Application of siRNA in preparation of medicine for treating hepatitis

Abstract

The invention discloses an application of siRNA (small interfering Ribonucleic Acid) in preparation of a medicine for treating hepatitis. A positive-sense strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO: 1, at least 85% of bases of an antisense strand of the siRNA and the positive-sense strand are complementary, and hepatitis is hepatitis D liver or hepatitis B and hepatitis D liver. The siRNA inhibits the duplication of liver D by inhibiting the concentration of HBV S protein in vivo.

Description

Technical Field

[0001] This invention relates to the use of siRNA in the preparation of drugs for treating hepatitis. Background Technology

[0002] Hepatitis D is an inflammation of the liver caused by the hepatitis D virus. This virus relies on the hepatitis B virus envelope to enter liver cells and replicate. Without hepatitis B virus, hepatitis D infection will not occur. Co-infection with hepatitis D virus and hepatitis B virus is considered the most severe form of chronic viral hepatitis because it accelerates the development of hepatocellular carcinoma and liver-related death.

[0003] Because the replication of the hepatitis D virus (HBV) requires the assistance of HBV to assemble into complete HBV particles, HBsAg carriers and hepatitis B patients can be both sources of HBV infection and susceptible individuals for the virus. Currently, there are no marketed drugs for treating hepatitis D.

[0004] The closest prior art to this invention is Hepcludex (Bulevirtide, Myrcludex B), a synthetic polypeptide derived from the pre-S1 domain of the HBsAg large envelope protein. It inhibits the entry of HBV and HDV into hepatocytes by binding to and inactivating NTCP. In 2020, this drug received conditional approval in the EU for the treatment of compensated chronic hepatitis D (fully approved in Russia), and is currently undergoing phase 3 clinical trials in the US. This product has received Breakthrough Therapy Designation and Orphan Drug Designation from the FDA for the treatment of hepatitis D. Summary of the Invention

[0005] This application aims to overcome the problem of limited available drugs for treating hepatitis D in the prior art by providing an application of siRNA in the preparation of drugs for treating hepatitis. The siRNA of this application, utilizing RNAi technology to silence the HBV gene, can not only effectively inhibit HBV replication but also induce abnormal biological function of HBV covalently closed circular DNA (cccDNA) during chronic infection, thereby fundamentally eliminating HBV. Simultaneously, it can directly inhibit the replication and expression of the HBV S protein in hepatitis B virus, blocking viral invasion of hepatocytes and breaking the cycle of persistent infection and reinfection in the liver; by inhibiting the concentration of HBV S protein in vivo, it inhibits the replication of hepatitis D, exhibiting a good inhibitory effect.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides an application of siRNA in the preparation of drugs for treating hepatitis;

[0008] The siRNA comprises a sense strand and an antisense strand forming an inverse complementary double-stranded region. The sense strand has a nucleotide sequence as shown in SEQ ID NO: 1, and the antisense strand of the siRNA is complementary to the sense strand by at least 85% of its bases.

[0009] The hepatitis mentioned is hepatitis D, or hepatitis B and hepatitis D.

[0010] In one embodiment, the antisense strand is complementary to the sense strand by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of bases.

[0011] In one embodiment, the antisense strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO: 2.

[0012] In this invention, the siRNA may be a modified siRNA.

[0013] In one embodiment, at least one nucleotide glycosyl group at the 2' position of the sense strand and / or antisense strand of the siRNA may be substituted, for example, by fluorine or methoxy substitution.

[0014] In one embodiment, the phosphate ester bonds between at least three adjacent nucleotides at the ends of the sense and / or antisense strands of the siRNA may be thiolated.

[0015] In one embodiment, the positive strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO: 1, which is as follows: GGGTTTTTCTTGTTGACAA.

[0016] In one embodiment, the positive strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO: 2, which is as follows: TTGTCAACAAGAAAAACCCTT.

[0017] In one embodiment, the sense strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO: 3, which is as follows: 5' -Gs Gs GU fU U fU fU fC UUGUUGA Cs As A-3';

[0018] Wherein, Gs is 2'-O-methylguanosine monophosphate, fU is 2'-fluorouridine monophosphate, fC is 2'-fluorocytidine monophosphate, Cs is 2'-O-methyl-3'-thiocytidine monophosphate, As is 2'-O-methyladenosine monophosphate, Us is 2'-O-methyl-3'-thiouridine monophosphate, and fA is 2'-fluoroadenosine monophosphate.

[0019] In one embodiment, the antisense strand of the siRNA has a sequence as shown in SEQ ID NO: 4, which is as follows: 5'-Us Us GUCA fA CAAG fA A fA AACC Cs Us U-3'.

[0020] In one embodiment, the siRNA further comprises 5'MVIP and / or 3'MVIP modified at the 5' and / or 3' ends, wherein the 5'MVIP and 3'MVIP are ligand structures with a liver-targeting specific ligand X, and further comprise a branched chain L, a linker B, and a connecting chain D.

[0021] In one embodiment, the 5'MVIP is coupled to the end of the justice chain and / or antisense chain 5', and it further includes a transition point R1 connected to the end of the justice chain or antisense chain 5';

[0022] The 3'MVIP is coupled to the end of the antisense chain and / or the justice chain 3', and includes a transition point R2 connected to the end of the justice chain or antisense chain 3'.

[0023] In one embodiment, the structure of the 5'MVIP is shown in general formula I, and the structure of the 3'MVIP is shown in general formula II.

[0024] , ,

[0025] in,

[0026] n and m are integers from 1 to 3, and n+m=2, 3 or 4;

[0027] The transition points R1 and R2 have -NH-, sulfur or oxygen atoms in their structures, and generally have at least one -NH-, sulfur or oxygen atom in their structures. R1 and R2 are connected to the 5'MVIP and 3'MVIP connecting chains D and the 5' and 3' ends of the sense chain and / or antisense chain respectively through the -NH-, sulfur or oxygen atoms in their structures.

[0028] In one implementation, R1 is -NH(CH2). x CH2O-, where x is an integer from 3 to 12, or R1 is -O(CH2)6O-, -S(CH2)6O- or -NH(CH2)6S-;

[0029] R2 is -NH(CH2) xl CH(OH)(CH2) x2CH2O-, where x1 is an integer from 1 to 4, x2 is an integer from 0 to 4, or R2 is , , or .

[0030] The liver-targeting specific ligand X is selected from galactose, galactosamine, N-acetylgalactosamine and its derivatives, and the liver-targeting specific ligand X is the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP;

[0031] The branched chain L contains -NH-, C=O, O, S, amide group, phosphoryl group, thiophosphoryl group, or C4-C group. 10 C4-C of aliphatic carbocyclic groups, phenyl groups, or combinations thereof 18 A straight chain, and the branch L is the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP;

[0032] The connector B is selected from the following structures:

[0033] ; ; ; ; ; ; ; ; ; ; ;

[0034] Among them, A l A2 and A2 are each independently C, O, S, -NH-, carbonyl, amide, phosphoryl, or thiophosphoryl, r is an integer from 0 to 4, and the connector B is the same or different between 5'MVIP and 3'MVIP;

[0035] The connecting chain D contains -NH-, C=O, O, S, amide group, phosphoryl group, thiophosphoryl group, aromatic hydrocarbon group, C4-C 10 Aliphatic carbocyclic groups, five- or six-membered heterocyclic groups containing 1-3 nitrogen atoms, or combinations of these groups in C3-C2. 18 Straight chain.

[0036] In one implementation, the structures of the 5'MVIP and 3'MVIP are as follows:

[0037]

[0038] In one embodiment, the liver-targeting specific ligand X is selected from N-acetylgalactosamine and its derivatives.

[0039] In one embodiment, C4-C in the branch L 18 The straight chain also has side chains of ethyl alcohols or carboxylic acids.

[0040] In one embodiment, the branched chain L is a C7-C containing an amide group or a six-membered aliphatic carbocyclic group. 18 Straight chain.

[0041] In one embodiment, C3-C in the connecting chain D 18 The straight chain also has side chains of methyl alcohol, methyl tert-butyl, methylphenol or C5-C6 aliphatic ring groups.

[0042] In one embodiment, the linker chain D is a C3-C chain containing two C=O groups, a six-membered aliphatic carbocyclic group, or a phenyl group. l0 Straight chain, preferably C3-C containing two C=O. l0 Straight chain.

[0043] In one implementation scheme, R l For -NH(CH2) x CH2O-, where x is an integer from 4 to 6, preferably -NH(CH2)6O-.

[0044] In one embodiment, the RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein R2 is... .

[0045] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for treating hepatitis, said pharmaceutical composition comprising siRNA as described in any of the preceding embodiments and pharmaceutically acceptable excipients.

[0046] In one implementation, the hepatitis is hepatitis D, or hepatitis B and hepatitis D.

[0047] The present invention also provides a method for preventing and / or treating hepatitis, the method comprising administering to a subject in need an effective amount of siRNA as described in any of the preceding protocols or a pharmaceutical composition as described in any of the preceding protocols; preferably, the effective amount is a therapeutically effective amount.

[0048] As used in this invention, the term "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, when every position in two polypeptide molecules is alanine, then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions × 100. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0049] As used in this article, “treatment” means therapeutic therapy. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0050] As used in this article, "pharmaceutical composition" refers to a composition containing a specified active ingredient that can be prepared into the same dosage form.

[0051] The term "pharmaceutical excipients" as used in this article refers to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. They are all substances contained in pharmaceutical preparations other than the active ingredient.

[0052] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0053] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available.

[0054] The positive and progressive effects of this invention are as follows: The siRNA of this invention can effectively treat hepatitis D, or hepatitis B and hepatitis D, can directly inhibit the replication and expression of HBV S protein in hepatitis B virus, can block the virus from invading hepatocytes, and break the cycle of infection and reinfection in the liver; it inhibits the replication of hepatitis D by inhibiting the concentration of HBV S protein in the body, and has a better inhibitory effect than the existing drug Myrcludex B which is intended to be used for hepatitis D. Attached Figure Description

[0055] Figure 1 The fitted curve of the inhibitory activity of HT-101 against HBV and HBsAg;

[0056] Figure 2The cell viability in the HT-101 HBV HBsAg assay;

[0057] Figure 3 The curve showing the fitted activity of HT-101 against HDV RNA;

[0058] Figure 4 The cell viability of HT-101 cells in the HDV RNA experiment. Detailed Implementation

[0059] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0060] AD66810 was purchased from Shanghai WuXi AppTec Co., Ltd.

[0061] Myrcludex B was purchased from Genscript Biotech Inc.

[0062] Example 1: Synthesis of HT-101

[0063] HT-101 was obtained by referring to the preparation method of ky-2208 in Example 1 of Patent CN113171371B.

[0064] The specific method is as follows: The positive and negative strands are obtained using the solid-phase phosphorus amide method. The positive and negative strands are then annealed to obtain the final product. The basic steps of the solid-phase phosphorus amide method include: 1) Deprotection: Removing the hydroxyl protecting group (DMTr) of the starting monomer Solid Supporty; 2) Coupling: Adding the first phosphorus amide monomer and coupling via the 3' to 5' direction; 3) Oxidation: Oxidizing the obtained nucleoside phosphite to a more stable nucleoside phosphate (i.e., oxidizing trivalent phosphorus to pentavalent phosphorus); 4) Capping: Capping the unreacted nucleotide monomer from the previous step with its 5'-OH group to prevent further reaction. The above steps are repeated until the last phosphorus amide monomer is added. Then, the ester bond between Solid Supporty and the starting monomer is cleaved using aqueous methylamine and ammonia, and the protecting groups cyanoethyl (P), benzoyl (mA, fA), and acetyl (mC) on the resulting oligonucleotide are removed. After HPLC separation and purification, the product is filtered for sterilization and lyophilized to obtain the corresponding positive or negative strand. Annealing was used to accurately determine the concentrations of the sense and antisense chains in the reconstituted solution. After mixing them at equimolar concentrations, 1 / 20 of the volume of 1M PBS solution was added and mixed again. The mixture was then heated to 95°C for 5 minutes and allowed to cool naturally for 3 hours to 40°C or room temperature. HPLC analysis was then performed. If the single-chain residue was <5%, the reaction was considered complete.

[0065] 1. Synthesis of 5'MVIP09:

[0066] 5'MVIP is the final phosphoramide monomer synthesized as the positive chain in a solid-phase synthesis. The general formula of the 5'MVIP phosphoramide monomer is as follows: , where n is 2.

[0067] 1.1 Synthesis of ERC-01-c1

[0068] Weigh 5.0 g (54.9 mmol) of 2-amino-1,3-propanediol and add it to 50 mL of DMSO and 5 mL of sodium hydroxide solution (1 g / mL). Cool to 0 °C. o C. Add tert-butyl acrylate (20 mL, 137.8 mol) dropwise over 2 hours, react at room temperature for 48 hours, add petroleum ether (100 mL), wash twice with saturated brine, and dry the organic layer. Pass through a chromatography column to obtain a colorless oily substance ERC-01-c1.

[0069] 1.2 Synthesis of ERC-01-c2

[0070] Weigh 6.2 g (17.9 mmol) of ERC-01-c1, add 50 mL of dichloromethane and 23 mL of sodium carbonate solution (25%), and add 8.2 mL (57.4 mmol) of benzyl chloroformate dropwise at room temperature over 2 hours. Let the mixture react overnight at room temperature, wash three times with saturated saline solution, dry with anhydrous sodium sulfate, evaporate the solvent, and pass the mixture through a chromatography column to obtain the oily substance ERC-01-c2.

[0071] 1.3 Synthesis of ERC-01-c3

[0072] Take 4.0 g, 8.3 mmol of ERC-01-c2 and add 12 mL of formic acid. React overnight at room temperature. Evaporate the solvent under reduced pressure to obtain ERC-01-c3.

[0073] 1.4 Synthesis of ERCd-01-c1

[0074] Compounds ERC-01-c3 (1.11 g, 3.0 mmol) and d1SANC-c4 (3.6 g, 8.04 mmol) were added to DMF (60 mL), followed by HOBt (2.24 g) and HBTU (3.36 g), and then DIEA (4.16 mL) was slowly added. The reaction mixture was stirred at room temperature for 3 hours. Water was then added, and the aqueous layer was extracted with dichloromethane (2 x 10 mL). The organic layers were combined and washed successively with saturated sodium bicarbonate (80 mL), water (2 x 60 mL), and saturated brine (60 mL). The mixture was dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by silica gel column chromatography. A pale yellow solid, ERCd-01-c1, was obtained.

[0075] 1.5 Synthesis of ERCd-01-c2

[0076] ERCd-01-c1 (3.24 g, 2.6 mmol) was dissolved in methanol (60 mL), and 10% palladium on carbon (0.3 g) and acetic acid (2.0 mL) were added. Hydrogen was then added under normal pressure, and the reaction was allowed to proceed overnight. The reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness under reduced pressure to obtain the oily substance ERCd01-c2.

[0077] 1.6 Synthesis of 5'MVIP09-ERCd-PFP-c1

[0078] Weigh ERCd-01-c2 (2.18 g, 2.0 mmol) and dissolve it in DMF (50 mL). Add monobenzyl glutarate (0.53 g, 2.4 mmol), DIPEA (0.78 g), and TBTU (0.84 g). Stir overnight at room temperature, quench with water (50 mL), extract with DCM (30 mL * 3), wash with 10% citric acid (50 mL * 3), 50 mL saturated sodium bicarbonate, and 100 mL pyridine. Dry with anhydrous sodium sulfate, filter, rotary evaporate, and purify by column chromatography to obtain product 5'MVIP09-ERCd-PFP-c1.

[0079] 1.7 Synthesis of 5'MVIP09-ERCd-PFP-c2

[0080] Weigh 5'MVIP09-ERCd-PFP-c1 (2.15 g, 1.66 mmol) and 10% palladium on carbon (0.21 g), add methanol (50 mL), stir and hydrogenate overnight at room temperature. After the reaction is complete, filter palladium on carbon with diatomaceous earth and rotary evaporate to obtain crude 5'MVIP09-ERCd-PFP-c2.

[0081] Synthesis of 1.8, 5'MVIP09-ERCd-PFP

[0082] Weigh 1.0 g (1.58 mmol) of crude 5'MVIP09-ERCd-PFP-c2 and dissolve it in DCM (60 mL). Add DIPEA (1.33 g), cool, add pentafluorophenol trifluoroacetate (2.21 g, 7.9 mmol), stir and react at room temperature for 2 h, then rotary evaporate. Dissolve again in DCM (60 mL), wash with saturated sodium bicarbonate (30 mL*3), 10% citric acid (30 mL*1), and saturated brine (50 mL*1), dry with anhydrous sodium sulfate, filter, and rotary evaporate to obtain crude 5'MVIP09-ERCd-PFP.

[0083] 1.9 Synthesis of 5'MVIP09 phosphorous amide monomer-c1

[0084] Crude 5'MVIP09-ERCd-PFP (2.35 g, 1.58 mmol) was dissolved in DCM (60 mL), and DIPEA (0.82 g, 6.32 mmol) and 6-amino-1-hexanol (0.37 g, 3.16 mmol) were added. The mixture was stirred overnight at room temperature. Extraction was performed with 10% citric acid (30 mL) and DCM (30 mL x 3). The extract was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, rotary evaporated, and purified by column chromatography to obtain the product 5'MVIP09 monomer-c1.

[0085] Synthesis of 1.10 and 5'MVIP09 phosphorous amide monomers

[0086] Weigh 1.3 g (1.0 mmol) of 5'MVIP09 phosphoridamide monomer-c1 and dissolve it in acetonitrile (30 mL). Add diisopropyltriazole (0.22 g) and add bis-(diisopropylamino)(2-cyanoethoxy)phosphine (0.36 g, 1.2 mmol) dropwise under ice bath. React at room temperature for 4 h. After the reaction is qualified by HPLC, concentrate and purify by column chromatography to obtain the product 5'MVIP09 monomer.

[0087] Synthesis of 2'MVIP09

[0088] 3'MVIP is the last phosphoramide monomer synthesized as an antisense chain in a solid-phase synthesis. The general formula of the 3'MVIP phosphoramide monomer is as follows: Where m is 2, It serves as a solid-phase carrier, such as macroporous aminomethyl resin.

[0089] 2.1 Synthesis of 3'MVIP09-c1

[0090] SANCd-01-c0 (0.824 g, 1.5 mmol) and ERCd-01-c2 (1.09 g, 1.0 mmol) were added sequentially to the reaction flask, followed by 10 mL of DCM. The mixture was stirred and dissolved, and then TBTU (0.963 g) and DIPEA (0.517 g) were added sequentially. The reaction was allowed to proceed overnight. Water was added, and the mixture was extracted with DCM. The organic phase was washed with saturated brine, dried, filtered, concentrated, and finally purified by silica gel column chromatography to obtain 3'MVIP09-c1.

[0091] 2.2, Synthesis of 3'MVIP09-c2

[0092] 3'MVIP09-c1 (1.62 g, 1 μmol) and 10 mL of DCM were added sequentially to the reaction flask and stirred at room temperature to dissolve. Then, DMAP (0.366 g) and succinic anhydride (0.2 g, 3 μmol) were added sequentially and stirred at room temperature. The reaction was analyzed by TLC. If the reaction was satisfactory, the DCM was concentrated, water was added, and the mixture was extracted with DCM. The organic phase was then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and finally purified by silica gel column chromatography to obtain 3'MVIP09-c2.

[0093] Solid Support synthesis of 2.3 and 3'MVIP09

[0094] Add 3'MVIP09-c2 (0.86 g, 0.5 μmol) and 10 mL LDM to the reaction flask in sequence, dissolve, then add HBTU (0.19 g), DIPEA (0.194 g) and macroporous aminomethyl resin (2.0 g) in sequence, shake on a shaker for 24 h, filter, wash the resin with 10% methanol / DCM, and then end-cap with 25% acetic acid / pyridine to a degree of substitution of 150 μmol / g.

[0095] The structure of the ky-2208 sequence is shown below:

[0096] .

[0097] Example 2: Evaluation of the anti-HBV activity of the compound

[0098] On day 0, the frozen PHH cells were thawed, and the cell density was adjusted to 6.00E+05 cells / mL. 450 μL (1.50E+05) cells were seeded into each well of a 48-well plate, and the culture medium was InvitroGRO CP Medium containing 10% FBS and 1% P / S. Six hours later, PHH cells were infected with type D HBV.

[0099] Day 1: Replace with fresh culture medium. Day 3: Infect PHH with HDV. Day 4: Replace with fresh culture medium. Day 6: Transfect cells with test compound HT-101 and control compound AD66810 using RNAiMax, in quadruple replicates (20 nM, 2 nM, 0.2 nM, 0.02 nM). Days 8 and 10: Replace with fresh culture medium. Day 13: Collect cell supernatant and assess cell viability using CCK8 assay; results are shown in Table 1.

[0100] A portion of the supernatant was used to detect HBsAg, and the remaining cell supernatant (Conditional Medium, CM) was frozen at -80℃ for later use.

[0101] Example 3: Evaluation of the compound's anti-HDV activity

[0102] On day 0, thawing cryopreserved PHH cells was performed, and the cell count was adjusted to 6.00E+05 cells / mL. 450 μL (1.50E+05) cells were seeded per well in 48-well plates using InvitroGRO CP Medium containing 10% FBS and 1% P / S. Six hours later, PHH cells were infected with CM supernatant (used in the compound anti-HBV activity evaluation experiment, day 13). Cells were pretreated with Myrcludex B for 1 hour as a control, and Myrcludex B was added simultaneously with CM supernatant infection. Myrcludex B was used in triple-well configurations at three concentrations (100 nM, 10 nM, 1 nM). Fresh medium was used on days 1, 3, and 5. On day 7, cell viability was assessed using CCK8 assays, cell samples were collected, RNA was extracted, and HDV RNA was detected by RT-PCR. The results are shown in Table 2.

[0103] Example 4: Detection of HBsAg content in cell culture supernatant by ELISA

[0104] Referring to the instructions for the Antu Bio HBsAg ELISA kit, the method is briefly described as follows: Equilibrate the kit at room temperature for 1 hour, add 50 µL of sample to each well, then add 50 µL of enzyme conjugate, and incubate at 37°C for 60 minutes. Wash the plate 6 times, add 50 µL of luminescent substrate, and incubate at room temperature in the dark for 10 minutes. Finally, detect the luminescence intensity.

[0105] Example 5: Detection of intracellular HDV RNA content using qPCR

[0106] RNA was extracted according to the instructions of the RNA extraction kit (Qiagen, 74182), and reverse transcribed into cDNA according to the instructions of the HiScript III RTSuperMix for qPCR (Vazyme, catalog number R323-01). The target gene cDNA was detected by qPCR. HDV plasmid pAAV-1.2X HDV was used as a standard, serially diluted 10-fold, with the standard range from 1.0 × 10⁻⁶. 7 The qPCR reaction program was as follows: heat at 95°C for 10 minutes, then enter cycling mode, heat at 95°C for 15 seconds, followed by 60°C for 1 minute, for a total of 40 cycles.

[0107] Example 6: CCK-8 cell viability assay

[0108] After removing the cell supernatant, add 0.20 ml of diluted CCK-8 (2% DMSO cell culture medium: CCK-8 = 9:1 dilution) to each well, incubate at 37°C for 2.5 hours, and detect the absorbance values ​​of OD450nm-630nm using a microplate reader.

[0109] Data Analysis:

[0110] The following formulas are used to calculate the percentage of cell viability and the inhibition rate.

[0111] Cell viability % = (value of test sample - mean of blank) / (mean of DMSO control - mean of blank) × 100%.

[0112] HDV RNA inhibition rate (%) = (1 - HDV RNA copy number in the compound group / HDV RNA copy number in the control group) × 100%

[0113] HBsAg inhibition rate (%) = (1 - HBsAg value of sample / HBsAg value of control group) × 100%

[0114] Cell viability % = [(Sample luminescence value - Culture medium control luminescence value) / (Control luminescence value - Culture medium control luminescence value)] × 100%

[0115] The EC50 value of the compound was obtained by nonlinear dose-response curve fitting analysis of the inhibition rate data of the compound using GraphPad Prism software.

[0116] The results showed that Myrcludex B had the expected inhibitory effect on HDV activity. At the three test concentrations of 100 nM, 10 nM, and 1 nM, the inhibitory rates against HDV RNA were 87.40 ± 0.92%, 54.62 ± 8.48%, and 29.62 ± 10.03%, respectively. The EC50 value for HDV inhibition by HT-101 was 0.16 nM.

[0117] HT-101, control compound AD66810, and Myrcludex B showed no significant cytotoxicity within the tested concentration range.

[0118] Table 1: Activity of compounds against HBV and HBsAg, EC50 values, and cell viability

[0119]

[0120] Table 2. Compound activity against HDV RNA, EC50 values, and cell viability.

[0121]

Claims

1. The application of a siRNA in the preparation of a drug for treating hepatitis; The siRNA comprises a sense strand and an antisense strand forming an inverse complementary double-stranded region, the sense strand having a nucleotide sequence as shown in SEQ ID NO: 1, and the antisense strand of the siRNA being complementary to the sense strand by at least 85% of its bases. The hepatitis mentioned is hepatitis D, or hepatitis B and hepatitis D.

2. The application as described in claim 1, characterized in that, The antisense strand has a nucleotide sequence as shown in SEQ ID NO: 2; And / or, the siRNA is a modified siRNA.

3. The application as described in claim 1 or 2, characterized in that, The modification satisfies one or more of the following: (1) At least one nucleotide sugar at the 2' position of the siRNA in the sense strand and / or antisense strand is substituted, for example, by fluorine or methoxy; (2) The phosphate ester bonds between at least three adjacent nucleotides at the end of the sense strand and / or antisense strand of the siRNA are thiolated.

4. The application as described in claim 1, characterized in that, The sense strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO: 3; and / or, the antisense strand of the siRNA has a nucleotide sequence as shown in SEQ ID NO:

4.

5. The application as described in any one of claims 1-4, characterized in that, The siRNA further comprises 5'MVIP and / or 3'MVIP modified at the 5' and / or 3' ends, wherein the 5'MVIP and 3'MVIP are ligand structures with liver-targeting specific ligand X, and further comprise a branched chain L, a linker B, and a connecting chain D; Best location: The 5'MVIP is coupled to the end of the positive chain and / or negative chain 5', and it also includes a transition point R1 connected to the end of the positive chain or negative chain 5'; The 3'MVIP is coupled to the end of the antisense chain and / or the justice chain 3', and includes a transition point R2 connected to the end of the justice chain or antisense chain 3'. Better: The structure of the 5' MVIP is shown in general formula I, and the structure of the 3' MVIP is shown in general formula II. 、 ， n and m are integers from 1 to 3, and n+m=2, 3 or 4; The transition points R1 and R2 contain -NH-, sulfur, or oxygen atoms, and generally have at least one -NH-, sulfur, or oxygen atom. R1 and R2 are connected to the 5'MVIP and 3'MVIP linking chains D, as well as the 5' and 3' ends of the sense and / or antisense chains, respectively, through the -NH-, sulfur, or oxygen atoms in their structures. For example: R1 is -NH(CH2) x CH2O-, where x is an integer from 3 to 12, or R1 is -O(CH2)6O-, -S(CH2)6O- or -NH(CH2)6S-; R2 is -NH(CH2) xl CH(OH)(CH2) x2 CH2O-, where x1 is an integer from 1 to 4, x2 is an integer from 0 to 4, or R2 is , , or ; The liver-targeting specific ligand X is selected from galactose, galactosamine, N-acetylgalactosamine and its derivatives, and the liver-targeting specific ligand X is the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP; The branched chain L contains -NH-, C=O, O, S, amide group, phosphoryl group, thiophosphoryl group, or C4-C group. 10 C4-C of aliphatic carbocyclic groups, phenyl groups, or combinations thereof 18 A straight chain, and the branch L is the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP; The connector B is selected from the following structures: 、 、 、 、 、 、 、 、 、 、 ， A l A2 and A2 are each independently C, O, S, -NH-, carbonyl, amide, phosphoryl, or thiophosphoryl, r is an integer from 0 to 4, and the connector B is the same or different between 5'MVIP and 3'MVIP; The connecting chain D contains -NH-, C=O, O, S, amide group, phosphoryl group, thiophosphoryl group, aromatic hydrocarbon group, or C4-C. 10 Aliphatic carbocyclic groups, five- or six-membered heterocyclic groups containing 1-3 nitrogen atoms, or combinations of these groups in C3-C2. 18 Straight chain.

6. The application as described in claim 4 or 5, characterized in that, The 5'MVIP and 3'MVIP satisfy one or more of the following: (1) The structures of 5' MVIP and 3' MVIP are shown below: ； (2) The liver-targeting specific ligand X is selected from N-acetylgalactosamine and its derivatives; (3) C4-C in the branch L 18 The straight chain also has side chains of ethyl alcohols or carboxylic acids; (4) The branched chain L is a C7-C containing an amide group or a six-membered aliphatic carbon ring group. 18 Straight chain; (5) C3-C in the connecting chain D 18 The straight chain also has side chains of methyl alcohol, methyl tert-butyl, methylphenol or C5-C6 aliphatic ring groups; (6) The connecting chain D is a C3-C chain containing two C=O groups, a six-membered aliphatic carbocyclic group, or a phenyl group. l0 Straight chain, preferably C3-C containing two C=O. l0 Straight chain; (7) R l For -NH(CH2) x CH2O-, where x is an integer from 4 to 6, preferably -NH(CH2)6O-; (8) R2 is .

7. Use of a pharmaceutical composition in the preparation of a medicament for treating hepatitis, said pharmaceutical composition comprising siRNA as described in any one of claims 1-6, and pharmaceutically acceptable excipients and / or carriers.

8. The application as described in claim 7, characterized in that, The hepatitis mentioned is hepatitis D, or hepatitis B and hepatitis D.

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