U.S. patent application number 10/467008 was filed with the patent office on 2004-06-17 for antisense modulation of protein phosphatase 2 catalytic subunit beta expression.
Invention is credited to Monia, Brett P, Wyatt, Jacqueline R.
Application Number | 20040116366 10/467008 |
Document ID | / |
Family ID | 37764002 |
Filed Date | 2004-06-17 |
United States Patent
Application |
20040116366 |
Kind Code |
A1 |
Monia, Brett P ; et
al. |
June 17, 2004 |
Antisense modulation of protein phosphatase 2 catalytic subunit
beta expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of Protein Phosphatase 2 catalytic
subunit beta. The compositions comprise antisense compounds,
particularly antisense oligonucleotides, targeted to nucleic acids
encoding Protein Phosphatase 2 catalytic subunit beta. Methods of
using these compounds for modulation of Protein Phosphatase 2
catalytic subunit beta expression and for treatment of diseases
associated within expression of Protein Phosphatase 2 catalytic
subunit beta are provided.
Inventors: |
Monia, Brett P; (Encinitas,
CA) ; Wyatt, Jacqueline R; (Encinitas, CA) |
Correspondence
Address: |
LICATA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
37764002 |
Appl. No.: |
10/467008 |
Filed: |
February 5, 2004 |
PCT Filed: |
January 31, 2002 |
PCT NO: |
PCT/US02/02805 |
Current U.S.
Class: |
514/44A ;
536/23.2 |
Current CPC
Class: |
C12N 2310/3341 20130101;
C12N 2310/346 20130101; C12N 2310/3525 20130101; C12N 2310/341
20130101; C12N 15/1137 20130101; C12N 2310/321 20130101; A61K 38/00
20130101; C12N 2310/321 20130101; C12Y 301/03016 20130101; Y02P
20/582 20151101; C12N 2310/315 20130101 |
Class at
Publication: |
514/044 ;
536/023.2 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. A compound 8 to 50 nucleobases in length targeted to a nucleic
acid molecule encoding Protein Phosphatase 2 catalytic subunit
beta, wherein said compound specifically hybridizes with and
inhibits the expression of Protein Phosphatase 2 catalytic subunit
beta.
2. The compound of claim 1 which is an antisense
oligonucleotide.
3. The compound of claim 2 wherein the antisense oligonucleotide
has a sequence comprising SEQ ID NO: 17, 20, 21, 22, 23, 25, 26,
27, 28, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 86, 87,
88, 92, 24, 52, 58, 95, 97, 98, 101, 102, 103, 106, 107, 108, 109,
111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,
125, 126, 129, 130, 132, 133 or 134.
4. The compound of claim 2 wherein the antisense oligonucleotide
comprises at least one modified internucleoside linkage.
5. The compound of claim 4 wherein the modified internucleoside
linkage is a phosphorothioate linkage.
6. The compound of claim 2 wherein the antisense oligonucleotide
comprises at least one modified sugar moiety.
7. The compound of claim 6 wherein the modified sugar moiety is a
2'-O-methoxyethyl sugar moiety.
8. The compound of claim 2 wherein the antisense oligonucleotide
comprises at least one modified nucleobase.
9. The compound of claim 8 wherein the modified nucleobase is a
5-methylcytosine.
10. The compound of claim 2 wherein the antisense oligonucleotide
is a chimeric oligonucleotide.
11. A compound 8 to 50 nucleobases in length which specifically
hybridizes with at least an 8-nucleobase portion of an active site
on a nucleic acid molecule encoding Protein Phosphatase 2 catalytic
subunit beta.
12. A composition comprising the compound of claim 1 and a
pharmaceutically acceptable carrier or diluent.
13. The composition of claim 12 further comprising a colloidal
dispersion system.
14. The composition of claim 12 wherein the compound is an
antisense oligonucleotide.
15. A method of inhibiting the expression of Protein Phosphatase 2
catalytic subunit beta in cells or tissues comprising contacting
said cells or tissues with the compound of claim 1 so that
expression of Protein Phosphatase 2 catalytic subunit beta is
inhibited.
16. A method of treating an animal having a disease or condition
associated with Protein Phosphatase 2 catalytic subunit beta
comprising administering to said animal a therapeutically or
prophylactically effective amount of the compound of claim 1 so
that expression of Protein Phosphatase 2 catalytic subunit beta is
inhibited.
17. The method of claim 16 wherein the disease or condition is a
hyperproliferative disorder.
18. The method of claim 17 wherein the hyperproliferative disorder
is cancer.
19. The method of claim 16 wherein the disease or condition is
aberrant insulin regulation.
20. The method of claim 16 wherein the disease or condition is
diabetes.
Description
FIELD OF THE INVENTION
[0001] The present invention provides compositions and methods for
modulating the expression of Protein Phosphatase 2 catalytic
subunit beta. In particular, this invention relates to compounds,
particularly oligonucleotides, specifically hybridizable with
nucleic acids encoding Protein Phosphatase 2 catalytic subunit
beta. Such compounds have been shown to modulate the expression of
Protein Phosphatase 2 catalytic subunit beta.
BACKGROUND OF THE INVENTION
[0002] The process of phosphorylation, defined as the attachment of
a phosphate moiety to a biological molecule through the action of
enzymes called kinases, represents one course by which
intracellular signals are propagated resulting finally in a
cellular response. Within the cell, proteins can be phosphorylated
on serine, threonine or tyrosine residues and the extent of
phosphorylation is regulated by the opposing action of
phosphatases, which remove the phosphate moieties. While the
majority of protein phosphorylation within the cell is on serine
and threonine residues (Wera and Hemings, Biochemistry Journal,
1995, 311, 17-29), tyrosine phosphorylation is modulated to the
greatest extent during oncogenic transformation and growth factor
stimulation (Zhang, Crit. Rev. Biochem. Mol. Biol., 1998, 33,
1-52).
[0003] Because phosphorylation is such a ubiquitous process within
cells and because cellular phenotypes are largely influenced by the
activity of these pathways, it is currently believed that a number
of disease states and/or disorders are a result of either aberrant
activation of, or functional mutations in, kinases and
phosphatases. Consequently, considerable attention has been devoted
recently to the characterization of these enzymes.
[0004] The enzyme protein phosphatase 2A (also known as PPP2A and
PP2A) is one of four major protein phosphatases identified in the
cytosol of eukaryotic cells which are responsible for the
dephosphorylation of serine and threonine residues in proteins.
These four enzymes have overlapping substrate specificities and are
distinguished by their regulation and dependence on metal ions.
Protein phosphatase 2A activity is independent of metal ions and
appears to play a role in the regulation of major metabolic
pathways, as well as the processes of translation, transcription,
platelet activation and control of the cell cycle (Goldberg,
Biochem. Pharmacol., 1999, 57, 321-328; Millward et al., Trends
Biochem. Sci., 1999, 24, 186-191; Toyoda et al., Thromb. Haemost.,
1996, 76, 1053-1062). More specifically, Protein Phosphatase 2A
participates as a negative regulator in many kinase signal
transduction pathways, including those involving MAP kinase, JNK
kinase, ERK kinase, CaM kinase, and casein kinase. In addition,
Protein Phosphatase 2A also interacts with many cellular and viral
proteins (Millward et al., Trends Biochem. Sci., 1999, 24,
186-191). The enzyme has been shown to be activated by ceramide, a
metabolic product of sphingomyelin hydrolysis and mediator of the
biological effects of hormones, cytokines and growth factors
(Dobrowsky et al., J. Biol. Chem., 1993, 268, 15523-15530).
[0005] The mammalian protein phosphatase 2A enzyme is a
heterotrimer composed of a catalytic subunit of 36 kD complexed to
two regulatory subunits, one of mass 65 kD and one of variable
mass. In addition, two isoforms of the catalytic subunit of protein
phosphatase 2A, alpha and beta, are demonstrable in many species.
The structures of these catalytic subunits show high evolutionary
conservation supporting the idea that they may serve crucial
functions (Goldberg, Biochem. Pharmacol., 1999, 57, 321-328;
Millward et al., Trends Biochem. Sci., 1999, 24, 186-191).
[0006] Protein Phosphatase 2 catalytic subunit beta (also known as
PPP2CB) was originally isolated from lung and lung fibroblast
libraries (Hemmings et al., Nucleic Acids Res., 1988, 16, 11366),
while the gene was isolated from a human leukocyte library
(Khew-Goodall et al., Biochemistry, 1991, 30, 89-97). Northern
analysis has revealed that the alpha subunit is expressed at
relatively high levels compared to the beta subunit in all tissues
examined. The structural characterization of the two genes implies
that this is due in part to the different strengths of the
promoters (Khew-Goodall et al., Biochemistry, 1991, 30, 89-97).
[0007] The catalytic subunit of Protein Phosphatase 2A has been
linked to both insulin signaling (Klarlund et al., J. Biol. Chem.,
1991, 266, 4052-4055; Kowluru et al., Endocrinology, 1996, 137,
2315-2323) and to retinoic acid-induced cellular differentiation of
HL-60 cells, an acute promyelocytic leukemia cell line (Nishikawa
et al., Cancer Res., 1994, 54, 4879-4884; Tawara et al., FEBS
Lett., 1993, 321, 224-228). The pharmacological modulation of the
catalytic subunit of Protein Phosphatase 2A activity and/or
expression may therefore be an appropriate point of therapeutic
intervention in pathological conditions such as diabetes and
cancer.
[0008] Currently, there are no known therapeutic agents which
effectively inhibit the synthesis of the beta isoform of Protein
Phosphatase 2A catalytic subunit, and to date, investigative
strategies aimed at modulating activity of Protein Phosphatase 2A
function have involved the use of antibodies, molecules that block
upstream entities and chemical inhibitors.
[0009] Disclosed in U.S. Pat. Nos. 5,925,660 and 5,700,821 are
compounds useful as phosphatase inhibitors and methods of making
such inhibitors (Lazo et al., 1999; Lazo et al., 1997). It has also
been reported that the compound, Fostriecin and compounds
structurally related to it are effective serine/threonine
phosphatase inhibitors. These are disclosed in the PCT publication
WO 98/14606 (Honkanen and Downey, 1998). Disclosed in the PCT
publication WO 99/27134 are antisense oligonucleotides targeting
serine/threonine phosphatases, PP5, PP4 and PP1.gamma.1 none of
which target or hybridize to the Protein Phosphatase 2A isoforms
(Honkanen and Dean, 1999).
[0010] In addition, at the protein level, there are compounds that
interact with and consequently modulate the activity of the Protein
Phosphatase 2A enzyme. These compounds and methods to identify
these compounds are disclosed in the PCT publication WO 97/37037
(Hemmings, 1997).
[0011] These strategies are untested as therapeutic protocols and
consequently there remains a long felt need for additional agents
capable of effectively inhibiting Protein Phosphatase 2A catalytic
subunit beta function.
[0012] Antisense technology is emerging as an effective means for
reducing the expression of specific gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications for the modulation of Protein Phosphatase
2A catalytic subunit beta expression.
[0013] The present invention provides compositions and methods for
modulating Protein Phosphatase 2A catalytic subunit beta
expression.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to compounds, particularly
antisense oligonucleotides, which are targeted to a nucleic acid
encoding Protein Phosphatase 2 catalytic subunit beta, and which
modulate the expression of Protein Phosphatase 2 catalytic subunit
beta. Pharmaceutical and other compositions comprising the
compounds of the invention are also provided. Further provided are
methods of modulating the expression of Protein Phosphatase 2
catalytic subunit beta in cells or tissues comprising contacting
said cells or tissues with one or more of the antisense compounds
or compositions of the invention. Further provided are methods of
treating an animal, particularly a human, suspected of having or
being prone to a disease or condition associated with expression of
Protein Phosphatase 2 catalytic subunit beta by administering a
therapeutically or prophylactically effective amount of one or more
of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention employs oligomeric compounds,
particularly antisense oligonucleotides, for use in modulating the
function of nucleic acid molecules encoding Protein Phosphatase 2
catalytic subunit beta, ultimately modulating the amount of Protein
Phosphatase 2 catalytic subunit beta produced. This is accomplished
by providing antisense compounds which specifically hybridize with
one or more nucleic acids encoding Protein Phosphatase 2 catalytic
subunit beta. As used herein, the terms "target nucleic acid" and
"nucleic acid encoding Protein Phosphatase 2 catalytic subunit
beta" encompass DNA encoding Protein Phosphatase 2 catalytic
subunit beta, RNA (including pre-mRNA and mRNA) transcribed from
such DNA, and also cDNA derived from such RNA. The specific
hybridization of an oligomeric compound with its target nucleic
acid interferes with the normal function of the nucleic acid. This
modulation of function of a target nucleic acid by compounds which
specifically hybridize to it is generally referred to as
"antisense". The functions of DNA to be interfered with include
replication and transcription. The functions of RNA to be
interfered with include all vital functions such as, for example,
translocation of the RNA to the site of protein translation,
translation of protein from the RNA, splicing of the RNA to yield
one or more mRNA species, and catalytic activity which may be
engaged in or facilitated by the RNA. The overall effect of such
interference with target nucleic acid function is modulation of the
expression of Protein Phosphatase 2 catalytic subunit beta. In the
context of the present invention, "modulation" means either an
increase (stimulation) or a decrease (inhibition) in the expression
of a gene. In the context of the present invention, inhibition is
the preferred form of modulation of gene expression and mRNA is a
preferred target.
[0016] It is preferred to target specific nucleic acids for
antisense. "Targeting" an antisense compound to a particular
nucleic acid, in the context of this invention, is a multistep
process. The process usually begins with the identification of a
nucleic acid sequence whose function is to be modulated. This may
be, for example, a cellular gene (or mRNA transcribed from the
gene) whose expression is associated with a particular disorder or
disease state, or a nucleic acid molecule from an infectious agent.
In the present invention, the target is a nucleic acid molecule
encoding Protein Phosphatase 2 catalytic subunit beta. The
targeting process also includes determination of a site or sites
within this gene for the antisense interaction to occur such that
the desired effect, e.g., detection or modulation of expression of
the protein, will result. Within the context of the present
invention, a preferred intragenic site is the region encompassing
the translation initiation or termination codon of the open reading
frame (ORF) of the gene. Since, as is known in the art, the
translation initiation codon is typically 5'-AUG (in transcribed
mRNA molecules; 5'-ATG in the corresponding DNA molecule), the
translation initiation codon is also referred to as the "AUG
codon," the "start codon" or the "AUG start codon". A minority of
genes have a translation initiation codon having the RNA sequence
5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been
shown to function in vivo. Thus, the terms "translation initiation
codon" and "start codon" can encompass many codon sequences, even
though the initiator amino acid in each instance is typically
methionine (in eukaryotes) or formylmethionine (in prokaryotes). It
is also known in the art that eukaryotic and prokaryotic genes may
have two or more alternative start codons, any one of which may be
preferentially utilized for translation initiation in a particular
cell type or tissue, or under a particular set of conditions. In
the context of the invention, "start codon" and "translation
initiation codon" refer to the codon or codons that are used in
vivo to initiate translation of an mRNA molecule transcribed from a
gene encoding Protein Phosphatase 2 catalytic subunit beta,
regardless of the sequence(s) of such codons.
[0017] It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences,
i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences
are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start
codon region" and "translation initiation codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation termination codon.
[0018] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Other target regions
include the 5' untranslated region (5'UTR), known in the art to
refer to the portion of an mRNA in the 5' direction from the
translation initiation codon, and thus including nucleotides
between the 5' cap site and the translation initiation codon of an
mRNA or corresponding nucleotides on the gene, and the 3'
untranslated region (3'UTR), known in the art to refer to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap. The
5' cap region may also be a preferred target region.
[0019] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence. mRNA
splice sites, i.e., intron-exon junctions, may also be preferred
target regions, and are particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular mRNA splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred targets. It has also been found that
introns can also be effective, and therefore preferred, target
regions for antisense compounds targeted, for example, to DNA or
pre-mRNA.
[0020] Once one or more target sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect.
[0021] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleoside or
nucleotide bases. For example, adenine and thymine are
complementary nucleobases which pair through the formation of
hydrogen bonds. "Complementary," as used herein, refers to the
capacity for precise pairing between two nucleotides. For example,
if a nucleotide at a certain position of an oligonucleotide is
capable of hydrogen bonding with a nucleotide at the same position
of a DNA or RNA molecule, then the oligonucleotide and the DNA or
RNA are considered to be complementary to each other at that
position. The oligonucleotide and the DNA or RNA are complementary
to each other when a sufficient number of corresponding positions
in each molecule are occupied by nucleotides which can hydrogen
bond with each other. Thus, "specifically hybridizable" and
"complementary" are terms which are used to indicate a sufficient
degree of complementarity or precise pairing such that stable and
specific binding occurs between the oligonucleotide and the DNA or
RNA target. It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and in the case of in vitro assays, under
conditions in which the assays are performed.
[0022] Antisense and other compounds of the invention which
hybridize to the target and inhibit expression of the target are
identified through experimentation, and the sequences of these
compounds are hereinbelow identified as preferred embodiments of
the invention. The target sites to which these preferred sequences
are complementary are hereinbelow referred to as "active sites" and
are therefore preferred sites for targeting. Therefore another
embodiment of the invention encompasses compounds which hybridize
to these active sites.
[0023] Antisense compounds are commonly used as research reagents
and diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
[0024] For use in kits and diagnostics, the antisense compounds of
the present invention, either alone or in combination with other
antisense compounds or therapeutics, can be used as tools in
differential and/or combinatorial analyses to elucidate expression
patterns of a portion or the entire complement of genes expressed
within cells and tissues.
[0025] Expression patterns within cells or tissues treated with one
or more antisense compounds are compared to control cells or
tissues not treated with antisense compounds and the patterns
produced are analyzed for differential levels of gene expression as
they pertain, for example, to disease association, signaling
pathway, cellular localization, expression level, size, structure
or function of the genes examined. These analyses can be performed
on stimulated or unstimulated cells and in the presence or absence
of other compounds which affect expression patterns.
[0026] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (reviewed in (To, Comb.
Chem. High Throughput Screen, 2000, 3, 235-41).
[0027] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense oligonucleotides have been employed as therapeutic
moieties in the treatment of disease states in animals and man.
Antisense oligonucleotide drugs, including ribozymes, have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
oligonucleotides can be useful therapeutic modalities that can be
configured to be useful in treatment regimes for treatment of
cells, tissues and animals, especially humans.
[0028] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
oligonucleotides composed of naturally-occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally-occurring portions which
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for nucleic acid target and increased stability in the
presence of nucleases.
[0029] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics such as are described below. The antisense
compounds in accordance with this invention preferably comprise
from about 8 to about 50 nucleobases (i.e. from about 8 to about 50
linked nucleosides). Particularly preferred antisense compounds are
antisense oligonucleotides, even more preferably those comprising
from about 12 to about 30 nucleobases. Antisense compounds include
ribozymes, external guide sequence (EGS) oligonucleotides
(oligozymes), and other short catalytic RNAs or catalytic
oligonucleotides which hybridize to the target nucleic acid and
modulate its expression.
[0030] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn the respective ends of this
linear polymeric structure can be further joined to form a circular
structure, however, open linear structures are generally preferred.
Within the oligonucleotide structure, the phosphate groups are
commonly referred to as forming the internucleoside backbone of the
oligonucleotide. The normal linkage or backbone of RNA and DNA is a
3' to 5' phosphodiester linkage.
[0031] Specific examples of preferred antisense compounds useful in
this 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, and as sometimes referenced in the
art, modified oligonucleotides that do not have a phosphorus atom
in their internucleoside backbone can also be considered to be
oligonucleosides.
[0032] Preferred modified oligonucleotide backbones include, for
example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriest- ers,
selenophosphates and boranophosphates having normal 3'-5' linkages,
2'-5' linked analogs of these, and those having inverted polarity
wherein one or more internucleotide linkages is a 3' to 3', 5' to
5' or 2' to 2' linkage. Preferred oligonucleotides having inverted
polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be a basic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included.
[0033] Representative United States patents that teach the
preparation of the above phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;
5,453,496;,5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;
5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain
of which are commonly owned with this application, and each of
which is herein incorporated by reference.
[0034] Preferred 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; riboacetyl 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.
[0035] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439, certain of which are commonly owned with
this application, and each of which is herein incorporated by
reference.
[0036] In other preferred 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 United States 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, 1991, 254,
1497-1500.
[0037] Most preferred embodiments 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
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0038] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' 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. Particularly preferred are
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.su- b.3)].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, 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 for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
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,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylamino-ethoxyethoxy (also known in the art as
2'-O-dimethylamino-ethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0039] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in WO
98/39352 and WO 99/14226.
[0040] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub- .2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 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 may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by reference
in its entirety.
[0041] 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 (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further modified nucleobases include tricyclic
pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazi- n-2(3H)-one),
phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin--
2(3H)-one), G-clamps such as a substituted phenoxazine cytidine
(e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b ]indol-2-one), pyridoindole
cytidine (H-pyrido[3', 2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. 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 O-6 substituted purines,
including 2-aminopropyl-adenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0042] Representative United States patents that teach the
preparation of certain of the above noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat Nos.:
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;
5,763,588; 6,005,096; and 5,681,941, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference, and U.S. Pat. No. 5,750,692, which is
commonly owned with the instant application and also herein
incorporated by reference.
[0043] Another modification of the oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. The
compounds of the invention can include conjugate groups covalently
bound to functional groups such as primary or secondary hydroxyl
groups. Conjugate groups of the invention include inter-calators,
reporter molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, phospholipids, biotin, phenazine, folate, phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and
dyes. Groups that enhance the pharmaco-dynamic properties, in the
context of this invention, include groups that improve oligomer
uptake, enhance oligomer resistance to degradation, and/or
strengthen sequence-specific hybridization with RNA. Groups that
enhance the pharmacokinetic properties, in the context of this
invention, include groups that improve oligomer uptake,
distribution, metabolism or excretion. Representative conjugate
groups are disclosed in International Patent Application
PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which
is incorporated herein by reference. Conjugate moieties include but
are not limited to lipid moieties such as a cholesterol moiety
(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,
6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,
1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309;
Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a
thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,
533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues
(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et
al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,
1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol
or triethylammonium 1,2-di-O-hexadecyl-rac-glyc-
ero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36,
3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a
polyamine or a polyethylene glycol chain (Manoharan et al.,
Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane
acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,
3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys.
Acta, 1995, 1264, 229-237), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the
invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbuta-zone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodo-benzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) which is incorporated herein by reference in its
entirety.
[0044] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference.
[0045] 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 antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0046] Chimeric antisense compounds of the invention may be formed
as composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797;
5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;
5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which
are commonly owned with the instant application, and each of which
is herein incorporated by reference in its entirety.
[0047] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0048] The antisense compounds of the invention are synthesized in
vitro and do not include antisense compositions of biological
origin, or genetic vector constructs designed to direct the in vivo
synthesis of antisense molecules. The compounds of the invention
may also be admixed, encapsulated, conjugated or otherwise
associated with other molecules, molecule structures or mixtures of
compounds, as for example, liposomes, receptor targeted molecules,
oral, rectal, topical or other formulations, for assisting in
uptake, distribution and/or absorption. Representative United
States patents that teach the preparation of such uptake,
distribution and/or absorption assisting formulations include, but
are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844;
5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020;
5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804;
5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978;
5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152;
5,556,948; 5,580,575; and 5,595,756, each of which is herein
incorporated by reference.
[0049] The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof. Accordingly,
for example, the disclosure is also drawn to prodrugs and
pharmaceutically acceptable salts of the compounds of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bioequivalents.
[0050] The term "prodrug" indicates a therapeutic agent that is
prepared in an inactive form that is converted to an active form
(i.e., drug) within the body or cells thereof by the action of
endogenous enzymes or other chemicals and/or conditions. In
particular, prodrug versions of the oligonucleotides of the
invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate]
derivatives according to the methods disclosed in WO 93/24510 to
Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S.
Pat. No. 5,770,713 to Imbach et al.
[0051] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto.
[0052] Pharmaceutically acceptable base addition salts are formed
with metals or amines, such as alkali and alkaline earth metals or
organic amines. Examples of metals used as cations are sodium,
potassium, magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19). The
base addition salts of said acidic compounds are prepared by
contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The
free acid form may be regenerated by contacting the salt form with
an acid and isolating the free acid in the conventional manner. The
free acid forms differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar solvents,
but otherwise the salts are equivalent to their respective free
acid for purposes of the present invention. As used herein, a
"pharmaceutical addition salts" includes a pharmaceutically
acceptable salt of an acid form of one of the components of the
compositions of the invention. These include organic or inorganic
acid salts of the amines. Preferred acid salts are the
hydrochlorides, acetates, salicylates, nitrates and phosphates.
Other suitable pharmaceutically acceptable salts are well known to
those skilled in the art and include basic salts of a variety of
inorganic and organic acids, such as, for example, with inorganic
acids, such as for example hydrochloric acid, hydrobromic acid,
sulfuric acid or phosphoric acid; with organic carboxylic,
sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,
for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic
acid, glucaric acid, glucuronic acid, citric acid, benzoic acid,
cinnamic acid, mahdelic acid, salicylic acid, 4-aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid,
nicotinic acid or isonicotinic acid; and with amino acids, such as
the 20 alpha-amino acids involved in the synthesis of proteins in
nature, for example glutamic acid or aspartic acid, and also with
phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 4-methylbenzenesulfonic acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid
(with the formation of cyclamates), or with other acid organic
compounds, such as ascorbic acid. Pharmaceutically acceptable salts
of compounds may also be prepared with a pharmaceutically
acceptable cation. Suitable pharmaceutically acceptable cations are
well known to those skilled in the art and include alkaline,
alkaline earth, ammonium and quaternary ammonium cations.
Carbonates or hydrogen carbonates are also possible.
[0053] For oligonucleotides, preferred examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
[0054] The antisense compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. For therapeutics, an animal, preferably a human,
suspected of having a disease or disorder which can be treated by
modulating the expression of Protein Phosphatase 2 catalytic
subunit beta is treated by administering antisense compounds in
accordance with this invention. The compounds of the invention can
be utilized in pharmaceutical compositions by adding an effective
amount of an antisense compound to a suitable pharmaceutically
acceptable diluent or carrier. Use of the antisense compounds and
methods of the invention may also be useful prophylactically, e.g.,
to prevent or delay infection, inflammation or tumor formation, for
example.
[0055] The antisense compounds of the invention are useful for
research and diagnostics, because these compounds hybridize to
nucleic acids encoding Protein Phosphatase 2 catalytic subunit
beta, enabling sandwich and other assays to easily be constructed
to exploit this fact. Hybridization of the antisense
oligonucleotides of the invention with a nucleic acid encoding
Protein Phosphatase 2 catalytic subunit beta can be detected by
means known in the art. Such means may include conjugation of an
enzyme to the oligonucleotide, radiolabelling of the
oligonucleotide or any other suitable detection means. Kits using
such detection means for detecting the level of Protein Phosphatase
2 catalytic subunit beta in a sample may also be prepared.
[0056] The present invention also includes pharmaceutical
compositions and formulations which include the antisense compounds
of the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical (including ophthalmic and
to mucous membranes including vaginal and rectal delivery),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. oligonucleotides with at least one
2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration.
[0057] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Preferred
topical formulations include those in which the oligonucleotides of
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Preferred lipids and liposomes
include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, diBtearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters
include but are not limited arachidonic acid, oleic acid,
eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic
acid, palmitic acid, stearic acid, linoleic acid, linolenic acid,
dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester (e.g. isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S.
patent application Ser. No. 09/315,298 filed on May 20, 1999 which
is incorporated herein by reference in its entirety.
[0058] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Preferred bile
acids/salts include chenodeoxydholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic
acid, deoxycholic acid, glucholic acid, glycholic acid,
glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid,
sodium tauro-24,25-dihydro-fusid- ate, sodium
glycodihydrofusidate,. Preferred fatty acids include arachidonic
acid, undecanoic acid, oleic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or
a pharmaceutically acceptable salt thereof (e.g. sodium). Also
preferred are combinations of penetration enhancers, for example,
fatty acids/salts in combination with bile acids/salts. A
particularly preferred combination is the sodium salt of lauric
acid, capric acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. oligonucleotide complexing agents
include poly-amino acids; polyimines; polyacrylates;
polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates;
cationized gelatins, albumins, starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates;
DEAE-derivatized polyimines, pollulans, celluloses and starches.
Particularly preferred complexing agents include chitosan,
N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,
polyspermines, protamine, polyvinylpyridine,
polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.
p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),
poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for oligonucleotides
and their preparation are described in detail in U.S. applications
Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul.
1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May
21, 1998) and 09/315,298 (filed May 20, 1999) each of which is
incorporated herein by reference in their entirety.
[0059] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0060] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0061] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0062] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0063] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compositions and formulations is generally
known to those skilled in the pharmaceutical and formulation arts
and may be applied to the formulation of the compositions of the
present invention.
[0064] Emulsions
[0065] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogenous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter. (Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising of two immiscible liquid phases
intimately mixed and dispersed with each other. In general,
emulsions may be either water-in-oil (w/o) or of the oil-in-water
(o/w) variety. When an aqueous phase is finely divided into and
dispersed as minute droplets into a bulk oily phase the resulting
composition is called a water-in-oil (w/o) emulsion. Alternatively,
when an oily phase is finely divided into and dispersed as minute
droplets into a bulk aqueous phase the resulting composition is
called an oil-in-water (o/w) emulsion. Emulsions may contain
additional components in addition to the dispersed phases and the
active drug which may be present as a solution in either the
aqueous phase, oily phase or itself as a separate phase.
Pharmaceutical excipients such as emulsifiers, stabilizers, dyes,
and anti-oxidants may also be present in emulsions as needed.
Pharmaceutical emulsions may also be multiple emulsions that are
comprised of more than two phases such as, for example, in the case
of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w)
emulsions. Such complex formulations often provide certain
advantages that simple binary emulsions do not. Multiple emulsions
in which individual oil droplets of an o/w emulsion enclose small
water droplets constitute a w/o/w emulsion. Likewise a system of
oil droplets enclosed in globules of water stabilized in an oily
continuous provides an o/w/o emulsion.
[0066] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0067] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0068] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0069] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0070] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0071] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0072] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of reasons of ease
of formulation, efficacy from an absorption and bioavailability
standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,
oil-soluble vitamins and high fat nutritive preparations are among
the materials that have commonly been administered orally as o/w
emulsions.
[0073] In one embodiment of the present invention, the compositions
of oligonucleotides and nucleic acids are formulated as
microemulsions. A microemulsion may be defined as a system of
water, oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0074] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0075] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0076] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or oligonucleotides. Microemulsions
have also been effective in the transdermal delivery of active
components in both cosmetic and pharmaceutical applications. It is
expected that the microemulsion compositions and formulations of
the present invention will facilitate the increased systemic
absorption of oligonucleotides and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular
uptake of oligonucleotides and nucleic acids within the
gastrointestinal tract, vagina, buccal cavity and other areas of
administration.
[0077] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
oligonucleotides and nucleic acids of the present invention.
Penetration enhancers used in the microemulsions of the present
invention may be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0078] Liposomes
[0079] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0080] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0081] In order to cross intact mammalian skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than
50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0082] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0083] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes. As the merging of the liposome and cell progresses, the
liposomal contents are emptied into the cell where the active agent
may act.
[0084] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0085] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis.
[0086] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0087] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0088] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0089] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g. as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0090] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/po- lyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).
[0091] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765). Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al.).
[0092] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos.
5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe
PEG-containing liposomes that can be further derivatized with
functional moieties on their surfaces.
[0093] A limited number of liposomes comprising nucleic acids are
known in the art. WO 96/40062 to Thierry et al. discloses methods
for encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al.
describes certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising antisense oligonucleotides targeted to the raf gene.
[0094] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g. they are self-optimizing (adaptive to the shape of pores
in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0095] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0096] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0097] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0098] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0099] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0100] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0101] Penetration Enhancers
[0102] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides, to the skin of animals. Most
drugs are present in solution in both ionized and nonionized forms.
However, usually only lipid soluble or lipophilic drugs readily
cross cell membranes. It has been discovered that even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
aiding the diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs.
[0103] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
p.92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail.
[0104] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of oligonucleotides through the mucosa is enhanced. In
addition to bile salts and fatty acids, these penetration enhancers
include, for example, sodium lauryl sulfate,
polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p.92); and perfluorochemical emulsions, such as FC-43.
Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
[0105] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,
1992, 44, 651-654).
[0106] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. The bile salts of the invention include, for example,
cholic acid (or its pharmaceutically acceptable sodium salt, sodium
cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic
acid (sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
[0107] Chelating Agents: Chelating agents, as used in connection
with the present invention, can be defined as compounds that remove
metallic ions from solution by forming complexes therewith, with
the result that absorption of oligonucleotides through the mucosa
is enhanced. With regards to their use as penetration enhancers in
the present invention, chelating agents have the added advantage of
also serving as DNase inhibitors, as most characterized DNA
nucleases require a divalent metal ion for catalysis and are thus
inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,
315-339). Chelating agents of the invention include but are not
limited to disodium ethylenediaminetetraacetate (EDTA), citric
acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14,
43-51).
[0108] Non-chelating Non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
oligonucleotides through the alimentary mucosa (Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This
class of penetration enhancers include, for example, unsaturated
cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0109] Agents that enhance uptake of oligonucleotides at the
cellular level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (Junichi et al, U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic
molecules, such as polylysine (Lollo et al., PCT Application WO
97/30731), are also known to enhance the cellular uptake of
oligonucleotides.
[0110] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0111] Carriers
[0112] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
coadministration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extracirculatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate oligonucleotide in hepatic tissue can be
reduced when it is coadministered with polyinosinic acid, dextran
sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
[0113] Excipients
[0114] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal. The excipient
may be liquid or solid and is selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc.).
[0115] Pharmaceutically acceptable organic or inorganic excipient
suitable for non-parenteral administration which do not.
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0116] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0117] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
[0118] Other Components
[0119] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0120] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0121] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more antisense compounds and (b)
one or more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphor- amide, 5-fiuorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the compounds of the invention,
such chemotherapeutic agents may be used individually (e.g., 5-FU
and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide
for a period of time followed by MTX and oligonucleotide), or in
combination with one or more other such chemotherapeutic agents
(e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and
oligonucleotide). 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. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-antisense chemotherapeutic agents are also within the
scope of this invention. Two or more combined compounds may be used
together or sequentially.
[0122] In another related embodiment, compositions of the invention
may contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Numerous examples of antisense compounds are known in the
art. Two or more combined compounds may be used together or
sequentially.
[0123] The formulation of therapeutic compositions and their
subsequent administration is believed to be within the skill of
those in the art. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.01 ug to 100 g per kg of body weight, and may be given
once or more daily, weekly, monthly or yearly, or even once every 2
to 20 years. Persons of ordinary skill in the art can easily
estimate repetition rates for dosing based on measured residence
times and concentrations of the drug in bodily fluids or tissues.
Following successful treatment, it may be desirable to have the
patient undergo maintenance therapy to prevent the recurrence of
the disease state, wherein the oligonucleotide is administered in
maintenance doses, ranging from 0.01 ug to 100 g per kg of body
weight, once or more daily, to once every 20 years.
[0124] While the present invention has been described with
specificity in accordance with certain of its preferred
embodiments, the following examples serve only to illustrate the
invention and are not intended to limit the same.
EXAMPLES
Example 1
[0125] Nucleoside Phosphoramidites for Oligonucleotide Synthesis
Deoxy and 2'-alkoxy amidites
[0126] 2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial sources (e.g.
Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.).
Other 2'-O-alkoxy substituted nucleoside amidites are prepared as
described in U.S. Pat. No. 5,506,351, herein incorporated by
reference. For oligonucleotides synthesized using 2'-alkoxy
amidites, the standard cycle for unmodified oligonucleotides was
utilized, except the wait step after pulse delivery of tetrazole
and base was increased to 360 seconds.
[0127] Oligonucleotides containing 5-methyl-2'-deoxycytidine
(5-Me--C) nucleotides were synthesized according to published
methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21,
3197-3203] using commercially available phosphoramidites (Glen
Research, Sterling Va. or ChemGenes, Needham Mass.).
[0128] 2'-Fluoro amidites
[0129] 2'-Fluorodeoxyadenosine amidites
[0130] 2'-fluoro oligonucleotides were synthesized as described
previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841]
and U.S. Pat. No. 5,670,633, herein incorporated by reference.
Briefly, the protected nucleoside
N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesized utilizing
commercially available 9-beta-D-arabinofuranosyladenine as starting
material and by modifying literature procedures whereby the
2'-alpha-fluoro atom is introduced by a S.sub.N2-displacement of a
2'-beta-trityl group. Thus
N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively
protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP)
intermediate. Deprotection of the THP and N6-benzoyl groups was
accomplished using standard methodologies and standard methods were
used to obtain the 5'-dimethoxytrityl-(DMT) and
5'-DMT-3'-phosphoramidite intermediates.
[0131] 2'-Fluorodeoxyguanosine
[0132] The synthesis of 2'-deoxy-2'-fluoroguanosine was
accomplished using tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguani- ne as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group was followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation was followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidi- tes.
[0133] 2'-Fluorouridine
[0134] Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by
the modification of a literature procedure in which
2,2'-anhydro-1-beta-D-ara- binofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3'phosphoramidites.
[0135] 2'-Fluorodeoxycytidine
[0136] 2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were
used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.
[0137] 2'-O-(2-Methoxyethyl) modified amidites
[0138] 2-O-Methoxyethyl-substituted nucleoside amidites are
prepared as follows, or alternatively, as per the methods of
Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
[0139]
2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]
[0140] 5-Methyluridine (ribosylthymine, commercially available
through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate
(90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were
added to DMF (300 mL). The mixture was heated to reflux, with
stirring, allowing the evolved carbon dioxide gas to be released in
a controlled manner. After 1 hour, the slightly darkened solution
was concentrated under reduced pressure. The resulting syrup was
poured into diethylether (2.5 L), with stirring. The product formed
a gum. The ether was decanted and the residue was dissolved in a
minimum amount of methanol (ca. 400 mL). The solution was poured
into fresh ether (2.5 L) to yield a stiff gum. The ether was
decanted and the gum was dried in a vacuum oven (60.degree. C. at 1
mm Hg for 24 h) to give a solid that was crushed to a light tan
powder (57 g, 85% crude yield). The NMR spectrum was consistent
with the structure, contaminated with phenol as its sodium salt
(ca. 5%). The material was used as is for further reactions (or it
can be purified further by column chromatography using a gradient
of methanol in ethyl acetate (10-25%) to give a white solid, mp
222-4.degree. C.).
[0141] 2'-O-Methoxyethyl-5-methyluridine
[0142] 2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160.degree. C. After heating for
48 hours at 155-160.degree. C., the vessel was opened and the
solution evaporated to dryness and triturated with MeOH (200 mL).
The residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 ml) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of product.
Additional material was obtained by reworking impure fractions.
[0143] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0144] 2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL). The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
[0145]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
[0146] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106
g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from
562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38
mL, 0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by TLC by first quenching the TLC
sample with the addition of MeOH. Upon completion of the reaction,
as judged by TLC, MeOH (50 mL) was added and the mixture evaporated
at 35.degree. C. The residue was dissolved in CHCl.sub.3 (800 mL)
and extracted with 2.times.200 mL of saturated sodium bicarbonate
and 2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/hexane(4:1). Pure product
fractions were evaporated to yield 96 g (84%). An additional 1.5 g
was recovered from later fractions.
[0147]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triaz-
oleuridine
[0148] A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methox-
yethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in
CH.sub.3CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M)
was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3CN, (1
L), cooled to -5.degree. C. and stirred for 0.5 h using an overhead
stirrer. POCl.sub.3 was added dropwise, over a 30 minute period, to
the stirred solution maintained at 0-10.degree. C., and the
resulting mixture stirred for an additional 2 hours. The first
solution was added dropwise, over a 45 minute period, to the latter
solution. The resulting reaction mixture was stored overnight in a
cold room. Salts were filtered from the reaction mixture and the
solution was evaporated. The residue was dissolved in EtOAc (1 L)
and the insoluble solids were removed by filtration. The filtrate
was washed with 1.times.300 mL of NaHCO.sub.3 and 2.times.300 mL of
saturated NaCl, dried over sodium sulfate and evaporated. The
residue was triturated with EtOAc to give the title compound.
[0149] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0150] A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and
NH.sub.4OH (30 mL) was stirred at room temperature for 2 hours. The
dioxane solution was evaporated and the residue azeotroped with
MeOH (2.times.200 mL). The residue was dissolved in MeOH (300 mL)
and transferred to a 2 liter stainless steel pressure vessel. MeOH
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (TLC showed complete
conversion). The vessel contents were evaporated to dryness and the
residue was dissolved in EtOAc (500 mL) and washed once with
saturated NaCl (200 mL). The organics were dried over sodium
sulfate and the solvent was evaporated to give 85 g (95%) of the
title compound.
[0151]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
[0152] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyl-cytidine (85
g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride
(37.2 g, 0.165 M) was added with stirring. After stirring for 3
hours, TLC showed the reaction to be approximately 95% complete.
The solvent was evaporated and the residue azeotroped with MeOH
(200 mL). The residue was dissolved in CHCl.sub.3 (700 mL) and
extracted with saturated NaHCO.sub.3 (2.times.300 mL) and saturated
NaCl (2.times.300 mL), dried over MgSO.sub.4 and evaporated to give
a residue (96 g). The residue was chromatographed on a 1.5 kg
silica column using EtOAc/hexane (1:1) containing 0.5% Et.sub.3NH
as the eluting solvent. The pure product fractions were evaporated
to give 90 g (90%) of the title compound.
[0153]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine--
3'-amidite
[0154]
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
(74 g, 0.10 M) was dissolved in CH.sub.2Cl.sub.2 (1 L). Tetrazole
diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (TLC showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1)
as the eluting solvent. The pure fractions were combined to give
90.6 g (87%) of the title compound.
[0155] 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites
[0156] 2'-(Dimethylaminooxyethoxy) nucleoside amidites
[0157] 2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known
in the art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
[0158]
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
[0159] O.sup.2-2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese,
Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013
eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient
temperature under an argon atmosphere and with mechanical stirring.
tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458
mmol) was added in one portion. The reaction was stirred for 16 h
at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a
complete reaction. The solution was concentrated under reduced
pressure to a thick oil. This was partitioned between
dichloromethane (1 L) and saturated sodium bicarbonate (2.times.1
L) and brine (1 L). The organic layer was dried over sodium sulfate
and concentrated under reduced pressure to a thick oil. The oil was
dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600
mL) and the solution was cooled to -10.degree. C. The resulting
crystalline product was collected by filtration, washed with ethyl
ether (3.times.200 mL) and dried (40.degree. C., 1 mm Hg, 24 h) to
149 g (74.8%) of white solid. TLC and NMR were consistent with pure
product.
[0160]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[0161] In a 2 L stainless steel, unstirred pressure reactor was
added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the
fume hood and with manual stirring, ethylene glycol (350 mL,
excess) was added cautiously at first until the evolution of
hydrogen gas subsided.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
(149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were
added with manual stirring. The reactor was sealed and heated in an
oil bath until an internal temperature of 160.degree. C. was
reached and then maintained for 16 h (pressure<100 psig). The
reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for
desired product and Rf 0.82 for ara-T side product, ethyl acetate)
indicated about 70% conversion to the product. In order to avoid
additional side product formation, the reaction was stopped,
concentrated under reduced pressure (10 to 1 mm Hg) in a warm water
bath (40-100.degree. C.) with the more extreme conditions used to
remove the ethylene glycol. [Alternatively, once the low boiling
solvent is gone, the remaining solution can be partitioned between
ethyl acetate and water. The product will be in the organic phase.]
The residue was purified by column chromatography (2 kg silica gel,
ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate
fractions were combined, stripped and dried to product as a white
crisp foam (84 g, 50%), contaminated starting material (17.4 g) and
pure reusable starting material 20 g. The yield based on starting
material less pure recovered starting material was 58%. TLC and NMR
were consistent with 99% pure product.
[0162]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne
[0163]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
as white foam (21.819 g, 86%).
[0164]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine
[0165]
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridi-
ne (3.1g, 4.5 mmol) was dissolved in dry CH.sub.2Cl.sub.2 (4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at
-10.degree. C. to 0.degree. C. After 1 h the mixture was filtered,
the filtrate was washed with ice cold CH.sub.2Cl.sub.2 and the
combined organic phase was washed with water, brine and dried over
anhydrous Na.sub.2SO.sub.4. The solution was concentrated to get
2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH
(67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1
eq.) was added and the resulting mixture was stirred for 1 h.
Solvent was removed under vacuum; residue-chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri-
dine as white foam (1.95 g, 78%).
[0166]
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-met-
hyluridine
[0167]
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-met-
hyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M
pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at
10.degree. C. under inert atmosphere. The reaction mixture was
stirred for 10 minutes at 10.degree. C. After that the reaction
vessel was removed from the ice bath and stirred at room
temperature for 2 h, the reaction monitored by TLC (5% MeOH in
CH.sub.2Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) was
added and extracted with ethyl acetate (2.times.20 mL). Ethyl
acetate phase was dried over anhydrous Na.sub.2SO.sub.4, evaporated
to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH
(30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and
the reaction mixture was stirred at room temperature for 10
minutes. Reaction mixture cooled to 10.degree. C. in an ice bath,
sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction
mixture stirred at 10.degree. C. for 10 minutes. After 10 minutes,
the reaction mixture was removed from the ice bath and stirred at
room temperature for 2 hrs. To the reaction mixture 5% NaHCO.sub.3
(25 mL) solution was added and extracted with ethyl acetate
(2.times.25 mL). Ethyl acetate layer was dried over anhydrous
Na.sub.2SO.sub.4 and evaporated to dryness The residue obtained was
purified by flash column chromatography and eluted with 5% MeOH in
CH.sub.2Cl.sub.2 to get
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluri-
dine as a white foam (14.6 g, 80%).
[0168] 2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0169] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was
dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept
over KOH). This mixture of triethylamine-2HF was then added to
5'-O-tert-butyldiphenylsil-
yl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4
mmol) and stirred at room temperature for 24 hrs. Reaction was
monitored by TLC (5% MeOH in CH.sub.2Cl.sub.2). Solvent was removed
under vacuum and the residue placed on a flash column and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 to get
2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).
[0170] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
[0171] 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17
mmol) was dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. It was then co-evaporated with anhydrous pyridine (20
mL). The residue obtained was dissolved in pyridine (11 mL) under
argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the
mixture and the reaction mixture was stirred at room temperature
until all of the starting material disappeared. Pyridine was
removed under vacuum and the residue chromatographed and eluted
with 10% MeOH in CH.sub.2Cl.sub.2 (containing a few drops of
pyridine) to get 5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5--
methyluridine (1.13 g, 80%).
[0172]
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2--
cyanoethyl)-N,N-diisopropylphosphoramidite]
[0173] 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08
g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the
residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was
added and dried over P.sub.2O.sub.5 under high vacuum overnight at
40.degree. C. Then the reaction mixture was dissolved in anhydrous
acetonitrile (8.4 mL) and
2-cyanoethyl-N,N,N.sup.1,N.sup.1-tetraisopropylphosphoramidite
(2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at
ambient temperature for 4 hrs under inert atmosphere. The progress
of the reaction was monitored by TLC (hexane:ethyl acetate 1:1).
The solvent was evaporated, then the residue was dissolved in ethyl
acetate (70 mL) and washed with 5% aqueous NaHCO.sub.3 (40 mL).
Ethyl acetate layer was dried over anhydrous Na.sub.2SO.sub.4 and
concentrated. Residue obtained was chromatographed (ethyl acetate
as eluent) to get 5'-O-DMT-2'-O-(2-N,N-dim-
ethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite] as a foam (1.04 g, 74.9%).
[0174] 2'-(Aminooxyethoxy) nucleoside amidites
[0175] 2'-(Aminooxyethoxy) nucleoside amidites [also known in the
art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as
described in the following paragraphs. Adenosine, cytidine and
thymidine nucleoside amidites are prepared similarly.
[0176]
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-
-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidi-
te]
[0177] The 2'-O-aminooxyethyl guanosine analog may be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside may be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside
along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside may be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO
94/02501 A1 940203.) Standard protection procedures should afford
2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'--
dimethoxytrityl)guanosine which may be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-hydroxyethyl)-5'-O-(4,4'-dim-
ethoxytrityl)guanosine. As before the hydroxyl group may be
displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the
protected nucleoside may phosphitylated as usual to yield
2-N-isobutyryl-6-O-diphen-
ylcarbamoyl-2'-O-([2-phthalmidoxy]ethyl)-5'-O-(4,4'-dimethoxytrityl)guanos-
ine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
[0178] 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites
[0179] 2'-dimethylaminoethoxyethoxy nucleoside amidites (also known
in the art as 2'-O-dimethylaminoethoxyethyl, i.e.,
2'-O--CH.sub.2--O--CH.sub.2--- N(CH.sub.2).sub.2, or 2'-DMAEOE
nucleoside amidites) are prepared as follows. Other nucleoside
amidites are prepared similarly.
[0180] 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl
uridine
[0181] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol)
is slowly added to a solution of borane in tetra-hydrofuran (1M, 10
mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves
as the solid dissolves. O.sup.2--,2'-anhydro-5-methyluridine (1.2
g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb
is sealed, placed in an oil bath and heated to 155.degree. C. for
26 hours. The bomb is cooled to room temperature and opened. The
crude solution is concentrated and the residue partitioned between
water (200 mL) and hexanes (200 mL). The excess phenol is extracted
into the hexane layer. The aqueous layer is extracted with ethyl
acetate (3.times.200 mL) and the combined organic layers are washed
once with water, dried over anhydrous sodium sulfate and
concentrated. The residue is columned on silica gel using
methanol/methylene chloride 1:20 (which has 2% triethylamine) as
the eluent. As the column fractions are concentrated a colorless
solid forms which is collected to give the title compound as a
white solid.
[0182]
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-me-
thyl uridine
[0183] To 0.5 g (1.3 mmol) of
2'-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5- -methyl uridine in
anhydrous pyridine (8 mL), triethylamine (0.36 mL) and
dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and
stirred for 1 hour. The reaction mixture is poured into water (200
mL) and extracted with CH.sub.2Cl.sub.2 (2.times.200 mL). The
combined CH.sub.2Cl.sub.2 layers are washed with saturated
NaHCO.sub.3 solution, followed by saturated NaCl solution and dried
over anhydrous sodium sulfate. Evaporation of the solvent followed
by silica gel chromatography using MeOH:CH.sub.2Cl.sub.2:Et.sub.3N
(20:1, v/v, with 1% triethylamine) gives the title compound.
[0184]
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-me-
thyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
[0185] Diisopropylaminotetrazolide (0.6 g) and
2-cyanoethoxy-N,N-diisoprop- yl phosphoramidite (1.1 mL, 2 eq.) are
added to a solution of 5'-O-dimethoxytrityl-2'-O-[2
(2-N,N-dimethylaminoethoxy)ethyl)]-5-methylu- ridine (2.17 g, 3
mmol) dissolved in CH.sub.2Cl.sub.2 (20 mL) under an atmosphere of
argon. The reaction mixture is stirred overnight and the solvent
evaporated. The resulting residue is purified by silica gel flash
column chromatography with ethyl acetate as the eluent to give the
title compound.
Example 2
[0186] Oligonucleotide Synthesis
[0187] Unsubstituted and substituted phosphodiester (P.dbd.O)
oligonucleotides are synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) using standard phosphoramidite
chemistry with oxidation by iodine.
[0188] Phosphorothioates (P.dbd.S) are synthesized as for the
phosphodiester oligonucleotides except the standard oxidation
bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one
1,1-dioxide in acetonitrile for the stepwise thiation of the
phosphite linkages. The thiation wait step was increased to 68 sec
and was followed by the capping step. After cleavage from the CPG
column and deblocking in concentrated ammonium hydroxide at
55.degree. C. (18 h), the oligonucleotides were purified by
precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl
solution. Phosphinate oligonucleotides are prepared as described in
U.S. Pat. No. 5,508,270, herein incorporated by reference.
[0189] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863, herein incorporated by reference.
[0190] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050,
herein incorporated by reference.
[0191] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. Nos., 5,256,775 or 5,366,878, herein incorporated by
reference.
[0192] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively), herein incorporated by reference.
[0193] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925, herein
incorporated by reference.
[0194] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243, herein incorporated by reference.
[0195] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated
by reference.
Example 3
[0196] Oligonucleoside Synthesis
[0197] Methylenemethylimino linked oligonucleosides, also
identified as MMI linked oligonucleosides,
methylenedi-methylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligo-nucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P.dbd.O or P.dbd.S linkages are
prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023,
5,489,677, 5,602,240 and 5,610,289, all of which are herein
incorporated by reference.
[0198] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564,
herein incorporated by reference.
[0199] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618, herein incorporated by
reference.
Example 4
[0200] PNA Synthesis
[0201] Peptide nucleic acids (PNAs) are prepared in accordance with
any of the various procedures referred to in Peptide Nucleic Acids
(PNA): Synthesis, Properties and Potential Applications, Bioorganic
& Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared
in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and
5,719,262, herein incorporated by reference.
Example 5
[0202] Synthesis of Chimeric Oligonucleotides
[0203] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[0204] [2'-O--Me]--[2'-deoxy]--[2'-O--Me] Chimeric Phosphorothioate
Oligonucleotides
[0205] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 380B, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
increasing the wait step after the delivery of tetrazole and base
to 600 s repeated four times for RNA and twice for 2'-O-methyl. The
fully protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 ammonia/ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hrs at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hrs at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to 1/2 volume
by rotovac before being desalted on a G25 size exclusion column.
The oligo recovered is then analyzed spectrophotometrically for
yield and for purity by capillary electrophoresis and by mass
spectrometry.
[0206] [2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)]
Chimeric Phosphorothioate Oligonucleotides
[0207] [2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxy-ethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[0208] [2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[0209] [2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxy
phosphorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidization with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0210] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065, herein
incorporated by reference.
Example 6
[0211] Oligonucleotide Isolation
[0212] After cleavage from the controlled pore glass column
(Applied Biosystems) and deblocking in concentrated ammonium
hydroxide at 55.degree. C. for 18 hours, the oligonucleotides or
oligonucleosides are purified by precipitation twice out of 0.5 M
NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were
analyzed by polyacrylamide gel electrophoresis on denaturing gels
and judged to be at least 85% full length material. The relative
amounts of phosphorothioate and phosphodiester linkages obtained in
synthesis were periodically checked by .sup.31P nuclear magnetic
resonance spectroscopy, and for some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
[0213] Oligonucleotide Synthesis--96 Well Plate Format
[0214] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a standard 96 well
format. Phosphodiester internucleotide linkages were afforded by
oxidation with aqueous iodine. Phosphorothioate internucleotide
linkages were generated by sulfurization utilizing 3,H-1,2
benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous
acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial vendors (e.g.
PE-Applied Biosystems, Foster City, Calif., or Pharmacia,
Piscataway, N.J.). Non-standard nucleosides are synthesized as per
known literature or patented methods. They are utilized as base
protected beta-cyanoethyldiisopropyl phosphoramidites.
[0215] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
[0216] Oligonucleotide Analysis--96 Well Plate Format
[0217] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96 well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
compounds utilizing electrospray-mass spectroscopy. All assay test
plates were diluted from the master plate using single and
multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the compounds on the plate were at
least 85% full length.
Example 9
[0218] Cell Culture and Oligonucleotide Treatment
[0219] The effect of antisense compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following 5 cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, Ribonuclease protection assays, or
RT-PCR.
[0220] T-24 Cells
[0221] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells were routinely cultured in complete
McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.)
supplemented with 10% fetal calf serum (Gibco/Life Technologies,
Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin
100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 7000 cells/well for use in
RT-PCR analysis.
[0222] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0223] A549 Cells
[0224] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Gibco/Life
Technologies, Gaithersburg, Md.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
[0225] NHDF Cells
[0226] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
[0227] HEK Cells
[0228] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
[0229] A10 Cells
[0230] The rat aortic smooth muscle cell line A10 was obtained from
the American Type Culture Collection (Manassas, Va.). A10 cells
were routinely cultured in DMEM, high glucose (American Type
Culture Collection, Manassas, Va.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were
routinely passaged by trypsinization and dilution when they reached
80% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 2500 cells/well for use in
RT-PCR analysis.
[0231] For Northern blotting or other analyses, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0232] Treatment with antisense compounds:
[0233] When cells reached 80% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 200 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Gibco BRL) and then treated with 130 .mu.L of OPTI-MEM.TM.-1
containing 3.75 .mu.g/mL LIPOFECTIN.TM. (Gibco BRL) and the desired
concentration of oligonucleotide. After 4-7 hours of treatment, the
medium was replaced with fresh medium. Cells were harvested 16-24
hours after oligonucleotide treatment.
[0234] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1,
a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold) with
a phosphorothioate backbone which is targeted to human H-ras. For
mouse or rat cells the positive control oligonucleotide is ISIS
15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2'-O-methoxyethyl
gapmer (2'-O-methoxyethyls shown in bold) with a phosphorothioate
backbone which is targeted to both mouse and rat c-raf. The
concentration of positive control oligonucleotide that results in
80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS
15770) mRNA is then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
H-ras or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments.
Example 10
[0235] Analysis of Oligonucleotide Inhibition of Protein
Phosphatase 2 Catalytic Subunit Beta Expression
[0236] Antisense modulation of Protein Phosphatase 2 catalytic
subunit beta expression can be assayed in a variety of ways known
in the art. For example, Protein Phosphatase 2 catalytic subunit
beta mRNA levels can be quantitated by, e.g., Northern blot
analysis, competitive polymerase chain reaction (PCR), or real-time
PCR (RT-PCR). Real-time quantitative PCR is presently preferred.
RNA analysis can be performed on total cellular RNA or
poly(A)+mRNA. Methods of RNA isolation are taught in, for example,
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,
Inc., 1993. Northern blot analysis is routine in the art and is
taught in, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley &
Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently
accomplished using the commercially available ABI PRISM.TM. 7700
Sequence Detection System, available from PE-Applied Biosystems,
Foster City, Calif. and used according to manufacturer's
instructions.
[0237] Protein levels of Protein Phosphatase 2 catalytic subunit
beta can be quantitated in a variety of ways well known in the art,
such as immunoprecipitation, Western blot analysis
(immunoblotting), ELISA or fluorescence-activated cell sorting
(FACS). Antibodies directed to Protein Phosphatase 2 catalytic
subunit beta can be identified and obtained from a variety of
sources, such as the MSRS catalog of antibodies (Aerie Corporation,
Birmingham, Mich.), or can be prepared via conventional antibody
generation methods. Methods for preparation of polyclonal antisera
are taught in, for example, Ausubel, F. M. et al., Current
Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John
Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies
is taught in, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley
& Sons, Inc., 1997.
[0238] Immunoprecipitation methods are standard in the art and can
be found at, for example, Ausubel, F. M. et al., Current Protocols
in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley
& Sons, Inc., 1998. Western blot (immunoblot) analysis is
standard in the art and can be found at, for example, Ausubel, F.
M. et al., Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
Example 11
[0239] Poly(A)+mRNA Isolation
[0240] Poly(A)+mRNA was isolated according to Miura et al., Clin.
Chem., 1996, 42, 1758-1764. Other methods for poly(A)+mRNA
isolation are taught in, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3,
John Wiley & Sons, Inc., 1993. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C. was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0241] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
Example 12
[0242] Total RNA Isolation
[0243] Total RNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 100 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 100 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 15
seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY
96.TM. plate and the vacuum again applied for 15 seconds. 1 mL of
Buffer RPE was then added to each well of the RNEASY 96.TM. plate
and the vacuum applied for a period of 15 seconds. The Buffer RPE
wash was then repeated and the vacuum was applied for an additional
10 minutes. The plate was then removed from the QIAVAC.TM. manifold
and blotted dry on paper towels. The plate was then re-attached to
the QIAVAC.TM. manifold fitted with a collection tube rack
containing 1.2 mL collection tubes. RNA was then eluted by
pipetting 60 .mu.L water into each well, incubating 1 minute, and
then applying the vacuum for 30 seconds. The elution step was
repeated with an additional 60 .mu.L water.
[0244] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13
[0245] Real-time Quantitative PCR Analysis of Protein Phosphatase 2
catalytic Subunit Beta mRNA Levels
[0246] Quantitation of Protein Phosphatase 2 catalytic subunit beta
mRNA levels was determined by real-time quantitative PCR using the
ABI PRISM.TM. 7700 Sequence Detection System (PE-Applied
Biosystems, Foster City, Calif.) according to manufacturer's
instructions. This is a closed-tube, non-gel-based, fluorescence
detection system which allows high-throughput quantitation of
polymerase chain reaction (PCR) products in real-time. As opposed
to standard PCR, in which amplification products are quantitated
after the PCR is completed, products in real-time quantitative PCR
are quantitated as they accumulate. This is accomplished by
including in the PCR reaction an oligonucleotide probe that anneals
specifically between the forward and reverse PCR primers, and
contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or
VIC, obtained from either Operon Technologies Inc., Alameda, Calif.
or PE-Applied Biosystems, Foster City, Calif.) is attached to the
5' end of the probe and a quencher dye (e.g., TAMRA, obtained from
either Operon Technologies Inc., Alameda, Calif. or PE-Applied
Biosystems, Foster City, Calif.) is attached to the 3' end of the
probe. When the probe and dyes are intact, reporter dye emission is
quenched by the proximity of the 3' quencher dye. During
amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. 7700 Sequence Detection System.
In each assay, a series of parallel reactions containing serial
dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0247] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0248] PCR reagents were obtained from PE-Applied Biosystems,
Foster City, Calif. RT-PCR reactions were carried out by adding 25
.mu.L PCR cocktail (1.times.TAQMAN.TM. buffer A, 5.5 mM MgCl.sub.2,
300 .mu.M each of dATP, dCTP and dGTP, 600 .mu.M of dUTP, 100 nM
each of forward primer, reverse primer, and probe, 20 Units RNAse
inhibitor, 1.25 Units AMPLITAQ GOLD.TM., and 12.5 Units MuLV
reverse transcriptase) to 96 well plates containing 25 .mu.L total
RNA solution. The RT reaction was carried out by incubation for 30
minutes at 48.degree. C. Following a 10 minute incubation at
95.degree. C. to activate the AMPLITAQ GOLD.TM., 40 cycles of a
two-step PCR protocol were carried out: 95.degree. C. for 15
seconds (denaturation) followed by 60.degree. C. for 1.5 minutes
(annealing/extension).
[0249] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
from Molecular Probes. Methods of RNA quantification by RiboGreen
are taught in Jones, L. J., et al, Analytical Biochemistry, 1998,
265, 368-374.
[0250] In this assay, 175 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 25 uL purified,
cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied
Biosystems) with excitation at 480 nm and emission at 520 nm.
[0251] Probes and primers to human Protein Phosphatase 2 catalytic
subunit beta were designed to hybridize to a human Protein
Phosphatase 2 catalytic subunit beta sequence, using published
sequence information (GenBank accession number M60484, incorporated
herein as SEQ ID NO:3). For human Protein Phosphatase 2 catalytic
subunit beta the PCR primers were:
[0252] forward primer: TCGTGCCCACCAGCTTGTA (SEQ ID NO: 4)
[0253] reverse primer: TAATTGGGTGCACTGAAAATGG (SEQ ID NO: 5)
and
[0254] the PCR probe was: FAM-CATTCCGATCATGACACCAATTGTATCCCT-TAMRA
(SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City,
Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye. For human
GAPDH the PCR primers were:
[0255] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)
[0256] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and
[0257] the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCCX-TAMRA 3'(SEQ
ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is
the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,
Foster City, Calif.) is the quencher dye.
[0258] Probes and primers to rat Protein Phosphatase 2 catalytic
subunit beta were designed to hybridize to a rat Protein
Phosphatase 2 catalytic subunit beta sequence, using published
sequence information (GenBank accession number M23591, incorporated
herein as SEQ ID NO:10). For rat Protein Phosphatase 2 catalytic
subunit beta the PCR primers were:
[0259] forward primer: GGACCAAAACGTGCCATACTAAT (SEQ ID NO:11)
[0260] reverse primer: TGGTTGACTCGGCTAGGACAGT (SEQ ID NO: 12)
and
[0261] the PCR probe was: FAM-ACTCAAGTTCACACCGTGCTGATGGCT-TAMRA
(SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City,
Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye. For rat GAPDH
the PCR primers were:
[0262] forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 14)
[0263] reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 15)
and
[0264] the PCR probe was: 5' JOE-TTGTGCAGTGCCAGCCTCGTCTCAX-TAMRA 3'
(SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City,
Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied
Biosystems, Foster City, Calif.) is the quencher dye.
Example 14
[0265] Northern Blot Analysis of Protein Phosphatase 2 Catalytic
Subunit Beta mRNA Levels
[0266] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared
following manufacturer's recommended protocols. Twenty micrograms
of total RNA was fractionated by electrophoresis through 1.2%
agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then
robed using QUICKHYB.TM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0267] To detect human Protein Phosphatase 2 catalytic subunit
beta, a human Protein Phosphatase 2 catalytic subunit beta specific
probe was prepared by PCR using the forward primer
TCGTGCCCACCAGCTTGTA (SEQ ID NO: 4) and the reverse primer
TAATTGGGTGCACTGAAAATGG (SEQ ID NO: 5). To normalize for variations
in loading and transfer efficiency membranes were stripped and
probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
RNA (Clontech, Palo Alto, Calif.).
[0268] To detect rat Protein Phosphatase 2 catalytic subunit beta,
a rat Protein Phosphatase 2 catalytic subunit beta specific probe
was prepared by PCR using the forward primer
GGACCAAAACGTGCCATACTAAT (SEQ ID NO:11) and the reverse primer
TGGTTGACTCGGCTAGGACAGT (SEQ ID NO: 12). To normalize for variations
in loading and transfer efficiency membranes were stripped and
probed for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA
(Clontech, Palo Alto, Calif.).
[0269] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 15
[0270] Antisense Inhibition of Human Protein Phosphatase 2
Catalytic Subunit Beta Expression by Chimeric Phosphorothioate
Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap
[0271] In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human Protein Phosphatase 2 catalytic subunit beta RNA, using
published sequences (GenBank accession number M60484, incorporated
herein as SEQ ID NO: 3). The oligonucleotides are shown in Table 1.
"Target site" indicates the first (5'-most) nucleotide number on
the particular target sequence to which the oligonucleotide binds.
All compounds in Table 1 are chimeric oligonucleotides ("gapmers")
20 nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by five-nucleotide "wings". The wings
are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines. The compounds were analyzed for their effect on
human Protein Phosphatase 2 catalytic subunit beta mRNA levels by
quantitative real-time PCR as described in other examples herein.
Data are averages from two experiments. If present, "N.D."
indicates "no data".
1TABLE 1 Inhibition of human Protein Phosphatase 2 catalytic
subunit beta mRNA levels by chimeric phosphorothioate
oligonucleotides having 2'-MOE wings and a deoxy gap TARGET TARGET
SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO 110188
3'UTR 3 2136 agcaggcaaactgttagact 76 17 119005 5'UTR 3 558
cctccgggtttccgtcatcg 0 18 119006 5'UTR 3 567 ctctcccctcctccgggttt
21 19 119007 5'UTR 3 596 gacttgtctttccccttctc 57 20 119008 5'UTR 3
624 gcccgcctcacgcctaccgg 52 21 119009 5'UTR 3 740
ccccgcccggccgcgcgccg 56 22 119010 5'UTR 3 763 ggaggagacccccgcccgcc
68 23 119011 5'UTR 3 806 gcgccccgcccaagcccagc 38 24 119012 5'UTR 3
862 ccgtcgccaggtcccacagg 60 25 119013 5'UTR 3 885
gccgctcccctctccctccg 64 26 119014 5'UTR 3 889 gggcgccgctcccctctccc
64 27 119015 5'UTR 3 890 cgggcgccgctcccctctcc 52 28 119016 5'UTR 3
904 ccccggccccggcccgggcg 38 29 119017 5'UTR 3 906
gcccccggccccggcccggg 31 30 119018 5'UTR 3 913 cccacccgcccccggccccg
41 31 119019 5'UTR 3 945 gagccccagcccggccgccg 58 32 119020 Start 3
983 cttgtcgtccatggcggccc 67 33 Codon 119021 Start 3 985
gccttgtcgtccatggcggc 49 34 Codon 119022 Coding 3 1042
ttcagctgcttacactcgtt 62 35 119023 Coding 3 1047
tctcgttcagctgcttacac 71 36 119024 Coding 3 1052
ttggttctcgttcagctgct 65 37 119025 Coding 3 1057
cgcacttggttctcgttcag 59 38 119026 Coding 3 1062
gcgtccgcacttggttctcg 61 39 119027 Coding 3 1067
gcaoagcgtccgcacttggt 68 40 119028 Coding 3 1072
ttctcgcacagcgtccgcac 71 41 119029 Coding 3 1096
tcttttgttaaaatttcctt 56 42 119030 Coding 3 1232
tacatagtcacccatgaata 73 43 119031 Coding 3 1279
aatgctacaagaagagtcac 60 44 119032 Coding 3 1264
cctttaatgctacaagaaga 71 45 119033 Coding 3 1363
tcataaaagccatatacttg 55 46 119034 Coding 3 1433
tggaagataatcaaagagat 62 47 119035 Coding 3 1438
gtaagtggaagataatcaaa 60 48 119036 Coding 3 1443
aagctgtaagtggaagataa 59 49 119037 Coding 3 1448
tactaaagctgtaagtggaa 63 50 119038 Coding 3 1453
ccatctactaaagctgtaag 69 51 119039 Coding 3 1458
tctgtccatctactaaagct 0 52 119040 Coding 3 1463 gaatatctgtccatctacta
49 53 119041 Coding 3 1468 aggcagaatatctgtccatc 57 54 119042 Coding
3 1492 atggatggagagaggccacc 60 55 119043 Coding 3 1546
tcatgtggaacttcctgtaa 55 56 119044 Coding 3 1563
gatcacacattgggccctca 60 57 119045 Coding 3 1627
gtgtagccagcaecacgtgg 44 58 119046 Coding 3 1632
caaatgtgtagccagcacca 66 59 119047 Coding 3 1637
ttgtccaaatgtgtagccag 74 60 119048 Coding 3 1642
atgtcttgtccaaatgtgta 69 61 119049 Coding 3 1647
cagaaatgtcttgtccaaat 76 62 119050 Coding 3 1795
atagcagcctggttcccaca 73 63 119051 Coding 3 1800
ccatgatagcagcctggttc 74 64 119052 Coding 3 1805
taattccatgatagcagcct 78 65 119053 Stop 3 1903 atttataggaagtagtctgg
53 66 Codon 119054 Stop 3 1909 ggagaaatttataggaagta 64 67 Codon
119055 3'UTR 3 1952 taaaaagccaggtatacttc 63 68 119056 3'UTR 3 1975
tttgtttttaaatacatata 14 69 119057 3'UTR 3 2069 gctcatcattagtatggcac
65 70 119058 3'UTR 3 2113 ctagaacatagtgtactaaa 66 71 119059 3'UTR 3
2132 ggcaaactgttagactgacc 70 72 119060 3'UTR 3 2164
tccaaaggaaaatggttact 68 73 119061 3'UTR 3 2202 ggagatgaagcagttagtta
68 74 119062 3'UTR 3 2257 ctattaatccatgccagtta 64 75 119063 3'UTR 3
2258 tctattaatccatgccagtt 77 76 119064 3'UTR 3 2260
actctattaatccatgccag 79 77 119065 3'UTR 3 2262 caactctattaatccatgcc
73 78 119066 3'UTR 3 2273 aaataaaactccaactctat 56 79 119067 3'UTR 3
2285 aatttttcttaaaaataaaa 22 80 119068 3'UTR 3 2296
gttagcttgtgaatttttct 64 81 119069 3'UTR 3 2305 ttagtggaagttagcttgtg
62 82 119070 3'UTR 3 2322 aaataaaggataatggatta 0 83 119071 3'UTR 3
2329 ttcaataaaataaaggataa 0 84 119072 3'UTR 3 2333
acatttcaataaaataaagg 0 85 119073 3'UTR 3 2370 tcccaagaagaaccttttct
78 86 119074 3'UTR 3 2383 tatgacaacatactcccaag 70 87 119075 3'UTR 3
2407 atgaagggaaatctctttaa 65 88 119076 3'UTR 3 2408
aatgaagggaaatctcttta 43 89 119077 3'UTR 3 2415 tagtttaaatgaagggaaat
28 90 119078 3'UTR 3 2444 aatatgcagatcaacataaa 36 91 119079 3'UTR 3
2482 caaataggatgcaagcactg 63 92 119080 3'UTR 3 2518
tttgtttaaaatgaaaagtt 16 93
[0272] As shown in Table 1, SEQ ID NOs 17, 20, 21, 22, 23, 25, 26,
27, 28, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, 50, 51, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 86, 87,
88 and 92 demonstrated at least 45% inhibition of human Protein
Phosphatase 2 catalytic subunit beta expression in this assay and
are therefore preferred. The target sites to which these preferred
sequences are complementary are herein referred to as "active
sites" and are therefore preferred sites for targeting by compounds
of the present invention.
Example 16
[0273] Antisense Inhibition of Rat Protein Phosphatase 2 Catalytic
Subunit Beta Expression By Chimeric Phosphorothioate
Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap
[0274] In accordance with the present invention, a second series of
oligonucleotides were designed to target different regions of the
rat Protein Phosphatase 2 catalytic subunit beta RNA, using
published sequences (GenBank accession number M23591, incorporated
herein as SEQ ID NO: 10). The oligonucleotides are shown in Table
2. "Target site" indicates the first (5'-most) nucleotide number on
the particular target sequence to which the oligonucleotide binds.
All compounds in Table 2 are chimeric oligonucleotides ("gapmers")
20 nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by five-nucleotide "wings". The wings
are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P.dbd.S)
throughout the oligonucleotide. All cytidine residues are
5-methylcytidines. The compounds were analyzed for their effect on
rat Protein Phosphatase 2 catalytic subunit beta mRNA levels by
quantitative real-time PCR as described in other examples herein.
Data are averages from two experiments. If present, "N.D."
indicates "no data".
2TABLE 2 Inhibition of rat Protein Phosphatase 2 catalytic subunit
beta mRNA levels by chimeric phosphorothicate oligonucleotides
having 2'-MOE wings and a deoxy gap TARGET TARGET SEQ ID ISIS #
REGION SEQ ID NO SITE SEQUENCE % INHIB NO 119011 5'UTR 10 211
gcgccccgcccaagcccagc 69 24 119021 Start 10 304 gccttgtcgtccatggcggc
75 34 Codon 119022 Coding 10 361 ttcagctgcttacactcgtt 61 35 119023
Coding 10 366 tctcgttcagctgcttacac 80 36 119024 Coding 10 371
ttggttctcgttcagctgct 51 37 119025 Coding 10 376
cgcacttggttctcgttcag 74 38 119026 Coding 10 381
gcgtccgcacttggttctcg 68 39 119027 Coding 10 386
gcacagcgtccgcacttggt 78 40 119028 Coding 10 391
ttctcgcacagcgtccgcac 88 41 119029 Coding 10 415
tcttttgttaaaatttcctt 61 42 119030 Coding 10 551
tacatagtcacccatgaata 76 43 119031 Coding 10 598
aatgctacaagaagagtcac 52 44 119032 Coding 10 603
cctttaatgctacaagaaga 35 45 119033 Coding 10 682
tcataaaagccatatacttg 62 46 119034 Coding 10 752
tggaagataatcaaagagat 57 47 119035 Coding 10 757
gtaagtggaagataatcaaa 58 48 119036 Coding 10 762
aagctgtaagtggaagataa 48 49 119037 Coding 10 767
tactaaagctgtaagtggaa 67 50 119038 Coding 10 772
ccatctactaaagctgtaag 69 51 119039 Coding 10 777
tctgtccatctactaaagct 49 52 119040 Coding 10 782
gaatatctgtccatctacta 43 53 119041 Coding 10 787
aggcagaatatctgtccatc 57 54 119042 Coding 10 811
atggatggagagaggccacc 63 55 119043 Coding 10 865
tcatgtggaacttcctgtaa 52 56 119044 Coding 10 882
gatcacacattgggccctca 37 57 119045 Coding 10 946
gtgtagccagcaccacgtgg 54 58 119046 Coding 10 951
caaatgtgtagccagcacca 55 59 119047 Coding 10 956
ttgtccaaatgtgtagccag 72 60 119048 Coding 10 961
atgtcttgtccaaatgtgta 71 61 119049 Coding 10 966
cagaaatgtcttgtccaaat 73 62 119050 Coding 10 1114
atagcagcctggttcccaca 77 63 119051 Coding 10 1119
ccatgatagcagcctggttc 70 64 119052 Coding 10 1124
taattccatgatagcagcct 63 65 119053 Stop 10 1222 atttataggaagtagtctgg
53 66 Codon 119055 3'UTR 10 1270 taaaaagccaggtatacttc 76 68 119066
3'UTR 10 1591 aaataaaactccaactctat 52 79 119085 5'UTR 10 1
gttctctcggcgctcgtgcc 0 94 119086 5'UTR 10 9 tggccgcggttctctcggcg 58
95 119087 5'UTR 10 13 gctctggccgcggttctctc 43 96 119088 5'UTR 10 23
gcctctccgcgctctggccg 50 97 119089 5'UTR 10 67 cggcccgacggcgcccgccc
73 98 119090 5'UTR 10 124 gcggcaaaaagggctcacgg 10 99 119091 5'UTR
10 195 cagcgggcagccgccggccc 41 100 119092 5'UTR 10 199
agcccagcgggcagccgccg 64 101 119093 5'UTR 10 221
cgggcagcccgcgccccgcc 54 102 119094 5'UTR 10 248
ccccggcgggaccaccgagc 79 103 119095 5'UTR 10 272
cccgccgcctcccccgccgc 34 104 119096 5'UTR 10 274
tccccgccgcctcccccgcc 42 105 119097 5'UTR 10 278
cgcgtccccgccgcctcccc 86 106 119098 Start 10 302
cttgtcgtccatggcggcga 71 107 Codon 119099 Coding 10 774
gtccatctactaaagctgta 81 108 119100 Stop 10 1229
gggaggaatttataggaagt 51 109 Codon 119101 3'UTR 10 1291
tatatatgtatatatatttt 0 110 119102 3'UTR 10 1335
acaatttgttacagagacac 78 111 119103 3'UTR 10 1339
tagcacaatttgttacagag 60 112 119104 3'UTR 10 1344
agacatagcacaatttgtta 66 113 119105 3'UTR 10 1383
agtatggcacgttttggtcc 92 114 119106 3'UTR 10 1410
ttcacaccgtgctgatggct 87 115 119107 3'UTR 10 1414
caagttcacaccgtgctgat 70 116 119108 3'UTR 10 1439
gttgactcggctaggacagt 86 117 119109 3'UTR 10 1450
gcggctgcctggttgactcg 59 118 119110 3'UTR 10 1483
gaaggacggctactacagca 77 119 119111 3'UTR 10 1498
cccttaaccagtcacgaagg 92 120 119112 3'UTR 10 1534
taagcgcaaaggagatgaag 50 121 119113 3'UTR 10 1575
ctataatccatgccagttaa 80 122 119114 3'UTR 10 1588
taaaactccaactctataat 59 123 119115 3'UTR 10 1594
taaaaataaaactccaactc 28 124 119116 3'UTR 10 1609
cagcttgtcaattcttaaaa 86 125 119117 3'UTR 10 1616
tggaagtcagcttgtcaatt 74 126 119118 3'UTR 10 1650
acatttcaacaaaataaagg 24 127 119119 3'UTR 10 1654
tcatacatttcaacaaaata 6 128 119120 3'UTR 10 1668
tcttcttcagttagtcatac 87 129 119121 3'UTR 10 1708
aaaggaaatcttagtgttat 53 130 119122 3'UTR 10 1714
aacttgaaaggaaatcttag 15 131 119123 3'UTR 10 1726
ttcagttcaggaaacttgaa 59 132 119124 3'UTR 10 1745
ggtcaacatccaacagtaat 63 133 119125 3'UTR 10 1755
agaatgtgcaggtcaacatc 53 134 119126 3'UTR 10 1798
tcagtacagcaaggaggatg 14 135
[0275] As shown in Table 2, SEQ ID NOs 24, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 56, 58, 59,
60, 61, 62, 63, 64, 65, 66, 68, 79, 95, 97, 98, 101, 102, 103, 106,
107, 108, 109, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 122, 123, 125, 126, 129, 130, 132, 133 and 134 demonstrated at
least 45% inhibition of rat Protein Phosphatase 2 catalytic subunit
beta expression in this experiment and are therefore preferred. The
target sites to which these preferred sequences are complementary
are herein referred to as "active sites" and are therefore
preferred sites for targeting by compounds of the present
invention.
Example 17
[0276] Western Blot Analysis of Protein Phosphatase 2 Catalytic
Subunit Beta Protein Levels
[0277] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a
16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to Protein Phosphatase 2 catalytic subunit beta
is used, with a radiolabelled or fluorescently labeled secondary
antibody directed against the primary antibody species. Bands are
visualized using a PHOSPHORIMAGER.TM. (Molecular Dynamics,
Sunnyvale Calif.).
Sequence CWU 1
1
135 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1
tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense
Oligonucleotide 2 atgcattctg cccccaagga 20 3 2574 DNA Homo sapiens
CDS (991)...(1920) 3 ttggggaggg gggtgcctag atggccccta agaggggtcc
ctgttctgtc tctcaataaa 60 tatttgttga atgaacaaat cattacaact
cagtacacat tgcagaaaat atagccaaga 120 gctctggagc tggaagggcc
acagattatc ctacagaaca atcatttcac ttttctaatg 180 tcgaaaggga
ggattcgaga tgttaggggc tacaggtgag gctggaaata attaatttac 240
tccatcaata ttgaatgcct gctattgagt gctagactct ggggagacaa tgttaaggga
300 agcccaagtt ttccaactcc ctgtccagag cgctgcaaag tactgcagga
agctaaagtg 360 aggacaaagt tcccagagat caggatattt aaagggagaa
ccagcagagc ttggtctggg 420 gcagggtggg gaaaagaggg accctggcct
cctcggaccg tttctccgcc aagccacgcg 480 agggcgctgt tctgctccta
gggcgccgtg tcccggcggc gccgcctgct cgccttttcc 540 cggcggaaat
gcccgagcga tgacggaaac ccggaggagg ggagagaaag agcgagagaa 600
ggggaaagac aagtcgggag aggccggtag gcgtgaggcg ggcctgaagc ggcagcgggc
660 ggccttcgtc cggcgagagc taggccgagg acccgcgccg cgctccccgg
cacctcaccg 720 cgtccttcac cgactcccgc ggcgcgcggc cgggcgggga
agggcgggcg ggggtctcct 780 ccaggctgcg cgctcggagc cgcctgctgg
gcttgggcgg ggcgcggggc ccgcggccgc 840 cctacccggc tcagtcctcc
ccctgtggga cctggcgacg gcggcggagg gagaggggag 900 cggcgcccgg
gccggggccg ggggcgggtg gggagggggg agggcggcgg ccgggctggg 960
gctcgggatc cgcatcggga tcgggccgcc atg gac gac aag gcg ttc acc aag
1014 Met Asp Asp Lys Ala Phe Thr Lys 1 5 gag ctg gac cag tgg gtc
gag cag ctg aac gag tgt aag cag ctg aac 1062 Glu Leu Asp Gln Trp
Val Glu Gln Leu Asn Glu Cys Lys Gln Leu Asn 10 15 20 gag aac caa
gtg cgg acg ctg tgc gag aag gca aag gaa att tta aca 1110 Glu Asn
Gln Val Arg Thr Leu Cys Glu Lys Ala Lys Glu Ile Leu Thr 25 30 35 40
aaa gaa tca aat gtg caa gag gtt cgt tgc cct gtt act gtc tgt gga
1158 Lys Glu Ser Asn Val Gln Glu Val Arg Cys Pro Val Thr Val Cys
Gly 45 50 55 gat gtg cat ggt caa ttt cat gat ctt atg gaa ctc ttt
aga att ggt 1206 Asp Val His Gly Gln Phe His Asp Leu Met Glu Leu
Phe Arg Ile Gly 60 65 70 gga aaa tca ccg gat aca aac tac tta ttc
atg ggt gac tat gta gac 1254 Gly Lys Ser Pro Asp Thr Asn Tyr Leu
Phe Met Gly Asp Tyr Val Asp 75 80 85 aga gga tat tat tca gtg gag
act gtg act ctt ctt gta gca tta aag 1302 Arg Gly Tyr Tyr Ser Val
Glu Thr Val Thr Leu Leu Val Ala Leu Lys 90 95 100 gtg cgt tat cca
gaa cgc att aca ata ttg aga gga aat cac gaa agc 1350 Val Arg Tyr
Pro Glu Arg Ile Thr Ile Leu Arg Gly Asn His Glu Ser 105 110 115 120
cga caa att acc caa gta tat ggc ttt tat gat gaa tgt ctg cga aag
1398 Arg Gln Ile Thr Gln Val Tyr Gly Phe Tyr Asp Glu Cys Leu Arg
Lys 125 130 135 tat ggg aat gcc aac gtt tgg aaa tat ttt aca gat ctc
ttt gat tat 1446 Tyr Gly Asn Ala Asn Val Trp Lys Tyr Phe Thr Asp
Leu Phe Asp Tyr 140 145 150 ctt cca ctt aca gct tta gta gat gga cag
ata ttc tgc ctc cat ggt 1494 Leu Pro Leu Thr Ala Leu Val Asp Gly
Gln Ile Phe Cys Leu His Gly 155 160 165 ggc ctc tct cca tcc ata gac
aca ctg gat cat ata aga gcc ctg gat 1542 Gly Leu Ser Pro Ser Ile
Asp Thr Leu Asp His Ile Arg Ala Leu Asp 170 175 180 cgt tta cag gaa
gtt cca cat gag ggc cca atg tgt gat ctg tta tgg 1590 Arg Leu Gln
Glu Val Pro His Glu Gly Pro Met Cys Asp Leu Leu Trp 185 190 195 200
tca gat cca gat gat cgt ggt gga tgg ggt att tca cca cgt ggt gct
1638 Ser Asp Pro Asp Asp Arg Gly Gly Trp Gly Ile Ser Pro Arg Gly
Ala 205 210 215 ggc tac aca ttt gga caa gac att tct gaa acc ttt aac
cat gcc aat 1686 Gly Tyr Thr Phe Gly Gln Asp Ile Ser Glu Thr Phe
Asn His Ala Asn 220 225 230 ggt ctc aca ctg gtt tct cgt gcc cac cag
ctt gta atg gag gga tac 1734 Gly Leu Thr Leu Val Ser Arg Ala His
Gln Leu Val Met Glu Gly Tyr 235 240 245 aat tgg tgt cat gat cgg aat
gtg gtt acc att ttc agt gca ccc aat 1782 Asn Trp Cys His Asp Arg
Asn Val Val Thr Ile Phe Ser Ala Pro Asn 250 255 260 tac tgt tat cgt
tgt ggg aac cag gct gct atc atg gaa tta gat gac 1830 Tyr Cys Tyr
Arg Cys Gly Asn Gln Ala Ala Ile Met Glu Leu Asp Asp 265 270 275 280
act tta aaa tat tcc ttc ctt caa ttt gac cca gcg cct cgt cgt ggt
1878 Thr Leu Lys Tyr Ser Phe Leu Gln Phe Asp Pro Ala Pro Arg Arg
Gly 285 290 295 gag cct cat gtt aca cgg cgc acc cca gac tac ttc cta
taa atttctcctg 1930 Glu Pro His Val Thr Arg Arg Thr Pro Asp Tyr Phe
Leu 300 305 310 ggaaacctgc ctttgtatgt ggaagtatac ctggcttttt
aaaatatatg tatttaaaaa 1990 caaaaagcaa cagtaatcta tgtgtttctg
taacaaattg ggatctgtct tggcattaaa 2050 ccacatcatg gaccaaatgt
gccatactaa tgatgagcat ttagcacaat ttgagactga 2110 aatttagtac
actatgttct aggtcagtct aacagtttgc ctgctgtatt tatagtaacc 2170
attttccttt ggactgttca agcaaaaaag gtaactaact gcttcatctc cttttgcgct
2230 tatttggaaa ttttagttat agtgtttaac tggcatggat taatagagtt
ggagttttat 2290 ttttaagaaa aattcacaag ctaacttcca ctaatccatt
atcctttatt ttattgaaat 2350 gtataattaa cttaactgaa gaaaaggttc
ttcttgggag tatgttgtca taacatttaa 2410 agagatttcc cttcatttaa
actaaattac tgttttatgt tgatctgcat atttctgtat 2470 atttgtcatg
acagtgcttg catcctattt ggtgtactca gcaaataaac ttttcatttt 2530
aaacaaaaca ttcatttatt gtgttgtgca ttaaatgaaa actt 2574 4 19 DNA
Artificial Sequence PCR Primer 4 tcgtgcccac cagcttgta 19 5 22 DNA
Artificial Sequence PCR Primer 5 taattgggtg cactgaaaat gg 22 6 30
DNA Artificial Sequence PCR Probe 6 cattccgatc atgacaccaa
ttgtatccct 30 7 19 DNA Artificial Sequence PCR Primer 7 gaaggtgaag
gtcggagtc 19 8 20 DNA Artificial Sequence PCR Primer 8 gaagatggtg
atgggatttc 20 9 20 DNA Artificial Sequence PCR Probe 9 caagcttccc
gttctcagcc 20 10 1843 DNA Rattus norvegicus CDS (310)...(1239) 10
ggcacgagcg ccgagagaac cgcggccaga gcgcggagag gcctgcgggc ggcgacggca
60 gcgggagggc gggcgccgtc gggccggagc ctcccccgag ccgcgccgcg
ctctggctcc 120 gagccgtgag ccctttttgc cgcgccccga gcgcgtggcc
gggggccggg cggggcgggc 180 gctcccggag gccggggccg gcggctgccc
gctgggcttg ggcggggcgc gggctgcccg 240 ctccgcggct cggtggtccc
gccgggggcc ggcggcgggg gaggcggcgg ggacgcgcgg 300 ctcgccgcc atg gac
gac aag gcg ttc acc aag gag ctg gac cag tgg gtg 351 Met Asp Asp Lys
Ala Phe Thr Lys Glu Leu Asp Gln Trp Val 1 5 10 gag cag ctg aac gag
tgt aag cag ctg aac gag aac caa gtg cgg acg 399 Glu Gln Leu Asn Glu
Cys Lys Gln Leu Asn Glu Asn Gln Val Arg Thr 15 20 25 30 ctg tgc gag
aag gct aag gaa att tta aca aaa gaa tca aat gta caa 447 Leu Cys Glu
Lys Ala Lys Glu Ile Leu Thr Lys Glu Ser Asn Val Gln 35 40 45 gag
gtt cgc tgt cct gtt acc gtc tgt gga gat gtg cat ggc caa ttc 495 Glu
Val Arg Cys Pro Val Thr Val Cys Gly Asp Val His Gly Gln Phe 50 55
60 cat gac ctt atg gaa ctc ttc aga att ggt gga aaa tca cca gac acc
543 His Asp Leu Met Glu Leu Phe Arg Ile Gly Gly Lys Ser Pro Asp Thr
65 70 75 aac tat cta ttc atg ggt gac tat gta gac aga gga tat tat
tct gtg 591 Asn Tyr Leu Phe Met Gly Asp Tyr Val Asp Arg Gly Tyr Tyr
Ser Val 80 85 90 gag acc gtg act ctt ctt gta gca tta aag gtg cgc
tat cca gag cgt 639 Glu Thr Val Thr Leu Leu Val Ala Leu Lys Val Arg
Tyr Pro Glu Arg 95 100 105 110 atc aca ata ttg cga gga aat cat gaa
agc cgg cag atc aca caa gta 687 Ile Thr Ile Leu Arg Gly Asn His Glu
Ser Arg Gln Ile Thr Gln Val 115 120 125 tat ggc ttt tat gat gaa tgc
cta cga aag tat ggg aac gcc aac gtg 735 Tyr Gly Phe Tyr Asp Glu Cys
Leu Arg Lys Tyr Gly Asn Ala Asn Val 130 135 140 tgg aaa tac ttt aca
gat ctc ttt gat tat ctt cca ctt aca gct tta 783 Trp Lys Tyr Phe Thr
Asp Leu Phe Asp Tyr Leu Pro Leu Thr Ala Leu 145 150 155 gta gat gga
cag ata ttc tgc ctc cac ggt ggc ctc tct cca tcc ata 831 Val Asp Gly
Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro Ser Ile 160 165 170 gat
aca ctg gat cac ata aga gcc ctg gat cgc tta cag gaa gtt cca 879 Asp
Thr Leu Asp His Ile Arg Ala Leu Asp Arg Leu Gln Glu Val Pro 175 180
185 190 cat gag ggc cca atg tgt gat ctc tta tgg tca gat cca gat gac
cgt 927 His Glu Gly Pro Met Cys Asp Leu Leu Trp Ser Asp Pro Asp Asp
Arg 195 200 205 ggt ggc tgg ggc att tct cca cgt ggt gct ggc tac aca
ttt gga caa 975 Gly Gly Trp Gly Ile Ser Pro Arg Gly Ala Gly Tyr Thr
Phe Gly Gln 210 215 220 gac att tct gaa aca ttt aac cat gcc aac ggc
ctc aca ctg gtg tcc 1023 Asp Ile Ser Glu Thr Phe Asn His Ala Asn
Gly Leu Thr Leu Val Ser 225 230 235 cgt gct cac cag ctt gta atg gaa
gga tat aat tgg tgc cat gat cgg 1071 Arg Ala His Gln Leu Val Met
Glu Gly Tyr Asn Trp Cys His Asp Arg 240 245 250 aat gtg gtc acc att
ttt agt gca ccc aat tac tgc tac cgc tgt ggg 1119 Asn Val Val Thr
Ile Phe Ser Ala Pro Asn Tyr Cys Tyr Arg Cys Gly 255 260 265 270 aac
cag gct gct atc atg gaa tta gac gac act tta aaa tac tct ttt 1167
Asn Gln Ala Ala Ile Met Glu Leu Asp Asp Thr Leu Lys Tyr Ser Phe 275
280 285 ctt cag ttt gac cca gca cct cgt cgt gga gag cct cat gtg acc
cgg 1215 Leu Gln Phe Asp Pro Ala Pro Arg Arg Gly Glu Pro His Val
Thr Arg 290 295 300 cgc acc cca gac tac ttc cta taa attcctcccc
aggacctgtc tttgtatgtt 1269 Arg Thr Pro Asp Tyr Phe Leu 305 310
gaagtatacc tggcttttta aaaaatatat atacatatat atatttaaaa acaacagtta
1329 tctgtgtgtc tctgtaacaa attgtgctat gtcttgacgt taaaacacat
catggaccaa 1389 aacgtgccat actaatggtg agccatcagc acggtgtgaa
cttgagtcca ctgtcctagc 1449 cgagtcaacc aggcagccgc ctgcccgcct
gcctgctgta gtagccgtcc ttcgtgactg 1509 gttaagggaa agggtcactg
gtggcttcat ctcctttgcg cttacttgga aatttagtta 1569 caagtttaac
tggcatggat tatagagttg gagttttatt tttaagaatt gacaagctga 1629
cttccactta aattcataac cctttatttt gttgaaatgt atgactaact gaagaagaga
1689 ttcttggagt atgttgtcat aacactaaga tttcctttca agtttcctga
actgaattac 1749 tgttggatgt tgacctgcac attctgtata tttgtcctga
cagtgttgca tcctccttgc 1809 tgtactgaac aaataaactt cccaatttag agag
1843 11 23 DNA Artificial Sequence PCR Primer 11 ggaccaaaac
gtgccatact aat 23 12 22 DNA Artificial Sequence PCR Primer 12
tggttgactc ggctaggaca gt 22 13 27 DNA Artificial Sequence PCR Probe
13 actcaagttc acaccgtgct gatggct 27 14 23 DNA Artificial Sequence
PCR Primer 14 tgttctagag acagccgcat ctt 23 15 21 DNA Artificial
Sequence PCR Primer 15 caccgacctt caccatcttg t 21 16 24 DNA
Artificial Sequence PCR Probe 16 ttgtgcagtg ccagcctcgt ctca 24 17
20 DNA Artificial Sequence Antisense Oligonucleotide 17 agcaggcaaa
ctgttagact 20 18 20 DNA Artificial Sequence Antisense
Oligonucleotide 18 cctccgggtt tccgtcatcg 20 19 20 DNA Artificial
Sequence Antisense Oligonucleotide 19 ctctcccctc ctccgggttt 20 20
20 DNA Artificial Sequence Antisense Oligonucleotide 20 gacttgtctt
tccccttctc 20 21 20 DNA Artificial Sequence Antisense
Oligonucleotide 21 gcccgcctca cgcctaccgg 20 22 20 DNA Artificial
Sequence Antisense Oligonucleotide 22 ccccgcccgg ccgcgcgccg 20 23
20 DNA Artificial Sequence Antisense Oligonucleotide 23 ggaggagacc
cccgcccgcc 20 24 20 DNA Artificial Sequence Antisense
Oligonucleotide 24 gcgccccgcc caagcccagc 20 25 20 DNA Artificial
Sequence Antisense Oligonucleotide 25 ccgtcgccag gtcccacagg 20 26
20 DNA Artificial Sequence Antisense Oligonucleotide 26 gccgctcccc
tctccctccg 20 27 20 DNA Artificial Sequence Antisense
Oligonucleotide 27 gggcgccgct cccctctccc 20 28 20 DNA Artificial
Sequence Antisense Oligonucleotide 28 cgggcgccgc tcccctctcc 20 29
20 DNA Artificial Sequence Antisense Oligonucleotide 29 ccccggcccc
ggcccgggcg 20 30 20 DNA Artificial Sequence Antisense
Oligonucleotide 30 gcccccggcc ccggcccggg 20 31 20 DNA Artificial
Sequence Antisense Oligonucleotide 31 cccacccgcc cccggccccg 20 32
20 DNA Artificial Sequence Antisense Oligonucleotide 32 gagccccagc
ccggccgccg 20 33 20 DNA Artificial Sequence Antisense
Oligonucleotide 33 cttgtcgtcc atggcggccc 20 34 20 DNA Artificial
Sequence Antisense Oligonucleotide 34 gccttgtcgt ccatggcggc 20 35
20 DNA Artificial Sequence Antisense Oligonucleotide 35 ttcagctgct
tacactcgtt 20 36 20 DNA Artificial Sequence Antisense
Oligonucleotide 36 tctcgttcag ctgcttacac 20 37 20 DNA Artificial
Sequence Antisense Oligonucleotide 37 ttggttctcg ttcagctgct 20 38
20 DNA Artificial Sequence Antisense Oligonucleotide 38 cgcacttggt
tctcgttcag 20 39 20 DNA Artificial Sequence Antisense
Oligonucleotide 39 gcgtccgcac ttggttctcg 20 40 20 DNA Artificial
Sequence Antisense Oligonucleotide 40 gcacagcgtc cgcacttggt 20 41
20 DNA Artificial Sequence Antisense Oligonucleotide 41 ttctcgcaca
gcgtccgcac 20 42 20 DNA Artificial Sequence Antisense
Oligonucleotide 42 tcttttgtta aaatttcctt 20 43 20 DNA Artificial
Sequence Antisense Oligonucleotide 43 tacatagtca cccatgaata 20 44
20 DNA Artificial Sequence Antisense Oligonucleotide 44 aatgctacaa
gaagagtcac 20 45 20 DNA Artificial Sequence Antisense
Oligonucleotide 45 cctttaatgc tacaagaaga 20 46 20 DNA Artificial
Sequence Antisense Oligonucleotide 46 tcataaaagc catatacttg 20 47
20 DNA Artificial Sequence Antisense Oligonucleotide 47 tggaagataa
tcaaagagat 20 48 20 DNA Artificial Sequence Antisense
Oligonucleotide 48 gtaagtggaa gataatcaaa 20 49 20 DNA Artificial
Sequence Antisense Oligonucleotide 49 aagctgtaag tggaagataa 20 50
20 DNA Artificial Sequence Antisense Oligonucleotide 50 tactaaagct
gtaagtggaa 20 51 20 DNA Artificial Sequence Antisense
Oligonucleotide 51 ccatctacta aagctgtaag 20 52 20 DNA Artificial
Sequence Antisense Oligonucleotide 52 tctgtccatc tactaaagct 20 53
20 DNA Artificial Sequence Antisense Oligonucleotide 53 gaatatctgt
ccatctacta 20 54 20 DNA Artificial Sequence Antisense
Oligonucleotide 54 aggcagaata tctgtccatc 20 55 20 DNA Artificial
Sequence Antisense Oligonucleotide 55 atggatggag agaggccacc 20 56
20 DNA Artificial Sequence Antisense Oligonucleotide 56 tcatgtggaa
cttcctgtaa
20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57
gatcacacat tgggccctca 20 58 20 DNA Artificial Sequence Antisense
Oligonucleotide 58 gtgtagccag caccacgtgg 20 59 20 DNA Artificial
Sequence Antisense Oligonucleotide 59 caaatgtgta gccagcacca 20 60
20 DNA Artificial Sequence Antisense Oligonucleotide 60 ttgtccaaat
gtgtagccag 20 61 20 DNA Artificial Sequence Antisense
Oligonucleotide 61 atgtcttgtc caaatgtgta 20 62 20 DNA Artificial
Sequence Antisense Oligonucleotide 62 cagaaatgtc ttgtccaaat 20 63
20 DNA Artificial Sequence Antisense Oligonucleotide 63 atagcagcct
ggttcccaca 20 64 20 DNA Artificial Sequence Antisense
Oligonucleotide 64 ccatgatagc agcctggttc 20 65 20 DNA Artificial
Sequence Antisense Oligonucleotide 65 taattccatg atagcagcct 20 66
20 DNA Artificial Sequence Antisense Oligonucleotide 66 atttatagga
agtagtctgg 20 67 20 DNA Artificial Sequence Antisense
Oligonucleotide 67 ggagaaattt ataggaagta 20 68 20 DNA Artificial
Sequence Antisense Oligonucleotide 68 taaaaagcca ggtatacttc 20 69
20 DNA Artificial Sequence Antisense Oligonucleotide 69 tttgttttta
aatacatata 20 70 20 DNA Artificial Sequence Antisense
Oligonucleotide 70 gctcatcatt agtatggcac 20 71 20 DNA Artificial
Sequence Antisense Oligonucleotide 71 ctagaacata gtgtactaaa 20 72
20 DNA Artificial Sequence Antisense Oligonucleotide 72 ggcaaactgt
tagactgacc 20 73 20 DNA Artificial Sequence Antisense
Oligonucleotide 73 tccaaaggaa aatggttact 20 74 20 DNA Artificial
Sequence Antisense Oligonucleotide 74 ggagatgaag cagttagtta 20 75
20 DNA Artificial Sequence Antisense Oligonucleotide 75 ctattaatcc
atgccagtta 20 76 20 DNA Artificial Sequence Antisense
Oligonucleotide 76 tctattaatc catgccagtt 20 77 20 DNA Artificial
Sequence Antisense Oligonucleotide 77 actctattaa tccatgccag 20 78
20 DNA Artificial Sequence Antisense Oligonucleotide 78 caactctatt
aatccatgcc 20 79 20 DNA Artificial Sequence Antisense
Oligonucleotide 79 aaataaaact ccaactctat 20 80 20 DNA Artificial
Sequence Antisense Oligonucleotide 80 aatttttctt aaaaataaaa 20 81
20 DNA Artificial Sequence Antisense Oligonucleotide 81 gttagcttgt
gaatttttct 20 82 20 DNA Artificial Sequence Antisense
Oligonucleotide 82 ttagtggaag ttagcttgtg 20 83 20 DNA Artificial
Sequence Antisense Oligonucleotide 83 aaataaagga taatggatta 20 84
20 DNA Artificial Sequence Antisense Oligonucleotide 84 ttcaataaaa
taaaggataa 20 85 20 DNA Artificial Sequence Antisense
Oligonucleotide 85 acatttcaat aaaataaagg 20 86 20 DNA Artificial
Sequence Antisense Oligonucleotide 86 tcccaagaag aaccttttct 20 87
20 DNA Artificial Sequence Antisense Oligonucleotide 87 tatgacaaca
tactcccaag 20 88 20 DNA Artificial Sequence Antisense
Oligonucleotide 88 atgaagggaa atctctttaa 20 89 20 DNA Artificial
Sequence Antisense Oligonucleotide 89 aatgaaggga aatctcttta 20 90
20 DNA Artificial Sequence Antisense Oligonucleotide 90 tagtttaaat
gaagggaaat 20 91 20 DNA Artificial Sequence Antisense
Oligonucleotide 91 aatatgcaga tcaacataaa 20 92 20 DNA Artificial
Sequence Antisense Oligonucleotide 92 caaataggat gcaagcactg 20 93
20 DNA Artificial Sequence Antisense Oligonucleotide 93 tttgtttaaa
atgaaaagtt 20 94 20 DNA Artificial Sequence Antisense
Oligonucleotide 94 gttctctcgg cgctcgtgcc 20 95 20 DNA Artificial
Sequence Antisense Oligonucleotide 95 tggccgcggt tctctcggcg 20 96
20 DNA Artificial Sequence Antisense Oligonucleotide 96 gctctggccg
cggttctctc 20 97 20 DNA Artificial Sequence Antisense
Oligonucleotide 97 gcctctccgc gctctggccg 20 98 20 DNA Artificial
Sequence Antisense Oligonucleotide 98 cggcccgacg gcgcccgccc 20 99
20 DNA Artificial Sequence Antisense Oligonucleotide 99 gcggcaaaaa
gggctcacgg 20 100 20 DNA Artificial Sequence Antisense
Oligonucleotide 100 cagcgggcag ccgccggccc 20 101 20 DNA Artificial
Sequence Antisense Oligonucleotide 101 agcccagcgg gcagccgccg 20 102
20 DNA Artificial Sequence Antisense Oligonucleotide 102 cgggcagccc
gcgccccgcc 20 103 20 DNA Artificial Sequence Antisense
Oligonucleotide 103 ccccggcggg accaccgagc 20 104 20 DNA Artificial
Sequence Antisense Oligonucleotide 104 cccgccgcct cccccgccgc 20 105
20 DNA Artificial Sequence Antisense Oligonucleotide 105 tccccgccgc
ctcccccgcc 20 106 20 DNA Artificial Sequence Antisense
Oligonucleotide 106 cgcgtccccg ccgcctcccc 20 107 20 DNA Artificial
Sequence Antisense Oligonucleotide 107 cttgtcgtcc atggcggcga 20 108
20 DNA Artificial Sequence Antisense Oligonucleotide 108 gtccatctac
taaagctgta 20 109 20 DNA Artificial Sequence Antisense
Oligonucleotide 109 gggaggaatt tataggaagt 20 110 20 DNA Artificial
Sequence Antisense Oligonucleotide 110 tatatatgta tatatatttt 20 111
20 DNA Artificial Sequence Antisense Oligonucleotide 111 acaatttgtt
acagagacac 20 112 20 DNA Artificial Sequence Antisense
Oligonucleotide 112 tagcacaatt tgttacagag 20 113 20 DNA Artificial
Sequence Antisense Oligonucleotide 113 agacatagca caatttgtta 20 114
20 DNA Artificial Sequence Antisense Oligonucleotide 114 agtatggcac
gttttggtcc 20 115 20 DNA Artificial Sequence Antisense
Oligonucleotide 115 ttcacaccgt gctgatggct 20 116 20 DNA Artificial
Sequence Antisense Oligonucleotide 116 caagttcaca ccgtgctgat 20 117
20 DNA Artificial Sequence Antisense Oligonucleotide 117 gttgactcgg
ctaggacagt 20 118 20 DNA Artificial Sequence Antisense
Oligonucleotide 118 gcggctgcct ggttgactcg 20 119 20 DNA Artificial
Sequence Antisense Oligonucleotide 119 gaaggacggc tactacagca 20 120
20 DNA Artificial Sequence Antisense Oligonucleotide 120 cccttaacca
gtcacgaagg 20 121 20 DNA Artificial Sequence Antisense
Oligonucleotide 121 taagcgcaaa ggagatgaag 20 122 20 DNA Artificial
Sequence Antisense Oligonucleotide 122 ctataatcca tgccagttaa 20 123
20 DNA Artificial Sequence Antisense Oligonucleotide 123 taaaactcca
actctataat 20 124 20 DNA Artificial Sequence Antisense
Oligonucleotide 124 taaaaataaa actccaactc 20 125 20 DNA Artificial
Sequence Antisense Oligonucleotide 125 cagcttgtca attcttaaaa 20 126
20 DNA Artificial Sequence Antisense Oligonucleotide 126 tggaagtcag
cttgtcaatt 20 127 20 DNA Artificial Sequence Antisense
Oligonucleotide 127 acatttcaac aaaataaagg 20 128 20 DNA Artificial
Sequence Antisense Oligonucleotide 128 tcatacattt caacaaaata 20 129
20 DNA Artificial Sequence Antisense Oligonucleotide 129 tcttcttcag
ttagtcatac 20 130 20 DNA Artificial Sequence Antisense
Oligonucleotide 130 aaaggaaatc ttagtgttat 20 131 20 DNA Artificial
Sequence Antisense Oligonucleotide 131 aacttgaaag gaaatcttag 20 132
20 DNA Artificial Sequence Antisense Oligonucleotide 132 ttcagttcag
gaaacttgaa 20 133 20 DNA Artificial Sequence Antisense
Oligonucleotide 133 ggtcaacatc caacagtaat 20 134 20 DNA Artificial
Sequence Antisense Oligonucleotide 134 agaatgtgca ggtcaacatc 20 135
20 DNA Artificial Sequence Antisense Oligonucleotide 135 tcagtacagc
aaggaggatg 20
* * * * *