U.S. patent application number 10/125181 was filed with the patent office on 2002-12-12 for insulin-like growth factor ii antisense oligonucleotide sequences and methods of using same to modulate cell growth.
This patent application is currently assigned to GENESENSE TECHNOLOGIES INC.. Invention is credited to Lee, Yoon S., Wright, Jim A., Young, Aiping H..
Application Number | 20020187954 10/125181 |
Document ID | / |
Family ID | 22173486 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187954 |
Kind Code |
A1 |
Wright, Jim A. ; et
al. |
December 12, 2002 |
Insulin-like growth factor II antisense oligonucleotide sequences
and methods of using same to modulate cell growth
Abstract
This invention relates to oligonucleotides complementary to the
IGF-II genes which modulate tumor cell growth in mammals This
invention is also related to methods of using such compounds in
inhibiting the growth of tumor cells in mammals This invention also
relates to pharmaceutical compositions comprising a
pharmaceutically acceptable excipient and an effective amount of a
compound of this invention.
Inventors: |
Wright, Jim A.; (Toronto,
CA) ; Young, Aiping H.; (Toronto, CA) ; Lee,
Yoon S.; (Don Mills, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
GENESENSE TECHNOLOGIES INC.
|
Family ID: |
22173486 |
Appl. No.: |
10/125181 |
Filed: |
April 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10125181 |
Apr 17, 2002 |
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09295593 |
Apr 22, 1999 |
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6417169 |
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60082791 |
Apr 23, 1998 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 35/00 20180101; C12N 15/1136 20130101; A61K 38/00 20130101;
C12N 2310/111 20130101; C12N 2310/315 20130101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 048/00 |
Claims
What is claimed:
1. A method for inhibiting the growth of a human tumor comprising,
administering to a human suspected of having the tumor an effective
amount of an antisense oligonucleotide comprising from about 20 to
100 nucleotides comprising a sequence selected from the group
consisting of SEQ ID NOs:17-31 in Table 2 under conditions such
that the growth of the tumor is inhibited.
2. The method according to claim 1 wherein the oligonucleotide is
nuclease resistant.
3. The method according to claim 1 further comprising the step of
administering to the human a chemotherapeutic agent.
4. A method for inhibiting the metastasis of a human tumor
comprising, administering to a human suspected of having a
metastatic tumor an effective amount of an antisense
oligonucleotide from about 20 to 100 nucleotides comprising a
sequence complementary to the 5' untranslated region consisting of
exons 4, 5 or 6 of human fetal IGF-II mRNA under conditions such
that metastasis of the tumor is inhibited.
5. The method according to claim 4 further comprising the step of
administering to the human a chemotherapeutic agent.
6. The method according to claim 4 wherein the oligonucleotide is
nuclease resistant.
7. The method according to claim 4 wherein the oligonucleotide
comprises a sequence selected from the group consisting of SEQ ID
NOs:1-15.
8. A method for inhibiting the metastasis of a human tumor
comprising, administering to a human suspected of having a
metastatic tumor an effective amount of an antisense
oligonucleotide from about 20 to 100 nucleotides comprising a
sequence selected from the group consisting of SEQ ID NOs:17-31
under conditions such that metastasis of the tumor is
inhibited.
9. The method according to claim 8 further comprising the step of
administering to the human a chemotherapeutic agent.
10. The method according to claim 9 wherein the oligonucleotide is
nuclease resistant.
11. The method according to claim 4, wherein the antisense
oligonucleotide comprises from about 20 to about 50 nucleotides.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/082,791 filed Apr. 23, 1998, which
application is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] This invention relates to oligonucleotides that are
complementary to mammalian insulin-like growth factor II (IGF II)
genes which oligonucleotides modulate tumor cell growth in mammals.
This invention is also related to methods of using such compounds
in inhibiting the growth of tumor cells in mammals. This invention
also relates to pharmaceutical compositions comprising a
pharmaceutically acceptable excipient and an effective amount of a
compound of this invention.
REFERENCES
[0004] The following publications, patent applications and patents
are cited in this application:
[0005] 1. Toretsky, J. A. and Helman, L. J. Involvement of IGF-II
in human cancer, J Endocrinol. 149: 367-72, 1996.
[0006] 2. Werner, H. and LeRoith, D. The role of the insulin-like
growth factor system in human cancer, Adv Cancer Res. 68: 183-223,
1996.
[0007] 3. Rogler, C. E., Yang, D., Rossetti, L., Donohoe, J., Alt,
E., Chang, C. J., Rosenfeld, R., Neely, K., and Hintz, R. Altered
body composition and increased frequency of diverse malignancies in
insulin-like growth factor-II transgenic mice, J Biol Chem. 269:
13779-84, 1994.
[0008] 4. Bates, P., Fisher, R., Ward, A., Richardson, L., Hill, D.
J., and Graham, C. F. Mammary cancer in transgenic mice expressing
insulin-like growth factor II (IGF-II) [see comments], Br J Cancer.
72: 1189-93, 1995.
[0009] 5. Cullen, K. J., Lippman, M. E., Chow, D., Hill, S., Rosen,
N., and Zwiebel, J. A. Insulin-like growth factor-II overexpression
in MCF-7 cells induces phenotypic changes associated with malignant
progression, Mol Endocrinol. 6: 91-100, 1992.
[0010] 6. Werner, H., Adamo, M., Roberts, C. T., Jr., and LeRoith,
D. Molecular and cellular aspects of insulin-like growth factor
action, Vitam Horm. 48: 1-58, 1994.
[0011] 7. Curcio, L. D., Bouffard, D. Y., and Scanlon, K. J.
Oligonucleotides as modulators of cancer gene expression, Pharmacol
Ther. 74: 317-32, 1997.
[0012] 8. Narayanan, R. and Akhtar, S. Antisense therapy, Curr Opin
Oncol. 8: 509-15, 1996.
[0013] 9. Ho, P. T. and Parkinson, D. R. Antisense oligonucleotides
as therapeutics for malignant diseases, Semin Oncol. 24: 187-202,
1997.
[0014] 10. Crooke, S. T. and Bennett, C. F. Progress in antisense
oligonucleotide therapeutics, Annu Rev Pharmacol Toxicol. 36:
107-29, 1996.
[0015] 11. Christofori, G., Naik, P., and Hanahan, D. A second
signal supplied by insulin-like growth factor II in oncogene-
induced tumorigenesis, Nature. 369: 414-8, 1994.
[0016] 12. El-Badry, O. M., Minniti, C., Kohn, E. C., Houghton, P.
J., Daughaday, W. H., and Helman, L. J. Insulin-like growth factor
II acts as an autocrine growth and motility factor in human
rhabdomyosarcoma tumors, Cell Growth Differ. 1: 325-31, 1990.
[0017] 13. Kim, K. W., Bae, S. K., Lee, O. H., Bae, M. H., Lee, M.
J., and Park, B. C. Insulin-like growth factor II induced by
hypoxia may contribute to angiogenesis of human hepatocellular
carcinoma, Cancer Res. 58: 348-51, 1998.
[0018] 14. Volpert, O., Jackson, D., Bouck, N., and Linzer, D. I.
The insulin-like growth factor II/mannose 6-phosphate receptor is
required for proliferin-induced angiogenesis, Endocrinology. 137:
3871-6, 1996.
[0019] 15. Lin, S. B., Hsieh, S. H., Hsu, H. L., Lai, M. Y., Kan,
L. S., and Au, L. C. Antisense oligodeoxynucleotides of IGF-II
selectively inhibit growth of human hepatoma cells overproducing
IGF-II, J Biochem (Tokyo). 122: 717-22, 1997.
[0020] 16. Steller, M. A., Delgado, C. H., Bartels, C. J.,
Woodworth, C. D., and Zou, Z. Overexpression of the insulin-like
growth factor-1 receptor and autocrine stimulation in human
cervical cancer cells, Cancer Res. 56: 1761-5, 1996.
[0021] 17. Steller, M. A., Delgado, C. H., and Zou, Z. Insulin-like
growth factor II mediates epidermal growth factor-induced
mitogenesis in cervical cancer cells, Proc Natl Acad Sci U S A. 92:
11970-4, 1995.
[0022] 18. Choy et al., "Molecular mechanisms of drug resistance
involving ribonucleotide reductase: hydroxyurea resistance in a
series of clonally related mouse cell lines selected in the
presence of increasing drug concentrations" Cancer Res.
48:2029-2035 (1988)
[0023] 19. Fan et al., "Ribonucleotide reductase R2 component is a
novel malignancy determinant that cooperates with activated
oncogenes to determine transformation and malignant potential"
Proc. Natl. Acad. Sci USA 93:14036-40 (1996)
[0024] 20. Huang and Wright, "Fibroblast growth factor mediated
alterations in drug resistance and evidence of gene amplification"
Oncogene 9:491-499 (1994)
[0025] 21. Uhlmann et al. Chem Rev. 90:534-583 (1990)
[0026] 22. Agrawal et al. Trends Biotechnol. 10:152-158 (1992)
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Company, Philadelphia Pa. 17.sup.th ed. (1985)
[0028] 24. Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York (1989, 1992)
[0029] 25. Ausubel et al., Current Protocols in Molecular Biology,
John Wiley and Sons, Baltimore Maryland (1989)
[0030] 26. Perbal, A Practical Guide to Molecular Cloning, John
Wiley & Sons, New York (1988)
[0031] 27. Hurta and Wright, "Malignant transformation by H-ras
results in aberrant regulation of ribonucleotide reductase gene
expression by transforming growth factor-beta" J. Cell Biochem
57:543-556 (1995)
[0032] 28. Dreeley et al., Science, 258:1650-1654 (1992)
[0033] 29. Nielsen et al.; Science (1991) 354:1497
[0034] 30. Good and Nielsen; "Inhibition of translation and
bacterial growth by peptide nucleic acid targeted to ribosomal
RNA", PNAS USA (1998) 95:2073-2076
[0035] 31. Buchardt, deceased, et al., U.S. Pat. No. 5,766,855
[0036] 32. Buchardt, deceased, et al., U.S. Pat. No. 5,719,262
[0037] 33. U.S. Pat. No. 5,034,506
[0038] 34. Altschul, et al. "Basic local alignment search tool", J.
Mol. Biol. (1990) 215:403-10;
[0039] 35. Devereux J. et al., "A comprehensive set of sequence
analysis programs for the VAX", Nucleic Acids Res. (1984)
12:387-395;
[0040] 36. Chang et al.; Somatic Gene Therapy, CRC Press, Ann Arbor
Mich. (1995);
[0041] 37. Vega et al.; Gene Targeting, CRC Press, Ann Arbor Mich.
(1995)
[0042] 38. Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Butterworths, Boston Mass. (1988)
[0043] 39. Sullivan, U.S. Pat. No. 5,225,347
[0044] 40. U.S. Pat. No. 5,023,252, issued Jun. 11, 1991
[0045] 41. Feigner et al., U.S. Pat. No. 5,580,859
[0046] All of the above publications, patent applications and
patents are herein incorporated by reference in their entirety to
the same extent as if each individual publication, patent
application or patent was specifically and individually indicated
to be incorporated by reference in its entirety.
[0047] State of the Art
[0048] Insulin-like growth factor II (IGF-II) is a 67 amino acid
polypeptide growth factor that is widely expressed in the
developing human embryonic tissues and is related to the growth and
differentiation of various tissues. After birth, the expression is
progressively extinguished in almost all human tissues. In adult
humans, serum levels of approximately 100 ng/ml are mainly produced
by the liver. The biological functions of IGF-II are mediated
through its binding to either the IGF-II receptor (related to
carbohydrate metabolism, motility of malignant cells and/or
tumor-induced angiogenesis) or the IGF-I receptor (related to
signal transduction pathway and mitogenesis).
[0049] IGF-II has been implicated in tumor progression and
metastasis by a variety of mechanisms in many tumors (reviewed in
(1, 2)). Tumors with extensive involvement of IGF-II include
childhood tumors such as rhabdomyosarcoma, Wilms' tumor and
neuroblastoma. These tumors demonstrate overexpression of IGF-II,
show existence of a paracrine or autocrine loop and result in
inhibition of tumor growth or metastasis upon blockage of the loop.
IGF-II contributes to tumor growth and metastasis to varying
degrees in a variety of tumors including osteosarcoma, breast
carcinoma, hepatoblastoma, germ cell tumors, hepatocellular
carcinoma, adrenocortical carcinoma, lung tumors, leiomyosarcoma,
brain tumors and colon carcinoma. Furthermore, the direct role of
IGF-II in oncogenesis has been elucidated by transgenic mice and
human cell lines overexpressing it (3-5).
[0050] The human IGF-II gene is located on chromosome 11p15 just
downstream of insulin gene and spans 30 kb (reviewed in (6);see
FIG. 1). It consists of 9 exons of which exons 7, 8 and part of 9
encode a precursor protein. Exons 1, 4, 5, and 6 are each preceded
by distinct promoters P1, P2, P3 and P4. Promoter P1 is active only
in adult liver, while P2-4 are active in most fetal tissues. There
are a few adult tissues that express low amount of transcripts from
P2, 3 and 4 (fetal transcripts). Four major mRNA species (6 Kb,
4.8-5 kb and 2.2 kb for fetal transcripts and 5.3 kb for adult
transcript) have been identified which are generated from distinct
promoters and by differential splicing. It appears that
overexpression of IGF-II observed in various primary cancers and
cell lines results from reactivation (in liver) or overexpression
(in other organs) of fetal mRNA species whose expression is mainly
derived from P3 and P4. These fetal transcripts contain unique 5'
untranslated regions (5'UTR containing exons 4 or 5 or 6) that are
absent in the adult transcript derived from P1(5'UTR containing
exons 1, 2 and 3).
[0051] Antisense oligonucleotides (AS-ODNs) have been widely
utilized to inhibit gene expression in a target-specific manner by
sequence-specific hybridization to target mRNA. In numerous
studies, antisense oligonucleotide-mediated repression of oncogenes
has revealed that these compounds are not only extremely useful for
delineating biochemical mechanisms governing oncogenesis (7), but
also considerably promising as novel therapeutic compounds for the
treatment of human cancer (8, 9). In addition, relatively less
toxicity has been attributed to oligonucleotide-based therapeutics
(10).
[0052] A few studies (11, 15-17) have shown that certain antisense
oligonucleotides targeted against human or mouse adult IGF-II
transcripts were effective in interfering with tumor cell
proliferation in vitro. In one study (15), the suppression of
IGF-II production by an antisense oligonucleotide targeting the
translation start site of human adult transcript has resulted in
growth inhibition of human hepatocellular carcinoma cell lines,
HuH-7 and HepG2. In another studies (16,17) utilizing human
cervical cancer cell line, an antisense oligonucleotide targeting
the protein coding region of IGF-II was shown to inhibit epidermal
growth factor (EGF)-induced mitogenic effect.
[0053] Therefore, it would be desirable to identify antisense
oligonucleotides directed against IGF-II which act to inhibit the
expression and production of IGF-II with higher specificity and
with less toxicity.
SUMMARY OF THE INVENTION
[0054] This invention is directed to antisense oligonucleotides
which modulate the expression of the IGF-II genes and production of
IGF-II in mammals and pharmaceutical compositions comprising such
antisense oligonucleotides. This invention is also related to
methods of using such antisense oligonucleotides for inhibiting
tumor growth and metastasis in mammals.
[0055] Accordingly, in one of its composition aspects, this
invention is directed to an antisense oligonucleotide, which
oligonucleotide from about 3 to about 100 nucleotides comprising
nucleotides complementary to the mammalian fetal IGF-II mRNA. The
antisense oligonucleotide may be nuclease resistant and may have
one or more phosphorothioate internucleotide linkages. The
antisense oligonucleotide may further comprise additional
nucleotides which are not complementary to the IGF-II mRNA. The
oligonucleotides may comprise a sequence selected from group
consisting of SEQ ID NOs:1 to 15 from Table 1.
[0056] This invention is also directed to an antisense
oligonucleotide, which oligonucleotide from about 20 to about 100
nucleotides comprising nucleotides complementary to the mammalian
adult IGF-II mRNA selected from the group consisting of SEQ ID
NOs:17-31 from Table 2.
[0057] In another of its composition aspects, this invention is
directed to a vector comprising an antisense oligonucleotide
sequence from about 3 to 100 nucleotides comprising a sequence
complementary to the 5' untranslated region of mammalian fetal
IGF-II mRNA.
[0058] In another of its composition aspects, this invention is
directed to a vector comprising an antisense oligonucleotide
sequence from about 20 to 100 nucleotides comprising a sequence
selected from the group consisting of SEQ ID NOs:1 7-31 in Table
2.
[0059] In still another of its composition aspects, this invention
is directed to a pharmaceutical composition comprising a
pharmaceutically acceptable excipient and an effective amount of an
antisense oligonucleotide from about 3 to about 100 nucleotides
comprising nucleotides complementary to the mammalian fetal IGF-II
mRNA. The oligonucleotides may comprise a sequence selected from
group consisting of SEQ ID NOs:1 to 15 from Table 1.
[0060] In still another of its composition aspects, this inveniton
is directed to a pharmaceutical composition comprising a
pharmaceutically acceptable excipient and an effective amount of an
antisense oligonucleotide from about 20 to about 100 nucleotides
comprising a sequence selected from the group consisting of SEQ ID
NOs:17-31 from Table 2.
[0061] In one of its method aspects, this invention is directed to
a method for inhibiting the growth of a mammalian tumor comprising,
administering to a mammal suspected of having the tumor an
effective amount of an antisense oligonucleotide from about 3
nucleotides to about 100 nucleotides complementary to mammalian
fetal IGF-II mRNA under conditions such that the growth of the
tumor is inhibited. The antisense oligonucleotide may be
administered with a chemotherapeutic agent. The oligonucleotide may
comprise a sequence selected from group consisting of SEQ ID NOs:1
to 15 from Table 1.
[0062] This invention is also directed to a method for inhibiting
the growth of a mammalian tumor comprising, administering to a
mammal suspected of having the tumor an effective amount of an
antisense oligonucleotide from about 20 nucleotides to about 100
nucleotides complementary to mammalian adult IGF-II mRNA selected
from the group consisting of SEQ ID NOs:17-31 from Table 2 under
conditions such that the growth of the tumor is inhibited.
[0063] In another of its method aspects, this invention is directed
to a method for inhibiting the metastasis of a mammalian tumor
comprising, administering to a mammal suspected of having a
metastatic tumor an effective amount of an antisense
oligonucleotide from about 3 nucleotides to about 100 nucleotides
complementary to the mammalian fetal IGF-II mRNA under conditions
such that the metastasis of the tumor is inhibited. The antisense
oligonucleotide may be administered with a chemotherapeutic agent.
The oligonucleotides may comprise a sequence selected from group
consisting of SEQ ID NOs:1 to 15 from Table 1.
[0064] This invention is also directed to a method for inhibiting
the metastasis of a mammalian tumor comprising, administering to a
mammal suspected of having a metastatic tumor an effective amount
of an antisense oligonucleotide from about 20 nucleotides to about
100 nucleotides complementary to the mammalian adult IGF-II mRNA
selected from the group consisting of SEQ ID NOs:1 7-31 from Table
2 under conditions such that the metastasis of the tumor is
inhibited.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 is map of the human IGF-II gene and alternatively
transcribed and spiced mRNAs. The numbered boxes (1-9) indicate the
exons of IGF-II gene. Four promoters (P1-P4) are also indicated
with arrows. Various IGF-II mRNA SPecies are depicted in the lower
part of the figure with their corresponding sizes. The solid boxes
represent coding regions of the IGF-II precursor protein.
[0066] FIGS. 2A-D are graphs of the percentage of inhibition of the
colony forming ability of different cell lines by administration of
the indicated antisense oligonucleotides.
[0067] FIG. 2A shows the percentage inhibition of the human
rhabdomyosarcoma cell line RD;
[0068] FIG. 2B shows percentage inhibition of the human prostate
cancer cell line PC-3;
[0069] FIG. 2C shows the percentage inhibition of the human
pancreatic cancer cell line AsPC-1:
[0070] FIG. 2D shows the percentage inhibition of the human
neuroblastoma cell line SK-N-AS.
[0071] FIG. 3 is an autoradiograph of Northern Blots of RNA from
either human neuroblastoma cell line SK-N-AS or rhabdomyosarcoma
cell line (RD) after administration with antisense
oligonucleotides: GTI4006 [SEQ ID NO:6] or GTI4011 [SEQ ID
NO:11]
[0072] FIG. 4 is a photograph of a Western Blot of IGF-II
expression in human neuroblastoma cells after treatment with
different antisense oligodeoxynucleotides.
[0073] FIG. 5 is a photograph of a Western Blot of IGF-II
expression in human rhabdomyosarcoma cells after treatment with
different antisense oligodeoxynucleotides.
[0074] FIG. 6A is a graph of the volume of a tumor following
injection of human neuroblastoma cells (SK-N-AS) in mice with
administration of various antisense oligonucleotides or without
(control).
[0075] FIG. 6B is a graph of the weight of a tumor 20 days after
injection of human neuroblastoma cells (SK-N-AS) in mice with
administration of various antisense oligonucleotides or without
(control).
[0076] FIG. 7A is a graph of the volume of a tumor following
injection of human Wilms' tumor cells (G401) in mice with
administration of various antisense oligonucleotides or without
(control).
[0077] FIG. 7B is a graph of the weight of a tumor 20 days after
injection of human Wilms' tumor cells (G401) in mice with
administration of various antisense oligonucleotides or without
(control).
[0078] FIG. 8 is an autoradiograph of a Northern Blot of IGF-II
mRNA levels in human neuroblastoma (SK-N-AS) tumors following
treatment with antisense oligonucleotide GTI4006 [SEQ ID NO:6].
[0079] FIG. 9 is a photograph of a Western blot of IGF-II protein
levels in human neuroblastoma (SK-N-AS) tumors following treatment
with various antisense oligonucleotides. The band below is a
photograph of the gel stained with India ink to show the total
protein loaded.
[0080] FIG. 10 is a graph of the average number of lung metastases
per mouse by the human melanoma cell line (C8161) after treatment
of the cell line with the various antisense oligonucleotides.
[0081] FIG. 11 is part of the nucleotide sequence of the human
IGF-II gene.
[0082] FIG. 11A is the sequence of exon 4 [SEQ ID NO:34],
[0083] FIG. 11B is the sequence of exon 5 [SEQ ID NO:35],
[0084] FIG. 11C is the sequence of exon 6 [SEQ ID NO:36] and
[0085] FIG. 11D is the sequence of exons 7-9 [SEQ ID NO:37].
DETAILED DESCRIPTION OF THE INVENTION
[0086] This invention relates to oligonucleotides that are
complementary to mammalian IGF II genes which oligonucleotides
modulate tumor cell growth in mammals. It appears that
overexpression of IGF-II observed in various human primary cancers
and cell lines results from reactivation (in liver) or
overexpression (in other organs) of fetal mRNA species.
Accordingly, antisense oligonucleotides designed to specifically
target fetal transcripts in the 5'UTR, leaving adult transcripts
intact, will be highly specific for targeting tumor cells.
[0087] Without being limited to a theory or mechanism, it is
believed that these antisense compounds will exert their antitumor
activity by not only suppressing autocrine growth of tumor cells
and possibly inducing apoptosis, but also inhibiting
autocrine/paracrine function of IGF-II, such as tumor cell motility
and/or induction of endothelial cell migration and
angiogenesis.
[0088] Definitions:
[0089] As used herein, the following terms have the following
meanings:
[0090] The term "antisense oligonucleotide" as used herein means a
nucleotide sequence that is complementary to the desired mRNA. The
antisense oligonucleotide is complementary to any portion of a
mammalian IGF-II mRNA that effectively acts as a target for
inhibiting IGF-II expression. Preferably, the antisense
oligonucleotide is complementary to the 5' untranslated region of
the IGF-II fetal transcript. More preferably, the antisense
oligonucleotide is complementary to the nucleotide sequence of
exons 4, 5 or 6 as set forth in FIGS. 11A-C.
[0091] Without being limited to any theory or mechanism, it is
generally believed that the activity of antisense oligonucleotides
depends on the binding of the oligonucleotide to the target nucleic
acid (e.g. to at least a portion of a genomic region, gene or mRNA
transcript thereof), thus disrupting the function of the target,
either by hybridization arrest or by destruction of target RNA by
RNase H (the ability to activate RNase H when hybridized to
RNA).
[0092] The term "oligonucleotide" refers to an oligomer or polymer
of nucleotide or nucleoside monomers consisting of naturally
occurring bases, sugars, and inter-sugar (backbone) linkages. The
term also includes modified or substituted oligomers comprising
non-naturally occurring monomers or portions thereof, which
function similarly. Such modified or substituted oligomers may be
preferred over naturally occurring forms because of the properties
such as enhanced cellular uptake, or increased stability in the
presence of nucleases. The term also includes chimeric
oligonucleotides which contain two or more chemically distinct
regions. For example, chimeric oligonucleotides may contain at
least one region of modified nucleotides that confer beneficial
properties (e.g. increased nuclease resistance, increased uptake
into cells) or two or more oligonucleotides of the invention may be
joined to form a chimeric oligonucleotide.
[0093] The antisense oligonucleotides of the present invention may
be ribonucleic or deoxyribonucleic acids and may contain naturally
occurring or synthetic monomeric bases, including adenine, guanine,
cytosine, thymine and uracil. The oligonucleotides may also contain
modified bases such as xanthine, hypoxanthine, 2-aminoadenine,
6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo
cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo
uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol
adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other
8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol
guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other
8-substituted guanines, other aza and deaza uracils, thymidines,
cytosines or guanines, 5-trifluoromethyl uracil and 5-trifluoro
cytosine. The modifications may also include attachment of other
chemical groups such as methyl, ethyl, propyl groups to the various
parts of the oligonucleotides including the sugar, base or backbone
components.
[0094] The antisense oligonucleotides of the invention may also
comprise modified phosphorus oxygen heteroatoms in the phosphate
backbone, short chain alkyl or cycloalkyl intersugar linkages or
short chain heteroatom or heterocyclic intersugar linkages. For
example, the antisense oligonucleotides may contain methyl
phosphonates, phosphorothioates, phosphorodithioates,
phosphotriesters, and morpholino oligomers. The antisense
oligonucleotides may comprise phosphorothioate bonds linking
between the four to six 3'-terminus nucleotides. The
phosphorothioate bonds may link all the nucleotides. The
phosphorothioate linkages may be mixed R.sub.P and S.sub.P
enantiomers, or they may be stereoregular or substantially
stereoregular in either R.sub.P or S.sub.P form.
[0095] The antisense oligonucleotides may also have sugar mimetics.
The oligonucleotide may have at least one nucleotide with a
modified base and/or sugar, such as a 2'-O-substituted
ribonucleotide. For purposes of the invention, the term
2'-O-substituted" means substitution of the 2' position of the
pentose moiety with an -O-lower alkyl group containing 1-6
saturated or unsaturated carbon atoms, or with an -O-aryl or allyl
group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl
group may be unsubstituted or may be substituted, e.g., with halo,
hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy,
carboxyl, carbalkoxyl, or amino groups. The oligonucleotides of the
invention may include four or five ribonucleotides 2'-O-alkylated
at their 5' terminus and/or four or five ribonucleotides
2'-O-alylated at their 3' terminus.
[0096] The antisense oligonucleotides of the invention may also
comprise nucleotide analogues wherein the structure of the
nucleotide is fundamentally altered. An example of such an
oligonucleotide analogue is a peptide nucleic acid (PNA) wherein
the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is
replaced with a polyamide backbone which is similar to that found
in peptides (Nielsen et al..sup.29; Good and Nielsen.sup.30;
Buchardt, deceased, et al..sup.31, U.S. Pat. No. 5,766,855;
Buchardt, deceased, et al..sup.32, U.S. Pat. No. 5,719,262). PNA
analogues have been shown to be resistant to degradation by enzymes
and to have extended lives in vivo and in vitro. PNAs also bind
more strongly to a complementary DNA sequence than to a naturally
occurring nucleic acid molecule due to the lack of charge repulsion
between the PNA strand and the DNA strand.
[0097] The oligonucleotides of the present invention may also
include other nucleotides comprising polymer backbones, cyclic
backbones, or acyclic backbones. For example, the nucleotides may
comprise morpholino backbone structures (U.S. Pat. No. 5,034,506
(33)).
[0098] The oligonucleotides of the present invention are "nuclease
resistant" when they have either been modified such that they are
not susceptible to degradation by DNA and RNA nucleases or
alternatively they have been placed in a delivery vehicle which in
itself protects the oligonucleotide from DNA or RNA nucleases.
Nuclease resistant oligonucleotides include, for example, methyl
phosphonates, phosphorothioates, phosphorodithioates,
phosphotriesters, and morpholino oligomers. Suitable delivery
vehicles for conferring nuclease resistance include, for example
liposomes.
[0099] The oligonucleotides of the present invention may also
contain groups, such as groups for improving the pharmacokinetic
properties of an oligonucleotide, or groups for improving the
pharmacodynamic properties of an oligonucleotide.
[0100] The antisense oligonucleotides are preferably selected from
the sequence complementary to the IGF-II gene such that the
sequence exhibits the least likelihood of showing duplex formation,
hair-pin formation, and homooligomer/sequence repeats but has a
high to moderate potential to bind to the IGF-II gene sequences.
These properties may be determined using the computer modeling
program OLIGO Primer Analysis Software, Version 5.0 (distributed by
National Biosciences, Inc., Plymouth, Minn.). This computer program
allows the determination of a qualitative estimation of these five
parameters.
[0101] Alternatively, the antisense oligonucleotides may also be
selected on the basis that the sequence is highly conserved for the
IGF-II gene between two or more mammalian species. These properties
may be determined using the BLASTN program (Altschul, et al.(34))
of the University of Wisconsin Computer group (GCG) software
(Devereux J. et al.(35)) with the National Center for Biotechnology
Information (NCBI) databases.
[0102] The antisense oligonucleotides may include mutations, such
as substitutions, insertions and deletions. Preferably there will
be less that 10% of the sequence having mutations.
[0103] The antisense oligonucleotides generally comprise from at
least about 3 nucleotides or nucleotide analogs, more preferably
they are at least about 5 nucleotides, more preferably they are at
least about 7 nucleotides, more preferably they are at least about
9 nucleotides and most preferably they are at least about 20
nucleotides. The antisense oligonucleotides are preferably less
than about 100 nucleotides or nucleotide analogs, more preferably,
less than about 50 nucleotides or nucleotide analogs, most
preferably less than about 35 nucleotide or nucleotide analogs.
[0104] Preferably, the antisense oligonucleotides are complementary
to the 5' untranslated region of the fetal IGF-II transcript. The
"untranslated region of the fetal IGF-II transcript" means that
part of the IGF-II gene which is transcribed in fetal cells to form
the major IGF-II transcript and which does not form part of the
adult IGF-II transcript (the major transcript in adult cells).
Preferably the "untranslated region of the fetal IGF-II transcript"
is exons 4, 5 and 6 of the IGF-II gene. Most preferably, the
"untranslated region of the fetal IGF-II transcript" is that
substnatially the sequence of exons 4, 5 and 6 as set forth in
FIGS. 11 A-C.
[0105] Preferably, the antisense oligonucleotides comprise the
sequences set forth in Tables 1 and 2 (below).
1TABLE 1 Antisense Sequences designed to target human IGF-II Fetal
mRNA SEQ ID .DELTA.G NO. Name Sequence 5'-3' Tm (.degree. C.)
(kcal/mol) 1 GT14001 GGC TCG CTG GGG CAG GAG GA 74.6 -46.5 2
GT14002 GCT GGT GGG CAG AGC GCG GG 78.0 -48.5 3 GT14003 TTG GTG TCT
ACA GCT CAG CA 57.8 -35.2 4 GT14004 CAG CGA GGC AGC GGG CGG CG 82.7
-52.5 5 GT14005 TCG GGC GAA GCG GGG ATG GG 79.0 -50.4 6 GT14006 CGG
GCC TCG GGA GGG GGA CA 78.2 -49.4 7 GT14007 GAC CGC GGG CGC CCA GCT
CG 81.7 -51.9 8 GT14008 ACG TCG AGG GGC CGG GGG AG 77.4 -49.3 9
GT14009 CGG GAG AAA GAG CGG GGG CC 75.1 -48.5 10 GT14010 CGA GAG
GGC GGG CGT GAG GG 77.0 -48.4 11 GT14011 CAG CGA GAG GCG GGC AGG CG
78.2 -49.0 12 GT14012 CGG GCT GTC TTC GGG CTG GG 74.9 -47.0 13
GT14013 GCG ACG GGG CAG AGC GGG GG 80.7 -51.4 14 GT14014 CGC TGC
CGC CCA CCT CCC TG 77.8 -48.5 15 GT14015 TTG GTG TCT GGA AGC CGG CG
72.0 -44.3
[0106] The antisense oligonucleotides were selected from the
sequence complementary to the human IGF-II mRNA such that the
sequence exhibits the least likelihood of showing duplex formation,
hairpin formation, and homooligomers/sequence repeats but has a
high potential to bind to the IGF-II mRNA sequence and contains a
GC clamp. In addition, false priming to other frequently occurring
or repetitive sequences in human and mouse was eliminated. These
properties were determined using the computer modeling program
OLIGO.RTM. Primer Analysis Software, Version 5.0 (distributed by
National Biosciences, Inc., Plymouth, Minn.).
2TABLE 2 Antisense oligonucleotides having a sequence complementary
to all regions of the human IGF-II mRNA SEQ ID .DELTA.G NO. Name
Sequence 5'-3' Tm (.degree. C.) (kcal/mol) 16 GT14016 TTC CCC ATT
GGG ATT CCC AT 66.8 -42.4 17 GT14017 GTC CAC CAG CTC CCC GCC GC
76.9 -47.9 18 GT14018 CGA TGC CAC GGC TGC GAC GG 77.6 -47.6 19
GT14019 ACG CAG GAG GGC AGG CAG GC 74.7 -46.5 20 GT14020 GCG AGC
ACG TGA CCC CGG CG 78.7 -48.6 21 GT14021 CGT GGG CGG GGT CTT GGG TG
75.4 -46.7 22 GT14022 TGT TTC GGG GAG GCG GGG CA 77.5 -48.8 23
GT14023 GCG GTA CGA GCG ACG TGC CC 73.8 -45.9 24 GT14024 CAA ATG
CCG CCG GCC GCA CA 79.7 -49.8 25 GT14025 CGC ATC AGT GCA CGG CCC CC
76.5 -46.9 26 GT14026 GTG CGG AAG GCG GCC ACC CT 76.4 -48.2 27
GT14027 CAG GGT GCT GAG GGG CGG GC 76.9 -48.0 28 GT14028 GCT CCG
GGG CCC AAG CAA CC 75.9 -48.3 29 GT14029 CCC TAG GCG CCG CGG TGG TG
77.6 -49.3 30 GT14030 TGG CAT GGA CGA CCC CCG GG 77.7 -48.1 31
GT14031 GGG CCG CAA GGT GGA CCG AG 74.8 -46.7
[0107] The antisense oligonucleotides were selected from the
sequence complementary to the human IGF-II mRNA such that the
sequence exhibits the least likelihood of showing duplex formation,
hairpin formation, and homooligomers/sequence repeats but has a
high potential to bind to the IGF-II mRNA sequence and contains a
GC clamp. In addition, false priming to other frequently occurring
or repetitive sequences in human and mouse was eliminated. These
properties were determined using the computer modeling program
OLIGO.RTM. Primer Analysis Software, Version 5.0 (distributed by
National Biosciences, Inc., Plymouth, Minn.).
[0108] In Tables 1 and 2 the "Tm" is the melting temperature of an
oligonucleotide duplex calculated according to the
nearest-neighbour thermodynamic values. At this temperature 50% of
nucleic acid molecules are in duplex and 50% are denatured. The
"AG" is the free energy of the oligonucleotide, which is a
measurement of an oligonucleotide duplex stability.
[0109] The term "alky" refers to monovalent alkyl groups preferably
having from 1 to 20 carbon atoms and more preferably 1 to 6 carbon
atoms. This term is exemplified by groups such as methyl, ethyl,
n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the
like.
[0110] The term "aryl" refers to an unsaturated aromatic
carbocyclic group of from 6 to 14 carbon atoms having a single ring
(e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl
or anthryl). Preferred aryls include phenyl, naphthyl and the
like.
[0111] The term "halo" or "halogen" refers to fluoro, chloro, bromo
and iodo and preferably is either fluoro or chloro.
[0112] As to any of the above groups which contain one or more
substituents, it is understood, of course, that such groups do not
contain any substitution or substitution patterns which are
sterically impractical and/or synthetically non-feasible. In
addition, the compounds of this invention include all
stereochemical isomers arising from the substitution of these
compounds.
[0113] The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s). The material is
compatible with a biological system such as a cell, cell culture,
tissue or organism.
[0114] The term "pharmaceutically acceptable salt" refers to salts
which retain the biological effectiveness and properties of the
antisense oligonucleotides of this invention and which are not
biologically or otherwise undesirable. In many cases, the antisense
oligonucleotides of this invention are capable of forming acid
and/or base salts by virtue of the presence of amino and/or
carboxyl groups or groups similar thereto.
[0115] Pharmaceutically acceptable base addition salts can be
prepared from inorganic and organic bases. Salts derived from
inorganic bases, include by way of example only, sodium, potassium,
lithium, ammonium, calcium and magnesium salts. Salts derived from
organic bases include, but are not limited to, salts of primary,
secondary and tertiary amines, such as alkyl amines, dialkyl
amines, trialkyl amines, substituted alkyl amines, di(substituted
alkyl)amines, tri(substituted alkyl)amines, alkenyl amines,
dialkenyl amines, trialkenyl amines, substituted alkenyl amines,
di(substituted alkenyl)amines, tri(substituted alkenyl)amines,
cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines,
substituted cycloalkyl amines, disubstituted cycloalkyl amine,
trisubstituted cycloalkyl amines, cycloalkenyl amines,
di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted
cycloalkenyl amines, disubstituted cycloalkenyl amine,
trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,
triaryl amines, heteroaryl amines, diheteroaryl amines,
triheteroaryl arnines, heterocyclic amines, diheterocyclic amines,
triheterocyclic amines, mixed di- and tri-amines where at least two
of the substituents on the amine are different and are selected
from the group consisting of alkyl, substituted alkyl, alkenyl,
substituted alkenyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,
heterocyclic, and the like. Also included are amines where the two
or three substituents, together with the amino nitrogen, form a
heterocyclic or heteroaryl group.
[0116] Examples of suitable amines include, by way of example only,
isopropylamine, trimethylamine, diethylamine, tri(iso-propyl)amine,
tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol,
tromethamine, lysine, arginine, histidine, caffeine, procaine,
hydrabamine, choline, betaine, ethylenediamine, glucosamine,
N-alkylglucamines, theobromine, purines, piperazine, piperidine,
morpholine, N-ethylpiperidine, and the like. It should also be
understood that other carboxylic acid derivatives would be useful
in the practice of this invention, for example, carboxylic acid
amides, including carboxamides, lower alkyl carboxamides, dialkyl
carboxamides, and the like.
[0117] Pharmaceutically acceptable acid addition salts may be
prepared from inorganic and organic acids. Salts derived from
inorganic acids include hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like. Salts
derived from organic acids include acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid,
and the like.
[0118] The term "IGF-II gene" or "insulin-like growth factor II"
refers to any gene which encodes a protein that is capable of
binding to the IGF-I or IGF-II receptor. Preferably the IGF-II gene
has one or more regions with a nucleotide sequence substantially
similar to the sequences of exons 4, 5, 6 or 7-9 as set forth in
FIGS. 11A-D.
[0119] The term "complementary to" means that the antisense
oligonucleotide sequence is capable of binding to the target
sequence, i.e. the IGF-II gene (or mRNA). Preferably, the antisense
oligonucleotide binds to the nucleic acid sequence under
physiological conditions, e.g. by Watson-Crick base pairing
(interaction between oligonucleotide and single-stranded nucleic
acid) or by Hoogsteen base pairing (interaction between
oligonucleotide and double-stranded nucleic acid) or by any other
means including in the case of an oligonucleotide binding to RNA,
causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen
base pairing under physiological conditions is measured as a
practical matter by observing interference with the function of the
nucleic acid sequence.
[0120] Preferably the antisense oligonucleotide sequence has at
least about 75% identity with the target sequence, preferably at
least about 90% identity and most preferably at least about 95%
identity with the target sequence allowing for gaps or mismatches
of several bases. Identity can be determined, for example, by using
the BLASTN program of the University of Wisconsin Computer Group
(GCG) software. Preferably the antisense oligonucleotide sequence
hybridizes to the IGF-II mRNA with a melting temperature of at
least 45.degree. C., more preferably at least about 50.degree. C.
and most preferably at least about 55.degree. C. as determined by
the OLIGO Primer Analysis Software, version 5.0 program described
herein.
[0121] The term "inhibiting growth" means a reduction or inhibition
in the growth of at least one tumor cell type, preferably by at
least 10%, more preferably of at least 50% and most preferably of
at least 75%. The inhibition of growth of tumors can be determined
by measuring the size of the tumor in nude mice or the inability of
the tumor cells to form colonies in vitro.
[0122] The term "inhibiting metastasis" means reducing or
inhibiting the number of metastatic tumors that develop, preferably
by at least 10% and more preferably by at least 50%. This can be
determined by the methods set forth in the Examples and other
methods known in the art.
[0123] The term "inhibiting expression of IGF-II" means that the
antisense oligonucleotide reduces the level of IGF-II mRNA or the
level of IGF-II protein produced by the cell when the
oligonucleotide is administered to the cell.
[0124] The term "mammal" or "mammalian" means all mammals including
humans, ovines, bovines, equines, swine, canines, felines and mice,
etc., preferably it means humans.
[0125] A "mammal suspected of having a tumor" means that the mammal
may have a proliferative disorder or tumor or has been diagnosed
with a proliferative disorder or tumor or has been previously
diagnosed with a proliferative disorder or tumor, the tumor has
been surgically removed and the mammal is suspected of harboring
some residual tumor cells.
[0126] Preparation of the Antisense Oligonucleotides
[0127] The antisense oligonucleotides of the present invention may
be prepared by conventional and well-known techniques. For example,
the oligonucleotides may be prepared using solid-phase synthesis
and in particular using commercially available equipment such as
the equipment available from Applied Biosystems Canada Inc.,
Mississauga, Canada. The oligonucleotides may also be prepared by
enzymatic digestion of the naturally occurring IGF-II gene by
methods known in the art.
[0128] These oligonucleotides can be prepared by the art recognized
methods such as phosphoramidate or H-phosphcate chemistry which can
be carried out manually or by an automated synthesizer as described
by Uhlmann et al.(21) and Agrawal et al. (22).
[0129] Isolation and Purification of the Antisense
Oligonucleotides
[0130] Isolation and purification of the antisense oligonucleotides
described herein can be effected, if desired, by any suitable
separation or purification such as, for example, filtration,
extraction, crystallization, column chromatography, thin-layer
chromatography, thick-layer chromatography, preparative low or
high-pressure liquid chromatography or a combination of these
procedures. However, other equivalent separation or isolation
procedures could, of course, also be used.
[0131] An expression vector comprising the antisense
oligonucleotide sequence may be constructed having regard to the
sequence of the oligonucleotide and using procedures known in the
art.
[0132] Vectors can be constructed by those skilled in the art to
contain all the expression elements required to achieve the desired
transcription of the antisense oligonucleotide sequences.
Therefore, the invention provides vectors comprising a
transcription control sequence operatively linked to a sequence
which encodes an antisense oligonucleotide. Suitable transcription
and translation elements may be derived from a variety of sources,
including bacterial, fungal, viral, mammalian or insect genes.
Selection of appropriate elements is dependent on the host cell
chosen.
[0133] Reporter genes may be included in the vector. Suitable
reporter genes include .beta.-galactosidase (e.g. lacZ),
chloramphenicol, acetyl-transferase, firefly luciferase, or an
immunoglobulin or portion thereof. Transcription of the antisense
oligonucleotide may be monitored by monitoring for the expression
of the reporter gene.
[0134] The vectors can be introduced into cells or tissues by any
one of a variety of known methods within the art. Such methods can
be found generally described in Sambrook et al..sup.24; Ausubel et
al..sup.25; Chang et al..sup.36; Vega et al..sup.37; and Vectors: A
Survey of Molecular Cloning Vectors and Their Uses.sup.38 and
include, for example, stable or transient transfection,
lipofection, electroporation and infection with recombinant viral
vectors.
[0135] Introduction of nucleic acids by infection offers several
advantages. Higher efficiency and specificity for tissue type can
be obtained. Viruses typically infect and propagate in specific
cell types. Thus, the virus' specificity may be used to target the
vector to specific cell types in vivo or within a tissue or mixed
culture of cells. Viral vectors can also be modified with specific
receptors or ligands to alter target specificity through receptor
mediated events.
[0136] It is contemplated that the oligonucleotide of this
invention may be a ribozyme which cleaves the mRNA. The ribozyme
preferably has a sequence homologous to a sequence of an
oligonucleotide of the invention and the necessary catalytic center
for cleaving the mRNA. For example, a homologous ribozyme sequence
may be selected which destroys the IGF-II mRNA. The ribozyme type
utilized in the present invention may be selected from types known
in the art. Several ribozyme structural families have been
identified including Group I introns, RNase P, the hepatitis delta
virus ribozyme, hammerhead ribozymes and the hairpin ribozyme
originally derived from the negative strand of the tobacco ringspot
virus satellite RNA (sTRSV) (Sullivar 1994, U.S. Pat. No.
5,225,347.sup.39). Hammerhead and hairpin ribozyme motifs are most
commonly adapted for trans cleavage of mRNAs for gene therapy
(Sullivan 1994). Hairpin ribozymes are preferably used in the
present invention. In general, the ribozyme is from 30 to 100
nucleotides in length.
[0137] The oligonucleotides of the invention may be insolubilized.
For example, the oligonucleotide may be bound to a suitable
carrier. Examples of suitable carriers are agarose, cellulose,
dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene,
filter paper, ion-exchange resin, plastic film, plastic tube, glass
beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino
acid copolymer, ethylene-maleic acid copolymer, nylon, silk etc.
The carrier may in the shape of, for example, a tube, test plate,
beads disc, sphere etc.
[0138] The insoubilized oligonucleotide may be prepared by reacting
the material with the suitable insoluble carrier using known
chemical or physical methods, for example, cyanogen bromide
coupling.
[0139] Pharmaceutical Formulations
[0140] When employed as pharmaceuticals, the antisense
oligonucleotides are usually administered in the form of
pharmaceutical compositions. These compounds can be administered by
a variety of routes including oral, rectal, transdermal,
subcutaneous, intravenous, intramuscular, and intranasal. These
compounds are effective as both injectable and oral compositions.
Such compositions are prepared in a manner well known in the
pharmaceutical art and comprise at least one active compound. The
pharmaceutical composition is, for example, administered
intravenously. It is contemplated that the pharmaceutical
composition may be administered directly into the tumor to be
treated.
[0141] This invention also includes pharmaceutical compositions
which contain, as the active ingredient, one or more of the
antisense oligonucleotides associated with pharmaceutically
acceptable carriers or excipients. In making the compositions of
this invention, the active ingredient is usually mixed with an
excipient, diluted by an excipient or enclosed within such a
carrier which can be in the form of a capsule, sachet, paper or
other container. When the excipient serves as a diluent, it can be
a solid, semi-solid, or liquid material, which acts as a vehicle,
carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges, sachets,
cachets, elixirs, suspensions, emulsions, solutions, syrups,
aerosols (as a solid or in a liquid medium), ointments containing,
for example, up to 10% by weight of the active compound, soft and
hard gelatin capsules, suppositories, sterile injectable solutions,
and sterile packaged powders.
[0142] In preparing a formulation, it may be necessary to mill the
active compound to provide the appropriate particle size prior to
combining with the other ingredients. If the active compound is
substantially insoluble, it ordinarily is milled to a particle size
of less than 200 mesh. If the active compound is substantially
water soluble, the particle size is normally adjusted by milling to
provide a substantially uniform distribution in the formulation,
e.g. about 40 mesh.
[0143] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, sterile water, syrup, and methyl cellulose. The
formulations can additionally include: lubricating agents such as
talc, magnesium stearate, and mineral oil; wetting agents;
emulsifying and suspending agents; preserving agents such as
methyl- and propylhydroxy-benzoates; sweetening agents; and
flavoring agents. The compositions of the invention can be
formulated so as to provide quick, sustained or delayed release of
the active ingredient after administration to the patient by
employing procedures known in the art.
[0144] The compositions are preferably formulated in a unit dosage
form, each dosage containing from about 1% to about 95%, more
usually about 5% to about 90% of the active ingredient. The term
"unit dosage forms" refers to physically discrete units suitable as
unitary dosages for human subjects and other mammals, each unit
containing a predetermined quantity of active material calculated
to produce the desired therapeutic effect, in association with a
suitable pharmaceutical excipient.
[0145] The antisense oligonucleotide is effective over a wide
dosage range and is generally administered in a pharmaceutically
effective amount. An effective amount is that amount which when
administered alleviates the symptoms. Preferably the effective
amount is that amount able to inhibit tumor cell growth. Preferably
the effective amount is from about 0.1 mg/kg body weight to about
20 mg/kg body weight. It will be understood, however, that the
amount of the antisense oligonucleotide actually administered will
be determined by a physician, in the light of the relevant
circumstances, including the condition to be treated, the chosen
route of administration, the actual compound administered, the age,
weight, and response of the individual patient, the severity of the
patient's symptoms, and the like. The course of therapy may last
from several days to several months or until diminution of the
disease is achieved. The antisense oligonucleotide may be
administered in combination with other known therapies. When
co-administered with one or more other therapies, the
oligonucleotide may be administered either simultaneously with the
other treatments(s), or sequentially. If administered sequentially,
the attending physician will decide on the appropriate sequence of
administering the oligonucleotide in combination with the other
therapy.
[0146] For preparing solid compositions such as tablets, the
principal active ingredient/antisense oligonucleotide is mixed with
a pharmaceutical excipient to form a solid preformulation
composition containing a homogeneous mixture of a compound of the
present invention. When referring to these preformulation
compositions as homogeneous, it is meant that the active ingredient
is dispersed evenly throughout the composition so that the
composition may be readily subdivided into equally effective unit
dosage forms such as tablets, pills and capsules.
[0147] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0148] The liquid forms in which the novel compositions of the
present invention may be incorporated for administration orally or
by injection include aqueous solutions, suitably flavored syrups,
aqueous or oil suspensions, and flavored emulsions with edible oils
such as corn oil, cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0149] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described herein. Preferably the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in preferably
pharmaceutically acceptable solvents may be nebulized by use of
inert gases. Nebulized solutions may be inhaled directly from the
nebulizing device or the nebulizing device may be attached to a
face mask tent, or intermittent positive pressure breathing
machine. Solution, suspension, or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0150] The pharmaceutical composition of the invention may be in
the form of a liposome, in which the oligonucleotide is combined,
in addition to other pharmaceutically acceptable carriers, with
amphipathic agents such as lipids which exist in aggregated form as
micells, insoluble monolayers, liquid crystals or lamellar layers
which are in aqueous solution. Suitable lipids for liposomal
formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin,
bile acids and the like. One particularly useful lipid carrier is
lipofectin. Preparation of such liposomal formulations is within
the skill in the art, for example, International Patent No.
W097/21808 (28) The pharmaceutical composition may further include
compounds such as cyclodextrins and the like which enhance delivery
of oligonucleotides into cells or slow release polymers.
[0151] Another preferred formulation employed in the methods of the
present invention employs transdermal delivery devices ("patches").
Such transdernal patches may be used to provide continuous or
discontinuous infusion of the antisense oligonucleotides of the
present invention in controlled amounts. The construction and use
of transdermal patches for the delivery of pharmaceutical agents is
well known in the art. See, for example, U.S. Pat.
5,023,252.sup.40, herein incorporated by reference. Such patches
may be constructed for continuous, pulsatile, or on demand delivery
of pharmaceutical agents.
[0152] Another preferred method of delivery involves "shotgun"
delivery of the naked antisense oligonucleotides across the dermal
layer. The delivery of "naked" antisense oligonucleotides is well
known in the art. See, for example, Felgner et al.,U.S. Pat. No.
5,580,859.sup.41. It is contemplated that the antisense
oligonucleotides may be packaged in a lipid vesicle before
"shotgun" delivery of the antisense oligonucleotide.
[0153] The following formulation examples illustrate representative
pharmaceutical compositions of the present invention.
FORMULATION EXAMPLE 1
[0154] Hard gelatin capsules containing the following ingredients
are prepared:
3 Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch
305.0 Magnesium stearate 5.0
[0155] The above ingredients are mixed and filled into hard gelatin
capsules in 340 mg quantities.
FORMULATION EXAMPLE 2
[0156] A tablet formula is prepared using the ingredients
below:
4 Quantity Ingredient (mg/tablet) Active Ingredient 25.0 Cellulose,
microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid
5.0
[0157] The components are blended and compressed to form tablets,
each weighing 240 mg.
FORMULATION EXAMPLE 3
[0158] A dry powder inhaler formulation is prepared containing the
following components:
5 Ingredient Weight % Active Ingredient 5 Lactose 95
FORMULATION EXAMPLE 4
[0159] Tablets, each containing 30 mg of active ingredient, are
prepared as follows:
6 Quantity Ingredient (mg/tablet) Active Ingredient 30.0 mg Starch
45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone 4.0
mg (as 10% solution in sterile water) Sodium carboxymethyl starch
4.5 mg Magnesium stearate 0.5 mg Talc 1.0 mg Total 120 mg
[0160] The active ingredient, starch and cellulose are passed
through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution
of polyvinylpyrrolidone is mixed with the resultant powders, which
are then passed through a 16 mesh U.S. sieve. The granules so
produced are dried at 50.degree. to 60.degree. C. and passed
through a 16 mesh U.S. sieve. The sodium carboxymethyl starch,
magnesium stearate, and talc, previously passed through a No. 30
mesh U.S. sieve, are then added to the granules which, after
mixing, are compressed on a tablet machine to yield tablets each
weighing 120 mg.
FORMULATION EXAMPLE 5
[0161] Capsules, each containing 40 mg of medicament are made as
follows:
7 Quantity Ingredient (mg/capsule) Active Ingredient 40.0 mg Starch
109.0 mg Magnesium stearate 1.0 mg Total 150.0 mg
[0162] The active ingredient, starch, and magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled into
hard gelatin capsules in 150 mg quantities.
FORMULATION EXAMPLE 6
[0163] Suppositories, each containing 25 mg of active ingredient
are made as follows:
8 Ingredient Amount Active Ingredient 25 mg Saturated fatty acid
glycerides to 2,000 mg
[0164] The active ingredient is passed through a No. 60 mesh U.S.
sieve and suspended in the saturated fatty acid glycerides
previously melted using the minimum heat necessary. The mixture is
then poured into a suppository mold of nominal 2.0 g capacity and
allowed to cool.
FORMULATION EXAMPLE 7
[0165] Suspensions, each containing 50 mg of medicament per 5.0 mL
dose are made as follows:
9 Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose
(89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and
Color q.v. Purified water to 5.0 mL
[0166] The active ingredient, sucrose and xanthan gum are blended,
passed through a No. 10 mesh U.S. sieve, and then mixed with a
previously made solution of the microcrystalline cellulose and
sodium carboxymethyl cellulose in water. The sodium benzoate,
flavor, and color are diluted with some of the water and added with
stirring. Sufficient water is then added to produce the required
volume.
FORMULATION EXAMPLE 8
[0167]
10 Quantity Ingredient (mg/capsule) Active Ingredient 15.0 mg
Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0 mg
[0168] The active ingredient, starch, and magnesium stearate are
blended, passed through a No. 20 mesh U.S. sieve, and filled into
hard gelatin capsules in 425.0 mg quantities.
FORMULATION EXAMPLE 9
[0169] A formulation may be prepared as follows:
11 A formulation may be prepared as follows: Ingredient Quantity
Active Ingredient 5.0 mg Corn Oil 1.0 mL
FORMULATION EXAMPLE 10
[0170] A topical formulation may be prepared as follows:
12 Ingredient Quantity Active Ingredient 1-10 g Emulsifying Wax 30
g Liquid Paraffin 20 g White Soft Paraffin to 100 g
[0171] The white soft paraffin is heated until molten. The liquid
paraffin and emulsifying wax are incorporated and stirred until
dissolved. The active ingredient is added and stirring is continued
until dispersed. The mixture is then cooled until solid.
[0172] Other suitable formulations for use in the present invention
can be found in Remington's Pharmaceutical Sciences (23).
[0173] The antisense oligonucleotides or the pharmaceutical
composition comprising the antisense oligonucleotides may be
packaged into convenient kits providing the necessary materials
packaged into suitable containers.
[0174] The antisense oligonucleotides of the invention in the form
of a therapeutic formulation are useful in treating diseases, and
disorders and conditions associated with tumor growth. In such
methods a therapeutic amount of a oligonucleotide effective in
inhibiting the expression of fetal transcripts of IGF-II is
administered to a cell. This cell may be part of a cell culture, a
tissue culture, or may be part of the whole body of a mammal such
as a human.
[0175] The oligonucleotides and ribozymes of the invention modulate
tumor cell growth. Therefore methods are provided for interfering
or inhibiting tumor cell growth in a mammal comprising contacting
the tumor or tumor cells with an antisense oligonucleotide of the
present invention.
[0176] The term "contact" refers to the addition of an
oligonucleotide, ribozyme, etc. to a cell suspension or tissue
sample or administering the oligonucleotides etc. directly or
indirectly to cells or tissues within an animal.
[0177] The methods may be used to treat proliferative disorders
including various forms of cancer or tumors such a leukemias,
lymphomas (Hodgkins and non-Hodgkins), sarcomas, melanomas,
adenomas, carcinomas of solid tissue, hypoxic tumors, squamous cell
carcinomas of the mouth, throat, larynx and lung, genitourinary
cancers such as cervical and bladder cancer, hematopoietic cancers,
colon cancer, breast cancer, pancreatic cancer, renal cancer, brain
cancer, skin cancer, liver cancer, head and neck cancers, and
nervous system cancers, as well as benign lesions such as
papillomas. Other proliferative disorders such as psoriasis and
those involving arthrosclerosis are also included.
[0178] The oligonucleotides of the invention may also be used to
treat drug resistant tumors. Examples of drug resistant tumors are
tumors resistant to such chemotherapeutic agents as 5-fluorouracil,
mitomycin C, methotrexate or hydroxyurea and tumors expressing high
levels of P-glycoprotein which is known to confer resistance to
multiple anticancer drugs such as colchicine, vinblastine and
doxorubicin; or tumors expressing multi-drug resistance protein as
described by Dreeley et al.(28). Accordingly, it is contemplated
that the oligonucleotides of the present invention may be
administered in conjunction with or in addition to known anticancer
compounds or chemotherapeutic agents. Chemotherapeutic agents are
compounds which may inhibit the growth of tumors. Such agents,
include, but are not limited to, 5-fluorouracil, mitomycin C,
methotrexate and hydroxyurea. It is contemplated that the amount of
chemotherapeutic agent may be either an effective amount, i.e. an
amount sufficient to inhibit tumor growth or a less than effective
amount.
[0179] The oligonucleotides of the present invention have been
found to reduce the growth of tumors that are metastatic such as
C8161 melanoma cells. In an embodiment of the invention, a method
is provided for reducing the growth of metastastic tumors in a
mammal comprising administering an effective amount of an
oligonucleotide from about 3 to about 100 nucleotides, comprising a
sequence complementary to the 5' untranslated region of mammalian
fetal IGF-II mRNA. The sequence may be selected from the group of
oligonucleotides shown in Table 1. In another embodiment, a method
is provided for reducing the growth of metastastic tumors in a
mammal comprising administering an effective amount of an
oligonucleotide from about 20 to about 100 nucleotides, comprising
a sequence selected from the group of SEQ ID NO: 17-31 set forth in
Table 2.
[0180] The oligonucleotides may be delivered using viral or
non-viral vectors. Sequences may be incorporated into cassettes or
constructs such that an oligonucleotide of the invention is
expressed in a cell. Preferably, the construct contains the proper
transcriptional control region to allow the oligonucleotide to be
transcribed in the cell.
[0181] Therefore, the invention provides vectors comprising a
transcription control sequence operatively linked to a sequence
which encodes an oligonucleotide of the invention. The present
invention further provides host cells, selected from suitable
eucaryotic and procaryotic cells, which are transformed with these
vectors.
[0182] Suitable vectors are known and preferably contain all of the
expression elements necessary to achieve the desired transcription
of the sequences. Phagemids are a specific example of such
beneficial vectors because they can be used either as plasmids or
as bacteriophage vectors. Examples of the vectors include viruses
such as bacteriophages, baculoviruses, retroviruses, DNA viruses,
liposomes and other recombination vectors. The vectors can also
contain elements for use in either procaryotic or eucaryotic host
systems. One of ordinary skill in the art will know which host
systems are compatible with a particular vector.
[0183] The vectors can be introduced into the cells by stable or
transient transfection, lipofection, electroporation and infection
with recombinant viral vectors.
[0184] Additional features can be added to the vector to ensure its
safety and/or enhance its therapeutic efficacy. Such features
include, for example, markers that can be used to negatively select
against cells infected with recombinant viruses. An example of such
a negative selection marker is the TK gene which confers
sensitivity to the antiviral gancyclovir. Features that limit
expression to particular cell types can also be included. Such
features include, for example, promoter and regulatory elements
that are specific for the desired cell type.
[0185] Retroviral vectors are another example of vectors useful for
the in vivo introduction of a desired nucleic acid because they
offer advantages such as lateral infection and targeting
specificity. Lateral infection is the process by which a single
infected cell produces many progeny virions that infect neighboring
cells. The result is that a large area becomes rapidly
infected.
[0186] A vector to be used in the methods of the invention may be
selected depending on the desired cell type to be targeted. For
example, if breast cancer is to be treated, then a vector specific
for epithelial cell may be used. Similarly, if cells of the
hematopoietic system are to be treated, then a viral vector that is
specific for blood cells is preferred.
[0187] Utility
[0188] The antisense oligonucleotides of the present invention may
be used for a variety of purposes. They may be used to inhibit the
expression of the IGF-II gene in a mammalian cell, resulting in the
inhibition of growth of that cell. The oligonucleotides may be used
as hybridization probes to detect the presence of the IGF-II mRNA
in mammalian cells. When so used the oligonucleotides may be
labeled with a suitable detectable group (such as a radioisotope, a
ligand, another member of a specific binding pair, for example,
biotin). Finally, the oligonucleotides may be used as molecular
weight markers.
[0189] In order to further illustrate the present invention and
advantages thereof, the following specific examples are given but
are not meant to limit the scope of the claims in any way.
EXAMPLES
[0190] In the examples below, all temperatures are in degrees
Celsius (unless otherwise indicated) and all percentages are weight
percentages (also unless otherwise indicated).
[0191] In the examples below, the following abbreviations have the
following meanings. If an abbreviation is not defined, it has its
generally accepted meaning:
[0192] AS antisense
[0193] cDNA=complementary deoxyribonucleic acid
[0194] ODN oligodeoxynucleotide
[0195] .mu.=micromolar
[0196] mM millimolar
[0197] M=molar
[0198] ml=milliliter
[0199] .mu.l=microliter
[0200] mg=milligram
[0201] .mu.g=microgram
[0202] PAGE=polyacrylamide gel electrophoresis
[0203] rpm=revolutions per minute
[0204] .DELTA.G=free energy, a measurement of oligonucleotide
duplex stability
[0205] kcal=kilocalories
[0206] FBS=fetal bovine serum
[0207] DTT=dithiothrietol
[0208] SDS=sodium dodecyl sulfate
[0209] PBS=phosphate buffered saline
[0210] PMSF=phenylmethylsulfonyl fluoride
[0211] GAPDH=glyceraldehyde-3-phosphate dehydrogenase
[0212] IgG=immunoglobulin G
[0213] kDa=kilodalton
[0214] PCR=polymerase chain reaction
[0215] Tris-HCl=Tris(hydroxymethyl)aminomethane-hydrochloric
acid
[0216] TRIzol=total RNA isolation reagent
[0217] ECL=western blotting detection reagents
[0218] IGF-I=insulin-like growth factor I
[0219] IGF-II=insulin-like growth factor II
[0220] UTR=untranslated region
[0221] General Methods in Molecular Biology:
[0222] Standard molecular biology techniques known in the art and
not specifically described were generally followed as in Sambrook
et al..sup.24; Ausubel et al..sup.25; and Perbal.sup.26.
[0223] Oligonucleotides
[0224] The antisense oligonucleotides were selected from the
sequence complementary to the IGF-II mRNA such that the sequence
exhibits the least likelihood of showing duplex formation, hairpin
formation, and homooligomers/sequence repeats but has a high
potential to bind to the IGF-II mRNA sequence. In addition, a false
priming to other frequently occurring or repetitive sequences in
human and mouse was eliminated. These properties were determined
using the computer modeling program OLIGO.RTM. Primer Analysis
Software, Version 5.0 International Biosciences, Inc. Plymouth
Minn.). Based on this analysis, phosphorothioate antisense
oligonucleotides were designed and then made by methods well known
in the art.
[0225] Cell Lines
[0226] Five different human cancer cell lines including embryonal
rhabdomyosarcoma (RD), neuroblastoma (SK-N-AS), Wilms' tumor
(G401), melanoma (C8161), human prostate adenocarcinoma (PC-3),
metastatic pancreatic adenocarcinoma (AsPC-1) were obtained from
American Type Culture Collection (ATCC). The cell lines were
maintained in cc-MEM medium (Gibco BRL, Gaithersburg, Md.)
supplemented with 10% fetal bovine serum (FBS).
Example 1
The Inhibition of Growth of Cancer Cell Lines by Antisense
Oligonucleotides Complementary to IGF-II
[0227] The colony forming ability of cancer cell lines treated with
different phosphorothioate antisense oligonucleotides was estimated
using a method previously described (Choy et al..sup.18).
Specifically, aliquots of a tumor cell suspension were seeded into
60 mm tissue culture dishes at a density of approximately
1.times.10.sup.4 and incubated overnight at 37.degree. C. in
.alpha.-MEM medium supplemented with 10% FBS. Cells were washed
once in 5 ml of PBS and treated with 0.2 .mu.M of the indicated
antisense oligonucleotides in the presence of cationic lipid
(Lipofectin reagent, final concentration, 5 .mu.g/ml, Gibco-BRL,
Gaithersburg, Md.) for 4 hours. The antisense oligonucleotides were
removed by washing the cells once with PBS and the cells were
cultured in growth medium (.alpha.-MEM medium supplemented with 10%
FBS) for 7 to 10 days at 37.degree. C. Colonies were stained with
methylene blue and scored by direct counting as described (Choy et
al. 18 and Huang and Wright.sup.20). Percent inhibition was
calculated by comparison with the number of colonies present in
cultures grown in the absence of antisense oligonucleotides. All
experiments were performed in quadruplicate.
[0228] The antisense oligonucleotides exerted inhibitory effects on
the colony forming ability of the human tumor cell lines. The
percent inhibition of each antisense oligonucleotide is shown in
FIG. 2A for rhabdomyosarcoma (RD); FIG. 2B for human prostate
cancer cell line (PC-3); FIG. 2C for human pancreatic cancer cell
line (AsPC-1); and FIG. 2D for human neuroblastoma cell line
(SK-N-AS).
Example 2
Decreased mRNA Levels Following Treatment with Antisense
Oligonucleotides Complementary to IGF-II
[0229] Human neuroblastoma cells (SK-N-AS) or rhabdomyosarcoma
cells (RD) were grown to subconfluency (70-80%) and were treated
with 0.2 .mu.M of phosphorothioate antisense oligonucleotides
complementary to IGF-II for 4 hours in the presence of cationic
lipid (Lipofectin reagent, final concentration, 5 .mu.g/ml,
Gibco-BRL) and Opti-MEM (Gibco-BRL). Cells were washed once with
PBS and incubated for 16 hours in .alpha.-MEM medium (Gibco-BRL)
containing 10% FBS. Total RNA was prepared in TRIzol reagent
(Gibco-BRL) and Northern blot analysis was performed as described
in Hurta and Wright(27) with some modifications. The blots were
hybridized with .sup.32P-labeled 389 bp PCR fragments synthesized
using forward primer (5'-TAC CGC CCC AGT GAG ACC CT-3') [SEQ ID
NO:32], reverse primer (5'-TGA CGT TTG GCC TCC CTG AA-3') [SEQ ID
NO:33] and the human colorectal adenocarcinoma 5'-stretch plus cDNA
library (Clonetech, Palo Alto Calif.) as a template. Human IGF-II
mRNA was expressed as a .sup.-6 kb nucleotide transcript (Werner et
al..sup.6). Equal RNA loading was demonstrated by methylene blue
staining of the blot prior to hybridization.
[0230] FIG. 3 shows that the antisense oligonucleotides reduce the
IGF-II mRNA levels to at least 50% of the control cells.
Example 3
Decreased IGF-II Protein Levels Following Treatment with Antisense
Oligonucleotides Complementary to IGF-II
[0231] Human neuroblastoma cells (SK-N-AS) or rhabdomyosarcoma
cells (RD) were grown to subconfluency (70-80%) and were treated
with 0.2 .mu.M of phosphorothioate antisense oligonucleotides
complementary to IGF-II for 4 hours in the presence of cationic
lipid (Lipofectin reagent, final concentration, 5 .mu.g/ml,
Gibco-BRL) and Opti-MEM (Gibco-BRL). Cells were washed once with
PBS and incubated for 20 hours in .alpha.-MEM medium (Gibco-BRL)
containing 10% FBS. The treatments and incubations were repeated
once more before the whole cell protein extracts were prepared in
2.times. sample loading buffer (100 mM Tris-HCl, pH 6.8, 0.2 M DTT,
4% SDS, 20% glycerol and 0.015% bromophenol blue).
[0232] Western blot analysis was performed as described previously
(Choy et al.(18); Fan et al. (19)) with some modification. The
expression of IGF-II was detected with anti-IGF-II antibody (1-2
.mu.g/ml) (Research Diagnostics Inc., Flanders N.J.) followed by
horseradish peroxidase-conjugated anti-goat IgG (sigma, St. Loius
Mo.) at a dilution of 1:7,000. Approximately 7.5 kDa protein was
visualized by ECL (Amersham, Arlington heights, Ill.)
[0233] FIG. 4 shows the reduction in IGF-II protein in human
neuroblastoma cells after treatment with various antisense
oligonucleotides.
[0234] FIG. 5 shows the reduction in IGF-II protein in human
rhabdomyosarcoma cells after treatment with various antisense
oligonucleotides.
Example 4
Inhibition of Human Tumor Cell Growth in Mice by Intravenous
Treatment with Antisense Oligonucleotides Complementary to
IGF-II
[0235] CD-1 athymic nude mice were purchased from Charles River
Laboratories (Montreal Canada). SK-N-AS human neuroblastoma cells
(typically 3.times.10.sup.6 cells in 100 .mu.l of PBS) were
subcutaneously injected into the right flank of 6-7 weeks old CD-1
athymic female nude mice. Each experimental group included 5 mice.
After the size of tumor reached an approximate volume of 100
mm.sup.3, typically 6 days post tumor cell injection, the various
antisense oligonucleotides were administered by bolus infusion into
the tail vein every other day at 10 mg/kg. Control animals received
saline alone for the same period. Treatments typically lasted 14
days thereafter.
[0236] FIG. 6A shows the effects of the various antisense
oligonucleotides on human neuroblastoma tumor growth in CD-1 nude
mice. Antitumor activities were estimated by the inhibition of
tumor volume, which was measured with a caliper on average of two
day intervals over the span of 14 days. Each point in the figure
represents mean tumor volume calculated from 5 animals per
experimental group. Analysis of covariance was used to compare the
regression curves of mice over time within each treatment group.
Specific hypothesis of equality of slopes, or equality of
intercepts when slopes are equal are derived from the analysis. All
analysis used the SAS (Statistical Analysis System) version 6.12.
When compared to the saline control, administration of the
antisense oligonucleotide inhibited the growth of the tumor with a
p value of <0.0001.
[0237] At the end of the treatment (usually 24 hours after the last
treatment) the animals were sacrificed and tumor weights were
measured. FIG. 6B shows the mean weight of the tumors. The
antisense oligonucleotides showed significant inhibitory effects on
tumor growth. One-way analysis of variance was used to compare the
means of groups of treatments. Where the overall group effect was
significant, a priori multiple comparisons using the least square
means was used to find the pairs of treatment groups that were
significantly different. When tumor weight was compared the
antisense oligonucleotides also showed statistically significant
inhibition when compared to the saline control.
Example 5
Inhibition of Human Tumor Cell Growth in Mice by Intravenous
Treatment with Antisense Oligonucleotides Complementary to
IGF-II
[0238] CD-1 athymic nude mice were purchased from Charles River
Laboratories (Montreal Canada). G401 human Wilms' tumor cells
(typically 3.times.10.sup.6 cells in 100 .mu.l of PBS) were
subcutaneously injected into the right flank of 6-7 weeks old CD-1
athymic female nude mice. Each experimental group included 5 mice.
After the size of tumor reached an approximate volume of 100
mm.sup.3, typically 8 days post tumor cell injection, the various
antisense oligonucleotides were administered by bolus infusion into
the tail vein every other day at 10 mg/kg. Control animals received
saline alone for the same period. Treatments typically lasted 18
days thereafter.
[0239] FIG. 7A shows the effects of the various antisense
oligonucleotides on human Wilms' tumor growth in CD-1 nude mice.
Antitumor activities were estimated by the inhibition of tumor
volume, which was measured with a caliper on average of two day
intervals over the span of 18 days. Each point in the figure
represents mean tumor volume calculated from 5 animals per
experimental group. Analysis of covariance was used to compare the
regression curves of mice over time within each treatment group.
Specific hypothesis of equality of slopes, or equality of
intercepts when slopes are equal are derived from the analysis. All
analysis used the SAS (Statistical Analysis System) version 6.12.
When compared to the saline control, administration of the
antisense oligonucleotide inhibited the growth of the tumor with a
p value of .ltoreq.0.0002.
[0240] At the end of the treatment (usually 24 hours after the last
treatment) the animals were sacrificed and tumor weights were
measured. FIG. 7B shows the mean weight of the tumors. The
antisense oligonucleotides showed significant inhibitory effects on
tumor growth. One-way analysis of variance was used to compare the
means of groups of treatments. Where the overall group effect was
significant, a priori multiple comparisons using the least square
means was used to find the pairs of treatment groups that were
significantly different When tumor weight was compared the
antisense oligonucleotides also showed statistically significant
inhibition when compared to the same control.
Example 6
Reduction in IGF-II mRNA Levels in Human Tumors in Mice by
Intravenous Treatment with Antisense Oligonucleotides Complementary
to IGF-II
[0241] CD-1 athymic nude mice were purchased from Charles River
Laboratories (Montreal Canada). SK-N-AS human neuroblastoma cells
(typically 3.times.10.sup.6 cells in 100 .mu.l of PBS) were
subcutaneously injected into the right flank of 6-7 weeks old CD-1
athymic female nude mice. Each experimental group included 5 mice.
After the size of tumor reached an approximate volume of 100
mm.sup.3, typically 6 days post tumor cell injection, the various
antisense oligonucleotides were administered by bolus infusion into
the tail vein every other day at 10 mg/kg. Control animals received
saline alone for the same period. Mice were sacrificed after 7
injections and excised tumor fragments of similar size were
immediately collected into TRIzol reagent (GIBCO BRL) and rapidly
homogenized for mRNA preparation.
[0242] To measure the effects of antisense oligonucleotides on
IGF-II mRNA levels, northern blot analysis was performed as
previously described (Hurta and Wright (27)) with some
modifications. The blots were hybridized with .sup.32P-labeled 389
bp PCR fragments synthesized using forward primer (5'-TAC CGC CCC
AGT GAG ACC CT-3') [SEQ ID NO:32], reverse primer (5'-TGA CGT TTG
GCC TCC CTG AA-3') [SEQ ID NO:33] and the human colorectal
adenocarcinoma 5'-stretch plus cDNA library (Clonetech, Palo Alto
Calif.) as a template. Human IGF-II mRNA was expressed as a .sup.-6
kb nucleotide transcript (Werner et al..sup.6) and its levels were
compared to glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNA
as previously described (Hurta and Wright (27)).
[0243] FIG. 8 shows that the level of IGF-II mRNA was reduced in
tumor treated with the antisense oligodeoxynucleotide GTI4006 [SEQ
ID NO:6].
Example 7
Reduction in IGF-II Protein Levels in Human Tumors in Mice by
Intravenous Treatment with Antisense Oligonucleotides Complementary
to IGF-II
[0244] CD-1 athymic nude mice were purchased from Charles River
Laboratories (Montreal Canada). SK-N-AS human neuroblastoma cells
(typically 3.times.10.sup.6 (cells in 100 .mu.l of PBS) were
subcutaneously injected into the right flank of 6-7 weeks old CD-1
athymic female nude mice. Each experimental group included 5 mice.
After the size of tumor reached an approximate volume of 100
mm.sup.3, typically 6 days post tumor cell injection, the various
antisense oligonucleotides were administered by bolus infusion into
the tail vein every other day at 10 mg/kg. Control animals received
saline alone for the same period. Mice were sacrificed after 7
injections and excised tumor fragments of similar size were
immediately collected into RIPA extraction buffer (50 mM Tris-HCl,
pH 7.5, 150 mM leupeptin) and rapidly homogenized for protein
preparation.
[0245] To measure the effects of antisense oligodeoxynucleotides on
IGF-II protein levels, western blot analysis was performed as
previously described (Choy et al. (18), Fan et al. (19)) with some
modification. The protein extracts (10-20 .mu.g) were fractionated
on a 15% SDS-PAGE gel and transferred to nitrocellulose membranes
and visualized by India ink staining. The expression of IGF-II was
detected with anti-IGF-II antibody (1-2 .mu.g/ml) (Research
Diagnostics Inc., Flanders N.J.) followed by horseradish
peroxidase-conjugated anti-goat IgG (sigma, St. Loius Mo.) at a
dilution of 1:7,000. Approximately 7.5 kDa protein was visualized
by ECL (Amersham, Arlington Heights, Ill.).
[0246] FIG. 9 shows the western blot of the protein extracted from
the tumor cells. Each of the antisense oligonucleotides tested
reduced the IGF-II protein levels in the tumors. A part of the blot
stained with India ink is shown underneath to demonstrate an equal
loading in each lane.
Example 8
Inhibition of Experimental Metastasis by Antisense
Oligonucleotides
[0247] Experimental metastasis of C8161 human melanoma cells
treated with different antisense oligonucleotides was estimated as
previously described (Fan et al., 1996.sup.19). Aliquots of cell
suspension were seeded into 100 mm tissue culture dishes at a
density of 2.times.10.sup.6 and incubated overnight at 37.degree.
C. in A-MEM medium supplemented with 10% FBS. Cells were washed
once in 10 ml of PBS and treated with 0.2 .mu.M of oligonucleotides
in the presence of cationic lipid (Lipofectin reagent, final
concentration, 5 .mu.g/ml, Gibco-BRL) for 4 hours. The antisense
oligonucleotides were removed by washing the cells once with PBS
and the cells were trypsinized. Cells were then collected by
centrifugation, and approximately 1.times.10.sup.5 cells suspended
in 0.1 ml of PBS were injected into the tail veins of 6-8 week old
CD-1 athymic female nude mice. Estimates of the number of lung
tumors were made 5 weeks later, after excised lungs from individual
mice were stained with picric acid dye solution (75% picric acid,
20% formaldehyde, 5% glacial acetic acid).
[0248] FIG. 10 shows the reduced number of lung tumors in the
female nude mice after treatment of the tumor cells with the
various antisense oligodeoxynucleotides.
Sequence CWU 0
0
* * * * *