WO2020037797A1 - Mir-221/222 and inhibitors thereof for use in preparing drugs and detection targets for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma - Google Patents

Abstract

Provided are miR-221/222 and inhibitors thereof for use in preparing drugs and detection targets for regulating liver fat deposition, liver fibrosis, and hepatocellular carcinoma. The effect is achieved by inhibiting the activity of miR-221 and/or miR-222 to reduce the liver fat infiltration level, the deposition of liver collagen fibers, plasma cholesterol level, serum transaminase and improve insulin resistance, thereby inhibiting malignant proliferation of liver cells.

Description

miR-221 / 222 and its inhibitors are used to prepare drugs and detection targets for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma Technical field

The invention belongs to the field of biological technology, and particularly relates to miR-221 / 222 and its inhibitor for preparing drugs and detection targets for regulating and controlling liver fat deposition, liver fibrosis, and hepatocellular carcinoma.

Background technique

MicroRNAs (microRNAs, miRNAs) are non-coding small RNAs (typically 21-23 nucleotides in length) that inhibit translation and / or messenger stability of target genes by binding to the 3 ′ untranslated region of a specific gene. There are currently more than 2,500 microRNAs discovered, and new microRNA candidate genes are still being discovered. Their expression is often developmental and / or tissue-specific, although some microRNAs are stably expressed throughout the body. miRNA is an important gene regulatory factor and is involved in regulating various basic processes of the body, such as cell proliferation and apoptosis, differentiation, development, organogenesis, differentiation, shaping, metabolism, stress response, stem cell differentiation, neurogenesis, angiogenesis, etc. . miRNA genes are not randomly arranged, some of them are clustered, and clustered genes are often co-expressed. miR-221 / 222 is a pair of miRNAs that are highly conserved in vertebrates and aggregated on the X chromosome. Because they have a consistent seed region, these two microRNAs regulate similar target genomes.

Summary of the Invention

The purpose of the present invention is to provide miR-221 / 222 and its inhibitors for preparing drugs and detecting targets for regulating liver fat deposition, fibrosis, and hepatocellular carcinoma.

The above object of the present invention is achieved by the following technical solutions:

In a first aspect of the present invention, miR-221 has a nucleoside base sequence as shown in SEQ ID NO: 1.

In a second aspect of the present invention, miR-222 has a nucleobase sequence as shown in SEQ ID NO: 2.

In a third aspect of the present invention, the precursor of miR-221 has a nucleoside base sequence as shown in SEQ ID NO: 3.

In a fourth aspect of the present invention, the precursor of miR-222 has a nucleobase sequence as shown in SEQ ID NO: 4.

In a fifth aspect of the present invention, the primer for detecting the expression level of miR-221 has a nucleobase sequence as shown in SEQ ID NO: 8 and SEQ ID NO: 9.

In a sixth aspect of the present invention, the primer for detecting the expression level of miR-222 has a nucleobase sequence as shown in SEQ ID NO: 10 and SEQ ID NO: 11.

According to a seventh aspect of the present invention, the primer for detecting the expression level of miR-221 is used for preparing a reagent for detecting the expression level of miR-221 target.

According to an eighth aspect of the present invention, the primers for detecting the expression level of miR-222 are used to prepare a reagent for detecting the expression level of miR-222 target.

According to a ninth aspect of the present invention, the miR-221 and the inhibitor thereof are used for preparing a medicament for regulating liver fat deposition, liver fibrosis, and hepatocellular carcinoma; more preferably, the miR-221 and the inhibitor thereof are used as Detection targets are used to prepare drugs that regulate liver fat deposition, liver fibrosis, and hepatocellular carcinoma.

According to a tenth aspect of the present invention, the miR-222 and the inhibitor thereof are used for preparing a medicament for regulating liver fat deposition, liver fibrosis, and hepatocellular carcinoma; more preferably, the miR-222 and the inhibitor thereof are used as Detection targets are used to prepare drugs that regulate liver fat deposition, liver fibrosis, and hepatocellular carcinoma.

According to an eleventh aspect of the present invention, a compound includes a modified oligonucleotide; wherein the modified oligonucleotide consists of 15 to 25 linked nucleosides and targets miR-221 and / or miR-222, and the nucleotide sequence of the nucleoside is complementary to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

Preferably, the modified oligonucleotide in the compound consists of any of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 linked nucleosides and targets miR- 221 and / or miR-222, and the nucleotide sequence of the nucleoside is complementary to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

Preferably, the oligonucleotide includes a nucleoside base sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

Preferably, the modified oligonucleotide further comprises at least one modified sugar; more preferably, each of the modified sugars is independently selected from 2'-O-methoxyethyl sugar, 2'-fluoro sugar, 2'-O-methyl sugar and bicyclic sugar moiety.

Preferably, the modified oligonucleotide further comprises at least one modified internucleoside linkage; more preferably, each of the modified internucleoside linkages is a phosphorothioate internucleoside linkage Or phosphorothioate internucleoside bonds.

In a twelfth aspect of the present invention, the use of the compound in the preparation of a medicament for the following uses:

(i) reducing the level of fatty infiltration in the subject's liver; or

(ii) preventing or delaying the appearance of liver collagen fibrous deposition in a subject; or

(iii) preventing or delaying the occurrence of hepatocellular carcinoma in a subject.

Preferably, the subject has at least one metabolic disorder of metabolic syndrome, obesity, diabetic dyslipidemia, hyperlipidemia, hypertriglyceridemia, hyperfatty acidemia, and hyperinsulinemia; and / Or hepatocellular carcinoma; more preferably, the subject's metabolic disorders include elevated blood lipid levels, elevated serum transaminase levels, liver B ultra-mild to severe fatty liver, liver fibrosis changes, elevated At least one of gluconeogenesis, insulin resistance, impaired glucose tolerance, and excess body fat.

Preferably, the medicine is used for:

(i) improving liver fatty infiltration in a subject; or

(ii) preventing or delaying the appearance of liver collagen fibrous deposition in a subject; or

(iii) preventing or delaying the occurrence of hepatocellular carcinoma in a subject.

Preferably, the medicament is realized by inhibiting the activity of miR-221 and / or miR-222 to reduce liver fat infiltration, reduce the degree of fibrosis, prevent or reduce malignant proliferation of liver cells, reduce plasma cholesterol levels, reduce serum transaminase and improve insulin resistance ; And / or by reducing the activity of gluconeogenesis in a subject by inhibiting the activity of miR-221 and / or miR-222.

Preferably, the medicine uses the above-mentioned compound as an active ingredient, and further includes a pharmaceutically acceptable auxiliary material or auxiliary ingredient.

Preferably, the mode of administration of the drug includes intravenous administration, subcutaneous administration, oral administration or parenteral administration.

Preferably, the applied dosage of the modified oligonucleotide in the medicine is 25-800 mg / kg.

According to a thirteenth aspect of the present invention, a pharmaceutical composition includes the compound containing a modified oligonucleotide, and further includes a pharmaceutically acceptable excipient or auxiliary ingredient; preferably, the modified oligonucleoside The acid is a sterile lyophilized oligonucleotide, and its application dose is 25-800 mg / kg.

In a fourteenth aspect of the present invention, a miR-221 / 222 detection target kit includes the primers for detecting the expression level of miR-221 and / or the primers for detecting the expression level of miR-222, further including the primer A compound comprising a modified oligonucleotide.

Supplementary note

In certain embodiments, the nucleobase sequence of the oligonucleotide is completely complementary to the nucleobase sequence of SEQ ID NO: 1, 2, 3, or 4; in certain embodiments, the The nucleobase sequence is at least 95% complementary to the nucleobase sequence of SEQ ID NO: 1, 2, 3, or 4; in certain embodiments, the nucleobase sequence of the oligonucleotide is identical to SEQ ID NO: Nucleotide base sequence of 1, 2, 3, or 4 is at least 90% complementary; nucleobase base sequence of the oligonucleotide is at least 85% complementary to the nucleotide base sequence of SEQ ID NO: 1, 2, 3, or 4 . In certain embodiments, the nucleobase sequence of the oligonucleotide does not mismatch with a nucleobase sequence selected from SEQ ID NO: 1, 2, 3, or 4; in certain embodiments, the oligo The nucleobase sequence of a nucleoside has a mismatch with a nucleobase sequence selected from SEQ ID NO: 1, 2, 3, or 4; in certain embodiments, the nucleobase sequence of an oligonucleotide Has no more than one mismatch with a nucleobase sequence selected from SEQ ID NO: 1, 2, 3, or 4; in certain embodiments, the nucleobase sequence of an oligonucleotide matches a NO sequence selected from SEQ ID : 1, 2, 3, or 4 nucleobase sequences have no more than two mismatches.

In certain embodiments, the oligonucleotide comprises at least one modified internucleoside linkage; in certain embodiments, the oligonucleotide comprises at least two modified internucleoside linkages; in certain embodiments In embodiments, the oligonucleotide comprises at least three modified internucleoside linkages; in certain embodiments, each internucleoside linkage of the oligonucleotide is a modified internucleoside linkage In certain embodiments, the first internucleoside linkage and the last internucleoside linkage of the oligonucleotide are modified internucleoside linkages; in certain embodiments, at least one modified The internucleoside linkage is a phosphorothioate internucleoside linkage; in certain embodiments, each nucleoside of the oligonucleotide comprises a modified sugar; in certain embodiments, the oligonucleoside The acid comprises at least three nucleosides comprising a modified sugar; in certain embodiments, the oligonucleotide comprises at least two nucleosides comprising a modified sugar; in certain embodiments, the oligonucleotide comprises At least one nucleoside comprising a modified sugar; in certain embodiments, each nucleoside of the oligonucleotide is 2'-O-methoxyethyl sugar-containing; in certain embodiments, the oligonucleotide comprises a plurality of 2'-O-methoxyethyl sugar-containing nucleosides and a plurality of 2'-fluoro Sugar-modified nucleosides; in certain embodiments, each modified sugar is independently selected from 2'-O-methoxyethyl sugar, 2'-fluoro sugar, 2'-O-methyl Sugars and bicyclic sugar moieties; in some embodiments, the bicyclic sugar moiety is LNA; in certain embodiments, the compound comprises a conjugate linked to an oligonucleotide; in certain embodiments, the conjugate The thing is cholesterol.

In certain embodiments, the modified oligonucleotide has the following modifications: each nucleoside is a 2'-O-methyl nucleoside, and the first two 5 'internucleoside linkages are phosphorothioate Each of the four 3 'terminal internucleoside linkages is a phosphorothioate, the remaining internucleoside linkages are phosphodiesters and the 3' terminal nucleoside is linked to cholesterol via a base prolinol linkage connection.

In certain embodiments, the oligonucleotide consists of 24 linked nucleosides; in certain embodiments, the oligonucleotide consists of 23 linked nucleosides; in certain embodiments, the oligonucleotide The acid consists of 22 linked nucleosides; in some embodiments, the oligonucleotide consists of 21 linked nucleosides; in certain embodiments, the oligonucleotide consists of 20 linked nucleosides; In certain embodiments, the oligonucleotide consists of 19 linked nucleosides; in certain embodiments, the oligonucleotide consists of 18 linked nucleosides; in certain embodiments, the oligonucleotide The acid consists of 17 linked nucleosides; in some embodiments, the oligonucleotide consists of 16 linked nucleosides; in certain embodiments, the oligonucleotide consists of 15 linked nucleosides; In certain embodiments, the oligonucleotide consists of 14 linked nucleosides; in certain embodiments, the oligonucleotide consists of 13 linked nucleosides; in certain embodiments, the oligonucleotide An acid consists of 12 linked nucleosides; in certain embodiments, an oligonucleotide consists of 11 linked nucleosides; In some embodiments, the oligonucleotide consists of 10 linked nucleosides; in certain embodiments, the oligonucleotide consists of 9 linked nucleosides; in certain embodiments, the oligonucleotide consists of It consists of 8 linked nucleosides; in certain embodiments, the oligonucleotide consists of 7 linked nucleosides.

In certain embodiments, the nucleobase sequence of the oligonucleotide comprises the nucleobase sequence of SEQ ID NO: 5, 6, or 7; in certain embodiments, the nucleobase of the oligonucleotide The sequence consists of the nucleobase sequence of SEQ ID NO: 5, 6, or 7.

In certain embodiments, it is achieved by inhibiting the activity of miR-221 and / or miR-222 to reduce liver cholesterol levels, reduce serum transaminase, and improve insulin resistance; in certain embodiments, by inhibiting miR-221 and / or miR The activity of -222 is reduced by the deposition of collagen fibers in the liver of a subject; in certain embodiments, it is achieved by inhibiting the activity of miR-221 and / or miR-222 to inhibit the malignant proliferation of liver cells.

The present invention provides methods for treating liver steatohepatitis, liver fibrosis, and hepatocellular carcinoma, and related conditions, comprising administering a compound comprising an oligonucleotide targeted to miR-221 and / or miR-222.

The present invention provides a method for reducing liver cholesterol deposition and plasma cholesterol level in a subject, which comprises administering to the subject a composition comprising 12 to 30 linked nucleosides and having complementarity to or miR-221 / 222 -221/222 is a compound of an oligonucleotide complementary to the nucleobase sequence of the precursor, and thereby reduces liver cholesterol deposition and plasma cholesterol levels in the subject. In certain embodiments, a subject is administered a compound comprising an oligonucleotide consisting of 7 to 12 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222, and thereby reduced Subject's liver cholesterol deposition and plasma cholesterol. In certain embodiments, the subject has elevated plasma cholesterol levels. In certain embodiments, the subject has mild to severe hepatic fatty deposits, and in certain embodiments, the method comprises selecting a test subject with mild to severe fatty liver or elevated plasma cholesterol levels By. In certain embodiments, the plasma cholesterol is LDL-cholesterol and / or VLDL-cholesterol.

The present invention provides a method for reducing a serum transaminase level in a subject, comprising administering to the subject a composition comprising 7 to 12 linked nucleosides and having a nucleobase base sequence complementary to miR-221 / 222 Oligonucleotide compounds, thereby reducing serum transaminase levels in a subject. In certain embodiments, the subject has elevated serum aminotransferase levels. In certain embodiments, the method comprises measuring a subject's serum transaminase level. In certain embodiments, the method includes selecting a subject having an elevated serum aminotransferase level.

The present invention provides a method for reducing liver fibrosis in a subject, comprising administering to the subject a composition comprising 12 to 30 linked nucleosides and having complementarity to or miR-221 / 222 Precursor compounds with complementary nucleobase sequences to oligonucleotides, and thereby reduce liver fibrosis in a subject. In certain embodiments, the subject has an elevated index of liver fibrosis level. In certain embodiments, administering to a subject a compound comprising an oligonucleotide consisting of 7 to 12 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 thereby reduces the test subject Level of liver fibrosis.

The present invention provides a method for preventing or delaying an increase in liver fatty infiltration levels in a subject at risk of elevated blood lipid levels, which comprises administering to the subject a composition comprising 12 to 30 linked nucleosides and Compounds having an nucleobase sequence that is complementary to miR-221 / 222 or a precursor of miR-221 / 222 thereby prevent or delay the occurrence of elevated levels of fatty infiltration in the liver of a subject. In certain embodiments, administration to a subject of a compound comprising an oligonucleotide consisting of 7 to 12 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 is thereby prevented or delayed Subjects had elevated levels of fatty infiltration in the liver.

The present invention provides a method for improving insulin sensitivity in a subject, comprising administering to the subject a composition comprising 12 to 30 linked nucleosides and having complementarity to or miR-221 / 222 The compounds of the oligonucleotides of the precursor complementary nucleobase sequence thus improve the subject's insulin sensitivity. In certain embodiments, the subject has insulin resistance. In certain embodiments, the method includes selecting a subject having insulin resistance. In certain embodiments, administering to a subject a compound comprising an oligonucleotide consisting of 7 to 12 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 thereby improves the test subject Insulin sensitivity.

The present invention provides a method for preventing or delaying the onset of insulin resistance in a subject at risk of developing membranin resistance, which comprises administering to the subject a composition comprising 12 to 30 linked nucleosides and having Compounds of oligonucleotides that are complementary to miR-221 / 222 or that are complementary to the precursors of miR-221 / 222 thereby prevent or delay the appearance of insulin resistance in a subject. In certain embodiments, the method includes selecting a subject at risk for developing insulin resistance. In certain embodiments, administration to a subject of a compound comprising an oligonucleotide consisting of 7 to 12 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 is thereby prevented or delayed Subject's insulin resistance appeared.

The present invention provides a method for preventing or inhibiting the occurrence of hepatocellular carcinoma in a subject, which comprises administering to the subject a composition comprising 12 to 30 linked nucleosides and having complementarity with miR-221 / 222 or with miR- The compounds of the 221/222 precursor complementary nucleobase sequence oligonucleotides thus inhibit the occurrence of liver cancer. In certain embodiments, the subject's blood lipid levels are elevated. In certain embodiments, the subject is a patient with mild to severe fatty liver. In certain embodiments, the subject is a patient with mild to severe liver fibrosis. In certain embodiments, the method comprises selecting a subject for liver cancer. A compound consisting of 7 to 12 nucleoside-linked oligonucleotides having a nucleobase sequence complementary to miR-221 / 222 thereby inhibits hepatocellular carcinoma occurrence.

In any of the above methods provided by the present invention, the subject may have a disorder of lipid metabolism, fatty liver, liver fibrosis, or hepatocellular carcinoma.

The present invention provides a method for preventing or delaying the appearance of at least one characteristic in a subject at risk of suffering from a disorder of lipid metabolism, fatty liver, liver fibrosis, or hepatocellular carcinoma, which comprises administering to the subject Compounds of modified oligonucleotides consisting of 12 to 30 linked nucleosides and having a nucleobase sequence that is complementary to miR-221 / 222 or a precursor of miR-221 / 222 are thereby prevented or delayed The subject had a disorder of lipid metabolism, fatty liver, liver fibrosis, or hepatocellular carcinoma. In certain embodiments, administration to a subject of a compound comprising a modified oligonucleotide consisting of 7 to 12 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 is thereby prevented Or delay the subject's lipid metabolism disorder, fatty liver, liver fibrosis or hepatocellular carcinoma.

The present invention provides a method for treating at least one of a lipid metabolism disorder, fatty liver, liver fibrosis, or hepatocellular carcinoma in a subject, comprising administering to a patient a composition comprising 12 to 30 linked nucleosides and having Compounds of oligonucleotides that are complementary to miR-221 / 222 or to the precursors of miR-221 / 222 thereby treat lipid disorders, fatty liver, liver fibrosis, or hepatocellular carcinoma. In certain embodiments, a patient is administered a compound comprising an oligonucleotide comprising 7 to 12 linked nucleosides and having a nucleobase base sequence complementary to the precursor complementary to miR-221 / 222. Disorders of lipid metabolism, fatty liver, liver fibrosis or hepatocellular carcinoma.

In certain embodiments, at least one metabolic disorder is pre-diabetes, diabetes, metabolic syndrome, obesity, diabetic dyslipidemia, hyperlipidemia, hypertriglyceridemia, hyperfatty acidemia, hypercholesterolemia.

In certain embodiments, administration includes parenteral administration. In certain embodiments, parenteral administration includes intravenous or subcutaneous administration. In certain embodiments, administration comprises oral administration.

In certain embodiments, administering comprises administering at least one additional therapy. In certain embodiments, at least one additional therapy is an agent that lowers blood lipids. In certain embodiments, the lipid-lowering agent is selected from drugs that affect lipid synthesis, metabolism, and clearance (nicotinic acid and its derivatives, clobetin and phenoxyacetic acid, methylolglutarate Coenzyme A (HMG-CoA) reductase inhibitors), drugs that affect cholesterol and bile acid absorption (cholic acid integrator, Probucol), polyene fatty acid drugs. In certain embodiments, at least one additional therapy is a lipid-lowering agent. In certain embodiments, at least one additional therapy is administered concurrently with the compound. In certain embodiments, at least one additional therapy is administered more frequently than the compound. In certain embodiments, at least one additional therapy is administered less frequently than the compound. In certain embodiments, at least one additional therapy is administered after the compound is administered. In certain embodiments, at least one additional therapy is administered before the compound is administered. In certain embodiments, at least one additional therapy is co-administered with the compound.

In certain embodiments, the compound is administered in the form of a pharmaceutical composition that uses the compound described above as the active ingredient and further includes a pharmaceutically acceptable excipient or auxiliary ingredient.

The present invention provides a method for identifying a subject in need of treatment, comprising comparing the amount of microRNA in a sample obtained from the subject to the amount of a negative control, wherein the microRNA is miR-221 / 222, and An elevated level of miR-221 / 222 in a sample obtained from the subject indicates that the subject is in need of treatment with a compound comprising a modified oligonucleotide complementary to miR-221 / 222. In certain embodiments, the sample is a liver sample. In certain embodiments, the sample is a serum sample. In certain embodiments, the subject is at risk of developing steatohepatitis, liver fibrosis, or hepatocellular carcinoma. In certain embodiments, the subject is suspected of having steatohepatitis, liver fibrosis, or hepatocellular carcinoma. In certain embodiments, the subject is treated with a compound comprising a modified oligonucleotide having a nucleobase that is complementary to or miR-221 / 222 or its precursor. The technical solutions and embodiments of the present invention are obvious in conjunction with the drawings, the description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Unless otherwise specified, wild-type male C57Bl / 6 mice (≌20g) were treated with PBS, anti-miR-221 / 222 (1 × 12.5mg / kg), and anti-miR-221 / 222 (2 × 12.5mg / kg) , Anti-miR-221 / 222 (2 × 15 / kg) or anti-miR-124 (2 × 15 / kg), while male ob / ob (45g) mice were treated with PBS, anti- (1 × 15mg / kg) ), Anti-miR-221 / 222 (2 × 15 mg / kg) or anti-miR-124 (2 × 15 mg / kg). Throughout the drawings, the labeling of anti-miR treatments is described in the table below.

Table 1

Figure 1 shows that miR-221 / 222 is up-regulated in a model of steatohepatitis; control diet (control diet), methionine-choline deficiency diet (MCD diet) mice (n = 3) miR- RNA expression level of 221/222; RNA expression level of miR-221 / 222 on total RNA of livers of control diet (ctrl) and high-fat diet (HFD) mice (n = 3).

Figure 2 shows the construction of miR-221 / 222 liver-specific knockout mice; hepatocyte-specific knockouts were obtained by mating LoRP transgenic mice with miR-221 / 222 and Alb-Cre mice driven by the albumin promoter. Mouse model of miR-221 / 222 (Liver-specific MiR-221 / 222knock out, MiR-221 / 222LKO); Real time-PCR results of control mice and MiR-221 / 222LKO liver tissue showed miR-221 and miR- 222 was successfully knocked out in the liver (n = 3).

Figure 3 shows the loss of miR-221 / 222 in hepatocytes of MCD mouse model, which can lead to reduced fat deposition such as cholesterol and triglycerides in the liver, and increased insulin sensitivity. Transmission electron microscopy images of control mice and MiR-221 / 222LKO liver tissue Staining with oil red O showed that knockdown of miR-221 / 222 by hepatocytes could reduce liver fat deposition.

Figure 4 shows that the expression of inflammatory factors in the liver of MiR-221 / 222LKO mice is reduced, and the aggregation of inflammatory cells is reduced; the real time-PCR results of control mice and liver tissue of MiR-221 / 222LKO in the MCD diet model show the inflammatory factors IL-1β, TNFα The expression levels of IL-6 and IL-6 were significantly decreased; H & E staining of liver sections showed a significant reduction in inflammatory cell infiltration.

Figure 5 shows increased collagen fiber deposition and increased expression of collagen family members in the liver of MiR-221 / 222LKO mice; liver Sirius Red and Maisong staining show that compared with control mice, the liver of MiR-221 / 222LKO mice on the MCD diet The positive staining area was reduced; Q-PCR showed that the expression of collagen family members in the liver of MiR-221 / 222LKO mice was reduced.

Figure 6 shows that adenovirus AD-miR-221 / 222 infected livers of MiR-221 / 222LKO mice and increased liver lipid deposition. MiR-221 / 222 was expressed by adenovirus in MiR-221 / 222LKO mice, and tail veins were found. Adenovirus AD-miR-221 / 222 injection can re-express miR-221 / 222 in mouse liver; the liver weight ratio and liver triglyceride of mice expressing miR-221 / 222 are higher than those of control mice.

Figure 7 shows that MiR-221 / 222LKO mice infected with adenovirus AD-miR-221 / 222 aggravated liver fibrosis; MiR-221 / 222LKO mice were injected with adenovirus AD-miR-221 / 222 and control AD-GFP through the tail vein. Sirius Red and Maisong staining showed that the area of Sirius Red and Maisong staining increased after miR-221 / 222LKO mice re-expressed miR-221 / 222, indicating that the collagen fiber deposition area increased.

FIG 8 is a miR-221 / 222inhibitors has good inhibitory effect in vitro; using locked nucleic acids Locked nucleicacids (LNA ^TM) modification were synthesized miR-221 / 222inhibitors (LNA- i-miR-221, LNA-i-miR-222 ); NC, LNA-i-miR-221 and LNA-i-miR-22250nM and 100nM were transfected with mouse liver cancer cell line hepa1-6 in vitro, Q-PCR detection showed LNA-i-miR-221, LNA-i-miR-222 has specific and good inhibitory effects on miR-221 and miR-222; compared with controls, LNA-i-miR-221 and LNA-i-miR-222 can significantly up-regulate the target gene P27 And TIMP3 protein levels.

Figure 9 shows that LNA-i-miR-221 and LNA-i-miR-222 have a better inhibitory effect on miR-221 and miR-222 in vivo; we compare LNA-i-miR-221 and LNA-i-miR -222 Intraperitoneal injection of MCD diet mice, it was found that LNA-i-miR-221 and LNA-i-miR-222 have a better inhibitory effect on miR-221 and miR-222 in vivo; control mice were intraperitoneally injected with NC, i-miR-221 and LNA-i-miR-222 (1, 4, 8, 15, 21 days), given the MCD diet at the same time, the livers of mice were taken at 25 days to identify the expression levels of miR-221 and miR-222, MiR -221 / 222LKO mice served as positive controls.

Figure 10 shows that LNA-i-miR-221 and LNA-i-miR-222 injections can significantly reduce the expression level of liver inflammatory factors and reduce inflammation infiltration caused by the MCD diet; LNA-i-miR-221 and LNA-i-miR The mRNA levels of Il6, Tnf, Il1b, and Adgre1 in mouse liver were reduced after -222 injection.

Figure 11 shows that LNA-i-miR-221 and LNA-i-miR-222 injection can significantly reduce the expression of liver collagen family members caused by MCD diet; LNA-i-miR-221 and LNA-i-miR-222 injection The mRNA levels of Sma, Col1a1, Col3a1, and Col5a3 in the liver of the mice were reduced after the mice.

Figure 12 shows that miR-221 and miR-222 are elevated in human fibrotic liver tissue; miR-221 and miR-222 expression levels are gradually increased in normal control human liver tissue, mild liver fibrosis tissue, and severe liver fibrosis tissue. high.

detailed description

Unless defined otherwise, all scientific terms and techniques used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless a specific definition is provided, the terms used in connection with the analytical chemistry, synthetic organic chemistry, medicinal, and medicinal chemistry described in the present invention, as well as its procedures and techniques, are those terms and procedures and techniques that are well known and commonly used in the art. If there are multiple definitions of the terms of the present invention, the ones in this section shall prevail. Unless otherwise stated, all patents, patent applications, published applications and publications, GenBank sequences, websites and other published materials mentioned in the entire disclosure of the present invention are incorporated by reference, if allowed.

Before disclosing and describing the compositions and methods of the present invention, it should be understood that the terminology used in the present invention is for the purpose of describing particular embodiments and is not intended to be limiting. It must be pointed out that, unless the context clearly indicates otherwise, the singular forms "a" and "the" used in the description and the appended claims include plural indicators.

definition

"Metabolic disorder" refers to a syndrome characterized by changes or disorders of one or more metabolic processes in the body, and is a risk factor for cardiovascular and cerebrovascular diseases leading to diabetes. Metabolic disorders include, but are not limited to, obesity, type 2 diabetes, metabolic syndrome, pre-diabetes, type 1 diabetes, diabetic dyslipidemia, and hyperinsulinemia.

"Obesity" is a chronic metabolic disease caused by many factors. It is characterized by an increase in the volume and number of adipocytes in the body leading to an abnormal increase in body fat as a percentage of body weight and excessive deposition in some areas. Body fat (or obesity) includes both fat distribution throughout the body and the size of adipose tissue accumulation. The distribution of body fat can be estimated by skin fold measurements, waist-to-hips ratio, or techniques such as ultrasound, computed tomography, or magnetic resonance imaging. Individuals with a body mass index (BMI) of 28 are considered clinically obese.

"Type 2 diabetes" refers to diabetes characterized by insulin resistance and relative insulin deficiency. Epidemiological studies have shown that obesity, high-calorie diets, insufficient physical activity, and aging are the most important environmental factors for type 2 diabetes, and factors such as hypertension and dyslipidemia also increase the risk of disease.

"Pre-diabetes", the development of diabetes is divided into three stages, the first stage is called "high-risk population", the second stage is called "pre-diabetes", and the third stage is called "diabetes." Pre-diabetes means that the subject's fasting blood glucose is> 6.1 mmol / L, or two hours after a meal, the blood glucose is> 7.8 mmol / L, but does not meet the diagnostic criteria for diabetes.

"Steatohepatitis" refers to hepatitis secondary to steatosis of bullous liver cells. Can be divided into alcoholic steatohepatitis and non-alcoholic steatohepatitis according to the cause. The liver histological changes of the two are basically similar. Both are manifested as hepatic balloon degeneration and mixed inflammatory cell infiltration mainly composed of neutrophils in the lobular on the basis of liver steatosis. Partial steatohepatitis is also accompanied by Mallory body and pericellular fibrosis and central vein fibrosis.

"Non-alcoholic fatty liver disease (nAFLD)" refers to a clinicopathological syndrome that is characterized primarily by excessive deposition of fat in the liver cells caused by alcohol and other well-defined liver damage factors. It is closely related to insulin resistance and genetic susceptibility. Metabolic stress-induced liver injury. Including simple fatty liver (SFL), non-alcoholic steatohepatitis (nASH) and related cirrhosis.

"Non-alcoholic steatohepatitis (nASH)", also known as metabolic steatohepatitis, is a clinical syndrome with pathological changes similar to alcoholic hepatitis but no history of excessive drinking. It occurs in middle-aged people, especially overweight and obese individuals. Non-alcoholic steatohepatitis is closely related to metabolic disorders such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia. It is mainly characterized by hepatocyte bullous steatosis with liver cell damage and inflammation. In severe cases, it can develop into liver There is no special treatment for sclerosis.

"Alcoholic steatohepatitis (ASH)" refers to a condition characterized by accumulation of fat in the liver and inflammation and epilepsy in the liver caused by long-term heavy drinking.

"B ultrasound diagnosis of fatty liver I": 1. The near-field echo in the liver area is diffusely enhanced (stronger than the kidney and spleen), and the far-field echo is gradually attenuated; 2. the intrahepatic duct structure is unclear; 3. the liver is mild to medium The degree of swelling is large, and the edge angle is round and blunt. 4. Color Doppler blood flow imaging indicates that the color blood flow signal in the liver is reduced or not easy to display. However, the intrahepatic blood vessels are normal. 5. The right liver capsule and diaphragm echo display. Ambiguous or incomplete. Those who have one of the above items l and 2 to 4 are mild fatty liver; those who have two of the above items l and 2-4 are moderate fatty liver; have the above items l and 2 to Two of 4 and 5 are severe fatty liver.

"Metabolic syndrome" refers to the pathological state of metabolic disorders of the body's proteins, fats, carbohydrates and other substances. It is a complex set of metabolic disorders and is a risk factor for diabetic cardiovascular and cerebrovascular diseases. A variety of metabolic disorders are combined, including obesity, hyperglycemia, hypertension, dyslipidemia, high blood viscosity, high uric acid, high fatty liver incidence, and hyperinsulinemia.

"Insulin sensitivity" refers to the ability of a cell to absorb glucose in response to the action of insulin.

"Insulin resistance" refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various reasons, and the body excessively secretes excessive insulin to produce hyperinsulinemia to maintain blood glucose stability. Insulin resistance can easily lead to metabolic syndrome and type 2 diabetes.

"Improving insulin resistance" refers to increasing the ability of a cell to produce a normal insulin response. In certain embodiments, improving insulin resistance in hepatocytes results in increased glucose storage in hepatocytes.

"Diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia" refers to a condition characterized by type 2 diabetes, decreased HDLC, increased serum triglycerides, and small, dense LDL particles.

"Steady degeneration" refers to the accumulation of triglycerides (neutral fats) in the cytoplasm, which is called steatosis or steatosis, and it occurs in tissues with strong metabolism and oxygen consumption, such as liver cells.

"Glucose tolerance test" or "GTT" refers to a glucose load test to understand the function of islet β cells and the body's ability to regulate blood glucose. It is a definite test for diagnosing diabetes and is widely used in clinical practice. "IPGTT" refers to GTT after intraperitoneal glucose injection. "OGTT" refers to GTT after oral glucose. In certain embodiments, GTT is used to test for pre-diabetes. In certain embodiments, GTT is used to identify a subject with diabetes. In certain embodiments, GTT is used to identify subjects at risk for developing diabetes. In certain embodiments, GTT is used to identify subjects with insulin resistance.

"Insulin tolerance test (ITT)" refers to a test that measures insulin sensitivity through a hormonal response to stress from low blood glucose levels. In normal people, 15-30 minutes after intravenous injection of insulin (0.1 μu / kg body weight), the blood glucose concentration is reduced by 50% compared with fasting; the fasting blood glucose level should be restored within 60 to 90 minutes. In certain embodiments, ITT is used to test for pre-diabetes. In certain embodiments, ITT is used to identify a subject with diabetes. In certain embodiments, ITT is used to identify subjects at risk for developing diabetes. In certain embodiments, ITT is used to identify subjects with insulin resistance.

"Anti-miR" refers to an oligonucleotide that has a nucleobase sequence complementary to a microRNA and targets the microRNA. In certain embodiments, the anti-miR is a modified oligonucleotide.

"Subject" refers to a human or non-human animal selected for treatment or therapy.

"At risk of ..." refers to a subject's tendency to develop a certain condition or disease. In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a lesser degree than is required to diagnose the condition or disease . In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not exhibit a sufficient number of symptoms to be diagnosed as the condition or disease.

"Administering" means providing an agent or composition to a subject and includes, but is not limited to, administration by a medical professional and self-administration.

"Subcutaneous administration" means administration directly under the skin.

"Intravenous administration" means administration into a vein.

"Parenteral administration" refers to administration by injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous, intravenous or intramuscular administration.

"Co-administration" means that at least two agents are administered to a subject in any manner that simultaneously exhibits a pharmacological effect in the subject. Co-administration does not require the two agents to be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. It is not necessary that the reagents exert their effects for the same time. The effects need only overlap for a period of time and do not need to continue together.

"Treatment" refers to the treatment of a disease. In certain embodiments, the therapy includes, but is not limited to, chemotherapy, surgical resection, liver transplantation, and / or chemoembolization.

"Treatment" refers to the application of one or more specific therapies to cure or ameliorate a disease. In certain embodiments, the specific therapy is the administration of one or more agents.

"Improving" means reducing the severity of at least one indicator of a condition or disease. The severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art. In certain embodiments, improvement includes delaying or slowing the progression of one or more indicators of the condition or disease.

"Prevention" refers to preventing the progression of a condition or disease in a subject at risk for the disease or condition. In certain embodiments, a subject at risk of developing a disease or condition receives treatment similar to that received by a subject already suffering from the disease or condition.

By "retarding" is meant delaying the progression of a condition or disease in a subject at risk for the disease or condition. In certain embodiments, a subject at risk of developing a disease or condition receives treatment similar to that received by a subject already suffering from the disease or condition.

"Therapeutic agent" refers to an agent used to cure, ameliorate, or prevent a disease.

"Dose" refers to a specified amount of an agent provided in a single administration. In certain embodiments, when subcutaneous administration is required, the necessary volume of the required dose is not conveniently provided by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, the dose may be administered by two or more injections to minimize injection site reactions in the individual.

"Dosage unit" refers to the form of the medicament provided. In certain embodiments, the dosage unit is a vial containing a lyophilized oligonucleotide. In certain embodiments, the dosage unit is a vial containing a reconstituted oligonucleotide.

A "therapeutically effective amount" refers to the amount of an agent that provides a therapeutic effect to an animal.

"Pharmaceutical composition" refers to a mixture of substances, including agents, suitable for administration to an individual. For example, the pharmaceutical composition may include a sterile aqueous solution.

"Pharmaceutical agent" refers to a substance that provides a therapeutic effect when administered to a subject.

"Active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that provides the desired effect.

"Improved liver function" refers to a change in liver function to a normal range. In certain embodiments, liver function is assessed by measuring molecules in the serum of a subject. For example, in certain embodiments, improved liver function is measured by a decrease in blood liver transaminase levels.

"Acceptable safety profile" refers to a pattern of side effects that is within clinically acceptable boundaries.

"Side effect" refers to a physiological response attributable to treatment other than the expected effect. In certain embodiments, side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal renal function, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathy. Such side effects can be detected directly or indirectly. For example, increased serum aminotransferase levels may indicate liver toxicity or liver function abnormalities. For example, increased bilirubin may indicate liver toxicity or liver function abnormalities.

"Injection site reaction" refers to skin inflammation or abnormal redness at the injection site of an individual.

"Subject compliance" means that the subject adheres to the recommended or prescribed therapy.

"Compliance" means that the subject adheres to the recommended therapy.

"Suggested therapy" means a therapy recommended by a medical professional for the treatment, amelioration, or prevention of a disease.

"Target nucleic acid" refers to a nucleic acid to which a designed oligomeric compound hybridizes.

"Targeting" refers to the process of designing and selecting a nucleobase sequence that will hybridize to a target nucleic acid.

"Modulation" refers to interference with function or activity. In certain embodiments, regulation refers to increasing gene expression. In certain embodiments, regulation refers to reducing gene expression.

"Expression" refers to any function or step that converts the encoded information of a gene into a structure that exists and operates in a cell.

The "5 'target site" refers to a nucleobase of a target nucleic acid that is complementary to the nucleobase of the 5' end of a specific oligonucleotide.

The "3 'target site" refers to a nucleobase of a target nucleic acid that is complementary to the nucleobase of the 3' end of a specific oligonucleotide.

"Region" refers to a portion of a nucleic acid linked to a nucleoside. In certain embodiments, there is a nucleobase sequence that is complementary to a target nucleic acid region. For example, in certain such embodiments, the region is complementary to a region of the miRNA stem-loop sequence. In certain such embodiments, the region is completely complementary to the region of the miRNA stem-loop sequence.

A "nucleoside base sequence" refers to the sequence of consecutive nucleobases in the 5 'to 3' direction, and is not related to any sugar, bonding, and / or nucleobase modification.

"Contiguous nucleobases" refer to nucleobases that are directly adjacent to each other in a nucleic acid.

"Complementary" refers to the ability of an oligomeric compound to hybridize to a target nucleic acid under stringent hybridization conditions.

By "fully complementary" is meant that each nucleobase of an oligomeric compound is capable of pairing with a nucleobase in each corresponding position of the target nucleic acid. For example, in certain embodiments, the oligomeric compound is fully complementary to the miRNA stem-loop sequence when each nucleobase is complementary to a nucleobase within a region of the miRNA stem-loop sequence.

"Nucleobase complementarity" refers to the ability of two nucleobases to non-covalently pair via hydrogen bonding.

"Percent complementarity" refers to the percentage of nucleobases of an oligomeric compound that is complementary to an equal length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of an oligomeric compound that is complementary to the nucleobase of the corresponding position of the target nucleic acid by the total length of the oligomeric compound. In certain embodiments, the percent complement refers to the number of nucleobases that are complementary to the target nucleic acid divided by the length of the modified oligonucleotide.

"Hybridization" refers to annealing of complementary nucleic acids that occur through nucleobase complementarity.

"Mismatch" means that the nucleobase of the first nucleic acid cannot be paired with the nucleobase of the corresponding position of the second nucleic acid.

"Identity" means having the same nucleobase sequence.

"MicroRNA" refers to non-coding RNA of 18 to 25 nucleobases in length and is found in a wide variety of organisms, from viruses to humans. These small RNAs bind to mRNA to block the expression of protein-coding genes, preventing them from being translated into proteins. Examples of mature miRNAs can be found in a miRNA database called miRBase (http://microrna.sanger.ac.uk/). In certain embodiments, microRNA is abbreviated as miRNA, or miR.

A precursor miRNA, a precursor miR, refers to a non-coding RNA with a hairpin structure, which is a cleavage product of a pri-miR by a double-stranded RNA-specific ribonuclease called Drosha.

A "stem loop sequence" refers to an RNA that has a hairpin structure and contains a mature miRNA sequence. The precursor miRNA sequence and the stem-loop sequence may overlap. Examples of stem-loop sequences can be found in a miRNA database called miRBase (http://microrna.sanger.ac.uk/).

"Pri-miRNA" or "pri-miR" refers to a non-coding RNA with a hairpin structure, which is a substrate for the double-stranded RNA-specific ribonuclease Drosha.

A "miRNA precursor" refers to a transcript derived from genomic DNA, and it contains a non-coding, structured RNA containing one or more miRNA sequences. For example, in certain embodiments, the miRNA precursor is a precursor miRNA. In certain embodiments, the miRNA precursor is a pri-miRNA.

"MiR-221" refers to a mature miRNA having the nucleotide sequence shown in SEQ ID NO: 1 (ACCUGGCAUACAAUGUAGAUUU).

"MiR-222" refers to a mature miRNA having the nucleotide base sequence shown in SEQ ID NO: 2 (AGCUACAUCUGGCUACUGGGU).

The "miR-221-1 stem-loop sequence" refers to a miR-221 precursor having the nucleobase sequence shown in SEQ ID NO: 3 (UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAUUUGUGUGUUCGUUAGGCAACAGCUACAUUGUCUGGGGGUUAGGCUACCUGGAAACAUGUUCUC).

The "miR-222-1 stem-loop sequence" refers to a miR-222 precursor having the nucleobase sequence shown in SEQ ID NO: 4 (GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUCAGUAGCCAGUGUAGAUCC UGUCUUUCGUAAUCAGCAGCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU).

"MiR-221 / 222" refers to a microRNA having a nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

A "monocistronic transcript" refers to a miRNA precursor comprising a single miRNA sequence.

"Polycistronic transcript" refers to a miRNA precursor containing two or more miRNA sequences.

"Seed sequence" refers to 2 to 6 or 2 to 7 core nucleotides from the 5 ' end of a mature miRNA sequence.

A "compound comprising an oligonucleotide comprising a plurality of linked nucleosides" refers to a compound comprising an oligonucleotide having a specified number of linked nucleosides. Therefore, the compound may contain additional substituents or conjugates. Unless otherwise stated, the compound does not contain any additional nucleosides other than those described.

"Oligonucleotide" refers to a nucleoside-linked polymer, each of which can be modified or unmodified independently of each other.

An "oligomeric compound" refers to a compound comprising a polymer of linked monomeric subunits.

"Naturally occurring internucleoside linkage" refers to a 3 'to 5' phosphodiester bond between nucleosides.

"Natural sugar" refers to a sugar that is found in DNA (2'-H) or RNA (2'-OH).

"Natural nucleobase" refers to a nucleobase that is unmodified relative to its naturally occurring form.

"Internucleoside linkage" refers to a covalent bond between adjacent nucleosides.

"Linked nucleoside" refers to a nucleoside linked by a covalent bond.

A "nucleoside base" refers to a heterocyclic moiety that is capable of non-covalent pairing with another nucleobase.

A "nucleoside" refers to a nucleoside base linked to a sugar.

A "nucleotide" refers to a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

"Modified oligonucleotide" refers to an oligonucleotide having one or more modifications relative to a naturally occurring end, sugar, nucleobase, and / or internucleoside linkage.

"Single-stranded modified oligonucleotide" refers to an oligonucleotide that has not hybridized to a complementary strand.

"Modified internucleoside linkage" refers to any change from a naturally occurring internucleoside linkage.

"Phosphorothioate internucleoside linkage" refers to a bond between nucleosides in which one non-bridged atom is a sulfur atom.

"Modified nucleobase" refers to any substitution and / or change other than the natural nucleobase.

"Modified sugar" means a substitution and / or any change other than a natural sugar.

"'2'-O-methyl sugar" or "2'-OMe sugar" means a sugar having an O-methyl modification at the 2' position.

"2'-O-methoxyethyl sugar" or "2'-MOE sugar" means a sugar having an O-methoxyethyl modification at the 2 'position.

"2'-O-fluoro" or "2'-F" refers to a sugar having a fluorine modification at the 2 'position.

"5-methylcytosine" refers to a methyl-modified cytosine attached to the 5 'position.

"Bicyclic sugar moiety" refers to a sugar modified by bridging two unpaired ring atoms.

"2'-O-methoxyethyl nucleoside" refers to a 2'-modified nucleoside having a 2'-O-methoxyethyl sugar modification.

"2'-fluoronucleoside" refers to a 2'-modified nucleoside having a 2'-fluorosaccharide modification.

"2'-O-methyl nucleoside" refers to a 2'-modified nucleoside having a 21-O-methyl sugar modification.

"Bicyclic nucleoside" refers to a 2'-modified nucleoside having a bicyclic sugar moiety.

"Motif" refers to a pattern of modified and / or unmodified nucleobases, sugars, and / or internucleoside linkages in an oligonucleotide.

By "fully modified oligonucleotide" is meant that each nucleobase, each sugar, and / or each internucleoside linkage is modified.

"Uniformly modified oligonucleotide" means that each nucleobase, each sugar, and / or each internucleoside linkage has the same modification throughout the modified oligonucleotide.

A "gapmer" refers to a modified oligonucleotide having an internal region linked to a nucleoside positioned between two external regions linked to a nucleoside, wherein the internal region of the nucleoside contains a sugar moiety that is different from each The sugar portion of the outer region of each nucleoside.

A "gapsegment" is an inner region of a gap body positioned between outer regions.

A "wing segment" is the outer region of the gap body positioned at the 5 'or 3' end of the inner region.

By "symmetric gapmer" is meant that each nucleoside of each outer region includes the same sugar modification.

"Asymmetric gapmer" means that each nucleoside of one outer region contains a first sugar modification and each nucleoside of another outer region contains a second sugar modification.

"Stabilizing modification" refers to the stability provided by a 2'-deoxynucleoside linked by a phosphodiester internucleoside linkage, such that the stability of the modified oligonucleotide in the presence of a nuclease is enhanced Nucleoside modification. For example, in certain embodiments, the stabilizing modification is a stabilizing nucleoside modification. In certain embodiments, the stabilizing modification is an internucleoside linkage modification.

"Stabilized nucleoside" refers to a modified nucleoside that provides enhanced nuclease stability to an oligonucleotide relative to the stability provided by 2'-deoxynucleoside. In one embodiment, the stabilized nucleoside is a 2'-modified nucleoside.

"Stabilized internucleoside linkage" refers to an internucleoside linkage that provides improved nuclease stability to an oligonucleotide relative to the stability provided by a phosphodiester internucleoside linkage. In one embodiment, the stabilized internucleoside linkage is a phosphorothioate internucleoside linkage.

Summary

Fatty hepatitis is a progressive chronic liver disease worldwide that can progress to cirrhosis, decompensated liver failure, and hepatocellular carcinoma. In recent decades, with the rising incidence of obesity, type 2 diabetes and metabolic syndrome, the prevalence of steatohepatitis has increased significantly. Its clinical features include hepatic steatosis, lobular inflammation, hepatocyte balloon dilation, and progressive pericyte fibrosis. Steatohepatitis affects millions of people worldwide and can be a life-threatening disease. Therefore, methods and compositions are needed to treat, prevent, or delay the appearance of metabolic disorders.

As shown in the present invention, administration of oligonucleotides complementary to miR-221 / 222 reduces liver fat deposition level, liver inflammation infiltration and liver collagen deposition, and prevents and / or delays the occurrence of hepatocellular carcinoma. These effects were observed in animal models of steatohepatitis. Oligonucleotides complementary to either or both of miR-221 / 222 can be used to achieve the phenotypic results described in the present invention.

Administration of a compound comprising an oligonucleotide complementary to miR-221 / 222 or a precursor thereof may lead to one or more clinically desirable results. Such clinically desirable results include, but are not limited to, reduced liver fat deposition, reduced liver inflammation infiltration, reduced liver fibrosis levels, and reduced liver tumorigenesis.

The invention provides a method and a composition for reducing liver fat deposition, reducing liver inflammation infiltration, reducing liver fibrosis level, and reducing liver tumorigenesis. The present invention also provides methods for treating, preventing or delaying the appearance of metabolic disorders associated with fatty liver, decreased insulin sensitivity, and increased plasma cholesterol. In certain embodiments, metabolic disorders include, but are not limited to, pre-diabetes, type 2 diabetes, metabolic syndrome, obesity, diabetic dyslipidemia, hyperglycemia, and hyperinsulinemia. In certain embodiments, a subject having a metabolic disorder also has a fatty liver disease. In certain embodiments, fatty liver disease includes, but is not limited to, non-alcoholic fatty liver disease, alcoholic fatty liver disease, and non-alcoholic fatty liver disease.

In certain embodiments, the invention provides a method for reducing liver fat deposition in a subject, comprising administering to the subject a nucleoside base comprising 12 to 30 linked nucleosides and having a complement to miR-221 / 222 A base sequence of an oligonucleotide compound.

In certain embodiments, the invention provides a method of reducing liver fat deposition in a subject, comprising administering to the subject a nucleoside base comprising 7 to 12 linked nucleosides and having a complement to miR-221 / 222 A base sequence of an oligonucleotide compound.

In certain embodiments, the method provided by the present invention includes B-ultrasound detection of liver fatty infiltration levels. Ultrasound detection of liver fatty infiltration levels may be performed before and / or after administration of a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222.

In certain embodiments, the ultrasound detects liver fatty infiltration levels when the subject is fasting for at least 8 hours.

In certain embodiments, the subject has mild fatty liver. In certain embodiments, the subject is identified as mild fatty liver. This identification is usually performed by a medical professional.

In certain embodiments, a subject with a metabolic disorder has moderate fatty liver. In certain embodiments, the subject is identified as having moderate fatty liver based on the subject's level of liver infiltration. Diagnosis of moderate fatty liver is usually performed by a medical professional.

In certain embodiments, the subject has severe fatty liver. In certain embodiments, the subject is identified as having severe fatty liver based on the subject's level of liver infiltration. Diagnosis is usually performed by a medical professional.

In certain embodiments, the present invention provides a method comprising monitoring the liver before administering a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 Fat infiltration level. In certain embodiments, the methods provided by the invention include measuring after administering a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 Liver fat infiltration level.

In certain embodiments, a method for reducing the level of fatty infiltration in the liver includes reducing the subject's liver imaging performance to normal liver imaging as determined by a medical institution such as the Chinese Medical Association Hepatology Branch or the World Health Organization which performed.

In certain embodiments, the invention provides a method of reducing liver fibrosis in a subject, comprising administering to the subject a nucleoside base comprising 12 to 30 linked nucleosides and having a complement to miR-221 / 222 A base sequence of an oligonucleotide compound.

In certain embodiments, the invention provides a method for reducing liver fibrosis in a subject, comprising administering to the subject a nucleoside base comprising 7 to 12 linked nucleosides and having a complement to miR-221 / 222 A base sequence of an oligonucleotide compound.

In certain embodiments, the method provided by the present invention includes detecting liver fibrosis levels using a Fibroscan. Fibroscan can detect liver fibrosis levels before and / or after administration of a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222.

In certain embodiments, Fibroscan detects liver fatty infiltration levels when the subject is fasting for at least 8 hours.

In certain embodiments, the subject has mild liver fibrosis. In certain embodiments, the subject is identified as mild liver fibrosis, and such identification is usually performed by a medical professional.

In certain embodiments, a subject with a metabolic disorder has moderate liver fibrosis. In certain embodiments, the subject is identified as having moderate liver fibrosis based on the subject's transient liver elasticity profile. Diagnosis of moderate liver fibrosis is usually performed by a medical professional.

In certain embodiments, the subject has severe liver fibrosis. In certain embodiments, the subject is identified as having severe liver fibrosis based on the subject's liver elasticity profile. Diagnosis is usually performed by a medical professional.

In certain embodiments, the present invention provides a method comprising monitoring the liver before administering a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 Instantaneous elasticity spectrum. In certain embodiments, the methods provided by the invention include measuring after administering a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222 Liver transient elasticity profile.

In certain embodiments, a method for reducing liver fibrosis includes reducing a subject's liver imaging performance to normal liver imaging as determined by a medical institution such as the Chinese Medical Association Hepatology Branch or the World Health Organization which performed.

In certain embodiments, the administration is performed at least once a week. In certain embodiments, the administration is performed every two weeks. In certain embodiments, the administration is performed every three weeks. In certain embodiments, the administration is performed every four weeks. The frequency of administration may be set by a medical professional.

In certain embodiments, a method of reducing hepatocellular carcinoma in a subject, comprising administering to the subject a nucleobase sequence comprising 12 to 30 linked nucleosides and having a complement to miR-221 / 222 Oligonucleotide compounds.

In certain embodiments, the present invention provides a method for reducing hepatocellular carcinoma in a subject, comprising administering to the subject a nucleoside comprising 7 to 12 linked nucleosides and having a complement to miR-221 / 222 Compounds of base sequence oligonucleotides.

In some embodiments, the method provided by the present invention includes B-ultrasound detection of liver tumorigenesis. The liver may be detected by B-ultrasound before and / or after administration of a compound comprising an oligonucleotide consisting of 7 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222.

In certain embodiments, the present invention provides a method for lowering plasma cholesterol in a subject, comprising administering to the subject a composition comprising 12 to 30 linked nucleosides and having miR-221 / 222 or a A compound of an oligonucleotide with a complementary nucleobase sequence. In certain embodiments, the subject has elevated plasma cholesterol. In certain embodiments, the subject is identified as having elevated plasma cholesterol. In certain embodiments, the administration reduces plasma cholesterol. In certain embodiments, the plasma cholesterol is plasma LDL-cholesterol. In certain embodiments, the plasma cholesterol is plasma VLDL-cholesterol.

In certain embodiments, the present invention provides a method for reducing plasma cholesterol in a subject, comprising administering to the subject a nucleoside comprising 7 to 12 linked nucleosides and having miR-221 / 222 Compounds of base sequence oligonucleotides.

In certain embodiments, the present invention provides a method for improving insulin resistance in a subject, comprising administering to the subject a composition comprising 12 to 30 linked nucleosides and having a precursor to miR-221 / 222 Compounds of oligonucleotides with complementary nucleobase sequences. In certain embodiments, the subject has insulin resistance. In certain embodiments, the method includes selecting a subject having insulin resistance.

In certain embodiments, the invention provides a method for improving insulin resistance in a subject, comprising administering to the subject a core comprising 7 to 12 linked nucleosides and having a core complementary to miR-221 / 222 Oligonucleotide sequence of compounds. In certain embodiments, a subject with elevated blood glucose levels has insulin resistance.

In certain embodiments, a subject having fatty liver has insulin resistance. In certain embodiments, a subject having liver fibrosis has insulin resistance.

In certain embodiments, the present invention provides a method for treating a metabolic disorder in a subject, comprising administering to the subject a composition comprising 12 to 30 linked nucleosides and having miR-221 / 222 and before A compound of an oligonucleotide with a complementary nucleobase sequence. In certain embodiments, the subject has a metabolic disorder. In certain embodiments, the subject is identified as having a metabolic disorder. In certain embodiments, metabolic disorders include, but are not limited to, pre-diabetes, type 2 diabetes, metabolic syndrome, obesity or diabetic dyslipidemia, hyperglycemia, and hyperinsulinemia. In certain embodiments, the subject is diagnosed with one or more metabolic disorders. After performing a medical test that is well known to those skilled in the medical arts, the subject may be diagnosed with a metabolic disorder.

In certain embodiments, the present invention provides a method for treating a metabolic disorder in a subject, comprising administering to the subject a compound comprising 7 to 12 linked nucleosides and having a complement to miR-221 / 222 Nucleotide base sequence of oligonucleotide compounds.

In certain embodiments, the present invention provides a method for preventing the occurrence of a metabolic disorder in a subject, which comprises administering to the subject a composition comprising 12 to 30 linked nucleosides and having miR-221 / 222 or Compounds of oligonucleotides with precursor complementary nucleobase sequences. In certain embodiments, the subject is at risk for developing a metabolic disorder. In certain embodiments, the subject is identified as being at risk for developing a metabolic disorder. In certain embodiments, the metabolic disorder is pre-diabetes, type 2 diabetes, metabolic syndrome, obesity or diabetic dyslipidemia, hyperglycemia, hyperinsulinemia.

In certain embodiments, the present invention provides a method for preventing the occurrence of a metabolic disorder in a subject, comprising administering to the subject a composition comprising 7 to 12 linked nucleosides and having a complement to miR-221 / 222 Nucleotide base sequence of oligonucleotide compounds.

In certain embodiments, the present invention provides a method for delaying the appearance of a metabolic disorder in a subject, comprising administering to the subject a composition comprising 12 to 30 linked nucleosides and having a miR-221 / 222 or Compounds of oligonucleotides with precursor complementary nucleobase sequences. In certain embodiments, the subject is at risk for developing a metabolic disorder. In certain embodiments, the subject is identified as being at risk for developing a metabolic disorder. In certain embodiments, metabolic disorders include, but are not limited to, pre-diabetes, type 2 diabetes, metabolic syndrome, obesity or diabetic dyslipidemia, hyperglycemia, and hyperinsulinemia.

In certain embodiments, the invention provides a method for delaying the appearance of a metabolic disorder in a subject, comprising administering to the subject a composition comprising 7 to 12 linked nucleosides and having a complement to miR-221 / 222 Nucleotide base sequence of oligonucleotide compounds.

In certain embodiments, the subject has one or more metabolic disorders. In certain embodiments, the subject is diagnosed with one or more metabolic disorders. After performing a medical test that is well known to those skilled in the medical arts, the subject may be diagnosed with a metabolic disorder.

The subject's response to treatment can be assessed by tests similar to those used to diagnose metabolic disorders, including blood lipid levels, blood and liver function levels, blood glucose level tests, glucose tolerance tests, and HbAlc tests. Response to treatment can also be assessed by comparing test results after treatment with test results before treatment.

In certain embodiments, the activity of miR-221 / 222 is inhibited by using a microRNA sponge, which includes one or more sequences having a nucleobase that is complementary to miR-221 / 222 . A "microRNA sponge" refers to a competitive inhibitor of microRNA in the form of a transcript that is strongly initiated and pre-expressed, which contains multiple tandem binding sites of the microRNA of interest. When vectors encoding these sponges were introduced into cells, the sponges at least as strongly inhibited microRNA targets as chemically modified antisense oligonucleotides. It specifically inhibits microRNAs with complementary seven-valent seeds so that a single sponge can be used to block the entire microRNA seed family. In certain embodiments, the microRNA seed family includes miR-221 / 222.

Certain compounds

The compound provided by the present invention is used for treating steatohepatitis, liver fibrosis and preventing the delay of hepatocellular carcinoma. In certain embodiments, the compound includes an oligonucleotide. In certain such embodiments, the compound consists of an oligonucleotide. In certain embodiments, the oligonucleotide is a modified oligonucleotide.

In certain such embodiments, the compound includes an oligonucleotide that hybridizes to a complementary strand, ie, the compound comprises a double-stranded oligomeric compound. In certain such embodiments, hybridization of the oligonucleotide to a complementary strand forms a blunt end at each end of the double-stranded oligomeric compound. In certain embodiments, hybridization of an oligonucleotide to a complementary strand forms at least one blunt end. In certain embodiments, the end of the oligonucleotide comprises one or more additional linked nucleosides relative to the number of linked nucleosides of the complementary strand. In certain embodiments, one or more additional nucleosides are at the 5 'terminus of the oligonucleotide. In certain embodiments, one or more additional nucleosides are at the 3 'terminus of the oligonucleotide. In certain embodiments, each nucleobase of each of the one or more additional nucleosides is complementary to the target RNA. In certain embodiments, at least one nucleobase of a nucleoside in one or more additional nucleosides is complementary to the target RNA. In certain embodiments, the end of the complementary strand comprises one or more additional linked nucleosides relative to the number of linked nucleosides of the oligonucleotide. In certain embodiments, one or more additional linked nucleosides are at the 5 'terminus of the complementary strand. In certain embodiments, one or more additional linked nucleosides are at the 3 'terminus of the complementary strand. In certain embodiments, two additional linking nucleosides are linked to the ends. In certain embodiments, an additional nucleoside is attached to the terminus.

In certain embodiments, the compound comprises an oligonucleotide that is conjugated to one or more partially enhanced activity, cell distribution, or cellular uptake of the antisense oligonucleotide produced. In certain such embodiments, the moiety is a cholesterol moiety or a lipid moiety. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, folic acid, phenazine, anthraquinone, phenanthridine, fluorescein, acridine, coumarin, rhodamine and dyes. In certain embodiments, the conjugate group is directly attached to the oligonucleotide. In certain embodiments, the conjugate group is attached to the oligonucleotide through a linking moiety selected from the group consisting of amino, hydroxyl, carboxylic acid, thiol, unsaturated moiety (e.g., double or triple bond), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC), 8-amino-3,6-dioxaoctanoic acid (ADO), 6-aminohexanoic acid (AHEX or AHA), substituted Cl-Cl0 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, the substituent is selected from hydroxy, amino, carboxy, alkoxy, benzyl, phenyl, nitro, mercapto, thioalkoxy, aryl, halogen, alkyl, alkene And alkynyl.

In certain such embodiments, the compound comprises an oligonucleotide having one or more stabilizing groups attached to one or both ends of the oligonucleotide to enhance properties such as nuclease stability. The stabilizing group contains a cap structure. These terminal modifications protect the oligonucleotide from exonuclease degradation and can help with delivery and / or localization within the cell. The cap may be present at the 5 'end (5' cap), or at the 3 'end (3' cap), or it may be present at both ends. The cap structure includes, for example, an inverted deoxybaseless cap.

Suitable cap structures include 4 ', 5'-methylene nucleotides, 4'-thionucleotides, 1- (β-D-erythrofuranosyl) nucleotides, and 1,5-dehydration Hexitol nucleotides, carbocyclic nucleotides, α-nucleotides, L-nucleotides, modified base nucleotides, threopentanosyl nucleotides, phosphorodithioate bonds Acyclic, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3 ', 4'-open ring nucleotide, 3'-3'-reverse nucleotide portion, acyclic 3,5-di Hydroxypentyl nucleotide, 3'-2'-reverse nucleotide portion, 3'-3'-reverse abasic portion, 1,4-butanediol phosphate, 3'-2'-reverse To abasic moiety, hexyl phosphate, 3 'phosphoramidate, 3'-phosphate, amino hexyl phosphate, dithiophosphate, 3'-thiophosphate, bridged methyl phosphate moiety, and non-bridged Methyl phosphate part, 1,3-diamino-2-propyl phosphate, 5'-amino-alkyl phosphate, 6-aminohexyl phosphate, 3-aminopropyl phosphate, hydroxypropyl phosphate , 1,2-aminododecyl phosphate, 5'-5'-reverse abasic portion, 5'-5'reverse nucleotide portion, 5'-thio Esters, 5'-phosphoramidate, 5'-amino, bridging and / or non-bridging 5'-phosphoramidate, phosphorothioate and 5'-mercapto moiety.

Nucleotide sequence

The nucleobase sequences listed in the present invention (including but not limited to those in the examples and sequence listings) are not related to any modification of the nucleic acid. Thus, a nucleic acid defined by SEQ ID NO may independently comprise one or more modifications to one or more sugar moieties, to one or more internucleoside linkages, and / or to one or more nucleobases.

In certain embodiments, the oligonucleotide has a sequence that is complementary to a miRNA or a precursor thereof. The nucleobase sequence of the mature miR-221 / 222 and its corresponding stem-loop sequence according to the present invention is a sequence existing in miRBase, miRBase is an online search database of miRNA sequences and annotations, see http: // microrna. sanger.ac.uk/. Entries in the miRBase sequence database represent the predicted hairpin portion (stem loop) of the miRNA transcript, as well as the location and sequence information of the mature miRNA sequence. The miRNA stem-loop sequence in the database is not a miRNA precursor (precursor miRNA) in the strict sense, and in some cases may include the precursor miRNA and some flanking sequences from the putative original transcript. The miR-221 / 222 nucleobase sequence according to the present invention comprises any form of miRNA, including the sequence described in the 22.0 version of the miRBase sequence database, and the sequence described in any earlier version of the miRBase sequence database. The release of a sequence database may lead to the renaming of certain miRNAs. The composition of the invention includes a modified oligonucleotide complementary to miR-221 / 222 in the form of any nucleobase sequence described herein.

In certain embodiments, the oligonucleotide has a nucleobase sequence that is complementary to miR-221 / 222 or a precursor thereof. Thus, in certain embodiments, the nucleobase sequence of an oligonucleotide may have one or more mismatched nucleobases relative to its target miR-221 / 222 or its precursor sequence, and still be capable of Hybridize to its target sequence. In certain embodiments, the oligonucleotide has a nucleobase sequence that is fully complementary to miR-221 / 222 or a precursor thereof.

In certain embodiments, the oligonucleotide has a sequence that is complementary to a nucleobase sequence selected from the stem-loop sequence of miR-221 / 222.

In certain embodiments, the oligonucleotide has a sequence complementary to the nucleobase sequence of the miRNA, wherein the nucleobase sequence of the miRNA is selected from SEQ ID NO: 1 or 2.

In certain embodiments, the oligonucleotide has a nucleobase sequence that is complementary to a region of the miR-221 / 222 stem-loop sequence (SEQ ID NO: 3 or 4).

In certain embodiments, the oligonucleotide has a nucleobase sequence that is complementary to the nucleobase sequence (SEQ ID NO: 1) of miR-221 / 222. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising a nucleobase sequence CAGCAGACAATGTAGC (SEQ ID NO: 5). In certain embodiments, the oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence AGTAGCCAGATGTAGC (SEQ ID NO: 6).

In certain embodiments, the oligonucleotide comprises a nucleobase sequence that is complementary to a seed sequence common between miR-221 / 222. Oligonucleotides of any length according to the invention may comprise a seed-matching sequence. In certain such embodiments, the modified oligonucleotide consists of 7 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 8 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 9 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 10 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 11 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 12 linked nucleosides.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence CAGCAGACAATGTAGC (SEQ ID NO: 5), which is the same as the nucleoside of miR-221 (SEQ ID NO: 1) Acids are complementary. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence AGTAGCCAGATGTAGC (SEQ ID NO: 6), which is complementary to the nucleotide of miR-222. Oligonucleotides with nucleobase sequences (SEQ ID NOs: 5 and 6) have been shown to inhibit the activity of miR-221 / 222. Some of these modified oligonucleotides have LNA sugar modifications at each nucleoside.

In certain embodiments, the oligonucleotide has a nucleobase that is complementary to a nucleobase sequence having at least 80% identity to a nucleobase sequence selected from the miR stem-loop sequences of SEQ ID NOs: 3 and 4. Base sequence. In certain embodiments, the oligonucleotide has at least 85%, at least 90%, at least 92%, at least 94%, a nucleobase sequence with a miR stem-loop sequence selected from SEQ ID NOs: 3 and 4. Nucleotide base sequences that are at least 96%, at least 98% identical, or 100% identical.

In certain embodiments, the oligonucleotide has a nucleobase sequence that is at least 80% identical to a nucleobase sequence of a miR selected from the nucleobase sequences of SEQ ID NOs: 1 and 2. Nucleotide base sequence. In certain embodiments, the oligonucleotide has a nucleoside base sequence that is at least 85%, at least 90%, at least 92%, at least 94% to the miRNA nucleobase sequence selected from SEQ ID NOs: 1 and 2. Nucleotide base sequences that are 100% identical, at least 96%, at least 98% identical, or 100% identical.

In certain embodiments, the nucleobase sequence of an oligonucleotide is completely complementary to a miRNA nucleobase sequence or precursor thereof as set forth herein. In certain embodiments, the oligonucleotide has a mismatched nucleobase sequence relative to the nucleobase sequence of a mature miRNA or a precursor thereof. In certain embodiments, the oligonucleotide has a nucleobase sequence with 2 mismatches relative to the nucleobase sequence of the mature miRNA or a precursor thereof. In certain such embodiments, the oligonucleotide has a nucleobase sequence with no more than 2 mismatches relative to the nucleobase sequence of the mature miRNA or a precursor thereof. In certain such embodiments, the mismatched nucleobases are continuous. In certain such embodiments, mismatched nucleobases are discontinuous.

In certain embodiments, the oligonucleotide consists of several linked nucleosides of equal length to a mature miR that is complementary to the linked nucleoside.

In certain embodiments, the number of linked nucleosides of the oligonucleotide is less than the length of the mature miRNA to which it is complementary. In certain such embodiments, the number of linked nucleosides of the oligonucleotide is one nucleoside less than the length of the mature miRNA to which it is complementary. In certain such embodiments, the oligonucleotide has one less nucleoside at the 3 ' end. In certain such embodiments, the oligonucleotide has one less nucleoside at the 5 'end. In certain such embodiments, the oligonucleotide has two fewer nucleosides at the 3 'end. In certain such embodiments, the oligonucleotide has two fewer nucleosides at the 5 'end. In the case where each nucleobase of an oligonucleotide is complementary to each nucleobase at a corresponding position in the miRNA, an oligonucleotide having a number of linked nucleosides smaller than the length of the miRNA is considered to have An oligonucleotide with a partially complementary nucleobase sequence.

In certain embodiments, the number of linked nucleosides of the oligonucleotide is greater than the length of the miRNA to which it is complementary. In certain such embodiments, the nucleobase of the additional nucleoside is complementary to the nucleobase of the miRNA stem-loop sequence. In certain embodiments, the number of linked nucleosides of the oligonucleotide is one nucleoside greater than the length of the miRNA to which it is complementary. In certain such embodiments, the additional nucleoside is at the 3 'end of the oligonucleotide. In certain such embodiments, the additional nucleoside is at the 5 'end of the oligonucleotide. In certain embodiments, the number of linked nucleosides of the oligonucleotide is two nucleosides greater than the length of the miRNA to which it is complementary. In certain such embodiments, the two additional nucleosides are at the 3 'terminus of the oligonucleotide. In certain such embodiments, the two additional nucleosides are at the 5 'terminus of the oligonucleotide. In certain such embodiments, an additional nucleoside is located at the 5 'end of the oligonucleotide and an additional nucleoside is located at the 3' end.

In certain embodiments, a portion of the nucleobase sequence of the oligonucleotide is completely complementary to the nucleobase sequence of the miRNA, but the entire modified oligonucleotide is not completely complementary to the miRNA. In certain such embodiments, the number of nucleosides of an oligonucleotide having a fully complementary portion is greater than the length of the miRNA. For example, in the case where the oligonucleotide consists of 24 linked nucleosides, and the nucleobases of nucleosides 1 to 23 are complementary to the corresponding positions of a miRNA with a length of 23 nucleobases, respectively, Nucleotides have 23 nucleoside moieties that are fully complementary to the nucleobase sequence of the miRNA and have approximately 96% overall complementarity to the nucleobase sequence of the miRNA.

In certain embodiments, the nucleobase sequence of the oligonucleotide is completely complementary to a portion of the nucleobase sequence of the miRNA. For example, in the case where the oligonucleotide consists of 22 linked nucleosides, and the nucleobases of nucleosides 1 to 22 are each complementary to the corresponding position of a miRNA with a length of 23 nucleobases, The nucleotides are completely complementary to the 22 nucleobase portions of the nucleobase sequence of the miRNA. Such oligonucleotides have approximately 96% overall complementarity to the nucleobase sequence of the entire miRNA and are 100% complementary to the 22 nucleobase portions of the miRNA.

In certain embodiments, a portion of a nucleobase sequence of an oligonucleotide is completely complementary to a portion of a nucleobase sequence of a miRNA or a precursor thereof. In certain such embodiments, the 24 consecutive nucleobases of the oligonucleotide are each complementary to the 24 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 23 consecutive nucleobases of the oligonucleotide are each complementary to the 23 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 22 consecutive nucleobases of the oligonucleotide are each complementary to the 22 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, each of the 21 consecutive nucleobases of the oligonucleotide is complementary to the 21 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 20 consecutive nucleobases of the oligonucleotide are each complementary to the 20 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 19 consecutive nucleobases of the oligonucleotide are each complementary to the 19 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 18 consecutive nucleobases of the oligonucleotide are each complementary to the 18 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 17 consecutive nucleobases of the oligonucleotide are each complementary to the 17 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, the 16 consecutive nucleobases of the oligonucleotide are each complementary to the 16 consecutive nucleobases of the miRNA or a precursor thereof. In certain such embodiments, each of the 15 consecutive nucleobases of the oligonucleotide is complementary to the 15 consecutive nucleobases of the miRNA or a precursor thereof.

Although the sequence listing accompanying this application identifies each nucleobase sequence as "RNA" or "DNA" as needed, in reality, these sequences can be modified with any combination of chemical modifications. Those skilled in the art will readily understand that names such as "RNA" or "DNA '" used to describe modified oligonucleotides are to some extent arbitrary. For example, containing a 2'-OH sugar moiety and thymine A base nucleoside oligonucleotide can be described as DNA with a modified sugar (2'-OH replaces the natural 2'-H of DNA), or as RNA with a modified base (thymine (Methylated uracil) a natural uracil that replaces RNA).

Therefore, the nucleic acid sequences provided in the present invention (including but not limited to those in the sequence listing) are expected to include nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to those having modified nucleobases. Nucleic acid. By way of further example, and without limitation, an oligomeric compound having a nucleobase sequence "TGTAGC" includes any oligomeric compound having such a nucleobase sequence, whether modified or unmodified, which includes (but Not limited to) Such compounds containing RNA bases, such as those with the sequence "TGTAGC" and those with some DNA bases and some RNA bases, such as "TGTAGC", and oligomers with other modified bases Compounds, "TmeGTAGC", where meG represents a guanine base containing a methyl group.

Certain modified oligonucleotides

In certain embodiments, the oligonucleotide consists of 21 to 24 linked nucleosides. In certain embodiments, the oligonucleotide consists of 19 to 24 linked nucleosides. In certain embodiments, the oligonucleotide consists of 15 to 30 linked nucleosides. In certain embodiments, the oligonucleotide consists of 12 to 30 linked nucleosides. In certain embodiments, the oligonucleotide consists of 7 to 11 linked nucleosides. In certain embodiments, the oligonucleotide consists of 7 to 25 linked nucleosides.

In certain embodiments, the oligonucleotide consists of 30 linked nucleosides. In certain embodiments, the oligonucleotide consists of 29 linked nucleosides. In certain embodiments, the oligonucleotide consists of 28 linked nucleosides. In certain embodiments, the oligonucleotide consists of 27 linked nucleosides. In certain embodiments, the oligonucleotide consists of 26 linked nucleosides. In certain embodiments, the oligonucleotide consists of 25 linked nucleosides. In certain embodiments, the oligonucleotide consists of 24 linked nucleosides. In certain embodiments, the oligonucleotide consists of 23 linked nucleosides. In certain embodiments, the oligonucleotide consists of 22 linked nucleosides. In certain embodiments, the oligonucleotide consists of 21 linked nucleosides. In certain embodiments, the oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the oligonucleotide consists of 19 linked nucleosides. In certain embodiments, the oligonucleotide consists of 18 linked nucleosides. In certain embodiments, the oligonucleotide consists of 17 linked nucleosides. In certain embodiments, the oligonucleotide consists of 16 linked nucleosides. In certain embodiments, the oligonucleotide consists of 15 linked nucleosides. In certain embodiments, the oligonucleotide consists of 14 linked nucleosides. In certain embodiments, the oligonucleotide consists of 13 linked nucleosides. In certain embodiments, the oligonucleotide consists of 12 linked nucleosides. In certain embodiments, the oligonucleotide consists of 11 linked nucleosides. In certain embodiments, the oligonucleotide consists of 10 linked nucleosides. In certain embodiments, the oligonucleotide consists of 9 linked nucleosides. In certain embodiments, the oligonucleotide consists of 8 linked nucleosides. In certain embodiments, the oligonucleotide consists of 7 linked nucleosides.

Certain modifications

In certain embodiments, the oligonucleotides provided by the present invention may comprise one or more modifications to nucleobases, sugars, and / or internucleoside linkages, and thereby become modified oligonucleotides . Because modified nucleobases, sugars, and / or internucleoside linkages can achieve desired properties, such as enhanced cellular uptake, enhanced affinity for their targeting oligonucleotides, or enhancement in the presence of nucleases Because of their stability, they are superior to the unmodified form.

In certain embodiments, a modified oligonucleotide comprises one or more modified nucleosides. In certain such embodiments, the modified nucleoside is a stabilized nucleoside. An example of a stabilized nucleoside is a sugar-modified nucleoside.

In certain embodiments, the modified nucleoside is a sugar-modified nucleoside. In certain such embodiments, the sugar-modified nucleoside may further comprise a natural or modified heterocyclic base moiety and / or a natural or modified internucleoside linkage, and may comprise further modifications unrelated to sugar modification . In certain embodiments, the sugar-modified nucleoside is a 2 ' -modified nucleoside, wherein the sugar ring is modified at the 2 ' -carbon from natural ribose or 2 ' -deoxyribose.

In certain embodiments, the 2 ' -modified nucleoside has a bicyclic sugar moiety. In certain embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration. In certain embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridging group between 2 'and 4'-carbon atoms. In certain embodiments, the bicyclic sugar moiety comprises 1 to 4 linking bis groups. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linking bis groups. In certain such embodiments, the bridging group comprises 1 to 8 linking diradicals. In certain embodiments, the bicyclic sugar moiety comprises 2 linking bis groups. In certain embodiments, the linking group is selected from bis -O -, - S -, - N (R1) -, - C (R 1) (R 2) -, - C (R 1) = C (R 1 )-, -C (R ₁ ) = N-, -C (= NR ₁ )-, -Si (R ₁ ) (R ₂ )-, -S (= O) _2- , -S (= O)- , -C (= O)-and -C (= S)-; wherein each of R ₁ and R _{2 is} independently H, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl , C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₅ -C ₂₀ aryl, substituted C ₅ -C ₂₀ aryl, heteroaryl, substituted heteroaryl, heterocyclic group, substituted heterocyclic group, C ₅ -C ₇ alicyclic group, substituted C ₅ -C ₇ Alicyclic group, substituted oxy (-O-), halogen, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, amino, substituted amino, CN, sulfonyl (S (= O) ₂ -H), substituted sulfonyl, sulfinyl (S = O) -H) sulfo, substituted sulfo, or substituted sulfinyl; and each substituent is independently halogen , C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, amino , Substituted amino, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl group, an amino C ₁ -C ₁₂ alkoxy, substituted C ₁ -C ₁₂ aminoalkyl, acyl, Substituted acyl, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ -C ₁₂ aminoalkoxy or protecting group.

In some embodiments, the bicyclic sugar moiety is bridged between 2 'and 4' carbon atoms by a diyl group selected from: -O-CH _2- , -O- (CH ₂ ) p-, -O-CH ( _{alkyl) -, - O-CH 2-} CH 2 -, - N ( _{alkyl) - (CH 2) p -} , - NH- (CH 2) p -, - (CH ( alkyl)) - (CH _{2) p -, - O-} CH -alkyl) -, - N _{(alkyl) -O- (CH 2) p -} , - NH-O- (CH 2) p- or -ON (alkyl) - ( CH ₂ ) p-, where p is 1, 2, 3, 4 or 5, and each alkyl group may be further substituted. In certain embodiments, p is 1, 2 or 3. In certain embodiments, the bicyclic sugar moiety is -O- (CH _2), also known as "locked nucleic acid" or "LNA '.

In certain embodiments, the 2'-modified nucleoside comprises a 2'-substituent group selected _{from: F, N3, NH 2,} OCF 3, O (CH 2) 3 NH 2, O-CH 3, CH 2 -CH = CH ₂ , O-CH ₂ -CH = CH ₂ , O (CH ₂ ) ₂ SCH ₃ , OCH ₂ CH ₂ OCH ₃ , -O (CH ₂ ) ₂ O (CH ₂ ) ₂ N (CH ₃ ) _2. O- (CH ₂ ) ₂ -ON (R _m ) (R _n ) and N-substituted acetamide (O-CH ₂ -C (= O) -N (R _m ) (R _n ), each of which R _m and R _{n are} independently H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ alkyl group.

In certain embodiments, the 2'-modified nucleoside comprises a 2'-substituent group selected from: halo group, an amino group, an allyl group, an amino _{stack, CN, SH, CF 3,} OCN, O-, OCF 3 , S- or N (R _m ) -alkyl; S-, O-, or N (R _m ) -alkenyl; S-, O-, or N (R _m ) -alkynyl; alkynyl, O- Alkenyl-O-alkyl, aralkyl, alkaryl, O-aralkyl, O-alkaryl, O (CH ₂ ) ₂ SCH ₃ , O- (CH ₂ ) ₂ -ON (R _m ) (R _n ) or O-CH ₂ -C (= O) -N (R _m ) (R _n ), where each R _m and R _{n is} independently H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl. These 2 'substituents may be further substituted with one or more substituents independently selected from the group consisting of amino, hydroxyl, carboxyl, alkoxy, benzyl, nitro (NO ₂ ), phenyl, and thioalkoxy (S-alkyl), mercapto, alkyl, halogen, alkenyl, aryl, and alkynyl.

In certain embodiments, the 2'-modified nucleoside comprises a 2'-substituent group selected from: F, OCH _3, and OCH ₂ CH ₂ OCH _3.

In certain embodiments, the 2'-modified nucleoside comprises a 2'-substituent selected from the group consisting of: F, O-CH ₃ , OCF ₃ , 2'-O (CH ₂ ) ₂ SCH ₃ , OCH ₂ CH ₂ OCH ₃ , O- (CH ₂ ) ₂ -ON (CH ₃ ) ₂ , -O (CH ₂ ) ₂ O (CH ₂ ) ₂ N (CH ₃ ) ₂ and O-CH ₂ -C (= O) N ( H) CH ₃ .

In certain embodiments, the sugar-modified nucleoside is a 4'-thio-modified nucleoside. In certain embodiments, the sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. 4'-thio-modified nucleosides have β-D-ribonucleosides in which 4'-O is replaced with 4'-S. A 4'-thio-2'-modified nucleoside is a 4'-thio-modified nucleoside in which 2'-OH is replaced with a 2'-substituent. Suitable substituents include _{2'-2'-OCH 3, 2'-O-} (CH 2) 2-OCH 3 , and 2'-F.

In certain embodiments, a modified oligonucleotide comprises one or more internucleoside modifications. In certain such embodiments, each internucleoside linkage of the oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage comprises a phosphorus atom.

In certain embodiments, the modified internucleoside linkage does not include a phosphorus atom. In certain such embodiments, the internucleoside linkage is formed by a cycloalkyl internucleoside linkage. In certain such embodiments, the internucleoside linkage is formed by a short-chain alkyl internucleoside linkage. In certain such embodiments, internucleoside linkages are formed by mixing heteroatoms and cycloalkyl internucleoside linkages. In certain such embodiments, the internucleoside linkage is formed by mixing a heteroatom and an alkyl internucleoside linkage. In certain such embodiments, the internucleoside linkage is formed by one or more heterocyclic internucleoside linkages. In certain such embodiments, the internucleoside linkage is formed by one or more short-chain heteroatom internucleoside linkages. In certain such embodiments, the internucleoside linkage has an amide backbone. In certain such embodiments, an internucleoside linkage part ₂ N, O, S and CH with mixed.

In certain embodiments, the modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, each cytosine of a modified oligonucleotide comprises 5-methylcytosine. In certain embodiments, the modified oligonucleotide comprises one or more 5-methylcytosines.

In certain embodiments, the modified nucleoside base is selected from the group consisting of 7-deaza adenine, 2-aminopyridine 7-deazaguanosine, and 2-pyridone. In certain embodiments, the modified nucleobase is selected from the group consisting of 7-deazaguanine, 5-hydroxymethylcytosine, and 7-deaza adenine. In certain embodiments, the modified nucleobase is selected from the group consisting of 5-substituted pyrimidine, 6-azapyrimidine and N-2, N-6 and O-6 substituted purines, including 2-aminopropyl adenine, 5-propynyluracil and 5propynylcytosine.

In certain embodiments, the modified nucleobase comprises a tricyclic heterocycle. In certain embodiments, the modified nucleobase comprises a polycyclic heterocyclic ring. In certain embodiments, the modified nucleobase comprises a phenoxazine derivative. In certain embodiments, phenoxazines may be further modified to form nucleobases known in the art as G-clamps.

Motif

Motifs for modified oligonucleotides suitable for the present invention include, but are not limited to, complete modifications, uniform modifications, localized modifications, and gapmers. Modified oligonucleotides with fully modified motifs including uniformly modified motifs can be designed to target mature miRNAs. Alternatively, modified oligonucleotides with fully modified motifs including uniformly modified motifs can be designed to target certain sites of pri-miRNAs or precursor miRNAs to prevent processing of miRNA precursors into mature miRNA. Modified oligonucleotides with fully modified or uniformly modified motifs are effective inhibitors of miRNA activity.

In certain embodiments, a fully modified oligonucleotide is modified at each internucleoside linkage. In certain such embodiments, each internucleoside linkage in a fully modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a fully modified oligonucleotide comprises a sugar modification at each nucleoside. In certain embodiments, each of the plurality of nucleosides is a 2 ' -O-methoxyethyl nucleoside, and each of the plurality of nucleosides is a bicyclic nucleoside. In certain such embodiments, the majority of the nucleosides are 2'-O-methoxyethyl nucleosides and the remaining nucleosides are 2'-fluoronucleosides. In certain such embodiments, each of the internucleoside linkages in a fully sugar-modified oligonucleotide is a modified internucleoside linkage. In certain such embodiments, the fully modified oligonucleotide further comprises at least one modified internucleoside linkage. In certain such embodiments, each of the internucleoside linkages in a fully sugar-modified oligonucleotide is a phosphorothioate internucleoside linkage. In certain embodiments, the fully sugar-modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage.

In certain embodiments, uniformly modified oligonucleosides have the same internucleoside linkage modification throughout. In certain such embodiments, each internucleoside linkage in the homogeneously modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a uniformly modified oligonucleotide comprises the same sugar modification at each nucleoside. In certain embodiments, each nucleoside in the modified oligonucleotide comprises a 2 ' -O-methyl sugar modification. In certain such embodiments, each nucleoside in the modified oligonucleotide comprises a 2 ' -O-methoxyethyl sugar modification. In certain such embodiments, the uniformly modified oligonucleotide further comprises at least one modified internucleoside linkage. In certain embodiments, each nucleoside in the modified oligonucleotide comprises a 2 ' fluorosaccharide modification. In certain embodiments, the uniform sugar-modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain such embodiments, each internucleoside linkage in a uniform sugar-modified oligonucleotide is a modified internucleoside linkage. In certain such embodiments, each internucleoside linkage in a uniform sugar-modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a locally modified oligonucleotide comprises several regions linked to a nucleoside, wherein each nucleoside of each region comprises the same sugar moiety, and wherein each nucleoside of each region comprises a Adjacent regions have different sugar moieties.

In certain embodiments, a locally modified oligonucleotide comprises at least 10 2 ' -fluoro modified nucleosides. Such a locally modified oligonucleotide can be represented by the following formula I:

5'-T _1- (Nu ₁ L ₁ ) n _1- (Nu ₂ L ₂ ) n ₂ -Nu ₂ (L ₃ -Nu ₃ ) n ₃ -T ₂ -3 ', where:

At least 10 Nu ₂ are 2'-fluoronucleosides;

Each of L ₁ , L ₂ and L _{3 is} independently an internucleoside linkage;

Each Nu ₁ and Nu _{3 are} independently stabilized nucleosides;

Each T ₁ and T _{2 is} independently H, a hydroxyl protecting group, an optionally attached conjugate group or a capping group;

n ₁ is 0 to about 3;

n ₂ is about 14 to about 22;

n ₁ is 0 to about 3, provided that if n ₁ is 0, T _{1 is} not H or a hydroxy protecting group, and if n ₃ is 0, T _{2 is} not H or a hydroxy protecting group.

In certain embodiments, the localization modification motif is represented by Formula II below, which represents a modified oligonucleotide consisting of a linked nucleoside:

T _1- (Nu ₁ ) n _1- (Nu ₂ ) n _2- (Nu ₃ ) n _3- (Nu ₄ ) n _4- (Nu ₅ ) n ₅ -T ₂ , where:

Nu ₁ and Nu _{5 are} independently 2 'stabilized nucleosides;

Nu ₂ and Nu ₄ are 2'-fluoronucleosides;

Nu ₃ is a 2'-modified nucleoside;

each of n ₁ and n ₅ is independently 0 to 3;

the sum of n ₂ and n ₄ is between 10 and 25;

n ₃ is 0 to 5; and

Each T ₁ and T _{2 is} independently H, a hydroxy protecting group, an optionally attached conjugate group or a capping group.

In certain embodiments, Nu ₁ is O- (CH ₂ ) ₂ -OCH ₃ , Nu ₃ is O- (CH ₂ ) ₂ -OCH ₃ , Nu ₅ is O- (CH ₂ ) ₂ -OCH ₃ , T ₁ is H and T ₂ is H.

In certain embodiments, the modified oligonucleotide that is complementary to the miRNA and consists of 16 linked nucleosides has formula II selected from Table 2, wherein each internucleoside linkage is a phosphorothioate nucleoside Between bonding. In certain embodiments, a modified oligonucleotide having Formula II selected from Table 2 has a nucleobase sequence of SEQ ID NO: 5.

Table 2

In certain embodiments, the modified oligonucleotide that is complementary to the miRNA and consists of 15 linked nucleosides has formula II selected from Table 3, wherein each internucleoside linkage is a phosphorothioate nucleoside Between bonding. In certain embodiments, a modified oligonucleotide having Formula II selected from Table 3 comprises 15 linked nucleosides of SEQ ID NO: 6.

table 3

n ₁ n ₂ n ₃ n ₄ n ₅ Nu ₁ Nu ₂ Nu ₅ T ₁ T ₂ 2 11 0 0 2 2’-MOE 2’-MOE 2’-MOE H H 2 2 2 7 2 2’-MOE 2’-MOE 2’-MOE H H 2 3 2 6 2 2’-MOE 2’-MOE 2’-MOE H H 2 4 2 5 2 2’-MOE 2’-MOE 2’-MOE H H 2 5 2 4 2 2’-MOE 2’-MOE 2’-MOE H H 2 6 2 3 2 2’-MOE 2’-MOE 2’-MOE H H 2 7 2 2 2 2’-MOE 2’-MOE 2’-MOE H H 2 2 3 6 2 2’-MOE 2’-MOE 2’-MOE H H 2 3 3 5 2 2’-MOE 2’-MOE 2’-MOE H H 2 4 3 4 2 2’-MOE 2’-MOE 2’-MOE H H 2 5 3 3 2 2’-MOE 2’-MOE 2’-MOE H H 2 6 3 2 2 2’-MOE 2’-MOE 2’-MOE H H

In certain embodiments, the compound is represented by Formula III:

(5 ') QxQz ¹ (Qy) _n Qz ² Qz ³ Qz ⁴ QL (3')

In certain embodiments, Q is a 2'-O-methyl modified nucleoside. In certain embodiments, x is a phosphorothioate. In certain embodiments, y is a phosphodiester. In certain ^{embodiments, z 1, z 2, z} 3 and z each independently a phosphorothioate or phosphodiester ^4. In certain embodiments, n is 6 to 17. In certain embodiments, L is cholesterol. In certain embodiments, n is 12 to 17.

A和B中的一个是S，而另一个是O；y是

z ¹、z ²、z ³和z ⁴中的每一个独立地为x或y；n＝6-17，L是

其中：x为N(CO)R ⁷或NR ⁷，R ¹、R ³和R ⁹中的每一个独立地为H、OH或－CH ₂OR ^b时，条件是R ¹、R ³和R ⁹中的至少一个为OH并且R ¹、R ³和R ⁹中的至少一个为－CH ₂0R ^b； In certain embodiments, x is

One of A and B is S and the other is O; y is

z ^1, z ^2, z ³ and z each independently x or y is ^4; n = 6-17, L is

Where: x is N (CO) R ⁷ or NR ⁷ , and each of R ¹ , R ^3, and R ⁹ is independently H, OH, or -CH ₂ OR ^b , provided that R ¹ , R ^3, and R ⁹ At least one of OH is OH and at least one of R ¹ , R ³ and R ⁹ is -CH ₂ 0R ^b ;

R ⁷ is R ^d or C ₁ -C ₂₀ alkyl substituted with NR ^c R ^d or NHC (O) R ^d :

R ^c is H or C ₁ -C ₆ alkyl:

R ^d is a carbohydrate group or a steroid group, which is optionally attached to at least one carbohydrate group: and

其中A和B中的一个是S，而另一个是O。 R ^b is

One of A and B is S and the other is O.

其中A和B中的一个是S，而另一个是O。在某些实施方案中，R ¹为－CH ₂OR ^b。在某些实施方案中，R ⁹为OH。在某些实施方案中，R ¹和R ⁹为反式。在某些实施方案中，R ¹和R ³为反式。在某些实施方案中，R ³为－CH ₂OR ^b。在某些实施方案中，R ¹为OH。在某些实施方案中，R ³和R ⁹为反式。在某些实施方案中，R ⁹为CH ₂OR ^b。在某些实施方案中，R ¹为OH。在某些实施方案中，X为NC(O)R ⁷。在某些实施方案中，R ⁷为－CH ₂(CH ₂) ₃CH ₂NHC(O)R ^d。 In certain embodiments, ^Rd is cholesterol. In certain ^{embodiments, z 1, z 2, z} 3 and z ⁴ each is a

One of A and B is S and the other is O. In certain embodiments, R ¹ is -CH ₂ OR ^b . In certain embodiments, R ⁹ is OH. In certain embodiments, R ¹ and R ⁹ are trans. In certain embodiments, R ¹ and R ³ are trans. In certain embodiments, R ³ is -CH ₂ OR ^b . In certain embodiments, R ¹ is OH. In certain embodiments, R ³ and R ⁹ are trans. In certain embodiments, R ⁹ is CH ₂ OR ^b . In certain embodiments, R ¹ is OH. In certain embodiments, X is NC (O) R ⁷ . In certain embodiments, R ⁷ is -CH ₂ (CH ₂ ) ₃ CH ₂ NHC (O) R ^d .

In certain embodiments, a modified oligonucleotide having a positioning modification motif comprises LNA. In certain embodiments, the modified oligonucleotide has a motif selected from one of the following motifs, where L = LNA nucleoside, d = DNA nucleoside, M = 2′-MOE nucleoside, and F = 2'-fluoronucleoside. In certain embodiments, the nucleosides in brackets are optionally included in the modified oligonucleotide, in other words, the motifs cover different lengths depending on the number of days of the nucleosides in the brackets included Modified oligonucleotide.

LdLdLLLddLLLdLL

LdLddLLddLdLdLL

LMLMLLLMMLLLMLL

LMLMMLLMMLMLMLL

LFLFLLLFFLLLFLL

LFLFFLLFFLFLFLL

dLddLddLdd (L) (d) (d) (L) (d) (d) (L)

LddLddLddL (d) (d) (L) (d) (d) (L) (d)

ddLddLddLd (d) (L) (d) (d) (L) (d) (d)

MLMMLMMLMM (L) (M) (M) (L) (M) (M) (L)

LMMLMMLMML (M) (M) (L) (M) (M) (L) (M)

MMLMMLMMLM (M) (L) (M) (M) (L) (M) (M)

FLFFLFFLFF (L) (F) (F) (L) (F) (F) (L)

LFFLFFLFFL (F) (F) (L) (F) (F) (L) (F)

FFLFFLFFLF (F) (L) (F) (F) (L) (F) (F)

LdLdLdLdL (d) (L) (d) (L) (d) (L) (d) (L)

dLdLdLdLdL (d) (L) (d) (L) (d) (L) (d)

LMLMLMLML (M) (L) (M) (L) (M) (L) (M) (L)

MLMLMLMLML (M) (L) (M) (L) (M) (L) (M)

LFLFLFLFL (F) (L) (F) (L) (F) (L) (F) (L)

FLFLFLFLFL (F) (L) (F) (L) (F) (L) (F)

A modified oligonucleotide having a gapmer motif may have an internal region composed of linked 2'-deoxynucleotides and an external region composed of linked 2'-modified nucleosides. This gap body can be designed to cause RNase H cleavage of the miRNA precursor. The internal 2 ' -deoxynucleoside region serves as a substrate for RNase H, allowing cleavage of the miRNA precursor targeted by the modified oligonucleotide. In certain embodiments, each nucleoside of each outer region comprises the same 2'-modified nucleoside. In certain embodiments, one outer region is uniformly composed of a first 2'-modified nucleoside and the other outer region is uniformly composed of a second 2'-modified nucleoside.

A modified oligonucleotide having a gapmer motif may have a sugar modification at each nucleoside. In certain such embodiments, the inner region is uniformly composed of 2'-fluoronucleosides, and each outer region is uniformly composed of 2'-O-methoxyethylnucleoside. In certain embodiments, the inner region is uniformly composed of the first 2'-modified nucleoside, and each outer region is uniformly composed of the second 2'-modified nucleoside.

In certain embodiments, each outer region of the gapmer is composed of 2 ' -O-methyl nucleoside. In certain embodiments, each outer region of the gapmer is composed of 2 ' -O-methoxyethyl nucleoside. In certain embodiments, each outer region of the gapmer consists of a linked bicyclic nucleoside. In certain embodiments, each outer region of the gapmer is composed of 2 ' -fluoronucleoside.

In certain such embodiments, each nucleoside of one outer region of the gapmer contains 2'-O-methoxyethyl nucleoside, and each nucleoside of another outer region contains 2'-O- Methyl nucleoside. In certain embodiments, each nucleoside of one outer region of the gapmer contains 2'-O-methoxyethyl nucleoside, and each nucleoside of another outer region contains a different 2'-modification. In certain such embodiments, each nucleoside of one outer region of the gapmer contains 2'-O-methyl nucleoside, and each nucleoside of the other outer region contains 2'-fluoronucleoside. In certain such embodiments, each nucleoside of one outer region of the gapmer contains 2'-O-methoxyethyl nucleoside, and each nucleoside of another outer region contains 2'-fluoronucleus Glucoside. In certain such embodiments, each nucleoside of one outer region of the interstitial body comprises a 2 ' -O-methyl nucleoside, and each nucleoside of another outer region comprises a bicyclic nucleoside. In certain such embodiments, each nucleoside of one outer region of the gapmer contains 2'-O-methoxyethyl nucleoside, and each nucleoside of the other outer region contains a bicyclic nucleoside.

In certain embodiments, a nucleoside of an external region comprises two or more sugar modifications. In certain embodiments, the nucleoside of each outer region comprises two or more sugar modifications. In certain embodiments, at least one nucleoside of the outer region comprises a 2 ' -O-methoxyethyl sugar, and at least one nucleoside of the same outer region comprises a bicyclic sugar moiety. In certain embodiments, at least one nucleoside of the external region comprises a 2 ' -O-methyl sugar, and at least one nucleoside of the same external region comprises a bicyclic sugar moiety. In certain embodiments, at least one nucleoside of the external region comprises 2'-O-methoxyethyl sugar, and at least one nucleoside of the same external region comprises 2'-fluorosaccharide. In certain embodiments, at least one nucleoside of an external region comprises a 2 ' -fluorosaccharide, and at least one nucleoside of the same external region comprises a bicyclic sugar moiety. In certain embodiments, at least one nucleoside of the external region comprises a 2'-O-methyl sugar, and at least one nucleoside of the same external region comprises a 2'-fluorosaccharide.

In certain embodiments, each outer region of the gapmer consists of the same number of linked nucleosides. In certain embodiments, one outer region of the gapmer consists of a different number of linked nucleosides than another outer region.

In certain embodiments, the external region independently comprises 1 to 6 nucleosides. In certain embodiments, the outer region comprises 6 nucleosides. In certain embodiments, the outer region comprises 5 nucleosides. In certain embodiments, the outer region comprises 4 nucleosides. In certain embodiments, the external region comprises 3 nucleosides. In certain embodiments, the outer region comprises 2 nucleosides. In certain embodiments, the outer region comprises 1 nucleoside. In certain embodiments, the internal region consists of 17 to 28 linked nucleosides. In certain embodiments, the internal region consists of 17 to 21 linked nucleosides. In certain embodiments, the internal region consists of 28 linked nucleosides. In certain embodiments, the internal region consists of 27 linked nucleosides. In certain embodiments, the internal region consists of 26 linked nucleosides. In certain embodiments, the internal region consists of 25 linked nucleosides. In certain embodiments, the internal region consists of 24 linked nucleosides. In certain embodiments, the internal region consists of 23 linked nucleosides. In certain embodiments, the internal region consists of 22 linked nucleosides. In certain embodiments, the internal region consists of 21 linked nucleosides. In some embodiments, the internal region consists of 20 linked nucleosides. In certain embodiments, the internal region consists of 19 linked nucleosides. In certain embodiments, the internal region consists of 18 linked nucleosides. In certain embodiments, the internal region consists of 17 linked nucleosides.

Some other therapy

The treatment of steatohepatitis, liver fibrosis, and hepatocellular carcinoma may involve more than one therapy. Accordingly, in certain embodiments, the present invention provides a method for treating steatohepatitis, liver fibrosis, and hepatocellular liver cancer, which comprises administering to a subject in need thereof a compound comprising miR-221 / 222 and / or Compounds of precursor complementary oligonucleotides, and also include administration of at least one additional agent.

In certain embodiments, the additional therapy is an anti-obesity agent. In certain embodiments, the anti-obesity agent is orlistat, inbryomin, or rimonabant.

In certain embodiments, the additional therapy is a therapeutic lifestyle change. In certain embodiments, changes in the therapeutic lifestyle include exercise regimen and / or diet.

In certain embodiments, the additional agent is a hypolipidemic agent. In certain embodiments, the hypolipidemic agent is a drug that affects lipid synthesis, metabolism, and clearance (nicotinic acid and its derivatives, clobetin and phenoxyacetic acids, methylolglutarate coenzyme A (HMG-CoA) reductase inhibitors), drugs that affect cholesterol and bile acid absorption (cholic acid integrator, Probucol), and polyene fatty acid drugs. In certain embodiments, the hypolipidemic agent is a PPAR agonist (a gamma agonist, a dual agonist, or a pan-agonist), a dipeptidyl peptidase (IV) inhibitor, a GLP-I analog, insulin, or an insulin analog , Insulin secretagogue, SGLT2 inhibitor, human amylin analog, biguanide, alpha-glucosidase inhibitor, meglitinide, thiazolidinedione or sulfonylurea.

In certain embodiments, the dose of the additional agent is the same as the dose that would be administered when the additional agent was administered alone. In certain embodiments, the dose of the additional agent is higher than the dose that would be administered when the additional agent was administered alone. In certain embodiments, the dose of the additional agent is lower than the dose that would be administered when the additional agent was administered alone. Other examples of additional agents include, but are not limited to, hypoglycemic agents (such as biguanides, sulfonylureas and non-sulfonylureas, alpha-glucosidase inhibitors, thiazolidinedione derivatives, DPP-4 enzymes Inhibitors, etc.); Analgesics (such as ethaminophen); Immunomodulators; Epinephrine modulators; Anti-inflammatory drugs, non-steroidal anti-inflammatory drugs (such as ibuprofen, co × 1 inhibitors and co × 2 inhibitors Salicylates; antibiotics; antivirals; antifungals; diuretics; hormones (such as anabolic steroids, androgens, androgens, calcitonin, progesterone, somatostatin, thyroid hormone); muscle relaxation Agents; antihistamines; osteoporosis agents (eg, bisphosphonates, calcitonin and estrogen); prostaglandins, antitumor drugs; psychotherapeutics; sedatives; poison sumac products; antibodies; and vaccines.

Certain pharmaceutical composition

The invention provides a pharmaceutical composition comprising an oligonucleotide. In certain embodiments, such pharmaceutical compositions are used to treat steatohepatitis, liver fibrosis, and hepatocellular carcinoma and related conditions. In certain embodiments, the pharmaceutical composition provided by the present invention comprises a compound comprising 12 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-221 / 222, or a precursor thereof Of oligonucleotides. In certain embodiments, the pharmaceutical composition provided by the present invention comprises a compound consisting of an oligonucleotide consisting of 12 to 30 linked nucleosides and having Somatic complementary nucleobase sequence.

Suitable routes of administration include, but are not limited to, oral, topical, suppositories, by inhalation, intrathecally, intraventricularly, intraperitoneally, intratumorally, and parenterally (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, the drug is administered intrathecally to achieve local rather than systemic exposure. For example, the pharmaceutical composition can be injected directly into the area of interest (eg, into the liver).

In certain embodiments, the pharmaceutical composition is administered in the form of a dosage unit (eg, a tablet, capsule, bolus, etc.). In certain embodiments, such pharmaceutical compositions comprise an oligonucleotide selected from the group consisting of 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg , 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg , 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 270mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg , 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg , 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg, 505mg, 510mg, 515mg, 520mg, 525mg, 530mg, 535mg, 540mg, 545mg, 550mg, 555mg, 560mg, 565mg, 570mg, 575mg, 580mg, 585mg, 590mg , 595mg, 600mg 605mg, 610mg, 615mg, 620mg, 625mg, 630mg, 635mg, 640mg, 645mg, 650mg, 655mg, 660mg, 665mg, 670mg, 675mg, 680mg, 685mg, 690mg, 695mg, 700mg, 705mg, 710mg, 715mg, 720mg, 725mg , 730mg, 735mg, 740mg, 745mg, 750mg, 755mg, 760mg, 765mg, 770mg, 775mg, 780mg, 785mg, 790mg, 795mg and 800mg. In certain such embodiments, the pharmaceutical composition comprises a modified oligonucleotide dose selected from: 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800mg.

In certain embodiments, the agent is a sterile lyophilized modified oligonucleotide that is reconstituted with a suitable diluent, such as sterile saline for injection. The reconstituted product is administered as a subcutaneous injection or intravenous infusion after dilution with saline. Lyophilized pharmaceutical products consist of oligonucleotides. During the preparation process, the oligonucleotides are prepared in saline for injection adjusted to pH 7.0-9.0 with acid or base, and then lyophilized. The lyophilized modified oligonucleotide may be an oligonucleotide of 25-800 mg. It should be understood that this includes 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600 , 625, 650, 675, 700, 725, 750, 775, and 800 mg of modified lyophilized oligonucleotides. The lyophilized drug product can be packaged in a 2mL type I clear glass vial (treated with ammonium sulfate), stoppered with a bromobutyl rubber stopper, and sealed with an aluminum FLIP-OFF cap.

In certain embodiments, the pharmaceutical composition provided by the present invention may additionally contain other auxiliary ingredients normally present in a pharmaceutical composition, which have a usage amount determined in the art. For example, the composition may include additional compatible pharmaceutically active materials, such as a local anesthetic or anti-inflammatory agent, or additional materials such as dyes, flavoring agents, which may be used to physically formulate various dosage forms of the composition of the present invention, Preservatives, antioxidants, sunscreens, thickeners and stabilizers. However, such materials should not interfere with the biological activity of the components of the composition of the invention when added. Formulations can be sterilized and, if required, mixed with adjuvants that do not interact with the formulated oligonucleotides in a harmful manner, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers , Salts, buffers, colorants, flavoring agents and / or aromatic substances that affect osmotic pressure.

The lipid portion has been used in nucleic acid therapy in a variety of ways. In the first method, the nucleic acid is introduced into a pre-made liposome or a lipoplex made of a mixture of cationic lipids and neutral lipids. In another method, a DNA complex with a mono- or polycationic lipid is formed in the absence of a neutral lipid. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent in a particular cell or tissue.

In certain embodiments, Intralipid injection (INTRALIPID) is used to prepare a pharmaceutical composition comprising an oligonucleotide. Intralipide injection is a fat emulsion prepared for intravenous administration. It consists of 10% soybean oil, 1.2% egg yolk phospholipid, 2.25% glycerol, and water for injection. In addition, sodium hydroxide was added to adjust the pH so that the pH of the final product ranged from 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided by the invention comprises one or more modified oligonucleotides and one or more excipients. In certain such embodiments, the excipient is selected from the group consisting of water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl fiber And polyvinylpyrrolidone.

In certain embodiments, the pharmaceutical compositions provided by the present invention are liquids (eg, suspensions, agents, and / or solutions). In certain such embodiments, the liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, ethylene glycol, oils, alcohols, flavoring agents, preservatives, and colorants.

In certain embodiments, the pharmaceutical compositions provided by the present invention are prepared using known techniques including, but not limited to, mixing, dissolving, granulating, dragee making, grinding, emulsifying, encapsulating, embedding or Tablet processing.

In certain embodiments, the pharmaceutical compositions provided by the present invention are solids (eg, powders, tablets, and / or capsules). In certain such embodiments, a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingredients known in the art including, but not limited to, starch, sugar, diluent, Granules, lubricants, binders and disintegrants.

In certain embodiments, the pharmaceutical compositions provided by the invention are prepared as a depot formulation. Certain such storage formulations are generally more prolonged than non-depot formulations. In certain embodiments, such formulations are administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, the depot formulation is prepared using a suitable polymer or hydrophobic material (e.g., an emulsion in an acceptable oil) or an ion exchange resin, or as a poorly soluble derivative, e.g., a poorly soluble salt.

In certain embodiments, a pharmaceutical composition provided by the invention comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems can be used to make certain pharmaceutical compositions, including those containing hydrophobic compounds. In certain embodiments, certain organic solvents are used, such as dimethylsulfoxide.

In certain embodiments, a pharmaceutical composition provided by the invention comprises one or more tissue-specific delivery molecules designed to deliver one or more agents of the invention to a particular tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises a liposome coated with a tissue-specific antibody.

In certain embodiments, the pharmaceutical compositions provided by the present invention comprise a sustained release system. A non-limiting example of such a sustained release system is a semi-permeable matrix of a solid hydrophobic polymer. In certain embodiments, depending on its chemical nature, a sustained release system can release an agent over hours, days, weeks, or months.

In certain embodiments, the pharmaceutical compositions provided herein are prepared for oral administration. In certain such embodiments, a pharmaceutical composition is formulated by combining one or more oligonucleotide-containing compounds with one or more pharmaceutically acceptable carriers. Certain such carriers are capable of formulating pharmaceutical compositions into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral administration by a subject. In certain embodiments, a pharmaceutical composition for oral administration is obtained by mixing an oligonucleotide with one or more solid excipients. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol: cellulose formulations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, xanthan, Methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidone (PVP). In certain embodiments, such a mixture is optionally ground, and an adjuvant is optionally added. In certain embodiments, a pharmaceutical composition is formed to obtain a tablet core or a dragee core. In certain embodiments, a disintegrant is added (eg, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate).

In certain embodiments, dragee cores have a coating. In certain such embodiments, concentrated sugar solutions may be used, which may contain gum arabic, talc, polyvinylpyrrolidone, carbomer gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable Organic solvents or solvent mixtures. Dyes or pigments can be added to tablets or dragee coatings.

In certain embodiments, the pharmaceutical composition for oral administration is a push-fit capsule made of gelatin. Some such push-fit capsules contain one mixed with one or more fillers (such as lactose), a binder (such as starch) and / or a lubricant (such as talc or stearic acid mold) and a stabilizer Or more agents of the invention. In certain embodiments, the oral pharmaceutical composition is a sealed soft capsule made of gelatin and a plasticizer, such as glycerol or sorbitol. In certain soft capsules, one or more agents of the invention are dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer may be added.

In certain embodiments, the pharmaceutical composition is prepared for oral administration. Certain such pharmaceutical compositions are tablets or lozenges formulated in a conventional manner.

In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution such as water or a physiologically compatible buffer such as a Hanks solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (eg, ingredients that aid dissolution or act as preservatives). In certain embodiments, suitable liquid carriers, suspensions, etc. are used to prepare suspensions for injection. Certain pharmaceutical compositions for injection are presented in unit dosage forms, such as in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Certain solvents in pharmaceutical compositions suitable for injection include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, and liposomes Aqueous injection suspensions may contain materials that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the agents to prepare Highly concentrated solution.

In certain embodiments, the pharmaceutical composition is prepared for transmucosal administration. In certain such embodiments, penetrants suitable for the barrier to be penetrated may be used in the formulation. Such penetrants are generally known in the art.

In certain embodiments, the pharmaceutical composition is prepared for administration by inhalation. Certain such pharmaceutical compositions for inhalation are prepared in the form of aerosol sprays in pressurized packages or nebulizers. Some such pharmaceutical compositions include a propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. In certain embodiments using a pressurized aerosol, the dosage unit may be determined by a valve that delivers a metered number. In certain embodiments, capsules and cartridges can be formulated for use in an inhaler or insufflator. Certain such formulations comprise a powder mixture of the agent of the invention and a suitable powder base such as lactose or starch.

In certain embodiments, a pharmaceutical composition provided by the invention comprises a therapeutically effective amount of an oligonucleotide. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or alleviate the symptoms of the disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides provided herein are formulated as a prodrug. In certain embodiments, the prodrug is chemically converted to a biologically, pharmaceutically or therapeutically more active form of the oligonucleotide after in vivo administration. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in some cases, the prodrug may be more bioavailable (e.g., by oral administration) than the corresponding active form. In some cases, the prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, the prodrug is less water soluble than the corresponding active form. In some cases, such prodrugs have superior transcellular membrane transport capabilities where water solubility is not conducive to movement. In certain embodiments, the prodrug is an ester. In certain such embodiments, the ester is metabolized to carboxylic acid upon administration. In some cases, the carboxylic acid-containing compound is the corresponding active form. In some embodiments, the prodrug comprises an acid-binding short peptide (polyamino acid). In certain such embodiments, the peptide is cleaved after administration to form the corresponding active form.

Certain kits

The invention also provides a kit. In some embodiments, the kit comprises detecting the expression level of the target miR-221 / 222, the level of mRNA and / or protein targeted by the miRNA, and the antisense inhibitory effect of the miRNA after the application of the modified oligonucleotide; An oligonucleotide of one or more compounds of the present invention, wherein the detection target kit comprises a detection miR-221 / 222 specific primer, a targeted mRNA primer, a targeted protein antibody, and a detection reagent. Can be used to detect cDNA libraries and proteins in serum or tissue samples. Antisense inhibition of miRNA is assessed by measuring the mRNA and / or protein levels of the miRNA target. The nucleobase sequence of the oligonucleotide is complementary to miR-221 / 222. The compound complementary to miR-221 / 222 may be any compound described in the present invention, and may have any modification described in the present invention. In some embodiments, a compound complementary to miR-221 / 222 may be present in a vial. Multiple (e.g., 10) vials may appear in, for example, a dispensing pack. In some embodiments, the vial is manufactured for easy syringe access. The kit may also include instructions for using a compound whose detection target is complementary to miR-221 / 222.

In some embodiments, the kit can be used to administer a compound complementary to miR-221 / 222 to a subject. In this case, in addition to the compounds complementary to miR-221 / 222, the kit may further include one or more of the following: a syringe, an alcohol swab, a cotton ball, and / or a gauze pad. In some embodiments, compounds that are complementary to miR-221 / 222 may be present in a pre-filled syringe (such as a single-dose syringe with, for example, a 27-inch 1 / 2-inch needle and needle guard), rather than in a vial in. Multiple (e.g., 10) pre-filled syringes may appear in, for example, a dispensing pack. The kit may also include instructions for detecting a compound whose target is complementary to the administration of miR-221 / 222.

Some experimental models

In certain embodiments, the invention provides methods for using and / or testing a modified oligonucleotide of the invention in an experimental model. Those skilled in the art will be able to select and modify experimental protocols for such experimental models to evaluate the agents of the invention.

Generally, modified oligonucleotides are first tested in cultured cells. Suitable cell types include those related to the cell type to which the oligonucleotide needs to be delivered in vivo. For example, cell types suitable for use in the research method of the present invention include primary hepatocytes and HepG2 cells.

In certain embodiments, the extent to which the oligonucleotide interferes with miRNA activity is assessed in cultured cells. In certain embodiments, inhibition of miRNA activity can be assessed by measuring miRNA levels. Alternatively, the level of a predicted or validated miRNA target can be measured. Inhibition of miRNA activity can result in increased mRNA and / or protein of the miRNA target. Moreover, in certain embodiments, certain phenotypic results can be measured. For example, appropriate phenotypic results include lipid and collagen deposition, tumor incidence, and the like.

Experimental animal models suitable for the test method of the present invention include: MCD diet mice (model for steatohepatitis), CCL4 mice (model for steatohepatitis), and high-fat-feeding C57BL6 / J small mouse.

Specific embodiment

The following examples are provided to more fully illustrate some embodiments of the invention. However, these examples should in no way be construed as limiting the broad scope of the invention. Throughout the examples, unless otherwise indicated, statistical significance is as follows: * = P <0.05; ** = P <0.01; *** = P <0.001.

Example 1: Expression of microRNA in liver tissue

In order to identify microRNAs involved in liver lipid metabolism, inflammatory infiltration and fibrosis, liver tissues were screened for microRNA expression. Through analysis to identify dysregulated microRNAs in the liver of MCD diet mice, CCl4-treated mice, and obese C57Bl6 / J mice induced by a high fat diet, all three mice are animal models of steatohepatitis. A pair of conserved and widely expressed microRNAs, miR-221 and miR-222 (see Figure 1), were found to be upregulated in the liver of these models. q-PCR results showed that miR-221 and miR-222 were up-regulated 2-3 times in the liver of MCD diet mice, and miR-221 and miR-222 were up-regulated in the liver of obese mice induced by high fat diet 1- 2-fold (see Table 4: miR-221 and miR-222 are up-regulated in the liver of MCD diet mice and high-fat diet-induced obese mice).

Table 4

Zh Control diet MCD diet Control diet High-fat diet Relative miR-221 expression 1 2.14 1 1.75 Relative miR-222 expression 1 2.83 1 1.6

MicroRNA expression was analyzed in liver biopsies from healthy individuals, human patients with non-alcoholic fatty liver disease (nAFLD), and non-alcoholic fatty liver disease (nASH). Increased levels of miR-221 / 222 and miR-221 / 222 in liver samples from subjects with nAFLD and nASH (see Table 5: miR-221 and miR-222 expression in liver samples from human subjects )

table 5

Zh Subject miR-221 relative expression value miR-222 relative expression value Contrast 5 1 1 nAFLD 6 2.33 2.65 nASH 5 3.94 4.28

Example 2: Knockout miR-221 and miR-222 relieve liver fat accumulation, inflammatory infiltration and collagen deposition, fibrosis formation in animals

MCD diet mice are often used as a model of steatohepatitis. Therefore, an MCD diet model was established in control mice and miR-221 / 222 knockout mice to evaluate the effect of miR-221 / 222 on steatohepatitis. Evaluation of the knockout effect of miR-221 / 222 in the liver of KO mice (see Table 6: miR-221 and miR-222 expression levels in the liver of control mice and miR-221 / 222-LKO mice).

Table 6

Zh Control mice miR-221 / 222-LKO mice miR-221 relative expression value 1 0.15 miR-222 relative expression value 1 0.11

Knockout of miR-221 and miR-222 reduced liver fat deposition in MCD diet mice (Figure 3). MiR-221 / 222-LKO mice were given the MCD diet for 6 weeks. Statistics of oil-red staining and oil-red area in liver slices showed that knockout of miR-221 and miR-222 significantly reduced liver lipid droplets caused by the MCD diet (Table). MiR-221 / 222-LKO mice were given the MCD diet for 6 weeks, and the lipid droplet area measured by transmission electron microscopy of liver sections showed that miR-221 / 222 knockdown significantly reduced the amount of hepatocyte lipid droplets caused by the MCD diet (Table 7: miR-221 / 222 knockout mice and control mice in an MCD diet model for lipid droplet analysis).

Table 7

Zh Control mice miR-221 / 222-LKO mice Relative amount of oil red O stained lipid droplets 1 0.26 Relative amount of lipid droplets in transmission electron microscope 1 0.21

Knockout of miR-221 and miR-222 reduced inflammatory infiltration of the liver in MCD diet mice (Figure 4). MiR-221 / 222-LKO mice were given MCD diet for 6 weeks. The expression levels of inflammatory cytokines were detected in liver tissues. It was found that the expression levels of IL-1β, TNFα and IL-6 were significantly reduced (Table 8: miR-221 / 222 knockout was small The expression levels of liver inflammatory factors IL-1β, TNFα and IL-6 in the MCD diet model of mice and control mice; the expression levels of markerF4 / 80 in inflammatory cells were significantly reduced (Table 9: miR-221 / 222 knockout mice and Control mice had markerF4 / 80 expression levels of hepatic inflammatory cells in the MCD diet model); H & E staining of liver sections showed a marked reduction in inflammatory cell infiltration (Figure 4).

Table 8

Zh Control mice miR-221 / 222-LKO mice IL-1β relative expression 1 0.46 Relative expression of TNFα 1 0.53 IL-6 relative expression 1 0.37

Table 9

Zh Control mice miR-221 / 222-LKO mice F4 / 80 relative expression 1 0.26 F4 / 80 relative protein level 1 0.31

Knockout miR-221 and miR-222 reduced collagen fiber deposition in the liver of MCD diet mice (Figure 5). MiR-221 / 222-LKO mice were given the MCD diet for 6 weeks. Sirius red staining and masson staining of liver sections were used to calculate the collagen fiber staining area. It was found that knocking out miR-221 and miR-222 can significantly reduce liver collagen caused by the MCD diet. Fibrous deposition (Table 10: miR-221 / 222 knockout mice and control mice in the MCD diet model liver Sirius Red and masson staining indicate collagen fiber positive area analysis).

MiR-221 / 222-LKO mice were given MCD diet for 6 weeks. The content of hydroxyproline, the main component of collagen tissue, was measured in liver tissues. It was found that knocking out miR-221 and miR-222 can significantly reduce the liver caused by MCD diet. Amount of hydroxyproline (Table 11: Analysis of hydroxyproline content of liver tissue in MCD diet model of miR-221 / 222 knockout mice and control mice).

MiR-221 / 222-LKO mice were given MCD diet for 6 weeks. The expression levels of collagen family members in liver tissue were detected in liver tissues. It was found that the expression levels of Col1a1, Col1a2 and Col3a1 were significantly reduced (Table 12: miR-221 / 222 knockout small Expression levels of liver collagen family Col1a1, Col1a2, and Col3a1 in the MCD diet model of mice and control mice; the expression levels of marker α-sma in stellate cells decreased significantly (Table 13: miR-221 / 222 knockout mice and controls were small The expression level of marker α-sma in liver stellate cells in mice in the MCD diet model); transmission electron microscopy of liver sections showed that liver cells saw a significant decrease in collagen fiber deposition (Figure 5).

Table 10

Zh Control mice miR-221 / 222-LKO mice Sirius red staining positive area (%) 1.43 0.4 masson staining positive area (%) 1.45 0.48

Table 11

Table 12

Zh Control mice miR-221 / 222-LKO mice Col1a1 relative expression 1 0.37 Col1a2 relative expression 1 0.31 Col3a1 relative expression 1 0.42

Table 13

Zh Control mice miR-221 / 222-LKO mice α-sma relative expression 1 0.34 α-sma relative protein levels 1 0.38

Knockout of miR-221 and miR-222 reduced liver damage and reduced transaminase levels in MCD diet mice. MiR-221 / 222-LKO mice were given MCD diet for 6 weeks, and the serum alanine aminotransferase levels in the mice were determined. miR-221 and miR-222 reduce liver damage caused by the MCD diet and reduce alanine aminotransferase levels (Table 14: serum alanine aminotransferase levels in miR-221 / 222 knockout mice and control mice in the MCD diet model) .

Table 14

Zh Control mice miR-221 / 222-LKO mice Serum ALT level 73.2 59.5

Example 3: MiR-221 / 222 overexpression worsens steatohepatitis caused by MCD diet, aggravates liver fat accumulation, inflammatory infiltration and collagen deposition, fibrosis formation

Adenovirus AD-miR-221 / 222 infected the liver of miR-221 / 222LKO mice to re-express miR-221 / 222. Adenovirus AD-miR-221 / 222 over-expressing miR-221 / 222 and control adenovirus AD-GFP (titer 1 * 10 ¹¹ ) were injected into tail vein (200 μl / head) of miR- 221 / 222LKO mice were fed the MCD diet for 6 weeks. After 6 weeks, the expression of miR-221 / 222 in liver tissues of mice was detected. It was found that AD-miR-221 / 222 can increase the expression of miR-221 / 222 in the liver of knockout mice (Table 15: miR-221 / 222LKO is small Rats were injected with AD-miR-221 / 222 and AD-GFP in the tail vein, and the expression of miR-221 / 222 in liver tissues was fed after 6-week MCD diet.

Table 15

Adenovirus AD-miR-221 / 222 infected liver of miR-221 / 222LKO mice and increased liver lipid deposition. After miR-221 / 222LKO mice re-expressed miR-221 / 222 and induced a steatohepatitis model using the MCD diet, the liver weight ratio and liver triglyceride of the mice were higher than those of control mice (Table 1 6: miR-221 / 222LKO mice were injected with AD-miR-221 / 222 and AD-GFP in the tail vein, and the liver weight ratio after 6 weeks of feeding on the MCD diet, Table 17: miR-221 / 222LKO mice were injected with AD-miR-221 / 222 in the tail vein And AD-GFP, liver triglyceride and cholesterol levels after a 6-week MCD diet).

Table 16

Table 17

Adenovirus AD-miR-221 / 222 infected the liver of miR-221 / 222LKO mice to increase liver inflammation infiltration; miR-221 / 222LKO mice re-expressed miR-221 / 222 and induced fatty liver model using the MCD diet.

The expression levels of inflammatory cytokines and markerF4 / 80 in mouse liver tissues were higher than those in control mice (Table 18: miR-221 / 222LKO mice were injected with AD-miR-221 / 222 and AD-GFP in the tail vein and fed for 6 weeks After the MCD diet,

Expression levels of inflammatory factors IL-1β, TNFα and IL-6 in liver tissues and markerF4 / 80 in inflammatory cells).

Table 18

Adenovirus AD-miR-221 / 222 infected liver of miR-221 / 222LKO mice and increased liver fibrosis. After miR-221 / 222LKO mice re-expressed miR-221 / 222 and induced a steatohepatitis model using the MCD diet, the area of collagen deposition in the liver of the mice increased (Figure 7, Table 19: tail vein injection of miR-221 / 222LKO mice AD-miR-221 / 222 and AD-GFP, after 6 weeks of feeding on the MCD diet, collagen deposition area of the liver tissue (Sirius red staining and masson staining analysis).

Table 19

Adenovirus AD-miR-221 / 222 infected liver of miR-221 / 222LKO mice and increased liver fibrosis factor. After miR-221 / 222LKO mice re-expressed miR-221 / 222 and used the MCD diet to induce a model of steatohepatitis, the expression levels of collagen family and marker α-sma in stellate cells of liver tissues of mice were higher than those of control mice ( Table 20: MiR-221 / 222LKO mice were injected with AD-miR-221 / 222 and AD-GFP in the tail vein. After 6 weeks of feeding on the MCD diet, the liver tissue collagen family and stellate cell markerα-sma expression levels).

Table 20

Example 4: antimiR-221 / 222 can achieve a higher effect of miR-221 / 222 inhibition in vitro

AntimiR-221 / 222 (LNA-i-miR-221, LNA-i-miR-222) was synthesized by using Locked nucleic acids (LNA ^™ ) modification technology. NC, LNA-i-miR-221 and LNA-i-miR-222 were transfected into mouse liver cancer cell line hepa1-6 at two concentrations of 50nM and 100nM. Cells were collected 48 hours later, and miR-221 / 222 expression level (Figure 8, Table 21: NC, LNA-i-miR-221 and LNA-i-miR-222 (50nM and 100nM) were transfected in hepa1-6 cells, and miR-221 was effectively inhibited after 48h / 222 expression level).

Table 21

Zh NC LNA-i-miR-221 LNA-i-miR-222 miR-221 relative expression value (50nM) 1 0.48 0.95 miR-222 relative expression value (50nM) 1 0.75 0.45 miR-221 relative expression value (100nM) 1 0.31 0.78 miR-222 relative expression value (100nM) 1 0.82 0.49

antimiR-221 / 222 can achieve a higher effect of up-regulating miR-221 / 222 target genes in vitro. antimiR-221 / 222 (100nM) was transfected into mouse liver cancer cell line hepa1-6, and cells were collected 48 hours later to detect the protein levels of miR-221 / 222's recognized target genes p27 and Timp3 (Figure 8, Table 22: Hepa1-6 cells were transfected with NC, LNA-i-miR-221 and LNA-i-miR-222 (100 nM), and after 48 hours, the protein levels of the target genes p27 and Timp3 of miR-221 / 222 were effectively increased).

Table 22

Zh NC LNA-i-miR-221 LNA-i-miR-222 Relative protein level of P27 1 3.13 2.85 Timp3 relative protein levels 1 2.15 2.79

Unless otherwise specified, the anti-miR used was modified as follows: anti-miR-221 has the sequence of SEQ ID NO: 5 with a 2'-O-methyl modification at each sugar, and the first 4 internucleoside linkages (5 'terminus) each has a phosphorothioate modification, each of the last 2 internucleoside linkages (3' terminus) has a phosphorothioate modification, and has a hydroxyproline bond Cholesterol connected to the 3 'end.

Anti-miR-222 has the sequence of SEQ ID NO: 6, 2'-O-methyl modification at each sugar, and thio group at each of the first 4 internucleoside linkages (5 'terminus) Phosphate modification, each of the last 2 internucleoside linkages (3 'terminus) has a phosphorothioate modification, and has cholesterol attached to the 3' terminus via a hydroxyprolinol bond.

The control anti-miR-Ctrl has the nucleobase sequence ACGTCTATACGCCCA (SEQ ID NO: 7), has a 2'-O-methyl modification at each sugar, and has each of the first 4 internucleoside linkages. Phosphorothioate modification, each of the last 2 internucleoside linkages has a phosphorothioate modification, and has cholesterol attached to the 3 'terminus via a hydroxyprolinol bond. Because miR-221 and miR-222 differ in nucleotides, anti-miR-Ctrl is mismatched relative to both miR-221 and miR-222.

Unless otherwise specified, experimental mice were 8-week-old control male mice and 8-week-old miR-221 / 222LKO male mice; and MCD diet mice were male mice that consumed the MCD diet for 6 weeks from 8 weeks of age. Anti-miR-221 (5 × 25mg / kg), anti-miR-222 (5 × 25mg / kg), anti-miR-221 + 222 (5 × 12.5 + 12.5mg / kg) or anti-miR-ctrl (5 × 25mg / kg).

Control mice received five 25 mg / kg anti-miR-221 or anti-miR-222 or five 12.5 mg / kg anti-miR-221 + 222 intraperitoneal injections. Anti-miR-ctrl was administered as a control treatment. Analysis of miR-221 and miR-222 RNA expression analysis showed that anti-miR-221 / 222 silenced miR-221 / 222 in the liver without affecting the expression of unrelated microRNAs. See Figure 9, Table 22.

After treatment, mice were tested for ALT levels and anti-miR-221 / 222 treatment did not cause significant toxicity (in mice treated with anti-miR-221, anti-miR-222, anti-miR-221 + 222, or anti-miR-ctrl (~ 20IU / L, ~ 21IU / L, ~ 19IU / L, ~ 20IU / L).

Example 5: antimiR-221 / 222 can effectively inhibit miR-221 / 222 expression levels in the liver of MCD diet mice

Control mice were randomly divided into 4 groups, and each group was given an intraperitoneal injection of 25 mg / kg on days 1, 4, 8, 15, and 22 respectively. LNA-i-miR-NC, LNA-i-miR-221, LNA-i- miR-222 or 12.5 mg / kg LNA-i-miR-221 and 12.5 mg / kg LNA-i-miR-222 (LNA-i-miR-221 + 222), and the MCD diet was started on the first day. One week after the last administration of anti-miR, mice were harvested, and the expression levels of miR-221 / 222 in mouse livers were analyzed (Table 23: anti-miR-221 / 222 intraperitoneally injected with MCD diet mice, miR-221 / 222 expression level).

Table 23

antimiR-221 / 222 can effectively inhibit lipid deposition and inflammatory infiltration in the liver of MCD diet mice (Figure 10). MCD diet mice were given antimiR-221 / 222. One week after the last injection of antimiR, the mice were harvested and analyzed for serum and liver lipid levels, lipid deposition in the liver, and inflammatory infiltration levels (Table 24: antimiR- (221/222 After intraperitoneal injection of MCD diet mice, TG levels, expression levels of inflammatory factors IL-1β, TNFα and IL-6, and markerF4 / 80 expression levels in inflammatory cells).

Table 24

antimiR-221 / 222 can effectively inhibit collagen deposition in livers of mice with MCD diet (Table 25: Analysis of Sirius red staining and masson staining area of mouse liver tissues after intraperitoneal injection of MCD diet mice with antimiR-221 / 222). MCD diet mice were given antimiR-221 / 222. One week after the last injection of antimiR, mice were harvested and analyzed for collagen deposition area (Sirius red staining and masson staining) in the liver tissue of the mice. It was found that antimiR-221 / 222 was effective Reduces collagen deposition in the liver of MCD model mice.

Table 25

antimiR-221 / 222 can effectively inhibit the expression of collagen family members in the liver of MCD diet mice and inhibit the activation of stellate cells. MCD diet mice are given antimiR-221 / 222. One week after the last injection of antimiR, mice are harvested and analyzed Expression levels of collagen family members in mouse liver tissues (Table 26: Marker analysis of collagen family members and stellate cells in mouse liver tissues after intraperitoneal injection of MCD diet mice in mice with antimiR-221 / 222), antimiR-221 / 222 was found It can effectively inhibit the expression of liver collagen family members and the expression of markerα-sma in stellate cells in MCD model mice.

Table 26

Example 6: miR-221 / 222 targets timp3 in hepatocytes

Through the prediction of miRNA target genes (such as Target Scan) and bioinformatics analysis, TIMP3, which regulates steatohepatitis, was initially identified as a candidate target gene. TIMP3 is a major regulator of TACE (TNF-α converting enzyme) activity and is an important regulator of inflammation, fibrosis, non-alcoholic fatty liver and liver cancer. TIMP3-deficient mice develop increased TNF-α activity and liver inflammation caused by adverse reactions to liver injury. Hepatocyte-specific overexpression of TIMP3 can prevent NAFLD and tumorigenesis by regulating ADAM17 activity. Macrophage-specific overexpression of TIMP3 protects mice against insulin resistance, NASH, and metabolic inflammation.

miR-221 / 222 directly regulates the transcription level of timp3 and 3'UTR in hepa1-6 cells. Transfect transfected miR-221 / 222 and timp3 3'UTR luciferase reporter plasmid or timp3 3'UTR seed sequence mutant luciferase reporter plasmid in hepa1-6 cells, and use the dual luciferase gene reporter system to detect miR -221/222 Regulation of timp3 and 3'UTR. It was found that miR-221 / 222 can significantly inhibit timp3 3'UTR wild-type luciferase activity, partially inhibit 1 tim3 3'UTR seed sequence mutants, and lose tim3 3'UTR 2 seed sequence mutants (Table 27) ).

Table 27

Relative ratio of luciferase reporting system NC miR-221 / 222 Timp3-WT 1 0.81 Timp3-mut1 1 0.90 Timp3-mut2 1 0.92 Timp3-mut1 + 2 1 1.08

Timp3 is upregulated in the liver of miR-221 / 222LKO mice on the MCD diet. The 8-week control and miR-221 / 222LKO mice were given a 6-week MCD diet, and the expression level of Timp3 in the liver was analyzed. It was found that the expression level of timp3 in the liver of miR-221 / 222LKO mice was increased (Table 28: 6-week MCD diet control MRNA and protein levels of Timp3 in liver of miR-221 / 222LKO mice)

Table 28

Zh Control mice miR-221 / 222-LKO mice Timp3 relative expression 1 2.45 Timp3 relative protein level 1 1.54

After miR-221 / 222LKO mice re-adenovirus expressed miR-221 / 222, the expression of Timp3 in liver decreased. The miR-221 / 222LKO mice were injected with adenovirus AD-miR-221 / 222 or control AD-GFP in the tail vein, and were given the MCD diet. After 6 weeks, the expression level of Timp3 in the liver was analyzed, and it was found that the expression level of timp3 in the liver of mice injected with the adenovirus AD-miR-221 / 222 decreased (Table 29)

Table 29

Transfection of LNA-antimiRs in hepa1-6 cells resulted in upregulation of timp3 expression. antimiR-221 / 222 (100nM) transfected mouse hepatocellular carcinoma cell line hepa1-6, cells were collected 48 hours later, and Timp3 levels of miR-221 / 222 were detected in the cells (Table 30: NC, LNA transfected in hepa1-6 cells -i-miR-221 and LNA-i-miR-222 (100nM), which can effectively up-regulate the mRNA and protein levels of the miR-221 / 222 target gene Timp3 after 48h).

Table 30

antimiR-221 / 222 can effectively increase the expression of timp3 in the liver of MCD diet mice (Table 30). Control mice were randomly divided into 4 groups, and each group was given an intraperitoneal injection of 25 mg / kg on days 1, 4, 8, 15, and 22 respectively. -222 or 12.5 mg / kg LNA-i-miR-221 and 12.5 mg / kg LNA-i-miR-222 (LNA-i-miR-221 + 222), and the MCD diet was started on the first day. One week after the last administration of antimiR, the mice were harvested and analyzed for the expression level of timp3 in the liver of the mice (Table 31: the expression level of timp3 in mouse liver after intraperitoneal injection of antimiR-221 / 222 to MCD diet mice).

Table 31

Example 7: Experimental method

Statistical analysis All bar graphs show mean ± STD. Significance was calculated using t-test (* p <0.05; ** p <0.01; *** p <0.001). Throughout the examples, unless otherwise indicated, statistical significance is indicated in the table: * p <0.05; ** p <0.01; *** p <0.001.

The RNA was extracted using Trizol reagent method (Invitrogen), and total RNA was extracted from mouse liver or hepa1-6 cells according to the instructions of miRNeasy MiniKit (Qiagen).

Reverse transcription into cDNA: The operation was performed according to the Promega instructions, and the reverse primer sequence was U6: random primer, miR-221: 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACG AAACCC-3 ’,

miR-222: 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACCCAGT-3 '.

Real-time PCR: According to the reverse transcription primer sequence, design the upstream and downstream primers of the Real-time PCR reaction. Through quantitative real-time PCR, use LightCycler 480SYBR Green Master I Mix (Roche) to measure mRNA expression. Transcription levels were normalized relative to U6, GAPDH or 36B4.

Detection of luciferase activity: The mouse timp3 and 3'UTR sequences were amplified by PCR with specific primers and inserted into the PRL-NULL empty vector with appropriate restriction enzymes. Hepa1-6 cells were cultured in a 24-well plate, and each well was transfected with 10 ng prl-null-3-utr (non-coding region), 100 ng PGL3-Control vector plasmid. Cells were harvested 24 hours after transfection and luciferase activity was measured. These values were normalized using the dual luciferase reporting analysis system according to the manufacturer's procedures.

All mouse models were bred in a SPF environment, maintained a 12h light / dark cycle, and had free access to water and food. MiR-221 / 222 ^{flox /} flox mice mate with hepatocyte-specific Alb-cre mice, and the obtained heterozygous mice miR-221 / 222 ^{flox / +} -Cre mate to produce hepatocyte-specific gene knockout mice miR -221/222 flox ^/ flox-Cre, miR-221 / 222-LKO and littermate control mice. Establish two mouse models of liver fibrosis, miR-221 / 222-LKO and control mice fed methionine choline deficiency diet (MCDD) or control diet for 6 weeks to obtain a diet model of steatohepatitis, the second model, MiR-221 / 222-LKO and control mice were injected intraperitoneally with CCl4 or a blank control for 6 weeks (0.5 ml / kg, twice a week). All mice used in the experiments were male. All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals published by the National Institutes of Health.

Adenovirus infection. Ad5CMVK-NpA vector was used to construct and package the adenovirus over-expressing miR-221 / 222. Adenovirus expressing green fluorescent protein was used as a control. MiR-221 / 222-LKO mice were given adenovirus 1 × 10 ¹¹ (plaque-forming units / 0.2 ml PBS) via tail vein. Adenovirus injection did not affect appetite in mice. Mice were sacrificed on day 5 after adenovirus injection.

LNA-antimiRs injection in vivo: AntimiRs LNA-i-miR-221 (sequence: CAGCAGACAATGTAGC) is an oligonucleotide of 16 DNA / LNA bases, and LNA-i-miR-222 (sequence: AGTAGCCAGATGTAGC) is 15 DNA / The LNA base mismatch oligonucleotide, LNA-i-miR-NC (sequence: ACGTCTATACGCCCA) was used as a control. All of these oligonucleotides have thio-modified backbones, Na ⁺ salt exchanged, lyophilized after HPLC purification. Control mice were randomly divided into 4 groups, and each group was given an intraperitoneal injection of 25 mg / kg LNA-i-miR-NC, LNA-i-miR-221, and LNA-i-miR on days 1, 4, 8, 15, and 22, respectively. -222 or 12.5 mg / kg LNA-i-miR-221 and 12.5 mg / kg LNA-i-miR-222 (LNA-i-miR-221 + 222), while the MCD diet was started on day 1. One week after the last administration of antimiR, mice were harvested.

Analysis of liver histology and immunohistochemical staining. Liver tissue was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Liver sections, hematoxylin-eosin staining, and horse pine staining solution (saturated picric acid Sirius Red contains 0.1% (w / v) Direct Red 80). The Sirius red positive area or Masson positive area was quantitatively analyzed by digital images. Results are expressed as the percentage of Sirius Red or Masson's positive area. For lipid droplet accumulation testing, liver sections were stained using Oil Red O (Sigma) according to standard procedures. Immunohistochemical staining was performed according to standard procedures. These images were acquired with an Olympus microscope system.

Biochemical analysis. Serum and liver total cholesterol (TC) and triglycerides (TG) were measured using a commercially available kit (BioVision) according to the manufacturer's instructions. Serum alanine aminotransferase (ALT) commercially available kit (BioVision) was measured according to the manufacturer's instructions. Blood glucose measurement uses a blood glucose meter to measure tail vein blood.

Determination of hydroxyproline content. The colorimetric determination of collagen-specific amino acid hydroxyproline was performed using a hydroxyproline assay kit (sigma). The hydroxyproline content is expressed as ng hydroxyproline per mg of liver.

Ultrastructure analysis. Analysis by transmission electron microscopy (TEM), fresh liver samples (1 cubic millimeter volume) were fixed to 2.5% glutaraldehyde and formaldehyde. Subsequently, the specimens were fixed with 2% osmium tetroxide for 1 h. After fixation, the tissue was dehydrated through a series of gradient alcohols and propylene oxide, and epoxy resin 812 was embedded to obtain ultra-thin sections. Stained with methylene blue in sodium borate, uranyl acetate and lead citrate, and photographed with an OPTONEM900 transmission electron microscope (Zeiss).

Cell culture, infection, and transfection: Hepa1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and placed in a 37 ° C, 5% CO2 humidified air incubator. . Cells were transfected with liposome 2000.

The foregoing description of specific embodiments so comprehensively demonstrates the general nature of the invention that others can easily modify and / or adapt such specific embodiments for use by applying current knowledge without undue experimentation and without departing from the general concepts. Various applications, therefore, such adaptations and modifications should and are intended to be included within the meaning and scope of equivalents of the disclosed embodiments. Although the invention has been described in connection with specific embodiments thereof, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. It is therefore intended to embrace all such alternative modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims (30)

miR-221 has a nucleoside base sequence as shown in SEQ ID NO: 1. miR-222 has a nucleoside base sequence as shown in SEQ ID NO: 2. The primers for detecting the expression level of miR-221 had nucleobase sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9. The primers for detecting the expression level of miR-222 have nucleobase sequences as shown in SEQ ID NO: 10 and SEQ ID NO: 11. The primer for detecting the expression level of miR-221 according to claim 3 is used to prepare a reagent for detecting the expression level of a target of miR-221. The primer for detecting the expression level of miR-222 according to claim 4 is used to prepare a reagent for detecting the expression level of miR-222 target. The miR-221 and its inhibitor according to claim 1 are used for preparing a medicament for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma. The use according to claim 7, characterized in that the miR-221 and its inhibitor are used as detection targets for preparing drugs for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma. The miR-222 and its inhibitor according to claim 2 are used for preparing a medicament for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma. The use according to claim 9, characterized in that the miR-222 and its inhibitor are used as a detection target to prepare a medicament for regulating liver fat deposition, liver fibrosis and hepatocellular carcinoma. The precursor of miR-221 has a nucleobase sequence as shown in SEQ ID NO: 3. The precursor of miR-222 has a nucleoside base sequence as shown in SEQ ID NO: 4. A compound comprising a modified oligonucleotide; wherein, The modified oligonucleotide is composed of 15 to 25 linked nucleosides and targets miR-221 and / or miR-222, and the base sequence of the nucleoside is the same as SEQ ID NO: 1, SEQ ID NO : 2, SEQ ID NO: 3 or SEQ ID NO: 4 nucleobase sequences are complementary. The compound according to claim 13, characterized in that the modified oligonucleotide in the compound comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 linked nucleosides Any one of them constitutes and targets miR-221 and / or miR-222, and the base sequence of the nucleoside is the same as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: The nucleotide sequence of 4 is complementary. The compound according to claim 13, wherein the oligonucleotide comprises a nucleoside base sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. The compound according to claim 13, wherein the modified oligonucleotide further comprises at least one modified sugar. The compound according to claim 16, wherein each of said modified sugars is independently selected from 2'-O-methoxyethyl sugar, 2'-fluoro sugar, 2'-O-methyl Sugars and bicyclic sugar moieties. The compound according to claim 13, wherein the modified oligonucleotide further comprises at least one modified internucleoside linkage. The compound according to claim 18, wherein each of the modified internucleoside linkages is a phosphorothioate internucleoside linkage or a phosphorothioate internucleoside linkage. Use of a compound according to any one of claims 13-19 in the manufacture of a medicament for the following uses: (i) reducing the level of fatty infiltration in the subject's liver; or (ii) preventing or delaying the appearance of liver collagen fibrous deposition in a subject; or (iii) preventing or delaying the occurrence of hepatocellular carcinoma in a subject. Use according to claim 20, wherein: The subject has at least one metabolic disorder of metabolic syndrome, obesity, diabetic dyslipidemia, hyperlipidemia, hypertriglyceridemia, hyper fatty acidemia, and hyperinsulinemia; and / or liver Cell liver cancer; The subject's metabolic disorders include elevated blood lipid levels, elevated serum transaminase levels, liver B-mild to severe fatty liver, liver fibrosis changes, elevated gluconeogenesis, insulin resistance, and reduced glucose At least one of tolerance and excess body fat. The use according to claim 20 or 21, wherein the medicine is used for: (i) improving liver fatty infiltration in a subject; or (ii) preventing or delaying the appearance of liver collagen fibrous deposition in a subject; or (iii) preventing or delaying the occurrence of hepatocellular carcinoma in a subject. The use according to claim 20, characterized in that, by inhibiting the activity of miR-221 and / or miR-222, liver fat infiltration is reduced, the degree of fibrosis is reduced, malignant proliferation of hepatocytes is prevented, plasma cholesterol level is reduced, Serum aminotransferase and improved insulin resistance are achieved. The use according to claim 20, wherein reducing gluconeogenesis in a subject is achieved by inhibiting the activity of miR-221 and / or miR-222. The use according to claim 20, characterized in that the medicament uses the compound according to any one of claims 13-19 as an active ingredient, and further comprises a pharmaceutically acceptable excipient or auxiliary ingredient. The use according to claim 20, wherein the mode of administration of the drug comprises intravenous, subcutaneous, oral or parenteral administration. The use according to claim 20, wherein the modified oligonucleotide in the medicine is applied at a dose of 25-800 mg / kg. A pharmaceutical composition comprising a compound comprising a modified oligonucleotide according to any one of claims 13-19, and further comprising a pharmaceutically acceptable excipient or auxiliary ingredient. The pharmaceutical composition according to claim 24, wherein the modified oligonucleotide is a sterile lyophilized oligonucleotide, and its application dose is 25-800 mg / kg. miR-221 / 222 detection target kit, comprising the primer for detecting miR-221 expression level according to claim 3 and / or the primer for detecting miR-222 expression level according to claim 4, further comprising A compound comprising a modified oligonucleotide according to any one of claims 13-19.

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