EP1817324A2 - Syntheses de conjugues polyaminiques d'arn interferents courts (arnic), et conjugues ainsi obtenus - Google Patents

Syntheses de conjugues polyaminiques d'arn interferents courts (arnic), et conjugues ainsi obtenus

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EP1817324A2
EP1817324A2 EP05825874A EP05825874A EP1817324A2 EP 1817324 A2 EP1817324 A2 EP 1817324A2 EP 05825874 A EP05825874 A EP 05825874A EP 05825874 A EP05825874 A EP 05825874A EP 1817324 A2 EP1817324 A2 EP 1817324A2
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polyamine
rna
group
conjugate
sirna
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Raul Andino
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • si-RNAs Small Interfering RNAs
  • 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.
  • RNA ribonucleic acids
  • siRNAs Short interfering RNAs
  • This enthusiasm is due to the fact that siRNAs have been shown to be efficacious in specifically suppressing the expression of targeted genes.
  • 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).
  • mRNAs messenger RNAs
  • 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 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.
  • polyethyleneimines are difficult to deliver into cells because they are high molecular- weight polymers.
  • 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).
  • 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.
  • 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.
  • spermidine 4 which is an aminoglycoside wherein the transporting residues are aminoguanidino groups.
  • modified spermine and spermidine groups are the transporting residues.
  • spermidine-conjugated antibiotics and antitumorals such as spergualin, laterosporamine, the edeins, glisperins A, B,and C, and glycocinamoylspermidines
  • a conjugated spermidine moiety is the transporter of the biologically active agent into the cells. Mention should also be made of the aminoglycoside antibiotics called kanamycins.
  • 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.
  • 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.
  • 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.
  • RISC RNA-inducing silencing complex
  • 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 notably 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. Sd (USA) (1992) 89:9186; Fernandez et al., (1994) Cell. MoI. Biol. 40:93).
  • Transfer RNAs are ribonucleotides roughly twice the size of siRNAs, but the ribonucleotide chains have similar structures.
  • 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.
  • 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).
  • RISC RNA-inducing silencing complex
  • Figs. 1OA and 1OB 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. 1OB
  • 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.
  • 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 RKA is an oligo-RNA, and most preferably that the RNA is a short interfering RNA (siRNA).
  • siRNA short interfering RNA
  • 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 Formula I:
  • A is selected from the group consisting OfC 2 - to C 6 -alkene and C 3 - to C 6 -cycloalkyl, cycloalkenyl, and cycloaryl;
  • B is independently selected from the group consisting of a single bond and Ci- to C 6 -alkyl and alkenyl;
  • D is independently selected from the group consisting OfC 1 - to C 6 -alkyl and alkenyl, and C 3 - to C 6 -cycloalkyl, cycloalkenyl, and cycloaryl;
  • E is methyl; and pharmaceutically-suitable salts thereof.
  • PROT is the protecting group.
  • the protecting group, PROT in both the Formula III intermediate and the Formula IV intermediate be a mesitylenesulfonyl moiety.
  • 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.
  • the RNA is conjugated to a polyamine selected from the group consisting of putrescine, spermine, spermidine, hirudonine, and derivatives thereof.
  • 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.
  • RNA ribonucleic acid
  • polyamine transporters are bound to the siRNAs by collapsible bonds that will release the ribonucleotides as they enter the cells.
  • the conjugates disclosed herein are highly useful for delivering siRNAs from outside a cell and into the cytoplasm and/or nucleus.
  • the K m values for the saturable putrescine and spermidine uptakes from plasma were 125 and 3.6 ⁇ M, respectively.
  • both Na+ and spermidine were shown to enter cells in a 1:1 relationship.
  • 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.
  • 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.
  • siRNAs 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.
  • siRNA pathway starts when a long double-stranded (ds) RNA is cleaved by the RNase m enzyme having the trivial name "Dicer,” into siRNAs in an ATP-dependent reaction. These siRNAs are then incorporated into the RNA-inducing silencing complex (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.
  • ds double-stranded
  • RISC RNA-inducing silencing complex
  • 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.
  • 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.
  • 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).
  • RNA synthesis 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) He/v. CMm 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 Fig.
  • a protected nucleoside such as shown in Fig. 1OA 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'-0-DMT. This is then followed by "TOMylation” at 2' oxygen to give the 2'-0-TOM derivative. Lastly, a "phosphitylation” step at the 3' oxygen using 2-cyanoethyl diisopropylphosphoramido chloridite yields the 3'-0-CEPA derivative shown in Fig. 1OA. The incorporation of the phosphoramidites into oligoribonucleotides is well documented. See Micura et al.
  • Fig. 1OB Another building block for oligoribonucleotides is depicted in Fig. 1OB.
  • 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. 1OB was the desire for a mildly acidic aqueous condition for the final deprotection at the 2'-0 group of the synthetic ribonucleotide. This cannot be achieved if the protecting group at 5'-0 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'-O- bis(2-acetoxyethyloxy)methyl (ACE) orthoester.
  • DOD 5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether
  • ACE 2-acetoxyethyloxymethyl
  • 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%.
  • the phosphate methyl protecting groups are removed with disodium 2-carbamoyl-2- cyanoethylene-l,l-dithiolate trihydrate (S 2 Na 2 ) 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.
  • 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 CH 3 NH 2 in ethanol/water, followed by Bu 4 NF in tetrahydrofuran. The process can be repeated up to about a 150-mer product before significant product degradation results.
  • 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 (Dl,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.
  • four semi-eclipsed conformational rotamers are possible around the diaminobutane segment.
  • the four have enantiomeric relationships.
  • Introduction of a bond between the C-I 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.
  • Cis and trans cyclopropyl analogs of spermine were prepared via the reactions illustrated in Schemes 1, 2, 3, 4, 4A, 5, and 5A, hereinbelow.
  • 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.
  • the trans cyclopropyl diester 1 was converted into the amide 14 by reaction with benzylamine (BnNH 2 ), 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.
  • Cis and trans unsaturated analogs of spermine were prepared via the reactions illustrated in Schemes 10, 10A, 11, and 1 IA.
  • the trans diester 49 was reduced to the dialcohol 50, which was then converted into the trans diamine 51.
  • the cis diamine 52 was obtained from the commercially available cis dialcohol 43'.
  • 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).
  • nucleoside A is a 2'-O- TOM nucleoside
  • nucleoside B is a 2'-O- ACE nucleoside
  • nucleoside C is a 5'- thiol nucleoside.
  • Nucleoside C is prepared from 5'- thioriboside that is acetylated in its nucleobase, converted into its 2'-0-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.
  • 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'-0 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.
  • 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, 9O 0 C) to form the duplex. This pairing is known as annealing the siRNAs.
  • 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'-0 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.
  • 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.
  • 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).
  • the conjugate 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.
  • a polyamide By reaction with a polyamine, a polyamide is formed. Ring opening of the anhydride with the 5'-0 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'-0-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.
  • Nucleoside A is preferred in this synthetic sequence and not nucleoside B because of the sensitivity of the orthoester protecting group at 2'-0 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 after 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
  • PGl could be ⁇ - FMOC
  • 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 Ia.
  • Cleavage of the protecting group at the ⁇ -amino residue gives Ib.
  • Compound Ib will undergo rapid hydrolysis in the presence of trypsin with release of p-nitroaniline.
  • the p-nitroaniline residue is replaced with nucleoside A via a simple displacement reaction (see Fig. 15).
  • 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.
  • 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.
  • 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.
  • the conjugates are useful for moving RNA, including siRNA from an extracellular space into the cytoplasm of a mallian 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 route.
  • the treatment method is also amenable to oral administration.
  • 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, i ⁇ jectables, 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.
  • the subject conjugates may be administered 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.
  • a physiologically- acceptable diluent or sterile liquid carrier such as water or oil, with or without additional surfactants or adjuvants.
  • carrier oils would include animal and vegetable oils (peanut oil, soy bean oil), petroleum-derived oils (mineral oil), and synthetic oils.
  • 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 ⁇ Mto 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.

Abstract

La présente invention concerne des conjugués comprenant une polyamine liée en covalence à un acide ribonucléique (ARN), de préférence un ARN interférent court.
EP05825874A 2004-11-04 2005-11-04 Syntheses de conjugues polyaminiques d'arn interferents courts (arnic), et conjugues ainsi obtenus Withdrawn EP1817324A2 (fr)

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ES2435420T3 (es) 2005-12-15 2013-12-19 Centre National De La Recherche Scientifique - Cnrs Oligonucleótidos catiónicos, procedimientos automáticos para preparar los mismos y sus usos
EP2075342A1 (fr) 2007-12-27 2009-07-01 PolyPlus Transfection Procédé d'hybridisation d'acides nucléiques
FR2926818B1 (fr) * 2008-01-30 2012-04-06 Centre Nat Rech Scient siRNA CATIONIQUES, SYNTHESE ET UTILISATION POUR L'ARN INTERFERENCE
CA2919088A1 (fr) * 2013-08-07 2015-02-12 Arrowhead Research Corporation Polyconjugues pour l'administration de declencheurs d'arni a des cellules tumorales in vivo

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AU2005304816A1 (en) 2006-05-18
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WO2006052854A3 (fr) 2006-10-12

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