U.S. patent application number 10/732218 was filed with the patent office on 2005-01-27 for modified antisense nucleotides complementary to a section of the human haras gene.
This patent application is currently assigned to Aventis Pharma Deutschland GmbH. Invention is credited to Chang, Esther, Peyman, Anuschirwan, Pirollo, Kathleen, Rait, Antonina, Uhlmann, Eugen, Will, David William.
Application Number | 20050020523 10/732218 |
Document ID | / |
Family ID | 8226765 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020523 |
Kind Code |
A1 |
Uhlmann, Eugen ; et
al. |
January 27, 2005 |
Modified antisense nucleotides complementary to a section of the
human haras gene
Abstract
The invention relates to a specific modified oligonucleotide
complementary to a section of the human Ha-ras gene and mRNA, and
its use to specifically regulate, modulate or inhibit expression of
the HA-ras gene, and its use as a pharmaceutical for the treatment
of conditions arising from the abnormal expression of the Ha-Ras
gene, in particular in combination with chemotherapy and
radiotherapy. The modified oligodeoxynucleotide according to the
invention has the sequence
5'-TxAxTxTxCxCxGxTxCxAxT-3'-O--PO.sub.2--O--R (SEQ ID NO:1),
wherein X is an internucleotide linkage of type o or s, with the
proviso that x is an s linkage at least 4 times and at most 9
times, and o means a phosphodiester internucleoside linkage, s
means a phosphorothioate internucleoside linkage, R means a
C.sub.8-C.sub.21 alkyl group,
--(CH.sub.2--CH.sub.2O)n-(CH.sub.2).sub.m--CH.sub.3, or
--CH.sub.2--CH(OH)CH.sub.2O--(CH.sub.2).sub.q--CH.sub.3 wherein n
is an integer from 1 to 6, m is an integer from 0 to 20 and q is an
integer from 7 to 20 and A is 2'-deoxyadenosine, G is
2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine.
Inventors: |
Uhlmann, Eugen; (Glashutten,
DE) ; Peyman, Anuschirwan; (Kelkheim, DE) ;
Will, David William; (Kriftel, DE) ; Chang,
Esther; (Chevy Chase, MD) ; Pirollo, Kathleen;
(Arlington, VA) ; Rait, Antonina; (Arlington,
VA) |
Correspondence
Address: |
ROSS J. OEHLER
AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Assignee: |
Aventis Pharma Deutschland
GmbH
Industriepark Hoechst
Frankurt Am Main
DE
|
Family ID: |
8226765 |
Appl. No.: |
10/732218 |
Filed: |
December 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10732218 |
Dec 11, 2003 |
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09423198 |
Feb 17, 2000 |
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6723706 |
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09423198 |
Feb 17, 2000 |
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PCT/EP98/02546 |
Apr 30, 1998 |
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Current U.S.
Class: |
514/44R ;
536/23.1 |
Current CPC
Class: |
C07H 21/02 20130101;
C12N 15/1135 20130101; C12N 2310/3531 20130101; C12N 2310/11
20130101; C12N 2310/315 20130101; C12N 2310/345 20130101; C12N
2310/322 20130101; A61P 35/00 20180101; A61K 38/00 20130101; C12N
2310/351 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 048/00; C07H
021/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 5, 1997 |
DE |
97107404.2 |
Claims
1-45. (Cancelled)
46. An oligodeoxynucleotide comprising SEQ ID NO:1
(5'-TxAxTxTxCxCxGxTxCxA- xT-3'-O--PO.sub.2--O--R), wherein x is an
internucleotide linkage of type o or s, with the proviso that x is
an s linkage at least 4 times and at most 9 times; o means a
phosphodiester internucleoside linkage; and s means a
phosphorothioate internucleoside linkage; R means a
C.sub.8-C.sub.2, alkyl group,
--(CH.sub.2--CH.sub.2O).sub.n--(CH.sub.2).s- ub.m--CH.sub.3 or
--CH.sub.2--CH(OH)CH.sub.2O--(CH.sub.2).sub.q--CH.sub.3, wherein n
is an integer from 1 to 6, m is an integer from 0 to 20, and q is
an integer from 7 to 20; A is 2'-deoxyadenosine; G is
2'-deoxyguanosine; C is 2'-deoxycytidine; and T is thymidine.
47. A method of treating a disease arising from the overexpression
and/or mutation of the Ha-ras gene, said method comprising
administering to a host an effective amount of the oligonucleotide
of claim 46.
48. A method of treating a disease arising from a
hyperproliferative disorder, said method comprising administering
to a host an effective amount of the oligodeoxynucleotide of claim
46.
49. The method of claim 48, wherein the disease is cancer.
50. The method of claim 49, further comprising administering
radiotherapy.
51. The method of claim 49, further comprising administering at
least one chemotherapeutic agent.
52. The method of claim 48, wherein the disease is restenosis.
53. The method of claim 48, wherein the disease is psoriasis.
54. A method of regulation, modulation, or inhibition of Ha-ras
gene expression, said method comprising administering to a host an
effective amount of the oligodeoxynucleotide of claim 46.
55. A method of inhibiting the proliferation of cancer cells, said
method comprising administering to a host an effective amount of
the oligodeoxynucleotide of claim 46.
56. A method of reversing the radio-resistance of cancer cells,
said method comprising administering to a host an effective amount
of the oligodeoxynucleotide of claim 46.
57. A method of reversing the chemo-resistance of cancer cells,
said method comprising administering to a host an effective amount
of the oligodeoxynucleotide of claim 46.
58. A method of preparing the oligodeoxynucleotide of claim 46,
said method comprising forming phosphorothioate linkages between
nucleotides of the oligodeoxynucleotide by sulfurization using
Beaucage reagent.
Description
[0001] This invention relates to a specific modified
oligonucleotide complementary to a section of the human Ha-ras gene
and mRNA, and its use to specifically regulate, modulate or inhibit
expression of the Ha-ras gene, and its use as a pharmaceutical for
the treatment of conditions arising from abnormal expression of the
Ha-ras gene.
[0002] Antisense oligonucleotides (AO) have proven to be specific
inhibitors of gene expression in a large number of systems, both in
vivo and in vitro. (Uhlmann and Peyman, Chem. Rev. 1990, 90,
543).
[0003] One of the major problems encountered when using unmodified
oligonucleotides containing only phosphodiester internucleoside
linkages (PO-oligonucleotides) is the rapid degradation of this
type of oligonucleotide in cells and biological fluids, such as,
for example, serum and cerebrospinal fluid, by a range of
nucleolytic activities. A wide range of chemical modifications to
oligonucleotides have been carried out in order to improve their
nucleolytic stability (Uhlmann and Peyman, Chem. Res. 1990, 90,
543). These modifications include the modification or replacement
of the phosphodiester internucleoside linkage, the sugar unit, the
nucleobase; or the sugar-phosphate backbone of the
oligonucleotides. The most thoroughly investigated type of
modification is alteration of the internucleoside linkage,
including phosphorothioate (PS), methylphosphonate (MeP) and
phosphorodithioate (PSS) linkages. It should be stressed that
modification of the oligonucleotide alters not only its nuclease
stability but also other characteristics of the oligonucleotide,
such as, for example, their cellular uptake, RNaseH activation, and
the strength and specificity of binding to their target nucleic
acid, and the like. It should be borne in mind that the stability
of the modified oligonucleotide in serum, frequently used to
determine the nuclease stability of the oligonucleotide, is not the
sole determinant of intracellular activity (P. D. Cook in
"Antisense Research and Applications", Crooke and Lebleu, Eds., CRC
Press, Boca Raton, 1993, Chap.9, p149 et seq.).
[0004] The phosphorothioate (PS) modified oligonucleotides are the
most widely used type of modified oligonucleotide. The following
strategies have been developed for the positioning of PS linkages
in antisense oligonucleotides:
[0005] (1) Replacement of All Phosphodiester Internucleoside
Linkages with Phosphorothioate Linkages.
[0006] The resulting all-phosphorothioate oligonucleotides are much
more stable to nucleases than PO-oligonucleotides (Monia et al. J.
Biol. Chem. 1996, 271, 14533). For example, degradation of all-PS
oligonucleotides by endonucleases is slowed down by a factor of
2-45 relative to a PO oligonucleotide (Stein et al. Nucleic Acids
Res. 1988, 16, 3209). In Xenopus oocytes or embryos, the
degradation of microinjected PO oligonucleotides proceeds with a
half-life of 30 minutes, while all-PS oligonucleotides have a
half-life of over 3 hours under the same conditions (Woolf et al.
Nucleic Acids Res. 1990, 18, 1763). All-PS oligonucleotides retain
their ability to activate RNaseH. The major disadvantages of all-PS
oligonucleotides are that their ability to form stable hybrids with
their target nucleic acid is reduced, and that they frequently give
rise to unspecific "non-antisense" effects (Monia et al., J. Biol.
Chem. 1996, 271, 14533).
[0007] (2) Oligonucleotides Containing Both Phosphorothioate and
Phosphodiester Internucleoside Linkages.
[0008] In an effort to overcome the non-antisense effects observed
with all-PS oligonucleotides, oligonucleotides containing both
phosphorothioate and phosphodiester internucleoside linkages have
been synthesized and tested for stability and biological
activity.
[0009] Ghosh et al. (Anticancer Drug Design 1993, 8, 15) describe a
PS-PO oligonucleotide containing various percentages of PS
linkages. Their construction follows, for example, the pattern
(PS-PO-PO-PO).sub.n, (PO-PO-PS).sub.n, (PS-PO).sub.n,
[(PO).sub.2--(PS).sub.2].sub.n, [PO-PS-PS].sub.n. They teach that a
PS linkage content of at least 50% is required for selective
translation inhibition in vitro and that activity drops drastically
when the PS content is less than 50%. More recently it has been
demonstrated that an oligonucleotide containing 50% PS-linkages
arranged in the pattern (PS-PO).sub.n showed no biological activity
in an assay system where an all-PS oligonucleotide and "end-capped"
PO-PS oligonucleotides (see below) of the same sequence were highly
active (Monia et al., J. Biol. Chem. 1996, 271, 14533).
[0010] (3) "End-Capped" Oligonucleotides, Where One, Two or Three
Internucleoside bridges on the 5' and/or the 3' End of the
Oligonucleotide are Phosphorothioate Modified. (Also Known as the
"Gap Technique")
[0011] This type of modification is designed primarily to protect
the oligonucleotide from degradation by exonucleases. In particular
modifications at the 3'-end of the oligonucleotide are desirable as
they offer protection from 3'-exonucleases, which are the most
abundant nucleases in serum (Uhlmann and Peyman, Chem. Rev. 1990,
90, 543).
[0012] An interesting comparison of strategies is found in Hoke et
al. (Nucleic Acids Res. 1991, 19, 5743). The authors compare the
activity of a range of antisense PS-oligonucleotides against HSV-1
in cell culture. Their findings confirm that 3', or 3'+5',
end-capped oligonucleotides (the first three internucleoside
linkages being modified in each case), similarly to all-PS
oligonucleotides are sufficiently protected against degradation by
nucleases in serum. In contrast internally modified (three PS
bridges) oligonucleotides and oligonucleotides in which only the
5'-end has been capped (again, the first three internucleoside
linkages being modified) are degraded rapidly. In contrast, the
authors found that neither 5' nor 3' end capping nor both are
sufficient for activity within the cell, and they drew the
conclusion that a uniform modification (all-PS) is required to
achieve sufficient stability to nucleases in cells.
[0013] More recently it has been discovered that pyrimidine
nucleosides are the most nuclease susceptible points in
oligonucleotides (Peyman, A. and Uhlmann, E., Biol. Chem.
Hoppe-Seyler 1996, 377, 67; EP 0 653 439 A2). It was found that a
combination of end-capping and PS protection of the pyrimidine
positions of oligonucleotides (the so called "minimal modification"
approach) is sufficient to make them highly resistant to nuclease
degradation. The biological activity (against Herpes simplex virus)
of an oligonucleotide with this type of PS modification pattern was
comparible to that of an all-PS oligonucleotide.
[0014] One of the major problems encountered when using AOs,
whether or not they are stabilized against degradation, is their
poor cellular uptake. Many approaches have been tried to attempt to
ameliorate this problem. Most of these approaches involve the
attachment of a variety of substances to the oligonucleotide.
Modifications include: the attachment of peptides to
oligonucleotides' (Lemaitre, M. et al. Proc. Natl. Acad. Sci. USA
1987, 84, 648) and the attachment of lipophilic residues, such as
alkyl chains or cholesterol, to oligonucleotides (Saison-Behmoaras,
T. et al. EMBO J. 1991, 10, 1111-1118; Will, D. W. and Brown, T.
Tetrahedron Lett. 1992, 33, 2729). It has been found, however, that
in many cases the introduction of a lipophilic group causes
biological effects which are independent of the sequence of the
oligonucleotide. Non-specific effects have been reported for
cholesterol-oligonucleotide conjugates (Henderson, G. B. and Stein,
C. A. Nucleic Acids Res. 1995, 23, 3726.), and for oligonucleotides
attached to alkyl chains (Shea, R. G. et al. Nucleic Acids Res.
1990, 18, 3777). Saison-Behmoaras et al (EMBO J. 1991, 10,
1111-1118; WO 96/34008) have reported that a 9mer all-PO
oligonucleotide derivatized with a 3'-dodecanol moiety and a
5'-acridine crosslinking agent, and antisense to mutated Ha-ras
inhibited T24 human bladder carcinoma cell proliferation. No
comparison of the antiproliferative activity of this
oligonucleotide with that of the corresponding oligonucleotide
without a dodecanol conjugate was made. The cellular uptake of the
dodecanol oligonucleotide was reported to be 4 times that of the
unmodified oligonucleotide in T24 cells. The acridine-dodecanol
modified oligonucleotide had no effect on the proliferation of a
human mammary cell line carrying an unmutated Ha-ras gene. This
effect can either be attributed to the sequence specificity of the
antisense oligonucleotide or to insufficient cellular uptake. Since
the uptake of the acridine-dodecanol oligonucleotide was not
determined in the human mammary cell line, the effect may be
entirely, or at least partly due to the fact that the
oligonucleotide was not taken-up by the human mammary cell line,
and thus did not have an opportunity to exhibit
non-sequence-specific effects on cell proliferation.
[0015] About 20% of human tumors have a mutation in one of the
three ras genes (Ha-ras, Ki-ras, and N-ras) leading to
over-expression of p21 protein which plays an important role in the
transformed phenotype (Bos, T. L. 1988 Mutation Res. 1988, 195,
255). It has been reported that inhibition of different ras genes
in different cell lines can either have no effect, or inhibit cell
proliferation depending on which of the ras genes is controlling
the cell proliferation (Chen et al. J. Biol. Chem., 1996, 271,
28259-65). It was suggested that differential modulation of
individual ras genes may be an approach to inhibit tumor growth
while minimising effects on normal cell growth. One approach to
devising successful therapy of Ha-ras-induced tumors is to
regulate, modulate or inhibit expression of the Ha-ras gene.
Antisense oligodeoxynucleotides (oligonucleotides) have been shown
to act as specific inhibitors of ras mRNA expression in cell-free
systems, in transformed cells in culture (T. Saison-Behmoaras et
al. EMBO J. 1991, 10, 1111-1118; Monia et al. J. Biol. Chem. 1996,
271, 14533), and in ras-activated tumors in vivo (Gray, G. D. et
al. Cancer Research, 1973, 53, 577).
[0016] In order to modulate the expression of the ras-gene in wild
type or in mutated form Brown et al. (Oncogene Res. 1989, 4,
243-252) propose the use of anti-ras oligodeoxy ribonucleoside
methylphosphonates which are complementary to the initiation codon
region of Balb-ras P21 mRNA (a mouse version of the human Ha-ras
Gene). However, it is well known that methyl phosphonates show some
major disadvantages compared to phosphorothioates, among other
their poor cellular uptake.
[0017] Monia et al. (J. Biol. Chem. 267, 19954-19962, 1992; WO
92/22651) disclose all-PS antisense oligonucleotides and antisense
oligonucleotides containing various percentages of PS linkages (WO
94/08003) directed to the translation initiation site or to codon
12 of the human Ha-ras gene, which however, have the
above-mentioned disadvantages concerning the cellular uptake and
their poor stability.
[0018] Pirollo et al. (Biochem. Biophys. Research Comm. 230,
196-201, 1997) propose the use of anti-ras all-PS
oligodeoxynucleotides in order to reverse radio resistance of
cancer cells. However, these antisense oligonucleotides show the
disadvantages (poor cellular uptake, unstability) as mentioned
above.
[0019] Therefore, this invention aims to provide an
oligonucleotide, modified to improve its stability and cell uptake,
complementary to Ha-ras mRNA which specifically regulates,
modulates or inhibits expression of the Ha-ras gene in the form of
its wild type as well as in its mutated forms, and which can be
used to inhibit the proliferation of cancer cells, to reverse
radio-resistance in cancer cells, and to treat conditions arising
from abnormal expression of the Ha-ras gene.
[0020] According to the invention, this problem is solved by
providing a modified oligodeoxynucleotide of the sequence SEQ ID
NO. 1 5'-TxAxTxTxCxCxGxTxCxAxT-3'-O--PO.sub.2--O--R
[0021] wherein
[0022] x is o or s
[0023] A is 2'-deoxyadenosine,
[0024] G is 2'-deoxyguanosine,
[0025] C is 2'-deoxycytidine and
[0026] T is thymidine.
[0027] The modified oligodeoxynucleotide according to the invention
is particularly characterized in that it has one of the following
sequences
[0028] (a) 5'-TsAsToToCsCoGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0029] (b) 5'-TsAsToToCsCoGsToCsAsT-3'-O--PO.sub.2--O--R,
[0030] (c) 5'-TsAoTsToCsCoGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0031] (d) 5'-TsAoTsTsCsCoGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0032] (e) 5'-TsAoTsToCsCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0033] (f) 5'-TsAoTsTsCsCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0034] (g) 5'-TsAsToToCsCsGsToCsAsT-3'-O--PO.sub.2--O--R,
[0035] (h) 5'-TsAoTsTsCsCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0036] (i) 5'-TsAsTsTsCsCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0037] (i) 5'-TsAsToToCoCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0038] (k) 5'-TsAsTsToCsCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0039] (l) 5'-TsAsTsToCsCoGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0040] (m) 5'-TsAsToTsCsCoGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0041] (n) 5'-TsAsToToCsCsGoTsCoAsT-3'-O--PO.sub.2--O--R,
[0042] (o) 5'-TsAsToTsCsCoGoTsCoAsT-3'-O--P.sub.2--O--R,
[0043] (p) 5'-TsAoTsTsCoCsGoTsCoAsT-3'-O--PO.sub.2--O--R,
[0044] (q) 5'-TsAsTsToCsCsGoToCsAsT-3'-O--PO.sub.2--O--R,
[0045] (r) 5'-TsAsTsToCoCsGoToCsAsT-3'-O--PO.sub.2--O--R,
[0046] (s) 5'-ToAoTsToCsCoGoToCsAsT-3'-O--PO.sub.2--O--R or
[0047] (t) 5'-TsAsTsTsCsCsGoTsCsAsT-3'-O--PO.sub.2--O--R,
[0048] wherein all the variables o, s, R, m, n and q and A, G, C
and T have the above-mentioned meanings.
[0049] The modified oligonucleotide according to the present
invention is complementary to the DNA or RNA deriving from the
human Ha-ras gene. The oligonucleotide is complementary to the
translation initiation region of Ha-ras mRNA. Therefore it inhibits
both wild-type and mutant ras expression resulting in inhibition of
tumor cell proliferation. The oligonucleotide is modified to
improve its stability and cell uptake characteristics. The
oligonucleotide contains four to nine phosphorothioate linkages at
certain positions which are especially vulnerable to attack by
nucleases. The oligonucleotide is modified at the 3'-end with a
C.sub.1-C.sub.21-alkyl or with a C.sub.1-C.sub.21-alkyl, preferably
C.sub.16-alkyl chain, covalently attached through either a
phosphodiester bridge, an oligoethyleneglycol phosphodiester
linkage, or a glyceryl ether phosphodiester linkage.
[0050] A particularly preferred embodiment of the present invention
is a modified oligodeoxynucleotide which has the sequence
5'-TsAsToToCsCoGoTsCsAsT-3'-O--PO.sub.2--O--R containing six
phosphorothioate linkages, wherein the variables o, s, R, n, m and
q and A, G, C and T have the above-mentioned meanings.
[0051] R is particularly preferably
--CH.sub.2CH(OH)CH.sub.2O(CH.sub.2).su- b.15CH.sub.3,
--(CH.sub.2).sub.15CH.sub.3 or --(CH.sub.2CH.sub.2O).sub.n---
(CH.sub.2).sub.15CH.sub.3, wherein n is an integer from 1 to 6;
preferably, n is 1, 2 or 3.
[0052] The invention further relates to the preparation of a
modified oligonucleotide. Preferably the oligonucleotide is
synthesized using standard phosphoramidite chamistry as outlind in
examples 1 and 2. Phosphorothioate linkages are for example
introduced by sulfurization using the Beaucage reagent.
[0053] Surprisingly it was found that particularly the glycerol
alkyl ether, i.e. the group
--CH.sub.2--CH(OH)CH.sub.2O--(CH.sub.2).sub.q--CH.s- ub.3, q being
an integer from 7 to 20, in particular the group
--CH.sub.2CH(OH)CH.sub.2O(CH.sub.2).sub.15CH.sub.3, linked by a
phosphodiester linkage to the 3'-end of the oligonucleotide renders
an oligonucleotide which no longer depends on the mixing with
uptake enhancers for optimal biological activity. This is the first
time that an oligonucleotide has been found to exhibit the same
biological activity with and without the addition of an uptake
enhancer.
[0054] The invention further relates to the preparation of a
modified oligonucleotide. Preferably the oligonucleotide is
synthesized using standard phosphoramidite chemistry as outlined in
examples 1 and 2. Phosphorsthioate linkages are for example
introduced sulfuization using the Beaucage reagent.
[0055] The modified oligodeoxynucleotide according to the present
invention as described above is particularly suitable for use as a
pharmaceutical. Therefore, a further object of the present
invention is the modified olidodeoxynucleotide as described above
for use as a pharmaceutical, in particular a pharmaceutical
formulation which contains an effective amount of at least one
modified oligodeoxynucleotide according to the invention. More
particularly, the oligodeoxynucleotide according to the present
invention is suitable for preparing a pharmaceutical for the
treatment of a disease arising from the overexpression and/or
mutation of the Ha-ras gene or diseases arising from a
hyperproliferative disorder. Such diseases are in particular
cancer, restenosis, or psoriasis.
[0056] Transforming activation of ras occurs in approximately
10-20% of human tumors by amplification of the ras gene at the DNA
level or by overexpression of the ras protein at the mRNA level.
Ras genes can be activated by point mutation at codon 12, 13, or 61
(Barbacid, M. Ann. Rev. Biochem. 1987, 56, 779-827; Bos, supra;
Lowy, supra). Point mutation of the Ha-ras gene at codon 12
converts the normally regulated protein to one that is continually
active. It is thought that the loss of regulation of normal ras
protein function is responsible for the transformation from normal
to malignant cell growth.
[0057] The modified oligodeoxynucleotide according to the invention
is particularly suitable for the regulation, modulation, or
inhibition of Ha-ras gene expression. The oligodeoxynucleotide is
complementary to the translation initiation region of Ha-ras mRNA.
Therefore, it inhibits both wild-type and mutant ras expression
resulting in inhibition of tumor cell proliferation. The
oligodeoxynucleotide according to the invention is further modified
to improve its stability and cell uptake characteristics. The
oligodeoxynucleotide contains four to nine, preferably six,
phosphorothioate linkages at certain positions which are especially
vulnerable to attack by nucleases. The cellular uptake of the
oligodeoxynucleotide is further improved by a
C.sub.8-C.sub.21-alkyl group or by an C.sub.1-C.sub.21-alkyl group
covalently attached through either a phosphodiester bridge, an
oligoethyleneglycol phosphodiester linkage, or a glyceryl ether
phosphodiester linkage. Due to the improved stability and to the
improved cellular uptake the oligodeoxynucleotide according to the
present invention depends no longer on the mixing with uptake
enhancers and is rendered for optimal biological activity.
[0058] Therefore, the oligodeoxynucleotide according to the present
invention is particularly suitable for preparing a pharmaceutical
which is directed to the regulation modulation or inhibition of
Ha-ras gene expression of both, of the Ha-ras wild type gene and
its mutants.
[0059] In case that the disease resulting from mutation of the
Ha-ras gene is cancer, the oligonucleotide according to the
invention is particularly suitable for preparing a pharmaceutical
which inhibits the proliferation of cancer cells.
[0060] However, on the other hand the oligodeoxy nucleotide is
advantageously used for preparing a pharmaceutical against cancer
which is used in combination with chemotherapy, in particular in
cases where the cancer cells have become resistant to chemotherapy,
since the oligodeoxynucleotide according to the invention reverses
chemoresistance in cancer cells. Therefore, a further object of the
present invention is the use of the modified oligodeoxynucleotide
as described above for preparing a pharmaceutical which reverses
the chemoresistance in cancer cells.
[0061] In particular due to this particular effect the
oligonucleotide according to the present invention is suitable for
preparing a pharmaceutical formulation which contains an effective
amount of at least one further chemotherapeutically effective
agent. Such chemotherapeutically effective agents are for example
cis-platinum and its derivatives, preferably cis-platinum, N-lost
derivatives, preferably cyclophosphamide, trofosfamide and
ifosfamide, aziridine derivatives, preferably thiothepa,
N-nitrosourea derivatives, folic acid antagonists, preferably
methotrexate, analogues of purine and pyrimidine bases, preferably
5-fluorouracil, cytostatically effective antibiotics, preferably
adriamycine, mitomycine and daunorubicine, estrogene antagonists,
preferably tamoxifene, and nucleoside derivatives, preferably MDL
101, 731 ((E)-2'-deoxy-2'-(fluoromethylene)cytidine; Cancer
Research 54, 1485-1490, 1994).
[0062] Ras genes are known to be involved in signal-transduction of
various factors for growth, differentiation and oncogenesis. Recent
studies have implicated the raf-1 oncogene, which is downstream
from ras in the signal transduction pathway, in the expression of
the radiation resistance phenotype. It can be shown that the
antisense oligonucleotide according to the present invention which
is directed to the initiation codon of Ha-ras reverses the
radiation resistance level of cancer cells.
[0063] Therefore, a further object of the present invention is the
use of the modified oligodeoxynucleotide according to the invention
for preparing a pharmaceutical for the treatment of cancer in
combination with radiotherapy. Particularly, the modified
oligodeoxynucleotide according to the present invention is suitable
for preparing a pharmaceutical which reverses the radioresistance
in cancer cells.
[0064] The modified oligonucleotide according to the invention is
prepared using an 1-alkanol and oligoethyleneglycol monoalkyl
ether, respectively, in conjunction with the 3'-derivatization
method described in EP 0 552 767 A2 and EP 0 552 766 A2; or a
glyceryl ether phosphodiester linkage, using the solid-support
described in Example 1.
EXAMPLES
[0065]
1TABLE 1 List of Oligonucleotides used in Examples. Modification
Code Partial SEQ ID NO. 2: PPS Phosphorothioate,
5'-TsAsToToCsCoGoTsCsAsT-3' Antisense. Partial SEQ ID NO. 3: PPS-S
Phosphorothioate, 5'-AsTsGoAoCsGoGoAsAsTsA-3' Sense. Partial SEQ ID
NO. 4: PPS-C16-S Phosphorothioate 5'-AsTsGoAoCsGoGoAsAsTsA-(PO.sub-
.2-- Sense + 3'-C16-
O--CH.sub.2CH(OH)CH.sub.2O(CH.sub.2).sub.15CH.- sub.3)-3' alkyl
Partial SEQ ID NO. 5: PPS-C16 Phosphorothioate
5'-TsAsToToCsCoGoTsCsAsT-(PO.sub.2--O-- Antisense + 3'-C16-
CH.sub.2CH(OH)CH.sub.2O(CH.sub.2).sub.15CH.sub.3)-3' alkyl
Example 1
[0066] Synthesis of Solid Support for the Synthesis of
Oligonucleotides Functionalised at the 3'-End with a Glycerol
Hexadecyl Ether Linked by a Phosphodiester Linkage.
[0067] DL-a-Hexadecylglycerol (1 mmol) was dried by coevaporation
with 3.times.5 ml anhydrous pyridine, then dissolved in anhydrous
pyridine (3 ml). The solution was cooled to 0.degree. C. and
4,4'-dimethoxytrityl chloride (1.15 mmol) was added. The reaction
was stirred overnight. The reaction was quenched by the addition of
water (100 ml) and evaporated to dryness. The residue was taken-up
in dichloromethane (20 ml) and extracted 3 times with 10 ml 0.1M
phosphate buffer pH7. The organic phase was dried over sodium
sulfate, filtered and evaporated. The residue was dried by
coevaporation with 2.times.5 ml anhydrous pyridine, then dissolved
in anhydrous pyridine (2 ml). 4,4-Dimethylaminopyridine (1.4 mmol),
and succinic anhydride (2.1 mmol) were added. The reaction was
stirred overnight. The reaction solution was evaporated to dryness
and coevaporated twice with toluene:dichloromethane/1:1. The
residue was taken-up in dichloromethane (20 ml), extracted with 10%
citric acid (11 ml) and washed 3 times with water (11 ml). Two
drops of triethylamine were added to the organic phase which was
then dried over sodium sulfate, filtered and evaporated. The
product was purified by silica gel chromatography using a gradient
of 1-4% methanol, 1% triethylamine in dichloromethane. The product
was obtained as a clear oil in 510 mg (62%) yield. The product
(0.056 mmol), N-ethylmorpholine (0.07 mmol) and TBTU (0.056 mmol)
were dissolved in DMF (2 ml) and added to aminopropyl
controlled-pore glass (CPG; 500 mg; 500 .ANG., Fluka). The mixture
was shaken for 4 h and the CPG was filtered-off and washed with
methanol and dichloromethane. The CPG was capped for 1 h using
acetic anhydride/N-methyl imidazole in THF, filtered and washed
with methanol, dichloromethane, THF and diethyl ether. After drying
in vacuo the loading of dimethoxytrityl groups was determined to be
80 mmolg.sup.-1.
Example 2
[0068] Oligonucleotide Synthesis
[0069] The oligonucleotides were synthesized using an Applied
Biosystems 394 DNA synthesizer (Applied Biosystems, Inc., Foster
City, USA) and standard phosphoramidite chemistry. After coupling,
phosphorothioate linkages were introduced where required by
sulfurization using the Beaucage reagent (Iyer et al., 1990)
followed by capping with acetic anhydride and N-methylimidazole in
tetrahydrofuran. After cleavage from the solid support and final
deprotection by treatment with conc. ammonia, the oligonucleotides
were purified by gel electrophoresis. For the synthesis of the
hexadecyl-modified oligonucleotides the solid support described in
Example 1 above was employed. All oligonucleotides were analyzed by
negative ion electrospray mass spectroscopy (Fisons Bio-Q) which in
all cases confirmed the calculated mass.
Example 3
[0070] Inhibition of In Vitro Translation of Ha-Ras mRNA.
[0071] 0.06 mg RNA was annealed with the appropriate
oligonucleotide by heating them together in 10 mM Tris-HCl, pH 7.5,
5 mM MgCl.sub.2, 25 mM KCl and 1 mM DTT at 90.degree. C. for 2
minutes and cooling slowly to room temperature. The translation
reaction mixture (10 .mu.l) consisted of 0.06 mg of RNA, varying
amounts (1, 5, 10 or 20 .mu.M) of oligonucleotide, 8 U of RNasin
Ribonuclease Inhibitors (40 U/.mu.l) (Promega Biotec), 20 .mu.M of
amino acid mixture minus methionine (Promega Biotec), 0.8 mCi of
.sup.35S-methionine (>1000 mCi/.mu.M, Amersham), 3-6 .mu.l of
nuclease-treated Rabbit Reticulocyte Lysate (Promega Biotec), and 1
.mu.M dithiothreitol (DTT). Translation proceeded for 1 hour at
30.degree. C.
[0072] After translation, 10 .mu.l of RIPA buffer (PBS, 1% NP40,
0.5% sodium deoxycholeate, 0.1% SDS) with fresh inhibitors (0.1
mg/.mu.l PMSF, 1 mM sodium orthovanadate, 30 ml/.mu.l aprotinin,
Sigma, Cat. #A6279) was added to each sample, and the mixture
incubated on ice for 20 minutes. 7 .mu.l of 4.times.SDS sample
buffer containing 1.25 M Tris-HCl pH 6.8, 6% SDS, 40% glycerol, 12%
2-mercaptoethanol and 0.001% bromphenol blue was added, and the
sample was boiled for 5 minutes. 10 .mu.l of each sample was
electrophoresed in a 5-12.5% polyacrylamide gel (PAGE). The
completed gel was fixed, dryed, and exposed to Kodak XAR-5 film
with an intensifying screen at -80.degree. C.
[0073] Results:
[0074] The claimed antisense oligonucleotide 5'-TsAsT T CsC G
TsCsAsT-(PO.sub.2--O--CH.sub.2CH(OH)CH.sub.2O(CH.sub.2).sub.15CH.sub.3)-3-
' (PPS-C16) inhibited translation of ras p21 protein by
approximately 10, 40, 50 and 80% at concentrations of 1, 5, 10, and
20 .mu.M respectively. Controls using the sense oligonucleotide
5'-AsTsG A CsG G
AsAsTsA-(PO.sub.2--O--CH.sub.2CH(OH)CH.sub.2O(CH.sub.2).sub.15CH.sub.3)-3-
' (PPS-C16-S) showed no inhibition of translation until 20 .mu.M at
which point an 11% inhibition was observed. This strongly suggests
that the C16 alkyl chain does not give rise to non-sequence
specific effects in this system.
Example 4
[0075] Inhibition of p21 Ras Protein Expression in Cell Culture
[0076] Cell Lines:
[0077] NIH3T3 mouse fibroblasts (Chang et al. Biochemistry 30
(1991) 8283-86) transformed by the human Ha-ras (RS485) were used
as an experimental cell line to evaluate the sequence-specific
inhibition of cell growth and p21 ras protein expression by
oligonucleotides directed to the first eleven nucleotides of the
initiation codon region of Ha-ras mRNA.
[0078] RS485 cells were grown in Dulbecco's Modified Eagle's Medium
(DMEM, Flow Labs) supplemented with 10% heat inactivated (30 min.
at 65.quadrature.C) fetal calf serum (FCS), antibiotics (50 U/.mu.l
each of penicillin, streptomycin, and neomycin), and 4 mM of
L-glutamine.
[0079] Oligonucleotide Treatment of RS485 Cells in Culture
[0080] RS485 cells were seeded in 6-well plates (105 cells per
well) and, after 20-24 hours (40-60% confluency), were treated with
oligonucleotides. Medium was removed from the wells, 1 ml of fresh
medium with oligonucleotides at various concentrations was added to
each well, and the plates were incubated at 37.degree. C. in an
humidified atmosphere containing 5% CO.sub.2/95% air for 24-48
hours.
[0081] Oligonucleotide Lipofection of RS485 Cells in Culture
[0082] RS485 cells (40-60% confluency) were transfected with
oligonucleotides using Lipofectin.TM. Reagent and the supplier's
protocol (from Life Technologies). Briefly, varying concentrations
of oligonucleotides were diluted in 100 .mu.l of serum-free medium.
In a separate tube Lipofectin was added to serum-free medium at a
concentration of 7 .mu.l/100 .mu.l. After 30 minutes at room
temperature, 100 .mu.l of Lipofectin solution was mixed with 100
.mu.l of oligonucleotide solution, incubated for an additional 15
minutes at room temperature, and mixed with 800 .mu.l of serum-free
medium. Cells were washed two times with serum-free medium, and the
total solution (1 ml) containing the oligonucleotide-Lipofectin
complex was overlayed onto the cells. After 6 hours, 1 ml of fresh
medium containing 8 mM L-glutamine and 20% calf serum was added,
and the cells were incubated for an additional 38 hours. Treated
cells were washed with PBS, harvested with trypsin, and cellular
proteins were extracted.
[0083] Results:
[0084] The effect of PPS and PPS-C16 oligonucleotides on p21
expression in RS485 cells was compared in the absence or presence
of Lipofectin (supra). After treatment total protein was extracted
and p21 levels were determined by Western Blot analysis
(infra).
[0085] Lipofectin alone had no effect on p21 expression. Both the
PPS- and PPS-C16-Lipofectin complexes almost completely inhibited
p21 expression in RS485 cells at 1 .mu.M concentration.
Surprisingly the PPS-C16 oligonucleotide without Lipofectin
completely inhibited p21 expression at 5 .mu.M concentration. The
PPS oligonucleotide without a C16 modification inhibited p21
expression by only 50% at the same concentration.
[0086] A comparison was then made of the effect of PPS-Lipofectin
complex and PPS-C16 alone on p21 expression in RS485 cells. The p21
level was decreased by 30% and 43% at 0.25 .mu.M and 0.5 .mu.M
PPS-Lipofectin concentrations respectively. Remarkably, the PPS-C16
oligonucleotide without any added Lipofectin showed approximately
the same level of inhibition, namely 33% at 0.25 .mu.M and 40% at
0.5 .mu.M. Complete (100%) inhibition of p21 expression was
observed at 1 .mu.M concentration, indicating that Lipofectin
delivery does not augment PPS-C16-mediated inhibition.
[0087] Finally, a comparison of the inhibition of p21 expression in
RS485 cell by PPS and PPS-C16 in the absence of Lipofectin was
made. PPS alone gave an inhibition of approximately 50% at 5 .mu.M.
However, at a concentration of only 0.75 .mu.M the PPS-C16
oligonucleotide exhibited nearly 60% inhibition of p21 expression
in RS485 cells.
Example 5
[0088] Inhibition of Cell Growth and p21 Ras Protein Synthesis in
Cell Culture
[0089] Cell Growth Inhibition Studies
[0090] Cells treated with antisense oligonucleotides, or cells
transfected with the oligonucleotide-Lipofectin complexes were
harvested and lysed in RIPA buffer. Total protein levels in each
well were determined spectrophotometrically. Inhibition of cell
growth was estimated as the ratio of protein (mg) in the
oligonucleotide treated cells compared with that in the control.
The control was either protein from cells treated with Lipofectin
only, or protein from untreated cells.
[0091] Western Blot Analysis of Ras Protein
[0092] Total cellular protein was prepared by lysing cells in RIPA
buffer with freshly added inhibitors (PMSF, aprotinin, sodium
orthovanadate) and homogenizing through 21-gauge needles according
to the protocol provided by Santa Cruz Biotechnology, Inc. The
extracts were cleaned by centrifugation at 15,000.times.g for 20
min at 4.degree. C. and the protein concentration of the
supernatant was determined. 40 .mu.g of protein was size
fractionated in 5-12.5% PAGE and electroblotted onto a
nitrocellulose membrane. The membrane was incubated with primary
antibody against Ha-Ras (C-20) (Santa Cruz Biotechnology, Inc.) and
then with Horseradish Peroxidase-conjugated goat anti-rabbit
immunoglobulin G. The ECL chemo-luminescent Western system
(Amersham, Arlington Heights, Ill.) was used to detect secondary
probes.
[0093] Results:
[0094] A series of experiments were performed to study the
sequence-sequence toxic effect of the PPS and PPS-C16
oligonucleotides on RS485 cells. FIG. 6A shows that treatment of
RS485 cells with 1-5 .mu.M of PPS and PPS-C16 in the presence of
Lipofectin (supra) reduces cell growth by approximately 35-50% over
a 2 day period. Lipofectin alone has a slight toxic effect (ca.
15%) on cells. Comparison of the Western blot analysis of p21
expression inside RS485 cells (supra) with the cell growth
inhibition by PPS and PPS-C16 demonstrates that a 1 .mu.M
oligonucleotide/Lipofectin complex concentration totally inhibits
p21 expression, but inhibits cell growth by only 35%.
[0095] FIG. 6B shows that PPS-C16 without Lipofectin inhibits cell
growth by 11% at 0.75 .mu.M while inhibiting p21 expression by 60%
(supra). PPS inhibits cell growth by 22% at a concentration of 5
.mu.M, a concentration at which it inhibits p21 expression by 50%.
FIG. 6C shows that Lipofectin exerts a toxic effect when used with
PPS at concentrations higher than 1 .mu.M and that the PPS-C16
oligonucleotide without Lipofectin is, in the concentration range
0.25-1 .mu.M, just as effective as the PPS oligonucleotide with
Lipofectin.
Example 6
[0096] Reversal of Radioresistance--Animal Study
[0097] Antitumor Effects of Antisense Oligonucleotides In Vivo
[0098] RS504 cells are NIH 3T3 cells transformed by the Ha-ras
oncogene isolated from EJ/T24, a human bladder carcinoma. Tumor
were induced by injecting 5.times.10.sup.6 RS504 cells subcutaneous
in female athymic NCr-nu mice. Forty-eight hours later, when tumors
were evident (-6 mm.sup.3), partially phosphorothioated
C16-modified [PPS-C16] antisense [AS] or sense [S] oligonucleorides
were injected directly into the tumors at a concentration of 50
.mu.l of 5 .mu.M solution (250 pmol) per tumor. After 24 hours,
tumors were irradiated with a 2.0 Gy dose (thin arrows, see FIG.
1). Irradiation was repeated every 48 hours until an accumulated
dose of 20 Gy had been delivered. Oligonucleotide injections were
repeated at 48 hours [50 .mu.l], 192 hours [50 .mu.l), and 240
hours [100 .mu.l] following the initial injection (bold arrows, see
FIG. 1). Tumor volume was recorded in mm.sup.3. Controls consisted
of tumors that were not injected with oligonucleotides and not
irradiated, tumors that were not injected with oligonucleotides but
were irradiated; and tumors injected with antisense PPS-C16
oligonucleotides but not irradiated.
[0099] Tumor Growth
[0100] Ras genes are known to be involved in signal-transduction of
various factors for growth, differentiation and oncogenesis. Recent
studies have implicated the raf-1 oncogene, which is downstream
from ras in the signal transduction pathway, in the expression of
the radiation resistence phenotype. It can be shown that the
antisense oligonucleotide according to the invention reverses the
radiation resistance level of the cells. To determine if there were
differences in the effects of radiation on ras-induced tumors
following treatment with oligonucleotides, RS 504-induced tumors in
nude mice were treated with antisense and sense PPS-C16
oligonucleotides. FIG. 1 shows that antisense PPS-C16
oligonucleotide treatment in conjunction with radiation was most
effective in inhibiting tumor growth during the 17 days of
observation and treatment.
Example 7
[0101] Reversion of Radioresistance in Cell Culture
[0102] Reversion of radioresistance phenotype in cell culture by
treatment with PPS-C16 Bladder (T24) carcinoma cell lines (obtained
from ATCC, Rockville, Md.) were maintained in McCoy's 5A medium
supplemented with 10% fetal bovine serum 50 .mu.g/ml each of
penicillin, streptomycin anfd neomycin and 2 mM L-glutamine.
[0103] For oligonucleotide treatment, the cells were plated at
1.times.10.sup.5 cells/well in 6-well tissue culture plates.
Twenty-four hours later, at approximately 40-60% confluency, the
cells were transfected with oligonucleotides, facilitated by
Lipofectin Reagent, using essentially the protocol supplied by the
manufacturer, Life Technologies, Inc. After 6 hours, the
lipofection solution was removed and the monolayer washed with
fresh medium containing 8 mM L-glutamine and 20% serum. The cells
were then incubated for an additional 16-18 hours in 1 ml of this
medium. Cellular response to radiation was evaluated by the colony
survival assay. Exponentially growing monolayer cultures of each
cell line were treated with the oligonucleotides as described
above. the cells were harvested 24-48 hours later, suspended in
fresh medium and irradiated at room temperature with graded doses
of .sup.137Cs.gamma. rays at a dose of approximately 36 Gy/minute
in a J.L. Shephard and Associates Mark I irradiator. Afterward, the
cells were diluted and plated at a concentration of 300 to 5000
cells per well in a 6-well tissue culture plate. Two to three days
after plating, the cells were supplemented with 0.5 ml of serum
plus 5 .mu.g/ml hydrocortisone. Approximately 7-14 days later, the
cells were stained with 1% crystal violet and colonies (comprising
50 or more cells of normal appearance) were scored. The
D.sub.10-value (radiation dose required to reduce survival of cells
to 10%) for antisense-treated (PPS-C16) T24 cells drops from the
highly resistant level of 5.5 (untreated or sense-treated
PPS-C16-S) to 4.5 Gy, a value dose to what is considered to be
radiosensitive. These results are shown in FIG. 2.
Sequence CWU 1
1
2 1 11 DNA Artificial Sequence Description of Artificial Sequence
synthetic DNA 1 tattccgtca t 11 2 11 DNA Artificial Sequence
Description of Artificial Sequence synthetic DNA 2 atgacggaat a
11
* * * * *