Note: Descriptions are shown in the official language in which they were submitted.
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Syntheses of Polyamine Conjugates of Small Interfering RNAs (si-RNAs)
and Conjugates Formed Thereby
CROSS-REFERENCE TO RELATED APPLICATIONS
Priority is hereby claimed to US provisional application Serial No.
60/624,906, filed 04 November 2004, incorporated herein by reference.
INCORPORATION BY REFERENCE
All of the papers and prior patents cited below are incorporated herein by
reference.
INTRODUCTION
The present invention fills a gap in crafting conjugates that contain
ribonucleic acids (RNA) in general, and short interfering RNA in particular,
and
using the conjugates as therapeutic agents. Short interfering RNAs (siRNAs)
have
been dubbed in the popular scientific literature as "one of the hottest things
in target
validation in the last five years." This enthusiasm is due to the fact that
siRNAs
have been shown to be efficacious in specifically suppressing the expression
of
targeted genes.
This sequence-specific post-transcriptional silencing of gene expression via
the action of siRNAs is a naturally occurring process first discovered in
fungi and
plants, and later shown to occur in bacterial and animal cells. Short
interfering
RNAs are short (ca. 21-25 nucleotides) RNA fragments, obtained either
enzymatically or by chemical synthesis. Short interfering RNAs function by
inducing sequence-specific degradation of targeted messenger RNAs (mRNAs).
The potential of siRNAs as potent antiviral and anticancer drugs is widely
accepted
in the scientific community. However, before this potential becomes a reality
in the
form of pharmaceutical compositions, fundamental problems of delivering the
siRNAs to their intended targets must be solved.
At the root of the delivery problem is that siRNAs are polyanions. Thus,
unassisted permeation of siRNAs across lipid bilayers is negligible. siRNAs
are
conventionally delivered to cells using cationic liposomes, or polyplexes with
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polyethyleneimines. Although the use of liposomes to deliver siRNAs has shown
some success, the major disadvantage of a liposome delivery vehicle is that a
number of cell types cannot be transfected using liposomes. Also, several cell
types
cannot be liposome-transfected with an efficiency that produces significant
biological effects. Moreover, in experiments using liposome delivery vehicles
several different manipulations of the cells are required. In short, the
process is
cumbersome.
Similarly, polyethyleneimines are difficult to deliver into cells because they
are high molecular-weight polymers. Thus, in vivo delivery of
polyethyleneimine-
siRNA polyplexes is plagued by all the obstacles inherent in the systemic
delivery of
a high molecular-weight cationic complex: the complexes must make their way to
the intended site, extravasate into the targeted tissue, etc. Therefore a
method of
moving siRNA molecules from the extracellular environment into the cytoplasm
of
target cells would be a significant breakthrough in the therapeutic use of
siRNAs to
silence genes in vitro (e.g., in cultured and somatic cells) and in vivo
(e.g., systemic
delivery of siRNAs as drugs).
SUMMARY OF THE INVENTION
The invention is thus a method to create an efficacious cell delivery system
for siRNAs that mimics a naturally occurring process, and the resulting siRNA
delivery system. Many antibiotics and low molecular-weight enzyme inhibitors
have been conjugated to amines. The amine moiety is often crucial for
increasing
biological activity. In many of these compounds, it is the amine moiety that
provides the structural elements required to ferry the conjugate into cells.
In
particular, the antibacterial activities of many antibiotics are due (in part)
to amines
or polyamines conjugated to glycosidic, aromatic, or polyketide moieties. The
cationic polyamine residues function to facilitate transport of the
antibiotics into the
cells. A fitting example is streptomycin (see Fig. 4), which is an
aminoglycoside
wherein the transporting residues are aminoguanidino groups. Similarly, in the
bleomycin-phleomycin group of antibiotics (see Fig. 2), modified spermine and
spermidine groups are the transporting residues. In spermidine-conjugated
antibiotics and antitumorals (such as spergualin, laterosporamine, the edeins,
glisperins A, B,and C, and glycocinamoylspermidines) (see Fig. 3), a
conjugated
spermidine moiety is the transporter of the biologically active agent into the
cells.
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Mention should also be made of the aminoglycoside antibiotics called
kanamycins.
In kanamycins, an amino moiety ferries the glycosides into the cells. This is
also
true of squalamine (see Fig. 7), a broad-spectrum, steroidal antibiotic
isolated from
the tissues of the dogfish shark. In these compounds, a sulfated bile acid is
fused to
the aminopropyl primary amine of spermidine. It is the spermidine portion of
the
molecule that acts to carry the remainder of the molecule across cell
membranes.
In the present invention, natural and/or synthetic polyamine groups are
covalently bonded to siRNA molecules via bonds that either: 1) are broken in
the
cytoplasm and set the siRNA moiety free; or 2) remain intact, while still
enabling
the siRNA portion of the conjugate to bind to the RNA-inducing silencing
complex
(RISC) molecule and thereby to cleave the specific mRNA targeted by the siRNA.
(See Dykxhoorn et al., Nature Reviews, RNAi Collection, p.7, December 2003).
The preferred polyamines for use in the present invention are preferably
spermidine and derivatives thereof, spermine and derivatives thereof, and
hirudonine
and derivatives thereof. See Figs. 1 and 6. Most cells take up polyamines by
carrier-mediated, energy-dependent mechanisms. Many cells (for instance, human
fibroblasts, mouse leukemia cells, rat Morris hepatoma cells, etc.) appear to
have a
single transporter for all polyamines. Thus, as a general proposition, the
specificity
of the transporter is not terribly stringent. For example, it is known from
previous
work that derivatives of polyamines substituted with alkyl substituents are
also
efficiently transported into cells by the same transporter system that
mobilizes the
natural polyamines.
Polyamines are also ideal carriers for siRNAs because of the large binding
affinity of polyamines to ribonucleic acids. Polyamines, especially spermine,
strongly bind to ribosomes, and are constitutive parts of transfer RNAs (t-
RNAs).
The strongly basic polyamines bind to t-RNAs by hydrogen bonds as well as by
electrostatic charges (Frydman et al., Proc. Natl. Acad. Sci (USA) (1992)
89:9186;
Fernandez et al., (1994) Cell. Mol. Biol. 40:93). Transfer RNAs are
ribonucleotides
roughly twice the size of siRNAs, but the ribonucleotide chains have similar
structures. Without being bound to any particular biological mechanism, in t-
RNA
complexes, spermine or spermidine bind to a loop of ribonucleotides; in siRNAs
the
polyamine could bind to and stabilize the annealed double-strand chain of the
siRNA until the siRNA binds to the RNA-inducing silencing complex.
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BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 depicts the chemical structures of putrescine, spermidine, and
spermine.
Fig. 2 depicts the chemical structures of the bleomycin class of compounds.
Fig. 3 depicts the chemical structures of spergualin and edeines A and B.
Fig. 4 depicts the chemical structure of streptomycin.
Fig. 5 depicts the chemical structures of 4-coumaroylagmatine and hordatine
A, B, and M.
Fig. 6 depicts the chemical structure of hirudonine.
Fig. 7 depicts the chemical structure of squalamine.
Fig. 8 is a schematic illustrating the siRNA-mediated, sequence-specific
cleavage of mRNA.
Fig. 9 is a schematic illustrating the siRNA-mediated, sequence-specific
cleavage of mRNA, and illustrating the RNA-inducing silencing complex (RISC).
Figs. 10A and 10B depict the chemical structures of protected
ribonucleosides that can be used to fabricate oligoribonucleotide siRNAs.
Fig. 11 is a reaction scheme illustrating phophoramadite-based
oligoribonucleotide synthesis.
Fig. 12 is a reaction scheme illustrating deprotection of the ribonucleoside
shown in Fig. lOB
Fig. 13 depicts the chemical structure of three different protected
ribonucleosides for use in the present invention.
Fig. 14 depicts a series of polyamine-siRNA conjugates according to the
present invention.
Fig. 15 depicts another series of polyamine-siRNA conjugates according to
the present invention.
Fig. 16 depicts yet another series of polyamine-siRNA conjugates according
to the present invention.
Fig. 17 depicts a general reaction scheme for fabricating polyamine-siRNA
conjugates according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
A first embodiment of the invention is directed to a conjugate comprising a
polyamine covalently bonded to a ribonucleic acid (RNA). It is preferred that
the
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RNA is an oligo-RNA, and most preferably that the RNA is a short interfering
RNA
(siRNA).
The polyamine portion of the conjugate can any polyamine, without
limitation. The preferred polyamines are those selected from the group
consisting of
putrescine, spermine, spermidine, hirudonine, and derivatives thereof.
Explicitly
included within those derivatives are the conformationally restricted
polyamine
compounds disclosed in U.S. Patent Nos. 6,392,098 and 6,794,545. More
specifically, for conformationally-restricted polyamines, the polyamine
portion of
the conjugate is preferably selected from compounds of Fonnula I:
E-NH-D-NH-B-A-B-NH-D-NH-E (I)
wherein A is selected from the group consisting of C2- to C6-alkene and C3-
to C6-cycloalkyl, cycloalkenyl, and cycloaryl;
B is independently selected from the group consisting of a single bond and
Ci- to C6-alkyl and alkenyl;
D is independently selected from the group consisting of C1- to C6-alkyl and
alkenyl, and C3- to C6-cycloalkyl, cycloalkenyl, and cycloaryl; and
E is methyl; and pharmaceutically-suitable salts thereof.
As detailed in greater detail hereinbelow, these conformationally-constrained
polyamines can be made according to the following general reaction scheme: A
compound of Formula II:
HO-B-A-B-OH (II)
is reacted with a protecting reagent, preferrably mesitylenesulfonyl chloride,
to yield
a compound of Formula III:
PROT-O-B-A-B-O PROT (III)
wherein PROT is the protecting group.
Then, the Formula III compound is reacted with a compound of Formula IV:
E-N(PROT) -D-NH-(PROT) (IV)
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to yield a compound of Formula V:
E-N(PROT) -D-N(PROT) -B-A-B-N(PROT) -D-N(PROT)-E (V)
It is much preferred that the protecting group, PROT, in both the Formula III
intermediate and the Formula IV intermediate be a mesitylenesulfonyl moiety.
The Formula V compound is then deprotected to yield a compound of
Formula I (E-NH-D-NH B-A-B-NH-D-NH-E).
A second embodiment of the invention is directed to a method of mobilizing
RNA into a living cell. The method comprises conjugating the RNA to a
polyamine
to yield a conjugate; and then contacting the conjugate to the living cell. It
is
preferred that the RNA is conjugated to a polyamine selected from the group
consisting of putrescine, spermine, spermidine, hirudonine, and derivatives
thereof.
It is also preferred that a siRNA is conjugated to the polyamine.
A third embodiment of the invention is directed to a composition of matter
for mobilizing RNA into a living cell, the composition comprising a polyamine
covalently bonded to a ribonucleic acid (RNA), in combination with a
pharmaceutically suitable carrier.
In the present invention, polyamine transporters are bound to the siRNAs by
collapsible bonds that will release the ribonucleotides as they enter the
cells.
Crafting discrete conjugates of polyamines with siRNAs that are devoid of
systemic
toxicity when administered in vivo, opens the way to study gene function by
modulating naturally occurring processes in mammalian cells, ultimately
leading to
new gene-specific therapeutic agents for treating disease. Thus, the
conjugates
disclosed herein are highly useful for delivering siRNAs from outside a cell
and into
the cytoplasm and/or nucleus.
1. Polyamine Transport of Natural Products into Cells:
The recognition of the ubiquity and essentiality of the polyamines in animal
tissues, as well as the ability to culture such tissues, has resulted in
numerous studies
of polyamine transport in mammalian cells. The inhibition of growth of tumor
cells
by specific inhibitors of polyamine synthesis and the restoration of growth by
exogenous polyamines has been a major stimulus to the study of polyamine
uptake.
For instance, several modes of uptake of [14C]- benzylamine are evident:
adsorption
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to cell surfaces (the adsorbed molecules are removable in large part by
washing); a
saturable, intracellular transport indicative of a specific process; and a
slower,
nonsaturable uptake, suggestive of a mix of diffusion and internal binding
(Cohen, A
Guide to the Polyamines, Oxford, 1998, pp. 467).
There are three natural polyamines present in mammalian cells; putrescine,
spermidine, and spermine (see Fig. 1). As summarized by Seiler and Dezeure
(1990) Int. J. Biochem, 22:211, polyamine uptake in mammalian cells is
generally
specific, saturable, requires energy, and is carrier-mediated. Polyamine
transport
can also be regulated in a process requiring RNA and protein synthesis. A
review of
the data on three membrane receptors of mammals has described marked
similarities
or homologies of these proteins to the numerous transporter proteins for amino
acids
and polyamines isolated from eubacteria and fungi (Reizer et al., Protein
Science
(1993) 2:20). These data suggest that the receptor proteins from mammal cells
may
be described as modified transport proteins sharing a common origin with the
transport proteins of these microbes.
Many cells have a single transporter for putrescine, spermidine, and
spermine, with Km values in the micromolar range. Both sodium-dependent and
sodium-independent systems have been detected. Stressing the therapeutic
importance of polyamine transport in cancer, reviewers pointed to the
decreased
virulence of a leukemia cell line lacking a transport system. Several new
inhibitors
of putrescine uptake inhibit the uptake of this diamine more actively than
that of
spermidine, see Minchin et al. (1991), Eur. J Biochem, 200:457. This also
indicates that the transport of these different polyamines in melanoma cells
are
different.
The uptake of putrescine in human platelets is saturable and energy-
dependent but appears complex. Studies with human erythrocytes, which are
unable
to synthesize the polyamines, demonstrated polyamine receptor sites at the
cell
surface and the uptake of putrescine, spermidine, and spermine largely (>95%)
into
an internal soluble compartment. Moulinoux et al. (1984), Biochimie, 66:385.
The
uptake was minimal at 4 C, and this related mainly to binding to cell stroma.
At
37 C, uptake of putrescine and spermidine was relatively rapid from serum
whereas
spermine entered slowly. The Km values for the saturable putrescine and
spermidine
uptakes from plasma were 125 and 3.6 pM, respectively.
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In one strain of mouse cells, both Na+ and spermidine were shown to enter
cells in a 1:1 relationship. Khan et al. (1990) Cell.Mol. Biol., 36:345. The
transport
appeared to be ATP-independent because it was unaffected by 2-deoxyglucose,
which depletes ATP. An examination of many more strains of mammalian cells
showed that spermidine uptake was affected by Na+ concentration, although
somewhat differently in each case. Khan et al. (1990), Pathobiology, 58:172.
The
uptake was generally inhibited by ionophores and some polyamine analogs.
Preloading the cells with asparagine accelerated putrescine uptake in two
strains, as
well as of putrescine uptake in neuroblastoma cells. Rinehart and Chen (1984)
J.
Biol. Chem. 259:4750. The uptake of putrescine by human colon cancer cells is
stimulated > 300-fold by asparagine. McCormack and Johnson (1991) Am. J
Physiol. 256:G868. The nature of the asparagine effect on putrescine uptake
appears
to be greatly exaggerated in some human cancer cells. It is known that
asparagine
activates a membrane Na+/H+ antiport, provokes an extrusion of H+, and causes
Na+- dependent intracellular alkalinization. Fong and Law (1988) Biochem.
Biophys. Res. Commun. 15 5: 937.
Studies with a wide variety of animal cells indicated the common responses
of polyamine transport mechanisms. Nevertheless, some tissue cells and cancer
cells appear to possess a single common mechanism. In contrast, others, like
the pig
renal LLC-PK cell line, revealed several more discriminating systems. The
latter
cells contained both sodium-dependent and sodium-independent transporters
localized in different cell areas. Van der Bosch et al. (1990), Biochem. J.
265:609.
By taking advantage of the aforementioned transport mechanism of
polyamines into animal cells, different drugs and chemical agents were
covalently
attached to the natural polyamines and were thus ferried into the cells. A
relatively
non-toxic compound, a nitroimidazole-spermidine conjugate entered cells and
inhibited uptake of the free spermidine, see Holley et al. (1992), Biochem.
Pharmacol. 43:763. It can also be mentioned that a spermine conjugate of an
iron
chelator is efficiently transported into tumor cells, even if the terminal
substituents
are quite large. See Bergeron et al. (2003), J. Med. Chem. 46:5478, 2003.
Polyamine conjugates with naphtalimides are also efficiently delivered into
the cells,
Lin et al. (2003), Biochem. Soc. Trans. 31(2):407; indenoisoquinolines
(topoisomerase I inhibitors) conjugated with polyamines are delivered into
cancer
cells, Nagarayan et al. (2003), J. Med. Chem. 46:5712; the spermine conjugate
of
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the protease inhibitor known as the Bowman-Birk inhibitor ("BBI," an 8000 Da
polypeptide) is very effective in localizing BBI in lung and liver, and has
none of the
unacceptable toxicity of the polylysine conjugate, Kennedy et al. (2003),
Pharm.
Research, 20(12):1908.
Natural products are a good sampler of polyamines as vectors for
intracellular delivery. Mention should be made of the bleomycins (see Fig. 2
for the
various chemical structures), with their wide array of polyamine vectors, as
well as
the spermidine conjugates spergualin and the edeins (see Fig. 3), and
streptomycin, a
diguanidino derivative (see Fig. 4). The aminoguanidines found in plants, such
as
cumaroylagmatine, and the hordatines (see Fig. 5) illustrate the importance of
the
strongly basic guanidine residue for transport, a residue also found in
hirudonine
(see Fig.6), an important bacterial and plant polyamine. Squalamine (see Fig.
7) is a
spermidine-steroid, where the spermidine moiety is the delivery vector into
the cells.
All of these natural products include a polyamine moiety.
The present inventor has found that the specificity of the polyamine transport
mechanism is surprisingly permissive. After surveying 24 polyamine-like
compounds, including spermine and homospermine analogues, pentamines and
different oligoamines, it was found that they are efficiently transported into
human
cells. The subject conjugate are thus expected to be efficiently transported
into
mammalian cells, including human cells.
2. Syntheses of Small Interfering RNAs (siRNAs):
Small interfering RNAs (siRNAs) are double-stranded fragments of about
21-23 ribonucleotides. It has been shown that siRNA molecules are the
mediators of
mRNA degradation, and that chemically synthesized duplexes with the fragment
pattern mentioned above are capable of guiding mRNA cleavage. Elbashir et al.
(2001), Genes and Development, 15:188. The currently accepted chain of events
in
the siRNA-mediated cleavage of mRNA is presented schematically in Fig. 8. As
shown in Fig. 8, siRNAs include a paired sense strand (red, shown in 5' to 3'
at the
top of Fig. 8) and an antisense strand (blue, shown 3' to 5' at the top of
Fig. 8), with
a 3' overhang, usually (TT) or (LTU), see reference "a" in Fig. 8. The siRNA
pathway starts when a long double-stranded (ds) RNA is cleaved by the RNase
III
enzyme having the trivial name "Dicer," into siRNAs in an ATP-dependent
reaction.
These siRNAs are then incorporated into the RNA-inducing silencing complex (
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RISC). Once uncoupled, the single-stranded antisense strand of the siRNA
guides
the RISC complex to messenger RNA (mRNA, which is single-stranded) and targets
a complementary sequence of the mRNA. This results in the endonucleolytic
cleavage of the targeted mRNA (as shown in reference "b" of Fig. 8). There are
also, however, data suggesting that in chemically synthesized siRNA duplexes,
both
the sense as well as the antisense strand target the mRNA. This process is
illustrated
schematically in Fig. 9. As shown in Fig. 9, a transfected siRNA is
incorporated
into the RISC, and either the sense or the antisense strand (they are not
delineated in
Fig. 9) can serve to recognize the complementary sequence in the targeted
mRNA.
See Duxbury et al. (2004), J. Surgical Res., 117:339.
Synthesis, purification and annealing of siRNAs by chemical processes is
becoming increasingly popular. See, for example, Micura (2002), Angew. Clrem.
Int. Ed., 41(13):2265; and Hobartner et al. (2003), Monatshefte fur Chemie,
134:851, 2003. Chemically synthesized RNA oligonucleotides are by key
components of siRNA technology. The coupling of the nucleosides is achieved by
conventional and well-known phosphoramidite chemistry as illustrated in Fig.
10.
Because the process is well-known to those skilled in the art, it will not be
described
in great detail. See the citations in the following paragraphs for a full
treatment.
There is also a thriving commercial market in custom RNA synthesis. For
example,
siRNAs of any specified sequence can be purchased from, for example, SynGen,
Inc. (San Carlos, California), Midland Certified Reagent Company, Inc.
(Midland,
Texas), and Dharmacon, Inc. (Boulder, Colorado), among many other companies.
Additionally, many university-based labs also sell custom RNA synthesis
services to
the public (e.g., The University of Wisconsin Biotechnology Center [Madison,
Wisconsin] and Kansas State University [Manhattan, Kansas], among many
others).
Improvements in the structure of suitable protecting groups has taken routine
RNA synthesis to the level of product quality and accessible oligonucleotide
length
as is the case for DNA synthesis. The need for robust RNA synthesis strategies
resulted in the crafting of new and sophisticated protecting groups. These
procedures were introduced in 1998 and are covered by various patents (see,
for
example, Pitsch et al., US Patent 5,986,084), and described in the scientific
literature
(Pitsch et al., (2001) Helv. Chim Acta, 84:3773. The most common procedure
(used
in commercial automated DNA synthesizers after some technical adjustments)
makes use of the "TOM" protecting group ( 2'-O-triisopropylsilyloxymethyl)
(see
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Fig. 10A, which is a protected ribonucleoside with the TOM protecting group at
the
2'-O position). This protecting group is superior to the classical 2'-O-tert-
butyldimethylsilyl (TBDMS) group used in DNA synthesis.
The construction of a protected nucleoside such as shown in Fig. 10A starts
with the N-acetylation at the exocyclic amino groups of the nucleobases of the
ribonucleosides, followed by tritylation at the 5' oxygen to give 5'-O-DMT.
This is
then followed by "TOMylation" at 2' oxygen to give the 2'-O-TOM derivative.
Lastly, a"phosphitylation" step at the 3' oxygen using 2-cyanoethyl
diisopropylphosphoramido chloridite yields the 3'-O-CEPA derivative shown in
Fig.
10A. The incorporation of the phosphoramidites into oligoribonucleotides is
well
documented. See Micura et al. (2001), Nucleic Acid Res. 29:3997; Hobartner et
al.
(2002), Angew. Clhem. Int. Ed. 41:605; Micura et al. (2000), Angew. Chem. Int.
Ed.
39:922; Micura et al. (2001), Nucleosides, Nucleotides, Nucleic Acids,
20:1287; and
Ebert et al. (2000), Helv. Chim. Acta, 83:2238).
Another building block for oligoribonucleotides is depicted in Fig. l OB.
This protected ribonucleoside was first reported in 1998 in U.S. Patent No.
6,111,086, see also Scaringe (2000), Methods in Enzymology, 317:3. The
rationale
behind the protected nucleoside shown in Fig. lOB was the desire for a mildly
acidic
aqueous condition for the final deprotection at the 2'-O group of the
synthetic
ribonucleotide. This cannot be achieved if the protecting group at 5'-O is
DMT,
which is itself labile to mild acidic conditions. DMT was therefore replaced
with the
5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD), together with the
2'-0-
bis(2-acetoxyethyloxy)methyl (ACE) orthoester. Herein the 3'-OH group is
derivatized as the methyl-N,N-diisopropylphosphoramidite because the
cyanoethyl
group (used in the TOM-protected nucleosides of Fig. 10A) proved to be
unstable to
the fluoride reagents needed to cleave DOD. The coupling yields with this
protected
ribonucleoside are higher than 99%.
After the oligonucleotide assembly, the phosphate methyl protecting groups
are removed with disodium 2-carbamoyl-2- cyanoethylene-1,1-dithiolate
trihydrate
(S2Na2) in DMF (see Fig.12). Then basic" conditions (40% aqueous methylamine)
cause oligonucleotide cleavage from the solid support, along with the removal
of the
acyl protecting groups on the exocyclic amino groups and the acetyl groups on
the
2'-orthoesters. The resulting 2'-O-bis(2-hydroxyethyloxy) methyl orthoesters
are
ten times more acid labile than before the removal of the acetyl groups. As a
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consequence, very mild conditions (pH 3.8, 30 min, 60 C) are all that is
required for
the final deprotection step. The 5'-O- DOD group is cleaved with fluoride
reagents.
The published HPLC chromatograms of the crude oligoribonucleotides obtained by
this procedure are impressive, exhibiting almost no by-products.
The general scheme of the automated synthesis of oligoribonucleotides is
depicted in Fig.11 (PG= protecting group). The synthesis starts from the 3'-
end by
attachment to the resin. Deprotection is achieved in two steps, without
degradation
of the RNA products, first with CH3NH2 in ethanol/water, followed by Bu4NF in
tetrahydrofuran. The process can be repeated up to about a 150-mer product
before
significant product degradation results.
3. Synthesis of Conformationally-Constrained Polyamines:
A host of naturally occurring polyamines, including putrescine, spermine,
spermidine, and hirudonine can be purchased commercially from a number of
worldwide suppliers such as Aldrich Chemical Co, Milwaukee, Wisconsin and
Fisher Scientific, Hampton, New Hampshire. The Aldrich catalog numbers are:
putrescine (D1,320-8), spermine (S383-6), and spermidine (S382-8). The Fisher
catalog number for hirudonine (diguanylspermidine, CAS No. 2465-97-6) is
ICN222595.
Conformationally-constrained polyamines suitable for use in the present
invention are preferably synthesized as disclosed in U.S. Patent Nos.
6,392,098 and
6,794,545. Briefly, various rigid moieties, either cyclic moieties or double-
or triple-
bonded moieties, are introduced into the backbone of a polyamine.
The first targeted location was the central 1,4-diaminobutane segment of a
polyamine. In its staggered conformation, four semi-eclipsed conformational
rotamers are possible around the diaminobutane segment. The four have
enantiomeric relationships. Introduction of a bond between the C-1 and C-3
positions or the C-2 and C-4 positions of the central diaminobutane segment
generates a cyclopropane ring. Introduction of an additional bond between the
C-2
and C-3 positions generates a conformationally restricted alkene derivative.
Cyclobutyl, cyclopentyl, and cyclohexyl moieties can be introduced into the
structure following the same strategy.
Using this approach four conformationally semi-rigid structures were
obtained which mimic the four semi-eclipsed conformational structures of
spermine.
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Two of the semi-rigid structures are epimers of the other two.
For purposes of the present invention, it is important to note that the cis
and
trans isomers of the subject compounds assume very distinct three-dimensional
conformations due to the restricted bond rotation afforded by the centrally-
located
ring structure or unsaturation. All geometric isomers (optically active or
otherwise),
including pure isolated cis forms and pure isolated trans forms of the
polyamines,
and mixtures thereof, are explicitly within the scope of the present
invention.
Additionally, all positional isomers of the subject compounds are explicitly
within
the scope of the present invention. When A or D of formula I is a cyclical
moiety,
the two B substituents or the amino moieties, respectively, may be oriented in
the
1,2 or 1,3 or 1,4 position with respect to each other.
(a) Spermine Analogs Containing a Cyclopropyl Ring:
Cis and trans cyclopropyl analogs of spermine were prepared via the
reactions illustrated in Schemes 1, 2, 3, 4, 4A, 5, and 5A, hereinbelow.
With reference to Schemes 1 and 2, the cyclopropyl diesters 1 and 2 were
first converted into their hydrazides 103 and 4, and the hydrazides converted
into the
diamines 5 and 6, respectively. The diamines 5 and 6 were then mesitylated to
give
the amides 7 and 8, and the amides were then alkylated with 9 to give 10 and
11,
respectively. Hydrolysis of the protective groups yielded the trans analog 12
and
the cis analog 13.
Referring now to Scheme 3, in a separate reaction, the trans cyclopropyl
diester 1 was converted into the amide 14 by reaction with benzylamine
(BnNH2),
the amide reduced to the amine 15, and the amine alkylated to 16. The phthalyl
residues were then cleaved with hydrazine to give 17. Compound 17 was then
either
deprotected by hydrogenolysis to give 18; or fully alklyated to 19, and the
benzyl
residues cleaved by hydrogenolysis to give 20.
With reference to Scheme 4, the amine 15 was also alkylated with 21 to give
22. Compound 22 was then deprotected to yield the trans cyclopropyl analog 23.
An alternative (and preferred) route io 23 is given in Scheme 4A. Here, 3-
ethylamino propionitrile 101 was converted into the corresponding amine 102,
which was then mesitylated to yield 3. In a parallel synthesis, the cis
diester 1 was
reduced to the dialcohol 15', which was then mesitylated to yield the
dimesityl
derivative 16'. Reacting 3 and 16' in the presence of sodium hydride yields
22'.
13
CA 02581869 2007-03-30
WO 2006/052854 PCT/US2005/040227
c
CT
.~~
PNl
v
N t~ ,2 z
:I.
n
w ~ U
w ~
U
~z xz
z
pH
x
w ~--~~
zcn zx
.-~
7~ zx
C
14
CA 02581869 2007-03-30
WO 2006/052854 PCT/US2005/040227
c~
~ 0
M S~ ~ y
z
sx5
~ -y
~.
z
-4xx
, z
N
~ z
8 v; d
U P ~ N ~
0
zcn zx
N N ~
z z'"'
x x
zw zx
N z~ zx
d'' (
61
O
SCIIEME 3
CON'HBn
C02Et BnNH~ LIAI[I~ BnHN NHBn v~i
EtO2C BnI3NOC 11 is
K;,CO3, BrN,"_~. NPth
DMPU
Bn /'~~h~iz IIzNNHy Bn 0
N Id ~- PthN~ /~ ~ N'~ NPth ~'
\~ L 15 Bn
H2N 17 Bn ti
N
Ui
CD
\112,Td C, AcOH and HCI ~
= EtBr,K2C03 0)
tD
0
0
I
0
W
Nn i;.~
4 B NiNHZ 4EIC1 0
ta
HZ/Pd on C, AcOH and HCI
y 4EIC1
20 H
CA 02581869 2007-03-30
WO 2006/052854 PCT/US2005/040227
SC'.HEMF 4
Ba 5
iN NEiBn +
21
KeCO;, DMPU
B II NIl N ~r \
/'',Z Ba Bn
I1Z/Pd on C, AcOfi aad IICI
H H N~~
/ N '~ ~ -~~- H
17
O
SCHEME 4A
~/\ H I,lA1H4 H MecSOZCI SO2Mcs
\/NHz + ~ CN ~/N\~~C?v 1HF ~/N~~NHz KOH ~/N\~~NHSOZ~ies
101 102 3
0
N
tn
OD
I--~ F
CA _ eD
C02Et LiA1FIi MessO2CI NaI3 ~
HO ' OII MesO2SO U502Mea DMF
P.tO2C 1 15 Pyridinc 16' 0
0
0
w
w
0
1 EIDr/As,OH, PhOll,
2 HCI SO2Mes S
ti N~\N~~ "1 N N
h
~23 H 4HCl Z~~~SOWCS ~/ SO2Mcs
22'
CA 02581869 2007-03-30
WO 2006/052854 PCT/US2005/040227
~
mU p
O
~ N N
O
g.~'-'~,~1s-
ze
x O~
a C~
az
W m
~ N\ wz
~zI
" V.
9z
~Y ~M gg
~~ wr
O~
sr~
. N, . ;f~ õ ,o z xz
19
O
SCHEME 5A
~ I,iA1H4 MesSO2C1
-~ HO OH PYndinO MasC}2S0 OSOzjvJes
EtO2C CO2Et ~
2 24 25'
0
N
Ln
OD
NaH p
OD
3 U~fF ~
N
0
0
0
1 HBr!AcOH, PhOH, w
2 HC1 0
Fi fi H H SO2Mes SO2Mes SOZtvfes SO2Mes
4HCI
28 z7
rA
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In the same fashion as Scheme 4, 22' was then deprotected to yield the trans
cyclopropyl analog 23.
Referring now to Scheme 5, in a separate reaction, the cis cyclopropyl diester
2 was reduced to the dialcohol 24. The dialcohol was then converted into the
amine
25, and the amine protected by mesitylation to 26. Compound 26 was then
alkylated
with 9 to yield 27, and then deprotected to yield the cis cyclopropyl
tetramine 28.
An alternative (and preferred) route to 28 is given in Scheme 5A. Here, the
cis cyclopropyl diester 2 is reduced to the dialcohol 24 in the same fashion
as in
Scheme 5. Compound 24 was then protected by mesitylation to yield 25'.
Compound 25 was then reacted with 3 to yield 27. Deprotecting yields the
tetramine
28.
(b) Spermine Analogs Containing a Cyclobutyl Ring:
Cis and trans cyclobutyl analogs of spermine were prepared via the reactions
illustrated in Schemes 6, 7, 8, 8A, 9, and 9A, hereinbelow.
Referring now to Schemes 6 and 7, the synthesis of the cyclobutyl
derivatives started with the trans and cis 1,2-diaminobutanes 29 and 30,
respectively. These compounds were first converted to the amides 31 and 32,
and
then alkylated to 33 and 34, respectively. Compounds 33 and 34 were then
deprotected to yield the trans tetramine 35 (Scheme 6) and the cis tetramine
36
(Scheme 7).
With reference to Schemes 8 and 9, in separate reactions, the trans
cyclobutyl diester 37 and the cis cyclobutyl diester 38 were reduced to the
respective
dialcohols 39 and 40, the dialcohols converted into the diamines 41 and 42.
The
diamines 41 and 42 were then protected by mesitylation to yield 43 and 44,
respectively. These compounds were then alkylated to give 45 and 46. The
protecting groups were then removed to yield the trans cyclobutyl tetramine 47
(Scheme 8) and the cis tetramine 48 (Scheme 8).
21
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SCHEME 6
MesSOlCI,
KOH,
A Dioxane, H20
H3C1 JIILH502Mes
olH3 'N MzsO2SSH\
29 31
SO2'vles
$r N~ NaH/DMfi
9
SO2Mes SO22v1es
~NN /
N\v v N\/
SO2N1es SO2Mes 33
~HI3rIAcOFr
HCI
4HCI
I H 35
22
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WO 2006/052854 PCT/US2005/040227
SC:}iEMf ?
.LfcsSO2Ci,
KOf-i,
Dioxane, Hy0
~ NE~SO2'vi~
elEi3 a N~f3Cl MesOyS
30 32
SOZMes
aI n~ NaEi,~DMF
9
~ ~ / / \ -~/~ =~~
SQZMe v S02Mos SOzt~4cs SOzMes
34
(IiBuAcOii
{i HCi
4HC!
i H 36
23
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WO 2006/052854 PCT/US2005/040227
6
o ~
r
zq,
0
U v
A u \I\
a ~
z ~Z w
~ zx
z-2
0
U
cl'
V q ~ ~y
2~ xz
0 ~
~
24
O
SC.HF.NIF 8A
CY>;E t Lir11I-Td MesSOZC!
HO OH Pyridinc mcs0-.~S0 OSOZr4cs o
E10Q,C
37 39 41' a~o
F-'
m
3 NaH ~
DMF
N
0
0
i HBr!AcOH,
w
H 2 H PH,
SO2Mes SC)%y1es 0
H
; H 4 HCI ~-- \/ N,~ SO2jvle o2Mes o
47 45
O
SCHEtviE 9
T,iAII-14 [:: HN3, PPh3
-~ -~
F.tO,-C C02El HO OH olH3FN NH3Cl
38 40 42
Me6502C1,
KOfL o
L)ioxane, H~O
m
N
SOzNics
tD
N
S02Mes SOZMes SO2?des SOZMes 9 o
NaH1DMF 10
VIesOnSHN NHSOZVies ,
46 44 ia
0
I HBr/AcOH
HCI
r' 4HCI 48
CA 02581869 2007-03-30
WO 2006/052854 PCT/US2005/040227
JJ \
V \/\P\
OZ
U
~ y
~
0 cn
Za
O
N
~ry
c'/-~N+
J ) ~
cf.,
rn x ~ :3
w 0 U
,G w C+ x
[Ll . N
xz
O
xz
Q
u
xz
w xz
., ~
27
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Alternative (and preferred) routes to 47 and 48 are given in Schemes 8A and
9A, respectively. The cis and trans diesters 37 and 38 were reduced to the
respective
dialcohols 39 and 40 in the same fashion as in Schemes 8 and 9. Compounds 39
and
40 were then mesitylated to yield 41' and 42', respectively. Reaction of 41'
and 42'
with 3 yields 45 (Scheme 8A) and 46 (Scheme 9A). Deprotecting yields the
desired
products 47 and 48.
(c) Spermine Analogs Containing an Unsaturation:
Cis and trans unsaturated analogs of spermine were prepared via the
reactions illustrated in Schemes 10, 10A, 11, and 1 lA.
Referring to Scheme 10, the trans diester 49 was reduced to the dialcoho150,
which was then converted into the trans diamine 51. Referring to Scheme 11,
the
cis diamine 52 was obtained from the commercially available cis dialcoho143'.
With reference to both Scheme 10 and Scheme 11, compounds 51 and 52 were
protected by mesitylation to give 53 and 54, respectively. Compounds 53 and 54
were alkylated to 55 and 56, and lastly deptrotected to yield the trans
tetramine 57
(Scheme 10) and the cis tetramine 58 (Scheme 11).
Alternative (and preferred) routes to 57 and 58 are given in Schemes l0A
and 11A, respectively. The cis and trans dialcohols 50' and 50 were obtained
in the
same fashion as in Schemes 10 and 11. Compounds 50' and 50 were then
mesitylated to yield 51' and 52', respectively. Reaction of 51' and 52' with 3
yields
55 (Scheme l0A) and 56 (Scheme 11A). Deprotecting yields the desired products
57 and 58.
Following the above general protocols, and using suitable and well known
starting reagents, all of the compounds of Formula I, including those where A
and D
are independently C5- or C6-cycloalkyl, cycloalkenyl, or cycloaryl, can be
readily
obtained. An illustrative listing of compounds of Formula I are presented in
Table
1.
28
O
SCHF3ME 10
HN3, PYh3,
~sE~ DIBALF H.O OH FI C~ CIHgN o 3CJ
EtUZC
49 50 51
MesSO2CI, ~
KOFI, 0
I)ioxane, H20
N
Ln
m
F-'
SOzMes ~
N
S02Mcs SO2vlcs q 0
N S);MSO,M Se NnH=/DMF Mes02SHN NHSOZMcs
0
55 53 w
w
0
f HBr/AcOH
t( HCl
H H
V ' ' \
ri V' Y N 4HCl
57
rA
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WO 2006/052854 PCT/US2005/040227
r
a
O
CO
1 M ~ N I N
Ve,
0
3
0
a~
y
~
-+ N
~
U
N x
zx
Q '~x
~ ( h
0
x7
J
~N
V_
,~. LLiFr,
I
30 _
O
ao
SCHEMF. 11
Mes5O2C.'1,
HN3, PPh3, KOH,
liCl Dioxane, H;O
HO OH 51H3N H3Ce1 --= MesOZSHN NHSO22vfcs
{0 52 54
~
SOZMes o
m
cn
F-'
9 rn
NaH/DMb' tD
N
0
0
SOzMcs S4zMcs SO2Mes SO2Mcs
56 0
HBr/AcOH
HCI
4HCI
58
= o
O
ao
SCHF.ME 17A
MesSO2C1
fI0 OH 50% KOH MesO"SO~ /OSOZMes
0
Sil' $G N
Ln
CD
INall ~
3~
N
0
1 HBr/AcOH, 0
II f EI EI 2 HCOI I~ SO2Mes SOZMes SOZMes SOZMes o
~~N~~/~~~~N~~~N,\ 4HC1 ~-- ~~N~~N~/-~~~N'~/~~N~~ w
w
0
58 56
= o
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TABLE .1
Compound
No.
12 H H SL-11027
~ /~ ~N~N v IIC1
N~ ~/ ~h
H H
13 SL-11033
El H H H 4HCI
2-1 SL-11038
H H
H 41-10
28 SL-11037
I-I Ii Ei EI
\/N~ ~ N
4HC1
35 H H SL-11028
N,,/4 HCI
fH H
36 JD" SL-1 1034
NN
EI I-I H H 4 E ECI
47 i.S-l 1il44
EIH 4EICI
48 SI,11043
H H H EI
N\/ N
N~~/Nj~~4HC1
57 H ET SL-11048
N H H 4HC1
58 H H H H SL-11047
N ~ /N --- N~NT~4 HCI
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The pure conjugates, as well as pharmaceutically-suitable salts thereof, are
explicitly within the scope of the present invention. By the term
"pharmaceutically-
suitable salts" is meant any salt form of the subject conjugates which renders
them
more amenable to administration by a chosen route. A wide range of such salts
are
well known to those of skill in the pharmaceutical art. The preferred
pharmaceutically-suitable salts are acid addition salts such as chlorides,
bromides,
iodides and the like.
4. Syntheses of Polyamine Conjugates of siRNAs:
Based on the above mentioned data, the three nucleosides shown in Fig. 13
can be prepared. Nucleoside A is a 2'-O- TOM nucleoside, nucleoside B is a 2'-
O-
ACE nucleoside, and nucleoside C is a 5'- thiol nucleoside. Nucleoside C is
prepared from 5'- thioriboside that is acetylated in its nucleobase, converted
into its
2'-O-ACE derivative and finally phosphitylated at 3' position.
Nucleoside B is attached to a polyamine chain at the 5'-O. The linker is
preferably a carbamate bond (as shown in Fig. 14). This linker can be affixed
either
via a chloroformate on the 5'-O, or by adding an the alcohol to an alkyl
isocyanate.
In either case, the polyamine residue will be attached to the ribose by a
carbamate
bond. The polyamine chains are protected with alkali-labile protecting groups,
such
as FMOC, trifluoroacetate, and the like. As the oligonucleotide chain grows
from
the 3'-end to the 5'-end (as shown in Fig. 11), the polyamine conjugated
nucleoside
will be attached in the last step of the synthesis. Release of the
oligonucleotide
from the resin under mild alkaline conditions (as shown in Fig. 10) will also
cleave
the protecting groups on the polyamine chain. The mild acid conditions
necessary to
free the 2'-O will not affect the carbamate bond, and an oligonucleotide
covalently
bound to a polyamine residue will be obtained. The resulting polyamine-RNA
conjugates are ferried into living cells in the same fashion as other
polyamine
conjugates are.
Regarding annealing, the strands of a double-strand siRNAs are constructed
independently; the sense strand is constructed with the desired sequence of
nucleotides, then the antisense strand is constructed with the corresponding
complementary bases. The single strands are incubated together (pH 7.4, 1 min,
90 C) to form the duplex. This pairing is known as annealing the siRNAs. In
the
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preferred embodiment of the present invention, the polyamine moiety is
attached to
the 5'-O of the sense strand (see Fig. 8).
Nucleoside C is constructed using 5'-thioribose. Polyamine derivatized
residues (see Fig. 15 for an exemplary list of preferred residues) are
attached to short
thioalkyl linkers. The N-thioethyl polyamine residues exemplified in Fig. 15
can be
obtained starting with S-benzylcysteinamine, and then building up the
polyamine
chain by successive alkylations following established procedures. Valasinas et
al.
(2003) and references therein), and finally by deprotecting the thiol group
using
hydrogenolysis to cleave the benzyl group.
The synthesis of the disulfide-linked polyamine to the 5'-S-
oligoribonucleoside is achieved by treatment of the mixture with diamide, a
known
thiol oxidant. The conjugated nucleoside is then attached to a sense
oligoribonucleotide chain as discussed hereinabove. The sense
oligoribonucleotide
chain is deprotected at the 2'-O position, the polyamine protecting groups are
cleaved, and the strand is thenannealed to the complementary antisense strand.
While not being limited to any particular biological mechanism or phenomenon,
the
rationale behind this approach is as follows: the polyamine will facilitate
the
transport across the plasma membrane of the siRNA duplex, and the conjugate
will
be freely translocated into the cytoplasm. The disulfide bond will then be
reduced in
the cytoplasm by thiols, thereby releasing the siRNA portion of the conjugate.
The
released siRNA will proceed to cause the sequence-specific mRNA degradation
that
it was designed to achieve based upon its pre-determined sequence.
Even if the conjugate remains intact after delivery into the cell, the two
strands of the siRNA will partially dissociate at the RISC after delivery of
the
conjugate to the cytoplasm. This will not affect the function of the siRNA
duplex,
as single-strand antisense siRNAs are able to silence endogenous gene
expression in
cells..
The third approach to the synthesis of a polyamine conjugate of an
oligoribonucleotide uses a linker that successfully mimics a peptidase and
sets free
amides and esters bound to it by way of an intramolecular-catalyzed cleavage.
The
polyamine chain and the nucleoside are linked through Kemp's triacid (Kemp and
Petrakis (1981), J. Org. Chem. 46:5140). This remarkable triacid has three
carboxylates in an all-axial orientation. One carboxyl is bound to an amine
(as an
amide), and a second carboxyl forms an ester with an alcohol. At a pH of about
6,
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the molecule will first release the amine residue via the intramolecular
formation of
an anhydride. The alcohol will then be released by rearrangement of the
intramolecular anhydride (see Fig. 16). When entering the cytoplasm, the
conjugate
will be confronted by a variety of peptidases and lysosomal enzymes that will
cleave
both the amide bond and the ester bond. This cleavage is assisted by the axial
geometry of the carboxylates. Relief of internal compression during anhydride
formation thus contributes to the enzyme-driven acceleration of hydrolysis.
The van
der Waals contact distances in Kemp's acid are very short, and energetically
costly
desolvation processes can thus be averted.
The synthesis uses Kemp's acid chloride-anhydride, see Fig. 16. By reaction
with a polyamine, a polyamide is formed. Ring opening of the anhydride with
the
5'-O alcohol of nucleoside A yields a Kemp acid substituted with an amide and
an
ester. The Kemp acid thus substituted with an amide and an ester of nucleoside
A is
then coupled to the growing edge of a sense ribonucleotide (e.g., see Fig. 11)
through the 3'-O-CEPA group. The TOM protecting group, the N-acetyl, and the
protecting groups on the polyamine are cleaved in mild alkali. The ester amide
ribonucleotide is then annealed with the antisense strand and transported into
the
cytoplasm.
It is expected that the polyamide linkage will be cleaved first by cytoplasmic
peptidases that will free the oligoribonucleotide from the polyamine chain.
Cleavage of the ester group by the cell's esterases will follow with release
of the
siRNA duplex. Even if the latter hydrolysis is slow ( hours), it should be
pointed out
that siRNAs linger in the cells for hours without degradation. Thus there is
enough
time to achieve hydrolysis of both amide and ester linkages. The disubstituted
Kemp acid is stable in plasma at pH 7.4. Nucleoside A is preferred in this
synthetic
sequence and not nucleoside B because of the sensitivity of the orthoester
protecting
group at 2'-O in nucleoside B to low pH conditions (and also to avoid cleavage
at
the substituted Kemp acid during deprotection of the conjugated
oligonucleotide).
The fourth approach to conjugate a polyamine with an oligoribonucleotide
will be based on the construction of a connector linkage that collapses affter
a
sequence of hydrolytic steps; the first involving an enzymatic cleavage and
the
second involving a solvolysis that proceeds spontaneously after the first step
occurs.
The connector linkage is constructed as shown in Fig. 17. A polyamine unit
(protected with acid stable groups; e.g., trifluoroacetate) will be bound to
the a-
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CA 02581869 2007-03-30
WO 2006/052854 PCT/US2005/040227
amino group of lysine (PG1 could be E- FMOC) through a carbamate bond; the
lysine is then converted into its corresponding amide by treatment with p-
aminobenzyl alcohol. Addition of the benzyl alcohol to p-nitrophenyl
isocyanate
results in the p-nitroanilide 1 a. Cleavage of the protecting group at the a-
amino
residue gives lb.
Compound lb will undergo rapid hydrolysis in the presence of trypsin with
release of p-nitroaniline. In the present invention, the p-nitroaniline
residue is
replaced with nucleoside A via a simple displacement reaction (see Fig. 15).
After
deprotection of PG1 at the a-amino group of the lysine residue to give 2a, the
amide
bond at the lysine residue can be hydrolyzed by trypsin and/or the lysosomal
proteases cathepsins B and L, thereby releasing benzyl carbamate 3 (see Fig.
17),
that will undergo spontaneous solvolysis to nucleoside A and p-aminobenzyl
alcohol.
In short, compound 2a is used as the last step of an oligonucleotide
construction to obtain a sense ribonucleotide strand bound through a
collapsible
linker to the polyamine chain. Cleavage of the TOM protecting groups, as well
as of
the polyamine protecting groups and PG1 at the a-lysine, followed by annealing
with the complementary RNA strand, affords a conjugated siRNA complex that is
delivered into living cell and then collapses into its component moieties
after
hydrolysis.
5. Administration and Pharmaceutical Unit Dosage Forms:
Because the above-described conjugates are effective to moblize RNAs into
living cells, the conjugates are suitable for therapeutically treating of
mammals in
vivo, including humans, and for treating mammalian cells in vitro, in any
treatment
regimen requiring the mobilization of RNA into mammalian cells. In short, the
conjugates are useful for moving RNA, including siRNA from an extracellular
space
into the cytoplasm of a mamallian cell.
Administration of the subject complexes to a human or non-human patient
can be accomplished by any means known in the pharmaceutical arts. The
preferred
administration route is parenteral, including intravenous administration,
intraarterial
administration, intratumor administration, intramuscular administration,
intraperitoneal administration, and subcutaneous administration, either neat
or in
combination with a pharmaceutical carrier suitable for the chosen
administration
37
CA 02581869 2007-03-30
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route. The treatment method is also amenable to oral administration.
It must be noted, as with all pharmaceuticals, the concentration or amount of
the polyamine-RNA conjugate administered will vary depending upon the severity
of the ailment being treated, the mode of administration, the condition and
age of the
subject being treated, and the particular polyamine-RNA conjugate or
combination
of conjugates being used.
The conjugates described herein are administratable in the form of tablets,
pills, powder mixtures, capsules, injectables, solutions, suppositories,
emulsions,
dispersions, food premixes, and in other suitable forms. The pharmaceutical
dosage
form which contains the conjugates described herein is conveniently admixed
with a
non-toxic pharmaceutical organic carrier or a non-toxic pharmaceutical
inorganic
carrier. Typical pharmaceutically-acceptable carriers include, for example,
mannitol, urea, dextrans, lactose, potato and maize starches, magnesium
stearate,
talc, vegetable oils, polyalkylene glycols, ethyl cellulose,
poly(vinylpyrrolidone),
calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium
carbonate, gelatin, potassium carbonate, silicic acid, and other
conventionally
employed acceptable carriers. The pharmaceutical dosage form may also contain
non-toxic auxiliary substances such as emulsifying, preserving, or wetting
agents,
and the like.
Solid forms, such as tablets, capsules and powders, can be fabricated using
conventional tabletting and capsule-filling machinery, which is well known in
the
art. Solid dosage forms may contain any number of additional non-active
ingredients known to the art, including excipients, lubricants, dessicants,
binders,
colorants, disintegrating agents, dry-flow modifiers, preservatives, and the
like.
Liquid forms for ingestion can be formulated using known liquid carriers,
including aqueous and non-aqueous carriers, suspensions, oil-in-water and/or
water-
in-oil emulsions, and the like. Liquid formulation may also contain any number
of
additional non-active ingredients, including colorants, fragrance, flavorings,
viscosity modifiers, preservatives, stabilizers, and the like.
For parenteral administration, the subject conjugates may be adminisfered as
injectable dosages of a solution or suspension of the conjugate in a
physiologically-
acceptable diluent or sterile liquid carrier such as water or oil, with or
without
additional surfactants or adjuvants. An illustrative list of carrier oils
would include
animal and vegetable oils (peanut oil, soy bean oil), petroleum-derived oils
(mineral
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WO 2006/052854 PCT/US2005/040227
oil), and synthetic oils. In general, for injectable unit doses, water,
saline, aqueous
dextrose and related sugar solutions, and ethanol and glycol solutions such as
propylene glycol or polyethylene glycol are preferred liquid carriers.
The pharmaceutical unit dosage chosen is preferably fabricated and
administered to provide a concentration of conjugate at the point of contact
with the
target cell of from, for example, about 1 M to about 10 mM. More preferred is
a
concentration of from about 1 Ea1VI to about 100 M. This concentration will,
of
course, depend on the chosen route of administration and the mass of the
subject
being treated. Concentrations above and below the above-stated ranges are
within
the scope of the invention.
39
