Note: Descriptions are shown in the official language in which they were submitted.
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
OLIGONUCLEOTIDES TARGETING PRION DISEASES
BACKGROUND OF THE INVENTION
[0001] The present invention concerns treatment or prevention of
traxlsmissible spongiform
encephalopathies, also referred to as prion diseases.
[0002] Transmissible spongiform encephalopathies (TSEs) encompass a group of
potentially fatal neurodegenerative diseases in animals and humans. The
etiology of
naturally occurnng TSEs seems to include horizontal and vertical transmission
as well as
genetic predisposition, yet for the majority of cases the etiology is unclear.
The onset of
clinical illness is preceded by a prolonged incubation period of months to
decades. Clinical
symptoms of TSEs include dementia and loss of movement and coordination.
Neuropathological examination in disease cases typically reveals gliosis and
the presence of
spongiform encaphalophy, sometimes accompanied by the formation of amyloid
deposits
(amyloid plaques).
[0003] TSEs, which include Creutzfeldt-Jacob Disease (CJD), variant CJD
(vCJD), fatal
familial insomnia (FFI), Gerstmann-Straussler-Scheinker Disease (GSS), kuru,
bovine
spongiform.encephalopathy (BSE), feline spongifonn encephalopathy (FSE),
transmissible
mink encephalopathy (TME), chronic wasting disease (CMD), and scrapie, are
characterized
by the accumulation of aggregates of the abnormal prion protein (PrPsc) in the
brain and
other infected tissues. The normal form, PrPc, which is dominated by alpha-
helices towards
the C-terminus, is most abundant in the central nervous system but its
physiological function
is unknown. The accumulation of the beta-structure rich isofonn, PrPsc, is
widely believed to
result from the ability of this isoform to stabilize thermodynamically,
similarly folded forms
during the folding of cellular PrPc. This process contributes to the formation
of increasing
numbers of misfolded prion proteins which upon aggregation, form the major
component of
amyloid plaques characteristic of TSE's.
[0004] Caughey and coworkers (1993) tested sulfated polyanions as inhibitors
of scrapie-
associated PrPsc accumulation in cultured cells. Pentosan polysulfate and the
amyloid-
binding dye Congo red potently inhibited the accumulation of PrPsc in cells
without apparent
effects on the metabolism of the normal isofonn PrPc. A comparision of the
activity of
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
pentosan polysulfate with that of sulfated glycans, non-sulfated polyanions,
dextran and
DEAF-dextran has suggested that the density of sulfation and molecular size
are factors
influencing anti-PrPsc activity of sulfated polyanions. Shyng and coworkers
(1995) also
reported that pentosan polysulfate and related compounds rapidly and
dramatically reduced
the amount of PrPc, the non-infectious precursor of PrPsc, present on the cell
surface.
[0005] Another study reported that treatment of TSE-infected animals with
certain cyclic
tetrapyrroles (porphyrins and phthalocyanines) increased survival time from 50
to 300%.
The significant inhibition of TSE disease by structurally dissimilar
tetrapyrroles identifies
these compounds as anti-TSE drugs (Priola et al., 2000).
[0006] Supatappone and coworkers (2001) demonstrated that exposure of scrapie-
infected
neuroblastoma cells to 3 micrograms of branched polyamines, including
polyamidoamine
and polypropyleneimine, for 4 weeks not only reduced PrPsc to a level
undetectable by
Western blot but also eradicated prion infectivity as determined by a bioassay
in mice. The
activity of branched polyamines ifz vitro was prion strain dependent.
[0007] Amphotericine B (AmB), a macrolide polyene antibiotic, is one of the
few drugs
that has shown therapeutic activity in scrapie-infected hamsters. A study
showed that
treatment with an AmB derivative delayed the progression of the disease,
possibly by
preventing the replication of the scrapie protein at the inoculation site
where the cells appear
to be the first producing abnormal PrP (Grigoriev et al. 2002)
[0008] Poli and collaborators (2003) demonstrated the ability of synthesized
Congo red
derivatives to prevent the prion protein conversion in cell-free and cellular
assays. However,
the most active compound in the cellular assay was also highly toxic at the
effective dose.
[0009] Another study reported that heparan sulfate mimetics could abolish
prion
propagation in scrapie-infected cells. PrPsc does not reappear for up to 50
days post-
treatment. When tested ih vivo, one compound hampered PrPsc accumulation in
scrapie- and
BSE-infected mice and prolonged significantly the survival time of scrapie-
infected hamsters
(Adjou et al. 2003).
[0010] Kocisko and coworkers (2003) are reported to have identified new
inhibitors of
PrPsc formation from a library of compounds. Several classes of compounds were
represented in the 17 most potent inhibitors, including naturally occurring
polyphenols (e.g.,
2
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
tannic acid and tea extracts), phenothiazines, antihistamines, statins, and
antimalarial
compounds.
[0011] Quinacrine was shown to hamper de novo generation of fibrillogeiuc
prion protein.
However, in vivo, no detectable effect was observed in an animal model,
consistent with other
recent studies and preliminary observations in humans. Despite its abilityto
cross the blood-
brain barrier, the use of quinacrine for the treatment of CJD is questionable
(Barret et al.
2003) (Nakajima et al, 2004).
[0012] The therapeutic efficacy of direct drug infusion into the brain was
assessed in
transgenic mice intracerebrally infected with the scrapie agent. Pentosan
polysulfate (PPS)
gave the most dramatic prolongation of the incubation period, and AmB had
intermediate
effects, but antimalarial drugs such as quinacrine gave no significant
prolongation. However,
at doses higher than that providing the maximal effects, intraventricular PPS
infusion caused
advexse effects such as hematoma formation in the experimental animals (Doh-
ura et al.,
2004).
[0013] The squalene synthase inhibitor squalestatin reduced the cholesterol
content of cells
and prevented the accumulation of PrPsc in three prion-infected cell lines.
Cells treated with
squalestatin were also protected against microglia-mediated killing. These
effects of
squalestatin were dose-dependent and were evident at nanomolar concentrations
(Bate et al.,
2004).
[0014] In a review article, Koster et al.(2003) described a number of possible
therapeutic
agents that have been tried and some reported to have activity against TSEs
but most of these
compounds have limitations in terms of toxicity and pharmacokinetics. Congo
red,
anthracyclines, and the polyanion dextran sulfate have limited ability to
cross the blood-brain
barner and may be toxic. The efficacy of polyene antibiotics seems to be
restricted to certain
scrapie strains. Tetrapyrroles and tetracyclines with low toxicities and
favorable
phannacokinetics could be useful in preventing PrPsc accumulation. Compounds
like
branched polyamines, Cp-60, analogs of Congo red, quinacrine and
chlorpromazine, beta-
sheet breaker peptides and inhibitory peptides, active immunization using
recombinant PrP
and passive immunization with anti-PrP aaztibodies, have potential use as
therapeutic agents
but will need fuz-ther research and clinical trials.
3
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0015] There is no currently available treatment to cure or prevent the
development of
transmissible spongiform encephalopathies and other prion-associated.diseases.
There is also
no treatment for animal or human tissue products to prevent transmission of
prion diseases. It
would be useful to have compounds, methods of treatment, and formulations to
treat, prevent
transmission and development of acid reverse progression in prion diseases.
[0016] Approximately 80 million units of blood are donated annually worldwide
(World
Health Organization, 2004). There have been chronic shortages of blood, partly
because of
increased demand from modern surgical techniques. For example, people who are
undergoing
aggressive cancer chemotherapy treatments require blood transfusions because
their own
body's ability to make blood cells diminishes. Premature infants may require
blood
transfusions to carry oxygen throughout their bodies. Medical treatments, such
as organ
transplants and cardiac bypass surgery, that require a large amount of blood,
were uncommon
30 yeaxs ago, yet today are routine. And the aging of the population means
that more people
live longer and are more likely to need medical treatments that require safe
blood and blood
products. Blood supplies are tested for several infectious agents and are
treated for such
agents when treatments are available. But no treatments are currently
available to safely
inactivate or destroy prions in blood and blood product supplies without
affecting the
required properties of such biological products.
[0017] The information provided and references cited herein is intended only
to assist the
understanding of the reader, and does not constitute an admission that any of
the information
or references constitutes prior art to the present invention.
SUMMARY OF THE INVENTION
[0018] The present invention concerns oligonucleotides that have anti-prion
activity, and
thus can be used in treatment, control, or prevention of one or more prion
diseases. Likewise, ,
such oligonucleotides can be used to treat~biological materials, e.g., to
prevent or reduce the
chance of infection following use of the biological material.
[0019] In addition, the inventors discovered that different length
oligonucleotides have
varying anti-prion effect, and fiu-ther that the length of anti-prion
oligonucleotide that
produces potent anti-prion effect is usually about 40 nucleotides or longer,
e.g., in the range
of 40-120 nucleotides. In view of the present discoveries concerning anti-
prion properties of
oligonucleotides, this invention provides oligonucleotide anti-prion agents
that can have
4
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
activity against several different prion disease agents, and can even be
selected as broad-
spectrum anti-prion agents. Such anti-prion agents are particularly
advantageous in view of
the limited anti-prion therapeutic options currently available. .
[0020] Therefore, the oligonucleotides of the present invention are useful in
therapy for
treating or preventing prion diseases and in treating or preventing other
diseases whose
etiology is prion-based.
[0021] Thus, the invention concerns anti-prion oligonucleotides and
oligonucletide
formulations that includes at least one anti-prion oligonucleotide, e.g., at
least 6 nucleotides
in length, adapted for use as an anti-prion agent. Preferably the anti-prion
activity of the
oligonucleotide occurs principally by a sequence independent mode of action.
Such a
formulation can include a mix of different oligonucleotides, e.g., at least 2,
3, 5, 10, 50, 100,
or even more.
[0022] A related aspect concerns an anti-prion oligonucleotide randomer
formulation,
where the anti-prion activity of the randomer occurs principally by a sequence
independent
mode of action. Such a randomer formulation can, for example, include a
mixture of
randomers of different lengths, e.g., at least 2, 3, 5, 10, or more different
lengths.
[0023] In another aspect, the invention provides an oligonucleotide having
anti-prion
activity against a prion disease, where the oligonucleotide is at least 29
nucleotides in length
(or in particular embodiments, at least 30, 32, 34, 36, 38, 40, 46, 50, 60,
70, 80, 90, 100, 110,
or 120 nucleotides in length). In particular embodiments, the sequence of the
oligonucleotide
is not complementary to any portion of the genome sequence of the animal
subject to the
particular prion disease of interest.
[0024] In another aspect, the invention provides an oligonucleotide
formulation, containing
at least one oligonucleotide having anti-prion activity against a prion
disease, where the
oligonucleotide is at least 6 nucleotides in length (in particular
embodiments, at least 10, 15,
18, 20, 22, 24, 26, 28, 29, 30, 32, 34, 36, 38, 40, 46, 50, 60, 70, 80, 90,
100, 110, or 120
nucleotides in length). In certain embodiments, the sequence of the
oligonucleotide is less
than 70% complementary to any portion of the genomic nucleic acid sequence of
the subj ect
animal for the particular prion disease and does not consist essentially of
polyA, polyC,
polyG, polyT, Gquartet, or a TG-rich sequence. In particular embodiments, the
oligonucleotide has less than 65%, 60%, 55%, 50%, 80% 90%, 95%, or 100%
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
complementarity to any portion of the genomic nucleic acid sequence of the
animal subject to
the particular prion disease.
[0025] Related aspects concern isolated, purified or enriched anti-prion
oligonucleotides as
described herein, e.g., as described for anti-prion oligonucleotide
formulations, as well as
other oligonucleotide preparations, e.g., preparations suitable for ira vivo
use.
[0026] Anti-prion oligonucleotides useful in the present invention can be of
various lengths,
e.g., at least 6, 10, 14, 15, 20, 25, 28, 29, 30, 35, 38, 40, 46, 50, 60, 70,
80, 90, 100, 110, 120,
140, 160, or more nucleotides in length. Likewise, the oligonucleotide can be
in a range, e.g.,
a range defined by taking any two of the preceding listed values as inclusive
end points of the
range, for example 10-20, 20-40, 30-50, 40-60, 40-80, 60-120, and 80-120
nucleotides. In
particular embodiments, a minimum length or length range is combined with any
other of the
oligonucleotide specifications listed herein for the present anti-prion
oligonucleotides.
[0027] The anti-prion nucleotide can include various modifications, e.g.,
stabilizing
modifications, and thus can include at least one modification in the
phosphodiester linkage
and/or on the sugar, and/or on the base. For example, the oligonucleotide can
include one or
more phosphorothioate linkages, phosphorodithioate linkages, and/or
methylphosphonate
linkages; modifications at the 2'-position of the sugar, such as 2'-O-methyl
modifications, 2'-
amino modifications, 2'-halo modifications such as 2'-fluoro; acyclic
nucleotide analogs, and
can also include at least one phosphodiester linkage. Other modifications are
also known in
the art and can be used. In oligos that contain 2'-O-methyl modifications, the
oligo should
not have 2'-O-methyl modifications throughout, as current results suggest that
such oligos do
not have suitable activity. In particular embodiments, the oligonucleotide has
modified
linkages throughout, e.g., phosphorothioate; has a 3'- and/or 5'-cap; includes
a terminal 3'-5'
linkage; the oligonucleotide is or includes a concatemer consisting of two or
more
oligonucleotide sequences joined by a linkers)
[0028] In particular embodiments, the oligonucleotide binds to one or more PrP
proteins;
the sequence of the oligonucleotide (or a portion thereof, e.g.; at least %2)
is derived from a
genome of a subject animal; the activity of an oligonucleotide with a sequence
derived from a
genome of a subj ect animal is not superior to a randomer oligonucleotide or a
random
oligonucleotide of the same length; the oligonucleotide includes a portion
complementary to
a genome of a subj ect animal and a portion not complementary to a genome of a
subj ect
animal; the sequence of the oligonucleotide is derived from a PrP sequence;
unless otherwise
6
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
indicated, the sequence of the oligonucleotide includes A(x), C(x), G(x),
T(x), AC(x), AG(x),
AT(x), CG(x), CT(x), or GT(x), where x is 2, 3, 4, 5, 6, ... 60 ... 120 ...;
the oligonucleotide
is single stranded (RNA or DNA); the oligonucleotide is double stranded (RNA
or DNA); the
oligonucleotide includes at least one Gquartet or CpG portion; the
oligonucleotide includes a
portion complementary to a mRNA of a subject animal; the oligonucleotide
includes at least
one non-Watson-Crick oligonucleotide and/or at least one nucleotide that
participates in non-
Watson-Crick binding with another nucleotide; the oligonucleotide is a random
oligonucleotide, the oligonucleotide is a randomer or includes a randomer
portion, e.g., a
randomer portion that has a length as specified above for oligonucleotide
length; the
oligonucleotide is linked or conjugated at one or more nucleotide residues to
a molecule that
modifies the characteristics of the oligonucleotide, e.g. to provide higher
stability (such as
stability in serum or stability in a particular solution), lower serum
interaction, higher cellular
uptake, improved ability to be formulated for delivery, a detectable signal,
improved
pharmacokinetic properties, specific tissue distribution, and/or lower
toxicity.
[0029) Oligonucleotides can also be used in combinations, e.g., as a mixture.
Such
combinations or mixtures can include, for example, at least 2, 4, 10, 100,
1000, 10000,
100,000, 1,000,000, or more different oligonucleotides. Such combinations or
mixtures can,
for example, be different sequences andlor different lengths and/or different
modifications
and/or different linked or conjugated molecules. In particular embodiments of
such
combinations or mixtures, a plurality of oligonucleotides have a minimum
length or are in a
length range as specified above for oligonucleotides. Tn particular
embodiments of such
combinations or mixtures, at least one, a plurality, or each of the
oligonucleotides can have
any of the other properties specified herein for individual anti-prion
oligonucleoties (which
can also be in any consistent combination).
[0030] The invention also provides an anti-prion pharmaceutical composition
that includes
a therapeutically effective amount of a pharmacologically acceptable, anti-
prion
oligonucleotide at least 6 nucleotides in length (or other length as listed
herein), and a
pharmaceutically acceptable carrier. Preferably the anti-prion activity of the
oligonucleotide
occurs principally by a sequence independent mode of action. In particular
embodiments, the
oligonucleotide or a combination or mixture of oligonucleotides is as
specified above for
individual oligonucleotides or combinations or mixtures of oligonucleotides.
In particular
embodiments, the pharmaceutical compositions are approved for administration
to a human,
or a non-human animal such as a non-human mammal.
7
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0031] In particular embodiments, the pharmaceutical composition is adapted
for the
treatment, control, or prevention of a disease with a prion etiology; adapted
for treatment,
control, or prevention of a prion disease; is adapted for delivery by
intraocular administration,
oral ingestion, enteric administration, inhalation, cutaneous, subcutaneous,
intramuscular,
intraperitoneal, intrathecal, intracerebral, intratracheal, or intravenous
injection, or topical
administration. In particular embodiments, the composition includes a delivery
system, e.g.,
targeted to specific cells or tissues;, a liposomal formulation, a penetration
enhancer, a
surfactant, another anti-prion drug, e.g., a non-nucleotide anti-prion
polymer, an antisense
molecule, an siRNA, or a small molecule drug.
[0032] In particular embodiments, the anti-prion oligonucleotide,
oligonucleotide
preparation, oligonucleotide formulation, or anti-prion pharmaceutical
composition has an
IC50 for a prion target (e.g., any of particular prion disease as indicated
herein) of 1.0, 0.50,
0.20, 0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03,
0.025, 0.02, 0.015, or
0.01 ~,M or less.
[0033] In particular embodiments of formulations, pharmaceutical compositions,
and
methods for prophylaxis or treatment, the composition or formulation is
adapted for
treatment, control, or prevention of a disease with priori etiology; is
adapted for the treatment,
control or prevention of a priori disease; is adapted for delivery by a mode
selected from the
group consisting of intraocular, oral ingestion, enterally, inhalation, or
cutaneous,
subcutaneous, intramuscular, intraperitoneal, intrathecal, intracerebral,
intratracheal ,
intraventricular, intracranial, topical or intravenous injection delivery;
further comprises a
delivery system, which can include or be associated with a molecule increasing
affinity with
specific cells; further comprises at least one other anti-priori drug in
combination (e.g.,
pentosan polysulfate); and/or further comprises an anti-priori polymer in
combination.
[0034] Tn another aspect, the invention provides a kit that includes at least
one anti-priori
oligonucleotide or oligonucleotide formulation in a labeled package, where the
anti-priori
activity of the oligonucleotide occurs principally by a sequence independent
mode of action
and the label on the package indicates that the anti-priori oligonucleotide
can be used against
at least one priori disease.
[0035] In particular embodiments the kit includes a pharmaceutical composition
that
includes at least one anti-priori oligonucletide as described herein; the anti-
priori
oligonucleotide is adapted for ira vivo use in an animal and/or the label
indicates that the
8
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
oligonucleotide or composition is acceptable and/or approved for use in an
animal; the animal
is a mammal, such as human, or a non-human mammal such as bovine,~porcine, a
ruminant,
ovine, or equine; the animal is a non-human animal; the kit is approved by a
regulatory
agency such as the U.S. Food and Drug Administration or equivalent agency.for
use in an
animal, e.g., a human; the kit is approved by the U.S. Food and Drug
Administration or
equivalent regulatory agency for an anti-prion indication; the kit includes
written instructions
for administration to a subject for an anti-prion indication.
[0036] In another aspect, the invention provides a method for selecting an
anti-prion
oligonucleotide, e.g, a sequence independent anti-prion oligonucleotide, for
use as an anti-
prion agent. The method involves synthesizing a plurality of different random
oligonucleotides, testing the oligonucleotides for activity in inhibiting the
ability of a PrP to
alter to PrPsc and/or to aggregate, and selecting an oligonucleotide having a
pharmaceutically
acceptable level of activity for use as an anti-prion agent.
[0037] In particular embodiments, the different random oligonucleotides
comprises
randomers of different lengths; the random oligonucleotides can have different
sequences or
'can have sequence in common, such as the sequence of the shortest oligos of
the plurality;
andlor the different random oligonucleotides comprise a plurality of
oligonucleotides
comprising a randomer segment at least 5 nucleotides in length or the
different random
oligonucleotides include a plurality of randomers of different lengths. Other
oligonucleotides, e.g., as described herein for anti-prion oligonucleotides,
can be tested in a
particular system.
[0038] W yet another aspect, the invention provides a method for the
prophylaxis or
treatment of a prion disease in a subj ect by administering to a subj ect in
need of such
treatment a therapeutically effective amount of at least one pharmacologically
acceptable
oligonucleotide as described herein, e.g., a sequence independent
oligonucleotide at least 6
nucleotides in length, or an anti-prion pharmaceutical composition or
formulation containing
such oligonucleotide. In particular embodiments, the prion disease can be any
of those listed
herein; the subject is a type of subject as indicated herein, e.g., human, non-
human animal,
non-human mammal, bovine, porcine, a ruminant, ovine, or equine; the treatment
is for a
prion disease or disease with a prion etiology. .
[0039] In particular embodiments, an anti-prion oligonucleotide (or
oligonucleotide
formulation or pharmaceutical composition) as described herein is
administered;
9
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
administration is a method as described herein; a delivery system or method as
described
herein is used.
[0040] In another aspect, the discovery that non-sequence dependent
interactions produce
effective anti-prion activity provides a method of screening to identify a
compound that alters
formation of PrPsc, e.g., binding of an oligonucleotide to a PrP. For example,
the method can
involve determining whether a test compound reduces the binding of
oligonucleotide to PrP.
[0041] In particular embodiments, any of a variety of assay formats and
detection methods
could be used to identify such alteration (e.g., alteration in binding), e.g.,
by contacting the
oligonucleotide .with the PrP (or cell in a cell-based assay) in the presence
and absence of a
compounds) to be screened (e.g., in separate reactions) and determining
whether a difference
occurs in binding of the oligo to PrP (or formation of PrPsc) in the presence
of the compound
compared to the absence of the compound. The presence of such a difference is
indicative
that the compound alters the binding of the random oligonucleotide to the PrP
(or formation
of PrPsc). Alternatively, a competitive displacement can be used, such that
oligonucleotide is
bound to the PrP and displacement by added test compound is determined, or
conversely test
compound is bound and displacement by added oligonucleotide is determined.
[0042] In particular embodiments, the oligonucleotide is as described herein
for anti-prion
oligonucleotides; the oligonucleotide is at least 6, 8, 10, 15, 20, 25, 29,
30, 32, 34, 36, 38, 40,
46, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides in length or at least
another length
specified herein for the anti-priori oligonucleotides, or is in a range
defined by taking any two
of the preceding values as inclusive endpoints of the range; the test
compounds) is a small
molecule; the test compound has a molecular weight of less than 400, 500, 600,
800, 1000,
1500, 2000, 2500, or 3000 daltons, or is in a range defined by taking any two
of the preceding
values as inclusive endpoints of the range; at least 100, 1000, 10,000,
20,000, 50,000, or
100,000 compounds are screened; the oligonucleotide has an IC50 of equal to or
less than
1.0, 0.500, 0.200, 0.100, 0.075, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02,
0.015, or 0.01 ~,M.
(0043] In a related aspect, the invention provides an anti-priori compound
identified by the
preceding method, e.g., a novel anti-priori compound.
[0044] In a fiuther aspect, the invention provides a method for purifying
oligonucleotides
binding to at least PrP from a pool of oligonucleotides by contacting the pool
with at least
PrP, e.g., bound to a stationary phase medium, and collecting oligonucleotides
that bind to
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
the PrP(s). Generally, the collecting involves displacing the oligonucleotides
from the PrP(s).
The method can also involve sequencing and/or testing anti-prion activity of
collected
oligonucleotides (i.e., oligonucleotides that bound to PrP)
[0045] In particular embodiments, the bound oligonucleotides of the pool are
displaced
from the stationary phase medium by any appropriate method, e.g., using an
ionic displacer,
and displaced oligonucleotides are collected. Typically for the various
methods of
displacement, the displacement can be performed in increasing stringent manner
(e.g., with
an increasing concentration of displacing agent, such as a salt concentration,
so that there is a
stepped or continuous gradient), such that oligonucleotides are displaced
generally in order of
increased binding affinity. .In many cases, a low stringency wash will be
performed to
remove weakly bound oligonucleotides, and one or more fractions will be
collected
containing displaced, tighter binding oligonucleotides. In some cases, it will
be desired to
select fractions that contain very tightly binding oligonucleotides (e.g.,
oligonucleotides in
fractions resulting from displacement by the more stringent displacement
conditions) for
further use.
[0046] Similarly, the invention provides a method for enriching
oligonucleotides from a
pool of oligonucleotides binding to at least one PrP, by contacting the pool
with one or more
PrP's, and amplifying oligonucleotides bound to the PrPs to provide an
enriched
oligonucleotide pool. The contacting and amplifying can be performed in
multiple rounds,
e.g., at least 1, 2, 3, 4, 5, 10, or more additional times using the enriched
oligonucleotide pool
from the preceding round as the pool of oligonucleotides for the next round.
The method can
also involve sequencing and testing anti-prion activity of oligonucleotides
'in the enriched
oligonucleotide pool following one or more rounds of contacting and
amplifying.
[0047] The method can involve displacing oligonucleotides from the PrP with
any of a
variety of teclnuques, such as those described above, e.g., using a
displacement agent. As
indicated above, it can be advantageous to select the tighter binding
oligonucleotides for
further use, e.g., in further rounds of binding and amplifying. The method can
further involve
selecting one or more enriched oligonucleotides, e.g., high affinity
oligonucleotides, for
further use. In particular embodiments, the selection can include eliminating
oligonucleotides that have sequences complementary to subject animal mRNA or
genomic
sequences for a particular prion disease of interest. Such elimination can
involve comparing
the oligonucleotide sequences) with sequences from the particular host in a
sequence
11
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
database(s), e.g., using a sequence alignment program (e.g., a BLAST search),
and
eliminating those oligonucleotides that have sequences identical or with a
particular level of
identity to a host sequence. Eliminating such host complementary sequences
and/or selecting
one or more oligonucleotides that are not complementary to host sequences can
also be done
for the other aspects of the present invention.
[0048] In the preceding methods for identifying, purifying, or enriching
oligonucleotides,
the oligonucleotides can be of types as described herein. The above methods
are
advantageous for identifying, purifying or enriching high affinity
oligonucleotides, e.g., from
an oligonucleotide randomer preparation.
[0049] In a related aspect, the invention concerns an anti-prion
oligonucleotide preparation
that includes one or more oligonucleotides identified using a method of any of
the preceding
methods for identifying, obtaining, or purifying anti-prion oligonucleotides
from an initial
oligonucleotide pool, where the oligonucleotides in the oligonucleotide
preparation exhibit
higher mean binding affinity with one or more PrP's than the mean binding
affinity of
oligonucletides in the initial oligonucleotide pool.
[0050] In particular embodiments, the mean binding affinity of the
oligonucleotides is at
least two-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater
than the mean
binding affinity of oligonucleotides in the initial oligonucleotide pool, or
even more; the
median of binding affinity is at least two-fold, 3-fold, 5-fold, 10-fold, 20-
fold, 50-fold, or
100-fold greater relative to the median of the binding affinity of the initial
oligo pool, where
median refers to the middle value.
[0051] In yet another aspect, the invention provides an anti-prion polymer mix
that includes
at least one anti-prion oligonucleotide and at least one non-nucleotide anti-
prion polymer. In
particular embodiments, the oligonucleotide is as described herein for anti-
prion
oligonucleotides and/or the anti-prion polymer is as described herein or
otherwise known in
the art or subsequently identified.
[0052] In yet another aspect, the invention provides an oligonucleotide
randomer, where the
randomer is at least 6 nucleotides in length. In particular embodiments the
randomer has a
length as specified above for anti-prion oligonucleotides; the randomer
includes at least one
phosphorothioate linkage; the randomer includes at least 50% phosphorothioate
linkages; the
randomer includes at least 80% phosphorothioate linkages; the randomer
includes all
12
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
phosphorothioate linkages; the randomer includes at least one
phosphorodithioate linkage or
other modification as listed herein; the randomer includes at least 20, 30,
40, 50, 60, 70, ~0,
or 90% modified linkages (e.g., of a type specified herein such as
phosphorothioate or
phosphorodithioate); the randomer oligonucleotides include at least one non-
randomer
segment (such as a segment complementary to a selected subject animal nucleic
acid
sequence), which can have a length. as specified above for oligonucleotides;
the randomer is
in a preparation or pool of preparations containing at least 5, 10, 15, 20,
50, 100, 200, 500, or
700 micromol, l, 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 mole of
randomer, or a
range defined by taking any two different values from the preceding as
inclusive end points,
or is synthesized at one of the listed scales or scale ranges.
[0053] Likewise, the invention provides a method for preparing anti-prion
randomers, by
synthesizing at least one randomer, e.g., a randomer as described above.
[0054] In yet another aspect, the invention provides a method for reducing
prion activity in
a biological material in oitro, by contacting the biological material with at
least one 'anti-prion
oligonucleotide, e.g., an anti-prion nucleotide as described herein.
[0055] In particular embodiments, the biological material is animal blood
(e.g., human,
bovine, or ovine blood); the biological materials is an animal blood product
(e.g., human,
bovine, or ovine blood product); the biological material is a mammalian tissue
(e.g., human,
bovine, or ovine tissue); the biological material is a mammalian organ (e.g.,
human, bovine,
or ovine organ).
[0056] In connection with modifying characteristics of an oligonucleotide by
linking or
conjugating with another molecule or moiety, the modifications in the
characteristics are
evaluated relative to the same oligonucleotide without the linked or
conjugated molecule or
moiety.
[0057] In the context of the present invention, unless specifically limited
the term
"oligonucleotide (ON)" means oligodeoxynucleotide (ODN) or
oligodeoxyribonucleotide or
oligoribonucleotide. Thus, "oligonucleotide" refers to an oligomer or polymer
of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term
includes
oligonucleotides composed of naturally-occurring nucleobases, sugars and
covalent
internucleoside (backbone) linkages as well as oligonucleotides having non-
naturally-
occurring portions which function similarly. Such modified or substituted
oligonucleotides
13
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
are often preferred over native forms because of desirable properties such as;
for example,
enhanced cellular uptake, enhanced affinity for a protein target and increased
stability in the
presence of nucleases. Examples of modifications that can be used axe
described herein.
Oligonucleotides that include backbone and/or other modifications can also be
referred to as
oligonucleosides.
[0058] The terms "prion", "prion protein", "infectious protein" and the like
are used
interchangeably herein to refer to the infectious PrPsc form of a PrP protein,
and is a
contraction of the words "protein" and "infection". Particles are comprised
largely, if not
exclusively, of PrPsc molecules encoded by a PrP gene. Prions are distinct
from bacteria,
viruses and viroids. Known prions infect animals to cause scrapie, a
transmissible,
degenerative disease of the nervous system of sheep and goats, as well as
bovine spongiform
encephalopathy (BSE), or "mad cow disease, "and feline spongiform
encephalopathy in cats.
Four prion diseases known to affect humans are: (1) kuru, (2) Creutzfeldt-
Jakob Disease
(CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal
familian insomnia
(FFI) (also referred to as fatal insomnia (FI)). Variant CJD (vCJD) is also
known, and is
related to human ingestion of material from animals infected with BSE.
[0059] As used herein "prion" includes all forms of prions causing all or any
of these
diseases or other diseases of similar pathology in any animals and in
particular in humans and
domesticated farm animals.
[0060] The terms "PrP protein", "PrP" and like are used interchangeably herein
and shall
mean both the infectious particle form PrPsc known to cause diseases
(spongiform
encephalopathies) in humans and animals and the noninfectious form PrPc which,
under
appropriate conditions is converted to the infectious PrPsc form.
[0061] The term "PrP gene" is used herein to describe genetic material which
encodes PrP
proteins including those with polymorphisms and pathogenic mutations (a number
of which
are known). The term "PrP gene" refers generally to any gene of any species
which encodes
any form of a prion protein.
[0062] As used herein in connection with anti-prion action of a material, the
phrase
"sequence independent mode of action" indicates that the mechanism by which
the material
exhibits an anti-prion effect is not due to hybridization of complementary
nucleic acid
sequences, e.g., an antisense effect. Furthermore, this term also implies that
the mechanism
14
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
of action is not due to a sequence dependent aptamer interaction with prion
proteins.
Conversely, a "sequence dependent mode of action" means that the anti-prion
effect of a
material involves hybridization of complementary nucleic acid sequences or the
specific
binding of a nucleic acid derived from its specific sequence. It also
describes a sequence
specific aptameric interaction between a nucleic acid sequence and a protein.
[0063] As used herein in connection with oligonucleotides or other materials,
the term
"anti-prion" refers to an effect which occurs in the presence of
oligonucleotides or other
agents which inhibit prion diseases by reducing or inhibiting the conversion
of PrPc to PrPsc
and/or reducing or inhibiting the accumulation of intracellular PrP or PrPsc
and/or PrPsc
aggregation into amyloid plaques and/or reducing the internalization of prion
protein andlor
reducing or inhibiting cell death induced by conversion of PrP or accumulation
of PrPsc.
[0064] The term "anti-prion oligonucleotide formulation" refers to a
preparation that
includes at least one anti-prion oligonucleotide that is adapted for use as an
anti-prion agent.
The formulation includes the oligonucleotide or oligonucleotides, and can
contain other
materials that do not interfere with use of this oligonucleotide as an anti-
prion agent ifz vivo.
Such other materials can include without restriction diluents, excipients,
carrier materials,
delivery systems and/or other anti-prion materials.
[0065] As used herein, the term "pharmaceutical composition" refers to an anti-
prion
oligonucleotide formulation that includes a physiologically or
pharmaceutically acceptable
caxrier or excipient. Such compositions can also include other components that
do not make ,
the composition unsuitable for administration to a desired subject, e.g., a
human. Typically
the composition is sufficiently sterile to be acceptable to a reasonable
medical practitioner for
administration to a human subject.
(0066] As used in connection with an anti-prion formulation, pharmaceutical
composition,
or other material, the phrase "adapted for use as an anti-prion agent"
indicates that the
material exhibits an anti-prion effect and does not include any component or
material that
makes it unsuitable for use in inhibiting a prion-associated disease in an ifa
vivo system, e.g.,
for administering to a subject such as a human subject.
[0067] As used herein in connection with administration of an anti-prion
material, the term
"subject" refers to a living higher organism, including, for example, animals
such as
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
mammals, e.g.,, humans, non-human primates, bovines, porcines, ovines,
equines, canines,
felines and birds.
[0068] In the present application, the term "randomer" is intended to mean a
single stranded
DNA having a wobble (I~ at every position, such as . Each base is
synthesized as a wobble such that this ON actually exists as a population of
different
randomly generated sequences of the same size.
[0069] As used herein in connection with oligonucleotide sequences, the term
"random"
characterizes a sequence or an ON that is not complementary to a mRNA of the
animal
subject to the particular prion disease of interest, and which is selected to
not form hairpins
and not to have palindromic sequences contained therein. When the term
"random" is used in
the context of anti-prion activity of an oligonucleotide toward a particular
prion disease, it
implies the absence of complementarity to a mRNA of animals subject to that
particular prion
disease.
[0070] The phrase "derived from a genome of a subject animal" indicates that a
particular
sequence has a nucleotide base sequence that has at least 85% identity to a
nucleotide
sequence of an animal subject to the particular prion disease, or its
complement, or is a
corresponding RNA sequence. In particular embodiments, the identity is at
least 90, 95, 98,
99, or 100%.
[0071] As used herein, the term "delivery system" refers to a component or
components
that, when combined with an oligonucleotide as described herein, increases the
amount of the
oligonucleotide that contacts the intended location in vivo, and/or extends
the duration of its
presence at the target, e.g., by at least 10, 20, 50, or 100%, or even more as
compared to the
amount and/or duration in the absence of the delivery system, and/or prevents
or reduces
interactions that cause side effects.
[0072] The term "therapeutically effective amount" refers to an amount that is
sufficient to
effect a therapeutically or prophylactically significant reduction, in prion
accumulation or
prion activity when administered to a typical subject of the intended type. In
aspects
involving administration of an anti-prion oligoriucleotide to a subj ect,
typically the
oligonucleotide, formulation, or composition should be administered in a
therapeutically
effective amount.
16
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0073] As used herein in connection with anti-prion oligonucleotides and
formulations, and
the like, in reference to a particular prion disease the term "targeted"
indicates that the
oligonucleotide is selected to inhibit development and/or aggregation of
PrPsc, and/or
development andlor progress of that particular prion disease. As used in
connection with a
particular tissue or cell type, the term indicates that the oligonucleotide,
formulation, or
delivery system is selected such that the oligonucleotide is preferentially
present and/or
preferentially exhibits an anti-prion effect in or proximal to the particular
tissue or cell type.
[0074] As used in connection with the present oligos, the term "TG-rich"
indicates that the
sequence of the anti-prion oligonucleotide consists of at least 70 percent T
and G nucleotides,
or if so specified, at least 80, 90, or 95% T and G, or even 100%.
Selected abbreviations
ON: Oligonucleotide
ODN: Oligodeoxynucleotide
PS: Phosphorothioate
TSE: Transmissible spongifonn encephalopathies
PrPsc: Abnormal isoform prion protein
PrPc: Normal host encoded prion protein
CJD: Creutzfeldt-Jacob Disease
BSE: Bovine spongifonn encephalopathy
CNS: Central Nervous System
[0075] Additional aspects and embodiments will be apparent from the following
Detailed
Description and from the claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. General
[0076] The present invention is concerned with the identification and use of
anti-prion ONs
that act by a sequence independent mechanism, and includes the discovery that
the anti-prion
activity is greater for larger ONs that are 10 bases in length; typically 20
bases or more in
length; and more preferably 40 and more bases in length (e.g., 20-60, 40-80,
60-100, 80-120.
17
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0077] As demonstrated by the results in Example 1, the anti-prion effect of
random PS-
ONs is not sequence specific or due to the action of an aptamer. Considering
the volumes
and concentrations of PS-ONs used in those tests, it is theoretically unlikely
that a particular
sequence is present at more than 1 copy in the mixture. This means than there
can be no
antisense or aptameric effect in these PS-ONs randomers. In all examples,
should the anti-
prion effect be caused by the sequence-specificity of the PS-ONs, such effect
would thus
have to be caused by only one molecule, a result that does not appear
plausible. For example,
for an ON randomer 40 bases in length, any particular sequence in the
population would
theoretically represent only 1/44° or .27X10-25 of the total fraction.
Given that 1 mole =
6.022X1023 molecules, and the fact that our largest synthesis is currently
done at the 15
micromole scale, all possible sequences will not be present and also, each
sequence is present
most probably as only one copy. Without limitation, a non-sequence dependent
mode of
action can be demonstrated by satisfying either Test 1 or Test 2 in Example 2.
[0078] Of course, one skilled in the art applying the teaching of the present
invention could
also use sequence specific ONs, but utilize the sequence independent activity
discovered in
the present invention. Accordingly, the present invention is not to be
restricted to sequence
independent ONs.
[0079] In the present invention, randomers (or other ONs) may inhibit prion
diseases by
several mechanisms, including but not limited to the following: inhibiting the
conversion of
PrPc to PrPsc, inhibiting the assembly of PrPsc, inhibiting the formation of
amyloid plaques,
inhibiting internalization of PrPc or PrPsc, rendering PrPsc sensitive to
intra or extracellular
proteases, preventing the precipitation of PrPsc and/or preventing the
polymerization of
PrPsc. While the preceding are suggested are potential mechausms, the present
invention is
not limited thereby.
II. Anti-prion ONs
[0080] According to the conclusions discussed above and the data reported
herein, ONs,
e.g., ON randomers such as ODN randoiners, have activity against the various
types of prion
disease.
18
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Chemical factors for inhibition of prion activity
[0081] In Example 1~ it is shown that PS modified ODN randomers exhibit potent
anti-
prion activity. This observation indicates that the anti-prion activity is
involves the protein
binding ability of the ON randomer.
[0082] One skilled in the art applying the teaching of the present invention
can also use
ONs with different chemical modifications. A modification of the ON, such as,
but not
limited to a PS modification, appears to be beneficial for anti-prion
activity. This is most
likely due to the effects of charge of ONs and/or to the requirement for
stabilization of
nucleic acids, e.g., DNA, both in the media and intracellularly, and/or the
fact that thioated
linkages promote protein binding. In addition, a specific chirality of each
thioated linkage (R
versus P) may also be important for PS-ON randomer anti-prion activity.
Design of non sequence-specific ONs
[0083] It can also be advantageous to design or select anti-prion ONs
demonstrating low
(preferably the lowest possible) homology with the human (or other subj ect
organism)
genome. The goal is to obtain an ON that will show the lowest toxicity due to
interactions
with human or animal genome sequences) and mRNAs. The first step is to produce
the
desired length sequence of the ON, e.g., by aligning nucleotides A, C, G, T in
a random
fashion, manually or, more commonly, using a computer program. The second step
is to
compare the ON sequence with a library of human sequences such as GenBank
and/or the
Ensemble Human Genome Database. The sequence generation and comparison can be
performed repetitively, if desired, to identify a sequence or sequences having
a desired low
homology level with the subject genome. It is desireable for the ON sequence
to have the
lowest homology possible with the entire genome or with mRNAs from the
organism, while
also minimizing self interaction. The last step is to test the ON in a prion
assay using the
suitable encapsulation to obtain anti-prion activity.
ONs combining non sequence-specific sequence with antisense sequence
[0084] In certain applications it can be desirable to couple a non-sequence
specific ON
sequence portions) with an antisense sequence portions) to increase the
activity of the final
ON. The non-sequence specific portion of the ONs is described in the present
invention. The
19
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
antisense portion is complementary to a mRNA of a gene involved in prion
disease. One aim
of this ON is to lower the expression of the PrP gene by combining a portion
complementary
to the mRNA of the Prp gene to the ON described herein.
ONs combining non-sequence-specific sequence with G-rich sequence
[0085] In another approach, non-sequence specific sequence portions) is/are
coupled with
a G-rich motif ON portions) to improve the activity of the final ON. The non-
specific
portion of the ON is described in the present invention. The G-rich motif
portion can, as non-
limiting examples, include, CpG, Gquartet, and/or CG that are described in the
literature as
stimulators of the immune system.
Non-Watson-Crick ONs
[0086] It can also be beyeficial to use an ON composed of one or more types of
non-
Watson-Crick nucleotides/nucleosides. Such ONs can mimic PS-ONs and other
modifications with some of the following characteristics similar to PS-ONs: a)
the total
charge; b) the space between the units; c) the length of the chain; d) a net
dipole with
accumulation of negative charge on one side; e) the ability to bind to
proteins f) the ability to
be encapsulate with delivery systems, h) an acceptable therapeutic index, i)
an anti-prion
activity. The ON has a preferred phosphorothioate backbone but is not limited
to it.
Examples of non-Watson-Crick nucleotides/nucleosides are described in Kool,
2002, Acc.
CIZen2. Res. 35:936-943; and Takeshita et al., (197) J. Biol. Chem. 262:10171-
10179 where
ONs containing synthetic abasic sites are described.
Linked ONs
[0087] In certain embodiments, ONs of the invention are modified in a number
of ways
without compromising their anti-prion activity. For example; the ONs are
linked or
conjugated, at one or more of their nucleotide residues, to another moiety.
Thus, modification
of the oligonucleotides of the invention can involve chemically linking to the
oligonucleotide
one or more moieties or conjugates which enhance the activity, cellular
distribution or
cellular uptake, increase transfer across cellular membranes specifically or
not, or protecting
against degradation or excretion, or providing other advantageous
characteristics. Such
advantageous characteristics can, for example, include lower serum
interaction, higher PrPsc
interaction, the ability to be formulated for delivery, a detectable signal,
improved
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
pharmacokinetic properties, and lower toxicity: Such conjugate groups can be
covalently
bound to functional groups such as primary or secondary hydroxyl groups. For
example,
conjugate moieties can include a steroid molecule, a non-aromatic lipophilic
molecule, a
peptide, cholesterol, bis-cholesterol, an antibody, PEG, a protein, a water
soluble vitamin, a
lipid soluble vitamin, another ON, or any other molecule improving the
activity and/or
bioavailability of ONs.
[0088] In greater detail, exemplary conjugate groups of the invention can
include
intercalators, reporter molecules, polyamines, polyamides, polyethylene
glycols, polyethers,
SATE, t-butyl-SATE, groups that enhance the pharmacodynamic properties of
oligomers,
and groups that enhance the pharmacokinetic properties of oligomers. Typical
conjugate
groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate,
phenanthridine,
anthraquinone, acridine, fluoresceins, rhodamines, coumarins, fluorescent
nucleobases, and
dyes.
[0089] Groups that enhance the pharmacodynamic properties, in the context of
this
invention, include groups that improve oligomer cellular uptake andlor enhance
oligomer
resistance to degradation and/or protect against serum interaction. Groups
that enhance the
pharmacokinetic properties, in the context of this invention, include groups
that improve
oligomer uptake, distribution, metabolism or excretion. Exemplary conjugate
groups are
described in International Patent Application PCT/IJS92/09196, filed Oct. 23,
1992, which is
incorporated herein by reference in its entirety.
[0090] Conjugate moieties can include but are not limited to lipid moieties
such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86,
6553-6556), cholic
acid (Manoharan et al., Bioorg. Med. C7zem. Let., 1994, 4, 1053-1060), a
thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acid. Sci., 1992, 660, 306-
309; Manoharan et
al., Bioo~g. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol
(Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues
(Saison-Behmoaras et at., EMBOJ., 1991, 10, 1111-1118; Kabanov et al.,
FEBSLett., 1990,
259, 327-330; Svinarchuk et at., Biochirnie, 1993, 75, 49-54), a phospholipid,
e.g., di-
hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-
phosphonate (Manoharan et at., Tetrahedf-ora Lett., 1995, 36, 3651-3654; Shea
et al., Nucl.
Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain
(Manoharan et
at., Nucleosides ~ Nucleotides, 1995, 14, 969-973), or adamantine acetic acid
(Manoharan et
21
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
at., TetYahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et
at., Biochim.
Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or
hexylaminocarbonyl-
oxycholesterol moiety (Crooke et al., J. Pharmacol Exp. They., 1996, 277, 923-
937.
[0091] The present oligonucleotides may also be conjugated to active drug
substances, for
example without limitation, aspirin, warfarin, phenylbutazone, ibuprofen,
suprofen, fenbufen,
ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-
triiodobenzoic acid,
flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a
diazepine, indomethicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial
or an antibiotic.
[0092] Exemplary U.S. patents that describe the preparation of exemplary
oligonucleotide
conjugates include, for example, U.S. Pat. Nos. 4,828,979; 4,948,882;
5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;
5,109,124;
5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;
4,587,044;
4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582;
4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022;
5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,
5,391,723;
5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941, each of
which is incorporated by reference herein in its entirety.
[0093] Another approach is to prepare anti-prion ONs as lipophilic pro-
oligonucleotides by
modification with enzymatically cleavable charge neutralizing adducts such as
s-acetylthio-
ethyl or s-pivasloylthio-ethyl (Vives et al., 1999, Nucl Acids Res 27: 4071-
4076). Such
modifications have been shown to increase the uptake of ONs into cells.
Oligonucleotide Modifications and Synthesis
[0094] As indicated above, modified oligonucleotides are useful in this
invention. Such
modified oligonucleotides include, for example, oligonucleotides containing
modified
backbones or non-natural intemucleoside linkages. Oligonucleotides having
modified
backbones include those that retain a phosphorus atom in the backbone and
those that do not
have a phosphorus atom in the backbone.
[0095] Such modified oligonucleotide backbones include, for example,
phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotri-
22
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
esters, methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene
phosphonates and chiral phosphonates, phosphinates, phosphoramidates including
3'-amino
phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, selenaphosphates,
carboranyl
phosphate and borano-phosphates having normal 3'-5' linkages, 2'-5' linked
analogs of
these, and those having inverted polarity wherein one or more internucleotide
linkages is a 3'
to 3', S' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity
typically include
a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single
inverted nucleoside
residue which may be abasic (the nucleobase is missing or has a hydroxyl group
in place
thereof). Various salts, mixed salts and free acid forms are also included.
[0096] Preparation of oligonucleotides with phosphorus-containing linkages as
indicated
above are described, for example, in U.S. Pat Nos. 3,687,808; 4,469,863;
4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;
5,321,131;
5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;
5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;
5,527,899;
5,721,218; 5,672,697 and 5,625,050, each of which is incorporated by reference
herein in its
entirety.
[0097] Some exemplary modified oligonucleotide backbones that do not include a
phosphorus atom have backbones that are formed by short chain alkyl or
cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages,
or one or more short chain heteroatomic or heterocyclic internucleoside
linkages. These
include those having morpholino linkages (formed in part from the sugar
portion of a
nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;
fonnacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones;
riboacetyl
backbones; alkene containing backbones; sulfamate backbones; methyleneimino
and
methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide
backbones; and
others having mixed N, 0, S and CHZ component parts. Particularly advantageous
are
backbone linkages that include one or more charged moieties. Examples of U.S.
patents
describing the preparation of the preceding oligonucleotides include U.S. Pat.
Nos.
5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;
5,264,564;
5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489, 677; 5,541,307; 5,561,225;
5,596,086;
5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312;
23
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is
incorporated by
reference herein in its entirety.
[0098] Modified oligonucleotides may also contain one or more substituted
sugar moieties.
For example, such oligonucleotides can include one of the following 2'-
modifications: OH;
F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-
alkyl,
wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1
to Clo allcyl or
Cz to Clo alkenyl and alkynyl, or 2'-O-(O-carboran-1-yl)methyl. Particular
examples are
O[(CHz)n0]",CH3, O(CHz)~OCH3, O(CHz)"NHz, O(CHz)"CH3, O(CHz)"ONHz, and
O(CHz)"ON [(CHz)"CH3)~z, where n and m axe from 1 to 10. Other exemplary
oligonucleotides include one of the following 2'-modifications: C1 to Clo
lower alkyl,
substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-
aralkyl, SH, SCH3,
OCN, Cl, Br, CN, CF3. OCF3, SOCH3, SOZCH3, ONOz, NOz, Ns, NHz,
heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a
reporter group, an
intercalator, a group for improving the pharmacokinetic properties of an
oligonucleotide, or a
group for improving the pharmacodynamic properties of an oligonucleotide.
Examples
include 2'-methoxyethoxy (2'-O--CH2CHZOCH~, also known as 2'-O-(2-
methoxyethyl) or
2'-MOE) (Martin et al., Helv. Claim. Acta, 1995, 78, 486-504) i.e., an
alkoxyalkoxy group;
2'-dimethy-laminooxyethoxy, i.e., a O(CHz)zON(CH3)z group, also known as 2'-
DMAOE;
and 2'-dimethylaminoethoxyethoxy (also known as 2'-O- dimethylaminoethoxyethyl
or 2'-
DMAEOE), i.e., 2'-O-CHz-O-CHz-N(CHz)z.
[0099] Other modifications include Locked Nucleic Acids (LNAs) in which the 2'-
hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby
forming a
bicyclic sugar moiety. The linkage can be a methelyne (-CHz-)~ group bridging
the 2'
oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation
thereof are
described in WO 98/39352 and WO 99/14226, which are incorporated herein by
reference in
their entireties.
[0100] Other modifications include sulfur-nitrogen bridge modifications, such
as locked
nucleic acid as described in Orum et al. (2001) Gury. Opifz. Mol. Then. 3:239-
243.
[0101] Other modifications include 2'-methoxy (2'-O-CH3), 2'-methoxyethyl (2'O-
CHz-
CH3 ), 2'-aminopropoxy (2'-OCHzCHzCHzNHz), 2'-allyl (2'-CHz-CH=CHz), 2'-O-
allyl
(2'-O-CHz-CH=CHz) and 2'-fluoro (2'-F). The 2'-modification may be in the
arabino (up)
position or ribo (down) position. Similar modifications may also be made at
other positions
24
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
on the oligonucleotide, particularly the 3' position of the sugar on the 3'
terminal nucleotide
or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal
nucleotide.
Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in
place of the
pentof~iranosyl sugar. Exemplary U.S. patents describing the preparation of
such modified
sugar structures include, for example, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080;
5,359,044; 5,393, 878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567, 811;
5,576,427;
5,591,722; 5,597,909; 5,610,300; 5,627, 053; 5,639,873; 5,646,265; 5,658,873;
5,670,633;
5,792, 747; and 5,700,920, each of which is incorporated by reference herein
in its entirety.
[0102] Still other modifications include an ON concatemer consisting of
multiple
oligonucleotide sequences joined by a linker(s). The linker may, for example,
consist of
modified nucleotides or non-nucleotide units. In some embodiments, the linker
provides
flexibility to the ON concatemer. Use of such ON concatemers can provide a
facile method
to synthesize a final molecule, by joining smaller oligonucleotide building
blocks to obtain
the desired length. For example, a 12 carbon linker (C12 phosphoramidite) can
be used to
join two or more ON concatemers and provide length, stability, and
flexibility.
[0103] As used herein, "unmodified" or "natural" bases (nucleobases) include
the purine
bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),
cytosine (C) and
uracil (L~. Oligonucleotides may also include base modifications or
substitutions. Modified
bases include other synthetic and naturally-occurring bases such as 5-
methylcytosine (5-me-
C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl
and other
alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives
of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-
propynyl(-C---C-CH3) uracil and cytosine and other alkynyl derivatives of
pyrimidine
bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-
thiouracil, 8-halo, 8-
arriino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo
particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-
methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine
and 8-
azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-
deazaadenine.
Additional modified bases include tricyclic pyrimidines such as phenoxazine
cytidine(1H-
pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-
pyrimido[5,4-
b] [1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine
cytidine (e.g. 9-
(2-aminoethoxy)-H-pyrimido [5,4-b][1,4]benzoxazin-2(3H)-one), carbazole
cytidine (2H-
pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-
pyrido[3',2':4,5]pyrrolo[2,3-
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
d]pyrimidin-2-one). Modified bases may also include those in which the purine
or
pyrimidine base is replaced with other heterocycles, for example 7-deaza-
adenine, 7-
deazaguanosine, 2-aminopyridine and 2-pyridone.. Further nucleobases include
those
described in U.S. Pat. No. 3,687,808, those disclosed in The Concise
Encyclopedia Df
Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John
Wiley & Sons,
1990, those disclosed by Englisch et al., Angewandte Chemie, International
Edition, 1991, 30,
613, and those disclosed by Sanghvi, Y. S., Chapter 1 S, Antisense Research
and Applications,
pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
[0104] Another modification includes phosphorodithioate linkages. Knowing that
phosphorodithioate ODNs (PS2-ODNs) and PS-ODNs have a similar binding affinity
to
proteins (Tonkinson et al. (1994) Antisense Res. Dev. 4 :269-278)(Cheng et al.
(1997) J. Nlol.
Recogn. 10:101-107) and knowing that a possible mechanism of action of ONs is
binding to
PrP, it could be desirable to include phosphorodithioate linkages on the anti-
prion ONs
described in this invention.
[0105] Another approach to modify ONs is to produce stereodefined or stereo-
enriched
ONs as described in Yu at al (2000) Bioorg. Med. Chem. 8:275-284 and in
Inagawa et al.
(2002) FEBSLett. 25:48-52. ONs prepared by conventional methods consist of a
mixture of
diastereomers by virtue of the asymmetry around the phosphorus atom involved
in the
internucleotide linkage. This may affect the stability of the binding between
ONs and PrP's.
Previous data showed that protein binding is significantly stereo-dependent
(Yu et al.). Thus,
using stereodefined or stereo-enriched ONs could improve their protein binding
properties
and improve their anti-prion efficacy. In particular embodiments, the
enrichment is at least 2-
fold, 4-fold, 6-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold or
even more.
[0106] The incorporation of modifications such as those described above can be
utilized in
many different incorporation patterns and levels. That is, a particular
modification need not
be included at each nucleotide or linkage in an oligonucleotide, and different
modifications
can be utilized in combination in a single bligonucleotide, or even in a
single nucleotide.
Oligonucleotide Synthesis
[0107] The present oligonucleotides can by synthesized using methods known in
the art.
For example, unsubstituted and substituted phosphodiester (P=O)
oligonucleotides can be
synthesized on an automated DNA synthesizer (e.g., Applied Biosystems model
380B) using
26
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
standard phosphoramidite chemistry with oxidation by iodine. Phosphorothioates
(P=S) can
be synthesized as for the phosphodiester oligonucleotides except the standard
oxidation bottle
can be replaced by 0.2 M solution of 311-1,2-benzodithiole-3-one 1,1-dioxide
in acetonitrile
for the step-wise thioation of the phosphite linkages. The thioation wait step
can be increased
to 68 sec, followed by the capping step. After cleavage from the CPG column
and deblocking
in concentrated ammonium hydroxide at 55°C. (18 h), the
oligonucleotides can be purified by
precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCI solution.
[0108] Phosphinate oligonucleotides can be prepared as described in U.S. Pat.
No.
5,508,270; alkyl phosphonate oligonucleotides can be prepared as described in
U.S. Pat. No.
4,469,863; 3'-Deoxy-3'-methylene phosphonate oligonucleotides can be prepared
as
described in U.S. Pat. Nos. 5,610,289 and 5,625,050; phosphoramidite
oligonucleotides can
be prepared as described in U.S. Pat. No. 5,256,775 and U.S. Pat. No.
5,366,878;
alkylphosphonothioate oligonucleotides can be prepared as described in
published PCT
applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and
WO
94/02499, respectively); 3'-Deoxy-3'-amino phosphoramidate oligonucleotides
can be
prepared as described in U.S. Pat. No. 5,476,925; Phosphotriester
oligonucleotides can be
prepared as described in U.S. Pat. No. 5,023,243; borano phosphate
oligonucleotides can be
prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198;
methylenemethylimino
linked oligonucleotides, also identified as MMI linked oligonucleotides,
methylenedimethyl-
hydrazo linked oligonucleotides, also identified as MDII linked
oligonucleotides, and
methylenecarbonylamino linked oligonucleotides, also identified as amide-3
linked
oligonucleotides, and methyleneaminocarbonyl linked oligo-nucleotides, also
identified as
amide-4 linked oligonucleo-sides, as well as mixed backbone compounds having,
for
instance, alternating MMI and P=O or P=S linkages can be prepared as described
in U.S. Pat.
Nos. 5,378, 825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289; formacetal and
thioformacetal linked oligonucleotides can be prepared as described in U.S.
Pat. Nos.
5,264,562 and 5,264,564; and ethylene oxide linked oligonucleotides can be
prepared as
described in U.S. Pat. No. 5,223,618. Each of the cited patents and patent
applications is
incorporated by reference herein in its entirety.
Concurrent use of anti-prion polymers with inhibition of PrP expression
[0109] The present oligonucleotides, e.g., ONs, can also be used concurrently
with an agent
that inhibits expression of PrPc. As known in the art, a variety of different
types of inhibitors
27
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
can be used, including, for example, ribozymes or other catalytic nucleic acid
molecules,
antisense, triple helix, and RNAi (e.g., using siRNA or shRNA which can be
prepared
synthetically or can be expressed intracellularly). RNAi, and specifically
siRNA is described
in numerous references, including for example, Fire et al., U.S. Pat.
6,506,559, issued Jan 14,
2003; Graham et al., U.S. Patent 6,573,099, issued June 3, 2003; Zernicka-
Goetz et al.; US
publ. 20030027783, published 2/6/03; appl. 10/150,426, filed 5/17/02; Tuschl
et al. (1)
published appl. 20020086356, appl. 09/821,832, filed 3/30/01, each of which is
incorporated
herein by reference in its entirety.
[0110] In such a concurrent approach, the anti-prion oligonucleotides and the
PrPc
expression inhibitor can be delivered together or separately, which can be by
the same or
different delivery routes and/or methods.
Polymers with prion inhibition properties
[0111] Another approach is to use a polymer mimicking the activity of ONs
described in
the present invention and encapsulate it with suitable delivery system in
order to provide
inhibition of prion activity. As described in the literature, several anionic
polymers were
shown to bind to proteins. These polymers belong to several classes: (1)
sulfate esters of
polysaccharides (dextrin and dextran sulfates, cellulose sulfate); (2)
polymers containing
sulfonated benzene or naphthalene rings and naphthalene sulfonate polymers;
(3)
polycarboxylates (acrylic acid polymers); and acetyl phthaloyl cellulose
(Neurath et al.
(2002) BMC Izzfect Dis 2:27); and (4) abasic oligonucleotides (Takeshita et
al., 1987, .I. Biol.
Chezzz. 262:10171-10179). Other examples of non-nucleotide protein binding
polymers are
described in the literature. The polymers described herein mimic ONs described
in this
invention and have the following characteristics similar to ONs: a) the length
of the chain; b)
a net dipole with accumulation of negative charge on one side; c) the ability
to bind to
proteins; d) the ability to be encapsulated by a delivery system, e) an
acceptable therapeutic
index, f) an anti-prion activity. In order to mimic the effect of an ON, the
anti-prion polymer
may preferably be a polyanion displaying similar space between its units as
compared to a
PS-ON. It may also have the ability to penetrate cells with a delivery system.
[0112] It may also be to possible to modify polymers which normally do not
have a anionic
character, for instance polyethylene imine, by the incorporation of sulfur and
or oxygen and
or other modifications which result in the conversion of the resultant polymer
from a neutral
or cationic polymer into a polyanion. This technique could be applied to any
and all suitable
28
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
polymers. Since we have evidence that the polyanionic nature of PS-ON
randomers forms
the basis of their anti-prion activity, we believe that any particular
molecule with a
polyanionic character (e.g., carbohydrate polymers or oligonucleotides) will
have anti-prion
activity.
Anti-prion activity of double-stranded ONs
[0113] According to our results described herein, an approach is to use double
stranded
ONs as effective anti-prion agent with or without an encapsulating agent to
deliver it.
Preferentially such ONs have a phosphorothioate backbone but may also have
other /
additional modifications which improve their pharmacokinetic behaviour and/or
anti-prion
activity and/or stability as described herein for single stranded ONs.
III. Treatment of Blood and Blood Products.
[0114] Conventional antiseptic compositions and antiseptic methodologies are
generally
insufficient for inactivating infectious proteins such as prions. Although
prions can be
inactivated by relatively high temperatures over very long periods of time,
the temperature
ranges and time periods generally used to kill bacteria and inactivate the
viruses are.
insufficient to inactivate prions. Temperature treatment may also alter or
destroy required
characteristics of blood and blood products.
[0115] Thus, the present invention also concerns the use of the ONs and
polyanions
described herein in methods to treat blood and blood products prior to
transfer to a human or
animal. Application of the ON or polyanion of the invention can render prions
non-infectious
and/or prevent prion formation and/or aid in the denaturation of prions from
blood and blood
products. An important aspect of the invention is that the active component be
able to
eliminate infectivity or denature an infectious protein such as PrP under
relatively mild
conditions in order to conserve the desired blood characteristics. The
protocol for treatment
includes a step where the ON or polyanion is put in contact with the blood or
blood product
for a determined amount of time. This treatment may also be done on whole
blood prior to
blood product processing steps or during any processing steps. ONs may also be
used in
combination with other physical or chemical blood treatments such as
temperature, radiation, .
and aseptic compositions.
29
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0116] Similarly, ONs or polyanions as described herein can be used to treat
tissue or
organs to be transplanted to humans or animals.
[0117] Likewise, immobilizd ONs or polyanions can be used in a method of for
removal of
PrP proteins from blood or blood products. The ON or polyanion immobilized on
a solid
phase support or membrane common in a variety of purification procedures can
be used to
remove prions from a biological material. A number of methods for use in the
present
invention are summarized as follows.
Methods of Purification .
[Oi 18] Another method that may be used to remove prions from a biological
sample
involves filtration. through a membrane. The membrane may have the ON or
polyanion
conjugated directly to the membrane or alternatively, the ~ON or polyanion may
be
compartmentalized in an area behind the membrane, which is inaccessible to the
larger
components of the biological materials, e. g. blood cells. In the latter
example, the ON or
polyanion cari be bound to an insoluble matrix behind the membrane. Suitable
materials for
the membrane include without limitation regenerated cellulose, cellulose
acetate, non-woven
acrylic copolymer, polysulphone, polyether sulphone, polyacrylonitrile,
polyamide and the
like. The ON or polyanion is immobilized in the pores and/or on the surface of
the side of the
membrane that faces away from the biological fluid.
[0119] Alternatively, the ON may be bound to a solid matrix and used on an
affinity
chromatography column. A number of matrices may be employed in the preparation
of
columns. Such matrices can include without limitation beads, and more
preferably spherical
beads, which serve as a support surface for the complexing agent of the
invention. Suggested
materials for the matrices include without limitation agarose, cross-linked
dextran,
polyhydroxyl ethyl methacrylate, polyacrylamide, polyurethane, cellulose,
cellulose acetate
and derivatives or combinations thereof. Those skilled in the use of such
materials are
familiar with techniques for binding or linking oligonucleotides and polymers
as described
herein to such matrices, and such techniques can be used with the present
invention.
References corresponding to abbreviated citations in text.
Adjou KT, Simoneau S, Sales N, Lamoury F, Dormont D, Papy-Garcia D, Barritault
D,
Deslys JP, Lasmezas CI. A novel generation of heparan sulfate mimetics for the
treatment of
prion diseases. J Gen Virol. 2003 Sep;84(Pt 9):2595-603.
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Banks WA, Farr SA, Butt W, Kumar VB, Franko MW, Morley JE. Delivery across the
blood-brain barrier of antisense directed against amyloid beta: reversal of
learning and
memory deficits in mice overexpressing amyloid precursor protein.. J Pharmacol
Exp Ther.
2001 Jun;297(3):1113-21.
Barret A, Tagliavini F, Forloni G, Bate C, Salmona M, Colombo L, De Luigi A,
Limido L,
Suardi S, Rossi G, Auvre F, Adjou KT, Sales N, Williams A, Lasmezas C, Deslys
JP.
Evaluation of quinacrine treatment for prion diseases. J Virol. 2003
Aug;77(15):8462-9.
Bate C, Salinona M, Diomede L, Williams A. Squalestatin cures prion-infected
neurons and
protects against prion neurotoxicity. J Biol Chem. 2004 Apr 9;279(15):14983-
90.
Brigger I, Morizet J, Aubert G, Chacun H, Terrier-Lacombe MJ, Couvreur P,
Vassal
Polyethylene glycol)-coated hexadecylcyanoacrylate nanospheres display a
combined effect
for brain tumor targeting. J Pharmacol Exp Ther. 2002 Dec;303(3):928-36.
Caughey B, Raymond GJ. Sulfated polyanion inhibition of scrapie-associated PrP
accumulation in cultured cells.) Virol. 1993 Feb;67(2):643-50.
Doh-ura K, Ishikawa K, Murakami-Kubo I, Sasaki K, Mohri S, Race R, Iwaki T.
Treatment
of transmissible spongiform encephalopathy by intraventricular drug infusion
in animal
models. J Virol. 2004 May;78(10):4999-5006.
Grigoriev VB, Adjou KT, Sales N, Simoneau S, Deslys JP, Seman M, Dormont D,
Fournier
JG. Effects of the polyene antibiotic derivative MS-8209 on the astrocyte
lysosomal system
of scrapie-infected hamsters. J Mol Neurosci. 2002 Jun;l8(3):271-81.
Huwyler J, Wu D, Pardridge WM. Brain drug delivery of small molecules using
immunoliposomes.Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14164-9.
Kocisko DA, Baron GS, Rubenstein R, Chen J, Kuizon S, Caughey B. New
inhibitors of
scrapie-associated prion protein formation in a library of 2000 drugs and
natural products. J
Virol. 2003 Oct;77(19):10288-94.
31
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Koster T, Singh K, Zimmermann M, Gruys E. Emerging therapeutic agents for
transmissible
spongiform encephalopathies: a review. J Vet Pharmacol Ther. 2003
Oct;26(5):315-26.
Nakajima M, Yamada T, Kusuhara T, Furukawa H, Takahashi M, Yamauchi A, Kataoka
Y.
Results of quinacrine administration to patients with Creutzfeldt-Jakob
disease. Dement
Geriatr Cogn Disord. 2004;17(3):158-63.
Omori N, Maruyama K, Jin G, Li F, Wang SJ, Hamakawa Y, Sato K, Nagano I, Shoji
M,
Abe K. Targeting of post-ischemic cerebral endothelium in rat by liposomes
bearing
polyethylene glycol-coupled transferrin. Neurol Res. 2003 Apr;25(3):275-9.
Poli G, Ponti W, Carcassola G, Ceciliani F, Colombo L, DalfAra P, Gervasoni M,
Giannino
ML, Martino PA, Pollera C, Villa S, Salmona M. In vitro evaluation of the anti-
prionic
activity of newly synthesized Congo red derivatives. Arzneimittelforschung.
2003;53(12):875-88.
Priola SA, Raines A, Caughey WS. Porphyrin and phthalocyanine antiscrapie
compounds.
Science. 2000 Feb 25;287(5457):1503-6.
Schmidt J, Metselaar JM, Wauben MH, Toyka KV, Storm G, Gold R. Drug targeting
by
long-circulating liposomal glucocorticosteroids increases therapeutic efficacy
in a model of
multiple sclerosis. Brain. 2003 Aug;126(Pt 8):1895-904.
Shyng SL, Lehmann S, Moulder KL, Harris DA. Sulfated glycans stimulate
endocytosis of
the cellular isoform of the prion protein, PrPC, in cultured cells. J Biol
Chem. 1995 Dec
15;270(50):30221-9.
Supattapone S, Wille H, Uyechi L, Safar J, Tremblay P, Szoka FC, Cohen FE,
Prusiner SB,
Scott MR. Branched polyamines cure prion-infected neuroblastoma cells. J
Virol. 2001
Apr;75(7):3453-61.
Vinogradov SV, Batrakova EV, Kabanov AV. Nanogels for oligonucleotide delivery
to the
brain. Bioconjug Chem. 2004 Jan-Feb;lS(1):50-60.
32
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Zhang X, Xie J, Li S, Wang X, Hou X. The study on brain targeting of the
amphotericin B
liposomes. J Drug Target. 2003 Feb; l l (2):117-22.
Zhang Y, Schlachetzki F, Pardridge WM. Global non-viral gene transfer to the
primate brain
following intravenous administration. Mol Ther. 2003 Jan;7(1):11-8.
IV. Pharmaceutical Compositions and Delivery
Pharmaceutical compositions
[0120] The ONs of the invention may be in the form of a therapeutic
composition or
formulation useful for treating (or prophylaxis of) a prior disease or
diseases, which can be
approved by a regulatory agency for use in humans or in non-human animals,
and/or against a
particular prior disease. These ONs may be used as part of a pharmaceutical
composition
when combined with a physiologically and/or pharmaceutically acceptable
carrier. The
characteristics of the carrier may depend on the route of administration. The
pharmaceutical
composition of the invention may also contain other active factors and/or
agents which
enhance activity.
[0121] Administration of the ONs of the invention used in the pharmaceutical
composition
or formulation or to practice the method of treating an animal can be carried
out in a variety
of conventional ways, such as intraocular, oral ingestion, enterally,
inhalation (using a wet or
dry aerosol), or cutaneous, subcutaneous, intramuscular, intraperitoneal,
intrathecal,
intratracheal, intracerebral, intracranial, intraventricular or intravenous
injection.
(0122] The pharmaceutical composition or oligonucleotide formulation of the
invention
may further contain other anti-prior agents, e.g., one or more PrPc expression
inhibitors.
[0123] The pharmaceutical composition or oligonucleotide formulation of the
invention
may further contain a polymer, such as, without restriction, polyanionic
agents, sulfated
polysaccharides, heparin, dextran sulfate, pentosan polysulfate,
polyvinylalcool sulfate,
acemannan, polyhydroxycarboxylates, cellulose sulfate, polymers containing
sulfonated
benzene or naphthalene rings and naphthalene sulfonate polymer, acetyl
phtha.loyl cellulose,
poly-L-lysine, sodium caprate, cationic amphiphiles, cholic acid.
33
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Oligonucleotide Formulations and Pharmaceutical Compositions
[0124] The present olig~nucleotides can be prepared in an oligonucleotide
formulation or
pharmaceutical composition. Thus, the present oligonucleotides may also be
admixed,
encapsulated, conjugated or otherwise associated with other molecules,
molecule structures
or mixtures of compounds, as for example, liposomes, receptor targeted
molecules, oral,
rectal, topical or other formulations, for assisting in uptake, distribution
and/or absorption.
Exemplary United States patents that describe the preparation of such uptake,
distribution
and/or absorption assisting formulations include, for example, U.S. Pat. Nos.
5,108,921;
5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020;
5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221;
5,356,633;
5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528;
5,534,259;
5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is incorporated
herein by
reference in its entirety.
[0125] The oligonucle~tides, formulations, and compositions of the invention
include any
pharmaceutically acceptable salts, esters, or salts of such esters, or any
other compound
which, upon administration to an animal including a human, is capable of
providing (directly
or indirectly) the biologically active metabolite or residue thereof.
Accordingly, for example,
the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts
of the
c~mpounds of the inventi~n, pharmaceutically acceptable salts of such
prodrugs, and other
bioequivalents.
[0126] The term "prodrug" indicates a therapeutic agent that is prepared in an
inactive form
that is converted to an active form (i.e., drug) within the body or cells
thereof by the action of
endogenous enzymes or other chemicals andlor conditions. In particular
embodiments,
prodrug versions of the present oligonucleotides are prepared as SATE [(S-
acetyl-2-thioethyl)
phosphate] derivatives according to the methods disclosed in Gosselin et al.,
WO 93/24510
and in Imbach et al., WO 94/26764 and U.S. Pat. No. 5,770,713, which are
hereby
incorporated by reference in their entireties.
[0127] The term "pharmaceutically acceptable salts" refers to physiologically
and
pharmaceutically acceptable salts of the present compounds: i.e., salts that
retain the desired
biological activity of the parent compound and do not impart undesired
toxicological effects
thereto. Many such pharmaceutically acceptable salts are known and can be used
in the
present inventi~n.
34
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0128] For oligonucleotides, useful examples of pharmaceutically acceptable
salts include
but are not limited to salts formed with cations such as sodium, potassium,
ammonium,
magnesium, calcium, polyamines such as spennine and spermidine, etc.; acid
addition salts
formed with inorganic acids, for example hydrochloric acid, hydrobromic acid,
sulfuric acid,
phosphoric acid, nitric acid and the like; salts formed with organic acids
such as, for example,
acetic acid, oxalic acid, tartaric acid, succinic acid, malefic acid, fuxnaric
acid, gluconic acid,
citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic
acid, alginic acid,
polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-
toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like; and salts
formed from
elemental aiuons such as chlorine, bromine, and iodine.
[0129] The present invention also includes pharmaceutical compositions and
formulations
which contain the anti-prion oligonucleotides of the invention. Such
pharmaceutical
compositions may be administered in a number of ways depending upon whether
local or
systemic treatment is desired and upon the area to be treated. For
examp°le, administration
may be topical (including ophthalmic and to mucous membranes including vaginal
and rectal
delivery); pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by
nebulizer; intratracheal; intranasal; epidermal and transdermal; oral; or
parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or
intramuscular injection or infusion; or intracranial, e.g., intrathecal or
intraventricular,
administration.
X0130] Pharmaceutical compositions and formulations for topical administration
may
include transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases,
thickeners and the like may be necessary or desirable. Coated condoms, gloves
and the like
may also be useful. Preferred topical formulations include those in which the
oligonucleotides of the invention are in admixture with a topical delivery
agent such as lipids,
liposomes, fatty acids, fatty acid esters, steroids, chelating agents and
surfactants. Preferred
lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE
ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative
(e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl
DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides may be
encapsulated within liposomes or may form complexes thereto, in particular to
cationc
liposomes. Alternatively, oligonucleotides may be complexed to lipids, in
particular to
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
cationic lipids. Preferred fatty' acids and esters include but are not limited
arachidonic acid,
oleic acid, eicosanoic acid, Laurie acid, caprylic acid, capric acid, myristic
acid, palmitic acid,
stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein,
dilaurin, glyceryl 1
-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine,
or a C1_io
allcyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or
pharmaceutically
acceptable salt thereof.
[0131] Compositions and formulations for oral administration include powders
or granules,
microparticulates, nanoparticulates, suspensions or solutions in water or non-
aqueous media,
capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring
agents, diluents,
emulsifiers, dispersing aids or binders may be desirable. Preferred oral
formulations are those
in which oligonucleotides of the invention are administered in conjunction
with one or more
penetration enhancers surfactants and chelators. Exemplary surfactants include
fatty acids
and/or esters or salts thereof, bile acids and/or salts thereof. Exemplary
bile acids/salts
include chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic acid
(UDCA),
cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic
acid,
glycodeoxycholic acid, taurocholic acid,'taurodeoxycholic acid, sodium tauro-
24,25-dihydro-
fusidate, sodium glycodihydrofusidate. Exemplary fatty acids include
arachidonic acid;
undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic
acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an
acylcholine, or
a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof
(e.g. sodium).
Also preferred are combinations of penetration enhancers, for example, fatty
acids/salts in
combination with bile acids/salts. A particularly preferred combination is the
sodium salt of
lauric acid, capric acid and UDCA. Further exemplary penetration enhancers
include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the
invention may be delivered orally in granular form including sprayed dried
particles, or
complexed to form micro or nanoparticles. Oligonucleotide complexing agents
include poly-
amino acids; polyimines; polyacrytates; polyalkylacrylates, polyoxethanes,
polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-
derivatized
polyimines, pollulans, celluloses, and starches. Particularly advantageous
complexing agents
include chitosan, N-trimethytchitosan, poly-L-lysine, polyhistidine,
polyorithine,
polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-
methylethylene
P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate),
36
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
poly(ethylcyanoacrylate), poly(butylcyanoacrylatc),
poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAF-methacrylate, DEAE-hexylacrylate, DEAF-
acrylamide,
DEAE-albumin and DEAF-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-
lactic
acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG).
[0132] Compositions and formulations for pareriteral, intracranial,
intracerebral, intrathecal
or intraventricular administration may include sterile aqueous solutions which
may also
contain buffers, diluents and other suitable additives such as, but not
limited to, penetration
enhancers, Garner compounds and other pharmaceutically acceptable carriers or
excipients.
[0133] Pharmaceutical compositions of the present invention include, but are
not limited to,
solutions, emulsions, and liposome-containing formulations. These compositions
may be
generated from a variety of components that include, but are not limited to,
preformed
liquids, self emulsifying solids and self emulsifying semisolids.
[0134] The pharmaceutical formulations of the present invention, which may
conveniently
be presented in unit dosage form, may be prepared according to conventional
techniques well
known in the pharmaceutical industry. Such techniques include the step of
bringing into
association the active ingredients with the pharmaceutical carriers) or
excipient(s). In general
the formulations are prepared by uniformly and intimately bringing into
association the active
ingredients with liquid carriers or finely divided solid carriers or both, and
then, if necessary,
shaking the product.
[0135] The compositions of the present invention may be formulated into any of
many
possible dosage forms such as, but not limited to, tablets, capsules, gel
capsules, liquid
syrups, soft gels, suppositories, and enemas. The compositions of the present
invention may
also be formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous
suspensions may further contain substances which increase the viscosity of the
suspension
including, for example, sodium carboxymethylcellulose, sorbitol and/or
dextran. The
suspension may also contain stabilizers.
[0136] In one embodiment of the present invention the pharmaceutical
compositions may
be formulated and used as foams. Pharmaceutical foams include formulations
such as, but not
limited to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar
in nature these formulations vary in the components and the consistency of the
final product.
The preparation of such compositions and formulations is generally known to
those skilled in
37
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
the pharmaceutical and formulation arts and may be applied to the formulation
of the
compositions of the present invention.
Emulsions
[0137] The formulations and compositions of the present invention may be
prepared and
formulated as emulsions. Emulsions are typically heterogenous systems of one
liquid
dispersed in another in the form of droplets usually exceeding 0.1 ~m in
diameter. (Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (lids.), 1988,
Marcel Dekker,
Inc., New York, N.Y., volume l, p. 199; Rosoff, in Pharmaceutical Dosage
Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., Volume
1, p. 245; Block in Pharmaceutical Dosage Forms" Lieberman, Rieger and Banker
(Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et at.,
in Remington's
Phaf°maceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.
301). Emulsions are
often biphasic systems comprising of two immiscible liquid phases intimately
mixed and
dispersed with each other. In general, emulsions may be either water-in-oil
(w/o) or of the
oil-in-water (o/w) variety. When an aqueous phase is finely divided into and
dispersed as
minute droplets into a bulk oily phase the resulting composition is called a
water-in-oil (w/o)
emulsion. Alternatively, when an oily phase is finely.divided into and
dispersed as minute
droplets into a bulk aqueous phase the resulting composition is called an oil-
in-water (o/w)
emulsion. Emulsions may contain additional components in addition to the
dispersed phases
and the active drug which may be present as a solution in either the aqueous
phase, oily phase
or itself as a separate phase. Pharmaceutical excipients such as emulsifiers,
stabilizers, dyes,
and anti-oxidants may also be present in emulsions as needed. Pharmaceutical
emulsions may
also be multiple emulsions that are comprised of more than two phases such as,
for example,
in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w)
emulsions. Such
complex formulations often provide certain advantages that simple binary
emulsions do not.
Multiple emulsions in which individual oil droplets of an o/w emulsion enclose
small water
droplets constitute a w/o/w emulsion. Likewise a system of oil droplets
enclosed in globules
of water stabilized in an oily continuous provides an o/w/o emulsion.
[0138] Emulsions are characterized by little or no thermodynamic stability.
Often, the
dispersed or discontinuous phase of the emulsion is well dispersed into the
external or
continuous phase and maintained in this form through the means of emulsifiers
or the .
viscosity of the formulation. Either of the phases of the emulsion may be a
semisolid or a
solid, as is the case of emulsion-style ointment bases and creams. Other means
of stabilizing
38
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
emulsions entail the use of emulsifiers that may be incorporated into either
phase of the
emulsion. Emulsifiers may broadly be classified into four categories:
synthetic surfactants,
naturally occurring emulsifiers, absorption bases, and finely dispersed solids
(Idson, in
Pha~maceutical.Dosage Fof°ms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199).
[0139] Synthetic surfactants, also known as surface active agents, have found
wide
applicability in the formulation of emulsions and have been reviewed in the
literature (Rieger,
in Pharmaceutical Dosage Fo~rns, Lieberman, Rieger and Banker (Eds.), 1988,
Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical
Dosage Forms,
Lieberman, Rieger and Banker (Eds.), Marcel Dekker, hlc., New York, N.Y.,
1988, volume
1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic
and a
hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of
the surfactant
has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool
in categorizing
and selecting surfactants in the preparation of formulations. Surfactants may
be classified into
different classes based on the nature of the hydrophilic group: non-ionic,
anionic, cationic
and amphoteric (Rieger, in Pharmaceutical Dosage Foams, Lieberman, Rieger and
Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
[0140] Naturally occurring emulsifiers used in emulsion formulations include
lanolin,
beeswax, phosphatides, lecithin and acacia. Absorption bases possess
hydrophilic properties
such that they can soak up water to form w/o emulsions yet retain their
semisolid
consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely
divided solids
have also been used as good emulsifiers especially in combination with
surfactants and in
viscous preparations. These include.polar inorganic solids, such as heavy
metal hydroxides,
nonswelling clays such as bentonite, attapulgite, hectorite, kaolin,
montmorillonite, colloidal
aluminum silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids
such as carbon or glyceryl tristearate.
[0141] A large variety of non-emulsifying materials are also included in
emulsion
formulations and contribute to the properties of emulsions. These include
fats, oils, waxes,
fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and
antioxidants (Block, in Phaf°maceutical Dosage FoYnas, Lieberman,
Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in
39
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marvel
Dekker,
Inc., New York, N.Y., volume l, p. 199).
[0142] Hydrophilic colloids or hydrocolloids include naturally occurring gums
and
synthetic polymers such as polysaccharides (for example, acacia, agar, alginic
acid,
carrageenan, guar gum, kaxaya gum, and tragacanth), cellulose derivatives (for
example,
caxboxyrnethylcellulose and carboxypropylcellulose), and synthetic polymers
(for example,
carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or
swell in water to
form colloidal solutions that stabilize emulsions by forming strong inter-
facial films around
the dispersed-phase droplets and by increasing the viscosity of the external
phase.
[0143] Since emulsions often contain a number of ingredients such as
carbohydrates,
proteins, sterols and phosphatides that may readily support the growth of
microbes, these
formulations often incorporate preservatives. Commonly used preservatives
included in
emulsion formulations include methyl paraben, propyl paraben, quaternary
ammonium salts,
benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid,
Antioxidants are
also commonly added to emulsion formulations to prevent deterioration of the
formulation.
Antioxidants used may be free radical scavengers such as tocopherols, alkyl
gallates,
butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as
ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric acid,
tartaric acid, and
lecithin.
[0144] The application of emulsion formulations via dermatological, oral and
parenteral
routes and methods for their manufacture have been reviewed in the literature
(Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marvel
Dekker,
Inc., New York, N.Y., volume l, p. 199). Emulsion formulations for oral
delivery have been
very widely used because of reasons of ease of formulation, efficacy from an
absorption and
bioavailabiity standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and
Banker (Eds.), 1988, Marvel Dekker, Inc., New York, N.Y., volume 1, p. 245;
ldson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Maxcel
Dekker,
Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-
soluble vitamins and
high fat nutritive preparations axe among the materials that have commonly
been
administered orally as olw emulsions.
[0145] In one embodiment of the present invention, the compositions of
oligonucleotides
are formulated as microemulsions. A microemulsion may be defined as a system
of water, oil
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
and amphiphile which is a single optically isotropic and thermodynamically
stable liquid
solution (Rosoff, in Plaarmaceutical Dosage Foams, Lieberman, Rieger and
Banker (Eds.),
1988, Marvel Dekker, hzc., New York, N.Y., volume 1, p. 245). Typically micro-
emulsions
are systems that are prepared by first dispersing an oil in an aqueous
surfactant solution and
then adding a sufficient amount of a fourth component, generally an
intermediate chain-
length alcohol to form a transparent system. Therefore, microemulsions have
also been
described as thermodynamically stable, isotropically clear dispersions of two
immiscible
liquids that are stabilized by interfacial films of surface-active molecules
(Leung and Shah,
in: Controlled Release of Ds°ugs: Polymers and Aggregate Systems,
Rosoff, M., Ed., 1989,
VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared
via a
combination of three to five components that include oil, water, surfactant,
cosurfactant and
electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-
in-water (o/w)
type is dependent on the properties of the oil and surfactant used and on the
structure and
geometric packing of the polar heads and hydrocarbon tails of the surfactant
molecules
(Schott, in Renaington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p.
271).
[0146] The phenomenological approach utilizing phase diagrams has been
extensively
studied and has yielded a comprehensive knowledge, to one skilled in the art,
of how to
formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and
Banker (Eds.), 1988, Marvel Dekker, Inc., New York, N.Y., volume 1, p. 245;
Block, in
Phaf°maceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marvel Dekker,
Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble drugs in a
formulation of
thermodynamically stable droplets that are formed spontaneously.
[0147] Surfactants used in the preparation of microemulsions include, but are
not limited to,
ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl
ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML31 O), tetraglycerol
monooleate (M0310),
hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500),
decaglycerol
monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol
sequioleate
(50750), decaglycerol decaoleate~(DA0750), alone or in combination with
cosurfactants. The
cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-
butanol, serves
to increase the interfacial fluidity by penetrating into the surfactant film
and consequently
creating a disordered filin because of the void space generated among
surfactant molecules.
41
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Microemulsions may, however, be prepared without the use of cosurfactants and
alcohol-free
self emulsifying microemulsion systems are known in the art. The aqueous phase
may
typically be, but is not limited to, water, an aqueous solution of the drug,
glycerol, PEG300,
PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
The oil phase
may include, but is not limited to, materials such as Captex 300, Captex 355,
Capmul MCM,
fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated
glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides,
saturated polyglycolized
C8-C10 glycerides, vegetable oils and silicone oil.
[0148] Microemulsions are particularly of interest from the standpoint of drug
solubilization and the enhanced absorption of drugs. Lipid based
microemulsions (both o/w
and w/o) have been proposed to enhance the oral bioavailability of drugs,
including peptides
(Constantinides et al., Pharmaceutical Reseay~ch, 1994, 11, 1385-1390;
Ritschet, Metli. Find.
Exp. Clin. PhannaacoL, 1993, 13, 205). Micro-emulsions afford advantages of
improved drug
solubilization, protection of drug from enzymatic hydrolysis, possible
enhancement of drug
absorption due to surfactant-induced alterations in membrane fluidity and
permeability, ease
of preparation, ease of oral administration over solid dosage forms, improved
clinical
potency, and decreased, toxicity (Constantinides et at., PIZanynaceutical
Reseal°ch, 1994, 11,
1385; Ho et al., J. Pharm. Set, 1996, 85, 138-143). Often microemulsions may
form
spontaneously when their components are brought together at ambient
temperature. This may
be particularly advantageous when formulating thermolabile drugs, peptides or
oligonucleotides. Microemulsions have also been effective in the transdermal
delivery of
active components in~both cosmetic and pharmaceutical applications. It is
expected that the
microemulsion compositions and formulations of the present invention will
facilitate the
increased systemic absorption of oligonucteotides and nucleic acids from the
gastrointestinal
tract, as well as improve the local cellular uptake of oligonucleotides and
nucleic acids within
the gastrointestinal tract, vagina, buccal cavity and other areas of
administration.
[0149] Microemulsions of the present invention may also contain additional
components
and additives such as sorbitan monostearate (Grill 3), Labrasol, and
penetration enhancers to
improve the properties of the formulation and to enhance the absorption of the
oligonucleotides and nucleic acids of the present invention. Penetration
enhancers used in the
microemulsions of the present invention may be classified as belonging to one
of five broad
categories - surfactants, fatty acids, bile salts, chelating agents, and non-
chelating non-
surfactants (Lee et al., Critical Reoiews in Therapeutic Drug
Caf°r~ief~ Systems, 1991, p. 92).
42
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Liposomes
[0150] There are many organized surfactant structures besides microemulsions
that have
been studied and used for the formulation of drugs. These include monolayers,
micelles,
bilayers and vesicles. Vesicles offer specificity and extended duration of
action for drug
delivery. Thus, as used herein, the term "liposome" refers to a vesicle
composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers, i.e.,
liposomes are unilamellax
or multilamellar vesicles which have a membrane formed from a lipophilic
material and an
aqueous interior. The aqueous portion typically contains the composition to be
delivered. In
order to cross intact mammalian skin, lipid vesicles must pass through a
series of fine pores,
each with a diameter less than 50 nm, under the influence of a suitable
transdermal gradient.
Therefore, it is desirable to use a liposome which is highly deformable and
able to pass
through such fine pores. Additional factors for liposomes include the lipid
surface charge,
and the aqueous volume of the liposomes.
[0151] Further advantages of liposomes include; liposomes obtained from
natural
phospholipids are biocompatible and biodegradable; liposomes can incorporate a
wide range
of water and lipid soluble drugs; liposomes can protect encapsulated drugs in
their internal
compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage
FoYms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.~., volume
1, p. 245).
[0152] For topical administration, there is evidence that liposomes present
several
advantages over other formulations. Such advantages include reduced side-
effects related to
high systemic absorption of the administered drug, increased accumulation of
the
administered drug at the desired target, and the ability to administer a wide
variety of drugs,
both hydrophilic and hydrophobic, into the skin. Compounds including
analgesics,
antibodies, hormones and high-molecular weight DNAs have been administered to
the skin,
generally resulting in targeting of the upper epidermis.
[0153] Liposomes fall into two broad classes. Cationic liposomes are
positively charged
liposomes which interact with the negatively charged DNA molecules to form a
stable
complex. The positively charged DNAlliposome complex binds to the negatively
charged cell
surface and is internalized in an endosome. Due to the acidic pH within the
endosome, the
liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang
et at.,
Biochena. Biophys. Res. Conatnuta., 1987, 147, 980-985).
43
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0154] Liposomes which are pH-sensitive or negatively-charged, entrap DNA
rather than
complex with it. Since both the DNA and the lipid are similarly charged,
repulsion rather
than complex formation occurs. The DNA is thus entrapped in the aqueous
interior of these
liposomes. pH-sensitive liposomes have been used, for example, to deliver DNA
encoding
the thymidine kinase gene to cell monolayers in culture (Zhou et al., Journal
of Cofzt~olled
Release, 1992, 19, 269-274).
[0155] One major type of liposomal composition includes phospholipids other
than
naturally-derived phosphatidylcholine. Neutral liposome compositions, for
example, can be
formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine
(DPPC). Anionic liposome compositions generally are formed from dimyristoyl
phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily
from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal composition is
formed from
phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another
type is
formed from mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0156] Several studies have assessed the topical delivery of liposomal drug
formulations to
the skin. Application of liposomes containing interferon to guinea pig skin
resulted in a
reduction of skin herpes sores while delivery of interferon via other means
(e.g. as a solution
or as an emulsion) were ineffective (Weiner et at., Journal ofDrug Targeting,
1992, 2, 405-
410). Further, an additional study tested the efficacy of interferon
administered as part of a
liposomal formulation to the administration of interferon using an aqueous
system, and
concluded that the liposomal formulation was superior to aqueous
administration (du Plessis
et al., A~ativi~al Research, 1992 18, 259-265).
[0157] Non-ionic liposomal systems have also been examined to determine their
utility in
the delivery of drugs to the skin, in particular systems comprising non-ionic
surfactant and
cholesterol. Non-ionic liposomal formulations comprising NovasoneTM I
(glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome~ II
(glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver
cyclosporin-A
into the dermis of mouse skin. Results indicated that such non-ionic liposomal
systems were
effective in facilitating the deposition of cyclosporin-A into different
layers of the skin (Hu et
at. S. T P. Plaa~rraa. Sci., 1994, 4, 6, 466).
[0158] Liposomes also include "sterically stabilized" liposomes, a term which,
as used
herein, refers to liposomes comprising one or more specialized lipids that,
when incorporated
44
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
into liposomes, result in enhanced circulation lifetimes relative to liposomes
lacking such
specialized lipids. Examples of sterically stabilized liposomes are those in
which part of the
vesicle-forming lipid portion of the liposome include one or more glycolipids,
such as
monosialoganglioside GM1, or is derivatized with one or more hydrophilic
polymers, such as
a polyethylene glycol (PEG) moiety. Without being bound by any particular
theory, it is
believed that for sterically stabilized liposomes containing gangliosides,
sphingomyelin, or
PEG-derivatized lipids, the increase in circulation half life of these
sterically stabilized
liposomes is due to a reduced uptake into cells of the reticuloendothelial
system (RES) (Allen
et at., FEBS Lett., 1987, 223, 42; Wu et al., CanceY Research, 1993, 53,
3765).
[0159] Various liposomes that include one or more glycolipids have been
reported in
Papahadjopoulos et al., Ann. N. Y. Acad. Sci., 1987, 507, 64
(monosiatoganglioside GM1,
galactocerebroside sulfate and phosphatidylinositol); Gabizon et at., P~oc.
Natl. Acad. Sci.
USA., 1988, 85, 6949,;Allen et al., US. Pat. No. 4,837,028 and International
Application
Publication WO 88/04924 (sphingomyelin and the ganglioside GMl or a
galactocerebroside
sulfate ester); Webb et al., U.S. Pat. No. 5,543,152 (sphingomyelin); Lim et
al., WO
97/13499 (1,2-sn-dimyristoylphosphatidylcholine).
[0160] Liposomes that include lipids derivatized with one or more hydrophilic
polymers,
and methods of preparation are described, for example, in Sunamoto et al:,
Bull. ~'Izem. Soc.
Jpn., 1980, 53, 2778 (a nonionic detergent, 2C1215G, that contains a PEG
moiety); Illum et
al., FEBS Lett., 1984, 167, 79 (hydrophilic coating of polystyrene particles
with polymeric
glycols); Sears, U.S. Pat. Nos. 4,426,330 and 4,534, 899 (synthetic
phospholipids modified
by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG));
Klibanov et al.,
FEBS Lett., 1990, 268, 235 (phosphatidylethanolamine (PE) derivatized with PEG
or PEG
stearate); Blume et al., Biochimica etBiophysica,Acta, 1990, 1029, 91 (PEG-
derivatized
phospholipids, e.g., DSPE-PEG, formed from the combination of
distearoylphosphatidylethanolamine (DSPE) and PEG); Fisher, European Patent
No. EP 0
445 131 Bl and WO 90/04384 (covalently bound PEG moieties on liposome external
surface); Woodle et al., U.S. Pat. Nos. 5,013,556 and 5,356,633, and Martin et
al., U.S. Pat.
No. 5,213,804 and European Patent No. EP 0 496 813 B 1 (liposome compositions
containing
1-20 mole percent of PE derivatized with PEG); Martin et al., WO 91/05545 and
U.S. Pat.
No. 5,225,212 and in Zalipsky et al., WO 94/20073 (liposomes containing a
number of other
lipid-polymer conjugates); Choi et al., WO 96/10391 (liposomes that include
PEG-modified
ceramide lipids); Miyazaki et al., U.S. Pat. No. 5,540,935, and Tagawa et al.,
U.S. Patent No.
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
5,556,948 (PEG-containing liposomes that can be further derivatized with
functional moieties
on their surfaces).
[0161] Liposomes that include nucleic acids have been described, for example,
in Thierry et
al., WO 96/40062 (methods for encapsulating high molecular weight nucleic
acids in
liposomes); Tagawa et al., U.S. Pat. No. 5,264,221 (protein-bonded liposomes
containing
RNA); Rahman et al., U.S. Pat. No. 5,665,710 (methods of encapsulating
oligodeoxynucleotides in liposomes); Love et al., WO 97/04787 (liposomes that
include
antisense oligonucleotides).
[0162] Another type of liposome, transfersomes are highly deformable lipid
aggregates
which are attractive for drug delivery vehicles. (Cevc et al., 1998, Bioclaim
Biophys Acta.
1368(2):201-15.) Transfersomes may be described as lipid droplets which are so
highly
deformable that they can penetrate through pores which are smaller than the
droplet.
Transfersomes are adaptable to the environment in which they are used, for
example, they are
shape adaptive, self repairing, frequently reach their targets without
fragmenting, and often
self loading. Transfersomes can be made, for example, by adding surface edge-
activators,
usually surfactants, to a standard liposomal composition.
Surfactants
[0163] Surfactants are widely used in formulations such as emulsions
(including
microemulsions) and liposomes. The most common way of classifying and ranking
the
properties of the many different types of surfactants, both natural and
synthetic, is by the use
of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group
(also known
as the "head") provides the most useful means for categorizing the different
surfactants used
in formulations (Rieger, in Pharmaceutical Dosage Fof°ms, Marcel
Dekker, Inc., New York,
N.Y., 1988, p. 285).
[0164] If the surfactant molecule is not ionized, it is classified as a
nonionic surfactant.
Nonionic surfactants are widely used in pharmaceutical and cosmetic products
and are usable
over a wide range of pH values, and with typical HLB values from 2 to about 18
depending
on structure. Nonionic surfactants include nonionic esters such as ethylene
glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan
esters, sucrose esters,
and ethoxylated esters; and nonionic alkanolamides and ethers such as fatty
alcohol
ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block
polymers are also
46
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
included in this class. The polyoxyethylene surfactants are the most commonly
used members
of the nonionic surfactant class.
[0165] Surfactant molecules that carry a negative charge when dissolved or
dispersed in
water are classified as anionic. Anionic surfactants include carboxylates such
as soaps, acyl
lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl
sulfates and
ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl
isothionates, acyl
laurates and sulfosuccinates, and phosphates. The allcyl sulfates and soaps
are the most
commonly used anionic surfactants.
[0166] Surfactant molecules that carry a positive charge when dissolved or
dispersed in
water are classified as cationic. Cationic surfactants include quaternary
ammonium salts and
ethoxylated amines, with the quaternary ammonium salts used most often.
[0167] Surfactant molecules that can carry either a positive or negative
charge are classified
as amphoteric. Amphoteric surfactants include acrylic acid derivatives,
substituted
alkylamides, N-alkylbetaines and phosphatides.
[0168] The use of surfactants in drug products, formulations and in emulsions
has been
reviewed in Rieger, in Pharmaceutical Dosage Forms, Marvel Dekker, Inc., New
York,
N.Y., 1988, p. 285).
Penetration Enhancers
[0169] In some embodiments, penetration enhancers are used in or with a
composition to
increase the delivery of nucleic acids, particularly oligonucleotides, to the
skin of animals.
Most drugs are present in solution in both ionized and nonionized forms.
However, usually
only lipid soluble or lipophilic drugs readily cross cell membranes. It has
been discovered
that even non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is
treated with a penetration enhancer. In addition to aiding the diffusion of
non-lipophilic drugs
across cell membranes, penetration enhancers also enhance the permeability of
lipophilic
drugs.
[0170] Exemplary penetration enhancers may be classified as belonging to one
of five
broad categories, i.e., surfactants, fatty acids, bile salts, chelating
agents, and non-chelating
nonsurfactants (Lee et al., Cf~itical Reviews ih TlaeYapeutic Drug Carrier
Systems, 1991,
p.92). Each of these classes of penetration enhancers is described below in
greater detail.
47
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0171] Surfactants: In connection with the present invention, surfactants (or
"surface-
active agents") are chemical entities which, when dissolved in an aqueous
solution, reduce
the surface tension of the solution or the interfacial tension between the
aqueous solution and
another liquid, with the result that absorption of oligonucleotides through
the mucosa is
enhanced. These penetration enhancers include, for example, sodium lauryl
sulfate,
polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et
at.,
Cr°iticalReviews in Therapeutic Drug Carrier' Systems, 1991, p.92); and
perfluorochemical
emulsions, such as FC-43. Takahashi et al., J. P7~arm. Pharmacol., 1988, 40,
252), each of
which is incorporated herein by reference in its entirety.
[0172] Fatty acids: Various fatty acids and their derivatives which act as
penetration
enhancers include, for example, oleic acid, lauric acid, capric acid (n-
decanoic acid), myristic
acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein
(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,
glycerol 1-
monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C1_lo alkyl esters
thereof (e.g., methyl, isopropyl and t-butyl), and mono- and diglycerides
thereof (i.e., oleate,
laurate, caprate, myristate, palinitate, stearate, linoleate, etc.) (Lee et
al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews itt
Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,
1992, 44, 651-
654), each of which is incorporated herein by reference in its entirety.
[0173] Bile salts: The physiological role of bile includes the facilitation of
dispersion and
absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman
& Gilman's
The Phartrtacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,
McGraw-Hill,
New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic
derivatives, act
as penetration enhancers. Thus the term "bile salts" includes any of the
naturally occurring
components of bile as well as any of their synthetic derivatives. The bile
salts of the invention
include, for example, cholic acid (or its pharmaceutically acceptable sodium
salt, sodium
cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium
deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium
glycocholate),
glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium
taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid
(sodium
chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-
fusidate
(STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE)
(Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92;
Swinyard, Chapter
48
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack
Publishing Co.,
Euston, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier
Systems, 1990, 7, 1-33; Yamamoto ct al., J. Pharm. Exp. Ther., 1992, 263, 25;
Yamashita et
al., J. Pharm:. Sci., 1990, 79, 579-583).
[0174] Chelating Agents: In the present context, chelating agents can be
regarded as
compounds that remove metallic ions from solution by forming complexes
therewith, with
the result that absorption of oligonucleotides through the mucosa is enhanced.
With regards
to their use as penetration enhancers in the present invention, chelating
agents have the added
advantage of also serving as DNase inhibitors, as most characterized DNA
nucleases require
a divalent metal ion for catalysis and are thus inhibited by chelating agents
(Jarrett, J.
Chromatogr., 1993, 618, 315-339). Without limitation, chelating agents include
disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium
salicylate, 5-
methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9
and N-amino
acyl derivatives of beta-diketones (enasnines)(Lee et al., Critical Reviews
ira Therapeutic
Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in
Therapeutic Drug
Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-
SI).
(0175] Non-chelating non-surfactants: As used herein, non-chelating non-
surfactant
penetration enhancing compounds are compounds that do not demonstrate
significant
chelating agent or surfactant activity, but still enhance absorption of
oligonucleotides through
the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems,
1990, 7, 1-33). Examples of such penetration enhancers include unsaturated
cyclic areas, 1 -
alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical
Reviews in
Therapeutic Drug Carrier' Systems, 1991, page 92); and nonsteroidal anti-
inflammatory
agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita
et al" J.
Pharm. Pharmacol., 1987, 39, 621-626).
[0176] Agents that enhance uptake of oligonucleotides at the cellular level
may also be
added to the pharmaceutical and other compositions and formulations of the
present
invention. For example, cationic lipids, such as lipofectin (Junichi et al,
U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic molecules, such as
polylysine
(Lollo et al., PCT Application WO 97130731), are also known to enhance the
cellular uptake
of oligonucleotides.
49
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0177] Other agents may be utilized to enhance the penetration of the
administered nucleic
acids, including glycols such as ethylene glycol and propylene glycol, pyrrols
such as 2-
pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
[0178] Certain compositions of the present invention also incorporate carrier
compounds in
the formulation. As used herein, "carner compound" or "carrier" can refer to a
nucleic acid,
or analog thereof, which is inert (i.e., does not possess biological activity
per se) but is
recognized as a nucleic acid by in vivo processes that reduce the
bioavailability of a nucleic
acid having biological activity by, for example, degrading the biologically
active nucleic acid
or promoting its removal from circulation. The coadministration of a nucleic
acid and a
Garner compound, often with an excess of the latter substance, can result in a
substantial
reduction of the amount of nucleic acid recovered in the liver, kidney or
other
extracirculatory reservoirs. For example, the recovery of a partially
phosphorothioate
oligonucleotide in hepatic tissue can be reduced when it is coadministered
with polyinosinic
acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-
2,2-disulfonic
acid (Miyao et al.,AhtisehseRes. Dev., 1995,5, 115-121; Takakura et al.,
Ahtisense & Nucl
Acid Drug Dev., 1996, 6, 177-183), each of which is incorporated herein by
reference in its
entirety.
Excipients
[0179] In contrast to a carrier compound, a "pharmaceutical Garner" or
"excipient" is a
pharmaceutically acceptable solvent, suspending agent or any other
pharmacologically inert
vehicle for delivering one or more nucleic acids to an animal, and is
typically liquid or solid.
A pharmaceutical carrier is generally selected to provide for the desired
bulk, consistency,
etc., when combined with a nucleic acid and the other components of a given
pharmaceutical
composition, in view of the intended administration mode. Typical
pharmaceutical carriers
include, but are not limited to, binding agents (e.g., pregelatinized maize
starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g.,
lactose and other
sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl
cellulose,
polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g.,
magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic stearates,
hydrogenated vegetable oils,
corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.);
disintegrants (e.g.,
starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium
lauryl sulphate, etc.).
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0180] Pharmaceutically acceptable organic or inorganic excipients suitable
for non-
parenteral administration which do not deleteriously react with nucleic acids
can also be used
to formulate the compositions of the present invention. Suitable
pharmaceutically acceptable
carriers include, but are not limited to, water, salt solutions, alcohols,
polyethylene glycols,
gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin,
hydroxymethylcellulose, polyvinylpyrrolidone and the like.
[0181] Formulations for topical administration of nucleic acids may include
sterile and non-
sterile aqueous solutions, non-aqueous solutions ire common solvents such as
alcohols, or
solutions of the nucleic acids in liquid or solid oil bases. The solutions may
also contain
buffers, diluents and other suitable additives. Pharmaceutically acceptable
organic or
inorganic excipients suitable for non-parenteral administration which do not
deleteriously
react with nucleic acids can be used. -
Other Pharmaceutical Composition Components
(0182] The present compositions may additionally contain other components
conventionally found in pharmaceutical compositions, at their art-established
usage levels.
Thus, for example, the compositions may contain additional, compatible,
pharmaceutically-
active materials such as, for example, antipruritics, astringents, local
anesthetics or anti-
inflammatory agents, or may contain additional materials useful in physically
formulating
various dosage forms of the compositions of the present invention, such as
dyes, flavoring
agents, preservatives, antioxidants, opacifiers, thickening agents and
stabilizers. However,
such materials, when added, should not unduly interfere with the biological
activities of the
components of the compositions of the present invention. The formulations can
be sterilized
and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting
agents, emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings
and/or axomatic substances and the like which do not deleteriously interact
with the nucleic
acids) of the formulation.
[0183] Aqueous suspensions may contain substances which increase the viscosity
of the
suspension including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran,
and/or stabilizers.
[0184] Certain embodiments of the invention provide pharmaceutical
compositions
containing (a) one or more anti-prion oligonucleotides and (b) one or more
other anti-prion
51
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
agents which function by a different mechanism, e.g., PrPc expression
inhibitors. Two or
more combined compounds. may be used together or sequentially.
CNS and other tissue delivery
[0185] All prion-related diseases axe characterized by neurological
dysfuntion. This is due
to the preferential accumulation of converted prion proteins in CNS neurons.
Prion-mediated
plaque formation in these neurons leads to altered neuronal function which is
the pathology
behind neurological impairment.
[0186] For any therapy against prion-related diseases to be effective, it must
be easily
delivered to the brain, the major site of prion accumulation. There is some
evidence to
indicate an active transport of ONs across the blood brain barrier (Banks et
al., 2001), but
naked ONs in general are not efficiently transported across the blood brain
barrier, so that
intrathecal, intraventricular, intracerebral, or intracranial injection can be
effective routes for
delivering an ON therapeutic. Since these routes of administration require
surgical
intervention, they are not preferable and axe not convenient for multiple dose
administration.
However, there are several technologies which can be used to either limit the
administration
to a single dose or to allow ONs to more efficiently cross the blood brain
barner, thus
opening up many other, preferable routes of administration (e.g., intravenous,
subcutaneous,
transdermal, inhalation).
[0187] Reservoirs of ONs (e.g. ALZET Osmotic Pumps, DURECT Corporation) can be
used intracranially to deliver ONs to the brain over long periods. However the
majority of
technologies successfully employed to increase the delivery of ONs across the
blood brain
barrier involves the use of cationic liposomes or polycationic polymers which
are known to
effectively encapsulate ONs. These technologies include but are not limited
to: pegylated
polyethyleneimine nanogels (Vinogradov et al., 2004), the use of pegylated
liposomes
conjugated to antibodies directed against the insulin receptor (Zhang et al.,
2003) or the
transferrin receptor (Huwyler et al., 1996), direct conjugation of pegylated
liposomes to
transfernn (Omori et a1.,2003), pegylated hexadecylcyanoacrylate
nanospheresBrigger et al.,
2002) or vasoactive peptide conjugated liposomes or pegylated liposomes (i.e.
RMP-7;
Zhang et al., 2003).
[0188] Since the PS-ON randomers described herein are compatible with all
these delivery
technologies or modifications, those technologies can be used to deliver PS-ON
randomers
across the blood brain barrier.
52
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0189] While the effects of PrPsc significantly relate to development of
amyloid plaques in
the CNS, it is advantageous to provide anti-prion activity to other tissues.
Thus, additional
delivery methods as described herein are also useful.
[0190] Thus, use of a delivery system can significantly increase the anti-
prion potency of
ON randomers. Additionally, they will serve to protect these compounds from
serum
interactions, reducing side effects and maximizing tissue and cellular
distribution.
[0191] Although PS-ONs axe more resistant to endogenous nucleases than natural
phosphodiesters, they are not completely stable and are slowly degraded in
blood and tissues.
A limitation in the clinical application ofPS oligonucleotide drugs is their
propensity to
activate complement on i.v. administration. In general, liposomes and other
delivery systems
enhance the therapeutic index of drugs, including ONs, by reducing drug
toxicity, increasing
residency time in the plasma, and delivering more active drug to tissue by
extravasation of the
carriers through hyperpermeable vasculature. Moreover in the case of PS-ON,
lipid
encapsulation prevents the interaction with potential protein-binding sites
while in circulation
(Klimuk et al. (2000) JPha~macol Exp They 292:480-488).
[0192] According to our results, an advantageous approach is to use a
delivery.system such
as, but without restriction, lipophilic molecules, polar lipids, liposomes,
monolayers, bilayers,
vesicles, programmable fusogenic vesicles, micelles, cyclodextrins, PEG,
iontophoresis,
powder injection, and nanoparticles (such as PIBCA, PIHCA, PHCA, gelatine, PEG-
PLA)
for the delivery of ONs described herein. The purpose of using such delivery
systems are to,
among other things, lower the toxicity of the active compound in animals and
humans,
increase cellular delivery, lower the IC50, increase the duration of action
from the standpoint
of drug delivery and protect the oligonucleotides from non-specific binding
with serum
proteins.
[0193] It is known in the art that one of the main therapeutic factors for
phosphorothioate
antisense oligonucleotides is their side effects due mainly to this increased
interaction with
proteins (specifically with serum proteins) as described by I~andimalla and co-
workers
(I~andimalla et al. (1998) Bioorg. Med. Chem. Lett. 8:2103-2108). Our data
suggests
substantial benefits by a suitable delivery system capable of delivering anti-
prion ONs into
the cell while preventing their interaction with serum proteins.
53
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0194] Another approach is to accomplish cell specific delivery by associating
the delivery
system with a molecules) that will increase affinity with specific cells, such
molecules being
without restriction antibodies, receptor ligands, vitamins, hormones and
peptides.
EXAMPLE 1: Demonstration of potent, size-dependent PS-ODN Randomer anti-prion
activity
[0195] The anti-PrP activity of PS-ODN randomers (prepared as single-stranded
randomers) was tested in a tissue culture model of PrP conversion. Three PS-
ODN
randomers were used: REP 2003 (10 mer), REP 2004 (20 mer), and REP 2006 (40
mer).
[0196] Approximately 20,000 RML or 22L scrapie-infected mouse neuroblastoma
cells
were added to each well of a 96 well plate in 100 ~,L of medium prior to the
addition of test
compounds. 22L-infected cells were developed by re-infection of RML-infected
mouse
neuroblastoma cells cured by 7 passages in medium containing 1 ~,g/mL pentosan
polysulfate. The cured cells were re-infected by incubation with PrPsc
purified from mouse
brains infected with 22L.strain of scrapie. The neuroblastoma cells reinfected
with 22L
scrapie have stably expressed PrPsc for over 70 passages. The cells were
allowed to settle for
4 hours before test compounds were added.
[0197] PS-ODN randomers were diluted into PBS prior to being introduced to the
cell
medium. 5 ~.L of solutions were added to the cell medium. After PS-ODN
randomers were
added, the cells were incubated for 5 days at 37° C in 5% C02 before
being lysed.
[0198] Prior to cell lysis, the cells were inspected by light microscopy for
toxicity, bacterial
contamination, and density compared to controls. After removal of the cell
media, 50 ~.L of
lysis buffer was added to each well. Lysis buffer was composed of 0.5% (w/v)
Triton X-100,
0.5% (w/v) sodium deoxycholate, 5 mM tris-HCI, pH 7.4 at 4°C, 5 mM
EDTA, and 150 rnM
NaCl. Five minutes after adding lysis buffer, 25 ~,L of 0.1 mg/mL PK
(Calbiochem) in TBS
was added to each well and incubated at 37° C for 50 minutes. 225 ~,L
of 1 mM Pefabloc
(Boehringer Mannheim) was then added to each well to inhibit PK activity. 250
~,L of 1 mM
Pefabloc was added to samples that were not PK-treated.
[0199] To detect the presence of converted (PK resistant) PrP protein, a 96
well dot blot
apparatus (Schleicher and Schuell) was set up with a sheet of 0.45 ~,m PVDF
Immobilon-P
54
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
(Millipore) membrane and each dot rinsed with 500 ~,L of TBS. Under vacuum,
the lysed
and PK-treated samples were added to the apparatus over the PVDF membrane and
rinsed
again with 500 ~.L of TBS. The PVDF membrane was then removed and covered with
3 M
GdnSCN (Fluka) for 10 minutes at ambient temperature. The GdnSCN was removed
by 5
PBS rinses and the membrane blocked in 5% (w/v) milk, 0.05% (v/v) Tween 20
(Sigma) in
TBS (TBST-milk) for 30 minutes. An appropriate dilution of a monoclonal
antibody 6B10,
an IgG 2a reactive against mouse, hamster, elk, and sheep PrP in irnrnunoblots
and ELISA
assays or ~ ~,g of purified 6H4 anti-PrP mouse monoclonal antibody (Prionics)
in 15 mL
TBST-milk was incubated with the membrane for 60 minutes. After rinsing with
TBST, a
solution of 500 ng of an alkaline phosphatase conjugated goat anti-mouse
linked antibody
(Zymed) in 15 mL TBST-milk was added for 45 minutes. After additional TBST
rinsing, the
membrane was treated with enhanced chemofluorescence agent (Amersham) for 10
minutes,
allowed to dry, and then scanned using a Storm Scanner (Molecular Dynamics).
The
intensity of the PrPsc signal from each well was quantitated using ImageQuant
software
(Molecular Dynamics).
[0200] We first tested REP 2006 activity against both 22L and RML strains of
mouse
scrapie (see Table 1)
Table 1. Inhibition of PrP conversion by REP 2006 (n=3)
PrP conversion
relative
to control
compound conc.Strain Strain
22L RML
Average Std. Dev.Average Std. Dev.
100006.84 9.15 -0.94 8.29
1000 -3.69 7.27 3.95 9.52
REP 2006
500 5.60 11.73 3.74 14.16
(nM)
100 15.61 12.01 5.95 7.00
~
50 -4.33 7.54 -6.43 8.63
Alexafluor10 107.98 29.95 115.03 41.25
(uM) 1 95.58 29.16 125.70 24.14
[0201] To determine where the IC50 of REP 2006's anti-PrP conversion activity
was, we
repeated this test using lower concentrations of REP 2006 including a sheep
strain of prion,
Rov-9 (see Table 2)
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
Table 2 Inhibition of PrP conversion by REP 2006 (low cone. range, n=3)
PrP
conversion
relative
to
control
cone.Strain Strain Strain
22L RML Rov-9
(nM) Std. Std. Std.
AverageDev. AverageDev. AverageDev.
500 -0.69 0.23 2.04 0.28 -0.35 5.69
100 1.19 0.86 1.79 1.69 29.55 12.40
50 2.45 1.89 5.15 2.24 55.66 21.05
48.54 11.72 87.35 17.16 69.22 21.45
5 62.41 2.31 89.72 9.51 nt nt
1 67.57 12.38 100.49 6.38 nt nt
0.5 90.42 11.31 92.45 11.29 nt nt
nt=not tested
[0202] We then tested to see if PS-ODN randomer inhibition of PrP conversion
was
dependent on randomer size. For this experiment, we tested PS-ODN randomers of
different
sizes (see Table 3).
Table 3. Inhibition of PrP Conversion by PS-ODN Randomers (n=3)
conversion
relative
to control
compound cone.Strain Strain
22L RML
Average Std. Dev.Average Std. Dev.
100 1.00 4.25 3.73 1.44
REP 2006
50 3.25 2.42 . 6.59 5.85
(nM)
10 105.15 7.58 121.70 5.53
1000 2.04 2.42 6.49 5.10
500 92.98 7.54 63.43 5.67
REP 2004
100 88.28 17.19 91.10 12.51
(nM)
50 77.32 17.05 101.48 9.60
10 70.22 9.99 97.60 9.88
REP 2003 1000 69.97 3.87 79.05 3.61
(nM) 500 88.92 15.61 92.94 2.29
.
100 80.06 7.54 91.45 11.83
56
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
conversion
relative
to control
compound conc.Strain Strain
22L RML
Average Std. Dev. Average Std. Dev.
50 83.12 5.91 100.72 3.59
10 86.97 4.90 96.10 7.15
[0203] These data show that PS-ODN randomers have a potent anti-PrP conversion
activity
against 22L, RML and Rov-9 strains of scrapie. This demonstrated potent
activity of REP
2006 against scrapie strains from different animals. Moreover, this activity
is dependent on
the size of the PS-ODN randomer used, with REP 2003 (10 mer) inactive, REP
2004 (20
mer) mildly active and REP 2006 (40 mer) highly potent (IC50 ~ l OnM).
[0204] Thus, these data show that PS-ODN randomers are active against prion
disease, and
thus can be used in anti-prion therapy useful in the treatment of prion-based
diseases in both
humans (e.g., CJD), in animals (e.g., BSE, foot and mouth disease) and in the
sterilization or
prophylactic treatment of humans, animals and of blood and feed products which
may be
tainted by prions.
Example 2: Tests for Determining if an Oligonucleotide Acts Predominantly by a
Sequence Independent Mode of Action
[0205] An ON, e.g., ODN, in question shall be considered to be acting
predominantly by a
sequence independent mode of action if it meets the criterion of any one of
the tests outlined
below.
TEST #1- Effect of partial de~eneracy on anti-prion efficacy
[0206] This test serves to measure the anti-prion activity of a particular ON
sequence when
part of its sequence is made degenerate. If the degenerate version of the ON
having the same
chemistry retains its activity as described below, is it deemed to be acting
predominantly by a
sequence independent mode of action. ONs will be made degenerate according to
the
following rule:
57
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
LoN = the number of bases in the original ON
X = the number of bases on each end of the oligo to be made degenerate (but
having the same chemistry as the original ON)
If LoN is even, then X=LoN/4
If LoN is odd, then X=integer (LoN/4)+1
[0207] Each degenerate base shall be synthesized according to any suitable
methodology,
e.g., the methodology described herein for the synthesis of PS-ON randomers.
[0208] The IC50 values shall be generated by a test of anti-prion efficacy
accepted by the
pharmaceutical industry. IC50 values shall be generated using a minimum of
seven
concentrations of compound, with three or more points in the linear range of
the dose
response curve. Using this test, the ICSO of said ON shall be compared to its
degenerate
counterpart. If the ICSO of the degenerate ON is less than 2-fold greater than
the original ON
for an ON of 25 bases and less, or is less than 10-fold greater than the
original ON for ONs
26 bases or more (based on minimum triplicate measurements, standard deviation
not to
exceed 15% of mean) then the ON shall be deemed to be functioning
predominantly by a
sequence independent mode of action.
TEST #2 - Comparison of efficacy with randomer
[0209] This test serves to compare the anti-prion efficacy of an ON with the
anti-prion
efficacy of a randomer ON of equivalent size and the same chemistry in the
same prion
disease.
[0210] The IC50 values shall be generated by a test of anti-prion efficacy
accepted by the
pharmaceutical industry. IC50 values shall be generated using a minimum of
seven
concentrations of compound, with three or more points in the linear range of
the dose
response curve. Using this test, the ICSO of the ON shall be compared to an ON
randomer of
equivalent size and the same chemistry. If the ICSO of the degenerate ON is
less than 2-fold
greater than the original ON for an ON of 25 bases and less, or is less than
10-fold greater
than the original ON for ONs 26 bases or more (based on minimum triplicate
measurements,
58
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
standard deviation not to exceed 15% of mean) then the ON shall be deemed to
be
functioning predominantly by a sequence independent mode of action.
[0212] One skilled in the art would readily appreciate that the present
invention is well
adapted to obtain the ends and advantages mentioned, as well as those inherent
therein. The
methods, variances, and compositions described herein as presently
representative of
preferred embodiments are exemplary and are not intended as limitations on the
scope of the
invention. Ghayges therein and other uses will occur to those skilled in the
art, which are
encompassed within the spirit of the invention, are defined by the scope of
the claims.
[0213] It will be readily apparent to one skilled in the art that varying
substitutions and
modifications may be made to the invention disclosed herein without departing
from the
scope and spirit of the invention. For example, variations can be made to
provide
oligonucleotides of various lengths and chemical modifications and/or various
methods of
administration can be used. Thus, such additional embodiments are within the
scope of the
present invention and the following claims.
[0214] The invention illustratively described herein suitably may be practiced
in the
absence of any element or elements, limitation or limitations which is not
specifically
disclosed herein. Thus, for example, in each instance herein any of the terms
"comprising",
"consisting essentially of and "consisting of may be replaced with either of
the other two
terms. The terms and expressions which have been employed axe used as terms of
description and not of limitation, and there is no intention that in the use
of such terms and
expressions of excluding any equivalents of the features shown and described
or portions
thereof, but it is recognized that various modifications are possible within
the scope of the
invention claimed. Thus; it should be understood that although the present
invention has
been specifically disclosed by preferred embodiments and optional features,
modification and
variation of the concepts herein disclosed may be resorted to by those skilled
in the art, and
that such modifications and variations axe considered to be within the scope
of this invention
as defined by the appended claims.
[0215] In addition, where features or aspects of the invention are described
in teens of
Markush groups or other grouping of alternatives, those skilled in the axt
will recognize that
the invention is also thereby described in terms of any individual member or
subgroup of
members of the Markush group or other group.
59
CA 02538245 2006-03-07
WO 2005/025487 PCT/IB2004/003740
[0216] Also, unless indicated to the contrary, where various numerical values
are provided
for embodiments, additional embodiments are described by taking any 2
different values as
the endpoints of a range. Such ranges are also within the scope of the
described invention.
[0217] Thus, additional embodiments are within the scope of the invention and
within the
following claims.
