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METHOD FOR VALIDATING/INVALIDATING
TARGETS) AND PATHWAYS
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to a method for validating target genes, e. g. central
nervous (CNS) genes
which utilizes anti-sense oligonucleotides (oligos) of low adenosine (low A)
or no adenosine (desA) content.
This method aids in the screening of target genes and their functions by
inhibiting the target gene's production
of its product. This method is particularly suited for in vivo applications
since many of a subject's functions
are responsive to adenosine, and adenosine's effects may mask the specific
target gene's detectable functions.
Description of the Background
The sequence of all human genes, approximately 100,000, is expected to become
known within the
next 1-3 years. This information will provide the opportunity to design new
medicines for treatrnent of
virtually all diseases which have in the past or presently afflict mankind.
This massive accumulation of
sequences, however, will not by itself be useful in the development of new
medicines until the technology
becomes available to discern the function of these genes, particularly with
respect to the potential effects, i.e.
therapeutic or toxicologic, of attenuating their function. For example, it is
imperative that novel methods be
designed for rapidly and accurately testing the function and, therefore,
usefulness of newly discovered gene
products as "targets" for drug discovery programs. In order to conserve drug
discovery resources, such
"targets" must be validated or invalidated as early as possible in the drug
discovery process. Even so, the
implementation of such methods will require the massive implementation of drug
discovery programs to
series of genes for which a sequence but not a function are known. One method
of considerable potential to
assess the value of newly discovered gene products as focal points for drug
discovery programs is the use of
anti-sense oligonucleotides to ablate the target gene function in vitro or in
vivo. While this method has great
theoretical importance, one problem is that anti-sense oligonucleotides have
the potential to be degraded in
vitro or in vivo, releasing their constitutive nucleotides. There is evidence
that one of these products of
oligonucleotide degradation, adenosine, is highly bioactive in certain
tissues. Adenosine mediates pleiotropic
effects including depression of neurotransmission, maintenance of thalamic
spindle rhythms, sleep induction,
antagonism of D 1 and D2 dopamine receptors, anti-nociception, mediation of
various effects of ethanol
including motor incoordination, autonomic control of cardiac function,
bronchoconstriction, negative
chronotropy, inotropy and dromotropy, anti-~3-adrenergic action, and renal
sodium retention. Clearly, the
liberation of even minute amounts of adenosine in certain tissues, e.g. CNS,
the hyper-responsive asthmatic
lung, heart, and kidney, among others, could locally activate adenosine
receptors. This would make it
impossible to interpret target validation data in a reliable, unambiguous
fashion. Anti-sense oligonucleotides
containing adenosine, thus, are not optimal to provide clear target validation
data since their breakdown could
cause pleiottopic adenosine-mediated effects.
Basic neuroscience research during the past few years has established a
relationship between
excitatory amino acid (EAA) central nervous system (CNS) transmitters, such as
glutamic and aspartic acid,
and various pathological states, e.g. stroke and CNS trauma. For example, a
major mechanism of neural tissue
degeneration following cerebral ischaemia, stroke or trauma, seems to involve
overactivity of the EAA system
in the brain, i.e. excessive release of glutamate and aspartate. This process
is called delayed excitatory
toxicity, and certain neural cell populations are selectively sensitive to
excitatory toxicity.
Adenosine, among other activities, has been found to inhibit the release of
EAA pre-synaptically and
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thus attenuate this excitatory toxicity, and its release would greatly
interfere with validation studies of this
system. Because the effects of adenosine are generally mediated by
extracellular receptors, the
pharmacologically relevant pool of adenosine is, therefore, that which is
outside the cell. Adenosine also has
neuro-behavioral effects, and acts as a CNS depressant, i.e. inhibits neural
activity. It is also a natural
anti-convulsant and sedative. New studies are revealing that, under normal
circumstances adenosine promotes
sleep and, therefore, may interfere with pathways involved in sleep related
responses, such as sleep apnea.
Now increasing evidence is confirming that adenosine is an important "fatigue
factor" and may also interfere
with validation studies on the molecular underpinnings of the sleep cycle. For
example in the brain, studies
indicate that the key receptors reside on nerve cells in brain arousal
networks. Some scientists believe that
adenosine promotes sleepiness by targeting arousal networks in the brain such
as the cholinergic system, such
as the cholinergic basal forebrain and the mesopontine cholinergic nuclei
which spur slumber. All these
effects of adenosine interfere with any validation study of targets in the
systems and pathways where it acts.
Accordingly, there is a definite need for a rapid and efficient method to
screen large numbers of
genes and their expression products to determine their functions and, thus,
their usefulness in the design of
therapeutic agents for treating diseases and conditions associated with the
target genes and/or their expressed
products. Moreover, there is a need for a method which is suitable for testing
individual gene functions while
avoiding triggering other gene functions which would obscure the
interpretation of the results.
SUMMARY OF THE INVENTION
The present invention relates to a method of validating\invalidating or
determining the existence of a
correlation between a function of a disease or condition and, a gene or mRNA
encoding a target polypeptide
suspected of being associated with a disease or condition. The method
generally comprises obtaining
oligonucleotides (oligos) consisting of up to about 15% adenosine (A), and
which is anti-sense to a target
selected from the group consisting of target genes and their corresponding
mRNAs, genomic and mRNA
flanking regions selected from the group consisting of 3' and 5' intron-exon
borders and the juxta-section
between coding and non-coding regions, and all mRNA segments encoding
polypeptides associated with a
pre-selected disease or condition; selecting amongst the oligos one that
significantly inhibits or ablates
expression of the polypeptide encoded by the mRNA upon in vitro hybridization
to the target mRNA;
administering to a subject an amount of the selected oligo effective for in
vivo hybridization to the target
mRNA; and assessing a subject's function that is associated with the disease
or condition before and after
administration of the oligo; wherein a change in the function's value greater
than about 70% indicates a
positive correlation, between about 40 and about 70% a possible correlation,
and below about 30% a lack of
correlation. Suitable applications for the present method are in the
elucidation of genes or networks of genes
which may be associated with diseases or conditions afflicting the lung,
brain, heart, kidney, tumor, blood,
immune system, skin, eye, nasal passages, scalp, testes, cervix, oral cavity,
pharynx, esophagus, intestine
(small and large), synovir tissues, muscle, ovaries, and ear canal, among
others, and in general any cells that
contain, or originate from, the target sites.
The invention will now be described in reference to specific drawings. Other
objects, advantages and
features of the present invention will become apparent to those skilled in the
art from the description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows an experiment where saline (control), adenosine (o), and dAMP
(P) were separately
administered to rabbits. Saline had no effect, and both adenosine and dAMP
affected a similar reduction
compliance in a dose dependent manner. These results show that nucleosides
such as dAMP either directly or
following degradation and/or metabolism to adenosine, have the physiological
effects of adenosine at
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adenosine receptors.
Figure 2 accompanying this patent demonstrates that oligonucleotides (oligos)
containing adenosine
(A), but not those which do not have adenosine (desA) release bioactive
adenosine. The released adenosine
activates adenosine receptors and causes biological responses which may
interfere with signals to be observed
in validation studies. Here, two 21-mer randomer phosphorothioate anti-sense
oligonucleotides (oligos), one
containing adenosine (triangles) and one desA oligo (circles), were
administered to asthmatic rabbits. The
adenosine containing oligonucleotide caused a significant loss of airway
compliance, reflecting A receptor
activity while the desA randomer oligo did not.
Figures 3 and 4 demonstrate that anti-sense oligonucleotides may be utilized
as effective agents in the
validation of targets associated with pulmonary or airway diseases. Figure 3
illustrates the effects of
oligonucleotides anti-sense to the adenosine A, receptor, and of mismatch
control anti-sense oligonucleotides
on the dynamic compliance of the bronchial airway in a rabbit model. Figure 3
illustrates the specificity of
oligonucleotides anti-sense to the A, adenosine receptor as indicated by the
A, and A= adenosine receptor
number present in A, adenosine receptor anti-sense oligonucleotide-treated
airway tissue.
The invention will be better understood in reference to the following
description of the preferred
embodiments.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention arose from a desire by the inventor to provide a novel
technology for the rapid and
efficient discovery of the function and, therefore, usefulness, of target
genes and their products. The inventor
surmised that he might successfully do this by applying his own prior
discovery that low adenosine anti-sense
oligonucleotides (oligos) may be administered in vivo to subjects without
eliciting undesirable side effects
mediated by adenosine receptors. The present method enables the assessment of
the value of newly
discovered genes and gene products as focal points for drug discovery programs
with the aid of anti-sense
oligonucleotides that ablate their function in vitro and/or in vivo. While the
utilization of anti-sense
oligonucleotides to ablate gene expression has had theoretical importance in
the past, the inventor's fording
that anti-sense oligonucleotides are degraded in vitro and in vivo, and
release their constitutive nucleotides
and nucleosides has hampered their successful application, explaining their
ineffectiveness. The present
inventor has evidence that one of the oligonucleoside degradation products,
adenosine monophosphate is, as
adenosine itself, highly bioactive in various tissues. Adenosine itself is
known to mediate pleiotropic effects
including depression of neurotransmission, sleep induction, antagonism of D 1
and D2 dopamine receptors,
anti-nociception, mediation of various effects of ethanol including motor
incoordination, autonomic control of
cardiac function, bronchoconstriction, negative chronotropy, inotcpy and
dromotropy, anti-p-adrenergic
action, and renal sodium retention, among others. Clearly, the liberation of
even minute amounts of adenosine
and as shown in the present examples adenosine nucleosides in certain tissues,
e.g. CNS, lung, heart, and
kidney, among others, could activate adenosine receptors in the local
environment. This would make it
impossible to interpret target validation data in a reliable, unambiguous
fashion. Examples 30 and 31 and
figures 1 and 2 accompanying this patent illustrate the break down of
adenosine-containing oligonucleotides
to release bioactive adenosine nucleosides. The figures show that
oligonucleotides (oligos) containing
adenosine (A), but not those without adenosine (desA), release bioactive
adenosine. In the exemplary
disclosure, two 21-mer randomer phosphorothioate anti-sense oligonucleotides
(oligos), one containing
adenosine (>) and one desA oligo (o), were administered to asthmatic rabbits.
The results showed that the
adenosine containing oligonucleotide caused a significant loss of airway
compliance, reflecting adenosine
receptor activity, while the desA randomer oligo did not. See, Example 31
below. In addition, an adenosine
3
CA 02366055 2001-08-31
WO 00/51621 PCT/US00/05643
nucleoside (dAMP) was shown to have an effect similar to adenosine at an
adenosine receptor. See, Example
30 below.
The work described here and results discussed in the examples accompanying
this patent clearly
shows that target validation is successfully attained by applying the method
of the invention to new targets as
they are discovered and/or to known targets as they become associated with
their functions. The experimental
work further indicates that the present method was found to be highly
selective and effective at
validating/invalidating targets when employing non-phosphodiester anti-sense
oligonucleotides specifically
targeted, by countering or reducing effects mediated by the target proteins,
and the like. 'That was shown for
all the anti-sense oligos targeting an adenosine A, receptor mRNA, the 1 anti-
sense oligo targeting an
adenosine A,b receptor mRNA, the 2 anti-sense oligos targeting an A, receptor
mRNA, and the 1 anti-sense
oligo targeting a bradykinin receptor, were shown to counter effects mediated
by the specific adenosine
receptors elicited by exogenously administered adenosine. The method of this
invention, moreover, is specific
in validating/invalidating the specifically selected target, and fails to
inhibit other targets, as shown with the
anti-sense oligos targeted to the adenosine A, and bradykinin genes and mRNAs.
In addition, the results show
that the method of the invention employing low or no adenosine containing
oligos results in extremely low or
non-existent deleterious side effects or toxicity. This represents 100%
success in providing a method that is
highly effective and specific in the validation of targets, as shown here for
the respiratory system. This
invention is broadly applicable in the same manner to all genes) and
corresponding mRNAs encoding
proteins of the respiratory/pulmonary system, involved in or associated with
airway diseases as well as targets
of other systems associated with specific diseases or conditions which may be
correlated with a specific
function or end point, e.g. the CNS. A comparison was also made of the
implementation of the method of the
invention with the aid of a phosphodiester oligo, and a version of the same
oligonucleotide wherein the
phosphodiester bonds are substituted with phosphorothioate bonds. The results
of the application of the
method of the invention evidenced an unexpected superiority for the
substituted over the phosphodiester
oligonucleotides. Anti-sense oligonucleotides with a high content of adenosine
(33%) thus are not suitable to
attain clear target validation data since their breakdown could cause
pleiotropic adenosine-mediated effects.
The low adenosine oligomers utilized by the present method are clearly free of
such side effects. This patent
describes a method which may be utilized to perform unambiguous "target
validation" that is enabling, for
example, in the respiratory tract or pulmonary system, the CNS, and other
organs or systems, where the
liberation of adenosine may cause pleiotropic effects. The method involves the
use of low A or desA anti-
sense oligos, i.e. oligos that have a low A content or lack adenosine
altogether, and which may not, therefore,
liberate amounts of significant bioactive adenosine upon degradation. The
present technology is enabled for
practice with virtually any gene, but has particular applicability for the
following gene subsets: G-protein
Coupled Receptors, Neurohormone Receptors, Neuropeptide Receptors,
Neurotransmitter Receptors, G
Proteins, Calcium Channel Proteins, Sodium Channel Proteins, Potassium Channel
Receptors, Chloride
Channel Receptors, i.e. virtually any gene expressed in normal or diseased
CNS, heart, lung, kidney, blood,
immune system, and many more in addition to those listed below. Thus, the
validation method described in
this patent may also be applied not only to genes and systems mentioned here,
but to other genuses and
subgenuses of genes and gene networks.
Successful drug discovery programs depend on a fast, accurate assessment of
the suitability of
candidate gene products as drug design targets. Traditionally, this process
has consumed an inordinate amount
of time, personnel and financial resources. This invention provides a rapid,
reliable method for target
validation/invalidation in various biological systems that utilizes
proprietary low or desAdenosine (desA) anti-
sense oligonucleotides (collectively called here desA-ASONs). Using desA-
ASONs, the present method may
4
CA 02366055 2001-08-31
WO 00/51621 PCT/CTS00/05643
validate/invalidate potential gene targets with a level of speed and accuracy
that has heretofore been
impossible using traditional technologies. DesA-ASONs provide greater levels
of accuracy and speed vs.
classical methods. DesA-ASONS cannot break down and release bioactive
adenosine which would obfuscate
target validation by causing adenosine-induced side effects. Among the ASONS
there is a subgroup intended
for respiratory administration herein referred to as RASONs. DesA-RASONs have
been used by the inventor
in practicing the method of this invention to validate adenosine A,, AZ and A,
targets. The present technology
is also applicable to CNS targets involving biochemical, behavioral,
physiological assessments following site
specific functional gene ablation in any region of the brain with desA-BASONs,
brain anti-sense
oligonucleotides (administered in situ in the brain) which cannot break down
to release adenosine, a major
modulator of brain physiology. In addition to the exemplary validation of
pulmonary targets, the present
method is illustrated below for CNS associated targets, and generally
comprises performing the following
several steps for target validation using desA anti-sense oligonucleotides.
The initial step requires the identification of a general system or area for
the target validation, e.g.
CNS, respiratory, renal, cardiac areas, etc. Then, the method turns to the aid
of public libraries such as
GenBank or other libraries in the public domain or private libraries such as
those proprietary to specific
companies. For example, a specific library such as one encompassing all G-
protein coupled receptors
(GPCRs) found in public and private data bases. Currently, there are about 250
GPCRs in existence. The
present target validation method, thus, is suitable for testing what
physiological, biophysical, biological,
behavioral, etc., events occur when the selected group of targets, e.g. GPCRs,
are individually attenuated
using appropriate desA anti-sense oligos. For this, desA anti-sense oligos are
designed as described below,
and synthesized for pre-selected target genes, e.g. the GPCRs referred to
above. The desA anti-sense oligos
are then tested, first in vitro, for example by using an appropriate cell
line, primary cell culture, or other cell
tissues. Such in vitro testing may be applied to determine which of several
desA anti-sense oligos designed
against a specific target are "most active" as anti-sense agents. That is,
which one best down regulates or
ablates gene expression. This may be done by using a biophysical, biological,
physiological, or other assay
which may be correlated with a specific activity of the cell. In other cases,
knocking out the target gene in the
in vitro system may itself provide useful target validation information. The
most active desA anti-sense oligos
are then selected and applied in vivo, for example, by direct instillation
into any brain region for CNS studies,
by administration into the lung targets associated with the respiratory
system, and the like. This may be done
by methods known in the art, such as via stereotactic implantation of cannulae
for brain targets, via inhalation
for respiratory targets, systemically for blood targets, by in situ
administration for organs and other localized
systems and for both parenchyma) cells and vascular heart cells, and
systemically via inhalation or via direct
instillation for renal targets. Behavioral, biophysical, physiological,
biochemical, immunological, and other
tests may be applied and data obtained on individual animals by administration
of the appropriate anti-sense
oligonucleotides to ablate one or more target genes. Behavioral functions or
endpoints such as food ingestion,
anxiety, libido, cognition, etc., or such physiological end points as
temperature, electroencephalograms
(ECG), electrocardiograms (EKG), glomerular filtration volume and content, ion
retention, protein loss, etc.,
may be assessed by methods known in the art. These steps are illustrated for
the CNS in the scheme shown
below.
CA 02366055 2001-08-31
WO 00/51621 PCT/US00/05643
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CA 02366055 2001-08-31
WO 00/51621 PCT/US00/05643
The present method relies on low A or desA-ASONs for application to
respiratory (desA-RASONs),
CNS (desA-BASONs), renal (desA-KASONs), cardiac (desA-CASONs), blood (desA-
SASONs), immune
system (desA-IASONs), malignant tissue (desA-MASONs), and other functions,
without releasing significant
bioactive amounts of adenosine. In this fashion, the present method prevents
the undesirable activation of
adenosine receptors in the lung, CNS, kidney, heart, blood, immune system,
malignant cell aggregates, etc.
Results obtained with presently available methods are less interpretable
because standard anti-sense constructs
contain normal (high) levels of adenosine about 25%, and activate adenosine
receptors. This effect blurs the
results and confuses the interpretation of functionality. In the lung, for
example, oligo-released adenosine will
cause changes in airway diameter (bronchoconstriction), inflammation, and
secretion of surfactant, all effects
that in the present case are unrelated to the validation of a different
target. Such "side effects" make the data
obtained with oligonucleotides or with ribozymes for that matter
uninterpretable. Similarly when an A-
containing anti-sense oligonucleotide is used in the brain, its effects
obscure a plurality of functions which
may be used as end points to validate targets. For example, adenosine causes
depression of neurotransmission,
sleep induction, anti-nociception, mediates various effects of ethanol
including lack of motor coordination,
autonomic control of cardiac function, antagonism of dopamine Dl and D2
receptors, alterations in CNS
blood flow, and a host of other effects. The presence of significant amounts
of adenosine clearly is
contraindicated when anti-sense oligos are used in target validation studies
in the hyper-responsive lung, CNS,
and other systems which contain adenosine receptors or are otherwise
responsive to adenosine. For these
reasons, the present method provides a superior target validation tool.
Particularly useful applications of the
present method are to investigate therapeutic areas related to the respiratory
tract, CNS, blood, malignant and
uncontrollably growing cells, and many other systems, which may be separately
targeted, and where one or
more functions associated with the targets are separately measured.
As the present method has been invented at a singular time in the history of
biological sciences, it
will permit a faster and more efficient utilization of the information
obtained from the sequencing of the
human genome. Knowing the sequence of every gene in the human genome has been
the Holy Grail of
modern medicine. This accomplishment will enable the correlation of specific
genes with their functions and
thereafter the creation of entirely new types of medicines that strike closer
to the heart of disease and lack the
side effects of currently available drugs. The flood of sequences that is
being banked currently provides
important targets. The present method provides an invaluable tool to identify
genes that are important in
body function and, thus, in disease, and to determine whether or not the
inhibition of their function is
therapeutically useful. The process of determining if inhibiting the function
of a gene is therapeutic is called
here "Target Validation". It is an important early step in the drug discovery
process, and it helps determine
whether or not critical resources are to be expended to develop new drugs for
inhibiting the function of a
target gene. In this context, it is just as important to validate a target as
it is to invalidate it, by showing that
inhibiting its function is without functional (therapeutic) effect. An early
invalidation of a target in the drug
discovery process prevents the waste of resources in unftuitful drug discovery
campaigns. This, in turn, will
permit a more rapid and focused investigation of more productive targets. The
present rapid and accurate
target validation method may make the difference between success and failure
in the therapeutic application
of large volumes of information, e.g. that was obtained from the human genome
project. The in vivo testing
of the anti-sense oligos in the method of the invention may be implemented,
for example, in vitro and in
animal models for important diseases, including models for respiratory
diseases such as asthma, hormonal
diseases, genetic diseases, obesity, and the like, including diseases of the
CNS, renal, cardiac, blood diseases,
etc. Assays known in the art may be utilized, such as whole body
plethysmographic techniques in the
conscious, unrestrained rodent, rabbit or primate, e.g. TruePrimateJ, or other
species, applied to practice this
7
CA 02366055 2001-08-31 ~~~ ~ ~ 0 5 6 4 3
- - EPI-149
A
invention. The present method, thus, helps to determine the existence of a
correlation between a function of a
disease or condition and a gene or mRNA encoding a target polypeptide
suspected of being associated with it.
The method itself generally comprises obtaining oligonucIeotides (oligos)
consisting of up to about 15%
adenosine (A), and which is anti-sense to a target selected from the group
consisting of target genes and their
corresponding mItNAs, genomic and mRNA flanking regions selected from the
group consisting of 3' arid 5'
intron-exon borders and the juxta-section between coding and non-coding
regions, and all mRNA segments
encoding polypeptides associated with a pre-selected disease or condition;
selecting amongst the oligos one
that sibnificantty inhibits or ablates expression of the polypcptide encoded
by the mRNA_ upon in vitro
hybridization to the target mRNA; administering to a subject an amount of the
selected oligo effective for in
vivo hybridization to the target mRNA; and assessing a subject's function that
is associated with the disease or
condition before and after administration of the oligo; wherein a change in
the function's value greater than
about 70% indicates a positive correlation, between about 40 and about 70% a
possible correlation, and below
about 30% a lack of correlation.
The anti-sense oIigos may be constructed by selecting fragments of a target
having at Least 4
contiguous nucleic acids selected from the group consisting of G and C and
obtaining a first oligonucleotide
' about 4, 6, 8, I O to about 15, 25, 45, 60 nucleotides long which comprises
the selected fragment and has a C
and G content of about 0%, about 3%, about 59~°, about IO%, about I2%
up to about I S%. Alternatively, the
target fragments may be selected by their type and/or extent of activity,
which may vary for pecific
purposes. Any number of adenosines may be substituted, if present, from ont to
all, by a "universal" or
alternative bass anch~as heteroaromatic bases which bind to a thymidine base
but have less than about 0.3 of
the adenosine base agonist activity at the adenosine A,, Ab, A~ and Al
raeptors, and heteroammatic bases
which have no activity at the adenosine A~, receptor. The heteroaromatic bases
may be pyrimidines and
purines, which may be substituted, for example, by O, halo, NHr, SH, SO, SOb
SOs, COOFi and branched and
fused primary and secondary amino, alkyl, alkcnyl, alkynyl, cycloalkyi,
heterocycloalkyI, aryl, heteroaryl,
alkoxy, alkenoxy, : acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, amyl,
arylthio, arylsttlfoxyl,
halocycloalkyI, alkyIcycloalkyl, alkenyIcycloalkyl, alkynylcydoallyl,
haloaryl, alkylaryl, alkenylaryl,
alkyaylaryl, arylalkyl, arylalkenyi, arylalkynyl, arylcycloaIkyl, which may be
further substituted by O, halo,
NH=, primary, secondary and tertiary amine, SH, S0, SO=, SO~, cycloalkyl,
heterocycloalkyl and heteroaryl.
Other compounds and other substituents, however, ,are also suitable for use
with the present method.
Typically, the pyrimidines and purines are substituted at positions 1, 2, 3,
4, 7 and 8, although other
substitutions are also encompassed. Examples of pyrimidines and purines are
theophylline, caffeine,
dyphylline, etophylline, acephyliine piperazine, bamifylline, enprofylline and
xanthine having the chemical
formula
O N
it t
RI 1N1 6 5 C/7 ~ 3
11
O:C~Ii/C~?~i
R''
wherein R' and R= are independently H, alkyl, alkenyl or allynyl and R' is H,
aryl, dicycIoalkyl,
dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-
cycloalkyl, O-cycloalkenyl, O-
cycloaIkynyl, NHs-alkylamino-ketoxyaIkyloxy-aryl and mono and
dialkylaminoa11,y1-N-alkylamino-SO= aryl.
8 . .
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Specific examples of universal or alternative bases are 3-nitropyrrole-2'-
deoxynucleoside, 5-nitro-indole, 2-
deoxyribosyl-(5-nitroindole), 2-deoxyribofuranosyl-(S-nitroindole), 2'-
deoxyinosine, 2'-deoxynebularine, 6H,
8H-3,4-dihydropyrimido [4,5-c] oxazine-7-one or 2-amino-6-methoxyaminopurine,
although others are also
suitable. Most preferred are adenosine analogs which have no activity at
adenosine receptors, i.e., which have
neither agonist nor antagonist properties at any adenosine receptor. The
method may also utilize, in another
preferred embodiment, a methylated cytocine ('"C) substituted in or for at
least one unmethylated C in a CpG
dinucleotide if present in the oligo(s), although many more or all may also be
substituted. Other C-5
modifications at pyrimidines are also useful, e.g., C-5 propyne, among others.
For practicing this method, one
or more or all linking residues of the anti-sense oligonucleotides are
preferably substituted or modified with a
residue selected from the group consisting of methylphosphonate,
phosphotriester, phosphorothioate,
phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether,
carbonate, carbamate, sulfate,
sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide,
hydroxylamine, 2'-
methylene(methyimino), (MMI), 2'-methoxymethyl (MOM), 2'-methoxyethyl (MOE),
2'-methyleneoxy
(methylimino) (MOMA), 2'-methoxy methyl (MOM), 2'-O-methyl, phosphoramidate,
and C-5 substitued (e.
g. C-5 propyne) residues and combinations thereof. Anti-sense oligos suitable
for practicing this method are
about 7, about 9, about 11, about 13, about 15, about 18, about 21 to about
25, about 28, about 30, about 35,
about 40, about 45, about 50, about 55, about 60 mononucleotides long,
although other lengths are also
suitable.
The method of the invention may also incorporate the use of an anti-sense
oligo that is linked to an
agent that is internalized or up-taken by cells as well as cell targeting
agents, such as transferrin,
asialoglycoprotein and streptavidin, among others known in the art. In one
embodiment the oligo is linked to a
vector, which may be prokaryotic or eukaryotic. Examples of vectors are known
in the art and need not be
described further in this patent. The amount of anti-sense oligo administered
is generally one that is effective
to reduce the production or availability, or to increase the degradation, of
the mRNA, or to reduce the amount
of the polypeptide present in situ. For example, when the gene to be validated
is associated with a respiratory
function, the anti-sense oligo may be administered directly to the lung (s).
When the gene and the function
are associated with another system, the nucleic acid is preferably
administered in situ to the affected region, e.
g. the brain, the heart, the kidney, the bladder, the gonads and the
reproductive system in general, the
respiratory and pulmonary systems, tumors in the case of cancer, the blood,
the immune system, the lung,
skin, eye, nasal passage, scalp, testes, cervix, oral cavity, larynx,
esophagus, small and large intestine,
synovial tissues, muscles, ovaries, ear canal, and many more, such as any
cells that originate from a selected
target site. In many cases, a disease or condition afflicts a certain system
or area of a system, such as those
described above. For example, a respiratory ailment may be associated with an
increase in
bronchoconstriction, inflammation, IgE-mediated allergies, surfactant
production, and other symptoms such as
in asthma, allergic rhinitis, COPD, lung tumors, ARDS, etc. Where a disease or
condition is associated with an
immunological dysfunction, the target may be selected amongst immunoglobulins
and antibody receptors,
cytokines and cytokine receptors, genes and other gene products, and
corresponding mRNAs encoding them
and other associated functions, the genes and mRNA flanking regions and intron
and exon borders, among
others. When the disease or condition is associated with a malignancy or
cancer, the target may be selected
from cancer related gene products, genes and mRNAs encoding therri, genes and
mRNAs associated with
oncogenes, genomic and mRNA flanking regions and exon and intron borders, etc.
The anti-sense oligos for use with the present method may be produced by
selection of a target from
the group consisting of polypeptides associated with a diseases) and/or
conditions) afflicting lung airways,
genes and RNAs encoding them, the genomic and mRNA flanking regions and the
genes) and mRNA(s)
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EPI-149
intron and exon borders, then obtaining the sequence of a mRNA(s) selected
from the group consisting of
mRNAs corresponding to the target genes) and mRNAs encoding the target
polypeptide(s), genomic and
mRNA flanking regions and the genes and mRNAs intron and exon borders;
selecting at least one segment of
the mRNA(s), synthesizing one or more oligo anti-sense to the selected mRNA
segment(s); and substituting, if
necessary, a universal or alternative bases) for one or more A(s) to reduce
the content of A present in the
oligo to up to about 10% of all nucleotides. Specific examples of the encoded
polypeptides associated with the
pulmonary system are NfxB Transcription Factor, Interleukin-8 Receptor (IL-8
R), Interleukin 5 Receptor
(IL-5 R), Interleukin 4 Receptor (IL-4 R), Interleukin 3 Receptor (IL-3 R),
Interleukin-1(3 (IL-1(3), Interleukin
1 ~i Receptor (IL- 1 (3 R), Eotaxin, Tryptase, Major Basic Protein, X32-
adrenergic Receptor Kinase, Endothelin
Receptor A, Endothelin Receptor B, Preproendothelin, Bradykinin B2 Receptor,
IgE High Affinity Receptor,
Interleukin 1 (IL-1), Interleukin 1 Receptor (IL-1 R), Interleukin 9 (IL-9),
Interleukin-9 Receptor (IL-9 R),
Interleukin 11 (IL-11), Interleukin-11 Receptor (IL-11 R), Inducible Nitric
Oxide Synthase, Cyclooxygenase
(COX), Intracellular Adhesion Molecule 1 (ICAM-1) Vascular Cellular Adhesion
Molecule (VCAM), Rantes,
Endothelial Leukocyte Adhesion Molecule (ELAM-1), Monocyte Activating Factor,
Neutrophil Chemotactic
Factor, Neutrophil Elastase, Defensin 1, 2 and 3, Muscarinic Acetylcholine
Receptors, Platelet Activating
Factor, Tumor Necrosis Factor a, 5-lipoxygenase, Phosphodiesterase IV,
Substance P, Substance P Receptor,
Histamine Receptor, Chymase, CCR-1 CC Chemokine Receptor, CCR-2 CC Chemokine
Receptor, CCR-3 CC
Chemokine Receptor, CCR-4 CC Chemokine Receptor, CCR-S CC Chemokine Receptor,
Prostanoid
Receptors, GATA-3 Transcription Factor, Neutrophil Adherence Receptor, MAP
Kinase, Interleukin-9 (IL-9),
NEAT Transcription Factors, STAT 4, MIP-1 a, MCP-2, MCP-3, MCP-4,
Cyclophillins, Phospholipase A2,
Basic Fibroblast Growth Factor, Metalloproteinase, CSBP/p38 MAP Kinase,
Tryptose Receptor, PDG2,
Interleukin-3 (IL-3), Interleukin-1~3 (IL-1(3), Cyclosporin A-Binding Protein,
FKS-Binding Protein, a4~31
Selectin, Fibronectin, a4~7 Selectin, Mad CAM-1, LFA-1 (CDlla/CD18), PECAM-1,
LFA-1 Selectin, C3bi,
PSGL-1, E-Selectin, P-Selectin, CD-34, L-Selectin, p150,95, Mac-1
(CDllb/CD18), Fucosyl transferase,
VLA-4, CD-18/CDlla, CDllb/CD18, ICAM2 and ICAM3, CSa, CCR3 (Eotaxin Receptor),
CCR1, CCR2,
CCR4, CCRS, LTB-4, AP-1 Transcription Factor, Protein kinase C, Cysteinyl
Leukotriene Receptor,
Tachychinnen Receptors (tach R), IxB Kinase 1 & 2, STAT 6, c-mas and NF-
Interleukin-6 (NF-IL-6).
Examples of polypeptides or genes associated with the CNS, ophthalmic,
cardiovascular and cardiopulmonary
systems are the family of G-protein coupled receptors (aproximately 250 known,
and approximately
750-1,000 more postulated and yet to be sequenced), Neuropeptide genes,
Neuropeptide receptor genes,
Excitatory amino acid receptor genes, Chloride channel genes, Calcium channel
genes, Purinergic receptor
genes, Adrenergic receptor genes, Serotonin receptor genes, Serotonin
transporter genes, Excitatory amino
acid transporter genes, Potassium channel genes, Tyrosine kinases,
Phosphorylases, Acetylcholine receptors,
Cholecystokinin receptors, Nitric Oxide synthase, Dopamine receptors,
Cholinergic receptors, Angiotensin,
Angiotensin receptors, Ion Channels including Potassium Channels, Structural
proteins including those related
to myelination/demyelination and axonic and dendritic structures,
Neurotransmitter release mediators and
structures, Opthalmic disorders, especially of the retina and asociated
structures, Calcitonin and its receptors,
Calcineurin and its receptors, CGRP and its receptors, Atrial natriuretic
peptide and its receptors, Brain
natriuretic peptide and its receptors, Bradykinin and its receptors,
Baroreceptors, GABA/GABA receptors,
Benzodiazepine receptors, Cholinesterases, Cannabinoid receptors, Calmodulin
and its receptors,
Calcium/Sodim exchange pump
Carbonic anhydrase, Catecholamines and their receptors, Histamine receptors,
Muscarinic receptors, Opioid
receptors, Chemokines, Choline acetyltransferase, Cholecalciferol, Mediators
of inflammation including
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cytokines, interleukins, interferons and their receptors, enzymes of the
lipoxygenase pathway, proteases,
DP(PGD2) receptors, Inositol phosphate associated enzymes, Endothelins and
their receptors, Enkephalinase,
Enkephalins, Benzodiazepine receptors, GABA transaminase, Galanin and its
receptors, Gastrin releasing
factor, Growth factors, Growth factor inhibitors, Cyclins, Nucleoside kinases,
Nucleotide kinases, Oncogenes'
Receptors, 5-hydroxytryptamine (SHT) receptors, ADH, IGF, Insulin receptors,
Lactamases, Kainate
receptors, Kallikreins and their recepotrs, Leukotriene-associated enzymes,
Lipocortins, L-NMMA and
receptors, Melanocyte stimulating hormone, Steroid transporters and metabolism
enzymes and synthases,
NMDA and receptors, Morphine receptors, MPTP, Neurokinins and their receptors,
Nicotinic receptors,
Phospholipases, Platelet activating factors, Signal transduction proteins,
PDGF, Dynorphins, Prostacyclins,
Prolactin and its receptors, Prolactin release inhibiting factor, Prohormones,
Prostanoids, Prostaglandins and
their receptors, Thrombin, Prothrombin, Pteroylglutamic acid and its
receptors, Lysergic acid receptors, Snake
and scorpion venom recertors, Renin angiotensin system components, Reverse
transcriptase, Second
messengers and associated enzymes, Sodium channels, Somatostatin and its
receptors, Somatotropin and its
receptors, Substance P, Substance k, Synatpic transmitters, Tachikinins,
Tetrodotoxin receptors,
Thromboxanes and their receotrs, Thyroid hormones, Hormones, Thyrotropin and
receptors, Protirelin, T4, T3
Topoisomerases, Tumor necrosis factors and their receptrors, TGFs and their
receptors, Xanthine oxidase,
Viral messenger RNAs, Bacterial mRNAs, Oxytocin and its receptors,
Chlecystokinin and its receptors,
Vasoactive intestinal peptide and its receptors, Monoamine oxidase, Tyrosine-
kinase linked receptors, and
many more. Other specific target genes are, for example, G-proteins and G-
protein coupled receptors, calcium
channel proteins and associated protein receptors, sodium channel proteins and
associated protein receptors,
potassium channel proteins and associated protein receptors, and chloride
channel proteins and associated
protein receptors, neurotransmitters and neurotransmitter receptors,
neurohormones and neurohormone
receptors, neuropeptides and neuropeptide receptors, and many others including
the ones listed throughout
this patent. Other target genes are, for example, G-proteins and G-protein
coupled receptor, calcium channel
proteins and associated protein receptors, sodium channel proteins and
associated protein receptors, potassium
channel proteins and associated protein receptors, and chloride channel
proteins and associated protein
receptors, neurotransmitters and neurotransmitter receptors, neurohormones and
neurohormone receptors,
neuropeptides and neuropeptide receptors, and many others.
In the present method, the composition may be administered in vitro, orally,
intracavitarily,
intranasally, intraanally, intravaginally, intrauterally, intracranially,
pulmonarily, intrarenally, intranodularly,
intraarticularly, intraotically, intralymphatically, transdermally,
intrabucally, intravenously, subcutaneously,
intramuscularly, intratumorously, intraglandularly, intraocularly,
intracranial, into an organ, intravascularly,
intrathecally, by implantation, by inhalation, intradermally,
intrapulmonarily, into the ear, onto the skin or
scalp or cervix (e.g. topically), into the heart, by slow release, by
sustained release and by a pump, and the
like. Examples of target genes and mRNAs associated with different systems and
diseases are genes and
mRNAs encoding polypeptides such as transcription factors, stimulating and
activating factors, cytokines and
their receptors, interleukins, interleukin receptors, chemokines, chemokine
receptors, endogenously produced
specific and non-specific enzymes, immunoglobulins, antibody receptors,
central nervous system (CNS) and
peripheral nervous and non-nervous system receptors, CNS and peripheral
nervous and non-nervous system
peptide transmitters, adhesion molecules, defensines, growth factors,
vasoactive peptides, peptide receptors
and binding protein and genes and mRNAs corresponding to oncogenes. The
administration of the oligo may
be conducted with an oral formulation having a liquid carrier such as
solutions, suspensions, and oil-in-water
and water-in-oil emulsions, and/or may be administered as a powder, dragees,
tablets, capsules, sprays,
aerosols, solutions, suspensions and emulsions. When administered as a topical
formulation, the carrier may
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be selected from creams, gels, ointments, sprays, aerosols, patches,
solutions, suspensions and emulsions.
When the formulation is injectable, the carrier may be selected from aqueous
and alcoholic solutions and
suspensions, oily solutions and suspensions and oil-in-water and water-in-oil
emulsions, among others. When
as a rectal formulation, it may be in the form of a suppository, when in the
form of a transdermal formulation,
the carrier is selected from aqueous and alcoholic solutions, oily solutions
and suspensions and oil-in-water
and water-in-oil emulsions, although others are also suitable. The transdermal
formulation, may be an
iontophoretic transdermal formulation, wherein the carrier is selected aqueous
and alcoholic solutions, oily
solutions and suspensions and oil-in-water and water-in-oil emulsions, and the
formulation may also contain a
transdermal transport promoting agent, of which many are known in the art.
Also suitable for prolonged
administration are implantable capsules or cartridges containing the
formulation. In this case, the carrier may
also be selected from aqueous and alcoholic solutions and suspensions, oily
solutions and suspensions and oil-
in-water and water-in-oil emulsions, be a hydrophobic carrier, such as lipid
vesicles or particles, e. g.
liposomes made of N-( 1-[ 2, 3-dioleoxyloxi] propyl) -N,N,N- trimethyl-
ammonium methylsulfate. and/or
other lipids, and microcrystals. For pulmonary applications, the formulation
is preferably a respirable or
inhalable formulation, e. g. in the form of an aerosol. For prolonged exposure
of a target area, the oligo may
be delivered through a localized implant, suppository, sublingual formulation,
and the like, all of which are
known in the art.
A factor which proves this method superior to other technology is the ability
to obtain data of greater
reliability and accuracy. Anti-sense ribozyme technology is unstable in in
vivo environments, and the
presence of adenosine in ribozyme and other oligonucleotides prevents the
attainment of reliable data, for
instance in the hyperactive respiratory tract, and other systems having a
substantial number of adenosine
receptors. No other method has proven, up to the present time, capable of
unambiguously attenuating targets
in A-containing systems, e.g. the respiratory tract while providing reliable
and accurate correlations. In
addition, the present method may be utilized to the elucidation of gene
networks, e.g. neuronal gene networks,
a quantum leap above the identification of single genes in isolation of their
broader context. This may be
done by selecting more than one target linked in a metabolic pathway and
testing them separately and in
conjunction with the others, that is by administration of one anti-sense oligo
at a time, then in twos, in threes,
etc., and comparing the results to ascertain whether or not there is linkage,
they work sequentially etc. The
present method permits the creation of a gene network data base suitable to
supplement a more elaborate
pharmoincentive discovery process to meet critical medical needs in areas such
as cognition, memory, pain,
anxiety, behavioral disturbances, ingestive behavior, hunger and satiety, and
neurological disease, among
others relating to the CNS. The present technology encompasses four basic
areas: functional genomics
applied to discerning neuronal gene networks of relevance to new drug
discovery, in situ hybridization to
understand the distribution of these networks within the brain, proprietary
Site Specific Functional Gene
Ablation (SSFGA) to determine the function of individual genes within network,
Multifactorial Behavioral
Analysis for qualitative and quantitative analysis of the participation of
genes and gene networks in various
behaviors of medical relevance, and physiological, biophysical, biochemical,
etc. analysis to discern the
effects of genes and gene networks upon extrapyramidal systems such as the
cardiovascular and pulmonary
systems.
The application of this method to the CNS may encompass areas such as pain,
satiety, anxiety/mood
disorders, libido, cognition/cognitive disorders, and sleep/sleep disorders,
among many others.The ability to
discern functional significance of identified novel genes and gene networks
relies on an assessment of their
spatial representation. In situ hybridization, for example, may be is used to
determine the precise three
dimensional (3-D) location of selected genes and gene networks within the
brain, and to enable the accurate
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targeting of gene ablation studies for assessing their significance as
candidates for drug discovery programs.
Site-specific functional gene ablation (SSFGA) provides a means to selectively
attenuate the expression of any
target gene in any desired region of a system, e.g. the brain. SSFGA for CNS
target validation may be
performed with the aid of the low A or desA anti-sense oligonucleotide
designed as described below. Other
approaches utilizing anti-sense oligonucleotides provide ambiguous data
because the oligonucleotides used
breakdown and release adenosine, one of the most bioactive autocoids, e.g. in
the CNS and in other target
systems containing adenosine receptors. The release of adenosine upon break-
down of oligonucleotides either
depresses or facilitates neurotransmission depending upon the system and the
specific area, e.g. the brain
region, where it is released, induces sleep, affects nociception, alters
thalamic spindle rhythms, mitigates or
potentiates myriad drug effects, affects autonomic control of cardiovascular
function and respiration, inhibits
Ca+ currents and presynaptic function of GABA, depresses both spontaneous and
evoked neuronal firing,
inhibits the release of neurotransmitters, decreases postsynaptic
excitability, inhibits long-term potentiation
postulated to be an underlying event in learning and memory, and induces
pleiotropic effects by causing
cephalic bronchodilation. Clearly, the release of significant amounts of
adenosine via break down of
oligonucleotides in the brain, or elsewhere, of the experimental animal is
contraindicated in target validation
studies. Neurological and behavioral tests suitable for application as end
points for CNS target validation are
known in the art. For example, tests for memory, three dimensional or spatial
ability, cognition, motor control,
sensitivity and responsiveness to exogenous triggers, vision, eye
coordination, skin sensory ability, gustation
and olfactory recognition, and many more. The testing of physiological
parameters is also known in the art
and may rely on the measurement of electrical conductivity such as EKGs and
EEGs, or other bodily
functions such as heart rate and rhythmicity, water voiding, sleep patterns,
etc. In many cases, side effects,
particularly cardiopulmonary, renal, and other side effects, constitute a
major reason for disqualifying
potential drug discovery CNS targets. And vice versa, CNS side effects often
disqualify otherwise suitable
therapeutic cardiopulmonary, renal, and other agents. It thus would be
advantageous to obtain evidence of
cardiopulmonary effects induced by attenuation of CNS targets as early as
possible in the development of a
drug. When such effects are discovered late in a development program they
cause the cancellation of a
program at a much later stage. The present method utilizes state of the art
analysis, significant surgical,
electrophysiological, and other types of tests, to assess any detrimental
effects, e.g. cardiovascular effects of
attenuating respiratory targets, and vice versa any detrimental side effects
of attenuating respiratory targets on
CNS or the cardiovascular system, among others. For example, lung fiznction
studies performed in conscious,
unrestrained animals, and cardiac function studies may provide further insight
on potential effects of
attenuating candidate CNS targets.
Target validation may proceed via SSFGA as described above using low A or desA
anti-sense
oligonucleotides targeting a variety of known receptors with suspected
function in human pathology, e.g.
neuropeptide Y (NPY)/leptin receptors and ingestive behavior, as well as novel
targets found from CNS gene
libraries. Animal models may be subjected to SSFGA targeting of a specific
receptor and then assessed for
changes in various behaviors, such as in ingestive behavior, analgesia,
locomotor behavior, sensimotor
reflexes, gating processes, sexual and reproductive behaviors, anxiety,
learning and memory, among many
others. This broad-based behavioral battery of tests provides a sensitive
measure of the effect of attenuating
specific CNS targets, and a critical knowledge in the initial stages of the
drug development decision process.
Combined with a physiological/biophysical/biochemical analysis, this method
provides for a rapid, intensive
determination of the potential of a candidate target as a worthwhile focus for
further drug discovery efforts.
The present method improves on prior methods for validation of gene and
protein targets in that desadenosine
anti-sense oligos targeted to genes associated with different systems are used
to inhibit the expression of a
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gene product and to test the effects and symptomatology and changes these
procedures. The present invention
is premised on the recent discovery by the inventor that, when
oligonucleotides are metabolized in vivo to
their mononucleotides, bioactive adenosine metabolites are released. Adenosine
(A)-containing
oligonucleotides break down and release adenosine metabolites which, in turn,
activate adenosine receptors
which, for example, in the lungs cause bronchoconstriction, inflammation, and
the like. The present
technology relies on the design of anti-sense oligos targeted to genes and
mRNAs associated with systems
involved in different functions, ailments, and pathology(ies). The oligos are
modified to reduce their
adenosine content to minimize the occurrence of undesirable side effects
caused by its release upon
breakdown. Doing so improves the statistical significance of the results
observed, particularly where
adenosine receptors may be involved in effects similar, or opposite to those
being observed, or indirectly by
producing changes in the experimental model's homeostasis of the system being
observed. In this manner, the
inventor targets a specific gene to design one or more anti-sense
oligonucleotide(s) (oligos) that selectively
binds) to the corresponding mRNA and, if necessary, reduces their content of
adenosine via substitution with
universal or alternative base or an adenosine analog incapable of activating
adenosine A,, Aza, Azb or A3
receptors. Based on his prior experience in the field, the inventor reasoned
that in addition to
"downregulating" or ablating specific genes, he could increase the accuracy of
the results by either selecting
segments of RNA that are devoid, or have a low content, of thymidine (T) or,
alternatively, substitute one or
more adenosine(s) present in the designed oligonucleotide(s) with other
nucleotide bases, so called universal
or alternative bases, which bind to thymidine but lack the ability to activate
adenosine receptors and otherwise
exercise the effect of adenosine in the lungs, etc. Given that adenosine (A)
is a nucleotide base complementary
to thymidine (T), when a T appears in the RNA, the anti-sense oligo will have
an A at the same position. For
consistency's sake, all RNAs and oligonucleotides are represented in this
patent by a single strand in the 5' to
3' direction, when read from left to right, although their complementary
sequences) is (are) also encompassed
within the four corners of the invention. In addition, all nucleotide bases
and amino acids are represented
utilizing the recommendations of the IUPAC-IUB Biochemical Nomenclature
Commission, or by the known
3-letter code (for amino acids).
The method of the present invention may be used to validate or invalidate any
number of target
genes, from single genes associated with one function in a subject, to
multiple single genes associated with a
network or pathway within a system. By validation/invalidation it is meant a
demonstration through
experimental evidence whether a specific gene is involved in any one of a
number of biophysical,
biochemical, physiological, behavioral, etc., functions. Even if a gene
appears initially not to have an effect, it
may be tested in conjunction with another gene target, as there may be a
requirement for the simultaneous
obliteration of their expression to observe an effect. The adenosine content
of the anti-sense agents) of the
invention have a reduced A content to prevent its liberation upon in vivo
degradation of the agent(s). For
example, if the system is the pulmonary or respiratory system, a large number
of genes is involved in different
functions, including those listed in Table 1 below.
Table 1: Pulmonary Disease or Condition Pulmonary and Inflammation Targets
NficB Transcription Factor Interleukin-8 Receptor (IL-8 R)
Interleukin-S Receptor (IL-SR) Interleukin-4 Receptor (IL-4R)
Interleukin-3 Receptor (IL-3R) Interleukin-1~3 (IL-1~3)
Interleukin-1~3 Receptor (IL-1~3R) Eotaxin
Tryptase Major Basic Protein
~i2-adrenergic Receptor Kinase Endothelin Receptor A
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Endothelin Receptor B Preproendothelin
Bradykinin B2 Receptor IgE (High Affinity Receptor)
(B2BR)
Interleukin-1 (IL-1) Interleukin 1 Receptor (IL-1 R)
Interleukin-9 (IL-9) Interleukin-9 Receptor (IL-9 R)
Interleukin-11 (IL-11) Interleukin-11 Receptor (IL-11 R)
Inducible Nitric Oxide Cyclooxygenase (COX)
Synthase
Intracellular Adhesion
Molecule 1 (ICAM-1)
Vascular Cellular Adhesion
Molec.Subst.P (VCAM)
Rantes Endothelial Leukocyte Adhesion Molecule
Endothelin ETA Receptor (ELAM-1)
Cyclooxygenase-2 (COX-2)GM-CSF, Endothelin-1
Monocyte Activating FactorNeutrophil Chemotactic Factor
Neutrophil Elastase Defensin 1,2,3
Muscarinic AcetylcholinePlatelet Activating Factor
Receptors
Tumor Necrosis Factor S-lipoxygenase
a
Phosphodiesterase IV Substance P
Substance P Receptor Histamine Receptor
Chymase CCR-1 CC Chemokine Receptor
Interleukin-2 (IL-2) Interleukin-4 (IL-4)
Interleukin-12 (IL-12) Interleukin-5 (IL-5)
Interleukin-6 (IL-6) Interleukin-7 (IL-7)
Interleukin-8 (IL-8) Interleukin-12 Receptor (IL-12R)
Interleukin-7 Receptor Interleukin-1 (IL-1)
(IL-7R)
Interleukin-14 Receptor Interleukin-14
(IL-14R)
CCR-2 CC Chemokine ReceptorCCR-3 CC Chemokine Receptor
CCR-4 CC Chemokine ReceptorCCR-5 CC Chemokine Receptor
Prostanoid Receptors GATA-3 Transcription Factor
Neutrophil Adherence MAP Kinase
Receptor
Interleukin-15 (IL-15) Interleukin-15 Receptor (IL-15R)
Interleukin-11 (IL-11) Interleukin-11 Receptor (IL-11R)
NEAT Transcription FactorsSTAT 4
MIP-1 a MCP-2
MCP-3 MCP-4
Cyclophillin (A, B, etc.)Phospholipase A2
Basic Fibroblast Growth Metalloproteinase
Factor
CSBP/p38 MAP Kinase Tryptase Receptor
PDG2 Interleukin-3 (IL-3)
Interleukin-10 (IL-10) Cyclosporin A - Binding Protein
FK506-Binding Protein a4(I1 Selectin
Fibronectin a4(37 Selectin
cMad CAM-1 LFA-1 (CDlla/CD18)
PECAM-1 LFA-1 Selectin
C3bi PSGL-1
E-Selectin P-Selectin
CD-34 L-Selectin
p150,95 Mac-1 (CDllb/CD18)
Fucosyl transferase VLA-4
STAT-1 STAT-2
CD-18/CD 11 a CD 11 b/CD 18
ICAM2 and ICAM3 CSa
CCR3 (Eotaxin Receptor) CCRl, CCR2, CCR4, CCRS
LTB-4 AP-1 Transcription Factor
Protein kinase C Cysteinyl Leukotriene Receptor
Tachykinnen Receptors IKB Kinase 1 & 2
(tach R)
Interleukin-2 Receptor (e.g., Substance P, NK-1 & NK-3 Receptors)
(IL-2R)
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WO 00/51621 PCT/US00/05643
STAT 6 c-mas
NF-Interleukin-6 (NF-IL-6)Interleukin-10 Receptor
(IL-lOR)
Interleukin-3 (IL-3) Interleukin-2 Receptor
(IL-2R)
Interleukin-13 (IL-13) Interleukin-12 Receptor
(IL-12R)
Interleukin-14 (IL-14) Interleukin-6 Receptor
(IL-6R)
Interleukin-16 (IL-16) Interleukin-13 Receptor
(IL-13R)
Medullasin Interleukin-16 Receptor
(IL-16R)
Adenosine A, Receptor Tryptase-I
(A, R)
Adenosine AZb Receptor Adenosine A3 Receptor (A3
(Azb R) R)
~3 Tryptase STAT-3
Adenosine Az~ Receptor IgE Receptor (3 Subunit
(AZa R) (IgE R (3)
Fc-epsilon receptor IgE Receptor a Subunit
CD23 antigen (IgE R a)
IgE Receptor Fc EpsilonFceR) Substance P Receptor
Receptor (IgER
Histidine decarboxylaseTryptase-1
Prostaglandin D SynthaseEosinophil Cationic Protein
Eosinophil Derived NeurotoxinEosinophil Peroxidase
Endothelial Nitric OxideEndothelial Monocyte Activating
Synthase Factor
Neutrophil Oxidase FactorCathepsin G
Macrophage InflammatoryInterleukin-8 Receptor
Protein-1- a Subunit (IL-8 Ra)
Alpha/Rantes Receptor Endothelin Receptor ET-B
These genes, and others, are involved in the normal functioning of respiration
as well as in diseases
associated with respiratory pathologies, including cystic fibrosis, asthma,
pulmonary hypertension and
vasoconstriction, chronic obstructive pulmonary disease (COPD), chronic
bronchitis, respiratory distress
syndrome CARDS), allergic rhinitis, lung cancer and lung metastatic cancers
and other airway diseases,
including those with inflammatory response. Anti-sense oligos to the adenosine
A,, A,~, Azb, and A,
receptors, CCR3 -(chemokine receptors), bradykinin 2B, CAM (vascular cell
adhesion molecule), and
eosinophil receptors, among others, have been shown to be effective in down-
regulating the expression of
their genes. Some of these act to alleviate the symptoms or reduce respiratory
ailments and/or inflammation,
for example, by "down regulation" of the adenosine A,, AZ~, A26, and/or A,
receptors and CCR3, bradykinin
2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors. These
agents may be utilized by the
present method alone or in conjunction with anti-sense oligos targeted to
other genes to validate pathway
and/or networks in which they are involved. For better results, the oligos are
preferably administered directly
into the respiratory system, e.g., by inhalation or other means, of the
experimental animal, so that they may
reach the lungs without widespread systemic dissemination. This permits the
use of low agent doses as
compared with those administered systemically or by other generalized routes
and, consequently, reduces the
number and degree of undesirable side effects resulting from the agent's
widespread distribution in the body.
The agents) of this invention has (have) been shown to reduce the amount of
receptor protein expressed by
the tissue. These agents, thus, rather than merely interacting with their
targets, e.g. a receptor, lower the
number of target proteins that other drugs may interact with. In this manner,
the present agents) affords)
extremely high efficacy with low toxicity.
The receptors discussed above are mere examples of the high power of the
present technology. In
fact, a large number of genes may be targeted in a similar manner by
practicing the present methods, to
significantly down-regulate or obliterate protein expression and observe any
changes wrought to one or more
functions within a system, e.g. the respiratory, CNS, cardiovascular, renal
and other systems. By means of
example, in the respiratory system, the functions tested may be ease of
breathing, bronchoconstriction,
inflammation, chronic bronchitis, surfactant production, and the like, and
others associated with diseases and
conditions such as chronic obstructive pulmonary disease (COPD), inhalation
burns, Acute Respiratory
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WO 00/51621 PCT/US00/05643
Distress Syndrome CARDS), cystic fibrosis, pulmonary fibrosis, radiation
pulmonitis, tonsilitis, emphysema,
dental pain, oral inflammation, joint pain, esophagitis, lung cancer and
esophageal cancer, among others.
These functions are of great interest because of their association with
respiratory dysfunction, as is the case in
asthma, allergies, allergic rhinitis, pulmonary bronchoconstriction and
hypertension, chronic obstructive
pulmonary disease (COPD), allergy, asthma, cystic fibrosis, Acute Respiratory
Distress Syndrome CARDS),
cancer, which either directly or by metastasis afflict the lung, the present
method may be applied to a list of
potential target mRNAs, which includes the targets listed in Table 1 above,
among others. In the CNS
system, functions that may be selected are food ingestion/satiety, mood
variation, anxiety, libido/sexual
dysfunction, cognition, sexual function/dysfunction, brain trauma, Alzheimer's
mediators, aneurism, etc.
The oligos of this invention may be obtained by first selecting fragments of a
target nucleic acid
having at least 4 contiguous nucleic acids selected from the group consisting
of G and C and/or having a
specific type and/or extent of activity, and then obtaining a first
oligonucleotide 4 to 60 nucleotides long
which comprises the selected fragment and has a thymidine (T) nucleic acid
content of up to and including
about 15%, preferably, about 12%, about 10%, about 7%, about 5%, about 3%,
about 1%, and more
preferably no thymidine. The latter step may be conducted by obtaining a
second oligonucleotide 4 to 60
nucleotides long comprising a sequence which is anti-sense to the selected
fragment, the second
oligonucleotide having an adenosine base content of up to and including about
15%, preferably about 12%,
about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no
adenosine. When the selected
fragment comprises at least one thymidine base, an adenosine base may be
substituted in the corresponding
anti-sense nucleotide fragment with a universal or alternative base selected
from the group consisting of
heteroaromatic bases which bind to a thymidine base but have less than about
bout 10%, preferably less than
about 1%, and more preferably less than about 0.3% of the adenosine base
agonist activity at the adenosine
A,, Az~, A26 and A3 receptors, and heteroaromatic bases which have no activity
at the adenosine Aza receptor,
when validating in the respiratory system. Other adenosine activities in other
systems may be determined in
other systems, as appropriate.
The analogue heteroaromatic bases may be selected from all pyrimidines and
purines, which may be
substituted by O, halo, NH,, SH, SO, SOZ, SO,, COOH and branched and fused
primary and secondary amino,
alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
alkoxy, alkenoxy, acyl, cycloacyl,
arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl,
halocycloalkyl, alkylcycloalkyl,
alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl,
alkynylaryl, arylalkyl, arylalkenyl,
arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH~,
primary, secondary and tertiary
amine, SH, SO, SOz, S03, cycloalkyl, heterocycloalkyl and heteroaryl. The
pyrimidines and purines may be
substituted at all positions as is known in the art, but preferred are those
which are substituted at positions 1, 2,
3, 4, 7 andlor 8. More preferred are pyrimidines and purines such as
theophylline, caffeine, dyphylline,
etophylline, acephylline piperazine, bamifylline, enprofylline and xantine
having the chemical formula
O H
n '
~y~ ! ~ ~ ; c' N \
" Rs
:c~ ~ ic..~/
o H H
R
wherein R' and RZ are independently H, alkyl, alkenyl or alkynyl and R' is H,
aryl, dicycloalkyl,
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WO 00/51621 PCT/US00/05643
dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-
cycloalkyl, O-cycloalkenyl, O-
cycloalkynyl, NHz-alkylamino-ketoxyalkyloxy-aryl, mono and dialkylaminoalkyl-N-
alkylamino-SOZaryI,
among others. Similar modifications in the sugar are also embodiments of this
invention. Reduced adenosine
content of the anti-sense oligos corresponding to the thymidines (T) present
in the target RNA serves to
prevent the breakdown of the oligos into products that free adenosine into the
system, e.g. the lung, brain,
heart, kidney, etc., tissue environment and, thereby, to prevent any unwanted
effects due to it.
By means of example, the NfxB transcription factor may be selected as a
target, and its mRNA or
DNA searched for low thymidine (T) or desthymidine (desT) fragments. Only desT
segments of the mRNA or
DNA are selected which, in turn, will produce desA anti-sense as their
complementary strand. When a
number of RNA desT segments are found, the sequence of the anti-sense segments
may be deduced.
Typically, about 10 to 30 and even larger numbers of desA anti-sense sequences
may be obtained. These anti-
sense sequences may include some or all desA anti-sense oligonucleotide
sequences corresponding to desT
segments of the mRNA of the target, such as anyone of those shown in Table 1
above, in Table 2 below, and
others associated with functions of the brain, cardiovascular and renal
systems, and many others. When this
occurs, the anti-sense oligonucleotides found are said to be 100% A-free. For
each of the original desA anti-
sense oligonucleotide sequences corresponding to the target gene, e.g. the
NFxB transcription factor, typically
about 10 to 30 sequences may be found within the target gene or RNA which have
a low content of thymidine
(RNA). In accordance with this invention, the selected fragment sequences may
also contain a small number
of thymidine (RNA) nucleotides within the secondary or tertiary or quaternary
sequences. In some cases, a
large adenosine content may suffice to render the anti-sense oligonucleotide
less active or even inactive
against the target. In accordance with this invention, these so called "non-
fully desA" sequences may
preferably have a content of adenosine of less than about 15%, about 12%,
about 10%, about 7%, about 5%,
and about 2% adenosine. Most preferred is no adenosine content (0%). In some
instances, however, a higher
content of adenosine is acceptable and the oligonucleotides still fail to show
detrimental "adenosine activity".
A particular important embodiment is that where the adenosine nucleotide is
"fixed" or replaced by a
"Universal or alternative" base that may base-pair with similar or equal
affinity to two or more of the four
nucleotide present in natural DNA: A, G, C, and T.
A universal or alternative base is defined in this patent as any compound,
more commonly an
adenosine analogue, which has substantial capacity to hybridize to thymidine,
reduced, or substantially lacks
ability to bind adenosine receptors or other molecules through which adenosine
may exert an undesirable side
effect in the experimental animal or in a cell system. Alternatively,
adenosine analogs which completely fail to
activate adenosine receptors, such as the adenosine A" Ate, AZb and/or A3
receptors, most preferably A,
receptors, may be used. One example of a universal or alternative base is a-
deoxyribofuranosol-(5-
nitroindole), and an artisan will know how to select others. This "fixing"
step generates further novel
sequences, different from those anti-sense to the ones found in nature, that
permits the anti-sense
oligonucleotide to bind, preferably equally well, with the target RNA. Other
examples of universal or
alternative bases are 2-deoxyribosyl-(5-nitroindole). Other examples of
universal or alternative bases are 3-
nitropyrrole-2'-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl-(5-
nitroindole), 2-deoxyribofuranosyl-(5-
nitroindole), 2'-deoxyinosine, 2'-deoxynebularine, 6H, 8H-3,4-dihydropyrimido
[4,5-c] oxazine-7-one and 2-
amino-6-methoxyaminopurine. In addition to the above, Universal or alternative
bases which may be
substituted for any other base although with somewhat reduced hybridization
potential, include 3-nitropyrrole
2'-deoxynucleoside 2-deoxyribofuranosyl-(5-nitroindole), 2'-deoxyinosine and
2'-deoxynebularine (Glen
Research, Sterling, VA). More specific mismatch repairs may be made using "P"
nucleotide, 6H, 8H-3,
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WO 00/51621 PCT/US00/05643
4-dihydropyrimido[4,5-c] [1,2] oxazin-7-one, which base pairs with either
guanine (G) or adenine (A) and
"K" nucleotide, 2-amino-6-methoxyaminopurine, which base pairs with either
cytidine (C) or thyniidine (T),
among others. Others which are known in the art or will become available are
also suitable. See, for example,
Loakes, D. and Brown, D. M., Nucl .Acids Res. 22:4039-4043 (1994); Ohtsuka, E.
et al., J. Biol.
Chem.260(5):2605-2608 (1985); Lin, P.K.T. and Brown, D. M., Nucleic Acids Res.
20(19):5149-5152 (1992;
Nichols, R. et al., Nature 369(6480): 492-493 (1994); Rahmon , M. S. and
Humayun, N. Z., Mutation
Research 377 (2): 263-8 (1997); Amosova, O., et al., Nucleic Acids Res. 25
(!0): 1930-1934 (1997); Loakes
D. & Brown, D. M., Nucleic Acids Res. 22 (20): 4039-4043 (1994), the entire
sections relating to universal or
alternative bases and their preparation and use in nucleic acid binding being
incorporated herein by reference.
When non-fully desT sequences are found in the naturally occurring target,
they typically are
selected so that about 1 to 3 universal or alternative base substitutions will
suffice to obtain a 100% "desA"
anti-sense oligonucleotide. Thus, the present method provides either anti-
sense oligonucleotides to different
targets which are low in, or devoid of, A content, as well as anti-sense
oligonucleotides where one or more
adenosine nucleotides, e. g. about 1 to 3, or more, may be "fixed" by
replacement with a "Universal or
alternative" base. Universal or alternative bases are known in the art and
need not be listed herein. An artisan
will know which bases may act as universal or alternative bases, and replace
them for A.
As used herein, the term "validate" or "validating" a target within a certain
system such as the
respiratory, inflammatory, CNS, cardiopulmonary, renal immune, and other
systems, refers to a process which
starts by selecting a therapeutic area within the system, for example, in the
CNS or the cardiovascular or
cardiopulmonary systems, among others. Areas that may be selected for study
within those systems are those
of anxiety, mood disorders, satiety and regulation of appetite, pain,
cognition, sleep induction and disorders,
regulation of temperature, and many others that are controlled by or regulated
through the brain, and others
exemplified in Table 2 below.
The next step is to select a gene sequence data base corresponding to the
appropriate system, e.g. a
CNS, lung, cardiac system, kidney/renal system, blood, immune system,
pulmonary and respiratory system,
sexual function/dysfunction, skin, eye, nasal passages, scalp, testes, cervix,
oral cavity, pharynx, esophagus,
small and large intestine, synvial tissues, ovaries, ear canal, and other
systems. Target genes are selected
amongst those known to be associated with the CNS, or with a certain area of
the CNS, and those of unlrnown
functions. Genes whose functions are known and might occur in the system may
also be selected. Anti-sense
oligos are then designed for these target genes or mRNA fragments as described
here. Finally, oligos may be
selected for their ability to ablate or significantly reduce the expression of
the target gene by in vitro
hybridization to cell and tissue DNA and RNA. Oligos that show high in vitro
down regulation, ablation or
expression inhibition are then applied in in vivo tests, preferably by site
specific administration, e.g. to a
region of the brain, heart, kidney, lung, etc., and pre-determined behavioral,
biophysical, biochemical,
cognitive, motor, sensory, physiological, and other functions, assessed to
establish whether or not a correlation
exists between the target and the function. In the respiratory system, for
example, a correlation may be shown
be decreasing the likelihood that the subject administered such treatment will
manifest symptoms of a
respiratory or inflammatory lung disease or other lung conditions, such as a
malignancy. As applied here, the
term "down-regulate" refers to inducing a decrease in production, secretion or
availability (and thus a decrease
in concentration) of the targeted intracellular protein, which may include a
complete ablation.
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Table 2: Examples of Diseases & Conditions Associated with Targets & Networks
Dementia Sttoke Anxiety Antinociception Analgesia
Cardiopulm. Behavioral Traumatic Organic Brain
Funct.
Autonomic ControlDisorders Brain Injury Disease
Degen. EncephalopathyDevelopmental Drug Sensitivity
CNS Disord. Vision
(Viral, etc.)
Abnorm.&Deform.
(Legal & Illegal)
Cognition Satiety Food Alcohol Sensitivity Heart Attacks
Learning Depression Ingestion Brain Inflammation
Anesthesia Hearing Olfaction Hypoxia Schizophrenia
Brain Cancer Cranial Def. Memory Neuropathy Neurogenic
Pain
Dental Pain Headache Sensation Motor Coord.
Mood disorders Mood Elevat. Bipolar Dis. Eating Dis.
Cachexia AneurismsCardiac & Vasc. Congestive
Cardiac & Vasc.
Cardiac & Vasc. Pain
Exudation Heart
Disease
Inflammation Stroke Angina Heart Failure
Ischemia Plaque FormationRestenosis Viral Infec.
Arrythmias Vascular Permeab. Arterial Degener.Libido
Angiogen. & Structural & Biochem. Defects Transplant.
Inhib. Anger Reject.
The present invention is concerned primarily with target validation in
vertebrates, and within this
group, of mammals, including human and non-human simians, wild and
domesticated animals, marine and
land animals, household pets, and zoo animals, for example, felines, canines,
equines, pachiderms, cetaceans,
and still more preferably to human subjects. One particularly suitable
application of this technology is for
veterinary purposes, and includes all types of small and large animals in the
care of a veterinarian, including
wild animals, marine animals, household animals, zoo animals, and the like.
Targeted genes and proteins are
preferably mammalian, and the sequences targeted are preferably of the same
species as the subject being
treated. Although in many instances, targets of a different species are also
suitable, particularly those segments
of the target RNA or gene that display greater than about 45% homology,
preferably greater than about 85%
homology, still more preferably greater than about 95% homology, with the
recipient's sequence. A preferable
group of agents is composed of desA anti-sense oligos. Another preferred group
is composed of non-fully
desA oligonucleotides, where one or more adenosine bases are replaced with
universal or alternative bases.
The terms "anti-sense" oligonucleotides generally refers to small, synthetic
oligonucleotides,
resembling single-stranded DNA, which in this patent are applied to the
inhibition of gene expression by
inhibition of a target messenger RNA (mRNA). See, Milligan, J. F. et al., J.
Med. Chem. 36(14), 1923-1937
(1993), the relevant portion of which is hereby incorporated in its entirety
by reference. The present method
utilizes anti-sense agents to inhibit gene expression of target genes,
including those listed in Table 1 above.
This is generally attained by hybridization of the anti-sense oligonucleotides
to coding (sense) sequences of a
targeted messenger RNA (mRNA), as is known in the art. The exogenously
administered agents of the
invention decrease the levels of mRNA and protein encoded by the target gene
and/or cause changes in the
growth characteristics or shapes of the thus tteated cells. See, Milligan et
al. (1993); Helene, C. and Toulme, J.
Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S. D., Ed.,
Oligodeoxynucleotides as Anti-sense
Inhibitors of Gene Expression; CRC Press: Boca Raton, FL ( 1987), the relevant
portion of which is hereby
incorporated in its entirety by reference. As used herein, "anti-sense
oligonucleotide" is generally a short
CA 02366055 2001-08-31
WO 00151621 PCT/US00/05643
sequence of synthetic nucleotide that (1) hybridizes to any segment of a mRNA
encoding a targeted protein
under appropriate hybridization conditions, and which (2) upon hybridization
causes a decrease in gene
expression of the targeted protein. The terms "desAdenosine" (desA) and "des-
thymidine" (desT) refer to
oligonucleotides substantially lacking either adenosine (desA) or thymidine
(desT). In some instances, the des
T sequences are naturally occurring, and in others they may result from
substitution of an undesirable
nucleotide (A) by another one lacking its undesirable activity. In the present
context, the substitution is
generally accomplished by substitution of A with a "universal or alternative
base", as is known in the art.
The mRNA sequence of the targeted protein may be derived from the nucleotide
sequence of the
gene expressing the protein, whether for existing targets or those to be found
in the future. Sequences for
many target genes of different systems are presently known. See, GenBank data
base, NIH, the entire
sequences of which are incorporated here by reference. The sequences of those
genes, whose sequences are
not yet available, may be obtained by isolating the target segments applying
technology known in the art.
Once the sequence of the gene, its RNA and/or the protein are known, anti-
sense oligonucleotides are
produced as described above and utilized to validate the target by in vivo
administration and testing for a
reduction of the production of the targeted protein in accordance with
standard techniques, and of specific
functions. In one aspect of this invention, the anti-sense oligonucleotide has
a sequence which specifically
binds to a portion or segment of an mRNA molecule which encodes a protein
associated with a disease or
condition of a specific system, e.g. CNS, respiratory, pulmonary, motor,
sensory, hormone regulatory, cardiac,
renal, immune, blood, cancer genes, and the like. One effect of this binding
is to reduce or even prevent the
translation of the corresponding mRNA and, thereby, reduce the available
amount of target protein in the
subject's lung.
In one preferred embodiment of this invention, one or more of the
phosphodiester residues of the
anti-sense oligonucleotide are modified or substituted. Chemical analogs of
oligonucleotides with modified or
substituted phosphodiester residues, e.g., to the methylphosphonate, the
phosphotriester, the phosphorothioate,
the phosphorodithioate, or the phosphoramidate, which increase the in vivo
stability of the oligonucleotide are
particularly preferred. The naturally occurring phosphodiester linkages of
oligonucleotides are susceptible to
some degree of degradation by cellular nucleases. Many of the residues
proposed herein, on the contrary, are
highly resistant to nuclease degradation. See Milligan et al., and Cohen, J.
S. D., supra. In another preferred
embodiment of the invention, the oligonucleotides may be protected from
degradation by adding a "3'-end
cap" by which nuclease-resistant linkages are substituted for phosphodiester
linkages at the 3' end of the
oligonucleotide. See, Tidd, D. M. and Warenius, H.M., Be. J. Cancer 60: 343-
350 (1989); Shaw, J.P. et al.,
Nucleic Acids Res. 19: 747-750 (1991), the relevant section of which are
incorporated in their entireties
herein by reference. Phosphoramidates, phosphorothioates, and
methylphosphonate linkages all function
adequately in this manner for the purposes of this invention. The more
extensive the modification of the
phosphodiester backbone the more stable the resulting agent, and in many
instances the higher their RNA
affinity and cellular permeation. See Milligan, et al., supra. Thus, the
number of residues which may be
modified or substituted will vary depending on the need, target, and route of
administration, and may be from
1 to all the residues, to any number in between. Many different methods for
replacing the entire
phosphodiester backbone with novel linkages are known. See, Millikan et al,
supra. Preferred analogue
residues for the base, the internucleotide linkage, or the sugar include
phosphorothioate, methylphosphonate,
phosphotriester, thioformacetal, phosphorodithioate, phosphoramidate,
formacetal boranophosphate, 3'-
thioformacetal, 5'-thioether, carbonate, S'-N-carbamate, sulfate, sulfonate,
sulfamate, sulfonamide, sulfone,
sulfite., 2'-O methyl, sulfoxide, sulfide, hydroxylamine, methoxy methyl
(MOM), methoxy ethyl (MOE),
methylene(methylimino) (MMI), and methyleneoxy(methylimino) (MOMI) residues.
Phosphorothioate and
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methylphosphonate-modified oligonucleotides are particularly preferred due to
their availability through
automated oligonucleotide synthesis. See, Millikan et al, supra. Where
appropriate, the agent of this invention
may be administered in the form of a pharmaceutically acceptable salt, or as a
mixture of the anti-sense
oligonucleotide and a salt. In another embodiment of this invention, a mixture
of different anti-sense
oligonucleotides or their pharmaceutically acceptable slats is administered.
The agents of this invention have
the capacity to attenuate the expression of one target mRNA and/or to enhance
or attenuate the activity of one
pathway. By means of example, the present method may be practiced by
identifying all possible
deoxyribonucleotide segments which are low in thymidine (T) or deoxynucleotide
segments low in adenosine
(A) of about 7 or more mononucleotides, preferably up to about 60
mononucleotides, more preferably about
to about 36 mononucleotides, and still more preferably about 12 to about 21
mononucleotides, in a target
mRNA or a gene, respectively. This may be attained by searching for
mononucleotide segments within a
target sequence which are low in, or lack thymidine (RNA), a nucleotide which
is complementary to
adenosine, or that are low in adenosine (gene), that are 7 or more nucleotides
long. In most cases, this search
typically results in about 10 to 30 such sequences, i.e. naturally lacking or
having less than about 40%
adenosine, anti-sense oligonucleotides of varying lengths for a typical target
mRNA of average length, i. e.,
about 1800 nucleotides long. Those with high content of T or A, respectively,
may be fixed by substitution of
a universal or alternative base for one or more As.
The agents) of this invention may be of any suitable length, including but not
limited to, about 7 to
about 60 nucleotides long, preferably about 12 to about 45, more preferably up
to about 30 nucleotides long,
and still more preferably up to about 21, although they may be of other
lengths as well, depending on the
particular target and the mode of delivery. The agents) of the invention may
be directed to any and all
segments of a target RNA. One preferred group of agents) includes those
directed to an mRNA region
containing a junction between an intron and an exon. Where the agent is
directed to an intron/exon junction, it
may either entirely overlie the junction or it may be sufficiently close to
the junction to inhibit the splicing-out
of the intervening exon during processing of precursor mRNA to mature mRNA,
e.g. with the 3' or 5' terminus
of the anti-sense oligonucleotide being positioned within about, for example,
within about 2 to 10, preferably
about 3 to 5, nucleotide of the intron/exon junction. Also preferred are anti-
sense oligonucleotides which
overlap the initiation codon, and those near the 5' and 3' termini of the
coding region. The anti-sense oligo
may have an adenosine content of about 0, about 3%, about S%, about 7% to
about 8%, about 10%, about
12%, about 15%, and any intermediate amounts and ranges of adenosine content.
In the present method, one
or more or all A may be substituted by a universal or alternative base such as
heteroaromatic bases which bind
to thymidine but have less than about 0.5, about 0.3, about 0.1 of the agonist
or antagonist activity of
adenosine at the adenosine A,, Aza, Aze and A3 receptors, and heteroaromatic
bases which have no activity at
the adenosine AZa receptor. The alternative base may be 3-nitropyrrole-2'-
deoxynucleoside, 5-nitro-indole, 2-
deoxyribosyl-(5-nitroindole), 2-deoxyribofuranosyl-(5-nitroindole), 2'-
deoxyinosine, 2'-deoxynebularine, 6H,
8H-3,4-dihydropyrimido [4,5-c] oxazine-7-one or 2-amino-6-methoxyaminopurine,
among others. The oligo
may have a methylated cytosine (°'C) substituted for one or more, or
all unmethylated C in a CpG dinucleotide
(s), if the latter is (are) present in the oligo(s). Other CS modifications to
pyrimidines are also embodiments of
the current invention, e.g. CS propyne, etc. One or more, or all nucleotide
linking residues of the oligos
suitable for use with the present method may be methylphosphonate,
phosphotriester, phosphorothioate,
phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether,
carbonate, carbamate, sulfate,
sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide,
hydroxylamine,
methylene(methyimino), (MMI), methoxymethyl (MOM), methoxyethyl (MOE),
methyleneoxy
(methylimino) (MOMA), methoxy methyl (MOM), 2'-O-methyl, phosphoramidate, and
C-S substituted (C-5
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propyne) residues and combinations thereof. The oligo used in the present
method may be linked to a vector,
such as a prokaryotic or eukaryotic vector, many of which are known in the
art. The method of the invention
prescribes the administration of an amount of anti-sense oligo effective to
reduce the production or
availability, or to increase the degradation, of the mRNA, or to reduce the
amount of the polypeptide present
in the lungs. The administration is preferably done in situ, e.g. directly
into the respiratory system or nasal
passage, e.g. by inhalation or applied to the subject's lungs, for respiratory
and pulmonary targets. The anti-
sense oligo may be administered directly into the brain, heart, kidney, tumor,
testes, eyes, ear passage, cervix,
nasal passage, scalp, oral cavity, muscle, pharynx, esophagus, intestines,
rectum, synovial tissue, ovaries, and
other localized tissues by injection, by stereotactic insertion, or in vitro,
among other methods. In addition, the
oligo may also be administered into the blood when the target is part and
parcel of the circulatory and immune
systems. In the latter case, wherein the disease or condition is associated
with an immunological dysfunction,
the target may be immunoglobulins, antibody receptors, cytokines, cytokine
receptors, genes) and the
corresponding mRNA(s) encoding them, the genes and mRNA flanking regions and
intron and exon borders,
among others. Wherein the disease or condition is associated with a malignancy
or cancer, the target may be
selected from growth regulation associated enzyme and other proteins,
immunoglobulins and antibody
receptors, genes) and mRNA(s) encoding them, genes and mRNAs associated with
oncogenes, and genomic
and mRNA flanking regions and exon and intron borders. Additionally, certain
genes of normal cells that are
involved in the cancer process, such as angiogenesis factors, adhesion
molecules and protease enzymes
involved in metastases and others are also part of the invention. The method
may be practiced, for example,
by administering the composition in vitro, orally, intracavitarily,
intranasally, intraanally, intravaginally,
intrauterally, intracranially, pulmonarily, intrarenally, intranodularly,
intraarticularly, intraotically,
intralymphatically, transdermally, intrabucally, intravenously,
subcutaneously, intramuscularly,
intratumorously, intraglandularly, intraocularly, intracranial, into an organ,
intravascularly, intrathecally, by
implantation, by inhalation, intradermally, intrapulmonarily, into the ear,
into the heart, by slow release, by
sustained release and by a pump. Other examples of targets are genes and mRNAs
encoding polypeptides
selected from the group consisting of transcription factors, stimulating and
activating factors, cytokines and
their receptors, interleukins, interleukin receptors, chemokines, chemokine
receptors, endogenously produced
specific and non-specific enzymes, immunoglobulins, antibody receptors,
central nervous system (CNS) and
peripheral nervous and non-nervous system receptors, CNS and peripheral
nervous and non-nervous system
peptide transmitters, adhesion molecules, defensines, growth factors,
vasoactive peptides, peptide receptors
and binding protein, and genes and mRNAs corresponding to oncogenes, G-protein
coupled receptors, etc.
The anti-sense oligo(s) may be produced by selection of a target from
polypeptides associated with diseases
and conditions afflicting lung airways, such as difficult respiratory activity
and malignancies, increased or
decreased surfactant secretion, and many others, genes and RNAs encoding them,
the genomic and mRNA
flanking regions and the genes) and mRNA(s) intron and exon borders; obtaining
the sequence of a mRNA(s)
selected from the group consisting of mRNAs corresponding to the target genes)
and mRNAs encoding the
target polypeptide(s), genomic and mRNA flanking regions and the genes and
mRNAs intron and exon
borders; selecting at least one segment of the mRNA(s); synthesizing one or
more oligo anti-sense to the
selected mRNA segment(s); and substituting, if necessary, an alternative
bases) capable of hybridizing to
thymidine (T) but having reduce or no agonist capacity at the adenosine
receptors (no or reduced adenosine
receptor activation) for one or more A(s) to reduce the content of A present
in the oligo to up to about 15% of
all nucleotides. As akeady indicated, suitable targets are target proteins,
genes and mRNAs encoding
polypeptides selected from transcription factors, stimulating and activating
factors, interleukins, interleukin
receptors, chemokines, chemokine receptors, endogenously produced specific and
non-specific enzymes,
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immunoglobulins, antibody receptors, central nervous system (CNS) and
peripheral nervous and non-nervous
system receptors, CNS and peripheral nervous and non-nervous system peptide
transmitters and their
receptors, adhesion molecules, defensines, growth factors, vasoactive peptides
and their receptors, and binding
proteins, and target genes and mRNAs corresponding to oncogenes, and their
flanking regions and intron and
exon borders. The encoded polypeptides may be selected from NficB
Transcription Factor, Interleukin-8
Receptor (IL-8 R), Interleukin 5 Receptor (IL-5 R), Interleukin 4 Receptor (IL-
4 R), Interleukin 3 Receptor
(IL-3 R), Interleukin-1 (3 (IL-1 (3), Interleukin 1 (3 Receptor (IL- 1 (3 R),
Eotaxin, Tryptase, Major Basic Protein,
~i2-adrenergic Receptor Kinase, Endothelin Receptor A, Endothelin Receptor B,
Preproendothelin, Bradykinin
B2 Receptor, IgE High Affinity Receptor, Interleukin 1 (IL-1), Interleukin 1
Receptor (IL-1 R), Interleukin 9
(IL-9), Interleukin-9 Receptor (IL-9 R), Interleukin 11 (IL-11), Interleukin-
11 Receptor (IL-11 R), Inducible
Nitric Oxide Synthase, Cyclooxygenase (COX), Intracellular Adhesion Molecule 1
(ICAM-1) Vascular
Cellular Adhesion Molecule (VCAM), Rantes, Endothelial Leukocyte Adhesion
Molecule (ELAM-1),
Monocyte Activating Factor, Neutrophil Chemotactic Factor, Neutrophil
Elastase, Defensin 1, 2 and 3,
Muscarinic Acetylcholine Receptors, Platelet Activating Factor, Tumor Necrosis
Factor a, 5-lipoxygenase,
Phosphodiesterase IV, Substance P, Substance P Receptor, Histamine Receptor,
Chymase, CCR-1 CC
Chemokine Receptor, CCR-2 CC Chemokine Receptor, CCR-3 CC Chemokine Receptor,
CCR-4 CC
Chemokine Receptor, CCR-5 CC Chemokine Receptor, Prostanoid Receptors, GATA-3
Transcription Factor,
Neutrophil Adherence Receptor, MAP Kinase, Interleukin-9 (IL-9), NEAT
Transcription Factors, STAT 4,
MIP-la, MCP-2, MCP-3, MCP-4, Cyclophillins, Phospholipase A2, Basic Fibroblast
Growth Factor,
Metalloproteinase, CSBP/p38 MAP Kinase, Tryptose Receptor, PDG2, Interleukin-3
(IL-3), Interleukin-1(3
(IL-1(i), Cyclosporin A-Binding Protein, FKS-Binding Protein, a4(31 Selectin,
Fibronectin, a4~37 Selectin,
Mad CAM-1, LFA-1 (CDlla/CD18), PECAM-1, LFA-1 Selectin, C3bi, PSGL-1, E-
Selectin, P-Selectin, CD-
34, L-Selectin, p150,95, Mac-1 (CDllb/CD18), Fucosyl transferase, VLA-4, CD-
18/CDlla, CDllb/CD18,
ICAM2 and ICAM3, CSa, CCR3 (Eotaxin Receptor), CCRl, CCR2, CCR4, CCRS, LTB-4,
AP-1
Transcription Factor, Protein kinase C, Cysteinyl Leukotriene Receptor,
Tachychinnen Receptors (tach R),
IxB Kinase 1 & 2, STAT 6, c-mas and NF-Interleukin-6 (NF-IL-6), and their
flanking regions and intron and
exon borders. However, this invention is primarily intended for application to
newly discovered genes not yet
available in public data bases. It is for this group of genes that target
validation is of most importance, for
without it their role in disease remains unknown.
Table 3 below provides a short list of targets to which the agents of the
invention are effectively
applied. These are by way of example only. The method is applicable to any
system in which target
validation would be discussed by virtue of bioactive adenosine release during
oligonucleotie degradation.
This is of importance because adenosine is anti-inflammatory and its release
would obscure cancer target
validation results.
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Table 3: Cancer Targets
Transforming Therapy
Oncogenes Targets
ras Thymidylate Synthetase
src Thymidylate Synthetase
myc Dihydrofolate Reductase
bcl-2 Thymidine Kinase
Deoxycytidine Kinase
Ribonucleotide Reductase
Angiogenesis Adhesion Molecules
factors
Oncogenes Folate Pathway Enzymes
DNA repair (One Carbon Pool)
genes
Telomerase
HMG CoA Reductase
Farnesyl Transferase
Glucose-6-Phosphate
Transferase
A group of preferred targets for the validation of cancer targets are genes
associated with different
types of cancers, or those generally known to be associated with malignancies,
whether they are regulatory or
involved in the production of RNA and/or proteins. Examples are transforming
oncogenes, targets which are
shown, among others, in Table 3 above. Other targets which present cancer
target validation agents are
directed to are various enzymes, primarily, although not exclusively,
thymidylate synthetase, dihydrofolate
reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase,
other gene products more
abundantly manufactured in cancer cells than in normal cells, and the like.
The present technology is
particularly useful in the validation/invalidation of cancer target genes
given that traditional cancer therapies
are not effective in selectively killing cancer cells while preserving normal
living cells from the devastating
effects of treatments such as chemotherapy, radiotherapy, and the like. That
is, present cancer treatments
cannot be selectively targeted to malignant cells. Any target validated by the
present method will provide the
ability of selectively attenuating a desired gene product and attenuating or
enhancing function, and its
pathway. This approach provides a significant advantage over standard cancer
treatments because it permits
the selection of a system, and within the system a pathway including multiple
targets, e.g. primary, secondary
and possibly tertiary targets, which may not be generally expressed
simultaneously in normal cells and
validate them separately and jointly. Thus, the present method will provide
targets for therapy. Once a target
is validated by the present method, a selective agent acting on the target may
be administered to a subject to
cause a selective increase in toxicity within tumor cells that, for instance,
express three targets while normal
cells that may expresses only one or two of the targets will be significantly
less affected or even spared. The
present method administers agents which are preferably designed to be anti-
sense to target genes and/or
mRNAs related in origin to the species to which it is to be administered. When
for validating targets in
humans, the agents are preferably designed to be anti-sense to a human gene or
RNA. The agents of the
invention encompass oligonucleotides which are anti-sense to naturally
occurring DNA and/or RNA
sequences, fragments thereof of up to a length of one ( 1 ) base less than the
targeted sequence, preferably at
least about 7 nucleotides long, oligos having only over about 0.1%, about 1%,
about 4% up to about 5%,
about 10%, about 15%, about 30%, or lacking adenosine altogether, and oligos
in which one or more of the
adenosine nucleotides have been replaced with so-called universal or
alternative bases, which may pair up
with thymidine nucleotides but fail to substantially trigger adenosine
receptor activity. Examples of human
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sequences and fragments, which are not limiting, of anti-sense oligonucleotide
of the invention are the
following fragments as well as shorter segments of the fragments and of the
full gene or mRNA coding
sequences, exons and intron-exon junctions encompassing preferably 7, 10, 15,
18 to 21, 24, 27, 30, n-1
nucleotides for each sequence, where n is the sequence's total number of
nucleotides. These fragments may
be selected from any portion of the longer oligo, for example, from the
middle, S'- end, 3'- end or starting at
any other site of the original sequence. Of particular importance are
fragments of low adenosine nucleotide
content, that is, those fragments containing less than or about 30%,
preferably less than or about 15%, more
preferably less than or about 10%, and even more preferably less than or about
5%, and most preferably those
devoid of adenosine nucleotide, either by choice or by replacement with a
universal or alternative base in
accordance with this invention. The agent of the invention includes as a most
preferred group sequences and
their fragments where one or more adenosines present in the sequence have been
replaced by a universal or
alternative base (B), as exemplified here. Similarly, also encompassed are all
shorter fragments of the B-
containing fragments designed by substitution of B(s) for adenosine(s) (A(s))
contained in the sequences,
fragments thereof or segments thereof, as described above.
The present method may utilize the agents by themselves or in the form of
pharmaceutical
compositions comprising an amount of the anti-sense oligonucleotide as given
above effective to reduce the
expression of a target protein. The anti-sense oligo must first pass through a
cell membrane to bind
specifically with mRNA encoding the protein in the cell and prevent its
translation. Such compositions are
provided in a suitable pharmaceutically acceptable carrier, e.g. sterile
pyrogen-free saline solution. The agent
of the invention may be formulated with a hydrophobic carrier capable of
passing through a cell membrane,
e.g. in a liposome, with the liposomes carried in a pharmaceutically
acceptable aqueous carrier, optionally and
alternatively with surfactant or lipid. In addition, the oligonucleotides may
be coupled to an agent which
inactivates mRNA, such as a ribozyme to attain a more complete inhibition of
translation. The pharmaceutical
formulation may also comprise chimeric molecules where the anti-sense oligos
are attached to molecules
which are known to be internalized by cells. These oligonucleotide conjugates
utilize cellular up-take
pathways to increase the intracellular concentrations of the oligonucleotide.
Examples of molecules used in
this manner are macromolecules including transferrin, asialoglycoprotein
(bound to oligonucleotides via
polylysine) and streptavidin, among others known in the art. The present
method may also utilize anti-sense
compounds in a pharmaceutical formulation, e.g. within a lipid particle or
vesicle, such as a liposome or
microcrystal. The particles may be of any suitable structure, such as
unilamellar or plurilamellar. The one
preferred embodiment, the anti-sense oligonucleotide is comprised within the
liposome. Positively charged
lipids such as N-[1-(2, 3 -dioleoyloxi) propyl] -N, N, N-
trimethylammoniumethylsulfate, or "DOTAP," are
particularly preferred for such particles and vesicles. However, others are
also suitable and may in fact be
more suitable. The preparation of such lipid particles is well known. See,
e.g., US Patent Nos. 4,880,635 to
Janoff et al., 4,906,477 to Kurono et al., 4,911,928 to Wallach, 4,917,951 to
Wallach, 4,920,016 to Allen et
al., 4,921,757 to Wheatley et al., the relevant sections of all of which are
herein incorporated in their entireties
by reference. The method of the invention provides for the administration of
the anti-sense oligos by any
means, preferably those which afford the least transport, e.g. in situ in the
brain, lungs, kidneys, heart, testes,
etc.
The administration of the agents) to the lungs may be done by any suitable
means, but preferably
through the respiratory system as a respirable formulation, more preferably in
the form of an aerosol
comprising respirable particles which, in turn, comprise the agent for
respiration or inhalation by the subject.
The respirable particles may be in gaseous, liquid or solid form, and they
may, optionally, contain other
therapeutic ingredients and formulation components. The particles of the
present invention are preferably
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particles of respirable size, preferably of a size sufficiently small to pass,
upon inhalation, through the mouth
and larynx and into the bronchi and alveoli of the lungs. In general,
particles ranging from about 0.5 to 10
microns in diameter are respirable. However, other sizes may also be suitable.
Particles of non-respirable size,
of considerably larger diameter, which are included in the respirable
formulation tend to deposit in the throat
and may be swallowed. Accordingly, it is desirable to minimize the quantity of
non-respirable particles in the
aerosol. For nasal administration, a particle size in the range of 10-500 :m
is preferred to ensure their retention
in the nasal cavity. Aerosols of liquid particles comprising the agent may be
produced by any suitable means,
such as with as insufflator or nebulizer. See, e.g., US Patent No. 4,501,729.
Suitable propellants include
solvents such as certain chlorofluorocarbon compounds, for example,
dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane and/or mixtures thereof.
Other propellants are suitable and
may be preferable when better suited for particular applications. The
formulation may additionally comprise
one or more co-solvents, for example, ethanol, surfactants, such as oleic acid
or sorbitan trioleate, antioxidants
and suitable flavoring agents. The anti-sense oligos may be administered to
the brain by stereotoxic
procedures or by injection to target isolated areas of the CNS, all methods
known in the art. Alternatively, the
oligos may be administered as a formulation that will cross the blood-brain
barrier, as is known in the art, e. g.
conjugates of streptavidin and a monoclonal antibody directed to the
transferrin receptor may be employed as
a universal carrier for the delivery of mono-biotinylated peptides, anti-sense
oligos (3'-biotinylation of
phosphodiesters or other derivatives) and peptide-oligos to the brain. See,
for example, Levy, R.M. et al., J.
Neuroviral 3 Suppl: 574-75 (1997); Wu-Pong and Gewirtz, BioPharm, pp. 32-38
(Jan. 1999); Boado, R. J., et
al., J. Pharm. Sci. 87 (11): 1308-1315 (1998). The administration to the
heart, liver and kidneys as well as
other organs, may be conducted by in situ administration techniques such as
catheterization, injection, and
regional diffusion, all of which are known in the art. See, for example,
Lewis, K.J. et al., J. Drug Target, 5(4):
291 (1998); Ayrin, M.A. et al., Cathet. Cardiovasc. Diagn, 41(3): 232-240
(1997); Luft, F.C., J. Molec. Med.
76(2): 75 ( 1998). These administrations are typically conducted with liquid,
solid or gaseous pharmaceutical
compositions of the agent, that may be prepared by combining the anti-sense
oligo with a suitable vehicle or
carrier, such as sterile pyrogen-free water, lipid, and/or other known
pharmaceutically or veterinarily
acceptable carrier. Other therapeutic compounds may be included as well as
other formulation ingredients as
is known in the art. Solid particulate compositions comprising dry particles
of, e.g. the micronized agent of
the invention may be prepared by grinding the dry anti-sense compound with a
mortar and pestle, and then
passing the thus ground, e.g. micronized composition through a screen, e.g.
400 mesh screen, to break up or
separate large agglomerates of particles. A solid particulate composition
comprising the anti-sense compound
may optionally also comprise a dispersant and other known agents, which serve
to facilitate the formation of a
mist or aerosol. A suitable dispersant is lactose, which may be blended with
the anti-sense compound in any
suitable ratio, about 1:1 w/w. Other ratios may be utilized as well, and other
therapeutic and formulation
agents may also be included. The relevant sections of the references cited in
this patent are intended for
incorporation to this text by referene, particularly of those publications and
patents which facilitate the
enablement and written description of the various aspects of the invention.
The dosage of the anti-sense compound administered generally varies with the
target, the function
and its amplification, and disease being investigated, the condition of the
subject, the particular formulation,
the route and site of administration, the timing of administration, etc. In
general, it is desirable to attain
intracellular concentrations of the oligonucleotide of from 0.05 to 50 :M, or
more particularly 0.2 to 5 :M.
However, a dose-response curve may suitably be determined to establish a
proper dose to observe a clear
response. The dosage utilized may be varied, e.g. from about 0.001, about
0.01, about 1 mg/kg to about 50,
about 100, and about 150 mg/kg are typically employed. Higher and lower doses
may also be administered as
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an artisan will see suitable for specific application. These amounts may be
administered once or over a period
of time, e.g. every 24 hrs where needed, although other regimens are also
suitable. The following examples
are provided to illustrate the present invention, and should not be construed
as limiting thereon. In these
examples, :M means micromolar, ml means milliliters, :m means micrometers, mm
means millimeters, cm
means centimeters, EC means degrees Celsius, :g means micrograms, mg means
milligrams, g means grams,
kg means kilograms, M means molar, and hrs. means hours.
EXAMPLES
Example 1: Design and Synthesis of Anti-sense Oligonucleotides
The design of anti-sense oligonucleotides against target receptors may require
the solution of the
complex secondary structure of the target receptor mRNA. Afrer generating this
structure, anti-sense
nucleotide are designed which target regions of mRNA which might be construed
to confer functional activity
or stability to the mRNA and which optimally may overlap the initiation codon.
Other target sites are readily
usable. As a demonstration of specificity of the anti-sense effect, other
oligonucleotides not totally
complementary to the target mRNA, but containing identical nucleotide
compositions on a w/w basis, are
included as controls in anti-sense experiments. For example, the mRNA
secondary structure of the adenosine
A, receptor was analyzed and used as described above. to design a
phosphorothioate anti-sense
oligonucleotide. The anti-sense oligonucleotide which was synthesized was
designated HAdA,AS and had the
following sequence: 5' -GAT GGA GGG CGG CAT GGC GGG-3' (SEQ ID NO:1). As a
control, a
mismatched phosphorothioate anti-sense nucleotide designated HAdAIMMI was
synthesized with the
following sequence: 5' -GTA GCA GGC GGG GAT GGG GGC-3' (SEQ ID N0:2). Each
oligonucleotide
had identical base content and general sequence structure. Homology searches
in GENBANK (release 85.0)
and EMBL (release 40.0) indicated that the anti-sense oligonucleotide was
specific for the human and rabbit
adenosine A, receptor genes, and that the mismatched control was not a
candidate for hybridization with any
known gene sequence. The secondary structure of the adenosine A, receptor mRNA
was similarly analyzed
and used as described above to design two phosphorothioate anti-sense
oligonucleotides. The first anti-sense
oligonucleotide (HAdA3AS 1 ) synthesized had the following sequence: 5' -GTT
GTT GGG CAT CTT GCC-
3' (SEQ ID N0:3). As a control, a mismatched phosphorothioate anti-sense
oligonucleotide (HAdA3MM1)
was synthesized, having the following sequence: 5' -GTA CTT GCG GAT CTA GGC-3'
(SEQ ID N0:4). A
second phosphorothioate anti-sense oligonucleotide (HAdA3AS2) was also
designed and synthesized, having
the following sequence: 5' -GTG GGC CTA GCT CTC GCC-3' (SEQ ID NO:S). Its
control oligonucleotide
(HAdA3MM2) had the sequence: 5' -GTC GGG GTA CCT GTC GGC-3' (SEQ ID N0:6).
Phosphorothioate
oligonucleotides were synthesized on an Applied Biosystems Model 396
Oligonucleotide Synthesizer, and
purified using NENSORB chromatography (DuPont, MD).
Example 2: In Vivo Testing of Adenosine Al
Receptor Anti-sense Oligos
The anti-sense oligonucleotide against the human A, receptor (SEQ ID NO:1)
described above. was
tested for efficacy in an in vitro model utilizing lung adenocarcinoma cells
HTB-54. HTB-54 lung
adenocarcinoma cells were demonstrated to express the A, adenosine receptor
using standard northern
blotting procedures and receptor probes designed and synthesized in the
laboratory. HTB-54 human lung
adenocarcinoma cells (106/100 mm tissue culture dish) were exposed to 5.0 :M
HAdAIAS or HAdAIMMI for
24 hours, with a fresh change of media and oligonucleotides after 12 hours of
incubation. Following 24 hour
exposure to the oligonucleotides, cells were harvested and their RNA extracted
by standard procedures. A 21-
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mer probe corresponding to the region of mRNA targeted by the anti-sense (and
therefore having the same
sequence as the anti-sense, but not phosphorothioated) was synthesized and
used to probe northern blots of
RNA prepared from HAdAIAS-treated, HAdAIMMI-treated and non-treated HTB-54
cells. These blots
showed clearly that HAdAIAS but not HAdAIMMI effectively reduced human
adenosine receptor mRNA by
>50%. This result showed that HAdAIAS is a good candidate for an anti-asthma
drug since it depletes
intracellular mRNA for the adenosine A, receptor, which is involved in asthma.
Example 3: In Vivo Efficacy of Adenosine Al
Receptor Anti-sense Oligos
A fortuitous homology between the rabbit and human DNA sequences within the
adenosine A, gene
overlapping the initiation codon permitted the use of the phosphorothioate
anti-sense oligonucleotides initially
designed for use against the human adenosine A, receptor in a rabbit model.
Neonatal New Zealand white
Pasteurella-free rabbits were immunized intraperitoneally within 24 hours of
birth with 312 antigen units/ml
house dustmite (D. farinae) extract (Berkeley Biologicals, Berkeley, CA),
mixed with 10% kaolin.
Immunizations were repeated weekly for the first month and then biweekly for
the next 2 months. At 3-4
months of age, eight sensitized rabbits were anesthetized and relaxed with a
mixture of ketamine
hydrochloride (44 mg/kg) and acepromazine maleate (0.4 mg/kg) administered
intramuscularly. The rabbits
were then laid supine in a comfortable position on a small molded, padded
animal board and intubated with a
4.0-mm intratracheal tube (Mallinkrodt, Inc., Glens Falls, NY). A polyethylene
catheter of external diameter
2.4 mm with an attached latex balloon was passed into the esophagus and
maintained at the same distance
(approximately 16 cm) from the mouth throughout the experiments. The
intratracheal tube was attached to a
heated Fleisch pneumotachograph (size 00; DOM Medical, Richmond, VA), and flow
was measured using a
Validyne differential pressure transducer (Model DP-45161927; Validyne
Engineering Coip., Northridge,
CA) driven by a Gould carrier amplifier (Model 11-4113; Gould Electronic,
Cleveland, OH). The esophageal
balloon was attached to one side of the differential pressure transducer, and
the outflow of the intratracheal
tube was connected to the opposite side of the pressure transducer to allow
recording of transpuhnonary
pressure. Flow was integrated to give a continuous tidal volume, and
measurements of total lung resistance
(RL) and dynamic compliance (Cdyn) were calculated at isovolumetric and flow
zero points, respectively,
using an automated respiratory analyzer (Model 6; Buxco, Sharon, CT). Animals
were randomized and on
Day 1 pretreatment values for PC50 were obtained for aerosolized adenosine.
Anti-sense (HAdAIAS) or
mismatched control (HAdAIMM) oligonucleotides were dissolved in sterile
physiological saline at a
concentration of 5000 :g (5 mg) per 1.0 ml. Animals were subsequently
administered the aerosolized anti-
sense or mismatch oligonucleotide via the intratracheal tube (approximately
5000 :g in a volume of 1.0 ml),
twice daily for two days. Aerosols of either saline, adenosine, or anti-sense
or mismatch oligonucleotides were
generated by an ultrasonic nebulizer (DeVilbiss, Somerset, PA), producing
aerosol droplets 80% of which
were smaller than 5 :m in diameter. In the first arm of the experiment, four
randomly selected allergic rabbits
were administered anti-sense oligonucleotide and four the mismatched control
oligonucleotide. On the
morning of the third day, PC50 values (the concentration of aerosolized
adenosine in mg/ml required to
reduce the dynamic compliance of the bronchial airway 50% from the baseline
value) were obtained and
compared to PC50 values obtained for these animals prior to exposure to
oligonucleotide. Following a 1 week
interval, animals were crossed over, with those previously administered
mismatch control oligonucleotide now
administered anti-sense oligonucleotide, and those previously treated with
anti-sense oligonucleotide now
administered mismatch control oligonucleotide. Treatment methods and
measurements were identical to those
employed in the first arm of the experiment. It should be noted that in six of
the eight animals treated with
anti-sense oligonucleotide, adenosine-mediated bronchoconstriction could not
be obtained up to the limit of
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solubility of adenosine, 20 mg/ml. For the purpose of calculation, PC50 values
for these animals were set at
20 mg/ml. The values given therefore represent a minimum figure for anti-sense
effectiveness. Actual
effectiveness was higher. The results of this experiment are illustrated in
Table 4 below.
Table 4: Effect of Adenosine A1 Receptor Anti-sense Oligo
upon PC50 Values in Asthmatic Rabbits
Mismatch Control A. RPI'PIltflY Snti-ennen WGr
__-__ ._-a_
Pre OligonucleotidePost OligonucleotidePre OligonucleotidePost Oligonucleotide
3.56 1.02 5.16 1.03 2.36 0.68 >19.5 + 0.34**
The results are presented as the mean (n=8) ~ SEM.
The significance was determined by repeated-measures analysis of variance
(ANOVA), and Tukey's protected test.
**Significantly different from all other groups, p<p.01.
In both arms of the experiment, animals receiving the anti-sense
oligonucleotide showed an order of
magnitude increase in the dose of aerosolized adenosine required to reduce
dynamic compliance of the lung
by 50%. No effect of the mismatched control oligonucleotide upon PC50 values
was observed. No toxicity
was observed in any animal receiving either anti-sense or control inhaled
oligonucleotide. These results show
clearly that the lung has exceptional potential as a target for anti-sense
oligonucleotide-based therapeutic
intervention in lung disease. They further show, in a model system which
closely resembles human asthma,
that down regulation of the adenosine A, receptor largely eliminates adenosine-
mediated bronchoconstriction
in asthmatic airways. Bronchial hyperresponsiveness in the allergic rabbit
model of human asthma is an
excellent endpoint for anti-sense intervention since the tissues involved in
this response lie near to the point of
contact with aerosolized oligonucleotides, and the model closely simulates an
important human disease.
Examule 4: Specificity of Al-adenosine Receptor
Anti-sense Oligonucleotide
At the conclusion of the cross-over experiment of Example 3 above, airway
smooth muscle from all
rabbits was quantitatively analyzed for adenosine A, receptor number. As a
control for the specificity of the
anti-sense oligonucleotide, adenosine A, receptors, which should not have been
affected, were also quantified.
Airway smooth muscle tissue was dissected from each rabbit and a membrane
fraction prepared according to
the method of Kleinstein et al. (Kleinstein, J. and Glossmann, H., Naunyn-
Schmiedeberg's Arch. Phatmacol.
305: 191-200 ( 1978)), the relevant portion of which is hereby incorporated in
its entirety by reference, with
slight modifications. Crude plasma membrane preparations were stored at 70EC
until the time of assay.
Protein content was determined by the method of Bradford (M. Bradford, Anal.
Biochem. 72, 240-254 ( 1976),
the relevant portion of which is hereby incorporated in its entirety by
reference). Frozen plasma membranes
were thawed at room temperature and were incubated with 0.2 U/ml adenosine
deaminase for 30 minutes at
37EC to remove endogenous adenosine. The binding of ['H] DPCPX (A, receptor-
specific) or ['H] CGS-
21680 (A, receptor-specific) was measured as previously described by Ali et
al. (Ali, S. et al., J. Pharmacol.
Exp. Ther. 268, Am. J. Physiol 266, L271-277 (1994), the relevant portion of
which is hereby incorporated in
its entirety by reference). The animals treated with adenosine A, anti-sense
oligonucleotide in the cross-over
experiment had a nearly 75% decrease in A, receptor number compared to
controls, as assayed by specific
binding of the A,-specific antagonist DPCPX. There was no change in adenosine
Az receptor number, as
assayed by specific binding of the A~ receptor-specific agonist 2- (p- (2-
carboxyethyl)-phenethylamino] -5' -
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(N-ethylcarboxamido) adenosine (CGS-21680). This is illustrated in Table 5
below. The results below
illustrate the effectiveness of anti-sense oligonucleotides in treating airway
disease. Since the anti-sense oligos
described above eliminate the receptor systems responsible for adenosine-
mediated bronchoconstriction, it
may be less imperative to eliminate adenosine from them. However, it would be
preferable to eliminate
adenosine from even these oligonticleotides to reduce the dose needed to
attain a similar effect. Described
above are other anti-sense oligonucleotides targeting mRNA of proteins
involved in inflammation. Adenosine
has been eliminated from their nucleotide content to prevent its liberation
during degradation.
Table 5: Specificity of Action of Adenosine Al
Receptor Anti-sense Oligonucleotide
Mismatch Control A, Anti-sense
Oligonucleotide Wli~nnn~lentirlP
A,-Specific Binding 1105 + 48** 293 + 18
Az-Specific Binding 302 + 22 442 + 171
Tho ~e~..it.. .,.. ...a
.._ .L_
_ _______ _._ ~.-..._..... ,." ........~..... ~u - v~ - ,,,i:,mt.
The significance was determined by repeated-measures analysis of variance
(ANOVA), and Tukey's protected test.
**Sigmficantly different from mismatch control, p<0.01.
Example 5: Anti-sense Oligos Directed to
Other Target Nucleic Acids
This work was conducted to demonstrate that the present invention is broadly
applicable to anti-sense
oligonucleotides ("oligos") specific to nucleic acid targets broadly. The
following experimental studies were
conducted to show that the method of the invention is broadly suitable for use
with anti-sense oligos designed
as taught by this application and targeted to any and all adenosine receptor
mltNAs. For this purpose, various
anti-sense oligos were prepared to adenosine receptor mltNAs exemplified by
the adenosine A,, AZb and A,
receptor ml2NAs. Anti-sense Oligo I was disclosed above (SEQ ID NO: 1).Five
additional anti-sense
phosphorothioate oligos were designed and synthesized as indicated above.
1- Oligo II (SEQ ID NO: 7) also targeted to the adenosine A, receptor,
but to a different region than Oligo I.
2- Oligo V (SEQ ID NO: 10) targeted to the adenosine Azb receptor.
3- Oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) targeted to
different regions of the adenosine A3 receptor.
4- Oligo I-PD (SEQ ID NO: 11)(a phosphodiester oligo of the same
sequence as Oligo I).
These anti-sense oligos were designed for therapy on a selected species as
described above and are
generally specific for that species, unless the segment of the target mltNA of
other species happens to contain
a similar sequences. All anti-sense oligos were prepared as described below,
and tested in vivo in a rabbit
model for bronchoconstriction, inflammation and allergy, which have breathing
difficulties and impeded lung
airways, as is the case in ailments such as asthma, as described in the above-
identified application.
Example 6: Design & Sequences of
Other Anti-sense Oligos
Six oligos and their effects in a rabbit model were studied and the results of
these studies are reported
and discussed below. Five of these oligos were selected for this study to
complement the data on Oligo I
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(SEQ ID NO: 1) provided in Examples 1 to 4 above. This oligo is anti-sense to
one region of the adenosine
A, receptor mRNA. The oligos tested are identified as anti-sense Oligos I (SEQ
ID NO: 1) and II (SEQ ID
NO: 7) targeted to a different region of the adenosine A, receptor mRNA, Oligo
V (SEQ ID NO: 8) targeted
to the adenosine AZb receptor mRNA, and anti-sense Oligos III and IV (SEQ ID
NOS: 9 and 10) targeted to
two different regions of the adenosine A3 receptor mRNA. The sixth oligo
(Oligo I-PD) is a phosphodiester
version of Oligo I (SEQ ID NO: 1). The design and synthesis of these anti-
sense oligos was performed in
accordance with Example 1 above.
(I) Anti-sense Oligo I
The anti-sense oligonucleotide I referred to in Examples 1 to 4 above is
targeted to the human A,
adenosine receptor mRNA (EPI 2010). Anti-sense oligo I is 21 nucleotide long,
overlaps the initiation codon,
and has the following sequence: 5'- GAT GGA GGG CGG CAT GGC GGG -3' (:SEQ ID
NO: 1). The
oligo I was previously shown to abrogate the adenosine-induced
bronchoconstriction in allergic rabbits, and to
reduce allergen-induced airway obstruction and bronchial hyperresponsiveness
(BHR), as discussed above
and shown by Nyce, J. W. & Metzger, W. J., Nature, 385:721 ( 1977), the
relevant portions of which reference
are incorporated in their entireties herein by reference.
(II) Anti-sense Oligo II
A phosphorothioate anti-sense oligo (SEQ ID NO: 7) was designed in accordance
with the invention
to target the rabbit adenosine A, receptor mRNA region +936 to +956 relative
to the initiation codon (start
site). The anti-sense oligo II is 21 nucleotide long, and has the following
sequence:5'-CTC GTC GCC GTC
GCC GGC GGG-3' (SEQ ID NO: 7).
(III) Anti-sense Oligo III
A phosphorothioate anti-sense oligo other than that provided in Example 1
above (SEQ ID NO: 8)
was designed in accordance with the invention to target the anti-sense A3
receptor mRNA region +3 to + 22
relative to the initiation codon start site. The anti-sense oligo III is 20
nucleotide long, and has the following
sequence: 5'-GGG TGG TGC TAT TGT CGG GC-3' (SEQ ID NO: 8).
(IV) Anti-sense Oligo IV
Yet another phosphorothioate anti-sense oligo (SEQ ID NO: 9) was designed in
accordance with the
invention to target the adenosine A3 receptor mRNA region + 386 to + 401
relative to the initiation codon
(start site). The anti-sense oligo IV is 15 nucleotide long, and has the
following sequence: 5'-GGC CCA GGG
CCA GCC-3' (SEQ ID NO: 9).
(V) Anti-sense Oligo V
A phosphorothioate anti-sense oligo (SEQ ID NO: 10) was designed in accordance
with the
invention to target the adenosine AZb receptor mRNA region -21 to -1 relative
to the initiation codon (start
site). The anti-sense oligonucleotide V is 21 nucleotide long, and has the
following sequence: 5'-GGC CGG
GCC AGC CGG GCC CGG-3' (SEQ ID NO: 10).
(VI) A1 Mismatch Oligos
Two different mismatched oligonucleotides having the following sequences were
used as
controls for anti-sense oligo I (SEQ ID NO: 1) described in Example 5 above:
A, MM2 5'-GTA
GGT GGC GGG CAA GGC GGG-3' (SEQ ID NO: 12), and A, MM3 5'-GAT GGA GGC GGG CAT
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GGC GGG-3' (SEQ ID NO: 13). Anti-sense oligo I and the two mismatch anti-sense
oligos had identical
base content and general sequence structure. Homology searches in GENBANK
(release 85.0) and EMBL
(release 40.0) indicated that the anti-sense oligo I was specific, not only
for the human, but also for the rabbit,
adenosine A, receptor genes, and that the mismatched controls were not
candidates for hybridization with any
known human or animal gene sequence.
(VII) Anti-sense Oligo Al-PD (Oligo VI)
A phosphodiester anti-sense oligo (Oligo VI; SEQ ID NO: 11) having the same
nucleotide sequence
as Oligo I was designed as disclosed in the above-identified application. Anti-
sense oligo I-PD is 21
nucleotide long, overlaps the initiation codon, and has the following
sequence: 5'- GAT GGA GGG CGG
CAT GGC GGG -3' (SEQ ID NO: 11 ).
(VIIn Controls
Each rabbit was administered 5.0 ml aerosolized sterile saline following the
same schedule as for the
anti-sense oligos in (II), (III), and (IV) above. The above are given as
examples of G-protein coupled
receptors. However, the method of reducing adenosine content is generally
applicable to any gene and any
target validation system in which the release of bioactive adenosine could
obscure experimental data for
actualizing adenosine receptors.
Example 7: Synthesis of Anti-sense Oligos
Phosphorothioate anti-sense oligos having the sequences described in (a)
above, were synthesized on
an Applied Biosystems Model 396 Oligonucleotide Synthesizer, and purified
using NENSORB
chromatography (DuPont, DE). TETD (tetraethylthiuram disulfide) was used as
the sulfurizing agent during
the synthesis. Anti-sense oligonucleotide II (SEQ ID NO: 7), anti-sense
oligonucleotide III (SEQ ID NO: 8)
and anti-sense oligonucleotide IV (SEQ ID NO: 9) were each synthesized and
purified in this manner.
Example 8: Preparation of Allergic Rabbits
Neonatal New Zealand white Pasturella-free rabbits were immunized
intraperitoneally within 24
hours of birth with 0.5 ml of 312 antigen unitslml house dust mite (D.
farinae) extract (Berkeley Biologicals,
Berkeley, CA) mixed with 10% kaolin as previously described (Metzger, W. J.,
in Late Phase Allergic
Reactions, Dorsch, W., Ed., CRC Handbook, pp. 347-362, CRC Press, Boca Raton
(1990); Ali, S., Metzger,
W. J. and Mustafa, S. J., Am. J. Resp. Crit. Care Med. 149: 908 ( 1994)), the
relevant portions of which are
incorporated in their entireties here by reference. Immunizations were
repeated weekly for the first month and
then biweekly until the age of 4 months. These rabbits preferentially produce
allergen-specific IgE antibody,
typically respond to aeroallergen challenge with both an early and late-phase
asthmatic response, and show
bronchial hyper responsiveness (BHR). Monthly intraperitoneal administration
of allergen (312 units dust
mite allergen, as above) continues to stimulate and maintain allergen-specific
IgE antibody and BHR. At 4
months of age, sensitized rabbits were prepared for aerosol administration as
described by Ali et al. (Ali, S.,
Metzger, W. J. and Mustafa, S. J., Am. J. Resp. Crit. Care Med. 149 (1994)),
the relevant section being
incorporated in its entirety here by reference.
DOSE-RESPONSE STUDIES
Example 9: Experimental Setup
Aerosols of either adenosine (0-20 mg/ml), or anti-sense or one of two
mismatch oligonucleotides (5
mg/ml) were separately prepared with an ultrasonic nebulizer (Model 646,
DeVilbiss, Somerset, PA), which
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produced aerosol droplets, 80% of which were smaller than S:m in diameter.
Equal volumes of the aerosols
were administered directly to the lungs via an intratracheal tube. The animals
were randomized, and
administered aerosolized adenosine. Day 1 pre-treatment values for sensitivity
to adenosine were calculated as
the dose of adenosine causing a 50% loss of compliance (PCS°
Adenosine). The animals were then
administered either the aerosolized anti-sense or one of the mismatch anti-
sense oligos via the intratracheal
tube (5 mg/1.0 ml), for 2 minutes, twice daily for 2 days (total dose, 20 mg).
Post-treatment PCS° values were
recorded (post-treatment challenge) on the morning of the third day. The
results of these studies are provided
in Example 21 below.
Example 10: Cross-over Experiments
For some experiments utilizing anti-sense oligo I (SEQ ID NO: 1) and a
corresponding mismatch
control oligonucleotide A1MM2, following a 2 week interval, the animals were
crossed over, with those
previously administered the mismatch control A,MM2, now receiving the anti-
sense oligo I, and those
previously treated with the anti-sense oligo I, now receiving the mismatch
control A,MM2 oligo. The number
of animals per group was as follows. For mismatch A,MM2 (Control 1), n=7,
since one animal was lost in the
second control arm of the experiment due to technical difficulties, for
mismatch A,MM3 n=4 (Control 2) and
for A,AS anti-sense oligo I, n=8. The A,MM3 oligo-treated animals were
analyzed separately and were not
part of the cross-over experiment. The treatment methods and measurements
employed following the cross-
over were identical to those employed in the first arm of the experiment. In 6
of the 8 animals treated with the
anti-sense oligo I (SEQ ID NO: 1), no PCS° value could be obtained for
adenosine doses of up to 20 mg/ml,
which is the limit of solubility of adenosine. Accordingly, the PCS°
values for these animals were assumed to
be 20 mg/ml for calculation purposes. The values given, therefore, represent a
minimum figure for the
effectiveness of the anti-sense oligonucleotides of the invention. Other
groups of allergic rabbits (n=4 for each
group) were administered 0.5 or 0.05 mg doses of the anti-sense oligo I (SEQ
ID NO: 1), or the A,MM2
oligo in the manner and according to the schedule described above (the total
doses being 2.0 or 0.2 mg). The
results of these studies are provided in Example 22 below.
Example 11: Anti-sense Oligo Formulation
Each one of anti-sense oligos were separately solubilized in an aqueous
solution and administered as
described for anti-sense oligo I (SEQ ID NO: 1) in (e) above, in four 5 mg
aliquots (20 mg total dose) by
means of a nebulizer via endotracheal tube, as described above. The results
obtained for anti-sense oligo I and
its mismatch controls confirmed that the mismatch controls are equivalent to
saline, as described in Example
19 below and in Table 1 of Nyce & Metzger, Nature 385, 721-725 (1997), the
contents of which are
incorporated herein by reference. Because of this fording, saline was used as
a control for pulmonary function
studies employing anti-sense oligos II, III and IV (SEQ ID NOS: 7, 8 and 9).
Examele 12: Specificity of Oligo I for Adenosine A1 Receptor
(Receptor Binding Studies)
Tissue from airway smooth muscle was dissected to primary, secondary and
tertiary bronchi from
rabbits which had been administered 20 mg oligo I (SEQ ID NO: 1) in 4 divided
doses over a period of 48
hours as described above. A membrane fraction was prepared according to the
method of Ali et al. (Ali, S., et
al., Am. J. Resp. Crit. Care Med. 149: 908 (1994), the relevant section
relating to the preparation of the
membrane fraction is incorporated in its entirety hereby by reference). The
protein content was determined by
the method of Bradford and plasma membranes were incubated with 0.2 U/ml
adenosine deaminase for 30
minutes at 37EC to remove endogenous adenosine. See, Bradford, M. M. Anal.
Biochem. 72, 240-254
(1976), the relevant portion of which is hereby incorporated in its entirety
by reference. The binding of
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['H]DPCPX, ['H]NPC17731, or ['H]CGS-21680 was measured as described by Jarvis
et al. See, Jarvis, M.F.,
et al., Pharmacol. Exptl. Ther. 251, 888-893 (1989), the relevant portion of
which is fully incorporated herein
by reference. Similar amounts of an oligo targeted to the bradykinin receptor
5'-
GGTGATGTTGAGCATTTCGGC-3' (SEQ ID NO: 14) were administered to another group of
animals. The
results of this study are shown in Table 6 and discussed in Example 20 below.
Example 13: Pulmonary Function Measurements
(Compliance cDyN and Resistance)
At 4 months of age, the immunized animals were anesthetized and relaxed with
1.5 ml of a mixture
of ketamine HCl (35 mg/kg) and acepromazine maleate (1.5 mg/kg) administered
intramuscularly. After
induction of anesthesia, allergic rabbits were comfortably positioned supine
on a soft molded animal board.
Salve was applied to the eyes to prevent drying, and they were closed. The
animals were then intubated with a
4.0 mm intermediate high-low cuffed Murphy 1 endotracheal tube (Mallinckrodt,
Glen Falls, NY), as
previously described by Zavala and Rhodes. See, Zavala and Rhodes, Proc. Soc.
Exp. Biol. Med. 144: 509-
512 (1973), the relevant portion of which is incorporated herein by reference
in its entirety. A polyethylene
catheter of OD 2.4 mm (Becton Dickinson, Clay Adams, Parsippany NJ) with an
attached thin-walled latex
balloon was passed into the esophagus and maintained at the same distance
(approximately 16 cm) from the
mouth throughout the experiment. The endotracheal tube was attached to a
heated Fleisch pneumotach (size
00; DEM Medical, Richmond, VA), and the flow (v) measured using a Validyne
differential pressure
transducer (Model DP-45-16-1927, Validyne Engineering, Northridge, CA), driven
by a Gould carrier
amplifier (Model 11-4113, Gould Electronics, Cleveland, OH). An esophageal
balloon was attached to one
side of the Validyne differential pressure transducer, and the other side was
attached to the outflow of the
endotracheal tube to obtain transpulmonary pressure (P,~). The flow was
integrated to yield a continuous tidal
volume, and the measurements of total lung resistance (Rt) and dynamic
compliance (Cdr,) were made at
isovolumetric and zero flow points. The flow, volume and pressure were
recorded on an eight channel Gould
2000 W high-frequency recorder and C~." was calculated using the total volume
and the difference in P~ at
zero flow, and . R, was calculated as the ratio of Ptp and V at midtidal lung
volumes. These calculations were
made automatically with the Buxco automated pulmonary mechanics respiratory
analyzer (Model 6, Buxco
Electronics, Sharon, CT), as previously described by Giles et al. See, Giles
et al., Arch. Int. Pharmacodyn.
Ther. 194: 213-232 (1971), the relevant portion of which describing these
calculations is incorporated in toto
hereby by reference. The results obtained upon administration of oligo II on
allergic rabbits are shown and
discussed in Example 26 below.
Example 14: Measurement of Bronchial Hyperresponsiveness (BHR)
Each allergic rabbit was administered histamine by aerosol to determine their
baseline
hyperresponsiveness. Aerosols of either saline or histamine were generated
using a DeVilbiss nebulizer
(DeVilbiss, Somerset, PA) for 30 seconds and then for 2 minutes at each dose
employed. The ultrasonic
nebulizer produced aerosol droplets of which 80% were <5 micron in diameter.
The histamine aerosol was
administered in increasing concentrations (0.156 to 80 mg/ml) and measurements
of pulmonary function were
made after each dose. The B4R was then determined by calculating the
concentration of histamine (mg/ml)
required to reduce the C~," 50% from baseline (PCSO H~S,a",~"~)
Example 15: Cardiovascular Effect of Anti-sense Oligo I
The measurement of cardiac output and other cardiovascular parameters using
CardiomaxJ utilizes
the principal of thermal dilution in which the change in temperature of the
blood exiting the heart after a
venous injection of a known volume of cool saline is monitored. A single rapid
injection of cool saline was
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made into the right atrium via cannulation of the right jugular vein, and the
corresponding changes in
temperature of the mixed injectate and blood in the aortic arch were recorded
via cannulation of the carotid
artery by a temperature-sensing miniprobe. Twelve hours after the allergic
rabbits had been treated with
aerosols of oligo I (EPI 2010; SEQ ID NO: 1) as described in (d) above, the
animals were anesthetized with
0.3 ml/kg of 80% Ketamine and 20% Xylazine. This time point coincides with
previous data showing
efficacy for SEQ ID NO: 1, as is clearly shown by Nyce & Metzger, (1997),
supra, the pertinent disclosure
being incorporated in its entirety here by reference. A thermocouple was then
inserted into the left carotid
artery of each rabbit, and was then advanced 6.5 cm and secured with a silk
ligature. The right jugular vein
was then cannulated and a length of polyethylene tubing was inserted and
secured. A thermodilution curve
was then established on a CardiomaxJ II (Columbus Instruments, Ohio) by
injecting sterile saline at 20EC to
determine the correctness of positioning of the thermocouple probe. After
establishing the correctness of the
position of the thermocouple, the femoral artery and vein were isolated. The
femoral vein was used as a portal
for drug injections, and the femoral artery for blood pressure and heart rate
measurements. Once constant
baseline cardiovascular parameters were established, CardiomaxJ measurements
of blood pressure, heart rate,
cardiac output, total peripheral resistance, and cardiac contractility were
made.
Example 16: Duration of Action of Oligo I
(SEQ ID NO: 1)
Eight allergic rabbits received initially increasing log doses of adenosine by
means of a nebulizer via
an infra-tracheal tube as described in (f) above, beginning with 0.156 mg/ml
until compliance was reduced by
50% (PCSO "~"~",~) to establish a baseline. Six of the rabbits then received
four 5 mg aerosolized doses of
(SEQ ID NO: 1) as described above. Two rabbits received equivalent amounts of
saline vehicle as controls.
Beginning 18 hours after the last treatment, the PCSO ,,~~;~~ values were
tested again. After this point, the
measurements were continued for all animals each day, for up to 10 days. The
results of this study are
discussed in Example 25 below.
Ezample 17: Reduction of Adenosine A2b Receptor
Number by Anti-sense Oligo V
Sprague Dawley rats were administered 2.0 mg respirable anti-sense oligo V
(SEQ ID NO: 10) three
times over two days using an inhalation chamber as described above. Twelve
hours after the last
administration, lung parenchyma) tissue was dissected and assayed for
adenosine Azb receptor binding using
[311 j-NECA as described by Nyce & Metzger ( 1997), supra. Controls were
conducted by administration of
equal volumes of saline. The results are significant at p<0.05 using Student's
paired t test, and are discussed in
Example 28 below.
Ezample 18: Comparison of Oligo I & Corresponding
Phosphodiester Oligo VI (SEQ ID NO: 11)
Oligo I (SEQ ID NO: 1) countered the effects of adenosine and eliminated
sensitivity to it for
adenosine amounts up to 20 mg adenosine/5.0 ml (the limit of solubility of
adenosine). Oligo VI (SEQ ID
NO: 11), the phosphodiester version of the oligonucleotide sequence, was
completely ineffective when tested
in the same manner. Both compounds have identical sequence, differing only in
the presence of
phosphorothioate residues in Oligo I (SEQ ID NO: 1), and were delivered as an
aerosol as described above
and in Nyce & Metzger (1997), supra. Significantly different at p<0.001,
Student's paired t test. The results
are discussed in Example 29 below.
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RESULTS OBTAINED FOR ANTI-SENSE OLIGO I - (SEQ ID NO: 1)
Example 19: Results of Prior Work
The nucleotide sequence and other data for anti-sense oligo I (SEQ ID NO: 1),
which is specific for
the adenosine A, receptor, were provided above. The experimental data showing
the effectiveness of oligo I
in down regulating the receptor number and activity were also provided above.
Further information on the
characteristics and activities of anti-sense oligo I is provided in Nyce, J.
W. and Metzger, W. J., Nature
385:721 (1997), the relevant parts of which relating to the following results
are incorporated in their entireties
herein by reference. The Nyce & Metzger ( 1997) publication provided data
showing that the anti-sense oligo I
(SEQ ID NO: 1):
( 1 ) The anti-sense oligo I reduces the number of adenosine A, receptors in
the
bronchial smooth muscle of allergic rabbits in a dose-dependent manner as may
be seen in
Table 6 below.
(2) Anti-sense Oligo I attenuates adenosine-induced bronchoconstriction and
allergen-
induced bronchoconstriction.
(3) The Oligo I attenuates bronchial hyperresponsiveness as measured by PCSo
histamine, a standard measurement to assess bronchial hyperresponsiveness.
This result
clearly demonstrates anti-inflammatory activity of the anti-sense oligo I as
is shown in
Tables 4, 5 and 6.
(4) As expected, because it was designed to target it, the anti-sense oligo I
is totally
specific for the adenosine A, receptor, and has no effect at all at any dose
on either the very
closely related adenosine A~ receptor or the related bradykinin B~ receptor.
This is seen in
Table 6 below.
(5) In contradistinction to the above effects of the Oligo I, the mismatch
control
molecules MM2 and MM3 (SEQ ID NO: 12 and SEQ ID NO: 13) which have identical
base composition and molecular weight but differed from the anti-sense oligo I
(SEQ ID
NO: 1) by 6 and 2 mismatches, respectively. These mismatches, which are the
minimum
possible while still retaining identical base composition, produced absolutely
no effect upon
any of the targeted receptors (A,, AZ or Bz).
These results, along with a complete lack of prior art on the use of anti-
sense oligonucleotides, such
as oligo I, targeted to the adenosine A, receptor, are unexpected results. The
showings presented in this patent
clearly enable and demonstrate the effectiveness, for its intended use, of the
validation method employing
agents and targeted to genes or mRNA associated with a function or end point
associated with pulmonary
functions, such as airway blockage, bronchoconstriction, pulmonary
inflammation and allergy(ies), and the
like.
Example 20: Oligo I Significantly Reduces Response to Adenosine Challenge
The receptor binding experiment is described in Example 12 above, and the
results shown in Table 6
below which shows the binding characteristics of the adenosine A,-selective
ligand [3H)DPCPX and the
bradykinin Bz-selective ligand ['H)NPC 17731 in membranes isolated from airway
smooth muscle of A,
adenosine receptor and Bz bradykinin receptor anti-sense- and mismatch-tteated
allergic rabbits.
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Table 6: Binding
Characteristics
of Three
Anti-Sense
Oligos
reatment , receptor 2 receptor
K
maz i7m~z
enosme , Receptor
0 mg . + , t , o es . f . . t , o es
2 mg 0.3810.030 nM 3212.56 fmoles* 0.410.02815.511.08 fmoles
nM
0.2 mg 0.3710.030 nM 4913.43 fmoles 0.3410.024l5.Ot1.06 fmoles
nM
A1MM1 (Control)
20 mg 0.3410.027 nM 52.Ot3.64 fmoles 0.3510.024l4.Ot1.0 fmoles
nM
2 mg 0.3710.033 nM 51.813.88 frnoles 0.38f0.02814.6f1.02 fmoles
nM
B=A (BradykininReceptor)
20 mg 0.360.028 nM 45.Ot3.15 fmoles 0.3810.0278.710.62 fmoles*
nM
2 mg 0.3910.035 nM 44.312.90 fmoles 0.3410.02411.910.76 fmoles**
nM
0.2 mg 0.4010.028 nM 47.Ot3.76 fmoles 0.3510.02815.111.05 fmoles
nM
BZMM (Control)
20 mg 0.3910.031 nM 42.02.94 frnoles 0.4110.029l4.Ot0.98 finoles
nM
2 mg 0.4110.035 nM 40.Ot3.20 fmoles 0.3710.03014.810.99 fmoles
nM
0.2 mg 0.3710.029 nM 43.Ot3.14 fmoles 0.3610.02515.111.35 fmoles
nM
Saline Control0.3710.041 46.Ot5.21 0.3910.047 nM 14.211.35 fmoles
' Refers to
total oligo
administered
in four equivalently
divided doses
over a 48
hour period.
Treatments
and analyses
were
pertormed n methods. Significance was determined
as described by repeated-measures analysis of
i variance (ANOVA),
'
and Tukey
s protected
t test. n
= 4-6 for
all groups.
' Significantly
different
from mismatch
control-
and saline-treated
groups, p<0.001;
..Significantly
different
from mismatch
control-
and saline-treated
groups, p<0.05.
Example 21: Dose-response Effect of Oligo I
Anti-sense oligo I (SEQ ID NO: 1) was found to reduce the effect of adenosine
administration to the
animal in a dose-dependent manner over the dose range tested as shown in Table
7 below.
Table 7: Dose-Response Effect to Anti-sense Oligo I
Total Dose PC50 Adenosine
(mg) (mg Adenosine)
Anti-sense Oligo I
0.2 8.32"7.2
2.0 14.0"7.2
20 19.5"0.34
A,MM2 oligo (control)
0.2 2.5110.46
2.0 3.131 0.71
20 3.25 t 0.34
The, above results were studied with the Student's paired t test and found to
be
starisrically different, p=0.05
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The oligo I (SEQ ID NO: 1), an anti-adenosine A, receptor oligo, acts
specifically on the adenosine
A, receptor, but not on the adenosine Az receptors. These results stem from
the treatment of rabbits with anti-
sense oligo I (SEQ ID NO: 1) or mismatch control oligo (SEQ ID NO: 12; A,MM2)
as described in Example
9 above and in Nyce & Metzger ( 1997), supra (four doses of 5 mg spaced 8 to
12 hours apart via nebulizer via
endotracheal tube), bronchial smooth muscle tissue excised and the number of
adenosine A, and adenosine AZ
receptors determined as reported in Nyce & Metzger (1997), supra.
Example 22: Specificity of Oligo I (SEQ ID NO: 1)
for Target Gene Product
Oligo I (SEQ ID NO: 1) is specific for the adenosine A, receptor whereas its
mismatch controls had
no activity. Figure 1 depicts the results obtained from the cross-over
experiment described in Example 10
above and in Nyce & Metzger (1997), supra. The two mismatch controls (SEQ ID
NO: 12 and SEQ ID NO:
13) evidenced no effect on the PCSO Adrn~ine value. On the contrary, the
administration of anti-sense oligo I
(SEQ ID NO: 1) showed a seven-fold increase in the PCS° A~"~;"~ value.
The results clearly indicate that the
anti-sense oligo I (SEQ ID NO: 1) reduces the response (attenuates the
sensitivity) to exogenously
administered adenosine when compared with a saline control. The results
provided in Table 6 above clearly
establish that the effect of the anti-sense oligo I is dose dependent (see,
column 3 of Table 6). The Oligo I
was also shown to be totally specific for the adenosine A, receptor, (see, top
3 rows of Table 6), inducing no
activity at either the closely related adenosine Az receptor or the bradykinin
Bz receptor (see, lines 8-10 of
Table 6 above). In addition, the results shown in Table 6 establish that the
anti-sense oligo I (SEQ ID NO: 1)
decreases sensitivity to adenosine in a dose dependent manner, and that it
does this in an anti-sense oligo-
dependent manner since neither of two mismatch control oligonucleotides
(A,MM2: SEQ ID NO: 12 and
A,MM3: SEQ ID NO: 13) show any effect on PCSO Aam~ine values or on attenuating
the number of adenosine
A, receptors.
Examule 23: Effect on Aeroallergen-induced
Bronchoconstriction & Inflammation
The Oligo I (SEQ ID NO: 1) was shown to significantly reduce the histamine-
induced effect in the
rabbit model when compared to the mismatch oligos. The effect of the and-sense
Oligo I (SEQ m No:l) and
the mismatch oligos (A,MM2, SEQ ID NO: 12 and A,MM3, SEQ ID NO: 12) on
allergen-induced airway
obstruction and bronchial hyperresponsiveness was assessed in allergic
rabbits. The effect of the anti-sense
oligo I (SEQ ID NO: 1) on allergen-induced airway obstruction was assessed. As
calculated from the area
under the plotted curve, the anti-sense oligo I significantly inhibited
allergen-induced airway obstruction when
compared with the mismatched control (55%, p<0.05; repeated measures ANOVA,
and Tukey's t test). A
complete lack of effect was induced by the mismatch oligo A,MM2 (Control) on
allergen induced airway
obstruction. The effect of the anti-sense oligo I (SEQ ID NO: 1) on allergen-
induced BHR was determined as
above. As calculated from the PCSO Hisou"ine value , the anti-sense oligo I
(SEQ ID NO: 1) significantly
inhibited allergen-induced BHR in allergic rabbits when compared to the
mismatched control (61%, p<0.05;
repeated measures ANOVA, Tukey's t test). A complete lack of effect of the
A,MM mismatch control on
allergen-induced BHR was observed. The results indicated that anti-sense oligo
I (SEQ ID NO: 1) is
effective to protect against aeroallergen-induced bronchoconstriction (house
dust mite). In addition, the anti-
sense oligo I (SEQ ID NO: i) was also found to be a potent inhibitor of dust
mite-induced bronchial hyper
responsiveness, as shown by its effects upon histamine sensitivity which
indicates anti- inflammatory activity
for anti-sense oligo I (SEQ ID NO: 1).
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Example 24: Low A Content Anti-sense Oligo I
is Free of Deleterious Side Effects
The Oligo I (SEQ ID NO: 1) was shown to be free of side effects that might be
toxic to the recipient.
No changes in arterial blood pressure, cardiac output, stroke volume, heart
rate, total peripheral resistance or
heart contractility (dPdT) were observed following administration of 2.0 or 20
mg oligo I (SEQ ID NO: 1).
The addition, the results of the measurement of cardiac output (CO), stroke
volume (SV), mean arterial
pressure (MAP), heart rate (HR), total peripheral resistance (TPR), and
contractility (dPdT) with a
CardiomaxJ apparatus (Columbus Instruments, Ohio) were assessed. These results
evidenced that oligo I
(SEQ ID NO: 1) has no detrimental effect upon critical cardiovascular
parameters. More particularly, this
oligo does not cause hypotension. This fording is of particular importance
because other phosphorothioate
anti-sense oligonucleotides have been shown in the past to induce hypotension
in some model systems.
Furthermore, the adenosine A, receptor plays an important role in sinoatrial
conduction within the heart.
Attenuation of the adenosine A, receptor by anti-sense oligo I (SEQ ID NO: 1)
might be expected to result,
therefore, in deleterious extrapulmonary activity in response to the down
regulation of the receptor. This is not
the case. The anti-sense oligo I (SEQ ID NO: 1 ) does not produce any
deleterious intrapulmonary effects and
renders the administration of the low doses of the present anti-sense oligo
free of unexpected, undesirable side
effects. This demonstrates that when oligo I (SEQ ID NO: 1) is administered
directly to the lung, it does not
reach the heart in significant quantities to cause deleterious effects. This
is in contrast to traditional adenosine
receptor antagonists like theophylline which do escape the lung and can cause
deleterious, even life-
threatening effects outside the lung.
Example 25: Long Lasting Effect of Oligo I
The Oligo I (SEQ ID NO: 1) evidenced a long lasting effect as evidenced by the
PCso and Resistance
values obtained upon its administration prior to adenosine challenge. The
duration of the effect was measured
for with respect to the PCSO of adenosine anti-sense oligo I when administered
in four equal doses of 5 mg
each by means of a nebulizer via an endotracheal tube, as described above. The
effect of the agent is
significant over days 1 to 8 after administration. When the effect of the anti-
sense oligo I (SEQ ID NO: 1)
had disappeared, the animals were administered saline aerosols (controls), and
the PCSO Aae~~~~~ values for all
animals were measured again. Saline-treated animals showed base line PCso
adenosine values (n=6). The
duration of the effect (with respect to Resistance) was measured for six
allergic rabbits which were
administered 20 mg of anti-sense oligo I (SEQ ID NO: 1) as described above,
upon airway resistance
measured as also described above. The mean calculated duration of effect was
8.3 days for both PCso
adenosine (p<0.05) and resistance (p<0.05). These results show that anti-sense
oligo I (SEQ ID NO: 1) has
an extremely long duration of action, which is completely unexpected.
Example 26: Adenosine-free Anti-sense Oligo II
Better than Anti-sense Oligo I
Anti-sense oligo II, targeted to a different region of the adenosine A,
receptor mRNA, was
found to be highly active against the adenosine A,-mediated effects. The
experiment measured the effect of
the administration of anti-sense oligo II (SEQ ID NO: 7) upon compliance and
resistance values when 20 mg
anti-sense oligo II or saline (control) were administered to two groups of
allergic rabbits as described above.
Compliance and resistance values were measured following an administration of
adenosine or saline as
described above in Example 13. The effect of the and-sense oligo of the
invention was different from the
control in a statistically significant manner, p<0.05 using paired t-test,
compliance; p<0.01 for resistance.
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The results showed that anti-sense oligo II (SEQ ID NO: 7), which targets the
adenosine A, receptor and
which contains no adenosine, effectively maintains compliance and reduces
resistance upon adenosine
challenge. In fact, the adenosine-free anti-sense Oligo II is more potent than
the low adenosine anti-sense
Oligo I (SEQ ID NO: 1). Because it contains no adenosine, it will not liberate
adenosine during degradation,
and hence it will not contribute to activating adenosine receptors.
Example 27: Anti-sense Oligos III and IV
Oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) were shown to be in fact
specifically targeted to
the adenosine A3 receptor by their effect on reducing inflammation and the
number of inflammatory cells
present upon separate administration of 20 mg of the anti-sense oligos III
(SEQ ID NO: 8) and IV (SEQ ID
NO: 9) to allergic rabbits as described above. The number of inflammatory
cells was determined in their
bronchial lavage fluid 3 hours later by counting at least 100 viable cells per
lavage. The effect of anti-sense
oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) upon granulocytes, and upon
total cells in bronchial
lavage were assessed following exposure to dust mite allergen. The results
showed that the anti-sense oligo IV
(SEQ ID NO: 9) and anti-sense oligo III (SEQ ID NO: 8) are very potent anti-
inflammatory agents in the
asthmatic lung following exposure to dust mite allergen. As is known in the
art, granulocytes, especially
eosinophils, are the primary inflammatory cells of asthma, and the
administration of anti-sense oligos III
(SEQ ID NO: 8) and IV (SEQ ID NO: 9) reduced their numbers by 40% and 66%,
respectively.
Furthermore, anti-sense oligos IV (SEQ ID NO: 9) and III (SEQ ID NO: 8) also
reduced the total number of
cells in the bronchial lavage fluid by 40% and 80%, respectively. This is also
an important indicator of anti-
inflammatory activity by the present anti-adenosine A3 agents of the
invention. Inflammation is known to
underlie bronchial hyperresponsiveness and allergen-induced
bronchoconstriction in asthma. Both anti-sense
oligonucleotides III (SEQ ID NO: 8) and IV (SEQ ID NO: 9), which are targeted
to the adenosine A,
receptor, are representative of an important new class of anti-inflammatory
agents which may be designed to
specifically target the lung receptors of each species.
Example 28: Anti-sense Oligo V
The anti-sense oligo V (SEQ ID NO: 10) , targeted to the adenosine A~b
adenosine receptor mRNA
was shown to be highly effective at countering adenosine A,b-mediated effects
and at reducing the number of
adenosine AZb receptors present to less than half.
Example 29: Unexpected Superiority of Substituted over
Phosphodiester-residue Oligo I-DS (SEQ ID NO: 11)
Oligos I (SEQ ID NO: 1) and I-DS (SEQ ID NO: 11) were separately administered
to allergic
rabbits as described above, and the rabbits were then challenged with
adenosine. The phosphodiester oligo I-
DS (SEQ ID NO: 11) was statistically significantly less effective in
countering the effect of adenosine
whereas oligo I (SEQ ID NO: 1) showed high effectiveness, evidencing a
PCSO,,~"~~°~ of 20 mg.
Example 30: Adenosine Containing Mononucleotides
have Adenosine Receptor Activity
This example demonstrates that in vivo break down products of anti-sense
oligonucleotides such as
ribonucleoside monophosphates, e.g. dAMP act at adenosine receptors. When
adenosine and adenosine
monophosphate (dAMP) were separately administered to experimental animals at
different doses of up to 10
mg/ml, both compounds have a similar effect in reducing % compliance as shown
in Figure 1. The effect in
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both cases increases with the dose whereas a saline control shows no effect.
These results show that
adenosine nucleosides as well as adenosine itself, interact with adenosine
receptors.
Example 31: Breakdown of Adenosine Containing Nucleic
Acid Produce Adenosine Receptor Activity
As a further test, randomer phosphorothioate anti-sense oligonucleosides were
administered to
rabbits to determine if they were degraded in vivo and released adenosine
nucleosides capable of interacting
with adenosine receptors. Asthmatic rabbits were separately administered
saline (control), an adenosine
containing randomer (?) and a desAdenosine randomer (C). The randomers used
were a desAdenosine
randomer consisting of random sequences of guanine, cytosine and thymidine and
an adenosine containing
randomer consisting of guanine, cytosine and adenosine. The results shown in
Figure 2 clearly indicate that
adenosine containing oligonucleotides release adenosine and/or adenosine
nucleosides upon degradation, and
that the adenosine compounds interact with adenosine receptors, while
desAdenosine oligonucleotides do not.
The release of adenosine nucleosides as degradation products of anti-sense
oligonucleotides, thus, would
confuse experimental results when assessing the effects of anti-sense knock
out experiments for Target
Validation. This experiment shows the necessity of using desAdenosine anti-
sense (no A or low A)
oligonucleotides in Target Validation studies.
The foregoing examples are illustrative of the present invention, and are not
to be construed as
limiting thereof. The invention is defined by the following claims, with
equivalents of the claims to be
included therein.
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