U.S. patent application number 12/436493 was filed with the patent office on 2010-11-11 for methods for increasing in vivo efficacy of oligonucleotides and inhibiting inflammation in mammals.
This patent application is currently assigned to TOPIGEN PHARMACEUTIQUE INC.. Invention is credited to Zoulfia ALLAKHVERDI, Mustapha ALLAM, Paolo RENZI.
Application Number | 20100286235 12/436493 |
Document ID | / |
Family ID | 23170407 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286235 |
Kind Code |
A1 |
RENZI; Paolo ; et
al. |
November 11, 2010 |
METHODS FOR INCREASING IN VIVO EFFICACY OF OLIGONUCLEOTIDES AND
INHIBITING INFLAMMATION IN MAMMALS
Abstract
The invention relates to the use of nucleotide substitutes for
increasing the in vivo efficacy of nucleic acid molecules and also
for inhibiting inflammation in mammals. More particularly, the
present invention relates to the use of 2'6' diaminopurine (DAP)
and analogs thereof per se in anti-inflammatory compositions, and
also for preparing nucleic acid molecules having an increased in
vivo physiological efficiency and a reduced toxicity as compared to
conventional oligos. The invention is particularly useful for the
preparation of antisense oligonucleotides for treating
pulmonary/respiratory diseases such as cystic fibrosis, asthma,
chronic bronchitis, chronic obstructive lung disease, eosinophilic
bronchitis, allergies, allergic rhinitis, pulmonary fibrosis, adult
respiratory distress syndrome, sinusitis, respiratory syncytial
virus or other viral respiratory tract infection and cancer.
Inventors: |
RENZI; Paolo; (Westmount,
CA) ; ALLAM; Mustapha; (Montreal, CA) ;
ALLAKHVERDI; Zoulfia; (Montreal, CA) |
Correspondence
Address: |
NIXON PEABODY, LLP
401 9TH STREET, NW, SUITE 900
WASHINGTON
DC
20004-2128
US
|
Assignee: |
TOPIGEN PHARMACEUTIQUE INC.
MONTREAL
CA
|
Family ID: |
23170407 |
Appl. No.: |
12/436493 |
Filed: |
May 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10482949 |
Aug 16, 2004 |
7745420 |
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PCT/CA02/01046 |
Jul 8, 2002 |
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12436493 |
|
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60303071 |
Jul 6, 2001 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
A61K 31/7076 20130101;
C07H 19/16 20130101; A61P 1/04 20180101; A61P 13/00 20180101; C12N
15/1138 20130101; A61P 37/02 20180101; C12N 2310/315 20130101; A61P
17/00 20180101; A61P 9/00 20180101; A61P 29/00 20180101; C07H 21/00
20130101; A61P 11/06 20180101; A61K 38/00 20130101; A61P 37/00
20180101; A61P 25/28 20180101; A61P 11/02 20180101; A61P 37/04
20180101; A61P 31/00 20180101; C12N 15/113 20130101; A61P 43/00
20180101; A61P 11/00 20180101; C07H 21/04 20130101; A61P 31/14
20180101; A61P 31/12 20180101; A61P 25/00 20180101; C07H 19/23
20130101; A61P 35/00 20180101; C12N 2310/333 20130101; A61P 27/02
20180101 |
Class at
Publication: |
514/44.A ;
536/23.1 |
International
Class: |
A61K 31/711 20060101
A61K031/711; C07H 21/04 20060101 C07H021/04; C07H 21/00 20060101
C07H021/00; C07H 21/02 20060101 C07H021/02; A61K 31/7105 20060101
A61K031/7105; A61P 37/00 20060101 A61P037/00 |
Claims
1. An oligonucleotide comprising an antisense oligonucleotide
against the common beta subunit of IL-3, IL-5 and GM-CSF, wherein
the oligonucleotide comprises at least one adenosine and said
adenosine has been replaced with a nucleotide substitute, wherein
the nucleotide substitute is 2-amino-2'-deoxyadenosine or a salt
thereof.
2. The oligonucleotide of claim 1, wherein the nucleotide
substitute is 2-amino-2'-deoxyadenosine.
3. The oligonucleotide of claim 2, wherein said oligonucleotide is
a DNA oligonucleotide.
4. The oligonucleotide of claim 2, wherein said oligonucleotide is
an RNA oligonucleotide.
5. The oligonucleotide of claim 2, wherein the oligonucleotide is
selected from the group consisting of SEQ ID NOs: 1-18, 23 and
26.
6. The oligonucleotide of claim 1, wherein the oligonucleotide
comprises SEQ ID NO. 8.
7. The oligonucleotide of claim 6, wherein the oligonucleotide
consists of SEQ ID NO. 8.
8. The oligonucleotide of claim 1, wherein the oligonucleotide has
at least one mononucleotide linking residue selected from the group
consisting of methylphosphonate, phosphotriester, phosphorothioate,
phosphodiester, phosphorodithioate, boranophosphate, formacetal,
thioformacetal, thioether, carbonate, carbamate, sulfate,
sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide,
sulfide, hydroxylamine, methylene (methyimino), methyleneoxy
(methylimino), and phosphoramidate residues.
9. A pharmaceutical composition comprising the oligonucleotide of
claim 1 and a pharmaceutically acceptable carrier.
10. A method for treating and/or preventing a disease, comprising
administering a pharmaceutical composition of claim 9 to a subject
in need thereof, wherein the disease is selected from the group
consisting of respiratory system diseases, neurological diseases,
cardiovascular diseases, rheumatological diseases, digestive
diseases, cutaneous diseases, ophtalmological diseases, urinary
system diseases, cancers, pathogen infections, genetic diseases,
hypereosinophilia, general inflammation, and cancers.
11. The composition of claim 9, wherein said oligonucleotide is
present in an amount of about 1% to about 90% of the
composition.
12. The composition of claim 9, further comprising an agent
selected from the group consisting of drugs, antioxidants,
surfactants, flavoring agents, volatile oils, buffering agents,
dispersants, propellants, preservatives, and combinations
thereof.
13. The method of claim 10, wherein the disease is a respiratory
system disease associated with an inflammation of the lungs, the
airways and/or the nose.
14. The method of claim 13, wherein the respiratory system disease
is selected from the group consisting of pulmonary fibrosis, adult
respiratory distress syndrome, cystic fibrosis, chronic obstructive
lung disease, chronic bronchitis, eosinophilic bronchitis, asthma,
allergy, allergic rhinitis, sinusitis and hypereosinophilia.
15. The composition of claim 9, wherein the composition is
contained in a pressurized aerosol dispenser, a nasal sprayer, a
nebulizer, a metered dose inhaler, a dry powder inhaler, or a
capsule.
Description
RELATED APPLICATION
[0001] This application claims priority of U.S. patent application
Ser. No. 10/482,949 filed Aug. 16, 2004, which claims priority of
Provisional Application 60/303,071 filed Jul. 6, 2001, the
disclosure of which is incorporated by reference herein in its
entirety.
BACKGROUND OF THE INVENTION
[0002] (a) Field of the Invention
[0003] The invention relates to the use of nucleotide substitutes
for increasing the in vivo efficacy of nucleic acid molecules and
also for inhibiting inflammation in mammals.
[0004] More particularly, the present invention relates to the use
of 2'6' diaminopurine (DAP) and analogs thereof per se in
anti-inflammatory compositions, and also for preparing nucleic acid
molecules having an increased in vivo physiological efficiency and
a reduced toxicity.
[0005] (b) Brief Description of the Prior Art
[0006] Therapeutic approaches based on the use of nucleic acid
molecules are becoming more and more popular. Gene-based therapies
and antisense based-therapies will probably change radically
medicine in a near future.
[0007] The problems to date with nucleic acid molecules as
therapeutics, and more particularly with antisense
oligonucleotides, have been toxicity (both systemic and topical),
stability, and non-specific binding to cell surface proteins. The
toxicity of antisense oligonucleotides seems to vary between
species, rats being the most sensitive, although the toxicity
appears at doses higher than those that are therapeutically
effective (see ST Crooke, Hematologic Pathology, 1995, 9:5972 for a
review). In pulmonary/respiratory diseases, nucleic acid molecules
toxicity associated with the administration of therapeutic
antisenses/genes include: an increase in immune stimulation, a
mononuclear cellular-inflammatory infiltrate into the lungs, and
possibly hypersensitivity and bronchoconstriction of the
airways.
[0008] Several solutions that are less than optimum have been
proposed up to date for circumventing the toxicity problem. Among
the most popular there is the preparation of nucleic acid molecules
containing various modified DNA bases, RNA bases, and/or a modified
backbone structure. For instance, WO 99/67378 describes antisense
oligonucleotides constructs based on modified sugars. Also, Nyce
has postulated, although not demonstrated, in WO 00/09525 and WO
00/62736 that the adenosine base included in antisense
oligonucleotides for treating respiratory diseases is a major cause
of toxicity in lungs. Accordingly, Nyce proposes low adenosine
oligonucleotides and oligonucleotides wherein the adenosine base
has been replaced by an analog of adenosine. However, none of the
low adenosine oligonucleotides and none of the adenosine analogs
proposed by Nyce have ever been tested for their biological
activity or their allegedly reduced toxicity.
[0009] 2',6'-diaminopurine nucleoside (2-amino-2'-deoxyadenosine;
DAP) was found to be present in DNA in place of adenosine by the
cyanophage S-2L (Cheng, X., Annu Rev Biophys Biomol Struct 24:
293-318, 1995); Khudyakov, I. Y., et al., Virology 88: 8-18, 1978).
Since then, 2',6'-diaminopurine nucleoside (DAP) has been widely
used and studied, notably as a chemical starting point for the
synthesis of antiviral compounds such as
2-amino-2',3'-dideoxy-adenosine (not DAP) which is capable of
selectively inhibiting human immunodeficiency virus (HIV)
replication in vitro (Balzarini, J. et al., Biochem. & Biophys.
Res. Communications 145:269-76 (1987). The use of DAP in antisense
oligonucleotides or in gene therapy methods has however never been
suggested.
[0010] Also, U.S. Pat. No. 5,925,624 and No. 5,889,178 describe
derivatives of 2,6-diaminopurine-beta-D-ribofuranuronamide.
Although these derivatives have an anti-inflammatory effect (mostly
against neutrophil superoxide release) and that they could be used
in the therapy of respiratory disease, they have a chemical formula
which is different from the formula of DAP and analogs thereof.
[0011] In summary, there has been up to date no suggestion nor any
evidence that DAP per se could be used in anti-inflammatory
compositions, nor any suggestion or example that DAP and analogs
thereof could be incorporated in nucleic acid molecules (gene
constructs and antisenses) for increasing the in vivo efficacy of
these oligos.
[0012] There is thus a need for more effective anti-inflammatory
compositions comprising 2'6-diaminopurine and/or analogs
thereof.
[0013] There is also a long felt need for nucleic acid molecules
that would remain stable in the body while exhibiting high
effectiveness and low toxicity.
[0014] There is more particularly a need for nucleic acid molecules
incorporating a nucleotide substitute such as 2'6' diaminopurine
(DAP) and analogs thereof, a need for composition comprising the
same and a need for methods of using these nucleic acid molecules,
particularly in gene and antisense therapies methods. No one has
ever tested whether replacement of base(s) by a nucleotide
substitute could affect the stability, binding, degradation
efficacy and toxicity of antisense oligonucleotides, nor have they
tested such modified antisense oligonucleotides for biological
activity in cells, in culture or in animals.
[0015] The present invention fulfils these needs and also other
needs which will be apparent to those skilled in the art upon
reading the following specification.
SUMMARY OF THE INVENTION
[0016] An object of the invention is to provide nucleic acid
molecules such as gene constructs and antisense oligonucleotides
that would remain stable in the body while exhibiting high
effectiveness and low toxicity.
[0017] According to an aspect of the invention, it is provided a
method for increasing in vivo efficacy of an nucleic acid molecule
that is administered to a mammal, comprising incorporating into the
nucleic acid molecule at least one nucleotide substitute. Such an
incorporation increases in vivo physiological effectiveness of the
nucleic acid molecule and also reduces its toxicity when
administered to a mammal, as compared to an nucleic acid molecule
not incorporating the nucleotide substitute. According to a
preferred embodiment, the nucleotide substitute is incorporated
into the nucleic acid molecule for substituting therein an
adenosine base. More preferably, the nucleotide substitute is
selected from the group consisting of 2'6'-diaminopurine and
analogs thereof. Preferred 2'6'-diaminopurine analogs include
2,6-diaminopurine hemisulfate,
2-amino-9-(B-D-2'-deoxyribofuranosyl) purine,
7-Deaza-2'-deoxyadenosine, N6-methyl-2'-deoxyadenosine,
2-aminoadenosine/2,6-diaminopurine riboside, salts thereof and
functional derivatives thereof.
[0018] The invention also relates to an improved method for the in
vivo administration of at least one nucleic acid molecule to a
mammal subject. The improvement consists of incorporating into the
nucleic acid molecule at least one 2'6'-diaminopurine and/or an
analog thereof. Preferably, 2'6'-diaminopurine or its analog is
incorporated into the nucleic acid molecule for substituting
therein an adenosine base.
[0019] According to another aspect of the invention, it is provided
an isolated or purified nucleic acid molecule selected from
antisense oligonucleotides and nucleic acid molecules comprising a
sequence coding for a therapeutic gene product, the nucleic acid
molecule according to the present invention comprising a nucleotide
substitute selected from the group consisting of 2'6'-diaminopurine
and analogs thereof.
[0020] According to another aspect of the invention, it is provided
a pharmaceutical composition comprising at least one nucleic acid
molecule as defined previously and a pharmaceutically acceptable
carrier. The composition of the invention may be useful for
treating and/or preventing a disease selected from respiratory
system diseases, neurological diseases, cardiovascular diseases,
rheumatological diseases, digestive diseases, cutaneous diseases,
ophtalmological diseases, urinary system diseases, cancers,
pathogen infections, and genetic diseases, hypereosinophilia,
general inflammation, and cancers.
[0021] According to a further aspect of the invention, it is
provided a method of antisense therapy, comprising the step of
administering, directly to the respiratory system of a mammal in
need thereof, an effective therapeutic or prophylactic amount of at
least one antisense oligonucleotide as defined previously. This
method is useful for preventing and/or treating respiratory system
diseases, cancers, pathogen infections, and genetic diseases, and
more particularly respiratory system diseases associated with an
inflammation of the lungs, the airways and/or the nose such as
pulmonary fibrosis, adult respiratory distress syndrome, cystic
fibrosis, chronic obstructive lung disease, chronic bronchitis,
eosinophilic bronchitis, asthma, allergy, sinusitis, respiratory
syncytial virus or other viral respiratory tract infection and
hypereosinophilia.
[0022] According to another aspect of the invention, it is provided
a method for inhibiting inflammation in a mammal, comprising the
use of a nucleotide substitute selected from the group consisting
of 2'6'-diaminopurine and analogs thereof. Typically, 2'6'
diaminopurine or its analogue(s) are administered to the mammal.
Preferably 2'6'-diaminopurine and its analogs are used as such in
an anti-inflammatory composition, but they may be also incorporated
into nucleic acid molecules. In a related aspect, the invention
concerns an anti-inflammatory composition comprising: an adenosine
antagonist compound selected from the group consisting of
2'6'-diaminopurine and analogs thereof; and a pharmaceutically
acceptable carrier. Another related aspect concerns the use of
2'6'-diaminopurine and/or an analog thereof for the preparation of
an anti-inflammatory composition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A, 1B, 1C, and 1D shows the chemical structures of
adenine, adenosine, inosine, 2'6'-diaminopurine
(2-amino-2'-deoxyadenosine; DAP) and different analogs of DAP.
[0024] FIG. 2A are pictures of semi-quantitative PCRs showing the
biological effectiveness of different antisenses to the common Beta
chain of human GM-CSF, IL-3 and IL-5 receptor in U937 cells. 1=Non
treated cells; 2=Cells treated with antisense AS107; 3=Cells
treated with antisense AS107 containing DAP instead of 2 adenosine
bases (AS107-DAP); and M=Molecular weights markers. G3PDH 450 bp=is
the number of bases at which the G3PDH housekeeping gene is found;
GM-CSFR.beta. 340 bp: is the number of bases at which the common
Beta chain band is found.
[0025] FIG. 2B is a picture of semi-quantitative PCRs showing the
biological effectiveness of antisense to the common Beta chain of
human GM-CSF, IL-3 and IL-5 receptor in TF-1 cells. 1=Non treated
cells; 2=Cells treated with antisense AS107; 3=Cells treated with
antisense AS107 containing DAP instead of 2 adenosine bases
(AS107-DAP); and M=Molecular weights markers. G3PDH 450 bp is the
number of bases at which the G3PDH housekeeping gene is found;
.beta. chain 340 bp is the number of bases at which the common Beta
chain band is found.
[0026] FIG. 3 is a picture of semi-quantitative PCRs showing the
biological ineffectiveness in TF-1 cells of replacing adenosine by
its analog inosine in the antisense to the common Beta chain of
human GM-CSF, IL-3 and IL-5 receptor. 1=TF-1 Control (Non treated
cells); 2=Cells treated with sense AS107; 3=Cells treated with
antisense AS107; 4=Cells treated with antisense AS107 containing
inosine instead of 2 adenosine bases (AS107-I); 5=Cells treated
with antisense AS107 having a one base mismatch; and M=Molecular
weights markers. G3PDH 450 bp is the number of bases at which the
G3PDH housekeeping gene is found; .beta. chain 340 bp is the number
of bases at which the common Beta chain band is found.
[0027] FIG. 4A is a graph showing the effects, on lung resistance
of sensitized Brown Norway rats, of intratracheal administration of
an antisense phosphorothioate oligonucleotide (AS141) directed
against the common Beta chain of rat GM-CSF, IL-3 and IL-5, as
compared to the effects of the same antisense containing DAP
instead of 2 adenosine bases (AS141-DAP). Lung resistance was
measured 0-2 h after administration of a dose of 60 .mu.g of each
oligonucleotide.
[0028] FIG. 4B is a graph showing the effects, on lung resistance
of sensitized Brown Norway rats, of intratracheal administration of
an antisense phosphorothioate oligonucleotide (AS141) directed
against the common Beta chain of rat GM-CSF, IL-3 and IL-5, as
compared to the effects of the same antisense containing inosine
instead of 2 adenosine bases (AS141-Inosine). Lung resistance was
measured 0-2 h after administration of a dose of 60 .mu.g of each
oligonucleotide.
[0029] FIG. 5 is a graph showing the effects, on lung resistance of
sensitized rat, of intratracheal instillation of adenosine, DAP
(2-amino-2' deoxyadenosine) and analogs thereof.
[0030] FIG. 6A is a bar graph showing that incorporation of DAP in
oligonucleotides antisense to the rat CCR3 and the common .beta.
chain of IL-3/IL-5/GM-CSF receptors increases the in vivo
physiological effectiveness of these antisenses. Biological
activity of the antisenses were measured in the rat model of
asthma: Control unchallenged; Control challenged; Rats treated with
200 .mu.g of antisense ASA4 and AS141 (18 nucleotides); Rats
treated with 200 .mu.g of antisense ASA4 and AS141 containing DAP
instead of adenosine bases (ASA4-DAP; 141-DAP); Rats treated with
200 .mu.g of mismatch antisense ASA4 and AS141; and Rats treated
with 200 .mu.g ASA4-DAP and AS141-DAP mismatch antisense.
Responsiveness to leukotriene D4 was measured 15 hours after
ovalbumin challenge.
[0031] FIG. 6B, is a bar graph showing that the combination of two
regular and two DAP containing oligonucleotides (total 50 .mu.g) is
more effective than 50 .mu.g of each oligonucleotide alone.
[0032] FIG. 7A, is a bar graph showing that oligonucleotides
against CCR3 containing DAP are more effective at inhibiting lung
inflammation in vivo after antigen challenge than oligonucleotides
without DAP.
[0033] FIG. 7B, is a bar graph showing that oligonucleotides
against the common .beta. chain of IL-3/IL-5/GM-CSF receptors
containing DAP are more effective at inhibiting lung inflammation
in vivo after antigen challenge than oligonucleotides without
DAP.
[0034] FIG. 7C, is a bar graph showing that the combination of two
DAP containing oligonucleotides (total 50 .mu.g) is more effective
at inhibiting lung inflammation in vivo after antigen challenge
than the combination of two regular oligonucleotides without
DAP.
[0035] FIG. 8 is a bar graph showing that adenosine selectively
increases eosinophil recruitment into the lungs of rats whereas DAP
does not, and that DAP is an antagonist of the pro-inflammatory
effects of adenosine.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention relates to nucleic acid molecules such
as gene constructs and antisenses that would remain stable in the
body while exhibiting high effectiveness and low toxicity. It also
relates to the use of 2'6'-diaminopurine and analogs for inhibiting
inflammation.
[0037] According to an aspect of the invention, there is provided a
method for increasing in vivo efficacy of an nucleic acid molecule
that is administered to a mammal. This method comprises the step of
incorporating into the nucleic acid molecule at least one
nucleotide substitute. As it will be shown in the examples herein
after, such an incorporation increases the in vivo physiological
effectiveness of the nucleic acid molecules and also reduces the
toxicity of the nucleic acid molecules when administered to a
mammal, as compared to nucleic acid molecules not incorporating the
nucleotide substitute.
[0038] The "reduced toxicity" of the nucleic acid molecules may be
evaluated using principles known in the art. According to a
preferred embodiment of the invention, the nucleotide substitute is
selected so that nucleic acid molecules incorporating the
nucleotide substitute exhibit lower in vivo inflammatory
properties, and thereby have a reduced toxicity. More preferably,
the nucleotide substitute is selected so that the nucleic acid
molecules incorporating this modification are capable of inhibiting
recruitment of lymphocytes, eosinophils, macrophages and/or
neutrophils at a site where these nucleic acid molecules are
administered and/or at a site of disease.
[0039] According to a preferred embodiment of the invention, the
nucleotide substitute is incorporated into the nucleic acid
molecule for substituting therein an adenosine base. Preferably,
the nucleotide substitute is 2'6'-diaminopurine (see FIG. 1) or an
analog thereof. As used herein, "analogs of 2'6'-diaminopurine"
include all compounds having a similar structure and substantially
the same biological activity/efficacy of 2'6'-diaminopurine.
Preferred 2'6'-diaminopurine analogs are also shown in FIG. 1 and
include: 2,6-diaminopurine hemisulfate (1H-purine-2,6-diamine,
sulfate (2:1); CAS 69369-16-0),
2-amino-9-(B-D-2'-deoxyribofuranosyl) purine (CAS 3616-24-8),
7-Deaza-2'-deoxyadenosine (CAS 60129-59-1),
N6-methyl-2'-deoxyadenosine (CAS 2002-35-9),
2-aminoadenosine/2,6-diaminopurine riboside (CAS 2096-10-08), and
salts thereof.
[0040] Among the 2'6'-diaminopurine analogs are also included all
functional derivatives of 2'6'-diaminopurine i.e. all compounds
that possess a biological activity/efficacy that is substantially
similar to the biological activity/efficacy of 2'6'-diaminopurine
and/or of the analogs thereof which are shown in FIG. 1.
[0041] According to another aspect of the invention, it is provided
an isolated or purified nucleic acid molecule comprising a
nucleotide substitute selected from the group consisting of
2'6'-diaminopurine and analogs thereof as defined previously. The
nucleic acid molecule of the invention may consists of an antisense
oligonucleotide, a double stranded RNA (as RNAi) or an nucleic acid
molecule comprising a sequence coding for a therapeutic gene
product. Preferably, the nucleotide substitute is incorporated into
the nucleic acid molecule for substituting therein an adenosine
base.
[0042] As used herein, the expression "nucleic acid molecule" means
any DNA, RNA sequence or molecule having one nucleotide or more,
including nucleotide sequences encoding a complete gene. The term
is intended to encompass all nucleic acids whether occurring
naturally or non-naturally in a particular cell, tissue or
organism. This includes DNA and fragments thereof, RNA and
fragments thereof, cDNAs and fragments thereof, expressed sequence
tags, artificial sequences including randomized artificial
sequences.
[0043] The nucleic acid molecules of the invention are synthesized
using methods well known in the art. They may be in the form of a
DNA, or an RNA, and they may comprise one or a plurality of
mononucleotide linking residue(s) such as methylphosphonate,
phosphotriester, phosphorothioate, phosphodiester,
phosphorodithioate, boranophosphate, formacetal, thioformacetal,
thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate,
sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine,
methylene (methyimino), methyleneoxy (methylimino), and
phosphoramidate residues.
[0044] DAP base and analogs thereof may be introduced chemically
into DNA and RNA sequences using conventional phosphoramidite
chemistry. Alternatively, DAP can be incorporated into DNA and RNA
by enzymatic methods via the use of DAP triphosphate and
polymerases as it is well known in the art. Interestingly,
DAP-triphosphate acts as a true analog of ATP for DNA synthetic
enzymes (Rackwitz, H. R., et al. Eur J Biochem 72: 191-200,
1977).
[0045] The nucleic acid molecules of the invention may also be
linked to a "carrier" molecule such as amino acids, peptides,
proteins, peptidomimetics, small chemicals, ligands, lipids,
nucleic acids, or carbohydrate moieties.
[0046] The size of the nucleic acid molecules of the invention will
vary depending on a desired use, the oligo having typically from 2
to about 10 000 nucleotides. More preferably, the size of antisense
nucleic acid molecules will vary from about 10 to about 100
nucleotides whereas the size for nucleic acid molecules comprising
a sequence coding for a therapeutic gene product would typically
vary from about 100 to about 10 000 nucleotides.
[0047] The nucleic acid molecules of the invention may be useful
for treating and/or preventing various diseases. Typical examples
of diseases that could benefit from the present nucleic acid
molecules include respiratory system diseases, neurological
diseases, cardiovascular diseases, rheumatological diseases,
digestive diseases, cutaneous diseases, ophtalmological diseases,
urinary system diseases, cancers, pathogen infections, genetic
diseases, hypereosinophilia, general inflammation, and cancers.
Most preferred nucleic acid molecules include the oligonucleotides
listed hereinafter in Table 1.
TABLE-US-00001 TABLE 1 Antisense oligonucleotides for treating or
preventing atopic diseases and neoplastic cell proliferation SEQ ID
Target Sequence NO: Antisense oligonucleotides agaccttcat
gttcccagag 1 inhibiting the common gttcccagag cttgccacct 2 subunit
of IL-4 and IL-13 cctgcaagac cttcatgtt 3 human receptor cgcccacagc
ccgcagagcc 4 ctccatgcag cctctcgcct 5 ccgccggcgc agagcagcag 6
cgcccccgcc cccgcccccg 7 Antisense oligonucleotides gggtctgcag
cgggatggt 8 inhibiting the common ggtctgcagc gggatggtt 9 subunit of
IL-3, IL-5 and agggtctgca gcgggatgg 10 GM-CSF human receptor
gcagggtctg cagcgggat 11 gcagcgggat ggtttcttc 12 cagcgggatg
gtttcttct 13 gtctgcagcg ggatggttt 14 Antisense oligonucleotides
ctgggccatc agtgctctg 15 inhibiting the CCR3 human ccctgacata
gtggatc receptor tagcatggcactgggc 16 ggagccagtcctagcgagc 17 18
[0048] Since the DAP-substituted nucleic acid molecules of the
invention have an improved efficacy and/or a reduced toxicity, they
could be used in gene therapy and DNA vaccination methods. For
instance, DAP-substituted nucleic acid molecules could be used as
therapeutics for inhibiting the multiplication of pathogens of the
respiratory system; as a therapeutic or vaccine to treat or to
prevent neoplastic cell proliferation in the lungs/airways/nose; as
therapeutics or vaccines to treat genetic diseases of the
lungs/airways/nose, such as cystic fibrosis; and as therapeutics or
vaccines for the treatment and/or prevention of asthma, allergy,
chronic obstructive lung disease, pulmonary fibrosis, chronic cough
and mucus production, the adult respiratory distress syndrome,
general inflammation, inflammatory diseases, cancer, pathogen
infections (e.g. sinusitis, respiratory syncytial virus or other
viral respiratory tract infection) genetic diseases, or any
diseases of the respiratory system. In addition DAP and its analogs
may be inserted into genes in place of adenine or any other base,
or be administered in association with the genes (e.g. incorporated
into a coding or non coding region) in order to decrease the immune
response that occurs during gene therapy and/or improve the
efficacy of gene therapy methods.
[0049] More particularly, DAP-substituted nucleic acid molecules
could be used to treat pathogen infections and/or prevent them from
occurring, by inhibiting replication of respiratory pathogens such
as respiratory syncytial virus (RSV), rhinovirus, influenza virus,
bacteria and other agents that cause diseases. Similarly,
DAP-substituted nucleic acid molecules that have anti-tumor
activity could be used for the treatment and prevention of lung or
other cancers. DAP-substituted DNA or genes would also be
particularly useful for therapeutic applications where an
inflammatory response to the gene is not desired such as in the
treatment of genetic diseases of the respiratory tract (e.g. cystic
fibrosis).
[0050] Depending on a desired use, it may be necessary that the
DAP-nucleic acid molecule be incorporated into a vector, such as a
plasmid or a virus, and that it comprises a sequence coding for a
therapeutic gene product.
[0051] According to a preferred embodiment of the invention, the
nucleic acid molecules are antisense oligonucleotides. As it will
be shown in the examples hereinafter, DAP-substituted antisense
oligonucleotides, have an improved efficacy and/or a reduced
toxicity. These antisenses could thus be used as therapeutic or
vaccine directed against at least one lung/airway/nose mediator or
receptor, as therapeutic for inhibiting the inflammatory reaction
that is present in asthma or allergic rhinitis, and as therapeutic
for preventing the development of allergy or asthma, or for
desensitizing patients with these diseases.
[0052] For instance, DAP-substituted antisense oligonucleotides
could be directed against nucleic acid sequences coding for
mediators and receptors, or other components of the inflammation
process, so that by inhibiting the expression of these proteins,
the inflammatory process could be turned off in the lungs/airways
(asthma, chronic obstructive lung disease therapeutic) or in the
nose (allergic rhinitis), or in the sinuses (chronic
sinusitis).
[0053] Therefore, a further aspect of the invention relates to a
method of antisense therapy, the method comprising the step of
administering to a mammal in need thereof, an effective therapeutic
or prophylactic amount of at least one antisense oligonucleotide as
defined previously. This method is particularly useful for
preventing and/or treating respiratory system diseases,
neurological diseases, cardiovascular diseases, rheumatological
diseases, digestive diseases, cutaneous diseases, ophtalmological
diseases, urinary system diseases, cancers, pathogen infections,
genetic diseases, general inflammation and cancer.
[0054] According to a preferred embodiment, the antisense
oligonucleotide is administered, directly to the respiratory system
for preventing and/or treating a respiratory system disease
associated with an inflammation of the lungs, the airways and/or
the nose such as pulmonary fibrosis, adult respiratory distress
syndrome, cystic fibrosis, chronic obstructive lung disease,
chronic bronchitis, eosinophilic bronchitis, asthma, allergy,
allergic rhinitis, sinusitis and hypereosinophilia.
[0055] Preferably, the nucleic acid molecules of the invention
would be incorporated in a pharmaceutical composition comprising at
least one of the nucleic acid molecules defined previously, and a
pharmaceutically acceptable carrier.
[0056] The amount of nucleic acid molecules present in the
composition of the present invention is a therapeutically effective
amount. A therapeutically effective amount of nucleic acid
molecules is that amount necessary so that the nucleic acid
molecule perform its biological function without causing, into the
host to which the composition is administered, overly negative
effects. The exact amount of nucleic acid molecules to be used and
composition to be administered will vary according to factors such
as the oligo biological activity, the type of condition being
treated, the mode of administration, as well as the other
ingredients in the composition. Typically, the composition will be
composed from about 1% to about 90% of nucleic acid molecule(s),
and about 20 .mu.g to about 20 mg of nucleic acid molecule will be
administered.
[0057] The pharmaceutically acceptable carrier of the composition
may be selected from the group consisting of solid carriers, liquid
carriers and gas phase carriers. Advantageously, the carrier is
selected from the group consisting of lipid particles, lipid
vesicles, microcrystals and surfactants.
[0058] Further agents can be added to the composition of the
invention. For instance, the composition of the invention may also
comprise agents such as drugs, antioxidants, surfactants, flavoring
agents, volatile oils, buffering agents, dispersants, propellants,
and preservatives. For preparing such pharmaceutical compositions,
methods well known in the art may be used.
[0059] The nucleic acid molecules and the composition of the
invention may be given via various routes of administration. For
instance, the composition may be administered in the form of
sterile injectable preparations, for example, as sterile injectable
aqueous or oleaginous suspensions. These suspensions may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparations may also be sterile injectable solutions or
suspensions in non-toxic parenterally-acceptable diluents or
solvents. They may be given parenterally, for example
intravenously, intramuscularly or sub-cutaneously by injection or
by infusion. The nucleic acid molecules and the composition of the
invention may also be formulated as creams or ointments for topical
administration. They may also be administered into the airways of a
subject by way of a pressurized aerosol dispenser, a nasal sprayer,
a nebulizer, a metered dose inhaler, a dry powder inhaler, or a
capsule. Suitable dosages will vary, depending upon factors such as
the amount of each of the components in the composition, the
desired effect (fast or long term), the disease or disorder to be
treated, the route of administration and the age and weight of the
individual to be treated. Anyhow, for administering the nucleic
acid molecules and the composition of the invention, methods well
known in the art may be used.
[0060] As mentioned previously, the present invention also relates
to the use of 2'6'-diaminopurine and analogs thereof for inhibiting
inflammation in a mammal. Therefore, the invention also provides an
anti-inflammatory composition comprising: 2'6'-diaminopurine and/or
an analog thereof; and a pharmaceutically acceptable carrier. The
invention also provides a method for inhibiting inflammation in a
mammal, comprising the use as such of 2'6'-diaminopurine and
analogs thereof and/or the use of this (these) compound(s) in
pharmaceutical compositions. 2'6'-diaminopurine and analogs thereof
may be administered as such or incorporated linked to a "carrier"
molecule such as amino acids, peptides, proteins, peptidomimetics,
small chemicals, ligands, lipids, nucleic acids, or carbohydrate
moieties. In a preferred embodiment, 2'6'-diaminopurine and/or its
analogs are incorporated into a nucleic acid molecule such that
degradation of the nucleic acid molecule by the body results in the
liberation of 2'6'-diaminopurine and/or its analogs.
[0061] In a related aspect, the invention concerns an
anti-inflammatory composition comprising an adenosine antagonist
compound selected from the group consisting of 2'6'-diaminopurine
and analogs thereof; and a pharmaceutically acceptable carrier.
Another related aspect concerns the use of 2'6'-diaminopurine
and/or an analog thereof for the preparation of an
anti-inflammatory composition.
[0062] The anti-inflammatory composition of the invention could be
particularly useful for the prevention and/or treatment of any
disease (topical or systemic) where activation of an adenosine
receptor is substantially toxic, and more particularly, systemic,
organ- or tissues-specific inflammation. More particularly, the
anti-inflammatory composition of the invention could be
particularly useful for the prevention and/or treatment of any
inflammation that is associated with and/or caused by a cancer, a
respiratory system disease, a neurological disease, a
cardiovascular disease, a rheumatological disease, a digestive
disease, a cutaneous disease, an ophtalmological disease and a
urinary system disease. More particular examples of respiratory
system diseases that could benefit from the anti-inflammatory
composition of the invention include pulmonary fibrosis, adult
respiratory distress syndrome, cystic fibrosis, chronic obstructive
lung disease, chronic bronchitis, eosinophilic bronchitis, asthma,
allergy, and hypereosinophilia.
[0063] As described hereinbefore, the amount of 2'6'-diaminopurine
and analogs thereof to be used in the composition of the invention,
the amount of the composition to be administered and its routes of
administration will vary according to various factors well known in
the art.
[0064] As it will now be demonstrated by way of examples
hereinafter: (1) the present invention provides a novel antisense
technology, based on analogs of 2,6 diaminopurine, substituted for
adenosine; (2) not all substitutes of adenosine are equally
effective, DAP and its analogs being surprisingly more effective
than others; (3) the nucleic acid molecules of the invention are
equally and surprisingly even more effective at inhibiting the
synthesis of target proteins than standard antisense
oligonucleotides; (4) that the DAP-based antisense technology
according to the present invention is more powerful and constitutes
a significant advance over existing technologies since DAP-based
antisenses have more significant anti-inflammatory effects than
conventional antisense oligonucleotides; (5) DAP-nucleic acid
molecules seem to exert their anti-inflammatory effects by a
mechanism that does not seem to be related to inhibition of
adenosine receptors; (6) the present nucleic acid molecules have
significantly reduced toxicity for any inflammatory disease and/or
the lungs/airways; (7) the use of the DAP-nucleic acid molecules,
compositions and methods of the invention would be more effective
than using regular antisenses (containing no DAP); and (8) finally,
2'6'-diaminopurine per se and its analogs have anti-inflammatory
activities.
EXAMPLES
[0065] The following examples are illustrative of the wide range of
applicability of the present invention and are not intended to
limit its scope. Modifications and variations can be made therein
without departing from the spirit and scope of the invention.
Although any method and material similar or equivalent to those
described herein can be used in the practice for testing of the
present invention, the preferred methods and materials are
described.
A) INTRODUCTION
[0066] Antisense oligonucleotides (AS) are a new class of
pharmaceuticals that have been extensively described in the
scientific and patent literature. This therapeutic strategy could
potentially be applied to any disease where an over-expression of
one or several genes is believed to cause the presence or
persistence of the disease. An increased efficacy and
anti-inflammatory efficacy would make AS an important therapeutic
strategy for every respiratory disease including asthma,
bronchiolitis, viral and other forms of infection, rhinitis, cystic
fibrosis, chronic bronchitis, chronic obstructive lung disease,
eosinophilic and other forms of cough, pulmonary fibrosis, adult
respiratory distress syndrome, conjunctivitis and other forms of
eye or skin inflammatory diseases.
[0067] A review of the systemic effects and toxicity of antisense
oligonucleotides has been summarized by ST Crooke (Hematologic
Pathology, 9: 5972; 1995). One way to circumvent the toxicity of
the PS oligonucleotides would be to administer them to the site of
the disease that they are designed to treat, minimizing systemic
distribution and thus the toxicity associated with it. PS AS
oligonucleotides have been nebulized to the lungs of mice or
rabbits (Templin M V et al. Antisense and nucleic acid drug
development, 10:359-368; 2000; Ali S et al. Am J Respir Critic Care
Med 163:989-993; 2001). Results have shown that there is very
little systemic distribution at doses that would be considered
therapeutically effective. However, at higher doses a multifocal
cellular infiltrate occurs in the lungs of mice, comprising
primarily lymphocytes and neutrophils, with a few macrophages and
monocytes. Although the adenosine base included in oligonucleotides
may have pro or anti-inflammatory effects, we have previously
reported in patent WO 99/66037 that an antisense oligonucleotide
directed against the CCR3 receptor and containing 5 adenosines per
18 mer (27.7% adenosine) was effective at inhibiting the asthmatic
response in vivo in rats.
[0068] No one has systematically tested whether replacement of
bases by analogs could affect the stability, binding, degradation
efficacy and toxicity of antisense oligonucleotides, nor have they
tested them for biological activity in cells in culture or in
animals. 2,6 diaminopurine (DAP) was found to be present in DNA in
place of adenosine by the cyanophage S-2L (Cheng, X., Annu Rev
Biophys Biomol Struct 24: 293-318, 1995); Khudyakov, I. Y., et al.,
Virology 88: 8-18, 1978). DAP alters the structure of duplex DNA
and introduces a third hydrogen bond in the D:T duplex (similar to
the three hydrogen bonds formed by cytosine and guanosine) when
compared with A:T duplex (Chollett, A. and Kawashima, E. of Biogen
SA (Geneva), Nucleic Acids Research 16:305-17, 1988). This extra
hydrogen bond leads to increase selectivity and hybridization
strength during DNA-DNA hybridization, as well as the inhibition of
cleavage of several restriction enzymes (Bailly, C. and Waring, M.
J., Nucleic Acids Research 23:885-92, 1995; Bailly, C. et al. PNAS
93:13623-8, 1996).
[0069] The additional N2 amino group of the C2 carbon in DAP is
used for base pairing. The additional bond causes increased DNA
duplex stability and renders the minor groove of both B- and Z-DNA
more hydrophilic. DAP substitution for adenosine causes an increase
in the Tm of DAP containing DNA, the temperature at which two
duplexed complementary DNA strands melt, of 1.5.degree. C. per DAP
residue (Hoheisel, J. D., Lehrach, H., FEBS Letters 274:103-6,
1990). DAP and its analogs have thus the potential to increase the
efficacy and anti-inflammatory activity of AS oligonucleotides when
included within the oligonucleotide either as substitution for a
base, in addition to the bases, when incorporated to gene therapy
or as seen below, when administered alone.
B) MATERIAL AND METHODS
Experiments with Cell Lines
[0070] Experiments were performed to assess whether antisense
oligonucleotides described in international application WO 99/66037
(incorporated herein) directed against the common beta sub-unit of
the IL-3, IL-5 and GM-CSF receptor, could inhibit the expression
and the function of this receptor when modified by replacing
adenosine by either 2-amino-2'-deoxyadenosine or inosine. TF-1 and
U937 cells express high levels of GM-CSF receptors. In addition,
TF-1 cells are dependent on GM-CSF for survival. These cells were
cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal
calf serum, penicillin, streptomycin and I-glutamine at 37.degree.
C. in 5% CO.sub.2 (the TF-1 cells were supplemented with GM-CSF).
For 12 hours they were either cultured in medium alone or medium
with sense (107S: 5'-ACCAT CCCGCTGCAGACCC-3' (SEQ ID NO:19) or
antisense (107A: 5'-GGGTCTGCAGCGGGATGGT-3'; SEQ ID NO:20)
oligonucleotides to the common beta sub-unit of the IL-3, IL-5 and
GM-CSF receptor. The sequence for 107A-DAP was:
5'-GGGTCTGCDapGCGGGDapTGGT-3' (SEQ ID NO:21); the sequence for
107A-inosine (107A-I) was: 5'-GGGTCTGCIGCGGGIT GGT-3' (SEQ ID
NO:22). The cells were retrieved and washed 3 times. RNA was then
retrieved and the presence of the beta chain of the receptor was
assessed by semi-quantitative RT-PCR.
Animals
[0071] Brown Norway rats 6-8 weeks of age and weighing 220-275 g
were obtained from Harlan-Sprague-Dawley (Walkerville, Md.). Rats
were maintained in conventional animal facilities.
Sensitization to Ovalbumin
[0072] Active sensitization of rats was performed by subcutaneous
injection of 1 ml of saline containing 1 mg of chicken egg
ovalbumin (OA) (Sigma, St. Louis, Mo.) and 3.5 mg of aluminum
hydroxide gel (BDH Chemicals, Poole, UK).
Ovalbumin Challenge
[0073] On day 14 after sensitization with ovalbumin, after general
anesthesia with 65 mg/Kg pentothal and endotracheal intubation,
ovalbumin challenge is performed by injecting 200 micrograms of
ovalbumin in 60 .mu.l intratracheally. After 8 hours or 15 hours,
the rats are again intubated after general anesthesia and a lung
lavage consisting of 5 times 5 ml instillation of 0.9% saline is
performed. Cells are washed, counted and centrifuged onto slides in
a Cytospin III.TM.. A differential cell count is performed.
Measurement of Airway Responses
[0074] The equipment and methodology for measuring pulmonary
resistance was as previously described (Renzi, P. M., et al. Am.
Rev. Respir. D is 146: 163-169, 1992). General anesthesia was
induced with either pentothal (50 mg/kg) or urethane (1.1 g/kg)
intra-peritoneally. Endo-tracheal intubation was then performed
using a 6 cm length of PE-240.TM. polyethylene catheter. A heating
pad was used to maintain constant body temperature and rectal
temperature was monitored continuously with an electronic
thermometer (Telethermometer.TM., Yellow Springs Instrument Co.,
Yellowsprings, Ohio). Lung resistance (RL) was measured during
spontaneous tidal breathing with the animals in the lateral
decubitus position. Flow was measured by placing the tip of the
tracheal tube inside a small Plexiglass.TM. box (265 ml in volume).
A Fleish.TM. No. 0 pneumotachograph coupled to a differential
pressure transducer (MP-45+2 cm H.sub.20; Validyne Corp,
Northridge, Calif.) was attached to the other end of the box to
measure airflow, and volume was obtained by numerical integration
of the flow signal. Changes in esophageal pressure were measured by
using a saline-filled catheter and a differential pressure
transducer (Sanborn 267 BC.TM.; Hewlett Packard, Waltham, Mass.).
The other port of the transducer was connected to the box. The
esophageal catheter consisted of polyethylene tubing (PE-200.TM.)
20-cm long attached to a shorter length (6 cm) of tubing
(PE-100.TM.). Transpulmonary pressure was computed as the
difference between esophageal and box pressure. The airway response
was evaluated from RL, which was determined by fitting the equation
of motion of the lung by multiple linear regression using
commercial software (RHT-Infodat Inc., Montreal, Quebec,
Canada).
Measurement of Lung Resistance Immediately after Administration of
Regular or Modified PS Antisense Oligonucleotides
[0075] On day 14 after sensitization with ovalbumin, after general
anesthesia with 65 mg/Kg pentothal and endotracheal intubation, 60
.mu.g of an PS AS oligonucleotide directed against the rat common
Beta chain of the GM-CSF, IL-3 and IL-5 receptor (AS141A:
5'-TGGCACTTTAGGTGGCTG-3'; SEQ ID NO:23) was injected
intratracheally. Lung resistance was measured at baseline, every
five minutes for 30 minutes and at 15 minutes intervals. The same
experiments were repeated with modified AS141 where adenosine has
been replaced by DAP, AS141-DAP (5-TGGCDapCTTTDapGGTGGCTG-3'; SEQ
ID NO:24) or by inosine, AS141-I (5'-TGGCICTTTIGGTGGCTG-3; SEQ ID
NO:25).
Experiments Assessing the Airway Responsiveness to Adenosine and
DAP Nucleoside and Other Specific DAP Analogs
[0076] On day 14 post-sensitization, rats were anesthetized with
pentothal (65 mg/kg), intubated, and baseline RL was measured. Rats
were given incremental doses intratracheally of adenosine (CAS
58-61-7), 2,6-diaminopurine hemisulfate salt (CAS 69369-16-0), DAP
(2-amino-2'-deoxyadenosine; CAS 4546-70-7),
2-amino-9-(B-D-2'-deoxyribo furanosyl)purine (CAS 3616-24-8),
7-Deaza-2' deoxyadenosine (CAS 60129-59-1),
N6-Methyl-2'-deoxyadenosine (CAS 2002-35-9),
2-aminoadenosine/2,6-diamunopurine riboside (CAS 2096-10-08) over
the dose range of 0.125 .mu.g to 100 .mu.g in 50 .mu.l of either
saline or saline plus acetic acid. Immediately after each dose RL
was measured. DAP was dissolved as follows: 3 mg of DAP was
combined with 100 .mu.l of acetic acid, adjusted to 1.5 to 3 ml by
the addition of saline, and heated to 70.degree. C. This gave a
final concentration of 1 to 2 .mu.g/.mu.l. Dilutions were performed
in the same buffer. Control animals received saline with acetic
acid at the same final concentration as indicated.
Experiments Assessing the Leukotriene D4 Responsiveness after
Antigen Challenge
[0077] We have previously shown that the antisense ASA4
(5'-ACTCATATTC ATAGGGTG-3'; SEQ ID NO:26) directed against the rat
CCR3 receptor was effective at inhibiting eosinophil influx into
the lungs after antigen challenge (see WO 99/66037). We employed
the same oligonucleotide sequences for these experiments. On day
14, the rats were intubated after general anesthesia with pentothal
(65 mg/kg) and received 200 .mu.g of ASA4,
ASA4-DAP(5'-DapCTCDapTDapTTCDapTDapGGGTG-3'; SEQ ID NO:27),
mismatch ASA4-DAP (5'-CDapTCDapT TDapTCATGDapGGTG-3'; SEQ ID
NO:28), AS141-DAP (5'-TGGCDapCTTTDapGGTGGCTG-3'; SEQ ID NO:29),
mismatch AS141-DAP (5'-GTGCCDapTTTGDapGTGGCTG-3'; SEQ ID NO:30),
combination of ASA4-DAP and AS141-DAP (total of 100 .mu.g) or
saline in 50 .mu.l of 0.9% NaCl intratracheally. Ten minutes later,
ovalbumin challenge was performed by injecting 200 micrograms of
ovalbumin in 50 .mu.l of 0.9% saline intratracheally. After 15
hours, the rats were again intubated after general anesthesia,
baseline lung resistance measured and doubling concentrations of
leukotriene D4 injected intratracheally (50 ng to 1600 ng) until
baseline lung resistance doubled (EC200).
Experiments Assessing the Cellular Influx into the Lungs after
Antigen Challenge
[0078] On day 14, the rats were intubated after general anesthesia
with pentothal (65 mg/kg) and received 200 .mu.g of ASA4, ASA4-DAP
(5'-DapCTCDapTDapTTCDapTDapGGGTG-3'; SEQ ID NO:27), AS141-DAP
(5'-TGGCDapCTTTDapGGTGGCTG-3'; SEQ ID NO:29), mismatch AS141-DAP
(5'-GTGCCDapTTTGDapGTGGCTG-3'; SEQ ID NO:30), combination of
ASA4-DAP and AS141-DAP (total of 100 .mu.g) or saline in 50 .mu.l
of 0.9% NaCl intratracheally. Twenty minutes later ovalbumin
challenge is performed by injecting 200 .mu.g of ovalbumin in 50
.mu.l of 0.9% saline intratracheally. After 15 hours, the rats were
again intubated after general anesthesia, and a lung lavage
consisting of 5 times 5 ml instillation was performed. Cells were
washed, counted and centrifuged onto slides in a Cytospin III.TM..
A differential cell count was finally performed.
Experiments Assessing the Cellular Influx into the Lungs after
Adenosine or DAP Administration
[0079] Fourteen days after sensitization, the rats were intubated
after anesthesia with pentothal 65 mg/kg, and 100 .mu.g of
adenosine or 2-amino-2'-deoxyadenosine or of 2-amino-2'
deoxyadenosine followed 10 minutes later by adenosine or of saline
was injected intratracheally in 50 .mu.l. Fifteen hours later, the
rats were intubated after general anesthesia with pentothal, and a
lung lavage was performed and the cells were counted as described
above.
C) RESULTS
Example 1
Replacing Adenosine by DAP is at Least as Effective In Vitro as a
Regular Phosphorothioate Antisense Oligonucleotide
[0080] A first set of experiments was designed in order to
determine whether replacement of adenosine by DAP affected the in
vitro efficacy of AS oligonucleotides. It is to be noted in FIG. 2A
that the biological effectiveness of antisense to the common Beta
chain of human GM-CSF, IL-3 and IL-5 receptor is not affected by
replacing adenosine by 2-amino-2' deoxyadenosine in U937 cells that
express this receptor. AS107 is a 19 mer oligonucleotide that
contains 2 adenosine bases. The adenosine bases were replaced by
DAP (AS107-DAP). This modified oligonucleotide was at least equally
effective at blocking the mRNA for the common Beta chain when
assessed by semi-quantitative PCR (with G3PDH as a housekeeping
gene) as AS107 containing adenosine (AS107).
[0081] To confirm the efficacy in another cell line, experiments
were repeated in TF1 cells that are dependent on GM-CSF for their
survival. It is to be noted in FIG. 2B that AS107-DAP to the common
Beta chain of human GM-CSF, IL-3 and IL-5 receptor was at least
equally effective at blocking mRNA expression as AS107 containing
adenosine (AS107). Replacing adenosine by DAP in antisense
oligonucleotides is effective in vitro.
Example 2
Not all Substitutes of Adenosine are Effective at Inhibiting Genes
when Incorporated into Phosphorothioate Antisense
Oligonucleotides
[0082] Experiments were performed in order to determine whether
substituting adenosine would affect the efficacy of antisense
oligonucleotides. It is to be noted in FIG. 3 that the
effectiveness of the same antisense oligonucleotide as described
above (AS107) is lost when both adenosines are replaced by inosine.
Antisenses containing inosine (AS107-I) was not effective at
inhibiting mRNA expression when assessed by semi-quantitative PCR
and compared to AS107 containing adenosine. Experiments were
performed by incubating U937 cells with medium alone, AS107 or
AS107-I at a concentration of 10 .mu.mol for six hours prior to
isolating RNA and performing semi-quantitative PCR.
Example 3
An Increase in Lung Resistance Occurs after Intratracheal Injection
of Phosphorothioate Antisense Oligonucleotides that is not Related
to Adenosine
[0083] Experiments were performed to assess the effect on lung
resistance after rapid intratracheal injection of phosphorothioate
antisense oligonucleotides contained in 50 .mu.l of saline.
[0084] FIG. 4A illustrates the effects of intratracheal
administration of an antisense phosphorothioate oligonucleotide
directed against the common Beta chain of rat GM-CSF, IL-3 and IL-5
(AS141, a 19 mer oligonucleotide that contains 2 adenosines) and
the effect of a DAP-substituted phosphorothioate antisense
oligonucleotide (AS141-DAP) of the same sequence, at a dose of 60
.mu.g each, on lung resistance of sensitized Brown Norway (BN)
rats. For these experiments and the following, sensitized Brown
Norway rats were employed as previously described (Renzi P M, Am
Rev Respir Dis 146:163-9; 1992). The injection of phosphate
buffered saline caused a mild increase in lung resistance 25%
maximal increase. Regular antisense, which included less than 15%
adenosine caused a moderate increase in lung resistance (87%). The
DAP-modified oligonucleotides caused a mild to moderate increase
(33%) in lung resistance. Nyce has suggested in WO 00/62736 and WO
00/09525 that the increase in lung resistance is caused by the
adenosine that is included in the oligonucleotide. However, the
oligonucleotides would not have found the time to degrade and
release adenosine (a few minutes), and the antisense
oligonucleotide that was employed contained only 10% adenosine
(which, according to WO 00/62736 and WO 00/09525, should not have
an effect on lung resistance).
[0085] An assay was performed to evaluate whether bronchospasms
were due to the 2 adenosines that were replaced, in AS141, by 2
inosines since it is known that inosine does not cause
bronchoconstriction of the lungs/airways compared to adenosine,
(Mann, J. C. et al., J Appl Physiol 61: 1667-76, 1986). As shown in
FIG. 4B, the intratracheal administration of the same antisense
phosphorothioate oligonucleotide directed against the common Beta
chain of rat GM-CSF, IL-3 and IL-5 (AS141) where inosine was
substituted for adenosine (AS141-Inosine) also caused a temporary
increase in lung resistance (by 108%). These results show that
intratracheal injection of antisense oligonucleotides temporarily
increases lung resistance by a mechanism that does not seem related
to adenosine.
Example 4
Effect of Different DAP Analogs on Lung Resistance
[0086] We assessed the effects of intratracheal administration of
DAP analogs and of adenosine on lung resistance in Brown Norway
rats. As shown in FIG. 5, adenosine, DAP, and five different
analogs of DAP were studied. For each compound, a minimum of six
rats were studied and the average % baseline lung resistance is
presented as a function of the intratracheal dose of DAP or its
analogs. As can be seen in this figure, lung resistance is
gradually increased to peak at a concentration of 5 .mu.g of
adenosine whereas this does not occur with 2-amino-2'
deoxyadenosine (DAP) or the analogs thereof under study. These
results thus show that, contrary to adenosine, DAP and its analogs
does not significantly increase lung resistance. Since
oligonucleotides are degraded progressively within the lungs it may
be unexpectedly advantageous to use these compounds instead of
adenosine.
Example 5
DAP-Modified Phosphorothioate Antisense Oligonucleotides are
Effective at Inhibiting the Airway Hyper-Responsiveness that Occurs
After Antigen Challenge In Vivo
[0087] The in vivo biological activity of DAP-modified antisense
directed against the rat CCR3 and the common .beta. chain of
IL-3/IL-5/GM-CSF receptors in the rat model of asthma is
demonstrated in FIG. 6A. ASA4 is an 18 mer phosphorothioate
antisense oligonucleotide that has been shown to inhibit the CCR3
receptor and inhibit the eosinophil influx that occurs after
antigen challenge (see WO 99/66037). AS141 is also an 18 mer
phosphorothioate antisense oligonucleotide that has been shown to
inhibit the common 13 chain of IL-3/IL-5/GM-CSF receptors and
inhibit the eosinophil influx that occurs after antigen challenge
(see WO 99/66037) ASA4 contains 5 adenosine bases (28% adenosine).
AS143 contains 2 adenosine bases. It is to be noted that ASA4-DAP
significantly decreased airway responsiveness to leukotriene D4 15
hours after ovalbumin challenge when compared to rats that received
no AS (control challenged; p<0.01) or DAP mismatch treated rats.
It is also to be noted that ASA4-DAP tended to be more effective
than unmodified ASA4 and was no different than results obtained
from unchallenged rats. AS141 also decreased significantly the
hyperresponsiveness to LTD.sub.4 (P<0.05) when compared to
control challenged and 141-DAP mismatch treated rats. Moreover,
airway responsiveness to LTD.sub.4 was significantly decreased in
the rats treated with the combination of CCR3 and the common .beta.
chain oligonucleotides compared to each antisense oligonucleotide
and this combination was as effective as the combination of DAP
oligonucleotides (total 50 .mu.g; FIG. 6B).
Example 6
DAP-Modified Phosphorothioate Antisense Oligonucleotides are More
Effective than Conventional Antisense Oligonucleotides at
Inhibiting the Airway Inflammation that Occurs after Antigen
Challenge in Vivo
[0088] These experiments were performed with antisense
oligonucleotides directed against the rat CCR3 receptor or the
common Beta chain of IL-3,5 and GM-CSF. Ovalbumin sensitized and
challenged rats were treated by intratracheal injection of saline,
200 .mu.g of regular ASA4 or 200 .mu.g of ASA4-DAP ten minutes
prior to ovalbumin challenge. After 15 hours, the rats were
anesthetized intubated and bronchoalveolar lavage was performed for
total cell count and differential. The results show that
administration of both regular ASA4 (FIG. 7A) and AS141 (FIG. 7B)
and both ASA4-DAP and AS141-DAP effectively inhibited the
recruitment of eosinophils (by 84% and 83% respectively; FIG. 7A).
However AS4DAP tended to decrease neutrophil and macrophage
recruitment and significantly decreased lymphocyte recruitment (by
74%). 141-DAP significantly decreased macrophage recruitment
(p<0.05) as well. The combination of two A4-DAP and 141-DAP
oligonucleotides (total 200 .mu.g) also significantly decreased the
recruitment of lymphocytes and macrophages (FIG. 7C). These results
show that DAP-modified oligonucleotides are not only effective but
also have a broader anti-inflammatory effect than regular
oligonucleotides.
Example 7
Adenosine has Pro-Inflammatory Effects in the Lungs that are
Specific for Eosinophil Recruitment and DAP is an Antagonist of the
Adenosine Pro-Inflammatory Effects
[0089] In another experiment, groups of six sensitized but
unchallenged Brown Norway rats were anesthetized with pentothal and
endotracheally intubated. The rats then received an intratracheal
injection of either (1) saline (control), (2) 100 .mu.g of
adenosine, (3) 100 .mu.g of 2-amino-2'-deoxyadenosine (DAP) or (4)
100 .mu.g of DAP followed by 100 .mu.g of adenosine 10 minutes
later. The rats were awakened, re-anesthetized and intubated 15
hours later for a lung lavage. Cells that were present in media
collected from the lavage, were counted and a differential was
obtained on Cytospin.TM. slides.
[0090] As shown in FIG. 8, adenosine is pro-inflammatory in the
lungs, leading to a selective recruitment of eosinophils (more than
10 fold increase), without significantly affecting other cell
types. To the opposite, DAP does not increase the cellularity of
lung lavage and completely inhibits the recruitment of eosinophils
that is induced by adenosine.
D) CONCLUSIONS
[0091] In view of the above, DAP-substituted antisense
oligonucleotides have the following advantages when compared to
unmodified antisenses or inosine-modified antisenses: [0092] a) As
shown in FIGS. 1 to 8 and documented in the present patent
application, the chemical structure and properties of DAP and DAP
analogs are different from adenosine. These different chemistries
cause antisense oligonucleotides containing DAP and/or DAP analogs
to have different chemistries, hybridization properties and
stability as compared to unmodified antisenses. [0093] b) Example 1
shows how DAP phosphorothioate antisenses are effective in
different cell lines in vitro. [0094] c) Example 2 shows that not
all substitutes of adenosine are effective at inhibiting genes when
incorporated into phosphorothioate antisense oligonucleotides (as
shown with inosine). [0095] d) Example 3 shows that an increase in
lung resistance occurs after intratracheal injection of
phosphorothioate antisense oligonucleotides, and that this increase
is not related to adenosine. Indeed, even though inosine does not
stimulate adenosine receptors an increase in lung resistance was
seen with inosine oligos. However, this increase was less important
with DAP-modified oligos. [0096] e) Example 4 shows that, although
different substitutes of adenosine, DAP and analogs of DAP have
different effects on lung resistance when injected intratracheally
in vivo, these compounds were all much less toxic than adenosine.
For instance, as shown at FIG. 5, at peak adenosine toxicity (5
.mu.g), the relative ranking of the compounds tested was
adenosine> N6-Methyl-2' deoxyadenosine> rest including DAP.
However at 5 fold lower concentration (1 .mu.g) the toxicity
ranking was different with adenosine> 2 amino-2
deoxyadenosine/DAP> rest. Free DAP base was used as a structure
control to determine whether the sugar was required for toxicity.
[0097] f) Example 5 shows that DAP-modified phosphorothioate
antisense oligonucleotides are effective at inhibiting the airway
hyper-responsiveness to leukotriene D4 that occurs after antigen
challenge in vivo and tend to be more effective than conventional
PS oligonucleotides. The combination of two DAP-modified
oligonucleotides is more effective than each oligonucleotide alone,
confirming synergy. [0098] g) Example 6 shows that DAP-modified
phosphorothioate antisense oligonucleotides are more effective than
conventional antisense oligonucleotides at inhibiting the airway
inflammation that occurs after antigen challenge in vivo. For ASA4
there were strong trends for decreases in neutrophils and
macrophages and also a significant decrease in lymphocytes, whereas
these effects were not encountered with conventional PS antisense
oligonucleotides. For AS141 there were strong trends for decreases
in neutrophils and also a significant decrease in lymphocytes and
macrophages, whereas these effects were not encountered with
conventional PS antisense oligonucleotides. For the combination of
ASA4 and AS141 there were strong trends for decreases in
neutrophils and also a significant decrease in lymphocytes and
macrophages, whereas these effects were not encountered with
conventional PS antisense oligonucleotides. [0099] h) Example 7
shows that adenosine has a pro-inflammatory effect in the lungs of
rats, selectively recruiting eosinophils and that it does not have
a significant effect on lymphocytes. At the same time, Example 7
shows that DAP blocks eosinophil influx, a demonstration that DAP
per se is an antagonist of adenosine.
[0100] The above 7 examples show that DAP-substituted antisenses
and antisenses with analogs of DAP, are inherently more effective
and much less toxic for the lungs/airways than free adenosine
nucleoside or unmodified antisense compounds containing
adenosine.
[0101] Also, contrary to what has been suggested in WO 00/09525 and
WO 00/62736, adenosines contained within the antisenses are not
pro-inflammatory since antisenses with up to 28% adenosine bases
were capable to inhibit eosinophil influx as much as antisenses
containing no adenosine but DAP (see FIG. 7). However, since the
oligonucleotides containing DAP also inhibited lymphocyte and
macrophage influx (FIG. 7), and adenosine does not affect
lymphocyte influx (FIG. 8), it seems that DAP contained in
antisenses does exert its effects through a mechanism that is not
related to the adenosine receptor(s).
[0102] In summary, DAP-antisenses thus provide an improved
technology platform for the development of antisenses therapeutics
and vaccines for the treatment and prevention of respiratory
diseases such as asthma, allergic rhinits, chronic obstructive
disease, eosinophilic cough, pulmonary fibrosis, cystic fibrosis,
pathogen infections, genetic diseases and lung cancer, and any
other disease where inflammation is a concern. Also, DAP per se and
analogs thereof have a strong potential in anti-inflammatory drugs
for inhibiting inflammation in mammals.
[0103] While several embodiments of the invention have been
described, it will be understood that the present invention is
capable of further modifications, and the present patent
application is intended to cover any variations, uses, or
adaptations of the invention, following in general the principles
of the invention and including such departures from the present
disclosure as to come within knowledge or customary practice in the
art to which the invention pertains.
Sequence CWU 1
1
30120DNAArtificial sequenceSequence is completely synthesized
1agaccttcat gttcccagag 20220DNAArtificial sequenceSequence is
completely synthesized 2gttcccagag cttgccacct 20319DNAArtificial
sequenceSequence is completely synthesized 3cctgcaagac cttcatgtt
19420DNAArtificial sequenceSequence is completely synthesized
4cgcccacagc ccgcagagcc 20520DNAArtificial sequenceSequence is
completely synthesized 5ctccatgcag cctctcgcct 20620DNAArtificial
sequenceSequence is completely synthesized 6ccgccggcgc agagcagcag
20720DNAArtificial sequenceSequence is completely synthesized
7cgcccccgcc cccgcccccg 20819DNAArtificial sequenceSequence is
completely synthesized 8gggtctgcag cgggatggt 19919DNAArtificial
sequenceSequence is completely synthesized 9ggtctgcagc gggatggtt
191019DNAArtificial sequenceSequence is completely synthesized
10agggtctgca gcgggatgg 191119DNAArtificial sequenceSequence is
completely synthesized 11gcagggtctg cagcgggat 191219DNAArtificial
sequenceSequence is completely synthesized 12gcagcgggat ggtttcttc
191319DNAArtificial sequenceSequence is completely synthesized
13cagcgggatg gtttcttct 191419DNAArtificial sequenceSequence is
completely synthesized 14gtctgcagcg ggatggttt 191519DNAArtificial
sequenceSequence is completely synthesized 15ctgggccatc agtgctctg
191617DNAArtificial sequenceSequence is completely synthesized
16ccctgacata gtggatc 171716DNAArtificial sequenceSequence is
completely synthesized 17tagcatggca ctgggc 161819DNAartificial
sequenceSequence is completely synthesized 18ggagccagtc ctagcgagc
191919DNAArtificial sequenceSequence is completely synthesized
19accatcccgc tgcagaccc 192019DNAArtificial sequenceSequence is
completely synthesized 20gggtctgcag cgggatggt 192119DNAArtificial
sequenceSequence is completely synthesized 21gggtctgcng cgggntggt
192219DNAArtificial sequenceSequence is completely synthesized
22gggtctgcng cgggntggt 192318DNAArtificial sequenceSequence is
completely synthesized 23tggcacttta ggtggctg 182418DNAArtificial
sequenceSequence is completely synthesized 24tggcnctttn ggtggctg
182518DNAArtificial sequenceSequence is completely synthesized
25tggcnctttn ggtggctg 182618DNAArtificial sequenceSequence is
completely synthesized 26actcatattc atagggtg 182718DNAArtificial
sequenceSequence is completely synthesized 27nctcntnttc ntngggtg
182818DNAArtificial sequenceSequence is completely synthesized
28cntcnttntc atgnggtg 182918DNAArtificial sequenceSequence is
completely synthesized 29tggcnctttn ggtggctg 183018DNAArtificial
sequenceSequence is completely synthesized 30gtgccntttg ngtggctg
18
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