Note: Descriptions are shown in the official language in which they were submitted.
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ANTISENSE INHIBITION OF ANGIOGENIN EXPRESSION
This application was funded in part by National Institutes of Health /
National Cancer
Institute grant no. RO1 CA60046.
This application claims priority to United States Provisional Application
Serial No.
60/041.182 filed March 21, 1997 hereby incorporated by reference in its
entirety.
1. Field of the Invention
Embodiments of the present invention relate in general to compositions and
methods
for inhibiting the expression of the angiogenin gene thereby reducing the
effects of angiogenin.
Embodiments of the present invention also relate to inhibition of angiogenin
gene expression
by antisense technologies including, but not limited to, the use of antisense
oligodeoxynucieotides and their derivatives. Embodiments of the present
invention are further
directed to compositions and methods for detecting the angiogenin gene, as
well as the
detection and diagnosis of abnormal expression of the angiogenin gene in cells
and tissues.
Embodiments of the present invention are also directed to methods for
inhibiting metastasis
of cells, such as human tumor cells. Furthermore, this invention is directed
to treatment of
conditions associated with abnormal angiogenesis, including cancer.
2. j2~,s~ip~l
Angiogenin is a potent inducer of angiogenesis [Felt, J. W., Strydom, D. J.,
Lobb, R. R.,
Alderman, E. M., Bethune, J. L., Riordan, J .F., and Vallee, B. L. (1985)
Biochemistry 24, 5480-
5486], a complex process of blood vessel formation that consists of several
separate but
interconnected steps at the cellular and biochemical level: (i) activation of
endothelial cells by
the action of an angiogenic stimulus, (ii) adhesion and invasion of activated
endothelial cells
into the surrounding tissues and migration toward the source of the angiogenic
stimulus, and
(iii) proliferation and differentiation of endothelial cells to form a new
microvasculature
1
im
CA 02293591 1999-12-02
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[Folkman, J., and Shing, Y. (1992) J. Biol. Chem. 267,10931-10934; Moscatelli,
D., and Rifkin, Y
D. B. (1988) Biochim. Biophys. Acta 948, 67-85]. Angiogenin has been
demonstrated to induce
most of the individual events in the process of angiogenesis including binding
to endothelial
cells [Badet, J., Soncin, F. Guitton, J. D., Lamare, O., Cartwright, T., and
Barritault, D. (1989)
Proc. Natl. Acad. Sci. U.S.A. 86, 8427-8431], stimulating second messengers
[Bicknell, R., and
Vallee, B. L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5961-5965], mediating
cell adhesion
[Soncin, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2232-2236], activating
cell-associated
proteases [Hu, G-F., and Riordan, J. F. (1993) Biochem. Biophys. Res.
Commun.197, 682-687],
inducing cell invasion (Hu, G-F., Riordan, J. F., and Vallee, B. L. (1994)
Proc. Natl. Acad. Sci.
U.S.A. 91, 12096-12100], inducing proliferation of endothelial cells [Hu, G-
F., Riordan, J. F.,
and Vallee, B. L. (1997) Proc. Natl. Acad Sci. U.SA. 94, 2204-2209] and
organizing the
formation of tubular structures from the cultured endothelial cells [Jimi, S-
L, Ito, K-I, Kohno,
K., Ono, M., Kuwano, M., Itagaki, Y., and Isikawa, H. (1995) Biochem. Biophys.
Res. Commun.
211, 476-483]. Angiogenin has also been shown to undergo nuclear translocation
in
endothelial cells via receptor-mediated endocytosis [Moroianu, J., and
Riordan, J. F. (1994)
Proc. Natl. Acad. Sci. U.SA. 91, 1677-1681] and nuclear localization sequence-
assisted nuclear
import [Moroianu, J., and Riordan, J. F. ( 1994) Biochem. Biophys. Res.
Commun. 203, 1765-
1772] .
Although originally isolated from medium conditioned by human colon cancer
cells
(Fett et al., 1985, ~yg~ and subsequently shown to be produced by several
other histologic
types of human tumors [Rybak, S. M., Fett, J. W., Yao, Q-Z., and Vallee, B. L.
(1987) Biochem.
Biophys. Res. Commun. 146, 1240-1248; Olsan, K. A., Fett, J. W., French, T.
C., Key, M. E.,
and Vallee, B. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 442-446],
angiogenin also is a
constituent of human plasma and normally circulates at a concentration of 250
to 360 ng/ml
[Shimoyama, S., Gansauge, F., Gansauge, S., Negri, G., Oohara, T., and Beger,
H. G. (1996)
Cancer Res. 56, 2703-2706; Blfiser, J., Triebl, S., Kopp, C., and Tschesche,
H. (1993) Eur. J.
Clin. Chem. Clin. Biochem. 31, 513-516].
While angiogenesis is a tightly controlled process under usual physiological
conditions,
abnormal angiogenesis can have devastating consequences as in pathological
conditions such
as arthritis, diabetic retinopathy and tumor growth. It is now well-
established that the growth
of virtually all solid tumors is angiogenesis dependent [Folkman, J. ( 1989)
J. Natl. Cancer Inst.
2
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82, 4-6]. Angiogenesis is also a prd'requisit~ fdi the development of
metastasis since it V.
provides the means whereby tumor cells disseminate from the original primary
tumor and
establish at distant sites [Mahadevan, V., and Hart, I. R. (1990) Rev. C~ncol.
3, 97-103; Blood
C. H., and Zetter B. R. (1990) Biochim. Biophys. Acta 1032, 89-118].
Therefore, interference
with the process of tumor-induced angiogenesis should be an effective therapy
for both
primary and metastatic cancers.
Several inhibitors of the functions of angiogenin have been developed. These
include:
(i) monoclonal antibodies (mAbs) [Felt, J. W., Olson, K. A., and Rybak, S. M.
(1994)
Biochemistry 33, 5421-5427], (ii) an angiogenin-binding protein (Hu, G-F,
Chang, S-I, Riordan
J. F., and Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A.. 88, 2227-2231;
Hu, G-F., Strydom,
D. J., Fett, J. W., Riordan, J. F., and Vallee B. L. (1993) Proc. Natl. Acad.
Sci. U.S.A. 90, 1217-
1221; Moroianu, J., Fett, J. W., Riordan, J. F., and Vallee B. L. (1993) Proc.
Natl. Acad. Sci.
U.S.A. 90, 3815-3819], (iii) the placental ribonuclease inhibitor (PRI)
[Shapiro, R., and Vallee,
B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2238-2241], (iv) peptides
synthesized based on
the C-terminal sequence of angiogenin [Rybak, S. M., Auld, D. S., St. Clair,
D. K., Yao, Q-Z.,
and Fett, J. W. (1989) Biochem. Biaphys. Res. Commun.162, 535-543], and (v)
inhibitory site-
directed mutants of angiogenin [Shapiro, R., and Vallee, B. L. (1989)
Biochemistry 28, 7401-
7408]. All inhibit angiogenin's activities but are not directly cytotoxic to
human tumor cells
grown in tissue culture.
mAbs or the angiogenin-binding protein when administered locally into
xenografts of
human tumor cells that were injected subcutaneously (s.c.) into athymic mice
are able to delay
or, remarkedly, completely prevent the appearance of colon, lung and
fibrosarcoma tumors
in these animals [Olson et al., 1995, , Olson, K. A., French, T. C., Vallee,
B. L., and Fett,
J. W. (1994) Cancer Res. 54, 4576-4579]. Histological examination revealed
that the
mechanism of tumor growth inhibition was via an anti-angiogenesis mechanism
(Olson et al. ,
1995, ). Thus, the inactivation of tumor-produced angiogenin or inhibition of
expression
of the angiogenin gene by tumor cells promise to be a powerful means of
managing cancer,
either alone or in combination with more conventional therapies (i.e.,
chemotherapy,
radiotherapy, immunotherapy, etc.).
Expression of specific genes may be suppressed by oligonucleotides having a
nucleotide
sequence complementary to the mRNA transcript of the target gene thereby
selectively
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impeding translation and has been termed an "antisense" methodology. In
addition, ~~
"antigene" or "triplex" methodologies may also suppress expression of genies
by using an
oligonucieotide which is complementary to a selected site of double stranded
DNA thereby
forming a triple-stranded complex to selectively inhibit transcription of the
gene. Both
"antisense" and "antigene" methodologies fend utility as molecular tools for
genetic analysis.
Antisense oligonucleotides have been extensively used to inhibit gene
expression in normal and
abnormal cells in studies of the.function of various cell proteins. Major
advances have been
made in the development of antisense or antigene reagents for the treatment of
disease states
in animals and humans ["Antisense Therapeutics" Agrawal, S. (ed.), Humana
Press, 1996;
Crooke, S. T., and Bennett, C. F. (1996) Annu. Rev. Pharmacol. Toxicol. 36,107-
129; "Prospects
for the Therapeutic Use of Antigene Oligonucleotides", Maher, L.J. ( 1996)
Cancer Investigation
14(1), 66-82 each hereby incorporated by reference in its entirety].
As therapeutics, oligonucleotides possess two major requirements for
successful drug
design - specificity and affinity. These are achieved by selectively targeting
particular DNA
or RNA sequences exploiting Watson-Crick base pairing with resulting
interference of protein
production whether through inhibition of gene transcription or translation of
mRNA. This
approach allows for rapid identification of lead compounds based on knowledge
of a relevant
gene target species. Recently, improvements have been made in increasing both
the stability
and affinity of these compounds. Phosphorothioate analogs of
oligodeoxynucleotides (ODNs),
in which nonbridging phosphoryl oxygens in the backbone of DNA are substituted
with sulfur,
abbreviated [S]ODNs, are substantially more stable than their native
phosphodiester
counterparts, while other derivatives, such as those alkylated on sugar oxygen
groups, show
enhanced target affinity. [S]ODNs possess good biological activity,
pharmacology,
pharmacokinetics and safety in vivo (Agrawal, 1996, , and references therein)
and have
been used successfully for anti-tumor therapy in animal models (Crooke and
Bennett, 1996,
Antisense reagents are now in clinical trials for treatment of cancers and
viral
infections (Agrawal, 1996, . Successful inhibition of specific gene function
has been
achieved by targeting various sites on specific mRNA sequences that include
the AUG
translational initiation codon, 5'-transcriptional start site, 3'-termination
codon and sequences
in both the 5'- and 3'-untranslated regions. Experience to date has indicated
that success has
been achieved by targeting these and other regions.
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As examples, U.S. Pat. N~. 5,0~~i~ ditected to antisense oligonucleotides Y
complementary to the c-myb oncogene and antisense oligonucleotide therapies
for certain
cancerous conditions. U.S. Pat. No. 5,135,917 provides antisense
oligonucleotides that inhibit
human interleukin-1 receptor expression. U.S. Pat. No. 5,087,617 provides
methods for treating
cancer patients with antisense oligonucleotides. U.S. Pat. No. 5,166.195
provides
oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810 provides oligomers
capable of
hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S
Pat. No.
5,194,428 provides antisense oligonucleotides having antiviral activity
against influenzavirus.
U.S. Pat. No. 4,806,463 provides antisense oligonucleotides and methods using
them to inhibit
HTLV-III replication. U.S. Pat. No. 5,286,717 is directed to a mixed linkage
oligonucleotide
phosphorothioates complementary to an oncogene. U.S. Pat. No. 5,276,019 and
U.S. Pat. No.
5,264,423 are directed to phosphorothioate oligonucleotide analogs used to
prevent replication
of foreign nucleic acids in cells.
The nucleic acid sequence of the entire angiogenin gene including the 5'- and
3'-flanking
regions has been determined [Karachi, K., Davie, E. W., Strydom, D. J.
Riordan, J. F. and
Vallee, B. L. (1985) Biochemistry 24, 5494-5499 hereby incorporated by
reference in its
entirety]. The native DNA segment coding for angiogenin, as all such mammalian
DNA strands,
has two strands; a sense strand and an antisense strand held together by
hydrogen bonding. The
messenger RNA coding for angiogenin has the same nucleotide sequence as the
sense strand
except that the DNA thymidine is replaced by uridine. Thus, synthetic
antisense nucleotide
sequences should bind with the DNA and RNA coding for angiogenin.
However, it is unknown whether antisense reagents will in fact be effective
for
inhibition of angiogenin expression. To date, no oligotrucleotide antisense
reagents have been
desig~d or demonstrated to be useful in the inhibition of the expression of
angiogenin.
Accordingly, a need exists to discover oligonucleotide antisense reagents
which can prove
useful in modulating or inhibiting the expression of angiogenin and to further
discover methods
by which such oligonucleotide antisense reagents can be used in methods of
diagnosis and
- treatment.
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Embodiments of the present invention are based on the discovery of
oligonucleotide
reagents capable of targeting nucleic acid sequences encoding angiogenin in a
manner to inhibit
(i.e., reduce, eliminate or otherwise interfere with) the expression of
angiogenin. Each
oligonucleotide, or analog thereof, has a nucleotide or base sequence which is
complementary,
i.e. capable of hybridizing with or binding to, at least a target portion of
the nucleic acid
encoding angiogenin, i.e. the angiogenin gene DNA or RNA, which has
significance in
expressing angiogenin. In accordance with one aspect of the present invention,
targeted RNA
or DNA, or cells containing it are contacted with oligonucleodde or analogs
thereof which are
configured to bind to the RNA or DNA in a manner to inhibit the expression of
angiogenin
whether by interfering with gene transcription as in an antigene strategy or
by interfering with
translation of mRNA as in an antisense strategy.
Embodiments of the present invention are further directed to methods for
inhibiting the
expression of angiogenin in a mammal by administering to or otherwise treating
the mammal
with an effective amount of an oligonucleotide or analog thereof having a base
sequence
complementary to a target portion of a nucleic acid encoding angiogenin so as
to inhibit the
expression of angiogenin. Embodiments of the present invention are also
directed to methods
for reducing size of tumors associated with angiogenesis in a mammal
comprising administering
to the mammal an effective amount of an oligonucleotide or analog thereof
having a base
sequence complementary to a target portion of a nucleic acid encoding
angiogenin so as to reduce
tumor size. Embodiments of the present invention are further directed to
methods for decreasing
production of angiogenin in a mammal comprising administering to the mammal an
effective
amount of an oligonucleotide or analog thereof having a base sequence
complementary to a
target portion of a nucleic acid encoding angiogenin so as to decrease
production of
angiogenin. Embodiments of the present invention are still further directed to
methods for
inhibiting metastasis of tumor cells in a mammal comprising administering to
the mammal an
effective amount of an oligonucleotide or analog thereof having a base
sequence
complementary to a target portion of a nucleic acid encoding angiogenin so as
to inhibit
metastasis of tumor cells. Embodiments of the present invention are even still
further directed
to methods for inhibiting the establishment of tumor cells in a mammal
comprising
administering to the mammal an effective amount of an oligonucleotide or
analog thereof
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angiogenin so as to inhibit establishment of tumor cells. Embodiments of tt~
present invention
are even still further directed to methods for inhibiting growth of tumors
associated with
angiogenesis in a mammal comprising administering to the mammal an effective
amount of an
oligonucleotide or analog thereof having a base sequence complementary to a
target portion
of a nucleic acid encoding angiogenin so as to inhibit tumor growth. The
oligonucleotides,
analogs thereof and methods described herein are therefore useful in methods
of therapeutically
treating a mammal, including a human, afflicted with pathological conditions
associated with
abnormal or unwanted angiogenesis, including cancer.
As an alternate embodiment of the present invention, labeled oligonucleotides
may also
be useful for diagnosing conditions associated with abnormal angiogenin
expression since the
labeled oligonucleotides of the present invention can also bind to the
angiogenin gene, DNA or
RNA and then can be detected and/or measured. Alternate embodiments of the
present invention
include methods detecting the presence of angiogenin in a sample comprising
contacting the
sample with a labeled oligonucleotide or analog thereof having a base sequence
complementary
to a target portion of a nucleic acid encoding angiogenin, allowing the
labeled oligonucleotide
or analog thereof to bind to the target portion of the nucleic acid encoding
angiogenin, and
detecting the labeled oligonucleotide or analog thereof. A further alternate
embodiment of the
present invention includes methods fox detecting the presence of angiogenin in
a mammal
comprising administering to the mammal a labeled oligonucleotide or analog
thereof having
a base sequence complementary to a target portion of a nucleic acid encoding
angiogenin,
allowing the labeled oligonucleotide or analog thereof to bind to the target
portion of the
nucleic acid encoding angiogenin, and detecting the labeled oligonucleotide or
analog thereof.
A still further alternate embodiment of the present invention includes methods
for diagnosing
conditions associated with abnormal angiogenesis in a mammal comprising
administering to
the mammal a labeled oligonucleodde or analog thereof having a base sequence
complementary
to a target portion of a nucleic acid encoding angiogenin, allowing the
labeled oligonucleotide
or analog thereof to bind to the target portion of the nucleic acid encoding
angiogenin,
detecting the labeled oligonucleotide or analog thereof, measuring the labeled
oligonucleotide
or analog thereof, and determining the abnormal cotxlition based on the
detecting and
measuring of the labeled oligonucleotide or analog thereof.
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These and other objects, features and advantages of the present invention will
become Y
apparent by reference to the remaining portions of the specification and the
attached drawings.
In the course of the detailed description of certain preferred embodiments to
follow,
reference will be made to the attached drawings, in which,
Fig. 1 depicts the nucleic acid sequence of the entire human angiogenin gene
including
the cDNA sequence as identified by arrows.
Fig. 2 is a graph depicting the inhibition by angiogenin antisense [S]ODN JF2S
of
angiogenin expression by PC-3 tumor cells in vitro and their subsequent growth
in vivo.
Fig. 3 is a graph depicting the inhibition by angiogenin antisense [S]ODN JF2S
of
angiogenin expression by HT-29 tumor cells in vitro and their subsequent
growth in vivo.
Fig. 4 is a graph showing treatment of HT-29 tumor cells in vitro with
antisense [S]ODN
JF2S and control sense [S]ODN JFI S and their subsequent growth in vivo.
Fig. 5 is a graph showing treatment of PC-3 tumor cells in vitro with
antisense [S]ODN
JF2S and control sense [S]ODN JF1 S and their subsequent growth in vivo.
Fig. 6 is a graph showing treatment of MDA-MB-435 tumor cells in vitro with
antisense
[S]ODN JF2S and control sense [S] ODN JF 1 S and their subsequent growth in
vivo.
Fig. 7 is a graph showing treatment of PC-3M tumor cells in vitro with
antisense [S]ODN
JF2S and control sense [S]ODN JF1 S and their subsequent growth in vivo.
Fig. 8 is a photograph showing the differences in the presence and size of
angiogenin
antisense [S]ODN (JF2S) and control (lipofectin)-treated PC-3 tumors excised
from athymic
mice.
Fig. 9 is a photograph showing the differences in the presence and size of
angiogenin
antisense [S]ODN (JF2S) and control (lipofectin}-treated HT-29 tumors excised
from athymic
mice.
Fig. 10 is a photograph showing the differences in the presence and size of
angiogenin
antisense [S]ODN (JF2S), control sense [S]ODN (JF1S) and control (lipofectin)-
treated HT-29
tumors excised from athymic mice.
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Fig. 11 is a photograph showing the differences in the presence and size of
angiogenin
antisense [SJODN (JF2S), control sense [SjODN (JF1S) and control (lipofectin)-
treated PC-3
tumors excised from athymic mice.
Fig. 12 is photograph showing the differences in the presence and size of
angiogenin
antisense [SJODN (JF2S), control sense [S]ODN (JF1 S) and control (lipofectin)-
treated MDA-
MB-435 tumors excised from athymic mice.
Fig. 13 is a photograph showing the differences in the presence and size of
angiogenin
antisense [S]ODN (JF2S), control sense [SJODN (JF1S) and control (lipofectin)-
treated PC-3M
tumors excised from athynuc mice.
Fig. 14 is a graph showing inhibition of the expression of angiogenin by PC-3
and PC-3M
tumor cell lines in culture using the two angiogenin antisense [SJODNs, 3F2S
and JF4S.
Fig. 15 is a graph showing in vivo therapy of PC-3 tumors with angiogenin
antisense
[S]ODN JF2S, control sense [SJODN JF1S and PBS diluent control in three
separate
experiments.
Fig. 16 is a graph showing in vivo therapy of MDA-MB-435 tumors with
angiogenin
antisense [SJODN JF2S, control sense [SJODN JF1S and PBS diluent control.
Fig. 17 is a graph showing in vivo therapy of MCF-7 tumors with angiogenin
antisense
[SJODN JF2S, control sense [SJODN JF1S and PBS diluent control.
nFTAtI.FD DE~CR1PTION OF CERTAIN PREFERRED EMBODIMENTS
The principles of the present invention may be advantageously applied to
produce novel
oligonucleotides or analogs thereof which bind to or otherwise target nucleic
acids encoding
angiogenin. The oligonucleotides or analogs thereof interfere with the normal
function of the
nucleic acids and otherwise inhibit the transcription, replication or
translation associated with the
expression of angiogenin.
Angiogenesis is prominent in solid tumor formation and metastasis. Angiogenic
factors
have been found associated with several solid tumors such as
rhabdomyosarcomas,
retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma. A tumor cannot
expand
without a blood supply to provide nutrients and remove cellular wastes. Tumors
in which
angiogenesis is important include solid tumors, and benign tumors such as
acoustic neuroma,
neurofibroma, trachoma and pyogenic granulomas. The present invention is
directed towards
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prevention of angiogenesis in the treatment of these and other angiogenesis
dependent tumors ~.
and the resultant damage to the mammal due to the presence of the tumor.
Angiogenesis is also associated with blood-born tumors such as leukemias, any
of various
acute or chronic neoplastic diseases of the bone marrow in which unrestrained
proliferation of
white blood cells occurs, usually accompanied by anemia, impaired blood
clotting, and
enlargement of the lymph nodes, liver, and spleen. It is believed that
angiogenesis plays a role
in the abnormalities in the bone marrow that gives rise to leukemia-like
tumors.
Angiogenesis is important in two stages of tumor metastasis. The first stage
where
angiogenesis stimulation is important is in the vascularization of the tumor
which allows cells
to enter the blood stream and to circulate throughout the body. After the
tumor cells have left
the primary site, and have settled into the secondary, metastasis site,
angiogenesis must occur
before the new tumor can grow and expand. Therefore, embodiments of the
present invention
are directed to the inhibition of angiogenesis as a treatment for the
prevention of metastasis of
tumors and containment of the neopiastic growth at the primary site.
Examples of diseases mediated by angiogenesis are disclosed in the prior art
such as
U.S.Patent No. 5,712,291 and include ocular neovascular disease as well as the
other diseases
to follow. Ocular neovascular disease is characterized by invasion of new
blood vessels into the
structure of the eye such as the retina or cornea. It is the most common cause
of blindness and
is involved in approximately twenty eye diseases. In age-related macular
degeneration, the
associated visual problems are caused by an ingrowth of choroidal capillaries
through defects in
Bruch's membrane with proliferation of fibrovascular tissue beneath the
retinal pigment
epithelium. Angiogenic damage is also associated with diabetic retinopathy,
retinopathy of
prematurity, corneal graft rejection, neovascular glaucoma and retrolental
fibroplasia. Other
diseases associated with corneal neovascularization include, but are not
limited to, epidemic
keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic
keratitis, superior
limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea,
phylectenulosis, syphilis,
mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers,
fungal ulcers,
Herpes simples infections, Herpes zoster infections, protozoan infections,
Kaposi sarcoma,
Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis,
rheumatoid arthritis,
systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, Scleritis,
Steven Johnson's disease,
periphigoid radical keratotomy, and corneal graph rejection.
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Diseases associated with retinal/choroidal neovascularization include, but are
not limited
to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid,
syphilis,
pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion,
carotid obstructive
disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,
systemic lupus
erythematosis, retinopathy of prematurity, Eales disease, Bechets disease,
infections causing a
retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease,
myopia, optic pits,
Stargarts disease, pans planitis, chronic retinal detachment, hyperviscosity
syndromes,
toxoplasmosis, trauma and post-laser complications. Other diseases include,
but are not limited
to, diseases associated with rubeosis (neovascularization of the angle) and
diseases caused by the
abnormal proliferation of fibrovascular or fibrous tissue including all forms
of proliferative
vitreoretinopathy.
Another disease in which angiogenesis is believed to be involved is rheumatoid
arthritis.
The blood vessels in the synovial lining of the joints undergo angiogenesis.
In addition to
forming new vascular networks, the endothelial cells release factors and
reactive oxygen species
that lead to pannus growth and cartilage destruction. The factors involved in
angiogenesis may
actively contribute to, and help maintain, the chronically inflamed state of
rheumatoid arthritis.
Factors associated with angiogenesis may also have a role in osteoarthritis.
The
activation of the chondrocytes by angiogenic-related factors contributes to
the destruction of the
joint. At a later stage, the angiogenic factors would promote new bone
formation. Therapeutic
intervention that prevents the bone destruction could halt the progress of the
disease and provide
relief for persons suffering from arthritis.
Chronic inflammation may also involve pathological angiogenesis. Such disease
states
as ulcerative colitis and Crohn's disease show histological changes with the
ingrowth of new
blood vessels into the inflamed tissues. Bartonellosis, a bacterial infection
found in South
America, can result in a chronic stage that is characterized by proliferation
of vascular
endothelial cells. Another pathological role associated with angiogenesis is
found in
atherosclerosis. The plaques formed with the lumen of blood vessels have been
shown to have
angiogenic stimulatory activity.
One of the most frequent angiogenic diseases of childhood is the hemangioma.
In most
cases, the tumors are benign and regress without intervention. In more severe
cases, the tumors
progress to large cavernous and infiltrative forms and create clinical
complications. Systemic
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forms of hemangiomas, the hemangiomatoses, have a high mortality rate. Therapy-
resistant 4~~
hemangiomas exist that cannot be treated with therapeutics currently in use.
Angiogenesis is also responsible for damage found in hereditary diseases such
as Osler-
Weber-Rendu disease, or hereditary hemorrhagic telangiectasia. This is an
inherited disease
characterized by multiple small angiomas, tumor of blood or lymph vessels. The
angiomas are
found in the skin and mucous membranes, often accompanied by epistaxis
(nosebleeds) or
gastrointestinal bleeding sometimes with pulmonary or hepatic arteriovenous
fistula.
Embodiments of the present invention further include treatment of the above
disease
states through the inhibition of angiogenesis
The relationship between an oligonucleotide and its complementary nucleic acid
target
to which it hybridizes is commonly referred to as "antisense" if the
complementary nucleic acid
target is single stranded or "antigene" or "triplex" if the complementary
nucleic acid target is
double stranded. It is to be understood that the oligonucleotides and methods
of the present
invention described herein are useful in both antisense or antigene
approaches. Accordingly,
those terms are used interchangeably herein.
In accordance with the teachings of the present invention, the oligonucleotide
employed
in the methods of the present invention will generally have a sequence that is
complementary to
the sequence of the target nucleic acid whether that be in the fonm of single
stranded RNA or
DNA or double stranded DNA. "Targeting" an oligonucleotide to a nucleic acid
sequence of the
angiogenin gene includes determining a site or sites within the nucleic acid
sequence for the
oligonucleotide interaction to occur such that the inhibition of the
expression of angiogenin will
result. "Inhibition of the expression of angiogenin" is herein defined as that
phrase is normally
understood and to also include the elimination of, prevention of, reduction of
or other
interference with the expression of angiogenin occurring prior to or in the
absence of the
interaction between the oligonucleotide and the nucleic acid sequence of the
angiogenin gene.
"Inhibition" itself is herein defined as the elimination of, prevention of,
reduction of or other
interference with the particular mechanism being interfered with. Once the
desired target site or
sites have been identified anywhere along the entire nucleic acid sequence of
the angiogenin
gene, one or more oligonucleotides are chosen which are sufficiently
complementary to the
target, i.e. hybridize sufficiently well and with sufficient specificity, to
inhibit the expression of
angiogenin.
12
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The terms "hybridization" or "to bind" as used herein means hydrogen bonding,
also
known as Watson-Crick base pairing, between complementary bases (i.e. purines
or
pyrimidines), usually on opposite nucleic acid strands or two regions of a
nucleic acid strand.
Guanine and cytosine are examples of complementary bases which are known to
form three
hydrogen bonds between them. Adenine and thymine are examples of complementary
bases
which form two hydrogen bonds between them.
The letters A, G; C, T, and U respectively indicate nucleotides in which the
nucleoside
is adenosine, guanosine, cytidine, thymidine, and uridine. As used herein,
oligonucleotides that
are antisense to the target angiogenin nucleic acid sense strand are
oligonucleotides which have
a nucleoside sequence complementary to the sense strand. Table 1 shows the
four possible sense
strand bases and their complements present in an antisense compound.
Table 1
Sense
Adenine Thymine
Guanine Cytosine
Cytosine Guanine
Thymine Adenine
"Specifically hybridizable" and "complementary" are terms which are used to
indicate
a sufficient degree of complementarity such that stable and specific binding
occurs between the
target nucleic acid and the oligonucleotide. It is to be understood that an
oligonucleotide need
not be 100% complementary to its target nucleic acid to be specifically
hybridizable, i.e. it may
lack one or more complements for certain nucleotides in the targeted nucleic
acid sequence. An
oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the target
nucleic acid inhibits the normal function of the target nucleic acid to cause
a loss of utility
whether of transcription or translation, and there is a sufficient degree of
complementarity to
avoid non-specific binding of the oligonucleotide 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, or in the case of in vitro assays, under conditions
in which the assays
are conducted. Accordingly, absolute complementarity is not required in the
practice of the
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present invention. In general, any oligonucleotide having sufficient
complementarity to form a 4
stable duplex with the target single stranded RNA or DNA or a stable triplex
with the target
double stranded DNA is considered to be suitable. Since stable duplex or
triplex formation
depends on the sequence and length of the hybridizing oligonucleotide and the
degree of
complementarity between the antisense oligonucleotide and the target sequence,
the system can
tolerate less fidelity {complementarity) when longer oligonucleotides are
used. In short, any
interaction or binding of an oligonucleotide or oligonucleotide analog with a
target nucleic acid
encoding angiogenin is believed to have the potential to inhibit the
expression of angiogenin.
In the context of this invention, the term "oligonucleotide" refers to a
plurality of joined
nucleotide units formed from naturally-occurring bases and ribofuranosyl
groups joined by native
phosphodiester bonds. This term effectively refers to naturally-occurring
species or synthetic
species fonmed from naturally-occurring subunits and includes both oligomers
of ribonucleotide
i.e., oligoribonucleotides, and oiigomers of deoxyribonucleotide i.e,
oligodeoxyribonucleotides
(also referred to herein as "oiigodeoxynucleotides"). As used herein, unless
otherwise indicated,
the term "oligonucleotide" also includes oligomers which may be large enough
to be termed
"polynucleotides". As further used herein, the terms "oligonucleotide" and
"oligodeoxynucleotide" include not only oligomers and polymers of the
biologically significant
nucleotides, i.e. nucleotides of adenine ("A"), deoxyadenine ("dA"}, guanine
("G"),
deoxyguanine ('dG"), cytosine ("C") deoxycytosine ("dC"), thymine ("T") and
uracil ("U"), but
also oligomers and polymers hybridizable to angiogenin DNA or RNA which may
contain other
nucleotides.
"Oligonucleotide analog" as that term is used in connection with this
invention, refers to
a compound having a modified internucleotide linkage, a modified purine or
pyrimidine
moiety, a modified sugar moiety, a modified 5' hydroxyl moiety, a modified 3'
hydroxyl
moiety or a modified 2' hydroxyl moiety. The analogs including the modified
moieties
function similarly to oligonucleotides in that they hybridize or otherwise
bind to target nucleic
acids but which have non naturally-occurring portions wherein one or more
purine or pyrimidine
moieties, sugar moieties or internucleotide phosphate linkages is chemically
modified, for
example, to improve stability and/or lipid solubility to enhance the ability
of the oligonucleotides
to penetrate into the region of cells where the RNA whose activity is to be
modulated is located.
For example, it is known that enhanced lipid solubility and/or resistance to
nuclease digestion
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results by substituting a methyl group or sulfur atom for a phosphate oxygen
in the Y
internucleotide phosphodiester linkage. Exemplary among these are the
phosphorothioate and
other sulfur containing species which are known in the art. Phosphorothioates
are compounds
in which one of the non-bridging oxygen atoms in the phosphate portion of the
nucleotide is
replaced by sulfur. These phosphorothioates are stable to cleavage by
nucleases, and since they
have the same number of charges as normal oligodeoxynucleotides, they have
good aqueous
solubility. Other modified oligonucleotides or analogs such as alkyl
phosphorothioate,
phosphodiester, phosphotriester, N-alkyl phosphoramidates,
phosphorodithioates, alkyl
phosphonates, and short chain alkyl or cycloalkyl structures may also be
useful. In accordance
with other preferred embodiments, one or more phosphodiester bonds are
substituted with
structures which are, at once, substantially non-ionic and non-chiral to
produce mixed linkage
oligonucleotides. Persons of ordinary skill in the art will be able to select
other linkages for use
in the practice of the invention.
Oligonucleotide analogs may also comprise altered base or sugar units, have
charged or
uncharged backbones, have additions at the ends of the oligonucleotide
molecule or other
modifications consistent with the spirit of this invention. Such analogs are
best described as
being functionally interchangeable with natural oligonucleotides (or
synthesized oligonucleotides
along natural lines), but which have one or more differences from natural
structure. All such
analogs are comprehended by this invention so long as they can function
effectively to bind to
selected portions of nucleic acids encoding angiogenin.
In accordance with the principles of the present invention, oligonucleotides
complementary to and hybridizable with any portion of nucleic acids
responsible for expression
of angiogenin whether human or animal are, in principle, effective for
inhibiting the expression
of angiogenin in the respective mammal. It is therefore to be understood that
the principles of
the present invention apply to all mammals, including humans, where inhibition
of the expression
of angiogenin is desired. For example, the nucleic acid sequence encoding
mouse angiogenin
is known. See Bond, M.D., and Vallee, B.L. (1990) Biochem. Biophys. Res.
Common. 171, 988-
995. The nucleic acid sequence for human angiogenin is shown in Fig. 1.
Accordingly, the
application of the principles of the present invention not only include human
uses, but animal
uses as well.
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Oligonucleotides according to certain embodiments of the present invention are
~.
represented by Formula I below although additional embodiments are described
throughout this
disclosure:
5,
B
R~OH2C O
O~ ~O ~RZ
iP~ f H2 B
Formula I
O
n
RIO R2
in which s'
X is O, S, or C,_4 alkyl;
B is adenine, guanine, cytosine, or thymine selected such that the
oligonucleotide has a
complementary base sequence with a portion of the nucleic acid strand coding
for angiogenin
thereby inhibiting expression thereof;
R, is H, C~_4 alkyl or substituted acridine;
R, is H, OH, SH, F, OCH3, OCN, or OCH6CH3; and n is 5 to 100.
Oligonucleotides within the scope of the present invention, including those
represented
by Formula I, include pharmaceutically acceptable salts or hydrates thereof.
Oligonucleotides
within the scope of the present invention optionally may include intercalating
molecules or
ribozyme sequences and may optionally have intervening sequences of other
nucleotides or non-
nucleotide molecules provided that such oligonucleotides hybridize with
angiogenin DNA or
RNA and inhibit its expression.
While any length oligonucleotide may be utilized in the practice of the
invention, such
as an oligonucleotide complementary to the entire angiogenin gene,
oligonucleotides having
between 5 to 100 subunits find utility and are preferred in the practice of
the present invention.
It is preferred that such oligonucleotides and analogs comprise at least about
5 subunits with
from about 8 to 50 subunits being more preferred. As will be appreciated, a
subunit is a base and
sugar combination suitably bound to adjacent subunits through phosphodiester
or other modified
bonds as previously discussed.
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Oligonucleotides shorter than 15 bases may be less specific in hybridizing to
the target ~.
angiogenin mRNA, and may be more easily destroyed by enzymatic digestion.
Hence,
oligonucleotides having 1 S or more nucleotides are preferred. Sequences
longer than 18 to 25
nucleotides may be somewhat less effective in inhibiting angiogenin
translation because of
decreased uptake by the target cell. Thus, oligomers of I S-25 nucleotides are
most preferred in
the practice of the present invention, particularly oligomers of 1 S-18
nucleotides.
It is to be understood that oligonucleotides having a sequence complementary
to any
region of the angiogenin gene find utility in the present invention, however
oligodeoxynucleotides complementary to a portion of (i) the "AUG"
translational start site, {ii)
the 5'-transcription initiation site, (iii) the 5 ="TATA" box site and , (iv)
the 3'-termination site
are particularly preferred. Random sequences in both the 5'-untranslated and
3'-untranslated
regions are also useful target nucleic acids for designing oligonucleotides
for the inhibition of
the expression of angiogenin.
Oligonucleotides of the present invention, including those represented by
Formula I,
hybridize or otherwise bind to target nucleic acids encoding for angiogenin,
the entire gene
sequence of which is shown in FIG. 1. When X in Formula 1 is oxygen, the
nucleotides are
connected by phosphodiester bonds. However, oligonucleotides of the present
invention include
analogs which differ from native DNA in that some or all of the phosphates in
the nucleotides
are replaced by phosphorothioates (in the case of X being sulfur),
methylphosphonates (in the
case of X being CH3) or other ~.~ alkylphosphonates such as ethyl, propyl,
butyl, methyl
phosphonate analogs disclosed by U.S. Pat. No. 4,469,863, phosphonate modified
oligodeoxynucleotides described by LaPlanche, et al. Nucleic Acid Research
14:9081 ( 1986)
and by Stec. et al. J. Am Chem. Soc. 106:6077 (1984), phosphodiesters and
phosphotriesters.
These compounds are referred to herein as having a modified oligonucleotide
linkage moiety.
Furthermore, recent advances in the production of oligoribonucleotide
analogues mean that other
agents may also be used for the purposes described here, e.g. 2'-
methylribonucleotides (Inoue et
al. Nucleic Acids Res. 15,6131, 1987) and chimeric oligonucleotides that are
composite RNA-
DNA analogues (moue et al. FEBS Lett. 215, 327, 1987).
S~cific examples of some preferred oligonucleotides envisioned for this
invention
may contain phosphorothioates, phosphotriesters; methyl phosphonates, short
chain alkyl or
cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic
intersugar
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WO 98I4Z722 PCTlI1S98105651
("backbone") linkages. Most preferred are phosphorothioates and those with CHI-
NH-O-
CH2, CH,-N(CH3)-O-CHZ , CH3-O-N(CH3)-CHz , CHI-N(CH3)-N(CH3)-CH, and O N(CH3)-
CHZ-CH~ backbones (where phosphodiester is O-P-O-CHz). Also preferred are
oligonucleotides having morpholino backbone structures. Summerton, J.E. and
Welter, D.D.,
U.S. Pat. No. 5,034,506. In other preferred embodiments, such as the protein-
nucleic acid or
peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the
oligonucleotide
may be replaced with a polyamide backbone, the bases being bound directly or
indirectly to
the aza nitrogen atoms of the polyamide backbone. P.E. Nielsen, M. Egholm,
R.H. Berg, O.
Buchardt, Science 1991, 154, 1497.
The oligonucleotides of Formula I optionally may be further differentiated
from
native DNA by replacing one or both of the free hydroxy groups with C,_4
alkoxy groups (in
the case of R, being C,_4 alkyl). As used herein, C,_4 alkyl means a branched
or unbranched
hydrocarbon having 1 to 4 carbon atoms.
Formula I oligonucleotides may also be substituted at the 3' and/or 5' ends by
R, being
an intercalating agent such as a "substituted acridine" which means any
acridine derivative
capable of intercalating nucleotide strands such as DNA. Preferred substituted
acridines are 2-
methoxy-6-chloro-9-pentylaminoacridine, N-(6-chloro-2-methoxyacridinyl)-O-
methoxydisopropylaminophosphinyl-3-aminopropanol and N-(6 chloro-2-
methoxyacridinyl)-O-
methoxydisopropylaminophosphinyl-5-aminopentanol. Other suitable acridine
derivatives are
readily apparent to persons skilled in the art.
Formula I oligonucleotides may also include ribozyme sequences inserted into
their
nucleotide sequence. The ribozyme sequences are inserted into Formula I
compounds such that
they are immediately preceded by AUC, UUC, GUA, GUU, GUC, or, preferably, CUC.
The
ribozyme sequence is any sequence which can be inserted and causes self
cleavage of messenger
RNA. The sequence CUG AUG AGU CCG UGA CGA A is preferred. Other such sequences
can be prepared as described by Haseloff and Gerlach, Nature (Aug. 18, 1988)
334; 585-591.
It is generally preferred for use in some embodiments of this invention that
the f
position of the linking sugar moieties in at least some of the subunits of the
oligonucleotides
or oligonucleotide analogs be substituted. Thus, 2' substituents such as RZ is
OH, SH, SCH2,
OCH3, F, OCN, OCH6CH3, OCH30CH3, OCH30(CHZ)" CH3, O(CH2)"NHZ or O (CHZ)"CH3
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where n is from 1 to about 10; C, to C,o lower alkyl, substituted lower alkyl,
alkaryl or
aralkyl; Ci; Br; CN; CF3; OCF3; O, S, or N-alkyl; O, S, or N-alkenyl; SOCH3;
SO,CH3; ONOZ;
NO2; N3; NHZ; heterocycloalkyl or alkaryl; aminoalkylamino; polyalkylamino;
substituted
silyl: an RNA cleaving group; a cholesteryl group; a conjugate; 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. Oligonucleotides having sugar mimetics
such as
cyclobutyls in place of the pentofuranosyl group are useful in the present
invention. Other
preferred embodiments may include at least one modified base form or
"universal base" such
as inosine.
The oligonucleotides used in accordance with this invention may be
conveniently and
routinely made through the well-known technique of solid phase synthesis on
automated nucleic
acid synthesizers, such as the Applied Biosystems 380B DNA Synthesizer which
utilizes ~i-
cyanoethyl phosphoramidite chemistry. Alternatively, the oligonucleotides of
the invention may
be synthesized by any of the known chemical oligonucleotide synthesis methods.
Such methods
are generally described, for example in Winnacker, From Genes to Clones:
Introduction to Gene
Technology. VCH Verlagsgesellshaft mbH (H. Ibelgaufts traps. 1987).
Any other means for such synthesis may also be employed; the actual synthesis
of the
oligonucleotides is well within the talents of the routineer. It is also well
known to use similar
techniques to prepare other oligonucleotides such as phosphorothioates and
alkylated derivatives.
For example, Formula I oligonucleotides in which one or more X is S are
prepared by published
procedures which are incorporated herein by reference. Stec., W.J. et al J.Am.
Chem. Soc. (1984)
106:6077-6079; Adams, S.P. et al. J. Am. Chem. Soc. ( 1983) 105:661;
Caruthers, M.H., et al,
Genetic Engineering; Settlow, J. Hollander. A. Eds; Plenum Press: New York (
1982) 4:1
Broido, M.S. et al; Biochem Riophys. Res. Commun. (1984) 119:663. It is also
well known to
use similar techniques and commercially available modified amidite and
controlled pore glass
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(CPG) products such as biotin, fiuorescein, acridine and psoralen-modified
au~idites and/or CPG
to synthesize fluorescently labeled, biotinylated or other modified
oligonucleotides.
Since the complete gene sequence of certain mamalian angiogenins are known,
including
human and mouse, oligonucleotides hybridizable with any portion of the gene
sequence or the
mRNA transcript may be prepared by the oligonucleotide synthesis methods known
to those
skilled in the art.
Overall, it is preferred to administer oligonucleotides or analogs thereof to
mammals
suffering from the effects of abnormal angiogenesis, such as tumor growth, in
either native form
or suspended in a carrier medium in amounts and upon treatment schedules which
are effective
to therapeutically treat the mammals to reduce the effects of abnormal
angiogenesis. One or
more different oligonucleotides or analogs thereof targeting different
sections of the nucleic acid
sequence of angiogenin may be administered together in a single dose or in
different doses and
at different amounts and times depending upon the desired therapy. The
oligonucleotides can
be administered to mammals in a manner capable of getting the oligonucleotides
initially into
the blood stream and subsequently into cells, or alternatively in a manner so
as to directly
introduce the oligonucleotides into the cells or groups of cells, for example
tumor cells, by such
means by electroporation or by direct injection into the tumor.
Oligonucleotides whose presence
in cells can inhibit transcription or protein synthesis can be administered by
intravenous
injection, intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection; orally or
rectally. Human phannacokinetics of certain antisense oligonucleotides have
been studied. See
Zhang et al. Clinical Pharmacology & Therapeutics (1995) 58(1), 44-53
incorporated by
reference in its entirety. It is within the scale of a person's skill in the
art to determine optimum
dosages and treatment schedules for such treatment regimens.
Doses of the oligonucleotides or analogs thereof of the present invention in a
pharmaceutical dosage unit will be an efficacious, nontoxic quantity selected
from the range of
0.1-100 mg/kg of body weight, preferably 0.1-50 mg/kg and more preferably 0.1
to 25 mg/kg.
The selected dose is administered to a human patient in need of inhibition of
angiogenin
expression from 1-6 or more times daily or every other day. Dosage is
dependent on severity and
responsiveness of the effects of abnormal angiogenesis to be treated, with
course of treatment
CA 02293591 1999-12-02
WO 98/42722 PCTII1S98~03651
lasting from several days to months or until a cure is effected or a reduction
of the effects is ~.~
achieved. Oral dosage units for human administration generally use lower
doses. The actual
dosage administered may take into account the size and weight of the patient,
whether the nature
of the treatment is prophylactic or therapeutic in nature, the age, weight,
health and sex of the
patient, the route of administration, and other factors.
Pharmaceutical compositions may contain suitable excipients and auxiliaries
which
facilitate processing of the oligonucleotides into preparations which can be
used
pharmaceutically. Preferably, the preparations, particularly those which can
be administered
orally and which can be used for the preferred type of administration, such as
tablets, dragees,
and capsules, and preparations which can be administered rectally, such as
suppositories, as well
as suitable solutions for administration parenterally or orally, and
compositions which can be
administered bucally or sublingually, including inclusion compounds, contain
from about 0.1 to
about 99 percent by weight of active ingredients, together with the excipient.
The pharmaceutical preparations of the present invention are manufactured in a
manner
which is itself well known in the art. For example, the pharmaceutical
preparations may be made
by means of conventional mixing, granulating, dragee-making, dissolving, or
lyophilizing
processes. The process to be used will depend ultimately on the physical
properties of the active
ingredient used.
Suitable excipients are, in particular, fillers such as sugars, for example,
lactose or
sucrose, mannitol or sorbitol, cellulose preparations and/or calcium
phosphates, for example,
tricalcium phosphate or calcium hydrogen phosphate as well as binders such as
starch, paste,
using, for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth,
methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose,
and/or
polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as
the above-
mentioned starches as well as carboxymethyl-starch, cross-linked polyvinyl
pyrrolidone, agar,
or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are
flow-regulating agents
and lubricants, for example, such as silica, talc, stearic acid or salts
thereof, such as magnesium
stearate or calcium stearate, and/or polyethylene glycol. Dragee cores may be
provided with
suitable coatings which, if desired, may be resistant to gastric juices. For
this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic, talc,
polyvinylpyrrolidone, polyethylene, glycol, and/or titanium dioxide, lacquer
solutions, and
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suitable organic solvents or solvent mixtures. In order to produce coatings
resistant to gastric
juices, solutions of suitable cellulose preparations such as acetyl-cellulose
phthalate or
hydrnxypmpylmethycellulose phthalate, are used. Dyestuffs and pigments may be
added to the
tablets of dragee coatings, for example, for identification or in order to
characterize different
combinations of active compound doses.
Other pharmaceutical preparations which can be used orally include push-fit
capsules
made of gelatin, as well as soft, sealed capsules made of gelatin and a
plasticizer such as glycerol
or sorbitoi. The push-fit capsules can contain the active compounds in the
form of granules
which may be mixed with filters such as lactose, binders such as starches,
and/or lubricants such
as talc or magnesium stearate and optionally, stabilizers. In soft capsules,
the active compounds
are preferably dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or
liquid polyethylene glycols. In additions, stabilizers may be added.
Possible pharmaceutical preparations which can be used rectally include, for
example,
suppositories, which consist of a combination of the active compounds with a
suppository base.
Suitable suppository bases are, for example, natural or synthetic
triglycerides, paraffin
hydrocarbons, polyethylene glycols or higher allcanols. In addition, it is
also possible to use
gelatin rectal capsules which consist of a combination of the active compounds
with a base.
Possible base materials include, for example liquid triglycerides,
polyethylene glycols, or
paraffin hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions
of the active
compounds in water-soluble or water-dispersible form. In addition, suspensions
of the active
compounds as appropriate oily injection suspensions may be administered.
Suitable lipophilic
solvents or vehicles include fatty oils, for example, ethyl oleate or
triglycerides. Aqueous
injection suspensions may contain substances which increase the viscosity of
the suspension
including, for example, sodium carboxymethyl cellulose, sorbitol, and/or
dextran. Optionally,
the suspension may also contain stabilizers.
Additionally, oligonucleotides of the present invention may also be
administered
encapsulated in liposomes or immunoliposomes, which are pharmaceutical
compositions wherein
the active ingredient is contained either dispersed or variously present in
corpuscles consisting
of aqueous concentric layers adherent to Iipidic layers. Liposomes are
especially active in
targeting the oligonucieotides to liver cells. The active ingredient,
depending upon its solubility.
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CA 02293591 1999-12-02
WO 98I4Z'122 PCTIUS98I~5651
may be present both in the aqueous layer and in the lipidic layer, or in what
is generally termed
a liposomic suspension. The hydrophobic layer, generally but not exclusively,
comprises
phospholipids such as lecithin and sphingomyelin, steroids such as
cholesterol, more or less ionic
surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid,
and/or other materials
of a hydrophobic nature. The diameters of the liposomes generally range from
about 1 Snm to
about 5 microns.
Antisensg in Combination with Other Thera
Published pharmacologic data indicate that phosphorothioate derivatives of
oligolrucleotides, when administered systemically, are taken up preferentially
by the liver (and
additionally by the kidney and bone marrow). Angiogenin is known to be a
normal compo~nt
of human plasma and serum synthesized predominantly by the adult liver.
Therefore,
oligonucleotides effective to inhibit the expression of angiogenin should
accumulate in the liver
and inhibit the endogenous synthesis of angiogenin and consequently lower its
concentration
in plasma and serum. The lower plasma/serum levels of angiogenin should then
allow for
more effective antitumor therapy using any of the angiogenin binding agents
described herein
that inhibit angiogenin's function by directly binding to the protein, since
they will not have
to first overcome binding to endogenous, circulating angiogenin before
reaching the tumor
itself. The use of oligonucleotides to inhibit the expression of angiogenin in
combination with
other angiogenin binding agents also lowers the potential for toxicity that
might result from
substantial amounts of circulating angiogenin inhibitor complexes due to the
reduced amount
of circulating angiogenin.
The oligonucleotides of the present invention are also envisioned to be useful
in
combination with other tumor targeted therapeutic maneuvers such as
chemotherapy,
immunotherapy, radiation therapy and the like so as to increase the overall
anticancer
therapeutic efficacy .
The oligonucleotides of the present invention are also useful for detection
and diagnosis
of angiogenin in clinical samples. For example, radio labeled oligonucleotides
can be prepared
by 32P labeling at the 5' end with polynucleotide kinase. Sambrook et al.
Molecular Cloning.
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CA 02293591 1999-12-02
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A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg.
10.59. Radio .
labeled oligonucleotides are then contacted with tissue or cell samples
suspected of containing
target nucleic acids and the sample is washed to remove unbound
oligonucleotide. Radioactivity
remaining in the sample indicates bound oligonucleotide (which in turn
indicates the presence
of target nucleic acids) and can be quantitated using a scintillation counter
or other routine
means. Abnormally high levels of target nucleic acids can be detected in this
way. Radio labeled
oligonucleotides can also be used to perform autoradiography of tissues to
determine the
localization, distribution and quantitation of target nucleic acids for
research, diagnostic or
therapeutic purposes. In such studies, tissue sections are treated with radio
labeled
oligonucleotide and washed as described above, then exposed to photographic
emulsion
according to routine autoradiology procedures. The emulsion, when developed,
yields an image
of silver grains over the regions expressing angiogenin.
Analogous assays for fluorescent detection of angiogenin expression can be
developed
using oligonucleotides of the invention which are conjugated with fluorescein
or other
fluorescent tag instead of radio labeling. Such conjugations are routinely
accomplished during
solid phase synthesis using fluorescently labeled amidites or CPG (c.g.
fluorescein labeled
amidites and CPG available from Gien Research, Sterling Va. Sec. 1993 Catalog
of Products
for DNA Research, Glen Research, Sterling Va, p. 21}.
Each of these assay formats is known in the art. One of skill could easily
adapt these
known assays for detection of target nucleic acids in accordance with the
teachings of the
invention providing a novel and useful means to detect levels of nucleic acids
encoding
angiogenin.
The following examples are set forth as representative of the present
invention. These
examples are not to be construed as limiting the scope of the invention as
these and other
equivalent embodiments will be apparent in view of the present disclosure,
figures, tables, and
accompanying claims.
EXAMPLE 1 "
Materials used in the following experimental examples were obtained as
follows.
Human PC-3 prostate and HT-29 colon tumor cells were obtained from the
American Type
24
CA 02293591 1999-12-02
wo ~4z7zz Pc~r~s9sros6si
Culture Collection. MDA-MB-435 human breast tumor and PC-3M human prostate
tumor cell Y
lines were obtained from Dr. Isaiah J. Fidler (M.D. Anderson Cancer Center).
Human MCF-
? breast cancer cells were obtained from Dr. Marc Lippman (Georgetown
University Medical
Center). Cell culture supplies were obtained as follow: all tissue culture
plastics were from
Costar; Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, MEM
Eagle
medium, trypsin-versene, and Hanks' buffered salt solution (HBSS) were
obtained from
BioWhittaker; fetal bovine serum (FBS) was from Hyclone. Materials for the
enzyme-linked
immunosorbent assay (ELISA) were as follows: human angiogenin was isolated
from an
Escherichia coli expression system [Shapiro, R., Harper, J. W., Fox, E. A.,
Jansen, H-W., Hein,
F., and Uhlmann, E. (1988) Anal. Biochem. 175, 450-461] and was provided by
Dr. Robert
Shapiro (Harvard Medical School); the anti-human angiogenin mAb 26-2F was
obtained by
us as described (Mahadevan and Hart, 1990, ; the rabbit polyclonai anti-human
angiogenin antibody 8113 was produced by immunization into a rabbit of human
angiogenin
together with Freund's adjuvant using classical techniques; ELISA plates were
from Costar;
bovine serum albumin {BSA) and p-nitrophenyl phosphate were from Sigma;
alkaline
phosphatase-labeled goat anti-rabbit igG was obtained from Kirkegaard and
Perry. Lipofectin
was from GibcoBRL. Slow release pellets containing 17 ~3-estradiol were
obtained from
Innovative Research of America. Custom-synthesized angiogenin sense and
antisense
[S]ODNs were from Promega or Boston BioSystems. Outbred, male and female
athymic
(nu/nu) mice were obtained from Charles River Laboratories and maintained
under specific
pathogen-free conditions in an environment strictly controlled for temperature
and humidity.
Matrigel basement membrane matrix was from Collaborative Biomedical Products.
EXAMPLE II
Cell Culture Growth Conditions
Cell cultures used in the following experimental examples are described as
follows.
All cells were maintained at 37° C in a humidified, 95 % airl5 ~ COZ
environment. HT-29 cells
were grown in DMEM containing 5 % FBS; PC-3M cells were gmwn in MEM Eagle
medium
containing 10% FBS and vitamins; PC-3 cells were cultured in Ham's F-12
containing 7%
FBS. All growth medium was supplemented with 2 mM L-glutamine and antibiotics
(gentamicin and fungizone). For experiments, cells were harvested with trypsin-
versene and
CA 02293591 1999-12-02
WO 98I427Z2 PCTIUS98n15651
counted with either a Coulter counter or by hemacytometry following staining
with Trypan -
blue for viability determination. Prior to injection into mice, cells were
fast washed twice
with HBSS.
EXAMPLE III
Angiogenin levels in medium conditioned by human tumor cells were measured by
a
double antibody ELISA as described [Newton, D. L., Xue, Y., Olson, K. A.,
Felt, J. W., and
Rybak, S. M. (1996) Biochemistry 35, 545-553]. Antihuman angiogenin mAb 26-2F
was coated
onto wells of an ELISA plate and blocked with BSA. Dilutions of medium to be
tested were
then added to the plate and incubated overnight. After washing, rabbit anti-
human angiogenin
antibody (R113) was added. Bound 8113 was detected by adding alkaline
phosphatase-labeled
goat anti-rabbit IgG followed by the addition of p-nitrophenyl phosphate. The
plates were read
at 405 ~1 with a computer-controlled Bio-Tek EL 311 ELISA reader using the
associated data
analysis program. Angiogenin levels in conditioned medium were quantitated by
comparison
with a standard curve of human angiogenin.
EXAMPLE IV
Angiogenin sense and andsense phosphorothioate oligodeoxynucleotides, [S]ODNs,
used in the following experiments were as follows. Two antisense 18-mer
[S]ODNs were
custom-synthesized by Promega based on the nucleic acid sequences of the
angiogenin gene
encompassing the AUG initiation codon and transcriptional start site regions
and labeled JF2S
and JF4S, respectively. In addition, an 18-mer control sense [S]ODN
complementary to JF2S
was synthesized and labeled JF1S. Their compositions are:
JF1S 5'- GAAGAGATGGTGATGGGC - 3'
JF2S 5'- GCCCATCACCATCTCTTC - 3'
JF4S 5'- ACACGGCATCATGAATCA - 3'
Other preferred oligonucleotides include the following:
JF6S 5'-CCAGGGGCCCGCTGGTTA-3'
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JFBS 5'-ACCAAATTTTATATTCTA-3'
JF10S 5'-CAGGCCCATCACCATCAC-3'
JF12S 5'-GCCCAGGCCCATCACCAT-3'
JF13S S'-TCTCTGACACGGCATCAT-3'
JF6S encompasses the 3'-termination site, JFBS encompasses the 5'-"TATA" box
site,
JF10S and JF12S encompass the "AUG" translational start site a~i comprise
variations of
sequence from JF2S, and JF13S encompasses the 5'-transcriptional start site
and comprises a
variation of sequence from JF4S.
It is to be understood that additional oligonucleotides within the scope of
the present
invention can be prepared by first selecting a target sequence anywhere along
the known
nucleic acid sequence of the angiogenin gene. An oligonucleotide complementary
to the target
sequence can then be prepared based upon the known complementary relationship
between
nucleic acids. In this manner, any number of oligonucleotides complementary to
target
seque~es of the angiogenin gene can be prepared and their ability to inhibit
the expression of
the angiogenin gene can than be determined based upon the teachings presented
herein.
EXAMPLE V
Anysense Oligod~nucleotides Inhibit Eanression of Anaiogenin
Experiments initially performed in vitro were aimed at assessing whether these
angiogenin antisense reagents were effective inhibitors of angiogenin
synthesis by prostatic
carcinoma cell lines. Efficient transfection of ODNs in vitro requires the
presence of a cationic
lipid, one of which, lipofectin, was obtained from GibcoBRL. Details of the
lipofectin
transfection procedure are provided by the manufacturer, GibcoBRL. In a first
experiment, the
results of which are shown in Fig. 2, panel A, PC-3 prostatic carcinoma cells
{5 x 105 cells in 35
mm dishes) were treated in vitro for 20 hr with lipofectin (5 ~,1) alone
(control, white bar) or
lipofectin plus JF2S (0.5 ~M) (black bar). The growth medium was then replaced
and the cells
allowed to recover for 24 hr. After that period the cells were harvested and
counted and the
conditioned medium was assayed for angiogenin levels by ELISA. The amount of
angiogenin
per cell number for the antisense-treated cultures in percent compared with
control-treated cells
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WO 98/42722 PCT/US98/05651
(100%) is plotted. The results of a second in vitro experiment under the same
conditions is
plotted in panel B of Fig. 2. The data indicate that angiogenin production in
vitro as a function
of cell number decreased by 18-33% by treating with the combination of
lipofectin plus JF2S in
comparison with the treatment with lipofectin alone.
Fig. 3 shows the results of a similar experiment using HT-29 colon
adenocarcinoma cell
line. In the first experiment, the results of which are plotted in Fig. 3
panel A, HT-29 cells (5 x
105 cells in 35 mm dishes) were treated in vitro for 20 hr with lipofectin (5
~1) alone (control,
white bar) or lipofectin plus JF2S (0.5 pM) (black bar). The growth medium was
then replaced
and the cells allowed to recover for 24 hr. After that period the cells were
harvested and counted
and the conditioned medium was assayed for angiogenin levels by ELISA. The
amount of
angiogenin per cell number for the antisense-treated cultures in percent
compared with control-
treated cells (100%) is plotted. The results of a second in vitro experiment
under the same
conditions is plotted in panel B of Fig. 3. The data indicates that angiogerun
production in vitro
as a function of cell number decreased by 30-38% by treating with the
combination of lipofectin
plus JF2S in comparison with the treatment with lipofectin alone.
The data demonstrates that for both PC-3 and HT-29 tumor cell types angiogenin
production in vitro as a function of cell number was decreased by treating
with the combination
of lipofectin plus JF2S in comparison to treatment with lipofectin alone.
EXAMPLE VI
Antisense O ' deo rnucleotides Reduce Tumor Size
The ex vivo-treated PC-3 tumor cells of Example V were injected s.c. into
athymic mice
(2.5 x 1 OS cells/mouse; 5 mice/group) with the usual co-administration of a
1:2 proportion of
Matrigel for reproducible cell growth of this cell line. After 8 days, by
which time the control
tumors had attained a size in excess of that supportable by diffusion and were
therefore
dependent upon angiogenesis, the mice were sacrificed and the tumors were
excised and
weighed. The average weight of the tumors resulting from injection of the
antisense-treated cells
in percent was compared with that of the control group's tumors (100%) and
shown in Fig. 2
(panels A & B, in vivo). Tumors arising from injection of JF2S-treated PC-3
cells were both 31-
54% smaller in average weight than the tumors which developed from their
respective control-
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treated cells. Of additional importance, in the experiment represented by Fig.
2 panel B, I P
mouse out of 5 did not develop an observable tumor by the time of sacrifice.
The ex vivo-treated HT-29 tumor cells were also injected s.c. into athymic
mice (2.5 x 1 OS
cells/mouse; S mice/group). After 15 days, by which time the control tumors
had attained a size
in excess of that supportable by diffusion and were therefore dependent upon
angiogenesis, the
mice were sacrificed and the tumors were excised and weighed. The average
weight of the
tumors resulting from injection of the antisense-treated cells in percent was
compared with that
of the control group's tumors (100%) and shown in Fig. 3 (panels A & B, in
vivo). Tumors
arising from injection of JF2S-treated PC-3 cells were both 53-66% smaller in
average weight
than the tumors which developed from their respective control-treated cells.
Of additional
importance, in the experiment represented by Fig. 3 panel A, 1 mouse out of S
did not develop
an observable tumor by the time of sacrifice.
These results indicate that a correlation exists between decreased tumor
growth in vivo
and decreased angiogenin production by tumor cells treated in vitro with JF2S.
Figs. 4, 5, 6 and 7 show the results of in vitro experiments in which HT-29,
PC-3, MDA-
MB-435 or PC-3M tumor cells, respectively, were treated with lipofectin alone
or lipofectin with
either antisense [S]ODN JF2S or control sense [S]ODN JF1 S. The amount of
angiogenin per
cell number for the antisense-treated (black bar) and sense-treated (grey bar)
cultures in percent
compared with control lipofectin-treated cells (100%) (white bar) is plotted.
Angiogenin
production in vitro as a function of cell number decreased by 39% (HT-29), 65%
(PC-3), 45%
(MDA-MB-435) and 48% (PC-3M) by treating with the combination of lipofectin
plus antisense
[S]ODN JF2S in comparison with treatment with lipofectin alone. Treatment with
control sense
[S)ODN JF1S plus lipofectin resulted in a decrease of 13% (HT-29), 44% (PC-3),
17% (MDA-
MB-435) and 26% (PC-3M) in comparison with treatment with lipofectin alone.
These same ex
vivo-treated tumor cells were subsequently injected into athymic mice [HT-29
cells: 2.5 x 105
cells/mouse (5 mice/group); PC-3 cells: 1.25 x 105 cells/mouse injected s.c.,
mixed with a 1:2
proportion of Matrigel (5 mice/group); MDA-MB-435 cells: 7.5 x 105
cells/mouse, injected s.c.
into the mammary fat pad (5 mice/group, except for the group receiving the
anfisense [S]ODN
JF2S-treated cells, in which there were 7 mice); PC-3M: 2.5 x 105 cells/mouse
injected s.c.,
mixed with a 1:2 proportion of Matrigel (5 mice/group)]. After 17 (HT-29), 25
(PC-3), 30
(MDA-MB-435) or 17 (PC-3M) days, by which time the control tumors had attained
a size in
29
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WO X8142722 PCTIUS98/05651
excess of that supportable by diffusion and were therefore dependent upon
angiogenesis, the
mice were sacrificed and the tumors were excised and weighed. The average
weight of the
tumors resulting from injection of antisense [S]ODN JF2S-treated cells in
percent was compared
with that of the control group's tumors (100%) and shown in Fig. 4 (HT-29),
Fig. 5 (PC-3), Fig.
6 (MDA-MB-435) and Fig. 7 {PC-3M) (in vivo). Tumors arising from injection of
antisense
[S]ODN JF2S-treated tumor cells were 53% (HT-29), 92% (PC-3), 59% (MDA-MB-435)
and
74% (PC-3M) smaller in average weight than the tumors which developed from the
cells treated
with lipofectin alone. Tumors arising from PC-3 cells treated with the control
sense [S]ODN
JF1 S were 16% (Fig. S, in vivo) smaller than those tumors which developed
from cells treated
with lipofectin alone. Tumors arising from HT-29, MDA-MB-435 or PC-3M cells
treated with
the control sense [S]ODN JF 1 S were actually 14%, 4% or 21 % larger,
respectively, than those
tumors which developed from the cells treated with Iipofectin alone. In all
three experiments all
mice receiving either control lipofectin or sense [S)ODN JF I S-treated cells
developed tumors.
Among those mice receiving cells treated with antisense [S]ODN JF2S, tumors
did not develop
by the time of termination of the experiments in 1 out of 5 (HT-29 cells), 4
out of 5 (PC-3 cells),
1 out of 7 (MDA-MB-435 cells) and 2 out of 5 (PC-3M cells) mice. These results
further
indicate that a correlation exists between decreased tumor growth in vivo and
decreased
angiogenin production by tumor cells treated in vitro with antisense [S]ODN
JF2S.
Fig. 8 is a photograph of the actual tumors excised from mice injected with PC-
3 cells
treated in vitro with either the antisense [S]ODN, JF2S, (bottom row) or
co~.itrol lipofectin (top
row) as described in Example V and as shown in Fig. 2 panel B. This shows the
differences
between these two groups of tumors in both size and occurrence. One mouse in
the antisense-
treated group (bottom row) did not develop a tumor while those that did
develop were on
average much smaller size than tumors arising from control-treated cells (top
row).
Fig. 9 is a photograph of the actual tumors excised from mice injected with HT-
29 cells
treated in vitro with either the antisense [S]ODN, JF2S {bottom row) or
control lipofectin (top
row) as described in Example V and as shown in Fig. 3 panel B. Once again the
differences
between these two groups of tumors in size is evident, with the tumors
developing from the
lipofectin plus JF2S-treated cells being much smaller on average than those
tumors developing
from HT-29 tumor cells treated with iipofectin alone. In particular, three of
the tumors
produced by HT-29 tumor cells treated with JF2S were extremely small in size.
CA 02293591 1999-12-02
WO 98142722 PCTIUS98105651
Photographs of the actual tumors excised from the mice in experiments shown in
Figs. -
4, 5, 6 and 7 are shown in Figs. 10, I1, 12 and 13, respectively. In each case
the photograph
shows the tumors resulting from injection of cells treated in vitro with
either antisense [S]ODN
JF2S {bottom row), sense control [S]ODN JF1S (middle row) or control
lipofectin {top row).
The average size of the two groups of control tumors arising from tumor cells
treated with
either the sense [S]ODN 3F1S or lipofectin alone is essentially equivalent,
while the average
size of those tumors arising from the tumor cells treated with the antisense
[S]ODN JF2S were
significantly smaller than either of these two control groups. In fact, tumors
did not develop
by the termination of the experiments in 1 out of 5 (HT-29 cells, Fig. 10), 4
out of 5 (PC-3
cells, Fig. 11), 1 out of 7 (MDA-MB-435 cells, Fig. I2) and 2 out of 5 (PC-3M
cells, Fig.
13) mice receiving cells treated with antisense [S]ODN JF2S.
From these studies the conclusion can again be drawn that angiogenin is indeed
critical
for the growth/establishment of tumors in this mouse model, further validating
the proposition
that anti-angiogenin therapies are effective for treatment of cancer
clinically.
EXAMPLE VII
In two further experiments, shown in Fig. 14 panels A and B, the amount of
angiogenin produced by PC-3 cells in vitro could be further decreased by
slightly adjusting the
conditions of transfecdon with antisense JF2S, The figure also shows that JF2S
can
additionally inhibit angiogenin production by PC-3M tumors cells and that
another angiogenin
antisense [S]ODN, JF4S, also can inhibit the synthesis of angiogenin by both
PC-3 and PC-3M
cells in vitro. PC-3 (panel A) or PC-3M {panel B) cells were treated for 20 hr
with HBSS as
diluent control (white bars), lipofectin (5 ~,1) alone (control, single cross-
hatched bars),
lipofectin plus JF2S [(0.5- (black bars) or 1.0 ~,M (dotted bars)], or
lipofectin plus JF4S [(0.5-
(grey bars) or 1.0 ~,M (double crossed hatched bars)] . The growth medium was
then replaced
and the cells allowed to recover for 48 hr at which time the cells were
harvested and counted
and the cotglitioned medium was assayed for angiogenin levels by ELiSA. The
amount of
angiogenin per cell number for each group in percent compared to that of the
HBSS-treated
control group (100 % ) is plotted. This shows that treatment with andsense
JF2S, under these
conditions, was now able to inhibit the synthesis of angiogenin by PC-3 cells
by about 87 %
as compared with HBBS-treated cells (panel A). JF2S also substantially
inhibits angiogenin
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WO 98/42722 PCT/US98105651
production by a third tumor cell line PC-3M (panel B). Pat~ls A & B also show
that a second -
angiogenin antisense reagent directed toward the transcriptional start site of
the angiogenin
gene, JF4S, also effectively interferes with angiogenin production by both PC-
3 and PC-3M
tumor cells. Lipofectin alone has essentially no effect on angiogenin levels
secreted by either
of the two cell types (panels A 8c B). Importantly, treatment with a control
"sense" sequence
[S)ODN complementary to JF2S, i.e. JF1S, did not result in decreased
angiogenin production
by PC-3 cells (not shown).
EXAMPLE VIII
Fig. 15 shows the results obtained in three separate therapy experiments. Each
experiment was conducted as follows. On day 0, mixtures of PC-3 tumor cells (1
x 10°
cells/mouse) with either antisense [S]ODN JF2S (200 ~,g/mouse), control sense
[S]ODN JF1S
(200 ~,g/mouse) or PBS (as diluent control) were injected s.c., together with
a 1:2 proportion
of Matrigel to other components into male athymic mice. Treatment was
continued for 48
days as follows: day 1-6, antisense [S]ODN JF2S (I00 ~cglmouse), control sense
[S]ODN JF1S
(100 ~,g/mouse) or PBS as diluent control injected daily s.c. 6 times per
week; days 7-20:
antisense [S]ODN JF2S (50 ~,g/mouse), control sense [S]ODN JF1S (50 ~g/mouse)
or PBS as
diluent control injected daily s.c. 6 times per week; days 21-49: antisense
[S]ODN JF2S (50
~g/mouse), control sense [S]ODN JFIS (50 ~,g/mouse) or PBS as diluent control
injected daily
s.c. 4 times per week. Mice were examined twice a week for the presence of a
palpable
tumor. After day 49, treatment was stopped.
Fig. 15, panels A, B, and C show the percentage of mice bearing a palpable
tumor in
each of the antisense [S]ODN JF2S-treated (black bar) and control sense [S]ODN
JF1S-treated
(grey bar) groups compared with the PBS diluent control-treated group (white
bar, 100 % ) for
three separate, independent experiments. In experiment 1 (panel A), no tumors
were observed
in any of the antisense [S)ODN JF2S-treated mice as of day 65, the day at
which the mice
were sacrificed. In contrast, all of the mice treated with either control
sense [S]ODN JF1S
or PBS as diluent control exhibited palpable tumors at the time of sacrifice
on day 65. In
experiment 2, the mice treated with control sense [S]ODN JF1S or PBS all
exhibited tumors
by day 49, and were subsequently sacrificed. However, no tumors were observed
at that time
in any of the mice treated with the antisense [S]ODN JF2S. These mice were
kept for
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WO 98/42722 PCTlUS98~D5651
observation with no further treatment until day 276; no tumors were observed
in any of these -
mice during this period. In experiment 3, the mice treated with control sense
[S]ODN JF1S
or PBS also all exhibited tumors by day 49 and were then sacrificed. The mice
treated in this
experiment with antisense [S]ODN JF2S did not develop tumors during a
subsequent
observation period, without fiurther treatment, until sacrifice on day 139. In
experiments 1 and
2 the [S]ODNs used for treatment were prepared by Promega Corp., while in
experiment 3
the [S]ODNs were prepared by Boston BioSystems. Thus in three separate
experiments
treatment with the antisense [S]ODN JF2S was shown to prevent the ap~arance of
PC-3
human tumors after injection of these tumor cells into athymic mice.
Figs. 16 and 17 show the results of similar en vivo therapy experiments using
human
breast tumor MDA-MB-435 or MCF-7 cells, respectively . The former cell Iine is
estrogen-
independent, while the latter is estrogen-dependent. In these experiments the
source of the
[S]ODNs used for therapy was Boston BioSystems. Tumor cells (5 x 105 MDA-MB-
435
cells/mouse or 2 x 106 MCF-7 cells/mouse) were injected into the surgically-
exposed
mammary fat pad behi~ the left front leg of female athymic mice. In the case
of the estrogen-
dependent MDF-7 cell line a slow release (60 day) estrogen pellet containing
0.72 mg of 17
(3-estradiol was inserted s.c. within 1 cm of the area of tumor cell injection
as an exogenous
source of estrogen. Within 5 minutes of the tumor cell injection, mice were
treated by s.c.
injection in the area of the tumor cell injection with either antisense [S]ODN
JF2S (200
~ug/mouse), control sense [S]ODN JF1S (200 ~cg/mouse) or PBS as diluent
control. The mice
were subsequently treated daily 6 times per week with either PBS as diluent
control or
antisense [S]ODN JF2S or control sense [S]ODN JF1S (100 ~,g of each
[S]ODN/mouse in the
experiment using MDA-MB-435 cells and 200 ~cg of each [S]ODN/mouse in the
experiment
using MCF-7 cells). The mice were checked for palpable tumors twice a week
until sacrifice
on day 28. Fig. 16 shows that all PBS diluent control (open circles)-and
control sense
[S]ODN JF1S (closed circles~treated mice developed palpable tumors by day 17.
In contrast,
at that time tumors had developed in only 29% of the antisense [S]ODN JF2S
(closed squares)-
treated mice by day 17 . On day 28, the day of termination of the experiment,
40 % of the
antisense [S]ODN JF2S-treated mice were still tumor-free. Fig. 17 shows that
100% of the
PBS diluent control (open circles)-and conxrol sense [S]ODN JF1S (closed
circles)-treated mice
developed palpable tumors by day 28, while only 22 % of those mice treated
with antisense
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WO 98/42722 PCT/US98/05651
[S]ODN JF2S (closed squares) exhibited palpable tumors at the time. Thus in
vivo treatment -
with antisense [S]ODN JF2S delayed and in a subset of mice completely
prevented the
appearance of tumors from two different human breast tumor cell lines injected
into athymic
mice.
EXAA~IPLE IX
The efficacy of antisense [S]ODN JF2S in preventing tumor metastasis was
investigated
using an orthotopic model of human prostate cancer metastasis in athymic mice.
Orthotopic
tumor models are those in which tumor cells are implanted into the mouse organ
equivalent
to the source organ from which the tumor cell line is derived. In the model
used to test
antisense [S]ODN JF2S, the human prostate tumor cell line PC-3M (3.75 x 105
cells/mouse)
was injected into one of the surgically exposed lobes of the prostate gland of
an athymic
mouse. The mouse was then treated 1 hour later by i.p. injection with either
antisense
[S]ODN JF2S (500 ~,g/mouse in the high dose gmup or 200 ~cg/mouse in the low
dose group),
co~rol sense [S]ODN JF1S (500 ~cg/mouse in the high dose gmup or 200 ~cg/mouse
in the low
dose group), anti-angiogenin monoclonal antibody 26-2F (300 ~,g/mouse;
included as a
positive control treatment group, since it has been previously determined that
this monoclonal
antibody is efficacious in preventing PC-3M tumor metastasis in the same
model), or PBS as
diluent control. The [S]ODN-and PBS-treated mice were subsequently injected
i.p. with the
same materials at the same above doses per mouse daily 6 times per week from
day 1-13,
followed by injections of the same dose of the same materials 4 times per week
until day 38.
Monoclonal antibody 26-2F was administered on the same schedule but at a
previously
determined optimal dose of 180 ~cg/mouse for days 1-38. On day 39 the mice
were sacrificed
and the prostate examined for evide~e of tumor. At that time all mice in the
experiment
contained a primary tumor in their prostate gland. The regional iliac lymph
nodes were
removed and preserved in phosphate-buffered formalin. These preserved lymph
nodes were
later dehydrated, embedded in paraffin, cut into 4 mm sections and stained
with hematoxylin
and eosin. The slides were then examined by a pathologist in a blinded fashion
for evidence
of metastasis. Table 2 below shows the results of this examination in terms of
the number of
mice in the indicated treatment group harboring metastasis in at least one of
the two iliac
34
CA 02293591 1999-12-02
WO 98/42722 PCT/US98I~5651
lymph nodes divided by the total number of mice in the treatment group. This
number is
expressed as a percentage in parentheses below the aforementioned fraction.
Table 2
incid~~~cp of Me~ct_s~~IC
' PBS mAb Sense Antisense Sense Antisense
{diluent 26-2F control JF2S control JF2S
control) (medium JF 1 S (high dose) JF 1 S {low dose)
dose) (high dose) (low dose)
6/6 4/9 9l9 5/1 0 6/6 4/5
{100%) (44%) (100%) (50%) (100%) (80%)
All of the mice treated with PBS, a diluent control, or control sense [S]ODN
JF1S (at
both high and low doses) developed metastasis in at least one of the regional
iliac lymph
nodes. Monoclonal antibody 26-2F protected 56% of the mice from developing
metastasis in
the regional lymph nodes, a percentage comparable to that obtained in previous
experiments.
A low dose of antisense [S]ODN JF2S protected 1 out of the 5 mice from
developing
metastasis. However, the high dose of antisense [S]ODN JF2S protected 50% of
the mice
from forming regional lymph node metastasis. Thus antisense [S]ODN JF2S is
effective in
preventing human tumor metastasis in an orthotopic model of prostate tumor
metastasis.
It is to be understood that the embodiments of the present invention which
have been
described are merely illustrative of some of the applications of the
principles of the invention.
Numerous modifications may be made by those skilled in the art based upon the
teachings
presented herein without departing from the true spirit and scope of the
invention.
