CN101292034A - Rna antagonist compounds for the inhibition of apo-b100 expression - Google Patents

Abstract

Oligonucleotides directed against the Apo-B100 gene are provided for modulating the expression of Apo-B100. The compositions comprise oligonucleotides, particularly antisense oligonucleotides, targeted to nucleic acids encoding the Apo-Bl00. Methods of using these compounds for modulation of Apo-Bl00 expression and for the treatment of diseases associated with either overexpression of Apo-Bl00, expression of mutated Apo-Bl00 or both are provided. Examples of diseases are cancer such as lung, breast, colon, prostate, pancreas, lung, liver, thyroid, kidney, brain, testes, stomach, intestine, bowel, spinal cord, sinuses, bladder, urinary tract or ovaries cancers. The oligonucleotides may be composed of deoxyribonucleosides or a nucleic acid analogue such as for example locked nucleic acid or a combination thereof.

Description

Suppress the RNA antagonist compound that APO-B100 expresses

Invention field

The invention provides composition and the method that APO-B100 expresses of regulating.Particularly, the present invention relates to can with the oligonucleotide compound of nucleic acid specificity hybridization of coding Apo-B100.This oligonucleotide compound has shown that can regulate Apo-B100 expresses, and discloses its pharmaceutical preparation and as the purposes to the treatment of Cancerous disease.

Background of invention

Apolipoprotein B (also claims ApoB, Apolipoprotein B-100; ApoB-100, Apolipoprotein B-48; ApoB-48 and Ag (x) antigen) be a big glycoprotein, it plays requisite effect in the picked-up of the transportation of the assembling of lipid and secretion and different classes of lipoprotein and acceptor-mediation with in sending.ApoB plays an important role in regulating the circulation lipoprotein levels, and therefore relevant with the atherosclerosis susceptibility, atherosclerosis susceptibility and the environmental concentration height correlation that contains the lipoprotein of apolipoprotein B.About being present in the ApoB of two kinds of forms in the Mammals, the further details of the medical importance of their structure and ApoB, referring to Davidson and Shelness (Annul Rev.Nutr., 2000,20,169-193).

Level rising and the danger of atherosclerosis and manifestation thereof that contains the lipoprotein Lp (a) of ApoB-100 in the blood plasma is increased relevant, and described manifestation can comprise hypercholesterolemia (Seed etc., N.Engl.J.Med., 1990,322,1494-1499), myocardial infarction (Sandkamp etc., Clin.Chew., 1990,36,20-23) and thrombosis (Nowak-Gottl etc., Pediatrics, 1997,99, Eli).

The plasma concentration of Lp (a) is influenced by inherited genetic factors strongly, and to most drug and dietary control reactionless (Katan and Beynen, Am.J.Epidemiol., 1987,125,387-399; Vessby etc., Atherosclerosis, 1982,44,61-71).Pharmacological treatment for Lp (a) level that raises has only obtained very little success, the separating plasma displacement remain the most effective therapeutic modality (Hajjar and Nachman, Annul Rev.Med., 1996,47,423-442).

The apolipoprotein B that has two kinds of forms in the Mammals.ApoB-100 representative comprises 4536 amino-acid residues, only in people's liver the synthetic full length protein (Davidson and Shelness, Annul Rev.Nutr., 2000,20,169-193).Clipped form and 2152 residue conllinear of N-terminal of being called as ApoB-48, and synthetic in all mammiferous small intestines (Davidson and Shelness, Annul Rev, Nutr., 2000,20,169-193).

The basis that the common structure gene of apolipoprotein B produces two kinds of different protein isoforms is a kind of process that is called as rna editing.A kind of site-specific cytosine(Cyt)-to the editor of-uridylic reacts and produces a UAA terminator codon, and the apolipoprotein B translation termination is to produce ApoB-48 (Davidson and Shelness, Annul Rev.Nutr., 2000,20,169-193}.

The medical significance of Mammals ApoB had used transgenic mice research (Kim and Young, J., Lipid Res., 1998,39, the 703-723 of expressing human ApoB; Nishina etc., J.Lipid Res., 1990,31,859-869) or ApoB knock-out mice (Farese etc., Proc.Natl.Acad.Sci.U.S.A., 1995,92,1774-1778; Kim and Young, J.Lipid Res., 1998,39,703-723) be verified.

Up to now, the strategy that is intended to suppress the apolipoprotein B function is confined to the displacement of Lp (a) separating plasma, antibody, antibody fragment and ribozyme.In addition, low biologically stable and/or low binding affinity antisense oligonucleotide at the open WO 00/97662 of PCT, are disclosed also claimed among WO 03/11887 and the WO 2004/44181.

Thereby, therefore still need other effectively antagonism apolipoprotein B function and reduce the promoting agent of blood plasma Lp (a) level.

The invention provides effective locked nucleic acid (LNA) oligomeric compounds and they and express application in the method for (comprise suppress apolipoprotein B or select isotype ApoB-48) regulating apolipoprotein B.

Brief summary of the invention

The invention provides composition and the method that apolipoprotein B (Apo-B100/Apo-B48) is expressed of regulating.Particularly, the present invention relates to comprise the oligonucleotide compound of the specificity motif of target apolipoprotein B.These motifs are SEQ ID NO:2-26, especially SEQ ID NO:2,3,10,11 and 21.The special design that comprises the oligonucleotide compound of LNA also is disclosed.Especially preferred compound is SEQ ID NO:29-47, particularly SEQ ID NO:29,30,31,36,37,38,40 and 42.Compound of the present invention is effective inhibitor of lipophorin mRNA and protein expression.External, SEQ ID NO:29 and 30 downward modulation ApoB express IC ₅₀About 1-5nM, and SEQ ID No 37 demonstrates the IC of about 0.5nM ₅₀In vivo, after SEQID NO:29 processing, ApoB-100mRNA is expressed in liver and the jejunum and is suppressed in the dose-dependently mode.Be accompanied by the ApoB-100 level of decline, total cholesterol reduces by 70% in the blood plasma.

Pharmaceutical composition and other composition of comprising oligonucleotide compound of the present invention also are provided.Further provide and regulated the method that apolipoprotein B is expressed in the cell or tissue, comprised making described cell or tissue contact one or more oligonucleotide compound or compositions of the present invention.Also disclose by one or more oligonucleotide compounds of the present invention or the composition of administering therapeutic or prevention significant quantity and treated the method for suffering from or easily suffering from the animal or human of disease relevant or illness with the apolipoprotein B expression.In addition, also provide the method that suppresses the apolipoprotein B expression disease relevant with the apolipoprotein B activity with the oligonucleotide compound with treatment.The example of these diseases is that dissimilar HDL/LDL cholesterol is unbalance; Dyslipidemia, for example, multiple lipoprotein type hyperlipidemia (FCHL), acquired hyperlipidaemia, hypercholesterolemia; To the drug-fast hypercholesterolemia of Statins; Coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis.

Brief Description Of Drawings

Figure 1A: the auxiliary picked-up of lipid SEQ ID NO:29, behind the siRNA that siRNA (unmodified) or cholesteryl are modified, the ApoB mRNA relative expression in the mouse liver cell (Hepa1-6 cell).

Figure 1B: after SEQ ID NO:29 and 30 processing, the ApoB relative expression in the BNLCL2 cell.Two kinds of compounds all have been effective ApoB-100mRNA inhibitor at 1nM or 5nM concentration.

Fig. 2 A: the siRNA (SEQ ID NO 50/49) that modifies with SEQ ID NO:29, siRNA (unmodified) (SEQ ID NO:48/49) or cholesteryl handles the relative expression of ApoB-100mRNA in (every day, intravenous administration continued 3 days) back liver.

Fig. 2 B: the siRNA (SEQ ID NO 50/49) that modifies with SEQ ID NO:29, siRNA (unmodified) (SEQ ID NO:48/49) or cholesteryl handles the relative expression of ApoB-100mRNA in (every day, intravenous administration continued 3 days) back jejunum.

Fig. 3: with SEQ ID NO:29, the level relatively of cholesterol in the mice plasma that the siRNA (SEQ ID NO:50/49) that siRNA (unmodified) (SEQ ID NO 48/49) or cholesteryl are modified handles.

Fig. 4: external, the ApoB-100 target downward modulation in BNCL or Hepa 1-6 cell.In the mouse cell lines, the dose response effect of SEQ ID NO:29 and 37 pairs of ApoB mRNA levels (with respect to the GapDH stdn).

Fig. 5 A: in vivo, the C57BL/6 mouse is the ApoB-100 silence in the liver after the LNA antisense is handled.In the C57BL/6 mouse, the LNA antisense molecule gives potion (6.25,12.5 or 25mg/kg), then logotype 3 days of siRNA (50mg/kg).By qPCR measurement ApoB-100 expression and with respect to the Gapdh stdn.Data represented mean value ± SD (n=7).

Fig. 5 B: in vivo, the C57BL/6 mouse is the ApoB-100 silence in the jejunum after the LNA antisense is handled.In the C57BL/6 mouse, the LNA antisense molecule gives potion (6.25,12.5 or 25mg/kg), then logotype 3 days of siRNA (50mg/kg).By qPCR measurement ApoB-100 expression and with respect to the Gapdh stdn.Data represented mean value ± SD (n=7).

Blood plasma cholesterol level after Fig. 6 A:LNA antisense is handled.In the C57BL/6 mouse, the LNA antisense molecule gives potion (6.25,12.5 or 25mg/kg), then logotype 3 days of siRNA (50mg/kg).The LDL-cholesterol levels uses the colorimetric reagent box to measure.Data represented mean value ± SD (n=7).

Blood plasma cholesterol level after Fig. 6 B:LNA antisense is handled.In the C57BL/6 mouse, the LNA antisense molecule gives potion (6.25,12.5 or 25mg/kg), then logotype 3 days of siRNA (50mg/kg).The total plasma cholesterol level uses the colorimetric reagent box to measure.Data represented mean value ± SD (n=7).

Fig. 7: the sequence of the reverse complementary sequence of the preferred sequence of demonstration ApoB target nucleic acid compares, and described sequence is used for design consideration oligomeric compounds of the present invention.

Fig. 8: in-vitro screening in Huh-7 (liver cell) cell of handling with different LNA antisense oligonucleotide and dose response (1,5 or 25nM) and as said target mrna (ApoB-100) downward modulation measure the effect of the oligonucleotide of (QPCR).

Fig. 9: the IC50 (50% suppresses the antisense oligonucleotide concentration that target (ApoB-100) is expressed) of the LNA antisense oligonucleotide of 7 selections of in the Huh-7 cell, measuring of analyzing by QPCR.

Figure 10 A: during execution in the 28th day, the ApoB-100mRNA level of measuring in the liver.The administration of C57BL/6 mouse is: weekly twice, and 2.5mg/kg/ agent (8 doses altogether), or weekly, 5mg/kg (4 doses altogether) continued for 4 weeks.

Figure 10 B: the plasma LDL levels of behind socket of the eye, measuring in the blood in totally 4 weeks once in a week.The administration of C57BL/6 mouse is: weekly twice, and 2.5mg/kg/ agent (8 doses altogether), or weekly, 5mg/kg (4 doses altogether) continued for 4 weeks.

Figure 11 A:, be determined as the acting duration of ApoB-100mRNA level in the liver when being condemned to death in 8,13 or 21 days the 3rd, 5.1,2 or 3 doses of C57BL/6 mouse administrations, 25mg/kg/ agent SEQ ID NO:37, continuous 1,2 or 3 day every day be potion respectively.

Figure 11 B: the 3rd, 5,8,13, or when being condemned to death in 21 days, the total plasma cholesterol of mensuration.1,2 or 3 doses of C57BL/6 female mice administrations, the SEQ ID NO:37 of 25mg/kg/ agent, continuous 1,2 or 3 day every day be potion respectively

Detailed description of the Invention

The present invention uses oligomeric compounds, and ASON particularly is used for regulating the function of the nucleic acid molecules of coding apolipoprotein B (such as Apo-B100 and/or ApoB-48). This regulates final so that the apolipoprotein B amount that produces changes. In the embodiment, this is by providing oligomeric compounds to finish, and hybridize with nucleic acid such as the mRNA of coding apolipoprotein B on described compound specificity ground. This regulates and preferably to cause inhibition that apolipoprotein B is expressed, that is to say, causes the quantity of the functional protein that produces to descend.

Fig. 1 shows that external the siRNA that comprises the LNA nucleotide analog is effective with single stranded antisense oligonucleotides in identical nanomole scope. Yet in vivo, 16 aggressiveness LNA ASONs of the present invention are better than siRNA unmodified and that cholesterol is puted together.

Fig. 2 A and 2B show in vivo, and the siRNA that LNA oligonucleotide of the present invention is puted together than cholesteryl is up to 8 times more effective (cf.).The LNA oligonucleotide reduces the total cholesterol level in the mice plasma, and siRNA handles then can not (Fig. 3).In addition, the LNA oligonucleotide has more biologically stable than siRNA.

Regulate oligomeric compounds that target expresses and be by experiment or identify by the technical skill of carrying out appropriate design based on target sequence information and how carry out oligonucleotide compound optimum design at desired target.The sequence of these compounds is the preferred embodiments of the invention.Similarly, these preferred oligonucleotide compounds with it the sequence motifs (being used as " focus ") in the complementary target be preferred target site.

Oligomeric compounds and oligonucleotide compound

Term " oligomeric compounds " can and term " oligonucleotide ", " oligomer " and " oligonucleotide compound " exchange, in the context of the invention, be meant a kind of oligomer, just nucleic acid polymers (for example, Yeast Nucleic Acid (RNA) or thymus nucleic acid (DNA) or nucleic acid analog as known in the art, preferred locked nucleic acid (LNA), or its mixture.The oligonucleotide that this term comprises the oligonucleotide that is connected to form by (main chain) between naturally occurring nucleic acid base, sugar and nucleosides and has the part that non-natural exists (its functional similarity or have specific improved function).The oligonucleotide of modifying wholly or in part or replacing is through being often better than natural form, because this class oligonucleotide has the characteristic of several hope, as the ability of permeates cell membranes, the good resistance of nuclease to born of the same parents in the outer or born of the same parents is to the high-affinity and the specificity of nucleic acid target.The LNA analogue is especially preferred, for example, and with regard to the characteristic of as above being mentioned.Therefore, in the highly preferred embodiment, according to the present invention, term " oligomeric compounds ", " oligonucleotide ", " oligomer " and " oligonucleotide compound " is such compound, its by Nucleotide and nucleotide analog unit for example the LNA cell formation to form compound (oligomer) between 12-50 Nucleotide/nucleotide analog.

Term " unit " is interpreted as monomer.

Oligomeric compounds of the present invention can with the apolipoprotein B messenger RNA(mRNA) and/or justice arranged or complementary Mammals apolipoprotein B (Apo-B) DNA chain hybridization.NCBI accession number NM_000384 provides the mRNA sequence of human apolipoprotein B.It is highly preferred that oligomeric compounds of the present invention can (comprise with human apolipoprotein or its reverse complemental thing by disclosed nucleic acid encoding among the NCBI accession number NM_000384, in a preferred embodiment, derive from the mRNA nucleic acid target of described human apolipoprotein) hybridization.

In a preferred embodiment, described oligonucleotide can with for example ApoBmRNA hybridization of target nucleic acid, form Tm and be at least 37 ℃, for example at least 40 ℃, at least 50 ℃, at least 55 ℃, or at least 60 ℃ duplex.In one aspect, Tm between 37 ℃-80 ℃, for example, between 50-70 ℃.

The mensuration of Tm

With 3 μ M compounds at 10mM sodium phosphate/100mM NaCl/0.1nM EDTA, solution among the pH7.0 and its complementary DNA or RNA oligonucleotide (3 μ M concentration, at 10mM sodium phosphate/100mM NaCl/0.1nM EDTA, among the pH7.0) mixed 1 minute at 90 ℃, and be cooled to room temperature.By in 25-95 ℃ scope,, measure absorbancy then, measure the melting curve of duplex at 260nm with the heating rate of 1 ℃/min.Tm is determined as the maximum value of melting curve first order derivative.

Oligomeric compounds is the antisense oligomeric compounds preferably, is also referred to as " antisense oligonucleotide " and " antisense inhibitor ".

Such antisense inhibitor is the compound that comprises with target nucleic acid complementary Nucleotide/nucleotide analog sequence, and can gets " siRNA ", " miRNA ", " ribozyme ", the form of " oligozyme ".Yet preferably, the Antisense Suppression thing is a single stranded oligonucleotide.This single stranded oligonucleotide preferably with the respective regions complementation of target nucleic acid.

Typically, strand ' antisense ' oligonucleotide interacts with the mRNA of target gene specifically, causes target degraded mRNA, for example by RNA enzyme H mechanism, perhaps otherwise stops translation.

In the embodiment,, justice or antisense DNA chain are for example arranged according to the oligomeric compounds of the present invention yard DNA of Mammals ApoB that can target delimits the organizational structure.Known siRNA can interact with target DNA.

Preferably comprise at least 3 nucleotide analogs according to oligomeric compounds of the present invention.Described at least three nucleotide analogs are the nucleotide analog of locked nucleic acid preferably, and the oligomeric compounds that comprises this class nucleotide analog is called as " LNA oligomeric compounds ", " LNA oligonucleotide compound " and " LNA oligonucleotide " herein.

Aptly, according to the present invention, term " oligonucleotide compound ", " oligomeric compounds ", " LNA oligomeric compounds " is oligonucleotide as defined herein, they can by for example through hydrogen bond combine with target nucleic acid and in the mankind the desired result of treatment of generation.

The present invention relates to oligomeric compounds, as oligonucleotide, by 8-50, for example, 10-50, a particularly 12-50 or 12-25 Nucleotide and/or nucleotide analog are formed, and wherein said compound comprises at least 8, for example at least 10, as at least 12, as at least 14, as at least 15, as the subsequence of 14,15,16 or 17 Nucleotide or nucleotide analog, described subsequence is positioned at the sequence (nucleic acid target sequence) of (promptly corresponding to) Apo-B100 and/or Apo-B48.Described nucleotide analog is sequence SEQ ID NO:2-26, especially SEQ ID NO:2,3,10,11 and the analogue of 21 each corresponding nucleotide.Therefore, the subsequence of The compounds of this invention is positioned at (promptly corresponding to) and is selected from SEQ ID NO:2-26, especially SEQ ID NO:2 is in 3,10,11 and 21 the sequence, perhaps comprise SEQ ID NO:2-26, especially SEQ ID NO:2,3,10, the analogue of the Nucleotide in 11 and 21 the sequence.

The preferred sequence set that the subsequence of compound is positioned at wherein (or subsequence comprises the analogue of Nucleotide wherein) comprises SEQ ID NO:2﹠amp; 3; SEQ ID NO:2﹠amp; 3﹠amp; 11; SEQID NO:10﹠amp; 11; SEQ ID No 21.

In the embodiment, the sequence set that the subsequence of compound is positioned at wherein (or subsequence comprises the analogue of Nucleotide wherein) comprises SEQ ID No 3.

In the embodiment, the sequence set that the subsequence of compound is positioned at wherein (or subsequence comprises the analogue of Nucleotide wherein) comprises and is selected from SEQ ID No 2, SEQ ID No 3, SEQID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ IDNo 15, SEQ ID No 16, SEQ ID No 17, SEQ ID No 27, SEQ ID No 28, the sequence of SEQ ID No 48 and SEQ ID No 50.

In the interesting embodiment, compound of the present invention comprises 8-50 Nucleotide, wherein said compound comprises the subsequence of at least 8 Nucleotide, described subsequence is positioned at the sequence that is selected from SEQ ID NO:2 and 3, wherein at least one Nucleotide substituted by corresponding nucleotide analog and wherein 3 ' hold to comprise Nucleotide rather than nucleotide analog.

In the embodiment of the The compounds of this invention that comprises 8-50 Nucleotide, wherein said compound comprises the subsequence of at least 8 Nucleotide, described subsequence is positioned at the sequence and the described Nucleotide that are selected from SEQ ID NO:2 and 3 and comprises the LNA nucleotide analog, this subsequence typically can comprise one section 2-6 LNA (as defined) here, thereafter being one section 4-12 Nucleotide, is one section 2-6 LNA (as defined here) after again.

Term " be positioned at ... interior " and " corresponding to " refer to the Nucleotide of oligomeric compounds of the present invention or its subsequence and the composite sequence and the i of nucleotide analog) the reverse complemental thing of apolipoprotein B nucleotide sequence (being nucleic acid target) and/or ii) respectively by SEQ ID NO:2-26, and the nucleotide sequence (being sequence motifs) that provided of 59-67 be equal to comparison between the nucleotide sequence (or its reverse complemental thing) in one embodiment.The Nucleotide that nucleotide analog directly is equal to it compares.

Subsequence can contain at least 8, for example at least 9, for example at least 10, for example at least 11, for example at least 12, for example at least 13, for example at least 14, for example at least 15, for example at least 16, for example at least 17, for example at least 18, for example at least 19, or at least 20 Nucleotide or nucleotide analog, it is corresponding to being selected from SEQ IDNo.63, SEQ ID No.64, SEQ ID No.65, SEQ ID No.66, the continuous nucleotide (see figure 7) of the equal number that exists in the nucleic acid of SEQ ID No.67 and SEQ ID No 68.

Preferably, at least 3 nucleotide analogs are arranged in described subsequence, randomly the continuous sequence of at least 3 nucleotide analogs of conduct, for example continuous sequence of 3,4,5 or 6 nucleotide analogs.

In a preferred embodiment, oligomeric compounds only is made up of a sub-sequence, and promptly the whole sequence of this oligomeric compounds sees in the corresponding sequence, for example is selected from the sequence of SEQ ID No 2-26 and SEQ ID No 59-62.

Preferably, do not have the Nucleotide or the nucleotide analog that form mispairing when related with the respective regions of ApoB target sequence, all Nucleotide that exist in the oligomer promptly of the present invention and nucleotide analog can both form continuous base pairing with the ApoB nucleic acid target sequence.

Yet, in the embodiment, in subsequence and nucleic acid target sequence, may have 1 mispairing or 2 mispairing.When mispairing took place, preferably mispairing was not between nucleotide analog and target sequence.

Yet in the gapmer that can raise RNA enzyme H " breach ", mispairing may cause recruiting the forfeiture of RNA enzyme H ability.Typically, need 5 or 6 continuous complementary nucleotides to guarantee competent RNA enzyme H activity.

In preferred embodiments, oligonucleotide compound of the present invention comprises the sequence corresponding to SEQID NO 59 and/or SEQ ID NO.60, and wherein said subsequence can randomly comprise 1 or 2 mispairing.

In the embodiment, oligonucleotide compound of the present invention comprises the sequence corresponding to SEQ IDNO.61 and/or SEQ ID NO.62, and wherein said subsequence can randomly comprise 1 or 2 mispairing.

In the preferred embodiment of the invention, subsequence comprises at least 8, for example at least 10, or at least 12, for example at least 14, for example 14,15,16,17,18,19 or 20 Nucleotide or nucleotide analogs, the continuous nucleotide of they are arranged in (promptly corresponding to) SEQ ID No 63 equal numbers, wherein said subsequence can randomly comprise 1 or 2 mispairing.

In other embodiment of the present invention, subsequence comprises at least 8, for example at least 10, or at least 12, for example at least 14, for example between 14-20, for example 14,15,16,17,18,19 or 20 Nucleotide or nucleotide analog, they are positioned at (promptly corresponding to) is selected from SEQ ID No 64, SEQ ID No 65, SEQ ID No 66, in the continuous nucleotide of equal number, wherein said subsequence can randomly comprise 1 or 2 mispairing in the nucleotide sequence of SEQ ID No 67 and SEQ ID No 68.

In the embodiment, oligomeric compounds of the present invention is a double chain oligonucleotide, wherein, every chain comprise (or by ... form) 16-30 Nucleotide and/or nucleotide analog altogether.Will be appreciated that a chain in the double-stranded complex body (oligonucleotide) corresponding to the oligonucleotide compound of definition herein, and another chain is the oligonucleotide with complementary sequence.

For example 8-50 Nucleotide and/or nucleotide analog mean 8-50 Nucleotide or 8-50 nucleotide analog or are no more than total 50 unitary its combinations of nucleosides altogether altogether.

Compound is preferably by 12-25 Nucleotide or nucleotide analog, and for example 13,14,15,16,17,18,19,20,21,22,23 or 24 Nucleotide or nucleotide analog are formed, for example 15-22 Nucleotide or nucleotide analog, for example 14-18 Nucleotide or nucleotide analog, more preferably 15 or 16 Nucleotide or nucleotide analog.

Herein, term " nucleosides " and " Nucleotide " use with their common implications.For example, it contains the 2-deoxyribosyl unit, and to nitrogenous base VITAMIN B4 (A), cytosine(Cyt) (C) is on one of thymus pyrimidine (T) or guanine (G) via the one carbon atom bonding for the latter.

Similarly, for example, in a preferred embodiment, when relating to The compounds of this invention, term " Nucleotide " refers to the 2-deoxyribosyl unit, it via the one carbon atom bonding to nitrogenous base VITAMIN B4 (A), cytosine(Cyt) (C), one of thymus pyrimidine (T) or guanine (G), and it is via its No. 5 carbon atom bondings to phosphoric acid between nucleosides (or in one embodiment, the group that is equal to, for example thiophosphoric acid group) or end group.Nucleotide also can, for example in one embodiment, comprise the ribose unit, for example RNA Nucleotide.

When this used, term " nucleotide analog " referred to the Nucleotide that non-natural exists, wherein, for example in a preferred embodiment, the ribose unit is different from 2-deoxyribosyl and/or nitrogenous base is different from A, and the phosphoric acid linking group is different between C, T and G and/or nucleosides.The object lesson of nucleoside analog is by Freier and Altmann Nucl.Acid Res., 1997,25,4429-4443 and Uhlmann; Curr.Opinion in Drug Development, 2000,3 (2), 293-213 and description in chart 1.

It is identical with nitrogenous base in the nucleoside/nucleotide that term " corresponding nucleoside/nucleotide analogue " and " corresponding nucleoside/nucleotide " mean the nucleoside/nucleotide analogue.For example, when the 2-deoxyribosyl unit of Nucleotide linked to each other with VITAMIN B4, " corresponding nucleoside analog " contained the pentose unit (being different from 2-deoxyribosyl) that links to each other with VITAMIN B4.

Term " nucleic acid " is defined as by 2 or the covalently bound molecule that forms of more a plurality of Nucleotide.Term " nucleic acid " and " polynucleotide " are used interchangeably at this.For example, DNA and RNA are nucleic acid.

Term " nucleic acid analog " refers to the nucleic acid binding compounds that non-natural exists, and promptly in a preferred embodiment, is a kind of compound, for example at least 1 Nucleotide and at least 1 nucleotide analog unitary sequence of LNA for example.These compounds are not natural to be seen in the mammalian organism (or in one embodiment, not known being found in the mammalian organism when of the present invention).

Preferred nucleotide analog is LNA, β-D-oxygen-LNA for example, α-L-oxygen-LNA, β-D-amino-LNA and β-D-sulphur-LNA, most preferably β-D-oxygen-LNA.The compounds of this invention is those compounds typically, and wherein said Nucleotide comprises the linking group that is selected from phosphate group, thiophosphoric acid group and borine phosphate (boranophosphate group), connect between nucleosides can be-O-P (O) ₂-O-, and-O-P (O, S)-O-, especially phosphate group and/or thiophosphoric acid group.In a special embodiment, all Nucleotide all comprises the thiophosphoric acid group.In the embodiment, some or all Nucleotide are connected to each other by the thiophosphoric acid group.Aptly, all Nucleotide are connected to each other by the thiophosphoric acid group.

Nucleotide typically is connected to each other by linking group.

Nucleotide analog and nucleic acid analog are at Freier﹠amp; And Altmann (Nucl.AcidRes., 1997,25,4429-4443) and Uhlmann (Curr.Opinion in Drug﹠amp; Developmen t (2000,3 (2): describe 293-213 ).Chart 1 and 2 illustrations be suitable for preparing the selection example of the nucleotide analog of nucleic acid:

Chart 1

In an interesting embodiment, compound comprises 3-12 nucleotide analog, for example 6 or 7 nucleotide analogs.In the most preferred embodiment up to the present, at least one described nucleotide analog is locked nucleic acid (LNA), as at least 2, or at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11 nucleotide analogs can be LNA, in one embodiment, whole nucleotide analogs can be LNA.

Term " LNA " refers to nucleotide analog, and it comprises one two cyclic nucleotide analogue, is also referred to as the LNA monomer.

" the LNA oligonucleotide " during this linguistic context, refers to an oligonucleotide that comprises one or more two rings nucleoside analog to term " LNA " when being used for.The locked nucleic acid (LNA) that uses in the oligonucleotide compound of the present invention has the structure of following general formula.

X and Y independently are selected from-O-,-S-, and-N (H)-,-N (R)-,-CH ₂-or-CH-(if being the part of two keys) ,-CH ₂-O-,-CH ₂-S-,-CH ₂-N (H)-,-CH ₂-N (R)-,-CH ₂-CH ₂-or-CH ₂-CH-(if being the part of two keys) ,-CH=CH-, wherein R is selected from hydrogen and C _1-4-alkyl; Z independently be selected between nucleosides with Z* be connected, end group or blocking group; B constitutes natural or non-natural nucleic acid base; And asymmetric group can be with any orientation.

Preferably, the locked nucleic acid (LNA) that uses in the oligonucleotide compound of the present invention comprises at least one locked nucleic acid according to following arbitrary general formula (LNA) unit.

Wherein, Y is-O--S-,-NH-or N (R ^H); Z independently be selected between nucleosides with Z* be connected, end group or blocking group, B constitutes natural or non-natural nucleic acid base, R ^HBe selected from hydrogen and C _1-4-alkyl.

Preferably, used locked nucleic acid (LNA) comprises to be selected between following nucleosides and connects in the oligonucleotide compound of the present invention :-O-P (O) ₂-O-, and-O-P (O, S)-O-,-O-P (S) ₂-O-,-S-P (O) ₂-O-, and-S-P (O, S)-O-,-S-P (S) ₂-O-,-O-P (O) ₂-S-, and-O-P (O, S)-S-,-S-P (O) ₂-S-,-O-PO (R ^H)-O-, O-PO (OCH ₃)-O-,-O-PO (NR ^H)-O-,-O-PO (OCH ₂CH ₂S-R)-and O-,-O-PO (BH ₃)-O-,-O-PO (NHR ^H)-O-,-O-P (O) ₂-NR ^H-,-NR ^H-P (O) ₂-O-,-NR ^H-CO-O-.Wherein, R ^HBe selected from hydrogen and C _1-4-alkyl.

As described, in an interesting embodiment of the present invention, the oligonucleotide compound comprises at least one chemical unit that is called LNA (locked nucleic acid).

Particularly preferred LNA unit is shown in chart 2.

Chart 2

Term " sulphur-LNA " comprises locking Nucleotide, in the wherein above-mentioned general formula among X or the Y at least one be selected from S or-CH ₂-S-.Sulphur-LNA can be β-D and α-L-configuration.

Term " amino-LNA " comprises locking Nucleotide, in the wherein above-mentioned general formula among X or the Y at least one be selected from-N (H)-, N (R)-, CH ₂-N (H)-,-CH ₂-N (R)-, wherein R is selected from hydrogen and C _1-4-alkyl.Amino-LNA can be β-D and α-L-configuration.

Term " oxygen-LNA " comprises locking Nucleotide, in the wherein above-mentioned general formula among X or the Y at least representative-O-or-CH ₂-O-.Oxygen-LNA can be β-D and α-L-configuration.

Term " ena-LNA " comprises locking Nucleotide, and the Y in the wherein above-mentioned general formula is-CH ₂-O-(wherein-CH ₂The Sauerstoffatom of-O-is connected to 2 '-position with respect to nucleic acid base B).

In a preferred embodiment, LNA is selected from β-D-oxygen-LNA, α-L-oxygen-LNA, β-D-amino-LNA and β-D-sulphur-LNA, especially β-D-oxygen-LNA.Nucleosides and/or LNA typically link together by phosphate group and/or thiophosphoric acid group.

Term " at least one " comprises the integer more than or equal to 1, and for example 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 etc.

Employed here term " target nucleic acid " has covered the DNA of coding Apo-B100, the RNA that transcribes from this DNA (before comprising-mRNA and mRNA and mRNA compilation) and derive from the cDNA of this RNA.

" target protein " is the Mammals apolipoprotein B, preferred human apolipoprotein B.People will appreciate that because ApoB-100 derives from identical genetic sequence with ApoB-48, therefore oligomeric compounds of the present invention can be used for reducing apolipoprotein Bs arbitrary or two kinds of forms, with the mRNA of coding ApoB-100, and the rna editing form of coding Apo-B48.

As used herein, term " gene " expression comprises the gene of exon, intron, non-coding 5 ' and 3 ' district and regulatory element and all present known its variants and any other variant that may be illustrated.

The present known mRNA transcript of employed here term " mRNA " expression target gene, and any other transcript that may be illustrated.

The increase (stimulation) or the reduction (inhibition) of employed here term " adjusting " expression genetic expression.Among the present invention, inhibition is that form is regulated in preferred genetic expression and mRNA is preferred target.

Employed here term " target " antisense compounds means by this way to specific target nucleic acid and provides antisense oligonucleotide to cell, animal or human, makes antisense compounds to expect target and regulate its function in conjunction with it.

Preferred nucleotide analog is LNA.

Further preferred nucleotide analog is that wherein to connect between nucleosides be thiophosphatephosphorothioate.

Preferred again nucleotide analog is that wherein Nucleotide is to have the LNA that thiophosphatephosphorothioate connects between nucleosides.

In an interesting embodiment, 3 ' end of The compounds of this invention comprises Nucleotide rather than nucleotide analog.

Preferably, oligomeric compounds of the present invention for example antisense oligonucleotide comprises at least one locked nucleic acid (LNA) unit, and for example 3,4,5,6,7,8,9 or 10 locked nucleic acids (LNA) unit, preferred 4-9 LNA unit, for example 6-9 LNA unit most preferably is 6,7 or 8 LNA unit.Preferably, the LNA unit comprises at least 1 β-D-oxygen-LNA unit, and for example 4,5,6,7,8,9 or 10 β-D-oxygen-LNA unit.Although think oligomeric compounds for example antisense oligonucleotide can comprise and surpass one type LNA unit, all LNA unit can all be β-D-oxygen-LNA unit.Aptly, oligomeric compounds can comprise one or more in β-D-oxygen-LNA unit and the following LNA unit: sulphur-LNA simultaneously, amino-LNA, and oxygen-LNA, ena-LNA and/or α-LNA are D-β or L-α configuration, or its combination.

Comprise nucleotide analog for example in the embodiment of LNA nucleotide analog at The compounds of this invention, subsequence typically can comprise one section 2-6 nucleotide analog (LNA nucleotide analog for example, as defined here), be one section 4-12 Nucleotide thereafter, connect again one section 2-6 nucleotide analog (LNA nucleotide analog for example, as herein defined).

Contain one section nucleotide analog for example the LNA nucleotide analog, thereafter be one section Nucleotide, be that the subsequence of one section nucleotide analog LNA is called gapmer after again.

Aptly, in such " gapmer " embodiment, described subsequence comprises one section 4 nucleotide analog (LNA nucleotide analog for example, as defined here), it is one section 8 Nucleotide thereafter, be one section 4 nucleotide analog (as defined LNA nucleotide analog here) again, randomly have a single Nucleotide at 3 ' end.

In other " gapmer " embodiment, described subsequence comprises one section 3 nucleotide analog (LNA nucleotide analog for example, as defined here), it is one section 9 Nucleotide thereafter, be one section 3 nucleotide analog (as defined LNA nucleotide analog here) again, randomly have a single Nucleotide at 3 ' end.It is very effective that people are surprised to find such design.

In another " gapmer " embodiment, described subsequence comprises one section 4 nucleotide analog (LNA nucleotide analog for example, as defined here), it is one section 8 Nucleotide thereafter, be one section 3 nucleotide analog (as defined LNA nucleotide analog here) again, randomly have a single Nucleotide at 3 ' end.

Preferably, oligomeric compounds, antisense oligonucleotide for example, can comprise LNA and dna single unit both.Preferably LNA and dna single unit add up to 14-20 altogether, for example between 15-18, and more preferably 16 or 17 LNA/DNA unit.Preferably, LNA that exists in the oligomeric compounds of the present invention and the ratio of DNA are between 0.3 and 1, more preferably between 0.4-0.9, for example between 0.6 and 0.8.

Preferably, oligomeric compounds, for example antisense oligonucleotide is gapmer according to the present invention, and it comprises the polynucleotide sequence (5 ' to 3 ') of following general formula, A-B-C (with D randomly), wherein, A (5 ' district) comprises at least one LNA unit or is made up of it, for example between 1-6 LNA unit, preferably between 2-5 LNA unit, 4 LNA unit most preferably; B (central field) preferably is close to the 3 ' end of A, comprises at least one DNA sugar unit or is made up of it, 1-12 dna single unit for example is preferably between 4-12 dna single unit, more preferably between 6-10 dna single unit, for example 7-9 dna single unit, most preferably 8 dna single units; C (3 ' district) preferably is close to the 3 ' end of B, comprises at least one LNA unit or is made up of it, for example 1-6 LNA unit, preferably 2-5 LNA unit, most preferably 4 LNA unit.Preferred gapmer design is open in WO2004/046160.

In the gapmer oligonucleotide, preferably any mispairing is not within the above-mentioned central field (B) that preferably comprises dna single unit or be made up of dna single unit.For RNA enzyme H digestion, need in the central field typically to find at least 5 continuous nucleotides (maybe RNA enzyme H can be raised the analogue of oligomer/target crossbred).Therefore, for gapmer, when central field surpasses 5 continuous nucleotides, envision 1 or may 2 mispairing may be acceptable, although this is also not preferred.。

In the embodiment of a gapmer oligonucleotide, can preferred any mispairing all be positioned at 5 ' or 3 ' end towards gamper.In such embodiments, preferably, in the gapmer oligonucleotide that comprises with the mispairing of said target mrna, such mispairing is positioned at 5 ' and/or 3 ' district, and/or described mispairing is between 5 ' or 3 ' the terminal nucleotide unit and target molecule of described gapmer oligonucleotide.

In one embodiment, the gapmer of general formula A-B-C also comprises further region D, it comprises 3 ' of one or more described oligomeric compounds and distinguishes (C) terminal DNA saccharide residue or form (preferably being made up of it) by described DNA saccharide residue, 1-3 DNA saccharide residue for example, comprise between 1-2 DNA saccharide residue, most preferably 1 DNA saccharide residue.

In one embodiment, for example in the antisense oligonucleotide, all LNA C residues all are 5 ' methyl-cytosine(Cyt)s at the oligomeric compounds that comprises LNA according to the present invention.

Make us especially in the interested embodiment at one, compound has following formula

5’-[(LNA) _3-4-(DNA/RNA) _8-9-(LNA) ₃-(DNA/RNA) ₁]-3’

Wherein, " LNA " indication LNA Nucleotide and " DNA " and " RNA " indicate deoxyribonucleotide and ribonucleotide respectively.

More specifically, compound can be selected from SEQ ID NO:29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46 and 47.Preferred compound can be selected from SEQ ID No 29,30,31,32,36,37,38,40, and 41 and 42 or be selected from SEQ ID No 30 and 31, and/or be selected from SEQ ID No 36,37 and 38, and/or be selected from SEQ ID No 41 and 42.Most preferred is that those are selected from SEQID NO:29 at present, the compound of SEQ ID NO:30 and SEQ ID NO:37.

Suitably, described Nucleotide and/or described LNA can be connected together by phosphate group and/or thiophosphoric acid group or its.

In one embodiment, described Nucleotide and/or described LNA preferably link together by the thiophosphoric acid group.

In one embodiment, the invention provides and comprise SEQ ID NO:29 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:30 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:31 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:32 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:33 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:34 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:35 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:36 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:37 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:38 or by its oligonucleotide compound of forming.

In embodiments, the invention provides an oligonucleotide compound that comprises or form by SEQ ID NO:39.

In one embodiment, the invention provides and comprise SEQ ID NO:40 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:41 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:42 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:43 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:44 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:45 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:46 or by its oligonucleotide compound of forming.

In one embodiment, the invention provides and comprise SEQ ID NO:47 or by its oligonucleotide compound of forming.

In the embodiment, when oligonucleotide of the present invention is the RNA oligonucleotide, for example SEQID No 48,4950 or 51 o'clock, 3 ' end comprises the ribonucleoside acid units that two common 2 '-O-methyl that connect are modified, with terminal ribonucleotide direct neighbor.

The preparation of oligonucleotide compound

LNA nucleotide analog component of thing (β-D-oxygen-LNA, β-D-sulphur-LNA, β-D-amino-LNA and α-L-oxygen-LNA) can follow the program of having delivered and the reference of wherein being quoted is prepared, referring to, for example, WO 03/095467A1; D.S.Pedersen, C.Rosenbohm, T.Koch (2002) Preparation of LNA Phosphoramidites, Synthesis 6,802-808; M.D.

L.

T.Bryld, A.E.

B.Verbeure, G.Gaubert, P.Herdewijn, J.Wengel (2002) α-L-ribo-configured Locked Nucleic Acid (α-1-LNA): Synthesis and Properties, J.Am.Chem.Soc., 124,2164-2176; S.K.Singh, R.Kumar, J.Wengel (1998) Synthesis of NovelBicyclo[2.2.1] Ribonucleosides:2 '-Amino-and 2 '-Thio-LNAMonomeric Nucleosides, J.Org.Chem.1998,63,6078-6079; C.Rosenbohm, S.M.Christensen, M.D.

D.S.Pedersen, L.E.Larsen, J.Wengel, T.Koch (2003) Synthesis of 2 '-amino-LNA:a new strategy, Org.Biomol.Chem.1,655-663; With WO 2004/069991A2.

The monomeric special example of thymidine LNA be (1S, 3R, 4R, 7S)-7-hydroxyl-1-methylol-3-(thymus pyrimidine-1 base)-2,5-two oxa-s-two ring [2: 2: 1] heptane.

The LNA oligonucleotide can be as in an embodiment with at WO 99/14226, and WO 00/56746, and WO 00/56748, and WO 00/66604, and WO 00/125248, and WO 02/28875, prepares like that described in WO2002/094250 and the WO 03/006475.Therefore, the LNA oligonucleotide can use the nucleic acid chemistry oligomerization technology production that the organic chemistry filed those of ordinary skill is known.Usually use the circulation of standard oligomerization (S.L.Beaucage and R.P.Iyer, Tetrahedron, 1993,49,6123 of phosphoramidite approach; S.L.Beaucage and R.P.Iyer, Tetrahedron, 1992,48,2223), but for example H-phosphonic acids chemical method, phosphotriester chemical method also can use.

For some monomers, coupling time of length, and/or multiple coupling and/or the more spissated coupling reagent of use may be essential or useful.

The phosphoramidite that uses typically carries out coupling with the substep productive rate of gratifying＞95%.Usually, with for example iodine/pyridine/H ₂O realizes the oxidation of three valent phosphors (III) to pentavalent phosphorus (V).Go after the protection, this can produce phosphodiester bond between natural nucleosides.Under the situation of phosphorothioate bond between the preparation nucleosides, the following vulcanisation step of carrying out: the common (iodine/pyridine/H for example that will be used for phosphodiester bond between synthetic nucleosides ₂O) oxidation replaces with and uses ADTT reagent ((0.01M is at acetonitrile: pyridine 9: 1 for xanthane hydride; Among the v/v) oxidation.Also can use other sulfuration reagent, for example Beaucage and PADS.Synthetic effectively thiophosphoric acid LNA oligonucleotide, substep coupling productive rate＞=98%.

The LNA oligonucleotide that comprises β-D-amino-LNA, β-D-sulphur-LNA and/or α-L-LNA also can use the phosphoramidite method synthetic effectively, substep coupling productive rate＞=98%.

Use disposable anti-phase purification column and/or reversed-phase HPLC and/or from ethanol or butanols, precipitate, can realize the purifying of LNA oligonucleotide.Use capillary gel electrophoresis, reversed-phase HPLC, MALDI-MS, and ESI-MS confirm the purity of synthetic LNA oligonucleotide.

Salt

The LNA oligonucleotide can use with multiple pharmacy acceptable salt.This term used herein is meant, can keep the expectation biological activity of LNA oligonucleotide and show the salt of the minimum toxicological effect of not expecting.The non-limiting example of this class salt can form with organic amino acid, base addition salt and metallic cation be formation such as zinc, calcium, bismuth, barium, magnesium, aluminium, copper, cobalt, nickel, cadmium, sodium, potassium for example, or with by ammonia, N, the positively charged ion that N-dibenzyl-ethylenediamin, D-glycosamine, Tetrylammonium or quadrol form forms; Or its combination, for example Weibull zinc salt etc.

This class salt is to be formed by the LNA oligonucleotide with phosphodiester group and/or thiophosphoric acid ester group, for example the salt that forms with appropriate base.These salt comprise, for example are derived from the nontoxic metal-salt of periodic table of elements Ia, Ib, IIa and IIB family metal, particularly suitable an alkali metal salt, for example lithium salts, sodium salt or sylvite, or alkaline earth salt, for example magnesium salts or calcium salt.They also comprise zinc salt and ammonium salt, and the salt that forms with the organic amine that is fit to, the for example unsubstituted or hydroxyl of described organic amine replace one, two or trialkylamine, particularly one, two or trialkylamine, or the salt that forms with quaternary ammonium compound, for example with N-methyl-N-ethylamine, diethylamine, triethylamine, single, two or three-(2-hydroxy lower alkyl) amine are for example single, two or three-(2-hydroxyethyl) amine, 2-hydroxyl-tert-butylamine or trihydroxymethylaminomethane, N, N-two low alkyl groups-N-(hydroxy lower alkyl) amine is N for example, and the salt that N-dimethyl-N-(2-hydroxyethyl) amine or three-(2-hydroxyethyl) amine or N-methyl D-glycosamine or quaternary ammonium compound form is 4-butyl ammonium for example.Preferred lithium salts, sodium salt, magnesium salts, zinc salt or sylvite, special particular certain cancers.

Prodrug

In one embodiment, the LNA oligonucleotide can be the form of prodrug.In fact, oligonucleotide is electronegative ion.Because the lipophilic character of cytolemma is compared with neutral or lipophilic Equivalent, the cellular uptake of oligonucleotide reduces.By use preceding regimen (referring to for example Crooke, R.M. (1998) in Crooke, S.T.Antisense research andApplication.Springer-Verlag, Berlin, Germany, vol.131, pp.103-140), can avoid this polarity " obstacle ".In this scheme, prepare the LNA oligonucleotide in shielded mode, make that the LNA oligonucleotide is a neutral when using.Can remove the mode of blocking group during with cellular uptake LNA oligonucleotide, design these blocking groups.The example of these blocking groups is S-ethanoyl thio-ethyl (SATE) or S-pivaloyl group thio-ethyl (tertiary butyls-SATE).These blocking groups are nuclease resistances, can optionally be removed in cell.

Conjugate

Another aspect of the present invention relates to conjugate, and it comprises the compound of this paper definition and at least one and is covalently bound to non-nucleotide or non-polynucleotide part on the described compound.

At a related aspect of the present invention, compound of the present invention is connected to and forms conjugate on the part, and described part is intended to increase the cellular uptake of conjugate with respect to antisense oligonucleotide.

Compound of the present invention or conjugate also can be puted together or further be conjugated on the active drug substance, for example acetylsalicylic acid, Ibuprofen BP/EP, sulfa drug, cholesterol-lowering agent, antidiabetic, antiseptic-germicide, chemotherapeutics or microbiotic.

In the context of the present invention, term " conjugate " is intended to represent heterogeneous molecule, it partly forms to one or more non-nucleotides or non-polynucleotide by covalently bound LNA oligonucleotide described herein (that is the compound that, comprises nucleosides or LNA nucleoside analog sequence).

Therefore, LNA oligonucleotide for example right and wrong Nucleotide or non-polynucleotide is partly puted together or is formed mosaic, described non-nucleotide or non-polynucleotide partly comprise peptide nucleic acid(PNA) (PNA), albumen (for example antibody of target protein), macromole, low-molecular-weight drug, fatty acid chain, saccharide residue, glycoprotein, polymkeric substance (for example polyoxyethylene glycol), micelle formation group, antibody, carbohydrate, the receptors bind group, steroide is cholesterol for example, polypeptide, intercalator is acridine derivatives for example, long-chain alcohol, branch-shape polymer, phosphatide and other lipophilic group or their combination etc. can be arranged with dimerization or dendritic structure as the LNA oligonucleotide.LNA oligonucleotide or conjugate also can be puted together or further be conjugated to active drug substance, and described active drug substance is acetylsalicylic acid, Ibuprofen BP/EP, sulfa drug, antidiabetic drug, antiseptic-germicide, chemotherapeutics or microbiotic for example.

This mode put together the beneficial property that has brought LNA oligonucleotide drug dynamic characteristic aspect.More specifically, puting together of this mode realized the cellular uptake that increases.

In one embodiment, the LNA oligonucleotide is connected to and forms conjugate on the part, and described part is in order to increase the cellular uptake of conjugate with respect to antisense LNA oligonucleotide.This put together can appear at terminal position 5 '/3 '-OH, but this part also can appear at sugar and/or base place.More specifically, the somatomedin that can put together of antisense LNA oligonucleotide can comprise Transferrins,iron complexes or folic acid.Can prepare Transferrins,iron complexes-polylysine-oligonucleotide complex or folic acid-polylysine-oligonucleotide complex, be used for by the cellular uptake of expressing high-caliber Transferrins,iron complexes or folacin receptor.Other example of conjugate/part is the cholesterol group, and the duplex intercalator is acridine for example, and poly-L-Lysine is with the linking group of one or more nuclease resistances single thiophosphate ester " end-blocking " etc. for example.

Wagner etc., Proc.Natl.Acad.Sci.USA87,3410-3414 (1990) has described the preparation of absorbing the transferrin complex of protein of the carrier in the cell as oligonucleotide.Low etc., United States Patent (USP) 5,108,921 have described by folacin receptor endocytosis cell and have sent folic acid-macromole conjugate, comprise and send antisense oligonucleotide.Also referring to, Leamon etc., Proc.Natl.Acad.Sci.88,5572 (1991).

Pharmaceutical composition

One of the present invention is made us interested aspect especially and relates to pharmaceutical composition, and it comprises the compound of this paper definition or conjugate and pharmaceutically acceptable thinner, carrier or the assistant agent of this paper definition.Make us especially in the interested embodiment at one, this pharmaceutical composition is suitable for oral administration.

The guidance of preparation of pharmaceutical compositions is found in " the Remington:The Science and Practice of Pharmacy " of Alfonso R.Gennaro and hereinafter.

Should be appreciated that the present invention also is particularly related to pharmaceutical composition, it comprises at least a antisense oligonucleotide construct of the present invention as activeconstituents.Should be appreciated that can randomly comprise pharmaceutical carrier according to pharmaceutical composition of the present invention, described pharmaceutical composition can randomly comprise other antisense compounds, chemotherapeutics, cholesterol-lowering agent, anti-inflammatory compound, antiviral compound and/or immunomodulatory compounds.

As described, pharmaceutical composition of the present invention can further comprise at least a treatment/preventative compound.Described compound typically is selected from: biliary salts resin (for example, Colestyramine, colestipol, with the hydrochloric acid colesevelam), HMGCoA-reductase inhibitor (for example, lovastatin, Cerivastatin, Pravastatin (prevastatin), Zarator, Simvastatin and fluvastatin), nicotinic acid, derivative is (for example in the special acid of shellfish (fibric acid), clofibrate, gemfibrozil, fenofibrate, bezafibrate and Win-35833), probucol, Xin Meisu, dextrothyroxine, plant-stanol ester, cholesterol absorption inhibitor (for example, ezetimibe), implitapide, the inhibitor of bile acid transport albumen (apical sodium dependency bile acid transport albumen), liver CYP7a conditioning agent, controversies in hormone replacement in the elderly (for example, tamoxifen) and antiphlogiston (for example, glucocorticosteroid).

Oligonucleotide compound that comprises among the present invention or conjugate can use by multiple pharmacy acceptable salt.This term used herein is meant that the expectation biological activity that can remain on this compounds identified also shows the salt of the minimum toxicological effect of not expecting, referring to " conjugate ".

In one embodiment of the invention, oligonucleotide compound or conjugate can be the form of prodrug, referring to " prodrug ".

The present invention also comprises the preparation of one or more oligonucleotide compounds disclosed herein or conjugate.Pharmaceutically acceptable tackiness agent and assistant agent can constitute the part of the medicine of being prepared.Capsule, tablet and pill etc. can contain for example following compounds: as Microcrystalline Cellulose, natural gum or the gelatin of tackiness agent; Starch or lactose as vehicle; Stearate as lubricant; Various sweeting agents or correctives.For capsule, dose unit can contain liquid vehicle, for example fatty oil.Equally, the dressing of sugar-coat or intestines agent can be the part of dose unit.Oligonucleotide agent also can be active pharmaceutical ingredient and the emulsion that can form the lipid of micelle emulsion.This class preparation is specially adapted to oral administration.

Oligonucleotide of the present invention can mix with any material that does not damage expectation function, or mixes with the material of additional expectation function.These materials can comprise other medicines, comprise other nucleotide compound.

For parenteral, subcutaneous, intracutaneous or topical application, preparation can comprise the conditioning agent and the antiseptic-germicide of sterile diluent, buffer reagent, tension force and ionic strength.Activated compound can prepare together with carrier, and this carrier can be protected and avoid degraded or protect avoiding discharging immediately from body, comprises implant or microcapsule with exhibit controlled release properties.Use for intravenously, preferred carrier is physiological saline or phosphate buffered saline (PBS).

Preferably, the oligonucleotide compound is to be enough to patient's delivery treatments significant quantity and not cause the amount of the patient's that treated serious side effects to be included in the unit formulation, for example in pharmaceutically acceptable carrier or thinner.

Pharmaceutical composition of the present invention can be used with multiple mode, and what this depended on expectation is part or whole body therapeutic and zone to be treated.Use can be (a) oral (b) through lung, for example pass through atomizer by being blown into or sucking powder or aerosol, comprising; In the tracheae, in the nose; (c) part comprises epidermis, transdermal, eye and comprises vagina and the mucosal delivery of rectum; Or (d) parenteral, comprise intravenously, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; Or encephalic, for example sheath is interior or Intraventricular is used.In one embodiment, target organ is used or be applied directly to activated LNA oligonucleotide by intravenously, intraperitoneal, per os, part or as bolus infusion.

The pharmaceutical composition and the preparation that are used for topical can comprise transdermal patch, ointment, lotion, breast frost, gel, drops, sprays, suppository, liquid agent and powder agent.Conventional medicine carrier, water-based, powder or oleaginous base, thickening material etc. may be essential or expectation.The condom of bag quilt, gloves etc. also are useful.Preferred topical formulations comprise oligonucleotide wherein of the present invention and local delivery agents mutually blended those, described local delivery agent for example is lipid, liposome, lipid acid, fatty acid ester, steroid, sequestrant and tensio-active agent.

Oral composition and preparation include but not limited to powder or particle, microparticle, nanoparticle, the suspension in water or non-aqueous media or solution, capsule, gel capsule, sachet, tablet or tabloid.Typically,

In the parenteral, sheath or the composition and the preparation of Intraventricular administration can comprise aseptic aqueous solution, it can also comprise buffer reagent, thinner and other suitable additive, such as but not limited to penetration enhancers, carrier compound and other pharmaceutically acceptable carrier or vehicle.

Pharmaceutical composition of the present invention includes but not limited to solution, emulsion and contains the preparation of liposome.These compositions can be formed by many components, and these components include but not limited to prefabricated solution (preformed liquid), self-emulsification solid and self-emulsifying semisolid.Medicine can be by the carrier mediated enhancing of sending to sending of hepatic tissue, and it includes but not limited to cationic-liposome, cyclodextrin, derivatives of porphyrin, side chain dendrimer (dendrimer), polyethyleneimine polymers, nano particle and microsphere (Dass CR.J Pharm Pharmacol 2002; 54 (1): 3-27).

Routine techniques according to pharmaceutical industry is known can prepare pharmaceutical preparation of the present invention, and it can exist as unit dosage easily.Such technology comprises makes the step of activeconstituents in conjunction with pharmaceutical carrier or vehicle.Usually,, where necessary product is shaped then, prepares preparation by making activeconstituents equably and the closely solid carrier of contact liq carrier or segmentation or the two.

Composition of the present invention can be mixed with any in the multiple possible formulation, such as but not limited to tablet, capsule, gel capsule, liquid syrups, soft gelifying agent and suppository.Composition of the present invention also can be mixed with the suspension in water-based, non-aqueous or blending agent.Waterborne suspension can further comprise the material that increases suspension agent viscosity, comprises for example Xylo-Mucine, Sorbitol Powder and/or dextran.This suspension also can contain stablizer.

The oligonucleotide compound that comprises LNA can be used for the multiple treatment that goes out as listed above and uses.Usually, methods of treatment of the present invention comprises to the Mammals oligonucleotide modified of the LNA-of people's administering therapeutic significant quantity particularly.

In a certain embodiment, the invention provides pharmaceutical composition, it comprises (a) one or more antisense compounds and (b) one or more other the cholesterol-lowering agent by antisense mechanism performance function.When using with compound of the present invention, these cholesterol-lowering agents can use (for example Zarator and oligonucleotide) separately, sequential use (for example using for some time Zarator and oligonucleotide to use another kind of medicament and oligonucleotide later on again) or with one or more other this class cholesterol-lowering agent combined utilization.All cholesterol-lowering agents known to those skilled in the art all here introduce as with the combination therapy of The compounds of this invention.

Antiphlogiston includes but not limited to NSAID (non-steroidal anti-inflammatory drug) and reflunomide, antiviral drug, and immunomodulator also can be united in the present composition.The compound of two or more associatings can be together or sequential application.

In another embodiment, composition of the present invention can comprise the antisense compounds of one or more target first nucleic acid antisense compounds, particularly oligonucleotide and one or more other target second nucleic acid target.The compound of two or more associatings can be together or sequential application.

Dosage depends on the seriousness and the reactivity of morbid state to be treated, and sustainable several days of the course of treatment is to some months, or until realizing curing or reaching alleviating of morbid state.Can the calculating optimum dosage regimen from the measurement that patient's drug disposition is accumulated.

Optimal dose can change with the relative potency of each oligonucleotide.Generally speaking, it can be according to finding effective EC on the animal model in vitro and in vivo ₅₀Estimate.Usually, dosage in the scope of per kilogram of body weight 0.01 μ g to 1g, and can every day, weekly, every month or use one or many every year, or even used once in per 2 to 10 years, or continuous infusion reached some months in several hours.The repetition rate of administration can be estimated according to the residence time and the concentration of the medicine of being measured in body fluid or tissue.Success may wish that the patient accepts to keep the recurrence of treatment with the preventing disease state after treating.

Methods of treatment

It will be recognized by those skilled in the art that the oligonucleotide compound that comprises LNA can be used to resist apolipoprotein B (Apo-B100) relative disease by multiple different principle, so it drops in the spirit of the present invention.

Described LNA oligonucleotide compound can be designed to siRNA, and it is used for the little double stranded rna molecule of reticent specific endogenous or foreign gene by still understanding very few " antisense sample " mechanism by cell.

Shown that β-D-oxygen-LNA does not support RNA enzyme H activity.Yet this can change by creating the chimeric oligonucleotide of being made up of β-D-oxygen-LNA and DNA that is called gapmer according to the present invention.Gapmer is based on the one section 4-12ntDNA in center that can be discerned and cut by the RNA enzyme or modifies monomer (breach), and typically its flank connects 1 to 6 β-D-oxygen-LNA residue (flank).Flank can also make up with the LNA derivative.Also have other according to chimeric construct body of the present invention, they can play a role through the mechanism of RNA enzyme H mediation.Headmer is by 5 ' end continuous one section β-D-oxygen-LNA or LNA derivative, after connect towards 3 ' can or the modifying monomer by the continuous section of DNA of RNA enzyme H identification and cutting and define of end, and tailmer can or modify monomer by the continuous section of DNA of RNA enzyme H identification and cutting by 5 ' end, after the continuous one section β-D-oxygen-LNA or the LNA derivative that connect towards 3 ' end define.According to other mosaic of the present invention, be called mixmers, by can being formed by the DNA of RNA enzyme H identification and cutting or the alternate group compound of modifying monomer and β-D-oxygen-LNA and/or LNA derivative, may also can H combination of mediate rna enzyme and cutting.Because it is active in to a certain degree that α-L-LNA raises RNA enzyme H, therefore may need and can or modify monomeric more small gap by the DNA of RNA enzyme H identification and cutting for the gapmer construct, and more flexible may introduce in mixmer makes up.

The clinical effectiveness of antisense oligonucleotide depends on their pharmacokinetics to a great extent, for example absorbs, and distributes cellular uptake, metabolism and drainage.These parameters are again by the size of potential chemical action and oligonucleotide and the significantly guiding of three-dimensional structure institute.

Regulating the pharmacokinetics character of LNA oligonucleotide of the present invention can further finish by connecting multiple different piece.For example, oligonucleotide can be by connection such as lipid part such as cholesterol moiety by the ability of cytolemma, thioether, and aliphatic chain, phosphatide or polyamines strengthen to this oligonucleotide.Similarly, the cellular uptake of LNA oligonucleotide can by put together to oligonucleotide with film intermediary guiding kytoplasm in the part of the interaction of molecules of transporting improve.

According to the present invention, can use improve oligomer picked-up, improve biologically stable for example improve oligomer to the resistance of degraded and/or increase oligonucleotide and target sequence for example the specificity of mRNA sequence hybridization feature and the group of avidity improve pharmacokinetics character.

Pharmaceutical composition of the present invention can be used for treating the illness relevant with the ApoB-100 abnormal level.

These examples of disorders have hyperlipoproteinemia, familial type 3 hyperlipoproteinemia (familial dysbetalipoproteinemia), and familial hyperalphalipoproteinemia; Hyperlipidaemia, combined hyperlipidemia familial, multiple lipoprotein type hyperlipidemia, and multiple lipoprotein type hyperlipidemia; Hypertriglyceridemia, familial hypertriglyceridemia and familial lipoprotein lipase; Hypercholesterolemia, to the drug-fast hypercholesterolemia of Statins, familial hypercholesterolemia, polygenic hypercholesterolemia and familial defective apolipoprotein B; Cardiovascular disorder comprises atherosclerosis and coronary artery disease; Thrombosis; Peripheral vascular disease; Glycogen storage disease (I type glycogenosis); Lipodystrophy (congenital type and acquisition type); CS; Sexual ateleiotic dwarfism (isolated growth hormone deficiency); Diabetes; Hyperthyroidism; Hypertension; Anorexia nervosa; Werner's syndrome; Acute intermittent porphyria; Primary biliary cirrhosis; Extrahepatic biliary passages blocks 5; Acute hepatitis; Liver cancer; Systemic lupus erythematous; MG (comprising myelomatosis, multiple myeloma, macroglobulinemia, and lymphoma); Incretopathy; Fat; Nephrotic syndrome; Metabolism syndrome; Inflammation; Thyroprivia; Uremia (hyperuricemia); Impotence; Obstructive liver disease; Idiopathic hypercalcemia; Dysglobulinemia; Insulin level raises; X syndrome; Dupuytren's contracture; AIDS; With Alzheimer and dementia.

The present invention also provides the method that reduces the illness risk, comprises step from the The compounds of this invention that is enough to suppress the amount that apolipoprotein B expresses to individuality that use, and described illness is selected from: gestation; Intermittent claudication; Gout; And mercury poisoning and mercury alloys disease.The present invention also provides and suppresses the method that the cholesterol particle is attached to blood vessel endothelium, comprise step from the The compounds of this invention that is enough to suppress the amount that apolipoprotein B expresses to individuality that use, as a result of, the present invention also provides the method that reduces following risk: (i) cholesterol particulate oxidation; (ii) monocyte and blood vessel endothelium combines; (iii) monocyte is divided into scavenger cell; (iv) scavenger cell is taken in lipid 30 particles of oxidation and is discharged cytokine (including but not limited to IL-1, TNF-α, TGF-β); (v) the thrombocyte of fibrous fabric fat infringement forms and inflammation; (vi) cause the endothelium infringement of blood clot; (blood clot that vii) causes myocardial infarction or apoplexy also comprises being administered to the step that individuality is enough to suppress the The compounds of this invention of the amount that apolipoprotein B expresses.

The present invention also provides and alleviates and alcoholism, smoking, use oral contraceptive, use glucocorticosteroid, use the Beta-3 adrenergic blocker, perhaps use the method for the relevant hyperlipidaemia of isotretinoin (13-cis tretinoin), comprise being administered to the step that individuality is enough to suppress the The compounds of this invention of the amount that apolipoprotein B expresses.

The present invention also provides the The compounds of this invention manufacturing to be used for the treatment of the purposes of the medicine of disclosed herein any and all illnesss in addition.

Generally speaking, one aspect of the present invention relates to treats the mammiferous method of suffering from or easily suffering from the disease relevant with the ApoB-100 abnormal level, comprises the oligonucleotide that comprises the unitary target Apo-B100 of one or more LNA to this administration treatment significant quantity.

One of the present invention interestingly relates to the purposes that the preparation of compound defined here or conjugate defined here is used for the treatment of the medicine of above-mentioned illness.

Method of the present invention is preferred for the disease for the treatment of or preventing to be caused by the ApoB-100 abnormal level.

In addition, invention described herein has covered the method for a kind of prevention or treatment disease, comprises the Apo-B100 adjustment oligonucleotide compound to people's administering therapeutic significant quantity of this kind of needs treatment, includes but not limited to the oligomer of high dosage.The present invention comprises that also short-term uses Apo-B100 adjustment oligonucleotide application of compound.

In one embodiment of the invention, the oligonucleotide compound is connected to part/conjugate.This is the mode that increases the cellular uptake antisense oligonucleotide.

Oligonucleotide compound of the present invention also can be conjugated on the active drug substance, for example acetylsalicylic acid, Ibuprofen BP/EP, sulfa drug, antidiabetic drug, antiseptic-germicide or microbiotic.

Perhaps, the present invention also relates to the method for the treatment of the ApoB-100 abnormal level in addition, described method comprises to the patient that these needs are arranged uses compound defined here or conjugate defined here or pharmaceutical composition defined here, and further comprises the chemotherapeutics of using other.Described further administration can be that other chemotherapeutics is conjugated on the The compounds of this invention, is present in the pharmaceutical composition, or uses with the preparation that separates.

The oligonucleotide compound of the LNA of containing of the present invention can also be used as the research reagent of diagnosis, treatment and prevention.Under study for action, antisense oligonucleotide can be used for suppressing specifically Apo-B100 gene synthetic in cell and laboratory animal, thereby promotes the functional analysis of target or estimate its intervenes target as treatment availability.Diagnosis aspect, described antisense oligonucleotide can be used for by through the Northern blotting, in situ hybridization or similar techniques detect and quantitatively the Apo-B100 in cell and the tissue express.For treatment, suspect that suffer from can Apo-B100 expresses the disease for the treatment of or the animal or human of illness treats by using antisense compounds of the present invention by regulating.Further provide by administering therapeutic or prevention one or more antisense compounds of the present invention of significant quantity or composition and treated especially mouse and rat and people's method of the animal that is suspected to have or easily suffers from disease relevant or illness with the Apo-B100 expression.

The present invention also relates to be used as the compound defined here or the conjugate of medicine.

The invention still further relates to the purposes of the medicine of defined compound or conjugate manufacturing treatment Apo-B100 abnormal level herein.Typically, the described Apo-B100 abnormal level form that is atherosclerosis, hypercholesterolemia or hyperlipidaemia.

In addition, the present invention relates to treat the method for the object of the disease of suffering from the atherosclerosis of being selected from, hypercholesterolemia and hyperlipidaemia or illness, this method comprises the step of the object that these needs are arranged being used pharmaceutical composition defined here.The preferred pharmaceutical compositions oral administration.

Embodiments more of the present invention

1, the compound of forming by 12-50 Nucleotide and/or nucleotide analog altogether, wherein said compound comprises the subsequence of at least 10 Nucleotide or nucleotide analog, described subsequence is positioned at and is selected from SEQ ID NO:SEQ ID No 2, SEQ ID No 3, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, SEQ ID No 10, SEQ ID No11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQID No 16, SEQ ID No 17, SEQ ID No 27, SEQ ID No 28, in the sequence of SEQ ID No48 and SEQ ID No 50, wherein said compound comprises at least 3 nucleotide analogs.

2, according to the compound of claim 1, form by double chain oligonucleotide, wherein every chain comprises altogether 16-30 Nucleotide and/or nucleotide analog, wherein said compound comprises the subsequence of 10 Nucleotide or nucleotide analog, described subsequence is positioned at and is selected from SEQ ID NO:27,28, in the sequence of (and/or 48 or 50), and wherein said compound comprises at least 3 nucleotide analogs.

3, according to the compound of embodiment 1, form by 12-25 Nucleotide or nucleotide analog.

4, according to the compound of embodiment 3, form by 15,16,17,18,19,20,21 or 22 Nucleotide or nucleotide analog.

5, according to the compound of embodiment 4, form by 16 Nucleotide or nucleotide analog.

6, according to the compound of any one embodiment of 1-5, wherein said Nucleotide comprises the linking group that is selected from phosphate, thiophosphoric acid base and borine phosphate, connects between nucleosides can be-O-P (O) ₂-O-, and-O-P (O, S)-O-.

7, according to the compound of embodiment 6, wherein said connection is a phosphate.

8, according to the compound of embodiment 6, wherein said connection is the thiophosphoric acid base.

9, according to the compound of embodiment 6, wherein all Nucleotide all comprise the thiophosphoric acid base.

10,, comprise 3-12 nucleotide analog according to the compound of embodiment 9.

11,, comprise 6 nucleotide analogs according to the compound of embodiment 10.

12, according to the compound of any one embodiment of 10-11, wherein at least one described nucleotide analog is locked nucleic acid (LNA).

13, according to the compound of any embodiment 12, wherein LNA is selected from β-D-oxygen-LNA, α-L-oxygen-LNA, β-D-amino-LNA or β-D-sulphur-LNA.

14, according to the compound of embodiment 13, wherein said nucleosides and/or LNA link together by phosphate.

15, according to the compound of embodiment 14, wherein said nucleosides and/or LNA link together by the thiophosphoric acid base.

16, according to the compound of embodiment 12, wherein said subsequence is SEQ ID NO:2.

17, according to the compound of embodiment 12, wherein said subsequence is SEQ ID NO:3.

18, according to the compound of any embodiment of 16-17, wherein 3 ' end LNA is substituted by corresponding natural nucleus glycoside.

19, the compound of forming by SEQ ID No 29.

20, the compound of forming by SEQ ID No 30.

21, comprise according to the compound of any embodiment of 1-20 and at least one non-nucleotide or non-polynucleotide partly the conjugate covalently bound with described compound.

22, pharmaceutical composition, it is included in the compound that defines in any embodiment of 1-20 or defined conjugate and pharmaceutically acceptable thinner, carrier or assistant agent in embodiment 21.

23,, also comprise the compound of at least a reducing cholesterol according to the pharmaceutical composition of embodiment 22.

24, according to the pharmaceutical composition of embodiment 23, wherein said compound is selected from: biliary salts resin (for example, Colestyramine, colestipol and hydrochloric acid colesevelam), the HMGCoA-reductase inhibitor is (for example, lovastatin, Cerivastatin, Pravastatin, Zarator, Simvastatin and fluvastatin), nicotinic acid, shellfish special acid derivative (for example, clofibrate, gemfibrozil, fenofibrate, bezafibrate and Win-35833), probucol, Xin Meisu, dextrothyroxine, plant-stanol ester, cholesterol absorption inhibitor (for example, ezetimibe), implitapide, the inhibitor of bile acid transport albumen (apical sodium dependency bile acid transport albumen), liver CYP7a conditioning agent, controversies in hormone replacement in the elderly is (for example, tamoxifen) and antiphlogiston (for example, glucocorticosteroid).

25, be used as the compound that in any embodiment of 1-20, defines of medicine or in embodiment 21 defined conjugate.

26, in any embodiment of 1-20 defined compound or in embodiment 21 purposes of the medicine of defined conjugate manufacturing treatment Apo-B100 abnormal level.

27, according to the purposes of embodiment 26, wherein said Apo-B100 abnormal level is the form of atherosclerosis, hypercholesterolemia or hyperlipidaemia.

Further set forth the present invention in non-limiting mode by the following example.

Embodiment

Embodiment 1: monomer is synthetic

Program of having delivered and the reference of wherein quoting are followed in the preparation of LNA monomer members and derivative thereof, referring to:

WO 03/095467A1

D.S.Pedersen，C.Rosenbohm，T.Koch(2002)Preparationof LNA Phosphoramidites，Synthesis 6，802-808.

M.D. L. T.Bryld，A.E.

B.Verbeure，G.Gaubert，P.Herdewijn，J.Wengel(2002)α-L-ribo-configured Locked Nucleic Acid(α-1-LNA)：Synthesisand Properties，J.Am.Chem.Soc.，124，2164-2176.

S.K.Singh，R.Kumar，J.Wengel(1998)Synthesis of NovelBicyclo[2.2.1]Ribonucleosides：2’-Amino-and 2’-Thio-LNAMonomeric Nucleosides，J.Org.Chem.1998，63，6078-6079.

C.Rosenbohm，S.M.Christensen，M.D. D.S.Pedersen，L.E.Larsen，J.Wengel，T.Koch(2003)Synthesis of2’-amino-LNA：a new strategy，Org.Biomol.Chem.1，655-663.

D.S.Pedersen，T.Koch(2003)Analogues of LNA(LockedNucleic Acid).Synthesis of the 2’-Thio-LNA Thymine and5-Methyl Cytosine Phosphoramidites，Synthesis 4，578-582.

Embodiment 2: oligonucleotide is synthetic

Go up use phosphoramidite method at Expedite 8900/MOSS synthesizer (Multiple OligonucleotideSynthesis System), with the scale synthetic oligonucleotide of 1 μ mol or 15 μ mol.For more extensive synthetic, use

Oligo Pilot.Synthetic last (having DMT), at room temperature used ammoniacal liquor 1-2 hour, oligonucleotide under the cracking from the solid support, and further 65 ℃ of following deprotections 4 hours.By this oligonucleotide of reversed-phase HPLC (RP-HPLC) purifying.After removing the DMT group, this oligonucleotide characterizes by AE-HPLC, RP-HPLC and CGE, and further confirms molecular weight by ESI-MS.More as follows detailed.

The preparation of LNA-solid support:

The preparation of LNA succinyl-half ester

5 '-O-Dmt-3 '-hydroxyl-LNA monomer (500mg), succinyl oxide (1.2 equivalent) and DMAP (1.2 equivalent) are dissolved among the DCM (35ml).This reactant at room temperature stirs and spends the night.NaH with 0.1M ₂PO ₄, after pH5.5 (2x) and salt solution (1x) extraction, use anhydrous Na ₂SO ₄Further dry organic layer filters and evaporation.Yield with 95% obtains this half ester derivatives, and it uses without any being further purified promptly.

The preparation of LNA-upholder

The half ester derivatives (90 μ mol) of above preparation is dissolved among the minimum DMF, adds DIEA and pyBOP (90 μ mol) and also mixed 1 minute together.Should preactivated mixture and LCAA-CPG (

300mg) is manually mixing and stirring in the synthesizer in 80-120 order footpath.After following 1.5 hours of the room temperature, filter upholder, and wash with DMF, DCM and MeOH.After the drying, last sample is defined as 57 μ mol/g and (sees Tom Brown, Dorcas J.S.Brown.Modernmachine-aided methods of oligodeoxyribonucleotide synthesis.In:F.Eckstein compiles, Oligonucleotides and Analogues APracticalApproach.Oxford:IRL Press, 1991:13-14).

The extension of oligonucleotide

By using 0.1M 5 ' in the acetonitrile-amidite solution of O-DMT-protection and the DCI (4 in the acetonitrile; 5-dicyan imidazoles) (0.25M) as activator; carry out phosphoramidite (A (bz), G (ibu), 5-methyl-C (bz)) or T-β-cyanoethyl-phosphoramidite) coupling.By using the xanthane muriate (at acetonitrile: the 0.01M in the pyridine 10%) carry out vulcanization reaction.Remaining reagent is those that use always during oligonucleotide synthesizes.The scheme that supplier provides is optimized suitably.

The RP-HPLC purifying:

Post: XterraRP ₁₈

Flow velocity: 3ml/min

Damping fluid: 0.1M ammonium acetate pH8 and acetonitrile

Abbreviation

DMT: dimethoxytrityl

DCI:4,5-dicyan imidazoles

The DMAP:4-Dimethylamino pyridine

DCM: methylene dichloride

DMF: dimethyl formamide

THF: tetrahydrofuran (THF)

DIEA:N, the N-diisopropylethylamine

PyBOP: phosphofluoric acid benzotriazole-1-base-oxygen base-tripyrrole Wan Phosphonium

Bz: benzoyl

Ibu: isobutyryl

Embodiment 3: the design of oligonucleotide compound

SiRNA is the sense strand (SEQ ID NO:27) of 21 Nucleotide and the antisense strand (SEQ ID NO:28) of 23 Nucleotide-the caused overhang at 2 Nucleotide of antisense strand 3 ' end.

ApoB-siRNA has justice 5 '-GUCAUCACACUGAAUACCAA*U-3 ' (SEQ ID NO:48), and ApoB-1-siRNA antisense strand 5 '-AUUGGUAUUCAGUGUGAUGAc*a*C-3 ' (SEQID NO:49) and ApoB-siRNA-Chol sense strand: 5 '-GUCAUCACACUGAAUACCAAU*Chol-3 ' (SEQ ID NO:50) are synthetic by RNATEC (Leuven).

In one embodiment of the invention, SEQ ID NO:2-26 comprises at least 3 LNA Nucleotide, and for example 6 or 7 LNA Nucleotide resemble in SEQ ID NO:29-47.

Table 1. oligonucleotide compound of the present invention

At SEQ ID NO:27, in 28,48,49,50, capitalization is represented the ribonucleoside acid unit, and on behalf of thiophosphatephosphorothioate, subscript " s " connect.

Test substances	Sequence	Target site
				SEQ ID NO：2	5-GGTATTCAGTGTGATG-3’	10169	The antisense motif
SEQ ID NO：3	5-ATTGGTATTCAGTGTG-3	10172	The antisense motif
				SEQ ID NO：4	5’-TTGTTCTGAATGTCCA-3’	3409	The antisense motif
SEQ ID NO：5	5’-TCTTGTTCTGAATGTC-3’	3411	The antisense motif
				SEQ ID NO：6	5-TGGTATTCAGTGTGAT-3		The antisense motif
SEQ ID NO：7	5-TTGGTATTCAGTGTGA-3		The antisense motif
				SEQ ID NO：8	5-CATTGGTATTCAGTGT-3	10173	The antisense motif
SEQ ID NO：9	5-GCATTGGTATTCAGTG-3	10174	The antisense motif
				SEQ ID NO：10	5-AGCATTGGTATTCAGT-3	10175	The antisense motif
SEQ ID NO：11	5-CAGCATTGGTATTCAG-3	10176	The antisense motif
				SEQ ID NO：12	5-TCAGCATTGGTATTCA-3		The antisense motif
SEQ ID NO：13	5-TTCAGCATTGGTATTC-3		The antisense motif
				SEQ ID NO：14	5-GTTCAGCATTGGTATT-3		The antisense motif
SEQ ID NO：15	5-AGTTCAGCATTGGTAT-3		The antisense motif
				SEQ ID NO：16	5-AAGTTCAGCATTGGTA-3		The antisense motif
SEQ ID NO：17	5-AAAGTTCAGCATTGGT-3		The antisense motif
				SEQ ID NO：18	5’-ATTTCCATTAAGTTCT-3’	10454	The antisense motif

Test substances	Sequence	Target site
				SEQ ID NO：19	5’-GGTATTTCCATTAAGT-3’	10457	The antisense motif
SEQ ID NO：20	5’-GACTCAATGGAAAAGT-3’	10594	The antisense motif
				SEQ ID NO：21	5’-ATGACTCAATGGAAAA-3’	10596	The antisense motif
SEQ ID NO：22	5’-GCTAACACTAAGAACC-3’	10998	The antisense motif
				SEQ ID NO：23	5’-CACTAAGAACCAGAAG-3’	11003	The antisense motif
SEQ ID NO：24	5’-CTAAGAACCAGAAGAT-3’	11005	The antisense motif
				SEQ ID NO：25	5’-TGAATCGGGTCGCATC-3’	252	The antisense motif
SEQ ID NO：26	5’-TGAATCGGGTCGCATT-3’	252	The antisense motif
				SEQ ID NO：27 SEQ ID NO：28	5-GUCAUCACACUGAAUACCAAU-3 5-AUUGGUAUUCAGUGUGAUGACAC-3		siRNA
SEQ ID NO：29	5-G sG sT sa st st sc sa sg st sg st sG sA sT sg-3’		Motif #2
				SEQ ID NO：30	5-A sT sT sg sg st sa st st sc sa sg sT sG sT sg-3’		Motif #3
SEQ ID NO：31	5-A sT sT sG sg st sa st st sc sa sg sT sG sT sg-3’		Motif #3
				SEQ ID NO：32	5’-T sT sG sT st sc st Sg sa sa st sg sT s MeC s MeC sa-3’		Motif #4
SEQ ID NO：33	5’-T s MeC sT sT sg st st sc st sg sa sa sT sG sT sc-3’		Motif #5
				SEQ ID NO：34	5- MeC sA sT sT sg sg st sa st st sc sa sG sT sG st-3		Motif #8
SEQ ID NO：35	5-G s MeC sA sT st sg sg st sa st st sc sA sG sT sg-3		Motif #9
				SEQ ID NO：36	5-A sG s MeC sA st st sg sg st sa st st s MeC sA sG st-3		Motif #10
SEQ ID NO：37	5- MeC sA sG sc sa st st sg sg st sa st sT s MeC sA sg-3		Motif #11
				SEQ ID NO：38	5- MeC sA sG s MeC sa st st sg sg st sa st sT s MeC sA sg-3		Motif #11
SEQ ID NO：39	5’-A sT sT sT sc sc sa st st sa sa sg sT sT s MeC st-3’		Motif #19
				SEQ ID NO：40	5’-G sG sT sA st st st sc sc sa st st sA sA sG st-3’		Motif #19
SEQ ID NO：41	5’-G sA s MeC sT sc sa sa st sg sg sa sa sA sA sG st-3’		Motif #20
				SEQ ID NO：42	5’-A sT sG sA sc st sc sa sa st sg sgA sA sA sa-3’		Motif #21
SEQ ID NO：43	5’-G s MeC sT sA sa sc sa sc st sa sa sg sA sA s MeC sc-3’		Motif #22
				SEQ ID NO：44	5’- MeC sA s MeC sT sa sa sg sa sa sc sc sa sG sA sA sg-3’		Motif #23

Test substances	Sequence	Target site
				SEQ ID NO：45	5’- MeC sT sA sA sg sa sa sc sc sa sg sa sA sG sA st-3’		Motif #24
SEQ ID NO：46	5’-T sG sA sA st sc sg sg sg st sc sg s MeC sA sT sc-3’		Motif #25
				SEQ ID NO：47	5’-T sG sA sA st sc sg sg sg st sc sg s MeC sA sT st-3’		Motif #26
SEQ ID NO：48 SEQ ID NO：49 ApoB-siRNA (RNA-TEC290.3/ RNA-TEC290.5)	5-GUCAUCACACUGAAUACCAAsU-3 (290.3) 5-AUUGGUAUUCAGUGUGAUGAcsasC-3 (290.5)		The non-siRNA that puts together
				SEQ ID NO：50 ApoB-siRNA-Chol (SEQ ID NO：51 RNA-TEC290.4/ RNA-TEC290.5)	5-GUCAUCACACUGAAUACCAAUs-Chol-3 (290.4) 5-AUUGGUAUUCAGUGUGAUGAcsasC-3 (290.5)		The siRNA that cholesteryl is puted together

SEQ ID No 30 is according to compound of interest of the present invention.

The stability of embodiment 4:LNA compound in people or mouse blood plasma

In blood plasma (also can be mouse, monkey or dog plasma), tested LNA oligonucleotide stability from people or rat.In 45 μ l blood plasma, add 5 μ l LNA oligonucleotide (final concentration 20 μ M).At 37 ℃, with oligomer incubation 0-96 hour (nuclease of test blood plasma reaches 96 hours, confirms not have nuclease shear mode difference) in blood plasma.In the specified time, with the sample quick freezing in liquid nitrogen.By adding sample dyestuff (Invitrogen) on 15 μ L water and the 3 μ L 6x, dilute the oligonucleotide of 2 μ L (equaling 40pmol) in blood plasma.Use 10bp ladder (Invitrogen 10821-015) thing as a token of.In 1 μ l ladder, add sample (dyestuff) and 4 μ l water on the 1 μ 16x.Biased sample is heated to 65 ℃ of 10min, and last sample arrives prerun gel (1x TBE is at 50 watts of prerun 1h for 16% acrylamide, 7M urea), and runs 2.5 hours at the 50-60 watt.Subsequently, be used in 1x SyBR gold (Molecular probes) among the 1x TBE to gel-colored 15min.Use is observed band from the phosphoimager of Biorad.

Embodiment 5: external model: cell cultures

Antisense compounds is to the influence of target nucleic acid expression, can be in the various kinds of cell type any in detect, condition is that target nucleic acid exists with measurable level.But target thing endogenous expression, or the instantaneous or stable transfection of the nucleic acid by the described nucleic acid of coding is expressed.

The expression level of target nucleic acid can use for example Northern engram analysis, quantitative PCR, ribonuclease protecting test to determine routinely.For the example purpose provides following cell type, but other cell type also can use routinely, and condition is that the target thing is expressed in selected cell type.

Cell cultures and remains on 37 ℃, 95-98% humidity and 5%CO in suitable culture medium as described below ₂In.Cell goes down to posterity 2-3 time weekly routinely.

BNCL-2:Mouse liver cell is that BNCL-2 is available from ATCC and be cultured among the DMEM (Sigma) that contains 10%FBS+Glutamax I+ non-essential amino acid+gentamicin.

Hepa1-6:Mouse liver cell is that Hepa1-6 is available from ATCC and be cultured among the DMEM (Sigma) that contains 10%FBS+Glutamax I+ non-essential amino acid+gentamicin.

HepG2:Human liver cell is that HepG2 is available from ATCC and be cultured among the Eagle MEM (Sigma) that contains 10%FBS+Glutamax I+ non-essential amino acid+gentamicin.

Embodiment 6: external model: handle with antisense oligonucleotide

Cell cultures and transfection: at 37 ℃ of (5%CO ₂), with BNCL-2 or Hepa1-6 cell inoculation the interpolation in the flat board of 12-hole 10%FBS, in the growth medium of Glutamax I and gentamicin.When cell 60-70% converges, use Lipofectamine 2000 (5 μ g/ml), with the oligonucleotide (0.04-25nM) of different concns in duplicate transfection they.Basically of (1994, JBC 269:16416-16424) such as Dean, carry out transfection.In brief, be used in the Lipofectamine incubation cell 10 minutes among the OptiMEM, add oligonucleotide subsequently to cumulative volume 0.5ml transfection mixture/hole.After 4 hours, remove transfection mixture, washed cell and 37 ℃ of about 20 hours of growths (mRNA analyzes and analysis of protein) in suitable growth medium.Then, harvested cell carries out albumen and RNA analysis.

Embodiment 7: external model: the extraction of RNA and cDNA are synthetic

Total RNA separates

Use the little test kit of RNeasy (Qiagen) to separate total RNA.Use the PBS washed cell, (RTL Qiagen) directly adds in the hand-hole with the cell lysis buffer solution of having added 1% mercaptoethanol.Behind the several minutes,, handle sample according to manufacturer's specification sheets.

First chain is synthetic

According to manufacturer's specification sheets (Qiagen), use OmniScript reversed transcriptive enzyme test kit or M-MLV reversed transcriptive enzyme (basically as described in the manufacturer (Ambion)) to carry out synthesizing of first chain.When using the OmniScript reversed transcriptive enzyme, to each sample, the total RNA of 0.5 μ g is adjusted to 12 μ l, and with 0.2 μ l many (dT) _12-18(0.5 μ g/ml) (Life Technologies), 2 μ l dNTP mixtures (every kind of 5mM), 2 μ l 10X RT damping fluids, 0.5 μ l RNAguardTMRNA enzyme inhibitors (33 units/ml, Amersham) and 1 μ l OmniScript reversed transcriptive enzyme mix, subsequently 37 ℃ of incubations 60 minutes, and 93 ℃ of hot deactivations 5 minutes.

When using ten aggressiveness and M-MLV reversed transcriptive enzyme at random to carry out synthetic (basically as manufacturer (Ambion) as described in) of first chain, at H ₂The total RNA of 0.25 μ g with each sample among the O is adjusted to 10.8 μ l.Add 2 μ l, ten aggressiveness and 2 μ l dNTP mixtures (every kind of 2.5mM).With sample be heated to 70 ℃ 3 minutes, and in frozen water, cool off immediately, add 3.25 μ l and contain (2 μ l 10xRT damping fluids; 1 μ l M-MLV reversed transcriptive enzyme; 0.25 mixture μ l RNA enzyme inhibitors).At 42 ℃ of synthetic cDNA 60min, 95 ℃ of hot inactivation step 10 minutes, be cooled to 4 ℃ at last subsequently.

Embodiment 8: external and body inner model: analyze the inhibition that oligonucleotide is expressed Apo-B100 by PCR in real time

Available several different methods known in the art is measured the antisense that Apo-B100 is expressed and is regulated.For example, the Apo-B100mRNA level can be come quantitatively by for example Northern engram analysis, competitive polymerase chain reaction (PCR) or PCR in real time.Real-time quantitative PCR is preferred at present.RNA analyzes and can carry out total cell RNA or mRNA.

RNA separates and the RNA analytical procedure, as the Northern engram analysis, is conventional in this area, and in the Current Protocols in MolecularBiology as John Wiley and Sons instruction is arranged.

The commercially available iQ multicolor real time PCR detection system that use can be purchased from BioRAD can realize real-time quantitative PCR easily.Real-time quantitative PCR is a technology well known in the art, and as people Real time quantitative PCR such as Heid, Genome Research (1996) has instruction among the 6:986-994.

The real-time quantitative PCR analysis of Apo-B100mRNA level

In order to measure the relative mouse ApoB mRNA level in treated and the untreated sample, in the quantitative PCR analysis of the iCycler that uses BioRad, use the cDNA that is produced.

In the cDNA of 5 times of 8 μ l (Gapdh and beta-actin) dilution, add 52 μ l mixtures, it contains 29.5 μ l Platinum qPCR Supermix-UDG (in-vitrogen), every kind of primer of 1030nM, 0.57X SYBR Green (Molecular probes) and 11.4nM fluorescein (Molecular probes).

25 μ l are used for Q-PCR:50 ℃ 120 seconds, 95 ℃ 120 seconds and 40 circulations [95 ℃ 30 seconds and 60 ℃ 60 seconds] in duplicate.

ApoB expresses cDNA and the standard Q-PCR scheme of using 50 times of dilutions and comes quantitatively.Primer (final concentration of forward and reverse primer is respectively 0.6 μ M and 0.9 μ M) and probe (final concentration is 0.1 μ M) and 2x Platinum Quantitative PCR SuperMix UDG (cat.#11730, Invitrogen) mix and add 3.3 μ l cDNA to final volume be 25 μ l.Each sample is to analyze in duplicate.The PCR program: 50 ℃ 2 minutes; 95 ℃ 10 minutes; 95 ℃ of 40 round-robin are 15 seconds then, 60 ℃ 1 minute.

ApoB mRNA expresses with respect to mouse beta-actin or Gapdh mRNA stdn, and the latter uses Q-PCR quantitative similarly.

Primer:

MGapdh:5 '-agcctcgtcccgtagacaaaat-3 ' (SEQ ID NO:51) and 5 '-gttgatggcaacaatctccacttt-3 ' (SEQ ID NO:52)

M beta-actin: 5 '-ccttccttcttgggtatggaa-3 ' (SEQ ID NO:53) and 5 '-gctcaggaggagcaatgatct-3 ' (SEQ ID NO:54)

MApoB:5 '-gcccattgtggacaagttgatc-3 ' (SEQ ID NO:55) and 5 '-ccaggacttggaggtcttgga-3 ' (SEQ ID NO:56)

MApoB Taqman probe: 5 '-fam-aagccagggcctatctccgcatcc-3 ' (SEQID NO:57)

To be used for the formation determination typical curve from 2 times of dilutions of untreated mouse liver cell (Hepa1-6 cell) synthetic cDNA of system (diluting 5 times and expression ApoB and beta-actin or Gapdh).Use iCycler iQ real-time detecting system software to determine the relative quantity of ApoB mRNA from the threshold cycle of calculating.

Embodiment 9: analyzed in vitro: the western blot analysis of Apo-B100 protein level

By Western blot, determine of the external influence of Apo-B100 oligomer to Apo-B100 protein level in the cells transfected.

Harvested cell, cracking in the 50mMTris-HCl pH6.8,10% glycerine, 2.5%SDS, 5mM DTT and the 6M urea that have added protease inhibitor cocktail (Roche).Use BCA protein determination kit (Pierce), measure total protein concentration.According to manufacturer's specification sheets (Invitrogen), on the 12%Bis-Tris gel in the MOPS damping fluid or on the 3-8%Tris acetate gel, run 20-100 μ g total protein, and trace is to pvdf membrane.After in sealing damping fluid (having added the PBS-T of 5% low fat milk powder), being incubated overnight, with anti-be incubated overnight of film with detection Apo-B100.As last sample contrast, use monoclonal antibody to detect tubulin or Actin muscle from Neomarker.Then film is resisted incubations with two, and use color development immunity detection reagent (Invitrogen) or chemoluminescence ECL ⁺Detection kit (Amersham) manifests Apo-B100.

Embodiment 10: analyzed in vitro: the Antisense Suppression of using antisense oligonucleotide that people Apo-B100 is expressed

According to the present invention, design the different zones that a series of oligonucleotide comes target people Apo-B100 RNA, see Table 1.Assessment oligonucleotide compound is assisted picked-up SEQ ID NO:29 at lipid in mouse liver cell (Hepa1-6 cell), knock down the potential (Figure 1A) of (knockdown) Apo-B100mRNA behind the siRNA that siRNA (unmodified) or cholesteryl are modified, and relatively in BNLCL2 2 kinds of LNA oligonucleotide SEQ ID No:29 and SEQ ID No:30 to Apo-B100 knock down (Figure 1B) and in the Hepa1-6 cell SEQ ID No:29 and SEQ ID No:37 to knock down (Fig. 4) of ApoB-100.

Data are expressed as the downward modulation per-cent with respect to the simulation transfectional cell.Through PCR in real time monitoring transcript steady state and with respect to the stdn of GAPDH transcript steady state.

Embodiment 11: contain the interior target downward modulation of body of the oligonucleotide compound of LNA

C57BL/6 mouse (20g) is accepted i.v.6.25 for three days on end, and 12.5,25 or 50mg/kg (every group of 7 mouse).Because molecular weight and the antisense oligonucleotide of siRNA-Chol are in a ratio of about 3: 1, thus our administration less antisense oligonucleotide.All siRNA and antisense oligonucleotide are dissolved in 0.9% salt solution (NaCl), and dosage is 10mL/kg body weight (the about 0.2ml of per injection).When putting to death, the record liver weight.Being used for measuring the tissue that ApoB mRNA expresses is stored among-20 ℃ of RNA later (Ambion) stand-by.To the mRNA of jejunum and liver analyze and blood plasma in total cholesterol be determined at last i.v. and inject and carried out (seeing Fig. 2 A and 2B) in back 24 hours.

Embodiment 12: cholesterol levels in the blood plasma

The colorimetric estimation Cholesterol CP of use ABX Pentra measures the total cholesterol level in the blood plasma.After enzymically hydrolyse and oxygenizement, measure cholesterol.The water that in 1.5 μ L blood plasma, adds 21.5 μ L.Add 250 μ L reagent, inherent 540nm wavelength was measured cholesterol level in 5 minutes.Measurement to each animal is all carried out in duplicate.Control compound (ABX Pentra N control) detection sensitivity and linearity with 2 times of dilutions.Determine relative cholesterol levels by subtracting background, and to present (see figure 3) with respect to the cholesterol levels in the brine treatment mice plasma.

Embodiment 13: the target of LNA oligonucleotide compound downward modulation in the body

C57BL/6 mouse (20g) is accepted i.v.6.25 for three days on end, and 12 or 25mg/kg (every group of 7 mouse).Antisense oligonucleotide (SEQ ID NO:29 and SEQ ID NO:37) is dissolved in 0.9% salt solution (NaCl), and dosage is 10mL/kg body weight (the about 0.2ml of per injection).Being used for measuring the tissue that ApoB mRNA expresses is stored among-20 ℃ of RNA later (Ambion) stand-by.To the mRNA of jejunum and liver analyze and blood plasma in total cholesterol and LDL cholesterol the last time i.v. inject and carried out (seeing Fig. 5 A, 5B, 6A and 6B) in back 24 hours.

Embodiment 14: give the oral LNA oligonucleotide of mouse compound

C57BL/6 mouse (20g) is accepted the preparation that 10mL/kg is the 0.2mL prepared fresh, described preparation be the oligonucleotide (SEQ ID NO:29 or SEQ IDNO:37) of 1.0ml in sterilized water (7.5mg/ml), 0.1ml tween 80,1.9ml sweet oil.The final concentration of oligonucleotide compound is 2.5mg/ml.This preparation is jolted 1 minute, supersound process 5 minutes (repeating 3 times).Do not observe negative reaction.

Embodiment 15: analyzed in vitro: the Antisense Suppression of the dose response in the cell culture (human liver cell Huh-7)/people Apo-B100 is expressed

According to the present invention, designed the different zones of a series of oligonucleotide with target people Apo-B100mRNA, see Table 1.Estimate the oligonucleotide compound auxiliary picked-up of lipid SEQ ID NO:31-32 in human liver cell (Huh-7 cell), knock down the potential (Fig. 8) of Apo-B100mRNA behind 36-38 and the 40-42.Described in embodiment 5-8, test.The result shows that all compounds are with the very strong downward modulation of 25nM (＞80%).Yet, with 1nM, have only 2 compounds to cause the Apo-B100mRNA downward modulation up to 70% (SEQ ID NO:37 and 40), this is very strong downward modulation (Fig. 8).

The IC of LNA antisense oligonucleotide in cell culture (human liver cell Huh-7) that embodiment 16:7 kind is selected ₅₀

7 kinds of antisense oligonucleotides that selection has best external downward modulation effect carry out IC ₅₀Research realizes that to determine antisense oligonucleotide 50% suppresses the concentration that ApoB-100mRNA expresses.Described in embodiment 5-8, test.Only SEQ ID NO:36 and 37 has the IC of about 1nM ₅₀, and the IC of SEQ ID NO:38 ₅₀Up to 5.7 (Fig. 9).0.5nM IC ₅₀Very high compound is renderd a service in indication, confirms by data (embodiment 17 and 18) in the body for SEQ ID NO:37.

Embodiment 17: the acting duration C57BL/6 mouse (20g) of administration SEQ ID NO 371 times, 2 times or 3 times was accepted i.p.6.25 or 25mg/ kilogram/agent (every group of 5 mouse) in continuous 1,2 or 3 day.All antisense oligonucleotides are dissolved in 0.9% salt solution (NaCl), and dosage is 10mL/kg body weight (the about 0.2ml of per injection).(the 3rd, 5,8,13 and 21 days) record liver weight when putting to death.Being used for measuring the tissue that ApoB mRNA expresses is stored among-20 ℃ of RNA later (Ambion) stand-by.During execution liver is carried out mRNA and analyzes, in the blood plasma LDL cholesterol and total cholesterol then the last time i.p. injected 2 or 3, and measured (seeing Figure 11) in 6,11 and 19 days back 24 hours.This studies show that ApoB-100mRNA downward modulation very strong after the SEQ ID NO:37 administration: 1 dose causes the 3rd day to the 8th day ApoB mRNA down-regulated expression 45-60% after the administration, and 3 doses cause the 13rd day the downward modulation 85-90%, about 70% downward modulation in the 21st day, show that the acting duration in liver was longer than 20 days when using 2 or 3 doses.Measuring ApoB-100mRNA described in embodiment 8 and 12 expresses and total cholesterol.

The usage of embodiment 18:SEQ ID NO 37

C57BL/6 mouse (20g) is accepted 2.mg/kg/ agent i.p. and continues 4 weeks or 5mg/kg/ agent weekly for 2 times and continue 4 weeks (every group of 5 mouse) 1 time weekly, to detect target (ApoB-100) mRNA is in harmonious proportion effect to blood plasma cholesterol level (collecting weekly 1 time) down.Antisense oligonucleotide is dissolved in 0.9% salt solution (NaCl), and dosage is 10mL/kg body weight (the about 0.2ml of per injection).(the 28th day) record liver weight during execution.Being used for measuring the tissue that ApoB mRNA expresses is stored among-20 ℃ of RNA later (Ambion) stand-by.During execution liver is carried out mRNA and analyze, the LDL cholesterol levels is measured (see figure 10) then the 7th, 14 in the blood plasma in the time of 21 and 28 days.The result shows that LDL cholesterol levels linearity in time descends, and weekly after 2 times, compares decline 30% in the time of the 28th day in 2.5 milligrams/kilogram/agent of administration with the salt solution group with the 7th day.When the antisense oligonucleotide that gives uniform amt but dosage regimen is 5 milligrams/kilogram/agent only obtains similar result weekly 1 time the time.In addition, irrelevant with dosage regimen (1 dose or 2 doses weekly), after 20 milligrams/kilogram of the administrations, the ApoB-100mRNA during execution in (the 28th day) liver demonstrates the downward modulation of 30-40% in 28 days.These results show even in the remarkable downward modulation of 37 couples of ApoB-100mRNA of low dosage SEQ ID NO, and this downward modulation reads influentially to treatment result, and it is measured as blood plasma LDL-cholesterol and descends 30%.Measuring ApoB-100mRNA described in embodiment 8 and 12 expresses and cholesterol levels.

Sequence table

<110>Santaris A/S

＜120〉suppress the RNA antagonist compound that APO-100 expresses

<130>16513PCT00

<160>68

<170>PatentIn version 3.3

<210>1

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉Scambled LNA oligonucleotide contrast

<400>1

cgtcagtatg cgaatc 16

<210>2

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>2

ggtattcagt gtgatg 16

<210>3

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>3

attggtattc agtgtg 16

<210>4

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>4

ttgttctgaa tgtcca 16

<210>5

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>5

tcttgttctg aatgtc 16

<210>6

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>6

tggtattcag tgtgat 16

<210>7

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>7

ttggtattca gtgtga 16

<210>8

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>8

cattggtatt cagtgt 16

<210>9

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>9

gcattggtat tcagtg 16

<210>10

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>10

agcattggta ttcagt 16

<210>11

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>11

cagcattggt attcag 16

<210>12

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>12

tcagcattgg tattca 16

<210>13

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>13

ttcagcattg gtattc 16

<210>14

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>14

gttcagcatt ggtatt 16

<210>15

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>15

agttcagcat tggtat 16

<210>16

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>16

aagttcagca ttggta 16

<210>17

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>17

aaagttcagc attggt 16

<210>18

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>18

atttccatta agttct 16

<210>19

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>19

ggtatttcca ttaagt 16

<210>20

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>20

gactcaatgg aaaagt 16

<210>21

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>21

atgactcaat ggaaaa 16

<210>22

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>22

gctaacacta agaacc 16

<210>23

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>23

cactaagaac cagaag 16

<210>24

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>24

ctaagaacca gaagat 16

<210>25

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>25

tgaatcgggt cgcatc 16

<210>26

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉antisense motif

<400>26

tgaatcgggt cgcatt 16

<210>27

<211>21

<212>RNA

＜213〉artificial

<220>

<223>siRNA

<400>27

gucaucacac ugaauaccaa u 21

<210>28

<211>23

<212>RNA

＜213〉artificial

<220>

<223>siRNA

<400>28

auugguauuc agugugauga cac 23

<210>29

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #2

<220>

＜221〉oligonucleotide of LNA modification

<222>(1)..(3)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉oligonucleotide of LNA modification

<222>(13)..(15)

<400>29

ggtattcagt gtgatg 16

<210>30

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #3

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(3)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>30

attggtattc agtgtg 16

<210>31

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #3

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>31

attggtattc agtgtg 16

<210>32

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #4

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(14)..(15)

<400>32

ttgttctgaa tgtcca 16

<210>33

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #5

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(2)..(2)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>33

tcttgttctg aatgtc 16

<210>34

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #8

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(1)..(1)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>34

cattggtatt cagtgt 16

<210>35

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #9

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(2)..(2)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>35

gcattggtat tcagtg 16

<210>36

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #10

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(3)..(3)

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(13)..(13)

<400>36

agcattggta ttcagt 16

<210>37

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #11

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(1)..(1)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(3)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(14)..(14)

<400>37

cagcattggt attcag 16

<210>38

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #11

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(1)..(1)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1).(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(4)..(4)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(14)..(14)

<400>38

cagcattggt attcag 16

<210>39

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #19

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉oligonucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(15)..(15)

<400>39

atttccatta agttct 16

<210>40

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #19

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>40

ggtatttcca ttaagt 16

<210>41

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #20

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(3)..(3)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>41

gactcaatgg aaaagt 16

<210>42

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #21

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>42

atgactcaat ggaaaa 16

<210>43

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #22

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(2)..(2)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(15)..(15)

<400>43

gctaacacta agaacc 16

<210>44

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #23

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(1)..(1)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(3)..(3)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>44

cactaagaac cagaag 16

<210>45

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #24

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(1)..(1)

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<400>45

ctaagaacca gaagat 16

<210>46

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #25

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(13)..(13)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<400>46

tgaatcgggt cgcatc 16

<210>47

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉motif #26

<220>

＜221〉Nucleotide of LNA modification

<222>(1)..(4)

<220>

＜221〉phosphorothioate bond

<222>(1)..(15)

<220>

＜221〉the LNA cytosine(Cyt) of 5-methyl modification

<222>(13)..(13)

<220>

＜221〉Nucleotide of LNA modification

<222>(13)..(15)

<400>47

tgaatcgggt cgcatt 16

<210>48

<211>21

<212>RNA

＜213〉artificial

<220>

＜223〉the non-siRNA that puts together

<400>48

gucaucacac ugaauaccaa u 21

<210>49

<211>23

<212>RNA

＜213〉artificial

<220>

＜223〉the non-siRNA that puts together

<400>49

auugguauuc agugugauga cac 23

<210>50

<211>21

<212>RNA

＜213〉artificial

<220>

＜223〉siRNA that puts together of cholesterol

<400>50

gucaucacac ugaauaccaa u 21

<210>51

<211>23

<212>RNA

＜213〉artificial

<220>

＜223〉siRNA that puts together of cholesterol

<400>51

auugguauuc agugugauga cac 23

<210>52

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>52

agcctcgtcc cgtagacaaa at 22

<210>53

<211>24

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>53

gttgatggca acaatctcca cttt 24

<210>54

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>54

ccttccttct tgggtatgga a 21

<210>55

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>55

gctcaggagg agcaatgatc t 21

<210>56

<211>22

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>56

gcccattgtg gacaagttga tc 22

<210>57

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉Oligonucleolide primers

<400>57

ccaggacttg gaggtcttgg a 21

<210>58

<211>24

<212>DNA

＜213〉artificial

<220>

＜223〉mApoB Taqman probe

<400>58

aagccagggc ctatctccgc atcc 24

<210>59

<211>8

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>59

gtattcag 8

<210>60

<211>8

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>60

ggtattca 8

<210>61

<211>8

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>61

tcagcatt 8

<210>62

<211>8

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>62

gcattggt 8

<210>63

<211>29

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>63

aaagttcagc attggtattc agtgtgatg 29

<210>64

<211>19

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>64

ggtatttcca ttaagttct 19

<210>65

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>65

atgactcaat ggaaaagt 18

<210>66

<211>24

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>66

cactaagaac cagaaccaga agat 24

<210>67

<211>16

<212>DNA

＜213〉artificial

<220>

＜223〉target motif

<400>67

tgaatcgggt cgcaty 16

<210>68

<211>18

<212>DNA

＜213〉artificial

<220>

＜223〉subsequence/preferred oligomer motif

<400>68

tcttgttctg aatgtcca 18

Claims (35)

1, oligomeric compounds is made up of 12-50 Nucleotide and/or nucleotide analog altogether, and wherein said compound comprises the subsequence of at least 10 Nucleotide or nucleotide analog, and described subsequence is corresponding to being selected from SEQ ID NO:2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 and 26 sequence, wherein said compound comprises at least 3 nucleotide analogs.

2, according to the compound of claim 1, be made up of double chain oligonucleotide, wherein every chain comprises altogether 16-30 Nucleotide and/or nucleotide analog, and wherein said compound comprises the subsequence of at least 10 Nucleotide or nucleotide analog, and described subsequence is positioned at and is selected from SEQ IDNO:2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, in 23,24,25 and 26 the sequence, wherein said compound comprises at least 3 nucleotide analogs.

3, according to the compound of claim 1 or 2, wherein said subsequence comprises described at least 3 nucleotide analogs.

4, according to the compound of aforementioned each claim, wherein said subsequence comprises one section 2-6 nucleotide analog, is thereafter one section 4-12 Nucleotide, is one section 2-6 nucleotide analog after again.

5, according to the compound of aforementioned each claim, wherein said subsequence and described SEQ ID NO:2,3,4,5,6,7,8,9 of being selected from, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 when comparing with the corresponding sequence in 26 the sequence, comprises 1 or 2 mispairing.

6, according to the compound of aforementioned each claim, form by 12-25 Nucleotide or nucleotide analog.

7, according to the compound of claim 6, by 15,16,17,18,19,20,21 or 22 Nucleotide or nucleotide analog are formed.

8, according to the compound of claim 7, form by 15 or 16 Nucleotide or nucleotide analog.

9, according to the compound of aforementioned each claim, wherein said Nucleotide comprises the linking group that is selected from phosphate, thiophosphoric acid base and borine phosphate, connects between nucleosides can be-O-P (O) ₂-O-, and-O-P (O, S)-O-.

10, according to the compound of claim 9, wherein said connection is a phosphate.

11, according to the compound of claim 9, wherein said connection is the thiophosphoric acid base.

12, according to the compound of claim 10, wherein all connections all are the thiophosphoric acid bases.

13,, comprise 3 to 12 nucleotide analogs according to the compound of aforementioned each claim.

14,, comprise 6 or 7 nucleotide analogs according to the compound of claim 13.

15, according to the compound of aforementioned each claim, wherein at least 1 described nucleotide analog is locked nucleic acid (LNA), for example at least 3, or all nucleotide analogs all are LNA.

16, according to the compound of claim 15, wherein LNA is selected from β-D-oxygen-LNA, α-L-oxygen-LNA, β-D-amino-LNA and β-D-sulphur-LNA.

17, according to the compound of claim 16, wherein said nucleosides and/or LNA link together by phosphoric acid or thiophosphoric acid group.

18, according to the compound of aforementioned each claim, subsequence wherein is selected from by SEQID No 63, SEQ ID NO:2, the group that SEQ ID No 3 and SEQ ID No 11 form.

19, according to the compound of claim 1-17, wherein said subsequence is selected from by SEQ IDNo 64, for example SEQ ID No 18 or 19; SEQ ID NO:65, for example SEQ ID No 20 or 21); SEQ ID No 66, SEQ ID No 22,23 for example, or 24; SEQ ID No 67, for example SEQ ID No 25 or 26) and SEQ ID No 68, for example SEQ ID No 4 or 5 groups of forming.

20, according to the compound of aforementioned each claim, it has following formula

5’-[(LNA) _2-6-(DNA/RNA) _4-12-(LNA) _2-6-(DNA/RNA) _0-1]-3’

Or

5’-[(LNA) _3-4-(DNA/RNA) _8-9-(LNA) ₃-(DNA/RNA) ₁]-3’

Wherein " LNA " indicates LNA Nucleotide, and " DNA " and " RNA " indicates deoxyribonucleotide and ribonucleotide respectively.

21, compound is selected from SEQ ID NO:29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46 and 47 groups of forming.

22, according to the compound of claim 20, it is SEQ ID NO:29.

23, according to the compound of claim 20, it is SEQ ID NO:30.

24, according to the compound of claim 20, it is SEQ ID NO:37.

25, conjugate, comprise according to claim 1-24 each compound and at least 1 non-nucleotide or the non-polynucleotide part covalently bound with described compound.

26, pharmaceutical composition comprises the conjugate of definition in compound that claim 1-24 defines in each or the claim 24 and pharmaceutically acceptable thinner, carrier or assistant agent.

27, according to the pharmaceutical composition of claim 26, it is suitable for oral administration.

28,, further comprise the compound of at least a reducing cholesterol according to the pharmaceutical composition of claim 26 or 27.

29, according to the pharmaceutical composition of claim 28, wherein said compound is selected from: biliary salts resin (for example, Colestyramine, colestipol and hydrochloric acid colesevelam), the HMGCoA-reductase inhibitor is (for example, lovastatin, Cerivastatin, Pravastatin, Zarator, Simvastatin and fluvastatin), nicotinic acid, shellfish special acid derivative (for example, clofibrate, gemfibrozil, fenofibrate, bezafibrate and Win-35833), probucol, Xin Meisu, dextrothyroxine, plant-stanol ester, cholesterol absorption inhibitor (for example, ezetimibe), implitapide, the inhibitor of bile acid transport albumen (apical sodium dependency bile acid transport albumen), liver CYP7a conditioning agent, controversies in hormone replacement in the elderly is (for example, tamoxifen) and antiphlogiston (for example, glucocorticosteroid).

30, in the compound that defines in each of claim 1-24 or the claim 25 conjugate of definition as medicine.

31, the conjugate of definition is used for the treatment of purposes in the medicine of Apo-B100 abnormal level in preparation in the compound that defines in each of claim 1-24 or the claim 25.

32, according to the purposes of claim 27, wherein said Apo-B100 abnormal level is relevant with the existence that is selected from following medical condition: atherosclerosis, hypercholesterolemia or hyperlipidaemia.

33, treatment suffers from the method for the object of the disease of the atherosclerosis of being selected from, hypercholesterolemia and hyperlipidaemia or illness, and this method comprises the step of the object that pharmaceutical composition that claim 26-29 is defined in each or conjugate are administered to these needs.

34, according to the method for claim 33, wherein said pharmaceutical composition or conjugate oral administration.

35, the method for downward modulation apolipoprotein B, this method comprise the step that pharmaceutical composition that claim 26-29 is defined in each or conjugate are administered to the object of definition in object such as the claim 33.

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