Hmgcr inhibitor and use thereof

Abstract

Provided are an HMGCR inhibitor and use thereof. The HMGCR inhibitor comprises a sense strand and an antisense strand, wherein the nucleotide sequence of the antisense strand comprises any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 4508, and the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region. An siRNA agent can significantly inhibit the expression of HMGCR and the corresponding mRNA in an ex vivo experiment.

Description

HMGCR inhibitors and their uses

[0001] Cross-references

[0002] This application claims priority to Chinese patent application No. 2025101186237, filed on January 24, 2025, entitled "HMGCR Inhibitor and Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedical technology, specifically to an HMGCR inhibitor and its uses. Background Technology

[0004] The HMGCR gene encodes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which catalyzes the synthesis of mevalonic acid (MVA) from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) during cholesterol synthesis. MVA is a precursor in cholesterol biosynthesis. HMG-CoA reductase is the rate-limiting enzyme in cholesterol biosynthesis; inhibiting HMG-CoA reductase in hepatocytes hinders cholesterol synthesis. Cholesterol levels in the body are mainly measured by low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TC). LDL-C is often called "bad cholesterol" because it leads to lipid deposition in blood vessels, plaque formation, or atherosclerosis, thus causing cardiovascular and cerebrovascular diseases. Statins, as competitive inhibitors of HMG-CoA reductase, are currently the main LDL-C-lowering drugs in clinical practice and are widely used for the prevention and treatment of cardiovascular diseases. However, statin treatment can lead to a compensatory increase in HMGCR protein, an effect that weakens the efficacy of statins and increases side effects. Some patients may experience muscle pain and abnormal liver enzymes; others may experience rare but serious adverse reactions, particularly statin-induced myopathy. Therefore, there is a need in the field to provide alternative therapies for subjects with lipid metabolism disorders.

[0005] Interfering RNA (siRNA), as an effective way to silence gene expression, can specifically degrade target gene mRNA. For example, HMGCR siRNA can degrade HMGCR mRNA, thereby directly inhibiting the protein expression of HMG-CoA reductase, achieving the goal of regulating cholesterol metabolism and effectively treating diseases. Summary of the Invention

[0006] To address the shortcomings of existing technologies, one objective of this application is to provide an siRNA agent that reduces HMGCR levels in subjects, comprising a sense strand and an antisense strand; the antisense strand comprises any one of the nucleotide sequences in SEQ ID NO:1 to SEQ ID NO:4508, or a sequence differing from it by no more than 1, 2, or 3 nucleotides; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region.

[0007] The antisense strand is complementary to a portion of the HMGCR mRNA; preferably, it is complementary to at least 15, 16, 17, 18, or 19 base sequences.

[0008] There are no more than 5 mismatches, preferably no more than 4, further preferably no more than 3, even more preferably no more than 2, and most preferably no more than 1 mismatch between the justice chain and the antisense chain.

[0009] Preferably, the nucleotide sequence is selected from any one of the nucleotide sequences in Table 1.

[0010] Preferably, the sense strand and / or antisense strand independently comprise one or more modified nucleotides.

[0011] Preferably, the modified nucleotide is at least one of 2′-fluorine modified nucleotide, 2′-O-methyl modified nucleotide, 2′-O-methoxyethyl modified nucleotide, 2′-O-alkyl modified nucleotide, 2′-O-allyl modified nucleotide, bicyclic nucleic acid, deoxyribonucleotide, reverse debasement residue (IB), EVP, UNA and GNA.

[0012] In some embodiments, the modified nucleotide is a nucleotide whose phosphate group is modified with thiophosphate, that is, the phosphate diester bond is replaced by a thiophosphate diester bond.

[0013] Preferably, the 5' and 3' ends of the sense and / or antisense chains independently contain 0, 1, or 2 thiophosphates, respectively.

[0014] Preferably, the nucleotide sequence is any one of the nucleotide sequences in Table 2.

[0015] Preferably, the sense strand and / or antisense strand of the siRNA each independently contain up to three unpaired nucleotides.

[0016] Furthermore, the sense and / or antisense strands of the siRNA may be the same or different in length.

[0017] Preferably, the sense strand and antisense strand of the siRNA each independently contain 20-25 nucleotides; preferably, the antisense strand of the siRNA contains 23 nucleotides and the sense strand of the siRNA contains 21 nucleotides.

[0018] Preferably, the positive chain sequence is further conjugated with a targeting ligand. "Conjugation" refers to the connection between two or more chemical structures via covalent bonds.

[0019] The targeting ligands are individually or simultaneously conjugated to the 3' or 5' end of the siRNA's positive strand.

[0020] The targeting ligand is one of the following: a delivery molecule containing N-acetylgalactosamine, a polypeptide molecule, a lipoprotein molecule, or an antibody molecule. Preferably, it is a delivery molecule containing N-acetylgalactosamine.

[0021] The delivery molecule containing N-acetylgalactosamine has the following structure:

[0022] Preferably, the siRNA agent comprises the siRNA sequence shown in Table 6.

[0023] Furthermore, the subject is a mammal, preferably a human.

[0024] The second aspect of this application also provides the use of the aforementioned siRNA agent in the preparation of medicaments for diseases or conditions caused by various lipid metabolism disorders.

[0025] Preferably, the disease or condition includes diseases or conditions related to ASCVD.

[0026] Preferably, the disease or condition is dyslipidemia, hypertriglyceridemia, hyperLDL-C, hypercholesterolemia, arterial plaque, or atherosclerosis.

[0027] Preferably, the drug comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises liposomes, microcapsules, or microspheres.

[0028] In some embodiments, the siRNA agent of the present invention is used in combination with other lipid-lowering drugs, including PCSK9 inhibitors, Lp(a) inhibitors, and ANGPTL3 inhibitors.

[0029] Preferably, the method includes administering an effective amount of the siRNA agent as described above to the target subject.

[0030] The beneficial effects of this invention are as follows: Experiments have shown that the HMGCR siRNA agent of this application can significantly inhibit the expression of HMGCR mRNA in in vitro experiments. Detailed Implementation

[0031] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention will be specifically described below with reference to specific embodiments.

[0032] The first aspect of this invention provides an siRNA agent for regulating HMGCR levels, comprising a sense strand and an antisense strand.

[0033] Specifically, the siRNA agent for regulating HMGCR levels described in this invention is primarily used to inhibit the expression of the HMGCR gene. After delivery to cells expressing the HMGCR gene, it inhibits or knocks down HMGCR expression in vitro and / or in vivo through the biological process of RNA interference (RNAi). As used herein, unless otherwise specifically indicated, HMGCR may refer to the HMGCR gene, HMGCR mRNA, or HMGCR protein, i.e., HMG-CoA reductase, where appropriate.

[0034] Specifically, the siRNA agent includes any one of the nucleotide sequences from HMGCR_1 to HMGCR_4508, or a sequence that differs from it by no more than 3 nucleotides (e.g., 0, 1, 2, 3).

[0035] Specifically, the nucleotide sequence can be in a modified state or in its original, unmodified state.

[0036] Specifically, the justice chain and the antisense chain are at least partially inversely complementary to form a dual-chain region.

[0037] Specifically, there are no more than 5, 4, 3, 2, or 1 mismatches between the justice chain and the antisense chain.

[0038] Specifically, the justice chain and the antisense chain can be completely opposite and complementary.

[0039] Specifically, the sense strand and / or antisense strand of the siRNA each independently contain unpaired nucleotides.

[0040] Furthermore, the sense strand and / or antisense strand of the siRNA each independently contain up to three unpaired nucleotides.

[0041] Specifically, the sense and / or antisense strands of the siRNA contain 20-25 nucleotides.

[0042] Furthermore, the antisense strand of the siRNA contains 23 nucleotides, and the sense strand of the siRNA contains 21 nucleotides.

[0043] Specifically, the sense and / or antisense strands of the siRNA may be the same or different in length.

[0044] Specifically, one or more of the sense and / or antisense strands are modified to form a modified nucleotide.

[0045] Specifically, the modified nucleotide is at least one of 2′-fluorine modified nucleotide, 2′-O-methyl modified nucleotide, 2′-O-methoxyethyl modified nucleotide, 2′-O-alkyl modified nucleotide, 2′-O-allyl modified nucleotide, bicyclic nucleic acid, deoxyribonucleotide, EVP, UNA and GNA.

[0046] Specifically, the siRNA has a nucleotide sequence as shown in any pair of sense and antisense strands in Table 1, Table 2 or Table 3.

[0047] The second aspect of this application also provides the use of the aforementioned siRNA agent in the preparation of a medicament for reducing the expression of HMGCR in mammals, or for preventing and / or treating related diseases or conditions, or for reducing the risk of diseases or conditions.

[0048] The diseases or conditions mentioned include those caused by various lipid metabolism abnormalities.

[0049] Specifically, the diseases or conditions caused by lipid metabolism abnormalities include obesity, dyslipidemia, non-alcoholic steatohepatitis, hypertension, stroke, coronary artery disease, and other atherosclerotic conditions.

[0050] The drug includes pharmaceutically acceptable excipients.

[0051] Specifically, pharmaceutically acceptable excipients include liposomes, microcapsules, and microspheres.

[0052] A third aspect of this application provides a method for preventing or treating a disease or condition, the method comprising administering an effective amount of the aforementioned siRNA agent or the aforementioned pharmaceutical composition to a target subject.

[0053] Specifically, the administration includes subcutaneous, intravenous, oral, rectal, or intraperitoneal administration.

[0054] The present application will be further explained below with reference to specific embodiments:

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available, and techniques not described in detail were performed according to standard methods well known to those skilled in the art.

[0056] Example 1: siRNA sequence design

[0057] The antisense strand of siRNA was generated by computer to complement the human HMGCR DNA sequence (NM_000859.3), and the sense strand was then generated by complementation. The nucleotide sequence composition is shown in Table 1.

[0058] Table 1 siRNA sequences

[0059] To screen for more efficient siRNA agents, computer analysis was used to predict the inhibitory efficiency of the siRNA sequences in Table 1. siRNA sequences with high inhibitory efficiency were selected and then modified. The modified double-stranded siRNA sequences targeting HMGCR are shown in Table 2.

[0060] Table 2. Sequences of modified double-stranded siRNA agents targeting HMGCR

[0061] In the table, A = adenosine-3'-phosphate.

[0062] C = Cytidine-3'-phosphate

[0063] G = Guanosine-3'-phosphate

[0064] U = uridine-3'-phosphate

[0065] Am = 2'-O-methyladenosine-3'-phosphate

[0066] Ams = 2'-O-methyladenosine-3'-thiophosphate

[0067] Cm = 2'-O-methylcytidine-3'-phosphate

[0068] Cms = 2'-O-methylcytidine-3'-thiophosphate

[0069] Gm = 2'-O-methylguanosine-3'-phosphate

[0070] Gms = 2'-O-methylguanosine-3'-thiophosphate

[0071] Um = 2'-O-methyluridine-3'-phosphate

[0072] Ums = 2'-O-methyluridine-3'-thiophosphate

[0073] Af = 2'-Fluoroadenosine-3'-phosphate

[0074] Afs = 2'-Fluoroadenosine-3'-Thiophosphate

[0075] Cf = 2'-Fluorocytidine-3'-phosphate

[0076] Cfs = 2'-Fluorocytidine-3'-Thiophosphate

[0077] Gf = 2'-Fluoroguanosine-3'-phosphate

[0078] Gfs = 2'-Fluoroguanosine-3'-Thiophosphate

[0079] Uf = 2'-fluorouridine-3'-phosphate

[0080] Ufs = 2'-fluorouridine-3'-thiophosphate

[0081] Example 2: Preliminary in vitro analysis of HMGCRRNAi agent

[0082] The HMGCR RNAi agents listed in Table 2 were synthesized in the solid phase, and their in vitro efficacy was screened. The results are shown in Table 3.

[0083] For screening purposes, the human HMGCR cDNA sequence (accession number NM_000859.3, synthesized by GenScript) was subcloned into the commercially available reporter-based screening plasmid psiCHECK2, generating Renalis luciferase / HMGCR fusion mRNA. To assess the efficacy of HMGCR RNAi agents in a human background, Huh7 cells (human hepatocellular carcinoma line) were seeded at approximately 10,000 cells / well in 96-well plates. Each HMGCR RNAi agent was mixed at two concentrations (10 nM and 1 nM) with 15 ng psiCHECK2-HMGCR plasmid DNA per well and 0.2 μL Lipofectamine 2000 per well. TM (Purchased from Invitrogen, cat#11668-019) Co-transfected together. Gene knockdown was determined by measuring the level of firefly luciferase (purchased from Promega, cat#E2940) using a dual luciferase reporter and normalizing it to the level of Renida luciferase, which is also present on the psiCHECK2 plasmid.

[0084] Table 3. Gene knockdown levels detected by Renal luciferase assay

[0085] Example 3: Screening of non-conjugated siRNA molecules by inhibiting HMGCR mRNA expression in human Huh7 cells.

[0086] Each HMGCR RNAi agent was mixed with 0.2 μL of Lipofectamine per well at two concentrations (10 nM and 1 nM). TMRNAiMAX (Invitrogen, cat#13778-150) was incubated in 96-well plates. Huh7 cells were seeded at approximately 10,000 cells / well in each 96-well plate. 24 hours after transfection, total RNA was extracted using the RNeasy 96 kit (QIAGEN, cat#74182). cDNA synthesis and real-time PCR were performed using a reverse transcription kit (Vazyme HiScript III RT SuperMix for qPCR, cat#R323-01) and a qPCR kit (TIANGEN, cat#FP209). HMGCR mRNA levels were determined by qRT-PCR, and the normalized percentage of housekeeping gene Actin mRNA expression relative to the corresponding samples was calculated. The results are shown in Table 4.

[0087] Table 4. HMGCR mRNA levels in human huh7 cells

[0088] Example 4: Inhibition of HMGCR mRNA expression in human Huh7 cells by different concentrations of siRNA molecules

[0089] Each HMGCR RNAi agent was mixed with 1 μL of Lipofectamine per well at various concentrations (10 pM-10 nM). TM RNAiMAX (Invitrogen, cat#13778-150) was incubated in 24-well plates. Huh7 cells were seeded at approximately 80,000 cells / well in each well. 24 hours after transfection, total RNA was extracted using the RNeasy 96 kit (QIAGEN, cat#74182). cDNA synthesis and real-time PCR were performed using a reverse transcription kit (Vazyme HiScript III RT SuperMix for qPCR, cat#R323-01) and a qPCR kit (TIANGEN, cat#FP209). HMGCR mRNA levels were determined by qRT-PCR, and the normalized percentage of housekeeping gene Actin mRNA expression relative to the corresponding samples was calculated. The results are shown in Table 5.

[0090] Table 5. HMGCR mRNA levels in human Huh7 cells

[0091] Example 5: Inhibition of HMGCR gene expression in mice injected with human HMGCR plasmid via tail vein hyperbaric injection using candidate HMGCR siRNA sequences of conjugated ligands.

[0092] Based on the results in Examples 2-4, the applicant selected several candidate HMGCR siRNA sequences with good inhibitory effects, conjugated them with ligands, and tested their inhibitory effects on HMGCR gene expression. The specific sequences of the conjugated HMGCR siRNAs are shown in Table 6, where XHY-2-009 is a sequence from the prior art, which serves as a positive control with known efficacy.

[0093] Table 6. siRNA sequences for conjugated target ligands

[0094] In this case, the lowercase 's' indicates that the molecules on both sides are linked by thiophosphate esters.

[0095] The structural formula for [Gal-6][Gal-6][Gal-6] is:

[0096] The L96 structural formula is:

[0097] On day -4 (D-4), male NOD-SCID mice were injected via high-pressure tail vein injection (HDI) with the human HMGCR plasmid carrying the luciferase gene (containing the human HMGCRNM_000859.3 cDNA sequence) to establish an HDI HMGCR-GLuc mouse model. On day 0 (D0), mice were divided into groups of four and received a single subcutaneous injection of 3 mg / kg of the test substance. Serum was collected two days before administration (D-2) and on days 2, 7, 14, 21, 28, and 35 after administration to detect luciferase expression levels. Normalized percentages were calculated by comparing these levels with those before administration (D-2) and in the PBS group to reflect the effect of the test substance on HMGCR gene expression. The experimental results are shown in Table 7.

[0098] Table 7. Luciferase Expression Results

[0099] The results showed that XHY-2-001, XHY-2-002, XHY-2-004, XHY-2-005, XHY-2-006, XHY-2-007, XHY-2-008 and XHY-2-009 had comparable inhibitory effects at D2. However, at D21 and D28, the siRNA agent with the conjugated ligand provided in this application had significantly better inhibitory effects than XHY-2-009, and even better than XHY-2-009 at D7 and D14.

[0100] Example 6: Effect of HMGCR mRNA expression in human primary hepatocytes

[0101] The HMGCR RNAi agent in Table 6 was mixed with 1 μM of Lipofectamine per well. TM RNAiMAX (Invitrogen, cat#13778-150) was used for incubation. Human primary hepatocytes were seeded at approximately 80,000 cells / well in 24-well plates onto the incubated transfection complex. Forty-eight hours post-transfection, total RNA was extracted using the RNeasy 96 kit (Qiagen, cat#74104). cDNA synthesis and real-time PCR were performed using a reverse transcription kit (Vazyme HiScript III RT SuperMix for qPCR, cat#R323-01) and a qPCR kit (TIANGEN, cat#FP209). HMGCR mRNA levels were determined by qRT-PCR relative to Actin mRNA expression (a housekeeping transcript) in the corresponding samples; the results are shown in Table 8.

[0102] Table 8. Remaining HMGCR mRNA Levels

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any other changes, modifications, substitutions, combinations and simplifications made under the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A siRNA agent that reduces HMGCR levels in subjects, comprising a sense strand and an antisense strand; The antisense strand includes any one of the nucleotide sequences in SEQ ID NO:1 to SEQ ID NO:4508, or a sequence that differs from it by no more than 3 nucleotides; The justice chain and the antisense chain are at least partially inversely complementary, forming a dual-chain region.

2. The siRNA agent of claim 1, wherein There are no more than 5 mismatches, preferably no more than 4, further preferably no more than 3, even more preferably no more than 2, and most preferably no more than 1 mismatch between the justice chain and the antisense chain.

3. The siRNA agent of claim 1, wherein The sense strand and / or antisense strand independently contain one or more modified nucleotides.

4. The siRNA agent of claim 3, wherein The modified nucleotide is at least one of 2′-fluorine modified nucleotide, 2′-O-methyl modified nucleotide, 2′-O-methoxyethyl modified nucleotide, 2′-O-alkyl modified nucleotide, 2′-O-allyl modified nucleotide, bicyclic nucleic acid, deoxyribonucleotide, EVP, UNA and GNA.

5. The siRNA agent of claim 4, wherein The nucleotide sequence is any one of the nucleotide sequences in Table 2.

6. The siRNA agent of claim 1, wherein The sense and / or antisense strands of the siRNA each independently contain up to three unpaired nucleotides.

7. The siRNA agent of claim 1, wherein The sense and / or antisense strands of the siRNA may be the same or different in length.

8. The siRNA agent of claim 1, wherein The sense and antisense strands of the siRNA each independently contain 20-25 nucleotides; preferably, the antisense strand of the siRNA contains 23 nucleotides and the sense strand of the siRNA contains 21 nucleotides.

9. The siRNA agent of any one of claims 1-8, wherein, The positive strand sequence is also linked to a ligand, and preferably, the siRNA agent comprises the siRNA sequence pairs shown in Table 6.

10. The siRNA agent of claim 1, wherein The subjects are mammals, preferably humans.

11. Use of the siRNA agent according to any one of claims 1-0 in the preparation of a medicament for diseases or conditions caused by various lipid metabolism disorders.

12. Use according to claim 11, characterized in that, The disease or condition mentioned includes diseases or conditions related to ASCVD.

13. Use according to claim 12, characterized in that, The disease or condition mentioned is dyslipidemia, primary dyslipoproteinemia, hypertriglyceridemia, or atherosclerosis.

14. Use according to any one of claims 11 to 13, characterized in that, The drug includes pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients include liposomes, microcapsules, or microspheres.

15. A method for reducing HMGCR expression in vivo or intracellularly in a subject, the method comprising administering an effective amount of the siRNA agent as described in any one of claims 1-10 to the target subject.

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