Pharmaceutical compositions comprising oligonucleotides

Abstract

The present disclosure relates to a pharmaceutical combination of oligonucleotides for inhibiting hepatitis B virus, comprising a biologically active ingredient oligonucleotide, and selected from hepatitis B antibodies and / or interferon, the combination described herein has a therapeutic advantage over the use of each alone, and can be used for treating or preventing diseases or conditions associated with hepatitis B virus (HBV) infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a drug combination containing oligonucleotides and other drugs for the prevention and treatment of hepatitis B, and its use, which can be used for the treatment of hepatitis B. 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 viral replication suppression 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 use of related small interfering RNA in combination with other drugs for the prevention and treatment of hepatitis B. This invention is an in-depth development of the patented technology, and some of the technical solutions therein are also applicable to this invention, and are therefore incorporated into this invention. Summary of the Invention

[0009] This invention provides a pharmaceutical combination containing oligonucleotides, the pharmaceutical combination comprising... (1) Oligonucleotides; and those selected from (2) Any one or a combination of hepatitis B antibody, interferon or nucleoside analogue.

[0010] The drug combination of the present invention contains oligonucleotides in an amount of 5-300 mg / minimum dose unit, preferably 100-250 mg / minimum dose unit.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In a preferred embodiment of the present invention, any one of the nucleic acids at the 2nd to 8th position of the 5'-terminus of the antisense strand 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, 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.

[0017] 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.

[0018] 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 A-2'-5' substitution occurs at the 7th nucleic acid position at the 5' end of the antisense strand.

[0019] 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.

[0020] 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.

[0021] 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: 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 some specific embodiments, the antibody described in this invention is a hepatitis B antibody, such as the antibody described in CN2021107808175 (e.g., antibody number 005 / 021) and the antibody described in CN2023118643457 (e.g., antibody number 055), which are hereby incorporated into this patent. One specific embodiment is that the antibody comprises HCDR and LCDR as described below: The sequence of HCDR1 is GYTFTGYY (SEQ ID NO: 12), the sequence of HCDR2 is INPNSGGT (SEQ ID NO: 13), the sequence of HCDR3 is ARDLWNDDVDYYGMDV (SEQ ID NO: 14); and the sequence of LCDR1 is QSISTY (SEQ ID NO: 15), the sequence of LCDR2 is AAS (SEQ ID NO: 16), and the sequence of LCDR3 is QQSYSTPLT (SEQ ID NO: 17) (021 antibody).

[0024] In one specific embodiment of the present invention, the interferon is a PEG-interferon, such as selected from Pegasys®, Pegasys®, Pegasys®, or Pegasys®, all of which are commercially available.

[0025] In one specific embodiment of the present invention, the nucleoside analogue is selected from adefovir, entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, tenofovir alafenamide fumarate, and lamivudine, preferably entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and tenofovir alafenamide fumarate, all of which are commercially available.

[0026] In yet another preferred embodiment of the invention, the combination may optionally further include a CAM anti-hepatitis B virus agent, a hepatitis B therapeutic vaccine, or an immunomodulator.

[0027] In one embodiment of the present invention, the drug combination is a combination package comprising: an oligonucleotide formulation, and a combination of hepatitis B antibody formulation, interferon formulation, or nucleotide drug product or a combination thereof. In a specific embodiment, the combination package contains: an oligonucleotide formulation at a concentration of 100-250 mg / ml, a hepatitis B antibody formulation at a concentration of 10-200 mg / ml and / or a PEG-IFN-α formulation at a concentration of 0.05-0.2 mg / ml, and a nucleoside analog formulation, wherein the nucleoside analog formulation is a commercially available unit dose.

[0028] In one embodiment of the present invention, a stable oligonucleotide combination is provided, comprising: (1) Oligonucleotides with a concentration of 5-300 mg / ml; (2) A buffer with a concentration of 5mM-50mM, wherein the buffer is selected from a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, a citrate-sodium citrate buffer system, a carbonate buffer system, a histidine-histidine hydrochloride buffer system, an acetate-sodium acetate buffer system or a sodium citrate buffer system. (3) Water for injection; Its pH is 6.0-7.0.

[0029] Chinese patent CN2022116602621 discloses a composition containing hepatitis B antibodies (such as O21 antibodies), which is incorporated herein by reference.

[0030] In one specific embodiment of the present invention, wherein, The oligonucleotide preparation comprises: (1) oligonucleotides at a concentration of 100-250 mg / ml, (2) a buffer system at a concentration of 10 mM-20 mM, wherein the buffer system is selected from the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, and (3) water for injection, wherein the pH of the oligonucleotide preparation is 6.0-7.0. The hepatitis B antibody preparation consists of: (1) hepatitis B antibody at a concentration of 20-200 mg / ml, and (2) histidine buffer at a concentration of 5-15 mmol / L. (3) sorbitol and glycine at a concentration of 10-20 mg / ml, (4) Tween 80 at a concentration of 0.1-0.5 mg / ml, and (5) methionine at a concentration of 0-10 mg / ml, and water for injection, wherein the composition has a pH of 6.0-6.3; The PEG-IFN-α formulation consists of: (1) PEG-IFN-α at a concentration of 0.05-5 mg / ml, (2) histidine hydrochloride at a concentration of 1-3 mg / ml, (3) sorbitol at a concentration of 20-60 mg / ml, (4) methionine at a concentration of 0.5-5 mg / ml, (5) Tween 80 at a concentration of 0.1-0.5 mg / ml, and water for injection at a pH of 5.0-6.0.

[0031] In another embodiment of the invention, the use of the combination described herein in the preparation of a medicament for treating HBV infection or hepatitis B, and / or hepatitis B virus co-infection with hepatitis D virus is provided.

[0032] In some implementations, the HBV infection is a chronic HBV infection.

[0033] In some implementations, the hepatitis B is chronic hepatitis B.

[0034] 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.

[0035] Unless otherwise stated, the scientific and technical terms used in this invention have the meanings commonly understood by those skilled in the art.

[0036] The minimum dosage unit described in this invention is the smallest unit of a drug used in clinical practice, usually in the form of a single vial, bottle, bag, or tablet. In clinical use, clinicians can determine the amount of the minimum dosage unit required for different patients based on actual clinical needs, such as 1, 2, 3, or 4 minimum dosage units.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Compared with the prior art, the present invention has the following beneficial effects: 1. The oligonucleotides of this invention have a significant inhibitory effect on HBsAg, HBV DNA, and HBeAg in HBV transgenic mice, with low toxicity. 2. The oligonucleotides of this invention, when combined with other hepatitis B prevention and treatment drugs, such as antibodies or interferon, exhibit even better efficacy. Attached Figure Description

[0044] Figure 1 The siRNA synthesis process is shown.

[0045] Figure 2 The inhibitory effects of compounds AL0107045, AL0107048, AL0107049, AL0107050, AL0107051, AL0107052, AL0107053, and AL0107054 on serum HBV DNA, HBeAg, and HBsAg in HBV transgenic mice after a single subcutaneous administration were demonstrated.

[0046] Figure 3 The inhibitory effects of compounds L0107045, AL0107057, and AL0107058 on serum HBV DNA, HBeAg, and HBsAg in HBV transgenic mice after a single subcutaneous administration were demonstrated.

[0047] Figure 4 The effects of different compounds on body weight changes in male and female SD rats are shown in the curves.

[0048] Figure 5 The effects of different compounds on the expression levels of aspartate aminotransferase (AST) in male and female SD rats were shown.

[0049] Figure 6 To investigate the effects of different drug combinations on HBsAg in hepatitis B mice.

[0050] Figure 7 To investigate the effects of different drug combinations on HBV DNA in hepatitis B mice. Detailed Implementation

[0051] The embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0052] In the specific implementation, L96 is purchased from the market by custom order (from a commercial company), or it can be prepared according to the method of CN104717982A.

[0053] Automated Oligonucleotide Synthesizer: Dr. Oligo 192XLc (Kunshan Berlik Precision Instruments Co., Ltd.)

[0054] 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.

[0055] 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. Example 1: Design of siRNA

[0056] First, candidate oligonucleotide sequences complementary to the Hepatitis B virus (HBV) D genotype (subtype ayw, V01460 J02203) were generated using a computer algorithm. Some sequences were also complementary to genotypes A, B, and C, or had no more than two mismatches. The siRNA was designed as a double strand with 19 / 21 pairings 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 were replaced with bases different from those in the HBV mRNA, and the corresponding bases in the antisense strand were modified accordingly to form a pair with the sense strand. In some sequences, bases at individual positions within the sense strand were substituted, forming mismatches with the corresponding positions in the antisense strand. In some sequences, deoxyribonucleotides were used to replace ribonucleotides at individual positions. In some sequences, hypoxanthine (I) was used to replace U, A, or G at individual positions in the antisense strand, forming a wobble pair with the sense strand. Table 1 lists the designed siRNA modified oligonucleotide sequences. Table 1 lists the modified oligonucleotide sequences optimized based on AL0107044. Table 1. siRNA sequences with vector Note: “G”, “C”, “A”, “U”, “T” and “I” usually represent nucleotides with guanine, cytosine, adenine, uracil, thymine and hypoxanthine as bases, respectively. 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). 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. With sequence 2 as a corresponding reference, sequences 3-11 replace the nucleic acid analogs at the corresponding positions. Wherein, L96 represents the vector. Alternatively, the structural formula of the ligand conjugated with the oligonucleotide is shown below: ; 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. Example 2: Preparation of siRNA

[0057] Abbreviations for nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in oligonucleotides, are interconnected by 5'-3' phosphodiester bonds unless otherwise stated. L96 can be purchased commercially or custom-made, or prepared according to methods disclosed in patents or literature in the prior art, such as CN104717982A, which discloses a method for preparing L96.

[0058] The preparation process of oligonucleotides is as follows.

[0059] (1) Preparation of siRNA

[0060] The siRNA sequence was synthesized separately on a solid support carrier via the sense strand (SS) and antisense strand (AS), and was obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0061] Solid-phase synthesis ( Figure 1The sense and antisense strands were synthesized separately on a solid-phase support using an automated oligonucleotide synthesizer, employing phosphoramide technology. The synthesizer, such as the AKTA Oligopilot (Cytiva) or Dr. Oligo192XLc (Kunshan Berlik Precision Instruments Co., Ltd.), was used. Solid-phase synthesis began at the 3' end of the sequence, with monomers sequentially coupled into the sequence. Each coupling of a phosphoramide monomer involved four chemical steps: 1) unblocking or deprotection (de-hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) end-capping. All phosphoramide monomers, reagents, and purification consumables used were commercially available, including various phosphoramide monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution and 28wt% ammonium hydroxide aqueous solution) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are described in US20130178612A1, US2015100197A1, etc.; the synthesis methods for VPUm and APU structural sequences are described in J. Med. Chem. 2018, 61, 734−744.

[0062] (2) Preparation of double-stranded RNA reagent

[0063] The process of synthesizing the justice chain is as follows: Oligonucleotides were synthesized using a solid-phase phosphoramidite 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 the solid-phase synthesizer, linking phosphoramidite 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 linked in each cycle, yielding an oligonucleotide sequence 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 and concentrated to obtain the crude positive chain. The crude product was purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the positive chain.

[0064] The synthesis process of the antisense chain is as follows: The synthesis of the antisense strand is similar to that of the sense strand. By controlling different pipelines with a solid-phase synthesizer, different raw materials, reagents and solvents are injected in the order from 3' to 5' of the sequence, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four-step cycles of DMT protecting group removal reaction, condensation reaction, oxidation or thiolation reaction, and capping reaction. Each cycle attaches one nucleotide unit, and an oligonucleotide sequence of 21 or 23 nucleotide units is obtained. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and then the synthesized sequence is cleaved from the solid-phase support carrier through an ammonolysis reaction, filtered, the filter cake is washed with ethanol, the filtrate and washing solution are collected, and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and lyophilized to obtain the target product, the antisense strand.

[0065] The preparation process of the siRNA duplex is as follows: The AS strand and the SS strand are respectively dissolved in injection water, mixed in a certain ratio (1.01:1.0 - 1.2:1.0), incubated at 30 - 50 °C for 30 - 90 min, and cooled to room temperature. After freeze-drying, the duplex siRNA product is obtained.

[0066] According to the same method, the duplex siRNA reagent conjugates in Table 1 and Table 2 above are prepared. Example 3 Evaluation of the anti-hepatitis B virus (HBV) activity of different compounds in HBV transgenic mice

[0067] SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice at 6 - 8 weeks old (Beijing Vitalstar Biotechnology Co., Ltd., animal production license SCXK (Beijing) 2019 - 0002) were used in the experiment. They were housed individually in single cages, and the use and detection of animal feed, bedding and drinking water were carried out in accordance with the specifications of GB14925 - 2010 "Laboratory Animal Environment and Facilities".

[0068] The animals were tested after one week of adaptive feeding. According to the quantitative detection results of mouse serum HBsAg (main) and HBV DNA (sub), the animals were randomly divided into 2 groups, with 5 animals in each group. The day of administration was recorded as D1. Blood was taken before administration on the day of administration for the determination of HBsAg, HBV DNA and HBeAg.

[0069] All animals were administered by subcutaneous injection on D1, all were single injections, at a dose of 3 mg / kg, and the administration volume was 5 mL / kg. Normal saline was used as the control group, and the same dose of normal saline was injected. The animals in each group were observed from 15 minutes to 1 hour after the last animal in each group was administered.

[0070] On the 8th, 15th, 22nd, 29th, 35th, and 42nd days after drug administration, 100 μL of blood was taken from the orbital cavity, serum was separated by centrifugation, and 10 μL of serum was diluted 50-fold with PBS and then sent for inspection. After dilution, HBsAg, HBeAg, and HBV DNA in the serum were tested by Beijing迪安医学检验实验室有限公司 (Beijing迪安Medical Laboratory Co., Ltd.).

[0071] The experimental data was statistically analyzed using Graphpad prism statistical analysis software. The calculation of the remaining inhibition rates of HBV DNA, HBsAg, and HBeAg was based on the average value of the two measurements before drug administration: Remaining inhibition rate = measured value at different times / baseline value before drug administration * 100%.

[0072] The experimental results are shown in Table 2. Table 2 Remaining rates of HBsAg, HBeAg, and HBV DNA in the serum of HBV transgenic mice after single subcutaneous administration

[0073] From the test results in Table 2, it can be seen that both compounds, AL0107045 and AL0107044, have significant inhibitory effects on HBsAg, HBV DNA, and HBeAg in HBV transgenic mice, and the effect of 7045 is better than that of 7044. Example 4 Evaluation of the anti-hepatitis B virus (HBV) activity of compounds in vivo using HBV transgenic mice

[0074] The experiment used SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice, 6 - 8 weeks old (Beijing维通达生物技术有限公司 (Beijing维通达Biotechnology Co., Ltd.), animal production certificate SCXK(Beijing)2019 - 0002). They were housed individually in single cages, and the use and detection of animal feed, bedding, and drinking water were carried out in accordance with the specifications of GB14925 - 2010 "Laboratory Animal Environment and Facilities".

[0075] The experiment was carried out one week after the animals were adapted to the feeding. According to the quantitative test results of HBsAg (main) and HBV DNA (sub) in the mouse serum, the animals were randomly divided into 9 groups, with 5 animals in each group. The day of drug administration was recorded as D1, and blood was taken before drug administration on the day of drug administration for the determination of HBsAg, HBV DNA, and HBeAg.

[0076] All animals were administered by subcutaneous injection on D1, all were single injections, the drug administration dose was 3 mg / kg, the drug administration volume was 5 mL / kg, and normal saline was used as the control group, injecting the same dose of normal saline. Each group of animals was observed 15 minutes to 1 hour after the last animal in the group was administered.

[0077] Blood samples of 100 μL were collected from the orbital cavity on days 8, 15, 22, and 29 after drug administration. Serum was separated by centrifugation, and 10 μL of serum was diluted 50-fold with PBS before being sent for testing. Serum HBsAg, HBeAg, and HBV DNA were analyzed by Beijing Dian Medical Laboratory Co., Ltd.

[0078] The experimental data were statistically analyzed using Graphpad Prism software. The residual inhibition rates of HBV DNA, HBsAg, and HBeAg were calculated based on the average of two pre-drug measurements: Residual inhibition rate = (Measurement value at different times / Pre-drug baseline value) * 100%.

[0079] The experimental results are shown in Table 3-5 and Figure 2 . Table 3. Inhibitory effect of a single subcutaneous administration on serum HBV DNA in HBV transgenic mice [Log] 10 HBV DNA (IU / mL) [Mean ± Standard Deviation] Table 4. Inhibitory effect of a single subcutaneous administration on serum HBeAg in HBV transgenic mice [Log] 10 HBeAg (IU / mL) [mean ± standard deviation] Table 5. Inhibitory effect of a single subcutaneous administration on serum HBsAg in HBV transgenic mice [Log] 10 HBsAg (IU / mL) [mean ± standard deviation]

[0080] From Table 3-5 and Figure 2 The test results showed that all compounds had significant inhibitory effects on HBsAg, HBV DNA and HBeAg in HBV transgenic mice, with compounds AL0107045 and AL0107048 showing the best inhibitory effects. The inhibitory efficiency of the optimized compounds (AL0107049-AL0107054) was slightly lower than that of the original compound AL0107048. Among the optimized compounds (AL0107049-AL0107054), AL0107049 and AL0107053 maintained relatively good inhibitory efficiency. Example 5: Evaluation of the in vivo anti-hepatitis B virus (HBV) activity of different compounds in HBV transgenic mice.

[0081] The experiment used SPF-grade male C57B / 6N-Tg(1.28HBV) / Vst mice at 6-8 weeks of age (Beijing Vitalstar Biotechnology Co., Ltd., animal production license SCXK(Jing)2019-0002). They were housed individually in single cages, and 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".

[0082] After the animals were acclimated for one week, the experiment was conducted. According to the quantitative test results of mouse serum HBsAg (primary) and HBV DNA (secondary), the animals were randomly divided into groups, with 5 animals in each group. The day of drug administration was recorded as D1. Blood was taken before drug administration on the day of drug administration for the determination of HBsAg, HBV DNA, and HBeAg.

[0083] All animals were administered by subcutaneous injection on D1, all were single injections, the dosing dose was 3 mg / kg, the dosing volume was 5 mL / kg, and normal saline was used as the control group, injecting the same dose of normal saline. On the 7th, 14th, 21st, 28th, and 35th days after drug administration, 100 μL of blood was taken from the orbital cavity, the serum was separated by centrifugation, and 10 μL of serum was diluted 50-fold with PBS and then sent for inspection. The diluted serum HBsAg, HBeAg, and HBV DNA were tested by Beijing迪安医学检验实验室有限公司 (Beijing迪安医学检验实验室有限公司 is not a standard English name and may need to be corrected to a proper English name if possible).

[0084] The experimental data were statistically analyzed using Graphpad prism statistical analysis software.

[0085] The experimental results are shown in Tables 6-8 and Figure 3 . Table 6. Inhibitory effect on serum HBV DNA of HBV transgenic mice after single subcutaneous administration [Log 10 HBV DNA (IU / mL)] (mean ± standard deviation) Table 7. Inhibitory effect on serum HBeAg of HBV transgenic mice after single subcutaneous administration [Log 10 HBeAg (IU / mL)] (mean ± standard deviation ) Table 8. Inhibitory effect on serum HBsAg of HBV transgenic mice after single subcutaneous administration [Log 10 HBsAg (IU / mL)] (mean ± standard deviation)

[0086] From Tables 6-8 and Figure 3The test results showed that compounds AL0107045, AL0107057, and AL0107058 all had significant inhibitory effects on HBsAg, HBV DNA, and HBeAg in HBV transgenic mice, with compound AL0107058 showing particularly significant effects. Compared to the original sequence AL0107045, AL0107058 unexpectedly enhanced the inhibitory effect on HBsAg, HBV DNA, and HBeAg, and this effect persisted throughout the entire experimental period. Example 6: Evaluation of in vivo toxicity of different concentrations of compounds using SD rats

[0087] 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. Animal 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. The experimental results are shown below. Figure 5-6 .

[0088] Results of weight change ( Figure 4 The results showed that, obviously, in all dose groups of AL0107045, the weight gain was slow compared to the saline group from days 7 to 14 of the experiment, and even negative weight gain was observed from days 12 to 14. The response was consistent between males and females, and the weight gain returned to normal from days 14 to 28. In contrast, there was no significant difference in weight gain between all dose groups of AL0107057 and AL0107058 and the saline group.

[0089] Aspartate aminotransferase (AST) Figure 5 The results showed that, obviously, 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.

[0090] Based on the changes in body weight and the results of aspartate aminotransferase (AST), it can be inferred that compound AL0107045 has significant toxic effects on rats, causing weight loss during the experiment and a significant increase in AST levels after the experiment, indicating that compound AL0107045 has a certain degree of liver toxicity. Compounds AL0107057 and AL0107058 were optimized based on AL0107045, and the results also showed no significant difference in weight gain and AST levels compared to the control group. It is obvious that compounds AL0107057 and AL0107058 optimized the toxic effects of AL0107045. Example 7: In vivo activity assay of oligonucleotides in combination with other hepatitis B treatment drugs

[0091] animal: Male C57BL / 6 mice, 5 weeks old and specific pathogen-free, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed in individually ventilated cages. Mice were housed and housed according to the experimental protocol approved by WuXi AppTec IACUC (IACUC #:ID01-013-2020v1.0). After a 4-day acclimatization period, mice were injected with AAV / HBV virus (i.e., recombinant AAV virus rAAV8-1.3HBV containing the complete genome of type D HBV; in this article, "AAV / HBV virus" and "rAAV8-1.3HBV" are used interchangeably).

[0092] Solvent: PBS.

[0093] Test compounds: Antibody 021 (prepared according to CN2021107808175, provided by Kain), PEG interferon (Paiyisheng®, provided by Kain), siRNA (AL0107058).

[0094] Recombinant rAAV8-1.3HBV: rAAV8-1.3HBV (type D, ayw) was provided by WuXi AppTec, batch number awy1-P4-200102, 1×10¹² viral genome (vg) / mL. It was diluted with sterile PBS to 5×10¹¹ v.g. / mL before the experiment. 200 μL was injected into each mouse, i.e., 1×10¹¹ v.g. per mouse.

[0095] Test method:

[0096] Establishment of AAV / HBV mouse model

[0097] AAV / HBV injection. rAAV8-1.3HBV was prepared in sterile PBS to a concentration of 1×10¹¹ v.g. / 200µL before injection. 200µL of rAAV8-1.3HBV solution was injected into 32 mice via the tail vein.

[0098] Blood was collected before grouping. On days 14, 21, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 112, 119, 126, and 163 post-injection, ~100 µL of blood was collected from the submandibular vein of all infected mice for plasma collection. The collected venous blood was anticoagulated with K2-EDTA, centrifuged at 4 °C, 7000 g / min for 10 minutes, and plasma was collected. HBV DNA was detected in the plasma by qPCR, and HBsAg was detected by ELISA. Plasma samples were stored at -80 °C until sent to the WuXi AppTec Biotechnology Department's in vitro laboratory for relevant testing.

[0099] Nineteen mice were selected from 23 mice for formal experiments and randomly divided into five groups, labeled as Group 1 to Group 6. Group 1 had 4 mice and the other groups had 3 mice. All mice were weighed before administration. The first administration was recorded as day 0. (1) PBS+PBS group: IV, QW, 10 mL / kg from day 0 to 63; SC, QW, 5 mL / kg from day 28 to 36; IP, QW, 5 mL / kg from day 70 to 77, once a week; (2) Antibody 021 group: IV, QW from day 0 to 63; IP, QW from day 70 to 77, all at a dose of 18 mpk, once a week; (3) siRNA (AL0107058) group: administered on day 0, 28 and 56, SC and QW, at a dose of 10 mpk; (4) Antibody 021 + Interferon group, Antibody 021, day 0-63, IV, QW, day 70-77, IP, QW, dose of 18mpk, once a week, Interferon, day 28-77, SC, QW, dose of 3mpk, once a week; (5) siRNA + Antibody 021 group, siRNA, injected on days 0, 28 and 56 (SC, QW), dose of 10mpk, Antibody 021, day 0-63, IV, QW, day 7 0-77 days, IP, QW, dose of 18mpk, once a week; (6) siRNA group + antibody 021 + interferon, siRNA, injected on days 0, 28 and 56 (SC, QW), dose of 10mpk, antibody 021, day 0-63, IV, QW, day 70-77, IP, QW, dose of 18mpk, once a week, interferon, day 28-77, SC, QW, dose of 3mpk, once a week. HBV DNA and HBsAg levels were measured based on plasma collected after viral injection, and the evaluation results were as follows. Figure 6 and 7 . Figure 6 and Figure 7The results showed that the combined use of siRNA and antibody, or siRNA, antibody, and interferon, was significantly more effective than using siRNA alone or antibody alone. After discontinuation of administration, the rebound rate of HBsAg or HBV DNA levels was significantly slower. Example 8 Formulation Table 9. Formulation composition per milliliter of preparation

[0100] Preparation process of buffer solution formulation:

[0101] Weigh 50% of the prescribed amount of water for injection into a preparation container. Add the weighed prescribed amounts of sodium dihydrogen phosphate (dihydrate) and disodium hydrogen phosphate (dihydrate) to the preparation container, stir, and visually dissolve. Add the weighed prescribed amount of oligonucleotide molecules to the preparation container, stir until completely dissolved, adjust the pH to 6.0 ± 0.2 using 0.15 mol / L phosphoric acid or 0.15 mol / L sodium hydroxide, stir, and weigh to the prescribed amount using water for injection. Table 10. Detection results of formulation-related items under different temperature conditions.

[0102] 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 drug combination, said drug combination containing (1) Oligonucleotides; and those selected from (2) Any one or a combination of hepatitis B antibodies, interferon, or nucleoside analogs. Preferably, in the drug combination, the oligonucleotide dose is 5-300 mg / minimum dosage unit, more preferably 100-250 mg / minimum dosage unit.

2. The drug combination 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 drug combination according to claim 2, wherein the 5' end of the antisense strand is modified or substituted at any of the 1st to 8th nucleic acid positions; preferably, the 5'-phosphate mimic is modified at the 5' end of the antisense strand, more preferably the 5'-phosphate mimic is modified at the 5' end of the antisense strand and the nucleic acid at any of the 2nd to 8th positions at the 5' end is substituted 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 nucleic acid position 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 nucleic acid position at the 5' end of the antisense strand.

4. The pharmaceutical combination according to claim 3, 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.

5. The pharmaceutical combination according to any one of claims 1-4, wherein the antibody comprises HCDR and LCDR as described below: The sequence of HCDR1 is GYTFTGYY (SEQ ID NO: 12), the sequence of HCDR2 is INPNSGGT (SEQ ID NO: 13), the sequence of HCDR3 is ARDLWNDDVDYYGMDV (SEQ ID NO: 14); and the sequence of LCDR1 is QSISTY (SEQ ID NO: 15), the sequence of LCDR2 is AAS (SEQ ID NO: 16), and the sequence of LCDR3 is QQSYSTPLT (SEQ ID NO: 17).

6. The combination according to any one of claims 1-4, wherein the interferon is a PEG-interferon, such as Pegasys. ® Peyron ® Paiyisheng ® or Pegbin ® Preferably, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and tenofovir alafenamide.

7. The pharmaceutical combination according to any one of claims 1-6, wherein the pharmaceutical combination optionally further comprises a CAM anti-hepatitis B virus agent, a hepatitis B therapeutic vaccine, or an immunomodulator.

8. The pharmaceutical combination according to any one of claims 1-7, wherein the pharmaceutical combination is a combination package, the combination package comprising: Oligonucleotide formulations with a concentration of 100-250 mg / ml, and formulations containing HBsAg hepatitis B antibodies with a concentration of 10-200 mg / ml and / or PEG-IFN-α formulations with a concentration of 0.05-0.2 mg / ml.

9. The pharmaceutical combination according to claim 8, wherein... The oligonucleotide formulation consists of: (1) oligonucleotides at a concentration of 5-250 mg / ml, (2) a buffer system at a concentration of 10 mM-20 mM, wherein the buffer system is selected from the disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, and (3) water for injection, wherein the pH of the oligonucleotide formulation is 6.0-7.

0. The hepatitis B antibody preparation comprises: (1) hepatitis B antibody at a concentration of 20-200 mg / ml, (2) histidine buffer at a concentration of 5-15 mmol / L, (3) sorbitol and glycine at a concentration of 10-20 mg / ml, (4) Tween 80 at a concentration of 0.1-0.5 mg / ml, and (5) methionine at a concentration of 0-10 mg / ml, and water for injection, wherein the pH of the composition is 6.0-6.3; The PEG-IFN-α formulation consists of: (1) PEG-IFN-α at a concentration of 0.05-5 mg / ml, (2) histidine hydrochloride at a concentration of 1-3 mg / ml, (3) sorbitol at a concentration of 20-60 mg / ml, (4) methionine at a concentration of 0.5-5 mg / ml, (5) Tween 80 at a concentration of 0.1-0.5 mg / ml, and water for injection at a pH of 5.0-6.

0.

10. Use of the pharmaceutical combination according to any one of claims 1-9 in the preparation of a medicament for treating HBV infection or hepatitis B, and / or hepatitis B virus co-infection with hepatitis D virus.

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