they were reconstituted as in Example 1. Once
complexed to liposomes, the TfRscFv protein will not be able
to enter the PAGE gel. Therefore, only the uncompiexed free
TfRscFv will be detected. It is difficult, under non-
denaturing and non-reducing conditions, to accurately
determine the amount of the free TfRscFv monomer. Thus,
uncomplexed samples containing 13.4 ng (10% of that in each
of the complexes), 26.8ng (20%) or 40.2ng (30%) of.the
single agent TfRscFv were-also run in the same gel as
concentration standards for a rough estimate of the amount
of the uncomplexed TfRscFv in each of the test complexes,
[062] The results indicated that approximately 10% or
less of the TfRscFv initially put into the complex is
present as free TfRscFv in the various fresh or lyophilized
preparations of TfRscFv-LipA-p53 complex even after storage
at 2-8°C for six months. These data suggest that the amount
of free, uncomplexed TfRscFv is quite consistent in all
preparations, and that this level does not change after
lyophilization and storage at 2-8°C for at least six months.
Example 9
Lyophilization of Licrand-Liposome-Nucleic Acid Complex
Containing an Antisense HER-2 Oligonucleotide
[063] The above studies used plasmid DNA in the complex.
Since plasmid DNA and oligonucleotides are not always
interchangeable, and can have different chemistries,
experiments also were carried out to demonstrate that the
lyophilization procedure could be applied to a ligand-
liposome complex containing an oligonucleotides (ODN). The
ODN used was a 15 mer phosphorothioated sequence specific
antisense HER-2 ODN complementary to the initiation codon
region of the HER-2 gene (AS HER-2) with the sequence 5'-TCC
ATG GTG CTC ACT-3'. Using MDA-MB-453 human breast cancer '
cell line as the assay system, cell killing by the TfRscFv-
lipA-AS HER-2 complex was evaluated after lyophilization
with different sugars at increasing ODN concentrations. The
complexes were prepared as described in Example 1 and were
composed of TfRscFv, Liposome A (D0TAP:D0PE at 1:1) and the
ODN at a ratio of 1 nmole to 15 nmole (ODN:liposome) and 1
ug to 30 ug (TfRscFv:liposome).
[064] The complexes to be lyophilized were prepared to
contain either 5% dextrose or 10% sucrose and compared to
freshly prepared comparable complex preparations comprising
10% sucrose. The complexes were lyophilized as described in
Example 1, stored overnight at 2-8°C and reconstituted in
endotoxins-free water as described in Example 1. 5 x 103
MDA-MB-453 cells were seeded/well of a 9 6-well plate. 24
hours later the cells were transfected with either the
freshly prepared or lyophilized and reconstituted complexes.
The cell viability XTT-based cytotoxicity assay (XTT=3'-[1-
phenyl-Amino-Carbonyl) -3, 4- [tetrazolium] -bis (4-methoxy-6-
nitro)benzene sulfonate) was performed in triplicate 48
hours post-transfection. As shown in Figure 9, at AS-HER-2
ODN concentrations above 0.25 µM, the lyophilized and
reconstituted complex containing 10% sucrose had the
greatest effect on cell killing. At the higher
concentrations of ODN, both the fresh and reconstituted 10%
sucrose-containing complexes were far superior to that with
5% dextrose and lyophilization had no adverse effect on the
cell-killing ability of the HER-2 antisense ODN contained in
the complex.
[065] To confirm that lyophilization and reconstitution
in the presence of 10% sucrose was not detrimental to the
efficacy of the complex, an XTT assay assessing the level of
chemosensitization to Gemzar in human pancreatic cancer
(PANC) 1 cells was preformed, comparing freshly prepared
complexes against comparable complexes that had been
lyophilized and reconstituted as described in Example 1.
The ratios of the components in the complex were 1 nmole :
15 nmole (ODN:liposome) and 1 ug : 30 ug
(TfRscFv: liposome) . 4 x 103 PANC-1 cells were seeded/well of
a 96 well plate and transfected 24 hours later with TfRscFv-
LipA-AS HER-2 (0.25^iM ODN) complex that was either freshly
prepared or had been mixed with sucrose to provide 10%
sucrose and lyophilized, stored refrigerated overnight at 2-
8°C and reconstituted. The chemotherapeutic drug Gemzar was
added 24 hours later. The cell viability XTT-based assay
was performed in triplicate 72 hours after drug addition.
The results are illustrated in Figure 10. As shown, the
survival curves of the samples were virtually identical. In
addition, the IC50 (the concentration of drug killing 50% of
the cells) values of the complexes are the same, if not
lower, than previously determined using a freshly prepared
complex with 5% dextrose. The preparation and storage
method of this invention thus also is amenable to use with
any antisense oligonucleotides since the target gene is
irrelevant to the process.
[066] These studies indicate that lyophilization of the
complete complex is feasible and that previous difficulties
with stability and shelf life.when using ODN as therapeutic
molecules can be overcome.
Example 10
Maintenance of Size, Zeta Potential and
Efficacy After Lyophilization of Complex Carrying
AS ODN and Storage for Six Months.
[067] The size., zeta potential and transfection activity
of the ligand-liposome-nucleic acid complexes containing AS
HER-2 ODN and prepared with 10% sucrose were examined .before
and after lyophilization. The size of the complex was found
to be essentially the same before and after lyophilization
and storage at -2 0°C for up to six months. For example:
Pre-lyophilization, the values for size (run) by intensity,
volume and number average for the fresh and six month
lyophilized complexes prepared as described in Example 9
were 410 (I), 454 (V) and 368 (N) vs 339 (I), 427 (V) and
397 (N), respectively. In addition, another oligonucleotide
that does not affect HER-2 levels (SC-ODN_ (5'-CTA GCC ATG
CTT GTC-3') was also complexed at the same ratio,
lyophilized, stored for up to six months at -20°C and
reconstituted as in Example 9. Here also lyophilization and
storage had no significant effect on size or zeta potential
of the complex. Thus, any ODN can be complexed and
lyophilized.
[068] The zeta potentials were -43.8 (fresh) and -47.7
(lyophilized) after six months storage. The transfection
efficiency of the lyophilized complex with 10% sucrose was
measured by assessing the ability of the TfRscFv-lip A-AS
HER-2 to down modulate HER-2 expression in vitro. After
preparation, lyophilization (as in Example 4), and storage
at -20°C for up to six months, the complex AS HER -2 ODN at
two different concentrations (0.3 or 0.6µM) or SC-ODN at
0.6uM, were used to transfect human breast cancer cell line
MDA-MS-435 cells. Freshly prepared complexes carrying AS
HER-2 or SC-ODN were used as controls. The SC-ODN had no
effect either before or after Lyophilization. However, there
was an AS HER-2 ODN dose dependent down-modulation of HER-2
expression by both freshly prepared and lyophilized
complexes (Figure 11) even after six months storage at -
2 0°C. Since the SC-ODN had no effect, the down-modulation
observed was not a result of any general cytotoxicity due to
lyophilization of the complex.
Example 11
Maintenance of Size and Zeta Potential
of Complex Carrying- siRNA After Lyophilization
[069] The stability of a complex of TfRscFv, Liposome A
and siRNA with 10% sucrose after lyophilization was assessed
by measuring the size of the complex and the zeta potential
before and after lyophilization. The complex was composed
of TfRscFv, Liposome A (DOTAP:DOPE at 1:1 mole ratio) and
siRNA at 33.3ug. Total volume of complex was 500 µL. The
ratio of the components was 1 ug to 7 nmole (siRNA: liposome)
and 1 ug to 30 µg (TfRscFv:liposome) . Sucrose was added to
the complex to a final concentration of 10%. The complex was
prepared and lyophilized as described in Example 1. After
lyophilization the complex was reconstituted as described in
Example 1 and size and zeta potential were measured using a
Malvern Zetasizer 3000H. The results are shown in Table 4.

Therefore, after Lyophilization there was no significant
change in size or zeta potential. If anything, the size by
intensity and volume are even smaller after lyophilization,
making the complex more efficient for in vivo use.
Example 12
Maintenance of Size Complex Made with
Peptide-liposome After Lyophilization
[070] To further demonstrate the general nature of this
invention a complex also was prepared that contained a
modified liposome. The liposome used to form the complex
was bound to a peptide. The peptide comprised histidine and
lysine and was a branched peptide 31 amino acids in length
and was composed of a combination of histidine and non-
histidine amino acids with the structure 5'-K[K(H)-K-K-K] 5-
K(H)-K-K-C-3'). The liposome in this study was comprised of
DOTAP:DOPE (1:1). The HK peptide was covalently conjugated
to the liposome through the terminal cysteine and a
maleimide group in the liposome. The complex consisted of
TfRscFv-HK-liposome-DNA where the ratios of the components
were as follows: TfRscFv to HK-liposome (µg: µg) of 1 µg:30
ug and DNA to HK-liposome (ug:nmole) of 1 ug: 14 nmole. The
DNA used was p53 (see Example 4) at 18 ug DNA for 3 00 uL of
total volume of complex. 10% sucrose was included in the
final complex. The complex was prepared and lyophilized as
described in Example 1. Post-lyophilization the complex was
stored at 2-8°C for 3 days and then reconstituted as
described in Example 1. The size of the complex before
lyophilization and after three days storage at 2-8°C was
measured on a Malvern Zetasizer 300OH. Prior to'
lyophilization the size (number average) was 601 nm. After
storage and reconstitution it was 588. Thus, once again
lyophilization of the complex using 10% sucrose did not
result in any significant change in the size of the complex
even with the inclusion of the HK peptide.
Example 13
In Vitro and In Vivo Assessment of Complex Carrying
a Different Therapeutic Gene (RB94)
After Lyophilization and Storage at 2°-8°C
[071] In addition to the Luciferase gene, a gene coding
for the enhanced green fluorescence protein, and the p53
gene, a liposome complex carrying other plasmid DNA can be
lyophilized and retain size and biological activity. To
further demonstrate, this complex was also prepared carrying
another therapeutic gene, the tumor suppressor gene RB94.
The complex was TfRscFv-liposome A-RB94 where liposome A is
D0TAP:D0PE (1:1). The ratio of the three components
(TfRscFV:Liposome:DNA) were 0.34 µg : 10 µg: µg. The
complex also contained 30 US' of RB94 plasmid DNA in a total
volume of 0.5mL, with 10% sucrose. The complex was prepared
as described in Example 1 and lyophilized using the method
described in Example 1 for the size and zeta potential
studies, or prepared as in Example 7 by Cardinal Health
using for use the in vitro and in vivo targeting studies.
Size and Zeta Potential
[072] The size and zeta potential of complex prepared as
in Example 1 and lyophilized, stored at 2-8°C for four days
and reconstituted as in Example 1 was compared before and
after lyophilization and storage using a Malvern Zetasizer
3000H. Prior to lyophilization the size (nm) was intensity
and 283 (Intensity) and 392 (Volume), while afterward it was
found to be 303 (Intensity) and 347 (Volume) . Thus, there
was no significant change in size after lyophilization and
storage for four days at 2°-8°C when 10% sucrose was
included. Similarly the zeta potential showed no major
difference, both being in the +20 to +30 range [19 (pre) and
30.7 (post)].
In Vitro and In Vivo Targeting
[073] The ability of the complex to specifically target
tumor cells and efficiently transfect them after
lyophilization and storage at 2-8°C for an extended period
of time was also tested in cell culture using human prostate
cell line DU145 and human bladder carcinoma cell line HTB-9.
Both cell lines were transfected in vitro using either
freshly prepared complex or complex that had been prepared
and lyophilized by the commercial contractor (Example 7) and
stored at 2°-8°C for approximately 4 months prior to
reconstitution as in Example 1. The level of RB94 protein
expression in the cells was determined by Western Analysis
using standard protocols known to one skilled in the art.
There was no significant difference in either human tumor
cell line between the amount of protein detected after
transfection with freshly prepared or lyophilized complex.
[074] Mice carrying human bladder carcinoma HTB-9
xenograft tumors were injected systemically (i.v. via the
tail vein) with the freshly prepared complex or complex that
had been prepared with 10% sucrose by stirring as in Example
1, lyophilized (Example 7), and stored at 2 °-8°C for almost
5 months prior to reconstitution in Example 1. The mice
received a total of three i.v. injections over 24 hours (40
µg of DNA in 0.67 mL per injection). Approximately 48 hours
after the last injection the animals were humanely
sacrificed, the tumors and liver excised, and protein
obtained and analyzed by Western Blot using an anti-RB94
monoclonal antibody (QED Biosciences, Inc) by means of a
common procedure as described by Xu, L., et al., tumor
Targeting 4:92-104 (1999). As with the in vitro studies
there was no significant difference in the level of RB94
protein evident in the tumors from the animals receiving the
fresh or the lyophilized complexes. If anything, the
expression was even higher in the tumors from the mice
injected with the lyophilized complex. Moreover, there was
virtually no expression in the"'livers in either group
demonstrating that the tumor targeting ability of the
complex was maintained after lyophilization in the presence
of 10% sucrose and storage at 2-8°C for at least 5 months.
WE CLATM :
1. A method for preparing a stable cell-targeting complex comprising a ligand and a cationic
liposome encapsulating a therapeutic agent, reporter gene or diagnostic agent which
comprises:
providing a complex comprising a ligand and a cationic liposome encapsulating a
diagnostic agent, reporter gene or therapeutic agent;
combining said liposome complex with an aqueous solution consisting essentially of a
stabilizing amount of sucrose in water or in a buffer to a final solution of about 1% to
about 80% sucrose; and
lyophilizing said solution of liposome complex and sucrose to obtain a lyophilized
preparation;
wherein, upon reconstitution, said preparation retains at least about 80% of its pre-
lyophilization activity.
2. The method as claimed in claim 1, wherein said solution consists essentially of sucrose in a
PBS buffer, in a HEPES buffer, in a TRIS buffer or in a TRIS/EDTA buffer.
3. The method as claimed in claim 1 or claim 2, wherein said preparation retains at least about
85% or at least about 90% or at least about 95% of its pre-lyophilization activity upon
reconstitution.
4. The method as claimed in claim 1 or claim 2, wherein said complex is combined with a
sucrose solution to a final concentration of about 1% to about 50% or about 1% to about
20% or about 5% to about 10% or about 10% sucrose.
5. The method as claimed in any one of claims 1 to 4, wherein said ligand comprises a ligand the
receptor for which is differentially expressed on a target cell.
6. The method as claimed in any one of claims 1 to 5, wherein said ligand is a protein.
7. The method as claimed in claim 5, wherein said ligand comprises transferrin, folate, an
antibody or an antibody fragment.
8. The method as claimed in claim 7, wherein said ligand comprises an anti-TfR monoclonal
antibody.
9. The method as claimed in claim 7. wherein said ligand comprises a single chain Fv
fragment of an antibody.
10. The method as claimed in claim 9, wherein said antibody fragment comprises an scFv
based on an anti-TfR monoclonal antibody.
11. The method as claimed in any one of claims 1 to 10, wherein said liposome comprises at
least one cationic lipid and at least one neutral or helper lipid.
12. The method as claimed in claim 11, wherein said cationic lipid comprises
dioleoyltrimethylammonium phosphate (DOTAP) or dimethyldioctadecylammonium
bromide (DDAB) and said neutral or helper lipid comprises
dioleoylphosphatidylethanolamine (DOPE) or cholesterol (chol).
13. The method as claimed in claim 11, wherein said liposome comprises a mixture of
DOTAP and DOPE.
14. The method as claimed in any one of claims 1 to 11, wherein the liposome is bound to a
peptide of at least about 10 amino acids, wherein said peptide is composed of about 5-
100% histidine and 0-95% non-histidine residues.
15. The method as claimed in claim 14. wherein at least 10% of said non-histidine residues of
said peptide are lysine residues.
16. The method as claimed in claim 15. wherein said peptide has the structure 5 '-K[K(H)-K-
K-K] 5-K (H)-K-K-C-3 '.
17. The method as claimed in any one of claims 1 to 16. wherein said therapeutic agent
comprises a gene, plasmid DNA. oligonucleotide, oligodeoxynucleotide. antisense
oligonucleotide or siRNA.
18. The method as claimed in any one of claims 1 to 16, wherein said diagnostic agent
comprises electron dense material, magnetic resonance imaging agents or
radiopharmaceuticals.
19. A lyophilized preparation comprising a complex of a ligand and a cationic liposome
encapsulating a therapeutic agent, or reporter gene or diagnostic agent which is stable
at a temperature in the range of about -80°C to 8°C for at least about six months while
retaining at least about 80% activity, said preparation comprising said complex and
about 1% to 80% sucrose to increase the stability of said complex.
20. The lyophilized preparation as claimed in claim 19, which comprises about 1% to about
50% or about 1% to about 20% or about 5% to about 10% or about 10% sucrose.
21. The lyophilized preparation as claimed in claim 19, which upon reconstitution retains at
least about 80% or about 85% or about 90% or about 95% of its pre-lyophilization
activity.
22. The lyophilized preparation as claimed in any one of claims 19 to 21, wherein said ligand,
said therapeutic agents, said diagnostic agent, and said liposome are as claimed in any
one of claims 5 to 18.


A method for preparing a stable cell-targeting complex comprising a ligand and a cationic liposome encapsulating
a therapeutic or diagnostic agent comprises (a) combining the complex with a solution comprising a stabilizing amount of sucrose
and (b) lyophilizing the resultant solution to obtain a lyophilized preparation; wherein, upon reconstitution, the preparation retains
at least about 80% of its pre-lyophilization activity.

Documents:

02616-kolnp-2005-abstract.pdf

02616-kolnp-2005-claims.pdf

02616-kolnp-2005-description complete.pdf

02616-kolnp-2005-drawings.pdf

02616-kolnp-2005-form 1.pdf

02616-kolnp-2005-form 3.pdf

02616-kolnp-2005-form 5.pdf

02616-kolnp-2005-international publication.pdf

2616-KOLNP-2005-ABSTRACT.pdf

2616-KOLNP-2005-AMENDED CLAIMS.pdf

2616-kolnp-2005-assignment-1.1.pdf

2616-kolnp-2005-assignment.pdf

2616-KOLNP-2005-CANCELLED PAGES.pdf

2616-kolnp-2005-correspondence-1.1.pdf

2616-kolnp-2005-correspondence.pdf

2616-KOLNP-2005-DESCRIPTION (COMPLETE).pdf

2616-KOLNP-2005-DRAWINGS.pdf

2616-kolnp-2005-examination report.pdf

2616-KOLNP-2005-FORM 1.pdf

2616-kolnp-2005-form 13-1.1.pdf

2616-KOLNP-2005-FORM 13.pdf

2616-kolnp-2005-form 18-1.1.pdf

2616-kolnp-2005-form 18.pdf

2616-kolnp-2005-form 3-1.1.pdf

2616-KOLNP-2005-FORM 3.pdf

2616-kolnp-2005-form 5.pdf

2616-KOLNP-2005-FORM-27.pdf

2616-kolnp-2005-gpa-1.1.pdf

2616-kolnp-2005-gpa.pdf

2616-kolnp-2005-granted-abstract.pdf

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2616-kolnp-2005-granted-letter patent.pdf

2616-kolnp-2005-granted-specification.pdf

2616-kolnp-2005-international search report.pdf

2616-kolnp-2005-others-1.1.pdf

2616-KOLNP-2005-OTHERS.pdf

2616-kolnp-2005-pct request form.pdf

2616-KOLNP-2005-PETITION UNDER RULE 137.pdf

2616-KOLNP-2005-REPLY TO EXAMINATION REPORT.pdf


Patent Number 245569
Indian Patent Application Number 2616/KOLNP/2005
PG Journal Number 04/2011
Publication Date 28-Jan-2011
Grant Date 25-Jan-2011
Date of Filing 16-Dec-2005
Name of Patentee GEORGETOWN UNIVERSITY
Applicant Address 37TH & O STREETS, N.W., WASHINGTON, DC 20057, UNITED STATES OF AMERICA
Inventors:
# Inventor's Name Inventor's Address
1 PIROLLO, KATHLEEN, F 547 ANDERSON AVENUE, ROCKVILLE, MA 20850, UNITED STATES OF AMERICA
2 CHANG, ESTHER, H 10244 DEMOCRACY BLVD., POTOMAC MA 20854, UNITED STATES OF AMERICA
PCT International Classification Number A61K 9/127
PCT International Application Number PCT/US2004/017695
PCT International Filing date 2004-06-04
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/475,500 2003-06-04 U.S.A.