U.S. patent application number 12/085894 was filed with the patent office on 2009-12-31 for cancer therapies and pharmaceutical compositions used therein.
Invention is credited to Neal Clifford Goodwin, J. Patrick McGovren.
Application Number | 20090324587 12/085894 |
Document ID | / |
Family ID | 38024274 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090324587 |
Kind Code |
A1 |
Goodwin; Neal Clifford ; et
al. |
December 31, 2009 |
Cancer Therapies and Pharmaceutical Compositions Used Therein
Abstract
The invention relates to compositions and methods to inhibit
gene expression. In particular, the invention provides co-therapies
comprising oligonucleotides plus other therapies to treat
cancer.
Inventors: |
Goodwin; Neal Clifford;
(Plainwell, MI) ; McGovren; J. Patrick;
(Kalamazoo, MI) |
Correspondence
Address: |
HONIGMAN MILLER SCHWARTZ & COHN LLP
444 WEST MICHIGAN AVENUE
KALAMAZOO
MI
49007-3714
US
|
Family ID: |
38024274 |
Appl. No.: |
12/085894 |
Filed: |
December 1, 2006 |
PCT Filed: |
December 1, 2006 |
PCT NO: |
PCT/US2006/046111 |
371 Date: |
April 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60741229 |
Dec 1, 2005 |
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60778304 |
Mar 2, 2006 |
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Current U.S.
Class: |
424/133.1 ;
424/144.1; 514/44R |
Current CPC
Class: |
A61K 31/4745 20130101;
A61K 2039/505 20130101; A61K 31/575 20130101; A61K 31/7088
20130101; A61K 31/573 20130101; A61K 31/7088 20130101; A61K 45/06
20130101; A61P 35/00 20180101; A61K 31/70 20130101; A61K 31/337
20130101; A61K 31/4745 20130101; A61K 2300/00 20130101; A61K 39/395
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 31/337 20130101; A61K 31/575 20130101;
C07K 16/2887 20130101; A61K 31/573 20130101; A61K 39/395 20130101;
A61K 31/70 20130101 |
Class at
Publication: |
424/133.1 ;
514/44.R; 424/144.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7052 20060101 A61K031/7052; A61P 35/00
20060101 A61P035/00 |
Claims
1. A pharmaceutical composition comprising: an oligonucleotide and
a chemotherapeutic or immunotherapeutic agent, or a combination
thereof, wherein the oligonucleotide is any oligomer of at least 10
nucleotides that hybridizes under physiological conditions to SEQ
ID NO:1249 or the complement thereof.
2. The composition of claim 1, wherein the chemotherapeutic agent
comprises an anti-metabolite, an anthracycline, an EGFR inhibitor,
a kinase inhibitor or combinations thereof.
3. The composition of claim 2, wherein the chemotherapeutic
agent-comprises methotrexate, 5-fluorouracil, gemcitabine,
6-mercaptopurine, 6-thioguanine, fludarabine, cladarabine,
cytarabine, daunorubicin, doxorubicin, idarubicin, epirubicin,
mitoxantrone, gefitinib. erlotinib, cetuximab, imatinib mesylate,
lefunomide, midostaurin or combinations thereof.
4-5. (canceled)
6. The composition of claim 1, wherein the chemotherapeutic agent
comprises a taxane.
7. The composition of claim 6, wherein the taxane is selected from
paclitaxel, docetaxel, or combinations thereof.
8. The composition of claim 1, wherein the chemotherapeutic agent
comprises a camptothecin.
9. The composition of claim 8, wherein the camptothecin is selected
from irinotecan, topotecan, etoposide, vincristine, vinblastine,
vinorelbine, or combinations thereof.
10-12. (canceled)
13. The composition of claim 1, wherein the immunotherapeutic agent
is selected from rituximab, tositumomab, ibritumomab, bevacizumab,
trastuzumab or combinations thereof.
14-15. (canceled)
16. The composition of claim 1, wherein the chemotherapeutic agent
comprises a cocktail comprising an immunotherapy, an alkylating
agent, an anthracycline, a camptothecin and prednisone.
17-18. (canceled)
19. The composition of claim 16, wherein the chemotherapeutic agent
comprises a cocktail comprising rituximab, cyclophosphamide,
doxorubicin, vincristine and prednisone.
20. (canceled)
21. The composition of claim 1 wherein the oligonucleotide
comprises an oligomer that hybridizes under physiological
conditions to nucleotides 500-1525 of SEQ ID NO:1249 or the
complement thereof.
22. (canceled)
23. The composition of claim 1 wherein the oligonucleotide
comprises an oligomer that hybridizes under physiological
conditions to nucleotides 900-1125 of SEQ ID NO:1249 or the
complement thereof.
24. (canceled)
25. The composition of claim 1 wherein the oligonucleotide
comprises an oligomer that hybridizes under physiological
conditions to nucleotides 970-1045 of SEQ ID NO:1249 or the
complement thereof.
26. The composition of claim 1 wherein the oligonucleotide
comprises an oligomer selected from the group consisting of SEQ ID
NOs:1250, 1251, 1252, 1253, 1267-1477 or the complements
thereof.
27-28. (canceled)
29. The composition of claim 1, wherein the oligonucleotide
comprises SEQ ID NO:1250 or 1251.
30-31. (canceled)
32. The composition of claim 1, further comprising an additional
oligonucleotide.
33. The composition of claim 32, wherein the additional oligomer
comprises any one of SEQ ID NOs:1250-1253, 1267-1477.
34. The composition of claim 32 wherein the additional oligomer
comprises SEQ ID NO: 940 or SEQ ID NO:943.
35. (canceled)
36. The composition of claim 1, wherein the length of the
oligonucleotide is about 15 to 35 nucleotides.
37. The composition of claim 1 wherein the oligonucleotide has a
phosphorothiolate backbone.
38. A method of treating cancer comprising: (a) administering to a
patient an effective amount of an oligonucleotide comprising an
oligomer of at least 10 nucleotides that hybridizes under
physiological conditions to SEQ ID NO:1249 or the complement
thereof; and (b) administering to the patient an effective amount
of a chemotherapeutic or immunotherapeutic agent or a combination
thereof.
39. The method of claim 38, wherein the chemotherapeutic agent
comprises a cocktail comprising rituximab, cyclophosphamide,
doxorubicin, vincristine and prednisone.
40. The method of claim 38, wherein the immunotherapeutic agent
comprises rituximab.
41. The method of claim 38, further comprising administering to the
patient a radiation therapy.
42. The method of claim 38, further comprising excising cancerous
tissue from a patient.
43. (canceled)
44. The method of claim 38 wherein the oligonucleotide comprises an
oligomer that hybridizes under physiological conditions to
nucleotides 500-1525 of SEQ ID NO:1249 or the complement
thereof.
45. (canceled)
46. The method of claim 38 wherein the oligonucleotide comprises an
oligomer that hybridizes under physiological conditions to
nucleotides 900-1125 of SEQ ID NO:1249 or the complement
thereof.
47. (canceled)
48. The method of claim 38 wherein the oligonucleotide comprises an
oligomer that hybridizes under physiological conditions to
nucleotides 970-1045 of SEQ ID NO:1249 or the complement
thereof.
49. The method of claim 38 wherein the oligonucleotide comprises an
oligomer selected from the group consisting of SEQ ID NOs:1250,
1251, 1252, 1253, 1267-1477 or the complements thereof.
50-51. (canceled)
52. The method of claim 38, wherein the oligonucleotide comprises
SEQ ID NO:1250 or 1251.
53. (canceled)
54. The method of claim 38, further comprising an additional
oligonucleotide.
55. The method of claim 54, wherein the additional oligonucleotide
comprises any one of SEQ ID NOs:1250-1253 and 1267-1477.
56. The method of claim 54 wherein the additional oligonucleotide
comprises SEQ ID NO:940 or 943.
57. (canceled)
58. The method claim 38, wherein the length of the oligonucleotide
is about 15 to 35 nucleotides.
59. The method of claim 38 wherein the oligonucleotide has a
phosphorothiolate backbone.
60. A method of treating cancer comprising: administering to a
patient an effective amount of an oligonucleotide comprising SEQ ID
NO:1251 and administering to the patient an effective amount of
rituximab.
61. The method of claim 38, wherein the chemotherapy agent
comprises an immunotherapeutic agent, an alkylating agent, an
anthracycline, a camptothecin and Prednisone.
62. The method of claim 38, wherein the chemotherapy agent
comprises a taxane or a camptothecin.
63. The method of claim 62, wherein the chemotherapy agent
comprises docetaxel.
64. The method of claim 62, wherein the chemotherapy agent
comprises vincristine.
65. The method of claim 38, wherein the wherein the oligonucleotide
comprises SEQ ID NO:1251 and the chemotherapy agent comprises
docetaxel.
66. The method of claim 38, wherein the oligonucleotide comprises
SEQ ID NO:1251 and the chemotherapy agent comprises
vincristine.
67. The method of claim 38, wherein the cancer is selected from
prostate cancer, lymphoma, non-Hodgkin's lymphoma, Burkitt's
lymphoma, melanoma, breast cancer, myeloma, ovarian cancer, colon
cancer, lung cancer, adenocarcinoma and gastric cancer.
68. The method of claim 38, wherein the cancer is selected from
prostate cancer, melanoma, lymphoma, non-Hodgkin's lymphoma, and
Burkitt's lymphoma.
69. The pharmaceutical composition of claim 1, wherein the
oligonucleotide comprises SEQ ID NO:1251 and the immunotherapeutic
agent comprises rituximab.
70. The pharmaceutical composition of claim 1, wherein the
oligonucleotide comprises SEQ ID NO:1251 and the chemotherapy agent
comprises docetaxel.
71. The pharmaceutical composition of claim 1, wherein the
oligonucleotide comprises SEQ ID NO:1251 and the chemotherapy agent
comprises vincristine.
Description
[0001] This application claims the benefit of the U.S. Provisional
application No. 60/741,229, filed on Dec. 1, 2005 and the U.S.
Provisional application No. 60/778,304, filed on Mar. 2, 2006, both
of which are herein incorporated by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to cancer therapies and
methods of using the same. In particular, the present invention
provides combination cancer therapies comprising oligomers and
another therapeutic agent and methods of using the same.
BACKGROUND OF THE INVENTION
[0003] Oncogenes have become the central concept in understanding
cancer biology and may provide valuable targets for therapeutic
drugs. In many types of human tumors, including lymphomas and
leukemias, oncogenes are over-expressed and may be associated with
tumorigenicity (Tsujimoto et al., Science 228:1440-1443 [1985]).
For instance, high levels of expression of the human bcl-2 gene
have been found in all lymphomas with a t(14; 18) chromosomal
translocations including most follicular B cell lymphomas and many
large cell non-Hodgkin's lymphomas. High levels of bcl-2 gene
expression have also been found in certain leukemias that do not
have a t(14; 18) chromosomal translation, including most cases of
chronic lymphocytic leukemia acute, many lymphocytic leukemias of
the pre-B cell type, neuroblastomas, nasophryngeal carcinomas, and
many adenocarcinomas of the prostate, breast and colon. (Reed et
al., Cancer Res. 51:6529 [1991]; Yunis et al., New England J. Med.
320:1047; Campos et al., Blood 81:3091-3096 [1993]; McDonnell et
al., Cancer Res. 52:6940-6944 [1992); Lu et al., Int. J Cancer
53:29-35 [1993]; Bonner et al., Lab Invest. 68:43A [1993]. Other
oncogenes include TGF-.alpha., c-ki-ras, ras, her-2 and c-myc.
[0004] Gene expression, including oncogene expression, can be
inhibited by molecules that interfere with promoter function.
Accordingly, the expression of oncogenes may be inhibited by single
stranded oligonucleotides.
[0005] Cancer treatment typically includes chemotherapeutic agents
and often radiation therapy. In many cases, however, the current
treatments are not efficacious or do not cure the cancer.
Consequently, there is a need for more effective cancer
treatments.
SUMMARY OF THE INVENTION
[0006] In general, the invention relates to co-therapies for
treating cancer and methods of using the same. In one aspect, the
present invention provides co-therapies comprising an
oligonucleotide compound that hybridizes to SEQ ID NO:1249 or the
complement thereof, and another cancer therapy (e.g., chemotherapy
agent, radiation, surgery, and the like).
[0007] In another aspect the invention provides a pharmaceutical
composition comprising an oligonucleotide compound and a
chemotherapy agent, wherein the oligonucleotide compound is an
oligomer that hybridizes under physiological conditions to SEQ ID
NO:1249, SEQ ID NO:936 or the complement thereof. In one
embodiment, the chemotherapy agent comprises an anti-metabolite.
The anti-metabolite can include methotraxate, 5-fluorouracil,
gemcitabine, 6-mercaptopurine, 6-thioguanine, fludarabine,
cladribine, cytarabine or combinations thereof.
[0008] In another embodiment, the chemotherapy agent comprises an
anthracycline. The anthracycline can comprise daunorubicin,
doxorubicin, idarubicin, epirubicin, mitoxantrone or combinations
thereof.
[0009] In yet another embodiment the chemotherapy agent comprises a
taxane. The taxane can include _paclitaxel, docetaxel,
Taxotere.TM., Taxol or combinations thereof.
[0010] In still another embodiment, the chemotherapy agent
comprises a camptothecin. The campothecin can include irinotecan,
topotecan, etoposide, vincristine, vinblastine, vinorelbine or
combinations thereof.
[0011] In still yet another embodiment, the chemotherapy agent
comprises an EGFR inhibitor. The EGFR inhibitor can include
gefitinib, erlotinib, cetuximab or combinations thereof.
[0012] In another embodiment, the chemotherapy agent comprises one
or more immunotherapies. The immunotherapies can include rituximab,
tositumomab, ibritumomab, bevacizumab or combinations thereof.
[0013] In an additional embodiment, the chemotherapy agent
comprises one or more kinase inhibitors. The tyrosine kinase
inhibitor can include imatinib mesylate, lefunomide and
midostaurin.
[0014] In a further embodiment the chemotherapy agent comprises a
cocktail that includes an immunotherapy, an alkylating agent, an
anthracycline, a camptothecin and Prednisone. The immunotherapy can
include rituximab, the alkylating agent can include
cyclophosphamide, the anthracycline can include doxorubicin and the
campothecin can include vincristine.
[0015] In another embodiment the oligomer can comprise an oligomer
that hybridizes under physiological conditions to nucleotides
500-2026, 500-1525, 800-1225, 900-1125, 950-1075 or 970-1045 of SEQ
ID NO:1249 or the complement thereof. In yet another embodiment the
oligomer can comprise SEQ ID NOs:1251, 1252, 1253, 1267-1477 or the
complement thereof. In an additional embodiment, the oligomer
includes an oligomer that hybridizes under physiological conditions
with nucleotides 1-650 of SEQ ID NO:936 or the complement thereof.
In another embodiment, the oligomer comprises SEQ ID NO:940, 943 or
the complement thereof.
[0016] In another embodiment, the oligomer includes an additional
oligomer. The additional oligomer can include any one of SEQ ID
NOs:1250-1253, 1267-1477, 2-281, 283-461, 463-935, 937-1080,
1082-1248 and the complements thereof.
[0017] In yet another embodiment, the oligonucleotides are between
15 and 35 base pairs in length. In still another embodiment, the
oligonucleotides have a phosphorothioate backbone.
[0018] In another aspect, the invention provides a method of
treating cancer including administering to a patient an effective
amount of an oligonucleotide compound and administering to the
patient an effective amount of a chemotherapy agent.
[0019] One embodiment of this aspect includes chemotherapy agents
including a cocktail having Rituximab, Cyclophosphamide, an
anthracycline, a camptothecin and Prednisone. In another
embodiment, the chemotherapy agent comprises rituximab. Another
embodiment further includes administering to the patient a
radiation therapy. Still another embodiment further includes
excising cancerous tissue from a patient.
[0020] Other embodiments of this aspect include an oligonucleotide
compound that can include any oligomer that hybridizes under
physiological conditions to SEQ ID NO: 1249, SEQ ID NO:936 or the
complement thereof. Another embodiment includes an oligomer that
hybridizes under physiological conditions to nucleotides 500-2026,
500-1525, 800-1225, 900-1125, 950-1075 or 970-1045 of SEQ ID
NO:1249 or the complement thereof. Still another embodiment
includes an oligomer selected from SEQ ID NOs:1250, 1251, 1252,
1253, 1267-1477, 2-281, 283-461, 463-935, 937-1080, 1082-1248 and
the complements thereof.
[0021] In another embodiment the method further includes
administering an additional oligomer. The additional oligomer can
comprise any one of SEQ ID NOs:1250-1253, 1267-1477, 2-281,
283-461, 463-935, 937-1080, 1082-1248 and the complements
thereof.
[0022] In yet another embodiment the oligonucleotides are between
15 and 35 base pairs in length. In still another embodiment, the
oligonucleotides have a phosphorothioate backbone.
[0023] In a third aspect the invention provides a method of
treating cancer comprising administering to a patient an effective
amount of an oligonucleotide compound including SEQ ID NO:1251 and
administering to the patient an effective amount of rituximab.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows mean tumor volume of tumors in the PC-3 GFP
prostate carcinoma subcutaneous model following treatment with SEQ
ID NO:1251 and Taxotere.TM..
[0025] FIG. 2 shows mean final tumor volume of tumors in the PC-3
GFP prostate carcinoma subcutaneous model following treatment with
SEQ ID NO:1251 and Taxotere.TM..
[0026] FIG. 3 shows percentage increase in tumor size in PC-3
xenografts following treatment with SEQ ID NO:1251 and
Taxotere.TM..
[0027] FIG. 4 shows the response of PC-3 tumors in mice to
liposomal PNT-100 and docetaxel.
[0028] FIG. 5 shows the response of PC-3 tumors in mice to
liposomal PNT-100 and docetaxel delivered by i.v. bolus
injection
[0029] FIG. 6 shows the response of PC-3 tumors in mice to
liposomal PNT-100 and docetaxel delivered by i.v. bolus injection
and slow infusion.
[0030] FIG. 7 shows the response of Daudi xenografts to PNT-100 and
rituximab.
[0031] FIG. 8 shows a Kaplan-Meier plot of the response of Daudi
xenografts to PNT-100 and rituximab.
[0032] FIG. 9 shows the body weight change of Daudi
xenograft-bearing mice treated with PNT-100 and/or rituximab.
DETAILED DESCRIPTION
I. Definitions
[0033] As used herein, a "chemotherapy agent" is a
non-oligonucleotide based cytotoxic drug or non-oligonucleotide
based cytotoxic cocktail of drugs that that are intended to destroy
or inhibit malignant cells and tissues.
[0034] As used herein, "patient" refers to a mammal, including a
human.
[0035] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to, humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0036] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates
such as rodents, non-human primates, ovines, bovines, ruminants,
lagomorphs, porcines, caprines, equines, canines, felines, aves,
etc. and non-vertebrate animals such as drosophila and
nematode.
[0037] As used herein, an effective amount is defined as the amount
required to confer a therapeutic effect on the treated patient, and
is typically determined based on age, surface area, weight and
condition of the patient. The interrelationship of dosages for
animals and humans (based on milligrams per meter squared of body
surface) is described by Freireich et al., Cancer Chemother. Rep.,
50: 219 (1966). Body surface area can be approximately determined
from height and weight of the patient. See, e.g., Scientific
Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
[0038] As used herein, the term "wherein said chemotherapy agent is
present at less than one half the standard dose" refers to a dosage
that is less than one half (e.g., less than 50%, less than 40%,
less than 10% or less than 1%) of the minimum value of the standard
dosage range used for dosing humans. In some embodiments, the
standard dosage range is the dosage range recommended by the
manufacturer. In other embodiments, the standard dosage range is
the range utilized by a medical doctor in the field. In still other
embodiments, the standard dosage range is the range considered the
normal standard of care in the field. The particular dosage within
the dosage range is determined, for example by the age, weight, and
health of the subject as well as the type of cancer being
treated.
[0039] As used herein, the term "under conditions such that
expression of said gene is inhibited" refers to conditions in which
an oligonucleotide of the present invention hybridizes to a gene
(e.g., a regulatory region of the gene) and inhibits transcription
of the gene by at least 10%, at least 25%, at least 50%, or at
least 90% relative to the level of transcription in the absence of
the oligonucleotide. The present invention is not limited to the
inhibition of expression of a particular gene. Exemplary genes
include, without limitation, c-ki-ras, c-Ha-ras, c-myc, her-2,
TGF-.alpha., and bcl-2.
[0040] As used herein, the term "under conditions such that growth
of said cell is reduced" refers to conditions where an
oligonucleotide of the present invention, when administered to a
cell (e.g., a cancer) reduces the rate of growth of the cell by at
least 10%, at least 25%, at least 50% or at least 90% relative to
the rate of growth of the cell in the absence of the
oligonucleotide.
[0041] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to,
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA.
[0042] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment is retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0043] As used herein, the "regulatory region" of a gene is any
part of a gene that regulates the expression of a gene, including,
without limitation, transcriptional and translational regulation.
The regions include without limitation the 5' and 3' regions of
genes, binding sites for regulatory factors, including without
limitation transcription factor binding sites. The regions also
include regions that are as long as 20,000 or more base pairs
upstream or downstream of translational start sites, so long as the
region is involved in any way in the regulation of the expression
of the gene. The region may be as short as 20 base pairs or as long
as thousands of base pairs.
[0044] As used herein, the term "heterologous gene" refers to a
gene that is not in its natural environment. For example, a
heterologous gene includes a gene from one species introduced into
another species. A heterologous gene also includes a gene native to
an organism that has been altered in some way (e.g., mutated, added
in multiple copies, linked to non-native regulatory sequences,
etc). Heterologous genes are distinguished from endogenous genes in
that the heterologous gene sequences are typically joined to DNA
sequences that are not found naturally associated with the gene
sequences in the chromosome or are associated with portions of the
chromosome not found in nature (e.g., genes expressed in loci where
the gene is not normally expressed).
[0045] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (i.e., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
mRNA. Gene expression can be regulated at many stages in the
process. "Up-regulation" or "activation" refers to regulation that
increases the production of gene expression products (i.e., RNA or
protein), while "down-regulation" or "repression" refers to
regulation that decrease production. Molecules (e.g., transcription
factors) that are involved in up-regulation or down-regulation are
often called "activators" and "repressors," respectively.
[0046] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences that are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters and enhancers that control
or influence the transcription of the gene. The 3' flanking region
may contain sequences that direct the termination of transcription,
post-transcriptional cleavage and polyadenylation.
[0047] The term "wild-type" refers to a gene or gene product
isolated from a naturally occurring source. A wild-type gene is
that which is most frequently observed in a population and is thus
arbitrarily designed the "normal" or "wild-type" form of the gene.
In contrast, the term "modified" or "mutant" refers to a gene or
gene product that displays modifications in sequence and or
functional properties (i.e., altered characteristics) when compared
to the wild-type gene or gene product. It is noted that naturally
occurring mutants can be isolated; these are identified by the fact
that they have altered characteristics (including altered nucleic
acid sequences) when compared to the wild-type gene or gene
product.
[0048] As used herein, the terms "an oligonucleotide having a
nucleotide sequence encoding a gene" and "polynucleotide having a
nucleotide sequence encoding a gene," means a nucleic acid sequence
comprising the coding region of a gene or in other words the
nucleic acid sequence that encodes a gene product. The coding
region may be present in a cDNA, genomic DNA or RNA form. When
present in a DNA form, the oligonucleotide or polynucleotide may be
single-stranded (i.e., the sense strand) or double-stranded.
Suitable control elements such as enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close
proximity to the coding region of the gene if needed to permit
proper initiation of transcription and/or correct processing of the
primary RNA transcript. Alternatively, the coding region utilized
in the expression vectors of the present invention may contain
endogenous enhancers/promoters, splice junctions, intervening
sequences, polyadenylation signals, etc. or a combination of both
endogenous and exogenous control elements.
[0049] As used herein, the term "oligonucleotide," refers to a
short length of single-stranded polynucleotide chain.
Oligonucleotides are typically less than 200 residues long (e.g.,
between 8 and 100), however, as used herein, the term is also
intended to encompass longer polynucleotide chains (e.g., as large
as 5000 residues). Oligonucleotides are often referred to by their
length. For example a 24 residue oligonucleotide is referred to as
a "24-mer." Oligonucleotides can form secondary and tertiary
structures by self-hybridizing or by hybridizing to other
polynucleotides. Such structures can include, but are not limited
to, duplexes, hairpins, cruciforms, bends, and triplexes.
[0050] In some embodiments, oligonucleotides are "antigenes." As
used herein, the term "antigene" refers to an oligonucleotide that
hybridizes to the promoter region of a gene. In some embodiments,
the hybridization of the antigene to the promoter inhibits
expression of the gene.
[0051] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, for the sequence "A-G-T," is complementary to the sequence
"T-C-A." Complementarity may be "partial," in which only some of
the nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, as well as
detection methods that depend upon binding between nucleic
acids.
[0052] As used herein, the term "completely complementary," for
example when used in reference to an oligonucleotide of the present
invention refers to an oligonucleotide where all of the nucleotides
are complementary to a target sequence (e.g., a gene).
[0053] As used herein, the term "partially complementary," for
example when used in reference to an oligonucleotide of the present
invention, refers to an oligonucleotide where at least one
nucleotide is not complementary to the target sequence. Exemplary
partially complementary oligonucleotides are those that can still
hybridize to the target sequence under physiological conditions.
The term "partially complementary" refers to oligonucleotides that
have regions of one or more non-complementary nucleotides both
internal to the oligonucleotide or at either end. Oligonucleotides
with mismatches at the ends may still hybridize to the target
sequence.
[0054] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is a nucleic acid
molecule that at least partially inhibits a completely
complementary nucleic acid molecule from hybridizing to a target
nucleic acid is "substantially homologous." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous nucleic acid molecule
to a target under conditions of low stringency. This is not to say
that conditions of low stringency are such that non-specific
binding is permitted; low stringency conditions require that the
binding of two sequences to one another be a specific (i.e.,
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target that is substantially
non-complementary (e.g., less than about 30% identity); in the
absence of non-specific binding the probe will not hybridize to the
second non-complementary target.
[0055] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described above.
[0056] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0057] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids. A single
molecule that contains pairing of complementary nucleic acids
within its structure is said to be "self-hybridized."
[0058] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other
references include more sophisticated computations that take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0059] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Under "low stringency conditions" a
nucleic acid sequence of interest will hybridize to its exact
complement, sequences with single base mismatches, closely related
sequences (e.g., sequences with 90% or greater homology), and
sequences having only partial homology (e.g., sequences with 50-90%
homology). Under "medium stringency conditions," a nucleic acid
sequence of interest will hybridize only to its exact complement,
sequences with single base mismatches, and closely related
sequences (e.g., 90% or greater homology). Under "high stringency
conditions," a nucleic acid sequence of interest will hybridize
only to its exact complement, and (depending on conditions such a
temperature) sequences with single base mismatches. In other words,
under conditions of high stringency the temperature can be raised
so as to exclude hybridization to sequences with single base
mismatches.
[0060] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times. Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0061] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4 H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times. Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0062] "Low stringency conditions" comprise conditions equivalent
to binding or hybridization at 42.degree. C. in a solution
consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l
NaH.sub.2PO.sub.4 H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4
with NaOH), 0.1% SDS, 5.times. Denhardt's reagent [50.times.
Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5
g BSA (Fraction V; Sigma)] and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 5.times.SSPE, 0.1%
SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0063] The present invention is not limited to the hybridization of
probes of about 500 nucleotides in length. The present invention
contemplates the use of probes between approximately 8 nucleotides
up to several thousand (e.g., at least 5000) nucleotides in length.
One skilled in the relevant understands that stringency conditions
may be altered for probes of other sizes (See e.g., Anderson and
Young, Quantitative Filter Hybridization, in Nucleic Acid
Hybridization [1985] and Sambrook et al., Molecular Cloning--A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 2001, and Current Protocols in Molecular Biology, M.
Ausubel et al., eds., (Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
and supplements through 2006).
[0064] It is well known in the art that numerous equivalent
conditions may be employed to comprise low stringency conditions;
factors such as the length and nature (DNA, RNA, base composition)
of the probe and nature of the target (DNA, RNA, base composition,
present in solution or immobilized, etc.) and the concentration of
the salts and other components (e.g., the presence or absence of
formamide, dextran sulfate, polyethylene glycol) are considered and
the hybridization solution may be varied to generate conditions of
low stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.) (See
definition above for "stringency").
[0065] As used herein, the term "physiological conditions" refers
to specific stringency conditions that approximate or are
conditions inside an animal (e.g., a human). Exemplary
physiological conditions for use in vitro include, but are not
limited to, 37.degree. C., 95% air, 5% CO.sub.2, commercial medium
for culture of mammalian cells (e.g., DMEM media available from
Gibco, Md.), 5-10% serum (e.g., calf serum or horse serum),
additional buffers, and optionally hormone (e.g., insulin and
epidermal growth factor).
[0066] As used herein, the term "isolated" when used in relation to
a nucleic acid, as in "an isolated oligonucleotide" or "isolated
polynucleotide" refers to a nucleic acid sequence that is
identified and separated from at least one component or contaminant
with which it is ordinarily associated in its natural source.
Isolated nucleic acid is such present in a form or setting that is
different from that in which it is found in nature. In contrast,
non-isolated nucleic acids as nucleic acids such as DNA and RNA
found in the state they exist in nature. For example, a given DNA
sequence (e.g., a gene) is found on the host cell chromosome in
proximity to neighboring genes; RNA sequences, such as a specific
mRNA sequence encoding a specific protein, are found in the cell as
a mixture with numerous other mRNAs that encode a multitude of
proteins. However, isolated nucleic acid encoding a given protein
includes, by way of example, such nucleic acid in cells ordinarily
expressing the given protein where the nucleic acid is in a
chromosomal location different from that of natural cells, or is
otherwise flanked by a different nucleic acid sequence than that
found in nature. The isolated nucleic acid, oligonucleotide, or
polynucleotide may be present in single-stranded or double-stranded
form. When an isolated nucleic acid, oligonucleotide or
polynucleotide is to be utilized to express a protein, the
oligonucleotide or polynucleotide will contain at a minimum the
sense or coding strand (i.e., the oligonucleotide or polynucleotide
may be single-stranded), but may contain both the sense and
anti-sense strands (i.e., the oligonucleotide or polynucleotide may
be double-stranded).
[0067] As used herein, the term "purified" or "to purify" refers to
the removal of components (e.g., contaminants) from a sample. For
example, recombinant polypeptides are expressed in bacterial host
cells and the polypeptides are purified by the removal of host cell
proteins; the percent of recombinant polypeptides is thereby
increased in the sample.
[0068] The term "epitope" as used herein refers to that portion of
an antigen that makes contact with a particular antibody.
[0069] When a protein or fragment of a protein is used to immunize
a host animal, numerous regions of the protein may induce the
production of antibodies which bind specifically to a given region
or three-dimensional structure on the protein; these regions or
structures are referred to as "antigenic determinants." An
antigenic determinant may compete with the intact antigen (i.e.,
the "immunogen" used to elicit the immune response) for binding to
an antibody.
[0070] As used herein, the term "western blot" refers to the
analysis of protein(s) (or polypeptides) immobilized onto a support
such as nitrocellulose or a membrane. The proteins are run on
acrylamide gels to separate the proteins, followed by transfer of
the protein from the gel to a solid support, such as nitrocellulose
or a nylon membrane. The immobilized proteins are then exposed to
antibodies with reactivity against an antigen of interest. The
binding of the antibodies may be detected by various methods,
including the use of radiolabeled antibodies.
[0071] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, transformed cell lines, finite cell lines (e.g.,
non-transformed cells), and any other cell population maintained in
vitro.
[0072] As used, the term "eukaryote" refers to organisms
distinguishable from "prokaryotes." It is intended that the term
encompass all organisms with cells that exhibit the usual
characteristics of eukaryotes, such as the presence of a true
nucleus bounded by a nuclear membrane, within which lie the
chromosomes, the presence of membrane-bound organelles, and other
characteristics commonly observed in eukaryotic organisms. Thus,
the term includes, but is not limited to such organisms as fungi,
protozoa, and animals (e.g., humans).
[0073] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments can consist of, but
are not limited to, test tubes and cell culture. The term "in vivo"
refers to the natural environment (e.g., an animal or a cell) and
to processes or reaction that occur within a natural
environment.
[0074] The terms "test compound" and "candidate compound" refer to
any chemical entity, pharmaceutical, drug, and the like that is a
candidate for use to treat or prevent a disease, illness, sickness,
or disorder of bodily function (e.g., cancer). Test compounds
comprise both known and potential therapeutic compounds. A test
compound can be determined to be therapeutic by screening using the
screening methods of the present invention. In some embodiments of
the present invention, test compounds include antisense
compounds.
[0075] As used herein, the term "chemotherapeutic agents" refers to
compounds that are useful in the treatment of disease (e.g.,
cancer). Exemplary chemotherapeutic agents affective against cancer
include, but are not limited to, daunorubicin, dactinomycin,
doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,
melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine,
cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR),
methotrexate (MTX), colchicine, vincristine, vinblastine,
etoposide, teniposide, cisplatin and diethylstilbestrol (DES).
[0076] As used herein, the term "sample" is used in its broadest
sense. In one sense, it is meant to include a specimen or culture
obtained from any source, as well as biological and environmental
samples. Biological samples may be obtained from animals (including
humans) and encompass fluids, solids, tissues, and gases.
Biological samples include blood products, such as plasma, serum
and the like. Environmental samples include environmental material
such as surface matter, soil, water, crystals and industrial
samples. Such examples are not however to be construed as limiting
the sample types applicable to the present invention.
[0077] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed.
Additionally, general principles of organic chemistry are described
in "Organic Chemistry", Thomas Sorrell, University Science Books,
Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed.,
Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York:
2001.
[0078] As used herein the term "aliphatic` encompasses the terms
alkyl, alkenyl, alkynyl, each of which being optionally substituted
as set forth below.
[0079] As used herein, an "alkyl" group refers to a saturated
aliphatic hydrocarbon group containing 1-8 (e.g., 1-6 or 1-4)
carbon atoms. An alkyl group can be straight or branched. Examples
of alkyl groups include, but are not limited to, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
n-pentyl, n-heptyl or 2-ethylhexyl. An alkyl group can be
substituted (i.e., optionally substituted) with one or more
substituents such as halo, cycloaliphatic, heterocycloaliphatic,
aryl, heteroaryl, alkoxy, aroyl, heteroaroyl,
cycloaliphaticcarbonyl, (heterocycloaliphatic)carbonyl, nitro,
cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl,
cycloaliphaticoxy, heterocycloaliphaticoxy, aryloxy, heteroaryloxy,
aralkyloxy, heteroarylalkoxy, or hydroxy. Without limitation, some
examples of substituted alkyls include carboxyalkyl (such as
HOOC-alkyl, alkoxycarbonylalkyl and alkylcarbonyloxyalkyl),
cyanoalkyl, hydroxyalkyl, alkoxyalkyl, acylalkyl, hydroxyalkyl,
aralkyl, (alkoxyaryl)alkyl, (sulfonylamino)alkyl (such as
(alkylsulfonylamino)alkyl), aminoalkyl, amidoalkyl,
(cycloaliphatic)alkyl, cyanoalkyl, or haloalkyl.
[0080] As used herein, an "alkenyl" group refers to an aliphatic
carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and
at least one double bond. Like an alkyl group, an alkenyl group can
be straight or branched. Examples of an alkenyl group include, but
are not limited to, allyl, isoprenyl, 2-butenyl and 2-hexenyl. An
alkenyl group can be optionally substituted with one or more
substituents such as halo, cycloaliphatic, heterocycloaliphatic,
aryl, heteroaryl, alkoxy, aroyl, heteroaroyl,
(cycloaliphatic)carbonyl, (heterocycloaliphatic)carbonyl, nitro,
cyano, amino, amido, acyl, sulfonyl, sulfinyl, sulfanyl, sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, carboxy, carbamoyl,
(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,
heteroaryloxy, aralkyloxy, (heteroaryl)alkoxy, or hydroxy.
[0081] As used herein, an "alkynyl" group refers to an aliphatic
carbon group that contains 2-8 (e.g., 2-6 or 2-4) carbon atoms and
has at least one triple bond. An alkynyl group can be straight or
branched. Examples of an alkynyl group include, but are not limited
to, propargyl and butynyl. An alkynyl group can be optionally
substituted with one or more substituents such as halo,
cycloaliphatic, heterocycloaliphatic, aryl, heteroaryl, alkoxy,
aroyl, heteroaroyl, (cycloaliphatic)carbonyl,
(heterocycloaliphatic)carbonyl, nitro, cyano, amino, amido, acyl,
sulfonyl, sulfinyl, sulfanyl, sulfoxy, urea, thiourea, sulfamoyl,
sulfamide, oxo, carboxy, carbamoyl, (cycloaliphatic)oxy,
(heterocycloaliphatic)oxy, aryloxy, heteroaryloxy, aralkyloxy,
(heteroaryl)alkoxy, or hydroxy.
[0082] As used herein, an "amido" encompasses both "aminocarbonyl"
and "carbonylamino". These terms when used alone or in connection
with another group refers to an amido group such as
N(R.sup.X).sub.2--C(O)-- or R.sup.YC(O)--N(R.sup.X).sub.2-- when
used terminally and --C(O)--N(R.sup.X)-- or --N(R.sup.X)--C(O)--
when used internally, wherein R.sup.X and R.sup.Y are defined
below. Examples of amido groups include alkylamido (such as
alkylcarbonylamino and alkylcarbonylamino),
(heterocycloaliphatic)amido, (heteroaralkyl)amido,
(heteroaryl)amido, (heterocycloalkyl)alkylamido, arylamido,
aralkylamido, (cycloalkyl)alkylamido, and cycloalkylamido.
[0083] As used herein, an "amino" group refers to --NR.sup.XR.sup.Y
wherein each of R.sup.X and R.sup.Y is independently hydrogen,
alkyl, cycloaliphatic, (cycloaliphatic)aliphatic, aryl,
araliphatic, heterocycloaliphatic, (heterocycloaliphatic)aliphatic,
heteroaryl, carboxy, sulfanyl, sulfinyl, sulfonyl,
(aliphatic)carbonyl, (cycloaliphatic)carbonyl,
((cycloaliphatic)aliphatic)carbonyl, arylcarbonyl,
(araliphatic)carbonyl, (heterocycloaliphatic)carbonyl,
((heterocycloaliphatic)aliphatic)carbonyl, (heteroaryl)carbonyl, or
(heteroaraliphatic)carbonyl, each of which being defined herein and
being optionally substituted. Examples of amino groups include
alkylamino, dialkylamino, and arylamino.
[0084] When the term "amino" is not the terminal group (e.g.,
alkylcarbonylamino), it is represented by --NR.sup.X--. R.sup.X has
the same meaning as defined above.
[0085] As used herein, an "aryl" group used alone or as part of a
larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl" refers
to monocyclic (e.g., phenyl); bicyclic (e.g., indenyl,
naphthalenyl, tetrahydronaphthyl, tetrahydroindenyl); and tricyclic
(e.g., fluorenyl tetrahydrofluorenyl, or tetrahydroanthracenyl,
anthracenyl). The bicyclic and tricyclic groups include benzofused
2-3 membered carbocyclic rings. For example, a benzofused group
includes phenyl fused with two or more C.sub.4-8 carbocyclic
moieties. An aryl is optionally substituted with one or more
substituents including aliphatic [e.g., alkyl, alkenyl, or
alkynyl]; cycloaliphatic; (cycloaliphatic)aliphatic;
heterocycloaliphatic; (heterocycloaliphatic)aliphatic; aryl;
heteroaryl; alkoxy; (cycloaliphatic)oxy; (heterocycloaliphatic)oxy;
aryloxy; heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy;
aroyl; heteroaroyl; amino; oxo (on a non-aromatic carbocyclic ring
of a benzofused bicyclic or tricyclic aryl); nitro; carboxy; amido;
acyl [e.g., aliphaticcarbonyl; (cycloaliphatic)carbonyl;
((cycloaliphatic)aliphatic)carbonyl; (araliphatic)carbonyl;
(heterocycloaliphatic)carbonyl;
((heterocycloaliphatic)aliphatic)carbonyl; and
(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl and
aminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,
aliphaticsulfanyl]; nitro; cyano; halo; hydroxyl; mercapto;
sulfoxy; urea; thiourea; sulfamoyl; sulfamide; and carbamoyl.
Alternatively, an aryl can be unsubstituted.
[0086] Non-limiting examples of substituted aryls include haloaryl
[e.g., mono-, di (such as p,m-dihaloaryl), and (trihalo)aryl];
(carboxy)aryl [e.g., (alkoxycarbonyl)aryl,
((arylalkyl)carbonyloxy)aryl, and (alkoxycarbonyl)aryl];
(amido)aryl [e.g., (aminocarbonyl)aryl,
(((alkylamino)alkyl)aminocarbonyl)aryl, (alkylcarbonyl)aminoaryl,
(arylaminocarbonyl)aryl, and (((heteroaryl)amino)carbonyl)aryl];
aminoaryl [e.g., ((alkylsulfonyl)amino)aryl and
((dialkyl)amino)aryl]; (cyanoalkyl)aryl; (alkoxy)aryl;
(sulfamoyl)aryl [e.g., (aminosulfonyl)aryl]; (alkylsulfonyl)aryl;
(cyano)aryl; (hydroxyalkyl)aryl; ((alkoxy)alkyl)aryl;
(hydroxyl)aryl, ((carboxy)alkyl)aryl; (((dialkyl)amino)alkyl)aryl;
(nitroalkyl)aryl; (((alkylsulfonyl)amino)alkyl)aryl;
((heterocycloaliphatic)carbonyl)aryl; ((alkylsulfonyl)alkyl)aryl;
(cyanoalkyl)aryl; (hydroxyalkyl)aryl; (alkylcarbonyl)aryl;
alkylaryl; (trihaloalkyl)aryl; p-amino-m-alkoxycarbonylaryl;
p-amino-m-cyanoaryl; p-halo-m-aminoaryl; and
(m-(heterocycloaliphatic)-o-(alkyl))aryl.
[0087] As used herein, an "araliphatic" such as an "aralkyl" group
refers to an aliphatic group (e.g., a C.sub.1-4 alkyl group) that
is substituted with an aryl group. "Aliphatic," "alkyl," and "aryl"
are defined herein. An example of an araliphatic such as an aralkyl
group is benzyl.
[0088] As used herein, a "bicyclic ring system" includes 8-12
(e.g., 9, 10, or 11) membered structures that form two rings,
wherein the two rings have at least one atom in common (e.g., 2
atoms in common). Bicyclic ring systems include bicycloaliphatics
(e.g., bicycloalkyl or bicycloalkenyl), bicycloheteroaliphatics,
bicyclic aryls, and bicyclic heteroaryls.
[0089] As used herein, a "cycloaliphatic" group encompasses a
"cycloalkyl" group and a "cycloalkenyl" group, each of which being
optionally substituted as set forth below.
[0090] As used herein, a "cycloalkyl" group refers to a saturated
carbocyclic mono- or bicyclic (fused or bridged) ring of 3-10
(e.g., 5-10) carbon atoms. Examples of cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
adamantyl, norbornyl, cubyl, octahydro-indenyl, decahydro-naphthyl,
bicyclo[3.2.1 ]octyl, bicyclo[2.2.2]octyl, bicyclo[3.3.1 ]nonyl,
bicyclo[3.3.2.]decyl, bicyclo[2.2.2]octyl, adamantyl,
azacycloalkyl, or ((aminocarbonyl)cycloalkyl)cycloalkyl. A
"cycloalkenyl" group, as used herein, refers to a non-aromatic
carbocyclic ring of 3-10 (e.g., 4-8) carbon atoms having one or
more double bonds. Examples of cycloalkenyl groups include
cyclopentenyl, 1,4-cyclohexa-di-enyl, cycloheptenyl, cyclooctenyl,
hexahydro-indenyl, octahydro-naphthyl, cyclohexenyl, cyclopentenyl,
bicyclo[2.2.2]octenyl, and bicyclo[3.3.1 ]nonenyl.
[0091] A cycloalkyl or cycloalkenyl group can be optionally
substituted with one or more substituents such as aliphatic [e.g.,
alkyl, alkenyl, or alkynyl], cycloaliphatic,
(cycloaliphatic)aliphatic, heterocycloaliphatic,
(heterocycloaliphatic)aliphatic, aryl, heteroaryl, alkoxy,
(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,
heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,
heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,
(cycloaliphatic)carbonylamino,
((cycloaliphatic)aliphatic)carbonylamino, (aryl)carbonylamino,
(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,
((heterocycloaliphatic)aliphatic)carbonylamino,
(heteroaryl)carbonylamino, and (heteroaraliphatic)carbonylamino],
nitro, carboxy [e.g., HOOC--, alkoxycarbonyl, and
alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl,
((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl,
(heterocycloaliphatic)carbonyl,
((heterocycloaliphatic)aliphatic)carbonyl, and
(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy,
mercapto, sulfonyl [e.g., alkylsulfonyl and arylsulfonyl], sulfinyl
[e.g., alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0092] As used herein, "cyclic moiety" includes cycloaliphatic,
heterocycloaliphatic, aryl, or heteroaryl, each of which has been
defined previously.
[0093] As used herein, the term "heterocycloaliphatic" encompasses
a heterocycloalkyl group and a heterocycloalkenyl group, each of
which being optionally substituted as set forth below.
[0094] As used herein, a "heterocycloalkyl" group refers to a 3-10
membered mono- or bicylic (fused or bridged) (e.g., 5- to
10-membered mono- or bicyclic) saturated ring structure, in which
one or more of the ring atoms is a heteroatom (e.g., N, O, S, or
combinations thereof). Examples of a heterocycloalkyl group include
piperidyl, piperazyl, tetrahydropyranyl, tetrahydrofuryl,
1,4-dioxolanyl, 1,4-dithianyl, 1,3-dioxolanyl, oxazolidyl,
isoxazolidyl, morpholinyl, thiomorpholyl, octahydro-benzofuryl,
octahydro-chromenyl, octahydro-thiochromenyl, octahydro-indolyl,
octahydro-pyrindinyl, decahydro-quinolinyl,
octahydro-benzo[b]thiopheneyl, 2-oxa-bicyclo[2.2.2]octyl,
1-aza-bicyclo[2.2.2]octyl, 3-aza-bicyclo[3.2.1]octyl, and
2,6-dioxa-tricyclo[3.3.1.0.sup.3,7]nonyl. A monocyclic
heterocycloalkyl group can be fused with a phenyl moiety such as
tetrahydroisoquinoline. A "heterocycloalkenyl" group, as used
herein, refers to a mono- or bicylic (e.g., 5- to 10-membered mono-
or bicyclic) non-aromatic ring structure having one or more double
bonds, and wherein one or more of the ring atoms is a heteroatom
(e.g., N, O, or S). Monocyclic and bicycloheteroaliphatics are
numbered according to standard chemical nomenclature.
[0095] A heterocycloalkyl or heterocycloalkenyl group can be
optionally substituted with one or more substituents such as
aliphatic [e.g., alkyl, alkenyl, or alkynyl], cycloaliphatic,
(cycloaliphatic) aliphatic, heterocycloaliphatic,
(heterocycloaliphatic)aliphatic, aryl, heteroaryl, alkoxy,
(cycloaliphatic)oxy, (heterocycloaliphatic)oxy, aryloxy,
heteroaryloxy, (araliphatic)oxy, (heteroaraliphatic)oxy, aroyl,
heteroaroyl, amino, amido [e.g., (aliphatic)carbonylamino,
(cycloaliphatic)carbonylamino, ((cycloaliphatic)
aliphatic)carbonylamino, (aryl)carbonylamino,
(araliphatic)carbonylamino, (heterocycloaliphatic)carbonylamino,
((heterocycloaliphatic)aliphatic)carbonylamino,
(heteroaryl)carbonylamino, and (heteroaraliphatic)carbonylamino],
nitro, carboxy [e.g., HOOC--, alkoxycarbonyl, and
alkylcarbonyloxy], acyl [e.g., (cycloaliphatic)carbonyl,
((cycloaliphatic) aliphatic)carbonyl, (araliphatic)carbonyl,
(heterocycloaliphatic)carbonyl,
((heterocycloaliphatic)aliphatic)carbonyl, and
(heteroaraliphatic)carbonyl], nitro, cyano, halo, hydroxy,
mercapto, sulfonyl [e.g., alkylsulfonyl and arylsulfonyl], sulfinyl
[e.g., alkylsulfinyl], sulfanyl [e.g., alkylsulfanyl], sulfoxy,
urea, thiourea, sulfamoyl, sulfamide, oxo, or carbamoyl.
[0096] A "heteroaryl" group, as used herein, refers to a
monocyclic, bicyclic, or tricyclic ring structure having 4 to 15
ring atoms wherein one or more of the ring atoms is a heteroatom
(e.g., N, O, S, or combinations thereof) and wherein one ore more
rings of the bicyclic or tricyclic ring structure is aromatic. A
heteroaryl group includes a benzofused ring system having 2 to 3
rings. For example, a benzofused group includes benzo fused with
one or two 4 to 8 membered heterocycloaliphatic moieties (e.g.,
indolizyl, indolyl, isoindolyl, 3H-indolyl, indolinyl,
benzo[b]furyl, benzo[b]thiophenyl, quinolinyl, or isoquinolinyl).
Some examples of heteroaryl are azetidinyl, pyridyl, 1H-indazolyl,
furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl,
tetrazolyl, benzofuryl, isoquinolinyl, benzthiazolyl, xanthene,
thioxanthene, phenothiazine, dihydroindole, benzo[1,3]dioxole,
benzo[b]furyl, benzo[b]thiophenyl, indazolyl, benzimidazolyl,
benzthiazolyl, puryl, cinnolyl, quinolyl, quinazolyl,cinnolyl,
phthalazyl, quinazolyl, quinoxalyl, isoquinolyl, 4H-quinolizyl,
benzo-1,2,5-thiadiazolyl, or 1,8-naphthyridyl.
[0097] Without limitation, monocyclic heteroaryls include furyl,
thiophenyl, 2H-pyrrolyl, pyrrolyl, oxazolyl, thazolyl, imidazolyl,
pyrazolyl, isoxazolyl, isothiazolyl, 1,3,4-thiadiazolyl,
2H-pyranyl, 4-H-pranyl, pyridyl, pyridazyl, pyrimidyl, pyrazolyl,
pyrazyl, or 1,3,5-triazyl. Monocyclic heteroaryls are numbered
according to standard chemical nomenclature.
[0098] Without limitation, bicyclic heteroaryls include indolizyl,
indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furyl,
benzo[b]thiophenyl, quinolinyl, isoquinolinyl, indazolyl,
benzimidazyl, benzthiazolyl, purinyl, 4H-quinolizyl, quinolyl,
isoquinolyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl,
1,8-naphthyridyl, or pteridyl. Bicyclic heteroaryls are numbered
according to standard chemical nomenclature.
[0099] A heteroaryl is optionally substituted with one or more
substituents such as aliphatic [e.g., alkyl, alkenyl, or alkynyl];
cycloaliphatic; (cycloaliphatic)aliphatic; heterocycloaliphatic;
(heterocycloaliphatic)aliphatic; aryl; heteroaryl; alkoxy;
(cycloaliphatic)oxy; (heterocycloaliphatic)oxy; aryloxy;
heteroaryloxy; (araliphatic)oxy; (heteroaraliphatic)oxy; aroyl;
heteroaroyl; amino; oxo (on a non-aromatic carbocyclic or
heterocyclic ring of a bicyclic or tricyclic heteroaryl); nitro;
carboxy; amido; acyl [e.g., aliphaticcarbonyl;
(cycloaliphatic)carbonyl; ((cycloaliphatic)aliphatic)carbonyl;
(araliphatic)carbonyl; (heterocycloaliphatic)carbonyl;
((heterocycloaliphatic)aliphatic)carbonyl; and
(heteroaraliphatic)carbonyl]; sulfonyl [e.g., aliphaticsulfonyl and
aminosulfonyl]; sulfinyl [e.g., aliphaticsulfinyl]; sulfanyl [e.g.,
aliphaticsulfanyl]; nitro; cyano; halo; hydroxyl; mercapto;
sulfoxy; urea; thiourea; sulfamoyl; sulfamide; or carbamoyl.
Alternatively, a heteroaryl can be unsubstituted.
[0100] Non-limiting examples of substituted heteroaryls include
(halo)heteroaryl [e.g., mono- and di-(halo)heteroaryl];
(carboxy)heteroaryl [e.g., (alkoxycarbonyl)heteroaryl];
cyanoheteroaryl; aminoheteroaryl [e.g.,
((alkylsulfonyl)amino)heteroaryl and ((dialkyl)amino)heteroaryl];
(amido)heteroaryl [e.g., aminocarbonylheteroaryl,
((alkylcarbonyl)amino)heteroaryl,
((((alkyl)amino)alkyl)aminocarbonyl)heteroaryl,
(((heteroaryl)amino)carbonyl)heteroaryl,
((heterocycloaliphatic)carbonyl)heteroaryl, and
((alkylcarbonyl)amino)heteroaryl]; (cyanoalkyl)heteroaryl;
(alkoxy)heteroaryl; (sulfamoyl)heteroaryl [e.g.,
(aminosulfonyl)heteroaryl]; (sulfonyl)heteroaryl [e.g.,
(alkylsulfonyl)heteroaryl]; (hydroxyalkyl)heteroaryl;
(alkoxyalkyl)heteroaryl; (hydroxyl)heteroaryl;
((carboxy)alkyl)heteroaryl; [((dialkyl)amino)alkyl]heteroaryl;
(heterocycloaliphatic)heteroaryl; (cycloaliphatic)heteroaryl;
(nitroalkyl)heteroaryl; (((alkylsulfonyl)amino)alkyl)heteroaryl;
((alkylsulfonyl)alkyl)heteroaryl; (cyanoalkyl)heteroaryl;
(acyl)heteroaryl [e.g., (alkylcarbonyl)heteroaryl];
(alkyl)heteroaryl, and (haloalkyl)heteroaryl [e.g.,
trihaloalkylheteroaryl].
[0101] A "heteroaraliphatic (such as a heteroaralkyl group) as used
herein, refers to an aliphatic group (e.g., a C.sub.1-4 alkyl
group) that is substituted with a heteroaryl group. "Aliphatic,"
"alkyl," and "heteroaryl" have been defined above.
[0102] As used herein, an "acyl" group refers to a formyl group or
R.sup.X--C(O)-- (such as -alkyl-C(O)--, also referred to as
"alkylcarbonyl") where R.sup.X and "alkyl" have been defined
previously. Acetyl and pivaloyl are examples of acyl groups.
[0103] As used herein, an "alkoxy" group refers to an alkyl-O--
group where "alkyl" has been defined previously.
[0104] As used herein, a "carbamoyl" group refers to a group having
the structure --O--CO--NR.sup.XR.sup.Y or
--NR.sup.X--CO--O--R.sup.Z wherein R.sup.X and R.sup.Y have been
defined above and R.sup.Z can be aliphatic, aryl, araliphatic,
heterocycloaliphatic, heteroaryl, or heteroaraliphatic.
[0105] As used herein, a "carboxy" group refers to --COOH,
--COOR.sup.X, --OC(O)H, --OC(O)R.sup.X when used as a terminal
group or --OC(O)-- or --C(O)O--; when used as an internal
group.
[0106] As used herein, a "haloaliphatic" group refers to an
aliphatic group substituted with 1-3 halogen. For instance, the
term haloalkyl includes the group --CF.sub.3.
[0107] As used herein, a "mercapto" group refers to --SH.
[0108] As used herein, a "sulfo" group refers to --SO.sub.3H or
--SO.sub.3R.sup.X when used terminally or --S(O)3- when used
internally.
[0109] As used herein, a "sulfamide" group refers to the structure
--NR.sup.X--S(O).sub.2--NR.sup.YR.sup.Z when used terminally and
--NR.sup.X--S(O).sub.2--NR.sup.Y-- when used internally, wherein
R.sup.X, R.sup.Y, and R.sup.Z have been defined above.
[0110] As used herein, a "sulfamoyl" group refers to the structure
--S(O).sub.2--NR.sup.XR.sup.Y or --NR.sup.X--S(O).sub.2--R.sup.Z
when used terminally or --S(O).sub.2--NR.sup.X-- or
--NR.sup.X--S(O).sub.2-- when used internally, wherein R.sup.X,
R.sup.Y, and R.sup.Z are defined above.
[0111] As used herein a "sulfanyl" group refers to --S--R.sup.X
when used terminally and --S-- when used internally, wherein
R.sup.X has been defined above. Examples of sulfanyls include
alkylsulfanyl.
[0112] As used herein a "sulfinyl" group refers to --S(O)--R.sup.X
when used terminally and --S(O)-- when used internally, wherein
R.sup.X has been defined above.
[0113] As used herein, a "sulfonyl" group refers to
--S(O).sub.2--R.sup.X when used terminally and --S(O).sub.2-- when
used internally, wherein R.sup.X has been defined above.
[0114] As used herein, a "sulfoxy" group refers to --O--SO--R.sup.X
or --SO--O--R.sup.X, when used terminally and --O--S(O)-- or
--S(O)--O-- when used internally, where R.sup.X has been defined
above.
[0115] As used herein, a "halogen" or "halo" group refers to
fluorine, chlorine, bromine or iodine.
[0116] As used herein, an "alkoxycarbonyl," which is encompassed by
the term carboxy, used alone or in connection with another group
refers to a group such as alkyl-O--C(O)--.
[0117] As used herein, an "alkoxyalkyl" refers to an alkyl group
such as alkyl-O-alkyl-, wherein alkyl has been defined above.
[0118] As used herein, a "carbonyl" refers to --C(O)--.
[0119] As used herein, an "oxo" refers to .dbd.O.
[0120] As used herein, an "aminoalkyl" refers to the structure
(R.sup.X).sub.2N-alkyl-.
[0121] As used herein, a "cyanoalkyl" refers to the structure
(NC)-alkyl-.
[0122] As used herein, a "urea" group refers to the structure
--NR.sup.X--CO--NR.sup.YR.sup.Z and a "thiourea" group refers to
the structure --NR.sup.X--CS--NR.sup.YR.sup.Z when used terminally
and --NR.sup.X--CO--NR.sup.Y-- or --NR.sup.X--CS--NR.sup.Y-- when
used internally, wherein R.sup.X, R.sup.Y, and R.sup.Z have been
defined above.
[0123] As used herein, a "guanidino" group refers to the structure
--N.dbd.C(N(R.sup.XR.sup.Y))N(R.sup.XR.sup.Y) wherein R.sup.X and
R.sup.Y have been defined above.
[0124] As used herein, the term "guanidino" group refers to the
structure --C.dbd.R.sup.X)N(R.sup.XR.sup.Y) wherein R.sup.X and
R.sup.Y have been defined above.
[0125] The terms "terminally" and "internally" refer to the
location of a group within a substituent. A group is terminal when
the group is present at the end of the substituent not further
bonded to the rest of the chemical structure. Carboxyalkyl, i.e.,
R.sup.XO(O)C-alkyl is an example of a carboxy group used
terminally. A group is internal when the group is present in the
middle of a substituent to at the end of the substituent bound to
the to the rest of the chemical structure. Alkylcarboxy (e.g.,
alkyl-C(O)O-- or alkyl-OC(O)--) and alkylcarboxyaryl (e.g.,
alkyl-C(O)O-aryl- or alkyl-O(CO)-aryl-) are examples of carboxy
groups used internally.
[0126] The phrase "optionally substituted" is used interchangeably
with the phrase "substituted or unsubstituted." As described
herein, compounds of the invention can optionally be substituted
with one or more substituents, such as are illustrated generally
above, or as exemplified by particular classes, subclasses, and
species of the invention. As described herein, the variables
contained herein encompass specific groups, such as alkyl and aryl.
Unless otherwise noted, each of the specific groups for the
variables contained herein can be optionally substituted with one
or more substituents described herein. Each substituent of a
specific group is further optionally substituted with one to three
of halo, cyano, oxoalkoxy, hydroxyl, amino, nitro, aryl, haloalkyl,
and alkyl. For instance, an alkyl group can be substituted with
alkylsulfanyl and the alkylsulfanyl can be optionally substituted
with one to three of halo, cyano, oxoalkoxy, hydroxyl, amino,
nitro, aryl, haloalkyl, and alkyl. As an additional example, the
cycloalkyl portion of a (cycloalkyl)carbonylamino can be optionally
substituted with one to three of halo, cyano, alkoxy, hydroxyl,
nitro, haloalkyl, and alkyl. When two alkoxy groups are bound to
the same atom or adjacent atoms, the two alkoxy groups can form a
ring together with the atom(s) to which they are bound.
[0127] In general, the term "substituted," whether preceded by the
term "optionally" or not, refers to the replacement of hydrogen
radicals in a given structure with the radical of a specified
substituent. Specific substituents are described above in the
definitions and below in the description of compounds and examples
thereof. Unless otherwise indicated, an optionally substituted
group can have a substituent at each substitutable position of the
group, and when more than one position in any given structure can
be substituted with more than one substituent selected from a
specified group, the substituent can be either the same or
different at every position. A ring substituent, such as a
heterocycloalkyl, can be bound to another ring, such as a
cycloalkyl, to form a spiro-bicyclic ring system, e.g., both rings
share one common atom. As one of ordinary skill in the art will
recognize, combinations of substituents envisioned by this
invention are those combinations that result in the formation of
stable or chemically feasible compounds.
[0128] The phrase "stable or chemically feasible," as used herein,
refers to compounds that are not substantially altered when
subjected to conditions to allow for their production, detection,
and preferably their recovery, purification, and use for one or
more of the purposes disclosed herein.
[0129] Unless otherwise stated, structures depicted herein are also
meant to include all isomeric (e.g., enantiomeric, diastereomeric,
and geometric (or conformational)) forms of the structure; for
example, the R and S configurations for each asymmetric center, (Z)
and (E) double bond isomers, and (Z) and (E) conformational
isomers. Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, and geometric (or conformational)
mixtures of the present compounds are within the scope of the
invention. Unless otherwise stated, all tautomeric forms of the
compounds of the invention are within the scope of the invention.
Additionally, unless otherwise stated, structures depicted herein
are also meant to include compounds that differ only in the
presence of one or more isotopically enriched atoms. For example,
compounds having the present structures except for the replacement
of hydrogen by deuterium or tritium, or the replacement of a carbon
by a .sup.13C- or .sup.14C-enriched carbon are within the scope of
this invention. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
[0130] As used herein, Co-therapies include any oligonucleotide
compounds that can be used alone or in combination with other
cancer therapies to treat cancer.
II. Cancer Therapies
[0131] Cancer-therapies of the present invention include
oligonucleotide compounds, chemotherapy agents, radiation therapy,
surgery, or combinations thereof.
[0132] A. Oligonucleotide Compounds
[0133] 1. Oncogene Targets
[0134] In some embodiments, the present invention provides antigene
inhibitors of oncogenes. The present invention is not limited to
the inhibition of a particular oncogene. Indeed, the present
invention encompasses antigene inhibitors to any number of
oncogenes including, but not limited to, those disclosed
herein.
[0135] a. Ras
[0136] One gene which has captured the attention of many scientists
is the human proto-oncogene, c-Ha-ras. This gene acts as a central
dispatcher, relaying chemical signals into cells and controlling
cell division. Ras gene alteration may cause the gene to stay in
the "on" position. The ras oncogene is believed to underlie up to
30% of cancer, including colon cancer, lung cancer, bladder and
mammary carcinoma (Bos, Cancer Res. 49:4682-4689 [1989]). The ras
oncogene has therefore become a target for therapeutic drugs.
[0137] There are several reports showing that oligonucleotides
complementary to various sites of ras mRNA can inhibit synthesis of
ras protein (p21), which decreases the cell proliferation rate in
cell culture (U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986;
Daska et al., Oncogene Res. 5:267-275 [1990]; Brown et al.,
Oncogene Res. 4:243-252 [1989]; Saison-Behmoaras et al., EMBO J.
10:1111-1116 [1991)]. Oligonucleotides complementary to the 5'
flanking region of the c-Ha-ras RNA transcript have shown to
inhibit tumor growth in nude mice for up to 14 days (Gray et al.,
Cancer Res. 53:577-580 [1993]). It was recently reported that an
antisense oligonucleotide directed to a point mutation (G>C) in
codon 12 of the c-Ha-ras mRNA inhibited cell proliferation as well
as tumor growth in nude mice when it was injected subcutaneously
(U.S. Pat. No. 5,576,208; U.S. Pat. No. 5,582,986; Schwab et al.,
Proc. Natl. Acad. Sci. USA 91:10460-10464 [1994]; each of which is
herein incorporated by reference). Researchers have also reported
that antisense drugs shrank ovarian tumors in small clinical trials
(Roush et al., Science 276:1192-1194 [1997]).
[0138] b. her-2
[0139] The her-2 (also known as neu oncogene or erbB-2) oncogene
encodes a receptor-like tyrosine kinase (RTK) that has been
extensively investigated because of its role in several human
carcinomas (Hynes and Stern, Biochim. et Biophy. Acta 1198:165-184
[1994]; Dougall et al., Oncogene 9:2109-2123 [1994]) and in
mammalian development (Lee et al., Nature 378:394-398 [1995]). The
sequence of the HER-2 protein was determined from a cDNA that was
cloned by homology to the epidermal growth factor receptor (EGFR)
mRNA from placenta (Coussens et al., Science 230:1132-1139 [1985])
and from a gastric carcinoma cell line (Yamamoto et al., Nature
319:230-234 [1986]). her-2 mRNA was shown to be about 4.5 kb
(Coussens et al., Science 230:1132-1139 [1985]; Yamamoto et al.,
Nature 319:230-234 [1986]) and encodes a transmembrane glycoprotein
of 185 kDa in normal and malignant human tissues (p185HER-2) (Hynes
and Steen, Biochim. et Biophys. Acta 1198:165-184 [1994]; Dougall
et al., Oncogene 9:2109-2123 [1994]). Overexpression of HER-2
causes phenotypic transformation of cultured cells (DiFiore et al.,
Science 237:178-182 [1987]; Hudziak et al., Proc. Natl. Acad. Sci.
USA 84:7159-7163 [1987]) and has been associated with aggressive
clinical progression of breast and ovarian cancer (Slamon et al.,
Science 235:177-182 [1987]; Slamon et al., Science 244:707-712
[1989]).
[0140] Her-2 is one of the most frequently altered genes in cancer.
It encodes a transmembrane receptor (also known as p185) with
tyrosine kinase activity and is a member of the epidermal growth
factor (EGF) family, and thus is related to the epidermal growth
factor receptor (EGFR or HER-1). Aberrant her-2 gene expression is
present in a wide variety of cancers and is most common in breast,
ovarian and gastric cancers. HER-2 is overexpressed in 25-30% of
all human breast and ovarian cancers. Levels of HER-2
overexpression correlate well with clinical stage of breast cancer,
prognosis and metastatic potential. Overexpression of HER-2 is
associated with lower survival rates, increased relapse rates and
increased metastatic potential. Tan et al., (Cancer Res., 57:1199
[1997]) have shown that overexpression of the HER-2 gene increases
the metastatic potential of breast cancer cells without increasing
their transformation ability.
[0141] Aberrant expression of HER-2 includes both increased
expression of normal HER-2 and expression of mutant HER-2.
Activation of the her-2 proto-oncogene can occur by any of three
mechanisms--point mutation, gene amplification and overexpression.
Gene amplification is the most common mechanism. Unlike the other
EGF family members for whom ligand activation is necessary for
promoting transformation, overexpression of HER-2 alone is
sufficient for transformation (Cohen, et al., J. Biol. Chem.,
271:30897 [1996]).
[0142] Several therapeutic approaches have been used to reduce
levels of the HER-2 gene product. The adenovirus type 5 gene
product E1A has been studied as a potential therapeutic using a
breast cancer model in nude mice. This gene product can repress
HER-2/neu overexpression by repressing her-2/neu promoter activity,
and suppress the tumorigenic potential of HER-2/neu-over-expressing
ovarian cancer cells. In mice bearing HER-2/neu-overexpressing
breast cancer xenografts, E1A delivered either by adenovirus or
liposome significantly inhibited tumor growth and prolonged mouse
survival compared with the controls (Chang et al., Oncogene 14:561
[1997])
[0143] Clinical trials have been conducted to evaluate a bispecific
antibody which targets the extracellular domains of both the
HER-2/neu protein product and Fc gamma RIII (CD16), the Fc gamma
receptor expressed by human natural killer cells, neutrophils, and
differentiated mononuclear phagocytes (Weiner et al., J.
Hematotherapy, 4:471 [1995]).
[0144] Overexpression of HER-2 has also been found to be associated
with increased resistance to chemotherapy. Thus, patients with
elevated levels of HER-2 respond poorly to many drugs. Methods used
to inhibit HER-2 expression have been combined with commonly used
chemotherapeutic agents (Ueno et al., Oncogone 15:953 [1997]).
Combining the adenovirus type 5 gene product, E1A, with taxol
showed a synergistic effect in human breast cancer cells. Zhang et
al., (Oncogene, 12:571 [1996]) demonstrated that emodin, a
tyrosine-specific inhibitor, sensitized non-small cell lung cancer
(NSCLC) cells to a variety of chemotherapeutic drugs, including
cisplatin, doxorubicin and etoposide. A HER-2 antibody was found to
increase the efficacy of tamoxifen in human breast cancer cells
(Witters et al., Breast Cancer Res. and Treatment, 42:1
[1997]).
[0145] Oligonucleotides have also been used to study the function
of HER-2. A triplex-forming oligonucleotide targeted to the HER-2
promoter, 42 to 69 nucleotides upstream of the mRNA transcription
start site was found to inhibit HER-2 expression in vitro
(Ebbinghaus et al., J. Clin. Invest., 92:2433 [1993]). Porumb et
al. (Cancer Res., 56:515 [1996]) also used a triplex-forming
oligonucleotide targeted to the same HER-2 promoter region.
Decreases in HER-2 mRNA and protein levels were seen in cultured
cells. Juhl et al. (J. Biol. Chem., 272:29482 [1997]) used
anti-HER-2 ribozymes targeted to a central region of the HER-2 RNA
just downstream of the transmembrane region of the protein to
demonstrate a reduction in HER-2 mRNA and protein levels in human
ovarian cancer cells. A reduction in tumor growth in nude mice was
also seen.
[0146] An antisense approach has been used as a potential
therapeutic for HER-2 over-expressing cancers. Pegues et al.
(Cancer Lett., 117:73 [1997]) cloned a 1.5 kb fragment of HER-2 in
an antisense orientation into an expression vector; transfecting of
this construct into ovarian cancer cells resulted in a reduction of
anchorage-independent growth. Casalini et al. (Int. J. Cancer
72:631 [1997]) used several human HER-2 antisense vector
constructs, containing HER-2 fragments from 151 bp to 415 bp in
length, to demonstrate reduction in HER-2 protein levels and
anchorage-independent growth in lung adenocarcinoma cells. Colomer
et al. (Br. J. Cancer, 70:819 [1994]) showed that phosphodiester
antisense oligonucleotides targeted at or immediately downstream
of, the translation initiation codon inhibited proliferation of
human breast cancer cells by up to 60%. Wiechen et al. (Int. J.
Cancer 63:604 [1995]) demonstrated that an 18-nucleotide
phosphorothioate oligonucleotide targeted to the coding region, 33
nucleotides downstream of the translation initiation codon, of
HER-2 reduced anchorage-independent growth of ovarian cancer cells.
Bertram et al. (Biochem. Biophys. Res. Commun., 200:661 [1994])
used antisense phosphorothioate oligonucleotides targeted to the
translation initiation region and a sequence at the 3' part of the
translated region of the mRNA which has high homology to a tyrosine
kinase consensus sequence, and demonstrated a 75% reduction in
HER-2 protein levels in human breast cancer cells. Liu et al.,
(Antisense and Nucleic Acid Drug Develop., 6:9 [1996]) used
antisense phosphorothioate oligonucleotides targeted to the 5' cap
site and coding region. The most effective oligonucleotide,
targeted to the 5' cap site, reduced HER-2 protein expression by
90%. Cell proliferation was also reduced by a comparable amount.
Vaughn et al. (Nuc. Acids. Res., 24:4558 [1996]) used
phosphorothioate, phosphorodithioate and chimeric antisense
oligonucleotides targeted at or adjacent to (either side) the
translation initiation region of HER-2. An alternating
dithioate/diester oligonucleotide targeted to the translation
initiation region worked slightly better than an all
phosphorothioate oligonucleotide. Brysch et al. (Cancer Gene Ther.,
1: 99 [1994]) used chemically modified antisense oligonucleotides
targeted to the translation initiation codon of HER-2 to reduce
protein levels and cause growth arrest of human breast cancer cell
line.
[0147] c. C-Myc
[0148] The c-myc gene product is encoded by an immediate early
response gene, the expression of which can be induced by various
mitogens. C-myc expression is involved in the signal transduction
pathways leading to cell division. Studies have demonstrated that
proliferating cells have higher levels of c-myc mRNA and c-myc
protein than do quiescent cells. Antibodies directed against the
human c-myc protein are known to inhibit DNA synthesis in nuclei
isolated from human cells. Conversely, constitutive expression of
c-myc produced by gene transfer inhibits induced differentiation of
several cell lines. Constitutive expression of c-myc predisposes
transgenic mice to the development of tumors.
[0149] Some studies have suggested that the c-myc gene product may
play a proliferative role in SMCs. Balloon de-endothelialization
and injury of rat aortas is known to increase c-myc mRNA expression
of vascular SMC prior to their subsequent proliferation and
migration. Also, SMCs in culture proliferate when exposed to
several mitogens, including PDGF, FGF, EGF, IGF-1 and to serum.
Each of these mitogens has been found to be capable of increasing
the expression in other cell lines of either c-myc protein, c-myc
mRNA, or both. Additionally, blood serum has been found to increase
c-myc mRNA levels in SMCs.
[0150] Harel-Bellan et al. (J. Immun. 140; 2431-2435 (1988))
demonstrated that antisense oligonucleotides complementary to c-myc
mRNA effectively inhibited the translation thereof in human T
cells. These T cells were prevented from entering the S phase of
cell division. c-myc proto-oncogene sequences are described in
Marcu et al., Ann. Rev. Biochem., 61:809-860 [1992]; Watt et al.,
Nature, 303:725-728 [1983)]; Battey et al., Cell, 34:779-787
(1983); and Epstein et al., NTIS publication PB93-100576
[0151] d. Bcl2
[0152] In many types of human tumors, including lymphomas and
leukemias, the human bcl-2 gene is overexpressed, and may be
associated with tumorigenicity (Tsujimoto et al., Science
228:1440-1443 [1985]). High levels of expression of the human bcl-2
gene have been found in all lymphomas with t (14; 18) chromosomal
translocations including most follicular B cell lymphomas and many
large cell non-Hodgkin's lymphomas. High levels of expression of
the bcl-2 gene have also been found in certain leukemias that do
not have a t(14; 18) chromosomal translation, including most cases
of chronic lymphocytic leukemia acute, many lymphocytic leukemias
of the pre-B cell type, neuroblastomas, nasophryngeal carcinomas,
and many adenocarcinomas of the prostate, breast and colon. (Reed
et al., Cancer Res. 51:6529 [1991]; Yunis et al., New England J.
Med. 320:1047; Campos et al., Blood 81:3091-3096 [1993]; McDonnell
et al., Cancer Res. 52:6940-6944 [1992); Lu et al., Int. J Cancer
53:29-35 [1993]; Bonner et al., Lab Invest. 68:43A [1993]).
[0153] e. TGF-.alpha.
[0154] Transforming Growth Factor Alpha (TGF-.alpha.) is a
polypeptide of 50 amino acids. It was first isolated from a
retrovirus-transformed mouse cell line and subsequently was
identified in human tumor cells, in early rat embryo cells and in
cell cultures from the human pituitary gland. TGF-.alpha. is
closely related to Epidermal Growth Factor (EGF), both structurally
and functionally, and both bind to the same receptor, i.e.,
Epidermal Growth Factor Receptor (EGFR).
[0155] The sequence and three dimensional structure of both EGF and
TGF-.alpha. have been determined (Campbell et al., Prog. Growth
Factor Res. 1:13 [1989]). TGF-.alpha. is a 50 amino acid
polypeptide having about 40% homology of residues with EGF. Both
peptides are characterized by three well defined loops (denoted A,
B and C) and have three intramolecular disulphide bonds.
[0156] Several growth factors, including TGF-.alpha. and EGF, are
believed to exert their biological effects via interaction with the
Epidermal Growth Factor Receptor (EGF Receptor). The EGF Receptor
is a Type 1 receptor tyrosine kinase. The EGF Receptor and its
ligands are of interest for their roles in normal physiological
processes as well as in hyperproliferative and neoplastic
diseases.
[0157] The in vivo precursor of TGF-.alpha. is a 160 amino acid
residue membrane-bound protein (pro-TGF-.alpha.) that is cleaved to
yield a soluble compound (Massague, J. Biol. Chem., 265:21393-21396
[1990]). This cleavage removes an extracellular portion comprised
of 50 amino acids with a molecular weight of 6 Kd and is considered
to be an important regulatory event (Pandiella et al., Proc. Natl.
Acad. Sci. USA, 88:1726-1730 [1990]) that can be stimulated by
phorbol esters acting via protein kinase C (Pandiella et al., J.
Biol. Chem., 266:5769-5773 [1991]).
[0158] Cultured human prostatic tumor lines contain elevated levels
of TGF-.alpha. mRNA and proliferate in response to TGF-.alpha.
(Wilding et al., The Prostate, 15:1-12 [1989]). TGF-.alpha. appears
to have both autocrine and paracrine function, stimulating
physiologic activities such as cell division and angiogenesis. When
induced in transgenic mice, TGF-.alpha. produced epithelial
hyperplasia and focal dysplastic changes that resembled carcinoma
in situ (Sandgren et al., Cell, 61:1121-1135 [1990]).
[0159] f. c-ki-Ras
[0160] The c-Ki-ras (KRAS) oncogene is expressed ubiquitously.
KRAS, with a length of more than 30 kb, is much larger than HRAS or
NRAS. Although the 3 ras genes, HRAS, KRAS, and NRAS, have
different genetic structures, all code for proteins of 189 amino
acid residues, generically designated p21. These genes acquire
malignant properties by single point mutations that affect the
incorporation of the 12th or 61st amino acid residue of their
respective p21. KRAS is involved in malignancy much more often than
is HRAS. In a study of 96 human tumors or tumor cell lines in the
NIH 3T3 transforming system, (Pulciani et al., Nature 300: 539
(1982) found a mutated HRAS locus only in T24 bladder cancer cells,
whereas transforming KRAS genes were identified in 8 different
carcinomas and sarcomas.
[0161] In a serious cystadenocarcinoma of the ovary, Feig et al.
(Science 223: 698 (1984)) showed the presence of an activated KRAS
oncogene not activated in normal cells of the same patient. The
transforming gene product displayed an electrophoretic mobility in
SDS-polyacrylamide gels that differed from the mobility of KRAS
transforming proteins in other tumors. Thus, a previously
undescribed mutation was responsible for activation of KRAS in this
ovarian carcinoma. To study the role of oncogenes in lung cancer,
Rodenhuis et al. (New Eng. J. Med. 317: 929 (1987)) used an assay
based on oligonucleotide hybridization following an in vitro
amplification step. Genomic DNA was examined from 39 tumor
specimens obtained at thoracotomy. The KRAS gene was found to be
activated by point mutations in codon 12 in 5 of 10
adenocarcinomas. Two of these tumors were less than 2 cm in size
and had not metastasized. No HRAS, KRAS or NRAS mutations were
observed in 15 squamous cell carcinomas, 10 large cell carcinomas,
1 carcinoid, 2 metastatic adenocarcinomas from primary tumors
outside the lung and 1 small cell carcinoma. An approximately
20-fold amplification of the unmutated KRAS gene was observed in a
tumor that proved to be a solitary lung metastasis of a rectal
carcinoma. Yanez et al. (Oncogene 1:315 (1987)) found mutations in
codon 12 of the KRAS gene in 4 of 16 colon cancers, 2 of 27 lung
cancers and 1 of 8 breast cancers; no mutations were found at
position 61. Of the 6 possible amino acid replacements in codon 12,
all but one were represented in the 7 mutations identified.
[0162] g. Other Oncogene Targets
[0163] The present invention is not limited to the oncogenes
described above. The methods of the present invention are suitable
for use with any oncogene with a known promoter region. Exemplary
oncogenes included, but are not limited to, BCR/ABL, ABL1/BCR, ABL,
BCL1, CD24, CDK4, EGFR/ERBB-1, HSTF1, INT1/WNT1, INT2, MDM2, MET,
MYB, MYC, MYCN, MYCL1, RAF1, NRAS, REL, AKT2, APC, BCL2-ALPHA,
BCL2-BETA, BCL3, BCR, BRCA1, BRCA2, CBL, CCND1, CDKN1A, CDKN1C,
CDKN2A, CDKN2B, CRK, CRK-II, CSF1R/FMS, DBL, DDOST, DCC,
DPC4/SMAD4, E-CAD, E2F1/RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3,
ERG, ETS1, ETS2, FER, FGR, FLI1/ERGB2, FOS, FPS/FES, FRA1, FRA2,
FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ERBB-4, HST2, INK4A,
INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS,
MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1,
PTC, RB1, RET, ROS1, SKI, SRC1, TAL1, TGFBR2, THRA1, THRB, TIAM1,
TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK/NPM1, AMI1, AXL, FMS,
GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, LMO-1,
LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST,
PAX-5, PMS-1, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TAN1,
TIAM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57/KIP2, PTEN,
HPC1, ATM, XPA/XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1/FLI1, FES,
FGF3, FGF4, FGF6, FANCA, FLI1/ERGB2, FOSL1, FOSL2, GLI, HRAS1,
HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MAS1, MCF2, MLLT1/MLL,
MLLT2/HR.sup.X, MTG8/RUNX1, MYCLK1, MYH11/CBFB, NFKB2, NOTCH1,
NPM1/ALK, NRG/REL, NTRK1, PBX1/TCF3, PML/RARA, PRCA1, RUNX1,
RUNX1/CBFA2T1, SET, TCF3/PBX1, TGFB1, TLX1, P53, WNT1, WNT2, WT1,
.alpha.v-.beta.3, PKC.alpha., TNF.alpha., Clusterin, Surviving,
TGF.beta., c-fos, c-SRC, and INT-1.
[0164] 2. Non-Oncogene Targets
[0165] The present invention is not limited to the targeting of
oncogenes. The methods and compositions of the present invention
are useful for targeting any gene that it is desirable to down
regulate its expression. For example, in some embodiments, the
genes to be targeted include, but are not limited to, an
immunoglobulin or antibody gene, a clotting factor gene, a
protease, a pituitary hormone, a protease inhibitor, a growth
factor, a somatomedian, a gonadotrophin, a chemotactin, a
chemokine, a plasma protein, a plasma protease inhibitor, an
interleukin, an interferon, a cytokine, a transcription factor, or
a pathogen target (e.g., a viral gene, a bacterial gene, a
microbial gene, a fungal gene).
[0166] Examples of specific genes include, but are not limited to,
ADAMTS4, ADAMTS5, APOA1, APOE, APP, B2M, COX2, CRP, DDX25, DMC1,
FKBP8, GH1, GHR, IAPP, IFNA1, IFNG, IL1, I110, IL12, IL13, IL2,
IL4, IL7, IL8, IPW, MAPK14, Mei1, MMP13, MYD88, NDN, PACE4, PRNP,
PSEN1, PSEN2, RAD51, RAD51C, SAP, SNRPN, TLR4, TLR9, TTR, UBE3A,
VLA-4, and PTP-1B, c-RAF, m-TOR, LDL, VLDL, ApoB-100, HDL, VEGF,
rhPDGF-BB, NADs, ICAM-1, MUC1, 2-dG, CTL, PSGL-1, E2F, NF-.kappa.B,
HIF, and GCPRs.
[0167] In other embodiments and gene from a pathogen is targeted.
Exemplary pathogens include, but are not limited to, Human
Immunodeficiency virus, Hepatitis B virus, hepatitis C virus,
hepatitis A virus, respiratory syncytial virus, pathogens involved
in severe acute respiratory syndrome, west nile virus and food
borne pathogens (e.g., E. coli).
[0168] 3. Oligonucleotides
[0169] In some embodiments, the present invention provides antigene
oligonucleotides for inhibiting the expression of oncogenes.
Exemplary design and production strategies for antigenes are
described below. The description below is not intended to limit the
scope of antigene compounds suitable for use in the present
invention and that other antigenes are within the scope of the
present invention.
[0170] a. Regulatory Regions of the Oncogenes
[0171] The bcl-2 gene has two promoters designated P1 and P2. P1
from which most bcl-2 mRNA is transcribed is located approximately
1.4 kb upstream of the translation initiation site and P2 is 1.3 kb
downstream of P1. (See Seto, M. et al. EMBO J. 7, 123-131 (1988).)
P1 is GC-rich, lacks a TATA box, has many transcription start sites
and includes seven consensus binding sites for the SP1
transcription factor. P2 includes a CCAAT box and a TATA box and
has two different transcription initiation sites. There are
multiple NF-.kappa.B recognition sites and an SV40 enhancer-like
octamer motif within P2. (See Heckman, C. A., et al. Oncogene 21,
3898-3908 (2002).) (See SEQ ID NO:1254). Most human follicular
lymphomas contain t(14; 18) chromosomal translocations that result
from 3'-bcl-2 gene region breakpoints. (See Tsujimoto, Y. et al.
Proc. Natl. Acad. Sci. U.S.A 84, 1329-1331 (1987).) These
translocations place bcl-2 expression under control of the
immunoglobulin heavy chain (IgH) locus enhancer resulting in
upregulation of BCL2 expression. Alternatively, there are 5'-bcl-2
breakpoint regions that result from fusions with either the IgH
locus or two different immunoglobulin light chain (IgL) loci that
are found in some DLCL lymphoma patient isolates. (See Yonetani, N.
et al. Jpn. J. Cancer Res. 92, 933-940 (2001).) These 5'-bcl-2
breakpoints have been mapped in separate heterogeneous patient
isolates to a region spanning 378 to 2312 bp upstream of the
translation initiation site. (See SEQ ID NOs:1255-1266.) Regions
around the breakpoints may be sequences that can be used for bcl-2
oligonucleotide design.
[0172] The upstream regions of TGF-.alpha., c-ki-ras, c-myc,
c-erb-2 (Her-2), and c-Ha-ras can also be investigated to find
regions to which oligonucleotides could bind based on preferred
design criteria.
[0173] b. Oligonucleotide Design
[0174] The oligonucleotides can include any oligomer that
hybridizes to the upstream regions of the c-ki-ras, c-Ha-ras,
c-myc, her-2, TGF-.alpha., or bcl-2 gene. For the purposes of this
invention, those upstream regions are defined as SEQ ID NO:1 (for
her-2, or c-erb-2), SEQ ID NO:282 (for c-ki-ras), SEQ ID NO:462
(for c-Ha-ras), SEQ ID NO:936 (for c-myc), SEQ ID NO:1081 (for
TGF-.alpha.) and SEQ ID NOs:1249 and 1254 (for bcl-2).
[0175] In some embodiments, oligonucleotides are designed based on
preferred design criteria. Such oligonucleotides can then be tested
for efficacy using the methods disclosed herein. For example, in
some embodiments, the oligonucleotides are methylated on at least
one, two or all of the CpG islands. In other embodiments, the
oligonucleotides contain no methylation. The present invention is
not limited to a particular mechanism. Indeed, an understanding of
the mechanism is not necessary to practice the present invention.
Nonetheless, it is contemplated that oligonucleotides in some
embodiments are those that have at least a 50% GC content and at
least two GC dinucleotides. Also, in some embodiments, the
oligonucleotides do not self hybridize. In further embodiments,
oligonucleotides are designed with at least 1 A or T to minimize
self hybridization. In yet further embodiments, commercially
available computer programs are used to survey oligonucleotides for
the ability to self hybridize. In still other embodiments,
oligonucleotides are at least 10, or 15 nucleotides and no more
than 100 nucleotides in length. In further embodiments,
oligonucleotides are 18-26 nucleotides in length. In additional
embodiments, oligonucleotides comprise the universal protein
binding sequences CGCCC and CGCG or the complements thereof.
[0176] In some embodiments, oligonucleotides hybridize to a
promoter region of a gene upstream from the TATA box of the
promoter. In further embodiments, oligonucleotides are designed to
hybridize to regions of the promoter region of an oncogene known to
be bound by proteins (e.g., transcription factors). In some
embodiments, oligonucleotide compounds are not completely
homologous to other regions of the human genome. The homology of
the oligonucleotide compounds of the present invention to other
regions of the genome can be determined using available search
tools (e.g., BLAST, available at the Internet site of NCBI).
[0177] The present invention is not limited to the oligonucleotides
described herein. Other suitable oligonucleotides may be identified
(e.g., using the criteria described above or other criteria).
Candidate oligonucleotides may be tested for efficacy using any
suitable method. For example, candidate oligonucleotides can be
evaluated for their ability to prevent cell proliferation at a
variety of concentrations. In some embodiments, oligonucleotides
inhibit gene expression or cell proliferation at a low
concentration (e.g., less that 20 .mu.M, or 10 .mu.M in in vitro
assays.).
[0178] c. Oligonucleotide Zones
[0179] In some embodiments, regions within the promoter region of
an oncogene are further defined as regions for hybridization of
oligonucleotides. In some embodiments, these regions are referred
to as "hot zones."
[0180] In some embodiments, hot zones are defined based on
oligonucleotide compounds that are demonstrated to be effective
(see above section on oligonucleotides) and those that are
contemplated to be effective based on the criteria for
oligonucleotides described above. In some embodiments, hot zones
encompass 10 bp upstream and downstream of each compound included
in each hot zone and have at least one CG or more within an
increment of 40 bp further upstream or downstream of each compound.
In further embodiments, hot zones encompass a maximum of 100 bp
upstream and downstream of each oligonucleotide compound included
in the hot zone. In additional embodiments, hot zones are defined
at beginning regions of each promoter. These hot zones are defined
either based on effective sequence(s) or contemplated sequences and
have a preferred maximum length of 200 bp. Based on the above
described criteria, exemplary hot zones were designed. These hot
zones are shown in Table 1.
TABLE-US-00001 TABLE 1 Exemplary Hot Zones Gene Hot Zones Bcl-2
679-720, 930-1050. 1070-1280. 1420-1760 c-erbB-2 206-346, 384-437
c-K-ras 1-290, 433-659 c-Ha-ras 21-220, 233-866, 1417-1536,
1637-1728 c-myc 71-263, 299-770 TGF-.alpha. 1-90, 175-219, 264-370,
434-934, 968-1183
[0181] d. Description
[0182] In one aspect, the oligonucleotides can be any oligomer that
hybridizes under physiological conditions to the following
sequences: SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID
NO:936, SEQ ID NO:1081, SEQ ID NO:1249 or SEQ ID NO:1254. In
another aspect, the oligonucleotides can be any oligomer that
hybridizes under physiological conditions to exemplary hot zones in
SEQ ID NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, SEQ ID
NO:1081 and SEQ ID NO:1249. Examples of oligomers include, without
limitation, those oligomers listed in SEQ ID NOs 2-281, 283-461,
463-935, 937-1080, 1082-1248, 1250-1253 and 1267-1477 and the
complements thereof. In another aspect, the oligonucleotides are
SEQ ID NOs 2-22, 283-301, 463-503, 937-958, 1082-1109, 1250-1254
and 1270-1477 and the complements thereof. In an embodiment of
these aspects, the oligonucleotides are from 15-35 base pairs in
length.
[0183] For the bcl-2 gene, the oligomer can be any oligomer that
hybridizes to SEQ ID NOs: 1249 or 1254. In another aspect, the
oligomer can be any oligomer that hybridizes to nucleotides
500-2026, nucleotides 500-1525, nucleotides 800-1225, nucleotides
900-1125, nucleotides 950-1075 or nucleotides 970-1045 of SEQ ID
NO:1249 or the complement thereof.
[0184] In one embodiment, the oligomer can be SEQ ID NO:1250, 1251,
1252, 1253, 1267-1477 or the complement thereof. In another
embodiment, the oligomer can be SEQ ID NO: 1250, 1251, 1267, 1268,
1276, 1277, 1285, 1286 or the complement thereof. In yet another
embodiment, the oligomer can be SEQ ID NOs 1250, 1251, 1289-1358 or
the complements thereof. In still another embodiment the oligomer
can be SEQ ID NO:1250 or 1251.
[0185] In a further embodiment of these aspects, the oligomer has
the sequence of the positive strand of the bcl-2 sequence, and
thus, binds to the negative strand of the sequence.
[0186] In other aspects, the oligomers can include mixtures of
bcl-2 oligonucleotides. For instance, the oligomer can include
multiple oligonucleotides each of which hybridizes to different
parts of SEQ ID NOs:1249 and 1254. Oligomers can hybridize to
overlapping regions on those sequences or the oligomers may
hybridize to non-overlapping regions. In other embodiments,
oligomers can be SEQ ID NOs:1250, 1251, 1252, 1253, 1267-1477 or
the complement thereof, wherein the mixture of bcl-2 oligomers
comprises oligomers of at least 2 different sequences.
[0187] In other embodiments, the oligomer can include a mixture of
oligomers, each of which hybridizes to a regulatory region of
different genes. For instance, the oligomer can include a first
oligomer that hybridizes to SEQ ID NO:1249 or 1254 and second
oligomer that hybridizes to a regulatory region of a second gene.
In some embodiments, the oligomer includes an oligomer of SEQ ID
NOs 1250-1254 and 1267-1477 or the complements thereof, and an
oligomer that hybridizes to SEQ ID NO:1, SEQ ID NO:282, SEQ ID
NO:462, SEQ ID NO:936, or SEQ ID NO:1081 or the complement thereof.
In other embodiments, the oligomer includes SEQ ID NO 1250 or 1251
or the complement thereof and an oligomer that hybridizes to SEQ ID
NO:1, SEQ ID NO:282, SEQ ID NO:462, SEQ ID NO:936, or SEQ ID
NO:1081 or the complement thereof. In yet other embodiments, the
oligomer includes SEQ ID NO:1250 or 1251 or the complement thereof
and any of SEQ ID NOs 2-281, 283-461, 463-935, 937-1080 and
1082-1248, or the complement thereof.
[0188] In some embodiments, the present invention provides
oligonucleotide therapeutics that are methylated at specific sites.
The present invention is not limited to a particular mechanism.
Indeed, an understanding of the mechanism is not necessary to
practice the present invention. Nonetheless, it is contemplated
that one mechanism for the regulation of gene activity is
methylation of cytosine residues in DNA. 5-methylcytosine (5-MeC)
is the only naturally occurring modified base detected in DNA
(Ehrlick et al., Science 212:1350-1357 (1981)). Although not all
genes are regulated by methylation, hypomethylation at specific
sites or in specific regions in a number of genes is correlated
with active transcription (Doerfler, Annu. Rev. Biochem. 52:93-124
[1984]; Christman, Curr. Top. Microbiol. Immunol. 108:49-78 [1988];
Cedar, Cell 34:5503-5513 [1988]). DNA methylation in vitro can
prevent efficient transcription of genes in a cell-free system or
transient expression of transfected genes. Methylation of C
residues in some specific cis-regulatory regions can also block or
enhance binding of transcriptional factors or repressors (Doerfler,
supra; Christman, supra; Cedar, Cell 34:5503-5513 (1988); Tate et
al., Curr. Opin. Genet. Dev. 3:225-231 [1993]; Christman et al.,
Virus Strategies, eds. Doerfler, W. & Bohm, P. (VCH, Weinheim,
N.Y.) pp. 319-333 [1993]).
[0189] Disruption of normal patterns of DNA methylation has been
linked to the development of cancer (Christman et al., Proc. Natl.
Acad. Sci. USA 92:7347-7351 [1995]). The 5-MeC content of DNA from
tumors and tumor derived cell lines is generally lower than normal
tissues (Jones et al., Adv. Cancer Res 40:1-30 [1983]).
Hypomethylation of specific oncogenes such as c-myc, c-Ki-ras and
c-Ha-ras has been detected in a variety of human and animal tumors
(Nambu et al., Jpn. J. Cancer (Gann) 78:696-704 [1987]; Feinberg et
al., Biochem. Biophys. Res. Commun. 111:47-54 [1983]; Cheah et al.,
JNCI73:1057-1063 [1984]; Bhave et al., Carcinogenesis (Lond)
9:343-348 [1988]. In one of the best studied examples of human
tumor progression, it has been shown that hypomethylation of DNA is
an early event in development of colon cancer (Goetz et al.,
Science 228:187-290 [1985]). Interference with methylation in vivo
can lead to tumor formation. Feeding of methylation inhibitors such
as L-methionine or 5-azacytodine or severe deficiency of
5-adenosine methionine through feeding of a diet depleted of
lipotropes has been reported to induce formation of liver tumors in
rats (Wainfan et al., Cancer Res. 52:2071s-2077s [1992]). Studies
show that extreme lipotrope deficient diets can cause loss of
methyl groups at specific sites in genes such as c-myc, ras and
c-fos (Dizik et al., Carcinogenesis 12:1307-1312 [1991]).
Hypomethylation occurs despite the presence of elevated levels of
DNA MTase activity (Wainfan et al., Cancer Res. 49:4094-4097
[1989]). Genes required for sustained active proliferation become
inactive as methylated during differentiation and tissue specific
genes become hypomethylated and are active. Hypomethylation can
then shift the balance between the two states. In some embodiments,
the present invention thus takes advantage of this naturally
occurring phenomena, to provide compositions and methods for site
specific methylation of specific gene promoters, thereby preventing
transcription and hence translation of certain genes. In other
embodiments, the present invention provides methods and
compositions for upregulating the expression of a gene of interest
(e.g., a tumor suppressor gene) by altering the gene's methylation
patterns.
[0190] The present invention is not limited to the use of
methylated oligonucleotides. Indeed, the use of non-methylated
oligonucleotides for the inhibition of gene expression is
specifically contemplated by the present invention. Experiments
conducted during the course of development of the present invention
(See e.g., Example 8) demonstrated that an unmethylated
oligonucleotide targeted toward Bcl-2 inhibited the growth of
lymphoma cells to a level that was comparable to that of a
methylated oligonucleotide.
[0191] 4. Preparation and Formulation of Oligonucleotides
[0192] Any of the known methods of oligonucleotide synthesis can be
used to prepare the modified oligonucleotides of the present
invention. In some embodiments utilizing methylated
oligonucleotides the nucleotide, dC is replaced by 5-methyl-dC
where appropriate, as taught by the present invention. The modified
or unmodified oligonucleotides of the present invention are most
conveniently prepared by using any of the commercially available
automated nucleic acid synthesizers. They can also be obtained from
commercial sources that synthesize custom oligonucleotides pursuant
to customer specifications.
[0193] While oligonucleotides are one form of compound, the present
invention comprehends other oligomeric oligonucleotide compounds,
including but not limited to oligonucleotide mimetics such as are
described below. The oligonucleotide compounds in accordance with
this invention typically comprise from about 18 to about 30
nucleobases (i.e., from about 18 to about 30 linked bases),
although both longer and shorter sequences may find use with the
present invention.
[0194] Specific examples of compounds useful with the present
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined in this
specification, oligonucleotides having modified backbones include
those that retain a phosphorus atom in the backbone and those that
do not have a phosphorus atom in the backbone. For the purposes of
this specification, modified oligonucleotides that do not have a
phosphorus atom in their internucleoside backbone can also be
considered to be oligonucleosides.
[0195] Modified oligonucleotide backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphorodithioates,
phosphotriesters, aminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates including 3'-amino
phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates having normal
3'-5' linkages, 2'-5' linked analogs of these, and those having
inverted polarity wherein the adjacent pairs of nucleoside units
are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed
salts and free acid forms are also included.
[0196] In some embodiments the oligonucleotides have a
phosphorothioate backbone having the following general
structure.
##STR00001##
[0197] Modified oligonucleotide backbones that do not include a
phosphorus atom therein have backbones that are formed by short
chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
[0198] In other oligonucleotide mimetics, both the sugar and the
internucleoside linkage (i.e., the backbone) of the nucleotide
units are replaced with novel groups. The base units are maintained
for hybridization with an appropriate nucleic acid target compound.
One such oligomeric compound, an oligonucleotide mimetic that has
been shown to have excellent hybridization properties, is referred
to as a peptide nucleic acid (PNA). In PNA compounds, the
sugar-backbone of an oligonucleotide is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone.
Representative patents that teach the preparation of PNA compounds
include, but are not limited to, U.S. Pat. Nos. 5,539,082;
5,714,331; and 5,719,262, each of which is herein incorporated by
reference. Further teaching of PNA compounds can be found in
Nielsen et al., Science 254:1497 (1991) and Neilsen, Methods in
Enzymology, 313, 156-164 (1999). PNA compounds can be obtained
commercially, for example, from Applied Biosystems (Foster City,
Calif., USA).
[0199] In some embodiments, oligonucleotides of the invention are
oligonucleotides with phosphorothioate backbones and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2, --NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a
methylene(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
exemplary are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0200] Oligonucleotides can also have sugars other than ribose and
deoxy ribose, including arabinofuranose (described in International
Publication number WO 99/67378, which is herein incorporated by
reference), xyloarabinofuranose (described in U.S. Pat. Nos.
6,316,612 and 6,489465, which are herein incorporated by
reference), .alpha.-threofuranose (Schoning, et al. (2000) Science,
290, 1347-51, which is herein incorporated by reference) and
L-ribofuranose. Sugar mimetics can replace the sugar in the
nucleotides. They include cyclohexene (Wang et al. (2000) J. Am.
Chem. Soc. 122, 8595-8602; Vebeure et al. Nucl. Acids Res. (2001)
29, 4941-4947, which are herein incorporated by reference), a
tricyclo group (Steffens, et al. J. Am. Chem. Soc. (1997) 119,
11548-11549, which is herein incorporated by reference), a
cyclobutyl group, a hexitol group (Maurinsh, et al. (1997) J. Org.
Chem, 62, 2861-71; J. Am. Chem. Soc. (1998) 120, 5381-94, which are
herein incorporated by reference), an altritol group (Allart, et
al., Tetrahedron (1999) 6527-46, which is herein incorporated by
reference), a pyrrolidine group (Scharer, et al., J. Am. Chem.
Soc., 117, 6623-24, which is herein incorporated by reference),
carbocyclic groups obtained by replacing the oxygen of the furnaose
ring with a methylene group (Froehler and Ricca, J. Am. Chem. Soc.
114, 8230-32, which is herein incorporated by reference) or with an
S to obtain 4'-thiofuranose (Hancock, et al., Nucl. Acids Res. 21,
3485-91, which is herein incorporated by reference, and/or
morpholino group (Heasman, (2002) Dev. Biol., 243, 209-214, which
is herein incorporated by reference) in place of the pentofuranosyl
sugar. Morpholino oligonucleotides are commercially available from
Gene Tools, LLC (Corvallis, Oreg., USA).
[0201] The oligonucleotides can also include "locked nucleic acids"
or LNAs. The LNAs can be bicyclic, tricyclic or polycyclic. LNAs
include a number of different monomers, one of which is depicted in
Formula I.
##STR00002## [0202] wherein [0203] B constitutes a nucleobase;
[0204] Z* is selected from an internucleoside linkage and a
terminal group; [0205] Z is selected from a bond to the
internucleoside linkage of a preceding nucleotide/nucleoside and a
terminal group, provided that only one of Z and Z* can be a
terminal group; [0206] X and Y are independently selected from
--O--, --S--, --N(H)--, --N(R)--, --CH.sub.2-- or --C(H).dbd.,
CH.sub.2--O--, --CH.sub.2--S--, --CH.sub.2--N(H)--,
--CH.sub.2--N(R)--, --CH.sub.2--CH.sub.2-- or
--CH.sub.2--C(H).dbd., --CH.dbd.CH--; provided that X and Y are not
both O.
[0207] In addition to the LNA
[2'-Y,4'-C-methylene-.beta.-D-ribofuranosyl] monomers depicted in
formula I (a [2,2,1] bicyclo nucleoside), an LNA nucleotide can
also include "locked nucleic acids" with other furanose or other 5
or 6-membered rings and/or with a different monomer formulation,
including 2'-Y,3' linked and 3'-Y,4' linked, 1'-Y,3 linked, 1'-Y,4'
linked, 3'-Y,5' linked, 2'-Y,5' linked, 1'-Y,2' linked
bicyclonucleosides and others. All the above mentioned LNAs can be
obtained with different chiral centers, resulting, for example, in
LNA [3'-Y-4'-C-methylene (or ethylene)-.beta. (or
.alpha.)-arabino-, xylo- or L-ribo-furanosyl] monomers. LNA
oligonucleotides and LNA nucleotides are generally described in
International Publication No. WO 99/14226 and subsequent
applications; International Publication Nos. WO 00/56746, WO
00/56748, WO 00/66604, WO 01/25248, WO 02/28875, WO 02/094250, WO
03/006475; U.S. Pat. Nos. 6,043,060, 6268490, 6770748, 6639051, and
U.S. Publication Nos. 2002/0125241, 2003/0105309, 2003/0125241,
2002/0147332, 2004/0244840 and 2005/0203042, all of which are
incorporated herein by reference. LNA oligonucleotides and LNA
analogue oligonucleotides are commercially available from, for
example, Proligo LLC, 6200 Lookout Road, Boulder, Colo. 80301
USA.
[0208] Oligonucleotides can also contain one or more substituted
sugar moieties. Oligonucleotides can comprise one of the following
at the 2' sugar position: OH; F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl, O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Yet other oligonucleotides comprise one of
the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide or a group
improving pharmacodynamic properties of an oligonucleotide and
other substituents having similar properties. One modification
includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta 78:486 [1995]) i.e., an alkoxyalkoxy group. A further
modification includes 2'-dimethylaminooxyethoxy (i.e., an
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group), also known as 2'-DMAOE,
and 2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2.
[0209] Other modifications include 2'-methoxy (2'-O--CH.sub.3),
2'-aminopropoxy(2'--OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and
2'-fluoro (2'-F). Similar modifications may also be made at other
positions on the oligonucleotide, particularly the 3' position of
the sugar on the 3' terminal nucleotide or in 2'-5' linked
oligonucleotides and the 5' position of 5' terminal nucleotide.
Oligonucleotides can also have sugar mimetics such as cyclobutyl
moieties in place of the pentofuranosyl sugar.
[0210] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,
5-propynyluracil, 5-propynylcytosine, 5-propynyl-6-fluoroluracil,
5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,
diaminopurine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,
3-deazaadenine, 8-azaguanine, 8-azaadenine,
7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine,
2-chloro-6-aminopurine, 4-acetylcytosine, 5-hydroxymethylcytosine,
8-hydroxy-N6-methyladenosine, aziridinylcytosine,
5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
N6-methyladenine, 7-methylguanine and other alkyl derivatives of
adenine and guanine, 2-propyl adenine and other alkyl derivatives
of adenine and guanine, 2-aminoadenine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,
queosine, 2-thiocytosine, 2-thiothymine, 5-halouracil,
5-halocytosine, 6-azo uracil, cytosine and thymine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
8-halo, 8-amino, 8-thiol, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-trifluoromethyl uracil and cytosine,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
queosine, xanthine, hypoxanthine, 2-thiocytosine and
2,6-diaminopurine. Further nucleobases include those disclosed in
U.S. Pat. No. 3,687,808. Certain of these nucleobases are
particularly useful for increasing the binding affinity of the
oligomeric compounds of the invention. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by -0.6-1.2.degree. C.
These are particularly effective when combined with
2'-O-methoxyethyl sugar modifications.
[0211] Another modification of the oligonucleotides of the present
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates that enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety, cholic acid, a thioether, (e.g.,
hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain, (e.g.,
dodecandiol or undecyl residues), a phospholipid, (e.g.,
di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or a
polyethylene glycol chain or adamantane acetic acid, a palmityl
moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol
moiety.
[0212] One skilled in the relevant art knows well how to generate
oligonucleotides containing the above-described modifications. The
present invention is not limited to the oligonucleotides described
above. Any suitable modification or substitution may be
utilized.
[0213] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes pharmaceutical compositions and
formulations that include the oligomeric compounds of the present
invention as described below.
[0214] 5. Cocktails
[0215] In some embodiments, the present invention provides
cocktails comprising two or more oligonucleotides directed toward
regulatory regions of genes (e.g., oncogenes). In some embodiments,
two or more oligonucleotides hybridize to different regions of a
regulatory region of the same gene. In other embodiments, the two
or more oligonucleotides hybridize to regulatory regions of two
different genes. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, it is
contemplated that the combination of two or more compounds of the
present invention provides an inhibition of cancer cell growth that
is greater than the additive inhibition of each of the compounds
administered separately.
[0216] 6. Index of SEQ IDs
TABLE-US-00002 SEQ ID NO: 1 c-erb-2 (her-2) upstream region SEQ ID
NOs: 2-281 c-erb-2 (her-2) oligonucleotides SEQ ID NO: 282 c-ki-ras
upstream region SEQ ID NOs: 283-461 c-ki-ras oligonucleotides SEQ
ID NO: 462 c-Ha-ras upstream region SEQ ID NOs: 463-935 c-Ha-ras
oligonucleotides SEQ ID NO: 936 c-myc upstream region SEQ ID NOs:
937-1080 c-myc oligonucleotides SEQ ID NO: 1081 TGF-.alpha.
upstream region SEQ ID NOs: 1082-1248 TGF-.alpha. oligonucleotides
SEQ ID NO: 1249 bcl-2 upstream region SEQ ID NO: 1250 PNT-100
oligonucleotide methylated SEQ ID NO: 1251 PNT-100 oligonucleotide
not methylated SEQ ID NO: 1252 bcl-2 oligonucleotide methylated SEQ
ID NO: 1253 bcl-2 oligonucleotide not methylated SEQ ID NO: 1254
bcl-2 secondary promoter sequence SEQ ID NOs: 1255-1266 bcl-2
sequences SEQ ID NOs: 1250-1254 bcl-2 oligonucleotides and
1267-1477 SEQ ID NOs: 1448-1461 bcl-2 control oligonucleotides
[0217] Oligonucleotide compounds of the present invention can be
used alone or in combination with a chemotherapy agent, radiation
therapy or surgery.
[0218] B. Chemotherapy Agents
[0219] Chemotherapy agents of the present invention can include any
suitable chemotherapy drug or combinations of chemotherapy drugs
(e.g., a cocktail). Exemplary chemotherapy agents include, without
limitation, alkylating agents, platinums, anti-metabolites,
anthracyclines, taxanes, camptothecins, nitrosoureas, EGFR
inhibitors, antibiotics, HER2/neu inhibitors, angiogenesis
inhibitors, kinase inhibitors, proteaosome inhibitors,
immunotherapies, hormone therapies, photodynamic therapies, cancer
vaccines, histone deacetylase inhibitors, sphingolipid modulators,
oligomers, other unclassified chemotherapy drugs and combinations
thereof.
[0220] 1. Alkylating Agents
[0221] Alkylating agents are chemotherapy agents that are thought
to attack the negatively charged sites on the DNA (e.g., the
oxygen, nitrogen, phosphorous and sulfur atoms) and bind to the DNA
thus altering replication, transcription and even base pairing. It
is also believed that alkylation of the DNA also leads to DNA
strand breaks and DNA strand cross-linking. By altering DNA in this
manner, cellular activity is effectively stopped and the cancer
cell will die. Common alkylating agents include, without
limitation, Procarbazine, Ifosphamide, Cyclophosphamide, Melphalan,
Chlorambucil, Decarbazine, Busulfan, Thiotepa, and the like.
Alkylating agents such as those mentioned above can be used in
combination with one or more other alkylating agents and/or with
one or more chemotherapy agents of a different class(es).
[0222] 2. Platinums
[0223] Platinum chemotherapy agents are believed to inhibit DNA
synthesis, transcription and function by cross-linking DNA
subunits. (The cross-linking can happen either between two strands
or within one strand of DNA.) Common platinum chemotherapy agents
include, without limitation, Cisplatin, Carboplatin, Oxaliplatin,
Eloxatin, and the like. Platinum chemotherapy agents such as those
mentioned above can be used in combination with one or more other
platinums and/or with one or more chemotherapy agents of a
different class(es).
[0224] 3. Anti-metabolites
[0225] Anti-metabolite chemotherapy agents are believed to
interfere with normal metabolic pathways, including those necessary
for making new DNA. Common anti-metabolites include, without
limitation, Methotraxate, 5-Fluorouracil (e.g., Capecitabine),
Gemcitabine (2'-deoxy-2',2'-difluorocytidine monohydrochloride
(.beta.-isomer), Eli Lilly), 6-mercaptopurine, 6-thioguanine,
fludarabine, Cladribine, Cytarabine, tegafur, raltitrexed, cytosine
arabinoside, and the like. Gallium nitrate is another
anti-metabolite that inhibits ribonucleotides reductase.
Anti-metabolites such as those mentioned above can be used in
combination with one or more other anti-metabolites and/or with one
or more chemotherapy agents of a different class(es).
[0226] 4. Anthracyclines
[0227] Anthracyclines are believed to promote the formation of free
oxygen radicals. These radicals result in DNA strand breaks and
subsequent inhibition of DNA synthesis and function. Anthracyclines
are also thought to inhibit the enzyme topoisomerase by forming a
complex with the enzyme and DNA. Common anthracyclines include,
without limitation, Daunorubicin, Doxorubicin, Idarubicin,
Epirubicin, Mitoxantrone, adriamycin, bleomycin, mitomycin-C,
dactinomycin, mithramycin and the like. Anthracyclines such as
those mentioned above can be used in combination with one or more
other anthracyclines and/or with one or more chemotherapy agents of
a different class(es).
[0228] 5. Taxanes
[0229] Taxanes are believed to bind with high affinity to the
microtubules during the M phase of the cell cycle and inhibit their
normal function. Common taxanes include, without limitation,
Paclitaxel, Docetaxel, Taxotere, Taxol, taxasm, 7-epipaclitaxel,
t-acetyl paclitaxel, 10-desacetyl-paclitaxel,
10-desacetyl-7-epipaclitaxel, 7-xylosylpaclitaxel,
10-desacetyl-7-epipaclitaxel, 7-N--N-dimethylglycylpaclitaxel,
7-L-alanylpaclitaxel and the like. Taxanes such as those mentioned
above can be used in combination with one or more other taxanes
and/or with one or more chemotherapy agents of a different
class(es).
[0230] 6. Camptothecins
[0231] Camptothecins are thought to complex with topoisomerase and
DNA resulting in the inhibition and function of this enzyme. It is
further believed that the presence of topoisomerase is required for
on-going DNA synthesis. Common camptothecins include, without
limitation, Irinotecan, Topotecan, Etoposide, vinca alkaloids
(e.g., Vincristine, Vinblastine or Vinorelbine), amsacrine,
teniposide and the like. Camptothecins such as those mentioned
above can be used in combination with one or more other
camptothecins and/or with one or more chemotherapy agents of a
different class(es).
[0232] 7. Nitrosoureas
[0233] Nitrosoureas are believed to inhibit changes necessary for
DNA repair. Common nitrosoureas include, without limitation,
Carmustine (BCNU), Lomustine (CCNU), semustine and the like.
Nitrosoureas such as those mentioned above can be used in
combination with one or more other nitrosoureas and/or with one or
more chemotherapy agents of a different class(es).
[0234] 8. EGFR Inhibitors
[0235] EGFR (i.e., epidermal growth factor receptor) inhibitors are
thought to inhibit EGFR and interfere with cellular responses
including cell proliferation and differentiation. EGFR inhibitors
include molecules that inhibit the function or production of one or
more EGFRs. They include small molecule inhibitors of EGFRs,
antibodies to EGFRs, antisense oligomers, RNAi inhibitors and other
oligomers that reduce the expression of EGFRs. Common EGFR
inhibitors include, without limitation, Gefitinib, Erlotinib
(Tarceva.RTM.), Cetuximab (Erbitux.RTM.), panitumumab
(Vectibix.TM..sup., Amgen) lapatinib (GlaxoSmithKline), CI1033 or
PD183805 or Canternib
(6-acrylamide-N-(3-chloro-4-flurorphenyl)-7-(3-morpholinopropoxy)quinazol-
in-4-amine, Pfizer), and the like. Other inhibitors include PKI-166
(4-[(1R)-1-phenylethylamino]-6-(4-hydroxyphenyl)-7H-pyrrolo[2,3-d]pyrimid-
ine, Novartis), CL-387785
(N-[4-(3-bromoanilino)quinazolin-6-yl]but-2-ynamide), EKB-569
(4-(3-chloro-4-fluroranilino)-3-cyano-6-(4-dimethylaminobut2(E)-enamido)--
7-ethozyquinoline, Wyeth), lapatinib (GW2016, GlaxoSmithKline),
EKB509 (Wyeth), Panitumumab (ABX-EGF, Abgenix), matuzumab (EMD
72000, Merck), and the monoclonal antibody RH3 (New York Medical).
EGFR inhibitors such as those mentioned above can be used in
combination with one or more other EGFR inhibitors and/or with one
or more chemotherapy agents of a different class(es).
[0236] 9. Antibiotics
[0237] Antibiotics are thought to promote the formation of free
oxygen radicals that result in DNA breaks leading to cancer cell
death. Common antibiotics include, without limitation, Bleomycin
and rapamycin and the like. The macrolide fungicide rapamycin (also
called RAP, Rapamune and Sirolimus) binds intracellularly to the to
the immunophilin FK506 binding protein 12 (FKBP12) and the
resultant complex inhibits the serine protein kinase activity of
mammalian target of rapamycin (mTOR). Rapamycin macrolides include
naturally occurring forms of rapamycin as well as rapamycin analogs
and derivatives that target and inhibit mTOR. Other rapamycin
macrolides include, without limitation, temsirolimus (CCI-779,
Wyeth)), Everolimus and ABT-578. Antibiotics such as those
mentioned above can be used in combination with one or more other
antibiotics and/or with one or more chemotherapy agents of a
different class(es).
[0238] 10. HER2/neu Inhibitors
[0239] HER2/neu Inhibitors are believed to block the HER2 receptor
and prevent the cascade of reactions necessary for tumor survival.
Her2 inhibitors include molecules that inhibit the function or
production of Her2. They include small molecule inhibitors of Her2,
antibodies to Her2, antisense oligomers, RNAi inhibitors and other
oligomers that reduce the expression of tyrosine kinases. Common
HER2/neu inhibitors include, without limitation, Trastuzumab
(Herceptin.RTM., Genentech) and the like. Other Her2/neu inhibitors
include bispecific antibodies MDX-210 (FC.gamma.R1-Her2/neu) and
MDX-447 (Medarex), pertuzumab (rhuMAb 2C4, Genentech), HER2/neu
inhibitors such as those mentioned above can be used in combination
with one or more other HER2/neu inhibitors and/or with one or more
chemotherapy agents of a different class(es).
[0240] 11. Angiogenesis Inhibitors
[0241] Angiogenesis inhibitors are believed to inhibit vascular
endothelial growth factor, i.e. VEGF, thereby inhibiting the
formation of new blood vessels necessary for tumor life. VEGF
inhibitors include molecules that inhibit the function or
production of one or more VEGFs. They include small molecule
inhibitors of VEGF, antibodies to VEGF, antisense oligomers, RNAi
inhibitors and other oligomers that reduce the expression of
tyrosine kinases. Common angiogenesis inhibitors include, without
limitation, Bevacizumab (Avastin.RTM., Genentech). Other
angiogenesis inhibitors include, without limitation, ZD6474
(AstraZeneca), Bay-43-9006, sorafenib (Nexavar, Bayer), semaxamib
(SU5416, Pharmacia), SU6668 (Pharmacia), ZD4190
(N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[2-(1H-1,2,3-triazol-1-yl)ethoxy]-
quinazolin-4-amine, Astra Zeneca), Zactima.TM. (ZD6474,
N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[2-(1H-1,2,3-triazol-1-yl)ethoxy]q-
uinazolin-4-amine, Astra Zeneca), Vatalanib, (PTK787, Novartis),
the monoclonal antibody IMC-1C11 (Imclone) and the like.
Angiogenesis inhibitors such as those mentioned above can be used
in combination with one or more other angiogenesis inhibitors
and/or with one or more chemotherapy agents of a different
class(es).
[0242] 12. Other Kinase Inhibitors
[0243] In addition to EGFR, HER2 and VEGF inhibitors, other kinase
inhibitors are used as chemotherapeutic agents. Aurora kinase
inhibitors include, without limitation, compounds such as 4-(4-N
benzoylamino)aniline)-6-methoxy-7-(3-(1-morpholino)propoxy)quinazoline
(ZM447439, Ditchfield et al., J. Cell. Biol., 161:267-80 (2003))
and Hesperadin (Haaf et al., J. Cell Biol., 161: 281-94 (2003)).
Other compounds suitable for use as Aurora kinase inhibitors are
described in Vankayalapati H, et al., Mol. Cancer Ther. 2:283-9
(2003). SRC/Abl kinase inhibitors include without limitation,
AZD0530
(4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)etho-
xy]-5-tetrahycropyran-4-yloxyquinazoline). Tyrosine kinase
inhibitors include molecules that inhibit the function or
production of one or more tyrosine kinases. They include small
molecule inhibitors of tyrosine kinases, antibodies to tyrosine
kinases and antisense oligomers, RNAi inhibitors and other
oligomers that reduce the expression of tyrosine kinases. CEP-701
and CEP-751 (Cephalon) act as tyrosine kinase inhibitors. Imatinib
mesylate is a tyrosine kinase inhibitor that inhibits bcr-abl by
binding to the ATP binding site of bcr-abl and competitively
inhibiting the enzyme activity of the protein. Although imatinib is
quite selective for bcr-abl, it does also inhibit other targets
such as c-kit and PDGF-R. FLT-3 inhibitors include, without
limitation, tandutinib (MLN518, Millenium), Sutent (SU11248,
5-[5-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,
4-dimethyl-1H-pyrrole-3-carboxylic acid [2-diethylaminoethyl]amide,
Pfizer), midostaurin (4'-N-Benzoyl Staurosporine,_Novartis),
lefunomide (SU101) and the like. MEK inhibitors include, without
limitation,
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzami-
de) (PD184352/CI-1044, Pfizer), PD198306 (Pfizer), PD98059
(2'-amino-3'-methoxyflavone), UO126 (Promega), Ro092210 from
fermented microbial extracts (Roche), the resorcyclic acid lactone,
L783277, also isolated from microbial extracts (Merck) and the
like. Tyrosine kinase inhibitors such as those mentioned above can
be used in combination with one or more other tyrosine kinase
inhibitors and/or with one or more chemotherapy agents of a
different class(es).
[0244] 13. Proteaosome Inhibitors
[0245] Proteaosome inhibitors are believed to inhibit the breakdown
of some of these proteins that have been marked for destruction.
This results in growth arrest or death of the cell. Common
proteaosome inhibitors include, without limitation, Bortezomib,
ortezomib and the like. Proteaosome inhibitors such as those
mentioned above can be used in combination with one or more other
proteaosome inhibitors and/or with one or more chemotherapy agents
of a different class(es).
[0246] 14. Immunotherapies
[0247] Immunotherapies are thought to bind to and block specific
targets, thereby disrupting the chain of events needed for tumor
cell proliferation. Common immunotherapies include, without
limitation, Rituximab and other antibodies directed against CD20,
Campath-1H and other antibodies directed against CD-50, epratuzmab
and other antibodies directed against CD-22, galiximab and other
antibodies directed against CD-80, apolizumab HU1D10 and other
antibodies directed against HLA-DR, and the like. Radioisotopes can
be conjugated to the antibody, resulting in radioimmunotherapy. Two
such anti-CD20 products are tositumomab (Bexxar) and ibritumomab
(Zevalin) Immunotherapies such as those mentioned above can be used
in combination with one or more other immunotherapies and/or with
one or more chemotherapy agents of a different class(es).
[0248] 15. Hormone Therapies
[0249] Hormone therapies are thought to block cellular receptors,
inhibit the in vivo production of hormones, and/or eliminate or
modify hormone receptors on cells, all with the end result of
slowing or stopping tumor proliferation. Common hormone therapies
include, without limitation, antiestrogens (e.g., tamoxifen,
toremifene, fulvestrant, raloxifene, droloxifene, idoxyfene and the
like), progestogens) e.g., megestrol acetate and the like)
aromatase inhibitors (e.g., Anastrozole, Letrozole, Exemestane,
vorazole, exemestane, fadrozole, aminoglutethimide, exemestane,
1-methyl-1,4-androstadiene-3,17-dione and the like), anti-androgens
(e.g., Bicalutimide, Nilutamide, Flutamide, cyproterone acetate,
and the like), luteinizing hormone releasing hormone agonist (LHRH
Agonist) (e.g., Goserelin, Leuprolide, buserelin and the like);
5-.alpha.-reductase inhibitors such as finasteride, and the like.
Hormone therapies such as those mentioned above can be used in
combination with one or more other hormone therapies and/or with
one or more chemotherapy agents of a different class(es).
[0250] 16. Photodynamic Therapies
[0251] Photodynamic therapies expose a photosensitizing drug to
specific wavelengths of light to kill cancer cells. Common
photodynamic therapies include, for example, porfimer sodium (e.g.,
Photofrin.RTM.) and the like. Photodynamic therapies such as those
mentioned above can be used in combination with one or more other
photodynamic therapies and/or with one or more chemotherapy agents
of a different class(es).
[0252] 17. Cancer Vaccines
[0253] Cancer vaccines are thought to utilize whole, inactivated
tumor cells, whole proteins, peptide fragments, viral vectors and
the like to generate an immune response that targets cancer cells.
Common cancer vaccines include, without limitation, modified tumor
cells, peptide vaccine, dendritic vaccines, viral vector vaccines,
heat shock protein vaccines and the like.
[0254] 18. Histone Deacetylase Inhibitors
[0255] Histone deacetylase inhibitors are able to modulate
transcriptional activity and consequently, can block angiogenesis
and cell cycling, and promote apoptosis and differentiation.
Histone deacetylase inhibitors include, without limitation, SAHA
(Suberoylanilide hydroxamic acid), depsipeptide (FK288) and
analogs, Pivanex (Titan), CI994 (Pfizer), MS275 PXD101 (CuraGen,
TopoTarget) MGCD0103 (MethylGene), LBH589, NVP-LAQ824 (Novartis)
and the like and have been used as chemotherapy agents. Histone
deacetylase inhibitors such as those mentioned above can be used in
combination with one or more other histone deacetylase inhibitors
and/or with one or more chemotherapy agents of a different
class(es).
[0256] 19. Sphingolipid Modulators
[0257] Modulators of Sphingolipid metabolism have been shown to
induce apoptosis. For reviews see N. S. Radin, Biochem J,
371:243-56 (2003); D. E. Modrak, et al., Mol. Cancer Ther,
5:200-208 (2006), K. Desai, et al., Biochim Biophys Acta,
1585:188-92 (2002) and C. P. Reynolds, et al. and Cancer Lett, 206,
169-80 (2004), all of which are incorporated herein by reference.
Modulators and inhibitors of various enzymes involved in
sphingolipid metabolism can be used as chemotherapeutic agents.
[0258] (a) Ceramide has been shown to induce apoptosis,
consequently, exogenous ceramide or a short chain ceramide analog
such as N-acetylsphingosine (C.sub.2-Cer), C.sub.6-Cer or
C.sub.8-Cer has been used. Other analogs include, without
limitation, Cer 1-glucuronide, poly(ethylene glycol)-derivatized
ceramides and pegylated ceramides.
[0259] (b) Modulators that stimulate ceramide synthesis have been
used to increase ceramide levels. Compounds that stimulate serine
palmitoyltransferase, an enzyme involved in ceramide synthesis,
include, without limitation, tetrahydrocannabinol (THC) and
synthetic analogs and anandamide, a naturally occurring mammalian
cannabinoid. Gemcitabine, retinoic acid and a derivative,
fenretinide [N-(4-hycroxyphenyl)retinamide, (4-HPR)], camptothecin,
homocamptothecin, etoposide, paclitaxel, daunorubicin and
fludarabine have also been shown to increase ceramide levels. In
addition, valspodar (PSC833, Novartis), a non-immunosuppressive
non-ephrotoxic analog of cyclosporin and an inhibitor of
p-glycoprotein, increases ceramide levels.
[0260] (c) Modulators of sphingomyelinases can increase ceramide
levels. They include compounds that lower GSH levels, as GSH
inhibits sphingomyelinases. For example, betathine (.beta.-alanyl
cysteamine disulphide), oxidizes GSH, and has produced good effects
in patients with myeloma, melanoma and breast cancer. COX-2
inhibitors, such as celecoxib, ketoconazole, an antifungal agent,
doxorubicin, mitoxantrone, D609 (tricyclodecan-9-yl-xanthogenate),
dexamethasone, and Ara-C (1-.beta.-D-arabinofuranosylcytosine) also
stimulate sphingomyelinases.
[0261] (d) Molecules that stimulate the hydrolysis of
glucosylceramide also raise ceramide levels. The enzyme, GlcCer
glucosidase, which is available for use in Gaucher's disease,
particularly with retinol or pentanol as glucose acceptors and/or
an activator of the enzyme can be used as therapeutic agents.
Saposin C and analogs thereof, as well as analogs of the
anti-psychotic drug, chloropromazine, may also be useful.
[0262] (e) Inhibitors of glucosylceramide synthesis include,
without limitation, PDMP
(N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyldecanamide]),
PMPP
(D,L-threo-(1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol),
P4 or PPPP
(D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol),
ethylenedioxy-P4, 2-decanoylamine-3-morpholinoprophenone,
tamixofen, raloxifene, mifepristone (RU486), N-butyl
deoxynojirimycin and anti androgen chemotherapy
(bicalutamide+leuprolide acetate). Zavesca,
(1,5-(butylimino)-1,5-dideoxy-D-glucitol) usually used to treat
Gaucher's disease, is another inhibitor of glucosylceramide
synthesis.
[0263] (f) Inhibitors of ceramidase include, without limitation,
N-oleoylethanolamine, a truncated form of ceramide, D-MAPP
(D-erythro-2-tetradecanoylamino-1-phenyl-1-propanol) and the
related inhibitor B13 (p-nitro-D-MAPP).
[0264] (g) Inhibitors of sphingosine kinase also result in
increased levels of ceramide. Inhibitors include, without
limitation, safingol (L-threo-dihydrosphingosine), N,N-dimethyl
sphingosine, trimethylsphingosine and analogs and derivatives of
sphingosine such as dihydrosphingosine, and myriocin.
[0265] (h) Fumonisins and fumonisin analogs, although they inhibit
ceramide synthase, also increase levels of sphinganine due to the
inhibition of de novo sphingolipid biosynthesis, resulting in
apoptosis.
[0266] (i) Other molecules that increase ceramide levels include,
without limitation, miltefosine (hexadecylphosphocholine).
Sphingolipid modulators, such as those mentioned above, can be used
in combination with one or more other sphingolipid modulators
and/or with one or more chemotherapy agents of a different
class(es).
[0267] 20. Oligomers
[0268] In addition to the oligonucleotides of the present
invention, other oligonucleotides have been used as cancer
therapies. They include Genasense (oblimersen, G3139, from Genta),
an antisense oligonucleotide that targets bcl-2 and G4460 (LR3001,
from Genta) another antisense oligonucleotide that targets c-myb.
Other oligomers include, without limitation, siRNAs, decoys, RNAi
oligonucleotides and the like. Oligonucleotides, such as those
mentioned above, can be used in combination with one or more other
oligonucleotide inhibitors and/or with one or more chemotherapy
agents of a different class(es).
[0269] 21. Other Chemotherapy Drugs
[0270] Additional unclassified chemotherapy agents are described in
Table 2 below.
TABLE-US-00003 TABLE 2 Additional unclassified chemotherapy agents.
Generic Name Brand Name Manufacturer Aldesleukin Proleukin Chiron
Corp., (des-alanyl-1, serine-125 human interleukin-2) Emeryville,
CA Alemtuzumab Campath Millennium and (IgG1.kappa. anti CD52
antibody) ILEX Partners, LP, Cambridge, MA Alitretinoin Panretin
Ligand (9-cis-retinoic acid) Pharmaceuticals, Inc., San Diego CA
Allopurinol Zyloprim GlaxoSmithKline,
(1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4- Research Triangle one
monosodium salt) Park, NC Altretamine Hexalen US Bioscience, West
(N,N,N',N',N'',N'',-hexamethyl-1,3,5-triazine-2, Conshohocken, PA
4,6-triamine) Amifostine Ethyol US Bioscience (ethanethiol,
2-[(3-aminopropyl)amino]-, dihydrogen phosphate (ester))
Anastrozole Arimidex AstraZeneca (1,3-Benzenediacetonitrile,
a,a,a',a'- Pharmaceuticals, LP,
tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl)) Wilmington, DE Arsenic
trioxide Trisenox Cell Therapeutic, Inc., Seattle, WA Asparaginase
Elspar Merck & Co., Inc., (L-asparagine amidohydrolase, type
EC-2) Whitehouse Station, NJ BCG Live TICE BCG Organon Teknika,
(lyophilized preparation of an attenuated strain Corp., Durham, NC
of Mycobacterium bovis (Bacillus Calmette- Gukin [BCG], substrain
Montreal) bexarotene capsules Targretin Ligand
(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl- Pharmaceuticals
2-napthalenyl) ethenyl] benzoic acid) bexarotene gel Targretin
Ligand Pharmaceuticals Carmustine with Polifeprosan 20 Implant
Gliadel Wafer Guilford Pharmaceuticals, Inc., Baltimore, MD
Celecoxib Celebrex Searle (as
4-[5-(4-methylphenyl)-3-(trifluoromethyl)- Pharmaceuticals,
1H-pyrazol-1-yl] England benzenesulfonamide) Chlorambucil Leukeran
GlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)
Cladribine Leustatin, 2- R. W. Johnson
(2-chloro-2'-deoxy-b-D-adenosine) CdA Pharmaceutical Research
Institute, Raritan, NJ Dacarbazine DTIC-Dome Bayer AG,
(5-(3,3-dimethyl-1-triazeno)-imidazole-4- Leverkusen, carboxamide
(DTIC)) Germany Dactinomycin, actinomycin D Cosmegen Merck
(actinomycin produced by Streptomyces parvullus,
C.sub.62H.sub.86N.sub.12O.sub.16) Darbepoetin alfa Aranesp Amgen,
Inc., (recombinant peptide) Thousand Oaks, CA Denileukin diftitox
Ontak Seragen, Inc., (recombinant peptide) Hopkinton, MA
Dexrazoxane Zinecard Pharmacia & Upjohn
((S)-4,4'-(1-methyl-1,2-ethanediyl)bis-2,6- Company
piperazinedione) dromostanolone propionate Dromostanolone Eli Lilly
& (17b-Hydroxy-2a-methyl-5a-androstan-3-one Company,
propionate) Indianapolis, IN dromostanolone propionate Masterone
Syntex, Corp., Palo injection Alto, CA Elliott's B Solution
Elliott's B Orphan Medical, Inc Solution Epoetin alfa Epogen Amgen,
Inc (recombinant peptide) Estramustine Emcyt Pharmacia & Upjohn
(estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3- Company
[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium
salt, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17-
(dihydrogen phosphate), disodium salt, monohydrate) Exemestane
Aromasin Pharmacia & Upjohn
(6-methylenandrosta-1,4-diene-3,17-dione) Company Filgrastim
Neupogen Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDR
Roche (2'-deoxy-5-fluorouridine) Fulvestrant Faslodex IPR
(7-alpha-[9-(4,4,5,5,5-penta Pharmaceuticals,
fluoropentylsulphinyl) nonyl]estra-1,3,5-(10)- Guayama, Puerto
triene-3,17-beta-diol) Rico Gemtuzumab Ozogamicin Mylotarg Wyeth
Ayerst (anti-CD33 hP67.6) Hydroxyurea Hydrea Bristol-Myers Squibb
Ifosfamide IFEX Bristol-Myers (3-(2-chloroethyl)-2-[(2- Squibb
chloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)
Imatinib Mesilate Gleevec Novartis AG, Basel,
(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4- Switzerland
methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-phenyl]benzamide
methanesulfonate) Interferon alfa-2a Roferon-A Hoffmann-La
(recombinant peptide) Roche, Inc., Nutley, NJ Interferon alfa-2b
Intron A Schering AG, Berlin, (recombinant peptide) (Lyophilized
Germany Betaseron) Irinotecan HCl Camptosar Pharmacia & Upjohn
((4S)-4,11-diethyl-4-hydroxy-9-[(4- Company
piperidinopiperidino)carbonyloxy]- 1H-pyrano[3',4':6, 7]
indolizino[1,2-b] quinoline-3,14(4H,12H)dione hydrochloride
trihydrate) Letrozole Femara Novartis
(4,4'-(1H-1,2,4-Triazol-1-ylmethylene)dibenzonitrile) Leucovorin
Wellcovorin, Immunex, Corp., (L-Glutamic acid,
N[4[[(2-amino-5-formyl- Leucovorin Seattle, WA
1,4,5,6,7,8-hexahydro-4oxo-6- pteridinyl)methyl]amino]benzoyl],
calcium salt (1:1)) Levamisole HCl Ergamisol Janssen Research
((-)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo Foundation, [2,1-b]
thiazole monohydrochloride Titusville, NJ
C.sub.11H.sub.12N.sub.2S.cndot.HCl) Lomustine CeeNU Bristol-Myers
(1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Squibb
Meclorethamine, nitrogen mustard Mustargen Merck
(2-chloro-N-(2-chloroethyl)-N- methylethanamine hydrochloride)
Megestrol acetate Megace Bristol-Myers
17.alpha.(acetyloxy)-6-methylpregna-4,6-diene- Squibb 3,20-dione
Melphalan, L-PAM Alkeran GlaxoSmithKline (4-[bis(2-chloroethyl)
amino]-L-phenylalanine) Mercaptopurine, 6-MP Purinethol
GlaxoSmithKline (1,7-dihydro-6H-purine-6-thione monohydrate) Mesna
Mesnex Asta Medica (sodium 2-mercaptoethane sulfonate) Methotrexate
Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6-
pteridinyl)methyl]methylamino]benzoyl]-L- glutamic acid)
Methoxsalen Uvadex Therakos, Inc., Way
(9-methoxy-7H-furo[3,2-g][1]-benzopyran-7- Exton, Pa one) Mitomycin
C Mutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen,
Inc., Dublin, CA Mitotane Lysodren Bristol-Myers
(1,1-dichloro-2-(o-chlorophenyl)-2-(p- Squibb chlorophenyl) ethane)
Mitoxantrone Novantrone Immunex (1,4-dihydroxy-5,8-bis[[2-[(2-
Corporation hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione
dihydrochloride) Nandrolone phenpropionate Durabolin-50 Organon,
Inc., West Orange, NJ Nofetumomab Verluma Boehringer Ingelheim
Pharma KG, Germany Oprelvekin Neumega Genetics Institute, (IL-11)
Inc., Alexandria, VA Pamidronate Aredia Novartis (phosphonic acid
(3-amino-1- hydroxypropylidene) bis-, disodium salt, pentahydrate,
(APD)) Pegademase Adagen Enzon ((monomethoxypolyethylene glycol
(Pegademase Pharmaceuticals, succinimidyl) 11-17-adenosine
deaminase) Bovine) Inc., Bridgewater, NJ Pegaspargase Oncaspar
Enzon (monomethoxypolyethylene glycol succinimidyl L-asparaginase)
Pegfilgrastim Neulasta Amgen, Inc (covalent conjugate of
recombinant methionyl human G-CSF (Filgrastim) and
monomethoxypolyethylene glycol) Pentostatin Nipent Parke-Davis
Pharmaceutical Co., Rockville, MD Pipobroman Vercyte Abbott
Laboratories, Abbott Park, IL Plicamycin, Mithramycin Mithracin
Pfizer, Inc., NY, NY (antibiotic produced by Streptomyces plicatus)
Quinacrine Atabrine Abbott Labs
(6-chloro-9-(1-methyl-4-diethyl-amine)butylamino-
2-methoxyacridine) Rasburicase Elitek Sanofi-Synthelabo,
(recombinant peptide) Inc., Sargramostim Prokine Immunex Corp
(recombinant peptide) Streptozocin Zanosar Pharmacia & Upjohn
(streptozocin 2-deoxy-2- Company
[[(methylnitrosoamino)carbonyl]amino]-a(and b)-D-glucopyranose and
220 mg citric acid anhydrous) Talc Sclerosol Bryan, Corp.,
(Mg.sub.3Si.sub.4O.sub.10(OH).sub.2) Woburn, MA Temozolomide
Temodar Schering (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-
tetrazine-8-carboxamide) Teniposide, VM-26 Vumon Bristol-Myers
(4'-demethylepipodophyllotoxin 9-[4,6-0-(R)-2- Squibb
thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac
Bristol-Myers (13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien- Squibb
17-oic acid [dgr]-lactone) Thioguanine, 6-TG Thioguanine
GlaxoSmithKline (2-amino-1,7-dihydro-6H-purine-6-thione) Thiotepa
Thioplex Immunex (Aziridine, 1,1',1''-phosphinothioylidynetris-, or
Corporation Tris (1-aziridinyl) phosphine sulfide) Topotecan HCl
Hycamtin GlaxoSmithKline ((S)-10-[(dimethylamino)
methyl]-4-ethyl-4,9- dihydroxy-1H-pyrano[3',4':6,7] indolizino
[1,2- b] quinoline-3,14-(4H,12H)-dione monohydrochloride)
Toremifene Fareston Roberts
(2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]- Pharmaceutical
phenoxy)-N,N-dimethylethylamine citrate (1:1)) Corp., Eatontown, NJ
Tositumomab, I 131 Tositumomab Bexxar Corixa Corp., (recombinant
murine immunotherapeutic Seattle, WA monoclonal IgG.sub.2a lambda
anti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))
Tretinoin, ATRA Vesanoid Roche (all-trans retinoic acid) Uracil
Mustard Uracil Mustard Roberts Labs Capsules Vairubicin,
N-trifluoroacetyladriamycin-14- Valstar Anthra --> Medeva
valerate ((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12- trihydroxy-7
methoxy-6,11-dioxo-[[4 2,3,6-
trideoxy-3-[(trifluoroacetyl)-amino-.alpha.-L-lyxo-
hexopyranosyl]oxyl]-2-naphthacenyl]-2- oxoethyl pentanoate)
Zoledronate, Zoledronic acid Zometa Novartis
((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid
monohydrate)
[0271] 22. Cocktails
[0272] Chemotherapy agents can include cocktails of two or more
chemotherapy drugs mentioned above. In several embodiments, a
chemotherapy agent is a cocktail that includes two or more
alkylating agents, platinums, anti-metabolites, anthracyclines,
taxanes, camptothecins; nitrosoureas, EGFR inhibitors, antibiotics,
HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors,
proteaosome inhibitors, immunotherapies, hormone therapies,
photodynamic therapies, cancer vaccines, sphingolipid modulators,
oligomers or combinations thereof.
[0273] In one embodiment, the chemotherapy agent is a cocktail that
includes an immunotherapy, an alkylating agent, an anthracycline, a
camptothecin and Prednisone. In other embodiments, the chemotherapy
agent is a cocktail that includes Rituximab, an alkylating agent,
an anthracycline, a camptothecin and Prednisone. In other
embodiments, the chemotherapy agent is a cocktail that includes
Rituximab, Cyclophosphamide, an anthracycline, a camptothecin and
Prednisone. In still other embodiments, the chemotherapy agent is a
cocktail that includes Rituximab, Cyclophosphamide, Doxorubicin,
Vincristine and Prednisone (e.g., R-CHOPS).
[0274] In another embodiment, the chemotherapy agent is a cocktail
that includes doxorubicin, ifosfamide and Mesna.
[0275] In other embodiments, the chemotherapy agent is a cocktail
that includes an anti-metabolite and a taxane. For example, the
chemotherapy agent includes Gemcitabine and Taxotere.
[0276] In other embodiments, the chemotherapy agent is a cocktail
that includes dacarbazine, Mitomycin, Doxorubicin and
Cisplatin.
[0277] In other embodiments, the chemotherapy agent is a cocktail
that includes Doxorubicin and Dacarbazine.
[0278] In alternative embodiments, the chemotherapy agent is a
cocktail that includes an alkylating agent, a camptothecins, an
anthracycline and dacarbazine. In other examples, the chemotherapy
agent includes cyclophosphamide, vincristine, doxorubicin and
dacarbazine.
[0279] In still other embodiments, the chemotherapy agent is a
cocktail that includes an alkylating agent, methotrexate, an
anti-metabolite and one or more anthracyclines. For example, the
chemotherapy agent includes 5-fluorouracil, methotrexate,
cyclophosphamide, doxorubicin and epirubicin.
[0280] In yet other embodiments, the chemotherapy agent is a
cocktail that includes a taxane and prednisone or estramustine. For
example, the chemotherapy agent can include docetaxel combined with
prednisone or estramustine.
[0281] In still yet another embodiment, the chemotherapy agent
includes an anthracycline and prednisone. For example, the
chemotherapy agent can include mitoxantrone and prednisone.
[0282] In other embodiments, the chemotherapy agent includes a
rapamycin macrolide and a kinase inhibitor. The kinase inhibitors
can be EGFR, Her2/neu, VEGF, Aurora kinase, SRC/Abl kinase,
tyrosine kinase and/or MEK inhibitors.
[0283] In another embodiment the chemotherapy agent includes two or
more sphingolipid modulators.
[0284] In still another embodiment the chemotherapy agent includes
an oligomer, such as Genasense and one or more alkylating agents,
platinums, anti-metabolites, anthracyclines, taxanes,
camptothecins, nitrosoureas, EGFR inhibitors, antibiotics, HER2/neu
inhibitors, angiogenesis inhibitors, kinase inhibitors, proteaosome
inhibitors, immunotherapies, hormone therapies, photodynamic
therapies, cancer vaccines, sphingolipid modulators or combinations
thereof.
[0285] Moreover, the chemotherapy drug or drugs composing the
chemotherapy agent can be administered in combination therapies
with other agents, or they may be administered sequentially or
concurrently to the patient.
[0286] C. Radiation Therapy
[0287] In several embodiments of the present invention, radiation
therapy is administered in addition to the administration of an
oligonucleotide compound. Radiation therapy includes both external
and internal radiation therapies.
[0288] 1. External Radiation Therapy
[0289] External radiation therapies include directing high-energy
rays (e.g., x-rays, gamma rays, and the like) or particles (alpha
particles, beta particles, protons, neutrons and the like) at the
cancer and the normal tissue surrounding it. The radiation is
produced outside the patient's body in a machine called a linear
accelerator. External radiation therapies can be combined with
chemotherapies, surgery or oligonucleotide compounds.
[0290] 2. Internal Radiation Therapy
[0291] Internal radiation therapies include placing the source of
the high-energy rays inside the body, as close as possible to the
cancer cells. Internal radiation therapies can be combined with
external radiation therapies, chemotherapies or surgery.
[0292] Radiation therapy can be administered with chemotherapy
simultaneously, concurrently, or separately. Moreover radiation
therapy can be administered with surgery simultaneously,
concurrently, or separately.
[0293] D. Surgery
[0294] In alternative embodiments, of the present invention,
surgery is used to remove cancerous tissue from a patient.
Cancerous tissue can be excised from a patient using any suitable
surgical procedure including, for example, laparoscopy, scalpel,
laser, scissors and the like. In several embodiments, surgery is
combined with chemotherapy. In other embodiments, surgery is
combined with radiation therapy. In still other embodiments,
surgery is combined with both chemotherapy and radiation
therapy.
III. Pharmaceutical Compositions
[0295] In one aspect of the present invention, a pharmaceutical
composition comprises one or more oligonucleotide compounds and a
chemotherapy agent. For example, a pharmaceutical composition
comprises an oligonucleotide compound having SEQ. ID NO. 1250,
1251, 1252, or 1253; and one or more of an alkylating agent, a
platinum, an anti-metabolite, an anthracycline, a taxane, a
camptothecins, a nitrosourea, an EGFR inhibitor, an antibiotic, a
HER2/neu inhibitor, an angiogenesis inhibitor, a proteaosome
inhibitor, an immunotherapy, a hormone therapy, a photodynamic
therapy, a cancer vaccine, other chemotherapy agents such as those
illustrated in Table 1, or combinations thereof.
[0296] In one embodiment, the pharmaceutical composition comprises
an oligonucleotide compound and a chemotherapy agent including an
immunotherapy, an alkylating agent, an anthracycline, a
camptothecin and Prednisone. For example, the pharmaceutical
composition comprises one or more oligonucleotide compounds
comprising SEQ ID NOs 2-281, 283-461, 463-935, 937-1080, 1082-1248,
1250-1254 and 1267-1477, and complements thereof; and a
chemotherapy agent including an immunotherapy, an alkylating agent,
an anthracycline, a camptothecin, and Prednisone. In other
embodiments, the pharmaceutical composition comprises an
oligonucleotide compound and a chemotherapy agent that includes
Rituximab, Cyclophosphamide, an anthracycline, a camptothecin and
Prednisone. In still other embodiments, the pharmaceutical
composition comprises an oligonucleotide and a chemotherapy agent
including Rituximab, Cyclophosphamide, Doxorubicin, Vincristine and
Prednisone (e.g., R-CHOPS).
[0297] Other embodiments of the invention provide pharmaceutical
compositions containing (a) one or more oligonucleotide compounds
and (b) a chemotherapy agent. Examples of such chemotherapeutic
agents include, without limitation, those listed above.
Anti-inflammatory drugs, including but not limited to nonsteroidal
anti-inflammatory drugs and corticosteroids, and antiviral drugs,
including but not limited to ribivirin, vidarabine, acyclovir and
ganciclovir, may also be combined in compositions of the invention.
Other non-oligonucleotide chemotherapeutic agents are also within
the scope of this invention. Two or more combined compounds may be
used together or sequentially.
[0298] Pharmaceutical compositions of the present invention can
optionally include medicaments such as anesthesia, nutritional
supplements (e.g., vitamins, minerals, protein and the like),
chromophores, combinations thereof, and the like.
[0299] A. Formulations, Administration and Uses
[0300] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, intraoccularly, buccally, vaginally, or via an
implanted reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques.
Preferably, the compositions are administered orally,
intraperitoneally or intravenously. Sterile injectable forms of the
compositions of this invention may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally-acceptable diluent or solvent, for example
as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium.
[0301] For this purpose, any bland fixed oil may be employed
including synthetic mono- or di-glycerides. Fatty acids, such as
oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0302] The pharmaceutically acceptable compositions of this
invention may be orally administered in any orally acceptable
dosage form including, but not limited to, capsules, tablets,
aqueous suspensions or solutions. In the case of tablets for oral
use, carriers commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried cornstarch. When aqueous suspensions are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
[0303] Alternatively, the pharmaceutically acceptable compositions
of this invention may be administered in the form of suppositories
for rectal administration. These can be prepared by mixing the
agent with a suitable non-irritating excipient that is solid at
room temperature but liquid at rectal temperature and therefore
will melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0304] The pharmaceutically acceptable compositions of this
invention may also be administered topically, especially when the
target of treatment includes areas or organs readily accessible by
topical application, including diseases of the eye, the skin or the
lower intestinal tract. Suitable topical formulations are readily
prepared for each of these areas or organs.
[0305] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0306] For topical applications, the pharmaceutically acceptable
compositions may be formulated in a suitable ointment containing
the active component suspended or dissolved in one or more
carriers. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutically acceptable compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0307] For ophthalmic use, the pharmaceutically acceptable
compositions may be formulated as micronized suspensions in
isotonic, pH adjusted sterile saline, or, preferably, as solutions
in isotonic, pH adjusted sterile saline, either with or without a
preservative such as benzylalkonium chloride. Alternatively, for
ophthalmic uses, the pharmaceutically acceptable compositions may
be formulated in an ointment such as petrolatum.
[0308] The pharmaceutically acceptable compositions of this
invention may also be administered by nasal aerosol or inhalation.
Such compositions are prepared according to techniques well-known
in the art of pharmaceutical formulation and may be prepared as
solutions in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other conventional solubilizing or dispersing
agents.
[0309] In several embodiments, the pharmaceutically acceptable
compositions of this invention are formulated for oral
administration.
[0310] The amount of the compounds of the present invention that
may be combined with the carrier materials to produce a composition
in a single dosage form will vary depending upon the host treated,
the particular mode of administration. Preferably, the compositions
should be formulated so that a dosage of between 0.01-100 mg/kg
body weight/day of the modulator can be administered to a patient
receiving these compositions.
[0311] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of a compound of the
present invention in the composition will also depend upon the
particular compound in the composition.
[0312] Depending upon the particular condition, or disease, to be
treated or prevented, additional therapeutic agents, which are
normally administered to treat or prevent that condition, may also
be present in the compositions of this invention. As used herein,
additional therapeutic agents normally administered to treat or
prevent a particular disease, or condition, are known as
"appropriate for the disease, or condition, being treated."
[0313] B. Delivery
[0314] The oligonucleotide compounds of the present invention may
be delivered using any suitable method. In some embodiments, naked
DNA is administered. In other embodiments, lipofection is utilized
for the delivery of nucleic acids to a subject. In still further
embodiments, oligonucleotides are modified with phosphothiolates
for delivery (See e.g., U.S. Pat. No. 6,169,177, herein
incorporated by reference).
[0315] In some embodiments, nucleic acids for delivery are
compacted to aid in their uptake (See e.g., U.S. Pat. Nos.
6,008,366, 6,383,811 herein incorporated by reference). In some
embodiments, compacted nucleic acids are targeted to a particular
cell type (e.g., cancer cell) via a target cell binding moiety (See
e.g., U.S. Pat. Nos. 5,844,107, 6,077,835, each of which is herein
incorporated by reference).
[0316] In some embodiments, oligonucleotides are conjugated to
other compounds to aid in their delivery. For example, in some
embodiments, nucleic acids are conjugated to polyethylene glycol to
aid in delivery (See e.g., U.S. Pat. Nos. 6,177,274, 6,287,591,
6,447,752, 6,447,753, and 6,440,743, each of which is herein
incorporated by reference). In yet other embodiments,
oligonucleotides are conjugated to protected graft copolymers,
which are chargeable" drug nano-carriers (PharmaIn), described in
U.S. Pat. No. 7,138,105, and U.S. publication numbers 2006/093660
and 2006/0239924, which are incorporated herein by reference. In
still further embodiments, the transport of oligonucleotides into
cells is facilitated by conjugation to vitamins (Endocyte, Inc,
West Lafayette, Ind.; See e.g., U.S. Pat. Nos. 5,108,921,
5,416,016, 5,635,382, 6,291,673 and WO 02/085908; each of which is
herein incorporated by reference). In other embodiments,
oligonucleotides are conjugated to nanoparticles (e.g., NanoMed
Pharmaceuticals; Kalamazoo, Mich.).
[0317] In still other embodiments, oligonucleotides are associated
with dendrimers. Dendrimers are synthetic macromolecules with
highly branched molecular structures. Representative dendrimeric
structures are cationic polymers such as starburst polyamidoamine
(PAMAM), one of which, SuperFect.RTM., is available from Qiagen
(Valencia, Calif.). Other dendrimers include polyester dentrimers
described by Gillies, et al., Mol. Pharm., 2:129-38, 2005, which is
incorporated herein by reference; phenylacetylene dendrimers,
described in Janssen and Meijer, eds, Synthesis of Polymers,
Materials science and technology series, Weinheim, Germany:
Wiley-VCH Verlag GMBH, Chapter 12, 1999, which is incorporated
herein by reference; poly(L-lysine) dendrimer-block-poly(ethylene
glycol)-block-poly(L-lysine) dendrimers described by Choi, et al.,
J. Am. Chem. Soc. 122, 474-80, 2000, which is incorporated herein
by reference; amphiphilic dendrimers, described by Joester, et al.,
Angew Chem Int. Ed. Engl., 42:1486-90, 2003, which is incorporated
herein by reference; polyethylene glycol star like conjugates,
described by Liu et al., Polym Chem, 37:3492-3503, 1999, which is
incorporated herein by reference; cationic phosphorus-containing
dendrimers described by Loup, et al., Chem Eur J, 5:3644-50, 1999,
which is incorporated herein by reference; poly(L-lysine)
dendrimers, described by Ohasaki, et al., Bioconjug Chem,
13:510-17, 2002, which is incorporated herein by reference and
amphipathic asymmetric dendrimers, described by Shah, et al., Int.
J. Pharm, 208:41-48, 2000, which is incorporated herein by
reference. Poly propylene imine dendrimers, described in Tack, et
al., J. Drug Target, 14;69-86, 2006, which is incorporated herein
by reference; and other dendrimers described above, can be
chemically modified to reduce toxicity, for example, as described
in Tack, et al.
[0318] Dendrimers complex with nucleic acids as do other cationic
polymers with high charge density. In general, the
dendrimer-nucleic acid interaction is based on electrostatic
interactions. Dendrimers can be conjugated with other molecules,
such as cyclodextrins to increase efficiency of systemic delivery
of dendrimer-nucleic acid complexes. (See Dufes, et al., Adv. Drug
Del. Rev, 57, 2177-2202, 2005, and Svenson and Tomalia, Adv. Drug
Del. Rev., 57, 2106-29, 2005, both of which are incorporated herein
by reference.) Some dendrimers have a flexible open structure that
can capture small molecules in their interior, and others have an
inaccessible interior. (See Svenson and Tomalia, Adv. Drug Del.
Rev., 57, 2106-29, 2005.)
[0319] In further embodiments, oligonucleotides are sequestered in
polymer vesicles. Polymer vesicles can be made from a number of
different materials, but in general are formed from block
copolymers, for example,
polystyrene.sub.40-poly(isocyano-L-alanine-L-alanine).sub.m. (See
for example, Discher, et al., Science, 297:967-73, 2002; Torchilin,
Cell. Mol. Life Sci, 61:2549-59, 2004; Taubert, et al., Curr Opin
Chem Biol, 8:598-603, 2004; Lee, et al., Pharm. Res., 22:1-10,
2005; and Gaucher, et al., J. Control. Rel, 109:169-88, 2005, each
of which is incorporated herein by reference.) Copolymer vesicles
are formed from a number of molecules, including, without
limitation, polyacrylic acid-polystyrene, nonionic
polyethyleneoxide-polybutadiene, the triblock
(polyethyleneoxide).sub.5-(poly[propyleneoxide]).sub.68-(polyethyleneoxid-
e).sub.5, polyethyleneoxide-poly(propylenesulfide),
polyethyleneoxide-polylactide, and polyethylene glycol-polylysine.
Many copolymers, particularly those of either amphiphilic or
oppositely charged copolymers, including
polystyrene.sub.40-poly(isocyano-L-alanine-L-alanine).sub.m, self
assemble into vesicles in aqueous conditions.
[0320] Oligonucleotides can be loaded into the polymer vesicles
using several methods. First, the block copolymer can be dissolved
along with the oligonucleotides in an aqueous solvent. This method
works well with moderately hydrophobic copolymers. Second, for
amphiphilic copolymers that are not readily soluble in water, and
where a solvent that solubilizes both the oligonucleotides and the
copolymer is available, the oligonucleotide and copolymer are
dissolved in the solvent and the mixture is dialyzed against water.
A third method involves dissolving both the oligonucleotides and
copolymer in a water/tert-butanol mixture and subsequent
lyophilization of the solvents. The oligonucleotide-loaded vesicles
are formed spontaneously when the lyophilized
oligonucleotide-copolymer is reconstituted in an injectable
vehicle. (Dufresne, et al., in Gurny, (ed.), B. T. Gattefosse, vol.
96, Gattefosse, Saint-Priest, p. 87-102, 2003, which is
incorporated herein by reference.)
[0321] Polymer vesicles can be targeted to specific cells by
tethering a ligand to the outer shell of vesicles by post
modification of a copolymer with a bifunctional spacer molecule or
by the direct synthesis of heterobifunctional block copolymers.
[0322] In some embodiments, oligonucleotides are enclosed in lipids
(e.g., liposomes or micelles) to aid in delivery (See e.g., U.S.
Pat. Nos. 6,458,382, 6,429,200; U.S. Patent Publications
2003/0099697, 2004/0120997, 2004/0131666, 2005/0164963, and
International Publication WO 06/048329, each of which is herein
incorporated by reference). Liposomes include, without limitation,
cardiolipin based cationic liposomes (e.g., NeoPhectin, available
from NeoPharm, Forest Lake, Ill.) and pH sensitive liposomes.
[0323] In some embodiments of the present invention, NeoPhectin is
utilized as the liposomal delivery vehicle. In some embodiments,
the NeoPhectin is formulated with the oligonucleotide so as to
reduce free NeoPhectin. In other embodiments, NeoPhectin is present
at a charge ratio 6:1 or less (e.g., 5:1, and 4:1) of NeoPhectin to
oligonucleotide.
[0324] In yet other embodiments, lipids, particularly phospholipids
that comprise some liposomes, are conjugated to polyethylene glycol
or a derivative thereof, to increase the time that the liposomes
circulate in the blood after intravenous injection. (See e.g.,
Moghimi, S. M. and Szebeni, J, Prog. Lipid Res., 42:463-78, 2003
and Li, W., et al., J. Gene Med., 7:67-79, 2005, which are
incorporated herein by reference.) Such liposomes, termed "stealth
liposomes" are able to avoid the reticuloentothelial system (RES),
resulting in half lives of more than 24 hours in some cases. In one
embodiment, the phospholipids in liposomes are conjugated to
polyethylene glycol-diorthoester molecules, as described in Li, W.,
et al., J. Gene Med., 7:67-79, 2005. In other embodiments, the
PEG-liposomes are targeted to specific cell receptors. For example,
haloperidol conjugated at the distal end of a PEG-linked
phospholipids in a cationic liposome targeted sigma receptors that
are overexpressed on some cancer cells as described in Mukherjee,
et al., J. Biol. Chem., 280, 15619-27, 2005, which is incorporated
herein by reference. Anisamide conjugated to PEG-linked
phospholipids in liposomes also targets the sigma receptor.
(Banerjee, et al., Int. J. Cancer, 112, 693-700, 2004, which is
incorporated herein by reference.)
[0325] In yet another embodiment, oligonucleotides can be
sequestered in hybrid liposome-copolymer vesicles, as described in
Ruysschaert, et. al., J. Am. Chem. Soc., 127, 6242-47, 2005, which
is incorporated herein by reference. For example, an amphiphilic
triblock copolymers, including
poly(2-methyloxazoline)-block-poly(dimethylsiloxan)-block-poly(2-methylox-
azoline) can interact with lipids, including phospholipids to form
hybrid liposome-copolymer vesicles.
[0326] In still further embodiments, oligonucleotides are complexed
with additional polymers to aid in delivery (See e.g., U.S. Pat.
Nos. 6,379,966, 6,339,067, 5,744,335; each of which is herein
incorporated by reference. For example, polymers of
N-2-hydroxypropyl methylacrylamide are described in U.S. patent
publication number 2006/0014695, which is incorporated herein by
reference. Similar cationic polymers are described in International
Patent Publication number WO 03/066054 and U.S. patent publication
number 2006/0051315, both of which are incorporated herein by
reference. Other polymers are described by Intradigm Corp.,
Rockville, Md.).
[0327] In still further embodiments, the controlled high pressure
delivery system developed by Mirus (Madison, Wis.) is utilized for
delivery of oligonucleotides. The delivery system is described in
U.S. Pat. No. 6,379,966, which is incorporated herein by
reference.
V. Examples of Cancer Therapies
[0328] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
Example 1
[0329] Administering to a patient an oligonucleotide compound; a
chemotherapy agent including Rituximab, Cyclophosphamide,
Doxorubicin, Vincristine, and Prednisone; and radiation
therapy.
Example 2
[0330] Administering to a patient an oligonucleotide compound,
radiation therapy and surgery.
Example 3
[0331] Administering to a patient an oligonucleotide compound, a
chemotherapy agent and radiation.
Example 4
[0332] Administering to a patient an oligonucleotide compound and a
chemotherapy agent.
Example 5
[0333] Administering to a patient an oligonucleotide compound, a
chemotherapy agent, radiation therapy and surgery.
Example 6
Inhibition of Tumor Growth in PC-3 Xenografts with PNT-100 and
Taxotere.TM.
[0334] Inhibition of tumor growth by PNT-100 (SEQ ID NO:1251) was
examined using the human PC-3 GFP prostate carcinoma subcutaneous
model. (See e.g., Yang et al., Cancer Research 59, 781-786, [1999];
Glinskii et al., Cancer Research 63, 4239-4243, [2003]; and Kalikin
et al., Cancer Biology and Therapy 2:6, 17-21 [2003].)
[0335] PC-3 cells were first transduced with the green fluorescent
protein (GFP) gene. A GFP expression vector, pLEIN, was purchased
from Clontech (Palo Alto, Calif.). The vector expresses enhanced
GFP and the neomycin resistance gene on the same bicistronic
message that contains an internal ribosome entry site. To produce
packaged GFP viral particles, PT67, an NIH3T3 derived packaging
cell line, expressing the 10 AI viral envelopes (Clontech) was
used. PT67 cells were cultured in DMEM supplemented with 10% fetal
bovine serum. PT67 cells, at 70% confluence, were incubated with a
precipitated mixture of
N-[1-(2,3-dioleoyloxyl)propyl]-N,N,-trimethylammoniummethyl sulfate
reagent and saturating amounts of pLEIN plasmid for 18 h. For
selection, the cells were cultured in the presence of 200-1000
.mu.g/ml G418 for 7 days. For GFP gene transduction, 20% confluent
PC-3 cells (ATCC, CRL 1435) were incubated with a 1:1 precipitated
mixture of retroviral supernatants of PT67 cells and Ham's F-12 K
containing 7% fetal bovine serum for 72 h. Fresh medium was
replenished at this time. PC-3 cells were harvested 72 h
posttransduction and subcultured at a ratio of 1:15 into selective
medium that contained G-418. The brightest PC-3 cell clones
expressing GFP were selected, combined, and then amplified and
transferred by conventional culture methods.
[0336] Tumor stocks were prepared by subcutaneously injecting
PC-3-GFP cells at a concentration of 5.times.10.sup.6 cells/200
.mu.l into the flank of nude mice (male athymic NCr nude mice
between 5 and 6 weeks of age (Taconic Quality Laboratory Animals
and Services for Research (Germantown, N.Y.)). Strong GFP
expression of tumors grown in the subcutis of mice was certified
before harvest. The tumor tissues harvested from subcutaneous
growth in nude mice were inspected and any grossly necrotic or
suspected necrotic or non GFP tumor tissues were removed. Tumor
tissues were subsequently cut into small fragments of approximately
2 mm.sup.3. A tumor stock of the prostate cancer PC-3 GFP was
established by subcutaneously injecting PC-3 GFP cells to the flank
of nude mice. The tumor was maintained in nude mice subcutaneously
as tumor stock prior to use. Before implantation, strong GFP
expression of the PC-3 GFP tumor tissue was confirmed by
fluorescent light. On the day of implantation, the tumor was
harvested from the subcutaneous site and placed in RPMI-1640
medium. Necrotic tissues were removed and viable tissues were cut
into 2 mm.sup.3 pieces. The tissue fragments were then implanted
subcutaneously to right flank of the nude nice. Tumor size was
measured by caliper monitoring. Approximate tumor volume was
calculated by the formula (Width.times.Length).times.1/2.
[0337] PNT100 (SEQ ID NO:1251) and PNT-1 (SEQ ID NO:1488) were
formulated with NeoPhectin-AT as follows. A 25 ml liposome delivery
vehicle (LDV) consisting of NeoPhectin-AT (NeoPharm, IL) bottle was
placed at room temperature for 15 min. The bottle was gently
swirled for 30 seconds to mix. 1000 .mu.l LDV was transferred to 50
ml sterile polypropylene tubes labeled: Day #PNT100. The PNT100
stock tube was vortexed and quickly centrifuged. 75 .mu.l PNT100
(Stock) was transferred to the Day #PNT100 tube and the mixture was
vortexed vigorously for 2 minutes. 5000 .mu.l dH2O was mixed with
5000 .mu.l 20% sucrose in a sterile 50 ml tube. 2150 .mu.l of the
diluted sucrose was added to the PNT100/Neophectin-AT solution and
mixed. An appropriate drug injection volume was transferred to a
1.5 ml polypropylene tube. The LDV control was generated by mixing
75 .mu.l RNAse/DNAse free water instead of PNT100 with 1000 .mu.l
LDV, 2150 10% sucrose was added and the mixture was injected.
[0338] Mice bearing 50-100 mm.sup.3 estimated tumor volume were
injected subcutaneously into the tumor with NeoPhectin-AT-PNT-100
(SEQ ID NO:1251) or PNT-1 (SEQ ID NO:1488) at a dose of 2.5-5.0
mg/kg daily for five days. A second group of mice received 5-10
mg/kg of Taxotere.TM. intravenously on days 2 and 5. A third group
of mice received 5 mg/kg of NeoPhectin-AT-PNT-100 (SEQ ID NO:1251)
injected subcutaneously into the tumor daily for five days and 5-10
mg/kg of Taxotere.TM. injected intravenously on days 2 and 5.
[0339] The study design is shown in Table 3
TABLE-US-00004 TABLE 3 Sub- group Dose ID Description (mg/kg)
Schedule Route N A PBS Control 200 .mu.l qd X 5 s.c 10 B PNT-C (5'-
5 qd X 5 s.c. 10 NNNNNNNNNNNNNN NNNNNNNNNN-3'; SEQ ID NO: 1448) +
LDV C PNT-100 (PhoMab12; 2.5 qd X 5 s.c. 10 SEQ ID NO: 1251) + LDV
D PNT-100 + LDV 5 qd X 5 s.c. 10 E TAXOTERE .TM. 10 and 5 Day 2
i.v. 10 and 5 F TAXOTERE .TM. + PNT- 10 and Day 2 i.v. + 10 100/LDV
5 + 5 and 5 + s.c. qd X 5
[0340] Tumor growth was monitored for 40 days. Twelve days after
implantation, whole body optical imaging of GFP-expressing tumors
was performed once per week using a fluorescence microscope. The
final tumor weights were taken after animals were sacrificed at the
forty-sixth day of the study.
[0341] Results are shown in FIGS. 1 and 2. FIG. 1 shows mean tumor
volume of tumors in the PC-3 GFP prostate carcinoma subcutaneous
model following treatment with PNT-100 and/or TAXOTERE.TM.. FIG. 2
shows mean final volume of tumors. The results indicate that
PNT-100+TAXOTERE.TM. is more effective than PNT-100 or TAXOTERE.TM.
alone.
Example 7
Inhibition of Tumor Growth in a Non-Hodgkin's Model with PNT-100
and Vincristine
[0342] A non-Hodgkin's-lymphoma model (NHL) was used. The
WSU-DLCL.sub.2 (Wayne State University diffuse large cell lymphoma)
model is a very robust model of chemoresistant aggressive human
diffuse large cell lymphoma. It was obtained from Dr. Ramzi
Mohammad and Dr. Al-Katib and colleagues at the Karmanos Cancer
Institute at Wayne State University. (See Al-Katib, A M, et al.,
Clin. Cancer Res. 4, 1305-1314 (1998); Mohammad, R, et al., Clin.
Cancer Res. 8, 1277-1283 (2002); Mohammad, R M, et al., Mol. Cancer
Ther., 4, 13-21 (2005); Mohammad, R M, et al., Clin. Cancer Res. 6,
4950-4956, (2000).) The study was designed to administer five daily
doses of 5 mg/kg PNT-100 (SEQ ID NO: 1251), and in certain cohorts,
combination therapy with vincristine. After one dose of PNT-100,
noticeable weight loss in the animals injected with PNT100 and
PNT-1 (SEQ ID NO:1488) was observed. The data shows decreased tumor
burden with combination therapy with PNT-100 and PNT-1 20 days post
WSU-DLCL2 transplantation. The results indicate that PNT-100, alone
and in combination with vincristine, decreases the growth tumors in
mice.
Example 8
Efficacy of PNT-100 and Taxotere.TM. Intravenous Delivery in the
PC-3 Xenograft Model
[0343] Xenografts were generated by subcutaneous injection of
2.times.10.sup.6 PC-3 cells in nude mice. A 6:1 PNT100:NeoPhectin
AT charge ratio was prepared as described in Example 6. Mice
bearing 50-100 mm.sup.3 xenografts were dosed intravenously with 1
mg/kg PNT-100+NeoPhectin AT, daily for 5 days, with 10 mg/kg on day
2 and with 5 m/kg on day 5 with Taxotere.TM.. Tumor response was
measured by caliper monitoring. Results are shown in FIG. 3, which
indicate PNT-100 with Taxotere.TM. is more efficacious than PNT-100
or Taxotere.TM. alone.
Example 9
Efficacy of Liposomal PNT-100 and Docetaxel in PC-3 Xenografts
[0344] Xenografts were generated by subcutaneous injection of
2.times.10.sup.6 PC-3 cells in nude mice. PNT-100 was formulated in
a lipid formulation of POPC/DOPE/MoChol/CHEMS in the molar ratio of
6/24/47/23. (See U.S. Patent application Nos. 2003/0099697,
2004/40120997, 2004/0131666, and International Application
Publication No. WO/05/094783, all of which are incorporated herein
by reference.) The mean size of the liposomes is less than 160
.eta.m, and the concentration of PNT-100 in the liposomal mixture
is about 2 mg/ml. Two different batches of liposomal PNT-100 were
used, 340.8 and 340.9. Mice bearing 50-200 mm3 xenografts were
dosed on day 1 with PNT-100 (SEQ ID NO:1251) or PNT100R (SEQ ID
NO:1288). Dosing was 10 mg/kg on days 1, 2, and 5 and 7.5 mg/kg on
days 3 and 4. Docetaxel dosing was 10 mg/kg on day 2 and 5 mg/kg on
day 5. Mann-Whitney analysis with a student t test was performed
with 95% confidence. N=5 except for 340.8+docetaxel, in which N=4.
Results are shown in FIG. 4 demonstrating a reduction in tumor size
with PNT-100+docetaxel compared to PNT-100 or docetaxel alone.
[0345] A repetition of the experiment gave similar results. One
batch of liposomal-PNT-100, called PNT-2253 was prepared with the
same properties as above. Xenograft bearing mice were administered
10 mg/kg of liposomal PNT-100 (PNT2253) or liposomal PNT-100R
(PNT2253R) by i.v. bolus injection daily for five days. Docetaxel
dosing was 10 mg/kg on day 2 and 5 mg/kg on day 5 by i.v. bolus
injection. Tumor volume was caliper measured. Studies were
concluded when control animal xenografts reached 2000 mm.sup.3.
Results are shown in FIGS. 5 and 6, showing an 80% tumor growth
inhibition for PNT-100+docetaxel. A second batch of liposomal
PNT-100 (PNT2252) was administered by i.v. slow infusion. Dosing
was 20 mg/kg daily for 5 days, and docetaxel was administered at 10
mg/kg on day 2 and 5 mg/kg on day 5 by i.v. bolus injection.
Results in FIG. 6 show 49% tumor growth inhibition for
PNT2252+docetaxel at 17 days after drug treatment.
Example 10
Efficacy of Liposomal PNT-100 and Rituximab in WSU-DLCL2
Xenografts
[0346] Xenografts with WSU-DLCL2 cells were generated in C.B-17
SCID mice between 4-6 weeks old as described in the previous
examples. Liposomal PNT-100 was formulated as in example 9 and has
similar properties and a concentration of 2 mg PNT-100 per ml.
Human pharmaceutical grade rituximab (Biogen Idec-Genentech) was
provided by Karmanos Cancer Institute. The mice were treated as in
Table 4.
TABLE-US-00005 TABLE 4 Liposomal PNT-100 Dose Volume Group (Per 25
g ID Description mouse) Schedule Route n A 10% Sucrose 125 .mu.l qd
.times. 5 i.v. 8 Control B 10 mg/kg Liposomal 125 .mu.l qd .times.
5 i.v. 7* PNT-100 C 20 mg/kg Rituxan NA Day 2 & Day 5 i.v. 8 D
10 mg/kg Liposomal 125 .mu.l qd .times. 5 i.v. 8 PNT-100 Day 2
& Day 5 20 mg/kg Rituxan Note: Dosage listed as mg/ml PNT100
*One animal did not develop palpable tumor.
[0347] Animals were checked three times weekly for tumor growth by
caliper measurements. An approximate tumor volume was calculated
using the formula 1/2(a.times.b.sup.2), where b is the smaller of
two perpendicular diameters. Animals were sacrificed when
individual animal tumor burden reached 2000 mm.sup.3 or when the
study was concluded 81 days
[0348] Rituximab at 20 mg/kg, administered on days 2 and 5 resulted
in complete regression of the tumor, i.e., tumor shrinkage below
measurable size for three consecutive time points, in seven out of
eight tumors and four out of eight showed complete regression
through the 81 day endpoint. Liposomal PNT-100 at 10 mg/ml,
administered daily for five days resulted in complete regression of
the tumor in one out of seven tumors and none of the tumors showed
complete regression through the 81 day endpoint. One out of seven
tumors had a partial regression, which is a less than 50% reduction
from initial tumor size for three consecutive time points.
Administration of Liposomal PNT-100 resulted in a slowing of the
growth rate of the tumor when compared to the sucrose control.
Liposomal PNT-100 administered along with rituximab (group D),
resulted in complete regression of the tumor in six out of eight of
the tumors, and 5 out of 8 tumors showed complete regression
through the 81 day endpoint. All eight tumors had partial
regressions. These results did not establish synergy of
rituximab+PNT-100, in WSU-DLCL.sub.2 xenografts, probably because
the rituximab levels administered were high.
Example 11
Efficacy of Liposomal PNT-100 and Rituximab in Daudi Xenografts
[0349] Daudi cells are a model of Burkett's lymphoma. Xenografts
with Daudi cells were generated in mice as described in the
previous examples. Liposomal PNT-100 was formulated as in example 9
and has similar properties and a concentration of 2.4 mg PNT-100
per ml. The mice were divided into 10 groups and treated as in
Table 5.
TABLE-US-00006 TABLE 5 Group ID Description Dose (mg/kg) Schedule
Route N 1 PBS Control 200 .mu.l qd X 5 i.v. 10 2 Rituximab 20 mg/kg
Schedule 2 i.v. 10 3 Liposomal 30 mg/kg Schedule 1 i.v. 10 PNT-100
4 Liposomal 20 mg/kg Schedule 1 i.v. 10 PNT-100 5 Liposomal 13.3
mg/kg Schedule 1 i.v. 10 PNT-100 6 Liposomal 8.89 mg/kg Schedule 1
i.v. 10 PNT-100 7 Liposomal 5.92 mg/kg Schedule 1 i.v. 10 PNT-100 8
Rituximab + 20 mg/kg RTX, Schedule 1- i.v. 10 Liposomal 20 mg/kg
rituximab, PNT-100 PNT-100 Schedule 2- PNT-100 9 Rituximab + 20
mg/kg RTX, Schedule 1- i.v. 10 Liposomal 13.3 mg/kg rituximab,
PNT-100 PNT-100 Schedule 2- PNT-100 Schedule 1 is 5 daily doses, 2
days off and then 5 daily doses, 2 days off, then 3 daily doses.
Schedule 2 is i.v. delivery of rituximab biweekly for 2.5 weeks for
a total of 5 injections.
[0350] Tumor volume was caliper measured. Studies were concluded
when control animal xenografts reached 2000 mm.sup.3. Results are
shown in FIGS. 8-10. FIG. 7 shows mean tumor volume up to 50 days.
FIG. 8 is a Kaplan-Meyer plot, showing the percent of mice whose
tumors have not yet reached 2000 mm.sup.3 each day. FIG. 9 shows
the change in body weight of the mice in each group. The results
show little effect with either rituximab or PNT-100 alone, but a
dramatic effect, when PNT-100 and rituximab are given together.
Indeed, in Daudi xenografts, the tumors shrink and disappear when
the mice bearing them are treated with PNT-100 and rituximab.
VI. Other Embodiments
[0351] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages and modifications are
within the scope of the following claims.
[0352] All references cited herein, are incorporated herein by
reference in their entirety.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090324587A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090324587A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References