Pharmaceutical Composition

a technology of pharmaceutical compositions and compositions, applied in the field of pharmaceutical compositions, can solve the problems of high dosage (80 mg/kg) of 2′-o-me antagomir for efficient silencing, low in vivo stability, and toxic to animals, and achieve the effect of reducing the repression of mrna

Inactive Publication Date: 2010-01-07
SANTARIS PHARMA AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]The present invention is based upon the discovery that the use of short oligonucleotides designed to bind...

Problems solved by technology

A drawback of this method is the need of high 2′-O-methyl oligonucleotide concentrations (100 micromolar) in transfection and injection experiments, which may be toxic to the animal.
Although these experiments resulted in effective silencing of endogenous miRNAs in vivo, which was found to be specific, efficient and long-lasting, a major drawback was the ne...

Method used

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  • Pharmaceutical Composition
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Examples

Experimental program
Comparison scheme
Effect test

example 1

Monomer Synthesis

[0460]The LNA monomer building blocks and derivatives thereof were prepared following published procedures and references cited therein, see, e.g. WO 03 / 095467 A1 and D. S. Pedersen, C. Rosenbohm, T. Koch (2002) Preparation of LNA Phosphoramidites, Synthesis 6, 802-808.

example 2

Oligonucleotide Synthesis

[0461]Oligonucleotides were synthesized using the phosphoramidite approach on an Expedite 8900 / MOSS synthesizer (Multiple Oligonucleotide Synthesis System) at 1 μmol or 15 μmol scale. For larger scale synthesis an Äkta Oligo Pilot (GE Healthcare) was used. At the end of the synthesis (DMT-on), the oligonucleotides were cleaved from the solid support using aqueous ammonia for 1-2 hours at room temperature, and further deprotected for 4 hours at 65° C. The oligonucleotides were purified by reverse phase HPLC (RP-HPLC). After the removal of the DMT-group, the oligonucleotides were characterized by AE-HPLC, RP-HPLC, and CGE and the molecular mass was further confirmed by ESI-MS. See below for more details.

[0462]Preparation of the LNA-Solid Support:

[0463]Preparation of the LNA succinyl hemiester

[0464]5′-O-Dmt-3′-hydroxy-LNA monomer (500 mg), succinic anhydride (1.2 eq.) and DMAP (1.2 eq.) were dissolved in DCM (35 mL). The reaction was stirred at room temperature...

example 3

Design of the LNA Anti-miR Oligonucleotides and Melting Temperatures

[0484]

Target microRNA:miR-122a:5′-uggagugugacaaugguguuugu-3′SEQ ID NO: 535miR-122a 3′ to 5′:3′-uguuugugguaacagugugaggu-5′(SEQ ID NO: 535 reverse orientation)

TABLE 1LNA anti-miR oligonucleotide sequences and Tm:SEQIDTmNO:Oligo IDSED IDSequence:(° C.)2SPC3370XxxXSEQ ID 5855′-cCatTgtCacActCca-PS75design3′backbone3SPC3372XxxXSEQ ID 5865′-ccAttGtcAcaCtcCa-PS69design3′backbone4SPC3375GapmerSEQ ID 5875′-PS69CCAttgtcacacTCCa-3′backbone5SPC354915-merSEQ ID 5885′-CcAttGTcaCaCtCC-PS783′backbone6SPC3550mismatchSEQ ID 5895′-CcAttCTgaCcCtAC-PS32control3′backbone7SPC3373mismatchSEQ ID 5905′-ccAttGtcTcaAtcCa-PS46control3′backbone8SPC354813-merSEQ ID 5915′-AttGTcaCaCtCC-3′PSbackbone

lower case: DNA, uppercase: LNA (all LNA C were methylated), underlined: mismatch

[0485]The melting temperatures were assessed towards the mature miR-122a sequence, using a synthetic miR-122a RNA oligonucleotide with phosphorothioate linkaged.

[0486]The LNA...

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Abstract

The invention provides pharmaceutical compositions comprising short single stranded oligonucleotides, of length of between 8 and 17 nucleobases which are complementary to human microRNAs. The short oligonucleotides are particularly effective at alleviating miRNA repression in vivo. It is found that the incorporation of high affinity nucleotide analogues into the oligonucleotides results in highly effective anti-microRNA molecules which appear to function via the formation of almost irreversible duplexes with the miRNA target, rather than RNA cleavage based mechanisms, such as mechanisms associated with RNaseH or RISC.

Description

FIELD OF THE INVENTION[0001]The present invention concerns pharmaceutical compositions comprising LNA-containing single stranded oligonucleotides capable of inhibiting disease-inducing microRNAs.BACKGROUND OF THE INVENTION[0002]MicroRNAs—Novel Regulators of Gene Expression[0003]MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs. The mature miRNAs are processed sequentially from longer hairpin transcripts by the RNAse III ribonucleases Drosha (Lee et al. 2003) and Dicer (Hutvagner et al. 2001, Ketting et al. 2001). To date more than 3400 miRNAs have been annotated in vertebrates, invertebrates and plants according to the miRBase microRNA database release 7.1 in October 2005 (Griffith-Jones 2004, Griffith-Jones et al. 2006), and many miRNAs that correspond to putative genes have also been identified.[0004]Most animal miRNAs recognize their target sites located in 3′-UT...

Claims

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Application Information

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IPC IPC(8): A61K31/7052A61P43/00C12N15/113
CPCC12N15/113C12N2310/113C12N2310/321C12N2310/322C12N2310/3231C12N2310/3515C12N2310/3521C12N2310/3533A61P1/16A61P11/00A61P15/00A61P21/00A61P25/00A61P29/00A61P3/00A61P3/10A61P31/04A61P31/12A61P31/14A61P35/00A61P3/06A61P35/02A61P37/02A61P43/00A61P9/10A61K48/00C12N15/11
Inventor ELMEN, JOACIMKEARNEY, PHILKAUPPINEN, SAKARI
Owner SANTARIS PHARMA AS
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