Double-stranded oligonucleotide containing terminal nucleotide modification and application thereof in inhibiting AGT gene expressionTechnical Field
The present disclosure relates to the technical field of nucleic acid pharmaceuticals, and in particular to a double-stranded oligonucleotide containing terminal nucleotide modification and an application thereof in inhibiting AGT gene expression.
Background
Renin (AGT), also known as SERPINA8 or ANHU, is a member of the serpin family and is a component of the renin-angiotensin-aldosterone system (RAAS), which plays a key role in blood pressure regulation. The juxtaglomerular cells of the kidney secrete renin into the circulation. It is produced mainly in the liver and released into the circulation where renin converts it to angiotensin I. Angiotensin I is then converted to angiotensin II by Angiotensin Converting Enzyme (ACE). Angiotensin II is a peptide hormone that causes vasoconstriction, which in turn can increase blood pressure. Angiotensin II also stimulates secretion of the hormone aldosterone in the adrenal cortex. Aldosterone causes the kidneys to increase reabsorption of sodium and water, resulting in an increase in the volume of fluid in the body, which in turn may increase blood pressure. Excessive stimulation or activity of the RAAS pathway can lead to high blood pressure. Chronic hypertension is known as hypertension. The high blood pressure of hypertensive patients requires the heart to make more effort to circulate blood through the blood vessels.
The World Health Organization (WHO) has identified hypertension as a major cause of cardiovascular morbidity. Hypertension is a major risk factor for a variety of diseases, disorders and conditions such as reduced life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysms), peripheral arterial disease, cardiac injury (e.g., enlarged or hypertrophic heart), and other cardiovascular-related diseases, disorders and/or conditions.
Despite the large number of anti-compression drugs available for the treatment of hypertension, more than two-thirds of subjects are not controlled with one anti-compression drug and require two or more anti-compression drugs selected from different drug classes. This further reduces the number of subjects with controlled blood pressure, as compliance and side effects increase with increasing medication. Accordingly, alternative therapies and combination therapies for subjects suffering from angiotensinogen related diseases remain to be investigated.
In view of this, the present disclosure is specifically proposed.
Disclosure of Invention
The present disclosure provides a double-stranded oligonucleotide that can induce silencing complex (RISC) -mediated cleavage of an RNA transcript of an Angiotensinogen (AGT) gene, inhibit expression of the AGT gene, and can be used to treat or prevent a disease or disorder mediated by the AGT gene. The disclosure also provides conjugates, pharmaceutical compositions, kits comprising the double stranded oligonucleotides and methods and uses thereof for inhibiting or reducing AGT gene expression or treating AGT gene-mediated diseases or conditions.
The technical scheme is as follows:
In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide for inhibiting the expression of an AGT gene, said double-stranded oligonucleotide comprising a sense strand and an antisense strand, said antisense strand comprising or being selected from a nucleotide sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides in any one of the sequences as shown in formula (A), or comprising a nucleotide sequence having a1, 2 or 3 nucleotide difference from said consecutive nucleotides, said sense strand comprising at least 17 nucleotides, and said sense strand being complementary or substantially complementary to said antisense strand to form a duplex region, said substantial complementarity meaning that the mismatch of said sense strand and said antisense strand in said duplex region does not exceed 3 nucleotides;
Formula (A) 5'-X1GUUUCUUCAUCCAGUUGA(X2)m(X3)n -3';
Wherein X1 is selected from A or U;
(X2)m(X3)n represents a 3' -terminal nucleobase, X2、X3 is independently selected from A, U, G, C or T, m and n are independently selected from 0 or 1, and m+n.gtoreq.1.
Further, at least 1 of the nucleotides X2、X3 is a [2'-R1-2'-R2 ] disubstituted modified nucleotide, which refers to a ribosyl 2' -hydroxy group of a nucleotide and a hydrogen-substituted nucleotide, wherein R1 and R2 are each independently selected from halogen (preferably F), optionally substituted C1-C6 alkyl (preferably methyl or ethyl), or optionally substituted C1-C6 alkoxy (preferably methoxy).
In some embodiments of the disclosure, X1 is selected from a. In other embodiments of the present disclosure, X1 is selected from U.
In some alternative embodiments of the present disclosure, (X2)m(X3)n is selected from GG, UU, TT, CC, AA, G, U, T, C or a. In other alternative embodiments of the present disclosure, (X2)m(X3)n is selected from UU or U. In some embodiments of the present disclosure, (X2)m(X3)n is selected from UU. In other embodiments of the present disclosure, (X2)m(X3)n is selected from U).
According to an embodiment of the disclosure, the antisense strand comprises or is selected from any one of the sequences shown in A1) -A3), or a nucleotide sequence having A1 or 2 nucleotide difference from any one of the above sequences:
A1)5'-AGUUUCUUCAUCCAGUUGAUU-3';
A2)5'-AGUUUCUUCAUCCAGUUGAU-3';
A3)5'-UGUUUCUUCAUCCAGUUGAUU-3'。
According to embodiments of the present disclosure, the sense strand comprises or is selected from a nucleotide sequence of at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides of any one of the sequences shown in B1) -B2), or comprises a nucleotide sequence having a1, 2 or 3 nucleotide difference from the consecutive nucleotides;
B1)5'-UCAACUGGAUGAAGAAACU-3';
B2)5'-UCAACUGGAUGAAGAAACA-3'。
in a specific embodiment of the disclosure, the antisense strand comprises or is selected from any one of the sequences set forth in A1) -A3), and the sense strand comprises or is selected from any one of the sequences set forth in B1) -B2).
In some embodiments of the disclosure, the double-stranded oligonucleotide is selected from one or more of the following:
1) The antisense strand has a nucleotide sequence as shown in A1 (SEQ ID NO. 3), and the sense strand has a nucleotide sequence as shown in B1 (SEQ ID NO. 1);
2) The antisense strand has a nucleotide sequence as shown in A2 (SEQ ID NO. 4) and the sense strand has a nucleotide sequence as shown in B1 (SEQ ID NO. 1);
3) The antisense strand has a nucleotide sequence as shown in A3 (SEQ ID NO. 5) and the sense strand has a nucleotide sequence as shown in B2 (SEQ ID NO. 2).
TABLE 1 naked sequence nucleotide information for double stranded oligonucleotides
In some embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from modified or unmodified nucleotides.
In some embodiments of the present disclosure, at least one nucleotide in the sense strand or the antisense strand of the double-stranded oligonucleotide is a modified nucleotide, for example, the modified nucleotide is a ribose group and optionally a phosphate group modified nucleotide group, but is not limited thereto.
According to an embodiment of the disclosure, substantially all of the nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides. Wherein "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most, but not all, of the nucleotides in the double-stranded oligonucleotide are modified nucleotides and may comprise no more than 5, 4, 3, 2 or 1 unmodified nucleotides.
According to an embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, the modification comprising one or both of single-or double-substitution modifications of the ribosyl 2' position of the nucleotide.
Wherein the unmodified nucleotide has the structural formula: Base stands for a nucleobase, and the nucleobase on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.
In alternative embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from the following modified nucleotides:
2 '-fluoro modified nucleotide, 2' -deoxy modified nucleotide, 2 '-O-methyl modified nucleotide, 2' -O- (CH2)x-O-R3) modified nucleotide, 2'-O-Si (R4)3 modified nucleotide, 2' -amino modified nucleotide, abasic nucleotide, nucleotide-like or [2'-F-2' -methyl ] disubstituted modified nucleotide.
The nucleotide-like is selected from one or more of peptide nucleic acid (peptide nucleic acid, PNA), morpholino nucleotide (Morpholino nucleic acid, MNA), bridge nucleic acid (bridged nucleic acid, BNA), locked nucleic acid (locked nucleic acid, LNA), ethylene glycol nucleic acid/glycerol nucleic acid (glycol nucleic acid, GNA), threose nucleic acid (threose nucleic acid, TNA) or unlocked nucleic acid (unlocked nucleic acid, UNA).
Wherein x is selected from 1 or 2, R3 is selected from optionally substituted C1-6 alkyl or optionally substituted C1-6 alkoxy, and if R3 contains a substituent, the substituent is selected from halogen, C1-6 alkoxy, hydroxy or amino.
R4 is independently selected from optionally substituted C1-6 alkyl, if R4 contains substituents selected from halogen, C1-C3 alkyl or C1-C3 alkoxy.
In the present disclosure, a 2'-O- (CH2)x-R3 modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group is replaced with-O- (CH2)x-R3. Wherein, when x is selected from 1, the 2'-O- (CH2)x-R3 modified nucleotide is selected from 2' -O-ethoxymethyl modified nucleotide or 2'-O-2, 2-trifluoroethoxymethyl modified nucleotide when x is selected from 2, the 2' -O- (CH2)x-R3 modified nucleotide is selected from 2 '-O-methoxyethyl modified nucleotide (also called 2' -O-moe modified nucleotide).
In some embodiments of the present disclosure, the 2' -O- (CH2)x-O-R3) -modified nucleotide is selected from a 2' -O-methoxyethyl-modified nucleotide or a 2' -O-ethoxymethyl-modified nucleotide.
In the present disclosure, "2' -O-Si (R4)3 modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with-O-Si (R4)3. Illustratively, a 2' -O-TBDMS modified nucleotide, a 2' -O-TIPS modified nucleotide, or a 2' -O-TOM modified nucleotide, wherein the TBDMS has the formulaThe structural formula of the TIPS isTOM has the structural formula of。
In some embodiments of the present disclosure, the 2'-O-Si (R4)3 modified nucleotide is selected from the group consisting of 2' -O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide, and 2' -O-TOM modified nucleotide).
In some embodiments of the present disclosure, the [2'-F-2' -methyl ] disubstituted modified nucleotide has the structural formulaBase represents nucleobase A, U, G, C or T.
According to an embodiment of the present disclosure, all of the nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, including ribosyl 2' -position single-substitution modification or 2' -position double-substitution modification of the nucleotides, each of the nucleotides in the double-stranded oligonucleotide is independently selected from at least three of a 2' -fluoro modified nucleotide, a 2' -O-methyl modified nucleotide, a 2' -O-methoxyethyl modified nucleotide, and a [2' -F-2' -methyl ] double-substitution modified nucleotide.
In some embodiments of the present disclosure, the antisense strand of the double-stranded oligonucleotide contains at least one 2' -O-methoxyethyl modified nucleotide.
In some embodiments of the disclosure, the nucleotide at position 20 and/or at position 21 of the antisense strand is a [2'-F-2' -methyl ] disubstituted modified nucleotide in the 5 'to 3' end direction.
In some embodiments of the disclosure, the nucleotide at position 19 of the sense strand is a [2'-F-2' -methyl ] disubstituted modified nucleotide in a 5 'to 3' end orientation.
In some embodiments of the present disclosure, at least 3 nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2 '-fluoro modified nucleotides, and the nucleotides at the remaining positions are independently selected from 2' -O-methyl modified nucleotides, in a 5 'to 3' end orientation.
In some embodiments of the present disclosure, at least 3 nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2 '-fluoro modified nucleotides, nucleotide 19 is selected from [2' -F-2 '-methyl ] disubstituted modified nucleotides, and the remaining positions are independently selected from 2' -O-methyl modified nucleotides, in a 5 'end to 3' end orientation.
According to an embodiment of the present disclosure, at least four nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2' -fluoro modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2' -fluoro modified nucleotides, the nucleotides at positions 20 and/or 21 are selected from [2' -F-2' -methyl ] disubstituted modified nucleotides, and the nucleotides at the remaining positions are selected from 2' -O-methyl modified nucleotides, according to the direction from the 5' end to the 3' end.
According to an embodiment of the present disclosure, at least four nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2 '-fluoro modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 are selected from 2' -fluoro modified nucleotides, the nucleotides at positions 8 and/or 15 are selected from 2 '-O-methoxyethyl modified nucleotides, the nucleotides at positions 20 and/or 21 are selected from [2' -F-2 '-methyl ] disubstituted nucleotides, and the nucleotides at the remaining positions are selected from 2' -O-methyl modified nucleotides.
According to an embodiment of the present disclosure, each nucleotide of the double-stranded oligonucleotide is independently selected from a modified nucleotide selected from any one of the following (1) - (2):
1) The nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2 '-fluoro modified nucleotides, the remaining positions are selected from 2' -O-methyl modified nucleotides, and the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2 '-fluoro modified nucleotides, the 20 and/or 21 nucleotides are selected from [2' -F-2 '-methyl ] disubstituted modified nucleotides, and the remaining positions are selected from 2' -O-methyl modified nucleotides, according to the direction from the 5 'end to the 3' end.
2) The nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2' -fluoro modified nucleotides, the remaining positions are selected from 2' -O-methyl modified nucleotides, and the nucleotides at positions 2, 6, 9, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2' -fluoro modified nucleotides, the 15 th nucleotide is selected from 2' -O-methoxyethyl modified nucleotides, the 20 th and/or 21 th nucleotides are selected from [2' -F-2' -methyl ] disubstituted modified nucleotides, and the remaining positions are selected from 2' -O-methyl modified nucleotides, according to the direction from the 5' end to the 3' end.
According to embodiments of the present disclosure, the sense strand and/or the antisense strand independently comprise one or more phosphorothioate linkages.
According to embodiments of the present disclosure, at least one or at least two of the following linkages between nucleotides of the sense strand are phosphorothioate linkages:
A linkage between nucleotide 1 and nucleotide 2 of the 5' end of the sense strand;
a linkage between the 2 nd and 3 rd nucleotides of the 5' end of the sense strand;
a linkage between nucleotide 1 and nucleotide 2 of the 3' end of the sense strand;
A linkage between the 2 nd and 3 rd nucleotides of the 3' end of the sense strand;
And
The linkage between the following nucleotides of the antisense strand is a phosphorothioate linkage:
A linkage between nucleotide 1 and nucleotide 2, and a linkage between nucleotide 2 and nucleotide 3, at the 5' end of the antisense strand;
the 3' end of the antisense strand is linked between nucleotide 1 and nucleotide 2, and between nucleotide 2 and nucleotide 3.
In some embodiments of the disclosure, the internucleoside linkage between nucleotide 10 and nucleotide 11 of the antisense strand is selected from a phosphorothioate linkage in the 5 'to 3' end direction.
According to an embodiment of the present disclosure, the sense strand of the double-stranded oligonucleotide comprises or is selected from any one of the modified nucleotide sequences shown in MB 1) -MB 4), in the 5 'end to 3' end direction:
MB1)UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsUm;
MB2)UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCmsUm;
MB3)UmsCmAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmsCms(NM);
MB4)UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmsAm。
according to an embodiment of the present disclosure, the antisense strand of the double-stranded oligonucleotide comprises or is selected from any one of the modified nucleotide sequences shown in MA 1) -MA 5), in the 5 'end to 3' end direction:
MA1)AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfGmUfUmGmAms(NM)s(NM);
MA2)AmsGfsUmUmUmCfUmUmCfAmsUmCmCmAfG(moe)UfUmGmAms(NM)s(NM);
MA3)AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmsAms(NM);
MA4)AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM);
MA5)UmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG(moe)UfUmGmAms(NM)s(NM)。
In some embodiments of the disclosure, the double stranded oligonucleotides comprise one or more of groups 1) -5) shown in table 2.
TABLE 2 nucleotide information of double-stranded oligonucleotide modified sequences
Wherein C, G, U, A, T represents cytidine-3 '-phosphate, guanosine-3' -phosphate, uridine-3 '-phosphate, adenosine-3' -phosphate, thymidine-3 '-phosphate, respectively, m represents that a nucleotide represented by one capital letter adjacent to the left side of the letter m is a 2' -O-methyl modified nucleotide, f represents that a nucleotide represented by one capital letter adjacent to the left side of the letter f is a2 '-fluorine modified nucleotide, (moe) represents that a nucleotide represented by one capital letter adjacent to the left side of the combination identifier (moe) is a 2' -O-methoxyethyl modified nucleotide, s represents that an internucleoside bond between one nucleotide adjacent to the left side and one nucleotide adjacent to the right side thereof is a phosphorothioate bond;
(NM) a nucleotide in which the hydroxyl group at the 2' -position of ribose and hydrogen are replaced by [2' -F-2' -methyl ] and the structural formula is。
In alternative embodiments, the double-stranded nucleotide is selected from the group consisting of siRNA.
In a second aspect of the present disclosure, the present disclosure provides a conjugate comprising a double-stranded oligonucleotide according to the first aspect of the present disclosure, and one or more ligands capable of binding to a cell receptor conjugated to the double-stranded oligonucleotide.
Wherein the ligand is capable of being covalently or otherwise chemically conjugated to the double stranded oligonucleotide and binding of the ligand to the cellular receptor is capable of facilitating specific targeting of the conjugate to the target cell and endocytosis of the conjugate into the target cell to inhibit translation of AGT mRNA into amino acids and conversion into proteins in the target cell, and is effective in alleviating, preventing and/or treating a disease or condition mediated by the Angiotensinogen (AGT) gene.
In some alternative embodiments of the present disclosure, the ligand is selected from the group consisting of asialoglycoprotein receptor ligands (ASGPR ligands).
In some alternative embodiments of the present disclosure, the ligand comprises at least one N-acetylgalactosamine (N-Acetyl Galactosamine, galNAc).
In some alternative embodiments of the present disclosure, the ligand is selected from the group consisting of structures represented by formula (I), or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof:
I
In formula (I), represents the conjugation site of the ligand to a double-stranded oligonucleotide, j is selected from 1, 2, 3 or 4;
in some alternative embodiments of the present disclosure, each Z is independently selected from hydroxyl or mercapto. In some embodiments of the present disclosure, each Z is hydroxy.
In some embodiments of the disclosure, each p is independently 1 or 2.
In some embodiments of the present disclosure, each p is 1.
In some embodiments of the disclosure, each q is independently 1 or 2.
In some embodiments of the present disclosure, each q is 1.
In some embodiments of the present disclosure, each p is 1 and each q is 1.
Each L is independently selected from C1-C30 alkylene orWherein each RL2a is independently selected from C1-C10 alkylene, each RL2b is independently selected from O, S, NH or-NH-C (O) -, and k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Further, in some embodiments of the present disclosure, the ligand has a structure as shown in formula (II), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:
II
In formula (II), j is selected from 1, 2,3 or 4;L independently selected from -CH2-、-CH2-CH2-、-CH2-CH2-CH2-、-(CH2)4-、-(CH2)5-、-(CH2)6-、-(CH2)7-、-(CH2)8-、-(CH2)9-、-(CH2)10-、-CH2-NH-CO-(CH2)5-、-(CH2)2-NH-CO-(CH2)4-、-(CH2)3-NH-CO-(CH2)3-、-(CH2)4-NH-CO-(CH2)2- or- (CH2)5-NH-CO-CH2 -).
In some alternative embodiments of the present disclosure, each L is independently selected from-CH2-CH2 -or- (CH2)2-NH-CO-(CH2)4 -.
In some embodiments of the disclosure, L is selected from-CH2-CH2 -.
In some embodiments of the present disclosure, L is selected from- (CH2)2-NH-CO-(CH2)4 -).
In some embodiments of the present disclosure, the ligand has a structure represented by formula (III), formula (IV), formula (V), or formula (VI), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof:
、、、。
according to an embodiment of the disclosure, the number of ligands is selected from one, one of the ligands is conjugated to the 3' end of the sense strand of the double stranded oligonucleotide.
According to an embodiment of the disclosure, the number of ligands is selected from two, two of which are conjugated to the 5 'end of the sense strand and the 3' end of the antisense strand of the double stranded oligonucleotide, respectively.
In some embodiments of the disclosure, the conjugates include one or more of R303079, R303082, R303086, R303087, R303088, R303089 shown in table 3.
Table 3 nucleotide information for conjugates
Wherein, (CR 01008 x 2), (CR 01008 x 3) and (CR 01008 x 4) each represent a ligand, meaning that the ligand conjugate is attached to the 5 'end of the sense strand if the ligand is at the 5' end of the sense strand, meaning that the ligand conjugate is attached to the 3 'end of the sense strand if the ligand is at the 3' end of the sense strand, and meaning that the ligand conjugate is attached to the 3 'end of the antisense strand if the ligand is at the 3' end of the antisense strand.
(CR 01008X 2) has the structural formula:;
(CR 01008X 3) has the structural formula:;
(CR 01008X 4) has the structural formula:。
In a third aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any of the following together with pharmaceutically acceptable excipients:
(I) The double-stranded oligonucleotide according to the first aspect of the present disclosure, and/or
(II) conjugates described in the second aspect of the disclosure.
Both the double-stranded oligonucleotide and the conjugate can degrade mRNA of AGT and inhibit expression of AGT. Thus, the pharmaceutical compositions of the present disclosure are effective in preventing and/or treating diseases or conditions mediated by the Angiotensinogen (AGT) gene.
The pharmaceutical compositions of the present disclosure include formulations suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the adjuvant materials to prepare a single dosage form is generally that amount of siRNA that produces a therapeutic effect.
In a fourth aspect of the present disclosure, the present disclosure provides the use of any of the following for the manufacture of a medicament for alleviating, preventing and/or treating an AGT gene-mediated disease or disorder:
(I) The double-stranded oligonucleotide according to the first aspect of the present disclosure, and/or
(II) conjugates of the second aspect of the disclosure, and/or
(III) pharmaceutical compositions according to the third aspect of the present disclosure.
According to embodiments of the present disclosure, the disease or condition includes hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertension risk, hypertension emergency status, isolated systolic and diastolic hypertension, pregnancy-associated hypertension, diabetic hypertension, refractory hypertension, paroxysmal hypertension, renal vascular hypertension, godbla's hypertension, ocular hypertension, glaucoma, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, and ventricular fibrosis.
The effective amount of the double-stranded oligonucleotides, conjugates, or pharmaceutical compositions described in the present disclosure may vary depending on the mode of administration, the severity of the disease to be treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc., the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times a day, e.g., as four times a day, three times a day, twice a day, once a day, or once every other day, or several times a day, may be proportionally reduced by the urgent need for the treatment of the condition.
The administration to the subject may be by any suitable route known in the art, including but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual), preferably intravenous.
In a fifth aspect of the disclosure, the disclosure provides a kit comprising any of the following:
(I) The double-stranded oligonucleotide according to the first aspect of the present disclosure, and/or
(II) conjugates of the second aspect of the disclosure, and/or
(III) pharmaceutical compositions according to the third aspect of the present disclosure.
The double-stranded oligonucleotides, conjugates, and pharmaceutical compositions provided herein can induce silencing complex (RISC) -mediated cleavage of the RNA transcript of the Angiotensinogen (AGT) gene, inhibit expression of the AGT gene, and facilitate treatment or prevention of a disease or condition mediated by the Angiotensinogen (AGT) gene.
Drawings
FIG. 1 shows the change in the level of AGT protein in cynomolgus monkey serum after administration of siRNA conjugate in example 1.
Detailed Description
The technical solutions of the present disclosure will be clearly and completely described below in connection with embodiments, and it is apparent that the described embodiments are some embodiments of the present disclosure, but not all embodiments. Based on the embodiments in this disclosure, all other embodiments that a person of ordinary skill in the art would obtain without making any inventive effort are within the scope of protection of this disclosure.
Interpretation of the terms
In this disclosure, the terms "comprises" and "comprising" are open-ended terms that include what is indicated in the disclosure, but do not exclude other aspects.
In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
In the present disclosure, "single substitution modification at 2 'position" means that the hydroxyl at 2' position of ribose in nucleotide is substituted.
In the present disclosure, "2' -disubstituted modification" means that the hydroxyl group at the 2' -position and the hydrogen at the 2' -position of ribose in a nucleotide are simultaneously substituted. For example [2'-F-2' -methyl ] substituted modified nucleotide having the structural formula。
In the present disclosure, the term "small interfering RNA (SMALL INTERFERING RNA, SIRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts of the RISC pathway by forming silencing complexes (RNA-induced silencing complex, RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting translation of mRNA into amino acids and conversion to proteins. For example, the siRNA can modulate (e.g., inhibit) expression of AGT in the cell.
In the present disclosure, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize under certain conditions to an oligonucleotide of a second sequence and form a double-stranded structure.
In the present disclosure, the terms "nucleotide difference" and "nucleotide base difference" and the term "difference in nucleotide sequence" may be used interchangeably. Refers to a change in the base type of the nucleotide at the same or corresponding position as compared with the original nucleotide sequence. For example, when one nucleotide base in the original nucleotide sequence is A, in the case where the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, or the like, it is considered that there is a difference in nucleotide sequence at that position. Here, in the case where a nucleotide at the same or corresponding position differs from the original nucleotide sequence only in the presence or absence of modification or the type of modification, the difference in nucleotide sequence at that position is not considered.
In the present disclosure, the term "overhang" refers to at least one unpaired nucleotide protruding from a double-stranded oligonucleotide duplex, as well as a nucleotide sequence in the siRNA structure other than the double-stranded region. For example, a nucleotide overhang is present when the 3 'end of one of the sense strand and/or the antisense strand extends beyond the 5' end of the other strand, or when the 5 'end of one of the sense strand and/or the antisense strand extends beyond the 3' end of the other strand. The overhang can comprise at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or consist of nucleotide/nucleoside analogues, including deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may occur at the 5 'end, the 3' end, or both ends of the antisense or sense strand.
In the present disclosure, the term "inhibiting the expression of an AGT gene" includes any level of inhibition of AGT gene, e.g., at least partial inhibition of AGT gene expression, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Wherein the expression of the AGT gene, e.g.mRNA level or protein level of AGT, can be evaluated based on the level of any variable related to the expression of the AGT gene. Inhibition may be assessed by a decrease in the absolute or relative level of one or more of these variables as compared to a control level. The control level may be any type of control level utilized in the art, e.g., a baseline level prior to administration, or a level determined from a similar subject, cell, or sample that has not been treated or treated with a control (e.g., a buffer-only control or an inactive agent control).
In the present disclosure, "conjugate" refers to two or more chemical moieties linked to each other by covalent linkage, and "conjugate" refers to a compound formed by covalent linkage between the respective chemical moieties, and "conjugate molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide by reaction, ultimately forming an oligonucleotide conjugate of the present disclosure.
In the present disclosure, "pharmaceutical composition" may refer to a composition for the treatment of a disease, as well as an in vitro culture experiment of cells. For the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of combining the active ingredient with adjuvants that constitute one or more adjunct ingredients. Generally, the compositions are prepared by uniformly and sufficiently combining the active siRNA with a liquid adjuvant, a finely divided solid adjuvant, or both.
In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used in the present disclosure refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.
In the present disclosure, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the siRNA of the present disclosure, such as any adverse biological effect produced or interactions with any other component of the pharmaceutically acceptable composition that occur in a deleterious manner, their use is also contemplated by the present disclosure.
In addition to any conventional adjuvants, the scope of incompatibility with the siRNA of the present disclosure, e.g., any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, is also contemplated by the present disclosure.
The present disclosure is further illustrated below by specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the present disclosure in any way.
Unless otherwise indicated, the siRNA sequences used in the present disclosure were all assigned to Kunststout Biotechnology Co., ltd, and the PCR primer synthesis used in the present disclosure was all assigned to Biotechnology engineering (Shanghai) Co., ltd.
The detection data of AGT protein at different time points of in vivo activity experiments related to the disclosure are summarized as follows:
;
in the context of the present disclosure, unless otherwise indicated, in vivo activity assay data are as followsThe experimental data were plotted and analyzed using GRAPHPAD PRISM 8.0.0 software.
In the context of the present disclosure, the reagent ratios provided below are calculated as volume ratios (v/v) unless otherwise specified.
Preparation of the Compounds
Unless otherwise indicated, reagents used in the preparation of the compounds of the present disclosure were purchased from Beijing coupling technologies Inc. The information of the main reagents is shown in Table 4.
TABLE 4 Table 4
Wherein CPG represents a controlled pore glass (Controlled Pore Glass) support.
Preparation of compound CR01008 of preparation 1:
The synthetic structure of compound CR01008 of (1-1) is shown below:
the synthetic route for compound CR01008 is shown below:
。
(1-1-1) Synthesis of Compound 2:
Compound 1 (trans-4- (Boc-amino) cyclohexylformaldehyde, 10.0g,1.0 eq) and an aqueous formaldehyde solution (8.9 g,37 mass%, 2.4 eq) were dissolved in 33ml of methanol, 13ml of an aqueous KOH solution having a concentration of 45.3 mass% was added dropwise, and after the addition, the reaction was stirred at 25℃for 30 minutes, warmed to 60℃and refluxed at 60℃for 2 hours. After the reaction is finished, the reaction solution is decompressed and evaporated to dryness after being cooled to room temperature, and a crude product in a white solid state is obtained. To the crude product was added a small amount of water to slurry, and filtered to give compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (M/z) =260 [ M+H ]+.
(1-1-2) Synthesis of Compound 3:
Compound 2 (9 g,1 eq) was dissolved in 70ml of 1, 4-dioxane, a solution of 1, 4-dioxane (45 ml, 4M) in hydrogen chloride was added, and the reaction was stirred at 25℃for 1 hour. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to give compound 3 (6.8 g, yield 100%) as a white solid.
(1-1-3) Synthesis of Compound 5:
Compound 3 (1.8 g,2.0 eq), compound 4 (5- [ [ (2R, 3R,4R,5R, 6R) -3-acetamido-4, 5-diacetoxy-6- (acetoxymethyl) -2-tetrahydropyranyl ] oxy ] pentanoic acid, 2.1g,1.0 eq) and DIEA (N, N-diisopropylethylamine, 3.5g,6.0 eq) were dissolved in 15ml of DMF, HBTU (1.9 g,1.1 eq) was added and the reaction stirred under N2 at 25℃for 3 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and purified by reverse phase column chromatography (22 vol% acetonitrile aqueous solution) to give compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m/z) =589 [ m+h ]+.
(1-1-4) Synthesis of Compound 6:
Compound 5 (1.54 g,1.0 eq) was dissolved in 15ml of pyridine, the reaction system was cooled to 0℃with an ice-water bath and DMTrCl (4, 4' -dimethoxytriphenylchloromethane, 1.32g,1.5 eq) was added at 0℃and reacted at 25℃for 3 hours, and 15ml of methanol was added to quench the reaction. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and purified by reverse phase column chromatography (60 vol% acetonitrile in water) to give compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (M/z) =891 [ M+H ]+.
(1-1-5) Synthesis of Compound CR 01008:
Compound 6 (1.08 g,1.0 eq) was dissolved in 20ml of anhydrous dichloromethane, DCI (115 mg,0.8 eq) and compound 7 (bis (diisopropylamino) (2-cyanoethoxy) phosphine, 730 mg,2.1 eq) were added separately, nitrogen was replaced 3 times, and the reaction was stirred at 25℃for 2 hours. After completion of the reaction, 20ml of saturated aqueous sodium hydrogencarbonate solution was added to the reaction mixture, the mixture was extracted 3 times with 20ml of methylene chloride (3X 20 ml), the organic phases were combined, evaporated to dryness under reduced pressure, and purified by reverse phase chromatography (72 vol% aqueous acetonitrile solution) and then dried in vacuo for 12 hours to give compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (M/z) =1091 [ M+Na ]+.
H NMR (400 MHz, DMSO-d6)δ1.05 (d,J= 6.7 Hz, 6H).1.14 (d,J= 6.7 Hz, 6H), 1.37 – 1.17 (m, 5H), 1.60 – 1.40 (m, 6H),1.68 – 1.62 (m, 1H),1.80 (s, 3H),1.80 (s, 3H),1.92 (s, 3H), 2.02 (s, 5H),2.13 (s, 3H),2.71 (t,J= 5.9 Hz, 2H), 2.79 (d,J= 8.4 Hz, 1H), 2.87 (d,J= 8.4 Hz, 1H),3.36 (s, 1H), 3.58 – 3.39 (m, 3H), 3.69 – 3.60 (m, 2H), 3.75 (s, 7H), 3.90 (dt,J= 11.2, 8.8 Hz, 1H), 4.05 (s, 3H),4.51 (d,J= 8.4 Hz, 1H),4.99 (dd,J= 11.3, 3.4 Hz, 1H), 5.24 (d,J= 3.4 Hz, 1H), 5.78 (s, 1H),6.93 – 6.87 (m, 4H),7.35 – 7.21 (m, 7H), 7.44 – 7.37 (m, 2H), 7.66 (d,J= 7.8 Hz, 1H), 7.84 (d,J= 9.2 Hz, 1H).
(1-2) Synthesis of Compound CR 01008Z:
the synthetic route for compound CR01008Z is shown below:
(1-2-1) Synthesis of Compound 9:
Compound 6 (500 mg) was dissolved in 10ml of methylene chloride, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg) and TEA (226.2 mg) were added, replaced with nitrogen 3 times, and reacted at 25℃for 16 hours with stirring, followed by flash purification to give compound 9 (300 mg, yield 53.6%). MS-ESI (M/z) =1013 [ m+na ]+.
(1-2-2) Synthesis of Compound CR 01008Z:
To a 20ml sample bottle, compound 9 (50 mg, amino CPG (1.25 g, 80. Mu. Mol/g,0.1 mmol), HBTU (27 mg), DIEA (12 mg) were added, followed by shaking for 16 hours, and after completion of the reaction, the reaction mixture was filtered to obtain a cake, which was washed once with 10ml of acetonitrile (1X 10 ml) and then dried under vacuum, to a 20ml sample bottle, was added dried cake, DMAP (3 mg), cap1 (10 ml, 200V) and Cap2 (1 ml, 20V), and shaking for 6 hours, after completion of the reaction, the reaction mixture was filtered to obtain a cake, which was washed once with 10ml of acetonitrile (1X 10 ml) and then dried under vacuum to obtain Compound CR01008Z (1.03 g, loading 20 to 30. Mu. Mol/g).
Cap1 and Cap2 are capping reagents, cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with the concentration of 20% by volume, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is an acetonitrile solution of acetic anhydride with the concentration of 20% by volume.
Synthesis of Compound NM054 of preparation 2:
In this preparation, the synthetic route for compound NM054 is shown below:
(2-1) Synthesis of Compound NM 054-2:
To a 500ml reaction vessel were added compound NM054-1 (3 g,11.54mmol,1.0eq, (2 ' R) -2' -deoxy-2 ' -fluoro-2 ' -methyluridine, CAS number 863329-66-2) and pyridine (30 ml), reduced to 0 ℃,4' -dimethoxytrityl chloride (4.29 g,12.7mmol,1.1 eq) was added in portions, nitrogen was displaced 3 times, and the reaction system was stirred under nitrogen atmosphere at 25℃for 3 hours, HPLC showed no starting material. After the completion of the reaction, the reaction mixture was concentrated, purified water (50 ml) and ethyl acetate (50 ml) were added to extract, and an organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound NM054-2 (2.7 g, yield 41.7%). MS ESI (M/z) = 563.0 [ m+h ]+.
(2-2) Synthesis of Compound NM 054:
to a 100ml reaction vessel was added compound NM054-2 (2.7 g,4.8mmol,1.0 eq), bis (diisopropylamino) (2-cyanoethoxy) phosphine (1.74 g,5.76mmol,1.2 eq), 4, 5-dicyanoimidazole (0.45 g,3.8mmol,0.8eq, abbreviated DCI, CAS number 1122-28-7) and dichloromethane (27 ml), the nitrogen was replaced 3 times, and the reaction system was stirred under nitrogen atmosphere at 25℃for 3 hours. After completion of the reaction, an aqueous sodium hydrogencarbonate solution (20 ml) was added to the reaction solution, an organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile/water=90/10, v/v) to give compound NM054 (3.0 g). MS ESI (M/z) =763 [ m+h ]+.
Preparation example 3 siRNA preparation:
(3-1) Synthesis of Sense Strand (SS):
By the method of phosphoramidite nucleic acid solid phase synthesis, nucleoside monomers are linked one by one according to the 3'-5' direction of nucleotide sequences by starting a cycle with a compound of a solid phase carrier. During the synthesis, compound NM054 was considered a nucleoside monomer.
Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The synthesis conditions were given as follows:
nucleoside monomers were formulated as an acetonitrile solution of nucleoside monomers at a concentration of 0.1M.
The deprotection conditions are the same for each step. The deprotection reaction was carried out at 25℃for 70 seconds with a molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support of 5:1 in the presence of a dichloromethane solution (3% by volume) of dichloroacetic acid as the deprotection reagent.
The conditions for each coupling reaction were identical. The conditions of the coupling reaction are that the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is an acetonitrile solution of 5-ethylthio-1H-tetrazole with the concentration of 0.5M, and the thio reagent is a mixed acetonitrile/pyridine solution of hydrogenated Huang Yuansu with the concentration of 0.2M (the volume ratio of acetonitrile to pyridine is 1:1).
The conditions for the capping reaction were the same for each step. The Cap reaction conditions are that the temperature is 25 ℃, the reaction time is 2 minutes, the Cap reagent solution is a mixed solution of Cap1 and Cap2 with the molar ratio of 1:1, cap1 is a pyridine/acetonitrile mixed solution of N-methylimidazole with the concentration of 20 volume percent, the volume ratio of pyridine to acetonitrile is 3:5, cap2 is an acetonitrile solution of acetic anhydride with the annual attack rate of 20 volume percent, and the molar ratio of N-methylimidazole in the Cap1 Cap reagent, acetic anhydride in the Cap2 Cap reagent and a nucleic acid sequence connected to a solid phase carrier is 1:1:1.
The conditions for each oxidation reaction are the same. The conditions of the oxidation reaction were 25℃for 3 seconds, 0.05M iodine water as the oxidizing agent, a molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling reaction of 30:1, and a water/pyridine mixed solvent (volume ratio of water to pyridine of 1:9). The conditions of the sulfidation reaction were 25℃for 360 seconds, 0.2M solution of pyridine hydrogenated Huang Yuansu in concentration of the thio reagent, 4:1 molar ratio of thio reagent to nucleic acid sequence attached to the solid support in the coupling reaction, and the thio reaction was carried out in a water/pyridine mixed solvent (volume ratio of water to pyridine: 1:9).
After the last nucleoside monomer is connected, the nucleic acid sequence connected on the solid phase carrier is sequentially cut, deprotected, purified and desalted, and then freeze-dried to obtain the sense strand, wherein:
The cleavage and deprotection conditions were such that the synthesized nucleotide sequence to which the solid support was attached was added to aqueous ammonia having a concentration of 25% by mass, the amount of aqueous ammonia was 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the solvent was removed, and concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved with 0.4 ml/. Mu.mol of N-methylpyrrolidone, followed by the addition of 0.3 ml/. Mu.mol of triethylamine and 0.6 ml/. Mu.mol of triethylamine-tricofluoride, relative to the amount of single-stranded nucleic acid, and the 2' -O-TBDMS protection on ribose was removed.
Purification and desalting conditions purification of nucleic acid was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the eluent 1 is 20mM sodium phosphate (pH=8.1), the solvent is a water/acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1), the eluent 2 is 1.5M sodium chloride, the solvent is 20mM sodium phosphate (pH=8.1), the solvent is a water/acetonitrile mixed solution (the volume ratio of water to acetonitrile is 9:1), and the elution gradient is eluent 1, eluent 2= (100:0) - (50:50). Collecting and combining product eluents, desalting by using a reversed phase chromatographic purification column, wherein the desalting conditions comprise desalting by using a sephadex column, eluting with deionized water, wherein the filler is sephadex G25.
Detecting, namely detecting the purity by using ion exchange chromatography (IEX-HPLC), detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the actual measurement value and the theoretical value of the molecular weight, wherein if the actual measurement value and the theoretical value are consistent, the sense strand of the siRNA is obtained.
(3-2) Synthesis of Antisense Strand (AS):
Antisense strands were synthesized using a universal solid support. The conditions of deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection conditions, purification and desalting in the solid phase synthesis method of antisense strand are the same as those of step (3-1) for synthesizing sense strand.
Detecting, namely detecting the purity by using ion exchange chromatography (IEX-HPLC), detecting the molecular weight by using liquid chromatography-mass spectrometry (LC-MS), and comparing the actual measurement value and the theoretical value of the molecular weight, wherein if the actual measurement value and the theoretical value are consistent, the antisense strand of the siRNA is obtained.
(3-3) Synthesis of siRNA:
the sense strand synthesized in step (3-1) and the antisense strand synthesized in step (3-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃, slowly cooled to room temperature and kept at room temperature for 10 minutes, and the sense strand and the antisense strand formed a double-stranded structure through hydrogen bonding, thereby obtaining siRNA having the sense strand and the antisense strand shown in table 2.
Preparation example 4 Synthesis of siRNA conjugate
(4-1) Synthesis of sense strand
By phosphoramidite nucleic acid solid phase synthesis method, using carrier compounds (such as Universal CPG carrier, universal PS carrier, compound CR 01008Z) start, according to the nucleotide sequence from 3 'end to 5' end direction connection nucleoside monomer. During the synthesis, compound CR01008, compound NM054 were each considered a nucleoside monomer.
Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. The conditions for deprotection, coupling, capping, oxidation or sulfidation reaction, cleavage and deprotection, purification and desalting in the synthesis of the sense strand of this preparation are the same as those for the synthesis of the sense strand of step (3-1).
During the synthesis of the sense strand, a two-cluster CR01008 vector (designated as (CR 01008X 2) or (CR 01008) X2), a three-cluster CR01008 vector (designated as (CR 01008X 3) or (CR 01008) X3), a four-cluster CR01008 vector (designated as (CR 01008X 4) or (CR 01008) X4), and the like were obtained.
The structural formula of the two clusters CR01008 is:。
the structural formula of the three clusters CR01008 is:
。
The structural formula of the four-cluster CR01008 is as follows:
。
(4-2) Synthesis of antisense strand
The antisense strand of this preparation was synthesized according to the method for synthesizing an antisense strand shown in step (3-2) of preparation example 3.
(4-3) The siRNA conjugate was synthesized according to the method shown in the step (3-3) in preparation example 3.
Wherein the structural formula of the siRNA conjugate in which the (CR 01008) x3 carrier is conjugated to the 3' -end of the sense strand is as follows:
Wherein, the method comprises the steps of,Indicating siRNA, SS representing the sense strand of siRNA, AS representing the antisense strand of siRNA. (CR 01008X 3) was conjugated to the 3' -end of the sense strand of siRNA via a phosphodiester linkage.
The structural formula of the siRNA conjugate of (CR 01008) x 4 vector conjugated to the 3' end of the sense strand is shown below:
Wherein, the method comprises the steps of,Indicating siRNA, SS representing the sense strand of siRNA, AS representing the antisense strand of siRNA. (CR 01008X 4) was conjugated to the 3' -end of the sense strand of siRNA via a phosphodiester linkage.
The structural formula of the siRNA conjugates of two (CR 01008) x 2 vectors conjugated to the 5 'end of the sense strand and the 3' end of the antisense strand, respectively, are shown below:
Wherein, the method comprises the steps of,Indicating siRNA, SS representing the sense strand of siRNA, AS representing the antisense strand of siRNA. Two (CR 01008X 2) were conjugated to the 5 'end of the sense strand and the 3' end of the antisense strand of the siRNA, respectively, via phosphodiester bonds.
Biological detection assay:
Unless otherwise indicated, reagent consumables and instrumentation used in biological assay experiments of the present disclosure are all derived from commercial products.
Example 1 in vivo efficacy comparison of siRNA conjugates in normal cynomolgus monkeys:
In this example, the expression of AGT protein in serum from cynomolgus monkeys at different time points after a single administration of RZ003089, R303079, R303082, R303086, R303087, R303088, R303089 was determined by ELISA.
The nucleotide information for conjugate RZ003089 is as follows:
the sense strand (5 '-3') UmsCmsAmAmCmUmGfGfAfUfGmAmAmGmAmAmAmCmUm _ (CR 01008X 3);
antisense strand (5 '-3') AmsGfsUmUmUmCfUmUmCfAmUmCmCmAfG (moe) UfUmGmAmsGmsGm.
Grouping animals, dosing and tissue sample collection:
Healthy cynomolgus monkeys of 3-5kg body weight were grouped according to serum AGT protein levels, 3 males per group. Each test group was given a predetermined dose of drug conjugate and the vehicle control group was increased. All animals were dosed on a weight basis in a single dose by subcutaneous injection in the back-scapular region, each drug conjugate being dosed as 6 mg (calculated as siRNA)/mL of 0.9% sodium chloride injection, with a dosing volume of 1:1 mL/kg (cynomolgus body weight), i.e. the dose of each drug conjugate was 6 mg (calculated as siRNA)/kg (cynomolgus body weight). Vehicle control group was given 0.9% sodium chloride injection 1 mL/kg (cynomolgus monkey body weight) without siRNA conjugate. Cynomolgus monkey serum was collected on day 1 (denoted D0, pre-dose), 7 days post-dose (denoted D7), 14 days (denoted D14), 21 days (denoted D21), 28 days (denoted D28), 35 days (denoted D35), 42 days (denoted D42) and assayed for AGT protein expression using the human Angiotensinogen (AGT) kit (IBL, 27412). The test is in progress.
TABLE 5 variation level of AGT protein in cynomolgus monkey serum after administration of the siRNA conjugates
The results of example 1 are shown in Table 5 and FIG. 1, and show that RZ003089, R303079, R303082, R303086, R303087, R303088 and R303089 can obviously reduce the AGT protein level in the serum of the cynomolgus monkey when the dose of 6mg/kg is given by single administration, and the effect of R303082 and R303089 on the serum AGT protein is slightly better than that of RZ003089 along with the prolonged observation time.
The above detailed description is illustrative of the present invention and is not meant to be limiting. Various modifications and improvements may be made by those skilled in the art without departing from the spirit and substance of the disclosure, and are also considered to be within the scope of the disclosure.