An oligonucleotide composition, a method of preparing the same, and use thereof

Abstract

The present invention relates to an oligonucleotide composition comprising an oligonucleotide and a pharmaceutically acceptable carrier. The oligonucleotide of the present invention and its composition are used for the treatment or prevention of a disease or disorder associated with a hepatitis B virus (HBV) infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an oligonucleotide composition, its preparation method, and its application. Background Technology

[0002] Hepatitis B is a viral hepatitis caused by infection with the hepatitis B virus (HBV), which is mainly transmitted through blood, sexual contact, and mother-to-child transmission (Nicolini LA, et al. Int. J. Environ. Res. PublicHealth 2019, 16, 3307).

[0003] The pathological basis of chronic hepatitis B (CHB) lies in the persistent covalently closed circular DNA (cccDNA) within hepatocytes, which serves as a stable template for viral replication and is difficult to eliminate. Furthermore, HBV DNA can randomly integrate into the host genome, and although it does not directly support viral replication, it can persistently express viral antigens such as hepatitis B surface antigen (HBsAg) and HBx protein (Suarez AAR, et al. Liver International. 2021;41, Suppl. 1:15-23). ​​The persistent presence of these viral proteins is a key factor leading to immune tolerance and disease progression.

[0004] Currently, the main clinical treatments include nucleoside (acid) analogs (NUCs, such as entecavir and tenofovir) and pegylated interferon-alpha (Peg-IFN-α). However, existing therapies struggle to completely eliminate cccDNA, resulting in extremely low HBsAg seroconversion rates. Patients require long-term or even lifelong medication to suppress viral replication, leading to a high risk of relapse after discontinuation. Therefore, achieving "functional cure" for CHB (i.e., persistent disappearance of HBsAg, undetectable HBV DNA, normalization of liver function, and improvement in liver histology after discontinuation of medication) has become the ultimate goal of current treatment. Among these, the disappearance of HBsAg is considered a core indicator of deep suppression of viral replication and safe discontinuation of medication. Developing innovative drugs that can directly target viral reservoirs or key pathogenic processes has become an urgent direction for addressing clinical needs.

[0005] In 1998, American scientists Andrew Fire and Craig Mello first revealed the phenomenon of RNA interference (RNAi), namely that double-stranded RNA (dsRNA) can specifically mediate the degradation of homologous mRNA (Fire A, et al. Nature 1998;391:806-811), and were awarded the 2006 Nobel Prize in Physiology or Medicine for this discovery. The core mechanism of RNAi is that exogenous or endogenous dsRNA is cleaved into small interfering RNA (siRNA) by the intracellular enzyme Dicer; subsequently, siRNA binds to the RNA-induced silencing complex (RISC), and the guide strand directs the RISC to specifically recognize and cleave complementary target mRNA, thereby efficiently and specifically silencing gene expression at the posttranscriptional level.

[0006] RNAi technology, due to its high specificity and effectiveness, has become an important tool for gene function research and drug development. Synthesized siRNAs can target specific pathogenic genes in humans, animals, or plants, activating endogenous RNAi pathways through cellular introduction to precisely inhibit the expression of target proteins. Currently, siRNA drugs targeting indications such as viral infections, cardiovascular diseases, and cancer have entered clinical development stages, with some already approved for marketing. Since the first siRNA drug was introduced in 2018, at least four siRNA therapies have been approved globally in Europe and the United States, marking the successful transformation of this technology into an effective treatment.

[0007] Despite the promising future of RNAi technology, the clinical application of siRNA faces numerous challenges. siRNA molecules are inherently unstable and easily degraded by nucleases; furthermore, as negatively charged macromolecules, they struggle to effectively penetrate cell membranes to reach their targets. In addition, factors such as drug stability, route of administration, in vivo distribution, and concentration directly determine their physicochemical properties and biological activity during manufacturing, storage, transportation, and in vivo delivery, thus affecting the efficacy and safety of treatment. Therefore, developing siRNA and its formulations with good stability, delivery efficiency, and low toxicity is an urgent need for the intervention of diseases such as chronic hepatitis B.

[0008] CN202411373939.2 (published on May 2, 2025) discloses small interfering RNA that inhibits hepatitis B virus, but the application does not involve the formulation and stability study of the relevant small interfering RNA. This invention is an in-depth development of the patented technology, and some of the technical solutions therein are also applicable to this invention, so they are introduced into this invention. Summary of the Invention

[0009] The purpose of this invention is to provide an oligonucleotide composition comprising an oligonucleotide and a pharmaceutically acceptable carrier.

[0010] In a preferred embodiment of the present invention, the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand is the sequence or fragment shown in SEQ ID NO: 1, and the antisense strand is the sequence or fragment shown in SEQ ID NO: 2.

[0011] In a preferred embodiment of the present invention, SEQ ID NO: 2 is modified or replaced by any one or a combination of adenosine-2' phosphate (A-2'-5'), guanosine diol nucleic acid (Ggn), adenosine diol nucleic acid (Agn), N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX), and 5'-phosphate ester mimics.

[0012] In a preferred embodiment of the present invention, the 5'-phosphate ester mimic is modified at the 5'-end of the antisense chain, and preferably the 5'-phosphate ester mimic is selected from any one or a combination of 5'-oxymethylphosphonate, 5'-vinylphosphonic acid (Vp), 5'-vinylphosphonate-2'-N-acetyl, and 5'-malonylphosphonate.

[0013] In a preferred embodiment of the present invention, any one of the nucleic acids at positions 1 to 8 of the 5' end of the antisense strand is modified or replaced.

[0014] In a preferred embodiment of the present invention, any one of the nucleic acids at the 2nd to 8th positions of the 5'-terminus of the antisense strand is replaced by any one or a combination of A-2'-5', Ggn, Agn, GNF-BX.

[0015] In a preferred embodiment of the present invention, either the 5th or 7th position of the 5' end of the antisense chain is replaced by any one or a combination of A-2'-5', Ggn, Agn, GNF-BX.

[0016] In a preferred embodiment of the present invention, Vp is modified at the 5'-end of the antisense strand sequence shown in SEQ ID NO: 2, and Ggn substitution occurs at the 5th nucleic acid position at the 5' end of the antisense strand.

[0017] In a preferred embodiment of the present invention, Vp is modified at the 5'-terminus of the antisense strand sequence shown in SEQ ID NO: 2, and A-2'-5' substitution occurs at the 7th nucleic acid position at the 5' end of the antisense strand.

[0018] In a preferred embodiment of the present invention, the nucleotide sequence of the antisense strand is selected from any of the sequences or fragments shown in SEQ ID NO: 2-SEQ ID NO: 11.

[0019] In a preferred embodiment of the present invention, the structure of the vinylphosphonate-modified uridine analog is as follows: Where U is a uracil base.

[0020] In a preferred embodiment of the present invention, the compound after the antisense strand 5' terminal nucleotide is modified with Vp has the following structure: ,

[0021] Where X is selected from -SH, Base is a uracil base, Y is another nucleoside on the oligonucleotide, and A is OCH3.

[0022] In a preferred embodiment of the present invention, the sense strand of the oligonucleotide is the sequence or fragment shown in SEQ ID NO: 1, and the antisense strand is selected from any one of the sequences or fragments shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

[0023] In a preferred embodiment of the present invention, the composition is a liquid formulation.

[0024] In a preferred embodiment of the present invention, the composition contains 0.3% to 30% oligonucleotides, preferably 0.4% to 2% or 10% to 25% by weight and volume percentage.

[0025] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is optionally selected from any one or a combination of buffers, osmotic pressure regulators.

[0026] In a preferred embodiment of the present invention, the composition has a pH of 5.0 to 8.5, preferably pH 5.5-8.0, and more preferably pH 6.0-pH 7.5.

[0027] In a preferred embodiment of the present invention, the composition contains a buffer of 5-50 mM, preferably 10-20 mM or 40-50 mM.

[0028] In a preferred embodiment of the present invention, the buffer is selected from any one or a combination of disodium hydrogen phosphate, citric acid, sodium citrate, carbonate, histidine, histidine hydrochloride, acetate, sodium citrate, and sodium citrate.

[0029] In a preferred embodiment of the present invention, the composition contains 10% to 25% oligonucleotides, 10mM to 20mM buffer, and water for injection, by weight and volume percentage. The composition has a pH of 6.0 to 7.0, wherein the buffer is disodium hydrogen phosphate.

[0030] In a preferred embodiment of the present invention, the composition contains 0.4%-2% oligonucleotides, 40mM-50mM buffer, and water for injection by weight-volume percentage, and the composition has a pH of 6.0-7.0, wherein the buffer is disodium hydrogen phosphate.

[0031] In a preferred embodiment of the present invention, the composition optionally contains an isotonic regulator.

[0032] In a preferred embodiment of the present invention, the osmotic pressure regulator is selected from either glucose or sodium chloride.

[0033] In a preferred embodiment of the present invention, the composition is selected from either intravenous or subcutaneous administration.

[0034] Another object of the present invention is to provide a method for preparing an oligonucleotide composition, wherein the required amount of oligonucleotide is uniformly mixed with a pharmaceutically acceptable carrier to obtain the composition.

[0035] In a preferred embodiment of the present invention, the composition is a liquid formulation.

[0036] In a preferred embodiment of the present invention, the composition contains 5-300 mg / mL of oligonucleotides, preferably 5-15 mg / mL or 100-200 mg / mL.

[0037] In a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is optionally selected from any one or a combination of buffers, osmotic pressure regulators.

[0038] In a preferred embodiment of the present invention, the composition has a pH of 5.0 to 8.5, preferably pH 5.5-8.0, and more preferably pH 6.0-pH 7.5.

[0039] In a preferred embodiment of the present invention, the buffer is selected from any one or a combination of disodium hydrogen phosphate, citric acid, sodium citrate, carbonate, histidine, histidine hydrochloride, acetate, sodium citrate, and sodium citrate.

[0040] In a preferred embodiment of the present invention, the composition contains a buffer of 5-50 mM.

[0041] In a preferred embodiment of the present invention, a required amount of oligonucleotide is added to a required amount of buffer solution and mixed evenly to obtain the final product.

[0042] Another object of the present invention is to provide a pharmaceutical combination package comprising the oligonucleotides of the present invention and other anti-HBV drugs.

[0043] In a preferred embodiment of the present invention, the other anti-HBV drugs are selected from any one or a combination of nucleoside analogs, hepatitis B virus entry inhibitors, capsid assembly inhibitors, HBsAg secretion inhibitors, hepatitis B therapeutic vaccines, host immune modulators, and apoptosis protein antagonists.

[0044] In a preferred embodiment of the present invention, the oligonucleotides and other anti-HBV drugs of the present invention are selected from any one of sequential or simultaneous drug administration.

[0045] Another object of the present invention is to provide the use of oligonucleotide compositions in the preparation of medicaments for reducing any one or a combination of HBsAg, HBeAg, and HBV DNA in the serum of a subject.

[0046] Another object of the present invention is to provide the use of oligonucleotide compositions in the preparation of medicaments for treating HBV infection, hepatitis B, or hepatitis B virus infection combined with hepatitis D virus infection or their complications.

[0047] In a preferred embodiment of the present invention, the HBV infection is a chronic HBV infection.

[0048] The methods described in this invention generally involve administering an effective amount (i.e., an amount capable of producing the desired therapeutic outcome) of an oligonucleotide to a subject. The appropriate dosage for the subject is related to factors such as the subject's body size, body surface area, age, the specific composition to be administered, one or more active ingredients in the composition, the time and route of administration, overall health, and other concurrently administered medications.

[0049] Unless otherwise stated, the scientific and technical terms used in this invention have the meanings commonly understood by those skilled in the art.

[0050] The "hepatitis B virus-related disease" or "HBV-related disease" as described in this invention refers to a disease or condition caused by or related to HBV infection or replication. The term "HBV-related disease" includes diseases, conditions, or symptoms that benefit from reduced HBV gene expression or replication. Non-limiting examples of HBV-related diseases include, for example, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0051] The term "complementarity" as used in this invention refers to a structural relationship between nucleotides (e.g., two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid chain) that allows nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposing nucleic acid can pair up with each other by forming hydrogen bonds.

[0052] In a preferred embodiment of the present invention, complementary nucleotides may be paired in a Watson-Crick manner or in any other manner that allows the formation of a stable double helix.

[0053] In a preferred embodiment of the present invention, the two nucleic acids may have nucleotide sequences that are complementary to each other in order to form complementary regions.

[0054] The nucleotide analogs in this invention can replace nucleotides on the main chain as nucleotide monomers. The nucleotide analogs include, but are not limited to: nucleotides modified with 2'-methoxyethyl (moe), nucleotides modified with 2'-methoxy (m), nucleotides modified with 2'-deoxy-2'-fluoro (f), nucleotides modified with thiophosphate (s), adenosine-2' phosphate (A-2'-5'), uridine-2' phosphate (U-2'-5'), guanosine-2' phosphate (G-2'-5'), guanosine-diol nucleic acid (Ggn), adenosine-diol nucleic acid (Agn), nucleotides modified with N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX), or nucleotides modified with 5'-phosphate mimics.

[0055] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0056] Compared with the prior art, the present invention has the following beneficial effects.

[0057] 1. The oligonucleotides of the present invention have significant inhibitory effects on HBsAg, HBV DNA and HBeAg in HBV transgenic mice, with low toxicity; the formulation is simple, which is more conducive to the safety of the product, and it can maintain long-term stability. Even at a concentration of about 200 mg / mL, it can remain stable at room temperature for 24 months or longer; activity.

[0058] 2. The preparation method provided by the present invention has the advantages of simple operation, mild conditions, green and environmentally friendly, no need for complex equipment, low production cost, and suitability for large-scale industrial production. Attached Figure Description

[0059] Figure 1 The present invention relates to a process for preparing oligonucleotides (siRNA).

[0060] Figure 2 The oligonucleotides of the present invention exhibit inhibitory effects on serum HBV DNA, HBeAg, and HBsAg in HBV transgenic mice after a single subcutaneous administration.

[0061] Figure 3 The oligonucleotides of the present invention exhibit inhibitory effects on serum HBV DNA, HBeAg, and HBsAg in HBV transgenic mice after a single subcutaneous administration.

[0062] Figure 4 The curves showing the effect of the oligonucleotides of this invention on the weight changes of male and female SD rats.

[0063] Figure 5 The effects of oligonucleotides of this invention on the expression levels of aspartate aminotransferase in male and female SD rats. Detailed Implementation

[0064] The embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0065] In the specific implementation, L96 is purchased custom-made from the market (from a commercial company) or prepared according to the method of CN104717982A.

[0066] Automated Oligonucleotide Synthesizer: Dr. Oligo 192XLc (Kunshan Berlik Precision Instruments Co., Ltd.)

[0067] All phosphoramidite monomers, reagents, and purification consumables used were commercially available reagents and consumables, such as various phosphoramidite monomers (e.g., 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (e.g., 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) purchased from Sigma-Aldrich LLC.

[0068] The siRNA synthesis and purification methods used in this invention are as described in US20130178612A1, US2015100197A1, etc.; the synthesis method of the VPUm and APU001 structural sequences is as described in J. Med. Chem. 2018, 61, 734−744.

[0069] Example 1: Design of the oligonucleotide (siRNA) of the present invention

[0070] Candidate oligonucleotide sequences complementary to the hepatitis B virus (Hepatitis B virus) D genotype (subtypeayw, V01460 J02203) were generated using computer algorithms. Some sequences were also complementary to genotypes A, B, C, etc., or had no more than two mismatches.

[0071] The siRNA is designed as a double strand with 19 / 21 pairs on the sense and antisense strands, with the antisense strand having two dangling ends complementary to the mRNA sequence. In some complementary pairing sequences, the 1st and / or 19th bases at the 5' end of the sense strand are replaced with bases different from those in HBV mRNA, and the corresponding bases in the antisense strand are modified accordingly to form a pair with the sense strand. In some sequences, bases at individual positions within the sense strand are replaced, forming mismatches with the corresponding positions in the antisense strand. In some sequences, deoxyribonucleotides are used to replace ribonucleotides at individual positions. In some sequences, hypoxanthine (I) is used to replace U, A, or G at individual positions in the antisense strand to form a wobbly pair with the sense strand. The modified oligonucleotide sequences of the siRNA designed in this invention and the modified oligonucleotide sequences optimized based on AL0107044 are shown in Table 1. Table 1. siRNA sequences of the present invention AL0107044 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm_L96 1 UmsGfsGmUmGmAfAmGfCmGmAmAfImUfGmCfAmCfAmsGmsGm 2 AL0107045 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm_L96 1 VpUmsGfsGmUmGmAfAmGfCmGmAmAfImUfGmCfAmCfAmsGmsGm 3 AL0107048 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm_L96 1 APU001sGfsGmUmGmAfAmGfCmGmAmAfImUfGmCfAmCfAmsGmsGm 4 AL0107049 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm_L96 1 APU001sGfsGmUm(Ggn)AfAmGfCmGmAmAfImUfGmCfAmCfAmsGmsGm 5 AL0107050 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm L96 1 APU001sGfsGmUmGmAf(Agn)GfCmGmAmAfImUfGmCfAmCfAmsGmsGm 6 AL0107051 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm L96 1 APU001sGfsGmUm(GNF-BX)AfAmGfCmGmAmAfImUfGmCfAmCfAmsGmsGm 7 AL0107052 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm L96 1 APU001sGfsGmUmGmAf(GNF-BX)GfCmGmAmAfImUfGmCfAmCfAmsGmsGm 8 AL0107053 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm L96 1 APU001sGfsGmUmGmAf(A-2’-5’)GfCmGmAmAfImUfGmCfAmCfAmsGmsGm 9 AL0107057 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm_L96 1 VpUmsGfsGmUm(Ggn)AfAmGfCmGmAmAfImUfGmCfAmCfAmsGmsGm 10 AL0107058 UmsGmsUmGmCmAmCfUmCfCfGmCmUmUmCmAmCmCmAm L96 1 VpUmsGfsGmUmGmAf(A-2'-5')GfCmGmAmAfImUfGmCfAmCfAmsGmsGm 11 Note: “G”, “C”, “A”, “U”, “T” and “I” usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively.

[0072] Modifications: d represents deoxynucleotide; m represents 2'-methoxy; f represents 2'-deoxy-2'-fluorine; Moe represents 2'-O-methoxyethyl; s represents 3'-thiophosphate; Vp represents 5'-vinylphosphonic acid (modified at the 5' end of the antisense chain).

[0073] A-2'-5' represents: adenosine-2' phosphate; U-2'-5' represents uridine-2' phosphate; G-2'-5' represents guanosine-2' phosphate; Ggn represents: guanosine-diol nucleic acid; Agn represents: adenosine-diol nucleic acid; Ugn represents: uridine-diol nucleic acid; GNF-BX represents: N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide; APU001 represents: 5' vinylphosphonate-2'-N-acetyl-uridine.

[0074] With sequence 2 as a corresponding reference, sequences 3-11 replace the nucleic acid analogs at the corresponding positions.

[0075] L96 indicates that the carrier structure is as follows:

[0076] Furthermore, the ligand is conjugated to the 3'-end of the sense strand of the oligonucleotide via a linker, forming the following conjugate: X is O.

[0077] Example 2 Preparation of the oligonucleotide (siRNA) of the present invention

[0078] Nucleic acid sequence representation uses abbreviations for nucleotide monomers. It will be understood that these monomers, when present in oligonucleotides, are interconnected by 5'-3' phosphodiester bonds unless otherwise stated.

[0079] The preparation of the oligonucleotides of the present invention includes the following steps:

[0080] 1. Preparation of siRNA The siRNA sequence is synthesized on a solid support carrier via a sense strand (SS) and an antisense strand (AS), and is obtained by deprotection, cleavage, purification, annealing, purification, and lyophilization.

[0081] Solid-phase synthesis process (see) Figure 1 The sense and antisense strands were synthesized separately on a solid-phase support using an automated oligonucleotide synthesizer with phosphoramide technology. Solid-phase synthesis started from the 3' end of the sequence and monomers were coupled into the sequence sequentially. Each coupling of a phosphoramide monomer involved four chemical steps: 1) unblocking or deprotection (deprotection of hydroxyl protecting groups); 2) coupling; 3) oxidation; and 4) end capping.

[0082] 2. Preparation of double-stranded RNA reagent

[0083] (1) The preparation of the justice chain includes the following steps:

[0084] Oligonucleotides were synthesized using a solid-phase phosphoramide method. A computer-controlled synthesizer was used, and the reaction was carried out in a stainless steel column. The positive chain synthesis started with a solid-phase support loaded with a targeting ligand (e.g., L96), or directly with the solid-phase support. Different starting materials, reagents, and solvents were injected sequentially from sequence 3' to 5' using different tubing within the solid-phase synthesizer, ligating phosphoramide nucleoside monomers one by one. The reaction process consisted of four cyclic steps: DMT protection removal, condensation, oxidation or thiolation, and end-capping. One nucleotide unit was ligated in each cycle, yielding oligonucleotide sequences of 19 or 21 nucleotides. After synthesis, the protecting group (2-cyanoethyl) was removed on the solid-phase column, and the synthesized sequence was cleaved from the solid-phase support via ammonolysis. The sequence was filtered, the filter cake was washed with ethanol, and the filtrate and washings were collected, concentrated, and the crude positive chain was purified by chromatography (SOURCE 15Q) and lyophilized to obtain the final product.

[0085] (2) The preparation of the antisense strand includes the following steps: The synthesis of the antisense strand is similar to that of the sense strand. A solid-phase synthesizer is used to control different pipelines to inject different raw materials, reagents and solvents in the sequence from 3' to 5', so as to connect the phosphoramidite nucleoside monomers one by one. The reaction process comprises a four-step cycle consisting of DMT protecting group removal reaction, coupling reaction, oxidation or thioation reaction, and capping reaction. Each cycle connects one nucleotide unit to obtain an oligonucleic acid sequence with 21 or 23 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, then the synthesized sequence is cleaved from the solid support carrier through ammonolysis reaction, followed by filtration, the filter cake is washed with ethanol, the filtrate and the washing liquid are collected and concentrated. The obtained crude antisense strand product is subjected to chromatographic purification (SOURCE 15Q), ultrafiltration and lyophilization to obtain the final product.

[0086] (3) The preparation of the siRNA duplex includes the following steps:

[0087] The AS strand and the SS strand are respectively dissolved in water for injection, mixed at a defined ratio (1.01:1.0-1.2:1.0), incubated at 30-50°C for 30-90 min, and then cooled to room temperature. After freeze-drying, the double-stranded siRNA product is obtained.

[0088] Example 3 Evaluation of the anti-hepatitis B virus (HBV) activity of the oligonucleotide of the present invention using HBV transgenic mice SPF grade male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Vitalstar Biotechnology Co., Ltd., animal production certificate SCXK (Beijing) 2019-0002) are used in the test. Each mouse is housed in a separate cage. The use and testing of animal feed, bedding and drinking water are carried out in accordance with the specifications of GB14925-2010 "Laboratory Animal Environment and Facilities".

[0089] The test is carried out after the animals are acclimatized for one week. According to the quantitative detection results of mouse serum HBsAg (primary) and HBV DNA (secondary), the animals are randomly divided into 2 groups with 5 mice in each group. The day of administration is recorded as D1. Blood is collected before administration on the day of administration to measure HBsAg, HBV DNA and HBeAg.

[0090] All animals are administered by subcutaneous injection on D1, all with a single injection at 3mg / kg, the administration volume is 5mL / kg. Normal saline is used as the control group, which is injected with the same dose of normal saline. The test animals are observed 15 minutes to 1 hour after the last animal in each group is administered.

[0091] On days 8, 15, 22, 29, 35, and 42 after test drug administration, 100 μL of blood was collected from the orbit, serum was separated by centrifugation, 10 μL of serum was diluted 50-fold with PBS, and serum HBsAg, HBeAg, and HBVDNA were tested by Beijing Dian Medical Laboratory Co., Ltd.

[0092] The test data were statistically analyzed using Graphpad prism statistical analysis software. The calculation of the residual inhibition rates of HBV DNA, HBsAg and HBeAg was based on the average value of two measurements before drug administration as the baseline value: Residual inhibition rate = measured value at different time points / baseline value before drug administration * 100%. The results are shown in Table 2.

[0093] Table 2 Residual rates of serum HBsAg, HBeAg and HBV DNA in HBV transgenic mice after single subcutaneous administration

[0094] It can be seen from Table 2 that both AL0107045 and AL0107044 have significant inhibitory effects on HBsAg, HBV DNA and HBeAg in HBV transgenic mice.

[0095] Example 4 Investigation of the anti-hepatitis B virus (HBV) activity of the oligonucleotide of the present invention using HBV transgenic mice SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Vitalstar Biotechnology Co., Ltd., animal production license SCXK (Beijing) 2019-0002) were used in the test. Each mouse was housed individually in a single cage. The use and testing of animal feed, bedding and drinking water were carried out in accordance with the specifications of GB14925-2010 "Laboratory Animal Environment and Facilities".

[0096] The test was carried out after the animals had adapted to feeding for one week. According to the quantitative test results of mouse serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into 9 groups, with 5 mice in each group. The day of administration was recorded as D1, and blood was collected before administration on the day of administration for the determination of HBsAg, HBV DNA and HBeAg.

[0097] All animals were administered by subcutaneous injection on D1, all as a single injection, with a administration dose of 3 mg / kg and a administration volume of 5 mL / kg. Normal saline was used as the control group, and an equal volume of normal saline was injected. The test animals were observed for 15 minutes to 1 hour after the last animal in each group was administered.

[0098] On days 8, 15, 22, and 29 after administration, 100 μL of blood was collected from the orbit, serum was separated by centrifugation, 10 μL of serum was diluted 50-fold with PBS, and serum HBsAg, HBeAg, and HBV DNA were tested by Beijing Dian Medical Laboratory Co., Ltd.

[0099] The test data was statistically analyzed using Graphpad prism statistical analysis software. The residual inhibition rates of HBV DNA, HBsAg and HBeAg were calculated based on the average value of the two measurements before administration: Residual inhibition rate = measured value at different time points / baseline value before administration * 100%. The results are shown in Tables 3-5 and Figure 2 .

[0100] Table 3 Inhibitory effect on serum HBV DNA in HBV transgenic mice after single subcutaneous administration [Log 10 HBV DNA (IU / mL)] (mean ± standard deviation)

[0101] Table 4. Inhibitory effect on serum HBeAg in HBV transgenic mice after single subcutaneous administration [Log 10 HBeAg (IU / mL)] (mean ± standard deviation)

[0102] Table 5 Inhibitory effect on serum HBsAg in HBV transgenic mice after single subcutaneous administration [Log 10 HBsAg (IU / mL)] (mean ± standard deviation)

[0103] As can be seen from Tables 3-5 and Figure 2 , the oligonucleotide of the present invention has a significant inhibitory effect on HBsAg, HBV DNA and HBeAg in HBV transgenic mice.

[0104] Example 5 Evaluation of the anti-hepatitis B virus (HBV) activity of the oligonucleotide of the present invention using HBV transgenic mice SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice aged 6-8 weeks (Beijing Vitalstar Biotechnology Co., Ltd., Animal Production License SCXK (Beijing) 2019-0002) were used in the test. Each mouse was housed in a separate cage. The use and testing of animal feed, bedding and drinking water were carried out in accordance with the specifications of GB14925-2010 "Laboratory Animal Environment and Facilities".

[0105] The test was carried out after the animals had adapted to feeding for one week. According to the quantitative detection results of serum HBsAg (primary) and HBV DNA (secondary) in the mice, the animals were randomly divided into 10 groups, with 5 mice in each group. The day of administration was recorded as D1. Blood was collected before administration on the day of administration for the determination of HBsAg, HBV DNA and HBeAg.

[0106] All animals received a single subcutaneous injection on day 1 at a dose of 3 mg / kg, administered in a volume of 5 mL / kg. A control group received the same volume of physiological saline. On days 7, 14, 21, 28, and 35 post-administration, 100 μL of blood was collected from the orbital sinus. Serum was separated by centrifugation, and 10 μL of serum was diluted 50-fold with PBS. Serum HBsAg, HBeAg, and HBV DNA were then measured by Beijing Dian Medical Laboratory Co., Ltd.

[0107] The experimental data were statistically analyzed using Graphpad Prism software. The results are shown in Tables 6-8 and 6-8. Figure 3 .

[0108] Table 6. Inhibitory effect of a single subcutaneous administration on serum HBV DNA in HBV transgenic mice [Log] 10 HBV DNA (IU / mL) [Mean ± Standard Deviation]

[0109] Table 7. Inhibitory effect of a single subcutaneous administration on serum HBeAg in HBV transgenic mice [Log] 10 HBeAg (IU / mL) [mean ± standard deviation]

[0110] Table 8. Inhibitory effect of a single subcutaneous administration on serum HBsAg in HBV transgenic mice [Log] 10 HBsAg (IU / mL) [mean ± standard deviation]

[0111] Example 6: Evaluation of in vivo toxicity of different concentrations of compounds using SD rats Eighty-six qualified SPF-grade SD rats (half male and half female) were selected for the experiment and randomly stratified by body weight into four groups: a control group (N group, 0.9% sodium chloride injection), AL0107045S group, AL7045M group, AL7045H group, AL0107057S group, AL0107057M group, AL0107057H group, AL0107058S group, AL0107058M group, and AL0107058H group, with eight animals in each group (half male and half female). After grouping, the test substance / solvent was administered subcutaneously three times (AL0107045, AL0107057, AL0107058 on day 1, day 15, and day 29). The day of the first administration of the test substance was designated as day 1. The test samples for groups S, M, and H were administered at doses of 50.0, 100.0, and 300.0 mg / kg / dose (body weight), respectively, while group N was administered 0.9% sodium chloride injection. The volume of each dose was 5.0 mL / kg / dose. Body weight was measured on days 1, 5, 7, 12, 14, 19, 21, 26, and 28. On the day of the last administration, all animals were fasted overnight, anesthetized via intraperitoneal injection, and blood was collected from the peritoneal vein for serum biochemical analysis. Results are shown below. Figure 5 .

[0112] See weight changes Figure 4 In AL0107045, all animal groups showed slow weight gain compared to the saline group from days 7 to 14 of the experiment, and even negative weight gain from days 12 to 14. Males and females showed the same response. Weight gain returned to normal from days 14 to 28. In AL0107057 and AL0107058, there was no significant difference in weight gain compared to the saline group.

[0113] Aspartate aminotransferase (AST) Figure 5 The results showed that all dose groups of AL0107045 had significantly higher levels than the saline group; all dose groups of AL0107057 and AL0107058 had no significant effect on aspartate aminotransferase (AST), and the weight gain and AST levels of AL0107057 and AL0107058 were not significantly different from the control group.

[0114] Example 7 Formulation composition Table 9. Composition of the formulation per milliliter Composition 1 AL0107058 200mg \ Add to 1 mL 6.0 Composition 2 AL0107058 200mg \ Add to 1 mL 7.0 Composition 3 AL0107058 100mg 4.75 mg / mL Add to 1 mL 6.0 The preparation method is as follows: Take the required amount of water for injection and NaCl into the preparation container and stir to dissolve; add the required amount of oligonucleotide to the above solution, stir until completely dissolved, and adjust the pH value to the target value to obtain the final product.

[0115] Table 10 Composition of the formulation per milliliter Composition 4 AL0107058 200mg Sodium dihydrogen phosphate dihydrate - Sodium dihydrogen phosphate dihydrate (10 mmol / L) \ Add to 1 mL 6.0 Composition 5 AL0107058 200mg Disodium hydrogen phosphate dihydrate - Sodium dihydrogen phosphate dihydrate (20 mmol / L) \ Add to 1 mL 6.0 Composition 6 AL0107057 200mg Sodium dihydrogen phosphate dihydrate - Sodium dihydrogen phosphate dihydrate (10 mmol / L) \ Add to 1 mL 6.0 Composition 7 AL0107057 100mg Sodium dihydrogen phosphate dihydrate - Sodium dihydrogen phosphate dihydrate (10 mmol / L) 4mg / mL Add to 1 mL 6.0 Composition 8 AL0107057 10mg Disodium hydrogen phosphate dihydrate - Sodium dihydrogen phosphate dihydrate (50 mmol / L) 6.4 mg / mL Add to 1 mL 6.5 Composition 9 AL0107058 5mg Disodium hydrogen phosphate dihydrate - Sodium dihydrogen phosphate dihydrate (50 mmol / L) 10mg / mL Add to 1 mL 6.5 Preparation method: Take the required amount of water for injection, phosphate buffer and NaCl (if available) into the preparation container and stir to dissolve; add the required amount of oligonucleotide to the above solution, stir until completely dissolved, and adjust the pH value to the required value to obtain the final product.

[0116] Table 11 Results of stability study of composition 1

[0117] Table 12 Results of stability study of composition 2

[0118] Table 13 Results of stability study of composition 3

[0119] Table 14 Results of stability study of composition 4

[0120] Table 15 Results of stability study of composition 5

[0121] Table 16 Results of stability study of composition 7

[0122] Table 17 Results of stability study of composition 9

[0123] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims.

Claims

1. An oligonucleotide composition comprising an oligonucleotide and a pharmaceutically acceptable carrier.

2. The composition according to claim 1, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein, The sense chain includes the sequence or fragment shown in SEQ ID NO: 1, and the antisense chain includes the sequence or fragment shown in SEQ ID NO: 2; Preferably, the antisense strand SEQ ID NO: 2 is optionally modified or replaced by any one or a combination of adenosine-2' phosphate (A-2'-5'), guanosine-diol nucleic acid (Ggn), adenosine-diol nucleic acid (Agn), N-(2,3-dihydroxypropyl)-3,5-bis(trifluoromethyl)benzamide (GNF-BX), and 5'-phosphate ester mimics; More preferably, the 5'-phosphate ester analogue is selected from any one or a combination of 5'-oxymethylphosphonate, 5'-vinylphosphonic acid (Vp), 5'-vinylphosphonate-2'-N-acetyl, and 5'-malonylphosphonate.

3. The composition according to claim 2, wherein the 5' end of the antisense strand is modified or substituted at any one of the nucleic acid positions from the 1st to the 8th position.

4. The composition according to claim 3, wherein the 5'-phosphate mimic is modified at the 5' end of the antisense strand, preferably the 5'-phosphate mimic is modified at the 5' end of the antisense strand and the nucleic acid at any position 2 to 8 at the 5' end is replaced by any one or a combination of A-2'-5', Ggn, Agn, GNF-BX; more preferably the 5'-vinylphosphonic acid is modified at the 5' end of the antisense strand and Ggn substitution occurs at the 5th position of the nucleic acid at the 5' end of the antisense strand, or the 5'-vinylphosphonic acid is modified at the 5' end of the antisense strand and A-2'-5' substitution occurs at the 7th position of the nucleic acid at the 5' end of the antisense strand.

5. The composition according to claim 4, wherein the sense strand of the oligonucleotide is the sequence or fragment shown in SEQ ID NO: 1, and the antisense strand is selected from any one of the sequences or fragments shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO:

11.

6. The composition according to any one of claims 1-5, wherein the composition is a liquid formulation, preferably containing 0.1% to 30% (mass / volume) oligonucleotides, more preferably having a pH of 5.00 to 8.

00.

7. The composition according to any one of claims 1-6, wherein the composition contains a buffer of 5 mM-50 mM, preferably selected from any one of disodium hydrogen phosphate-sodium dihydrogen phosphate, citric acid-sodium citrate, carbonate, histidine-histidine hydrochloride, acetate-sodium acetate or sodium citrate.

8. The composition according to any one of claims 1-7, wherein the composition contains 10%-25% (mass / volume) oligonucleotides, 10mM-20mM buffer, and water for injection, and the composition has a pH of 6.0-7.0; Alternatively, the composition contains 0.4%-2% (w / v) oligonucleotides, 30mM-50mM buffer, and water for injection, wherein the composition has a pH of 6.0-7.

0. The buffer is selected from disodium hydrogen phosphate-sodium dihydrogen phosphate.

9. Use of the composition according to any one of claims 1-8 in the preparation of a medicament for treating HBV infection, hepatitis B, or hepatitis B virus infection combined with hepatitis D virus infection or its complications.

10. A combination package comprising the composition of any one of claims 1-8, and other active pharmaceutical ingredients for the prevention or treatment of hepatitis B.

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