WO2012061810A1 - Base modified oligonucleotides - Google Patents
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- WO2012061810A1 WO2012061810A1 PCT/US2011/059588 US2011059588W WO2012061810A1 WO 2012061810 A1 WO2012061810 A1 WO 2012061810A1 US 2011059588 W US2011059588 W US 2011059588W WO 2012061810 A1 WO2012061810 A1 WO 2012061810A1
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Definitions
- the present invention relates to modified oligonucleotides with enhanced binding affinity towards complementary polynucleotides.
- miRs have been implicated in a number of biological processes including regulation and maintenance of cardiac function (Van Rooij et al., "'MicroRNAs: Powerful New Regulators of Heart Disease and Proactive Therapeutic Targets," J. Clin. Invest. 117(9):2369-2376 (2007); Chien KR, "Molecular Medicine: MicroRNAs and the Tell-tale Heart,” Nature 447:389-390 (2007)). Therefore, miRs represent a relatively new class of therapeutic targets for conditions such as cardiac hypertrophy, myocardial infarction, heart failure, vascular damage, and pathologic cardiac fibrosis, among others.
- miRs are small, non-protein coding RNAs of about, 18 to about 25 nucleotides in length, and act as repressors of target mRNAs by promoting their degradation, when their sequences are perfectly complementary, or by inhibiting translation, when their sequences contain mismatches.
- the mechanism involves incorporation of the mature miRNA. strand into the RNA-induced silencing complex (RISC), where it associates with its target RNAs by base-pair complementarity.
- RISC RNA-induced silencing complex
- miRNA function may be targeted therapeutically by antisense polynucleotides or by polynucleotides that mimic miRNA. function ("miRNA mimetic").
- miRNA mimetic targeting miRNAs therapeutically with oligomieleoti de-based agents poses several challenges, including RNA-binding affinity and specificity, efficiency of cel lular uptake, and nuclease resistance. For example, when polynucleotides are introduced into intact cells they are attacked and degraded by nucleases leading to a loss of activity.
- Oligonucleotide chemistry patterns or motifs for miRNA inhibitors have the potential to improve the delivery, stability, potency, specificity, and/or toxicity profile of the inhibitors, and as such are needed for effectively targeting miRNA function in a therapeutic context.
- the present invention relates to oligonucleotides comprising at least one nucleotide having a 2' modification and at least one nucleotide having an amino carbonyl modified base, as well as pharmaceutical compositions comprising the modified oligonucleotides, and methods of use and synthesis for these oligonucleotides.
- the present invention provides oligonucleotides comprising at least one nucleotide having a 2' modification and at least one nucleotide having an amino carbonyl modified base.
- the oligonucleotides provide advantages in duplex binding affinity, among other advantages, such as efficiency in RNA knockdown.
- the oligonucleotide comprises a nucleotide sequence that is at least substantially complementary to a nucleotide sequence of human miRNA .
- the oligonucleotide is at least substantially complementary to a mammalian transcript, other than a miRNA, and is therefore useful for antisense inhibition of gene expression, in still other embodiments, the oligonucleotide comprises the sequence of a human miRNA, and thereby mimics miRNA function, in still other embodiments, the oligonucleotide is a detection probe for in vitro detection or quantification of nucleic acids in a sample, using any conventional platform.
- the base modification is an amino carbonyl, such as a carboxamino, carbamoyl, or carbamide group.
- the modification in various embodiments is at the C-5 position of a pyrimidine base or C-8 of a purine base.
- the modifying amino carbonyl group of the instant oligonucleotide contains a radical or substituent which can be, without limitation, C-.-C] 8 alkyl, C-.-Cjg alkenyl, cycloalkyl, aryl, heteroaryi, heterocyclyl, and -(C33 ⁇ 4) n -NRiR 2 , wherein n is an integer from 1 to 6 and R 3 ⁇ 4 and R 2 are independently H or Ci -Cealkyi.
- moieties include piperidine, piperazine, morpholino, or imidazole, each of which may be substituted or unsubstituted.
- the substituent is from C4 to C20 alkyl or alkenyl, phenyl, or an amine,
- the oligonucleotide further comprises at least one nucleotide with a 2' modification.
- the 2' modifications may be independently selected from CI -6 alkyl, 2' 0-aikyl(Cl -C6), F, CI, N3 ⁇ 4, CN, or Si I.
- Other potential 2' modifications are described elsewhere herein.
- An exemplary 2' modification is 2' Q-Me, which may provide synergistic enhancements of the oligonucleotide's T m , together with the base modification.
- at least, one nucleotide has a 2' modification that is a 2' - 4' bridge locking the sugar in the C3 endo configuration. Unmodified 2' positions may be hydrogen.
- the number of nucleotides having a modified base may vary, but in certain embodiments is at least 25% of nucleotides, or at least 50% of nucleotides, or at least 75% of nucleotides or 100% of nucleotides.
- the enhancement of T m may be accomplished with relatively few base-modified nucleotides, such as less than 50% of nucleotides or less than 25% of nucleotides.
- the oligonucleotide contains only 1 , 2, 3, or 4 base-modified nucleotides.
- the base modified nucleotides in these embodiments may be pyrimidine bases, such as uridine or thymine, and/or may contain a modification such as 2' O'Me. That is, the oligonucleotide (e.g., of about 16 nucleotides) may have a sragle incorporation of a nucleotide having the base modification and 2' OMe modification, with unmodified 2' positions being hydrogen, or alternatively independently selected from LNA.
- a modification such as 2' O'Me. That is, the oligonucleotide (e.g., of about 16 nucleotides) may have a sragle incorporation of a nucleotide having the base modification and 2' OMe modification, with unmodified 2' positions being hydrogen, or alternatively independently selected from LNA.
- the oligonucleotide further comprises backbone chemistries such as cap modifications and phosphorothioate linkages.
- the invention includes the discovery that novel base modified 2 '-OMe-pyrimidines show enhancements of duplex binding affinity with their complementary sequences when incorporated into antisense oligonucleotides. Additionally, these pyrimidine base modified 2'-OMe nucleotides with phosphorothioate backbone modifications show biological activity against their microRNA target sequences in cell culture, even without the use of transfection reagents. In vivo activity is also demonstrated herein using a model in vivo system showing knockdown of target miRNA in cardiac tissue.
- the present invention provides a method of reducing or inhibiting RNA expression or activity in a ceil, a method of preventing or treating a condition in a subject associated with or mediated by RNA or expression thereof, the method using the base modified oligonucleotides described herein.
- Figure 1 is a table showing the amount of T m enhancement for various base modifications of 2'-OMe-uridine oligonucleotides. Base modifications were carboxamido linkages at C5.
- Figure 2 illustrates the synthesis of modified monomeric nucleosides and corresponding phosphoramidites for incorporation into oligonucleotides.
- Figure 3 illustrates hydrophilic and hydrophobic nucleoside modifications synthesized via the scheme in Figure 2 and shows some example incorporation patterns in oligonucleotides.
- Figure 4 compares T M measurements for several base modifications against LNA/DI A, 2'-OMe phoshorothioate, and DNA oligonucleotide. The base modification pattern is shown.
- Figure 5 is a table of experimental T m measurements for modified anti-miR-208a when duplexed with unmodified miR-208a RNA. All oligonucleotides contain phosphorothioate linkages; +U stands for base modified nucleotide with 2'OMe; m stands for 2'OMe, yU stands for C 18 base modification and 2'OMe ribose; 1 stands for LNA modification; d stands for DNA.
- Figure 6 shows a miR-208a knockdown by modified antimiR-208a in rat primary neonatal cardiomyocytes without lipid transfection reagent.
- Figure 7 shows the miR-2()8a knockdown data in Figure 6 superimposed on bMHC levels.
- Figure 8 is a plot of in vivo efficacy of base modified oligonucleotides in C57BL/6 mice. The plot shows the fold-change relative to saline injections for some modified oligonucleotides.
- Figure 10 is a graph of AT m against number of base modifications and 2' modifications, and shows the synergistic effect.
- Figure 1 1 is a graph of T m effect of a select base modification with respect to number of modifications and backbone chemistry.
- the present invention relates to oligonucleotides comprising at least one nucleotide having a 2' modification and at least one nucleotide having an amino carbonvl modified base.
- the present invention further relates to methods of use and synthesis for these oligonucleotides.
- nucleoside base modification has been largely limited to investigations of effects on gene expression.
- Certain nucleobase derivatives, especially C-5 propynylated pyrimidines have exhibited only modest gains in affinity/duplex stability for DNA'RNA duplexes (Znosko et al, J. Am. Chem. Soc.,125(20):6090-6097 2003)).
- More complex pendant functional groups are considered less likely to increase oligonucleotide affinity, given the potential competing effects of hydrophobicity or steric effects (Hashimoto et al, J. Am.Chem.Soc, 115( 16): 7128-7134 (1993)).
- base modification may potentially change the overall hydrophobicity and hydrogen bonding characteristic of an oligonucleotide bearing the modification, and might even lead to non-canonical base pairing interactions (Vaught et al, J, Am. Chem. Soc, 132(12):4141-4151 (2010)), an effect that is not desirable for sequence-specific RNA inhibition.
- the present invention provides oligonucleotides comprising at least one nucleotide having a 2' modification and at least one nucleotide having an amino carbonyl modified base.
- the oligonucleotides provide advantages in duple binding affinity, among other advantages, such as efficiency in RNA knockdown.
- the oligonucleotide comprises a nucleotide sequence that is at least substantially complementary to a nucleotide sequence of human miRNA.
- the oligonucleotide is substantially complementary or fully complementary to a mammalian transcript, other than a miRNA, and is therefore useful for antisense inhibition of gene expression.
- the oligonucleotide comprises a sequence of a human miRNA sufficient to mimic of miRNA function.
- the oligonucleotide is a detection probe for in vitro detection or quantification of nucleic acids in a sample, using any conventional platform, such as a microarray or other hybridization-based platform.
- the oligonucleotide is from about 6 to about 22 nucleotides in length.
- the oligonucleotides having one or more of the base, sugar, and/or backbone modifications disclosed herein can be, for example, from 8 to 18 nucleotides in length, or from 12 to 16 nucleotides in length.
- the oligonucleotide is about 8 nucleotides in length, about 9 nucleotides in length, about 10 nucleotides in length, about 11 nucleotides in length, about 12 nucleotides in length, about 13 nucleotides in length, about 14 nucleotides in length, about 15 nucleotides in length, or about 16 nucleotides in length.
- the oligonucleotide may have the sequence CTTTTTGCTCGTCTTA (SEQ ID O:64).
- the base modification is generally an amino carbonyl, such as a. carboxamino, carbamoyl, or carbamide group.
- the modification in various embodiments is at the C-5 position of one or more pyrimidine bases, and/or at C-8 of one or more purine bases.
- the modifying amino carbonyl group of the instant oligonucleotide contains a radical or substituent which can be, without limitation, Ci-Qs alkyl, Ci-Cjg alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -(CH2) r .- RiR.2, wherein n is an integer from 1 to 6 and Rj and R 2 are independently H or Cj-Cealkyl.
- the radical or substituent is a nitrogen- containing heterocyele, such as, for example, piperidine, piperazine, morpholino, or imidazole, each of which may be substituted or unsubstituted with one, two, or three alkyl or alkenyl substituents (e.g., Cl -8 or Cl-4).
- heterocyele such as, for example, piperidine, piperazine, morpholino, or imidazole, each of which may be substituted or unsubstituted with one, two, or three alkyl or alkenyl substituents (e.g., Cl -8 or Cl-4).
- alkyl or alkenyl substituents e.g., Cl -8 or Cl-4
- Examples include 2-ethyl, 1-methyl- imidazole, 3-propyl imidazole, and propyl morpholino, which are depicted in Figure 1.
- the radical or substituent is a carbocyclic group, such as a cycloalkyl (e.g., C5 to C8) or phenyl, which may optionally be substituted with one or more (e.g., 1, 2, or 3) alkyl or alkenyl substituents (e.g., CI -8 or Cl-4).
- a cycloalkyl e.g., C5 to C8
- phenyl which may optionally be substituted with one or more (e.g., 1, 2, or 3) alkyl or alkenyl substituents (e.g., CI -8 or Cl-4).
- examples include Benzyl as shown in Figure 6,
- the radical or substituent is a secondary or tertiary amine, for example, having one or two alkyl or alkenyl substituents (e.g., Cl -8, or Cl-4).
- Examples include propyl dimethyl amino, and ethyl dimethyl amino, as shown in Figure 1.
- the modifying amino carbonyl group contains a lipophilic or hydrophilic substituent, and in some embodiments, the substituent is cationic. Examples include C6 and CI 8 alkyl as shown in Figure 1.
- the radical or substituent is bound to the C5 position of a pyrimidine base through a carboxamino linkage, optionally having a linking group of from 1 to 4 carbon units. The radical or substituent may be as described elsewhere herein.
- the base modification contains a group that is positively charged, and optionally having multiple positive charges, under physiological conditions, such as a pipirazine.
- Primary, secondary and quaternary amines can also be used as suitable base modifications.
- the base modification contains a peptide linkage, which are more likely to be metabolized into less toxic iiucleobases.
- the base modified nucleotides are incorporated in the middle of the sequence.
- the modified nucleotides are not incorporated at the last 1 , 2, or 3 nucleotides on the 5' and 3 ' ends.
- Moieties that are cationic under physiological conditions can provide substantial increases in T m .
- imidazole and morpholine derivatives that have pKa's in the range of 6,5-7.5 provide substantial binding and biological activity.
- Trialkylamines are also shown herein to be effective.
- Other cationic species of interest include guanidine type derivatives and hydrazines or hydroxylamines.
- substituted piperazmes moieties that often act pharmacologically similar to morpholines due to similar pKa's, but that have two cationic centers.
- Hydrophobic substitutions such as benzyl and alkyl moieties may also enhance T m , provide nuclease resistance, and/or aid in cytosolic delivery.
- the biological activity and T m enhancement may be due in-part to an increase in enthalpic binding, and therefore, the modified oligonucleotides have the potential to enhance mismatch discrimination, and are thus useful as probes for diagnostic applications.
- the oligonucleotide further comprises at least one nucleotide with a 2' modification.
- the term "2' modification” includes any 2' group other than H or OH.
- the 2' modifications may be independently selected from Cl-6 alkyl, 2' 0-alkyl(Cl-C6), F, CI, NH 2 , CN, or SH.
- Other potential 2' modifications are described elsewhere herein.
- An exemplar ⁇ ' 2' modification is 2' O-Me, which may provide synergistic enhancements of the oligonucleotide's T in , together with the base modification (e.g., when incorporated in the same nucleotide).
- at least one nucleotide has a 2' modification that is a 2' - 4' bridge locking the sugar in the C3 endo configuration.
- the oligonucleotide contains a 2' modification selected from alkyl, alkenyl, alkynyl, and alkoxvalkyl, where the alkyl (including the alkyl portion of alkoxy), alkenyl and alkynyl may be substituted or unsubstituted.
- the alkyl, alkenyl, and alkynyl may be CI to CIO alkyl, alkenyl, or alkynyl, such as CI, C2, or C3,
- the hydrocarbon substituents may include one or two or three non-carbon atoms, which may be independently selected from N, O, and/or S.
- the 2' modifications may further include the alkyl, alkenyl, and alkynyl as O-aikyl, O-alkenyl, and O-alkynyl.
- exemplary 2' modifications in accordance with the invention include 2'-0- alkyl (Cl-3 alkyl, such as 2'OMe or 2'OEt), 2'-0-methoxyethyl (2'-0-MOE), 2'-0- aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0- dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyI (2'-G-DMAEGE), or 2'-0-N-methylacetamido (2'-Q-NMA) substitutions,
- the oligonucleotide may have several nucleotides with the base modification as described, such as from 1 to about 10, or about 2 to about 9 nucleotides.
- the oligonucleotide contains (exactly) 1 , 2 or 3 nucleotides having the modified base.
- the oligonucleotide may also, independently, have several nucleotides modified at the 2' position. That is, the base modified nucleotides may also contain a 2' modification as described, such as a 2'OMe modification.
- at least one or two nucleotides have both a modified base and modified 2' position, each as described above.
- the oligonucleotide comprises a nucleotide with a base modification shown in Figure 1, together with a 2'OMe modification.
- the oligonucleotide in certain embodiments has exactly one, two, or three of such modified nucleotides.
- the 2' modification may be locked nucleic acid (LNA).
- LNAs are described, for example, in US Provisional Application Serial No. 61 /495,224, US Patent 6,268,490, US Patent 6,316,198, US Patent 6,403,566, US Patent 6,770,748, US Patent 6,998,484, US Patent 6,670,461, and US Patent 7,034,133, all of which are hereby incorporated by reference in their entireties.
- LNAs are modified nucleotides or ribonucleotides that contain an extra bridge between the 2' and 4' carbons of the ribose sugar moiety resulting in a "locked" conformation, and/or bicvclic structure.
- the oligonucleotide contains one or more LNAs having the structure shown by structure A below.
- the oligonucleotide may contain one or more LNAs having the structure shown by structure B below.
- the oligonucleotide contains one or more LNAs having the structure shown by structure C below.
- Suitable locked nucleotides that can be incorporated in the oligonucleotides of the invention include those described in US Patent 6,403,566 and US Patent, 6,833,361, both of which are hereby incorporated by reference in their entireties.
- the oligonucleotide may contain at, least 3, at least 5, or at least 7 locked nucleotides, and in various embodiments is not fully comprised of locked nucleotides.
- the number and position of locked nucleotides may be as described in 61/495,224, which is hereby incorporated by reference, and particularly for miR-208 family inhibitors.
- the oligonucleotide may have one or more 2'-deoxy nucleotides, and in some embodiments, contains from 2 to about 10 2'-deoxy nucleotides, in some embodiments, at least one, or all, base-modified nucleotides are 2' deoxy.
- the number of nucleotides having a modified base may vary, but in certain embodiments is at least 25% of nucleotides, or at least 50% of nucleotides, or at least 75% of nucleotides, or 100% of nucleotides.
- the enhancement of T m may be accomplished with relatively few base-modified nucleotides, such as less than 50% of nucleotides or less than 25% of nucleotides in some embodiments.
- the oligonucleotide contains only 1 , 2, 3, or 4 base-modified nucleotides (e.g., as shown in Figure 1), and such base-modified nucleotides may also contain a 2' modification such as 2'OMe.
- the base modified nucleotides in these embodiments may be pyrimidine bases, such as uridine or thymine in some embodiments.
- the oligonucleotide contains a single incorporation of a base-modified oligonucleotide having a 2'OMe.
- the oligonucleotide contains at least 6, or at least 9 nucleotides having a. 2'-QMe.
- all nucleotides may be 2' O-Me.
- the cationic class of C-5 modified bases exhibited substantial T m enhancement (as shown herein), in addition to some lipophillic enhancements to the C-5 position of 2'- OMe-Uridine.
- T m enhancement as shown herein
- mixtures of modifications containing both lipophillic and cationic moieties may have a larger effect on miRNA 's already associated with intracellular enzymes and proteins that regulate the miRNA's activity.
- These chimeric nucleotides may not only associate with their complementary target sequence, but also interact with hydrophobic or hydrophilic regions of the protein associated with the miRNA.
- the oligonucleotide further comprises at least one terminal modification or "cap”.
- the cap may be a 5' and/or a 3 '-cap structure.
- the terms "cap” or “end-cap” include chemical modifications at either terminus of the oligonucleotide (with respect to terminal ribonucleotides), and including modifications at the linkage between the last two nucleotides on the 5 ' end and the last two nucleotides on the 3' end.
- the cap structure as described herein may increase resistance of the oligonucleotide to exonucleases without compromising molecular interactions with the RNA target or cellular machinery. Such modifications may be selected on the basis of their increased potency in vitro or in vivo.
- the cap can be present at the 5'-terminus (5'-cap) or at the 3'-terminus (3 - cap) or can be present on both ends.
- the 5' ⁇ and/or 3 '-cap is independently selected from phosphorothioate monophosphate, abasic residue (moiety), phosphorothioate linkage, 4' ⁇ thio nucleotide, carbocyclic nucleotide, phosphorodithioate linkage, inverted nucleotide or inverted abasic moiety (2'-3' or 3'-3'), phosphorodithioate monophosphate, and methylphosphonate moiety.
- the phosphorothioate or phosphorodithioate Imkage(s), when part of a cap structure, are generally positioned between the two terminal nucleotides on the 5' end and the two terminal nucleotides on the 3' end.
- the oligonucleotide has at, least one terminal phosphorothioate monophosphate.
- the phosphorothioate monophosphate may be at the 5' and/or 3' end of the oligonucleotide.
- a phosphorothioate monophosphate is defined by the following structures, where B is base, and R is a 2' modification as described above:
- Phosphorothioate linkages may be present in some embodiments, such as between the last two nucleotides on the 5' and the 3' end (e.g., as part of a cap structure), or as alternating with phosphodiester bonds.
- the oligonucleotide may contain at least one terminal abasic residue at either or both the 5' and 3' ends.
- An abasic moiety does not contain a commonly recognized purine or pyrimidine nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
- abasic moieties lack a nucleotide base or have other non-nucleotide base chemical groups at the ⁇ position.
- the abasic nucleotide may be a reverse abasic nucleotide, e.g., where a reverse abasic phosphoramidite is coupled via a 5' amidite (instead of 3' amidite) resulting in a 5 '-5' phosphate bond.
- the stmcture of a reverse abasic nucleoside for the 5' and the 3' end of a polynucleotide is shown below.
- the oligonucleotide may contain one or more phosphorothioate linkages. Phosphorothioate linkages have been used to render oligonucleotides more resistant to nuclease cleavage.
- the polynucleotide may be partially phosphorothioate- linked, for example, phosphorothioate linkages may alternate with phophodiester linkages. In certain embodiments, however, the oligonucleotide is fully phosphorothioate-linked. In other embodiments, the oligonucleotide has from one to five or one to three phosphate linkages.
- the invention includes the discovery that novel base modified 2'-OMe-pyrimidines show enhancements of duplex binding affinity with their complementary sequences when incorporated into 2'-GMe nucleotides (See Figure 1). Additionally, these pyrimidine base modified 2'-OMe nucleotides with phosphorothioate backbone modifications show biological activity against their microRNA. target, sequences in cell culture, even without the use of transfection reagents, a characteristic that unconjugated 2'-OMe phosphorothioate nucleotides do not exhibit without the use of special 3' and 5' ⁇ conjugates.
- pyrimidine base modified 2'-OMe nucleotides with phosphorothioate backbone modifications exhibit knockdown of target rniRN.A in cardiac tissue following saline injection ( Figure 8).
- Figure 8 A series of model compounds were synthesized where the pendant modification on the C-5 base position were either hydrophobic or hydrophilic. Structures are included in Figures 1 -3.
- Anti-miRNA oligonucleotides containing only C-5 hydrophobic modifications of ail of the 2'-OMe-uridine nucleosides modestly increases the T m of a duplex compared to the nucleotides with unmodified 2'-OMe-uridine.
- nucleotides did not provide substantial benefit with respect to miRNA inhibition in cell culture experiments, both with and without lipid transfection reagents ( Figures 6 and 7).
- anti-miRNA nucleotides containing hydrophilic, amine (catioiiic) containing pendant, groups alone on C-5 of all uridines showed large increases in T m ( Figures 4-6).
- cell culture experiments with nucleotides containing these modifications show unique biological properties such as the ability to inhibit, miRNA. targets, even in the absence of lipid transfection reagents or conjugates. It should also be noted that some nucleotides with combinations of hydrophobic and cationic base modifications showed good anti-miRNA activity.
- pyrimidine base modifications enhance binding affinity through interaction with the polar major groove of the resulting RNA.
- duplexes The nucleosides described herein are modified, for example, via carboxamido modifications that are cross conjugated to the pyrimidine base and provide additional hydrogen bonding sites, either to another nucleobase or to the polar major groove. This is a distinct mode of duplex stabilization than commonly used sugar modifications, such as bridged nucleosides and 2 '-modifications, that favor A-form conformations of the nucleobase which enhance binding to RNA.
- C-5 carboxamido-modified nucleobases will act at least additively to the binding enhancement provided by sugar modification.
- C-5 carboxamido-modified nucleosides that also contain a 2'-4'-bridged sugar can also be employed to achieve enhanced binding of the oligonucleotides to their target, including the bridge structure shown below.
- Oligonucleotides incorporating the 2'-CBBN nucleosides are described in US Provisional Application No. 61/532,738, which is hereby incorporated by reference.
- R represents the carboxamido modification described herein
- R' and R" represent the 5' and 3 ' ends.
- Nucleotides incorporating the modified nucleobases described herein display enhanced binding affinity to their complementary nucleotides. Increases in T m have been measured as high as 5 °C/incorporation ( Figure 5), comparable to the bicyciic LNA monomers that, to this point, have been observed to be the most effective and widely used affinity enhancing modification. The enhancement of T m may be especially effective in creating more active and potent microRNA inhibitors.
- nucleobase modifications likely enhance cellular uptake by either masking some of the negatively charged phosphates, in the case of cationic moieties, or, in the case of lipophillie modifications, by shielding the backbone from nucleases and creating an aniphiphiliic nucleotide ( Figures 6, 7, and 8).
- the modifications may be used in oligonucleotides designed to mimic miRNA sequences, and may comprise any one of the mature miRNA sequences in Table 1 below.
- Such antisense and sense sequences may be incorporated into shRNAs or other R A structures containing stem and loop portions, for example.
- Such sequences are useful for, among other things, mimicking or targeting miRN A function for treatment or ameliorating cardiac hypertrophy, myocardial infarction, heart failure (e.g., congestive heart failure), vascular damage, and/or pathologic cardiac fibrosis, among others.
- Exemplary miRNA therapeutic utilities are disclosed in the US and PCT patent references listed in Table 1 below, each of which is hereby incorporated by reference in its entirety.
- the mature and pre-processed forms of miRNAs are disclosed in the patent references listed below, and such descriptions are also hereby incorporated by reference.
- the oligonucleotide targets a miR-208 family miRNA, such as miR-208a or miR-208b, or alternatively miR- 15b or miR-21 .
- the oligonucleotide has a sequence and structure shown in Figure 5. "m” refers to 2'OMe modification, and “+” refers to base-modified nucleotide with 2'OMe. Descriptions of abbreviations are found in Figure 1 and Figure 5.
- the oligonucleotide may be incorporated within a variety of macromolecular assemblies or compositions.
- Such complexes for delivery may include a variety of liposomes, nanopartic!es, and micelles, formulated for delivery to a patient.
- the complexes may include one or more fusogenic or lipophilic molecules to initiate cellular membrane penetration.
- fusogenic or lipophilic molecules to initiate cellular membrane penetration.
- the oligonucelotide may further comprise a pendant lipophilic group to aid cellular delivery, such as those described in WO 2010/129672, which is hereby incorporated by reference.
- the present invention relates to a pharmaceutical composition which comprises an effective amount of the oligonucleotide of the present invention or a its pharmaceutically-acceptable, and a pharmaceutically-acceptable carrier or diluent.
- the composition or formulation may employ a plurality of therapeutic oligonucleotides, including at least one described herein.
- the composition or formulation may employ at least 2, 3, 4, or 5 miRNA inhibitors described herein.
- the oligonucleotides of the invention may be formulated as a variety of pharmaceutical compositions. Pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
- Exemplary delivery/ formulation systems include colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to cardiac and skeletal muscle tissues include Intralipid®, liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar lipid emulsions.
- a preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.
- Exemplary formulations are also disclosed in LIS 5,981 ,505; US 6,217,900; US 6,383,512; US 5,783,565; US 7,202,227; US 6,379,965; US 6,127,170; US 5,837,533; US 6,747,014; and WO03/093449, which are hereby incorporated by reference in their entireties,
- compositions and formulations may employ appropriate salts and buffers to render delivery vehicles stable and allow for uptake by target cells.
- Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle comprising the inhibitor oligonucleotide (e.g. liposomes or other complexes), dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
- pharmaceutically acceptable or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
- pharmaceutically acceptable carrier may include one or more solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
- pharmaceuticals such as pharmaceuticals suitable for administration to humans.
- the use of such media and agents for pharmaceutically active substances is well known in the art.
- Supplementary active ingredients also can be incorporated into the compositions.
- Administration or deliver ⁇ ' of the pharmaceutical compositions according to the present invention may be via any route so long as the target tissue is available via that route.
- administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into target tissue (e.g., cardiac tissue).
- target tissue e.g., cardiac tissue.
- the stability and/or potency of the oligonucleotides disclosed herein allows for convenient routes of administration, including subcutaneous, intradermal, and intramuscular.
- Pharmaceutical compositions comprising miRNA inhibitors may also be administered by catheter systems or systems that isolate coronary circulation for delivering therapeutic agents to the heart.
- catheter systems for delivering therapeutic agents to the heart and coronary vasculature are known in the art.
- compositions or formulations may also be administered parenterally or intraperitoneally.
- solutions of the conjugates as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- these preparations are sterile and fluid to the extent that easy injectabiiity exists.
- Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyoi (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions may be prepared by incorporating the conjugates in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
- the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredients) plus any additional desired ingredient from, a previously sterile-filtered solution thereof.
- solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
- the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
- aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
- a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclvsis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
- Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
- the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards,
- the present invention provides a method of reducing or inhibiting RNA expression or activity in a cell.
- the method comprises contacting the ceil with a modified oligonucleotide (or pharmaceutical composition thereof) having a chemistry pattern described herein, where the oligonucleotide hybridizes (e.g., is at least substantially complementary to) an RNA transcript expressed by the cell.
- the RN A is a miRNA.
- the present invention provides a method of preventing or treating a condition in a subject associated with or mediated by RNA or expression thereof.
- the RNA is a miRNA.
- the method of prevention or treatment according to the present invention involves administering to the subject a pharmaceutical composition which comprises an effective amount of the base-modified oligonucleotide or a its pharmaceutically-acceptable composition thereof.
- the invention provides a method for delivering the modified oligonucleotides to a mammalian cell (e.g., as part of a composition or formulation described herein), and methods for treating, ameliorating, or preventing the progression of a condition in a mammalian patient.
- a mammalian cell e.g., as part of a composition or formulation described herein
- the oligonucleotide or pharmaceutical composition may be contacted in vitro or in vivo with a target cell (e.g., a mammalian cell).
- the cell may be a heart, cell.
- the method generally comprises administering the oligonucleotide or composition comprising the same to a mammalian patient or population of target cells.
- the oligonucleotide may be a miRNA inhibitor (e.g., having a nucleotide sequence designed to inhibit expression or activity of a miRNA).
- the miRNA inhibiter is an inhibitor of a miR-208 family miRNA
- the patient may have a condition associated with, mediated by, or resulting from, miR-208 family expression.
- Such conditions include, for example, cardiac hypertrophy, myocardial infarction, heart failure (e.g., congestive heart failure), vascular damage, restenosis, or pathologic cardiac fibrosis, cancer, or other miRNA associated disorder, including those disorders described in the patent publication listed in Table L
- heart failure e.g., congestive heart failure
- vascular damage e.g., vascular damage, restenosis, or pathologic cardiac fibrosis, cancer, or other miRNA associated disorder, including those disorders described in the patent publication listed in Table L
- the invention provides a use of the modified oligonucleotides and compositions of the invention for treating such conditions, and for the preparation of medicaments for such treatments.
- the patient e.g., human patient
- the patient has one or more risk factors including, for example, long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congestive heart failure, congenital predisposition to heart disease and pathological hypertrophy.
- the patient may have been diagnosed as having a. genetic predisposition to, for example, cardiac hypertrophy, or may have a familial history of, for example, cardiac hypertrophy.
- the present invention may provide for an improved exercise tolerance, reduced hospitalization, better quality of life, decreased morbidity, and/or decreased mortality in a patient with heart failure or cardiac hypertrophy.
- the activity of micoRNA in cardiac tissue, or as determined in patient serum, is reduced or inhibited.
- the pharmaceutical composition is administered by parenteral administration or by direct injection into heart tissue.
- the parenteral administration may be intravenous, subcutaneous, or intramuscular.
- the composition is administered by oral, transdermal, sustained release, controlled release, delayed release, suppository, catheter, or sublingual administration.
- the oligonucleotide is administered at, a dose of 25 mg/kg or less, or a dose of 10 mg/kg or less, or a dose of 5 mg/kg or less.
- the oligonucleotide or composition may be administered by intramuscular or subcutaneous injection, or intravenously.
- the methods further comprise scavenging or clearing the miR A inhibitors following treatment.
- a oligonucleotide having a nucleotide sequence that is complementary to the inhibitor may be administered after therapy to attenuate or stop the function of the inhibitor.
- Example 1 Gesseral Procedure For Preparation of S-posltioo-modified 2'-0- methyhiridine ssiic!eoside phosphoramidites
- 5-Iodo-2'-0-methyluridine was readily synthesized by known methods, and is also commercially available.
- the 5'- and 3 '-hydroxy 1 groups of the nucleoside are protected by standard 4,4'-Dimethoxytritylation and acetylation methods, respectively.
- This doubly protected nucleoside was then subjected to carboxamidation by dissolving the nucleoside in a 1 : 1 mixture of anhydrous THF and ⁇ , ⁇ -dimethylacetamide in a 50 mL borosiiicate boston round bottle.
- 5 equivalents of TEA and 3 equivalents of a primary amine or amine hydrochloride were added to the mixture followed by addition of 0.1 equiv.
- the Bottle is placed into a 250 mL Parr Bomb, which is sealed and evacuated through the needle valve.
- the Bomb is then pressurized to 60 psi with Carbon Monoxide.
- the bomb is then evacuated under high vacuum and re-charged with Carbon Monoxide (60psi).
- the bomb is resealed and placed in an oil bath heated to 70 °C For 17h.
- the bomb is cooled to rt and the pressure released slowly.
- the bottle is removed from the bomb and the solvent is removed in vacuo (Vaught et al., J. Am. Chem. Soc, 132(12):4141 -4151 (2010) which is hereby incorporated by reference in its entirety).
- TLC revealed that the reaction was complete.
- the reaction mixture was diluted with sat NaHC0 3 ( 100 mL) and the aqueous phase was extracted with DC (3 x 50 mL).
- the organic phases were combined and dried with a brine wash (1 x 50 mL) and addition of Na S0 4 .
- the organic phase was filtered and concentrated.
- Carboxamido-substituents for modifications were chosen from both hydrophilic and hydrophobic groups.
- Hydrophilic groups were preferentially chosen for the following reasons: Their ability to create new hydrogen bonding interactions with other nucleobases; the lack of exchangeable protons or sensitive functional groups that would require extra protecting groups under standard oligonucleotide synthesis; the cationic nature of these groups at physiological pH.
- Hydrophobic groups were chosen to attempt to exploit pi- stacking interactions between nucleobases and to create new hydrophobic regions in the nucleotide. Creating new hydrophobic and cationic/hydrophilic regions on a nucleotide may also create enhanced binding to serum proteins that enhance cell permeability.
- Pendant hydrophobic groups such as sterols and straight chain lipids
- nucleotides with 2 '-hydrophobic modifications can enhance cellular uptake by increasing interaction with serum lipoprotein particles.
- counteracting the very anionic nucleotide backbone with highly charged cationic species also enhances cellular uptake.
- Short strands of oligonucleotides bearing sugar and base modifications can be prepared once the modified nucleoside is synthesized and the free 5' and 3 '-hydroxy! groups are masked with appropriate reactive groups to become a nucleotide monomer.
- oligonucleotide synthesis a series of nucleotide monomers are sequentially attached, via their phosphoramidite derivatives, in a predetermined order to either, depending on the direction of chain extension, the 5'-funetional group or the 3'- functional group of the growing oligonucleotide strand.
- the oligonucleotide strand is anchored to an insoluble moiety such as controlled pore glass or polystyrene resin beads.
- the method of attachment of each monomer is generally comprised of the following steps 1-5.
- Step 1 involves the protection of the reactive functionality.
- the common reactive functionality is the 5 -hydroxyl group of the terminal nucleoside. This functionality is usually protected with a 4,4'-dimethoxytrityl (DMT) moiety that can be removed via acid treatment.
- DMT 4,4'-dimethoxytrityl
- One of the attractive features of the DMT moiety is that it forms a bright orange DMT cation during acid deprotection.
- Step 2 involves the coupling by addition of a phosphoramidite derivative and an activator.
- the phosphoramidite derivative is usually a nucleoside phosphoramidite, however, it may also be a phosphoramidite derivatized with a different organic moiety.
- Step 3 involves the capping of unreacted terminal functional groups. This step introduces an inert protective group that prevents further coupling to failure sequences.
- Step 4 involves oxidation of the newly formed phosphorous nucleotide backbone linkage from the trivending phosphite to the stable pentavalent state.
- This oxidation step can be performed with either an oxygen-based oxidant that results in a phosphate nucleotide or a sulfurizing oxidant that results in a phosphorothioate nucleotide.
- Step 5 involves a repetition of the process the after a washing step.
- Truncated, 16 nucleotide sequence complementary to a nucleotide sequence of human mi -208a was synthesized in 1 iimol scale on an ABI Expedite 8909 Automated Nucleic Acid Synthesis System, The synthesizer was operated using standard detritylation and capping solutions, known to those skilled in the art, single couplings of 420 seconds for each base and oxidation with 0.2M PADS oxidation solution after each coupling cycle.
- the unmodified anti-208a RNA sequence incorporates nine uridine residues which were fully replaced with nine modified nucleobases.
- the balance of the nucleotides were comprised of 2'-0-methyl-nueleotides.
- One exception was the incorporation of oieyl- carboxamido derivative, where there is a single incorporation on base position 15 of 16 where the nucleoside amidite was incorporated via a double coupling of 420 seconds each.
- Phosphoramidite Reagent (3c) in the Synthesis of the Base Modified Oligonucleotide was used.
- the oligodeoxynucleotide was synthesized using an ABI Expedite (Model 8909) DNA'RNA synthesizer. The synthesis was performed according to the manufacturer's recommendations in DMT-ON mode employing commercial synthesis reagents, exchanging 0.2M PADS in 1 : 1 Pyridine/ ACN for the oxidizing solution.
- the phosphoramidite reagent was added as a 0.1 M solution in acetonitrile during the appropriate coupling cycle.
- the cleavage of the oligonucleotide from the support was accomplished either by the method of described in US Patent 5,750,672 (which is hereby incorporated by reference in its entirety) or via heating of the CPG bound oligonucleotide with a solution of 40% aqueous methyl amine at 55 °C for 30 minutes.
- the resultant aqueous solution of oligonucleotide was further purified by loading the crude DMT-ON oligonucleotide solution on a Waters Sep-Pak® Vac CI 8 cartridge and eluting using a standard DMT-ON oligonucleotide desalting procedure known to those knowledgeable in the art.
- the characterization of product was performed by MALDI-TOF mass spectrometry utilizing 3-hydroxypieoiinie acid as matrix and standard methods known to those knowledgeable in the art: calcd 6922.4, found 6920.7.
- Compound M- 10708 ( Figure 5) was synthesized with amidite 3e in the uridine position in exactly the manner described above. The characterization of product was performed by ESI mass spectrometry on a Waters SQD mass detector in 200 niM HFIP/8.15 niM TEA buffer gradient with MeOH: calcd 6597.6, found 6599.1.
- Compound M- 10768 ( Figure 5) was synthesized with 2'-0-methyluridine in its amidite position and amidite 3d in the auxiliary amidite position in exactly the manner described above.
- the characterization of product was performed by ESI mass spectrometry (negative mode) on a Waters SQD mass detector in 200 mM HFIP/8.15 mM TEA buffer gradient with MeOH: calcd 6003.9, found 6005.2.
- Compound M- 10772 ( Figure 5) was synthesized with amidite 3i in the uridine position in exactly the manner described above. The characterization of product was performed by ESI mass spectrometry (negative mode) on a Waters SQD mass detector in 200 mM HFIP/8.15 mM TEA buffer gradient with MeOH: calcd 6552.8, found 6553.4.
- Compound M- 10774 ( Figure 5) was synthesized with 2'-0-methyluridine in its amidite position and amidite 3i in the auxiliary amidite position in exactly the manner described above.
- the characterization of product was performed by ES mass spectrometry (negative mode) on a Waters SQL ) mass detector in 200 mM HFIP/8.15 mM TEA buffer gradient with MeOH: calcd 5912.0, found 5912.8.
- Compound M- 10876 ( Figure 5) was synthesized with amidite 3b in the uridine position in exactly the manner described above. The characterization of product was performed by ESI mass spectrometry (negative mode) on a Waters SQD mass detector in 200 mM HFIP/8.15 mM TEA buffer gradient with MeOH: calcd 6931.2, found 6931.9.
- the characterization of product was performed by ESI mass spectrometry (negative mode) on a Waters SQD mass detector in 200 mM HFIP/8.15 mM TEA buffer gradient with MeOH: calcd 7056.0, found 7056.5.
- Compound M- 10878 ( Figure 5) was synthesized with 2'-0-methyluridine in its amidite position and amidite 3b in the auxiliary amidite position in exactly the manner described above.
- the characterization of product was performed by ESI mass spectrometry (negative mode) on a Waters SQD mass detector in 200 mM HFIP/8. 5 mM TEA buffer gradient with MeOH: calcd 6080.1, found 6081.
- T m Melting temperature enhancement was determined on a per incorporation basis by determining the difference between the melting temperature of the modified strand and that of the identical sequence utilizing either a phosphorothioate DNA nucleotide or a phosphorothioate 2'-0-methyl RNA nucleotide.
- the modified anti-208a oligonucleotides were annealed to the complementary sequence, twenty-two nucleotides in length, comprised of RNA nucleosides and a phosphate backbone.
- the complementary sequence was identical to the endogenous miRNA.
- Thermal denaturation temperatures (T m ) were measured as a maximum of the first derivative plot of melting curvex (A 260 vs. Temp).
- the duplexes were constituted at ⁇ in a 0.9% NaCl buffer. Temperature was ramped from 25 °C to 95 °C at 4 °C/min and OD's at 260 nm were read once per minute. T m values are averages of at least two measurements.
- Duplex melting temperatures for various modifications of a 16 nucleotide sequence complementary to a nucleotide sequence of human miR-208a Modifications included a mixed 9 LNA/7 DNA phosphorothioate, fully substituted 2'-0-methyl- nucleotide phosphorothioate, fully 2'-deoxynucleotide phosphorothioate and various substitution patterns of fully 2'-0-methyl-nucieotides with 5-carboxamide substituents.
- the 5-(2-(2 -methyl- lH-imidazo 1-1 -yi)- ethylca.rboxamido)-2'-0-methyluridine nucleotide variant shows inhibition of miR-208a when incorporated in a 16-nucieotide 2'-0-methyl phosphorothioate anti-208a nucleotide sequence where either 4 or 9 of the total 9 natural uridine nucleotide positions are substituted. It is only the oligonucleotide that has 4 substitutions that shows effective bMHC mRN A regulation.
- oligonucleotides were studied in vivo in C57BL/6 mice (10941 , 10876, 10711). A scrambled control containing the comparable bases of each oligo were also injected (11091, 11087, 1 108(5). The oligonucleotides were dosed with a 25 nig/kg delivered via subcutaneous injection on Day 1. Cardiac tissue was harvested 4 days after dosing and miR-208a levels were determined via. real time PCR. There was neither injection site reaction nor any visible organ damage following take down of the mice.
- Figure 10 presents data from figure 9 as AT m / er modification, counting both sugar and base modifications.
- Multiple incorporations of 5-carboxyamido-2'-0- methyl uridine nucleosides unexpectedly give a greater stabilization per sugar and base modification than either the base or sugar do alone.
- This evidence indicates that 5- carboxamido in conjunction with modifications that favor a 3'-endo sugar pucker of nucleosides are more than additive. They work synergistically to give greater duplex stabilit than either modification alone. Increased duplex stability, subject to limits, is likely desirable for certain oligonucleotide based therapeutics, such as mieroRNA inhibitors.
- these types of modifications may also protect from enzymatic degradation, cellular delivery due to decreased electrostatic charge and enhanced pharmacokinetic and/or pharmacodynamic properties.
- modifications disclosed in this invention can be used alone, as single or multiple incorporations, or in conjunction with other sugar modifications, as single or multiple incorporations, to obtain a therapeutic oligonucleotide with desirable duplexing properties, duplex-protein binding properties, or along with desirable pharmacokinetic and/or pharmacodynamic properties.
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Abstract
Description
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AU2011323085A AU2011323085B2 (en) | 2010-11-05 | 2011-11-07 | Base modified oligonucleotides |
JP2013537906A JP5995855B2 (en) | 2010-11-05 | 2011-11-07 | Base-modified oligonucleotide |
US13/883,363 US9416360B2 (en) | 2010-11-05 | 2011-11-07 | Base modified oligonucleotides |
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CN (1) | CN103492569B (en) |
AU (1) | AU2011323085B2 (en) |
CA (1) | CA2817002C (en) |
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Publication number | Publication date |
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EP2635681A1 (en) | 2013-09-11 |
EP2635681B8 (en) | 2017-10-04 |
EP2635681B1 (en) | 2017-08-23 |
AU2011323085B2 (en) | 2014-10-30 |
AU2011323085A1 (en) | 2013-03-21 |
US9416360B2 (en) | 2016-08-16 |
EP2635681A4 (en) | 2015-01-14 |
CA2817002A1 (en) | 2012-05-10 |
CN103492569B (en) | 2020-04-07 |
US20130296402A1 (en) | 2013-11-07 |
CN103492569A (en) | 2014-01-01 |
JP5995855B2 (en) | 2016-09-21 |
JP2014503192A (en) | 2014-02-13 |
CA2817002C (en) | 2019-01-15 |
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