U.S. patent application number 11/010158 was filed with the patent office on 2006-06-29 for liposomes containing oligonucleotides.
This patent application is currently assigned to Georgetown University. Invention is credited to Anatoly Dritschilo, Prafulla Gokhale, Usha Kasid, Aquilur Rahman, Chuanbo Zhang.
Application Number | 20060141020 11/010158 |
Document ID | / |
Family ID | 26717905 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060141020 |
Kind Code |
A1 |
Kasid; Usha ; et
al. |
June 29, 2006 |
Liposomes containing oligonucleotides
Abstract
It is possible to radiosensitize tumor cells by administration
of compositions containing the Human antisense c-raf-1
oligodeoxyribonucleotide (ODN/oligo) sequence:
5'-GTGCTCCATTGATGC-3' (SEQ. ID. NO: 1) wherein only the end bases
are phosphorothioated is a preferred embodiment. Antisense
sequences of up to 40 bases which contain this sequence, such as
5'-CCTGTATGTGCTCCATTGATGCAGC-3' (SEQ ID NO: 2) may be used in
accord with the teachings of this disclosure. Compositions
comprising a cationic liposome of dimethyldioctadecyl ammonium
bromide, phosphatidylcholine and cholesterol may be used as a
carrier system. The liposomes provide a new carrier system that is
particularly useful for administration of sequences for
therapy.
Inventors: |
Kasid; Usha; (Rockville,
MD) ; Gokhale; Prafulla; (Oak Hill, VA) ;
Zhang; Chuanbo; (Rockville, MD) ; Dritschilo;
Anatoly; (Bethesda, MD) ; Rahman; Aquilur;
(Potomac, MD) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
Georgetown University
Washington
DC
|
Family ID: |
26717905 |
Appl. No.: |
11/010158 |
Filed: |
December 10, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09930283 |
Aug 16, 2001 |
|
|
|
11010158 |
Dec 10, 2004 |
|
|
|
09354109 |
Jul 15, 1999 |
|
|
|
09930283 |
Aug 16, 2001 |
|
|
|
08957327 |
Oct 24, 1997 |
6126965 |
|
|
09354109 |
Jul 15, 1999 |
|
|
|
60041192 |
Mar 21, 1997 |
|
|
|
Current U.S.
Class: |
424/450 ;
435/458; 514/44A |
Current CPC
Class: |
C12N 15/88 20130101;
A61K 9/127 20130101; A61K 9/0019 20130101; C12N 2310/345 20130101;
Y10S 436/829 20130101; A61K 9/1272 20130101; C12N 2310/315
20130101; C12N 15/1135 20130101; C07H 21/00 20130101; A61K 38/00
20130101 |
Class at
Publication: |
424/450 ;
514/044; 435/458 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 9/127 20060101 A61K009/127; C12N 15/88 20060101
C12N015/88 |
Claims
1. A composition comprising a cationic liposome comprising a
cationic liposome containing a cationic lipid, phosphatidylcholine,
cholesterol and an antisense oligonucleotide comprising a raf
antisense oligonucleotide wherein the antisense oligonucleotide
comprises a sequence of the formula 5'-CCTGTATGTGCTCCATTGATGCAGC-3'
(SEQ ID NO: 2) and wherein only the terminal bases of said
oligonucleotide are phosphorothioated.
2. (canceled)
3. (canceled)
4. (canceled)
5. The composition of claim 1 further comprising a pharmaceutically
acceptable carrier.
6. (canceled)
7. The composition of claim 5, wherein the pharmaceutically
acceptable carrier is isotonic.
8. The composition of claim 7, wherein the pharmaceutically
acceptable carrier is a buffered isotonic solution.
9. A method of radiosensitizing tumor tissue by administration of a
radiosensitizing effective amount of least one antisense
oligonucleotide of no more than 40 bases comprising the sequence
5'-CCTGTATGTGCTCCATTGATGCAGC-3' (SEQ ID NO: 2), wherein the
radiosensitizing effective amount comprises a dosage that delivers
a serum concentration of about 1 .mu.g/mL to 1000 .mu.g/mL.
10. The method of claim 9, wherein the oligonucleotide is
phosphorothioated at only the end nucleotides.
11. (canceled)
12. The method of claim 9, wherein the oligonucleotide is
administered intravenously.
13. The method of claim 9, wherein the oligonucleotide is
administered directly to the target tissue.
14. The method of claim 9, wherein the oligonucleotide is
administered into the arterial supply to the target tissue.
15. (canceled)
16. (canceled)
17. (canceled)
Description
[0001] This application takes priority from Provisional Application
60/041,192 filed Mar. 21, 1997. This work was supported by grants
from the National Institutes of Health. The United States
Government has certain rights in this invention.
FIELD OF THE INVENTION
[0002] This invention is related to use antisense of sequences of
.ltoreq.40 bases for enhancing radiosensitivity of
radiation-resistant tumors and to cationic liposomes which are
particularly useful as carriers for antisense sequences.
BACKGROUND OF THE INVENTION
[0003] Radiation therapy is an important treatment modality of
cancer. However, therapeutic management may be limited by the
inherent relative resistance of some cancers to the cytotoxic
effects of ionizing radiation. Recently, several lines of
investigation have coalesced to demonstrate a link between certain
oncogenes (ras, raf, cot, mos, myc), growth factors (PDGF, FGF) and
the phenomenon of cellular resistance to ionizing radiation.
[0004] It was previously reported that expression of antisense
c-raf-1 cDNA results in reduced expression (RNA) of c-raf-1 gene, a
cause of delayed tumor growth in athymic mice and in enhanced
radiation sensitivity of relatively radioresistant laryngeal
squamous carcinoma cells, SQ-20B (Kasid et al., Science
243:1354-1356, 1989).
SUMMARY OF THE INVENTION
[0005] It is possible to radiosensitize tumor cells by
administration of compositions containing the Human antisense
c-raf-1 oligodeoxyribonucleotide (ODN/oligo) sequence:
[0006] 5'-GTGCTCCATTGATGC-3' (seq. #1) wherein only the end bases
are phosphorylated is a preferred sequence. Antisense sequences of
up to 40 bases which containing this sequence may be used in accord
with the teachings of this disclosure. A composition of the 25-mer
oligo:
5'-CCTGTATGTGCTCCATTGATGCAGC-3' (seq. #2) wherein the sequence is
also effective. Compositions comprising cationic liposomes
containing at least one non-toxic cationic lipid,
phosphatidylcholine and cholesterol may be used as a carrier
system.
DESCRIPTION OF THE INVENTION
[0007] The search for clinically useful radiation sensitizers for
treatment of cancers which fail to respond to radiation therapy has
been actively pursued. This invention provides specific sequences
which, while inducing radiation sensitivity on tumor cells, is
non-toxic to normal tissue. As little as 10 .rho.mol/.mu.l of the
sequences encapsulated in liposomes is effective when tumor cells
are contacted with the compositions. It was found that the
expression and enzymatic activity of Raf-1 protein are inhibited in
cells exposed to raf antisense oligodeoxyribonucleotide (As-ODNs)
directed against the translation initiation site of human c-raf-1
cDNA. In contrast, treatment of cells with an equimolar
concentration of raf sense oligodeoxyribonucleotide (S-ODNS) had no
effect on the expression and activity of Raf-1. Furthermore, it was
observed radiosensitization of raf As-ODNs-treated SQ-20B cells.
The dose modifying factor of As-ODNs treatment was 1.4. This
demonstrates that raf As-ODNs is a DNA sequence-specific
radiosensitizer which may have potential for use in the radiation
therapy of cancers. Hence, the method of the invention comprises
administration of a radiosensitizing effective amount of at least
one antisense nucleotide of no more than 40 bases containing the
sequence 5'-GTGCTCCATTGATGC-3'.
[0008] This invention provides new liposomal compositions which
provide means of enhancing the effect of oligonucleotides
encapsulated in the novel liposomes. The invention is exemplified
using encapsulated raf oligodeoxyribonucleotides. The novel
cationic liposomes of the invention were prepared using
dimethyldioctadecyl ammonium bromide, phosphatidylcholine and
cholesterol. However, other nontoxic cationic lipids such as
N-(2,3-(dioleoyloxy)propyl)-N,N,N-trimethyl ammonium chloride or
1-[2-(9(Z)-octadecenoyloxy)-ethyl]-2-(8(Z)heptadecenyl)-3-(2-hydroxyethyl-
)-imidazolinium chloride may be used. These liposomes provide
protection from degradation in plasma and normal tissues to protect
the oligonucleotides while they are reaching their intended target
cells. Hence, smaller amounts of oligonucleotides are needed to
obtain desired results.
[0009] Cationic liposomes have been used to deliver genes in vitro
and in vivo (Felgner, Editorial, Human Gene Therapy 7:1791-1793,
1996). The novel formulation of the cationic liposomes to
encapsulate antisense raf oligonucleotides has been tested and
found effective. It has been found that these liposomes encapsulate
>90% oligos. Liposomal encapsulation provides protection of
antisense raf oligonucleotide from degradation in plasma, and
normal tissues, and that tumor cells treated with the
liposome-encapsulated antisense raf oligo (LE-ATG-AS raf ODN) are
significantly radiosensitive compared to control or sense raf
oligo-treated cells. It is now disclosed herein that LE-ATG-AS raf
ODN inhibits Raf-1 protein expression in solid tumors. (Gokhale et
al., "Antisense 97: Targeting the Molecular Basis of Disease,"
Cambridge Symposium Meeting, May 1997). The liposomal compositions
of the invention disclosed herein are believed to be particularly
useful as radiosensitizers in solid tumors.
Materials and Methods
oligodeoxyribonucleotides
[0010] The sense and antisense raf ODNs were designed against the
translation initiation site of human c-raf-1 cDNA in accord with
the teachings of Bonner (Bonner et al., Nucleic Acids Res.,
14:1009-1015, 1986), and have the following sequence: sense ODN
(ATG-S raf), 5'-GCATCAATGGAGCAC-3' (seq. #3); antisense ODN (ATG-AS
raf), 5'-GTGCTCCATTGATGC-3' (seq. #1), Only two of the bases, one
at each end, are phosphorothioated. While antisense sequences of
raf of up to 40 bases containing seq. #1 may be used, the larger
sequences may be less effective. The fully phosphorothioated
sequences may also be effective, but are more likely to cause toxic
effects. That the sequences having only the end bases
phosphorothioated are non-toxic to normal cells greatly enhances
the value of such sequences for use in targeting malignant
cells.
Synthesis and Purification of ODNs
[0011] Oligodeoxyribonucleotide synthesis was performed at
Lofstrand Labs Limited, Gaithersburg, using Beta-Cyanoethyl
Phosphoramidite chemistry on Biosearch 8750 DNA synthesizers.
Desired base linkages were modified to phosphorothioate groups
using 3H-1,2-benzodithiole-3-1,1,1-dioxide as the sulfurizing
agent. Oligos synthesized at the 15 .mu.mol scale were cleaved and
deprotected in 30% ammonium hydroxide for 24 hours at room
temperature and purified over reverse phase chromatography columns.
Deprotected DMT-on (trytl on) oligos retained by the support column
as failure sequences were washed off in basic aqueous solution.
Full length product was detritylated using 2% trifluoro-acetic
acid, washed with sterile, deionized water and eluted with 20%
acetonitrile, dried and resuspended in sterile, deionized water.
For quality control, a small aliquot of each oligo preparation was
.sup.32P-end labeled and visualized by polyacrylamide gel
electrophoresis (20% acrylamide and 5% bis) followed by
densitometer scanning of the labeled products.
Source of Cells
[0012] The SQ-20B tumor cells used were established in culture from
the laryngeal squamous carcinoma of a patient who had failed a full
course of radiation therapy. In vitro radiation survival analysis
has confirmed that these tumor cells are relatively radioresistant.
Previously, it had been demonstrated that the transfection of
antisense human c-raf-1 cDNA into SQ-20B cells leads to the
down-regulation of endogenous raf-1 gene expression, delayed tumor
growth in athymic mice, and enhanced radiation sensitivity compared
with the sense c-raf-1 cDNA transfectants, and the untransfected
tumor cells. Hence a "dual" role was proposed for Raf-1 in these
tumor cells; a direct role in the expression of the malignant
phenotype, and an indirect role in cellular responses to radiation
damage.
Cell Culture, Cell Viability and Cell Cycle Assay
[0013] SQ-20B stock cultures were grown and maintained in complete
Dulbecco's modified MEM (GIBCO/BRL) containing 20% heat inactivated
fetal bovine serum (FBS), 2 mM glutamine, 0.1 mM non-essential
amino acids, 0.4 .mu.g/ml hydrocortisone, 100 .mu.g/ml streptomycin
and, 100 U/ml penicillin. For cell viability and cell cycle
analysis, logarithmically growing cells were cultured in T-25
flasks, complete medium was replaced with 1% FBS containing medium
in the presence of a desired concentration of ODNs followed by
continued incubation for various times. Control cells were grown in
1% FBS containing medium without ODNs. Cells were collected by
trypsinization and viability was determined by the trypan blue dye
exclusion assay. Duplicate samples were analyzed by the FACS method
to determine the % distribution of cells in different phases of the
cell cycle.
Intracellular Uptake and Stability of ODNs
[0014] Stock solutions of oligos were prepared by reconstitution of
the lyophilized compounds in sterile phosphate buffered saline
(PBS) just before use. Oligos (10 pmol/.mu.l) were 5'-end labeled
with [.gamma.-.sup.32P]ATP (50 .mu.Ci, 3000 Curies/mmol) and T4
polynucleotide kinase (10 U/.mu.l), and purified on ChromaSpin-10
column (Clontech). Logarithmically growing cells were rinsed with
Hank's balanced salt solution and fresh medium containing 1% FBS,
100 pmol/.mu.l ODNs, and radiolabeled ODNs (2.times.10.sup.6 cpm)
was added. Following incubation at 37.degree. C. in a humidified,
5% CO.sub.2 atmosphere for the desired time, cells were collected,
washed three times in PBS, and lysed for 2 hours at 37.degree. C.
in the buffer containing 10 mM Tris-HCl (pH 7.5), 1% SDS, and 200
.mu.g/ml proteinase K. DNA isolation was performed using the
phenol:chloroform:isoamyl-alchohol (25:24:1) extraction procedure.
ODNs uptake was determined by liquid scintillation counting of the
aqueous phase, and was expressed as a percentage of the total
radioactivity applied to the cells. Aliquots of the cell extracts
(5.times.10.sup.3 cpm per sample) were resuspended in sample buffer
(95% formamide, 0.05% xylene cyanol, 0.05% bromophenol blue) and
electrophoresed on 8 M Urea-20% polyacrylamide gel. The dried gel
was auto-radiographed to visualize the oligos.
Raf-1 Immunoprecipitation, Immunoblotting, and In Vitro Kinase
Activity Assays
[0015] Cells were washed twice with cold PBS and lysed at 4.degree.
C. for 10 min. in RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5%
sodium deoxycholate, 100 mM NaCl, 1 mM phenylmethylsulfonyl
fluoride, 20 .mu.g/ml aprotinin, 20 .mu.g/ml leupeptin). Insoluble
material was removed by centrifugation at 4.degree. C. for 30 min
at 9,000.times.g and protein concentration was determined (Pierce).
Immunoprecipitation was performed by incubating the lysate with
polyclonal Raf-1 antibody directed against the last 12 amino acids
of human Raf-1 and conjugated with Protein A-Agarose (Santa Cruz).
Immunoprecipitates were washed once with the lysis buffer, twice
with 0.5M LiCl-0.1M Tris (pH7.4), and once with 10 mM Tris (pH
7.4). For immunoblotting, the immune-complex was boiled in Lammeli
sample buffer and electrophoresed on a 7.5% SDS-polyacrylamide gel,
followed by western blotting using Raf-1 antibody and detection of
Raf-1 protein by the ECL method according to the manufacturer's
protocol (Amersham). Raf-1 protein kinase activity was determined
in vitro using a pseudosubstrate peptide (Syntide 2, Santa Cruz) as
substrate. The phosphotransferase assay was performed by incubating
Raf-1 immunoprecipitates along with the exogenous substrate for 20
min at 30.degree. C. in 40 .mu.l of reaction buffer containing 25
mM HEPES (pH7.4), 25 mM 6 glycerol phosphate, 1 mM DTT, 10 mM
MnCl.sub.2, 100 .mu.M ATP, and 10 .mu.Ci of [.gamma.-.sup.32P]ATP.
The assay was terminated by spotting 20 .mu.l of the reaction mix
onto 2 cm.times.3 cm pieces of Whatman P81 phosphocellulose paper.
The filters were washed four times for 15 min in a solution of
0.85% phosphoric acid. The Syntide 2-associated .sup.32P
radioactivity bound to the filters was quantitated by Cerenkov
counting.
Clonogenic Radiation Survival Assay, and Data Analysis
[0016] The appropriate number of tumor cells were seeded into T-25
flasks in complete medium containing 20% FBS. Cells were allowed to
attach for 8 hr and the medium was replaced with medium containing
1% FBS and 100 pmol/.mu.l of raf S- or As-ODNs. Control cells were
incubated with complete medium containing 1% FBS. Oligo treatment
lasted for 10 hr, followed by exposure of the cells to the
indicated graded doses of .gamma.-radiation. Irradiations were
performed using a .sup.137Cs gamma irradiator (JL Shepard MARK I
irradiator) at a dose rate of 3.83 Gy/min. The irradiated cells
were then maintained under these incubation conditions for an
additional 2 hr. The growth medium in all flasks then was replaced
with complete medium containing 20% FBS and the cells were
incubated for 7-10 days. Surviving colonies were fixed and stained
with 1% methylene blue. Colonies greater than 50 cells were scored
and the data were fitted to the Albright's computer-generated
single-hit multitarget and linear-quadratic models of radiation
survival response.
Liposome Preparation
[0017] Liposome-encapsulated raf oligodeoxyribonucleotides,
LE-ATG-S raf ODN and LE-ATG-AS raf ODN, were prepared using
dimethyldioctadecyl ammonium bromide, phosphatidylcholine and
cholesterol (Avanti Polar Lipids, Inc., Alabaster, Ala., USA) in a
molar ratio of 1:3.2:1.6. Briefly, the lipids dissolved in
chloroform or methanol were evaporated to dryness in a
round-bottomed flask using a rotatory vacuum evaporator. The dried
lipid film was hydrated overnight at 4.degree. C. by adding 1 ml of
ODN at 1.0 mg/ml in phosphate buffered saline (PBS). The film was
dispersed by vigorous vortexing and the liposome suspension was
sonicated for 5 min in a bath type sonicator (Laboratory Supplies
Co. Inc., Hicksville, NY, USA). The ODN to lipid ratio was 30 .mu.g
ODN/mg of lipid. The unencapsulated ODN was removed by washing the
liposomes by centrifugation (3 times at 75,000 g for 30 min) in
PBS. The encapsulation efficiency was determined by the
scintillation counting of an aliquot of the preparation in which
traces of .sup.32P-end labeled ODN were added to the initial ODN.
The entrapment efficiency was found to be >90% (n=10). The
liposome encapsulated ODN were stored at 4.degree. C. and used
within 2 weeks of preparation. Blank liposomes were prepared
exactly as described above but without ODN.
Animals
[0018] Male Balb/c nu/nu mice, 10-12 weeks old, were maintained in
the RRF facility of the Georgetown University according to
accredited procedure and fed purina chow and water ad libitum.
Pharmacological Disposition Studies
[0019] The pharmacological disposition of free (ATG-AS) or
liposome-encapsulated antisense raf oligodeoxyribonucleotide
(LE-ATG-AS) was carried out in Balb/c nu/nu mice. Male Balb/c nu/nu
mice were injected intravenously via tail vein with 30 mg/kg of
ATG-AS raf ODN or LE-ATG-AS raf ODN. At 5 min, 15 min, 30 min, 1 h,
2 h, 4 h, 8 h, 24 h and 48 h after injection, one animal in each
group was bled from the retro-orbital sinus into heparinized tubes
and sacrificed by cervical dislocation. The blood was centrifuged
immediately at 2000 r.p.m for 10 min at 4.degree. C. to separate
the plasma. The liver, spleen, kidney, heart and lung were rapidly
excised and rinsed in ice-cold normal saline. The 5' organs and
plasma were stored frozen at -70.degree. C. until analysis.
[0020] ODN was isolated from plasma samples using the
phenol/chloroform extraction method, and from tissues using a DNA
extraction kit (Stratagene, La Jolla, Calif., USA). The extracts
were then loaded onto 20% polyacrylamide/8 M urea gels and
electrophoresed in TBE buffer. The gel was electroblotted onto
nylon membrane in 0.5.times.TBE buffer at 20 V for 1 h. The blots
were probed with .sup.32P-labeled sense raf ODN (ATG-S raf ODN) in
Quickhyb buffer (Stratagene, La Jolla, Calif., USA) at 30.degree.
C. overnight. The ODN concentration standard was prepared by
spiking known amount of the ATG-AS raf ODN in blank plasma or blank
tissue samples, followed by extraction as described above. The
autoradiographs were scanned using a computer program (ImageQuant
software version 3.3, Molecular Dynamics), and the amounts of
ATG-AS raf ODN in various samples were calculated by comparison to
standards.
SQ-20B Tumor Xenograft Studies
[0021] Logarithmically growing SQ-20B cells (2.times.10.sup.6) were
injected subcutaneously in the flank region on both sides in male
Balb/c nu/nu mice under mild anesthesia. Tumors were allowed to
grow to a mean tumor volume of 115 mm.sup.3 before initiation of
ODN treatment.
[0022] For intratumoral delivery of LE-ATG-AS raf ODN or LE-ATG-S
raf ODN, mice were randomly divided into 3 groups, Three mice in
each group received intratumoral injections of 4 mg/kg LE-ATG-AS
raf ODN on the right flank, and LE-ATG-S raf ODN on the left flank.
The ODN was administered intratumorally daily for 7 days. Control
groups received normal saline or blank liposomes. Mice were
sacrificed 24 hours after the last dose of ODN, and the organs were
rapidly excised, rinsed in ice-cold normal saline and stored at
-70.degree. C. until analysis. Raf-1 protein expression was
analyzed from tissue homogenates by immunoprecipitation and
immunoblotting and guantified using the ImageQuant computer program
as described above.
Results
Effects of raf ODNs on Cell Viability and Cell Cycle Distribution,
and Intracellular Uptake
[0023] ODNs sequences were directed towards the translation
initiation site of human c-raf-1 cDNA (15-mer,
ends-phosphorothioated, S/As; 25-mer, fully-phosphorothioated,
S-F/As-F). Since cell survival in response to .gamma.-radiation is
regulated during the cell cycle, and since several reports have
suggested the possibility of a cell cycle control of Raf-1 protein
kinase activity, it was important to first determine the effects of
As-ODNs on the cell cycle distribution pattern of SQ-20B cells.
Logarithmically growing cells were exposed to 25-100 pmol/.mu.l of
various ODNs for 4-12 hr, and the cell viability and cell cycle
distribution patterns were measured. Sense and antisense ODNs
showed toxicity as compared to the control cells in 1% FBS-medium
without oligos. The cell cycle distributions of ODNs-treated cells
were also found to differ as compared to control cells. These data
are in general agreement with previous reports suggesting
non-specific effects due to the introduction of oligos per se.
Remarkably, ends-modified As-ODNs was neither cytotoxic nor had any
significant effect on the cell cycle distribution profile of SQ-20B
cells as compared to sense ODNs (S-ODNs). Similar observations were
made with As-F and S-F ODNs. The intracellular uptakes of ODNs were
examined. A linear increase in the intracellular level of ODNs was
seen at between 2-12 hr post-treatment. Approximately 4%
(equivalent to 4 pmol/.mu.l) of the total extracellular or applied
ODNs was taken up by these tumor cells by 12 hr. Intracellular ODNs
were also found to be stable as 15-mers.
Specificity of Inhibition of Raf-1.
[0024] To ascertain the specificity of inhibition of Raf-1 protein
kinase by raf As-ODNs, all experiments were performed, in parallel,
using two controls: i) cells were exposed to raf S-ODNs using
treatment conditions identical to As-ODNs to rule out the influence
of non-specific effects due to ODNs; and ii) control cells were
treated with 1% FBS containing medium (without ODNs) to determine
the base-line levels of the Raf-1 expression and enzymatic activity
in SQ-20B cells.
[0025] The dose-dependence and time-course of inhibition of Raf-1
protein expression by As-ODNs was studied using a combination of
Raf-1 immunoprecipitation and immunoblotting assays. Densitometric
analysis of the Raf-1 band (.sup..about.75 kDa) showed that about
40% inhibition of Raf-1 protein expression occurred at 12 hr when
100 pmol/.mu.l As-ODNs was applied to the cells. Raf-1 protein
expression in the S-ODNs-treated cells (extracellular 100
pmol/.mu.l, 12 hr) was found to be identical to the expression in
untreated control cells, and in cells exposed to 25 pmol/.mu.l
extracellular As-ODNs. Specificity of the Raf-1 antibody was
confirmed by elimination of the .sup..about.75 kDa band observed in
control cells when immunoprecipitation was performed with
antigen-blocked antibody. Time-course experiments revealed that
approximately 50% inhibition of Raf-1 protein was achieved by 12 hr
post-incubation with 100 pmol/.mu.l extracellular As-ODNs. The
inhibitory effect of As-ODNs appeared to diminish by 18 hr. This
recovery of the Raf-1 expression may be attributed to the apparent
degradation of As-ODNs over time and to the synthesis of new
protein. Nevertheless, three independent studies indicated that
approximately 50% inhibition of Raf-1 protein occurred by 12 hr in
the As-ODNs-treated cells as compared to cells treated with
equimolar concentration of S-ODNs.
[0026] Having established 100 pmol/.mu.l as a non-toxic and
inhibitory extracellular dose of raf As-ODNs in SQ-20B cells
relative to the equimolar concentration of S-ODNs, the effect of
As-ODNs on in vitro phosphotransferase activity of Raf-1 protein
kinase was examined. Consistent with immunoblotting data,
approximately 50% inhibition of the in vitro Raf-1 protein kinase
activity was noted in Raf-1 immunoprecipitates of As-ODNs-treated
cells as compared to the S-ODNs-treated cells (applied ODNs: 100
pmol/.mu.l, 12 hours) and the untreated control cells. Experiments
were also performed to measure the Raf-1 immune-complex-associated
in vitro kinase activity in tumor cells exposed to the fully
modified ODNs (S-F/As-F, applied dose 100 pmol/.mu.l, 12 hr).
Inhibition of Raf-1 activity was observed in the As-F ODNs-treated
cells as compared to the S-F ODNs response. It is noteworthy that
the expression and activity of Raf-1 were observed to be similar in
the control (C, without oligo) and raf S-ODNs-treated cells (S).
This finding along with the concurrent inhibition of the expression
and activity of Raf-1 noted in the raf As-ODNs-treated cells (As),
implied that the inhibition of Raf-1 protein kinase was
sequence-specific, and not due to the non-specific effects of
ODNs.
raf As-ODNs is a Biologic Radiosensitizer of SQ-20B Cells
[0027] Radiation survival dose responses of SQ-20B cells exposed to
S- and As-ODNs (100 pmol/.mu.l, 12 hr) were evaluated. (S- and
As-ODNs used in this study do not contain the G-quartet or CpG
motifs previously shown to be responsible for
non-antisense-specific effects such as enhanced affinity for
protein or interference with the immune response.) The plating
efficiencies indicated that the As-ODNs treatment had no effect on
cell viability as compared to S-ODNs-treated cells (Table 1). These
data are also in agreement with the S- and As-ODNs effects on the
viability of logarithmically growing cells discussed earlier.
Radiation survival dose responses of the control (without oligo)
and S-ODNs-treated cells were almost identical. Most important,
As-ODNs treatment resulted in decreases of the shoulder and the
slope of the survival curve. The radiobiological parameters were
obtained by fitting the data (surviving number of colonies) to the
single-hit multitarget (D.sub.0, D.sub.q, n) and linear-quadratic
(.alpha., .beta.) models of radiation survival response. In
addition, the value of a model-free parameter, mean inactivation
dose (D) was calculated (14) (Table 1). Based on a ratio of the
mean inactivation dose, the dose modifying factor (DMF) of As-ODNs
treatment was .sup..about.1.4. Significant decreases observed in
the values of radiobiological parameters, D, D.sub.q, and D.sub.0
of SQ-20B cells following treatment with the raf As-ODNs indicate a
good correlation between the DNA sequence-specific inhibition of
Raf-1 protein kinase and the radiosensitization of these relatively
radioresistant tumor cells.
[0028] The studies indicated that greater than 50% inhibition of
Raf-1 expression could be achieved with only 10 .rho.mol.mu.l of
the liposome-encapsulated raf As-ODNs.
Liposomal Encapsulation Protects ATG-AS raf ODN In Vivo
[0029] The plasma concentration-time profile of LE-ATG-AS raf ODN
was studied. Dosage of 30 mg/kg LE-ATG-AS raf ODN or ATG-AS raf ODN
was administered i.v. in Balb/c nu/nu mice. Blood samples were
collected from retro-orbital sinus at indicated times after
injection and the ODN in plasma samples was extracted by
phenol:chloroform. The samples were electrophoresed on 20%
polyacrylamide/8 M urea gel and electroblotted on nylon membrane.
The blots were probed with .sup.32P-labeled ATG-S raf ODN.
Auto-radiographs were scanned using a computer program (ImageQuant
software version 3.3, Molecular Dynamics). St, standard prepared by
spiking known concentration of ATG-AS raf ODN in blank plasma.
Quantification data were calculated based on a known concentration
of the standard sample, and then normalized against dilution
factors at various time points.
[0030] Following intravenous administration, the peak plasma
concentration of 6.39 .mu.g/ml was achieved and intact ODN could be
detected up to 24 h. The decrease in plasma concentration of
LE-ATG-AS raf ODN followed a biexponential pattern with an initial
half-life (t.sub.1/2.alpha.) of 24.5 min and a terminal half-life
(t.sub.1/2.beta.) of 11.36 h. The area under the plasma
concentration-time curve for LE-ATG-AS raf ODN was 5.99 .mu.gh/ml,
with total body clearance of 75.94 ml/min/kg and volume of
distribution of 74.67 L/kg. In contrast, intact free ODN (ATG-AS
raf ODN) was detectable only at 5 min; the plasma concentration
being 9.75 .mu.g/ml. These observations indicate that ATG-AS ODN
was either rapidly cleared from the circulation, or extensively
degraded in plasma due to nuclease activity.
[0031] Normal tissue distribution profiles of LE-ATG-AS raf ODN
were studied. Tissue samples were collected at indicated times
after i.v. administration of 30 mg/kg LE-ATG-AS raf ODN. ODN was
extracted from homogenized tissues using a DNA extraction kit
(Stratagene). Samples were electrophoresed and electroblotted. The
blots were probed with .sup.32P-labeled ATG-S raf ODN and
autoradiographs were analyzed. Quantification data were calculated
based on a known concentration of the standard sample, and then
normalized against the weight of the tissue sample collected.
[0032] Intact ODN could be detected in all organs examined up to 48
hours after administration. Interestingly, following ATG-AS raf ODN
administration, intact ODN could be detected only at 5 minutes
post-administration in all the organs examined and degradation
products were observed at all other times. These findings, along
with the plasma data for LE-ATG-AS raf ODN, suggests that ODN with
only the end bases phosphorothioated is rapidly degraded in vivo
and that liposome encapsulation using the liposomes of the
invention protects it from degradation for at least 48 hours.
TABLE-US-00001 TABLE 1 Radiation survival parameters of SQ-20B
cells treated with raf oligodeoxyribonucleotides No. of D.sub.0 Dq
.alpha. .beta. D raf ODNs Expts. (Gy) (Gy) n (Gy.sup.-1)
(Gy.sup.-2) (Gy) Sense/Control* 6 3.028 1.265 1.519 0.2373 0.0050
3.694 Antisense 3 2.374 0.551 1.261 0.3614 0.0038 2.628
[0033] The appropriate number of cells were seeded into two replica
T-25 flasks per dose in each experiment. Control cells were
cultured in medium containing 1% FBS without oligo. Plating
efficiencies of the S-ODNs-treated, As-ODNs-treated and control
cells were in the range of 13-58%, 28-61% and 41-67%, respectively.
Clonogenic survival data were computer-fitted to the single-hit
multitarget and the linear-quadratic models of radiation survival
dose response. Composite values of the various parameters were
obtained from the three experiments performed with the
S-ODNs-treated and the three experiments using the control
cells.
Specificity of Inhibition of Raf-1 Protein Expression and Activity
In Vitro
[0034] Initially, the possibility of cytotoxic effects of liposomes
in SQ-20B cells was examined. Blank liposomes, at concentration
equivalent to 10 .mu.M LE-ATG-AS raf ODN, were found to be
non-cytotoxic as determined by the clonogenic and trypan blue dye
exclusion methods. However, blank liposomes showed cytotoxicity at
doses higher than 20 .mu.M. Therefore, a dose of 10 .mu.M or less
was used for in vitro experiments. Further, 10 .mu.M LE-ATG-AS raf
ODN or LE-ATG-S raf ODN was non-toxic to SQ-20B cells.
[0035] Specificity of inhibition of Raf-1 protein expression by
LE-ATG-AS raf ODN. Logarithmically growing SQ-20B cells were
treated with 10 .mu.M LE-ATG-AS raf ODN (AS), 10 .mu.M LE-ATG-S raf
ODN (S) or blank liposomes (BL) for indicated time in 1% FBS
containing medium. Untreated control cells (C) were simultaneously
switched to 1% FBS containing medium for 8 hours. Whole cell lyates
were normalized for total protein content and immunoprecipitated
with agarose-conjugated polyclonal anti-Raf-1 antibody (Santa
Cruz). Immune-complexes were resolved on 7.5% SDS-PAGE and Raf-1
protein expression was detected by immuno-blotting with polyclonal
anti-Raf-1 antibody (Santa Cruz), followed by the ECL detection
protocol (Amersham). Results from three independent experiments
were quantified using a computer program (ImageQuant, Molecular
Dynamics), and data are expressed relative to the level of Raf-1 in
LE-ATG-S raf ODN-treated cells (bottom). Panel B: Dose-response
analysis. Logarithmically growing SQ-20B tumor cells were treated
with indicated concentrations of LE-ATG-AS raf ODN (AS) or LE-ATG-S
raf ODN (S) in 1% FBS containing medium for 8 h. Normalized cell
lysates were analyzed for Raf-1 protein expression.
[0036] Logarithmically growing SQ-20B cells were treated with 10
.mu.M LE-ATG-AS raf ODN (AS), or 10 .mu.M LE-ATG-S raf ODN (S) for
8 h in 1% FBS containing medium. Control cells (C) were
simultaneously switched to 1% FBS containing medium for 8 hours.
Whole cell lysates were normalized for protein content, and Raf-1
was immunoprecipitated. Phosphotransferase activity of Raf-1 in
immune-complexes was assayed in vitro using a physiologic substrate
MKK1. Radiolabeled reaction products were separated by
electrophoresis and autoradiographed.
[0037] Time-course experiments revealed that a maximum inhibition
(52.3.+-.5.7%) of Raf-1 protein expression (.sup..about.74 kDa)
occurred at 8 hours post-incubation of cells with 10 .mu.M
LE-ATG-AS raf ODN. The inhibitory effect of LE-ATG-AS raf ODN was
maintained up to 24 h (45.6.+-.9.8%). The level of Raf-1 protein
was comparable in the control untreated cells (C), blank
liposome-treated cells (BL), and LE-ATG-S ODN-treated cells,
indicating the LE-ATG-AS raf ODN specifically inhibited the Raf-1
protein expression in SQ-20B cells. Dose response studies showed
that 35.94.+-.16.8% and 52.3.+-.5.7% inhibition of Raf-1 expression
occurred with 5 .mu.M and 10 .mu.M LE-ATG-AS raf ODN treatment for
8 hours.
[0038] The effects of LE-ATG-AS raf ODN on the enzymatic activity
of Raf-1 protein kinase using mitogen-activated protein kinase
kinase (MKK1) as substrate were studied. In concurrence with Raf-1
protein inhibition data, it was found that 10 .mu.M LE-ATG-AS raf
ODN treatment for 8 hours inhibited 62.6.+-.9.0% in vitro
phosphotransferase activity of Raf-1 protein. LE-ATG-S raf ODN did
not have any effect on the Raf-1 protein kinase activity as
compared with the untreated control cells.
LE-ATG-AS raf ODN is a Biological Radiosensitizer
[0039] Radiation survival dose responses of SQ-20B cells exposed to
LE-ATG-AS raf ODN, LE-ATG-S raf ODN, and blank liposomes are
presented in Table 2. Comparison of the radiation survival dose
response of SQ-20B cells treated with LE-ATG-AS raf ODN (AS),
LE-ATG-S raf ODN (S), or blank liposomes (BL). The appropriate
number of cells were seeded in duplicate to obtain 40-60 colonies
per T-25 flask (Costar) for each radiation dose. The clonogenic
survival data were computer-fitted to the single-hit multitarget
model of radiation survival dose response. Representative data from
one experiment performed for each treatment category were
evaluated.
[0040] The plating efficiencies of cells treated with S/AS ODN or
blank liposomes were comparable (Table 2). Radiation survival dose
responses of the blank liposome-treated (BL) and LE-ATG-S raf
ODN-treated cells were also comparable. LE-ATG-AS raf ODN treatment
resulted in a significant radiosensitization (Table 2). Based on a
ratio of the mean inactivation dose, the dose modifying factor
(DMF) of LE-ATG-AS raf ODN treatment was -1.6. Significant
decreases observed in the values of radiobiological parameters,
{overscore (D)}, D.sub.q, and D.sub.0 of SQ-20B cells following
treatment with the LE-ATG-AS raf ODN indicate a good correlation
between the DNA sequence-specific inhibition of Raf-1 protein
kinase and the radiosensitization of these relatively
radioresistant tumor cells.
LE-ATG-AS raf ODN is a Specific Inhibitor of Raf-1 Protein
Expression in Solid Tumor
[0041] The effects of intratumoral administration of LE-ATG-AS raf
ODN and LE-ATG-S raf ODN on the expression of Raf-1 protein was
examined in a SQ-20B tumor xenograft model. Mice with tumors on
both flanks received intratumorally LE-ATG-AS raf ODN on the right
flank, and LE-ATG-S ODN on the left flank. Results demonstrated a
significant inhibition of Raf-1 protein in tumor tissue following
treatment with LE-ATG-AS raf ODN compared with LE-ATG-S raf ODN
(60.3.+-.6.4%).
Inhibition of Raf-1 Protein Expression by LE-ATG-AS raf ODN in
SQ-20B Tumor Xenografts
[0042] SQ-20B tumors were established subcutaneously in both hind
limbs of nude mice, Balb C nu/nu (mean tumor volume 115 mm.sup.3)
Each animal then received intratumoral injections of LE-ATG-AS raf
ODN (AS) on the right flank and LE-ATG-S raf ODN (S) on the left
flank at a dose of 4 mg/kg daily for 7 days. Tumor tissue was
excised 24 h after the last treatment, and Raf-1 protein expression
in tumor samples was analyzed. ECL images were quantified using a
computer program (ImageQuant, Molecular Dynamics), and
quantification data from three mice are expressed as the level of
Raf-1 protein expression in LE-ATG-AS raf ODN-treated tumors
relative to LE-ATG-S raf ODN-treated tumors.
[0043] Liposomes prepared in accord with the teachings of the
invention are non-toxic both in culture and in animals. While only
specific liposomes are disclosed herein, the novel carriers may be
used for delivery of a variety of DNA-based compounds for delivery.
TABLE-US-00002 TABLE 2 Radiation Survival Parameters of SQ-20B
Cells Treated with LE-ATG-S/AS raf ODN No. of raf ODN Expts.
D.sub.0(Gy) D.sub.q(Gy) {overscore (n)} .alpha.(Gy.sup.-1)
.beta.(Gy.sup.-2) {overscore (D)}(Gy) Blank liposomes/ 5 2.795 .+-.
0.38 1.445 .+-. 1.22 2.012 .+-. 1.34 0.2184 .+-. 0.11 0.0087 .+-.
0.00 3.659 .+-. 0.02 LE-ATG-S* LE-ATG-AS 3 2.287 .+-. 0.23 0.051
.+-. 0.05 1.021 .+-. 0.19 0.4385 .+-. 0.05 0.0000 .+-. 0.00 2.280 +
0.00
The appropriate number of cells were seeded in duplicate T25 flasks
per dose in each experiment. Plating efficiencies of the blank
liposome-treated, LE-ATG-S raf ODN-treated, and LE-ATG-AS raf
ODN-treated cells were in the range of 65-79%, 52-83%, and 59-90%
respectively. Clonogenic survival data were computer-fitted to the
single-hit multitarget and the linear-quadratic models of radiation
survival dose response. *Composite values of the various parameters
were obtained from the three experiments performed with LE-ATG-S
raf ODN-treated cells and two experiments performed with the blank
liposome-treated cells.
[0044] The liposomes of the invention provide significant
protection of antisense oligonucleotides against degradation in
blood and normal tissue. The formulation may replace the need for
complete modification of all bases of the antisense
oligonucleotides for therapeutic uses. Compositions comprising
oligonucleotides may be administered in many ways, depending on the
target tissue.
[0045] The particular method used to deliver compositions of the
invention to the tissues of the intact animal will depend on the
particular tissue to which it is administered. For example,
compositions of the invention can be administered intrathecally to
facilitate contact of the active agent with neuronal tissue. For
administration to the lung, the liposomal compositions may be
administered transbronchially as a spray or mist. Liposomal
compositions may also be administered to tissue locally during
surgery. For example, the compositions could be administered into
the peritoneal cavity as a mist during surgery.
[0046] The liposomes may also be injected into the target tissue or
into the arterial blood supply to the target tissue. When the
target tissue is the lining of a hollow organ, they may be
introduced into the lumen of the organ.
[0047] The dosage required will depend on the agent and the subject
being treated with the liposomal compositions. For example, when a
radiosensitizing oligonucleotide is administered by means of
liposomes, a radiosensitizing amount of oligonucleotides must reach
the target organ.
[0048] The radiosensitizing oligonucleotides may also be
administered systemically. A preferred method of administration is
by intravenous injection.
[0049] Acceptable carriers include, for example, glucose, saline,
phosphate buffered saline. Carriers may also contain other
substances frequently found in pharmaceuticals such as
preservatives, emulsifiers and surfactants.
[0050] Dosage will depend on the extent to which it is possible to
present the active agents to the target tissue. The appropriate
dosage should deliver a serum concentration of about 1 .mu.g/ml to
1000 .mu.g/ml. In some instances, this dosage can be delivered into
the target tissue directly. Hence, that level of dosage need not be
achieved in the total blood volume.
Sequence CWU 1
1
3 1 15 DNA Artificial Synthetic 1 gtgctccatt gatgc 15 2 25 DNA
Artificial Synthetic 2 cctgtatgtg ctccattgat gcagc 25 3 15 DNA
Artificial Synthetic 3 gcatcaatgg agcac 15
* * * * *